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Molecular and Cellular Biology, March 2001, p. 1621-1632, Vol. 21, No. 5
School of Life Sciences, Wellcome Trust
Biocenter, University of Dundee, Dundee DD1 5EH,
Scotland,1 and Istituto
Ricerche di Biologia Molecolare Angeletti, Pomezia,
Italy2
Received 17 July 2000/Returned for modification 3 October
2000/Accepted 8 December 2000
We generated mice carrying a STAT3 allele amenable to Cre-mediated
deletion and intercrossed them with Mx-Cre transgenic mice, in which
the expression of Cre recombinase can be induced by type I interferon.
Interferon-induced deletion of STAT3 occurred very efficiently (more
than 90%) in the liver and slightly less efficiently (about 70%) in
the bone marrow. Analysis of the induction of liver acute-phase genes
in response to bacterial lipopolysaccharide unequivocally identifies
STAT3 as a fundamental mediator of their induction. The different
degrees of defectiveness displayed by the various genes allowed us to
differentiate them into three separate groups according to their degree
of dependence on STAT3. Induction was totally defective for group I
genes, defective at 24 h but almost normal at earlier time points
for group II genes, and only slightly defective for group III genes.
This division was in good agreement with the known structures of the
respective promoters. We also found that the overall induction of the
transcription factors C/EBP The acute-phase (AP) proteins are liver plasma
proteins whose levels of expression are either positively or negatively
regulated by cytokines during inflammation, chiefly through the
regulation of the activities of their cognate genes (13).
Interleukin 1 (IL-1) and IL-6 are the main inflammatory mediators
involved in this transcriptional induction (27), acting
synergistically to activate a subset of AP genes known as class I. In
contrast, class II genes are solely responsive to IL-6-type cytokines.
In addition, corticosteroid hormones are induced by inflammatory cytokines and participate in the induction of most AP genes, being required for their optimal induction and in turn exerting an inhibitory effect on cytokine production, thus activating a negative-feedback loop
(3, 4, 35). Two main kinds of cytokine-responsive elements
have been characterized on the promoters of AP genes (recently reviewed
in reference 36). Type I IL-6-responsive elements
(IL-6REs) are binding sites for CAAT/enhancer binding protein (C/EBP)
transcription factors, which can mediate transcriptional induction by
both IL-6 and IL-1, and have been identified on the promoters of most
class I genes, such as those for haptoglobin (HP) (25),
IL-6-type cytokines, which share the receptor signaling subunit gp130,
are known to elicit the activation of two major signaling pathways
through the activation of kinases belonging to the JAK family: tyrosine
phosphorylation and activation of STAT factors, mainly STAT3 (19,
41), and activation of the mitogen-activated protein kinase
(MAPK) pathway through recruitment of the SH2-containing protein
tyrosine phosphatase 2 (SHP-2) as a molecular adapter (14,
26). Both pathways lead to the activation of transcription factors involved in the regulation of AP genes: STAT3-APRF is activated
directly by JAK family kinases, while C/EBP STAT3 can be activated by many cytokines and growth factors in addition
to gp130 cytokines (see reference 1 for a recent review),
and indeed, STAT3 inactivation by gene targeting leads to early
embryonic lethality (44). Cre-mediated inactivation of
STAT3 in different cell types has been recently described, demonstrating important roles for this factor in mediating functions of
IL-6, IL-2, epidermal growth factor (EGF), and prolactin (9, 40,
42, 43). We describe here the independent generation of
conditional STAT3 mutant mice in which inactivation of the gene can be
induced in several tissues by treatment with synthetic double-stranded
RNA [poly(I · C)], which triggers the expression of an interferon
(IFN)-inducible Cre recombinase (28). Although STAT3
deletion obtained through repeated poly(I · C) treatments eventually
triggers the development of a fulminant form of ulcerative colitis (T. Alonzi et al., unpublished data), short-term treatment causes
preferential deletion in the liver and macrophages. We made use of this
model to directly assess in vivo the role of STAT3 in regulating
transcription of AP genes in response to treatment with recombinant
IL-6 or bacterial lipopolysaccharide (LPS). We demonstrate that STAT3
is indeed essential for the induction of all tested genes downstream of
IL-6. On the other hand, STAT3 also plays an important role in the
activation of most genes in response to LPS, which triggers the
production of a much more complex repertoire of inflammatory cytokines,
including IL-6, IL-1, and tumor necrosis factor alpha (TNF- Generation of the targeting vector and of the targeted ES cells
and mice.
A genomic library from the 129/SV mouse strain
(Stratagene Cloning Systems, La Jolla, Calif.) was screened with a cDNA
clone for the mouse STAT3, and several overlapping positive clones were identified. A SalI-StuI fragment of approximately
10 kb, containing exons 6 to 14, was subcloned into a Bluescript
plasmid. An 88-bp fragment containing a single loxP site, engineered to
contain an EcoRV restriction site, was obtained by PCR from
the plasmid pGEM-30 (18) and cloned into the
EcoRI site in intron 14. A SalI-XbaI
fragment containing a pMC1/Neo expression cassette flanked by loxP
sites from the plasmid pL2-Neo (17) was inserted into the
EcoRI site of intron 11. Finally, a pMC1 herpes simplex
virus thymidine kinase cassette (33) was inserted
downstream of the 3' homology region to generate the targeting vector.
