From the Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical
School, Suita, Osaka 565-0871, Japan
The signal transducers and activators of transcription (STAT) family members have been implicated in regulating the growth, differentiation, and death of normal and transformed cells in response to either extracellular stimuli, including cytokines and growth factors, or intracellular
tyrosine kinases. c-myc expression is coordinately regulated by multiple signals in these diverse cellular responses. We show that STAT3 mostly mediates the rapid activation of the c-myc gene
upon stimulation of the interleukin (IL)-6 receptor or gp130, a signal transducing subunit of
the receptor complexes for the IL-6 cytokine family. STAT3 does so most likely by binding to
cis-regulatory region(s) of the c-myc gene. We show that STAT3 binds to a region overlapping
with the E2F site in the c-myc promoter and this site is critical for the c-myc gene promoter-
driven transcriptional activation by IL-6 or gp130 signals. This is the first identification of the
linkage between a member of the STAT family and the c-myc gene activation, and also explains
how the IL-6 family of cytokines is capable of inducing the expression of the c-myc gene.
Key words:
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Introduction |
Signal transducers and activators of transcription (STAT)1
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
Dictyostelium to mammals (1). STAT proteins are activated
not only by receptor-associated Janus tyrosine kinases (JAK)
(5), but also by receptor type tyrosine kinases (10)
and by the oncogenic tyrosine kinases v-src and v-abl (14- 16). The involvement of STAT proteins in cell survival,
growth, transformation, and differentiation has been reported in a number of instances. A role for STAT5 has
been suggested in IL-2-mediated cell growth signals in
murine pro-B BAF/B03 cell lines expressing a variety of
mutant IL-2 receptors (17), and it is also partially responsible for IL-3-induced cell growth in a pro-B BAF/B03 cell line (18). STAT3 activity plays a critical role in mediating gp130 signals leading to both growth arrest and macrophage differentiation in M1 leukemic cells (19, 20) as well as
to cell survival in BAF/B03 cells stably expressing chimeric
gp130 receptors (21). Lymphocytes from mice with a disruption in their stat6 or stat4 genes lose their proliferative
responses to IL-4 and IL-12, respectively, indicating critical
roles for these STAT molecules in cytokine-induced cell
growth (22). Moreover, a disruption of the stat3 genes causes embryonic lethality around embryonic day E7.5 (27),
suggesting a role for STAT3 in cell proliferation or survival
in early embryonic stages. Recently, a comparison of the responses of lymphocytes from normal and gene-disrupted
mice deficient in STAT6 or STAT4 led to the suggestion
that STAT6 and STAT4 control lymphocyte proliferation
by downregulating the levels of p27Kip1 protein (28). However, STAT5a is also involved in IL-2-induced lymphocyte
proliferation via induction of the IL-2 receptor
chain, as
shown by comparing lymphocytes from STAT5a null mice with those from normal mice (29). Regarding the roles of
STAT proteins in oncogenesis, STAT1 activation is correlated with cellular transformation by Eyk (30), and recently
STAT3 was shown to be involved in the transformation of
NIH3T3 cells by v-src (31, 32). In other cases, STAT1
protein plays a role in IFN-
-induced growth arrest and
apoptosis (33). All of these indicate the complexity of the
multiple roles of STAT family proteins. However, regarding the roles for STAT family proteins in cell proliferation, there is no report showing a linkage between STAT proteins and the direct regulation of genes critically involved
in the cell cycle progression or in cellular transformation.
The product of the c-myc gene has been shown to be a
critical regulator of cell growth, especially for cell cycle progression from the G1 to S phase (for review see reference 34)
and for the induction of cdc25A (35). The c-myc gene is
commonly activated during responses to the proliferative signals elicited by extracellular stimuli such as serum, epidermal
growth factor, platelet-derived growth factor (PDGF) (36),
nerve growth factor (37), colony stimulating factor 1 (38),
and a variety of cytokines including IL-1, IL-3, GM-CSF,
IL-5, IL-2, IL-4, IL-7, IL-6, IL-9, and IL-12 (39).
Among the molecules known to be involved in growth factor and cytokine signaling, the nonreceptor tyrosine kinases
c-src (46), syk (47), and JAK (48) have been shown to
activate the c-myc gene. Oncogenic nonreceptor tyrosine kinases, v-src and v-abl, bcr-abl, and the oncogenic serine/
threonine kinase v-akt have also been shown to induce c-myc
mRNA expression (51). STAM (the signal transducing
adaptor molecule), which is phosphorylated on tyrosine residues after stimulation with a variety of cytokines such as IL-2,
IL-3, GM-CSF, and epidermal growth factor appears to be
involved in IL-2- and GM-CSF-induced activation of the
c-myc gene promoter (55). However, very little is known
about the mechanisms by which these different effector molecules activate the c-myc gene. Only E2F molecules, composed of members of the E2F family and DP1 or DP2, have
been identified as the final common target molecules that affect transcriptional activation of the c-myc gene upon stimulation with serum, PDGF (56), v-abl (57), bcr-abl (54),
and phosphatidyl inositol 3-kinase/c-akt (60, 61).
The IL-6 family of cytokines, which includes IL-6, ciliary
nerve trophic factor, leukemia inhibitory factor, oncostatin
M, IL-11, and cardiotrophin-1, is variably involved in cell
growth, differentiation, and survival in a variety of tissues and
cells (62). In particular, IL-6 and IL-11 are potent growth factors for multiple myelomas (63, 64). The receptors for the
IL-6 family of cytokines share gp130 as a signal transducing
receptor subunit, which is capable of activating a variety of
signal transduction pathways, i.e., the STAT3-mediated pathway, the SH2 domain containing phosphatase (SHP)-2/Ras/
mitogen-activated protein kinase-mediated pathway, and
other poorly characterized pathways (6). IL-6 is essential for
cell growth of regenerating murine hepatocytes after partial
hepatectomy, causing the rapid activation of STAT3 and the
rapid induction of c-myc mRNA expression (65). Stimulation of gp130 activates STAT3, and induces c-myc mRNA expression and cell proliferation in BAF/B03 cells (21). One of
the important questions to be resolved is how cytokines, such
as those in the IL-6 family, are capable of inducing the expression of the c-myc gene, which plays an essential role in
cell fate, growth, and differentiation. Here, we characterize
the signaling pathways leading to the IL-6-induced full c-myc
gene activation and show that STAT3 is involved in the rapid
activation of the c-myc gene at least partly by directly binding
to a site overlapping with the c-myc E2F binding site in the
c-myc gene P2 promoter.
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Materials and Methods |
Cell Lines and Cell Culture.
The murine proB cell line BAF/
B03 cells were maintained in RPMI 1640 medium (GIBCO
BRL) supplemented with 10% FCS, 0.1 ng/ml recombinant
mouse IL-3, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere with 5% CO2. BAF transformants
expressing the chimeric receptors containing the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) and the transmembrane and cytoplasmic domains of gp130 have been described previously (21). A cell sorter
(FACScan®; Becton Dickinson) was used to check the expression
levels of the chimeric receptors which were labeled with an anti-
G-CSFR antibody. One representative clone for each type of
transformant (BAF-G277, BAF-G133, BAF-G68, BAF-G133F2,
and BAF-G133F3) expressing similar levels of the chimeric receptors was used in this study. To make stable BAF-G133 transformants expressing the dominant-negative STAT3s, 50 µg of
each expression vector encoding either the dominant-negative STAT3F or STAT3D (pCAGGS-Neo hemagglutinin [HA]Stat3D
and pCAGGS-Neo HAStat3F) along with 5 µg of pMIK-Hyg was
transfected by electroporation, and the transformants were selected with 200 µg/ml hygromycin. The expression levels of
STAT3D and STAT3F were analyzed by immunoblotting with
an anti-HA mAb, (12CA5; Boehringer Mannheim). The KT-3
human T cell lymphoma cells were grown in RPMI 1640 medium
supplemented with 10% FCS and 10 ng/ml recombinant human
IL-6. The human HepG2 hepatoma cells were grown in DME
(GIBCO BRL) supplemented with 10% FCS and antibiotics.
Northern Blot Analysis.
Total RNA was extracted using the
TRIzol reagent (GIBCO BRL) according to the procedure recommended by the manufacturer. Total RNA (20 µg per sample)
was separated by electrophoresis in 1% agarose formaldehyde gels
and transferred to Hybond N+ (Amersham Corp.) nylon membranes. Membranes were hybridized overnight at 65°C with 32P-labeled cDNA fragments, washed three times with 0.1× SSC,
0.1% SDS at 58°C for 20 min, and subjected to autoradiography.
The amount of loaded RNA was verified with the levels of
CHO-B mRNA intensity. The probes used here were the human c-myc cDNA (2.0 kb, EcoRV-EcoRI fragment), junB (2.1 kb, EcoRI fragment), and CHO-B (0.6 kb, EcoRI-BamHI fragment).
