The Phosphatidylinositol 3-Kinase-Akt Pathway Limits Lipopolysaccharide Activation of Signaling Pathways and Expression of Inflammatory Mediators in Human Monocytic Cells
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m203298200
ISSN1083-351X
Autores Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoMonocytes 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. 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. lipopolysaccharide cytomegalovirus dominant negative enzyme-linked immunosorbent assay extracellular signal-regulated kinase glycogen synthase kinase c-Jun N-terminal kinase luciferase mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase MEK kinase nuclear factor-κB peripheral blood mononuclear cell(s) phosphatidylinositol 3-kinase tissue factor tumor necrosis factor-alpha 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 (1Steinemann S. Ulevitch R.J. Mackman N. 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LPS activation of the CD14·TLR4·MD2 complex results in the expression of tumor necrosis factor-alpha (TNF-α) and tissue factor (TF) (1Steinemann S. Ulevitch R.J. Mackman N. Arterioscler. Thromb. 1994; 14: 1202-1209Crossref PubMed Scopus (48) Google Scholar, 8Ulevitch R.J. Tobias P.S. Curr. Opin. Immunol. 1999; 11: 19-22Crossref PubMed Scopus (487) Google Scholar, 9Liu M.K. Herrera-Velit P. Browsney R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar, 10Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar). The signaling pathways that positively regulate TNF-α and TF gene expression in LPS-stimulated monocytes/macrophages are well characterized (2van der Bruggen T. Nijenhuis S. van Raaij E. Verhoef J. van Asbeck B.S. Infect. Immun. 1999; 67: 3824-3829Crossref PubMed Google Scholar, 3Swantek J.L. Christerson L. Cobb M.H. J. Biol. 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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 (16Lee J. Mira-Arbibe L. Ulevitch R.J. J. Leukocyte Biol. 2000; 68: 909-915PubMed Google Scholar, 17Pahan K. Raymond J.R. Singh I. J. Biol. Chem. 1999; 274: 7528-7536Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 18Park Y.C. Lee C.H. Kang H.S. Chung H.T. Kim H.D. Biochem. Biophys. Res. Commun. 1997; 240: 692-696Crossref PubMed Scopus (98) Google Scholar), 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 (19Scheid M.P. Woodgett J.R. Curr. Biol. 2000; 10: R191-R194Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar, 21Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar). 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 (22Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1305) Google Scholar, 23Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). 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 (18Park Y.C. Lee C.H. Kang H.S. Chung H.T. Kim H.D. Biochem. Biophys. Res. Commun. 1997; 240: 692-696Crossref PubMed Scopus (98) Google Scholar), and activation of PI3K-Akt suppressed LPS-induced lipoprotein lipase expression in J774 macrophages (24Tengku-Muhammad T.S. Hughes T.R. Cryer A. Ramji D.P. Cytokine. 1999; 11: 463-468Crossref PubMed Scopus (21) Google Scholar). Induction of nitric-oxide synthase in C6 glial cells and rat primary astrocytes was also regulated negatively by activation of PI3K (17Pahan K. Raymond J.R. Singh I. J. Biol. Chem. 1999; 274: 7528-7536Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and a constitutively active PI3K inhibited induction of nitric-oxide synthase gene expression in human astrocytes (25Pahan K. Liu X. Wood C. Raymond J.R. FEBS Lett. 2000; 472: 203-207Crossref PubMed Scopus (32) Google Scholar). Angiopoeitin-1, a potent activator of PI3K, negatively regulated vascular endothelial growth factor- and TNF-α-induced TF expression in endothelial cells (26Kim I., Oh, J.L. Ryu Y.S., So, J.N. Sessa W.C. Walsk K. Koh G.Y. FASEB J. 2002; 16: 126-128PubMed Google Scholar). Finally, in endothelial cells the PI3K-Akt pathway limited vascular endothelial growth factor activation of the p38 MAPK pathway and TF gene expression (27Blum 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: 33428-33434Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). 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 (28Thomas K.W. Monick M.M. Stabler J.M. Yarovinsky T. Carter A.B. Hunninghake G.W. J. Biol. Chem. 2002; 277: 492-501Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). 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 (29Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar). 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 (30Reddy S.A.G. Huang J.H. Liao W.S.-L. J. Immunol. 2000; 164: 1355-1363Crossref PubMed Scopus (168) Google Scholar, 31Madrid L.V. Wang C.-Y. Guttridge D.C. Schottelius A.J.G. