GTPase Activating Proteins in Aging and Osteoarthritis

描述(申请人提供)：项目摘要：在衰老和骨关节炎(OA)中，关节软骨对合成生长因子胰岛素样生长因子-L(IGF-I)的反应减弱。我们发现IGF-I通过上调GTP酶激活蛋白(GAP)的活性来抑制软骨细胞中小G蛋白CDC42(细胞分裂周期42)的激活，并且这种信号机制与软骨细胞的表型表达有关。我们还记录了在软骨细胞老化过程中IGF-I的丢失诱导了对CDC42活性的调节。这一建议的前提是，阐明IGF~&gt；cdc42--&gt；在衰老和OA中的表型是理解OA发病机制的关键，并可能为预防或治疗OA提供新的方法。这项提案的主要目标是研究IGF-I在衰老和骨关节炎过程中对软骨细胞中CDC42的调节。这一提议的假设是：“IGF-I调节的GAP将被识别，保持对该GAP活性的正常调节对于在衰老和骨关节炎期间维持正常的软骨细胞表型至关重要。”在特定的目标1中，我们将识别和表征软骨细胞的间隙。我们将使用免疫沉淀、亲和柱纯化和酵母2-杂交方法来吸引与活性Cdc42结合的间隙。假定的空隙将使用质谱仪进行测序。对于具有GAP同源结构域的蛋白质，我们将确认功能GAP活性，并使用[？-32P]GTP水解法确定哪些GAP受IGF-I调控。在具体目标2中，我们将通过功能获得和丧失分析来确定间隙对软骨基质动态平衡的影响。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，然后用定量PCR检测由此产生的软骨分子aggrecan、II型胶原和基质金属蛋白酶3和13的mRNA表达。然后，我们将在衰老和骨关节炎软骨细胞中表达GAP突变体，用IGF-I处理细胞，并测量蛋白多糖中[35S]硫酸盐的掺入，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。这项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-&GT；CDC42信号通路的理解，以寻求确定IGF-I在关节软骨老化中无反应的分子机制。我们预计，这些发现将有助于识别目标分子，以开发治疗OA的方法。

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.