From the INSERM U564, 4 rue Larrey, Centre Hospitalier Universitaire Angers, 49033 Angers Cedex, France
Received for publication, October 11, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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Activated forms of STAT3 transcription factors
are often found in various cancers and tumor cell lines, indicating
that this signaling pathway is involved in tumorogenesis. At the
molecular level, STAT3 proteins function as transcriptional activators
and up-regulate several growth-promoting genes such as myc,
pim-1, or cyclin D1.
However, these transcription factors have also proapoptotic functions
and can activate the expression of the cell-cycle inhibitor p21waf1, suggesting that STAT3 can also block cell-cycle
progression and prevent abnormal cell proliferation. To reconcile these
observations, one would predict that the STAT3-mediated activation of
p21waf1 is lost during cell transformation. In this study, we
show that upon IL-6 stimulation of glioblastoma cells, STAT3 does not
activate the expression of the p21waf1 gene, whereas the
expression of the myc gene remains unaltered. Chromatin
immunoprecipitation experiments show that STAT3 and its cofactor
NcoA/SRC1a are effectively recruited to the p21waf1 promoter
but that this is not followed by the association of the CREB-binding
protein (CBP) histone acetylase and the type II RNA polymerase
as normally seen on the myc promoter. Whereas the PI-3K/Akt pathway is
constitutively activated in these cells, inactivation of this pathway
restores the loading of CBP and the RNA polymerase and the expression
of the p21waf1 gene without having any effect on
myc regulation. Moreover, this effect was recapitulated in
HepG2 cells expressing an activated form of the Akt kinase. In these
cells, the kinase blocked the STAT3-mediated expression of the
p21waf1 gene by inhibiting the recruitment of CREB-binding
protein and the type II RNA polymerase, without having any effects on
the loading of STAT3 and its cofactor NcoA/SRC1a. Together, these findings suggest that the phosphatidylinositol 3-kinase/Akt
pathway inhibits the transcriptional activation of the p21waf1
gene by STAT3 proteins without altering the regulation of the myc promoter.
STAT3 proteins are cytoplasmic transcription factors that are
phosphorylated upon activation, translocate into the nucleus, and
activate target genes. A large number of growth factors activates these
transcription factors since they play an important role in a wide
variety of physiological pathways such as cell proliferation, differentiation, and apoptosis. Among the
STAT1 family, STAT3 is the
only gene for which ablation leads to embryonic lethality (1), and
tissue-specific ablation of the transcription factor yields important
defects in hepatocytes, macrophages, keratinocytes, and thymic or
mammary epithelial cells (2, 3). Besides these normal functions, STAT3
participates in cellular transformation and tumorigenesis. Constitutive
activation of STAT3 has been reported in several primary cancers, in
tumor cell lines, and in many oncogene-transformed cells, and
inactivation of STAT3 in these cell lines leads to an inhibition of
cell proliferation (4-6). Moreover, a constitutively activated form of
STAT3 induces cell transformation, growth in soft agar, and tumors in
nude mice, further confirming the importance of the activated form
detected in tumors (7). Together, these observations raise important
questions concerning the gene program activated by STAT3 proteins,
because the target genes activated by its oncogenic form probably favor
cell transformation. Effectively, STAT3 activates several genes
involved in cell cycle progression such as fos, cyclin
D, myc or, pim-1, and up-regulates
antiapoptotic genes such as Bcl-2 and
Bcl-XL (7-11) Therefore, through a combined inhibition of
apoptosis and activation of cell-cycle progression, constitutively
activated forms of STAT3 are believed to participate in cell
transformation (4, 12). However, these observations are complicated by
the fact that STAT3 is involved in contradictory cell responses.
Conditional inactivation of STAT3 shows that it has proapoptotic
functions during mammary-gland involution (2) and that it inhibits
neutrophil proliferation (13). STAT3 activation leads to
down-regulation of myc genes and induction of
junB and IRF1, thereby inducing cell-cycle arrest
of monocytic cells (3). Inhibition of melanoma cells proliferation also
relies on STAT3 activity, and, importantly, STAT3 activates the
expression of the cell-cycle inhibitor p21waf1 (14-16). How a
single transcription factor can mediate such contradictory responses is
an important issue to be resolved because STAT3-mediated cell
transformation is probably related to a specific set of activated genes.
