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J Biol Chem, Vol. 273, Issue 48, 32222-32229, November 27, 1998
From the Departments of Surgery and Medicine, Toronto Hospital,
General Division and the University of Toronto,
Toronto M5G 2C4, Canada
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) limited fgl-2 functional activity but had little to no effect on
fgl-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 both
in vitro and in vivo. Although the ERK and p38
MAPK proteins have discordant effects at the level of fgl-2
expression, both converge at the level of its activity, suggesting that
targeted MAPK inhibition may ultimately be useful in the modulation of
viral hepatitis.
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 (1). 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 (1, 2).
Fulminant hepatitis induced by this virus is characterized by the
presence of sinusoidal thrombosis and associated hepatocellular
necrosis (3-5). 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 the
fgl-2 gene, has the ability to activate directly the
coagulation cascade, an ability expressed as procoagulant activity
(PCA) and measured by standard clotting assays (6, 7). 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 the
fgl-2 gene and its protein product is observed in the
sinusoidal lining cells (8). By contrast, other organs simultaneously
infected with MHV-3 fail to express Fgl-2 protein and remain uninjured
(8). Importantly, pretreatment with a neutralizing monoclonal antibody
directed against MHV-3-induced PCA prevents sinusoidal fibrin
deposition, hepatocellular necrosis, and mortality in infected mice
(5). Thus, there is good reason to conclude that an fgl-2
up-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 (9), which may
contribute to inducible endothelial PCA (10, 11).
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 (2, 5, 12). 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 (13, 14). Moreover, the resistant A/J strains will
develop both fulminant hepatitis and macrophage PCA following
pretreatment with corticosteroids, which stabilize fgl-2
mRNA (14). 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 (2, 15). 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 (16, 17) 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 (17). 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 (18).
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 (19-25). We therefore
hypothesized a role for these proteins in MHV-induced macrophage
fgl-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.
Animals, Buffers, and Reagents
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.
Cell and MHV-3 Preparation
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 (26). Cells were incubated for 60 min at
37 °C, 5% CO2 prior to experimentation. MHV-3 was
obtained and purified as described previously (18). 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.).
Cell Activation
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 µM
SB203580 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.
Measurement of PCA
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 Western Blot Analysis
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 mM
phenylmethylsulfonyl 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).
Immunoprecipitations and Kinase Assays
ERK-2 and p38 Immunoprecipitations--
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.
ERK-2 and p38 Kinase Assays--
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 [ RNA Extraction and Northern Blot Analysis--
Total RNA from
10 × 106 PEM was obtained using the
guanidinium-isothiocyanate method (27). RNA was denatured,
electrophoresed through a 1.2% formaldehyde-agarose gel, and
transferred to nylon membrane. Hybridization was carried out using a
32P-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.
Immunofluorescence
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.
In Vivo PEM Stimulation
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.
Statistical Analysis
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.
Induction of ERK Tyrosine Phosphorylation and Activity by
MHV-3--
Two approaches were used to determine whether the ERK MAP
kinase was targeted by the response to MHV-3. Fig.
1A 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. 1B, 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 vitro
kinase assays were performed to evaluate its activation (Fig.
1C). MHV induced ERK2 activation with a time course
consistent with its pattern of tyrosine phosphorylation.
Induction of p38 Tyrosine Phosphorylation and Activity by
MHV-3--
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
(28), 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. 2C). 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 (21, 24), 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-2 expression, whereas LPS does not. Taken together, these results argue
that our findings are not due to LPS contamination.
Inhibition of Virus-induced ERK and p38 Activation Using PD98059
and SB203580--
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 (29, 30), whereas the bicyclic imidazole
SB203580 directly inhibits p38 kinase activity (31, 32). Prior to
evaluating the role of these MAP kinases in virus-induced
fgl-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.
3A), although most
consistently at 50 µM. Confirming its selectivity,
PD98059 failed to inhibit the MHV-3-induced p38 activation (Fig.
3B, 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.
3B). As shown in Fig. 3C, 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.
Induction of fgl-2 by MHV-3: Differential Roles of p38 and
ERK--
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. 4A 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 µM
failed 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-2
expression.
