Identification of Mechanoresponsive Promoter Elements in Chondrogenesis

描述（由申请人提供）：软骨经常受到包括压缩、拉伸和剪切在内的机械力的作用。这些作用力转化为生理反应，对软骨基质组成、细胞存活和祖细胞分化至关重要。细胞对机械力的反应差异很大，范围从增殖到分化，到细胞凋亡死亡，从基质合成到酶促基质降解。为了软骨组织的适当维护，必须调用适当的细胞反应。不适当的细胞对机械负荷的反应有助于在生长和衰老过程中的病理结果。更清楚地了解机械转导途径在许多临床应用中具有巨大的潜力。机械转导事件的传统研究主要集中在细胞膜相关蛋白和细胞质信号转导蛋白上。这些方法已经成功地鉴定了整合素、黏附复合物、细胞骨架以及作为机械转导因子的激酶途径。然而，我们仍然没有确定基因启动子中赋予基因机械反应性的DNA元件。软骨机械转导途径的靶基因包括聚集蛋白、II型胶原和软骨寡聚基质蛋白（COMP）等细胞外基质基因。COMP在软骨中胶原原纤维的正常组装中起作用。COMP在软骨内成骨过程中由软骨细胞表达，并在成熟软骨的软骨细胞中继续表达。重要的是，在软骨细胞和骨髓干细胞中，COMP转录对机械刺激高度敏感。我们的初步数据表明，人类COMP启动子的近3kb足以在机械刺激下激活COMP转录。在这项研究中，我们将在3kb的人类COMP启动子中鉴定机械反应性DNA元件。我们期望发现介导细胞对机械力反应的新途径，我们预计COMP机械反应途径将在其他基因中保守。这个项目是我们长期战略的一个组成部分，以深入了解机械转导途径，提高我们的组织工程和软骨修复能力。直接源于这项工作的未来目标包括1)确定机械反应元件的小分子抑制剂/激活剂，2)确定其他机械反应基因和途径，以及3)允许非破坏性测量机械转导活性的体内模型。

DESCRIPTION (provided by applicant): Cartilage is constantly subjected to mechanical forces including compression, tension, and shear. These forces are converted into physiological responses that are critical to cartilage ma trix composition, cellular survival, and progenitor cell differentiation. Cellular responses to me- chanical forces vary greatly, and can range from proliferation, to differentiation, to apoptotic cell death, and from matrix synthesis to enzymatic matrix degradation. For the proper maintenance of cartilage tissue, the appropriate cellular response must be invoked. Inappropriate cellular re- sponse to mechanical loading contributes to pathological outcomes during growth and in aging. A clearer understanding of the mechanotransduction pathway has tremendous potential in many clinical applications. Traditional studies of mechanotransduction events have focused on cell membrane-associated proteins and cytosolic signal transduction proteins. These approaches have successfully identi- fied integrins, focal adhesion complexes, the cytoskeleton, and select kinase pathways as me- diators of mechanotransduction. However, we have still not identified the DNA elements in a gene promoter that confer the mechanoresponsiveness to the gene. Target genes of mechanotransduction pathways in cartilage include extracellular matrix genes such as Aggrecan and type II collagen and Cartilage Oligomeric Matrix Protein (COMP). COMP functions in the proper assembly of collagen fibrils in cartilage. COMP is expressed by chondro- cytes in endochondral bone formation, and continues to be expressed chondrocytes of mature cartilage. Importantly, COMP transcription is highly sensitive to mechanical stimulation both in chondrocytes and bone marrow stem cells. Our preliminary data demonstrates that the proximal 3kb of the human COMP promoter is suffi- cient to activate COMP transcription in response to mechanical stimulation. In this study we will identify the mechanoresponsive DNA element within the 3kb human COMP promoter. We ex- pect to identify new pathways that mediate the cellular response to mechanical forces, and we anticipate that the COMP mechanoresponse pathways will be conserved in other genes. This project is an integral part of our long-term strategy to gain insight into mechanotransduction pathways and improve our tissue engineering and cartilage repair abilities. Future goals directly stemming from this work include 1) identification of small molecule inhibitors/activators of the mechanoresponse element, 2) identification of additional mechanoresponsive genes and path- ways, and 3) an in vivo model that allows non-destructive measurement of mechanotransduc- tion activity.