O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway.
OBJECTIVE
Glycosylation with β-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway.


RESULTS
Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 μM) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan.


CONCLUSIONS
We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.
Department of Physiology, Medical College of Georgia, 1120 Fifteenth Street, CA-3141, Augusta, GA 30912-3000, USA. vlima@mcg.edu
Victor V
VV
Lima
Fernanda R
FR
Giachini
Fernando S
FS
Carneiro
Maria Helena C
MH
Carvalho
Zuleica B
ZB
Fortes
R Clinton
RC
Webb
Rita C
RC
Tostes
D000117:Acetylglucosamine / Q000378:metabolism*; D000215:Acylation / Q000502:physiology; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002318:Cardiovascular Diseases / Q000378:metabolism; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D019332:Endothelin-1; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000117:Acetylglucosamine