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A Novel Pathway for Tumor Necrosis Factor-α and Ceramide Signaling Involving Sequential Activation of Tyrosine Kinase, p21ras, and Phosphatidylinositol 3-Kinase*

  • Atef N. Hanna
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  • Edmond Y.W. Chan
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  • James Xu
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  • James C. Stone
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  • David N. Brindley
    Correspondence
    Recipient of a Medical Scientist Award from the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Signal Transduction Laboratories, Lipid and Lipoprotein Research Group, and Dept. of Biochemistry, University of Alberta, 357 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada. Tel.: 403-492-2078; Fax: 403-492-3383
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  • Author Footnotes
    * This work was supported by grants from the Medical Research Council of Canada, the Canadian Diabetes Foundation (in honor of Helen Margaret Clery), and the Heart and Stroke Foundation of Alberta (to D. N. B.) and a grant from the National Cancer Institute of Canada (to J. C. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ¶ Recipient of a Research Fellowship from the Alberta Heritage Foundation for Medical Research.
Open AccessPublished:April 30, 1999DOI:https://doi.org/10.1074/jbc.274.18.12722
      Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-α (TNFα) increased phosphatidylinositol (PI) 3-kinase activity by 3–6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFα, sphingomyelinase, and C2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFα was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFα that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.
      Ceramides are important lipid second messengers that are generated through sphingomyelin hydrolysis by sphingomyelinases (
      • Hannun Y.A.
      ). Agonists such as γ-interferon, TNFα (
      • Kim M.-Y.
      • Linardic C.
      • Obeid L.
      • Hannun Y.
      ),
      The abbreviations used are: TNFα, tumor necrosis factor-α; DMEM, Dulbecco's minimum essential medium; EGF, epidermal growth factor; FAK, focal adhesion kinase; IRS-1, insulin receptor substrate-1; MAP, mitogen-activated protein (Erk); PI, phosphatidylinositol; PDGF, platelet-derived growth factor; PP1, 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo{3,4-d}pyrimidine; SH, Src homology domain; d-MAPP, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol
      1The abbreviations used are: TNFα, tumor necrosis factor-α; DMEM, Dulbecco's minimum essential medium; EGF, epidermal growth factor; FAK, focal adhesion kinase; IRS-1, insulin receptor substrate-1; MAP, mitogen-activated protein (Erk); PI, phosphatidylinositol; PDGF, platelet-derived growth factor; PP1, 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo{3,4-d}pyrimidine; SH, Src homology domain; d-MAPP, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol
      interleukin-1 (
      • Mathias S.
      • Younes A.
      • Kan C.-C.
      • Orlow I.
      • Joseph C.
      • Kolesnick R.N.
      ), Fas ligand (
      • Cifone M.G.
      • De Maria R.
      • Roncaioli P.
      • Rippo M.R.
      • Azuma M.
      • Lanier L.L.
      • Santoni A.
      • Testi R.
      ), and nerve growth factor (
      • Dobrowsky R.T.
      • Werner M.H.
      • Castellino A.M.
      • Chao M.V.
      • Hannun Y.A.
      ) activate sphingomyelinases leading to ceramide accumulation. The ability of ceramides to initiate apoptosis in myeloid and lymphoid tumor cell lines as well as in normal lymphocytes is well known (
      • Obeid L.M.
      • Linardic C.M.
      • Karolak L.A.
      • Hannun Y.A.
      ,
      • De Maria R.
      • Boirivant M.
      • Cifone M.G.
      • Roncaioli P.
      • Hahne M.
      • Tschopp J.
      • Pallone F.
      • Santoni A.
      • Testi R.
      ). Depending upon the cell type, however, ceramides can display other effects. Ceramides play an important role in the differentiation of HL-60 cells induced by vitamin D3 (
      • Okazaki T.
      • Bell R.M.
      • Hannun Y.A.
      ), TNFα, and γ-interferon (
      • Kim M.-Y.
      • Linardic C.
      • Obeid L.
      • Hannun Y.
      ). Furthermore, TNFα and ceramides can cause cell proliferation depending on the target cells (
      • Sugarman B.
      ,
      • Aggarwal B.B.
      • Singh S.
      • LaPushin R.
      • Totpal K.
      ,
      • Battegay E.J.
