From the Division of Molecular Oncology, Department of Oncology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita City, Osaka 565, Japan
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ABSTRACT |
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STAT3 (signal transducer and activator of transcription 3) is a key transcription factor mediating the signals for a variety of cytokines, including interleukin-6 (IL-6). The Stat3 gene itself is activated by IL-6 signals. We show that the region of the signal-transducing subunit, gp130, essential for STAT3 activation, is also required for activation of the Stat3 gene. To elucidate the mechanisms activating the Stat3 gene, we identified an IL-6 response element (IL-6RE) in the Stat3 gene promoter containing both a low affinity STAT3-binding element and a cAMP-responsive element (CRE). Electrophoretic mobility shift assays showed that IL-6 induced a slowly migrating complex on the IL-6RE containing a STAT3 homodimer and an unidentified CRE-binding protein. With the combination of transient transfection assays using mutant Stat3 promoter-reporter constructs and electrophoretic mobility shift assays, we found that the formation of a slowly migrating complex was required for full activation of the Stat3 gene. Thus, STAT3 activates the Stat3 gene in cooperation with an unidentified CRE-binding protein. This regulatory mechanism is similar to that of the junB gene, which is activated by IL-6 through the junB IL-6RE, which contains a low affinity STAT3-binding site and a CRE-like site.
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INTRODUCTION |
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STAT (signal transducer and
activator of transcription) proteins have been
shown to play pivotal roles in cytokine signaling pathways, which are
involved in regulating cell growth and differentiation in systems
ranging from Drosophila to mammals (1-3). In mammals, the
STAT family consists of at least six members, designated STAT1-6 (3).
Different STAT family members are activated by a variety of cytokines,
including interferon-/
, interferon-
, the interleukin-6 (IL-6)1 family, growth
hormone, erythropoietin, and leptin in a manner dependent on the Janus
kinase family of tyrosine kinases (4-12). Furthermore, several growth
factors (such as epidermal growth factor, platelet-derived growth
factor, and fibroblast growth factor) and non-receptor tyrosine kinases
(including c-Src, c-Abl, and their v-oncogene products) have been shown
to activate STAT proteins (13-19). Thus, three different types of
tyrosine kinases, Janus kinases, receptor-type tyrosine kinases, and
certain Src family tyrosine kinases, can all activate STAT proteins,
probably by directly phosphorylating the tyrosine residue critical for causing the STAT molecules to form homo- or heterodimers through their
SH2 domains (20-25). Tyrosine-phosphorylated STAT dimers enter the
nucleus and activate the target genes by binding to their specific
target DNA sequences (26). The cis-acting DNA sequences
recognized by STAT dimers, the STAT-binding element (SBE), have the
general structure TTN5AA (1, 27). The sequence and the size
of the spacer region affect the binding of the respective STAT dimers
(27). In certain cases, STAT proteins bind to the target DNA in
combination with other DNA-binding proteins. For instance, the STAT3
homodimer forms a complex with p36 CRE-binding protein for an IL-6
response element in the junB gene (JRE-IL6) (28) or with
c-Jun for the
2-macroglobulin APRE (29). The STAT1
homodimer has been shown to make a complex with Sp1 for the
interferon-
response element of the intercellular adhesion molecule-1 gene (30). Stocklin et al. (31) showed that the glucocorticoid receptor can act as a coactivator and enhance
STAT5-dependent transcription by making complexes with
STAT5. STAT dimers even form complexes with other STAT dimers through
their amino-terminal regions on the repeated low affinity SBEs in the
interferon-
gene intron (32). Therefore, in addition to the sequence
of a SBE itself, the sequence outside the SBE is also critical for determining the target gene specificity because the complex formation with other proteins or other STAT dimers sometimes changes the binding
specificity or increases the affinity of the complex for the DNA (21,
28, 32). Other important determinants for STAT function might be the
intensity and duration of the STAT activity and the amounts of STAT
proteins ready to be activated in the cells. All of these factors
eventually determine the range of target genes and the duration and
intensity of target gene activation and thereby often determine the
outcome, such as differentiation, growth, and cell survival or
death.
