(Received for publication, August 31, 1996, and in revised form, March 24, 1997)
From the Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263
Haptoglobin (HP) is one of the major acute phase
plasma proteins in the mouse, and its synthesis is additively induced
by interleukin (IL)-6 and glucocorticoids. STAT3 serves as the mediator of the IL-6 receptor signal and appears to contribute to the
transcriptional induction of acute phase protein genes. The
carboxyl-terminal region of STAT3, consisting of an acidic domain and
containing a serine phosphorylation site, has been proposed to
contribute to the induction process. To assess the role of STAT3 in the
transcriptional control of the HP promoter, we applied two mutant forms
of STAT3: one with a deletion of the carboxyl-terminal 55 amino acid
residues, STAT355C, and the other with a substitution of serine 727 to alanine, STAT3SA. Like the wild-type STAT3, both mutant STAT3 forms
are activated by the signal-transducing subunit of the IL-6 receptor,
gp130, or by co-transfected IL-3 receptor. Ectopic expression and
activation of wild-type STAT3 or STAT3SA in HepG2 hepatoma cells
similarly enhance transcription through the IL-6-response element of
the HP promoter. This enhancement is specific for STAT3 and cannot be
reproduced by STAT1 or STAT5. In contrast, STAT3
55C inhibits
IL-6-induced transcriptional activation. Interestingly, whereas
receptor-activated STAT3 also enhances stimulation of the haptoglobin
promoter by dexamethasone through the glucocorticoid receptor,
activated STAT3
55C reduces the regulation below the level achieved
by the glucocorticoid receptor alone. This transdominant action by
STAT3
55C is dependent on a functional IL-6-responsive element. The
data suggest that the carboxyl-terminal domain, but not its serine
phosphorylation site of STAT3, is required for transcription as part of
the hematopoietin receptor signaling as well as for cooperation with
other transcription factors such as the glucocorticoid receptor.
Acute phase response is elicited by the various forms of
inflammatory condition, tissue injury, and infection (1). The recruitment of the systemic defense mechanisms is initiated by many
cell types at the site of local inflammation and includes macrophages,
mast cells, fibroblasts, and endothelial cells and involves the
production, release, and distribution of several cytokines. A major
component of the systemic acute phase response is the enhanced
production of several plasma proteins in the liver that are
collectively called acute phase plasma proteins
(APPs)1 (1). In most vertebrate species,
haptoglobin (HP) is one of the core sets of APPs, and its synthesis is
generally increased severalfold. In the mouse, the increase is 30-fold
and exceptionally high (2). HP functions as a hemoglobin-binding
protein and is most effective in clearing free hemoglobin from the
circulation (3). IL-6 is the major inducer of the hepatic production of most APPs, whereas IL-1 and tumor necrosis factor- act only on a
subset of APPs (4). Glucocorticoids or dexamethasone enhance the
production of many APPs through yet-to-be defined mechanisms. The mouse
HP gene is an example of APPs that are strongly induced by both IL-6
and dexamethasone (2).
Signaling by the IL-6 receptor is mediated by the signal-transducing
common subunit gp130 through Janus kinases associated with the
cytoplasmic domain containing the Box-1 and Box-2 motifs (5). Following
phosphorylation on the tyrosine residue in the Box-3 motifs of gp130,
STAT3 is recruited to the gp130 and is activated by receptor-associated
Janus kinases (JAKs) through phosphorylation (6). Activated STAT3
dimerizes, translocates to the nucleus, and binds to specific DNA
sequences (7). The interaction of STAT3 with specific
cis-acting elements has been correlated with enhanced
transcription of the responsive target genes including APPs (8, 9).
Recent analyses have suggested that additional modifications of STAT3
protein alter its function as a transcriptional activator.
Phosphorylation on serine 727 has been shown to enhance DNA binding
affinity and activation of transcription (10, 11). Deletion of the
carboxyl-terminal region, as found in the STAT3 form, rendered the
protein inactive as an activator of transcription and as a regulator of
differentiation of the myeloid cell line M1 in response to IL-6
(12-14). Although characterization of STAT3 has indicated structural
domains relevant for gene induction, the action of critical STAT3
mutant forms, in the context of an IL-6-sensitive gene promoter, has
not been described. The promoter of the mouse HP gene has been selected as a model to define the relevance of the carboxyl-terminal peptide and
the serine 727 phosphorylation site for regulation by IL-6R and other
hematopoietin receptors in hepatic cells.
