From the Institut für Biochemie, RWTH Aachen, 52057 Aachen,
Germany, the ¶ Institut für Pharmakologie und Toxikologie,
RWTH Aachen, 52057 Aachen, Germany, the Department of Molecular
Pharmacology, Stanford University School of Medicine, Stanford,
California 94305, the § Molecular Oncology Program, H. Lee
Moffitt Cancer Center and Research Institute, Tampa, Florida 33613, the
** Deutsches Institut für Ernährungsforschung,
D-14558 Potsdam-Rehbrücke, Germany, and the
Abteilung für Endokrinologie,
Heinrich-Heine-Universität,
D-40225 Düsseldorf, Germany
Received for publication, May 31, 2002, and in revised form, October 24, 2002
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ABSTRACT |
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The phosphatidylinositol 3-kinase/Akt pathway
plays an important role in the signaling of insulin and other growth
factors, which reportedly attenuate the interleukin-6 (IL-6)-mediated
stimulation of acute phase plasma protein genes. We investigated the
effect of the protein kinase Akt on IL-6-mediated transcriptional
activation. The transient expression of constitutively active Akt
inhibited the IL-6-dependent activity of the
IL-61 is the major
regulator of acute phase protein (APP) synthesis by the liver during
the inflammatory response (1). It exerts its actions through binding to
the receptor complex consisting of a ligand-specific IL-6R The STAT3-dependent action of IL-6 appears to be modulated
by a variety of stimuli, including insulin and epidermal growth factor
that have been reported to inhibit the APP production by cultured
hepatic cells (4, 5). The potential inhibitory role of growth promoting
signals on the IL-6-inducible Jak/STAT3 pathway corresponds well with
the suppressed acute phase response in regenerating liver (6). However,
the mechanisms whereby growth factors mediate this effect remained
unclear. One of the major effects of signaling via the insulin receptor
and other growth factor receptors is the activation of PI 3-kinase (7). Generation of PI 3-phosphorylated lipids in the plasma membrane leads
to phosphorylation and activation of the serine/threonine kinase Akt
(also called protein kinase B) by
phosphatidylinositol-dependent kinase 1 (PDK1).
Activated Akt has been described to translocate to the nucleus
(8) and to directly phosphorylate members of the forkhead family of
transcription factors (9-11). Phosphorylation of FKHR or closely
related FKHRL1 and AFX by Akt results in their transcriptional
inactivation and retention in the cytoplasm (9, 12).
In the present study, we investigated a potential cross-talk between
the PI 3-kinase/Akt signaling and the IL-6-inducible Jak/STAT3 pathway.
We have identified FKHR as a specific transcriptional coactivator of
STAT3. This functional interaction reflects the association of both
proteins and their colocalization in nuclear regions of HepG2 cells.
Cytokines and Reagents--
Recombinant human IL-6 (2 × 106 B-cell stimulatory factor-2 units/mg) together with
soluble human IL-6 receptor was used as described previously (13).
Recombinant insulin, tumor growth factor- Plasmids--
Wild type Akt, constitutively active Akt
(iSH2Akt) in pECE, pSVL-STAT3, pGL3- Transient Transfections and Luciferase Reporter Gene
Assays--
HepG2 cells were transiently transfected using FuGene6
reagent (Roche Molecular Biochemicals) or by the calcium phosphate method as described previously (16). 24 h after transfection, cells were stimulated with cytokines for another 18 h. Luciferase activities were determined with the Luciferase Assay System (Promega), and the data were normalized according to co-expressed
Northern Blots--
Total RNA was extracted from HepG2 cells
with the RNeasy kit from Qiagen. 10 µg of RNA were separated by 1%
agarose gel electrophoresis and transferred onto nylon membranes
(Nytran; Schleicher and Schüll). Detection with the
32P-labeled Cell Fractionation, Immunoprecipitation, and Western Blot
Analysis--
Nuclear extracts were prepared as described (13) with
modifications. Cells were lysed in hypotonic buffer A (10 mM Hepes/KOH, pH 7.9, 1.5 mM MgCl2,
10 mM KCl, 1 mM Na3VO4,
0.2 mM phenylmethylsulfonyl fluoride) for 10 min at 4 °C
and centrifuged at 300 × g for 2 min at 4 °C. The
crude nuclei were incubated in lysis buffer B (1% (w/v) BRIJ-97, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM sodium fluoride, 1 mM EDTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin) for 30 min at
4 °C. Short sonication and centrifugation at 14,000 × g for 2 min at 4 °C yielded the nuclear extracts. Total
cellular lysates were prepared by direct lysis of the harvested cells
in buffer B and processed as described above. Protein extracts were
incubated overnight at 4 °C with polyclonal antiserum against STAT3
(C-20), SMAD2/3 (N-19), or extracellular signal-regulated kinase 2 (C-14; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or monoclonal antibodies against STAT5 (MGF; Transduction Laboratories). The precipitates were later collected with Protein A-Sepharose (Amersham Biosciences), washed three times with lysis buffer, and resolved on
7.5% SDS-PAGE gels. After transfer to polyvinylidene difluoride membranes (GelmanSciences), the blots were probed with the respective antibodies and detected for signals using the ECL system (Amersham Biosciences).
Akt Kinase Assay--
Cells were lysed with lysis buffer (50 mM Hepes, pH 7.6, 150 mM NaCl, 10% (v/v)
glycerol, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM
Na3VO4, 30 mM
Na4P2O7, 10 mM NaF, 1 mM EDTA, 1 mM dithiothreitol, 100 nM ocadaic acid), and the Akt activity was assayed as
described previously (19). The phosphopeptide spots were quantified
using a Cyclone phosphor imager and the Optiquant software (Packard).
