Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) is an intracellular enzyme that catalyzes the irreversible degradation of S1P. We found SPL to be highly expressed in murine ESCs (mESCs). To investigate the role of SPL in mESC biology, we silenced SPL in mESCs via stable transfection with a lentiviral SPL-specific short hairpin RNA (shRNA) construct. SPL-knockdown (SPL-KD) mESCs showed a 5-fold increase in cellular S1P levels, increased proliferation rates and high expression of cell surface pluripotency markers SSEA1 and OCT4 compared to vector control cells. Compared to control mESCs, SPL-KD cells showed robust activation of STAT3 and a 10-fold increase in S1P 2 expression. Inhibition of S1P 2 or STAT3 reversed the proliferation and pluripotency phenotypes of SPL-KD mESCs. Further, inhibition of S1P 2 attenuated, in a dose-dependent fashion, the high levels of OCT4 and STAT3 activation observed in SPL-KD mESCs. Finally, we showed that SPL-KD cells are capable of generating embryoid bodies from which muscle stem cells, called satellite cells, can be isolated. These findings demonstrate an important role for SPL in ESC homeostasis and suggest that SPL inhibition could facilitate ex vivo ESC expansion for therapeutic purposes.
Maceyka, M.; Harikumar, K.B.; Milstien, S.; Spiegel, S. Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol. 2012, 22, 50–60, doi:10.1016/j.tcb.2011.09.003.
[5]
Kluk, M.J.; Hla, T. Role of the sphingosine 1-phosphate receptor edg-1 in vascular smooth muscle cell proliferation and migration. Circ. Res. 2001, 89, 496–502, doi:10.1161/hh1801.096338.
[6]
Cuvillier, O.; Pirianov, G.; Kleuser, B.; Vanek, P.G.; Coso, O.A.; Gutkind, S.; Spiegel, S. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 1996, 381, 800–803, doi:10.1038/381800a0.
[7]
Spiegel, S.; Milstien, S. The outs and the ins of sphingosine-1-phosphate in immunity. Nat. Rev. Immunol. 2011, 11, 403–415, doi:10.1038/nri2974.
[8]
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.
[9]
Nagata, Y.; Partridge, T.; Matsuda, R.; Zammit, P. Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling. J. Cell Biol. 2006, 174, 245–253, doi:10.1083/jcb.200605028.
[10]
Seitz, G.; Boehmler, A.M.; Kanz, L.; Mohle, R. The role of sphingosine 1-phosphate receptors in the trafficking of hematopoietic progenitor cells. Ann. NY Acad. Sci. 2005, 1044, 84–89.
[11]
Pebay, A.; Bonder, C.S.; Pitson, S.M. Stem cell regulation by lysophospholipids. Prostaglandins Other Lipid Mediat. 2007, 84, 83–97, doi:10.1016/j.prostaglandins.2007.08.004.
[12]
Pyne, N.J.; Long, J.S.; Lee, S.C.; Loveridge, C.; Gillies, L.; Pyne, S. New aspects of sphingosine 1-phosphate signaling in mammalian cells. Adv. Enzyme Regul. 2009, 49, 214–221, doi:10.1016/j.advenzreg.2009.01.011.
[13]
Saba, J.D.; Nara, F.; Bielawska, A.; Garrett, S.; Hannun, Y.A. The bst1 gene of saccharomyces cerevisiae is the sphingosine-1-phosphate lyase. J. Biol. Chem. 1997, 272, 26087–26090, doi:10.1074/jbc.272.42.26087.
[14]
Rodgers, A.; Mormeneo, D.; Long, J.S.; Delgado, A.; Pyne, N.J.; Pyne, S. Sphingosine 1-phosphate regulation of extracellular signal-regulated kinase-1/2 in embryonic stem cells. Stem Cells Dev. 2009, 18, 1319–1330, doi:10.1089/scd.2009.0023.
[15]
Inniss, K.; Moore, H. Mediation of apoptosis and proliferation of human embryonic stem cells by sphingosine-1-phosphate. Stem Cells Dev. 2006, 15, 789–796, doi:10.1089/scd.2006.15.789.
[16]
Pebay, A.; Wong, R.C.; Pitson, S.M.; Wolvetang, E.J.; Peh, G.S.; Filipczyk, A.; Koh, K.L.; Tellis, I.; Nguyen, L.T.; Pera, M.F. Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem Cells 2005, 23, 1541–1548, doi:10.1634/stemcells.2004-0338.
[17]
Harada, J.; Foley, M.; Moskowitz, M.; Waeber, C. Sphingosine-1-phosphate induces proliferation and morphological changes of neural progenitor cells. J. Neurochem. 2004, 88, 1026–1039, doi:10.1046/j.1471-4159.2003.02219.x.
He, X.; H'Ng S, C.; Leong, D.T.; Hutmacher, D.W.; Melendez, A.J. Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells. J. Mol. Cell Biol. 2010, 2, 199–208, doi:10.1093/jmcb/mjq011.
[20]
Weinreb, M.; Shamir, D.; Machwate, M.; Rodan, G.A.; Harada, S.; Keila, S. Prostaglandin e2 (pge2) increases the number of rat bone marrow osteogenic stromal cells (bmsc) via binding the ep4 receptor, activating sphingosine kinase and inhibiting caspase activity. Prostaglandins Leukot. Essent. Fatty Acids 2006, 75, 81–90, doi:10.1016/j.plefa.2006.06.004.
[21]
Wong, R.C.; Tellis, I.; Jamshidi, P.; Pera, M.; Pebay, A. Anti-apoptotic effect of sphingosine-1-phosphate and platelet-derived growth factor in human embryonic stem cells. Stem Cells Dev. 2007, 16, 989–1001, doi:10.1089/scd.2007.0057.
