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Molecular and Cellular Biology, December 2001, p. 8301-8317, Vol. 21, No. 24
Department of Molecular and Cellular
Biochemistry, College of Medicine,1
Section of Oral Biology, College of
Dentistry,2 and Department of Internal
Medicine, College of Medicine,3 The Ohio
State University, Columbus, Ohio 43210
Received 10 July 2001/Returned for modification 28 August
2001/Accepted 19 September 2001
Metallothionein I (MT-I) and MT-II have been implicated in the
protection of cells against reactive oxygen species (ROS), heavy
metals, and a variety of pathological and environmental stressors.
Here, we show a robust increase in MT-I/MT-II mRNA level and MT
proteins in the livers and lungs of C57BL/6 mice exposed to the
influenza A/PR8 virus that infects the upper respiratory tract and
lungs. Interleukin-6 (IL-6) had a pronounced effect on the induction of
these genes in the liver but not the lung. Treatment of the animals
with RU-486, a glucocorticoid receptor antagonist, inhibited induction
of MT-I/MT-II in both liver and lung, revealing a direct role of
glucocorticoid that is increased upon infection in this induction
process. In vivo genomic footprinting (IVGF) analysis demonstrated
involvement of almost all metal response elements, major late
transcription factor/antioxidant response element (MLTF/ARE),
the STAT3 binding site on the MT-I upstream promoter, and
the glucocorticoid responsive element (GRE1), located upstream of the MT-II gene, in the induction process in the
liver and lung. In the lung, inducible footprinting was also identified at a unique gamma interferon (IFN- Viral infection of the respiratory
tract remains a leading cause of morbidity and mortality worldwide.
Influenza virus infection causes approximately 20,000 deaths and
110,000 hospitalizations per year in the United States
(13). Influenza virus A is a member of the orthomyxovirus
family of enveloped, segmented, negative-strand RNA viruses. This virus
replicates in the epithelial cells lining the upper respiratory tract
of humans and in both the upper and lower respiratory tract of mice.
The infection and initial replication cycle stimulate the production
and release of antiviral and proinflammatory cytokines such as alpha,
beta, and gamma interferon (IFN) and interleukin-6 (IL-6) (32,
38). The cytokines limit viral replication as well as stimulate
the innate immune response, leading to recruitment of activated
monocytes/macrophages.
These immune cells use a variety of mechanisms to limit viral
replication until the host can generate a cell-mediated,
antigen-specific response. One such mechanism involves macrophage
phagocytosis, which generates reactive oxygen species. These oxygen
species contribute to the immune-mediated pathology associated with the infection. Successful resolution of the infection requires viral clearance as well as restriction of immune-mediated damage.
Experimental influenza virus infection also induces expression of a set
of cellular genes that include acute-phase proteins in the liver.
Metallothionein I (MT-I) and MT-II are stress response proteins that
are coordinately induced at a very high level in response to variety of
pathological conditions, including inflammation, bacterial infection,
restraint stress, anticancer drugs, heavy metals, and agents that
generate reactive oxygen species (for reviews, see references 5
and 21). The unique metal-thiolate bonds of these cysteine-rich,
heavy-metal-binding proteins can scavenge most potent hydroxyl and
other free radicals very efficiently (60, 64). MT-I and
MT-II are expressed in all eukaryotes and are conserved throughout
evolution, whereas the isoforms MT-III and MT-IV are expressed only in
mammals (58). Unlike MT-I and MT-II, which are ubiquitous
(21, 53), MT-III and MT-IV are expressed primarily in the
brain and stratified squamous epithelium (58), respectively.
MT-I and MT-II have been implicated in the scavenging of toxic metals,
such as cadmium and mercury, as well as in maintaining homeostasis of
biologically essential metals, e.g., zinc and copper (42,
43). Recent studies, however, suggest a significant role for
MT-I and MT-II in the maintenance of redox balance (51), controlling the activity of zinc-containing enzymes (37,
52), modulating mitochondrial respiration (67), and
scavenging free radicals (64). Studies have demonstrated a
protective role of MT-I and MT-II against agents that generate free
radicals, e.g., NO, UV radiation, and cadmium (45, 46).
Recent investigations with transgenic mice overexpressing MT
selectively in the heart have shown that MT can protect cardiac tissues
from injuries caused by the potent anticancer drug doxorubicin
(39, 40).
In general, cells refractory to heavy metals and reactive oxygen
species appear to tolerate these insults by producing relatively high
levels of MT. The genetic evidence that MT is a free radical scavenger
was demonstrated in the yeast Saccharomyces cerevisiae in
which Cu-Zn superoxide dismutase (SOD) mutant cells are very sensitive
to free-radical generators, (e.g., H2O2 and
paraquat), and mammalian or yeast MT could replace the function of SOD
in these cells (63). Similarly, we have recently shown
that the MT level is significantly elevated in the livers of Cu-Zn
SOD-null mice (24).
Most of the agents with which MT-I and MT-II interact (e.g., heavy
metals and ROS) are also potent inducers of these genes. The key
transcription factor MTF-1 mediates activation of these genes in
response to these agents (5, 59). Studies with MTF-1-null embryonic stem (ES) cells have shown that this transcription factor is
essential for the basal as well as induced expression of MT-I in
response to heavy metals and ROS (28, 35). In vivo genomic footprinting (IVGF) of the MT-I promoter showed occupancy of
the metal response elements (MREs) after induction with zinc or
cadmium, indicating the involvement of the same DNA-binding protein
(25, 49, 50). The mechanism of activation of MTF-1 by
heavy metals other than zinc and free radicals has yet to be explored.
Apart from MTF-1, glucocorticoid receptor and STAT3 play important
roles in the expression of MT genes in response to restraint
stress (26, 34) and lipopolysaccharides (47), respectively.
In recent years, our laboratory has been involved in the identification
and characterization of factors that upregulate (6, 7) and
downregulate (23, 48) MT expression. The molecular mechanisms of activation of MT genes in response to
different physiological and pathological stimuli have not yet been
fully explored.
