CCN proteins in cartilage formation and maintenance

描述(由申请人提供)：CCN家族的成员是疾病过程的关键媒介。CCN2/CTGF(结缔组织生长因子)是迄今研究的所有纤维化条件下ECM过度合成的主要介质，而CCN1/Cyr61在肿瘤进展中具有强大的促血管生成活性。这些蛋白被认为是基质细胞蛋白，通过ECM-整合素与其他途径相互作用来协调信号。然而，这些蛋白质的许多体外活性中，哪些是与生理相关的尚不清楚，因为它们在正常组织发育/维持中的功能尚不清楚。对靶向小鼠的研究表明，CCN2/CTGF在软骨形成中是必不可少的。CCN2是否通过作用来调节其效应 作为整合素的配体，影响ECM及其调节剂的合成，和/或改变 软骨中的信号通路将在目标一中进行研究。我们将研究Ccn2缺失对生长板离散区域基因表达的影响，以检验Ccn2对细胞外基质含量具有全局性影响的假设，并测试特定信号通路的输出是否受到CCN2缺失的影响。控制软骨中CCN2表达的信号通路将在目标2中确定。由于Ccn2-L-小鼠与Sox9小鼠有惊人的相似之处，Ccn2是否是SOX因子的直接靶标将被调查。由于Ccn2-/-小鼠的主要表型之一是由于软骨细胞肥大导致的骨量减少，这些研究也将阐明肥大的软骨控制后续骨形成的能力所必需的信号通路。关于基质细胞蛋白作用的一个关键问题是它们在多大程度上完成重叠的功能。体外研究表明，CCNS的活性大致相似。然而，没有证据表明CCN1和CCN2具有重叠的功能。通过对CCN1基因缺失小鼠的分析，揭示了CCN1在软骨形成和关节形成中的独特作用。目的三是研究这种独特的关节表型，并使用CCN1的等位基因来确定CCN1在软骨中的作用。对双突变体和复合突变体的分析将直接测试CCN1和CCN2完成重叠功能的程度。最后，有令人信服的证据表明，多个CCN家族成员对成人关节软骨的维持是必不可少的。CCN2是成人关节软骨中含量最丰富的转录本之一，关节软骨细胞对CCN2产生反应，导致关节损伤的修复。在人类中，相关基因CCN6的丢失会导致进行性假性类风湿性发育不良，这是一种严重的关节炎形式，这表明CCN蛋白的主要共同功能是维持关节软骨。我们使用Ccn2等位基因和他莫昔芬诱导的Cre重组酶验证了CCN2对成人软骨的维持至关重要的假设。

DESCRIPTION (provided by applicant): Members of the CCN family are key mediators of disease processes. CCN2/CTGF (Connective Tissue Growth Factor) is the major mediator of excess ECM synthesis in all fibrotic conditions studied to date, and CCN1/Cyr61 has potent proangiogenic activities in tumor progression. These proteins are proposed to act as matricellular proteins, coordinating signals mediated by ECM-integrin interactions with other pathways. However, which of the many in vitro activities of these proteins are physiologically relevant is unknown, because their functions in normal tissue development /maintenance are undefined. Studies of targeted mice demonstrate that CCN2/CTGF is essential for chondrogenesis. Whether CCN2 mediates its effects by acting as a ligand for integrins, affecting synthesis of ECM and its modulators, and/or altering the outputs of signaling pathways in cartilage will be investigated in aim one. The consequences of loss of Ccn2 on gene expression in discrete regions of the growth plate will be investigated to test the hypotheses that Ccn2 has global effects on ECM content, and to test whether output of specific signaling pathways is impacted by loss of CCN2. Signaling pathways controlling CCN2 expression in cartilage will be identified in aim two. As Ccn2-l- mice bear a striking resemblance to Sox9 mice, whether CCN2 is a direct target of Sox factors will be investigated. As one of the major phenotypes of Ccn2-/- mice is osteopenia as a result of defective chondrocyte hypertrophy, these studies will also shed light on signaling pathways essential for the ability of hypertrophic cartilage to control subsequent bone formation. A key question regarding the roles of matricellular proteins is the extent to which they fulfill overlapping functions. In vitro studies suggest broadly similar activities for CCNs. However, in no case has it been shown that CCN1 and CCN2 have overlapping functions. A unique role of CCN1 in chondrogenesis and in joint formation is revealed by analysis of Ccn1 null mice. In aim three this unique joint phenotype is investigated and a Ccn1 floxed allele is used to define the role of CCN1 in cartilage. Analysis of double and compound mutants will test directly the degree to which CCN1 and 2 fulfill overlapping functions. Finally, there is compelling evidence implicating multiple CCN family members as essential for the maintenance of adult articular cartilage. Ccn2 is one of the most abundant transcripts in adult articular cartilage, and articular chondrocytes respond to CCN2, leading to repair of articular defects. In humans, loss of a related gene, CCN6, leads to a progressive pseudorheumatoid dysplasia, a severe form of arthritis, suggesting that a major shared function for CCN proteins is to maintain articular cartilage. We test the hypothesis that CCN2 is vital for the maintenance of adult cartilage using a floxed Ccn2 allele and a tamoxifen-inducible Cre recombinase.