Activation of ETB receptors regulates the abundance of ET-1 mRNA in vascular endothelial cells.
OBJECTIVE
The factors that influence the cellular levels of endothelin-1 (ET-1) include transcription, mRNA localization, stability and translation, post-translational maturation of preproET-1 and degradation of ET-1. We investigated the regulation of ET-1 mRNA abundance by extracellular ET-1 in porcine aortic endothelial cells (PAECs).


METHODS
Passsage one cultures of PAECs were incubated in starving medium in the presence or absence of ET-1 and antagonists or pharmacological inhibitors. PreproET-1 mRNA, endothelin-1 promoter activity, Erk and p38 MAPK activation were determined.


RESULTS
Exogenous ET-1 reduced cellular ET-1 mRNA content: a reduction of 10 000-fold was observed after 4 h. ET-1 simultaneously reduced the stability of ET-1 mRNA and increased the loading of RNA Polymerase II at the endothelin-1 promoter. In the absence of exogenous ET-1, the ETB-selective antagonist, BQ788, increased ET-1 mRNA. An ETA-selective antagonist had no effect. ET-1 mRNA returned to control levels within 24 h. Whereas activation of p38 MAPK induced by ET-1 peaked at 30 min and returned to control levels within 90 min, Erk1/2 remained active after 4 h of stimulation. Inhibition of p38 MAPK prevented the ET-1-induced decrease in ET-1 mRNA. In contrast, Erk1/2 inhibition increased ET-1 mRNA. Similarly, inhibition of receptor internalization increased ET-1 mRNA in the presence or absence of exogenous ET-1.


CONCLUSIONS
These results suggest that extracellular ET-1 regulates the abundance of ET-1 mRNA in PAECs, in an ETB receptor-dependent manner, by modulating both mRNA stability and transcription via mechanisms involving receptor endocytosis and both ERK and p38 MAPK pathways.
Farhat
N
N
Department of Biochemistry, University of Montreal, Montreal, Quebec, Canada.
Matouk
C C
CC
Mamarbachi
A M
AM
Marsden
P A
PA
Allen
B G
BG
Thorin
E
E
D000818:Animals; D001011:Aorta / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D042783:Endothelial Cells / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D020871:RNA Stability / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D044023:Receptor, Endothelin B / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D014158:Transcription, Genetic / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D019332:Endothelin-1; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D012319:RNA Polymerase II