Oxidative stress has been implicated in the pathogenesis of certain
diseases that include atherosclerosis, postischemic reoxygenation injury, chronic inflammation, and certain viral and parasitic infections (12, 14, 31). In particular, potentially fatal acute influenza virus infection causes edema, hemorrhage, and massive
infiltration of inflammatory cells in the upper respiratory tract and
the lungs. The molecular mechanisms of viral pathogenesis are not fully
understood. Based on the activation of immune cells to produce reactive
oxygen species and the known involvement of activated phagocytes in the
initiation and promotion of tissue damage via oxidative and proteolytic
processes, a role for reactive oxygen species has been proposed in the
pathogenesis of influenza virus infection (57). The role
of oxidative stress in influenza virus pathogenesis and dietary
intervention with selenium have been demonstrated by Beck et al.
(8). Because agents that produce reactive oxygen
intermediates are known to induce metallothionein, one should
anticipate a marked induction of MT during influenza virus infection,
particularly in the lung. In the present study, we have demonstrated a
robust induction of MT-I and MT-II in the liver and lung of mice
infected with the influenza A virus and have further explored the
molecular mechanisms of this process.
Animals.
C57BL/6 male mice 4 to 8 weeks of age and free of
virus antibodies were obtained from Charles River, Inc. IL-6 knockout
mice generated on C57/BL6 background were purchased from Jackson
Laboratories. The animals were housed five per cage at 22 ± 2°C
on a 12-h light-12-h dark cycle (lights on at 06:00) and were allowed
to acclimatize for at least 1 week before experimentation.
Infection of mice and treatment with RU-486.
Mice were
infected intranasally with 16 hemagglutinating units (HAU) in 50 µl
of influenza A/PR8 virus diluted in phosphate-buffered saline (PBS).
Prior to infection, mice were anesthetized with an intramuscular
injection (200 µl) of xylazine diluted in ketamine (Vedco, St.
Joseph's, Mo.). Infection was verified by seroconversion (by
enzyme-linked immunosorbent assay [ELISA]) as previously published (13, 53). For RU-486 treatment, animals were injected
subcutaneously with the vehicle (polyethylene glycol) or the drug (25 mg/kg of body weight) every day, starting 1 day prior to infection.
Mice were sacrificed at different days postinfection by cervical
dislocation, and tissues were frozen in liquid N2 for RNA
isolation or processed immediately for the isolation of nuclei.
Isolation of RNA and Northern blot analysis.
Total RNA was
isolated using the guanidinium thiocyanate-acid phenol method
(15) and subjected to Northern blot analysis following a
published protocol (22).
Estimation of metallothionein proteins in tissue extract and
serum.
A cadmium hemoglobin-binding assay was used to estimate MT
proteins (19). Frozen tissues were pulverized in liquid
N2 by using a tissue meizer and extracted with the
homogenization buffer (50 mM Tris [pH 7.4], 1 mM EDTA, protease
inhibitor cocktail, and 1 mM dithioerythritol) (4 ml of buffer/g of
tissue). The homogenate was centrifuged at 40,000 × g
for 30 min at 4°C, and the supernatant was snap-frozen in liquid
N2 in small aliquots and stored at Isolation of nuclei and preparation of nuclear extract.
Nuclei were isolated from the liver and the lung by homogenizing in
high-density sucrose buffer (2 M sucrose, 25 mM HEPES [pH 7.9], 25 mM
KCl, 1 mM EDTA, 10% glycerol, 0.15 mM spermine, 0.5 mM spermidine, and
protease and phosphatase inhibitor cocktails [Sigma]) and isolated by
sucrose density gradient centrifugation following Gorski et al.
(27). The nuclei were resuspended in 3 volumes of buffer A
(25 mM HEPES [pH 7.9], 1.5 mM MgCl2, 100 mM KCl,
25% glycerol, 1 mM dithiothreitol [DTT] along with protease and
phosphatase inhibitor cocktails) and lysed by adding 1.2 M KCl dropwise
to a final concentration of 0.4 M (65). After 30 min on
ice, the soluble proteins were collected by centrifuging at
100,000 × g for 30 min at 4°C. The extracts were
snap-frozen in small aliquots in liquid N2 and stored at
EMSA.
Nuclear extracts were prepared from the livers of
control and infected mice as described earlier (26) and
incubated with IVGF.
We followed our published protocol for IVGF in tissue
nuclei (25). The nuclei (5 × 106 to
10 × 106 in 500 µl of PBS) were mixed with 500 µl
of PBS containing 0.4% dimethyl sulfate for 2 min at room temperature,
and the nuclear suspension was immediately diluted to 50 ml with
ice-cold PBS and sedimented by centrifugation at 1,500 × g for 2 min. The pellet was washed twice with the same buffer.
Next, the nuclei were subjected to proteinase K digestion, and DNA was
purified by phenol-chloroform extraction and ethanol precipitation. The
resultant DNA was dissolved in Tris-EDTA (TE) and treated with
piperidine (1 M) for 30 min at 95°C to cleave the methylated
guanosines. The DNA fragments were collected by ethanol
precipitation and analyzed on agarose gels to ensure fragment sizes of
<500 nucleotides. Next, equal amounts of DNA (1 to 2 µg) from
each sample were subjected to LM-PCR with gene-specific primers.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.24.8301-8317.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Influenza Virus Infection Induces Metallothionein Gene
Expression in the Mouse Liver and Lung by Overlapping but
Distinct Molecular Mechanisms
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) response element (-IRE) and
at Sp1 sites. The mobility shift analysis showed activation of STAT3
and the glucocorticoid receptor in the liver and lung nuclear extracts,
which was consistent with the IVGF data. Analysis of the newly
synthesized mRNA for cytokines in the infected lung by real-time PCR
showed a robust increase in the levels of IL-10 and IFN- mRNA that
can activate STAT3 and STAT1, respectively. A STAT1-containing complex
that binds to the -IRE in vitro was activated in the infected lung.
No major change in MLTF/ARE DNA binding activity in the liver and lung
occurred after infection. These results have demonstrated that MT-I and
MT-II can be induced robustly in the liver and lung following
experimental influenza virus infection by overlapping but distinct
molecular mechanisms.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C.
80°C. These extracts were used for detection of DNA-binding
proteins by electrophoretic mobility shift assay (EMSA).
-32P-labeled specific oligonucleotides in
the binding buffer containing 20 mM HEPES (pH 7.9), 75 to 90 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 µg of
poly(dI-dC)/µg of protein, and 10 to 12% glycerol. For the
supershift assay, the nuclear extract was incubated in binding buffer
on ice with antibodies before addition of the labeled oligonucleotide.
