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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 SCIENCE in THE FACULTY OF G R A D U A T E STUDIES (Department of Medicine; Experimental Medicine Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 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. The prototypic proinflammatory agonist interleukin-1(3 (IL-1 p), signals through the interleukin-1 receptor (IL-1 R), the founding member of the IL-1 R superfamily, and robustly activates this pro-inflammatory cascade. Synthesis of many downstream chemokines in response to IL-1|5, requires the activation of the transcription factor N F K B . The objective of the work presented here is to characterize the regulatory pathways responsible for chemokine synthesis in lECs in response to IL-1p\ and to examine their relationship with N F K B . Three important pathways in IL-1 (3 signaling were examined. Initially the MAPK family of pathways was examined. IL-1 p treatment led to activation of ERK, p38 MAPK, as well as JNK. Inhibition of E R K had no effect on either chemokine synthesis, or N F K B activation. 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 and posttranscriptional regulation. JNK 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. CK2 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. As a result, CK2 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 activation, although they had differing regulatory roles. PI3K and PKB regulated N F K B transactivation, through an undetermined mechanism. 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 DNA 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, and thus have implications for chronic inflammatory conditions such as IBD. TABLE OF CONTENTS A B S T R A C T ii TABLE OF C O N T E N T S iv LIST OF T A B L E S vii LIST OF F IGURES viii ABBREVIATIONS xi A C K N O W L E D G E M E N T S xiv C H A P T E R 1 - G E N E R A L INTRODUCTION 1 1.1 INTESTINAL EPITHELIAL C E L L S 1 1.1.1 Biology 1 1.1.2 Innate Immune Response 1 1.2 CHEMOKINES 4 1.2.1 Structure, Expression, and Function 4 1.2.2 IL-8 6 1.3 N F K B 8 1.3.1 Structure 8 1.3.2 Regulation 9 1.3.3 Function 10 1.4 IEC SIGNALING 12 1.4.1 IL-1 signaling 12 1.4.2 MAP Kinases 14 1.4.3 Protein Kinase CK2 16 1.4.4 PI3K Pathway 17 1.4.4 IKK complex 19 1.5 INFLAMMATORY BOWEL DISEASE 21 1.5.1 Pathology 21 1.5.2 Pathogenesis 22 1.5.3 The role of lECs, chemokines, and N F K B 24 C H A P T E R 2 - MATERIALS AND METHODS 29 2.1 MATERIALS 29 2.1.1 Cell lines and Cell Culture 29 2.1.2 Reagents, Enzymes, and Chemicals 29 2.1.3 Plasmids and cDNAs 30 2.1.4 Primers 30 2.2 METHODS 31 2.2.1 Nuclear Preps 31 2.2.2 Electrophoretic Mobility Shift Assay (EMSA) 32 2.2.3 Immunoblotting 33 2.2.4 CK2 Phosphotransferase Activity Assays 34 2.2.5 Transient Transfections 35 2.2.6 Luciferase Assay 35 2.2.7 Isolation of RNA and RT-PCR 36 2.2.8 Tissue Procurement and Immunohistochemistry 36 2.2.9 ELISA assays for IL-8 and MCP-1 37 iv 2.2.10 M A P K A P K 2 assay 37 2.2.11 Immunoprecipitation 38 2.2.12 Immune complex assays for PKB and IKK signalsome 38 C H A P T E R 3 - M A P K REGULATION OF IL-1 P SIGNALING 40 3.1 RATIONALE AND HYPOTHESIS 40 3.2 R E S U L T S 40 3.2.1 IL-1 p activates protein tyrosine phosphorylation and MA P K s in lECs 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 MAPK 42 3.2.4 N F K B DNA binding and transactivation are independent of p38 MAPK activation 42 3.2.5 IL-8 and MCP-1 messages are regulated by p38 in lECs 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 JNK 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 - CK2 regulation of IEC N F K B Activation and Chemokine Synthesis 62 4.1 RATIONALE AND HYPOTHESIS 62 4.2 R E S U L T S 63 4.2.1 CK2 activity is increased in patients with active IBD 63 4.2.2 CK2 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 CK2 inhibits IL-1 p induced N F K B activation 64 4.2.5 CK2 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 CK2 activity is increased with IL-1 p stimulation 66 4.2.8 Inhibition of CK2 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 R E S U L T S 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 PKB 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 PKB 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 PKB 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 C H A P T E R 6 - G E N E R A L CONCLUSIONS 107 R E F E R E N C E S 109 vi LIST OF TABLES Table 1 - Activators and Inducers of N F K B in lECs vii LIST OF FIGURES Figure 1. Signaling pathways activated by IL-ip 26 Figure 2. N F K B family members 27 Figure 3. Detailed activation of N F K B , including important regulatory phosphorylations 28 Figure 4. IL-ip stimulation of lECs results in the activation of ERK, JNK, and p38 47 Figure 5. Inhibition of p38 MAPK 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 DNA binding 52 Figure 10. p38 M A P K regulates IL-ip induced IL-8 and MCP-1 mRNA expression 53 Figure 11. IL-1 p induced activation of the IL-8 promoter is dependent on p38 M A P K 54 Figure 12. Selective JNK inhibitor S P 600125 inhibits JNK activity, however does not regulate N F K B DNA binding 55 Figure 13. Selective JNK 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 UC 69 Figure 15. D S S induced colitis in mice increases CK2 activity in actively inflamed tissue 70 Figure 16. Inhibition of CK2 using a selective inhibitor prevents IL-1 (5 induced activation of N F K B in intestinal epithelial cells 71 Figure 17. Overexpression of CK2 a or a ' modulates both basal and IL-1 p induced N F K B activation 72 Figure 18. CK2 regulation of IL-1 p induced N F K B activation is at the level of transactivation through p65 serine 529 73 Figure 19. 