Sphingosine 1-phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production.
Sphingosine 1-phosphate (S1P) can prevent endothelial cell apoptosis. We investigated the molecular mechanisms and signaling pathways by which S1P protects endothelial cells from serum deprivation-induced apoptosis. We show here that human umbilical vein endothelial cells (HUVECs) undergo apoptosis associated with increased DEVDase activity, caspase-3 activation, cytochrome c release, and DNA fragmentation after 24 h of serum deprivation. These apoptotic markers were suppressed by the addition of S1P, the NO donor S-nitroso-N-acetylpenicillamine (100 micrometer), or caspase-3 inhibitor z-VAD-fmk. The protective effects of S1P were reversed by the nitric-oxide synthase (NOS) inhibitor N-monomethyl-l-arginine, but not by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]-quanoxaline-1-one, suggesting that NO, but not cGMP, is responsible for S1P protection from apoptosis. Furthermore, S1P increased NO production by enhancing Ca(2+)-sensitive NOS activity without changes in the eNOS protein level. S1P-mediated cell survival and NO production were suppressed significantly by pretreatment with antisense oligonucleotide of EDG-1 and partially by EDG-3 antisense. S1P-mediated NO production was suppressed by the addition of pertussis toxin, an inhibitor of G(i) proteins, the specific inhibitor of phospholipase C (PLC), and the Ca(2+) chelator BAPTA-AM. These findings indicate that S1P protects HUVECs from apoptosis through the activation of eNOS activity mainly through an EDG-1 and -3/G(i)/PLC/Ca(2+) signaling pathway.
Kwon
Y G
YG
Department of Biochemistry, College of Natural Sciences, Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwon-do 200-701, Korea. ygkwon@cc.kangwon.ac.kr
Min
J K
JK
Kim
K M
KM
Lee
D J
DJ
Billiar
T R
TR
Kim
Y M
YM
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013110:Sphingosine / Q000494:pharmacology*
D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D009569:Nitric Oxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D013110:Sphingosine