The DNA-protein complexes were separated by polyacrylamide (4 to 6%
acrylamide) gel electrophoresis with 0.25× to 0.5× TBE
(Tris-borate-EDTA) as running buffer (depending on the nature of the
complex), and analyzed by autoradiography or in a Storm system
(Molecular Dynamics). The sequences of the deoxyoligonucleotides used
in EMSA were as follows: 1, mMT-I STAT 3, 5'-GATCGAGTTCTCGTAAACTC; 2, MRE-s,
5'-GATCCAGGGAGCTCTGCACACGGCCCGAAAAGTA; 3, MLTF/ARE,
5'-GATCCGCGGGGCGCGTGACTATGCGTGGGCTGGA; 4, mutMLTF/ARE, 5'-GATCCGCGGGGGCCGTGACTATGCGTGGGCTGGA; 5, mMT-I GRE-1,
5'-GATCAAGTGGGGAAACACCATGTACCTCCAG; 6, mMT-I--IRE,
5'-CGGCTCTGCCAGGACGCGGGGCG; and 7, Sp1,
5'-GATCATTCGATCGGGGCGGGGCGAG.
Reverse transcription.
Total RNA was isolated from the
pooled samples as described above. RNA was reverse transcribed using a
commercially available kit (Promega, Madison, Wis.) Briefly, 2 µg of
RNA was combined with 5 mM MgCl2, 1 mM each of the for
deoxynucleoside triphosphates, reverse transcription buffer, 1 U of
RNasin, 15 U of avian myeloblastosis virus reverse transcriptase, and
primed with 0.5 µg of random hexamers per µg. After a 10-min
incubation at room temperature, samples were heated to 42°C for
1 h, placed in boiling water for 5 min, and then put in ice for
another 5 mins. Diethyl pyrocarbonate-treated water was added to the
samples to bring the final volume to 100 µl. The resulting cDNA was
then stored at 70°C until use in the real-time PCR.
Quantitation of cytokine gene expression using real-time
PCR.
Increased expressions of IFN-,-
, and - and IL-6,
-10, -12, and -15 due to infection was assessed with total RNA isolated from lung using the real-time PCR technique. The primer and probe sequences for each cytokine are given below. The PCR was run in sealed
96-well microtiter plates. The reaction consisted of cDNA, primers, and
fluorescently labeled probes for the cytokine of interest, 18S RNA
(internal positive control), and Taqman Universal Master Mix (PE
Applied Biosystems, Foster City, Calif.) for a final volume of 25 µl.
The final concentrations of primer and probe were 900 nM and 100 nM,
respectively, for each of the cytokines and the 18S control.
primers were forward (5'-TGCAACCCTCCTAGACTCATTCT),
reverse, (5'-CCAGCAGGGCGTCTTCCT), and probe
(5'-CTGCATCAGACAGCCTTGCAGGTCATT). The IFN- primers were
forward (5'-TGAATGGAAAGATCAACCTCACCTA), reverse
(5'-CTCTTCTGCATCTTCTCCGTCA), and probe
(5'-AGGGCGGACTTCAAGATCCCTATGGA). The IFN- primers were
forward (5'-AGCAACAGCAAGGCGAAAA), reverse (5'-CTGGACCTGTGGGTTGTTGA), and probe
(5'-CCTCAAACTTGGCAATACTCATGAATGCATCC). The IL-6 primers were
forward (5'-TATGAAGTTCCTCTCTGCAAGAGA), reverse (5'-TAGGGAAGGCCGTGGTT), and probe
(5'-CCAGCATCAGTCCCAAGAAGGCAACT). The IL-10 primers were
forward (5'-TTTGAATTCCCTGGGTGAGAA), reverse (5'-ACAGGGGAGAAATCGATGACA), and probe
(5'-TGAAGACCCTCAGGATGCGGCTG). The IL-12 primers were forward
(5'-AGCTAACCATCTCCTGGTTTGC), reverse (5'-CCACCTCTACAACATAAACGTCTTTC), and probe
(5'-TGCTGGTGTCTCCACTCATGGCCA). The IL-15 primers were
forward (5'-TCATATTGACACCACTTTATACACTGACA), reverse
(5'-GCAATTCCAGGAGAAAGCAGTT), and probe
(5'-CTTTCATCCCAGTTGCAAAGTTACTGCAATG).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression of MT-I and MT-II genes is
upregulated in livers and lungs of mice infected with influenza A
virus.
A mouse model for influenza A virus infection has
been used to study the role of the hypothalamus-pituitary-adrenal
axis in the pathophysiology of infection and the effect of
restraint stress on immunomodulation during the course of infection
(18, 33). In this model, mice are infected intranasally
with the strain of virus (A/PR8) that selectively infects the upper
respiratory tract and the lung. The virus infection caused induction of
an array of cytokines, e.g., IL-1, IL-1, IL-6, IL-10, IL-12,
IL-15, tumor necrosis factor alpha, IFN-, IFN-, and IFN-. Some
of these cytokines, e.g., IL-6, transport to the brain and activate the
hypothalamus-pituitary-adrenal axis to secrete glucocorticoids into the
circulation. IL-6 also causes activation of genes for the acute-phase
response proteins in the liver such as 2-macroglobulin, serum
amyloid A, and C-reactive proteins. This lytic virus also induces the
formation of reactive oxygen species in the infected cells that leads
to cytopathic effect. The host cells, e.g., macrophages, also generate
reactive oxygen-species as an inflammatory response against the virus.
|
MT induction by influenza A virus infection is significantly
inhibited in liver but not in lung of IL-6 null mice.
Many
cytokine genes are upregulated during influenza virus infection. Among
these genes, the proinflammatory cytokine IL-6 mediates induction of
acute-phase proteins, including MTs, in the liver (30,
38). Analysis of the MT-I promoter (Fig.
2A) revealed the existence of potential
type I and type IL-6 response elements (REs) (41, 47). To
explore the role of IL-6 in influenza virus-induced MT-I and MT-II
expression, we used mice with a targeted disruption of the
IL-6 gene (IL-6
/) along with
wild-type C57BL/6 (IL-6+/+) mice. Despite normal
development, IL-6 null mice are unable to activate the acute-phase
response in the liver, and their innate immune function is compromised
to some extent (2, 10, 56). Five null and five wild-type
mice were infected with the virus and sacrificed on day 5 postinfection, and the level of MT-I and MT-II mRNA in the livers
and lungs of individual animals was measured using
32P-labeled MT-I cDNA (this probe hybridizes to
both isoforms of MT).
|
i) (that detects both isoforms) and
GAPDH cDNA.
