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BackgroundAdenosine is the effector molecule mediating the antirheumatic effects of methotrexate, but the contributions of synoviocyte adenosine receptors (AdoRs) are unknown. Matrix metalloproteinase 3 (MMP-3) is released by fibroblast-like synoviocytes in response to inflammatory signaling, and serum MMP-3 is elevated during active rheumatoid arthritis. To elucidate the therapeutic mechanisms of methotrexate, we investigated the effects of A2A AdoR activation and inhibition on tumor necrosis factor-alpha (TNFa)-induced MMP-3 release by MH7A human rheumatoid synovial cells.MethodsCultured MH7A cells were treated with 25 pg/ml TNFa or vehicle for 24 h in the presence or absence of the selective A2A AdoR agonist HENECA (10, 50, or 100 nM), and MMP-3 release was measured in the culture supernatant. In other experiments, cells were treated with TNFa plus the cAMP analog dbcAMP or pretreated with the A2A AdoR antagonist ZM241385 prior to TNFa with or without HENECA. Activation of the p38 MAPK signaling pathway was evaluated by western blotting of phosphorylated signaling proteins. Treatment group means were compared by independent samples t-tests.ResultsMH7A cells constitutively expressed membrane-associated A2A AdoRs, and HENECA enhanced intracellular cAMP, indicating that these receptors are functionally coupled to adenylyl cyclase. Stimulation with TNFa markedly enhanced release of MMP-3 from MH7A cells, whereas HENECA partially and dose-dependently inhibited TNFa-evoked MMP-3 release. Similarly, dbcAMP partially inhibited TNFa-induced MMP-3 release. Pretreatment with ZM241385 reversed the inhibitory effects of HENECA. Further, TNFa induced p38 MAPK and ATF-2 phosphorylation, whereas HENECA suppressed p38 MAPK and ATF-2 phosphorylation.ConclusionsAdenosine signaling via A2A AdoRs, adenylyl cyclase, and cAMP reduces (although does not completely block) TNFa-induced MMP-3 production, by interfering with p38 MAPK/ATF-2 activity. Activation of A2A AdoR pathway and suppression of MMP-3 release may explain the antirheumatic effects of methotrexate. |
