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）：项目摘要：在衰老和骨关节炎（OA）期间，关节软骨表现出对合成代谢生长因子胰岛素样生长因子L（IGF-I）的反应性降低。我们已经表明，IGF-I通过上调GTPase激活蛋白（GAP）活性来抑制软骨细胞中小的G蛋白CDC42（细胞 - 分离周期42）的激活，并且该信号传导机制与软骨细胞表型表达相关。我们还记录了在软骨细胞衰老过程中IGF-I诱导的CDC42活性的调节。该提案的前提是阐明衰老和OA期间IGF〜> cdc42-->表型的信号传导对于理解OA的发病机理至关重要，并可能提供对预防或治疗OA的新方法的见解。该提案的广泛目的是检查IGF-I在衰老和OA期间对软骨细胞中CDC42的调节。该提议的假设是：“将确定IGF-I调节的差距，并且保留对该间隙活性的正常调控对于维持衰老和OA期间正常软骨细胞型至关重要。”在特定目标1中，我们将识别和表征软骨细胞中的差距。我们将使用免疫沉淀，亲和力柱纯化和酵母2杂交方法来吸引与活动CDC42结合的间隙。推定的间隙将使用质谱法测序。对于具有间隙 - 同源域的蛋白质，我们将使用[？-32P] GTP水解测定法确定功能间隙活性，并确定哪些差距受IGF-I调节。在特定的目标2中，我们将通过功能分析的增益和损失来确定差距对软骨基质体内稳态的影响。我们将在年轻和老年软骨细胞中瞬时表达野生型，活性和显性负间隙突变体，然后使用定量PCR使用定量PCR测试软骨分子Aggrecan，II型和基质金属蛋白酶3＆13的mRNA表达。然后，我们将在老化和OA软骨细胞中表达间隙突变体，用IGF-I处理细胞，并测量[35S]硫酸盐掺入蛋白聚糖中，以确定间隙表达是否可以从IGF-I无反应状态中拯救软骨细胞。这项研究与公共荒地的相关性：这些研究将进一步了解软骨细胞中的IGF-I〜> gap--> cdc42信号传导途径，以追求确定IGF-I在衰老关节骨骼中不反应的分子机制。我们预计这些发现将有助于鉴定目标分子以开发OA的治疗方法。