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ß Mediates the Lipopolysaccharide-Induced Activation of Transcription Factor Stat1 in Mouse Macrophages: Pivotal Role of Stat1 in Induction of the Inducible Nitric Oxide Synthase Gene1
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Wilkinson Laboratory of the Kansas Cancer Institute and Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160; and
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the induction by LPS of
the mouse inducible nitric oxide synthase (EC 1.14.13.39) gene. LPS
induced both the tyrosine phosphorylation of Stat1 and the
production of nitric oxide in a time- and dose-dependent manner. The
phosphorylation of Stat1 elicited by LPS differed from that observed
using IFN-
or IFN-ß, in that LPS induced less phosphorylated
protein and the time course of induction was much delayed (2–4 h
compared with 30 min). Cycloheximide inhibited LPS-mediated Stat1
phosphorylation. In addition, cell culture supernatants derived from
macrophages treated with LPS for 4 h could be transferred to naive
macrophage cultures resulting in rapid (30 min), rather than delayed (4
h), phosphorylation of Stat1. Together, these results implicated an
autocrine/paracrine effector protein(s) in the phosphorylation process.
LPS stimulated phosphorylation of Stat1 in peritoneal macrophages
derived from IFN--knockout mice, negating any possibility that
IFN- was the mediator. By contrast, neutralizing Ig raised against
mouse IFN-ß inhibited both the delayed LPS-mediated
phosphorylation of Stat1 and the rapid induction of phosphorylation
induced by supernatants from LPS-stimulated cultures. Collectively,
these results show that LPS-induced IFN-ß production, Stat1
activation, and nitrite accumulation closely parallel one another,
suggesting that indirect activation of transcription factor Stat1 by
IFN-ß is a critical determinant of LPS-mediated inducible nitric
oxide synthase gene expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to produce the enzyme
inducible nitric oxide synthase
(iNOS).3 This, in turn,
catalyzes the production of nitric oxide (NO), which helps mediate the
cytotoxic function of macrophages against tumor cells and a variety of
microbes (1, 2, 3). LPS, by itself, induces dose-dependent low level
synthesis of iNOS, and these levels are synergistically augmented by
the presence of IFN- (4, 5, 6, 7). IFN- alone fails to induce the
production of iNOS by macrophages from certain strains of mice (7, 8, 9).
The combination of LPS and IFN- has the effect of accelerating the
kinetics of induction of iNOS gene transcription (7), mRNA accumulation
(7), protein synthesis (10), and NO production (10, 11) compared with
the kinetics of induction elicited by LPS alone.
It has been reported that the combination of LPS plus IFN- induces
iNOS gene expression through the activation of transcription factors
that bind to LPS and IFN response elements in the iNOS promoter. Among
these elements, two 
B sites (12, 13, 14), an octamer (13, 15) (our
unpublished observations), and two juxtaposed IFN-stimulated response
elements (ISREs) (16, 17, 18) are functional in regulating iNOS gene
expression. We have recently identified a sixth element, an
IFN--activated site (GAS), that is also required for iNOS promoter
activity (18). The iNOS GAS is bound by transcription factor Stat1
after stimulation of RAW 264.7 macrophages with IFN-, LPS, or LPS
plus IFN- (18). IFN- or the combination of LPS plus IFN-
induces GAS binding of Stat1 within 30 min of exposure. In contrast,
LPS induction of GAS binding activity requires approximately 2 h
and is quantitatively less than that induced by IFN- alone or LPS
plus IFN- (18).
Induction of the iNOS gene results from a signal transduction cascade
that activates/induces the aforementioned transcription factors. Thus,
we hypothesized that the reduced and delayed production of iNOS and NO
by LPS stimulation alone is due to rate- and amplitude-limiting steps
in the activation or induction of these transcription factors.
Consistent with this hypothesis, it has previously been demonstrated
that induction by LPS alone of iNOS protein production (19) and NO
synthesis (the latter measured as nitrite accumulation) (20, 21) is
dependent upon the prior autocrine/paracrine synthesis of IFN-ß.