      • Raines E.W.
      • Colbert T.
      • Ross R.
      ,
      • Olivera A.
      • Buckley N.E.
      • Spiegel S.
      ,
      • Hauser J.M.L.
      • Buehrer B.M.
      • Bell R.M.
      ). For example, ceramides stimulate cell division in confluent quiescent Swiss 3T3 fibroblasts (
      • Olivera A.
      • Buckley N.E.
      • Spiegel S.
      ,
      • Hauser J.M.L.
      • Buehrer B.M.
      • Bell R.M.
      ). TNFα-induced proliferation of fibroblasts has been implicated in the pathogenesis of diseases such as rheumatoid arthritis (
      • Gerritsen M.E.
      • Shen C.-P.
      • Perry C.A.
      ), neuroma formation after peripheral nerve damage (
      • Lu G.
      • Beuerman R.W.
      • Zhao S.
      • Sun G.
      • Nguyen D.H.
      • Ma S.
      • Kline D.G.
      ), pulmonary fibrosis (
      • Miyazaki Y.
      • Araki K.
      • Vesin C.
      • Garcia I.
      • Kapanci Y.
      • Whitsett J.A.
      • Piquet P.F.
      ), and chronic intestinal inflammatory disorders such as ulcerative colitis and Crohn's disease (
      • Jobson T.M.
      • Billington C.K.
      • Hall I.P.
      ).
      There are several proposed downstream targets for ceramide action including ceramide-activated protein phosphatase (
      • Dobrowsky R.T.
      • Hannun Y.A.
      ), ceramide-activated protein kinase (
      • Liu J.
      • Mathias S.
      • Yang Z.
      • Kolesnick R.N.
      ), and protein kinase C-ζ (
      • Müller G.
      • Ayoub M.
      • Storz P.
      • Rennecke J.
      • Fabbro D.
      • Pfizenmaier K.
      ). Additionally, we and others found that ceramides inhibit the agonist-induced activation of phospholipase D (
      • Gómez-Muñoz A.
      • Martin A.
      • O'Brien L.
      • Brindley D.N.
      ,
      • Gómez-Muñoz A.
      • Abousalham A.
      • Kikuchi Y.
      • Waggoner D.W.
      • Brindley D.N.
      ). We recently showed that treatment of 3T3-L1 adipocytes for 12 h with C2-ceramide increased the PI 3-kinase activity that was physically associated with IRS-1, and this increased glucose uptake in the absence of insulin (
      • Wang C.-N.
      • O'Brien L.
      • Brindley D.N.
      ). These effects of ceramides mimic those of TNFα, which can increase the tyrosine phosphorylation of IRS-1, its binding of PI 3-kinase (
      • Guo D.
      • Donner D.B.
      ), the synthesis of GLUT1 (
      • Wang C.-N.
      • O'Brien L.
      • Brindley D.N.
      ,
      • McGowam K.M.
      • Police S.
      • Winslow J.B.
      • Pekala P.H.
      ), and the basal uptake of glucose by cells (
      • Evans D.A.
      • Jacobs D.O.
      • Wilmore D.W.
      ).
      PI 3-kinase phosphorylates the 3-position of the inositol ring to produce a family of 3-phosphoinositides that play important roles in cell signaling. In various cell types, PI 3-kinase is implicated in regulating cell growth and inhibiting apoptosis (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Philpott K.L.
      • McCarthy M.J.
      • Klippel A.
      • Rubin L.L.
      ), intracellular vesicle trafficking and secretion (
      • Davidson H.W.
      ,
      • Brown W.J.
      • DeWald D.B.
      • Emr S.D.
      • Plutner H.
      • Balch W.E.
      ,
      • Jones S.M.
      • Howell K.E.
      ), and cytoskeletal organization (
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ,
      • Wymann M.
      • Arcaro A.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ). PI 3-kinase exists as a heterodimer consisting of a p110 catalytic subunit and a p85 regulatory subunit. The p85 subunit contains two SH2 domains, one SH3 domain, a Bcr homology domain, and proline-rich sequences (for review see Refs.
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Kapeller R.
      • Cantley L.C.
      ,
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ), which suggests that PI 3-kinase is regulated by multiple mechanisms. Tyrosine phosphorylated proteins such as the EGF and PDGF receptors IRS-1 and CD28 bind the SH2 domains of the p85 subunit (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Kapeller R.
