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A Phosphatidylinositol 3-Kinase/Akt Pathway, Activated by Tumor Necrosis Factor or Interleukin-1, Inhibits Apoptosis but Does Not Activate NFκB in Human Endothelial Cells*

  • Lisa A. Madge
    Affiliations
    Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06511
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  • Jordan S. Pober
    Correspondence
    To whom correspondence should be addressed: Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Tel.: 203-737-2292; Fax: 203-737-2293
    Affiliations
    Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06511
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  • Author Footnotes
    * This work was supported by Grant HL-36007 from the National Institutes of Health and ISIS Pharmaceuticals.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.
Open AccessPublished:May 19, 2000DOI:https://doi.org/10.1074/jbc.M001237200
      Tumor necrosis factor (TNF) and interleukin-1 (IL-1) activate the transcription of both anti-apoptotic and pro-inflammatory gene products in human endothelial cells (EC) via NFκB. Here we report that both TNF and IL-1 activate the anti-apoptotic protein kinase Akt in growth factor and serum-deprived EC, assessed by Western blotting for phospho-Akt. Phosphorylation of Akt is blocked by LY294002 or wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase). Consistent with these biochemical observations, TNF and IL-1 reduce apoptosis caused by growth factor and serum deprivation, and this action is also blocked by LY294002. Although Akt has been reported to activate NFκB, LY294002 does not prevent TNF- or IL-1-induced degradation of IκBα, β, or ε, transcription of NFκB-dependent E-selectin or ICAM-1 promoter-reporter genes, or surface expression of E-selectin or ICAM-1 in human EC. LY294002 potentiates the activation of mitogen-activated protein kinases and stress-activated protein kinases by TNF and IL-1, suggesting Akt inhibits these responses. We conclude that TNF and IL-1 activate a PI 3-kinase/Akt anti-apoptotic pathway and that the anti-apoptotic effects of Akt are independent of NFκB. Moreover, the PI 3-kinase/Akt pathway does not play a major role in the pro-inflammatory responses of EC to TNF or IL-1.
      PDGF
      platelet-derived growth factor
      EC
      endothelial cell
      ECGF
      endothelial cell growth factor
      Erk
      extracellular receptor-activated kinase
      FADD
      Fas-associated death domain protein
      FGF
      fibroblast growth factor
      ICAM-1
      intercellular adhesion molecule-1
      IKK
      IκB kinase
      IL-1
      interleukin-1
      IL-1R1
      IL-1 receptor type 1
      IRAK
      IL-1 receptor-associated kinase
      IκB
      inhibitor of NFκB
      JNK
      c-Jun N-terminal kinase
      MAPK
      mitogen-activated protein kinase
      MEK
      MAPK kinase, MEKK, MEK kinase
      NFκB
      nuclear factor κB
      NIK
      NFκB-inducing kinase
      PI 3-kinase
      phosphatidylinositol-3 kinase
      RIP
      receptor-interacting protein
      SAPK
      stress-activated protein kinase
      SH
      Src homology
      TNF
      tumor necrosis factor
      TNFR1
      TNF receptor 1
      TNFR2
      TNF receptor 2
      TRADD
      TNF receptor-associated death domain protein
      TRAF
      TNF receptor associated factor
      VCAM-1
      vascular cell adhesion molecule-1
      PBS
      phosphate-buffered saline
      FACS
      fluorescence-activated cell sorter
      Many of the anti-apoptotic responses initiated by polypeptide growth factors such as PDGF1or FGF are mediated by phosphatidylinositol 3-kinase (PI 3-kinase), which is a ubiquitous heterodimeric lipid-modifying enzyme consisting of a p85 regulatory and p110 catalytic subunit (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). Growth factor binding causes autophosphorylation of tyrosine residues located within the intracellular portion of the receptor polypeptide. One or more of these phosphorylated tyrosine residues then serves as a binding site for the Src homology 2 (SH2) domain of the p85 regulatory subunit (
      • Klippel A.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      ). An increase in catalytic activity of PI 3-kinase results from a combination of this docking of PI 3-kinase in the proximity of the plasma membrane, where its lipid substrate is located, plus allosteric regulation of p110 activity by the receptor-bound p85 subunit. Activated p110 catalyzes the phosphorylation of membrane phosphatidylinositol 4,5-bisphosphate in the D3 position to generate phosphatidylinositol 3,4,5-trisphosphate (
      • Carpenter C.L.
      • Cantley L.C.
      ). Phosphatidylinositol 3,4,5-trisphosphate and its phospholipid phosphatase product, phosphatidylinositol 3,4-bisphosphate, accumulate in the membrane, creating docking sites for two lipid-binding protein kinases, namely phosphatidylinositol-dependent kinase 1 and protein kinase B (more commonly known as Akt) which bind to these lipids via their pleckstrin homology domains (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ). Akt becomes fully activated as a result of this plasma membrane localization and by its phosphorylation on both Thr308 and Ser473. These phosphorylation events are catalyzed by phosphatidylinositol dependent kinase 1 and an unidentified but provisionally named phosphatidylinositol-dependent kinase 2, respectively (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ). Once activated, Akt can inhibit apoptosis by a number of actions including phosphorylation and inactivation of the pro-apoptotic Bcl-2 homolog Bad (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ), of the apoptosis-initiating enzyme caspase 9 (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), and of the forkhead family transcription factor FKHR1 that mediates transcription of pro-apoptotic gene products (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Kops G.J.