We have previously shown that STAT3 is critical for IL-6-induced gene regulation, including the repression of c-myb and c-myc, the induction of junB and IRF1, and IL-6-induced growth arrest and terminal macrophage differentiation in M1 leukemic cells (33, 34). Moreover, STAT3 activity is critical for the gp130-mediated anti-apoptotic signal in murine pro-B BaF/B03 cells (35). To understand how STAT3-mediated signals affect growth, differentiation, and cell death or survival in different types of cells, it is important to study the regulatory mechanisms of Stat3 gene expression itself. In this study, we characterize the signals required for Stat3 gene activation and show that STAT3 activates the Stat3 gene promoter through a novel IL-6 response element containing two DNA motifs, a low affinity SBE and a CRE. This is similar to the mechanism for JRE-IL6, a previously characterized IL-6 response element in the junB promoter (36).
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EXPERIMENTAL PROCEDURES |
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Cell Lines-- M1 murine myeloid leukemic cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2. M1 transformants expressing the chimeric receptors containing the extracellular domain of the GHR and the transmembrane and cytoplasmic domains of gp130 have been described previously (34). Of those, seven clones for each (M1-GHR277, M1-GHR133, M1-GHR108, M1-GHR68, M1-GHR133F2, M1-GHR133F3, and M1-GHR133F2/3) were used in this study. HepG2 hepatoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). MH60 B cell hybridoma cells (37) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 0.2 ng/ml recombinant human IL-6.
Northern Blot Analysis-- Total RNA was extracted using the TRIzol reagent (Life Technologies, Inc.) according to the procedures recommended by the manufacturer. Total RNA (15 µg/sample) was separated by electrophoresis in formaldehyde-containing 1% agarose gels and transferred to Hybond N+ nylon membranes (Amersham Corp.). Membranes were hybridized with 32P-labeled cDNA fragments overnight, washed three times with 0.1× SSC and 0.1% SDS at 56 °C, and subjected to autoradiography. The amount of loaded RNA was verified by ethidium bromide staining or by measuring the expression level of CHO-B mRNA. The probes used were the 2.5-kb SalI-BamHI fragment of pBSHA-STAT3 containing a full-length STAT3 cDNA and the 0.6-kb EcoRI-BamHI fragment of CHO-B cDNA (a gift from J. E. Darnell, Jr.).
Isolation of the 5'-Flanking Region of the Mouse Stat3 Gene and Plasmid Construction-- A mouse Stat3 5'-flanking region was obtained by PCR using the Promoter Finder DNA Walking kit (CLONTECH). Two gene-specific primers, 5'-CTCAGCGATCCGGTTAGGGCTCGTT-3' (GSP1) and 5'-CAGGTTCCCCCTCCCTGTC TACACT-3' (GSP2), were made based on the sequence of the mouse Stat3 cDNA reported by Akira et al. (38). Primary PCR and secondary nested PCR were done using the GSP1 and GSP2 primers, the primers (AP1 and AP2) corresponding to the adaptor sequences, and adaptor-ligated genomic libraries. The resulting 2.2-kb PCR fragment was subcloned using the TA cloning vector pCR2.1 (Invitrogen). DNA sequencing was carried out using an automated sequencer, and data were obtained for both strands. A series of 5'-deletion mutations of the Stat3 5'-flanking region were made by subcloning the EcoRV-BamHI, SspI-BamHI, ScaI-BamHI, AflII-BamHI, StyI-BamHI, and PvuII-BamHI fragments into the reporter construct pSPLuc, containing a luciferase gene and SV40 poly(A) signal inserted into pSP72 between the PvuII and BamHI sites.
Synthetic Oligonucleotides-- The oligonucleotides used as probes or competitors were as follows: Stat3-IL-6RE-WT (where WT is wild-type), 5'-GTGTCTTGACGTCACGCACTGCCAGGAACT-3' and 3'-AGAACTGCAGTGCGTGACGGTCCTTGAGTCG-5'; Stat3-IL-6RE-mSTAT, 5'-GTGTCTTGACGTCACGCACTGCCAGGTCCT-3' and 3'-AGAACTGCAGTGCGTGACGGTCCAGGAGTCG-5'; Stat3-IL-6RE-mCRE, 5'-GTGTCTTGTCATCACGCACTGCCAGGAACT-3' and 3'-AGAACAGTAGTGCGTGACGGTCCTTGAGTCG-5'. The underlined bases are the mutated ones. The APRE and somatostatin CRE probes were synthesized as reported previously (28).
