Sphingosine-1-phosphate inhibition of placental trophoblast differentiation through a Gi-coupled receptor response

The failure of placental trophoblasts to differentiate properly is thought to play an important role in the cause of pregnancy disorders such as preeclampsia. We looked at the effects of the bioactive lipid sphingosine-1-phosphate (S1P) on the differentiation of primary human cytotrophoblasts (CTs) into syncytiotrophoblasts (STs) in culture. We found that S1P inhibited CT differentiation measured by human chorionic gonadotropin (hCG) secretion and the expression of placental alkaline phosphatase but had no effect on their fusion into multinucleated syncytialized cells. G-protein-linked S1P receptors 1, 2, and 3 were found in CTs by reverse transcriptase-polymerase chain reaction, and receptor 1 was found by Western blot analysis. Disruption of Gi signaling with pertussis toxin reversed the inhibitory effects of S1P. S1P reduced intracellular cAMP, and the addition of 8-bromo-cAMP reversed S1P inhibition of hCG secretion.Therefore, we suggest that S1P inhibits the differentiation of CTs into STs through Gi-coupled S1P receptor interaction(s), leading to the inhibition of adenylate cyclase and reduced production of intracellular cAMP. This is the first reported effect of S1P on placental trophoblast function. The failure of placental trophoblasts to differentiate properly is thought to play an important role in the cause of pregnancy disorders such as preeclampsia. We looked at the effects of the bioactive lipid sphingosine-1-phosphate (S1P) on the differentiation of primary human cytotrophoblasts (CTs) into syncytiotrophoblasts (STs) in culture. We found that S1P inhibited CT differentiation measured by human chorionic gonadotropin (hCG) secretion and the expression of placental alkaline phosphatase but had no effect on their fusion into multinucleated syncytialized cells. G-protein-linked S1P receptors 1, 2, and 3 were found in CTs by reverse transcriptase-polymerase chain reaction, and receptor 1 was found by Western blot analysis. Disruption of Gi signaling with pertussis toxin reversed the inhibitory effects of S1P. S1P reduced intracellular cAMP, and the addition of 8-bromo-cAMP reversed S1P inhibition of hCG secretion. Therefore, we suggest that S1P inhibits the differentiation of CTs into STs through Gi-coupled S1P receptor interaction(s), leading to the inhibition of adenylate cyclase and reduced production of intracellular cAMP. This is the first reported effect of S1P on placental trophoblast function. Sphingosine-1-phosphate (S1P) is a member of an important group of signaling sphingolipids now recognized to play a role in a diverse array of cellular processes, such as apoptosis, cell motility, calcium signaling, differentiation, and proliferation (1Maceyka M. Payne S.G. Milstien S. Spiegel S. Sphingosine kinase, sphingosine-1-phosphate, and apoptosis.Biochim. Biophys. Acta. 2002; 1585: 193-201Google Scholar). It is particularly interesting because it appears to have dual functions inside and outside cells (2Spiegel S. Milstien S. Exogenous and intracellularly generated sphingosine 1-phosphate can regulate cellular processes by divergent pathways.Biochem. Soc. Trans. 2003; 31: 1216-1219Google Scholar). The intracellular signaling of S1P is less well characterized because an intracellular target has yet to be identified in mammals, but this mode of action is strongly supported (1Maceyka M. Payne S.G. Milstien S. Spiegel S. Sphingosine kinase, sphingosine-1-phosphate, and apoptosis.Biochim. Biophys. Acta. 2002; 1585: 193-201Google Scholar). Signaling by S1P outside the cell is mediated by extracellular G-protein-coupled membrane receptors, five of which have been identified and characterized (S1P1 to S1P5) (3Siehler S. Manning D.R. Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors.Biochim. Biophys. Acta. 2002; 1582: 94-99Google Scholar). Each receptor signals to multiple downstream responses by coupling to different G-proteins: S1P1 exclusively links to Gi, S1P2 and S1P3 link to Gi, Gq, and G12/13, and S1P4 and S1P5 link to Gi and Gq. Downstream, S1P1/Gi inhibits adenylate cyclase (3Siehler S. Manning D.R. Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors.Biochim. Biophys. Acta. 2002; 1582: 94-99Google Scholar) and stimulates the Ras small GTPase, possibly phospholipase C (4Okamoto H. Takuwa N. Gonda K. Okazaki H. Chang K. Yatomi Y. Shigematsu H. Takuwa Y. EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a Gi/o to multiple signaling pathways, including phospholipase C activation, Ca2+ mobilization, Ras-mitogen-activated protein kinase activation, and adenylate cyclase inhibition.J. Biol. Chem. 1998; 273: 27104-27110Google Scholar), and phosphatidylinositol 3-kinase (5Neves S.R. Ram P.T. Iyengar R. G protein pathways.Science. 2002; 296: 1636-1639Google Scholar). S1P2/Gq linkage leads to calcium mobilization through phospholipase C (6Kupperman E. An S. Osborne N. Waldron S. Stainier D.Y. A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development.Nature. 2000; 406: 192-195Google Scholar). In contrast to S1P1 and S1P3, S1P2 inhibits cell migration by inhibiting Rac (7Ishii I. Fukushima N. Ye X. Chun J. Lysophospholipid receptors: signaling and biology.Annu. Rev. Biochem. 2004; 73: 321-354Google Scholar) despite coupling to the same G-proteins (Gi, Gq, and G12/13) as S1P3. It may also link to Gs, because it stimulates rather than inhibits adenylate cyclase (8Gonda K. Okamoto H. Takuwa N. Yatomi Y. Okazaki H. Sakurai T. Kimura S. Sillard R. Harii K. Takuwa Y. The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways.Biochem. J. 1999; 337: 67-75Google Scholar). S1P3 promotes cell migration through Gi (leading to Rac stimulation) and G12/13 (leading to Rho stimulation) as well as calcium mobilization through Gq; unlike S1P2, it inhibits adenylate cyclase (3Siehler S. Manning D.R. Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors.Biochim. Biophys. Acta. 2002; 1582: 94-99Google Scholar). The distribution of S1P receptors varies across cell types, with S1P1 to S1P3 being widespread and S1P4 and S1P5 being more restricted to lymphocytes, lung, and the central nervous system (7Ishii I. Fukushima N. Ye X. Chun J. Lysophospholipid receptors: signaling and biology.Annu. Rev. Biochem. 2004; 73: 321-354Google Scholar). How S1P functions through receptors 1–3 is thought to depend on the relative concentration of receptor type in individual cells and the corresponding activity of the signaling pathways triggered by their respective G-proteins (9Hla T. Signaling and biological actions of sphingosine 1-phosphate.Pharmacol. Res. 2003; 47: 401-407Google Scholar). The majority of work on S1P actions has centered on its roles in chemotaxis and apoptosis control. However, recently, a role was proposed in cell differentiation (10Kihara A. Ikeda M. Kariya Y. Lee E.Y. Lee Y.M. Igarashi Y. Sphingosine-1-phosphate lyase is involved in the differentiation of F9 embryonal carcinoma cells to primitive endoderm.J. Biol. Chem. 2003; 278: 14578-14585Google Scholar). The villous trophoblast separates maternal from fetal blood in the placenta and comprises immature replicative cytotrophoblasts (CTs) that fuse with and mature into functional but proliferatively senescent syncytiotrophoblasts (STs) (11Benirschke K. Kaufmann P. Pathology of the Human Placenta. 4th edition. Springer Verlag, New York2000Google Scholar). Abnormal differentiation is one of the features of the villous trophoblast of placentas from pregnancies complicated by preeclampsia and the intrauterine growth restriction that accompanies it (12Redman C.W. The placenta, pre-eclampsia and chronic villitis.in: Redman C.W. Sargent I.L. Starkey P.M. The Human Placenta. Blackwell Scientific Publications, London1993: 433-467Google Scholar, 13Arnholdt H. Meisel F. Fandrey K. Lohrs U. Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies.Virchows Arch. B Cell Pathol. 1991; 60: 365-372Google Scholar, 14Mayhew T.M. Ohadike C. Baker P.N. Crocker I.P. Mitchell C. Ong S.S. Stereological investigation of placental morphology in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction.Placenta. 