The Pennsylvania State University The Graduate School MEPRIN METALLOPROTEASES MODULATE EPITHELIAL BARRIER INTEGRITY AND MONOCYTE MIGRATION A Dissertation in Biochemistry and Molecular Biology by Jialing Bao © 2012 Jialing Bao Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2012 The dissertation of Jialing Bao was reviewed and approved* by the following: Judith S. Bond Evan Pugh and Distinguished Professor, Chair of Biochemistry and Molecular Biology Dissertation Advisor Co-Chair of Committee Gail L. Matters Associate Professor of Biochemistry and Molecular Biology Co-Chair of Committee Sergei A. Grigoryev Professor of Biochemistry and Molecular Biology W. Brian Reeves Professor of Medicine Harriet C. Isom Distinguished Professor of Microbiology and Immunology * Signatures are on file in the Graduate School ii Abstract Meprin metalloproteases have been implicated in a number of normal developmental and pathological processes. However, it has been difficult to establish the cellular and molecular basis for the biological role of meprin metalloproteases in health and disease, possibly because of the large number of metalloproteases, many having overlapping substrate specificities. A number of important biological molecules have been demonstrated to be meprin substrates in vitro, such as extracellular matrix and cytokines. With the development of congenic mice lacking meprin activity, it has been possible to relate in vitro results with in vivo data to examine cellular and molecular processes. The hypothesis of the thesis work is that meprins relax epithelial barriers by cleaving tight junction proteins, and facilitate monocyte migration. The study demonstrated that homomeric meprin A and meprin B cleaved the tight junction protein occludin, but not claudin-4, in membrane fractions from MDCK cells. Meprin A, but not meprin B, added exogenously to MDCK monolayers cleaved occludin. Meprin A, but not meprin B, cleaved recombinant occludin extracellular loops, and the cleavage site was determined in the first extracellular loop of occludin. Different cleavage site preferences at extracellular regions explain the different results between meprin A and meprin B to cleave occludin in intact cells and cell extacts. The biological relevance of the in vitro experiments was demonstrated by studies in cell culture, in vivo and ex vivo. Meprin A disrupted the immunostaining of tight junction proteins occludin and ZO-1 on MDCK monolayers. In addition, meprin A impaired the barrier function of MDCK monolayers, as shown by increased small molecule flux and decreased transepithelial electrical resistance. To elucidate the role of meprin A in acute urinary tract infections, meprin A was infused into the mouse bladder and the effects on bladder epithelium wall was investigated. The results showed that active meprin A increased the permeability of the epithelium as demonstrated by the influx of a fluorescene dye. The hypothesis that meprin A disrupts epithelial barriers and facilitates monocyte migration was further investigated by co-culturing monocytes with MDCK monolayers and measuring monocyte migration. Monocytes from mice lacking iii meprin A (meprin αKO) were less able to migrate through MDCK monolayers than monocytes from wild-type mice. It is concluded that the capability of meprin A to disrupt epithelial barriers is one important factor by which meprin A modulates inflammation. iv Table of Contents List of Figures………………………………………………………………………...ix List of Tables………………………………………………………...……………….xii List of Abbreviations………………………………………………...…………… xiii Acknowledgements………………………………………………………...……….xvi Chapter 1. Introduction 1.1 Overview…………………………………………………………………………….1 1.2 Proteases……………………………………………………………….....……..…...1 1.2.1 Protease classification………………………………………………………..1 1.2.2 Classification and structure of metalloprotease……………………………2 1.2.3 Function of metalloprotease………………………………………………....2 1.3 Meprin metalloproteases…………………………………………………………….4 1.3.1 History and classification of meprin………………………………………..4 1.3.2 Meprin domain structure and oligomerization…………………………….5 1.3.3 Meprin tissue expression…………………………………………………….7 1.3.4 Activation and inhibition of meprins……………………………………….9 1.3.5 Meprin peptide bond and substrate specificities ……………………….…9 1.3.6 Meprins in inflammation………………………………………………......11 1.4 Epithelial barrier and leukocyte migration……………………………………….12 1.4.1 Epithelial barrier functions……………………………...………………...12 1.4.2 Tight junctions and component proteins…………………………...…….13 1.4.3 Tight junctions and leukocyte migration……………………………...….16 1.5 Rationale for this work………………………………………………………...….17 Chapter 2. Materials and methods 2.1 Meprins………………………………………………………………………...…...18 2.1.1 Meprin purification, activation and activity assays……………..