Extracellular export of sphingosine kinase-1 enzyme. Sphingosine 1-phosphate generation and the induction of angiogenic vascular maturation.
The enzyme sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that acts extracellularly on G protein-coupled receptors of the S1P(1)/EDG-1 subfamily. Although S1P is formed in the cytosol of various cells, S1P release is not understood and is controversial because this lipid mediator is also regarded as a second messenger. In this report, we describe the existence of an extracellular S1P-generating system in vascular endothelial cells. Endothelial cells release SK constitutively and form S1P in the range of receptor stimulation. Levels of sphingosine but not ATP in the extracellular environment are rate-limiting. Treatment of endothelial cells with small interfering RNA for SK-1 transcript specifically inhibited SK export, and SK-1-transfected human embryonic kidney 293 cells exhibited enhanced release of SK-1. The export of SK-1 is constitutive and is inhibited by cytochalasin D and treatment at 4 degrees C but not by brefeldin A or nocodazole, suggesting that a nonclassical secretory pathway that requires the actin cytoskeleton dynamics is involved. Because S1P regulates angiogenesis and vascular maturation, we overexpressed SK-1 using an adenoviral vector in vivo in the Matrigel system of angiogenesis. Overexpression of SK-1 resulted in enhanced release of SK activity and induced angiogenesis and vascular maturation. These findings suggest that S1P is made in the extracellular milieu and that extracellular export of SK contributes to the action of S1P in the vascular system.
Ancellin
Nicolas
N
Center for Vascular Biology, Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030-3501, USA.
Colmont
Chantal
C
Su
Joseph
J
Li
Qin
Q
Mittereder
Nanette
N
Chae
Sung-Suk
SS
Stefansson
Steingrimur
S
Liau
Gene
G
Hla
Timothy
T
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D017077:Culture Media, Conditioned; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008246:Lysophospholipids; D018919:Neovascularization, Physiologic; D009865:Oocytes / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering; D022661:RNA, Untranslated / Q000235:genetics; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D012097:Repressor Proteins / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D014162:Transfection; D014981:Xenopus; D029867:Xenopus Proteins; D000074165:Zinc Finger E-box Binding Homeobox 2; D000099157:Sphingosine Kinase
D002118:Calcium; D017077:Culture Media, Conditioned; D018398:Homeodomain Proteins; D017874:Immediate-Early Proteins; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D012097:Repressor Proteins; D013110:Sphingosine; D029867:Xenopus Proteins; D000074165:Zinc Finger E-box Binding Homeobox 2; D000099157:Sphingosine Kinase; C492364:ZEB2 protein, Xenopus; C492363:ZEB2 protein, human; C060506:sphingosine 1-phosphate