TLR4‐dependent induction of vascular adhesion molecule‐1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A2α/cyclooxygenase‐2

Lipopolysaccharide (LPS)/Toll‐like receptor 4 (TLR4)‐mediated signaling pathways have caught the attention of strategies designed for rheumatoid arthritis (RA). In this study, we identified that cPLA₂α acted as a modulator of LPS‐induced VCAM‐1 expression and THP‐1 (human acute monocytic leukemia cell line) adherence. Treatment of RA synovial fibroblasts (RASFs) with LPS, a TLR4 agonist, promoted the VCAM‐1 expression and THP‐1 adherence which were decreased by pretreatment with a selective cytosolic phospholipase A₂ (cPLA₂) inhibitor (AACOCF₃), implying the involvement of cPLA₂α in these responses. This notion was further confirmed by knockdown of cPLA₂α expression by transfection with cPLA₂α small interfering RNA (siRNA) leading to a decrease in VCAM‐1 expression and THP‐1 adherence induced by LPS. Subsequently, the LPS‐stimulated cPLA₂α phosphorylation was attenuated by pretreatment with a MEK1/2 inhibitor (U0126), suggesting that LPS‐stimulated cPLA₂α phosphorylation and activity are mediated through an ERK‐dependent mechanism. Moreover, COX‐2‐derived PGE₂ production appeared to involve in LPS‐induced VCAM‐1 expression which was attenuated by pretreatment with selective COX‐2 inhibitors (NS‐398 and celecoxib), transfection with COX‐2 siRNA, or PGE₂ receptor antagonists. In addition, pretreatment with ecosapentaenoic acid (EPA), a substrate competitor of arachidonic acid (AA), also blocked LPS‐induced VCAM‐1 mRNA and protein expression, and THP‐1 adherence. Collectively, these results suggest that LPS‐induced VCAM‐1 expression and adhesion of THP‐1 cells are mediated through the TLR4/ERK/cPLA₂α phosphorylation and COX‐2 expression/PGE₂ synthesis in RASFs. J. Cell. Physiol. 223: 480–491, 2010. © 2010 Wiley‐Liss, Inc.