Hou Chun-Han, Tang Chih-Hsin, Hsu Chin-Jung, Hou Sheng-Mon, Liu Ju-Fang
Arthritis Res Ther. 2013 Jan 23;15(1):R19. doi: 10.1186/ar4151.
Osteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.
CCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.
Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor (LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.
Our results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.
骨关节炎（OA）是最常见的退行性关节疾病，涉及关节软骨的降解。OA的确切病因尚未完全明确。CCN4与骨关节炎患者软骨中的上调有关。先前的研究表明CCN4可能与OA的发病机制有关，但CCN4介导滑膜成纤维细胞（SF）中白细胞介素-6（IL-6）表达的确切信号通路在很大程度上尚不清楚。因此，我们探讨了人滑膜成纤维细胞中CCN4诱导IL-6产生所涉及的细胞内信号通路。
采用定量实时荧光定量PCR和酶联免疫吸附测定法评估CCN4诱导的IL-6产生。通过蛋白质免疫印迹法研究CCN4在不同信号通路中的作用机制。使用整合素中和抗体阻断整合素信号通路。荧光素酶测定法用于研究IL-6和核因子κB（NF-κB）启动子活性。免疫细胞化学法用于检测p65的转位活性。
骨关节炎滑膜成纤维细胞（OASF）显示出CCN4的显著表达，且该表达高于正常SF。用CCN4刺激OASF可诱导IL-6产生呈浓度和时间依赖性增加。用αvβ5整合素抗体而非α5β1和αvβ3整合素抗体预处理OASF可降低CCN4诱导的IL-6产生。CCN4介导的IL-6产生被磷脂酰肌醇-3激酶（PI3K）抑制剂（LY294002和渥曼青霉素）、蛋白激酶B（Akt）抑制剂（Akti）和NF-κB抑制剂（吡咯烷二硫代氨基甲酸盐和N-对甲苯磺酰-L-苯丙氨酰氯甲基酮）减弱。用CCN4刺激细胞也可增加PI3K、Akt和NF-κB的激活。
我们的结果表明，CCN4激活αvβ5整合素、PI3K、Akt和NF-κB通路，导致IL-6产生上调。根据我们的结果，CCN4可能是未来OA药物干预的合适靶点。