This was linearized with SalI and electroporated into E14
embryonic stem (ES) cells (20) according to standard
protocols. Clones resistant to both G418 and ganciclovir were screened
for homologous recombination by Southern blotting of
EcoRV-digested genomic DNA and probed with a cDNA fragment
containing exons 15 to 24, not included in the region of homology. The
predicted fragment sizes were as follows: wild-type, allele, 15 kb;
replaced allele, 11 kb. By using a 5' probe (not shown), correct
recombination was confirmed to have also occurred at the 5' side. Three
targeted clones were transiently transfected with pMC1-Cre
(18) to delete the Neo cassette. Clones which had became
sensitive to G418 were further analyzed by Southern blotting upon
digestion with EcoRV and hybridization with a 2.1-kb EcoRV/EcoRI fragment from intron 11 (upstream of
the Neo insertion) as a probe. The predicted sizes for the different
alleles were as follows: wild type, 11 kb; replaced, 5.2 kb; floxed,
4.1 kb; deleted, 2.2 kb. Four STAT3 clones carrying the floxed allele, derived from two distinct replaced clones, were microinjected into
CB6F1 (C57BL/6 × BALB/c) blastocysts to generate chimeric mice.
Germ line transmission was obtained from all of them.
STAT3fl/+ mice derived from two distinct clones
were then intercrossed to obtain mice homozygous for the floxed
mutation (STAT3fl/fl). After verifying that both
STAT3fl/fl lines were viable and fertile, the
one derived from clone 138 was chosen for further studies.
Animals and treatments.
STAT3fl/fl
mice generated as described above were crossed with MX-Cre mice
(28). MX+ (i.e., deletable) and
MX
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1621-1632.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Essential Role of STAT3 in the Control of the
Acute-Phase Response as Revealed by Inducible Gene Activation in
the Liver
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and -
was only minimally defective in
the absence of STAT3. Finally, even though corticosterone levels and
action were found to be normal in the conditional-mutant mice,
production of both proinflammatory and antiinflammatory cytokines was
increased and prolonged, probably as a result of STAT3 deletion in macrophages.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1-acid glycoprotein (AGP) (38, 47), hemopexin (Hpx)
(37), complement component 3 (C3) (24),
C-reactive protein (CRP) (34), and serum amyloids A (SAA)
(21) and P (SAP) (34). On the other hand,
class II genes, such as -2 macroglobulin (2M) (45)
and fibrinogens (FBs) (10, 32, 50), appear to be mainly
regulated by type II IL-6REs, which are binding sites for members of
the signal transducers and activators of transcription (STAT) family of
transcription factors and particularly for STAT3-APRF (45). Type II IL-6RE-STAT3 sites have also been
identified on class I gene promoters.
and -, the two C/EBP
family members that are induced during inflammation, are activated
through the MAPK pathway (see reference 36 and references
therein). However, on the basis of a number of in vitro data, STAT3 has
been proposed to be the main mediator of AP gene induction downstream
of IL-6 and other gp130 cytokines (29, 30), and indeed, in
IL-6-deficient turpentine-treated mice, failure to activate the AP
genes correlated with defective STAT3 activation (2, 12).
On the other hand, the analysis of C/EBP-deficient mice has failed
to reveal any dramatic defect in the activation of AP genes, although a
final conclusion on the overall role of C/EBPs in the regulation of AP
promoters cannot be drawn from these data, since C/EBP might well be
able to compensate for the absence of C/EBP (8).
). In
this case, however, its relative functional relevance varied in
agreement with the structures of the different promoters. Moreover, we
show that STAT3 is only marginally involved in the transcriptional
induction of C/EBP and - and that corticosterone (CS) production
is normal in STAT3 conditional-mutant mice.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
(i.e., nondeletable littermate controls)
STAT3fl/fl mice for the experiments were
generated by mating MX+ STAT3fl/fl
males with MX STAT3fl/fl females.
Genetic screening for the Cre transgene was performed by PCR using the
following oligonucleotides: CRE1, 5'-AGGCGTTTTCTGAGCATACC-3'; CRE10, 5'-TAGCTGGCTGGTGGCAGATG-3'.
STAT3fl/fl mice were injected intraperitoneally
(i.p.) once with 250 µg of poly(I · C) 4 days before LPS treatment.
LPS (Escherichia coli serotype O26:B6; Sigma Chemical Co.,
St. Louis, Mo.) was resuspended in sterile pyrogen-free saline solution
and injected i.p. at a dose of 1 mg/kg of body weight. Human
recombinant IL-6 was injected at a dose of 10 µg/mouse as described
previously (2), and dexamethasone (Decadron Shock Pack;
MSD, Brussels, Belgium) (750 µg/mouse) was injected i.p. 30 min prior
to LPS treatment. The mice were sacrificed by CO2
asphyxiation, blood was collected by cardiac puncture, and the livers
were immediately removed.
Total liver protein extraction and Western blot analysis. The frozen livers were lysed by homogenization in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, 1 mM sodium vanadate, and a 40-µg/ml protease inhibitor cocktail (Sigma) and cleared by centrifugation. The protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, Calif.). Proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The antibodies used were as follows: anti-STAT3 monoclonal antibody directed against the amino-terminal part of the protein and anti-STAT1 polyclonal serum (Signal Transduction Laboratories, San Diego, Calif.) and anti-phospho-STAT3 (Tyr 705), anti-phospho-p44/42 MAPK, and anti-p44/42 MAPK (New England Biolabs, Beverly, Mass.).
Liver nuclear extracts and electrophoretic mobility shift assays (EMSAs). Nuclear extracts were prepared after LPS or saline treatment from freshly removed livers as described previously (16) with modifications. Briefly, 1 g of liver was homogenized in 2.5 ml of homogenization buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM EDTA, 2 M sucrose, 10% glycerol, 0.5 mM spermidine, 0.15 mM spermine, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 1% aprotinin) using a glass-Teflon Dounce homogenizer. The nuclei were pelleted by ultracentrifugation over a cushion of the same buffer and directly lysed in 10 mM HEPES (pH 7.9), 400 mM NaCl, 0.1 mM EGTA, 5% glycerol, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 1% aprotinin at 4°C for 30 min. The lysates were cleared by centrifugation and frozen in liquid nitrogen. The protein concentration was determined by Bradford assay.
