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Immunomodulatory effects of LL-37 in the epithelia Filewod, Niall Christopher Jack 2008

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IMMUNOMODULATORY EFFECTS OF LL-37 IN THE EPITHELIA by Niall Christopher Jack Filewod B.Sc.(H), Queen’s University, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA Vancouver June 2008 © Niall Christopher Jack Filewod, 2008  ABSTRACT The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 µg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.  ii  TABLE OF CONTENTS Abstract................................................................................................................................ii List of Tables .......................................................................................................................v List of Figures.....................................................................................................................vi Acknowledgements: ........................................................................................................ viii Dedication:..........................................................................................................................ix Co-authorship statement ......................................................................................................x Chapter I ..............................................................................................................................1 Introduction .....................................................................................................................1 Host defence peptides ......................................................................................................2 Induced expression of host defence peptides...................................................................4 Pro-inflammatory effects of host defence peptides .........................................................7 Wound-healing activities of host defence peptides .........................................................9 Theme and hypothesis ...................................................................................................11 Literature Cited..............................................................................................................13 Chapter II ...........................................................................................................................23 Introduction ...................................................................................................................23 Materials and Methods ..................................................................................................27 Cell cultivation: .........................................................................................................27 Reagents: ...................................................................................................................27 Cell stimulation: ........................................................................................................28 Assays:.......................................................................................................................28 Inhibitor studies: ........................................................................................................29 Western blotting: .......................................................................................................29 Results: ..........................................................................................................................31 LL-37 increased IL-8 production by subconfluent keratinocytes in response to pro-inflammatory stimuli........................................................................31 LL-37 increased IL-8 production by calcium-differentiated keratinocytes in response to pro-inflammatory stimuli. ............................................34 LL-37 increased IL-8 production by bronchial epithelial cells in response to pro-inflammatory stimuli........................................................................36 Co-stimulation of bronchial epithelial cells with LL-37 and poly(I:C) elicited a rapid IL-8 response and delayed cytotoxicity. ...........................................39 Inhibition of the synergistic increase in IL-8 production by inhibitors of Src-family kinase signalling or NF-κb translocation ............................................43 Stimulation of keratinocytes with LL-37 and flagellin resulted in a strong increase in the phosphorylation of the transcription factor CREB.........................................................................................................................45 Discussion......................................................................................................................46 Acknowledgements: ......................................................................................................49 Literature cited:..............................................................................................................50 Chapter III..........................................................................................................................54 Introduction ...................................................................................................................54 Materials and Methods ..................................................................................................56 Cell cultivation ..........................................................................................................56 Reagents.....................................................................................................................56 RNA isolation ............................................................................................................56 Microarray analysis ...................................................................................................57 iii  Bioinformatic analysis ...............................................................................................58 Results: ..........................................................................................................................59 Treatment with low doses of LL-37 resulted in altered gene expression ..................................................................................................................59 The differentially expressed genes might have been co-regulated by a set of common transcription factors. .........................................................................67 Pathway and gene ontology over-representation analysis suggested that LL-37 selectively activates genes involved in protein synthesis, tissue remodelling, and innate immune responses.....................................................68 Discussion:.....................................................................................................................76 Literature cited:..............................................................................................................79 Chapter IV .........................................................................................................................81 Literature cited:..............................................................................................................87 Appendix ...........................................................................................................................91  iv  LIST OF TABLES Table 3.1: Selected genes showing differential expression in keratinocytes 1 hour post-treatment with 3 µg/ml LL-37. .......................................................................59 Table 3.2: Selected genes showing differential expression in keratinocytes 2 hours post-treatment with 3 µg/ml LL-37. .....................................................................61 Table 3.3: Selected genes showing differential expression in keratinocytes 4 hours post-treatment with 3 µg/ml LL-37. .....................................................................63 Table 3.4: Transcription factor binding sites overrepresented in the promoter regions of genes showing differential expression after stimulation with 3 µg/ml LL-37. .......................................................................................67 Table 3.5: Results of pathway over-representation analysis. ............................................69 Table 3.6: Over-represented gene ontology terms associated with differentially expressed genes at 1, 2, and 4 hours after stimulation with 3 µg/ml LL-37.......................................................................................................................73 Supplementary Table 7: Differentially expressed genes 1 hour poststimulation with 3 µg/ml LL-37. .......................................................................................91 Supplementary Table 8: Differentially expressed genes 2 hours poststimulation with 3 µg/ml LL-37. .....................................................................................108 Supplementary Table 9: Differentially expressed genes 4 hours poststimulation with 3 µg/ml LL-37. .....................................................................................119  v  LIST OF FIGURES Figure 2.1: LL-37 alters IL-8 production by subconfluent keratinocytes in response to flagellin...........................................................................................................32 Figure 2.2: LL-37 alters of IL-8 production by subconfluent keratinocytes in response to IL-1β...........................................................................................................32 Figure 2.3: Low doses of LL-37 synergistically increase IL-8 production by subconfluent keratinocytes in response to flagellin and IL-1β.....................................33 Figure 2.4: LL-37 alters IL-8 production by differentiated keratinocytes in response to flagellin...........................................................................................................34 Figure 2.5: LL-37 alters IL-8 production by differentiated keratinocytes in response to IL-1β...............................................................................................................35 Figure 2.6: Low doses of LL-37 synergistically increase IL-8 production by differentiated keratinocytes in response to flagellin and IL-1β....................................35 Figure 2.7: LL-37 alters IL-8 production by bronchial epithelial cells in response to flagellin...........................................................................................................37 Figure 2.8: LL-37 alters IL-8 production by bronchial epithelial cells in response to IL-1β...............................................................................................................37 Figure 2.9: LL-37 alters IL-8 production by bronchial epithelial cells in response to PAM3CSK4....................................................................................................38 Figure 2.10: Low doses of LL-37 synergistically increase IL-8 production by bronchial epithelial cells in response to flagellin and IL-1β ........................................39 Figure 2.11: Co-stimulation of bronchial epithelial cells with LL-37 and poly(I:C) elicits a rapid IL-8 response...............................................................................40 Figure 2.12: Co-stimulation of bronchial epithelial cells with LL-37 and poly(I:C) results in rapid and pronounced cytotoxicity.....................................................41 Figure 2.13: Low doses of LL-37 synergistically increase IL-8 production by bronchial epithelial cells in response to poly(I:C)........................................................42 Figure 2.14: Low doses of LL-37 synergistically increase cytotoxicity subsequent to treatment with poly(I:C). ............................................................................42 Figure 2.15: The Src-family kinase inhibitors PP2 and SU6656 suppress IL-8 production by keratinocytes in response to co-stimulation with LL-37 and flagellin. ......................................................................................................................44  vi  Figure 2.16: The NF-κB inhibitor Bay11 suppresses IL-8 production by keratinocytes in response to co-stimulation with LL-37 and flagellin ..............................45 Figure 2.17: Low doses of LL-37 do not alter the increased phosphorylation of CREB observed after keratinocytes are stimulated with flagellin ..............................................................................................................................46 Figure 3.1: Genes within the 'Prostate cancer' pathway showing altered regulation after stimulation with 3 µg/ml LL-37...............................................................73  vii  ACKNOWLEDGEMENTS First and foremost I would like to acknowledge the kindness and support of my supervisor, Dr. Bob Hancock, and my committee members, Dr. Pauline Johnson and Dr. Mike Gold. Without their help it would have been impossible to complete this thesis on the timeline I have followed. I would like to thank the entire Hancock lab for their help over the last two yearsparticularly, I am indebted to Sheena Tam for her help with ELISAs, to Yue Xin Li for having helped me find my feet, and to Jelena Pistolic for her constant guidance. Shaan Gellatly, Aaron Wyatt, and Laurence Madera have been great co-workers and even better friends. Outside of the lab, I would like to thank the friends who have become my family in Vancouver. They know who they are, but I would like to single out Peter and Terry Herd, who helped me make this city home, and Shawn Northwood, who did everything from lending me his car at midnight to handing me a beer when I just needed a break.  viii  DEDICATION  This thesis is dedicated to my grandmother, Dr. A. A. Crowder, because I still know the difference between a grass and a sedge.  ix  CO-AUTHORSHIP STATEMENT The research program from which these results were generated was collaboratively designed by Dr. R. E. W. Hancock and N. C. J. Filewod. Experiments were designed, performed, and analysed by NCJF. The manuscript was written by NCJF and REWH. A version of Chapter II will be submitted for publication as follows: Filewod, N. C. J., Pistolic, J. and R. E. W. Hancock. Low physiological doses of the human host defence peptide LL-37 alter the responses of keratinocytes and bronchial epithelial cells to pro-inflammatory stimuli. A version of Chapter III will eventually be submitted for publication as follows: Filewod, N. C. J., Falsafi, R., Gardy, J., and R. E. W. Hancock. Title to be determined.  x  CHAPTER I  INTRODUCTION The innate immune system forms the basis of the body’s defence against invasive microorganisms. Due to the frequency with which microbes are encountered, innate immunity is, through necessity, both highly reactive and highly regulated; inflammation is a necessary response to infection and injury, but unnecessary or uncontrolled inflammation can have extremely deleterious effects. The regulation of differential responses to both harmless and pathogenic microbes is accordingly a subject of great interest, as an improved understanding of the processes governing innate immune responses might lead to novel therapeutics to combat infection (1). The epithelia are one of the frontlines of this complex defence network. Surfaces such as the skin and the bronchial epithelium are not merely physical barriers, but play roles as sentinels of the innate immune system, activating further host defences upon infection or injury. Both keratinocytes (the major cell type present in the skin) and bronchial epithelial cells release chemokines that attract other immune cells after exposure to pathogen-associated signature molecules such as flagellin, the protein monomer that is the basic unit of bacterial flagella (2, 3), while keratinocytes have been suggested to both directly attack extracellular bacteria via the production of antimicrobials (4) and to destroy internalized bacteria via autophagy (5). Hence, the skin and bronchial epithelium are capable of rapid and effective responses to pathogens. One such response is the production of host defence peptides, a diverse array of innate immune molecules that play a role in both the initial inflammatory response to infection and injury and the eventual restoration of tissue homeostasis via wound-healing 1  activities. This chapter reviews the role of host defence peptides in the skin and bronchial epithelium, and discusses how their immunomodulatory properties render them fundamentally important to epithelial innate immunity.  HOST DEFENCE PEPTIDES Host defence peptides are an evolutionarily ancient component of innate immunity. The number and structural diversity of described peptides (6) suggests that they may be ubiquitous to complex life. As a class, however, animal host defence peptides share common characteristics: being generally short (12 – 50 amino acids long) and positively charged (7). Their cationic and amphipathic nature enables host defence peptides to interact with negatively charged lipids integral to all biological membranes. This enables many peptides to either destroy the permeability barrier or attack anionic cytoplasmic targets and exert direct antimicrobial activity against both Gram-negative and Gram-positive bacteria and fungi (8). Considerable research activity has been devoted to the development of modified host defence peptides as ‘natural antibiotics’ (9). More recently, however, many host defence peptides have been shown to modulate systemic immune responses and provoke wound healing in epithelial tissues (10-12), suggesting that their in vivo role may extend beyond the direct killing of pathogens. Several classes of host defence peptides are present in human epithelia, of which the defensin family and the cathelicidin LL-37 are the most important. Defensins are characterized by a three-stranded β-sheet (the ‘defensin fold’) and the presence of three internal disulphide bonds, the arrangement of which distinguishes α- and β-defensins (13). (A third group, the θ-defensins, are cyclic molecules that are not expressed in humans (14)). Of the α-defensins, the Human Neutrophil Peptides 1 – 3 are employed by neutrophils in the killing of microbes, while Human Defensins 5 and 6 are expressed by 2  the Paneth cells of the small intestine (15) and in the epithelia of the female reproductive tract (16). The four well-characterized β-defensins play an important role in epithelial defence. Human β-defensin-1 (hBD1) is constitutively expressed in a variety of epithelial tissues, including bronchial epithelium and skin (15, 17), while hBD2 – 4 show inducible expression in both tissues (18-23). The host defence peptide LL-37 is the sole member of the cathelicidin family expressed in humans. LL-37, which is an amphipathic α-helical peptide, is a cleavage product of a larger protein, the 18-kDa Human Cathelidin Protein (hCAP18) (24). hCAP18 was originally isolated from the specific granules of neutrophils, where it has been proposed that LL-37 plays a similar role to the Human Neutrophil Peptides in the direct killing of phagocytosed bacteria (25), although there is little evidence of large amounts of the cleaved product LL-37 inside cells and hCAP18 has no direct antimicrobial  activity.  In  addition  to  neutrophils,  LL-37  is  expressed  by  monocytes/macrophages, NK cells, γδ T cells, B cells (26), and mast cells (27). Expression patterns vary between epithelial tissues; LL-37 is constitutively expressed by lung epithelial cells, and secreted into airway fluids (28), and constitutively expressed by the non-keratized squamous epithelia of the buccal mucosa, tongue, esophagus, cervix and vagina (29). Keratinocytes, however, have been suggested to express LL-37 only following induction by pro-inflammatory compounds or during disease states (30). The value of the expression of host defence peptides by epithelial cells was originally thought to rely upon the ability of both defensins and LL-37 to act as ‘natural antibiotics’ by impairing the integrity of lipid membranes, a non-specific antimicrobial activity that allows them to be effective against both Gram-negative and Gram-positive bacteria, fungi, and enveloped viruses (8, 18, 19, 31, 32). This antimicrobial activity, however, is extremely salt-sensitive, and some controversy exists over its in vivo 3  relevance; a convincing body of work suggests that defensins and LL-37 are unlikely to show significant antimicrobial activity in the presence of physiological concentrations of mono- and divalent cations (10, 33), except at very high concentrations (e.g. α-defensins in the phagolysosomes of neutrophils or β-defensins in the crypts of the intestine). However some authors maintain that these host defence peptides do in fact retain antimicrobial activity in tissue culture medium (4). While the defensins and LL-37 might show direct antimicrobial activity at epithelial surfaces at areas of high local concentrations (e.g. immediately following release from the granules of neutrophils), it seems unlikely that they would show significant antimicrobial activity in the epithelia itself. Instead, I hypothesize that their primary importance is dependent on their ability to mediate responses to infection and wounding.  INDUCED EXPRESSION OF HOST DEFENCE PEPTIDES The ability of epithelial cells to produce host defence peptides in response to the presence of pathogens relies on their expression of conserved families of receptor molecules specific for pathogen ‘signature’ molecules (also termed Pathogen Associated Molecular Patterns); that is, molecules present, and loosely conserved, in the structure of bacteria and viruses that do not normally appear in eukaryotic cells. Toll-like and Nodlike Receptors (TLRs and NLRs, respectively) both fulfill this function, as do RIG-1-like RNA helicases (RLRs). TLRs are membrane-associated receptors (34), while NLRs and RLRs are cytosolic (35, 36). Although they use different mechanisms of signal transduction, the stimulation of either TLRs or NLRs can result in increased host defence peptide expression in epithelial cells (37, 38). As the work in this thesis concerns TLR signalling, the topic will be discussed briefly here.  4  Since the recognition of the importance of TLRs to innate immunity (39), much has been discovered about their specificities and mechanisms of signal transduction [reviewed in (34) and (40)]. The TLRs can be broken down into groups based on their ligand specificity. Various lipid-based microbial molecules are recognized by TLR4 (as a homodimer) or TLR2 (as a heterodimer with TLR1 or TLR6), while TLR3 homodimers and various homo- and heterodimers of TLR7, TLR8 and TLR9 recognize nucleic-acids and related molecules. TLR5 is a receptor for the bacterial protein flagellin. TLRs are presumed to form low-affinity dimers, which undergo a change in configuration when their extramembrane domains interact with ligand. This conformational shift allows the recruitment of specific adaptors, which bind the cytosolic face of the TLR complex via the interaction of Toll/IL-1 Receptor (TIR) domains. Five such adaptors have been characterized (MyD88, MAL, TRIF, TRAM, and SARM); they are differentially recruited to various TLR complexes, allowing for variation in downstream responses. With the exception of TLR3 signalling and some TLR4 responses, however, the involvement of MyD88 in the signalling complex leads to the recruitment of IRAK4 and, through a signal transduction cascade, the eventual activation of the transcription factor NF-κB (among others) or the activation of the MAP kinases p38 and c-Jun aminoterminal kinase (JNK). In the epithelia, these responses are fundamentally important to the mobilization of innate immune defences and the activation of adaptive immunity (4143), and play a central role in the control of the inducible expression of hDB2-4. Wounding can also increase the expression of host defence peptides in infected epithelial tissues. After skin is wounded, vitamin D3 signalling results in increased expression of hCAP18 (44, 45); similarly, the promotor region of hCAP18 contains several elements responsive to IL-6, a cytokine produced by epithelial cells in response to both damaged cell parts and infection (29). The expression of hBD2 is also increased 5  in wounded skin and chronic wounds (46), although it exhibits decreased expression in burned tissue (47). Both LL-37 and hBD2 are thus inducible by wounding. Both wounding and the presence of pathogen signature molecules can increase the expression of host defence peptides by epithelial cells; the quantification of actual concentrations of peptide in vivo, however, is technically difficult. Some data is available on systemic concentrations of the hBDs: hBD2 is present in plasma at concentrations of about 8.3 fmol/ml, which increase almost 4-fold during bacterial pneumonia (48), while hBD3 has been observed to have a normal serum concentration of about 140 pg/ml, which rises to about 250 pg/ml during bacterial pneumonia (49). The relevance of these findings to the epithelia is uncertain. Human β-defensin 2, however, has been described to increase from barely detectable levels to almost 100 pg/ml in the bronchoalveolar lavage of patients with diffuse panbronchiolitis, a condition characterized by inflammation and recurrent infection by Pseudomonas aeruginosa (50). The in vivo concentrations of LL-37 are better characterized. Tracheal aspirates from healthy neonates contain concentrations of approximately 5 µg/ml and concentrations increase 2to 3-fold during systemic or pulmonary infections (51). Bronchoalveolar lavage fluids from adult cystic fibrosis patients contain concentrations of LL-37 ranging from 0 – 16 µg/ml (52). In the skin, concentrations can range widely; keratinocytes do not usually express LL-37 (30), however LL-37 is present in sweat at concentrations averaging about 5 µg/ml (53), and has been suggested to be present in concentrations as high as 1 mg/ml in psoriatic scales (54). Taken together, these findings suggest that inflammation and wounding can induce increases in the expression of host defence peptides, creating localized areas of high concentration where they can act to promote immune responses, and, eventually, tissue repair.  6  PRO-INFLAMMATORY EFFECTS OF HOST DEFENCE PEPTIDES The β-defensins and LL-37 possess many immunomodulatory effects, allowing them to recruit immune cells to the site of epithelial invasion, stimulate the release of chemical messengers by epithelial cells, and activate immune cells to better respond to the presence of pathogens. Various β-defensins are chemoattractants for monocytes, neutrophils, T cells, immature dendritic cells, and mast cells (55-58), while LL-37 can recruit T cells, neutrophils, monocytes (59), and mast cells (27). The expression of host defence peptides in wounded or infected tissue can therefore help facilitate an immediate immune response. Host defence peptides can also elicit the release of a variety of cytokines by epithelial cells, thereby inducing a number of secondary effects on the innate immune response. In in vitro experiments, hBD2-4 and LL-37 stimulate the release of IL-8, IL18, IL-6, IL-10, IP-10, MCP-1, MIP-3α, and RANTES by keratinocytes (11, 60). Similarly, LL-37 induces the production of IL-8 by bronchial epithelial cells (61, 62), and airway smooth muscle cells (63). The secretion of this plethora of chemical messengers aids in the recruitment of immune cells and may allow other pleiotropic effects. In addition, both β-defensins and LL-37 can alter the responses of immune cells, allowing them to modulate the immune response in ways that may facilitate the resolution of infection. A prototypic example of these effects is the ability of LL-37 injection to block sepsis in both rat and mouse models (61, 64); intriguingly, there is a detectable protective effect even when the peptide is administered 12 h prior to the induction of sepsis (65). In humans, LL-37 can dramatically reduce the production of TNF-α by peripheral blood mononuclear cells (PBMCs) treated with LPS (66). While this response may not always be relevant to epithelial infection, it is interesting that LL7  37 and the human β-defensins alter mast cell behaviour. Exposure to host defence peptides results in increased expression of TLRs and production of proinflammatory cytokines (67), and can also provoke mast cell degranulation (27, 68), accordingly increasing the permeability of skin vasculature (69). Both hBD3 and LL-37 can also antagonize neutrophil apoptosis, which has been suggested to enable a more prolonged response to invading pathogens (70 , 71). The expression of inducible host defence peptides thus appears to be an excellent alarm signal, resulting in the rapid mobilization of immune cells and provoking local inflammation. Unfortunately no single mechanism can explain these effects. For instance, βdefensins recruit dendritic and T cells using the CCR6 receptor (57), but CCR6 is not required for the chemotaxis of macrophages and mast cells (72). Similarly, the receptor FPRL-1 mediates LL-37-induced chemotaxis of T cells, neutrophils and monocytes (59), but is not involved in mast cell chemotaxis (27). Nor is there a clear consensus on how host defence peptides elicit cytokine production by epithelial cells. A number of candidate receptors have been suggested, but the fact that a synthetic form of LL-37 composed entirely of D amino acids can also provoke IL-8 release from keratinocytes argues against a specific conventional receptor (73). The observation of differential responses to LL-37 treatment has led some authors to suggest that host defence peptides possess a complex mode of action with multiple points of intervention (66). Regardless, the ability of host defence peptides to mobilize epithelial defences is of considerable importance in vivo. Overexpression of HBD2 in rat lung tissues is protective against Pseudomonas aeruginosa infection and sepsis-induced lung injury (74). Similarly, mice inoculated with cancerous cells that overexpress HBD2 are more resistant to bacterial infections (75). The creation of a total β-defensin knockout mouse has not yet been reported, but a knockout of the murine homologue of HBD1 has been 8  created. The resultant phenotype is mild, perhaps due to the diversity of murine defensins, which may create considerable redundancy in function, but is associated with delayed clearance of pulmonary Haemophilus influenzae infection (76) and increased proclivity to colonization of the bladder by Staphylococcal bacteria (77). Similarly, mice deficient in the murine homolog of LL-37, CRAMP, are more prone to skin infections (78). These effects have been suggested to be the result of direct antimicrobial action by the host defence peptide in question, but could equally well result from improved or impaired activation of the immune response associated with altered peptide expression. The importance of host defence peptides to epithelial homeostasis is also strongly suggested by their association with human disease states. For instance, dysregulation of defensin expression coincides with Crohn’s disease, an inflammatory bowel syndrome (79), whereas increased genomic β-defensin copy number is associated with the incidence of psoriasis, an inflammatory disorder of the skin (80). The LL-37 precursor hCAP18 is selectively induced in keratinocytes in a variety of inflammatory skin disorders, including psoriasis, subacute lupus erythematosis, nickel allergy challenge, and atopic dermatitis (30). LL-37 is also induced in verruca vulgaris, a condition caused by Human Papilloma Virus (HPV) infection (81), and in fungal infections of the skin (82) Multiple lines of evidence reaffirm the role of host defence peptides as pluripotent effectors of innate immunity in both skin and lung tissues, able to activate responses to pathogens and mediate an eventual return to homeostasis.  WOUND-HEALING ACTIVITIES OF HOST DEFENCE PEPTIDES As might be expected of molecules secreted by damaged tissue, host defence peptides have the ability to stimulate the migration and proliferation of epithelial cells, and the formation of new blood vessels, a process known as angiogenesis. The 9  importance of this activity in vivo is demonstrated by the association of improperly regulated host defence peptide expression with disease states involving chronic infection and hyper-proliferation. In addition to eliciting inflammatory responses to pathogens, host defence peptides facilitate the eventual return to tissue homeostasis. Relatively little work has been done to characterize the wound-healing abilities of β-defensins, although keratinocytes treated with the growth factors insulin-like growth factor 1 and TGF-α have been shown to increase their expression of hBD3 (83), and hBD2-4 are known to stimulate keratinocyte proliferation and migration (60). LL-37, in contrast, has been extensively studied as a growth-stimulating agent. LL-37 induces cell migration, cell proliferation, and the healing of mechanically induced wounds in cultured bronchial epithelial cells at a concentration of 1 µg/ml (84) and acts as a growth factor for lung cancer cells at concentrations as low as 5 ng/ml (85). The peptide also induces migration and proliferation in primary keratinocytes (60, 86) and keratinocyte cell lines (87). A number of studies have implicated EGFR signalling in the ability of LL-37 to stimulate cell proliferation and migration (84-86). Activation of EGFR leads to signal transduction by STAT1 and STAT3 (60, 86) and, in the HaCaT keratinocyte cell line, results in the induction of the transcription factors Snail and Slug, which control proliferative responses, and the activation of matrix metalloproteases, which play roles in tissue remodelling (87). Wound repair also requires the formation of new blood vessels; LL-37 has been shown to stimulate angiogenesis in a rabbit hind limb model, via an FPRL-1 receptor dependent mechanism (88). LL-37 is thus an important factor in epithelial wound repair. The in vivo relevance of LL-37 stimulated wound healing has been demonstrated in a number of studies. Adenoviral gene transfer of hCAP18 stimulates wound healing in diabetic mice (87) and it has been observed that LL-37 is lacking in chronic ulcers in 10  human skin (44). Furthermore, treatment of excised wounded keratinocytes with an antiLL-37 antibody inhibits cell proliferation (44). Psoriasis, which in addition to being an inflammatory disorder is also marked by increased keratinocyte proliferation, is also associated with increased expression of hBD2 and LL-37 (54, 89), although it is unclear whether this is a result of the disorder or a contributing factor to its etiology. In conclusion, there is good evidence that LL-37 (and possibly also hBD2-4) act as growth factors for epithelial cells both in vitro and in vivo.  THEME AND HYPOTHESIS In the skin and lung epithelia, LL-37 plays an important role in the regulation of tissue homeostasis; at high concentrations it mediates a pro-inflammatory response that combats infection, whereas at lower concentrations it stimulates wound healing. Systemically, LL-37 exhibits potent immunodulatory activity, but LL-37-mediated immunomodulation has not been observed in epithelial tissues. Taken together, these observations have led me to address the question: what role do low, physiologically relevant concentrations of LL-37 exert on epithelial innate immune responses? Although LL-37 accumulates to high concentration during inflammation, its role in the initiation of inflammation, when concentrations are presumably much lower, is poorly understood. As LL-37 may be involved in the etiology of psoriasis (90), a medical condition that involves dysregulated epithelial inflammation, the results of these studies might have medical relevance. I hypothesized that low, physiologically relevant concentrations of LL-37, such as would be expected to be present in epithelial tissues prior to inflammation, would alter TLR-mediated IL-8 responses in epithelial cells, and that the altered response would be reliant on MAPK signalling. This hypothesis was investigated here using cultured 11  primary keratinocytes and bronchial epithelial cells by exposing these cells to proinflammatory stimuli in the presence or absence of low concentrations of LL-37, and initially assessing IL-8 production will be assayed by ELISA. This research thus aimed to increase our understanding of the role of host defence peptides in the regulation of inflammation in the epithelia.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Gilliet. 2007. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449:564-569.  22  CHAPTER II1  Low physiological doses of the human host defence peptide LL-37 alter the responses of keratinocytes and bronchial epithelial cells to pro-inflammatory stimuli  INTRODUCTION The cationic host defence (antimicrobial) peptide LL-37, the sole member of the cathelicidin family expressed in humans (1), is important for innate immune responses in both the skin and the lung epithelium. LL-37 is present in low concentrations at the surface of both tissues; bronchial epithelial cells constitutively express LL-37 and secrete it into the airway surfactant (2), and while it is thought that keratinocytes do not normally express LL-37 (1), the peptide is detectable in human sweat at concentrations of about 5 µg/ml (3). LL-37 is a cleavage product of the 18-kDa human cathelicidin protein (hCAP18) (4); expression of this precursor has been shown to increase in both keratinocytes and bronchial epithelium in response to pro-inflammatory stimuli and wounding (1, 5-8). This enhanced expression underlies the increased concentration of LL-37 observed in epithelial infections. For instance, the peptide is present at concentrations of about 5 µg/ml in tracheal aspirates from healthy neonates but increases 2- to 3-fold in concentration as a result of systemic or pulmonary infection (9). Similarly, concentrations of 0-16 µg/ml were observed in bronchoalveolar lavage from adults with cystic fibrosis; increased expression of LL-37 correlated with the severity of degenerative lung disease (10). 1  A version of this chapter will be submitted for publication. Filewod, N. C. J., Pistolic, J. and R. E. W. Hancock. Low physiological doses of the human host defence peptide LL-37 alter the responses of keratinocytes and bronchial epithelial cells to pro-inflammatory stimuli.  23  The value of this inducible expression of LL-37 has been previously attributed to the ability of LL-37 to act as a ‘natural antibiotic’ with direct killing activity against a variety of pathogens (2, 11). It has been convincingly demonstrated, however, that LL-37, even at concentrations exceeding those observed at most epithelial surfaces, lacks direct antimicrobial activity at physiological salt concentrations (12), indicating the possibility that this peptide might play some other role in the restoration of tissue homeostasis. Indeed, at concentrations of around 25-40 µg/ml, LL-37 can elicit the release of cytokines and chemokines by epithelial cells, act as a growth factor, and alter the function of other immune cells to allow more effective responses to pathogens. LL-37 stimulates the production of IL-8, IL-18, IL-6, IL-10, IP-10, MCP-1, MIP-3α and RANTES by keratinocytes (13, 14) and IL-8 by bronchial epithelial cells (15, 16). It promotes keratinocyte proliferation and migration (14, 17, 18), wound healing responses by bronchial epithelial cells (19), proliferation of lung cancer cells (20), and angiogenesis in a rabbit hind limb model (21). Treatment of excised skin samples with an antibody directed against LL-37 impairs wound healing ex vivo (7). LL-37 can also alter the function of other cell types important for epithelial defence. The peptide promotes cellular recruitment at low concentrations (1-5 µg/ml), being both directly chemotactic for T cells, neutrophils, monocytes (22), and mast cells (23). Higher concentrations of LL-37 (15-40 µg/ml) stimulate the production of chemokines in monocytic cells (15, 16) and neutrophils (24 , 25). LL-37 increases the longevity of neutrophils by antagonizing apoptosis, which would help to increase the ability of the epithelium to resist infectious agents (24 , 25). It also activates mast cells, increasing their expression of Toll-like receptors and their production of proinflammatory cytokines (26), and provoking degranulation (23, 27), which would increase the permeability of the skin vasculature to 24  enable an influx of immune effector cells. LL-37 is accordingly a pluripotent effector of innate immunity in the epithelia.  Toll-like receptor (TLR) signalling is a fundamental mechanism by which epithelial cells recognize the presence of microbes. The TLR family are so-called pattern recognition receptors for conserved ‘signature molecules’ (sometimes referred to as pathogen associated molecular patterns or PAMPs) that occur in microbes but not in mammalian cells. TLRs are transmembrane receptors, variously located on the cell surface or contained within cytosolic vacuoles, and form homo- and hetero- dimers that undergo a conformational change when they bind ligand, stimulating signal transduction through an assortment of adaptor molecules (28) resulting, typically, in the activation of the transcription factor NF-κB, among others, and the mitogen activated protein kinases (MAPK) p38, Erk1/2 and JNK (29). Various TLRs recognize different classes of signature molecules. For example, TLR5 recognizes the bacterial protein flagellin (30), while a heterodimer of TLR2/1 recognizes bacterial lipoprotein (31), and TLR3 homodimerizes within cytosolic vacuoles to bind viral dsRNA (32). Along with other conserved pattern receptors such as NOD-like receptors and RIG-I-like RNA helicases, TLRs constitute a mainstay of epithelial innate immunity.  In other cell types, LL-37 can profoundly alter cytokine responses to TLR ligands. LL-37 treatment suppresses production of the quintessential proinflammatory cytokine TNF-α by PBMCs in response to lipopolysaccharide (LPS) (33), and the peptide is protective against endotoxemia in rat and mouse models (15, 34). LL-37 can also increase cytokine responses. For instance, peripheral blood mononuclear cells treated with either IL-1β or  25  GM-CSF show synergistic increases in IL-8 production when co-stimulated with LL-37 (35). LL-37 is thus able to alter responses important to the regulation of innate immunity. LL-37 plays an important anti-infective role in the epithelia. Mice deficient in CRAMP, the murine homolog of LL-37, show increased morbidity following streptococcal skin infection (36). Furthermore, human skin disorders such as psoriasis, which is associated with increased LL-37 expression, are typically associated with a decreased frequency of skin infections, while those in which LL-37 expression is reduced (such as atopic dermatitis) show an increased frequency of infection (37, 38). These findings are intriguing, as LL-37 does not appear to possess direct antimicrobial activity at physiological salt concentrations (12). It was thus of interest to determine whether LL-37 might activate innate immunity in some other manner. Accordingly, we investigated the possibility that LL-37 might alter epithelial responses to TLR ligands.  Herein we report that low, physiologically-relevant concentrations of LL-37 profoundly alter chemokine production by normal human keratinocytes and bronchial epithelial cells in response to pro-inflammatory stimuli. The addition of LL-37 at 1-3 µg/ml led to a synergistic increase in IL-8 production in response to IL-1β and agonists of TLR5, TLR3, and TLR2/1, but not GM-CSF. This synergistic response was suppressed by pretreatment of keratinocytes with the Src kinase inhibitors PP2 and Su6656. Treatment of keratinocytes with flagellin and peptide increased the phosphorylation of the transcription factor CREB. We hypothesize that the collaboration of LL-37 with endogenous and exogenous immune stimulants might represent a novel mechanism for the regulation of inflammatory responses in the epithelia.  26  MATERIALS AND METHODS Cell cultivation: Normal primary adult keratinocytes were obtained from Cascade Biologics (Portland, OR) and maintained in their proprietary Epilife medium with the addition of a Human Keratinocyte Growth Supplement that contained bovine pituitary extract, bovine insulin, hydrocortisone, bovine transferrin, and human epidermal growth factor. Unsupplemented Epilife contained 0.65 µM calcium. The medium was changed every two days and cells were passaged prior to confluence to avoid differentiation. Cultures were only used for a maximum of six passages.  Normal primary adult bronchial epithelial cells were obtained from Cambrex BioScience Inc. (Walkersville, MD) and cultivated in their proprietary BEGM basal medium with the addition of ‘Singlequot’ growth supplements, comprising human epidermal growth factor, triiodothyronine, bovine pituitary extract, epinephrine, transferrin, insulin, hydrocortisone, gentamycin/amphotericin, and retinoic acid. The cells were maintained for up to 6 passages as described for the keratinocyte cultures. All cells were cultivated in a 37° C incubator containing 5% CO2.  Reagents: Human LL-37 peptide (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized at the Nucleic acid/Protein synthesis unit at the University of British Columbia, using F-Moc chemistry. The synthesized peptide was re-suspended in endotoxin-free water (Sigma-Aldrich, Oakville, ON) and stored at -20°C until further use.  27  Cell stimulation: Keratinocytes were seeded into tissue-culture-treated 24-well plates (Corning Inc. Life Sciences, Acton, MA) at a density of 7000 cells/cm2 and cultivated in supplemented medium until they attained the desired level of confluence. Subconfluent keratinocytes were used at about 70% confluence, while calcium-differentiated keratinocytes were grown to confluence and cultivated for 2 days in unsupplemented Epilife medium containing 1.35 mM calcium. Bronchial epithelial cells were seeded into tissue-culture-treated 24-well plates at a density of 10,000 cells/cm2 and cultivated in supplemented medium until confluence was attained.  Once cells had reached the appropriate level of confluence or had been differentiated, the medium was replaced with fresh unsupplemented medium (1 ml/well). After a two-hour rest, the cells were treated with LL-37 and/or pro-inflammatory stimuli. Salmonella typhimurium flagellin, poly(I:C), and PAM3CSK4 were obtained from InvivoGen (San Diego, CA), while recombinant IL-1β and GM-CSF were purchased from Research Diagnostics (Flanders, NJ). Twenty-four hours later, supernatants were collected. Supernatants were stored at 4°C until assayed for LDH activity, and then frozen until assayed for IL-8 concentration.  Assays: Cytotoxicity was monitored using a Cytotoxicity Detection Kit (Roche, ON), which measures LDH activity in collected supernatants. Results were normalized using a negative control (untreated cells) and a positive control (cells treated with unsupplemented media containing 2% Triton-X), according to the following formula: % cytotoxicity = (sample – negative control)/(positive control – negative control) x 100. IL8 concentrations in supernatants were assessed by Enzyme Linked Immunosorbent Assay  28  (ELISA), as per the manufacturer’s instructions (Biosource International, Camarillo, CA).  Apoptosis was detected by the terminal uridine deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (Promega, Madison, WI). Bronchial epithelial cells were seeded at 10,000 cell/cm2 into Lab-Tek 8-chambered slides (Nalge Nunc Interational, Rochester, NY) and allowed to grow to confluence before being rested in unsupplemented medium for two hours and treated with peptide and poly(I:C). TUNEL was performed at 6 and 24 hours. Slides were visualized using an Eclipse 7E2000-S fluorescence microscope (Nikon, ON) equipped with a Photometric Coolsnap ES camera (Roper Scientific, AZ). Images were processed using the Image-Pro Plus software package (Media Cybernetics Inc., MD).  Inhibitor studies: Experiments involving inhibitors were performed as previously described, with the addition of the appropriate inhibitor to the fresh unsupplemented medium that the cells received two hours prior to treatment with peptide and/or proinflammatory stimuli. PP2 and SU6656 were purchased from Biosource and used at concentrations of 10 and 5 µM, respectively. Bay11-7085 was purchased from Calbiochem (Mississauga, ON) and used at a concentration of 2 µM. As a control, some cells received unsupplemented medium containing concentrations of DMSO at levels equivalent to or exceeding those present in the inhibitor solutions.  Western blotting: Keratinocytes were seeded into 60 mm tissue-culture-treated Petri dishes (Corning Life Science) at a density of 7000 cells/cm2, and cultivated as previously described until they reached around 70% confluency. The cells received fresh 29  unsupplemented medium and were rested two hours prior to stimulation with peptide and/or flagellin. Thirty minutes after treatment, the cells were washed with ice-cold PBS containing 1 mM sodium orthovanadate and lysed with NP-40 lysis buffer (1% NP-40, 20 mM Tris-HCl pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Lysis continued for 30 min on ice, following which the cells were scraped and the lysates were centrifuged for 10 min at 15,000 rpm at 4 °C. Protein concentration in the lysates was assayed using a BCA assay (Pierce, Rockford, IL), after which the lysates were denatured by addition of SDSPAGE sample buffer and heating at 95° C for 5 minutes and then resolved on a 12% acrylamide gel. Protein was then transferred to a PVDF Immuno-blot membrane (BioRad, Hercules, CA) via the application of a 100 V potential for 1 hour. The membrane was rendered hydrophobic by drying three times with methanol, and then incubated with the appropriate primary antibody in 5% non-fat milk TBST solution for 1 h. The membrane was then washed four times for ten minutes each time in TBST and incubated with the appropriate secondary antibody in 5% non-fat milk TBST solution for thirty minutes, then washed a further four times in TBST. The blot was then visualized using an ECL chemiluminescence kit (Bio-Rad); images were exposed on X-ray film (Kodak, Rochester, NY). The monoclonal anti-phospho-CREB antibody and the monoclonal horse radish peroxidase-conjugated anti-rabbit-IgG antibody (Cell Signaling Technology, Danvers, MA) were used at concentrations of 1:1000 and 1:5000, respectively. The monoclonal anti-GAPDH antibody (Fitzgerald, Concord, MA) and monoclonal antimouse-IgG antibody (Amersham, Piscataway, NJ) were used at concentrations of 1:1000 and 1:5000, respectively.  30  RESULTS: LL-37 increased IL-8 production by subconfluent keratinocytes in response to proinflammatory stimuli. LL-37 is a potent immunomodulatory agent, although many of these effects have only been demonstrated at pathological concentrations (25 µg/ml). Even at low physiological concentrations (<5 µg/ml), however, LL-37 almost completely inhibits TNF-α production by LPS-stimulated PBMCs (33), and synergistically increases the production of IL-1β-induced cytokines by PBMCs (35). LL-37-mediated immunomodulation has not, however, been previously demonstrated in epithelial cells at low physiological concentrations. To investigate the possibility that such immunomodulatory effects would only be observed in synergy with other agents, we treated subconfluent primary keratinocytes with LL-37 in combination with IL-1β, GM-CSF, and a variety of TLR agonists that were chosen based on the relative expression of TLRs in epithelial cells (39). These studies employed LL-37 at concentrations of <3 µg/ml; these low concentrations of LL-37 did not, in isolation, markedly increase IL-8 production over a twenty-four hour time period (Fig. 2.1-2.3). However when the keratinocytes were costimulated with LL-37 and pro-inflammatory stimuli, the presence of 3 µg/ml of LL-37 resulted in a 2-fold increase in IL-8 production in response to flagellin (Fig. 2.1) and a 2.6-fold change in IL-8 production in response to IL-1β (Fig. 2.2), but did not significantly alter IL-8 production in response to PAM3CSK4 or GM-CSF (data not shown). After background subtraction, it was revealed that the increase in IL-8 due to these combinations was greater than the sum of the individual treatments, implying that co-stimulation of the cells with LL-37 and either flagellin or IL-1β resulted in a synergistic increase in IL-8 production (Fig. 2.3).  31  Figure
 2.1:
 LL­37
 alters
 IL­8
 production
 by
 subconfluent
 keratinocytes
 in
 response
 to
 flagellin.