Plasmid Construction.
PHXL (55), a gift from Dr. K. Sugamura
and T. Arita (Tohoku University School of Medicine, Sendai,
Japan), is a luciferase reporter plasmid containing the human c-myc
gene with the region spanning from
2309 to +532 bp relative to
the transcription initiation site of the P1 promoter. This construct
was called
2309/+532 Luc in this paper for simplification. A
series of 5' deletion mutants were made as follows. To make
1398/+532 Luc and
349/+532 Luc, respectively, fragments
extending from a BglII site in the PHXL multicloning site to a SpeI
or PvuII site in the c-myc promoter were deleted. The resultant
constructs were then made blunt-ended and religated. For
101/
+532 Luc and +68/+532 Luc, SmaI and the XhoI fragments,
respectively, were deleted from PHXL. To make +102/+532
constructs, the region containing +102/+532 was amplified by PCR using the oligonucleotides 5'-AACTCGAGAAAAAGAACGGAGGGAGGGA-3' and 5'-GCCGGGCCTTTCTTTATGTT-3' as primers. The XhoI-HindIII fragment of the PCR
product was subcloned into the XhoI, HindIII site of PHXL, from
which the longer XhoI-HindII fragment, containing the c-myc region upstream of the HindIII site had been deleted. To make 3 × c-myc E2F Luc and 3 × adenovirus (Ad) E2 E2F Luc, three repeats
of c-myc E2F and Ad type 5 E2 E2F oligonucleotides were inserted
in front of the minimal mouse junB promoter Luc (66, 67). The
sequences of the oligonucleotides were as follows: c-myc E2F
5'-TTGGCGGGAAAAA-3' and Ad E2 promoter E2F 5'-GTTTCGCGCC-CTTTCTCAA-3' (68) with an additional SalI and
KpnI site at each end.
Site-directed Mutagenesis by PCR.
Mutations were introduced
in the c-myc E2F binding site in the context of the intact c-myc
promoter with the upstream region to
2309 bp (
2309/+532
mE2F Luc) and with that to
101 bp (
101/+532 mE2F Luc)
by the overlap extension technique, using PCR. The primers used for these mutations were 5'-AGGCTTGGAAGTTAAAAGAACGGAGGGAGGATC-3', 5'-TTTAACTTCCAAGCCTC-TGAGAAGCCCTG-3', and reverse and universal primers for
pBluescriptII (Stratagene). The underlined bases are the mutated
ones. pBluescriptII containing the XhoI-HindIII fragment of the
c-myc promoter was used as a template. After the PCR products were subcloned into pBluescriptII SK+ (Stratagene), their sequences were verified by DNA sequencing. The PCR product
digested with XhoI and HindIII was then inserted at the proper
position of
2309/+532 Luc and
101/+532 Luc to make
2309/+532 mE2F Luc and
101/+532 mE2F Luc.
Transient Transfection Assay.
For transfection experiments,
HepG2 cells were transfected with DNA mixtures using the calcium phosphate coprecipitation method. Typically, 1.2 µg of one
of the reporter plasmids containing the firefly luciferase gene, and
1 µg of pEFLacZ, a pEF-BOS expression vector containing the
-lacZ gene encoding
-galactosidase as an internal control for
transfection efficiency, were used. 3 µg of either pCAGGS-Neo,
an expression vector without an insert (control), or pCAGGS-NeoHAStat3F, an expression vector containing a cDNA encoding HA-STAT3F (20) was cotransfected in some experiments. Cells were incubated with DNA precipitates for 12 h, washed
with PBS, fed with DME containing 0.1% FCS for 20-24 h, and
stimulated with 100 ng/ml of IL-6 for the last 6 h. Approximately 42 h after transfection, cells were collected in 120 µl lysis
buffer and subjected to assays for luciferase and
-galactosidase
activity as described (69).
Electromobility Shift Assay (EMSA).
EMSAs were performed
according to the procedure published previously (70). The oligonucleotides used as probes or competitors for the EMSA were as
follows: c-myc E2F, 5'-GACGCTTGGCGGGAAAAAG-3' and
5'-GGCTT-TTTCCCGCCAAG-3'; Ad E2 E2F, 5'-GACGTT-TCGCGCCCTTTCT-3' and 5'-GGAGAAAGGGCGCGAAA-3' (68); STAT5, 5'-GCGAGATTTCTAGGAATTCAAT-3' and
5'-GGATTG-AATTCCTAGAAATCT-3' (71). The acute phase
response elements (APRE) were synthesized as reported previously
(70). Nuclear extracts (10 µg) were incubated in a final volume of
20 µl (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
cold oligonucleotides for 5 min before the addition of labeled oligonucleotide. For antibody interaction studies, antiserum or a
mAb specific to each STAT family member was included in the binding reaction during a 30-min preincubation on ice. The antibodies used were anti-STAT1 mAb recognizing the STAT1
NH2 terminus from Transduction Laboratories (anti-ISGF3
G16920); anti-STAT3 polyclonal antibody recognizing the
COOH terminus of STAT3 (70); and anti-STAT5 polyclonal antibody recognizing both STAT5a and STAT5b (sc-835X; Santa
Cruz Biotechnology).
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Results |
gp130-mediated Rapid Activation of the c-myc Gene without
Requiring De Novo Protein Synthesis.
We first characterized
the nature of the gp130-mediated c-myc mRNA induction
in BAF-G277, a BAF-B03 pro-B cell line expressing the
chimeric receptor containing the extracellular domain of
the G-CSF receptor and the transmembrane and cytoplasmic domains of gp130 (21). Total RNA was obtained from
BAF-G277 cells which had been deprived of IL-3 for 12 h
and then stimulated with 100 ng/ml of G-CSF for up to 15 h.
The levels of mRNAs for the c-myc gene and a housekeeping gene, CHO-B, are shown (Fig. 1 A). The c-myc
mRNA levels normalized with those of CHO-B were
plotted (Fig. 1 B). G-CSF increased immediately the levels
of c-myc mRNA by around sixfold with a peak at 1 h after
stimulation, followed by a gradual decrease until 12 h and a
slight increase at 15 h (Fig. 1 A, lanes 1-7; Fig. 1 B). This
induction is due to the activation of the chimeric receptor
since G-CSF did not increase c-myc mRNA level in the
parental BAF/B03 cells (Fig. 1 A, lanes 8-10). Pretreatment of BAF-G277 with cycloheximide did not inhibit but
rather enhanced the c-myc mRNA level at 1 h after stimulation with G-CSF, indicating that at least the initial phase
of c-myc mRNA induction by gp130-mediated signals did
not require de novo protein synthesis in BAF-G277 cells
(Fig. 1 C, lanes 1-4).

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Fig. 1.
Induction of c-myc mRNA expression via gp130 signaling in
BAF-G277 cells. (A) Northern blot analysis for c-myc mRNA expression
in G-CSF-stimulated BAF/B03 transfectants expressing a chimeric receptor consisting of the G-CSFR extracellular domain and the transmembrane and cytoplasmic domains of gp130, including the full-length, 277 amino acid-long cytoplasmic domain (BAF-G277). Total RNAs were
extracted from BAF-G277 cells (lanes 1-7) and parental BAF/B03 cells
(lanes 8-10) which had been deprived of IL-3 for 12 h and then stimulated with 100 ng/ml G-CSF for the indicated periods of time and subjected to Northern blot analysis for detection of c-myc mRNA (top) and
CHO-B mRNA (bottom), the latter being controls for the amounts of
loaded RNA. (B) Kinetic changes in the c-myc mRNA levels. Values for
c-myc mRNA levels were normalized to those for the corresponding
CHO-B mRNA levels and were plotted. Means ± SD of data from three
independent experiments are shown. (C) c-myc mRNA induction in the
presence of an inhibitor for protein synthesis. Total RNAs were extracted
from BAF-G277 cells, which had been deprived of IL-3 for 12 h and
then stimulated with 0 (lanes 1 and 3) or 100 ng/ml of G-CSF for 1 h
(lanes 2 and 4) in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of
30-min pretreatment of cells with 10 µg/ml of cycloheximide, and were
subjected to Northern blot analysis for c-myc and CHO-B mRNA expression as in A.
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The Involvement of STAT3 in gp130-mediated Rapid and
Full Activation of the c-myc Gene.
To investigate which signals were responsible for the c-myc gene activation by the
IL-6 cytokine family, we determined which region of
gp130 was required for full c-myc mRNA induction. To do
this, we selected BAF transformants with similar expression levels of the chimeric receptors for BAF-G277, BAF-G133, BAF-G133F2, BAF-G133F3, and BAF-G68 (Fig.
