Mechanisms by which matrix stiffness regulates Rho

描述(申请人提供)：在细胞-基质黏附领域，一个新兴的概念是细胞对三维配置的基质的反应不同于已经研究了几年的经典的二维配置。随着我们对细胞-基质相互作用的了解的加深，细胞不仅识别基质的组成和3D构型，而且还识别周围细胞外基质环境的顺应性和硬度。我们是最早证明乳腺上皮细胞通过Rho及其效应器ROCK收缩顺应性基质的人之一，并且这种收缩对于体外乳腺导管的形态形成是必要的(Wozniak等，2003)。重要的是，基质的刚性反馈调节Rho的活动，但这种机械信号发生的机制尚不清楚。此外，尽管关于Rho在ECM的一系列反应中的作用已知很多，但令人惊讶的是，关于哪些Rho GEF和间隙可能与机械信号有关，人们知之甚少。我们假设Rho是细胞感知其环境的僵硬并对其作出反应的中心管道。更具体地说，我们假设在基质刚性条件下，Rho的调节是通过特定GEF的激活来实现的，而在基质顺应性的条件下，Rho的调节是通过特定间隙的失活来实现的。因此，本方案的目的是研究这些分子如何调节Rho对基质刚性的反应：目的1：确定p190RhoGAP-B对顺应性基质的调节机制。我们发现p190RhoGAP-B，而不是-A，在顺应性基质中介导Rho下调和导管形态发生。我们将验证p190RhoGAP-B是侵袭抑制因子的假设，以及它通过与p120连环蛋白和Rac 1结合来调节Rho的假设。目的2：鉴定和研究特定的Rho GEF在激活Rho对刚性基质的反应中的作用。将确定全环基金-H1和p115Rho全环基金对调节RHO的贡献。除了这一候选方法外，还将对所有RhoGEF进行siRNA筛选，以确定其他调节Rho的GEF对基质刚性的反应。通过这些方法确定的RhoGEF将被评估它们在将Rho与下游效应联系起来的作用：细胞收缩、基质重塑和肿瘤侵袭。目的3：确定丝素通过Rho和侵袭3D基质在体内外调节收缩的机制。丝素可增加细胞的收缩能力和基质重塑，并调节细胞对僵硬基质的反应。我们将确定细丝在体外和体内是否增强侵袭，并研究细丝与其伙伴Trio、ROCK或PAK的结合是否介导其对Rho和收缩的调节。 公共卫生相关性：了解细胞外基质的物理特性在癌症进展中所起的作用具有重大的健康相关性，因为乳房密度导致癌症风险增加4-6倍。这些实验旨在了解致密的细胞外基质调节乳腺细胞行为的潜在分子机制，并可能为未来的治疗目标提供建议。

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. PUBLIC HEALTH RELEVANCE: Understanding the role the physical properties of the extracellular matrix plays in cancer progression is of great health relevance as breast density accounts for a 4-6 fold increase in carcinoma risk. These experiments are designed to understand the underlying molecular mechanisms by which the dense extracellular matrix regulates breast cell behavior and could suggest future targets for therapy.