Molecular Analysis of Extracellular Matrix Assembly

描述(由申请人提供)：细胞外基质(ECM)组装是发育、组织动态平衡、衰老、伤口愈合以及疾病开始和发展的重要组成部分。细胞外基质的物理和机械性质通过其对细胞组织和信号的影响而成为这些过程的关键调节因子。在发育过程中，以及在癌症、纤维化和结缔组织异常等病理过程中，ECM的组成和物理状态可能会有很大的变化。为了开发预防或逆转异常ECM影响的治疗方法，我们需要了解组装适合组织的基质的分子相互作用，以及这些相互作用在疾病中是如何受到影响的。这项建议的总体目标是确定细胞周围基质的组装如何支持和指导其他ECM成分的后续组装，以构建适合组织的最终基质。纤维连接蛋白(FN)是一种普遍存在的细胞外基质蛋白，在细胞表面受体和其他细胞外基质成分之间起着重要的连接作用。FN通过细胞介导的过程组装成纤维状ECM，该过程涉及FN构象变化以促进FN-FN相互作用，并最终形成最终的FN基质，该基质可以通过洗涤剂脱氧胆酸盐(DOC)中的不溶性来识别。了解适合组织的ECM组装的一个关键问题是顺应性的影响，因为基质组装发生在硬度显著不同的组织中。在目标1中，我们将测试这一假设，即细胞周围ECM的硬度通过调节组装过程中的一个或多个关键步骤来调控FN基质组装。我们将分析使用定义弹性模数的聚丙烯酰胺凝胶底物组装的主要步骤(整合素结合、FN构象变化和DOC不溶性)，并将使用构象敏感抗体检验在不同刚性条件下组装的基质中FN构象不同的想法。不溶的FN基质形成了组装其他ECM蛋白的基础，包括一些胶原蛋白，但我们对其结构的了解有限。在目标2中，我们将检验这样一个假设，即特定的Fn-Fn相互作用介导了最终的DOC不溶基质组装的最后步骤。突变的重组FN将被用来识别重要的相互作用残基，一种新的质谱屏将被应用于识别参与DOC不溶性相互作用的其他FN结构域。为了了解FN组装、组织硬度和其他ECM蛋白的整合之间的相互作用，在目标3中，我们将在体外软骨形成的组织分化模型中测试FN基质控制细胞命运和胶原沉积的假设。我们将调控不溶性FN基质的水平及其硬度，并确定在骨髓间充质干细胞向软骨分化过程中对分化标记物和胶原组装的影响。通过拟议的研究获得的信息将为正常基质组装的机制提供新的见解，并将提出疾病改变组装导致异常ECM聚集的方法，如纤维化、疤痕形成和癌症。

DESCRIPTION (provided by applicant): Extracellular matrix (ECM) assembly is an essential part of development, tissue homeostasis, aging, wound healing, and initiation and progression of disease. The physical and mechanical properties of the ECM are emerging as critical regulators of these processes through their effects on cell organization and signaling. During development and in pathologies such as cancer, fibrosis, and connective tissue abnormalities, the composition and the physical state of the ECM can vary dramatically. To develop treatments to prevent or reverse the effects of abnormal ECM, we need to understand the molecular interactions that assemble a tissue-appropriate matrix and how these are affected in disease. The overall goal of this proposal is to determine how the assembly of the pericellular matrix supports and directs the subsequent assembly of additional ECM components to build a tissue-appropriate definitive matrix. Fibronectin (FN) is a ubiquitous ECM protein that makes an essential connection between cell surface receptors and other ECM components. FN is assembled into a fibrillar ECM via a cell-mediated process that involves FN conformational changes to promote FN-FN interactions and culminates with the definitive FN matrix that can be identified by insolubility in the detergent deoxycholate (DOC). A critical question for understanding tissue-appropriate ECM assembly is the effect of compliance since matrix assembly occurs in tissues that differ significantly in stiffness. In Aim 1 we will test the hypothesis that stiffness of the pericellular ECM governs FN matrix assembly by regulating one or more key steps in the assembly process. We will analyze the major steps of assembly (integrin binding, FN conformational changes, and DOC insolubility) using polyacrylamide gel substrates of defined elastic modulus and will test the idea that FN conformation differs in matrices assembled under different stiffness conditions using a conformation-sensitive antibody. The insoluble FN matrix forms the foundation for assembly of other ECM proteins, including some collagens, yet we have limited understanding of its structure. In Aim 2, we will test the hypothesis that specific FN-FN interactions mediate the final step in assembly of the definitive DOC- insoluble matrix. Mutant recombinant FNs will be used to identify important interacting residues and a novel mass spectrometry screen will be applied to identify other FN domains involved in DOC-insoluble interactions. To understand the interplay between FN assembly, tissue stiffness, and incorporation of other ECM proteins, in Aim 3 we will test the hypothesis that FN matrix controls both cell fate and deposition of collagens in a tissue differentiation model, in vitro chondrogenesis. We will manipulate the level of insoluble FN matrix and its stiffness and determine the effects on differentiation markers and assembly of collagens during chondrogenic differentiation of mesenchymal stem cells. The information obtained through the proposed studies will provide novel insights into the mechanisms of normal matrix assembly and will suggest ways in which disease can change assembly to cause abnormal ECM accumulation as in fibrosis, scar formation, and cancer.