Efforts in prevention and control of tuberculosis suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. The exploitation of host cell signaling pathways to the benefit of the pathogen is a phenomenon that deserves to be looked into in detail. We have tested the hypothesis that lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signaling pathways linked to cell survival. The Bcl-2 family member Bad is a proapoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. We demonstrate that man-LAM stimulates Bad phosphorylation in a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. LAM-stimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI-3K (Δp85), indicating that activation of PI-3K is sufficient to trigger phosphorylation of Bad by LAM. Since phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase Akt, the effect of LAM on Akt kinase activity was tested. Man-LAM could activate Akt as evidenced from phosphorylation of Akt at Thr308 and by the phosphorylation of the exogenous substrate histone 2B. Akt activation was abrogated in cells transfected with Δp85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of Akt. These results establish that LAM-mediated Bad phosphorylation occurs in a PI-3K/Akt-dependent manner. It is therefore the first demonstration of the ability of a mycobacterial virulence factor to up-regulate a signaling pathway involved in cell survival. This is likely to be one of a number of virulence-associated mechanisms by which bacilli control host cell apoptosis. Efforts in prevention and control of tuberculosis suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. The exploitation of host cell signaling pathways to the benefit of the pathogen is a phenomenon that deserves to be looked into in detail. We have tested the hypothesis that lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signaling pathways linked to cell survival. The Bcl-2 family member Bad is a proapoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. We demonstrate that man-LAM stimulates Bad phosphorylation in a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. LAM-stimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI-3K (Δp85), indicating that activation of PI-3K is sufficient to trigger phosphorylation of Bad by LAM. Since phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase Akt, the effect of LAM on Akt kinase activity was tested. Man-LAM could activate Akt as evidenced from phosphorylation of Akt at Thr308 and by the phosphorylation of the exogenous substrate histone 2B. Akt activation was abrogated in cells transfected with Δp85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of Akt. These results establish that LAM-mediated Bad phosphorylation occurs in a PI-3K/Akt-dependent manner. It is therefore the first demonstration of the ability of a mycobacterial virulence factor to up-regulate a signaling pathway involved in cell survival. This is likely to be one of a number of virulence-associated mechanisms by which bacilli control host cell apoptosis. lipoarabinomannan phosphatidylinositol phosphatidylinositol 3-kinase Despite the potential role of the macrophage in the eradication of microbes, pathogenic Mycobacterium species have survived down the ages as some of the most successful in evading macrophage surveillance mechanisms in a manner that ensures their survival and replication inside the macrophage. A variety of mechanisms contribute to the survival of Mycobacterium tuberculosiswithin macrophages (1Fenton M.J. Vermeulen M.W. Infect. Immun. 1996; 64: 683-690Crossref PubMed Google Scholar) including inhibition of phagosome-lysosome fusion (2Meresse, S., Steele-Mortimer, O., Moreno, E., Desjardin, M., Finlay, B., and Gorvel, J.-P. (1999) Nat. Cell Biol. E183–E188Google Scholar), inhibition of the acidification of phagosomes (3Sturgill-Koszycki S. Schlesinger P.H. Chakraborty P. Haddix P.L. Collins H.L. Fok A.K. Allen R.D. Gluck S.L. Heuser J. Russell D.G. Science. 1994; 263: 678-691Crossref PubMed Scopus (1057) Google Scholar), and resistance to killing by reactive oxygen (4Jacket P.S. Andrew P.W. Lowrie D.B. Adv. Exp. Med. Biol. 1982; 155: 687-693Crossref PubMed Scopus (6) Google Scholar) and reactive nitrogen intermediates (5MacMicking J. North R.J. La Course R. Mudgett J.S. Shah S.K. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5243-5248Crossref PubMed Scopus (902) Google Scholar, 6Chan J. Xing Y. Magliozzo R.S. Bloom B.R. J. Exp. Med. 1992; 175: 1111-1112Crossref PubMed Scopus (858) Google Scholar). M. tuberculosis produces large quantities of lipoarabinomannan (LAM).1 LAM can inhibit macrophage activation and triggering and represents a virulence factor contributing to the persistence of mycobacteria within macrophages. LAM is a complex molecule consisting of a phosphatidylinositol (PI) moiety that anchors a large mannose core to the mycobacterial cell wall (7Chatterjee D. Khoo K.