2003; 24: 219-226Google Scholar, 15Lee X. Keith Jr., J.C. Stumm N. Moutsatsos I. McCoy J.M. Crum C.P. Genest D. Chin D. Ehrenfels C. Pijnenborg R. et al.Downregulation of placental syncytin expression and abnormal protein localization in pre-eclampsia.Placenta. 2001; 22: 808-812Google Scholar, 16Crocker I.P. Cooper S. Ong S.C. Baker P.N. Differences in apoptotic susceptibility of cytotrophoblasts and syncytiotrophoblasts in normal pregnancy to those complicated with preeclampsia and intrauterine growth restriction.Am. J. Pathol. 2003; 162: 637-643Google Scholar). Platelet activation is another characteristic of preeclampsia (17Trudinger B.J. Platelets and intrauterine growth retardation in pre-eclampsia.Br. J. Obstet. Gynaecol. 1976; 83: 284-286Google Scholar) and is a major source of intravascular S1P (18Yatomi Y. Ruan F. Hakomori S. Igarashi Y. Sphingosine-1-phosphate: a platelet-activating sphingolipid released from agonist-stimulated human platelets.Blood. 1995; 86: 193-202Google Scholar). Indeed, plasma S1P levels are increased in preeclampsia patients (19Hemmings D.G. Brindley D.N. Davidge S.T. Sphingosine-1-phosphate levels are elevated in plasma and serum samples from preeclamptic women.J. Soc. Gynecol. Investig. 2003; 10: 280AGoogle Scholar). We tested the hypothesis that altered S1P levels or signaling are responsible for the abnormal placental trophoblast differentiation seen in preeclampsia by determining whether S1P affects the differentiation of trophoblasts from uncomplicated placentas. Differentiation of highly purified villous CTs (20Guilbert L.J. Winkler-Lowen B. Sherburne R. Rote N.S. Li H. Morrish D.W. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.Placenta. 2002; 23: 175-183Google Scholar) was measured by three independent methods: fusion into syncytia, expression of placental alkaline phosphatase (PLAP; expressed in the apical membrane of the ST), and secretion of human chorionic gonadotropin (hCG; expressed by the ST). We show that S1P inhibits the functional markers of differentiation (PLAP and hCG), but not fusion, in a 5 day culture with epidermal growth factor (EGF). S1P inhibits through a Gi-coupled S1P receptor response that results in adenylate cyclase inhibition and lower intracellular cAMP. S1P (10 μM; Biomol, Plymouth Meeting, PA), lysophosphatidic acid (LPA) at 1 and 10 μM , the cell-permeable cAMP analog 8-bromo-cAMP (1 mM; Sigma, Oakville, Ontario, Canada), and pertussis toxin (200 ng/ml; Sigma) were added to cell cultures 2–4 h after cell plating. All other chemicals were of the purest grades available commercially. Placentas were obtained with ethics approval of the Capital Health Authority after elective cesarean or normal term delivery from uncomplicated pregnancies at the Royal Alexandra Hospital, Edmonton. Villous CTs (>99.99% pure) (20Guilbert L.J. Winkler-Lowen B. Sherburne R. Rote N.S. Li H. Morrish D.W. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.Placenta. 2002; 23: 175-183Google Scholar) were isolated by trypsin-DNase digestion of minced chorionic tissue and immunoabsorption onto Ig-coated glass bead columns as described previously (20Guilbert L.J. Winkler-Lowen B. Sherburne R. Rote N.S. Li H. Morrish D.W. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.Placenta. 2002; 23: 175-183Google Scholar, 21Yui J. Garcia-Lloret M.I. Brown A.J. Berdan D.W. Morrish D.W. Wegmann T.G. Guilbert L.J. Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells.Placenta. 