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Interleukin-Iβ signaling in intestinal epithelial cells Parhar, Kuljit Singh 2003

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INTERLEUKIN-ip SIGNALING IN INTESTINAL EPITHELIAL CELLS by KULJIT SINGH P A R H A R B.ScH, The University of British Columbia, 2001  A thesis submitted in partial fulfillment of the requirements for the degree of M A S T E R ' S OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Medicine; Experimental Medicine Program)  W e accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A October 2003 © Kuljit Singh Parhar, 2003  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his  or  her representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ABSTRACT  Although long regarded as providing a passive physical barrier to the contents of the lumen, a large body of evidence implicates intestinal epithelial cells (lECs) as playing a central role in the regulation of the gut immune response. One of the major immune functions of intestinal epithelial cells is the production of chemotactic cytokines, known as chemokines, which are responsible for the recruitment and activation of the underlying mononuclear cells, neutrophils, and dendritic cells. Several soluble agonists, including bacterial products as well as host-derived cytokines, are capable of driving this response. through  the  The prototypic proinflammatory interleukin-1 receptor  (IL-1 R),  agonist interleukin-1(3 the founding  member  (IL-1 p), signals of the  IL-1 R  superfamily, and robustly activates this pro-inflammatory cascade. Synthesis of many downstream  chemokines in  transcription factor N F K B .  response to  IL-1|5, requires the  activation  of  the  The objective of the work presented here is to characterize  the regulatory pathways responsible for chemokine synthesis in l E C s in response to IL1p\  and to examine their relationship with N F K B .  Three important pathways in IL-1 (3  signaling were examined. Initially the M A P K family of pathways was examined. IL-1 p treatment led to activation of ERK,  p38 M A P K , as well as J N K . Inhibition of ERK  effect on either chemokine synthesis, or N F K B activation.  had no  Inhibition of p38 M A P K  caused a 50% reduction in IL-1 p induced chemokine release. This effect was due to regulation of the IL-8 promoter, and independent of N F K B regulation.  and posttranscriptional  J N K inhibition, using curcumin or a pharmacological inhibitor, attenuated  N F K B  activation and IL-8 promoter activation; however, only curcumin was able to  inhibit chemokine release. The next chapter focuses on regulation by protein kinase CK2.  C K 2 was found increased in tissues of ulcerative colitis patients when compared  to normal uninflamed tissue. Its activity was required for the activation of N F K B , and this was through regulation of p65 transactivation in the nucleus of the cell, via serine 529.  A s a result, C K 2 inhibition leads to attenuation of chemokine synthesis. Finally  the phosphatidylinositol-3 kinase (PI3K) pathway was examined. Activation of PI3K, protein kinase B (PKB) and 3'-phosphoinositide dependant kinase (PDK1) were all required for chemokine synthesis as well as N F K B differing regulatory roles. undetermined mechanism.  activation, although they had  PI3K and P K B regulated N F K B transactivation, through an PDK1 regulated the IKK complex, potentially through a  phosphorylation at serine residues 180/181 on IKKa and IKK(3. This resulted in PDK1 regulation of N F K B D N A binding, as well as p65 transactivation through phosphorylation of serine 536. The work presented here has examined the signaling pathways important in the regulation of IEC chemokine release. This may provide potential targets for in vivo studies of  chronic  inflammation,  inflammatory conditions such as IBD.  and thus  have  implications for  chronic  TABLE OF CONTENTS ABSTRACT T A B L E OF C O N T E N T S LIST OF T A B L E S LIST O F F I G U R E S ABBREVIATIONS ACKNOWLEDGEMENTS C H A P T E R 1 - G E N E R A L INTRODUCTION 1.1 INTESTINAL EPITHELIAL C E L L S 1.1.1 Biology 1.1.2 Innate Immune Response 1.2 CHEMOKINES 1.2.1 Structure, Expression, and Function 1.2.2 IL-8 1.3  ii iv vii viii xi xiv 1 1 1 1 4 4 6 8  N F K B  1.3.1 Structure 1.3.2 Regulation 1.3.3 Function 1.4 IEC SIGNALING 1.4.1 IL-1 signaling 1.4.2 M A P Kinases 1.4.3 Protein Kinase C K 2 1.4.4 PI3K Pathway 1.4.4 IKK complex 1.5 INFLAMMATORY BOWEL DISEASE 1.5.1 Pathology 1.5.2 Pathogenesis 1.5.3 The role of lECs, chemokines, and N F K B CHAPTER 2 - MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 Cell lines and Cell Culture 2.1.2 Reagents, Enzymes, and Chemicals 2.1.3 Plasmids and c D N A s 2.1.4 Primers 2.2 METHODS 2.2.1 Nuclear Preps 2.2.2 Electrophoretic Mobility Shift Assay (EMSA) 2.2.3 Immunoblotting 2.2.4 C K 2 Phosphotransferase Activity Assays 2.2.5 Transient Transfections 2.2.6 Luciferase Assay 2.2.7 Isolation of R N A and R T - P C R 2.2.8 Tissue Procurement and Immunohistochemistry 2.2.9 ELISA assays for IL-8 and MCP-1 iv  8 9 10 12 12 14 16 17 19 21 21 22 24 29 29 29 29 30 30 31 31 32 33 34 35 35 36 36 37  2.2.10 2.2.11 2.2.12  M A P K A P K 2 assay Immunoprecipitation Immune complex assays for P K B and IKK signalsome  37 38 38  C H A P T E R 3 - M A P K R E G U L A T I O N O F IL-1 P SIGNALING 40 3.1 RATIONALE AND HYPOTHESIS 40 3.2 RESULTS 40 3.2.1 IL-1 p activates protein tyrosine phosphorylation and M A P K s in l E C s 40 3.2.2 S B 203580 attenuates IL-8 and MCP-1 production by IL-1 p stimulated lECs 41 3.2.3 I K B phosphorylation and degradation are independent of p38 M A P K 42 3.2.4 N F K B DNA binding and transactivation are independent of p38 M A P K activation 42 3.2.5 IL-8 and MCP-1 messages are regulated by p38 in l E C s 44 3.2.6 p38 is involved in activation of the IL-8 promoter 44 3.2.7 Pharmacological inhibitor S P 600125 inhibits IL-1 induced J N K activity however has no effect on p65 DNA binding 45 3.2.8 S P 600125 inhibits IL-1 (3 induced N F K B activation and IL-8 promoter activation 46 3.3 DISCUSSION 57 C H A P T E R 4 - C K 2 regulation of IEC N F K B Activation and Chemokine Synthesis 62 4.1 RATIONALE AND HYPOTHESIS 62 4.2 RESULTS 63 4.2.1 C K 2 activity is increased in patients with active IBD 63 4.2.2 C K 2 activity is increased in a murine model of colitis 63 4.2.3 Inhibition of CK2 prevents IL-1 p induced N F K B activation 64 4.2.4 Overexpression of a kinase inactive C K 2 inhibits IL-1 p induced N F K B activation 64 4.2.5 C K 2 regulates the transactivation of p65 65 4.2.6 C K 2 a associates with p65, exclusively in the nucleus 66 4.2.7 p65 associated C K 2 activity is increased with IL-1 p stimulation 66 4.2.8 Inhibition of C K 2 prevents IL-1 p induced activation of the IL-8 proximal promoter 67 4.2.9 Inhibition of CK2 prevents the message synthesis of N F K B downstream targets 67 4.2 DISCUSSION 78 C H A P T E R 5 - PI3K and PDK1 regulation of IL-1 (3 induced N F K B and chemkokine production 81 5.1 RATIONALE 81 5.2 RESULTS 81 5.2.1 Inhibition of both PI3K and PDK1 results in attenuation of IL-1 p induced N F K B activation 81 5.2.2 Overexpression of PDK1 or P K B is sufficient to drive N F K B activation. ...82 5.2.3 IL-1 p induces P K B serine 473 phosphorylation and also its specific activity. 82 5.2.4 Overexpression of PDK1 but not P K B activates the IKK complex 83 v  5.2.5 Pharmacological inhibition of PDK1 but not PI3K results in the inhibition of p65 DNA binding 84 5.2.6 Pharmacological inhibition of P K B results in the attenuation of p65 transactivation 84 5.2.7 Pharmacological inhibition of PDK1 results in the attenuation of p65 transactivation 85 5.2.8 Overexpression of P K B or PDK1 is sufficient to induce p65 transactivation 85 5.2.9 PDK1 and P K B regulate p65 transactivation through unique sites 85 5.2.10 PDK1 and IKK coassociate 86 5.2.11 IKK a/p ser 177/181 is sensitive to T P C K , and is a potential PDK1 phosphorylation site 87 5.2.12 Both PI3K and PDK1 are required for the activation of the IL-8 promoter 87 5.2.13 Overexpression of PDK1 is sufficient to activate the IL-8 promoter 87 5.2.14 Overexpression of PDK1 is sufficient to IL-8 message synthesis 88 5.3 DISCUSSION 104 CHAPTER 6 - GENERAL CONCLUSIONS 107 REFERENCES 109  vi  LIST OF TABLES  Table 1 - Activators and Inducers of N F K B in l E C s  vii  LIST OF FIGURES  Figure 1.  Signaling pathways activated by IL-ip  Figure 2.  N F K B  Figure 3.  Detailed activation of N F K B , including important regulatory  family members  27  phosphorylations Figure 4.  26  28  IL-ip stimulation of l E C s results in the activation of E R K , J N K , and p38  47  Figure 5.  Inhibition of p38 M A P K attenuates IL-ip induced IL-8 production  48  Figure 6.  IL-ip induced degradation of I K B is independent of p38  49  Figure 7.  IL-ip induced activation of N F K B is independent of p38  50  Figure 8.  IL-ip induced activation of N F K B is not modulated by overexpression of kinase inactive p38  51  Figure 9.  Inhibition of p38 attenuates IL-ip induced AP-1 D N A binding  52  Figure 10.  p38 M A P K regulates IL-ip induced IL-8 and MCP-1 m R N A expression  Figure 11.  53  IL-1 p induced activation of the IL-8 promoter is dependent on p38 M A P K  Figure 12.  54  Selective J N K inhibitor S P 600125 inhibits J N K activity, however does not regulate N F K B DNA binding  Figure 13.  55  Selective J N K inhibitor S P 600125 inhibits N F K B and IL-8 promoter activation  56  viii  Figure 14.  C K 2 a is overexpressed in patients with active U C  Figure 15.  D S S induced colitis in mice increases C K 2 activity in actively inflamed tissue  Figure 16.  70  Inhibition of C K 2 using a selective inhibitor prevents IL-1 (5 induced activation of N F K B in intestinal epithelial cells  Figure 17.  72  C K 2 regulation of IL-1 p induced N F K B activation is at the level of transactivation through p65 serine 529  Figure 19.  71  Overexpression of C K 2 a or a ' modulates both basal and IL-1 p induced N F K B activation  Figure 18.  69  73  C K 2 a associates with p65, and this association occurs exclusively in the nucleus  74  Figure 20.  p65 bound C K 2 a activity increase with I L - i p treatment  75  Figure 21.  Inhibition of C K 2 prevents IL-1 p induced activation of the IL-8 proximal promoter  Figure 22.  76  Inhibition of C K 2 prevents the synthesis of IL-1 p induced I K B and chemokines  77  Figure 23.  Inhibition of PI3K inhibits IL-1 p induced N F K B activation  89  Figure 24.  Inhibition of PDK1 inhibits IL-1 p induced N F K B activation  90  Figure 25.  Overexpression of PDK1 or P K B is sufficient for the activation of N F K B  91  Figure 26.  IL-1 p activates P K B and the phosphorylation of I K B and p65  92  Figure 27.  Overexpression of P D K 1 , but not P K B , results in the activation of IKK activity  93 ix  Figure 28.  Inhibition of PDK1 N F K B  but not PI3K results in the inhibition of  nuclear translocation and subsequent D N A binding  94  Figure 29.  Inhibition of PI3K inhibits IL-1 p induced N F K B transactivation  95  Figure 30.  Inhibition of PDK1  96  Figure 31.  Overexpression of PDK1  inhibits IL-1 p induced N F K B transactivation and P K B results in increased  transactivation Figure 32.  97  PDK1 mediated increased transactivation is dependant on p65  serine 536  98  Figure 33.  PDK1 coassociates with the IKK signalsome  Figure 34.  Ser 180/181 on IKKa/p are potential PDK1 phosphorylation sites and are sensitive to T P C K  Figure 35.  Inhibition of PI3K or PDK1  100  inhibits IL-1 p induced proximal  IL-8 promoter activation Figure 36.  Overexpression of PDK1  101 or PKB  results in the activation  of the IL-8 proximal promoter Figure 37.  Overexpression of PDK1  99  102  but not P K B results in increased  IL-8 message synthesis  103  x  ABBREVIATIONS Abbreviation  Definition  3' UTR  3' untranslated region activator protein 1 AU-rich cis regulatory elements activating transcription factor-2 C A A T / enhancer binding protein calmodulin activated kinase IV C R E B binding protein Crohn's disease cellular inhibitor of FLICE C / E B P homologous protein cellular inhibitor of apoptosis cyclooxygenase-2 CCCTC-binding factor 5,6 dichloro-ribifuranosylbenzimidazole dextran sodium sulfate enzyme linked immunosorbent assay electrophoretic mobility shift assay epithelial cell derived neutrophil attractant 78 extracellular signal regulated kinase empty vector fetal bovine serum growth related oncogene a histone acetyl transferase heat shock protein inhibitor of K B inflammatory bowel disease intestinal epithelial cells inhibitor of K B kinase interleukin-ip interleukin 18 receptor interleukin-1 receptor interleukin-1 receptor antagonist interleukin-1 receptor, type 1 interkeukin-1 receptor, type 2 integrin linked kinase inducible nitric oxide synthase interferon y inducible protein interkeukin-1 receptor associated kinase insulin receptor substrate interferon inducible T cell attractant c-jun NH2-terminal kinase  AP-1 ARE ATF-2 C/EBP C A M K IV CBP CD cFLIP CHOP clAP COX-2 CTCF DRB DSS ELISA EMSA ENA-78 ERK EV FBS GROcc  HAT HSP IKB  IBD IEC IKK  IL-1P IL-18R IL-1R IL-1RA IL-1 RI IL-1RII ILK iNOS  IP-10 IRAK IRS  l-TAC JNK  xi  KD LPMNC LPS LTBR MAPK MAPKAPK2 MAPKK MAPKKK MCP MDC MEKK MHC MIG MIP MLK3 MSK1 N F K B  NIK NRE NRF p90 RSK PAMP PCR PDK1 PI(3,4,5)P3 PI(4,5)P2 PI3K PKA PKB PKC PRR RT-PCR SDS-PAGE SH2 TAD TAK1 TGFp THi TH tir TNFa TNFR1 TPCK TRAF-6 TWEAK UC 2  kinase dead lamina propria mononuclear cell lipopolysaccharide leukotriene p receptor mitogen activated protein kinase mitogen activated protein kinase activated protein kinase-2 mitogen activated protein kinase kinase mitogen activated protein kinase kinase kinase monocyte chemoattracant protein macrophage derived chemokine MEK kinase major histocompatibility complex monokine induced by interferon y macrophage inflammatory protein mixed Unease kinase-3 mitogen and stress activated kinase-1 nuclear factor K B N F K B inducing kinase negative regulatory element nuclear factor K B repressing factor p90 ribosomal S6 kinase pathogen associated molecular pattern polymerase chain reaction 3' phosphoinositide dependant kinase-1 phosphoinositol(3,4,5)triphosphate phosphoinositol(4,5)biphosphate phoshphatidylinositol-3 kinase protein kinase A protein kinase B protein kinase C pathogen recognition receptor reverse transcriptase-polymerase chain reaction sodium dodecyl sulfate-polyacrylamide gel eletrophoresis src-homology 2 transactivation domain transforming growth factor p activated kinase transforming growth factor p T helper cell, type 1 T helper cell, type 2 toll/IL-1 R receptor tumor necrosis factor a tumor necrosis factor receptor 1 n-tosyl phenylalanyl chloromoethyl ketone tumor necrosis factor receptor associated factor-6 tumor necrosis factor weak inducer of apoptosis ulcerative colitis xii  WT  wildtype  xiii  ACKNOWLEDGEMENTS  I would like to dedicate this thesis to my parents, who have always undeniably supported me in all of my educational pursuits. I would also like to thank my family and my friends for believing in me, and also that I could possibly finish this huge accomplishment. I would like to thank my supervisor Dr. Bill Salh, for his mentorship, as well as Dr. Alice Mui and Dr Vince Duronio for their excellent supervision of this thesis work.  Finally I would like to acknowledge the National Sciences and Engineering  Research Council, and the Michael Smith Foundation for Health Research, for salary support throughout this work.  xiv  CHAPTER 1 - GENERAL INTRODUCTION 1.1  INTESTINAL EPITHELIAL CELLS  1.1.1 Biology  The intestinal epithelium is a rapidly renewing tissue for which homeostasis means striking not only a balance between proliferation and cell death, but also balancing communications and interactions between the myriad of cell types with which it interacts. Cells arise at the base of the crypt, where the stem cell compartment is located, and they become progressively more differentiated as they proceed along the villus to the top of the crypt, before being shed into the lumen(1). Due to their location intestinal epithelial cells (lECs) will interact with cells of myeloid, lymphoid, and as well as mesenchymal lineage, while they travel up the crypt. This crucial single layer of cells functions to provide an important physical barrier, separating the host from what is often deemed hostile contents of the gut lumen. The past few years have led to an ever increasing body of evidence that shows that in addition to providing a passive barrier, lECs are in fact important players in the activation of gut inflammatory response.  1.1.2 Innate Immune Response  As mentioned before, the role of lECs has evolved from a passive bystander to more of a sentinel of the intestinal immune system. lECs are able to respond to a milieu of adherent and invasive bacteria, as well as evolutionarily conserved soluble bacterial antigens that are known as pathogen associated molecular patterns (PAMPs). PAMPs include such things as CpG DNA, lipopolysacharide (LPS), flagellin, and cell wall 1  polymers(2).  A number of recent studies have reported that l E C s can express  receptors that are capable of recognizing these P A M P s . These receptors are highly conserved throughout evolution, and known as pathogen recognition receptors (PRRs). They include extracellular receptors that belong to the toll-like receptor family (TLR)(39), as well as soluble internal receptors such as CARD4/Nod1 and CARD15/Nod2(1014). In addition l E C s have the ability to respond to proinflammatory cytokines such as interleukin-1p (IL-ip) and tumor necrosis factor a (TNFa)(15).  A s a result of IEC  activation, a powerful and robust pro-inflammatory cascade is set off, including the resultant  expression  of  proinflammatory  cytokines,  chemoattractants  (such  as  chemokines), as well as the induction of adhesion molecules(16-23). This results in the activation and recruitment of adjacent lamina propria mononuclear cells (LPMNCs), dendritic cells, mast cells, as well as the recruitment of peripheral blood mononuclear cells (PBMNCs), and polymorphonuclear cells (PMNs). Such a robust activation of the inflammatory cascade highlights the need for equally strong regulatory mechanisms that maintain a state of hyporesponsiveness between l E C s and the commensal bacteria of the gut.  These mechanisms are not completely understood, however may involve T-  regulatory cells, as l E C s have been shown to activate and expand T-regulatory cells(24, 25), and may also involve probiotic commensal bacteria of the G l flora, which have been shown to suppress intestinal inflammation(26).  