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• W2017101260 abstract "The Pasteurella multocida toxin (PMT) is highly mitogenic and has potential carcinogenic properties. PMT causes porcine atrophic rhinitis that is characterized by bone resorption and loss of nasal turbinates, but experimental nasal infection also leads to excess proliferation of bladder epithelial cells. PMT acts intracellularly and activates phospholipase C-linked signals and MAPK pathways via the heterotrimeric Gαq and Gα12/13 proteins. We found that PMT induces activation of STAT proteins, and we identified STAT1, STAT3, and STAT5 as new targets of PMT-induced Gαq signaling. Inhibition of Janus kinases completely abolished STAT activation. PMT-dependent STAT phosphorylation remained constitutive for at least 18 h. PMT caused down-regulation of the expression of the suppressor of cytokine signaling-3, indicating a novel mechanism to maintain activation of STATs. Moreover, stimulation of Swiss 3T3 cells with PMT increased transcription of the cancer-associated STAT-dependent gene cyclooxygenase-2. Because constitutive activation of STATs has been found in a number of cancers, our findings offer a new mechanism for a carcinogenic role of PMT. The Pasteurella multocida toxin (PMT) is highly mitogenic and has potential carcinogenic properties. PMT causes porcine atrophic rhinitis that is characterized by bone resorption and loss of nasal turbinates, but experimental nasal infection also leads to excess proliferation of bladder epithelial cells. PMT acts intracellularly and activates phospholipase C-linked signals and MAPK pathways via the heterotrimeric Gαq and Gα12/13 proteins. We found that PMT induces activation of STAT proteins, and we identified STAT1, STAT3, and STAT5 as new targets of PMT-induced Gαq signaling. Inhibition of Janus kinases completely abolished STAT activation. PMT-dependent STAT phosphorylation remained constitutive for at least 18 h. PMT caused down-regulation of the expression of the suppressor of cytokine signaling-3, indicating a novel mechanism to maintain activation of STATs. Moreover, stimulation of Swiss 3T3 cells with PMT increased transcription of the cancer-associated STAT-dependent gene cyclooxygenase-2. Because constitutive activation of STATs has been found in a number of cancers, our findings offer a new mechanism for a carcinogenic role of PMT. Pasteurella multocida is a Gram-negative bacterium that causes serious diseases in animals and humans. It has been isolated from chronic respiratory infections in various animals, and it is principally associated with atrophic rhinitis in pigs, a disease characterized by bone loss of nasal turbinates and inflammation of the nasal mucosa (1Kimman T.G. Löwik C.W.G.M. van de Wee-Pals L.J.A. Thesingh C.W. Defize P. Kamp E.M. Bijvoet O.L.M. Infect. Immun. 1987; 55: 2110-2116Crossref PubMed Google Scholar). Recently, the pathogen has been linked to cancer development, as natural infection with P. multocida or injection of its major virulence factor P. multocida toxin (PMT) 2The abbreviations used are: PMT, P. multocida toxin; STAT, signal transducer and activator of transcription; SOCS, suppressor of cytokine signaling; COX-2, cyclooxygenase-2; IFN-γ, interferon-γ; LHRE, lactogenic hormone-responsive element; IRF7, interferon regulatory factor 7; MAP, mitogen-activated protein; GST, glutathione S-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF, mouse embryonic fibroblast; qPCR, quantitative PCR. 2The abbreviations used are: PMT, P. multocida toxin; STAT, signal transducer and activator of transcription; SOCS, suppressor of cytokine signaling; COX-2, cyclooxygenase-2; IFN-γ, interferon-γ; LHRE, lactogenic hormone-responsive element; IRF7, interferon regulatory factor 7; MAP, mitogen-activated protein; GST, glutathione S-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF, mouse embryonic fibroblast; qPCR, quantitative PCR. causes proliferation of the bladder epithelium in the absence of an inflammatory reaction (2Ward P.N. Miles A.J. Sumner I.G. Thomas L.H. Lax A.J. Infect. Immun. 