      • de Ruiter N.D.
      • De Vries-Smits A.M.
      • Powell D.R.
      • Bos J.L.
      • Burgering B.M.
      ).
      The mitogenic response of growth factors is typically mediated by a distinct intracellular pathway involving a kinase cascade resulting in the activation of mitogen-activated protein kinases (MAPKs). Stimulation of this MAPK cascade by growth factors is initiated by recruitment of the adapter protein Grb2 via its SH2 domain either directly to the receptor (
      • Lowenstein E.J.
      • Daly R.J.
      • Batzer A.G.
      • Li W.
      • Margolis B.
      • Lammers R.
      • Ullrich A.
      • Skolnik E.Y.
      • Bar-Sagi D.
      • Schlessinger J.
      ,
      • Arvidsson A.K.
      • Rupp E.
      • Nanberg E.
      • Downward J.
      • Ronnstrand L.
      • Wennstrom S.
      • Schlessinger J.
      • Heldin C.H.
      • Claesson-Welsh L.
      ) or to tyrosine-phosphorylated Shc linker protein (
      • Pelicci G.
      • Lanfrancone L.
      • Grignani F.
      • McGlade J.
      • Cavallo F.
      • Forni G.
      • Nicoletti I.
      • Grignani F.
      • Pawson T.
      • Pelicci P.G.
      ,
      • Pronk G.J.
      • de Vries-Smits A.M.
      • Buday L.
      • Downward J.
      • Maassen J.A.
      • Medema R.H.
      • Bos J.L.
      ). Grb2 also contains a SH3 domain that recruits the guanine nucleotide exchange factor Sos to the receptor complex (
      • Egan S.E.
      • Giddings B.W.
      • Brooks M.W.
      • Buday L.
      • Sizeland A.M.
      • Weinberg R.A.
      ). Receptor-bound Sos catalyzes the activation of Ras by facilitating GDP/GTP exchange, and GTP·Ras binds to and activates the serine-threonine protein kinase Raf. Activated Raf phosphorylates MEK-1 (
      • Kyriakis J.M.
      • App H.
      • Zhang X.F.
      • Banerjee P.
      • Brautigan D.L.
      • Rapp U.R.
      • Avruch J.
      ) which in turn phosphorylates extracellular receptor-activated kinase-1 and -2 (Erk-1 and -2) (
      • Nakielny S.
      • Cohen P.
      • Wu J.
      • Sturgill T.
      ), prototypic MAPKs implicated in cell cycle entry (
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ,
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ). Certain inhibitors of PI 3-kinase block activation of Erk, suggesting a regulatory role for the PI 3-kinase pathway in the MAPK cascade (
      • Cross D.A.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • Wennstrom S.
      • Downward J.
      ). However, some investigators (
      • Kwon T.
      • Kwon D.Y.
      • Chun J.
      • Kim J.H.
      • Kang S.S.
      ,
      • Zimmermann S.
      • Moelling K.
      ,
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ) have actually reported an increase in MAPK activities in some cell types in response to PI 3-kinase inhibitors, implying a negative regulatory role for this pathway.
      Cytokines such as TNF and IL-1 play an important role in a different set of cellular responses from those triggered by growth factors, namely activation of inflammatory functions and, in the case of TNF, initiation of apoptosis. The pro-apoptotic activity of TNF is mediated by the autocatalytic activation of caspase 8 by a ligand-receptor complex involving the type 1 TNF receptor (TNFR1) and the adapter proteins TNF receptor-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD) (
      • Boldin M.P.
      • Goncharov T.M.
      • Goltsev Y.V.
      • Wallach D.
      ,
      • Hsu H.
      • Shu H.B.
      • Pan M.G.
      • Goeddel D.V.
      ). Most untransformed cell types are not susceptible to killing by TNF unless mRNA or protein synthesis is blocked. This observation is largely attributed to the capacity of TNF to initiate NFκB-dependent synthesis of anti-apoptotic gene products such as the signal inhibitor A20 (
      • Opipari Jr., A.W.
      • Hu H.M.
      • Yabkowitz R.
      • Dixit V.M.
      ) and the Bcl-2 homolog A1 (
      • Karsan A.
      • Yee E.
      • Harlan J.M.
      ). Direct evidence for the role of NFκB as an inhibitor of TNF-mediated apoptosis has been observed in transfected cells (
      • Wang C.Y.
      • Mayo M.W.
      • Baldwin Jr., A.S.
      ,
      • Van Antwerp D.J.
      • Martin S.J.
      • Kafri T.
      • Green D.R.
      • Verma I.M.
      ,
      • Beg A.A.
      • Baltimore D.
      ) and is strongly supported by the observation that mice lacking the p65 subunit of NFκB die in utero unless they are crossed with TNF knockout animals (
      • Doi T.S.
      • Marino M.W.
      • Takahashi T.
      • Yoshida T.
      • Sakakura T.
      • Old L.J.
      • Obata Y.
      ).
      The initial steps in the major pathway utilized by TNF to activate NFκB are fairly well understood. NFκB activation is usually initiated by the interaction of TNF with TNFR1 (also called p55), one of two distinct surface receptors, the other being TNFR2 (also called p75) (reviewed in Ref.