 Supernatants
 were
 collected
 at
 24
 hours.
 Error
 bars
 show
 S.E.M.
 of
 at
 least
 three
 independent
experiments.
Statistical
comparisons
were
performed
using
a
2­tailed
Student’s
 T­test.
  Figure
2.2:
LL­37
alters
of
IL­8
production
by
subconfluent
keratinocytes
in
response
to
IL­1β.
 Supernatants
were
collected
at
24
hours.
Error
bars
show
S.E.M.
of
at
least
three
independent
 experiments.
Statistical
comparisons
were
performed
using
a
2­tailed
Student’s
T­test.
  32  Figure
2.3:
Low
doses
of
LL­37
synergistically
increase
IL­8
production
by
subconfluent
 keratinocytes
in
response
to
flagellin
and
IL­1β.
Supernatants
were
collected
at
24
hours.
 Error
bars
show
S.E.M.
of
at
least
three
independent
experiments.
Statistical
comparisons
 were
performed
using
a
2­tailed
Student’s
T­test;
*
indicates
p<0.05.
A
background
 subtraction
was
performed
on
the
data.
  The ability of low doses of LL-37 to dramatically increase IL-8 production in response to low concentrations of pro-inflammatory stimuli demonstrated that LL-37 had immunomodulatory activity. As not all keratinocytes in the skin are in a proliferative state, however, but differentiate as they move upwards through the skin strata, we wished to repeat the experiment in a system that modelled terminal differentiation.  33  LL-37 increased IL-8 production by calcium-differentiated keratinocytes in response to pro-inflammatory stimuli. Increased extracellular calcium concentrations provoke differentiation and stratification in cultured keratinocytes (40). To better model upper skin strata, we grew keratinocytes to confluence and then maintained them for two days in growth-factor-free media containing 1.35 mM Ca2+. The cells were co-stimulated with LL-37 and proinflammatory stimuli; the presence of 3 µg/ml LL-37 resulted in a 2-fold increase in IL-8 production in response to flagellin (Fig. 2.4) and a 1.6-fold increase in IL-8 production in response to IL-1β (Fig. 2.5), a less pronounced effect than was observed in subconfluent cells. LL-37 did not significantly alter IL-8 production by differentiated cells in response to PAM3CSK4 or GM-CSF (data not shown). Co-stimulation of the cells with LL-37 and either flagellin or IL-1β resulted in a synergistic increase in IL-8 production (Fig. 2.6).  Figure
 2.4:
 LL­37
 alters
 IL­8
 production
 by
 differentiated
 keratinocytes
 in
 response
 to
 flagellin.
 Supernatants
 were
 collected
 at
 24
 hours.
 Error
 bars
 show
 S.E.M.
 of
 at
 least
 three
 independent
experiments.
Statistical
comparisons
were
performed
using
a
2­tailed
Student’s
 T­test.
  34  Figure
2.5:
LL­37
alters
IL­8
production
by
differentiated
keratinocytes
in
response
to
IL­1β.
 Supernatants
were
collected
at
24
hours.
Error
bars
show
S.E.M.
of
at
least
three
independent
 experiments.
Statistical
comparisons
were
performed
using
a
2­tailed
Student’s
T­test.
  Figure
 2.6:
 Low
 doses
 of
 LL­37
 synergistically
 increase
 IL­8
 production
 by
 differentiated
 keratinocytes
 in
 response
 to
 flagellin
 and
 IL­1β.
 Supernatants
 were
 collected
 at
 24
 hours.
  35  Error
 bars
 show
 S.E.M.
 of
 at
 least
 three
 independent
 experiments.
 Statistical
 comparisons
 were
 performed
 using
 a
 2­tailed
 Student’s
 T­test;
 *
 indicates
 p<0.05.
 A
 background
 subtraction
was
performed
on
the
data.
  These findings indicated that LL-37 was able to alter innate immune responses in both proliferating and differentiated keratinocytes. This was intriguing since LL-37 is only produced by keratinocytes following infection and injury (1, 7). Therefore, it was of interest to see if similar effects would be observed in a cell type that consistently encounters LL-37.  LL-37 increased IL-8 production by bronchial epithelial cells in response to proinflammatory stimuli. Bronchial epithelial cells constitutively produce LL-37 and secrete it into the airway surfactant (2). Accordingly, we tested the ability of LL-37 to alter IL-8 release in this cell type. The presence of 3 µg/ml LL-37 resulted in a 3-fold increase in IL-8 production in response to flagellin (Fig. 2.7), a 4-fold increase in IL-8 production in response to IL-1β (Fig. 2.8), and a 2.2-fold increase in IL-8 release in response to PAM3CSK4 (Fig. 2.9), stronger responses than those observed in keratinocytes. Co-stimulation of the cells with LL-37 and either flagellin or IL-1β resulted in a synergistic increase in IL-8 production (Fig. 2.10).  36  Figure
2.7:
LL­37
alters
IL­8
production
by
bronchial
epithelial
cells
in
response
to
flagellin.
 Supernatants
were
collected
at
24
hours.
Error
bars
show
S.E.M.
of
at
least
three
independent
 experiments.
Statistical
comparisons
were
performed
using
a
2­tailed
Student’s
T­test.
  
 Figure
 2.8:
 LL­37
 alters
 IL­8
 production
 by
 bronchial
 epithelial
 cells
 in
 response
 to
 IL­1β.
 Supernatants
were
collected
at
24
hours.
Error
bars
show
S.E.M.
of
at
least
three
independent
 experiments.
Statistical
comparisons
were
performed
using
a
2­tailed
Student’s
T­test.
  37  Figure
 2.9:
 LL­37
 alters
 IL­8
 production
 by
 bronchial
 epithelial
 cells
 in
 response
 to
 PAM3CSK4.
Supernatants
were
collected
at
24
hours.
Error
bars
show
S.E.M.
of
at
least
three
 independent
experiments.
Statistical
comparisons
were
performed
using
a
2­tailed
Student’s
 T­test;
*
indicates
p<0.05.
  38  Figure
 2.10:
 Low
 doses
 of
 LL­37
 synergistically
 increase
 IL­8
 production
 by
 bronchial
 epithelial
 cells
 in
 response
 to
 flagellin
 and
 IL­1β.
 Supernatants
 were
 collected
 at
 24
 hours.
 Error
 bars
 show
 S.E.M.
 of
 at
 least
 three
 independent
 experiments.
 Statistical
 comparisons
 were
 performed
 using
 a
 2­tailed
 Student’s
 T­test;
 *
 indicates
 p<0.05.
 A
 background
 subtraction
was
performed
on
the
data.
  Co-stimulation of bronchial epithelial cells with LL-37 and poly(I:C) elicited a rapid IL-8 response and delayed cytotoxicity. In addition to the above agonists of surface TLRs, potential synergy with an intracellular TLR was investigated. Bronchial epithelial cells encounter infectious viral particles in vivo and are accordingly responsive to the presence of virus-associated signature molecules such as dsRNA (Guillot 2005) that interact with intracellular TLRs. To investigate whether LL-37 would show synergy with a viral signature molecule, bronchial epithelial cells were stimulated with the TLR3 agonist poly(I:C) in the presence or absence of 3 µg/ml LL-37, and supernatants were collected at 3, 6, and 24 39  hours. Co-stimulation with poly(I:C) and peptide elicited a strong, rapid IL-8 response (Fig. 2.11), followed by substantial cytotoxicity (Fig. 2.12). LL-37 did not induce notable cytotoxicity in the absence of poly(I:C), and poly(I:C) was minimally cytotoxic at early timepoints. However co-stimulation with poly(I:C) and peptide resulted in a synergistic increase in both IL-8 release (Fig. 2.13) and cytotoxicity (Fig. 2.14). TUNEL assays performed on bronchial epithelial cells treated with both poly(I:C) and peptide did not detect any increase in apoptosis at 6 h or 24 h (data not shown). As the IL-8 response preceded the cytotoxicity, it was concluded that the increased IL-8 production in these cells was a result of LL-37-mediated immunomodulation.  Figure
2.11:
Co­stimulation
of
bronchial
epithelial
cells
with
LL­37
and
poly(I:C)
elicits
a
rapid
 IL­8
 response.
 Error
 bars
 show
 S.E.M.
 of
 at
 least
 three
 independent
 experiments.
 Statistical
 comparisons
 were
 performed
 using
 a
 2­tailed
 Student’s
 T­test;
 *
 indicates
 p<0.05
 when
 compared
to
null.
  40  Figure
 2.12:
 Co­stimulation
 of
 bronchial
 epithelial
 cells
 with
 LL­37
 and
 poly(I:C)
 results
 in
 rapid
and
pronounced
cytotoxicity
as
measured
by
LDH
release.
Error
bars
show
S.E.M.
of
at
 least
 three
 independent
 experiments.
 For
 clarity,
 the
 results
 of
 statistical
 analysis
 are
 omitted.

  41  Figure
 2.13:
 Low
 doses
 of
 LL­37
 synergistically
 increase
 IL­8
 production
 by
 bronchial
 epithelial
cells
in
response
to
poly(I:C).
Error
bars
show
S.E.M.
of
at
least
three
independent
 experiments.
 Statistical
 comparisons
 were
 performed
 using
 a
 2­tailed
 Student’s
 T­test;
 *
 indicates
p<0.05.
  Figure
2.14:
Low
doses
of
LL­37
synergistically
increase
cytotoxicity
subsequent
to
treatment
 with
 poly(I:C).
 Error
 bars
 show
 S.E.M.
 of
 at
 least
 three
 independent
 experiments.
 Statistical
 comparisons
were
performed
using
a
2­tailed
Student’s
T­test;
*
indicates
p<0.05.
 
  42  Inhibition of the synergistic increase in IL-8 production by inhibitors of Src-family kinase signalling or NF-κB translocation In bronchial epithelial cells, Src-family kinases can regulate the activation of p38 and ERK1/2, and the subsequence release of IL-8 (41). Similarly, the inhibition of Src-family kinases inhibits LL-37-induced IL-8 release in airway smooth muscle cells (42). Additionally, the production of IL-8 by bronchial epithelial cells in response to TLR ligands has been shown to be dependent upon EGFR signalling (43); in keratinocytes, SRC family kinases are activated downstream of EGFR (44). To investigate the possibility that the observed synergistic response was dependent on Src-family kinase activity, keratinocytes were treated with the inhibitors PP2 and SU6656. The presence of either inhibitor resulted in a 70% decrease in IL-8 release relative to that typically observed when cells were treated with flagellin and LL-37 (Fig. 2.15).  43  Figure
 2.15:
 The
 Src­family
 kinase
 inhibitors
 PP2
 and
 SU6656
 suppress
 IL­8
 production
 by
 keratinocytes
in
response
to
co­stimulation
with
LL­37
and
flagellin.
Cells
were
pretreated
for
 2
hours
with
10
μM
PP2
or
5
μM
SU6656.
Error
bars
show
S.E.M.
of
at
least
three
independent
 experiments.
 Statistical
 comparisons
 were
 performed
 using
 a
 2­tailed
 Student’s
 T­test;
 *
 indicates
p<0.05.
  As the translocation of NF-κB is required for LL-37-mediated cytokine production in PBMCs (33, 35), we investigated the effects of the IκB inhibitor Bay11 on IL-8 responses to LL-37/flagellin co-stimulation. Keratinocytes pretreated with Bay11 exhibited an 80% reduction in IL-8 production after flagellin/LL-37 co-stimulation (Fig. 2.16).  44  Figure
 2.16:
 The
 NF­κB
 inhibitor
 Bay11
 suppresses
 IL­8
 production
 by
 keratinocytes
 in
 response
 to
 co­stimulation
 with
 LL­37
 and
 flagellin.
 Error
 bars
 show
 S.E.M.
 of
 at
 least
 three
 independent
experiments.
  Stimulation of keratinocytes with LL-37 and flagellin resulted in a strong increase in the phosphorylation of the transcription factor CREB. Having determined that the observed synergistic responses were dependent upon the activation of Src-family kinases and NF-κB, the impact of TLR stimulation on known effects of LL-37 treatment was investigated. In PBMC, LL-37 stimulation results in increased phosphorylation of the MAP kinase ERK-1(45), which is known to phosphorylate the transcription factor CREB, stimulating its translocation into the nucleus (46). Interestingly, co-stimulation of PBMC with LL-37 and IL-1β led to 45  stronger increases in the phosphorylation of CREB than were detectable following stimulation with either alone (35). To determine if a similar effect would result from costimulation of keratinocytes with flagellin and LL-37, cells were treated with LL-37 and/or flagellin and lysed after 30 min; the low doses of LL-37 used in the experiment did not markedly increase CREB phosphorylation either in isolation or when cells were co-stimulated with flagellin (Fig. 2.17).  Figure
2.17:
Low
doses
of
LL­37
do
not
alter
the
increased
phosphorylation
of
CREB
observed
 after
 keratinocytes
 are
 stimulated
 with
 flagellin.
 Cells
 were
 lysed
 30
 minutes
 post­ stimulation.
Bands
are,
from
top
to
bottom:
phospho­CREB,
phospho­ATF­1,
and
GAPDH.
The
 blot
shown
is
representative
of
three
independent
experiments.

  DISCUSSION We have demonstrated here a novel immunomodulatory role for LL-37 in its interactions with epithelial cells. Low doses of LL-37 that were insufficient to elicit strong increases in IL-8 production on their own were able to synergistically increase IL-8 production by keratinocytes and bronchial epithelial cells in response to pro-inflammatory stimuli.  46  The exact mechanism underlying these effects is not completely clear. Previous studies have shown that the inhibition of Src-family kinases can reduce IL-8 production in response to LL-37 treatment of smooth muscle cells (42); similarly, the ability of LL-37 to alter epithelial cell responses was shown here to be suppressed by pre-treatment with Src-family kinase inhibitors. It seems possible that this effect is a consequence of the ability of Src-family kinases to regulate the phophorylation state of the MAP kinases p38 and ERK1/2 (41), the activation of which is necessary for LL-37-stimulated IL-18 production in keratinocytes (13) and a variety of peptide-mediated effects in other cell types (16, 45, 47). The translocation of the transcription factor NF-κB is also necessary for IL-8 production by keratinocytes (48). Thus as expected, an inhibitor of NF- κB translocation blocked cellular IL-8 production in response to co-stimulation by LL-37 and flagellin. These results indicate that the ability of LL-37 to alter epithelial cell responses to pro-inflammatory stimuli may be dependent upon altered MAPK signalling and NF-κB activation.  The ability of LL-37 to induce synergistic increases in IL-8 production and cytotoxicity in cells treated with the TLR3 agonist poly(I:C) was intriguing. Rapid secretion of IL-8, followed by a widespread loss of membrane integrity, was observed in cells that were treated with poly(I:C) and LL-37 and although cells treated with poly(I:C) alone also showed increases in cytotoxicity, the effect was not as rapid nor as pronounced. The mechanism underlying this effect is unclear, but it is presumed that the cells underwent necrotic cell death as we were unable to detect increased levels of apoptosis using a TUNEL assay. As poly(I:C) is anionic and LL-37 is cationic, their ability to induce strong responses in epithelial cells might result in part from complex formation. LL-37 has been previously shown to form complexes with human DNA, allowing a strong and 47  immunologically unusual activation of plasmacytoid dendritic cells (49). The potential ability of LL-37 to form complexes with nucleic acids and alter TLR signalling is an intriguing topic for further investigation. As dsRNA like poly(I:C) is thought to reflect a viral activating signal for TLR3, it seems possible that the induction of cell death might actually represent a viral defence signal, as indeed may the induction of IL-8 that would recruit neutrophils to the vicinity of the LL-37 stimulated epithelium.  As the effects described here are mediated by low concentrations of LL-37, equivalent to those normally present in sweat and airway surfactant (3, 10), we postulate that LL-37mediated increases in IL-8 release may be important to the regulation of epithelial inflammation in vivo. Both keratinocytes and bronchial epithelial cells show increased expression of LL-37 as a consequence of wounding (7, 8) and inflammation (50), suggesting that local increases in LL-37 concentrations at the epithelial surface, together with an appropriate additional endogenous or exogenous signal, may serve as an alarm signal, resulting in increased IL-8 production in response to pro-inflammatory stimuli and a concomitant increase in immune cell recruitment. The ability of LL-37 to alter epithelial cell responses to pro-inflammatory stimuli also suggests new possibilities in the etiology of psoriasis, an auto-immune disorder that results in localized hyperproliferative areas of skin inflammation. Although classically regarded as a disorder of the adaptive immune system, it has been suggested that psoriasis might result from the abnormal over-expression of host defence peptides in the epithelium (51). As LL-37 is expressed at high concentrations in psoriatic plaques (37), the ability of LL-37 to increase keratinocyte IL-8 responses may be important to the pathophysiology of psoriasis.  48  The complex defences of innate immunity are vital to the maintenance of epithelial homeostasis. The observation here that modest concentrations of LL-37 can alter epithelial IL-8 production indicates that LL-37 plays a broader role than previously suspected in the regulation of the inflammatory response. An improved understanding of the mechanisms underlying these responses might facilitate the development of novel anti-infective therapeutics.  ACKNOWLEDGEMENTS: We gratefully acknowledge the invaluable guidance of Jelena Pistolic and the technical assistance of Sheena Tam. NCJF was the recipient of an NSERC CGS M award and a MSFHR Junior Trainee Award. We gratefully acknowledge the support of Genome BC and Genome Prairie for the 'Pathogenomics of Innate Immunity' research program.  49  LITERATURE CITED: 1.  2.  3. 4.  5. 6. 7.  8.  9. 10. 11. 12. 13.  14.  Frohm, M., B. Agerberth, G. Ahangari, M. Stahle-Backdahl, S. Liden, H. Wigzell, and G. H. Gudmundsson. 1997. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. The Journal of biological chemistry 272:15258-15263. Bals, R., X. Wang, M. Zasloff, and J. M. Wilson. 1998. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proceedings of the National Academy of Sciences 95:9541-9546. Murakami, M., T. Ohtake, R. A. Dorschner, B. Schittek, C. Garbe, and R. L. Gallo. 2002. Cathelicidin Anti-Microbial Peptide Expression in Sweat, an Innate Defense System for the Skin. J Invest Dermatol 119:1090-1095. Gudmundsson, G. H., B. Agerberth, J. Odeberg, T. Bergman, B. Olsson, and R. Salcedo. 1996. The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. European journal of biochemistry / FEBS 238:325-332. Conner, K., K. Nern, J. Rudisill, T. O'Grady, and R. L. Gallo. 2002. The antimicrobial peptide LL-37 is expressed by keratinocytes in condyloma acuminatum and verruca vulgaris. J Am Acad Dermatol 47:347-350. Lopez-Garcia, B., P. H. Lee, and R. L. Gallo. 2006. Expression and potential function of cathelicidin antimicrobial peptides in dermatophytosis and tinea versicolor. The Journal of antimicrobial chemotherapy 57:877-882. Heilborn, J. D., M. F. Nilsson, G. Kratz, G. Weber, O. Sorensen, N. Borregaard, and M. Stahle-Backdahl. 2003. The Cathelicidin Anti-Microbial Peptide LL-37 is Involved in Re-Epithelialization of Human Skin Wounds and is Lacking in Chronic Ulcer Epithelium. J. Invest. Dermatol. 120:379-389. Schauber, J., R. A. Dorschner, A. B. Coda, A. S. Buchau, P. T. Liu, D. Kiken, Y. R. Helfrich, S. Kang, H. Z. Elalieh, A. Steinmeyer, U. Zugel, D. D. Bikle, R. L. Modlin, and R. L. Gallo. 2007. Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism. The Journal of clinical investigation 117:803-811. Schaller-Bals, S., A. Schulze, and R. Bals. 2002. Increased Levels of Antimicrobial Peptides in Tracheal Aspirates of Newborn Infants during Infection. Am. J. Respir. Crit. Care Med. 165:992-995. Chen, C. I. U., S. Schaller-Bals, K. P. Paul, U. Wahn, and R. Bals. 2004. [beta]defensins and LL-37 in bronchoalveolar lavage fluid of patients with cystic fibrosis. Journal of Cystic Fibrosis 3:45-50. Braff, M. H., M. Zaiou, J. Fierer, V. Nizet, and R. L. Gallo. 2005. Keratinocyte production of cathelicidin provides direct activity against bacterial skin pathogens. Infect Immun 73:6771-6781. Bowdish, D. M., D. J. Davidson, Y. E. Lau, K. Lee, M. G. Scott, and R. E. Hancock. 2005. Impact of LL-37 on anti-infective immunity. J Leukoc Biol 77:451-459. Niyonsaba, F., H. Ushio, I. Nagaoka, K. Okumura, and H. Ogawa. 2005. The human beta-defensins (-1, -2, -3, -4) and cathelicidin LL-37 induce IL-18 secretion through p38 and ERK MAPK activation in primary human keratinocytes. J Immunol 175:1776-1784. Niyonsaba, F., H. Ushio, N. Nakano, W. Ng, K. Sayama, K. Hashimoto, I. Nagaoka, K. Okumura, and H. Ogawa. 2007. Antimicrobial peptides human beta50  15. 16.  17.  18.  19.  20.  21.  22.  23. 24. 25.  defensins stimulate epidermal keratinocyte migration, proliferation and production of proinflammatory cytokines and chemokines. J Invest Dermatol 127:594-604. Scott, M. G., D. J. Davidson, M. R. Gold, D. Bowdish, and R. E. W. Hancock. 2002. The Human Antimicrobial Peptide LL-37 Is a Multifunctional Modulator of Innate Immune Responses. J Immunol 169:3883-3891. Tjabringa, G. S., J. Aarbiou, D. K. Ninaber, J. W. Drijfhout, O. E. Sorensen, N. Borregaard, K. F. Rabe, and P. S. Hiemstra. 2003. The antimicrobial peptide LL37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. J Immunol 171:6690-6696. Tokumaru, S., K. Sayama, Y. Shirakata, H. Komatsuzawa, K. Ouhara, Y. Hanakawa, Y. Yahata, X. Dai, M. Tohyama, H. Nagai, L. Yang, S. Higashiyama, A. Yoshimura, M. Sugai, and K. Hashimoto. 2005. Induction of Keratinocyte Migration via Transactivation of the Epidermal Growth Factor Receptor by the Antimicrobial Peptide LL-37. J Immunol 175:4662-4668. Carretero, M., M. J. Escamez, M. Garcia, B. Duarte, A. Holguin, L. Retamosa, J. L. Jorcano, M. del Rio, and F. Larcher. 2007. In vitro and In vivo Wound Healing-Promoting Activities of Human Cathelicidin LL-37. J Invest Dermatol 128:223-236. Shaykhiev, R., C. Beisswenger, K. Kandler, J. Senske, A. Puchner, T. Damm, J. Behr, and R. Bals. 2005. Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure. American journal of physiology 289:L842-848. von Haussen, J., R. Koczulla, R. Shaykhiev, C. Herr, O. Pinkenburg, D. Reimer, R. Wiewrodt, S. Biesterfeld, A. Aigner, F. Czubayko, and R. Bals. 2008. The host defence peptide LL-37/hCAP-18 is a growth factor for lung cancer cells. Lung Cancer 59:12-23. Koczulla, R., G. von Degenfeld, C. Kupatt, F. Krotz, S. Zahler, T. Gloe, K. Issbrucker, P. Unterberger, M. Zaiou, C. Lebherz, A. Karl, P. Raake, A. Pfosser, P. Boekstegers, U. Welsch, P. S. Hiemstra, C. Vogelmeier, R. L. Gallo, M. Clauss, and R. Bals. 2003. An angiogenic role for the human peptide antibiotic LL-37/hCAP-18. The Journal of clinical investigation 111:1665-1672. Yang, D., Q. Chen, A. P. Schmidt, G. M. Anderson, J. M. Wang, J. Wooters, J. J. Oppenheim, and O. Chertov. 2000. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 192:1069-1074. Niyonsaba, F., K. Iwabuchi, A. Someya, M. Hirata, H. Matsuda, H. Ogawa, and I. Nagaoka. 2002. A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis. Immunology 106:20-26. Nagaoka, I., F. Niyonsaba, Y. Tsutsumi-Ishii, H. Tamura, and M. Hirata. 2008. Evaluation of the effect of human {beta}-defensins on neutrophil apoptosis. Int. Immunol. 20:543-553. Barlow, P. G., Y. Li, T. S. Wilkinson, D. M. E. Bowdish, Y. E. Lau, C. Cosseau, C. Haslett, A. J. Simpson, R. E. W. Hancock, and D. J. Davidson. 2006. The human cationic host defense peptide LL-37 mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system. 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Rocchi, V. Saba, M. Zanetti, and G. Scalise. 2006. LL-37 Protects Rats against Lethal Sepsis Caused by GramNegative Bacteria. Antimicrob. Agents Chemother. 50:1672-1679. Yu, J., N. Mookherjee, K. Wee, D. M. Bowdish, J. Pistolic, Y. Li, L. Rehaume, and R. E. Hancock. 2007. Host defense peptide LL-37, in synergy with inflammatory mediator IL-1beta, augments immune responses by multiple pathways. J Immunol 179:7684-7691. Nizet, V., T. Ohtake, X. Lauth, J. Trowbridge, J. Rudisill, R. A. Dorschner, V. Pestonjamasp, J. Piraino, K. Huttner, and R. L. Gallo. 2001. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414:454-457. Ong, P. Y., T. Ohtake, C. Brandt, I. Strickland, M. Boguniewicz, T. Ganz, R. L. Gallo, and D. Y. Leung. 2002. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. The New England journal of medicine 347:11511160. de Jongh, G. J., P. L. J. M. Zeeuwen, M. Kucharekova, R. Pfundt, P. G. van der Valk, W. Blokx, A. Dogan, P. S. Hiemstra, P. C. van de Kerkhof, and J. Schalkwijk. 2005. High Expression Levels of Keratinocyte Antimicrobial Proteins in Psoriasis Compared with Atopic Dermatitis. J Investig Dermatol 125:1163-1173. Kollisch, G., B. N. Kalali, V. Voelcker, R. Wallich, H. Behrendt, J. Ring, S. Bauer, T. Jakob, M. Mempel, and M. Ollert. 2005. Various members of the Toll52  40. 41. 42. 43. 44. 45.  46. 47.  48.  49.  50.  51.  like receptor family contribute to the innate immune response of human epidermal keratinocytes. Immunology 114:531-541. Bikle, D. D., and S. Pillai. 1993. Vitamin D, calcium, and epidermal differentiation. Endocr Rev 14:3-19. Ovrevik, J., M. Lag, P. Schwarze, and M. Refsnes. 2004. p38 and Src-ERK1/2 Pathways Regulate Crystalline Silica-Induced Chemokine Release in Pulmonary Epithelial Cells. Toxicol. Sci. 81:480-490. Zuyderduyn, S., D. K. Ninaber, P. S. Hiemstra, and K. F. Rabe. 2006. The antimicrobial peptide LL-37 enhances IL-8 release by human airway smooth muscle cells. Journal of Allergy and Clinical Immunology 117:1328-1335. Koff, J. L., M. X. G. Shao, I. F. Ueki, and J. A. Nadel. 2008. Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium. American journal of physiology 294:L1068-1075. Ayli, E. E., W. Li, T. T. Brown, A. Witkiewicz, R. Elenitsas, and J. T. Seykora. 2008. Activation of Src-family tyrosine kinases in hyperproliferative epidermal disorders. Journal of Cutaneous Pathology 35:273-277. Bowdish, D. M. E., D. J. Davidson, D. P. Speert, and R. E. W. Hancock. 2004. The Human Cationic Peptide LL-37 Induces Activation of the Extracellular Signal-Regulated Kinase and p38 Kinase Pathways in Primary Human Monocytes. J Immunol 172:3758-3765. Xing, J., D. D. Ginty, and M. E. Greenberg. 1996. Coupling of the RAS-MAPK Pathway to Gene Activation by RSK2, a Growth Factor-Regulated CREB Kinase. Science (New York, N.Y 273:959-963. Chen, X., F. Niyonsaba, H. Ushio, I. Nagaoka, S. Ikeda, K. Okumura, and H. Ogawa. 2006. Human cathelicidin LL-37 increases vascular permeability in the skin via mast cell activation, and phosphorylates MAP kinases p38 and ERK in mast cells. Journal of Dermatological Science 43:63-66. Dai, X., K. Yamasaki, Y. Shirakata, K. Sayama, and K. Hashimoto. 2004. AllTrans-Retinoic Acid Induces Interleukin-8 via the Nuclear Factor-[kappa]B and p38 Mitogen-Activated Protein Kinase Pathways in Normal Human Keratinocytes. J Investig Dermatol 123:1078-1085. Lande, R., J. Gregorio, V. Facchinetti, B. Chatterjee, Y.-H. Wang, B. Homey, W. Cao, Y.-H. Wang, B. Su, F. O. Nestle, T. Zal, I. Mellman, J.-M. Schroder, Y.-J. Liu, and M. Gilliet. 2007. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449:564-569. Frohm Nilsson, M., B. Sandstedt, O. Sorensen, G. Weber, N. Borregaard, and M. Stahle-Backdahl. 1999. The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6. Infect Immun 67:2561-2566. Buchau, A. S., and R. L. Gallo. 2007. Innate immunity and antimicrobial defense systems in psoriasis. Clinics in Dermatology 25:616-624.  53  CHAPTER III2 INTRODUCTION Although often considered as two separate processes, the inflammatory and woundhealing responses of the skin are intimately interlinked (1). The cationic host defence peptide LL-37 is a pleiotropic effector molecule of innate immunity that exerts both proinflammatory and growth-stimulating effects upon keratinocytes, suggesting that it might be important in the restoration of tissue homeostasis after wounding and infection. The expression by keratinocytes of both the LL-37 precursor hCAP18 and LL-37 itself is increased during inflammation. Indeed, even though keratinocytes are thought to not normally express LL-37, increased concentrations of the peptide are detectable in inflamed skin both as a result of atopic and autoimmune responses (2, 3), and as a consequence of fungal and viral infections (4, 5). The treatment of cultivated keratinocytes with pathological doses of LL-37 elicits the production of IL-8 (2), IL-6, IL-10, IP-10, MCP-1, MIP-3α, RANTES (7), and IL-18 (8). Furthermore, LL-37 is chemotactic for a variety of immune cells, including monocytes, T cells and mast cells (9, 10). The importance in vivo of these activities is suggested by the phenotype of mice deficient in the murine LL-37 homolog CRAMP, which exhibit moderate increased susceptibility to skin infection by Group A Streptococcal bacteria (3).  Low doses of LL-37, however, stimulate wound healing. LL-37 provokes keratinocyte migration and proliferation (7, 12). These activities involve activation of the Epidermal Growth Factor Receptor (EGFR), downstream signalling through the Stat3 pathway, and  2
A
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Filewod,
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Falsafi,
R.,
Gardy,
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  R.
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  54  the activation of the transcription factors Snail and Slug (12, 13). Similarly, the treatment of excised skin samples with an anti-LL-37 blocking antibody has been shown to inhibit re-epithelialisation in a concentration-dependent manner (4). LL-37 is also an angiogenic factor, able to stimulate the development of capillaries in a rabbit hind limb model (15) and elicit the secretion of vascular endothelial growth factor by keratinocytes (5). LL-37 is thus thought to be involved in both the initial responses to infection and eventual tissue repair.  It is increasingly apparent that Toll-like receptor (TLR) signalling plays an important role in the maintenance of epithelial tissue homeostasis and integrity. MyD88-dependent signalling induced by commensal microbes has been shown to be necessary for appropriate responses to epithelial injury in a murine model of colitis (6); similarly, the stimulation of TLR2 and TLR5 provokes growth and reduces apoptosis in bronchial epithelial cells (7). These effects have been suggested to be dependent upon EGFR signalling and independent of the production of pro-inflammatory cytokines (7). While the relevance of these findings to the skin has yet to be confirmed, the possibility of a common mechanism in TLR- and LL-37-mediated growth responses aroused my curiosity. LL-37 is able to alter responses to TLR stimulation in a variety of cell types (19-22), including keratinocytes (Chapter II). Accordingly, I hypothesized that the presence of TLR2 and TLR5 ligands might alter keratinocyte growth responses to LL-37 treatment in vitro. In this Chapter, preliminary evidence is presented that indicates that the ability of LL-37 to stimulate keratinocyte proliferation relies upon the secondary production of growth factors by peptide-stimulated cells. Furthermore, genes were identified that showed altered regulation after LL-37 treatment. If continued as suggested  55  in the discussion, these studies may improve our understanding of the interplay of innate immunity and commensal microflora in the repair of epithelial injury.  MATERIALS AND METHODS Cell cultivation: Normal primary adult keratinocytes were obtained from Cascade Biologics (Portland, OR) and maintained in their proprietary Epilife medium with the addition of a Human Keratinocyte Growth Supplement that contained bovine pituitary extract, bovine insulin, hydrocortisone, bovine transferrin, and human epidermal growth factor. Unsupplemented Epilife contained 0.65 µM calcium. The medium was changed every two days and cells were passaged prior to confluence to avoid differentiation. Cultures were only used for a maximum of six passages. The cells were cultivated in a 37° C incubator containing 5% CO2.  Reagents: Human peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized at the Nucleic acid/Protein synthesis unit at the University of British Columbia, using F-Moc chemistry. The synthesized peptide was re-suspended in endotoxin-free water (Sigma-Aldrich, Oakville, ON) and stored at -20°C until further use.  RNA isolation: Keratinocytes were seeded into tissue-culture-treated 6-cm Petri dishes (Corning Inc. Life Sciences, Acton, MA) at a density of 7000 cells/cm2 and cultivated in supplemented medium until they reached about 70% confluence. The medium was then replaced with unsupplemented Epilife (Cascade Biologics) and the cells were rested for 2 hours prior to stimulation with 3 µg/ml LL-37 or a vehicle control. RNA was isolated at  56  1, 2 and 4 hours using an RNeasy Mini-kit (Qiagen, Mississauga, ON). Eluted RNA was treated with an RNAse inhibitor (Ambion, Austin, TX) and stored at -80° until use.  Microarray analysis: RNA purity and integrity were assessed using an Agilent 2100 Bioanalyzer using RNA 6000 Nano kits (Agilent Technologies, Santa Clara, CA). The RNA was amplified using an amPULSE RNA Amplification kit from Kreatech (Amsterdam, The Netherlands), as per the manufacturer’s instructions. Briefly, the RNA was reverse transcribed to make sCDNA, from which dsCDNA was synthesized. The dsCDNA was then amplified via In Vitro Transcription (IVT), yielding aRNA. This aRNA was labeled with Cyanine 3 and Cyanine 5, and the labeled product was cleaned using the columns provided. Yield and fluorophore incorporation were measured using a NanoDrop 1000 fluorometer/spectrophotometer (Nanodrop, Wilmington, DE). Microarray slides were printed using the human genome 21K Array-Ready Oligo Set (Qiagen) at the Jack Bell Research Centre (Vancouver, BC). The slides were prehybridised for 45 minutes at 48° C in prehybridization buffer containing 5 x SSC (Ambion), 0.1% (w/v) SDS, and 0.2% (w/v) BSA. Equivalent (20 pmol) cyanine-labelled samples from control and treated cells were then mixed and hybridized on the array slides, in Ambion SlideHyb buffer no. 2 (Ambion) for 18 hours at 50°C in a hybridization oven. Following hybridization, the slides were washed twice in 2x SSC/0.2% SDS for 15 minutes at 65°C, and then once in 2x SSC for 15 minutes at 42°C, followed by washing in 0.2% SSC for 15 minutes at room temperature. Slides were then centrifuged for 2 minutes at 2000 x g, dried, and scanned using a ScanArray Express software/scanner (PerkinElmer). The images were quantified using Imagene software (BioDiscovery, El Segundo, CA).  57  Bioinformatic analysis: Assessment of slide image quality, data normalization, detection of differential gene expression, and statistical analysis were conducted using the ArrayPipe (www.pathogenomics.ca/arraypipe) web-based software package (8). The following steps were applied: markers were flagged and excluded, background correction was applied using the Limma ‘normexp’ method, spot intensities within each subgrid were normalized using the Limma Loess method, a Limma eBayes modified t-test was performed, and an annotated list of genes and fold changes was obtained. A list of differentially expressed genes was obtained for each timepoint using a cutoff p-value of 0.05 and a minimal fold change calculated by ArrayPipe to allow a 40% chance of false discovery based on an analysis of variation between the biological repeats (this high false discovery rate was chosen so as not to miss any of the important dysregulated genes, reasoning that downstream analyses will eventually decipher which of the results are most reliable; in typical confirmatory experiments from the Hancock lab usually more than 70% of genes can be confirmed and this number rises as the extent of differential expression rises above 2-fold). Transcription factor binding site over-representation analysis was performed using the oPossum web-based software package (http://burgundy.cmmt.ubc.ca/oPOSSUM) (9) using the default settings. Pathway overrepresentation analysis was performed using the InnateDB database and web-based software package using a p-value cutoff of 0.05 and a fold change cutoff of 1.5 (http://innatedb.ca) (publication in press); pathway diagrams were produced using the Cerebral Cytoscape plug-in (http://innatedb.ca/resources.jsp) (10). Gene ontology overrepresentation analysis was performed using the GOTree machine web-based software package (http://genereg.ornl.gov/gotm/) (11).  58  RESULTS: Treatment with low doses of LL-37 resulted in altered gene expression. As LL-37 is known to provoke keratinocyte migration and proliferation (7, 12), we wished to investigate altered gene expression in response to low dose LL-37 stimulation. Keratinocytes were grown to about 70% confluence and stimulated with 3 µg/ml of LL37; RNA was collected 1, 2 and 4 hours post-stimulation. Microarray analysis revealed differences in gene expression between LL-37-treated and control cells at all timepoints. Two hundred and sixty-five genes showed significant differential expression at the 1hour timepoint, 150 at the 2 hour timepoint, and 592 at 4 hours. Selected results of relevance to wound healing and innate immunity are presented in Table 3.1 (1 hour timepoint), Table 3.2 (2 hour timepoint), and Table 3.3 (4 hour timepoint); complete results are presented in Supplementary Tables 7-9, respectively. Table
3.1:
Selected
genes
showing
differential
expression
in
keratinocytes
1
hour
post­ treatment
with
3
μg/ml
LL­37.
Genes
were
excluded
if
they
showed
<2.5­fold
change,
and
 gene
lists
were
manually
curated
for
potential
relevance
to
wound
healing
and
innate
 immune
responses.
