Keio University School of Medicine, Tokyo, Japan.
Yamaguchi University Graduate School of Medicine, Ube, Japan.
Arthritis Rheumatol. 2015 Jun;67(6):1557-67. doi: 10.1002/art.39078.
ADAMTS-4, also called aggrecanase 1, is considered to play a key role in aggrecan degradation in human osteoarthritic (OA) cartilage, but information about regulators of ADAMTS-4 aggrecanase activity remains limited. We undertook this study to search for molecules that modulate ADAMTS-4 activity.
Molecules copurified with ADAMTS-4 from ADAMTS-4-transfected chondrocytic cells were sequenced by nanoscale liquid chromatography tandem mass spectrometry. Binding activity was determined by immunoprecipitation and solid-phase binding assay. Effects on ADAMTS-4 activity were examined by aggrecan digestion assay. Expression of the binding molecule in OA cartilage and chondrocytes was examined by immunohistochemistry and reverse transcription-polymerase chain reaction.
We identified CCN1 (Cyr61) as an ADAMTS-4-binding protein and showed specific binding to the ADAMTS-4 cysteine-rich domain. Aggrecanase activity of ADAMTS-4 was inhibited by interaction with CCN1. Expression of messenger RNA for CCN1 was significantly higher in human OA cartilage than in normal cartilage. CCN1 was immunolocalized to chondrocytes in OA cartilage, showing direct correlations of immunoreactivity with the Mankin score of cartilage lesions and chondrocyte cloning. CCN1 and ADAMTS-4 were commonly coexpressed in clustered chondrocytes. CCN1 expression in OA chondrocytes was down-regulated by interleukin-1α (IL-1α) and up-regulated by transforming growth factor β (TGFβ). ADAMTS-4 expression was induced by treatment with IL-1α or TGFβ, but aggrecanase activity was detected only under stimulation with IL-1α. TGFβ-treated chondrocytes exhibited aggrecanase activity when CCN1 expression was knocked down.
Our findings provide the first evidence that CCN1 suppresses ADAMTS-4 activity and that CCN1 overexpression is directly correlated with chondrocyte cloning in OA cartilage. Our results suggest that the TGFβ/CCN1 axis plays a role in chondrocyte cluster formation through inhibition of ADAMTS-4.
ADAMTS-4，也称为聚集素酶 1，被认为在人类骨关节炎（OA）软骨中聚集素的降解中发挥关键作用，但关于 ADAMTS-4 聚集素酶活性的调节剂的信息仍然有限。我们进行了这项研究，以寻找调节 ADAMTS-4 活性的分子。
通过纳米级液相色谱串联质谱对从 ADAMTS-4 转染的软骨细胞中与 ADAMTS-4 共纯化的分子进行测序。通过免疫沉淀和固相结合测定法确定结合活性。通过聚集素消化测定法检查对 ADAMTS-4 活性的影响。通过免疫组织化学和逆转录聚合酶链反应检查结合分子在 OA 软骨和软骨细胞中的表达。
我们将 CCN1（Cyr61）鉴定为 ADAMTS-4 结合蛋白，并显示与 ADAMTS-4 富含半胱氨酸的结构域特异性结合。CCN1 与 ADAMTS-4 的相互作用抑制了聚集素酶的活性。CCN1 的信使 RNA 在人类 OA 软骨中的表达明显高于正常软骨。CCN1 在 OA 软骨中的软骨细胞中免疫定位，其免疫反应性与软骨病变的 Mankin 评分和软骨细胞克隆呈直接相关性。CCN1 和 ADAMTS-4 共同表达在聚集的软骨细胞中。白细胞介素 1α（IL-1α）下调 OA 软骨细胞中的 CCN1 表达，转化生长因子β（TGFβ）上调 CCN1 表达。用 IL-1α 或 TGFβ 处理后诱导 ADAMTS-4 表达，但仅在 IL-1α 刺激下检测到聚集素酶活性。当 CCN1 表达被敲低时，TGFβ 处理的软骨细胞表现出聚集素酶活性。
我们的研究结果首次提供了证据表明 CCN1 抑制 ADAMTS-4 活性，并且 CCN1 过表达与 OA 软骨中的软骨细胞克隆直接相关。我们的结果表明，TGFβ/CCN1 轴通过抑制 ADAMTS-4 在软骨细胞簇形成中发挥作用。
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