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. 2009 Feb 23;14(3):424–441. doi: 10.2478/s11658-009-0009-1

Chronic increases in sphingosine kinase-1 activity induce a pro-inflammatory, pro-angiogenic phenotype in endothelial cells

Vidya Limaye 1,2,3,✉, Pu Xia 6, Chris Hahn 2, Malcolm Smith 4, Mathew A Vadas 6, Stuart M Pitson 2,5, Jennifer R Gamble 6
PMCID: PMC6275620  PMID: 19238330

Abstract

Sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (ECSK) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. ECSK showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and further showed a more augmented VCAM-1 and E selectin response to TNF compared with empty vector control cells (ECEV). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in ECSK. By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.

Key words: Inflammation, Angiogenesis, Endothelial cells, Sphingosine kinase, TNF

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Abbreviations used

DMS

N′N′-dimethylsphingosine

ECEV

endothelial cells infected with EV

ECG82D

endothelial cells over-expressing dominant-negative SK1

ECSK

endothelial cells over-expressing SK1

E selectin

endothelial selectin

EV

empty vector

GPCR

G-protein coupled receptor

HUVEC

human umbilical vein endothelial cells

IL-1

interleukin 1

PTX

pertussis toxin

S1P

sphingosine 1 phosphate

SK

sphingosine kinase 1

TNF

tumour necrosis factor

VCAM-1

vascular cell adhesion molecule-1

VEGF

vascular endothelial growth factor

References

  • 1.Prendergast R.A., Lutty G.A., Dinarello C.A. Interleukin-1 induces corneal neovascularisation. Fed. Proc. 2002;46:1200. [Google Scholar]
  • 2.Koch A.E., Harlow L.A., Haines G.K., Amento E.P., Unemori E.N., Wong W.L., Pope R.M., Ferrara N. Vascular endothelial growth factor. A cytokine modulating endothelial function in rheumatoid arthritis. J. Immunol. 1994;152:4149–4156. [PubMed] [Google Scholar]
  • 3.Kraling B.M., Razon M.J., Boon L.M., Zurakowski D., Seachord C., Darveau R.P., Mulliken J.B., Corless C.L., Bischoff J. E-selectin is present in proliferating endothelial cells in human hemangiomas. Am. J. Pathol. 1996;148:1181–1191. [PMC free article] [PubMed] [Google Scholar]
  • 4.Nguyen M., Strubel N.A., Bischoff J. A role for sialyl Lewis-X/A glycoconjugates in capillary morphogenesis. Nature. 1993;365:267–269. doi: 10.1038/365267a0. [DOI] [PubMed] [Google Scholar]
  • 5.Koch A.E., Halloran M.M., Haskell C.J., Shah M.R., Polverini P.J. Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1. Nature. 1995;376:517–519. doi: 10.1038/376517a0. [DOI] [PubMed] [Google Scholar]
  • 6.Pyne S., Pyne N.J. Sphingosine 1-phosphate signalling in mammalian cells. Biochem. J. 2000;349:385–402. doi: 10.1042/0264-6021:3490385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Johnson K.R., Johnson K.Y., Becker K.P., Bielawski J., Mao C., Obeid L.M. Role of human sphingosine-1-phosphate phosphatase 1 in the regulation of intra- and extracellular sphingosine-1-phosphate levels and cell viability. J. Biol. Chem. 2003;278:34541–34547. doi: 10.1074/jbc.M301741200. [DOI] [PubMed] [Google Scholar]
  • 8.Xia P., Gamble J.R., Rye K.A., Wang L., Hii C.S., Cockerill P., Khew-Goodall Y., Bert A.G., Barter P.J., Vadas M.A. Tumor necrosis factoralpha induces adhesion molecule expression through the sphingosine kinase pathway. Proc. Natl. Acad. Sci. USA. 1998;95:14196–14201. doi: 10.1073/pnas.95.24.14196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Xia P., Wang L., Gamble J.R., Vadas M.A. Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells. J. Biol. Chem. 1999;274:34499–34505. doi: 10.1074/jbc.274.48.34499. [DOI] [PubMed] [Google Scholar]
  • 10.Olivera A., Spiegel S. Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens. Nature. 1993;365:557–560. doi: 10.1038/365557a0. [DOI] [PubMed] [Google Scholar]
  • 11.Mazurek N., Megidish T., Hakomori S., Igarashi Y. Regulatory effect of phorbol esters on sphingosine kinase in BALB/C 3T3 fibroblasts (variant A31): demonstration of cell type-specific response — a preliminary note. Biochem. Biophys. Res. Commun. 1994;198:1–9. doi: 10.1006/bbrc.1994.1001. [DOI] [PubMed] [Google Scholar]
  • 12.Edsall L.C., Pirianov G.G., Spiegel S. Involvement of sphingosine 1-phosphate in nerve growth factor-mediated neuronal survival and differentiation. J. Neurosci. 1997;17:6952–6960. doi: 10.1523/JNEUROSCI.17-18-06952.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rius R.A., Edsall L.C., Spiegel S. Activation of sphingosine kinase in pheochromocytoma PC12 neuronal cells in response to trophic factors. FEBS Lett. 1997;417:173–176. doi: 10.1016/S0014-5793(97)01277-5. [DOI] [PubMed] [Google Scholar]
  • 14.Choi O.H., Kim J.H., Kinet J.P. Calcium mobilization via sphingosine kinase in signalling by the Fc epsilon RI antigen receptor. Nature. 1996;634:634–636. doi: 10.1038/380634a0. [DOI] [PubMed] [Google Scholar]
  • 15.Melendez A., Floto R.A., Gillooly D.J., Harnett M.M., Allen J.M. Fcgamma RI coupling to phospholipase D initiates sphingosine kinasemediated calcium mobilization and vesicular trafficking. J. Biol. Chem. 1998;273:9393–9402. doi: 10.1074/jbc.273.16.9393. [DOI] [PubMed] [Google Scholar]
  • 16.Wu W., Shu X., Hovsepyan H., Mosteller R.D., Broek D. VEGF receptor expression and signaling in human bladder tumors. Oncogene. 2003;29:3361–3370. doi: 10.1038/sj.onc.1206285. [DOI] [PubMed] [Google Scholar]
  • 17.Wang F., Van Brocklyn J.R., Hobson J.P., Movafagh S., Zukowska-Grojec Z., Milstien S., Spiegel S. Sphingosine 1-phosphate stimulates cell migration through a G(i)- coupled cell surface receptor. Potential involvement in angiogenesis. J. Biol. Chem. 1999;274:35343–35350. doi: 10.1074/jbc.274.50.35343. [DOI] [PubMed] [Google Scholar]
  • 18.Lee M.J., Van Brocklyn J.R., Thangada S., Liu C.H., Hand A.R., Menzeleev R., Spiegel S., Hla T. Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science. 1998;279:1552–1555. doi: 10.1126/science.279.5356.1552. [DOI] [PubMed] [Google Scholar]
  • 19.Lee M.J., Thangada S., Claffey K.P., Ancellin N., Liu C.H., Kluk M., Volpi M., Sha’afi R.I., Hla T. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell. 1999;99:301–312. doi: 10.1016/S0092-8674(00)81661-X. [DOI] [PubMed] [Google Scholar]
  • 20.An S., Zheng Y., Bleu T. Sphingosine 1-phosphate-induced cell proliferation, survival, and related signaling events mediated by G proteincoupled receptors Edg3 and Edg5. J. Biol. Chem. 2000;275:288–296. doi: 10.1074/jbc.275.1.288. [DOI] [PubMed] [Google Scholar]
  • 21.Kimura T., Watanabe T., Sato K., Kon J., Tomura H., Tamama K., Kuwabara A., Kanda T., Kobayashi I., Ohta H., Ui M., Okajima F. Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3. Biochem. J. 2000;348:71–76. doi: 10.1042/0264-6021:3480071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kwon Y.G., Min J.K., Kim K.M., Lee D.J., Billiar T.R., Kim Y.M. Sphingosine 1-phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production. J. Biol. Chem. 2001;276:10627–10633. doi: 10.1074/jbc.M011449200. [DOI] [PubMed] [Google Scholar]
  • 23.Morales-Ruiz M., Lee M.J., Zollner S., Gratton J.P., Scotland R., Shiojima I., Walsh K., Hla T., Sessa W.C. Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells. J. Biol. Chem. 2001;276:19672–19677. doi: 10.1074/jbc.M009993200. [DOI] [PubMed] [Google Scholar]
  • 24.Spiegel S. Sphingosine 1-phosphate: a ligand for the EDG-1 family of Gprotein-coupled receptors. Ann. N. Y. Acad. Sci. 2000;905:54–60. doi: 10.1111/j.1749-6632.2000.tb06537.x. [DOI] [PubMed] [Google Scholar]
  • 25.Ghosh T.K., Bian J., Gill D.L. Intracellular calcium release mediated by sphingosine derivatives generated in cells. Science. 1990;248:1653–1656. doi: 10.1126/science.2163543. [DOI] [PubMed] [Google Scholar]
  • 26.Desai N.N., Zhang H., Olivera A., Mattie M.E., Spiegel S. Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation. J. Biol. Chem. 1992;267:23122–23128. [PubMed] [Google Scholar]
