REGULATION OF COLLAGEN IV GENE TRANSCRIPTION BY TGF-BETA

描述（改编自申请人的摘要）：肾功能衰竭的进展 疾病与IV型胶原蛋白的异常沉积有关， 其他基质大分子。 体液和细胞因子调节 正常和患病肾脏中的IV型胶原合成尚未被证实。 定义明确。 TGF-β 1是一种有效的瘢痕介质， 刺激纤维胶原蛋白（胶原蛋白I和胶原蛋白III）的合成， 许多细胞系统。 最近的研究表明，TGF-β 1是 在实验性肾小球肾炎中产生，并可能在 肾小球和间质中IV型胶原的积聚 据推测，TGF-β 1在疾病进展中起主导作用， 肾损伤进展中的作用，通过刺激肾功能衰竭 IV型胶原基因的转录。 本项目的主要目标是 研究的目的是确定TGF-β 1 增加IV型胶原基因的转录。TGF-β 1介导 将评估α 1（IV）和α 2（IV）胶原mRNA的诱导 在来自大鼠肾小球、肾小管上皮和肾小球细胞中。 将通过核连续试验测量IV型胶原基因转录。 由于IV型胶原的转录调控有两个水平， 启动和转录延长已经确定，的作用 TGF-β 1在刺激转录起始和延伸中的作用将 在NIH-3 T3细胞中表征，NIH-3 T3细胞是一种易于操作的细胞系， 对TGF-β 1的反应是α 1（IV）和α 2（IV）增加 胶原mRNA和IV型胶原基因转录。 α 1（IV）和 α 2（IV）胶原基因共享130个碱基对的双向启动子; α 1（IV）基因的第一内含子内的增强子元件， 这是IV型胶原基因组织特异性表达所必需的。的 这些和其他潜在顺式作用因子在TGF-β 1中的作用 IV型胶原转录的介导诱导将被定义为 将嵌合IV型胶原启动子/增强子基因构建体转染入 TGF-β 1处理的NIH-3 T3细胞。 凝胶迁移率变动分析和DNA酶 I足迹研究将用于确定假定的反式作用 与细胞内的关键调节元件相互作用的因素 IV型胶原基因。 顺式和反式作用因子的解析 TGF-β 1介导IV型胶原基因诱导中重要作用 转录可以为开发更好的策略提供基础， 用于治疗干预，目的是阻断肾脏疾病的进展， 终末期肾病的损伤。

DESCRIPTION (Adapted from Applicant's Abstract): Progression of renal disease is associated with the abnormal deposition of collagen IV and other matrix macromolecules. Humoral and cellular factors that regulate collagen IV synthesis in the normal and diseased kidney have not been well defined. TGF- beta1 is a potent cicatricial mediator that markedly stimulates fibrillar collagen (collagen I and collagen III) synthesis in many cell systems. Recent studies have demonstrated that TGF-beta1 is produced during experimental glomerulonephritis and may play a role in both glomerular and interstitial accumulation of collagen IV during disease progression.It is hypothesized that TGF-beta1 plays a dominant role in the progression of renal injury to renal failure by stimulating transcription of the collagen IV genes. The major objective of this grant application is to determine the mechanism by which TGF-beta1 increases transcription of the collagen IV genes. TGF-beta1 mediated induction of alpha1(IV) and alpha2(IV) collagen mRNA will be assessed in cell derived from rat glomeruli, tubular epithelium, and interstitium. Collagen IV gene transcription will be measured by nuclear run-on assays. Since two levels of transcriptional regulation of collagen IV, transcript initiation and transcript elongation have been identified, the role of TGF- beta1 in stimulation of transcript initiation and elongation will be characterized in NIH-3T3 cells, a readily manipulable cell line that responds to TGF-beta1 with increases in alpha1(IV) and alpha2(IV) collagen mRNA and collagen IV gene transcription. The alpha1(IV) and alpha2(IV) collagen genes share a 130 base pair bidirectional promoter; an enhancer element within the first intron of the alpha1(IV) gene is necessary for tissue-specific expression of the collagen IV genes. The role of these and other potential cis-acting factors in TGF-beta1 mediated induction of collagen IV transcription will be defined by transfecting chimeric collagen IV promoter/enhancer gene constructs into TGF-beta1 treated NIH-3T3 cells. Gel mobility shift assays and DNAse I footprinting studies will be used to identify putative trans-acting factors that interact with critical regulatory elements within the collagen IV genes. Elucidation of the cis- and trans- acting factors important in TGF-beta1 mediated induction of collagen IV gene transcription may provide the basis for development of better strategies for therapeutic interventions aimed at blocking the progression of renal injury to end stage renal disease.