The PINCH-ILK-parvin complexes in glomerular cells

描述(申请人提供)：这项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、黏附、细胞骨架和细胞凋亡的分子机制。整合素连接的激酶(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。我们在上一次资助期间的研究表明，ILK与Pinch-1和α-Parvin形成了一个复合体。这种三元蛋白质复合体似乎是调节肾小球细胞行为的细胞-基质粘连的细胞机制的关键部分。建议的研究集中在肾小球细胞中这种蛋白质复合体的三个方面，即其上游调节因子、下游效应因子和体内功能。目的1是研究该复合体在系膜细胞和足细胞中的调控机制。我们最近的研究发现，转化生长因子-β1是该复合体的上游调节因子，但其机制尚不清楚。我们将实时分析该复合体在活肾小球细胞中的调节，并探讨转化生长因子-β1调节该复合体的机制。目的2研究Pinch-1-ILK-α-Parvin复合体调节系膜纤维连接蛋白基质沉积、足细胞-基质黏附、细胞骨架和存活的下游效应。我们和其他人最近的研究确定了几个候选者。我们将使用遗传、分子和细胞方法的组合来确定它们在肾小球细胞中Pinch-1-ILK-α-Parvin复合体介导的过程中的作用。目的3确定PINCH-1在体内足细胞中的功能。根据我们和其他人最近的研究，我们假设在体内丢失Pinch-1将损害足细胞的细胞骨架调节、基质组织、黏附和生存信号。我们将通过在体内选择性地灭活小鼠足细胞中的Pinch-1基因来检验这一假设。此外，我们将产生条件永生化的PINCH-1/FLOX足细胞，并用它们进一步研究PINCH-1在足细胞中的作用机制。肾小球损害是肾功能衰竭的主要原因之一。因此，拟议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，而且还可能导致缓解进行性肾小球衰竭的新的治疗方法。

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.