From the School of Human and Biomedical Sciences,
Division of Science and Design, University of Canberra, Canberra,
ACT 2601, the ** Department of Pathology, Division of Faculty
of Medicine, Blackburn Building, D06, University of Sydney, New
South Wales 2006, the § Viral Engineering and Cytokines
Group, Division of Immunology and Cell Biology, the
Medical Molecular Biology Group and ¶ Leukocyte
Signalling and Regulation Group, Division of Biochemistry and
Molecular Biology, The John Curtin School of Medical Research, The
Australian National University, Canberra, ACT 2601, Australia
Received for publication, June 30, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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MuMig or Mig
(murine monokine induced by
interferon Interferons (IFNs)1 are
an important group of cytokines that have biological activities,
including antiviral effects, regulation of cell growth and cell
differentiation, and modulation of the immune response (1, 2). IFNs
mediate many of their biological effects through regulation of specific
RNA and protein expression in the responding cell (2). Knowledge of the
ways in which the IFNs regulate gene transcription is necessary to our
understanding of the basic mechanisms of their action. Although type I
(IFN- Both type I and type II IFNs signal through pathways that employ
STAT-1. IFN- Differential screening of a cDNA library from IFN- It was shown recently that IFN- Mice--
Six- to eight-week-old specific pathogen-free C57BL/6J
(B6) wild type (WT) mice and IFN- Virus--
The virulent vaccinia virus, Western-Reserve strain
(VV-WR ATCC VR-119), was propagated in CV-1 cells and used after
purification on a sucrose density gradient as described elsewhere (18).
Mice were injected intraperitoneal on day 0 with 106
plaque-forming units (PFU) of the virus.
Neutralization of IFN- Isolation and Purification of mRNA--
RNase-free plastic
and water were used throughout the assay. Tissues (approximately 50 mg)
were homogenized in 1 ml of RNAzol B (Biotecx, Friendswood, TX), and
total RNA was isolated as recommended by the manufacturer. The RNA was
resuspended in diethylpyrocarbonate-treated water containing 1 mM EDTA and quantitated spectrophotometrically. All samples
were DNase-treated to remove genomic DNA. RNA gel electrophoresis was
performed to confirm that RNA was intact and that the concentration had
been determined correctly as described previously (20).
RT-PCR Detection of Cytokine and Chemokine mRNA
Transcripts--
A reverse transcriptase (RT)-PCR procedure was
performed as described previously (21) with some modification (22) to
determine relative quantities of mRNA for Mig, IFN- Nuclear and Cytosol Extracts--
Liver tissues were obtained
from groups of WT B6, IFN- Western Blot Analysis--
Tris-glycine-SDS-polyacrylamide gel
electrophoresis of protein samples from cytosolic extracts was
performed with 12% acrylamide gels according to the method of Laemmli
(26). As a positive control, concentrated supernatants from CV-1 cells
(monkey kidney cell line) infected with recombinant VV (rVV) expressing
Mig was analyzed in parallel with liver cytosolic extracts.
Construction of rVV encoding Mig has been described in detail elsewhere
(24). Immunoblotting was performed as described previously (27).
Anti-Mig serum, JH48, was kindly provided by Dr. Joshua Farber,
National Institutes of Health (Bethesda, MD).
Gel Electrophoretic Mobility Shift Assay (EMSA)--
This was
performed using double-stranded oligonucleotides radiolabeled by
fill-in reaction using the Klenow fragment of DNA polymerase I as
described previously (28). The double-stranded oligonucleotides used
were the Mig mRNA Is Expressed at High Levels in Organs of B6 WT
Mice--
Organs were harvested at days 3, 6, and 10 after infection
of B6 WT mice with 106 PFU of VV-WR. Organs from uninfected
mice were used as controls. Ovaries were pooled from groups of four
mice for each time point, whereas other organs were analyzed
individually. Fig. 1A shows that Mig mRNA is expressed at high levels in all organs
examined during the course of infection but was not detected in organs of uninfected mice. The time course of infection indicates that Mig mRNA is expressed at high levels in all organs on
days 3 and 6, but declines by day 10. Levels of expression were highest
in the liver followed by ovaries, uterus, and spleen. Expression of
IFN- Mig mRNA and Protein Are Expressed in Mice Lacking IFN-
It was also important to determine whether the expression of
Mig mRNA was associated with the presence of Mig
protein. We performed Western blot analysis on cytosolic extracts from
livers pooled from groups of four B6 WT mice and four
IFN- Mig mRNA Is Not Induced in Mice Lacking Both IFN-
Next, we utilized mice deficient in receptors for both IFN- Transcription Factor
Besides AAF Is Induced in IFN-
When GAS sites were used as probes, increasing amounts of the
competitor (unlabeled GAS fragment) in binding reactions resulted in
diminished levels of AAF-labeled GAS complex formation (Fig. 6A). A reduction in AAF·GAS
complex formation was observed when the unlabeled
On the other hand, when the radiolabeled
In competition experiments involving nuclear extracts from B6 WT mice
probed with labeled NF- Cytokine-mediated intracellular signaling pathways have provided a
general paradigm for the molecular mechanisms by which extracellular
signals induce transcription of target genes. A number of cytokines,
growth factors, and hormones trigger phosphorylation of latent
cytoplasmic transcription factors termed STATs via one or more members
of the Janus family of protein-tyrosine kinases (Jak) (2).