Effect of p38 and ERK Inhibition on MHV-induced Fgl-2
Activity--
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 independent of factors VII and X, indicative of
prothrombinase activity rather than tissue factor or factor X activator
(12). Monoclonal antibodies directed against the MHV-3-induced
prothrombinase, which do not cross-react with tissue factor, completely
abolish PCA following infection of macrophages with MHV-3 both in
vitro and in vivo (5, 7). Finally, MHV-3 infection of
macrophages clearly leads to fgl-2 mRNA and protein
expression, and the transient expression of this protein in RAW 264.7 cells induces PCA and prothrombin cleavage activity (6, 7, 12). Taken
together these studies demonstrate that MHV-3 infection of murine
macrophages stimulates the expression of the Fgl-2 prothrombinase,
which in turn is responsible for virally induced PCA.
As shown in Fig. 6, SB203580 caused a
dose-dependent inhibition of the MHV-3-induced PCA at
concentrations shown to reduce expression of fgl-2.
Interestingly, PD98059 at 10 and 50 µM caused a 40 and
80% reduction, respectively, in PCA compared with MHV-3 alone. Since
these concentrations were able to inhibit MHV-3-stimulated ERK2
activation, but had no effect on fgl-2 expression, these findings suggest either a post-transcriptional effect of ERK2 activation on functional Fgl-2 activity or an effect on an as yet
unknown fgl-2 cofactor.
Effect of ERK Inhibition on Fgl-2 Protein Localization--
Recent
studies have suggested that the ERK MAP kinase proteins can be
associated with cytoskeletal elements such as microtubules and
phosphorylate regulatory proteins such as dynamin, suggesting that they
may play some role in intracellular transport (33-35). We postulated
that one mechanism through which ERK inhibition could be affecting
Fgl-2 activity is through inhibition of its transport to its
biologically active location in the plasma membrane. To address this
issue PEM were infected with MHV-3 and then assessed for Fgl-2
localization using immunofluorescence. As demonstrated in Fig.
7, PEM pretreatment with a 50 mM dose of the ERK-selective PD98059 had no gross effect on
either the amount or localization of the Fgl-2 protein. When considered
together with the studies outlined above, these results suggest that
the ERK MAP kinase is not essential for either the expression or the
intracellular transport of Fgl-2.
Effect of UV Irradiation on MHV-3-induced PCA and p38
Activation--
By having determined that p38 MAP kinase activation is
essential for fgl-2 synthesis, we performed studies using
UV-irradiated virus in an effort to dissect whether this activation is
sufficient for macrophage prothrombinase expression. As shown in Fig.
8A, UV irradiation eliminated
the ability of MHV-3 to stimulate functional PCA. These data are
consistent with the finding that UV-irradiated MHV-3 does not induce
fgl-2 mRNA.2
However, the induction of phosphorylated p38 MAP kinase was unchanged (Fig. 8B). These data suggest that p38 MAP kinase activation
is necessary but not sufficient for fgl-2 synthesis.
In Vivo p38 and ERK Tyrosine Phosphorylation in Murine PEM by
MHV-3--
To determine whether MHV-3 was able to stimulate ERK and
p38 phosphorylation in vivo, animals were inoculated
intraperitoneally with MHV-3 or medium vehicle at 5 days after PEM
elicitation with thioglycollate. At various times following MHV-3
injection, cells were harvested from animals and subjected to Western
blot analysis. Compared with cells recovered from mice injected with
medium alone, cells from MHV-3-treated animals exhibited a significant
increase in the tyrosine-phosphorylated forms of p38 and ERK (Figs.
9, A and B,
respectively). The lower panel in each figure confirms equivalent loading of the indicated protein among lanes.
The present data provide a detailed assessment of the cellular
mechanisms by which a pathogenic virus, MHV-3, causes the activation of
physiologic intracellular signaling cascades and leads to the production of a biologically active protein responsible for disease. Expression of the prothrombinase encoded by the fgl-2 gene
is central to the pathogenesis of hepatitis caused by MHV-3 (5). A
previous report from our group (18) demonstrated that induction of
tyrosine phosphorylation by MHV-3 was necessary for macrophage expression of this molecule. In the present studies, components of the
signaling cascade leading to fgl-2 expression are further defined. Specifically, we demonstrate that MHV-3 is able to rapidly induce the tyrosine phosphorylation and activation of two members of
the MAP kinase family, p38 and ERK. By using specific inhibitors of
both pathways, p38 activation is shown to be required for induction of
fgl-2 gene expression and elaboration of its protein
product. Despite being activated by MHV-3, ERK does not appear to be
essential for fgl-2 gene induction, although it may
participate in the post-translational modification of the protein or
alternatively in the action of a cofactor required for its biological
activity. When considered in conjunction with the in vivo
data demonstrating tyrosine phosphorylation of both these kinases in
peritoneal macrophages following intraperitoneal injection of MHV-3,
these findings strongly support the idea that these pathways are
critical in the development of hepatitis following MHV-3 infection.
