The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells. We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter.

Abstract-Type II secreted phospholipase A 2 (sPLA 2 ) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA 2 , which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA 2 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 sPLA 2 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-␤ 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 sPLA 2 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 sPLA 2 mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-␤ and -␦ acting through a protein kinase A signaling pathway. (Arterioscler

The inflammation that occurs during rheumatoid arthritis or atherosclerosis is characterized by the release of large amounts of sPLA(2) (group IIA secretory phospholipase A(2)). We have shown previously that the sPLA(2) promoter in SMC (smooth-muscle cells) is activated by interleukin-1beta and cAMP-signalling pathways, through the interplay of multiple transcription factors [Antonio, Brouillet, Janvier, Monne, Bereziat, Andreani, and Raymondjean (2002) Biochem. J. 368, 415-424]. In the present study, we have investigated the regulation of sPLA(2) gene expression in rat aortic SMCs by oxysterols. We found that oxysterol ligands that bind to the LXR (liver X receptor), including 25-HC (25-hydroxycholesterol) and 22( R )-HC, cause the accumulation of sPLA(2) mRNA and an increased enzyme activity. Transient transfection experiments demonstrated that the sPLA(2) promoter is synergistically activated by 22( R )-HC in combination with 9- cis -retinoic acid, a ligand for the LXR heterodimeric partner RXR (retinoid X receptor). Promoter activity was also increased in a sterol-responsive fashion when cells were co-transfected with LXRalpha/RXRalpha or LXRbeta/RXRalpha. Mutagenesis studies and gel mobility-shift assays revealed that LXR/RXR heterodimers regulate sPLA(2) transcription directly, by interacting with a degenerated LXRE (LXR response element) at position [-421/-406] of the sPLA(2) promoter. Chromatin immunoprecipitation revealed the in vivo occupancy of LXR on the sPLA(2) promoter. In addition, the orphan nuclear receptor LRH-1 (liver receptor homologue-1) potentiated the sterol-dependent regulation of the sPLA(2) promoter by binding to an identified promoter element (TCAAGGCTG). Finally, we have demonstrated that oxysterols act independent of interleukin-1beta and cAMP pathways to activate the sPLA(2) promoter. In the present study, we have identified a new pathway activating sPLA(2) gene expression in SMCs.