      • Cantley L.C.
      ,
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ). This binding increases PI 3-kinase activity and, in some situations, causes translocation of PI 3-kinase to plasma membranes to bring it into proximity with its lipid substrates (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.-A.
      • Williams L.T.
      ). Other possible mechanisms regulating PI 3-kinase activation include binding of the SH3 domains of the Src family of tyrosine kinases to the proline-rich sequences on the p85 subunit (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ,
      • Prasad K.V.S.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Liu X.
      • Marengere L.E.M.
      • Koch C.A.
      • Pawson T.
      ), interaction of Cdc42 or Rac with the Bcr homology domain (
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ,
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ), tyrosine phosphorylation of p85, and autophosphorylation of p85 by the p110 kinase at serine 608 (
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). In addition, binding of Ras-GTP to the catalytic domain p110 increases PI 3-kinase activity (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ).
      In this work we investigated whether ceramides are able to stimulate PI 3-kinase in rat2 fibroblasts. We demonstrated that C2-ceramide but not dihydro-C2-ceramide activates PI 3-kinase transiently after 5–20 min through a pathway that involves tyrosine kinase activity and the activation of Ras. Treatment of the fibroblasts with TNFα or sphingomyelinase for 20 min also stimulated PI 3-kinase activity through activation of tyrosine kinase activity and Ras. This work therefore identifies a novel TNFα and ceramide signaling pathway that could contribute to cellular responses such as proliferation of fibroblasts.

      DISCUSSION

      PI 3-kinase can be activated in different cell types by protein-tyrosine kinase- and Ras-dependent pathways (for review see Refs.
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Kapeller R.
      • Cantley L.C.
      ,
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ). These two mechanisms can activate PI 3-kinase synergistically (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). The present work establishes that TNFα, sphingomyelinase, and C2-ceramide all activate PI 3-kinase in rat2 fibroblasts. This process is dependent on tyrosine kinase activity, and it involves an increase in the amount of Ras-GTP. The novelty of the present study is that TNFα, sphingomyelinase, and ceramides should initiate such a signaling pathway.
      The activation of PI 3-kinase observed after incubating rat2 fibroblasts for 10 min with 5–80 μmC2-ceramide was demonstrated by assaying the activity of the enzyme after immunoprecipitation with anti-p85 or anti-phosphotyrosine antibodies. Furthermore, ceramide caused p85 to translocate to membranes where its associated p110 subunit would have access to its lipid substrates. C2-ceramide also increased the tyrosine phosphorylation of p85. The activation of PI 3-kinase was ascribed to C2-ceramide itself rather than metabolism to sphingosine because this rate of conversion was very low (estimated as <0.02% over the 10 min incubations), and the ceramide effect was not diminished by inhibitors of ceramidases. The specificity of the ceramide effect was confirmed because dihydro-C2-ceramide had no significant effect on PI 3-kinase activity.
      Tyrosine kinases could contribute to the activation of PI 3-kinase by several mechanisms. For example, binding the p85 SH2 domains to tyrosine phosphorylated receptors or nonreceptor proteins increases PI 3-kinase activity. This binding also localizes PI 3-kinase to the membrane bringing it into proximity with its lipid substrates thus contributing to the increased PI 3-kinase activity in vivo(
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.-A.
      • Williams L.T.
      ). Alternatively, or additionally, tyrosine phosphorylation events can cause the Ras guanyl nucleotide exchange protein, Sos, to be recruited to the plasma membrane where it can activate Ras and facilitate binding of Ras to p110. Tyrosine phosphorylation of p85 might represent yet another mechanism of PI 3-kinase activation by protein-tyrosine kinases, but this remains controversial. The role of tyrosine kinases in the C2-ceramide-induced activation of PI 3-kinase was confirmed by the inhibition of the ceramide-induced activation by genistein and PP1 and also our observation of increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase (Fig.3 B), FAK, and various proteins in cell lysates after treatment with C2-ceramide (Fig. 6, B andC).
      There is growing evidence linking PI 3-kinase to Ras-mediated signaling. Ras interacts with the p110 catalytic subunit in a GTP-dependent manner (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ), and PI 3-kinase co-immunoprecipitates with Ras (
      • Sjölander A.