In general, high expression of p21waf1 inhibits the cdk2 kinase
to block cell-cycle progression (17). This predicts that this gene
should be a frequent target of mutations or inactivations in the
neoplastic process. Although p21waf1 mutations are extremely
rare, its promoter is effectively silenced during carcinogenesis
through CpG hypermethylation (18, 19). In light of these observations,
we hypothesized that STAT3 should be prevented from activating
p21waf1 in tumor cell lines. In this study, we report that
STAT3 does not activate the expression of the p21waf1 gene in
T98G glioblastoma cells because of the constitutive expression of the
PI3K/Akt signaling pathway. Using chromatin immunoprecipitation experiments, we found that STAT3 and its transcriptional cofactor NcoA/SRC1a are normally recruited to the promoter of p21waf1
but that the histone acetylase CBP and the type II RNA polymerase do
not associate with the promoter. Inactivation of PI3K/Akt reinduces the
binding of CBP, the binding of the polymerase, and the expression of
the p21waf1 mRNA. Importantly, the transcriptional events
leading to the activation of the myc gene remains normal in
glioblastoma cells, suggesting that the inhibition of the
p21waf1 gene is specific.
Reagents--
Polyclonal antibodies against STAT3 (C20),
phospho-STAT3-Tyr705, phospho-STAT3-Ser727,
NcoA/SRC1 (M-341), CBP (A-22), and type II RNA polymerase (N-20) were
obtained from Santa Cruz Biotechnology. Antibodies against phospho-Akt-Ser473 were from Cell Signaling Technology.
Transient Transfections and Preparation of Nuclear
Extracts--
Transfection experiments and nuclear extracts were done
as described before (16) using the calcium phosphate precipitation method and were repeated at least five times. The amount of transfected DNA was kept constant by addition of appropriate amounts of the parental empty, expression vector.
Electrophoretic Mobility-shift Assay, Western Blot, and
Northern Blot Analysis--
Nuclear extracts were preincubated for 5 min at room temperature in 25 mM NaCl, 10 mM
Tris pH 7.5, 1 mM MgCl2, 5 mM EDTA
pH 8, 5% glycerol, and 1 mM dithiothreitol with 1 µg of
poly(dI-dC) as a nonspecific competitor. Where indicated, 5-µg
extracts were preincubated for 1.5 h at 4 °C with 1 µg of
polyclonal antibodies (C20) directed against STAT3. 10 pg of probe
(5'-CATTTCCCGTAAATCTTGTCG-3', 20,000 cpm) was then added to the protein
mixture for 15 min followed by loading on a 5% polyacrylamide gel
(30:1) and separated by electrophoresis in 50 mM Tris, 0.38 M glycine, 1 mM EDTA (pH 8.5). Western blot and luciferase
assays were performed as described (16). Northern blots were performed
with 20 µg of RNA using either a human p21 or myc cDNA
probes labeled with [32P]dCTP using the random-priming
labeling kit from Amersham Biosciences (20).
Chromatin Immunoprecipitation Assay--
Cells, grown to 60%
confluence, were serum-starved for 2 days. After stimulation (20 min,
IL-6 20 ng/ml), cells were washed and then cross-linked with 1%
formaldehyde at room temperature for 10 min. Cells were washed
sequentially two times with one ml of ice-cold phosphate-buffered
saline, centrifuged, resuspended in 0.5 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin), and sonicated three times for 15 s each at the
maximum setting. Supernatants were then recovered by centrifugation at
12,000 rpm for 10 min at 4 °C, diluted 3-10 times in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM
NaCl, 20 mM Tris-HCl pH 8.1) and subjected to one round of
immunoclearing for 2 h at 4 °C with 2 µg of sheared
salmon-sperm DNA, 2.5 µg of preimmune serum, and 20 µl of protein
A-Sepharose (of 50% slurry). Immunoprecipitation was performed
overnight with specific antibodies, and then 2 µg of sheared
salmon-sperm DNA and 20 µl of protein A-Sepharose (of 50% slurry)
were further added for 1 h at 4 °C. Note that the CBP and
NcoA/SRC1a immunoprecipitations were performed respectively in the
presence of 1% Brij 97 or 1% Nonidet P-40. Immunoprecipitates were
washed sequentially for 10 min each in TSE I (0.1% SDS, 1% Triton
X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), and buffer III (0.25 M LiCl, 1%
Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1). Bead precipitates were then washed
three times with TE buffer and eluted three times with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated at
65 °C overnight to reverse the formaldehyde cross-linking.
Supernatants were then incubated for 1 h at 45 °C with
proteinase K (80 µg each), and DNA was precipitated using classical
procedures. For PCR, 10 µl from a 50-µl DNA preparation were used
for 30 cycles of amplifications. The following primers were used:
region The IL-6 Signaling Pathway Does Not Activate the Expression of the
p21waf1 Gene in Glioblastoma Cells--
To characterize STAT3
activity in glioblastoma cells, we first analyzed the expression of the
p21waf1 mRNA after IL-6 stimulation of glioblastoma T98G or
control cell lines. As previously reported (14-16), Northern blot
analysis showed that IL-6 induced a significant induction of the
p21waf1 mRNA in HepG2 and NGP cells (Fig.
