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Requires Stat1 Activation
Tularik, Inc., South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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antagonizes many physiological responses mediated by IL-4,
including the inhibition of IL-4-induced IgE production. This event is
largely mediated at the level of transcription. We observed that the
IL-4 response element of the germline epsilon promoter is sufficient to
confer IFN--mediated repression onto a reporter construct. The
inhibitory effects were observed in both lymphoid and nonlymphoid cell
lines. Stat1, which is activated by IFN-, cannot recognize the
Stat6-specific IL-4 response element in the 
promoter. Hence,
competitive DNA binding does not seem to be the underlying mechanism
for the inhibitory effect. This is supported by the observation that
inhibition is not seen at early time points, but requires prolonged
IFN- treatment. IFN- stimulation results in a loss of
IL-4-induced Stat6 tyrosine phosphorylation, nuclear translocation, and
DNA binding. Using the fibrosarcoma cell line U3A, which lacks Stat1,
we demonstrated that the transcription activation function of Stat1 is
required for the IFN--mediated repression. Repression was restored
by overexpression of Stat1
, but not Stat1ß, in U3A cells.
Treatment with IFN-, but not IL-4, specifically up-regulates the
expression of SOCS-1 (silencer of cytokine signaling), a recently
characterized inhibitor of cytokine signaling pathways, such as IL-6
and IFN-. Overexpression of SOCS-1 effectively blocks IL-4-induced
Stat6 phosphorylation and transcription. This suggests that
IFN--mediated repression of IL-4-induced transcription is at least
in part mediated by SOCS-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are two crucial cytokines involved in the regulation of
immune and inflammatory responses (1, 2). Among the many physiological
responses mediated by IL-4 is its ability to promote the
differentiation of T helper precursors toward the Th2 lineage while
inhibiting Th1 cells development (3, 4, 5, 6). IL-4 stimulation of B cells
triggers Ig switching to the IgE isotype (7). In contrast, IFN-,
secreted by T cells of the Th1 lineage, represses the development of
Th2 cells (4, 5) and inhibits the class-switching event mediated by
IL-4 in B cells (8, 9, 10, 11, 12, 13).
Both, IL-4 and IFN- signal through the JAK/STAT pathway. Upon ligand
binding to the cognate cytokine receptor, latent proteins known as
STATs are tyrosine phosphorylated in the cytoplasm by members of the
JAK5 family. Activated STATs
dimerize, migrate to the nucleus, bind to specific
cis-acting elements, and activate transcription of distinct
sets of cytokine-responsive genes (14, 15, 16, 17). IFN- and IL-4
stimulation leads to the activation of Stat1 and Stat6, respectively
(18, 19, 20). Activated Stat1 dimers bind to a cis-acting
element, known as -activated sequence (GAS) which is a palindrome
spaced by three nucleotides (TTCN3GAA; N3 site). This
element is able to drive IFN--induced gene expression when fused to
a truncated promoter (21, 22, 23). Activated Stat6 dimers also recognize
the N3 site, but an optimal Stat6 binding site contains four (N4 site)
instead of three nucleotides in the center and is not bound by Stat1
(22, 24). Furthermore, DNA binding of Stat6 is not sufficient to
trigger transcription activation, since neither an N3 nor an N4 site
will potentiate IL-4-induced gene expression when fused to a truncated
promoter (21). Instead, Stat6 needs to cooperate with adjacently bound
transcription factors to promote IL-4-induced transcription. The IL-4
response element found in the germline epsilon promoter is composed of
an authentic N4 Stat6 binding site flanked by a C/EBP site (21, 25).
IL-4 induced transcription depends on the integrity of both sites, and
activation of this promoter is essential for class switching to the IgE
isotype. B cells derived from Stat6 knockout mice do not produce IgE in
response to IL-4 treatment, illustrating that Stat6 is absolutely
required for this event (26, 27, 28).
IL-4 and IFN- act antagonistically in many immune scenarios, which
at least in part can be explained by the opposite effects of these two
cytokines on gene transcription (8, 13, 29). The underlying mechanism
by which IL-4 and IFN- antagonize gene expression is not completely
understood and may be different for individual genes or cell types. For
example, transcription of the IFN--induced gene IRF-1 is strongly
repressed by IL-4 in macrophages (29). The mechanism of this repression
appears to be mediated by a competition between Stat1 and Stat6 for the
GAS element (N3 site) in the IRF-1 promoter.