The signaling pathways upstream of the MHV-3-induced ERK and p38
activation remain to be determined. The rapid activation of ERK and p38
activity is consistent with the conclusion that viral replication
per se is not required for the early signaling events that
ultimately contribute to fgl-2 expression. This is further
supported by the observation that UV-irradiated MHV-3 similarly induces
p38 phosphorylation. The receptor for MHV-3 is a 110-kDa glycoprotein,
which is a member of the murine carcinoembryonic antigen family (16).
Its short intracellular domain lacking tyrosine residues precludes its
ability to function as a receptor tyrosine kinase or act as a binding
site for SH2 domains of nonreceptor tyrosine kinases. However, variants
of the receptor with a long cytoplasmic tail containing tyrosine
residues, derived by alternative mRNA splicing, have been reported
to serve as MHV receptors (16). Phosphorylation of one of these carcino
embryonic antigen-related glycoproteins may have participated directly
in the signaling pathways (36, 37). In this regard, members of the
carcino embryonic antigen-related glycoprotein family have been
reported to associate with molecules that could feed into downstream
MAP kinases. Tyrosine-phosphorylated biliary glycoprotein can
reversibly associate with the protein tyrosine phosphatase SHP-1 (38), whereas CD66a, a human homologue, can be tyrosine-phosphorylated and
associate with pp60c-src, leading to increased
c-Src activity in vitro (39, 40). Activated c-Src
is a classic inducer of the Ras and Raf proteins, both potentially upstream of ERK (41, 42), and it is clear that tyrosine phosphatase activity can regulate both the ERK and p38 MAP kinases (43-47). An
analogous situation may be found in the human immunodeficiency virus
tyrosine phosphorylation response, in which gp120 binds to the T-cell
CD4 antigen, leading to the release of the CD4-associated, src family
tyrosine kinase p56lck and the subsequent
phosphorylation of the MAP kinase kinase kinase Raf-1 (48). Consistent
with the notion of the role of c-Src in the MHV-3 ERK signal, we
have found that PP-1, an src family inhibitor (49), inhibits
MHV-3-dependent ERK tyrosine phosphorylation (data not
shown). Alternatively, it is possible that the extracellular domains of
MHV receptor lacking a cytoplasmic tail might associate with a
transmembrane receptor capable of initiating an intracellular signal,
as has been reported for the interleukin-6 receptor (50). It is
interesting to speculate that the different responses to MHV-3 in
susceptible and non-susceptible mice might be partially due to
differences in receptor-mediated signaling, since Bgp variants derived
from alternative mRNA splicing are expressed differently in
susceptible and non-susceptible mouse strains (17).
Inhibition of Fgl-2 protein by selective p38 inhibition with SB203580
appears to be at the level of transcription. Although the decrease in
mRNA levels may be due to decreased transcription rates, other
groups have also described a role for p38 MAP kinase in the maintenance
of mRNA transcript stability (51). This finding is consistent with
the presence of AUUU-rich regions in the 3'-region of the
fgl-2 mRNA transcript.2 By contrast, ERK
inhibition did not affect fgl-2 mRNA or protein expression but abolished its activity as reflected in the PCA clotting
assay. Control studies testing the effect of PD98059 on PCA assay
itself indicated that this inhibition was not related to a direct
effect of the compound on clotting per se. As demonstrated in Fig. 7, selective ERK inhibition did not appear to affect Fgl-2 localization within macrophages. Rather, the inhibition appears to be
either posttranslational or related to the synthesis of a necessary
cofactor. In this regard, our findings are consistent with previous
observations made during evaluation of the effect of prostaglandin
E2 on Fgl-2 function. PGE2 inhibited
Fgl-2-dependent PCA and liver necrosis (2) but had no
effect on Fgl-2 protein levels (15). A recent study found that
PGE2 reduced the induction of ERK activity by
platelet-derived growth factor and epidermal growth factor in rat
mesangial cells (52), suggesting that the effect of PGE2 on
MHV-3-stimulated ERK activity may, in part, be due to inhibition of the
ERK pathway. Consistent with increased cyclic AMP leading to decreased
ERK activation, adenosine simultaneously increased cyclic AMP and
inhibited the tyrosine phosphorylation of ERK-2 in human cultured mast
cells following Fc epsilon RI receptor cross-linking (53). The role of
the ERK MAP kinase in regulating Fgl-2 activity deserves further
investigation. The fact that ERK inhibition does not affect Fgl-2
protein migration by PAGE argues against protein cleavage as a
post-translational modification; however, Fgl-2 phosphorylation
(should it occur) could be under the influence of ERK. It is possible
that a cofactor protein is necessary for Fgl-2 function, in a
manner analogous to the recently described effector cell protease
receptor-1 protein and the classical factor Xa-Va prothrombinase
complex (54). The expression or activity of such a protein could be
directly influenced by ERK activity. In any case, our data suggest that the ERK and p38 MAP kinase pathways act in a coordinated fashion to
regulate MHV-3-induced prothrombinase activity.