Site-directed Mutagenesis by PCR-- Mutations were introduced at the STAT3-binding element or the CRE site of the Stat3 IL-6RE in the context of the intact Stat3 promoter with the 2.2-kb upstream region by the overlap extension technique using PCR. The primers used for these mutations were as follows: for mSTAT, 5'-ACTGCCAGGTCCTCAGCTGAGTTTTCAG-3' and 5'-AGCTGAGGACCTGGCAGTGCGTGACGT-3'; for mCRE, 5'-GTGTCTTGTCATCACGCACTGCCAGGAACT-3' and 5'-AGTGCGTGATGACAAGACACTTTGAATGCCCT-3'; for the 5'-primer, 5'-CCCAAATGCTTAAGTGGGGTGACACCT-3'; and for the 3'-primer, 5'-AGCTCGGATCCACTAGTAACGGCCG-3'. The underlined bases are the mutated ones. After the PCR products were subcloned into pCR2.1, the sequences of the PCR products were verified by sequencing the products. The PCR products were digested with SacI and inserted at the proper position of p2166-Stat3-Luc-WT to make p2166-Stat3-Luc-mSTAT and p2166-Stat3-Luc-mCRE. The primers used to make the p478/229-Luc series were as follows: for the 5'-primer, 5'-AACGCTGCAGTTAAGTGGGGTGACACCTGG; and for the 3'-primer, 5'-CCAGGTACCCCAAGGGACGCGCAGAGGCC. The PCR products were digested with PstI and KpnI and inserted upstream of the minimal junB promoter at the PstI-KpnI sites of pSPBLuc.
Transient Transfection Assay--
For transfection experiments,
HepG2 cells were transfected with DNA using the calcium phosphate
coprecipitation method (36). Typically, 1.2 µg of one of the reporter
plasmids containing the firefly luciferase gene and 1 µg of
pEF-lacZ, an expression vector containing the
lacZ gene encoding -galactosidase as an internal control
for transfection efficiency, were used. Three µg of pCAGGS-Neo (an
expression vector without an insert; control) or pCAGGS-NeoHA-STAT1F or
pCAGGS-NeoHA-STAT3F (expression vectors containing a cDNA encoding either HA-STAT1F or HA-STAT3F (33), respectively) was cotransfected in
some experiments. Cells were incubated with DNA precipitates for
16 h, washed with phosphate-buffered saline, fed Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum for 20-24 h, and stimulated with 100 ng/ml IL-6 for the last 16 h.
Approximately 40-45 h after transfection, cells were collected in 120 µl of lysis buffer and subjected to assays for luciferase and
-galactosidase activities as described (28).
Electrophoretic Mobility Shift Assay-- This was performed according to the procedure published previously (28). Briefly, nuclear extracts (10 µg) were incubated in a final volume of 20 µl of 10 mM HEPES, pH 7.9, 80 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 1 mM EDTA, and 100 µg/ml poly(dI-dC)·poly(dI-dC) with each 32P-labeled probe (10,000 cpm, 0.5-1 ng) for 20 min at room temperature. The protein-DNA complexes were resolved on a 4.5% nondenaturing polyacrylamide gel containing 2.5% glycerol in 0.25× TBE (1× TBE is 0.13 M Tris base, 0.12 M boric acid, and 2.0 mM EDTA, pH 8.8) at room temperature and autoradiographed. For competition analysis, extracts were preincubated with a 50- or 250-fold molar excess of unlabeled oligonucleotides for 5 min before the addition of labeled oligonucleotide. For antibody interaction studies, antisera or monoclonal antibody specific to several transcription factors was included in the binding reaction during a 30-min preincubation on ice. The antibodies used were as follows: anti-STAT1 N terminus (amino acids 1-194 of human STAT1; anti-ISGF3 G16920) monoclonal antibody (Transduction Laboratories, Lexington, KY); anti-STAT3 C terminus (amino acids 702-770 of human STAT3) polyclonal antibody (28); anti-CREB rabbit polyclonal antibody (9192; New England Biolabs Inc.); anti-CRE-BP1 (ATF2) rabbit polyclonal antibody (SC187X; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-c-Jun/AP-1 goat polyclonal antibody recognizing the DNA-binding domains of c-Jun, JunB, and JunD (SC44X; Santa Cruz Biotechnology, Inc.); anti-ATF3 rabbit polyclonal antibody (SC188X; Santa Cruz Biotechnology, Inc.); and anti-ATF4 mouse monoclonal antibody (SC244X; Santa Cruz Biotechnology, Inc.). The effectiveness of the antibodies including anti-STAT1, anti-STAT3, anti-c-Jun, anti-ATF2, and anti-CREB was verified in electrophoretic mobility shift assays using the Stat3 IL-6RE probe and appropriate nuclear extracts containing the endogenous proteins or exogenously expressed ATF2, c-Jun, or CREB (data not shown). Anti-ATF3 and anti-ATF4 antibodies for gel shift assays were used following the manufacturer's instructions.