2 A). The constructs of the chimeric receptors are described in the legend to Fig. 2. Previously, we showed that
the membrane-proximal region of gp130, consisting of
133-amino acid residues, is necessary and sufficient for
ligand-induced cell growth (21). This region of gp130 contains two tyrosines, Y759 and Y767, which are required
for activation of the SHP-2/RAS/MAPK pathway and
STAT3, respectively (21, 72). In BAF-G133 cells, G-CSF
induced c-myc mRNA expression at a level somewhat less
than that in BAF-G277, whereas BAF-G68 showed c-myc
mRNA expression at a much lower level (Fig. 2 B). The
low-level c-myc mRNA expression in the BAF-G68 clone
was repeatedly observed and estimated as one-seventh and
one-fifth of those of BAF-G277 and BAF-G133, respectively. These results suggested that although a poorly characterized and weak signal to the c-myc gene was generated
from the membrane-proximal region between 1 and 68, the region between 68 and 133 is necessary for the full activation of the c-myc gene. The use of the other transformants, BAF-G133F2 and BAF-G133F3, which express the
truncated chimeric receptors with a tyrosine to phenylalanine mutation at the second (Y759) and third (Y767) tyrosine residues in the cytoplasmic domain, respectively, indicated that the third tyrosine residue, Y767, in G133 was
required for the full activation of the c-myc gene (Fig. 2 B).
Since this requirement is the same as for STAT3 activation
(19, 72), it seemed likely that STAT3 is involved in the full
c-myc induction. To test this directly, we used two other
BAF transformants expressing both truncated chimeric receptors, G133, and one of the two dominant-negative
STAT3 mutants (20), BAF-G133 STAT3F and BAF-G133
STAT3D. Expression of either dominant-negative STAT3
almost completely inhibited the gp130-mediated expression of both c-myc and junB mRNA (Fig. 3), the latter of
which has been known to be one of IL-6-inducible immediate early response genes (66, 70, 73). The three cell
lines, BAF-G133, BAF-G133 STAT3F, and BAF-G133
STAT3D, could respond to IL-3 by expressing c-myc
mRNA with levels similar in each cell line but much
higher than that of gp130-induced c-myc mRNA, indicating that the poor gp130-mediated induction of c-myc
mRNA seen in the dominant-negative STAT3-expressing
cell lines was due to the specific effect of the exogenous
STAT3F and STAT3D. These results indicated a critical role for STAT3 in the gp130-mediated c-myc and junB
gene activation. Considering that the c-myc mRNA induction through gp130 signals occurred without requiring de
novo protein synthesis (Fig. 1 C) and that the STAT3 transcription factor was involved, it seemed likely that STAT3
directly activated transcription of the c-myc gene by binding to a cis-regulatory region(s) of the gene.

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Fig. 2.
Induction of c-myc
mRNA expression in BAF transformants expressing various
G-CSFR-gp130 chimeric receptors. (A) FACS® analysis for the
expression levels of the chimeric
receptors on the BAF transformants. Cell lines tested were as follows: BAF-G277, BAF/B03 transformants
bearing a chimeric receptor, the extracellular domain of the G-CSFR and
the truncated gp130 including the transmembrane and cytoplasmic 133-
amino acid residues, referred to as G133 (shown as BAF-G133), G133F2
with a tyrosine (Y) to phenylalanine (F) mutation at the second tyrosine,
Y759 (BAF-G133F2), G-133F3, with a Y to F mutation at the third
tyrosine Y767 (BAF-G133F3), and G-68, bearing the 68 membrane-proximal amino acid residues of the cytoplasmic region of gp130 (BAF-G68). (B) Northern blot analysis for c-myc mRNA expression in BAF
transformants expressing various G-CSFR-gp130 chimeric receptors.
Total RNAs extracted from the various BAF transformants which had
been deprived of IL-3 for 12 h and stimulated with nothing or G-CSF at
100 ng/ml for the indicated periods of time were subjected to Northern
blot analysis for c-myc mRNA expression. Cell lines tested were as follows: BAF-G277 (lanes 1-3), BAF-G133 (lanes 4-6), BAF-G133F2 (lanes
7-9), G-133F3 (lanes 10-12), and BAF-G68 (lanes 13-15).
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Fig. 3.
Inhibition of gp130-mediated c-myc and junB mRNA
expression by dominant-negative
STAT3s. Total RNAs extracted
from the IL-3-deprived various
BAF-G133 transformants unstimulated or stimulated for indicated
hours with G-CSF at 100 ng/ml
(top) or recombinant mouse IL-3
at 0.2 ng/ml (bottom) were subjected to Northern blot analysis
for c-myc, junB, and CHO-B mRNA expression. Cell lines used were as
follows: BAF-G133 (lanes 1-3), BAF-G133 stably expressing HA-tagged
STAT3F (BAF-G133 STAT3F; lanes 4-6), and BAF-G133 stably expressing HA-tagged STAT3D (BAF-G133 STAT3D; lanes 7-9).
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Rapid Activation of the c-myc Promoter by STAT3.
We
tested whether IL-6 or gp130 activation could induce
c-myc mRNA expression in cells other than BAF transformants. IL-6 increased transiently the c-myc mRNA levels
by ~2.5-3-fold in HepG2 at 1 h after stimulation (Fig. 4 A),
as for BAF-G277. We then took advantage of the easy
transfectability of HepG2 cells to test if IL-6 can activate
the c-myc gene promoter. First we transiently transfected
HepG2 cells with a luciferase gene construct containing the
human c-myc gene with the region spanning from
2.3 kb
to +530 bp relative to the P1 initiation site, and tested for
IL-6 responsiveness. As shown in Fig. 4 B, IL-6 increased the reporter gene expression by ~3.0-fold. This activation
of the c-myc promoter-driven transcription was effectively
inhibited by the dominant-negative STAT3F, indicating
that a STAT3-responsive element(s) resides in the upstream
or promoter region of the c-myc gene. Fig. 4 C also shows
that only the chimeric receptors containing the gp130 region capable of activating STAT3, that is, G277, G133, and
G133F2, but not G133F3 or G68, could activate the c-myc
promoter driven-reporter gene expression, fully consistent
with the c-myc mRNA expression pattern observed in BAF
transformants. However, this does not rule out the possible
involvement of other region(s) outside the upstream and promoter region of the c-myc gene used in this study. Enhancement of induction was observed with the G133F2 expression
vector. This may be explained by an inhibitory effect of
SHP-2, which is activated through the tyrosine phosphorylation module at Y759, on the STAT3 activity (74).

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Fig. 4.
Signals through
gp130 activate the c-myc promoter-driven transcription in
HepG2. (A) Rapid induction of
c-myc mRNA in HepG2 by IL-6.
Total RNAs extracted from
HepG2 which had been cultured
in the presence of 0.1% serum
for 24 h (serum-starved) and
then stimulated with IL-6 at 100 ng/ml for the indicated periods
(lanes 1-3) were subjected to
Northern blot analysis for c-myc
and CHO-B mRNA expression.
Normalized values for c-myc
mRNA level relative to the
CHO-B mRNA level are shown
below the blots. (B) Activation
of human c-myc promoter-
driven luciferase gene expression by IL-6 in HepG2 cells.
HepG2 cells were transiently
transfected with 1.2 µg of the
human c-myc promoter luciferase
construct containing the upstream and downstream region
from 2309 to +532 bp relative
to the transcription initiation site
of the c-myc P1 promoter, 3 µg of either an expression vector, pCAGGS-Neo or pCAGGS-Neo bearing cDNA encoding the dominant-negative
HA-tagged STAT3F, pCAGGS-Neo HAStat3F, and 1 µg of pEFLacZ.
Transfected cells were serum starved for 24 h and then stimulated with
100 ng/ml of IL-6 for the last 6 h (black bars) or left unstimulated (open
bars). Cells were then harvested and subjected to assays for luciferase and
-galactosidase activity. Values were normalized to transfection efficiency
and represent the means of three different experiments. The SDs of the
mean values are indicated by error bars. The numbers at the right of the
bars indicate the fold increases in response to IL-6. (C) The signal derived
from the third tyrosine module of G133 is necessary for gp130-mediated
activation of the c-myc gene promoter. HepG2 cells were transfected with
3 µg of expression vectors for chimeric receptors (G277, G133, G133F2,
G133F3, and G68) together with 1.2 µg 2309/+532 Luc and 1 µg of
pEFLacZ. Transfected cells were serum starved for 24 h and stimulated
with 100 ng/ml of G-CSF (black bars) or left unstimulated (open bars) for
6 h. The luciferase assay was performed as described in Materials and
Methods. Values represent the means of three different experiments, and
the SDs of the mean values are indicated by error bars. The numbers at
the right of the bars indicate the fold increases in response to IL-6.
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STAT3 Responsive Element Resides in the Proximal Region
of the c-myc P2 Promoter.
