 |
INTRODUCTION |
Regulation of inflammatory and immune responses involves
intercellular communication through a network of secreted cytokines (1). Cytokines derived from T helper
(Th)1 1 cells (prototypically
IFN
) promote the development of cellular immunity, while Th2-derived
cytokines such as IL-4 promote humoral immunity and antagonize
Th1-dependent activities (2-5). At least a portion of the
anti-inflammatory function of IL-4 is targeted to mononuclear
phagocytes where expression of inducible genes encoding cytokines
(e.g. TNF
, IL-1, IL-1
, various chemokines) (6-16)
and cell surface molecules (FcR and ICAM-1) (17, 18) is suppressed,
while expression of other anti-inflammatory products such as the IL-1
receptor antagonist is amplified (9-11, 19, 20).
The molecular mechanisms mediating cytokine-induced gene transcription
have been extensively characterized in recent studies on the Janus
kinase (JAK) family of protein tyrosine kinases and signal
transducers and activators of
transcription (STATs) (21-25). IFN has been shown to
induce phosphorylation of STAT1, which, in homodimeric form, binds to
the IFN activation sequence (GAS) found in many IFN-inducible
genes (26-29). In similar fashion, IL-4 stimulates tyrosine
phosphorylation of STAT6 (30-32), which can bind to GAS motifs as well
as IL-4-responsive STAT binding elements (SBEs) found in IL-4-inducible
genes (20, 33-37). Interestingly, STAT6 exhibits transactivating
function only in the context of a subset of SBE sequence motifs (20,
34, 37-40).
IL-4 and IFN exhibit antagonistic effects on macrophage gene
expression (14-18). We have previously observed that IFN-mediated induction and IL-4-mediated suppression utilize the same regulatory sequences (the ISRE in the IP-10 gene and the GAS motif in the IRF-1
gene) (16, 40). Furthermore, IL-4 does not inhibit the activation of
STAT1 by IFN and IL-4-induced STAT6 is able to bind the
IFN-responsive site without transactivation (40). Recent studies
have shown that STAT1-dependent transcription requires transcriptional coactivators p300 and CREB-binding protein (CBP), which
may be present in limiting amounts (41, 42). On the basis of these
lines of evidence, we postulated that the inhibitory effect of IL-4 may
result, at least in part, from the direct action of IL-4-induced STAT6
on IFN-induced STAT1-dependent transcription. The
present study was undertaken to determine if IL-4-induced STAT6 is
required for the suppressive action of IL-4 on IFN-induced gene
transcription and to extend our analysis to include the
IFN-responsive element (RE), which controls expression of the MIG
chemokine gene (43, 44), a third form of IFN-sensitive nucleotide
regulatory motif. The results demonstrate that
IL-4-dependent suppression of IFN-induced MIG gene
expression is abolished in peritoneal macrophages from mice in which
the STAT6 gene has been deleted (45). Both the MIG gene promoter and
the RE from the MIG promoter are sensitive to the stimulatory action
of IFN and the inhibitory effects of IL-4. Furthermore, we find that
STAT6 as well as STAT1 can interact with the coactivator CBP. These
results suggest that these two factors may compete for coactivator
and/or occupancy of the RE site with opposite functional
consequences.
| |
EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
Recombinant mouse IFN and IL-4
were obtained from Life Technologies, Inc. and R & D Systems, Inc.
(Minneapolis, MN), respectively. Rabbit polyclonal antibodies to mouse
STAT1 (M-23), STAT6 (M-20), CBP (C-20), NF
B1(NLS), and RelA (A)
were obtained from Santa Cruz Biotechnology (Hercules, CA). Normal
rabbit IgG was obtained from Sigma.
Mice--
Homozygous STAT1 mutant mice (Stat1
/)
in which the Stat1 gene has been deleted by homologous
recombination were kindly provided by Dr. Robert D. Schreiber
(Washington University School of Medicine, St. Louis, MO) (46).
Stat6/ mice were kindly provided by Dr. James N. Ihle
(St. Jude Children's Research Hospital, Memphis, TN) (45). Specific
pathogen-free C57Bl/6 mice 9-12 weeks of age were purchased from the
Trudeau Institute (Saranac Lake, NY).
