ROLE OF RHO PROTEINS IN CHONDROCYTE DIFFERENTIATION

一个MCSDA会让我作为一个兽医和博士，扩大我的软骨生物学知识，并进一步了解软骨细胞代谢，以寻求预防或治疗骨关节炎的方式。我选择的导师是国际公认的细胞内信号传导，并有良好的记录，培训科学家成为独立的研究人员。在这个提案中概述的研究检查Rho亚家族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（Cdc 42、Rac、RhoA）调节肌动蛋白细胞骨架结构，并参与诱导软骨细胞去分化和软骨降解的白细胞介素-1（IL-1）信号传导。胰岛素样生长因子-I（IGF-I）促进分化软骨细胞的表型表达，并可减轻IL-1和肿瘤坏死因子（TNF）的降解作用。因此，分化的软骨细胞表型的稳定性和预防骨关节炎的关键可能在于通过Cdc 42、Rac或RhoA的IGF-I调节的细胞因子信号传导。本研究的主要目的是确定IGF-I在IL-1/TNF诱导的去分化调节中的作用。在第一阶段的培训中，我将获得生物化学和基于分子的方法来研究软骨细胞中Rho蛋白信号通路的经验。目的1将研究IGF-I、IL-1和TNF如何影响Cdc 42、Rac和RhoA的活化和定位。目的2将采用显微注射方法，以确定是否突变（组成型活性或失活）Rho蛋白可以促进形态脱分化。目标3将研究牛痘病毒表达的突变（组成型活性或非活性）Rho蛋白刺激表型去分化的能力。在第二阶段，我将开始发展一个独立的研究计划，这将进一步扩大我的知识，Rho蛋白信号在软骨细胞中的重点是确定参与软骨细胞分化的Rho靶蛋白。我将专注于Rho激酶，RhoA的特异性靶点，和PAK-2（p21激活的激酶），Cdc 42和Rac的特异性靶点。牛痘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确定用于开发骨关节炎药物干预或基因治疗模式的靶分子。这种培训将使我能够追求学术生涯作为一个临床科学家与独特的资格，以扩大基础和临床研究之间的相互作用。

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.