Breakdown of the homeostasis  can result in chronic inflammatory conditions such as inflammatory bowel disease (IBD). It should be noted that although  lECs  have been reported to express major  histocompatibility complex (MHC) class II, they are not capable of expressing the  2  appropriate co-stimulatory molecule and thus can not fully activate T cells, but instead annergize T cells to promote local tolerance to intestinal antigens(27, 28). Inducers of N F K B in l E C s Cytokines and Growth Factors lnterleukin-1 lnterleukin-2 lnterleukin-17 lnterleukin-18 Leukotriene B4 Tumor necrosis factor Platelet-derived growth factor  Oxidative Stress Hydrogen peroxide Ozone Reactive oxygen intermediates Viruses and Viral Products Adenovirus  Bacteria and Bacteria Products Salmonella Shigella Enteropathogenic E. coli Listeria monocytogenes LPS Peptidoglycan-polysaccharide Toxic shock syndrome toxin 1  Molecules regulated bv N F K B in l E C s Cytokines and chemokines lnterleukin-1 p lnterleukin-6 lnterleukin-8 GROa/p RANTES Macrophage inflammatory protein-2  Cell surface receptor lnterleukin-2R CD95/APO-1 (Fas) Adhesion molecule ICAM-1 Inflammatory enzyme Inducible nitric oxide synthase Cyclooxygenase-2  Stress proteins Complement factors B, C 3 , C 4  Immunoregulatory molecules Major histocompatibility complex I and II  Table 1 - Activators and Inducers of  NFKB  3  in lECs.  1.2  CHEMOKINES  1.2.1  Structure, Expression, and Function One of the hallmarks of the inflammatory response is the migration of leukocytes  from the blood to sites of active inflammation.  Chemotactic cytokines, also known as  chemokines, are small polypeptides (8-14 kDa) that play an essential role in the homing of immune cells to areas of active inflammation.  There are over 50 chemokines that  can be subdivided into four families based upon their N-terminal motif of cysteines. The two main superfamilies are the C - C chemokines and C - X - C chemokines, along with two smaller families, the C and the C X C family(29, 30). 3  Although many of these  chemokines play a role in the recruiting immune cells during inflammation, chemokines are also responsible for homing during normal homeostasis, as well some chemokines can  also  control  angiogenesis,  cell  growth,  gastrointestinal  (Gl)  and  cardiac  organogenesis(31, 32). Under normal conditions the large and small bowel have large numbers of cellular infilitrates that include B and T lymphocytes as well as monocytes, dendritic cells, and a small number of mast cells and eosinophils. A s a result of this, the intestine is considered to be physiologically inflamed, and this would be considered pathological inflammation in any other tissue setting.  The expression of chemokines is highly  regulated, being restricted to specific cell and tissue types. l E C s are important in the activation of the gut inflammatory response, and as a result can synthesize and release numerous chemokines and cytokines, including members of both the C - C , and C - X - C group of chemokines, thus playing an important role in the recruitment of these 4  infiltrating immune cells.  Macrophage and neutrophil chemoattractants that can be  produced by l E C s include IL-8/CXCL8, epithelial-cell derived neutrophil attractant (ENA78/CXCL5), growth related oncogene a (Groa/CXCL1), monocyte chemoattractant protein-1 (MCP-1/CCL2), and macrophage inflammatory protein-1 a (MIP-1a/CCL3), however this list is not exhaustive(20-22). l E C s also produce macrophage inflammatory protein-3a (MIP-3a) which is important for chemoattracting C D 4 5 R O T cells as well as +  immature dendritic cells(18). l E C s also produce a large body of chemoattractants for C D 4 T cells. The large proportion of T cells in the lamina propria are of T H i phenotype +  (interferon y (IFNy) producing cells), and l E C s are capable of producing interferongamma  inducible  protein  (IP-10/CXCL10), monokine  induced  by  interferon  y  (MIG/CXCL9), as well as interferon inducible T cell attractant (I-TAC/CXCL11), which can attract T helper type 1 cells (TH-i) phenotype T cells expressing chemokine receptor CXCR3(19, 33). A small proportion of C D 4 T cells in the bowel are T helper type 2 +  cells (TH ) (which produce anti-inflammatory cytokines such as IL-4 and IL-10), and 2  lECs are capable of producing macrophage derived chemoattractant (MDC/CCL20) that is important for attracting T H phenotype T cells that express CCR4(17). The balance 2  between a large proportion of proinflammatory T H i C D 4 T cells, and small proportion of +  anti-inflammatory T H C D 4 T cells maintains this basal inflammation of the intestine in +  2  a controlled fashion. A s a result chemokine regulation of this balance by l E C s plays an essential role in intestinal homeostasis.  5  IL-8  1.2.2  IL-8 is a very well studied prototypic C X C chemokine involved in the recruitment of neutrophils, macrophages, and T lymphocytes(21). Sources of IL-8 have been reported to include CD14+ macrophages(34), neutrophils(34), endothelial cells(35), fibroblasts(36), T lymphocytes, and epithelial cells. In normal homeostatic tissue, IL-8 is often undetectable, however during inflammation it is robustly induced, resulting in a ten to 100 fold increase in concentrations(37). The  regulation of IL-8 production has been studied extensively, and often used  as a paradigm for the regulation of other chemokine family members. The transcription of IL-8 relies on the activation of an essential core promoter region spanning (positions -1 to -133) within the 5' proximal promoter(38-40). Within this core promoter there are binding sites for a number or transcription factors, including N F K B , activator protein-1 (AP-1), and CAAT/enhancer binding protein (3(C/EBP-p), which is also known as NF-IL6.  Promoter analysis studies have revealed that binding of N F K B  is essential for  transcriptional activation of IL-8 promoter, however AP-1 and C / E B P are dispensable. In order to get full gene activation however all three transcription factors are required(37, 39-43). A  number of other signaling pathways also regulate the transcription of IL-8,  through a variety of transcriptional interactions. In some systems the transcription of IL8 requires the activation the Jun NH2-terminal kinase (JNK) pathway. This activation is independent of the requirement of N F K B ( 4 4 ) . N F K B  NRF  There is also a role for the repressor,  repressing factor (NRF) as a negative transcription regulator of the IL-8 promoter. binds to a negative regulatory element (NRE), which overlaps the N F K B  6  binding  site. Depletion of this factor using antisense R N A , leads to spontaneous IL-8 synthesis(45). N R F thus has a dual role in that it is required for the stimulus-induced activation of IL-8, and requires the activation of J N K in order to do this(45).  Another  transcriptional interaction is that of transcriptional co-activator creb binding protein (CBP)/p300. CBP/p300 has histone acetyl transferase (HAT) activity and binds many of these transcription factors to form an enhancesome that can mediate  efficient  transcription of the IL-8 gene(46, 47). A further epigenetic level of regulation of IL-8 transcription is also present. Signaling pathways can modify histone protein complexes, which in turn regulate the availability of specific genes to the appropriate transcription factor. In the case of IL-8, the p38 mitogen activated protein kinase (p38 M A P K ) pathway has been shown to regulate the phosphorylation and acetylation of histone H3, and thus allow the promoter of IL-8 to be available for access to the transcription factor N F K B ( 4 8 ) . Once transcribed, IL-8 also is privy to post transcriptional regulation. During a state of homeostasis, IL-8 is not present due to the lack of both transcription of the message, as well as the instability of the message. IL-8 contains AU-rich cis regulatory elements (AREs) in the 3' untranslated region (3' UTR). The 3' UTR can be regulated and stabilized by many signaling pathways.  A signaling pathway that has been  implicated to regulate this in some systems is the p38 M A P K pathway. Inhibition of p38 during stimulus-induced IL-8 activation results in a marked reduction in the half life of IL8 message, and as a result the rapid destabilization and degradation of IL-8(49-52).  7  1.3  NFKB  1.3.1 Structure N F K B  is a family of inducible heterodimeric transcription factors (Fig 2), including  p65/relA, relB, c-rel, N F K B 1 , and N F K B 2 ( 5 3 - 5 5 ) . These proteins are structurally related through their evoluntionarily conserved Rel homology domain. p65/RelA, relB, and c-rel are all synthesized in their mature form, while N F K B 1 and N F K B 2 are processed from p100 to p52, and p105 to p50, respectively.  p100 and p105 also share a region of  homology in their ankyrin repeats, which are homologous to those found on I K B , the cytoplasmic binding partner of N F K B ( 5 3 - 5 5 ) .  Upon activation, p65 mainly associates  with p50. In addition, c-rel also associates with p50, however much less efficiently. Interestingly p100, in its unprocessed form, associates with and sequesters relB. p50 is constitutively processed whereas p52 is inducibly processed(56, 57). Upon activation and  processing, they form p52/relB heterodimers. p65/p50 heterodimers are the most  common form found in IECs(58). In addition to being synthesized in their mature form, p65,  relB, and c-rel are all related because they all contain at least one transactivation  domain. Another group of proteins, the inhibitor of kappa B ( I K B ) proteins, bind to N F K B family members and keep them sequestered in the cytoplasm. These are a group of 5 members including k B a , k B p , k B y , k B s , and BCL-3, in addition to the processed k B portions of p100 and p105. They all contain 6-7 ankyrin repeats, which mediates their binding to rel homology domains(53-55).  8  1.3.2  Regulation Activation of N F K B  usually occurs through one of two pathways, the classical/  canonical pathway, or the recently defined alternate pathway; however in both cases the I K B kinase complex is paramount in the convergence of upstream activators of N F K B  and the subsequent activation of the appropriate N F K B response(53, 54, 57). The classical pathway is responsible for the activation p65:p50 heterodimers.  These individual proteins are normally found sequestered in the cytoplasm, by the inhibitory protein I K B . Upon receiving a pro-inflammatory stimulus such as T N F a or ILi p , I K B is quickly phosphorylated by the inhibitor of kappa-B kinase (IKK) complex at serine residues 32 and 36 (detailed upstream regulation of IKK will be discussed later). This is a process that requires IKKp, and that does not require IKKa(59-63). Phosphorylated I K B is polyubiquitinated by the E 3 - S C F " P  with the  p-TRCP  Polyubiquitinated  complex functioning IKB  is  targeted  for  as the  T R C P  ubiquitin ligase, complex  I K B specific ubiquitin  subsequent  26S  proteasomal  ligase(64). mediated  degradation. Loss of I K B binding reveals the p65 nuclear localization signal allows it to proceed to the nucleus. Before p65 can become transcriptionally active it requires post-translational modifications including both phosphorylations and acetylations.  These are crucial  regulatory steps in p65 mediated transactivation. Many molecules have been shown to regulate these post translational modifications. CBP/p300, which contains HAT activity, provides a necessary acetylation of p65 on lysines 218, 221, and 310, with lysine 310 being an essential acetylation for full transcriptional activity of p65(65, 66). Similarly, protein phosphorylations at serines 276, 311, 529, 535 and 536 have all been shown to 9  be  required for full transcriptional acitivity of p65. Several protein kinases have been  implicated in the regulation of some of these sites including mitogen and stress activated protein kinase (MSK) and protein kinase A (PKA) for ser 276(67-69), protein kinase C zeta ( P K C Q for ser 311(70), protein kinase C K 2 for ser 529(71-73), calmodulin-dependent kinase IV (CaMKIV) for ser 535(74), and IKKp, protein kinase B (PKB), phosphatidylinositol-3 kinase (PI3K), and N F K B 536(75-84).  inducing kinase (NIK) for ser  These post-translation modifications can affect which co-activators are  capable of associating with p65. The  alternative pathway requires a signal from ligands that are usually distinct  from those that activate the classical pathway.  These include T N F superfamily  members leukotriene-p receptor (LTpR), CD40 ligand, as well as T N F like weak inducer of apoptosis (TWEAK)(85-88). These signals converge on the IKK signaling complex and  activate it, so that the p100 subunit may get phosphorylated.  classical pathway, this process requires IKKa, not IKKp(57). p 100  In contrast to the  Once phosphorylated  is processed into p52 and an I K B portion that is targeted for polyubiquitination and  subsequent proteasome mediated degradation. p52 dimerizes with RelB and has been noted to be a slower N F K B  response than the classical, however it is a far more  sustained response(89).  1.3.3  Function N F K B  has typically been associated with providing a protective cellular response.  This can take a variety of shapes and forms, however for the most part can be divided into two groups; a protective anti-apoptotic/proliferative response(53), or a protective  10  innate immune response(55). In this thesis we will be discussing the protective innate immune response, for the most part, however it is important to note that N F K B is equally a potent regulator of the anti-apoptotic response, through its ability to respond to proapoptotic signals such as fas ligand, T N F a , and tumor necrosis factor weak inducer of apoptosis (TWEAK), and its regulation of the expression of many  anti-apoptotic  molecules such as B C L X L , cellular inhibitor of flice (cFLIP), and cellular inhibitors of apoptosis (clAPs), as well as proliferative proteins such as cyclin D1(53). N F K B  can respond to a variety of immunogenic agents.  These range from  cytokines and growth factors (such as IL-1 p, T N F , M-CSF) to bacteria (Salmonella, and enteropathogenic E. coli) or bacterial soluble antigens, some of which include LPS, DNA,  CpG  peptidoglycan. In addition viral products such as double stranded R N A can also  activate N F K B .  Oxidative stress including hydrogen peroxide and reactive oxygen  intermediates can also activate N F K B ( 1 5 , 55). Likewise the N F K B  family plays an integral role in the transcription of many  important mediators of the innate immune response. These include the very cytokines and chemokines that are capable of activating N F K B itself, such as IL-1 (3 and T N F a , as well as the majority of the chemokines that have been described in the previous section. In addition, the expression of a wide variety of cell surface receptors, adhesion molecules,  and  complement  cascade proteins  are  also  regulated  by  N F K B .  Inflammatory enzymes that also potentiate inflammation such as inducible nitric oxide synthase  (iNOS)  and  cyclo-oxygenase-2  transcription(15, 55).  11  (cox-2)  are  also  targets  of  N F K B  Studies using genetic knockouts have provided much insight into the function of specific members of the N F K B family. Knockout mice lacking the IKK1 (encodes IKKa) are viable however have severe defects in keratinocyte differentiation, bone and limb development, and lack mature B cells(63, 90-92). These mice are incapable of p100 processing(57). Likewise knockout of the N F K B 1 gene (encodes p100) results in mice that have no B cell development(93). In contrast, knockout of either N E M O (encodes IKKy) or IKK2 (encodes IKKp) is embryonic lethal at E11.5-E12.5 due to T N F a dependant liver apoptosis(59, 60, 94, 95). Mouse embryonic fibroblasts (MEFs) from these embryos lack the ability to activate p65. Similarity p65 knockout in mice is also embryonic lethal at E15.5-E16.5, due to T N F a dependant liver apoptosis(96).  RelB  knockout mice die postnatally from multiorgan inflammation(97), and c-rel knockout mice have no developmental defects, but instead have defective lymphocyte and macrophage function(98). Disruption of p50 results in viable mice however these mice have defects in lymphocyte function(99).  1.4  IEC SIGNALING  1.4.1  IL-1 signaling IL-1 is a very important pro-inflammatory cytokine that is responsible for the  synthesis of many inflammatory mediators, including enzymes, adhesion molecules, chemokines, tissue degrading molecules, and acute phase proteins.  There are two  types of IL-1: IL-1 a, IL-1 p, as well as a closely related protein IL-1 receptor antagonist (IL-1RA) that acts as an antagonist to the type 1 IL-1 receptor (IL-1 RI). Both IL-1 a and IL-1 p signal through the type 1 IL-1 receptor(100). 12  The type I IL-1 receptor is a member of the IL-1 receptor/toll like receptor (IL1R/TLR) superfamily.  This family consists of over 33 members that are all related  based on a homologous cytoplasmic domain named the toll IL-1 R (tir) domain.  The  members of the IL-1R/TLR are divided into 3 families based upon their extracellular domains. The first group is based on those family members that have Ig domains in their extracellular domain(100). receptor (IL-18R)(100, 101).  This group includes the IL-1 RI and the interleukin-18 A n interesting member of this subgroup is also the  interleukin-1 receptor type II (IL-1RII) that acts as a decoy receptor to inhibit IL-1 RI signaling(102). The IL-1 R11 does not contain a tir domain, and thus can not participate in downstream cellular signaling, even though it is capable of binding and internalizing IL-1 (103).  The second group has family members that contain leucine-rich repeats  (LRRs) in the extracellular domains, as opposed to Ig domains, in addition to containing a cytoplasmic tir domain. These include a variety of members that act as P R R s , such as a large group of toll like receptors (TLRs 1-9)(2). A small third group contains family member that contain only cytoplasmic tir domains.  These proteins act largely as  adaptors to facilitate downstream signaling. Members of this family include MyD88, and mal(104-106). Due to the conserved nature of the tir domain between family members of this group, they evoke similar downstream responses. Binding of IL-1 to the IL-1 R activates numerous cell signaling pathways (many of which will be discussed below). This results in the formation of a complex including the adapter protein MyD88(104, 107, 108).  