EMSAs were carried out by incubating 6 µg of each extract in a 20-µl final volume of a solution of 20 mM HEPES (pH 7.9), 50 mM NaCl containing 3 µg of poly(dI-dC), and 2 µg of salmon sperm DNA for 10 min on ice. 32P-labeled double-stranded oligonucleotides (2 × 104 cpm) were added, and the mixture was incubated for 15 min at room temperature. DNA-protein complexes were separated by electrophoresis on a 6% polyacrylamide gel in 0.25× TBE buffer (25 mM Tris, 25 mM boric acid, 0.6 mM EDTA) and visualized by autoradiography. Antibodies for supershift experiments were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.) and added to the preincubation mixture for 30 min on ice prior to addition of the probe. The double-stranded oligonucleotides were labeled by filling in with Klenow polymerase. The sequences of the upper strands were as follows: NF-
B binding site, 5'-GATCCGCTGGGGACTTTCCAGGCG-3'; STAT
site, 5'-GATCGATTTCCCCGAAAT-3'; C/EBP site,
5'-GGGCATAGTGGCGCAAACTCCCTTACTG-3'.
Slot blot analysis. Total RNA was prepared from frozen livers using a Qiagen kit according to the manufacturer's instructions. Five or 20 µg, respectively, of total RNA was analyzed by slot blotting or by Northern blotting as previously described (12). The different cDNA probes used have been previously described (8, 12) and were labeled by random priming. The relative abundances of the different mRNAs were measured by phosphorimager analysis and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase).
Cytokine and CS measurements.
Cytokines were measured by
enzyme-linked immunosorbent assay (ELISA) using kits purchased from
PharMingen (San Diego, Calif.) according to the manufacturer's
instructions. IL-1 levels were assayed by a two-sided sandwich ELISA
using antibody pairs purchased from R&D Systems (Minneapolis, Minn.).
CS was measured by radioimmunoassay using a kit from ICN (Costa Mesa,
Calif.) according to the manufacturer's instructions.
Statistical analysis. Results were analyzed by the analysis of variables test using the Statview computer program (Abacus Concept). A P value of <0.05 was considered statistically significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation of a mouse line amenable to Cre-mediated conditional
inactivation of STAT3.
A targeting vector was constructed in which
the region corresponding to exons 12 to 14 (encoding the putative DNA
binding domain) was flanked with two loxP sites, while a loxP-Neo
cassette was inserted into intron 11 (Fig.
1A). Homologous recombinant E14 embryonic
stem cell clones were identified by Southern blot analysis (Fig. 1B and
C) and subjected to transient transfection with a Cre-encoding plasmid.
Colonies which had lost resistance to G418 were amplified and analyzed
by Southern blotting, thus identifying those clones which had the
Neor cassette but not the exonic region deleted (Fig. 1D).
The resulting STAT3 floxed allele (STAT3fl) was
expected to be functional but amenable to inactivation by Cre-mediated
recombination through removal of the region corresponding to exons 12 to 14 and generation of a STAT3 "deleted" (
) allele. Transcription from this STAT3
allele generates a
shorter, frameshifted mRNA that should be unable to encode a functional
protein. Embryonic stem cells carrying a floxed allele were injected
into recipient blastocysts, and the resulting chimeras were crossed to
BALBC/A mice to generate mice carrying the
STAT3fl allele in their germ line.
STAT3fl/fl mice were obtained from heterozygous
matings at a Mendelian ratio and were phenotypically indistinguishable
from wild-type or heterozygous littermates, indicating that the
STAT3fl allele is functional.
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Inducible STAT3 inactivation.
In order to generate animals in
which the STAT3 gene could be inactivated in an inducible way,
STAT3fl/fl mice were bred to Mx-Cre transgenic
mice, which express the Cre recombinase under the control of the
IFN-responsive Mx-1 promoter (28), thus generating
MX+ (i.e., where the STAT3 allele is deletable) or
MX (control) STAT3fl/fl
mice. Cre expression was induced in adult mice by a single
injection of a synthetic double-stranded RNA [poly(I · C)], which
is known to induce a strong and transient production of type I IFN. The efficiency of Cre-mediated deletion in different tissues was evaluated by Southern blot analysis of genomic DNA extracted 2 days after the
treatment and was found to be almost 100% in the liver and 70 to 80%
in the adipose tissue and bone marrow (not shown). Western blot
analysis of liver protein extracts 4 (not shown) and 5 (Fig. 1E) days
after poly(I · C) treatment confirmed that liver STAT3 protein
levels were greatly reduced, indicating that the deletion allele is
unable to encode a detectable protein. Therefore, MX+
STAT3fl/fl mice that have been treated with
poly(I · C) as described above will hereafter be referred
to as STAT3 conditional-mutant mice. STAT3 levels were slightly
induced by LPS treatment in both kinds of mice (Fig. 1E and F).
In addition, STAT3 activation as detected in liver nuclear extracts by
specific anti-phosphotyrosine STAT3 antibodies was strongly induced by
LPS in the MX control mice at both 4.5 and 9 after LPS
treatment (Fig. 1F). In contrast, STAT3 phosphorylation was barely
detectable at 4.5 h and not at all by 9 h in the STAT3
conditional-mutant mice (Fig. 1F), thus confirming that very low levels
of active STAT3 were present even under the inflamed conditions.
