TGF-beta and Cytoskeletal Signaling in Mesangial Cell

描述（由申请人提供）：临床和实验数据表明，进行性肾小球疾病的两个促成因素是转化生长因子（TGF）-β表达和肾小球高血压。我们的初步数据表明，TGF-β迅速刺激系膜细胞骨架的重排，细胞骨架信号在TGF-β 1诱导的系膜细胞I型胶原蛋白表达中起作用。从这些观察结果中，我们认为肾小球内高血压引起的系膜牵张模拟了TGF-β激活系膜细胞后的物理事件。因此，肾小球内高血压可加速肾小球硬化的一种方式是通过放大细胞外基质积累的TGF-β刺激信号。然而，在肾小球硬化症的物理和生化细胞事件的机制还没有很好地理解。这些物理和化学信号之间的一个潜在联系是Rho家族GTP酶（RGTPases）。这个探索性的R21建议是基于这样的总体假设，即RGT 3活性，独立地或通过对细胞骨架重排的作用，有助于TGF-β刺激的胶原蛋白表达。提出了两个具体目标。首先，我们将确定TGF-β 1在系膜细胞中激活RGTPases的途径。我们将对RGTPases RhoA、Rac 1和Cdc 42的活性进行测定，然后确定TGF-β是否通过我们先前发现的TGF-β 1在系膜细胞中诱导的信号通路激活这些RGTPases：磷脂酰肌醇-3-激酶（PI 3 K）、蛋白激酶C（PKC）、粘着斑激酶（FAK）和Smad。在第二个目标中，我们将确定是否RGTPases影响TGF-β 1刺激的胶原蛋白I基因的表达仅通过细胞骨架重排，或也有信号的影响，是独立的细胞骨架重排。我们将描述TGF-β 1诱导的系膜细胞形状和粘着斑的变化，并确定胶原反应是否需要细胞内的刚性或张力。使用微图案化的表面来限制TGF-β刺激的粘着斑形成，我们将确定是否通过限制细胞骨架重排来降低胶原蛋白表达。相反，我们将通过拉伸细胞来增加细胞骨架张力，并确定当RGT 3活性被阻断时，这是否足以支持TGF-β 1刺激的胶原蛋白表达。这些研究将确定RGT 4对胶原蛋白表达的影响是否需要增加细胞骨架张力。

DESCRIPTION (provided by applicant): 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.