Advertisement

The Phosphatidylinositol 3-Kinase-Akt Pathway Limits Lipopolysaccharide Activation of Signaling Pathways and Expression of Inflammatory Mediators in Human Monocytic Cells*

  • Mausumee Guha
    Affiliations
    From the Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037
    Search for articles by this author
  • Nigel Mackman
    Correspondence
    To whom correspondence should be addressed: Depts. of Immunology and Cell Biology, C-204, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8594; Fax: 858-784-8480;
    Affiliations
    From the Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037
    Search for articles by this author
  • Author Footnotes
    * This work was supported by National Institutes of Health Grant HL 48872 (to N. M.).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:June 06, 2002DOI:https://doi.org/10.1074/jbc.M203298200
      Monocytes and macrophages express cytokines and procoagulant molecules in various inflammatory diseases. In sepsis, lipopolysaccharide (LPS) from Gram-negative bacteria induces tumor necrosis factor-alpha (TNF-α) and tissue factor (TF) in monocytic cells via the activation of the transcription factors Egr-1, AP-1, and nuclear factor-κB. However, the signaling pathways that negatively regulate LPS-induced TNF-α and TF expression in monocytic cells are currently unknown. We report that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway enhances LPS-induced activation of the mitogen-activated protein kinase pathways (ERK1/2, p38, and JNK) and the downstream targets AP-1 and Egr-1. In addition, inhibition of PI3K-Akt enhanced LPS-induced nuclear translocation of nuclear factor-κB and prevented Akt-dependent inactivation of glycogen synthase kinase-β, which increased the transactivational activity of p65. We propose that the activation of the PI3K-Akt pathway in human monocytes limits the LPS induction of TNF-α and TF expression. Our study provides new insight into the inhibitory mechanism by which the PI3K-Akt pathway ensures transient expression of these potent inflammatory mediators.
      LPS
      lipopolysaccharide
      CMV
      cytomegalovirus
      dn
      dominant negative
      ELISA
      enzyme-linked immunosorbent assay
      ERK
      extracellular signal-regulated kinase
      GSK
      glycogen synthase kinase
      JNK
      c-Jun N-terminal kinase
      LUC
      luciferase
      MAPK
      mitogen-activated protein kinase
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      MEKK
      MEK kinase
      NF-κB
      nuclear factor-κB
      PBMC(s)
      peripheral blood mononuclear cell(s)
      PI3K
      phosphatidylinositol 3-kinase
      TF
      tissue factor
      TNF-α
      tumor necrosis factor-alpha
      TLR
      toll receptor
      Regulation of proinflammatory gene expression in a biological system is a balance between positive and negative signal transduction pathways. Lipopolysaccharide (LPS),1 the outer membrane component of Gram-negative bacteria, induces expression of many proinflammatory mediators in monocyte/macrophages, one of the key cell types involved in sepsis (
      • Steinemann S.
      • Ulevitch R.J.
      • Mackman N.
      ,
      • van der Bruggen T.
      • Nijenhuis S.
      • van Raaij E.
      • Verhoef J.
      • van Asbeck B.S.
      ,
      • Swantek J.L.
      • Christerson L.
      • Cobb M.H.
      ,
      • Coleman D.L.
      • Bartiss A.H.
      • Sukhatme V.P.
      • Liu J.
      • Rupprecht H.D.
      ,
      • Hall A.J.
      • Vos H.L.
      • Bertina R.M.
      ,
      • Scherle P.A.
      • Jones E.A.
      • Favata M.F.
      • Daulerio A.J.
      • Covington M.B.
      • Nurnberg S.A.
      • Magolda R.L.
      • Trzaskos J.M.
      ,
      • Guha M.
      • Mackman N.
      ). LPS activation of the CD14·TLR4·MD2 complex results in the expression of tumor necrosis factor-alpha (TNF-α) and tissue factor (TF) (
      • Steinemann S.
      • Ulevitch R.J.
      • Mackman N.
      ,
      • Ulevitch R.J.
      • Tobias P.S.
      ,
      • Liu M.K.
      • Herrera-Velit P.
      • Browsney R.W.
      • Reiner N.E.
      ,
      • Beutler B.
      ). The signaling pathways that positively regulate TNF-α and TF gene expression in LPS-stimulated monocytes/macrophages are well characterized (
      • van der Bruggen T.
      • Nijenhuis S.
      • van Raaij E.
      • Verhoef J.
      • van Asbeck B.S.
      ,
      • Swantek J.L.
      • Christerson L.
      • Cobb M.H.
      ,
      • Hall A.J.
      • Vos H.L.
      • Bertina R.M.
      ,
      • Hambleton J.
      • Weinstein S.L.
      • Lem L.
      • DeFranco A.L.
      ,
      • Fischer C.
      • Page S.
      • Weber M.
      • Eisele T.
      • Neumeier D.
      • Brand K.
      ,
      • Yao J.
      • Mackman N.
      • Edgington T.S.
      • Fan S.-T.
      ,
      • Mackman N.
      • Brand K.
      • Edgington T.S.
      ,
      • Guha M.
      • O'Connell M.A.
      • Pawlinski R.
      • Hollis A.
      • McGovern P.
      • Yan S.-F.
      • Stern D.
      • Mackman N.
      ). However, mechanisms and signaling pathways that limit the magnitude of the induction of these genes are poorly understood.
      Recent evidence suggests that activation of phosphatidylinositol 3-kinase (PI3K), a ubiquitous lipid-modifying enzyme, may modulate positively acting signaling pathways. PI3K is a heterodimeric protein consisting of a p85 regulatory subunit and a p110 catalytic subunit. LPS stimulation of monocytes/macrophages activates the PI3K pathway (
      • Lee J.
      • Mira-Arbibe L.
      • Ulevitch R.J.
      ,
      • Pahan K.
      • Raymond J.R.
      • Singh I.
      ,
      • Park Y.C.
      • Lee C.H.
      • Kang H.S.
      • Chung H.T.
      • Kim H.D.
      ), although the steps between the CD14·TLR4·MD2 complex and activation of PI3K have not been characterized. Activation of PI3K appears to occur via phosphorylation of tyrosine residues in the Src homology 2 domain of p85, which permits docking of PI3K to the plasma membrane and allows allosteric modifications that increase its catalytic activity (
      • Scheid M.P.
      • Woodgett J.R.
      ,
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ). Activated PI3K catalyzes the phosphorylation of membrane inositol lipids and the accumulation of phosphatidylinositol 3,4,5-trisphosphate and its phospholipid phosphatase product phosphatidylinositol 3,4-bisphosphate in the membrane. These membrane changes allow docking of the lipid kinases phosphatidylinositol-dependent kinase 1 and protein kinase B/Akt. After membrane recruitment Akt is activated by dual phosphorylation of Ser473 and Thr308 by phosphatidylinositol-dependent kinase 1 and possibly phosphatidylinositol-dependent kinase 1-related kinase-2 (
