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• W2015540930 abstract "The clinical syndrome of acute liver failure produced by fulminant viral hepatitis can be reproduced in mice by infection with murine hepatitis virus strain 3 (MHV-3). Although it is clear that MHV-3-induced hepatitis depends upon macrophage activation and the expression of a specific prothrombinase,fgl-2, the signaling pathways involved in virally stimulated cell activation are unclear. Since we had previously found that MHV-3 induces the tyrosine phosphorylation of cellular proteins, we investigated the roles of the mitogen-activated protein kinase (MAPK) proteins. In a series of Western blots, immunoprecipitation and in vitro kinase assay studies, we found that both the extracellular signal-related kinase (ERK) and p38 MAPK proteins are tyrosine-phosphorylated and activated following exposure of murine peritoneal exudative macrophages (PEM) to MHV-3. Although p38 phosphorylation and activity are induced soon after MHV-3 exposure, peaking by 1–5 min, ERK phosphorylation and activity increase more gradually, peaking at 20–30 min and gradually fading thereafter. Interestingly, whereas selective p38 inhibition with SB203580 (1–20 μm) abolished the virally stimulated induction of fgl-2 mRNA, protein, and functional activity, selective ERK inhibition with PD98059 (1–50 μm) limitedfgl-2 functional activity but had little to no effect onfgl-2 mRNA or protein levels. Moreover, whereas inhibition of ERK had no effect on p38 activity, p38 inhibition consistently increased MHV-3-induced ERK activity. To ensure that these pathways were relevant in vivo, MHV-3 was injected intraperitoneally, and peritoneal exudative macrophages were collected. Again, MHV-3 exposure led to increased p38 and ERK tyrosine phosphorylation. These data argue that MHV-3 induces tightly interconnected ERK and p38 MAPK cascades in the macrophage bothin vitro and in vivo. Although the ERK and p38 MAPK proteins have discordant effects at the level of fgl-2expression, both converge at the level of its activity, suggesting that targeted MAPK inhibition may ultimately be useful in the modulation of viral hepatitis. The clinical syndrome of acute liver failure produced by fulminant viral hepatitis can be reproduced in mice by infection with murine hepatitis virus strain 3 (MHV-3). Although it is clear that MHV-3-induced hepatitis depends upon macrophage activation and the expression of a specific prothrombinase,fgl-2, the signaling pathways involved in virally stimulated cell activation are unclear. Since we had previously found that MHV-3 induces the tyrosine phosphorylation of cellular proteins, we investigated the roles of the mitogen-activated protein kinase (MAPK) proteins. In a series of Western blots, immunoprecipitation and in vitro kinase assay studies, we found that both the extracellular signal-related kinase (ERK) and p38 MAPK proteins are tyrosine-phosphorylated and activated following exposure of murine peritoneal exudative macrophages (PEM) to MHV-3. Although p38 phosphorylation and activity are induced soon after MHV-3 exposure, peaking by 1–5 min, ERK phosphorylation and activity increase more gradually, peaking at 20–30 min and gradually fading thereafter. Interestingly, whereas selective p38 inhibition with SB203580 (1–20 μm) abolished the virally stimulated induction of fgl-2 mRNA, protein, and functional activity, selective ERK inhibition with PD98059 (1–50 μm) limitedfgl-2 functional activity but had little to no effect onfgl-2 mRNA or protein levels. Moreover, whereas inhibition of ERK had no effect on p38 activity, p38 inhibition consistently increased MHV-3-induced ERK activity. To