-H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (297) Google Scholar, 8Hunter S.W. Brennan P.J. J. Biol. Chem. 1990; 265: 9272-9279Abstract Full Text PDF PubMed Google Scholar, 9Hunter S.W. Gaylord H. Brennan P.J. J. Biol. Chem. 1986; 261: 12345-12351Abstract Full Text PDF PubMed Google Scholar). The mannose core consists of multiple branched, arabinofuranosyl side chains. M. tuberculosis and M. leprae modify the nonreducing end of the arabinofuranosyl chains with mannose residues yielding man-LAM, whereas rapidly growing mycobacterial species have nonreducing termini of two types, the linear Ara4 and the branched Ara6 motifs, thereby giving rise to ara-LAM (7Chatterjee D. Khoo K.-H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (297) Google Scholar, 10Khoo K.-H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 1996; 270: 12380-12389Abstract Full Text Full Text PDF Scopus (160) Google Scholar). LAM exhibits a wide array of immunomodulatory functions including inhibition of interferon-γ-induced functions such as macrophage microbicidal and tumoricidal activity (11Sibley L.D. Hunter S.W. Brennan P.J. Krahenbuhl J.L. Infect. Immun. 1988; 56: 1232-1236Crossref PubMed Google Scholar), scavenging of potentially cytotoxic oxygen free radicals (12Chan J. Fan X. Hunter S.W. Brennan P.J. Bloom B.R. Infect. Immun. 1991; 59: 1755-1761Crossref PubMed Google Scholar), inhibition of protein kinase C activity (13Chan, S. D., Fan, Hunter, S. W., Brennan, P. J., and Bloom, B. R. (1991)Google Scholar), and evocation of a number of cytokines such as tumor necrosis factor-α (14Barnes P.F. Chatterjee D. Brennan P.J. Rea T.H. Modlin R.L. Infect. Immun. 1992; 60: 1441-1446Crossref PubMed Google Scholar). The early response genes c-fos, KC, and JE are induced by ara-LAM but not by man-LAM (15Roach T.A. Barton C.H. Chatterjee D. Blackwell J.M. J. Immunol. 1993; 60: 1886-1896Google Scholar). The ability of man-LAM to impair responsiveness to interferon-γ and to attenuate tumor necrosis factor-α and interleukin-12 mRNA production through effects on the protein phosphatase SHP-1 has been suggested to be a major mechanism by which man-LAM promotes intracellular survival (16Knutson K.L. Hmaama Z. Herrera-Velit P. Rochford R. Reiner N.E. J. Biol. Chem. 1998; 273: 645-652Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Despite the recent advances, the intracellular signaling following interaction of the mycobacterium with host cells and the role of LAM in these processes is incompletely understood. The survival of M. tuberculosis in macrophages probably involves more than one mechanism. In this study, we have tested whether LAM plays a role in triggering a signaling pathway that suppresses the intrinsic cell death machinery of phagocytic cells. The regulation of programmed cell death, apoptosis, is an exceptionally complicated process that involves a myriad of proteins. The family of proteins that includes Bcl-2 comprises members that are both antiapoptotic and those that are proapoptotic such as Bax and Bad. Bad interacts with Bcl-2 and Bcl-XL, sequestering these proteins, and thus promotes apoptosis (17Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1888) Google Scholar). Phosphorylation of Bad at either of two sites, serine residues 112 and 136 (numbering based on the sequence of murine Bad) creates consensus sites for interaction with the 14-3-3 protein. Bad is then bound to 14-3-3 instead of Bcl-2 or Bcl-XL, resulting in the liberation of the antiapoptotic proteins and promotion of cell survival. Interleukin-3 (18Yao R. Cooper G.N. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1290) Google Scholar) and other survival factors promote cell survival through their ability to stimulate phosphatidylinositol 3-kinase (PI-3K) (19Yao R. Minshall C. Arkins S. Freund G.G. Kelley K.W. J. Immunol. 1996; 156: 939-947PubMed Google Scholar) and inactivate the apoptotic factor Bad (20Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2246) Google Scholar, 21Songyang Z. Baltimore D. Cantley L. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11345-11350Crossref PubMed Scopus (322) Google Scholar). The PI-3K-sensitive pathway involves the activation of the serine/threonine kinase Akt or protein kinase B (22Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2215) Google Scholar, 23Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar, 24Kaufmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1069) Google Scholar, 25Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1182) Google Scholar) and direct phosphorylation of Bad at serine 136 (26Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Goto, Y., and Nunez, G. (1997) 91, 231–241Google Scholar, 27del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1981) Google Scholar, 28Eves E.M. Xiong W. Bellacosa A. Kennedy S.G. Tsichlis P.N. Rosner M.R. Hay N. Mol. Cell. Biol. 1998; 18: 2143-2152Crossref PubMed Scopus (175) Google Scholar, 29Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Abstract Full Text Full Text PDF PubMed Google Scholar). Akt/protein kinase B is the major downstream target of receptor tyrosine kinases that signal via the PI-3K. Activated protein kinase B has been implicated in glucose metabolism, transcriptional control, and the regulation of apoptosis in many cell types (30Galetic I. Andjelkovic M. Meier R. Brodbeck D. Park J. Hemmings B.A. Pharmacol. Ther. 1999; 82: 409-425Crossref PubMed Scopus (95) Google Scholar, 31Datta S. Brunet A. Greenberg M. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3711) Google Scholar). In addition to Bad, Akt also phosphorylates caspase 9 (32Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2722) Google Scholar), forkhead transcription factor (33Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar, 34Kops G.J.P.L. de Ruiter N.D. Vries-Smits A.M.M. Powell D.R. Bos J.L. Burgering B.M.T. Nature. 1999; 398: 630-634Crossref PubMed Scopus (949) Google Scholar), and the IκB kinase, thereby activating NF-κB (35Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1883) Google Scholar, 36Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1664) Google Scholar). In addition to phosphorylation at serine 136 mediated by protein kinase B, Bad undergoes protein kinase A- (37Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Korsmeyer S.J. Mol. Cell. 1999; 3: 413-422Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar) and p90RSK- (38Tan Y. Ruan H. Demeer M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) mediated phosphorylation on serine 112. The Ca2+-activated protein phosphatase calcineurin can dephosphorylate Bad, reversing the phosphorylation at both serine 112 and serine 136 (39Wang H.-G. Pathan N. Ethell I. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (960) Google Scholar). A third phosphorylation site at serine 155 has recently been identified (40Lizcano J.M. Morrice N. Cohen P. Biochem. J. 2000; 349: 547-557Crossref PubMed Scopus (256) Google Scholar, 41Zhou X.-M. Liu Y. Payne G. Lutz R.J. Chittenden T. J. Biol. Chem. 2000; 275: 25046-25051Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 42Tan Y. Demeter M.R. Ruan H. Comb M.J. J. Biol. Chem. 2000; 275: 25865-25869Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 43Datta S.R. Katsov A. Hu L. Petros A. Fesik S.W. Yaffe M.B. Greenberg M.E. Mol. Cell. 2000; 6: 41-51Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). When Bad is bound to prosurvival Bcl-2 family members, Bad serine 155 phosphorylation requires the prior phosphorylation at serine 136, which recruits 14-3-3 proteins that then function to increase the accessibility of serine 155 to survival-promoting kinases (43Datta S.R. Katsov A. Hu L. Petros A. Fesik S.W. Yaffe M.B. Greenberg M.E. Mol. Cell. 2000; 6: 41-51Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). Bad is not a ubiquitously expressed protein. Nevertheless, several major signaling pathways influence cell survival through their effects on the phosphorylation state of Bad (44Downward J. Nat. Cell Biol. 1999; 1: E33-E35Crossref PubMed Scopus (203) Google Scholar). We demonstrate that man-LAM from the virulent Erdman strain of M. tuberculosis promotes phosphorylation of Bad at serine 136 through a PI-3K/Akt pathway in the human cell line THP-1 and hypothesize that this probably represents one of the mechanisms by which man-LAM promotes cell survival to the benefit of the pathogen. Histone 2B was purchased from Roche Molecular Biochemicals. Phorbol 12-myristate 13-acetate was purchased from Sigma. Protein A/G Plus-agarose, rabbit anti-p85 phosphoinositide 3-kinase, goat anti-Akt, and rabbit anti-Bad antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phophotyrosine antibody was from Life Technologies, Inc. Wortmannin, LY294002, PD98059, and SB203580 were from Calbiochem. Phosphorylation state-specific antibodies Bad (Ser112) and (Ser136) and Akt (Thr308) antibodies and the Phototope-HRP Chemiluminescent Western Detection Kit were from New England Biolabs, Inc. (Beverly, MA). All other chemicals were from Sigma. THP-1 cells (derived from a patient with acute monocytic leukemia) are mature cells from the monocyte/macrophage lineage. These were obtained from the National Center for Cell Science (Pune, India). Media and supplements were obtained from Life Technologies, Inc. The cell line was maintained in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mmglutamine, and 20 mm sodium bicarbonate. The cells were incubated at 5% CO2 and 95% humidity in a 37 °C chamber. THP-1 cells were treated with phorbol 12-myristate 13-acetate to induce maturation of the monocytes to a macrophage-like, adherent phenotype. The cells were washed three times with culture medium without fetal bovine serum and resuspended to a concentration of 2 × 106 cells/ml. Cell viability was determined to be >95% by the trypan blue dye exclusion method. 