1994; 15: 231-246Google Scholar) using anti-CD9 (monoclonal antibody 50H.19; house preparation), anti-major histocompatibility complex (MHC) class I (W6/32; Harlan Sera-Lab, Crawley Down, Sussex, UK), and anti-MHC class II (clone 7H3; house preparation) antibodies for immunoelimination. The purified cells were routinely cryopreserved and after thawing were washed twice in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with 10% FBS (GIBCO) and antibiotics (end The cells were at well of in tissue culture and well in and for h at in a of in The cells and were with was with the and the were to the with for culture for the for individual The was obtained from Brindley of and as described previously A. A. Brindley D.N. the of and phospholipase activity by and in Biol. Chem. 1994; Scholar) with the In the h of the h to the cells were to (10 Sigma) and in The cells were with for at washed with and with a of the antibody for with cells were to for and to and washed with were as described cells on were with (10 or 10 not to after with cells were with serum to for 1 h and to a primary antibody for 10 Sigma, or 10 at The cells were washed with and to 1 for washed with and with of for 10 at were obtained with an with a and a of well were obtained with and We Ontario, as and for analysis. was by of cells. was after the at the of in as and the relative of cultures was with was isolated from three trophoblast cultures using (GIBCO) to the was on of using the reverse μM μM and reverse in a of was using the μM and in a of using and of for S1P2, S1P3, and S1P5 were at the of to from the and S1P3 C. R. M. K. Human and multiple J. 2001; Scholar), S1P1 and S1P5 Y. M. B. Sphingosine 1 the chemotaxis of human cells. for G and 3 J. 2002; the for a from the of Health UK), were and were on and of the were and for using a Ontario, was as a for S1P4 and which were not in trophoblast to protein were in with using a serum protein was in by for 5 and on 10% was as described previously using a II system were for in and were onto which were with a in with and for a of 1 The were at with washed twice with the and with antibodies for h at washed and using on to S1P2 and S1P3 using Western were because of the failure of antibodies to S1P2 and S1P3 in Cell were at for 5 at and at A system was to levels with on a cAMP levels were obtained with a were for with (10 and with or without S1P. 1 trophoblasts were with 1 of and cAMP was were obtained the for were at three on trophoblasts isolated from different placentas. Differences for the cell or were by with multiple using the were at term CTs pure) mature in culture to a of trophoblast differentiation E. J.M. and in differentiation of cytotrophoblasts from human term Scholar), with and without FBS D.W. D. H. growth factor differentiation and secretion of human chorionic gonadotropin and placental in normal human Scholar). purified CTs (>99.99% also differentiate in to hCG and fuse in culture to multinucleated cells L.J. Winkler-Lowen B. Sherburne R. Rote N.S. Li H. Morrish D.W. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.Placenta. 2002; 23: 175-183Google Scholar), to a than the less purified cells. The addition of S1P to CTs with serum results in the inhibition of differentiation, as by markers of hCG production and The effect was independent of cell and apoptosis which were not different the not hCG production and were also inhibited by S1P and are to inhibition of the respective differentiation with We have previously that CTs in culture to multinucleated but that it was not for the of hCG production D.W. J. Li H. J. R. Sherburne R. Berdan Guilbert L.J. In human term a for normal primary cells a differentiation that for of Scholar). However, highly purified CTs fuse without to a is whether S1P inhibited the fusion in with its effects on hCG and Therefore, we measured levels of with and S1P and found that was no and cells In strongly fusion, but S1P not reverse this fusion with S1P also its effects through a of G-protein-coupled receptors that are expressed E. of receptors for sphingosine-1-phosphate, and receptors for Biochem. 2004; Scholar). Therefore, we tested at 1 and 10 μM for its effect on proliferation A. A. Brindley D.N. the of and phospholipase activity by and in Biol. Chem. 1994; Scholar) and on trophoblast production of hCG and fusion into multinucleated cells. The was 1 μM 10 μM of cells We that not hCG secretion from trophoblasts at 1 and 10 μM not the of cell fusion in a 5 day We that the of the not trophoblast We identified a of action of S1P. In we show that trophoblasts S1P2, and S1P3 receptor but show no of S1P4 and S1P5 that expression of one of the S1P receptors leads to protein we the of S1P1 protein S1P2, and S1P3 with Gi (5Neves S.R. Ram P.T. Iyengar R. G protein pathways.Science. 