      • 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.
      ).
      The PI3K-Akt pathway has been shown to regulate negatively NF-κB and the expression of inflammatory genes. Wortmannin, a specific inhibitor of PI3K, enhanced LPS-induced nitric-oxide synthase in murine peritoneal macrophages (
      • Park Y.C.
      • Lee C.H.
      • Kang H.S.
      • Chung H.T.
      • Kim H.D.
      ), and activation of PI3K-Akt suppressed LPS-induced lipoprotein lipase expression in J774 macrophages (
      • Tengku-Muhammad T.S.
      • Hughes T.R.
      • Cryer A.
      • Ramji D.P.
      ). Induction of nitric-oxide synthase in C6 glial cells and rat primary astrocytes was also regulated negatively by activation of PI3K (
      • Pahan K.
      • Raymond J.R.
      • Singh I.
      ), and a constitutively active PI3K inhibited induction of nitric-oxide synthase gene expression in human astrocytes (
      • Pahan K.
      • Liu X.
      • Wood C.
      • Raymond J.R.
      ). Angiopoeitin-1, a potent activator of PI3K, negatively regulated vascular endothelial growth factor- and TNF-α-induced TF expression in endothelial cells (
      • Kim I., Oh, J.L.
      • Ryu Y.S., So, J.N.
      • Sessa W.C.
      • Walsk K.
      • Koh G.Y.
      ). Finally, in endothelial cells the PI3K-Akt pathway limited vascular endothelial growth factor activation of the p38 MAPK pathway and TF gene expression (
      • Blum S.
      • Issbrhker K.
      • Willuweit A.
      • Hehlgans S.
      • Lucerna M.
      • Mechtcheriakova D.
      • Walsh K.
      • von der Ahe D.
      • Hofer E.
      • Clauss M.
      ).
      In contrast to studies showing that the PI3K-Akt pathway negatively regulates expression of inflammatory genes in macrophages, a recent study demonstrated that the PI3K-Akt pathway positively regulated nuclear factor (NF)-κB-dependent gene expression in HepG2 cells via phosphorylation and increased transactivation activity of p65 (
      • Thomas K.W.
      • Monick M.M.
      • Stabler J.M.
      • Yarovinsky T.
      • Carter A.B.
      • Hunninghake G.W.
      ). Overexpression of a constitutively active form of Akt also increased NF-κB-dependent gene expression in 3T3 fibroblasts via the activation of I-κB kinase and the p38 MAPK (
      • Madrid L.V.
      • Mayo M.W.
      • Reuther J.Y.
      • Baldwin Jr., A.S.
      ). Activation of PI3K-Akt has been implicated in playing a pivotal role in cytokine-induced transcriptional activation of NF-κB- and AP-1-dependent gene expression and in inhibiting apoptosis (
      • Reddy S.A.G.
      • Huang J.H.
      • Liao W.S.-L.
      ,
      • Madrid L.V.
      • Wang C.-Y.
      • Guttridge D.C.
      • Schottelius A.J.G.
      • Baldwin Jr., A.S.
      • Mayo M.W.
      ,
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Béraud C.
      • Henzel W.J.
      • Baeuerle P.A.
      ,
      • Reddy S.A.G.
      • Huang J.H.
      • Liao W.S.-L.
      ). Finally, activation of the TLR2 receptor in human monocytic cells by Gram-positive bacteria activated the PI3K-Akt pathway and increased the transactivation activity of p65 (
      • Arbibe L.
      • Mira J.-P.
      • Teusch N.
      • Kline L.
      • Guha M.
      • Mackman N.
      • Godowski P.J.
      • Ulevitch R.J.
      • Knaus U.G.
      ).
      The PI3K-Akt pathway has been shown to regulate negatively many kinases including Raf-1 and glycogen synthase kinase (GSK)-3β, which mediate induction of inflammatory genes. We and others have shown that LPS-induced TNF-α and TF expression in monocytic cells is mediated, in part, via the activation of the Raf-MEK-ERK1/2 pathway (
      • van der Bruggen T.
      • Nijenhuis S.
      • van Raaij E.
      • Verhoef J.
      • van Asbeck B.S.
      ,
      • Scherle P.A.
      • Jones E.A.
      • Favata M.F.
      • Daulerio A.J.
      • Covington M.B.
      • Nurnberg S.A.
      • Magolda R.L.
      • Trzaskos J.M.
      ,
      • Yao J.
      • Mackman N.
      • Edgington T.S.
      • Fan S.-T.
      ,
      • Guha M.
      • O'Connell M.A.
      • Pawlinski R.
      • Hollis A.
      • McGovern P.
      • Yan S.-F.
      • Stern D.
      • Mackman N.
      ). Activation of Akt has been shown to regulate negatively the serine/threonine kinase Raf-1 and the downstream MEK-ERK1/2 signaling pathway (
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Zimmermann S.
      • Moelling K.
      ). Akt induces an inhibitory phosphorylation of Ser259 in the N-terminal CR2 domain of Raf-1, which increases its association with 14-3-3 protein and masks the accessibility of residues in the kinase domain of Raf-1 necessary for its activation (
      • Morrison D.K.
      • Cutler Jr., R.E.
      ). Dephosphorylation of Ser259 and phosphorylation of Ser338 and Tyr341 in the C-terminal kinase domain are required for Raf-1 activation and its interaction with downstream substrates (
      • Chaudhary A.
      • King W.G.
      • Mattaliano M.D.
      • Frost J.A.
      • Diaz B.
      • Morrison D.K.
      • Cobb M.H.
      • Marshall M.S.
      • Brugge J.S.
      ,
      • Dhillon A.S.
      • Meikle S.
      • Yazici Z.
      • Eulitz M.
      • Kolch W.
      ).
      GSK-3β is another serine/threonine kinase that is inhibited by Akt-dependent phosphorylation. Akt phosphorylates Ser9 in the N terminus of GSK-3β and inactivates the kinase (
      • Cohen P.
      • Frame S.
      ). The phenotype of GSK-3β−/− embryos is similar to that of RelA−/− embryos, suggesting that GSK-3β may regulate the transactivational activity of p65 (
      • Hoeflich K.P.
      • Luo J.
      • Rubie E.A.
      • Tsao M.-S.
      • Jin O.
      • Woodgett J.R.
      ). LiCl is a potent inhibitor of GSK-3β and inactivates GSK-3β by inducing its phosphorylation on Ser9 in its N terminus (
      • Frame S.
      • Cohen P.
      ). Indeed, the effect of LiCl on wingless signaling in wild-type cells mimicked the phenotype observed in GSK-3β null cells (
      • Cohen P.
      • Frame S.
      ). The role of GSK-3β in the regulation of inflammatory genes in monocytes is currently undefined.
      Our study demonstrates that inhibition of the PI3K-Akt pathway enhances LPS-induced TNF-α and TF gene expression via increased activation of Egr-1-, AP-1, and NF-κB. Inhibition of PI3K also enhanced TNF-α and TF gene expression, in part, by increasing the transactivational activity of p65 by inhibiting Akt-dependent inactivation of GSK-3β. Therefore, activation of the PI3K-Akt pathway in LPS-treated human monocytes and THP-1 cells limits the induction of TNF-α and TF gene expression.