Cell Culture--
Thioglycollate-elicited peritoneal macrophages
were prepared as described previously (47) and cultured in RPMI 1640 containing L-glutamine, penicillin, streptomycin, and 5%
fetal bovine serum. The RAW264.7 or ANA-1 (48) mouse macrophage-like
cell lines were cultured in Dulbecco's modified Eagle's medium
containing L-glutamine, penicillin-streptomycin, and 10%
fetal bovine serum.
Preparation of RNA and Northern Hybridization
Analysis--
Total cellular RNA was extracted by the guanidine
isothiocyanate-cesium chloride method (49). Northern hybridization
analysis and cDNA probes for mouse MIG and rat GAPDH were described
previously (47, 50). Northern blots were also quantified using
phosphorescence detection. The relative magnitude of expression was
determined for each gene and normalized to values for GAPDH expression
in the same experiment.
Nuclear Run-on Assay--
Cultures of 6 × 107
RAW264.7 cells in 150-mm Petri dishes were treated as indicated in the
text for 90 min, and nuclei were isolated as described previously (51).
Transcription initiated in intact cells was allowed to complete in the
presence of [-32P]UTP (NEN Life Science Products), and
the RNA was isolated and hybridized to slot-blotted plasmid containing
specific cDNA insert (7 µg of DNA/slot) as described elsewhere
(52).
Reporter Plasmids--
A sequence encoding the 5'-flanking
promoter/enhancer region of the MIG gene was cloned by PCR using Ultima
DNA polymerase (Perkin-Elmer) from a mouse genomic DNA using a set of
primers corresponding to the mouse MIG genomic sequence (53) (upstream primer; 5'-TCGCCATATAGTGTCATGTCCC-3', downstream primer;
5'-CTGTGTTGGAGTGAAGTCCG-3'). The amplified PCR fragment was subcloned
into luciferase reporter construct (pGL2-B, Promega) and the nucleotide
sequence confirmed. The sequences corresponded to nucleotide residues
328 through +34 (53). The 5'-half-site of the RE motif at position
198 to 189 of the MIG promoter was mutated by using two-step PCR amplification (54). The mutant sequence utilized was
TCCCggAggATccACT. Lowercase letters represent the mutant
sequence, and the underlined sequence is the 5'-half-site of the RE.
One copy of the RE motif (sense strand:
5'-gATCCCTTACTATAAACTCCCCGTTTATGTGAAATGGA-3') was placed in front of a
heterologous thymidine kinase promoter (55) linked to the luciferase
gene (pTK-81Luc). The luciferase control plasmid under control of the
cytomegalovirus promoter was provided by Dr. G. Sen (Dept. of Molecular
Biology, Cleveland Clinic Foundation).
Transient Transfection--
Luciferase reporter gene plasmids
were transiently transfected into RAW264.7 cells by the DEAE-dextran
method as described previously (20, 56). Briefly, 15 µg of reporter
plasmid DNA and 3 µg of reference plasmid (pRL-TK, Promega) were
transfected in the presence of DEAE-dextran (300 µg/ml). To
standardize transfection efficiencies, the transfected cells were
harvested, pooled, and seeded in four 60-mm culture dishes. After
24 h, the cells were stimulated with IFN and/or IL-4 for 8 h prior to analysis of luciferase activity. Firefly and
Renilla luciferase activities were assayed using reagents
provided by Promega according to the manufacturer's instructions.
Twenty µg of extract protein were utilized in each assay.
Immunoprecipitation and Western Blot Analysis--
Cultures of
6 × 107 RAW264.7 cells in 150-mm Petri dishes were
treated as indicated in the text. After stimulation, RAW264.7 cells
were washed with ice-cold phosphate-buffered saline, harvested, and
resuspended in Lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4, and 10 µg/ml
leupeptin, antipain, aprotinin, and pepstatin) for 10 min on ice. After
pre-clearing the lysate with normal rabbit IgG (Sigma) and Protein
G-Sepharose (Amersham Pharmacia Biotech), whole cell lysate (~500
µg of protein) were incubated with anti-CBP (1 µg) or normal rabbit
IgG and protein G-Sepharose (50% slurry) for 16 h at 4 °C. The
immunoprecipitates were washed six times with 1 ml of lysis buffer,
eluted with SDS-PAGE sample buffer, resolved on 7.5% SDS-PAGE, and
analyzed by Western blot as descried previously (40).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared as described previously using a modified method
of Dignam et al. (20, 40, 57). The following oligonucleotides were used in EMSA: RE:
5'-GATCCCTTACTATAAACTCCCCGTTTATGTGAAATGGA-3'; mut5'RE:
5'-GATCCCggAggATccACTCCCCGTTTATGTGAAATGGA-3'; 5'-half-site: 5'-ATCCCTTACTATAAACTCCC-3'; 3'-half-site:
5'-CCCCGTTTATGTGAAATGGA-3.