1A, lanes 5 and
8). Surprisingly, IL-6 stimulation of T98G cells did not
lead to a detectable p21waf1 mRNA expression (Fig.
1A, compares lane 1 and 2).
Importantly, the same effect was also observed in different
glioblastoma cell lines (data not shown). As a control and as
previously reported (20), serum stimulation induced p21waf1
mRNA expression to the same extent in the HepG2 and T98G cell lines
(Fig. 1A, lanes 3 and 6). Equal
loading was verified by visualization of 18S ribosomal RNA expression
(Fig. 1A, bottom). To investigate whether the
regulation of p21waf1 mRNA expression occurred at the
transcriptional level, we monitored the expression of a p21waf1
promoter luciferase construct (20) after IL-6 or 10% serum stimulation. As shown in Fig. 1B, we found that the
p21waf1 promoter-driven luciferase activity was up-regulated
4-fold in HepG2 cells (Fig. 1B, lanes 3-4),
however, this activation was not observed in T98G cells after IL-6
stimulation (Fig. 1B, lanes 1-2) but remained functional
upon serum stimulation (Fig. 1B, lanes 5-6). To
extend these results, we evaluated the expression of the
p21waf1 protein in these cell lines. Using nuclear cell
extracts, we found that the p21waf1 protein was induced in
HepG2 cells after IL-6 stimulation (Fig. 1C, lanes
3-4). However, under the same conditions, this expression was not
detectable in T98G cells (Fig. 1C, lanes 1-2).
We conclude from these results that IL-6 stimulation did not activate
to a detectable level the expression of the p21waf1 promoter,
mRNA, and protein in T98G cells.
IL-6 Stimulation Induces the Expression of c-myc
mRNA and Promoter in T98G Cells--
To determine whether
STAT3-mediated gene activation was inhibited as a general phenomenon or
whether this observation was only related to the p21waf1 gene,
we evaluated the activation of myc, a second target gene of
the transcription factor (8). As previously reported, IL-6 induced a
significant induction of the c-myc mRNA in HepG2 cells, and the same effect was observed in T98G cells (Fig.
2A, lanes 2-5 and
7). Maximal c-myc mRNA expression was
observed after 1 h of IL-6 stimulation, a time during which the
expression of the p21waf1 mRNA was not detected in both
cell lines (data not shown). To confirm these results at the
transcriptional level, we monitored the expression of the
c-myc HBM promoter luciferase construct (21) after
IL-6 stimulation. As shown in Fig. 2B, the c-myc promoter activity was up-regulated to the same extent, 6- to 8-fold, in
T98G or HepG2 cells.
Altogether, these results suggest that STAT3 activates the
c-myc gene to the same extent in HepG2 and T98G cells,
suggesting that the inhibition of STAT3 signaling is specific to the
p21waf1 promoter.
IL-6 Activates STAT3 Phosphorylation and DNA-binding Activity in
T98G Cells--
To explain the absence of p21waf1 expression
in T98G cells, we first hypothesized that the signaling pathway leading
to STAT3 activation was modified. To this end, STAT3 nuclear
phosphorylation was analyzed in nuclear extracts of T98G cells as
compared with NGP and HepG2 control cell lines. After starvation and
IL-6 stimulation, STAT3 translocated to the nucleus and was
phosphorylated on Tyr705 and Ser727 in each
cell lines (Fig. 3, A and
B, lanes 3-4). Hence, neither the nuclear
localization nor the phosphorylation status of STAT3 was inhibited in
T98G cells. In addition, electrophoretic mobility-shift assay
experiments indicated that endogeneous STAT3 associated with DNA upon
IL-6 stimulation in T98G cells (Fig. 3C, lanes
1-2). The presence of STAT3 in the complex was verified
by supershift with a polyclonal STAT3 antibody (Fig. 3C,
lanes 5-6). Thus, we conclude from these results that STAT3
activation is functional in T98G cells.
Occupancy of the p21waf1 and myc Promoters by
STAT3, CBP, NcoA/SRC1a, and Type II RNA Polymerase--
Using
chromatin immunoprecipitation (ChIP), we have recently shown that the
transcriptional activity of STAT3 on the p21waf1 promoter
depends on the recruitment of the NcoA/SRC1a and CBP coactivators (16).
