Nieminen Riina, Korhonen Riku, Moilanen Teemu, Clark Andrew R, Moilanen Eeva
The Immunopharmacology Research Group, University of Tampere Medical School and Tampere University Hospital, Tampere, Finland.
Arthritis Rheum. 2010 Jun;62(6):1650-9. doi: 10.1002/art.27409.
Aurothiomalate is a disease-modifying antirheumatic drug that suppresses inflammation and retards cartilage degradation and bone erosion in arthritis. The molecular mechanisms of action of aurothiomalate are not known in detail. MAPK pathways are major signaling pathways in inflammation that regulate the production of many inflammatory and destructive factors in arthritis. The purpose of the present study was to investigate the effects of aurothiomalate on the activity of p38 MAPK and on the expression of MAPK phosphatase 1 (MKP-1), cyclooxygenase 2 (COX-2), matrix metalloproteinase 3 (MMP-3), and interleukin-6 (IL-6) in immortalized murine H4 chondrocytes and in intact human and murine cartilage.
Protein expression was examined by Western blotting or by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was examined by real-time reverse transcription-polymerase chain reaction analysis. The mediator role of MKP-1 was investigated by using small interfering RNA (siRNA) methods to down-regulated MKP-1 expression in chondrocytes in culture and by comparing the responses in intact cartilage from MKP-1-deficient and wild-type mice. The effects of aurothiomalate were also confirmed in human rheumatoid cartilage by using tissue samples obtained at the time of total knee replacement surgery.
Aurothiomalate inhibited IL-1beta-induced COX-2 expression and prostaglandin E(2) production by destabilizing COX-2 mRNA, as did the p38 MAPK inhibitor SB203580. Interestingly, aurothiomalate also increased the expression of MKP-1 and reduced the IL-1beta-induced phosphorylation of p38 MAPK. Knockdown of MKP-1 by siRNA significantly impaired the ability of aurothiomalate to inhibit the phosphorylation of p38 MAPK and the expression of COX-2, MMP-3, and IL-6. Likewise, aurothiomalate reduced COX-2, MMP-3, and IL-6 expression in articular cartilage from patients with rheumatoid arthritis, as well as in articular cartilage from wild-type mice but not from MKP-1(-/-) mice.
Our findings indicate a novel mechanism for the antiinflammatory and antierosive actions of aurothiomalate, through increased expression of MKP-1, which leads to reduced activation of p38 MAPK and suppressed expression of COX-2, MMP-3, and IL-6. The results suggest that manipulation of MKP-1 levels is a promising new mechanism to be directed in the search and development of novel antiinflammatory and antierosive compounds that have the good efficacy of gold compounds but not their toxicity.
金硫代苹果酸是一种改善病情的抗风湿药物，可抑制炎症并延缓关节炎中的软骨降解和骨侵蚀。金硫代苹果酸的分子作用机制尚不清楚。丝裂原活化蛋白激酶（MAPK）通路是炎症中的主要信号通路，可调节关节炎中许多炎症和破坏因子的产生。本研究的目的是探讨金硫代苹果酸对永生化小鼠H4软骨细胞以及完整的人及小鼠软骨中p38 MAPK活性、MAPK磷酸酶1（MKP-1）、环氧化酶2（COX-2）、基质金属蛋白酶3（MMP-3）和白细胞介素-6（IL-6）表达的影响。
通过蛋白质印迹法或酶联免疫吸附测定法检测蛋白质表达，通过实时逆转录-聚合酶链反应分析检测信使核糖核酸（mRNA）表达。通过使用小干扰RNA（siRNA）方法下调培养的软骨细胞中MKP-1的表达，并比较MKP-1缺陷型和野生型小鼠完整软骨中的反应，研究MKP-1的介导作用。还通过使用全膝关节置换手术时获得的组织样本，在人类风湿性软骨中证实了金硫代苹果酸的作用。
金硫代苹果酸通过使COX-2 mRNA不稳定来抑制IL-1β诱导的COX-2表达和前列腺素E2的产生，p38 MAPK抑制剂SB203580也有此作用。有趣的是，金硫代苹果酸还增加了MKP-1的表达，并减少了IL-1β诱导的p38 MAPK磷酸化。通过siRNA敲低MKP-1显著损害了金硫代苹果酸抑制p38 MAPK磷酸化以及COX-2、MMP-3和IL-6表达的能力。同样，金硫代苹果酸降低了类风湿性关节炎患者关节软骨以及野生型小鼠而非MKP-1（-/-）小鼠关节软骨中COX-2、MMP-3和IL-6的表达。
我们的研究结果表明，金硫代苹果酸的抗炎和抗侵蚀作用有一个新机制，即通过增加MKP-1的表达，导致p38 MAPK的激活减少以及COX-2、MMP-3和IL-6的表达受到抑制。结果表明，调节MKP-1水平是一种有前景的新机制，可用于寻找和开发具有金化合物的良好疗效但无其毒性的新型抗炎和抗侵蚀化合物。