/ mice (Fig.
2B) but no significant inhibition in the lung (Fig. 2C). Reprobing the
blot with the antisense MT-II oligonucleotide showed that expression of
this isoform was also inhibited in the liver but not in the lung of
IL-6 null mice (data not shown). These results indicate that the
signaling mechanism activated by virus infection leading to MT
induction in the liver is distinct from that in the lung. This is
consistent with the report that the primary target site for IL-6 is the
liver, where it mediates acute-phase responses by activating certain
genes, including that for MT-I, at a very high level.
Glucocorticoid receptor antagonist RU-486 inhibits MT
induction in liver and lung of both wild-type and IL-6 knockout mice in
response to influenza A virus infection.
The unabated expression
of MT-I in the lung of IL-6 null mice in response to
influenza virus infection indicates the involvement of a different
physiological mediator(s) in this tissue. Furthermore, MT-I
expression in the livers of IL-6 null mice was not completely abolished, indicating interplay of multiple pathways for
MT-I gene activation in the liver. One potential candidate
is the glucocorticoid hormone, whose level is increased dramatically in
a biphasic manner after virus infection (33).
Glucocorticoids are known to upregulate MT-I gene
expression, and GREs are located 7 kb upstream of the MT-I
gene and 1 kb upstream of the MT-II gene (Fig.
3A). These two GREs can independently
impart glucocorticoid responsiveness to both the MT-I and
MT-II downstream promoters (44).
|
/ mice (Fig. 3B, lanes 2 and 6) but not
in the lung (Fig. 3D, lanes 2 and 6). Treatment of the animals with
RU-486 further inhibited MT-I expression in the livers of
both wild-type and IL-6/ mice by 50 to 60%
(Fig. 3B, lanes 4 and 8). The GR antagonist also inhibited
MT-I induction in the lung of both wild-type and IL-6 null mice by 40 to 50% (Fig. 3D, lanes 4 and 8). Quantitative analyses of the three
such Northern blots are presented in Fig. 3C
and 3E, respectively. These results clearly indicate that in addition
to IL-6, glucocorticoids play a critical role in MT induction in the liver, whereas in the lung they appear to be a key mediator of
MT induction in response to an influenza virus infection.
IVGF studies demonstrate that potential IL-6 type II response
element and some MREs on the immediate upstream promoter of
MT-I as well as GRE1 located upstream of the
MT-II promoter are occupied in liver in response to
infection.
Experiments with IL-6 null mice and GR antagonists have
indicated the involvement of multiple signaling mechanisms in influenza virus-induced MT-I expression (Fig. 2 and 3). IL-6 can
activate target genes by two distinct mechanisms. Transcriptional
activation of genes is mediated through the C/EBP family of proteins
that bind to type I IL-6 RE located in the promoter regions of proteins that include haptoglobulin, C-reactive protein, and serum amyloid A. Type II IL-6 RE is the cis element through which STAT3
induces expression of a different group of acute-phase proteins, such as 2-macroglobulin and fibrinogens (4). Infection also
causes activation of the hypothalamus-pituitary-adrenal axis, leading to an increase in serum glucocorticoid level. Glucocorticoid then binds
to the cytoplasmic receptor in target cells, and the activated receptor
then translocates to the nucleus and binds to the GRE of target genes,
resulting in transcriptional induction. In target cells as well as in
residential macrophages virus infection also generates reactive oxygen
species that in turn activate redox-sensitive transcription factors,
e.g., NF-
B and MTF-1. To identify the cis
elements that are involved in upregulating MT-I gene
expression by interacting with transcription factors in the liver and
lung during virus infection, we performed IVGF.
300 of the
mouse MT-I promoter (which harbors most of the elements required for basal as well as inducible expression of the gene) and the
region upstream of the MT-II gene that harbors two GREs. Sp1
and MLTF stimulate basal expression of the MT-I gene
(21, 29). Among the six MREs, MRE-c and MRE-d are occupied
for the basal expression of the gene (Fig. 2A), and all MREs are bound by the essential transcription factor MTF-1 in response to heavy metals
and oxidative stress (11, 59). Analysis of the
MT-I promoter reveals the existence of both type I and type
II IL-6 REs (see Fig. 2A) (41, 47).
We designed a series of LM-PCR primers to analyze in vivo footprinting
at these cis elements in liver and lung in response to virus
infection. IVGF was performed using tissue nuclei from the control and
infected animals as described in Materials and Methods. The naked
genomic DNA from both the liver and lung was treated with dimethyl
sulfate (DMS) and piperidine and amplified to provide the corresponding
genomic G- ladder. The differential availability of guanine residues to
the methylating reagent in the tissue nuclei from the control or
infected mice, under similar conditions, will reflect the presence or
absence of DNA-protein interaction at a specific site within the
promoter. DNA-protein interaction can result in either protection or
hypersensitivity of a G residue towards methylation and subsequent
cleavage compared to the naked DNA.
To detect footprinting at the potential IL-6-responsive element located
at the MT-I promoter, we used different sets of primers (mMT1-STAT3) that allowed us to read regions upstream of MREs (Fig.
2A). Significant protection was detected at the potential IL-6 type II
RE located at 297 bp in the lower strand of the MT-I
promoter amplified from the infected liver DNA (Fig.
4A, lane 3). No footprinting was observed
in the upper strand spanning this site in the liver nuclei from the
infected mice (data not shown). This is most likely because the
transcription factor contacts the cis element in a way that
leads to hypersensitivity or protection of G residues only in one
strand. No constitutive footprinting was detected at the corresponding
sites in either strand of the MT-I promoter in the control
liver nuclei (lane 2). These results indicate that the inducible
binding of transcription factors, probably STATs, occurred at the IL-6
type II RE located at 297 bp in the liver in response to virus
infection. No constitutive or inducible footprinting was observed at
the putative type I IL-6 RE (Fig. 4B).
|
84
and 190. These results demonstrate that virus infection resulted in
inducible binding of MTF-1 to different MREs. We have observed
constitutive occupancy of Sp1 and MLTF/ARE under noninduced condition
in both cells in culture as well as in control rat liver
(50).
