Regulation of chondrocytes by extracellular matrix protein.

描述（由申请人提供）：F-反应蛋白是一个蛋白家族的成员，该蛋白家族共同属于TSR（血小板反应蛋白）I型分子亚组。我们已经发现，F-spondin的表达显着增加，在骨关节炎软骨以及啮齿类动物骨关节炎模型。初步研究表明，F-spondin对人软骨细胞代谢有显著影响，并且还在胚胎生长板的肥大区域中表达，在该区域中，F-spondin起调节矿化和软骨内骨形成的作用。该提案旨在表征F-脊椎蛋白的功能作用，其对OA的进展具有重大且先前未被认识的影响。我们将测试两个中心假设：1）F-spondin通过未识别的途径调节胶原降解，包括TGF-β的激活和MMP的诱导，2）F-spondin诱导关节软骨细胞的肥大分化，并在矿化和软骨内骨形成的调节中发挥重要作用。具体目的旨在测试这些假设如下：在特定目的1中，我们将研究F-spondin对体外培养模型中人关节软骨细胞肥大和矿化活性的调节的影响。将对软骨标本进行组织学检查，以研究F-脊椎蛋白与OA软骨细胞的其他特征性肥大/骨化标志物之间的联系。在特定目的2中，我们将研究OA软骨中F-脊椎蛋白介导的胶原降解的分子机制。外植体或细胞培养物将用于a）鉴定由F-脊椎蛋白诱导的MMP并检查它们在F-脊椎蛋白介导的胶原降解中的作用B）确立TGF-β在调节F-脊椎蛋白功能中的作用和c）比较全长F-脊椎蛋白分子相对于其蛋白水解片段的功能活性。在特定目的3中，我们将鉴定和表征F-脊椎蛋白的相互作用蛋白。a）研究F-脊椎蛋白与潜伏性TGF-β复合物的潜伏相关肽（LTP）的相互作用，和B）利用酵母2杂交和蛋白质组学技术鉴定可调节其在关节软骨中的活性的新型F-脊椎蛋白结合蛋白（蛋白酶、受体、基质分子）。在SPECIFIC AIM 4中，我们将研究体内软骨中F-spondin的表达和功能。我们将研究F-脊椎蛋白在体内软骨中的表达，i）在软骨内骨发育期间，ii）在OA的小鼠骨切除模型中。我们将产生一个F-spondin基因敲除小鼠，并表征软骨内骨发育和OA疾病进展过程中软骨表型的变化。了解F-spondin对软骨细胞功能的调节可能会导致软骨修复和骨关节炎疾病改善治疗的新策略。公共卫生相关性：骨关节炎影响超过20%的60岁以上的人口，并且对于数百万人来说是残疾的重要原因。这项研究提案将集中在一种蛋白质，F-spondin，以前从未在软骨中描述过，它调节在骨关节炎关节恶化中重要的过程。拟议的实验旨在确定以前未被识别的病理事件，这些事件导致骨关节炎的新治疗方法，从而防止疾病进展，降低残疾的可能性并改善公众健康。

DESCRIPTION (provided by applicant): F-spondin is a member of a family of proteins that collectively belong to a subgroup of TSR (thrombospondin) type I class molecules. We have discovered that F-spondin expression is significantly increased in osteoarthritic cartilage as well as in rodent meniscectomy models of OA. Preliminary studies indicate that F-spondin has significant effects on human chondrocyte metabolism and is also expressed in the hypertrophic regions of embryonic growth plates where it acts to regulate mineralization and endochondral bone formation. This proposal is designed to characterize the functional effects of F-spondin, which have major and previously unrecognized implications for the progression of OA. We will test two central hypotheses: 1) F-spondin modulates collagen degradation via unrecognized pathways that include activation of TGF-¿ and induction of MMPs and 2) F-spondin induces hypertrophic differentiation of articular chondrocytes and plays an essential role in the regulation of mineralization and endochondral bone formation. The specific aims designed to test these hypotheses are as follows: In SPECIFIC AIM 1 we will investigate the effects of F-spondin on the regulation of hypertrophy and mineralizing activity in human articular chondrocytes in vitro culture models. Cartilage specimens will be examined histologically to investigate the link between F-spondin and other characteristic hypertrophic/ossification markers of OA chondrocytes. In SPECIFIC AIM 2 we will investigate the molecular mechanism(s) of F-spondin-mediated collagen degradation in OA cartilage. Explant or cell cultures will be used to a) identify MMPs induced by F- spondin and examine their role in F-spondin-mediated collagen degradation b) establish the role of TGF-¿ in modulation of F-spondin functions and c) compare functional activity of the full length F-spondin molecule relative to its proteolytic fragments. In SPECIFIC AIM 3 we will identify and characterize the interacting proteins of F-spondin. a) investigate the interaction of F-spondin with the Latency-associated peptide (LAP) of the latent TGF-¿ complex and b) utilize yeast 2 hybrid and proteomic technologies to identify novel F-spondin binding proteins (proteases, receptors, matrix molecules) that may regulate its activity in articular cartilage. In SPECIFIC AIM 4 we will investigate the expression and function of F-spondin in cartilage in vivo. We will investigate F-spondin expression in cartilage in vivo i) during endochondral bone development and ii) in the mouse meniscectomy model of OA. We will generate an F-spondin knockout mouse and characterize the changes in cartilage phenotype during endochondral bone development and OA disease progression. Understanding the regulation of chondrocyte functions by F-spondin could lead to novel strategies for cartilage repair and disease modifying treatments for osteoarthritis. PUBLIC HEALTH RELEVANCE: Osteoarthritis affects more than 20% of the population over age 60 and for millions of people is a significant cause of disability. This research proposal will focus on a protein, F-spondin, never before described in cartilage, which regulates processes that are important in joint deterioration in osteoarthritis. The proposed experiments are designed to identify previously unrecognized pathological events that lead to new treatments for osteoarthritis, which prevent the progression of disease, reduce the likelihood of disability and improve the public health.