Further evidence for the close interaction of the ERK and p38 MAP
kinase pathways comes from the fact that selective p38 inhibition with
SB203580 consistently increased ERK tyrosine phosphorylation and
activity in response to MHV-3. This finding suggests an inhibitory feedback of ERK by p38 MAP kinase activation. These data are consistent with the recent finding of a similar p38-mediated inhibition of ERK in
mast cells stimulated by IgE aggregation (55). Although the mechanism
for such cross-talk is unclear, feedback modulation of ERK activity has
been previously described to proceed via the mSOS adaptor protein (56),
ERK-dependent expression or modulation of phosphatases (57,
58), inactivation of the MAP kinase kinase kinase Raf-1 by a
GTP-sensitive tyrosine phosphatase (47), and possibly cytosolic
phospholipase A2-mediated signaling events (55). The
functional significance of this cross-talk between MAP kinase pathways
following MHV-3 stimulation is unclear. Further elucidation of the
significance of ERK activation in response to MHV-3 may clarify this issue.
Our results have important implications both for viral hepatitis and
for virally induced inflammatory responses. Several viruses have been
demonstrated to stimulate tyrosine phosphorylation and through this
contribute to aspects of the acute inflammatory response as well as
virus-induced cellular transformation. For example, both the increased
tumor necrosis factor- The relative roles of the ERK and p38 MAP kinase pathways in the
pathogenesis of viral hepatitis in vivo remain to be
determined. We have demonstrated that both ERK and p38 are
tyrosine-phosphorylated in vivo in PEM exposed to MHV-3 by
intraperitoneal injection. Since it has been previously demonstrated
that inhibition of fgl-2 PCA by specific monoclonal antibody
greatly attenuates MHV-3-induced liver necrosis and mortality (5), the
data suggest that selective inhibition of p38 MAP kinase activation,
and possibly of ERK, might be of benefit in vivo. In favor
of this notion is recent work using the Tyrphostin family of
nonspecific tyrosine kinase inhibitors, resulting in improved survival
and organ function in a lethal endotoxemia model in mice and an
intra-abdominal sepsis model in dogs (69-71). Preliminary work in our
laboratory has suggested that pretreatment of mice with Tyrphostin
AG126 markedly inhibits the increase in hepatic fgl-2
mRNA following infection with MHV-3. Future studies to define the
effect of nonspecific and selective inhibition of tyrosine
phosphorylation signaling routes in this murine model of fulminant
viral hepatitis may ultimately suggest novel treatment strategies for
the clinical disease.
*
This work was supported by the Medical Research Council of
Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a Medical Research Council of Canada fellowship.
¶
To whom correspondence should be addressed: Toronto Hospital,
General Division, 200 Elizabeth St., EN9-232, Toronto, Ontario, Canada
M5G 2C4. Tel.: 416-340-9888; Fax: 416-595-9486; E-mail: ori.rotstein{at}utoronto.ca.
The abbreviations used are:
MHV-3, murine
hepatitis virus strain-3; DTT, dithiothreitol; ERK, extracellular
signal-related kinase; FCS, fetal calf serum; LPS, lipopolysaccharide; MAP, mitogen-activated protein; MBP, myelin basic protein; m.o.i., multiplicity of infection; pAb, polyclonal antibody; PAGE, polyacrylamide gel electrophoresis; PCA, procoagulant activity; PEM, peritoneal exudative macrophages; HBSS, Hanks' buffered saline
solution; PBS, phosphate-buffered saline; PGE2, prostaglandin E2.