Phosphorylated STATs assemble in dimeric or oligomeric form,
translocate to the nucleus, and bind to specific DNA sequence motifs or
SBEs (34). IFN receptors are coupled to the Jak/STAT signal
transduction machinery (2, 38), which transcriptionally regulates a
panel of genes that mediate the effects of IFNs, including their
antiviral, antiproliferative, and immunomodulatory activities (39, 40).
IFN- The present study was undertaken to clarify some molecular mechanism(s)
involved in transcriptional regulation of Mig gene expression in the IFN- To define a more precise role for IFN- To ascertain whether IFN- The DNA binding factor We have established the presence of an additional To investigate the mechanism by which IFN- Other potential regulatory elements have been identified in the Mig
promoter region, which includes a sequence with similarity to the
NF- Taken together, our data suggest that the STAT-1) is a CXC chemokine whose
induction is thought to be strictly dependent on interferon
(IFN-
). Here we have studied the expression of this chemokine
gene in various organs of mice infected with vaccinia virus. We
have employed animals deficient in either IFN-
(IFN-
/
), or receptors for IFN-
/
, IFN-
, or
both IFN-
/
and IFN-
(DR
/
) to dissect out the
role of interferons in the induction of Mig during the host
response to virus infection. Our data show that Mig
mRNA and protein are expressed in organs of vaccinia virus-infected IFN-
/
mice, albeit at lower levels compared with
infected, wild-type animals. In the DR
/
mice and in
IFN-
/
mice treated with a neutralizing antibody to
IFN-
/
, Mig mRNA transcripts were completely
absent. Our data indicate that, in vaccinia virus-infected
IFN-
/
mice, Mig mRNA expression is
mediated through the interaction between IFN-
responsive element 1 (
RE-1) and IFN-
/
-induced STAT-1 complex referred to as
IFN-
response factor 2 (
RF-2). Further, our findings support the
view that
RF-2 is the IFN-
/
induced STAT-1 complex,
IFN-
-activated factor. We have found that, in the absence of
IFN-
, IFN-
/
are able to induce Mig in response to a viral
infection in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IFN-
) and type II (IFN-
) IFNs bind to different cell
surface receptors, they can induce the expression of both an
overlapping as well as distinct sets of genes (2, 3). The study of
genes activated by both type I and type II IFNs has led to the
identification of two well-characterized promoter elements, namely, the
IFN-stimulated response element (ISRE) (4) and the IFN-
activation
sequence (GAS) (5).
promotes the activation of early response genes through
a STAT-1 homodimer, GAF (IFN-
-activated factor), which binds to GAS
elements (2, 6). IFN-
stimulation leads to the formation of a STAT-1
homodimer (AAF; IFN-
-activated factor) and heterodimer composed of
STAT-1 and STAT-2. This STAT-1:STAT-2 dimer interacts with a
"non-STAT" protein (for example, p48) to form an active
transcription factor ISGF3 (7). The AAF and ISGF3 complexes translocate
to the nucleus and bind to GAS elements and ISRE elements, respectively.
-activated
macrophages led to the identification of the murine CXC
chemokine gene Mig (8). Chemokines constitute a family of
small cytokines that are produced during the process of inflammatory
and immune reactions. They regulate efficient and orchestrated
recruitment of leukocytes to the site of inflammation (9-11). Mig is
chemotactic for natural killer cells and T lymphocytes. Studies using
macrophage cell lines and monocytes showed that both murine and human
Mig were inducible only by recombinant IFN-
in these cells (8, 12).
A fragment from the 5'-flanking region of
235 to
167 of the
Mig gene was able to mediate dramatic induction of a
heterologous promoter by recombinant IFN-
and this element termed
RE-1 has similarities to the IFN-
activation sequence
(GAS) (13). Furthermore, the presence of NF-
B sites in the
Mig gene was shown to be important for gene transcription
involving a synergistic interaction between IFN-
and TNF (14).
is necessary for induction of Mig in
multiple organs of mice infected with Toxoplasma gondii, Plasmodium yeolli, or vaccinia virus (VV) (15). In the
present report, we demonstrate that Mig mRNA is induced
in multiple organs, albeit at low levels, in IFN-
/
mice after infection with VV. Treatment of VV-infected
IFN-
/
mice with an antibody to IFN-
/
completely abolished Mig induction in the liver, indicating that
expression of the chemokine in VV-infected IFN-
/
mice is most likely mediated by IFN-
/
. This is further supported by our findings that Mig mRNA transcripts are absent in
VV-infected mice lacking functional receptors for both type I and type
II IFNs. Our data indicate that Mig mRNA induction in
livers of VV-infected IFN-
/
mice is most probably
mediated via a STAT-1 complex (
RF-2) binding to a STAT binding
element (
RE-1) in the Mig promoter. The transcription factor
(
RF-2) appears to be a STAT-1 homodimer complex.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene knockout
(IFN-
/
) mice (16) on the B6 background were obtained
from the Animal Breeding Establishment, John Curtin School of Medical
Research (ABE, JCSMR), Canberra, Australia. Mice lacking functional
type I (
/
) receptors (IFN-
/
R
/
), type II
(
) receptors (IFN-
R
/
), or both type I and type II
IFN receptors (DR
/
) (17) on the 129/SvJ background also
bred at the ABE, JCSMR, were used at 6-12 weeks of age. WT 129/SvJ
mice were used as controls.