For binding reactions, nuclear extracts (5 µg of protein) were
incubated in 12.5 µl of total volume containing 20 mM
HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 200 µg/ml bovine serum
albumin, and 1.25 µg of poly(dI-dC) for 15 min at room temperature.
32P-Labeled oligonucleotide (0.5 ng, 5 × 105 cpm) was then added to the reaction mixture and
incubated for 15 min at room temperature. The reaction products were
analyzed by electrophoresis in a 5% polyacrylamide gel with 0.25× TBE
buffer (22.3 mM Tris, 22.2 mM borate, 0.5 mM EDTA). In some experiments, rabbit antibody to NFB1
(p50), RelA (p65), STAT1, and STAT6 were added prior to
electrophoresis. The dried gels were analyzed by autoradiography and by
phosphorescence detection.
| |
RESULTS |
IL-4 Suppresses IFN-induced Expression of MIG
mRNA--
IL-4 is known to suppress IFN-induced gene expression
in mononuclear phagocytes (14-18). The MIG gene encodes a C-X-C
Chemokine known to be transcriptionally regulated by IFN (43, 44,
53). To determine whether IL-4 can suppress MIG expression,
thioglycollate-elicited peritoneal macrophages were pretreated with
IL-4 (10 ng/ml) for 15 min, followed by stimulation with different
doses of IFN for 4 h prior to preparation of total RNA and
Northern hybridization analysis. IFN induced and IL-4 suppressed
levels of MIG mRNA (Fig.
1A). IL-4 did not alter the
kinetics of IFN-induced MIG mRNA expression but reduced the
steady state mRNA levels over the entire time course (Fig.
1B). Quantitatively and qualitatively similar effects of
IFN and IL-4 on MIG mRNA were seen in the macrophage-like cell
lines RAW264.7 (Fig. 1C) and ANA-1 (Fig. 1D). The
suppression by IL-4 was mediated predominantly by inhibiting transcription as assessed by nuclear run-on assay in RAW264.7 cells
(Fig. 2).
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 1.
IL-4 suppresses IFN-induced MIG mRNA
expression in macrophages. A, thioglycollate
broth-elicited peritoneal macrophages were pretreated with IL-4 (10 ng/ml) for 15 min, followed by stimulation with different
concentrations of IFN for 4 h prior to preparation of total RNA
and analysis of specific mRNA levels by Northern hybridization as
described under "Experimental Procedures." Five µg of total RNA
were analyzed in each lane. Similar results were obtained in two
separate experiments. B, time dependence of MIG mRNA
expression in IFN- and/or IL-4-stimulated macrophages.
Thioglycollate broth-elicited peritoneal macrophages were pretreated
with IL-4 (10 ng/ml) for 15 min, followed by stimulation with IFN
(100 units/ml) for the indicated times prior to analysis of specific
mRNA levels as described above. Northern blots were quantified by
phosphorimage analysis, and relative mRNA levels are presented as
percentage of maximum expression as described under "Experimental
Procedures." C and D, RAW264.7 (C)
or ANA-1 (D) macrophages were unstimulated or pretreated
with IL-4 (10 ng/ml) for 15 min prior to stimulation with increasing
concentrations of IFN as indicated for 4 h. Levels of MIG
mRNA were determined by Northern hybridization as described above.
