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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  ii 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.     iii 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  iv 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                                     v 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 post- stimulation with 3 µg/ml LL-37. .......................................................................................91  Supplementary Table 8: Differentially expressed genes 2 hours post- stimulation with 3 µg/ml LL-37. .....................................................................................108  Supplementary Table 9: Differentially expressed genes 4 hours post- stimulation with 3 µg/ml LL-37. .....................................................................................119                       vi 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   vii 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                                          viii 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 years- particularly, 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.                                 ix DEDICATION      This thesis is dedicated to my grandmother, Dr. A. A. Crowder, because I still know the difference between a grass and a sedge.                                           x 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.                                1  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  2 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  3 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  4 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 Nod- like 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.  5 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 amino- terminal kinase (JNK). In the epithelia, these responses are fundamentally important to the mobilization of innate immune defences and the activation of adaptive immunity (41- 43), 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  6 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 2- to 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.   7 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, IL- 18, 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 LL-  8 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, β- defensins 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  9 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  10 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  11 human skin (44). Furthermore, treatment of excised wounded keratinocytes with an anti- LL-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  12 primary keratinocytes and bronchial epithelial cells by exposing these cells to pro- inflammatory 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.         23 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.  24  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  25 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  26 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 pre- treatment 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.    27 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.   28 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. IL- 8 concentrations in supernatants were assessed by Enzyme Linked Immunosorbent Assay  29 (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 pro- inflammatory 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  30 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 SDS- PAGE 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 (Bio- Rad, 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 anti- mouse-IgG antibody (Amersham, Piscataway, NJ) were used at concentrations of 1:1000 and 1:5000, respectively.    31 RESULTS: LL-37 increased IL-8 production by subconfluent keratinocytes in response to pro- inflammatory 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 co- stimulated 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).  32  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.
  33  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.      34 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 pro- inflammatory 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.
  35  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.
  36 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 pro- inflammatory 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).  37  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.
  38  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.
   39  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  40 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.
  41  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.

  42  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.
 
  43 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).  44  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).  45  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  46 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 co- stimulation 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.   47 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  48 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-37- mediated 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 hyper- proliferative 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.   49 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.                 50 LITERATURE CITED: 1. 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. 2. 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. 3. 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. 4. Gudmundsson, G. H., B. Agerberth, J. 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Fukuishi, Y. Kubo, H. Yamanobe, K. Ohsaki, Y. Kawasoe, M. Murata, A. Ishizumi, Y. Nishii, N. Matsui, and M. Akagi. 2008. Human cathelicidin CAP18/LL-37 changes mast cell function toward innate immunity. Biological & pharmaceutical bulletin 31:212-216. 27. Niyonsaba, F., A. Someya, M. Hirata, H. Ogawa, and I. Nagaoka. 2001. Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D2 production from mast cells. European Journal of Immunology 31:1066-1075. 28. O'Neill, L. A. J., and A. G. Bowie. 2007. The family of five: TIR-domain- containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7:353-364. 29. O'Neill, L. A. 2006. How Toll-like receptors signal: what we know and what we don't know. Curr Opin Immunol 18:3-9. 30. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett, J. K. Eng, S. Akira, D. M. Underhill, and A. Aderem. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099-1103. 31. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, R. L. Modlin, and S. Akira. 2002. Cutting Edge: Role of Toll-Like Receptor 1 in Mediating Immune Response to Microbial Lipoproteins. J Immunol 169:10-14. 32. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-[kappa]B by Toll-like receptor 3. Nature 413:732-738. 33. 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. Hancock. 2006. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol 176:2455-2464. 34. 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 Gram- Negative Bacteria. Antimicrob. Agents Chemother. 50:1672-1679. 35. 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. 36. 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. 37. 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 347:1151- 1160. 38. 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. 39. 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 Toll-  53 like receptor family contribute to the innate immune response of human epidermal keratinocytes. Immunology 114:531-541. 40. Bikle, D. D., and S. Pillai. 1993. Vitamin D, calcium, and epidermal differentiation. Endocr Rev 14:3-19. 41. 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. 42. 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. 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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. 48. Dai, X., K. Yamasaki, Y. Shirakata, K. Sayama, and K. Hashimoto. 2004. All- Trans-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. 49. 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. 50. 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. 51. Buchau, A. S., and R. L. Gallo. 2007. Innate immunity and antimicrobial defense systems in psoriasis. Clinics in Dermatology 25:616-624.      54 CHAPTER III2 INTRODUCTION Although often considered as two separate processes, the inflammatory and wound- healing 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 pro- inflammatory 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
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Filewod,
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  55 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  56 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 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPR- TES) 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  57 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).   58 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).    59 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 LL- 37; 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 1- hour 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 Change p Value SEM4A Inhibits axonal extension by providing local signals to specify territories inaccessible for growing axons 4.70 0.0023 E2F2 Transcription activator that binds DNA cooperatively with DP proteins through the E2 recognition site. 4.33 0.0029 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 receptors for fibronectin. 3.44 0.0056 AKT2 General protein kinase capable of phosphorylating several known proteins 3.35 0.006 PLCB4 The production of the second messenger molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) is mediated by activated phosphatidylinositol-specific phospholipase C enzymes. 2.83 0.0106 ONEC2 Transcriptional activator. Activates the transcription of a number of liver genes such as HNF3B 2.77 0.0114 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 Cysteine protease required for autophagy. 2.53 0.0157 CLTR1 Receptor for cysteinyl leukotrienes mediating bronchoconstriction of individuals with and without asthma. -2.50 0.0166 BKRB2 Receptor for bradykinin.  -2.53 0.0157  60 Gene Name Gene Description Fold Change p Value 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 -2.66 0.0012 NP060218.1 Histone H5 -2.67 0.0129 PO2F3 Transcription factor that binds to the octamer motif (5'- ATTTGCAT-3'). -2.76 0.0115 NFASC Cell adhesion, ankyrin-binding protein which may be involved in neurite extension, axonal guidance, synaptogenesis, myelination and neuron-glial cell interactions. -2.80 0.0109 DLX5 Homeobox protein DLX-5 -2.87 0.01 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. -2.90 0.0097 CASR Senses changes in the extracellular concentration of calcium ions. -2.93 0.0094 TLE4 Transcriptional corepressor that binds to a number of transcription factors. -2.95 0.0091 CELR1 Receptor that may have an important role in cell/cell signaling during nervous system formation -3.25 0.0067 VNN2 Probable hydrolase. Involved in the thymus homing of bone marrow cells. May regulate beta-2 integrin-mediated cell adhesion, migration and motility of neutrophil -3.43 0.0056 PLOD2 Forms hydroxylysine residues in -Xaa-Lys-Gly- sequences in collagens. -3.73 0.0044 SL9A8 Involved in pH regulation. Plays an important role in signal transduction -3.75 0.0043 RTC1 Catalyzes the conversion of 3'-phosphate to a 2',3'-cyclic phosphodiester at the end of RNA. -3.81 0.0003 TIGD6 Tigger transposable element-derived protein 6 -3.89 0.0039 F261 Synthesis and degradation of fructose 2,6-bisphosphate -3.93 0.0002 K22E Probably contributes to terminal cornification. Associated with keratinocyte activation, proliferation and keratinization -4.52 0.0026 DB118 Has antibacterial activity  -4.56 0.0025 RNAS1 Endonuclease that catalyzes the cleavage of RNA on the 3' side of pyrimidine nucleotides. Acts on single stranded and double stranded RNA -4.90 0.0021 ZN134 May be involved in transcriptional regulation -5.67 0.0015 FGF2 The heparin-binding growth factors are angiogenic agents in vivo and are potent mitogens for a variety of cell types in vitro. -7.71 0.0008 ATRX Could be a global transcriptional regulator. -9.95 0.0005 ZHANG Strongly activates transcription when bound to HCFC1.  -12.16 0.0003  
 
   61 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 Change p Value TRI16 May play a role in the regulation of keratinocyte differentiation 7.67 0.004 MIF; MMP11 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. 6.36 0.0054 FOS Nuclear phosphoprotein which forms a tight but non-covalently linked complex with the JUN/AP-1 transcription factor. 4.57 0.0099 TIE2 This protein is a protein tyrosine-kinase transmembrane receptor for angiopoietin 1. 4.35 0.011 GBG8 Guanine nucleotide-binding proteins (G proteins) are involved as a modulator or transducer in various transmembrane signaling systems. 4.24 0.0116 RT12 28S ribosomal protein S12, mitochondrial precursor; S12mt; MRP-S12; MT-RPS12 4.20 0.0118 KS6A2 Serine/threonine kinase that may play a role in mediating the growth-factor and stress induced activation of the transcription factor CREB 3.92 0.0137 NPT2B May be involved in actively transporting phosphate into cells via Na(+) cotransport.  May have a role in the synthesis of surfactant in lungs' alveoli. 3.13 0.0162 ATE1 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. 3.06 0.0244 TRIO Promotes the exchange of GDP by GTP. Could play a role in coordinating cell-matrix and cytoskeletal rearrangements necessary for cell migration and cell growth. 3.06 0.0244 NP115589.2 Engulfment and cell motility. 3.05 0.0247 PPIL2 PPIases accelerate the folding of proteins. It catalyzes the cis- trans isomerization of proline imidic peptide bonds in oligopeptides 3.02 0.0252 NGLY1 Specifically deglycosylates the denatured form of N-linked glycoproteins in the cytoplasm and assists their proteasome- mediated degradation. 2.99 0.0258 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 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 2.53 0.0406 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 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. -2.64 0.0449 SIRPG Probable immunoglobulin-like cell surface receptor. On binding with CD47, mediates cell-cell adhesion. -2.70 0.0339  62 Gene Name Gene Description Fold Change p Value SG1D1 May bind androgens and other steroids, may also bind estramustine, a chemotherapeutic agent used for prostate cancer. -2.72 0.0332 SMURF2 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 -2.80 0.0307 DLG1 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 -2.87 0.0287 RNF34 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 -2.89 0.0283 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 -3.36 0.0067 LAIR1 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. 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. -3.48 0.0178 PLOD2 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 -3.94 0.0135 CELR3 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 -5.25 0.0446 CX04A May have an important role of cell protection in inflammation reaction -5.66 0.0067 BPAEA 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 -6.43 0.0053 ENSG00- 000117114 Brain-specific angiogenesis inhibitor. -6.58 0.0051 RRP5 Involved in the biogenesis of rRNA. -6.63 0.0051  63 Gene Name Gene Description Fold Change p Value FGF2 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 -8.32 0.0011 MUC16 Thought to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces -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 Change p Value EGF EGF stimulates the growth of various epidermal and epithelial tissues in vivo and in vitro and of some fibroblasts in cell culture 7.03 0.0004 DUS9 Inactivates MAP kinases. Has a specificity for the ERK family; Required for the uptake of creatine in muscles and brain 5.99 0.0006 IFNA1 Produced by macrophages, IFN-alpha have antiviral activities. Interferon stimulates the production of two enzymes: a protein kinase and an oligoadenylate synthetase 5.52 0.0008 SMAD6 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. 5.07 0.001 INHBC 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 4.99 0.0011 CARD9 Activates NF-kappa-B via BCL10 4.60 0.0014 DACH2 Transcription factor that is involved in regulation of organogenesis. 3.63 0.0034 IGF1R This receptor binds insulin-like growth factor 1 (IGF1) with a high affinity and IGF2 with a lower affinity. 3.63 0.0035 SULF1 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 3.07 0.0068 KCC1D 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 2.99 0.0076  64 Gene Name Gene Description Fold Change p Value kinase MAPK3 (By similarity). In vitro, phosphorylates transcription factor CREM isoform Beta and probably CREB1 ZN174 Transcriptional repressor 2.99 0.0077 HXD11 Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis 2.97 0.0079 HMGB3 Binds preferentially single-stranded DNA and unwinds double stranded DNA 2.80 0.0433 ISCU Involved in the assembly or repair of the [Fe-S] clusters present in iron-sulfur proteins. Binds iron 2.79 0.0103 SIM2 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 2.73 0.0114 LECT1 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 2.68 0.0123 PCOC1 Binds to the C-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity 2.64 0.0132 HSPA1 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 2.60 0.0141 TGFB3 Involved in embryogenesis and cell differentiation 2.50 0.0433 CLC4E 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 -2.51 0.0165 TLR5 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. -2.56 0.0153 TNR19 Can mediate activation of JNK and NF-kappa-B. May promote caspase-independent cell death -2.64 0.0131 CBX8 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 -2.69 0.0068 E2F3 Transcription activator. -2.72 0.0116  65 Gene Name Gene Description Fold Change p Value KU70 Single stranded DNA-dependent ATP-dependent helicase.  -2.73 0.0114 NDRF Appears to mediate neuronal differentiation -2.73 0.0113 RABP2 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 -2.77 0.001 IKKB 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 -2.78 0.0104 DUSP4 Regulates mitogenic signal transduction by dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2 -2.82 1.71E- 05 SMAD7 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. -2.84 0.0095 DDFL1 Promotes cell proliferation -2.87 0.009 TCF21 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 -2.88 0.009 TENS1 May be involved in cell migration, cartilage development and in linking signal transduction pathways to the cytoskeleton -2.93 0.0083 PO3F2 Transcription factor. -2.94 0.0081 EPO Erythropoietin is the principal hormone involved in the regulation of erythrocyte differentiation and the maintenance of a physiological level of circulating erythrocyte mass -2.97 0.0124 AN32A 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 -2.97 0.0039 NFKB2 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. -3.02 0.0073 STYK1 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 -3.10 0.0065 CLD5 Plays a major role in tight junction-specific obliteration of the intercellular space -3.13 4.38E- 05  66 Gene Name Gene Description Fold Change p Value FOSL2N Fos-related antigen 2 -3.13 0.0063 PGM5 Component of adherens-type cell-cell and cell-matrix junctions. Lacks phosphoglucomutase activity -3.14 0.0062 CIDEA Activates apoptosis -3.24 0.0055 ZN703 May function as a transcriptional repressor -3.26 0.0053 SEM3D Induces the collapse and paralysis of neuronal growth cones. Could potentially act as repulsive cues toward specific neuronal populations. Binds to neuropilin -3.27 0.0053 LTBP3 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) -3.42 4.30E- 03 HS2ST Heparan sulfate 2-O-sulfotransferase -3.43 0.0043 TRAF1 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 -3.70 0.0032 DOCK4 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 -4.03 0.0023 PLOD2 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 -4.05 0.0021 DOK3 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 -4.07 0.0022 FAN 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 -4.81 0.0012 CAD12 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 -5.12 0.001 FGF2 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 -5.27 0.0008 CAN11 Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction -5.41 0.0008 PSD12 Acts as a regulatory subunit of the 26S proteasome which is involved in the ATP-dependent degradation of ubiquitinated proteins -6.44 0.0005 NP004849.2 HCO3- transporter, cytoplasmic; HCO3- transporter, eukaryote; HCO3-transporter, c-terminal; Na+/HCO3- transporter -6.60 0.0004 DEF Regulates the p53 pathway to control the expansion -6.98 0.0103  67 Gene Name Gene Description Fold Change p Value growth of digestive organs MFSD4 Major facilitator superfamily domain-containing protein 4 -7.32 0.0003 CDC6 Involved in the initiation of DNA replication. Also participates in checkpoint controls that ensure DNA replication is completed before mitosis is initiated -7.58 0.0003 PLAL1 Shows weak transcriptional activatory activity. Transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase-activating polypeptide -7.76 0.0079 EYA4 Thought to play a role in transcription regulation during organogenesis through its intrinsic protein phosphatase activity. -118.41 1.61E- 06  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.
