Phospholipase A2 Metabolites Mediate Endothelin Stimulation of the Human Brain Natriuretic Peptide Promoter
Brain natriuretic peptide (BNP) gene expression accompanies cardiac hypertrophy and heart failure. The vasoconstrictor endothelin-1 (ET)may be involved in the development of these diseases. ET has also been shown to activate phospholipase A2 (PLA2). Thus we studied whether ET and PLA2 metabolites regulate BNP gene expression. The hBNP promoter (-1818 to + 100) coupled to a luciferase reporter gene was transferred into neonatal ventricular myocytes (NVM),and luciferase activity was measured as an index of promoter activity. ET (10-7M)induced BNP mRNA in NVM as assessed by Northern blot. It also stimulated the hBNP promoter 4-fold vs control, an effect completely inhibited by actinomycin D. To test the involvement of different PLA2 isoforms, transfected cells were treated with the Ca++-independent PLA2 (iPLA2)inhibitor bromoenol lactone (BEL), the cytosolic PLA2 inhibitor methyl arachidonyl fluorophosphonate, or the secretory PLA2 inhibitor ONO-RS-082 prior to stimulation with ET. Only the iPLA2 inhibitor BEL prevented ET-stimulated hBNP promoter activity. The PLA2 metabolite lysophosphatidic acid (LPA) also activated the hBNP promoter (2.2-fold; n = 3), but lysophosphatidylcholine did not. To test whether arachidonic acid metabolites are involved in ET’s effect, cells were pretreated with either a lipoxygenase (LO), cyclooxygenase, or p450 monooxygenase inhibitor. Only the LO inhibitor baicalein prevented ET stimulation of the hBNP promoter. Finally, we studied the involvement of cis elements in ET-stimulated hBNP promoter activity. Deletion of BNP promoter sequences from -1818 to -408 and from -408 to -40 reduced ET’s effect by 54% and 78%, respectively. Moreover, ET-stimulated luciferase activity was reduced by 53% when the GATA element (at position -85 relative to the start site of transcription) was mutated. These data suggest that: 1) ET activates the hBNP promoter through a transcriptional mechanism; 2) LPA, perhaps generated by a BEL-sensitive iPLA2, is involved in ET’s effect; 3) a LO pathway may also mediate ET signaling; and 4) ET regulation of the hBNP promoter targets both distal and proximal cis elements, including GATA.