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，影响着数千万患者的生活质量。来自人类骨性关节炎和几种动物骨性关节炎模型的研究证据表明，在骨性关节炎进展过程中，软骨细胞表型成熟的再现类似于胚胎软骨内成骨。在之前的资助期内，我们确定了许多在OA软骨中表达的表型成熟特征，包括PTHrP、MMP9、MMP13、Ihh和BMP6。关节软骨细胞在培养过程中很难诱导这种成熟级联反应，并且对生长因子的反应相对较弱。然而，用氮扎胞苷治疗，允许抑制基因去甲基化，诱导正常级联的软骨细胞成熟，导致X型胶原蛋白和其他肥厚标志物的表达。对相关特定信号通路的研究表明Smad泛素化连接酶Smurf2是将信号优势从tgf - β转移到BMP Smad通路的潜在关键调控步骤，从而导致软骨细胞成熟。发现Smurf2在人OA中表达，而在正常关节软骨中不表达，并在培养中被氮胞苷处理诱导。Smurf2刺激或过表达导致Smad2和Smad3降解，tgf - β信号下调，BMP信号上调。这足以启动整个级联的软骨细胞成熟和肥大的培养。特异性目的(1):Smurfs在体外调节软骨细胞表型中的作用研究将利用培养的关节软骨细胞和生长板软骨细胞，研究Smurf1和2对Smad信号通路和软骨细胞表型的影响。相关生长因子和细胞因子对Smurf表达的调控也将被研究。具体目的(2)：研究Smurf2在体内软骨细胞成熟和OA中的作用，将利用小鸡肢体芽系统分析Smurf2在发育过程中对软骨形成和成熟的影响。我们将建立一个软骨靶向Smurf2过表达的转基因小鼠模型，并利用体内软骨细胞的病毒转导来确定Smurf2过表达是诱导OA，还是抑制OA。最后，我们将检测Smurf1和Smurf2在人正常软骨和OA软骨中的表达。这些研究应该为蓝精灵在调节正常和OA软骨表型方面的功能提供一个全面的图景。

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.