To identify a STAT3 responsive
element in the c-myc promoter, we made a series of deletion mutants linked to the luciferase gene, as depicted in
Fig. 5. We transfected HepG2 cells with these constructs
and tested for IL-6 responsiveness. Whereas the promoter constructs bearing deletions
1398,
349,
101, and
+68 bp relative to the P1 transcription initiation site responded to IL-6 by 2.7-3.8-fold over baseline transcription
levels, the promoter truncated at +102 bp lost IL-6 responsiveness (Fig. 5), suggesting the existence of a STAT3
responsive element just upstream of the P2 promoter.

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Fig. 5.
IL-6 responsiveness of the various c-myc gene promoters. A
series of the luciferase reporter constructs bearing various lengths of the 5'
upstream and promoter region of the c-myc gene was used to search for
the IL-6 response region. The locations of restriction sites and numbers
relative to the transcription initiation site of the P1 promoter are shown,
as are the locations of the initiation site of the P2 promoter and E2F binding site. Each 5' deletion mutant of the c-myc gene promoter-luciferase
construct was transfected into HepG2 cells, and IL-6 responsiveness of
the promoter construct was assayed. Luciferase activities normalized to
the transfection efficiency are shown as described in the legend to Fig. 4.
Values are from four independent experiments. The numbers at the right
of the bars indicate the fold increases in response to IL-6.
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A Region Overlapping with the c-myc E2F Binding Site Is
Responsible for STAT3-mediated Activation of the c-myc Promoter.
Since the promoter region between +68 and
+102 bp from the P1 initiation site contained two known
functional sites, Me1a2 and part of the E2F binding site, we
hypothesized that the major determinant for IL-6-induced
and STAT3-mediated activation might be the c-myc E2F
site, located at +98 to +106 bp. To test if this site was
responsible for IL-6-induced and STAT3-mediated activation of the promoter, we made point mutations at this
c-myc E2F site, changing from TTGGCGGGAAA to TTGGAAGTTAA, in the context of the full-length promoter
construct (
2309/+532 mE2F Luc) and in the truncated
version of the c-myc promoter construct (
101/+532
mE2F Luc), and tested for IL-6 responsiveness. The mutations severely compromised the IL-6 responsiveness of
both constructs (Fig. 6 A), indicating that this site is really
required for IL-6 responsiveness in the intact promoter.
Then we proceeded to test whether the STAT3-mediated
activation can be seen in E2F binding site in general or specifically in the c-myc E2F binding site. We constructed two
reporter gene constructs, one containing three repeats of the c-myc E2F site, TTGGCGGGAAAAAG, and the other
containing three repeats of a typical E2F binding site,
GTTTCGCGCCCTTTCTCAA, from the Ad E2 promoter (68, 75) inserted upstream of the heterologous minimal junB promoter linked to the luciferase gene (66, 67), and tested them for IL-6 responsiveness in HepG2 cells
(Fig. 6 B). Interestingly, IL-6 dramatically activated the
c-myc E2F site-driven transcription, but not the wild-type
E2F site-driven expression, and the IL-6-induced activation of the c-myc E2F site was inhibited by the coexpression of the dominant-negative STAT3F (Fig. 6 B). From
these results, we concluded that STAT3 activated the c-myc gene promoter by specifically activating the c-myc E2F
binding site or an overlapping region. These results also indicated that the STAT3-mediated activation of the c-myc
gene promoter occurs independently of E2F activity. The
basal promoter activities of the c-myc promoters with the
mutated E2F site (
2309/+532 mE2F Luc and
101/
+532 mE2F Luc) were reproducibly higher than those of
the corresponding intact c-myc promoters (
2309/+532
Luc and
101/+532 Luc), consistent with the existence of
a repressor activity bound to the c-myc E2F site in the serum-starved HepG2 cells.

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Fig. 6.
The c-myc E2F site is crucial for the activation of the c-myc
gene promoter-driven transcription by STAT3. (A) The c-myc E2F site is
necessary for the IL-6 responsiveness of the c-myc gene promoter. Point
mutations were made in the E2F binding site region (see Materials and
Methods). The two promoter constructs with the mutated E2F site as
well as the original constructs were assayed for IL-6 responsiveness in
HepG2 cells. Normalized luciferase activities are shown as described
above. Values are from four independent experiments. The numbers at
the right of the bars indicate fold increases in response to IL-6. (B)
STAT3-dependent IL-6 activation of the c-myc E2F site. The reporter
construct containing three repeats of the c-myc E2F site inserted upstream
of the minimal junB promoter-luciferase gene (66) (shown as 3 × c-myc
E2F), the similar construct with three repeats of a typical E2F site from
the adenovirus E2 gene (3 × Ad E2 E2F), and the minimal junB promoter construct were transfected and tested for IL-6 responsiveness. The
dominant-negative STAT3 (STAT3F) expression vector was included in
some experiments using the 3 × c-myc E2F construct (3 × c-myc E2F + DN STAT3F). Normalized luciferase activities with standard deviations
from three independent experiments are shown as in Fig. 4. The numbers
at the right of the bars indicate fold increases in response to IL-6.
|
|
Direct Binding of STAT3 to the c-myc E2F Binding Site.
Since the sequence of the c-myc E2F binding site, TTGGCGGGAAA, is distinct from the known STAT3 binding
sites (76), we tested whether STAT3 and other STAT
proteins bind directly to the c-myc E2F site or indirectly
through interactions with other proteins. To do this, we
used EMSA, testing nuclear extracts from BAF-G277 and
HepG2 cells stimulated with either G-CSF or IL-6. The
extracts were incubated with labeled oligonucleotides bearing the c-myc E2F site and specific antibodies to each
STAT protein in some experiments and unlabeled oligonucleotides as competitors in other experiments. The nuclear
extracts from BAF-G277 cells stimulated with G-CSF for
15 min (Fig. 7 A, lanes 1-5) and HepG2 cells stimulated with IL-6 for 15 min (Fig. 7 B, lanes 1-4) have prominent
c-myc E2F site binding activity. In the nuclear extracts from
BAF-G277 cells stimulated with G-CSF, most of the complexes on the c-myc E2F site were shifted by anti-STAT3
serum (Fig. 7 B, lane 4), indicating that STAT3 is the major component bound to the c-myc E2F site in BAF-G277
cells, which have very little E2F activity, either free E2F or
complexes of E2F and one of the retinoblastoma protein
family members. The inducible complexes in the IL-6-
stimulated HepG2 nuclear extracts were most likely to
contain STAT3, STAT3/STAT1, and STAT1 (indicated
by arrows in Fig. 7 B), since anti-STAT3 serum shifted the
two upper bands and anti-STAT1 antibody shifted the
lower band. Interestingly, the mobilities of the complexes with the c-myc E2F probe are exactly the same as those of
complexes seen using an oligonucleotide probe containing
an APRE from
2-macroglobulin, a typical STAT binding
site (compare lane 2 with lane 4 in Fig. 7 C), suggesting
that these inducible complexes with c-myc E2F probe are
dimers of STAT3, STAT3/STAT1, and STAT1, although
c-myc E2F probe has less affinity than APRE probe. These
inducible complexes of STAT1 and 3 with the c-myc E2F
probe were competed by an oligonucleotide containing
APRE (Fig. 7 D, lanes 7 and 8) five times as efficiently as
by a control c-myc E2F oligonucleotide (Fig. 7 D, lanes 3 and 4), but not by the typical E2F binding site, TTTCGCGC, taken from the adenovirus E2 promoter (Fig. 7 D,
lanes 5 and 6). These results suggested that activated STAT
proteins may directly bind to the c-myc E2F site with
around fivefold less affinity than to APRE and that in spite
of the low affinity to the c-myc E2F site, the binding activities of STAT proteins were much stronger than those of
E2F-containing complexes.

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Fig. 7.
The nature of the gp130-signal-induced DNA binding activities at the
c-myc promoter E2F site. Nuclear extracts
from (A) IL-3-deprived BAF-G277 cells
and (B) serum-starved HepG2 cells either
untreated (A and B, lane 1) or treated with
G-CSF (A, lanes 2-5) or IL-6 (B, lanes 2-4)
at 100 ng/ml for 15 min were preincubated
without (A and B, lanes 1 and 2) or with
anti-STAT1 mAb (A and B, lane 3), anti-STAT3 antibody (A and B, lane 4), or anti-STAT5 antibody (A, lane 5) for 30 min on
ice before the addition of 32P-labeled c-myc
E2F oligonucleotides, and were then subjected to electrophoresis and autoradiography. The positions of the gp130-signal
inducible complexes containing STAT3,
STAT3/STAT1, and STAT1 are indicated.