Because both type I and type II IFNs induce tyrosine phosphorylation of
Stat1 (22), we predicted that activated Stat1 would be the
limiting transcription factor in LPS-stimulated vs LPS- plus
IFN--stimulated macrophages. The data presented here confirm that
these hypotheses are correct; autocrine/paracrine IFN-ß is the
LPS-induced feedback activator of Stat1, a transcription factor
critical for LPS-mediated induction of the mouse iNOS gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 wild-type mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). IFN--knockout mice were provided by
Genentech (South San Francisco, CA) and obtained through the National
Institutes of Health (Bethesda, MD) after backcross with the C57BL/6
strain. Each strain was used at the age of 6 to 8 wk. Mice were
injected i.p. with 3 ml of 4% Brewer thioglycolate (Difco, Detroit,
MI), and peritoneal macrophages were harvested 5 days later by lavage
with cold HEPES-buffered (25 mM) RPMI 1640 growth medium (Life
Technologies, Grand Island, NY). They were then seeded at 1 x
107 cells/100-mm culture dish and incubated overnight in
growth medium supplemented with 10% fetal clone I (HyClone, Logan,
UT), 2 mM glutamine (JRH Biosciences, Lenexa, KS), 100 µg/ml
streptomycin (Sigma, St. Louis, MO), and 100 U/ml penicillin
(Apothecon, Princeton, NJ) at 37°C in a humidified, 5%
CO2 environment before stimulation.
Natural mouse IFN-ß and rabbit anti-mouse-IFN-ß antiserum
were purchased from Lee BioMolecular Research Laboratory (San Diego,
CA). Recombinant murine IFN- was obtained from Schering-Plough
through the American Cancer Society (Atlanta, GA). The lipid A-rich
fraction II of LPS, extracted from Escherichia coli O111:B4
with phenol, was obtained from List Biological Laboratories (Campbell,
CA). Anti-Stat1 (N terminus) mAbs (used for Western blot analyses) were
purchased from Transduction Laboratories (Lexington, KY). Anti-Stat1
84/91 polyclonal Abs (used for immunoprecipitation) were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Protein A-Sepharose
conjugate (used for Ig purification and immunoprecipitation), rabbit
anti-mouse IgG-horseradish peroxidase Ab conjugate (used for
Western blots), and normal rabbit serum (used for Ab neutralization and
immunoprecipitation studies) were purchased from Sigma.
Anti-phosphotyrosine (anti-P-tyr) Ab mixture (PY-Plus)
was obtained from Zymed (South San Francisco, CA). Endotoxin was
undetectable in culture medium or reagents, as determined by the
Limulus amebocyte lysate assay (Associates of Cape Cod,
Woods Hole, MA), at a sensitivity of 50 pg/ml.
Cell extract preparation and electrophoretic mobility shift assays (EMSAs)
After overnight culture, cells were washed twice and then incubated in medium alone or in medium that contained appropriate stimuli for the times indicated in the figures. Whole cell extracts were prepared as described by Andrews and Faller (23), with slight modification. Cells were washed twice with cold PBS and once with cold PBS containing 1 mM sodium orthovanadate, and lysed in high salt buffer that contained 20 mM HEPES (pH 7.9 with KOH), 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0 with KOH), 420 mM KCl, 25% glycerol, 1 mM DTT, and the following protease/phosphatase inhibitors: 0.5 mM PMSF, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 10 µg/ml leupeptin, and 1 mM sodium orthovanadate. Cell lysates were centrifuged (23), and supernatants from each lysate were aliquoted and frozen at -70°C until needed. Nuclear extracts were prepared by a modification of the method of Shapiro et al. (24). Cells were washed as described above and then washed once with hypotonic buffer (24) that contained the same protease/phosphatase inhibitors described above. Cells were scraped into 1 ml of hypotonic buffer plus protease/phosphatase inhibitors and disrupted with 10 strokes of a tight-fitting Dounce pestle (Kontes, Vineland, NJ). Immediately after breaking the cells, 0.1 vol of 75% sucrose were added with mixing. Nuclei were pelleted in an Eppendorf microcentrifuge (model 5415 C, Eppendorf, Fremont, CA) at maximal speed for 2 min at 4°C. The supernatant was discarded, and the nuclear pellet was resuspended in 60 µl of the high salt buffer used for whole cell extracts (see above). The nuclear suspension was incubated on ice for 30 min with periodic gentle mixing. Nuclear debris were removed by centrifugation, as described above, for 30 min. The supernatant was removed to a new tube, and the extract was aliquoted and stored frozen (-70°C) until needed. EMSAs and supershift assays were performed using nuclear extracts prepared in this manner, exactly as described by Gao et al. (18).