2 × 106cells were plated in each well of six-well plates. Cells were deprived of serum by culturing in RPMI without fetal bovine serum for 12–16 h, before treatment with LAM. Transfections were carried out on adherent THP-1 cells (2 × 106 cells/well in six-well plates). Cells were transfected with 2 μg of plasmid (recombinants or empty vectors) using the Effectene Reagent (Qiagen) in RPMI with 10% fetal bovine serum according to the manufacturer's instructions. The dominant-negative mutant of p85 was deleted in the inter-SH2 region of wild type p85α (Δp85), which abolishes binding to the p110 subunit of PI 3-kinase. The kinase-deficient mutant of Akt (Akt.KD) carried the mutation K179M. Endotoxin-free man-LAM and ara-LAM were kindly provided by Dr. John Belisle (Colorado State University, Ft. Collins, CO, through NIAID, National Institutes of Health, Contract NO1-AI-75320). Man-LAM was derived from the virulent Erdman strain of M. tuberculosis. LAM was dissolved at a concentration of 1 mg/ml in pyrogen-free water and diluted with medium before each experiment. To assay for Akt protein kinase activity, cells were lysed in lysis buffer (45Datta K. Franke T.F. Chan T.O. Makris A. Yang S.-I. Kaplan D.R. Morrison D.K. Golemis E.A. Tsichlis P.N. Mol. Cell. Biol. 1995; 15: 2304-2310Crossref PubMed Scopus (156) Google Scholar); lysates were incubated with anti-Akt antibody, and kinase assays were carried out with the immunoprecipitates using histone 2B as substrate (45Datta K. Franke T.F. Chan T.O. Makris A. Yang S.-I. Kaplan D.R. Morrison D.K. Golemis E.A. Tsichlis P.N. Mol. Cell. Biol. 1995; 15: 2304-2310Crossref PubMed Scopus (156) Google Scholar). After incubation at room temperature for 30 min, the reaction was stopped by adding protein gel denaturing buffer, and the mixture was separated by SDS-polyacrylamide gel electrophoresis, followed by autoradiography. Following immunoprecipitation of cell lysates with anti-p85 PI-3K antibody, immunoprecipitates were washed, and PI-3K assays were performed (46Whitman M. Kaplan D. Roberts T. Cantley L. Biochem. J. 1987; 247: 165-174Crossref PubMed Scopus (188) Google Scholar). Reactions were carried out for 15 min at room temperature, phosphorylated lipid products were extracted and separated on TLC plates (47Fukui Y. Hanafusa H. Mol. Cell. Biol. 1989; 9: 1651-1658Crossref PubMed Scopus (131) Google Scholar), and incorporated radioactivity was measured by liquid scintillation counting. Cells after treatment without or with LAM were washed, resuspended in buffer A (137 mmNaCl, 2.7 mm KCl, 8.1 mmNa2HPO4, 1.5 mmKH2PO4, 2.5 mm EDTA, 1 mm EDTA, 1 mm dithiothreitol, and 0.1 mm pefabloc), sonicated twice for 10 s each, and centrifuged for 5 min at 800 × g. After discarding the nuclei and unbroken cells, membranes were prepared by ultracentrifugation at 250,000 × g for 60 min. The pellets (membranes) were washed and suspended in buffer A. Cell lysates were prepared as described above and immunoprecipitated using anti-Akt, anti-Bad, or the respective phosphospecific antibodies. The immunoprecipitates were fractionated on SDS-polyacrylamide (10% for Akt and 12% for Bad) gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk, incubated with primary antibody overnight at 4 °C, followed by goat anti-rabbit secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature, and finally visualized using the Phototope-HRP Western detection kit. Following treatment of THP-1 cells with man-LAM, the phosphorylation status of Bad was examined using phosphospecific antibodies. Man-LAM from the virulent Erdman species of M. tuberculosis promoted the phosphorylation of Bad at Ser136 in a time-dependent manner (Fig.1 A). LAM from a fast growing species (ara-LAM) did not exhibit an effect equivalent to that of man-LAM (data not shown). Phosphorylation of Bad at serine 112 was not observed. The phosphorylation of Bad at serine 136 occurs through a PI-3K/Akt-dependent pathway (26Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Goto, Y., and Nunez, G. (1997) 91, 231–241Google Scholar, 27del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1981) Google Scholar, 28Eves E.M. Xiong W. Bellacosa A. Kennedy S.G. Tsichlis P.N. Rosner M.R. Hay N. Mol. Cell. Biol. 1998; 18: 2143-2152Crossref PubMed Scopus (175) Google Scholar, 29Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Abstract Full Text Full Text PDF PubMed Google Scholar). Cells were pretreated with selective inhibitors prior to stimulation with man-LAM in order to test their ability to block Bad phosphorylation. The PI-3K inhibitors wortmannin and LY294002 inhibited Bad phosphorylation at serine 136 in a dose-dependent manner (Fig.1 B). The MEKK1 inhibitors PD90859 and SB20358 did not influence Bad phosphorylation (data not shown). It was inferred that man-LAM signals through PI-3K to phosphorylate Bad. Since man-LAM-induced Bad phosphorylation was abrogated by PI-3K inhibitors, in vitro PI-3K activity was tested after treatment with man-LAM. Man-LAM was found to stimulate PI-3K activity (Fig. 2 A) in a time-dependent manner. Control experiments showed that equal amounts of precipitated PI-3K were used to assay kinase activity. Activation of heterodimeric (p85-p110) PI-3K is often promoted by recruitment to the plasma membrane through interaction of SH2 domains in p85 with tyrosine-phosphorylated proteins. Treatment with man-LAM caused a time-dependent increase in the amount of the p85 subunit detected in the membrane fraction, indicating translocation from the cytosol (Fig. 2 B). Tyrosine phosphorylation of p85 might represent another mechanism of PI-3K activation (48Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White M. Cantley L. Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF PubMed Scopus (410) Google Scholar). Cell lysates were immunoprecipitated with anti-p85 and analyzed by Western blotting with anti-phosphotyrosine antibody. man-LAM treatment increased the tyrosine phosphorylation of the p85α subunit of PI-3K in a time-dependent manner (Fig. 2 C). The PI-3K-sensitive pathway of Bad phosphorylation involves the activation of the protein kinase Akt (or protein kinase B) and the phosphorylation of Bad at serine 136. The interaction of the amino-terminal pleckstrin homology domain of Akt with the phospholipid product of PI-3K induces a conformational change in Akt, making it a more efficient substrate for the phosphatidylinositide-dependent kinase 1 (49Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 50Alessi 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, 51Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar). Stimulus-induced Akt phosphorylation on Thr308 by phosphatidylinositide-dependent kinase 1 and on Ser473 is required for maximal activity. To determine whether Akt is a downstream effector of man-LAM-induced PI-3K signaling in THP-1 cells, Akt phosphorylation on Thr308 was measured using immunoblotting with phosphospecific Thr308 anti-Akt antibody, followed by densitometric analysis of the autoradiograms. Man-LAM-stimulated phosphorylation of Akt at Thr308 in a dose-dependent (Fig.3 A) and time-dependent (Fig. 3 B) manner. LAM stimulated histone 2B phosphorylation in a time-dependent manner (Fig.3 C), indicating LAM-induced stimulation of Akt kinase activity. Histone phosphorylation stimulated by man-LAM was inhibited by the PI-3K inhibitors wortmannin and LY294002 (Fig. 3 D). Unlike man-LAM, exposure of cells to ara-LAM for 60 min did not result in stimulation of Akt kinase activity (Fig. 3 D,lanes g–i). To establish whether Akt is the serine/threonine kinase that is downstream of PI-3K in the man-LAM signaling pathway, the effect of expression of the dominant negative mutant of p85α on LAM-induced Akt kinase activation was assessed. Thr308 phosphorylation of Akt was abrogated by expression of the Δp85 mutant of PI-3K but not by the control empty vector (Fig.3 E). In harmony with this, Bad phosphorylation at serine 136 was also abrogated in cells expressing Δp85 (data not shown). In order to establish a connection between Akt kinase activation and the phosphorylation of Bad at serine 136 induced by LAM, we transfected THP-1 cells with a kinase-dead mutant (K179M) of Akt and assessed the phosphorylation status of Bad. Akt.KD abrogated the phosphorylation of Bad following stimulation by LAM, whereas control empty vector had no such effect (Fig.3 F). The molecular basis of the pathogenicity of M. tuberculosis is poorly understood . M. tuberculosis is a facultative intracellular pathogen. Whereas the normal function of macrophages is to engulf and destroy microorganisms, mycobacteria have evolved ways to circumvent the defense mechanisms of macrophages. Central to the ability of M. tuberculosis to infect the host and cause active or latent disease is the propensity of the tubercle bacillus to enter the host mononuclear phagocyte and survive and multiply within macrophages. Macrophage apoptosis contributes to host defense against M. tuberculosis infection. Human alveolar macrophages undergo apoptosis in response to M. tuberculosisinfection (52Keane J. Balcewicz-Sablinska M.K. Remold H.G. Chupp G.L. Meek B.B. Fenton M.J. Kornfeld H. Infect. Immun. 1997; 65: 298-304Crossref PubMed Google Scholar). Very recently it has been demonstrated that bacillary control of host cell apoptosis is a virulence-associated phenotype of M. tuberculosis, with virulent strains having the ability to evade apoptosis of infected macrophages (53Keane J. Remold H.G. Kornfeld H. J. Immunol. 