/
in Vivo--
Purified rabbit
polyclonal antibody to murine IFN-
/
was purchased from Lee
Biomolecular Research Laboratories, Inc. (San Diego, CA). Groups of
IFN-
/
mice were given (0.2 mg) 300 neutralizing
units of anti-IFN-
/
on days
1, 1, and 2. One neutralizing unit
of anti-IFN-
/
is defined as the reciprocal of the dilution of
antibody, which completely neutralizes 10 units of the IFN activity
in vitro (19). The mice were infected intraperitoneally with
106 PFU of VV-WR on day 0 and sacrificed on day 3. Mice
similarly treated with 0.2 mg of rabbit IgG (Calbiochem, La Jolla, CA)
were used as controls.
, IFN-
1,
IFN-
, and hypoxanthine-guanine
phosphoribosyltransferase. The primers and probes for all genes
were prepared by the Biomolecular Resource Facility, JCSMR, on a DNA
synthesizer (Applied Biosystems, Foster City, CA). Primer and probe
sequences for Mig, IFN-
, IFN-
1, IFN-
, and hypoxanthine-guanine
phosphoribosyltransferase have been described previously (21, 23, 24).
The cycle numbers used for amplification of each gene product are as
follows: Mig, 24 cycles; IFN-
, 25 cycles; IFN-
1 and IFN-
, 27 cycles; and hypoxanthine-guanine phosphoribosyltransferase, 23 cycles.
After the appropriate number of PCR cycles, the amplified DNA was
analyzed by gel electrophoresis, Southern blotting, and detected using the ECL detection system as recommended by the manufacturer (Amersham Pharmacia Biotech).
/
, and DR
/
mice 3 days after infection with VV-WR. Liver tissues were also obtained from virus-infected IFN-
/
mice treated with
antibody against IFN-
/
. To prepare cell suspension, the liver was
teased apart and passed through a metal sieve into approximately 20 ml
of ice-cold suspension buffer (250 mM sucrose, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and
2 µg/ml aprotinin). The cell suspension was centrifuged at 1500 rpm
for 5 min at 4 °C, the supernatant was aspirated, and the resultant
pellet was resuspended in ice-cold suspension buffer. Red blood cells
were removed from the suspension using red blood cell lysis buffer, pH
7.3 (10 mM potassium bicarbonate, 0.15 M
ammonium chloride, 0.1 mM EDTA, 5% fetal calf serum). The
suspension was then spun at 1500 rpm for 5 min and washed twice with
ice-cold suspension buffer. Nuclear and cytosol extracts were then
prepared from the cell suspension according to the method of Dignam
et al. (25).
RE-1 sequence (
200 to
167) and NF-
B sequence (
145
to
154) of the Mig promoter (28, 29) and the GAS sequence (
126 to
101) of the guanylate-binding protein promoter (5). The ISRE sequence
(
224 to
211) of the murine Crg-2 gene was used as a
nonspecific competitor (30). Protein concentration was measured by the
method of Bradford (31) using the protein dye reagent (Bio-Rad,
Richmond, CA). Binding reaction mixtures (25 µl) contained nuclear
extracts (5 µg of protein), 2 µg of denatured salmon sperm DNA in
binding buffer (20 mM HEPES, pH 7.9, 50 mM
NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5%
glycerol, 200 µg/ml bovine serum albumin) and were incubated for 15 min at 4 °C. The 32P-labeled oligonucleotide (5 × 105 cpm) was then added to the reaction mixture and
incubated for 20 min at room temperature. Where indicated, unlabeled
double-stranded oligonucleotides containing
RE-1 element, GAS
element, or ISRE element (competitors) were added to the binding
reaction simultaneously during addition of radiolabeled fragment. In
some experiments, antibody against STAT-1
(specific for p91;
obtained from Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) was
included in the reaction mixture. Following incubation, 5 µl of 0.1%
bromphenol blue in binding buffer was then added and the mixture was
immediately loaded on a 6% polyacrylamide gel. Electrophoresis was
performed in 0.25× TBE buffer (TBE buffer is 89 mM Tris
base, 89 mM boric acid, and 2 mM EDTA, pH 8) at
175 V with buffer recirculation. The gels were dried and analyzed by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was highest in the ovaries followed by uterus, spleen, and
liver. Thus, although a strong correlation between the kinetics and
expression of Mig induction and that of IFN-
was observed in the
spleen, ovaries, and uterus, this was not the case in the liver where
we observed high levels of Mig and comparatively low levels of IFN-
mRNA. It is possible that Mig in the liver is up-regulated by the
elevated serum IFN-
levels following infection with VV (data not
shown). It is also possible that some other factor(s) are responsible
for Mig induction in the liver.
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Fig. 1.
