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Regulation of Innate Immune Responses by The UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS BIOLÓGICAS Departamento de Bioquímica y Biología Molecular I REGULACIÓN DE LA RESPUESTA INMUNE INNATA POR LA PROTEÍNA DE MATRIZ EXTRACELULAR TROMBOESPONDINA-1 MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR María Gema Martín Manso Madrid, 2008 • ISBN: 978-84-692-2767-1 REGULATION OF INNATE IMMUNE RESPONSES BY THE EXTRACELLULAR MATRIX PROTEIN THROMBOSPONDIN-1 I REGULATION OF INNATE IMMUNE RESPONSES BY THE EXTRACELLULAR MATRIX PROTEIN THROMBOSPONDIN-1 A THESIS DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY UNIVERSIDAD COMPLUTENSE DE MADRID BY María Gema Martín Manso Laboratory of Pathology National Cancer Institute, NIH Bethesda II Dedicada a las niñas de mis ojos, María y Beatriz. A mi Juanma, a mi hermano Ricardo y a mis queridos padres. III Contents Acknowledgments……………………………………………XIV Abbreviations………………………………………………….XVII I A.INTRODUCCIÓN..............................................................1 1. Las Trombospondinas.…………………………………….2 1.1. La Trombospondina-1……………………………………..8 IV 1.1.1. Organización estructural ……………………………....8 1.1.1. A. Dominio N-terminal ……………………………….9 1.1.1. B. Dominio trimérico de oligomerización …………11 1.1.1. C. Dominio con el Factor de von Willebrand (vWF) de tipo C……………………………………………………...11 1.1.1. D. Dominios de repetición o TSRs………………...12 1.1.1. E Dominio de identidad………….………………….13 1.1.2. Principales sitios de expresión……………………….15 1.1.3. Conexiones moleculares……………………………….16 1.1.4. Funciones de Trombospondina-1…………………….18 1.1.4.A. Regulación de las Respuestas Inmunes………..26 1.1.4.B. Regulación de las Respuestas Inmunes Innatas………………………………………………………..30 1.1.4.C. Regulación de la Respuesta Inmune Innata mediada por macrófagos contra el tumor………………...35 V B.INTRODUCTION.............................................................37 1. Thrombospondins…………………………………………..37 1.1. Thrombospondin-1………………………………………...43 1.1.1. Structural organization………………………………….43 1.1.1. A. N-terminal domain………………………………...44 1.1.1. B. Trimeric oligomerization domain………………...45 1.1.1. C. von Willebrand Factor (vWF) type C domain…..46 1.1.1. D. Thrombospondin repeats………………………...47 1.1.1. E Signature domain………….……………………….48 1.1.2. Major sites of expression……………………………….49 1.1.3. Molecular Connections………………………………….51 1.1.4. Roles of Thrombospondin-1…………………………...52 1.1.4.A. Regulation of Immune Responses……………….59 1.1.4.B. Regulation of Innate Immune Responses……….62 1.1.4.C. Regulation of Macrophage-mediated Innate Immune Response against tumors………………………...65 VI REFERENCES…………………………………………...........67 II A.OBJETIVOS…………………………………………...........90 B.STATEMENT OF PURPOSE……………………….......93 VII III A.RESULTS………………………………………….........……94 (Gema Martin-Manso, Susana Galli, Lisa A. Ridnour, Maria Tsokos, David A. Wink, and David D. Roberts. Thrombospondin1 promotes Tumor Macrophage Recruitment and Enhances Tumor Cell Cytotoxicity of Differentiated U937 Cells. Cancer Res. 2008; 68(17): 7090-9) 1. TSP1 Over Expression Increases Tumor Macrophage Recruitment in vivo…………………………………........CR-7092 2. Role of MCP-1 and PAI-1 in TSP1-dependent Tumor- Associated Macrophage Recruitment………………...CR-7092 3. Increased M1 Macrophage Recruitment into TSP1 Over- Expressing Tumors………………………………………CR-7094 4. TSP1 Stimulates Macrophage Cytotoxicity towards Breast Carcinoma and Melanoma Cells………………CR-7094 5. TSP1 Increases Extracellular Release of Superoxide Anion….......................................................................…CR-7096 VIII 6. TSP1-stimulated Superoxide Production in Macrophages Requires Intracellular Ca2+………….…CR-7096 B.ADDITIONAL RESULTS...............................................96 1. Elevated production of proinflammatory cytokines and chemokines by TSP1-activated human monocytic cells…96 2. Role of TGF-β1 in TSP1-mediated cytokine and chemokine expression by human monocytic cells………104 3. Identification of NF-κB as a downstream effector of TSP1-mediated cytokine and chemokine expression…...109 REFERENCES………………………………………..........…111 IX IV A.DISCUSIÓN……………………………........……………..114 B.DISCUSSION.................................................................127 REFERENCES……………………………………........