Interstitial collagenase is an enzyme produced by synovial fibroblasts which contributes to the pathophysiology of rheumatoid arthritis. In this disease, synovial fibroblasts are activated by inflammatory signals, such as interleukin-1 (IL-1 and produce inappropriately high levels of callagenase, contributing to the degradation of cartilage, ligament and bone. Despite the importance of collagenase gene activation in RA, the signaling pathways leading to increased collagenase transcrption have not been defined. Recent studies I have conducted demonstrate that in addition to IL-1 by v-src is a potent activator of collagenase transcription. Furthermore, IL-1 and v-src can work synergistically to activate the collagenase promoter, suggesting these stimuli share common signaling intermediates. To begin to define the pathway (s) leading to collagenase transcription, the transcription factors activated b v-src and interact with the proximal collagenase promoter will be characterized. Downstream mediators of v-src, such as Map kinases will be identified and the function of these downstream kinases will be assayed in synovial fibroblasts expressing v-src. The synergism between v-src and IL-1 may be due to a src-related kinase which is an intermediate in the the IL-1 dependent pathway. To explore this possibility, a dominant negative v-src willbe tested as a specific inhibito of IL-1 induced collagenase transcription. Alternatively, transcription factors which are activated by v-src and IL-1 may physically interact in the nucleus to cooperatively recruit RNA polymerase II to the collagenase promoter. Thus, potential protein-protein interactions between v-src and IL-1 induced transcription factors will be tested through use of the yeast two hybrid system. This approach will also be used to identify signal transducing proteins, such as Map kinases which interact with activate these transcription factors. This work will, for th first time, descrive the specific protein kinases and transcription factor which interact the signaling pathways involved in collagenase transcription will lead to a better understanding of the molecular mechanisms of connective tissue disease.
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