Mechanisms by which matrix stiffness regulates Rho

描述（由申请人提供）：在细胞-基质粘附领域，一个新兴的概念是，与已经研究了几年的“经典”2D构型相比，细胞对三维构型的基质的反应不同。随着我们对细胞-基质相互作用的理解的增加，越来越明显的是，细胞不仅识别基质组成和3D构型，而且识别周围细胞外基质环境的顺应性与刚度。我们是最早证明乳腺上皮细胞通过Rho及其效应物ROCK收缩顺应性基质的人之一，并且这种收缩是体外乳腺导管形态发生所必需的（Wozniak等人，2003）。重要的是，基质的刚度反馈调节Rho活性，但这种机械信号传导发生的机制尚不清楚。此外，尽管人们对Rho在对ECM的许多反应中的作用了解很多，但令人惊讶的是，人们对哪些Rho GEF和GAP可能与机械信号有关知之甚少。我们假设Rho是细胞感知其环境的硬度并对该硬度做出反应的中心管道。更具体地说，我们假设Rho的调节发生在基质硬度条件下由于特定GEF的激活，以及基质顺应性条件下特定GAP的失活。因此，本提案的目标是研究这些分子如何发挥作用来调节Rho以响应基质刚度：目的1：确定P190 RhoGAP-B响应顺应性基质的Rho调节机制。我们发现，p190 RhoGAP-B，而不是-A，调解Rho下调和导管形态发生在一个兼容的矩阵。我们将检验p190 RhoGAP-B是侵袭抑制因子，并且它通过与p120连环蛋白和Rac 1结合来调节Rho的假设。目的2：识别和研究特定的Rho GEF在激活Rho以响应刚性基质中的作用。将确定GEF-H1和p115 RhoGEF对Rho调节的贡献。除了这种候选方法之外，还将对所有RhoGEF进行siRNA筛选，以鉴定响应基质硬度调节Rho的其他GEF。将评估通过这些方法鉴定的RhoGEF在将Rho与下游效应联系起来方面的作用：细胞收缩性、基质重塑和肿瘤侵袭。目的3：确定细丝蛋白通过Rho调节收缩性的机制，并在体外和体内侵入3D基质。细丝蛋白增加细胞收缩性和基质重塑，并调节细胞对刚性基质的反应。我们将确定细丝蛋白是否在体外和体内增强侵袭，并研究细丝蛋白与其伴侣Trio，ROCK或PAK的结合是否介导其对Rho和收缩性的调节。

DESCRIPTION (provided by applicant): In the field of cell-matrix adhesion, an emerging concept is that cells respond differently to matrices that are configured in three-dimensions, compared to the "classic" 2D configuration that has been studied for several years. As our understanding of cell-matrix interactions increases, it is becoming apparent that cells recognize not only the matrix composition and the 3D configuration, but also the compliance vs. stiffness of the surrounding extracellular matrix environment. We were among the first to demonstrate that breast epithelial cells contract compliant matrices through Rho and its effector, ROCK, and that this contraction is necessary for mammary ductal morphogenesis in vitro (Wozniak et al, 2003). Importantly, the stiffness of the matrix feeds back to regulate Rho activity, but the mechanism by which this mechano-signaling occurs is unknown. Moreover, although much is known about the role of Rho in a host of responses to the ECM, surprisingly little is known about which Rho GEFs and GAPs might be linked to mechanical signals. We hypothesize that Rho is the central conduit by which cells sense the stiffness of their environment, and respond to that stiffness. More specifically, we hypothesize that regulation of Rho occurs by activation due to specific GEFs under conditions of matrix stiffness, and the inactivation by specific GAPs under conditions of matrix compliance. Therefore, the goal of this proposal is to investigate how these molecules function to regulate Rho in response to matrix stiffness: Aim 1: Determine mechanisms of Rho regulation by p190RhoGAP-B in response to compliant matrices. We find that p190RhoGAP-B, but not-A, mediates Rho down-regulation and ductal morphogenesis in a compliant matrix. We will test the hypothesis that p190RhoGAP-B is a suppressor of invasion, and that it regulates Rho via binding to p120 Catenin and Rac 1. Aim 2: Identify and investigate the role of specific Rho GEFs in activating Rho in response to stiff matrices. The contribution of GEF-H1 and p115RhoGEF to regulation of Rho will be determined. In addition to this candidate approach, an siRNA screen of all RhoGEFs will be performed to identify additional GEFs that regulate Rho in response to matrix stiffness. RhoGEFs identified by these means will be assessed for their role in linking Rho to downstream effects: cellular contractility, matrix remodeling, and tumor invasion. Aim 3: Determine mechanisms by which filamin regulates contractility through Rho and invasion into 3D matrices in vitro and in vivo. Filamin increases cellular contractility and matrix remodeling, and tunes cellular response to a stiff matrix. We will determine whether filamin enhances invasion in vitro and in vivo, and investigate whether binding of filamin to its partners Trio, ROCK, or PAK mediates its regulation of Rho and contractility.