1998; 66: 5636-5642Crossref PubMed Google Scholar).PMT is a 146-kDa protein toxin that after entering the cell acts through activation of intracellular signaling cascades related to cell growth (reviewed in Ref. 3Lax A.J. Grigoriadis A.E. Int. J. Med. Microbiol. 2001; 291: 261-268Crossref PubMed Scopus (36) Google Scholar). It is a potent mitogen in vivo and in vitro and is therefore thought to have high carcinogenic potential (4Lax A.J. Pullinger G.D. Baldwin M.R. Harmey D. Grigoriadis A.E. Lakey J.H. Int. J. Med. Microbiol. 2004; 293: 505-512Crossref PubMed Scopus (30) Google Scholar, 5Lax A.J. Nat. Rev. Microbiol. 2005; 3: 343-349Crossref PubMed Scopus (109) Google Scholar). Under in vitro conditions, picomolar concentrations of PMT are sufficient to promote re-entry of quiescent cells into the cell cycle (6Higgins T.E. Murphy A.C. Staddon J.M. Lax A.J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4240-4244Crossref PubMed Scopus (47) Google Scholar, 7Rozengurt E. Higgins T. Chanter N. Lax A.J. Staddon J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 123-127Crossref PubMed Scopus (108) Google Scholar). PMT exerts its function through activation of the heterotrimeric G proteins Gαq and Gα12/13 (8Orth J.H. Lang S. Taniguchi M. Aktories K. J. Biol. Chem. 2005; 280: 36701-36707Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), although the exact mechanism of activation has not yet been elucidated. Eventually signals related to phospholipase Cβ (9Wilson B.A. Ponferrada V.G. Vallance J.E. Ho M.F. Infect. Immun. 1999; 67: 80-87Crossref PubMed Google Scholar), the small GTPase RhoA (10Lacerda H.M. Lax A.J. Rozengurt E. J. Biol. Chem. 1996; 271: 439-445Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11Dudet L.I. Chailler P. Dubreuil D. Martineau-Doize B. J. Cell. Physiol. 1996; 168: 173-182Crossref PubMed Scopus (31) Google Scholar), and mitogen-activated protein (MAP) kinase cascades are activated (10Lacerda H.M. Lax A.J. Rozengurt E. J. Biol. Chem. 1996; 271: 439-445Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Although some of the pathways that are activated by PMT are linked to known oncogenes, such as RhoA, Src, or Erk kinases, to date there are no data available whether chronic P. multocida infection may facilitate oncogenesis.Even though a number of viruses are now known to be connected to cancer, the relationship between bacterial pathogens and carcinogenesis has been established only recently. The discovery that chronic infection with Helicobacter pylori can cause gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma suggested that bacteria may have a certain impact on the development of cancer (12Court M. Robinson P.A. Dixon M.F. Crabtree J.E. J. Infect. Dis. 2002; 186: 1348-1352Crossref PubMed Scopus (42) Google Scholar, 13Nardone G. Rocco A. Malfertheiner P. Aliment. Pharmacol. Ther. 2004; 20: 261-270Crossref PubMed Scopus (118) Google Scholar, 14Uemura N. Okamoto S. Yamamoto S. Matsumura N. Yamaguchi S. Yamakido M. Taniyama K. Sasaki N. Schlemper R.J. N. Engl. J. Med. 2001; 345: 784-789Crossref PubMed Scopus (3612) Google Scholar), and since then other pathogens have been discussed to cause cancer (15Lax A.J. Thomas W. Trends Microbiol. 2002; 10: 293-299Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). One possible connection between infections caused by bacteria and cancer is chronic inflammation that supports anti-apoptotic or chronic proliferative conditions eventually allowing tumor onset or promotion (5Lax A.J. Nat. Rev. Microbiol. 2005; 3: 343-349Crossref PubMed Scopus (109) Google Scholar). Cytotoxic necrotizing factor from Escherichia coli, for example, is known to up-regulate the expression of proteins of the Bcl2 family that have an anti-apoptotic effect (16Oswald E. Nougayrede J.P. Taieb F. Sugai M. Curr. Opin. Microbiol. 2005; 8: 83-91Crossref PubMed Scopus (122) Google Scholar), and H. pylori stimulates the production of pro-inflammatory cytokines as well as the expression of cyclooxygenase-2 (COX-2) (17Juttner S. Cramer T. Wessler S. Walduck A. Gao F. Schmitz F. Wunder C. Weber M. Fischer S.M. Schmidt W.E. Wiedenmann B. Meyer T.F. Naumann M. Hocker M. Cell Microbiol. 