Nongenomic mechanisms of endothelial nitric oxide synthase activation by the selective estrogen receptor modulator raloxifene.
BACKGROUND
Nontranscriptional signaling through estrogen receptors (ERs) is important in the cardiovascular system. In particular, estrogen stimulates endothelial NO synthase (eNOS) via the phosphatidylinositol 3-kinase (PI3K) pathway. The selective estrogen receptor modulator (SERM) raloxifene is effective for the treatment of postmenopausal osteoporosis, but its ability to activate eNOS via PI3K is unknown.


RESULTS
Human umbilical vein endothelial cells were cultured in estrogen-deprived, phenol red-free medium. Raloxifene stimulated eNOS in a concentration- and time-dependent manner. Activation of eNOS by raloxifene was blocked by the PI3K inhibitor wortmannin and by the ER antagonist ICI 182,780 but not by transcriptional or translational inhibitors. Coimmunoprecipitation studies demonstrated that, in a ligand-dependent manner, raloxifene increased ERalpha-associated p85alpha, p110alpha, and PI3K activity. This correlated temporally with increases in the serine and threonine phosphorylation and activation of protein kinase Akt.


CONCLUSIONS
Our findings indicate that nongenomic ER signaling triggered by a SERM leads to a rapid activation of NO synthesis in human endothelial cells. The ability of raloxifene to facilitate ERalpha-PI3K interaction may provide additional insight into the structure-function relationship of specific SERMs, which promote the nontranscriptional effects of ER.
Simoncini
Tommaso
T
Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Italy.
Genazzani
Andrea R
AR
Liao
James K
JK
D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004967:Estrogens / Q000172:deficiency; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D020849:Raloxifene Hydrochloride / Q000494:pharmacology*; D011960:Receptors, Estrogen / Q000187:drug effects* / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D004791:Enzyme Inhibitors; D004967:Estrogens; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020849:Raloxifene Hydrochloride; D011960:Receptors, Estrogen; C496314:NOS3 protein, human; C494918:AKT1 protein, human