Time course expression of Mig
and IFN- mRNA in
(A) B6 WT and (B)
IFN-
/
mice during infection
with VV. Total RNA was prepared from organs of uninfected mice
(D0) as well as mice infected for 3 (D3), 6 (D6), and 10 (D10) days with VV. Specific
mRNA species (Mig, IFN-
, and hypoxanthine-guanine
phosphoribosyltransferase) were amplified by RT-PCR. The amplification
products were blotted onto nylon membranes and hybridized to
fluorescein-labeled oligonucleotide probes specific for the PCR
product. Filters were exposed for 5 min in all cases. These experiments
were repeated twice with comparable results. Groups of four mice were
used in these experiments. RT co, RT control; PCR
co, PCR control.
Function--
To establish the role of IFN-
in Mig induction during
VV infection, we used IFN-
/
mice on the B6
background. Organs were harvested at days 3, 6, and 10 after virus
infection while organs from uninfected mice were used as controls. Fig.
1B shows that Mig mRNA was not detected in
uninfected IFN-
/
mice. Unexpectedly, however, low
levels of Mig mRNA transcripts were detected in all
organs at days 3 and 6 post-infection and expression was most prominent
in the liver. We next investigated the expression of Mig during the
course of VV infection in IFN-
R
/
mice (126/SvJ
background). Fig. 2A shows
that Mig mRNA was expressed in the livers of VV-infected
IFN-
R
/
mice and that the levels of expression are
comparable with those in IFN-
/
mice (B6 background).
This also indicates that, at least for the two strains of mice studied
(B6 and 129), the genetic background of the animals did not influence
Mig expression in the absence of IFN-
function.
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Fig. 2.
Mig mRNA and protein
expression in mice lacking IFN- function.
A, total RNA was prepared from livers of
IFN-
/
mice (B6 background) and IFN-
R
/
(129/SvJ) mice 3 days after infection
with VV. Specific mRNA species were amplified by RT-PCR in
VV-infected IFN-
/
mice (B6 background)
and VV-infected IFN-
R
/
mice (129/SvJ
background). B, cytosolic extracts were prepared from
livers pooled from groups of 4 IFN-
/
mice
(lane 3) and 4 WT B6 mice (lane 2) 3 days after
infection. Cell culture supernatant from CV-1 cells infected with rVV
expressing Mig protein was included as a positive control (lane
1). Samples were resolved by Tris-glycine-SDS-polyacrylamide gel
electrophoresis, and Western blot analysis of Mig protein was carried
out using anti-Mig serum JH48. This experiment was repeated twice with
similar results.
/
mice 3 days after infection with VV. Clearly,
Mig protein was detected in cytosolic extracts of
IFN-
/
mice, but the level was significantly lower
compared with that of B6 WT mice (Fig. 2B). Culture
supernatant from CV-1 cells infected with rVV expressing Mig was used
as a positive control. These data show that Mig protein is expressed
consonant with mRNA levels.
and
IFN
/
Function--
In the preceding section we have shown that
Mig mRNA and protein are present in VV-infected
IFN-
/
mice and that the expression of this chemokine
was most prominent in the liver. Likely candidate(s) responsible for
the induction of Mig in IFN-
/
mice are the type I
IFNs. A time course study revealed that the expression of
Mig mRNA also paralleled that of IFN-
1 and IFN-
in
livers of VV-infected IFN-
/
mice (Fig.
3A). It was therefore of
interest to determine whether the expression of Mig in
IFN-
/
mice was mediated through the activity of
IFN
/
. We used a polyclonal antibody to IFN
/
to inhibit its
function in vivo. As expected, Mig mRNA
expression in VV-infected IFN-
/
mice treated with an
antibody to IFN-
/
was completely abolished (Fig. 3B).
However, in VV-infected IFN-
/
R
/
mice, where
IFN-
function is intact but IFN-
/
function is compromised,
Mig mRNA expression was similar to that of WT mice (data
not shown). This finding is consistent with the view that IFN-
alone
is a potent inducer of Mig gene transcription (13), however,
in the absence of IFN-
, IFN-
/
can induce Mig expression.
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Fig. 3.
A, time course expression of Mig,
IFN- 1, and IFN-
mRNAs in livers of IFN-
/
mice 3 (D3), 6 (D6), and 8 (D8) days
after infection with VV. Presence of specific mRNA was analyzed by
RT-PCR as described in the legend to Fig. 1. Controls are as described
in the legend to Fig. 1. B, expression of Mig
mRNA in the livers of IFN-
/
mice and
IFN-
/
mice treated with anti-IFN-
/
or control
antibody (IgG), 3 days after virus infection. C,
expression of Mig mRNA in VV-infected
DR
/
mice. WT mice on the 129/SvJ (129)
background were included for comparison. Total RNA was prepared from
livers of mice 3 days after infection with VV. The experiment was
repeated twice with comparable results.
/
and
IFN-
(DR
/
). Because the DR
/
mice are
on a 129/SvJ background, the 129/SvJ wild-type mice were used as
controls in these experiments. Livers were harvested 3 days after
infection with VV and analyzed for Mig expression. Fig. 3C
shows a complete absence of Mig mRNA transcripts in
these mice, whereas in the control, 129/SvJ WT mice, the expression was
similar to that observed in B6 WT animals (Fig. 1A). These results, and those from the preceding sections, confirm that IFN-
is
important for the optimal induction of Mig expression in
vivo. Interestingly, however, they also indicate that IFNs
/
are able to induce Mig mRNA, albeit at a low level, in
VV-infected IFN-
/
mice.
