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Roles of Phosphatidylinositol 3-Kinase and Rac in the Nuclear Signaling by Tumor Necrosis Factor-α in Rat-2 Fibroblasts*

Open AccessPublished:August 20, 1999DOI:https://doi.org/10.1074/jbc.274.34.24372
      We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-α leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-α. Similarly, LY294002 significantly diminished TNF-α-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-α is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-α to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-α signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-α.
      PI 3-kinase
      phosphatidylinositol 3-kinase
      TNF-α
      tumor necrosis factor-α
      JNK
      c-Jun amino-terminal kinase
      PLA2
      phospholipase A2
      cPLA2
      cytosolic phospholipase A2
      DMEM
      Dulbecco's modified Eagle's medium
      PBS
      phosphate-buffered saline
      SRE
      serum response element
      PBS-T
      PBS plus Tween 20
      TNFR1
      tumor necrosis factor receptor-1
      TRAF2
      TNFR-associated factor-2
      FBS
      fetal bovine serum
      Phosphatidylinositol 3-kinase (PI 3-kinase)1 is a lipid kinase involved in mitogenic signal transduction and cellular transformation (
      • Vanhaesebroeck B.
      • Leevers S.
      • Panayotou G.
      • Waterfield P.
      ). Evidence from intact cells suggests that PI 3-kinase is activated by a variety of growth factors and exerts its cellular effects by elevating of phosphatidylinositol (3,4,5)-triphosphate levels (
      • Vanhaesebroeck B.
      • Leevers S.
      • Panayotou G.
      • Waterfield P.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield P.
      • Downward J.
      ). In mammalian cells, PI 3-kinase is required for growth factor-induced changes of the actin cytoskeleton that are mediated by Rac, a member of Rho family GTPases (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ,
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ,
      • Nobes C.D.
      • Hawkins P.
      • Stephens L.
      • Hall A.
      ). For example, an inhibition of PI 3-kinase was shown to block growth factor induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ,
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ). Thus, Rac appears to lie downstream of PI 3-kinase within a signaling pathway that controls actin remodeling.
      Rac is also crucially involved in the regulation of signal transduction cascades to the nucleus evoked by environmental stresses and proinflammatory cytokines; elements of such cascades include c-Jun amino-terminal kinase (JNK) (
      • Coso O.A.
      • Chiariello M., Yu, J.C.
      • Teramoto H.
      • Crespo P.
      • Xu N.
      • Miki T.
      • Gutkind J.S.
      ,
      • Minden A.
      • Lin A.
      • Claret F.X.
      • Abo A.
      • Karin M.
      ), c-fos serum response element (SRE) (
      • Hill C.S.
      • Wynne J.
      • Treisman R.
      ,
      • Kim B.C.
      • Kim J.H.
      ,
      • Kim J.H.
      • Kwack H.J.
      • Choi S.E.
      • Kim B.C.
      • Kim Y.S.
      • Kang I.J.
      • Kumar C.C.
      ), p70S6 kinase (
      • Chou M.M.
      • Blenis J.
      ), and the transcription factor NF-κB (
      • Perona R.
      • Montaner S.
      • Saniger L.
      • Sanchez-Perez I.
      • Bravo R.
      • Lacal J.C.
      ). For instance, in response to exogenous application of H2O2 or ceramide, a second messenger product of sphingomyelin hydrolysis by sphingomyelinase (