 Gene Name Gene Description Fold p Value Change SEM4A Inhibits axonal extension by providing local signals to specify 4.70 0.0023 territories inaccessible for growing axons E2F2 Transcription activator that binds DNA cooperatively with DP 4.33 0.0029 proteins through the E2 recognition site. CAD19 Cadherins are calcium dependent cell adhesion proteins. 3.55 0.005 ITA4 Integrins alpha-4/beta-1 (VLA-4) and alpha-4/beta-7 are 3.44 0.0056 receptors for fibronectin. AKT2 General protein kinase capable of phosphorylating several 3.35 0.006 known proteins PLCB4 The production of the second messenger molecules 2.83 0.0106 diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) is mediated by activated phosphatidylinositol-specific phospholipase C enzymes. ONEC2 Transcriptional activator. Activates the transcription of a 2.77 0.0114 number of liver genes such as HNF3B FGD1  Activates CDC42, a member of the Ras-like family of Rho-and Rac proteins, by exchanging bound GDP for free GTP. Plays a role in regulating the actin cytoskeleton and cell shape  2.76  0.0115  ATG4D CLTR1  Cysteine protease required for autophagy. Receptor for cysteinyl leukotrienes mediating bronchoconstriction of individuals with and without asthma.  2.53 -2.50  0.0157 0.0166  BKRB2  Receptor for bradykinin.  -2.53  0.0157  59  Gene Name  Gene Description  CUL5  Component of E3 ubiquitin ligase complexes, which mediate the ubiquitination and subsequent proteasomal degradation of target proteins. Seems to be involved poteosomal degradation of p53/TP53 stimulated by adenovirus E1B-55 kDa protein. May form a cell surface vasopressin receptor NP060218.1 Histone H5 PO2F3 Transcription factor that binds to the octamer motif (5'ATTTGCAT-3'). NFASC Cell adhesion, ankyrin-binding protein which may be involved in neurite extension, axonal guidance, synaptogenesis, myelination and neuron-glial cell interactions. DLX5 Homeobox protein DLX-5 PIAS1 Plays a crucial role as a transcriptional coregulation in various cellular pathways, including the STAT pathway, the p53 pathway and the steroid hormone signaling pathway. CASR TLE4 CELR1 VNN2 PLOD2 SL9A8 RTC1 TIGD6 F261 K22E DB118 RNAS1 ZN134 FGF2 ATRX ZHANG  Fold p Value Change -2.66 0.0012  -2.67 -2.76  0.0129 0.0115  -2.80  0.0109  -2.87 -2.90  0.01 0.0097  Senses changes in the extracellular concentration of calcium ions. Transcriptional corepressor that binds to a number of transcription factors. Receptor that may have an important role in cell/cell signaling during nervous system formation Probable hydrolase. Involved in the thymus homing of bone marrow cells. May regulate beta-2 integrin-mediated cell adhesion, migration and motility of neutrophil Forms hydroxylysine residues in -Xaa-Lys-Gly- sequences in collagens. Involved in pH regulation. Plays an important role in signal transduction Catalyzes the conversion of 3'-phosphate to a 2',3'-cyclic phosphodiester at the end of RNA. Tigger transposable element-derived protein 6 Synthesis and degradation of fructose 2,6-bisphosphate Probably contributes to terminal cornification. Associated with keratinocyte activation, proliferation and keratinization Has antibacterial activity Endonuclease that catalyzes the cleavage of RNA on the 3' side of pyrimidine nucleotides. Acts on single stranded and double stranded RNA May be involved in transcriptional regulation The heparin-binding growth factors are angiogenic agents in vivo and are potent mitogens for a variety of cell types in vitro.  -2.93  0.0094  -2.95  0.0091  -3.25  0.0067  -3.43  0.0056  -3.73  0.0044  -3.75  0.0043  -3.81  0.0003  -3.89 -3.93 -4.52  0.0039 0.0002 0.0026  -4.56 -4.90  0.0025 0.0021  -5.67 -7.71  0.0015 0.0008  Could be a global transcriptional regulator. Strongly activates transcription when bound to HCFC1.  -9.95 -12.16  0.0005 0.0003  
 
  60  Table
3.2:
Selected
genes
showing
differential
expression
in
keratinocytes
2
hours
post­ treatment
with
3
μg/ml
LL­37.
Genes
were
excluded
if
they
showed
<2.5­fold
change,
and
 gene
lists
were
manually
curated
for
potential
relevance
to
wound
healing
and
innate
 immune
responses.
 Gene Name Gene Description Fold p Value Change TRI16 May play a role in the regulation of keratinocyte differentiation 7.67 0.004 MIF; May play an important role in the progression of epithelial 6.36 0.0054 MMP11 malignancies; The expression of MIF at sites of inflammation suggest a role for the mediator in regulating the function of macrophage in host defense. FOS Nuclear phosphoprotein which forms a tight but non-covalently 4.57 0.0099 linked complex with the JUN/AP-1 transcription factor. TIE2 This protein is a protein tyrosine-kinase transmembrane 4.35 0.011 receptor for angiopoietin 1. GBG8 Guanine nucleotide-binding proteins (G proteins) are involved 4.24 0.0116 as a modulator or transducer in various transmembrane signaling systems. RT12 28S ribosomal protein S12, mitochondrial precursor; S12mt; 4.20 0.0118 MRP-S12; MT-RPS12 KS6A2 Serine/threonine kinase that may play a role in mediating the 3.92 0.0137 growth-factor and stress induced activation of the transcription factor CREB NPT2B May be involved in actively transporting phosphate into cells 3.13 0.0162 via Na(+) cotransport. May have a role in the synthesis of surfactant in lungs' alveoli. ATE1 Involved in the posttranslational conjugation of arginine to the 3.06 0.0244 N-terminal aspartate or glutamate of a protein. This arginylation is required for degradation of the protein via the ubiquitin pathway. TRIO Promotes the exchange of GDP by GTP. Could play a role in 3.06 0.0244 coordinating cell-matrix and cytoskeletal rearrangements necessary for cell migration and cell growth. NP115589.2 Engulfment and cell motility. 3.05 0.0247 PPIL2 PPIases accelerate the folding of proteins. It catalyzes the cis3.02 0.0252 trans isomerization of proline imidic peptide bonds in oligopeptides NGLY1 Specifically deglycosylates the denatured form of N-linked 2.99 0.0258 glycoproteins in the cytoplasm and assists their proteasomemediated degradation. RET7 Intracellular transport of retinol 2.96 0.0264 ABI2 May be involved in cytoskeletal reorganization. 2.66 0.0352 HSH2D May be a modulator of the apoptotic response through its 2.53 0.0406 ability to affect mitochondrial stability (By similarity). Adapter protein involved in tyrosine kinase and CD28 signaling. Seems to affect CD28-mediated activation of the RE/AP element of the interleukin-2 promoter RND1 Controls rearrangements of the actin cytoskeleton. 2.52 0.041 TRIC May play a role in the formation of the epithelial barrier -2.60 0.0374 PVRL3 Plays a role in cell-cell adhesion. Also involved in the -2.64 0.0449 formation of cell-cell junctions, including adherens junctions and synapses. Induces endocytosis-mediated down-regulation of PVR from the cell surface, resulting in reduction of cell movement and proliferation. SIRPG Probable immunoglobulin-like cell surface receptor. On -2.70 0.0339 binding with CD47, mediates cell-cell adhesion.  61  Gene Name SG1D1 SMURF2  DLG1  RNF34  CUL5  LAIR1  PLOD2  CELR3  CX04A BPAEA  Gene Description May bind androgens and other steroids, may also bind estramustine, a chemotherapeutic agent used for prostate cancer. E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. I Essential multidomain scaffolding protein required for normal development (By similarity). Recruits channels, receptors and signaling molecules to discrete plasma membrane domains in polarized cells. May play a role in adherens junction assembly, signal transduction, cell proliferation, synaptogenesis and lymphocyte activation Has E3 ubiquitin-protein ligase activity. Regulates the levels of CASP8 and CASP10 by targeting them for proteasomal degradation. Protects cells against apoptosis induced by TNF. Binds phosphatidylinositol-5-phosphate and phosphatidylinositol-3-phosphate Component of E3 ubiquitin ligase complexes, which mediate the ubiquitination and subsequent proteasomal degradation of target proteins. Seems to be involved poteosomal degradation of p53/TP53 stimulated by adenovirus E1B-55 kDa protein. May form a cell surface vasopressin receptor Functions as an inhibitory receptor that plays a constitutive negative regulatory role on cytolytic function of natural killer (NK) cells, B-cells and T-cellsModulates cytokine production in CD4+ T-cells, downregulating IL2 and IFNG production while inducing secretion of transforming growth factor beta. Downregulates also IgG and IgE production in B-cells as well as IL8, IL10 and TNF secretion. Inhibits proliferation and induces apoptosis in myeloid leukemia cell lines as well as prevents nuclear translocation of NF-kappa-B p65 subunit/RELA and phosphorylation of I-kappa-B alpha/CHUK in these cells. Forms hydroxylysine residues in -Xaa-Lys-Gly- sequences in collagens. These hydroxylysines serve as sites of attachment for carbohydrate units and are essential for the stability of the intermolecular collagen cross-links Has an important role in stress fiber formation induced by active diaphanous protein homolog 1 (DRF1). Induces microspike formation, in vivo (By similarity). In vitro, stimulates N-WASP-induced ARP2/3 complex activation in the absence of CDC42 (By similarity). May play an important role in the maintenance of sarcomeres and/or in the assembly of myofibrils into sarcomeres. Implicated in regulation of actin polymerization and cell adhesion May have an important role of cell protection in inflammation reaction Cytoskeletal linker protein. Anchors keratin-containing intermediate filaments to the inner plaque of hemidesmosomes. The proteins may self-aggregate to form filaments or a two-dimensional mesh Brain-specific angiogenesis inhibitor.  ENSG00000117114 RRP5 Involved in the biogenesis of rRNA.  Fold p Value Change -2.72 0.0332 -2.80  0.0307  -2.87  0.0287  -2.89  0.0283  -3.36  0.0067  -3.48  0.0178  -3.94  0.0135  -5.25  0.0446  -5.66  0.0067  -6.43  0.0053  -6.58  0.0051  -6.63  0.0051  62  Gene Name FGF2  MUC16  Gene Description The heparin-binding growth factors are angiogenic agents in vivo and are potent mitogens for a variety of cell types in vitro. There are differences in the tissue distribution and concentration of these 2 growth factors Thought to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces  Fold p Value Change -8.32 0.0011  -8.92  0.0032  Table
3.3:
Selected
genes
showing
differential
expression
in
keratinocytes
4
hours
post­ treatment
with
3
μg/ml
LL­37.
Genes
were
excluded
if
they
showed
<2.5­fold
change,
and
 gene
lists
were
manually
curated
for
potential
relevance
to
wound
healing
and
innate
 immune
responses.
 Gene Name Gene Description Fold p Change Value EGF EGF stimulates the growth of various epidermal and 7.03 0.0004 epithelial tissues in vivo and in vitro and of some fibroblasts in cell culture DUS9 Inactivates MAP kinases. Has a specificity for the ERK 5.99 0.0006 family; Required for the uptake of creatine in muscles and brain IFNA1 Produced by macrophages, IFN-alpha have antiviral 5.52 0.0008 activities. Interferon stimulates the production of two enzymes: a protein kinase and an oligoadenylate synthetase SMAD6 Antagonist of signaling by TGF-beta (transforming growth 5.07 0.001 factor) type 1 receptor superfamily members; has been shown to inhibit selectively BMP (bone morphogenetic proteins) signaling. INHBC Inhibins and activins inhibit and activate, respectively, the 4.99 0.0011 secretion of follitropin by the pituitary gland. Inhibins/activins are involved in regulating a number of diverse functions such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, nerve cell survival, embryonic axial development or bone growth, depending on their subunit composition. Inhibins appear to oppose the functions of activins CARD9 Activates NF-kappa-B via BCL10 4.60 0.0014 DACH2 Transcription factor that is involved in regulation of 3.63 0.0034 organogenesis. IGF1R This receptor binds insulin-like growth factor 1 (IGF1) with 3.63 0.0035 a high affinity and IGF2 with a lower affinity. SULF1  KCC1D  Exhibits arylsulfatase activity and highly specific endoglucosamine-6-sulfatase activity. It can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Diminishes HSPG (heparan sulfate proteoglycans) sulfation, inhibits signaling by heparin-dependent growth factors, diminishes proliferation, and facilitates apoptosis in response to exogenous stimulation Calcium/calmodulin-dependent protein kinase belonging to a proposed calcium-triggered signaling cascade. May regulate calcium-mediated granulocyte function. May play a role in apoptosis of erythroleukemia cells. Activates MAP  3.07  0.0068  2.99  0.0076  63  Gene Name  Gene Description  Fold Change  p Value  2.99 2.97  0.0077 0.0079  2.80  0.0433  2.79  0.0103  2.73  0.0114  2.68  0.0123  2.64  0.0132  2.60  0.0141  2.50 -2.51  0.0433 0.0165  -2.56  0.0153  -2.64  0.0131  -2.69  0.0068  -2.72  0.0116  kinase MAPK3 (By similarity). In vitro, phosphorylates transcription factor CREM isoform Beta and probably CREB1 ZN174 HXD11 HMGB3 ISCU SIM2  LECT1  PCOC1 HSPA1  TGFB3 CLC4E  TLR5  TNR19 CBX8  E2F3  Transcriptional repressor Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis Binds preferentially single-stranded DNA and unwinds double stranded DNA Involved in the assembly or repair of the [Fe-S] clusters present in iron-sulfur proteins. Binds iron Transcription factor that may be a master gene of CNS development in cooperation with Arnt. It may have pleiotropic effects in the tissues expressed during development Bifunctional growth regulator that stimulates the growth of cultured chondrocytes in the presence of basic fibroblast growth factor (FGF) but inhibits the growth of cultured vascular endothelial cells. May contribute to the rapid growth of cartilage and vascular invasion prior to the replacement of cartilage by bone during endochondral bone development Binds to the C-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity In cooperation with other chaperones, Hsp70s stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. These chaperones participate in all these processes through their ability to recognize nonnative conformations of other proteins. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage Involved in embryogenesis and cell differentiation May play a role in the response to inflammatory stimuli in peritoneal macrophages. May be involved in immune surveillance processes under transcriptional control of CEBPB Participates in the innate immune response to microbial agents. Mediates detection of bacterial flagellins. Acts via MyD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Can mediate activation of JNK and NF-kappa-B. May promote caspase-independent cell death Component of the Polycomb group (PcG) multiprotein PRC1 complex, a complex required to maintain the transcriptionally repressive state of many genes, including Hox genes, throughout development. PcG PRC1 complex acts via chromatin remodeling and modification of histones; it mediates monoubiquitination of histone H2A 'Lys-119', rendering chromatin heritably changed in its expressibility Transcription activator.  64  Gene Name KU70 NDRF RABP2  IKKB  DUSP4 SMAD7  DDFL1 TCF21  TENS1 PO3F2 EPO  AN32A  NFKB2  STYK1  CLD5  Gene Description Single stranded DNA-dependent ATP-dependent helicase. Appears to mediate neuronal differentiation Cytosolic CRABPs may regulate the access of retinoic acid to the nuclear retinoic acid receptors. CRABP2 may participate in a regulatory feedback mechanism to control the action of retinoic acid on cell differentiation Phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. Also phosphorylates NCOA3 Regulates mitogenic signal transduction by dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2 Antagonist of signaling by TGF-beta (transforming growth factor) type 1 receptor superfamily members; has been shown to inhibit TGF-beta (Transforming growth factor) and activin signaling by associating with their receptors thus preventing SMAD2 access. Promotes cell proliferation Involved in epithelial-mesenchymal interactions in kidney and lung morphogenesis that include epithelial differentiation and branching morphogenesis. May play a role in the specification or differentiation of one or more subsets of epicardial cell types May be involved in cell migration, cartilage development and in linking signal transduction pathways to the cytoskeleton Transcription factor. Erythropoietin is the principal hormone involved in the regulation of erythrocyte differentiation and the maintenance of a physiological level of circulating erythrocyte mass Implicated in a number of cellular processes, including proliferation, differentiation, caspase-dependent and caspase-independent apoptosis, suppression of transformation (tumor suppressor), inhibition of protein phosphatase 2A, regulation of mRNA trafficking and stability in association with ELAVL1, and inhibition of acetyltransferases as part of the INHAT (inhibitor of histone acetyltransferases) complex Appears to have dual functions such as cytoplasmic retention of attached NF-kappa-B proteins and generation of p52 by a cotranslational processing. The proteasomemediated process ensures the production of both p52 and p100 and preserves their independent function. p52 binds to the kappa-B consensus sequence 5'-GGRNNYYCC-3', located in the enhancer region of genes involved in immune response and acute phase reactions. Probable tyrosine protein-kinase, which has strong transforming capabilities on a variety of cell lines. When overexpressed, it can also induce tumor cell invasion as well as metastasis in distant organs. May act by activating both MAP kinase and phosphatidylinositol 3'-kinases (PI3K) pathways Plays a major role in tight junction-specific obliteration of the intercellular space  Fold Change -2.73 -2.73 -2.77  p Value 0.0114 0.0113 0.001  -2.78  0.0104  -2.82  1.71E05  -2.84  0.0095  -2.87 -2.88  0.009 0.009  -2.93  0.0083  -2.94 -2.97  0.0081 0.0124  -2.97  0.0039  -3.02  0.0073  -3.10  0.0065  -3.13  4.38E05  65  Gene Name  Gene Description  FOSL2N PGM5  Fos-related antigen 2 Component of adherens-type cell-cell and cell-matrix junctions. Lacks phosphoglucomutase activity  CIDEA ZN703 SEM3D  Activates apoptosis May function as a transcriptional repressor Induces the collapse and paralysis of neuronal growth cones. Could potentially act as repulsive cues toward specific neuronal populations. Binds to neuropilin May be involved in the assembly, secretion and targeting of TGFB1 to sites at which it is stored and/or activated. May play critical roles in controlling and directing the activity of TGFB1. May have a structural role in the extra cellular matrix (ECM) Heparan sulfate 2-O-sulfotransferase Adapter protein and signal transducer that links members of the tumor necrosis factor receptor family to different signaling pathways by association with the receptor cytoplasmic domain and kinases. Mediates activation of NF-kappa-B and JNK and is involved in apoptosis. The TRAF1/TRAF2 complex recruits the apoptotic suppressors BIRC2 and BIRC3 to TNFRSF1B/TNFR2 Involved in regulation of adherens junction between cells. Functions as a guanine nucleotide exchange factor (GEF), which activates Rap1 small GTPase by exchanging bound GDP for free GTP Forms hydroxylysine residues in -Xaa-Lys-Gly- sequences in collagens. These hydroxylysines serve as sites of attachment for carbohydrate units and are essential for the stability of the intermolecular collagen cross-links Docking proteins interact with receptor tyrosine kinases and mediate particular biological responses. DOK3 is a negative regulator of JNK signaling in B-cells through interaction with INPP5D/SHIP. May modulate Abl function Couples the p55 TNF-receptor (TNF-R55 / TNFR1) to neutral sphingomyelinase (N-SMASE). Specifically binds to the N-smase activation domain of TNF-R55. May regulate ceramide production by N-SMASE Cadherins are calcium dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types The heparin-binding growth factors are angiogenic agents in vivo and are potent mitogens for a variety of cell types in vitro. There are differences in the tissue distribution and concentration of these 2 growth factors Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction Acts as a regulatory subunit of the 26S proteasome which is involved in the ATP-dependent degradation of ubiquitinated proteins HCO3- transporter, cytoplasmic; HCO3- transporter, eukaryote; HCO3-transporter, c-terminal; Na+/HCO3transporter Regulates the p53 pathway to control the expansion  LTBP3  HS2ST TRAF1  DOCK4  PLOD2  DOK3  FAN  CAD12  FGF2  CAN11 PSD12 NP004849.2 DEF  Fold Change -3.13 -3.14  p Value 0.0063 0.0062  -3.24 -3.26 -3.27  0.0055 0.0053 0.0053  -3.42  4.30E03  -3.43 -3.70  0.0043 0.0032  -4.03  0.0023  -4.05  0.0021  -4.07  0.0022  -4.81  0.0012  -5.12  0.001  -5.27  0.0008  -5.41  0.0008  -6.44  0.0005  -6.60  0.0004  -6.98  0.0103  66  Gene Name  MFSD4 CDC6 PLAL1 EYA4  Gene Description growth of digestive organs Major facilitator superfamily domain-containing protein 4 Involved in the initiation of DNA replication. Also participates in checkpoint controls that ensure DNA replication is completed before mitosis is initiated Shows weak transcriptional activatory activity. Transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase-activating polypeptide Thought to play a role in transcription regulation during organogenesis through its intrinsic protein phosphatase activity.  Fold Change  p Value  -7.32 -7.58  0.0003 0.0003  -7.76  0.0079  -118.41  1.61E06  The differentially expressed genes might have been co-regulated by a set of common transcription factors. As a large number of genes showed altered expression, I was curious to determine if some of them might be potentially co-regulated. Accordingly, a transcription factor binding site overrepresentation analysis was performed (Table 3.4). In this analysis, the frequency with which predicted transcription factor binding sites appear within the promoter regions of differentially expressed genes is compared with the frequency expected due to chance alone; a high Z score and low Fisher score suggest that the transcription factor binding site appears more often than can be explained by chance. This analysis revealed several transcription factors that might mediate downstream effects of LL-37 stimulation. Transcription factors that were implicated at multiple timepoints are presented in bold and may merit further investigation; these include Foxd1, Foxd3, FoxI1, HMG-IY, Nkx2-5, Prrx2, and SRY. Foxd1 and HMG-1Y are known to alter NF-kB responses (12, 13), making them expecially interesting targets. Table
3.4:
Transcription
factor
binding
sites
overrepresented
in
the
promoter
regions
of
 genes
showing
differential
expression
after
stimulation
with
3
µg/ml
LL­37.
  1 hour  Transcription factor ATHB5 ELF5 EMBP1  TF class HOMEO-ZIP ETS bZIP  Z-score 6.091 3.067 6.34  Fisher score 2.36E-03 2.03E-01 2.47E-02  67  Transcription factor FOXD1 Foxd3 HMG-IY MYC-MAX NFYA  Z-score 3.657 3.994 4.124 5.283 5.244  Fisher score 4.06E-02 9.94E-02 3.52E-02 1.73E-02 2.43E-02  NR3C1 TLX1-NFIC  TF class FORKHEAD FORKHEAD HMG bHLH-ZIP CAAT-BOX NUCLEAR RECEPTOR HOMEO/CAAT  6.561 3.419  5.27E-02 7.94E-02  2 hours  Athb-1 Broad-complex_1 Broad-complex_3 FOXD1 FOXI1 HMG-IY Nkx2-5 Prrx2 SOX9 SRY  HOMEO-ZIP ZN-FINGER, C2H2 ZN-FINGER, C2H2 FORKHEAD FORKHEAD HMG HOMEO HOMEO HMG HMG  11.91 11.33 12.12 10.17 10.94 12.74 12.76 11.51 11.39 15.73  8.51E-03 3.74E-06 4.42E-03 2.89E-03 2.58E-02 6.82E-03 5.47E-01 2.79E-01 1.44E-03 1.90E-01  4 hours  Broad-complex_4 Foxa2 Foxd3 FOXI1 Hunchback IRF1 Nkx2-5 Prrx2 Sox5 SRY  ZN-FINGER, C2H2 FORKHEAD FORKHEAD FORKHEAD ZN-FINGER, C2H2 TRP-CLUSTER HOMEO HOMEO HMG HMG  25.37 15.98 18.62 21.85 21.07 16.29 19.1 21.26 22.33 21.54  8.78E-06 2.53E-03 1.79E-03 2.39E-03 3.89E-04 9.90E-04 4.70E-02 1.47E-03 3.43E-06 4.43E-03  Pathway and gene ontology over-representation analysis suggested that LL-37 selectively activates genes involved in protein synthesis, tissue remodelling, and innate immune responses. Having identified genes showing altered expression after LL-37 treatment, as well as a number of transcription factors that might be putative effectors of LL-37-mediated downstream effects, I wished to gain insight into the potential biological consequences of altered gene expression after LL-37 treatment. Accordingly, I performed a pathway overrepresentation analysis (Table 3.5), which is intended to identify signalling pathways 68  whose component proteins show non-random changes in expression as a result of the experimental treatment. Such a test generates two sets of statistics: a p-value, representing the probability that the number of genes found to shown altered reguation amongst the pathway components occurred by chance alone, and a second p-value corrected for multiple testing, representing the probabilility that the first statistical test resulted from chance alone, given the number of tests being performed. No pathways were found to be significantly overrepresented amongst the differentially regulated genes at any timepoint when results were corrected for multiple testing; accordingly, a corrected p-value of 0.3 was chosen as a cutoff for further analysis. This corrected pvalue represents a 30% probability that the obtained results were due to chance alone. No pathways showed downregulation with a corrected p value of less than 0.3. The upregulated pathways were predominantly involved in mRNA translation and protein synthesis. Notably, almost all results were found to originate from the 4 hour dataset, perhaps as a result of the larger number of differentially expressed genes detected at that timepoint. 
 Table
3.5:
Results
of
pathway
over­representation
analysis.
  InnateDB Pathway ID Condition LL37_4hr 1958  LL37_4hr  1710  LL37_4hr LL37_4hr LL37_4hr LL37_4hr LL37_4hr LL37_4hr  1376 1319 1373 1248 1464 1535  Pathway L13a-mediated translational silencing of Ceruloplasmin expression Processing of Intronless PremRNAs Formation of the Editosome mRNA Editing: C to U Conversion Eukaryotic Translation Elongation Peptide chain elongation mRNA 3`-end processing SLBP Dependent Processing of Replication-Dependent Histone  p-Value, corrected Genes for Uppmultiple regulated value testing 6 0.001 0.158  7  0.001  0.167  7 7 5 5 7 7  0.003 0.003 0.003 0.003 0.003 0.001  0.168 0.168 0.172 0.172 0.175 0.178  69  InnateDB Pathway Condition ID LL37_4hr  1630  LL37_4hr  1923  LL37_4hr  1919  LL37_4hr  1527  LL37_4hr  1251  LL37_4hr LL37_4hr  1921 1354  LL37_4hr  1505  LL37_4hr  1361  LL37_4hr  1706  LL37_4hr LL37_4hr  474 1655  LL37_1hr LL37_4hr  1980 1611  LL37_4hr  973  LL37_4hr  1685  LL37_4hr  1088  LL37_4hr  544  Pathway Pre-mRNAs Cap-dependent Translation Initiation Formation of a pool of free 40S subunits GTP hydrolysis and joining of the 60S ribosomal subunit Ribosomal scanning and start codon recognition Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S Viral mRNA Translation SLBP independent Processing of Histone Pre-mRNAs Transport of Mature mRNA Derived from an Intronless Transcript Transport of Mature mRNA derived from an Intron-Containing Transcript Eukaryotic Translation Termination Ribosome Cleavage of Growing Transcript in the Termination Region Formation of PAPS Transport of the SLBP Dependant Mature mRNA Skeletal muscle hypertrophy is regulated via akt-mtor pathway Translation initiation complex formation Noncanonical Wnt signaling pathway Prostate cancer  p-Value, corrected Genes for Uppmultiple regulated value testing 6  0.002  0.181  6  0.002  0.181  6  0.002  0.181  6  0.002  0.181  6  0.002  0.183  5 7  0.002 0.001  0.184 0.193  7  0.001  0.198  7  0.002  0.201  5  0.002  0.206  5 7  0.001 0.004  0.209 0.235  2 7  0 0.001  0.243 0.243  3  0.005  0.278  4  0.005  0.282  3  0  0.296  5  0.006  0.299  The results of this analysis suggested that LL-37 treatment might upregulate a number of genes involved in transcription, translation and growth responses. To identify specific genes that might be important to the mode of action of LL-37, some of these overrepresented pathways were visualized using Cerebral. The ‘prostate cancer’ pathway aroused my interest; although I am not interested in prostate biology, I hypothesized that 70  genes important to oncogenesis might also be involved in growth responses and wound repair. Indeed, visualization of the ‘Prostate cancer’ pathway revealed that LL-37 treatment alters keratinocyte expression of growth factors and growth factor receptors (Fig. 3.1), including EFG and insulin. In combination with the apparent upregulation of pathways involved in protein synthesis, this suggested LL-37 might selectively upregulate genes involved in tissue remodelling.  71  72  Figure
3.1:
Genes
within
the
'Prostate
cancer'
pathway
showing
altered
regulation
after
 stimulation
with
3
µg/ml
LL­37.
Red
indicates
upregulation;
green,
downregulation.
Results
 were
excluded
unless
the
absolute
value
of
the
fold
change
was
>1.5
and
the
result
was
 statistically
significant
(p
≤
0.05).

  To further clarify the biological consequences of LL-37 treatment of keratinocytes, I performed a gene ontology over-representation analysis. In this analysis the observed number of genes associated with a specific gene ontology term is compared with the expected number (based on the size of the dataset) in order to identify ontological categories that appear more often than can be explained by chance alone. The results of this analysis (Table 3.6) provided a chronology of the cellular response: at one hour poststimulation, a variety of genes involved in the determination of tissue morphology were upregulated, as were genes having transcription co-activator activity. At two hours poststimulation, genes involved in intracellular signalling and cytoskeletal organization were selectively upregulated. Four hours post-stimulation, genes involved in cell differentiation and the innate immune response showed selective enrichment. Overall, these results support a role for low doses of LL-37 in wound repair and the regulation of innate immunity.  1 hour timepoint  Table
3.6:
Over­represented
gene
ontology
terms
associated
with
differentially
expressed
 genes
at
1,
2,
and
4
hours
after
stimulation
with
3
μg/ml
LL­37.
Notable
findings
are
 presented
in
bold
font.

