CCN proteins in cartilage formation and maintenance

描述（由申请人提供）：CCN家族成员是疾病过程的关键介质。CCN 2/CTGF（结缔组织生长因子）是迄今为止研究的所有纤维化条件下过量ECM合成的主要介质，CCN 1/Cyr 61在肿瘤进展中具有强效促血管生成活性。这些蛋白质被认为是基质细胞蛋白，协调ECM-整合素相互作用介导的信号与其他途径。然而，这些蛋白质的许多体外活性中哪些是生理学相关的是未知的，因为它们在正常组织发育/维持中的功能是不确定的。靶向小鼠的研究表明，CCN 2/CTGF是软骨形成所必需的。CCN 2是否通过作用于 作为整联蛋白的配体，影响ECM及其调节剂的合成，和/或改变ECM的输出， 目标一将研究软骨中的信号传导途径。将研究Ccn 2缺失对生长板离散区域基因表达的影响，以检验Ccn 2对ECM含量具有全局影响的假设，并检验特定信号传导途径的输出是否受到CCN 2缺失的影响。控制软骨中CCN 2表达的信号通路将在目标二中鉴定。由于CCN 2 - 1-小鼠与Sox 9小鼠具有惊人的相似性，因此将研究CCN 2是否是Sox因子的直接靶标。由于Ccn 2-/-小鼠的主要表型之一是由于缺陷性软骨细胞肥大导致的骨质减少，这些研究还将阐明肥大软骨控制随后骨形成的能力所必需的信号通路。关于基质细胞蛋白的作用的一个关键问题是它们实现重叠功能的程度。体外研究表明CCN的活性大致相似。然而，在任何情况下都没有显示CCN 1和CCN 2具有重叠的功能。CCN 1基因敲除小鼠的分析揭示了CCN 1在软骨形成和关节形成中的独特作用。在第三个目标中，研究了这种独特的关节表型，并使用CCN 1 floxed等位基因来定义CCN 1在软骨中的作用。双重和复合突变体的分析将直接测试CCN 1和2实现重叠功能的程度。最后，有令人信服的证据表明，多个CCN家族成员对维持成人关节软骨至关重要。CCN 2是成人关节软骨中最丰富的转录本之一，关节软骨细胞对CCN 2产生应答，导致关节缺损的修复。在人类中，相关基因CCN 6的缺失导致进行性假性关节炎发育不良，这是一种严重的关节炎，表明CCN蛋白的主要共同功能是维持关节软骨。我们测试的假设，CCN 2是至关重要的成人软骨使用floxed CCN 2等位基因和他莫昔芬诱导的Cre重组酶的维护。

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.