Previously, Xu and Rothman showed that expression of the germline
epsilon transcript in the mouse B cell line M12.4.1. can be induced by
IL-4 and LPS treatment, and the expression is inhibited by
costimulation with IFN- (13). Here, we demonstrate that
transcription of the human gene is induced by IL-4 in the B
lymphoma BJAB, and the expression is antagonized by IFN- treatment.
We also show that the IL-4 response element of the promoter, which
is bound by Stat6, is a target for IFN--mediated repression. IFN-
treatment inhibits IL-4-induced tyrosine phosphorylation, nuclear
translocation, and DNA binding activity of Stat6. Stat1, although
incapable of binding to the Stat6-specific N4 site, is essential for
the inhibitory effect mediated by IFN-. We show that IFN- induces
the expression of the inhibitory protein SOCS-1, which interferes with
IL-4-dependent Stat6 tyrosine phosphorylation and transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human B lymphoma BJAB cells were cultured in RPMI 1640 (Meditech, Herndon, VA) supplemented with 10% FBS (Meditech, Herndon, VA) and 10 µM 2-ME. HepG2 human hepatic carcinoma and HEK293 cells were cultured in DMEM/Ham’s F-12 (Meditech) supplemented with 10% FBS. The human fibrosarcoma U3A cells (a gift from Dr. George Stark) were cultured in DMEM (Meditech) supplemented with 10% FBS.
Cytokines
Human IL-4 (4.1 x 107 U/mg) and IFN-
(4.75 x 107 unit/mg) were purchased from R&D Systems
(Minneapolis, MN).
Northern blot analysis
BJAB or HepG2 cells were treated with cytokines as indicated in
the figure legends. The final concentration of both IL-4 and IFN-
was 10 ng/ml. Total RNA were prepared using Trizol reagent (Life
Technologies, Gaithersburg, MD) according to the manufacturer’s
protocol. Northern blots were probed with randomly primed DNA probes
(Megaprime DNA Labeling System, Amersham, Arlington Heights, IL),
corresponding to exons 2, 3, and 4 of the germline transcript, the
full-length SOCS-1 cDNA (30), or GAPDH cDNA.
Transfection
The IL-4-inducible luciferase reporter construct carrying four
copies of the C/EBP-N4 site has been described previously (21). A
IL-1-responsive luciferase reporter construct containing the E-selectin
promoter (E-selectin-Luc) has also been described (31). Full-length
Stat1 (Stat1) was amplified by PCR using the following primers:
sense strand, 5'-GTT GGG GCA CAA GGA TCC AGG ATG TCT CAG TGG TAC-3';
and antisense strand, 5'-ATT CAT GCT GCG GCC GCA CTA TAC TGT GTT CAT
CAT-3'. Mutant Stat1 lacking the C-terminal activation domain
(Stat1ß) was synthesized by PCR amplification using the following
primers: sense strand, 5'-GTT GGG GCA CAA GGA TCC AGG ATG TCT CAG TGG
TAC-3' and antisense strand, 5'-AGA AGG GTG GCG GCC GCA TCA AAC TTC AGA
CAC AGA AAT-3'. The resulting fragments were cloned into the
BamHI and NotI sites of pcDNA3. The integrity of
the DNA fragment was confirmed by DNA sequence analysis. The SOCS-1
expression plasmid (TIF3) has been described previously (30). HepG2,
HEK293, or U3A cells were transfected using calcium phosphate
precipitation (Promega, Madison, WI). BJAB cells were transiently
transfected with the IL-4-inducible reporter construct (21) using
electroporation as described previously (32). For the experiments shown
in Figs. 2
and 5, cells were transfected overnight with the appropriate
plasmids. The next day, cells were split into six-well plates, and
cytokines were added as described for each experiment. Cells were
harvested at the indicated times after cytokine treatment. A control
plasmid carrying the ß-galactosidase gene under the CMV promoter was
cotransfected in each experiment. Luciferase and ß-galactosidase
activities were determined using the Promega assay system (Promega).