 Biological Process microspike biogenesis filopodium formation mitotic recombination detection of chemical stimulus detection of calcium ion amine transport neutral amino acid transport monoamine transport morphogenesis of a branching structure mammary gland development  Observed  Expected  Relative enrichment  p Value  2 2 2 2 2 5 2 2  0.11 0.08 0.08 0.13 0.1 0.65 0.15 0.08  18.18 25 25 15.38 20 7.69 13.33 25  0.0051 0.0028 0.0028 0.0065 0.0039 0.0005 0.0097 0.0028  2 2  0.04 0.04  50 50  0.0006 0.0006  73  Biological Process tube morphogenesis gland development tube development embryonic pattern specification regulation of Ras GTPase activity regulation of Rho GTPase activity regulation of Cdc42 GTPase activity Molecular Function transcription coactivator activity phospholipase C activity phosphoinositide phospholipase C activity inositol or phosphatidylinositol phosphodiesterase activity amine transporter activity monoamine transporter activity calcium channel activity calcium-activated potassium channel activity  2 hour timepoint  Cellular Component ruffle integral to plasma membrane intrinsic to plasma membrane plasma membrane part plasma membrane Biological Process signal transduction intracellular signaling cascade small GTPase mediated signal transduction ARF protein signal transduction regulation of ARF protein signal transduction regulation of small GTPase mediated signal transduction regulation of Rho protein signal transduction Rho protein signal transduction regulation of signal transduction cell maturation neuron maturation neuron remodeling neurogenesis generation of neurons neuron differentiation  Observed  Expected  Relative enrichment  p Value  2 2 2 2 2 2  0.08 0.11 0.15 0.15 0.1 0.1  25 18.18 13.33 13.33 20 20  0.0028 0.0051 0.0097 0.0097 0.0039 0.0039  2  0.1  20  0.0039  7 3  2.2 0.37  3.18 8.11  0.0066 0.0059  3  0.33  9.09  0.0042  3 5 2 4  0.33 0.73 0.07 0.76  9.09 6.85 28.57 5.26  0.0042 0.0008 0.0018 0.0069  2  0.15  13.33  0.0096  3 31 31 35 36  0.33 16.75 16.89 20.34 24.2  9.09 1.85 1.84 1.72 1.49  0.0040 0.0005 0.0006 0.0009 0.0085  Observed  Expected  Relative enrichment  p Value  35 17  24.23 8.82  1.44 1.93  0.0097 0.0062  9 2  2.63 0.13  3.42 15.38  0.0013 0.0069  2  0.13  15.38  0.0069  6  0.95  6.32  0.0004  4 4  0.54 0.69  7.41 5.8  0.0021 0.0051  8 2 2 2 4 4 4  2.57 0.15 0.06 0.02 0.69 0.68 0.63  3.11 13.33 33.33 100 5.8 5.88 6.35  0.0041 0.0096 0.0013 0.0001 0.0049 0.0047 0.0036  74  4 hour timepoint  Biological Process neuron development retrograde vesicle-mediated transport\, Golgi to ER cytoskeleton organization and biogenesis actin filament-based process actin cytoskeleton organization and biogenesis actin filament organization protein amino acid acylation protein amino acid acetylation developmental maturation  Observed  Expected  Relative enrichment  p Value  4  0.53  7.55  0.0019  2  0.11  18.18  0.0053  9 6  2.93 1.31  3.07 4.58  0.0026 0.0020  6 3 2 2 2  1.18 0.19 0.15 0.13 0.15  5.08 15.79 13.33 15.38 13.33  0.0012 0.0008 0.0096 0.0077 0.0096  2 2 7  0.12 0.05 2.13  16.67 40 3.29  0.0067 0.0011 0.0055  7  0.99  7.07  0.0001  2  0.12  16.67  0.0067  4  0.56  7.14  0.0024  Molecular Function potassium channel regulator activity cytoskeletal adaptor activity GTPase regulator activity guanyl-nucleotide exchange factor activity ARF guanyl-nucleotide exchange factor activity Ras guanyl-nucleotide exchange factor activity Rho guanyl-nucleotide exchange factor activity small GTPase regulator activity channel or pore class transporter activity alpha-type channel activity ion channel activity anion channel activity chloride channel activity ion transporter activity  4 6  0.49 1.12  8.16 5.36  0.0014 0.0009  8 8 8 3 3 11  2.77 2.68 2.46 0.42 0.37 4.66  2.89 2.99 3.25 7.14 8.11 2.36  0.0065 0.0054 0.0032 0.0087 0.0061 0.0068  Cellular Component cell projection lamellipodium leading edge basolateral plasma membrane cell junction  4 3 3 3 5  0.82 0.16 0.28 0.2 1.05  4.88 18.75 10.71 15 4.76  0.0093 0.0005 0.0026 0.0010 0.0039  Observed  Expected  Relative enrichment  p Value  3 24 15  0.39 13.83 7.43  7.69 1.74 2.02  0.0063 0.0062 0.0078  2 2 28 10  0.1 0.1 16.53 4.11  20 20 1.69 2.43  0.0034 0.0034 0.0044 0.0084  Biological Process response to nutrient cell differentiation intracellular protein transport fructose 2,6-bisphosphate metabolism peptide hormone processing ion transport anion transport  75  Biological Process inorganic anion transport chloride transport localization establishment of localization innate immune response Molecular Function anion binding chloride ion binding protein homodimerization activity fructose-2,6-bisphosphate 2phosphatase activity intramolecular transferase activity intramolecular transferase activity\, phosphotransferases 6-phosphofructo-2-kinase activity phosphotransferase activity\, alcohol group as acceptor nitric-oxide synthase regulator activity receptor signaling protein activity receptor signaling protein serine/threonine kinase signaling protein activity transmembrane receptor protein serine/threonine kinase signaling protein activity transforming growth factor beta receptor\, cytoplasmic mediator activity transforming growth factor beta receptor\, inhibitory cytoplasmic mediator activity chloride channel activity voltage-gated chloride channel activity ion transporter activity anion transporter activity iodide transporter activity  Observed  Expected  Relative enrichment  p Value  9 5 77 77 6  3.38 1.02 58.11 57.55 1.8  2.66 4.9 1.33 1.34 3.33  0.0069 0.0034 0.0042 0.0032 0.0092  5 5 7  1.23 1.23 2.14  4.07 4.07 3.27  0.0076 0.0076 0.0056  2 3  0.14 0.39  14.29 7.69  0.0076 0.0066  3 2  0.3 0.09  10 22.22  0.0030 0.0031  26  15.47  1.68  0.0068  2 10  0.09 3.25  22.22 3.08  0.0031 0.0016  2  0.12  16.67  0.0051  2  0.14  14.29  0.0076  2  0.12  16.67  0.0051  2 5  0.05 1.18  40 4.24  0.0005 0.0064  3 25 10 2  0.44 14.82 2.76 0.05  6.82 1.69 3.62 40  0.0091 0.0075 0.0005 0.0005  Cellular Component No terms enriched  DISCUSSION: To date, only one microarray experiment has previously been performed on LL-37stimulated primary keratinocytes and in that experiment a much higher concentration of LL-37 was used (about 50 µg/ml) (2). While the complete results of that experiment are 76  not publicly available, the published findings suggest that such concentrations result in a marked upregulation of a variety of pro-inflammatory cytokines. In contrast, in our experiment, low concentrations of LL-37 altered the expression of a variety of genes apparently involved in growth responses, tissue remodelling, and RNA and protein processing, indicating a very different response. While these results have yet to be confirmed, they suggest a number of interesting directions for future work.  The results of this microarray should be confirmed by an independent experiment. Ideally this experiment would confirm some of the more interesting results using the more accurate technique qRT-PCR and examine the effects of TLR stimulation, LL-37 stimulation, and combinatorial TLR and LL-37 stimulation, upon genes of interest that showed differential expression in the microarray. This would allow some insight into potential interplay between TLR signalling and LL-37-mediated growth responses. The observation that LL-37 appears to upregulate the expression of a number of growth factors, including EGF and insulin, merits further investigation, as was the exciting observation of effects on genes of innate immunity at 4 hours (DUS9, IFNA1, SMAD6, CARD9, HMGB3, HSPA1, TGFB3, CLC4E, TLR5,TNR19, RABP2, IKKB, DUSP4, SMAD7, TCF21, NFKB2, STYK1, TRAF1, DOCK4, DOK3, FAN, although many of these represented a down regulation of innate immunity - genes in italics). Should these effects be confirmed, it would be interesting to investigate their relevance to LL-37-mediated growth and pro- and anti-inflammatory responses.  The results of the transcription factor binding site over-representation analysis also suggest new targets for investigation. Many of the transcription factors identified by the transcription factor binding site overrepresentation analysis have been previously 77  identified as effectors of wound healing or inflammation. For instance, Foxd1 regulates the activity of NF-AT and NF-κB in T cells (12), while Foxd3 has been shown to be induced by Snail in Xenopus embryos (14). HMG-1/Y helps NF-κB to activate transcription (13), and NKx2-5 has been implicated in the regulation of genes involved in bladder responses to wounding and infection (15). Prrx2 plays an important role in the regulation of wound repair by fetal fibroblasts (16), and SRY has been identified as a potential downstream effector of p38 in a previous keratinocyte microarray experiment (17). The potential role of these transcription factors in the mechanism of action of LL37 merits further investigation.  To complement these studies, this microarray experiment should be repeated in a model of the bronchial epithelium. LL-37 has been shown to stimulate wound-healing responses in bronchial epithelial cells via EGFR activation (18). A comparative analysis of genes showing altered regulation in response to LL-37 in both keratinocytes and bronchial epithelial cells might reveal a conserved set of ‘wound response genes’ and facilitate the identification of downstream effectors of LL-37-mediated effects. These studies, when complete, should increase our understanding of epithelial wound responses and the interplay between TLR and LL-37-mediated responses in the maintenance of epithelial homeostasis.  78  LITERATURE CITED: 1. Eming, S. A., T. Krieg, and J. M. Davidson. 2007. Inflammation in Wound Repair: Molecular and Cellular Mechanisms. J Invest Dermatol 127:514-525. 2. Braff, M. H., M. i. A. Hawkins, A. D. Nardo, B. Lopez-Garcia, M. D. Howell, C. Wong, K. Lin, J. E. Streib, R. Dorschner, D. Y. M. Leung, and R. L. Gallo. 2005. Structure-Function Relationships among Human Cathelicidin Peptides: Dissociation of Antimicrobial Properties from Host Immunostimulatory Activities. J Immunol 174:4271-4278. 3. Nizet, V., T. Ohtake, X. Lauth, J. Trowbridge, J. Rudisill, R. A. Dorschner, V. Pestonjamasp, J. Piraino, K. Huttner, and R. L. Gallo. 2001. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414:454-457. 4. Heilborn, J. D., M. F. Nilsson, G. Kratz, G. Weber, O. Sorensen, N. Borregaard, and M. Stahle-Backdahl. 2003. The Cathelicidin Anti-Microbial Peptide LL-37 is Involved in Re-Epithelialization of Human Skin Wounds and is Lacking in Chronic Ulcer Epithelium. J. Invest. Dermatol. 120:379-389. 5. Rodriguez-Martinez, S., J. C. Cancino-Diaz, L. M. Vargas-Zuniga, and M. E. Cancino-Diaz. 2008. LL-37 regulates the overexpression of vascular endothelial growth factor (VEGF) and c-IAP-2 in human keratinocytes. International Journal of Dermatology 47:457-462. 6. Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, and R. Medzhitov. 2004. Recognition of Commensal Microflora by Toll-Like Receptors Is Required for Intestinal Homeostasis. Cell 118:229-241. 7. Shaykhiev, R., J. Behr, and R. Bals. 2008. Microbial Patterns Signaling via Toll-Like Receptors 2 and 5 Contribute to Epithelial Repair, Growth and Survival. PLoS ONE 3:1393. 8. Hokamp, K., F. M. Roche, M. Acab, M. E. Rousseau, B. Kuo, D. Goode, D. Aeschliman, J. Bryan, L. A. Babiuk, R. E. Hancock, and F. S. Brinkman. 2004. ArrayPipe: a flexible processing pipeline for microarray data. Nucleic Acids Res 32:W457-459. 9. Ho Sui, S. J., J. R. Mortimer, D. J. Arenillas, J. Brumm, C. J. Walsh, B. P. Kennedy, and W. W. Wasserman. 2005. oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes. Nucleic Acids Res 33:31543164. 10. Barsky, A., J. L. Gardy, R. E. Hancock, and T. Munzner. 2007. Cerebral: a Cytoscape plugin for layout of and interaction with biological networks using subcellular localization annotation. Bioinformatics (Oxford, England) 23:1040-1042. 11. Zhang, B., D. Schmoyer, S. Kirov, and J. Snoddy. 2004. GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies. BMC Bioinformatics 5:16. 12. Lin, L., and S. L. Peng. 2006. Coordination of NF-{kappa}B and NFAT Antagonism by the Forkhead Transcription Factor Foxd1. J Immunol 176:4793-4803. 13. Lehming, N., D. Thanos, J. M. Brickman, J. Ma, T. Maniatis, and M. Ptashne. 1994. An HMG-like protein that can switch a transcriptional activator to a repressor. Nature 371:175-179. 14. Aybar, M. J., M. A. Nieto, and R. Mayor. 2003. Snail precedes Slug in the genetic cascade required for the specification and migration of the Xenopus neural crest. Development 130:483-494. 79  15. Saban, R., C. Simpson, R. Vadigepalli, S. Memet, I. Dozmorov, and M. R. Saban. 2007. Bladder inflammatory transcriptome in response to tachykinins: neurokinin 1 receptor-dependent genes and transcription regulatory elements. BMC urology 7:7. 16. White, P., D. W. Thomas, S. Fong, E. Stelnicki, F. Meijlink, C. Largman, and P. Stephens. 2003. Deletion of the Homeobox Gene PRX-2 Affects Fetal but Not Adult Fibroblast Wound Healing Responses. 120:135-144. 17. Gazel, A., R. I. Nijhawan, R. Walsh, and M. Blumenberg. 2008. Transcriptional profiling defines the roles of ERK and p38 kinases in epidermal keratinocytes. Journal of cellular physiology 215:292-308. 18. Shaykhiev, R., C. Beisswenger, K. Kandler, J. Senske, A. Puchner, T. Damm, J. Behr, and R. Bals. 2005. Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure. American journal of physiology 289:L842-848.  80  CHAPTER IV In this thesis, I have demonstrated that low, physiologically relevant doses of the host defence peptide LL-37 can substantially increase the amount of IL-8 released by epithelial cells exposed to pro-inflammatory stimuli. As epithelial concentrations of LL37 increase in response to infection or wounding (1-5), the ability of LL-37 to boost innate immune responses suggests a potential regulatory role for LL-37 in the epithelial inflammatory response.  Prior to this work, considerable evidence identified LL-37 as both an immunomodulatory agent and an important component of epithelial defence. Administration of LL-37 rescues rats and mice from systemic endotoxaemia (6, 7), suppresses TNF-α production by peripheral blood mononuclear cells in response to lipopolysaccharide (8), and increases cytokine and chemokine production by peripheral blood mononuclear cells in response to IL-1β and GM-CSF (9). In the epithelia, LL-37 has been shown to have a generally pro-inflammatory effect, eliciting the production of IL-8, IL-18, IL-6, IL-10, IP-10, MCP-1, MIP-3α, and RANTES by keratinocytes (10, 11) and the production of IL-8 (6, 12) and IL-6 (Pistolic and Hancock, unpublished data) by bronchial epithelial cells. Studies involving the effects of LL-37 treatment upon epithelial cells, however, have generally sought to replicate in vivo concentrations of LL-37 during infection through the use of 20 – 50 µg/ml concentrations of peptide; in inflammation, concentrations of LL-37 have been suggested to increase to as much as 50 µg/ml in the lung (13) and mg/ml concentrations in the skin (14). The work presented in this thesis is novel as it demonstrates that LL-37 can alter epithelial cell responses at concentrations far lower than those thought to be present during inflammation. Indeed, concentrations of  81  LL-37 exceeding those used in these studies are thought to be present in normal human sweat (15) and airway surfactant (13). This finding has some interesting implications.  One of the great challenges of epithelial immunology is to better understand the mechanisms that regulate differential responses to commensal and pathogenic microbes. Toll-like receptor (TLR) ligands, for instance, are often referred to as Pathogen Associated Molecular Patterns (PAMPs), a name that overlooks the fact that the same molecules are found in a variety of microbial species that do not elicit inflammation. It would be an error to overlook the influence of microbial manipulation of host defence in the maintenance of such immunological cease-fires (16), but the mechanisms used by the body to distinguish friend from foe are of primary interest due to their potential to reveal new targets for therapeutic intervention. My thesis advances this field by revealing a novel mechanism that might allow differential responses to microbes based on epithelial integrity; areas in which the epithelial layer is compromised would be expected to exhibit increased local concentrations of LL-37, which would strongly increase the proinflammatory responses of epithelial cells and facilitate a more rapid return to homeostasis.  Accordingly, these findings suggest several avenues of investigation. One goal of future work should be to confirm that LL-37 alters epithelial cell responses in vivo. While cultured primary cells are a useful model system, it would be interesting to expand these studies into ex vivo and in vivo experiments. One potential model system would be the collection of skin samples from volunteers using a punch biopsy; excised skin could then be maintained in tissue culture medium and stimulated with TLR ligands or live bacteria in the presence and absence of LL-37. Transcriptional profiling of the stimulated cells 82  should provide additional evidence that LL-37 regulates epithelial inflammation in vivo, and might also provide some evidence as to the mechanisms underlying those effects. Complementary studies could be pursued in a mouse model. Mice deficient in the murine LL-37 homologue CRAMP are susceptible to skin infection by Group A Streptococci (17), a finding which was attributed to the purported ‘direct killing’ ability of host defence peptides. As LL-37 is thought to lack direct antimicrobial activity at physiological salt concentrations (18), it would be exceedingly interesting to study the CRAMP knockout mouse in order to determine the actual mechanism underlying its propensity to skin infection. For instance, do increased concentrations of CRAMP alter the responses of murine keratinocytes to bacteria? If so, it would be interesting to examine potential differences in cytokine responses elicited from wild-type and CRAMP-deficient epithelial cells by pro-inflammatory stimuli. One might also study the ability of exogenous CRAMP or LL-37 to alter the innate immune responses of the knockout mouse. For instance, could topical application of a host defence peptide increase immune cell recruitment and inflammation in infected epithelial tissues? The results of such studies would dramatically expand our understanding of the role of host defence peptides in epithelial inflammation.  Future studies should also address the mechanism by which LL-37 modulates its immunomodulatory effects in epithelial cells. While it would be ideal to identify a specific receptor for LL-37, a number of lines of evidence suggest that such a receptor may not exist. For instance, in the lung epithelial cell line A549 LL-37 is rapidly taken up and translocated to the perinuclear region (19), suggesting that interaction with a cellsurface receptor may be less relevant. Similarly, the ability of a D-amino-acid form of LL-37 to elicit IL-8 release from keratinocytes (20) suggests that LL-37 does not mediate 83  its effects via a traditional ‘lock and key’ receptor. Two lines of investigation accordingly suggest themselves. First, I am interested in the possibility that LL-37 mediates its wide variety of effects via the general activation of cellular stress response pathways. LL-37 is cytotoxic to human cells when present in high concentrations, and I hypothesize that it causes local alterations in membrane chemistry even when at tolerated concentrations. Local disruptions in membrane integrity might activate diverse stress response pathways, which would then mediate downstream responses such as cell proliferation, migration and angiogenesis by provoking the compensatory production of growth factors. Transitory alteration of membrane integrity might also serve as a ‘danger signal’; the release of cytoplasmic contents into the extracellular environment might alter the responses of neighbouring cells to pro-inflammatory stimuli. This hypothesis would be difficult to test; however transcriptional profiling of epithelial cell responses to LL-37 might reveal stress-response pathways that could be further investigated as host defence peptide targets. A second line of investigation would address the possibility that LL-37 can mediate its immunomodulatory effects by altering the binding of TLR ligands to TLR receptors. I am intrigued by the ability of LL-37/poly(I:C) co-stimulation to provoke rapid IL-8 and extreme cytotoxicity in bronchial epithelial cells, a phenomenon which merits further investigation as a potential anti-viral defence mechanism in vivo. The activation of TLR3 allows innate immune responses to a number of viral infections (21); as LL-37 concentrations are expected to increase at sites of infection, the increases in IL-8 release and cytotoxicity observed when bronchial epithelial cells were costimulated with LL-37 and poly(I:C) might represent an adaptation to limit viral replication and spread. It would be interesting to investigate these responses are in fact TLR-dependent, or instead rely on other pathways, such as the RIG-1-like helicases, which also detect dsRNA. Similarly, as LL-37 is able to form complexes with human 84  DNA and activate plasmacytoid dendritic cells (22), it would be interesting to investigate whether the presence of LL-37 could allow human DNA to elicit an inappropriate inflammatory response in the epithelia—were this to prove the case, it might have profound implications for the etiology of autoimmune conditions of the skin such as psoriasis. An improved understanding of the dramatic response observed to dsRNA and peptide might inform the design of future immunostimulants, or be potentially relevant to the design of vaccine adjuvants.  As low doses of peptide, in conjunction with appropriate additional stimuli, can alter cytokine production by epithelial cells, it would also be interesting to investigate the ability of host defense peptides to alter TLR-mediated growth responses. In the murine colonic epithelium, TLR stimulation is necessary for the maintenance of epithelial repair and homeostasis; mice deficient in TLR signaling show dramatically worsened symptoms in a colitis model (23). As LL-37 is a known growth factor for a variety of epithelial cell types (10, 24, 25), it would be interesting to investigate potential interactions between LL-37 and TLR ligands in the alteration of epithelial growth responses. Previously, a synthetic peptide based on the sequence of LL-37 has been shown to alter the proliferative responses of bronchial epithelial cells to LPS and LTA (26). Accordingly, I suggest that the microarray analysis presented in Chapter III be continued, as it might allow the identification of proliferative pathways activated in epithelial cells by LL-37 treatment. Once genes of interest have been identified, qPCR could be used to determine if they showed altered expression in cells that were stimulated with both LL-37 and a TLR ligand. These studies might increase our understanding of how host defense peptides facilitate epithelial growth responses and speed the return to tissue homeostasis. 85  Ultimately, the goal of these studies is the identification of targets for therapeutic intervention. An improved understanding of host defense peptide-mediated epithelial immunomodulation might eventually translate into the development of both novel immunostimulants and anti-inflammatories. For instance, if LL-37 can complex with nucleic acids and provoke a strong immune response, perhaps syntheic peptide/nucleic acid complexes might be useful therapeutic agents for papillomaviral skin infection, which typically does not trigger inflammation. Alternately, understanding the mechanism of action by which LL-37 alters pro-inflammatory responses in the epithelia might allow for the design of therapies to block those processes in autoimmune conditions such as psoriasis. In order to proceed, however, a better understanding of the interaction between LL-37 and other regulators of inflammation is required. For instance, 1’25’-dihydroxy Vitamin D (calcitriol), an important signaling molecule in the epithelia, strongly induces increased expression of the LL-37 precursor hCAP-18 in keratinocytes (27), yet Vitamin D3 analogues are typically used as psoriasis therapies, where they reduce inappropriate inflammation (28). Given these apparently contradictory indications, it would be interesting to investigate the effects of Vitamin D3 treatment on the ability of LL-37 to alter epithelial cell responses to pro-inflammatory stimuli. Such studies might improve our understanding of the regulation of epithelial inflammation.  To conclude, the skin and bronchial epithelium are complex immune organs. It is to be hoped that an improved understanding of the role of host defense peptides in epithelial innate immunity will someday allow the design of new strategies both to boost innate immunity in order to resolve infections, and to restrain it when it goes awry.  86  LITERATURE CITED: 1.  Conner, K., K. Nern, J. Rudisill, T. O'Grady, and R. L. Gallo. 2002. The antimicrobial peptide LL-37 is expressed by keratinocytes in condyloma acuminatum and verruca vulgaris. J Am Acad Dermatol 47:347-350.  2.  Lopez-Garcia, B., P. H. Lee, and R. L. Gallo. 2006. Expression and potential function of cathelicidin antimicrobial peptides in dermatophytosis and tinea versicolor. The Journal of antimicrobial chemotherapy 57:877-882.  3.  Frohm, M., B. Agerberth, G. Ahangari, M. Stahle-Backdahl, S. Liden, H. Wigzell, and G. H. Gudmundsson. 1997. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. The Journal of biological chemistry 272:15258-15263.  4.  Heilborn, J. D., M. F. Nilsson, G. Kratz, G. Weber, O. Sorensen, N. Borregaard, and M. Stahle-Backdahl. 2003. The Cathelicidin Anti-Microbial Peptide LL-37 is Involved in Re-Epithelialization of Human Skin Wounds and is Lacking in Chronic Ulcer Epithelium. J. Invest. Dermatol. 120:379-389.  5.  Schauber, J., R. A. Dorschner, A. B. Coda, A. S. Buchau, P. T. Liu, D. Kiken, Y. R. Helfrich, S. Kang, H. Z. Elalieh, A. Steinmeyer, U. Zugel, D. D. Bikle, R. L. Modlin, and R. L. Gallo. 2007. Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism. The Journal of clinical investigation 117:803-811.  6.  Scott, M. G., D. J. Davidson, M. R. Gold, D. Bowdish, and R. E. W. Hancock. 2002. The Human Antimicrobial Peptide LL-37 Is a Multifunctional Modulator of Innate Immune Responses. J Immunol 169:3883-3891.  7.  Cirioni, O., A. Giacometti, R. Ghiselli, C. Bergnach, F. Orlando, C. Silvestri, F. Mocchegiani, A. Licci, B. Skerlavaj, M. Rocchi, V. Saba, M. Zanetti, and G. Scalise. 2006. LL-37 Protects Rats against Lethal Sepsis Caused by GramNegative Bacteria. Antimicrob. Agents Chemother. 50:1672-1679.  8.  Mookherjee, N., K. L. Brown, D. M. Bowdish, S. Doria, R. Falsafi, K. Hokamp, F. M. Roche, R. Mu, G. H. Doho, J. Pistolic, J. P. Powers, J. Bryan, F. S. Brinkman, and R. E. W. Hancock. 2006. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol 176:2455-2464. 87  9.  Yu, J., N. Mookherjee, K. Wee, D. M. Bowdish, J. Pistolic, Y. Li, L. Rehaume, and R. E. W. Hancock. 2007. Host defense peptide LL-37, in synergy with inflammatory mediator IL-1beta, augments immune responses by multiple pathways. J Immunol 179:7684-7691.  10.  Niyonsaba, F., H. Ushio, N. Nakano, W. Ng, K. Sayama, K. Hashimoto, I. Nagaoka, K. Okumura, and H. Ogawa. 2007. Antimicrobial peptides human betadefensins stimulate epidermal keratinocyte migration, proliferation and production of proinflammatory cytokines and chemokines. J Invest Dermatol 127:594-604.  11.  Niyonsaba, F., H. Ushio, I. Nagaoka, K. Okumura, and H. Ogawa. 2005. The human beta-defensins (-1, -2, -3, -4) and cathelicidin LL-37 induce IL-18 secretion through p38 and ERK MAPK activation in primary human keratinocytes. J Immunol 175:1776-1784.  12.  Tjabringa, G. S., J. Aarbiou, D. K. Ninaber, J. W. Drijfhout, O. E. Sorensen, N. Borregaard, K. F. Rabe, and P. S. Hiemstra. 2003. The antimicrobial peptide LL37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. J Immunol 171:6690-6696.  13.  Schaller-Bals, S., A. Schulze, and R. Bals. 2002. Increased Levels of Antimicrobial Peptides in Tracheal Aspirates of Newborn Infants during Infection. Am. J. Respir. Crit. Care Med. 165:992-995.  14.  Ong, P. Y., T. Ohtake, C. Brandt, I. Strickland, M. Boguniewicz, T. Ganz, R. L. Gallo, and D. Y. Leung. 2002. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. The New England journal of medicine 347:11511160.  15.  Murakami, M., T. Ohtake, R. A. Dorschner, B. Schittek, C. Garbe, and R. L. Gallo. 2002. Cathelicidin Anti-Microbial Peptide Expression in Sweat, an Innate Defense System for the Skin. 119:1090-1095.  16.  Menendez, A., and B. B. Finlay. 2007. Defensins in the immunology of bacterial infections. Curr Opin Immunol 19:385-391.  17.  Nizet, V., T. Ohtake, X. Lauth, J. Trowbridge, J. Rudisill, R. A. Dorschner, V. Pestonjamasp, J. Piraino, K. Huttner, and R. L. Gallo. 2001. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414:454-457.  88  18.  Bowdish, D. M., D. J. Davidson, Y. E. Lau, K. Lee, M. G. Scott, and R. E. W. Hancock. 2005. Impact of LL-37 on anti-infective immunity. J Leukoc Biol 77:451-459.  19.  Lau, Y. E., A. Rozek, M. G. Scott, D. L. Goosney, D. J. Davidson, and R. E. W. Hancock. 2005. Interaction and Cellular Localization of the Human Host Defense Peptide LL-37 with Lung Epithelial Cells. Infect. Immun. 73:583-591.  20.  Braff, M. H., M. i. A. Hawkins, A. D. Nardo, B. Lopez-Garcia, M. D. Howell, C. Wong, K. Lin, J. E. Streib, R. Dorschner, D. Y. M. Leung, and R. L. Gallo. 2005. Structure-Function  Relationships  among  Human  Cathelicidin  Peptides:  Dissociation of Antimicrobial Properties from Host Immunostimulatory Activities. J Immunol 174:4271-4278. 21.  Vercammen, E., J. Staal, and R. Beyaert. 2008. Sensing of Viral Infection and Activation of Innate Immunity by Toll-Like Receptor 3. Clin. Microbiol. Rev. 21:13-25.  22.  Lande, R., J. Gregorio, V. Facchinetti, B. Chatterjee, Y.-H. Wang, B. Homey, W. Cao, Y.-H. Wang, B. Su, F. O. Nestle, T. Zal, I. Mellman, J.-M. Schroder, Y.-J. Liu, and M. Gilliet. 2007. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449:564-569.  23.  Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, and R. Medzhitov. 2004. Recognition of Commensal Microflora by Toll-Like Receptors Is Required for Intestinal Homeostasis. Cell 118:229-241.  24.  Shaykhiev, R., C. Beisswenger, K. Kandler, J. Senske, A. Puchner, T. Damm, J. Behr, and R. Bals. 2005. Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure. American journal of physiology 289:L842-848.  25.  Carretero, M., M. J. Escamez, M. Garcia, B. Duarte, A. Holguin, L. Retamosa, J. L. Jorcano, M. d. Rio, and F. Larcher. 2007. In vitro and In vivo Wound HealingPromoting Activities of Human Cathelicidin LL-37. J Invest Dermatol 128:223236.  26.  Vonk, M. J., P. S. Hiemstra, and J. J. Grote. 2008. An Antimicrobial Peptide Modulates Epithelial Responses to Bacterial Products. The Laryngoscope.  89  27.  Schauber, J., R. A. Dorschner, K. Yamasaki, B. Brouha, and R. L. Gallo. 2006. Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli. Immunology 118:509-519.  28.  Baumgarth, N., and C. L. Bevins. 2007. Autoimmune disease: Skin deep but complex. Nature 449:551-553.  90  APPENDIX SUPPLEMENTARY TABLES: Supplementary
Table
7:
Differentially
expressed
genes
1
hour
post­stimulation
with
3
µg/ml
 LL­37.

 Gene Name SEM4A E2F2  KCE1L NP_073590.2 CAD19  NP_659002.1 ITA4  SCUB2 AKT2 ZN566  Fold Change 4.70  p Value 0.0023  4.33  0.0029  3.94  0.0037  3.91  0.0038  3.55  0.005  Antifreeze protein, type I; Pollen allergen Poa pIX/Phl pVI, C-terminal; Zinc finger, CCCH-type Integrins alpha-4/beta-1 (VLA-4) and alpha-4/beta-7 are receptors for fibronectin. They recognize one or more domains within the alternatively spliced CS-1 and CS-5 regions of fibronectin. They are also receptors for VCAM1. Integrin alpha-4/beta-1 recognizes the sequence Q-I-D-S in VCAM1. Integrin alpha-4/beta-7 is also a receptor for MADCAM1. It recognizes the sequence L-D-T in MADCAM1. On activated endothelial cells integrin VLA-4 triggers homotypic aggregation for most VLA-4-positive leukocyte cell lines. It may also participate in cytolytic T-cell interactions with target cells  3.48  0.0054  3.44  0.0056  Signal peptide, CUB and EGF-like domain-containing protein 2 precursor; Protein CEGP1 General protein kinase capable of phosphorylating several known proteins May be involved in transcriptional regulation  3.36  0.006  3.35  0.006  3.03  0.0203  Gene Description Inhibits axonal extension by providing local signals to specify territories inaccessible for growing axons Transcription activator that binds DNA cooperatively with DP proteins through the E2 recognition site, 5'TTTC[CG]CGC-3' found in the promoter region of a number of genes whose products are involved in cell cycle regulation or in DNA replication. The DRTF1/E2F complex functions in the control of cellcycle progression from g1 to s phase. E2F-2 binds specifically to RB1 protein, in a cell-cycle dependent manner Potassium voltage-gated channel subfamily E member 1-like protein; AMME syndrome candidate gene 2 protein; AMMECR2 protein Acc:NP_073590]; S100P binding protein isoform a [Source:RefSeq_peptide Cadherins are calcium dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types  91  Gene Name KCMB1  RNF40  Q8NHH4 IRK15  NP_000924.2  CASP ZN214 TTBK1 ONEC2 FGD1  K1199  Gene Description Regulatory subunit of the calcium activated potassium KCNMA1 (maxiK) channel. Modulates the calcium sensitivity and gating kinetics of KCNMA1, thereby contributing to KCNMA1 channel diversity. Increases the apparent Ca(2+)/voltage sensitivity of the KCNMA1 channel. It also modifies KCNMA1 channel kinetics and alters its pharmacological properties. It slows down the activation and the deactivation kinetics of the channel. Acts as a negative regulator of smooth muscle contraction by enhancing the calcium sensitivity to KCNMA1. Its presence is also a requirement for internal binding of the KCNMA1 channel opener dehydrosoyasaponin I (DHS-1) triterpene glycoside and for external binding of the agonist hormone 17-beta-estradiol (E2). Increases the binding activity of charybdotoxin (CTX) toxin to KCNMA1 peptide blocker by increasing the CTX association rate and decreasing the dissociation rate E3 ubiquitin ligase protein that mediates monoubiquitination of 'Lys-120' of histone H2B. H2B 'Lys-120' ubiquitination gives a specific tag for epigenetic transcriptional activation and is also prerequisite for histone H3 'Lys-4' and 'Lys-79' methylation. Forms a ubiquitin ligase complex in cooperation with the E2 enzyme UBE2E1/UBCH6. It thereby plays a central role in histone code and gene regulation. Required for transcriptional activation of Hox genes Pistil-specific extensin-like protein; Proline-rich region Inward rectifier potassium channels are characterized by a greater tendency to allow potassium to flow into the cell rather than out of it. Their voltage dependence is regulated by the concentration of extracellular potassium; as external potassium is raised, the voltage range of the channel opening shifts to more positive voltages. The inward rectification is mainly due to the blockage of outward current by internal magnesium The production of the second messenger molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) is mediated by activated phosphatidylinositolspecific phospholipase C enzymes. This form has a role in retina signal transduction May be involved in intra-Golgi retrograde transport May be involved in transcriptional regulation Serine/threonine kinase which is able to phosphorylate TAU on serine, threonine and tyrosine residues. Induces aggregation of TAU Transcriptional activator. Activates the transcription of a number of liver genes such as HNF3B Activates CDC42, a member of the Ras-like family of Rho-and Rac proteins, by exchanging bound GDP for free GTP. Plays a role in regulating the actin cytoskeleton and cell shape May be involved in hearing  Fold Change 3.00  p Value 0.0087  2.95  0.0091  2.95 2.86  0.0092 0.0102  2.83  0.0106  2.78 2.78 2.77  0.0064 0.0112 0.0113  2.77  0.0114  2.76  0.0115  2.75  0.0024  92  Gene Name TDGF1 PARC BSN  EST1A  NP_064614.2 SRR O15420 B3GN2 ATG4D  TMPS5 NP_060105.3 S10A6 RASN MAST1  Gene Description Could play a role in the determination of the epiblastic cells that subsequently give rise to the mesoderm Cytoplasmic anchor protein in p53-associated protein complex. Regulates the subcellular localization of p53 and subsequent function Is thought to be involved in the organization of the cytomatrix at the nerve terminals active zone (CAZ) which regulates neurotransmitter release. Seems to act through binding to ERC2/CAST1. Essential in regulated neurotransmitter release from a subset of brain glutamatergic synapses. Involved in the formation of the retinal photoreceptor ribbon synapses Component of the telomerase ribonucleoprotein (RNP) complex that is essential for the replication of chromosome termini. May have a general role in telomere regulation. Promotes in vitro the ability of TERT to elongate telomeres. Overexpression induces telomere uncapping, chromosomal end-to-end fusions (telomeric DNA persists at the fusion points) and did not perturb TRF2 telomeric localization. Dephosphorylates RENT1. Plays a role in nonsensemediated mRNA decay. May function as endonuclease. Degrades single-stranded RNA (ssRNA), but not ssDNA or dsRNA PR domain-containing protein 11 Catalyzes the synthesis of D-serine from L-serine Acc:O15420]; CAGH1 alternate open reading frame. [Source:Uniprot/SPTREMBL Catalyzes the initiation and elongation of poly-Nacetyllactosamine chains Cysteine protease required for autophagy, which cleaves the C-terminal part of either MAP1LC3, GABARAPL2 or GABARAP, allowing the liberation of form I. A subpopulation of form I is subsequently converted to a smaller form (form II). Form II, with a revealed C-terminal glycine, is considered to be the phosphatidylethanolamine (PE)-conjugated form, and has the capacity for the binding to autophagosomes Transmembrane protease, serine 5; Spinesin SET Protein S100-A6; S100 calcium-binding protein A6; Calcyclin; Prolactin receptor-associated protein; PRA; Growth factor-inducible protein 2A9; MLN 4 Ras proteins bind GDP/GTP and possess intrinsic GTPase activity Appears to link the dystrophin/utrophin network with microtubule filaments via the syntrophins. Phosphorylation of DMD or UTRN may modulate their affinities for associated proteins  Fold Change 2.71  p Value 0.0123  2.67  0.013  2.66  0.013  2.65  0.0132  2.61 2.60 2.57  0.0141 0.0142 0.0149  2.54  0.0155  2.53  0.0157  2.52 2.45 2.45  0.016 0.0179 0.0237  2.45  0.0183  2.43  0.0183  93  Gene Name HEY1  NPBW2  NP_001018068.1 SEL1L  TCRG1  CCG4 ZN598 ELN  NEO1  ADDB  ACE2  NM_014069.1 ABHD9 ZN225 NP_056368.1  Gene Description Downstream effector of Notch signaling which may be required for cardiovascular development. Transcriptional repressor which binds preferentially to the canonical E box sequence 5'-CACGTG-3'. Represses transcription by the cardiac transcriptional activators GATA4 and GATA6 Interacts specifically with a number of opioid ligands. Receptor for neuropeptides B and W, which may be involved in neuroendocrine system regulation, food intake and the organization of other signals Basic helix-loop-helix dimerisation region bHLH May play a role in Notch signaling (By similarity). May be involved in the endoplasmic reticulum quality control (ERQC) system also called ER-associated degradation (ERAD) involved in ubiquitin-dependent degradation of misfolded endoplasmic reticulum proteins Transcription factor that binds RNA polymerase II and inhibits the elongation of transcripts from target promoters. Regulates transcription elongation in a TATA box-dependent manner. Necessary for TATdependent activation of the human immunodeficiency virus type 1 (HIV-1) promoter Thought to stabilize the calcium channel in an inactivated (closed) state Zinc finger protein 598 Major structural protein of tissues such as aorta and nuchal ligament, which must expand rapidly and recover completely. Molecular determinant of the late arterial morphogenesis, stabilizing arterial structure by regulating proliferation and organization of vascular smooth muscle May be involved as a regulatory protein in the transition of undifferentiated proliferating cells to their differentiated state. May also function as a cell adhesion molecule in a broad spectrum of embryonic and adult tissues Membrane-cytoskeleton-associated protein that promotes the assembly of the spectrin-actin network. Binds to calmodulin. Calmodulin binds preferentially to the beta subunit Carboxypeptidase which converts angiotensin I to angiotensin 1-9, a peptide of unknown function, and angiotensin II to angiotensin 1-7, a vasodilator. Also able to hydrolyze apelin-13 and dynorphin-13 with high efficiency. May be an important regulator of heart function. In case of human coronaviruses SARS and HCoV-NL63 infections, serve as functional receptor for the spike glycoprotein of both coronaviruses Proline-rich region Abhydrolase domain-containing protein 9 precursor May be involved in transcriptional regulation Pleckstrin-like  Fold Change 2.43  p Value 0.0185  2.42  0.0187  2.42 2.41  0.0187 0.019  2.40  0.0192  2.39  0.0196  2.39 2.37  0.0097 0.0201  2.36  0.0207  2.35  0.0208  2.35  0.0211  2.34 2.33 2.33 2.33  0.0214 0.0217 0.0217 0.0218  94  Gene Name AICDA  NP_079093.2 DPOLQ TLX3 JAD1C  MKL2 NP_060478.2 CRSP3 Q96MH6-2 ASB3 ARNT  TMG2 Q8IYI7  GSTT1  ENSG00000211744  Gene Description RNA-editing deaminase involved in somatic hypermutation, gene conversion, and class-switch recombination. Required for several crucial steps of B-cell terminal differentiation necessary for efficient antibody responses ADAM-TS Spacer 1 Could be involved in the repair of interstrand crosslinks T-cell leukemia homeobox protein 3; Homeobox protein Hox-11L2 Histone