Purification of Abs
Rabbit Ig against mouse IFN-ß and normal rabbit Ig were
purified by protein A-Sepharose column chromatography. After
purification, Igs were sterilized by filtration through a Millex-GV
filter (Millipore, Bedford, MA) and assayed to determine the
concentration of protein. Either normal (control) or
anti-IFN-ß IgG was then used for IFN-ß neutralization
studies, as indicated in Results.
Western blot analysis
Proteins for analysis were separated via SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were incubated overnight at 4°C in blocking buffer (1% BSA, 10 mM Tris-HCl (pH 7.5), and 100 mM NaCl), blotted with anti-Stat1 (N terminus) mAbs or anti-P-Tyr Abs in blocking buffer that contained 0.1% Tween-20 for 1 h, washed five times in wash buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.5% Tween 20) for 6 min each time, and blotted for 1 h with a rabbit anti-mouse IgG-horseradish peroxidase conjugate in wash buffer that contained 5% nonfat milk. Each membrane was then washed five times in wash buffer for 6 min each. Specific binding of anti-Stat1 (N terminus) or anti-P-Tyr was detected using the Renaissance chemiluminescence detection kit (DuPont, Boston, MA). Autoradiograms were scanned, where indicated, with a Molecular Dynamics (Sunnyvale, CA) Personal Densitometer SI and analyzed with the ImageQuaNT software package (Molecular Dynamics) using a Windows NT (Microsoft, Redmond, WA) format.
To strip and reprobe PVDF membranes, they were incubated in a solution
containing 7 M guanidine hydrochloride, 50 mM glycine (pH 10.8), 0.05
mM EDTA, 0.1 M KCl, and 20 mM ß-ME for 30 min at room temperature.
Membranes were then washed twice with water for 10 min each, blocked,
reprobed as indicated in Figure 2
, and then developed as described
above.
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Nitrite concentrations were measured in cell culture
supernatants using the Greiss reagent in a previously described
colorimetric assay (25). Absorbance (at 570 nM) was determined using a
Dynatech MR700 plate reader (Chantilly, VA). Sodium nitrite, dissolved
in RPMI 1640, was used to generate a standard concentration
curve. Cell culture supernatants were assayed for antiviral activity
(IFN-ß) by the plaque reduction method using monolayers of
vesicular stomatitis virus-infected mouse L-929 cells. The assay was
performed as previously described (26), except that 96-well tissue
culture dishes were used. Each supernatant dilution was assayed in
triplicate. IFN activity is expressed in terms of National Institutes
of Health reference units, where 1 U of activity is defined as the
amount of IFN that causes a 50% reduction in viral plaques. Mouse
IFN-ß (Lee BioMolecular Research Laboratory) was used as the
reference standard.
Immunoprecipitation
Peritoneal macrophages were cultured overnight (2 x
107 cells/150-mm culture dish), washed twice with culture
medium, and then incubated with medium that contained appropriate
stimuli for the times indicated in Figure 2
. Cells were then washed
three times with cold PBS, incubated in cold lysis buffer (PBS, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 µg/ml PMSF,
10 µg/ml aprotinin, and 1 mM sodium orthovanadate) with shaking for
10 min, and disrupted by repeated aspiration/ejection through a
23-gauge needle. Cellular debris were removed by centrifugation in an
Eppendorf microcentrifuge (1 min, 4°C, maximum speed), and the
supernatant was precleared by adding 1 µg of normal rabbit Ig with 20
µl of protein A-Sepharose conjugate followed by a 10-min incubation
at 4°C and another brief centrifugation. Two micrograms of polyclonal
anti-Stat1 84/91 Abs were added to the cellular lysate, which was
then incubated overnight at 4°C. After this, 100 µl of protein
A-Sepharose conjugate was added, and the incubation was continued for
approximately 4 h at 4°C. The immunoprecipitate was collected
after a brief centrifugation. The pelleted beads were washed four times
with cold lysis buffer, resuspended in 100 µl of SDS-sample buffer,
and boiled for 3 min. After another brief centrifugation, an aliquot
was used for Western blot analysis, as described above. Remaining
sample was stored at -70°C for future use.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in mouse macrophages
Previous results from our laboratory (18) demonstrated a delayed
induction of the binding of Stat1 to oligonucleotides that mimic
either the iNOS or IFN regulatory factor (IRF-1) GAS binding sites.