After STAT3 binding to DNA, the recruitment of a SRC1a-CBP complex is
believed to lead to the activation of STAT3-responsive genes. The
recruitment of these complexes was analyzed on the STAT3-responsive
regions of the p21waf1 (Fig.
4A) and myc (Fig.
4B) promoters in T98G cells. ChIPs experiments show that
STAT3 and NcoA/SRC1a were recruited to the p21waf1 and
c-myc promoters after IL-6 stimulation of glioblastoma cells (Fig. 4, A and B, lanes 4 and
6). As expected, the c-Myc occupancy by STAT3 and NcoA/SRC1a
was associated with the recruitment of CBP and the RNA polymerase II
(Fig. 4B, lanes 8 and 10). However, under the same conditions (i.e. the same extraction), STAT3
activation failed to induce any CBP or RNA polymerase II association
with the p21waf1 promoter (Fig. 4A, lanes
7-8 and 11-12). By contrast, serum stimulation induced the recruitment of CBP and RNA polymerase II to the
p21waf1 and myc promoters (Fig. 4C,
lanes 2 and 4 for p21waf1, lane
8 for myc). Finally, as a control for DNA sonication
efficiency, PCR analysis did not detect any increase in the STAT3
occupancy of a region 2 kb upstream of the STAT3-responsive region of
the p21waf1 promoter (Fig. 4A, lanes
13-22; Fig. 4C, lanes 5-6). Taken
together, these results suggest that STAT3 and NcoA/SRC1a are recruited to the promoter of the p21waf1 gene in T98G cells, however, the
CBP histone acetylase and the RNA polymerase II are not recruited. By
contrast, the recruitment of these complexes remains normal on the
myc promoter.
The Akt Pathway Inhibits STAT3 Transcriptional
Activity--
PTEN is a tumor suppressor that inhibits tumor
cell proliferation through inactivation of the PI3K/Akt signaling
pathway. Loss of heterozygosity at the 10q23 PTEN locus occurs
frequently in glioblastoma (22) and has been reported in T98G cells to induce the constitutive activation of Akt (23). In our hands, Western
blot experiments effectively confirmed that Akt is constitutively phosphorylated on Ser473 in T98G cells (Fig.
5A, lanes 1-2) and
that IL-6 did not further activate the kinase. By contrast, IL-6
stimulation was necessary to activate Akt in control HepG2 cells (Fig.
5A, lanes 3-4). Because Akt down-regulates STAT3
transcriptional activity (10, 24), we explored the possible role of
this kinase in the modification of STAT3 transcriptional activity in
T98G cells. To this end, we first confirmed in HepG2 cells the effect
of a constitutively active form of Akt (Akt-CA; Ref. 25) on STAT3
transcriptional activity. As previously reported (10, 24), we found
that Akt-CA inhibits the activity of a Gal4-STAT3 fusion protein (Fig.
5B, compare lanes 2 and 4). These
results suggested that Akt might regulate the STAT3 functions in T98G
cells. To test this hypothesis, we then monitored the effect of the
LY294002 pharmacological inhibitor of the PI3K/Akt pathway on the
STAT3-mediated regulation of the p21waf1 gene in T98G cells.
ChIP analysis indicated that treatment of T98G cells with LY294002
before IL-6 stimulation restored the association of CBP and the RNA
polymerase II with the p21waf1 promoter (Fig.
6A, compare lanes
1-2 and 9-10 with lanes 3-4 and
11-12). PCR analysis did not detect any increase in the
STAT3 occupancy of a control p21waf1 DNA region (Fig.
6A, lanes 5-8 and 13-16). We then
evaluated the activity of the p21waf1 promoter after IL-6
stimulation and LY294002 pretreatment. Results showed that PI3K/Akt
inhibition allowed a 3-fold activation of the p21waf1 promoter
in response to STAT3 activation, whereas it remained otherwise inactive
in the absence of drug pretreatment (Fig. 6B, compare
lanes 3 and 6). Further confirming these
observations, Northern blot experiments showed that inactivation of the
PI3K/Akt signaling pathway restored the expression of the
p21waf1 mRNA upon STAT3 activation, whereas this was not
observed under control conditions (Fig. 6C, compare
lanes 5 and 6). Finally, expression of the
p21waf1 protein was also recovered when STAT3 was activated
after LY294002 pretreatment (Fig. 6D, compare lanes
5 and 6). Importantly, inactivation of the PI3K/Akt
signaling pathway by LY294002 had no significant effect on the
expression of myc mRNA in T98G cells (Fig.
