Tumor Necrosis Factor-α-stimulated Cell Proliferation Is Mediated through Sphingosine Kinase-dependent Akt Activation and Cyclin D Expression

Abstract

Tumor necrosis factor-α (TNF-α) has been shown to activate sphingosine kinase (SphK) in a variety of cell types. The extent to which SphK signaling mediates the pleiotropic effects of TNF-α is not entirely clear. The current study examined the role of SphK activity in TNF-α-stimulated cell proliferation in 1321N1 glioblastoma cells. We first demonstrated that pharmacological inhibitors of SphK markedly decrease TNF-α-stimulated DNA synthesis. Signaling mechanisms through which SphK mediated the effect of TNF-α on DNA synthesis were then examined. Inhibition of Rho proteins with C3 exoenzyme or of Rho kinase with Y27632 attenuated TNF-α-stimulated DNA synthesis. However, RhoA activation by TNF-α was not blocked by SphK inhibition. ERK activation was also required for TNF-α-stimulated DNA synthesis but likewise TNF-α-induced ERK activation was not blocked by inhibition of SphK. Thus, neither RhoA nor ERK activation are the SphK-dependent transducers of TNF-α-induced proliferation. In contrast, TNF-α-stimulated Akt phosphorylation, which was also required for DNA synthesis, was attenuated by SphK inhibition or SphK1 knockdown by small interfering RNA. Furthermore, cyclin D expression was increased by TNF-α in a SphK- and Akt-dependent manner. Additional studies demonstrated that TNF-α effects on DNA synthesis, ERK, and Akt phosphorylation are not mediated through cell surface Gi -coupled S1P receptors, because none of these responses were inhibited by pertussis toxin. We conclude that SphK-dependent Akt activation plays a significant role in TNF-α-induced cyclin D expression and cell proliferation. Tumor necrosis factor-α (TNF-α) has been shown to activate sphingosine kinase (SphK) in a variety of cell types. The extent to which SphK signaling mediates the pleiotropic effects of TNF-α is not entirely clear. The current study examined the role of SphK activity in TNF-α-stimulated cell proliferation in 1321N1 glioblastoma cells. We first demonstrated that pharmacological inhibitors of SphK markedly decrease TNF-α-stimulated DNA synthesis. Signaling mechanisms through which SphK mediated the effect of TNF-α on DNA synthesis were then examined. Inhibition of Rho proteins with C3 exoenzyme or of Rho kinase with Y27632 attenuated TNF-α-stimulated DNA synthesis. However, RhoA activation by TNF-α was not blocked by SphK inhibition. ERK activation was also required for TNF-α-stimulated DNA synthesis but likewise TNF-α-induced ERK activation was not blocked by inhibition of SphK. Thus, neither RhoA nor ERK activation are the SphK-dependent transducers of TNF-α-induced proliferation. In contrast, TNF-α-stimulated Akt phosphorylation, which was also required for DNA synthesis, was attenuated by SphK inhibition or SphK1 knockdown by small interfering RNA. Furthermore, cyclin D expression was increased by TNF-α in a SphK- and Akt-dependent manner. Additional studies demonstrated that TNF-α effects on DNA synthesis, ERK, and Akt phosphorylation are not mediated through cell surface Gi -coupled S1P receptors, because none of these responses were inhibited by pertussis toxin. We conclude that SphK-dependent Akt activation plays a significant role in TNF-α-induced cyclin D expression and cell proliferation. Tumor necrosis factor-α (TNF-α) 4The abbreviations used are: TNF-α, tumor necrosis factor-α; SphK, sphingosine kinase; S1P, sphingosine 1 phosphate; ERK, extracellular signal-regulated kinase; PTX, pertussis toxin; DMS, N,N-dimethylsphingosine; SKI, sphingosine kinase inhibitor; PI3K, phosphoinositide 3-kinase; siRNA, small interfering RNA. is a pleiotropic cytokine that can affect diverse cellular responses. Although TNF-α is cytotoxic in many systems and can promote apoptosis when protein synthesis is inhibited, TNF-α can also act as an anti-apoptotic signal and promote cell survival and proliferation. This is particularly evident in cancer cells, which tend to proliferate in response to TNF-α and are often resistant to TNF-α-induced apoptosis (1Ferrajoli A. Keating M.J. Manshouri T. Giles F.J. Dey A. Estrov Z. Koller C.A. Kurzrock R. Thomas D.A. Faderl S. Lerner S. O'Brien S. Albitar M. Blood. 