      MATERIALS AND METHODS

      LPS (Escherichia coli serotype 0111:B4) and the PI3K inhibitors wortmannin and LY294002 were obtained from Calbiochem. LiCl and NaCl were obtained from Sigma.

      Cell Culture

      The human monocytic cell line THP-1 was obtained from American Type Culture Collection. THP-1 cells were cultured in RPMI 1640 (Invitrogen) with 8% fetal calf serum (Omega Scientific, Tarzana, CA), l-glutamine (Invitrogen), and 2-mercaptoethanol (Sigma). Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood from healthy volunteers by buoyant density gradient centrifugation on low endotoxin Ficoll-Hypaque (
      • Boyum A.
      ).

      TNF-α ELISA

      To study the effect of the PI3K inhibitors on TNF-α production, THP-1 cells or PBMCs (1 × 106) were preincubated with 100 nm wortmannin or 10 μm LY294002 for 1 h at 37 °C before the addition of LPS for 5 h at 37 °C. The effect of inhibition of GSK-3β on TNF-α release was studied by preincubating THP-1 cells with 20 or 50 mm LiCl for 1 h at 37 °C before stimulating with LPS for 5 h at 37 °C. 20 or 50 mm NaCl served as an osmolarity control for LiCl. TNF-α protein levels were measured using a commercial ELISA kit (R&D Systems, MN).

      TF Activity

      THP-1 or PBMC cell pellets (1 × 106) were solubilized at 37 °C for 15 min using 15 mm octyl-β-d-glucopyranoside. TF activity in cell lysates was measured using a one-stage clotting assay as described by Morrissey et al. (
      • Morrissey J.H.
      • Fair D.S.
      • Edgington T.S.
      ) with the PT program on theStart 4 clotting machine (Diagnostica Stago, Asnieres, France). Clotting times were converted to milliunits of TF activity by comparison with a standard curve established with purified human brain TF.

