Universidad Autónoma de Madrid Departamento de Bioquímica Identification of the substrates of the protease MT1-MMP in TNFα- stimulated endothelial cells by quantitative proteomics. Analysis of their potential use as biomarkers in inflammatory bowel disease. Agnieszka A. Koziol Tesis doctoral Madrid, 2013 2 Departamento de Bioquímica Facultad de Medicina Universidad Autónoma de Madrid Identification of the substrates of the protease MT1-MMP in TNFα- stimulated endothelial cells by quantitative proteomics. Analysis of their potential use as biomarkers in inflammatory bowel disease. Memoria presentada por Agnieszka A. Koziol licenciada en Ciencias Biológicas para optar al grado de Doctor. Directora: Dra Alicia García Arroyo Centro Nacional de Investigaciones Cardiovasculares (CNIC) Madrid, 2013 3 4 ACKNOWLEDGEMENTS - ACKNOWLEDGEMENTS - First and foremost I would like to express my sincere gratitude to my supervisor Dr. Alicia García Arroyo for giving me the great opportunity to study under her direction. Her encouragement, guidance and support from the beginning to the end, enabled me to develop and understand the subject. Her feedback to this manuscript, critical reading and corrections was inappreciable to finish this dissertation. Next, I would like to express my gratitude to those who helped me substantially along the way. To all members of MMPs’ lab, for their interest in the project, teaching me during al these years and valuable contribution at the seminary discussion. Without your help it would have not been possible to do some of those experiments. Thank you all for creating a nice atmosphere at work and for being so good friends in private. I would like to also thank all of the collaborators and technical units for their support and professionalism. Finally, I would like to acknowledge Ministerio de Educación, Cultura y Deporte for providing me the financial support for my research, CNIC and Universidad Autonoma de Madrid for all the economical and technological facilities supporting my thesis work and coursework. 5 6 SUMMARY - SUMMARY - Understanding the molecular mechanism regulating angiogenesis is an essential aspect of pathology of many diseases. Recent evidences implicate membrane-type 1 matrix metalloprotease (MT1-MMP), a potent pericellular enzyme expressed by vascular cells, in inflammation and angiogenesis. This work aimed to explore the MT1-MMP’s contribution to inflammatory-induced angiogenesis, formation of new vessels from preexisting ones. To accomplish this, we applied quantitative proteomic technique named SILAC to TNFα-activated endothelial cells derived from wildtype and MT1-MMP null mice to identify the substrate repertoire of this protease and identify candidates that might affect capillary sprouting within the inflammatory context. We found that TNFα activated endothelial cells and induced expression of several markers characteristic for endothelial tip cells, the main effectors of sprouting angiogenesis. Moreover, MT1-MMP expression was upregulated after TNFα pulse, accompanied by differences in supernatant glycoproteome between wildtype and MT1-MMP null endothelial cells. Glycoproteins identified as putative MT1-MMP substrates include key extracellular matrix, matricellular and secreted proteins such TSP1, NID1, CYR61, SEM3C or SLIT2 as well as cytokines and growth factors like EGFR and CSF1. Bioinformatic analysis revealed a combinatorial MT1-MMP proteolytic program, in which combined rather than single substrate processing would determine biological decisions by activated endothelial cells, including chemotaxis, cell motility and adhesion, and vasculature development. Processing of TSP1, CYR61, NID1 and SEM3C was impaired in MT1-MMP- deficient animals leading to reduce angiogenic response. The relevance of this MT1-MMP-driven proteolytic program was observed in pathology in aberrant angiogenesis related to chronic inflammatory disorders as inflammatory bowel disease (IBD). Using an experimental model of mouse colitis we showed soluble levels of TSP1 and NID1 were increased and positively correlated with MT1-MMP expression. Impaired processing of these substrates was also observed in human serum samples from patients with low/moderate activity of ulcerative colitis or Crohn’s disease. Taken together, these data illustrate that MT1-MMP is an important regulator of inflammatory-induced angiogenic response in endothelial cells and that substrates processed by this protease, such as TSP1 and NID1 might be used as a biomarkers for the diagnosis or prognosis of chronic inflammatory disorders. 