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• W2023802571 abstract "Anthrax lethal toxin (LeTx) is a virulence factor causing immune suppression and toxic shock of Bacillus anthracis infected host. It inhibits cytokine production and cell proliferation/differentiation in various immune cells. This study showed that a brief exposure of LeTx caused a continual MEK1 cleavage and prevented tumor necrosis factor-α (TNF) production in response to lipopolysaccharide (LPS) in non-proliferating cells such as human peripheral blood mononuclear cells or mouse primary peritoneal macrophages. In human monocytic cell lines U-937 and THP-1, LeTx induced cell cycle arrest in G0-G1 phase by rapid down-regulation of cyclin D1/D2 and checkpoint kinase 1 through MEK1 inhibition. However, THP-1 cells adaptively adjusted to LeTx and overrode cell cycle arrest by activating the phosphatidylinositol 3-kinase/Akt signaling pathway. Inhibitory Ser-9 phosphorylation of glycogen synthase kinase 3β (GSK3β) by Akt prevented proteasome-mediated cyclin D1 degradation and induced cell cycle progress in LeTx-intoxicated THP-1 cells. Recovery from cell cycle arrest was required before recovering from on-going MEK1 cleavage and suppression of TNF production. Furthermore, pretreatment with LeTx or the GSK3-specific inhibitor SB-216763, or transfection with dominant active mutant Akt or degradation-defected mutant cyclin D1 protected cells from LeTx-induced cell cycle arrest, on-going MEK1 cleavage and suppression of TNF production. These results indicate that modulation of phosphatidylinositol 3-kinase/Akt/GSK3β signaling cascades can be beneficial for protecting or facilitating recovery from cellular LeTx intoxication in cells that depend on basal MEK1 activity for proliferation. Anthrax lethal toxin (LeTx) is a virulence factor causing immune suppression and toxic shock of Bacillus anthracis infected host. It inhibits cytokine production and cell proliferation/differentiation in various immune cells. This study showed that a brief exposure of LeTx caused a continual MEK1 cleavage and prevented tumor necrosis factor-α (TNF) production in response to lipopolysaccharide (LPS) in non-proliferating cells such as human peripheral blood mononuclear cells or mouse primary peritoneal macrophages. In human monocytic cell lines U-937 and THP-1, LeTx induced cell cycle arrest in G0-G1 phase by rapid down-regulation of cyclin D1/D2 and checkpoint kinase 1 through MEK1 inhibition. However, THP-1 cells adaptively adjusted to LeTx and overrode cell cycle arrest by activating the phosphatidylinositol 3-kinase/Akt signaling pathway. Inhibitory Ser-9 phosphorylation of glycogen synthase kinase 3β (GSK3β) by Akt prevented proteasome-mediated cyclin D1 degradation and induced cell cycle progress in LeTx-intoxicated THP-1 cells. Recovery from cell cycle arrest was required before recovering from on-going MEK1 cleavage and suppression of TNF production. Furthermore, pretreatment with LeTx or the GSK3-specific inhibitor SB-216763, or transfection with dominant active mutant Akt or degradation-defected mutant cyclin D1 protected cells from LeTx-induced cell cycle arrest, on-going MEK1 cleavage and suppression of TNF production. These results indicate that modulation of phosphatidylinositol 3-kinase/Akt/GSK3β signaling cascades can be beneficial for protecting or facilitating recovery from cellular LeTx intoxication in cells that depend on basal MEK1 activity for proliferation. Bacillus anthracis is a Gram-positive spore forming bacterium and systemic infection of B. anthracis is often fatal when inhaled spores germinate inside the host (1Dixon T.C. Meselson M. Guillemin J. Hanna P.C. N. Engl. J. Med. 1999; 341: 815-826Crossref PubMed