A s mentioned before MyD88 contains a tir  domain, as well as a death domain, through which MyD88 can dimerize with other molecules. Attraction of MyD88 allows the recruitment of the serine threonine kinase  13  interleukin receptor associated kinase-1 (IRAK-1)(107, 109). Prior to associating with MyD88, IRAK-1 is phosphorylated and activated by IRAK-4 which releases it from the cytoplasmic inhibitor tollip(109, 110). Activated IRAK-1 recruits tumor necrosis factor associated factor-6 (TRAF-6)(111). T R A F - 6 then signals down to transforming growth factor activated kinase-1 (TAK1). At this branch point, several signaling pathways are activated including the M A P K family, as well as the IKK pathway.  1.4.2  MAP Kinases The M A P kinase signaling cascades are very evolutionarily conserved, and  perhaps some of the most ancient signaling pathways. These pathways play a very important role in the activation of the innate immune response. There are three main families, including the extracellular signal regulated protein kinases (ERK)(112), the cJun NH2-terminal kinases (JNK)(113, 114), as well as the p38 MAPKs(115). The activation of these three members requires a dual phosphorylation on a Thr-X-Tyr motif present on all three of these members. This phosphorylation occurs via a M A P K kinase (MKK). These M K K s are in turn activated by M K K kinases (MKKK), which themselves are thought to be activated by small G proteins.  1.4.2.1 p38MAPK p38 M A P K family is activated by M A P K K s , M K K 3 and M K K 6 . These in turn are activated by upstream Rho family of GTPases, through mixed lineage kinase-3 (MLK3) and TAK1 (115). Although the role of p38 M A P K in the gut inflammatory response is not well known, several reports show that is plays a very important role in other systems. Inflammatory mediators such as IFNy, iNOS, C O X - 2 , IL-6 and T N F a , have all 14  been shown to be subject to regulation via p38 MAPK(116-120). In addition, a few reports have also described p38 being an upstream regulator of the activation of N F K B , being able to modulate the phosphorylation and transactivation of N F K B .  p38 also has  the ability to regulate the transcription factor AP-1 by regulating activating transcription factor-2 (ATF-2) phosphorylation. A third line of evidence that implicates p38 M A P K in the regulation of the inflammatory response is its ability to regulate message stability of several cytokines/chemokines through their 3' UTR. In the gut inflammatory response, the role of p38 M A P K is largely unexplored.  1.4.2.2 JNK J N K s consist of over 10 isoforms encoded as splice variants from 3 separate gene loci.  Activation of J N K s occur via the M A P K K s , M K K 4 and MKK7. These are  activated through the upstream Rho family of G T P a s e s through the M E K kinase 1 and 4 (MEKK1/4) and MLK3.  The role of J N K in proinflammatory signaling has been  controversial, however it is clear that J N K is able to activate and regulate the transcription factor A P - 1 , through its ability to regulate c-jun(113, 114). This is important as AP-1 has the ability to regulate numerous chemokines, such as IL-8, as previously discussed.  1.4.2.3 ERK E R K s are robustly activated by growth and differentiation factors, as well as cytokines. The activation of E R K s occurs via the M A P K K s , MKK1 and MKK2, which are activated by Ras through the Raf family of MKKKs(112).  15  The two major isoforms of  ERK  are ERK1/p44 and ERK2/p42.  E R K s have previously been described to be  responsible for growth and differentiation of IECs(121, 122), however their functional role in mediating the gut inflammatory response has been largely unexplored.  1.4.3  Protein Kinase CK2 Protein kinase C K 2 (formerly known as casein kinase 2) is a ubiquitously  expressed protein kinase that has been linked to over 160 cellular substrates(123). In spite of the numerous cellular targets that have been identified, its regulation and cellular function remain largely enigmatic.  The C K 2 holoenzyme is composed of 2  catalytic subunits ( a or a') with two regulatory subunits (P)(124-126). C K 2 is found to have basal activity even in the absence of stimulation, supporting the notion that it is a constitutively active kinase, however its specific activity can be modulated by growth and stress factors(127-129).  C K 2 has been co-localized in all cellular compartments  including the nucleus, where its nuclear-matrix bound activity has previously been shown to be important for the regulation of apoptosis(130). It has also been linked to the regulation of many transcription factors, including C / E B P homologous protein (CHOP), CCCTC-binding factor (CTCF), c-myc, c-fos, p-catenin as well as N F - K B ( 1 3 1 - 1 3 6 ) . role in activating N F - K B  has been largely controversial, with different  Its  regulatory  mechanisms in different cell systems. In mammary epithelial cells it has been linked to direct association with the IKK complex(135, 136), whereas other groups have shown it to regulate the transactivation of N F K B ,  through a direct phosphorylation of serine  529(71, 72). The role of protein kinase C K 2 in the context of the gut inflammatory response is largely uncharacterized.  16  1.4.4  PI3K Pathway PI3Ks compose a family of kinases that catalyse the 3' phosphorylation on  inositol rings.  These produce a variety of phosphoinositide products, including most  notably the second messenger phosphatidylinositol(3,4,5)triphosphate  (PI(3,4,5)P3).  PI(3,4,5)P3 acts as a second messenger to regulate many cellular events including cell growth, cell survival, cytoskeletal remodeling, and intracellular organelle trafficking (137143). The PI3K family contains four classes: l , I B , II, and III, based on their structure A  and specificity for substrates. Class l PI3Ks are responsible for catalyzing the reaction A  from phosphatidylinositol(4,5)biphosphate PI(4,5)P to PI(3,4,5)P (140-143). Class l 2  3  A  PI3K is most often composed of a regulatory subunit (p85) as well as a catalytic subunit (p110)(140-143).  The p85 subunit contains 2 src-homology-2 (SH2) domains that  facilitates the recruitment of tyrosine phosphorylated substrates at the membrane to the p110 subunit, which then can be activated, as recruitment of adaptor proteins (such as insulin receptor substrate (IRS)) relieves the constitutive inhibition in the p85-p110 complex(140-143). Other downstream effectors include P K B , some P K C isoforms, 3'phosphoinositide dependant kinase 1 (PDK1), as well as others. There is strong evidence for a role for PI3K in regulating the immune response. Cytokines such as IL-1 p, IL-2, IL-3, IL-6, IL-7, IL-15, T N F a , and granulocyte/monocytecolony stimulating factor (GM-CSF), have all been shown to activate PI3K(140-143). PI3Ks have been shown to act downstream of these cytokine receptors as well as T and B cell receptors and as a result regulate numerous immune functions including aspects of both the innate and adaptive immune response. In the IEC system the role of PI3K is largely unexplored however it has been shown to be a negative regulator of IEC  17  differentiation, a negative regulator of T N F a induced cox-2 synthesis, as well as being important for mediating cellular proliferation(144-147). Although it is well accepted that PI3K can modulate N F K B signaling, its precise role, and that of one of its downsteam effector kinases, P K B , has been controversial. No work has yet been done to characterize these pathways in the IEC cell system.  1.4.4.1 PKB P K B (also known as A K T or Rac) is known largely as a prosurvival kinase, regulating numerous proliferative and survival effectors including B A D , caspase-9, and the forkhead family of transcription factors(148). localization in a PI(3,4,5)P phosphorylations(148).  3  Its activation requires its membrane  dependant fashion, where it can undergo activating  The initial phosphorylation comes from as yet unidentified  kinase termed phosphoinositide-dependant kinase 2 (PDK2) in its hydrophobic motif at serine 473.  Some candidate kinases for this phosphorylation include P K B itself via  autophosphorylation,  integrin  linked kinase (ILK),  or an  unidentified  membrane  associated kinase (149-152). Once phosphorylated at serine 473, this allows threonine 308 to be phosphorylated by P D K 1 , to induce its kinase activity fully(148).  It is  generally agreed that both phosphorylations are required for full activity, however examination of the phosphorylation status at serine 473 is not sufficient to assess kinase activity. The role of P K B in the immune response is largely unexplored. P K B has been linked to the activation of N F K B ,  but the precise mechanism of activation has been  controversial. There are reports that P K B is a direct regulator of IKK activity, through direct phosphorylation of IKKa, and thus being able to regulate the ability of p65 to 18  separate from k B and translocate to the nucleus, in a PI3K dependant fashion(153, 154).  More interestingly a number of reports have linked the PI3K/PKB pathway to the  regulation of N F K B  through regulation of p65 transactivation.  This suggests that  regulation of N F K B by PI3K, and P K B may be stimulus and system specific.  1.4.4.2 3'-Phosphoinositide dependant kinase 1 PDK1  is a nodal kinase regulating several downstream signaling pathways  including several A G C kinases such as p70 S6 kinase, p90 ribosomal s6 kinase (p90 RSK),  P K C , and PKB(155). PDK1 itself is an A G C kinase, and becomes activated  through an essential phosphorylation of tyrosine 9 and trans-autophosphorylation of serine 241 (156, 157). PDK1 provides a critical phosphorylation on the activation loop of its downstream targets also(155). It is found to have high constitutive activity. Despite this several potential mechanisms exist to regulate its cellular activity, including regulation by PI(3,4,5)P , binding to heat shock protein 90 (HSP90), binding to 14-3-3, 3  and subcellular distribution(155, 158, 159). Also there is some evidence that PDK1 can function in PI3K independent ways(160). Its role in the IEC cell system is largely unexplored, especially with respect to chemokine synthesis and N F K B activation.  1.4.4  IKK complex Signal induced activation of IKK remains the rate limiting step in the activation of  N F K B ,  IKK  thus its understanding is fundamental to understanding N F K B activation.  The  signalsome is composed of a homodimer of the IKKy/NEMO regulatory subunit with  either a catalytic IKKa/IKKp heterodimer or an IKKa homodimer(54, 161, 162). Some  19  specific functions have been ascribed for each of the subunits. IKKa has been shown to be essential for p100 processing (57), and regulating chromatin remodeling(163, 164). IKKp has been shown to be essential for I K B phosphorylation on serines 32 and 36(94), as well as p65 phosphorylation on serine 536(81). Activation of IKK requires phosphorylation of the catalytic subunits on their activation loops. phosphorylation  on  serine  residues  176  and  180,  whereas  IKKa requires IKKp  requires  phosphorylations on serine residues 177 and 181(94). The mechanism through which upstream activation of these phosphorylations occurs is controversial.  One possible  mechanism is that a potential "IKK kinase" exists that phosphorylates these sites. Based on genetic knockout experiments, several candidate kinases have been implicated, depending on the cell system studied. implicated(165).  In HeLa cells, using siRNA, TAK1 has been  M E K K 3 has been shown to be essential in MEFs(166).  PKCq has  been implicated only in mouse lungs cells but the same was not true in MEFs(167). A second possibility is that the IKK catalytic subunits trans-autophosphorylate themselves. This notion is supported by the observation that recombinantly expressed IKK a or p purified from insects or mammalian cells is constitutively active, due to phosphorylations on their active loops(168-173). In addition several reports have shown that homotypic oligomerizations results in trans-autophosphorylations(168). It is still unclear how these oligomerizations occur in the absence of overexpression, and where the initial phosphorylation comes from. It is hypothesized that there is a small pool of IKK that is active and capable of phosphorylating the remainder IKK molecules when there is ligand. Other factors that may play a role in the activation of IKK include HSP90 and cdc42(174).  20  1.5  INFLAMMATORY BOWEL DISEASE  1.5.1  Pathology  Inflammatory bowel disease (IBD) collectively refers to 2 conditions, Crohn's disease (CD), and ulcerative colitis (UC), which are characterized by a chronic idiopathic relapsing and remitting inflammation of the large or small bowel, and often leads to an irreversible impairment of gastrointestinal function. The North American prevalence of IBD is roughly 10-200 per 100,000 people. Although similar, C D and U C are defined based upon some markedly different characteristics.  C D affects the terminal ileum,  cecum, and large bowel, and often has patch lesions in between normal areas. U C on the other hand affects the  rectum and continues proximal and is continuous.  Inflammation in C D is transmural, affecting all muscle layers, and is characterized by lymphocyte infiltration as well submucosal fibrosis. U C inflammation is more superficial, however has a large lymphocytic and granulocytic infiltration, as well as a loss of goblet cells. In both cases this results in severe diarrhea, as well as blood loss. C D can lead to strictures, as well as bowel obstructions. U C can lead to loss of peristaltic function and rigidity of colon wall, as well as eventual toxic megacolon.  Long term chronic  inflammation as a result of IBD also increases the risk of colon carcinomas. Current treatments for IBD include 5-ASA compounds which target the activation of the transcription factor N F K B , corticosteroids, azathioprine/6-MP which target Rac and T cell apoptosis, surgery as well as monoclonal antibody therapy to T N F a for CD(175).  21  1.5.2  Pathogenesis The pathogenesis of IBD still remains largely enigmatic, however it is generally  well accepted that both C D and UC are complex genetic disorders.  Despite this,  studies using numerous murine models of IBD have brought forth a number of emerging concepts important to understanding human IBD. Currently there are several murine models of IBD; some of these are spontaneous, some require a haptenating agent, and while others require adoptive transfer of populations of T-cells. A role for genetics is highlighted by murine models that develop spontaneous colitis, and thus highlight a very important principle that there are several gene products, that once dysregulated, are sufficient  to  result  inflammation(176).  in  the  development  of  very  similar  models  of  intestinal  Another important tenet is that the host background plays a  paramount role in determining disease susceptibility.  IL-10 knockout mice develop  spontaneous colitis only on certain inbred backgrounds, whereas other  inbred  backgrounds are completely resistant(177). This underscores the notion of a complex interplay between a number of different gene products as a requirement for the pathogenesis of IBD. A second and very important concept is the requirement of normal gut flora for the development and sustenance of colitis. Mice kept in germ-free conditions generally do not develop experimental colitis(178). Despite this there has not been one organism, or one group of organisms that has been found responsible for experimental colitis. In addition, a group of bacteria also seem to have protective effects have been termed probiotic bacteria(26).  22  A third emerging concept is that an imbalance between effector T-cell function and regulatory T-cell function can lead to IBD. Many models of murine colitis rely on an overproduction of pro-inflammatory  cytokines to induce inflammation (eg tri-nitro  benzene sulphonic acid (TNBS) induced colitis is dependant upon an IL-12 response to L P S in the gut).  Likewise, murine models that are defective in the generation of  regulatory T cells (Tgs26 mice), or defective in regulatory cytokine signaling (IL-10 -/-, transforming growth factor (TGFp -/-) develop colitis as well(179, 180). A fourth emerging concept is the imbalance of profile of T cells that occurs in IBD. Polarization to either T H i or to TH2 leads to a major driving force for inflammation. Thus a critical balance between T H i and T H CD4+ T cells must be maintained. C D is 2  characterized by a T H i profile of cytokines, with IL-2, IL-12, IFNy, and T N F a being very important players. In contrast, U C is often characterized by a T H profile of cytokines, 2  with increases in IL-4, IL-5, and IL-13.  To further support this notion, experimental  colitis (eg T N B S induced colitis) that is driven by a THi  response, histologically  resembles C D . Likewise, experimental colitis driven by a T H response (eg Oxazalone 2  colitis), histologically resembles U C . The role of the mucosal epithelium is an emerging concept in the pathogenesis and is the fifth concept. Maintenance of the barrier integrity of the mucosal epithelium is essential, due to the number of infiltrating lymphocytes that are basally present in the underlying lamina propria. Some of the best evidence for this comes from a transgenic model where a dominant negative N-cadherin was expressed. These mice had severe barrier dysfunction and as a result acquired colitis(181). In addition l E C s are capable of expressing many P R R s such as T L R s and NOD receptors.  23  These are important in  normal host defense and normally inert to the contents of the lumen. Dysregulation of this hyporesponsiveness may play an important role in the initiation of inflammation in IBD,  as l E C s are the first to encounter intestinal antigens, and damage. In addition  defects in the innate immune response may be important as mutations in the intracellular signaling receptor NOD2 are responsible for between 5-15% of all people with CD(182, 183).  