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RESULTS |
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Increased Stat3 Gene Expression in Response to IL-6-- Akira et al. (38) reported that treatment with IL-6 increases the level of Stat3 mRNA severalfold in liver. Also, we showed that IL-6 treatment of M1 cells increases the protein level of STAT3 and that the increased level is sustained for >48 h (33). To begin a deeper analysis of STAT3 regulation, we first determined a detailed time course for the IL-6-induced mRNA expression of the Stat3 gene in M1, HepG2, and IL-6-dependent MH60 B cell hybridoma cells. In M1 cells, the level of Stat3 mRNA started to increase at 1 h in response to IL-6 and reached its maximum level at 3 h. After reaching its maximum, Stat3 mRNA levels showed a slight transient decrease at 6 h and then increased again for over 48 h (Fig. 1A, lanes 1-6). The induction of Stat3 mRNA was also tested in the presence of cycloheximide, a protein synthesis inhibitor. Treatment of M1 cells with cycloheximide failed to inhibit the IL-6-induced activation of the Stat3 gene (Fig. 1A, lanes 7-9), indicating that the induction of Stat3 by IL-6 did not require new protein synthesis. The rapid induction of Stat3 mRNA by IL-6 was also observed with HepG2 cells (Fig. 1B) and MH60 cells (data not shown).
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The Cytoplasmic Region from Amino Acids 108 to 133, Especially the Third Tyrosine (Tyr-3) of gp130, Is Required for Regulating the Expression of Stat3-- We next analyzed which part of gp130 was necessary for the induction of Stat3 gene expression. Using M1 transformants expressing chimeric receptors consisting of the extracellular domain of the GHR and the transmembrane and cytoplasmic domains of gp130 with progressive C-terminal truncations and point mutations at the tyrosine residues (34), we proceeded to identify the cytoplasmic region of gp130 required for Stat3 gene transactivation. Growth hormone enhanced Stat3 gene expression in the transformants expressing the chimeric receptors containing at least 133 amino acid residues of the gp130 cytoplasmic domain (Fig. 2, lanes 1-6). Chimeric receptors with further truncation of gp130 to 108 or 68 amino acid residues (GHR108 and GHR68) were unable to induce Stat3 gene expression (Fig. 2, lanes 7-12). GHR133F2, which contains a Tyr-to-Phe mutation at the second tyrosine (Tyr-2), could activate the Stat3 gene (Fig. 2, lanes 13-15), but GHR133F3, which has a Tyr-to-Phe mutation at the third tyrosine (Tyr-3), and GHR133F2/3, which has Tyr-to-Phe mutations at both Tyr-2 and Tyr-3, did not activate the Stat3 gene (lanes 16-21). These results show that the cytoplasmic region of gp130 from amino acids 108 to 133, with an intact YXXQ motif at Tyr-3, is required for activating the Stat3 gene. As this region corresponds to the region required for activating the STAT3 protein, it is likely that STAT3 participates in the transcriptional activation of its own promoter.