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (587) Google Scholar, 32Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 33Béraud C. Henzel W.J. Baeuerle P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 429-434Crossref PubMed Scopus (264) Google Scholar, 34Reddy S.A.G. Huang J.H. Liao W.S.-L. J. Biol. Chem. 1997; 272: 29167-29173Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). 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 (35Arbibe 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: 533-540Crossref PubMed Scopus (568) Google Scholar). 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 (2van der Bruggen T. Nijenhuis S. van Raaij E. Verhoef J. van Asbeck B.S. Infect. Immun. 1999; 67: 3824-3829Crossref PubMed Google Scholar, 6Scherle 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: 5681-5686PubMed Google Scholar, 13Yao J. Mackman N. Edgington T.S. Fan S.-T. J. Biol. Chem. 1997; 272: 17795-17801Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar,15Guha M. O'Connell M.A. Pawlinski R. Hollis A. McGovern P. Yan S.-F. Stern D. Mackman N. Blood. 2001; 98: 1429-1439Crossref PubMed Scopus (328) Google Scholar). Activation of Akt has been shown to regulate negatively the serine/threonine kinase Raf-1 and the downstream MEK-ERK1/2 signaling pathway (36Rommel C. Clarke B.A. Zimmermann S. Nunez L. Rossman R. Reid K. Moelling K. Yancopoulos G.D. Glass D.J. Science. 1999; 286: 1738-1741Crossref PubMed Scopus (663) Google Scholar, 37Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (909) Google Scholar). 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 (38Morrison D.K. Cutler Jr., R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (537) Google Scholar). 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 (39Chaudhary 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: 551-554Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 40Dhillon A.S. Meikle S. Yazici Z. Eulitz M. Kolch W. EMBO J. 2002; 21: 64-71Crossref PubMed Scopus (229) Google Scholar). 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 (41Cohen P. Frame S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776Crossref PubMed Scopus (1302) Google Scholar). The phenotype of GSK-3β−/− embryos is similar to that of RelA−/− embryos, suggesting that GSK-3β may regulate the transactivational activity of p65 (42Hoeflich K.P. Luo J. Rubie E.A. Tsao M.-S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1223) Google Scholar). LiCl is a potent inhibitor of GSK-3β and inactivates GSK-3β by inducing its phosphorylation on Ser9 in its N terminus (43Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1280) Google Scholar). Indeed, the effect of LiCl on wingless signaling in wild-type cells mimicked the phenotype observed in GSK-3β null cells (41Cohen P. Frame S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776Crossref PubMed Scopus (1302) Google Scholar). 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. LPS (Escherichia coli serotype 0111:B4) and the PI3K inhibitors wortmannin and LY294002 were obtained from Calbiochem. LiCl and NaCl were obtained from Sigma. 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 (44Boyum A. Scand. J. Clin. Lab. Invest. Suppl. 1968; 97: 77-89PubMed Google Scholar). 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). 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. (45Morrissey J.H. Fair D.S. Edgington T.S. Thromb. Res. 1988; 52: 247-261Abstract Full Text PDF PubMed Scopus (113) Google Scholar) 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. Whole cell lysates and cytosolic and nuclear extracts were prepared from THP-1 cells (5 × 106) (16Lee J. Mira-Arbibe L. Ulevitch R.J. J. Leukocyte Biol. 2000; 68: 909-915PubMed Google Scholar, 45Morrissey J.H. Fair D.S. Edgington T.S. Thromb. Res. 1988; 52: 247-261Abstract Full Text PDF PubMed Scopus (113) Google Scholar). 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 (39Chaudhary 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: 551-554Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 40Dhillon A.S. Meikle S. Yazici Z. Eulitz M. Kolch W. EMBO J. 2002; 21: 64-71Crossref PubMed Scopus (229) Google Scholar) (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). 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 (41Cohen P. Frame S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776Crossref PubMed Scopus (1302) Google Scholar). 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. Nuclear extracts were prepared from THP-1 cells (5 × 106) as described previously (46Oeth P.A. Parry G.C.N. Kunsch C. Nantermet P. Rosen C.A. Mackman N. Mol. Cell. Biol. 1994; 14: 3772-3781Crossref PubMed Google Scholar). 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. 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) (47Oeth P. Parry G.C. Mackman N. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 365-374Crossref PubMed Scopus (127) Google Scholar). 