ROLE OF RHO PROTEINS IN CHONDROCYTE DIFFERENTIATION

作为一名兽医和博士，MCSDA将使我能够扩展我的软骨生物学知识，并在寻找骨关节炎的预防或治疗方法时进一步了解软骨细胞的新陈代谢。我选择的导师在细胞内信号方面得到了国际认可，并在培训科学家成为独立研究人员方面有着良好的记录。这项研究概述了Rho亚家族GTP结合蛋白信号在软骨细胞分化中的作用。Rho结合蛋白(CDC42、Rac、RhoA)调节肌动蛋白的细胞骨架结构，并参与白细胞介素1(IL-1)信号转导，诱导软骨细胞去分化和软骨降解。胰岛素样生长因子-I(IGF-I)促进分化的软骨细胞表型表达，并可减轻IL-1和肿瘤坏死因子(TNF)的降解作用。因此，维持分化的软骨细胞表型，以及预防骨关节炎的关键，可能在于IGF-I通过CDC42、RAC或RhoA调节细胞因子信号。这项研究的主要目的是确定IGF-I在调节IL-1/TNF诱导的去分化中所起的作用。在第一阶段的培训中，我将获得生化和分子方法方面的经验，以研究软骨细胞中的Rho蛋白信号通路。目标1将研究IGF-I、IL-1和肿瘤坏死因子如何影响CDC42、RAC和RhoA的激活和定位。目的2将使用显微注射方法来确定突变的(结构性活性或非活性)Rho蛋白是否可以促进形态去分化。目标3将研究痘苗病毒表达、突变(成分活性或非活性)的Rho蛋白刺激表型去分化的能力。在第二阶段，我将开始开发一项独立的研究计划，进一步扩大我对软骨细胞中Rho蛋白信号转导的了解，重点是识别参与软骨细胞分化的Rho靶蛋白。我将重点介绍Rho激酶，RhoA的特异性靶标，以及PAK-2(p21激活的激酶)，CDC42和RAC的特异性靶标。痘苗病毒系统将被用来表达Rho激酶和PAK-2的组成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。人们的期望是，这些研究将为软骨细胞分化的调控提供重要的见解，而最终目标是利用所获得的知识来识别靶分子，以开发治疗骨关节炎的药物干预或基因治疗方法。这一培训将使我能够从事临床科学家的学术生涯，具有扩大基础研究和临床研究之间互动的独特资格。

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.