      • Verheij M.
      • Bose R.
      • Lin X.H.
      • Yao B.
      • Jarvis W.D.
      • Grant S.
      • Birrer M.J.
      • Szabo E.
      • Zon L.I.
      • Kyriakis J.M.
      • Haimovitz-Friedman A.
      • Fuks Z.
      • Kolesnick R.N.
      ), c-fos SRE, was activated via a Rac-dependent signaling pathway, suggesting a role of Rac in stress-induced gene regulation (
      • Kim B.C.
      • Kim J.H.
      ,
      • Kim J.H.
      • Kwack H.J.
      • Choi S.E.
      • Kim B.C.
      • Kim Y.S.
      • Kang I.J.
      • Kumar C.C.
      ). Although the role of PI 3-kinase in the regulation of Rac-mediated membrane ruffling has been well studied (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ,
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ,
      • Nobes C.D.
      • Hawkins P.
      • Stephens L.
      • Hall A.
      ), almost nothing is known about the potential role of PI 3-kinase in Rac-mediated gene regulation in response to environmental stress or proinflammatory cytokines.
      Tumor necrosis factor (TNF)-α is one of the most pleiotropic proinflammatory cytokines, signaling a large number of cellular responses, including cytotoxicity, antiviral activity, fibroblast proliferation, and the transcriptional regulation of various genes (
      • Tartaglia L.A.
      • Ayres T.M.
      • Wong G.H.W.
      • Goeddel D.V.
      ). It is known that a large majority of the pleiotropic activities of TNF are signaled by the TNF receptor-1 (TNFR1; Refs.
      • Engelmann H.
      • Holtmann H.
      • Brakebush C.
      • Avni Y.S.
      • Sarov I.
      • Nophar Y.
      • Hadas E.
      • Leitner O.
      • Wallach D.
      ,
      • Espevik T.
      • Brockhaus M.
      • Loetscher H.
      • Nonstad U.
      • Shalaby R.
      ,
      • Wong G.H.W.
      • Tartaglia L.A.
      • Lee M.S.
      • Goeddel D.V.
      ). TNF engagement of TNFR1 leads to the recruitment of TNFR1-associated death domain protein, receptor-interacting protein, and TNFR-associated factor-2 (TRAF2) leads to the formation of a receptor complex (
      • Hsu H.
      • Xiang J.
      • Goeddel D.V.
      ,
      • Hsu H.
      • Huang J.
      • Shu H.-B.
      • Baichwai V.
      • Goeddel D.V.
      ,
      • Hsu H.
      • Shu H.B.
      • Pan M.-G.
      • Goeddel D.V.
      ) within which receptor-interacting protein and TRAF2, respectively, transduce signals required for TNF-mediated activation of NF-κB (
      • Kelliher A.
      • Grimm S.
      • Ishida Y.
      • Kuo F.
      • Stanger B.Z.
      • Leder P.
      ) and JNK (
      • Lee S.Y.
      • Reichlin A.
      • Santana A.
      • Sokol K.A.
      • Nussenzweig M.C.
      • Choi Y.
      ,
      • Yeh W-C.
      • Shahinian A.
      • Speiser D.
      • Kraunus J.
      • Billia F.
      • Wakeham A.
      • de la Pompa J.L.
      • Ferrick D.
      • Hum B.
      • Iscove N.
      • Ohashi P.
      • Rothe M.
      • Goeddel D.V.
      • Mak T.W.
      ,
      • Reinhard C.
      • Shamoon B.
      • Shyamala V.
      • Williams L.T.
      ). Nonetheless, little is known about the intracellular signaling mediating activation of nuclear transcription factors. In particular, the roles of PI 3-kinase and Rac in the nuclear signaling by TNF-α are as yet unclear. In the present study, we investigated the extent to which PI 3-kinase and Rac are involved in the TNF-α-induced activation of c-fos SRE and JNK. Our findings suggest that both PI 3-kinase and Rac have crucial functions within the intracellular signaling cascade triggered by TNF-α in Rat-2 fibroblasts.

