Transcriptional regulation of the rat type IIA phospholipase A2 gene by cAMP and interleukin-1β in vascular smooth muscle cells: interplay of the CCAAT/enhancer binding protein (C/EBP), nuclear factor-κB and Ets transcription factors

Abstract

The abundant secretion of type IIA secreted phospholipase A2 (sPLA2) is a major feature of the inflammatory process of atherosclerosis. sPLA2 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 sPLA2 transcription by cAMP and interleukin-1β (IL-1β), and shown that the 500bp region upstream of the transcription start site of the rat sPLA2 gene is implicated in activation by synergistically acting cAMP and IL-1β. 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 sPLA2—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 sPLA2 promoter activation. We find evidence for the first time that activation of the sPLA2 promoter by IL-1β 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-κB 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 sPLA2 promoter.