Baker Natasha, Sharpe Paul, Culley Kirsty, Otero Miguel, Bevan Damon, Newham Peter, Barker Wendy, Clements Kristen M, Langham Caroline J, Goldring Mary B, Gavrilović Jelena
School of Biological Sciences, University of East Anglia, Norwich, UK.
Arthritis Rheum. 2012 Jul;64(7):2289-99. doi: 10.1002/art.34411.
Wnt-1-inducible signaling pathway protein 3 (WISP-3)/CCN6 is mutated in progressive pseudorheumatoid dysplasia and may have effects on cartilage homeostasis. The aim of this study was to ascertain additional roles for WISP-3/CCN6 by determining its expression in osteoarthritic (OA) cartilage and by investigating its effects on cartilage-relevant metalloproteinase expression in immortalized (C-28/I2) and primary chondrocytes.
Cartilage steady-state levels of WISP-3/CCN6 messenger RNA and protein production were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. WISP-3/CCN6 was overexpressed in C-28/I2 cells, and the resultant clones were analyzed by quantitative RT-PCR. The stable clones were analyzed by RT-PCR for metalloproteinase expression, and the signaling pathways involved were investigated using pharmacologic inhibition. The effects of WISP-3/CCN6 on metalloproteinase expression in primary chondrocytes were investigated using a small interfering RNA approach.
WISP-3/CCN6 was highly expressed in OA cartilage compared with undamaged cartilage, at both the RNA and protein levels. WISP-3/CCN6 overexpression in C-28/I2 cells resulted in unexpected dual regulation of metalloproteinases; expression of the potent aggrecanase ADAMTS-5 was down-regulated 9-fold, while expression of MMP-10 was up-regulated 14-fold, and these responses were accentuated in the WISP-3/CCN6 clones grown in suspension. MMP-10 up-regulation was dependent on several MAPKs, but WISP-3/CCN6-mediated ADAMTS-5 repression was independent of these pathways and was partially relieved by activation of β-catenin signaling. WISP-3/CCN6 also suppressed ADAMTS-5 expression in C-28/I2 cells treated with cytokines. In cytokine-treated primary chondrocytes, gene silencing of WISP-3/CCN6 resulted in enhanced ADAMTS-5 expression, while MMP-10 expression was suppressed.
WISP-3/CCN6 was highly expressed in end-stage OA cartilage, suggesting a role for this growth factor in cartilage homeostasis. WISP-3/CCN6-induced repression of ADAMTS-5 expression and regulation of MMP-10 expression suggest complex and context-dependent roles for WISP-3/CCN6 in cartilage biology.
在进行性假类风湿性发育异常中，Wnt-1诱导信号通路蛋白3（WISP-3）/CCN6发生突变，可能对软骨内环境稳定产生影响。本研究旨在通过确定WISP-3/CCN6在骨关节炎（OA）软骨中的表达，并研究其对永生化（C-28/I2）和原代软骨细胞中与软骨相关的金属蛋白酶表达的影响，来确定WISP-3/CCN6的其他作用。
分别通过实时定量逆转录聚合酶链反应（RT-PCR）和免疫组织化学法测定WISP-3/CCN6信使核糖核酸的软骨稳态水平和蛋白质生成。在C-28/I2细胞中过表达WISP-3/CCN6，并通过定量RT-PCR分析所得克隆。通过RT-PCR分析稳定克隆的金属蛋白酶表达，并使用药理学抑制研究其中涉及的信号通路。使用小干扰RNA方法研究WISP-3/CCN6对原代软骨细胞中金属蛋白酶表达的影响。
与未受损软骨相比，WISP-3/CCN6在OA软骨的RNA和蛋白质水平上均高表达。在C-28/I2细胞中过表达WISP-3/CCN6导致金属蛋白酶出现意外的双重调节；强效聚糖酶ADAMTS-5的表达下调9倍，而基质金属蛋白酶-10（MMP-10）的表达上调14倍，并且在悬浮培养的WISP-3/CCN6克隆中这些反应更加明显。MMP-10的上调依赖于几种丝裂原活化蛋白激酶（MAPK），但WISP-3/CCN6介导的ADAMTS-5抑制独立于这些途径，并且通过β-连环蛋白信号的激活而部分缓解。WISP-3/CCN6还抑制了用细胞因子处理的C-28/I2细胞中ADAMTS-5的表达。在细胞因子处理的原代软骨细胞中，WISP-3/CCN6的基因沉默导致ADAMTS-5表达增强，而MMP-10表达受到抑制。
WISP-3/CCN6在终末期OA软骨中高表达，表明这种生长因子在软骨内环境稳定中发挥作用。WISP-3/CCN6诱导的ADAMTS-5表达抑制和MMP-10表达调节表明WISP-3/CCN6在软骨生物学中具有复杂且依赖于环境的作用。
