2-carba-cyclic Phosphatidic Acid Suppresses Expression of Cartilage Degrading Enzymes such as MMP-3, MMP-13 and ADAMTS-4 in Inflammatory Synovial Fibroblasts and Articular Chondrocytes Induced by IL-1 Beta and/or TNF Alfa

Purpose: Cyclic phosphatidic acid (cPA) is one of bioactive lipid, has been implicated as a mediator of various biological effects including inhibitory effects of proliferation, invasion and metastasis of cancer cells. cPA is naturally occurring mediator even exists in human serum. 2-carba-cPA (2ccPA) is structurally modified formula of cPA and has shown improved bioactivity. We have previously confirmed that 2ccPA stimulated HAS-2 production on human osteoarthritic chondrocytes and synovial fibroblasts (SFs) in vitro. Furthermore, intra-articular administration of 2ccPA has shown its suppressing effect of pain, swelling, and articular cartilage destruction in rabbit experimental osteoarthritis. We have shown that 2ccPA might had direct inhibitory effect of cartilage degrading enzymes on rheumatoid synovial fibroblasts (SFs) in vitro. Inflammatory arthritis such as rheumatoid arthritis and early stage of OA involves synovial inflammation and subsequent production of cartilage degrading enzymes also from chondrocytes. 2ccPA may be possible another therapeutic option for degenerative arthritis. The aim of this study was to evaluate the direct effects of 2ccPA on cartilage matrix degrading enzymes using SFs and articular chondrocytes under influence of inflammatory cytokines. Methods: In vitro studies were performed using SFs and chondrocytes obtained from arthritis patients at joint replacement surgery. 2ccPA 0–25 μM was added to cell cultures and effects of 2ccPA on ADAMTS-4, -5, MMP-3, 9, -13 expression was assessed by real time PCR using specific primers to corresponding genes. Beta-actin was used as endogenous expression control for PCR. SFs or chondrocytes were also pre-cultured with IL-1β (1 ng/ml) and/or TNF-α (10 ng/ml) for 24 hours, then added 10 μM 2ccPA to study attenuated effect of 2ccPA. Newly synthesized MMP-3, 9, -13 from SFs or chondrocytes in cultured media after 24 hours of 2ccPA addition were measured by sandwich ELISA. Results: 2ccPA itself repressed cartilage degrading enzymes, ADAMTS-4, ADAMTS-5, MMP-3, MMP-9, and MMP-13 expression in both SFs and chondrocytes was all repressed by low dose of 2ccPA. Even after cells had been stimulated by cytokines, 10 μM 2ccPA repressed expression of cartilage degenerating enzymes in SF or chondrocytes. Expression of MMP-13 were repressed more in chondrocytes by 2ccPA. ELISA results also confirmed the inhibitory effect of 2ccPA on MMP-13 production in SF or chondrocytes. Conclusions: The in vitro results confirmed that 2ccPA had suppressing effect of cartilage degrading enzymes on SFs and chondrocytes, supports the hypothesis that 2ccPA might have played direct role to suppress inflammation and also protect articular cartilage in arthritic condition. Molecular mechanism of 2ccPA to prevent cartilage degradation remains to be elucidated, however, further study should be warranted for 2ccPA as a novel candidate for therapeutic agent of arthritis.