      Western Blotting

      Whole cell lysates and cytosolic and nuclear extracts were prepared from THP-1 cells (5 × 106) (
      • Lee J.
      • Mira-Arbibe L.
      • Ulevitch R.J.
      ,
      • Morrissey J.H.
      • Fair D.S.
      • Edgington T.S.
      ). Protein concentrations were measured using a Bio-Rad protein assay kit. Proteins were separated by SDS-PAGE and transferred to Hybond-enhanced chemiluminescence membrane (AmershamBiosciences). Activation of Akt, ERK1/2, p38, and JNK was assessed using a 1:1,000 dilution of anti-phosphospecific antibodies (New England Biolabs). Inactivated GSK-3β was detected using a 1:1,000 dilution of an antibody that recognizes GSK-3β phosphorylated at Ser9. Activation of Raf-1 was monitored using a 1:2,000 dilution of an antibody that recognizes Raf-1 phosphorylated at Ser338 (
      • Chaudhary A.
      • King W.G.
      • Mattaliano M.D.
      • Frost J.A.
      • Diaz B.
      • Morrison D.K.
      • Cobb M.H.
      • Marshall M.S.
      • Brugge J.S.
      ,
      • Dhillon A.S.
      • Meikle S.
      • Yazici Z.
      • Eulitz M.
      • Kolch W.
      ) (United Biotechnology Inc., PA). When whole cell or cytosolic extracts were used, blots were stripped and reprobed using a 1:1,000 dilution of antibodies against the nonphosphorylated forms of each protein to monitor protein loading. Levels of p65 were monitored in the nuclear extracts using a 1:1,000 dilution of an anti-N-terminal RelA antibody (Santa Cruz Biotechnology). Egr-1 was visualized in nuclear extracts using a 1:1,000 dilution of an anti-Egr-1 antibody (Santa Cruz Biotechnology). To ensure equal protein loading, blots with nuclear extracts were stripped and reprobed with a 1:1000 dilution of an anti-histone antibody (Santa Cruz Biotechnology).

      Northern Blotting

      Total cellular RNA was isolated from THP-1 cells (5 × 106) stimulated with 10 μg/ml LPS using Trizol Reagent (Invitrogen). 10 μg of RNA was analyzed by Northern blotting (
      • Cohen P.
      • Frame S.
      ). A 641-bp human TF cDNA fragment, a 800-bp human TNF-α cDNA fragment, or a 1,500-bp Egr-1 cDNA fragment was labeled with RNA [α-32P]dCTP (ICN, Costa Mesa, CA) using a Prime-It Kit (Strategene). Blots were rehybridized with the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (CLONTECH). Bands were visualized by autoradiography.

      Electrophoretic Mobility Shift Assays

      Nuclear extracts were prepared from THP-1 cells (5 × 106) as described previously (
      • Oeth P.A.
      • Parry G.C.N.
      • Kunsch C.
      • Nantermet P.
      • Rosen C.A.
      • Mackman N.
      ). Nuclear extracts were incubated with radiolabeled double-stranded oligonucleotide probes (Operon Technologies, Alameda, CA) containing the immunoglobulin IgκB site (underlined), 5′-CAGAGGGGACTTTCCGAGA-3′; an AP-1 site (underlined), 5′-CTGGGGTGAGTCATCCCTT-3′; or a Sp1 site (underlined), 5′-ATTCGATCGGGGCGGGGCGAGC-3′. Protein-DNA complexes were separated from free DNA probe by electrophoresis through 6% nondenaturing acrylamide gels (Invitrogen) in 0.5 × Tris borate EDTA (TBE) buffer. Gels were dried, and protein-DNA complexes were visualized by autoradiography.

      Plasmids

      pTF-LUC contains 2,106 bp of the human TF promoter. pTNF-α-LUC contains 615 bp of the human TNF-α promoter, and pEgr-1-LUC contains 1,200 bp of the murine Egr-1 promoter. p(κB)5-LUC contains five copies of an NF-κB site, and p(AP-1)4-LUC contains four copies of an AP-1 site. These sites were cloned upstream of the minimal simian virus 40 (SV40) promoter expressing the firefly luciferase (LUC) reporter gene in pGL2-Promoter (Promega, Madison, WI) (
      • Oeth P.
      • Parry G.C.
      • Mackman N.
      ). A plasmid expressing dominant-negative (dnAkt) (S308A/S473A) was kindly provided by G. Bokoch (The Scripps Research Institute, La Jolla, CA). The control plasmid pFA-CMV expresses the GAL4 DNA binding domain alone and was obtained from Stratagene. pFR-Luc (pGAL4-LUC) contains 5XGAL4 binding sites upstream of a minimal promoter. pGAL4-p65 contains the transactivation domain (amino acids 386–551) of p65 fused to the DNA binding portion of GAL4 (
      • Arbibe L.
      • Mira J.-P.
      • Teusch N.
      • Kline L.
      • Guha M.
      • Mackman N.
      • Godowski P.J.
      • Ulevitch R.J.
      • Knaus U.G.
      ). pcDNA3 (Invitrogen) was used as a control plasmid for transfections when expression plasmids were used.