ensure that these pathways were relevant in vivo, MHV-3 was injected intraperitoneally, and peritoneal exudative macrophages were collected. Again, MHV-3 exposure led to increased p38 and ERK tyrosine phosphorylation. These data argue that MHV-3 induces tightly interconnected ERK and p38 MAPK cascades in the macrophage bothin vitro and in vivo. Although the ERK and p38 MAPK proteins have discordant effects at the level of fgl-2expression, both converge at the level of its activity, suggesting that targeted MAPK inhibition may ultimately be useful in the modulation of viral hepatitis. murine hepatitis virus strain-3 dithiothreitol extracellular signal-related kinase fetal calf serum lipopolysaccharide mitogen-activated protein myelin basic protein multiplicity of infection polyclonal antibody polyacrylamide gel electrophoresis procoagulant activity peritoneal exudative macrophages Hanks' buffered saline solution phosphate-buffered saline prostaglandin E2. The mortality rate associated with fulminant hepatitis remains in excess of 25–45%, despite the use of liver transplantation as an acceptable form of therapy (1Pappas S.C. Gastroenterol. Clin. North Am. 1995; 24: 161-173Abstract Full Text PDF PubMed Google Scholar). Studies using a model of viral hepatitis induced by infection with murine hepatitis virus strain 3 (MHV-3)1 have provided significant insights into the mechanisms underlying the pathogenesis of this disease and have suggested novel approaches to therapy (1Pappas S.C. Gastroenterol. Clin. North Am. 1995; 24: 161-173Abstract Full Text PDF PubMed Google Scholar, 2Abecassis A. Falk J.A. Makowka L. Dindzans V.J. Falk R.E. Levy G.A. J. Clin. Invest. 1987; 80: 881-889Crossref PubMed Scopus (100) Google Scholar). Fulminant hepatitis induced by this virus is characterized by the presence of sinusoidal thrombosis and associated hepatocellular necrosis (3Levy G.A. MacPhee P.J. Fung L.S. Fisher M.M. Rappaport A.M. Hepatology. 1983; 3: 964-973Crossref PubMed Scopus (34) Google Scholar, 4Levy G. Abecassis M. Rev. Infect. Dis. 1989; 11: 712-721Crossref Scopus (16) Google Scholar, 5Li C. Fung L.S. Chung S. Crow A. Myers-Mason N. Phillips M.J. Leibowitz J.L. Cole E. Ottaway C.A. Levy G. J. Exp. Med. 1992; 176: 689-697Crossref PubMed Scopus (64) Google Scholar). These findings occur concomitant with the expression of a virus-induced procoagulant molecule in the sinusoidal lining cells of the liver. This prothrombinase protein, encoded by thefgl-2 gene, has the ability to activate directly the coagulation cascade, an ability expressed as procoagulant activity (PCA) and measured by standard clotting assays (6Parr R.L. Fung L. Reneker J. Myers-Mason N. Leibowitz J.L. Levy G. J. Virol. 1995; 69: 5033-5038Crossref PubMed Google Scholar, 7Fung L.S. Neil G. Leibowitz J. Cole E.H. Chung S. Crow A. Levy G.A. J. Biol. Chem. 1991; 266: 1789-1795Abstract Full Text PDF PubMed Google Scholar). Accumulated evidence suggests that the virus-induced PCA plays a central role in the pathological changes observed in this disease. Following infection with MHV-3, hepatocellular necrosis is seen to occur in regions of sinusoidal fibrin deposition, where concomitant expression of thefgl-2 gene and its protein product is observed in the sinusoidal lining cells (8Ding J.W. Ning Q. Liu M.F. Lai A. Leibowitz J. Peltekian K.M. Cole E.H. Fung L.S. Holloway C. Marsden P.A. Yeger H. Phillips M.J. Levy G.A. J. Virol. 1997; 71: 9223-9230Crossref PubMed Google Scholar). By contrast, other organs simultaneously infected with MHV-3 fail to express Fgl-2 protein and remain uninjured (8Ding J.W. Ning Q. Liu M.F. Lai A. Leibowitz J. Peltekian K.M. Cole E.H. Fung L.S. Holloway C. Marsden P.A. Yeger H. Phillips M.J. Levy G.A. J. Virol. 