Hypoxia-induced upregulation of endothelial small G protein RhoA and Rho-kinase/ROCK2 inhibits eNOS expression.
The small G protein RhoA and its downstream effector Rho-kinase/ROCK2 play an important role in regulation of various vasculature cellular functions. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important mediator of vascular homeostasis and cerebral blood flow. Using the human endothelial cell line HUVEC, the present study investigated the role of RhoA and Rho-kinase in endothelial eNOS protein expression under hypoxic conditions as an in vitro model of ischemia. RhoA protein levels in HUVEC were low under normoxic conditions, but were significantly increased after 5h of hypoxia. Endothelial Rho-kinase expression was not detected until after 3h of hypoxia; such expression remained significantly increased after 5h. On the other hand, endothelial eNOS expression was similar after 3h of hypoxia, but was significantly decreased after 5h. The hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of RhoA and significantly inhibited by suppression of RhoA expression by small interfering RNA. The hypoxia-induced decrease in eNOS expression was significantly inhibited when endogenous Rho-kinase activation was inhibited by Rho-binding domain expression. Furthermore, the hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of Rho-kinase. Since expression and activation of RhoA and Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and Rho-kinase by multiple drugs, such as statins or Rho-kinase inhibitors, might provide endothelial and cardiovascular benefits through upregulation of eNOS.
Jin
Hong Guo
HG
Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Hiroshima 734-8551, Japan.
Yamashita
Hiroshi
H
Nagano
Yoshito
Y
Fukuba
Hiromasa
H
Hiji
Masanori
M
Ohtsuki
Toshiho
T
Takahashi
Tetsuya
T
Kohriyama
Tatsuo
T
Kaibuchi
Kozo
K
Matsumoto
Masayasu
M
D015687:Cell Hypoxia; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D015854:Up-Regulation; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein