From the Department of Gastroenterology and
Hepatology, Medizinische Hochschule Hannover, 30625 Hannover and the
Institute of Clinical Immunology, Universität Leipzig,
04129 Leipzig, Germany
Received for publication, October 11, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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LAP/C/EBP CAAT/enhancer-binding proteins
(C/EBPs)1 are a family of
leucine zipper transcription factors currently comprising six members involved in the regulation of various aspects of cellular
differentiation and function in multiple tissues. Three closely related
members of the C/EBP family, C/EBP Several inflammatory signals, including lipopolysaccharides (LPS),
interleukin 6 (IL-6), interleukin 1 (IL-1), tumor necrosis factor Of the numerous cytokines and growth factors that are involved in
regulating the acute phase response, IL-6 plays a major role (reviewed
in Ref. 13). Binding of IL-6 to its receptor induces homodimerization
of the signal-transducing component gp130 (14), which results in the
activation of constitutive associated JAK kinases and phosphorylation
of gp130 at different tyrosine residues (15, 16). Tyrosine
phosphorylation creates specific docking sites for signaling molecules
containing SH2 domains (17). In the case of gp130 at least three
signaling cascades, the STAT1, the STAT3, and the MAP kinase pathway
are activated (18-21). Activation of STAT3 is the most prominent
pathway during this process (15, 22, 23). Phosphorylation of STAT3
through JAK kinases results in its homo- or heterodimerization and
nuclear translocation. STAT3 then binds to target sequences in
different promoters and enhances gene transcription. Binding sites for
STAT3 are located in most promoters of acute phase genes, and
originally STAT3 was identified as acute phase response factor
(24-26).
Analysis of the C/EBP In this analysis we show that IL-6 activates LAP/C/EBP Stimulation of Mice and Preparation of Total RNA and of Liver
Nuclear Extracts--
C3H mice were stimulated intraperitoneally with
40 µg of human recombinant IL-6. At different time points after
injection, the livers were removed and preparation of RNA and of liver
nuclear extracts was performed. At each time point at least three
animals were used in parallel. RNA was isolated by the guanidinium
isothiocyanate method (30). For preparation of nuclear extracts the
pooled livers were rinsed in ice-cold phosphate-buffered saline, and liver nuclear proteins were prepared as described previously (30). All
steps were performed at 4 °C. Nuclear proteins were aliquoted and
frozen immediately in liquid nitrogen.
Northern Blot Analysis--
Northern blot analysis was performed
as described before, according to standard procedures (30). 30 µg of
total RNA was analyzed through a 1% agarose formaldehyde gel followed
by transfer to Hybond N+ membranes (Amersham Pharmacia
Biotech, Braunschweig, Germany). The LAP/C/EBP LAPC/EBP Expression Vectors for STAT1, STAT3, and Chimeric STAT
Proteins--
A series of expression vectors for truncated STAT3
proteins and various STAT3-STAT1 chimeric proteins were constructed as described elsewhere (22, 31). For the construction of cDNAs coding
for STAT3-STAT1 chimeric proteins, additional unique restriction sites
were introduced into the STAT cDNAs by site-directed mutagenesis or
authentic restriction sites were used. The chimeric STAT cDNAs were
constructed by exchanging the respective DNA fragments within the
pBluescript vector context. The cDNAs were then subcloned into
the pRc/CMV vector (Invitrogen, Groningen, The Netherlands). The STAT3D
(EE STAT-Gal4 Fusion Constructs--
To create the 5'Gal4-STAT1NT
and 5'Gal4-STAT3NT fusion constructs, the respective STAT
amino-terminal NotI/SphI fragments were cloned
into pSG424-Gal4(1-147) (33) cleaved with EcoRI and
XbaI. Klenow fill-in reactions and mung bean digestions were used to clone the constructs in-frame. To create the STAT1NT-3'Gal4 and
the STAT3NT-3'Gal4 fusion constructs the Gal4-(1-147) fragment of the pSG424-Gal4-(1-147) vector was amplified by polymerase chain
reaction to introduce 5'-SphI and 3'-ApaI
restriction sites, verified by sequencing, and then cloned into the
respective pRcCMV-STAT expression vector behind the STAT amino-terminal
NotI/SphI fragments.
Other Plasmids--
The pCMV Cell Culture, Transfection Experiments, and Luciferase
Assays--
HepG2 cells (ATCC) were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. DNA
transfection was performed using a modified calcium phosphate
precipitation method as described previously (35). HepG2 cells were
grown on 60-mm dishes to about 50% confluence when used for
transfection experiments. The amount of reporter and expression vectors
is indicated in the figure legends. The total amount of DNA was kept constant in each transfection experiment by adding pBSK+DNA
(Stratagene, La Jolla, CA) to 5 µg. All transfections contained 0.1 µg of the
To measure luciferase activity, cells were washed twice with
phosphate-buffered saline and lysed by adding 350 µl of extraction buffer (25 mM Tris-H3PO4, pH 7.8, 2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, and 2 mM DTT) for 10 min. The lysates were cleared by
centrifugation. 50 µl of the supernatant was assayed by addition of
300 µl of measuring buffer (25 mM glycylglycine, 15 mM MgSO4, and 5 mM ATP). The light
emission was measured in duplicate for 10 s in a Lumat LB 9501 (Berthold) by injecting 100 µl of 250 µM luciferin.
Data are represented as the mean ± S.D. of triplicate experiments
and are representative for three independent experiments.
Preparation of Nuclear Extracts--
HepG2 nuclear extracts were
prepared by the modified Dignam C method (5). 48 h after
transfection, cells were washed twice with ice-cold phosphate-buffered
saline, scraped into microcentrifuge tubes, and centrifuged for 5 min
at 4000 × g, 4 °C. Cell pellets were resuspended in
hypotonic buffer (10 mM Tris, pH 7.4, 2 mM MgCl2, 140 mM NaCl, 1 mM DTT, 0.5 mM PMSF, and 0.5% Triton X-100) at 4 °C for 10 min (100 µl for 5 × 106 cells), transferred onto
one volume of 50% sucrose in hypotonic buffer, and centrifuged at
14,000 × g and 4 °C for 10 min. Nuclei were
resuspended in Dignam C buffer (20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF) (50 µl for 5 × 106 cells) and
gently rocked at 4 °C for 30 min. Nuclear debris was removed by
centrifugation at 14,000 × g and 4 °C for 10 min.
The extracts were aliquoted and stored at Gel Retardation Assays--
For gel retardation assays nuclear
extracts were used as indicated in the figure legends. Binding buffer
consisted of 25 mM HEPES, pH 7.6, 5 mM
MgCl2, 34 mM KCl, 2 mM DTT, 0,2 mM PMSF, 50 ng of poly(dI-dC)/µl, and 100 ng of bovine
serum albumin/µl. The binding reaction was performed for 30 min on
ice. Free DNA and DNA·protein complexes were resolved on a 6%
polyacrylamide gel.