Immunocytochemistry and Confocal Fluorescence
Microscopy--
Cells grown on coverslips to subconfluence were
serum-deprived for the last 12 h, fixed, and permeabilized as
described before (20). For intracellular staining, polyclonal
anti-STAT3 antibodies (C-20; Santa Cruz Biotechnology) or rabbit
antiserum against FKHR and then secondary donkey polyclonal fluorescein
isothiocyanate- or rhodamine-coupled antibodies were used (Santa Cruz
Biotechnology). After mounting, fluorescence images were visualized by
confocal laser-scanning microscopy (LSM 510; Apochromat ×63 objective
lens; Zeiss). Argon and helium-neon lasers were switched between the excitation wavelengths for fluorescein ( Activation of PDK1/Akt Signaling Inhibits the
IL-6-mediated Gene Activation through a FKHR-dependent
Mechanism--
In agreement with previous studies (4, 5), we found
that insulin inhibits the IL-6-induced expression of several acute phase plasma proteins, including
The protein kinase Akt is known to directly phosphorylate and inhibit
transcription factors of the forkhead family (9, 21). Therefore, we
asked whether FKHR (FOXO1a), a forkhead transcription factor that has
been reported to be regulated in an insulin-dependent manner in hepatic cells (22), is involved in
The inactivation of FKHR by Akt results from a direct phosphorylation
of three regulatory sites Thr24, Ser256, and
Ser319 (10, 23, 24), which in turn leads to cytoplasmic
retention of FKHR (9, 12). In order to investigate the influence of FKHR phosphorylation on IL-6 Does Not Induce Akt Activity and FKHR Phosphorylation in HepG2
Cells--
IL-6 has been reported to activate Akt in Hep3B and
different cells from myeloma patients (25, 26). In this case, IL-6 stimulation should result in nuclear translocation of activated STAT3
and concomitant repression and nuclear exclusion of FKHR. However,
whereas insulin induced a robust and sustained activation of Akt, IL-6
did not significantly stimulate Akt kinase activity over its basal
level in HepG2 cells (Fig.
3A). The low Akt activity after IL-6 stimulation was reflected by the lack of its phosphorylation on two critical regulatory residues, Thr308 and
Ser473 (Fig. 3B). This also correlated with the
low phosphorylation status of Thr24 and Ser256,
two residues of FKHR, which become directly targeted by Akt (Fig.
3C). In contrast, Akt and FKHR were both strongly
phosphorylated after treatment of the cells with insulin, and their
activation was not significantly affected by the presence of IL-6.
However, as expected, IL-6 strongly induced tyrosine phosphorylation of STAT3, the major transcription factor mediating
IL-6-dependent signal transduction (2, 3) (Fig.
3D). The pattern of Akt, FKHR, and STAT3 phosphorylation in
HepG2 cells after 16 h of IL-6 and/or insulin treatment was
qualitatively similar, although the phosphorylation of STAT3 was less
prominent (data not shown). We conclude that in HepG2 cells, IL-6
cannot induce Akt activity and consequently does not repress FKHR.
Functional Interaction between FKHR and STAT3 in Transcriptional
Regulation--
In order to clarify the role of FKHR for the
regulation of the
The stimulatory effect of FKHR on the The Complete C Terminus of FKHR Is Crucial for the Coactivation of
IL-6-dependent Gene Expression--
The PAX3-FKHR fusion
protein from alveolar rhabdomyosarcoma was shown to possess a strong
transactivation domain localized in its C-terminal FKHR-derived part
(29, 30). We found that the complete C-terminal part of FKHR was also
required for its effect on the FKHR and STAT3 Colocalize in the Nuclei of HepG2 Cells--
In
order to estimate if both proteins may interact in vivo, we
assessed the localization of endogenous STAT3 and FKHR in HepG2 cells.
We found by indirect immunofluorescence and confocal microscopy that in
unstimulated cells the localization of FKHR (Fig.
5, right panels)
was predominantly nuclear, whereas STAT3 (Fig. 5, left panels) was distributed equally in the nuclear and
cytoplasmic regions. It is noteworthy that, although STAT3 was present
in the nuclei of untreated HepG2 cells, it did not bind DNA as measured in gel shift experiments (data not shown). Consistent with our previous
observations, IL-6 stimulation did not affect FKHR localization but
induced STAT3 migration to the nucleus, so that both factors accumulated in the nuclear regions of IL-6-treated HepG2 cells. In
contrast, insulin treatment with or without IL-6 costimulation led to
partial nuclear exclusion of FKHR. However, this effect was
significantly weaker in HepG2 cells than in HeLa cells used as positive
controls (27) (data not shown). Taken together, these results suggest
that FKHR might associate with STAT3 in the nuclei of IL-6-stimulated
HepG2 cells.
Physical Association of FKHR and STAT3--
Since FKHR
specifically contributes to the activation of
STAT3-dependent promoters, we investigated whether this
effect reflects a physical interaction between these two proteins.
Western blot analysis indicated that FKHR was expressed in HepG2 cells
at a low level and could be visualized only in highly concentrated total cellular lysates (Fig. 3C; compare to Figs.
2D and 6A). In contrast, FKHR was easily
detectable in immunoprecipitates obtained with anti-STAT3 antibodies
from total cell lysates of HepG2 cells (Fig.
6A), indicating the
association of both proteins. The reverse immunoprecipitation
experiments performed using antiserum against FKHR yielded similar
results and led to coprecipitation of STAT3 from total cellular
lysates, confirming the previous results (Fig. 6A). To
further consolidate the specificity of the interaction between FKHR and
STAT3, we carried out several additional control immunoprecipitations.