(C) Nuclear extracts from serum-starved
HepG2 either unstimulated (lanes 1 and 3) or stimulated with IL-6 for 15 min (lanes 2 and 4) were subjected to EMSA with c-myc E2F oligonucleotide
probe (lanes 1 and 2) and APRE oligonucleotide probe (lanes 3 and 4). (D) Binding specificity of IL-6-inducible c-myc E2F site-binding complexes. Nuclear extracts from serum-starved HepG2 cells either untreated (lane 1) or treated with IL-6 (lanes 2-8) were preincubated with unlabeled oligonucleotide competitors (50- or 250-fold molar excess as indicated) for 5 min, followed by incubation with labeled c-myc E2F probes. The competitors used
were unlabeled oligonucleotides containing the c-myc E2F site (lanes 3 and 4), the adenovirus E2 E2F site (lanes 5 and 6), or the 2 macroglobulin
APRE (lanes 7 and 8).
|
|
Binding Specificity of STAT Proteins for the c-myc E2F
Site.
Next we tested the binding specificity of other
STAT proteins for the c-myc E2F site. For this experiment,
we first tested the nuclear extracts from IL-2-stimulated
KT-3 cells (79) for STAT5 activity using EMSA with an
oligonucleotide containing the STAT5 binding site (71)
and a specific antibody to STAT5 that recognizes both
STAT5a and 5b. As shown in Fig. 8, IL-2-stimulated KT-3
nuclear extracts showed abundant STAT5 complexes on
the STAT5 probe recognizable by anti-STAT5 antibody
(lane 10) and a small amount of STAT3-containing complex, which was recognized by an anti-STAT3 antibody
(lane 9). The same extracts showed inducible binding activity to the c-myc E2F site (Fig. 8, lane 2). Interestingly, the
anti-STAT5 antibody did not cause any shift of the complexes (Fig. 8, lane 5), whereas most of the complexes were
shifted by the anti-STAT3 antibody (Fig. 8, lane 4). These
results indicated that the c-myc E2F site binds preferentially
to STAT3 and STAT1, but not to STAT5, and that the
nuclear extracts from IL-2-stimulated KT-3 cells had
STAT3 bound to the c-myc E2F binding site. Although we
do not know the role of STAT3 in the IL-2 signaling pathway, this binding specificity is at least consistent with the
lack of a role for STAT5 in the IL-2-induced activation of
the c-myc gene.

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Fig. 8.
Binding of STAT3
but not STAT5 to the c-myc E2F
site in IL-2-stimulated KT-3 nuclear extracts. Nuclear extracts
from IL-6-deprived KT-3 cells
either untreated (lanes 1 and 6)
or treated with IL-2 at 10 ng/ml
for 15 min (lanes 2-5 and 7-10)
were preincubated without (lanes
1, 2, 6, and 7) or with an anti-STAT1 mAb (lanes 3 and 8),
anti-Stat3 antibody (lanes 4 and
9), or anti-Stat5 antibody (lanes
5 and 10) for 30 min on ice before the addition of 32P-labeled
oligonucleotide containing the
c-myc promoter E2F site (lanes 1-5) or 32P-labeled oligonucleotide containing a Stat5 binding site (lanes 6-10), and were then subjected to electrophoresis and autoradiography. The positions of the complexes containing STAT3 or STAT5 are indicated.
|
|
 |
Discussion |
Rapid Activation of the c-myc Gene by STAT3.
E2F activity has been shown to be involved in the activation of
the c-myc gene in response to serum or PDGF (56). It is also likely that E2F activity is elevated in cycling cells in the
G1 to S phase transition, including BAF-G277 cells. However, this mechanism does not account for the immediate
induction of c-myc mRNA observed in BAF-G277 and
HepG2 cells, since the increases in CDK4/6 kinase activity
were only apparent 4 h after gp130 stimulation in BAF-G277 cells (data not shown), and no apparent increase in E2F activity was detected in IL-6-stimulated HepG2 cells,
as exemplified by the lack of activation of the reporter gene
containing three repeats of the wild-type E2F binding sites
upstream of the minimal junB promoter (Fig. 6 B). Instead,
we suggested that STAT3 is essential for the rapid IL-6-
induced or gp130-mediated induction of the c-myc mRNA
expression and showed that STAT3 rapidly activated the
c-myc gene promoter by binding to a site overlapping with the c-myc E2F site. However, at the present time we do
not know whether STAT3 cooperates with other proteins
to activate the c-myc promoter-driven transcription or
whether STAT3 synergistically enhances the c-myc mRNA
expression with other uncharacterized pathway(s) or mechanisms affecting the levels of transcriptional elongation or
mRNA stability. The putative STAT3 binding site and the
E2F binding site in the c-myc promoter are depicted and compared with the typical E2F binding site in the adenovirus E2 promoter (Fig. 9). As shown in Fig. 9, a STAT3
binding site overlaps with the E2F binding site in the c-myc
P2 promoter, while a typical E2F site, such as the E2F
binding site in the adenovirus E2 promoter, does not have
a STAT3 binding site. The point mutations at the c-myc
E2F site, from TTGGCGGGAAA to TTGGAAGTTAA,
compromised the IL-6 responsiveness of the c-myc promoter (Fig. 6 A). Although these point mutations were
originally intended to abolish the binding activity to E2F
and E2F complexes, the mutations actually inhibited the
STAT binding activity (data not shown), consistent with
the notion that this site also works as a functional STAT binding element. The role of STAT proteins in activating
the c-myc gene is crucial, especially for the IL-6 family of
cytokines, which mainly activate STAT3 and weakly activate the c-myc gene as shown in Fig. 3. Mouse hepatocytes,
when responding in vivo to partial hepatectomy, show a
very rapid increase in STAT3 activity as well as junB and
c-myc mRNA expression, in a manner dependent on IL-6
(65). The rapid increase in c-myc mRNA expression in regenerating hepatocytes may be explained by the direct effect of STAT3 on the c-myc gene activation shown here.
Role of the E2F Site in Regulating the c-myc Gene.
The
c-myc E2F site is also a target for free E2F and E2F complexes with the products of the retinoblastoma gene (pRb)
family (80, 81). It has been shown that at quiescent and
early G1 phases, pRb and related members, complexed
with E2F, repress the transcriptional activity of the genes
with E2F binding sites in their cis-regulatory elements (82,
83), and, during the progression of the cell cycle, E2F freed
from pRb family members can activate the genes required
for S phase progression (56). In this context, the role of
STAT3, which is rapidly activated and efficiently binds to
the c-myc E2F site, may be dual. First, transcriptionally active STAT3 can rapidly activate the c-myc gene by directly
binding to the c-myc E2F site. Second, STAT3 may work
only through excluding the repressor complexes from the
E2F site, although this possibility remains to be examined.
It is noteworthy that this mechanism can only be applied to
the specific genes that contain the E2F binding site with an
affinity for both STAT and E2F proteins.
Cytokine Signals Regulating c-myc Expression.
Upon binding to a ligand, most cytokine receptors initiate a variety
of signaling pathways by activating JAK tyrosine protein kinases (6, 84, 85). Some signaling pathways appear to activate the c-myc gene. For instance, IL-2 has been shown to activate the c-myc gene through at least three distinct pathways, the tyrosine kinase syk (47), STAM (55), and phosphatidyl inositol 3-kinase/Akt, protein kinase B (60, 61).
The last pathway was shown to increase the E2F activity by
phosphorylating and removing pRb from E2F (61). As for
the STAT proteins, STAT5, activated by IL-2 or by IL-3R
c, has been shown not to be responsible for c-myc mRNA
induction (18, 86, 87). Mui et al. showed that carboxy-terminally truncated dominant-negative STAT5 inhibited the
IL-3-induced mRNA expression for the cis and pim-1
genes but not that of the c-myc gene (18). The absence of a
role for STAT5 in c-myc activation is consistent with our
result showing that STAT5 does not bind to the c-myc E2F
site (Fig. 8). This is quite a contrast to the critical role for
STAT3 in activation of the c-myc gene through gp130 signaling, and shows that each STAT molecule has different
target genes. Although STAT1 can bind to the STAT binding site overlapping with E2F site in the c-myc promoter, the contribution of STAT1 in IL-6-induced or
gp130-mediated c-myc gene activation may not be important considering the following observations. First, the
amount of STAT1 is much smaller than that of STAT3 in
BAF-G277 cells. Second, two types of dominant-negative STAT3 efficiently inhibited the gp130-mediated c-myc
mRNA induction in BAF-G133 cells. Third, dominant-negative STAT3, but not dominant-negative STAT1 (data
not shown), inhibited IL-6-induced activation of the c-myc
gene promoter activity in HepG2 cells (Fig. 4 B). Our results indicate that different cytokine receptor systems have
their own strategy to cause the similar outcomes, survival
of cells and induction of the genes required for cell cycle
progression, including the c-myc gene. It is also noteworthy
that STAT3 activates the c-myc gene in some cells shown
here but in other cells, e.g., M1 leukemic cells, the same
STAT3 is involved in repression of c-myc gene expression
with slower kinetics (20). The role of STAT1 in regulating
the c-myc gene expression in response to other cytokines,
including IFN
, should be examined carefully in this context.
