Abstract
P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.
Highlights
P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS)
We have explored the role of the Group V (GV) phospholipase A2 (PLA2) in the production of PGE2 upon long-term stimulation of P388D1 cells with LPS
Specific second-generation antisense oligonucleotide (AS-ON) directed against GV PLA2 reduced the expression of the enzyme
Summary
P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). We show that treatment of P388D1 cells with AS-ONs resulted in a marked decrease of basal GV PLA2 mRNA, prevented the LPS-induced increase in GV PLA2 mRNA as measured with real-time quantitative PCR (Q-PCR), and caused a significant decrease in basal and stimulated GV PLA2 protein and activity levels. Treatment of the cells with the novel GIVA PLA2 inhibitor pyrrophenone resulted in a 60–70% inhibition of PGE2 production but did not affect COX-2 protein levels.
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