A key feature of osteoarthritis (OA) is the erosion of articular cartilage, which is caused by degradative proteinases, such as matrix metalloproteinases (MMPs) and aggrecanases, which are produced abnormally by certain OA chondrocytes. Chondrocyte-dependent matrix-degrading activities lie at the heart of OA pathology and prevention may halt or slow down the inevitable progression of OA. This project will concentrate on the regulation of the gene encoding the major collagenase involved in OA, namely MMP-13. In vivo, gene expression is not only controlled by transcriptional activators and repressors, but also depends on the epigenetic DNA methylation of the promoter, which is the mechanism whereby genes that are never expressed by a somatic cell are permanently silenced. This study will be the first to take into account the DNA methylation status of the MMP-13 promoter to investigate regulation of expression of this gene in a manner that replicates what takes place in vivo. We will test the following hypothesis: MMP-13 promoter activity is determined by the interactions between DNA methylation status and transcription factor binding to cognate binding sites. Thus, the presence of methylation correlates, on the whole, with gene silencing by preventing the binding of some transcription factors.
Specific aims are: 1) Determine the methylation status of all 14 CpG sites in the MMP-13 promoter in chondrocytes derived from non-OA, superficial and deep zones of OA patients, quantify mRNA expression and % methylation for selected CpG sites. 2) Investigate the effects of inflammatory cytokines and a demethylation agent on MMP-13 expression and methylation status. 3) Compare the effects of transcription factors on MMP-13 promoter activity in non-methylated and methylated promoter constructs. 4) Investigate specific binding of transcription factors to the MMP-13 promoter in normal vs cytokine-induced chondrocytes. After providing complete in vivo data regarding the changes in methylation status in OA, we will determine how the methylation status of normal chondrocytes is changed by inflammatory cytokines. Our sub-hypothesis is that these cytokines will initiate a demethylation of the MMP-13 promoter, thereby increasing accessibility to transcription factors and thus transcription of MMP-13. In the promoter construct studies, we will focus initially on transcription factors that are known to regulate MMP-13 promoter activity, including ETS factors, AP-1, Runx2 and NFkB. Our sub- hypothesis is that DNA methylation will prevent binding of some, but not all, transcription factors. We will then investigate which transcription factors are actually bound to the MMP-13 promoters in MMP-13 expressing vs non-expressing cells in cotransfections using non-methylated and methylated MMP-13 promoter constructs and in EMSA and ChIP assays. This will allow us to correlate functional responses with the proximity of CpG sites and methylation status. The first two aims will be investigated by the Co-PI in Southampton, where previous experience in the techniques exists. The second two aims build on the experience of the PI with respect to the transcriptional regulation. Previous findings by the Southampton group indicate that demethylation underlies the abnormal MMP-13 expression in OA. The proposed studies will advance considerably our understanding about the importance of DNA methylation in the pathology of OA. ? ? ? ? ? ?
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