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 (35Arbibe 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: 533-540Crossref PubMed Scopus (568) Google Scholar). pcDNA3 (Invitrogen) was used as a control plasmid for transfections when expression plasmids were used. THP-1 cells were transfected using DEAE-dextran (14Mackman N. Brand K. Edgington T.S. J. Exp. Med. 1991; 174: 1517-1526Crossref PubMed Scopus (331) Google Scholar). 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. 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. We examined the role of the PI3K-Akt pathway in LPS induction of TNF-α and TF in human monocytic cells using two different pharmacological inhibitors (LY294002 and wortmannin) that block the activation of PI3K by different mechanisms. LPS induction of TNF-α and TF in PBMCs was measured in the presence and absence of LY294002 (Fig.1A). LY294002 enhanced LPS induction of TNF-α and TF expression 2.0- and 3.3-fold, respectively. THP-1 cells represent a well established model of human monocytes. LY294002 also enhanced LPS induction of TNF-α and TF expression in THP-1 cells 2.8- and 2.0-fold, respectively (Fig. 1B). Similar results were observed in THP-1 cells with wortmannin (Fig.1B). These results indicate that the PI3K-Akt pathway is a negative regulator of LPS-induced TNF-α and TF expression in human monocytic cells. Because the response to LY294002 was similar in PBMCs and THP-1 cells, we used THP-1 cells to determine further the mechanism by which activation of PI3K-Akt negatively regulates LPS-induced TNF-α and TF expression. GSK-3β is a kinase that is negatively regulated by Akt-dependent phosphorylation. However, the role of GSK-3β in the regulation of inflammatory genes is currently unknown. We wished to determine whether GSK-3β played a role in LPS induction of TNF-α and TF gene expression. We used LiCl to inhibit GSK-3β kinase activity (41Cohen P. Frame S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776Crossref PubMed Scopus (1302) Google Scholar, 42Hoeflich K.P. Luo J. Rubie E.A. Tsao M.-S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1223) Google Scholar). LiCl reduced LPS-induced TNF-α and TF expression in a dose-dependent manner (Fig.1C). NaCl was used as an osmolarity control. These results indicate that GSK-3β positively regulates TNF-α and TF expression in LPS-stimulated monocytic cells and may be a potential target for negative regulation by the PI3K-Akt signaling pathway. The role of the PI3K-Akt pathway in the LPS induction of TNF-α and TF mRNA expression was determined by Northern blot analysis (Fig.2). LPS induced maximal levels of TNF-α and TF mRNAs at 1 h. LY294002 enhanced the LPS induction of TNF-α at 1 h by 4.2-fold. However, LY294002 had only a minor effect on LPS-induced levels of TF mRNA at 1 h but increased TF mRNA expression at 2 h by 5.8-fold. The slower migrating band represents a differentially spliced, nonfunctional TF transcript (48Brand K. Fowler B.J. Edgington T.S. Mackman N. Mol. Cell. Biol. 1991; 11: 4732-4738Crossref PubMed Scopus (123) Google Scholar). These results indicate that activation of PI3K limits the LPS induction of TNF-α and TF mRNA expression in monocytic cells. Next, we evaluated the role of PI3K-Akt signaling in the LPS-induced TNF-α and TF promoter activity. Wortmannin significantly enhanced LPS-induced TNF-α (p = 0.0008) and TF (p = 0.037) promoter activity (Fig.3A). Importantly, cotransfection of cells with a plasmid expressing dnAkt also enhanced LPS induction of TNF-α and TF promoter activity (Fig. 3B). The results using a pharmacological inhibitor and dnAkt indicate that LPS-induced TNF-α and TF gene expression is negatively regulated by activation of Akt at the level of transcription. Activation of PI3K-Akt differentially regulates downstream effectors in different cell types (21Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar, 24Tengku-Muhammad T.S. Hughes T.R. Cryer A. Ramji D.P. Cytokine. 1999; 11: 463-468Crossref PubMed Scopus (21) Google Scholar, 25Pahan K. Liu X. Wood C. Raymond J.R. FEBS Lett. 2000; 472: 203-207Crossref PubMed Scopus (32) Google Scholar, 26Kim I., Oh, J.L. Ryu Y.S., So, J.N. Sessa W.C. Walsk K. Koh G.Y. FASEB J. 2002; 16: 126-128PubMed Google Scholar, 27Blum 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: 33428-33434Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 28Thomas K.W. Monick M.M. Stabler J.M. Yarovinsky T. Carter A.B. Hunninghake G.W. J. Biol. Chem. 2002; 277: 492-501Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 29Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 30Reddy S.A.G. Huang J.H. Liao W.S.-L. J. Immunol. 2000; 164: 1355-1363Crossref PubMed Scopus (168) Google Scholar). LPS stimulation of THP-1 cells resulted in a time-dependent phosphorylation of Akt which was maximal at 1 h (Fig.4A). 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