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Cell Type-specific Expression of the IκB Kinases Determines the Significance of Phosphatidylinositol 3-Kinase/Akt Signaling to NF-κB Activation*

  • Jason A. Gustin
    Footnotes
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • Osman N. Ozes
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • Hakan Akca
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • Roxana Pincheira
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • Lindsey D. Mayo
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • Qiutang Li
    Affiliations
    Laboratory of Genetics, Salk Institute, La Jolla, California 92037
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  • Javier Rivera Guzman
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • Chandrashekhar K. Korgaonkar
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • David B. Donner
    Correspondence
    To whom correspondence should be addressed: Walther Oncology Center, Indiana University School of Medicine, 950 West Walnut St., Indianapolis, IN 46202
    Affiliations
    Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and Walther Cancer Institute, Indianapolis, Indiana 46208
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants CA67891 and 73023 (to D. B. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by the Indiana University Cancer Center and the Indiana University Diabetes Training Program.
Open AccessPublished:October 29, 2003DOI:https://doi.org/10.1074/jbc.M306976200
      Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-κB through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-κB activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-κB DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-κB is retained in the cytoplasm by inhibitory proteins, IκBs, which are phosphorylated and targeted for degradation by IκB kinases (IKKα and IKKβ). Expression and the ratios of IKKα and IKKβ, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKKα (the IKK kinase activated by Akt) to IKKβ were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKKβ diminished the capacity of the inhibitors to block NF-κB DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-κB DNA binding in Ikkβ–/– but not Ikkα–/– or wild-type cells in which the ratio of IKKα to IKKβ is low. Thus, noncoordinate expression of IκB kinases plays a role in determining the cell type-specific role of Akt in NF-κB activation.
      The NF-κB family of transcription factors plays a fundamental role in development, maintenance of the immune system, and cell viability (
      • Baeuerle P.A.
      • Baltimore D.
      ,
      • Ghosh S.
      • May M.J.
      • Kopp E.B.
      ,
      • Verma I.M.
      • Stevenson J.K.
      • Schwarz E.M.
      • Van Antwerp D.
      • Miyamoto S.
      ). NF-κB is composed of heterodimers of DNA-binding subunits (p50 and p52) and subunits with transcriptional activity (p65 (RelA), RelB, or c-Rel). In unstimulated cells, binary complexes of these subunits are restricted to the cytoplasm by interaction with members of a family of inhibitory proteins, inhibitors of κB (IκBs)
      The abbreviations used are: IκB, inhibitor of κB; IKK, IκB kinase; MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1; TNF, tumor necrosis factor; PI, phosphatidylinositol; HUVEC, human umbilical vein endothelial cells; MEF, mouse embryo fibroblast; EMSA, electrophoretic mobility shift assay; PTEN, phosphatase and tensin homolog deleted on chromosome ten.
      1The abbreviations used are: IκB, inhibitor of κB; IKK, IκB kinase; MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1; TNF, tumor necrosis factor; PI, phosphatidylinositol; HUVEC, human umbilical vein endothelial cells; MEF, mouse embryo fibroblast; EMSA, electrophoretic mobility shift assay; PTEN, phosphatase and tensin homolog deleted on chromosome ten.
      (
      • Mercurio F.
      • Manning A.M.
      ,
      • Baldwin Jr, A.S.
      ). In response to extracellular stimuli such as cytokines or UV radiation, IκB proteins are phosphorylated, polyubiquitinated, and then degraded by the 26 S proteasome (
      • Baldi L.
      • Brown K.
      • Franzoso G.
      • Siebenlist U.
      ,
      • Brockman J.A.
      • Scherer D.C.
      • McKinsey T.A.
      • Hall S.M.
      • Qi X.
      • Lee W.Y.
      • Ballard D.W.
      ,
      • Brown K.
      • Gerstberger S.
      • Carlson L.
      • Franzoso G.
      • Siebenlist U.
      ,
      • Traenckner E.B.
      • Pahl H.L.
      • Henkel T.
      • Schmidt K.N.
      • Wilk S.
      • Baeuerle P.A.
      ,
      • Traenckner E.B.
      • Baeuerle P.A.
      ,
      • Whiteside S.T.
      • Ernst M.K.
      • LeBail O.
      • Laurent-Winter C.
      • Rice N.
      • Israel A.
      ,
      • Scherer D.C.
      • Brockman J.A.
      • Chen Z.
      • Maniatis T.
      • Ballard D.W.
      ,
      • Palombella V.J.