2000; 164: 2016-2020Crossref PubMed Scopus (447) Google Scholar). In light of these observations, understanding the virulence factors that may modulate host cell apoptosis is necessary. We chose to study whether man-LAM from a virulent strain of M. tuberculosis could modulate cell signaling pathways known to control cell survival. The phosphorylation of Bad is one of the mechanisms of protection of cells from programmed cell death. We have tested the hypothesis that man-LAM from the virulent species of M. tuberculosisprotects cells from apoptosis at least partly through phosphorylation of Bad. Man-LAM was found to stimulate Bad phosphorylation on serine 136 in THP-1 cells. Bad phosphorylation at serine 136 is believed to occur via a PI-3K/Akt signaling pathway. Consistent with this, the PI-3K inhibitors wortmannin and LY294002 inhibited the LAM-stimulated phosphorylation of Bad. Activation of PI-3K was observed after stimulation of cells with LAM. Furthermore, LAM caused p85 to translocate to the membrane, a phenomenon associated with enhanced lipid kinase activity of this enzyme. Tyrosine phosphorylation of p85 may represent another mechanism of PI-3K activation. LAM stimulated tyrosine phosphorylation of p85. The fact that PI-3K activation is sufficient for Bad phosphorylation upon treatment of cells with LAM was demonstrated by the fact that transfection of THP-1 cells with the dominant negative mutant (Δp85) of PI-3K abrogated LAM-mediated Bad phosphorylation. One of the major functions of Akt is protection of cells from programmed cell death. This protection has been demonstrated for several cell types including COS cells (23Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar), fibroblasts (24Kaufmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1069) Google Scholar), and neuronal cells (55Philpott K.L. McCarthy M.J. Klippel A. Rubin L.L. J. Cell Biol. 1997; 139: 809-815Crossref PubMed Scopus (220) Google Scholar). This is at least in part due to the ability of Akt to phosphorylate Bad at serine 136. We attempted to establish a connection between PI-3K, Akt, and the phosphorylation of Bad. Akt phosphorylation at Thr308 was stimulated by LAM in a PI-3K-sensitive manner. Akt kinase activity assessed by using histone 2B as exogenous substrate was also stimulated by LAM. Transfection of THP-1 with the Δp85α mutant of PI-3K abolished the LAM-stimulated phosphorylation of Akt at Thr308 as well as the ability of Akt to phosphorylate histone 2B, establishing that Akt is a downstream effector of LAM-mediated PI-3K signaling. The connection between Akt and Bad was similarly established by transfecting cells with a kinase-deficient mutant of Akt (Akt.KD) and assessing the phosphorylation of Bad after stimulation with LAM. LAM-mediated Bad phosphorylation was abolished in cells transfected with Akt.KD. In summary, we demonstrate that man-LAM from the virulent Erdman strain of M. tuberculosis activates Akt in a PI-3K-sensitive manner, leading to the phosphorylation of Bad at serine 136, suggesting that this may be one of the mechanisms by which LAM directly promotes macrophage cell survival. Inhibition of macrophage apoptosis would allow the mycobacteria to escape from being packaged into apoptotic bodies. Uptake of bacilli packaged in this way is suggested to result in more effective microbicidal processing (54Fratazzi C. Arbeit C. Carini C. Remold H.G. J. Immunol. 1997; 158: 4320-4327PubMed Google Scholar). This is the first demonstration of a mycobacterial virulence factor having the capability of up-regulating a macrophage survival signaling pathway, thereby creating an environment favorable for the survival of the pathogen. We are grateful to Drs. Robert Farase and Kenneth Walsh for the gifts of the Δp85 mutant of PI-3K and Akt.KD, respectively.
The N-terminal cytoplasmic domain of the anion exchanger 1 (AE1 or band 3) of the human erythrocyte associates with peripheral membrane proteins to regulate membrane-cytoskeleton interactions, with glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase and aldolase, with the protein-tyrosine kinase p72syk, with hemoglobin and with hemichromes. We have demonstrated that the N-terminal cytoplasmic domain of band 3 (CDB3) is a substrate of the apoptosis executioner caspase 3 (1). CDB3 has two non-conventional caspase 3 cleavage sites, TATD45 and EQGD205 (2). In vitro treatment of recombinant CDB3 with caspase 3 generated two fragments, which could be blocked by pretreatment with the caspase 3 inhibitor Z-DEVD-fmk (3). Recombinant CDB3 in which the caspase 3 cleavage sites Asp45 and Asp205 were mutated, was resistant to proteolysis (4). Proteolytically derived fragments crossreactive with polyclonal anti-band 3 antibody appeared with simultaneous cleavage of poly (ADP-ribose) polymerase and procaspase 3 in staurosporine (STS)-treated HEK293 cells transiently transfected with CDB3 (5). In vivo cleavage of CDB3 could be blocked by pretreatment of cells with Z-DEVD-fmk or in cells transfected with mutant CDB3 (D45A, D205A) (6). Co-transfection experiments showed that STS-mediated cleavage of CDB3 diminished its interaction with the N-terminal domain of protein 4.2, confirming that such cleavage interferes with the interaction of CDB3 with cytoskeletal proteins (7). Active caspase 3 was observed in aged red cells but not in young cells. This red cell caspase 3 could cleave band 3 present in inside-out vesicles prepared from young erythrocytes arguing in favor of a physiological role of caspase 3 in aged erythrocytes.