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 CK2 prevents IL-1 p induced activation of the IL-8 proximal promoter 76 Figure 22. Inhibition of CK2 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 PKB 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 PDK1, but not PKB, results in the activation of IKK activity 93 ix Figure 28. Inhibition of PDK1 but not PI3K results in the inhibition of N F K B nuclear translocation and subsequent DNA binding 94 Figure 29. Inhibition of PI3K inhibits IL-1 p induced N F K B transactivation 95 Figure 30. Inhibition of PDK1 inhibits IL-1 p induced N F K B transactivation 96 Figure 31. Overexpression of PDK1 and P K B results in increased transactivation 97 Figure 32. PDK1 mediated increased transactivation is dependant on p65 serine 536 98 Figure 33. PDK1 coassociates with the IKK signalsome 99 Figure 34. Ser 180/181 on IKKa/p are potential PDK1 phosphorylation sites and are sensitive to T P C K 100 Figure 35. Inhibition of PI3K or PDK1 inhibits IL-1 p induced proximal IL-8 promoter activation 101 Figure 36. Overexpression of PDK1 or P K B results in the activation of the IL-8 proximal promoter 102 Figure 37. Overexpression of PDK1 but not P K B results in increased IL-8 message synthesis 103 x ABBREVIATIONS Abbreviation Definition 3' UTR 3' untranslated region AP-1 activator protein 1 A R E AU-rich cis regulatory elements ATF-2 activating transcription factor-2 C /EBP CAAT/ enhancer binding protein CAMK IV calmodulin activated kinase IV C B P C R E B binding protein CD Crohn's disease cFLIP cellular inhibitor of FLICE C H O P C / E B P homologous protein c lAP cellular inhibitor of apoptosis COX-2 cyclooxygenase-2 CTCF CCCTC-binding factor DRB 5,6 dichloro-ribifuranosylbenzimidazole DSS dextran sodium sulfate ELISA enzyme linked immunosorbent assay EMSA electrophoretic mobility shift assay ENA-78 epithelial cell derived neutrophil attractant 78 ERK extracellular signal regulated kinase EV empty vector FBS fetal bovine serum GROcc growth related oncogene a HAT histone acetyl transferase HSP heat shock protein I K B inhibitor of K B IBD inflammatory bowel disease IEC intestinal epithelial cells IKK inhibitor of K B kinase IL-1P interleukin-ip IL-18R interleukin 18 receptor IL-1R interleukin-1 receptor IL-1RA interleukin-1 receptor antagonist IL-1 RI interleukin-1 receptor, type 1 IL-1RII interkeukin-1 receptor, type 2 ILK integrin linked kinase i N O S inducible nitric oxide synthase IP-10 interferon y inducible protein IRAK interkeukin-1 receptor associated kinase IRS insulin receptor substrate l-TAC interferon inducible T cell attractant JNK c-jun NH2-terminal kinase xi KD kinase dead LPMNC lamina propria mononuclear cell LPS lipopolysaccharide LTBR leukotriene p receptor MAPK mitogen activated protein kinase MAPKAPK2 mitogen activated protein kinase activated protein kinase-2 MAPKK mitogen activated protein kinase kinase MAPKKK mitogen activated protein kinase kinase kinase MCP monocyte chemoattracant protein MDC macrophage derived chemokine MEKK MEK kinase MHC major histocompatibility complex MIG monokine induced by interferon y MIP macrophage inflammatory protein MLK3 mixed Unease kinase-3 MSK1 mitogen and stress activated kinase-1 N F K B nuclear factor K B NIK N F K B inducing kinase NRE negative regulatory element NRF nuclear factor K B repressing factor p90 RSK p90 ribosomal S6 kinase PAMP pathogen associated molecular pattern PCR polymerase chain reaction PDK1 3' phosphoinositide dependant kinase-1 PI(3,4,5)P3 phosphoinositol(3,4,5)triphosphate PI(4,5)P2 phosphoinositol(4,5)biphosphate PI3K phoshphatidylinositol-3 kinase PKA protein kinase A PKB protein kinase B PKC protein kinase C PRR pathogen recognition receptor RT-PCR reverse transcriptase-polymerase chain reaction SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel eletrophoresis SH2 src-homology 2 TAD transactivation domain TAK1 transforming growth factor p activated kinase TGFp transforming growth factor p THi T helper cell, type 1 TH 2 T helper cell, type 2 tir toll/IL-1 R receptor TNFa tumor necrosis factor a TNFR1 tumor necrosis factor receptor 1 TPCK n-tosyl phenylalanyl chloromoethyl ketone TRAF-6 tumor necrosis factor receptor associated factor-6 TWEAK tumor necrosis factor weak inducer of apoptosis UC 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 lECs 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)(3-9), as well as soluble internal receptors such as CARD4/Nod1 and CARD15/Nod2(10-14). In addition lECs have the ability to respond to proinflammatory cytokines such as interleukin-1p (IL-ip) and tumor necrosis factor a (TNFa)(15). As 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 lECs and the commensal bacteria of the gut. These mechanisms are not completely understood, however may involve T-regulatory cells, as lECs have been shown to activate and expand T-regulatory cells(24, 25), and may also involve probiotic commensal bacteria of the Gl 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 lECs Cytokines and Growth Factors Oxidative Stress lnterleukin-1 Hydrogen peroxide lnterleukin-2 Ozone Reactive oxygen intermediates lnterleukin-17 lnterleukin-18 Viruses and Viral Products Leukotriene B4 Adenovirus Tumor necrosis factor Platelet-derived growth factor Bacteria and Bacteria Products Salmonella Shigella Enteropathogenic E. coli Listeria monocytogenes L P S Peptidoglycan-polysaccharide Toxic shock syndrome toxin 1 Molecules regulated bv N F K B in lECs Cytokines and chemokines lnterleukin-1 p lnterleukin-6 lnterleukin-8 GROa/p RANTES Macrophage inflammatory protein-2 Stress proteins Complement factors B, C3 , C4 Cell surface receptor lnterleukin-2R CD95/APO-1 (Fas) Adhesion molecule ICAM-1 Inflammatory enzyme Inducible nitric oxide synthase Cyclooxygenase-2 Immunoregulatory molecules Major histocompatibility complex I and II Table 1 - Activators and Inducers of N F K B in lECs. 3 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 3 C family(29, 30). 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. As 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. lECs 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 lECs include IL-8/CXCL8, epithelial-cell derived neutrophil attractant (ENA-78/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). lECs 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). lECs 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 THi phenotype (interferon y (IFNy) producing cells), and lECs are capable of producing interferon-gamma 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 (TH2) (which produce anti-inflammatory cytokines such as IL-4 and IL-10), and lECs are capable of producing macrophage derived chemoattractant (MDC/CCL20) that is important for attracting T H 2 phenotype T cells that express CCR4(17). The balance between a large proportion of proinflammatory THi C D 4 + T cells, and small proportion of anti-inflammatory T H 2 C D 4 + T cells maintains this basal inflammation of the intestine in a controlled fashion. As a result chemokine regulation of this balance by lECs plays an essential role in intestinal homeostasis. 