Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation.
OBJECTIVE
Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.


METHODS
We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation.


RESULTS
We showed that exposure of EA.hy926 cells to Deta-NO (125-1000 microM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 microM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1.


CONCLUSIONS
These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis.
Schwalm
Stephanie
S
Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt am Main, Germany.
Pfeilschifter
Josef
J
Huwiler
Andrea
A
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D009569:Nitric Oxide / Q000378:metabolism / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014226:Triazenes / Q000494:pharmacology; D015854:Up-Regulation; D000099157:Sphingosine Kinase
D006152:Cyclic GMP; D049409:Luciferases, Firefly; D009569:Nitric Oxide; D020030:Nitric Oxide Donors; D017853:Phosphotransferases (Alcohol Group Acceptor); D034741:RNA, Small Interfering; D014226:Triazenes; D000099157:Sphingosine Kinase; C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene