Molecular Mechanisms of Mesangial Sclerosis

描述（由申请人提供）：层粘连蛋白（LN）亚型的时空表达对肾小球发育至关重要，并有助于糖尿病肾病和其他进行性肾脏疾病中细胞功能的改变。然而，很少有人知道的机制，调节LN亚型表达。我们提出胰岛素样生长因子结合蛋白-5（IGFBP-5）导致形成基于细丝蛋白的核穿梭体，其结合转录因子（例如sox 9、GKLF、Sp1、Smad）和转录共激活因子（例如，IGFBP-5和LIM结构域蛋白如FHL 2）并调节肾小球系膜细胞（MC）中层粘连蛋白（LN）同种型表达。这一假说提出了一种新的机制，即在细胞-基质界面的扰动调节基因表达。具体目的1：表征用IGFBP-5处理MC后细丝蛋白核穿梭的产生和组成。首先，我们将证明细丝蛋白片段的形成和易位到细胞核。其次，我们将描述被招募到复合物中的转录因子;最后，我们将评估转录共激活因子LIM结构域蛋白FHL 2和IGFBP-5在复合物中的作用。具体目的2：证明细丝蛋白的核积累是IGFBP-5介导的LN基因表达变化所必需的。首先，我们将证明细丝蛋白是必需的IGFBP-5介导的LN基因表达的影响。其次，我们将表明，细丝蛋白穿梭招募LN基因位点，它特异性地修改LN基因转录。具体目标3：确定细丝蛋白核穿梭激活LN基因转录的机制。首先，我们将确定FLN是否直接与LN启动子相互作用，以及它是否驱动转录。第二，我们将定义穿梭中转录控制所需的元件。这些研究将确定基于细丝蛋白的核穿梭在介导IGFBP-5对层粘连蛋白基因表达的影响中的作用。了解LN亚型表达的转录调控将为LN在正常肾小球发生中的作用以及在肾小球疾病中的紊乱提供新的见解。

DESCRIPTION (provided by applicant): Temporal and spatial expression of laminin (LN) isoforms is critical to glomerular development and contributes to altered cell function in diabetic nephropathy and other progressive renal diseases. Yet, little is known about the mechanisms that regulate LN isoform expression. We propose that insulin-like growth factor binding protein-5 (IGFBP-5) leads to formation of a filamin-based nuclear shuttle that binds transcription factors (e.g. sox9, GKLF, Sp1, Smad) and transcriptional co-activators (e.g., IGFBP-5 and LIM domain proteins such as FHL2) and regulates laminin (LN) isoform expression in mesangial cells (MC). This hypothesis suggests a novel mechanism whereby perturbations in the cell-matrix interface regulate gene expression. This hypothesis will be examined by: Specific Aim 1: To characterize the generation and composition of the filamin nuclear shuttle following treatment of MC with IGFBP-5. First, we will demonstrate that a fragment of filamin forms and translocates to the nucleus. Second, we will characterize the transcription factors that are recruited to the complex; and finally, we will evaluate the role of transcriptional co-activators, LIM domain protein FHL2 and IGFBP-5, in the complex. Specific Aim 2: To demonstrate that nuclear accumulation of filamin is required for IGFBP-5-mediated changes in LN gene expression. First, we will demonstrate that filamin is required for IGFBP-5-mediated effects on LN gene expression. Second, we will show that the filamin shuttle is recruited to LN gene loci where it specifically modifies LN gene transcription. Specific Aim 3: To define the mechanisms whereby the filamin nuclear shuttle activates LN gene transcription. First, we will determine if FLN interacts directly with the LN promoter and if it drives transcription. Second, we will define the elements in the shuttle that are required for transcriptional control. These studies will define the role of a filamin-based nuclear shuttle in mediating the effects of IGFBP-5 on laminin gene expression. Understanding transcriptional control of LN isoform expression will add new insights into the role that LN plays in normal glomerulogenesis and that becomes disordered in glomerular disease.