Involvement of MAPKs, NF-κB and p300 co-activator in IL-1β-induced cytosolic phospholipase A₂ expression in canine tracheal smooth muscle cells

Cytosolic phospholipase A₂ (cPLA₂) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during stimulation with interleukin-1β (IL-1β). However, the mechanisms underlying IL-1β-induced cPLA₂ expression and PGE₂ synthesis by canine tracheal smooth muscle cells (CTSMCs) have not been defined. IL-1β induced cPLA₂ protein and mRNA expression, PGE₂ production, and phosphorylation of p42/p44 MAPK, p38 MAPK (ATF₂), and JNK (c-Jun) in a time- and concentration-dependent manner, determined by Western blotting, RT-PCR, and ELISA, which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), or transfection with dominant negative mutants of MEK1/2, p38, and JNK, respectively. Furthermore, IL-1β-induced cPLA₂ expression and PGE₂ synthesis was inhibited by a selective NF-κB inhibitor (helenalin) or transfection with dominant negative mutants of NF-κB inducing kinase (NIK), IκB kinase (IKK)-α, and IKK-β. Consistently, IL-1β stimulated both IκB-α degradation and NF-κB translocation into nucleus in these cells. NF-κB translocation was blocked by helenalin, but not by U0126, SB202190, and SP600125. MAPKs together with NF-κB-activated p300 recruited to cPLA₂ promoter thus facilitating the binding of NF-κB to cPLA₂ promoter region and expression of cPLA₂ mRNA. IL-1β-induced cPLA₂ expression and PGE₂ production was inhibited by actinomycin D and cycloheximide, indicating the involvement of transcriptional and translational events in these responses. These results suggest that in CTSMCs, IL-1β-induced cPLA₂ expression and PGE₂ synthesis was independently mediated through activation of MAPKs and NF-κB pathways and was connected to p300 recruitment and activation.