Scopus (882) Google Scholar). The virulence of B. anthracis is primarily attributed to two secreted exotoxins, lethal toxin (LeTx) 3The abbreviations used are:LeTxlethal toxinPAprotective antigenLFlethal factorEFedema factorMAPKmitogen-activated protein kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseCFSEcarboxyfluorescein succinimidyl esterNALP1bNACHT-leucine-rich repeat and pyrin domain-containing protein 1bChk1checkpoint kinase 1TNFtumor necrosis factor-αLPSlipopolysaccharidePI3Kphosphatidylinositol 3-kinaseGSK3βglycogen synthase kinase 3βJNKc-Jun N-terminal kinaseERKextracellular signal-regulated kinasePBMCperipheral blood mononuclear cellMOPS4-morpholinepropanesulfonic acidPIpropidium iodideFACSfluorescence-activated cell sortingSBSB202190LyLY294002Aktmyrmyristoylated Akt. 3The abbreviations used are:LeTxlethal toxinPAprotective antigenLFlethal factorEFedema factorMAPKmitogen-activated protein kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseCFSEcarboxyfluorescein succinimidyl esterNALP1bNACHT-leucine-rich repeat and pyrin domain-containing protein 1bChk1checkpoint kinase 1TNFtumor necrosis factor-αLPSlipopolysaccharidePI3Kphosphatidylinositol 3-kinaseGSK3βglycogen synthase kinase 3βJNKc-Jun N-terminal kinaseERKextracellular signal-regulated kinasePBMCperipheral blood mononuclear cellMOPS4-morpholinepropanesulfonic acidPIpropidium iodideFACSfluorescence-activated cell sortingSBSB202190LyLY294002Aktmyrmyristoylated Akt. and edema toxin, and an exterior capsule, encoded in two large plasmids (2Green B.D. Battisti L. Koehler T.M. Thorne C.B. Ivins B.E. Infect. Immun. 1985; 49: 291-297Crossref PubMed Google Scholar, 3Mikesell P. Ivins B.E. Ristroph J.D. Dreier T.M. Infect. Immun. 1983; 39: 371-376Crossref PubMed Google Scholar). The capsule comprises poly-d-glutamic acid, which protects bacteria from phagocytosis by the host immune cells and contributes to bacterial dissemination (4Drysdale M. Heninger S. Hutt J. Chen Y. Lyons C.R. Koehler T.M. EMBO J. 2005; 24: 221-227Crossref PubMed Scopus (139) Google Scholar). LeTx and edema toxin are binary A:B toxins comprising protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. PA is a molecular transporter allowing receptor-mediated entry and release of LF or EF into the cytosol. PA binds to either of two surface receptors: anthrax receptor 1 (also known as the tumor endothelial marker 8) and anthrax receptor 2 (also known as the capillary morphogenesis gene-2). Anthrax receptor 1 is expressed in high levels in macrophages, endothelial cells and several tumor cells (5Bradley K.A. Mogridge J. Mourez M. Collier R.J. Young J.A. Nature. 2001; 414: 225-229Crossref PubMed Scopus (750) Google Scholar, 6Bonuccelli G. Sotgia F. Frank P.G. Williams T.M. de Almeida C.J. Tanowitz H.B. Scherer P.E. Hotchkiss K.A. Terman B.I. Rollman B. Alileche A. Brojatsch J. Lisanti M.P. Am. J. Physiol. 2005; 288: C1402-C1410Crossref PubMed Scopus (95) Google Scholar, 7Rmali K.A. Al-Rawi M.A. Parr C. Puntis M.C. Jiang W.G. Int. J. Mol. Med. 2004; 14: 75-80PubMed Google Scholar); whereas, anthrax receptor 2 is widely distributed in human tissues (8Scobie H.M. Rainey G.J. Bradley K.A. Young J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5170-5174Crossref PubMed Scopus (521) Google Scholar). EF has adenylate cyclase activity (9Leppla S.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3162-3166Crossref PubMed Scopus (760) Google Scholar), whereas LF is a zinc metalloprotease that cleaves and inactivates the N-terminal end of the mitogen-activated protein kinase (MAPK) kinases (MEK) 1 to 7, except MEK5, resulting in the inactivation of most their downstream signaling cascades. Both LF and EF cause severe defects in immune responses and contribute proliferation and dissemination of B. anthracis in the host (10Li Y. Sherer K. Cui X. Eichacker P.Q. Expert. Opin. Biol. Ther. 2007; 7: 843-854Crossref PubMed Scopus (14) Google Scholar, 11Tournier J.N. Quesnel-Hellmann A. Cleret A. Vidal D.R. Cell Microbiol. 