…….138 V A.CONCLUSIONES ………………………………….........142 B.CONCLUSIONS ……………………………………........146 X VI A.MATERIALS AND METHODS………………………...148 (Gema Martin-Manso, Susana Galli, Lisa A. Ridnour, Maria Tsokos, David A. Wink, and David D. Roberts. Thrombospondin1 promotes Tumor Macrophage Recruitment and Enhances Tumor Cell Cytotoxicity of Differentiated U937 Cells. Cancer Res. 2008; 68(17): 7090-9) 1. Proteins and peptides..............................................CR-7090 2. Reagents………………………………………………...CR-7091 3. THBS1-transfected cells……………………………...CR-7091 4. Cell culture and differentiation………………………CR-7091 5. Tumorigenesis assay in nude mice………………...CR-7091 6. Immunohistochemical evaluation…………………..CR-7091 7. Measurement of monocyte chemotactic protein-1, PAI-1, and IL-10………………………………………………...CR-7091 8. Western blotting………………………………………..CR-7091 XI 9. Real-time quantitative reverse transcription-PCR analysis……………………………………………………..CR-7091 10. U937 and ANA-1 cell-mediated cytolysis………...CR-7091 11. Cytotoxicity assay……………………………………CR-7092 12. Superoxide production……………………………...CR-7092 13. Flow cytometry analysis…………………………….CR-7092 14. Measurement of intracellular free Ca2+…………...CR-7092 15. Statistical analysis…………………………………...CR-7092 B.ADDITIONAL MATERIALS AND METHODS…………………………………………………...…148 1. Proteins and peptides......................................................149 2. Reagents...........................................................................149 3. Cell culture and differentiation.......................................150 4. Real-time quantitative reverse transcription-PCR analysis.................................................................................150 XII 5. Measurement of human cytokines and chemokines....151 6. Plasmids...........................................................................151 7. Transient transfection and Luciferase assay................152 REFERENCES…………………………………………….….153 XIII Acknowledgments There are a great number of people that I need to thank for providing me with the professional, technical and emotional support that I so genuinely appreciated and without which the completion of this work would not have been possible. To my mentor, Dr. David D. Roberts. I would like to express my sincere gratitude for your support, patience and encouragement over the years. I have learned a great deal from you and I thank you for the guidance and for the enthusiasm for science that you have instilled in me. To my friend and colleague, Dr. Elizabeth Perruccio. I have learned so much from you and I have appreciated all your help and support. It has been a pleasure working with you and I am lucky that our paths have crossed. XIV To Dr. Svetlana A. Kuznetsova, I want to thank you for your encouragement and the generosity of your time and help, which have resulted in a valuable contribution to this work. To Dr. María J. Calzada, I also owe a debt of gratitude for the contribution of your time. I am grateful for your generosity and appreciated all the helpful discussions. I would like to thank Dr. Jeff S. Isenberg, Dr. Justin B. Maxhimer, Dr. Thomas W. Miller, John M. Sipes, Dr. Michael L. Pendrak, Dr. Dhammika Navarathna, Dr. Yifeng Jia, and Jo Anne Cashel. I respect you for the accomplished scientist that you are and I genuinely appreciate all your help, advice and support that you have given me. To Mónica Feijoó Cuaresma, Dr. Guillermo Velasco, and Dr. Álvaro Martínez del Pozo. I appreciate the encouragement and support you have shown me. I am especially grateful for your input and help. XV To Dr. Mary L. Stracke, Dr. Malgorzata M. Ptaszynska, Dr. Timothy Clair, Dr. Elliot Schiffmann and Russel W. Bandle, I also owe a debt of gratitude for the contribution of your time and equipment. I am grateful for your generosity and appreciated all the helpful discussions. Thanks also to Dr. David A. Wink, Dr. Lisa A. Ridnour, and Dr. Douglas D. Thomas for the use of your lab equipment and advice over the years which significantly helped me to accomplish various goals. I am grateful for your help and constant encouragement. I was lucky to have Dr. Maria Tsokos and Dr. Susana Galli as next door neighbors in the department where I was able to frequently drop in and ask for expert advice and also use the lab equipment. Thank you for the numerous valuable discussions. XVI Abbreviations ADAMTS proteins containing a disintegrin and a metalloproteinase with TSPs motifs BSA albumin bovine serum Ca2+ calcium COMP cartilage oligomeric matrix protein CTL cytotoxic T lymphocyte DAB 3,3–diaminobenzidine DCs dendritic cells ECM extracellular matrix EGF epidermal growth factor FBS fetal bovine serum FGF fibroblast growth factor FMLP n-formyl-methionyl-leucyl-phenylalanine GM-CSF granulocyte-macrophage-colony-stimulating factor HBD heparin-binding domain XVII HBSS Hanks’ balanced salt solution HSPG heparan sulphate proteoglycans H2O2 hydrogen peroxide ICAM-1 intracellular cell adhesion molecule-1 IFNγ interferon-γ IL interleukin LAP latency-associated protein LDL low density lipoprotein LFA leukocyte function associated antigen LPS lipopolysaccharide LRP LDL receptor-related protein MCP-1 monocyte chemotactic protein-1 MCP-2 monocyte chemotactic protein-2 MMPs matrix metalloproteinases NMR nuclear magnetic resonance NO nitric oxide - O2 superoxide
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