As shown in Fig. 6B, FKHR did not associate with STAT5 or
other unrelated signaling molecules like SMAD2/3 or extracellular
signal-regulated kinase 2. This allows us to conclude that the
functional interaction of FKHR and STAT3 reflects a specific binding of
both proteins. Although STAT3 and FKHR were found to be associated also
in total cell lysates from untreated cells, stimulation with IL-6
strongly enhanced the binding of both factors in the nuclear fraction
of HepG2 cells (Fig. 6C). Furthermore, in agreement with our
results on insulin-mediated inhibition of FKHR Interacts with STAT3 and Augments IL-6-dependent
Gene Expression--
The major finding of the present study is that
FKHR (FOXO1a), a member of the forkhead family of transcription
factors, can augment IL-6-dependent transcriptional
activity by interacting with STAT3. This conclusion is based on three
lines of evidence. First, FKHR expression enhanced the
IL-6-dependent activation of the
Our results suggest that FKHR can act as a coactivator in
STAT3-mediated transcriptional activation of acute phase protein genes.
Since FKHR did not significantly induce basal activity of the
FKHR was originally identified in human rhabdomyosarcomas as a fusion
protein composed of the transactivation domain of FKHR combined with
the intact DNA binding domain of the transcription factor PAX3 (40).
The C-terminal half of FKHR turned out to be very potent in
transcriptional activation, although PAX3-FKHR proteins showed impaired
DNA binding (41). Our results indicate that the C-terminal part of FKHR
was also required for its effect on the
Plausible mechanisms of the costimulatory action of FKHR on
STAT3-mediated gene expression can include 1) enhanced STAT3
activation, 2) facilitated nuclear migration of STAT3, and 3)
recruitment of additional coactivator proteins to the STAT3
transcriptional complex. Enhanced STAT3 activation does not seem
likely, since gel shift experiments did not reveal an effect of FKHR on
DNA-binding activity of STAT3 (data not shown). Our immunofluorescence
results do not support the second possibility of facilitated
translocation of STAT3 to the nucleus in the presence of FKHR. We did
not observe any effect of insulin on IL-6-induced nuclear STAT3
staining. Another possibility could be that FKHR augments IL-6-mediated signaling by recruiting additional coactivator molecules to the STAT3-containing transcriptosome. In HepG2 cells, FKHR has recently been shown to interact with the coactivator, p300/CBP, of the constitutive transcription machinery (31). Interestingly, STAT3 has
also been found to associate with p300/CBP via its carboxyl terminus.
This interaction as well as the level of transactivation are relatively
weak compared with other STATs such as STAT2 (32). Therefore, the
transcriptional function of STAT3 might be reinforced by FKHR acting as
an accessory factor directing coactivator complexes to STAT3 binding
sites in the promoter of respective target genes.
Activation of PDK1/Akt Signaling Modulates IL-6-mediated
Gene Expression--
The present results suggest a novel mechanism of
the modulation of IL-6-dependent gene expression by the PI
3-kinase/Akt signaling pathway. We have demonstrated that
down-regulation of Akt activity by a kinase-defective mutant of PDK1
and constitutive activation of Akt induce opposite effects on the
IL-6-responsive
Phosphorylation of FKHR by Akt on Thr24,
Ser256, and Ser319 was reported to attenuate
its nuclear import possibly by binding of FKHR to 14-3-3 proteins (9,
12, 27). Several groups demonstrated the resistance of FKHR
threonine/serine mutants to both Akt-mediated phosphorylation and PI
3-kinase-stimulated nuclear export (23, 24). This could well explain
the enhanced potency of the mutated FKHR variants T24A, T24A/S256A, and
T24A/S256A/S319A to augment IL-6-induced Conclusions--
In the present study, we have investigated the
potential cross-talk between the PI 3-kinase/Akt and Jak/STAT3
signaling pathways at the level of transcriptional regulation. Our
results reveal a novel function of FKHR, suggesting that it acts as a
specific coactivator of STAT3. This functional interaction correlates
with the physical association of both proteins and their colocalization in the nuclear regions of human HepG2 hepatoma cells. Taken together, our data demonstrate that activation of the PI 3-kinase/Akt pathway can
modulate IL-6 signaling by targeting and inactivating FKHR. This
can apply to APP genes like the 2-macroglobulin promoter in HepG2 cells,
whereas expression of an inactive mutant of
phosphatidylinositol-dependent kinase 1 had the opposite
effect. Since Akt is known to regulate gene expression through
inactivation of the transcription factor FKHR
(forkhead in rhabdomyosarcoma), we
examined the effect of FKHR on STAT3-mediated transcriptional
regulation. Indeed, the overexpression of FKHR specifically enhanced
the activity of STAT3-dependent promoters but not
that of a STAT5-responsive promoter. The effect of FKHR required the
presence of functional STAT3 and was abrogated by the expression of
dominant negative STAT3 mutants. Furthermore, FKHR and STAT3 were shown
to coimmunoprecipitate and to colocalize in the nuclear regions of
IL-6-treated HepG2 cells. Our results indicate that FKHR can modulate
the IL-6-induced transcriptional activity by acting as a coactivator of STAT3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain
(gp80) and two signal-transducing
-subunits (gp130). Activation of
the gp130-associated Janus kinases Jak1, Jak2, and Tyk2 results in the
tyrosine phosphorylation of several cellular substrates, including
signal transducer and activator of transcription 3 (STAT3), the major
mediator of IL-6-induced signaling (2, 3). Phosphorylated STAT3
dimerizes and translocates to the nucleus, where it regulates the
transcription of multiple target genes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and interferon-
were
from Roche Molecular Biochemicals, R&D Systems, and PeproTech;
erythropoietin was a kind gift of J. Burg and K.-H. Sellinger
(Roche Molecular Biochemicals). LY294002 was from Calbiochem.
Polyclonal rabbit FKHR antiserum was generated using purified
glutathione S-transferase-FKHR as antigen.