2002; 296: 1636-1639Google Scholar). toxin Gi signaling by the (5Neves S.R. Ram P.T. Iyengar R. G protein pathways.Science. 2002; 296: 1636-1639Google Scholar) and inhibits the actions of S1P mediated through the S1P linkage M. T. The G protein-coupled receptor signals via the protein kinase Biol. Chem. Scholar). toxin not the differentiation of as by hCG it reversed the inhibitory effects of S1P results suggest that Gi coupling is not important in differentiation to an but that it the inhibitory action of toxin the inhibitory effect of S1P on hCG secretion. were as described in the to 1 and with pertussis toxin or S1P (10 with medium 5 the hCG levels were and expressed as a of control. are of independent with cells from three different placentas. with different or are different of Gi inhibits the intracellular production of cAMP by cyclase Lee R. A. S. et actions of extracellular through the Gi-coupled receptor and intracellular to regulate proliferation and Cell Biol. 1998; Scholar). cAMP trophoblast differentiation in M. C. The central role of human chorionic gonadotropin in the of human placental 2003; Scholar), we that S1P inhibited differentiation through Gi inhibition of in reduced intracellular cAMP. this we cAMP production with and measured intracellular cAMP in the and of S1P. The addition of S1P to CTs for 1 h in a in intracellular cAMP levels by we found that a cell-permeable analog of cAMP, was to reverse the inhibitory effects of S1P on hCG production added during a 5 day culture that actions are downstream of S1P of 8-bromo-cAMP the inhibitory effect of S1P secretion of were as described in the to 1 and with or without 8-bromo-cAMP (1 and S1P (10 as with medium day hCG levels in the were measured and to the cAMP are of three independent on cells from three this is the first to show a role for S1P in trophoblast results suggest that this bioactive lipid may and the differentiation of CTs into the which as the maternal and fetal is of it be from the replicative CTs pregnancy (11Benirschke K. Kaufmann P. Pathology of the Human Placenta. 4th edition. Springer Verlag, New York2000Google Scholar). that this less may the and the of placental leading to the reduced fetal that is seen in intrauterine growth restriction and of preeclampsia K. A. A. A. in pregnancy and at Scholar). are that S1P is increased in maternal serum in pregnancies complicated by preeclampsia (19Hemmings D.G. Brindley D.N. Davidge S.T. Sphingosine-1-phosphate levels are elevated in plasma and serum samples from preeclamptic women.J. Soc. Gynecol. Investig. 2003; 10: 280AGoogle Scholar) but no on fetal S1P the majority of serum S1P from (18Yatomi Y. Ruan F. Hakomori S. Igarashi Y. Sphingosine-1-phosphate: a platelet-activating sphingolipid released from agonist-stimulated human platelets.Blood. 1995; 86: 193-202Google Scholar) and in preeclampsia is altered in maternal (17Trudinger B.J. Platelets and intrauterine growth retardation in pre-eclampsia.Br. J. Obstet. Gynaecol. 1976; 83: 284-286Google Scholar) and fetal S.C. Chang The effect on fetal by with as well as 1994; Scholar) the trophoblasts CTs and are to S1P levels in pregnancies complicated by preeclampsia. We have that S1P inhibited of three markers of and trophoblast differentiation secretion and expression but not cellular 1, The expression of hCG was to be for or differentiation by hCG of CTs M. C. The central role of human chorionic gonadotropin in the of human placental 2003; Scholar). expression on the of trophoblasts J. of human alkaline phosphatase via phosphatidylinositol to and cell plasma J. Biochem. Scholar), whether this is linked to hCG production or other of the differentiation is The of of the in multinucleated after 5 the addition of was by S1P. This may that the S1P inhibition of hCG secretion