Nitric Oxide Regulates Shear Stress-Induced Early Growth Response-1: Expression via the Extracellular Signal-Regulated Kinase Pathway in Endothelial Cells

Endothelial cells (ECs) subjected to shear stress constantly release nitric oxide (NO). The effect of NO on shear stress-induced endothelial responses was examined. ECs subjected to shear stress induced a transient and shear force-dependent increase in early growth response-1 (Egr-1) mRNA levels. Treatment of ECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1), inhibited this shear stress-induced Egr-1 expression. Conversely, an NO synthase inhibitor to ECs, N-monomethyl-L-arginine, augmented this Egr-1 expression. NO modulation of Egr-1 expression was demonstrated by functional analysis of Egr-1 promoter activity using a chimera containing the Egr-1 promoter region (−698 bp) and reporter gene luciferase. In contrast to the enhanced promoter activity after N-monomethyl-L-arginine treatment, shear stress-induced Egr-1 promoter activity was attenuated after ECs were treated with an NO donor. ECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK) inhibited shear stress-induced Egr-1 promoter activity. NO modulation of the signaling pathway was shown by its inhibitory effect on shear stress-induced ERK1/ERK2 phosphorylation and activity. This inhibitory effect was further substantiated by the inhibition of NO on both the shear stress-induced transcriptional activity of Elk-1 (an ERK substrate) and the promoter activity of a reporter construct containing serum response element. NO-treated ECs resulted in a reduction of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. These results indicate that shear stress-induced Egr-1 expression is modulated by NO via the ERK signaling pathway in ECs. Our findings support the importance of NO as a negative regulator in endothelial responses to hemodynamic forces.