Analysis of the AP response in the STAT3 conditional-mutant
mice.
In order to compare the induction of AP mRNAs in the
presence and absence of STAT3 in the liver, MX+ and
MX STAT3fl/fl mice were treated
once with poly(I · C) to trigger STAT3 inactivation and injected
with LPS after 4 days. Liver and blood samples were then collected at
different times, and total RNA from the liver was subjected to slot
blot analysis with cDNA probes directed to different AP mRNAs. The
results were quantified and normalized to GAPDH as an internal control,
and the normalized arbitrary levels are plotted in Fig.
2. It is worth noticing that poly(I · C) treatment did not increase the basal levels of the different AP
mRNAs tested, which were equivalent in the saline-treated mice injected
and not injected with poly(I · C) (not shown). All mRNAs tested
were increased by severalfold in the control mice, although with
slightly different time courses. Induction of all tested AP mRNAs was
affected in the STAT3 conditional-mutant mice, and the respective genes
could be divided into three groups. Group I comprised genes whose
activation was always totally defective in the absence of STAT3,
including those for SAP, one of the major AP reactant in the mouse, and
FB and -
. Group II comprised genes whose activation was
comparable to that of the controls at early time points but declined
much faster and was not detectable at 24 h, including those for HP,
AGP, and FB. Finally, group III comprised genes whose activation was
only slightly defective and whose mRNAs were induced at levels
comparable to those of the controls 6 or 12 h after LPS treatment
and remained only slightly defective at 24 h. The SAA, Hpx, and C3
genes belonged to this category. The SAA probe used in this experiment
recognized all three major forms of SAA, which in the mouse are encoded
by three homologous genes, SAA1, -2, and
-3. To study the specific contributions of the different SAA
forms to the total SAA RNA level, probes preferentially recognizing
each of the three isoforms were used. As shown at the bottom of Fig. 2,
SAA3 induction was the least affected by the absence of STAT3, being
normal at 6 and 12 h and only reduced by about 50% at 24 h.
In contrast, SAA1 induction was almost totally defective, and the SAA2
mRNA showed an intermediate behavior, being induced at 6 h but
failing to increase any further.
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Defective AP mRNA induction is not due to lack of C/EBP or -
activation.
Transcription factors belonging to the C/EBP family,
and in particular C/EBP and -, are believed to play an important
role in mediating the cytokine inducibility of most AP genes
(36). In addition, transcription of both factors is
activated during inflammation and in response to IL-6 both in vivo and
in vitro, and this activation has been proposed, at least for C/EBP,
to be dependent on STAT3 (7). We therefore analyzed the
induction of both C/EBP and - mRNAs in the livers of the STAT3
conditional-mutant and control mice upon treatment with either LPS or
recombinant IL-6. As shown in Fig. 4A and
B, the overall inductions of both genes
were similar in the conditional-mutant and control mice upon both LPS
and IL-6 treatment. This suggests that STAT3 is not strictly required
for transcriptional induction of C/EBP or - in the liver during
inflammation and that the defective expression of AP mRNAs in the
conditional-mutant mice is directly due to lack of STAT3 function and
is not mediated by defective C/EBP induction. Interestingly, however,
induction of C/EBP and C/EBP by LPS was slightly but
significantly blunted at 1.5 and 6 h after LPS treatment but then
increased and was still apparent at 24 h, when it had almost
returned to basal levels in the control mice. In contrast, the
inductions of both mRNAs were completely comparable in response to
recombinant IL-6. These observations suggest that STAT3 may indeed play
a role in mediating full induction of both genes at early time points
but apparently not in response to IL-6. It is worth noting that the
mRNA levels of C/EBP, a family member thought not to be involved in
the regulation of AP genes and whose transcription is actually reduced
by LPS and IL-6, were equivalent in untreated STAT3 conditional-mutant
and control mice and decreased to similar extents upon LPS or IL-6 treatment (data not shown).
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, negative; +, positive. (D) EMSA analysis of C/EBP
binding activities. Nuclear extracts from mice either untreated or
treated with LPS for 4.5 h were used in an EMSA with a
double-stranded oligonucleotide carrying a C/EBP binding site. Where
indicated, antisera against different C/EBP proteins were preincubated
with the extracts.
and - proteins and
their DNA binding capacities reflected the abundance of the respective
mRNAs, liver nuclear extracts were analyzed by Western blotting. As
shown in Fig. 4C, the levels of both C/EBP and - were strongly
increased 4.5 h after LPS treatment and slightly decreased by
9 h, and the overall abundances of the two proteins were
comparable in the conditional-mutant and control mice. C/EBP occurs
in three different forms, the full-length form, a slightly shorter form
called LAP, and a truncated form termed LIP, which lacks the activation
domain and is thought to act as a dominant negative (11).
