Molecular Mechanisms of Mesangial Sclerosis

描述（由申请人提供）：层粘连蛋白（LN）异构体的时空表达对肾小球的发育至关重要，并有助于改变糖尿病肾病和其他进行性肾脏疾病的细胞功能。然而，人们对调控LN异构体表达的机制知之甚少。我们提出胰岛素样生长因子结合蛋白-5 （IGFBP-5）导致基于丝蛋白的核梭形成，该核梭结合转录因子（如sox9， GKLF, Sp1, Smad）和转录共激活因子（如IGFBP-5和LIM结构域蛋白如FHL2）并调节层粘连蛋白（LN）异构体在系膜细胞（MC）中的表达。这一假设提出了一种新的机制，即细胞-基质界面的扰动调节基因表达。具体目的1：描述IGFBP-5治疗MC后丝蛋白核穿梭的产生和组成。首先，我们将证明丝蛋白片段形成并易位到细胞核。其次，我们将描述被招募到复合体的转录因子；最后，我们将评估转录共激活因子，LIM结构域蛋白FHL2和IGFBP-5在复合体中的作用。特异性目的2：证明igfbp -5介导的LN基因表达变化需要丝蛋白的核积累。首先，我们将证明丝蛋白是igfbp -5介导的LN基因表达效应所必需的。其次，我们将证明丝蛋白穿梭被招募到LN基因位点，在那里它特异性地修饰LN基因的转录。特异性目的3：明确丝蛋白核穿梭激活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。其次，我们将定义梭体中转录控制所需的元素。这些研究将确定基于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异构体表达的转录控制将为LN在正常肾小球发生和肾小球疾病中紊乱所起的作用提供新的见解。

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.