  Transcription factor TF class Z-score Fisher score ATHB5 HOMEO-ZIP 6.091 2.36E-03 ELF5 ETS 3.067 2.03E-01 1 hour EMBP1 bZIP 6.34 2.47E-02  68  Transcription factor TF class Z-score Fisher score FOXD1 FORKHEAD 3.657 4.06E-02 Foxd3 FORKHEAD 3.994 9.94E-02 HMG-IY HMG 4.124 3.52E-02 MYC-MAX bHLH-ZIP 5.283 1.73E-02 NFYA CAAT-BOX 5.244 2.43E-02 NR3C1 NUCLEAR RECEPTOR 6.561 5.27E-02  TLX1-NFIC HOMEO/CAAT 3.419 7.94E-02  Athb-1 HOMEO-ZIP 11.91 8.51E-03 Broad-complex_1 ZN-FINGER, C2H2 11.33 3.74E-06 Broad-complex_3 ZN-FINGER, C2H2 12.12 4.42E-03 FOXD1 FORKHEAD 10.17 2.89E-03 FOXI1 FORKHEAD 10.94 2.58E-02 HMG-IY HMG 12.74 6.82E-03 Nkx2-5 HOMEO 12.76 5.47E-01 Prrx2 HOMEO 11.51 2.79E-01 SOX9 HMG 11.39 1.44E-03 2 hours SRY HMG 15.73 1.90E-01  Broad-complex_4 ZN-FINGER, C2H2 25.37 8.78E-06 Foxa2 FORKHEAD 15.98 2.53E-03 Foxd3 FORKHEAD 18.62 1.79E-03 FOXI1 FORKHEAD 21.85 2.39E-03 Hunchback ZN-FINGER, C2H2 21.07 3.89E-04 IRF1 TRP-CLUSTER 16.29 9.90E-04 Nkx2-5 HOMEO 19.1 4.70E-02 Prrx2 HOMEO 21.26 1.47E-03 Sox5 HMG 22.33 3.43E-06 4 hours SRY HMG 21.54 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 over- representation analysis (Table 3.5), which is intended to identify signalling pathways  69 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 p- value 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 up- regulated 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.
 Condition InnateDB Pathway ID Pathway Genes Up- regulated p- value p-Value, corrected for multiple testing LL37_4hr 1958 L13a-mediated translational silencing of Ceruloplasmin expression 6 0.001 0.158 LL37_4hr 1710 Processing of Intronless Pre- mRNAs 7 0.001 0.167 LL37_4hr 1376 Formation of the Editosome 7 0.003 0.168 LL37_4hr 1319 mRNA Editing: C to U Conversion 7 0.003 0.168 LL37_4hr 1373 Eukaryotic Translation Elongation 5 0.003 0.172 LL37_4hr 1248 Peptide chain elongation 5 0.003 0.172 LL37_4hr 1464 mRNA 3`-end processing 7 0.003 0.175 LL37_4hr 1535 SLBP Dependent Processing of Replication-Dependent Histone 7 0.001 0.178  70 Condition InnateDB Pathway ID Pathway Genes Up- regulated p- value p-Value, corrected for multiple testing Pre-mRNAs LL37_4hr 1630 Cap-dependent Translation Initiation 6 0.002 0.181 LL37_4hr 1923 Formation of a pool of free 40S subunits 6 0.002 0.181 LL37_4hr 1919 GTP hydrolysis and joining of the 60S ribosomal subunit 6 0.002 0.181 LL37_4hr 1527 Ribosomal scanning and start codon recognition 6 0.002 0.181 LL37_4hr 1251 Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S 6 0.002 0.183 LL37_4hr 1921 Viral mRNA Translation 5 0.002 0.184 LL37_4hr 1354 SLBP independent Processing of Histone Pre-mRNAs 7 0.001 0.193 LL37_4hr 1505 Transport of Mature mRNA Derived from an Intronless Transcript 7 0.001 0.198 LL37_4hr 1361 Transport of Mature mRNA derived from an Intron-Containing Transcript 7 0.002 0.201 LL37_4hr 1706 Eukaryotic Translation Termination 5 0.002 0.206 LL37_4hr 474 Ribosome 5 0.001 0.209 LL37_4hr 1655 Cleavage of Growing Transcript in the Termination Region 7 0.004 0.235 LL37_1hr 1980 Formation of PAPS 2 0 0.243 LL37_4hr 1611 Transport of the SLBP Dependant Mature mRNA 7 0.001 0.243 LL37_4hr 973 Skeletal muscle hypertrophy is regulated via akt-mtor pathway 3 0.005 0.278 LL37_4hr 1685 Translation initiation complex formation 4 0.005 0.282 LL37_4hr 1088 Noncanonical Wnt signaling pathway 3 0 0.296 LL37_4hr 544 Prostate cancer 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 over- represented pathways were visualized using Cerebral. The ‘prostate cancer’ pathway aroused my interest; although I am not interested in prostate biology, I hypothesized that  71 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.   72   73  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 post- stimulation, a variety of genes involved in the determination of tissue morphology were upregulated, as were genes having transcription co-activator activity. At two hours post- stimulation, 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. 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 Observed Expected Relative enrichment p Value microspike biogenesis 2 0.11 18.18 0.0051 filopodium formation 2 0.08 25 0.0028 mitotic recombination 2 0.08 25 0.0028 detection of chemical stimulus 2 0.13 15.38 0.0065 detection of calcium ion 2 0.1 20 0.0039 amine transport 5 0.65 7.69 0.0005 neutral amino acid transport 2 0.15 13.33 0.0097 monoamine transport 2 0.08 25 0.0028 morphogenesis of a branching structure 2 0.04 50 0.0006 1 ho ur  ti m ep oi nt  mammary gland development 2 0.04 50 0.0006  74 Biological Process Observed Expected Relative enrichment p Value tube morphogenesis 2 0.08 25 0.0028 gland development 2 0.11 18.18 0.0051 tube development 2 0.15 13.33 0.0097 embryonic pattern specification 2 0.15 13.33 0.0097 regulation of Ras GTPase activity 2 0.1 20 0.0039 regulation of Rho GTPase activity 2 0.1 20 0.0039 regulation of Cdc42 GTPase activity 2 0.1 20 0.0039  Molecular Function transcription coactivator activity 7 2.2 3.18 0.0066 phospholipase C activity 3 0.37 8.11 0.0059 phosphoinositide phospholipase C activity 3 0.33 9.09 0.0042 inositol or phosphatidylinositol phosphodiesterase activity 3 0.33 9.09 0.0042 amine transporter activity 5 0.73 6.85 0.0008 monoamine transporter activity 2 0.07 28.57 0.0018 calcium channel activity 4 0.76 5.26 0.0069 calcium-activated potassium channel activity 2 0.15 13.33 0.0096  Cellular Component ruffle 3 0.33 9.09 0.0040 integral to plasma membrane 31 16.75 1.85 0.0005 intrinsic to plasma membrane 31 16.89 1.84 0.0006 plasma membrane part 35 20.34 1.72 0.0009  plasma membrane 36 24.2 1.49 0.0085  Biological Process Observed Expected Relative enrichment p Value signal transduction 35 24.23 1.44 0.0097 intracellular signaling cascade 17 8.82 1.93 0.0062 small GTPase mediated signal transduction 9 2.63 3.42 0.0013 ARF protein signal transduction 2 0.13 15.38 0.0069 regulation of ARF protein signal transduction 2 0.13 15.38 0.0069 regulation of small GTPase mediated signal transduction 6 0.95 6.32 0.0004 regulation of Rho protein signal transduction 4 0.54 7.41 0.0021 Rho protein signal transduction 4 0.69 5.8 0.0051 regulation of signal transduction 8 2.57 3.11 0.0041 cell maturation 2 0.15 13.33 0.0096 neuron maturation 2 0.06 33.33 0.0013 neuron remodeling 2 0.02 100 0.0001 neurogenesis 4 0.69 5.8 0.0049 generation of neurons 4 0.68 5.88 0.0047 2 ho ur  ti m ep oi nt  neuron differentiation 4 0.63 6.35 0.0036  75 Biological Process Observed Expected Relative enrichment p Value neuron development 4 0.53 7.55 0.0019 retrograde vesicle-mediated transport\, Golgi to ER 2 0.11 18.18 0.0053 cytoskeleton organization and biogenesis 9 2.93 3.07 0.0026 actin filament-based process 6 1.31 4.58 0.0020 actin cytoskeleton organization and biogenesis 6 1.18 5.08 0.0012 actin filament organization 3 0.19 15.79 0.0008 protein amino acid acylation 2 0.15 13.33 0.0096 protein amino acid acetylation 2 0.13 15.38 0.0077 developmental maturation 2 0.15 13.33 0.0096  Molecular Function potassium channel regulator activity 2 0.12 16.67 0.0067 cytoskeletal adaptor activity 2 0.05 40 0.0011 GTPase regulator activity 7 2.13 3.29 0.0055 guanyl-nucleotide exchange factor activity 7 0.99 7.07 0.0001 ARF guanyl-nucleotide exchange factor activity 2 0.12 16.67 0.0067 Ras guanyl-nucleotide exchange factor activity 4 0.56 7.14 0.0024 Rho guanyl-nucleotide exchange factor activity 4 0.49 8.16 0.0014 small GTPase regulator activity 6 1.12 5.36 0.0009 channel or pore class transporter activity 8 2.77 2.89 0.0065 alpha-type channel activity 8 2.68 2.99 0.0054 ion channel activity 8 2.46 3.25 0.0032 anion channel activity 3 0.42 7.14 0.0087 chloride channel activity 3 0.37 8.11 0.0061 ion transporter activity 11 4.66 2.36 0.0068  Cellular Component cell projection 4 0.82 4.88 0.0093 lamellipodium 3 0.16 18.75 0.0005 leading edge 3 0.28 10.71 0.0026 basolateral plasma membrane 3 0.2 15 0.0010  cell junction 5 1.05 4.76 0.0039  Biological Process Observed Expected Relative enrichment p Value response to nutrient 3 0.39 7.69 0.0063 cell differentiation 24 13.83 1.74 0.0062 intracellular protein transport 15 7.43 2.02 0.0078 fructose 2,6-bisphosphate metabolism 2 0.1 20 0.0034 peptide hormone processing 2 0.1 20 0.0034 ion transport 28 16.53 1.69 0.0044 4 ho ur  ti m ep oi nt  anion transport 10 4.11 2.43 0.0084  76 Biological Process Observed Expected Relative enrichment p Value inorganic anion transport 9 3.38 2.66 0.0069 chloride transport 5 1.02 4.9 0.0034 localization 77 58.11 1.33 0.0042 establishment of localization 77 57.55 1.34 0.0032 innate immune response 6 1.8 3.33 0.0092  Molecular Function anion binding 5 1.23 4.07 0.0076 chloride ion binding 5 1.23 4.07 0.0076 protein homodimerization activity 7 2.14 3.27 0.0056 fructose-2,6-bisphosphate 2- phosphatase activity 2 0.14 14.29 0.0076 intramolecular transferase activity 3 0.39 7.69 0.0066 intramolecular transferase activity\, phosphotransferases 3 0.3 10 0.0030 6-phosphofructo-2-kinase activity 2 0.09 22.22 0.0031 phosphotransferase activity\, alcohol group as acceptor 26 15.47 1.68 0.0068 nitric-oxide synthase regulator activity 2 0.09 22.22 0.0031 receptor signaling protein activity 10 3.25 3.08 0.0016 receptor signaling protein serine/threonine kinase signaling protein activity 2 0.12 16.67 0.0051 transmembrane receptor protein serine/threonine kinase signaling protein activity 2 0.14 14.29 0.0076 transforming growth factor beta receptor\, cytoplasmic mediator activity 2 0.12 16.67 0.0051 transforming growth factor beta receptor\, inhibitory cytoplasmic mediator activity 2 0.05 40 0.0005 chloride channel activity 5 1.18 4.24 0.0064 voltage-gated chloride channel activity 3 0.44 6.82 0.0091 ion transporter activity 25 14.82 1.69 0.0075 anion transporter activity 10 2.76 3.62 0.0005 iodide transporter activity 2 0.05 40 0.0005  Cellular Component  No terms enriched  DISCUSSION: To date, only one microarray experiment has previously been performed on LL-37- stimulated 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  77 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  78 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 LL- 37 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.        79 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 33:3154- 3164. 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.  80 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.                                  81 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 LL- 37 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  82 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 pro- inflammatory 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  83 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 cell- surface 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  84 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 co- stimulated 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  85 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.  86  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.   87 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 Gram- Negative 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. 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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 LL- 37 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 347:1151- 1160. 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.  89 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 Healing- Promoting Activities of Human Cathelicidin LL-37. J Invest Dermatol 128:223- 236. 26. Vonk, M. J., P. S. Hiemstra, and J. J. Grote. 2008. An Antimicrobial Peptide Modulates Epithelial Responses to Bacterial Products. The Laryngoscope.  90 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.                              91 APPENDIX SUPPLEMENTARY TABLES:  Supplementary
Table
7:
Differentially
expressed
genes
1
hour
post­stimulation
with
3
µg/ml
 LL­37.