Similar results were obtained in two separate experiments.
|
|
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
IL-4 suppresses rate of the IFN-induced
MIG transcription. RAW264.7 cells were untreated (UT)
or pretreated with IL-4 (10 ng/ml) for 15 min, followed by followed by
stimulation with or without IFN (100 units/ml) for 90 min prior to
isolate nuclei. Transcription was assessed by nuclear run-on assay as
described under "Experimental Procedures." Radiolabeled RNA
transcripts were hybridized with slot blotted cDNA encoding MIG or
GAPDH as indicated. Similar results were obtained in two separate
experiments.
|
|
The Effects of IFN and IL-4 on MIG Expression Require STAT1 and
STAT6, Respectively--
STAT1 is believed to be important for most if
not all IFN-induced gene expression (46, 58), while STAT6 is a
primary signaling molecule linked with response to IL-4 (45, 59, 60). To examine the role of IFN-induced STAT1 and IL-4-activated STAT6 in
control of MIG expression, macrophages from Stat1/ and
Stat6/ mice were used to measure each response (45, 46)
(Fig. 3). As expected, IFN-induced MIG
mRNA expression was completely abolished in peritoneal macrophages
from Stat1/ mice (Fig. 3A) but remained intact in macrophages from Stat6/ (Fig. 3B).
Whereas IFN-induced MIG mRNA was inhibited by IL-4 in wild type
macrophages, the suppressive effect of IL-4 was entirely lost in
macrophages from the Stat6/ mice (Fig. 3, B
and C).
View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
STAT6 is indispensable for the IL-4-mediated
suppression of the IFN-induced, STAT1-dependent
expression of MIG mRNA. A, thioglycollate
broth-elicited peritoneal macrophages from wild type mice or from
Stat1/ mice were untreated (UT) or stimulated
with IFN (100 units/ml) for 4 h. Levels of murine MIG mRNA
were determined by Northern hybridization. Five µg of total RNA were
analyzed in each lane. B, thioglycollate broth-elicited
peritoneal macrophages from wild type mice or from
Stat6/ mice were untreated (UT) or pretreated
with IL-4 (10 ng/ml) for 15 min, followed by stimulation with IFN
(100 units/ml) for 4 h prior to analysis of specific mRNA
levels by Northern hybridization. Five µg of total RNA were analyzed
in each lane. C, Northern blots were quantified by
phosphorimage analysis and relative mRNA levels are presented as
percentage of the IFN-induced expression. Each column and
bar represents the mean ± S.E. from three independent
experiments.
|
|
The RE Motif in the MIG Promoter Is Sensitive to Both IFN and
IL-4--
IFN-induced transcription of the MIG gene has been shown
to depend upon the RE motif located at positions 198 to 171 in the MIG promoter (44). To determine whether the suppression of
IFN-induced MIG gene expression by IL-4 is dependent upon the RE
motif, 328 nt of the 5'-flanking sequence of the MIG gene were linked
to the luciferase reporter gene and transiently transfected into
RAW264.7 cells. Twenty-four hours after transfection, the cultures were
stimulated with IFN alone or in the presence of IL-4 for 8 h
prior to analysis of luciferase activity. Although IL-4 alone had no
effect on luciferase activity, reporter gene expression was markedly
elevated in cells stimulated with IFN (Fig.
4A). IL-4 added along with
IFN suppressed the induced luciferase activity by approximately
50%. IL-4 did not modulate the luciferase activity from the cells
transfected with cytomegalovirus promoter-driven luciferase construct
(data not shown).
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of IFN and IL-4 on transcription
from the MIG promoter. A, the luciferase reporter
construct (shown schematically) was transiently transfected into
RAW264.7 cells as described under "Experimental Procedures."
Twenty-four h after transfection, the cells were pretreated with IL-4
(10 ng/ml) for 15 min prior to stimulation with IFN (100 units/ml)
for 8 h, followed by analysis of cell extracts for luciferase
activity. The relative luciferase activity is presented as a percentage
of activity obtained in cells stimulated with IFN (100 units/ml)
alone. Each column and bar represents the
mean ± S.E. from three independent experiments. B,
RAW264.7 cells were transiently transfected with a luciferase reporter
plasmid containing one copy of the MIG RE motif linked to the TK
promoter (TK-81) as shown schematically. After 24 h, the cells
were either unstimulated (UT) or treated with IL-4 (10 ng/ml) for 15 min, followed by stimulation with IFN (100 units/ml)
for 8 h prior to measurement of luciferase activity. The relative
luciferase activity is presented as percentage of maximum activity
obtained in cells transfected with the reporter plasmid stimulated with
IFN (100 units/ml). Each column and bar
represents the mean ± S.E. from three independent experiments.