Because the activation of Stat1 is accompanied by a mobility shift
in SDS-polyacrylamide gels (27), we sought to determine, by Western
blot analysis, the kinetics of Stat1 activation in response to
treatment of C57BL/6 peritoneal macrophages with IFN-, IFN-ß, or
LPS. Figure 1 shows a Western blot that
investigates these kinetics. Both IFN- and IFN-ß induced the rapid
appearance (within 30 min) of the slower migrating form of activated
Stat1 (A and B). Although 100-fold more
IFN-ß than IFN- was used on a per unit basis, conversion of
Stat1 to its activated form was more complete with IFN- than with
IFN-ß (compare the 0.5 h lanes, A and
B). Levels of activated Stat1 declined after 0.5 h
of treatment and were not detectable 4 h poststimulation.
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activation, but to a lesser extent than did either IFN- or IFN-ß
(Fig. 1C). Furthermore, maximal levels of activated Stat1
were reached only after approximately 4 h of stimulation. The
amount of detectable activated Stat1 decreased thereafter and was
barely detectable by 8 h poststimulation. Thus, at the
concentrations of mediators used in this study, LPS caused the
activation of Stat1 in peritoneal macrophages, but with slower
kinetics and at amounts consistently less than those achieved with
IFN- or IFN-ß.
In addition to these changes in Stat1 activation, the abundance of
Stat1 protein itself was also affected by the stimuli used in Figure 1. Each of these agents increased, by approximately 50%, the level of
Stat1 protein found in whole cell extracts (compare lanes
M with lanes 12 in A–C; densitometric
scanning data not shown). The significance of the stimulatory effect of
these agents on Stat1 production, as it may relate to secondary gene
induction, is currently not known.
LPS induces the tyrosine phosphorylation of Stat1
The activation of Stat1 and the SDS-PAGE mobility shift that
accompanies such are the results of tyrosine phosphorylation of residue
Y701 of this protein (28). Because tyrosine phosphorylation
of Stat1 is essential for its transcriptional activity (22, 27, 28),
we sought to determine whether LPS also induced tyrosine
phosphorylation of Stat1. To this end, immunoprecipitation and
Western blot experiments were performed. Cellular lysates were first
immunoprecipitated with anti-Stat1 polyclonal Abs. Next,
electrophoretically separated proteins were blotted and reacted
sequentially with the anti-P-Tyr Ab mixture and then
with the anti-Stat1 mAbs (see Materials and Methods).
The results of such experiments are shown in Figure 2. Untreated macrophages contained no
detectable tyrosine-phosphorylated Stat1 (Fig. 2, top
panel, lane M). However, IFN-, LPS, and IFN-ß each
induced the appearance of a new band that reacted with the
anti-P-Tyr Abs (Fig. 2, top panel,
lanes , L, and ß, respectively).
In this experiment treatment with the IFNs was performed for 30 min,
while LPS treatment was conducted for 4 h. As expected, LPS
stimulated consistently less tyrosine-phosphorylated Stat1 than that
induced by IFN- and required a longer period of stimulation before
it was detectable. These results validated an assumption made in Figure 1 that the Stat1 mobility shift caused by LPS treatment was the
result of tyrosine phosphorylation of Stat1 similar to that observed
with the IFNs, but with different kinetics.