RF-1 Is Only Detected in B6 WT
Mice--
Recent reports have shown that the IFN-
-induced
transcription factor,
RF-1, is involved in IFN-
-mediated
transcriptional regulation through
RE-1, a major regulatory element
in the Mig promoter (13, 28). We have found that nuclear extracts from the livers of virus-infected B6 WT mice abundantly express
RF-1 (Fig. 4A, lane 2).
This is consistent with the high levels of Mig mRNA
found in this group (Fig. 1A).
RF-1 was not detected in
nuclear extracts from livers of uninfected B6 WT mice (Fig. 4A, lane 1). Time course studies revealed that
the induction of
RF-1 in the liver of virus-infected B6 WT mice
paralleled the expression of Mig mRNA (Fig.
4B). Given this association of
RF-1 and Mig expression in
B6 WT mice, we were interested in determining whether the expression of
Mig in VV-infected IFN-
/
mice was also related to
RF-1 complex formation. However,
RF-1 was not detected in nuclear
extracts of VV-infected IFN-
/
mice (Fig.
4A, lane 3), in mice lacking functional IFN
receptors (DR
/
) (Fig. 4A, lane
4), or in IFN-
/
mice treated with antibody to
IFN-
/
(Fig. 4A, lane 5). This may explain,
at least in part, the reduced levels or complete absence of
Mig mRNA expression in these groups of mice.
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Fig. 4.
A, DNA binding activity of RF-1,
RF-2, and AAF in B6 WT, IFN-
/
,
DR
/
mice, and in IFN-
/
mice treated
with antibody to IFN-
/
. Nuclear extracts were prepared from
livers pooled from groups of three mice, either uninfected (
), or 3 days after infection (+) with VV. Gel electrophoretic mobility shift
assay was performed as described under "Experimental Procedures."
Kinetics of Mig mRNA expression and
RF-1 induction in
WT mice (B) and IFN-
/
mice (C)
during VV infection. Livers from three mice were pooled for each time
point. Mig mRNA was analyzed by RT-PCR as described in
Fig. 1, and
RF-1 was detected as described in A. Five
micrograms of each nuclear extract was analyzed for the presence of
these complexes by EMSA.
RF-1, an additional
RE-1 binding protein was also
detected by EMSA and is referred to here as
RF-2. The
RF-2
complex demonstrated higher mobility than
RF-1 and was detected in
both B6 WT and IFN-
/
mice, but the levels were
clearly higher in the latter (Fig. 4A, lanes 2 and 3, respectively). This complex was not detected in the
DR
/
mice and in IFN-
/
mice treated
with antibody to IFN-
/
(Fig. 4A, lanes 4 and 5). In addition, the time course studies show a similar
pattern of expression of Mig mRNA and
RF-2 in the
livers of VV-infected IFN-
/
mice (Fig.
4C). These findings are consistent with a role for
RF-2
in the induction of Mig expression in IFN-
/
mice
infected with VV.
/
Mice--
The preceding
sections strongly suggested that IFN-
/
is responsible for the low
level Mig expression in VV-infected IFN-
/
mice. It
is possible that the transcription factor
RF-2, most probably
induced by IFN-
/
, could have mediated this effect, because
RF-1 is not detected in these animals (Fig. 4A). IFN-
can directly induce some of the genes activated by IFN-
through STAT-1 complexes such as AAF or ISGF3 (33). A comparison of the
RE-1
element, which binds to
RF-1, with the GAS element, which binds to
STAT-1 complexes GAF·AAF, showed partial homology (7 of 15 base
matches). It was therefore possible that AAF may also contribute to the
transcriptional activation of Mig mRNA in the
IFN-
/
mice. Indeed, we have detected AAF in
VV-infected B6 WT (Fig. 4A, lane 8) and
IFN-
/
mice (Fig. 4A, lane 7)
but not in the VV-infected DR
/
mice or
IFN-
/
mice treated with antibody to IFN-
/
(Fig. 4A, lanes 9 and 10). In
addition, STAT-1 complexes, detected by Western blot analysis, were not
present in extracts obtained from VV-infected DR
/
mice
or from VV-infected IFN-
/
mice treated with
anti-IFN-
/
(data not shown).
RF-2 and AAF Share a Common Component--
Our results suggest
that, although
RF-1 is required for optimal induction of
Mig mRNA in B6 WT mice,
RF-2 is responsible for the
low level Mig expression in VV-infected IFN-
/
mice.
Guyer and colleagues (13) recently reported that the
RF-1 complex
consists of three proteins, one of which is related antigenically to
STAT-1
. It is possible that
RF-2 also contains a STAT-1 subunit,
which binds to the
RE-1 element of the Mig promoter. In gel shift
analysis, the addition of anti-STAT-1
antibody resulted in reduced
mobility (super-shift) of
RF-2 and AAF, suggesting that these
complexes are antigenically related to STAT-1
(Fig.