Previously, we have found that the 237-bp HP promoter contains
cis-acting elements for mediating induction by IL-6 and
dexamethasone, and includes two C/EBP binding sites ("A" element at
positions 213 to
198 and "C" element at
137 to
123), the
IL-6 response element (IL-6RE) ("B" element at positions
165 to
152), and the PEA-3 binding site (at positions
110 to
87) (see
Fig. 6A; Ref. 2). Although no glucocorticoid receptor (GR)
binding site could be identified in the mouse HP promoter, the
dexamethasone response appears to be dependent on the presence of the
GR and requires the cytokine response region at positions
184 to
106 of the HP promoter (2).
Here we report the specificity with which STAT3 contributes to transcription through the IL-6RE of the HP gene and the relevance of the IL-6RE and STAT3 in mediating induction by IL-6 and dexamethasone.
COS-1 and HepG2 cells (15) were maintained in minimal essential medium supplemented with 5 and 10% fetal calf serum, respectively. Mouse hepatoma cells, Hepa-1 (16), were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cells were treated with serum-free medium containing 100 ng/ml IL-6, (Genetics Institute), IL-3 (Sandoz), or GCSF (Immunex Corp). Where necessary, 1 µM dexamethasone was used.
DNA ConstructsExpression vector encoding the human
GCSFR-gp130 chimeric receptor (GCSFR-gp130(133)) (17), IL-3 receptor
(18) and
(19), rat glucocorticoid receptor (pRSVGR) (20), and
rat STAT3 (9) have been described. Plasmid pmHP(237)-CAT contains the promoter of the Mus domesticus HP gene (from positions
237
to +20 relative to the transcription start site) linked to the CAT gene
in pCT (2). In plasmid pmHP(237)MB-CAT, the IL-6RE sequence, the B
element (
163TTACTGGAA
155) was mutated to
TAAGTGGCA using the mutagenesis kit
(CLONTECH). Plasmid mHP(IL-6RE)-CAT contained four
tandem copies of the IL-6RE region of HP promoter (
172 to
151; see
Fig. 6A) in the BglII site of pCAT (Promega). The
serine 727 to alanine mutation in STAT3SA was introduced by replacing
codon 727, UCC, with GCG. STAT3
55C was constructed by adding two
stop codons after amino acid residue 715. All of the mutations were
verified by DNA sequence analysis, and the cDNA forms were cloned
into the human cytomegalovirus-based vector pDC (21).
COS-1 cells were transfected by
the DEAE-dextran method (22). After a 36-h recovery, the cells were
maintained in minimal essential medium without serum for 16 h.
After treatment with cytokines for 15 min, whole cell extracts were
prepared (23). HepG2 cells were transfected by the modified calcium
phosphate method (24). This method yielded 2.5 ± 5.6%
(n = 12) of transfected cells in the culture as judged
from in situ staining for the expression of the transfection
marker -galactosidase. The standard plasmid mixtures contained a
final concentration of 20-25 µg/ml and included 2 µg of pIE-MUP as
an internal marker (25). After a 24-h recovery, cells were treated with
the cytokines for 24 h, and serial dilutions of cell extracts were
used to measure CAT activity within the linear range of the assay.
Conversion of chloramphenicol to acetylated products was determined.
The values were normalized to the expression of the co-transfected
major urinary protein (25) and calculated relative to the untreated
control (defined as 1.0). Mean ± S.D. of at least three
independently performed experimental series are reproduced in the
figures.
An aliquot of the whole cell extracts containing 5 µg of protein was used for EMSA. The double-stranded, high affinity sis-inducible element SIEm67 (23) served as binding substrate for STAT1 and STAT3, and TB2 served as substrate for STAT5 (26). From the same whole cell extracts, aliquots containing 30 µg were also separated on 6 or 7.5% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membrane and were reacted with anti-STAT1 (Transduction Laboratories), anti-STAT3 (C-20, Santa Cruz Biotechnology), monoclonal STAT3 antibody (Transduction Laboratories), STAT5 antibody (Santa Cruz Biotechnology), or phosphotyrosine 705-specific anti-STAT3 (New England Biolabs). Similarly, total cell lysates (30 µg) were analyzed with anti-human GCSFR (Springville Laboratories, RPCI). Immune complexes were visualized by enhanced chemiluminescence reaction (Amersham Corp.).