1997; 71: 9223-9230Crossref PubMed Google Scholar). Importantly, pretreatment with a neutralizing monoclonal antibody directed against MHV-3-induced PCA prevents sinusoidal fibrin deposition, hepatocellular necrosis, and mortality in infected mice (5Li C. Fung L.S. Chung S. Crow A. Myers-Mason N. Phillips M.J. Leibowitz J.L. Cole E. Ottaway C.A. Levy G. J. Exp. Med. 1992; 176: 689-697Crossref PubMed Scopus (64) Google Scholar). Thus, there is good reason to conclude that an fgl-2up-regulation is essential to the lethal hepatitis induced by MHV-3. These considerations become all the more relevant with the recent discovery of a human fgl-2 analogue (9Ruegg C. Pytela R. Gene (Amst.). 1995; 160: 257-262Crossref PubMed Scopus (36) Google Scholar), which may contribute to inducible endothelial PCA (10Sekiya F. Usui H. Inoue K. Fukudome K. Morita T. J. Biol. Chem. 1994; 269: 32441-32445Abstract Full Text PDF PubMed Google Scholar, 11Liu L. Rodgers G.M. Blood. 1996; 88: 2989-2994Crossref PubMed Google Scholar). MHV-3 infection of murine macrophages represents an excellent model to study the induction of fgl-2 for several reasons. First, the degree of hepatocellular necrosis following infection correlates well with the induction of macrophage PCA (2Abecassis A. Falk J.A. Makowka L. Dindzans V.J. Falk R.E. Levy G.A. J. Clin. Invest. 1987; 80: 881-889Crossref PubMed Scopus (100) Google Scholar, 5Li C. Fung L.S. Chung S. Crow A. Myers-Mason N. Phillips M.J. Leibowitz J.L. Cole E. Ottaway C.A. Levy G. J. Exp. Med. 1992; 176: 689-697Crossref PubMed Scopus (64) Google Scholar, 12Pope M. Rotstein O. Cole E. Sinclair S. Parr R. Cruz B. Fingerote R. Chung S. Gorczynski R. Fung L. Leibowitz J. Rao Y.S. Levy G.A. J. Virol. 1995; 69: 5252-5260Crossref PubMed Google Scholar). Macrophages from susceptible mouse strains (Balb/cJ) infected with MHV-3 exhibit a marked increase in PCA, whereas those recovered from resistant mice (A/J) fail to do so (13Chung S. Gorczynski R. Cruz B. Fingerote R. Skamene E. Perlman S. Leibowitz J. Fung L. Flowers M. Levy G. Immunology. 1994; 83: 353-361PubMed Google Scholar, 14Fingerote R.J. Abecassis M. Phillips M.J. Rao Y.S. Cole E.H. Leibowitz J. Levy G.A. J. Virol. 1996; 70: 275-4282Crossref Google Scholar). Moreover, the resistant A/J strains will develop both fulminant hepatitis and macrophage PCA following pretreatment with corticosteroids, which stabilize fgl-2mRNA (14Fingerote R.J. Abecassis M. Phillips M.J. Rao Y.S. Cole E.H. Leibowitz J. Levy G.A. J. Virol. 1996; 70: 275-4282Crossref Google Scholar). Second, administration of exogenous prostaglandin E2 completely abrogates viral induction of macrophage PCA both in vitro and in vivo and prevents the development of fulminant hepatitis (2Abecassis A. Falk J.A. Makowka L. Dindzans V.J. Falk R.E. Levy G.A. J. Clin. Invest. 1987; 80: 881-889Crossref PubMed Scopus (100) Google Scholar, 15Chung S.W. Sinclair S.B. Fung L.S. Cole E.H. Levy G.A. Prostaglandins. 1991; 42: 501-511Crossref PubMed Scopus (6) Google Scholar). Considered together, these findings suggest that macrophage PCA may serve both as a marker of disease and contribute to the pathogenesis of the process by virtue of the ability of the cells to sequester in the infected liver. The cellular mechanisms underlying the induction of this protein in macrophages as well as in other cells are presently being defined. Detailed studies by Holmes and colleagues (16Dveksler G.S. Dieffenbach C.W. Cardellichio C.B. McCuaig K. Pensiero M.N. Jiang G.S. Beauchemin N. Holmes K.V. J. Virol. 1993; 67: 1-8Crossref PubMed Google Scholar, 17Nedellec P. Dveksler G.S. Daniels E. Turbide C. Chow B. Basile A.A. Holmes K.V. Beauchemin N. J. Virol. 