2003; 5: 821-834Crossref PubMed Scopus (77) Google Scholar) that is well known for its rapid up-regulation in response to cytokines and to inflammation (18O'Banion M.K. Winn V.D. Young D.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4888-4892Crossref PubMed Scopus (800) Google Scholar).Binding of cytokines to their cell surface receptors activates receptor-associated members of the Janus kinases (JAKs), JAK1, JAK2, JAK3, and Tyk2 and leads to their auto-phosphorylation and the subsequent phosphorylation of tyrosine residues on the cytoplasmic tails of the receptors. These phosphotyrosines can then serve as docking sites for Src homology 2 domain-containing proteins, such as signal transducer and activator of transcription proteins (STATs). STATs are latent cytoplasmic transcription factors that translocate to the nucleus in their phosphorylated and dimerized state (19Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene (Amst.). 2002; 285: 1-24Crossref PubMed Scopus (894) Google Scholar). It is now clear that STAT proteins are also potential targets of oncogenes (20Garcia R. Jove R. J. Biomed. Sci. 1998; 5: 79-85Crossref PubMed Scopus (183) Google Scholar). From knock-out studies it was possible to allocate most STAT proteins to specific cytokine-induced signaling pathways. Stat3 is the only gene whose knock-out leads to embryonic lethality (21Takeda K. Noguchi K. Shi W. Tanaka T. Matsumoto M. Yoshida N. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3801-3804Crossref PubMed Scopus (1089) Google Scholar), although this cannot be sufficiently explained by its functions in gp130-like receptor signaling. It is generally accepted that aberrant STAT activation contributes to malignant transformation of cells through promotion of cell cycle progression and cell survival (20Garcia R. Jove R. J. Biomed. Sci. 1998; 5: 79-85Crossref PubMed Scopus (183) Google Scholar). Oncogenic activity is particularly associated with constitutive activation of STAT3 and STAT5, and in various human cancers STAT3 is persistently activated (20Garcia R. Jove R. J. Biomed. Sci. 1998; 5: 79-85Crossref PubMed Scopus (183) Google Scholar, 22Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2472) Google Scholar). In cell culture experiments, constitutive STAT3 activation can be connected to growth deregulation that enhances transformation or blocks apoptosis of cells. Transcriptional events that are controlled by STAT proteins and that might play a role in cell transformation are cyclin D1, cyclin D2, Bcl-XL, c-Myc, and p21 WAF (23Bowman T. Garcia R. Turkson J. Jove R. Oncogene. 2000; 19: 2474-2488Crossref PubMed Scopus (1576) Google Scholar, 24Martino A. Holmes J.H. Lord J.D. Moon J.J. Nelson B.H. J. Immunol. 2001; 166: 1723-1729Crossref PubMed Scopus (95) Google Scholar).EXPERIMENTAL PROCEDURESCell Culture—Mouse embryonic fibroblasts (MEFs) derived from Gαq/Gα11 or Gα12/Gα13 gene-deficient or wild-type (WT) mice were cultured as described previously (25Zywietz A. Gohla A. Schmelz M. Schultz G. Offermanns S. J. Biol. Chem. 2001; 276: 3840-3845Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 26Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 27Offermanns S. Simon M.I. Oncogene. 1998; 17: 1375-1381Crossref PubMed Scopus (41) Google Scholar).Dual Luciferase Assays—Activation of STAT proteins was measured by luciferase activity of 293-derived Phoenix eco cells transfected with pGRR5-Luc for general STAT activation (28Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar), pLHRE-Luc (lactogenic hormone-responsive element) for STAT5 activation (29Sotiropoulos A. Moutoussamy S. Renaudie F. Clauss M. Kayser C. Gouilleux F. Kelly P.A. Finidori J. Mol. Endocrinol. 