Kinase ReactionWhole cell extracts from COS-1 cells
transiently expressing STAT3 or STAT3SA served as substrates for the
MAPK reaction. Twenty µg of whole cell protein in a total of 100 µl
of reaction buffer (20 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 20 mM MnCl2,
150 mM NaCl, 8% glycerol, and 50 mM NaF) were
incubated with 10 µCi of [-32P]ATP and 20 µl of
activated MAPK-agarose conjugate (Upstate Biotechnology, Inc.) for 30 min at 37 °C. Reactions were stopped by removing the MAPK beads by
brief centrifugation. STAT3 was immunoprecipitated from the supernatant
fraction with STAT3 antibody (C-20, Santa Cruz Biotechnology) and then
subjected to 7.5% SDS-polyacrylamide gel electrophoresis for
autoradiography and Western analysis.
To characterize the role of STAT3 in the regulation of
mouse HP gene elements, we constructed two key mutant forms of STAT3: STAT3SA with a mutation of the serine 725 to alanine at its
carboxyl-terminal kinase substrate site, and STAT355C with a
truncation of the acidic carboxyl-terminal fragment as observed in
STAT3
(12, 13) but lacking the latter's additional 7 residues
encoded by the extra exon sequence. We verified the expression of the
STAT proteins by transiently transfecting COS-1 cells (Fig.
1A). Western blot analysis (bottom
part, mAb) demonstrated that each STAT form was
produced in approximately equal amounts, yielding levels that exceeded
severalfold that of the endogenous STAT3 (Fig. 1A,
Ctrl). The major portion of the immunodetectable STAT
proteins migrated with the expected sizes. However, a minor fraction of
STAT3wt and STAT3SA migrated with an approximately 5000 smaller
molecular size. These forms probably represent proteolytically modified STAT3 proteins (see similar forms detected in HepG2 cell
extracts; Fig. 2A).
Effect of STAT proteins on gene induction
through the HP promoter. A, HepG2 cells were transfected
with a combination of expression vectors for GCSFR-gp130(133) (5 µg/ml), the indicated STAT proteins (10 µg/ml), and empty expression vector to a
total of 20 µg/ml. In this experiment, rat STAT1 instead of
STAT1
was used for easier detection of the transfected protein in
the presence of the relatively abundant endogenous STAT1. After 1 day
of recovery, cells were divided in 6-well plates. The cells were
treated with IL-6 (Vector Control) or GCSF for 15 min. One part of the collected cells was dissolved directly in SDS sample buffer
for Western blot analysis of GCSFR-gp130 (only cytokine-treated cells),
and the remaining part was used for preparing whole cell extract.
Aliquots containing 5 and 30 µg of protein were used for EMSA and
Western analysis, respectively. For EMSA, SIE (all lanes
except last lane) or TB2 (last lane with STAT5B)
served as binding substrate. For STAT protein Western analyses,
extracts were electrophoresed in quadruplicate, and proteins on the
nitrocellulose membrane were reacted with the indicated antibodies.
B, for detecting CAT gene regulation, HepG2 cells were
transfected with a similar combination of plasmids as in A,
consisting of the reporter mHP(237)-CAT (15 µg/ml) and expression
vector for GCSFR-gp130(133) (1 µg/ml) and indicated STAT proteins (5 µg/ml). In this case, rat STAT1
was used. After 1 day of recovery,
the cell cultures were divided and treated for 24 h with the
indicated cytokine or dexamethasone. The thin layer pattern of one
reaction is shown. The -fold stimulation measured in this experimental
series is indicated above the autoradiogram. C,
HepG2 cells were transfected with a 15 µg/ml of mHP(237)-CAT (upper panel) or mHP(IL-6RE)-CAT (lower panel)
and the increasing amounts of expression vector for STAT3, STAT3SA, or
STAT3
55C as marked. The DNA amount in each transfection was adjusted
to a total of 25 µg/ml with pUC13 DNA. Cells were treated with IL-6 for 24 h, and CAT activities were determined. All values are
expressed relative to control culture without IL-6 treatment and STAT3
transfection.
The activation of the transfected STAT proteins by gp130 signals, was
demonstrated by the co-transfected chimeric receptor, GCSFR-gp130(133).