1.5.3  The role of lECs, chemokines, and  NFKB  Although a large majority of work on IBD pathogenesis is looking at the breakdown of adaptive immune response, there is growing body of evidence that suggests that l E C s , the transcription factor N F K B , and chemokines play critical roles. Breakdown in the regulation of these components may be instigating factors in the pathogenesis of IBD. and  Overproduction of the potent pro-inflammatory cytokines IL-1 p  T N F a in the intestinal mucosa of patients with IBD is well documented(184-190). In  cell culture systems, IL-1 p and T N F a can robustly activate the transcription factor N F K B , thus it is not surprising that in vivo examination of IBD patients shows increased activation of N F K B  in patients with active inflammation when compared to normal  uninflamed control tissue(191, 192). In addition, a study examining localization of this N F K B  activation reveals that it is limited to l E C s and macrophages(191). One of the  functional consequences of N F K B  activation is that it can drive the expression of  numerous inflammatory mediators, but most notably chemokines. There have been numerous studies examining levels of chemoattractants in tissues from patients with inflammatory bowel disease.  These studies have shown that there are significant  24  increases in a variety of chemokines including IL-8, M C P - 1 , M C P - 2 , M C P - 3 , MIP-1a, MIP-1p\ IP-10, R A N T E S , as well as G R O a , in actively inflamed tissues when compared to normal uninflamed tissues(193-198).  A recent study examining over 500 biopsy  samples from patients with IBD, used immunohistochemistry  to characterize the  localization of these chemoattractants. Epithelial cells were determined to be one of the major producers of chemokines(198).  Thus it can be hypothesized that a potential  dysregulation of chemokine expression through activation of N F K B in l E C s may be an important mechanism in the pathogenesis of IBD.  25  Figure 1. Signaling pathways activated by IL-1 p  -26-  431  551  p65/RelA 619  c-REL 579  RELB |  p105/p50 969  435  BBS 3333 | GRR  p100/p52 900  405  mmHHH  317 fcBa  356  607 IKBY  500  AIAIABAIAIA  IKBE  446  ggg 3B3H Figure 2.  NFKB  family members  -27-  BCL-3  IL-1  III  529  311  \  Ubiquitination and Degradation  • * LJ  WW Figure 3. Detailed activation of N F K B , including important regulatory phosphorylations -28-  CHAPTER 2 - MATERIALS AND METHODS  2.1  MATERIALS  2.1.1 Cell lines and Cell Culture HCT-116, of IEC origin, were a kind gift of Bert Vogelstein (Johns Hopkins, Baltimore, Maryland). HCT-116 cells were cultured in McCoys 5A Medium  (Gibco, Burlington,  Ontario) containing 10% heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, Utah) with 100 U/mL of penicillan and 100 ug/mL of streptomycin (Gibco, Burlington, Ontario). Caco-2 cells and HT29 cells, of IEC origin, were acquired from the American Type Cell Culture (ATCC, Manassas, VA). Caco-2 and HT-29 cells were cultured in M199 Medium containing 10% F B S with 100 U/mL of penicillin and 100 ug/mL of streptomycin (Gibco, Burlington, Ontario). H E K 293T cells, of human embryonic kidney cell origin, were a kind gift of Alice Mui (University of British Columbia, Vancouver, BC). They were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat inactivated F B S with 100 U/mL of penicillan and 100 ug/mL of streptomycin. All cell lines were grown at 37°C with 5% C 0 (v/v). 2  2.1.2 Reagents, Enzymes, and Chemicals PD 98059, S B 203580, S P 600125, 5,6-dichloro-ribifuranosylbenzimidazole (DRB), apigenin, LY294002, wortmannin, actinomycin-D, and N-tosyl phenylalanyl chloromethyl ketone were purchased from Calbiochem (San Diego, California). All chemical stock solutions were made up in dimethylsulfoxide (DMSO) except for D R B in 100% ethanol,  29  and T P C K in methanol.  All restriction enzymes were purchased from New England  Biolabs (Missisauga, Ontario, Canada). All other chemicals were purchased from Sigma-Aldrich (Oakville, Ontario, Canada).  2.1.3  Plasmids and cDNAs  Numerous plasmids and cDNAs were generously provided for use in this work: WT and KD FI_AG-p38  J . Han  Scripps Institute (San Diego, California)  WT and KD H A - C K 2 a  D. Litchfield  University of W . Ontario (London, Ontario)  WT and KD H A - C K 2 a'  D. Litchfield  University of W. Ontario (London, Ontario)  WT and KD M Y C - P D K 1  J S . Park  Friedrich Miescher Instit (Basel, Switzerland)  WT A N D KD H A - P K B  Kinetek Pharmaceuticals (Vancouver, BC)  4XKB-LUC  Kinetek Pharmaceuticals (Vancouver, BC)  IL8-LUC  G. Wu  University of Pensylvania (Philadelphia, PA)  pfr-Luc  Stratagene  (La Jolla, California)  pTADI-lll  BR Cullen  Duke University, (Durham, North Carolina)  pTADIII  BR Cullen  Duke University, (Durham, North Carolina)  pM-p65 (521-551)  T. Okamoto  Nagoya University (Nagoya, Japan)  pM-p65 (521-551, S529A) T. Okamoto  Nagoya University (Nagoya, Japan  pM-p65 (521-551, S536A) T. Okamoto  Nagoya University (Nagoya, Japan)  2.1.4  Primers  Primers used in R T - P C R are as follows: IL-8 Forward: IL-8 Reverse:  5'-TCTGCAGCTCTGBTGTGAAGGTGCAGTT-3' S'-TTCCTTTGACCCACGTCTCCCAA-S'  MCP-1 Forward:  5'-TCTGTGCCTGCTGCTCATAGC-3' 30  MCP-1 Reverse:  5'-GGGTAGAACTGTGGTTCAAGAGG-3'  Actin Forward: Actin Reverse:  5'-CCAACCGCGAGAAGATGACC-3' 5'-GATCTTCATGAGGTAGTCAGT-3'  I K B Forward I K B Reverse  5'-TACACCTTGCCTGTGAGCAG-3' 5'-AGGATTTTGCAGGTCCACTG-3'  GROcc Forward G R O a Reverse  5-ACTCAAGAATGGGCGGAAAG-3' 5'-TGGCATGTTGCAGGCTCCT-3'  T N F a Forward T N F a Reverse  5-CGGGACGTGGAGCTGGCCGAGGAG-3' 5'-CACCAGCTGGTTATCTCTCAGCTC-3'  iNOS Forward iNOS Reverse  5'-CGGTGCTGTATTTCCTTACGAGGCGAAGAAGG-3' 5'-GGTGCTGCTTGTTAGGAGGTCAAGTAAAGGGC-3'  R A N T E S Forward R A N T E S Reverse  5'-CCAACCCAGCAGTCGTCTTTG-3' 5'-CTCCCAAGCTAGGACAAGAGC-3'  2.2  METHODS  2.2.1  Nuclear Preps  Cells were seeded onto 60 mm plates and grown to confluence, and then pretreated with the appropriate inhibitor for 1 h, before being stimulated with IL-1 (3 for 30 min. Cells were washed once with ice cold phosphate buffered saline (PBS), scraped into 1 ml of P B S before being centrifuged at 14,000 R P M . The pellet was resuspended in 200 uL Buffer A (10 mM Hepes pH 7.9, 10 mM KCI, 0.1 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 0.5 mM P M S F ) for 15 min, before adding 13 uL of 10 % Nonidet NP-40. The cell suspension was vortexed for 10 s before being centrifuged again at 14,000 R P M for 30 s. The supernatant was removed and the nuclear pellet resuspended in 30 uL of Buffer C (20 mM Hepes pH 7.9, 0.4 M NaCI, 1 mM EDTA, 1 mM E G T A , 2 5 % glycerol, 1 mM 31  DTT, 1 mM P M S F ) . The sample was vigorously rocked for 15 min and subsequently centrifuged for 5 min at 14,000 R P M . The supernatant was retained and the protein concentration determined  by the Bradford assay (Bio-Rad,  Mississuagua, Ont).  Samples were stored at -80°C until use.  2.2.2  Electrophoretic Mobility Shift Assay (EMSA) A  synthetic Kb oligonucleotide was cloned into the  cloning vector, p B S  (Stratagene, La Jolla, CA), using the EcoRI and H/ndlll sites, to create pBS-EMSAicb. To radiolabel the probe, it was excised from pBS-EMSAicb using EcoRI and HindUl, and labelled using [y- P]dCTP (Amersham, Montreal, Quebec, Canada) and the Klenow 32  fragment of DNA polymerase (New England Biolabs, Missisauga, Ont., Canada). The probe was then purified by running on a 5% non-denaturing gel, cutting out the fragment, and incubating the gel slice in elution buffer [(0.6 M ammonium acetate, 1 mM EDTA, 0-1% sodium dodecyl sulphate (SDS)] overnight. Briefly, 10 ug of nuclear extract were preincubated in binding buffer (20 mM Hepes pH 7.9, 100 mM KCI, 10% glycerol, 1 mM DTT) and 1 ug of poly dldC (Amersham, Montreal, Quebec), for 15 min. 20,000 C P M of hot probe was then added, and the reaction mixture incubated at room temperature for 30 min, and subsequently resolved on a 5% non-denaturing polyacrylamide gel in 0.25 x T B E at 200V for 1.5 h. The gel was then dried for 45 min before phosphoimaging analysis using a Bio-Rad molecular imager F X developed).  (or alternatively exposed to film overnight at  -80°C and then  For supershift or cold competitor reactions, the nuclear extract was  preincubated with 1 ug of anti-p65 antibody (Calbiochem, San Diego, California), or 32  100-fold excess of unlabelled probe with binding buffer and poly dldC for 30 min before adding the radiolabelled probe. Alternatively, AP-1 oligonucleotides were labeled using polynucleotide kinase, and [ P]ATP. Labelled probe was cleaned as described above. 32  The oligonucleotide sequence used were: C G C T T G A T G A C T C A G C C G G A A , Human AP-1 E M S A Forward T T C C G G C T G A G T C A T C A A G C G , Human AP-1 E M S A Reverse  2.2.3  Immunoblotting  Cells were washed once with ice cold P B S , resuspended in homogenization buffer (20 mM M O P S , 50 mM b-glycerophosphate, 5 mM E G T A , 50 mM NaF, 1 mM DTT, 1 mM sodium vanadate, 1% NP-40 and 1 mM PMSF) for 30 min, sonicated for three 5 s intervals on ice at 30% output, before being centrifuged at 14,000 R P M for 15 min. The protein concentration in the supernatant was determined by the Bradford assay (BioRad, Mississauga, Ont). 50 mg of protein from each sample was resolved using 10% S D S - P A G E before transferring to nitrocellulose membranes (Bio-Rad). The blots were blocked in 5% skim milk in T B S T (20 mM Tris-HCI pH 7.4, 250 mM NaCI, 0.05% Tween-20) for 1 h before probing for 2-4 h using the appropriate primary antibody. The blots were washed with T B S T for 10 min three times, before being incubated with the appropriate secondary antibody for 1.5 h. Following 3 further washes in TBS-T, they were developed using the enhanced chemiluminescence detection system (ECL, Amersham, Montreal, Quebec).  P h o s p h o - k B , k B a , phospho-IKK, phospho-p38,  phospho-JNK, J N K , phospho-ERK, E R K , phospho-PKB, phospho-PDK1, phospho-p65 antibodies were purchased from Cell Signaling (Missisaugua, Ont). Antibodies to P K B 33  kinase (PDK1), p65, IKKy, IKKp, C K 2 a and a ' were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), or were a kind gift of Steve Pellech (UBC, Vancouver, B C , Canada).  Antibody to p38 M A P K was purchased from Stressgen  Biotechnologies (Victoria, BC).  2.2.4  CK2 Phosphotransferase Activity Assays  CK2 assays were carried out as previously described (129).  Briefly 5 ug of protein  lysate was incubated in a final volume of 25 uL with 5 ug of partially phosphorylated casein or the specific C K 2 substrate R R A D D S D D D D D and 100 DM [ P ] G T P (2.5 32  • C i / a s s a y in Buffer C (12 mM M O P S (pH 7.2), and 15 mM MgCI ) for 15 min at 30°C. 2  The phosphorylation of casein or the specific substrate was quantitated by spotting 20 ml on to a 1.5 c m piece of Whatman P-81 phosphocellulose paper. The papers were 2  washed extensively in 1% (w/v) phosphoric acid, transferred into 6-ml plastic vials containing 0.5 ml of Ecolume (ICN) scintillation fluid, and the incorporated radioactivity was measured in a Wallace (LKB) scintillation counter. C K 2 immunocomplex assays were also carried out as previously described. Immunoprecipitations were performed by incubating 500 ug of whole lysate with 4 ug of C K 2 a polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) for overnight and collected with 20 ul of protein A Sepharose (preblocked with 1% BSA) for 3-4 h at 4°C. The beads were washed four times with 20 mM Tris-HCI (pH 7.4), 150 mM NaCI, 1% NP-40. The immunoprecipitated beads were washed one additional time with buffer A (12 mM M O P S (pH 7.2) and 15 mM MgCI2), and incubated with either 5 mg of casein of the specific C K 2 substrate R R A D D S D D D D D . Reactions were stopped by the addition of S D S - P A G E sample 34  buffer.  After boiling, the samples were subjected to sodium dodecyl sulfate-poly  acrylamide gel electrophoresis ( S D S - P A G E ) for autoradiography with X-ray film or by immunoblotting analysis.  2.2.5  Transient Transfections  Cells were seeded at a confluency of 75%. 24 hours later cells were transfected using Lipfectamine 2000, as per the manufacturers instructions, at a ratio of 2:1 (liposome u L D N A ug).  1 ug of plasmid DNA per 60 mm plate was used. Cells were incubated  overnight, and cell medium was aspirated and fresh medium was added. Cells were allowed to express the proteins for over 24 hours, before the experiment was carried out.  2.2.6  Luciferase Assay  0.5 ug of an NFicB-dependant reporter containing 4 repeats of the Kb consensus sequence from the IL-ip gene (4xKb-Luc), was cotransfected with a LacZ plasmid that constitutively encodes a p-galactosidase gene (kindly donated by William Jia, U B C ) . Cells were pretreated for 1 h with the appropriate inhibitor, stimulated with IL-1 p for 6 h, before being harvested.  Luciferase and p-galactosidase activities were measured  according to the manufacturer's instructions (Promega, Madison, WI).  Light emission  was measured using a luminometer, and the luciferase readings were normalized using p-galactosidase activity.  35  2.2.7  Isolation of RNA and RT-PCR  R N A was isolated using the TRIZOL method (Life Technologies, Burlingtion, Ontario). The purity of the R N A was determined by running 1 mg of R N A on a 1 % agarose gel for 1.5 h at 75 volts. 1 mg of R N A was reverse transcribed using 0.5 mg of oligo (dT)1218 (Amersham, Montreal, Quebec), 1 ml of 10 mM d N T P s , 2 ml of 0.1 M DTT, 40 units of  RNA-guard (Amersham, Montreal,  Quebec)  in  1x first  strand  buffer  (Life  Technologies, Burlington, Ontario) using 200 units of M-MLV reverse transcriptase, by incubating the reaction mixture for 50 min at 37°C. 2 ml of c D N A was used in each subsequent polymerase chain reaction (PCR) reaction. For each 50 uL P C R reaction, 2 U of T A Q (PE Biosystems, Branchburg, New Jersey), 1 x P C R Buffer (PE Biosystems, Branchburg, New Jersey), 10 pmol of each primer, 1 ml of 10 mM dNTPs, and 3 ml of 25 mM MgCI2 were used. The P C R temperatures used were: 94°C denaturing for 45 s, 56°C annealing for 45 s, and 72°C extension for 1 min. 10 uL aliquots of the reaction were electrophoresed on a 1.5% agarose gel containing ethidium bromide.  Negative  controls for c D N A synthesis were run without template, and also without RT. Linearity of P C R reactions was determined in the range between 20-40 cycles. Linearity of the reaction was further determined by template dilutions (1 in 10 and 1 in 100). Densitometry was performed using Bio-Rad Quantity-One software.  2.2.8  Tissue Procurement and Immunohistochemistry A total of 18 cases of normal or active IBD were obtained through Dr. D. Owen  from the Division of Anatomical Pathology at Vancouver Hospital and Health Sciences Centre (VH&HSC).  Paraffin-embedded colonic tissue samples were de-waxed in  xylene twice for 5 min, rehydrated in a series of ethanol (100%-70%)for 3 min each 36  followed by rehydration in P B S for 30 min. After rehydration the endogenous peroxidase was blocked with 0.3 % hydrogen peroxide followed by antigen retrieval by microwaving sections in citrate buffer pH 6.0 (10 mM Na-citrate). Following antigen retrieval, the sections were stained using the above mentioned kit according to manufacturer 's recommendations but with the following modifications. Sections were incubated with the primary antibody at 4° C overnight at the indicated dilutions: C K 2 a (1:100). Sections were stained with Vectastain A B C elite kit and DAB secondary detection kit (Vector Laboratories, C A , USA). Each section had its own control using the secondary antibody only. Pre-immune serum was initially used to ensure specificity of the signal with each of the antibodies.  2.2.9  ELISA assays for IL-8 and MCP-1  Cells were pretreated with the appropriate inhibitors and stimulated for 36 hr with IL-1 p. The supernatants were sampled and the chemokine concentration was determined, in triplicate, by using enzyme-linked immunosorbent assay (ELISA) (BD Pharmingen, Missisaugua, Ont., Canada), as per the manufacturer's instructions.  If required,  supernatants were stored at -80°C prior to use. The sensitivity of the MCP-1 and IL-8 ELISA assays was 1.0 pg/mL and .8 pg/mL, respectively. This was determined b y the manufacturer and defined as two standard deviations above the mean optical density of 20 replicates of the zero standard.  2.2.10 MAPKAPK2 assay This was carried out as an in vitro assay using crude lysate. Briefly, Caco-2 cells were preincubated with S B 203580 for 2 h, stimulated with IL-1 p (2 ng/ml) for 30 min and  37  then  harvested  in  homogenization  buffer  (described  under  section  2.2.3  Immunoblotting). Ten uL (equal to 10 ug of protein) of cell lysate was then used in a kinase assay, using heat shock protein 27 (HSP 27) (1 ug; Stressgen Biotechnology) as the substrate and 0.5 ug of A T P (250 UM A T P , 1 uCi [V*- P]-labeled), for 20 min at 30°C. 32  Samples were then boiled in 5x sample buffer and resolved on S D S - P A G E . After transfer to nitrocellulose membranes, the substrate bands were visualized using autoradiography. Ponceau staining was carried out to demonstrate equal loading.  2.2.11 Immunoprecipitation Lysates were homogenized and normalized for protein using Bradford assay as described under immunoblotting.  400 ug of soluble protein in a final volume of 500 uL  of homogenization buffer was used. 5 uL of antibody was added and the tubes placed on a rotator overnight at 4°C. The following morning, 30 uL of 1:1 slurry of protein A / G (Sigma, Oakville, Ontario) beads were added and then rotated for 1 h at 4°C. Following this the beads were washed twice with homogenization buffer and twice with Kll buffer (12.5 mM -glycerol phosphate, 20 mM M O P S pH 7.2, 5 m M E G T A , 7.5 m M MgCI 2, 50 mM NaF and 0.25 mM DTT). The beads were then resuspended in 15 uL of Kll and subjected to an immune complex kinase assay as described below.  2.2.12 Immune complex assays for PKB and IKK signalsome 10 uL of the substrate cocktail, containing either histone H2B (1 ug), G S T - I K B (1 ug), or GST-p65  (1 ug)  in assay dilution  buffer  (20 m M M O P S ,  pH 7.2, 25 mM -  glycerophosphate, 20 m M MgCI 2, 5 mM E G T A , 2 m M EDTA, 1 mM DTT and 1 m M sodium vanadate), were added to the washed beads, and the reaction carried out as  38  above for 20 min using 5 uL of the A T P cocktail (250 mM A T P , 1 Ci [y32 P] ATP). 10 uL of 5X sample buffer were added to the beads and after boiling for 5 min. The samples were resolved by 10% S D S - P A G E , and stained with coomasie blue. The gel was then dried for 45 min before phosphoimaging analysis using a Bio-Rad molecular imager F X (or alternatively exposed to film overnight at -80°C and then developed).  39  CHAPTER 3 - MAPK REGULATION OF IL-1 p SIGNALING 3.1  RATIONALE AND HYPOTHESIS As  mentioned  previously,  there  is strong  evidence for  MAPK  signaling  downstream of IL-1 (5. The functional role of each of the M A P K family members in l E C s is not known, especially with respect to activation of N F K B , and subsequent synthesis of chemokines. In other systems, recent work has shown a role for p38 in the synthesis of many other inflammatory mediators. These observations include regulation of IFNy in lymphocytes, iNOS  in astrocytes and glial cells, and cox-2, IL-6, and T N F a  in  monocytes(116-120). Also the food agent curcumin has been shown to be an inhibitor of JNK(199). Preliminary reports have shown that curcumin can in fact modulate both N F K B  signaling and chemokine production(200).  