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Probes corresponding to the Stat6 binding site (N4) and the Stat1 binding probe (N3) have been described previously (21). Nuclear extracts were prepared at the indicated time points, and EMSAs were performed as described previously (19, 33).
Immunoprecipitation and Western blot analysis
HepG2 cells were stimulated with cytokines as indicated in the figure legends. For experiments involving SOCS-1, HEK293 cells were transiently transfected with the His-tagged Stat6 (provided by Dr. Tim Hoey, Tularik, South San Francisco, CA) in the presence or the absence of the SOCS-1 expression plasmid. HEK293 cells were stimulated with IL-4 (10 ng/ml) for 30 min before cell lysis. Cytoplasmic extracts were prepared by lysing the cells in buffer B (0.1% Nonidet-P 40, 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, and 1 mM PMSF) for 10 min on ice. Detergent-insoluble material was removed by centrifugation at 14,000 rpm for 10 min. Five hundred micrograms of soluble proteins were immunoprecipitated with either 1 µl of anti-Stat6 antiserum (24) or 6x His mAb (Clontech, Palo Alto, CA) and 40 µl of protein A agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were separated on 7.5% SDS-PAGE. Immunoblots were probed with horseradish peroxidase-conjugated recombinant antiphosphotyrosine Ab (RC20H; Transduction Laboratories, Lexington, KY) as described previously (34). Proteins were visualized using the enhanced chemiluminescence reagent (Amersham). The same blots were stripped and reprobed with anti-Stat6 antiserum (1/10,000) followed by horseradish peroxidase-conjugated anti-rabbit Ab (Amersham).
For nuclear translocation studies of Stat6 or Stat1, 30 µg of total nuclear proteins were subjected to Western blot analysis using anti-Stat6 (24) or anti-Stat1 (Santa Cruz Biotechnology) antiserum.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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germline transcript expression
by IFN-
Previously it was demonstrated that the IL-4-induced IgE
production in normal lymphocytes can be suppressed by IFN- (8, 9, 10, 11, 12, 13).
Expression of the sterile transcript was a prerequisite for isotype
switching. To determine whether IFN- affected the IL-4-induced
expression of the germline epsilon transcript, we analyzed a panel of
human B lymphoma lines for their ability to properly regulate the
promoter in response to IL-4 and IFN-. Fig. 1A showed that the human B
lymphoma line, BJAB, behaved like normal B cells in its responsiveness
toward IL-4 and IFN-. Expression of the IRF-1 mRNA was observed
3 h after IFN- treatment (data not shown) and remained elevated
for 28 h following stimulation (Fig. 1A, lane
3). IL-4 treatment had no effect on IRF-1 transcription
(lane 2), and costimulation with IL-4 and IFN-
resulted in a slight reduction of the IRF-1 mRNA level
(lane 4). The inhibitory effect was less pronounced
than previously observed in macrophages, which may be due to the
different cell types used in the two studies (29). These data showed
that the IFN- signaling pathway is active in the BJAB B lymphoma
line.
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germline transcript in BJAB cells
was observed 7 h poststimulation (Fig. 1
B, lane
2). No detectable RNA was observed at earlier time points (data
not shown). The transcript continued to accumulate over time, and much
higher amounts were seen after 21 and 28 h (lanes
5 and 8). IFN- alone could not activate the
promoter (lanes 3, 6, and 9).
However, cotreatment of BJAB cells with IL-4 and IFN- resulted in a
great reduction of the IL-4-induced germline transcript level at
the later time points (21 and 28 h; lanes 7 and
10). Costimulation with IFN- had no effect at earlier
time points (7 h, lane 4). These observations demonstrate
that BJAB cells were a suitable model system to study the regulation of
the germline epsilon gene.