1994; 68: 4525-4537Crossref PubMed Google Scholar) have characterized the MHV receptor as a 110–120-kDa glycoprotein in the carcinoembryonic antigen family of glycoproteins. Expression of this receptor in hamster cell lines confers susceptibility to MHV infection (17Nedellec P. Dveksler G.S. Daniels E. Turbide C. Chow B. Basile A.A. Holmes K.V. Beauchemin N. J. Virol. 1994; 68: 4525-4537Crossref PubMed Google Scholar). Subsequent events in the signaling pathway(s) leading to expression of fgl-2 in MHV-infected macrophages have not been clearly elucidated. Our group previously demonstrated that incubation of macrophages with MHV-3 caused the rapid accumulation of tyrosine phosphoproteins over a range of molecular masses from 33 to 91 kDa. In addition, the nonspecific tyrosine kinase inhibitors genistein, herbimycin, and tyrphostin AG51 inhibited virus-induced PCA, both at the functional level and at the level of gene expression (18Dackiw A.P.B. Zakrzewski K. Nathens A.B. Cheung P.Y.C. Fingerote R. Levy G.A. Rotstein O.D. J. Virol. 1995; 69: 5824-5828Crossref PubMed Google Scholar). Together, these findings suggest that tyrosine kinase activation in response to MHV-3 stimulation is an important component of the signaling cascade leading to fgl-2 expression. In this regard, a clustering of tyrosine phosphorylation around the 38–44-kDa region was suggestive of activation of members of the MAP kinase family, specifically p38/CSBP/reactivating kinase and extracellular signal-related kinase (ERK)-1 and ERK2. These proteins, which undergo dual phosphorylation on tyrosine and threonine residues during their activation, are known to be involved in the response of cells to a variety of infectious and inflammatory stimuli (19McGilvray I.D. Lu Z. Bitar R. Dackiw A.P.B. Davreux C.J. Rotstein O.D. J. Biol. Chem. 1997; 272: 10287-10294Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 20Liu M.K. Brownsey R.W. Reiner N.E. Infect. Immun. 1994; 62: 2722-2727Crossref PubMed Google Scholar, 21Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar, 22Geng Y. Gulbins E. Altman A. Lotz M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8602-8606Crossref PubMed Scopus (132) Google Scholar, 23Durden D.L. Kim H.M. Calore B. Liu Y. J. Immunol. 1995; 154: 4039-4047PubMed Google Scholar, 24Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2390) Google Scholar, 25Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (676) Google Scholar). We therefore hypothesized a role for these proteins in MHV-induced macrophagefgl-2 expression. In the present studies, we demonstrate that MHV-3 induces the phosphorylation and activation of both ERK and p38. However, although both of these kinases are activated, the use of specific inhibitors clearly demonstrates that p38, but not ERK, is integral to the induction of fgl-2 mRNA and its protein product. Pathogen-free female Swiss-Webster mice aged 6–7 weeks were obtained from Taconic Farms and were chow fed and allowed to acclimatize for 1 week prior to experiments. 3% thioglycollate (Life Technologies, Inc.) was prepared as per the manufacturer's instructions. Endotoxin-free RPMI and HBSS were purchased from Life Technologies, Inc.; fetal calf serum (FCS) was from HyClone. The p38-selective inhibitor SB203580 was the kind gift of Dr. J. C. Lee (SmithKline Beecham) and was prepared in Me2SO to a 20 mm solution. The selective MAP kinase kinase-1 inhibitor PD98059 (Research Biochemicals International) was prepared in Me2SO to a 10 mm stock solution. Peritoneal exudative macrophages (PEM) were harvested in ice-cold HBSS 5–6 days after the intraperitoneal injection of 2 ml of sterile thioglycollate. The cells were washed twice in cold HBSS and resuspended in RPMI, 2% FCS, l-Gln at 1–10 × 106 cells/ml. This procedure consistently yields a >96% macrophage cell population by Wright's stain, with >97% viability by trypan blue exclusion (26Brisseau G.F. Dackiw A.P.B. Cheung P.Y.C. Christie N. Rotstein O.D. Blood. 