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.
Cartilage is constantly subjected to mechanical forces including compression, tension, and shear. These forces are converted into cellular responses that activate genes required for main- tenance of healthy cartilage. At the gene level, the DNA elements that confer sensitivity to me- chanical forces are unknown. It is important to identify these elements, since inappropriate cel- lular response to mechanical force contributes to disease both during growth and in aging. COMP is an important component of cartilage, and the COMP gene is activated by mechanical forces. We report preliminary evidence that the DNA element responsible for mechanical acti- vation of the COMP gene lies somewhere within the first 3000 base-pairs of its promoter. In this proposal we will systematically dissect the 3000bp COMP promoter to identify the specific DNA element. This will provide new insight into how healthy cartilage is maintained, and lays the foundation for more effective approaches to engineering cartilage tissue and halting cartilage degradation in arthritis.
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