6C, lanes 7-11, compare the top and
middle panels). Moreover, the activity of the
p21waf1 promoter was not affected by the inhibitor in HepG2
cells (Fig. 6B, compare lanes 10 and
12).
Therefore, we concluded from these results that the PI3K/Akt signaling
pathway inhibits the activation of the p21waf1 gene but not of
myc in T98G cells. To further confirm these results, we made
the hypothesis that we should be able to recapitulate the inactivation
of the p21waf1 gene in HepG2 cells by using the constitutively
active form of Akt, Akt-CA. To this end, HepG2 cells were stably
transfected with an expression vector encoding the activated form of
Akt, Akt-CA, or the corresponding parental plasmid. Following G418 selection, Akt-CA was clearly overexpressed and phosphorylated in
Akt-CA expressing cells as compared with control clones (Fig. 7A, lanes 1-2). Given that
the PI3K/Akt signaling pathway inhibited the transcriptional activity
of STAT3 in T98G cells, we hypothesized that the expression of
p21waf1 should be down-regulated in Akt-CA-expressing clones.
Effectively, although IL-6 and 10% serum stimulation significantly
induced p21waf1 mRNA in control HepG2 cells (Fig.
7B, lanes 1-3), the STAT3-mediated induction was
not detected in CA-Akt-expressing cells (Fig. 7B, lanes 4-5). Importantly, the kinase did not affect the
serum-mediated activation of the p21waf1 gene, further
indicating the specificity of the kinase effects toward the STAT3
transcriptional activity. Equal loading was verified with control 18S
ribosomal RNA staining. ChIPs experiments were then performed to
determine whether the constitutive expression of the kinase could mimic
the observations made on the transcriptional cofactor recruitment in
T98G cells. As described for the glioblastoma cell line, STAT3 and
NcoA/SRC1a were recruited to the p21waf1 promoter after IL-6
stimulation to the same extent in control or in Akt-CA-expressing cell
lines (Fig. 7C, compare lanes 1-2 and
3-4, middle panels). Importantly, although STAT3
activation was associated with the recruitment of CBP and RNA
polymerase II in control cell lines, these proteins were not recruited
by STAT3 on the p21waf1 promoter in cells expressing the
constitutive form of the Akt kinase (Fig. 7C, compare
lanes 1-2 and 3-4, bottom panel, and compare lanes 5-6 and 7-8). Therefore, we
conclude from these results that Akt blocks the STAT3-mediated
activation of the p21waf1 gene by inhibiting the recruitment of
the histone acetylase CBP and the RNA polymerase II to the
promoter.
STAT3 proteins are mediators of cell survival and proliferation
that play important roles in cellular transformation and tumorigenesis (4-6). Therefore, it is important to determine the subset of target
genes that are differentially activated by the oncogenic form of the
transcription factor as opposed to the genes activated by STAT3 during
normal growth conditions. One could predict that oncogenic forms of
STAT3 activate growth-promoting genes either directly involved in
cell-cycle progression or in prosurvival functions. However, STAT3 has
also been reported to activate the expression of p21waf1, which
inhibits cell-cycle progression when highly expressed. Therefore, this
inhibitor theoretically should block the effect of the oncogenic form
of STAT3 on cell proliferation. In this study, we have shown that the
activation of p21waf1 by STAT3 is inhibited by the
constitutively activated form of Akt present in T98G cells. Moreover,
these results were extended to a different model, because HepG2 cells
that overexpress a constitutively activated form of the kinase do not
activate the expression of p21waf1 upon STAT3 activation.
Importantly, activation of Akt is a common event in glioblastomas as a
consequence of LOH at 10q23, the locus of the PTEN phosphatase.
Confirming this study, STAT3 activation of p21waf1 is also lost
during progression of human malignant melanomas (26), whereas Akt
confers resistance to the antiproliferative effect of IL-6 on these
cells (27). Finally, the regulation of the myc gene remained
unaltered in T98G cells, suggesting that Akt specifically targets the
inhibitory functions of STAT3 on cell-cycle progression.
A few possibilities can be raised concerning the molecular mechanisms
whereby activation of the p21waf1 gene is inhibited. First,
methylation of the p21waf1 proximal promoter could lead to
silencing of the gene. We consider this possibility unlikely because
mRNA expression is induced after serum stimulation, and preliminary
experiments in our laboratory indicate that the promoter is not
methylated in T98G cells. Alternatively, we have recently shown that
cyclin D1 inhibits the transcriptional activity of STAT3, and it is
known that Akt activates the expression of the cyclin through protein
stabilization (28). Therefore, the effect of Akt could be indirect and
mediated through an enhanced interaction between cyclin D1 and STAT3.
