Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells.
Raloxifene is a tissue-selective estrogen receptor modulator. The effect of estrogen on cardiovascular disease is mainly dependent on direct actions on the vascular wall involving activation of endothelial nitric oxide synthase (eNOS) via Akt and extracellular signal-regulated protein kinase (ERK) cascades. Although raloxifene is also known to activate eNOS in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), the raloxifene analog LY117018 caused acute phosphorylation of eNOS that was unaffected by actinomycin D and was blocked by the pure estrogen receptor antagonist ICI182,780. Activation of Akt by raloxifene reached a plateau at 15-30 min and declined thereafter, a similar time frame to that of Akt activation by 17beta-estradiol. On the other hand, both activation and phosphorylation of ERK by raloxifene showed a biphasic pattern (peaks at 5 min and 1 h), whereas ERK activation and phosphorylation by 17beta-estradiol reached a plateau at 5 min and declined thereafter. A MEK inhibitor, PD98059, had no effect on the raloxifene-induced Akt activity, suggesting an absence of cross-talk between the ERK and Akt cascades. Either exogenous expression of a dominant-negative Akt or pretreatment of TRLECs with PD98059 decreased the raloxifene-induced eNOS phosphorylation. Moreover, raloxifene stimulated the activation of Akt, ERK, and eNOS in Chinese hamster ovary cells expressing estrogen receptor alpha but not Chinese hamster ovary cells expressing estrogen receptor beta. Our findings suggest that raloxifene-induced eNOS phosphorylation is mediated by estrogen receptor alpha via a nongenomic mechanism and is differentially mediated by Akt- and ERK-dependent cascades.
Hisamoto
K
K
Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
Ohmichi
M
M
Kanda
Y
Y
Adachi
K
K
Nishio
Y
Y
Hayakawa
J
J
Mabuchi
S
S
Takahashi
K
K
Tasaka
K
K
Miyamoto
Y
Y
Taniguchi
N
N
Murata
Y
Y
D000818:Animals; D016466:CHO Cells; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D004967:Estrogens / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D000077267:Fulvestrant; D006801:Humans; D008168:Lung / Q000166:cytology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011759:Pyrrolidines / Q000494:pharmacology*; D020849:Raloxifene Hydrochloride / Q000494:pharmacology*; D051381:Rats; D011959:Receptors, Estradiol / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology*; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology
D004791:Enzyme Inhibitors; D004965:Estrogen Antagonists; D004967:Estrogens; D005419:Flavonoids; D011518:Proto-Oncogene Proteins; D011759:Pyrrolidines; D011959:Receptors, Estradiol; D013876:Thiophenes; D000077267:Fulvestrant; D020849:Raloxifene Hydrochloride; D004958:Estradiol; C025430:LY 117018; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one