  • 27.Limaye V., Li X., Hahn C., Xia P., Berndt M.C., Vadas M.A., Gamble J.R. Sphingosine kinase-1 enhances endothelial cell survival through a PECAM-1- dependent activation of PI-3K/Akt and regulation of Bcl-2 family members. Blood. 2005;105:3169–3177. doi: 10.1182/blood-2004-02-0452. [DOI] [PubMed] [Google Scholar]
  • 28.Litwin M., Clark K., Noack L., Furze J., Berndt M., Albelda S., Vadas M., Gamble J.R. Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31) J. Cell Biol. 1997;139:219–228. doi: 10.1083/jcb.139.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Pitson S.M., D’andrea R.J., Vandeleur L., Moretti P.A., Xia P., Gamble J.R., Vadas M.A., Wattenberg B.W. Human SK: purification, molecular cloning and characterization of the native and recombinant enzymes. Biochem. J. 2000;350:429–441. doi: 10.1042/0264-6021:3500429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pitson S.M., Moretti P.A., Zebol J.R., Xia P., Gamble J.R., Vadas M.A., D’andrea R.J., Wattenberg B.W. Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase. J. Biol. Chem. 2000;275:33945–33950. doi: 10.1074/jbc.M006176200. [DOI] [PubMed] [Google Scholar]
  • 31.Zannettino A.C., Rayner J.R., Ashman L.K., Gonda T.J., Simmons P.J. A powerful new technique for isolating genes encoding cell surface antigens using retroviral expression cloning. J. Immunol. 1996;156:611–620. [PubMed] [Google Scholar]
  • 32.Roberts J.L., Moretti P.A.B., Darrow A.L., Derian C.K., Vadas M.A., Pitson S.M. An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes. Anal. Biochem. 2004;331:122–129. doi: 10.1016/j.ab.2004.03.030. [DOI] [PubMed] [Google Scholar]
  • 33.Leavesley D.I., Schwartz A., Rosenfeld M., Cheresh D.A. Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J. Cell. Biol. 1993;121:163–170. doi: 10.1083/jcb.121.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Edsall L.C., Spiegel S. Enzymatic measurement of sphingosine 1-phosphate. Anal. Biochem. 1999;272:80–86. doi: 10.1006/abio.1999.4157. [DOI] [PubMed] [Google Scholar]
  • 35.Gamble J.R., Harlan J.M., Klebanoff S.J., Vadas M.A. Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc. Natl. Acad. Sci. USA. 1985;82:8667–8671. doi: 10.1073/pnas.82.24.8667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Gamble J.R., Khew-Goodall Y., Vadas M.A. Transforming growth factor-beta inhibits E-selectin expression on human endothelial cells. J. Immunol. 1993;150:4494–4503. [PubMed] [Google Scholar]
  • 37.Sadeghi M.M., Collinge M., Pardi R., Bender J.R. Simvastatin modulates cytokine-mediated endothelial cell adhesion molecule induction: involvement of an inhibitory G protein. J. Immunol. 2000;165:2712–2718. doi: 10.4049/jimmunol.165.5.2712. [DOI] [PubMed] [Google Scholar]
  • 38.Yamauchi J., Miyamoto Y., Kokubu H., Nishii H., Okamoto M., Sugawara Y., Hirasawa A., Tsujimoto G., Itoh H. Endothelin suppresses cell migration via the JNK signaling pathway in a manner dependent upon Src kinase, Rac1, and Cdc42. FEBS Lett. 2002;11:284–288. doi: 10.1016/S0014-5793(02)03231-3. [DOI] [PubMed] [Google Scholar]
  • 39.Okamoto H., Yatomi Y., Ohmori T., Satoh K., Matsumoto Y., Ozaki Y. Sphingosine 1-phosphate stimulates G(i)- and Rho-mediated vascular endothelial cell spreading and migration. Thromb. Res. 2000;99:259–265. doi: 10.1016/S0049-3848(00)00251-6. [DOI] [PubMed] [Google Scholar]
  • 40.Ancellin N., Colmont C., Su J., Li Q., Mittereder N., Chae S.S., Stefansson S., Liau G., Hla T. Extracellular export of sphingosine kinase-1 enzyme. Sphingosine 1- phosphate generation and the induction of angiogenic vascular maturation. J. Biol. Chem. 2002;277:6667–6675. doi: 10.1074/jbc.M102841200. [DOI] [PubMed] [Google Scholar]
