Endothelin-1 impairs nitric oxide signaling in endothelial cells through a protein kinase Cdelta-dependent activation of STAT3 and decreased endothelial nitric oxide synthase expression.
In an ovine model of persistent pulmonary hypertension of the newborn (PPHN), endothelin-1 (ET-1) expression is increased, while endothelial nitric oxide synthase (eNOS) expression is decreased. However, the molecular mechanisms by which ET-1 attenuates eNOS expression in endothelial cells are not completely understood. Thus, the goal of this study was to determine if the overexpression of ET-1 decreases eNOS expression in pulmonary arterial endothelial cells isolated from fetal lambs. To increase the ET-1 expression, cells were transfected with a plasmid coding for Prepro-ET-1, a precursor of ET-1. After overexpression of Prepro-ET-1, ET-1 levels in the culture medium were significantly increased (control = 805.3 +/- 69.8; Prepro-ET-1 overexpression = 1351 +/- 127.9). eNOS promoter activity, protein levels, and NO generation were all significantly decreased by the overexpression of Prepro-ET-1. The decrease in transcription correlated with increased activity of protein kinase Cdelta (PKCdelta) and STAT3. Further, DNA binding activity of STAT3 was also increased by Prepro-ET-1 overexpression. The increase in STAT3 activity and decrease in eNOS promoter activity were inhibited by the overexpression of dominant negative mutants of PKCdelta or STAT3. Further, a 2 bp mutation in the STAT3 binding site in the eNOS promoter inhibited STAT3 binding and led to enhanced promoter activity in the presence of Prepro-ET-1 overexpression. In conclusion, ET-1 secretion is increased by Prepro-ET-1 overexpression. This results in activation of PKCdelta, which phosphorylates STAT3, increasing its binding to the eNOS promoter. This in turn decreases eNOS promoter activity, protein levels, and NO production. Thus, ET-1 can reduce eNOS expression and NO generation in fetal pulmonary artery endothelial cells through PKCdelta-mediated activation of STAT3.
Sud
Neetu
N
Program in Pulmonary Vascular Disease, Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912, USA.
Black
Stephen M
SM
D000818:Animals; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism*; D006976:Hypertension, Pulmonary / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D012756:Sheep; D012757:Sheep Diseases / Q000378:metabolism; D015398:Signal Transduction
D019332:Endothelin-1; D050796:STAT3 Transcription Factor; D052250:Nitric Oxide Synthase Type III; D051745:Protein Kinase C-delta