      Transfections

      THP-1 cells were transfected using DEAE-dextran (
      • Mackman N.
      • Brand K.
      • Edgington T.S.
      ). After transfection, cells were incubated in complete medium for 46 h at 37 °C before stimulating with 10 μg/ml LPS for 5 h at 37 °C. In some experiments cells were incubated with 100 nm wortmannin or 10 μm LY294002 for 1 h at 37 °C before stimulation with LPS. In other experiments cells were incubated with 50 mm LiCl or 50 mmNaCl for 1 h at 37 °C before stimulation with LPS. Cell lysates were assayed for luciferase activity as described in the manufacturer's protocol (Promega) using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Cells were cotransfected with pRLTK, which expresses Renilla luciferase (Promega). Renilla luciferase was measured according to the manufacturer's protocol (Promega) and used to normalize the activity of the firefly luciferase.

      Data Analysis

      The number of experiments analyzed is indicated in each figure. Band intensity was quantified by densitometric analyses using a Personal Densitometer (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. Data were collected using a minimum of three experiments and used to calculate the mean ± S.D. Statistical significance was calculated using an unpaired Student's t test and was considered significant atp values ≤ 0.05.

      DISCUSSION

      Our study demonstrates that LPS-induced expression of TNF-α and TF in human monocytic cells is regulated by both positive and negative pathways. We provide multiple lines of evidence and new insights to support the contention that LPS-induced activation of the PI3K-Akt pathway produces a “limiting” effect on TNF-α and TF gene expression in PBMCs and THP-1 monocytic cells. We show that LPS-induced activation of PI3K-Akt negatively regulates the transcription factors Egr-1, AP-1, and NF-κB. The net inhibitory effect on the activation of all three transcription factors reduces LPS induction of TNF-α and TF expression in monocytic cells, which is a key cell type in sepsis.
      This study demonstrates that LPS-induced activation of PI3K-Akt in monocytic cells negatively regulates Raf-1. Two targets of the Raf-1 pathway (ERK1/2 and Egr-1) were also negatively regulated by LPS-induced activation of PI3K-Akt. Egr-1 is required for maximal induction of both TNF-α and TF in human monocytic cells treated with LPS (
      • Yao J.
      • Mackman N.
      • Edgington T.S.
      • Fan S.-T.
      ,
      • Guha M.
      • O'Connell M.A.
      • Pawlinski R.
      • Hollis A.
      • McGovern P.
      • Yan S.-F.
      • Stern D.
      • Mackman N.
      ). Enhancement of LPS-induced TF mRNA expression in the presence of LY294002 was delayed (2 h) relative to the enhancement of TNF-α mRNA expression (1 h). This difference may be the result of a greater contribution of Egr-1 to the induction of TF gene expression because the TF promoter contains three Egr-1 sites. We also found that inhibition of PI3K enhanced LPS-induced activation of p38 and JNK. Our data are consistent with a recent study demonstrating that inhibition of the PI3K-Akt pathway in endothelial cells enhanced TF expression by increasing the activation of p38 (
      • Blum S.
      • Issbrhker K.
      • Willuweit A.
      • Hehlgans S.
      • Lucerna M.
      • Mechtcheriakova D.
      • Walsh K.
      • von der Ahe D.
      • Hofer E.
      • Clauss M.
      ). We found that LY294002 and dnAkt increased LPS-induced AP-1-dependent gene expression. Gratton and colleagues (
      • Gratton J.P.
      • Morales-Ruiz M.
      • Kureishi Y.
      • Fulton D.
      • Walsh K.
      • Sessa W.C.
      ) have shown recently that Akt-dependent phosphorylation of MEKK3 reduces its kinase activity and inhibits the MEKK3/6-p38 pathway. These data suggest that Akt can negatively regulate multiple signaling pathways.
      Studies on the role of the PI3K-Akt pathway in NF-κB-dependent gene expression are controversial. The PI3K-Akt pathway has been shown to act both positively and negatively on NF-κB-dependent gene expression. These differences may reflect the use of different cell types and different agonists. In addition, overexpression of a constitutively active form of Akt may override normal regulatory pathways. Our study demonstrates that the PI3K-Akt pathway negatively regulates NF-κB in LPS-stimulated monocytic cells. Inhibition of PI3K- Akt enhanced LPS-induced nuclear translocation of p65, increased NF-κB binding, and increased NF-κB-dependent gene expression.
      Recent studies showed that both TLR2 and TLR4 signaling activates the PI3K-Akt pathway in human monocytic cells and macrophages (
      • Arbibe L.
      • Mira J.-P.
      • Teusch N.
      • Kline L.
      • Guha M.
      • Mackman N.
      • Godowski P.J.
      • Ulevitch R.J.
      • Knaus U.G.
      ,
      • Jones B.W.
      • Heldwein K.A.
      • Means T.K.
      • Saukkonen J.J.
      • Fenton M.J.
      ). We show that LPS-TLR4 signaling in human monocytic cells activates Akt. Importantly, macrophages exhibited different patterns of cellular responses after stimulation with TLR2 and TLR4 agonists, which suggested that different intracellular signaling pathways were activated by TLR2 and TLR4 (
