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(申请人提供)：骨关节炎(OA)的一个关键特征是关节软骨的侵蚀，这是由某些骨关节炎软骨细胞异常产生的降解性蛋白酶引起的，如基质金属蛋白酶(MMPs)和聚集酶(Agrecanase)。软骨细胞依赖的基质降解活动是骨性关节炎病理的核心，预防可以阻止或减缓骨性关节炎不可避免的进展。这个项目将集中于编码参与骨性关节炎的主要胶原酶的基因的调控，即基质金属蛋白酶-13。在体内，基因的表达不仅受转录激活因子和抑制因子的控制，还依赖于启动子的表观遗传DNA甲基化，这是体细胞从未表达的基因永久沉默的机制。这项研究将首次考虑到基质金属蛋白酶-13启动子的DNA甲基化状态，以复制体内发生的事情的方式来研究该基因的表达调节。我们将检验以下假设：MMP-13启动子的活性是由DNA甲基化状态和转录因子与同源结合位点结合的相互作用决定的。因此，总的来说，甲基化的存在与基因沉默有关，因为它阻止了一些转录因子的结合。具体目标是：1)检测非骨性关节炎、骨性关节炎患者浅层和深层软骨细胞中基质金属蛋白酶-13启动子14个CpG位点的甲基化状态，定量检测选定的CpG位点的mRNA表达和甲基化百分率。2)探讨炎性细胞因子和去甲基化药物对基质金属蛋白酶-13表达及甲基化状态的影响。3)比较非甲基化和甲基化启动子结构中转录因子对基质金属蛋白酶-13启动子活性的影响。4)研究正常软骨细胞和细胞因子诱导的软骨细胞中转录因子与基质金属蛋白酶-13启动子的特异性结合。在提供了有关骨性关节炎甲基化状态变化的完整体内数据后，我们将确定炎性细胞因子如何改变正常软骨细胞的甲基化状态。我们的假设是，这些细胞因子将启动基质金属蛋白酶-13启动子的去甲基化，从而增加对转录因子的可及性，从而增加基质金属蛋白酶-13的转录。在启动子构建的研究中，我们首先将重点放在已知调节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甲基化将阻止部分但不是全部转录因子的结合。然后，我们将研究在使用非甲基化和甲基化的MMP-13启动子构建的共转染中，以及在EMSA和CHIP检测中，哪些转录因子实际上与MMP-13的启动子结合。这将使我们能够将功能反应与CpG位点的接近程度和甲基化状态联系起来。前两个目标将由南安普敦的Co-PI进行调查，那里有以前的技术经验。第二个目的是建立在PI在转录调控方面的经验的基础上。南安普顿研究小组先前的发现表明，去甲基化是骨性关节炎中异常表达的基质金属蛋白酶-13的基础。建议的研究将极大地提高我们对DNA甲基化在骨性关节炎病理中的重要性的理解。