The oligonucleotides were purchased from Eurogentec (Seraing, Belgium)
and used as 32P-labeled probes. Oligonucleotides A to M
were derived from the LAP/C/EBP Southwestern Analysis--
Southwestern analysis was performed
as described with some modifications (36, 37). 20 µg of liver nuclear
extracts prepared before and 1 h after stimulation of C3H mice
with 40 µg of IL-6 was heated for 30 min at 60 °C in SDS sample
buffer and separated on a 10% SDS-polyacrylamide gel. After
electrophoresis the gel was incubated in renaturing buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 50 mM
NaCl, 1 mM DTT, 4 M urea, adjusted to pH 7.5)
for 4 h at room temperature and then blotted onto a nitrocellulose
membrane (Millipore, Bedford, MA) in a solution containing 25 mM Tris and 192 mM glycine (adjusted to pH 8.3)
at 4 °C for 2 h at a constant current of 200 mA. The membranes
were prehybridized in binding buffer (10 mM Tris, pH 7.5, 0.1 mM EDTA, 1 mM DTT, 50 mM NaCl, 2× Denhardt's solution, adjusted to pH 7.2) at 4 °C overnight and
then incubated with 2.5 × 106 cpm
32P-labeled oligonucleotide A for 1 h at room
temperature. After incubation the membranes were washed several times
in binding buffer and exposures were made to x-ray films.
Western Blot Analysis--
Nuclear extracts were separated on a
10% SDS-polyacrylamide gel (38) and blotted onto nitrocellulose
membrane (Millipore) in 1% SDS, 20% methanol, 400 mM
glycine, 50 mM Tris, pH 8.3, at 4 °C for 2 h at 200 mA. The antibody directed against Gal4-(1-147) was purchased from
Santa Cruz Biotechnology, the antibody directed against phospho-STAT3
was purchased from New England BioLabs (Beverly, MA). The
antigen·antibody complexes were visualized using the ECL detection
system as recommended by the manufacturer (Amersham Pharmacia Biotech,
Braunschweig, Germany).
IL-6-induced LAP/C/EBP IL-6-induced LAP/C/EBP STAT3 Activation Is Essential for IL-6-mediated LAP/C/EBP
After binding of IL-6 to gp130 at least three intracellular signaling
cascades, STAT1, STAT3, and the Map kinase pathway (reviewed in Ref.
40), are activated. Participation of these signaling pathways in
IL-6-dependent LAP/C/EBP IL-6 Induces Binding of a 68-kDa Protein to the CRE-like Sites in
the LAP/C/EBP
In time course experiments, formation of the IL-6-stimulated complex
was studied. Oligonucleotide A (see Fig. 4A) represents the
sequence between nucleotide
Because binding of this IL-6-inducible factor occurred at the CRE-like
site in the LAP/C/EBP
Next, Southwestern analysis was performed to characterize the molecular
weight of the IL-6-induced factor (Fig. 4F). In control as
well as in IL-6-stimulated liver nuclear extracts we found p30,
corresponding to the lower gel shift band, and p43, corresponding to
CREB. An additional p68 protein corresponding to the upper band was
only found in IL-6-stimulated extracts (Fig. 4F). In parallel UV cross-linking, experiments showing very weak signals (data
not shown) confirmed our results indicating a molecular mass of 68 kDa
for the IL-6-inducible factor binding to both CRE-like sites in the
LAP/C/EBP Both CRE-like Sites in the LAP/C/EBP The Amino-terminal Domain of STAT3 Is Essential for IL-6-mediated
LAP/C/EBP
After receptor stimulation, the SH2 domain of the STAT proteins
interacts with specific phosphorylated tyrosines in the intracellular receptor domain (19, 22). Substitution of the STAT3 by the STAT1 SH2
domain (Stat 3/1 (SH2)) prevented IL-6-mediated LAP/C/EBP
These data indicate that interaction with the basal transcriptional
machinery and, thereby, activation of LAP/C/EBP Overexpression of the Amino-terminal Domain of STAT3 Prevents
IL-6-mediated Transcription of the LAP/C/EBP
Comparable expression of the respective fusion proteins after
transfection of HepG2 cells was found by Western blot analysis of
nuclear extracts (Fig. 7B). In luciferase reporter gene
experiments cotransfection of 5'Gal4-STAT3NT with LAPPRO 8 inhibited
the IL-6-mediated LAP/C/EBP Most of the acute phase genes are activated on a
transcriptional level through IL-6-responsive elements in their
promoter regions. The best studied example for a transcription factor
involved in this mechanism is STAT3 (23, 25); however, C/EBP family members also contribute to this regulation (2, 24). Induction of the
acute phase response in hepatocytes enhances the expression of
LAP/C/EBP The STAT3 Signaling Cascade Is Involved in IL-6-mediated
LAP/C/EBP The IL-6-responsive Element in the LAP/C/EBP
Mutations in the CRE-like sites of the LAP/C/EBP A Model for Tethering STAT3 to the LAP/C/EBP
The STAT3/STAT1 amino terminus (NT) domain swap mutants and the
Gal4-STAT3NT fusion constructs indicated that the amino-terminal part
of STAT3 is very crucial for mediating the link between the basal
machinery and the two CRE-like sites. This observation implies that the
amino terminus most likely does not bind to a more general factor,
which would be a part of the general RNA polymerase machinery, but to
one of the proteins, e.g. p68, binding to the CRE-like sites
in the LAP/C/EBP
In recent reports there is evidence that several transcription factors
activate transcription without DNA binding. In specific situations a
pre-existing complex of transcription factors bound to DNA allows the
association with an additional transcription factor without DNA
binding. This mechanism has also been called tethering, and meanwhile
several examples are known. ATF6 can be tethered to the c-Fos and to
the atrial natriuretic factor promoter by serum response factor bound
to the serum response element, without direct binding of ATF6 to the
serum response element (51, 52). Ubeda et al. (53) reported
the recruitment of CHOP to an AP1·DNA complex without DNA binding of
CHOP. Glucocorticoid receptors can also act as transcriptional
coactivators for STAT5 without binding to a glucocorticoid response
element (54, 55). The involvement of STAT1 in tumor necrosis
factor-
The close correlation between DNA binding of p68 to the CRE-like
elements and the relevance of STAT3 for the increase in LAP/C/EBP Importance for an Interplay of Different Regulatory Sites and for
Interaction of Different Activators with the Same Sites in the
LAP/C/EBP
Besides the DNA binding elements in the promoter region other more cell
type-specific mechanisms, e.g. starvation, proliferation, determine gene regulation in a certain cellular background. Analysis of
the LAP/C/EBP is a member of the C/EBP family of
transcription factors and contributes to the regulation of the acute
phase response in hepatocytes. Here we show that IL-6 controls
LAP/C/EBP
gene transcription and identify an IL-6
responsive element in the LAP/C/EBP
promoter, which contains no
STAT3 DNA binding motif. However, luciferase reporter gene
assays showed that STAT3 activation through the gp130 signal transducer
molecule is involved in mediating IL-6-dependent
LAP/C/EBP
transcription. Southwestern analysis indicated that
IL-6 induces binding of a 68-kDa protein to the recently characterized
CRE-like elements in the LAP/C/EBP
promoter. Transfection
experiments using promoter constructs with mutated CRE-like elements
revealed that these sites confer IL-6 responsiveness. Further analysis
using STAT1/STAT3 chimeras identified specific domains of the protein
that are required for the IL-6-dependent increase in
LAP/C/EBP
gene transcription. Overexpression of
the amino-terminal domain of STAT3 blocked the IL-6-mediated response, suggesting that the STAT3 amino terminus has an important function in
IL-6-mediated transcription of the LAP/C/EBP
gene. These
data lead to a model of how tethering STAT3 to a DNA-bound complex contributes to IL-6-dependent LAP/C/EBP
gene
transcription. Our analysis describes a new mechanism by which STAT3
controls gene transcription and which has direct implication for the
acute phase response in liver cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, LAP/C/EBP
, and C/EBP
, are
known to contribute to liver-specific gene transcription. Specific
functions of these transcription factors have been described in the
regulation of the acute phase response and during liver regeneration,
where they seem to be involved in the process of hepatocyte
proliferation and differentiation (reviewed in Ref. 1).