2-M-215luc, pECE-STAT5A,
pSVL-Eg-YLVLD, and pGL3-
-casein-luc have been described before
(14-17). Wild type PDK1 and kinase-dead PDK1 in pCMV5 were a kind gift
from Dario Alessi (Dundee, UK). pSIE-tk-luc was kindly provided
by Hugues Gascan (Angers, France). STAT3F and STAT3D in pCAGGS
were from Koichi Nakajima and Toshio Hirano (Osaka, Japan). The
FKHR cDNA (a kind gift from Frank Rauscher) was subcloned
into pcDNA3.1/His C (Invitrogen) or pEGFP-C1
(Clontech). The pEGFP-FKHR expression plasmid was
further digested with AccI, Van91I,
BpiI, or HindIII in combination with
BamHI (Roche Molecular Biochemicals), filled in with Klenow,
and religated to obtain FKHR
357,
505,
560, and
639,
respectively. The FKHR point mutants (T24A, S256A, and S319A) were
generated by PCR-based site-directed mutagenesis. The resulting
constructs were verified by sequencing.
-galactosidase (pCH110) or Renilla luciferase (pRLTK)
activities. All of the experiments were repeated at least three times
with similar results. Shown are the means ± S.D. of one
representative experiment performed in triplicate.
2-macroglobulin
(
2-M) probe was performed as described previously (18),
and the signals were detected using a Personal Molecular Imager FX
(Bio-Rad).
ex = 488 nm) and
rhodamine (
ex = 543 nm) fluorescences, which were
detected using a 505-530-nm band pass or 560-nm long pass emission
filter, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-M in hepatoma cells.
The levels of
2-M mRNA in IL-6-treated HepG2
hepatoma cells were significantly decreased in the presence of insulin
(Fig. 1A). One of the well known mediators of insulin signaling is PI 3-kinase, which further downstream activates the serine/threonine kinase Akt (7).
Interestingly, the inhibitory effect of insulin on
2-M
expression could be partially relieved by an inhibitor of PI 3-kinase,
wortmannin (Fig. 1A). As shown in Fig. 1B,
similar effects were also observed on the level of
2-M
promoter activity using a fragment of the 5'-regulatory sequence of the
rat
2-M gene, which is known for its high sensitivity to
STAT3-mediated signaling (16). Therefore, we decided to investigate a
potential cross-talk between the PI 3-kinase/Akt pathway and the
IL-6-inducible Jak/STAT3 signal transduction cascade. Transfection of
HepG2 cells with a constitutively active variant of Akt resulted in a
significant reduction of the IL-6-mediated activation of a transfected
2-M promoter-luciferase reporter gene construct (Fig.
2A), comparable with the
degree of inhibition by insulin (Fig. 1B). Consistently,
overexpression of a kinase-dead variant of PDK1 exerted the opposite
effect and augmented the reporter gene activity. The effects observed
using wild type forms of both kinases were less pronounced (data not
shown). These results suggested that Akt kinase activity can negatively
influence IL-6-dependent gene expression.
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Fig. 1.
Insulin inhibits the IL-6-induced
2-M gene expression. A,
Northern blot analysis of HepG2 cells treated overnight with IL-6 (200 units/ml), insulin (1 µM), and wortmannin (0.1 µM) in serum-free medium. 10 µg of RNA were separated
by agarose gel electrophoresis and blotted onto nylon membranes.
2-M mRNA was detected with a 32P-labeled
specific cDNA probe. An ethidium bromide staining of the RNA is
shown as a loading control. B,
2-M promoter
activity in HepG2 cells treated as above with IL-6 (200 units/ml),
insulin (1 µM), and wortmannin (0.1 µM).
Shown are the means ± S.D. of a representative experiment
performed in triplicate. RLU, relative luciferase units.
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Fig. 2.
Activation of PDK1/Akt signaling inhibits the
IL-6-mediated activation of the
2-M promoter through a
FKHR-dependent mechanism. A, effect of
constitutively active Akt (ca-Akt) and kinase-dead PDK1
(kd-PDK1) on the
2-M promoter activity in
HepG2 cells. Cells were transiently transfected with plasmids encoding
constitutively active Akt or kinase-dead PDK1 and incubated with IL-6
(black bars) or left untreated (open
bars). The IL-6-induced promoter activity of the
mock-transfected controls was set as 100% in all experiments.
B, dose-dependent effect of expressed
FKHR-cDNA on the
2-M promoter activity in HepG2
cells. C, effect of PDK1 (left panel)
and Akt (right panel) on the
2-M
promoter activity in HepG2 cells overexpressing FKHR. Cells were
transfected with combinations of expression plasmids encoding FKHR and
PDK1 (wild type or kinase-dead) or Akt (wild type or constitutively
active) and with reporter plasmids as before. D, the effect
of FKHR T24A, S256A, and S319A mutations on the
2-M
promoter activity. HepG2 cells were transfected with reporter plasmids
as above together with the expression vectors encoding wild type or
point-mutated variants of green fluorescent protein-tagged FKHR
(FKHR-T24A, FKHR-T24A/S256A, FKHR-T24A/S256A/S319A) or with an empty
vector, and the relative luciferase activity was determined
(left panel). The right
panel shows a Western blot (WB) with total cell
lysates (10 µg/lane) probed with anti-FKHR antibody to control the
expression level of the different constructs. The upper
arrow indicates a nonspecific band, whereas the
lower arrow labels a band at 80 kDa presumably
corresponding to endogenous FKHR. RLU, relative
luciferase units.
2-M
promoter regulation. Transfection of HepG2 cells with increasing
amounts of FKHR cDNA led to a marked and dose-dependent
increase of the IL-6 responsiveness of the
2-M promoter
(Fig. 2B) without a significant effect on the promoter
activity in untreated cells. In order to confirm that FKHR links Akt to
IL-6 signaling, we examined whether its overexpression affects the
inhibitory action of PDK1 and Akt. As shown in Fig. 2C
(left panel), wild type PDK1 decreased the IL-6-induced reporter gene activity by about 50% in
FKHR-overexpressing cells, whereas the kinase-dead PDK1 mutant produced
the opposite effect. In addition, overexpression of FKHR remarkably
enhanced the effect of Akt. Whereas wild type Akt, similarly to wild
type PDK1, decreased IL-6-mediated gene expression, expression of
constitutively active Akt further reduced the reporter gene activity to
about 25% of the control level (Fig. 2C, right
panel).