2002; 100: 1215-1219Crossref PubMed Google Scholar, 2Kirstein M. Baglioni C. J. Cell. Physiol. 1988; 134: 479-484Crossref PubMed Scopus (30) Google Scholar, 3Liu R.Y. Fan C. Liu G. Olashaw N.E. Zuckerman K.S. J. Biol. Chem. 2000; 275: 21086-21093Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 4Wu S. Boyer C.M. Whitaker R.S. Berchuck A. Wiener J.R. Weinberg J.B. Bast Jr., R.C. Cancer Res. 1993; 53: 1939-1944PubMed Google Scholar). We studied a human glioblastoma cell line (1321N1) in which TNF-α acts in this manner and examined the basis for its proliferative effects. TNF-α (5Xia P. Wang L. Moretti P.A. Albanese N. Chai F. Pitson S.M. D'Andrea R.J. Gamble J.R. Vadas M.A. J. Biol. Chem. 2002; 277: 7996-8003Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 6Vann L.R. Payne S.G. Edsall L.C. Twitty S. Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 12649-12656Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 7Osawa Y. Banno Y. Nagaki M. Brenner D.A. Naiki T. Nozawa Y. Nakashima S. Moriwaki H. J. Immunol. 2001; 167: 173-180Crossref PubMed Scopus (144) Google Scholar) and a range of agonists, including tyrosine kinase growth factors (8Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (828) Google Scholar, 9Olivera A. Edsall L. Poulton S. Kazlauskas A. Spiegel S. FASEB J. 1999; 13: 1593-1600Crossref PubMed Scopus (74) Google Scholar), G protein-coupled receptor ligands (10Young K.W. Bootman M.D. Channing D.R. Lipp P. Maycox P.R. Meakin J. Challiss R.A. Nahorski S.R. J. Biol. Chem. 2000; 275: 38532-38539Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 11Meyer zu Heringdorf D. Lass H. Alemany R. Laser K.T. Neumann E. Zhang C. Schmidt M. Rauen U. Jakobs K.H. van Koppen C.J. EMBO J. 1998; 17: 2830-2837Crossref PubMed Scopus (202) Google Scholar), and antigen receptor agonists (12Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 13Choi O.H. Kim J.H. Kinet J.P. Nature. 1996; 380: 634-636Crossref PubMed Scopus (386) Google Scholar), have been shown to activate SphK. SphK has emerged as an important mediator of cell proliferation, particularly in the context of cancer. There is considerable evidence suggesting that SphK can act as an oncogene, contributing to cell transformation (14Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A. Wattenberg B.W. D'Andrea R.J. Vadas M.A. Curr. Biol. 2000; 10: 1527-1530Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Overexpression of SphK1 increases proliferation of C6 glioma (6Vann L.R. Payne S.G. Edsall L.C. Twitty S. Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 12649-12656Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and MCF-7 breast cancer cells (15Nava V.E. Hobson J.P. Murthy S. Milstien S. Spiegel S. Exp. Cell Res. 2002; 281: 115-127Crossref PubMed Scopus (200) Google Scholar) and increases cell cycle progression and promotes tumors in mice (15Nava V.E. Hobson J.P. Murthy S. Milstien S. Spiegel S. Exp. Cell Res. 2002; 281: 115-127Crossref PubMed Scopus (200) Google Scholar). In addition, siRNA-mediated blockade of SphK1 attenuates cell proliferation and arrests the cell cycle in breast cancer cells (16Sarkar S. Maceyka M. Hait N.C. Paugh S.W. Sankala H. Milstien S. Spiegel S. FEBS Lett. 2005; 579: 5313-5317Crossref PubMed Scopus (175) Google Scholar) and in U-1242 MG and U-87 MG glioblastoma cell lines (17Van Brocklyn J.R. Jackson C.A. Pearl D.K. Kotur M.S. Snyder P.J. Prior T.W. J. Neuropathol. Exp. Neurol. 2005; 64: 695-705Crossref PubMed Scopus (289) Google Scholar). Although SphK activation is clearly linked to abnormal cell growth, potential involvement of SphK in TNF-α-induced cell proliferation has not been clarified nor has the mechanism by which SphK enhances proliferation been fully elucidated. Sphingosine 1 phosphate (S1P) is formed when SphK is activated. S1P, similar to TNF-α, can mediate diverse cellular responses, including cell proliferation and protection from apoptosis as well as cell migration and cytoskeletal reorganization (18Maceyka M. Payne S.G. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Crossref PubMed Scopus (508) Google Scholar). S1P can be released from cells to act on cell surface S1P receptors, of which five distinct subtypes coupled to multiple heterotrimeric G proteins have been described (19Sanchez T. Hla T. J. Cell Biochem. 2004; 92: 913-922Crossref PubMed Scopus (416) Google Scholar). S1P has also been suggested to serve as an intracellular signal working through receptor-independent pathways (9Olivera A. Edsall L. Poulton S. Kazlauskas A. Spiegel S. FASEB J. 1999; 13: 1593-1600Crossref PubMed Scopus (74) Google Scholar, 20Olivera A. Rosenfeldt H.M. Bektas M. Wang F. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 46452-46460Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Van 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. J. Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (450) Google Scholar). The studies reported here have explored the role of SphK activation in the proliferative effects of TNF-α, examined intracellular signaling mechanisms underlying the requirement for SphK, and asked whether SphK-mediated TNF-α signaling involves cell surface S1P receptors. Materials—The plasmid for the glutathione S-transferase fusion proteins of the Rho binding domain of the Rho effector rhotekin was provided by Dr. M. Schwartz (22Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1374) Google Scholar). Human TNF-α was purchased from R & D Systems and used at 100 ng/ml. S1P was purchased from Avanti Polar Lipids and used at 5 μm. Pertussis toxin (PTX) was from Calbiochem and used at 100 ng/ml. C3 exoenzyme was from Cytoskeleton, Inc., and was used at 40 μg/ml. N,N-Dimethylsphingosine (DMS) was from Biomol and used at 1 μm. Sphingosine kinase inhibitor (SKI) and PD98059 were purchased from Calbiochem and used at 1 and 5 μm, respectively. Akt inhibitors III and V were also purchased from Calbiochem and used at 10 and 1 μm, respectively. Y27632 was from Calbiochem and used at 10 μm. Anti-RhoA and anti-cyclin D antibodies were from Santa Cruz Biotechnology. Antibodies to phosphorylated p42/44 ERK, total p42/44 ERK, phosphorylated Akt (Ser-473), and total Akt were all purchased from Cell Signaling Technology. [γ-32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Silica gel 60A plates were from Whatman (Florham Park, NJ). Cell Culture—1321N1 human astrocytoma cells were originally isolated from primary cultures of a human cerebral glioblastoma multiforme. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a 37 °C 10% CO2-humidified environment, as described in several previous publications (23Seasholtz T.M. Radeff-Huang J. Sagi S.A. Matteo R. Weems J.M. Cohen A.S. Feramisco J.R. Brown J.H. J. Neurochem. 2004; 91: 501-512Crossref PubMed Scopus (27) Google Scholar, 24Majumdar M. Seasholtz T.M. Goldstein D. de Lanerolle P. Brown J.H. J. Biol. Chem. 1998; 273: 10099-10106Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). DNA Synthesis—Cells were plated in 24-well dishes and serum-starved for 48 to were for in the or of were then with 1 of for with and with were with a of the of 1 for and then 1 were and was by RhoA cells were to on plates and serum-starved for to the were in the or of inhibitors for the were with and 10% 1 100 5 10 and 10 was The cells were and of the was for total RhoA and the was with Rho binding domain to Rho for at and proteins were then to a of with and was to the to on as described with a RhoA RhoA was and to total Cell and were to on plates and serum-starved for to the of the with agonists, the cells were with and then was were and at °C with for were was and were for 5 cell were to proteins were to which were then with for antibodies were at and was at for 1 at were and were to and The cells were with the to the V and human SphK1 were from The that were used were and The was also which a and a were for effects and used for 48 was and was the III first synthesis for to the were used to human SphK1 human or human in with