      • Rando O.J.
      • Goldberg A.L.
      • Maniatis T.
      ). Dissociation from IκBs unmasks the nuclear localization sequence of NF-κB, permitting it to move into the nucleus, bind the promoters of target genes, and alter gene expression and cell function (
      • Traenckner E.B.
      • Baeuerle P.A.
      ,
      • Palombella V.J.
      • Rando O.J.
      • Goldberg A.L.
      • Maniatis T.
      ,
      • Chen Z.
      • Hagler J.
      • Palombella V.J.
      • Melandri F.
      • Scherer D.
      • Ballard D.
      • Maniatis T.
      ). The demonstration that phosphorylation of IκB proteins initiates events necessary for activation of NF-κB led to the discovery of IκB kinase (IKK) complexes composed of IKKα, IKKβ, and IKKγ (NEMO) (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandi E.
      • Karin M.
      ,
      • Regnier C.H.
      • Song H.Y.
      • Gao X.
      • Goeddel D.V.
      • Cao Z.
      • Rothe M.
      ,
      • Mercurio F.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Young D.B.
      • Li J.W.
      • Pascual G.
      • Motiwala A.
      • Zhu H.
      • Mann M.
      • Manning A.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.
      ). IKKα and IKKβ are serine-threonine kinases, and IKKγ is a scaffolding protein essential for the function of IKKα and IKKβ. IKKα and IKKβ share a high degree of amino acid homology and domain organization. The kinases are composed of an N-terminal kinase domain, a leucine zipper that facilitates homo- and heterodimerization, and a helix-loop-helix domain (
      • Karin M.
      • Ben-Neriah Y.
      ).
      IKKα and IKKβ can be activated by diverse kinases, among which are NF-κB-inducing kinase (NIK), MEKK1, Cot, NF-κB-activating kinase (NAK/TBK), protein kinases Cβ and Cδ, and MEKK3 (
      • Lee F.S.
      • Hagler J.
      • Chen Z.J.
      • Maniatis T.
      ,
      • Lee F.S.
      • Peters R.T.
      • Dang L.C.
      • Maniatis T.
      ,
      • Lin X.
      • Mu Y.
      • Cunningham Jr., E.T.
      • Marcu K.B.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Lin X.
      • Cunningham Jr., E.T.
      • Mu Y.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Lin X.
      • O'Mahony A.
      • Mu Y.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Malinin N.L.
      • Boldin M.P.
      • Kovalenko A.V.
      • Wallach D.
      ,
      • Woronicz J.D.
      • Gao X.
      • Cao Z.
      • Rothe M.
      • Goeddel D.V.
      ,
      • Lallena M.J.
      • Diaz-Meco M.T.
      • Bren G.
      • Paya C.V.
      • Moscat J.
      ). However, interest remains sustained in identifying other kinases that affect IKK complexes and NF-κB activity. One of these is the Akt serine-threonine kinase, a downstream target for activated phosphatidylinositol 3-kinase (PI 3-kinase) (
      • Brazil D.P.
      • Hemmings B.A.
      ,
      • Toker A.
      • Cantley L.C.
      ,
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ,
      • Testa J.R.
      • Bellacosa A.
      ). Akt is activated by mitogens and cytokines that function as survival factors. Akt mediates its functions by phosphorylating substrates that decrease the activity of pro-apoptotic proteins or increase the activity of anti-apoptotic proteins (
      • Testa J.R.
      • Bellacosa A.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ,
      • 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.
      ,
      • Collado M.
      • Medema R.H.
      • Garcia-Cao I.
      • Dubuisson M.L.
      • Barradas M.
      • Glassford J.
      • Rivas C.
      • Burgering B.M.
      • Serrano M.
      • Lam E.W.
      ,
      • Zhou B.P.
      • Liao Y.
      • Xia W.
      • Spohn B.
      • Lee M.H.
      • Hung M.C.
      ,
      • Mayo L.D.
      • Donner D.B.
      ,
      • Mayo L.D.
      • Donner D.B.
      ,
      • Ashcroft M.
      • Ludwig R.L.
      • Woods D.B.
      • Copeland T.D.
      • Weber H.O.
      • MacRae E.J.
      • Vousden K.H.
      ,
      • Zhou B.P.
      • Liao Y.
      • Xia W.
      • Zou Y.
      • Spohn B.
      • Hung M.C.
      ).