      • Jones B.W.
      • Heldwein K.A.
      • Means T.K.
      • Saukkonen J.J.
      • Fenton M.J.
      ,
      • Jones B.W.
      • Means T.K.
      • Heldwein K.A.
      • Keen M.A.
      • Hill P.J.
      • Belisle J.T.
      • Fenton M.J.
      ,
      • Hirschfeld M.
      • Weis J.J.
      • Toshchakov V.
      • Salkowski C.A.
      • Cody M.J.
      • Ward D.C.
      • Qureshi N.
      • Michalek S.M.
      • Vogel S.N.
      ,
      • Carl V.S.
      • Brown-Steinke K.
      • Nicklin M.J.
      • Smith Jr., M.F.
      ,
      • Toshchakov V.
      • Jones B.W.
      • Perera P.Y.
      • Thomas K.
      • Cody M.J.
      • Zhang S.
      • Williams B.R.
      • Major J.
      • Hamilton T.A.
      • Fenton M.J.
      • Vogel S.N.
      ). In human monocytic cells, TLR2-dependent activation of the PI3K-Akt pathway positively regulated the transactivational activity of p65 (
      • Arbibe L.
      • Mira J.-P.
      • Teusch N.
      • Kline L.
      • Guha M.
      • Mackman N.
      • Godowski P.J.
      • Ulevitch R.J.
      • Knaus U.G.
      ). In contrast, we show that TLR4-dependent activation of the PI3K-Akt pathway negatively regulates the transactivational activity of p65. These differences probably reflect the activation of different signaling pathways that modulate the effect of the PI3K-Akt pathway on the transactivational activity of p65.
      The increased transactivational activity of p65 in the presence of wortmannin correlated with the inhibition of LPS-induced, Akt-dependent inactivation of GSK-3β. In parallel, we showed that inhibition of GSK-3β with LiCl reduced LPS induction of TNF-α and TF in monocytic cells, suggesting that GSK-3β positively regulates these genes by increasing NF-κB activity. LiCl did not affect LPS-induced nuclear translocation of NF-κB but decreased the transactivational activity of p65. Therefore, inhibition of GSK-3β at later times via Akt-dependent phosphorylation may represent at least one mechanism by which monocytic cells limit the expression of NF-κB-dependent genes.
      Several kinases have been implicated in the control of p65 transcriptional activity, but the most compelling data are derived from studies of various knockout mice. GSK-3β and T2K are both necessary for TNF-α and IL-1 signaling, whereas NIK is selective to the LT-βR pathway (
      • Hoeflich K.P.
      • Luo J.
      • Rubie E.A.
      • Tsao M.-S.
      • Jin O.
      • Woodgett J.R.
      ,
      • Bonnard M.
      • Mirtsos C.
      • Suzuki S.
      • Graham K.
      • Huang J., Ng, M.
      • Itie A.
      • Wakeham A.
      • Shahinian A.
      • Henzel W.J.
      • Elia A.J.
      • Shillinglaw W.
      • Mak T.W.
      • Cao Z.
      • Yeh W.C.
      ,
      • Yin L., Wu, L.
      • Wesche H.
      • Arthur C.D.
      • White J.M.
      • Goeddel D.V.
      • Schreiber R.D.
      ,
      • Matsushima A.
      • Kaisho T.
      • Rennert P.D.
      • Nakano H.
      • Kurosawa K.
      • Uchida D.
      • Takeda K.
      • Akira S.
      • Matsumoto M.
      ). Interestingly, protein kinase Cζ deficiency impairs p65 transcriptional activity in response to TNF-α, interleukin-1, and lymphotoxin-β, which suggests that protein kinase Cζ may be downstream of GSK-3β, NF-κB-inducing kinase, and T2K (
      • Leitges M.
      • Sanz L.
      • Martin P.
      • Duran A.
      • Braun U.
      • Garcia J.F.
      • Camacho F.
      • Diaz-Meco M.T.
      • Rennert P.
      • Moscat J.
      ). Indeed, protein kinase Cζ is the only kinase that has been shown to interact directly with p65.
      A model of LPS induction of TNF-α and TF in monocytic cells is shown in Fig. 8. The current study demonstrates that LPS stimulation of monocytic cells leads to an activation of the PI3K-Akt pathway, which inactivates MAPK pathways (ERK1/2, p38, and JNK) and the NF-κB pathway by phosphorylation of Raf-1, I-κB kinase, GSK-3β, and other upstream kinases, such as MEKK3. Inhibition of these pathways limits the activation of the transcription factors NF-κB, AP-1, and Egr-1, all of which cooperatively regulate TNF-α and TF gene expression. Thus, the PI3K-Akt pathway imposes a “braking” mechanism to limit the expression of TNF-α and TF in LPS-stimulated monocytes and ensure transient expression of these inflammatory mediators.
      Figure thumbnail gr8
      FIG. 8Activation of the PI3K-Akt pathway in monocytic cells limits LPS-induced TNF-α and TF gene expression. Binding of LPS to the CD14 and TLR4/MD2 complex activates the PI3K-Akt signaling pathway. Akt directly or indirectly inactivates the MAPK (ERK1/2, p38, and JNK) and the NF-κB pathway by negatively regulating upstream kinases including Raf-1, MEKK3, and I-κB kinase. LPS activation of PI3K-Akt also inactivates GSK-3β, which reduces the transactivational activity of p65. Akt-dependent inactivation of these pathways limits the activation of the transcription factors NF-κB, AP-1, and Egr-1, all of which cooperatively regulate TNF-α and TF gene expression.Wort, wortmannin; LY, LY294002.