Interestingly, while the intermediate form, LAP, was already abundant
in the liver extracts of untreated mice and was slightly increased only
at 4.5 h, the other two forms were strongly induced at 4.5 h
and still well above basal levels at 9 h. Finally, we have
analyzed by EMSA the C/EBP DNA binding activities present in the liver
nuclear extracts from mice either untreated or treated with LPS for
4.5 h, when the levels of both the C/EBP and - proteins were
highest. Using a double-stranded oligonucleotide carrying a C/EBP
binding site, the DNA-protein complexes formed with the extracts from
untreated mice were equivalent in the conditional-mutant and control
mice (Fig. 4D). In both cases, most complexes were supershifted by
antibodies against either C/EBP or C/EBP, suggesting that under
untreated conditions, C/EBP and - homo- and heterodimers are
responsible for the vast majority of the detected C/EBP DNA binding
activities. Upon LPS treatment, the mobilities of the complexes were
greatly increased, and no C/EBP binding activity was detected
anymore by supershift analysis. In contrast, all complexes detected
appeared to contain C/EBP, since everything was supershifted by
anti-C/EBP antibodies. Again, no difference was detected between
extracts from STAT3 conditional-mutant mice and controls. Surprisingly,
we were unable to detect any C/EBP DNA binding activity by
supershift analysis despite having attempted it with two different
sources of anti-C/EBP antibodies expected to possess supershifting
activity, including those that readily detected the protein by Western
blotting. Perhaps the proportion of C/EBP isoforms present in the
extracts is too high for C/EBP to detectably bind, at least in
vitro, under these conditions.
Hyperproduction of cytokines and susceptibility to endotoxic shock
in the STAT3 conditional-mutant mice.
As already mentioned, STAT3
inactivation induced by poly(I · C) was not limited to the liver but
occurred at good efficiency in other compartments as well, and most
notably in the bone marrow (data not shown) and in bone marrow-derived
macrophages (Alonzi et al., unpublished data). Since macrophages are
the major cytokine-producing inflammatory cells, it was important to
assess their functionality by measuring the levels of circulating
inflammatory cytokines produced upon LPS injection in the STAT3
conditional-mutant mice compared to those in the controls.
Interestingly, the induction of all proinflammatory cytokines tested
was found to be stronger and more sustained in the STAT3 conditional
mutants (Fig. 5A). IL-6 was still
increasing between 6 and 12 h after injection, at a time when it was
reduced to basal levels in the control mice. IL-1 followed a very
similar pattern of induction, although its levels were below the
detection limits of our assay in the control mice. Similarly, TNF-
was still increasing at 6 h, while it had become undetectable
after 3 h in the control mice. Taken together, these data indicate
that the defective activation of AP genes detected in the
conditional-mutant mice in response to LPS is most definitely not due
to impaired cytokine production, and if anything they emphasize the
fundamental role of STAT3, which is absolutely required for the
induction of most AP genes even in the presence of abnormally high
levels of cytokines.
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hyperproduction. Our results are in agreement with
those reported by S. Akira and colleagues in mice in which STAT3 had
been inactivated specifically in macrophages and granulocytes (42) and can be explained by their observation that STAT3
is required for responsiveness to IL-10, a cytokine known for its potent deactivating effects on macrophages. IL-10, is also known to
exert an inhibitory effect on its own synthesis, and apparently this
action is also dependent on STAT3, since IL-10 levels in the
conditional-mutant mice were abnormally high 12 h after LPS treatment (Fig. 5A).
CS production and action are normal in the STAT3 conditional-mutant
mice.
The activation of the hypothalamic-pituitary-adrenal axis by
inflammatory cytokines and the consequent production of glucocorticoid hormones play an important dual role in the inflammatory response by
participating as coactivators in the transcriptional induction of
several AP genes (3) while at the same time providing
negative feedback for cytokine production (4, 35). STAT3
deletion occurring in tissues other than the liver may therefore
interfere either with cytokine-induced CS production or with the action of CS itself on target cells. We have therefore measured CS levels in
the serum of mice treated with LPS for different lengths of time and
found no differences between the STAT3 conditional mutant and the
control mice (Fig. 5B), suggesting that the activation of the
hypothalamic-pituitary-adrenal axis is not compromised. Moreover,
administration of dexamethasone 30 min prior to LPS injection was able
to significantly reduce IL-6 and TNF- levels in the blood of both
the control and the conditional-mutant mice (Fig. 5C), thus
demonstrating that glucocorticoid hormone action is not impaired by
STAT3 inactivation.
Increased NF-B and STAT1 activation in the livers of the STAT3
conditional-mutant mice.
As shown in Fig. 4, the overall induction
of both C/EBP and - in the conditional mutant mice was comparable
to that in the control mice, and accordingly, C/EBP DNA binding
activities in liver nuclei before and after LPS injection were also
equivalent. Other transcription factors that are activated by LPS and
that may play a role in the induction of AP genes are NF-B and
STAT1. NF-B is the main target of TNF- and IL-1, while STAT1 is
activated along with STAT3 by IFN- and gp130 cytokines. LPS
injection triggered the activation of NF-B DNA binding activity,
which was abundant after 4.5 h and slightly decreased after 9 h in the livers of the control mice (Fig.
6A, top). The same complexes, only much more abundant, were also formed by using nuclear extracts from the
STAT3 conditional-mutant mice, suggesting a stronger and more sustained
NF-B activation.
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and - mRNAs. Indeed, phosphorylation of ERK1 and ERK2 was
more elevated in the absence of STAT3, although by 15 h after LPS
treatment the activation levels were equivalent to those found in the
control mice, and by 24 h phosphorylation levels were back to their
basal values in both cases (Fig. 6B).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our previous observation that in IL-6-deficient mice impaired and normal induction of AP genes in response to different stimuli correlated with defective and normal activation of STAT3, respectively (2, 12), pointed towards a fundamental role for STAT3 in the induction of AP genes in vivo. Indeed, we found that deletion of STAT3 in the livers of adult mice virtually abolished the ability of IL-6 to induce all AP mRNAs measured. More importantly still, AP gene induction was also defective upon LPS injection, thus largely confirming the pivotal role of STAT3 even under conditions that mimic a systemically induced inflammatory reaction and lead to the production of a broad variety of cytokines.
Promoter structure correlates with functional predominance of STAT3
for induction of AP genes.