Next, we analyzed footprinting at two GREs as RU-486 blocked
virus-mediated MT-I induction (Fig. 3). Each of these GREs can activate
a reporter gene in response to glucocorticoids (44). The
IVGF data demonstrated the hypersensitivity of three consecutive guanines spanning and flanking the GRE1 sequence in the upper strand in
the infected liver (Fig. 4C, lane 3), indicating the involvement of
this site in infection-induced MT-I gene expression in the
liver. No footprinting was, however, detected at GRE2 in either strand
(lane 3) in control or infected liver. These results indicate that
although there are two potential GREs upstream of the MT-I
and -II promoters, there is preferential in vivo occupancy of GRE1 in the liver in response to virus infection.
IVGF analysis of MT-I promoter in the lung reveals
induced occupancy at a potential IL-6 type II RE, Sp1 sites, MREs, GRE1
and a unique footprinting at -IRE in response to
infection.
We also analyzed the IVGF profile of the
MT-I promoter in the nuclei from the control and infected
mouse lung. Northern blot analysis has shown that MT-I was induced in
the lung in response to viral infection in both wild-type and IL-6 null
mice, which was blocked by RU-486 treatment of the animals (Fig. 2 and
3). We explored the possibility of differential footprinting on the MT-I promoter, particularly at the IL-6 type II RE located
at 297 bp in the infected lung. To our surprise, we observed
footprinting at this site (Fig. 5A, lane
3) on the MT-I promoter, the pattern of which is identical
to that in the liver. These results indicate that in the lung of
infected mice, STAT3 is activated via an IL-6-independent pathway, as
MT-I was activated in the lung of IL-6 null mice (see Fig. 2
and 3). Indeed, STAT3 can be activated by cytokines other than IL-6,
e.g., IL-10, IFN-, IFN-, and a variety of growth factors
(1).
|
response element (-IRE) located between MRE-c
and MLTF/ARE. This site was identified as the IFN--responsive element in the major histocompatibility complex class II gene DPA (66). This unique footprinting is
not observed in the liver from the infected animals (Fig. 4B, lane 3),
which might be due to activation of an IFN- signaling pathway
exclusively at the site of infection. Alternatively, the mononuclear
cells migrating to the lungs in response to infection, such as natural
killer (NK) cells, could be responsible for IFN- production.
Constitutive footprinting was not observed at the MREs and Sp1 sites in
the lung (Fig. 5B). However, in contrast to the liver, we observed
induced binding of Sp1 and occupancy of the ARE site in the upper
strand of the MT-I promoter in the infected lung (lane 3).
The infection-induced occupancy of MRE-b and MRE-d was observed in
the upper strand of the MT-I promoter in the lung (Fig. 5B,
lane 3). In the lower strand, we detected virus infection-induced footprinting at MRE-d and MRE-c in the nuclei from infected
animals (lane 6). Analysis of the footprinting at the two GREs in the lung revealed inducible footprinting at GRE1, which showed
hypersensitivity to DMS treatment, as observed in the infected liver
(Fig. 5C). These results indicate that in the lung, virus infection
causes inducible occupancy at STAT3, MREs, GRE1, ARE, and -IRE
sites, implicating activation of multiple transcription factors.
DNA-binding activities of STAT3 and GR are significantly elevated
in liver and lung nuclear extracts from infected mice.
To identify
the transcription factors that bind to the cis elements of
the MT-I promoter in response to virus infection, we performed EMSA with nuclear extracts prepared from both control and
infected tissues. We compared the DNA-binding activities of several
transcription factors that included STAT3/STAT1, GR, MTF-1, and
MLTF/ARE binding proteins. To identify the transcription factor that
binds to IL-6 RE type II, the liver nuclear extract was incubated with
an -32P-labeled oligonucleotide spanning from bp 300
to 277 of the MT-I promoter, where protection was detected
in IVGF analysis (Fig. 5A). This oligonucleotide differs from the
consensus STAT site at two bases (Fig.
6A). A specific complex
was detected in the nuclear extract from the wild-type mice that
increased three- to fivefold in the extract from infected animals (Fig.
6B, compare lanes 2 and 3). The upper complex appeared to be the
specific one, as its formation was inhibited by a molar excess of
unlabeled oligonucleotide (lanes 8 to 11). That STAT3 but not STAT1 was a component of this complex was revealed by supershift of this complex
with only anti-STAT3 antibodies (lanes 6 and 7). Minimal activation of
the STAT3 complex was observed in the nuclear extract from IL-6 null
mice (lanes 4 and 5), which is consistent with the inability of these
null mice to express MT-I (Fig. 2).
|
and isoforms of
GR (Fig. 6C). The specific complex was supershifted with
anti-STAT3 antibodies (lane 7) but not with anti-GR (lane
11) or anti-STAT1 (lane 9) antibodies, indicating that neither GR nor
STAT1 is a component of the complex. These results demonstrate that
STAT3 can indeed bind to the potential IL-6 type II RE on the mouse MT-I promoter and that its activity was significantly
increased in the liver of infected wild-type mice, leading to inducible binding and gene activation.
Next, we measured the DNA-binding activity of GR, as IVGF analysis
demonstrated occupancy at GRE1 located far upstream of the
MT-I promoter. A specific complex was detected in the liver nuclear extract, the activity of which increased two- to threefold in
the extract from infected mice (Fig. 6D, lanes 2 and 3). The specificity of the complex was evident by competition with unlabeled oligonucleotide (lanes 4 and 5) and supershift with anti-GR antibodies (lanes 6 to 9) but not with STAT3 antibodies (lanes 10 and 11). GR
and probably GR are part of this complex, as antibodies against both
isoforms could specifically supershift the complex. Activation of GR in
the extract from infected animals was more evident from the
quantitation of the signal from the supershifted complex. These results
clearly show that virus infection causes activation of the
glucocorticoid receptors that then occupy GRE1 of the MT promoter, leading to transcriptional activation of the gene in the
liver nuclei.
We also measured the DNA-binding activity of MTF-1. This key
transcription factor is required for basal as well as induced expression of the MT-I gene in response to heavy metals and
reactive oxygen species (28, 35). Among these agents, only
zinc can directly activate MTF-1. The mechanism of its activation by
inducers other than zinc is still an enigma. To determine whether
influenza virus infection has any effect on MTF-1 activity, we
performed EMSA with a 32P-labeled MRE-s oligonucleotide, to
which MTF-1 specifically binds (59). Liver nuclear
extracts from the control mice showed high MTF-1 activity (Fig. 6E,
lane 2) that did not increase significantly after infection (lane 3).
