Background Cyclic phosphatidic acid (cPA) is one of bioactive lipid, has been implicated as an mediator of various biological effects including inhibitory effects of proliferation, invasion and metastasis of cancer cells1. On human skin fibroblasts, cPA stimulates high molecular hyaluronic acid (HA) production through up-regulating HA synthase-2 (HAS-2)2. We have previously confirmed that cPA also stimulated HAS-2 production on human osteoarthritic chondrocytes and synovial fibroblasts (SFs) in vitro. Furthermore, intra-articular administration of cPA has shown its suppressing effect of pain, swelling, and articular cartilage destruction in rabbit experimental osteoarthritis. These compelling results lead to a hypothesis that cPA may have direct role on anti-inflammation and protection of cartilage in arthritic condition. Inflammatory arthritis such as rheumatoid arthritis and early stage of OA involves synovial inflammation and subsequent production of cartilage degrading enzymes. cPA is naturally occurring mediator even exists in human serum. cPA may be possible another therapeutic option for arthritis.
Objectives The aim of this study was to evaluate the direct effects of cPA on cartilage matrix degrading enzymes using rheumatoid SFs, which are under more severe inflammatory condition than osteoarthritis.
Methods In vitro studies were performed using SFs obtained from rheumatoid arthritis patients at joint replacement surgery. cPA 0-25 μM was added to SFs cultures and effects of cPA on HAS-1-3, HYAL-1, -2, ADAMTS-4, -5, MMP-3, -13, TIMP-3 expression were assessed at 24 and 48hrs by real time PCR using specific primers to corresponding genes. SFs were also cultured with IL-1β (2.5ng/ml) and/or TNF-α (10 ng/ml), to study attenuated effect of cPA. Beta-actin was used as endogenous expression control for PCR. Newly synthesized HA, MMP-3, -13, TIMP-3 from SFs in cultured media were measured by sandwich ELISA.
Results cPA stimulated endogenous HA synthesis from SFs as time and dose-dependent manner in vitro. HAS-2 gene was up-regulated as dose–dependent manner. Cytokine (IL-1β and/or TNF-α) addition increased HAS-2 expression, and addition of cPA further stimulated. On the other hand, cPA itself repressed HYAL-1 and -2 expression, and cPA repressed HYAL expression stimulated by cytokines. Not only with HYAL, other cartilage degrading enzymes, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13 expression in SFs was all repressed by low dose of cPA, even after these expressions was stimulated by cytokines. ELISA results also confirmed the inhibitory effect of cPA on MMP-3, MMP-13, and PGE2 production.
Conclusions The in vitro results confirmed that cPA had stimulatory effects on HA synthesis by rheumatoid SFs. In addition, the suppressing effect of HYAL, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13 on rheumatoid SFs by cPA, supports the hypothesis that cPA might have played direct role to suppress inflammation and also protect articular cartilage in arthritic condition. Molecular mechanism of cPA to prevent cartilage degradation remains to be elucidated, however, further study should be warranted for cPA as a novel candidate for therapeutic agent of arthritis.
Baker D et al. J Biol Chem 2006; 281: 22786-93.
Maeda-Sano K et al. Biochim Biophys Acta 2014; 1814: 1256-63.
Disclosure of Interest None declared
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