      • Tartaglia L.A.
      • Goeddel D.V.
      ). Ligand-induced clustering of TNFR1 leads to the recruitment of TRADD (but not FADD) to the intracellular portion of the receptor (
      • Hsu H.
      • Xiong J.
      • Goeddel D.V.
      ). Receptor-bound TRADD recruits both the protein kinase designated receptor-interacting protein (RIP) and the ring/zinc finger protein TNF receptor-associated factor 2 (TRAF2) (
      • Hsu H.
      • Shu H.B.
      • Pan M.G.
      • Goeddel D.V.
      ,
      • Hsu H.
      • Huang J.
      • Shu H.B.
      • Baichwal V.
      • Goeddel D.V.
      ). These two adapter proteins lead to the activation of NFκB as well as the activation of the stress-activated protein kinases (SAPKs) such as c-Jun N-terminal kinase (JNK) and p38 MAPK. IL-1 also activates NFκB and SAPKs using a similar pathway initiated by ligand binding to the type 1 IL-1 receptor (IL-1R1) and involving adapter proteins MyD88, IL-1 receptor-associated kinase (IRAK), and TRAF6 (
      • Wesche H.
      • Henzel W.J.
      • Shillinglaw W.
      • Li S.
      • Cao Z.
      ,
      • Cao Z.
      • Xiong J.
      • Takeuchi M.
      • Kurama T.
      • Goeddel D.V.
      ). The precise connection between RIP·TRAF2 or IRAK·TRAF6 complexes and NFκB are less well established but are thought to involve the MAPK kinase kinase family members MEKK-1 or NFκB-inducing kinase (NIK) (
      • Lee F.S.
      • Hagler J.
      • Chen Z.J.
      • Maniatis T.
      ,
      • Karin M.
      • Delhase M.
      ). Both of these protein kinases can directly phosphorylate and activate IκB kinase (IKK) (
      • Karin M.
      • Delhase M.
      ,
      • Malinin N.L.
      • Boldin M.P.
      • Kovalenko A.V.
      • Wallach D.
      ,
      • Regnier C.H.
      • Song H.Y.
      • Gao X.
      • Goeddel D.V.
      • Cao Z.
      • Rothe M.
      ) which is a multiprotein enzyme complex consisting of IKK-1, IKK-2, and Nemo (also known as IKKγ) subunits (
      • Mercurio F.
      • Zhu H.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Li J.
      • Young D.B.
      • Barbosa M.
      • Mann M.
      • Manning A.
      • Rao A.
      ,
      • Woronicz J.D.
      • Gao X.
      • Cao Z.
      • Rothe M.
      • Goeddel D.V.
      ,
      • Zandi E.
      • Rothwarf D.M.
      • Delhase M.
      • Hayakawa M.
      • Karin M.
      ,
      • Rothwarf D.M.
      • Zandi E.
      • Natoli G.
      • Karin M.
      ,
      • Yamaoka S.
      • Courtois G.
      • Bessia C.
      • Whiteside S.T.
      • Weil R.
      • Agou F.
      • Kirk H.E.
      • Kay R.J.
      • Israel A.
      ). IKK-1 and IKK-2 phosphorylate members of the IκB family (i.e. IκBα, -β, -ε) which share a common mechanism for regulation of NFκB. In the resting cell, IκB proteins bind to and mask the nuclear localization sequence of NFκB resulting in the retention of the transcription factor in the cytosol (
      • Verma I.M.
      • Stevenson J.K.
      • Schwarz E.M.
      • Van Antwerp D.
      • Miyamoto S.
      ). Phosphorylation of IκB by IKK targets this protein for rapid ubiquitination and degradation by the proteosome. This process releases NFκB which then translocates to the nucleus and induces transcription (
      • Miyamoto S.
      • Maki M.
      • Schmitt M.J.
      • Hatanaka M.
      • Verma I.M.
      ,
      • Traenckner E.B.
      • Wilk S.
      • Baeuerle P.A.
      ). MEKK-1, but not NIK, catalyzes the phosphorylation and activation of yet other MAPK kinases (e.g. MEK-2, -4, -6, and -7) and is probably responsible for the parallel activation of SAPKs (
      • Lin A.
      • Minden A.
      • Martinetto H.
      • Claret F.X.
      • Lange-Carter C.
      • Mercurio F.
      • Johnson G.L.
      • Karin M.
      ).
      Recently, an alternative signaling pathway has been described in some cells in which Akt directly phosphorylates IKK leading to the activation of NFκB, independent of MEKK-1 and NIK (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ,
      • Romashkova J.A.
      • Makarov S.S.
      ). This pathway thus allows growth factors such as PDGF to activate NFκB (
      • Romashkova J.A.
      • Makarov S.S.
      ). Moreover, TNF and IL-1 have been found to activate the PI 3-kinase/Akt pathway (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ,
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ,
      • Kim B.C.
      • Lee M.N.
      • Kim J.Y.
      • Lee S.S.
      • Chang J.D.
      • Kim S.S.
      • Lee S.Y.
      • Kim J.H.
      ,
      • Hanna A.N.
      • Chan E.Y.
      • Xu J.