2007; 9: 555-565Crossref PubMed Scopus (64) Google Scholar). lethal toxin protective antigen lethal factor edema factor mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase carboxyfluorescein succinimidyl ester NACHT-leucine-rich repeat and pyrin domain-containing protein 1b checkpoint kinase 1 tumor necrosis factor-α lipopolysaccharide phosphatidylinositol 3-kinase glycogen synthase kinase 3β c-Jun N-terminal kinase extracellular signal-regulated kinase peripheral blood mononuclear cell 4-morpholinepropanesulfonic acid propidium iodide fluorescence-activated cell sorting SB202190 LY294002 myristoylated Akt lethal toxin protective antigen lethal factor edema factor mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase carboxyfluorescein succinimidyl ester NACHT-leucine-rich repeat and pyrin domain-containing protein 1b checkpoint kinase 1 tumor necrosis factor-α lipopolysaccharide phosphatidylinositol 3-kinase glycogen synthase kinase 3β c-Jun N-terminal kinase extracellular signal-regulated kinase peripheral blood mononuclear cell 4-morpholinepropanesulfonic acid propidium iodide fluorescence-activated cell sorting SB202190 LY294002 myristoylated Akt Monocytes and macrophages are key innate immune cells and likely the first immune cells encountering the spores and germinating bacteria (12Cote C.K. Rea K.M. Norris S.L. van Rooijen N. Welkos S.L. Microb. Pathog. 2004; 37: 169-175Crossref PubMed Scopus (66) Google Scholar, 13Cote C.K. Van Rooijen N. Welkos S.L. Infect. Immun. 2006; 74: 469-480Crossref PubMed Scopus (123) Google Scholar, 14Hu H. Sa Q. Koehler T.M. Aronson A.I. Zhou D. Cell Microbiol. 2006; 8: 1634-1642Crossref PubMed Scopus (66) Google Scholar). LeTx induces rapid necrosis-like cell death of macrophages prepared from certain strains of inbred mice (15Friedlander A.M. J. Biol. Chem. 1986; 261: 7123-7126Abstract Full Text PDF PubMed Google Scholar, 16Welkos S.L. Keener T.J. Gibbs P.H. Infect. Immun. 1986; 51: 795-800Crossref PubMed Google Scholar, 17Kim S.O. Jing Q. Hoebe K. Beutler B. Duesbery N.S. Han J. J. Biol. Chem. 2003; 278: 7413-7421Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Muehlbauer S.M. Evering T.H. Bonuccelli G. Squires R.C. Ashton A.W. Porcelli S.A. Lisanti M.P. Brojatsch J. Cell Cycle. 2007; 6: 758-766Crossref PubMed Scopus (67) Google Scholar). A genetic study investigating LeTx-sensitivity trait loci in different strains of mice identified the NACHT-leucine-rich repeat and pyrin domain-containing protein 1b (NALP1b) as a host factor that confers rapid LeTx cytotoxicity (19Boyden E.D. Dietrich W.F. Nat. Genet. 2006; 38: 240-244Crossref PubMed Scopus (644) Google Scholar). Caspase-1–/– macrophages with LeTx-susceptible genetic backgrounds were resistance to LeTx. LeTx also rapidly induces the release of interleukin-1β and -18 in LeTx-susceptible macrophages (20Cordoba-Rodriguez R. Fang H. Lankford C.S. Frucht D.M. J. Biol. Chem. 2004; 279: 20563-20566Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), indicating that LeTx induces Nalp1b- and caspase-1-mediated cell death. However, human macrophages lack NALP1b and are resistant to rapid necrotic cell death by LeTx. Instead, LeTx was shown to cause delayed apoptotic cell death of differentiated macrophages and inhibit cell proliferation/differentiation, which are probably mediated through MAPK inhibition (18Muehlbauer S.M. Evering T.H. Bonuccelli G. Squires R.C. Ashton A.W. Porcelli S.A. Lisanti M.P. Brojatsch J. Cell Cycle. 