5 1.2.2 IL-8 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-IL-6. 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 IL-8 requires the activation the Jun NH2-terminal kinase (JNK) pathway. This activation is independent of the requirement of N F K B ( 4 4 ) . There is also a role for the repressor, N F K B repressing factor (NRF) as a negative transcription regulator of the IL-8 promoter. NRF binds to a negative regulatory element (NRE), which overlaps the N F K B binding 6 site. Depletion of this factor using antisense RNA, leads to spontaneous IL-8 synthesis(45). NRF thus has a dual role in that it is required for the stimulus-induced activation of IL-8, and requires the activation of JNK 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 MAPK) 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 IL-8 message, and as a result the rapid destabilization and degradation of IL-8(49-52). 7 1.3 N F K B 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 , kBy, 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 IL-ip , 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 " T R C P ubiquitin ligase, complex with the p-TRCP complex functioning as the I K B specific ubiquitin ligase(64). Polyubiquitinated I K B is targeted for subsequent 26S proteasomal 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 CK2 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 inducing kinase (NIK) for ser 536(75-84). 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 TNF superfamily members leukotriene-p receptor (LTpR), CD40 ligand, as well as TNF 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. In contrast to the classical pathway, this process requires IKKa, not IKKp(57). Once phosphorylated p 100 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 pro-apoptotic signals such as fas ligand, TNFa , 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, TNF, M-CSF) to bacteria (Salmonella, and enteropathogenic E. coli) or bacterial soluble antigens, some of which include LPS, CpG DNA, peptidoglycan. In addition viral products such as double stranded RNA 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 TNFa , 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 (cox-2) are also targets of N F K B transcription(15, 55). 11 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 NEMO (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 (IL-1R/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). This group includes the IL-1 RI and the interleukin-18 receptor (IL-18R)(100, 101). An 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 PRRs , 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). As 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). TRAF-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 MAP 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 c-Jun 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 MAPK kinase (MKK). These MKKs are in turn activated by MKK kinases (MKKK), which themselves are thought to be activated by small G proteins. 1.4.2.1 p38MAPK p38 MAPK family is activated by MAPKKs , MKK3 and MKK6. 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, COX-2 , IL-6 and TNFa, 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 MAPK 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 JNKs consist of over 10 isoforms encoded as splice variants from 3 separate gene loci. Activation of JNKs occur via the MAPKKs , MKK4 and MKK7. These are activated through the upstream Rho family of GTPases through the M E K kinase 1 and 4 (MEKK1/4) and MLK3. The role of JNK in proinflammatory signaling has been controversial, however it is clear that JNK is able to activate and regulate the transcription factor AP-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 ERKs are robustly activated by growth and differentiation factors, as well as cytokines. The activation of E R K s occurs via the MAPKKs , MKK1 and MKK2, which are activated by Ras through the Raf family of MKKKs(112). The two major isoforms of 15 ERK are ERK1/p44 and ERK2/p42. ERKs 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 CK2 (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 CK2 holoenzyme is composed of 2 catalytic subunits (a or a') with two regulatory subunits (P)(124-126). CK2 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). CK2 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 ) . Its role in activating N F - K B has been largely controversial, with different 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 CK2 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 (137-143). The PI3K family contains four classes: l A , I B , II, and III, based on their structure and specificity for substrates. Class l A PI3Ks are responsible for catalyzing the reaction from phosphatidylinositol(4,5)biphosphate PI(4,5)P 2 to PI(3,4,5)P 3 (140-143). Class l 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 PKB , 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, TNFa , and granulocyte/monocyte-colony 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 PKB (also known as AKT or Rac) is known largely as a prosurvival kinase, regulating numerous proliferative and survival effectors including BAD, caspase-9, and the forkhead family of transcription factors(148). Its activation requires its membrane localization in a PI(3,4,5)P 3 dependant fashion, where it can undergo activating phosphorylations(148). 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 PDK1, 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. PKB 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 PKB 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 3, binding to heat shock protein 90 (HSP90), binding to 14-3-3, 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 , thus its understanding is fundamental to understanding N F K B activation. The IKK 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. IKKa requires phosphorylation on serine residues 176 and 180, whereas 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. In HeLa cells, using siRNA, TAK1 has been implicated(165). 