A functional analysis of the known
structure of the corresponding promoters, whose schematic
representation is shown in Fig. 7,
indicates a surprisingly good correlation between known functional promoter organization and the differential role played by STAT3 in
their induction. The scheme focuses particularly on C/EBP and STAT
sites, which are the two main regulatory elements common to most AP
genes, but other sites characterized as functionally important are
indicated as well. Where functional data were not available for the
mouse promoters, either the rat or the human promoter is shown.
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and -) (32,
50); genes carrying both STAT and C/EBP sites of equivalent
functional importance on their promoters, whose induction was also
totally defective at 24 h but was almost normal at earlier time
points (group II, including HP [25], AGP [38, 47], and FB [25]); and finally, genes with no
characterized STAT site on their promoters, which were only minimally
affected by the absence of STAT3 (group III, including SAA3
[21] and C3 [24, 46]). The only two
exceptions to this rule involved the SAP and Hpx genes, each of which
carries one STAT and one C/EBP site (22, 34) and yet was
totally or minimally defective, respectively. In both cases however,
one of the sites was found to be dominant over the other (34,
37), which suggests that perhaps in vivo this functional
predominance may be more stringent. Particularly in the case of Hpx,
while the C/EBP site has been shown to be dominant in the human
promoter (37), the reverse is true for the rat promoter
(23). Although no functional data are available for the
mouse gene, sequence comparison indicates that the C/EBP site on the
mouse promoter is more similar to the human site than to the rat site,
and in contrast to the rat site, it can be predicted to be a strong
binding site for C/EBP proteins (Fig. 7B) (F. Altruda, personal
communication). The relative independence from STAT3 shown by the SAA3
and C3 genes is in agreement with previous data obtained with
C/EBP-deficient mice, where in contrast, Hpx induction was
completely uncompromised (8). This could perhaps reflect a
dominant role for C/EBP rather than - in the induction of this
gene. Some weak and therefore not-yet-characterized STAT element might
likewise be present on the mouse SAA3 and C3 promoters, since their
induction is slightly but reproducibly impaired in the STAT3
conditional-mutant mice. However, these two promoters appear to be
functionally different, since SAA3 was strongly induced by recombinant
IL-6 while C3 was not.
The regulation of the SAA family revealed some unexpected
characteristics. C/EBP and NF-B sites have been shown to play a major role in the transcriptional induction of all characterized SAA
genes (mouse SAA3 [21], rabbit SAA [39],
rat SAA1 [31], and human SAA2 [5]), which
therefore would be expected not to be affected by the absence of STAT3.
As discussed above, this was indeed the case for SAA3, whose induction
by LPS was only partially reduced in the STAT3 conditional-mutant mice.
In contrast, induction of both SAA1 and SAA2 was profoundly impaired.
Although the mouse SAA1 and -2 gene promoters have not been
characterized, computer-assisted alignment including the human SAA1
promoter revealed two conserved potential STAT elements but no C/EBP or NF-B sites (6, 48), which is in good agreement with the substantially defective activation of these two genes in the absence of
STAT3. It is worth noting that, although SAA3 induction was only
partially impaired by STAT3 inactivation, IL-6-dependent induction was
totally defective, suggesting that the portion of SAA3 induction due to
IL-6 action is dependent on STAT3. On the other hand, the low but
reproducible two- to threefold induction displayed by the Hpx gene in
response to IL-6 seems to suggest that IL-6 is indeed capable of
inducing certain AP genes via a STAT3-independent pathway, most likely
involving C/EBP and - activation, which was normal in the STAT3
conditional-mutant mice.
C/EBP and C/EBP induction are not dependent on STAT3.
STAT3 has been proposed to play an important role in the induction of
C/EBP by IL-6, and C/EBP has been shown to carry a functionally
active STAT binding site (7). It was therefore conceivable
that the defective AP gene induction in the absence of STAT3 might be
at least partly mediated by impaired induction of these two C/EBP
family members. Our data indicate that STAT3 may play a marginal role
in the induction of both C/EBP and - mRNAs, mostly at early time
points, but suggest that its function is not strictly required, since
overall induction was only partially blunted. Moreover, both protein
levels and DNA binding activities were comparable in STAT3
conditional-mutant and control mice, thus confirming that defective
C/EBP activity could not be an indirect cause of defective AP gene
induction. The reason why C/EBP factors alone are not able to at least
weakly activate genes such as those for AGP, HP, and FB while they
are apparently able to do so for Hpx, C3, and SAA3 is at present not
clear. Possibly this difference originates from the structures of the
respective promoters. For example, efficient induction of a given AP
gene by C/EBP or - might require cooperation with at least
another type of cytokine-inducible factor. Indeed, in addition to the C/EBP site(s), the C3 promoter carries an NF-B-like IL-1RE
(24), and the murine SAA3 promoter carries a binding site
for SEF-1 (SAA enhancer factor), which is also required for induction
(21). In contrast, only C/EBP and STAT sites are involved
in the induction of the HP, AGP, and FB gene promoters (10,
25, 38, 47).
Cytokine overproduction and prolonged C/EBP and MAPK
activation.