      Akt may affect NF-κB through multiple mechanisms. We demonstrated previously that TNF activates Akt, which phosphorylates and activates IKKα, thus promoting NF-κB function (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ). TNF and interleukin-1 can also increase the transactivation potential of the RelA/p65 subunit of NF-κB through a mechanism in which Akt has been implicated (
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Sizemore N.
      • Lerner N.
      • Dombrowski N.
      • Sakurai H.
      • Stark G.R.
      ,
      • Madrid L.V.
      • Wang C.Y.
      • Guttridge D.C.
      • Schottelius A.J.
      • Baldwin Jr., A.S.
      • Mayo M.W.
      ). PI 3-kinase activated by phorbol esters or lipopolysaccharide and PI 3-kinase/Akt signaling induced by signaling through CD40, interleukin-1, or G protein-coupled receptors activates NF-κB (
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Andjelic S.
      • Hsia C.
      • Suzuki H.
      • Kadowaki T.
      • Koyasu S.
      • Liou H.C.
      ,
      • Manna S.K.
      • Aggarwal B.B.
      ,
      • Xie P.
      • Browning D.D.
      • Hay N.
      • Mackman N.
      • Ye R.D.
      ). However, PI 3-kinase/Akt signaling induced by TNF in human umbilical vein endothelial cells inhibits apoptosis without playing a significant role in activation of NF-κB (
      • Madge L.A.
      • Pober J.S.
      ). Furthermore, Akt can activate a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, Cot, and indirectly affect IKK activity and NF-κB (
      • Kane L.P.
      • Mollenauer M.N.
      • Xu Z.
      • Turck C.W.
      • Weiss A.
      ). Thus, PI 3-kinase/Akt signaling is upstream of diverse pathways that activate NF-κB. However, the mechanisms through which PI 3-kinase/Akt signaling activates NF-κB are cell type-specific, and surrogate pathways also affect NF-κB.
      The goal of the present study was to determine the basis for the cell type specificity with which PI 3-kinase/Akt signaling activates NF-κB. We demonstrate that the ability of PI 3-kinase inhibitors to impair TNF-induced DNA binding of NF-κB is cell type-specific. Such specificity does not necessarily result from alterations in the ability of TNF to activate Akt, as this capacity was observed in seven different cell types, including primary cells. Rather, the proportion of IKKα to IKKβ varies among cells, and those cells in which the ratio of IKKα, the target for Akt phosphorylation, to IKKβ is high are most susceptible to the ability of PI 3-kinase inhibitors to impair NF-κB DNA binding.

      EXPERIMENTAL PROCEDURES

      Materials—Recombinant human tumor necrosis factor was a gift from Genentech Inc. (South San Francisco, CA). Antibodies to IKKα and IKKβ were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Akt and anti-Akt were from Cell Signaling, Inc. (Beverly, MA).
      Cell Culture—293 cells were purchased from the ATCC. 293, ME-180, HeLa, NIH 3T3, H1299, and MCF-7 cells and wild-type, Ikk1–/– (Ikkα–/–), and Ikk2–/– (Ikkβ–/–) mouse embryo fibroblasts (MEFs) were grown in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum supplemented with penicillin/streptomycin and l-glutamine at 37 °C under 5% CO2. Human umbilical vein endothelial cells (HUVEC) were grown in endothelial growth media (EGM, Clonetics, Inc.).
      Transfections and Preparation of Cell Lysates—293 cells were transfected using the calcium phosphate procedure. After transfection, cells were washed twice with ice cold phosphate-buffered saline and lysed in 50 mm HEPES, pH 7.0, 150 mm NaCl, 10% glycerol, 1.2% Triton X-100, 1.5 mm MgCl2, 1 m EGTA, 1 mm EDTA, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 0.15 unit/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 1 mm sodium orthovanadate, and 1 mm dithiothreitol. Lysates were centrifuged (13,000 rpm, 4 °C, 5 min), and equal amounts of supernatant protein was fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes.
      NF-κB DNA Binding—Electrophoretic mobility shift assays (EMSAs) were conducted using 6 μg of protein isolated from cells lysed in 40 mm HEPES, pH 7.0, 100 mm KCl, 1% Nonidet P-40, 1 mm dithiothreitol, 1mm phenylmethylsulfonyl fluoride, 1 μg of aprotinin, 1 μg of pepstatin A, and 1 μg of leupeptin. Cellular protein was incubated with the double-stranded 32P-labeled κB probe 5′-GTTGAGGGACTTTCCCAGG-3′ in 1× Tris-EDTA, 1 mm KCl, 10% glycerol, 1 mm dithiothreitol, and 1 mg/ml polydeoxycytosine-deoxyinosine for 30 min at room temperature. DNA protein complexes were size-fractionated on native 5% polyacrylamide gels, dried, and exposed to film for autoradiography.