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, CD and UC are defined based upon some markedly different characteristics. CD affects the terminal ileum, cecum, and large bowel, and often has patch lesions in between normal areas. UC on the other hand affects the rectum and continues proximal and is continuous. Inflammation in CD is transmural, affecting all muscle layers, and is characterized by lymphocyte infiltration as well submucosal fibrosis. UC 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. CD can lead to strictures, as well as bowel obstructions. UC 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 CD 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 in the development of very similar models of intestinal inflammation(176). 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 LPS 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 THi or to TH2 leads to a major driving force for inflammation. Thus a critical balance between THi and T H 2 CD4+ T cells must be maintained. CD is characterized by a THi profile of cytokines, with IL-2, IL-12, IFNy, and T N F a being very important players. In contrast, UC is often characterized by a T H 2 profile of cytokines, 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 CD. Likewise, experimental colitis driven by a T H 2 response (eg Oxazalone colitis), histologically resembles UC. 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 lECs are capable of expressing many P R R s such as TLRs and NOD receptors. These are important in 23 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 lECs 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 N F K B 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 lECs, 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. Overproduction of the potent pro-inflammatory cytokines IL-1 p and 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 lECs 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 , MCP-2 , MCP-3 , MIP-1a, MIP-1p\ IP-10, RANTES, 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 lECs 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 | 435 p105/p50 969 BBS 3333 | 405 G R R p100/p52 900 mmHHH 317 fcBa 356 607 IKBY 500 AIAIABAIAIA IKBE 446 ggg 3B3H BCL-3 Figure 2. N F K B family members -27-IL-1 III 529 311 \ Ubiquitination • and * Degradation LJ W W 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% FBS with 100 U/mL of penicillin and 100 ug/mL of streptomycin (Gibco, Burlington, Ontario). HEK 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 2 (v/v). 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 DRB 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 HA-CK2 a D. Litchfield University of W. Ontario (London, Ontario) WT and KD HA-CK2 a' D. Litchfield University of W. Ontario (London, Ontario) WT and KD MYC-PDK1 J S . Park Friedrich Miescher Instit (Basel, Switzerland) Kinetek Pharmaceuticals (Vancouver, BC) Kinetek Pharmaceuticals (Vancouver, BC) G. Wu University of Pensylvania (Philadelphia, PA) Stratagene (La Jolla, California) BR Cullen Duke University, (Durham, North Carolina) BR Cullen Duke University, (Durham, North Carolina) 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) WT AND KD HA-PKB 4 X K B - L U C I L 8 - L U C pfr-Luc pTADI-lll pTADIII pM-p65 (521-551) 2.1.4 Primers Primers used in R T - P C R are as follows: IL-8 Forward: IL-8 Reverse: 5 ' - T C T G C A G C T C T G B T G T G A A G G T G C A G T T - 3 ' S ' - T T C C T T T G A C C C A C G T C T C C C A A - S ' MCP-1 Forward: 5 ' - T C T G T G C C T G C T G C T C A T A G C - 3 ' 30 MCP-1 Reverse: 5 ' - G G G T A G A A C T G T G G T T C A A G A G G - 3 ' Actin Forward: Actin Reverse: 5 ' - C C A A C C G C G A G A A G A T G A C C - 3 ' 5 ' -GATCTTCATGAGGTAGTCAGT-3 ' I K B Forward I K B Reverse 5 ' - T A C A C C T T G C C T G T G A G C A G - 3 ' 5 ' -AGGATTTTGCAGGTCCACTG-3 ' GROcc Forward G R O a Reverse 5 - A C T C A A G A A T G G G C G G A A A G - 3 ' 5 ' - T G G C A T G T T G C A G G C T C C T - 3 ' TNFa Forward T N F a Reverse 5 - C G G G A C G T G G A G C T G G C C G A G G A G - 3 ' 5 ' - C A C C A G C T G G T T A T C T C T C A G C T C - 3 ' iNOS Forward iNOS Reverse 5 ' - C G G T G C T G T A T T T C C T T A C G A G G C G A A G A A G G - 3 ' 5 ' - G G T G C T G C T T G T T A G G A G G T C A A G T A A A G G G C - 3 ' RANTES Forward 5 ' - C C A A C C C A G C A G T C G T C T T T G - 3 ' RANTES Reverse 5 ' - C T C C C A A G C T A G G A C A A G A G C - 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 PMSF) 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 EGTA, 25% glycerol, 1 mM 31 DTT, 1 mM PMSF) . 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, pBS (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- 3 2P]dCTP (Amersham, Montreal, Quebec, Canada) and the Klenow 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 FX (or alternatively exposed to film overnight at -80°C and then developed). 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 [ 3 2 P]ATP. Labelled probe was cleaned as described above. 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 EGTA, 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 (Bio-Rad, 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 TBST (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 TBST 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). Phospho-kB, k B a , phospho-IKK, phospho-p38, phospho-JNK, JNK, phospho-ERK, ERK, 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, CK2 a and a ' were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), or were a kind gift of Steve Pellech (UBC, Vancouver, BC, Canada). Antibody to p38 MAPK 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 CK2 substrate RRADDSDDDDD and 100 DM [ 3 2 P]GTP (2.5 •C i /assay in Buffer C (12 mM M O P S (pH 7.2), and 15 mM MgCI 2) for 15 min at 30°C. The phosphorylation of casein or the specific substrate was quantitated by spotting 20 ml on to a 1.5 cm 2 piece of Whatman P-81 phosphocellulose paper. The papers were 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. CK2 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 CK2 substrate RRADDSDDDDD. 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 (SDS-PAGE) 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, UBC). 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 RNA was isolated using the TRIZOL method (Life Technologies, Burlingtion, Ontario). The purity of the RNA was determined by running 1 mg of RNA on a 1 % agarose gel for 1.5 h at 75 volts. 