 Gene Name Gene Description Fold Change p Value SEM4A  Inhibits axonal extension by providing local signals to specify territories inaccessible for growing axons 4.70 0.0023 E2F2  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 cell- cycle progression from g1 to s phase. E2F-2 binds specifically to RB1 protein, in a cell-cycle dependent manner 4.33 0.0029 KCE1L  Potassium voltage-gated channel subfamily E member 1-like protein; AMME syndrome candidate gene 2 protein; AMMECR2 protein 3.94 0.0037 NP_073590.2 Acc:NP_073590]; S100P binding protein isoform a [Source:RefSeq_peptide 3.91 0.0038 CAD19  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 3.55 0.005 NP_659002.1  Antifreeze protein, type I; Pollen allergen Poa pIX/Phl pVI, C-terminal; Zinc finger, CCCH-type 3.48 0.0054 ITA4  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 3.44 0.0056 SCUB2  Signal peptide, CUB and EGF-like domain-containing protein 2 precursor; Protein CEGP1 3.36 0.006 AKT2   General protein kinase capable of phosphorylating several known proteins 3.35 0.006 ZN566  May be involved in transcriptional regulation 3.03 0.0203  92 Gene Name Gene Description Fold Change p Value KCMB1  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 3.00 0.0087 RNF40  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 2.95 0.0091 Q8NHH4 Pistil-specific extensin-like protein; Proline-rich region 2.95 0.0092 IRK15  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 2.86 0.0102 NP_000924.2  The production of the second messenger molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) is mediated by activated phosphatidylinositol- specific phospholipase C enzymes. This form has a role in retina signal transduction 2.83 0.0106 CASP  May be involved in intra-Golgi retrograde transport 2.78 0.0064 ZN214  May be involved in transcriptional regulation 2.78 0.0112 TTBK1  Serine/threonine kinase which is able to phosphorylate TAU on serine, threonine and tyrosine residues. Induces aggregation of TAU 2.77 0.0113 ONEC2  Transcriptional activator. Activates the transcription of a number of liver genes such as HNF3B 2.77 0.0114 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 K1199  May be involved in hearing 2.75 0.0024  93 Gene Name Gene Description Fold Change p Value TDGF1  Could play a role in the determination of the epiblastic cells that subsequently give rise to the mesoderm 2.71 0.0123 PARC   Cytoplasmic anchor protein in p53-associated protein complex. Regulates the subcellular localization of p53 and subsequent function 2.67 0.013 BSN  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 2.66 0.013 EST1A   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 nonsense- mediated mRNA decay. May function as endonuclease. Degrades single-stranded RNA (ssRNA), but not ssDNA or dsRNA 2.65 0.0132 NP_064614.2  PR domain-containing protein 11 2.61 0.0141 SRR  Catalyzes the synthesis of D-serine from L-serine 2.60 0.0142 O15420 Acc:O15420]; CAGH1 alternate open reading frame. [Source:Uniprot/SPTREMBL 2.57 0.0149 B3GN2  Catalyzes the initiation and elongation of poly-N- acetyllactosamine chains 2.54 0.0155 ATG4D  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 2.53 0.0157 TMPS5  Transmembrane protease, serine 5; Spinesin 2.52 0.016 NP_060105.3 SET 2.45 0.0179 S10A6  Protein S100-A6; S100 calcium-binding protein A6; Calcyclin; Prolactin receptor-associated protein; PRA; Growth factor-inducible protein 2A9; MLN 4 2.45 0.0237 RASN  Ras proteins bind GDP/GTP and possess intrinsic GTPase activity 2.45 0.0183 MAST1  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 2.43 0.0183  94 Gene Name Gene Description Fold Change p Value HEY1   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 2.43 0.0185 NPBW2  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 2.42 0.0187 NP_001018068.1  Basic helix-loop-helix dimerisation region bHLH 2.42 0.0187 SEL1L  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 2.41 0.019 TCRG1  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 TAT- dependent activation of the human immunodeficiency virus type 1 (HIV-1) promoter 2.40 0.0192 CCG4  Thought to stabilize the calcium channel in an inactivated (closed) state 2.39 0.0196 ZN598  Zinc finger protein 598 2.39 0.0097 ELN   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 2.37 0.0201 NEO1  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 2.36 0.0207 ADDB   Membrane-cytoskeleton-associated protein that promotes the assembly of the spectrin-actin network. Binds to calmodulin. Calmodulin binds preferentially to the beta subunit 2.35 0.0208 ACE2  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 2.35 0.0211 NM_014069.1 Proline-rich region 2.34 0.0214 ABHD9  Abhydrolase domain-containing protein 9 precursor 2.33 0.0217 ZN225  May be involved in transcriptional regulation 2.33 0.0217 NP_056368.1 Pleckstrin-like 2.33 0.0218  95 Gene Name Gene Description Fold Change p Value AICDA  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 2.31 0.0223 NP_079093.2 ADAM-TS Spacer 1 2.31 0.0224 DPOLQ  Could be involved in the repair of interstrand cross- links 2.31 0.0226 TLX3  T-cell leukemia homeobox protein 3; Homeobox protein Hox-11L2 2.30 0.0228 JAD1C  Histone demethylase that specifically demethylates 'Lys-4' of histone H3, thereby playing a central role in histone code. Does not demethylate histone H3 'Lys- 9', H3 'Lys-27', H3 'Lys-36', H3 'Lys-79' or H4 'Lys- 20'. Demethylates trimethylated and dimethylated but not monomethylated H3 'Lys-4'. Participates in transcriptional repression of neuronal genes by recruiting histone deacetylases and REST at neuron- restrictive silencer elements 2.29 0.0232 MKL2  Acts as a transcriptional coactivator of serum response factor (SRF). Required for skeletal myogenic differentiation 2.29 0.0233 NP_060478.2 Zinc finger, C2H2-subtype; Zinc finger, C2H2-type 2.29 0.0233 CRSP3   Plays a role in transcriptional coactivation 2.27 0.0149 Q96MH6-2 Acc:NP_689630]; transmembrane protein 68 [Source:RefSeq_peptide 2.26 0.032 ASB3  Ankyrin repeat and SOCS box protein 3; ASB-3 2.26 0.0245 ARNT   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 2.26 0.0033 TMG2  Transmembrane gamma-carboxyglutamic acid protein 2 precursor; Proline-rich Gla protein 2; Proline-rich gamma-carboxyglutamic acid protein 2 2.25 0.0248 Q8IYI7   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 2.25 0.0251 GSTT1  Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles. Acts on 1,2-epoxy-3-(4- nitrophenoxy)propane, phenethylisothiocyanate 4- nitrobenzyl chloride and 4-nitrophenethyl bromide. Displays glutathione peroxidase activity with cumene hydroperoxide 2.23 0.0258 ENSG00000211744 Immunoglobulin V-set; Immunoglobulin-like 2.23 0.0259  96 Gene Name Gene Description Fold Change p Value NDN 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 2.23 0.003 SLC14A1  Specialized low-affinity urea transporter. Mediates urea transport in erythrocytes 2.22 0.0265 SGCD 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 2.20 0.0273 NP_940905.2  Blood group Rhesus C/E and D polypeptide; Calcium- activated BK potassium channel, alpha subunit; EAG/ELK/ERG potassium channel; Ion transport 2 2.20 0.0275 NP_001546.2  Immunoglobulin; Immunoglobulin V-set; Immunoglobulin-like; Interleukin-1 receptor, type I and type II; Vascular endothelial growth factor receptor, VEGFR, N-terminal 2.19 0.028 Q6NUT1 Eukaryotic translation initiation factor 4E (eIF-4E) 2.18 0.0282 YTDC2  YTH domain-containing protein 2 2.18 0.0285 LIPG  Gastric triacylglycerol lipase precursor; Gastric lipase; GL 2.18 0.0285 KCNB1 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 2.17 0.0083 Q5VYN8  Regulator of G protein signalling 2.17 0.0289 GLCI1  Glucocorticoid-induced transcript 1 protein 2.17 0.029 FGD2   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 2.16 0.0297 ST5  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 2.15 0.03 GLI4   Zinc finger protein GLI4; Krueppel-related zinc finger protein 4; Protein HKR4 2.15 0.0302 PHF20  Possible transcription factor 2.15 0.0303 HNRPM  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 2.14 0.0309  97 Gene Name Gene Description Fold Change p Value Q6ZS65  Acc:NP_001009909]; leucine zipper protein 2 [Source:RefSeq_peptide 2.14 0.0309 Q96MM7-2 Heparan sulphate 6-sulfotransferase 2.13 0.031 VPP4  Part of the proton channel of the V-ATPase that is involved in normal vectorial acid transport into the urine by the kidney 2.11 0.0129 CCNF  Likely to be involved in the control of the cell cycle during S phase and G2 2.11 0.0325 ZP3  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 2.11 0.0328 KREM1  Receptor for Dickkopf protein. Cooperates with Dickkopf to block Wnt/beta-catenin signaling 2.09 0.0342 EXOC5   Component of the exocyst complex involved in the docking of exocystic vesicles with fusion sites on the plasma membrane 2.08 0.0101 Q5VZE3  Acc:Q8NBJ4]; Golgi phosphoprotein 2 (Golgi membrane protein GP73). [Source:Uniprot/SWISSPROT 2.07 0.0351 SMAD6  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 2.07 0.0355 RAD50  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 2.06 0.0112 TAAR8  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 2.06 0.0349  98 Gene Name Gene Description Fold Change p Value FKB1B   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 2.06 0.0364 SMC2  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 2.06 0.0364 CTBP2  Corepressor targeting diverse transcription regulators 2.05 0.037 RAP2B  Ras-related protein Rap-2b precursor 2.05 0.0371 NP_061889.1 Eggshell protein; Intermediate filament protein; Keratin, type I 2.04 0.0375 F104B  Protein FAM104B 2.04 0.0377 Q9Y2I9  Peptidase M14, carboxypeptidase A; RabGAP/TBC 2.03 0.0384 PI2R  Receptor for prostacyclin (prostaglandin I2 or PGI2). The activity of this receptor is mediated by G(s) proteins which activate adenylate cyclase 2.03 0.0387 Q9BTA9-2  WW domain-containing adapter protein with coiled- coil 2.00 0.0055 PSYR  Receptor for the glycosphingolipid psychosine (PSY) and several related glycosphingolipids. May have a role in activation-induced cell death or differentiation of T-cells 1.99 0.0416 ASPX  Acrosomal protein SP-10 precursor; Acrosomal vesicle protein 1 1.99 0.0417 VMAT1  Involved in the vesicular transport of biogenic amines 1.99 0.0423 NP_001010984.1  Peptidase M, neutral zinc metallopeptidases, zinc- binding site 1.98 0.0425 EPDR1  Mammalian ependymin-related protein 1 precursor; MERP-1; UCC1 protein 1.98 0.0483 IFT74  Intraflagellar transport 74 homolog; Coiled-coil domain-containing protein 2; Capillary morphogenesis protein 1; CMG-1 1.98 0.0098 RAE1L  Binds mRNA. May function in nucleocytoplasmic transport and in directly or indirectly attaching cytoplasmic mRNPs to the cytoskeleton 1.97 0.0033 CCDC13 Coiled-coil domain-containing protein 13 1.96 0.045 GRP75  Implicated in the control of cell proliferation and cellular aging. May also act as a chaperone 1.95 0.0454 ZN434  May be involved in transcriptional regulation 1.95 0.0454 FRGL  Putative FRG1-like protein C20orf80 1.95 0.0455  99 Gene Name Gene Description Fold Change p Value ENP1   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 1.95 0.0456 TRI65  Tripartite motif-containing protein 65 1.95 0.0462 EST1A  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 nonsense- mediated mRNA decay. May function as endonuclease. Degrades single-stranded RNA (ssRNA), but not ssDNA or dsRNA 1.94 0.0465 PELI1  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 1.94 0.0469 PSN2  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 (beta- amyloid 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 1.93 0.0479 PIGZ  Mannosyltransferase involved in glycosylphosphatidylinositol-anchor biosynthesis. Transfers a fourth mannose to some trimannosyl- GPIs 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 1.93 0.0217 NP_653217.1 Proline-rich region 1.93 0.0274 USP6  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 1.92 0.0487 SYNP2  Has an actin-binding and actin-bundling activity 1.92 0.0489 PP1RA  Inhibitor of PPP1CA and PPP1CC phosphatase activities. Has inhibitory activity on PPP1CA only when phosphorylated. Binds to mRNA, single- stranded DNA (ssDNA), poly(A) and poly(G) homopolymers 1.92 0.049 NP_002411.3 Ion transport 1.91 0.0191 NP_056993.2 Ribosomal protein S14 1.90 0.0087 DNPEP  Likely to play an important role in intracellular protein and peptide metabolism 1.90 0.0086  100 Gene Name Gene Description Fold Change p Value BCAT2  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 1.90 0.0316 H2B2E  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 post- translational modifications of histones, also called histone code, and nucleosome remodeling 1.90 0.0215 TBCA  Tubulin-folding protein; involved in the early step of the tubulin folding pathway 1.89 0.0084 Q8NBT7  Acc:Q8NBT7]; CDNA FLJ90757 fis, clone SKNMC1000014 (FLJ90757 protein). [Source:Uniprot/SPTREMBL -1.92 0.0099 S12A5  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 -1.93 0.0486 ITBA1  Transmembrane protein 187; Protein ITBA1 -1.93 0.0228 NP_060170.1 DH; cAMP/cGMP-dependent protein kinase -1.94 0.0471 CA021  Uncharacterized protein C1orf21; Cell proliferation- inducing gene 13 protein -1.94 0.0471 PTN18  Differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues -1.94 0.0469 CG034  Uncharacterized protein C7orf34 precursor; MSSP- binding protein CTM-1 -1.94 0.0203 MKRN3  Makorin-3; Zinc finger protein 127; RING finger protein 63 -1.94 0.0464 OCRL  Converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. Also converts inositol 1,4,5-trisphosphate to inositol 1,4- bisphosphate 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 -1.94 0.0414 IBP5  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 -1.95 0.046 GATA1  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 -1.95 0.043 DUSP4  Regulates mitogenic signal transduction by dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2 -1.95 0.0392  101 Gene Name Gene Description Fold Change p Value GNS  N-acetylglucosamine-6-sulfatase precursor; G6S; Glucosamine-6-sulfatase -1.95 0.0457 GRIK2   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-cyano- 7-nitroquinoxaline-2,3-dione &gt; L-glutamate = 6,7- dinitroquinoxaline-2,3-dione &gt; dihydrokainate -1.96 0.0452 ENSG00000171914 Adenosine/AMP deaminase active site; Band 4.1; I/LWEQ -1.96 0.0236 M3K8  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 -1.97 0.0434 NP_006537.3 KH, type 1; RNA-binding region RNP-1 (RNA recognition motif) -1.98 0.043 ALKB1  Alkylated DNA repair protein alkB homolog 1 -1.98 0.0377 Q86V25-2  Angiogenesis inhibitor. Inhibits network formation by endothelial cells -1.99 0.0422 ENSG00000132463 RNA-binding region RNP-1 (RNA recognition motif) -1.99 0.013 O75800-2  Zinc finger MYND domain-containing protein 10; BLu protein -1.99 0.0417 NP_659453.2 Aralkyl acyl-CoA:amino acid N-acyltransferase; Aralkyl acyl-CoA:amino acid N-acyltransferase, C- terminal -2.00 0.0413 GPSM3   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 -2.00 0.0412 Q6P578   Tetratricopeptide repeat protein 3; TPR repeat protein 3; TPR repeat protein D; RING finger protein 105 -2.00 0.0019 NP_872354.1  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 -2.01 0.0276 SCF  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 -2.01 0.0045  102 Gene Name Gene Description Fold Change p Value NP_056477.1 WD-40 repeat -2.02 0.0392 Q8TAC0 Acc:Q8TAC0]; MGC27345 protein. [Source:Uniprot/SPTREMBL -2.02 0.0057 DNAS1  Among other functions, seems to be involved in cell death by apoptosis. Binds specifically to G-actin and blocks actin polymerization -2.03 0.0339 NP_064618.3  Metallophosphoesterase -2.03 0.0387 SERC3  May be involved in cellular transformation -2.03 0.0384 TF2H4  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 -2.03 0.0382 PDE4C  cAMP-specific 3',5'-cyclic phosphodiesterase 4C; DPDE1; PDE21 -2.04 0.0326 G3P   Glyceraldehyde-3-phosphate dehydrogenase; GAPDH -2.04 0.0123 RBL2  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 -2.05 0.0112 NP_872339.2 Acc:Q6ZRT9]; CDNA FLJ46112 fis, clone TESTI2035962 (Novel protein). [Source:Uniprot/SPTREMBL -2.06 0.0361 NP_001005472.1 Ribosomal protein S2 -2.06 0.0358 NP_078886.2 Transcription factor jumonji -2.07 0.0356 CACB3  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 -2.08 0.0347 ECHD1   Enoyl-CoA hydratase domain-containing protein 1 -2.09 