The IL-4 response element in the epsilon promoter was a target for
IFN--mediated repression
IL-4-mediated transcription activation of the epsilon promoter
depends on a cis-acting element, which was composed of a
Stat6 (N4 site) and a C/EBP binding site (21, 25). This element, when
fused to a truncated promoter, was sufficient to drive IL-4-induced
expression in either lymphoid or nonlymphoid cells (21). We were
interested to determine whether this element was also sufficient for
the IFN--mediated inhibition. Therefore, a reporter construct
carrying the luciferase gene under the control of a truncated thymidine
kinase promoter and four copies of the IL-4 response element was
transiently transfected into BJAB cells. Strong inducible luciferase
activity was observed 8 and 12 h after IL-4 stimulation. The
activity declined to some extent 16 and 20 h post-treatment (Fig. 2A). Stimulation by IFN-
alone did not result in an increase in luciferase activity. However,
cotreatment of BJAB cells with IL-4 and IFN- resulted in a 50–70%
reduction of the IL-4-induced luciferase activity. This level of
repression was only observed 16 and 20 h after cytokine treatment
(Fig. 2A). Repression was less pronounced at earlier time
points (8 and 12 h post-treatment). This time-dependent inhibition
was consistent with the observations made for the endogenous germline
epsilon transcript. These observations suggest that the repression seen
with IFN- is at least in part mediated by the IL-4 response element
of the epsilon promoter.
IFN- inhibited IL-4-mediated transcription activity in
nonlymphoid cell lines
The inhibition mediated by IFN- was clearly observed in B
cells. To explore possible underlying mechanisms, we investigated other
cell lines for their ability to mediate IFN--dependent inhibition of
IL-4-induced transcription. Previously, we demonstrated strong
IL-4-dependent promoter activity in the hepatic carcinoma line HepG2
using our reporter construct (21). To investigate the inhibitory effect
mediated by IFN-, we transiently transfected our reporter into HepG2
cells. IL-4-induced luciferase activity was detected 2 h following
cytokine treatment, and the activity increased steadily after 4, 8, and
22 h (Fig. 2B). Pretreatment of the transfected HepG2
cells with IFN- for 1 h before the addition of IL-4 had very
little effect at the shorter time points (2 and 4 h
poststimulation). However, a drastic reduction of IL-4-induced
luciferase activity was seen when the cells were exposed to
both cytokines for longer than 8 h. These results are consistent
with the observations made in BJAB cells.
In another set of experiments, we wished to explore the effect of
IFN- when given before IL-4. Extended pretreatment of the cells with
IFN- not only reduced the IL-4-induced luciferase activity, which
was seen after 8 h, but also inhibited the IL-4-induced activity
at earlier time points (2 and 4 h post-IL-4 treatment; Fig. 2C). In general, the inhibitory effect of IFN- was
greater in HepG2 cells than in B cells. However, for both cell types
the data clearly show that the inhibition was time dependent and,
hence, cannot be explained by the direct competition of Stat1 and Stat6
for the cis-regulatory IL-4 response element.
IFN- inhibited IL-4-induced Stat6 DNA binding activity
We next investigated the DNA binding activity of Stat6 in cells
that had been stimulated either with IL-4 alone or with IL-4 and
IFN-. Using the epsilon Stat6 binding site as a probe, we performed
mobility shift assays with extracts prepared from HepG2 cells that were
treated for 1 h either with IL-4 or with IL-4 plus IFN- (Fig. 3A). No DNA binding was
observed in untreated cells (lane 1). Stat6 DNA
binding was strongly induced after 1 h of IL-4 treatment
(lane 2). No change in DNA binding was seen with
extracts prepared from cells that had been stimulated for 1 h with
IL-4 and IFN- (lane 3). These data demonstrated
that the DNA binding activity of Stat6 was not immediately affected by
IFN-. Hence, a prolonged period of IFN- stimulation may be
required to inhibit the DNA binding activity of Stat6.
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for 1 h
before adding IL-4. IL-4 stimulation was then conducted for 1, 2, 4,
14, and 28 h (Fig. 3B). Again, Stat6 DNA binding was
absent in untreated cells (lane 1) and was strongly
induced in response to IL-4 after 1 h (lane 2).
Stat6 DNA binding activity persisted for at least 14 h following
IL-4 treatment (lanes 2–5) and was absent after
28 h (lane 6). In contrast, 1-h pretreatment
with IFN- resulted in a significant reduction in IL-4-induced Stat6
DNA binding activity (lanes 7–11). Some inhibition
was apparent at earlier time points (1 and 2 h; lanes 7
and 8), but it became more obvious 4 h or more after
IL-4 treatment (lanes 9–11). These data showed that
the DNA binding activity of Stat6 decreased more rapidly in cells that
had been treated with IFN- in addition to IL-4.