      RESULTS

      We previously showed that PI 3-kinase/Akt signaling induces NF-κB DNA binding in 293 cells (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ). However, this observation has not been observed in human umbilical vein endothelial cells (
      • Madge L.A.
      • Pober J.S.
      ). Experiments were therefore conducted to test whether the effect of PI 3-kinase/Akt signaling on NF-κB DNA binding is cell type-specific. To accomplish this, we used EMSAs to test the effect of the inhibitors of PI 3-kinase, LY294002 and wortmannin, on TNF-induced NF-κB DNA binding in seven different cell types. As illustrated in Fig. 1, NF-κB DNA binding was induced by TNF in each cell type. The NF-κB DNA binding complex was identified by supershifting with an antibody to p65 (RelA) (Fig. 1, far right lane in each EMSA). LY294002 or wortmannin variably affected TNF-induced NF-κB DNA binding. The inhibitors completely blocked TNF-induced NF-κB DNA binding in 293 cells, partially blocked binding in MCF-7, ME-180, HeLa, and NIH 3T3 cells, but had little effect on binding in H1299 cells and HUVEC.
      Figure thumbnail gr1
      Fig. 1Effect of inhibition of PI 3-kinase on NF-κB DNA binding. Serum-starved 293, HeLa, MCF-7, NIH 3T3, HUVEC, and H1299 cells were treated with Me2SO, 20 μm LY294002 (Ly), or 100 nm wortmannin (Wort) for 1 h prior to stimulation with vehicle or 1 nm TNF for 30 min at 37 °C. NF-κB DNA binding was assayed by an EMSA of equal amounts of protein from cell lysates. Anti-p65 was used to supershift DNA-binding complexes containing NF-κB. Ab, antibody.
      The variable efficacy with which PI 3-kinase inhibitors blocked TNF-induced NF-κB DNA binding led us to test whether Akt activation might be attenuated or defective in some cell types. Akt activity was assayed by probing Western blots prepared from lysates of TNF-treated cells with an antibody that exclusively recognizes the Akt, which is phosphorylated on serine 473 and therefore is active. TNF activated Akt in each cell type used to characterize the effect of PI 3-kinase inhibitors on NF-κB DNA binding in Fig. 1, although the kinetics and extent of activation varied from one cell type to another (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Activation of Akt by TNF. Western blots prepared from lysates of serum-starved cells treated with 1 nm TNF for various times were probed with anti-phospho-Ser473 (active) Akt or anti-Akt to normalize for protein loading.
      Activation of Akt by TNF could not account for the varied cellular sensitivity to LY294002. Because our previous work suggested that IKKα is the IKK kinase upon which Akt acts (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ), we focused on the possibility that expression of the IKK kinases might be a determinant in the capacity of PI 3-kinase inhibitors to affect NF-κB DNA binding. Expression of IKKα and IKKβ in cell lines was therefore examined. Western blots showed that the amount of IKKα and IKKβ varies among cell types (Fig. 3a). Additionally, we reproducibly observed faster and slower migrating forms of IKKβ in cells. Although the basis for this heterogeneity is not yet defined, it might result from phosphorylation of IKKβ by diverse kinases in cells. Densitometry quantified the proportion of IKKα to IKKβ (Fig. 3b). In 293 cells the proportion of IKKα to IKKβ was highest among the cell types tested. H1299 cells and HUVEC contain a very low proportion of IKKα to IKKβ. In ME180, HeLa, MCF-7, and NIH 3T3 cells, the proportion of IKKα to IKKβ favors IKKβ but not so greatly as in H1299 cells and HUVEC. These observations show that the proportion of IKKα to IKKβ differs among cells. Also, a high proportion of IKKα to IKKβ correlates with the ability of PI 3-kinase inhibitors to block TNF-induced NF-κB DNA binding.
      Figure thumbnail gr3
      Fig. 3Cellular expression of IKKα and IKKβ. a, Western blots prepared from lysates of various cell types were probed with antibodies directed against IKKα, IKKβ, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). b, densitometry assayed the relative levels of the IKK subunits, and the bar graph represents IKKα/IKKβ.
