Open AccessEndothelin-1 Impairs Nitric Oxide Signaling in Endothelial Cells Through a Protein Kinase Cδ-Dependent Activation of STAT3 and Decreased Endothelial Nitric Oxide Synthase Expression
Author(s) -
Neetu Sud,
Stephen M. Black
Publication year - 2009
Publication title -
dna and cell biology
Language(s) - English
Resource type - Journals
eISSN - 1557-7430
pISSN - 1044-5498
DOI - 10.1089/dna.2009.0865
Subject(s) - enos , biology , microbiology and biotechnology , endothelin 1 , protein kinase c , nitric oxide synthase , transfection , nitric oxide synthase type iii , nitric oxide , stat3 , cell culture , phosphorylation , endocrinology , biochemistry , receptor , genetics
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.