This is the first report showing the linkage between
STAT family proteins and c-myc gene activation. This result implies that other growth factor receptors, or nonreceptor type tyrosine kinases, or oncogene products that are
capable of activating STAT3 also induce c-myc mRNA expression, at least in part through STAT3 activation.
Address correspondence to Toshio Hirano, Division of Molecular Oncology, Biomedical Research Center,
Osaka University Medical School, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-879-3880;
Fax: 81-6-879-3889; E-mail: hirano{at}molonc.med.osaka-u.ac.jp
We thank Drs. K. Sugamura and T. Arita for the gift of the human c-myc promoter construct, and Drs. K. Ishihara, H. Maeda, K. Nishida and Miss. J. Ishikawa for technical assistance.
This work was supported by Grants-in-Aid for COE Research, Scientific Research (B), and on Priority
Areas from the Ministry of Education, Science, Sports, and Culture in Japan, the Special Coordination Fund
from the Science and Technology Agency of the Japanese Government, the Osaka Foundation for Promotion of Clinical Immunology, and the Ryoichi Naito Foundation for Medical Research.
1.
|
Darnell, J.J.,
I.M. Kerr, and
G.R. Stark.
1994.
Jak-STAT
pathways and transcriptional activation in response to IFNs
and other extracellular signaling proteins.
Science.
264:
1415-1421
[Medline].
|
2.
|
Hou, X.S., and
N. Perrimon.
1997.
The JAK-STAT pathway in Drosophila.
Trends Genet.
13:
105-110
[Medline].
|
3.
|
Kawata, T.,
A. Shevchenko,
M. Fukuzawa,
K.A. Jermyn,
N.F. Totty,
N.V. Zhukovskaya,
A.E. Sterling,
M. Mann, and
J.G. Williams.
1997.
SH2 signaling in a lower eukaryote: a
STAT protein that regulates stalk cell differentiation in dictyostelium.
Cell.
89:
909-916
[Medline].
|
4.
|
Yan, R.,
S. Small,
C. Desplan,
C.R. Dearolf, and
J.E. Darnell Jr..
1996.
Identification of a Stat gene that functions in
Drosophila development.
Cell.
84:
421-430
[Medline].
|
5.
|
Darnell, J.E. Jr..
1997.
STATs and gene regulation.
Science.
277:
1630-1635
[Abstract/Free Full Text].
|
6.
|
Hirano, T.,
K. Nakajima, and
M. Hibi.
1997.
Signaling
mechanisms through gp130: a model of the cytokine system.
Cytokine Growth Factor Rev.
8:
241-252
.
[Medline] |
7.
|
Ihle, J.N.,
T. Nosaka,
W. Thierfelder,
F.W. Quelle, and
K. Shimoda.
1997.
Jaks and Stats in cytokine signaling.
Stem
Cells.
15:
105-111
[Medline].
|
8.
|
O'Shea, J.J..
1997.
Jaks, STATs, cytokine signal transduction,
and immunoregulation: are we there yet?
Immunity.
7:
1-11
[Medline].
|
9.
|
Taniguchi, T..
1995.
Cytokine signaling through nonreceptor
protein tyrosine kinases.
Science.
268:
251-255
[Medline].
|
10.
|
David, M.,
L. Wong,
R. Flavell,
S.A. Thompson,
A. Wells,
A.C. Larner, and
G.R. Johnson.
1996.
STAT activation by
epidermal growth factor (EGF) and amphiregulin. Requirement for the EGF receptor kinase but not for tyrosine phosphorylation sites or JAK1.
J. Biol. Chem.
271:
9185-9188
[Abstract/Free Full Text].
|
11.
|
Park, O.K.,
T.S. Schaefer, and
D. Nathans.
1996.
In vitro activation of Stat3 by epidermal growth factor receptor kinase.
Proc. Natl. Acad. Sci. USA.
93:
13704-13718
[Abstract/Free Full Text].
|
12.
|
Quelle, F.W.,
W. Thierfelder,
B.A. Witthuhn,
B. Tang,
S. Cohen, and
J.N. Ihle.
1995.
Phosphorylation and activation
of the DNA binding activity of purified Stat1 by the Janus
protein-tyrosine kinases and the epidermal growth factor receptor.
J. Biol. Chem.
270:
20775-20780
[Abstract/Free Full Text].
|
13.
|
Vignais, M.L.,
H.B. Sadowski,
D. Watling,
N.C. Rogers, and
M. Gilman.
1996.
Platelet-derived growth factor induces
phosphorylation of multiple JAK family kinases and STAT
proteins.
Mol. Cell. Biol.
16:
1759-1769
[Abstract].
|
14.
|
Cao, X.,
A. Tay,
G.R. Guy, and
Y.H. Tan.
1996.
Activation
and association of Stat3 with Src in v-Src-transformed cell
lines.
Mol. Cell. Biol.
16:
1595-1603
[Abstract].
|
15.
|
Danial, N.N.,
A. Pernis, and
P.B. Rothman.
1995.
Jak-STAT signaling induced by the v-abl oncogene.
Science.
269:
1875-1877
[Medline].
|
16.
|
Yu, C.L.,
D.J. Meyer,
G.S. Campbell,
A.C. Larner,
C. Carter-Su,
J. Schwartz, and
R. Jove.
1995.
Enhanced DNA-binding activity of a Stat3-related protein in cells transformed
by the Src oncoprotein.
Science.
269:
81-83
[Medline].
|
17.
|
Friedmann, M.C.,
T.S. Migone,
S.M. Russell, and
W.J. Leonard.
1996.
Different interleukin 2 receptor beta-chain tyrosines couple to at least two signaling pathways and synergistically mediate interleukin 2-induced proliferation.
Proc.
Natl. Acad. Sci. USA.
93:
2077-2082
[Abstract/Free Full Text].
|
18.
|
Mui, A.L.,
H. Wakao,
T. Kinoshita,
T. Kitamura, and
A. Miyajima.
1996.
Suppression of interleukin-3-induced gene
expression by a C-terminal truncated Stat5: role of Stat5 in
proliferation.
EMBO (Eur. Mol. Biol. Organ.) J.
15:
2425-2433
[Abstract].
|
19.
|
Yamanaka, Y.,
K. Nakajima,
T. Fukada,
M. Hibi, and
T. Hirano.
1996.
Differentiation and growth arrest signals are
generated through the cytoplasmic region of gp130 that is essential for Stat3 activation.
EMBO (Eur. Mol. Biol. Organ.) J.
15:
1557-1565
[Abstract].
|
20.
|
Nakajima, K.,
Y. Yamanaka,
K. Nakae,
H. Kojima,
M. Ichiba,
N. Kiuchi,
T. Kitaoka,
T. Fukada,
M. Hibi, and
T. Hirano.
1996.
A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells.
EMBO (Eur. Mol. Biol. Organ.) J.
15:
3651-3658
[Abstract].
|
21.
|
Fukada, T.,
M. Hibi,
Y. Yamanaka,
M. Takahashi-Tezuka,
Y. Fujitani,
T. Yamaguchi,
K. Nakajima, and
T. Hirano.
1996.
Two signals are necessary for cell proliferation induced
by a cytokine receptor gp130: involvement of Stat3 in anti-apoptosis.
Immunity.
5:
449-460
[Medline].
|
22.
|
Kaplan, M.H.,
Y.L. Sun,
T. Hoey, and
M.J. Grusby.
1996.
Impaired IL-12 responses and enhanced development of Th2
cells in Stat4-deficient mice.
Nature.
382:
174-177
[Medline].
|
23.
|
Kaplan, M.H.,
U. Schindler,
S.T. Smiley, and
M.J. Grusby.
1996.
Stat6 is required for mediating responses to IL-4 and
for development of Th2 cells.
Immunity.
4:
313-319
[Medline].
|
24.
|
Shimoda, K.,
J. van Deursen,
M.Y. Sangster,
S.R. Sarawar,
R.T. Carson,
R.A. Tripp,
C. Chu,
F.W. Quelle,
T. Nosaka,
D.A. Vignali, et al
.
1996.
Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6
gene.
Nature.
380:
630-633
[Medline].
|
25.
|
Takeda, K.,
T. Tanaka,
W. Shi,
M. Matsumoto,
M. Minami,
S. Kashiwamura,
K. Nakanishi,
N. Yoshida,
T. Kishimoto, and
S. Akira.
1996.
Essential role of Stat6 in IL-4 signalling.
Nature.
380:
627-630
[Medline].
|
26.
|
Thierfelder, W.E.,
J.M. van Deursen,
K. Yamamoto,
R.A. Tripp,
S.R. Sarawar,
R.T. Carson,
M.Y. Sangster,
D.A. Vignali,
P.C. Doherty,
G.C. Grosveld, and
J.N. Ihle.
1996.
Requirement for Stat4 in interleukin-12-mediated responses of
natural killer and T cells.
Nature.
