ROLE OF RHO PROTEINS IN CHONDROCYTE DIFFERENTIATION

RHO 蛋白在软骨细胞分化中的作用

• 批准号: 6497165
• 负责人: LISA A FORTIER
• 金额: $ 11.52万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497165
• 关键词: biological signal transduction; cell differentiation; cell growth regulation; chondrocytes; gene expression; guanine nucleotide binding protein; horses; immunofluorescence technique; insulinlike growth factor; interleukin 1; microinjections; osteoarthritis; protein localization; protein structure function; tumor necrosis factor alpha; vaccinia virus

A MCSDA would allow me as a veterinary surgeon and Ph.D., to expand my knowledge of cartilage biology and further understand chondrocyte metabolism in the search of preventive or treatment modalities for osteoarthritis. The mentor I have chosen is internationally recognized in intracellular signaling and has a well documented record of training scientists to become independent researchers. The studies outlined in this proposal examine the role of Rho-subfamily GTP-binding protein signaling in chondrocyte differentiation. Rho-binding proteins (Cdc42, Rac, RhoA) regulate actin cytoskeletal architecture and are involved in interleukin-1 (IL-1) signaling which induces chondrocyte dedifferentiation and cartilage degradation. Insulin-like growth factor-I (IGF-I) promotes the phenotypic expression of differentiated chondrocytes and can alleviate the degradative effects of IL-1 and tumor necrosis factor (TNF). Thus, the maintainance of a differentiated chondrocytes phenotype, and the key to prevention of osteoarthritis, may lie in IGF- I-regulated cytokine signaling through Cdc42, Rac, or RhoA. The broad objective of this study is to define the role IGF-I plays in the regulation of IL-1/TNF-induced dedifferentiation. During Phase I training, I will gain experience in biochemical and molecular-based approaches to study Rho-protein signaling pathways in chondrocytes. Aim 1 will examine how IGF-I, IL-1 and TNF influence the activation and localization of Cdc42, Rac, and RhoA. Aim 2 will employ microinjection methods to determine if mutated (constitutively active or inactive) Rho-proteins can prompt morphologic dedifferentiation. And Aim 3 will investigate the ability of vaccinia virus-expressed, mutated (constitutively active or inactive) Rho-proteins to incite phenotypic dedifferentiation. In Phase II, I will begin to develop an independent research program which will further extend my knowledge of Rho-proteins signaling in chondrocytes with emphasis on identifying Rho-target proteins involved in chondrocyte differentiation. I will focus on Rho kinase, a specific target for RhoA, and PAK-2 (p21-activated kinase), a specific target for Cdc42 and Rac. The vaccinia virus system will be used to express constitutively active or kinase-dead mutants of Rho kinase an PAK-2 to determine the effects of these kinases on chondrocyte differentiation. The expectation is that these studies will provide important insights into the regulation of chondrocyte differentiation while the ultimate goal is to use the knowledge gained to identify target molecules for the development of drug intervention or gene therapy modalities for osteoarthritis. This training would allow me to pursue an academic career as a clinician scientist with the unique qualifications to expand the interactions between basic and clinical research.

MCSDA可以让我作为兽医和博士学位，扩大我对软骨生物学的了解，并进一步了解软骨细胞代谢，以寻求预防性或治疗骨关节炎的治疗方式。我选择的导师在细胞内信号传导中得到了国际认可，并具有培训科学家的良好记录，以成为独立研究人员。该提案中概述的研究研究了Rho-Subfimily GTP结合蛋白信号传导在软骨细胞分化中的作用。 Rho结合蛋白（CDC42，RAC，RHOA）调节肌动蛋白细胞骨架结构，并参与白介素-1（IL-1）信号传导，从而诱导软骨细胞分裂和软骨降解。胰岛素样生长因子I（IGF-I）促进分化软骨细胞的表型表达，并可以减轻IL-1和肿瘤坏死因子（TNF）的降解作用。因此，通过CDC42，RAC或RhoA的IGF I调节的细胞因子信号传导，可以在分化的软骨细胞表型的维持以及预防骨关节炎的关键。这项研究的广泛目的是定义IGF-I在调节IL-1/TNF诱导的去分化的调节中所起的作用。在I期训练中，我将获得基于生化和分子的方法来研究软骨细胞中的Rho蛋白信号通路的经验。 AIM 1将检查IGF-I，IL-1和TNF如何影响Cdc42，rac和Rhoa的激活和定位。 AIM 2将采用显微注射方法来确定突变（组成性活性或无活性）Rho蛋白会促使形态学去分化。 AIM 3将研究疫苗病毒表达，突变（组成性活性或无活性）Rho蛋白促进表型去分化的能力。在第二阶段，我将开始制定一个独立的研究计划，该计划将进一步扩展我对软骨细胞中Rho蛋白信号传导的了解，重点是鉴定参与软骨细胞分化的Rho-Target蛋白。我将重点介绍Rho激酶，Rho激酶是RhoA的特定靶标，PAK-2（P21激活激酶）是CDC42和RAC的特定靶标。离子病毒系统将用于表达Rho激酶和PAK-2的组成型活性或激酶死亡突变体，以确定这些激酶对软骨细胞分化的影响。期望这些研究将为调节软骨细胞分化的调节提供重要的见解，而最终目标是利用所获得的知识来鉴定靶分子来开发药物干预或基因治疗方式来骨关节炎。这项培训将使我能够从事临床科学家的学术职业，并拥有独特的资格，以扩大基础研究和临床研究之间的相互作用。