2007; 6: 758-766Crossref PubMed Scopus (67) Google Scholar, 21Popov S.G. Villasmil R. Bernardi J. Grene E. Cardwell J. Popova T. Wu A. Alibek D. Bailey C. Alibek K. FEBS Lett. 2002; 527: 211-215Crossref PubMed Scopus (77) Google Scholar, 22Kassam A. Der S.D. Mogridge J. Cell Microbiol. 2005; 7: 281-292Crossref PubMed Scopus (62) Google Scholar). Because sustained MAPK activation is required for cell cycle progress of many different cells and the prevalence in expression of PA receptors, it is expected that LeTx causes cell cycle arrest in many cells and tissues of infected hosts. In fact, LeTx was shown to inhibit cell proliferation/differentiation in other cell types, including melanocytes (23Koo H.M. VanBrocklin M. McWilliams M.J. Leppla S.H. Duesbery N.S. Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3052-3057Crossref PubMed Scopus (136) Google Scholar), B lymphocytes (24Fang H. Xu L. Chen T.Y. Cyr J.M. Frucht D.M. J. Immunol. 2006; 176: 6155-6161Crossref PubMed Scopus (67) Google Scholar), and T lymphocytes (25Paccani S.R. Tonello F. Ghittoni R. Natale M. Muraro L. D'Elios M.M. Tang W.J. Montecucco C. Baldari C.T. J. Exp. Med. 2005; 201: 325-331Crossref PubMed Scopus (138) Google Scholar, 26Fang H. Cordoba-Rodriguez R. Lankford C.S. Frucht D.M. J. Immunol. 2005; 174: 4966-4971Crossref PubMed Scopus (59) Google Scholar, 27Comer J.E. Chopra A.K. Peterson J.W. Konig R. Infect. Immun. 2005; 73: 8275-8281Crossref PubMed Scopus (89) Google Scholar), which contribute to the host immune paralysis. To date the signaling mechanism of cell proliferation inhibition by LeTx and its contribution in the pathogenesis of anthrax remains largely unknown. This study examined further details on the effects of LeTx on cell proliferation. LeTx induced cell cycle arrest in G0-G1 phase via down-regulating key cell cycle progress proteins cyclin D1, D2, and checkpoint kinase 1 (Chk1) via MEK1 inhibition in human monocytic cell lines. LF incorporated inside cells had almost permanent MEK1-cleaving activity and completely blocked tumor necrosis factor-α (TNF) production in response to a bacterial component lipopolysaccharide (LPS). Recovery from the prolong MEK-cleaving LF activity and the TNF-suppressing effects required cell proliferation, which was mediated through adaptive response of THP-1 cells by inducing the phosphatidylinositol 3-kinase (PI3K)/Akt/GSK3β signaling pathway. Activation of PI3K/Akt by pretreating cells with LeTx or a chemical GSK3-inhibitor protected cell cycle arrest and facilitated recovery from cellular LeTx intoxication. Cell Culture and Reagents—The human monocytic leukemia cell line THP-1 and U937 cells were grown in complete RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (Sigma), 10 mm minimal essential medium non-essential amino acids solution, 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 1 mm sodium pyruvate. Cells were grown at 37 °C in the humidified atmosphere of 5% CO2. LF and PA were prepared in the laboratory as previously described (28Wesche J. Elliott J.L. Falnes P.O. Olsnes S. Collier R.J. Biochemistry. 1998; 37: 15737-15746Crossref PubMed Scopus (172) Google Scholar, 29Miller C.J. Elliott J.L. Collier R.J. Biochemistry. 