      ACKNOWLEDGEMENTS

      We thank C. Johnson for preparing the manuscript; D. Navamani for technical help; and R. Pawlinski, M. Riewald, and U. Knaus for a critical reading of the manuscript.

      REFERENCES

        • Steinemann S.
        • Ulevitch R.J.
        • Mackman N.
        Arterioscler. Thromb. 1994; 14: 1202-1209
        • van der Bruggen T.
        • Nijenhuis S.
        • van Raaij E.
        • Verhoef J.
        • van Asbeck B.S.
        Infect. Immun. 1999; 67: 3824-3829
        • Swantek J.L.
        • Christerson L.
        • Cobb M.H.
        J. Biol. Chem. 1999; 274: 11667-11671
        • Coleman D.L.
        • Bartiss A.H.
        • Sukhatme V.P.
        • Liu J.
        • Rupprecht H.D.
        J. Immunol. 1992; 149: 3045-3051
        • Hall A.J.
        • Vos H.L.
        • Bertina R.M.
        J. Biol. Chem. 1999; 274: 376-383
        • Scherle P.A.
        • Jones E.A.
        • Favata M.F.
        • Daulerio A.J.
        • Covington M.B.
        • Nurnberg S.A.
        • Magolda R.L.
        • Trzaskos J.M.
        J. Immunol. 1998; 161: 5681-5686
        • Guha M.
        • Mackman N.
        Cell. Signal. 2001; 13: 85-94
        • Ulevitch R.J.
        • Tobias P.S.
        Curr. Opin. Immunol. 1999; 11: 19-22
        • Liu M.K.
        • Herrera-Velit P.
        • Browsney R.W.
        • Reiner N.E.
        J. Immunol. 1994; 153: 2642-2652
        • Beutler B.
        Curr. Opin. Immunol. 2000; 12: 20-26
        • Hambleton J.
        • Weinstein S.L.
        • Lem L.
        • DeFranco A.L.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2774-2778
        • Fischer C.
        • Page S.
        • Weber M.
        • Eisele T.
        • Neumeier D.
        • Brand K.
        J. Biol. Chem. 1999; 274: 24625-24632
        • Yao J.
        • Mackman N.
        • Edgington T.S.
        • Fan S.-T.
        J. Biol. Chem. 1997; 272: 17795-17801
        • Mackman N.
        • Brand K.
        • Edgington T.S.
        J. Exp. Med. 1991; 174: 1517-1526
        • Guha M.
        • O'Connell M.A.
        • Pawlinski R.
        • Hollis A.
        • McGovern P.
        • Yan S.-F.
        • Stern D.
        • Mackman N.
        Blood. 2001; 98: 1429-1439
        • Lee J.
        • Mira-Arbibe L.
        • Ulevitch R.J.
        J. Leukocyte Biol. 2000; 68: 909-915
        • Pahan K.
        • Raymond J.R.
        • Singh I.
        J. Biol. Chem. 1999; 274: 7528-7536
        • Park Y.C.
        • Lee C.H.
        • Kang H.S.
        • Chung H.T.
        • Kim H.D.
        Biochem. Biophys. Res. Commun. 1997; 240: 692-696
        • Scheid M.P.
        • Woodgett J.R.
        Curr. Biol. 2000; 10: R191-R194
        • Datta S.R.
        • Brunet A.
        • Greenberg M.E.
        Genes Dev. 1999; 13: 2905-2927
        • 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
        • Tengku-Muhammad T.S.
        • Hughes T.R.
        • Cryer A.
        • Ramji D.P.
        Cytokine. 1999; 11: 463-468
        • Pahan K.
        • Liu X.
        • Wood C.
        • Raymond J.R.
        FEBS Lett. 2000; 472: 203-207
        • Kim I., Oh, J.L.
        • Ryu Y.S., So, J.N.
        • Sessa W.C.
        • Walsk K.
        • Koh G.Y.
        FASEB J. 2002; 16: 126-128
        • Blum S.
        • Issbrhker K.
        • Willuweit A.
        • Hehlgans S.
        • Lucerna M.
        • Mechtcheriakova D.
        • Walsh K.
        • von der Ahe D.
        • Hofer E.
        • Clauss M.
        J. Biol. Chem. 2001; 276: 33428-33434
        • Thomas K.W.
        • Monick M.M.
        • Stabler J.M.
        • Yarovinsky T.
        • Carter A.B.
        • Hunninghake G.W.
        J. Biol. Chem. 2002; 277: 492-501
        • Madrid L.V.
        • Mayo M.W.
        • Reuther J.Y.
        • Baldwin Jr., A.S.
        J. Biol. Chem. 2001; 276: 18934-18940
        • Reddy S.A.G.
        • Huang J.H.
        • Liao W.S.-L.
        J. Immunol. 2000; 164: 1355-1363
        • Madrid L.V.
        • Wang C.-Y.
        • Guttridge D.C.
        • Schottelius A.J.G.
        • Baldwin Jr., A.S.
        • Mayo M.W.
        Mol. Cell. Biol. 2000; 20: 1626-1638
        • Sizemore N.
        • Leung S.
        • Stark G.R.
        Mol. Cell. Biol. 1999; 19: 4798-4805