To determine whether IFN- treatment could also inhibit Stat6 DNA
binding in B cells, we conducted a similar experiment using the B
lymphoma line BJAB (Fig. 3C). Stat6 DNA binding was induced
very rapidly in these cells (data not shown) and persisted up to
20 h post-IL-4 stimulation (lanes 6–9, top
panel). IFN- stimulation alone did not activate Stat6
(lanes 2–5). However, costimulation with IFN-
reduced the IL-4-induced Stat6 DNA binding activity by about 50%
(lanes 10–13). These data showed that the inhibitory
effect mediated by IFN- is also seen in BJAB B lymphoma. The level
of inhibition was less pronounced than that in HepG2 cells, but closely
correlated with the level of inhibition of IL-4-induced luciferase
activity observed under the same conditions. To explore whether the
inhibitory effect was specific for Stat6, we assayed the extracts with
a probe carrying an N3 GAS element. Under these conditions we clearly
observed IFN--induced Stat1 binding (bottom panel, lanes
2–5). Stat1 DNA binding activity was absent in extracts from
untreated or IL-4-stimulated cells (lanes 1 and
6–9). Costimulation with both cytokines did not affect
Stat1 binding to DNA (lanes 10–13). Hence, the
inhibitory activity had no effect on Stat1 DNA binding and appeared to
be specific for Stat6.
IL-4-induced Stat6 tyrosine phosphorylation and nuclear
translocation is inhibited by IFN-
The reduced DNA binding activity of Stat6 in the presence of
IFN- could be due to a reduction in IL-4-induced Stat6 tyrosine
phosphorylation and/or nuclear translocation. To address this issue, we
first examined whether the IL-4-dependent nuclear translocation of
Stat6 was blocked by IFN- treatment. Total nuclear extracts were
immunoblotted to detect either Stat6 or Stat1 with their corresponding
Abs (Fig. 4A). Stat6 was not
detected in the nuclear fraction of unstimulated cells
(lane 1, top panel). Nuclear translocation of Stat6
was seen 2, 4, and 8 h after IL-4 stimulation (lanes
2–4). Similarly, Stat6 was also detected in the nuclear fraction
of cells that had been simultaneously treated with IFN- and IL-4 for
shorter time periods (2 h; lane 5). However, significantly
less Stat6 was seen in the nuclear fraction of cells that had been
stimulated with both cytokines for longer periods of time
(lanes 6 and 7). These data show that
IFN- treatment affected the IL-4-induced nuclear translocation of
Stat6. No Stat6 was seen in the nuclear fraction that had been
stimulated with IFN- alone (lanes 8–10). In
contrast, IFN- induced the nuclear translocation of Stat1, which
remained unaffected upon costimulation with IL-4 (lanes
5–10, bottom panel).
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treatment is due to a reduction in Stat6 tyrosine
phosphorylation. Cytoplasmic Stat6 was immunoprecipitated from extracts
prepared from HepG2 cells that had been stimulated with IL-4, IFN-,
or both cytokines. Immunoprecipitates were subjected to Western blot
analysis using antiphosphotyrosine Abs. As shown in Fig. 4B,
no tyrosine-phosphorylated Stat6 was seen in unstimulated cells
(lane 1, top panel). Prominent tyrosine
phosphorylation was observed after 2, 4, and 8 h of IL-4 treatment
(lanes 2–4). Phosphorylation was effectively
inhibited when the cells were pretreated with IFN- for 1 h
before 4 h of IL-4 treatment (lanes 8–10). In
contrast, IFN- alone did not induce Stat6 tyrosine phosphorylation
(lanes 10–13). Stripping and reprobing of the same
blot with anti-Stat6 Ab (bottom panel) showed that
IFN- did not alter the levels of Stat6 protein during these
treatments. These results show that prolonged treatment of HepG2 cells
with IFN- was necessary to inhibit IL-4-induced Stat6 tyrosine
phosphorylation and nuclear translocation.