      To demonstrate whether the proportion of IKKα to IKKβ affects the role of PI 3-kinase in NF-κB DNA binding, the proportion of the kinases was altered by transient transfection of 293 cells, in which the IKKα/IKKβ proportion is high. These, or vector-transfected control cells, were treated with wortmannin and stimulated with TNF or medium, and NF-κB DNA binding was assayed. Wortmannin abrogated TNF-induced NF-κB DNA binding in 293 cells transfected with empty vector but had lesser effect in cells transfected with IKKβ (Fig. 4a). Quantitation of the observations just described showed that inhibition of PI 3-kinase in cells transfected with empty vector diminished NF-κB DNA binding by 66%, whereas NF-κB DNA binding was diminished by only 33% in cells transfected with IKKβ. Reproducibly, expression of IKKβ in 293 cells increased DNA binding, an effect that may relate to its ability to phosphorylate IκBα more effectively than IKKα (
      • Lee F.S.
      • Peters R.T.
      • Dang L.C.
      • Maniatis T.
      ,
      • Woronicz J.D.
      • Gao X.
      • Cao Z.
      • Rothe M.
      • Goeddel D.V.
      ,
      • Li J.
      • Peet G.W.
      • Pullen S.S.
      • Schembri-King J.
      • Warren T.C.
      • Marcu K.B.
      • Kehry M.R.
      • Barton R.
      • Jakes S.
      ,
      • 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.
      ). Western blot analysis confirmed that the cellular content of IKKβ was greatly increased in cells transfected with IKKβ (Fig. 4b). This experiment showed that by altering the proportion of IKKα to IKKβ in favor of the latter, the sensitivity of NF-κB DNA binding to PI 3-kinase inhibition is diminished. These observations support the conclusion that the proportion of IKKα to IKKβ determines the sensitivity of NF-κB DNA binding to blockade by PI 3-kinase inhibitors.
      Figure thumbnail gr4
      Fig. 4Alteration of the proportion of IKKα to IKKβ changes the sensitivity of NF-κB DNA binding to inhibition by wortmannin (Wort). 293 cells were transfected with empty vector or IKKβ, and 24 h thereafter the cells were subjected to serum starvation for 24 h, incubated in the absence or presence of 100 nm wortmannin for 1 h, and then treated with 1 nm TNF for 30 min. a, NF-κB DNA binding was assayed by EMSA using equal amounts of protein from cell lysates. b, Western blot analysis demonstrated overexpression of IKKβ in cells transfected with this kinase. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. This autoradiograph was developed for a shorter period than that in to permit visualization of the extent to which IKKβ was overexpressed in transfected cells.
      To support these observations, wild-type MEFs or MEFs from IKKα- or IKKβ-deficient mice were used to assess the sensitivity of NF-κB DNA binding to inhibitors of PI 3-kinase. Serum-starved wild-type, Ikkα –/–, and Ikkβ –/– MEFs treated for 1 h with Me2SO or 20 μm LY294002 were incubated with phosphate-buffered saline or 1 nm TNF for 30 min. TNF increased DNA binding in the parental or knockout MEFs, but LY294002 inhibited NF-κB DNA binding only in Ikkβ –/– cells (Fig. 5a). Supershifting using anti-p65 showed that NF-κB DNA binding complexes from parental, Ikkβ–/–, and Ikkα–/– MEFs contained this subunit. These observations causally relate the effect of PI 3-kinase inhibitors on NF-κB DNA binding and the proportion and expression of IKKα and IKKβ.
      Figure thumbnail gr5
      Fig. 5Effect of PI 3-kinase inhibition on NF-κB DNA binding in MEFs. a, serum-starved MEFs were treated with Me2SO or 20 μm LY294002 for 1 h prior to stimulation with vehicle or 1 nm TNF for 30 min at 37 °C. NF-κB DNA binding was assayed by EMSA using equal amounts of protein from lysates. WT, wild type; ab, antibody. b, equal amounts of protein from lysates of wild-type, Ikkα–/–, or Ikkβ–/– mouse embryo fibroblasts were fractionated by SDS-PAGE, and a Western blot was probed with antibodies to IKKα, IKKβ, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). c, a Western blot prepared from lysates of H1299 cells and wild-type mouse embryo fibroblasts was probed with antibodies to IKKα, IKKβ, and glyceraldehyde-3-phosphate dehydrogenase.
      Our previous observations (Ref.