      DISCUSSION

      In the present study, we provide evidence supporting novel roles for PI 3-kinase and Rac in the nuclear signaling cascade triggered by TNF-α in Rat-2 fibroblasts. TNF-α was previously reported to rapidly induce protooncogene c-fos in the adipogenic TA1 cell line, although the exact target promoter sequences by which TNF-α stimulates c-fos transcription remain unknown (
      • Haliday E.M.
      • Ramesha C.S.
      • Ringold G.
      ). Our results clearly indicate that SRE is at least one of the nuclear target sequences by which TNF-α stimulates c-fos expression. Consistent with this conclusion, c-fos SRE is also reported to be a nuclear target of ceramide, a putative second messenger for certain stresses (e.g. ultraviolet and x-rays) and inflammatory cytokines such as TNF-α (
      • Kim B.C.
      • Kim J.H.
      ). In addition, our results suggest a role for cPLA2 that is in good agreement with the earlier report of Haliday et al. (
      • Haliday E.M.
      • Ramesha C.S.
      • Ringold G.
      ) showing that AA and its lipoxygenase-generated metabolite are downstream elements in the TNF-α signaling pathway to c-fos. The function of AA as a downstream mediator of TNF-α signaling was also demonstrated in stromal cells, where AA mediates TNF-α-induced activation of JNK (
      • Rizzo M.T.
      • Carlo-Stella C.
      ).
      The involvement of PI 3-kinase in TNF-α-induced signaling to c-fos SRE was confirmed by the significant inhibitory effects of LT294003 and wortmannin, specific PI 3-kinase antagonists, and of transient transfection with pSG5-Δp85 encoding a dominant negative PI 3-kinase mutant. Consistent with this conclusion, JNK activation by TNF-α was dramatically inhibited by LY294002, implying PI 3-kinase functions broadly as a downstream TNF-α mediator in the signaling pathways leading to SRE and JNK activation. That TNF-α stimulates PI 3-kinase activity in vitro lends additional support to this idea. We do not yet know the TNF-α target molecule(s) that mediates PI 3-kinase activation; nonetheless, since the mode of action of C2-ceramide is quite similar to that of TNF-α, especially with respect to inhibition by LY294002, we postulate that enhanced production of ceramide might be involved. On the other hand, although further characterization is needed for confirmation, our evidence suggests the role of TRAF2 in the TNF-α signaling to SRE or JNK is minimal. For example, a dominant negative mutant of TRAF2 does not inhibit activation of either JNK or SRE in cells exposed to TNF α (data not shown). This finding is in contrast to previous reports (
      • Lee S.Y.
      • Reichlin A.
      • Santana A.
      • Sokol K.A.
      • Nussenzweig M.C.
      • Choi Y.
      ,
      • Reinhard C.
      • Shamoon B.
      • Shyamala V.
      • Williams L.T.
      ) in which TRAF2 was shown to be essential for TNF-α-induced JNK activation in lymphocytes, suggesting the function of TRAF2 differs in Rat-2 fibroblasts and lymphocytes. In any event, our present findings make us confident that PI 3-kinase is essential for mediating the nuclear signaling cascades triggered by TNF-α or ceramide, which is consistent with increasing evidence indicating that PI 3-kinase is activated by environmental stresses and growth factors (
      • Kimura K.
      • Miyake S.
      • Makuuchi M.
      • Morita R.
      • Usui T.
      • Yoshida M.
      • Horinouchi S.
      • Fukui Y.
      ,
      • Lin R.Z.
      • Hu Z.W.
      • Chin J.H.
      • Hoffman B.B.
      ,
      • Yuan Z.M.
      • Utsugisawa T.
      • Huang Y.
      • Ishiko T.
      • Nakada S.
      • Kharbanda S.
      • Weichselbaum R.
      • Kufe D.
      ,
      • Logan S.K.
      • Falasca M.
      • Hu P.
      • Schlessinger J.
      ).
      We also found evidence for the role of Rac in TNF-α signaling to the nucleus, which is consistent with earlier findings demonstrating an essential role of Rac in the nuclear signaling by C2-ceramide, cytokines and environmental stresses (
      • Coso O.A.
      • Chiariello M., Yu, J.C.
      • Teramoto H.
      • Crespo P.
      • Xu N.
      • Miki T.
      • Gutkind J.S.
      ,
      • Minden A.
      • Lin A.
      • Claret F.X.
      • Abo A.
      • Karin M.
      ,
      • Kim B.C.
      • Kim J.H.
      ). Thus, the present study shows that TNF-α stimulates c-fos SRE and JNK via a signaling cascade involving PI 3-kinase and Rac. Although precise determination of the mechanisms of action of PI 3-kinase and Rac will require further study, we postulate a hierarchical relationship among these proteins (TNF-α → PI 3-kinase → Rac), whereby Rac serves as a PI 3-kinase downstream molecule in a TNF-α-triggered nuclear signaling pathway. Future studies elucidating the linkage between PI 3-kinase and Rac will likely be pivotal to a complete understanding of TNF-α-evoked intracellular signaling.

      ACKNOWLEDGEMENTS

      We thank Dr. D.-M. Jue and Dr. A. Hall for providing recombinant human TNF-α and expression plasmids (pEXV, pEXV-RacV12, and pEXV-RhoV14), respectively. We also thank Dr. J. Downward for providing us pSG5-Δp85 plasmid.

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