SIGNAL PATHWAY--INTERSTIT COLLAGE TRANSCRIPT

信号通路--校际拼贴成绩单

• 批准号: 6171333
• 负责人: MATTHEW P VINCENTI
• 金额: $ 9.26万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171333
• 关键词: DNA binding protein; biological signal transduction; collagenase; enzyme activity; enzyme biosynthesis; fibroblasts; gel mobility shift assay; gene expression; genetic promoter element; immunoprecipitation; interleukin 1; interstitial; laboratory rabbit; mitogen activated protein kinase; molecular cloning; pathologic process; polymerase chain reaction; protein kinase; protein protein interaction; rheumatoid arthritis; site directed mutagenesis; southern blotting; synovial fluid; transcription factor; transfection; yeast two hybrid system

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.

间质胶原酶是由滑膜成纤维细胞产生的酶 其有助于类风湿性关节炎的病理生理学。在这 疾病时，滑膜成纤维细胞被炎症信号激活， 作为白细胞介素-1（IL-1），并产生不适当的高水平的 愈伤组织胶原酶，有助于软骨，韧带和 骨头 尽管胶原酶基因激活在RA中的重要性， 导致胶原酶转录增加的信号传导途径 被定义。 我最近进行的研究表明， 通过v-src添加到IL-1是胶原酶的有效激活剂 转录。 此外，IL-1和v-src可以协同作用， 激活胶原酶启动子，表明这些刺激共享 常见的信号中间体。 开始定义路径 导致胶原酶转录，转录因子激活b B v-src并与近端胶原酶启动子相互作用， 表征了 v-src的下游介质，如Map激酶， 这些下游激酶的功能将被确定， 在表达V-src的滑膜成纤维细胞中测定。 之间的协同作用 v-src和IL-1可能是由于src相关激酶， IL-1依赖性途径中的中间体。 探索这个 可能性，显性阴性v-src将作为特异性抑制剂进行检测。 IL-1诱导的胶原酶转录。 或者，转录 由v-src和IL-1激活的因子可以在体内物理相互作用， 细胞核合作招募RNA聚合酶II到 胶原酶启动子。 因此，潜在的蛋白质-蛋白质相互作用 将测试v-src和IL-1诱导的转录因子之间的关系 通过使用酵母双杂交系统。 这种方法也将 用于鉴定信号转导蛋白，如MAP激酶， 与激活这些转录因子相互作用。 这项工作将为 首次描述了特异性蛋白激酶和转录因子 与胶原酶的信号通路相互作用 转录将导致更好地了解分子 结缔组织病的发病机制。