the and by the and of Sphingosine activity was a M. S. Payne S.G. G. 2005; PubMed Scopus Google Scholar). cells were in and then in 1 10 μg/ml 1 100 and 10 μg/ml and at °C with for were and of was with 10 of 1 sphingosine in 5% and 10 The was at at 37 °C and by the of of of and were and were by The in the were by on Silica 60A with and by The to S1P were with a and with to the as of formed of whether TNF-α cell proliferation through a that involves SphK, pharmacological inhibitors with mechanisms of were SphK by with its sphingosine Y. F. T. T. S. Y. 1996; PubMed Scopus Google Scholar), is a inhibitor of the binding of SphK R.S. Lee Y. Cancer Res. 2003; Google Scholar). were with 1 or 1 or for and then with 100 TNF-α for DNA synthesis was and that TNF-α increased and that this response was attenuated with 1 or 1 with SphK inhibitors not decrease DNA synthesis and nor inhibitor apoptosis not the effects of these inhibitors on required for DNA synthesis, examined the effects of and on the response to nor attenuated Thus, SphK to be at an in the signaling by which TNF-α DNA synthesis. We have shown that the small G protein Rho with to cell cycle progression and DNA synthesis in 1321N1 cells M. Seasholtz T.M. Goldstein D. de Lanerolle P. Brown J.H. J. Biol. Chem. 1998; 273: 10099-10106Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, L.R. Goldstein D. Brown J.H. Feramisco J.R. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). The role of Rho in DNA synthesis was by Rho through with C3 exoenzyme van T. N. S. J. Cell Biol. PubMed Scopus Google Scholar). C3 attenuated TNF-α-stimulated Rho TNF-α-induced DNA synthesis was also attenuated when the Rho effector Rho kinase was inhibited with Y27632 RhoA activation was examined by the RhoA the Rho effector TNF-α not activate RhoA at or at but RhoA activation was at and Thus, RhoA activation is not an in TNF-α signaling but be a response through TNF-α signaling whether SphK activation was TNF-α-induced RhoA activation was with the SphK inhibitors or TNF-α-stimulated RhoA activation was not attenuated SphK inhibition with or not Thus, RhoA activation by TNF-α not SphK or its RhoA activation is not SphK. 1321N1 cells were with 10 TNF-α for or or Rho was as described was a of cells were with 10 TNF-α for 1 Rho was as described are of We whether ERK activation was in DNA synthesis whether TNF-α-stimulated ERK signaling was Inhibition of protein signal-regulated kinase the kinase of with 5 PD98059 attenuated TNF-α-stimulated TNF-α increased ERK phosphorylation at and This response was not in cells with or Thus, ERK signaling is in the proliferative effects of TNF-α, ERK activation is not the SphK-dependent mediator of TNF-α-induced DNA synthesis. Akt signaling has been shown to be in human A. G. 2002; PubMed Google Scholar) and can a role in cell proliferation by cell cycle progression J. J.M. Cell 2003; PubMed Scopus Google Scholar), examined the involvement of Akt in TNF-α-induced cell proliferation. Akt inhibitors III and and the inhibitor were used to whether Akt signaling was in the TNF-α-stimulated proliferative the inhibitor and Akt inhibitors III and V were shown to TNF-α-stimulated Akt phosphorylation Akt inhibitors also attenuated TNF-α-stimulated as the inhibitor and Akt activation in TNF-α-stimulated DNA synthesis. whether Akt be the of SphK activation by TNF-α, examined the effects of the SphK inhibitors on TNF-α-stimulated Akt phosphorylation In to was for ERK and Rho inhibition of SphK with or attenuated TNF-α-induced Akt phosphorylation of from demonstrated a inhibition of TNF-α-stimulated Akt phosphorylation by and SphK knockdown with was used as an to SphK and that TNF-α effects on Akt phosphorylation were were first to for knockdown of SphK1 expression or distinct SphK1 were of which demonstrated significant knockdown the not this was used in SphK1 attenuated SphK1 expression with the with significant effect on with to of SphK1 were in that knockdown of SphK in the of SphK activity was of SphK1 of SphK1 in a significant decrease of SphK activity TNF-α-stimulated Akt phosphorylation