2-2--(hydroxynitrosohydrazono)bis-ethanamine and sphingosine-kinase

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.. 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.. 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.. 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.

Topics: Blotting, Western; Cell Culture Techniques; Cell Line; Cell Movement; Cyclic GMP; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Luciferases, Firefly; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide Donors; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Time Factors; Transfection; Triazenes; Up-Regulation