……...18 2.1.2 Meprin activity inhibition………………………………………………..19 v 2.2 Cell culture……………….……………………………………………………..….19 2.3 Animal model……………………………..…………………………….………….19 2.3.1 Generation and validation of meprin αKO mice………………………..19 2.3.2 Anesthesia..………………………………………………………………...20 2.3.3 Transurethral catheterization…………………………………………….20 2.3.4 Assessment of bladder permeability……………………………………...21 2.4 Preparation of recombinant occludins……………………………………….…...21 2.4.1 Recombinant occludin (49-290 amino acids)……………………………21 2.4.2 MBP conjugated occludin extracellular loops…………………………..21 2.4.2.1 Construction of occludin extracelluar loops expressing vectors…………………...…………………………..21 2.4.2.2 Induction and purification of MBP-occludin extracellular loops………..……………………………………………………..22 2.5 Assays………………………………………………..………..…………………….22 2.5.1 MDCK epithelial barrier set-up………………………………………….22 2.5.2 Immunocytochemistry and confocal microscopy of tight junction proteins……………………………………………………………………..24 2.5.3 Permeability assay………………………………………………………...24 2.5.4 Transepithelial electrical resistance (TER)……………………………...24 2.5.5 Cell viability………………………………………………………………..25 2.5.6 Preparation of membrane-enriched fractions of MDCK cells…………25 2.5.7 Western blot analysis……………………………………………………...26 2.5.8 Isolation of monocytes from mouse bone marrow………………………26 2.5.9 Transmigration of monocyte through MDCK monolayers……….……..26 2.6 Statistical analysis and graphing software……………………………..………..27 Chapter 3. Meprin A weakens epithelial barriers and facilitates monocyte migration 3.1 Meprin A impairs MDCK monolayer’s barrier function……….…….………..28 3.2 Meprin A increases mouse bladder permeability……………………….............32 3.3 Impaired barrier function was not due to cell toxicity of meprin A….……….34 vi 3.4 Meprin A disrupts TJs on MDCK monolayers……………..…………..….……35 3.5 Occludin in MDCK monolayer is degraded by exogenous meprin A….……....39 3.6 Claudin-4 in MDCK monolayer is not degraded by exogenous meprin A….....43 3.7 Meprin A does not disrupt claudin-4 staining on MDCK monolayers………...43 3.8 Occludin in MDCK cell extracts is cleaved by meprin A…...……..….………...46 3.9 Claudin-4 in MDCK cell extracts is not degraded by meprin A…...……….......48 3.10 Recombinant occludin is cleaved by meprin A…………………………….…...49 3.11 Meprin A regulates monocyte transmigration…………………………………54 Chapter 4. Determine the cleavage site(s) of meprin A on occludin 4.1 Construction of recombinant maltose binding protein (MBP) conjugated occludin extracelluar loops……….….……………...…………………………….56 4.2 Occludin extracellular loops are cleaved by meprin A……………..……..…….56 4.3 Determination of the occludin cleavage site(s) by Mass-spectrometric analysis……………………………………………………………………...………60 Chapter 5. Meprin B weakens epithelial barriers 5.1 Meprin B impairs MDCK monolayer’s barrier function……....……………62 5.2 Meprin B disrupts TJs on MDCK monolayers…………………....………...….65 5.3 Meprin B has little proteolytic activity on occludin in MDCK monolayer…...65 5.4 Occludin in MDCK cell extracts is cleaved by meprin B………………………69 5.5 Claudin-4 in membrane fractions of MDCK cells is not degraded by meprin B…………………………………………………………………...……..71 Chapter 6. General conclusions and discussion 6.1 Meprins in inflammation: acute and chronic conditions…….………..….…….72 6.2 Meprins cleave certain tight junction proteins…………..….………….……….75 6.3 Meprins modulate epithelial barrier functions…………………….………...….76 6.4 Meprins facilitate leukocyte migration……………………………….……...…..79 6.5 Meprins modulate cytokine profiles in inflammation………………….……….80 vii 6.6 Meprins participation in wound healing and tissue repair……………...….….80 6.7 Closing……………………………………………………………………………..81 Appendix: Characterization of immune-cell derived meprin A in a model of inflammatory bowel disease A.1 Overview……………………………………………………………...……………85 A.2 Methods………………………………………………………………...…………..85 A.2.1 Bone marrow transplantation………………………………..…...…….85 A.2.2 Induction of experimental Ulcerative Colitis…………………..………86 A.2.3 Measurement of weight loss and disease activity index…………..…...86 A.2.4 Myeloperoxidase assay………………………………………………….86 A.3 Results……………………………………………………………………..………87 A.3.1 Mixed background meprin αKO showed greater weight loss after DSS treatment………………………………………………………….87 A.3.2 Congenic meprin αKO and wild-type mice lost similar percentage of weight after DSS treatment…………………………………………….87 A.3.3 Bone marrow transplantation……………………………………..……90 A.3.4 Meprin αKO recipient mice show greater weight loss after DSS challenge………….……………………………………………………..90 A.3.5 Meprin WT to αKO chimeras have higher DAI scores after DSS challenge…………….…………………………………………………..95 A.3.6 Meprin αKO recipientmice and WT recipient mice have comparable levels