The complexes formed with the extracts of the control and infected mice
were competed with unlabeled MRE-s oligonucleotide (lanes 4 and 5) and
supershifted with antibodies against MTF-1 (lanes 6 and 7). These
results indicate that, unlike STAT3 and GR, the constitutive
DNA-binding activity of MTF-1 was significantly higher in the liver
nuclear extract because of its role in basal MT expression and was not
further elevated during infection.
The complexes formed at the composite major late transcription
factor/antioxidant response element (MLTF/ARE) site were measured in
the liver nuclear extract from control and infected mice (Fig. 6F).
Among the two specific complexes formed, the slower-migrating one was
MLTF/upstream stimulatory factor (USF), as its formation could
be competed by MLTF consensus oligonucleotide (lanes 3 and 4) and the
complex could be supershifted with anti-USF1 antibodies (lane 8). The
faster-migrating complex appears to be ARE binding protein, as its
formation could be competed by a mutant MLTF/ARE oligonucleotide (lanes
5 and 6). The formation of both of these complexes was unaltered in the
liver nuclear extract from the infected mice (lanes 1 and 2). These
results indicate that the basal activity of the proteins that bind to
MLTF/ARE was high in the liver nuclear extract and these activities
were not changed to any significant level after viral infection.
We also measured the DNA-binding activities of the
trans-acting factors in the lung nuclear extract (Fig.
7). EMSA results revealed formation of a
specific complex at the potential site located between 297 and 277
that was indeed activated three- to fourfold in the lung nuclear
extract (Fig. 7A, lanes 2 and 3). Supershift of this complex with
anti-STAT3 but not with anti-STAT1 antibodies confirmed its identity as
STAT3. These results demonstrate that the nature of the complex formed
at this site was identical to that formed in the liver. Comparable
activation of STAT3 was observed after infection when nuclear extracts
from IL-6 null mice were analyzed (compare lanes 8 and 9 with lanes 2 and 3, respectively). This data corroborated the IVGF results.
Similarly, the binding of GR at GRE1 was activated two- to threefold in
the lung nuclear extract from the infected mice (Fig. 7B, lanes 2 and
3). The specificity of the complex was determined by supershift with
anti-GR antibodies (Fig. 7B, lane 6). As observed in the liver, the
constitutive activity of MTF-1 in the lung nuclear extract was high
(Fig. 7C, lane 2) and did not increase significantly after infection
(lane 3). EMSA studies showed that MLTF activity decreased in the lung
nuclear extract of infected mice compared to the control, whereas ARE
binding activity remained unchanged (Fig. 7D, lanes 1 and 2). The
reduced activity of MLTF was probably due to its instability during
isolation of nuclei and extraction of proteins from the lung of
infected mice.
|
-IRE site in the proximal promoter. This site is
involved in IFN--mediated gene expression, as determined by
transient-transfection studies (66). To the best of our
knowledge, the trans-acting factor binding to this site has
not been identified. We attempted to identify the complex that binds to
-IRE by EMSA and subsequent supershift with relevant antibodies.
Nuclear extract was prepared from both control and infected lung and
incubated with -32P-labeled -IRE (bp 123 to 100)
of the mouse MT-I gene. A specific complex, C2, was detected
in the nuclear extract from the infected mice (Fig. 7E, lane 2), which
was barely detectable in the control lung nuclear extract (lane 1).
This complex appeared to be specific, as it was competed out by a molar
excess of the unlabeled oligonucleotide. Supershift analysis showed
that STAT1 but not STAT3 might be involved in the formation of C2
complex, as only the antibodies against STAT1 were able to destabilize
the C2 complex (lanes 7 and 8). Further investigation is necessary to
decipher the exact nature of the C2 complex that plays an important
role in activating the MT-I gene in the infected lung.
Another complex, C1, was detected in the control lung, which was
significantly reduced in the infected lung (lanes 1 and 2). This
complex was partially competed out by unlabeled -IRE oligonucleotide
and was not supershifted by either STAT1 or STAT3 antibody. The nature
and role of this complex in MT-I induction remain to be studied.
Real-time PCR analysis of lung tissue reveals increased expression
of cytokines that signal through STAT1 or STAT3 pathway.
As the
IVGF and EMSA data from the lung demonstrated, MT-I expression is
increased in response to viral infection via STAT1 and STAT3, so we
assessed the expression of cytokines that signal through these
pathways. Total RNA from the pooled lungs was reverse transcribed, and
the resulting cDNA was used in the real-time PCR (see Materials and
Methods for details). The relative increase in the expression
(mRNA) of each of the cytokines in the infected tissues was
determined using the comparative Ct method, with corresponding tissue
from the noninfected animals as the baseline (Fig.
8). The data (representative of three
different experiments) show increased expression of the cytokines at
different days postinfection. Although the fold increase in cytokine
expression varied among experiments due to variability of the
infection, the pattern of expression remained similar in each case.
|
/ are important in limiting the early
replication and spread of the virus. In particular, RNA viruses are
strong inducers of IFN- and IFN- due to formation of
double-stranded RNA during the replication cycle (17).
During early stages of infection (on day 3), the level of IFN-
increased 20-fold, after which it started to decline (Fig. 8). The
level of IFN-, a Th1 cytokine vital for the development of
cell-mediated immunity, started to increase on day 3 and attained a
level greater than 150-fold on day 7 postinfection. The observed
increase in IFN- expression offers a potential explanation for the
footprinting observed at the -IRE in the lung (Fig. 5C) and suggests
its role in the increase in STAT1 activity observed in the lungs.
The next set of cytokines that we examined signal through the STAT3
pathway. The IL-6 mRNA level increased in the lungs of infected
animals during early stages of infection (on day 3), after which it
started to decline. Its expression was, however, not critical for the
induction of MT expression in the lung, as demonstrated by the IL-6
knockout experiment (Fig. 2). Because IL-6 is not critical for the
induction of MT in the lungs of infected mice, other cytokines (IL-10,
IL-12, and IL-15) that utilize the STAT3 pathway are likely to promote
MT gene expression. The level of IL-10, a Th2 cytokine
important for humoral immunity, started to increase on day 3 postinfection, and its level increased more than 260-fold compared to
the uninfected control lung on day 7 postinfection. On the contrary,
the levels of IL-12 and IL-15, produced by macrophages to activate NK
cells, were not markedly elevated following viral infection. The large
increase in IL-10 expression suggests that IL-10 is responsible for the
observed increase in lung STAT3 activity in response to viral infection.