1 mg of RNA was reverse transcribed using 0.5 mg of oligo (dT)12-18 (Amersham, Montreal, Quebec), 1 ml of 10 mM dNTPs, 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 cDNA was used in each subsequent polymerase chain reaction (PCR) reaction. For each 50 uL P C R reaction, 2 U of TAQ (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 cDNA 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, CA, 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 ATP , 1 uCi [V*-32P]-labeled), for 20 min at 30°C. 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 mM EGTA, 7.5 mM 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 mM M O P S , pH 7.2, 25 mM -glycerophosphate, 20 mM MgCI 2, 5 mM EGTA, 2 mM EDTA, 1 mM DTT and 1 mM sodium vanadate), were added to the washed beads, and the reaction carried out as 38 above for 20 min using 5 uL of the ATP cocktail (250 mM ATP, 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 FX (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 lECs 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 MAPK family members was first examined. The data indicate that all three MAPKs 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 MAPK the selective inhibitor SB 203580 was used(201). To confirm that the SB 203580 inhibited signaling through to downstream mitogen-activated protein kinase-activated protein k inase 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 MAPK inhibitor, SB 203580, on the production of an a -chemokine (IL-8) and a p-chemokine (MCP-1) by lECs 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 SB 203580 led to a significant reduction in IL-8 production (Fig. 5A). A similar inhibitory effect of the p38 MAPK 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 MAPK is activated in lECs in response to IL-1 p and is involved in the production of important immunoregulatory chemokines. 41 3.2.3 I K B phosphorylation and degradation are independent of p38 M A P K As previously mentioned N F K B has been reported to be a major regulator of IL-8 and 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 CK2 has been shown to be downstream of p38 M A P K in response to stress(129). Therefore the possibility that p38 MAPK or downstream CK2 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 DRB, a specific inhibitor of protein kinase CK2 at the concentrations used, did not prevent IL-1 p induced phosphorylation and degradation of I K B (Fig. 6A). 3.2.4 N F K B D N A binding and transactivation are independent of p38 M A P K 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 SB 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. As 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 MAPK 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 (Fig. 7C). Pretreatment with S B 203580 did not attenuate the activation of N F K B . Curcumin was found to attenuate N F K B 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 MAPK, and investigated their influence upon DNA 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 MAPK constructs upon N F K B reporter activation were examined. The data indicated (Fig. 8B) that despite the small changes observed in the DNA-binding study (Fig. 8A), this is not translated into a reduction in transactivation. We believe that this complements the data obtained with the SB 203580 inhibitor, and establishes that p38 MAPK 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 MAPK protein was overexpressed. In contrast to the data obtained for N F K B , we were able to show a 43 reduction in p induced AP-1 DNA binding using SB 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 MAPK 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, RT-PCR 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 counter-regulatory 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 JNK 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 7C AND 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 JNK (Fig 12A). When the phosphorylation status of JNK was examined, it clear that it inhibits JNK 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 N F K B 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 , using the 4 X K B - I U C construct. S P 600125 inhibited N F K B 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 113 -92 -53 -29 SjjSl 5B8B> SSm i:infa~' m JI.LUI fir i lim « ( M N » < M* • — « S « H W «™* «| mm jy W V P IHR WW ^mmm m)Mw '-mm* B 5 10 15 30 45 60 Time (minutes) p-ERK p-JNK GAPDH p-Tyrosine Figure 4. IL-ip* stimulation of lECs results in the activation of ERK, 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 SB 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 SDS-PAGE, 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 MAPK attenuates IL-1B induced IL-8 production. Caco-2 were pretreated with SB 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 SB 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 SB 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 SDS-PAGE and immunoblotted with the appropriate antibody. The results are representative of at least four independent experiments. -49-DRB SB IL-16 - + -- - + + + + B SB Supershift Bound probe Free probe Probe IL-16 Anti-p65 Cold Comp + + + + + + - + -- + 3 H cu w CO cu l _ CJ o LL Z> _J 4 4XKB-IUC 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 SB 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 B Interleukin 1 (I) Empty vector p38 (wt) p38 (kd) C IL-1 p Vector p38 WT p38 K D Bound probe p38 MAPK + - + - + - + - + + - - - -- - - + + - -+ - + - - + + 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 SB 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-1 + S B 203580 IL-8 mm mm — — ~ — — I MCP-1 mm mm mm mm -OJL^  ^r^j^w- ^g^^jtt 1 •••^•i!