This receptor consists of the extracellular domain of the human GCSFR
fused to the transmembrane and the cytoplasmic domain of 133 residues
of human gp130. The chimeric receptor is considered to undergo a
GCSF-dependent dimerization that also includes the
cytoplasmic gp130 domains. An analogous dimerization process has been
predicted for the bona fide ligand-activated IL-6R (27, 28). Expression
of the chimeric receptor provides the advantage that signaling function
of gp130 can be analyzed specifically in transfected cells and
independently of the endogenous gp130 (17, 29). Extracts from cells
treated with or without GCSF indicated that each STAT form was
activated by the receptor and yielded comparable SIE-binding activity
(Fig. 1A, upper panel). As shown previously (9)
in COS-1 cells not transfected with any STAT expression vectors,
GCSFR-gp130 activated detectable SIE-binding activity of endogenous
STAT proteins consisting primarily of STAT1. In the presence of
overproduced STAT3, however, the predicted SIE binding activities
characteristic for STAT3 homodimers (SIF-A complexes, Ref. 23) were
obtained. Only a minor fraction of heterodimers between STAT3 and
endogenous STAT1 (SIF-B complex) appeared as an additional band below
the STAT3 homodimer complex. As predicted, the SIE complexes formed
with wild-type STAT3 and STAT3SA were recognized by the C-20 anti-STAT3
antibody, yielding a supershifted EMSA pattern (Fig. 1A,
upper panel, lanes 7 and 8). The
STAT355C-containing complex was not recognized by C-20 antibody
(lane 9) but showed a characteristic slower mobility probably due to the loss of the acidic C-terminal fragment that bears
the epitope for the C-20 antibody (see smaller size on Western blot in
Fig. 1A). Only STAT3
55C showed minor SIE-binding activity in untreated cells, which is in agreement with previous data (12, 13).
The reaction with anti-phosphotyrosine STAT3 (Fig. 1A, lower panel) illustrated that the phosphorylation at
tyrosine 705 in each STAT3 form correlated with the DNA binding
activity of the protein.
To show in our experimental system that overexpressed STAT355C forms
heterodimers with STAT1 or STAT3 (see also Ref. 13) we transfected
varying amounts of expression vector for STAT3
55C, STAT3, and STAT1
into COS-1 cells. DNA binding activity of the STAT proteins was
activated through GCSFR-gp130 and determined by EMSA.
Dose-dependent formation of heterodimers between
STAT3
55C and STAT1 (Fig. 1B) or STAT3 (Fig.
1C) was clearly evident. Moreover, a shift toward
predominant STAT3
55C homodimers at high STAT3
55C expression level
was achieved. Analyses of subcellular fractions indicated that
activated STAT3
55C was translocated to the nucleus, and this
translocation was indistinguishable from the wild-type STAT3 or STAT3SA
(data not shown).
Observing that STAT3SA, contrary to previous predictions (10), was expressed at the same level as wild-type STAT3 and bound to SIE with apparently comparable affinity, we determined whether the point mutation had indeed modified the substrate behavior of STAT3 for MAPK. COS cell extracts containing overexpressed STAT3 or STAT3SA were reacted in vitro with MAPK and [32P]ATP. Immunoprecipitation and gel electrophoresis showed that wild-type STAT3, but not STAT3SA, was prominently labeled (data not shown), indicating that the serine to alanine mutation had effectively removed a principle phosphorylation site in STAT3.
Taken together, these results confirmed the expected physicochemical properties of the two mutant STAT3 forms and also indicated that these proteins were essentially indistinguishable in their activation by the gp130 signal, complex formation, and binding to DNA substrates. Hence, these mutant forms fulfilled the preconditions for their application in probing STAT3 function as an inducer of transcription through the HP promoter.