The real-time PCR analysis of lung RNA demonstrated a significant
increase in the levels of cytokines that signal through the STAT1 and
STAT3 pathways. These results reinforce the IVGF and EMSA data (Fig. 5
and 7) from the lung by demonstrating robust expression of IFN- and
IL-10, which signal via STAT1 and STAT3, respectively. Although the
IL-6 level increased as high as 130-fold on day 3 postinfection, its
effect on MT-I induction was minimal, as IL-6 null mice could induce
lung MT-I to the same extent as the wild-type mice (Fig. 2 and 3).
| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Infection by influenza A virus causes an acute infection of the upper respiratory tract (in humans) and the lung (in rodents). This lytic infection activates resident phagocytes in the tissues to generate reactive oxygen species as part of the host's defense mechanism against virus replication. In order to protect the cells from virus-induced oxidative stress, host cells must have evolved some antioxidant mechanism that effectively scavanges the reactive oxygen species. Because MT has been implicated in such scavenging, it was of interest to investigate whether this gene was activated following an influenza virus infection. The present study clearly demonstrates that this is indeed the case.
Both MT genes (MT-I and MT-II) were coordinately induced in a time-dependent manner in the lung and liver, with maximal expression attained within 5 days following infection. To our knowledge, the present study is the first demonstration of the activation of MT genes in response to an experimental viral infection in an animal model and elucidation of the molecular mechanisms for the induction process. One recent study using DNA microarray technology has identified MT-IIA as one of the genes induced in HeLa cells infected with influenza A virus (20). This study does not, however, address the molecular mechanisms of MT induction in specific tissues of organisms.
Although induction of MT-I in the liver and lung involves
overlapping mechanisms, there are unique differences in the signaling mechanisms in the induction of the same gene in the two tissues. It is
known that infection of mice with influenza A virus evokes an
acute-phase response in the liver and activates the
hypothalamus-pituitary-adrenal axis, leading to increased serum
glucocorticoid levels (32, 36). Viral infection also
induces the expression of proinflammatory cytokines, e.g., IL-1, IL-6,
and IL-10, some of which activate the genes involved in the generation
of reactive oxygen species. In the present study, we have observed that
the expression of several cytokines, IL-6, IL-10, IL-12, and IL-15 as
well as IFN-, IFN-, and IFN-, was elevated in the lung during
viral infection (Fig. 8). We have shown the involvement of multifarious
signaling mechanisms that include cytokines, glucocorticoids, and
perhaps zinc and reactive oxygen species (Fig.
9) in the induction of MT in response to
virus infection. It appears that simultaneous participation of several
transcription factors, such as STAT3, STAT1, GR, MTF -1, Sp1, MLTF, and
ARE binding proteins, leads to robust induction of MT gene
expression in the liver and lung.
|
An important difference in the profile of MT induction in the liver versus lung is that MT expression in the lung continued to rise through day 7 postinfection, in contrast to the peak induction on day 5 in the liver (Fig. 1). This difference in the kinetics of MT induction may be due to trafficking of immune cells to the site of infection. These incoming cells (e.g., macrophages, NK cells, and T cells) will produce cytokines that are not necessarily formed in the resident cells. The prolonged expression of the MT genes in the lung relative to liver may therefore arise from the trafficking cells and the higher levels of cytokines that they produce.
Another noteworthy observation is the ability of IL-6 null mice to
express the MT-I gene in the lung but not in the liver. Among the proinflammatory cytokines, IL-6 can directly activate the
MT-I promoter in hepatocytes (54, 61). IL-6 had
a more profound effect on the activation of MT genes in the
liver relative to the lung following infection by influenza virus. This
differential action of IL-6 is probably due to a higher concentration
of the IL-6 receptor complex on the surface of liver cells compared to lung cells. Indeed, administration of radiolabeled-IL-6 to mice led to
its maximal accumulation in the liver (1). Alternatively, IL-6 deficiency in the lung of null mice may be compensated for by
other cytokines with overlapping signaling mechanisms. The cytokines
that are expressed at a significantly high level following viral
infection
IL-10, IL-12, and IL-15can activate STAT3 (1, 30,
36). In addition to this, some growth factors, e.g., granulocyte colony-stimulating factor, produced during viral infection can also
activate STAT3 (28, 33).
Quantitative analysis of the expression profiles of various cytokines by real-time PCR in the lung revealed an extremely high level of IL-10 during infection (Fig. 8). This is probably responsible for STAT3 activation and MT induction in the lung. Viral infection caused a significant increase in IL-6 mRNA in the lung, particularly at the early stage of infection (day 3). Interestingly, the IL-10 level started to rise on day 3, when the IL-6 level began to decline (Fig. 8). We cannot rule out the potential role of IL-6 in MT induction during early stages of infection (days 1 to 3), when the IL-10 level is significantly low.
To decipher the mechanism of activation of the MT-I gene in liver and lung, we analyzed the MT-I-proximal promoter as well as the region upstream of the MT-II gene that harbors relevant cis elements (see Fig. 2 and 3). Because IL-6 plays a key role in MT-I gene induction in the liver but not in the lung, we explored the characteristics of footprinting at the IL-6 response elements in the liver and lung. IVGF analysis revealed occupancy of the same STAT3 site in both tissues. This observation validates our assumption that STAT3 was indeed activated in the lung by a cytokine(s) other than IL-6.
IL-6 activates acute-phase response genes in the liver by two distinct
mechanisms mediated through different cis elements (3). Analysis of the MT-I promoter reveals the
existence of putative type I and type II IL-6 REs. IVGF analysis
demonstrated occupancy of only type II IL-6 RE located between 297
and 277 bp in response to viral infection (Fig. 4A and 5A). This
observation correlates well with a significant increase in the
DNA-binding activity of STAT3 in the liver nuclear extract from the
infected wild-type mice but not from IL-6 null mice. On the
contrary, IVGF analysis did not show inducible occupancy of IL-6
type I RE in either liver or lung, which is consistent with the lack of
inducible binding of any factor to the oligonucleotide spanning this
site in EMSA using tissue nuclear extract from the infected mice (data not shown).