382:
171-174
[Medline].
|
27.
|
Takeda, K.,
K. Noguchi,
W. Shi,
T. Tanaka,
M. Matsumoto,
N. Yoshida,
T. Kishimoto, and
S. Akira.
1997.
Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality.
Proc. Natl. Acad. Sci. USA.
94:
3801-3804
[Abstract/Free Full Text].
|
28.
|
Kaplan, M.H.,
C. Daniel,
U. Schindler, and
M.J. Grusby.
1998.
Stat proteins control lymphocyte proliferation by regulating p27Kip1 expression.
Mol. Cell. Biol.
18:
1996-2003
[Abstract/Free Full Text].
|
29.
|
Nakajima, H.,
X.W. Liu,
A. Wynshaw-Boris,
L.A. Rosenthal,
K. Imada,
D.S. Finbloom,
L. Hennighausen, and
W.J. Leonard.
1997.
An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2
receptor alpha chain induction.
Immunity.
7:
691-701
[Medline].
|
30.
|
Zong, C.,
R. Yan,
A. August,
J.E. Darnell Jr., and
H. Hanafusa.
1996.
Unique signal transduction of Eyk: constitutive
stimulation of the JAK-STAT pathway by an oncogenic receptor-type tyrosine kinase.
EMBO (Eur. Mol. Biol. Organ.)
J.
15:
4515-4525
[Abstract].
|
31.
|
Bromberg, J.F.,
C.M. Horvath,
D. Besser,
W.W. Lathem, and
J.E. Darnell Jr..
1998.
Stat3 activation is required for cellular transformation by v-src.
Mol. Cell. Biol.
18:
2553-2558
[Abstract/Free Full Text].
|
32.
|
Turkson, J.,
T. Bowman,
R. Garcia,
E. Caldenhoven,
R.P. De Groot, and
R. Jove.
1998.
Stat3 activation by Src induces
specific gene regulation and is required for cell transformation.
Mol. Cell. Biol.
18:
2545-2552
[Abstract/Free Full Text].
|
33.
|
Chin, Y.E.,
M. Kitagawa,
W.C. Su,
Z.H. You,
Y. Iwamoto, and
X.Y. Fu.
1996.
Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated
by STAT1.
Science.
272:
719-722
[Abstract].
|
34.
|
Henriksson, M., and
B. Luscher.
1996.
Proteins of the Myc
network: essential regulators of cell growth and differentiation.
Adv. Cancer Res.
68:
109-182
[Medline].
|
35.
|
Galaktionov, K.,
X. Chen, and
D. Beach.
1996.
Cdc25 cell-cycle phosphatase as a target of c-myc.
Nature.
382:
511-517
[Medline].
|
36.
|
Kelly, K.,
B.H. Cochran,
C.D. Stiles, and
P. Leder.
1983.
Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor.
Cell.
35:
603-610
[Medline].
|
37.
|
Greenberg, M.E.,
A.L. Hermanowski, and
E.B. Ziff.
1986.
Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription.
Mol. Cell.
Biol.
6:
1050-1057
[Medline].
|
38.
|
Roussel, M.F.,
J.L. Cleveland,
S.A. Shurtleff, and
C.J. Sherr.
1991.
Myc rescue of a mutant CSF-1 receptor impaired in
mitogenic signalling.
Nature.
353:
361-363
[Medline].
|
39.
|
Conscience, J.F.,
B. Verrier, and
G. Martin.
1986.
Interleukin-3-dependent expression of the c-myc and c-fos proto-
oncogenes in hemopoietic cell lines.
EMBO (Eur. Mol. Biol.
Organ.) J.
5:
317-323
[Abstract].
|
40.
|
Harel-Bellan, A., and
W.L. Farrar.
1987.
Modulation of
proto-oncogene expression by colony stimulating factors.
Biochem. Biophys. Res. Commun.
148:
1001-1008
[Medline].
|
41.
|
Kessler, D.J.,
M.P. Duyao,
D.B. Spicer, and
G.E. Sonenshein.
1992.
NF-kappa B-like factors mediate interleukin 1 induction of c-myc gene transcription in fibroblasts.
J. Exp.
Med.
176:
787-792
[Abstract].
|
42.
|
Klemsz, M.J.,
L.B. Justement,
E. Palmer, and
J.C. Cambier.
1989.
Induction of c-fos and c-myc expression during B cell
activation by IL-4 and immunoglobulin binding ligands.
J.
Immunol.
143:
1032-1039
[Abstract/Free Full Text].
|
43.
|
Morrow, M.A.,
G. Lee,
S. Gillis,
G.D. Yancopoulos, and
F.W. Alt.
1992.
Interleukin-7 induces N-myc and c-myc expression in normal precursor B lymphocytes.
Genes Dev.
6:
61-70
[Abstract].
|
44.
|
Nabata, T.,
S. Morimoto,
E. Koh,
T. Shiraishi, and
T. Ogihara.
1990.
Interleukin-6 stimulates c-myc expression and
proliferation of cultured vascular smooth muscle cells.
Biochem. Int.
20:
445-453
[Medline].
|
45.
|
Reed, J.C.,
D.E. Sabath,
R.G. Hoover, and
M.B. Prystowsky.
1985.
Recombinant interleukin 2 regulates levels of
c-myc mRNA in a cloned murine T lymphocyte.
Mol. Cell.
Biol.
5:
3361-3368
[Medline].
|
46.
|
Barone, M.V., and
S.A. Courtneidge.
1995.
Myc but not Fos
rescue of PDGF signalling block caused by kinase-inactive
Src.
Nature.
378:
509-512
[Medline].
|
47.
|
Minami, Y.,
Y. Nakagawa,
A. Kawahara,
T. Miyazaki,
K. Sada,
H. Yamamura, and
T. Taniguchi.
1995.
Protein tyrosine kinase Syk is associated with and activated by the IL-2
receptor: possible link with the c-myc induction pathway.
Immunity.
2:
89-100
[Medline].
|
48.
|
Kawahara, A.,
Y. Minami,
T. Miyazaki,
J.N. Ihle, and
T. Taniguchi.
1995.
Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos
and c-myc, but not bcl-2, gene induction.
Proc. Natl. Acad. Sci.
USA.
92:
8724-8728
[Abstract].
|
49.
|
Sakamaki, K.,
I. Miyajima,
T. Kitamura, and
A. Miyajima.
1992.
Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3, and IL-5 receptors for
growth signal transduction and tyrosine phosphorylation.
EMBO (Eur. Mol. Biol. Organ.) J.
11:
3541-3549
[Abstract].
|
50.
|
Watanabe, S.,
T. Itoh, and
K. Arai.
1996.
JAK2 is essential
for activation of c-fos and c-myc promoters and cell proliferation through the human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells.
J. Biol. Chem.
271:
12681-12686
[Abstract/Free Full Text].
|
51.
|
Cleveland, J.L.,
M. Dean,
N. Rosenberg,
J.Y. Wang, and
U.R. Rapp.
1989.
Tyrosine kinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells through signaling pathways involving c-myc: conditional regulation of c-myc
transcription by temperature-sensitive v-abl.
Mol. Cell. Biol.
9:
5685-5695
[Medline].
|
52.
|
Kuchino, Y.,
K. Nemoto,
S. Kawai, and
S. Nishimura.
1985.
Activation of c-myc gene transcription by Rous sarcoma virus
infection.
Jpn. J. Cancer Res.
76:
75-78
[Medline].
|
53.
|
Sovova, V.,
R. Friis,
H. Fidlerova, and
I. Hlozanek.
1993.
c-myc gene activation as a permanent trait of RSV-infected
quail cells.
Int. J. Cancer.
53:
983-987
[Medline].
|
54.
|
Stewart, M.J.,
S. Litz-Jackson,
G.S. Burgess,
E.A. Williamson,
D.S. Leibowitz, and
H.S. Boswell.
1995.
Role for E2F1
in p210 BCR-ABL downstream regulation of c-myc transcription initiation. Studies in murine myeloid cells.
Leukemia.
9:
1499-1507
[Medline].
|
55.
|
Takeshita, T.,
T. Arita,
M. Higuchi,
H. Asao,
K. Endo,
H. Kuroda,
N. Tanaka,
K. Murata,
N. Ishii, and
K. Sugamura.
1997.
STAM, signal transducing adaptor molecule, is associated with Janus kinases and involved in signaling for cell
growth and c-myc induction.
Immunity.
6:
449-457
[Medline].
|
56.
|
Mudryj, M.,
S.W. Hiebert, and
J.R. Nevins.
1990.
A role for
the adenovirus inducible E2F transcription factor in a proliferation dependent signal transduction pathway.
EMBO (Eur.
Mol. Biol. Organ.) J.
9:
2179-2184
[Abstract].
|
57.
|
Birchenall-Roberts, M.C.,
Y.D. Yoo,
D.C. Bertolette III,
K.H. Lee,
J.M. Turley,
O.S. Bang,
F.W. Ruscetti, and
S.J. Kim.
