The PINCH-ILK-parvin complexes in glomerular cells

描述（由申请人提供）：这个竞争性延续申请的长期目标是阐明肾小球细胞控制细胞外基质沉积、粘附、细胞骨架和凋亡的分子机制。整合素连接激酶（ILK）是细胞-基质粘连的细胞质成分，与进行性肾小球衰竭有关。我们在之前资助期的研究表明，ILK与PINCH-1和α -parvin形成复合物。这种三元蛋白复合物似乎是调节肾小球细胞行为的细胞-基质粘附的细胞机制的关键部分。拟开展的研究主要集中在肾小球细胞中该蛋白复合物的上游调控物、下游效应物和体内功能三个方面。目的1是研究该复合物在系膜细胞和足细胞中的调控机制。我们最近的研究发现tgf - β是该复合物的上游调节因子，但其机制尚不清楚。我们将实时分析这种复合物在活肾小球细胞中的调节，并研究tgf - β 1调节这种复合物的机制。目的2是表征pinchi -1- ilk - α -parvin复合物调节系膜纤维连接蛋白基质沉积、足细胞-基质粘附、细胞骨架和存活的下游效应物。我们和其他人最近的研究已经确定了几个候选者。我们将结合遗传、分子和细胞方法来确定它们在肾小球细胞中pinch -1- ilk - α -parvin复合物介导的过程中的作用。目的3是确定PINCH-1在足细胞体内的功能。根据我们和其他人最近的研究，我们假设体内PINCH-1的缺失会损害足细胞细胞骨架调节、基质组织、粘附和生存信号。我们将通过在小鼠足细胞中选择性灭活PINCH-1基因来验证这一假设。此外，我们将生成有条件永生化的PINCH-1flox/flox足细胞，并利用它们进一步研究PINCH-1在足细胞中的作用机制。肾小球损害是肾衰竭的主要原因。因此，所提出的研究不仅将促进我们对控制肾小球细胞行为的分子机制的理解，而且还可能导致新的治疗方法来缓解进行性肾小球衰竭。

DESCRIPTION (provided by applicant): The long term goal of this competing continuation application is to elucidate the molecular mechanisms by which glomerular cells control extracellular matrix deposition, adhesion, cytoskeleton and apoptosis. Integrin-linked kinase (ILK) is a cytoplasmic component of the cell-matrix adhesions implicated in progressive glomerular failure. Our studies during the previous funding period have demonstrated that ILK forms a complex with PINCH-1 and alpha-parvin. This ternary protein complex appears to function as a key part of the cellular machinery at cell-matrix adhesions that regulates glomerular cell behavior. The proposed studies focus on three aspects of this protein complex in glomerular cells, namely its upstream regulators, downstream effectors, and in vivo functions. Aim 1 is to investigate the mechanism by which the complex is regulated in mesangial cells and podocytes. Our recent studies identify TGF-beta1 as an upstream regulator of the complex but the mechanisms are not known. We will analyze in real time the regulation of this complex in live glomerular cells, and to investigate the mechanisms whereby TGF-beta1 regulates this complex. Aim 2 is to characterize the downstream effectors through which the PINCH-1-ILK-alpha-parvin complex regulates mesangial fibronectin matrix deposition, podocyte-matrix adhesion, cytoskeleton and survival. Recent studies by us and others have identified several candidates. We will use a combination of genetic, molecular and cellular approaches to define their roles in the PINCH-1-ILK-alpha-parvin complex-mediated processes in glomerular cells. Aim 3 is to determine the functions of PINCH-1 in podocytes in vivo. Based on recent studies by us and others, we hypothesize that loss of PINCH-1 in vivo will compromise podocyte cytoskeletal regulation, matrix organization, adhesion, and survival signaling. We will test this hypothesis by selectively inactivating the PINCH-1 gene in mouse podocytes in vivo. Furthermore, we will generate conditionally immortalized PINCH-1flox/flox podocytes, and use them to further investigate the mechanism by which PINCH-1 functions in podocytes. Glomerular damage is a main reason of renal failure. The proposed studies, therefore, not only will advance our understanding of the molecular mechanisms that govern glomerular cell behavior but also may lead to novel therapeutic approaches that alleviate progressive glomerular failure.