Clinical and experimental data suggest that two contributors to progressive glomerular disease are transforming growth factor (TGF)-beta expression and glomerular hypertension. Our preliminary data indicate that TGF- beta rapidly stimulates rearrangement of the mesangial cell cytoskeleton, and that cytoskeletal signaling plays a role in TGF- beta1-induced mesangial cell type I collagen expression. We believe from these observations that mesangial stretch resulting from intraglomerular hypertension mimics the physical events following TGF-beta activation of mesangial cells. Thus, one way in which intraglomerular hypertension could accelerate glomerulosclerosis is by amplifying TGF-beta-stimulated signals for extracellular matrix accumulation. However, the mechanisms linking the physical and biochemical cellular events in glomerulosclerosis are not well understood. One potential link between these physical and chemical signals is the Rho-family GTPases (RGTPases). This exploratory R21 proposal is based on the overall hypothesis that RGTPase activity, either independently or through actions on cytoskeletal rearrangement, contributes to TGF-beta-stimulated collagen expression.
Two Specific Aims are proposed. First, we will determine the pathways by which RGTPases are activated by TGF-beta1 in mesangial cells. We will perform assays for activity of the RGTPases RhoA, Rac1 and Cdc42 and then determine whether TGF-beta activates these RGTPases through signaling pathways that we previously have found to be induced by TGF-beta1 in mesangial cells: phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), focal adhesion kinase (FAK) and Smad. In the second aim, we will determine whether RGTPases affect TGF-beta1-stimulated collagen I gene expression solely through cytoskeletal rearrangement, or also have effects on signaling that are independent of cytoskeletal rearrangement. We will characterize the changes in mesangial cell shape and focal adhesions induced by TGF-beta1, and determine whether intracellular rigidity or tension is required for the collagen response. Using micropatterned surfaces to limit TGF-beta-stimulated focal adhesion formation, we will determine whether collagen expression is decreased by restricting cytoskeletal rearrangement. Conversely, we will increase cytoskeletal tension by stretching the cells and determine whether this is sufficient to support TGF-beta1-stimulated collagen expression when RGTPase activity is blocked. These studies will determine whether increased cytoskeletal tension is required for RGTPase effects on collagen expression.