The reason why induction of C/EBP and - is
prolonged in the absence of STAT3 will require further study. One
possible cause could be the prolonged and increased production of
inflammatory cytokines that occurs as a likely consequence of the lack
of responsiveness to IL-10 of STAT3-deficient macrophages
(42). This could also explain the stronger and prolonged
NF-B and ERK activation detected in the conditional-mutant mice. On
the other hand, abnormally prolonged activation of the MAPK pathway,
involved in the induction of C/EBP factors, has already been shown to
occur in vitro when a mutant form of gp130 unable to activate STAT
factors is used (30). This has been proposed to occur
through physical interference of STAT3, bound to its membrane-proximal
docking site, with the association of SHP-2 to its own docking site,
which is situated in close proximity. It is tempting to speculate that,
in analogy to the dual role of SHP-2 as activator of ERKs and
down-regulator of the STAT activity, STAT3 activation might in turn
exert an inhibitory effect on the MAPK pathway, the absence of which
could explain the prolonged induction of the C/EBP factors.
genes were
activated in the absence of STAT3 6 h after LPS treatment but were
totally defective after 24 h raises the question of which other
transcription factor(s) might be involved in the early induction. Even
though IL-6REs are exquisitively responsive to STAT3, some have been
shown to be able to respond to STAT1 as well, although at a lower
efficiency (49). It is therefore tempting to propose that
perhaps the abnormally high STAT1 levels present in the
conditional-mutant mice, particularly at earlier time points (4.5 h),
might be able to partially compensate for the absence of STAT3 and
could account for the initial burst of AP gene activation, perhaps in
conjunction with C/EBP factors. It has been shown that two of the four
gp130 YXXQ domains that act as docking sites for STAT factors are
specific for STAT3 while two can recruit both STAT3 and STAT1 with
similar affinities (15). It is therefore likely that the
absence of STAT3 releases competition for the docking site and favors
recruitment and activation of STAT1.
| |
ACKNOWLEDGMENTS |
|---|
We are grateful to W. Müller, U. Betz, and K. Rajewsky for providing the Mx-Cre transgenic mice; to H. Baumann, S. Maeda, U. Müller-Eberhard, J. Sipe, W. Liao, K. Yamamoto, S. McKnight, and J. E. Darnell for the gift of plasmids; to H. van der Putten for providing the E14 ES cells; to F. Altruda for sharing unpublished sequences; to Ian Newton for technical help; and to L. Malone and V. Murray-Tait for expert mouse care. STAT3 targeted mice were generated at the Istituto Ricerche di Biologia Molecolare. Angeletti by V.P. and T.A.
This work was supported by the Wellcome Trust (Senior Research Fellowship to V.P.). T.A. and B.G. were the recipients of EC Marie Curie fellowships.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: School of Life Sciences, Wellcome Trust Biocenter, University of Dundee, Dow St., Dundee DD1 5EH, Scotland. Phone: 44-1382-345787. Fax: 44-1382-345893. E-mail: v.poli{at}dundee.ac.uk.
Present address: Laboratory of Gene Expression, I. R. C. C. S. "L. Spallanzani," Rome, Italy.
Present address: Department of Molecular Biology, University of
Ghent, 9000 Ghent, Belgium.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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(2005). C/EBP{delta} is a crucial regulator of pro-apoptotic gene expression during mammary gland involution. Development
132: 4675-4685
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Ki, S. H., Cho, I. J., Choi, D. W., Kim, S. G.
(2005). Glucocorticoid Receptor (GR)-Associated SMRT Binding to C/EBP{beta} TAD and Nrf2 Neh4/5: Role of SMRT Recruited to GR in GSTA2 Gene Repression. Mol. Cell. Biol.
25: 4150-4165
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Debidda, M., Wang, L., Zang, H., Poli, V., Zheng, Y.
(2005). A Role of STAT3 in Rho GTPase-regulated Cell Migration and Proliferation. J. Biol. Chem.
280: 17275-17285
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Severgnini, M., Takahashi, S., Tu, P., Perides, G., Homer, R. J., Jhung, J. W., Bhavsar, D., Cochran, B. H., Simon, A. R.
(2005). Inhibition of the Src and Jak Kinases Protects against Lipopolysaccharide-induced Acute Lung Injury. Am. J. Respir. Crit. Care Med.
171: 858-867
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Xu, J., Ji, S., Venable, D. Y, Franklin, J. L, Messina, J. L
(2005). Prolonged insulin treatment inhibits GH signaling via STAT3 and STAT1. J Endocrinol
184: 481-492
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Carrithers, M., Tandon, S., Canosa, S., Michaud, M., Graesser, D., Madri, J. A.
(2005). Enhanced Susceptibility to Endotoxic Shock and Impaired STAT3 Signaling in CD31-Deficient Mice. Am. J. Pathol.
166: 185-196
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Srisodsai, A., Kurotani, R., Chiba, Y., Sheikh, F., Young, H. A., Donnelly, R. P., Kimura, S.
(2004). Interleukin-10 Induces Uteroglobin-related Protein (UGRP) 1 Gene Expression in Lung Epithelial Cells through Homeodomain Transcription Factor T/EBP/NKX2.1. J. Biol. Chem.
279: 54358-54368
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Dave, V., Childs, T., Whitsett, J. A.
(2004). Nuclear Factor of Activated T Cells Regulates Transcription of the Surfactant Protein D Gene (Sftpd) via Direct Interaction with Thyroid Transcription Factor-1 in Lung Epithelial Cells. J. Biol. Chem.
279: 34578-34588
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Hilfiker-Kleiner, D., Hilfiker, A., Fuchs, M., Kaminski, K., Schaefer, A., Schieffer, B., Hillmer, A., Schmiedl, A., Ding, Z., Podewski, E., Podewski, E., Poli, V., Schneider, M. D., Schulz, R., Park, J.-K., Wollert, K. C., Drexler, H.
(2004). Signal Transducer and Activator of Transcription 3 Is Required for Myocardial Capillary Growth, Control of Interstitial Matrix Deposition, and Heart Protection From Ischemic Injury. Circ. Res.