Stat1 was required for the IFN--mediated repression
Based on the time-dependent inhibition, we speculated that IFN-
activates Stat1-dependent genes, which encode repressor molecules, and
these molecules would interfere with the IL-4 signaling pathway. To
address the Stat1 requirement in this inhibitory pathway, we used the
fibrosarcoma cell line, U3A, which lack Stat1 (35). First, we
investigated whether Stat6 could be activated in U3A cells. Fig. 5A showed an IL-4 induced DNA
binding activity in extracts prepared from U3A cells (lane
2), which was absent in untreated cells (lane
1). The activity was inhibited by anti-Stat6 Abs
(lane 3) and comigrated with the IL-4-inducible
activity from BJAB cells (lane 4). Hence, U3A cells
express Stat6, and the protein is activated upon IL-4 stimulation.
Next, we addressed whether the IL-4-inducible reporter construct could
be activated in U3A cells in response to IL-4 and inhibited upon
IFN- treatment. The reporter was transiently introduced into U3A
cells, and luciferase activity was determined in either untreated cells
or cells that had been stimulated with cytokine for 5, 8, and 20 h
(Fig. 5B). The reporter was completely inactive in
unstimulated cells. A significant increase in luciferase activity was
seen after IL-4 treatment. However, costimulation with IFN- had no
effect on the IL-4-induced luciferase activity. These data suggested
that Stat1 was required for the inhibitory effect seen with IFN- in
other cell types. To prove this concept, we cotransfected a Stat1
expression plasmid (Stat1) into U3A cells together with the
IL-4-inducible reporter construct. Again, cells were stimulated for 5,
8, and 20 h with IL-4 alone or in the presence of IFN- (Fig. 5C). Interestingly, in the presence of Stat1, the
inhibitory effect of IFN- was restored. Slight inhibition was seen
8 h postcytokine treatment. The effect was significantly stronger
after 20 h, which is consistent with the observations made for
HepG2 and BJAB cells. These data demonstrated that Stat1 is required
for IFN--mediated inhibition of the IL-4 response element.
To investigate whether the transcription activation function of Stat1
was required, we cotransfected Stat1ß (which lacked the activation
domain) along with the IL-4-responsive reporter. The luciferase
activities obtained after IL-4 treatment under these conditions were
comparable to those observed with the parental U3A cells (Fig. 5D). Our results showed that Stat1ß was unable to restore
the inhibitory effect mediated by IFN-. Equivalent expression of
Stat1 and Stat1ß was demonstrated by Western analysis (Fig. 5E). These results clearly show that the transcription
activation function of Stat1 is essential for the IFN--mediated
repression of the IL-4 signaling pathway.
SOCS-1 inhibited IL-4-induced transcription and Stat6 tyrosine phosphorylation
Recent studies have identified a family of inhibitory proteins,
termed SOCS proteins, that suppresses cytokine signaling mediated by
the JAK/STAT pathway (36). Members of this family are induced by a
variety of cytokines, including IFN-, IL-4, IL-1, IL-3, and IL-7
(36). Among these molecules, expression of SOCS-1 was up-regulated by
IFN-, but not IL-4, in bone marrow-derived cells (37). Hence, we
examined SOCS-1 expression in HepG2 cells. Cells were stimulated with
IFN-, IL-4, or both cytokines, and RNA was isolated at different
time points following cytokine treatment. SOCS-1 mRNA was not detected
in unstimulated HepG2 cells (Fig. 6,
lane 1). Treatment with IFN- for 2, 4, and 8 h
resulted in the prominent appearance of the SOCS-1 transcript
(lanes 2–4). However, IL-4 treatment did not induce
SOCS-1 expression during these periods (lanes 5–7).
Costimulation of IL-4 and IFN- resulted in SOCS-1 expression, and
the levels were similar to those seen with IFN- stimulation alone
(lanes 7–10). These results suggest that the
differential up-regulation of SOCS-1 by IFN- may contribute to the
inhibitory effects of this cytokine on the IL-4 response element.
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A). In the presence of
SOCS-1, a dose-dependent inhibition of the IL-4-induced luciferase
activity was seen (up to 100% inhibition). SOCS-1 did not affect basal
luciferase activity. Furthermore, this effect was specific for the
IL-4-inducible reporter system because SOCS-1 failed to inhibit the
IL-1 induced luciferase activity mediated through the E-selectin
promoter (Fig. 7B).
