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 A(2) (cPLA(2)) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during stimulation with interleukin-1 beta (IL-1 beta). However the mechanisms underlying IL-1 beta-induced cPLA(2) expression and PGE(2) synthesis by canine tracheal smooth muscle cells (CTSMCs) have not been defined. IL-1 beta induced cPLA(2) protein and mRNA expression, PGE(2) production, and phosphorylation of p42/p44 MAPK, P38 MAPK (ATF(2)) 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-1 beta-induced cPLA(2) expression and PGE2 synthesis was inhibited by a selective NF-kappa B inhibitor (helenalin) or transfection with dominant negative mutants of NF-kappa B inducing kinase (NIK), I kappa B kinase (IKK)-alpha, and IKK-beta. Consistently, IL-1 beta stimulated both I kappa B-alpha degradation and NF-kappa B translocation into nucleus in these cells. NF-kappa B translocation was blocked by helenalin, but not by U0126, SB202190, and SP600125. MAPKs together with NF-kappa B-activated p300 recruited to cPLA(2) promoter thus facilitating the binding of NF-kappa B to cPLA(2) promoter region and expression of cPLA(2) mRNA. IL-1 beta-induced cPLA(2) expression and PGE(2) 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-1 beta-induced cPLA(2) expression and PGE(2) synthesis was independently mediated through activation of MAPKs and NF-kappa B pathways and was connected to p300 recruitment and activation. (C) 2008 Elsevier Inc. All rights reserved.