Zhang Qiuyu, Wu Juanjuan, Cao Qi, Xiao Lianbo, Wang Li, He Dongyi, Ouyang Guilin, Lin Jinpiao, Shen Baihua, Shi Yuan, Zhang Yan, Li Dangsheng, Li Ningli
Shanghai Jiao Tong University School of Medicine, and Guanghua Rheumatology Hospital, Shanghai, China.
Arthritis Rheum. 2009 Dec;60(12):3602-12. doi: 10.1002/art.24999.
Fibroblast-like synoviocytes (FLS) are a major component of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Cyr61 (CCN1) is a product of a growth factor-inducible immediate early gene and is involved in cell adhesion, proliferation, and differentiation. However, the role that Cyr61 plays in FLS proliferation has remained undetermined. The aim of this study was to identify the role of Cyr61 in regulating the proliferation of FLS derived from patients with RA.
Expression of Cyr61 in synovial tissue (ST) and in FLS was determined simultaneously using immunohistochemistry, real-time polymerase chain reaction, and Western blotting. Cyr61 levels in synovial fluid (SF) were determined by enzyme-linked immunosorbent assay. FLS proliferation stimulated by SF, Cyr61, and interleukin-17 (IL-17) was measured by thymidine incorporation. Activation of signal transduction pathways was determined by Western blotting and confocal microscopy.
Cyr61 was overexpressed in ST, FLS, and SF samples from RA patients as compared with samples from normal controls. Elevated levels of Cyr61 in RA SF promoted the proliferation of FLS, an effect that was abrogated by a neutralizing monoclonal antibody against human Cyr61. Furthermore, in samples from RA patients, Cyr61 was found to protect FLS from apoptosis and to sustain the expression of Bcl-2 in FLS. Most importantly, the expression of Cyr61 in FLS was regulated by IL-17 mainly via the p38 MAPK and NF-kappaB signaling pathways. Knockdown of expression of the Cyr61 gene inhibited IL-17-stimulated FLS proliferation.
Our findings indicate that Cyr61 plays a critical role in IL-17-mediated proliferation of FLS in RA and likely contributes to hyperplasia of synovial lining cells and eventually to joint destruction in patients with RA.
成纤维样滑膜细胞（FLS）是增生性滑膜血管翳的主要组成部分，在类风湿关节炎（RA）病程中会侵袭软骨和骨。Cyr61（CCN1）是一种生长因子诱导的即早基因产物，参与细胞黏附、增殖和分化。然而，Cyr61在FLS增殖中所起的作用尚未明确。本研究旨在确定Cyr61在调节RA患者来源的FLS增殖中的作用。
采用免疫组织化学、实时聚合酶链反应和蛋白质印迹法同时检测滑膜组织（ST）和FLS中Cyr61的表达。采用酶联免疫吸附测定法测定滑液（SF）中Cyr61的水平。通过胸腺嘧啶核苷掺入法检测SF、Cyr61和白细胞介素-17（IL-17）刺激的FLS增殖。通过蛋白质印迹法和共聚焦显微镜检测信号转导通路的激活情况。
与正常对照样本相比，RA患者的ST、FLS和SF样本中Cyr61表达上调。RA患者SF中升高的Cyr61水平促进了FLS的增殖，抗人Cyr61中和单克隆抗体可消除该作用。此外，在RA患者样本中，发现Cyr61可保护FLS免于凋亡并维持FLS中Bcl-2的表达。最重要的是，FLS中Cyr61的表达主要通过p38丝裂原活化蛋白激酶和核因子κB信号通路受IL-17调节。敲低Cyr61基因表达可抑制IL-17刺激的FLS增殖。
我们的研究结果表明，Cyr61在RA中IL-17介导的FLS增殖中起关键作用，可能导致滑膜衬里细胞增生，并最终导致RA患者关节破坏。