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Gao, H., Guo, R.-F., Speyer, C. L., Reuben, J., Neff, T. A., Hoesel, L. M., Riedemann, N. C., McClintock, S. D., Sarma, J. V., Van Rooijen, N., Zetoune, F. S., Ward, P. A.
(2004). Stat3 Activation in Acute Lung Injury. J. Immunol.
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Severgnini, M., Takahashi, S., Rozo, L. M., Homer, R. J., Kuhn, C., Jhung, J. W., Perides, G., Steer, M., Hassoun, P. M., Fanburg, B. L., Cochran, B. H., Simon, A. R.
(2004). Activation of the STAT pathway in acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol.
286: L1282-L1292
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Shi, C.-S., Kehrl, J. H.
(2004). Pyk2 Amplifies Epidermal Growth Factor and c-Src-induced Stat3 Activation. J. Biol. Chem.
279: 17224-17231
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Gervois, P., Kleemann, R., Pilon, A., Percevault, F., Koenig, W., Staels, B., Kooistra, T.
(2004). Global Suppression of IL-6-induced Acute Phase Response Gene Expression after Chronic in Vivo Treatment with the Peroxisome Proliferator-activated Receptor-{alpha} Activator Fenofibrate. J. Biol. Chem.
279: 16154-16160
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Mackey, S. L., Darlington, G. J.
(2004). CCAAT Enhancer-binding Protein {alpha} Is Required for Interleukin-6 Receptor {alpha} Signaling in Newborn Hepatocytes. J. Biol. Chem.
279: 16206-16213
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Kamaraju, A. K., Adjalley, S., Zhang, P., Chebath, J., Revel, M.
(2004). C/EBP-{delta} Induction by gp130 Signaling: ROLE IN TRANSITION TO MYELIN GENE EXPRESSING PHENOTYPE IN A MELANOMA CELL LINE MODEL. J. Biol. Chem.
279: 3852-3861
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Cui, Y., Huang, L., Elefteriou, F., Yang, G., Shelton, J. M., Giles, J. E., Oz, O. K., Pourbahrami, T., Lu, C. Y. H., Richardson, J. A., Karsenty, G., Li, C.
(2004). Essential Role of STAT3 in Body Weight and Glucose Homeostasis. Mol. Cell. Biol.
24: 258-269
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Shen, Y., Schlessinger, K., Zhu, X., Meffre, E., Quimby, F., Levy, D. E., Darnell, J. E. Jr.
(2004). Essential Role of STAT3 in Postnatal Survival and Growth Revealed by Mice Lacking STAT3 Serine 727 Phosphorylation. Mol. Cell. Biol.
24: 407-419
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Duan, H. O., Simpson-Haidaris, P. J.
(2003). Functional Analysis of Interleukin 6 Response Elements (IL-6REs) on the Human {gamma}-Fibrinogen Promoter: BINDING OF HEPATIC Stat3 CORRELATES NEGATIVELY WITH TRANSACTIVATION POTENTIAL OF TYPE II IL-6REs. J. Biol. Chem.
278: 41270-41281
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Minoguchi, M., Minoguchi, S., Aki, D., Joo, A., Yamamoto, T., Yumioka, T., Matsuda, T., Yoshimura, A.
(2003). STAP-2/BKS, an Adaptor/Docking Protein, Modulates STAT3 Activation in Acute-phase Response through Its YXXQ Motif. J. Biol. Chem.
278: 11182-11189
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Wuestefeld, T., Klein, C., Streetz, K. L., Betz, U., Lauber, J., Buer, J., Manns, M. P., Muller, W., Trautwein, C.
(2003). Interleukin-6/Glycoprotein 130-dependent Pathways Are Protective during Liver Regeneration. J. Biol. Chem.
278: 11281-11288
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Hall, M. A., Curtis, D. J., Metcalf, D., Elefanty, A. G., Sourris, K., Robb, L., Gothert, J. R., Jane, S. M., Begley, C. G.
(2003). The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12. Proc. Natl. Acad. Sci. USA
100: 992-997
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Li, W., Liang, X., Kellendonk, C., Poli, V., Taub, R.
(2002). STAT3 Contributes to the Mitogenic Response of Hepatocytes during Liver Regeneration. J. Biol. Chem.
277: 28411-28417
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Brasier, A. R., Recinos, A. III, Eledrisi, M. S.
(2002). Vascular Inflammation and the Renin-Angiotensin System. Arterioscler. Thromb. Vasc. Bio.
22: 1257-1266
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Seshadri, V., Fox, P. L., Mukhopadhyay, C. K.
(2002). Dual Role of Insulin in Transcriptional Regulation of the Acute Phase Reactant Ceruloplasmin. J. Biol. Chem.
277: 27903-27911
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Costa-Pereira, A. P., Tininini, S., Strobl, B., Alonzi, T., Schlaak, J. F., Is'harc, H., Gesualdo, I., Newman, S. J., Kerr, I. M., Poli, V.
(2002). Mutational switch of an IL-6 response to an interferon-gamma -like response. Proc. Natl. Acad. Sci. USA
99: 8043-8047
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Feister, H. A., Auerbach, B. J., Cole, L. A., Krause, B. R., Karathanasis, S. K.
(2002). Identification of an IL-6 response element in the human LCAT promoter. J. Lipid Res.
43: 960-970
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Ghoshal, K., Majumder, S., Zhu, Q., Hunzeker, J., Datta, J., Shah, M., Sheridan, J. F., Jacob, S. T.
(2001). Influenza Virus Infection Induces Metallothionein Gene Expression in the Mouse Liver and Lung by Overlapping but Distinct Molecular Mechanisms. Mol. Cell. Biol.
21: 8301-8317
[Abstract] [Full Text]
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