Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism.
Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.
pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Huwiler@em.uni-frankfurt.de
Andrea
A
Huwiler
Frauke
F
Döll
Shuyu
S
Ren
Sabine
S
Klawitter
Anna
A
Greening
Isolde
I
Römer
Svetlana
S
Bubnova
Luise
L
Reinsberg
Josef
J
Pfeilschifter
D001158:Arteries / Q000166:cytology*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D003513:Cycloheximide / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006632:Histamine / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D000099157:Sphingosine Kinase
D003513:Cycloheximide; D004791:Enzyme Inhibitors; D006632:Histamine; D007527:Isoenzymes; D017853:Phosphotransferases (Alcohol Group Acceptor); D051571:Protein Kinase C-alpha; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D013755:Tetradecanoylphorbol Acetate; D000099157:Sphingosine Kinase