1995; 85: 1025-1035Crossref PubMed Google Scholar). Cells were incubated for 60 min at 37 °C, 5% CO2 prior to experimentation. MHV-3 was obtained and purified as described previously (18Dackiw A.P.B. Zakrzewski K. Nathens A.B. Cheung P.Y.C. Fingerote R. Levy G.A. Rotstein O.D. J. Virol. 1995; 69: 5824-5828Crossref PubMed Google Scholar). Virus was grown to titers of 10–50 × 106 plaque-forming units/ml RPMI on confluent 17CL cells with a strictly aseptic technique. For studies using dead virus, MHV-3 preparations were irradiated under UV light for 20 min (UVG-11 ultraviolet lamp; Ultra-Violet Products Inc.). Cells were incubated at 37 °C in 5% CO2 in the presence or absence of MHV-3 for times ranging from 1 min to 6 h. Unless otherwise indicated, a multiplicity of infection (m.o.i.) of 5:1 was employed. In some studies, PEM were preincubated in the presence or absence of 1–50 μm PD98059 or 1–20 μmSB203580 for 45 min at 37 °C, 5% CO2. Control cells were exposed to vehicle, 0.1% Me2SO, during the preincubation period. At the end of the incubation period, reactions were stopped by placing the cells on ice. PEM were pelleted 6 h after exposure to viral particles and resuspended at 1 × 106 cells/ml RPMI. Following a single freeze-thaw cycle at −70 °C, PCA was measured by single stage recalcification clotting assay. PCA was expressed as milliunits/106 cells by comparison to rabbit brain thromboplastin as described previously (18Dackiw A.P.B. Zakrzewski K. Nathens A.B. Cheung P.Y.C. Fingerote R. Levy G.A. Rotstein O.D. J. Virol. 1995; 69: 5824-5828Crossref PubMed Google Scholar, 26Brisseau G.F. Dackiw A.P.B. Cheung P.Y.C. Christie N. Rotstein O.D. Blood. 1995; 85: 1025-1035Crossref PubMed Google Scholar). Previous work has established that MHV-3-induced PCA is entirely dependent on the induction of the fgl-2 prothrombinase (5Li C. Fung L.S. Chung S. Crow A. Myers-Mason N. Phillips M.J. Leibowitz J.L. Cole E. Ottaway C.A. Levy G. J. Exp. Med. 1992; 176: 689-697Crossref PubMed Scopus (64) Google Scholar, 6Parr R.L. Fung L. Reneker J. Myers-Mason N. Leibowitz J.L. Levy G. J. Virol. 1995; 69: 5033-5038Crossref PubMed Google Scholar, 7Fung L.S. Neil G. Leibowitz J. Cole E.H. Chung S. Crow A. Levy G.A. J. Biol. Chem. 1991; 266: 1789-1795Abstract Full Text PDF PubMed Google Scholar, 12Pope M. Rotstein O. Cole E. Sinclair S. Parr R. Cruz B. Fingerote R. Chung S. Gorczynski R. Fung L. Leibowitz J. Rao Y.S. Levy G.A. J. Virol. 1995; 69: 5252-5260Crossref PubMed Google Scholar); for details, see “Results.” At various times after virion exposure, PEM were pelleted and lysed in ice-cold cell lysis buffer. Whole cell lysates were prepared with 2× Laemmli, 0.1 m dithiothreitol (DTT) buffer followed by immediate boiling at 100 °C for 5 min. Cytosolic fractions were isolated with 1% Triton X-100, 150 mm NaCl, 10 mm Tris-HCl (pH 7.4), 2 mm sodium orthovanadate, 10 μg/ml leupeptin, 50 mm NaF, 5 mm EDTA, 1 mm EGTA, and 1 mmphenylmethylsulfonyl fluoride. Postnuclear supernatants were collected following centrifugation at 10,000 × g for 5 min and diluted with 2× Laemmli buffer, 0.1 m dithiothreitol (DTT). Lysates prepared from 100,000 cells were separated on 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Blots were then probed with polyclonal rabbit anti-phosphotyrosine (Transduction Laboratories), anti-phospho-ERK or anti-phospho-p38 (New England Biolabs) antibody, or rabbit anti-fgl-2 antibody (Dr. G. Levy, University of Toronto). Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech), blots were developed using an ECL-based system (Amersham Pharmacia Biotech). Cells (3 × 106) were lysed as above and the postnuclear supernatants precleared with protein G-Sepharose (Amersham Pharmacia Biotech). Cellular proteins were immunocomplexed using rabbit polyclonal anti-ERK-2 or anti-p38 antibody (Santa Cruz Biotechnologies) for 1 h at 4 °C. Protein G-Sepharose was added and incubated at 4 °C for 1 h. The resulting immune complexes were washed five times with cold phosphate-buffered saline, 0.01% Tween 20, and then separated from beads by 2× Laemmli buffer, 0.1 m DTT and boiling at 100 °C for 5 min. Beads were then sedimented by ultracentrifugation, and the supernatant was collected for Western blot analysis. ERK2 or p38 immunocomplexes were washed with 5 changes of cold phosphate-buffered saline, 0.01% Tween 20 and then incubated for 30 min at 30 °C with 20 μg of ultra-pure myelin basic protein (MBP, Upstate Biotechnology, Inc.) or 5 μg of recombinant activating transcription factor-2 (Santa Cruz Biotechnologies), respectively, in kinase assay buffer composed of 0.4 mm cold and 0.4 mm [γ-32P]ATP (NEN Life Science Products), 50 mm Tris-HCl (pH 7.4), and 10 mm MgCl2. Reactions were stopped with the addition of 2× Laemmli buffer, 0.1 m DTT and boiling at 100 °C for 5 min. Equal volumes were loaded and run on 10% SDS-PAGE. Autoradiograms developed by exposure of the dried gels to Kodak BIOMAX MR film. Total RNA from 10 × 106 PEM was obtained using the guanidinium-isothiocyanate method (27Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 109-113Crossref Scopus (62909) Google Scholar). RNA was denatured, electrophoresed through a 1.2% formaldehyde-agarose gel, and transferred to nylon membrane. Hybridization was carried out using a32P-labeled, random-primed murine fgl-2 cDNA probe, after which the blots were stripped and probed for 18 S RNA or glyceraldehyde-3-phosphate dehydrogenase mRNA to ensure equal loading. PEM were allowed to adhere to autoclaved glass coverslips for 1 h at 37 °C, 5% CO2, incubated in the presence or absence of 50 μm PD98059, and then infected with MHV-3 at an m.o.i. of 1. Following a 6-h incubation with the virus, the cells were fixed in fresh 4% paraformaldehyde (Sigma) for 20 min at room temperature, quenched for 10 min with 100 mm glycine, and blocked overnight at 4 °C with 5% normal goat serum (Sigma) in PBS (pH 7.4). Rabbit anti-Fgl-2 primary antibody was diluted 1:100 in PBS, 1% normal goat serum and incubated with fixed PEM for 2 h at room temperature. Cells were washed with five changes of PBS and then incubated with a 1:500 dilution of Cy3-conjugated goat anti-rabbit pAb (Jackson ImmunoResearch) for 1 h at room temperature. After five washes in PBS coverslips were mounted using SlowFade antifade reagents (Molecular Probes) and evaluated by confocal microscopy using a Bio-Rad MRC 600 confocal microscope with Comos 7.0 software. At day 5 following intraperitoneal injection of thioglycollate, mice were infected with 50 × 106 virions in 500 μl of RPMI, 2% FCS, l-Gln at 37 °C by intraperitoneal injection. Control animals were injected with 500 μl of medium alone, pre-warmed to 37 °C. At 10, 20, and 30 min after injection of the virus, mice were euthanized by cervical dislocation and PEM collected by peritoneal lavage using 10 ml of ice-cold HBSS. Cells were immediately placed on ice, sedimented, and prepared for Western blot analysis as described above. Protein amounts were standardized by Bradford protein assay (Bio-Rad) prior to gel loading. Continuous