      • Yamamoto K.
      • Huber B.E.
      • Lapetina E.G.
      ,
      • Sjölander A.
      • Lapetina E.G.
      ). The involvement of Ras in the activation of PI 3-kinase by C2-ceramide is established by: 1) the lack of PI 3-kinase activation in cells expressing dominant-negative (N17) Ras, 2) the ceramide-induced increase in Ras-GTP in rat2 fibroblasts stably overexpressing wild type Ha-Ras, and 3) the ceramide-induced increase in the physical association of Ras with PI 3-kinase. This last point was demonstrated both by co-precipitation of Ras with PI 3-kinase using the anti-p85 antibody and also by the increase in PI 3-kinase activity found in anti-Ras immunoprecipitates. The idea that PI 3-kinase activation by ceramide involves an association with both tyrosine phosphorylated cellular proteins and Ras-GTP is significant. Our results show that C2-ceramide causes maximal activation of Ras after 5 min. However, the maximum PI 3-kinase activity found in anti-Ras immunoprecipitates occurs after 20 min. This could be because Ras activation is necessary, but alone is not enough to cause a detectable increase in PI 3-kinase activity. It may require concomitant tyrosine phosphorylation of proteins, such as FAK, which is maximal after a 20-min incubation with C2-ceramide (Fig. 6 C). Such a dual regulatory mechanism in other systems causes the synergistic stimulation of PI 3-kinase activity (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ).
      Our results are not limited to the effects of exogenously added ceramide but probably reflect the normal biological signaling events downstream of cell activation by TNFα. This cytokine exerts its effects, in part, by stimulating sphingomyelinase with subsequent accumulation of ceramides (
      • Kim M.-Y.
      • Linardic C.
      • Obeid L.
      • Hannun Y.
      ). TNFα and sphingomyelinase stimulated PI 3-kinase, and these effects were blocked by the tyrosine kinase inhibitors, genistein and PP1. In addition, activation of PI 3-kinase by sphingomyelinase was completely abolished in fibroblasts that expressed N17 Ras, as was the case for C2-ceramide. However, the activation of PI 3-kinase by TNFα in the same cell line was inhibited by only 70%. This indicates that a maximum of about 30% of the TNFα-induced increase in PI 3-kinase is probably mediated by a sphingomyelinase-independent pathway and the formation of ceramides and Ras-GTP. Taken together, our results demonstrate that tyrosine kinase stimulation and formation of Ras-GTP is upstream of PI 3-kinase activation by TNFα as well as by C2-ceramide and long chain ceramide.
      The complete inhibition of the ceramide-induced activation of PI 3-kinase in rat2 cells expressing N17 Ras is striking. Warner et al. (
      • Warner L.C.
      • Hack N.
      • Egan S.E.
      • Goldberg H.J.
      • Weinberg R.A.
      • Skorecki K.L.
      ) showed that stable expression of N17 Ras in rat1 fibroblasts using the same vector as described here causes a significant but partial block to MAP kinase activation in response to EGF treatment. However, in these cells, N17 Ras expression completely blocked activation of phospholipase A2 by EGF. By contrast, Burgering et al. (
      • Burgering B.M.T.
      • Vries-Smits A.M.M.
      • Medema R.H.
      • Weeren P.C.
      • Tertoolen L.G.J.
      • Bos J.L.
      ) found that N17 Ras expressed by means of a vaccinia vector in rat1 fibroblasts did not block EGF stimulation of MAP kinase. Burgering et al. argued that rat fibroblasts possess both Ras-dependent and Ras-independent pathways that function in EGF signaling to MAP kinase. Likewise, we found that N17 Ras expression does not block PI 3-kinase activation by EGF, although there was a 80% inhibition of the PDGF activation of PI 3-kinase (Fig. 5 A). Also N17 Ras expression in rat2 cells decreased the stimulation of MAP kinase by 0.1–100 ng/ml EGF by only about 8%.