Interestingly, both cyclin D1 and Akt affect the activation domain of
STAT3, and both proteins have the same effect on the recruitment of
STAT3 transcriptional cofactors
(29).2 ChIP experiments are
actually conducted to determine whether cyclin D1 is present on the
promoter of the p21waf1 gene but not on myc in
glioblastoma cells.
It remains also to be determined why Akt inhibits the activity of STAT3
on the p21waf1 without having any effect on the myc
gene. Most enhancers contains DNA-binding sites for multiple
transcription factors so that differential gene expression is activated
by the combination of the corresponding proteins. The presence or
absence of these factors and their regulated association in a
three-dimensional complex maintained by architectural proteins
(enhanceosome) achieve a specific pattern of gene expression. For
instance, viral induction of the interferon- Altogether, these results elucidate a new pathway induced by oncogene
activation that would displace the transcriptional activity of STAT3
toward the activation of growth-promoting genes at the expense of
cell-cycle inhibitory proteins. We propose that this could be a
necessary step in the activation of the STAT3 oncogenic potential.
Inactivation of p21waf1 would remove a major inhibitory
signaling pathway responsible for cell-cycle arrest, so that oncogenic
STAT3, in cooperation with other oncogenes such as Akt, could activate
only growth-promoting genes (Fig. 8).
Because this hypothesis is for the moment based on the regulation of
only two genes, further experiments are needed to determine whether
these observations are related to a general phenomena or are only
specific to the myc and p21waf1 promoters.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
879 to
593 of the p21waf1 promoter (STAT3 and
CBP binding), 5'-TTCAGGAGACAGACAACTCACTCG-3'(forward primer),
5'-GACACCCCAACAAAGCATCTTG-3'(reverse primer); region
262 to
70
of the p21waf1 promoter (RNA polymerase II binding),
5'-GAACTGACTTCGGCAGCTGC-3'(forward primer),
5'-CCGGCTGGCCTGCTGGAACT-3'(reverse primer), region
2760 to
2486 of
the p21waf1 promoter (control), 5'-TTGTGCCACTGCTGACTTTGTC-3'
(forward primer), 5'-AGCCTGAAGAAGGAGGATGTGAGG-3' (reverse primer);
region
333 to
44 (relative to P2) of the myc promoter
(binding of all proteins tested in this study),
5'-CCGAAAACCGGCTTTTATAC-3'(forward primer), 5'-CTGAGTCTCCTCCCCACCTT-3'
(reverse primer).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
T98G glioblastoma cells are refractory to
STAT3 activation of the p21waf1 gene. A, Northern
blot analysis of p21waf1 expression in T98G (lanes
1-3), HepG2 (lanes 4-6), or NGP cells (lanes
7-8). Cells were serum-starved for 2 days and stimulated for
6 h with IL-6 (20 ng/ml) or 10% serum as indicated. Total RNA was
prepared, and 20 µg of RNA was subjected to Northern blot analysis
using a human cDNA probe. The membrane was stripped and reprobed
with a 18S oligonucleotide (bottom panel).
B, cells were transfected with 500 ng of a vector encoding
the p21waf1 promoter linked to a luciferase reporter gene.
After transfection, cells were serum-starved for 24 h and
stimulated with IL-6 (20 ng/ml, lanes 2, 4) or
10% serum (lane 6) for 6 h. Cytoplasmic extracts were
then prepared and processed to measure luciferase activity. The mean of
5 transfections is shown. C, T98G or HepG2 cells were
serum-starved for 48 h and stimulated with IL-6 (20 ng/ml,
lanes 2, 4) for 15 h. After stimulation,
nuclear extracts were prepared, and p21waf1 expression was
analyzed by Western blot with polyclonal antibodies directed against
the protein.
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Fig. 2.
IL-6 activates the expression of the
c-myc gene in T98G glioblastoma cells. A,
Northern blot analysis of c-myc expression in T98G
(lanes 1-5) or HepG2 cells (lanes 6-8) after
IL-6 stimulation. B, cells were transfected with 500 ng of
the HBM-Luc vector encoding the myc promoter linked to a
luciferase reporter gene. After transfection, cells were serum-starved
for 24 h and stimulated with IL-6 (20 ng/ml, lanes 2,
4) for 6 h. Cytoplasmic extracts were then prepared and
processed to measure luciferase activity. The mean of 5 transfections
is shown.
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Fig. 3.