-*?' ^(PWP: q^^^P i^^^PW- 1 ACTIN — mm mm mm mm mm mm mm mm mm 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Minutes 1.40 0 30 60 90 120 150 180 Time (minutes) — • — IL-1 — • — IL-1 + SB Minutes 0 30 60 90 120 150 180 Time (minutes) — • — IL-1 — • — IL-1 + SB IL-1 + Act D — IL-8 IL-1 +SB+ Act D 0 15 30 60 90 120 IL-8 (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 transcriptase-polymerase chain reaction (RT-PCR) was performed and PCR 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 PCR 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 MAPK 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 S P IL-1 IL-1 (20 u M ) + SP(20uM) B pJNK JNK p-p38 p38 C SP IL-1 IL-1 (20 uM) + SP (20 uM) IL-1 S P 2 u M SP 20 uM Figure 12. Selective J N K inhibitor SP 600125 inhibits J N K activity, however does not regulate N F K B D N A binding. HCT-116 cells were pretreated with SP 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 GST-ATF2 as a substrate. Phosphorylated substrate was resolved using SDS-PAGE, and visualized using autoradiography. LC-Lysate and non specific IgG control, C-control, SP-SP 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 4 X K B - L U C Control SP 2 uM SP 20 uM IL-1 IL-1 + SP 2 uM IL-1 + SP 20 uM B IL8-LUC 12.0 -r 10.0 -8.0 -6.0 -4.0 -2.0 -0.0 -IL-1 • S P 2 uM IL-1 + S P 20 uM 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 JNK activity using curcumin or selective JNK 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, MCP-1 , Groa, and R A N T E S (data not shown). E R K 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 ERK 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 TNF induced IL-8 production in IECs(209). This report showed that ERKs 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 MAPK family members, the intermediates, 57 such as the adaptor proteins that are recruited immediately following receptor activation, can be completely different. As 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 TNFR superfamily are important. The question of the functional role of ERK 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 lECs has never been investigated, although the production of IL-8 in response to invasion of lECs 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 MAPK 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 N F K B 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 MAPKs , 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 MAPK plays a role distinct from that of regulating N F K B activation. p38 MAPK 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 mRNA 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 AP-1 . Although it is not required for chemokine synthesis, it can play an important role in modulating the maximum production of chemokines. As 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 MAPK 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 JNK 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 JNK 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 JNK 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 JNK 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 PD 98059 and SB 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 JNK inhibitor, due to the high degree of homology between MAPK family members. Further investigation using molecular and genetic approaches is required to fully delineate the mechanism of JNK 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 CK2 is highly conserved throughout evolution, thus underscoring its important biological role. Enigmatically, however, the precise role of this kinase has yet to be defined. An area in which CK2 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 CK2 and IEC chemokine production, some interesting reports in other cell systems do set a very interesting precedence. CK2 has been linked to the regulation of N F K B , as previously discussed. This regulation however has been controversial, with different 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 phosphorylat ion^-73). In addition, a recent report has also linked CK2 to IFNy mediated signaling(228). Thus it can be hypothesized that CK2 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 CK2 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 CK2 kinase assays, using G T P and a CK2 specific peptide substrate. There was a significant increase in the activity of CK2 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). As 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 I L - 1 p (2ng/mL). Pretreatment with apigenin led to a dose dependant decrease in the IL-1B 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 I L - 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 CK2 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. Pretreatment of cells with apigenin(229) or a second CK2 inhibitor, 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 N F K B was examined (by EMSA), pretreatment of cells with either CK2 inhibitor had no effect (Fig 18B). The ability of p65 itself to undergo post-translational modifications provides an important mechanism to regulate its transactivation. We examined the ability of CK2 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 CK2 inhibitors prevented this activation (Fig 18C). Previously it has been shown that CK2 can regulate the C terminus transactivation domain via serine 529. When CK2 was overexpressed with a WT 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 CK2 regulates IL-1 p induced activation of N F K B 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. C K 2 a 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 CK2 is able to associate with p65, and the amount of this association remained constant, the specific activity of this p65 bound pool of CK2 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 CK2 assays, using casein as a substrate and GTP as the phosphate donor. p65 bound CK2 activity was increased at 30 minutes of stimulation (Fig 20). When compared to the kinetics of degradation of k B , the p65 bound CK2 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 CK2 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 CK2 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 CK2 inhibitors dose dependency attenuated this activation (Fig 21). 4.2.9 Inhibition of CK2 prevents the message synthesis of N F K B downstream targets To further confirm CK2 was able to regulate downstream N F K B targets we examined the IL-1 (3 induced message synthesis of N F K B downstream targets. k B 67 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. C K 2 a is overexpressed in patients with active U C . 