      • Stone J.C.
      • Brindley D.N.
      ), potentially providing an alternative pathway for TNF and IL-1 to activate NFκB that is independent of NIK or MEKK-1 (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ). The importance of this alternative NFκB pathway compared with the previously described TRADD/RIP/TRAF2 or MyD88/IRAK/TRAF6 cytokine signaling pathways is unknown and may vary with the cell type. TNF and IL-1 also activate MAPK (
      • Saklatvala J.
      • Dean J.
      • Finch A.
      ), but this pathway is even less well defined. Furthermore, TNF and IL-1 are typically anti-mitogenic in EC (
      • Stolpen A.H.
      • Guinan E.C.
      • Fiers W.
      • Pober J.S.
      ), despite their stimulation of MAPK activity.
      Vascular endothelial cells (EC) are a highly relevant cell type in which to assess the significance of inflammatory cytokine-activated PI 3-kinase/Akt, NFκB, and MAPK pathways. These cells are among the principal physiological targets of the pro-inflammatory actions of TNF and IL-1. Specifically, the activation of NFκB and SAPKs by TNF or IL-1 results in de novo transcription of leukocyte-binding adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) (
      • Collins T.
      • Read M.A.
      • Neish A.S.
      • Whitley M.Z.
      • Thanos D.
      • Maniatis T.
      ). Cytokine-induced expression of these adhesion molecules is essential for the development of inflammatory reactions (
      • DeLisser H.M.
      • Albelda S.M.
      ). In the present study we have confirmed that TNF and IL-1 stimulate the phosphorylation of Akt in cultured human EC, and we have shown that a pharmacological inhibitor of PI 3-kinase, LY294002, completely abrogates this response. This same agent blocks the anti-apoptotic effects of these cytokines that are revealed by conditions of serum and growth factor withdrawal. However, LY294002 has no measurable effect on cytokine-mediated IκB degradation or on NFκB activation. LY294002 treatment fails to inhibit MAPK and JNK responses and actually potentiates MAPK and JNK activation by TNF and IL-1, consistent with a negative regulatory role for Akt in these pathways. Furthermore, LY294002 has little effect on adhesion molecule expression. Collectively, these data suggest that the Akt pathway is functionally activated by TNF or IL-1 in human EC but that it is not a major mediator of pro-inflammatory functions. They also establish that the Akt pathway mediates anti-apoptotic actions of TNF (and IL-1) that are independent of NFκB.

      DISCUSSION

      In this study we have observed that pro-inflammatory cytokines TNF and IL-1 stimulate the phosphorylation of the anti-apoptotic serine/threonine kinase Akt in human EC and that this phosphorylation is dependent on the activation of PI 3-kinase. Several other studies have reported similar observations in different cell types (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ,
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ,
      • Kim B.C.
      • Lee M.N.
      • Kim J.Y.
      • Lee S.S.
      • Chang J.D.
      • Kim S.S.
      • Lee S.Y.
      • Kim J.H.
      ). Little is known of the mechanism of activation of PI 3-kinase by TNF and IL-1, although several modes of activation have been described in response to other stimuli. For example stimulation of growth factor receptors, such as the PDGF receptor, which contain intrinsic tyrosine kinase activity result in the activation of PI 3-kinase by recruitment of the enzyme to the intracellular region of the autophosphorylated receptor (
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ). The SH2 domain of p85 specifically recognizes and associates with phosphotyrosine containing YXXM or YVXV motifs (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      ). Although neither TNFR1 nor IL-1R1 have any intrinsic kinase activity, one such motif has been reported to account for the recruitment of p85 to the IL-1R1 (
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ). To date, the kinase responsible for phosphorylation of IL-1R1 remains unidentified. Neither of the SH2 interacting motifs are present in TNFR1, and activation of PI 3-kinase by TNF may require either phosphorylation of an intermediate scaffold protein or an alternative mechanism of activation. Interactions between many proteins are dependent on the binding of proline-rich motifs with SH3 domains, and both such motifs have been identified in p85 (
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ,
      • Pleiman C.M.
      • Clark M.R.
      • Gauen L.K.
      • Winitz S.
      • Coggeshall K.M.
      • Johnson G.L.
      • Shaw A.S.
      • Cambier J.C.
      ). In one recent report a proline-rich region within TNFR1 was shown to mediate an interaction with the adapter protein Grb2 via its SH3 domain (
      • Hildt E.
      • Oess S.
      ). PI 3-kinase may therefore similarly associate with TNFR1 via an interaction between the SH3 domain of p85 and proline-rich region of TNFR1. Another region of p85 described in the activation of PI 3-kinase is the Rho-GAP (or Bcr) homology domain. This domain lacks GTPase promoting activity but binds Rac and Cdc42 (
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ). Although the importance of this interaction in the activation of PI 3-kinase remains questionable (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ), activated Cdc42 and Rac can potentiate TNF-dependent signaling in EC and could therefore play a role in TNF-dependent activation of PI 3-kinase (
      • Min W.
      • Pober J.S.
      ,
      • Wojciak-Stothard B.
      • Entwistle A.
      • Garg R.
      • Ridley A.J.
      ). Yet another mechanism for activation of PI 3-kinase, described to synergize with the recruitment of the enzyme to membrane receptors, is dependent on Ras interactions with the catalytic subunit of PI 3-kinase (
      • Downward J.