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The 478/
229 Region of the Stat3 Promoter Shows IL-6
Responsiveness in a Manner Dependent on STAT3--
To elucidate the
molecular mechanisms activating the Stat3 gene promoter, we
obtained a 2.2-kb fragment of the Stat3 gene 5'-flanking
region by genomic PCR. The sequence of the proximal region up to
478
bp with the known DNA motifs is shown in Fig. 3A. The sequence published by
Shi et al. (39) is missing a 0.25-kb region between
positions
424 and
169. We first constructed a reporter gene
containing the 2.2-kb Stat3 gene 5'-flanking region linked
to the luciferase reporter gene and tested whether this 2.2-kb fragment
showed IL-6 responsiveness by transfecting the reporter gene construct
into HepG2 cells, followed by IL-6 stimulation for 16 h.
Dominant-negative STAT3 and STAT1 expression vectors were also
cotransfected with the promoter-reporter construct to test whether
STAT3 is involved in the IL-6 activation of the Stat3 gene
promoter. As shown in Fig. 3B, IL-6 increased the promoter activity of the 2.2-kb 5'-flanking region by ~5-fold, and this induction was effectively inhibited by dominant-negative STAT3 (DN-Stat3), but not by dominant-negative STAT1 (DN-Stat1), indicating that the IL-6 activation of the 2.2-kb Stat3 gene promoter
was dependent on STAT3. Next, to localize the region containing the IL-6 response element, a series of 5'-deletion mutations of the Stat3 gene promoter constructs were made and tested for IL-6
responsiveness as described above. The promoter regions retained a
similar level of IL-6 responsiveness when the promoter contained up to
position
478 (Fig. 3C). However, deletion up to position
229 substantially decreased the IL-6 responsiveness, suggesting that
an IL-6 response element(s) resides in the
478/
229 region (Fig.
3C).
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Both STAT3 and CRE-binding Proteins Are Necessary for the
IL-6-induced Transcriptional Activation of the Stat3 Gene--
To test
whether the 478/
/229 region alone has the ability to render a
heterologous minimal promoter responsive to IL-6, we assayed the IL-6
responsiveness of a luciferase construct containing the
478/
229 DNA
fragment inserted upstream of the minimal junB promoter-luciferase gene construct (p478/229-Luc). As shown in Fig.
4A, IL-6 increased the
transcription of the reporter gene driven by the
478/
229 fragment
by ~10-fold. As expected, dominant-negative STAT3
(DN-Stat3), but not STAT1 (DN-Stat1), effectively
inhibited the IL-6 responsiveness of the
478/
229 region. These
results indicate that STAT3 activates its own transcription through the
478/
229 region. We next searched for STAT3-binding sequence(s) within the
478/
229 DNA region. Although we could not find a typical
STAT3-binding sequence (a TT-AA motif with a spacing of 5 bp)
within the
478/
229 DNA region, instead we found an atypical SBE (TGCCTGGAA) and a CRE (TGACGTCA) with a 5-bp spacing between the
motifs, which is very similar to the junB response element for IL-6 (JRE-IL6) (Fig. 3A) (28). To investigate whether
both the SBE and CRE were responsible for IL-6 responsiveness, we made two different p478/229-Luc mutants containing mutations at the SBE
(p478/229-Luc-mSTAT) or the CRE (p478/229-Luc-mCRE), as illustrated in
Fig. 4B, and tested them for IL-6 responsiveness. Mutations at the putative SBE reduced the IL-6 responsiveness by >90%, and mutations at the CRE reduced it by >80% (Fig. 4B). These
results indicated that both DNA motifs were required for IL-6
responsiveness. To assess the role of the two DNA motifs in the intact
2.2-kb Stat3 gene promoter, we introduced the same mutations
as described above into the Stat3 IL-6RE in the intact
2.2-kb Stat3 promoter (p2166-Stat3-Luc-mSTAT and
p2166-Stat3-Luc-mCRE). As shown in Fig. 4C,
mutations at the putative STAT3-binding site in the 2.2-kb Stat3 promoter (p2166-Stat3-Luc-mSTAT) severely
reduced the IL-6 responsiveness of the Stat3 promoter by
>80%, and mutations at the CRE reduced the IL-6 responsiveness of the
promoter by ~60%. The weaker inhibition by the mutations at the CRE
in the otherwise intact promoter compared with that by the mutations at
the CRE in the context of the
478/
229 region alone may be due to
the existence of unidentified region(s) cooperatively working with STAT3 outside the
478/
229 region. These results, in any case, indicate that the two DNA motifs in the Stat3 IL-6RE are the
major determinants in the 2.2-kb Stat3 promoter for IL-6
responsiveness.
