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，进展性肾小球疾病的两个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快速刺激系膜细胞骨架的重排，细胞骨架信号在TGF- β诱导的系膜细胞I型胶原表达中发挥作用。我们认为，从这些观察结果来看，肾小球内高血压引起的肾小球系膜拉伸模拟了肾小球系膜细胞tgf - β活化后的物理事件。因此，肾小球内高血压加速肾小球硬化的一种方式是通过放大tgf - β刺激的细胞外基质积累信号。然而，连接肾小球硬化的物理和生化细胞事件的机制尚不清楚。这些物理和化学信号之间的一个潜在联系是rho家族GTPases （RGTPases）。这个探索性的R21建议是基于RGTPase活性的整体假设，无论是独立的还是通过对细胞骨架重排的作用，有助于tgf - β刺激的胶原表达。提出了两个具体目标。首先，我们将确定系膜细胞中tgf - β 1激活rgtpase的途径。我们将检测rgtpase RhoA， Rac1和Cdc42的活性，然后确定tgf - β是否通过我们之前发现的系膜细胞中由tgf - β诱导的信号通路激活这些rgtpase：磷脂酰肌醇-3激酶（PI3K），蛋白激酶C (PKC)，局灶粘附激酶（FAK）和Smad。在第二个目标中，我们将确定rgtpase是否仅通过细胞骨架重排影响tgf - β 1刺激的I型胶原基因表达，还是也对独立于细胞骨架重排的信号传导产生影响。我们将描述tgf - β 1诱导的系膜细胞形状和局灶粘连的变化，并确定胶原反应是否需要细胞内刚性或张力。使用微图案表面来限制tgf - β刺激的局灶粘连形成，我们将确定是否通过限制细胞骨架重排来减少胶原表达。相反，我们将通过拉伸细胞来增加细胞骨架张力，并确定当RGTPase活性被阻断时，这是否足以支持tgf - β 1刺激的胶原表达。这些研究将确定RGTPase对胶原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.