      ). Several reports have demonstrated that the Ras-Raf pathway is activated in response to TNF (
      • Hildt E.
      • Oess S.
      ,
      • Muller G.
      • Storz P.
      • Bourteele S.
      • Doppler H.
      • Pfizenmaier K.
      • Mischak H.
      • Philipp A.
      • Kaiser C.
      • Kolch W.
      ,
      • Xu X.S.
      • Vanderziel C.
      • Bennett C.F.
      • Monia B.P.
      ), but the significance of Ras in the activation of PI 3-kinase in response to either TNF or IL-1 has yet to be demonstrated.
      To evaluate the functional significance of PI 3-kinase/Akt activation by TNF and IL-1, the effect of inhibition of PI 3-kinase using the pharmacological inhibitor LY294002 on the pro-inflammatory and anti-apoptotic actions of these cytokines was examined. LY294002 effectively blocks TNF and IL-1-mediated phosphorylation of Akt so that any effect of TNF or IL-1 in the presence of this drug must be independent of the PI 3-kinase/Akt pathway. We believe the actions of LY294002 are also specific for PI 3-kinase in EC because it does not effect the activation of MAPK activities by cytokines (e.g.IL-11) which do not activate Akt in human EC.2 Moreover, the effects of LY294002 can be repeated with wortmannin, a structurally unrelated inhibitor of PI 3-kinase.
      Akt is well described as a mediator of cell survival, and activation of Akt has been shown to be protective against apoptosis induced by growth factor or serum withdrawal (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Gerber H.P.
      • McMurtrey A.
      • Kowalski J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ). In this study we considered whether TNF- and IL-1-dependent activation of Akt could similarly promote cell survival in opposition to serum and growth factor withdrawal. TNF, IL-1, and ECGF, a growth factor control, all effectively inhibited apoptosis to a similar extent to that observed in EC treated with VEGF (
      • Gerber H.P.
      • McMurtrey A.
      • Kowalski J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ). The protective effect of all three agents was reversed by pretreatment with LY294002. These observations indicate that the PI 3-kinase/Akt pathway is functionally involved in TNF- and IL-1-mediated protection from serum withdrawal-induced apoptosis. We have also observed that LY294002, like protein and RNA synthesis inhibitors, reveals TNF-mediated apoptosis in the presence of serum and growth factor. However, under these conditions, TNF is not the only activator of Akt.
      Other potential targets for regulation by PI 3-kinase/Akt are the transcription factor NFκB and the SAPKs, which are central to the proinflammatory actions of TNF and IL-1. Recent studies focused on the interplay between NFκB and PI 3-kinase/Akt have yielded conflicting results. An Akt-dependent pathway for activation of NFκB by TNF, independent of NIK or MEKK-1, has been described (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ), but this pathway does not seem to be active for all cell types or stimuli (
      • Romashkova J.A.
      • Makarov S.S.
      ,
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ,
      • Beraud C.
      • Henzel W.J.
      • Baeuerle P.A.
      ). In human EC, inhibition of PI 3-kinase using LY294002 effectively inhibited TNF or IL-1-dependent activation of Akt without affecting the degradation of IκBα, IκBβ, or IκBε. Similarly, the activities of the E-selectin promoter-reporter gene, which is dependent on p50/p65, or the ICAM-1 promoter-reporter, which is dependent on p65/c-Rel (
      • Collins T.
      • Read M.A.
      • Neish A.S.
      • Whitley M.Z.
      • Thanos D.
      • Maniatis T.
      ), were not affected by inhibition of PI 3-kinase. Notably, we also observed that ECGF, although able to stimulate PI 3-kinase/Akt, was not an effective stimulator of NFκB in EC (data not shown). Together these results indicate that PI 3-kinase/Akt does not contribute to the activation of NFκB in EC and that the major pathway for NFκB activation by TNF and IL-1 is mediated by the previously described TRADD/RIP/TRAF2- or MyD88/IRAK/TRAF6-NIK/MEKK-1-dependent phosphorylation of IKK.
      The activation of MAPK and SAPKs may be positively or negatively regulated by the activation of PI 3-kinase (
      • Cross D.A.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • Wennstrom S.
      • Downward J.
      ,
      • Kwon T.
      • Kwon D.Y.
      • Chun J.
      • Kim J.H.
      • Kang S.S.
      ,
      • Zimmermann S.
      • Moelling K.
      ,
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Logan S.K.
      • Falasca M.
      • Hu P.
      • Schlessinger J.
      ,
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ). In EC, inhibition of PI 3-kinase/Akt resulted in an increase in the TNF- and IL-1-stimulated activity of both MAPK and JNK. The negative regulation of JNK and MAPK pathways may occur by phosphorylation and inactivation either of the upstream kinase Raf (
      • Zimmermann S.
      • Moelling K.
      ,
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ) or the GTPase Rac (
      • Kwon T.
      • Kwon D.Y.
      • Chun J.
      • Kim J.H.
      • Kang S.S.