2-M promoter activity, we
compared the effects of wild type FKHR and mutated FKHR variants
lacking the first, the first two, or all three phosphorylation sites
(Fig. 2D). All three examined FKHR mutants were more
efficient than wild type FKHR in augmenting the IL-6-induced
2-M promoter activity. Interestingly, mutation of
threonine 24 to alanine was already sufficient for a nearly maximal
effect; introduction of additional point mutations (S256A and S319A,
respectively) did not lead to a significant further increase in
promoter activity. These results indicate that PDK1/Akt signaling can
modulate the transcriptional activation of IL-6-responsive genes by
targeting FKHR.
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Fig. 3.
IL-6 does not induce Akt activity and FKHR
phosphorylation in HepG2 cells. A, stimulation of HepG2
cells with IL-6 does not affect Akt activity. Cells were kept in
serum-free medium and stimulated for 15 min with either insulin (1 µM), IL-6 (200 units/ml), or both in combination. The
data show the quantitation of incorporated 32P into the
GSK-3 substrate peptide of an in vitro kinase assay. Shown
are the means ± S.D. of one representative experiment performed
in triplicate with the basal Akt activity set as 100%.
B-D, determination of the phosphorylation state of critical
regulatory residues in Akt, FKHR, and STAT3 following stimulation with
either insulin, IL-6, or both in combination as before. 100 µg of
total cell lysate were subjected to SDS-PAGE, blotted onto
polyvinylidene difluoride membranes, and probed with specific
antibodies. B, phosphorylation of Akt on residues
Thr308 and Ser473 (P-Thr and
P-Ser). C, phosphorylation of FKHR on residues
Thr24 and Ser256. D, phosphorylation
of STAT3 on Tyr705. Single representative blots are
shown.
2-M promoter, we investigated whether
FKHR modifies the function of STAT3, the crucial mediator of IL-6
signaling (2, 3). Coexpression of STAT3 and FKHR in HepG2 cells indeed
synergistically increased the responsiveness of the
2-M
promoter to IL-6 stimulation (Fig.
4A). In contrast, FKHR
expression had no effect on the transcriptional activation of the
-casein promoter by STAT5, another member of the STAT family of
transcription factors (Fig. 4B). Likewise, FKHR did not
enhance the activity of transcription factors unrelated to STATs such
as SMADS, the mediators of tumor growth factor-
signaling (data not
shown). Hence, FKHR appears to be a specific transcriptional
coactivator of STAT3-responsive promoters.
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Fig. 4.
Functional interaction between FKHR and STAT3
in transcriptional regulation. A and B, FKHR
contributes specifically to the function of STAT3. A,
synergistic effect of FKHR and STAT3 on the IL-6-induced
2-M promoter activity. HepG2 cells were transiently
transfected as described in the legend to Fig. 1 together with FKHR
and/or wild type STAT3 expression vectors. B, FKHR has no
effect on the STAT5-mediated activation of the
-casein promoter.
HepG2 cells were transiently transfected with a vector encoding a
chimeric receptor (Eg-YLVLD) specifically activating STAT5
together with vectors encoding the
-casein promoter, FKHR, and/or
STAT5A. Cells were treated with EPO (black bars)
or left in serum-free medium (open bars).
C and D, the effect of FKHR on the
transcriptional regulation depends on the presence of functional STAT3.
C, the effect of dominant negative STAT3 mutants on
FKHR-induced
2-M promoter activity. HepG2 cells were
transfected with reporter plasmids together with FKHR alone or in
combination with dominant negative STAT3D or STAT3F. D, FKHR
enhances the IL-6-induction of the STAT3-dependent
SIE-tk-luc reporter gene construct. E and F, the
complete C terminus of FKHR is crucial for its function as a
coactivator of the
2-M promoter. E, schematic
representation of the truncated variants of FKHR. DB, DNA
binding domain; NES, nuclear export sequence. F,
HepG2 cells were transfected with vectors encoding the different FKHR
mutants and the
2-M promoter reporter construct
and were incubated with IL-6 (black bars)
or left untreated (open bars).
RLU, relative luciferase units.
2-M promoter
activation essentially depends on the presence of functional STAT3,
since overexpression of dominant negative STAT3 factors (STAT3F or
STAT3D, respectively) (28) almost completely abrogated the
FKHR-mediated induction of
2-M promoter activity (Fig.
4C). This is also consistent with the observation that
expression of FKHR efficiently up-regulates IL-6 responsiveness of an
artificial promoter, comprising a tandem of isolated STAT3 consensus
binding sites (Fig. 4D). These results suggest that the
transcriptional effects of FKHR are indirect and result from an
enhanced activity of STAT3.
2-M promoter activation
(Fig. 4, E and F). FKHR was recently shown to
interact in HepG2 cells with the coactivator p300/CREB-binding protein
(CBP), which is also essential for its transcriptional activity (31).
STAT3 is also capable of recruiting p300/CBP, but this interaction and
the level of transactivation are relatively weak in comparison with
other STATs like STAT2 (32). It is widely accepted that activating
proteins like the STATs most often do not act alone but rather in
combination with other site-specific or more general DNA-binding
proteins as well as with coactivators of transcription (33, 34).
Therefore, the transcriptional function of STAT3 might be further
enhanced by FKHR acting as an accessory factor that directs coactivator complexes to STAT3 sites in the promoter.
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Fig. 5.
Subcellular localization of STAT3
(left panel) and FKHR (right
panel) in response to IL-6 and insulin in HepG2
cells. Cells were serum-starved for 24 h and incubated
in the presence of LY294002 for the last 1 h. After starvation,
cells were washed extensively and stimulated for 10 min with either
insulin (1 µM), IL-6 (200 units/ml), or both in
combination, fixed, and analyzed by confocal laser-scanning microscopy.