5). Because both AAF and
RF-2
exhibited identical mobility and are reactive to STAT-1 antibody, it is
possible that
RF-2 is in fact AAF. Supporting this possibility is
the recent finding of Ohmori et al. that the
RF-2
complex, which appears to be a STAT-1 homodimer, binds to the
3'-half-site of the
RE-1 motif (Ref. 42 and T. Hamilton, personal
communication). In addition, our UV cross-linking studies on
RF-2
and AAF show two identical bands corresponding to molecular mass
of 95 kDa (data not shown), consistent with the idea that
RF-2
complex may indeed be a STAT-1 homodimer. Although our data show that
AAF and
RF-2 are identical (STAT-1 homodimers), we have used the
term AAF in gel shift assays where the DNA binding element is labeled
GAS element. On the other hand, the term
RF-2 is used in assays
where the DNA binding element is
RE-1.
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Fig. 5.
Supershift of electrophoretic mobility
of RF-2 and AAF DNA complexes by
anti-STAT-1
antibody. Nuclear extracts
prepared from livers of VV-infected IFN-
/
mice were
incubated for 2 h at 4 °C in the absence (
) or presence (+)
of anti-STAT-1
antibody. These samples were then analyzed for DNA
binding (to
RE-1 or GAS) activity by EMSA as described under
"Experimental Procedures." The arrows indicate the
position of the super-shifted complexes.
RF-2 Binds
RE-1 and GAS Elements with Differential
Affinity--
We next examined the binding affinity of
RF-2,
induced in IFN-
/
mice, to
RE-1 and GAS elements.
A competition assay was prepared in which increasing amounts of
unlabeled oligonucleotide containing one of the elements GAS
(GBP gene),
RE-1 (Mig gene), or ISRE (Crg-2 gene) were added to assays in which the complex was
formed with radiolabeled oligonucleotides containing either the
RE-1 or GAS sites.
RE-1 fragment was
increased by 10-fold or more molar excess. The intensity of the complex
formation was quantified with a densitometer and plotted against
inhibitor concentration (data not shown). It was estimated that the
apparent dissociation constant (the concentration of competitor DNA
required to reduce AAF·GAS complex formation by 50%) for GAS was 0.4 nM and
RE-1 was 1.5 nM.
View larger version (42K):
[in a new window]
Fig. 6.
Binding affinity of
RF-1,
RF-2, and AAF to GAS
and
RE-1 DNA elements. Nuclear extracts
were prepared from livers pooled from 3 IFN-
/
(A and B) and B6 WT (C) mice. All
samples were taken at day 3 after infection with VV. Binding affinity
to GAS (A) or
RE-1 (B and C) DNA
elements was determined by EMSA, as described under "Experimental
Procedures," using radiolabeled GAS or
RE-1 elements in a
competition assay using severalfold excess of unlabeled
oligonucleotides, as indicated. An ISRE element of the murine
Crg-2 gene served as a nonspecific oligonucleotide
probe.
RE-1 element was used as a
probe and competed with a 1-fold molar excess of unlabeled GAS element,
there was approximately a 50-fold reduction in
RF-2·
RE-1 complex formation as analyzed by densitometry (Fig. 6B). In
contrast, 1-fold molar excess of unlabeled
RE-1 resulted in a
20-fold reduction in
RF-2·
RE-1 complex formation (Fig.
6B).
RE-1, a 1-fold molar excess of unlabeled GAS
element resulted in loss of
RF-2·
RE-1 complex formation (Fig.
6C). Furthermore, a reduction in
RF-1·
RE-1 complex formation was observed when unlabeled GAS element was increased by
20-fold molar excess. When a 1-fold molar excess of unlabeled
RE-1
was used,
RF-2·
RE-1 complex formation was reduced markedly (Fig. 6C). These results demonstrate that the
RF-2 complex displays a higher binding affinity for GAS element than for
RE-1 element.
B Complex Is Induced in the IFN-
/
Mice--
The presence of NF-
B sites in the Mig gene was
previously shown to be important for transcription of Mig
mRNA involving a synergistic interaction between IFN-
and TNF
(14). Because it is well documented that TNF is a potent inducer of
NF-
B, it is possible that NF-
B motifs may also contribute to the
low levels of Mig mRNA in the IFN-
/
mice. However, levels of mRNA for TNF and NF-
B (data not shown) and of NF-
B protein·DNA complexes (Fig.
7) in livers of VV-infected B6 WT,
IFN-
/
, and DR
/
mice were similar.
Treatment of VV-infected IFN-
/
mice with
neutralizing antibodies to TNF did not result in loss of Mig expression
(data not shown). These results suggest that TNF and NF-
B complexes
do not play a significant role in the transcriptional activation of
Mig mRNA in VV-infected IFN-
/
mice.
View larger version (26K):
[in a new window]
Fig. 7.
Activation of NF- B1
and NF-
B1/RelA in livers of uninfected and
virus-infected mice. Nuclear extracts were prepared from livers
pooled from groups of three mice, 3 days after infection with VV.
Samples were from uninfected B6 WT mice (lane 1),
VV-infected B6 WT (lane 2), IFN-
/
(lane 3), and DR
/
(lane 4) mice.