,
and interferon gamma (IFN-
) contribute to the regulation of the
acute phase response in hepatocytes. The activities of the C/EBP family
members are differentially modulated in response to the various
inflammatory stimuli on the mRNA and protein level. Upon induction
of the acute phase response C/EBP
mRNA levels decrease in the
liver, whereas LAP/C/EBP
and C/EBP
gene
transcription is enhanced. For the regulation of LAP/C/EBP
, however,
post-translational mechanisms were described in addition to
transcriptional activation (reviewed in Ref. 2). Several
phosphorylation sites in the LAP/C/EBP
protein have been shown to be
functionally relevant (3-6). Phosphorylation occurs in
vitro in response to an intracellular Ca2+ increase,
through activation of the protein kinase C pathway and through
activation of the mitogen-activated protein (MAP) kinase following
induction of the Ras pathway. Although all these events lead to
increased transactivation of LAP/C/EBP
-dependent genes,
only the MAP kinase pathway can be linked to the acute phase response.
Moreover, stimulation with lipopolysaccharides, IL-6, IL-1,
dexamethasone, and glucagon strongly induces LAP/C/EBP
expression
(7-10). Initially, LAP/C/EBP
has also been cloned because it binds
to IL-6-responsive elements in the promoters of acute phase response
genes (11, 12) and thus was originally named NF-IL6 (nuclear factor
involved in the IL-6 gene expression).
promoter revealed that STAT3 contributes to
higher gene transcription after IL-6 stimulation (27, 28). Besides DNA
binding, cooperative interaction of STAT3 with Sp1 is essential for the
regulation of the C/EBP
promoter. In contrast, no information is
available about the molecular mechanisms involved in the
IL-6-dependent increase of LAP/C/EBP
gene
transcription. Therefore, we were interested in investigating how IL-6
controls the activation of this gene.
gene transcription using the two already characterized CRE-like sites in the LAP/C/EBP
promoter (29). STAT3 activation through the gp130
signal transducer is essential for increased LAP/C/EBP
transcription
despite the lack of sequence-specific STAT DNA binding sites in this
promoter region. Enhanced IL-6-dependent transcription of
the LAP/C/EBP
gene correlates with the binding of a new
IL-6-inducible factor with a molecular mass of 68 kDa to the
CRE-like sites in the LAP/C/EBP
promoter. Functional dissection of
the gp130 signal transducer molecule and the use of STAT1/STAT3
chimeras further demonstrated the essential role of STAT3 and
identified specific regions of the protein that are required for the
IL-6-dependent increase of LAP/C/EBP
gene
transcription. These results lead to a model of how tethering STAT3 to
the promoter region contributes to IL-6-dependent gene
transcription without direct DNA binding and thus indicate a new
mechanism for STAT3-dependent gene transcription.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes were
labeled with [
-32P]CTP according to the instructions
for Rediprime (Amersham Pharmacia Biotech). The hybridization procedure
was performed as described previously (30). Blots were exposed for
autoradiography and exposed to an imaging plate (Fuji) for
quantification. LAP/C/EBP
signals were normalized to the GAPDH
signals and set to 1 for untreated animals. The values for IL-6
treatment were shown as -fold stimulation.
Promoter Constructs--
The LAPPRO 1, 2, 3, 7, 8, and 9 constructs corresponding to increasing deletions in the
5'-flanking region of the LAP/C/EBP
open reading frame linked to a
luciferase reporter gene were described previously (29). The LAPPRO
8WT
construct carries a deletion of 27 nucleotides (
103 to
76)
between the two CRE-like sites and the LAPPRO 8 MUT I+II construct
carries mutations in both CRE-like sites (
109 to
107 = ACG
GTT and
65 to
61 = TGACG
GATCC) as described elsewhere
(29).
AA) construct was kindly provided by Toshio Hirano (32).
Gal vector was used as internal
standard. The pRcCMV-PIAS3 expression vector was kindly provided by
Chan Chung (34). The Epo-receptor/gp130 chimeras were described before
(19). They consisted of the extracellular domain of the murine
Epo-receptor fused to the human transmembrane domain and different
cytoplasmic tyrosine modules of the IL-6 signal transducer
gp130. The EpoR/MAP site corresponds to the Tyr-759 module and
the EpoR/STAT3 site corresponds to the Tyr-767 module. The EpoR/STAT1
site comprised only the membrane-proximal part of gp130, fused to a
peptide covering the Tyr-440 tyrosine motif of the IFN-
R.
-galactosidase reporter pCMV
Gal as an internal standard. For stimulation experiments, cells were starved with 1%
fetal calf serum for 24 h after transfection. Stimulation was performed with human recombinant IL-6 (Strathmann Biotech, Hannover, Germany) in the amounts and for the time points indicated or with 7 units/ml human recombinant erythropoietin (Epo) (Roche Molecular Biochemicals, Mannheim, Germany) for 4 h. 1 unit of IL-6 is
equivalent to 0.005 ng (Strathmann Biotech).
70 °C.
promoter. Oligonucleotide A
corresponds to
123 to
95 (GCG GCC GGG CAA TGA CGC GCA CCG ACC CG),
oligonucleotide B to
102 to
75 (CCG ACC CGG CGG CGG GGC GGC GGG AGG
G), oligonucleotide C to
90 to
68 (CGG GGC GGC GGG AGG GGC CCC GG),
oligonucleotide D to
75 to
43 (GGC CCC GGC GTG ACG CAG CCC GTT GCC
AGG CGC), oligonucleotide E to
123 to
107 (GCG GCC GGG CAA TGA CG),
oligonucleotide F to
110 to
87 (GAC GCG CAC CGA CCC GGC GGC GGG),
and oligonucleotide M to
123 to
99, mutated in
109 to
107 (GCG
GCC GGG CAA TGG TTC GCA). The SIE consensus oligonucleotide corresponds
to the sequence: 5'-GTG CAT TTC CCG TAA ATC TTG TCT ACA-3'. Supershift experiments were performed with antibodies against STAT3 or CREB1 (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclear extracts were modified in Fig. 4 as indicated. Prior to the incubation with the
32P-labeled probe, nuclear extracts were incubated with
0.08% desoxycholic acid (DOC) for 20 min at 4 °C, then
0.15% Nonidet P-40 was added. For the depletion assay 2.5 µl of
antibody to CREB1 (Santa Cruz Biotechnology) was coupled to 10 µl of
protein A-Sepharose, saturated with 1 µg/µl bovine serum albumin,
in TNE buffer (10 mM Tris, pH 8.0, 100 mM NaCl,
1 mM EDTA, 2 mM DTT, 2 mM Pefabloc,
1% Nonidet P-40) for 4 h at 4 °C, washed twice in TNE buffer,
and twice in gel shift sample buffer. Incubation with 3 µg of nuclear
extract was performed at 4 °C overnight. Protein A-Sepharose without
antibody was treated under the same conditions and served as control.