0.0339 MPIP2   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 -2.10 0.0333 SYFB  Phenylalanyl-tRNA synthetase beta chain; Phenylalanine--tRNA ligase beta chain; PheRS -2.10 0.0333 CU045  Uncharacterized protein C21orf45; FAPP1-associated protein 1 -2.10 0.0488 K1279  May play a role in both peripheral and central nervous system -2.10 0.0207 TMM58  Transmembrane protein 58 precursor -2.11 0.0044  103 Gene Name Gene Description Fold Change p Value PYGL  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 -2.12 0.0245 MDM4  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 -2.13 0.031 LRP4  Potential cell surface endocytic receptor, which binds and internalizes extracellular ligands for degradation by lysosomes -2.14 0.0304 RIPK1  Promotes apoptosis and activation of NF-kappa-B. Required for TNFRSF1A mediated activation of NF- kappa-B -2.15 0.0302 NP_073742.1 Acc:Q5VW35]; Novel protein (FLJ12806). [Source:Uniprot/SPTREMBL -2.15 0.0062 MA2C1   Alpha-mannosidase 2C1; Alpha-D-mannoside mannohydrolase; Mannosidase alpha class 2C member 1; Alpha mannosidase 6A8B -2.16 0.0297 DSG2  Component of intercellular desmosome junctions. Involved in the interaction of plaque proteins and intermediate filaments mediating cell-cell adhesion -2.16 0.0294 ORAI2  Key regulator or component of store-operated Ca(2+) channel and transcription factor NFAT nuclear import -2.20 0.0276 SLIK1  Enhances neuronal dendrite outgrowth -2.21 0.0271 G3P   Glyceraldehyde-3-phosphate dehydrogenase; GAPDH -2.22 0.0012 SOX30 Transcriptional activator. Binds to the DNA sequence 5'-ACAAT-3' and shows a preference for guanine residues surrounding this core motif -2.23 0.0134 LAT3  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 -2.24 0.0254 Q6UX34 ASCL830 (UNQ830), mRNA [Source:RefSeq_dna; Acc:NM_206895] -2.26 0.0247 PRP39  Involved in pre-mRNA splicing -2.26 0.0247 Q96NM2  Acc:NM_001004325]; keratin associated protein 5-2 (KRTAP5-2), mRNA [Source:RefSeq_dna -2.28 0.0238 Q9HAJ0 Acc:Q9HAJ0]; CDNA FLJ11556 fis, clone HEMBA1003079. [Source:Uniprot/SPTREMBL -2.28 0.0237 S38A3  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 -2.30 0.023  104 Gene Name Gene Description Fold Change p Value SSXT  SSXT protein; Synovial sarcoma, translocated to X chromosome; SYT protein -2.31 0.0226 GBRG2   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 -2.31 0.0223 NP_001073963.1 Tetratricopeptide TPR_2 -2.32 0.0329 Q6ZNA8   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 DNA- dependent ATPase activity. Underwinds duplex DNA and forms helical nucleoprotein filaments -2.32 0.0221 Q9H4F8-2 Calcium-binding EF-hand; Protease inhibitor, Kazal- type; Proteinase inhibitor I1, Kazal; Thyroglobulin type-1 -2.33 0.0216 OBSCN  Immunoglobulin; Immunoglobulin C1-set; Immunoglobulin I-set; Immunoglobulin V-set; Immunoglobulin-like; Proline-rich region; Protein kinase; Tyrosine protein kinase, active site -2.37 0.0202 ARSB  Arylsulfatase B precursor; ASB; N- acetylgalactosamine-4-sulfatase; G4S -2.38 0.0188 NP_071425.2 Type II fibronectin, collagen-binding -2.39 0.0197 MSRE  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) -2.39 0.0197 L2HDH   L-2-hydroxyglutarate dehydrogenase, mitochondrial precursor; Duranin -2.41 0.019 DNM3B  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 -2.44 0.0181 Q9H9G5   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 -2.44 0.0181 SAM14  Sterile alpha motif domain-containing protein 14 -2.45 0.0179 RSU1  Potentially plays a role in the Ras signal transduction pathway. Capable of suppressing v-Ras transformation in vitro -2.47 0.0173 NP_001007793.1  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 3- kinase, 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 -2.49 0.0104  105 Gene Name Gene Description Fold Change p Value CLTR1  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 phosphatidylinositol- calcium second messenger system. The rank order of affinities for the leukotrienes is LTD4 &gt;&gt; LTE4 = LTC4 &gt;&gt; LTB4 -2.50 0.0166 C43BP  Phosphorylates on Ser and Thr residues the Goodpasture autoantigen (in vitro). Isoform 2 seems to be less active -2.52 0.0161 WDR55  WD repeat protein 55 -2.52 0.016 BKRB2  Receptor for bradykinin. It is associated with G proteins that activate a phosphatidylinositol-calcium second messenger system -2.53 0.0157 NP_055654.2 Phosphoesterase, PA-phosphatase related -2.53 0.0157 ACSL5  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 -2.54 0.0154 LR37A   Leucine-rich repeat-containing protein 37A; Leucine- rich repeat-containing protein 37B precursor; C66 SLIT-like testicular protein -2.59 0.0002 Q9NSC5-3 EVH1 -2.61 0.0003 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 -2.66 0.0012 NP_060218.1 Histone H5 -2.67 0.0129 TRIPB  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 cis- Golgi structure -2.69 0.0126 INHBC  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 -2.71 0.0122 METTL6 Probable methyltransferase -2.75 0.0116 PO2F3  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 -2.76 0.0115  106 Gene Name Gene Description Fold Change p Value GBF1  Promotes guanine-nucleotide exchange on ARF5. Promotes the activation of ARF5 through replacement of GDP with GTP -2.79 0.0111 NFASC  Cell adhesion, ankyrin-binding protein which may be involved in neurite extension, axonal guidance, synaptogenesis, myelination and neuron-glial cell interactions -2.80 0.0109 CT121  May act as a protein that binds a hydrophobic ligand  -2.82 0.0485 DAAM1   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 -2.86 0.0343 DLX5  Homeobox protein DLX-5 -2.87 0.01 ZNF19  May be involved in transcriptional regulation -2.89 0.0039 PIAS1  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 -2.90 0.0097 CASR   Senses changes in the extracellular concentration of calcium ions. The activity of this receptor is mediated by a G-protein that activates a phosphatidylinositol- calcium second messenger system -2.93 0.0094 TLE4  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 -2.95 0.0091 Q8NBC0  Orphan nuclear receptor, HMR type -3.00 0.0442 T2R16  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 -3.05 0.0082 FBXL7  Probably recognizes and binds to some phosphorylated proteins and promotes their ubiquitination and degradation -3.05 0.0081 CATG  Cathepsin G precursor; CG -3.15 0.0073 ASAHL  Degrades bioactive fatty acid amides to their corresponding acids, with the following preference: N- palmitoylethanolamine &gt; N-myristoylethanolamine &gt; N-lauroylethanolamine = N-stearoylethanolamine &gt; N-arachidonoylethanolamine &gt; N- oleoylethanolamine. Also exhibits weak hydrolytic activity against the ceramides N-lauroylsphingosine and N-palmitoylsphingosine -3.16 0.0073 SC6A3  Amine transporter. Terminates the action of dopamine by its high affinity sodium-dependent reuptake into presynaptic terminals -3.20 0.007  107 Gene Name Gene Description Fold Change p Value MBOA5  Membrane-bound O-acyltransferase domain- containing protein 5; O-acyltransferase domain- containing protein 5 -3.24 0.0006 CELR1  Receptor that may have an important role in cell/cell signaling during nervous system formation -3.25 0.0067 F102A   May play a role in estrogen action -3.30 0.0064 VNN2  Probable hydrolase. Involved in the thymus homing of bone marrow cells. May regulate beta-2 integrin- mediated cell adhesion, migration and motility of neutrophil -3.43 0.0056 NP_659462.1  Chaperonin clpA/B; Disease resistance protein -3.56 0.0003 NP_116250.2 Acc:NP_116250]; serine active site containing 1 [Source:RefSeq_peptide -3.65 0.0047 EVI2B  EVI2B protein precursor; Ecotropic viral integration site 2B protein homolog; EVI-2B -3.70 0.0045 AGPAT5 Converts lysophosphatidic acid (LPA) into phosphatidic acid by incorporating an acyl moiety at the sn-2 position of the glycerol backbone -3.72 0.0044 PLOD2 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 -3.73 0.0044 SL9A8  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 -3.75 0.0043 RTC1  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 RNA- N3'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 -3.81 0.0003 TIGD6  Tigger transposable element-derived protein 6 -3.89 0.0039 F261  Synthesis and degradation of fructose 2,6- bisphosphate -3.93 0.0002 AAT1   Isoform 4 may play a role in spermatogenesis -4.18 0.0032 ATP7B  Involved in the export of copper out of the cells, such as the efflux of hepatic copper into the bile -4.40 0.0028 K22E  Probably contributes to terminal cornification. Associated with keratinocyte activation, proliferation and keratinization -4.52 0.0026 NP_115510.1 Acc:NP_115510]; glutamine rich 2 [Source:RefSeq_peptide -4.53 0.0026 DB118  Has antibacterial activity  -4.56 0.0025 BCL7A  B-cell CLL/lymphoma 7 protein family member A -4.62 0.0024 MAGBA  Melanoma-associated antigen B10; MAGE-B10 antigen -4.66 0.0024  108 Gene Name Gene Description Fold Change p Value Q7Z3S7  Cache; VWA N-terminal; von Willebrand factor, type A -4.69 0.0023 RNAS1  Endonuclease that catalyzes the cleavage of RNA on the 3' side of pyrimidine nucleotides. Acts on single stranded and double stranded RNA -4.90 0.0021 GABARAPL3 Gamma-aminobutyric acid receptor-associated protein-like 3; GABA(A) receptor-associated protein- like 3 -5.16 0.0018 ENSG00000184956 Orphan nuclear receptor, HMR type -5.44 0.0016 ZN134  May be involved in transcriptional regulation -5.67 0.0015 OR2S1   Putative odorant receptor -6.58 0.0011 FGF2  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 -7.71 0.0008 ATRX  Could be a global transcriptional regulator. Modifies gene expression by affecting chromatin. May be involved in brain development and facial morphogenesis -9.95 0.0005 ZHANG  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 CREBZF- expressing cells -12.16 0.0003 ENSG00000199437 Acc:RF00416]; Small nucleolar RNA ACA43 [Source:RFAM -13.06 0.0003 ZNF613 May be involved in transcriptional regulation -47.09 5.4E- 05  Supplementary
Table
8:
Differentially
expressed
genes
2
hours
post­stimulation
with
3
µg/ml
 LL­37.

 Gene Name Gene Description Fold Change p Value NP_001032407.1  May play a role in the regulation of keratinocyte differentiation 7.67 0.004 TMEM33  Transmembrane protein 33; DB83 protein 7.24 0.0044 MIF   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 6.36 0.0054 TSN13  Tetraspanin-13; Tspan-13; Transmembrane 4 superfamily member 13; Tetraspan NET-6 5.47 0.0071 ENSG00000203581 Acc:NR_002169]; olfactory receptor, family 1, subfamily F, member 2 (OR1F2) on chromosome 16 [Source:RefSeq_dna 5.19 0.0078  109 Gene Name Gene Description Fold Change p Value KCNH2   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 4.82 0.009 FOS  Nuclear phosphoprotein which forms a tight but non- covalently 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 4.57 0.0099 TIE2  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 4.35 0.011 GBG8  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 protein- effector interaction 4.24 0.0116 RT12  28S ribosomal protein S12, mitochondrial precursor; S12mt; MRP-S12; MT-RPS12 4.20 0.0118 FGD5  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 3.93 0.0135 KS6A2  Serine/threonine kinase that may play a role in mediating the growth-factor and stress induced activation of the transcription factor CREB 3.92 0.0137 IDH2 Plays a role in intermediary metabolism and energy production. It may tightly associate or interact with the pyruvate dehydrogenase complex 3.72 0.0153 Q9BSF7  Acc:Q9BSF7]; MGC13008 protein. [Source:Uniprot/SPTREMBL 3.65 0.016 Q6I9Y3  Chromo; Post-SET zinc-binding region; Pre-SET zinc-binding region; SET 3.58 0.0167 SP4  Binds to GT and GC boxes promoters elements. Probable transcriptional activator 3.52 0.0174 NPT2B  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 3.13 0.0162 NP_116256.2 Proline-rich region; Tropomyosin 3.10 0.0235 BT2A1   Butyrophilin subfamily 2 member A1 precursor; Butyrophilin subfamily 2 member A3 precursor 3.07 0.0242  110 Gene Name Gene Description Fold Change p Value ATE1   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 3.06 0.0244 TRIO  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 3.06 0.0244 NP_115589.2 Engulfment and cell motility, ELM 3.05 0.0247 PPIL2  PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides 3.02 0.0252 NGLY1   Specifically deglycosylates the denatured form of N- linked 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 2.99 0.0258 RET7  Intracellular transport of retinol 2.96 0.0264 NP_997646.1 AT-rich interaction region 2.94 0.027 BAALC May play a synaptic role at the postsynaptic lipid rafts by interacting with CAMK2A 2.87 0.0287 LDHC  Possible role in sperm motility 2.83 0.0298 ABI2   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 2.66 0.0352 NP_006537.3 KH, type 1; RNA-binding region RNP-1 (RNA recognition motif) 2.66 0.0353 NP_079174.2 Adenovirus fibre protein; DENN; dDENN; uDENN 2.63 0.0363 GPX7  Glutathione peroxidase 7 precursor; CL683 2.62 0.0368 ZN485  May be involved in transcriptional regulation 2.61 0.0371 CAH3  Reversible hydration of carbon dioxide 2.61 0.0373 ARHG4  DH; Guanine-nucleotide dissociation stimulator, CDC24; Pleckstrin-like; Src homology-3; Variant SH3 2.56 0.0391 CHST6  Catalyzes the transfer of sulfate to position 6 of non- reducing 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 2.56 0.0393  111 Gene Name Gene Description Fold Change p Value SNX23 May be involved in several stages of intracellular trafficking. Probable microtubule-dependent motor protein 2.54 0.04 P48067-2  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 2.54 0.0401 HSH2D  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 CD28- mediated activation of the RE/AP element of the interleukin-2 promoter 2.53 0.0406 RND1  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 2.52 0.041 RFIP2  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 clathrin- coated vesicles. Binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) and phosphatidic acid (PA) 2.51 0.0417 PDZD2  PDZ domain-containing protein 2; PDZ domain- containing protein 3; Activated in prostate cancer protein 2.50 0.042 BIN3  Involved in cytokinesis and septation where it has a role in the localization of F-actin 2.45 0.0445 KCMB1  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 2.42 0.0462  112 Gene Name Gene Description Fold Change p Value NP_079038.2 KRAB box; Zinc finger, C2H2-subtype; Zinc finger, C2H2-type 2.39 0.0476 MCR   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 2.39 0.0477 FARP2  Rho-guanine nucleotide exchange factor that activates RAC1. Plays a role in the response to class 3 semaphorins and remodeling of the actin cytoskeleton 2.39 0.0479 NP_060156.1 Appr-1-p processing 2.38 0.0485 CPN2   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 2.38 0.0486 TNFC  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 2.38 0.0487 ABD12   PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides; This is a calcium- activated, phospholipid-dependent, serine- and threonine-specific enzyme. May play a role in cell motility by phosphorylating CSPG4 2.37 0.0026 MYST3  Histone acetyltransferase which may act as a transcriptional coactivator for RUNX1 and RUNX2 2.36 0.0496 NP1L2  Acidic protein which may be involved in interactions with other proteins or DNA 2.30 0.0081 VWF  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 2.28 0.0092 AMAC1L1  Protein AMAC1; Transmembrane protein 21A; Protein AMAC1L2 2.17 0.0114 RMD5B  RMD5 homolog B -2.19 0.0148 DHB3  Favors the reduction of androstenedione to testosterone. Uses NADPH while the two other EDH17B enzymes use NADH -2.29 0.0476 Q5T6R2   Could be involved in signal transduction -2.34 0.0257  113 Gene Name Gene Description Fold Change p Value Q96GD3-2  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 -2.36 0.0497 PRB4S  Basic salivary proline-rich protein 4 allele S precursor; Salivary proline-rich protein Po; Parotid o protein; Protein N1; Glycosylated protein A -2.38 0.0487 UBAP1  Ubiquitin-associated protein 1; UBAP -2.38 0.0485 COPE  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 -2.40 0.0473 Q75VX8  Proline-rich region -2.40 0.0471 Q5K675  Acc:Q5K675]; Osteoligament factor. [Source:Uniprot/SPTREMBL -2.41 0.0466 MYPN  Component of the sarcomere that tethers together nebulin (skeletal muscle) and nebulette (cardiac muscle) to alpha-actinin, at the Z lines -2.42 0.0461 NALDL   NAALADase-like