Prostaglandin E₂ downregulates TNF-α-induced production of matrix metalloproteinase-1 in HCS-2/8 chondrocytes by inhibiting Raf-1/MEK/ERK cascade through EP4 prostanoid receptor activation

Matrix metalloproteinase-1 (MMP-1, collagenase-1) plays a pivotal role in the process of joint destruction in degenerative joint diseases. We have examined the regulation of MMP-1 production in human chondrocytic HCS-2/8 cells stimulated by tumor necrosis factor-α (TNF-α). In response to TNF-α, MMP-1 is induced and actively released from HCS-2/8 cells. The induction of MMP-1 expression correlates with activation of ERK1/2, MEK, and Raf-1,and is potently prevented by U0126, a selective inhibitor of MEK1/2 activation. In contrast, SB203580, a selective p38mitogen-activated protein kinases (MAPK) inhibitor, had no effects on TNF-α-induced MMP-1 release. A serine/threonine kinase. Akt was not activated in TNF-α-stimulated HCS-2/8 cells. TNF-α-stimulated the production of PGE₂ in addition to MMP-1 in HCS-2/8 cells. Exogenously added PGE₂ potently inhibited TNF-α-induced both MMP-1 production and activation of ERK1/2. The effects of PGE₂ were mimicked by ONO-AE1-329, a selective EP4 receptor agonist but not by butaprost, a selective EP2 agonist. In contrast, blockade of endogenously produced PGE₂ signaling by ONO-AE3-208, a selective EP4 receptor antagonist, enhanced TNF-α-induced MMP-1 production. Furthermore, the suppression of MMP-1 production by exogenously added PGE₂ was reversed by ONO-AE3-208. Activation of EP4 receptor resulted in cAMP-mediated phosphorylation of Raf-1 on Ser259, a negative regulatory site, and blocked activation of Raf-1/MEK/ERK cascade. Taken together, these findings indicate that Raf-1/MEK/ERK signaling pathway plays a crucial role in the production of MMP-1 in HCS-2/8 cells in response to TNF-α, and that the produced PGE₂ downregulates the expression of MMP-1 by blockage of TNF-α-induced Raf-1 activation through EP4-PGE₂ receptor activation.