1996; 10: 998-1009Crossref PubMed Scopus (125) Google Scholar), or pIRF7-Luc (Interferon Regulatory Factor) for STAT1 activation (30Dumoutier L. Tounsi A. Michiels T. Sommereyns C. Kotenko S.V. Renauld J.C. J. Biol. Chem. 2004; 279: 32269-32274Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). As an internal control, the pRL-TK vector (Promega) was used. Phoenix cells were seeded in 96-well plates at 5 × 104 cells/well and transfected with 100 ng of the reporter cDNAs using calcium phosphate transfection. After 4 h medium was changed, and cells were either preincubated for 1 h with 500 nm JAK inhibitor I (Calbiochem) or stimulated directly with 2 μg/ml PMTWT or PMTC1165S. Twenty four hours after transfection, cells were lysed, and luciferase assays were performed using the dual luciferase reporter assay kit (Promega) and a Microlumat Plus luminometer (Berthold, Pforzheim, Germany).Further transfections included the use of MEF cells and were performed using the nucleofection system (Amaxa Biosystems, Cologne, Germany) according to the general protocol for nucleofection of mouse embryonic fibroblasts. Briefly, 1 × 106 cells were resuspended in 100 μl of Nucleofector Solution (MEF2) together with 5 μg of reporter plasmids and then electroporated. After nucleofection, cells were recovered by addition of 800 μl of Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum and seeded into 8 wells of a 96-well plate. After 4 h, inhibitors or toxins were added, and the cells were treated as described before.Whole Cell Extracts and Immunoprecipitation—One confluent T175 flask of Swiss 3T3 cells was stimulated overnight with 2 μg/ml PMT or left untreated. Cells were detached by using a cell scraper. After washing with cold phosphate-buffered saline, cells were lysed in 750 μl of Nonidet P-40 lysis buffer containing 1% Nonidet P-40, 150 mm NaCl, 20 mm Tris (pH 7.4), 10 mm NaF, 1 mm EDTA, 1 mm MgCl2, 1 mm Na3VO4, and 10% glycerol for 45 min at 4 °C. After centrifugation, lysates were either immunoprecipitated overnight with 1.5 μg of JAK1 or JAK2 antibody (Cell Signaling Technology and Upstate Biotechnology, Inc., respectively) bound to protein G-Sepharose (Sigma). Samples were separated on 10% SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies specific for the tyrosine-phosphorylated forms of JAK1 and JAK2 (Cell Signaling Technology, Danvers, MA) at a dilution of 1:1000, followed by incubation with horseradish peroxidase-coupled anti-rabbit antibody (Cell Signaling Technology, Danvers, MA) at a dilution of 1:1000 and detection by enhanced chemiluminescence.Cytosolic and Nuclear Extracts—Cytosolic and nuclear extracts from human Swiss 3T3 cells were prepared as described (31Haspel R.L. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10188-10193Crossref PubMed Scopus (165) Google Scholar). Briefly, cells were detached with a cell scraper in cold phosphate-buffered saline. Washed cells were pelleted and resuspended in cold hypotonic lysis buffer (20 mm Hepes (pH 7.9), 10 mm KCl, 1 mm EDTA, 10% glycerol, 0.2% Nonidet P-40, protease inhibitors) and allowed to swell on ice for 10 min. After centrifugation for 1 min, supernatants containing cytosolic fractions were used for direct immunoblotting or frozen at -80 °C. Nuclear pellets were resuspended in cold high salt lysis buffer (20 mm Hepes (pH 7.9), 10 mm KCl, 1 mm EDTA, 20% glycerol, 420 mm NaCl, 0.2% Nonidet P-40, protease inhibitors). Nuclear extracts were collected after a 10-min centrifugation at maximum speed, and the protein content was determined by Bradford protein assay.Protein samples from cytosolic or nuclear fractions (50 μg) were migrated on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was blocked with TBST containing 5% milk powder for 1 h at room temperature and then incubated overnight at 4 °C with the appropriate STAT antibodies (Cell Signaling Technology, Danvers, MA) at a dilution of 1:1000. An anti-rabbit IgG horseradish peroxidase-linked antibody (Cell Signaling Technology) was used at a dilution of 1:1000, and signals were detected by enhanced chemiluminescence.Reverse Transcription-PCR—Serum-starved mouse embryonic fibroblasts were treated with or without PMTWT, PMTC1165S (each 1 μg/ml, 18 h), IFN-γ (20 ng/ml, 2 h). RNA was extracted with RNeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Total RNA was quantified spectrophotometrically. Samples were stored at -80 °C until analysis. Then cDNA was prepared using QuantiTect (Qiagen, Hilden, Germany). In brief, 700 ng of total RNA was reverse-transcribed (30 min, 42 °C) in a total volume of 20 μl. The reaction was stopped by heating to 93 °C for 3 min.Aliquots of the cDNA were used for quantitative PCR analysis with the Mx3000P qPCR system (Stratagene) and