The -fold induction of stimulated versus unstimulated
samples is also indicated over the column.
|
|
To determine if the RE motif of the MIG gene was the target site for
IL-4, one copy of this motif was placed in front of the TK promoter
(TK-81) linked to the luciferase reporter gene and transiently
transfected in RAW264.7 cells (Fig. 4B). As was observed for
the natural MIG promoter, IFN stimulated luciferase expression. IL-4
alone had no effect but suppressed the response to IFN by
approximately 50%. Luciferase activity from TK-81 alone was not
affected by IFN and/or IL-4 (data not shown).
IL-4-induced STAT6 Interacts with Coactivator CBP--
The
promoter analysis of the MIG gene indicates that the RE motif is
essential for both the IFN-induced transcriptional activation of the
MIG promoter and the suppressive effect of IL-4. Furthermore, STAT6 is
required for the suppressive activity of IL-4. Since
STAT1-dependent transcription has been demonstrated to
require the presence of the coactivator p300 or CBP (41, 42), it is
conceivable that suppression might involve competition between STAT1
and STAT6 for limiting quantities of the coactivator. To explore this
possibility, we assessed whether STAT6 can interact with CBP in
vivo using a co-immunoprecipitation analysis (Fig. 5). Consistent with previous results (41,
42), STAT1 was present in immunoprecipitates using antibody to CBP in
whole cell lysates from RAW264.7 cells, and the association appeared to
be independent of stimulation (Fig. 5A). The same CBP
immunoprecipitates also contained STAT6 (Fig. 5B). These
results indicate that STAT6 as well as STAT1 binds CBP and are
consistent with the possibility that STAT6 may compete for coactivator
CBP.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Association of STAT1 and STAT6 with CBP in
RAW267.4 cells. Whole cell extracts were prepared from RAW264.7
cells either untreated (UT) or pretreated with IL-4 (10 ng/ml) for 15 min, followed by stimulation with or without IFN (100 units/ml) for 30 min before lysis the cells. Whole cell extracts
(~500 µg of protein) were immunoprecipitated (IP) with
anti-CBP antibody or normal rabbit IgG as indicated. CBP-associated
proteins were then eluted, and half of the eluted protein was
individually separated by 7.5% SDS-PAGE. One µg of whole cell
extract was also loaded onto the gel (Input). Proteins were
transferred to membrane and each blots were developed with
anti-STAT1 (A) and anti-STAT6 (B) antibodies,
respectively. The blot in A was subsequently stripped and
immunoblotted with anti-CBP antibody (C). Similar results
were obtained in two separate experiments.
|
|
IL-4-induced STAT6 Binds to the RE Motif--
An alternative
possibility is that IL-4-induced STAT6 might compete for binding of
STAT1 to the RE motif. Since STAT6 can bind to a number of sequence
motifs without transactivating function (20, 38, 40), the occupancy of
the RE site by STAT6 might result in suppression and not
transactivation. Indeed, although IFN could stimulate reporter gene
expression from constructs containing the RE linked to the TK
promoter, treatment with IL-4 was entirely inactive. To further test
this hypothesis, we determined the ability of STAT1 or STAT6 to bind
with the RE motif. Nuclear extracts were prepared from untreated,
IFN-treated, and/or IL-4-treated RAW264.7 cells and analyzed by EMSA
using a radiolabeled oligonucleotide corresponding to the RE motif
(Fig. 6A). Several
constitutive RE binding activities were detected in nuclear extracts
from untreated cells (designated as I and II). IFN induced two
additional RE binding activities (lane 2, a low and a
high mobility complex designated as RF-1 and RF-2, respectively).
Interestingly, IL-4 induced a single binding complex on the RE motif
(lane 3). When the cells were co-stimulated with IFN and
IL-4, the pattern of complex formation was essentially the same as seen
in nuclear extracts from the cells treated with individual stimuli. As
reported previously, IL-4 did not effect the tyrosine phosphorylation
or nuclear localization of STAT1 in response to IFN or vice
versa (40).
View larger version (32K):
[in this window]
[in a new window]
|
Fig. 6.