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. DSS colitis was induced in mice using 2.0% DSS (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 CK2 specific substrate and GTP. -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 CK2 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 CK2 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), CK2a 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 B IL-1 Apigenin DRB + + + - + -- - + 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 I K B (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 - 7 3 -IP: CK2a IP:p65 IB p65 IB CK2a IB CK2a IBp65 AC LC 0 15 AC LC 0 15 B CYTO NUC W IP p65 IB C K 2 a IP p65 IB C K 2 a ' L A 0 5 15 0 5 15 - J J I" L """" m IB p65 IP C K 2 a IB C K 2 a L A 0 5 15 0 5 15 CYTO N U C _ Histone H3 0 5 15 0 5 15 Figure 19. CK2a 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 SDS-PAGE, 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 SDS-PAGE, 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-—mm- A mm :mm™w ^^K^^m^ ^ ^ ^ M P ^ P ' ^ ^ ^ ^ ^ ^ ^ ^ | B B | M ^ p-kB 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 CK2a 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 IKB (B) p65 was immunoprecipitated overnight before immunocomplexes were collected with protein A /G, CK2 kinase assays were performed using GTP and casein as a substrate. The reactions were then resolved by SDS-PAGE, coomasie stained, followed by exposure to film for 6h at -80 C. -75-"L-1 _ + + + Apigenin (uM) _ . 7 5 20 DRB 2 0 Figure 21. Inhibition of CK2 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 I K B I L - 1 Apigenin Figure 22. Inhibition of CK2 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 , MCP -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 lECs. In actively inflamed tissue there seems to be an increase in CK2 expression and also activity. The functional role of CK2 in lECs was then examined and a regulatory role for CK2 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 CK2 that appears to occur by a pool of CK2 that is constitutively bound to p65. Upon the addition of IL-1 p this pool increases in specific activity. Although CK2 has been described to be elevated in many solid tumors and rapidly proliferating tissues, the precise functional consequence of CK2 overexpression has not yet been determined(126, 231). The data presented here provides some evidence for the potential functional consequences of overexpression of CK2. In an inflammatory setting, this may lead to elevated basal N F K B activity, or a hyperactive N F K B response, especially in response to proinflammatory cytokines. Although normally considered a constitutively active kinase, CK2 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 TNFa , 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 CK2, 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 CK2 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 CK2 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 CK2 in cancer cells can regulate apoptosis in response to anticancer agents(130). Although CK2 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 PEST sequence of IKB by CK2 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 CK2 and site directed mutation of the CK2 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 CK2 is able to phosphorylate IKB in vitro, however this report failed to show the activation of CK2 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 CK2 may be able to modulate basal N F K B activation (Fig 17). Interestingly, the functional differences between CK2 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 CK2 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 TNFa and it was shown to be due to CK2. 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 TBP, TFIIB. As 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 C H A P T E R 5 - P I 3 K and P D K 1 regulation of IL-1|3 induced N F K B and chemkokine production 5.1 R A T I O N A L E The PI3K pathway, including PDK1 and PKB, 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 NFKB (80 , 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, PKB, and PDK1 was examined. Due to their previous roles in regulating N F K B , it can be hypothesized that PI3K and PKB 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 R E S U L T S 5.2.1 Inhibition of both PI3K and P D K 1 results in attenuation of IL-1 p induced N F K B activation. 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 LY and wortmannin were inhibiting PI3K, the phosphorylation status of PKB (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 S6 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 P K B is sufficient to drive N F K B activation. To further confirm the role of PDK1 and PKB in the activation of N F K B , empty vector, PDK1, or P K B were overexpressed with the 4 X K B - I U C construct. Compelling evidence from three different cell lines was acquired (Fig 25 A/B/C) . 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 HEK293T (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 PKB in response to IL-1 p, the kinetics of serine 473 phosphorylation were compared with PKB 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 PKB 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 PKB were regulating N F K B , the activity of the IKK complex was first examined. 293T cells were transfected with vector, PDK1, 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 PDK1, but not PKB, 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 PDK1, but not PKB, induced endogenous IKB phosphorylation (Fig 27B). In addition, pretreatment with TPCK, but not LY and wortmannin, led to prevention of IL-1 p induced IKB degradation (Fig 27C). This meant that PDK1 and PKB were activating N F K B in distinct ways, and that PKB 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 EMSA, the effects of TPCK, LY, and wortmannin on p65 DNA binding were thus examined. Not surprisingly, when cells were pretreated with TPCK, 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 HCT-116 cells. 