Cell-Surface Association Between Progelatinases and Beta2 Integrins: Role of the Complexes in Leukocyte Migration

Cell•Surface Association between Progelatinases andb2 Integrins: Role of the Complexes in Leukocyte Migration MICHAEL STEFANIDAKIS Department of Biological and Environmental Sciences, Division of Biochemistry and Turku Graduate School “In vitro diagnostics”, Faculty of Biosciences, University of Helsinki, Finland Academic dissertation To be presented for public criticism, with the permission of the Faculty of Biosciences, University of Helsinki In the auditorium 1041 at Viikki Biocenter, Viikinkaari 5, Helsinki On February 3rd, 2006, at 12 o’clock noon 1 Supervised by: Docent Erkki Koivunen Department of Biological and Environmental Sciences Division of Biochemistry University of Helsinki Finland and Professor Carl G. Gahmberg Department of Biological and Environmental Sciences Division of Biochemistry University of Helsinki Finland Reviewed by: Professor Jari Ylänne Department of Biological and Environmental Sciences University of Jyväskylä Finland and Docent Jukka Westermarck Center for Biotechnology University of Turku & Abo Academi University Finland Opponent: Professor Antti Vaheri Department of Virology Haartman Institute University of Helsinki Finland ISSN: 1795•7079 ISBN: 952•10•2838•6 (printed) 952•10•2839•4 (PDF) Yliopistopaino Helsinki 2005 2 Integrins: connecting MMPs 3 To my Family 4 TABLE OF CONTENTS ORIGINAL PUBLICATIONS ABBREVIATIONS ABSTRACT REVIEW OF THE LITERATURE LEUKOCYTE ADHESION AND MIGRATION THE LEUKOCYTE MIGRATION CASCADE NEUTROPHIL FUNCTION AND ADHESION DURING INFLAMMATION Granulopoiesis and subsets of neutrophil granules INTEGRINS AND THEIR LIGANDS Structure and function of leukocyteb2 integrins Activation of leukocyte b2 integrins MATRIX METALLOPROTEINASES CLASSIFICATION OF MMPs STRUCTURAL FEATURES OF MMPs REGULATION OF MMPS Transcriptional regulation Zymogen activation Inhibition of enzymatic activity ROLE OF INTEGRINS AND MMPs IN LEUKOCYTE MIGRATION REGULATION OF LEUKOCYTE ADHESION AND MIGRATION CELL•SURFACE ASSOCIATION OF MMPs AND OTHER PROTEASES ROLE OF INTEGRINS AND GELATINASES IN CANCER AND INFLAMMATION INTEGRINS AND GELATINASES in tumor angiogenesis and growth INTEGRINS AND GELATINASES in invasion and metastasis INTEGRINS AND GELATINASES in cancer associated•inflammation ROLE OF INTEGRINS AND GELATINASES IN ACUTE LEUKEMIAS THERAPEUTIC INTERVENTION WITH MMP AND INTEGRIN INHIBITORS AIMS OF THE PRESENT STUDY MATERIALS AND METHODS 5 RESULTS IDENTIFICATION OF DDGW, AN aMb2 INTEGRIN•BINDING PEPTIDE THAT INHIBITS LEUKOCYTE MIGRATION IN VITRO (I). BLOCKING THE PROGELATINASE/ȕ2 INTEGRIN COMPLEX INHIBITS MIGRATION OF PMNs TO AN INFLAMMATORY SITE IN VIVO (II) IDENTIFICATION OF ANaMb2 INTEGRIN•SMALL BINDING MOLECULE WHICH INHIBITS INTEGRIN•DEPENDENT LEUKEMIA CELL MIGRATION (III) INHIBITION OF HUMAN LEUKEMIA TUMOR XENOGRAFTS BY BLOCKING THE INTERACTION BETWEEN INTEGRINS AND PROGELATINASES (IV) DISCUSSION ACKNOWLEDGEMENTS REFERENCES 6 ORIGINAL PUBLICATIONS This thesis is based on the following original publications, referred to in the text by their Roman numerals I• IV: I Stefanidakis, M., Björklund, M., Ihanus, E., Gahmberg, C. G., and Koivunen, E. Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte b2 integrins. Journal of Biological Chemistry, 278, 34674•84, 2003. II Stefanidakis, M., Ruohtula, T., Borregaard, N., Gahmberg, C. G., and Koivunen, E. Intracellular and cell surface localization of a complex betweenaMb2 integrin and promatrix metalloproteinase•9 progelatinase in neutrophils. Journal of Immunology, 172, 7060•68, 2004. III Björklund, M., Aitio, O.,Stefanidakis, M., Suojanen, J., Salo, T., Sorsa, T., Koivunen, E. Stabilization of the activated aMb2 integrin by a small molecule inhibits leukocyte migration and recruitment.Biochemistry, In press, 2006. IV Stefanidakis, M., Gahmberg, C. G., Jaalouk, D., Pasqualini, R., Arap, W., and Koivunen, E. Disruption of a cell surface MMP•9/b2 integrin interaction inhibits leukemia medullary extravasation and extramedullary tissue invasion. Submitted, 2006. 7 ABBREVIATIONS ADAM a disintegrin and metalloproteinase ADAMTS a disintegrin and metalloproteinase with a thrombospondin motif a2•M a2•macroglobulin AML acute myelocytic leukemia APMA aminophenyl mercuric acetate APC antigen presenting cell BFGF basic fibroblast growth factor BPI bactericidal/permeability•increasing protein CTT gelatinase inhibitor peptide CTTHWGFTLC DDGW aM/L I domain ligand peptide ADGACILWMDDGWCGAAG ECM extracellular matrix EGF epidermal growth factor EM electron microscopy EMMPRIN extracellular matrix metalloproteinase inducer FAK focal adhesion kinase fMLP N•formyl•methionyl•leucylphenylalanine FnIII fibronectin type III GST glutathione•S•transferase ICAM intercellular adhesion molecule IgSF