The green fluorescence corresponds to anti-STAT3
staining, and the red fluorescence corresponds to
anti-FKHR staining. A single representative confocal section is shown.
Scale bar, 10 µm.
2-M gene
expression, costimulation with insulin abrogated the IL-6-induced
interaction between STAT3 and FKHR in the nucleus. Therefore, the
negative regulation of IL-6-dependent gene expression by
the activation of the PI 3-kinase/Akt pathway appears to result from
the loss of cooperation between STAT3 and its transcriptional partner
FKHR and depends on the subcellular localization of both proteins.
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Fig. 6.
Physical association of FKHR and
STAT3. A, coimmunoprecipitation of FKHR and STAT3.
HepG2 cells were serum-starved for 24 h and incubated in the
presence of LY294002 (100 µM) for the last 1 h.
Thereafter, cells were washed with medium and stimulated for 10 min
with IL-6 (200 units/ml), lysed, and incubated with polyclonal
anti-STAT3 (left panel) or polyclonal anti-FKHR
antibodies (right panel). The precipitates
together with 30 µg of total cell lysates (TCL) were
separated by SDS-PAGE, blotted onto polyvinylidene difluoride
membranes, and detected with anti-FKHR or anti-STAT3 (upper
panels). After stripping, the blots were redeveloped with
the antibodies used for the immunoprecipitations (IP) to
control loading (lower panels). B,
FKHR does not interact with signaling molecules other than STAT3.
Starved HepG2 cells were stimulated with IL-6 (200 units/ml),
interferon- (1000 units/ml), tumor growth factor-
(10 units/ml),
or 10% fetal calf serum, lysed, and incubated with anti-STAT3,
anti-STAT5, anti-SMAD2/3, or anti-extracellular signal-regulated kinase
2 antibodies, respectively. The precipitates together with a fraction
of total cell lysates collected before the immunoprecipitation were
separated by SDS-PAGE and analyzed as described above. Upper
panel, sections of the same blot developed with anti-FKHR
serum. Lower panel, control detection of the
stripped membranes with the respective antibodies used for the
immunoprecipitations. C, treatment with IL-6 enhances the
association of FKHR and STAT3 in nuclear fractions of HepG2 cells.
Cells were serum-starved and pretreated with LY294002 as previously and
then stimulated with IL-6 (200 units/ml) or IL-6 together with insulin
(1 µM). After fractionation, nuclear extracts were
incubated with polyclonal anti-STAT3 antibodies or control normal
rabbit serum (NRS). The precipitates were separated by
SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and
detected with anti-FKHR (upper panel). After
stripping, the blots were redeveloped with the antibodies used for the
immunoprecipitations to control loading (lower
panel). WB, Western blot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin promoter in HepG2 cells and showed a
synergistic action together with STAT3. Second, we observed the
physical association of both factors in nuclear extracts of HepG2
cells, which could be further enhanced by IL-6 stimulation and
abrogated in the presence of insulin. Third, STAT3 showed similar to
FKHR a nuclear distribution in IL-6-treated HepG2 cells.
2-M promoter and its positive influence on gene
expression was dependent on the presence of activated STAT3, we
conclude that FKHR indirectly augments IL-6-induced transcriptional
activation. To our knowledge, this is the first report demonstrating
the cooperation between both factors. However, it is widely accepted
that activating proteins like the STATs most often do not act alone but
rather in combination with other site-specific or more general
DNA-binding proteins as well as with coactivators of transcription (33, 34). Examples of such a cooperativity include STAT1 and Sp1 (35), STAT5 and the glucocorticoid receptor (36), and
STAT3 and c-Jun (37). The cooperation between STAT3 and
c-Jun has been well documented for a number of different genes (34),
but the initial observation concerned their interaction on the
2-M promoter (37). Therefore, it would be of interest to
assess whether c-Jun could also participate in the interaction between STAT3 and FKHR. A recent report (38) documented that STAT3 can act in
concert with hepatocyte nuclear factor 1 to enhance the hepatocyte
nuclear factor 1-mediated transactivation of hepatic gene expression in
HepG2 cells and murine livers. It seems conceivable that this finding
represents a more general mechanism whereby tissue-specific and
inducible transcription factors cooperate in response to external
signals. The results of our study suggest a different scenario, where
FKHR transcription factors, closely related to the liver-specific
hepatocyte nuclear factor 3 (39), support the
STAT3-dependent gene expression.
2-M promoter
activation, but in this case the lack of the 16 last amino acid
residues already significantly inhibited the coactivating
function of FKHR. This finding implies that the complete C terminus of
FKHR is required for an efficient cooperation with STAT3.
2-M promoter in human HepG2 hepatoma
cells. Moreover, these effects could be associated with the expression
level and the phosphorylation status of FKHR transcription factors.
FKHR has been reported to be regulated by insulin in several cell lines
of hepatic origin including SV40-transformed murine hepatocytes (22),
rat hepatomas (42), and human HepG2 hepatoma cells (43). Recently, also
IL-6 has been shown to activate Akt in a significant proportion of
multiple myeloma cell lines (26) as well as in human Hep3B hepatoma
cells (25, 44). In both cell types, activation of the PI 3-kinase/Akt
pathway was suggested to play a role in IL-6-dependent
protection against apoptosis. The activation of Akt signaling should
result in the direct phosphorylation of three regulatory sites
(Thr24, Ser256, and Ser319) in FKHR
and subsequent transcriptional inactivation and relocation to the
cytoplasm (10, 23, 24). However, it is not known whether the reported
transient activation of Akt in multiple myeloma and Hep3B hepatoma
cells is sufficient for the permanent exclusion of FKHR from nuclei of
treated cells. We found that IL-6 neither induced kinase activity of
Akt nor stimulated its phosphorylation on the critical regulatory
residues Thr308 and Ser473 (7).
