Protein kinase A-dependent stimulation of rat type II secreted phospholipase A2 gene transcription involves C/EBP-β and -δ in vascular smooth muscle cells

Abstract

Type II secreted phospholipase A2 (sPLA2) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA2, which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA2 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-dependent increase in sPLA2 gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter 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-β and -δ antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA2 promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA2 mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-β and -δ acting through a protein kinase A signaling pathway.