Thus there is strong evidence to  support the notion that M A P K family will regulate IEC chemokine production.  3.2  RESULTS  3.2.1  IL-1 (3 activates protein tyrosine phosphorylation and MAPKs in lECs The effect of IL-ip stimulation on the temporal characteristics of the activation of  the M A P K family members was first examined. The data indicate that all three M A P K s were activated within 10-15 min, with p38 M A P K exhibiting the earliest activation (Fig. 4A). In addition it is apparent that the activation was sustained for at least 60 min. The lowest panel shows that proteins corresponding to M A P K s (with a molecular mass of 40-44 kDa) become tyrosine phosphorylated at similar times. 40  To address the role of p38 M A P K the selective inhibitor S B 203580 was used(201). To confirm that the S B 203580 inhibited signaling through to downstream mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), in vitro phosphorylation reactions were performed using its substrate, heat shock protein (HSP 27). The data indicates that preincubation with the p38 inhibitor attenuated activation of this downstream target (Fig. 4B).  3.2.2  SB 203580 attenuates IL-8 and MCP-1 production by IL-1 (3 stimulated lECs Next the effect of the p38 M A P K inhibitor, S B 203580, on the production of an a -  chemokine (IL-8) and a p-chemokine (MCP-1) by l E C s was examined. This inhibitor acts by binding to the ATP-binding pocket of p38(201). Pretreatment of both Caco-2 cells and HT-29 with S B 203580 led to a significant reduction in IL-8 production (Fig. 5A). A similar inhibitory effect of the p38 M A P K inhibitor was observed on MCP-1 production in Caco-2 cells (Fig. 5B). HT-29 cells do not produce any MCP-1 in response to IL-1 p alone, as previously reported(202). PD 98059 (an inhibitor of E R K via upstream activator-dependent phosphorylation) had no effect upon the production of either chemokine in the Caco-2 cell line (lane 4). The data indicates that p38 M A P K is activated in l E C s in response to IL-1 p and is involved in the production of important immunoregulatory chemokines.  41  3.2.3  IKB  As and  phosphorylation and degradation are independent of p38  MAPK  previously mentioned N F K B has been reported to be a major regulator of IL-8  MCP-1 transcription. The phosphorylation and subsequent degradation of I K B is an  integral step in the activation of N F K B . Previously, protein kinase C K 2 has been shown to be downstream of p38 M A P K in response to stress(129). Therefore the possibility that p38 M A P K or downstream C K 2 were involved in the phosphorylation and degradation of I K B was explored. However, as the data indicates, pretreatment of cells with either S B  203580 or D R B , a specific inhibitor of protein kinase C K 2 at the  concentrations used, did not prevent IL-1 p induced phosphorylation and degradation of IKB  (Fig. 6A).  3.2.4  NFKB  DNA  binding and transactivation are independent of p38  MAPK  activation N F K B  is regulated by its ability to translocate to the nucleus and bind to its  consensus sequence. Therefore, the potential role of p38 M A P K on this aspect of N F K B activation was investigated. Electrophoretic mobility  shift  assays (EMSAs)  were  performed on nuclei isolated from Caco-2 cells. Pretreatment of cells with S B 203580 or DRB  did not prevent nuclear translocation or the ability of N F K B to bind to its consensus  sequence (Fig. 7A). It can be seen that there was no effect upon the bound probe signal with either of the two inhibitors. In order to confirm that in fact p65 was the major N F K B family member binding to the N F K B  consensus sequence, a supershift assay was  performed, by preincubating nuclear extracts with the anti-p65 antibody before addition 42  of the probe.  A s seen, the p65/RelA band was almost entirely shifted up with the  addition of the antibody (Fig. 7B). DNA binding alone does not confer transcriptional activation, as N F K B  requires multiple transactivating phosphorylations in order to be  functionally active once bound to DNA(203).  To investigate the possibility that p38  M A P K may be phosphorylating and regulating the transactivation of p65, we performed luciferase reporter assays with four synthetics repeats of a consensus K B site fused to a luciferase gene(4xKB-l_uc). The data indicates that there is a threefold increase in N F K B activation upon exposure of Caco-2 cells to IL-1 p 203580 did not attenuate the activation of N F K B . N F K B  (Fig. 7C). Pretreatment with S B Curcumin was found to attenuate  activation, in accordance with a previous report(200). This data excludes an  effect of p38 M A P K upon N F K B activation in this cell system. To  confirm that there was no involvement of p38 M A P K upon N F K B DNA binding,  we transiently transfected Caco-2 cells with both the wild-type and kinase-dead versions of p38 M A P K , and investigated their influence upon D N A binding in response to stimulation with IL-1 p. These interventions were found to have only a marginal effect upon this parameter (Fig. 8A). In addition the effects of transient transfections of the p38 M A P K constructs upon N F K B (Fig.  reporter activation were examined. The data indicated  8B) that despite the small changes observed in the DNA-binding study (Fig. 8A),  this is not translated  into a reduction in transactivation. W e believe that this  complements the data obtained with the S B 203580 inhibitor, and establishes that p38 M A P K is not involved at any step of N F K B activation within the Caco-2 cell system. The  control western immunoblot (Fig. 8C) shows that the p38 M A P K protein was  overexpressed. In contrast to the data obtained for N F K B , 43  we were able to show a  reduction in  p induced AP-1 DNA binding using S B 203580 (Fig. 9). This reduction  was limited to IL-1 p induced AP-1 binding and had no effect on basal constitutive AP-1 DNA binding.  3.2.5  IL-8 and MCP-1 messages are regulated by p38 in lECs Next the role of the p38 M A P K inhibitor on the relative amounts of both IL-8 and  MCP-1 message was investigated. A time-course investigation using semiquantitative reverse transcriptase P C R (RT-PCR) was carried out, and after resolving the P C R products the bands were quantified by densitometry and corrected for using actin expression. The data indicate (Fig. 10A and 10B) that there was a reduction in the message for both of the chemokines investigated. The magnitude of this reduction was 40% for IL-8 and 50% for M C P - 1 . In order to verify or exclude a role for message stability being the mechanism for reduction of chemokine expression, R T - P C R assays were  repeated  using actinomycin-D to  stop  active transcription,  as  previously  reported(204). The possibility of message stability being the mechanism for reduction of chemokine expression was excluded by our findings (Fig. 10C). More specifically, whereas Wang et al. were able to show an almost 80% reduction in L P S stimulated messages for both T N F a and IL-6 in human monocytes within 20 min, our findings showed negligible changes, even as late as 120 min into the assay.  3.2.6  p38 is involved in activation of the IL-8 promoter Our findings did not confirm an effect on chemokine message stability, and thus  further studies were performed with the previously characterized IL-8 luciferase (IL8-  44  Luc) promoter construct to determine whether p38 was involved at this level(41). The data clearly indicate that using S B 203580 resulted in a significant attenuation of promoter activation (Fig 11A). The data also indicate that the p38 kinase dead (p38KD) construct reduces IL-8 promoter activation when compared to cells transfected with the empty vector (EV). Unexpectedly there was also a smaller reduction of activation using the wildtype (p38WT) construct when compared with the empty vector (Fig. 1 1 B ) . The explanation for this is not clear and may be a result of the activation of counterregulatory pathways by a constitutively active p38 signal.  3.2.7  Pharmacological inhibitor SP 600125 inhibits IL-1 induced JNK activity  however has no effect on p65 DNA binding To expand on the previous reports using curcumin, as well as our own work that have successfully reproduced the curcumin induced inhibition of chemokine production (data not shown), the selective J N K inhibitor S P 600125 was used(205).  Using  curcumin we have observed inhibition of IL-8 promoter activation, as well as inhibition of N F K B  activation (Fig 7 C A N D 8A). Thus this work was repeated using S P 600125.  Treatment of cells with 20 uM S P 600125, prevented the IL-1 (3 induced activation of J N K (Fig 12A). When the phosphorylation status of J N K was examined, it clear that it inhibits J N K phosphorylation (Fig 12B).  Interestingly, p38 phosphorylation was also  inhibited, suggesting a degree of nonspecificity. When E R K was examined, there was no effect on its I L - 1 p induced phosphorylation (data not shown). When the DNA binding of p65 was examined, S P 600125 pretreatment had no effect (Fig 12C). Similarity there was no effect on I L - 1 p induced I K B degradation (data not shown).  45  3.2.8  SP 600125 inhibits IL-1 p induced  NFKB  activation and IL-8 promoter  activation Since S P 600125 had no effect on N F K B nuclear translocation and D N A binding, we tested its ability to regulate the complete activation of N F K B , construct.  S P 600125 inhibited N F K B  using the 4 X K B - I U C  activation by 50% (FIG 13A).  When IL-8  promoter activation was examined using S P 600125, it also attenuated IL-8 promoter activation by 50% (FIG 13B).  Interestingly when R T - P C R was performed and the  mRNA of several chemokines was examined, including IL-8, there was no effect (data not shown).  46  A  SjjSl 5B8B> SSm i:inf~' a  JI.LUI fir  m  -  «S«HW  53  -  W  B  ^mmm  «(MN»  <  p-JNK  —  113 92  29  lim  M*  • «™*  i  p-ERK  GAPDH  jy  «| mm V P IHR WW m)M  '-mm*  5  10  w  p-Tyrosine  15  30  45  60  Time (minutes)  Figure 4. IL-ip* stimulation of l E C s results in the activation of E R K , J N K and p38. (A) Caco-2 cells were stimulated with IL-1 p (2 ng/ml) for the indicated times, before being harvested. Western immunoblotting was carried out. (B) Caco-2 cells were preincubated with S B 203580 for 2 h before being stimulated with IL-1 (2 ng/ml) for 30 min and harvested. Kinase assays were performed, with HSP27 as substrate. Samples were resolved by S D S - P A G E , transferred to nitrocellulose membranes, and visualized using autoradiography. The panel on the left is an autoradiogram and the one on the right is a Ponceau stain of the same membrane, showing co-localization of the HSP27 band. The results shown are representative of at least three independent experiments. -47-  1 = Control 2 = 1L-1P 3 = IL-ip + SB 203580 (10 uM) 4 = IL-ip + PD 98059 (25 uM)  Figure 5. Inhibition of p38 M A P K attenuates IL-1B induced IL-8 production. Caco-2 were pretreated with S B 203580 (10 uM) or PD 98059 (25 uM) for 2 h, stimulation with IL-1 p (2 ng/ml) for 36 h. Supernatants were collected and analysed for the presence of IL-8 (A) or monocyte chemotactic protein-1 (MCP-1) (B) by ELISA. (Lane 1, control; lane 2, IL-16; lane 3, IL-18 with S B 203580; lane 4, IL-1 with PD 98059.) The results shown are representative of at least three independent experiments. The results are expressed as mean ± standard error of the mean (SEM) (* P <001). -48-  IL-16 SB DRB  .  .  . -  . +  + + -  -  + +  + + -  Figure 6. IL-16 induced degradation of I K B is independent of p38. Caco-2 cells were preincubated with DRB (10 uM) or S B 203580 (10 uM) for 2 h. Cells were stimulated with IL-1 p (2 ng/ml) for 30 min, then harvested. Cells were lysed, resolved by S D S - P A G E and immunoblotted with the appropriate antibody. The results are representative of at least four independent experiments.  -49-  Supershift  SB  B  Bound probe  Free probe DRB SB IL-16  - + - - + + + +  Probe IL-16 Anti-p65 Cold Comp  + + + + + +  -  +  -  - +  4XKB-IUC cu  w CO  4  3H  cu l_  CJ  o LL  Z> _J  IL-18 SB Curcumin  + +  Figure 7. IL-1 p induced activation of N F K B is independent of p38. Caco-2 cells were preincubated overnight with DRB (10 uM) or S B 203580 (10 uM) for 2 h before being stimulated with IL-1 p (2 ng/ml) for 30 min. (A) Nuclear extracts were prepared and assayed for N F K B binding by using electromobility shift assays (EMSAs). (B) Specificity of N F K B binding was determined by antibody supershifting or competition with cold probe. Nuclear extracts were preincubated with an anti-Rel A antibody or 100-fold excess of cold unlabelled probe, before adding radiolabeled probe. (C) Cells were co-transfected with the N F K B reporter 4 X K B - I U C and LacZ. 24 hours later cells were preincubated with the appropriate inhibitor for 2 h, before being stimulated with IL-1 p (2 ng/ml) for 6 h. Cells were harvested and the luciferase activity was determined. Results were normalized for transfection efficiency using LacZ activity. Results are representative of at least five independent experiments. -50-  Vector p38WT p38 K D IL-1  + _ _  Bound probe  B  Interleukin 1 (I) Empty vector p38 (wt) p38 (kd) C p38 MAPK IL-1 p Vector p38 WT p38 K D  +  -  +  -  +  +  -  +  -  +  +  -  -  -  +  -  -  -  +  - -  +  -  - -  +  +  Figure 8. IL-1 p induced activation of N F K B is not modulated by overexpression of kinase inactive p38. Caco-2 cells were transfected with the indicated plasmids (p38wt = p38 wild-type, p38kd = p38 kinase-dead) and then stimulated with IL-1p for 30 min ( A ) or 6 h ( B ) . ( A ) D N A binding was carried out using E M S A , as previously described. ( B ) In addition to p38wt and p38kd, cells were also transfected with the N F K B reporter 4 X K B - I U C Cells were harvested and the luciferase activity was determined. Results were normalized for transfection efficiency using LacZ activity for p38 expression. (C) Expression of both native and transfected p38 M A P K is shown by immunoblotting. Results are representative of at least five independent experiments. -51-  Figure 9. Inhibition of p38 attenuates IL-1B induced AP-1 DNA binding. Caco-2 cells were pretreated with S B 203580 (10uM) for 2 h before being DNA binding was assessed using EMSA, as previously described. Results are representative of at least two independent experiments. -52-  IL-1 IL-8 mm  MCP-1  ACTIN  mm  — 0  30  IL-1 + S B 203580 mm  mm —  mm  mm mm  ~ ——  —  -OL J^  ^r^j^w-  ^g^^jtt  •••^•i!-*?'  ^(PWP:  q^^^P  90  120  I 1 i^^^PW-  1  mm mm mm mm mm mm mm mm 60  90  120  150  180  0  30  60  Minutes  150  180  Minutes  1.40  0  30  60  90  120 150  180  0  30  Time (minutes) — • — IL-1  60  90  120  150 180  Time (minutes)  — • — IL-1 + S B  — • — IL-1  IL-1 + Act D  — • — IL-1 + S B  IL-8  —  IL-8  IL-1 +SB+ Act D 0  15  30  60  90  120  (minutes after Actinomycin D treatment) Figure 10. p38 MAPK regulates IL-1 p induced IL-8 and MCP-1 mRNA expression. (A) Caco-2 cells were preincubated with the appropriate inhibitor for 2 h, before being stimulated with IL-16 for 3 h. Total RNA was isolated, and semiquantitative reverse transcriptasepolymerase chain reaction (RT-PCR) was performed and P C R products were resolved using agarose gel electrophoresis. (B) Densitometry was performed using Bio-Rad Quantity One Software. (C) Cells were stimulated with IL-1 for 3 h and then cultures were adjusted so that they contained 10ug/ml of actinomycin D (Act D), either alone or together with 10 urn SB 203580 (SB). The samples were then harvested at the indicated time-points, total RNA extracted and semiquantitative P C R for IL-8 was performed. The data shown are representative of three independent experiments. -53-  20  Figure 11. IL-1 p induced activation of the IL-8 promoter is dependent on p38 M A P K regulation. (A) Caco-2 cells were co-transfected with the IL-8 promoter and LacZ for 24 h. Cells were preincubated with the appropriate inhibitor for 2 hr, before being stimulated with IL-1 p (2 ng/ml) for 6 h. (B) Alternatively, the IL-8 promoter was co-transfected with the p38 MAPK constructs (p38WT = p38 wild-type, p38KD = p38 kinase-dead) and then stimulated with IL-1 p. Cells were harvested and the luciferase activity determined. Results were normalized for transfection efficiency using LacZ activity. Results are representative of at least three independent experiments. -54-  GST-ATF2  LC  C  SP  IL-1  (20 u M )  IL-1 + SP(20uM)  B  pJNK  JNK p-p38 p38 C  SP (20 uM)  IL-1  IL-1 + S P (20 uM)  IL-1 SP2uM S P 20 uM  Figure 12. Selective J N K inhibitor S P 600125 inhibits J N K activity, however does not regulate N F K B D N A binding. HCT-116 cells were pretreated with S P 600125 at the indicated concentration for 2 h before being stimulated with IL-1 p for 30 min. (A) JNK activity was determined by immunoprecipitating JNK and using GSTATF2 as a substrate. Phosphorylated substrate was resolved using S D S - P A G E , and visualized using autoradiography. LC-Lysate and non specific IgG control, C-control, S P - S P 600125 at 20 uM ( B ) Immunoblotting was performed to examine phosphoryaltion status of p38 and JNK. (C) EMSA was performed to assess N F K B DNA binding. Results are representative of at least 3 independent experiments.  -55-  A 4XKB-LUC  Control  SP 2 uM  SP 20 uM  IL-1  IL-1 + SP 2 uM  IL-1 + SP 20 uM  IL-1 • S P 2 u M  IL-1 + S P 20 u M  B IL8-LUC 12.0 -r 10.0 8.0 6.0 4.0 2.0 0.0 -  Figure 13. Selective J N K inhibitor SP600125 inhibits N F K B activation and IL-8 promoter activation. HCT-116 cells were transfected with the K B - L U C (A) or IL8-Luc ( B ) for 24 h. Cells were then pretreated with the SP600125 at the indicated concentration for 2 hours before being stimulated with IL-1 p for 6 h. Results are representative of at least three independent experiments.  -56-  3.3  DISCUSSION This section of work was devoted to the investigation of the role of M A P K  family members in the regulation of IL-1 p induced IEC chemokine release. Inhibition of ERK  had no effect on the activation of N F K B , and subsequently no effect on IL-8 or  MCP-1  production. Inhibition of p38 had significant effects on IL-8 and MCP-1  production; however, this effect was not through a p38 dependant regulation of either message stability or N F K B .  