      A. N. Hanna and D. N. Brindley, unpublished work.
      In contrast, stimulation of MAP kinase activity by 5 ng/ml PDGF was decreased by 33–43% in rat2 fibroblasts expressing N17 Ras (results not shown). The existence of Ras-independent pathways of MAP kinase activation might explain why expression of N17 Ras had only a modest effect on the growth rate of rat2 cells in our studies. In any case, our results with N17 Ras indicate that sphingomyelinase and C2-ceramide activate PI 3-kinase by a pathway that depends absolutely on Ras-GTP in rat2 fibroblasts. This conclusion is supported by our observation that ceramide treatment leads to activation of wild type Ras.
      The activation of PI 3-kinase by ceramides and TNFα could play an important role in regulating several cellular functions. For example, we demonstrated that cell-permeable ceramides increase PI 3-kinase activity associated with IRS-1 in 3T3-L1 adipocytes (
      • Wang C.-N.
      • O'Brien L.
      • Brindley D.N.
      ). We have also shown that treatment of rat2 cells for 20 min with either C2-ceramide or TNFα leads to activation of MAP kinase.2 These effects are substantially blocked by Ly 294002 and by expression of N17 Ras. Other workers demonstrated that ceramides can activate MAP kinase in some cell types through the stimulation of a ceramide-dependent kinase that activates Raf (
      • Yao B.
      • Zhang Y.
      • Dellkat S.
      • Mathias S.
      • Basu S.
      • Kolesnick R.
      ). Our studies provide an alternative pathway for the ceramide-induced activation of MAP kinase that involves the stimulation of tyrosine kinases, Ras and PI 3-kinase. The conclusion that ceramides can activate mitogenic enzymes such as PI 3-kinase and MAP kinase in confluent rat2 fibroblasts may appear counter-intuitive because ceramides are often associated with producing apoptosis. In an attempt to explain this contradiction, Kolesnick and Fuks (
      • Kolesnick R.
      • Fuks Z.
      ) suggested that the cellular responses to ceramides depend on the genetic component of cells as well as the microenvironment in which the signal is generated.
      The stimulation of fibroblast proliferation by TNFα plays an important role in the pathogenesis of many autoimmune and chronic inflammatory diseases (
      • Gerritsen M.E.
      • Shen C.-P.
      • Perry C.A.
      ,
      • Lu G.
      • Beuerman R.W.
      • Zhao S.
      • Sun G.
      • Nguyen D.H.
      • Ma S.
      • Kline D.G.
      ,
      • Miyazaki Y.
      • Araki K.
      • Vesin C.
      • Garcia I.
      • Kapanci Y.
      • Whitsett J.A.
      • Piquet P.F.
      ,
      • Jobson T.M.
      • Billington C.K.
      • Hall I.P.
      ). TNFα-induced fibroblast proliferation has been reported to be dependent on PDGF secretion (
      • Battegay E.J.
      • Raines E.W.
      • Colbert T.
      • Ross R.
      ), stimulation of c-raf-1 kinase (
      • Belka C.
      • Wiegmann K.
      • Adam D.
      • Holland R.
      • Neuloh M.
      • Herrmann F.
      • Krönke M.
      • Brach M.A.
      ), and MAP kinase activation (
      • Lu G.
      • Beuerman R.W.
      • Zhao S.
      • Sun G.
      • Nguyen D.H.
      • Ma S.
      • Kline D.G.
      ). Our results provide another mechanism for the TNFα-induced proliferation of fibroblasts, which is dependent on PI 3-kinase activation. PI 3-kinase plays a key role in many cell processes such as growth (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Philpott K.L.
      • McCarthy M.J.
      • Klippel A.
      • Rubin L.L.
      ), intracellular vesicle trafficking, secretion (
      • Davidson H.W.
      ,
      • Brown W.J.
      • DeWald D.B.
      • Emr S.D.
      • Plutner H.
      • Balch W.E.
      ,
      • Jones S.M.
      • Howell K.E.
      ), and regulation of the cytoskeleton (
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ,
      • Wymann M.
      • Arcaro A.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ). The demonstration that PI 3-kinase is activated by TNFα and ceramides in a tyrosine kinase- and Ras-GTP-dependent manner identifies a pathway that may contribute to signal transduction by cytokines and other agonists that stimulate sphingomyelinase activities.

      Acknowledgments

      We thank Drs. Y. Hannun and A. Bielawska for their gift of d-MAPP and Dr. S. E. Egan for the vector containing N17 Ras. We are also grateful to Dr. S. Bourgoin for helpful advice and David Li for experimental assistance.

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