Molecular Mechanisms of Mesangial Sclerosis

描述(申请人提供)：层粘连蛋白(LN)异构体的时间和空间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细胞功能的改变。然而，对调控LN亚型表达的机制知之甚少。我们认为，胰岛素样生长因子结合蛋白-5(IGFBP-5)可导致在系膜细胞(MC)中形成以丝胺为基础的核穿梭，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)亚型的表达。这一假说提出了一种新的机制，即细胞-基质界面的扰动调节基因的表达。这一假设将通过以下具体目标1来检验：用IGFBP-5处理MC后，表征丝胺核航天飞机的生成和组成。首先，我们将证明一段细丝蛋白形成并移位到细胞核。其次，我们将鉴定被招募到该复合体中的转录因子；最后，我们将评估转录共激活因子LIM结构域蛋白FHL2和IGFBP-5在该复合体中的作用。具体目的2：证明IGFBP-5介导的LN基因表达变化需要丝素的核积累。首先，我们将证明丝氨酸是IGFBP-5介导的对LN基因表达的影响所必需的。其次，我们将证明丝胺穿梭被招募到LN基因位点，在那里它特异性地修改LN基因的转录。具体目的3：明确丝素核穿梭激活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，我们将定义航天飞机中转录控制所需的元件。这些研究将确定以丝胺为基础的核航天飞机在介导IGFBP-5对层粘连蛋白基因表达的影响中所起的作用。了解LN亚型表达的转录调控将增加对LN在正常肾小球形成中所起的作用以及在肾小球疾病中变得紊乱的新的见解。

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.