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, lanes 1 and 3).
However, IL-4 treatment for 30 min resulted in a prominent induction of
Stat6 tyrosine phosphorylation (lane 2).
Interestingly, coexpression of SOCS-1 completely inhibited Stat6
tyrosine phosphorylation (lane 4). These results
strongly suggest that the IFN--inducible SOCS-1 protein inhibits
IL-4-induced transcription at least in part by preventing Stat6
tyrosine phosphorylation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment
inhibits the IL-4-induced expression of the germline epsilon
transcript, and this inhibition depends on the integrity of the IL-4
response element located in the epsilon promoter. Numerous studies
demonstrated that the IL-4-induced IgE production is suppressed by
IFN- (8, 9, 10, 11, 12, 13). However, the underlying mechanism is not well
understood.
In general, IL-4 and IFN- are well documented to act in an
antagonistic fashion with respect to a number of immune responses.
Stimulation with IL-4 and IFN- leads to the rapid activation of
Stat6 and Stat1, respectively. Down-regulation or deactivation of
individual STATs occurs with different kinetics in individual cell
types, and different mechanisms have been proposed for the inactivation
of tyrosine-phosphorylated STAT molecules. Proteolysis and
dephosphorylation have been shown to be important for the spontaneous
decline of STAT activity (40, 41). However, IFN--mediated inhibition
of IL-4 function appears to be mediated by a more direct and specific
mechanism. Expression of the endogenous germline epsilon transcript is
induced by IL-4 in the human B lymphoma line BJAB. Using Northern
analysis, the transcript can be detected as early as 6 h
poststimulation and continues to accumulate over time. Inhibition by
IFN- is not seen at earlier time points (7 h), but is very obvious
at later times (after 20 h). The inhibitory effect is also seen
with a reporter construct that was under the control of the IL-4
response element derived from the germline epsilon promoter.
IL-4-induced luciferase activity is down-regulated by IFN-
treatment, demonstrating that IL-4-induced activation and
IFN--induced inhibition are mediated by the same regulatory element.
As observed for the endogenous transcript, down-regulation of the
luciferase activity in B cells is significant, but not complete. In
contrast, the inhibitory effect is more pronounced in HepG2 cells.
However, in both cell types, down-regulation of the reporter activity
is time dependent as observed for the endogenous gene. This difference
may be due to the significantly greater amount of Stat6 in lymphoid vs
nonlymphoid cells (21). Lymphoid cells use a heterodimeric IL-4R
complex that engages JAK3 and JAK1, whereas nonlymphoid cells often
only require JAK1 (42). The difference in these two receptor complexes
may also explain the more pronounced inhibition by IFN- in HepG2 vs
BJAB cells.
Repression of transcription correlated with an inhibition of Stat6
activation. The spontaneous decline of IL-4-induced Stat6 DNA binding
in the absence of IFN- is observed in HepG2 cells after 28 h.
However, under those conditions the IL-4-induced promoter appeared to
still be active, which may reflect the accumulation of luciferase mRNA
or protein over time. In the presence of IFN-, the IL-4-induced DNA
binding activity of Stat6 is diminished after 4 h. IFN-
treatment seems to affect Stat6 DNA binding activity more rapidly than
IL-4-induced transcription, suggesting that Stat6 activation may only
be required for the initial steps in IL-4-induced promoter activity.
Previously, Ohmori and Hamilton showed that IFN--induced IRF-1
expression in macrophages is down-regulated in response to IL-4
treatment (29). The GAS element (N3 site) in the IRF-1 promoter can be
bound be Stat1 and Stat6, and the inhibitory effect was explained by
competition of Stat1 and Stat6 for the GAS element. However, in our
study such a mechanism is unlikely for the following three reasons.
First, the IL-4 response element (N4 site) of the promoter is only
bound by Stat6 and not by Stat1. Second, competitive DNA binding would
result in a more rapid inhibition, in contrast to the more gradual
repression which we observed over time. Third, we found that
IL-4-induced Stat6 tyrosine phosphorylation and nuclear translocation
is inhibited by IFN- at later time points. In contrast, Ohmori and
Hamilton showed that the IFN--induced Stat1 activation and nuclear
translocation are not affected by IL-4 treatment (29). Our studies are
in agreement with a previous report that demonstrated that pretreatment
of peripheral blood monocytes with IFN- inhibits IL-4-induced Stat6
tyrosine phosphorylation and nuclear translocation (43). However, the
time course of Stat6 inhibition appears to be somewhat different in
monocytes vs HepG2 or BJAB cells.