the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) with the following primers: GAPDH, sense, 5′-tcccattcttccacctttgatg-3′, and antisense, 5′-gtccaccaccctgttgctgta-3′; S12, sense, 5′-ggcatagctgctggaggtgtaa-3′, and antisense, 5′-gggcttggcgcttgtctaa-3′; COX-2, sense, 5′-aaaggcctccattgaccagag-3′, and antisense, 5′-gccatttccttctctcctgtaagttc-3′; SOCS-3, sense, 5′-cgcgggcacctttcttatc-3′, and antisense, 5′-tcacactggatgcgtaggttct-3′. The results were analyzed using the MxPro qPCR software (Stratagene). For further analysis results were exported to Excel (Microsoft) and calculated by qCalculator 1.0 (32Gilsbach R. Kouta M. Bonisch H. Bruss M. BioTechniques. 2006; 40: 173-177Crossref PubMed Scopus (118) Google Scholar). All results were normalized with respect to the housekeeping genes (Gapdh and S12).Expression and Purification of PMT Proteins—Recombinant PMT and PMTC1165S were expressed as glutathione S-transferase (GST) fusion proteins and purified according to the manufacturer's instructions (GE Healthcare). In brief, GST fusion proteins were isolated by affinity chromatography with glutathione-Sepharose, followed by proteolytic cleavage using 3.25 units of thrombin/mg of recombinant GST fusion protein. Thrombin was removed by incubation with benzamidine-Sepharose (Amersham Biosciences) (33Busch C. Orth J. Djouder N. Aktories K. Infect. Immun. 2001; 69: 3628-3634Crossref PubMed Scopus (55) Google Scholar).RESULTSPMT Activates Transcriptional Activity of STAT Proteins— PMT is known to interact with various intracellular host signaling pathways to exert its mitogenic activity. To investigate whether PMT is able to activate latent cytoplasmic transcription factors of the STAT family, we transiently transfected human 293-derived Phoenix cells with the STAT-specific reporter plasmid pGRR5-Luc (28Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). Transcriptional activity was assessed in a luciferase assay after overnight incubation with 2 μg/ml of either PMTWT or a biologically inactive mutant PMTC1165S (2Ward P.N. Miles A.J. Sumner I.G. Thomas L.H. Lax A.J. Infect. Immun. 1998; 66: 5636-5642Crossref PubMed Google Scholar, 33Busch C. Orth J. Djouder N. Aktories K. Infect. Immun. 2001; 69: 3628-3634Crossref PubMed Scopus (55) Google Scholar). Fig. 1A shows that stimulation of cells with PMTWT leads to an increase in activity of about 4-fold when compared with untreated cells. To exclude unspecific activation caused by the preparation of recombinant PMT, an inactive mutant, PMTC1165S, was employed. Because this mutant toxin is not able to activate STAT activity, the increase in luciferase activity through wild-type PMT is a specific effect of this toxin.To further investigate PMT-induced STAT activation, luciferase assays were performed in a dose-dependent manner (Fig. 1B). Cells were incubated overnight with PMT at concentrations ranging from 0.005 to 5 μg/ml. Already at the lowest concentrations, a small but reproducible increase in luciferase activity could be monitored. STAT activation mounted rapidly with increasing PMT concentrations, and more than half-maximal induction was attained at 0.05 μg/ml PMT.PMT-mediated STAT Activation Depends on Gαq Protein— Because it is well known that PMT can signal via two families of heterotrimeric G proteins, Gα12/13 or Gαq, we wanted to determine whether STAT activation by PMT depends on either of these proteins. For that purpose we made use of a mouse embryonic fibroblast (MEF) cell line that is deficient for Gα12/13 (25Zywietz A. Gohla A. Schmelz M. Schultz G. Offermanns S. J. Biol. Chem. 2001; 276: 3840-3845Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 26Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 27Offermanns S. Simon M.I. Oncogene. 