Correspondingly, phosphorylation of FKHR on Thr24 and
Ser256 was not increased during IL-6 treatment of HepG2
cells for up to 16 h (Fig. 3C and data not
shown). Our initial observation that expression of kinase-dead PDK1
(Fig. 2) and treatment with wortmannin (Fig. 1B) can enhance
the IL-6-induced
2-M promoter activity may result from
an inhibition of the basal, IL-6-independent Akt activity. These
results were further corroborated by immunofluorescence data showing no
change in nuclear localization of endogenous FKHR as well as of
overexpressed green fluorescent protein-tagged FKHR upon stimulation of
HepG2 cells with IL-6 (Fig. 5 and data not shown). Therefore, we
conclude that FKHR during IL-6 treatment remains active and may
participate in IL-6-induced gene expression.
2-M promoter
activity as a result of nuclear retention of these proteins. Our
observations indicate that the single mutation of Thr24 is
sufficient for maximal transcriptional stimulation. They are in
agreement with previous reports on the crucial role of
Thr24, a residue that lies within a 14-3-3 consensus motif,
involved in the interaction with 14-3-3 proteins (9, 45).
2-macroglobulin gene,
which are negatively regulated by insulin and growth factors.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Hubertus Axer and Bernd Giese for assistance in confocal microscopy, Dieter Schmoll for helpful discussions, and Marlies Kaufmann for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by Fonds der Chemischen Industrie (Frankfurt a.M.), Deutsche Forschungsgemeinschaft (Bonn) (to I. B. and P. C. H.), National Institutes of Health Grant DK 34926 (to R. A. R.), a grant from the medical faculty (RWTH Aachen), and a Feodor-Lynen-Fellowship of the Alexander von Humboldt-Stiftung (both to A. B.).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.
These authors contributed equally to this work.
§§ To whom correspondence should be addressed: Abteilung für Endokrinologie, Heinrich-Heine-Universität, Moorenstr. 5, D-40225 Düsseldorf, Germany. Tel.: 49-211-8117810; Fax: 49-211-8117860; E-mail: Andreas.Barthel@uni-duesseldorf.de.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M205403200
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ABBREVIATIONS |
---|
The abbreviations used are:
IL-6, interleukin-6;
2-M,
2-macroglobulin;
APP, acute phase
protein;
CBP, CREB-binding protein;
CREB, cAMP-response element-binding
protein;
PDK1, 3-phosphoinositide-dependent protein
kinase-1;
PI, phosphatidylinositol;
STAT, signal transducer and
activator of transcription.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Heinrich, P. C., Castell, J. V., and Andus, T. (1990) Biochem. J. 265, 621-636[Medline] [Order article via Infotrieve] |
2. | Hirano, T., Ishihara, K., and Hibi, M. (2000) Oncogene 19, 2548-2556[CrossRef][Medline] [Order article via Infotrieve] |
3. | Heinrich, P. C., Behrmann, I., Muller-Newen, G., Schaper, F., and Graeve, L. (1998) Biochem. J. 334, 297-314[Medline] [Order article via Infotrieve] |
4. | Campos, S. P., and Baumann, H. (1992) Mol. Cell. Biol. 12, 1789-1797[Abstract] |
5. | Wang, Y., Ripperger, J., Fey, G. H., Samols, D., Kordula, T., Wetzler, M., Van Etten, R. A., and Baumann, H. (1999) Hepatology 30, 682-697[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Milland, J.,
Tsykin, A.,
Thomas, T.,
Aldred, A. R.,
Cole, T.,
and Schreiber, G.
(1990)
Am. J. Physiol.
259,
G340-G347 |
7. |
Datta, S. R.,
Brunet, A.,
and Greenberg, M. E.
(1999)
Genes Dev.
13,
2905-2927 |
8. |
Meier, R.,
Alessi, D. R.,
Cron, P.,
Andjelkovic, M.,
and Hemmings, B. A.
(1997)
J. Biol. Chem.
272,
30491-30497 |
9. | Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857-868[Medline] [Order article via Infotrieve] |
10. |
Rena, G.,
Guo, S.,
Cichy, S. C.,
Unterman, T. G.,
and Cohen, P.
(1999)
J. Biol. Chem.
274,
17179-17183 |
11. |
Takaishi, H.,
Konishi, H.,
Matsuzaki, H.,
Ono, Y.,
Shirai, Y.,
Saito, N.,
Kitamura, T.,
Ogawa, W.,
Kasuga, M.,
Kikkawa, U.,
and Nishizuka, Y.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11836-11841 |
12. |
Cahill, C. M.,
Tzivion, G.,
Nasrin, N.,
Ogg, S.,
Dore, J.,
Ruvkun, G.,
and Alexander-Bridges, M.
(2001)
J. Biol. Chem.
276,
13402-13410 |
13. | Kortylewski, M., Heinrich, P. C., Mackiewicz, A., Schniertshauer, U., Klingmuller, U., Nakajima, K., Hirano, T., Horn, F., and Behrmann, I. (1999) Oncogene 18, 3742-3753[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Kohn, A. D.,
Takeuchi, F.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
21920-21926 |
15. |
Hemmann, U.,
Gerhartz, C.,
Heesel, B.,
Sasse, J.,
Kurapkat, G.,
Grotzinger, J.,
Wollmer, A.,
Zhong, Z.,
Darnell, J. E.,
Graeve, L.,
Heinrich, P. C.,
and Horn, F.