5.2.6 Pharmacological inhibition of PKB results in the attenuation of p65 transactivation A potential mechanism for PKB 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 LY and wortmannin (Fig 29A). Thus PKB 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 PKB 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 CK2 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 WT, GST-p65 S529A, or GST-p65 S536A. As 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 PKB should not regulate serine 536 because it does not regulate IKK. Thus using the one-hybrid system with a mutant version of p65 TAD 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 WT 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 PDK1, they were co-immunoprecipitated and immunoblotted for each other. As 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 PKB 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 LY 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 RNA was transcribed as a result of promoter activation. Overexpression of PKB 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 (4XKB-Luc), 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 SDS-PAGE, and probed for phospho-PKB (serine 473). -89-B p P D K 1 p S 6 p E R K Figure 24. Inhibition of PDK1 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 ( 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 SDS-PAGE, and probed for phospho-PDK1, phospho-S6, or phospho-ERK. -90-Vector Vector + PDK-1 PDK-1 AKTWT AKTWT + IL-1 WT WT + IL-1 IL-1 6.5 B 5.5 a) 4.5 1.5 0.5 Vector Vector+ 1-1 PDK-1 WT PDK-1 WT + AKTWT AKT WT + IL-1 IL-1 1 1 -1- 1 1 1 1 Vector Vector + IL- PDK-1 WT PDK-1 WT AKTWT AKTWT + 1 + IL-1 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 IL-1 Stimulation (min) pPKB 15 30 60 B 6.0 Time (Minutes) Figure 26. IL-1B activates P K B and the phosphorylation of IKB and p65. HCT-116 colonic epithelial cells were stimulated with IL-1 p (10 ng/mL) for varying lengths of time. Proteins were subsequently resolved by SDS-PAGE, 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 PCDNA3 EV + + Myc-PDK1 HA-PKB IL- ip - + • + + + + + + GST IkB B V V+l P P+l A A+l IB: p k B V=Vector P=PDK1 A=PKB I B : I K B C T10 T50 T100 IL-1 I+T10 I+T50 I+T100 T=TPCK L=LY W=Wortmannin I B : I K B C LY WM IL-1 l+L l+W Figure 27. Overexpression of PDK1, but not PKB , results in the activation of IKK activity. (A) 293T cells were transfected with either empty PCDNA3 vector, myc-PDK1, 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 TPCK (50-100 uM) , for 1 h before being stimulated with IL-16 (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 s e r 5 3 6 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 SDS-PAGE, 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 TPCK (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 TPCK (50-100 uM), stimulated with IL-1B (2ng/mL) for 30 min. Proteins were subsequently resolved by SDS-PAGE, and probed for phospho-p65. -96-Vector Vector + IL-1 PDK-1 WT PDK-1 WT + AKT WT AKT WT + IL-1 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 c 5 - . . -........ *HPN P C D N A 3 E V + + - - - -Myc-PDK1 - - + + - -HA-PKB - - - - + + IL-1 - + - + - + 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, myc-PDK1, 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 HA-PKB. 24 h post transfection cells were stimulated with IL-16 for 30 min. Proteins were subsequently resolved by SDS-PAGE, 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 IL-1 l+L l+W pIKK Ser 180/181 C T50 T100 IL-1 I+T50 I+T100 L=LY W=wortmannin T=TPCK B PKB SGK p70SGK p90RSK PKA PRK PKC PDK1 K D G A T M K E H N S T T S H D G T V T H D H E K K A Y R V K G R T W G Y G D R T S M D G V T T R S K Q A R A N IKK ALPHA D Q G S L C T IKK BETA D Q G S L C T F C G T P E Y L A F C G T V E Y M A F C G T I E Y M A F C G T V E Y M A L C G T P E Y L A F C G T P E F L A F C G T P D Y I A F V G T A Q Y V S P P P P P P P P E E E E E E E 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 TPCK. (A) Alignment of consensus PDK1 phosphorylation sites. (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 SDS-PAGE, and probed for phospho-IKK (serine 180/181). -100-10.5 8.5 CO TO § 6.5 c i 4.5 2.5 0.5 Control LY25 Wm 100 IL-1 IL-1 + LY 25 IL-1 + Wm 100 12.0 B 10.0 8 8.0 03 0 | 6.0 1 4.0 2.0 0.0 I j I Hi Control TPCK TPCK TPCK IL-1 10 50 100 IL-1 + IL-1 + IL-1 + TPCK TPCK TPCK 10 50 100 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 TPCK (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 AKT WT AKTWT + 1 + IL-1 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 RT-PCR 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 IKB 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 PDK1. The regulatory activating phosphorylation of IKKa and IKKB were potential PDK1 phosphorylation sites, and were sensitive to TPCK. 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 PKB 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, designed to allow the study of the serine-protease chymotrypsin(242). 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 RSK, S6K, 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. The same group then reported that the cellular target of T P C K was PDK1(239). 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 PDK1, 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 HSP90 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 phosphorylat ion^). Our data clearly shows that PDK1, not PKB, 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 PKB 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 TNF induced liver apoptosis(247). When examined, GSK3B was found to regulate N F K B in a method independent of I K B 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 lECs leading to the synthesis of chemokines, in response to IL-1 p. Three important pathways were examined (MAPK, protein kinase CK2 , 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 PKB. 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 GSK3p. Upon entering the nucleus p65 is further phosphorylated by protein kinase CK2 at serine 529. p65, now being fully active, binds its consensus DNA 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 IL-8 promoter, however independently of N F K B . Another important transcription factor, A P -1 is required for maximal transcriptional activation. Its DNA binding ability appears to be regulated by p38 MAPK, and this may be one the ways that p38 M A P K regulates chemokines. At this point JNK may play a role in interacting with functional p65 and thus regulating both p65 activity and the IL-8 promoter. This allows the promoter to be regulated by multiple inputs, all downstream of the IL-1 R. 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