Five micrograms of each nuclear extract was analyzed for the presence
of these complexes by EMSA as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
induces tyrosine phosphorylation of STAT-1
, and a
homodimeric form of STAT-1
binds to GAS (33), a SBE that has been
identified as a critical sequence motif involved in the transcriptional
activation of many IFN-inducible genes, including the IRF-1,
ICAM, and Mig genes (35-37). Some genes are
stimulated transcriptionally by only one type of IFN, whereas others
like the guanylate-binding protein (GBP) gene
respond to IFN-
and IFN-
(5, 33). The molecular pathways that
allow two different ligand-receptor pairs to cause a similar biological
response are of major interest in the study of intracellular signaling.
/
mice. IFN-
has previously
been shown to be necessary for Mig mRNA expression in
macrophages in vitro (8, 12). More recently, a reduction in
Mig mRNA transcripts was demonstrated in vivo
using a neutralizing antibody against IFN-
in BALB/c mice infected with VV (15). In this report, we have analyzed the expression of Mig in
response to VV infection in normal B6 WT mice and gene knockout mice
lacking IFN function. The high levels of expression of this chemokine
in spleens, ovaries, and uteri of normal, WT mice early after infection
paralleled that of IFN-
, IFN-
, and IFN-
expression (Figs. 1
and 3A). The high levels of Mig and IFN-
expression in
the ovaries and uterus also correlate with a previous report, which
showed that VV replicates to high titers in these organs (32). These
IFNs are induced early in infection and are known to play a critical
role in the defense against poxvirus infections (19). Because the IFNs
confer resistance to many types of viruses by activating a set of
IFN-inducible genes, we speculated that Mig mediated some of the
antiviral effects of IFNs. Indeed, we have recently demonstrated that
Mig expressed by an rVV exhibited antiviral activity in vivo
(24).
in the induction of Mig, we
analyzed profiles of the chemokine in IFN-
/
mice
after VV infection. Clearly, mRNA transcripts for Mig were detected
in organs of these mice, albeit at reduced levels compared with normal
B6 WT mice (Fig. 1B), indicating that factor(s) other than
IFN-
contributed to the expression of Mig in mice lacking the
cytokine. This is the first demonstration of Mig mRNA
expression in vivo in the complete absence of IFN-
.
Previous reports have shown that treatment of the macrophage cell line
RAW 264.7 with cycloheximide and IFN-
augmented Mig transcription
(29), whereas treatment with cycloheximide alone does not (8). It was
suggested that, in addition to increasing transcription, IFN-
might
also affect the stability of Mig mRNA (8, 29). Thus
there was a possibility that the Mig mRNA expressed in
the IFN-
-deficient mice was unstable and did not translate to
protein. However, our data indicate that, along with mRNA, Mig
protein is also present in the liver extracts from VV-infected
IFN-
/
mice (Fig. 2B).
and/or -
were responsible for induction
of low levels of Mig expression in these animals, two approaches were
taken. First, antibody to IFN-
/
was used to neutralize activity.
In antibody-treated, VV-infected IFN-
/
mice,
expression of this chemokine was completely abolished (Fig. 3B). Second, mice deficient in receptors for both
IFN-
/
and IFN-
(DR
/
) were employed. The
overall phenotype of the DR
/
mice is identical to that
of STAT-1
/
mice in that they both display a global
deficiency in their ability to respond to either IFN-
or IFN-
/
(41). We confirmed the absence of STAT-1 protein complexes in the
DR
/
mice by immunoblotting with antibody specific for
STAT-1 (data not shown). We found that Mig was not induced in
DR
/
mice following infection with VV (Fig.
3C). In contrast, IFN-
/
R
/
mice, with
intact IFN-
function, expressed Mig at similar levels to WT mice
(data not shown). This was expected as IFN-
has been shown to induce
high levels of Mig mRNA through the induction of
transcription factor
RF-1 (28). In VV-infected mice with intact
IFN-
/
function, but deficient in IFN-
receptors (IFN-
R
/
), liver Mig mRNA levels were similar
to those observed in VV-infected IFN-
/
mice, but
lower than VV-infected WT controls (Fig. 2A). Taken together, our data indicate that Mig can be induced in vivo
by IFN-
/
in mice deficient for IFN-
function, however, the
levels are significantly lower.
RF-1 is involved in IFN-
-mediated
transcriptional regulation through
RE-1, a major regulatory element in the Mig promoter (28). The
RF-1 complex is distinct from the
IFN-
-responsive transcription factor GAF but contains closely related members of the STAT protein family (13). The rapid and preferential induction of
RF-1 by IFN-
in a variety of cells suggests that this factor is responsible for controlling a subset of
IFN-
-mediated responses, including Mig expression (13) even though,
to date, the
RE-1 element has only been found in the Mig
gene promoter. In this study, the levels and kinetic expression of
RF-1 detected in the liver is consistent with Mig expression in
VV-infected B6 WT mice (Fig. 4, A and B).
However, despite the absence of
RF-1 in VV-infected
IFN-
/
mice (Fig. 4A), Mig
mRNA was induced (Fig. 1B and Fig. 2A).
RE-1-binding
protein, which we have referred to as
RF-2 (Fig. 4A).
This complex exhibited higher mobility than
RF-1 and was present in VV-infected B6 WT and IFN-
/
mice but not in
VV-infected DR
/
or in IFN-
/
mice
treated with antibody to IFN-
/
. Furthermore,
RF-2 induction paralleled Mig mRNA expression in livers of infected
IFN-
/
animals (Fig. 4C). These findings
are consistent with the hypothesis that
RF-2 is responsible for
Mig mRNA expression in the absence of IFN-
function.