Cycloheximide (CHX) inhibits the LPS-induced phosphorylation of
Stat1
The delay in LPS-induced tyrosine phosphorylation of Stat1
raised the possibility that an autocrine/paracrine mediator of Stat1
activation might be induced by LPS. If the proposed autocrine/paracrine
mediator were a protein factor, then its induction should be ablated by
the protein synthesis inhibitor, CHX. Thus, macrophage cultures were
preincubated for 30 min in the presence or the absence of CHX, after
which cultures were either stimulated with 100 ng/ml LPS or left
untreated for 2 h (in the continued presence of CHX where
appropriate). The CHX concentration used was previously determined to
inhibit >95% of cellular protein synthesis (data not shown). As shown
in Figure 3, CHX did not affect the
Stat1 detected in untreated macrophages (compare lane 2
with lane 1). However, the LPS-induced activation of
Stat1 (lane 3) was completely abolished by
treatment with CHX (lane 4). Thus, protein synthesis
is required for Stat1 phosphorylation to develop, supporting the
hypothesis that autocrine/paracrine protein(s) mediates LPS-induced
Stat1 phosphorylation. In addition, the LPS-induced increase in
Stat1 protein accumulation, observed in Figure 1C after
0.5 to 2 h of stimulation, was also inhibited by CHX treatment
(compare the band intensity observed in lane 4 with that
observed in lane 3).
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phosphorylation
In an approach complementary to the CHX experiments, we asked
whether supernatants from LPS-stimulated macrophage cultures, when
transferred to naive cultures, could induce rapid (30 min), rather than
delayed (4 h), activation of Stat1. As shown in Figure 4, when a primary macrophage culture was
treated for 4 h with 100 ng/ml LPS and the culture supernatant was
then transferred to a fresh, secondary culture of macrophages,
phosphorylation of Stat1 in this secondary culture was observed
within 30 min (Fig. 4, lane 3). The observed result was
similar to that seen when macrophage cultures were treated with IFN-
alone for 30 min (compare lanes 2 and 3) or with
LPS alone for 4 h (compare lane 3 with lane
4 of Fig. 1C). Treatment of secondary macrophage
cultures for 4 h with supernatants from LPS-treated primary
cultures showed results similar to those obtained with a 30-min
treatment (Fig. 4, lane 4).
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activation
is IFN-ß
LPS is known to induce production of several cytokines by mouse
macrophages (29). A number of these could interact with the producer
macrophages and cause activation of transcription factor Stat1 (30).
The prototypic activators of Stat1 are IFN- and IFN-ß.
Because the production of both IFN- (31) and IFN-ß (20, 21, 32, 33, 34, 35, 36) by mouse macrophages can be induced by LPS, we wanted to
determine whether either was the mediator of LPS-induced Stat1
activation.
The potential role of IFN- was tested by examining the ability of
LPS to stimulate Stat1 phosphorylation in macrophages from
IFN--knockout mice. LPS treatment of peritoneal macrophages isolated
from both wild-type and IFN--knockout C57BL/6 mice resulted in
equivalent levels of Stat1 phosphorylation (Fig. 5, compare lanes 2 and
4). Therefore, production of IFN- was not required for
LPS-induced phosphorylation of Stat1.
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ß in this system was investigated using
neutralizing Ig raised against these IFNs. Preliminary studies showed
that anti-IFN-ß Ig, used at a concentration of 0.33 µg
protein/ml, inhibited LPS-induced nitrite accumulation by C57BL/6
peritoneal macrophages by approximately 90% (in two experiments an
average of 2.3 µM nitrite was generated in the presence of
anti-IFN-ß Ig, compared with 22 µM generated in the presence
of normal rabbit Ig). This same concentration of Ig also inhibited,
nearly completely, the LPS-induced phosphorylation of Stat1 (Fig. 6, compare lanes 2 and
6). In contrast, normal rabbit Ig, which reduced by an
average of 20% LPS-induced nitrite accumulation by C57BL/6
macrophages, caused little or no change in the LPS-induced
phosphorylation of Stat1 (Fig. 6, lane 4).
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(Fig. 6, lane 7), but neutralizing Ig raised
against IFN-ß nearly abolished rapid activation (lane
8). In contrast to these results, when naive macrophage cultures
were exposed for 4 h to supernatants from LPS-treated primary
cultures, anti-IFN-ß-Ig failed to block the activation of
Stat1 completely (lane 10). This observation
probably resulted from levels of autocrine/paracrine IFN-ß
exceeding those of neutralizing Ig in the latter experiment due to a
combined presence of IFN-ß induced by LPS in the primary culture
and by the remaining LPS transferred to the secondary culture. Finally,
lane 9 illustrates that the anti-IFN-ß-Ig itself
did not inhibit the induction of Stat1 phosphorylation in secondary
cultures that were additionally stimulated with IFN- during the last
30 min of the 4-h treatment with primary culture supernatant.
