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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2559.)
© 2000 American Heart Association, Inc.

Vascular Biology

Protein Kinase A–Dependent Stimulation of Rat Type II Secreted Phospholipase A₂ Gene Transcription Involves C/EBP-ß and - in Vascular Smooth Muscle Cells

Cyril Couturier; Valérie Antonio; Arthur Brouillet; Gilbert Béréziat; Michel Raymondjean; Marise Andréani

From Unité Propre de Recherche de l’Université Pierre et Marie Curie, associée au CNRS (ESA7079).

Correspondence to Marise Andréani, Université Pierre et Marie Curie, ESA 7079, Université Pierre et Marie Curie, Case 256, 7 quai Saint Bernard 75005 Paris, France. E-mail andreani{at}ccr.jussieu.fr

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-ß and - 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-ß and - acting through a protein kinase A signaling pathway.

Key Words: gene regulation • protein kinase A • secreted type II phospholipase A₂ • smooth muscle cells • C/EBP

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