ACTIVATION OF CHONDROCYTE MATURATION IN OSTEOARTHRITIS

描述（由申请人提供）：骨关节炎（OA）是一种广泛且日益流行的疾病，影响数千万患者的生活质量。来自人类OA和几种动物OA模型研究的证据表明，在OA进展期间，软骨细胞表型成熟的重演发生类似于胚胎软骨内骨化。在之前的资助期间，我们确定了许多表型成熟特征，这些特征在OA软骨中表达，包括PTHrP，MMP 9，MMP 13，Ihh和BMP 6。关节软骨细胞在培养中对诱导这种成熟级联反应极其顽固，并且对生长因子相对无反应。然而，用氮杂胞苷治疗，它允许抑制基因的去甲基化，诱导软骨细胞成熟的正常级联反应，导致X型胶原蛋白和其他肥大标志物的表达。对所涉及的特定信号传导途径的研究表明，Smad泛素化连接酶Smurf 2是将信号优势从TGF-β转移到BMP Smad途径的潜在关键调节步骤，导致软骨细胞成熟。发现Smurf 2在人OA中表达，但在正常关节软骨中不表达，并且在培养物中由氮杂胞苷处理诱导。Smurf 2刺激或过表达导致Smad 2和Smad 3降解、TGF-β信号转导下调和BMP信号转导上调。这足以启动培养中软骨细胞成熟和肥大的整个级联反应。具体目标（1）：研究Smurfs在体外调节软骨细胞表型中的作用将利用培养的关节和生长板软骨细胞来研究Smurf 1和2对Smad信号传导和软骨细胞表型的影响。还将研究相关生长因子和细胞因子对Smurf表达的调节。具体目标（二）：研究Smurf 2在体内软骨细胞成熟和OA中的作用将使用鸡肢芽系统来分析Smurf 2在发育过程中对软骨发生和成熟的影响。将创建软骨靶向Smurf 2过表达的转基因鼠模型，并将利用体内软骨细胞的病毒转导来确定Smurf 2过表达是否可以诱导或其抑制抑制OA。最后，将检查Smurf 1和Smurf 2在人正常和OA软骨中的表达。这些研究应提供蓝精灵在调节正常和OA软骨表型的功能的全面图片。

DESCRIPTION (provided by applicant): Osteoarthritis (OA) is a widespread and increasingly prevalent disorder impairing quality of life for tens of millions of patients. Evidence from study of human OA and several animal OA models, suggests that during OA progression a recapitulation of chondrocyte phenotypic maturation occurs resembling embryonic endochondral ossification. In the prior funding period we identified a number of phenotypic maturational characteristics, which are expressed in OA cartilage, including PTHrP, MMP9, MMP13, Ihh, and BMP6. Articular chondrocytes are extremely refractory in culture to induction of this maturational cascade and are relatively unresponsive to growth factors. However, treatment with azacytidine, which allows demethylation of suppressed genes, induces the normal cascade of chondrocyte maturation, resulting in expression of type X collagen and other hypertrophic markers. Study of the specific signaling pathways involved have implicated a Smad ubiquitinating ligase, Smurf2, as a potential key regulatory step in shifting signal dominance from the TGF-beta to the BMP Smad pathway, leading to chondrocyte maturation. Smurf2 was found to be expressed in human OA but not normal articular cartilage, and is induced by azacytidine treatment in culture. Smurf2 stimulation or over-expression leads to Smad2 and Smad3 degradation, down-regulation of TGF-beta signaling, and up-regulation of BMP signaling. This is sufficient to initiate the entire cascade of chondrocyte maturation and hypertrophy in culture. Specific Aim (1): An investigation of the role of Smurfs in regulation of chondrocyte phenotype in vitro will utilize articular and growth plate chondrocytes in culture to study Smurf1 and 2 effects on Smad signaling and chondrocyte phenotype. The regulation of Smurf expression by relevant growth factors and cytokines will also be studied. Specific Aim (2): Investigation of the role of Smurf2 in chondrocyte maturation and OA in vivo will use a chick limb bud system to analyze effects of Smurf2 on chondrogenesis and maturation during development. A transgenic murine model of cartilage targeted Smurf2 over-expression will be created, and viral transduction of chondrocytes in vivo will be utilized to determine if Smurf2 over-expression can induce, or its inhibition suppress, OA. Finally, the expression of Smurf1 and Smurf2 in human normal and OA cartilage will be examined. These studies should provide a comprehensive picture of the function of Smurfs in regulating normal and OA cartilage phenotype.