2
Dr. G. Levy, unpublished observations.
Murine Hepatitis Virus Strain 3 Induces the Macrophage
Prothrombinase fgl-2 through p38 Mitogen-activated Protein
Kinase Activation*
,
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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 (18, 26). Previous work has
established that MHV-3-induced PCA is entirely dependent on the
induction of the fgl-2 prothrombinase (5-7, 12); for
details, see "Results."
-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.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (58K):
[in a new window]
Fig. 1.
Induction of ERK tyrosine phosphorylation and
activity by MHV-3. Following activation with MHV-3, cytosolic PEM
proteins were prepared for Western blot analysis or immunoprecipitation
of the p42 ERK2 as described under "Experimental Procedures."
A, Western blot (WB) staining PEM lysates with
pAb specific to the tyrosine-phosphorylated form of ERK (New England
Biolabs). Note the bands of tyrosine-phosphorylated ERK migrating at 42 and 44 kDa and corresponding to ERK2 and ERK1, respectively.
B, immunoprecipitated (IP) ERK2 was probed for
tyrosine-phosphorylated residues using polyclonal anti-phosphotyrosine
pAb. To control for loading, the blot was stripped and probed for ERK2
protein, as shown in the lower panel. C,
immunoprecipitated ERK2 was allowed to phosphorylate ultra-pure MBP
(Upstate Biotechnology Inc.) for 30 min at 30 °C, using
[32P]ATP. A typical autoradiogram of the 20-kDa band is
shown.
View larger version (49K):
[in a new window]
Fig. 2.
Induction of p38 tyrosine phosphorylation and
activity by MHV-3. PEM were activated by treatment with MHV-3,
lysed at the indicated times, and prepared for Western blot
(WB) analysis or p38 MAP kinase immunoprecipitation
(IP). A, PEM lysates were probed with an antibody
specific to the tyrosine-phosphorylated form of the p38 MAP kinase (New
England Biolabs). Note the early induction of phosphorylated p38, in
marked contrast to the time course of ERK tyrosine phosphorylation.
B, immunoprecipitated p38 was probed with polyclonal
anti-phosphotyrosine antibody. The lower panel is the same
blot, stripped and probed for p38 protein to control for loading.
C, immunoprecipitated p38 was allowed to phosphorylate
purified ATF2 (Santa Cruz Biotechnology) for 30 min at 37 °C, using
[32P]ATP. A typical autoradiogram of the 60-kDa band is
shown.
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Fig. 3.
Selective inhibition of the ERK and p38
pathways: PD98059 and SB203580. A, in a series of ERK2
in vitro kinase assays, preincubation of PEM with increasing
doses of PD98059 abrogated the MHV-3 induction of ERK2 activity. A
typical autoradiogram of the 20-kDa 32P-MBP band is
presented; all points are taken at 20 min after treatment with MHV-3.
B, PEM preincubated in the presence or absence of PD98059 or
SB203580 were treated with MHV-3 as before, and in vitro
kinase assays were performed on immunoprecipitated p38 MAP kinase. Note
that whereas a 10 µM dose of SB203580 greatly decreased
p38 activity, a similar dose of PD98059 had no effect. A typical
autoradiogram of the 60-kDa 32P-ATF2 band is shown.
C, following pretreatment with 50 µM PD98059
or 20 µM SB203580, PEM were exposed to MHV-3 for 20 min,
after which ERK2 protein was immunoprecipitated and an in
vitro kinase assay performed. Note that although ERK2 activity was
effectively eliminated by PD98059, it was consistently increased by
SB203580. Similar results were seen by in vitro ERK2 kinase
assay (data not shown). IP, immunoprecipitated.
View larger version (60K):
[in a new window]
Fig. 4.
Abrogation of prothrombinase protein
expression by p38 MAP kinase inhibition. PEM
preincubated in the presence or absence of PD98059 or SB20380 were
exposed to MHV-3 for 4 h, lysed, and prepared for Western blot
(WB) analysis with polyclonal anti-fgl-2
antibody. Note the large induction of the fgl-2
prothrombinase that followed treatment with MHV-3. Although 50 and 100 µM doses of PD98059 only minimally blunted the increase
in prothrombinase expression, SB 203580 (20 µM)
effectively eliminated it.