demethylase that specifically demethylates 'Lys-4' of histone H3, thereby playing a central role in histone code. Does not demethylate histone H3 'Lys9', H3 'Lys-27', H3 'Lys-36', H3 'Lys-79' or H4 'Lys20'. Demethylates trimethylated and dimethylated but not monomethylated H3 'Lys-4'. Participates in transcriptional repression of neuronal genes by recruiting histone deacetylases and REST at neuronrestrictive silencer elements Acts as a transcriptional coactivator of serum response factor (SRF). Required for skeletal myogenic differentiation Zinc finger, C2H2-subtype; Zinc finger, C2H2-type Plays a role in transcriptional coactivation Acc:NP_689630]; transmembrane protein 68 [Source:RefSeq_peptide Ankyrin repeat and SOCS box protein 3; ASB-3 Required for activity of the Ah (dioxin) receptor. This protein is required for the ligand-binding subunit to translocate from the cytosol to the nucleus after ligand binding. The complex then initiates transcription of genes involved in the activation of PAH procarcinogens. The heterodimer with HIF1A or EPAS1/HIF2A functions as a transcriptional regulator of the adaptive response to hypoxia Transmembrane gamma-carboxyglutamic acid protein 2 precursor; Proline-rich Gla protein 2; Proline-rich gamma-carboxyglutamic acid protein 2 Component of the COPII coat, that covers ER-derived vesicles involved in transport from the endoplasmic reticulum to the Golgi apparatus. COPII acts in the cytoplasm to promote the transport of secretory, plasma membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles. Acts on 1,2-epoxy-3-(4nitrophenoxy)propane, phenethylisothiocyanate 4nitrobenzyl chloride and 4-nitrophenethyl bromide. Displays glutathione peroxidase activity with cumene hydroperoxide Immunoglobulin V-set; Immunoglobulin-like  Fold Change 2.31  p Value 0.0223  2.31 2.31  0.0224 0.0226  2.30  0.0228  2.29  0.0232  2.29  0.0233  2.29 2.27 2.26  0.0233 0.0149 0.032  2.26 2.26  0.0245 0.0033  2.25  0.0248  2.25  0.0251  2.23  0.0258  2.23  0.0259  95  Gene Name NDN  SLC14A1 SGCD  NP_940905.2 NP_001546.2  Q6NUT1 YTDC2 LIPG KCNB1  Q5VYN8 GLCI1 FGD2  ST5  GLI4 PHF20 HNRPM  Gene Description Growth suppressor that facilitates the entry of the cell into cell cycle arrest. Functionally similar to the retinoblastoma protein it binds to and represses the activity of cell-cycle-promoting proteins such as SV40 large T antigen, adenovirus E1A, and the transcription factor E2F. Necdin also interacts with p53 and works in an additive manner to inhibit cell growth. Functions also as transcription factor and binds directly to specific guanosine-rich DNA sequences Specialized low-affinity urea transporter. Mediates urea transport in erythrocytes Component of the sarcoglycan complex, a subcomplex of the dystrophin-glycoprotein complex which forms a link between the F-actin cytoskeleton and the extracellular matrix Blood group Rhesus C/E and D polypeptide; Calciumactivated BK potassium channel, alpha subunit; EAG/ELK/ERG potassium channel; Ion transport 2 Immunoglobulin; Immunoglobulin V-set; Immunoglobulin-like; Interleukin-1 receptor, type I and type II; Vascular endothelial growth factor receptor, VEGFR, N-terminal Eukaryotic translation initiation factor 4E (eIF-4E) YTH domain-containing protein 2 Gastric triacylglycerol lipase precursor; Gastric lipase; GL Mediates the voltage-dependent potassium ion permeability of excitable membranes. Channels open or close in response to the voltage difference across the membrane, letting potassium ions pass in accordance with their electrochemical gradient Regulator of G protein signalling Glucocorticoid-induced transcript 1 protein May activate CDC42, a member of the Ras-like family of Rho- and Rac proteins, by exchanging bound GDP for free GTP. May play a role in regulating the actin cytoskeleton and cell shape May be involved in cytoskeletal organization and tumorgenicity. Isoform 1 seems to be involved in a signaling transduction pathway leading to activation of MAPK1/ERK2. Isoform 3 may block ERK2 activation stimulated by ABL1. Isoform 3 may alter cell morphology and cell growth Zinc finger protein GLI4; Krueppel-related zinc finger protein 4; Protein HKR4 Possible transcription factor Pre-mRNA binding protein in vivo, binds avidly to poly(G) and poly(U) RNA homopolymers in vitro. Involved in splicing. Acts as a receptor for carcinoembryonic antigen in Kupffer cells, may initiate a series of signaling events leading to tyrosine phosphorylation of proteins and induction of IL-1 alpha, IL-6, IL-10 and tumor necrosis factor alpha cytokines  Fold Change 2.23  p Value 0.003  2.22  0.0265  2.20  0.0273  2.20  0.0275  2.19  0.028  2.18 2.18 2.18  0.0282 0.0285 0.0285  2.17  0.0083  2.17 2.17 2.16  0.0289 0.029 0.0297  2.15  0.03  2.15  0.0302  2.15 2.14  0.0303 0.0309  96  Gene Name Q6ZS65 Q96MM7-2 VPP4 CCNF ZP3  KREM1 EXOC5 Q5VZE3 SMAD6  RAD50  TAAR8  Gene Description Acc:NP_001009909]; leucine zipper protein 2 [Source:RefSeq_peptide Heparan sulphate 6-sulfotransferase Part of the proton channel of the V-ATPase that is involved in normal vectorial acid transport into the urine by the kidney Likely to be involved in the control of the cell cycle during S phase and G2 The mammalian zona pellucida, which mediates species-specific sperm binding, induction of the acrosome reaction and prevents post-fertilization polyspermy, is composed of three to four glycoproteins, ZP1, ZP2, ZP3, and ZP4. ZP3 is essential for sperm binding and zona matrix formation Receptor for Dickkopf protein. Cooperates with Dickkopf to block Wnt/beta-catenin signaling Component of the exocyst complex involved in the docking of exocystic vesicles with fusion sites on the plasma membrane Acc:Q8NBJ4]; Golgi phosphoprotein 2 (Golgi membrane protein GP73). [Source:Uniprot/SWISSPROT Antagonist of signaling by TGF-beta (transforming growth factor) type 1 receptor superfamily members; has been shown to inhibit selectively BMP (bone morphogenetic proteins) signaling by competing with the co-SMAD SMAD4 for receptor-activated SMAD1. SMAD6 is an inhibitory SMAD (I-SMAD) or antagonistic SMAD. Binds to regulatory elements in target promoter regions Component of the MRN complex, which plays a central role in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity and meiosis. The complex possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity, which are provided by MRE11A. RAD50 may be required to bind DNA ends and hold them in close proximity. This could facilitate searches for short or long regions of sequence homology in the recombining DNA templates, and may also stimulate the activity of DNA ligases and/or restrict the nuclease activity of MRE11A to prevent nucleolytic degradation past a given point. The complex may also be required for DNA damage signaling via activation of the ATM kinase. In telomeres the MRN complex may modulate t-loop formation Orphan receptor. Could be a receptor for trace amines. Trace amines are biogenic amines present in very low levels in mammalian tissues. Although some trace amines have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Trace amines are likely to be involved in a variety of physiological functions that have yet to be fully understood  Fold Change 2.14  p Value 0.0309  2.13 2.11  0.031 0.0129  2.11  0.0325  2.11  0.0328  2.09  0.0342  2.08  0.0101  2.07  0.0351  2.07  0.0355  2.06  0.0112  2.06  0.0349  97  Gene Name FKB1B  SMC2  CTBP2 RAP2B NP_061889.1 F104B Q9Y2I9 PI2R Q9BTA9-2 PSYR  ASPX VMAT1 NP_001010984.1 EPDR1 IFT74 RAE1L CCDC13 GRP75 ZN434 FRGL  Gene Description Associates with the ryanodine receptor (RYR-2) in cardiac muscle sarcoplasmic reticulum and may play a unique physiological role in excitation-contraction coupling in cardiac muscle. There are four molecules of FKBP12.6 per heart muscle RYR. Has the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin. PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides Central component of the condensin complex, a complex required for conversion of interphase chromatin into mitotic-like condense chromosomes. The condensin complex probably introduces positive supercoils into relaxed DNA in the presence of type I topoisomerases and converts nicked DNA into positive knotted forms in the presence of type II topoisomerases Corepressor targeting diverse transcription regulators Ras-related protein Rap-2b precursor Eggshell protein; Intermediate filament protein; Keratin, type I Protein FAM104B Peptidase M14, carboxypeptidase A; RabGAP/TBC Receptor for prostacyclin (prostaglandin I2 or PGI2). The activity of this receptor is mediated by G(s) proteins which activate adenylate cyclase WW domain-containing adapter protein with coiledcoil Receptor for the glycosphingolipid psychosine (PSY) and several related glycosphingolipids. May have a role in activation-induced cell death or differentiation of T-cells Acrosomal protein SP-10 precursor; Acrosomal vesicle protein 1 Involved in the vesicular transport of biogenic amines Peptidase M, neutral zinc metallopeptidases, zincbinding site Mammalian ependymin-related protein 1 precursor; MERP-1; UCC1 protein Intraflagellar transport 74 homolog; Coiled-coil domain-containing protein 2; Capillary morphogenesis protein 1; CMG-1 Binds mRNA. May function in nucleocytoplasmic transport and in directly or indirectly attaching cytoplasmic mRNPs to the cytoskeleton Coiled-coil domain-containing protein 13 Implicated in the control of cell proliferation and cellular aging. May also act as a chaperone May be involved in transcriptional regulation Putative FRG1-like protein C20orf80  Fold Change 2.06  p Value 0.0364  2.06  0.0364  2.05 2.05 2.04  0.037 0.0371 0.0375  2.04 2.03 2.03  0.0377 0.0384 0.0387  2.00  0.0055  1.99  0.0416  1.99  0.0417  1.99 1.98  0.0423 0.0425  1.98  0.0483  1.98  0.0098  1.97  0.0033  1.96 1.95  0.045 0.0454  1.95 1.95  0.0454 0.0455  98  Gene Name ENP1  TRI65 EST1A  PELI1  PSN2  PIGZ  NP_653217.1 USP6  SYNP2 PP1RA  NP_002411.3 NP_056993.2 DNPEP  Gene Description In the nervous system, could hydrolyze ATP and other nucleotides to regulate purinergic neurotransmission. Could also be implicated in the prevention of platelet aggregation. Hydrolyzes ATP and ADP equally well Tripartite motif-containing protein 65 Component of the telomerase ribonucleoprotein (RNP) complex that is essential for the replication of chromosome termini. May have a general role in telomere regulation. Promotes in vitro the ability of TERT to elongate telomeres. Overexpression induces telomere uncapping, chromosomal end-to-end fusions (telomeric DNA persists at the fusion points) and did not perturb TRF2 telomeric localization. Dephosphorylates RENT1. Plays a role in nonsensemediated mRNA decay. May function as endonuclease. Degrades single-stranded RNA (ssRNA), but not ssDNA or dsRNA Scaffold protein involved in the IL-1 signaling pathway via its interaction with the complex containing IRAK kinases and TRAF6. Required for NF-kappa-B activation and IL-8 gene expression in response to IL1 Probable catalytic subunit of the gamma-secretase complex, an endoprotease complex that catalyzes the intramembrane cleavage of integral membrane proteins such as Notch receptors and APP (betaamyloid precursor protein). Requires the other members of the gamma-secretase complex to have a protease activity. May play a role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane. May function in the cytoplasmic partitioning of proteins Mannosyltransferase involved in glycosylphosphatidylinositol-anchor biosynthesis. Transfers a fourth mannose to some trimannosylGPIs during GPI precursor assembly. The presence of a fourth mannose in GPI is facultative and only scarcely detected, suggesting that it only exists in some tissues Proline-rich region Has an ATP-independent isopeptidase activity, cleaving at the C-terminus of the ubiquitin moiety. In vitro, isoform 2, but not isoform 3, shows deubiquitinating activity Has an actin-binding and actin-bundling activity Inhibitor of PPP1CA and PPP1CC phosphatase activities. Has inhibitory activity on PPP1CA only when phosphorylated. Binds to mRNA, singlestranded DNA (ssDNA), poly(A) and poly(G) homopolymers Ion transport Ribosomal protein S14 Likely to play an important role in intracellular protein and peptide metabolism  Fold Change 1.95  p Value 0.0456  1.95 1.94  0.0462 0.0465  1.94  0.0469  1.93  0.0479  1.93  0.0217  1.93 1.92  0.0274 0.0487  1.92 1.92  0.0489 0.049  1.91 1.90 1.90  0.0191 0.0087 0.0086  99  Gene Name BCAT2  H2B2E  TBCA Q8NBT7 S12A5  ITBA1 NP_060170.1 CA021 PTN18 CG034 MKRN3 OCRL  IBP5  GATA1  DUSP4  Gene Description Catalyzes the first reaction in the catabolism of the essential branched chain amino acids leucine, isoleucine, and valine. May also function as a transporter of branched chain alpha-keto acids Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of posttranslational modifications of histones, also called histone code, and nucleosome remodeling Tubulin-folding protein; involved in the early step of the tubulin folding pathway Acc:Q8NBT7]; CDNA FLJ90757 fis, clone SKNMC1000014 (FLJ90757 protein). [Source:Uniprot/SPTREMBL Mediates electroneutral potassium-chloride cotransport in mature neurons. Transport occurs under isotonic conditions, but is activated 20-fold by cell swelling. Important for Cl(-) homeostasis in neurons Transmembrane protein 187; Protein ITBA1 DH; cAMP/cGMP-dependent protein kinase Uncharacterized protein C1orf21; Cell proliferationinducing gene 13 protein Differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues Uncharacterized protein C7orf34 precursor; MSSPbinding protein CTM-1 Makorin-3; Zinc finger protein 127; RING finger protein 63 Converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. Also converts inositol 1,4,5-trisphosphate to inositol 1,4bisphosphate and inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. May function in lysosomal membrane trafficking by regulating the specific pool of phosphatidylinositol 4,5-bisphosphate that is associated with lysosomes IGF-binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth promoting effects of the IGFs on cell culture. They alter the interaction of IGFs with their cell surface receptors Transcriptional activator which probably serves as a general switch factor for erythroid development. It binds to DNA sites with the consensus sequence [AT]GATA[AG] within regulatory regions of globin genes and of other genes expressed in erythroid cells Regulates mitogenic signal transduction by dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2  Fold Change 1.90  p Value 0.0316  1.90  0.0215  1.89  0.0084  -1.92  0.0099  -1.93  0.0486  -1.93 -1.94 -1.94  0.0228 0.0471 0.0471  -1.94  0.0469  -1.94  0.0203  -1.94  0.0464  -1.94  0.0414  -1.95  0.046  -1.95  0.043  -1.95  0.0392  100  Gene Name GNS GRIK2  ENSG00000171914 M3K8  NP_006537.3 ALKB1 Q86V25-2 ENSG00000132463 O75800-2 NP_659453.2 GPSM3  Q6P578 NP_872354.1  SCF  Gene Description N-acetylglucosamine-6-sulfatase precursor; G6S; Glucosamine-6-sulfatase Receptor for glutamate. L-glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system. The postsynaptic actions of Glu are mediated by a variety of receptors that are named according to their selective agonists. May be involved in the transmission of light information from the retina to the hypothalamus. This receptor binds domoate &gt; kainate &gt; quisqualate &gt; 6-cyano7-nitroquinoxaline-2,3-dione &gt; L-glutamate = 6,7dinitroquinoxaline-2,3-dione &gt; dihydrokainate Adenosine/AMP deaminase active site; Band 4.1; I/LWEQ Able to activate NF-kappa-B 1 by stimulating proteasome-mediated proteolysis of NF-kappa-B 1/p105. Plays a role in the cell cycle. The longer form of cot has some transforming activity, although it is much weaker than the activated cot oncoprotein KH, type 1; RNA-binding region RNP-1 (RNA recognition motif) Alkylated DNA repair protein alkB homolog 1 Angiogenesis inhibitor. Inhibits network formation by endothelial cells RNA-binding region RNP-1 (RNA recognition motif) Zinc finger MYND domain-containing protein 10; BLu protein Aralkyl acyl-CoA:amino acid N-acyltransferase; Aralkyl acyl-CoA:amino acid N-acyltransferase, Cterminal Functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 to regulate cell-fate determination. Upon ligand activation through the released notch intracellular domain (NICD) it forms a transcriptional activator complex with RBP-J kappa and activates genes of the enhancer of split locus. Affects the implementation of differentiation, proliferation and apoptotic programs. May regulate branching morphogenesis in the developing vascular system Tetratricopeptide repeat protein 3; TPR repeat protein 3; TPR repeat protein D; RING finger protein 105 Plays a role in mediating Ca(2+) influx following depletion of intracellular Ca(2+) stores. Acts as Ca(2+) sensor in the endoplasmic reticulum via its EF-hand domain. Upon Ca(2+) depletion, translocates from the endoplasmic reticulum to the plasma membrane where it activates the Ca(2+) release-activated Ca(2+) (CRAC) channel subunit, TMEM142A/ORAI1 Stimulates the proliferation of mast cells. Able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow culture. Mediates also cell-cell adhesion. Acts synergistically with other cytokines, probably interleukins  Fold Change -1.95  p Value 0.0457  -1.96  0.0452  -1.96  0.0236  -1.97  0.0434  -1.98  0.043  -1.98 -1.99  0.0377 0.0422  -1.99 -1.99  0.013 0.0417  -2.00  0.0413  -2.00  0.0412  -2.00  0.0019  -2.01  0.0276  -2.01  0.0045  101  Gene Name NP_056477.1 Q8TAC0 DNAS1 NP_064618.3 SERC3 TF2H4  PDE4C G3P RBL2  NP_872339.2 NP_001005472.1 NP_078886.2 CACB3  ECHD1 MPIP2  SYFB CU045 K1279 TMM58  Gene Description WD-40 repeat Acc:Q8TAC0]; MGC27345 protein. [Source:Uniprot/SPTREMBL Among other functions, seems to be involved in cell death by apoptosis. Binds specifically to G-actin and blocks actin polymerization Metallophosphoesterase May be involved in cellular transformation Component of the core-TFIIH basal transcription factor involved in nucleotide excision repair (NER) of DNA and, when complexed to CAK, in RNA transcription by RNA polymerase II cAMP-specific 3',5'-cyclic phosphodiesterase 4C; DPDE1; PDE21 Glyceraldehyde-3-phosphate dehydrogenase; GAPDH Key regulator of entry into cell division. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV420H1 and SUV420H2, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Probably acts as a transcription repressor by recruiting chromatin-modifying enzymes to promoters. Potent inhibitor of E2F-mediated trans-activation, associates preferentially with E2F5. Binds to cyclins A and E. Binds to and may be involved in the transforming capacity of the adenovirus E1A protein. May act as a tumor suppressor Acc:Q6ZRT9]; CDNA FLJ46112 fis, clone TESTI2035962 (Novel protein). [Source:Uniprot/SPTREMBL Ribosomal protein S2 Transcription factor jumonji The beta subunit of voltage-dependent calcium channels contributes to the function of the calcium channel by increasing peak calcium current, shifting the voltage dependencies of activation and inactivation, modulating G protein inhibition and controlling the alpha-1 subunit membrane targeting Enoyl-CoA hydratase domain-containing protein 1 Tyrosine protein phosphatase which functions as a dosage-dependent inducer of mitotic progression. Directly dephosphorylates CDC2 and stimulates its kinase activity. The three isoforms seem to have a different level of activity Phenylalanyl-tRNA synthetase beta chain; Phenylalanine--tRNA ligase beta chain; PheRS Uncharacterized protein C21orf45; FAPP1-associated protein 1 May play a role in both peripheral and central nervous system Transmembrane protein 58 precursor  Fold Change -2.02 -2.02  p Value 0.0392 0.0057  -2.03  0.0339  -2.03 -2.03 -2.03  0.0387 0.0384 0.0382  -2.04  0.0326  -2.04  0.0123  -2.05  0.0112  -2.06  0.0361  -2.06 -2.07 -2.08  0.0358 0.0356 0.0347  -2.09 -2.10  0.0339 0.0333  -2.10  0.0333  -2.10  0.0488  -2.10  0.0207  -2.11  0.0044  102  Gene Name PYGL  MDM4  LRP4 RIPK1 NP_073742.1 MA2C1 DSG2 ORAI2 SLIK1 G3P SOX30 LAT3  Q6UX34 PRP39 Q96NM2 Q9HAJ0 S38A3  Gene Description Phosphorylase is an important allosteric enzyme in carbohydrate metabolism. Enzymes from different sources differ in their regulatory mechanisms and in their natural substrates. However, all known phosphorylases share catalytic and structural properties Inhibits p53- and p73-mediated cell cycle arrest and apoptosis by binding its transcriptional activation domain. Inhibits degradation of MDM2. Can reverse MDM2-targeted degradation of p53 while maintaining suppression of p53 transactivation and apoptotic functions Potential cell surface endocytic receptor, which binds and internalizes extracellular ligands for degradation by lysosomes Promotes apoptosis and activation of NF-kappa-B. Required for TNFRSF1A mediated activation of NFkappa-B Acc:Q5VW35]; Novel protein (FLJ12806). [Source:Uniprot/SPTREMBL Alpha-mannosidase 2C1; Alpha-D-mannoside mannohydrolase; Mannosidase alpha class 2C member 1; Alpha mannosidase 6A8B Component of intercellular desmosome junctions. Involved in the interaction of plaque proteins and intermediate filaments mediating cell-cell adhesion Key regulator or component of store-operated Ca(2+) channel and transcription factor NFAT nuclear import Enhances neuronal dendrite outgrowth Glyceraldehyde-3-phosphate dehydrogenase; GAPDH Transcriptional activator. Binds to the DNA sequence 5'-ACAAT-3' and shows a preference for guanine residues surrounding this core motif Sodium-independent, high affinity transport of large neutral amino acids. Has narrower substrate selectivity compared to SLC7A5 and SLC7A8 and mainly transports branched-chain amino acids and phenylalanine. Plays a role in the development of human prostate cancer, from prostatic intraepithelial neoplasia to invasive prostate cancer ASCL830 (UNQ830), mRNA [Source:RefSeq_dna; Acc:NM_206895] Involved in pre-mRNA splicing Acc:NM_001004325]; keratin associated protein 5-2 (KRTAP5-2), mRNA [Source:RefSeq_dna Acc:Q9HAJ0]; CDNA FLJ11556 fis, clone HEMBA1003079. [Source:Uniprot/SPTREMBL Sodium-dependent amino acid/proton antiporter. Mediates electrogenic cotransport of glutamine and sodium ions in exchange for protons. Also recognizes histidine, asparagine and alanine. May mediate amino acid transport in either direction under physiological conditions. May play a role in nitrogen metabolism and synaptic transmission  Fold Change -2.12  p Value 0.0245  -2.13  0.031  -2.14  0.0304  -2.15  0.0302  -2.15  0.0062  -2.16  0.0297  -2.16  0.0294  -2.20  0.0276  -2.21 -2.22  0.0271 0.0012  -2.23  0.0134  -2.24  0.0254  -2.26  0.0247  -2.26 -2.28  0.0247 0.0238  -2.28  0.0237  -2.30  0.023  103  Gene Name SSXT GBRG2  NP_001073963.1 Q6ZNA8  Q9H4F8-2 OBSCN  ARSB NP_071425.2 MSRE  L2HDH DNM3B  Q9H9G5  SAM14 RSU1 NP_001007793.1  Gene Description SSXT protein; Synovial sarcoma, translocated to X chromosome; SYT protein GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel Tetratricopeptide TPR_2 May participate in a common DNA damage response pathway associated with the activation of homologous recombination and double-strand break repair. Binds to single and double stranded DNA and exhibits DNAdependent ATPase activity. Underwinds duplex DNA and forms helical nucleoprotein filaments Calcium-binding EF-hand; Protease inhibitor, Kazaltype; Proteinase inhibitor I1, Kazal; Thyroglobulin type-1 Immunoglobulin; Immunoglobulin C1-set; Immunoglobulin I-set; Immunoglobulin V-set; Immunoglobulin-like; Proline-rich region; Protein kinase; Tyrosine protein kinase, active site Arylsulfatase B precursor; ASB; Nacetylgalactosamine-4-sulfatase; G4S Type II fibronectin, collagen-binding Membrane glycoproteins implicated in the pathologic deposition of cholesterol in arterial walls during atherogenesis. Two types of receptor subunits exist. These receptors mediate the endocytosis of a diverse group of macromolecules, including modified low density lipoproteins (LDL) L-2-hydroxyglutarate dehydrogenase, mitochondrial precursor; Duranin Required for genome wide de novo methylation and is essential for development. DNA methylation is coordinated with methylation of histones. Isoforms 4 and 5 are probably not functional due to the deletion of two conserved methyltransferase motifs Shows growth cone collapsing activity on dorsal root ganglion (DRG) neurons in vitro. May be a stop signal for the DRG neurons in their target areas, and possibly also for other neurons. May also be involved in the maintenance and remodeling of neuronal connections Sterile alpha motif domain-containing protein 14 Potentially plays a role in the Ras signal transduction pathway. Capable of suppressing v-Ras transformation in vitro Required for high-affinity binding to nerve growth factor (NGF), neurotrophin-3 and neurotrophin-4/5 but not brain-derived neurotrophic factor (BDNF). Known substrates for the Trk receptors are SHC1, PI 3kinase, and PLC-gamma-1. Has a crucial role in the development and function of the nociceptive reception system as well as establishment of thermal regulation via sweating. Activates ERK1 by either SHC1- or PLC-gamma-1-dependent signaling pathway  Fold Change -2.31  p Value 0.0226  -2.31  0.0223  -2.32 -2.32  0.0329 0.0221  -2.33  0.0216  -2.37  0.0202  -2.38  0.0188  -2.39 -2.39  0.0197 0.0197  -2.41  0.019  -2.44  0.0181  -2.44  0.0181  -2.45 -2.47  0.0179 0.0173  -2.49  0.0104  104  Gene Name CLTR1  C43BP WDR55 BKRB2 NP_055654.2 ACSL5  LR37A Q9NSC5-3 CUL5  NP_060218.1 TRIPB  INHBC  METTL6 PO2F3  Gene Description Receptor for cysteinyl leukotrienes mediating bronchoconstriction of individuals with and without asthma. Stimulation by LTD4 results in the contraction and proliferation of smooth muscle, edema, eosinophil migration and damage to the mucus layer in the lung. This response is mediated via a G-protein that activates a phosphatidylinositolcalcium second messenger system. The rank order of affinities for the leukotrienes is LTD4 &gt;&gt; LTE4 = LTC4 &gt;&gt; LTB4 Phosphorylates on Ser and Thr residues the Goodpasture autoantigen (in vitro). Isoform 2 seems to be less active WD repeat protein 55 Receptor for bradykinin. It is associated with G proteins that activate a phosphatidylinositol-calcium second messenger system Phosphoesterase, PA-phosphatase related Activation of long-chain fatty acids for both synthesis of cellular lipids, and degradation via beta-oxidation. Utilizes a wide range of saturated fatty acids with a preference for C16-C18 unsaturated fatty acids Leucine-rich repeat-containing protein 37A; Leucinerich repeat-containing protein 37B precursor; C66 SLIT-like testicular protein EVH1 Component of E3 ubiquitin ligase complexes, which mediate the ubiquitination and subsequent proteasomal degradation of target proteins. Seems to be involved poteosomal degradation of p53/TP53 stimulated by adenovirus E1B-55 kDa protein. May form a cell surface vasopressin receptor Histone H5 Binds the ligand binding domain of the thyroid receptor (THRB) in the presence of triiodothyronine and enhances THRB-modulated transcription. Golgi auto-antigen; probably involved in maintaining cisGolgi structure Inhibins and activins inhibit and activate, respectively, the secretion of follitropin by the pituitary gland. Inhibins/activins are involved in regulating a number of diverse functions such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, nerve cell survival, embryonic axial development or bone growth, depending on their subunit composition. Inhibins appear to oppose the functions of activins Probable methyltransferase Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3'). Regulated the expression of a number of genes such as SPRR2A or placental lactogen  Fold Change -2.50  p Value 0.0166  -2.52  0.0161  -2.52 -2.53  0.016 0.0157  -2.53 -2.54  0.0157 0.0154  -2.59  0.0002  -2.61 -2.66  0.0003 0.0012  -2.67 -2.69  0.0129 0.0126  -2.71  0.0122  -2.75 -2.76  0.0116 0.0115  105  Gene Name GBF1 NFASC  CT121 DAAM1  DLX5 ZNF19 PIAS1  CASR  TLE4  Q8NBC0 T2R16  FBXL7 CATG ASAHL  SC6A3  Gene Description Promotes guanine-nucleotide exchange on ARF5. Promotes the activation of ARF5 through replacement of GDP with GTP Cell adhesion, ankyrin-binding protein which may be involved in neurite extension, axonal guidance, synaptogenesis, myelination and neuron-glial cell interactions May act as a protein that binds a hydrophobic ligand Binds to disheveled (Dvl) and Rho, and mediates Wnt-induced Dvl-Rho complex formation. May play a role as a scaffolding protein to recruit Rho-GDP and Rho-GEF, thereby enhancing Rho-GTP formation Homeobox protein DLX-5 May be involved in transcriptional regulation Functions as an E3-type small ubiquitin-like modifier (SUMO) ligase, stabilizing the interaction between UBE2I and the substrate, and as a SUMO-tethering factor. Plays a crucial role as a transcriptional coregulation in various cellular pathways, including the STAT pathway, the p53 pathway and the steroid hormone signaling pathway. The effects of this transcriptional coregulation, transactivation or silencing, may vary depending upon the biological context Senses changes in the extracellular concentration of calcium ions. The activity of this receptor is mediated by a G-protein that activates a phosphatidylinositolcalcium second messenger system Transcriptional corepressor that binds to a number of transcription factors. Inhibits the transcriptional activation mediated by PAX5, and by CTNNB1 and TCF family members in Wnt signaling. The effects of full-length TLE family members may be modulated by association with dominant-negative AES Orphan nuclear receptor, HMR type Gustducin-coupled receptor implicated in the perception of bitter compounds in the oral cavity and the gastrointestinal tract. Signals through PLCB2 and the calcium-regulated cation channel TRPM5 Probably recognizes and binds to some phosphorylated proteins and promotes their ubiquitination and degradation Cathepsin G precursor; CG Degrades bioactive fatty acid amides to their corresponding acids, with the following preference: Npalmitoylethanolamine &gt; N-myristoylethanolamine &gt; N-lauroylethanolamine = N-stearoylethanolamine &gt; N-arachidonoylethanolamine &gt; Noleoylethanolamine. Also exhibits weak hydrolytic activity against the ceramides N-lauroylsphingosine and N-palmitoylsphingosine Amine transporter. Terminates the action of dopamine by its high affinity sodium-dependent reuptake into presynaptic terminals  Fold Change -2.79  p Value 0.0111  -2.80  0.0109  -2.82 -2.86  0.0485 0.0343  -2.87 -2.89 -2.90  0.01 0.0039 0.0097  -2.93  0.0094  -2.95  0.0091  -3.00 -3.05  0.0442 0.0082  -3.05  0.0081  -3.15 -3.16  0.0073 0.0073  -3.20  0.007  106  Gene Name  Gene Description  MBOA5  Membrane-bound O-acyltransferase domaincontaining protein 5; O-acyltransferase domaincontaining protein 5  CELR1  Receptor that may have an important role in cell/cell signaling during nervous system formation May play a role in estrogen action Probable hydrolase. Involved in the thymus homing of bone marrow cells. May regulate beta-2 integrinmediated cell adhesion, migration and motility of neutrophil Chaperonin clpA/B; Disease resistance protein Acc:NP_116250]; serine active site containing 1 [Source:RefSeq_peptide EVI2B protein precursor; Ecotropic viral integration site 2B protein homolog; EVI-2B Converts lysophosphatidic acid (LPA) into phosphatidic acid by incorporating an acyl moiety at the sn-2 position of the glycerol backbone Forms hydroxylysine residues in -Xaa-Lys-Glysequences in collagens. These hydroxylysines serve as sites of attachment for carbohydrate units and are essential for the stability of the intermolecular collagen cross-links Involved in pH regulation to eliminate acids generated by active metabolism or to counter adverse environmental conditions. Major proton extruding system driven by the inward sodium ion chemical gradient. Plays an important role in signal transduction Catalyzes the conversion of 3'-phosphate to a 2',3'cyclic phosphodiester at the end of RNA. The mechanism of action of the enzyme occurs in 3 steps: (A) adenylation of the enzyme by ATP; (B) the enzyme acts on RNA-N3'P to produce RNAN3'PP5'A; (C) a non catalytic nucleophilic attack by the adjacent 2'hydroxyl on the phosphorus in the diester linkage to produce the cyclic end product. The biological role of this enzyme is unknown but it is likely to function in some aspects of cellular RNA processing Tigger transposable element-derived protein 6 Synthesis and degradation of fructose 2,6bisphosphate Isoform 4 may play a role in spermatogenesis  F102A VNN2  NP_659462.1 NP_116250.2 EVI2B AGPAT5 PLOD2  SL9A8  RTC1  TIGD6 F261 AAT1 ATP7B K22E NP_115510.1 DB118 BCL7A MAGBA  Involved in the export of copper out of the cells, such as the efflux of hepatic copper into the bile Probably contributes to terminal cornification. Associated with keratinocyte activation, proliferation and keratinization Acc:NP_115510]; glutamine rich 2 [Source:RefSeq_peptide Has antibacterial activity B-cell CLL/lymphoma 7 protein family member A Melanoma-associated antigen B10; MAGE-B10 antigen  Fold Change -3.24  p Value 0.0006  -3.25  0.0067  -3.30 -3.43  0.0064 0.0056  -3.56 -3.65  0.0003 0.0047  -3.70  0.0045  -3.72  0.0044  -3.73  0.0044  -3.75  0.0043  -3.81  0.0003  -3.89 -3.93  0.0039 0.0002  -4.18 -4.40  0.0032 0.0028  -4.52  0.0026  -4.53  0.0026  -4.56 -4.62 -4.66  0.0025 0.0024 0.0024  107  Gene Name Q7Z3S7 RNAS1 GABARAPL3 ENSG00000184956 ZN134 OR2S1 FGF2  ATRX  ZHANG  ENSG00000199437  Gene Description Cache; VWA N-terminal; von Willebrand factor, type A Endonuclease that catalyzes the cleavage of RNA on the 3' side of pyrimidine nucleotides. Acts on single stranded and double stranded RNA Gamma-aminobutyric acid receptor-associated protein-like 3; GABA(A) receptor-associated proteinlike 3 Orphan nuclear receptor, HMR type May be involved in transcriptional regulation Putative odorant receptor The heparin-binding growth factors are angiogenic agents in vivo and are potent mitogens for a variety of cell types in vitro. There are differences in the tissue distribution and concentration of these 2 growth factors Could be a global transcriptional regulator. Modifies gene expression by affecting chromatin. May be involved in brain development and facial morphogenesis Strongly activates transcription when bound to HCFC1. Suppresses the expression of HSV proteins in cells infected with the virus in a HCFC1-dependent manner. Also suppresses the HCFC1-dependent transcriptional activation by CREB3 and reduces the amount of CREB3 in the cell. Able to down-regulate expression of some cellular genes in CREBZFexpressing cells Acc:RF00416]; Small nucleolar RNA ACA43 [Source:RFAM  ZNF613  Fold Change -4.69  p Value 0.0023  -4.90  0.0021  -5.16  0.0018  -5.44 -5.67 -6.58 -7.71  0.0016 0.0015 0.0011 0.0008  -9.95  0.0005  -12.16  0.0003  -13.06  0.0003  -47.09  5.4E05  May be involved in transcriptional regulation  Supplementary
Table
8:
Differentially
expressed
genes
2
hours
post­stimulation
with
3
µg/ml
 LL­37.

 Gene Name NP_001032407.1 differentiation TMEM33 MIF  TSN13 ENSG00000203581  Gene Description May play a role in the regulation of keratinocyte Transmembrane protein 33; DB83 protein May play an important role in the progression of epithelial malignancies; The expression of MIF at sites of inflammation suggest a role for the mediator in regulating the function of macrophage in host defense. Also acts as a phenylpyruvate tautomerase Tetraspanin-13; Tspan-13; Transmembrane 4 superfamily member 13; Tetraspan NET-6 Acc:NR_002169]; olfactory receptor, family 1, subfamily F, member 2 (OR1F2) on chromosome 16 [Source:RefSeq_dna  Fold Change 7.67  p Value 0.004  7.24 6.36  0.0044 0.0054  5.47  0.0071  5.19  0.0078  108  Gene Name KCNH2  FOS  TIE2  GBG8  RT12 FGD5  KS6A2 IDH2 Q9BSF7 Q6I9Y3 SP4 NPT2B  NP_116256.2 BT2A1  Gene Description Pore-forming (alpha) subunit of voltage-gated inwardly rectifying potassium channel. Channel properties are modulated by cAMP and subunit assembly. Mediates the rapidly activating component of the delayed rectifying potassium current in heart (IKr). Isoform 3 has no channel activity by itself, but modulates channel characteristics when associated with isoform 1 Nuclear phosphoprotein which forms a tight but noncovalently linked complex with the JUN/AP-1 transcription factor. In the heterodimer, c-fos and JUN/AP-1 basic regions each seems to interact with symmetrical DNA half sites. Has a critical function in regulating the development of cells destined to form and maintain the skeleton. It is thought to have an important role in signal transduction, cell proliferation and differentiation This protein is a protein tyrosine-kinase transmembrane receptor for angiopoietin 1. It may constitute the earliest mammalian endothelial cell lineage marker. Probably regulates endothelial cell proliferation, differentiation and guides the proper patterning of endothelial cells during blood vessel formation Guanine nucleotide-binding proteins (G proteins) are involved as a modulator or transducer in various transmembrane signaling systems. The beta and gamma chains are required for the GTPase activity, for replacement of GDP by GTP, and for G proteineffector interaction 28S ribosomal protein S12, mitochondrial precursor; S12mt; MRP-S12; MT-RPS12 May activate CDC42, a member of the Ras-like family of Rho- and Rac proteins, by exchanging bound GDP for free GTP. May play a role in regulating the actin cytoskeleton and cell shape Serine/threonine kinase that may play a role in mediating the growth-factor and stress induced activation of the transcription factor CREB Plays a role in intermediary metabolism and energy production. It may tightly associate or interact with the pyruvate dehydrogenase complex Acc:Q9BSF7]; MGC13008 protein. [Source:Uniprot/SPTREMBL Chromo; Post-SET zinc-binding region; Pre-SET zinc-binding region; SET Binds to GT and GC boxes promoters elements. Probable transcriptional activator May be involved in actively transporting phosphate into cells via Na(+) cotransport. It may be the main phosphate transport protein in the intestinal brush border membrane. May have a role in the synthesis of surfactant in lungs' alveoli Proline-rich region; Tropomyosin Butyrophilin subfamily 2 member A1 precursor; Butyrophilin subfamily 2 member A3 precursor  Fold Change 4.82  p Value 0.009  4.57  0.0099  4.35  0.011  4.24  0.0116  4.20  0.0118  3.93  0.0135  3.92  0.0137  3.72  0.0153  3.65  0.016  3.58  0.0167  3.52  0.0174  3.13  0.0162  3.10 3.07  0.0235 0.0242  109  Gene Name ATE1  TRIO  NP_115589.2 PPIL2 NGLY1  RET7 NP_997646.1 BAALC LDHC ABI2  NP_006537.3 NP_079174.2 GPX7 ZN485 CAH3 ARHG4 CHST6  Gene Description Involved in the posttranslational conjugation of arginine to the N-terminal aspartate or glutamate of a protein. This arginylation is required for degradation of the protein via the ubiquitin pathway. Does not arginylate cysteine residues Promotes the exchange of GDP by GTP. Together with leukocyte antigen-related (LAR) protein, it could play a role in coordinating cell-matrix and cytoskeletal rearrangements necessary for cell migration and cell growth Engulfment and cell motility, ELM PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides Specifically deglycosylates the denatured form of Nlinked glycoproteins in the cytoplasm and assists their proteasome-mediated degradation. Cleaves the beta-aspartyl-glucosamine (GlcNAc) of the glycan and the amide side chain of Asn, converting Asn to Asp. Prefers proteins containing high-mannose over those bearing complex type oligosaccharides. Can recognize misfolded proteins in the endoplasmic reticulun that are exported in the cytosol to be destroyed and deglycosylate them, while it has no activity toward native proteins. Deglycosylation is prerequisite for subsequent proteasome-mediated degradation of some, but not all, misfolded glycoproteins Intracellular transport of retinol AT-rich interaction region May play a synaptic role at the postsynaptic lipid rafts by interacting with CAMK2A Possible role in sperm motility May act in regulation of cell growth and transformation by interacting with nonreceptor tyrosine kinases ABL1 and/or ABL2. May be involved in cytoskeletal reorganization. Regulates ABL1/c-Abl-mediated phosphorylation of MENA KH, type 1; RNA-binding region RNP-1 (RNA recognition motif) Adenovirus fibre protein; DENN; dDENN; uDENN Glutathione peroxidase 7 precursor; CL683 May be involved in transcriptional regulation Reversible hydration of carbon dioxide DH; Guanine-nucleotide dissociation stimulator, CDC24; Pleckstrin-like; Src homology-3; Variant SH3 Catalyzes the transfer of sulfate to position 6 of nonreducing N-acetylglucosamine (GlcNAc) residues of keratan. Mediates sulfation of keratan in cornea. Keratan sulfate plays a central role in maintaining corneal transparency. Acts on the nonreducing terminal GlcNAc of short and long carbohydrate substrates that have poly-N-acetyllactosamine structures  Fold Change 3.06  p Value 0.0244  3.06  0.0244  3.05 3.02  0.0247 0.0252  2.99  0.0258  2.96 2.94 2.87  0.0264 0.027 0.0287  2.83 2.66  0.0298 0.0352  2.66  0.0353  2.63 2.62 2.61 2.61 2.56  0.0363 0.0368 0.0371 0.0373 0.0391  2.56  0.0393  110  Gene Name SNX23 P48067-2  HSH2D  RND1  RFIP2  PDZD2 BIN3 KCMB1  Gene Description May be involved in several stages of intracellular trafficking. Probable microtubule-dependent motor protein Terminates the action of glycine by its high affinity sodium-dependent reuptake into presynaptic terminals. May play a role in regulation of glycine levels in NMDA receptor-mediated neurotransmission May be a modulator of the apoptotic response through its ability to affect mitochondrial stability (By similarity). Adapter protein involved in tyrosine kinase and CD28 signaling. Seems to affect CD28mediated activation of the RE/AP element of the interleukin-2 promoter Lacks intrinsic GTPase activity. Has a low affinity for GDP, and constitutively binds GTP. Controls rearrangements of the actin cytoskeleton. Induces the Rac-dependent neuritic process formation in part by disruption of the cortical actin filaments. Causes the formation