activity unknown. Has no NAAG hydrolyzing activity. Exhibits a dipeptidyl-peptidase IV type activity. In vitro, cleaves Gly-Pro-AMC -2.43 0.0457 CCG6  Thought to stabilize the calcium channel in an inactivated (closed) state -2.45 0.0446 NP_277045.1 Sterol-sensing 5TM box -2.45 0.0443  114 Gene Name Gene Description Fold Change p Value O94813-3  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 growth- stimulatory 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 -2.49 0.0425 XPA  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 -2.50 0.0419 Q8NHQ8-2  Ras association domain-containing protein 8; Carcinoma-associated protein HOJ-1 -2.53 0.0406 PDE4C  cAMP-specific 3',5'-cyclic phosphodiesterase 4C; DPDE1; PDE21 -2.54 0.0399 CP2J2  This enzyme metabolizes arachidonic acid predominantly via a NADPH-dependent olefin epoxidation to all four regioisomeric cis- epoxyeicosatrienoic acids. One of the predominant enzymes responsible for the epoxidation of endogenous cardiac arachidonic acid pools -2.55 0.0397 KIF5C  Kinesin is a microtubule-associated force-producing protein that may play a role in organelle transport -2.57 0.0389 SF04  Plays a role in premRNA splicing -2.58 0.0384 S26A2  Sulfate transporter. May play a role in endochondral bone formation -2.58 0.0384 MARVELD2  May play a role in the formation of the epithelial barrier -2.60 0.0374 RHGXX  GTPase activator for the Rho-type GTPases by converting them to an inactive GDP-bound state -2.62 0.0369  115 Gene Name Gene Description Fold Change p Value ADCY6  Membrane-bound, calcium-inhibitable adenylyl cyclase -2.62 0.0368 ARMET  Protein ARMET precursor; Arginine-rich protein -2.64 0.0359 PVRL3  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 down- regulation of PVR from the cell surface, resulting in reduction of cell movement and proliferation. Plays a role in the morphology of the ciliary body -2.64 0.0449 GDE5  Putative glycerophosphodiester phosphodiesterase 5 -2.65 0.0356 BHLH2  May function as a transcriptional factor to modulate chondrogenesis in response to the cAMP pathway -2.66 0.0351 DOCK8  Potential guanine nucleotide exchange factor (GEF). GEF proteins activate some small GTPases by exchanging bound GDP for free GTP -2.67 0.0347 FASP1 Uncharacterized protein C21orf45; FAPP1- associated protein 1 -2.68 0.0345 TMUB1  Transmembrane and ubiquitin-like domain- containing protein 1; Ubiquitin-like protein SB144 -2.68 0.0344 SIRPG  Probable immunoglobulin-like cell surface receptor. On binding with CD47, mediates cell-cell adhesion. Engagement on T-cells by CD47 on antigen- presenting cells results in enhanced antigen-specific T-cell proliferation and co-stimulates T-cell activation -2.70 0.0339 SG1D1  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 -2.72 0.0332 METTL6 Probable methyltransferase -2.73 0.0329 SMURF2 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 -2.80 0.0307 KCNK7  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 -2.81 0.0303  116 Gene Name Gene Description Fold Change p Value ZDH11  Probable palmitoyltransferase ZDHHC11; Zinc finger DHHC domain-containing protein 11; DHHC-11; Zinc finger protein 399 -2.84 0.0296 MULK  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 -2.86 0.029 DLG1   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 -2.87 0.0287 RNF34  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 -2.89 0.0283 RSU1  Potentially plays a role in the Ras signal transduction pathway. Capable of suppressing v-Ras transformation in vitro -2.90 0.0279 ENP5  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 -2.93 0.0271 CYH4  Promotes guanine-nucleotide exchange on ARF1 and ARF5. Promotes the activation of ARF through replacement of GDP with GTP -2.95 0.0028 Q9NWT9  Calcium-binding EF-hand -2.96 0.0265 LRC8B  Leucine-rich repeat-containing protein 8B; T-cell activation leucine repeat-rich protein -3.01 0.0255 SFXN2  Potential iron transporter -3.03 0.0108 GBF1  Promotes guanine-nucleotide exchange on ARF5. Promotes the activation of ARF5 through replacement of GDP with GTP -3.04 0.0248 GUAD  Catalyzes the hydrolytic deamination of guanine, producing xanthine and ammonia -3.04 0.0247 Q9H6U4  Glycoside hydrolase, family 85 -3.06 0.0243 HHEX  Recognizes the DNA sequence 5'-ATTAA-3'. May play a role in hematopoietic differentiation -3.09 0.0238 Q495Z4  Adrenergic receptor, beta 1; Proline-rich region -3.14 0.0228 NP_079280.1 Acc:NP_079280]; coiled-coil domain containing 15 [Source:RefSeq_peptide -3.15 0.0227 MAGB1  Melanoma-associated antigen B1; MAGE-B1 antigen; MAGE-XP antigen; DSS-AHC critical interval MAGE superfamily 10; DAM10 -3.16 0.0224  117 Gene Name Gene Description Fold Change p Value 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 -3.36 0.0067 FGF3  Could be involved in ear development -3.39 0.0189 LAIR1   Functions as an inhibitory receptor that plays a constitutive negative regulatory role on cytolytic function of natural killer (NK) cells, B-cells and T- cells. 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 -3.48 0.0178 FA49A  Protein FAM49A -3.53 0.0173 Q9NSC5-3 EVH1 -3.58 0.0132 F261  Synthesis and degradation of fructose 2,6- bisphosphate -3.64 0.0161 FA53C  Protein FAM53C -3.65 0.0163 SEM6A  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 -3.72 0.0154 DPYL1  Dihydropyrimidinase-related protein 1; DRP-1; Collapsin response mediator protein 1; CRMP-1 -3.79 0.0147 PLOD2 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 -3.94 0.0135 NARGL  May belong to a complex displaying N-terminal acetyltransferase activity -3.97 0.0133 C43BP  Phosphorylates on Ser and Thr residues the Goodpasture autoantigen (in vitro). Isoform 2 seems to be less active -3.98 0.0156 TRFM  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 -4.03 0.0128 RB11B  Possesses GTPase activity -4.08 0.0125 SPAG1  Plays a role in fertilization. Binds GTP and has GTPase activity -4.20 0.0118  118 Gene Name Gene Description Fold Change p Value ANR11  May recruit HDACs to the p160 coactivators/nuclear receptor complex to inhibit ligand-dependent transactivation -4.23 0.0116 HRSL1  HRAS-like suppressor -4.52 0.0101 Q69YY3  DKFZ -4.55 0.01 OVOL1  Putative transcription factor. Involved in hair formation and spermatogenesis. May function in the differentiation and/or maintenance of the urogenital system -4.66 0.0096 CLCN4  Voltage-gated chloride channel. Chloride channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport -4.72 0.0093 BSND  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 -4.77 0.0091 PHF14  PHD finger protein 14 -4.99 0.0084 TRBV6-1  Immunoglobulin V-set; Immunoglobulin-like -5.20 0.0078 CELR3   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 -5.25 0.0446 TULP4  Tubby-related protein 4; Tubby-like protein 4; Tubby superfamily protein -5.56 0.0069 CX04A  May have an important role of cell protection in inflammation reaction -5.66 0.0067 APOB  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 -5.72 0.0065 ELAV4   May play a role in neuron-specific RNA processing. Protects CDKN1A mRNA from decay by binding to its 3'-UTR -6.31 0.0055 BPAEA   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 -6.43 0.0053 KPBB  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 -6.43 0.0053  119 Gene Name Gene Description Fold Change p Value ENSG00000117114 Brain-specific angiogenesis inhibitor; CD97 antigen; D-galactoside/L-rhamnose binding SUEL lectin; EMR1 hormone receptor; GPCR, family 2, secretin- like; GPS; Hormone receptor, extracellular; Latrophilin receptor; Latrophilin, C-terminal; Olfactomedin-like -6.58 0.0051 RRP5  Involved in the biogenesis of rRNA -6.63 0.0051 TMG1  Transmembrane gamma-carboxyglutamic acid protein 1 precursor; Proline-rich Gla protein 1; Proline-rich gamma-carboxyglutamic acid protein 1 -6.82 0.0049 ENSG00000086200 Importin-beta, N-terminal -6.86 0.0048 FGF2  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 -8.32 0.0011 GLRA2  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) -8.57 0.0034 MUC16  Thought to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces -8.92 0.0032 LIPA2  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 -9.95 0.0028 IGS21  Immunoglobulin superfamily member 21 precursor -10.15 0.0027 NP_079354.2 Cystinosin/ERS1p repeat -10.21 0.0027 NP_036401.1 EGF-like region; Glycoside hydrolase, family 56; Glycoside hydrolase, family 56, sperm surface protein PH20; Multicopper oxidase, copper-binding site -14.31 0.0017 ENSG00000199437 Acc:RF00416]; Small nucleolar RNA ACA43 [Source:RFAM -20.72 0.0011  
 
  Supplementary
Table
9:
Differentially
expressed
genes
4
hours
post­stimulation
with
3
µg/ml
 LL­37.

 Gene Name Gene Description Fold Change p Value Q9H6U4  Glycoside hydrolase, family 85 10.07 0.0001 RBP17  May function as a nuclear transport receptor 9.32 0.0002  120 Gene Name Gene Description Fold Change p Value EGF  EGF stimulates the growth of various epidermal and epithelial tissues in vivo and in vitro and of some fibroblasts in cell culture 7.03 0.0004 CLCN5  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 6.26 0.0065 DUS9   Inactivates MAP kinases. Has a specificity for the ERK family; Required for the uptake of creatine in muscles and brain 5.99 0.0006 ENPP5  May play a role in neuronal cell communication. Lacks nucleotide pyrophosphatase and lysopholipase D activity 5.93 0.0006 IFNA1  Produced by macrophages, IFN-alpha have antiviral activities. Interferon stimulates the production of two enzymes: a protein kinase and an oligoadenylate synthetase 5.52 0.0008 Q96NK6  Peptidase S1 and S6, chymotrypsin/Hap 5.49 0.0008 GPR25  Orphan receptor 5.21 0.0009 SMAD6  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 5.07 0.001 NP_001025226.1  GTPase-activating protein for Rho family members. May play a role in the reduction of the p21rasGTPase-activating potential of p120GAP 4.99 0.0011 INHBC  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 4.99 0.0011 CCG4  Thought to stabilize the calcium channel in an inactivated (closed) state 4.79 0.0012 Q8N370  Major facilitator superfamily MFS_1 4.79 0.0012 NP_060845.2 Sodium:dicarboxylate symporter; Zinc/iron permease 4.72 0.0013 DAB1  Adapter molecule functioning in neural development. May regulate SIAH1 activity 4.63 0.0014 CARD9  Activates NF-kappa-B via BCL10 4.60 0.0014 ADCK1  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) 4.54 0.0015 GIPR  This is a receptor for GIP. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase 4.53 0.0015  121 Gene Name Gene Description Fold Change p Value DIAP2   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 4.29 0.0018 NP_001032671.1  Fatty acid desaturase; Fatty acid desaturase, type 1 4.25 0.0019 FOXP1  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 4.16 0.0021 ZN566  May be involved in transcriptional regulation 4.05 0.0195 NP_076955.1 Acc:XR_019463]; similar to CG31855-PA (LOC401770), mRNA [Source:RefSeq_dna 3.99 0.0024 AP3B1  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 3.86 0.0108 TPBG  Trophoblast glycoprotein precursor; 5T4 oncofetal trophoblast glycoprotein; 5T4 oncotrophoblast glycoprotein; 5T4 oncofetal antigen; M6P1 3.73 0.0004 Q502X4   Enzyme which catalyzes the acetylation of polyamines. Substrate specificity: norspermidine &gt; spermidine = spermine &gt;&gt; N(1)acetylspermine = putrescine 3.70 0.0032 AP3S2 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 3.68 0.0033 GRPEL1  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 ATP- dependent manner. Seems to control the nucleotide- dependent binding of mitochondrial HSP70 to substrate proteins 3.68 0.0033 NP_005178.4  EDG-8 sphingosine 1-phosphate receptor; Proline- rich region; TAFH/NHR1; Treacher Collins syndrome protein Treacle; Zinc finger, MYND-type 3.65 0.0034  122 Gene Name Gene Description Fold Change p Value DACH2   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 cyclin- dependent 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 3.63 0.0034 IGF1R  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 IGF1- stimulated downstream signaling cascade 3.63 0.0035 AT131   Probable cation-transporting ATPase 13A1 3.59 0.0036 NP_001480.3 Nuclear hormone receptor, DNA-binding; Nuclear hormone receptor, ligand-binding; Retinoid X receptor; Steroid hormone receptor; Vitamin D receptor 3.53 0.0038 N4BP2  Has 5'-polynucleotide kinase and nicking endonuclease activity. May play a role in DNA repair or recombination 3.52 0.0039 NET4  May play an important role in neural, kidney and vascular development. Promotes neurite elongation from olfactory bulb explants 3.49 0.0415 GP126   Orphan receptor 3.45 0.0042 CYB5  Cytochrome b5 is a membrane bound hemoprotein which function as an electron carrier for several membrane bound oxygenases 3.44 0.0134 M10L1  Putative RNA helicase. Isoform 1 may play a role in male germ cell development 3.38 0.0207 RBM16  DH; Guanine-nucleotide dissociation stimulator, CDC24; PDZ/DHR/GLGF; Pleckstrin-like; Raf-like Ras-binding; Spectrin/pleckstrin-like 3.33 0.0048 DNS2A  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 3.23 0.0055 Q9H9G5  Plexin; Semaphorin/CD100 antigen 3.22 0.0056  123 Gene Name Gene Description Fold Change p Value PZP  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 3.22 0.0056 NIPBL  Probably plays a structural role in chromatin. Involved in sister chromatid cohesion, possibly by interacting with the cohesin complex 3.18 0.0058 Q5JSH9  Antifreeze protein, type I; Orphan nuclear receptor, NOR1 type; Proline-rich region; Ribosomal protein P2; Salmonella/Shigella invasin protein C; Zinc finger, MIZ-type 3.17 0.006 NP_036256.1 CHORD; CS 3.17 0.006 TLX3  T-cell leukemia homeobox protein 3; Homeobox protein Hox-11L2 3.12 0.0064 RAI3  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 3.09 0.0067 SFRIP  Plays a role in pre-mRNA alternative splicing by regulating spliceosome assembly 3.09 0.0067 SULF1  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 3.07 0.0068 NP_114158.2  Acc:NP_114158]; spermatogenesis associated 9 isoform a [Source:RefSeq_peptide 3.03 0.0072 KLDC2  Kelch domain-containing protein 2; Hepatocellular carcinoma-associated antigen 33; Host cell factor homolog LCP 3.02 0.0073 TRI50  Tripartite motif-containing protein 50 3.01 0.0075 KCC1D  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 2.99 0.0076 ZN174  Transcriptional repressor 2.99 0.0077  124 Gene Name Gene Description Fold Change p Value HXD11   Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior- posterior axis 2.97 0.0079 PKCB1   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 2.93 0.0083 ZFP95  May be involved in transcriptional regulation 2.91 0.0085 MAP4  Non-neuronal microtubule-associated protein. Promotes microtubule assembly 2.88 0.0089 MAGA5  Not known, though may play a role tumor transformation or progression 2.88 0.0089 GABARAPL3 Gamma-aminobutyric acid receptor-associated protein-like 3; GABA(A) receptor-associated protein- like 3 2.88 0.0033 GUF1  GTP-binding protein GUF1 homolog 2.86 0.0092 AOAH  Removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides 2.84 0.0095 ASPM  Probable role in mitotic spindle regulation and coordination of mitotic processes (By similarity). May have a preferential role in regulating neurogenesis 2.83 0.0096 NP_064614.2  PR domain-containing protein 11 2.82 0.0098 NP_062536.2  Proline-rich region 2.82 0.0099 HMGB3  Binds preferentially single-stranded DNA and unwinds double stranded DNA 2.80 0.0433 ISCU  Involved in the assembly or repair of the [Fe-S] clusters present in iron-sulfur proteins. Binds iron 2.79 0.0103 Q9BSC4-2 NUC153 2.78 0.0105 PAPOA  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 -2.76 0.0109 GBRD  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 -2.76 0.0108 RABP2  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 -2.77 0.001 GBRG3  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 -2.77 0.0105 FRMD5  FERM domain-containing protein 5 -2.78 0.0105 IKKB  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 -2.78 0.0104  125 Gene Name Gene Description Fold Change p Value DOK5   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 -2.81 0.01 CLCN3   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 -2.82 0.0098 DUSP4  Regulates mitogenic signal transduction by dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2 -2.82 1.71E- 05 ZN659  Zinc finger protein 659 -2.83 0.0097 SMAD7  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 -2.84 0.0095 G7D   Involved in meiotic recombination. Facilitate crossovers between homologs during meiosis -2.84 0.0095 C1QA  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 -2.86 0.0093 SP1  Binds to GC box promoters elements and selectively activates mRNA synthesis from genes that contain functional recognition sites. Can interact with G/C- rich motifs from serotonin receptor promoter -2.87 0.0091 GLE1  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) -2.87 0.0091 DDFL1  Promotes cell proliferation -2.87 0.009 TCF21  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 -2.88 0.009 LYZL6  Lysozyme-like protein 6 precursor -2.88 0.009  126 Gene Name Gene Description Fold Change p Value IMA4   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 GTP- and 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 -2.88 0.0089 O95394-2 Phosphoglucomutase/phosphomannomutase; Phosphoglucomutase/phosphomannomutase C- terminal; Phosphoglucomutase/phosphomannomutase alpha/beta/alpha domain I -2.89 0.0089 Q96FJ0-2 Mov34/MPN/PAD-1 -2.89 0.0088 Q59GS4  IQ calmodulin-binding region; Myosin head, motor region -2.91 0.0117 NP_653185.1 Acc:Q5T8I8]; Novel protein (FLJ30525) (Fragment). [Source:Uniprot/SPTREMBL -2.91 0.0086 NP_055772.2 DENN; WD-40 repeat; dDENN -2.92 0.0084 NOX1  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 -2.92 0.0241 TENS1  May be involved in cell migration, cartilage development and in linking signal transduction pathways to the cytoskeleton -2.93 0.0083 RM40  39S ribosomal protein L40, mitochondrial precursor; L40mt; MRP-40; Nuclear localization signal- containing protein deleted in velocardiofacial syndrome; Up-regulated in metastasis -2.93 0.0083 PO3F2  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 -2.94 0.0081  127 Gene Name Gene Description Fold Change p Value RPIA  Ribose-5-phosphate isomerase; Phosphoriboisomerase -2.95 0.0081 EPO  Erythropoietin is the principal hormone involved in the regulation of erythrocyte differentiation and the maintenance of a physiological level of circulating erythrocyte mass -2.97 0.0124 NP_079038.2 KRAB box; Zinc finger, C2H2-subtype; Zinc finger, C2H2-type -2.97 0.0079 O60696  Zinc finger, PHD-type -2.97 0.0079 RMD5B  RMD5 homolog B -2.97 0.0189 AN32A   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 -2.97 0.0039 ATN1  Atrophin-1; Dentatorubral-pallidoluysian atrophy protein -2.97 0.0016 OR5V1  Putative odorant receptor -2.99 0.0077 PI51B  Mediates RAC1-dependent reorganization of actin filaments (By similarity). Participates in the biosynthesis of phosphatidylinositol-4,5- bisphosphate -3.00 0.0076 PDZD1  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 -3.00 0.0075 TPD52  Tumor protein D52; Protein N8 -3.01 0.0074  128 Gene Name Gene Description Fold Change p Value NFKB2   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 -3.02 0.0073 Q5TF39  Cytochrome c-type biogenesis protein CcbS; Major facilitator superfamily MFS_1 -3.03 0.0072 NP_640332.1 Ankyrin; Proline-rich region -3.04 0.0071 DUSP7  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 PYST2- L although it is unclear whether this is by competing for transcription, translation or activation factors -3.04 0.0019 STB5L  May play a role in vesicle trafficking and exocytosis  -3.04 0.0071 OR2H1   Putative odorant receptor -3.05 0.007 MAP7  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 -3.06 0.0069 WD51A  WD repeat protein 51A -3.07 0.0069 NP_060554.3 Tropomyosin -3.07 3.2E- 05 ZC12B  Protein ZC3H12B -3.07 0.0068 ENSG00000180150 High mobility group protein HMG14 and HMG17; Histone H5 -3.07 0.0068 LOC197350 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 -3.09 0.0218 ENSG00000117707 Homeobox prospero-like -3.10 0.0066 STYK1  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 -3.10 0.0065 GAGE10  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 -3.11 0.0065  129 Gene Name Gene Description Fold Change p Value VATG3  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 -3.11 0.0065 SNG4  Synaptogyrin-4 -3.11 0.0003 Q9BT26  Acc:Q9BT26]; MGC10981 protein. [Source:Uniprot/SPTREMBL -3.12 0.0064 KBTB3  Kelch repeat and BTB domain-containing protein 3; BTB and kelch domain-containing protein 3 -3.13 0.0063 FOSL2   Fos-related antigen 2 -3.13 0.0063 CLD5  Plays a major role in tight junction-specific obliteration of the intercellular space -3.13 4.38E- 05 PGM5  Component of adherens-type cell-cell and cell-matrix junctions. Lacks phosphoglucomutase activity -3.14 0.0062 ENSG00000174672 Proline-rich region; Protein kinase; Serine/threonine protein kinase, active site; Tyrosine protein kinase -3.15 0.0003 NP_001276.2 ATPase, F1 complex, gamma subunit; Chloride channel calcium-activated; von Willebrand factor, type A -3.21 0.0057 GOG8A   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 -3.22 0.0056 F261  Synthesis and degradation of fructose 2,6- bisphosphate -3.24 0.0003 ABCA2   Probable transporter, its natural substrate has not been found yet. May have a role in macrophage lipid metabolism and neural development -3.24 0.0044 CIDEA   Activates apoptosis -3.24 0.0055 ACV1B  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 -3.25 0.0054 LPP3  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 -3.25 0.0054 ZN703  May function as a transcriptional repressor -3.26 0.0053 SEM3D  Induces the collapse and paralysis of neuronal growth cones. Could potentially act as repulsive cues toward specific neuronal populations. Binds to neuropilin -3.27 0.0053 Q4LDG9  Leucine-rich repeat -3.27 0.0052 NP_112579.2 GSG1-like; Voltage-dependent calcium channel gamma -3.30 0.0051 RNAS1  Endonuclease that catalyzes the cleavage of RNA on the 3' side of pyrimidine nucleotides. Acts on single stranded and double stranded RNA -3.33 0.0403  130 Gene Name Gene Description Fold Change p Value Q8TEE9  Proline-rich region -3.39 0.001 ZN445 May be involved in transcriptional regulation -3.41 7.32E- 06 NP_006324.1 Exosome-associated -3.42 0.0044 LTBP3 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) -3.42 4.30E- 03 HS2ST  Heparan sulfate 2-O-sulfotransferase -3.43 0.0043 Q5T7M9  Acc:Q5T7M9]; Novel protein possible ortholog of mouse RIKEN cDNA 2900024C23 gene. [Source:Uniprot/SPTREMBL -3.44 0.0042 WIF1  Binds to WNT proteins and inhibits their activities. May be involved in mesoderm segmentation -3.45 0.0042 LRIG1   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 -3.45 0.0042 M4K3  May play a role in the response to environmental stress. Appears to act upstream of the JUN N- terminal pathway -3.45 0.0042 MCHL1   Pro-MCH-like protein 1; Pro-melanin-concentrating hormone-like protein 1; Pro-MCH variant; Pro-MCH- like protein 2; Pro-melanin-concentrating hormone- like protein 2 -3.51 0.0039 NP_203131.1  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 -3.55 0.0038 TMM98  Transmembrane protein 98; Protein TADA1 -3.57 0.0037 PAK1   The activated kinase acts on a variety of targets. Likely to be the GTPase effector that links the Rho- related 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 -3.57 0.0003 NP_733936.1  Succinate semialdehyde dehydrogenase, mitochondrial precursor; NAD(+)-dependent succinic semialdehyde dehydrogenase -3.57 0.0037 NNAT  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 -3.57 0.0002 MAMC2  MAM domain-containing protein 2 precursor -3.59 0.0036 ENSG00000110077  CD20/IgE Fc receptor beta subunit -3.59 0.0036 SOX18  Binds to the consensus sequence 5'-AACAAAG-3' and is able to trans-activate transcription via this site -3.62 0.0002  131 Gene Name Gene Description Fold Change p Value UBP37  Ubiquitin carboxyl-terminal hydrolase 37; Ubiquitin thioesterase 37; Ubiquitin-specific-processing protease 37; Deubiquitinating enzyme 37 -3.68 0.0033 TRAF1  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 -3.70 0.0032 ODFP  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 -3.74 0.0031 C43BP  Phosphorylates on Ser and Thr residues the Goodpasture autoantigen (in vitro). Isoform 2 seems to be less active -3.75 0.0002 Q9NSC5-3 EVH1 -3.75 0.0025 Q8WVI0 Acc:Q8WVI0]; SNHG8 protein (Fragment). [Source:Uniprot/SPTREMBL -3.76 0.003 SYLC  Leucyl-tRNA synthetase, cytoplasmic; Leucine-- tRNA ligase; LeuRS -3.83 0.0221 TWST1  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 -3.83 0.0028 Q53RY2  Acc:P15336]; Cyclic AMP-dependent transcription factor ATF-2 (Activating transcription factor 2) (cAMP response element-binding protein CRE- BP1) (HB16). [Source:Uniprot/SWISSPROT -3.84 0.0028 NP_659415.1 Acc:Q96M89]; CDNA FLJ32745 fis, clone TESTI2001511, weakly similar to MYOSIN II HEAVY CHAIN, NON MUSCLE. [Source:Uniprot/SPTREMBL -3.88 0.0027 NP_116277.2 Collagen triple helix repeat; Dopamine D4 receptor; Fibrillar collagen, C-terminal; Pistil-specific extensin- like protein; Proline-rich region -3.94 0.0025 AN30A   Ankyrin repeat domain-containing protein 30A; Serologically defined breast cancer antigen NY-BR-1 -3.95 0.0025 PLPL Uncharacterized protein C14orf162; Myelin proteolipid protein-like protein -3.98 0.0024 ENSG00000166965 Regulator of chromosome condensation, RCC1 -4.00 0.0024  132 Gene Name Gene Description Fold Change p Value 5HT1E  This is one of the several different receptors for 5- hydroxytryptamine (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 -4.00 0.0024 DOCK4  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 -4.03 0.0023 PLOD2 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 -4.05 0.0021 DOK3   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 -4.07 0.0022 ACCN3   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 -4.14 0.0021 DCLK1  Probable kinase that may be involved in a calcium- signaling pathway controlling neuronal migration in the developing brain. May also participate in functions of the mature nervous system -4.28 0.0019 ZCHC4  May be a methyltransferase -4.34 0.0018 GRID2  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 -4.35 0.0018 NP_055483.2 Acc:NP_149081]; GREB1 protein isoform b [Source:RefSeq_peptide -4.36 0.0025 ENSG00000162105 Ankyrin; PDZ/DHR/GLGF; Src homology-3; Variant SH3 -4.37 0.0017 SMO  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) -4.37 0.0017 ENSG00000196229 HMG-I and HMG-Y, DNA-binding; Molluscan rhodopsin C-terminal tail; Proline-rich region -4.43 0.0016  133 Gene Name Gene Description Fold Change p Value SSR1  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 -4.47 0.0016 Q5VTT3   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 -4.55 0.0015 ABHD4  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 -4.56 0.0015 IGHV3-11  Immunoglobulin; Immunoglobulin V-set; Immunoglobulin-like -4.76 0.0013 SUCR1  Receptor for succinate -4.81 0.0012 FAN  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 -4.81 0.0012 NP_001020026.1  May be involved in transcriptional regulation -5.04 0.001 CAD12  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 -5.12 0.001 FGF2  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 -5.27 0.0008 PNMA1  Paraneoplastic antigen Ma1; Neuron- and testis- specific protein 1; 37 kDa neuronal protein -5.28 0.0009 Q8NBC0 Orphan nuclear receptor, HMR type -5.36 3.25E- 05 NP_996803.2 Low density lipoprotein-receptor, class A; MAM -5.37 0.0008 Q6ZN87 KRAB box -5.40 0.0008 CAN11  Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction -5.41 0.0008  134 Gene Name Gene Description Fold Change p Value NP_001025186.1  Photoreceptor required for regulation of circadian rhythm. Contributes to pupillar reflex and other non- image forming responses to light. May be able to isomerize covalently bound all-trans retinal back to 11-cis retinal -5.43 0.0008 FXYD6  FXYD domain-containing ion transport regulator 6 precursor -5.59 0.0007 CLGN  Probably plays an important role in spermatogenesis. Binds calcium ions -5.78 0.0007 ADCK3  May be a chaperone-like protein essential for the proper conformation and functioning of protein complexes in the respiratory chain -5.98 0.0006 PSD12  Acts as a regulatory subunit of the 26S proteasome which is involved in the ATP-dependent degradation of ubiquitinated proteins -6.44 0.0005 NP_004849.2  HCO3- transporter, cytoplasmic; HCO3- transporter, eukaryote; HCO3-transporter, c-terminal; Na+/HCO3- transporter -6.60 0.0004 Q6ICE7 Acc:Q6ICE7]; CN5H6.4 protein (OTTHUMP00000028648). [Source:Uniprot/SPTREMBL -6.86 1.56E- 05 TMG1  Transmembrane gamma-carboxyglutamic acid protein 1 precursor; Proline-rich Gla protein 1; Proline-rich gamma-carboxyglutamic acid protein 1 -6.87 0.0093 DEF  Regulates the p53 pathway to control the expansion growth of digestive organs -6.98 0.0103 MFSD4  Major facilitator superfamily domain-containing protein 4 -7.32 0.0003 CDC6  Involved in the initiation of DNA replication. Also participates in checkpoint controls that ensure DNA replication is completed before mitosis is initiated -7.58 0.0003 PLAL1  Shows weak transcriptional activatory activity. Transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase-activating polypeptide -7.76 0.0079 Q99490-2 Ankyrin; Arf GTPase activating protein; Miro-like; Pleckstrin-like; Ras; Ras GTPase -8.10 0.0035 ENSG00000199437 Acc:RF00416]; Small nucleolar RNA ACA43 [Source:RFAM -8.25 0.0163 Q86SZ9   WD repeat protein 23 -8.87 0.0002 GMDS  Conversion of GDP-D-mannose to GDP-4-keto-6-D- deoxymannose -8.94 0.0002 TRBV6-1  Immunoglobulin V-set; Immunoglobulin-like -9.27 0.0002 SC5A6  Transports pantothenate, biotin and lipoate in the presence of sodium -12.33 8.34E- 05 EYA4  Thought to play a role in transcription regulation during organogenesis through its intrinsic protein phosphatase activity. May be involved in development of the eye -118.41 1.61E- 06 ENP1   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 2.74 0.0112 JHDM1D  JmjC domain-containing histone demethylation protein 1D 2.73 0.0113  135 Gene Name Gene Description Fold Change p Value SIM2  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 2.73 0.0114 B3GN2  Catalyzes the initiation and elongation of poly-N- acetyllactosamine chains 2.71 0.0116 CCDC5  Regulator of spindle function and integrity during the metaphase-anaphase transition 2.71 0.0117 Q14591-2 Zinc finger, C2H2-subtype; Zinc finger, C2H2-type 2.70 0.0119 Q86V61  GAT; VHS 2.69 0.0121 LECT1  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 2.68 0.0123 ENSG00000196547 Glycoside hydrolase, family 38; Glycosyl hydrolases 38, C-terminal 2.67 0.0125 DKKL1  Dickkopf-like protein 1 precursor; Soggy-1 protein; SGY-1 2.67 0.0126 CR2   Receptor for complement C3Dd, for the Epstein-Barr virus on human B-cells and T-cells and for HNRPU. Participates in B lymphocytes activation 2.67 0.0126 GBRG3  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 2.65 0.0129 ENSG00000186660 Antifreeze protein, type I; Zinc finger, C2H2-type 2.64 0.0131 ZNF490  May be involved in transcriptional regulation 2.64 0.0132 PCOC1  Binds to the C-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity 2.64 0.0132 ZNF506 May be involved in transcriptional regulation 2.62 0.0069 Q4ZG89  Major facilitator superfamily; Major facilitator superfamily MFS_1; Sugar transporter superfamily; Tetracycline resistance protein 2.61 0.0138 ATP7B  Involved in the export of copper out of the cells, such as the efflux of hepatic copper into the bile 2.61 0.0138 HSPA1A  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 2.60 0.0141  136 Gene Name Gene Description Fold Change p Value 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 2.59 0.0247 IG2AS  Putative insulin-like growth factor 2 antisense gene protein; IGF2-AS; PEG8/IGF2AS protein 2.58 0.0099 CP4F2   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 2.56 0.0152 Q8IY89   Acc:Q9H7C2]; CDNA: FLJ21062 fis, clone CAS01044. [Source:Uniprot/SPTREMBL 2.54 0.0156 NP_116216.1 Basic helix-loop-helix dimerisation region bHLH; Proline-rich region 2.53 0.0001 S29A2  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 2.53 0.016 PO4F1  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 2.53 0.0161 SCN4A  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 2.51 0.0167 TGFB3  Involved in embryogenesis and cell differentiation 2.50 0.0433 NP_071768.2 Ubiquitin 2.50 0.0293 EIF2AK4  Can phosphorylate the alpha subunit of EIF2 and may mediate translational control 2.49 0.0172 HXB5  Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior- posterior axis 2.48 0.0175 NP_659002.1  Antifreeze protein, type I; Pollen allergen Poa pIX/Phl