The effect was determined to be at the proximal IL-8  promoter, potentially through disruption of AP-1 activation.  Inhibition of J N K activity  using curcumin or selective J N K inhibitor S P 600125 reduced IL-8 promoter activation as well as N F K B  activation.  Curcumin prevented chemokine expression; however,  using S P 600125, enigmatically, there was no effect on message production of a number of chemokines including IL-8, M C P - 1 , Groa, and R A N T E S (data not shown). ERK  activation in response to IL-1 p is well documented(206-208). Many reports  in several different systems have dissociated E R K activation from both N F K B activation, and chemokine synthesis, so the lack of E R K regulation on chemokine synthesis in the IEC system was not surprising. Interestingly at the same time these studies were completed, a report was published looking at T N F induced IL-8 production in IECs(209). This report showed that E R K s did in fact play a role in the IL-8 synthesis.  This is  possible because due to the difference in downstream signaling pathways induced by TNF and IL-1 p. Although both the IL-1 R and tumor necrosis factor receptor-1 (TNFR1) are capable of activating N F K B as well as the M A P K family members, the intermediates,  57  such as the adaptor proteins that are recruited immediately following receptor activation, can be completely different.  A s a result, differences in the cytoplasmic portion of  different families of receptors, such as those seen here between the IL-1 R superfamily and the T N F R superfamily are important. The question of the functional role of E R K activation in response to IL-1 p remains, and it may be responsible for things such as IEC migration, and epithelial cell restitution, which are important processes during the inflammatory response. A role for p38 M A P K in functional responses to IL-1 p in human l E C s has never been investigated, although the production of IL-8 in response to invasion of l E C s with Salmonella typhimurium has been previously demonstrated(207). IL-1 p regulation of MCP-1 has been investigated, and these reports show a regulatory effect of p38 M A P K on MCP-1 production^ 10-212). In many other systems p38 M A P K has been shown to regulate chemokine production. Several mechanisms occur to explain this regulation, and these appear to be system and stimulus specific. Several reports have indicated that p38 M A P K is involved in the transactivation of  NFKB  as one  potential  mechanism(213-215). This  may  occur via  indirect  mechanisms, including modulation of p65 transactivation and phosphorylation of the associated TATA-binding protein, required for transcriptional activation. Additionally, a hypothesized (but unproven) second signaling pathway involving M A P K s , which may allow fine-tuning of transcriptional responses within the nucleus, has been invoked to explain this effect. Perhaps the most compelling evidence for cross-talk between these two integral signalling pathways is derived from data in cardiac myocytes, where there is evidence of an interaction between MKK6 and IKK(216). With particular reference to  58  the IEC system, our study clearly contrasts these observations by showing that p38 M A P K plays a role distinct from that of regulating N F K B activation. p38 M A P K did not significantly influence the ability of N F K B to bind to DNA, or to phosphorylate IKB, nor did it affect the ability of translocated p65 to activate a target gene.  Regulation of m R N A transcript levels is probably one of the major determinants of cytosolic levels of protein, and this is a potential second mechanism. The transcripts of many short-lived cytokines and proto-oncogenes are often characterized by A R E s found in their 3' UTR, and it is these A R E s that mediate their subsequent destabilization and decay(217, 218).  IL-8 and MCP-1 both feature A R E s , which provide stimulus-  specific regulation of their decay through the MKK6/p38 pathway(51, 219). Knockout mice that have A R E s deleted from the T N F a gene exhibit increased levels of T N F a and develop chronic inflammatory arthritis and Crohn's-like IBD(220). Predictably, peritoneal macrophages from these animals were found to be unresponsive to modulation by the p38 pathway using S B 203580 at concentrations similar to those used in our study (10 uM). The studies presented here preclude this mechanism, despite the strong evidence of this regulation in other systems. A final mechanism may be the regulation of the chemokine promoters as we have presented here. Another candidate transcription factor that is regulated by p38 is A P - 1 . Although it is not required for chemokine synthesis, it can play an important role in modulating the maximum production of chemokines. A s such it has been previously reported that mutation of the AP-1 binding site results in an approximate 50% decline in promoter activity(41), similar to what was seen here. This does not preclude a role for  59  p38 in other aspects of promoter regulation. The coactivator p300/CBP is essential for the formation of the transcriptional activation complex. Although no work has looked at p38 regulation of this coactivator, a recent study looking at IL-2 promoter assembly showed that inhibition of p38 led to an inability to form a functional A P - 1 , N F K B , C R E B transcriptional complex(221).  Inhibition of p38 prevented the ability of C R E B to  associate with CBP/p300 and rendered the complex inactive. This also presents the final aspect of promoter assembly, p38 M A P K may regulate chromatin remodeling and access to chemokine promoters, as previously reported(48). Our initial studies looking at histone proteins shown to be under p38 regulation, showed a lack of increased phosphorylation of histone H3 at serine 9, as well as a lack of histone H3 dual phosphorylation and acetylation in response to IL-1 p (data not shown). Pretreatment of S B 203580 was not able to modulate histone H3 modification both basally and in response to IL-1 p. The role of J N K in chemokine synthesis is well explored in some systems, however still remains largely enigmatic in the IEC system. Two approaches were taken in this work to examine the potential role of J N K in IEC chemokine release. Curcumin which has been shown to inhibit both signals upstream of IKK activation(200) as well as JNK(199, 222), was used as well as the selective J N K inhibitor S P 600125. Both the inhibitors attenuated N F K B activation as well as IL-8 promoter activation. Many reports have shown a role for J N K in regulating the IL-8 promoter by directly binding to it and being an important part of the transcriptional assemble complex. Alternatively both of these inhibitors may have additional effects on other kinases and regulatory pathways. Recently both P D 98059 and S B 203580 were shown to have a non-selective inhibitory  60  effect on the related MSK1 and this led to inhibition of both N F K B activation as well as chemokine release(69, 223). The same may be happening with the J N K inhibitor, due to the high degree of homology between M A P K family members.  Further investigation  using molecular and genetic approaches is required to fully delineate the mechanism of J N K control on promoter activation.  61  CHAPTER 4 - CK2 regulation of IEC N F K B Activation and Chemokine Synthesis  4.1  RATIONALE AND HYPOTHESIS Protein kinase C K 2 is highly conserved throughout evolution, thus underscoring  its important biological role. Enigmatically, however, the precise role of this kinase has yet to be defined.  A n area in which C K 2 may play a significant role, but yet to be  explored, is its role in IEC inflammatory signaling. Although there has been no formal connection between C K 2 and IEC chemokine production, some interesting reports in other cell systems do set a very interesting precedence. C K 2 has been linked to the regulation of N F K B , as previously discussed. controversial, with different  This regulation however has been  mechanisms in different cell systems. Some describe  regulation of the basal stability of IKB(224-227), some describe regulation of IKK activity(135, 136), whereas some describe regulation of p65 phosphorylation^-73). In addition, a recent report has also linked C K 2 to IFNy mediated signaling(228). Thus it can be hypothesized that C K 2 will play a role in chemokine expression in lECs, through its regulation of N F K B .  62  4.2  RESULTS  4.2.1  CK2 activity is increased in patients with active IBD Patient samples were acquired from both normal uninflamed tissue as well as  inflamed tissue from people suffering from IBD.  These were immunohistochemically  stained for C K 2 a protein expression. Staining of normal tissue revealed a relatively ubiquitous expression of CK2oc (Fig 14A).  It was present in both epithelial cells and  infiltrating lymphocytes, however absent from mucous containing vacuoles of goblet cells. Staining of actively inflamed tissues showed an increase in C K 2 a staining. There was increased CK2ct staining in each epithelial cell cytoplasm. Infiltrating lymphocytes also stained with a higher intensity.  4.2.2  CK2 activity is increased in a murine model of colitis In order to assess C K 2 kinase activity, normal and inflamed tissue was examined  in mice induced with colitis by treating with 2 % dextran sodium sulphate (DSS). Tissue homogenates were subjected to C K 2 kinase assays, using G T P and a C K 2 specific peptide substrate. There was a significant increase in the activity of C K 2 in tissue from inflamed mice, as compared to their normal control counterparts (Fig 15). Similar results were also seen in an alternate murine model of colitis, the T N B S model (data not shown).  A s with the human samples, immunohistochemistry analysis revealed that  there was a concomitant increase in the protein expression of C K 2 a (data not shown).  63  4.2.3  Inhibition of C K 2 prevents IL-1 (3 induced N F K B activation N F K B  plays a very important role in the activation of the IEC inflammatory  response, thus the role of CK2 in regulating N F K B was next investigated. The IEC cell lines Caco-2, and HCT-116 were used as they have been well characterized, can form tight junctions, as well as release numerous cytokines and chemokines. CK2 activity was inhibited using the selective CK2 inhibitor, apigenin, as previously reported(229). Cells were transfected with a K B dependant reporter (4XKB-IUC) for 24 h before being pretreated with the inhibitor at the appropriate concentration for 1 h, and stimulated with IL-1 p (2ng/mL). Pretreatment with apigenin led to a dose dependant decrease in the IL1B induced activation of N F K B  (Fig 16).  Data from Caco-2 is presented, and is  representative of that from HCT-116 (data not shown).  4.2.4  Overexpression of a kinase inactive C K 2 inhibits IL-1 (3 induced N F K B  activation  The catalytic dimer in CK2 holoenzyme can be made up of either a or a', so the effect of overexpression of CK2 a or  a ' on  N F K B  luciferase reporter activity was  examined next. Overexpression of CK2a was sufficient to activate the N F K B reporter (4XKB-IUC) (Fig 17). Interestingly this was not repeated by overexpression of the CK2a'. Upon treatment with IL-1 p, both a and a ' overexpression led to a mild hyperactivation of the N F K B dependant reporter above empty vector stimulated, however this effect was additive, and not synergistic. Overexpression of either kinase inactive a or a ' led to both an inhibition of basal N F K B activity, as well as I L - 1 p induced activity.  64  4.2.5  CK2 regulates the transactivation of p65 Next the mechanism of regulation of C K 2 was investigated in more depth. The  phosphorylation and subsequent degradation of IKB, the first regulatory step in activation of N F K B , was examined by immunoblotting. apigenin(229)  or a second C K 2 inhibitor,  Pretreatment of cells with  5,6-dichloro-ribifuranosylbenzimidazole  (DRB)(230), had no effect on IL-ip induced phosphorylation and degradation of IKB (Fig 18A). Subsequently, when the IL-1 p induced nuclear translocation and DNA binding of NFKB  was examined (by E M S A ) , pretreatment of cells with either C K 2 inhibitor had no  effect (Fig 18B). The ability of p65 itself to undergo post-translational modifications provides an important mechanism to regulate its transactivation.  W e examined the  ability of C K 2 to regulate the IL-1 p induced transactivation of the p65 subunit.  We  employed the one-hybrid system whereby a portion of the C-terminal transactivation domain (amino acids 501-551, or 521-551) was fused to the yeast Gal4 DNA binding domain. This construct was co-expressed with Gal4 responsive luciferase reporter to assess signal specific activation of the p65 transactivation domain-1 (TAD1). IL-1 p was able to activate the C-term transactivation domain, and the pretreatment of C K 2 inhibitors prevented this activation (Fig 18C). Previously it has been shown that C K 2 can regulate the C terminus transactivation domain via serine 529. When C K 2 was overexpressed with a W T construct, it was able to induce its activation, however when overexpressed with a serine 529 to alanine mutant construct, it was unable to  65  transactivate this construct (Fig 18D). Thus C K 2 regulates IL-1 p induced activation of NFKB  through the regulation of transactivation domain 1 (TAD1) via serine 529.  4.2.6  CK2a associates with p65, exclusively in the nucleus The possibility that there may be an association of C K 2 a with p65 was next  examined. HCT-116 cells were stimulated with IL-1 p for 15 minutes, and subsequently immunoprecipitated for p65 and C K 2 a , and immunoblotted for the other protein.  CK2a  was found to associate with p65 (Fig 19A). Interestingly the amount of association did not change with IL-1 p stimulation, even when time points as long as one h were examined. The cellular localization of this association was examined, by fractionating the cellular components into cytoplasm and nucleus, before immunoprecipitating them for p65 and CK2oc.  C K 2 a and p65 were found to associate exclusively in the nucleus,  suggesting that this association was found to be after IKB has released p65 and revealed its nuclear localization signal (Fig 19B). Also when p65 immunoprecipitates were immunoblotted for C K 2 a ' , it was found to associate with p65 as well. To confirm that the nuclear and cytoplasmic separations were clean, whole cell lysate was immunoblotted for histone H3, a protein marker found only in the nucleus (Fig 19C).  4.2.7  p65 associated CK2 activity is increased with IL-1 p stimulation Since a pool of C K 2 is able to associate with p65, and the amount of this  association remained constant, the specific activity of this p65 bound pool of C K 2 was examined. HCT-116 cells were stimulated with IL-1 p for varying lengths of time, before 66  being immunoprecipitated for p65. p65 immunocomplexes were then subjected to C K 2 assays, using casein as a substrate and G T P as the phosphate donor. p65 bound C K 2 activity was increased at 30 minutes of stimulation (Fig 20). When compared to the kinetics of degradation of k B , the p65 bound C K 2 was activated most likely at the point when it entered the nucleus.  Similar results were also achieved using a specific  substrate instead of casein. When total cellular C K 2 activity was examined, there was no significant change with IL-1 p stimulation (data not shown).  4.2.8  Inhibition of CK2 prevents IL-1 p induced activation of the IL-8 proximal  promoter Next we examined if C K 2 could regulate the activation of the downstream N F K B target, IL-8, through its regulation of N F K B transactivation. N F K B has been shown to essential for the activation of the proximal IL-8 promoter(41). Cells were transfected with a luciferase reporter gene construct under the control of the IL-8 promoter.  IL-1 p  robustly activated this construct, and pretreatment with the C K 2 inhibitors dose dependency attenuated this activation (Fig 21).  4.2.9  Inhibition of CK2 prevents the message synthesis of  NFKB  downstream  targets To  further confirm C K 2 was able to regulate downstream N F K B  examined the IL-1 (3 induced message synthesis of N F K B  67  targets we  downstream targets.  kB  synthesis one of the simplest N F K B target promoters.  Pretreatment with apigenin  prevented the resynthesis of IKB message that is required for the resynthesis of IKB following its degradation (Fig 22A). In addition, we examined several chemokines and cytokines. Many of these had been attenuated by pretreatment with apigenin (Fig 22).  68  Figure  14.  CK2a  is  overexpressed  in  patients  with  active  UC.  Immunohistochemical staining of C K 2 a in a patient with normal uninflamed colon (A/D) or in actively inflamed tissue from a patient diagnosed with U C (B/E). Immunohistochemical staining of uninflamed with non-specific rabbit IgG (C).  -69-  Figure 15. DSS induced colitis in mice increases CK2 activity in actively inflamed tissue. D S S colitis was induced in mice using 2.0% D S S (3 animals per group). 48 h later, mice were sacrificed, and inflamed or normal tissue from the large bowel was collected and homogenized for protein. Samples were normalized for protein concentration before performing CK2 kinase assays using C K 2 specific substrate and G T P .  -70-  8.0  i  CONTROL  IL-1 2ng/mL  IL-1 + Api 7.5 uM  IL-1 + Api 80 uM  Figure 16. Inhibition of C K 2 using a selective inhibitor prevents IL-1p induced activation of N F K B in intestinal epithelial cells. Caco-2 cells were transfected with an N F K B responsive luciferase reporter (wxKb-Luc). 36 h later cells were pretreated with the selective C K 2 inhibitor apigenin at various concentrations before being stimulated with IL-1 (3 (2 ng/mL) for 6 h. Cells were lysed and measured for luminescence. Results are representative of at least four independent experiments.  -71-  Figure 17. Overexpression of C K 2 a or a ' modulates both basal and IL-1B induced N F K B activation. HCT-116 colonic epithelial cells were transfected with an N F K B responsive luciferase reporter (4XKD-LUC) and with 1 ug of either empty PCDNA3 vector (EV), CK2ct wt (a WT), CK2a" wt (a' WT), C K 2 a kinase dead (a KD), or CK2 (a' KD). 36 h later cells were stimulated with IL-1 (3 (2 ng/mL) for 6 h. Cells were lysed and measured for luminescence. Results are representative of at least three independent experiments.  -72-  IKB  IL-1  +  +  +  Apigenin  -  +  -  DRB  -  -  +  B  IL-1 Apigenin DRB  Figure 1 8 . CK2 regulation of IL-1 p induced N F K B activation is at the level of transactivation through p65 serine 529. Caco-2 cells were pretreated for 1 h with the selective CK2 inhibitors apigenin (80 uM) and DRB (20 uM), before being stimulated with IL-1(3 (2ng/mL) for 30 min. Cells were lysed for total proteins and subsequently resolved by SDS-PAGE and probed for IKB (A) or lysed for nuclear proteins and subjected to electrophoretic mobility shift assay (EMSA), using a probe containing a consensus K B binding site (B). (C) To assess transactivation, cells were transfected with a Gal4 responsive luciferase plasmid (pfr-Luc) along with p65 transactivating domain 1 - Gal4 fusion (amino acids 501-550) together. 36 h later cells were preincubated with DRB and stimulated with IL-1 p (2 ng/mL) for 6 h. Cells were lysed and measured for luminescence. (D) Transactivation was assessed by transfecting cells with a Gal4 responsive luciferase plasmid (pfr-Luc) along with p65 transactivating domain 1 - Gal4 fusion (amino acids 501-550, either wildtype or ser529ala mutant) together along with empty vector or wildtype CK2 for 36 h. Cells were lysed and measured for luminescence. Results are representative of at least 3 independent experiments. (D) CK2  -73-  IP: CK2a  AC LC  IP:p65  0  IB p65  IB CK2a  IB CK2a  IBp65  15  AC LC  CYTO  B  NUC  0  15  W IP p65 IB C K 2 a  IP p65 IB C K 2 a '  L A  0  5 15  0  5  15  - " """" JJI  L  m  IB p65 IP C K 2 a IB C K 2 a  L A  0  5 15  0  CYTO  5  15  NUC_ Histone H3  0  5 15  0  5  15  Figure 19. C K 2 a associates with p65. and this association occurs exclusively in the nucleus. (A) HCT-116 colonic epithelial cells were serum starved for 6 h before being stimulated with IL-1 p (10 ng/mL) for the indicated lengths of time. p65 or C K 2 a was immunoprecipitated overnight before immunocomplexes were collected with protein A / G , subjected to S D S - P A G E , and immunoblotted for C K 2 a , C 2 a ' , or p65. (B/C) HCT-116 colonic epithelial cells were serum starved for 6 h before being stimulated with IL-16 (10 ng/mL) for the indicated lengths of time. Cells were fractionated into nuclear and cytoplasmic fractions. p65 was immunoprecipitated overnight before immunocomplexes were collected with protein A / G , subjected to S D S - P A G E , and immunoblotted for C K 2 a , C K 2 a ' , or p65. (D) Fractions from the nuclear and cytoplasm extracts were immunoblotted for histone H3. AC= Ab and beads control, LC=lysate and non specific IgG control.  -74-  w  mm  A  —mmmm™  :  ^^K^^m^ ^ ^ ^ M P ^ P '  ^^^^^^^  p-kB  ^|BB|M^  IKB  p-p65 Ser 536 IL-1  0  2  5  15  30  60  (min)  B  p-casein  L A  0  2  5  15  30 60  Figure 20. p65 bound C K 2 a activity increase with IL-1B treatment. HCT-116 colonic epithelial cells were serum starved for 6 h before being stimulated with IL-13 (10 ng/mL) for the indicated lengths of time. (A) Cells were immunoblotted for phospho-p65, p k B , or I K B (B) p65 was immunoprecipitated overnight before immunocomplexes were collected with protein A / G , CK2 kinase assays were performed using G T P and casein as a substrate. The reactions were then resolved by S D S - P A G E , coomasie stained, followed by exposure to film for 6h at -80 C.  -75-  "L-1  _  Apigenin (uM) DRB  _  +  .  +  +  75  20 20  Figure 21. Inhibition of C K 2 prevents IL-1B induced activation of the IL-8 proximal promoter. HCT-116 cells were transfected with the IL-8 promoter luciferase reporter (IL8-Luc). 36 h post-transfection cells were pretreated for 1 h with the selective CK2 inhibitors apigenin and DRB at the appropriate concentrations, before being stimulated with IL-1 (3 (2ng/mL) for 6 h. Cells were lysed and measured for luminescence. Results are representative of at least 4 independent experiments.  -76-  A  IKB IL-1  Apigenin  Figure 22. Inhibition of C K 2 prevents the synthesis of IL-1B induced IKB and chemokines. Caco-2 cells were pretreated with apigenin for 1 h before being stimulated with I L - 1 B for 3 h. RNA was isolated and subsequent RT-PCR was performed for I K B (A), as well as I L - 8 , M C P - 1 , iNOS, and GROct ( B ) . Results are representative of three independent experiments.  -77-  4.2  DISCUSSION This section of work presents a number of novel observations about inflammatory  signaling in l E C s .  In actively inflamed tissue there seems to be an increase in C K 2  expression and also activity. The functional role of C K 2 in l E C s was then examined and a regulatory role for C K 2 in the regulation and activation of the p65 subunit of N F K B was observed.  This activation is required for the subsequent downstream activation of  several N F K B dependant promoters, including most notably IL-8. The activation of p65 is due to the phosphorylation of p65 at serine 529 by C K 2 that appears to occur by a pool of C K 2 that is constitutively bound to p65.  Upon the addition of IL-1 p this pool  increases in specific activity. Although C K 2 has been described to be elevated in many solid tumors and rapidly proliferating tissues, the precise functional consequence of C K 2 overexpression has not yet been determined(126, 231). The data presented here provides some evidence for the potential functional consequences of overexpression of C K 2 . In an inflammatory setting, this may lead to elevated basal N F K B activity, or a hyperactive NFKB  response, especially in response to proinflammatory cytokines. Although normally considered a constitutively active kinase, C K 2 modulation by  growth factors and stress factors has been described(129, 232). Activation of its activity by cytokines has also been described in response to T N F a , and IFNy(129, 228). This is the first report of its activity being modulated by IL-1 (3.  In addition due to the large  number of substrates for protein kinase C K 2 , the mechanism through which it can  78  regulate these substrates is not known. A possible mechanism through which it may accomplish this is by distinct pools of C K 2 that are capable of acting independently of one another. In the situation presented here a nuclear pool, bound specifically to p65, regulates p65 transactivation potential, and its ability to become activated.  When  compared to total C K 2 activity, there is only a negligible change in total cellular activity (data not shown). This phenomenon of pools has been described before, as nuclear matrix bound pools of C K 2 in cancer cells can regulate apoptosis in response to anticancer agents(130). Although C K 2 previously has been reported to regulate N F K B activation, its method of regulation has been controversial. The c-terminus P E S T sequence is a major point of regulation of IKB, as proteins that have regions of sequence with a high proportion of proline (P), glutamic acid (E), serine (S), and threonine (T) residues are degraded much quicker (233). Regulation here is mediated by the phosphorylation of the P E S T sequence of IKB by C K 2 at serine 283, threonine 288, serine 291, and serine 293, however preferentially at serine 293(224-227).  This phosphorylation does not  affect the cytokine mediated degradation of IKB, however it regulates the steady state turn over of IKB by decreasing its stability. An important point to consider is that in the absence of stimulation, there is constitutive translocation of N F K B to the nucleus, due to the short basal half-life of IKB. Loss of function experiments involving the depletion of C K 2 and site directed mutation of the C K 2 phosphorylation sites results in increased IKB  stability and decreased constitutive N F K B translocation (225, 227, 234-236).  An  independent study has also cited that C K 2 is able to phosphorylate IKB in vitro, however this report failed to show the activation of C K 2 in response to T N F a , a potent activator 79  of the N F K B cascade (237). The data presented here in this report provides some preliminary evidence that overexpression of C K 2 may be able to modulate basal N F K B activation (Fig 17). Interestingly, the functional differences between C K 2 a and a' are not well characterized or reported, however, in this situation only a overexpression, not a' overexpression, was capable of inducing basal N F K B activation. This is one the few functional differences that has ever been observed thus far, between C K 2 a and a'. A more direct role has recently been elucidated for C K 2 in regulating N F K B as it has been shown to directly phosphorylate the p65 subunit on serine 529 and regulate its transactivation potential(71, 73). This site is exclusively phosphorylated in response to T N F a and it was shown to be due to C K 2 . Phosphorylation at this site did not modulate the ability of p65 to translocate to the nucleus, only its transactivation potential. Interestingly this is located in transactivation domain-1 of the C-terminal of p65, the area that interacts with other transcriptional coactivators such as C B P , and p300 as well as T B P , TFIIB. A s a result phosphorylation at this site may be responsible for interaction with other transcription factors, or with the ability of N F K B to disrupt chromatin, suggesting a specific role for each promoter. This report now shows for the first time that this site is also important for mediating IL-1 p induced p65 transactivation.  80  and P D K 1 regulation of chemkokine production  CHAPTER 5 -  5.1  PI3K  IL-1|3  induced  NFKB  and  RATIONALE  The PI3K pathway, including PDK1 and P K B , is perhaps one of the most intriguing cell signaling pathways. It has been implicated in the regulation of numerous cell functions including viability, apoptosis, cell metabolism, migration, and endocytosis, just to name a few(140). PI3K and P K B have been strongly implicated in the inflammatory response, by regulating N F K B ( 8 0 , 83, 153, 154). The mechanism of regulation is different in different systems. No work to this date has examined the role of PDK1 regulation of N F K B , or chemokine production, especially in lECs. Thus in this section the precise regulation of chemokines and N F K B by PI3K, P K B , and PDK1 was examined. Due to their previous roles in regulating N F K B , it can be hypothesized that PI3K and P K B will regulate N F K B activation, as well as chemokine synthesis. Also it can be hypothesized that PDK1 can regulate N F K B , although its role is yet completely undefined.  5.2  RESULTS  5.2.1  Inhibition of both P I 3 K and  NFKB  activation.  PDK1  results in attenuation of  IL-1  p induced  Initially using the N F K B reporter ( 4 X K B - I U C ) , cells were pretreated with inhibitors to PI3K (LY 294002 (LY) or wortmannin)(238) or a recently identified inhibitor to PDK1 (N-tosyl-L-phenylalanyl chloromethyl ketone/TPCK)(239), and stimulated with IL-1 p for 81  6 h.  The results show that inhibition of either PI3K (Fig 23A) or PDK1 (Fig 24A)  attenuates IL-1 (3 induced N F K B activation. To confirm that L Y and wortmannin were inhibiting PI3K, the phosphorylation status of P K B (serine 473) was examined, and its IL-1 (3 induced activation was in fact attenuated by the inhibitors (Fig 23B). Similarly T P C K inhibited both basal and IL-1 p induced PDK1 trans-autophosphorylation on serine 241, as well as phosphorylation of downstream target S 6 ribosomal protein (Fig 24B). As  previously  reported  inhibition  of  PDK1 led to  a  mild  activation  in E R K  phosphorylation(239). These data thus confirmed the selectivity of the PDK1 inhibitor, TPCK.  5.2.2  Overexpression of PDK1 or  PKB  is sufficient to drive  NFKB  activation.  To further confirm the role of PDK1 and P K B in the activation of N F K B , empty vector, P D K 1 , or P K B were overexpressed with the 4 X K B - I U C construct. evidence from three different cell lines was acquired (Fig 25 A/B/C).  Compelling  In all cases the  overexpression of P K B and PDK1 was sufficient to drive both basal and IL-1 p induced activation of N F K B .  Most importantly this data was reproducible in the non-tumorigenic  cell line H E K 2 9 3 T (Fig 25C).  5.2.3  IL-1 p induces P K B serine 473 phosphorylation and also its specific activity. Since serine 473 phosphorylation is not sufficient to assess activation of P K B in  response to IL-1 p, the kinetics of serine 473 phosphorylation were compared with P K B activity.  IL-1 p activated P K B in a biphasic manner, with the initial activity coming very  quickly at 2 min, and a second increase in activity at 30 min (Fig 26B). Maximal activity coincided with serine 473 phosphorylation, although serine 473 phosphorylation was not 82  detected until 30 minutes (Fig 26A). PDK1 activity is absolutely required for P K B activation(240), thus it can be inferred that PDK1 is also activated very early in this system. This data was reproducible in both HCT-116 cells and Caco-2 cells.  5.2.4  Overexpression of PDK1 but not PKB activates the IKK complex In order to examine the mechanism through which PDK1 and P K B were  regulating N F K B , the activity of the IKK complex was first examined. 293T cells were transfected with vector, P D K 1 , or P K B and 24 h later immunoprecipitated for IKKy, which pulls down a complete IKK complex including IKKy, IKKa, and IKKp (data not shown).  Immunocomplexes were then used in a radioactive kinase assay with either  the GST-p65 or G S T - k B substrate, because both of these molecules are endogenous substrates of the IKK signalsome. Although both P K B and PDK1 were capable of activating N F K B (Fig 25), surprisingly only overexpression of P D K 1 , but not P K B , was sufficient to activate the IKK complex (Fig 27A). To further confirm this, the effects of overexpressing  PDK1  and  PKB  on  IKB  phosphorylation  were  examined.  Overexpression of P D K 1 , but not P K B , induced endogenous IKB phosphorylation (Fig 27B).  In addition, pretreatment with T P C K , but not LY and wortmannin, led to  prevention of IL-1 p induced IKB degradation (Fig 27C). This meant that PDK1 and P K B were activating N F K B in distinct ways, and that P K B was acting at a more distal regulatory location.  83  5.2.5  Pharmacological inhibition of PDK1 but not PI3K results in the inhibition of  p65 DNA binding Since PDK1 regulates IKK activation, it should therefore also regulate N F K B DNA binding. Using E M S A , the effects of T P C K , LY, and wortmannin on p65 DNA binding were thus examined. Not surprisingly, when cells were pretreated with T P C K , there was attenuation of DNA binding (Fig 28A/B).  Pretreatment of cells with LY and  wortmannin had no effect on DNA binding (Fig 28A/B), also confirming the previous data that PDK1 was regulating the IKK complex, whereas P K B was regulating some other distal regulatory point.  Overexpression of PDK1 but not P K B was sufficient to  induced K B DNA binding (Fig 28C). This data was reproducible in both 293T and H C T 116 cells.  5.2.6  Pharmacological inhibition of PKB results in the attenuation of p65  transactivation A potential mechanism for P K B regulation of N F K B may be through regulation of p65 transactivation. To assess this we used the one-hybrid system (p65 TADI-III fused to Gal4 DNA binding, transfected along with a Gal4 responsive luciferase reporter). Inhibition of p65 transactivation was observed with pretreatment of both L Y and wortmannin (Fig 29A). Thus P K B was regulating N F K B activation through regulation of p65 transactivation. When p65 phosphorylation at serine 536 was examined, there was no effect of LY or wortmannin on IL-1 (3 induced p65 phosphorylation at serine 536 (Fig 29B).  84  5.2.7 Pharmacological inhibition of PDK1 results in the attenuation of p65 transactivation The IKK signalsome has two main regulatory functions.  The first is that it  regulates the phosphorylation of IKB. The second function is that it phosphorylates p65 and regulates its transactivation.  Due to the fact that PDK1 was regulating the IKK  complex, this would mean that PDK1 would also regulate the transactivation of p65. We tested this by using the Gal4 one-hybrid system. Pretreatment of cells with T P C K resulted in attenuation of IL-1 p induced p65 transactivation (Fig 30A). Thus PDK1 can also regulate p65 transactivation.  When p65 serine 536 was examined, T P C K did  inhibit IL-1 p mediated serine 536 phosphorylation on p65 (Fig 30B).  5.2.8 Overexpression of PKB or PDK1 is sufficient to induce p65 transactivation To confirm the abilities of P K B and PDK1 to regulate p65 transactivation, they were overexpressed with the Gal4 one-hybrid system.  Both P K B and PDK1 were  sufficient to activate the Gal4-dependant luciferase reporter (Fig 31). This confirmed the data presented with the pharmacological inhibitors, that both P K B and PDK1 were able to regulate p65 transactivation; however PDK1 was regulating transactivation via IKK, and P K B was not.  5.2.9 PDK1 and PKB regulate p65 transactivation through unique sites The IKK signalsome regulates p65 transactivation by phosphorylating p65 at serine 536, in an IKKp dependant fashion(81). To confirm this in our system, mutant GST-constructs that were mutated at serine 529 or serine 536 and changed to alanines were generated. Serine 529 is the C K 2 regulated site and this mutant was used as a  85  control (Chapter 4). The IKK complex was assayed by immunoprecipitating IKKy, and performing radioactive IKK assays using either GST-p65 W T , GST-p65 S529A, or G S T p65 S536A. A s is clearly shown (Fig 32A), mutation of serine 536, but not serine 529 attenuates p65 phosphorylation.  Thus only the serine 536 can be phosphorylated by  the IKK signalsome in our system. Since PDK1 regulates the IKK signalsome complex, we hypothesized that PDK1 should regulate p65 transactivation through serine 536. Similarly P K B should not regulate serine 536 because it does not regulate IKK. Thus using the one-hybrid system with a mutant version of p65 T A D that contains a serine 536 to alanine mutation, both P K B and PDK1 were overexpressed. Overexpression of PDK1 and P K B both resulted in transactivation of the W T p65 construct.  Mutation of  serine 536 to alanine completely attenuated the ability of PDK1 to transactivate the p65 construct, whereas P K B was still able to transactivate the p65 construct (Fig 32B). This further substantiates the notion that PDK1 is regulating p65 via IKK and serine 536, whereas P K B is regulating p65 through an as of yet unknown site. To further confirm this, endogenous phospho-p65 (serine 536) was examined by immunoblotting.  This  shows that overexpression of PDK1 but not P K B is sufficient to induce serine 536 phosphorylation (Fig 32C).  5.2.10 PDK1 and IKK coassociate To see if there was a physical association between IKK and P D K 1 , they were coimmunoprecipitated and immunoblotted for each other.  