To confirm and extend the link between the activation of Stat1 by
LPS-induced autocrine/paracrine IFN-ß and induction of the iNOS
gene, we performed EMSAs using an oligonucleotide containing the iNOS
gene’s GAS and the nuclear extracts used in lanes 7 and
8 of Figure 6. Nuclear extracts, prepared from secondary
macrophage cultures that were treated with primary culture supernatant
for 30 min in the presence of control Ig, contained an iNOS GAS binding
activity (Fig. 7, lane 4, the
position of the specific binding complex is marked by the filled
arrowhead) that was not detected in extracts from untreated macrophage
cultures (lane 1). This complex was similar to that
observed in extracts prepared from IFN--treated cultures
(lane 2), although binding induced by IFN- was
greater than that induced in the supernatant transfer experiment, as
expected. In contrast with these EMSAs, nuclear extracts prepared from
secondary macrophage cultures, which had been treated for 30 min with
primary culture supernatant that additionally contained
anti-IFN-ß-Ig, showed little specific DNA-binding complex
formation (lane 6).
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observed in Figure 6. A previous report
from this laboratory (18) showed that LPS treatment by itself required
2 h before sufficient DNA binding activity could be observed by
EMSA. Thus, the results shown in Figure 7 demonstrate rapid activation
of iNOS GAS binding activity by the transferred culture supernatant.
Figure 7 further shows that the specific DNA binding complexes detected
in lanes 2 and 4, could be supershifted by adding
anti-Stat1 mAbs to the EMSA reaction. The binding of this
anti-Stat1 Ab appeared to increase the interaction of Stat1 with
the iNOS GAS-containing oligonucleotide and to sharpen the resultant
band that was seen upon autoradiography (see Fig. 7) (18). Thus, the
small amount of residual GAS binding activity observed in lane
6 was more readily detected in the supershift experiment shown in
lane 7. The supershift experiments confirmed that the EMSA
activity detected in this figure was, in fact, due to the binding of
transcription factor Stat1 to the iNOS GAS.
The activation of Stat1, production of IFN-ß, and
accumulation of nitrite parallel one another in a LPS dose-dependent
fashion
The data presented here suggest that the production of
autocrine/paracrine IFN-ß, the activation of transcription factor
Stat1, and the elaboration of NO in LPS-stimulated peritoneal
macrophage are coupled components of a LPS-induced regulatory cascade.
To investigate this further, we performed the LPS dose-response
experiment shown in Figure 8. Peritoneal
macrophage cultures were treated with the indicated doses of LPS for
4 h. Cell culture supernatants were then assayed for the presence
of IFN bioactivity, while nuclear extracts prepared from these same
cultures were assayed for Stat1 activation. A parallel set of
macrophage cultures was treated for 24 h, followed by the assay of
culture supernatants for nitrite. The upper portion of
Figure 8 shows that the activation of Stat1 was dependent upon the
dose of LPS used; 1 ng/ml LPS elicited barely detectable levels of
phosphorylated Stat1, while levels increased using 10 ng/ml and
increased yet again using 100 ng/ml. The graph in the bottom
portion of Figure 8 illustrates that the production of IFN-ß
and nitrite increased in parallel with the accumulation of
phosphorylated Stat1 when each was plotted as a function of the LPS
concentration used.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß. Second, our work identifies the molecular targets of
this autocrine/paracrine effector. These are the transcription factor
Stat1 and, as demonstrated here and in previous work (18), the
cognate binding element for phosphorylated Stat1, a GAS, which is
located in the iNOS gene’s enhancer. Third, we have ascertained, on
more molecular grounds, the basis for quantitative and temporal
differences in iNOS gene activation by LPS vs LPS plus IFN-. Last,
this work reinforces observations that two independent signals are
required for optimal iNOS gene activation, one provided by the IFNs and
the other provided by LPS-related signals distinct from those induced
by autocrine/paracrine IFN-ß.