Of note is the finding that the
RF-2 complex was more prominent in
the IFN-
/
mice compared with WT mice. This may be
due to the absence of
RF-1 in liver nuclear extracts of
IFN-
/
mice, which can compete with
RF-2 for
binding to
RE-1. Recently, Ohmori and colleagues (42) have reported
that
RF-2 complex binds to the 3'-half-site of the
RE-1 motif and
is likely to be a STAT-1 homodimer complex. In addition, our UV
cross-linking studies show two bands corresponding to molecular mass of
95 kDa (data not shown) similar to AAF, suggesting that
RF-2 is also a STAT-1 homodimer.
mediates Mig
mRNA expression in the absence of IFN-
, candidate transcription factors ISGF3, AAF, and IRF-1 were considered. The role of ISGF3 was
ruled out, because there was no cross competition between
RE-1 and
ISRE of the GBP gene by EMSA (data not shown). Furthermore, the absence of IRF-1 binding sites in the Mig promoter excludes the
involvement of this transcription factor. A comparison of the "GAS
core site" consensus sequence TT(C/A)CNNNAA with
RE-1 reveals
homology to both halves of the
RE-1 imperfect palindrome (28). The
significance of this homology is unclear, but it may play a role in the
binding to STAT-1 protein. There is a distinct possibility that the
expression of Mig in VV-infected IFN-
/
mice is
mediated through AAF, which is able to activate GAS driven genes (33).
Supporting this idea is the observation that IFN-
, like IFN-
,
stimulates the appearance of a nuclear GBP-1 GAS binding activity (5,
33). This mechanism would allow IFN-
to directly induce some of the
genes activated by IFN-
. Here we have shown that AAF was activated
in infected IFN-
/
mice but not in infected
DR
/
mice (Fig. 4A). Consistent with this,
low affinity binding of AAF to
RE-1 did occur between the two
components as demonstrated in competition experiments using EMSA (Fig.
6A). We have at least four lines of evidence that suggest
that
RF-2 is indeed AAF. First,
RF-2 and AAF exhibit identical
mobility on EMSA (Fig. 4A). Second, both factors are
reactive with STAT-1 antibody (Fig. 5). Third, formation of
RF-2·
RE-1 complex was abolished when competed with a 1-fold
molar excess of unlabeled GAS element (which is specifically bound by
AAF with high affinity) (Fig. 6C) and
RF-2 binds with
higher affinity to GAS elements than to
RE-1 element (Fig.
6C). Fourth, UV cross-linking studies for both factors revealed the presence of two bands with a molecular mass of
approximately 95 kDa, which correspond to that of STAT-1 (data not shown).
B binding site located at position
145 to
154 and an AP-2
binding site at position
214 to
221 (29). The finding of an
NF-
B-like site raised the possibility that the Mig gene might also be regulated by TNF. However, our results do not support a
role for TNF with respect to transcription of Mig in VV-infected IFN-
/
mice. There were no differences in levels of
expression of TNF mRNA, NF-
B mRNA (data not shown), or
NF-
B complexes (Fig. 7) in livers of WT, IFN-
/
,
and DR
/
mice. These findings suggest that
Mig mRNA expression in infected IFN-
/
mice is not mediated through NF-
B complexes.
subunit of
RF-2
binds to
RE-1 in the Mig promoter and drives the transcription of
Mig gene in the IFN-
/
mice. In this
regard, a recent report showed that IFN-
treatment of fibroblasts
from STAT-1-deficient mice failed to induce Mig expression, indicating
a requirement for STAT-1 protein for transcription of this chemokine
(14).
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Joshua Farber for kindly providing the Mig primer sequences and the Mig antiserum (JH48). We also thank Drs. Michael Crouch and Frances Shannon for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was funded in part by the National Center for HIV Virology Research, the Medical Foundation of the University of Sydney, and the National Health and Medical Research Council (to G. K.).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.
A recipient of a Human Frontiers Foundation Postdoctoral Fellowship.
¶¶ A Medical Foundation Fellow of the University of Sydney and an International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Pathology, Host Defence Group, Blackburn Bldg., D06, University of Sydney, New South Wales 2006, Australia. Tel.: 61-2-9351-6151; Fax: 61-2-9351-3429; E-mail: gunak@med.usyd.edu.au.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M005773200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IFN, interferon;
Mig, murine Mig;
Crg-2, cytokine responsive gene-2;
ISRE, IFN-stimulated response element;
GAS, IFN- activation sequence;
RE-1, IFN-
-responsive element;
GAF, IFN-
-activated factor;
AAF, IFN-
-activated factor;
ISGF3, IFN-stimulated gene factor 3;
RF-1/2, IFN-
responsive factors 1 and 2;
STAT, signal transducer
and activator of transcription;
EMSA, electrophoretic mobility shift
assay;
RT-PCR, reverse transcriptase-polymerase chain reaction;
NF-
B, nuclear factor
B;
TNF, tumor necrosis factor;
VV, vaccinia
virus;
, rVV, recombinant vaccinia virus;
WT, wild-type;
PFU, plaque-forming unit(s);
SBE, STAT binding element.
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