1997.
The p120-v-Abl protein interacts with E2F-1
and regulates E2F-1 transcriptional activity.
J. Biol. Chem.
272:
8905-8911
[Abstract/Free Full Text].
|
58.
|
Wong, K.K.,
X. Zou,
K.T. Merrell,
A.J. Patel,
K.B. Marcu,
S. Chellappan, and
K. Calame.
1995.
v-Abl activates c-myc
transcription through the E2F site.
Mol. Cell. Biol.
15:
6535-6544
[Abstract].
|
59.
|
Zou, X.,
S. Rudchenko,
K. Wong, and
K. Calame.
1997.
Induction of c-myc transcription by the v-Abl tyrosine kinase
requires Ras, Raf1, and cyclin-dependent kinases.
Genes Dev.
11:
654-662
[Abstract].
|
60.
|
Ahmed, N.N.,
H.L. Grimes,
A. Bellacosa,
T.O. Chan, and
P.N. Tsichlis.
1997.
Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase.
Proc.
Natl. Acad. Sci. USA.
94:
3627-3632
[Abstract/Free Full Text].
|
61.
|
Brennan, P.,
J.W. Babbage,
B.M. Burgering,
B. Groner,
K. Reif, and
D.A. Cantrell.
1997.
Phosphatidylinositol 3-kinase
couples the interleukin-2 receptor to the cell cycle regulator
E2F.
Immunity.
7:
679-689
[Medline].
|
62.
|
Hirano, T..
1998.
Interleukin 6 and its receptor: ten years
later.
Int. Rev. Immunol.
16:
249-284
[Medline].
|
63.
|
Kawano, M.,
T. Hirano,
T. Matsuda,
T. Taga,
Y. Horii,
K. Iwato,
H. Asaoku,
B. Tang,
O. Tanabe,
H. Tanaka, et al
.
1988.
Autocrine generation and requirement of BSF-2/IL-6
for human multiple myelomas.
Nature.
332:
83-85
[Medline].
|
64.
|
Zhang, X.G.,
J.J. Gu,
Z.Y. Lu,
K. Yasukawa,
G.D. Yancopoulos,
K. Turner,
M. Shoyab,
T. Taga,
T. Kishimoto,
R. Bataille, and
B. Klein.
1994.
Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are
growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130.
J. Exp. Med.
179:
1337-1342
[Abstract].
|
65.
|
Cressman, D.E.,
L.E. Greenbaum,
R.A. DeAngelis,
G. Ciliberto,
E.E. Furth,
V. Poli, and
R. Taub.
1996.
Liver failure
and defective hepatocyte regeneration in interleukin-6-deficient mice.
Science.
274:
1379-1383
[Abstract/Free Full Text].
|
66.
|
Nakajima, K.,
T. Kusafuka,
T. Takeda,
Y. Fujitani,
K. Nakae, and
T. Hirano.
1993.
Identification of a novel interleukin-6 response element containing an Ets-binding site and a
CRE-like site in the junB promoter.
Mol. Cell. Biol.
13:
3027-3041
[Abstract].
|
67.
|
Nakae, K.,
K. Nakajima,
J. Inazawa,
T. Kitaoka, and
T. Hirano.
1995.
ERM, a PEA3 subfamily of Ets transcription
factors, can cooperate with c-Jun.
J. Biol. Chem.
270:
23795-23800
[Abstract/Free Full Text].
|
68.
|
Kovesdi, I.,
R. Reichel, and
J.R. Nevins.
1986.
Identification of a cellular transcription factor involved in E1A trans-activation.
Cell.
45:
219-228
[Medline].
|
69.
|
Ichiba, M.,
K. Nakajima,
Y. Yamanaka,
N. Kiuchi, and
T. Hirano.
1998.
Autoregulation of the Stat3 gene through cooperation with a cAMP-responsive element-binding protein.
J. Biol. Chem.
273:
6132-6138
[Abstract/Free Full Text].
|
70.
|
Kojima, H.,
K. Nakajima, and
T. Hirano.
1996.
IL-6-inducible complexes on an IL-6 response element of the junB promoter contain Stat3 and 36 kDa CRE-like site binding protein(s).
Oncogene.
12:
547-554
[Medline].
|
71.
|
Chaturvedi, P.,
S. Sharma, and
E.P. Reddy.
1997.
Abrogation of interleukin-3 dependence of myeloid cells by the v-src
oncogene requires SH2 and SH3 domains which specify activation of STATs.
Mol. Cell. Biol.
17:
3295-3304
[Abstract].
|
72.
|
Stahl, N.,
T.J. Farruggella,
T.G. Boulton,
Z. Zhong,
J.J. Darnell, and
G.D. Yancopoulos.
1995.
Choice of STATs and
other substrates specified by modular tyrosine-based motifs in
cytokine receptors.
Science.
267:
1349-1353
[Medline].
|
73.
|
Nakajima, K., and
R. Wall.
1991.
Interleukin-6 signals activating junB and TIS11 gene transcription in a B-cell hybridoma.
Mol. Cell. Biol.
11:
1409-1418
[Medline].
|
74.
|
Kim, H.,
T.S. Hawley,
R.G. Hawley, and
H. Baumann.
1998.
Protein tyrosine phosphatase 2 (SHP-2) moderates signaling by gp130 but is not required for the induction of
acute-phase plasma protein genes in hepatic cells.
Mol. Cell.
Biol.
18:
1525-1533
[Abstract/Free Full Text].
|
75.
|
Hiebert, S.W.,
M. Lipp, and
J.R. Nevins.
1989.
E1A-dependent trans-activation of the human MYC promoter is mediated by the E2F factor.
Proc. Natl. Acad. Sci. USA.
86:
3594-3598
[Abstract].
|
76.
|
Decker, T.,
P. Kovarik, and
A. Meinke.
1997.
GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression.
J. Interferon Cytokine Res.
17:
121-134
[Medline].
|
77.
|
Horvath, C.M.,
Z. Wen, and
J.E. Darnell Jr..
1995.
A STAT
protein domain that determines DNA sequence recognition
suggests a novel DNA-binding domain.
Genes Dev.
9:
984-994
[Abstract].
|
78.
|
Seidel, H.M.,
L.H. Milocco,
P. Lamb,
J.E. Darnell Jr.,
R.B. Stein, and
J. Rosen.
1995.
Spacing of palindromic half sites as
a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity.
Proc. Natl. Acad. Sci. USA.
92:
3041-3045
[Abstract].
|
79.
|
Shimizu, S.,
T. Hirano,
R. Yoshioka,
S. Sugai,
T. Matsuda,
T. Taga,
T. Kishimoto, and
S. Konda.
1988.
Interleukin-6
(B-cell stimulatory factor 2)-dependent growth of a Lennert's lymphoma-derived T-cell line (KT-3).
Blood.
72:
1826-1828
[Abstract].
|
80.
|
Chellappan, S.P.,
S. Hiebert,
M. Mudryj,
J.M. Horowitz, and
J.R. Nevins.
1991.
The E2F transcription factor is a cellular target for the RB protein.
Cell.
65:
1053-1061
[Medline].
|
81.
|
Nevins, J.R.,
S.P. Chellappan,
M. Mudryj,
S. Hiebert,
S. Devoto,
J. Horowitz,
T. Hunter, and
J. Pines.
1991.
E2F
transcription factor is a target for the RB protein and the cyclin A protein.
Cold Spring Harbor Symp. Quant. Biol.
56:
157-162
[Medline].
|
82.
|
Weintraub, S.J.,
C.A. Prater, and
D.C. Dean.
1992.
Retinoblastoma protein switches the E2F site from positive to negative element.
Nature.
358:
259-261
[Medline].
|
83.
|
Weintraub, S.J.,
K.N. Chow,
R.X. Luo,
S.H. Zhang,
S. He, and
D.C. Dean.
1995.
Mechanism of active transcriptional
repression by the retinoblastoma protein.
Nature.
375:
812-815
[Medline].
|
84.
|
Ihle, J.N..
1995.
The Janus protein tyrosine kinase family and
its role in cytokine signaling.
Adv. Immunol.
60:
1-35
[Medline].
|
85.
|
Ihle, J.N., and
I.M. Kerr.
1995.
Jaks and Stats in signaling by
the cytokine receptor superfamily.
Trends Genet.
11:
69-74
[Medline].
|
86.
|
Azam, M.,
C. Lee,
I. Strehlow, and
C. Schindler.
1997.
Functionally distinct isoforms of STAT5 are generated by
protein processing.
Immunity.
6:
691-701
[Medline].
|
87.
|
Wang, D.,
D. Stravopodis,
S. Teglund,
J. Kitazawa, and
J.N. Ihle.
1996.
Naturally occurring dominant negative variants of
Stat5.
Mol. Cell. Biol.
16:
6141-6148
[Abstract].
|