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.
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