Phospholipase A 2 Metabolites Mediate Endothelin Stimulation of the Human Brain Natriuretic Peptide Promoter

Abstract

P155 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 A 2 (PLA 2 ). Thus we studied whether ET and PLA 2 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 -7 M)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 PLA 2 isoforms, transfected cells were treated with the Ca ++ -independent PLA 2 (iPLA 2 )inhibitor bromoenol lactone (BEL), the cytosolic PLA 2 inhibitor methyl arachidonyl fluorophosphonate, or the secretory PLA 2 inhibitor ONO-RS-082 prior to stimulation with ET. Only the iPLA 2 inhibitor BEL prevented ET-stimulated hBNP promoter activity. The PLA 2 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 iPLA 2 , 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.