Studies with RU-486, the type II glucocorticoid receptor antagonist, indicate a critical role of glucocorticoids in MT-I induction. It is known that serum glucocorticoid level is elevated upon viral infection (33). Interestingly, IVGF analysis demonstrated footprinting at one (GRE1) of the two GREs (44) in both the liver and lung in response to viral infection. To our knowledge, this is the first report of genomic footprinting at a GRE site that allowed us to functionally distinguish the two GREs in vivo during viral infection. The reason for the preferential occupancy of GRE1 in both tissues in response to infection is not evident. Although both GREs can impart glucocorticoid sensitivity to a reporter gene in a transient-transfection assay, GRE1 may be the functional element in the chromatin context. Alternatively, it is conceivable that the corticosterone level induced in response to viral infection is optimal for binding to GRE1. It is also known that the glucocorticoid receptor and STAT3 can activate a gene synergistically using either the STAT3 binding site or GRE as the cis element (62, 68). Such a mechanism does not appear to be involved in MT induction following viral infection, as the DNA-protein complex formed between STAT3 and IL-6 type II RE could not be supershifted by anti-GR antibodies. Similarly, the complex formed with GRE1 and GR could not be supershifted with anti-STAT3 antibodies. These results imply that STAT3 and GR transactivate the MT-I promoter in response to infection by influenza virus by binding to the individual cognate sites rather than by protein-protein interaction.
Another significant finding is the unique footprinting in the lung at a
potential IFN- RE (-IRE) located between 105 and 115 of the
MT-I promoter (Fig. 5B). This is the first demonstration of
such a cis-acting element on the MT-I promoter.
The occupancy of this site in vivo has never been observed when the
MT-I gene is induced in cells in culture or in the liver by
other agents that include heavy metals (50), hydrogen
peroxide (16), and lipopolysaccharides (47).
This specific footprinting of the MT-I promoter clearly
demonstrates that MT-I induction by different agents follows
distinct signaling pathways. The lack of -IRE footprinting in the
liver in response to infection suggests that the expression and
activity of the interferons are specific to the lung, the site of
infection, and that a different signaling pathway is activated in this
tissue. The transcription factors that are activated upon interferon
signaling include STAT1, STAT3, and interferon response factor, capable
of binding to this site. An in vitro binding assay demonstrated the
function of a distinct complex containing STAT1 in the infected lung
nuclear extract and the -IRE oligonucleotide. Further study is
necessary to determine the exact nature of the complex.
Although we considered cytokines and glucocorticoids as the major players in MT-I induction during viral infection, the importance of MTF-1, the key and indispensable transcription factor, cannot be overlooked. IVGF analysis of the liver and lung nuclei from the infected mice demonstrated characteristic footprinting of a majority of the MREs in both tissues. These results suggest that MREs mediate not only heavy-metal-induced expression of MT-I, but also influenza virus-induced expression. In the lung, free radicals are generated following viral replication and the host's immune response to the infection that results in the activation of MTF-1 and its occupancy of MREs. It is conceivable that the systemic release of proinflammatory cytokines produced in response to viral infection leads to oxidative stress in the host liver as well. Alternatively, the footprinting at MREs in the liver might be due to mobilization of zinc following infection. Indeed, preliminary analysis of the zinc level in the liver during infection has shown a 25 to 40% increase in the level compared to that in the uninfected control mice of the same age (W. Erdahl and K. Ghoshal, unpublished data). Another possibility is the activation of MTF-1 during the acute-phase response. EMSA results showed that the constitutive DNA-binding activity of MTF-1 is quite high in these tissues and is not activated further following infection. MTF-1 shows similar behavior in response to almost all inducers, except zinc, that directly activate MTF-1 by binding to its six zinc fingers (59). In MTF-1 null cells, the basal MT-I level is significantly low and it cannot be induced (35), pointing to an absolute requirement of MTF-1 for MT-I expression.
Our recent studies have shown that IVGF profiles of the MT-I promoter after cadmium and zinc treatment are similar, and cadmium is a more potent inducer than zinc (50). The MTF-1 activity in the nuclear extract from the cadmium-treated cells is, however, very similar to that of the control cells. These results strongly argue for MTF-1 activation in response to various inducers in vivo and its loss with the exception of zinc in the cell-free system. Elucidation of the signaling mechanism of MTF-1 activation in the liver and lung is of immense importance.
It is possible that the activation of MTF-1 in lung and liver follows distinct pathways. The tissue damage at the site of infection (the lung) caused by viral replication and host immune response will generate a battery of free radicals locally that can activate the MT-I gene. Alternatively, proinflammatory cytokines may activate MTF-1 by an as yet unknown mechanism. Similarly, EMSA results demonstrate that more than one protein binds to the composite MLTF/ARE, none of which are increased in the extract prepared from the livers and lungs of infected animals (Fig. 6F and 7D). It is possible that in vivo binding of MTF-1 to the MREs can facilitate occupancy of MLTF/ARE by USF and ARE binding proteins. Alternatively, infection results in posttranslational modifications of these factors outside the DNA-binding domain, resulting in their recruitment to MLTF/ARE. Identification of the protein(s) that binds to ARE will be of considerable interest.
We propose that MT is a part of the cellular antioxidant mechanism that protects the host from the cytopathic effect of free radicals generated upon viral infection and that the balance between the host cell pro- and antioxidant activity and removal of free radicals determines the severity of the infection. It is conceivable that the therapeutic use of zinc lozenges, which significantly reduce the manifestation of the influenza virus infection, is probably due to MT induction. It is therefore logical to expect that MT-I and MT-II knockout mice will be more susceptible to influenza virus infection than genetically matched wild-type animals. It would be of interest to explore whether the morbidity and mortality of the null mice are higher than those of the wild-type mice. Studies along these lines are in progress.
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ACKNOWLEDGMENTS |
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We thank Richard Palmiter and Walter Schaffner for mouse MT-I minigene and anti-MTF-1 antibodies, respectively; Jaharul Haque for valuable suggestions in assaying STAT3 activity; and Aaron Fawn for critically reading the manuscript.
This research was supported, in part, by grants ES10874 and CA81024 (S.T.J.) and MH46801-09 (J.F.S).
Kalpana Ghoshal and Sarmila Majumder contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, 333 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Phone: (614) 688-5494. Fax: (614) 688-5600. E-mail: jacob.42{at}osu.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
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Molecular and Cellular Biology, December 2001, p. 8301-8317, Vol. 21, No. 24
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.24.8301-8317.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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