View larger version (76K):
[in a new window]
Fig. 5.
fgl-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 for
fgl-2 as described under "Experimental Procedures." Note
that although PD98059 had little to no effect on MHV-3-induced
fgl-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 larger version (24K):
[in a new window]
Fig. 6.
Effect of ERK and p38 MAP kinase inhibition
on Fgl-2 activity. 6 h after exposure to MHV-3, PEM
were washed, resuspended at 106 cells/ml RPMI, and
freeze-thawed at 70 °C. Fgl-2 activity, manifested as PCA, was
determined by single stage recalcification assay. Both PD98059 and
SB203580 were able to dose-dependently inhibit PCA. Data
mean ± S.E., n 
3/group. Stats: analysis of
variance with post hoc Tukey, ***p < 0.001, *p < 0.05 versus control.
View larger version (79K):
[in a new window]
Fig. 7.
Effect of ERK inhibition on Fgl-2
prothrombinase localization. PEM adherent to glass coverslips were
infected with MHV-3 (m.o.i. of 1), incubated for 6 h at 37 °C,
5% CO2, then fixed and prepared for immunofluorescence
using polyclonal anti-Fgl-2 antibody. After counterstaining with
Cy.3-labeled goat anti-rabbit secondary antibody, cells were visualized
with a Bio-Rad MRC 600 confocal microscope. The following conditions
were tested. A, control PEM, the open arrows
indicate cell positions; B, PEM infected with MHV-3;
C, PEM pretreated with a 50 µM dose of PD98059
and then infected with MHV-3; D, control PEM treated with a
50 µM dose of PD98059.
View larger version (19K):
[in a new window]
Fig. 8.
UV irradiation of MHV-3. MHV-3 was
killed by 20 min of UV irradiation as described under "Experimental
Procedures." A, comparison of live and UV-irradiated virus
in their ability to stimulate 6-h PCA, using an m.o.i. of 1:1. Points
are taken in duplicate and are representative of results obtained on
three separate experiments. * p < 0.05 versus control (analysis of variance with post
hoc Tukey). B, following incubation in the presence or
absence of live or UV-irradiated MHV-3, PEM were lysed and prepared for
Western blot (WB) analysis with anti-phospho-p38 MAP kinase
antibody. Both live and irradiated viruses were able to induce p38
phosphorylation.
View larger version (60K):
[in a new window]
Fig. 9.
Tyrosine phosphorylation of the p38 and ERK
MAP kinases in vivo. Five days after intraperitoneal
injection of sterile 3% thioglycollate, 100 µl of RPMI, 2% FCS or
50 × 106 plaque-forming units MHV-3 in 100 µl of
RPMI, 2% FCS was instilled intraperitoneally. Mice were euthanized by
cervical dislocation 10, 20, and 30 min after virus injection, and PEM
was rapidly harvested and prepared for Western blot (WB)
analysis as described under "Experimental Procedures."
A, cell lysates probed with pAb specific to the
phosphorylated form of p38. The same blot, stripped and probed for p38
protein, is shown in the lower panel to control for loading.
B, cell lysates probed with pAb specific to phosphorylated
ERK. Note the induction of both p42 ERK2 and p44 ERK1 protein. In the
lower panel the same blot has been stripped and probed for
ERK2 protein to control for loading.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
expression by astrocytomas exposed to
Newcastle disease virus and the increased c-myc and c-jun expression which follows simian virus 40 binding to
growth-arrested cells occur in a tyrosine kinase-dependent
fashion (58, 59). Hepatitis B has been shown to activate MAP kinase
cascades via the virus-specific HBx protein (60-62), and human
immunodeficiency virus infection of lymphocytic cells leads to the
prompt activation of both the p38 MAP kinase and the MAP kinase kinase
Raf-1 (48, 63). Although MAP kinase cascades have been suggested to be important for virally induced cell cycle events (64), their role in
virally induced inflammatory events remains unclear. The finding that
the p38 MAP kinase in particular is essential for the viral induction
of the fgl-2 prothrombinase, an important inflammatory
mediator in viral hepatitis, adds to a growing body of evidence
suggesting that this kinase can play an integral role in the
inflammatory response elicited by a number of stimuli (24, 25,
65-68).
FOOTNOTES
Recipient of a Medical Research Council of Canada fellowship and
the Bayer fellowship for Research in Surgical Infectious Disease.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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