Phosphorylation and DNA-binding activity of
STAT3 in T98G cells. T98G, HepG2 or NGP cells were serum-starved
for 2 days and stimulated for 20 mn with IL-6 (20 ng/ml). A
and B, after stimulation, nuclear cell extracts were
prepared and activation of STAT3 was analyzed using antibodies directed
against STAT3 (Fig. 3A, bottom panel),
its Tyr705 (Fig. 3A, top panel), or
Ser727 (Fig. 3B), phosphorylated forms.
C, STAT3 DNA-binding activity present in T98G cells was
determined by electrophoretic mobility-shift assay after IL-6
stimulation as described above (lanes 2, 3,
5, and 6). Supershift of STAT3 was performed
(lane 5) with the addition of 2 µg of polyclonal STAT3
antibodies. Note that the gel was run for a longer time on lanes
3-6 to detect the supershift so that the free probe is no longer
apparent.
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Fig. 4.
ChIPs analysis of the recruitment of STAT3,
NcoA/SRC1a, CBP, and RNA polymerase II on the p21waf1 and
myc promoters. Soluble chromatin was prepared
from cells treated with IL-6 or untreated for 10-20 min and
immunoprecipitated with the indicated antibodies. Final DNA extractions
were amplified using two pairs of primers that cover the STAT3
(A, lanes 1-8) and RNA polymerase II binding
sites of p21waf1 (A, lanes 9-12), or one
pair of primers that covers both sites on the myc promoter
(B, lanes 1-10). As a control, a distal region
of the p21waf1 promoter was analyzed (A, lanes
13-22 and C, lanes 5-6). Amplifications
shown in C were done with the same pair of primers,
respectively, except that the cells were stimulated with 10% serum.
Note that RNA polymerase II association was detected after 20 min of
IL-6 or serum stimulation, whereas the other complexes are present as
early as 10 min.
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Fig. 5.
Akt inhibits the transcriptional activity of
a Gal4-STAT3 fusion protein. A, T98G or HepG2 cells were
serum-starved for 2 days and stimulated or untreated for 30 min with
IL-6 (20 ng/ml). Nuclear extracts were prepared and activation of Akt
was analyzed using antibodies directed against Akt (A,
lanes 1-2 top), or its
Ser473-phosphorylated form (A, lanes
1-2 bottom and lanes 3-4). B,
HepG2 cells were co-transfected with vectors expressing a Gal4
luciferase reporter gene (100 ng) together with vectors expressing a
Gal4 fusion protein linked to the STAT3 activation domain (300 ng,
lanes 2 and 4, with a plasmid encoding an
activated form of the Akt kinase (Akt-CA, lanes 3-4). After
transfection, cells were serum-starved for 24 h and stimulated for
6 h with IL-6 (20 ng/ml). Cytoplasmic extracts were then prepared
and processed to measure luciferase activity. The mean of 5 transfections is shown.
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Fig. 6.
Inhibition of the PI3K/Akt-signaling pathway
restores the activation of p21waf1 in T98G cells. A,
serum-starved T98G cells were pretreated for 1 h with LY294002 (50 µM) or with Me2SO (vehicle) and were then
stimulated with IL-6 for 10-20 min. The association of CBP
(lanes 9-16) and RNA polymerase II (lanes 1-8)
on the p21waf1 promoter was then analyzed by ChIPs analysis as
described above. B, T98G (lanes 1-6) or HepG2
cells (lanes 7-12) were transfected with 500 ng of a vector
encoding the p21waf1 promoter linked to a luciferase reporter
gene. Cells were then serum-starved for 24 h, pretreated for
1 h with LY294002 (lanes 3, 6, 9,
and 12; 50 µM) or with Me2SO
(vehicle, lanes 2, 5, 8,
and 11) and were then stimulated with IL-6 for 6 h.
Cytoplasmic extracts were then prepared and processed to measure
luciferase activity. The mean of 5 transfections is shown.
C, Northern blot analysis of the expression of the
p21waf1 (lanes 1-6) or myc mRNAs
(lanes 7-11) after IL-6 stimulation of T98 G cells, in the
absence (lanes 1-2, 4-5, lanes 7-11
middle panel) or presence (lanes 3 and
6, lanes 7-11 top panel) of LY294002
pretreatment. D, Western blot analysis of the expression of
the p21waf1 protein. T98G cells were serum-starved for 48 h and stimulated with IL-6 (20 ng/ml, lanes 4-6) for
15 h in the absence (lanes 1-2, 4-5) or
presence (lanes 3, 6) of LY294002. After stimulation,
nuclear extracts were prepared and p21waf1 expression was
analyzed by Western blot with polyclonal antibodies directed against
the protein. Equal loading was verified using antibodies directed
against tubulin.
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Fig. 7.
