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 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.

Autocrine and Paracrine Transcriptional Regulation of Type IIA Secretory Phospholipase A2 Gene in Vascular Smooth Muscle Cells

“…[Ϫ488;ϩ46] sPLA2-luc and mutant constructs were obtained by site-directed mutagenesis as previously described. 23 …”

“…We have previously shown that the best characterized activators of gene expression during inflammation, the nuclear factor-B (NF-B), CAAT/enhancer-binding protein (C/EBP), and winged helix-turn-helix transcription factors (Ets-1), are involved in the stimulation of rat sPLA2-IIA gene transcription by IL-1␤ and cAMP. 23 Therefore, we investigated whether these transcription factors were involved in the induction of the sPLA2-IIA transcription by the hsPLA2-IIA supernatant using [-488;ϩ46] luc constructs mutated for the transcription factor binding sites. The luciferase/␤-galactosidase activity ratio stimulation obtained with the hsPLA2-IIA supernatant was abolished with the mutant constructs, whereas forskolin still induced the transcription of the mutant Ets and mutant NF-kB constructs (Figure 4).…”

“…23 We first reproduced these inductions by cotransfecting VSMCs with a [Ϫ488;ϩ46] sPLA2-luciferase reporter gene and a pCMV-␤-galactosidase gene (cytomegalovirus promoter/enhancer) (used to normalize transfection efficiency) (Figure 4). The luciferase activity measured 24 hours after induction reflected the efficiency of the sPLA2-IIA transcription.…”

“…20 VSMCs transfections and luciferase assays were realized as described previously. 23 Ni and V5 Depletions of the Supernatants 8 g of the V5 antibody (In Vitrogen) were incubated 1 night with 50 L of proteins A/G agarose beads (Santa Cruz). Supernatants were depleted by a 2-hour incubation with these beads or Ni-NTA agarose nickel-charged beads (Quiagen), filtered, and used to induce VSMCs.…”

Alteration in the activation state of new inflammation-associated targets by phospholipase A2-activating protein (PLAA)

“…I). Sp1 is a transcription factor known to regulate the activities of proteins involved in eicosanoid production, such as cPLA 2 [55], sPLA 2 [56], platelet-activating factor acetylhydrolase (PAF-AH) [57], and 12(S)-lipoxygenase [58].…”