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      and figures herein) suggested that IKKα is the target for Akt in IκB kinase complexes. The inability of PI 3-kinase inhibitors to affect NF-κB DNA binding in wild-type MEFs therefore led us to predict that this cell type would have a low proportion of IKKβ relative to IKKα when compared with other cell types. To evaluate this prediction, we first confirmed the phenotypes of wild-type, Ikkα–/–, and Ikkβ–/– MEFs insofar as IκB kinase expression is concerned. Western blot analysis of the expression of the IκB kinases revealed that wild-type MEFs express IKKα and IKKβ, Ikkα–/– MEFs express IKKβ but not IKKα, and Ikkβ–/– MEFs express IKKα but not IKKβ (Fig. 5b). Having confirmed the phenotypes of the cells in this study, we compared the expression of IKKα to IKKβ in H1299 cells and wild-type MEFs. It was observed (Fig. 5c) that the proportion of IKKα to IKKβ was similarly low in H1299 cells and wild-type MEFs. Also, IKKα in human H1299 cells displayed a somewhat greater apparent molecular weight than IKKα in murine wild-type MEFs. The results obtained from these experiments are consistent with the conclusion that in comparisons among different cell types, a high proportion of IKKβ relative to IKKα diminishes the ability of inhibitors of PI 3-kinase to block NF-κB DNA binding.

      DISCUSSION

      A high molecular weight IκB kinase complex called a signal-some (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandi E.
      • Karin M.
      ,
      • Lee F.S.
      • Hagler J.
      • Chen Z.J.
      • Maniatis T.
      ,
      • 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.
      ,
      • Maniatis T.
      ), which can be composed of IKKα, IKKβ, and IKKγ, is activated by a group of serine-threonine kinases (
      • Lee F.S.
      • Hagler J.
      • Chen Z.J.
      • Maniatis T.
      ,
      • Lee F.S.
      • Peters R.T.
      • Dang L.C.
      • Maniatis T.
      ,
      • Lin X.
      • Mu Y.
      • Cunningham Jr., E.T.
      • Marcu K.B.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Lin X.
      • Cunningham Jr., E.T.
      • Mu Y.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Lin X.
      • O'Mahony A.
      • Mu Y.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Malinin N.L.
      • Boldin M.P.
      • Kovalenko A.V.
      • Wallach D.
      ,
      • Woronicz J.D.
      • Gao X.
      • Cao Z.
      • Rothe M.
      • Goeddel D.V.
      ,
      • Lallena M.J.
      • Diaz-Meco M.T.
      • Bren G.
      • Paya C.V.
      • Moscat J.
      ,
      • Chu Z.L.
      • DiDonato J.A.
      • Hawiger J.
      • Ballard D.W.
      ,
      • Geleziunas R.
      • Ferrell S.
      • Lin X.
      • Mu Y.
      • Cunningham Jr., E.T.
      • Grant M.
      • Connelly M.A.
      • Hambor J.E.
      • Marcu K.B.
      • Greene W.C.
      ,
      • Uhlik M.
      • Good L.
      • Xiao G.
      • Harhaj E.W.
      • Zandi E.
      • Karin M.
      • Sun S.C.
      ,
      • Yin M.J.
      • Christerson L.B.
      • Yamamoto Y.
      • Kwak Y.T.
      • Xu S.
      • Mercurio F.
      • Barbosa M.
      • Cobb M.H.
      • Gaynor R.B.
      ). Activated signalsomes phosphorylate IκB proteins, promoting their dissociation from NF-κB (
      • Karin M.
      • Ben-Neriah Y.
      ). Once released from the inhibitory proteins, NF-κB translocates into the nucleus and activates target genes. The present study addressed the cell type specificity with which PI 3-kinase/Akt signaling affects NF-κB. Investigation of transformed, immortalized, and primary cells shows that pharmacological blockade of PI 3-kinase can entirely inhibit, partially impair, or have minimal effect on NF-κB DNA binding induced by TNF. In some cell types, the failure of pharmacological blockade can result from an inability of TNF to activate Akt. For example, in a comparison of PC-3 and DU-145 prostate cancer cells, we observed that TNF activated Akt in the former but not the latter cell type (
      • Gustin J.A.
      • Maehama T.
      • Dixon J.E.
      • Donner D.B.
      ). This distinction was associated with the absence of PTEN expression in PC-3 cells and robust expression of PTEN in DU-145 cells. PTEN dephosphorylates the lipid mediators that mediate PI 3-kinse function and thereby blocks Akt activation (
      • Mayo L.D.
      • Donner D.B.
      ,
      • Maehama T.
      • Dixon J.E.
      ,
      • Maehama T.
      • Dixon J.E.
      ).