The supernatants were analyzed in gel shift experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mRNA Expression in the Liver
Correlates with STAT3 Activation--
LAP/C/EBP
was originally
identified, because an increase in DNA binding was evident after IL-6
treatment in human hepatoma cells (11, 12). IL-6 stimulates
LAP/C/EBP
at the transcriptional and post-translational level (2,
7). However, the molecular mechanisms responsible for IL-6-mediated
LAP/C/EBP
gene transcription are unknown. In an initial
experiment, C3H mice were stimulated with IL-6 and LAP/C/EBP
mRNA expression was studied by Northern blot analysis (Fig.
1A). LAP/C/EBP
signals were
normalized to the GAPDH signals and set to 1 for untreated animals.
Quantification revealed an increase in LAP/C/EBP
mRNA expression
already after 30 min while maximum levels (3.8-fold) were found 6 h after stimulation (Fig. 1A). At later time points
LAP/C/EBP
mRNA expression decreased again. After binding of IL-6
to its receptor, several pathways are activated. However, STAT3
activation has been shown to stimulate transcription of many
acute-phase genes. Therefore, activation of STAT3, as already described
before (39), was studied. IL-6 induced an increase in nuclear
expression of tyrosine-phosphorylated STAT3 30 min after injection as
shown by Western blot analysis (Fig. 1B). The nuclear
expression of phospho-STAT3 remained high for the first 4 h and
was no more detectable after 12 h. Gel shift experiments with an
SIE element as STAT3 target sequence showed activation of DNA binding
30 min after IL-6 injection (Fig. 1C). DNA binding remained
high for up to 3 h and then subsequently decreased. No complex
formation was found after 12 h. The specificity of STAT3 in the
new appearing complex was verified by supershift experiments (data not
shown) and has been shown before using a STAT3 DNA target sequence
derived from the
2-macrolgobulin promoter (39). Thus, these
experiments show a close correlation between an increase in
LAP/C/EBP
mRNA expression and STAT3 activation after IL-6
stimulation in the liver in vivo.
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Fig. 1.
IL-6-induced LAP/C/EBP
mRNA expression in the liver correlates with STAT3
activation. A, C3H mice were stimulated with 40 µg of
IL-6, total RNA was isolated from the liver before and at different
time points after stimulation (as indicated), and Northern blot
analysis with a 32P-labeled DNA for LAP/C/EBP
(upper panel) and for GAPDH were performed. The ratio
(lower panel) between the LAP/C/EBP
signal and the GAPDH
signal was calculated and set to 1 for the untreated probe. Changes
after IL-6 treatment are shown as -fold stimulation. B,
Western blot analysis was performed with 10 µg of liver nuclear
extract prepared before and at different time points after stimulation
(as indicated) of C3H mice with 40 µg of IL-6. Nuclear expression of
tyrosine-phosphorylated STAT3 (P-STAT3) was detected with an
antibody directed against tyrosine-phosphorylated STAT3. C,
gel shift experiments were performed with 3 µg of liver nuclear
extract prepared before and at different time points after stimulation
(as indicated) of C3H mice with 40 µg of IL-6. Complex formation was
performed with the 32P-labeled STAT3-specific
oligonucleotide comprising the SIE site (Santa Cruz
Biotechnology).
Transcription Is Mediated by a Region in
Proximity to the TATA Box--
As LAP/C/EBP
mRNA expression
increased after IL-6 stimulation in vivo, we performed
experiments to identify regulatory sequences in the LAP/C/EBP
promoter that mediate IL-6 induction. In transfection experiments
luciferase reporter constructs with increasing deletions in the
5'-flanking region, located upstream of the start site of transcription
in the LAP/C/EBP
gene, were analyzed in HepG2 cells. The
constructs were cotransfected with 50 ng of STAT3 expression vector,
and cells were treated with 1000 units/ml IL-6 for 4 h (Fig.
2A). The relative luciferase
activity of the respective LAPPRO reporter construct without
stimulation was set to 1, and the changes after IL-6 treatment were
shown as -fold stimulation. Strong induction (up to 20-fold) of the
luciferase reporter gene was observed when the whole 1.4-kb 5'-region
of the promoter was used (LAPPRO 1). IL-6-dependent
stimulation of the 5'-flanking region was still found when deletions up
to nucleotide
121 were analyzed. No increase in reporter gene
activity after IL-6 treatment was evident with the LAPPRO 9 construct
(truncated to position
71, see Fig. 2A). These data
indicate that the region located between nucleotides
121 and
71 in
close proximity to the TATA box is involved in mediating
IL-6-dependent transcription of the LAP/C/EBP
gene. The IL-6-dependent effect on the LAP/C/EBP
promoter was further investigated by dose and time-kinetic experiments (Fig. 2, B and C). This analysis showed that a
maximal effect on LAP/C/EBP
promoter activity was found with 1000 units/ml IL-6 for 4 h.
View larger version (18K):
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Fig. 2.
IL-6-induced LAP/C/EBP
transcription is mediated by a region in proximity to the TATA
box. A, increasing deletions were introduced in the
5'-flanking region of the LAP/C/EBP
gene. LAPPRO
1 corresponds to the whole 1.4-kb 5'-region located between the
StuI and AvaII sites. The AvaII site
is located 16 bp downstream of the start site of transcription in the
LAP/C/EBP
gene.
, potential STAT3 binding sites.
Restriction sites in the 1.4-kb fragment were used to create increasing
5'-deletions as indicated (LAPPRO 2, 3,
7, 8, and 9). All fragments were
linked to a luciferase reporter gene. HepG2 cells were cotransfected
with 2 µg of the respective LAPPRO luciferase reporter constructs and
with 50 ng of a STAT3 expression plasmid. 24 h after transfection
cells were starved with 1% fetal calf serum, and then stimulation was
performed with 1000 units of IL-6/ml for 4 h. The relative
luciferase activity of the respective LAPPRO luciferase reporter
construct without stimulation was set to 1, and the changes after IL-6
stimulation are shown as -fold stimulation. B and
C, HepG2 cells were cotransfected with 2 µg of the LAPPRO
8 luciferase reporter construct and without or with 50 ng of a STAT3
expression plasmid. Stimulation was performed for 4 h with
increasing units IL-6/ml as indicated (B) or with 1000 units
of IL-6/ml for different time points as indicated (C). The
relative luciferase activity without stimulation was set to 1, and the
changes after IL-6 treatment are shown as -fold stimulation.
