REGULATION OF TYPE IV COLLAGEN GENE AND TRANSFORMING GROWTH FACTOR BETA

细胞外基质的不可逆和不可阻挡的积累是 以各种获得性和遗传性肾病为特征， 导致固定的器官功能障碍或衰竭。 的最新证据 几个实验室表明，TGF-β，一种多效性瘢痕， 在肾小球疾病的发病机制中起着重要的作用 疤痕（硬化）。 alpha1（I）和alpha1（IV）mRNA是急性 在肾小球外和肾小球隔室早期增加， 兔肾小球肾炎新月体模型的病程。 与此同时，TGF-β mRNA和活性 蛋白 抗TGF-β抗体中和了这种活性， 被其他人证明可以阻止或改善 免疫性系膜损伤模型中的肾小球硬化。 因此，TGF- β可能在肾小球疾病的发展中起重要作用。 硬化和间质纤维化。 虽然TGF-β的作用 间质胶原的合成已被充分研究， 已知TGF-β对基底细胞代谢的作用， 聚集在肾小球基质中的膜成分。 IV型胶原，肾小球基底的主要结构成分 膜，是由至少5个基因不同的 多肽。 alpha1（IV）和alpha2（IV）链通过以下方法合成： 体外培养肾小球细胞。 TGF-β增加IV型胶原的生物合成 不依赖于细胞增殖，这是由于增加了 alpha1（IV）和alpha2（IV）基因，导致细胞增殖增加3 - 5倍， IV型胶原mRNA的稳态水平。 TGF-β的作用机制 IV型胶原基因转录的诱导变化是未知的。 胶原 IV基因的转录依赖于两种独立的机制，转录 引发和延伸。 α 1（IV）和α 2（IV）基因是 由130 bp双向启动子分隔， 在嵌入结构中的增强子序列的调控下， 基因. 转录本衰减的机制尚不清楚。 以来 TGF-β增加已经启动的细胞中的IV型胶原mRNA 在接近最大水平的转录，它被假定为改变 转录物延伸效率（衰减）。 为了测试这种 假设，提出以下研究：1）胶原IV 转录物起始和延伸将通过核连续（run-on）来评估 静止期和TGF-β刺激的小鼠肾小球中的转录 系膜细胞 将绘制转录本衰减位点 使用α 1（IV）基因的片段; 2）顺式作用的功能 假定减弱转录的序列将被 通过转染嵌合基因构建体在静止和静止细胞中进行评估， TGF-β刺激的细胞; 3）核蛋白， 与顺式作用序列的相互作用将在粗品中鉴定 静止和TGF-β刺激的细胞的提取物及其结合 位点表征; 4）独特的DNA结合蛋白将被表征 通过从表达文库中克隆cDNA。 这些研究将提供 深入了解TGF-β对IV型胶原蛋白作用的分子机制 基因转录和细胞外基质异常积累 肾脏疾病 有了这些信息，治疗策略可能 改变肾脏疾病的进程。

The irreversible and inexorable accumulation of extracellular matrix is characteristic of a variety of acquired and hereditary renal disorders, leading to fixed organ dysfunction or failure. Recent evidence from several laboratories suggests that TGF-beta, a pleiotropic cicatricial mediator, plays an important role in the pathogenesis of glomerular scarring (sclerosis). alpha1(I) and alpha1(IV) mRNA are acutely increased in the extraglomerular and glomerular compartments early in the course of a crescentic model of glomerulonephritis in the rabbit. Concomitant with this change, was expression of TGF-beta mRNA and active protein. Anti-TGF-beta antibodies neutralized this activity and have been shown by others to block or ameliorate the development of glomerular sclerosis in a model of immune mesangial injury. Thus, TGF- beta likely plays an important role in the development of glomerular sclerosis and interstitial fibrosis. While the action of TGF-beta on synthesis of interstitial collagens has been well studied, little is known about the action of TGF-beta on the metabolism of basement membrane components which accumulate in sclerotic glomerular matrices. Collagen IV, the major structural component of glomerular basement membranes, is assembled from at least 5 genetically distinct polypeptides. The alpha1(IV) and alpha2(IV) chains are synthesized by glomerular cells in vitro. TGF-beta increases collagen IV biosynthesis independent of cell proliferation due to increased transcription of the alpha1(IV) and alpha2(IV) genes leading to a 3-5 fold increase in the steady-state level of collagen IV mRNA. The mechanism of TGF-beta- induced changes in collagen IV gene transcription are unknown. Collagen IV gene transcription depends on two independent mechanisms, transcript initiation and elongation. The alpha1(IV) and alpha2(IV) genes are separated by a 130 bp bidirectional promoter which initiates transcripts under the regulation of enhancer sequences embedded in the structural genes. The mechanism of transcript attenuation is not known. Since TGF-beta increases collagen IV mRNA in cells already initiating transcripts at a near maximal level, it is postulated to alter transcript elongation efficiency (attenuation). In order to test this hypothesis, the following studies are proposed: 1) Collagen IV transcript initiation and elongation will be assessed by nuclear run-on transcription in quiescent and TGF-beta stimulated murine glomerular mesangial cells. The site(s) of transcript attenuation will be mapped using fragments of the alpha1(IV) gene; 2) The function of cis-acting sequences which are postulated to attenuate transcription will be assessed by transfection of chimeric gene constructs in quiescent and TGF-beta stimulated cells; 3) Nuclear proteins which specifically interact with the cis-acting sequence will be identified in crude extracts of quiescent and TGF-beta stimulated cells and their binding site characterized; 4) Unique DNA-binding proteins will be characterized by cDNA cloning from an expression library. These studies will provide insights into the molecular mechanism of TGF-beta action on collagen IV gene transcription and aberrant extracellular matrix accumulation in renal diseases. With this information therapeutic strategies may be developed to alter the course of renal disease.