1998; 17: 1375-1381Crossref PubMed Scopus (41) Google Scholar, 34Orth J. Lang S. Aktories K. J. Biol. Chem. 2004; 279: 34150-34155Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The STAT reporter construct pGRR5-Luc was transfected into these cells using the nucleofection system (Amaxa Biosystems). Freshly transfected cells were grown in medium containing 0.5% fetal bovine serum and were stimulated with PMT overnight (2 μg/ml) or left untreated. In Gα12/13-deficient cells PMT-dependent luciferase activity could still be monitored, and an increase in transcriptional activity of about 5-fold compared with the negative control was observed (Fig. 2). This result indicates that Gα12/13 is not necessary for PMT-mediated induction of STAT activation. Interestingly, when we preincubated these cells with the Gαq/11-specific inhibitor YM-254890 (35Takasaki J. Saito T. Taniguchi M. Kawasaki T. Moritani Y. Hayashi K. Kobori M. J. Biol. Chem. 2004; 279: 47438-47445Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar) before addition of PMT, STAT activity was completely abolished. These data suggest that Gαq but not Gα12/13 is essential for PMT-mediated STAT activation. To corroborate this result, we repeated the described experiment in Gαq/11-deficient MEF cells to determine their ability to activate STAT proteins after PMT incubation, and we found that the Gαq/11 gene knock-out does completely abrogate the activation of STAT proteins in luciferase assays. We therefore conclude that the heterotrimeric Gαq protein is necessary and sufficient for PMT-induced transcriptional activity of STAT proteins.FIGURE 2Analysis of STAT activation in G protein-deficient cells. Gα12/13- and Gαq/11-deficient (inset) mouse embryonic fibroblasts were assayed for STAT transcriptional activity using the pGRR5-Luc plasmid and pRL-TK as a control. Cells were either preincubated with the Gαq/11 specific inhibitor YM-254890 for 60 min or left untreated and then intoxicated overnight with 2 μg/ml PMTWT. Results were obtained from one representative experiment performed in triplicate ± S.D. RLU, relative light units; unstim, unstimulated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nuclear Extracts Show Constitutive Activation of STAT1, STAT3, and STAT5—Because the pGRR5-Luc reporter plasmid contains the γ-interferon-activated site, a DNA-binding site recognized by all STAT proteins, we wanted to investigate in more detail which STAT proteins were activated by PMT. Thus we prepared cytosolic and nuclear extracts from Swiss 3T3 cells. To determine whether STAT proteins were expressed in these cells, cytoplasmic extracts were prepared, and proteins were separated on 10% SDS-PAGE, transferred to membranes, and blotted with antibodies specific for STAT1, STAT3, and STAT5. All three proteins and their known splice variants could be detected (Fig. 3A).FIGURE 3Analysis of STAT protein activation and phosphorylation through PMT. A, Swiss 3T3 cells were starved in serum-free medium for 4 h before addition of 2 μg/ml PMTWT for overnight incubation. Cytosolic extracts of Swiss 3T3 were prepared, and proteins were separated by 10% SDS-PAGE. Expression of STAT1, STAT3, and STAT5 proteins was detected using specific antibodies and enhanced chemiluminescence. B, nuclear extracts of Swiss 3T3 cells were prepared, and 50 μg of protein were loaded onto a 10% SDS-polyacrylamide gel. Proteins were separated by SDS-PAGE, and STAT proteins were detected in their activated states by immunoblotting using antibodies specific for Tyr(P)-701 STAT1, Tyr(P)-705 STAT3, Ser(P)-727 STAT3, and Tyr(P)-674 STAT5 as indicated. C, quantification of STAT5 transcriptional activity by a luciferase assay. Phoenix cells were transfected with pLHRE-Luc and pRL-TK, and cells were stimulated overnight with 2 μg/ml PMTWT or left untreated. Results were obtained from one representative experiment performed in triplicate ± S.D. D, STAT1 transcriptional activity quantified by a luciferase assay. Phoenix cells were transfected with pIRF7-Luc and pRL-TK, and cells were stimulated overnight with 2 μg/ml PMTWT or left unstimulated (unstim). Results were obtained from one representative experiment performed in triplicate ± S.D. E, time course of PMT-dependent STAT3 phosphorylation. Swiss 3T3 cells were left untreated or stimulated with 2 μg/ml PMTWT for the times indicated. Cytosolic extracts were prepared; proteins were separated by 10% SDS-PAGE, and tyrosine phosphorylation of STAT3 was detected by immunoblotting and enhanced chemiluminescence. As a loading control the amount of β-actin was assessed using a specific antibody