IL-6 Response through the HP Promoter Is Enhanced by STAT3To identify the role of STAT proteins in the IL-6 regulation of the mouse HP promoter, we needed to test the IL-6-responsive mHP(237)-CAT construct in hepatic cells, because COS-1 cells failed to reproduce a liver-like regulation of that construct (data not shown). Since no mouse hepatoma cells with IL-6-regulatable endogenous HP gene expression have been identified, we selected HepG2 cells as the experimental system for their prominent IL-6 responsiveness (2, 9, 17). These cells were transfected with pHP(237)-CAT alone or together with expression vectors for the various STAT isoforms to demonstrate the specificity of these proteins to modify signaling by endogenous IL-6R and co-transfected GCSFR-gp130. Although we applied a transient transfection protocol that had been optimized for hepatoma cells (24), we could detect only a relative low percentage of transfected HepG2 cells in the culture (see "Experimental Procedures") and lower protein expression than in COS-1 cells (data not shown). Nonetheless, the transfection efficiency was sufficient to verify the presence of the overexpressed STAT proteins by Western blotting and activation of their DNA binding activity through receptor signals by EMSA (Fig. 2A). By comparing the Western blot and EMSA signals for STAT3 in STAT3-transfected cells with that in control or non-STAT3 transfected cells, and by taking into consideration the low transfection efficiency, we estimated that the amount of ectopically expressed STAT3 proteins exceeded the level of endogenous STAT3 by at least 10-fold. Although the low level production of GCSFR-gp130 rendered detection of the receptor protein in the transfected culture difficult, we could determine by Western blot analysis that the expression of GCSFR-gp130 was relatively comparable among separately transfected cell cultures (Fig. 2A, bottom part).
The quantitation of CAT activities (Fig. 2B) revealed that
the expression of the mHP(237)-CAT construct was increased 2-5-fold by
the action of GCSFR-gp130 and endogenous IL-6R. The cytokine (e.g. IL-6) response was further enhanced by co-treatment
with dexamethasone. Co-expression of STAT1 or STAT5B was ineffective in modulating the regulation. STAT3, as well as STAT3SA, however, enhanced induction in GCSF and IL-6-treated cells by 2-3-fold. In
contrast, STAT3
55C abrogated induction by the receptors, yet did not
significantly affect basal activity of the pHP237-CAT construct. Since
overexpressed receptors containing the extracellular domain of GCSFR
were noted previously to reduce by unknown mechanisms the signaling by
IL-6R in HepG2 cells (17), subsequent analyses of IL-6 action were
determined only in cells without co-transfected GCSFR-gp130.
The influence of the STAT3 forms on mHP-CAT gene regulation through the
endogenous IL-6R (Fig. 2C, upper panel) or
GCSFR-gp130 (data not shown) was dependent on the dose of
co-transfected STAT3 expression vectors. Generally, a maximal
enhancement of the cytokine signal by wild-type STAT3 and STAT3SA was
attained with 1-5 µg/ml transfected expression vector. With
increasing amounts of STAT355C expression vector, the cytokine
receptor-mediated induction of the reporter gene declined, reaching
basal level in some instances.
The B element located at
positions 165 to
152) of the HP promoter is conserved among HP
genes from rodents and primates and has been identified as an
IL-6-responsive element (IL-6RE) (33). Since, for unknown reasons, a
binding of the isolated HP IL-6RE sequence to STAT3 could not be
detected by the standard EMSA conditions as used in Fig. 1, we verified
by functional cell assay that this element was the target of the
regulation by STAT3. Four tandem copies of the core sequence of the
mouse HP IL-6RE (see sequence in Fig. 6A) inserted it into a
heterologous promoter-CAT construct, and this construct was transfected
into HepG2 cells, along with STAT3 forms (Fig. 2C,
lower panel). The results revealed that the element was
responsive to IL-6 treatment and that both STAT3 and STAT3SA similarly
enhanced the induction. As noted for the HP promoter construct,
STAT3
55C was inhibitory.
The
signaling functions of the overexpressed STAT3 forms, as shown in Fig.
2C, appear to be superimposed on those exerted by the
endogenous signal-transducing components, including the resident STAT
proteins that are utilized by IL-6R or GCSFR-gp130 (Fig.
2A). A more direct demonstration of the signaling action of
the transfected STAT3 forms was possible through activation by
co-transfected IL-3R. IL-3R lacks a Box-3 sequence motif in its
cytoplasmic domains and, therefore, is ineffective in recruiting and
activating endogenous STAT3, but it is able to activate STAT3 proteins
when overexpressed (Fig. 3A; Ref. 30). As
shown in Fig. 3B, the IL-3R did not detectably induce the HP
promoter or HP-IL-6RE construct. However, in combination with either
wild-type STAT3 or STAT3SA, a strong induction was obtained. In the
same experimental systems, STAT355C was ineffective.
STAT3
The HP promoter also mediated a severalfold induction by
dexamethasone in the absence of IL-6 (Fig.
