GTPase Activating Proteins in Aging and Osteoarthritis

衰老和骨关节炎中的 GTP 酶激活蛋白

• 批准号: 7096245
• 负责人: LISA A FORTIER
• 金额: $ 19.73万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7096245
• 关键词: G protein; affinity chromatography; aging; biological signal transduction; chondrocytes; chondroitin sulfates; enzyme activity; guanine nucleotide binding protein; guanosinetriphosphatase activating protein; guanosinetriphosphatases; horses; hydrolysis; immunoprecipitation; insulinlike growth factor; mass spectrometry; osteoarthritis; polymerase chain reaction; protein structure function; proteoglycan; yeast two hybrid system

DESCRIPTION (provided by applicant): Project Summary: During aging and in osteoarthritis (OA), articular cartilage exhibits diminished responsiveness to the anabolic growth factor insulin-like growth factor-l (IGF-I). We have shown that IGF-I suppresses activation of the small G-protein Cdc42 (cell-division-cycle 42) in chondrocytes by upregulation of GTPase activating protein (GAP) activity, and that this signaling mechanism is linked to chondrocyte phenotypic expression. We have also documented loss of IGF-I induced regulation of Cdc42 activity during aging in chondrocytes. The premise of this proposal is that elucidating the signaling underlying IGF~>Cdc42- -> phenotype during aging and in OA is critical toward understanding the pathogenesis of OA, and may provide insight into novel approaches for the prevention or treatment of OA. The broad objectives of this proposal are to examine the regulation of Cdc42 in chondrocytes by IGF-I during aging and OA. The hypothesis for this proposal is: "The IGF-I regulated GAP will be identified, and retaining normal regulation of this GAP's activity will be critical for maintenance of a normal chondrocyte phenotype during aging and OA". In Specific Aim 1, we will identify and characterize GAPs in chondrocytes. We will use immunoprecipitation, affinity column purification, and yeast 2-hybrid approaches to attract GAPs which bind to active Cdc42. Putative GAPs will be sequenced using mass spectrometry. For proteins with GAP-homology domains, we will confirm functional GAP activity and determine which GAPs are regulated by IGF-I using [?-32P]GTP hydrolysis assays. In Specific Aim 2, we will determine the impact of GAPs on cartilage matrix homeostasis through gain and loss of function assays. We will transiently express wild-type, active, and dominant negative GAP mutants in young and aged chondrocytes and then test for resulting mRNA expression of the cartilage molecules aggrecan, collagen type II, and matrix metalloproteinases 3 &13 using quantitative PCR. We will then express GAP mutants in aged and OA chondrocytes, treat the cells with IGF-I, and measure [35S]sulfate incorporation into proteoglycans to determine if GAP expression can rescue chondrocytes from an IGF-I unresponsive state. Relevance of this research to Public Heath: These studies will further our understanding of the IGF-I~>GAP- ->Cdc42 signaling pathway in chondrocytes in pursuit of identifying the molecular mechanism of IGF-I unresponsiveness in aging articular cartilage. We anticipate that these findings will facilitate identification of target molecules for development of therapeutics modalities for OA.

描述（由申请人提供）：项目总结：在衰老和骨关节炎（OA）期间，关节软骨对合成代谢生长因子胰岛素样生长因子-I（IGF-I）的反应性降低。我们已经表明，IGF-I抑制激活的小G蛋白Cdc 42（细胞分裂周期42）在软骨细胞通过上调GT3活化蛋白（GAP）的活性，这种信号传导机制与软骨细胞表型表达。我们还记录了软骨细胞老化过程中IGF-I诱导的Cdc 42活性调节的丧失。该建议的前提是阐明衰老过程中和OA中IGF~> Cdc 42- ->表型的潜在信号传导对于理解OA的发病机制至关重要，并可能为预防或治疗OA提供新的方法。本提案的主要目的是研究衰老和OA过程中IGF-1对软骨细胞Cdc 42的调节作用。该提案的假设是：“IGF-I调节的GAP将被鉴定，并且保持该GAP活性的正常调节对于在衰老和OA期间维持正常软骨细胞表型至关重要”。在特定目标1中，我们将识别和表征软骨细胞中的缺口。我们将使用免疫沉淀，亲和柱纯化，酵母双杂交方法来吸引GAP结合到活性Cdc 42。将使用质谱法对推定的GAP进行测序。对于具有GAP同源结构域的蛋白质，我们将确认功能性GAP活性，并确定哪些GAP受IGF-I的调节，使用[？] 32 P]GTP水解测定。在特定目标2中，我们将通过功能获得和丧失测定来确定GAP对软骨基质稳态的影响。我们将在年轻和老年软骨细胞中瞬时表达野生型、活性和显性负性GAP突变体，然后使用定量PCR测试软骨分子聚集蛋白聚糖、II型胶原和基质金属蛋白酶3和13的mRNA表达。然后，我们将在老化和OA软骨细胞中表达GAP突变体，用IGF-I处理细胞，并测量[35 S]硫酸盐掺入蛋白聚糖以确定GAP表达是否可以将软骨细胞从IGF-I无反应状态中拯救出来。本研究与公共卫生的相关性：这些研究将进一步加深我们对软骨细胞中IGF-I~>GAP- -> Cdc 42信号通路的理解，以确定老化关节软骨中IGF-I无反应性的分子机制。我们预计，这些发现将有助于确定目标分子的治疗方法的OA的发展。