      ). A consensus site for phosphorylation by Akt has also been identified in JNK itself, although a direct relationship between JNK and Akt has yet to be demonstrated. The increase in the activity of both JNK and MAPK observed in EC following inhibition of PI 3-kinase could theoretically contribute to the slight increase in surface expression of both E-selectin and ICAM-1 which was observed following treatment LY294002. However, the same response was not observed with E-selectin and ICAM reporter genes, suggesting that PI 3-kinase/Akt may instead effect a post-transcriptional process.
      In summary, TNF and IL-1 activate the PI 3-kinase/Akt pathway in EC, and this pathway functionally regulates associated cell cycle events and growth factor withdrawal-induced apoptosis. This anti-apoptotic effect appears to be independent of NFκB since blockade of PI 3-kinase does not prevent NFκB activation or NFκB-dependent gene transcription. Our studies also suggest that PI 3-kinase/Akt may negatively regulate both MAPK and JNK in human EC. Collectively these results suggest that activation of PI 3-kinase/Akt by TNF or IL-1 is not a pro-inflammatory signal in EC and that blockade of this pathway is not likely to inhibit inflammatory processes. In fact, the negative regulation of MAPK and especially JNK may actually limit pro-inflammatory responses to TNF and IL-1.

      Acknowledgments

      We thank Walter Fiers and Werner Lesslauer for provision of TNF muteins. We also thank Louise Camera Benson and Gwendoline Davis for excellent assistance in cell culture.

      REFERENCES

        • Fruman D.A.
        • Meyers R.E.
        • Cantley L.C.
        Annu. Rev. Biochem. 1998; 67: 481-507
        • Klippel A.
        • Escobedo J.A.
        • Fantl W.J.
        • Williams L.T.
        Mol. Cell. Biol. 1992; 12: 1451-1459
        • Carpenter C.L.
        • Cantley L.C.
        Curr. Opin. Cell Biol. 1996; 8: 153-158
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        • Toker A.
        Science. 1997; 275: 665-668
        • Alessi D.R.
        • James S.R.
        • Downes C.P.
        • Holmes A.B.
        • Gaffney P.R.
        • Reese C.B.
        • Cohen P.
        Curr. Biol. 1997; 7: 261-269
        • Datta S.R.
        • Dudek H.
        • Tao X.
        • Masters S.
        • Fu H.
        • Gotoh Y.
        • Greenberg M.E.
        Cell. 1997; 91: 231-241
        • Cardone M.H.
        • Roy N.
        • Stennicke H.R.
        • Salvesen G.S.
        • Franke T.F.
        • Stanbridge E.
        • Frisch S.
        • Reed J.C.
        Science. 1998; 282: 1318-1321
        • Brunet A.
        • Bonni A.
        • Zigmond M.J.
        • Lin M.Z.
        • Juo P.
        • Hu L.S.
        • Anderson M.J.
        • Arden K.C.
        • Blenis J.
        • Greenberg M.E.
        Cell. 1999; 96: 857-868
        • Kops G.J.
        • de Ruiter N.D.
        • De Vries-Smits A.M.
        • Powell D.R.
        • Bos J.L.
        • Burgering B.M.
        Nature. 1999; 398: 630-634
        • Lowenstein E.J.
        • Daly R.J.
        • Batzer A.G.
        • Li W.
        • Margolis B.
        • Lammers R.
        • Ullrich A.
        • Skolnik E.Y.
        • Bar-Sagi D.
        • Schlessinger J.
        Cell. 1992; 70: 431-442
        • Arvidsson A.K.
        • Rupp E.
        • Nanberg E.
        • Downward J.
        • Ronnstrand L.
        • Wennstrom S.
        • Schlessinger J.
        • Heldin C.H.
        • Claesson-Welsh L.
        Mol. Cell. Biol. 1994; 14: 6715-6726
        • Pelicci G.
        • Lanfrancone L.
        • Grignani F.
        • McGlade J.
        • Cavallo F.
        • Forni G.
        • Nicoletti I.
        • Grignani F.
        • Pawson T.
        • Pelicci P.G.
        Cell. 1992; 70: 93-104
        • Pronk G.J.
        • de Vries-Smits A.M.
        • Buday L.
        • Downward J.
        • Maassen J.A.
        • Medema R.H.
        • Bos J.L.
        Mol. Cell. Biol. 1994; 14: 1575-1581
        • Egan S.E.
        • Giddings B.W.
        • Brooks M.W.
        • Buday L.
        • Sizeland A.M.
        • Weinberg R.A.
        Nature. 1993; 363: 45-51
        • Kyriakis J.M.
        • App H.
        • Zhang X.F.
        • Banerjee P.
        • Brautigan D.L.
        • Rapp U.R.
        • Avruch J.
        Nature. 1992; 358: 417-421
        • Nakielny S.
        • Cohen P.
        • Wu J.
        • Sturgill T.
        EMBO J. 1992; 11: 2123-2129
        • Lavoie J.N.
        • L'Allemain G.
        • Brunet A.
        • Muller R.
        • Pouyssegur J.
        J. Biol. Chem. 1996; 271: 20608-20616
        • Weber J.D.
        • Raben D.M.
        • Phillips P.J.
        • Baldassare J.J.
        Biochem. J. 1997; 326: 61-68
        • Cross D.A.
        • Alessi D.R.
        • Vandenheede J.R.
        • McDowell H.E.
        • Hundal H.S.
        • Cohen P.