The autocrine/paracrine production of IFN-ß has been shown to
affect macrophage activation for nitrite accumulation both negatively
and positively (19, 20, 21, 37). Both exogenously added and endogenously
produced IFN-ß (the latter induced with poly(I-C)) inhibited
IFN--induced nitrite accumulation in peritoneal macrophages from
C3H/HeSlc mice (37). Conversely, other reports (19, 20, 21) demonstrated a
positive effect of autocrine/paracrine IFN-ß on LPS-induced
production of iNOS protein (19) and nitrite accumulation (20, 21). In
these studies, anti-IFN-ß Abs nearly eliminated LPS-induced
nitrite accumulation in peritoneal macrophages derived from C3HeB/FeJ
mice (20) and significantly inhibited LPS-induced nitrite accumulation
and iNOS production in RAW 264.7 (19, 20) and J774 (21) mouse
macrophage cell lines. The contrasting effects of IFN-ß on LPS- vs
IFN--induced nitrite accumulation most likely result from the
different fates of Stat1 in IFN-- vs IFN-ß-stimulated cells.
In IFN--treated cells, phosphorylated Stat1 exists almost
exclusively as GAS-binding homodimers. In IFN-ß-treated cell
cultures Stat1 is apportioned between the transcription factor
complex, ISGF-3 (which contains Stat1, Stat2, and ISGF-3 (also
called p48)), and complexes of phosphorylated Stat1 homodimers.
Consequently, levels of Stat1 homodimers are lower in cells
stimulated with IFN-ß than in those stimulated with IFN- (38)
(J. J. Gao and M. J. Fultz, unpublished observations). Thus,
IFN--induced nitrite accumulation would be expected to be diminished
by IFN-ß treatment because Stat1 homodimers would be diminished
by the IFN-ß-induced formation of ISGF-3. Conversely,
autocrine/paracrine IFN-ß positively affects LPS-mediated
induction of nitrite accumulation because it generates Stat1
homodimers, despite their lower concentration relative to that induced
by IFN-, that are essential for gene activation.
The activation of Stat1 correlated well with the observed activation
of mouse macrophages for nitrite accumulation, implicating Stat1 as
essential for controlling the ultimate level of iNOS gene induction.
Concentrations of LPS <10 ng/ml stimulated little or no nitrite
accumulation and, similarly, stimulated little or no IFN-ß
production or Stat1 activation. On the other hand, 10 and 100 ng/ml
LPS induced progressively more nitrite accumulation, IFN-ß
production, and Stat1 activation. The combination of LPS and IFN-
induces maximal Stat1 activation, as judged by the GAS binding
activity of Stat1 homodimers (18), and, consequently, maximal
nitrite accumulation (7). This contrasts sharply with the activation of
transcription factor NF-B by LPS, where as little as 1 ng/ml is
sufficient to achieve maximal NF-B binding to synthetic
oligonucleotides yet induces no nitrite accumulation (14).
The information reported here, along with that accumulated in many
publications investigating transcriptional induction of the mouse
macrophage iNOS gene, have prompted us to propose the gene activation
model outlined in Figure 9. When both LPS
and IFN- are used as coinducers, the former stimulus effects the
release of transcription factor NF-B from its inhibitor protein
I-B. LPS also activates, without need for prior protein synthesis,
the IRF-1 gene (39), which was shown previously to be necessary for
iNOS gene induction (16, 17, 18). IFN-, on the other hand, stimulates
the tyrosine phosphorylation of Stat1, allowing the predominant
formation of homodimers. Stat1 homodimers also participate in
trans activation of the IRF-1 gene by binding the IRF-1
gene’s GAS, leading to increased abundance of the transcription factor
IRF-1 (40). The accumulation of these three transcription factors
(NF-B, IRF-1, and Stat1) in the nucleus leads to their binding,
along with one of the constitutively present octamer binding factors
(our unpublished observations), to their cognate sequence elements
upstream of the iNOS gene. This, in turn, leads to the initiation of
iNOS gene transcription.
