Identification of Mechanoresponsive Promoter Elements in Chondrogenesis

描述(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。这些力量被转化为对软骨基质组成、细胞存活和祖细胞分化至关重要的生理反应。细胞对机械力的反应差异很大，从增殖到分化，从细胞凋亡到细胞死亡，从基质合成到酶基质降解。为了适当地维持软骨组织，必须启动适当的细胞反应。在生长和衰老过程中，细胞对机械负荷的不适当反应会导致病理结果。更清楚地了解机械转导通路在许多临床应用中具有巨大的潜力。传统的机械信号转导研究主要集中在细胞膜相关蛋白和胞浆信号转导蛋白上。这些方法已经成功地识别了整合素、粘着斑复合体、细胞骨架和选择的激酶通路作为机械转导的介体。然而，我们仍然没有确定基因启动子中的DNA元件，这些元件赋予了对基因的机械反应。软骨力学转导的靶基因包括细胞外基质基因，如聚集素、II型胶原和软骨低聚基质蛋白(COMP)。COMP在软骨中胶原纤维的正确组装中起作用。COMP在软骨内成骨过程中由软骨细胞表达，在成熟软骨中继续表达。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启动子近端3kb足以激活COMP转录，以响应机械刺激。在这项研究中，我们将确定3kb的人COMP启动子中的机械响应DNA元件。我们期望找到新的途径来调节细胞对机械力的反应，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，目的是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。这项工作直接产生的未来目标包括：1)识别机械反应元件的小分子抑制物/激活剂，2)识别额外的机械反应基因和途径，以及3)允许非破坏性测量机械传递活性的体内模型。

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.