[22]
Avery, K.; Avery, S.; Shepherd, J.; Heath, P.R.; Moore, H. Sphingosine-1-phosphate mediates transcriptional regulation of key targets associated with survival, proliferation, and pluripotency in human embryonic stem cells. Stem Cells Dev. 2008, 17, 1195–1205, doi:10.1089/scd.2008.0063.
[23]
Burdon, T.; Stracey, C.; Chambers, I.; Nichols, J.; Smith, A. Suppression of shp-2 and erk signalling promotes self-renewal of mouse embryonic stem cells. Dev. Biol. 1999, 210, 30–43, doi:10.1006/dbio.1999.9265.
[24]
Aaronson, D.S.; Horvath, C.M. A road map for those who don't know jak-stat. Science 2002, 296, 1653–1655, doi:10.1126/science.1071545.
[25]
Boeuf, H.; Hauss, C.; Graeve, F.D.; Baran, N.; Kedinger, C. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells. J. Cell Biol. 1997, 138, 1207–1217, doi:10.1083/jcb.138.6.1207.
[26]
Abe, K.; Hirai, M.; Mizuno, K.; Higashi, N.; Sekimoto, T.; Miki, T.; Hirano, T.; Nakajima, K. The yxxq motif in gp 130 is crucial for stat3 phosphorylation at ser727 through an h7-sensitive kinase pathway. Oncogene 2001, 20, 3464–3474, doi:10.1038/sj.onc.1204461.
[27]
Lee, H.; Deng, J.; Kujawski, M.; Yang, C.; Liu, Y.; Herrmann, A.; Kortylewski, M.; Horne, D.; Somlo, G.; Forman, S.; et al. Stat3-induced s1pr1 expression is crucial for persistent stat3 activation in tumors. Nat. Med. 2010, 16, 1421–1428, doi:10.1038/nm.2250.
[28]
Niwa, H.; Burdon, T.; Chambers, I.; Smith, A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of stat3. Genes Dev. 1998, 12, 2048–2060, doi:10.1101/gad.12.13.2048.
[29]
Kidder, B.L.; Yang, J.; Palmer, S. Stat3 and c-myc genome-wide promoter occupancy in embryonic stem cells. PLoS One 2008, 3, e3932, doi:10.1371/journal.pone.0003932.
[30]
Newbigging, S.; Zhang, M.; Saba, J.D. Immunohistochemical analysis of sphingosine phosphate lyase expression during murine development. Gene Expr. Patterns 2012, 13, 21–29, doi:10.1016/j.gep.2012.09.001.
[31]
Furusawa, T.; Ohkoshi, K.; Honda, C.; Takahashi, S.; Tokunaga, T. Embryonic stem cells expressing both platelet endothelial cell adhesion molecule-1 and stage-specific embryonic antigen-1 differentiate predominantly into epiblast cells in a chimeric embryo. Biol. Reprod. 2004, 70, 1452–1457, doi:10.1095/biolreprod.103.024190.
[32]
Smith, K.P.; Luong, M.X.; Stein, G.S. Pluripotency: Toward a gold standard for human es and ips cells. J. Cell. Physiol. 2009, 220, 21–29, doi:10.1002/jcp.21681.
[33]
Niwa, H.; Miyazaki, J.; Smith, A.G. Quantitative expression of oct-3/4 defines differentiation, dedifferentiation or self-renewal of es cells. Nat. Genet. 2000, 24, 372–376, doi:10.1038/74199.
[34]
Lee, C.W.; Rivera, R.; Gardell, S.; Dubin, A.E.; Chun, J. Gpr92 as a new g12/13- and gq-coupled lysophosphatidic acid receptor that increases camp, lpa5. J. Biol. Chem. 2006, 281, 23589–23597, doi:10.1074/jbc.M603670200.
[35]
Loh, K.C.; Leong, W.I.; Carlson, M.E.; Oskouian, B.; Kumar, A.; Fyrst, H.; Zhang, M.; Proia, R.L.; Hoffman, E.P.; Saba, J.D. Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a s1pr2/stat3 signaling pathway. PLoS One 2012, 7, e37218, doi:10.1371/journal.pone.0037218.
[36]
Donati, C.; Cencetti, F.; Nincheri, P.; Bernacchioni, C.; Brunelli, S.; Clementi, E.; Cossu, G.; Bruni, P. Sphingosine 1-phosphate mediates proliferation and survival of mesoangioblasts. Stem Cells 2007, 25, 1713–1719, doi:10.1634/stemcells.2006-0725.
[37]
Bandhuvula, P.; Fyrst, H.; Saba, J. A rapid fluorescent assay for sphingosine-1-phosphate lyase enzyme activity. J. Lipid Res. 2007, 48, 2769–2778, doi:10.1194/jlr.D700010-JLR200.
[38]
Spelke, D.P.; Ortmann, D.; Khademhosseini, A.; Ferreira, L.; Karp, J.M. Methods for embryoid body formation: the microwell approach. Methods Mol. Biol. 2011, 690, 151–162, doi:10.1007/978-1-60761-962-8_10.
[39]
Chang, H.; Yoshimoto, M.; Umeda, K.; Iwasa, T.; Mizuno, Y.; Fukada, S.; Yamamoto, H.; Motohashi, N.; Miyagoe-Suzuki, Y.; Takeda, S.; et al. Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells. FASEB J. 2009, 23, 1907–1919, doi:10.1096/fj.08-123661.