4A). Moreover, the combination of
dexamethasone and IL-6 acted additively. The dexamethasone response,
but not the IL-6 response, was dose-dependently increased by co-transfected GR (Fig. 4B). A maximal, 3-fold enhanced
dexamethasone action was achieved with 2 µg/ml expression vector. At
higher GR doses, a substantially reduced responsiveness was detected, probably in part due to squelching of overexpressed GR (32). The
stimulatory effect of the GR suggests a direct action GR in the
induction process through the HP promoter. Conversely, overexpressed wild-type STAT3, which had proven to enhance the IL-6 response (Figs.
2C and 3B), neither appreciably influenced the
dexamethasone effect nor magnified the additive response to IL-6 and
dexamethasone (Fig. 4, A and C, left
panel). Similarly, overexpressed STAT355C had no significant
effect on dexamethasone alone; however, when activated by IL-6
treatment, it reduced the dexamethasone response to 50%.
In the presence of overexpressed GR, the induction of the HP promoter
CAT construct enhanced by dexamethasone was virtually unaffected by
either STAT3 wild-type or STAT355C (Fig. 4C, right panel). In contrast, when treated with the combination of IL-6 and
dexamethasone, STAT3
55C, but not STAT3 wild-type, attenuated gene
regulation below the level of that achieved by dexamethasone alone
(Fig. 4C, right panel). This negative
interference of STAT3
55C was specific to the 237-bp HP promoter
construct. The mHP(IL-6RE)-CAT construct did not appreciably respond to
dexamethasone even in the presence of overexpressed GR (Fig.
4A, right panel), thus excluding the possibility
that the GR acted indirectly through the STAT proteins interacting with
the IL-6RE.
To provide evidence that dexamethasone treatment is capable of regulating not only episomal plasmid constructs but also the chromosomal mouse HP gene (the HP gene in HepG2 cells does not respond to dexamethasone; Ref. 33), we turned to the mouse hepatoma Hepa-1 cell line. Previous studies have shown that Hepa-1 cells respond to dexamethasone by increased production of haptoglobin (34), although the same cells failed to respond to any IL-6-type cytokines. Hence, these cells were of limited value as an experimental system for characterizing the cytokine response elements of the HP gene. As shown in Fig. 4D, a 2-h treatment of Hepa-1 cells led to a prominent increase of HP mRNA, and this action was largely maintained in cells with cycloheximide-inhibited protein synthesis. We conclude from these results that dexamethasone, via the GR, induced HP gene transcription. The transfection experiments in HepG2 cells suggested that the 237-bp promoter, but not its IL-6RE, is the target of the GR.
Functional Cooperativity between STAT3 and GRTo assess the
specific contribution of wild-type and mutant STAT3 to the action of GR
on HP promoter, we determine the STAT3 dose dependence of the
regulation by utilizing the IL-3R-mediated STAT activation process
introduced in Fig. 3. Maximal induction of the reporter gene construct
was attained with ~1.5 µg/ml of wild type STAT3 expression vector
(Fig. 5A). When using a submaximal concentration (0.5 µg/ml) of wild-type STAT3 and an increasing dose
of STAT355C expression vector, we detected an enhancing effect of a
low dose of STAT3
55C in IL-3- as well as IL-3 plus dexamethasone-treated cells (Fig. 5B). At a higher dose of
STAT3
55C, the transdominant inhibitory action of the STAT protein
prevailed. This dual effect of STAT3
55C on HP promoter
activity in IL-3- and dexamethasone-treated cells was even more
pronounced in the presence of co-expressed GR (Fig. 5C). We
interpret these results to mean that at low dose and approximately
equal expression, levels of wild type and STAT3
55C heterodimer
between the two STAT proteins are formed (see Fig. 1C) and
that these, like the wild type STAT3 homodimer, mediate
transactivation. At high STAT
55C concentration, STAT3
55C
homodimer predominates (Fig. 1C) and acts as a competitive inhibitor. The data also suggest that a cooperative action between GR
and STAT3 is established that in part would explain the additive induction. The carboxyl-terminal portion of STAT3 is probably involved
in that cooperativity, and if absent as in STAT3
55C dimers, an
active inhibition is obtained.