A s the data shows they are  constitutively bound, and their association does not change with IL-1 p stimulation (Fig 33). When we examined overall cellular PDK1 activity there was no change, nor was there a change in IKKy bound PDK1 specific activity (data not shown). To confirm that 86  IKKy (and the IKK signalsome) was successfully immunoprecipitated, the same membrane was stripped and reprobed for IKKp. Thus there is constitutive association between PDK1 and IKK.  5.2.11 IKK a/p ser 177/181 is sensitive to TPCK, and is a potential PDK1 phosphorylation site In order to explore the mechanism through which PDK1 regulates IKK, we looked at the activating phosphorylations of IKKa and p (serine 180/181). When cells were pretreated with T P C K , this activating phosphorylation was inhibited (Fig 34A). Close examination of the IKK a and p activation loop phosphorylation site reveals that it is a potential PDK1 phosphorylation site, as it matches those sites in other PDK1 downstream targets quite closely (Fig 34B).  5.2.12 Both PI3K and PDK1 are required for the activation of the IL-8 promoter Now that a clear role for PDK1 and P K B regulation in the activation of the N F K B has been established, the activation of downstream N F K B target, IL-8, was examined. We used an IL-8 promoter attached to a luciferase reporter (IL8-luc) to examine IL-8 promoter activation. Cells pretreated with either L Y or wortmannin, were unable to activate the reporter in response to IL-1 p (Fig 35A).  Likewise cells pretreated with  T P C K were also unable to activate this reporter (Fig 35B).  5.2.13 Overexpression of PDK1 is sufficient to activate the IL-8 promoter To further confirm the pharmacological data in Fig 35, P K B and PDK1 were overexpressed with the IL8-luc reporter.  PDK1 was sufficient to activate the IL-8  87  proximal promoter.  PDK1 robustly activated this promoter construct with a 20 fold  activation basally, and almost a 200 fold activation with IL-1B stimulation (Fig 36). Overexpression of P K B was not sufficient to activate the IL-8 promoter construct; however, the addition of IL-1 p led to a synergistic activation.  5.2.14 Overexpression of PDK1 is sufficient to IL-8 message synthesis The expression of the chemokine IL-8 was examined by R T - P C R in order to see if R N A was transcribed as a result of promoter activation. Overexpression of P K B led to a very mild increase in IL-8 message, however PDK1 overexpression resulted in a very significant increase in IL-8 message (Fig 37). This increase in IL-8 message from PDK1 overexpression was almost equivalent to that of IL-1 p induced IL-8 message  88  Figure 23. Inhibition of PI3K inhibits IL-1B induced N F K B activation. (A) HCT-116 colonic epithelial cells, transfected with an N F K B responsive luciferase reporter (4XKBLuc), were pretreated with LY (25 uM), wortmannin (100 nM), for 1 h before being stimulated with IL-1 p (2 ng/mL) for 6 h. (B) HCT-116 colonic epithelial cells were pretreated with LY (25 uM), wortmannin (100 nM), stimulated with IL-1 p (2 ng/mL) for 30 min. Proteins were subsequently resolved by S D S - P A G E , and probed for phosphoPKB (serine 473).  -89-  B  pPDK1 pS6 pERK  Figure 24. Inhibition of PDK1 inhibits IL-1B induced N F K B activation. (A) HCT116 colonic epithelial cells, transfected with an N F K B responsive luciferase reporter ( 4 X K B - L U C ) , were pretreated with TPCK (10-100 uM) for 1 h before being stimulated with IL-16 (2 ng/mL) for 6 h. ( B ) HCT-116 colonic epithelial cells were pretreated with TPCK (50-100 uM) for 1 h, stimulated with IL-16 (2 ng/mL) for 30 min. Proteins were subsequently resolved by S D S - P A G E , and probed for phospho-PDK1, phospho-S6, or phospho-ERK.  -90-  Vector  PDK-1 PDK-1 AKTWT AKTWT + WT WT + IL-1 IL-1  Vector +  IL-1 6.5  B 5.5 a) 4.5  1.5 0.5 Vector  -1-  Vector  Vector+ 1-1  PDK-1 WT  1  1  PDK-1 WT + IL-1  1  Vector + IL- PDK-1 WT PDK-1 WT 1 + IL-1  AKTWT  AKT WT + IL-1  1 1 1 AKTWT  AKTWT + IL-1  Figure 25. Overexpression of PDK1 or P K B is sufficient for the activation of N F K B . HCT-116 colonic epithelial cells (A) Caco-2 colonic epithelial cells (B) or 293T human embryonic kidney cells (C) were transfected with an N F K B responsive luciferase reporter (4XKB-LUC) as well as either empty PCDNA3 vector, myc-PDK1, or HA-PKB. 24 h post transfection cells were stimulated with IL-1 (3 for 6 h.  -91-  A  pPKB IL-1 Stimulation (min)  B  15  30  60  6.0  Time (Minutes)  Figure 26. IL-1B activates P K B and the phosphorylation of IKB and p65. HCT116 colonic epithelial cells were stimulated with IL-1 p (10 ng/mL) for varying lengths of time. Proteins were subsequently resolved by S D S - P A G E , and probed for phospho-PKB (A) or were immunoprecipitated with an antibody against PKB, and a PKB immune complex kinase assay was performed (B).  -92-  GST p65  GST IkB  •  PCDNA3 EV  +  +  +  Myc-PDK1  + +  HA-PKB  IL-ip  -  +  +  +  +  B  IB: p k B V=Vector  P=PDK1  A=PKB V  V+l  P  P+l  A  A+l  IB:  C  T10  T50  T100  IL-1  IKB  I+T10 I+T50 I+T100  T=TPCK L=LY  W=Wortmannin  IB:  C  LY  WM  IL-1  l+L  IKB  l+W  Figure 27. Overexpression of PDK1, but not P K B , results in the activation of IKK activity. (A) 293T cells were transfected with either empty PCDNA3 vector, mycPDK1, or HA-PKB. 24 h post transfection cells were stimulated with IL-16 for 30 min. (A) IKK was subsequently immunoprecipitated and kinase assays were performed. (B) Lysates were immunoblotted for phospho-kB. (C) Cells were pretreated with TPCK (T) or 25 uM LY294002 (L) or 100 nM wortmannin (W). Cell lysates were immunoblotted for protein k B . -93-  Figure 28. Inhibition of PDK1 but not PI3K results in the inhibition of N F K B nuclear translocation and subsequent DNA binding. HCT-116 colonic epithelial cells (A) or 293T human embryonic kidney cells (B) were pretreated with LY (25 uM), wortmannin (100 nM), or T P C K (50-100 uM) , for 1 h before being stimulated with IL16 (2 ng/mL) for 30 min. Nuclear proteins were isolated and electrophoretic mobility shift assay (EMSA) was performed. (C) HCT-116 cells were transfected with empty PCDNA3 vector, myc-PDK1, or HA-PKB. 24 h post transfection cells were harvested for nuclear proteins and analyzed for p65 DNA binding by EMSA. -94-  B  p-p65 ser 536  Figure 29. Inhibition of PI3K inhibits IL-1 p induced N F K B transactivation. (A) HCT-116 colonic epithelial cells, transfected with a GAL4 responsive luciferase reporter (Gal4-Luc), along with pTADI-lll (transactivation domains l-lll fused to a Gal4 DNA binding domain), were pretreated with LY (25 uM), wortmannin (100 nM), for 1 h before being stimulated with IL-1 p (2 ng/mL) for 6 h. (B) HCT-116 colonic epithelial cells were pretreated with LY (25 uM), wortmannin (100 nM), stimulated with IL-16 (2 ng/mL) for 30 min. Proteins were subsequently resolved by S D S - P A G E , and probed for phospho-p65.  -95-  Figure 30. Inhibition of PDK1 inhibits IL-1B induced N F K B transactivation. (A) HCT-116 colonic epithelial cells, transfected with a GAL4 responsive luciferase reporter (Gal4-Luc), along with pTADI-lll (transactivation domains I-lll fused to a Gal4 DNA binding domain), were pretreated with T P C K (50-100 uM) for 1 h before being stimulated with IL-1 p (2 ng/mL) for 6 h. (B) HCT-116 colonic epithelial cells were pretreated with T P C K (50-100 uM), stimulated with IL-1B (2ng/mL) for 30 min. Proteins were subsequently resolved by S D S - P A G E , and probed for phospho-p65.  -96-  Vector  Vector + IL-1  PDK-1 WT  PDK-1 WT + IL-1  AKT WT  AKT WT + IL-1  Figure 31. Overexpression of PDK1 and P K B results in increased transactivation. (A) HCT-116 colonic epithelial cells, transfected with a GAL4 responsive luciferase reporter (Gal4-Luc), along with pTADI-lll (transactivation domains l-lll fused to a Gal4 DNA binding domain) and with either empty PCDNA3 vector, myc-PDK1, or HA-PKB. 24 h post transfection cells were stimulated with IL-16 for 6 h.  -97-  WT  S529A  S536A  B  Vector  PDK1WT  AKT WT  • pm-p65(521-551) 0 pm-p65(521-551 )s536a  5-..-  c  ........  PCDNA3EV  +  Myc-PDK1  -  HA-PKB  -  IL-1  -  *HPN  +  -  -  -  + +  -  + -  -  -  +  -  -  +  +  -  +  Figure 32. PDK1 mediated increased transactivation is dependant on p65 serine 536. (A) HCT-116 cells were stimulated with IL-1 p for 15 min. IKK was subsequently immunoprecipitated and kinase assays were performed, using WT GST-p65, serine 529 to alanine GST-p65, or serine 536 to alanine GST-p65. (B) HCT-116 colonic epithelial cells, transfected with a GAL4 responsive luciferase reporter (Gal4-Luc), along with pTADIII (transactivation domain III fused to a Gal4 DNA binding domain - WT or S536A) and with either empty PCDNA3 vector, mycPDK1, or HA-PKB. 24 h post transfection cells were stimulated with IL-1 p for 6 h. (C) 293T cells were transfected with either empty PCDNA3 vector, myc-PDK1, or HAPKB. 24 h post transfection cells were stimulated with IL-16 for 30 min. Proteins were subsequently resolved by S D S - P A G E , and probed for phospho-p65. -98-  IP: IKKy IB: PDK1  IP: IKKy IB: IKKp Ab  LC  C  IL-1  Figure 33. PDK1 coassociates with IKK signalsome. HCT-116 cells were stimulated with IL-1 p for 15 min. IKK was subsequently immunoprecipitated and immunoblotted for either PDK1 or IKKp. Ab = antibody and beads control, LC = lysate and non-specific IgG control  -99-  pIKK Ser 180/181 LY  WM  T50  T100  IL-1  l+L  l+W  pIKK Ser 180/181  L=LY  C  IL-1  I+T50 I+T100  W=wortmannin T=TPCK  B  PKB SGK p70SGK p90RSK PKA PRK PKC PDK1  K D E H H D D H R V G Y MD S K  G A TMK N S T T S G T V T H E K K A Y K G R TW G D R T S G V T T R Q A R A N  IKK ALPHA D Q G S L C T IKK BETA D Q G S L C T  F F F F L F F F  C C C C C C C V  G G G G G G G G  T P E Y L A P E T V E Y M A P E T I E Y M A P E T V E Y M A P E T P E Y L A P E T P E F L A P E T P D Y I A P E T A Q Y V S P E  F V G T L Q Y L A P E F V G T L Q Y L A P E  Figure 34. Ser 180/181 on IKKcc/6 are potential PDK1 phosphorylation sites and are sensitive to T P C K . (A) Alignment of consensus PDK1 phosphorylation sites. (B) HCT-116 colonic epithelial cells were pretreated with T P C K (50-100 uM) for 1 h, stimulated with IL-16 (2 ng/mL) for 30 min. Proteins were subsequently resolved by S D S - P A G E , and probed for phospho-IKK (serine 180/181).  -100-  10.5 8.5 CO  TO § c  i  6.5 4.5 2.5 0.5  Control  LY25  Wm 100  IL-1 + LY 25 IL-1 + Wm  IL-1  100  12.0 B  8  10.0 8.0  03 0  |  6.0  1  4.0  I j  2.0 0.0 Control  TPCK 10  TPCK 50  TPCK 100  IL-1  I  IL-1 + TPCK 10  Hi  IL-1 + TPCK 50  IL-1 + TPCK 100  Figure 35. Inhibition of PI3K or PDK1 inhibits IL-16 induced proximal IL-8 promoter activation. (A) HCT-116 colonic epithelial cells, transfected with an IL-8 luciferase reporter (IL8-Luc), were pretreated with LY (25uM), wortmannin (100 nM) (A), or T P C K (50-100 uM) (B) for 1 h before being stimulated with IL-16 (2 ng/mL) for 6h. -101-  Vector  Vector+IL- PDK-1 WT PDK-1 WT 1 + IL-1  AKT WT  AKTWT + IL-1  Figure 36. Overexpression of PDK1 or P K B results in the activation of the IL-8 proximal promoter. HCT-116 colonic epithelial cells, transfected with an IL-8 luciferase reporter (IL8-Luc), along with either empty PCDNA3 vector, myc-PDK1, or HA-PKB. 24 h post transfection cells were stimulated with IL-ip for 6 h.  -102-  V=Vector P=PDK1 A=PKB  IL8 RT-PCR  V  A  P  V+l  A+l  P+l  Figure 37. Overexpression of PDK1 but not P K B results in increased IL-8 message synthesis. HCT-116 cells were transfected with either empty PCDNA3 vector, myc-PDK1, or HA-PKB. 24 h post transfection cells were harvested for RNA and subjected to R T - P C R to examine IL-8 message.  -103-  5.3  DISCUSSION In this section the PI3K, P K B axis was examined and compared with PDK1 with  respect to its ability to regulate N F K B and chemokine synthesis. Both P K B and PDK1 were capable of regulating chemokine synthesis. In addition, both P K B and PDK1 were able to regulate N F K B .  Interestingly however, although P K B and PDK1 are considered  to be in the same pathway, they had distinct mechanisms of regulating N F K B , perhaps suggesting in the context of IL-1B signaling they work independently. PDK1 was shown to regulate the IKK complex, and as a result could modulate I K B phosphorylation at serine 32/36 as well as p65 transactivation through serine 536. Alternatively through the PI3K and P K B pathway, N F K B transactivation was being regulated, however in a serine 536 independent fashion. Interestingly, on closer examination, IKK was capable of co-associating with P D K 1 . The regulatory activating phosphorylation of IKKa and IKKB were potential PDK1 phosphorylation sites, and were sensitive to T P C K . The mechanism of regulation of N F K B by the PI3K pathway has been very contentious, and disputed. Some reports observe that PI3K and P K B can regulate the IKK complex, and thus regulate IKB phosphorylation and degradation(153, 154). Some reports instead observe that PI3K and P K B instead play a very important role in regulating the transactivation of p65(80, 83). Other reports argue any role at all for PI3K and PKB(241). Many of these studies are confounded by the diversity of cell systems used, along with the variety of stimuli used in the studies. The data presented here  104  shows in the IEC system, that PI3K regulates the transactivation potential of p65, and not IKB phosphorylation and degradation. T P C K has only been recently identified as a PDK1 inhibitor. It was discovered as an  in  vitro  inhibitor,  chymotrypsin(242).  designed  to  allow  the  study  of  the  serine-protease  T P C K was initially observed to inhibit tumorigenesis, reduce the  rate of proliferation, despite the lack of knowledge as to its in vivo cellular target(243, 244).  A study then reported that T P C K can inhibit R S K , S 6 K , and PKB(245). Many of  these observations were not reproducible with the use of other chymotrypsin inhibitors, thus suggesting that the cellular target of T P C K was not a chymotrypsin but some other pathway. PDK1(239).  The same group then reported that the cellular target of T P C K was T P C K has also been long regarded as an inhibitor of N F K B ( 1 7 3 , 246).  Inhibition of N F K B has been attributed to an attenuation of I K B phosphorylation, thus regulating a signal upstream of IKK. This observation is similar to our observations of the IKK complex being sensitive to T P C K signaling. Thus these reports provided a link between the previously inhibition of PDK1 by T P C K , and N F K B . Much evidence supports the notion that PDK1 may be regulating IKK. Numerous studies have described the ability of IKK a and B to trans-autophosphorylate each other(168-173). At the same time these reports all leave the same question as to how the initial pool of IKK become phosphorylated and activated. The studies presented here show a constitutively bound PDK1 to IKK. Due to the constitutive activity of P D K 1 , this would allow IKK to become initially phosphorylated, allowing the IKK kinase subunits to trans-autophosphorylate each other. This may be mediated by a signal induced change in the IKK signalsome, either conformational, or with the addition of  105  loss of another protein.  A candidate protein for this may be the cellular chaperone  H S P 9 0 that has been shown to associate and regulate both IKK as well as associate and regulate PDK1(159, 174). The data presented thus suggests a small pool of PDK1 may exist in the cell, that is bound to IKK, and that is not regulated by PI3K. PI3K independent activation of PDK1 has been previously reported(160). Previously, P K B has been linked to regulation of serine 536 phosphorylation^). Our data clearly shows that P D K 1 , not P K B , regulates this site in an IKK dependant fashion.  This then leaves the question as to how P K B may be regulating p65  transactivation between residues 501-551. One potential mechanism may be that P K B directly phosphorylates p65. Another mechanism may be that P K B is working through another downstream effector.  A candidate molecule for this may be GSK3B.  Gene  knockout of this molecule results in embryonic lethality due to T N F induced liver apoptosis(247).  When examined, GSK3B was found to regulate N F K B in a method  independent of IKB phosphorylation and degradation, similar to the mechanism through which P K B regulates N F K B in our system.  106  CHAPTER 6 - GENERAL CONCLUSIONS The body of work presented here has examined the signaling pathways that are activated in l E C s leading to the synthesis of chemokines, in response to IL-1 p. Three important pathways were examined (MAPK, protein kinase C K 2 , and PI3K/PDK1). Their relationship with respect to activation of N F K B activation was also examined. IL-1 p, upon binding to its receptor, activated all three M A P K family member, as well as protein kinase C K 2 and P K B . N F K B , which is required for the synthesis of many chemokines, was found to be a point of convergence of many of these pathways. Activation of the IKK complex is required for the signal induced phosphorylation and degradation of I K B .  PDK1 was found to regulate the IKK complex, in a PI3K  independent fashion, potentially by phosphorylating IKKa and p at serines 180/181. IKK also phosphorylates the p65 subunit at serine 536 to allow it to become functionally active.  PDK1 was found to regulate this phosphorylation at serine 536, in a process  dependant upon IKKp. In addition, further phosphorylations of p65 are required for full transactivational activity. P K B regulated the transactivation of p65 in a serine 536 independent fashion. This may occur by a direct phosphorylation of p65 or indirectly through a downstream effector, a potential candidate being G S K 3 p .  Upon entering the nucleus p65 is further  phosphorylated by protein kinase C K 2 at serine 529. p65, now being fully active, binds its consensus D N A sequence and forms part of the transcriptional complex. For promoters such as IL-8, it will synergize with other trans-acting regulatory factors such 107  as other transcription factors and coactivators. p38 M A P K was found to regulate the IL8 promoter, however independently of N F K B . Another important transcription factor, A P 1 is required for maximal transcriptional activation. Its D N A binding ability appears to be regulated by p38 M A P K , and this may be one the ways that p38 M A P K regulates chemokines. At this point J N K may play a role in interacting with functional p65 and thus regulating both p65 activity and the IL-8 promoter.  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