        Biochem. J. 1994; 303: 21-26
        • Duckworth B.C.
        • Cantley L.C.
        J. Biol. Chem. 1997; 272: 27665-27670
        • Wennstrom S.
        • Downward J.
        Mol. Cell. Biol. 1999; 19: 4279-4288
        • Kwon T.
        • Kwon D.Y.
        • Chun J.
        • Kim J.H.
        • Kang S.S.
        J. Biol. Chem. 1999; 275: 423-428
        • Zimmermann S.
        • Moelling K.
        Science. 1999; 286: 1741-1744
        • Rommel C.
        • Clarke B.A.
        • Zimmermann S.
        • Nunez L.
        • Rossman R.
        • Reid K.
        • Moelling K.
        • Yancopoulos G.D.
        • Glass D.J.
        Science. 1999; 286: 1738-1741
        • Boldin M.P.
        • Goncharov T.M.
        • Goltsev Y.V.
        • Wallach D.
        Cell. 1996; 85: 803-815
        • Hsu H.
        • Shu H.B.
        • Pan M.G.
        • Goeddel D.V.
        Cell. 1996; 84: 299-308
        • Opipari Jr., A.W.
        • Hu H.M.
        • Yabkowitz R.
        • Dixit V.M.
        J. Biol. Chem. 1992; 267: 12424-12427
        • Karsan A.
        • Yee E.
        • Harlan J.M.
        J. Biol. Chem. 1996; 271: 27201-27204
        • Wang C.Y.
        • Mayo M.W.
        • Baldwin Jr., A.S.
        Science. 1996; 274: 784-787
        • Van Antwerp D.J.
        • Martin S.J.
        • Kafri T.
        • Green D.R.
        • Verma I.M.
        Science. 1996; 274: 787-789
        • Beg A.A.
        • Baltimore D.
        Science. 1996; 274: 782-784
        • Doi T.S.
        • Marino M.W.
        • Takahashi T.
        • Yoshida T.
        • Sakakura T.
        • Old L.J.
        • Obata Y.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2994-2999
        • Tartaglia L.A.
        • Goeddel D.V.
        Immunol. Today. 1992; 13: 151-153
        • Hsu H.
        • Xiong J.
        • Goeddel D.V.
        Cell. 1995; 81: 495-504
        • Hsu H.
        • Huang J.
        • Shu H.B.
        • Baichwal V.
        • Goeddel D.V.
        Immunity. 1996; 4: 387-396
        • Wesche H.
        • Henzel W.J.
        • Shillinglaw W.
        • Li S.
        • Cao Z.
        Immunity. 1997; 7: 837-847
        • Cao Z.
        • Xiong J.
        • Takeuchi M.
        • Kurama T.
        • Goeddel D.V.
        Nature. 1996; 383: 443-446
        • Lee F.S.
        • Hagler J.
        • Chen Z.J.
        • Maniatis T.
        Cell. 1997; 88: 213-222
        • Karin M.
        • Delhase M.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9067-9069
        • Malinin N.L.
        • Boldin M.P.
        • Kovalenko A.V.
        • Wallach D.
        Nature. 1997; 385: 540-544
        • Regnier C.H.
        • Song H.Y.
        • Gao X.
        • Goeddel D.V.
        • Cao Z.
        • Rothe M.
        Cell. 1997; 90: 373-383
        • Mercurio F.
        • Zhu H.
        • Murray B.W.
        • Shevchenko A.
        • Bennett B.L.
        • Li J.
        • Young D.B.
        • Barbosa M.
        • Mann M.
        • Manning A.
        • Rao A.
        Science. 1997; 278: 860-866
        • Woronicz J.D.
        • Gao X.
        • Cao Z.
        • Rothe M.
        • Goeddel D.V.
        Science. 1997; 278: 866-869
        • Zandi E.
        • Rothwarf D.M.
        • Delhase M.
        • Hayakawa M.
        • Karin M.
        Cell. 1997; 91: 243-252
        • Rothwarf D.M.
        • Zandi E.
        • Natoli G.
        • Karin M.
        Nature. 1998; 395: 297-300
        • Yamaoka S.
        • Courtois G.
        • Bessia C.
        • Whiteside S.T.
        • Weil R.
        • Agou F.
        • Kirk H.E.
        • Kay R.J.
        • Israel A.
        Cell. 1998; 93: 1231-1240
        • Verma I.M.
        • Stevenson J.K.
        • Schwarz E.M.
        • Van Antwerp D.
        • Miyamoto S.
        Genes Dev. 1995; 9: 2723-2735
        • Miyamoto S.
        • Maki M.
        • Schmitt M.J.
        • Hatanaka M.
        • Verma I.M.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12740-12744
        • Traenckner E.B.
        • Wilk S.
        • Baeuerle P.A.
        EMBO J. 1994; 13: 5433-5441
        • Lin A.
        • Minden A.
        • Martinetto H.
        • Claret F.X.
        • Lange-Carter C.
        • Mercurio F.
        • Johnson G.L.
        • Karin M.
        Science. 1995; 268: 286-290
        • Ozes O.N.
        • Mayo L.D.
        • Gustin J.A.
        • Pfeffer S.R.
        • Pfeffer L.M.
        • Donner D.B.