Transcription--
Our next interest was to analyze the relevance of
the STAT3 signaling cascade for IL-6-mediated LAP/C/EBP
transcription despite the finding that there is no STAT consensus
sequence located in the LAP/C/EBP
promoter region, which confers
IL-6 inducibility. Therefore cotransfection experiments were performed
with the LAPPRO 8 reporter construct and an empty control vector or
expression vectors for STAT1 or STAT3. These studies showed that IL-6
stimulated the promoter construct 2-fold, whereas cotransfection of the
STAT3 expression vector enhanced this effect (Fig.
3A). In contrast, cotransfection of a STAT1 expression vector had no influence on the
IL-6 inducibility compared with cotransfection of the empty control
vector (Fig. 3A). In further experiments, the STAT3-specific inhibitor PIAS3 (protein inhibitor of activated STAT3) (14) was used to
characterize the STAT3-dependent mechanism. Cotransfection experiments of the LAPPRO 8 constructs and increasing amounts of an
expression vector for PIAS3 were performed with or without an
expression vector for STAT3. Under both conditions PIAS3 inhibited the
IL-6-dependent increase in LAP/C/EBP
gene
transcription in a concentration-dependent manner (Fig.
3B).
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Fig. 3.
STAT3 activation is essential for
IL-6-mediated LAP/C/EBP transcription.
A, HepG2 cells were cotransfected with 2 µg of the LAPPRO
8 luciferase reporter construct and with 50 ng of an empty expression
vector (
), 50 ng of a STAT1 expression vector (Stat1), or
50 ng of a STAT3 expression vector (Stat3). Cells were
starved as indicated in Fig. 2 and then stimulation was performed with
1000 units of IL-6/ml for 4 h. The relative luciferase activity of
the respective transfection without stimulation was set to 1, and the
changes after IL-6 treatment are shown as -fold stimulation.
B, HepG2 cells were cotransfected with 2 µg of the LAPPRO
8 luciferase reporter construct, without (
) or with (+) 100 ng of a
STAT3 expression vector (Stat3) and with increasing amounts
of a PIAS3 expression plasmid (PIAS) as indicated. Cells
were starved as indicated in Fig. 2 and then stimulation was performed
with 1000 units of IL-6/ml for 4 h. The relative luciferase
activity of the transfection without stimulation and without PIAS3
cotransfection was set to 1 for the samples without and with STAT3
cotransfection, respectively, and the changes after IL-6 treatment are
shown as -fold stimulation. C, EpoR/gp130 chimeras. The
receptor chimeras consisted of the extracellular domain of murine EpoR
fused to the transmembrane domain and different cytoplasmic tyrosine
modules of the IL-6 signal transducer gp130 (EpoR/Map site,
EpoR/STAT3 site). The EpoR/Stat1 site comprised
only the membrane-proximal part of gp130, fused to a peptide covering
the Tyr-440 tyrosine motif of the IFN-
R. D, HepG2 cells
were cotransfected with 2 µg of the LAPPRO 8 luciferase reporter
construct, 2 µg of the respective expression vector coding for the
receptor chimeras as indicated, and with 50 ng of a STAT3 expression
vector. Cells were starved as indicated in Fig. 2 and then stimulation
was performed with 7 units of Epo/ml for 4 h. The relative
luciferase activity of the respective transfection without stimulation
was set to 1, and the changes after Epo treatment are shown as -fold
stimulation.
transcription was further analyzed by using chimeric receptors. These chimeras consisted of the
extracellular domain of the erythropoietin receptor (EpoR) fused to the
transmembrane domain and different cytoplasmic tyrosine modules
specifically either activating STAT3 (gp130 Y767, EpoR/STAT3 site), the
MAP kinase pathway (gp130 Tyr-759, EpoR/Map site), or STAT1 (IFN
R
Tyr-440/EpoR/STAT1 site) (Fig. 3C). Stimulation of HepG2
cells with erythropoietin (Epo) had no effect on LAPPRO 8 activity
(data not shown). Epo stimulation after cotransfection of HepG2 cells
with the LAPPRO 8 construct and the EpoR/Map site or the EpoR/STAT1
site fusion receptors also resulted in no increase of reporter gene
activity (Fig. 3D). However, when the EpoR/STAT3 site
chimera was used, a 3-fold induction of LAP/C/EBP
gene
transcription was found. Therefore these data indicate that of the
three pathways studied only STAT3 can mediate
IL-6-dependent transcription of the LAP/C/EBP
gene.
Promoter--
The cotransfection experiments
indicated that STAT3 is involved in mediating
IL-6-dependent LAP/C/EBP
transcription. However, even
computer-assisted analysis showed no potential STAT DNA binding site
(41) between nucleotide
121 and the TATA box in the LAP/C/EBP
promoter (for the whole sequence see Ref. 29). To exclude the possibility that STAT3 may bind to a yet unknown target sequence in
this region, gel shift experiments were performed using mouse liver
nuclear extracts prepared before and 2 h after IL-6 treatment when
STAT3 binding to its consensus sequence occurred. Overlapping oligonucleotides derived from the LAP/C/EBP
promoter
(A-M, Fig. 4A)
were used as 32P-labeled probes. As shown in Fig.
4B, complex formation of two complexes was only found with
oligonucleotide A and D containing the two CRE-like sites of the
LAP/C/EBP
promoter. Complex formation was blocked when the mutant
oligonucleotide M (mutation of the first CRE-like site) was used as a
32P-labeled probe. In contrast to unstimulated extracts, a
third, new complex was detected in IL-6-stimulated liver nuclear
extracts, when the intact CRE-like site was included in the
oligonucleotides.
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Fig. 4.
IL-6 induces binding of a 68-kDa protein to
the CRE-like sites in the LAP/C/EBP
promoter. A, oligonucleotides A,
B, C, D, E, F,
and M derived from the LAP/C/EBP
promoter and used for
gel shift experiments are shown. The two CRE-like binding sites in the
LAP/C/EBP
promoter (TGACGCGC,
111 to
104 and TGACGCAG,
65 to
58) are marked as circles. B, gel shift
experiments were performed with 3 µg of liver nuclear extract
prepared before (
) and 2 h after stimulation (+) of C3H mice
with 40 µg of IL-6. Complex formation was performed with the
respective oligonucleotide as 32P-labeled probe.
IL-6-induced complex formation is marked with an arrowhead.
C, gel shift experiments were performed with 3 µg of liver
nuclear extracts prepared before and at different time points after
stimulation (as indicated) of C3H mice with 40 µg of IL-6. Complex
formation was performed with oligonucleotide A (
123 to
95) derived from the LAP/C/EBP
promoter as 32P-labeled
probe. IL-6-induced complex formation is marked with an
arrowhead. D, for supershift/binding inhibition
analysis antibodies against STAT3 or CREB were added to the 2-h value
after IL-6 stimulation (w/o = without antibody).
IL-6-induced complex formation is marked with an arrowhead.
E, gel shift experiments were performed with 3 µg of liver
nuclear extract prepared before (
) and 2 h after stimulation (+)
of C3H mice with 40 µg of IL-6 and oligonucleotide A
(
123 to
95) as 32P-labeled probe. Before complex
formation was performed, the extracts were modified as follows:
Doc = incubation with detergent (desoxycholic acid and
Nonidet P-40), Prot. A = incubation with protein
A-Sepharose beads, Prot. A/CREB = incubation with CREB antibody coupled protein A-Sepharose beads.