(1996)
J. Biol. Chem.
271,
12999-13007 |
16. | Yuan, J., Wegenka, U. M., Lutticken, C., Buschmann, J., Decker, T., Schindler, C., Heinrich, P. C., and Horn, F. (1994) Mol. Cell. Biol. 14, 1657-1668[Abstract] |
17. | May, P., Gerhartz, C., Heesel, B., Welte, T., Doppler, W., Graeve, L., Horn, F., and Heinrich, P. C. (1996) FEBS Lett. 394, 221-226[CrossRef][Medline] [Order article via Infotrieve] |
18. | Siewert, E., Bort, R., Kluge, R., Heinrich, P. C., Castell, J., and Jover, R. (2000) Hepatology 32, 49-55[Medline] [Order article via Infotrieve] |
19. |
Barthel, A.,
Okino, S. T.,
Liao, J.,
Nakatani, K., Li, J.,
Whitlock, J. P., Jr.,
and Roth, R. A.
(1999)
J. Biol. Chem.
274,
20281-20286 |
20. | Kortylewski, M., Heinrich, P. C., Kauffmann, M. E., Bohm, M., Mackiewicz, A., and Behrmann, I. (2001) Biochem. J. 357, 297-303[CrossRef][Medline] [Order article via Infotrieve] |
21. | Kops, G. J., de Ruiter, N. D., De, Vries-Smits, A. M., Powell, D. R., Bos, J. L., and Burgering, B. M. (1999) Nature 398, 630-634[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Nakae, J.,
Park, B. C.,
and Accili, D.
(1999)
J. Biol. Chem.
274,
15982-15985 |
23. |
Biggs, W. H., III,
Meisenhelder, J.,
Hunter, T.,
Cavenee, W. K.,
and Arden, K. C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7421-7426 |
24. |
Tang, E. D.,
Nunez, G.,
Barr, F. G.,
and Guan, K. L.
(1999)
J. Biol. Chem.
274,
16741-16746 |
25. |
Chen, R. H.,
Chang, M. C., Su, Y. H.,
Tsai, Y. T.,
and Kuo, M. L.
(1999)
J. Biol. Chem.
274,
23013-23019 |
26. |
Tu, Y.,
Gardner, A.,
and Lichtenstein, A.
(2000)
Cancer Res.
60,
6763-6770 |
27. |
Brownawell, A. M.,
Kops, G. J.,
Macara, I. G.,
and Burgering, B. M.
(2001)
Mol. Cell. Biol.
21,
3534-3546 |
28. | Nakajima, K., Yamanaka, Y., Nakae, K., Kojima, H., Ichiba, M., Kiuchi, N., Kitaoka, T., Fukada, T., Hibi, M., and Hirano, T. (1996) EMBO J. 15, 3651-3658[Abstract] |
29. |
Lam, P. Y.,
Sublett, J. E.,
Hollenbach, A. D.,
and Roussel, M. F.
(1999)
Mol. Cell. Biol.
19,
594-601 |
30. | Bennicelli, J. L., Fredericks, W. J., Wilson, R. B., Rauscher, F. J., III, and Barr, F. G. (1995) Oncogene 11, 119-130[Medline] [Order article via Infotrieve] |
31. |
Nasrin, N.,
Ogg, S.,
Cahill, C. M.,
Biggs, W.,
Nui, S.,
Dore, J.,
Calvo, D.,
Shi, Y.,
Ruvkun, G.,
and Alexander-Bridges, M. C.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
10412-10417 |
32. |
Paulson, M.,
Pisharody, S.,
Pan, L.,
Guadagno, S.,
Mui, A. L.,
and Levy, D. E.
(1999)
J. Biol. Chem.
274,
25343-25349 |
33. | Bromberg, J., and Darnell, J. E., Jr. (2000) Oncogene 19, 2468-2473[CrossRef][Medline] [Order article via Infotrieve] |
34. | Shuai, K. (2000) Oncogene 19, 2638-2644[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Look, D. C.,
Pelletier, M. R.,
Tidwell, R. M.,
Roswit, W. T.,
and Holtzman, M. J.
(1995)
J. Biol. Chem.
270,
30264-30267 |
36. | Stocklin, E., Wissler, M., Gouilleux, F., and Groner, B. (1996) Nature 383, 726-728[CrossRef][Medline] [Order article via Infotrieve] |
37. | Schaefer, T. S., Sanders, L. K., and Nathans, D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9097-9101[Abstract] |
38. |
Leu, J. I.,
Crissey, M. A.,
Leu, J. P.,
Ciliberto, G.,
and Taub, R.
(2001)
Mol. Cell. Biol.
21,
414-424 |
39. | Kops, G. J., and Burgering, B. M. (1999) J. Mol. Med. 77, 656-665[CrossRef][Medline] [Order article via Infotrieve] |
40. | Galili, N., Davis, R. J., Fredericks, W. J., Mukhopadhyay, S., Rauscher, F. J., 3rd, Emanuel, B. S., Rovera, G., and Barr, F. G. (1993) Nat. Genet. 5, 230-235[Medline] [Order article via Infotrieve] |
41. | Fredericks, W. J., Galili, N., Mukhopadhyay, S., Rovera, G., Bennicelli, J., Barr, F. G., and Rauscher, F. J., III (1995) Mol. Cell. Biol. 15, 1522-1535[Abstract] |
42. |
Tomizawa, M.,
Kumar, A.,
Perrot, V.,
Nakae, J.,
Accili, D.,
Rechler, M. M.,
and Kumaro, A.
(2000)
J. Biol. Chem.
275,
7289-7295 |
43. |
Guo, S.,
Rena, G.,
Cichy, S., He, X.,
Cohen, P.,
and Unterman, T.
(1999)
J. Biol. Chem.
274,
17184-17192 |
44. | Kuo, M. L., Chuang, S. E., Lin, M. T., and Yang, S. Y. (2001) Oncogene 20, 677-685[CrossRef][Medline] [Order article via Infotrieve] |
45. | Rena, G., Prescott, A. R., Guo, S., Cohen, P., and Unterman, T. G. (2001) Biochem. J. 354, 605-612[CrossRef][Medline] [Order article via Infotrieve] |