Regulation of intestinal inflammation by mitogen activated protein kinase phosphatase 3

REGULATION OF INTESTINAL INFLAMMATION BY MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE-3 SUZAN SAIDIN (Bachelor of Science (Biotechnology) (Hons.), Monash University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENT First and foremost I would like to thank my supervisor Dr Zhang Yongliang for giving me an opportunity to work on this project and for his guidance throughout the duration of my study I would also like to express my deep gratitude towards Dr Png Chin Wen for his mentorship, advice and suggestions, which contributed significantly to this project I would like to thank Fiona and Chein Sze who have kindly given permission for their work to be included in the supplementary figures I would like to thank Weiliang and Emi for their excellent administrative support Thank you to Huipeng, Jenny, Madhu, Wu Di, Danke, Jennifer, Heng Boon, Mei Xing, Hong Ying, Siyuan, Edward, Han Jian and Yi Xiong for their great company in the lab I am very lucky to have known Hoey Lit, Yen-ling and Narisa who have been very supportive friends Their sheer company provided pleasant distractions from the unexplainable results and failed experiments I would like to thank Hong Ting for making sure to feed me with a little bit of alcohol whenever I am distressed and also Ban Xiong for reading this thesis I am sincerely grateful to Eng Kwan, Dana and Grace for helping me in going through my difficult times Last but not least I would like to thank my parents and my sisters for their support i TABLE OF CONTENTS ACKNOWLEDGEMENT i! TABLE OF CONTENTS ii! LIST OF ABBREVIATIONS iv! ABSTRACT viii! 1.! Introduction 1! 1.1.! Inflammatory Bowel Diseases (IBD) 1! 1.1.1.! Causes and Pathogenesis of IBD 2! 1.1.2.! Dextran Sulphate Sodium (DSS)-induced Murine Model of Colitis 5! 1.2.! The Role of Immune Response in Intestinal Inflammation 5! 1.3.! The Role of Intestinal Epithelium in Intestinal Inflammation 7! 1.4.! Mitogen-Activated Protein Kinases (MAPKs) 10! 1.4.1.! The Role of MAPKs in the Immune Response 12! 1.4.2.! The Role of MAPKs in Cell Proliferation and Survival 14! 1.5.! MAPK Phosphatases (MKPs) .16! 1.5.1.! MAPK Phosphatase-3 (MKP-3) .18! 1.6.! Study Rationale and Objectives .19! 2.! Materials and Methods .21! 2.1.! Cell Culture 21! 2.2.! RNA Isolation and Analysis 22! 2.3.! Western Blotting 24! 2.3.1.! Protein Extraction .24! 2.3.2.! SDS-PAGE and Protein Transfer .25! 2.3.3.! Protein Detection and Analysis 25! 2.4.! Wound Healing Assay .26! ii 2.5.! Proliferation Assay 27! 2.6.! Animal Studies 27! 2.6.1.! Histological Analysis 28! 2.6.2.! Ki67 Immunohistochemistry 29! 2.7.! Statistical Analysis 29! 3.! Results 30! 3.1.! MKP-3 Negatively Regulates Inflammatory Response in CMT93 cells .30! 3.2.! MKP-3 Regulates Epithelial Cell Proliferation and Migration In Vitro 32! 3.3.! Loss of MKP-3 Results in Less Severe Colitis .36! 3.4.! Increase in IEC-Associated Proliferation Genes in MKP-3-/- Mice after DSS Treatment 40! 4.! Discussion 43! 5.! Future Directions 50! 6.! Conclusion 52! REFERENCES 53! APPENDICES 65! iii LIST OF ABBREVIATIONS AOM Azoxymethane APC Antigen presenting cells ASK1 Apoptosis signal-regulating kinase BAD B-cell lymphoma-2-associated death protein BAX B-cell lymphoma-2-associated X protein Bcl-2 B-cell lymphoma-2 BMDM Bone marrow-derived macrophages BSA Bovine serum albumin CD Crohn’s disease CDK Cyclin-dependent kinase Cox2 Cyclooxygenase-2 DEPC Diethylpyrocarbonate DLK Dual leucine zipper-bearing kinase DMEM Dulbecco’s Modified Eagle’s Medium DNA Deoxyribonucleic acid DSS Dextran sodium sulphate DUSP Dual-specificity phosphatase DUSP2-/- DUSP-2 knockout EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor ER Endoplasmic reticulum ERK Extracellular signal-regulated kinases FasL Fas ligand FBS Fetal bovine serum iv FGF Fibroblast growth factor Foxp3 Forkhead box P3 GDP Guanosine diphosphate Grb-2 Growth factor receptor-bound protein-2 GM-CSF Granulocyte macrophage colony-stimulating factor GTP Guanosine triphosphate GWAS Genome-wide association studies H and E Hematoxylin and Eosin HRP Horse radish peroxidase IBD Inflammatory bowel diseases IEC Intestinal epithelial cells IFN-! Interferon-! IL Interleukin iNOS Inducible nitric oxide synthase IRF3 Interferon regulatory transcription factor JNK c-Jun NH2-terminal kinases KLF Krüppel-like transcription factor KLF4-/- KLF4 knockout KLF5+/- Heterozygous KLF5 knockout LPS Lipopolysaccharide M1 Macrophage MAPK Mitogen-activated protein kinase MCP-1 Monocyte chemotactic protein-1 MKK Mitogen-activated protein kinase kinase MKKK Mitogen-activated protein kinase kinase kinase v MKP Mitogen-activated protein kinase phosphatase MKP-1-/- MKP-1 knockout MKP-3-/- MKP-3 knockout MKP-5-/- MKP-5 knockout MLK3 Mixed lineage kinase mRNA Messenger ribonucleic acid MSK Mitogen- and stress-activated protein kinase MyD88 Myeloid differentiation primary response 88 NF-"B Nuclear factor kappa-light-chain-enhancer of activated B cells PAMP Pathogen-associated molecular pattern PCR Polymerase chain reaction PRR Pattern recognition receptor PVDF Polyvinylidene fluoride RO Reverse osmosis ROS Reactive oxygen species SDS Sodium dodecyl sulphate SOS Son of sevenless SPF Specific pathogen-free TAK1 Transforming growth factor-# activated kinase TBS Tris buffered saline TBST Tris buffered saline tween TCR T cell receptor TIRAP Toll-interleukin receptor domain containing adaptor protein TPL2 Tumour Progression Locus vi TRAM Toll/interleukin-1 receptor-domain-containing adapter-inducing interferon-!-related adaptor molecule TRIF Toll/interleukin-1 receptor-domain-containing adapter-inducing interferon-! TGF-# Transforming growth factor-# Th T helper cells TLR Toll-like receptor TNF-$ Tumour necrosis factor-$ Treg Regulatory T cells UC Ulcerative colitis UPR Unfolded protein response vii ABSTRACT During intestinal inflammation, the disruption of the intestinal mucosa barrier enabled the luminal microbiota to come into direct contact with the underlying immune cells, which results in inflammatory response The recognition of the luminal microbiota by the Toll-like receptors (TLRs) activates downstream signalling pathways such as the mitogen-activated protein kinase (MAPK) The MAPK pathway is essential in regulating immune response and its negative regulation is controlled by MAPK phosphatases (MKPs) MKP-3, also known as DUSP6, is a MAPK phosphatase with high specificity towards ERK, which is known to regulate cell proliferation The role of MKPs in immune cells has been widely studied but the role of MKPs in the intestinal epithelial cells (IEC) during intestinal inflammation has yet to be explored In this study, we utilised CMT93 cells overexpressing MKP-3 and dextran sodium sulphate (DSS)-induced colitis model in mice deficient in MKP-3 to investigate the role of MKP-3 in intestinal inflammation The results showed that overexpression of MKP-3 downregulated the phosphorylation of all three major groups of MAPKs (ERK, JNK and p38) and pro-inflammatory gene expression upon DSS and LPS stimulation In addition, the overexpression of MKP-3 retarded cell proliferation and wound healing ability of CMT93 cells MKP-3-/- mice subjected to days of DSS treatment developed less severe colitis compared to the wildtype mice mRNA and protein analysis showed that the expression of pro-inflammatory genes was reduced and the phosphorylation of ERK was increased in the colon of MKP-3-/mice The expression of proliferation genes and Krüppel-like transcription factor (KLF5) protein was also elevated in the colon of MKP-3-/- mice In addition, Ki67 staining showed increased IEC proliferation 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mucosal damage Goblet Cell Loss = normal 50% Inflammatory Cell Infiltration = occasional infiltration = increasing leukocytes in lamina propria = confluence of leukocytes extending to submucosa = transmural extension of inflammatory infiltrates Comments 65 Supplementary Figure MKP-3 expression was induced in CMT93 cells in response to (A) 2% DSS and (B) LPS (10 µg/ml) stimulation CMT93 cells were stimulated with 2% DSS and 10 µg/ml and the expression of MKP-3 was examined at mRNA and protein level via qPCR and Western blot, respectively Supplementary Figure Overexpression of KLF5 in CMT93 cell line confirms the specificity of the antibody used for Western blotting CMT93 cells were transiently transfected with pcDNA empty vector and pcDNA harboring KLF5 cDNA sequence Cells were subsequently harvested for protein analysis via Western blot 66 Supplementary Figure Basal expression level of several (A) inflammatory and (B) proliferation genes in the colon was comparable between wildtype and MKP3-/- mice, except for Cxcl1 The colon tissues of eight-week old untreated wildtype and MKP-3-/- mice were harvested and the expression level of several inflammatory and proliferation genes were measured via qPCR Dot plots show individual values, means and standard deviations from two (male) and three (female) mice from each group * P < 0.05 with unpaired t test 67 Supplementary Figure Expression of pro-inflammatory genes in BMDM upon LPS (100 ng/ml) stimulation Bone marrow-derived macrophages from wildtype and MKP-3-/- mice were stimulated with 100 ng/ml LPS, followed by the measurement of the expression of pro-inflammatory genes via qPCR 68 ... Mitogen- activated protein kinase MCP-1 Monocyte chemotactic protein- 1 MKK Mitogen- activated protein kinase kinase MKKK Mitogen- activated protein kinase kinase kinase v MKP Mitogen- activated protein. .. 29! 3. ! Results 30 ! 3. 1.! MKP -3 Negatively Regulates Inflammatory Response in CMT 93 cells .30 ! 3. 2.! MKP -3 Regulates Epithelial Cell Proliferation and Migration In Vitro 32 ! 3. 3.!... cascade of kinases The MAPKs are activated by MAPK kinase (MKKs), which in turn are activated by MAPK kinase kinases (MKKKs) 46,47 Once activated, MAPKs will then act on their substrate (eg protein