CREB = no modification, supershift reaction with CREB
antibody. IL-6-induced complex formation is marked with an
arrowhead. F, Southwestern analysis. 20 µg of
liver nuclear extract prepared before (
) and 1 h after
stimulation (+) of C3H mice with 40 µg of IL-6 were separated by
SDS-polyacrylamide gel electrophoresis, blotted on nitrocellulose,
incubated with 32P-labeled oligonucleotide A (
123 to
95), and exposed to autoradiography. The arrows mark the
proteins in the stimulated extracts (p68, p43,
p30) corresponding to the three gel shift bands. Molecular
mass markers are shown in kDa on the left.
123 and
95 of the LAP/C/EBP
promoter
that was used as the 32P-labeled probe. After IL-6
stimulation the intensity of the two complexes found already in
untreated animals did not change (Fig. 4C). Formation of the
IL-6-inducible third complex was found 1 h after IL-6 injection.
Maximal DNA binding was evident after 2 h. At later time points
complex formation decreased again (Fig. 4C). Supershift
experiments were performed to study whether STAT3 binds to the CRE-like
sequence in the LAP/C/EBP
promoter after IL-6 stimulation (Fig.
4D). Antibodies directed against CREB and STAT3 were used as
described before (30, 39). However, DNA binding of the
IL-6-dependent complex was not changed by using a STAT3
antibody, whereas DNA binding of one of the two unstimulated complexes
could be inhibited with a CREB antibody (Fig. 4D). The protein of the second unstimulated complex is not known (38).
promoter, we were interested in examining
whether binding of the IL-6-induced factor to the CRE-like binding site
occurs only in the presence of CREB or if the IL-6-induced factor is
able to bind independently of CREB. Therefore, gel shift experiments
with modified liver nuclear extracts as described under "Experimental
Procedures" were performed. We used detergent incubation with
desoxycholic acid and Nonidet P-40 (DOC) to inhibit protein·protein
interaction. However, DNA binding of the IL-6-induced factor was
unchanged after DOC treatment (Fig. 4E). Complete depletion
of liver nuclear extracts with an anti-CREB antibody coupled to protein
A-Sepharose (Prot.A/CREB) also had no effect on DNA binding
of the IL-6-induced factor (Fig. 4E). Thus, these data
indicate that the IL-6-induced factor binds to the CRE-like site in the
LAP/C/EBP
promoter independently of CREB.
promoter. The Southwestern analysis revealed weak binding
of an additional IL-6-stimulated factor with a molecular mass of ~45 kDa.
Promoter Are Essential for
IL-6-dependent Induction--
To further characterize the
significance of the two CRE-like sites for IL-6 induction, we analyzed
mutant LAP/C/EBP
promoter constructs in HepG2 cells (Fig.
5A). Mutations in both
CRE-like sites (LAPPRO 8 MUT I+II) led to a strong decrease in
IL-6-dependent transcription (Fig. 5B). No
stimulation was observed after transfection of the LAPPRO 9 construct
that contains only one CRE-like site. Recently Cantwell et
al. (27) described the cooperative function of STAT3 and Sp1 in
IL-6-mediated C/EBP
induction. We therefore analyzed the deletion
construct LAPPRO 8 WT
, which carries a deletion of 27 nucleotides
between the two CRE-like sites in the GC-rich sequence with possible
Sp1 binding sites. This deletion caused a marked decrease in
IL-6-dependent transcription (Fig. 5B). The data
show that both CRE-like sites contribute to IL-6-mediated LAP/C/EBP
induction and indicate that the spacer sequence between them might be
of functional relevance.
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Fig. 5.
Both CRE-like sites in the
LAP/C/EBP promoter are essential for the
IL-6-dependent induction. HepG2 cells were
cotransfected with 2 µg of the respective LAPPRO luciferase reporter
constructs and with 100 ng of a STAT3 expression plasmid. Cells were
starved as indicated in Fig. 2 and then stimulation was performed with
1000 units of IL-6/ml for 4 h. The relative luciferase activity of
the respective transfection without stimulation was set to 1, and the
changes after IL-6 treatment are shown as -fold stimulation.
A, the following constructs were used: the LAPPRO 8 WT
construct; the LAPPRO 8 MUT I+II construct, where the two CRE-like
sites were mutated; the LAPPRO 8 WT
construct, where a part of the
spacer between the two CRE-like sites is deleted; and the LAPPRO 9 construct, which contains only the second CRE-like site. B,
following IL-6 stimulation the relative changes in luciferase activity
compared with the unstimulated construct are shown as -fold
stimulation.
Transcription--
Our results indicate that STAT3
contributes to IL-6-dependent LAP/C/EBP
transcription
without direct DNA binding to the promoter region. To better understand
the role of different domains of STAT3 in this context, cotransfection
experiments were performed with the LAPPRO8 reporter construct and
expression vectors for different STAT3/STAT1 domain swap mutants and
analyzed after IL-6 stimulation (Fig. 6).
The expression level and the characterization of the domain swap
mutants have been shown before (15).
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Fig. 6.
The amino-terminal domain of STAT3 is
essential for IL-6-mediated LAP/C/EBP
transcription. STAT3/STAT1 domain swap mutants combining
portions of murine STAT3 and human STAT1
are shown. Open
and closed bars represent domains derived from STAT1
and
STAT3, respectively. The numbers on top according to the STAT3 amino
acid sequence indicates boundaries between the domains. In the chimera
nomenclature, parts derived from STAT1
are indicated in brackets:
NT = amino terminus, C-C = coiled-coil
domain, D = DNA-binding domain, Linker = Linker domain, SH2 = SH2 domain, Y = tyrosine phosphorylation site, CT = carboxyl terminus.
HepG2 cells were cotransfected with 2 µg of the LAPPRO 8 luciferase
reporter construct and 100 ng of the respective STAT domain swap mutant
expression vector, starved as described in Fig. 2, and stimulated with
1000 units of IL-6/ml for 4 h. The relative luciferase activity of
the respective transfection without stimulation was set to 1, and the
changes after treatment were determined as -fold stimulation and
presented as mean ± S.D. (left column). No stimulation
(
) corresponds to the effect without cotransfection of any STAT
expression vector, stimulation (+) corresponds to a STAT effect
(right column).
transcription (Fig. 6). Therefore, the SH2 region of STAT3 is essential
for the IL-6 effect, as already implicated by the EpoR transfection
experiments (Fig. 3D). The exchange of the tyrosine region
(Stat 3/1 (Y)), responsible for dimerization of STAT
proteins after phosphorylation by members of the JAK kinase family,
maintained the IL-6 effect (Fig. 6). The amino-terminal part
(NT) of the STAT proteins contributes to several
protein·protein interactions (reviewed in Ref. 41). Substitution to
STAT1 NT (Stat 3/1 (NT)) abolished the IL-6 effect (Fig. 6).