Gastric infection, as well as inflammation, caused by Helicobacter pylori, activates the production of cytokines and chemokines by mononuclear cells; interleukin-8 (IL-8) is one of the major inflammatory chemokines. Since H. pylori does not invade mucosal tissue, we observed the effect of the water extract of H. pylori (HPE), containing shed factors, on the production of IL-8 by human peripheral blood monocytes and the human monocyte cell line THP-1. HPE-treatment induced activation of the mitogen-activated protein kinases (MAPKs) ERK (extracellular signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase), an effect which was not dependent on the presence of the cag pathogenicity island. p38 MAPK activation was sustained. The specific inhibitors, U0126 (for ERK1/2 signalling) and SB203580 (for p38 MAPK signalling), both abrogated IL-8 secretion from HPE-treated THP-1. Dominant-negative mutants of the upstream kinases MEK1 (MAPK/ERK kinase 1), MKK (MAPK kinase) 6 and MKK7 also inhibited IL-8 secretion, pointing to a role of all three MAPKs in HPE-mediated IL-8 release. The inhibitory effects of polymyxin B and anti-CD14 antibody suggested that the effect of HPE on MAPKs was mediated by H. pylori lipopolysaccharide (LPS). By analysis of IL-8-promoter-driven luciferase gene expression, we observed that the effects of HPE-induced nuclear factor-κB (NF-κB) activation and MAPK signalling were mediated at the level of the IL-8 promoter. While ERK1/2 activation could be linked to enhanced DNA binding of activator protein-1 (AP-1), p38 MAPK signalling did not affect AP-1 DNA binding. Taken together, these results provide the first evidence that LPS from H. pylori stimulates IL-8 release from cells of the monocytic lineage through activation of NF-κB and signalling along MAPK cascades. The stimulation of MAPK signalling in macrophages by LPS of H. pylori amplifies the inflammatory response associated with gastric H. pylori infection and needs to be taken into consideration when developing therapeutics based on these signalling pathways.
Matrix metalloproteinases (MMPs) contribute to the matrix-degrading phenotype of mycobacterial diseases. Considering that MMPs could contribute to the mutual exacerbation of both Mycobacterium avium and HIV in coinfections, it is of importance to understand the mechanisms of M. avium-induced MMP induction. Focusing on MMP-9, our work demonstrates that a cyclooxygenase-2 (COX-2)-dependent signalling loop is critical for activation of MMP-9 transcription in RAW264.7 cells and murine bone marrow-derived macrophages. M. avium-stimulated MMP-9 induction involves the p65 and p50 subunits of NF-κB and the c-Fos and c-jun subunits of AP-1. The c-Fos gene is upregulated in a MEK1-dependent manner in M. avium-challenged macrophages. M. avium-induced MMP-9 gene induction requires the histone acetyltransferase p300 and chromatin modifications involving phosphorylation of p65 at serine 276 and its acetylation at lysines 221 and 310. At the same time, histone H3 modified by mitogen and stress-activated protein kinase 1 (MSK1)-dependent phosphorylation on serine 10 and by acetylation on lysine 14, typical signatures linked to transcriptional activation, also associates with the MMP-9 promoter following M. avium challenge. Taken together, our results show that co-ordinated post-translational modifications of p65 and histone H3 involving phosphorylation and acetylation drive COX-2-dependent transcriptional activation of the MMP-9 gene in response to challenge of macrophages with M. avium.