of many neuritic processes from the cell body with disruption of the cortical actin filaments A Rab11 effector protein acting in the regulation of the transport of vesicles from the endosomal recycling compartment (ERC) to the plasma membrane. Also involved in receptor-mediated endocytosis and membrane trafficking of recycling endosomes, probably originating from clathrincoated vesicles. Binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) and phosphatidic acid (PA) PDZ domain-containing protein 2; PDZ domaincontaining protein 3; Activated in prostate cancer protein Involved in cytokinesis and septation where it has a role in the localization of F-actin Regulatory subunit of the calcium activated potassium KCNMA1 (maxiK) channel. Modulates the calcium sensitivity and gating kinetics of KCNMA1, thereby contributing to KCNMA1 channel diversity. Increases the apparent Ca(2+)/voltage sensitivity of the KCNMA1 channel. It also modifies KCNMA1 channel kinetics and alters its pharmacological properties. It slows down the activation and the deactivation kinetics of the channel. Acts as a negative regulator of smooth muscle contraction by enhancing the calcium sensitivity to KCNMA1. Its presence is also a requirement for internal binding of the KCNMA1 channel opener dehydrosoyasaponin I (DHS-1) triterpene glycoside and for external binding of the agonist hormone 17-beta-estradiol (E2). Increases the binding activity of charybdotoxin (CTX) toxin to KCNMA1 peptide blocker by increasing the CTX association rate and decreasing the dissociation rate  Fold Change 2.54  p Value 0.04  2.54  0.0401  2.53  0.0406  2.52  0.041  2.51  0.0417  2.50  0.042  2.45  0.0445  2.42  0.0462  111  Gene Name NP_079038.2 MCR  FARP2  NP_060156.1 CPN2  TNFC  ABD12  MYST3 NP1L2 VWF  AMAC1L1 RMD5B DHB3 Q5T6R2  Gene Description KRAB box; Zinc finger, C2H2-subtype; Zinc finger, C2H2-type Receptor for both mineralocorticoids (MC) such as aldosterone and glucocorticoids (GC) such as corticosterone or cortisol. Binds to mineralocorticoid response elements (MRE) and transactivates target genes. The effect of MC is to increase ion and water transport and thus raise extracellular fluid volume and blood pressure and lower potassium levels Rho-guanine nucleotide exchange factor that activates RAC1. Plays a role in the response to class 3 semaphorins and remodeling of the actin cytoskeleton Appr-1-p processing Involved in DNA repair and mitotic recombination. May play an active role in recombination processes in concert with other members of the RAD52 epistasis group; May function in chaperone-mediated protein folding; The 83 kDa subunit binds and stabilizes the catalytic subunit at 37 degrees Celsius and keeps it in circulation. Under some circumstances it may be an allosteric modifier of the catalytic subunit Cytokine that binds to LTBR/TNFRSF3. May play a specific role in immune response regulation. Provides the membrane anchor for the attachment of the heterotrimeric complex to the cell surface. Isoform 2 is probably non-functional PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides; This is a calciumactivated, phospholipid-dependent, serine- and threonine-specific enzyme. May play a role in cell motility by phosphorylating CSPG4 Histone acetyltransferase which may act as a transcriptional coactivator for RUNX1 and RUNX2 Acidic protein which may be involved in interactions with other proteins or DNA Important in the maintenance of hemostasis, it promotes adhesion of platelets to the sites of vascular injury by forming a molecular bridge between sub-endothelial collagen matrix and platelet-surface receptor complex GPIb-IX-V. Also acts as a chaperone for coagulation factor VIII, delivering it to the site of injury, stabilizing its heterodimeric structure and protecting it from premature clearance from plasma Protein AMAC1; Transmembrane protein 21A; Protein AMAC1L2 RMD5 homolog B Favors the reduction of androstenedione to testosterone. Uses NADPH while the two other EDH17B enzymes use NADH Could be involved in signal transduction  Fold Change 2.39  p Value 0.0476  2.39  0.0477  2.39  0.0479  2.38 2.38  0.0485 0.0486  2.38  0.0487  2.37  0.0026  2.36  0.0496  2.30  0.0081  2.28  0.0092  2.17  0.0114  -2.19 -2.29  0.0148 0.0476  -2.34  0.0257  112  Gene Name Q96GD3-2  PRB4S UBAP1 COPE  Q75VX8 Q5K675 MYPN NALDL CCG6 NP_277045.1  Gene Description Component of the Polycomb group (PcG) multiprotein PRC1 complex, a complex required to maintain the transcriptionally repressive state of many genes, including Hox genes, throughout development. PcG PRC1 complex acts via chromatin remodeling and modification of histones; it mediates monoubiquitination of histone H2A 'Lys119', rendering chromatin heritably changed in its expressibility Basic salivary proline-rich protein 4 allele S precursor; Salivary proline-rich protein Po; Parotid o protein; Protein N1; Glycosylated protein A Ubiquitin-associated protein 1; UBAP The coatomer is a cytosolic protein complex that binds to dilysine motifs and reversibly associates with Golgi non-clathrin-coated vesicles, which further mediate biosynthetic protein transport from the ER, via the Golgi up to the trans Golgi network. Coatomer complex is required for budding from Golgi membranes, and is essential for the retrograde Golgi-to-ER transport of dilysine-tagged proteins. In mammals, the coatomer can only be recruited by membranes associated to ADP-ribosylation factors (ARFs), which are small GTP-binding proteins; the complex also influences the Golgi structural integrity, as well as the processing, activity, and endocytic recycling of LDL receptors Proline-rich region Acc:Q5K675]; Osteoligament factor. [Source:Uniprot/SPTREMBL Component of the sarcomere that tethers together nebulin (skeletal muscle) and nebulette (cardiac muscle) to alpha-actinin, at the Z lines NAALADase-like activity unknown. Has no NAAG hydrolyzing activity. Exhibits a dipeptidyl-peptidase IV type activity. In vitro, cleaves Gly-Pro-AMC Thought to stabilize the calcium channel in an inactivated (closed) state Sterol-sensing 5TM box  Fold Change -2.36  p Value 0.0497  -2.38  0.0487  -2.38 -2.40  0.0485 0.0473  -2.40 -2.41  0.0471 0.0466  -2.42  0.0461  -2.43  0.0457  -2.45  0.0446  -2.45  0.0443  113  Gene Name O94813-3  XPA  Q8NHQ8-2 PDE4C CP2J2  KIF5C SF04 S26A2 MARVELD2 RHGXX  Fold Change -2.49  p Value 0.0425  -2.50  0.0419  -2.53  0.0406  -2.54  0.0399  -2.55  0.0397  Kinesin is a microtubule-associated force-producing protein that may play a role in organelle transport Plays a role in premRNA splicing Sulfate transporter. May play a role in endochondral bone formation May play a role in the formation of the epithelial barrier  -2.57  0.0389  -2.58 -2.58  0.0384 0.0384  -2.60  0.0374  GTPase activator for the Rho-type GTPases by converting them to an inactive GDP-bound state  -2.62  0.0369  Gene Description Thought to act as molecular guidance cue in cellular migration, and function appears to be mediated by interaction with roundabout homolog receptors. During neural development involved in axonal navigation at the ventral midline of the neural tube and projection of axons to different regions. SLIT1 and SLIT2 seem to be essential for midline guidance in the forebrain by acting as repulsive signal preventing inappropriate midline crossing by axons projecting from the olfactory bulb. In spinal chord development may play a role in guiding commissural axons once they reached the floor plate by modulating the response to netrin. In vitro, silences the attractive effect of NTN1 but not its growthstimulatory effect and silencing requires the formation of a ROBO1-DCC complex. May be implicated in spinal chord midline post-crossing axon repulsion. In vitro, only commissural axons that crossed the midline responded to SLIT2. In the developing visual system appears to function as repellent for retinal ganglion axons by providing a repulsion that directs these axons along their appropriate paths prior to, and after passage through, the optic chiasm. In vitro, collapses and repels retinal ganglion cell growth cones. Seems to play a role in branching and arborization of CNS sensory axons, and in neuronal cell migration. In vitro, Slit homolog 2 protein N-product, but not Slit homolog 2 protein C-product, repels olfactory bulb (OB) but not dorsal root ganglia (DRG) axons, induces OB growth cones collapse and induces branching of DRG axons. Seems to be involved in regulating leukocyte migration Involved in DNA excision repair. Initiates repair by binding to damaged sites with various affinities, depending on the photoproduct and the transcriptional state of the region Ras association domain-containing protein 8; Carcinoma-associated protein HOJ-1 cAMP-specific 3',5'-cyclic phosphodiesterase 4C; DPDE1; PDE21 This enzyme metabolizes arachidonic acid predominantly via a NADPH-dependent olefin epoxidation to all four regioisomeric cisepoxyeicosatrienoic acids. One of the predominant enzymes responsible for the epoxidation of endogenous cardiac arachidonic acid pools  114  Gene Name ADCY6 ARMET PVRL3  GDE5 BHLH2 DOCK8 FASP1 TMUB1 SIRPG  SG1D1  METTL6 SMURF2  KCNK7  Fold Change -2.62  p Value 0.0368  -2.64 -2.64  0.0359 0.0449  -2.65  0.0356  May function as a transcriptional factor to modulate chondrogenesis in response to the cAMP pathway Potential guanine nucleotide exchange factor (GEF). GEF proteins activate some small GTPases by exchanging bound GDP for free GTP Uncharacterized protein C21orf45; FAPP1associated protein 1  -2.66  0.0351  -2.67  0.0347  -2.68  0.0345  Transmembrane and ubiquitin-like domaincontaining protein 1; Ubiquitin-like protein SB144 Probable immunoglobulin-like cell surface receptor. On binding with CD47, mediates cell-cell adhesion. Engagement on T-cells by CD47 on antigenpresenting cells results in enhanced antigen-specific T-cell proliferation and co-stimulates T-cell activation May bind androgens and other steroids, may also bind estramustine, a chemotherapeutic agent used for prostate cancer. May be under transcriptional regulation of steroid hormones Probable methyltransferase E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. Interacts with SMAD1, SMAD2 and SMAD7 in order to trigger their ubiquitination and proteasome-dependent degradation. Enhances the inhibitory activity of SMAD7 and reduces the transcriptional activity of SMAD2. Coexpression of SMURF2 with SMAD1 results in considerable decrease in steady-state level of SMAD1 protein and a smaller decrease of SMAD2 level Probable potassium channel subunit. No channel activity observed in vitro as protein remains in the endoplasmic reticulum. May need to associate with an as yet unknown partner in order to reach the plasma membrane  -2.68  0.0344  -2.70  0.0339  -2.72  0.0332  -2.73 -2.80  0.0329 0.0307  -2.81  0.0303  Gene Description Membrane-bound, calcium-inhibitable adenylyl cyclase Protein ARMET precursor; Arginine-rich protein Plays a role in cell-cell adhesion through heterophilic trans-interactions with nectin-like proteins or nectins, such as trans-interaction with PVRL2/nectin-2 at Sertoli-spermatid junctions. Trans-interaction with PVR induces activation of CDC42 and RAC small G proteins through common signaling molecules such as SRC and RAP1. Also involved in the formation of cell-cell junctions, including adherens junctions and synapses. Induces endocytosis-mediated downregulation of PVR from the cell surface, resulting in reduction of cell movement and proliferation. Plays a role in the morphology of the ciliary body Putative glycerophosphodiester phosphodiesterase 5  115  Gene Name ZDH11 MULK  DLG1  RNF34  RSU1 ENP5  CYH4 Q9NWT9 LRC8B SFXN2 GBF1 GUAD Q9H6U4 HHEX Q495Z4 NP_079280.1 MAGB1  Gene Description Probable palmitoyltransferase ZDHHC11; Zinc finger DHHC domain-containing protein 11; DHHC-11; Zinc finger protein 399 Lipid kinase that can phosphorylate both monoacylglycerol and diacylglycerol to form lysophosphatidic acid (LPA) and phosphatidic acid (PA), respectively. Does not phosphorylate sphingosine. Overexpression increases the formation and secretion of LPA, resulting in transactivation of EGFR and activation of the downstream MAPK signaling pathway, leading to increased cell growth Essential multidomain scaffolding protein required for normal development (By similarity). Recruits channels, receptors and signaling molecules to discrete plasma membrane domains in polarized cells. May play a role in adherens junction assembly, signal transduction, cell proliferation, synaptogenesis and lymphocyte activation Has E3 ubiquitin-protein ligase activity. Regulates the levels of CASP8 and CASP10 by targeting them for proteasomal degradation. Protects cells against apoptosis induced by TNF. Binds phosphatidylinositol-5-phosphate and phosphatidylinositol-3-phosphate Potentially plays a role in the Ras signal transduction pathway. Capable of suppressing v-Ras transformation in vitro Likely to promote reglycosylation reactions involved in glycoproteins folding and quality control in the endoplasmic reticulum. Hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate Promotes guanine-nucleotide exchange on ARF1 and ARF5. Promotes the activation of ARF through replacement of GDP with GTP Calcium-binding EF-hand Leucine-rich repeat-containing protein 8B; T-cell activation leucine repeat-rich protein Potential iron transporter Promotes guanine-nucleotide exchange on ARF5. Promotes the activation of ARF5 through replacement of GDP with GTP Catalyzes the hydrolytic deamination of guanine, producing xanthine and ammonia Glycoside hydrolase, family 85 Recognizes the DNA sequence 5'-ATTAA-3'. May play a role in hematopoietic differentiation Adrenergic receptor, beta 1; Proline-rich region Acc:NP_079280]; coiled-coil domain containing 15 [Source:RefSeq_peptide Melanoma-associated antigen B1; MAGE-B1 antigen; MAGE-XP antigen; DSS-AHC critical interval MAGE superfamily 10; DAM10  Fold Change -2.84  p Value 0.0296  -2.86  0.029  -2.87  0.0287  -2.89  0.0283  -2.90  0.0279  -2.93  0.0271  -2.95  0.0028  -2.96 -3.01  0.0265 0.0255  -3.03 -3.04  0.0108 0.0248  -3.04  0.0247  -3.06 -3.09  0.0243 0.0238  -3.14 -3.15  0.0228 0.0227  -3.16  0.0224  116  Gene Name CUL5  FGF3 LAIR1  FA49A Q9NSC5-3 F261 FA53C SEM6A  DPYL1 PLOD2  NARGL C43BP TRFM  RB11B SPAG1  Gene Description Component of E3 ubiquitin ligase complexes, which mediate the ubiquitination and subsequent proteasomal degradation of target proteins. Seems to be involved poteosomal degradation of p53/TP53 stimulated by adenovirus E1B-55 kDa protein. May form a cell surface vasopressin receptor Could be involved in ear development Functions as an inhibitory receptor that plays a constitutive negative regulatory role on cytolytic function of natural killer (NK) cells, B-cells and Tcells. Activation by Tyr phosphorylation results in recruitment and activation of the phosphatases PTPN6 and PTPN11. It also reduces the increase of intracellular calcium evoked by B-cell receptor ligation. May also play its inhibitory role independently of SH2-containing phosphatases. Modulates cytokine production in CD4+ T-cells, downregulating IL2 and IFNG production while inducing secretion of transforming growth factor beta. Down-regulates also IgG and IgE production in B-cells as well as IL8, IL10 and TNF secretion. Inhibits proliferation and induces apoptosis in myeloid leukemia cell lines as well as prevents nuclear translocation of NF-kappa-B p65 subunit/RELA and phosphorylation of I-kappa-B alpha/CHUK in these cells. Inhibits the differentiation of peripheral blood precursors towards dendritic cells Protein FAM49A EVH1 Synthesis and degradation of fructose 2,6bisphosphate Protein FAM53C Can act as repulsive axon guidance cues. May play a role in channeling sympathetic axons into the sympathetic chains and controlling the temporal sequence of sympathetic target innervation Dihydropyrimidinase-related protein 1; DRP-1; Collapsin response mediator protein 1; CRMP-1 Forms hydroxylysine residues in -Xaa-Lys-Glysequences in collagens. These hydroxylysines serve as sites of attachment for carbohydrate units and are essential for the stability of the intermolecular collagen cross-links May belong to a complex displaying N-terminal acetyltransferase activity Phosphorylates on Ser and Thr residues the Goodpasture autoantigen (in vitro). Isoform 2 seems to be less active Involved in iron cellular uptake. Seems to be internalized and then recycled back to the cell membrane. Binds a single atom of iron per subunit. Could also bind zinc Possesses GTPase activity Plays a role in fertilization. Binds GTP and has GTPase activity  Fold Change -3.36  p Value 0.0067  -3.39 -3.48  0.0189 0.0178  -3.53 -3.58 -3.64  0.0173 0.0132 0.0161  -3.65 -3.72  0.0163 0.0154  -3.79  0.0147  -3.94  0.0135  -3.97  0.0133  -3.98  0.0156  -4.03  0.0128  -4.08 -4.20  0.0125 0.0118  117  Gene Name ANR11 HRSL1 Q69YY3 OVOL1  CLCN4  BSND  PHF14 TRBV6-1 CELR3  TULP4 CX04A APOB  ELAV4 BPAEA  KPBB  Gene Description May recruit HDACs to the p160 coactivators/nuclear receptor complex to inhibit ligand-dependent transactivation HRAS-like suppressor DKFZ Putative transcription factor. Involved in hair formation and spermatogenesis. May function in the differentiation and/or maintenance of the urogenital system Voltage-gated chloride channel. Chloride channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport Functions as a beta-subunit for CLCNKA and CLCNKB chloride channels. In the kidney CLCNK/BSND heteromers mediate chloride reabsorption by facilitating its basolateral efflux. In the stria, CLCNK/BSND channels drive potassium secretion by recycling chloride for the basolateral SLC12A2 cotransporter PHD finger protein 14 Immunoglobulin V-set; Immunoglobulin-like Has an important role in stress fiber formation induced by active diaphanous protein homolog 1 (DRF1). Induces microspike formation, in vivo (By similarity). In vitro, stimulates N-WASP-induced ARP2/3 complex activation in the absence of CDC42 (By similarity). May play an important role in the maintenance of sarcomeres and/or in the assembly of myofibrils into sarcomeres. Implicated in regulation of actin polymerization and cell adhesion Tubby-related protein 4; Tubby-like protein 4; Tubby superfamily protein May have an important role of cell protection in inflammation reaction Apolipoprotein B is a major protein constituent of chylomicrons (apo B-48), LDL (apo B-100) and VLDL (apo B-100). Apo B-100 functions as a recognition signal for the cellular binding and internalization of LDL particles by the apoB/E receptor May play a role in neuron-specific RNA processing. Protects CDKN1A mRNA from decay by binding to its 3'-UTR Cytoskeletal linker protein. Anchors keratincontaining intermediate filaments to the inner plaque of hemidesmosomes. The proteins may selfaggregate to form filaments or a two-dimensional mesh Phosphorylase b kinase catalyzes the phosphorylation of serine in certain substrates, including troponin I. The beta chain acts as a regulatory unit and modulates the activity of the holoenzyme in response to phosphorylation  Fold Change -4.23  p Value 0.0116  -4.52 -4.55 -4.66  0.0101 0.01 0.0096  -4.72  0.0093  -4.77  0.0091  -4.99 -5.20 -5.25  0.0084 0.0078 0.0446  -5.56  0.0069  -5.66  0.0067  -5.72  0.0065  -6.31  0.0055  -6.43  0.0053  -6.43  0.0053  118  Gene Name  Gene Description  ENSG00000117114  Brain-specific angiogenesis inhibitor; CD97 antigen; D-galactoside/L-rhamnose binding SUEL lectin; EMR1 hormone receptor; GPCR, family 2, secretinlike; GPS; Hormone receptor, extracellular; Latrophilin receptor; Latrophilin, C-terminal; Olfactomedin-like Involved in the biogenesis of rRNA Transmembrane gamma-carboxyglutamic acid protein 1 precursor; Proline-rich Gla protein 1; Proline-rich gamma-carboxyglutamic acid protein 1 Importin-beta, N-terminal The heparin-binding growth factors are angiogenic agents in vivo and are potent mitogens for a variety of cell types in vitro. There are differences in the tissue distribution and concentration of these 2 growth factors The glycine receptor is a neurotransmitter-gated ion channel. Binding of glycine to its receptor increases the chloride conductance and thus produces hyperpolarization (inhibition of neuronal firing) Thought to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces Alters PTPRF cellular localization and induces PTPRF clustering. May regulate the disassembly of focal adhesions. May localize receptor-like tyrosine phosphatases type 2A at specific sites on the plasma membrane, possibly regulating their interaction with the extracellular environment and their association with substrates Immunoglobulin superfamily member 21 precursor Cystinosin/ERS1p repeat EGF-like region; Glycoside hydrolase, family 56; Glycoside hydrolase, family 56, sperm surface protein PH20; Multicopper oxidase, copper-binding site Acc:RF00416]; Small nucleolar RNA ACA43 [Source:RFAM  RRP5 TMG1 ENSG00000086200 FGF2  GLRA2  MUC16 LIPA2  IGS21 NP_079354.2 NP_036401.1  ENSG00000199437  Fold Change -6.58  p Value 0.0051  -6.63 -6.82  0.0051 0.0049  -6.86 -8.32  0.0048 0.0011  -8.57  0.0034  -8.92  0.0032  -9.95  0.0028  -10.15 -10.21 -14.31  0.0027 0.0027 0.0017  -20.72  0.0011  
 
  Supplementary
Table
9:
Differentially
expressed
genes
4
hours
post­stimulation
with
3
µg/ml
 LL­37.

 Gene Name Q9H6U4 RBP17  Gene Description Glycoside hydrolase, family 85 May function as a nuclear transport receptor  Fold Change 10.07 9.32  p Value 0.0001 0.0002  119  Gene Name EGF CLCN5  DUS9 ENPP5 IFNA1  Q96NK6 GPR25 SMAD6  NP_001025226.1 INHBC  CCG4 Q8N370 NP_060845.2 DAB1 CARD9 ADCK1 GIPR  Gene Description EGF stimulates the growth of various epidermal and epithelial tissues in vivo and in vitro and of some fibroblasts in cell culture Voltage-gated chloride channel. Chloride channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport. May play an important role in renal tubular function Inactivates MAP kinases. Has a specificity for the ERK family; Required for the uptake of creatine in muscles and brain May play a role in neuronal cell communication. Lacks nucleotide pyrophosphatase and lysopholipase D activity Produced by macrophages, IFN-alpha have antiviral activities. Interferon stimulates the production of two enzymes: a protein kinase and an oligoadenylate synthetase Peptidase S1 and S6, chymotrypsin/Hap Orphan receptor Antagonist of signaling by TGF-beta (transforming growth factor) type 1 receptor superfamily members; has been shown to inhibit selectively BMP (bone morphogenetic proteins) signaling by competing with the co-SMAD SMAD4 for receptor-activated SMAD1. SMAD6 is an inhibitory SMAD (I-SMAD) or antagonistic SMAD. Binds to regulatory elements in target promoter regions GTPase-activating protein for Rho family members. May play a role in the reduction of the p21rasGTPase-activating potential of p120GAP Inhibins and activins inhibit and activate, respectively, the secretion of follitropin by the pituitary gland. Inhibins/activins are involved in regulating a number of diverse functions such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, nerve cell survival, embryonic axial development or bone growth, depending on their subunit composition. Inhibins appear to oppose the functions of activins Thought to stabilize the calcium channel in an inactivated (closed) state Major facilitator superfamily MFS_1 Sodium:dicarboxylate symporter; Zinc/iron permease Adapter molecule functioning in neural development. May regulate SIAH1 activity Activates NF-kappa-B via BCL10 The function of this protein is not yet clear. It is not known if it has protein kinase activity and what type of substrate it would phosphorylate (Ser, Thr or Tyr) This is a receptor for GIP. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase  Fold Change 7.03  p Value 0.0004  6.26  0.0065  5.99  0.0006  5.93  0.0006  5.52  0.0008  5.49 5.21 5.07  0.0008 0.0009 0.001  4.99  0.0011  4.99  0.0011  4.79  0.0012  4.79 4.72 4.63  0.0012 0.0013 0.0014  4.60 4.54  0.0014 0.0015  4.53  0.0015  120  Gene Name DIAP2  NP_001032671.1 FOXP1  ZN566 NP_076955.1 AP3B1  TPBG Q502X4  AP3S2  GRPEL1  NP_005178.4  Gene Description Absolutely required for simian virus 40 DNA replication in vitro. It participates in a very early step in initiation. RP-A is a single-stranded DNA-binding protein; Could be involved in oogenesis. Involved in the regulation of endosome dynamics. Implicated in a novel signal transduction pathway, in which isoform 3 and CSK are sequentially activated by RHOD to regulate the motility of early endosomes through interactions with the actin cytoskeleton Fatty acid desaturase; Fatty acid desaturase, type 1 Transcriptional repressor that plays an important role in the specification and differentiation of lung epithelium. Can act with CTBP1 to synergistically repress transcription but CTPBP1 is not essential. Essential transcriptional regulator of B cell development May be involved in transcriptional regulation Acc:XR_019463]; similar to CG31855-PA (LOC401770), mRNA [Source:RefSeq_dna Subunit of non-clathrin- and clathrin-associated adaptor protein complex 3 that plays a role in protein sorting in the late-Golgi/trans-Golgi network (TGN) and/or endosomes. The AP complexes mediate both the recruitment of clathrin to membranes and the recognition of sorting signals within the cytosolic tails of transmembrane cargo molecules. AP-3 appears to be involved in the sorting of a subset of transmembrane proteins targeted to lysosomes and lysosome-related organelles Trophoblast glycoprotein precursor; 5T4 oncofetal trophoblast glycoprotein; 5T4 oncotrophoblast glycoprotein; 5T4 oncofetal antigen; M6P1 Enzyme which catalyzes the acetylation of polyamines. Substrate specificity: norspermidine &gt; spermidine = spermine &gt;&gt; N(1)acetylspermine = putrescine Part of the AP-3 complex, an adapter-related complex which is not clathrin-associated. The complex is associated with the Golgi region as well as more peripheral structures. It facilitates the budding of vesicles from the Golgi membrane and may be directly involved in trafficking to lysosomes Essential component of the PAM complex, a complex required for the translocation of transit peptide-containing proteins from the inner membrane into the mitochondrial matrix in an ATPdependent manner. Seems to control the nucleotidedependent binding of mitochondrial HSP70 to substrate proteins EDG-8 sphingosine 1-phosphate receptor; Prolinerich region; TAFH/NHR1; Treacher Collins syndrome protein Treacle; Zinc finger, MYND-type  Fold Change 4.29  p Value 0.0018  4.25 4.16  0.0019 0.0021  4.05 3.99  0.0195 0.0024  3.86  0.0108  3.73  0.0004  3.70  0.0032  3.68  0.0033  3.68  0.0033  3.65  0.0034  121  Gene Name DACH2  IGF1R  AT131 NP_001480.3  N4BP2 NET4 GP126 CYB5 M10L1 RBM16 DNS2A  Q9H9G5  Gene Description Transcription factor that is involved in regulation of organogenesis. Seems to be a regulator for SIX1 and SIX6. Seems to act as a corepressor of SIX6 in regulating proliferation by directly repressing cyclindependent kinase inhibitors, including the p27Kip1 promoter. Is recruited with SIX6 to the p27Kip1 promoter in embryonal retina. SIX6 corepression seems also to involve NCOR1, TBL1, HDAC1 and HDAC3. May be involved together with PAX3, SIX1, and EYA2 in regulation of myogenesis. In the developing somite, expression of DACH2 and PAX3 is regulated by the overlying ectoderm, and DACH2 and PAX3 positively regulate each other's expression (By similarity). Probably binds to DNA via its DACHbox-N domain This receptor binds insulin-like growth factor 1 (IGF1) with a high affinity and IGF2 with a lower affinity. It has a tyrosine-protein kinase activity, which is necessary for the activation of the IGF1stimulated downstream signaling cascade Probable cation-transporting ATPase 13A1 Nuclear hormone receptor, DNA-binding; Nuclear hormone receptor, ligand-binding; Retinoid X receptor; Steroid hormone receptor; Vitamin D receptor Has 5'-polynucleotide kinase and nicking endonuclease activity. May play a role in DNA repair or recombination May play an important role in neural, kidney and vascular development. Promotes neurite elongation from olfactory bulb explants Orphan receptor Cytochrome b5 is a membrane bound hemoprotein which function as an electron carrier for several membrane bound oxygenases Putative RNA helicase. Isoform 1 may play a role in male germ cell development DH; Guanine-nucleotide dissociation stimulator, CDC24; PDZ/DHR/GLGF; Pleckstrin-like; Raf-like Ras-binding; Spectrin/pleckstrin-like Hydrolyzes DNA under acidic conditions with a preference for double-stranded DNA. Plays a major role in the degradation of nuclear DNA in cellular apoptosis during development. Necessary for proper fetal development and for definitive erythropoiesis in fetal liver, where it degrades nuclear DNA expelled from erythroid precursor cells Plexin; Semaphorin/CD100 antigen  Fold Change 3.63  p Value 0.0034  3.63  0.0035  3.59 3.53  0.0036 0.0038  3.52  0.0039  3.49  0.0415  3.45 3.44  0.0042 0.0134  3.38  0.0207  3.33  0.0048  3.23  0.0055  3.22  0.0056  122  Gene Name PZP  NIPBL Q5JSH9  NP_036256.1 TLX3 RAI3  SFRIP SULF1  NP_114158.2 KLDC2 TRI50 KCC1D  ZN174  Gene Description Is able to inhibit all four classes of proteinases by a unique 'trapping' mechanism. This protein has a peptide stretch, called the 'bait region' which contains specific cleavage sites for different proteinases. When a proteinase cleaves the bait region, a conformational change is induced in the protein which traps the proteinase. The entrapped enzyme remains active against low molecular weight substrates (activity against high molecular weight substrates is greatly reduced). Following cleavage in the bait region a thioester bond is hydrolyzed and mediates the covalent binding of the protein to the proteinase Probably plays a structural role in chromatin. Involved in sister chromatid cohesion, possibly by interacting with the cohesin complex Antifreeze protein, type I; Orphan nuclear receptor, NOR1 type; Proline-rich region; Ribosomal protein P2; Salmonella/Shigella invasin protein C; Zinc finger, MIZ-type CHORD; CS T-cell leukemia homeobox protein 3; Homeobox protein Hox-11L2 Unknown. This G-protein coupled receptor could be involved in modulating differentiation and maintaining homeostasis of epithelial cells. The comparable expression level in fetal lung and kidney with adult tissues suggests a possible role in embryonic development and maturation of these organs. This retinoic acid-inducible GPCR provide evidence for a possible interaction between retinoid and G-protein signaling pathways Plays a role in pre-mRNA alternative splicing by regulating spliceosome assembly Exhibits arylsulfatase activity and highly specific endoglucosamine-6-sulfatase activity. It can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Diminishes HSPG (heparan sulfate proteoglycans) sulfation, inhibits signaling by heparin-dependent growth factors, diminishes proliferation, and facilitates apoptosis in response to exogenous stimulation Acc:NP_114158]; spermatogenesis associated 9 isoform a [Source:RefSeq_peptide Kelch domain-containing protein 2; Hepatocellular carcinoma-associated antigen 33; Host cell factor homolog LCP Tripartite motif-containing protein 50 Calcium/calmodulin-dependent protein kinase belonging to a proposed calcium-triggered signaling cascade. May regulate calcium-mediated granulocyte function. May play a role in apoptosis of erythroleukemia cells. Activates MAP kinase MAPK3 (By similarity). In vitro, phosphorylates transcription factor CREM isoform Beta and probably CREB1 Transcriptional repressor  Fold Change 3.22  p Value 0.0056  3.18  0.0058  3.17  0.006  3.17 3.12  0.006 0.0064  3.09  0.0067  3.09  0.0067  3.07  0.0068  3.03  0.0072  3.02  0.0073  3.01 2.99  0.0075 0.0076  2.99  0.0077  123  Gene Name HXD11  PKCB1  ZFP95 MAP4 MAGA5 GABARAPL3 GUF1 AOAH ASPM NP_064614.2 NP_062536.2 HMGB3 ISCU Q9BSC4-2 PAPOA  GBRD  RABP2  GBRG3  FRMD5 IKKB  Gene Description Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anteriorposterior axis Protein kinase C-binding protein 1; Rack7; Cutaneous T-cell lymphoma-associated antigen se14-3; CTCL tumor antigen se14-3; Zinc finger MYND domain-containing protein 8 May be involved in transcriptional regulation Non-neuronal microtubule-associated protein. Promotes microtubule assembly Not known, though may play a role tumor transformation or progression Gamma-aminobutyric acid receptor-associated protein-like 3; GABA(A) receptor-associated proteinlike 3 GTP-binding protein GUF1 homolog Removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides Probable role in mitotic spindle regulation and coordination of mitotic processes (By similarity). May have a preferential role in regulating neurogenesis PR domain-containing protein 11 Proline-rich region Binds preferentially single-stranded DNA and unwinds double stranded DNA Involved in the assembly or repair of the [Fe-S] clusters present in iron-sulfur proteins. Binds iron NUC153 Polymerase that creates the 3' poly(A) tail of mRNA's. Also required for the endoribonucleolytic cleavage reaction at some polyadenylation sites. May acquire specificity through interaction with a cleavage and polyadenylation specificity factor (CPSF) at its C-terminus GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel Cytosolic CRABPs may regulate the access of retinoic acid to the nuclear retinoic acid receptors. CRABP2 may participate in a regulatory feedback mechanism to control the action of retinoic acid on cell differentiation GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel FERM domain-containing protein 5 Phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappaB complex and ultimately the degradation of the inhibitor. Also phosphorylates NCOA3  Fold Change 2.97  p Value 0.0079  2.93  0.0083  2.91 2.88  0.0085 0.0089  2.88  0.0089  2.88  0.0033  2.86 2.84  0.0092 0.0095  2.83  0.0096  2.82 2.82 2.80  0.0098 0.0099 0.0433  2.79  0.0103  2.78 -2.76  0.0105 0.0109  -2.76  0.0108  -2.77  0.001  -2.77  0.0105  -2.78 -2.78  0.0105 0.0104  124  Gene Name DOK5  CLCN3  DUSP4 ZN659 SMAD7  G7D C1QA  SP1  GLE1  DDFL1 TCF21  LYZL6  Gene Description Docking proteins interact with receptor tyrosine kinases and mediate particular biological responses. DOK5 functions in RET-mediated neurite outgrowth and plays a positive role in activation of the MAP kinase pathway. Putative link with downstream effectors of RET in neuronal differentiation Voltage-gated chloride channel. Chloride channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport. May play an important role in neuronal cell function through regulation of membrane excitability by protein kinase C. It could help neuronal cells to establish short-term memory Regulates mitogenic signal transduction by dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2 Zinc finger protein 659 Antagonist of signaling by TGF-beta (transforming growth factor) type 1 receptor superfamily members; has been shown to inhibit TGF-beta (Transforming growth factor) and activin signaling by associating with their receptors thus preventing SMAD2 access. Functions as an adaptor to recruit SMURF2 to the TGF-beta receptor complex. SMAD7 is an inhibitory SMAD (I-SMAD) or antagonistic SMAD whose inhibitory activity is enhanced by SMURF2 Involved in meiotic recombination. Facilitate crossovers between homologs during meiosis C1q associates with the proenzymes C1r and C1s to yield C1, the first component of the serum complement system. The collagen-like regions of C1q interact with the Ca(2+)-dependent C1r(2)C1s(2) proenzyme complex, and efficient activation of C1 takes place on interaction of the globular heads of C1q with the Fc regions of IgG or IgM antibody present in immune complexes Binds to GC box promoters elements and selectively activates mRNA synthesis from genes that contain functional recognition sites. Can interact with G/Crich motifs from serotonin receptor promoter Required for the export of mRNAs containing poly(A) tails from the nucleus into the cytoplasm. May be involved in the terminal step of the mRNA transport through the nuclear pore complex (NPC) Promotes cell proliferation Involved in epithelial-mesenchymal interactions in kidney and lung morphogenesis that include epithelial differentiation and branching morphogenesis. May play a role in the specification or differentiation of one or more subsets of epicardial cell types Lysozyme-like protein 6 precursor  Fold Change -2.81  p Value 0.01  -2.82  0.0098  -2.82  1.71E05  -2.83 -2.84  0.0097 0.0095  -2.84  0.0095  -2.86  0.0093  -2.87  0.0091  -2.87  0.0091  -2.87 -2.88  0.009 0.009  -2.88  0.009  125  Gene Name IMA4  O95394-2  Q96FJ0-2 Q59GS4 NP_653185.1 NP_055772.2 NOX1  TENS1 RM40  PO3F2  Gene Description Functions in nuclear protein import as an adapter protein for nuclear receptor KPNB1. Binds specifically and directly to substrates containing either a simple or bipartite NLS motif. Docking of the importin/substrate complex to the nuclear pore complex (NPC) is mediated by KPNB1 through binding to nucleoporin FxFG repeats and the complex is subsequently translocated through the pore by an energy requiring, Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to importin-beta and the three components separate and importin-alpha and -beta are re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran from importin. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTPand GDP-bound forms of Ran between the cytoplasm and nucleus. In vitro, mediates the nuclear import of human cytomegalovirus UL84 by recognizing a non-classical NLS. In vitro, mediates the nuclear import of human cytomegalovirus UL84 by recognizing a nonclassical NLS Phosphoglucomutase/phosphomannomutase; Phosphoglucomutase/phosphomannomutase Cterminal; Phosphoglucomutase/phosphomannomutase alpha/beta/alpha domain I Mov34/MPN/PAD-1 IQ calmodulin-binding region; Myosin head, motor region Acc:Q5T8I8]; Novel protein (FLJ30525) (Fragment). [Source:Uniprot/SPTREMBL DENN; WD-40 repeat; dDENN NOH-1S is a voltage-gated proton channel that mediates the H(+) currents of resting phagocytes and other tissues. It participates in the regulation of cellular pH and is blocked by zinc. NOH-1L is a pyridine nucleotide-dependent oxidoreductase that generates superoxide and might conduct H(+) ions as part of its electron transport mechanism, whereas NOH-1S does not contain an electron transport chain May be involved in cell migration, cartilage development and in linking signal transduction pathways to the cytoskeleton 39S ribosomal protein L40, mitochondrial precursor; L40mt; MRP-40; Nuclear localization signalcontaining protein deleted in velocardiofacial syndrome; Up-regulated in metastasis Transcription factor that binds preferentially to the recognition sequence which consists of two distinct half-sites, ('GCAT') and ('TAAT'), separated by a nonconserved spacer region of 0, 2, or 3 nucleotides. Positively regulates the genes under the control of corticotropin-releasing hormone (CRH) and CRH II promoters  Fold Change -2.88  p Value 0.0089  -2.89  0.0089  -2.89 -2.91  0.0088 0.0117  -2.91  0.0086  -2.92 -2.92  0.0084 0.0241  -2.93  0.0083  -2.93  0.0083  -2.94  0.0081  126  Gene Name RPIA EPO  NP_079038.2 O60696 RMD5B AN32A  ATN1 OR5V1 PI51B  PDZD1  TPD52  Gene Description Ribose-5-phosphate isomerase; Phosphoriboisomerase Erythropoietin is the principal hormone involved in the regulation of erythrocyte differentiation and the maintenance of a physiological level of circulating erythrocyte mass KRAB box; Zinc finger, C2H2-subtype; Zinc finger, C2H2-type Zinc finger, PHD-type RMD5 homolog B Implicated in a number of cellular processes, including proliferation, differentiation, caspasedependent and caspase-independent apoptosis, suppression of transformation (tumor suppressor), inhibition of protein phosphatase 2A, regulation of mRNA trafficking and stability in association with ELAVL1, and inhibition of acetyltransferases as part of the INHAT (inhibitor of histone acetyltransferases) complex Atrophin-1; Dentatorubral-pallidoluysian atrophy protein Putative odorant receptor Mediates RAC1-dependent reorganization of actin filaments (By similarity). Participates in the biosynthesis of phosphatidylinositol-4,5bisphosphate A scaffold protein that connects plasma membrane proteins and regulatory components, regulating their surface expression in epithelial cells apical domains. May be involved in the coordination of a diverse range of regulatory processes for ion transport and second messenger cascades. In complex with SLC9A3R1, may cluster proteins that are functionally dependent in a mutual fashion and modulate the trafficking and the activity of the associated membrane proteins. May play a role in the cellular mechanisms associated with multidrug resistance through its interaction with ABCC2 and PDZK1IP1. May potentiate the CFTR chloride channel activity. May function to connect SCARB1 with the cellular machineries for intracellular cholesterol transport and/or metabolism. May be involved in the regulation of proximal tubular Na(+)-dependent inorganic phosphate cotransport therefore playing an important role in tubule function Tumor protein D52; Protein N8  Fold Change -2.95  p Value 0.0081  -2.97  0.0124  -2.97  0.0079  -2.97 -2.97 -2.97  0.0079 0.0189 0.0039  -2.97  0.0016  -2.99 -3.00  0.0077 0.0076  -3.00  0.0075  -3.01  0.0074  127  Gene Name NFKB2  Q5TF39 NP_640332.1 DUSP7  STB5L OR2H1 MAP7  WD51A NP_060554.3 ZC12B ENSG00000180150 LOC197350  ENSG00000117707 STYK1  GAGE10  Gene Description Appears to have dual functions such as cytoplasmic retention of attached NF-kappa-B proteins and generation of p52 by a cotranslational processing. The proteasome-mediated process ensures the production of both p52 and p100 and preserves their independent function. p52 binds to the kappa-B consensus sequence 5'-GGRNNYYCC-3', located in the enhancer region of genes involved in immune response and acute phase reactions. p52 and p100 are respectively the minor and major form; the processing of p100 being relatively poor. Isoform p49 is a subunit of the NF-kappa-B protein complex, which stimulates the HIV enhancer in synergy with p65 Cytochrome c-type biogenesis protein CcbS; Major facilitator superfamily MFS_1 Ankyrin; Proline-rich region Regulates the activity of the MAP kinase family in response to changes in the cellular environment. PYST2-S may act as a negative regulator of PYST2L although it is unclear whether this is by competing for transcription, translation or activation factors May play a role in vesicle trafficking and exocytosis Putative odorant receptor Microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells. Associates with microtubules in a dynamic manner. May play a role in the formation of intercellular contacts. Colocalization with TRPV4 results in the redistribution of TRPV4 toward the membrane and may link cytoskeletal microfilaments WD repeat protein 51A Tropomyosin Protein ZC3H12B High mobility group protein HMG14 and HMG17; Histone H5 2Fe-2S ferredoxin, iron-sulfur binding site; Caspase, p20 subunit; Peptidase C14, caspase catalytic; Peptidase C14, caspase non-catalytic subunit p10; Peptidase C14, caspase precursor p45 Homeobox prospero-like Probable tyrosine protein-kinase, which has strong transforming capabilities on a variety of cell lines. When overexpressed, it can also induce tumor cell invasion as well as metastasis in distant organs. May act by activating both MAP kinase and phosphatidylinositol 3'-kinases (PI3K) pathways Antigen, recognized on melanoma by autologous cytolytic T-lymphocytes; Antigen, recognized on melanoma by autologous cytolytic T-lymphocytes. Completely silent in normal adult tissues, except testis  Fold Change -3.02  p Value 0.0073  -3.03  0.0072  -3.04 -3.04  0.0071 0.0019  -3.04 -3.05 -3.06  0.0071 0.007 0.0069  -3.07 -3.07 -3.07 -3.07  0.0069 3.2E05 0.0068 0.0068  -3.09  0.0218  -3.10 -3.10  0.0066 0.0065  -3.11  0.0065  128  Gene Name VATG3  SNG4 Q9BT26 KBTB3 FOSL2 CLD5 PGM5 ENSG00000174672 NP_001276.2 GOG8A  F261 ABCA2 CIDEA ACV1B  LPP3  ZN703 SEM3D  Q4LDG9 NP_112579.2 RNAS1  Fold Change -3.11  p Value 0.0065  -3.11 -3.12  0.0003 0.0064  Kelch repeat and BTB domain-containing protein 3; BTB and kelch domain-containing protein 3 Fos-related antigen 2 Plays a major role in tight junction-specific obliteration of the intercellular space  -3.13  0.0063  -3.13 -3.13  Component of adherens-type cell-cell and cell-matrix junctions. Lacks phosphoglucomutase activity Proline-rich region; Protein kinase; Serine/threonine protein kinase, active site; Tyrosine protein kinase ATPase, F1 complex, gamma subunit; Chloride channel calcium-activated; von Willebrand factor, type A Adapter protein that may provide indirect link between the endocytic membrane traffic and the actin assembly machinery. May regulate the formation of clathrin-coated vesicles; May be involved in maintaining Golgi structure Synthesis and degradation of fructose 2,6bisphosphate Probable transporter, its natural substrate has not been found yet. May have a role in macrophage lipid metabolism and neural development Activates apoptosis On ligand binding, forms a receptor complex consisting of two type II and two type I transmembrane serine/threonine kinases. Type II receptors phosphorylate and activate type I receptors which autophosphorylate, then bind and activate SMAD transcriptional regulators Catalyzes the conversion of phosphatidic acid (PA) to diacylglycerol (DG). In addition it hydrolyzes lysophosphatidic acid (LPA), ceramide-1-phosphate (C-1-P) and sphingosine-1-phosphate (S-1-P). The relative catalytic efficiency is LPA = PA &gt; C-1-P &gt; S-1-P. May be involved in cell adhesion and in cell-cell interactions May function as a transcriptional repressor Induces the collapse and paralysis of neuronal growth cones. Could potentially act as repulsive cues toward specific neuronal populations. Binds to neuropilin Leucine-rich repeat GSG1-like; Voltage-dependent calcium channel gamma Endonuclease that catalyzes the cleavage of RNA on the 3' side of pyrimidine nucleotides. Acts on single stranded and double stranded RNA  -3.14  0.0063 4.38E05 0.0062  -3.15  0.0003  -3.21  0.0057  -3.22  0.0056  -3.24  0.0003  -3.24  0.0044  -3.24 -3.25  0.0055 0.0054  -3.25  0.0054  -3.26 -3.27  0.0053 0.0053  -3.27 -3.30  0.0052 0.0051  -3.33  0.0403  Gene Description Catalytic subunit of the peripheral V1 complex of vacuolar ATPase (V-ATPase). V-ATPase is responsible for acidifying a variety of intracellular compartments in eukaryotic cells Synaptogyrin-4 Acc:Q9BT26]; MGC10981 protein. [Source:Uniprot/SPTREMBL  129  Gene Name Q8TEE9 ZN445 NP_006324.1 LTBP3  HS2ST Q5T7M9 WIF1 LRIG1  M4K3 MCHL1  NP_203131.1  TMM98 PAK1  NP_733936.1 NNAT  MAMC2 ENSG00000110077 SOX18  Gene Description Proline-rich region May be involved in transcriptional regulation Exosome-associated May be involved in the assembly, secretion and targeting of TGFB1 to sites at which it is stored and/or activated. May play critical roles in controlling and directing the activity of TGFB1. May have a structural role in the extra cellular matrix (ECM) Heparan sulfate 2-O-sulfotransferase Acc:Q5T7M9]; Novel protein possible ortholog of mouse RIKEN cDNA 2900024C23 gene. [Source:Uniprot/SPTREMBL Binds to WNT proteins and inhibits their activities. May be involved in mesoderm segmentation Act as a feedback negative regulator of signaling by receptor tyrosine kinases, through a mechanism that involves enhancement of receptor ubiquitination and accelerated intracellular degradation May play a role in the response to environmental stress. Appears to act upstream of the JUN Nterminal pathway Pro-MCH-like protein 1; Pro-melanin-concentrating hormone-like protein 1; Pro-MCH variant; Pro-MCHlike protein 2; Pro-melanin-concentrating hormonelike protein 2 Inhibits signal transduction by increasing the GTPase activity of G protein alpha subunits thereby driving them into their inactive GDP-bound form. Preferentially binds to G(o)-alpha and G(i)-alpha-3 Transmembrane protein 98; Protein TADA1 The activated kinase acts on a variety of targets. Likely to be the GTPase effector that links the Rhorelated GTPases to the JNK MAP kinase pathway. Activated by CDC42 and RAC1. Involved in dissolution of stress fibers and reorganization of focal complexes. Involved in regulation of microtubule biogenesis through phosphorylation of TBCB. Activity is inhibited in cells undergoing apoptosis, potentially due to binding of CDC2L1 and CDC2L2 Succinate semialdehyde dehydrogenase, mitochondrial precursor; NAD(+)-dependent succinic semialdehyde dehydrogenase May participate in the maintenance of segment identity in the hindbrain and pituitary development, and maturation or maintenance of the overall structure of the nervous system. May function as a regulatory subunit of ion channels MAM domain-containing protein 2 precursor CD20/IgE Fc receptor beta subunit Binds to the consensus sequence 5'-AACAAAG-3' and is able to trans-activate transcription via this site  Fold Change -3.39 -3.41 -3.42 -3.42  p Value 0.001 7.32E06 0.0044 4.30E03  -3.43 -3.44  0.0043 0.0042  -3.45  0.0042  -3.45  0.0042  -3.45  0.0042  -3.51  0.0039  -3.55  0.0038  -3.57 -3.57  0.0037 0.0003  -3.57  0.0037  -3.57  0.0002  -3.59 -3.59 -3.62  0.0036 0.0036 0.0002  130  Gene Name UBP37 TRAF1  ODFP  C43BP Q9NSC5-3 Q8WVI0 SYLC TWST1  Q53RY2  NP_659415.1  NP_116277.2 AN30A PLPL ENSG00000166965  Gene Description Ubiquitin carboxyl-terminal hydrolase 37; Ubiquitin thioesterase 37; Ubiquitin-specific-processing protease 37; Deubiquitinating enzyme 37 Adapter protein and signal transducer that links members of the tumor necrosis factor receptor family to different signaling pathways by association with the receptor cytoplasmic domain and kinases. Mediates activation of NF-kappa-B and JNK and is involved in apoptosis. The TRAF1/TRAF2 complex recruits the apoptotic suppressors BIRC2 and BIRC3 to TNFRSF1B/TNFR2 Component of the outer dense fibers (ODF) of spermatozoa. ODF are filamentous structures located on the outside of the axoneme in the midpiece and principal piece of the mammalian sperm tail and may help to maintain the passive elastic structures and elastic recoil of the sperm tail Phosphorylates on Ser and Thr residues the Goodpasture autoantigen (in vitro). Isoform 2 seems to be less active EVH1 Acc:Q8WVI0]; SNHG8 protein (Fragment). [Source:Uniprot/SPTREMBL Leucyl-tRNA synthetase, cytoplasmic; Leucine-tRNA ligase; LeuRS Probable transcription factor, which seems to be involved in the negative regulation of cellular determination and in the differentiation of several lineages including myogenesis, osteogenesis, and neurogenesis. Inhibits myogenesis by sequestrating E proteins, inhibiting trans-activation by MEF2, and inhibiting DNA-binding by MYOD1 through physical interaction. This interaction probably involves the basic domains of both proteins (By similarity). Also represses expression of proinflammatory cytokines such as TNFA and IL1B Acc:P15336]; Cyclic AMP-dependent transcription factor ATF-2 (Activating transcription factor 2) (cAMP response element-binding protein CRE- BP1) (HB16). [Source:Uniprot/SWISSPROT Acc:Q96M89]; CDNA FLJ32745 fis, clone TESTI2001511, weakly similar to MYOSIN II HEAVY CHAIN, NON MUSCLE. [Source:Uniprot/SPTREMBL Collagen triple helix repeat; Dopamine D4 receptor; Fibrillar collagen, C-terminal; Pistil-specific extensinlike protein; Proline-rich region Ankyrin repeat domain-containing protein 30A; Serologically defined breast cancer antigen NY-BR-1 Uncharacterized protein C14orf162; Myelin proteolipid protein-like protein Regulator of chromosome condensation, RCC1  Fold Change -3.68  p Value 0.0033  -3.70  0.0032  -3.74  0.0031  -3.75  0.0002  -3.75 -3.76  0.0025 0.003  -3.83  0.0221  -3.83  0.0028  -3.84  0.0028  -3.88  0.0027  -3.94  0.0025  -3.95  0.0025  -3.98  0.0024  -4.00  0.0024  131  Gene Name 5HT1E  DOCK4  PLOD2  DOK3  ACCN3  DCLK1  ZCHC4 GRID2  NP_055483.2 ENSG00000162105 SMO  ENSG00000196229  Gene Description This is one of the several different receptors for 5hydroxytryptamine (serotonin), a biogenic hormone that functions as a neurotransmitter, a hormone, and a mitogen. The activity of this receptor is mediated by G proteins that inhibit adenylate cyclase activity Involved in regulation of adherens junction between cells. Functions as a guanine nucleotide exchange factor (GEF), which activates Rap1 small GTPase by exchanging bound GDP for free GTP Forms hydroxylysine residues in -Xaa-Lys-Glysequences in collagens. These hydroxylysines serve as sites of attachment for carbohydrate units and are essential for the stability of the intermolecular collagen cross-links Docking proteins interact with receptor tyrosine kinases and mediate particular biological responses. DOK3 is a negative regulator of JNK signaling in Bcells through interaction with INPP5D/SHIP. May modulate Abl function Cation channel with high affinity for sodium, which is gated by extracellular protons and inhibited by the diuretic amiloride. Generates a biphasic current with a fast inactivating and a slow sustained phase. In sensory neurons is proposed to mediate the pain induced by acidosis that occurs in ischemic, damaged or inflamed tissue. May be involved in hyperalgesia. May play a role in mechanoreception. Heteromeric channel assembly seems to modulate channel properties Probable kinase that may be involved in a calciumsignaling pathway controlling neuronal migration in the developing brain. May also participate in functions of the mature nervous system May be a methyltransferase Receptor for glutamate. L-glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system. The postsynaptic actions of Glu are mediated by a variety of receptors that are named according to their selective agonists Acc:NP_149081]; GREB1 protein isoform b [Source:RefSeq_peptide Ankyrin; PDZ/DHR/GLGF; Src homology-3; Variant SH3 G protein-coupled receptor that probably associates with the patched protein (PTCH) to transduce the hedgehog's proteins signal. Binding of sonic hedgehog (SHH) to its receptor patched is thought to prevent normal inhibition by patched of smoothened (SMO) HMG-I and HMG-Y, DNA-binding; Molluscan rhodopsin C-terminal tail; Proline-rich region  Fold Change -4.00  p Value 0.0024  -4.03  0.0023  -4.05  0.0021  -4.07  0.0022  -4.14  0.0021  -4.28  0.0019  -4.34 -4.35  0.0018 0.0018  -4.36  0.0025  -4.37  0.0017  -4.37  0.0017  -4.43  0.0016  132  Gene Name SSR1  Q5VTT3  ABHD4  IGHV3-11 SUCR1 FAN  NP_001020026.1 CAD12  FGF2  PNMA1  Gene Description Receptor for somatostatin with higher affinity for somatostatin-14 than -28. This receptor is coupled via pertussis toxin sensitive G proteins to inhibition of adenylyl cyclase. In addition it stimulates phosphotyrosine phosphatase and Na(+)/H(+) exchanger via pertussis toxin insensitive G proteins Tyrosine-protein kinase receptor which may be involved in the early formation of the chondrocytes. It seems to be required for cartilage and growth plate development Lysophospholipase selective for N-acyl phosphatidylethanolamine (NAPE). Contributes to the biosynthesis of N-acyl ethanolamines, including the endocannabinoid anandamide by hydrolyzing the sn-1 and sn-2 acyl chains from N-acyl phosphatidylethanolamine (NAPE) generating glycerophospho-N-acyl ethanolamine (GP-NAE), an intermediate for N-acyl ethanolamine biosynthesis. Hydrolyzes substrates bearing saturated, monounsaturated, polyunsaturated N-acyl chains. Shows no significant activity towards other lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylserine Immunoglobulin; Immunoglobulin V-set; Immunoglobulin-like Receptor for succinate Couples the p55 TNF-receptor (TNF-R55 / TNFR1) to neutral sphingomyelinase (N-SMASE). Specifically binds to the N-smase activation domain of TNF-R55. May regulate ceramide production by N-SMASE May be involved in transcriptional regulation Cadherins are calcium dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types The heparin-binding growth factors are angiogenic agents in vivo and are potent mitogens for a variety of cell types in vitro. There are differences in the tissue distribution and concentration of these 2 growth factors Paraneoplastic antigen Ma1; Neuron- and testisspecific protein 1; 37 kDa neuronal protein  Q8NBC0 NP_996803.2 Q6ZN87 CAN11  Orphan nuclear receptor, HMR type Low density lipoprotein-receptor, class A; MAM KRAB box Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction  Fold Change -4.47  p Value 0.0016  -4.55  0.0015  -4.56  0.0015  -4.76  0.0013  -4.81 -4.81  0.0012 0.0012  -5.04 -5.12  0.001 0.001  -5.27  0.0008  -5.28  0.0009  -5.36  3.25E05 0.0008 0.0008 0.0008  -5.37 -5.40 -5.41  133  Gene Name NP_001025186.1  FXYD6 CLGN ADCK3 PSD12 NP_004849.2 Q6ICE7 TMG1 DEF MFSD4 CDC6 PLAL1 Q99490-2 ENSG00000199437 Q86SZ9 GMDS TRBV6-1 SC5A6 EYA4  ENP1  JHDM1D  Gene Description Photoreceptor required for regulation of circadian rhythm. Contributes to pupillar reflex and other nonimage forming responses to light. May be able to isomerize covalently bound all-trans retinal back to 11-cis retinal FXYD domain-containing ion transport regulator 6 precursor Probably plays an important role in spermatogenesis. Binds calcium ions May be a chaperone-like protein essential for the proper conformation and functioning of protein complexes in the respiratory chain Acts as a regulatory subunit of the 26S proteasome which is involved in the ATP-dependent degradation of ubiquitinated proteins HCO3- transporter, cytoplasmic; HCO3- transporter, eukaryote; HCO3-transporter, c-terminal; Na+/HCO3- transporter Acc:Q6ICE7]; CN5H6.4 protein (OTTHUMP00000028648). [Source:Uniprot/SPTREMBL Transmembrane gamma-carboxyglutamic acid protein 1 precursor; Proline-rich Gla protein 1; Proline-rich gamma-carboxyglutamic acid protein 1 Regulates the p53 pathway to control the expansion growth of digestive organs Major facilitator superfamily domain-containing protein 4 Involved in the initiation of DNA replication. Also participates in checkpoint controls that ensure DNA replication is completed before mitosis is initiated Shows weak transcriptional activatory activity. Transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase-activating polypeptide Ankyrin; Arf GTPase activating protein; Miro-like; Pleckstrin-like; Ras; Ras GTPase Acc:RF00416]; Small nucleolar RNA ACA43 [Source:RFAM WD repeat protein 23 Conversion of GDP-D-mannose to GDP-4-keto-6-Ddeoxymannose Immunoglobulin V-set; Immunoglobulin-like Transports pantothenate, biotin and lipoate in the presence of sodium Thought to play a role in transcription regulation during organogenesis through its intrinsic protein phosphatase activity. May be involved in development of the eye In the nervous system, could hydrolyze ATP and other nucleotides to regulate purinergic neurotransmission. Could also be implicated in the prevention of platelet aggregation. Hydrolyzes ATP and ADP equally well JmjC domain-containing histone demethylation protein 1D  Fold Change -5.43  p Value 0.0008  -5.59  0.0007  -5.78  0.0007  -5.98  0.0006  -6.44  0.0005  -6.60  0.0004  -6.86  1.56E05  -6.87  0.0093  -6.98  0.0103  -7.32  0.0003  -7.58  0.0003  -7.76  0.0079  -8.10  0.0035  -8.25  0.0163  -8.87 -8.94  0.0002 0.0002  -9.27 -12.33  0.0002 8.34E05 1.61E06  -118.41  2.74  0.0112  2.73  0.0113  134  Gene Name SIM2  B3GN2 CCDC5 Q14591-2 Q86V61 LECT1  ENSG00000196547 DKKL1 CR2 GBRG3  ENSG00000186660 ZNF490 PCOC1 ZNF506 Q4ZG89 ATP7B HSPA1A  Gene Description Transcription factor that may be a master gene of CNS development in cooperation with Arnt. It may have pleiotropic effects in the tissues expressed during development Catalyzes the initiation and elongation of poly-Nacetyllactosamine chains Regulator of spindle function and integrity during the metaphase-anaphase transition Zinc finger, C2H2-subtype; Zinc finger, C2H2-type GAT; VHS Bifunctional growth regulator that stimulates the growth of cultured chondrocytes in the presence of basic fibroblast growth factor (FGF) but inhibits the growth of cultured vascular endothelial cells. May contribute to the rapid growth of cartilage and vascular invasion prior to the replacement of cartilage by bone during endochondral bone development Glycoside hydrolase, family 38; Glycosyl hydrolases 38, C-terminal Dickkopf-like protein 1 precursor; Soggy-1 protein; SGY-1 Receptor for complement C3Dd, for the Epstein-Barr virus on human B-cells and T-cells and for HNRPU. Participates in B lymphocytes activation GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel Antifreeze protein, type I; Zinc finger, C2H2-type May be involved in transcriptional regulation Binds to the C-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity May be involved in transcriptional regulation Major facilitator superfamily; Major facilitator superfamily MFS_1; Sugar transporter superfamily; Tetracycline resistance protein Involved in the export of copper out of the cells, such as the efflux of hepatic copper into the bile In cooperation with other chaperones, Hsp70s stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. These chaperones participate in all these processes through their ability to recognize nonnative conformations of other proteins. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stressinduced damage  Fold Change 2.73  p Value 0.0114  2.71  0.0116  2.71  0.0117  2.70 2.69 2.68  0.0119 0.0121 0.0123  2.67  0.0125  2.67  0.0126  2.67  0.0126  2.65  0.0129  2.64 2.64 2.64  0.0131 0.0132 0.0132  2.62 2.61  0.0069 0.0138  2.61  0.0138  2.60  0.0141  135  Gene Name  Gene Description  Fold Change 2.59  p Value 0.0247  ANXA1  Calcium/phospholipid-binding protein which promotes membrane fusion and is involved in exocytosis. This protein regulates phospholipase A2 activity. It seems to bind from two to four calcium ions with high affinity  IG2AS  Putative insulin-like growth factor 2 antisense gene protein; IGF2-AS; PEG8/IGF2AS protein Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics; Cytochromes P450 are a group of heme-thiolate monooxygenases. This enzyme requires molecular oxygen and NADPH for the omega-hydroxylation of LTB4, a potent chemoattractant for polymorphonuclear leukocytes Acc:Q9H7C2]; CDNA: FLJ21062 fis, clone CAS01044. [Source:Uniprot/SPTREMBL Basic helix-loop-helix dimerisation region bHLH; Proline-rich region Mediates equilibrative transport of purine, pyrimidine nucleosides and the purine base hypoxanthine. Less sensitive than SLC29A1 to inhibition by nitrobenzylthioinosine (NBMPR), dipyridamole, dilazep and draflazine Probable transcription factor which may play a role in the regulation of specific gene expression within a subset of neuronal lineages. May play a role in determining or maintaining the identities of a small subset of visual system neurons This protein mediates the voltage-dependent sodium ion permeability of excitable membranes. Assuming opened or closed conformations in response to the voltage difference across the membrane, the protein forms a sodium-selective channel through which Na(+) ions may pass in accordance with their electrochemical gradient. This sodium channel may be present in both denervated and innervated skeletal muscle Involved in embryogenesis and cell differentiation Ubiquitin Can phosphorylate the alpha subunit of EIF2 and may mediate translational control Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anteriorposterior axis  2.58  0.0099  2.56  0.0152  2.54  0.0156  2.53  0.0001  2.53  0.016  2.53  0.0161  2.51  0.0167  2.50 2.50 2.49  0.0433 0.0293 0.0172  2.48  0.0175  Antifreeze protein, type I; Pollen allergen Poa pIX/Phl pVI, C-terminal; Zinc finger, CCCH-type Melanoma-derived leucine zipper-containing extranuclear factor Has antibacterial activity  2.48  0.0176  2.47  0.0177  2.47  0.0177  CP4F2  Q8IY89 NP_116216.1 S29A2  PO4F1  SCN4A  TGFB3 NP_071768.2 EIF2AK4 HXB5  NP_659002.1 MLZE DB118  136  Gene Name PA24B  GPR17  ARHGF NLK  DUFFY  FBX24 HEM4 CLC3A ADR2  PO2F3  Gene Description Calcium-dependent phospholipase A2 that selectively hydrolyzes glycerophospholipids in the sn-2 position with a preference for arachidonoyl phospholipids. Has a much weaker activity than PLA2G4A. Isoform 3 has calcium-dependent activity against palmitoyl-arachidonylphosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine Dual specificity receptor for uracil nucleotides and cysteinyl leukotrienes (CysLTs). Signals through G(i) and inhibition of adenylyl cyclase. May mediate brain damage by nucleotides and CysLTs following ischemia Specific GEF for RhoA activation and the regulation of vascular smooth muscle contractility Role in cell fate determination, required for differentiation of bone marrow stromal cells. Acts downstream of MAP3K7 and HIPK2 to negatively regulate the canonical Wnt/beta-catenin signaling pathway and the phosphorylation and destruction of the MYB transcription factor. May suppress a wide range of transcription factors by phosphorylation of the coactivator, CREBBP Non-specific receptor for many chemokines such as IL-8, GRO, RANTES, MCP-1 and TARC. It is also the receptor for the human malaria parasites Plasmodium vivax and Plasmodium knowlesi Substrate-recognition component of the SCF (SKP1CUL1-F-box protein)-type E3 ubiquitin ligase complex Uroporphyrinogen-III synthase; UROS; Uroporphyrinogen-III cosynthetase; Hydroxymethylbilane hydrolyase [cyclizing]; UROIIIS C-type lectin domain family 3 member A precursor; C-type lectin superfamily member 1; Cartilagederived C-type lectin Receptor for globular and full-length adiponectin (APM1), an essential hormone secreted by adipocytes that acts as an antidiabetic. Probably involved in metabolic pathways that regulate lipid metabolism such as fatty acid oxidation. Mediates increased AMPK, PPARA ligand activity, fatty acid oxidation and glucose uptake by adiponectin. Has some intermediate-affinity receptor activity for both globular and full-length adiponectin Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3'). Regulated the expression of a number of genes such as SPRR2A or placental lactogen  Fold Change 2.46  p Value 0.0183  2.45  0.0185  2.44  0.0471  2.44  0.0034  2.43  0.0191  2.42  0.0194  2.41  0.0198  2.41  0.02  2.40  0.0202  2.40  0.0204  137  Gene Name P2RX1  GNS Q99919 MINK1  Q9Y3K2 TEAD1  MRCKB  ARL5A  Q9NZS4 PODO ENSG00000135365 APOL1  H11 S26A2 ENSG00000104915  Gene Description Ligand gated ion channel with relatively high calcium permeability. Binding to ATP mediates synaptic transmission between neurons and from neurons to smooth muscle. Seems to be linked to apoptosis, by increasing the intracellular concentration of calcium in the presence of ATP, leading to programmed cell death N-acetylglucosamine-6-sulfatase precursor; G6S; Glucosamine-6-sulfatase Glycoside hydrolase family 2, TIM barrel Serine/threonine kinase that may play a role in the response to environmental stress. Appears to act upstream of the JUN N-terminal pathway. May play a role in the development of the brain Glutathione S-transferase, C-terminal; Glutathione S-transferase, N-terminal Binds specifically and cooperatively to the SPH and GT-IIC "enhansons" (5'-GTGGAATGT-3') and activates transcription in vivo in a cell-specific manner. The activation function appears to be mediated by a limiting cell-specific transcriptional intermediary factor (TIF). Involved in cardiac development. Binds to the M-CAT motif May act as a downstream effector of CDC42 in cytoskeletal reorganization. Contributes to the actomyosin contractility required for cell invasion, through the regulation of MYPT1 and thus MLC2 phosphorylation Endoglycosidase which is a cell surface and extracellular matrix-degrading enzyme. Cleaves heparan sulfate proteoglycans (HSPGs) into heparan sulfate side chains and core proteoglycans. Also implicated in the extravasation of leukocytes and tumor cell lines. Due to its contribution to metastasis and angiogenesis, it is considered to be a potential target for anti-cancer therapies; Lacks ADP-ribosylation enhancing activity HECT Plays a role in the regulation of glomerular permeability, acting probably as a linker between the plasma membrane and the cytoskeleton Proline-rich region; Zinc finger, PHD-type May affect the movement of lipids in the cytoplasm or allow the binding of lipids to organelles; May play a role in lipid exchange and transport throughout the body. May participate in reverse cholesterol transport from peripheral cells to the liver Histones H1 are necessary for the condensation of nucleosome chains into higher order structures Sulfate transporter. May play a role in endochondral bone formation Target SNARE coiled-coil region  Fold Change 2.40  p Value 0.0205  2.38  0.0211  2.38 2.37  0.0212 0.0214  2.37  0.0215  2.37  0.0215  2.37  0.0217  2.36  0.0218  2.36 2.35  0.0222 0.0225  2.34 2.34  0.0025 0.023  2.34  0.023  2.34  0.0231  2.33  0.0235  138  Gene Name BSN  CXA3  DARS2 NP_203752.1 GNS ZN501 Q5VT18 NP_612390.1  NP_057423.1 MTL5 TBCE  NP_079136.1 NP_055148.2  CLK3  TMPS6 TXD13 NP_113634.3 Q9H252-2  Gene Description Is thought to be involved in the organization of the cytomatrix at the nerve terminals active zone (CAZ) which regulates neurotransmitter release. Seems to act through binding to ERC2/CAST1. Essential in regulated neurotransmitter release from a subset of brain glutamatergic synapses. Involved in the formation of the retinal photoreceptor ribbon synapses One gap junction consists of a cluster of closely packed pairs of transmembrane channels, the connexons, through which materials of low MW diffuse from one cell to a neighboring cell Aspartyl-tRNA synthetase, mitochondrial precursor; Aspartate--tRNA ligase; AspRS Ankyrin; Tetratricopeptide TPR_1; Tetratricopeptide TPR_2; Tetratricopeptide region N-acetylglucosamine-6-sulfatase precursor; G6S; Glucosamine-6-sulfatase May be involved in transcriptional regulation Guanylate kinase; L27; PDZ/DHR/GLGF; Protein kinase; Src homology-3; Variant SH3 Ferric reductase, NAD binding; Flavoprotein pyridine nucleotide cytochrome reductase; NADH:cytochrome b5 reductase (CBR); Oxidoreductase FAD/NAD(P)-binding; Phenol hydroxylase reductase Guanine-nucleotide dissociation stimulator CDC25; RA May have a role in spermatogenesis Tubulin-folding protein; involved in the second step of the tubulin folding pathway. Seems to be implicated in the maintenance of the neuronal microtubule network. Involved in regulation of tubulin heterodimer dissociation SET CD80-like, immunoglobulin C2-set; Immunoglobulin; Immunoglobulin I-set; Immunoglobulin V-set; Immunoglobulin-like; Vascular cell adhesion molecule-1 Phosphorylates serine- and arginine-rich (SR) proteins of the spliceosomal complex. May be a constituent of a network of regulatory mechanisms that enable SR proteins to control RNA splicing. Phosphorylates serines, threonines and tyrosines May play a specialized role in matrix remodeling processes in liver Thioredoxin domain-containing protein 13 precursor Acc:Q6GW03]; HBV X-transactivated protein 13. [Source:Uniprot/SPTREMBL WD repeat protein 68; WD repeat protein An11 homolog  Fold Change 2.32  p Value 0.0236  2.32  0.0239  2.32  0.0241  2.31  0.0243  2.31  0.0243  2.30 2.29  0.0246 0.004  2.29  0.0256  2.28  0.0258  2.28 2.27  0.026 0.0262  2.27 2.27  0.0263 0.0264  2.27  0.0264  2.27  0.0266  2.27 2.26  0.0267 0.0268  2.26  0.027  139  Gene Name PELI1  Q9NXB7 NP_001034976.1  CEAM3  CM35A NP_057732.2 ATG4A  EGR1  ABI2  Q8TC23 O60729-3 MFGM  Gene Description Scaffold protein involved in the IL-1 signaling pathway via its interaction with the complex containing IRAK kinases and TRAF6. Required for NF-kappa-B activation and IL-8 gene expression in response to IL-1 Peptidase M14, carboxypeptidase A May be involved in several stages of intracellular trafficking. Could play an important role in the regulation of glucose transport by insulin. May act as a downstream effector of RHOQ/TC10 in the regulation of insulin-stimulated glucose transport Major granulocyte receptor mediating recognition and efficient opsonin-independent phagocytosis of CEACAM-binding microorganisms, including Neissiria, Moxarella and Haemophilus species, thus playing an important role in the clearance of pathogens by the innate immune system. Responsible for RAC1 stimulation in the course of pathogen phagocytosis CMRF35-A antigen precursor; CMRF-35; CD300c antigen Lupus La protein; RNA-binding protein Lupus La; RNA-binding region RNP-1 (RNA recognition motif) Cysteine protease required for autophagy, which cleaves the C-terminal part of either MAP1LC3, GABARAPL2 or GABARAP, allowing the liberation of form I. A subpopulation of form I is subsequently converted to a smaller form (form II). Form II, with a revealed C-terminal glycine, is considered to be the phosphatidylethanolamine (PE)-conjugated form, and has the capacity for the binding to autophagosomes. Preferred substrate is GABARAPL2 followed by MAP1LC3A and GABARAP Transcriptional regulator. Recognizes and binds to the DNA sequence 5'-CGCCCCCGC-3'(EGR-site). Activates the transcription of target genes whose products are required for mitogenesis and differentiation May act in regulation of cell growth and transformation by interacting with nonreceptor tyrosine kinases ABL1 and/or ABL2. May be involved in cytoskeletal reorganization. Regulates ABL1/c-Abl-mediated phosphorylation of MENA Negatively modulates RNA polymerase II function by binding to RPB5 Dual specificity protein phosphatase; Tyrosine specific protein phosphatase and dual specificity protein phosphatase Specific ligand for the alpha-v/beta-3 and alphav/beta-5 receptors. Also binds to phosphatidylserineenriched cell surfaces in a receptor-independent manner. Zona pellucida-binding protein which may play a role in gamete interaction. Binds specifically to rotavirus and inhibits its replication  Fold Change 2.26  p Value 0.027  2.26 2.25  0.0271 0.0277  2.25  0.0277  2.25  0.0278  2.25  0.0278  2.24  0.028  2.24  0.0282  2.23  0.0286  2.21  0.0304  2.20  0.0305  2.20  0.0305  140  Gene Name KCD12 GP116 Q5TCU6 S12A2 HDAC5  ZN746 KCP2 NP_071769.1 NP_005194.3 G137B MESD2 CP2C9  SO2B1  ENSA  Q9NZ71-2 FCHO2 DBC1  Gene Description BTB/POZ domain-containing protein KCTD12; Pfetin; Predominantly fetal expressed T1 domain May have a role in the regulation of acid-base balance Band 4.1; I/LWEQ; Proline-rich region; Small prolinerich; Vinculin/alpha-catenin Electrically silent transporter system. Mediates sodium and chloride reabsorption. Plays a vital role in the regulation of ionic balance and cell volume Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes. Involved in muscle maturation by repressing transcription of myocyte enhancer MEF2C. During muscle differentiation, it shuttles into the cytoplasm, allowing the expression of myocyte enhancer factors May be involved in transcriptional regulation Keratinocytes-associated protein 2; KCP-2 Guanylate-binding protein Collagen triple helix repeat; Galanin 3 receptor; Proline-rich region Integral membrane protein GPR137B; Transmembrane 7 superfamily member 1 Mesoderm development candidate 2; Renal carcinoma antigen NY-REN-61 Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. This enzyme contributes to the wide pharmacokinetics variability of the metabolism of drugs such as S-warfarin, diclofenac, phenytoin, tolbutamide and losartan Mediates the Na(+)-independent transport of organic anions such as taurocholate, the prostaglandins PGD2, PGE1, PGE2, leukotriene C4, thromboxane B2 and iloprost Endogenous ligand for sulfonylurea receptor. By inhibiting sulfonylurea from binding to the receptor, it reduces K(ATP) channel currents and thereby stimulates insulin secretion Decoy receptor for the cytotoxic ligands TNFS14/LIGHT and TNFSF6/FASL. Protects against apoptosis; Probable ATP-dependent DNA helicase FCH domain only protein 2 Inhibits cell proliferation by negative regulation of the G1/S transition. Mediates cell death which is not of the classical apoptotic type and regulates expression of components of the plasminogen pathway  Fold Change 2.20  p Value 0.0308  2.20  0.0308  2.20  0.031  2.20  0.031  2.19  0.0314  2.18 2.18 2.17 2.17  0.0321 0.0324 0.0328 0.0331  2.16  0.0336  2.16  0.034  2.15  0.034  2.15  0.0344  2.14  0.0348  2.14  0.0348  2.14 2.13  0.0348 0.0356  141  Gene Name HMOX1  NUD16 GTR14  GLPE GCNT1 CTDP1  Q8N8K0 NP_003311.2 U2AFL  LRC56 STXB2 GTF2E1  NP_037422.2 H2B2E  AQP10  Gene Description Heme oxygenase cleaves the heme ring at the alpha methene bridge to form biliverdin. Biliverdin is subsequently converted to bilirubin by biliverdin reductase. Under physiological conditions, the activity of heme oxygenase is highest in the spleen, where senescent erythrocytes are sequestrated and destroyed NUDIX hydrolase Facilitative glucose transporter (By similarity). May have a specific function related to spermatogenesis; Facilitative glucose transporter. Probably a neuronal glucose transporter This protein is a minor sialoglycoprotein in human erythrocyte membranes Forms critical branches in O-glycans Processively dephosphorylates 'Ser-2' and 'Ser-5' of the heptad repeats YSPTSPS in the C-terminal domain of the largest RNA polymerase II subunit. This promotes the activity of RNA polymerase II Myb, DNA-binding Functions in signal transduction from heterotrimeric G protein-coupled receptors. Could be involved in the hypothalamic regulation of body weight U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit-related protein 1; U2(RNU2) small nuclear RNA auxiliary factor 1-like 1; CCCH type zinc finger, RNA-binding motif and serine/arginine rich protein 1 Leucine-rich repeat-containing protein 56 Involved in the protein trafficking from the Golgi apparatus to the plasma membrane Recruits TFIIH to the initiation complex and stimulates the RNA polymerase II C-terminal domain kinase and DNA-dependent ATPase activities of TFIIH. Both TFIIH and TFIIE are required for promoter clearance by RNA polymerase Tat binding protein 1-interacting Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of posttranslational modifications of histones, also called histone code, and nucleosome remodeling Forms a water channel. Not permeable to urea and glycerol. May contribute to water transport in the upper portion of small intestine  Fold Change 2.13  p Value 0.036  2.13 2.12  0.0362 0.0365  2.12  0.037  2.12 2.11  0.0371 0.038  2.10 2.10  0.0385 0.0388  2.09  0.0397  2.09 2.08  0.0397 0.0399  2.08  0.0399  2.08 2.08  0.0088 0.04  2.08  0.0405  142  Gene Name H32  NP_001028751.1 RNF34  HIPK1  UBCE7IP1  BCAS1 SLC2B MPRD  ANC4  NP_064533.2 RBM6 GLYC  Gene Description Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of posttranslational modifications of histones, also called histone code, and nucleosome remodeling IQ calmodulin-binding region; Myosin head, motor region Has E3 ubiquitin-protein ligase activity. Regulates the levels of CASP8 and CASP10 by targeting them for proteasomal degradation. Protects cells against apoptosis induced by TNF. Binds phosphatidylinositol-5-phosphate and phosphatidylinositol-3-phosphate May play a role as a corepressor for homeodomain transcription factors. Phosphorylates DAXX in response to stress, and mediates its translocation from the nucleus to the cytoplasm. May be involved in malignant squamous cell tumor formation Isoform 1 acts as an E3 ubiquitin ligase, which accepts ubiquitin from specific E2 ubiquitinconjugating enzymes, and then transfers it to substrates promoting their degradation by the proteasome. Promotes degradation of TLR4 amd TLR9. Isoform 3/ZIN inhibits TNF and IL-1 mediated activation of NF-kappa-B. Promotes TNF and RIP mediated apoptosis Breast carcinoma amplified sequence 1; Novel amplified in breast cancer 1; Amplified and overexpressed in breast cancer May act as Rab effector protein and play a role in vesicle trafficking Transport of phosphorylated lysosomal enzymes from the Golgi complex and the cell surface to lysosomes. Lysosomal enzymes bearing phosphomannosyl residues bind specifically to mannose-6-phosphate receptors in the Golgi apparatus and the resulting receptor-ligand complex is transported to an acidic prelyosomal compartment where the low pH mediates the dissociation of the complex Component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle Acc:NP_064533]; spire homolog 1 [Source:RefSeq_peptide Specifically binds poly(G) RNA homopolymers in vitro Interconversion of serine and glycine  Fold Change 2.07  p Value 0.0415  2.07  0.0417  2.06  0.0422  2.06  0.0424  2.06  0.0424  2.06  0.0425  2.06  0.0427  2.05  0.0429  2.05  0.0429  2.05  0.01  2.05  0.0433  2.05  0.0433  143  Gene Name UGCGL1  RHBL2  NP_061889.1 ADRBK2 NP_006693.3 ATXN7 OR2B6 DEN1A HXA9  NP_612147.1 RRS1 PAK6 FAM126A  NP_001020951.1 MAFG  S26A4  Gene Description Recognizes glycoproteins with minor folding defects. Reglucosylates single N-glycans near the misfolded part of the protein, thus providing quality control for protein folding in the endoplasmic reticulum. Reglucosylated proteins are recognized by calreticulin for recycling to the endoplasmic reticulum and refolding or degradation Involved in regulated intramembrane proteolysis and the subsequent release of functional polypeptides from their membrane anchors. Known substrate: EFNB3 Eggshell protein; Intermediate filament protein; Keratin, type I Specifically phosphorylates the agonist-occupied form of the beta-adrenergic and closely related receptors Cyclic nucleotide-binding; Patatin; Protein of unknown function UPF0028; cAMP/cGMPdependent protein kinase Involved in neurodegeneration Putative odorant receptor DENN domain-containing protein 1A Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anteriorposterior axis RUN Involved in ribosome biogenesis The activated kinase acts on a variety of targets May have a role in the beta-catenin/Lef signaling pathway. May have a role in the process of myelination of the central and peripheral nervous system Phospholipase/Carboxylesterase Since they lack a putative transactivation domain, the small Mafs behave as transcriptional repressors when they dimerize among themselves. However, they seem to serve as transcriptional activators by dimerizing with other (usually larger) basic-zipper proteins and recruiting them to specific DNA-binding sites. Small Maf proteins heterodimerize with Fos and may act as competitive repressors of the NF-E2 transcription factor. Transcription factor, component of erythroid-specific transcription factor NF-E2. Activates globin gene expression when associated with NF-E2 Sodium-independent transporter of chloride and iodide  Fold Change 2.05  p Value 0.0436  2.05  0.0437  2.05  0.0438  2.04  0.0442  2.04  0.0429  2.04 2.03 2.03 2.03  0.0445 0.0449 0.0449 0.0451  2.03 2.03 2.02 2.02  0.0454 0.0458 0.0461 0.0462  2.02 2.02  0.0463 0.0467  2.02  0.027  144  Gene Name IL1F5  MTLR K1HB Q9UJT2-2 CEP41 RNF39 SC5A5 ANGL2 CN159 ESR1  NP_055721.3  Q6WBX8-3 BACE2  PGAM2  DLL1  Gene Description Is a highly and a specific antagonist of the IL-1 receptor-related protein 2-mediated response to interleukin 1 family member 9 (IL1F9). Could constitute part of an independent signaling system analogous to interleukin-1 alpha (IL-1A), beta (IL-1B) receptor agonist and interleukin-1 receptor type I (IL1R1), that is present in epithelial barriers and takes part in local inflammatory response Receptor for motilin Keratin, type I cuticular Ha3-II; Hair keratin, type I Ha3-II May play a role in testicular physiology, most probably in the process of spermatogenesis or spermiogenesis Centrosomal protein of 41 kDa; Protein Cep41; Testis-specific gene A14 protein May play a role in prolonged long term-potentiation (LTP) maintenance Mediates iodide uptake in the thyroid gland Induces sprouting in endothelial cells through an autocrine and paracrine action UPF0317 protein C14orf159, mitochondrial precursor Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues Component of the ESCRT-II complex, which is required for multivesicular bodies (MVBs) formation and sorting of endosomal cargo proteins into MVBs. The MVB pathway mediates delivery of transmembrane proteins into the lumen of the lysosome for degradation. The ESCRT-II complex is probably involved in the recruitment of the ESCRT-III complex. Its ability to bind ubiquitin probably plays