immunoglobulin superfamily LAD leukocyte adhesion deficiency LFA leukocyte function•associated antigen LPS lipopolysaccharide LRP low•density lipoprotein receptor•related protein MAbs monoclonal antibodies MadCAM mucosal addressin cell adhesion molecule MAPK mitogen•activated protein kinase MCP•3 monocyte chemotactic protein•3 MIDAS metal ion•dependent adhesion site MMP matrix metalloproteinase MPO myeloperoxidase NGAL neutrophil gelatinase•associated lipocalin NMR nuclear magnetic resonance PECAM platelet•endothelial cell adhesion molecule RECK reversion•inducing cysteine rich protein with kazal motifs RME receptor•mediated endocytosis ROI reactive oxygen intermediate SDF•1 stromal•cell derived factor•1 TNF•a tumor necrosis factor•alpha tPA tissue•type plasminogen activator uPA urokinase type plasminogen activator uPAR urokinase type plasminogen activator receptor VCAM vascular cell adhesion molecule VEGF vascular endothelial growth factor VLA very late antigen TIMP tissue inhibitor of metalloproteinases 8 ABSTRACT Leukocyte motility is known to be dependent on bothb2•integrins and matrix metalloproteinases MMP•2/•9 or gelatinases, capable of mediating leukocyte adhesion and the proteolysis needed for invasion, respectively. We have used phage display technology to identify peptide sequences interacting with theaM integrin I domain, an about 200 amino acid residue sequence known to be responsible for ligand binding in b2 integrins. One of the peptides contained a sequence very similar to the conserved DELW(S/T)LG sequence found in MMP•2 and –9. In several binding, migration and mutation analysis studies, we showed that the integrin recognition sequence mapped to the MMP catalytic domain, specifically bound to theaM I domain, and it inhibited migration of leukocytesin vitro. Subcellular fractionation experiments revealed that the proMMP•9/aMb2 complex was formed intracellularly and could be translocated to the cell surface upon cell activation. This interaction was efficiently blocked by a peptide sequence derived from the catalytic domain of MMP•9. Also, a novel small•molecule ligand to atheM I domain, identified by screening a combinatorial library, inhibited DDGW•phage binding to the I domain and reduced leukocyte infiltration to an inflammatory site in vivo. The concept that MMPs associate with integrins, as well as its importance in some physiological and pathological conditions has been advanced previously but has not been examined on leukocytes. Gelatinases not only play an important role in cell migration, tissue remodelling and angiogenesis during development, but are also involved in the progression and invasiveness of many cancers, including leukemias. We showed that MMP•9 association with b2 integrins seems to play an important role in leukemia growth and dissemination in vivo, as inhibition of complex formation significantly improved the survival of mice that developed leukemia. These findings suggest that the integrin/MMP•9 complex may serve as a functional target for intervention in human acute leukemias. 9 REVIEW OF THE LITERATURE LEUKOCYTE ADHESION AND MIGRATION THE LEUKOCYTE MIGRATION CASCADE Leukocytes or white blood cells (WBC) are bone marrow•derived cells and principal components of the immune system. They circulate in the bloodstream as passive, non•polarized cells and function by destroying “nonself” substances, including invading microbes, bacteria, and viruses. Over the past two decades, much progress has been made towards elucidating the molecular basis of leukocyte migration from the bloodstream to the tissues (reviewed in Butcher, 1991; Springer, 1995; Carlos and Harlan; 1994; von Andrian and Mackay, 2000; McIntyre et al., 2003). Recruitment of neutrophils from the blood to the inflamed tissues requires a sequence of adhesion and activation events which are mediated by several adhesion molecules, including mainly selectins (that bind to their carbohydrate•based ligands) and integrins (that interact with cell adhesion molecules or CAMs) (Vestweber and Blanks, 1999; von Andrian and Mackay, 2000) (Figure 1). At least four steps of adhesion and activation events are required for a succesful extravasation of leukocytes from the vascular lumen to the tissues: (I) “Thethering and rolling” is the initial and essential event in leukocyte recruitment. It describes a process of weak adhesive interactions between the surfaces of the neutrophil and the endothelial cell, largely mediated by three members of the selectin family and their highly glycosylated ligands. Weak adhesive interactions between selectins and their ligands tether neutrophils to the vascular endothelium, and under shear flow, causes them to crawl along it. Such interactions can also initiate signals which promote the opening

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