Stocking density is one of the important aspects of housing management. Increased stocking density can influence the skeletal development of broilers negatively and can increase the leg abnormalities thereby generating welfare issue for the same. To study the effect of stocking density, an experiment was conducted at poultry farm, DUVASU, Mathura. Week old turkey poults were distributed randomly on uniform body weight basis in three treatment groups- T-1: standard stocking density (2.5 ft2 per bird); T-2: high stocking density (1.25 ft2 per bird) and T-3: low stocking density (5 ft2 per bird). Tibia and femur were separated from the sacrificed birds and dried in oven at 1050C for 72 hours. There after cooling down to room temperature, the bones were weighed. Digital radiographs of the bones were taken. The dimensions and thickness of the bones were measured. Average femur length was found to be significantly higher (p<0.01) in standard and high SD treatments than the low SD treatment. Radiographic density of Tibia medulla was found to be significantly higher (p<0.01) in low SD treatment than the other two SD treatments.
Abstract Helicobacter pylori infection causes inflammation and increases the expression of IL-8 in human gastric epithelial cells. H. pylori activates NF-κB and AP-1, essential transcriptional factors in H. pylori-induced IL-8 gene transcription. Although colonization creates a local oxidative stress, the molecular basis for the transition from infection to the expression of redox-sensitive cytokine genes is unknown. We recently reported that the expression of apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE-1/Ref-1), which repairs oxidative DNA damage and reductively activates transcription factors including AP-1 and NF-κB, is increased in human gastric epithelia during H. pylori infection. In this study, we examine whether APE-1/Ref-1 functions in the modulation of IL-8 gene expression in H. pylori-infected human gastric epithelial cells. Small interfering RNA-mediated silencing of APE-1/Ref-1 inhibited basal and H. pylori-induced AP-1 and NF-κB DNA-binding activity without affecting the nuclear translocation of these transcription factors and also reduced H. pylori-induced IL-8 mRNA and protein. In contrast, overexpression of APE-1/Ref-1 enhanced basal and H. pylori-induced IL-8 gene transcription, and the relative involvement of AP-1 in inducible IL-8 promoter activity was greater in APE-1/Ref-1 overexpressing cells than in cells with basal levels of APE-1/Ref-1. APE-1/Ref-1 inhibition also reduced other H. pylori-induced chemokine expression. By implicating APE-1/Ref-1 as an important regulator of gastric epithelial responses to H. pylori infection, these data elucidate a novel mechanism controlling transcription and gene expression in bacterial pathogenesis.
Abstract Background The hypoxic microenvironment is a key component of the gastric tumour niche. Carcinoembryonic antigen‐related cell adhesion molecule 6 (CEACAM6) is upregulated in gastric cancer and is considered a novel biomarker for the disease. However, no prior studies have elaborated on the status of CEACAM6 and its role in the hypoxic gastric cancer niche. Methods In this short study, we evaluated the effect of hypoxia in modulating CEACAM6 level in gastric cancer cells (GCCs) through western blotting and determined the effect of CEACAM6 upregulation on gastric cancer progression through clonogenicity, cell proliferation and migration assays. The wound‐healing ability of GCCs was downregulated by siRNA‐mediated CEACAM6 silencing. Human gastric cancer biopsy samples were examined by immunofluorescence microscopy to assess the level of a hypoxia marker HIF1α and CEACAM6. The effect of empty vector or CEACAM6‐ expression on peripheral blood‐derived mononuclear cell (PBMC)‐derived macrophage polarization under normoxia or hypoxia was studied by incubating macrophages in conditioned medium collected from GCC cultures. Macrophage polarization status was observed using flow cytometry and fluorescence microscopy. Reactive oxygen species (ROS) generation by macrophages was evaluated using fluorescence microscopy. Results We identified that hypoxia promoted CEACAM6 in GCCs, and these cells acquired increased proliferative potential and migration ability. Moreover, the cell culture supernatant from hypoxia‐exposed CEACAM6‐overexpressing cells promoted an M2‐like macrophage population and discouraged the M1 phenotype. Conclusion This study established that hypoxia increased CEACAM6 which promoted gastric cancer progression by influencing GCC proliferation and motility as well as macrophage polarization.
'%3e%3ccircle r='20' fill='%23008'/%3e%3ccircle r='17.5' fill='%23fff'/%3e%3ccircle r='3.5' fill='%23008'/%3e%3cg id='d'%3e%3cg id='c'%3e%3cg id='b'%3e%3cg id='a' fill='%23008'%3e%3ccircle r='.875' transform='rotate(7.5 -8.75 133.5)'/%3e%3cpath d='M0 17.5.6 7 0 2l-.6 5L0 17.5z'/%3e%3c/g%3e%3cuse xlink:href='%23a' transform='rotate(15)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(30)'/%3e%3c/g%3e%3cuse xlink:href='%23c' transform='rotate(60)'/%3e%3c/g%3e%3cuse xlink:href='%23d' transform='rotate(120)'/%3e%3cuse xlink:href='%23d' transform='rotate(-120)'/%3e%3c/g%3e%3c/svg%3e)