data are represented as the mean ± S.E. of the indicated number of experiments. Where representative studies are shown, these are indicative of at least three equivalent studies performed independently. Statistical comparisons were made using one-way analysis of variance with post hoc Tukey. Two approaches were used to determine whether the ERK MAP kinase was targeted by the response to MHV-3. Fig. 1 A shows the time course of the appearance of tyrosine-phosphorylated p44 ERK1 and p42 ERK2 in response to MHV-3 stimulation. MHV-3 induced a rise in phospho-ERK as early as 5 min, reaching a peak at 20–30 min, and fading over the ensuing 30 min. As confirmation of this effect, ERK2 was immunoprecipitated and evaluated for tyrosine-phosphorylated residues. As shown in Fig. 1 B, tyrosine phosphorylation of ERK2 peaked at 20–30 min after exposure to MHV-3 and persisted through 45 min. By having shown the tyrosine phosphorylation of ERK2, in vitrokinase assays were performed to evaluate its activation (Fig. 1 C). MHV induced ERK2 activation with a time course consistent with its pattern of tyrosine phosphorylation. MHV-3 also induced rapid tyrosine phosphorylation of p38. Fig. 2, A and B,illustrates the time course of phosphorylation of p38 using the phosphospecific anti-p38 antibody and immunoprecipitation, respectively. It should be noted that in our work and that of others (28Krump E. Sanghera J.S. Pelech S.L. Furuya W. Grinstein S. J. Biol. Chem. 1997; 272: 937-944Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), the p38 MAP kinase migrates at 42 kDa. In contrast to the activation of the ERK pathway, MHV-3 induces a much faster and briefer tyrosine phosphorylation of p38. Phosphorylation was seen as early as 1 min, generally peaking by 1–5 min, and fading over the next 10–20 min. The time course of p38 tyrosine phosphorylation was reflected directly in p38 activity, as revealed by in vitro kinase assay (Fig. 2 C). Considered together with the results in Fig. 1, these studies demonstrate that MHV-3 induces both ERK and p38 activation, albeit with markedly different time courses. Although endotoxin (LPS) has been shown to activate both ERK and p38 in cells of the monocyte/macrophage lineage (21Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar, 24Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2390) Google Scholar), it is very unlikely that LPS contamination contributed to our results for the following reasons. All media and culture materials were endotoxin-free, both by commercial testing and Limulus assay, and strict attention was paid to sterile techniques. Moreover, endotoxin induces a very different pattern of ERK and p38 activation in PEM; a 1 μg/ml dose of Escherichia coli O111:B4 LPS leads to a more profound tyrosine phosphorylation of the ERK1 and ERK2 proteins that persists through 60 min and beyond and induces p38 tyrosine phosphorylation that peaks at 20–30 min and persists to 60 min (data not shown). Furthermore, pretreatment of cells with 50 μg/ml LPS-complexing polymyxin B greatly attenuated LPS-induced PEM activation as assessed by PCA but had no effect on MHV-3-dependent PCA (data not shown). Finally, MHV-3 preparations induced fgl-2expression, whereas LPS does not. Taken together, these results argue that our findings are not due to LPS contamination. Two compounds have recently been described that act as selective inhibitors of the ERK and p38 pathways. PD98059 selectively inhibits MAP kinase kinase-1, the tyrosine kinase immediately upstream of ERK (29Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 30Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2582) Google Scholar), whereas the bicyclic imidazole SB203580 directly inhibits p38 kinase activity (31Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1971) Google Scholar, 32Kumar S. McDonnell P.C. Gum R.J. Hand A.T. Lee J.C. Young P.R. Biochem. Biophys. Res. Commun. 