IFN and IL-4 stimulate different RE
binding activities in RAW264.7 cells. A, nuclear
extracts were prepared from RAW264.7 cells either untreated
(UT) or pretreated with IL-4 (10 ng/ml) for 15 min, followed
by stimulation with or without IFN (100 units/ml) for 30 min prior
to the preparation of nuclear extracts. Five µg of nuclear extract
protein were utilized for assay of DNA binding activities by EMSA using
radiolabeled oligonucleotides containing the RE element from the MIG
gene. Constitutive binding activities (designated as I and
II) and IFN- and IL-4-induced binding activities are
indicated as filled and open arrows,
respectively. Nonspecific binding activity is designated as
ns. B, IL-4-induced RE binding complex
contains STAT6. Five µg of nuclear extract protein prepared from
RAW264.7 cells were utilized in each reaction with or without addition
of antibody (1 µg) to STAT1, STAT6, NFB1, or RelA and assayed for
DNA binding activity as described above.
|
|
To identify the proteins participating in formation of the RE
binding complexes, antibody supershift assays were performed (Fig.
6B). The constitutive binding activities (I and II) in
nuclear extracts from untreated cells were reactive with antibodies to NFB1 (p50) and RelA (p65) (lanes 4 and
5). This result is consistent with previous observations of
low level of NFB binding activity in nuclear extracts from
unstimulated macrophages (61) and that NFB1 (p50) and RelA (p65)
recognize and bind to some SBE motifs with low affinity (50, 62). The
most slowly migrating constitutive complex represents nonspecific
binding since specific competitor could not eliminate the complex (data
not shown). When nuclear extracts from IFN-treated cells were
analyzed, antibody to STAT1 supershifted the IFN-induced RE
binding complexes without affecting NFB1 and RelA (lane
7). A combination of antibodies to STAT1 and NFB1
supershifted the inducible and constitutive binding complexes
(lane 11). When nuclear extracts from
IL-4-treated cells were analyzed, antibody to STAT6 supershifted the
IL-4-induced complex (lane 15). The residual
binding activity in nuclear extracts treated with anti-STAT6 and
anti-NFB1 (lane 19) appears to be the
supershifted-NFB1 as seen in lanes 4 and
11. These results indicate that both IL-4-induced STAT6 and
IFN-induced STAT1 recognize the RE motif. Two different complexes
containing STAT1 were identified; the more rapidly migrating complex
(RF-2) is likely to be STAT1 homodimers, whereas the more slowly
migrating complex (RF-1) could be a tetrameric form of STAT1 (63,
64).
STAT6 Binds to the 3'-Half-site of RE-1 Motif--
The RE
motif consists of two GAS-like motifs (Fig.
7D), and both sites have been
shown to be required for IFN-induced transcription of the MIG gene
(44). To determine whether the binding of IL-4-induced STAT6 to the
RE motif also requires both 3'- and 5'-half-sites, the 5'-half-site
of the RE motif was mutated and tested for binding activity by EMSA
(Fig. 7A). Although the IFN-induced RF-1 binding complex was markedly reduced on the mutant oligonucleotide
(lane 6), IL-4-induced STAT6 binding remained
intact (lane 7). Interestingly, the
IFN-induced high mobility complex (RF-2) substantially increased when the mutant oligonucleotide was used as the probe (lane
6). The IFN-induced transcriptional activity of the RE
was almost completely abolished when a luciferase reporter construct
containing a mutant 5'-half-site was analyzed by transient transfection
(Fig. 7B), indicating that the formation of the RF-1
complex is essential for IFN-induced MIG promoter activity. The
residual IFN-induced promoter activity in the mutant luciferase
construct was still sensitive to IL-4. To determine if the 3'-half-site
of the RE motif alone is sufficient for STAT6 binding,
oligonucleotides corresponding to the 3' or the 5'-half-site of the
RE motif were independently synthesized and tested for IFN- and
IL-4-induced binding activity by EMSA. Consistent with the results
shown in Fig. 7A, IL-4-induced STAT6 was able to form a
complex on the 3'-half-site (lane 15), while
IFN stimulated primarily the formation of the high mobility complex
RF-2 (lane 14). Interestingly, when the
5'-half-site was used as probe (lanes 17-20),
neither IFN nor IL-4 induced any detectable complex formation. These
results indicate that while the 3'-half-site of the RE motif is
sufficient for STAT6 binding and for RF-2, formation of RF-1
requires both the 3'- and 5'-half-sites.
View larger version (47K):
[in this window]
[in a new window]
|
Fig. 7.
