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Open Banerjee Thesis.Pdf The Pennsylvania State University The Graduate School College of Medicine MEPRIN METALLOPROTEASES MODULATE INTESTINAL HOST RESPONSE A Dissertation in Biochemistry and Molecular Biology by Sanjita Banerjee © 2008 Sanjita Banerjee Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2008 The thesis of Sanjita Banerjee was reviewed and approved* by the following: Judith S Bond Professor and Chair of Department of Biochemistry and Molecular Biology Thesis Adviser Chair of Committee Kristin A. Eckert Professor of Biochemistry and Molecular Biology Sergei A. Grigoryev Associate Professor of Biochemistry and Molecular Biology Harriet C. Isom Distinguished Professor of Microbiology and Immunology Cara-Lynne Schengrund Professor of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School ii ABSTRACT Meprins, zinc metalloproteases of the astacin family, are composed of two independently expressed subunits meprin α and β which associate to form homo and heter-oligomers, termed meprin A and B. Meprin isoforms are enriched in human as well as rodent intestine. They have also been reported in kidney, leukocytes, lung and skin. Considerable research has been conducted regarding the in vitro functions of these proteases, however the physiological roles of these proteases are unknown. Generation of meprin β, α and αβ knock-out mice were steps towards filling this void. This work reports the general characterization of the meprin α knock-out mouse and investigates the role of meprin proteases in the mouse intestine using a model of colitis. Several polymorphisms have been identified in the human meprin α gene that significantly correlate with ulcerative colitis and Crohn’s disease, which make this mouse model relevant to the human pathology. Human and rodent intestines have high but non-uniform meprin expression and hence allow one to study the contributions of different meprin isoforms. To understand the role of meprin A, colitis was induced in wild-type and meprin α knock-out mice by dextran sulfate sodium administration. Meprin α knock-out mice showed greater susceptibility to injury and had a phenotype of heightened inflammation as evidenced by different markers of inflammation at macroscopic as well as molecular level. Further investigations showed a role for mucosal meprin A in the process of tissue repair. To understand the phenotype of increased inflammation, meprin interaction with immune-mediators was studied at greater detail. This led to the first known documentation of an in vivo interaction of meprins with an immune-mediator, interleukin–18. Subsequent experiments using meprin αβ knock-out mice elucidated the contribution of meprin B in the phenotype in this model of inflammatory bowel disease. These experiments collectively demonstrated a novel function of meprins in immuno-modulation. This thesis furthers our knowledge about the roles of meprin metalloproteases in the rodent intestine and also elucidates the importance of proper distribution of meprin isoforms. iii TABLE OF CONTENTS List of Figures ix List of Tables xii List of Abbreviations xiii Acknowledgements xvi Chapter 1: Introduction 1 1.1 Overview 1 1.1.1 Proteases: In Life and Death 1 1.1.2 The story of Meprin – Metalloendopeptidase from Renal Tissue 3 1.1.2.1 Discovery of Meprin 3 1.1.2.2 Characterization of Meprin 4 1.1.2.3 Meprins’ Unique Oligomerization 4 1.1.2.4 A first attempt at elucidating the function 5 1.1.2.5 Meprin Classification 7 1.1.2.6 Meprin localization 9 1.1.2.7 Domains of Meprins 10 1.1.2.7.1 The NH2 & COOH Domains 10 1.1.2.7.2 The I Domain 10 1.1.2.7.3 The Interaction Domains 12 1.1.2.8 The higher order structure of meprins 14 1.1.2.9 Meprin Substrates 16 1.1.2.10 Meprins in Disease 18 1.1.2.11 In vivo studies 18 1.1.2.12 Meprin distribution in the intestine 19 1.1.2.13 Inflammatory Bowel Disease 20 1.1.2.14 Rationale for this thesis work 24 Chapter 2: Characterization of Meprin α Knockout Mice 26 2.1 Overview 26 2.2 Experimental Procedures 28 2.2.1 Generation and validation of αKO mice 28 2.2.2 Urine and serum analyses 30 2.2.3 End-Point PCR 30 iv 2.2.4 Immunoblotting of urinary samples 32 2.2.5 Immunohistochemistry 32 2.2.6 Ileum brush-border membrane preparation 33 2.2.7 Meprin β activity assay 33 2.2.8 Statistical Analysis 34 2.3 Results 34 2.3.1 Meprin αKO mice did not express meprin α mRNA or protein 34 2.3.2 General Parameters 34 2.3.3 Litter size 37 2.3.4 Meprin β mRNA and protein is unaffected in αKO mice 37 2.4 Discussion 42 Chapter 3: Delineating the role of meprin A in a Model of 44 Inflammatory Bowel Disease 3.1 Overview 44 3.2 Experimental Procedures 47 3.2.1 Induction of Experimental Colitis by DSS 47 3.2.2 Histological Scoring 48 3.2.3 Myeloperoxidase Assay 48 3.2.4 Collection