Protein kinase A-dependent stimulation of rat type II secreted phospholipase A(2) gene transcription involves C/EBP-beta and -delta in vascular smooth muscle cells

Type II secreted phospholipase A(2) (sPLA(2)) releases precursors of import ant inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA(2), which has been implicated in chronic inflammat ory conditions such as arthritis, contributes to atherosclerosis in the art erial wall. sPLA, was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-depe ndent increase in sPLA(2) gene expression. Semiquantitative reverse transcr iptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent in hibition of forskolin-induced mRNA by protein kinase A inhibitor. Electroph oretic mobility shift analysis of nuclear proteins from forskolin-treated a nd db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oli gonucleotides from the rat promoter revealed greater binding than in contro l VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, a lso blocked the binding of nuclear C/EBP to the C/EBP site of the rat promo ter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-b eta and -delta antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat co pies of the C/EBP element from the rat sPLA, promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression v ector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP, H89 inhibit ed this activations. We therefore conclude that the increases in sPLA2 mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a m echanism involving nuclear C/EBP-beta and -delta acting through a protein k inase A signaling pathway.