The IL-6RE Is Critical for HP Gene Induction by IL-6 and Dexamethasone
Previous analysis of the mouse HP promoter (2)
indicated a contributing role of C/EBP isoforms and PEA-3 for
determining expression levels. The present results propose a role of
STAT3 in mediating IL-6 induction, most probably through the IL-6RE (Figs. 2C and 3B). To prove the functional
relevance of the IL-6RE in the 237-bp promoter context, we mutated the
core sequence of this IL-6RE at three positions (MB) as indicated in
Fig. 6A. Wild-type and MB constructs were
introduced into HepG2 cells (Fig. 6B, upper panel). The mutation had two striking effects: 1) basal expression was reduced ~10-fold; and 2) stimulation by both IL-6 and
dexamethasone had been abolished. The mutation was outside of the
two principle C/EBP binding sites (Fig. 6A; Ref. 2);
therefore, we could still demonstrate transactivation by overexpressed
C/EBP (data not shown). These results suggest that the IL-6RE
sequence plays a critical role in determining basal expression and in
mediating the induction by dexamethasone and IL-6.
The influence of MB mutation in the HP promoter on the specific
transactivating function of STAT3 and GR was determined in HepG2 cells
overexpressing either transcription factor (Fig. 6B, lower panel). STAT3 was activated by co-expressed IL-3R, but
no effect on the HP-CAT construct was detected. In contrast, GR
produced a dexamethasone-mediated induction of the MB construct. The GR action was severalfold lower than seen with wild-type HP promoter (Fig.
4B) and was also not influenced by co-expressed and
IL-3R-activated wild-type STAT3 or STAT355C (data not shown). The
data support the model that STAT3 through interaction with IL-6RE
establishes a functional cooperativity with GR and that the C-terminal
portion of STAT3 is necessary for communication with this
transcriptional machinery.
Acute phase regulation of hepatic HP gene expression is primarily mediated by IL-6-type cytokines (35). However, additional regulatory factors need to be considered, such as growth factors and endocrine hormones, including glucocorticoids to explain the full acute phase induction (2, 4, 34). Several signaling pathways with distinct transcription-controlling factors have been proposed for IL-6R (36), some of which may act on the HP gene (2, 37, 38). The results of our study document an active role of STAT3 in mediating hematopoietin receptor signal, in particular that of gp130 as part of the IL-6R complex to the mouse HP gene promoter in hepatic cells. We approached the characterization by using two mutant STAT3 proteins that had been predicted to act as specific inhibitors and applied these to dissect the IL-6R signal mechanisms.
The STAT3SA mutant, by lacking the serine phosphorylation site at residue 727, was described as having a reduced DNA binding activity (10) and lower potency as a transactivator (11). Our data show that STAT3SA was used as signaling molecules by gp130 or IL-3R in hepatic cells like wild-type STAT3. We interpret the differences between our work and previous publications as indicating that the hematopoietin receptor systems, utilizing STAT3 as a signal transducer in hepatic cells, accomplish gene induction without the involvement of the modification of this particular serine residue. By extension, we assume that receptor-mediated activation of MAPK or any protein kinase C isoforms that might act upon serine 727 (11, 39) does not play a critical role in HP gene induction. This assumption is supported in part by our earlier observation that treatment of hepatic cells with reagents that activate the MAPK pathway or protein kinase C independently of the hematopoietin receptor signal, e.g. by insulin or phorbol ester, could neither mimic nor enhance the action of IL-6 on APP genes or transfected reporter constructs (40, 41). In fact, insulin treatment that activates MAPK (42) and increases serine phosphorylation of STAT3 (43) reduces the IL-6 induction of APP genes (40, 41). We conclude that phosphorylation at serine 727 may not be an important modification for achieving signaling toward APP genes, but we cannot rule out that phosphorylation at other sites influences transcription-inducing activity or cellular turnover of STAT3 (10, 43).
The second mutant STAT3, STAT355C, has been predicted (based on the
precedents of STAT1
(10) or STAT5B
40C (44)) to be inactive as an
inducer of transcription and to act either as a transdominant or
competition inhibitor of wild-type STAT3. The mode of action differs
from the dominant negative action of the tyrosine 705 mutant STAT3
that, by virtue of being unable to dimerize, acts as a terminator of
the signaling at the receptor level (45). STAT3
55C has an
essentially wild-type function in respect to recruitment to the
receptor, activation by receptor-associated kinases, and DNA binding.
Evidently, STAT3
55C terminates signaling by failing to stimulate
transcription in cis. This implies that the
carboxyl-terminal acidic fragment may have stimulatory properties analogous to the acidic domains of other transcription factors such as
GCN4 or VP16, which interact with general transcription factors TFIIB
and TFIID (46).