The IFN--mediated repression of IL-4-induced Stat6 tyrosine
phosphorylation could be mediated at the level of IL-4R expression.
Using Northern blots we showed that the amount of IL-4R -chain mRNA
is not affected in HepG2 cells upon IFN- treatment (data not shown).
These data rule out that IFN- treatment down-regulates the
expression of the IL-4R signaling chain, which is consistent with the
observations made in monocytes (43).
Our data suggest that de novo protein synthesis may be required for the
inhibition mediated by IFN-. Using the fibrosarcoma line, U3A, we
demonstrate that Stat1 is absolutely essential for the repression
mediated by IFN-. No IFN--mediated inhibition of the
IL-4-inducible reporter is seen in U3A cells, which lack Stat1.
However, overexpression of wild-type Stat1 protein restores the
inhibitory effect. In contrast, overexpression of Stat1ß, lacking the
transcription activation domain, has no effect. Based on these data, we
envision that IFN- treatment leads to the activation of an
inhibitory molecule that modulates the IL-4 signaling pathway.
To date, three classes of molecules have been described that negatively
regulate the JAK/STAT pathways. First, molecules known as PIAS1 and
PIAS3 (protein inhibitor of activated STAT) have been identified that
specifically bind to activated Stat1 and Stat3 and inhibit their DNA
binding activity (44, 45). A similar molecule that specifically targets
activated Stat6 may exist. However, based on their proposed mechanism
of action we would assume that PIAS proteins do not affect the
phosphorylation and nuclear translocation of STAT proteins, as we
observed for IFN--mediated inhibition. Second, tyrosine phosphatase
activity induced by IFN- may contribute to the repression of IL-4
signaling. Pervanadate treatment of cells leads to the activation of
Stat6 (46), suggesting that IL-4 signaling can be down-regulated by a
tyrosine phosphatase. If IFN- inducible, this tyrosine phosphatase
could help to explain the observed inhibitory effect on IL-4-induced
Stat6 tyrosine phosphorylation. Based on our existing data we cannot
rule out the existence of an IFN--inducible phosphatase. Third, the
SOCS family (also referred to as JAB or STAT-induced STAT inhibitor)
includes members that contain an Src homology 2 (SH2) domain (36, 37, 38, 47, 48). Expression of several members of the SOCS family can be
induced by IFN- (37, 48). In this study we show that SOCS-1
expression is up-regulated by IFN-, but not by IL-4. The induction
of SOCS-1 closely correlates with its ability to effectively inhibit
IL-4-induced Stat6 tyrosine phosphorylation and transcription. We
speculate that the mechanism of SOCS-1-mediated inhibition of Stat6
phosphorylation may be similar to those reported for IFN- and IL-6
signaling. In those cases SOCS-1 interacts with phosphotyrosine
residues on activated JAK kinases and down-regulates their kinase
activities (36, 48). Hence, the inhibitory effect of IFN- could be
mediated by the expression of SOCS-1, which subsequently interferes
with the IL-4 signaling pathway.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, Berlex Biosciences, 15049 San Pablo Ave., Richmond, CA 94804-0099.
3 Current address: Department of Molecular Biology, Jichi Medical School, 3311-1 Yakushiji, Minami-Kawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan.
4 Address correspondence and reprint requests to Dr. Ulrike Schindler, Tularik Inc., Two Corporate Dr., South San Francisco, CA 94080. E-mail address:
5 Abbreviations used in this paper: JAK, Janus kinase; GAS, -activated sequence; C/EBP, CAAT/enhancer binding protein; IRF-1, insulin response factor-1; JAB, Janus kinase-binding protein; SOCS, silencer of cytokine signaling; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; PIAS, protein inhibitor of activated signal transducer and activator of transcription; SH2, src homology domain 2.
Received for publication April 14, 1998. Accepted for publication December 28, 1998.
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