  • 41.Hoang M.V., Whelan M.C., Senger D.R. Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Proc. Natl. Acad. Sci. USA. 2004;101:1874–1879. doi: 10.1073/pnas.0308525100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Bayless K.J., Davis G.E. Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices. Biochem. Biophys. Res. Commun. 2003;26:903–913. doi: 10.1016/j.bbrc.2003.11.017. [DOI] [PubMed] [Google Scholar]
  • 43.Van Brocklyn J.R., Lee M.J., Menzeleev R., Olivera A., Edsall L., Cuvillier O., Thomas D.M., Coopman P.J., Thangada S., Liu C.H., Hla T., Spiegel S. Dual actions of sphingosine-1-phosphate: extracellular through the Gi- coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J. Cell. Biol. 1998;142:229–240. doi: 10.1083/jcb.142.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Yoon M.J., Cho C.H., Lee C.S., Jang I.H., Ryu S.H., Koh G.Y. Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells. Biochem. Biophys. Res. Commun. 2003;308:101–105. doi: 10.1016/S0006-291X(03)01341-X. [DOI] [PubMed] [Google Scholar]
  • 45.Haskell H., Natarajan M., Hecker T.P., Ding Q., Stewart J., Jr., Grammer J.R., Gladson C.L. Focal adhesion kinase is expressed in the angiogenic blood vessels of malignant astrocytic tumors in vivo and promotes capillary tube formation of brain microvascular endothelial cells. Clin. Cancer Res. 2003;9:2157–2169. [PubMed] [Google Scholar]
  • 46.Li X., Stankovic M., Bonder C.S., Hahn C.N., Parsons M., Pitson S.M., Proia R.L., Vadas M.A., Gamble J.R. Basal and angiopoietin-1-mediated endothelial permeability is regulated by sphingosine kinase-1. Blood. 2008;111:3489–3497. doi: 10.1182/blood-2007-05-092148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Esser S., Lampugnani M.G., Corada M., Dejana E., Risau W. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J. Cell. Sci. 1998;111:1853–1865. doi: 10.1242/jcs.111.13.1853. [DOI] [PubMed] [Google Scholar]
  • 48.Ferrero E., Villa A., Ferrero M.E., Toninelli E., Bender J.R., Pardi R., Zocchi M.R. Tumor necrosis factor -induced vascular leakage involves PECAM1 phosphorylation. Cancer Res. 1996;56:3211–3215. [PubMed] [Google Scholar]
  • 49.Kitano M., Hla T., Sekiguchi M., Kawahito Y., Yoshimura R., Miyazawa K., Iwasaki T., Sano H., Saba J.D., Tam Y.Y. Sphingosine 1-phosphate/sphingosine 1-phosphate receptor 1 signaling in rheumatoid synovium: regulation of synovial proliferation and inflammatory gene expression. Arthritis Rheum. 2006;54:742–753. doi: 10.1002/art.21668. [DOI] [PubMed] [Google Scholar]
  • 50.Pi X., Tan S.Y., Hayes M., Xiao L., Shayman J.A., Ling S., Holoshitz J. Sphingosine kinase 1-mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells. Arthritis Rheum. 2006;54:754–764. doi: 10.1002/art.21635. [DOI] [PubMed] [Google Scholar]
  • 51.Tan S.Y., Xiao L., Pi X., Holoshitz J. Aberrant Gi protein coupled receptor-mediated cell survival signaling in rheumatoid arthritis B cell lines. Front Biosci. 2007;12:1651–1660. doi: 10.2741/2177. [DOI] [PubMed] [Google Scholar]
  • 52.Michaud J., Kohno M., Proia R.L., Hla T. Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice. FEBS Lett. 2006;580:4607–4612. doi: 10.1016/j.febslet.2006.07.035. [DOI] [PubMed] [Google Scholar]
  • 53.Maines L.W., Fitzpatrick L.R., French K.J., Zhuang Y., Xia Z., Keller S.N., Upson J.J., Smith C.D. Suppression of Ulcerative Colitis in Mice by Orally Available Inhibitors of Sphingosine Kinase. Dig. Dis. Sci. 2008;53:997–1012. doi: 10.1007/s10620-007-0133-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Shu X., Wu W., Mosteller R.D., Broek D. Sphingosine kinase mediated vascular endothelial growth-factor-induced activation of ras and mitogen-activated protein kinases. Mol. Cell Biol. 2002;22:7758–7768. doi: 10.1128/MCB.22.22.7758-7768.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Limaye, V., Vadas, M.A., Pitson, S.M. and Gamble, J.R. The effects of markedly raised intracellular sphingosine kinase-1 activity in endothelial cells. Cell. Mol. Biol. Lett.14 (2009) in press, DOI: 10.2478/s11658-009-0008-2. [DOI] [PMC free article] [PubMed]

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