by expression by not inhibition to is that hCG secretion and expression are from cell In of the we that with promotes cellular S1P inhibits hCG but not fusion In a receptor antibody fusion but not hCG expression in trophoblasts D.W. J. Li H. J. R. Sherburne R. Berdan Guilbert L.J. In human term a for normal primary cells a differentiation that for of Scholar). It has been that CT differentiation is linked to activation and the of apoptosis S. M. Kaufmann P. B. N. B. fusion of human trophoblast on 2004; Scholar). However, stimulates CT fusion D.W. D. H. growth factor differentiation and secretion of human chorionic gonadotropin and placental in normal human Scholar, M. C. The central role of human chorionic gonadotropin in the of human placental 2003; Scholar) and promotes CT to apoptosis M. J. Winkler-Lowen B. Guilbert L.J. growth factor inhibits apoptosis of primary human Scholar). production of S1P is by and S1P is an for CTs or S1P activation apoptosis is S1P receptors have not previously been identified on Therefore, we first for S1P1 to S1P5 by and identified S1P1 to S1P3. three have been to link to Gi (3Siehler S. Manning D.R. Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors.Biochim. Biophys. Acta. 2002; 1582: 94-99Google Scholar), we pertussis a of Gi (3Siehler S. Manning D.R. Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors.Biochim. Biophys. Acta. 2002; 1582: 94-99Google Scholar, K. Okamoto H. Takuwa N. Yatomi Y. Okazaki H. Sakurai T. Kimura S. Sillard R. Harii K. Takuwa Y. The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways.Biochem. J. 1999; 337: 67-75Google Scholar, M. T. The G protein-coupled receptor signals via the protein kinase Biol. Chem. Scholar), to whether the inhibitory effect of S1P on trophoblast production of hCG was through toxin the inhibitory effects of S1P on hCG production but not the We that S1P but not signaling of hCG production is coupled to are a of that by to cell receptors linked to G-proteins E. of receptors for sphingosine-1-phosphate, and receptors for Biochem. 2004; Scholar). we are not to of S1P signaling the we have that at 1 and 10 μM not or trophoblast differentiation more than We are the expression of receptors to on primary and are we of the effect of on trophoblast of the actions of Gi is to adenylate the in cAMP (3Siehler S. Manning D.R. Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors.Biochim. Biophys. Acta. 2002; 1582: 94-99Google Scholar). cAMP levels were with we found that S1P reduced CTs with a cell-permeable of cAMP stimulates fusion E. K. D. protein and human trophoblast cell differentiation in Cell 1998; Scholar) and differentiation to cells M. C. The central role of human chorionic gonadotropin in the of human placental 2003; Scholar) through with the cAMP response leading to the of hCG M. H. P. and of the in term villous 1999; Scholar), and the leading to the of hCG M. S. B. H. P. H. of the human chorionic during villous trophoblast 2004; Scholar). We found that CTs with 8-bromo-cAMP reversed the inhibitory effects of S1P on hCG that S1P signaling is of purified villous trophoblasts to differentiate into and to hCG E. J.M. and in differentiation of cytotrophoblasts from human term Scholar, D.W. D. H. growth factor differentiation and secretion of human chorionic gonadotropin and placental in normal human Scholar). purified trophoblasts also differentiate in but more and to a L.J. Winkler-Lowen B. Sherburne R. Rote N.S. Li H. Morrish D.W. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.Placenta. 2002; 23: 175-183Google Scholar). However, highly purified cells fuse to in a medium for cells R. Chang D. Guilbert L.J. trophoblasts by multiple with but after Scholar). differentiation and of trophoblasts in culture to depend on the of cells the and and on the medium effects of and are and but can be The of primary trophoblast differentiation by S1P is of a of differentiation in this The the of and for tissue and the for Health for this is a and of the for

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