Therefore, our experiments indicate that the amino-terminal region of
STAT3 is required for IL-6-mediated LAP/C/EBP
transcription. The
carboxyl-terminal part (CT) of STAT proteins mediates the
transcriptional activation of target genes (42, 43). The exchange of
the carboxyl-terminal region (Stat 3/1 (CT)) or a mutation
of serine 727 to alanine (Stat 3S/A) maintained IL-6
stimulation (Fig. 6). However, no IL-6-dependent
transcription was found when cotransfection experiments were performed
with carboxyl-terminal deletion mutants (Stat3
715, Stat3
711). A mutation
in the DNA binding region (Stat 3D) as well as substitution
of the STAT3 DNA binding region to STAT1 (Stat 3/1
(D,Linker)) abolished IL-6 induction (Fig. 6).
transcription is
mediated by the STAT3 carboxyl-terminal region and that this function
is exchangeable toward STAT1. In contrast, the amino-terminal part and
the DNA binding region of STAT3 are absolutely required to mediate IL-6
induction, which shows that in addition to the SH2 site these two
domains of STAT3 mediate specificity during this process.
Promoter--
The
experiments using the chimeric STAT3/STAT1 constructs suggested that
interactions at the amino terminus of STAT3 are crucial to mediate
IL-6-dependent transcription of the LAP/C/EBP
promoter. To confirm this hypothesis we fused the amino-terminal part of STAT3 or
STAT1 to the Gal4 DNA binding domain to provide this region with a
nuclear translocation signal and investigated whether overexpression of
the STAT3 construct might act as a dominant-negative inhibitor of
IL-6-dependent LAP/C/EBP
induction (Fig.
7A). Fusion to the Gal4 DNA
binding domain was performed with the amino-terminal (5'Gal4-Stat1NT, 5'Gal4-Stat3NT) as well as with
the carboxyl-terminal domain (Stat1NT-3'Gal4,
Stat3NT-3'Gal4) of the respective STAT amino terminus (Fig.
7A). Both approaches were chosen, because it would minimize
the possible risk of misfolding in the tertiary structure.
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Fig. 7.
Expression of the amino-terminal domain of
STAT3 prevents the IL-6-mediated transcription of the
LAP/C/EBP promoter. A, the
fusion constructs of the amino-terminal domain of STAT1 or STAT3
(corresponding to the natural occurring restriction sites
NotI and SphI) with the DNA binding domain of the
Gal4 protein (nuclear localization signal) are shown. The Gal4 domain
was either cloned 5' of the respective STAT amino terminus
(5'Gal4-Stat1NT and 5'Gal4-Stat3NT) or 3' of the
respective STAT amino terminus (Stat1NT-3'Gal4 and
Stat3NT-3'Gal4). B, expression of the respective
fusion proteins was shown by Western blot analysis. HepG2 cells were
transfected with pBS, 2 µg of 5'Gal4-STAT1NT, 2 µg of
5'Gal4-STAT3NT, 1 µg of STAT1NT-3'Gal4, or 1 µg of STAT3NT-3'Gal4
as indicated. Cells were harvested 48 h after transfection;
nuclear extracts were prepared and analyzed by Western blotting with a
Gal4-specific antibody. C, HepG2 cells were cotransfected
with 2 µg of the LAPPRO 8 luciferase reporter construct, without (
)
or with (+) 100 ng of a STAT3 expression vector (Stat3), and
with increasing amounts (100, 500, 1000, and 2000 ng) of the
5'Gal4-STAT3NT, the 5'Gal4-STAT1NT, or the Gal4 construct. Cells were
starved as indicated in Fig. 2 and then stimulation was performed with
1000 units of IL-6/ml for 4 h. The relative luciferase activity
without stimulation for the respective transfection was set to 1, and
the changes after IL-6 treatment are shown as -fold stimulation.
D, HepG2 cells were cotransfected with 2 µg of the LAPPRO
8 luciferase reporter construct, without (
) or with (+) 100 ng of a
STAT3 expression vector (Stat3) and with increasing amounts
(10, 25, 50, and 100 ng) of the STAT3NT-3'Gal4 or the STAT1NT-3'Gal4
construct. Cells were starved as indicated in Fig. 2 and then
stimulation was performed with 1000 units of IL-6/ml for 4 h. The
relative luciferase activity without stimulation for the respective
transfection was set to 1, and the changes after IL-6 treatment are
shown as -fold stimulation.
induction in a
concentration-dependent manner, whereas the 5'Gal4-STAT1NT
and the Gal4 alone showed no effect (Fig. 7C). The same
results were found with the 3' fusion constructs. STAT3NT-3'Gal4 prevented IL-6 induction, whereas STAT1NT-3'Gal4 had no influence (Fig.
7D). However, the STAT3NT-3'Gal4 constructs were
significantly more effective (concentration range 10-100 ng) compared
with the 5'Gal4-STAT3NT constructs (concentration range 100-1000 ng)
(Fig. 7, C and D). These experiments clearly show
that the amino-terminal domain of STAT3 is able to act as a
dominant-negative inhibitor, and therefore, these results suggest that
the STAT3 amino terminus has an important function in IL-6-mediated
transcription of the LAP/C/EBP
gene.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and C/EBP
, which then activate transcription of several
acute phase response genes. Earlier results showed that the C/EBP
promoter contains two Sp1 binding sites, an STAT3 binding site, and a
CRE-like binding site in close proximity to the TATA-box. For the
IL-6-dependent increase in C/EBP
gene
transcription, the STAT3 and the Sp1 DNA binding sites function
cooperatively, whereas the CRE-like site does not participate in this
regulation (27, 28). STAT3 is also supposed to contribute to
IL-6-dependent transcription of the LAP/C/EBP
gene (2, 44).
Transcription--
Our in vivo and in
vitro experiments demonstrated that the IL6/gp130/STAT3 pathway
induces higher LAP/C/EBP
gene transcription. An increase
in LAP/C/EBP
gene transcription was also reported after
lipopolysaccharide (LPS) stimulation and hepatocyte growth factor treatment. This is mediated by an autoregulatory loop in which phosphorylation of LAP/C/EBP
increases transactivation and
thus higher LAP/C/EBP
gene transcription. The
LAP/C/EBP
binding site(s), which contribute to this regulation, were
mapped outside the IL-6-responsive region characterized in our study (45, 46). IL-6 also stimulates the MAP kinase pathway, which has been
shown before to lead to post-translational activation of LAP/C/EBP
by its phosphorylation (4). This mechanism could be further excluded,
because the EpoR/Map site chimera was unable to induce LAP/C/EBP
transcription, indicating that an autoregulatory loop does not explain
our observations.