a role in endosomal sorting of ubiquitinated cargo proteins by ESCRT complexes. The ESCRT-II complex may also play a role in transcription regulation, possibly via its interaction with ELL Cell cycle checkpoint control protein RAD9B homolog; RAD9 homolog B; hRAD9B Beta-secretase 2 precursor; Beta-site APP-cleaving enzyme 2; Aspartyl protease 1; Asp 1; ASP1; Membrane-associated aspartic protease 1; Memapsin-1; Aspartic-like protease 56 kDa; Down region aspartic protease Interconversion of 3- and 2-phosphoglycerate with 2,3-bisphosphoglycerate as the primer of the reaction. Can also catalyze the reaction of EC 5.4.2.4 (synthase) and EC 3.1.3.13 (phosphatase), but with a reduced activity Acts as a ligand for Notch receptors. Blocks the differentiation of progenitor cells into the B-cell lineage while promoting the emergence of a population of cells with the characteristics of a Tcell/NK-cell precursor  Fold Change 2.02  p Value 0.047  2.01 2.01  0.0473 0.0475  2.01  0.0475  2.01  0.0477  2.00  0.0484  2.00 -2.01  0.0485 0.0175  -2.01  0.048  -2.01  0.0476  -2.01  0.0003  -2.01  0.0472  -2.01  0.0472  -2.02  0.031  -2.02  0.0468  145  Gene Name P25A  P73  CDN2D PROD RFX2 KIF17 RGS20  NLGN3  ADIPO  EMAL2 MMP12  RBTN1  Gene Description Promotes in vitro the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundled stabilized microtubules blocks. When overexpressed, inhibits mitotic spindle assembly and nuclear envelope breakdown, apparently without affecting other cellular events Participates in the apoptotic response to DNA damage. When overproduced, activates transcription from p53-responsive promoters and induces apoptosis. May be a tumor suppressor protein Interacts strongly with CDK4 and CDK6 Converts proline to delta-1-pyrroline-5-carboxylate DNA-binding protein RFX2 Transports vesicles containing N-methyl-D-aspartate (NMDA) receptor 2B along microtubules Inhibits signal transduction by increasing the GTPase activity of G protein alpha subunits thereby driving them into their inactive GDP-bound form. Binds selectively to G(z)-alpha and is inhibited by phosphorylation and palmitoylation of the G-protein Neuronal cell surface protein thought to be involved in cell-cell-interactions by forming intercellular junctions through binding to beta-neurexins. May play a role in formation or maintenance of synaptic junctions. May also play a role in glia-glia or glianeuron interactions in the developing peripheral nervous system Important adipokine involved in the control of fat metabolism and insulin sensitivity, with direct antidiabetic, anti-atherogenic and anti-inflammatory activities. Stimulates AMPK phosphorylation and activation in the liver and the skeletal muscle, enhancing glucose utilization and fatty-acid combustion. Antagonizes TNF-alpha by negatively regulating its expression in various tissues such as liver and macrophages, and also by counteracting its effects. Inhibits endothelial NF-kappa-B signaling through a cAMP-dependent pathway. May play a role in cell growth, angiogenesis and tissue remodeling by binding and sequestering various growth factors with distinct binding affinities, depending on the type of complex, LMW, MMW or HMW May modify the assembly dynamics of microtubules, such that microtubules are slightly longer, but more dynamic May be involved in tissue injury and remodeling. Has significant elastolytic activity. Can accept large and small amino acids at the P1' site, but has a preference for leucine. Aromatic or hydrophobic residues are preferred at the P1 site, with small hydrophobic residues (preferably alanine) occupying P3 May be involved in gene regulation within neural lineage cells potentially by direct DNA binding or by binding to other transcription factors  Fold Change -2.02  p Value 0.0464  -2.02  0.0323  -2.02 -2.03 -2.04 -2.04  0.0463 0.0165 0.0194 0.0446  -2.04  0.0445  -2.04  0.0443  -2.04  0.0442  -2.04  0.0441  -2.04  0.0198  -2.05  0.0318  146  Gene Name HNF4G FTHFD CENPN  FAM3C GBGT1  IFIH1  Q8NI38 MOSC2  ACV1B  Gene Description Transcription factor. Has a lower transcription activation potential than HNF4-alpha 10-formyltetrahydrofolate dehydrogenase; 10FTHFDH; Aldehyde dehydrogenase 1 family member L1 Component of the CENPA-NAC (nucleosomeassociated) complex, a complex that plays a central role in assembly of kinetochore proteins, mitotic progression and chromosome segregation. The CENPA-NAC complex recruits the CENPA-CAD (nucleosome distal) complex and may be involved in incorporation of newly synthesized CENPA into centromeres Protein FAM3C precursor; Protein GS3786 Catalyzes the formation of some glycolipid via the addition of N-acetylgalactosamine (GalNAc) in alpha-1,3-linkage to some substrate. Glycolipids probably serve for adherence of some pathogens RNA helicase that, through its ATP-dependent unwinding of RNA, may function to promote message degradation by specific RNases. Seems to have growth suppressive properties. Involved in innate immune defense against viruses. Upon interaction with intracellular dsRNA produced during viral replication, triggers a transduction cascade involving MAVS/IPS1, which results in the activation of NF-kappa-B, IRF3 and IRF7 and the induction of the expression of antiviral cytokines such as IFNbeta and RANTES (CCL5). ATPase activity is specifically induced by dsRNA. Essential for the production of interferons in response to picornaviruses Ankyrin; Proline-rich region Catalytic component of the benzamidoxime prodrugconverting complex, a complex required to reduce Nhydroxylated structures, such as benzamidoxime prodrug. Benzamidoxime is an amidine prodrug produced by N-hydroxylation which is used to enhance bioavailability and increase intestinal absorption. It is then reduced into benzamidine, its active amidine, by the benzamidoxime prodrugconverting complex On ligand binding, forms a receptor complex consisting of two type II and two type I transmembrane serine/threonine kinases. Type II receptors phosphorylate and activate type I receptors which autophosphorylate, then bind and activate SMAD transcriptional regulators; On ligand binding, forms a receptor complex consisting of two type II and two type I transmembrane serine/threonine kinases. Type II receptors phosphorylate and activate type I receptors which autophosphorylate, then bind and activate SMAD transcriptional regulators. Receptor for TGF-beta. May bind activin as well  Fold Change -2.05  p Value 0.0104  -2.05  0.0433  -2.05  0.0405  -2.06 -2.06  0.023 0.0348  -2.06  0.0421  -2.06 -2.06  0.042 0.042  -2.06  0.042  147  Gene Name NP_060340.2 EP15  AIFM2  ABI2  SIRT6 ENSG00000168827  68MP ENSG00000130224 COF1  UDA1  NP_997208.1 NP_067081.2 OTOR STON1  LRC17  Gene Description Acc:NP_060340]; transmembrane protein 132A isoform a [Source:RefSeq_peptide Involved in cell growth regulation. May be involved in the regulation of mitogenic signals and control of cell proliferation. Involved in the internalization of ligandinducible receptors of the receptor tyrosine kinase (RTK) type, in particular EGFR Oxidoreductase, which may play a role in mediating a TP53/p53-dependent apoptosis response. Probable oxidoreductase that acts as a caspaseindependent mitochondrial effector of apoptotic cell death. Binds to DNA in a sequence-independent manner. May contribute to genotoxin-induced growth arrest May act in regulation of cell growth and transformation by interacting with nonreceptor tyrosine kinases ABL1 and/or ABL2. May be involved in cytoskeletal reorganization. Regulates ABL1/c-Abl-mediated phosphorylation of MENA Mono-ADP-ribosyltransferase sirtuin-6; SIR2-like protein 6 Protein synthesis factor, GTP-binding; Translation elongation factor EFG/EF2, C-terminal; Translation elongation factor EFG/EF2, domain IV; Translation elongation factor EFTu/EF1A, domain 2 6.8 kDa mitochondrial proteolipid Calponin-like actin-binding; Leucine-rich repeat Controls reversibly actin polymerization and depolymerization in a pH-sensitive manner. It has the ability to bind G- and F-actin in a 1:1 ratio of cofilin to actin. It is the major component of intranuclear and cytoplasmic actin rods UDPGT is of major importance in the conjugation and subsequent elimination of potentially toxic xenobiotics and endogenous compounds. This isoform is active on odorants and seems to be involved in olfaction; it could help clear lipophilic odorant molecules from the sensory epithelium Acc:NP_997208]; dpy-19-like 3 [Source:RefSeq_peptide Pistil-specific extensin-like protein Otoraplin precursor; Fibrocyte-derived protein; Melanoma inhibitory activity-like protein May be involved in the endocytic machinery; May function as a testis specific transcription factor. Binds DNA in conjunction with GTF2A2 and TBP (the TATA-binding protein) and together with GTF2A2, allows mRNA transcription Leucine-rich repeat-containing protein 17 precursor; p37NB  Fold Change -2.06  p Value 0.0162  -2.07  0.0064  -2.07  0.0417  -2.07  0.0417  -2.07  0.0057  -2.07  0.0415  -2.07 -2.07 -2.07  0.0412 0.0412 0.0076  -2.07  0.0411  -2.07  0.0408  -2.08 -2.08  0.0408 0.0406  -2.08  0.0372  -2.08  0.0403  148  Gene Name 7B2  SORT  EGFR  FABD  MASP2  NP_055451.1 PAX9  UT14C  Gene Description Acts as a molecular chaperone for PCSK2/PC2, preventing its premature activation in the regulated secretory pathway. Binds to inactive PCSK2 in the endoplasmic reticulum and facilitates its transport from there to later compartments of the secretory pathway where it is proteolytically matured and activated. Also required for cleavage of PCSK2 but does not appear to be involved in its folding. Plays a role in regulating pituitary hormone secretion. The Cterminal peptide inhibits PCSK2 in vitro Functions as a sorting receptor in the Golgi compartment and as a clearance receptor on the cell surface. Required for protein transport from the Golgi apparatus to the lysosomes by a pathway that is independent of the mannose-6-phosphate receptor (M6PR). Also required for protein transport from the Golgi apparatus to the endosomes. Promotes neuronal apoptosis by mediating endocytosis of the proapoptotic precursor forms of BDNF (proBDNF) and NGFB (proNGFB). Also acts as a receptor for neurotensin. May promote mineralization of the extracellular matrix during osteogenic differentiation by scavenging extracellular LPL. Probably required in adipocytes for the formation of specialized storage vesicles containing the glucose transporter SLC2A4/GLUT4 (GLUT4 storage vesicles, or GSVs). These vesicles provide a stable pool of SLC2A4 and confer increased responsiveness to insulin. May also mediate transport from the endoplasmic reticulum to the Golgi Receptor for EGF, but also for other members of the EGF family, as TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30 and vaccinia virus growth factor. Is involved in the control of cell growth and differentiation Catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of the mitochondrial ACP protein (NDUFAB1). This suggests the existence of the biosynthesis of fatty acids in mitochondrias Serum protease that plays an important role in the activation of the complement system via mannosebinding lectin. After activation by auto-catalytic cleavage it cleaves C2 and C4, leading to their activation and to the formation of C3 convertase Guanine-nucleotide dissociation stimulator CDC25; Pleckstrin-like Transcription factor required for normal development of thymus, parathyroid glands, ultimobranchial bodies, teeth, skeletal elements of skull and larynx as well as distal limbs Essential for spermatogenesis. May be required specifically for ribosome biogenesis and hence protein synthesis during male meiosis  Fold Change -2.08  p Value 0.0402  -2.08  0.0401  -2.08  0.0399  -2.09  0.0396  -2.09  0.0395  -2.09  0.0394  -2.09  0.0083  -2.09  0.0367  149  Gene Name H2B2E  QSK MALT1  CASB NP_061959.2 BBC3 Q13862 DDX23 NP_065976.2 INGR1 DYN1  COPE  MK  ARRD3 Q5T5W6 ORML3  Gene Description Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of posttranslational modifications of histones, also called histone code, and nucleosome remodeling Serine/threonine-protein kinase QSK Enhances BCL10-induced activation of NF-kappa-B. Involved in nuclear export of BCL10. Binds to TRAF6, inducing TRAF6 oligomerization and activation of its ligase activity. Has ubiquitin ligase activity Important role in determination of the surface properties of the casein micelles Immunoglobulin; Immunoglobulin I-set; Immunoglobulin V-set; Immunoglobulin-like Essential mediator of p53-dependent and p53independent apoptosis Acc:Q13862]; DNA-binding protein (Hypothetical protein SPBPBP). [Source:Uniprot/SPTREMBL Probably involved in pre-mRNA splicing Cache; VWA N-terminal; von Willebrand factor, type A Receptor for interferon gamma. Two receptors bind one interferon gamma dimer Microtubule-associated force-producing protein involved in producing microtubule bundles and able to bind and hydrolyze GTP. Most probably involved in vesicular trafficking processes, in particular endocytosis The coatomer is a cytosolic protein complex that binds to dilysine motifs and reversibly associates with Golgi non-clathrin-coated vesicles, which further mediate biosynthetic protein transport from the ER, via the Golgi up to the trans Golgi network. Coatomer complex is required for budding from Golgi membranes, and is essential for the retrograde Golgi-to-ER transport of dilysine-tagged proteins. In mammals, the coatomer can only be recruited by membranes associated to ADP-ribosylation factors (ARFs), which are small GTP-binding proteins; the complex also influences the Golgi structural integrity, as well as the processing, activity, and endocytic recycling of LDL receptors Has heparin binding activity, and growth promoting activity. Involved in neointima formation after arterial injury, possibly by mediating leukocyte recruitment. Also involved in early fetal adrenal gland development Arrestin domain-containing protein 3 Myb, DNA-binding ORM1-like protein 3  Fold Change -2.10  p Value 0.039  -2.10 -2.10  0.0388 0.0159  -2.10  0.0386  -2.10  0.0385  -2.10  0.0089  -2.10  0.0382  -2.10 -2.11  0.0228 0.0376  -2.11  0.0065  -2.11  0.0047  -2.12  0.0371  -2.12  0.0046  -2.12 -2.12 -2.12  0.0029 0.037 0.0025  150  Gene Name ARL3 CP39A UBR1  FAT3  OAT4 Q5VZI7  AGGF1  ENSG00000204124 NP_071375.1 DNAL4 MLPH F262 PLMN  Gene Description Does not act as an allosteric activator of the cholera toxin catalytic subunit Involved in the bile acid metabolism. Has a preference for 24-hydroxycholesterol, and converts it into a 7-alpha-hydroxylated product E3 ubiquitin-protein ligase which is a component of the N-end rule pathway. Recognizes and binds to proteins bearing specific amino-terminal residues that are destabilizing according to the N-end rule, leading to their ubiquitination and subsequent degradation. May be involved in pancreatic homeostasis Aspartic acid and asparagine hydroxylation site; Cadherin; EGF-like; EGF-like calcium-binding; EGFlike region; EGF-like, laminin; EGF-like, type 2; EGFlike, type 3; Laminin G; Laminin G, subdomain 1; Laminin G, subdomain 2; Proline-rich region Mediates saturable uptake of estrone sulfate, dehydroepiandrosterone sulfate and related compounds Cooperates with LY96 and CD14 to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MyD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response Promotes angiogenesis and the proliferation of endothelial cells. Able to bind to endothelial cells and promote cell proliferation, suggesting that it may act in an autocrine fashion Doublecortin AAA ATPase, core; ATPase associated with various cellular activities, AAA-5; Proline-rich region Force generating protein of respiratory cilia. Produces force towards the minus ends of microtubules. Dynein has ATPase activity Rab effector protein involved in melanosome transport. Serves as link between melanosomebound RAB27A and the motor protein MYO5A Synthesis and degradation of fructose 2,6bisphosphate Plasmin dissolves the fibrin of blood clots and acts as a proteolytic factor in a variety of other processes including embryonic development, tissue remodeling, tumor invasion, and inflammation; in ovulation it weakens the walls of the Graafian follicle. It activates the urokinase-type plasminogen activator, collagenases and several complement zymogens, such as C1 and C5. It cleaves fibrin, fibronectin, thrombospondin, laminin and von Willebrand factor. Its role in tissue remodeling and tumor invasion may be modulated by CSPG4  Fold Change -2.12  p Value 0.0365  -2.12  0.0365  -2.12  0.0365  -2.12  0.0364  -2.13  0.0002  -2.13  0.0359  -2.13  0.0358  -2.13 -2.14  0.0355 0.0204  -2.14  0.0351  -2.15  0.0344  -2.15  0.0143  -2.16  0.0333  151  Gene Name RIP  NP_478126.1 GCYA2  GNAO1  PADI2 PREP  NRK2  ETV6 PRMT7 IF MAGBI TTC5 TRAIP NP_001070249.1 NBEA  Gene Description Mediates the import of RPA complex into the nucleus, possibly via some interaction with importin beta. Isoform 2 is sumoylated and mediates the localization of RPA complex into the PML body of the nucleus, thereby participating in RPA function in DNA metabolism Exoribonuclease Has guanylyl cyclase on binding to the beta-1 subunit. The alternatively spliced isoform alpha-2-I acts as a negative regulator of guanylyl cyclase activity as it forms non-functional heterodimers with the beta subunits Guanine nucleotide-binding proteins (G proteins) are involved as modulators or transducers in various transmembrane signaling systems. The G(o) protein function is not clear Catalyzes the deimination of arginine residues of proteins ATP-independent protease that degrades mitochondrial transit peptides after their cleavage. Also degrades other unstructured peptides. Specific for peptides in the range of 10 to 65 residues. Able to degrade amyloid beta A4 (APP) protein when it accumulates in mitochondrion, suggesting a link with Alzheimer disease. Shows a preference for cleavage after small polar residues and before basic residues, but without any positional preference Reduces laminin matrix deposition and cell adhesion to laminin, but not to fibronectin. Involved in the regulation of PXN at the protein level and of PXN tyrosine phosphorylation. May play a role in the regulation of terminal myogenesis. Catalyzes the synthesis of nicotinamide nucleotide (NMN) from nicotinamide riboside Transcriptional repressor; binds to the DNA sequence 5'-CCGGAAGT-3' Probably methylates the guanidino nitrogens of arginyl residues in some proteins Promotes absorption of the essential vitamin cobalamin (Cbl) in the ileum by specific receptormediated endocytosis Melanoma-associated antigen B18; MAGE-B18 antigen Tetratricopeptide repeat protein 5; TPR repeat protein 5 Inhibits activation of NF-kappa-B mediated by TNF Zinc finger, C2H2-subtype; Zinc finger, C2H2-type Binds to type II regulatory subunits of protein kinase A and anchors/targets them to the membrane. May anchor the kinase to cytoskeletal and/or organelleassociated proteins  Fold Change -2.17  p Value 0.0328  -2.17 -2.17  0.0265 0.0325  -2.17  0.0017  -2.19  0.0313  -2.19  0.0313  -2.19  0.0312  -2.19  0.0312  -2.20  0.0309  -2.20  0.0004  -2.20  0.0005  -2.20  0.0306  -2.20 -2.21 -2.21  0.0304 0.0301 0.03  152  Gene Name Q7Z5G2  NP_116107.2 Q53RC4  ZN343 WBS14 IPP P04278-2  Q8N157-3 RB33A FPRL1  NP_055873.1 Q7Z5Q7 SEC62 LMA2L KCD17  Gene Description Serine/threonine protein kinase involved in both mRNA surveillance and genotoxic stress response pathways. Recognizes the substrate consensus sequence [ST]-Q. Involved in nonsense-mediated decay (NMD) of mRNAs containing premature stop codons by phosphorylating UPF1/RENT1. Also acts as a genotoxic stress-activated protein kinase that displays some functional overlap with ATM. Can phosphorylate TP53/p53 and is required for optimal TP53/p53 activation after cellular exposure to genotoxic stress. Its depletion leads to spontaneous DNA damage and increased sensitivity to ionizing radiation (IR). May activate PRKCI but not PRKCZ Major facilitator superfamily; Major facilitator superfamily MFS_1; Sugar transporter superfamily; Tetracycline resistance protein Fodrin, which seems to be involved in secretion, interacts with calmodulin in a calcium-dependent manner and is thus candidate for the calciumdependent movement of the cytoskeleton at the membrane May be involved in transcriptional regulation Transcriptional repressor. Binds to the canonical and non-canonical E box sequences 5'-CACGTG-3' May play a role in organizing the actin cytoskeleton Functions as an androgen transport protein, but may also be involved in receptor mediated processes. Each dimer binds one molecule of steroid. Specific for 5-alpha-dihydrotestosterone, testosterone, and 17-beta-estradiol. Regulates the plasma metabolic clearance rate of steroid hormones by controlling their plasma concentration G-protein, beta subunit; Src homology-3; Variant SH3; WD-40 repeat Ras-related protein Rab-33A; Small GTP-binding protein S10 Low affinity receptor to N-formyl-methionyl peptides, which are powerful neutrophils chemotactic factors. Binding of FMLP to the receptor causes activation of neutrophils. This response is mediated via a Gprotein that activates a phosphatidylinositol-calcium second messenger system. The activation of LXA4R could result in an anti-inflammatory outcome counteracting the actions of proinflammatory signals such as LTB4 (leukotriene B4) ATPase associated with various cellular activities, AAA-5; von Willebrand factor, type A Acc:Q7Z5Q7]; Lung cancer oncogene 5. [Source:Uniprot/SPTREMBL Required for preprotein translocation May be involved in the regulation of export from the endoplasmic reticulum of a subset of glycoproteins. May function as a regulator of ERGIC-53 BTB/POZ domain-containing protein KCTD17  Fold Change -2.21  p Value 0.0299  -2.22  0.0296  -2.22  0.0161  -2.22 -2.22  0.0295 0.0292  -2.22 -2.23  0.0292 0.029  -2.23  0.0048  -2.23  0.0287  -2.23  0.0287  -2.24  0.003  -2.24  0.0283  -2.24 -2.24  0.0051 0.0042  -2.24  0.0116  153  Gene Name GALA  Q9Y4E5-2 ZN460 REXO1 YETS2 CAN5 NP_940905.2  C8AP2  CAN13 MGST3 RHES  PHF6 NAPSA NP_057593.2 TM107 ITF2  NP_065805.1 ARP8  Gene Description Contracts smooth muscle of the gastrointestinal and genitourinary tract, regulates growth hormone release, modulates insulin release, and may be involved in the control of adrenal secretion May be involved in transcriptional regulation. Coactivator for steroid receptors May be involved in transcriptional regulation Seems to have no detectable effect on transcription elongation in vitro YEATS domain-containing protein 2 Calpain-5; nCL-3; htra-3 Blood group Rhesus C/E and D polypeptide; Calcium-activated BK potassium channel, alpha subunit; EAG/ELK/ERG potassium channel; Ion transport 2 Participates in TNF-alpha-induced blockade of glucocorticoid receptor (GR) transactivation at the nuclear receptor coactivator level, upstream and independently of NF-kappa-B. Suppresses both NCOA2- and NCOA3-induced enhancement of GR transactivation. Involved in TNF-alpha-induced activation of NF-kappa-B via a TRAF2-dependent pathway. Acts as a downstream mediator for CASP8-induced activation of NF-kappa-B. Required for the activation of CASP8 in FAS-mediated apoptosis Probable non-lysosomal thiol-protease Also functions as a glutathione peroxidase Binds to GTP and possesses intrinsic GTPase activity. May play a role in mediating signal transduction (By similarity). May be involved in mediating the insulin secretory response to efaroxan May play a role in transcriptional regulation May be involved in processing of pneumocyte surfactant precursors C-type lectin; Type II antifreeze protein Transmembrane protein 107 Transcription factor that binds to the immunoglobulin enchancer Mu-E5/KE5-motif. Binds to the E-box present in the somatostatin receptor 2 initiator element (SSTR2-INR) to activate transcription (By similarity). Preferentially binds to either 5'ACANNTGT-3' or 5'-CCANNTGG-3' Proline-rich region; RhoGAP Actin-related protein 8  Fold Change -2.24  p Value 0.0281  -2.25  0.0277  -2.25 -2.26  0.0274 0.0273  -2.26 -2.26 -2.26  0.027 0.0269 0.0269  -2.26  0.0269  -2.26 -2.26 -2.28  0.0269 0.0268 0.0262  -2.28 -2.29  0.0053 0.0256  -2.29 -2.30 -2.30  0.0252 0.0251 0.025  -2.30 -2.30  0.0248 0.0248  154  Gene Name COA1  Gene Description Can act on substrates such as myelin basic protein and histone 2A on serine and threonine residues; Catalyzes the rate-limiting reaction in the biogenesis of long-chain fatty acids. Carries out three functions: biotin carboxyl carrier protein, biotin carboxylase and carboxyltransferase; Essential ion channel and serine/threonine-protein kinase. Crucial for magnesium homeostasis. Has an important role in epithelial magnesium transport and in the active magnesium absorption in the gut and kidney. Isoforms of the type M6-kinase lack the ion channel region; F-box-like protein involved in the recruitment of the ubiquitin/19S proteasome complex to nuclear receptor-regulated transcription units. Plays an essential role in transcription activation mediated by nuclear receptors. Probably acts as integral component of the N-Cor corepressor complex that mediates the recruitment of the 19S proteasome complex, leading to the subsequent proteosomal degradation of N-Cor complex, thereby allowing cofactor exchange, and transcription activation; May be involved in the synthesis of gangliosides GD1c, GT1a, GQ1b and GT3 from GD1a, GT1b, GM1b and GD3 respectively; RNA-binding protein implicated in the regulation of several post-transcriptional events. Involved in pre-mRNA alternative splicing, mRNA translation and stability. Mediates exon inclusion and/or exclusion in pre-mRNA that are subject to tissue-specific and developmentally regulated alternative splicing. Specifically activates exon 5 inclusion of cardiac isoforms of TNNT2 during heart remodeling at the juvenile to adult transition. Acts as both an activator and repressor of a pair of coregulated exons: promotes inclusion of the smooth muscle (SM) exon but exclusion of the non-muscle (NM) exon in actinin pre-mRNAs. Activates SM exon 5 inclusion by antagonizing the repressive effect of PTB. Promotes exclusion of exon 11 of the INSR pre-mRNA. Increases translation and controls the choice of translation initiation codon of CEBPB mRNA. Increases mRNA translation of CEBPB in aging liver (By similarity). Increases translation of CDKN1A mRNA by antagonizing the repressive effect of CALR3. Mediates rapid cytoplasmic mRNA deadenylation. Recruits the deadenylase PARN to the poly(A) tail of EDEN-containing mRNAs to promote their deadenylation. Required for completion of spermatogenesis (By similarity). Binds to (CUG)n triplet repeats in the 3'-UTR of transcripts such as DMPK and to Bruno response elements (BREs). Binds to muscle-specific splicing enhancer (MSE) intronic sites flanking the alternative exon 5 of TNNT2 pre-mRNA. Binds to AU-rich sequences (AREs or EDEN-like) localized in the 3'-UTR of JUN and FOS mRNAs. Binds to the 5'-region of CDKN1A and CEBPB mRNAs. Binds with the 5'-region of CEBPB mRNA in aging liver  Fold Change -2.30  p Value 0.0027  155  Gene Name K1C19 DEF4 CC28A ITCH  CCAR1  Q8TCQ1 KCNN3  NP_071746.1 NP_006171.2  ACHA2  GPR161 Q5TCU6  GO45 SFRIP NP_060340.2  Gene Description Involved in the organization of myofibers. Together with KRT8, helps to link the contractile apparatus to dystrophin at the costameres of striated muscle This peptide has antibiotic and anti-fungi activity Coiled-coil domain-containing protein 28A; CCRL1AP E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. Regulates the transcriptional activity of several transcription factors, and probably plays an important role in the regulation of immune response May be involved in apoptosis signaling in the presence of the reinoid CD437. Apoptosis induction involves sequestration of 14-3-3 protein(s) and mediated altered expression of multiple cell cycle regulatory genes including MYC, CCNB1 and CDKN1A. Plays a role in cell cycle progression and/or cell proliferation Zinc finger, RING-type Forms a voltage-independent potassium channel activated by intracellular calcium. Activation is followed by membrane hyperpolarization. Thought to regulate neuronal excitability by contributing to the slow component of synaptic afterhyperpolarization. The channel is blocked by apamin Antibiotic biosynthesis monooxygenase; Calciumbinding EF-hand Receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-3 and neurotrophin-4/5 but not nerve growth factor (NGF). Involved in the development and/or maintenance of the nervous system. This is a tyrosine-protein kinase receptor. Known substrates for the TRK receptors are SHC1, PI-3 kinase, and PLC-gamma-1 After binding acetylcholine, the AChR responds by an extensive change in conformation that affects all subunits and leads to opening of an ion-conducting channel across the plasma membrane Orphan receptor Probably involved in connections of major cytoskeletal structures to the plasma membrane. High molecular weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts Required for normal Golgi structure and for protein transport from the endoplasmic reticulum (ER) through the Golgi apparatus to the cell surface Plays a role in pre-mRNA alternative splicing by regulating spliceosome assembly Acc:NP_060340]; transmembrane protein 132A isoform a [Source:RefSeq_peptide  Fold Change -2.31  p Value 0.0013  -2.31 -2.32  0.0243 0.0241  -2.32  0.0241  -2.32  0.024  -2.32 -2.32  0.0136 0.0142  -2.32  0.0239  -2.33  0.0052  -2.33  0.0234  -2.33 -2.34  0.0234 0.0008  -2.34  0.0229  -2.35  0.0132  -2.35  0.0224  156  Gene Name STK4  SIGIR  NP_777577.1 STML1 CRLD2  NP_689704.3 TNF18  DCTN3 NP_542398.2 ZN179 PER1  NP_001073884.1 GLT12  Gene Description Stress-activated, pro-apoptotic kinase which, following caspase-cleavage, enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation. Phosphorylates 'Ser-14' of histone H2B during apoptosis. Phosphorylates FOXO3 upon oxidative stress, which results in its nuclear translocation and cell death initiation Acts as a negative regulator of the Toll-like and IL1R receptor signaling pathways. Attenuates the recruitment of receptor-proximal signaling components to the TLR4 receptor, probably through an TIR-TIR domain interaction with TLR4. Through its extracellular domain interferes with the heterodimerization of Il1R1 and IL1RAP AMP-dependent synthetase and ligase Stomatin-like protein 1; SLP-1; Stomatin-related protein; STORP; EPB72-like 1; UNC24 homolog Cysteine-rich secretory protein LCCL domaincontaining 2 precursor; LCCL domain-containing cysteine-rich secretory protein 2; Cysteine-rich secretory protein 11; CRISP-11 Peptidase M20; Peptidase M20, dimerisation Cytokine that binds to TNFRSF18/AITR/GITR. Important for interactions between activated Tlymphocytes and endothelial cells and may modulate T-lymphocyte survival in peripheral tissues Together with dynein may be involved in spindle assembly and cytokinesis Acc:NP_542398]; coiled-coil domain containing 104 [Source:RefSeq_peptide Zinc finger protein 179; Brain finger protein; RING finger protein 112 Component of the circadian clock mechanism which is essential for generating circadian rhythms. Negative element in the circadian transcriptional loop. Influences clock function by interacting with other circadian regulatory proteins and transporting them to the nucleus. Negatively regulates CLOCK|NPAS2-BMAL1|BMAL2-induced transactivation Lymphocyte-specific protein; Proline-rich region; RNA-binding region RNP-1 (RNA recognition motif); SWAP/Surp Catalyzes the initial reaction in O-linked oligosaccharide biosynthesis, the transfer of an Nacetyl-D-galactosamine residue to a serine or threonine residue on the protein receptor. Has activity toward non-glycosylated peptides such as Muc5AC, Muc1a and EA2, and no detectable activity with Muc2 and Muc7. Displays enzymatic activity toward the Gal-NAc-Muc5AC glycopeptide, but no detectable activity to mono-GalNAc-glycosylated Muc1a, Muc2, Muc7 and EA2. May play an important role in the initial step of mucin-type oligosaccharide biosynthesis in digestive organs  Fold Change -2.35  p Value 0.0029  -2.36  0.0219  -2.38 -2.38  0.0213 0.021  -2.40  0.0202  -2.40 -2.41  0.0012 0.0201  -2.41  0.0198  -2.42  0.0004  -2.42  0.0196  -2.43  0.0435  -2.43  0.0313  -2.44  0.0188  157  Gene Name SFXN5 NP_006429.1 SIP1 APBA2  SHOC2 MYL4 OPCML TCF3  MPDZ  Q5VTQ0 P66A NTCP2 CLC4E  GBRL1  NAPG  Gene Description Transports citrate. Potential iron transporter C-type lectin; Collagen triple helix repeat Transcriptional inhibitor that binds to DNA sequence 5'-CACCT-3' in different promoters. Represses transcription of E-cadherin Putative function in synaptic vesicle exocytosis by binding to STXBP1, an essential component of the synaptic vesicle exocytotic machinery. May modulate processing of the beta-amyloid precursor protein (APP) and hence formation of beta-APP Leucine-rich repeat protein SHOC-2; Ras-binding protein Sur-8 Regulatory light chain of myosin. Does not bind calcium Binds opioids in the presence of acidic lipids; probably involved in cell contact Heterodimers between TCF3 and tissue-specific basic helix-loop-helix (bHLH) proteins play major roles in determining tissue-specific cell fate during embryogenesis, like muscle or early B-cell differentiation. Dimers bind DNA on E-box motifs: 5'CANNTG-3'. Binds to the kappa-E2 site in the kappa immunoglobulin gene enhancer Interacts with HTR2C and provokes its clustering at the cell surface (By similarity). Member of the NMDAR signaling complex that may play a role in control of AMPAR potentiation and synaptic plasticity in excitatory synapses Peptidase S26A, signal peptidase I; Tetratricopeptide TPR_2; Tetratricopeptide region Transcriptional repressor Plays a critical role in the sodium-dependent reabsorption of bile acids from the lumen of the small intestine. Plays a key role in cholesterol metabolism May play a role in the response to inflammatory stimuli in peritoneal macrophages. May be involved in immune surveillance processes under transcriptional control of CEBPB Gamma-aminobutyric acid receptor-associated protein-like 1; GABA(A) receptor-associated proteinlike 1; Glandular epithelial cell protein 1; GEC-1; Early estrogen-regulated protein Required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus  Fold Change -2.44 -2.44 -2.45  p Value 0.0187 0.0187 0.0186  -2.45  0.0185  -2.45  0.0184  -2.46  0.018  -2.47  0.0179  -2.47  0.0179  -2.48  0.0175  -2.48  0.0175  -2.48 -2.49  0.0484 0.0171  -2.51  0.0165  -2.52  0.0003  -2.52  0.0164  158  Gene Name PACS2  Q6ZN28 LAT3  TLR5  NP_001001552.3 PRG3  RHBT2 Q6ZNV0 WDR55 DHX35 CDS1  Q6PJS5 TSN9 CLCN4  TMCC3 NP_060615.1 NP_115510.1  Gene Description Multifunctional sorting protein that controls the endoplasmic reticulum (ER)-mitochondria communication, including the apposition of mitochondria with the ER and ER homeostasis. In addition, in response to apoptic inducer, translocates BIB to mitochondria, which initiates a sequence of events including the formation of mitochondrial truncated BID, the release of cytochrome c, the activation of caspase-3 thereby causing cell death. May also involved in ion channel traficking, directing acidic cluster-containing ion channels to distict subcellular compartements Variant SH3 Sodium-independent, high affinity transport of large neutral amino acids. Has narrower substrate selectivity compared to SLC7A5 and SLC7A8 and mainly transports branched-chain amino acids and phenylalanine. Plays a role in the development of human prostate cancer, from prostatic intraepithelial neoplasia to invasive prostate cancer Participates in the innate immune response to microbial agents. Mediates detection of bacterial flagellins. Acts via MyD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response Lamino-associated polypeptide 2/emerin Possesses similar cytotoxic and cytostimulatory activities to PRG2/MBP. In vitro, stimulates neutrophil superoxide production and IL8 release, and histamine and leukotriene C4 release from basophils Rho-related BTB domain-containing protein 2; Deleted in breast cancer 2 gene protein; p83 Ribosomal protein L22/L17 WD repeat protein 55 May be involved in pre-mRNA splicing Provides CDP-diacylglycerol an important precursor for the synthesis of phosphatidylinositol (PtdIns), phosphatidylglycerol, and cardiolipin. Overexpression may amplify cellular signaling responses from cytokines. May also play an important role in the signal transduction mechanism of retina and neural cells Zinc finger, B-box; Zinc finger, RING-type Tetraspanin-9; Tspan-9; Tetraspan NET-5 Voltage-gated chloride channel. Chloride channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport Transmembrane and coiled-coil domains protein 3 Acc:NP_060615]; family with sequence similarity 82, member C [Source:RefSeq_peptide Acc:NP_115510]; glutamine rich 2 [Source:RefSeq_peptide  Fold Change -2.52  p Value 0.0162  -2.53 -2.54  0.0016 0.0157  -2.56  0.0153  -2.57 -2.57  0.0052 0.0149  -2.57  0.0149  -2.57 -2.58 -2.58 -2.58  0.0147 0.0147 0.0146 0.0145  -2.59 -2.60 -2.60  0.027 0.0163 0.0141  -2.60 -2.60  0.005 0.0039  -2.61  0.0018  159  Gene Name Q9NSG0-5 ANK3  CUL5  DOCK5 Q9NS68-2 ATP2C2 COG2 NP_659456.2 CT038  PRND NP_001074014.1 GBRG2  SMYD4 NUP54 PEVR2 CBX8  Gene Description GTPase activator for the Rho-type GTPases by converting them to an inactive GDP-bound state Membrane-cytoskeleton linker. The neural-specific isoforms may participate in the maintenance/targeting of ion channels and cell adhesion molecules at the nodes of Ranvier and axonal initial segments Component of E3 ubiquitin ligase complexes, which mediate the ubiquitination and subsequent proteasomal degradation of target proteins. Seems to be involved poteosomal degradation of p53/TP53 stimulated by adenovirus E1B-55 kDa protein. May form a cell surface vasopressin receptor Protein phosphatase 2A regulatory subunit PR55 Can mediate activation of JNK and NF-kappa-B. May promote caspase-independent cell death This magnesium-dependent enzyme catalyzes the hydrolysis of ATP coupled with the transport of calcium Required for normal Golgi morphology and function Acc:NM_145019]; family with sequence similarity 124A (FAM124A), mRNA [Source:RefSeq_dna Binds Gram-positive and Gram-negative bacteria; DNA primase is the polymerase that synthesizes small RNA primers for the Okazaki fragments made during discontinuous DNA replication; Receptor for both mineralocorticoids (MC) such as aldosterone and glucocorticoids (GC) such as corticosterone or cortisol. Binds to mineralocorticoid response elements (MRE) and transactivates target genes. The effect of MC is to increase ion and water transport and thus raise extracellular fluid volume and blood pressure and lower potassium levels Not known Protein phosphatase inhibitor, 1DARPP-32 GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel SET and MYND domain-containing protein 4 Component of the nuclear pore complex, a complex required for the trafficking across the nuclear membrane Acts as receptor for porcine endogenous retrovirus subgroup A (PERV-A) Component of the Polycomb group (PcG) multiprotein PRC1 complex, a complex required to maintain the transcriptionally repressive state of many genes, including Hox genes, throughout development. PcG PRC1 complex acts via chromatin remodeling and modification of histones; it mediates monoubiquitination of histone H2A 'Lys119', rendering chromatin heritably changed in its expressibility  Fold Change -2.62  p Value 0.0257  -2.63  0.0135  -2.63  0.0002  -2.64 -2.64  0.0132 0.0131  -2.65  0.0129  -2.65 -2.66  0.0493 0.0128  -2.66  0.0127  -2.67 -2.67 -2.67  0.0126 0.0018 0.0124  -2.67 -2.68  0.0124 0.0122  -2.69  0.012  -2.69  0.0068  160  Gene Name PTPRD NP_758438.1 TESK2  E2F3  SPG20 KU70  NEUROD2 NP_112196.2 TNR9 AIFM1  Gene Description Receptor-type tyrosine-protein phosphatase delta precursor; Protein-tyrosine phosphatase delta; RPTP-delta Transcription factor AP-2, C-terminal Dual specificity protein kinase activity catalyzing autophosphorylation and phosphorylation of exogenous substrates on both serine/threonine and tyrosine residues. Phosphorylates cofilin at 'Ser-3'. May play an important role in spermatogenesis Transcription activator that binds DNA cooperatively with DP proteins through the E2 recognition site, 5'TTTC[CG]CGC-3' found in the promoter region of a number of genes whose products are involved in cell cycle regulation or in DNA replication. The DRTF1/E2F complex functions in the control of cellcycle progression from G1 to S phase. E2F-3 binds specifically to RB1 protein, in a cell-cycle dependent manner May be implicated in endosomal trafficking, or microtubule dynamics, or both Single stranded DNA-dependent ATP-dependent helicase. Has a role in chromosome translocation. The DNA helicase II complex binds preferentially to fork-like ends of double-stranded DNA in a cell cycle-dependent manner. It works in the 3'-5' direction. Binding to DNA may be mediated by p70. Involved in DNA nonhomologous end joining (NHEJ) required for double-strand break repair and V(D)J recombination. The Ku p70/p86 dimer acts as regulatory subunit of the DNA-dependent protein kinase complex DNA-PK by increasing the affinity of the catalytic subunit PRKDC to DNA by 100-fold. The Ku p70/p86 dimer is probably involved in stabilizing broken DNA ends and bringing them together. The assembly of the DNA-PK complex to DNA ends is required for the NHEJ ligation step. Required for osteocalcin gene expression Appears to mediate neuronal differentiation N2,N2-dimethylguanosine tRNA methyltransferase; Zinc finger, C2H2-type Receptor for TNFSF14/4-1BBL. Possibly active during T cell activation Probable oxidoreductase that acts as a caspaseindependent mitochondrial effector of apoptotic cell death. Extramitochondrial AIF induces nuclear chromatin condensation and large scale DNA fragmentation (in vitro)  Fold Change -2.70  p Value 0.0118  -2.71 -2.71  0.0212 0.0116  -2.72  0.0116  -2.73  0.0114  -2.73  0.0114  -2.73 -2.74  0.0113 0.0112  -2.75  0.011  -2.75  0.011  161  

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