1999; 38: 10432-10441Crossref PubMed Scopus (224) Google Scholar). LY294002, Akt inhibitor II, MG132, Ac-YVAD-CMK, SB202190, U0126, and JNK inhibitor I were purchased from Calbiochem (EMD Biosciences, La Jolla, CA). Chk1 inhibitor (SB218078) and GSK3 inhibitor (SB216763) was obtained from Sigma. Pan and phospho-site specific antibodies toward Akt, p38 MAPK, ERK1/2, MEK1 (COOH terminus of MEK1), and GSK3α/β were obtained from Cell Signaling Technologies (Pickering, Ontario, Canada). The antibody raised against the N terminus of MEK1 was from QED Bioscience Inc. MEK3 and Chk1 antibodies were purchased form Santa Cruz Biotechnology, and cyclin D1 and LF antibodies were from Neo Markers (Fremont, CA) and List Biological Laboratories (Campbell, CA), respectively. Preparation of Human and Murine Macrophages—Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque Plus (Amersham Biosciences). Blood obtained from volunteers were diluted with 3 volumes of phosphate-buffered saline and layered on top of the Ficoll at a 3 to 1 ratio. Blood cells were separated into different strata, and the lymphocyte layer was extracted. Isolated PBMCs were washed three times with phosphate-buffered saline and plated with complete RPM1 1640 media. For isolation of mouse peritoneal macrophages, mice received intraperitoneal injections of 3% thioglycollate 4 days before the preparation of peritoneal macrophages. Macrophages were harvested from mice by injecting 5 ml of saline into peritoneal cavity and re-extracted by withdrawal with a syringe. Cells were put through a cell strainer (40 μm, BD Biosciences) and incubated at 37 °C overnight in a humidified atmosphere of 5% CO2. Bone marrow-derived macrophages were obtained from C57BL/6 mice as previously described (30Falk L.A. Vogel S.N. J. Leukoc. Biol. 1988; 43: 148-157Crossref PubMed Scopus (43) Google Scholar), except culturing bone marrow cells with IL-3 (10 ng/ml) for 5 days. Total Cell Lysate Preparation and Western Immunoblot Analysis—Total cell lysate extraction and Western blotting analysis were performed as previously described (17Kim S.O. Jing Q. Hoebe K. Beutler B. Duesbery N.S. Han J. J. Biol. Chem. 2003; 278: 7413-7421Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Briefly, cells were lysed in ice-cold cell lysis buffer containing 20 mm MOPS, 15 mm EGTA, 2 mm EDTA, 1 mm Na3VO4, 1 mm dithiothreitol, 75 mm β-glycerophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 10 μg/ml pepstatin A, 1 μg/ml leupeptin, and 1% Triton X-100 and then sonicated on ice. Cell extracts were obtained by centrifuging the homogenate at 18,000 × g for 10 min. These extracts were electrophoretically resolved in ready-made 10% SDS-PAGE gels (Bio-Rad), followed by transfer onto nitrocellulose membranes. Membranes were subsequently blocked with 5% skim milk for 30 min, immunoblotted with antibodies, and developed using an enhanced chemiluminescence detection system (ECL, Pierce Bioscience). Total cell lysates were obtained from THP-1 cells 48 h after LeTx (250 ng of LF and 500 ng of PA for 5 h) exposure and Kinetworks™ KCCP-1.0 Cell Cycle Protein multi-immunoblotting analysis was performed at Kinexus Bioinformatics Inc. (Vancouver, Canada) as described on the Kinexus website. Quantitative Real-time PCR—mRNAs expression were quantified on the Rotor-Gene RG3000 quantitative multiplex PCR instrument using the Brilliant SYBR Green PCR Master Mix (Applied Biosystems). Total cellular RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. Briefly, 4 μg of total RNA was reverse transcribed by using oligo(dT) primers and the M-MuLV reverse transcriptase (New England Biotechnology) according to the manufacturer's recommendations. Oligonucleotide primers were the following: for cyclin D1, 5′-CCCTCGGTGTCCTACTTCAA-3′ (5′ primer) and 5′-AGGAAGCGGTCCAGGTAGTT-3′ (3′ primer); for cyclin D2, 5′-GTCTCAAAGCTTGCCAGGAG-3′ (5′ primer) and 5′-ATATCCCGCACGTCTGTAGG-3′ (3′ primer); for cyclin B, 5′-CAAGCCCAATGGAAACATCT-3′ (5′ primer) and 5′-GGATCAGCTCCATCTTCTGG-3′ (3′ primer); for cyclin E, 5′-ATCCTCCAAAGTTGCACCAG-3′ (5′ primer) and 5′-AGGGGACTTAAACGCCACTT-3′ (3′ primer); for CHK1, 5′-CTGAAGAAGCAGTCGCAGTG-3′ (5′ primer) and 5′-TTGCCTTCTCTCCTGTGACC-3′ (3′ primer); for glyceraldehyde-3-phosphate dehydrogenase, 5′-ACCCACTCCTCCACCTTTG-3′ (5′ primer) and 5′-CTCTTGTGCTCTTGCTGGG-3′ (3′ primer). Cell Cycle and Proliferation Analysis—Analyses of intracellular fluorescence by carboxyfluorescein succinimidyl ester (CFSE) and DNA content using propidium iodide (PI) were performed using CellQuest software on a FACSCalibur flow cytometer (Becton Dickinson). For cell proliferation studies by CFSE, cells were incubated with CFSE of 1 μm for 10 min at 37 °C, and reaction was quenched on an ice bath for 2 min. Cells were washed with 3 volumes of complete media and plated. Cells were harvested daily for FACS analysis. For DNA content analysis by PI, 1.0 × 106 cells were harvested at the indicated time points, fixed by dropwise addition of ice-cold 70% ethanol after three times washing with 1× phosphate-buffered saline containing 0.1% glucose, and were stored at 4 °C. Subsequently, cell were pelleted by centrifugation and resuspended in staining solution containing 50 μg of PI/ml and 100 units of RNase A/ml. After 60 min of incubation at room temperature, the cell was loaded in a FACSCalibur flow cytometer. The data were analyzed using CellQuest and ModFit LT 3.0 software (BD Biosciences). For the purpose of analysis, acquired events were gated to eliminate cell aggregates and debris. Aktmyr and AktK179M Retroviruses Generation and Infection—The virus preparation and infection were performed as described (31Kim S.O. Ha S.D. Lee S. Stanton S. Beutler B. Han J. BioTechniques. 2007; 42: 493-501Crossref PubMed Scopus (6) Google Scholar). Briefly, constitutively active myristoylated Akt (Aktmyr) and dominant negative Akt (Akt K179M) retroviruses were generated in Phoenix Amphotropic producer cells using the calcium phosphate method. Empty retroviral vector was transfected by same procedure for control virus. Cells were cultured for 24 h at 37 °C and for another 24 h were incubated at 32 °C for collection of viruses. Virus was collected and filtered through a 0.45-μm filter. THP-1 cells of 1 × 106 were suspended with viruses solution containing 4 μg of Polybrene per milliliter and plated in 6-well plates. The plates were centrifuged at 2,500 rpm for 45 min at 32 °C, and fresh RPMI 1640 media were added into the each well after 3-h incubation at 37 °C. Viruses expressing cells were selected by green fluorescent protein 48 h after infection and treated with the LeTx for cell cycle analysis. pCDNA-Cyclin D1T286A Construction and Transfection—pCDNA3 containing human mutant cyclin D1-T286A was made by digesting a pFlex-D1-T286A (32Diehl J.A. Zindy F. Sherr C.J. Genes Dev. 1997; 11: 957-972Crossref PubMed Scopus (646) Google Scholar) with the EcoR1 restriction enzyme and ligated the resulting fragment into the pCDNA3.1 vector (Invitrogen). The construct was verified by DNA sequencing. The stable transfection of constructed plasmids was performed by electrophoresis using Gene Pulser Xcell (Bio-Rad) in THP-1 cell line, and mutant cyclin D1 expression cells were selected by G418 antibiotics. LeTx Induces G1 Cell Cycle Arrest—Previous studies have shown that LeTx inhibits proliferation and differentiation of immune cells (22Kassam A. Der S.D. Mogridge J. Cell Microbiol. 2005; 7: 281-292Crossref PubMed Scopus (62) Google Scholar, 24Fang H. Xu L. Chen T.Y. Cyr J.M. Frucht D.M. J. Immunol. 2006; 176: 6155-6161Crossref PubMed Scopus (67) Google Scholar, 27Comer J.E. Chopra A.K. Peterson J.W. Konig R. Infect. Immun. 