        Nature. 1999; 401: 82-85
        • Romashkova J.A.
        • Makarov S.S.
        Nature. 1999; 401: 86-90
        • Sizemore N.
        • Leung S.
        • Stark G.R.
        Mol. Cell. Biol. 1999; 19: 4798-4805
        • Reddy S.A.
        • Huang J.H.
        • Liao W.S.
        J. Biol. Chem. 1997; 272: 29167-29173
        • Kim B.C.
        • Lee M.N.
        • Kim J.Y.
        • Lee S.S.
        • Chang J.D.
        • Kim S.S.
        • Lee S.Y.
        • Kim J.H.
        J. Biol. Chem. 1999; 274: 24372-24377
        • Hanna A.N.
        • Chan E.Y.
        • Xu J.
        • Stone J.C.
        • Brindley D.N.
        J. Biol. Chem. 1999; 274: 12722-12729
        • Saklatvala J.
        • Dean J.
        • Finch A.
        Biochem. Soc. Symp. 1999; 64: 63-77
        • Stolpen A.H.
        • Guinan E.C.
        • Fiers W.
        • Pober J.S.
        Am. J. Pathol. 1986; 123: 16-24
        • Collins T.
        • Read M.A.
        • Neish A.S.
        • Whitley M.Z.
        • Thanos D.
        • Maniatis T.
        FASEB J. 1995; 9: 899-909
        • DeLisser H.M.
        • Albelda S.M.
        Am. J. Respir. Cell. Mol. Biol. 1998; 19: 533-536
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Slowik M.R.
        • De Luca L.G.
        • Fiers W.
        • Pober J.S.
        Am. J. Pathol. 1993; 143: 1724-1730
        • Karmann K.
        • Min W.
        • Fanslow W.C.
        • Pober J.S.
        J. Exp. Med. 1996; 184: 173-182
        • Min W.
        • Pober J.S.
        J. Immunol. 1997; 159: 3508-3518
        • Hu P.
        • Margolis B.
        • Skolnik E.Y.
        • Lammers R.
        • Ullrich A.
        • Schlessinger J.
        Mol. Cell. Biol. 1992; 12: 981-990
        • Songyang Z.
        • Shoelson S.E.
        • Chaudhuri M.
        • Gish G.
        • Pawson T.
        • Haser W.G.
        • King F.
        • Roberts T.
        • Ratnofsky S.
        • Lechleider R.J.
        Cell. 1993; 72: 767-778
        • Prasad K.V.
        • Janssen O.
        • Kapeller R.
        • Raab M.
        • Cantley L.C.
        • Rudd C.E.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7366-7370
        • Pleiman C.M.
        • Hertz W.M.
        • Cambier J.C.
        Science. 1994; 263: 1609-1612
        • Pleiman C.M.
        • Clark M.R.
        • Gauen L.K.
        • Winitz S.
        • Coggeshall K.M.
        • Johnson G.L.
        • Shaw A.S.
        • Cambier J.C.
        Mol. Cell. Biol. 1993; 13: 5877-5887
        • Hildt E.
        • Oess S.
        J. Exp. Med. 1999; 189: 1707-1714
        • Zheng Y.
        • Bagrodia S.
        • Cerione R.A.
        J. Biol. Chem. 1994; 269: 18727-18730
        • Tolias K.F.
        • Cantley L.C.
        • Carpenter C.L.
        J. Biol. Chem. 1995; 270: 17656-17659
        • Rodriguez-Viciana P.
        • Warne P.H.
        • Dhand R.
        • Vanhaesebroeck B.
        • Gout I.
        • Fry M.J.
        • Waterfield M.D.
        • Downward J.
        Nature. 1994; 370: 527-532
        • Wojciak-Stothard B.
        • Entwistle A.
        • Garg R.
        • Ridley A.J.
        J. Cell. Physiol. 1998; 176: 150-165
        • Downward J.
        Adv. Second Messenger Phosphoprotein Res. 1997; 31: 1-10
        • Muller G.
        • Storz P.
        • Bourteele S.
        • Doppler H.
        • Pfizenmaier K.
        • Mischak H.
        • Philipp A.
        • Kaiser C.
        • Kolch W.
        EMBO J. 1998; 17: 732-742
        • Xu X.S.
        • Vanderziel C.
        • Bennett C.F.
        • Monia B.P.
        J. Biol. Chem. 1998; 273: 33230-33238
        • Gerber H.P.
        • McMurtrey A.
        • Kowalski J.
        • Yan M.
        • Keyt B.A.
        • Dixit V.
        • Ferrara N.
        J. Biol. Chem. 1998; 273: 30336-30343
        • Beraud C.
        • Henzel W.J.
        • Baeuerle P.A.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 429-434
        • Logan S.K.
        • Falasca M.
        • Hu P.
        • Schlessinger J.
        Mol. Cell. Biol. 1997; 17: 5784-5790
        • Klippel A.
        • Reinhard C.
        • Kavanaugh W.M.
        • Apell G.
        • Escobedo M.A.
        • Williams L.T.
        Mol. Cell. Biol. 1996; 16: 4117-4127
        • Lopez-Ilasaca M.
        • Crespo P.
        • Pellici P.G.
        • Gutkind J.S.
        • Wetzker R.
        Science. 1997; 275: 394-397