IL-4-induced STAT6 preferentially binds to
the 3'-half-site of the RE motif. A, nuclear
extracts were prepared from RAW264.7 cells as described in the legend
of Fig. 6. Five µg of nuclear extract protein were utilized for assay
of DNA binding activities in EMSA using radiolabeled oligonucleotides
containing the RE or a RE containing a mutant 5'-half-site
(mut 5'RE) as shown schematically in
D. B, The RF-1 complex is required for the
IFN-induced transcriptional activation of the MIG promoter. RAW264.7
cells were transiently transfected with the wild type promoter
(MIG-328) or the mutant construct (mut
5'RE) in which the 5'-half-site of the RE has
been mutated as indicated in D. After 24 h, the cells
were either unstimulated (UT) or treated with IL-4 (10 ng/ml) for 15 min, followed by stimulation with different concentration
of IFN for 8 h prior to measurement of luciferase activity. The
relative luciferase activity is presented as percentage of maximum
activity obtained in cells transfected with the MIG-328 reporter
plasmid and stimulated with IFN (100 units/ml). Each
column and bar represents the mean ± S.E.
from three independent experiments. The -fold induction of stimulated
versus unstimulated samples is also indicated. C,
nuclear extracts were analyzed for the DNA binding activities by EMSA
using radiolabeled oligonucleotides containing the RE-1 or
3'-half-site or 5'-half-site of the RE motif as indicated. Similar
results were obtained in two separate experiments. D,
nucleotide sequences of wild type and mutant RE-1 oligonucleotides
used in this study. The 3'- and the 5'-half-sites of the RE-1 motif
are boxed. Mutated nucleotides in the mutant 5'RE are
indicated in lowercase. Consensus sequences for the GAS are
shown (M, A or C; K, G or T).
|
|
Taken together with the previous observation that the RE is not
responsive to IL-4 alone (see Fig. 4), the data from the association of
STAT6 with CBP and the RE binding suggest two potential
possibilities for the antagonistic functions of STAT6 on IFN-induced
STAT1-dependent transcription of the MIG gene. 1)
IL-4-induced STAT6 could compete for the limiting amount of coactivator
CBP, which is necessary for the STAT1-dependent
transcription; and/or 2) STAT6 could reduce the frequency of
IFN-induced transcription by competing for occupancy of the RE by
STAT1.
| |
DISCUSSION |
The initiation, magnitude, and resolution of immune-mediated
inflammation are regulated at least in part through the action of pro-
and anti-inflammatory cytokines (1, 2). For example, the Th1-derived
cytokine IFN promotes the pro-inflammatory function of macrophages
through stimulation of new gene expression, and this is often
antagonized by the Th2-derived cytokine IL-4 (2-5). IFN and IL-4
are both known to activate distinct members of the STAT family of
transcription factors, each of which may exhibit differential
transactivating potential depending upon the sequence motif to which
they bind (20, 34, 37-40). In consideration of this, we postulated
that the antagonistic action of IL-4 on IFN-stimulated gene
transcription is mediated, at least part, through a direct antagonistic
action of IL-4-activated STAT6. The results from the present study
demonstrate that STAT6 is indispensable for the negative regulatory
function of IL-4 on IFN-induced, STAT1-dependent
transcription of the MIG gene in mouse macrophages, and both factors
appear to mediate their action through the RE motif found in the MIG
gene promoter. These conclusions are based on the following
observations. 1) IFN-induced MIG mRNA expression was suppressed
by IL-4 in primary macrophages and in ANA-1 or RAW264.7 macrophage-like
cell lines. 2) IFN-induced MIG gene expression was abolished in
peritoneal macrophages from Stat1/ mice, while the
suppressive effect of IL-4 was abolished in macrophages from
Stat6/ mice. 3) IL-4 suppressed the IFN-induced
transcription of a luciferase reporter gene linked to either a 328-base
pair fragment flanking the transcriptional start site of the MIG gene or to a single copy of the RE motif from the MIG gene in the context
of a heterologous promoter; IL-4 alone showed no independent transactivating function on either construct.
Our findings provide support for two possible mechanisms through which
STAT6 may function to directly suppress IFN-induced gene expression.
One mechanism of STAT6-mediated suppression may involve the
participation of transcriptional co-activators such as CBP or p300. It
has been reported recently that STAT1-dependent transcription requires either CBP and/or p300 (41, 42). Thus the
finding that STAT6 can interact with CBP raises the possibility that
STAT6 might sequester limiting quantities of CBP and thereby antagonize
the STAT1-dependent transcription (41). The association of
both STAT1 and STAT6 with CBP was not altered by stimulation with
either IFN or IL-4. Thus, these data do not provide any direct
evidence of competition between the two STATs for CBP. Nevertheless,
further studies of the functional consequences of STAT6-CBP interaction
will be required to determine the physiological significance.