and enhanced chemiluminescence. F, quantification of STAT3 tyrosine phosphorylation. The intensity of the Tyr(P)-STAT3 bands obtained in Fig. 3E was determined using the MultiGauge software (Fujifilm, Düsseldorf, Germany). The figure displays one representative example of three independent experiments. RLU, relative light units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Once STAT proteins are activated and tyrosine-phosphorylated in the cytosol, they dimerize and translocate to the nucleus. To monitor nuclear STAT proteins, Swiss 3T3 cells were starved, and nuclear extracts were prepared from unstimulated cells and cells that had been incubated overnight with 2 μg/ml PMT. Equal amounts of protein were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with specific antibodies recognizing STAT proteins in their tyrosine- or serine-phosphorylated state, respectively. As can be seen in Fig. 3B, PMT is able to induce activation of STAT1, STAT3, and STAT5 as detected by phosphorylation of tyrosine 701 of STAT1 and tyrosine phosphorylation of residue 694 of STAT5. In the case of STAT3 phosphorylation of tyrosine 705 and serine 727 was detected, whereas extracts from unstimulated cell extracts showed only a weak activation for all STAT proteins examined.To further strengthen these data, we performed luciferase assays with reporter constructs containing the interferon regulatory factor-7 site that is specific for STAT1 and STAT2 (pIRF7-Luc (30Dumoutier L. Tounsi A. Michiels T. Sommereyns C. Kotenko S.V. Renauld J.C. J. Biol. Chem. 2004; 279: 32269-32274Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar)) or the lactogenic hormone-responsive element that is specific for STAT5 (pLHRE-Luc (29Sotiropoulos A. Moutoussamy S. Renaudie F. Clauss M. Kayser C. Gouilleux F. Kelly P.A. Finidori J. Mol. Endocrinol. 1996; 10: 998-1009Crossref PubMed Scopus (125) Google Scholar)). Transfected Phoenix cells were intoxicated overnight with 2 μg/ml PMT or left unstimulated, and cell lysates were then assayed for luciferase activity. As Fig. 3, C and D, shows, an increase in PMT-induced luciferase activity of about 5-fold could be detected with both reporter plasmids, confirming the specific activation of these two transcription factors.Time Course of STAT3 Activation—Because cytokine-induced STAT activation is a transient event that occurs within minutes after binding of a cytokine to its cognate receptor, we wanted to determine the kinetics of PMT-induced STAT3 activation. It is known that there is delay of about 1 h between addition of PMT and the earliest cellular events caused by the receptor-mediated uptake of PMT (7Rozengurt E. Higgins T. Chanter N. Lax A.J. Staddon J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 123-127Crossref PubMed Scopus (108) Google Scholar, 36Wilson B.A. Aminova L.R. Ponferrada V.G. Ho M. Infect. Immun. 2000; 68: 4531-4538Crossref PubMed Scopus (28) Google Scholar).To study STAT3 tyrosine phosphorylation in a time-dependent manner, Swiss 3T3 cells were starved overnight in the absence of fetal bovine serum to suppress serum-induced STAT activation. Cells were left unstimulated or were stimulated with 2 μg/ml PMTWT, and cell lysates of stimulated cells were prepared at the indicated times (Fig. 3E). One additional time point was taken after overnight stimulation with PMTWT, corresponding to an 18-h incubation. STAT activation was then analyzed by Western blotting. Fig. 3, E and F, illustrate that PMT-induced STAT3 activation can be seen as early as 2 h after stimulation, with a peak in activity after 3 h. The signal then slightly decreases but remains stable on a lower level and can still be detected at the same intensity 18" @default.
• W2017101260 created "2016-06-24" @default.
• W2017101260 creator A5029891202 @default.
• W2017101260 creator A5072111511 @default.
• W2017101260 creator A5084370353 @default.
• W2017101260 date "2007-02-01" @default.
• W2017101260 modified "2023-10-12" @default.
• W2017101260 title "Modulation of Host Cell Gene Expression through Activation of STAT Transcription Factors by Pasteurella multocida Toxin" @default.
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