REGULATION OF COLLAGEN IV GENE TRANSCRIPTION BY TGF-BETA

TGF-β 对 IV 型胶原蛋白基因转录的调控

• 批准号: 2144496
• 负责人: JOSEPH PETER GRANDE
• 金额: $ 10.28万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144496
• 关键词: 3T3 cells; DNA footprinting; collagen; genetic enhancer element; genetic promoter element; genetic transcription; glomerulonephritis; kidney cell; laboratory rat; nuclear runoff assay; protein biosynthesis; regulatory gene; renal failure; renal glomerulus; transcription factor; transfection; transforming growth factors

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.

描述（改编自申请人的摘要）：肾脏的进展 疾病与胶原蛋白IV和 其他基质大分子。 调节的体液和细胞因子 正常肾脏和患病的胶原蛋白IV合成尚未 定义很好。 tgf- beta1是一种有效的塞卡特介质，明显 刺激原纤维胶原蛋白（胶原蛋白I和胶原蛋白III）合成 许多细胞系统。 最近的研究表明TGF-BETA1是 在实验性肾小球肾炎期间产生，可能在 胶原蛋白IV期间的肾小球积累 疾病进展。假设TGF-BETA1占主导地位 通过刺激肾脏损伤的肾脏损伤进展 胶原蛋白IV基因的转录。 主要目标 授予应用是确定TGF-BETA1的机制 增加胶原蛋白IV基因的转录。 TGF-BETA1介导 将评估α1（IV）和α2（IV）胶原mRNA的诱导 在源自大鼠肾小球，管状上皮和间质的细胞中。 胶原蛋白IV基因转录将通过核跑分析测量。 由于胶原蛋白IV的两个级别的转录调控，转录本 已经确定了启动和笔录伸长， tgf-beta1在刺激转录引发和伸长率时将 在NIH-3T3细胞中表征，这是一种容易操纵的细胞系 随着α1（IV）和alpha2（iv）的增加而对TGF-BETA1做出响应 胶原mRNA和胶原蛋白IV基因转录。 alpha1（iv）和 Alpha2（IV）胶原蛋白基因具有130个基对双向启动子； Alpha1（IV）基因的第一个内含子中的增强子元件是 胶原蛋白IV基因的组织特异性表达所必需的。这 这些和其他潜在的顺式作用因子在TGF-BETA1中的​​作用 胶原蛋白IV转录的介导的诱导将由 转染嵌合胶原蛋白IV启动子/增强子基因构建体 TGF-BETA1处理的NIH-3T3细胞。 凝胶迁移分析和DNase I足迹研究将用于识别推定的跨性别作用 与关键调节元素相互作用的因素 胶原蛋白IV基因。 阐明顺式和转变因子 在TGF-BETA1介导的胶原蛋白IV基因的诱导中很重要 转录可以为制定更好的策略提供基础 旨在阻止肾脏进展的治疗干预措施 末期肾脏疾病受伤。