pVI, C-terminal; Zinc finger, CCCH-type 2.48 0.0176 MLZE Melanoma-derived leucine zipper-containing extranuclear factor 2.47 0.0177 DB118  Has antibacterial activity  2.47 0.0177  137 Gene Name Gene Description Fold Change p Value PA24B  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-arachidonyl- phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine 2.46 0.0183 GPR17  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 2.45 0.0185 ARHGF  Specific GEF for RhoA activation and the regulation of vascular smooth muscle contractility 2.44 0.0471 NLK  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 2.44 0.0034 DUFFY  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 2.43 0.0191 FBX24  Substrate-recognition component of the SCF (SKP1- CUL1-F-box protein)-type E3 ubiquitin ligase complex 2.42 0.0194 HEM4  Uroporphyrinogen-III synthase; UROS; Uroporphyrinogen-III cosynthetase; Hydroxymethylbilane hydrolyase [cyclizing]; UROIIIS 2.41 0.0198 CLC3A  C-type lectin domain family 3 member A precursor; C-type lectin superfamily member 1; Cartilage- derived C-type lectin 2.41 0.02 ADR2  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 2.40 0.0202 PO2F3  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 2.40 0.0204  138 Gene Name Gene Description Fold Change p Value P2RX1  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 2.40 0.0205 GNS  N-acetylglucosamine-6-sulfatase precursor; G6S; Glucosamine-6-sulfatase 2.38 0.0211 Q99919 Glycoside hydrolase family 2, TIM barrel 2.38 0.0212 MINK1   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 2.37 0.0214 Q9Y3K2  Glutathione S-transferase, C-terminal; Glutathione S-transferase, N-terminal 2.37 0.0215 TEAD1  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 2.37 0.0215 MRCKB  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 2.37 0.0217 ARL5A   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 2.36 0.0218 Q9NZS4  HECT 2.36 0.0222 PODO   Plays a role in the regulation of glomerular permeability, acting probably as a linker between the plasma membrane and the cytoskeleton 2.35 0.0225 ENSG00000135365 Proline-rich region; Zinc finger, PHD-type 2.34 0.0025 APOL1   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 2.34 0.023 H11  Histones H1 are necessary for the condensation of nucleosome chains into higher order structures 2.34 0.023 S26A2  Sulfate transporter. May play a role in endochondral bone formation 2.34 0.0231 ENSG00000104915 Target SNARE coiled-coil region 2.33 0.0235  139 Gene Name Gene Description Fold Change p Value BSN  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 2.32 0.0236 CXA3  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 2.32 0.0239 DARS2 Aspartyl-tRNA synthetase, mitochondrial precursor; Aspartate--tRNA ligase; AspRS 2.32 0.0241 NP_203752.1 Ankyrin; Tetratricopeptide TPR_1; Tetratricopeptide TPR_2; Tetratricopeptide region 2.31 0.0243 GNS  N-acetylglucosamine-6-sulfatase precursor; G6S; Glucosamine-6-sulfatase 2.31 0.0243 ZN501  May be involved in transcriptional regulation 2.30 0.0246 Q5VT18  Guanylate kinase; L27; PDZ/DHR/GLGF; Protein kinase; Src homology-3; Variant SH3 2.29 0.004 NP_612390.1 Ferric reductase, NAD binding; Flavoprotein pyridine nucleotide cytochrome reductase; NADH:cytochrome b5 reductase (CBR); Oxidoreductase FAD/NAD(P)-binding; Phenol hydroxylase reductase 2.29 0.0256 NP_057423.1 Guanine-nucleotide dissociation stimulator CDC25; RA 2.28 0.0258 MTL5  May have a role in spermatogenesis 2.28 0.026 TBCE  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 2.27 0.0262 NP_079136.1  SET 2.27 0.0263 NP_055148.2  CD80-like, immunoglobulin C2-set; Immunoglobulin; Immunoglobulin I-set; Immunoglobulin V-set; Immunoglobulin-like; Vascular cell adhesion molecule-1 2.27 0.0264 CLK3  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 2.27 0.0264 TMPS6  May play a specialized role in matrix remodeling processes in liver 2.27 0.0266 TXD13  Thioredoxin domain-containing protein 13 precursor 2.27 0.0267 NP_113634.3 Acc:Q6GW03]; HBV X-transactivated protein 13. [Source:Uniprot/SPTREMBL 2.26 0.0268 Q9H252-2  WD repeat protein 68; WD repeat protein An11 homolog 2.26 0.027  140 Gene Name Gene Description Fold Change p Value PELI1  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 2.26 0.027 Q9NXB7  Peptidase M14, carboxypeptidase A 2.26 0.0271 NP_001034976.1  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 2.25 0.0277 CEAM3   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 2.25 0.0277 CM35A  CMRF35-A antigen precursor; CMRF-35; CD300c antigen 2.25 0.0278 NP_057732.2 Lupus La protein; RNA-binding protein Lupus La; RNA-binding region RNP-1 (RNA recognition motif) 2.25 0.0278 ATG4A  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 2.24 0.028 EGR1  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 2.24 0.0282 ABI2   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 2.23 0.0286 Q8TC23   Negatively modulates RNA polymerase II function by binding to RPB5 2.21 0.0304 O60729-3 Dual specificity protein phosphatase; Tyrosine specific protein phosphatase and dual specificity protein phosphatase 2.20 0.0305 MFGM  Specific ligand for the alpha-v/beta-3 and alpha- v/beta-5 receptors. Also binds to phosphatidylserine- enriched 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 2.20 0.0305  141 Gene Name Gene Description Fold Change p Value KCD12  BTB/POZ domain-containing protein KCTD12; Pfetin; Predominantly fetal expressed T1 domain 2.20 0.0308 GP116  May have a role in the regulation of acid-base balance 2.20 0.0308 Q5TCU6   Band 4.1; I/LWEQ; Proline-rich region; Small proline- rich; Vinculin/alpha-catenin 2.20 0.031 S12A2  Electrically silent transporter system. Mediates sodium and chloride reabsorption. Plays a vital role in the regulation of ionic balance and cell volume 2.20 0.031 HDAC5  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 2.19 0.0314 ZN746  May be involved in transcriptional regulation 2.18 0.0321 KCP2   Keratinocytes-associated protein 2; KCP-2 2.18 0.0324 NP_071769.1  Guanylate-binding protein 2.17 0.0328 NP_005194.3 Collagen triple helix repeat; Galanin 3 receptor; Proline-rich region 2.17 0.0331 G137B  Integral membrane protein GPR137B; Transmembrane 7 superfamily member 1 2.16 0.0336 MESD2  Mesoderm development candidate 2; Renal carcinoma antigen NY-REN-61 2.16 0.034 CP2C9  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 2.15 0.034 SO2B1  Mediates the Na(+)-independent transport of organic anions such as taurocholate, the prostaglandins PGD2, PGE1, PGE2, leukotriene C4, thromboxane B2 and iloprost 2.15 0.0344 ENSA   Endogenous ligand for sulfonylurea receptor. By inhibiting sulfonylurea from binding to the receptor, it reduces K(ATP) channel currents and thereby stimulates insulin secretion 2.14 0.0348 Q9NZ71-2  Decoy receptor for the cytotoxic ligands TNFS14/LIGHT and TNFSF6/FASL. Protects against apoptosis; Probable ATP-dependent DNA helicase 2.14 0.0348 FCHO2  FCH domain only protein 2 2.14 0.0348 DBC1  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 2.13 0.0356  142 Gene Name Gene Description Fold Change p Value HMOX1  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 2.13 0.036 NUD16  NUDIX hydrolase 2.13 0.0362 GTR14   Facilitative glucose transporter (By similarity). May have a specific function related to spermatogenesis; Facilitative glucose transporter. Probably a neuronal glucose transporter 2.12 0.0365 GLPE  This protein is a minor sialoglycoprotein in human erythrocyte membranes 2.12 0.037 GCNT1  Forms critical branches in O-glycans 2.12 0.0371 CTDP1  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 2.11 0.038 Q8N8K0 Myb, DNA-binding 2.10 0.0385 NP_003311.2  Functions in signal transduction from heterotrimeric G protein-coupled receptors. Could be involved in the hypothalamic regulation of body weight 2.10 0.0388 U2AFL  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 2.09 0.0397 LRC56  Leucine-rich repeat-containing protein 56 2.09 0.0397 STXB2  Involved in the protein trafficking from the Golgi apparatus to the plasma membrane 2.08 0.0399 GTF2E1  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 2.08 0.0399 NP_037422.2 Tat binding protein 1-interacting 2.08 0.0088 H2B2E  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 post- translational modifications of histones, also called histone code, and nucleosome remodeling 2.08 0.04 AQP10  Forms a water channel. Not permeable to urea and glycerol. May contribute to water transport in the upper portion of small intestine 2.08 0.0405  143 Gene Name Gene Description Fold Change p Value H32  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 post- translational modifications of histones, also called histone code, and nucleosome remodeling 2.07 0.0415 NP_001028751.1  IQ calmodulin-binding region; Myosin head, motor region 2.07 0.0417 RNF34  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 2.06 0.0422 HIPK1  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 2.06 0.0424 UBCE7IP1  Isoform 1 acts as an E3 ubiquitin ligase, which accepts ubiquitin from specific E2 ubiquitin- conjugating 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 2.06 0.0424 BCAS1   Breast carcinoma amplified sequence 1; Novel amplified in breast cancer 1; Amplified and overexpressed in breast cancer 2.06 0.0425 SLC2B  May act as Rab effector protein and play a role in vesicle trafficking 2.06 0.0427 MPRD  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 2.05 0.0429 ANC4  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 2.05 0.0429 NP_064533.2 Acc:NP_064533]; spire homolog 1 [Source:RefSeq_peptide 2.05 0.01 RBM6  Specifically binds poly(G) RNA homopolymers in vitro 2.05 0.0433 GLYC  Interconversion of serine and glycine 2.05 0.0433  144 Gene Name Gene Description Fold Change p Value UGCGL1  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 2.05 0.0436 RHBL2  Involved in regulated intramembrane proteolysis and the subsequent release of functional polypeptides from their membrane anchors. Known substrate: EFNB3 2.05 0.0437 NP_061889.1 Eggshell protein; Intermediate filament protein; Keratin, type I 2.05 0.0438 ADRBK2  Specifically phosphorylates the agonist-occupied form of the beta-adrenergic and closely related receptors 2.04 0.0442 NP_006693.3 Cyclic nucleotide-binding; Patatin; Protein of unknown function UPF0028; cAMP/cGMP- dependent protein kinase 2.04 0.0429 ATXN7  Involved in neurodegeneration 2.04 0.0445 OR2B6  Putative odorant receptor 2.03 0.0449 DEN1A  DENN domain-containing protein 1A 2.03 0.0449 HXA9  Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior- posterior axis 2.03 0.0451 NP_612147.1 RUN 2.03 0.0454 RRS1  Involved in ribosome biogenesis 2.03 0.0458 PAK6  The activated kinase acts on a variety of targets 2.02 0.0461 FAM126A  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 2.02 0.0462 NP_001020951.1  Phospholipase/Carboxylesterase 2.02 0.0463 MAFG  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 2.02 0.0467 S26A4  Sodium-independent transporter of chloride and iodide 2.02 0.027  145 Gene Name Gene Description Fold Change p Value IL1F5  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 (IL- 1R1), that is present in epithelial barriers and takes part in local inflammatory response 2.02 0.047 MTLR  Receptor for motilin 2.01 0.0473 K1HB  Keratin, type I cuticular Ha3-II; Hair keratin, type I Ha3-II 2.01 0.0475 Q9UJT2-2  May play a role in testicular physiology, most probably in the process of spermatogenesis or spermiogenesis 2.01 0.0475 CEP41  Centrosomal protein of 41 kDa; Protein Cep41; Testis-specific gene A14 protein 2.01 0.0477 RNF39  May play a role in prolonged long term-potentiation (LTP) maintenance 2.00 0.0484 SC5A5  Mediates iodide uptake in the thyroid gland 2.00 0.0485 ANGL2  Induces sprouting in endothelial cells through an autocrine and paracrine action -2.01 0.0175 CN159   UPF0317 protein C14orf159, mitochondrial precursor -2.01 0.048 ESR1  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 -2.01 0.0476 NP_055721.3  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 -2.01 0.0003 Q6WBX8-3  Cell cycle checkpoint control protein RAD9B homolog; RAD9 homolog B; hRAD9B -2.01 0.0472 BACE2  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 -2.01 0.0472 PGAM2  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 -2.02 0.031 DLL1  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 T- cell/NK-cell precursor -2.02 0.0468  146 Gene Name Gene Description Fold Change p Value P25A  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 -2.02 0.0464 P73  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 -2.02 0.0323 CDN2D  Interacts strongly with CDK4 and CDK6 -2.02 0.0463 PROD  Converts proline to delta-1-pyrroline-5-carboxylate -2.03 0.0165 RFX2  DNA-binding protein RFX2 -2.04 0.0194 KIF17   Transports vesicles containing N-methyl-D-aspartate (NMDA) receptor 2B along microtubules -2.04 0.0446 RGS20  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 -2.04 0.0445 NLGN3   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 glia- neuron interactions in the developing peripheral nervous system -2.04 0.0443 ADIPO  Important adipokine involved in the control of fat metabolism and insulin sensitivity, with direct anti- diabetic, 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 -2.04 0.0442 EMAL2  May modify the assembly dynamics of microtubules, such that microtubules are slightly longer, but more dynamic -2.04 0.0441 MMP12  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 -2.04 0.0198 RBTN1  May be involved in gene regulation within neural lineage cells potentially by direct DNA binding or by binding to other transcription factors -2.05 0.0318  147 Gene Name Gene Description Fold Change p Value HNF4G  Transcription factor. Has a lower transcription activation potential than HNF4-alpha -2.05 0.0104 FTHFD  10-formyltetrahydrofolate dehydrogenase; 10- FTHFDH; Aldehyde dehydrogenase 1 family member L1 -2.05 0.0433 CENPN  Component of the CENPA-NAC (nucleosome- associated) 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 -2.05 0.0405 FAM3C  Protein FAM3C precursor; Protein GS3786 -2.06 0.023 GBGT1  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 -2.06 0.0348 IFIH1  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 IFN- beta and RANTES (CCL5). ATPase activity is specifically induced by dsRNA. Essential for the production of interferons in response to picornaviruses -2.06 0.0421 Q8NI38  Ankyrin; Proline-rich region -2.06 0.042 MOSC2   Catalytic component of the benzamidoxime prodrug- converting complex, a complex required to reduce N- hydroxylated 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 prodrug- converting complex -2.06 0.042 ACV1B   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 -2.06 0.042  148 Gene Name Gene Description Fold Change p Value NP_060340.2 Acc:NP_060340]; transmembrane protein 132A isoform a [Source:RefSeq_peptide -2.06 0.0162 EP15  Involved in cell growth regulation. May be involved in the regulation of mitogenic signals and control of cell proliferation. Involved in the internalization of ligand- inducible receptors of the receptor tyrosine kinase (RTK) type, in particular EGFR -2.07 0.0064 AIFM2  Oxidoreductase, which may play a role in mediating a TP53/p53-dependent apoptosis response. Probable oxidoreductase that acts as a caspase- independent mitochondrial effector of apoptotic cell death. Binds to DNA in a sequence-independent manner. May contribute to genotoxin-induced growth arrest -2.07 0.0417 ABI2   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 -2.07 0.0417 SIRT6  Mono-ADP-ribosyltransferase sirtuin-6; SIR2-like protein 6 -2.07 0.0057 ENSG00000168827 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 -2.07 0.0415 68MP  6.8 kDa mitochondrial proteolipid -2.07 0.0412 ENSG00000130224 Calponin-like actin-binding; Leucine-rich repeat -2.07 0.0412 COF1  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 -2.07 0.0076 UDA1  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 -2.07 0.0411 NP_997208.1 Acc:NP_997208]; dpy-19-like 3 [Source:RefSeq_peptide -2.07 0.0408 NP_067081.2 Pistil-specific extensin-like protein -2.08 0.0408 OTOR  Otoraplin precursor; Fibrocyte-derived protein; Melanoma inhibitory activity-like protein -2.08 0.0406 STON1   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 -2.08 0.0372 LRC17  Leucine-rich repeat-containing protein 17 precursor; p37NB -2.08 0.0403  149 Gene Name Gene Description Fold Change p Value 7B2  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 C- terminal peptide inhibits PCSK2 in vitro -2.08 0.0402 SORT  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 -2.08 0.0401 EGFR  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 -2.08 0.0399 FABD  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 -2.09 0.0396 MASP2  Serum protease that plays an important role in the activation of the complement system via mannose- binding lectin. After activation by auto-catalytic cleavage it cleaves C2 and C4, leading to their activation and to the formation of C3 convertase -2.09 0.0395 NP_055451.1 Guanine-nucleotide dissociation stimulator CDC25; Pleckstrin-like -2.09 0.0394 PAX9  Transcription factor required for normal development of thymus, parathyroid glands, ultimobranchial bodies, teeth, skeletal elements of skull and larynx as well as distal limbs -2.09 0.0083 UT14C  Essential for spermatogenesis. May be required specifically for ribosome biogenesis and hence protein synthesis during male meiosis -2.09 0.0367  150 Gene Name Gene Description Fold Change p Value H2B2E  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 post- translational modifications of histones, also called histone code, and nucleosome remodeling -2.10 0.039 QSK  Serine/threonine-protein kinase QSK -2.10 0.0388 MALT1  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 -2.10 0.0159 CASB  Important role in determination of the surface properties of the casein micelles -2.10 0.0386 NP_061959.2 Immunoglobulin; Immunoglobulin I-set; Immunoglobulin V-set; Immunoglobulin-like -2.10 0.0385 BBC3  Essential mediator of p53-dependent and p53- independent apoptosis -2.10 0.0089 Q13862 Acc:Q13862]; DNA-binding protein (Hypothetical protein SPBPBP). [Source:Uniprot/SPTREMBL -2.10 0.0382 DDX23  Probably involved in pre-mRNA splicing -2.10 0.0228 NP_065976.2 Cache; VWA N-terminal; von Willebrand factor, type A -2.11 0.0376 INGR1  Receptor for interferon gamma. Two receptors bind one interferon gamma dimer -2.11 0.0065 DYN1  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 -2.11 0.0047 COPE  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 -2.12 0.0371 MK  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 -2.12 0.0046 ARRD3  Arrestin domain-containing protein 3 -2.12 0.0029 Q5T5W6   Myb, DNA-binding -2.12 0.037 ORML3  ORM1-like protein 3 -2.12 0.0025  151 Gene Name Gene Description Fold Change p Value ARL3  Does not act as an allosteric activator of the cholera toxin catalytic subunit -2.12 0.0365 CP39A  Involved in the bile acid metabolism. Has a preference for 24-hydroxycholesterol, and converts it into a 7-alpha-hydroxylated product -2.12 0.0365 UBR1 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 -2.12 0.0365 FAT3  Aspartic acid and asparagine hydroxylation site; Cadherin; EGF-like; EGF-like calcium-binding; EGF- like region; EGF-like, laminin; EGF-like, type 2; EGF- like, type 3; Laminin G; Laminin G, subdomain 1; Laminin G, subdomain 2; Proline-rich region -2.12 0.0364 OAT4  Mediates saturable uptake of estrone sulfate, dehydroepiandrosterone sulfate and related compounds -2.13 0.0002 Q5VZI7   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 -2.13 0.0359 AGGF1  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 -2.13 0.0358 ENSG00000204124 Doublecortin -2.13 0.0355 NP_071375.1  AAA ATPase, core; ATPase associated with various cellular activities, AAA-5; Proline-rich region -2.14 0.0204 DNAL4  Force generating protein of respiratory cilia. Produces force towards the minus ends of microtubules. Dynein has ATPase activity -2.14 0.0351 MLPH  Rab effector protein involved in melanosome transport. Serves as link between melanosome- bound RAB27A and the motor protein MYO5A -2.15 0.0344 F262  Synthesis and degradation of fructose 2,6- bisphosphate -2.15 0.0143 PLMN   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 -2.16 0.0333  152 Gene Name Gene Description Fold Change p Value RIP  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 -2.17 0.0328 NP_478126.1 Exoribonuclease -2.17 0.0265 GCYA2   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 -2.17 0.0325 GNAO1   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 -2.17 0.0017 PADI2  Catalyzes the deimination of arginine residues of proteins -2.19 0.0313 PREP   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 -2.19 0.0313 NRK2  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 -2.19 0.0312 ETV6  Transcriptional repressor; binds to the DNA sequence 5'-CCGGAAGT-3' -2.19 0.0312 PRMT7  Probably methylates the guanidino nitrogens of arginyl residues in some proteins -2.20 0.0309 IF  Promotes absorption of the essential vitamin cobalamin (Cbl) in the ileum by specific receptor- mediated endocytosis -2.20 0.0004 MAGBI  Melanoma-associated antigen B18; MAGE-B18 antigen -2.20 0.0005 TTC5  Tetratricopeptide repeat protein 5; TPR repeat protein 5 -2.20 0.0306 TRAIP  Inhibits activation of NF-kappa-B mediated by TNF -2.20 0.0304 NP_001070249.1 Zinc finger, C2H2-subtype; Zinc finger, C2H2-type -2.21 0.0301 NBEA   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 organelle- associated proteins -2.21 0.03  153 Gene Name Gene Description Fold Change p Value Q7Z5G2   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 -2.21 0.0299 NP_116107.2 Major facilitator superfamily; Major facilitator superfamily MFS_1; Sugar transporter superfamily; Tetracycline resistance protein -2.22 0.0296 Q53RC4   Fodrin, which seems to be involved in secretion, interacts with calmodulin in a calcium-dependent manner and is thus candidate for the calcium- dependent movement of the cytoskeleton at the membrane -2.22 0.0161 ZN343  May be involved in transcriptional regulation -2.22 0.0295 WBS14  Transcriptional repressor. Binds to the canonical and non-canonical E box sequences 5'-CACGTG-3' -2.22 0.0292 IPP   May play a role in organizing the actin cytoskeleton -2.22 0.0292 P04278-2  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 -2.23 0.029 Q8N157-3 G-protein, beta subunit; Src homology-3; Variant SH3; WD-40 repeat -2.23 0.0048 RB33A  Ras-related protein Rab-33A; Small GTP-binding protein S10 -2.23 0.0287 FPRL1  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 G- protein 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) -2.23 0.0287 NP_055873.1  ATPase associated with various cellular activities, AAA-5; von Willebrand factor, type A -2.24 0.003 Q7Z5Q7 Acc:Q7Z5Q7]; Lung cancer oncogene 5. [Source:Uniprot/SPTREMBL -2.24 0.0283 SEC62  Required for preprotein translocation -2.24 0.0051 LMA2L  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 -2.24 0.0042 KCD17   BTB/POZ domain-containing protein KCTD17 -2.24 0.0116  154 Gene Name Gene Description Fold Change p Value GALA  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 -2.24 0.0281 Q9Y4E5-2  May be involved in transcriptional regulation. Coactivator for steroid receptors -2.25 0.0277 ZN460  May be involved in transcriptional regulation -2.25 0.0274 REXO1  Seems to have no detectable effect on transcription elongation in vitro -2.26 0.0273 YETS2  YEATS domain-containing protein 2 -2.26 0.027 CAN5  Calpain-5; nCL-3; htra-3 -2.26 0.0269 NP_940905.2  Blood group Rhesus C/E and D polypeptide; Calcium-activated BK potassium channel, alpha subunit; EAG/ELK/ERG potassium channel; Ion transport 2 -2.26 0.0269 C8AP2  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 -2.26 0.0269 CAN13  Probable non-lysosomal thiol-protease -2.26 0.0269 MGST3  Also functions as a glutathione peroxidase -2.26 0.0268 RHES  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 -2.28 0.0262 PHF6  May play a role in transcriptional regulation -2.28 0.0053 NAPSA  May be involved in processing of pneumocyte surfactant precursors -2.29 0.0256 NP_057593.2 C-type lectin; Type II antifreeze protein -2.29 0.0252 TM107  Transmembrane protein 107 -2.30 0.0251 ITF2   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' -2.30 0.025 NP_065805.1 Proline-rich region; RhoGAP -2.30 0.0248 ARP8  Actin-related protein 8 -2.30 0.0248  155 Gene Name Gene Description Fold Change p Value COA1   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 -2.30 0.0027  156 Gene Name Gene Description Fold Change p Value K1C19  Involved in the organization of myofibers. Together with KRT8, helps to link the contractile apparatus to dystrophin at the costameres of striated muscle -2.31 0.0013 DEF4  This peptide has antibiotic and anti-fungi activity -2.31 0.0243 CC28A  Coiled-coil domain-containing protein 28A; CCRL1AP -2.32 0.0241 ITCH   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 -2.32 0.0241 CCAR1  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 -2.32 0.024 Q8TCQ1  Zinc finger, RING-type -2.32 0.0136 KCNN3  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 -2.32 0.0142 NP_071746.1 Antibiotic biosynthesis monooxygenase; Calcium- binding EF-hand -2.32 0.0239 NP_006171.2  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 -2.33 0.0052 ACHA2  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 -2.33 0.0234 GPR161 Orphan receptor -2.33 0.0234 Q5TCU6   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 -2.34 0.0008 GO45  Required for normal Golgi structure and for protein transport from the endoplasmic reticulum (ER) through the Golgi apparatus to the cell surface -2.34 0.0229 SFRIP  Plays a role in pre-mRNA alternative splicing by regulating spliceosome assembly -2.35 0.0132 NP_060340.2 Acc:NP_060340]; transmembrane protein 132A isoform a [Source:RefSeq_peptide -2.35 0.0224  157 Gene Name Gene Description Fold Change p Value STK4  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 -2.35 0.0029 SIGIR  Acts as a negative regulator of the Toll-like and IL- 1R 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 -2.36 0.0219 NP_777577.1  AMP-dependent synthetase and ligase -2.38 0.0213 STML1  Stomatin-like protein 1; SLP-1; Stomatin-related protein; STORP; EPB72-like 1; UNC24 homolog -2.38 0.021 CRLD2  Cysteine-rich secretory protein LCCL domain- containing 2 precursor; LCCL domain-containing cysteine-rich secretory protein 2; Cysteine-rich secretory protein 11; CRISP-11 -2.40 0.0202 NP_689704.3 Peptidase M20; Peptidase M20, dimerisation -2.40 0.0012 TNF18  Cytokine that binds to TNFRSF18/AITR/GITR. Important for interactions between activated T- lymphocytes and endothelial cells and may modulate T-lymphocyte survival in peripheral tissues -2.41 0.0201 DCTN3  Together with dynein may be involved in spindle assembly and cytokinesis -2.41 0.0198 NP_542398.2 Acc:NP_542398]; coiled-coil domain containing 104 [Source:RefSeq_peptide -2.42 0.0004 ZN179  Zinc finger protein 179; Brain finger protein; RING finger protein 112 -2.42 0.0196 PER1   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 -2.43 0.0435 NP_001073884.1 Lymphocyte-specific protein; Proline-rich region; RNA-binding region RNP-1 (RNA recognition motif); SWAP/Surp -2.43 0.0313 GLT12  Catalyzes the initial reaction in O-linked oligosaccharide biosynthesis, the transfer of an N- acetyl-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 -2.44 0.0188  158 Gene Name Gene Description Fold Change p Value SFXN5  Transports citrate. Potential iron transporter -2.44 0.0187 NP_006429.1 C-type lectin; Collagen triple helix repeat -2.44 0.0187 SIP1  Transcriptional inhibitor that binds to DNA sequence 5'-CACCT-3' in different promoters. Represses transcription of E-cadherin -2.45 0.0186 APBA2  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 -2.45 0.0185 SHOC2  Leucine-rich repeat protein SHOC-2; Ras-binding protein Sur-8 -2.45 0.0184 MYL4  Regulatory light chain of myosin. Does not bind calcium -2.46 0.018 OPCML  Binds opioids in the presence of acidic lipids; probably involved in cell contact -2.47 0.0179 TCF3  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 -2.47 0.0179 MPDZ   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 -2.48 0.0175 Q5VTQ0  Peptidase S26A, signal peptidase I; Tetratricopeptide TPR_2; Tetratricopeptide region -2.48 0.0175 P66A  Transcriptional repressor -2.48 0.0484 NTCP2   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 -2.49 0.0171 CLC4E  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 -2.51 0.0165 GBRL1  Gamma-aminobutyric acid receptor-associated protein-like 1; GABA(A) receptor-associated protein- like 1; Glandular epithelial cell protein 1; GEC-1; Early estrogen-regulated protein -2.52 0.0003 NAPG  Required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus -2.52 0.0164  159 Gene Name Gene Description Fold Change p Value PACS2  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 -2.52 0.0162 Q6ZN28  Variant SH3 -2.53 0.0016 LAT3  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 -2.54 0.0157 TLR5  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 -2.56 0.0153 NP_001001552.3 Lamino-associated polypeptide 2/emerin -2.57 0.0052 PRG3  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 -2.57 0.0149 RHBT2  Rho-related BTB domain-containing protein 2; Deleted in breast cancer 2 gene protein; p83 -2.57 0.0149 Q6ZNV0 Ribosomal protein L22/L17 -2.57 0.0147 WDR55  WD repeat protein 55 -2.58 0.0147 DHX35  May be involved in pre-mRNA splicing -2.58 0.0146 CDS1  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 -2.58 0.0145 Q6PJS5  Zinc finger, B-box; Zinc finger, RING-type -2.59 0.027 TSN9  Tetraspanin-9; Tspan-9; Tetraspan NET-5 -2.60 0.0163 CLCN4  Voltage-gated chloride channel. Chloride channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport -2.60 0.0141 TMCC3  Transmembrane and coiled-coil domains protein 3 -2.60 0.005 NP_060615.1 Acc:NP_060615]; family with sequence similarity 82, member C [Source:RefSeq_peptide -2.60 0.0039 NP_115510.1 Acc:NP_115510]; glutamine rich 2 [Source:RefSeq_peptide -2.61 0.0018  160 Gene Name Gene Description Fold Change p Value Q9NSG0-5  GTPase activator for the Rho-type GTPases by converting them to an inactive GDP-bound state -2.62 0.0257 ANK3  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 -2.63 0.0135 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 -2.63 0.0002 DOCK5   Protein phosphatase 2A regulatory subunit PR55 -2.64 0.0132 Q9NS68-2  Can mediate activation of JNK and NF-kappa-B. May promote caspase-independent cell death -2.64 0.0131 ATP2C2  This magnesium-dependent enzyme catalyzes the hydrolysis of ATP coupled with the transport of calcium -2.65 0.0129 COG2  Required for normal Golgi morphology and function -2.65 0.0493 NP_659456.2 Acc:NM_145019]; family with sequence similarity 124A (FAM124A), mRNA [Source:RefSeq_dna -2.66 0.0128 CT038   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 -2.66 0.0127 PRND  Not known -2.67 0.0126 NP_001074014.1 Protein phosphatase inhibitor, 1DARPP-32 -2.67 0.0018 GBRG2   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 -2.67 0.0124 SMYD4  SET and MYND domain-containing protein 4 -2.67 0.0124 NUP54  Component of the nuclear pore complex, a complex required for the trafficking across the nuclear membrane -2.68 0.0122 PEVR2  Acts as receptor for porcine endogenous retrovirus subgroup A (PERV-A) -2.69 0.012 CBX8  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 -2.69 0.0068  161 Gene Name Gene Description Fold Change p Value PTPRD  Receptor-type tyrosine-protein phosphatase delta precursor; Protein-tyrosine phosphatase delta; R- PTP-delta -2.70 0.0118 NP_758438.1 Transcription factor AP-2, C-terminal -2.71 0.0212 TESK2  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 -2.71 0.0116 E2F3  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 cell- cycle progression from G1 to S phase. E2F-3 binds specifically to RB1 protein, in a cell-cycle dependent manner -2.72 0.0116 SPG20  May be implicated in endosomal trafficking, or microtubule dynamics, or both -2.73 0.0114 KU70   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 -2.73 0.0114 NEUROD2  Appears to mediate neuronal differentiation -2.73 0.0113 NP_112196.2 N2,N2-dimethylguanosine tRNA methyltransferase; Zinc finger, C2H2-type -2.74 0.0112 TNR9  Receptor for TNFSF14/4-1BBL. Possibly active during T cell activation -2.75 0.011 AIFM1  Probable oxidoreductase that acts as a caspase- independent mitochondrial effector of apoptotic cell death. Extramitochondrial AIF induces nuclear chromatin condensation and large scale DNA fragmentation (in vitro) -2.75 0.011   

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