was then examined 48 of with SphK1 or In cells, TNF-α an in Akt phosphorylation In cells that were with SphK1 siRNA, TNF-α-induced Akt phosphorylation was with that in cells. The effect on TNF-α-stimulated Akt phosphorylation the with pharmacological inhibition of SphK in a significant of SphK1 to TNF-α-induced Akt The effect of Akt on cell proliferation has been suggested to on cell cycle proteins as and cyclin D J. J.M. Cell 2003; PubMed Scopus Google Scholar). We asked whether the by TNF-α expression of these cell cycle TNF-α of the cell cycle inhibitor but SphK inhibitors effect on this response not However, TNF-α also the expression of cyclin a protein that mediates to cell cycle progression SphK inhibition with or attenuated of cyclin D by TNF-α with the Akt inhibitors III and V of TNF-α-stimulated cyclin D expression The a role for SphK signaling and Akt activation in the proliferative response to mechanism by which this is the effects of S1P on G protein-coupled S1P receptors. the of S1P to 1321N1 cells to a similar extent as TNF-α and S1P also ERK and Akt phosphorylation to a similar extent as TNF-α Inhibition of Gi signaling by pertussis toxin (PTX) blocked all of the responses to S1P that extracellular S1P DNA synthesis, ERK, and Akt through coupled to Gi TNF-α S1P, which these responses S1P receptors, then TNF-α also be to demonstrated in TNF-α-stimulated DNA synthesis was to TNF-α-stimulated ERK and Akt phosphorylation were likewise by the that TNF-α S1P, which in ERK, and DNA synthesis through extracellular S1P receptors. evidence that responses to TNF-α not signaling through S1P is that S1P phosphate and in 1321N1 cells, TNF-α not not The here that DNA synthesis in 1321N1 astrocytoma cells involves several signaling including ERK, and of ERK signaling in DNA synthesis is well F. Biochem. J. 1996; PubMed Scopus Google Scholar), and considerable from (23Seasholtz T.M. Radeff-Huang J. Sagi S.A. Matteo R. Weems J.M. Cohen A.S. Feramisco J.R. Brown J.H. J. Neurochem. 2004; 91: 501-512Crossref PubMed Scopus (27) Google Scholar, 24Majumdar M. Seasholtz T.M. Goldstein D. de Lanerolle P. Brown J.H. J. Biol. Chem. 1998; 273: 10099-10106Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and C.J. Nature. 1998; PubMed Scopus Google Scholar, P. A. J. Cell Biol. 2000; PubMed Scopus Google Scholar) Rho signaling in cell proliferation. In contrast, the involvement of SphK1 as a mediator of TNF-α-induced cell proliferation, and in its to affect this response to TNF-α through Akt and cyclin is and Akt is often for its effects on cell but a role for Akt in cell proliferation is well in cancer cells J. J.M. Cell 2003; PubMed Scopus Google Scholar, J.R. A. U. S. A. 2001; PubMed Scopus Google Scholar). this has been linked to the of Akt to the expression of or the kinase inhibitors and in cell cycle progression J. J. T. R. Lee J.H. S. C. R. E. J.M. 2002; PubMed Scopus Google Scholar, Y. M. Cancer Res. 2002; Google Scholar). Akt has also been shown to to cell cycle progression by its to cyclin D for M. C.J. 1998; PubMed Scopus Google Scholar, J.R. M. 2000; PubMed Scopus Google Scholar). cyclin D the to M. C.J. 1998; PubMed Scopus Google Scholar). from the current study that cyclin D is a of Akt through SphK and previous that of SphK1 increases cyclin D expression (15Nava V.E. Hobson J.P. Murthy S. Milstien S. Spiegel S. Exp. Cell Res. 2002; 281: 115-127Crossref PubMed Scopus (200) Google Scholar) by that this can in response to SphK activation by an of signaling is evident in several of human including which have been shown to have in the that to L.C. U. S. A. 1999; PubMed Scopus Google Scholar). SphK is also in cancer cells. SphK1 is in cells with cells T. J. T. M.J. D. FASEB J. PubMed Scopus Google Scholar). In addition, expression of SphK1 was with survival in with (17Van Brocklyn J.R. Jackson C.A. Pearl D.K. Kotur M.S. Snyder P.J. Prior T.W. J. Neuropathol. Exp. Neurol. 