Akt inhibits the STAT3-mediated activation of
p21waf1 in HepG2 cells. A, HepG2 cells were stably
transfected with an expression vector encoding a constitutively
activated form of Akt (Akt-CA, lane 2) or the corresponding
parental plasmid (lane 1). After G418 selection,
overexpression was verified using nuclear extracts analyzed by Western
blot and polyclonal antibodies against the
Ser473-phosphorylated form of the kinase. B,
HepG2 cells described in A were serum-starved for 48 h
and then stimulated with IL-6 (10 ng/ml, lanes 2 and
5) or 10% serum for 4 h. Northern blot analysis of
p21waf1 expression was then performed in control clones
(lanes 1-3) or in Akt-CA-expressing cells (lanes
4-6). C, soluble chromatin was prepared from stable
cell lines treated with IL-6 or untreated and immunoprecipitated with
the indicated antibodies. Final DNA extractions were amplified using
pairs of primers that cover the STAT3 (lanes 1-4) and RNA
polymerase II-binding sites (lanes 5-8) of the
p21waf1 promoter as described above.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gene requires the
coordinate activation of NF
B, interferon regulatory factors, and ATF-2/c-Jun dimers with the architectural protein HMGI(Y) to form
an enhanceosome that recruits the transcriptional machinery (30-33).
Such an enhanceosome remains to be characterized in the case of the
STAT3-regulation of the p21waf1 and myc genes.
However, STAT3 cooperates with c-jun to achieve a maximal
expression of the
2-macroglobulin gene (34, 35), and the same
complex regulates transcription from the Fas promoter. Interestingly,
in this different experimental model, the STAT3-jun complex is also
inhibited by the PI3K/Akt-signaling pathway (10, 24). In light
of these observations, it is tempting to speculate that the regulation
of the p21waf1 promoter relies on the formation of a
c-Jun-STAT3 heterodimer that is negatively regulated by Akt. By
contrast, the activation of myc would not rely on such a
complex. Alternatively, we also found that Akt blocks the
STAT3-mediated activation of the p21waf1 gene by inhibiting the
recruitment of CBP and the RNA polymerase II to the promoter, whereas
the loading of STAT3 and its cofactor NcoA/SRC1a is not modified. The
NcoA/SRC1a coactivator is thought to contribute to transcriptional
activation by recruiting the CBP histone acetylase to transcription
factors. Activation of the p21waf1 gene probably relies on the
recruitment of a SRC1a-CBP complex that initiates the loading of the
initiation complex, either through histone acetylation and chromatin
remodeling or through a direct contact with the RNA helicase A/RNA
polymerase II complex (36). We can speculate that the PI3K/Akt
signaling pathway would block the association between NcoA/SRC1a and
CBP on the p21waf1 promoter, thereby releasing the histone
acetylase and inhibiting the association with the polymerase complex on
the p21waf1 promoter. Finally, it is also possible that the
PI3K/Akt signaling pathway activates a transcriptional repressor that
would inhibit only the p21waf1 promoter without having any
effect on the myc gene. Alternatively, these kinases could
also target an unknown transcription factor activated by IL-6 that
would be different from STAT3 and that would specifically regulate the
p21waf1 gene but not the myc promoter.
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Fig. 8.
Proposed model for STAT3-mediated activation
of the p21waf1 gene during carcinogenesis. Under normal
conditions, the STAT3-mediated activation of the p21waf1 and
myc genes relies on the formation of a STAT3/SRC1a/CBP
complex that participates in the promoter loading of the type II RNA
polymerase. After constitutive activation of the PI3K/Akt signaling
pathway, the association of CBP with STAT3/SRC1a is inhibited and the
RNA polymerase II does not bind to the p21waf1 promoter
anymore. By contrast, the regulation of the myc gene remains
normal. The expression of p21waf1 is prevented, which might
lead to an enhanced proliferation.
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Brunet, M.E Greenberg, L. Penn, and R. A Roth for the gift of various expression vectors.
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FOOTNOTES |
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* This work was supported by a grant from the Ligue Nationale Pour la Recherche Sur le Cancer as a "Equipe Labelisée La Ligue Contre le Cancer."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.
To whom correspondence should be addressed. Tel.:
33-2-41-35-47-33; Fax: 33-2-41-73-16-30; E-mail:
olivier.coqueret@univ-angers.fr.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M210422200
2 F. Bienvenu and O. Coqueret, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: STAT, signal transducers and activators of transcription; ChIP, chromatin immunoprecipitation; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; PTEN, phosphatase and tensin homolog deleted on chromosome ten; IL, interleukin; Akt-CA, constitutively active Akt; PI3K, phosphatidylinositol 3-kinase.
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