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Characteristics of Stat3 IL-6RE-binding Proteins--
We next
examined the nature of the Stat3 IL-6RE-binding complexes in
the nuclear extracts from IL-6-stimulated M1, HepG2, and MH60 cells by
electrophoretic mobility shift assays using an oligonucleotide
containing the Stat3 IL-6RE as a probe. We also used another
oligonucleotide containing the 2-macroglobulin APRE (40)
as a control for STAT binding. Nuclear extracts from IL-6-stimulated
MH60 cells showed a prominent IL-6-inducible Stat3 IL-6RE-binding complex on the Stat3 IL-6RE probe (indicated
as Stat3IL6RE-BC), which appeared rapidly at 15 min,
declined transiently at 3 h, and returned to the level of 15 min
at 6 h (Fig. 5A,
lanes 2-5). The mobility of the IL-6-induced
Stat3 IL-6RE-binding complex is slower than that of the
APRE-binding complex containing a STAT3 homodimer, called APRF (40)
(Fig. 5A, lane 7). More important, the level of
the IL-6-inducible complex is equivalent to that of APRF (Fig.
5A, compare lanes 2 and 7). This
IL-6-inducible complex could be seen more clearly in MH60 nuclear
extracts than in M1 and HepG2 nuclear extracts, although similar
IL-6-inducible complexes were present in the nuclear extracts from all
three cell lines (data not shown). Therefore, only results with MH60 nuclear extracts are shown. To investigate the binding sites and binding specificities of the Stat3 IL-6RE-binding complex,
we used two mutant Stat3 IL-6RE oligonucleotides containing
mutations either at the CRE site (Stat3-IL-6RE-mCRE) or at
the STAT3-binding element (Stat3-IL-6RE-mSTAT) as probes and
other oligonucleotides as competitors, including the APRE, JRE-IL6, and
somatostatin CRE. The Stat3-IL-6RE-mSTAT probe did not show
any IL-6-inducible Stat3 IL-6RE-binding complexes (Fig.
5A, lanes 8 and 9). On the other hand,
the Stat3-IL-6RE-mCRE probe showed an IL-6-inducible complex
with the same mobility as that of APRF (Fig. 5A, lanes 10 and 11), and this complex was likely to be a STAT3
homodimer since anti-STAT3 antibody shifted the complex (data not
shown). Both the APRE and JRE-IL6 oligonucleotides effectively competed with the Stat3 IL-6RE probe for forming the IL-6-inducible
Stat3 IL-6RE-binding complex (Fig. 5B,
lanes 2-5), whereas the somatostatin CRE oligonucleotides
competed for all of the CRE-binding complexes and the Stat3
IL-6RE-binding complex, but made the appearance of a fast migrating
IL-6RE-binding complex (lanes 6 and 7). The complex is likely to be a STAT3 homodimer since the complex migrated as
APRF on the APRE probe, and anti-STAT3 antibody shifted the complex
(data not shown). This STAT3 homodimer could barely be seen on the
wild-type Stat3 IL-6RE probe (Fig. 5A). These
results indicate that formation of the Stat3 IL-6RE-binding
complex requires both the STAT-binding element and the CRE and suggest
that the STAT-binding element has a low affinity for STAT3 homodimers
alone.