2005; 64: 695-705Crossref PubMed Scopus (289) Google Scholar). Although is not whether 1321N1 cells have that activation of Akt or SphK the of TNF-α to signal through these pathways that a significant role in TNF-α signaling studies have shown that TNF-α SphK and that this in the of has in cells (5Xia P. Wang L. Moretti P.A. Albanese N. Chai F. Pitson S.M. D'Andrea R.J. Gamble J.R. Vadas M.A. J. Biol. Chem. 2002; 277: 7996-8003Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), C6 glioma cells (6Vann L.R. Payne S.G. Edsall L.C. Twitty S. Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 12649-12656Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and human Y. Banno Y. Nagaki M. Brenner D.A. Naiki T. Nozawa Y. Nakashima S. Moriwaki H. J. Immunol. 2001; 167: 173-180Crossref PubMed Scopus (144) Google Scholar), SphK is and S1P is increased of TNF-α lines of evidence that S1P from SphK activation can act on cell surface S1P Y. Banno Y. Nagaki M. Brenner D.A. Naiki T. Nozawa Y. Nakashima S. Moriwaki H. J. Immunol. 2001; 167: 173-180Crossref PubMed Scopus (144) Google Scholar, 20Olivera A. Rosenfeldt H.M. Bektas M. Wang F. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 46452-46460Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, A. S. Y. FEBS Lett. 2005; 579: PubMed Scopus Google Scholar). The study to is that of Y. Banno Y. Nagaki M. Brenner D.A. Naiki T. Nozawa Y. Nakashima S. Moriwaki H. J. Immunol. 2001; 167: 173-180Crossref PubMed Scopus (144) Google Scholar), that activation of cell surface was required for TNF-α to activate Akt in However, in to these have demonstrated that DNA synthesis, Akt phosphorylation, and ERK phosphorylation but not responses by Thus, in 1321N1 glioblastoma cells, the of TNF-α to activate Akt signaling SphK to be of the cell surface by from study are with that SphK1 is for cell proliferation, but its effects of G protein-coupled S1P receptors. Olivera (9Olivera A. Edsall L. Poulton S. Kazlauskas A. Spiegel S. FASEB J. 1999; 13: 1593-1600Crossref PubMed Scopus (74) Google Scholar, 20Olivera A. Rosenfeldt H.M. Bektas M. Wang F. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 46452-46460Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) that of SphK1 promotes cell survival and proliferation when and are as well as with PTX, and in from mice with of S1P have been is evidence that S1P can intracellular or affect zu Heringdorf D. Lass H. Alemany R. Laser K.T. Neumann E. Zhang C. Schmidt M. Rauen U. Jakobs K.H. van Koppen C.J. EMBO J. 1998; 17: 2830-2837Crossref PubMed Scopus (202) Google Scholar, C.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google not S1P in the SphK-dependent effects of is that the of TNF-α on SphK of SphK Sphingosine kinase has been described as a the of proliferative and through of sphingosine and to S1P (18Maceyka M. Payne S.G. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Crossref PubMed Scopus (508) Google Scholar). and sphingosine have been shown to mediate apoptosis O. Biochim. Biophys. Acta. 2002; 1585: PubMed Scopus Google Scholar, R. Biochem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). has also been reported to growth and apoptosis by Akt D. M. N. T. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, L. M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, in the activation of SphK decrease of its sphingosine and Akt activation and cell proliferation. inhibition of SphK sphingosine However, TNF-α not apoptosis in 1321N1 cells when SphK was inhibited not Thus, the SphK-dependent effects of TNF-α are a of SphK-mediated Akt phosphorylation and cyclin D expression effects to increased cellular or The of a cell to or to and the extent to which proliferative are activated. TNF-α is to cell in and proliferation in The that TNF-α and in cancer cells is by this We that is the of SphK and Akt activation that this in of survival and proliferation in 1321N1 glioblastoma cells. that TNF-α can act as a for K.W. M. J. Immunol. Google Scholar, S. J. Full Text PDF PubMed Scopus Google not increased DNA synthesis in primary with this not the proliferative effects of TNF-α, mediated through Akt signaling and cyclin be in cancer cells. We Weems for We also Dr. Payne for a and considerable with of the sphingosine kinase