      In most cell types that we have tested, however, the different responsiveness of NF-κB DNA binding to blockade by inhibitors of PI 3-kinse did not arise from inability of TNF to activate Akt. Rather, distinct cellular sensitivities arose from varied proportions of IKKα to IKKβ. In 293 cells the proportion of IKKα to IKKβ favors the former, relative to most other cell types, in which this relationship is reversed. In HUVEC, H1299 cells, and mouse embryo fibroblasts the proportion greatly favors IKKβ. Cells containing a high proportion of IKKα to IKKβ are most susceptible to inhibition of NF-κB DNA binding by PI 3-kinase inhibitors. This is explained by our demonstration that Akt associates with, phosphorylates, and activates IKKα but not IKKβ (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ). Two additional observations causally demonstrate that IKKα is the target for PI 3-kinase/Akt signaling in the IKK complex. Alteration of the proportion of IKKα to IKKβ in 293 cells by transient expression of IKKβ all but eliminated the ability of wortmannin to inhibit NF-κB DNA binding. In addition, studies with mouse embryo fibroblasts showed that inhibitors of PI 3-kinase had little or no effect on TNF-induced DNA binding in wild type or Ikkα –/– cells, the former having low expression and the latter no expression of IKKα. However, LY294002 abrogated NF-κB DNA binding induced by TNF in Ikkβ –/– cells. The demonstration that PI 3-kinase/Akt signaling is directed toward IKKα explains a report (
      • Madge L.A.
      • Pober J.S.
      ), confirmed here, that Akt does not play a role in the activation of NF-κB by TNF in HUVEC, as these cells express little IKKα.
      IKKα and IKKβ are homologous but have different functions. Homozygous deletion of the IKKβ gene decreases cytokine-induced NF-κB activation and results in embryonic lethality in mice, because of severe apoptosis in the liver (
      • Li Z.W.
      • Chu W.
      • Hu Y.
      • Delhase M.
      • Deerinck T.
      • Ellisman M.
      • Johnson R.
      • Karin M.
      ,
      • Li Q.
      • Van Antwerp D.
      • Mercurio F.
      • Lee K.F.
      • Verma I.M.
      ,
      • Tanaka M.
      • Fuentes M.E.
      • Yamaguchi K.
      • Durnin M.H.
      • Dalrymple S.A.
      • Hardy K.L.
      • Goeddel D.V.
      ). Cytokine-induced NF-κB activity is modestly diminished or unaffected in cells from IKKα knockout animals (
      • Hu Y.
      • Baud V.
      • Delhase M.
      • Zhang P.
      • Deerinck T.
      • Ellisman M.
      • Johnson R.
      • Karin M.
      ,
      • Takeda K.
      • Takeuchi O.
      • Tsujimura T.
      • Itami S.
      • Adachi O.
      • Kawai T.
      • Sanjo H.
      • Yoshikawa K.
      • Terada N.
      • Akira S.
      ,
      • Li Q.
      • Lu Q.
      • Hwang J.Y.
      • Buscher D.
      • Lee K.F.
      • Izpisua-Belmonte J.C.
      • Verma I.M.
      ), but they die shortly after birth because of severe skin and bone defects. IKKα but not IKKβ induces processing of NF-κB2 (p100) to p52 (
      • Senftleben U.
      • Cao Y.
      • Xiao G.
      • Greten F.R.
      • Krahn G.
      • Bonizzi G.
      • Chen Y.
      • Hu Y.
      • Fong A.
      • Sun S.C.
      • Karin M.
      ) and is a component of a pathway that controls mammary epithelial cell proliferation in response to receptor activator of NF-κB (RANK) signaling (
      • Cao Y.
      • Bonizzi G.
      • Seagroves T.N.
      • Greten F.R.
      • Johnson R.
      • Schmidt E.V.
      • Karin M.
      ).
      Both kinases phosphorylate IκBα in vitro, but IKKβ is far more effective than IKKα (
      • Mercurio F.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Young D.B.
      • Li J.W.
      • Pascual G.
      • Motiwala A.
      • Zhu H.
      • Mann M.
      • Manning A.M.
      ,
      • Lee F.S.
      • Peters R.T.
      • Dang L.C.
      • Maniatis T.
      ,
      • Woronicz J.D.
      • Gao X.
      • Cao Z.
      • Rothe M.
      • Goeddel D.V.
      ,
      • Li J.
      • Peet G.W.
      • Pullen S.S.
      • Schembri-King J.
      • Warren T.C.
      • Marcu K.B.
      • Kehry M.R.
