REGULATION OF COLLAGEN IV GENE TRANSCRIPTION BY TGF-BETA

描述(摘自申请者摘要)：肾脏进展 疾病与IV型胶原的异常沉积和 其他基质大分子。调节体液和细胞因子 正常和疾病肾脏中IV型胶原的合成尚未得到 定义得很清楚。转化生长因子-β1是一种有效的瘢痕介导剂，显著 促进纤维状胶原(I型和III型)的合成 许多细胞系统。最近的研究表明，转化生长因子-β1是 在实验性肾小球肾炎期间产生，并可能在 IV型胶原在肾小球和间质中的积聚 疾病进展。假设转化生长因子-β1起主导作用 刺激在肾损伤进展为肾功能衰竭中的作用 IV型胶原基因的转录。这项工作的主要目标是 GRANT的应用是确定转化生长因子-β1的作用机制 增加IV型胶原基因的转录。转化生长因子-β1介导的 将评估α1(IV)和α2(IV)胶原mRNA的诱导 在来自大鼠肾小球、肾小管上皮和间质的细胞中。 IV型胶原基因的转录将通过核连续分析来测量。 由于IV型胶原的转录调控有两个水平，转录本 已经确定了启动和转录延长，其作用是 转化生长因子-β1在刺激转录起始和延长意志中的作用 以NIH-3T3细胞为特征，这是一种易于操作的细胞系 对转化生长因子-β1的应答：α1(IV)和α2(IV)升高 胶原mRNA和IV型胶原基因转录。字母1(IV)和 α2(IV)胶原基因共有130个碱基对的双向启动子； 甲型1(IV)基因第一内含子内的增强子元件是 是组织特异性表达IV型胶原基因所必需的。这个 这些和其他潜在的顺式作用因子在转化生长因子-β1中的作用 介导的IV型胶原转录的诱导将被定义为 IV型胶原启动子/增强子嵌合基因载体的构建 转化生长因子-β1作用于NIH-3T3细胞。凝胶迁移率变化分析与DNA酶 I足迹研究将用于确定推定的反式作用 与内部关键监管要素相互作用的因素 IV型胶原基因。顺式和反式作用因子的阐明 在转化生长因子-β1诱导IV型胶原基因表达中的重要作用 转录可能为制定更好的策略提供基础 用于旨在阻止肾脏进展的治疗干预 损伤至终末期肾病。

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.