of blood samples 49 3.2.5 Measurement of Serum Nitrite levels 49 3.2.6 FITC-Dextran Oral Gavage 50 3.2.7 Measurement of Colon and Serum Cytokines 50 3.2.8 Statistical Analysis 51 3.3 Results 51 3.3.1 Meprin αKO mice show greater susceptibility to 51 DSS-induced colitis 3.3.2 DSS-treatment causes greater colon injury in meprin αKO mice 55 3.3.3 Meprin αKO colons show heightened inflammation 58 3.3.4 Meprin A is not involved in maintaining the epithelial barrier 62 function 3.3.5 Mucosal meprin A plays a role in tissue repair and remodeling 62 3.3.6 DSS-induced colitis elicits greater systemic inflammation in 66 v αKO mice 3.4 Discussion 72 Chapter 4: Delving into the interaction of Meprins with Interleukin-18 78 4.1 Overview 78 4.2 Experimental Procedures 81 4.2.1 Construction of proIL-18 expression vector 81 4.2.1.1 Generation of pET30b::His6-proIL-18 81 4.2.1.2 Generation of pET28a::His6-proIL-18 82 4.2.2 Preparation of BL21(DE3)-RIL Competent Cells 84 4.2.3 Induction and purification of His6-proIL-18 84 4.2.4 Thrombin cleavage and proIL-18 purification 87 4.2.5 Activation of recombinant meprins 88 4.2.6 Meprin – proIL-18 reaction 88 4.2.7 Identification of IL-18 site of cleavage 89 4.2.8 Kinetic measurements 90 4.2.9 Meprin B and IL-18 interaction in MDCK cells 90 4.2.10 NF-κB activation in EL-4 cells by IL-18 91 4.2.11 ELISA of serum IL-18 91 4.2.12 Statistical Analysis 92 4.3 Results 92 4.3.1 Generation of recombinant murine proIL-18 92 4.3.2 Cleavage of proIL-18 by meprins 93 4.3.3 Biochemical characterization of proIL-18 cleavage by meprins 99 4.3.4 Identification of IL-18 cleavage site 99 4.3.5 Meprin B and proIL-18 interaction in a cell-culture system 102 4.3.6 Meprin B cleavage results in active IL-18 105 4.3.7 Corroboration of Meprin B – IL-18 interaction in vivo 105 4.4 Discussion 109 Chapter 5: Elucidating a role for meprin B in the DSS-induced 114 colitis model 5.1 Overview 114 vi 5.2 Experimental Procedures 115 5.2.1 Induction of Experimental Colitis by DSS 115 5.2.2 Colon myeloperoxidase assay 115 5.2.3 Collection of blood samples for serum nitrite measurement 115 5.2.4 Statistical Analysis 116 5.3 Results 116 5.3.1 Meprin αβKO mice show greater susceptibility to 116 DSS-induced colitis 5.3.2 Meprin αβKO do not exhibit a higher degree of inflammation 120 than WT 5.4 Discussion 124 Chapter 6: Conclusions and Discussion 128 6.1 Overview 128 6.1.1 Meprins: similar yet different from MMPs 128 6.1.2 Advancement of meprin knowledge 129 6.1.2.1 Meprin Redundancy 129 6.1.2.2 Meprins in the intestine 130 6.1.2.3 Meprins expressed by immune-mediators 133 6.1.3 Possible roles for meprins 137 6.1.3.1 Meprin distribution in different leukocytic populations 137 6.1.3.2 Meprin involvement in MΦ - T cell cross talk 138 6.1.3.3 Meprin signalling in innate immune response 139 6.2 Closing remarks 142 Appendix: Characterization of meprin α expression in a human 145 hepatocellular carcinoma cell-line, HepG2 A.1 Overview 145 A.2 Experimental Procedures 146 A.2.1 End-point PCR 146 A.2.2 Immunoblotting of HepG2 culture medium 146 A.2.3 HepG2 growth assay 147 A.2.4 Matrigel assay 147 vii A.3 Results 147 A.3.1 HepG2 cells over-express meprin α mRNA and protein 147 A.3.2 Meprin α inhibition does not affect HepG2 growth 148 A.3.3 HepG2 invasiveness unaffected by the loss of meprin α activity 148 A.4 Discussion 148 Bibliography 154 viii List of Figures Figure 1.1 Meprin metalloproteases show unique oligomerization 6 Figure 1.2 Schematic view of the catalytic centre of the metzincins 8 Figure 1.3 Domain structure of meprin subunits 11 Figure 1.4 Importance of different domains in the transport of meprin 13 subunits Figure 1.5 Multimeric structures of meprin metalloproteases 15 Figure 1.6 Differences in substrate bond specificities between the two 17 subunits Figure 1.7 Different mouse models of colitis and their utilities 22 Figure 2.1 Strategy for Mep1a gene disruption 31 Figure 2.2 Validation of meprin αKO mice at mRNA and protein level 35 Figure 2.3 The αKO and WT mice show comparable growth rates 38 Figure 2.4 Meprin αKO mice have smaller litters 40 Figure 2.5 Meprin β is unaffected in the αKO mice 41 Figure 3.1A Meprin αKO mice severely affected by four day DSS 52 treatment Figure 3.1B Meprin αKO mice have higher occult blood scores at day 7 53 Figure 3.1C Meprin αKO mice have higher DAI scores 54 Figure 3.2A Significant colon shortening seen in meprin αKO mice 56 on day 7 Figure 3.2B Meprin αKO mice show greater colon injury than the 57 WT mice Figure 3.2C Meprin αKO mice have higher colon injury scores 59 Figure 3.3 Higher inflammation and leukocyte infiltration seen in 60 αKO mice Figure 3.4 Colon cytokines in αKO mice significantly more elevated 64 than WT mice Figure 3.5 Lack of meprin A does
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