CCN6通过IGF-1通路调节软骨基质代谢平衡的机制研究

Mechanism of articular cartilage matrix turnover is not clear and which contributes to the poor treatment effectiveness of medicines to osteoarthritis(OA), since the key pathological change is the progressive destruction of cartilage matrix, and how to regulate the homeostasis of cartilage matrix is very important. Ours previously studies showed that Wnt1-inducible signaling pathway 3 (Wisp3), also called CCN6, was involved in the insulin-like growth factor 1 (IGF-1)pathway and regulated the synthesis and secretion of collagen, the mutation of CCN6 will lead to the Pseudo-rheumatoid osteoarthritis (PPD), over-expression or very low-expression of CCN6 will lead to the cartilage destruction seen in OA with high levels of matrix degradation enzymes and PPD with little matrix degrading enzymes respectively. Then we hypothesis that CCN6 could regulate the homeostasis of cartilage matrix and its degrading enzyme precisely through IGF-1 pathway mediated by insulin-like growth factor binding protein(IGFBP) domain. To validate this hypothesis, we are going to study relative quantitative expression in normal cartilage matrix and its degrading enzyme system, and changes of expression caused by over or lack of CCN6 expression, and try to determine the CCN6 protein concentration needed to maintain cartilage homeostasis; and to determine whether CCN6 regulates differential expression of matrix and its degrading enzymes through IGF-1 signal pathway mediated by IGFBP domain , to discover the mechanism of CCN6 on extracellular matrix related proteins, and to establish a cornerstone for finding new target of therapeutic medicine for OA and PPD.

骨关节炎（OA）的病理本质是软骨基质进行性破坏，揭示软骨如何维持基质转换平衡及基质破坏导致OA的调控机制,是在OA防治药物上取得突破的关键。我们前期发现CCN6基因IGFBP结构域突变可导致基质退化降解为特征的假性关节炎（PPD），其过高或过低表达分别与基质降解酶过多的OA和基质降解酶过少的PPD密切相关。由此我们假设：CCN6可通过其IGFBP结构域介导的IGF-1信号实现对软骨细胞外基质/基质降解酶系的精确调节。为此我们将研究正常软骨基质及其降解酶谱的相对定量表达及CCN6过表达或缺失所引起其表达的改变，确定维持软骨稳态所需的CCN6蛋白量；判定CCN6是否通过其IGFBP结构域与IGF-1结合调节IGF-1的分泌，进一步调节Wnt/β-catenin或MAPK信号通路来调节基质及基质降解酶的差异表达，揭示CCN6调节细胞外基质相关蛋白的作用机理，为发现骨关节炎药物防治新靶点奠定基础。

骨关节炎（OA）的病理本质是软骨基质进行性破坏，揭示软骨如何维持基质转换平衡及基质破坏导致OA的调控机制,是在OA防治药物上取得突破的关键。Wnt1诱导分泌蛋白（Wisp3/CCN6）通过胰岛素受体因子-1（IGF-1）通路，参与胶原合成和分泌过程。我们前期发现CCN6基因IGFBP结构域突变可导致基质退化降解为特征的假性关节炎（PPD），其过高或过低表达分别与基质降解酶过多的OA和基质降解酶过少的PPD密切相关。本次试验我们构建了CCN6过表达或缺失的软骨细胞系模型，发现CCN6 过表达或缺失能引起软骨细胞外基质和基质降解酶谱的改变。我们利用软骨细胞三维沉淀培养技术在体外培养形成软骨，通过不同CCN6浓度梯度的设置，发现50ng/ml的CCN6或800ng/ml以上 CCN6均可造成基质金属蛋白酶的表达相对与正常软骨细胞出现明显异常。于是我们进一步对CCN6调节细胞外基质相关因子的功能区进行定位，发现IGFBP 结构域的缺失后，用IGF-1（200ng/ml）去干预细胞，Akt的表达及磷酸化明显增高，PI3K 活性增强。本次研究确立CCN6通过其IGFBP结构域与IGF-1结合调节IGF-1的分泌，揭示CCN6调节细胞外基质相关蛋白的作用机理，为发现骨关节炎药物防治新靶点奠定基础。