Epigenetic Regulation of MMP-13

MMP-13 的表观遗传调控

• 批准号: 7385654
• 负责人: MARY B GOLDRING
• 金额: $ 17.11万
• 依托单位: HOSPITAL FOR SPECIAL SURGERY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7385654
• 关键词: Accounting; Adherent Culture; Binding; Binding Sites; Biological Assay; Cartilage; Cartilage Matrix; Cells; Chondrocytes; Collagen Type II; Condition; DNA; DNA Methylation; Data; Decitabine; Degenerative polyarthritis; Depth; Disease Progression; EMSA; Endopeptidases; Enhancers; Enzymes; Epigenetic Process; Fracture; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Head; Heart; Histone Deacetylase Inhibitor; Human; In Vitro; Individual; Inflammatory; Interleukin-1; Luciferases; Matrix Metalloproteinases; Mediating; Methylation; New York; Nuclear; Nuclear Extract; Nucleic Acid Regulatory Sequences; Pathology; Patients; Peptide Hydrolases; Polymerase Chain Reaction; Prevention; Proliferating; Promoter Regions; RNA; Regulation; Reporter; Sampling; Severities; Site; Somatic Cell; Source; SssI methylase; Structure of neck of femur; Synovial Membrane; TNF gene; TNFRSF5 gene; Techniques; Testing; Time; Transcription Coactivator; Transcription Factor AP-1; Transcriptional Regulation; Trichostatin A; Validation; activating transcription factor; articular cartilage; collagenase; collagenase 3; cytokine; demethylation; experience; expression vector; in vitro Assay; in vivo; mRNA Expression; oncostatin M; p65; prevent; promoter; response; transcription factor; vector

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：骨关节炎（OA）的关键特征是关节软骨的侵蚀，该软骨是由降解蛋白酶（例如基质金属蛋白酶（MMP）（MMP）和脂肪酶引起的，这些蛋白酶由某些OA Chondrocytes异常产生。软骨细胞依赖性基质降解活性位于OA病理和预防的核心，可能会停止或减慢不可避免的OA进展。该项目将集中于编码与OA相关的主要胶原酶的基因调节，即MMP-13。在体内，基因表达不仅受转录激活因子和阻遏物的控制，而且还取决于启动子的表观遗传DNA甲基化，这是该机制永久沉默的机制。这项研究将是第一个考虑MMP-13启动子的DNA甲基化状态，以复制体内发生的方式调节该基因的表达。我们将检验以下假设：MMP-13启动子活性取决于DNA甲基化状态与转录因子与同源结合位点的结合之间的相互作用。因此，总体上，甲基化的存在与基因沉默相关联，通过防止某些转录因子的结合。具体目的是：1）确定在MMP-13启动子中所有14个CpG位点的甲基化状态，这些软骨细胞衍生自OA患者的非OA，浅层和深区域，对所选CpG位点的MRNA表达和甲基化％进行量化mRNA表达和％。 2）研究炎症细胞因子和脱甲基化剂对MMP-13表达和甲基化状态的影响。 3）比较转录因子对非甲基化和甲基化启动子构建体中MMP-13启动子活性的影响。 4）研究转录因子与正常与细胞因子诱导的软骨细胞中MMP-13启动子的特异性结合。在提供有关OA甲基化状态变化的完整体内数据之后，我们将确定如何通过炎症细胞因子改变正常软骨细胞的甲基化状态。我们的子假设是，这些细胞因子将启动MMP-13启动子的脱甲基化，从而增加对转录因子的可访问性，从而增加MMP-13的转录。在启动子构建研究中，我们最初将重点放在已知可以调节MMP-13启动子活性的转录因子上，包括ETS因子AP-1，RUNX2和NFKB。我们的假设是DNA甲基化将防止某些但不是全部转录因子的结合。然后，我们将使用非甲基化和甲基化的MMP-13启动子构建体以及EMSA和CHIP测定法中的MMP-13中实际上与MMP-13启动子与非表达细胞的MMP-13启动子结合。这将使我们能够将功能响应与CPG位点的接近性和甲基化状态相关联。前两个目标将由南安普敦的Co-Pi进行调查，该技术先前的技术存在。第二个目标是基于PI在转录调节方面的经验。南安普敦组的先前发现表明，脱甲基是OA中MMP-13异常表达的基础。拟议的研究将大大提高我们对DNA甲基化在OA病理学中的重要性的理解。