        • Béraud C.
        • Henzel W.J.
        • Baeuerle P.A.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 429-434
        • Reddy S.A.G.
        • Huang J.H.
        • Liao W.S.-L.
        J. Biol. Chem. 1997; 272: 29167-29173
        • Arbibe L.
        • Mira J.-P.
        • Teusch N.
        • Kline L.
        • Guha M.
        • Mackman N.
        • Godowski P.J.
        • Ulevitch R.J.
        • Knaus U.G.
        Nat. Immunol. 2000; 1: 533-540
        • 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
        • Zimmermann S.
        • Moelling K.
        Science. 1999; 286: 1741-1744
        • Morrison D.K.
        • Cutler Jr., R.E.
        Curr. Opin. Cell Biol. 1997; 9: 174-179
        • Chaudhary A.
        • King W.G.
        • Mattaliano M.D.
        • Frost J.A.
        • Diaz B.
        • Morrison D.K.
        • Cobb M.H.
        • Marshall M.S.
        • Brugge J.S.
        Curr. Biol. 2000; 10: 551-554
        • Dhillon A.S.
        • Meikle S.
        • Yazici Z.
        • Eulitz M.
        • Kolch W.
        EMBO J. 2002; 21: 64-71
        • Cohen P.
        • Frame S.
        Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776
        • Hoeflich K.P.
        • Luo J.
        • Rubie E.A.
        • Tsao M.-S.
        • Jin O.
        • Woodgett J.R.
        Nature. 2000; 406: 86-90
        • Frame S.
        • Cohen P.
        Biochem. J. 2001; 359: 1-16
        • Boyum A.
        Scand. J. Clin. Lab. Invest. Suppl. 1968; 97: 77-89
        • Morrissey J.H.
        • Fair D.S.
        • Edgington T.S.
        Thromb. Res. 1988; 52: 247-261
        • Oeth P.A.
        • Parry G.C.N.
        • Kunsch C.
        • Nantermet P.
        • Rosen C.A.
        • Mackman N.
        Mol. Cell. Biol. 1994; 14: 3772-3781
        • Oeth P.
        • Parry G.C.
        • Mackman N.
        Arterioscler. Thromb. Vasc. Biol. 1997; 17: 365-374
        • Brand K.
        • Fowler B.J.
        • Edgington T.S.
        • Mackman N.
        Mol. Cell. Biol. 1991; 11: 4732-4738
        • Gratton J.P.
        • Morales-Ruiz M.
        • Kureishi Y.
        • Fulton D.
        • Walsh K.
        • Sessa W.C.
        J. Biol. Chem. 2001; 276: 30359-30365
        • Jones B.W.
        • Heldwein K.A.
        • Means T.K.
        • Saukkonen J.J.
        • Fenton M.J.
        Ann. Rheum. Dis. 2001; 60: 6-12
        • Jones B.W.
        • Means T.K.
        • Heldwein K.A.
        • Keen M.A.
        • Hill P.J.
        • Belisle J.T.
        • Fenton M.J.
        J. Leukocyte Biol. 2001; 69: 1036-1044
        • Hirschfeld M.
        • Weis J.J.
        • Toshchakov V.
        • Salkowski C.A.
        • Cody M.J.
        • Ward D.C.
        • Qureshi N.
        • Michalek S.M.
        • Vogel S.N.
        Infect. Immunol. 2001; 69: 1477-1492
        • Carl V.S.
        • Brown-Steinke K.
        • Nicklin M.J.
        • Smith Jr., M.F.
        J. Biol. Chem. 2002; 277: 17448-17456
        • Toshchakov V.
        • Jones B.W.
        • Perera P.Y.
        • Thomas K.
        • Cody M.J.
        • Zhang S.
        • Williams B.R.
        • Major J.
        • Hamilton T.A.
        • Fenton M.J.
        • Vogel S.N.
        Nat. Immunol. 2002; 3: 392-398
        • Bonnard M.
        • Mirtsos C.
        • Suzuki S.
        • Graham K.
        • Huang J., Ng, M.
        • Itie A.
        • Wakeham A.
        • Shahinian A.
        • Henzel W.J.
        • Elia A.J.
        • Shillinglaw W.
        • Mak T.W.
        • Cao Z.
        • Yeh W.C.
        EMBO J. 2000; 19: 4976-4985
        • Yin L., Wu, L.
        • Wesche H.
        • Arthur C.D.
        • White J.M.
        • Goeddel D.V.
        • Schreiber R.D.
        Science. 2001; 291: 2162-2165
        • Matsushima A.
        • Kaisho T.
        • Rennert P.D.
        • Nakano H.
        • Kurosawa K.
        • Uchida D.
        • Takeda K.
        • Akira S.
        • Matsumoto M.
        J. Exp. Med. 2001; 193: 631-636
        • Leitges M.
        • Sanz L.
        • Martin P.
        • Duran A.
        • Braun U.
        • Garcia J.F.
        • Camacho F.
        • Diaz-Meco M.T.
        • Rennert P.
        • Moscat J.
        Mol. Cell. 2001; 8: 771-780