7 SUMMARY 8 SUMMARY - RESUMEN - Entender los mecanismos moleculares que regulan la angiogénesis es un aspecto esencial en la patología de numerosas enfermedades. La metaloproteasa de matriz extracelular de membrana tipo 1 (MT1-MMP) es una potente enzima pericelular expresada por las células vasculares. Este trabajo ha explorado la contribución de MT1-MMP en la angiogénesis, formación de nuevos vasos a partir de vasculatura pre-existente, en el contexto inflamatorio. Para ello, se ha aplicado la técnica de proteómica cuantitativa denominada SILAC en células endoteliales derivadas de ratones de genotipo silvestre o deficientes en MT1-MMP; estas células endoteliales se han activado con la citoquina inflamatoria TNFα con objeto de identificar el repertorio de sustratos de MT1-MMP e identificar candidatos que podrían afectar la respuesta angiogénica en inflamación. Hemos demostrado que TNFα activaba las células endoteliales e inducía la expresión de varios marcadores característicos de células endoteliales líder (tip). El tratamiento con TNFα también aumentaba la expresión de la proteasa MT1-MMP en las células endoteliales lo que correlacionaba con diferencias en el glicoproteoma del sobrenadante de cultivo de células endoteliales de genotipo silvestre versus deficientes en MT1-MMP. Las glicoproteínas identificadas como sustratos potenciales de MT1-MMP incluyen proteínas de la matriz extracelular, proteínas matricelulares y proteínas secretadas como TSP1, CYR61, NID1, SEM3C o SLIT2 así como citoquinas y factores de crecimiento como EGFR y CSF1. El análisis bioinformático de los datos proteómicos ha desvelado un programa proteolítico combinatorial, en el cual el procesamiento combinado de varios sustratos por MT1-MMP en vez del procesamiento de sustratos individuales es el que determina las decisiones biológicas de las células endoteliales activadas tales como quimiotaxis, motilidad y adhesión celular, y desarrollo vascular. Hemos confirmado que el procesamiento de TSP1, CYR61, NID1 y SEM3C está disminuido tanto en células como en ratones deficientes en MT1-MMP lo que correlaciona con una reducción de la respuesta angiogénica. Por último, hemos investigado la importancia de este programa proteolítico mediado por MT1-MMP en patologías que cursan con angiogénesis inflamatoria, en concreto en enfermedad inflamatoria intestinal. Utilizando un modelo de colitis experimental en ratón hemos observado que los niveles solubles en suero de los sustratos TSP1 y NID1 aumentan en correlación con la expresión de MT1-MMP a lo largo de la severidad de la colitis. Además, en muestras de suero procedentes de pacientes con colitis ulcerosa o enfermedad de Crohn con actividad baja/moderada también se observa un aumento en los niveles de TSP1 y NID1. En conclusión, nuestros datos demuestran la importancia de MT1-MMP como proteasa reguladora de la angiogénesis inflamatoria en células endoteliales e indican que sustratos procesados por MT1-MMP como TSP1 o NID1 pueden servir como biomarcadores para el diagnóstico o pronóstico de enfermedades inflamatorias crónicas. 9 SUMMARY 10 CONTENTS CONTENTS ACKNOWLEDGEMENTS - .................................................................. 5 SUMMARY - ...................................................................................... 7 RESUMEN - ....................................................................................... 9 NON-STANDARD ABBREVIATIONS AND ACRONYMS - ....................... 15 INTRODUCTION - ............................................................................ 15 - MORPHOGENESIS OF THE VASCULAR SYSTEM - .................................... 17 Principal cellular players in angiogenesis .................................................................... 17 Molecular regulation of angiogenesis .......................................................................... 19 Inflammation-driven angiogenesis .............................................................................. 20 - MMP FAMILY OF PROTEASES - ............................................................. 21 MMPs as multifunctional enzymes ............................................................................ 22 Role of MMPs in inflammation and angiogenesis ....................................................... 22 MMPs in human disease models ................................................................................ 23 Domain structure and classification ............................................................................ 24 MT1-MMP as a paradigm of membrane-tethered MMP ............................................. 26 Regulatory mechanisms controlling MT1-MMP expression ........................................ 26 MT1-MMP subcellular location ................................................................................. 27 Cleavage preferences and targets of MT1-MMP .......................................................... 29 MT1-MMP in physiology and pathology ...................................................................