1997; 235: 533-538Crossref PubMed Scopus (447) Google Scholar). Prior to evaluating the role of these MAP kinases in virus-inducedfgl-2 expression, initial studies were performed to determine the effect of these inhibitors on activation of ERK2 and p38 following MHV-3 stimulation. Pretreatment of cells with PD98059 caused a dose-dependent decrease in ERK2 activity, with complete inhibition of ERK2 activity generally achieved at a 10–50 μm dose (Fig. 3 A), although most consistently at 50 μm. Confirming its selectivity, PD98059 failed to inhibit the MHV-3-induced p38 activation (Fig. 3 B, compare lane 4 to lane 6). Pretreatment of PEM with 10 μm SB203580 markedly attenuated the activation of p38 following treatment with MHV-3 (Fig. 3 B). As shown in Fig. 3 C, SB20380 did not cross-inhibit ERK2 activation and, in fact, caused a consistent augmentation. Neither inhibitor caused cellular toxicity, as evidenced by >95% trypan blue exclusion after 6 and 24 h of incubation. By having defined the ability of the two agents to inhibit their respective kinase activities following MHV-3 stimulation, studies were performed to evaluate their effect on fgl-2 expression. Fig. 4 A is a representative Western blot examining the effect of PD98059 and SB203580 on Fgl-2 protein in response to MHV-3. At concentrations shown to completely inhibit ERK2 kinase activation, PD98059 had little inhibitory effect on MHV-induced Fgl-2 protein levels. By contrast, SB203580 (20 μm) caused a marked attenuation of Fgl-2 protein expression. Consistent with the effect on Fgl-2 protein, PD98058 failed to reduce the MHV-stimulated increase in fgl-2 mRNA levels, whereas SB203580 caused almost complete inhibition (Fig. 5 presents results obtained with a 20 μm dose of PD98059; doses up to 50 μmfailed to inhibit fgl-2 mRNA). These data therefore suggest that although MHV-3 activates both the ERK and p38 pathways, it appears that only the latter is required for fgl-2expression.Figure 5fgl-2 mRNA expression is selectively abrogated by inhibition of p38 MAP kinase. Following pretreatment with PD98059 (20 μm) or SB203580 (20 μm), PEM were exposed to MHV-3 and incubated for 4 h at 37 °C, 5% CO2. The mRNA from 10 × 106 cells was isolated, separated, and probed forfgl-2 as described under “Experimental Procedures.” Note that although PD98059 had little to no effect on MHV-3-inducedfgl-2 mRNA expression, the increase was blocked by selective p38 MAP kinase inhibition with SB203580. A typical Northern blot is shown and is representative of results obtained in at least four independent experiments. Note that doses of PD98059 up to 50 μm had no effect on fgl-2 mRNA expression (data not shown). g3pdh, glyceraldehyde-3-phosphate dehydrogenase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of the specific MAP kinase inhibitors on functional PCA was evaluated. Previous work from our group has established that MHV-3-induced PCA is entirely dependent on the Fgl-2 prothrombinase, as distinct from the other major macrophage procoagulant, tissue factor. In brief, MHV-3-induced PCA is dependent on factor II but in" @default.
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• W2015540930 date "1998-11-01" @default.
• W2015540930 modified "2023-10-18" @default.
• W2015540930 title "Murine Hepatitis Virus Strain 3 Induces the Macrophage Prothrombinase fgl-2 through p38 Mitogen-activated Protein Kinase Activation" @default.
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