      • Barton R.
      • Jakes S.
      ). IKKα and IKKβ homo- and heterodimerize, and heterogeneous signalsomes have been isolated from cells (
      • Mercurio F.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Young D.B.
      • Li J.W.
      • Pascual G.
      • Motiwala A.
      • Zhu H.
      • Mann M.
      • Manning A.M.
      ). IKKα/IKKβ heterodimers are components of a 900-kDa multicomponent complex, which additionally contains NEMO/IKKγ/IKKAP1, a protein that lacks kinase activity but appears to be necessary for proper organization of the complex (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandi E.
      • Karin M.
      ,
      • Mercurio F.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Young D.B.
      • Li J.W.
      • Pascual G.
      • Motiwala A.
      • Zhu H.
      • Mann M.
      • Manning A.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.
      ,
      • 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.
      ,
      • Cohen L.
      • Henzel W.J.
      • Baeuerle P.A.
      ). In heterodimers, which form in preference to homodimers, IKKα inhibits basal IKKβ activity. TNF, lipopolysaccharide, and ectopically expressed NIK (NF-κB-inducing kinase), Cot, MEKK1, and Tax activate heterodimeric signalsomes (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandi E.
      • Karin M.
      ,
      • Regnier C.H.
      • Song H.Y.
      • Gao X.
      • Goeddel D.V.
      • Cao Z.
      • Rothe M.
      ,
      • Lee F.S.
      • Hagler J.
      • Chen Z.J.
      • Maniatis T.
      ,
      • Lin X.
      • Mu Y.
      • Cunningham Jr., E.T.
      • Marcu K.B.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Lin X.
      • Cunningham Jr., E.T.
      • Mu Y.
      • Geleziunas R.
      • Greene W.C.
      ,
      • Chu Z.L.
      • DiDonato J.A.
      • Hawiger J.
      • Ballard D.W.
      ,
      • Geleziunas R.
      • Ferrell S.
      • Lin X.
      • Mu Y.
      • Cunningham Jr., E.T.
      • Grant M.
      • Connelly M.A.
      • Hambor J.E.
      • Marcu K.B.
      • Greene W.C.
      ,
      • Uhlik M.
      • Good L.
      • Xiao G.
      • Harhaj E.W.
      • Zandi E.
      • Karin M.
      • Sun S.C.
      ,
      • Yin M.J.
      • Christerson L.B.
      • Yamamoto Y.
      • Kwak Y.T.
      • Xu S.
      • Mercurio F.
      • Barbosa M.
      • Cobb M.H.
      • Gaynor R.B.
      ,
      • O'Connell M.A.
      • Bennett B.L.
      • Mercurio F.
      • Manning A.M.
      • Mackman N.
      ) by relieving repression of IKKβ activity by IKKα (
      • O'Mahony A.
      • Lin X.
      • Geleziunas R.
      • Greene W.C.
      ). IKKα homodimers can be isolated from high molecular weight multimeric complexes, whereas IKKβ homodimers are recovered from lower molecular weight complexes (
      • Mercurio F.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Young D.B.
      • Li J.W.
      • Pascual G.
      • Motiwala A.
      • Zhu H.
      • Mann M.
      • Manning A.M.
      ). Our demonstration that the expression of IKKα to IKKβ in cells is variable and that the proportions of the kinases to one another varies among cell types is important in understanding the cell type-specific mechanisms through which NF-κB can be activated. HeLa cells express a high proportion of IKKβ to IKKα, which is consistent with the isolation of IKKα-IKKβ heterodimers and IKKβ homodimers from these cells (
      • Mercurio F.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Young D.B.
      • Li J.W.
      • Pascual G.
      • Motiwala A.
      • Zhu H.
      • Mann M.
      • Manning A.M.
      ). Thus, the proportion of IKKα to IKKβ, defined by Western blot analysis in this study, finds its counterpart in the composition of signalsomes in cells. That Akt acts on and through IKKα but not IKKβ explains the cell type specificity with which PI 3-kinase/Akt signaling affects NF-κB induction. The substrate specificity of Akt is consistent with its initiating activation of IKKα-IKKβ heterodimers and its having the capacity to activate IKKα but not IKKβ homodimers. Variable expression of IKKα and IKKβ, as well as the presence of upstream kinases that preferentially activate one or the other IκB kinase, identifies a mechanism that can account for cell type-specific regulation of NF-κB activity.

      Acknowledgments

      We thank Dr. Inder Verma for advice and assistance during the course of these studies.

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