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B, induction of the IRF-1
gene, and increased synthesis of IFN-ß. The genes encoding IFN-
and IFN-ß are constitutively transcribed in peritoneal macrophages
from C57BL/6 (35) and C3H/HeN mice (33). However, their mRNAs are
unstable (our unpublished observations) and, therefore, present in low
abundance in unstimulated cells (35). LPS treatment causes a
dose-dependent stabilization of IFN-ß mRNA without a change in the
IFN-ß gene transcription rate (33), resulting in IFN-ß synthesis
and secretion into the culture medium. While the mechanism of IFN-
induction in mouse macrophages is unknown, these cells can clearly
produce both types of IFN, although IFN-ß predominates (34, 36). As
IFN-ß accumulates, it interacts with its receptor, causing the
tyrosine phosphorylation of Stat1. IFN-ß treatment also causes
the tyrosine phosphorylation of transcription factor Stat2, which
together with Stat1 and ISGF-3 (p48) form the transcription
factor complex ISGF-3. Excess Stat1 can homodimerize, resulting in
GAS binding activity less than that induced by IFN-. Active
Stat1, generated in LPS-stimulated macrophages, should allow further
transcriptional activation of the IRF-1 gene. As NF-B, Stat1
homodimers, and IRF-1 accumulate in the nucleus and bind DNA in concert
with octamer binding protein, trans activation of the iNOS
gene occurs. The synthesis of autocrine/paracrine IFN-ß is also
induced in LPS- plus IFN--treated mouse macrophages, but because the
appearance of IFN-ß lags behind the initial exposure to IFN-,
the level of Stat1 homodimers is apparently not influenced
dramatically.
Not conveyed in the model depicted in Figure 9 is the fact that each of
the transcription factors that bind to the iNOS promoter’s six
transcriptional elements apparently must be present simultaneously to
achieve activation of this gene. Indications that this is a
prerequisite come from four types of observations. The first is similar
to that shown in this work, where threshold doses of LPS, despite their
ability to activate NF-B (14) and induce IRF-1 (39), do not
partially activate the iNOS gene in the absence of an IFN signal.
Second, the work of Pace et al. (10) and Alley et al. (19) show that
exposure of a population of macrophages to exogenous IFN- or
endogenously produced IFN-ß, respectively, in the presence of a
threshold dose of LPS increases the number of individual cells in that
population that are maximally synthesizing iNOS protein compared with
LPS treatment alone. Third, targeted disruption of genes encoding
either Stat1 (41) or IRF-1 (9, 16) results in an inability to induce
in macrophages, by either LPS plus IFN- or LPS plus IFN-ß, the
accumulation of either nitrite or iNOS mRNA. Finally, the
octamer-binding transcription factors Oct-1 and Oct-2 are
constitutively present in the nucleus of mouse macrophages but are
unable to bind the iNOS gene’s octamer site until a proper stimulus is
administered (our unpublished observations). Each of these observations
is consistent with the hypothesis that the nuclear presence of subsets
of those transcription factors necessary for full induction of the iNOS
gene does not result in DNA binding or partial activation of the gene
in mouse macrophages.
The model outlined in Figure 9 is consistent with that which is known
about LPS-induced vs LPS- plus IFN--induced expression of the mouse
iNOS gene and provides a framework upon which a more thorough
understanding of the regulation of this gene can be achieved. It also
underscores the need for two signaling pathways to achieve optimal gene
induction. While LPS treatment alone can cause the activation of
NF-B and induction of IRF-1, either IFN- or IFN-ß is essential
for the activation of transcription factor Stat1, which binds the
iNOS enhancer-linked GAS. Stat1 also trans activates the
IRF-1 gene, the product of which binds the iNOS enhancer-linked ISRE.
This dual stimulus requirement apparently assures that the iNOS gene
will be activated only under those circumstances that warrant the
synthesis of the potentially toxic NO molecule.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William J. Murphy, Wilkinson Laboratory of the Kansas Cancer Institute, 1008 Wahl Hall West, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7184. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible nitric oxide synthase; NO, nitric oxide; ISRE, IFN-stimulated response element; GAS, IFN--activated site; EMSA, electrophoretic mobility shift assay; anti-P-Tyr, anti-phosphotyrosine; PVDF, polyvinylidene difluoride; IRF-1, IFN regulatory factor-1; CHX, cycloheximide; ISGF-3, IFN-stimulated gene factor-3.
Received for publication February 5, 1998. Accepted for publication July 1, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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