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DISCUSSION |
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In this study, we showed that STAT3 rapidly induced
transcriptional activation of the Stat3 gene through an
IL-6RE located at positions 335 to
314 in the Stat3 gene
promoter. This IL-6RE was shown to consist of a low affinity SBE
(TGCCAGGAA) and a CRE (TGACGTCA). The functional role of the two DNA
motifs in the Stat3 IL-6RE was confirmed in two ways. First,
mutations at either the SBE or CRE effectively inhibited the IL-6
responsiveness of the
478/
229 fragment linked to a heterologous
promoter. Second, the same mutations at the DNA motifs in the otherwise
intact 2.2-kb Stat3 promoter also effectively inhibited the
IL-6 responsiveness of the promoter. The functional roles of the two
DNA motifs were further confirmed by the demonstration that IL-6
rapidly induced the formation of complexes with the Stat3
IL-6RE. A slowly migrating complex with the IL-6RE required both the
SBE and CRE. We showed that this complex contained a STAT3 dimer and
unidentified CRE-binding protein(s) distinct from CREB, ATF1,
ATF2/CRE-BP1, ATF3, ATF4, or the c-Jun/AP-1 family of proteins. Ternary
complex formation on other IL-6 response elements containing a SBE and
CRE has been shown by us (28), including the IL-6 response elements
seen in the junB promoter, JRE-IL6 (36), and the
IRF1 promoter, IR/IRF1 (41). Table
I compares the sequences of such IL-6
response elements with that of the Stat3 IL-6RE. The size of
the spacing between the two DNA motifs also varies from 1 to 5 bp. The
combination of the DNA motifs varies: a low affinity SBE with an
atypical CRE (JRE-IL6), a high affinity SBE with an atypical CRE
(IR/IRF1), and a low affinity SBE with a typical CRE (Stat3
IL-6RE). Considering the binding specificity of the Stat3
IL-6RE-binding complex shown here and the JRE-IL6-binding complexes
shown by Kojima et al. (28), similar CRE-like site-binding
protein(s) may make complexes with the STAT3 homodimer on the
Stat3 IL-6RE DNA. The identification of CRE-binding
protein(s) would help prove such a structure.
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The results of the kinetics study of the IL-6 induction of Stat3 mRNA in wild-type M1 cells (Fig. 1A) are fully consistent with the levels of STAT3 activity detected by tyrosine phosphorylation of STAT3 and its DNA binding activity shown previously by us (33), supporting the notion of the STAT3-mediated activation of the Stat3 gene. It is remarkable that three of the IL-6 target genes inducible in M1 cells, junB, IRF1, and Stat3, contain very similar IL-6 response elements in their promoters. Interestingly, these genes can be activated by a low dose of IL-6 (such as 1 ng/ml) in M1 cells.2 In contrast, in another case of IL-6-induced gene regulation, the IL-6 repression of c-myc and c-myb is turned on only by higher doses of IL-6 in the same cell line.2 The difference in sensitivity to IL-6 concentration may be explained by CRE or CRE-like site-binding proteins that may be crucial in recruiting small amounts of activated STAT3 to the IL-6 response element by making complexes with STAT3 and DNA. The identification and functional role of the unidentified CRE-binding protein would help to prove this model.
It has been noted that in IL-6-stimulated M1 cells, STAT3 activity
persists at a high level for >48 h (33). This activation pattern is
not detected in other cell lines. The sustained STAT3 activity at high
levels in M1 cells may be important in causing growth arrest and
terminal differentiation. Therefore, it is likely that the activation
of the Stat3 gene by STAT3 in conjunction with the
cooperative CRE-binding protein forms an autoregulatory loop, and this
may be one of the mechanisms by which the persistent activation of
STAT3 protein in M1 cells is induced. Recently, O'Brien and Manolagas
(42) showed that transcription of the signal-transducing -subunit of
the IL-6 receptor complex (gp130) is also activated by STAT3 through a
STAT-binding element in the promoter. Their findings and ours suggest
the existence of autoregulatory mechanisms in the IL-6
signal-transducing system at the levels of both the receptor and the
signal-transducing transcription factor.
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ACKNOWLEDGEMENT |
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We thank J. Ishikawa for technical assistance.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan; the Special Coordination Fund of the Science and Technology Agency of the Japanese Government; the Yamanouchi Foundation for Research on Metabolic Disorders; the Osaka Foundation for Promotion of Clinical Immunology; and the Sankyo Foundation of Life Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ and GenBankTM/EMBL Data Banks with accession number AB008160.
To whom correspondence should be addressed. Tel.: 81-6-879-3880;
Fax: 81-6-879-3889; E-mail: hirano{at}molonc.med.osaka-u.ac.jp.
1 The abbreviations used are: IL-6, interleukin-6; IL-6RE, interleukin-6 response element; SBE, STAT-binding element; CRE, cAMP-responsive element; APRE, acute-phase response element; GHR, growth hormone receptor; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; APRF, acute phase response factor.
2 M. Ichiba, K. Nakajima, Y. Yamanaka, N. Kiuchi, and T. Hirano, unpublished data.
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REFERENCES |
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