The present results are also consistent with a scenario in which STAT6
and STAT1 compete for occupancy of the RE regulatory site, as has
been suggested previously in studies of the effects of IL-4 on
IFN-stimulated IRF-1 expression (40). Because STAT6 has poor
transactivating function on the MIG promoter, occupancy of the STAT1
binding site by STAT6 would result in reduced transcription. The nature
of the regulatory sequence motif that determines both induction and
suppression of the MIG gene (RE) provides some unique features to
the potential interactions between SBEs and STAT1 or STAT6. First, the
formation of the RF-1 complex requires both the 3'- and
5'-half-sites of the RE motif while STAT6 only recognizes the
3'-half-site. Second, the mutant 5'-half-site version of the RE
motif will form RF-2 but not RF-1 and is functionally inactive in
supporting IFN-driven reporter gene transcription. Thus, the RF-1
complex but not the RF-2 complex is essential for the IFN-induced
transcriptional activity of the MIG gene. Finally, IL-4-induced STAT6
is able to bind to the RE (3'-half-site) without transactivating
function. These findings suggest that binding of STAT6 on the
3'-half-site may prevent the formation of the RF-1 complex,
resulting in decreased transcriptional frequency.
STAT6 exhibits the broadest DNA binding specificity of all STAT family
members (20, 33-40). However, binding of STAT6 to IFN-responsive
motifs does not lead to transcriptional activation despite the fact
that STAT6 can interact with CBP. In contrast, STAT6 functions as a
transcriptional activator when bound to a subset of SBEs including
those found in the IgE or IL-1 receptor antagonist genes (20, 34).
Thus, the nucleotide sequence of the DNA binding site is a critical
determinant of the transcriptional activating function of STAT6. This
may be an important distinguishing feature of STAT6, which allows both
positive and negative effects on gene transcription.
The ability of STAT6 to compete with STAT1 for binding to the RE was
not detectable by EMSA (Figs. 6 and 7). This result is, however, not
surprising since the oligonucleotide concentration in the reaction is
in great excess relative to the concentration of STAT1 or STAT6, a
condition under which competition between the protein factors will not
occur. Reduction of the oligonucleotide concentration to a level at
which competition might occur does not allow detection of the complexes
(data not shown). Thus EMSA analysis of RE binding activities using
nuclear extracts from macrophages treated with IFN and IL-4 would
not be expected show reduced formation of the RF-1 complex.
STAT1 has been shown to exhibit a cooperative binding activity with two
or more low affinity SBE motifs, which is mediated by the N-terminal
region of the protein (63, 64). Thus, it is likely that the
IFN-induced RF-1 complex is a tetrameric form of STAT1. The
higher mobility RF-2 complex appears to be a STAT1 homodimer, which
binds to the 3'-half-site of the RE motif. Interestingly, STAT6
formed only a single complex despite its ability to bind a relatively
wide range of SBE motifs (20, 33-40). Whether STAT6 lacks
cooperativity in the N-terminal region or the RE motif is
insufficient for the cooperative binding of STAT6 remains to be
determined.
Although IL-4-mediated anti-inflammatory function has been found to
include both post-transcriptional and translational events (8, 12, 66),
the majority of reports have identified transcription as a primary
molecular target (8, 14, 16, 40, 67-71). The data in the present study
further demonstrate that STAT6 is essential for IL-4-mediated
suppression of IFN-induced MIG gene expression. Indeed, we have also
observed that IL-4-mediated suppression of IFN-induced IP-10, IRF-1,
ICAM-1, and inducible nitric-oxide synthase gene expression are also
abolished in peritoneal macrophages from Stat6/ mice
(data not shown). IL-13-mediated suppression of inducible nitric-oxide
synthase gene expression has also been shown to depend upon STAT6 (72).
This collection of studies indicates that IL-4- or IL-13-induced STAT6
can provide negative regulatory function, which is necessary for the
anti-inflammatory cytokine action.
and
Top
Abstract
Introduction
To whom correspondence should be addressed: Dept. of Immunology,
Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Ave., Cleveland, Ohio 44195. Tel.: 216-444-4669; Fax: 216-444-9329.
