LAMININ GENE EXPRESSION IN GLOMERULAR CELLS

描述（由申请人提供）：肾小球硬化症是进行性肾病最严重的后遗症。肾小球硬化症是由蛋白质的异常积累引起的，其中一些蛋白质通常构成肾小球基底膜（GBM）和系膜基质（MM）。层粘连蛋白是GBM和MM的组分之一，其在肾小球疾病进展期间在肾小球内积聚。许多生长因子，包括转化生长因子-β（TGF-β），激活肾小球细胞，导致层粘连蛋白的异常积累。尽管层粘连蛋白在肾小球硬化的发生发展中起关键作用，但其调控层粘连蛋白链表达的机制还不完全清楚。用TGF-β和其他生长因子处理肾小球细胞可增加层粘连蛋白γ 1链的mRNA水平。人和啮齿动物层粘连蛋白γ 1链（LAMC 1）基因启动子含有由转录因子uE 3（TFE 3）激活的关键bcn-1元件。在肾小球系膜细胞中，通过LAMC 1启动子的Smad结合元件（SBE）和TGF-β信号通路，Smad蛋白极大地增强了TFE 3介导的bcn-1元件的激活。通过TFE 3和Smad蛋白的协同作用，LAMC 1启动子的bcn-1元件依赖性激活提供了对TGF-β如何介导这些细胞中内源性LAMC 1基因激活的深入了解。转录辅激活因子CREB结合蛋白（CBP）的表达增加了系膜细胞中LAMC 1启动子的活性。目前的建议的目标是确定负责TFE 3和Smad依赖性激活LAMC 1基因转录的分子机制，该转录由TGF-β在肾小球细胞中诱导。为探讨这些机制，提出了以下具体目标。我们将确定CBP和其他共激活因子在介导TFE 3-Smad依赖性TGF-β诱导的LAMC 1基因表达中的作用。我们将确定与TFE 3蛋白相互作用的基础转录机制的组成部分，并确定它们在介导TGF-β诱导的基因表达中的作用。我们将确定负责TFE 3-Smad 3协同激活LAMC 1基因的分子机制，以响应TGF-β。这些研究将提供深入了解TGF-β用于指导肾小球细胞中TFE 3和Smad依赖性LAMC 1基因转录的转录机制。这些研究的结果预期对理解TGF-β触发细胞外基质成分编码基因和肾小球细胞中表达的其他基因的转录的基本机制具有广泛的影响。

DESCRIPTION (provided by applicant): Glomerulosclerosis is the most serious sequela of progressive renal disease. Glomerulosclerosis results from an abnormal accumulation of proteins, some of which, that normally make up the glomerular basement membrane (GBM) and mesangial matrix (MM). Laminin is one of the components of GBM and MM that accumulates within glomeruli during the progression of glomerular disease. A number of growth factors, including transforming growth factor-beta (TGF-beta), activate glomerular cells resulting in abnormal accumulation of laminin. Although laminin is known to play a key role in the development of glomerulosclerosis, the mechanisms that regulate expression of laminin chains are incompletely understood. Treatment of glomerular cells with TGF-beta and other growth factors increases mRNA levels of laminin gamma1 chain. The human and rodent laminin gamma1 chain (LAMC1) gene promoters contain the critical bcn-1 element that is activated by transcription factor uE3 (TFE3). In glomerular mesangial cells, the TFE3-mediated activation of the bcn-1 element is greatly augmented by Smad proteins, acting through the LAMC1 promoter's Smad binding elements (SBE), and by the TGF-beta-signaling pathways. The bcn-1-element-dependent activation of the LAMC1 promoter by the synergistic action of TFE3 and Smad proteins provides insight into how TGF-beta mediates activation of the endogenous LAMC1 gene in these cells. Expression of the transcriptional co-activator, CREB binding protein (CBP), increased the activity of the LAMC1 promoter in mesangial cells. The goal of the current proposal is to define the molecular mechanisms responsible for the TFE3- and Smad-dependent activation of LAMC 1 gene transcription that is induced by TGF-beta in glomerular cells. The following specific aims are proposed to explore these mechanisms. We will define the role of CBP and other co-activators in mediating the TFE3-Smad-dependent TGF-beta-induced LAMC1 gene expression. We will identify components of the basal transcriptional machinery that interact with TFE3 protein and define their role in mediating TGF-beta-induced gene expression. We will define the molecular mechanisms responsible for the TFE3-Smad3 synergistic activation of the LAMC1 gene in response to TGF-beta. These studies will provide insight into transcriptional mechanisms utilized by TGF-beta to direct TFE3- and Smad-dependent LAMC1 gene transcription in glomerular cells. The results of these studies are anticipated to have a broad impact on understanding the basic mechanisms by which TGF-beta triggers transcription of genes encoding components of extracellular matrix and of other genes expressed in glomerular cells.