STAT355C was modeled after STAT3
(12) but differs by not having
an extra 7-amino acid extension. In agreement with the previous report
(13), STAT3
55C is unlike STAT3
in two important properties: 1)
STAT3
55C does not display appreciable DNA binding activity in cells
not treated with inducing cytokines, e.g. IL-6 or IL-3; and
2) STAT3
55C shows normal DNA binding activity but fails to induce
transcription. The fact that carboxyl-terminal-truncated STAT3 forms
naturally occur demands consideration of their potential biological
relevance.
The inhibitory activity of STAT355C was readily detectable on the
co-transfected HP gene construct. A similar action is also expected to
occur on all endogenous IL-6-responsive APP genes that are also
sensitive to STAT3. Due to the technical limitation of the transient
transfection system, such an inhibition could not be determined in the
HepG2 cells. We attempted by retroviral vector transduction to generate
HepG2 cells stably overexpressing STAT3
55C.2 Although many clonal lines
were obtained, none yielded a STAT3
55C expression level that was
sufficient to eliminate the action of the endogenous STAT3. However,
lines were identified in which the IL-6 induction of HP was reduced by
50% relative to the parental line.
Our data confirm a competitive inhibitory action of STAT355C in the
context of an APP gene promoter (Fig. 5). Furthermore, we show an
inhibitory role for STAT3
55C in the case of GR-enhanced transcription through the 237-bp HP promoter (Figs. 4C and
5). This function of STAT3
55C needs to be activated by IL-6
treatment, suggesting that STAT3
55C may interact with the IL-6RE of
the HP promoter and thereby attenuate transcription-inducing action of
the GR. Thus, the carboxyl-terminal peptide of STAT3, by its deletion,
appears to determine the inhibitor function. We have not been able to
detect a direct binding of the glucocorticoid receptor with the HP
promoter sequence or an interaction of the glucocorticoid receptor with
STAT3. We cannot, therefore, exclude the possibility that the
glucocorticoid receptor acts at a site (or sites) distant from the DNA
(32). The results with Hepa-1 cells (Fig. 4D) at least rule
out an indirect mechanism that depends on new protein synthesis.
Combining all information, the following working model for regulation
through the HP promoter is proposed. As shown in Fig. 6A,
the promoter contains functionally defined interaction and/or response
elements that include two sites (sites A and C) for C/EBP that flank
the single STAT response element (B or IL-6RE) and a more proximally
located PEA-3 site that is characteristic to the mouse HP gene (2). A
site for the interaction of the GR within the dexamethasone-responsive
BC-containing segment (2) has not been found. IL-6RE is sensitive to
STAT3 but appears also to be a target of a factor(s) necessary for
establishing basal expression and executing the dexamethasone response
(Fig. 6B). IL-6 treatment has two effects. One effect is the
immediate activation of STAT3 that interacts with the IL-6RE or
associated proteins or replaces the factors already bound (31). The
other effect is enhancing the production and nuclear concentration of
C/EBP
(37, 38, 47). C/EBP
binds to the HP promoter, stimulates HP
transcription (2), and appears to enhance the effects of STAT3 and
GR.2 Although STAT3 alone is sufficient to induce
transcription through the isolated IL-6RE (Fig. 3B), to
obtain an appreciable magnitude of induction that is equal to that of
STAT3 through the HP promoter, an oligomerization of the IL-6RE is
required. Thus, we speculate that the combination of STAT3 and
C/EBP
, as predicted for the HP promoter, may be as effective as a
complex of several STAT3s, as predicted for the IL-6RE oligomer. The GR
is directly or indirectly interacting with the promoter and, in
combination with the factor associated with IL-6RE, stimulates
transcription. If STAT3, as a consequence of IL-6 treatment, interacts
with IL-6RE, a higher transcription is achieved than through the factor
that is at this site in cells not treated with IL-6 (hypothetical basal
factors). Since STAT3
55C is as effective in binding to DNA as STAT3,
it similarly interacts with IL-6RE and replaces the basal factors but
is unable to cooperate with the GR, thus blocking the GR action.
We thank Drs. J. Ripperger and G. Fey for providing STAT3 cDNA, Karen K. Kuropatwinski and Yanping Wang for technical assistance; C. Steger for critical reading of the manuscript; and Marcia Held and Lucy Scere for secretarial work.