2005; 73: 8275-8281Crossref PubMed Scopus (89) Google Scholar). We further examined the mechanism of cell cycle arrest in the human monocytic cells lines U937 and THP-1. Proliferation of these cells after brief exposure of LeTx (250 ng/ml LF and 500 ng/ml PA for 5 h) was analyzed for 4 days by measuring the dilution of intracellular fluorescence dye CFSE in dividing cells. LeTx temporarily inhibited cell proliferation of both U937 and THP-1 cells for up to 2 and 3 days, respectively (Fig. 1A). Cell cycle analysis using PI chromosome staining showed that number of cells in G0-G1 phase of cell cycle was increased, whereas numbers in S and G2-M phase were greatly diminished in LeTx-treated THP-1 and U937 cells (Fig. 1B). LeTx Causes a Rapid Down-regulation of Cyclin D1, Cyclin D2, and ChK1 mRNA Levels through MEK1 Inhibition—To identify signaling molecules involved in the LeTx-induced cell cycle arrest, we examined 25 different cell cycle-related proteins using Kinetworks™ multi-immunoblotting analysis in LeTx-treated THP-1 cells. Among them, protein levels of cyclin D1 and Chk1 were most significantly diminished by LeTx (Fig. 2A). Both protein and mRNA levels of cyclin D1 rapidly dropped in 3 h and maximally diminished within 6 h; whereas, the decrease in protein and mRNA levels of Chk1 were less dramatic and maximally decreased 24–48 h after an LeTx treatment (Fig. 2, B and C). The rapid decrease of cyclin D1 in mRNA and protein levels was likely due to inhibition by LF at the transcriptional levels and the short half-life of the mRNA and protein. It is unlikely that cyclin D1 was directly targeted by LF, because cells treated with the proteasome inhibitor MG132 (data not shown) or stably transfected with proteasome-mediated degradation-defected mutant cyclin D1 (cyclin D1T286A) were resistant to LeTx-induced cyclin D1 down-regulation (Fig. 2D). Also cyclin D1 mRNA levels in cells treated with the transcription inhibitor actinomycin D were down-regulated with the same rate in both non-treated and LeTx-treated cells (data not shown). Because LeTx cleaves the N-terminal end of MEKs and inhibits most downstream MAPKs, we examined whether inhibition of MAPKs could cause cell cycle arrest in THP-1 cells. Treatment of the MEK1 inhibitor U0126 (10 μm) for 24 h caused accumulation of cells at G0-G1 phase and prevented entering into S phase; whereas, the p38 MAPK inhibitor SB202190 (SB, 10 μm) had no effects on S phase progress, but it significantly prevented cells entering G2 phase (Fig. 3A). Treatment of both U0126 and SB additively blocked cell cycle progression. A JNK inhibitor (10 μm) had no effect on cell cycle progress of THP-1 cells. These results indicate that LeTx prevents cells entering S phase and G2 phase by inhibiting ERK1/2 and p38 MAPK, respectively. Similar results were detected in U937 cells. When THP-1 cells were treated with U0126, SB, or JNK inhibitor, only U0126 induced down-regulation of both cyclin D1 and Chk1, suggesting that the down-regulation of both cyclin D1 and ChK1 by LF was at least in part mediated through inhibiting MEK1 (Fig. 3B). In addition to cyclin D1, mRNA levels of cyclin D2 were similarly down-regulated by U0126 and LeTx, but not by SB or JNK inhibitor. LeTx had no effects on mRNA levels of cyclin B and cyclin E (Fig. 3C). Inhibition of MAPKs and TNF Production in Response to LPS by LeTx Were Permanent in Non-proliferating Cells—We have previously shown that LF reside inside cells and continuously cleaves MEK1 for prolong periods (∼4–5 days) in murine macrophage cell line RAW246.7 cells (33Ha S.D. Ng D. Lamothe J. Valvano M.A. Han J. Kim S.O. J. Biol. Chem. 2007; 282: 26275-26283Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). To examine the duration of LF activity in non-proliferating cells, PBMCs were briefly exposed to LeTx for 5 h, and cells were cultured in fresh media for up to 6 days. Each day, LeTx-exposed and non-exposed cells were treated with LPS (1 μg/ml) for 6 h, and TNF production was measured using a TNF bioassay as previously described (34Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6401) Google Scholar) and MEK-1 N-terminal cleavage was examined using Western blots. Throughout the cell culture period, a brief exposure of LeTx continuously cleaved MEK1 (Fig. 4A, upper panel) and prevented TNF production in responses to LPS (Fig. 4A, middle panel). Because PBMCs are a heterogeneous population of cells, including monocytes, T cells, and B cells, we further characterized TNF-producing cells in PBMCs and found that almost all TNF-producing cells in day 1 were CD13+ myeloids and in day 6 were mostly CD3+ lymphocytes (data not shown). Most of the CD13+ cells were short-lived and did not survive after 24-h culture in both LeTx-treated and non-treated PBMCs (data not shown). To specifically examine in non-proliferating macrophages, mouse peritoneal macrophages were isolated from C57BL/6 mice harboring LeTx-resistant macrophages (17Kim S.O. Jing Q. Hoebe K. Beutler B. Duesbery N.S. Han J. J. Biol. Chem. 2003; 278: 7413-7421Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and treated with LeTx. MEK1 was continuously cleaved by a brief exposure of LeTx for the next 6 days (Fig. 4A, lower panel) by when cells started dying by senescence. TNF production in response to LPS was also abolished for the next 6 days (data not shown). Cell Cycle Inhibition by LeTx Was Recovered in the Absence of ERK1/2 Activity in Monocytic Cells—The effect of LeTx is almost permanent in non-proliferating cells. If LeTx causes cell cycle arrest, the effect of LeTx should be permanent in these cells. However, when we examined MEK1 N-terminal cleavage and TNF production in response to LPS in LeTx-treated THP-1 cells, both MEK1 and TNF production were returned to normal levels in ∼5 days after LeTx treatments (Fig. 4B). Based on Western blot analysis, LF inside cells was slowly degraded over >3 days (Fig. 4C). LF was no longer detectible after 4 days of a LeTx treatment. We then examined whether cell cycle arrest by LeTx was alleviated before MEK1/ERKs activities were recovered. In fact, THP-1 cells resumed cell cycle before the third day even when LF was readily detectible by Western blots (Fig. 4C), and TNF production in response to LPS was completely blocked (Fig. 4B, lower panel). Cell cycle arrest was released after the second day and returned to almost at normal levels after 3 days of a LeTx treatment (Fig. 4D). These results indicate that LeTx-exposed THP-1 cells underwent cell cycle progression in the absence of MAPK activities. Cells Pre-exposed with LeTx Were Protected from Cell Cycle Arrest by Subsequent LeTx Treatment through Activation of PI3K—If cells adaptively adjusted themselves to LeTx and resumed cell cycle even in the absence of MEK activities, these cells should continue to proliferate even in the presence of further LeTx challenges. To examine whether these cells were indeed resistant to LeTx-induced cell cycle arrest, we briefly pre-treated cells for 5 h with LeTx and then treated them again 2 days after the pretreatment. Consistent with previous results, numbers of cells at S and G2-M phases were dramatically reduced by LeTx in 2 days, but LeTx-pretreated cells were resistant to cell cycle arrest by a subsequent LeTx-treatment (Fig. 5A, left panel). To examine whether th" @default.
• W2023802571 created "2016-06-24" @default.
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• W2023802571 date "2007-12-01" @default.
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• W2023802571 title "Critical Role of the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase-3β Signaling Pathway in Recovery from Anthrax Lethal Toxin-induced Cell Cycle Arrest and MEK Cleavage in Macrophages" @default.
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