Synergistic transcriptional regulation of collagenase gene expression in chondrocytes Christopher Macdonald BSc (Hons) A thesis submitted to the Faculty of Medical Sciences, Newcastle University, in partial fulfilment of the requirements for the degree of Doctor of Philosophy Institute of Cellular Medicine February 2013 Abstract The proteolytic degradation of articular cartilage in load-bearing joints is a key pathological step in the progression of arthritis, a process mediated by enzymes called collagenases (specifically MMP-1 and MMP-13). My research has focused on the transcriptional regulation of these enzymes in cartilage cells (chondrocytes) in response to a pro-catabolic stimulus which mimics the complex milieu of elevated cytokines found within the arthritic joint. Activating Protein (AP)-1 transcription factors, specifically the c-Fos/c-Jun heterodimer, have previously been shown to be crucial in collagenase gene regulation. c-Fos/c-Jun gene expression, protein production and collagenase promoter enrichment studies identified a temporal deficit between transient c-Fos/c-Jun peak following 1 hour stimulation and the initiation of collagenase gene transcription following 6 hours stimulation. Protein synthesis inhibitor studies indicated that although c-Fos/c-Jun are indeed important, they are not the sole regulators of collagenase gene expression. DNA microarray studies highlighted a number of genes that contributed to transcriptional regulation early within this temporal deficit. Collagenase gene expression was assessed following the siRNA-mediated silencing of these factors. This confirmed that new factors, not previously associated with collagenase gene regulation, were demonstrated to have a significant role in initiating their transcription. This included factors such as activating transcription factor (ATF)3 and early growth response (EGR)2 which demonstrated differential regulation of the collagenase genes, with their silencing affecting MMP13 expression alone. Having identified a number of contributing factors, I then assessed their temporal gene expression and protein production. Comparisons to c-Fos/c-Jun induction confirmed that a number of these factors were transient, similar to AP-1, yet they peaked following longer durations of stimulation. Subsequent siRNA gene silencing of c-Fos and c-Jun led to decreased expression of some of these factors. This demonstrated that these factors may be regulating collagenase expression indirectly by controlling the expression of other transcription factors that, themselves contribute to the regulation of collagenase gene. The present study improves our understanding of how collagenases are regulated in chondrocytes in response to pro-catabolic stimuli. With an improved knowledge of regulation it may be possible to specifically abrogate aberrant collagenases expression in disease. Moreover, by exploiting the differential regulation of collagenases exhibited by some of these factors, there is the potential to mitigate the side effects associated with broad-spectrum collagenase inhibition, thereby removing the barrier to successful treatment. II Acknowledgements Firstly, I would like to thank my supervisors, Professor Drew Rowan and Dr Nick Europe-Finner, for their advice and encouragement over the past four years. I would also like to thank Dr Gary Litherland, Dr Jelena Mann, Dr Matt Barter and Dr Harriet Purvis for their advice and discussion towards successful completion of this thesis. Thanks also to all the members of the Musculoskeletal Research Group for their help, support and friendship throughout my PhD. I would like to thank the Oliver Bird Rheumatism Programme and the Nuffield foundation for their funding. Finally I would like to thank my family for the support and encouragement they have provided during the course of my studies. Without these people, I would not be where I am today. Thank you. Declaration This thesis is based on research performed in the Musculoskeletal Research Group (Rheumatology), Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK. Except for commonly held concepts, and where specific reference is made to other work, the content of the thesis is original. No part of this thesis has been submitted for the award of any other degree. III Table of Contents 1 Chapter 1: Introduction ............................................................................................. 1 1.1 Articular Cartilage ............................................................................................... 1 1.1.1 Structure and Function ............................................................................... 1 1.1.2 Chondrocytes .............................................................................................. 2 1.1.3 Extracellular Matrix ..................................................................................... 2 1.2 Arthritis ............................................................................................................... 4 1.2.1 Rheumatoid arthritis ................................................................................... 5 1.2.2 Osteoarthritis .............................................................................................. 6 1.3 Enzymes and cartilage degradation ................................................................... 7 1.4 Matrix Metalloproteinases ................................................................................. 8 1.5 Collagenases ....................................................................................................... 9 1.5.1 Collagenases in arthritis ............................................................................ 10 1.6 Pro-Inflammatory Cytokines ............................................................................ 12 1.6.1 Interleukin-1 .............................................................................................. 13 1.6.2 Pro-Inflammatory Cytokine Synergy ......................................................... 14 1.7 IL-1- and OSM-mediated cell signalling pathways ........................................... 16 1.7.1 MAPK ......................................................................................................... 16 1.7.2 Jak/STAT .................................................................................................... 17 1.7.3 Phosphatidylinositol 3-kinase (PI3K)/Akt pathway ................................... 18 1.7.4 PI3K/Protein Kinase C pathway ................................................................. 18 1.7.5 NF-κB ......................................................................................................... 19 1.8 Cis-regulatory elements and trans-activators .................................................. 20 1.8.1 Immediate early genes .............................................................................. 21 1.8.2 Activator Protein-1 .................................................................................... 22 IV 1.8.3 AP-1 and collagenase gene regulation ...................................................... 24 1.8.4 Other tran-activators and collagenase gene regulation ........................... 26 1.9 Epigenetic regulation ........................................................................................ 26 1.9.1 Chromatin remodelling ............................................................................. 27 1.9.2 The Transcriptional Cycle .......................................................................... 28 1.9.3 Epigenetic regulation of collagenase genes .............................................. 29 1.10 Summary and aims of the study ....................................................................... 30 2 Chapter 2: Materials and Methods .......................................................................... 32 2.1 Materials ........................................................................................................... 32 2.1.1 Antibodies ................................................................................................. 32 2.1.2 Cytokines ................................................................................................... 32 2.1.3 Cell and Tissue Culture Reagents .............................................................. 33 2.1.4 Commercially available kits ....................................................................... 33 2.1.5 Immunoblotting Reagents ........................................................................ 33 2.1.6 Cell Lines .................................................................................................... 34 2.1.7 Biochemical Reagents ............................................................................... 34 2.1.8 Molecular Biology Reagents ...................................................................... 34 2.2 Methods ........................................................................................................... 35 2.2.1 Cartilage Sample Collection ...................................................................... 35 2.2.2 Chondrocyte Extraction and Culture......................................................... 35 2.2.3 Cell Culture ................................................................................................ 36 2.2.4 Real-time PCR ............................................................................................ 37 2.2.5 RNA extraction and reverse transcription ...............................................