Promoter Contains
No STAT3 Consensus Sequence and Is Regulated through Two CRE-like
Binding Sites--
Analysis of the whole 1.4-kb LAP/C/EBP
promoter
by transfection experiments revealed that the three potential STAT DNA
binding sites are not involved in mediating the increase in
IL6-dependent gene transcription. In contrast, the recently
described CRE-like sites (
111 to
104 and
65 to
58) in the
promoter control this effect, and IL-6 stimulates DNA binding of a
68-kDa protein to these sequences. Induction of CRE-like site binding
proteins after IL-6 stimulation was already reported for the STAT3 (47)
and JunB promoter (48). However, these proteins were not further characterized. Meanwhile, the ATF/CREB family comprises a growing number of proteins that recognize the CRE-consensus or CRE-like binding
sites (reviewed in Ref. 49). The mechanism for the induction of the DNA
binding activity of the CRE-like site binding proteins after IL-6
stimulation is not known.
promoter resulted
in a lack of p68 binding and in a decrease in
IL-6-dependent transcription. Mutation of the first site
had a stronger effect on IL-6 inducibility than that of the second site
(data not shown), but strongest inhibition was observed with the double
mutant construct. Additionally, our results indicate that the spacer
region between the two CRE sites is important in mediating
IL-6-dependent gene transcription. To exclude the
possibility that other transcription factors are involved in this
regulation, we subcloned the two CRE sites with the wild type or an
unrelated spacer region in a heterologous promoter. These results
showed that IL-6 inducibility is not dependent on the sequence of the
spacer region (data not shown), indicating that only the spacing
between the CRE sites and no factors binding to this region are crucial
to confer IL-6 inducibility of the LAP/C/EBP
promoter.
Promoter--
Our results suggested that STAT3 contributes to
higher IL-6-dependent LAP/C/EBP
gene
transcription without DNA binding. Further analyses with STAT3/STAT1
domain swap mutants suggested that the amino-terminal part, the SH2
domain, and the DNA binding domain of STAT3 are crucial for the
IL-6-dependent increase in LAP/C/EBP
gene
transcription. The essential role of the STAT3 DNA binding domain for
this regulation was unexpected. Our mutation analysis in the
LAP/C/EBP
promoter clearly showed that only the CRE-like sites are
relevant for IL-6 inducibility. Consequently, STAT3 would have to bind
to this region in a nonspecific manner. Thus we cannot completely rule
out the possibility that STAT3 contributes to p68 DNA binding through a
yet undefined mechanism, which is not detected by gel shift
experiments. To prove this possibility we also depleted IL-6-induced
nuclear extracts from STAT3 as shown for CREB. However, STAT3-depleted
extracts had no influence on DNA binding of p68 (data not shown).
Therefore we favor the possibility that the DNA binding domain of STAT3
contributes to protein·protein interaction. In a recent report (50),
parts of the STAT3 DNA binding domain are shown to be involved in
mediating the interaction between STAT3 and c-Jun.
promoter. Thus this interaction seems to mediate
specificity during the regulation. The Gal4 part was fused to both the
carboxyl-terminal and the amino-terminal end of the STAT3 amino
terminus to minimize steric effects. Fusion to the carboxyl-terminal
end (STAT3NT-3'Gal4) resulted in a 10-fold more potent
dominant-negative inhibitor than fusion to the amino-terminal end
(5'Gal4-STAT3NT). The difference between the two constructs might be
best explained by the localization of the interacting region.
-mediated apoptosis, where SH2 mutants can restore wild-type
function in STAT1 knockout cells, lead to the model that STAT1 might be
recruited to a promoter through protein·protein interaction with a
DNA-bound partner (56, 57). STAT3 has also been reported as a
transcriptional coactivator without association with its DNA binding
motif. IL-6-activated STAT3 associates with ligand-bound glucocorticoid
receptor to form a transactivating complex bound to a glucocorticoid
response element (58). Besides the model of the tethered coactivator function for STAT3, there is evidence for its role during other mechanisms not related to DNA binding. An adapter function for STAT3
has been implicated in the recruitment of phosphatidylinositol 3-kinase
to the IFN-
receptor (59).
transcription indicates that these mechanisms could be directly linked
to each other. Therefore, based on our results we would like to propose
a hypothetical model explaining the role of STAT3 during
IL-6-dependent activation of LAP/C/EBP
gene
transcription. We suggest that IL-6 activates STAT3 and DNA binding of
p68. STAT3 is tethered to the p68-containing complex at the CRE-like
site in the LAP/C/EBP
promoter. In this regulation, STAT3 would act as transcriptional coactivator whereby the carboxyl-terminal region interacts with the basal transcription machinery and activates LAP/C/EBP
transcription while the amino terminus may interact with
p68 or other related factors. It is conceivable that certain steric
conditions are necessary for tethering STAT3 by p68 bound to both
CRE-like sites. This would explain the critical distance between the
two sites for IL-6 induction. DNA binding can affect the quaternary
structure of transcriptional regulators and thereby determine
heterodimerization partners (60). This conformational change would
allow tethering of STAT3 to p68 after binding to the CRE-like site in
the LAP/C/EBP
promoter.
Promoter--
Among mouse, rat, and humans the two
CRE-like sites and the GC-rich linker sequence are highly conserved
regulatory elements in the LAP/C/EBP
promoter (61). Recently, we
have reported that basal and protein kinase A-inducible LAP/C/EBP
promoter activity in cells of hepatic and neuronal origin requires the synergistic activity of the two CRE-like sites (29). Since then additional examples for the relevance of a cAMP-dependent
regulation of LAP/C/EBP
through these sites were described during
memory formation in hippocampal neurons (62), in human endometrial stroma cells as part of prolactin induction (63), in sertoli cells
(64), for the gonadotropin response in granulosa cells (65, 66), and
during adipocyte differentiation (67).
promoter in monocytic cells showed the relevance of an
interplay between an Sp1 site and the second CRE-like site for basal
activity and for PMA or LPS stimulation, whereas the first CRE-like
site was not involved in this regulation (61). In hepatocytes both
CRE-like sites are required and recognized by different activators and
coactivators, which contribute to the regulation of
LAP/C/EBP
gene transcription during the acute phase
response and during liver regeneration. The increase in promoter
activity varied after protein kinase A stimulation (29) and as shown
here after IL-6 treatment between 6- and 20-fold. The mechanisms, which
contribute to these differences, are currently unknown. However, for
example, after IL-6 stimulation, binding of an additional factor p45 as
shown by Southwestern analysis could be detected, which might be a
possible heterodimerization partner for p68 modulating its activity.
Additionally, the proliferative state of the cell may directly modulate
the basal activity of the LAP/C/EBP
promoter. Therefore, to better
understand all the different transcriptional control mechanisms under
various physiological conditions, it might be necessary in the future
to further characterize the transcription factors binding to the
CRE-like sites and to identify other possible interacting coactivators.
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ACKNOWLEDGEMENT |
---|
We thank Valeria Poli, Dundee, UK, for carefully reviewing the manuscript and for valuable comments.
![]() |
FOOTNOTES |
---|
* This work was supported by the Sonderforschungsbereich 566, project B08.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.
This work is dedicated on the occasion of Prof. Dr. W. Gerok's 75th birthday.
§ Present address: Fraunhofer-Institut, D-30625 Hannover, Germany.
¶ Present address: Dianova, D-20354 Hamburg, Germany.
** To whom correspondence should be addressed: Dept. of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. Tel.: 49-511-532-3489; Fax: 49-511-532-4896; E-mail: trautwein.christian@mh-hannover.de.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M009284200
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ABBREVIATIONS |
---|
The abbreviations used are:
C/EBP, CAAT/enhancer-binding protein;
LPS, lipopolysaccharide;
IL-1, interleukin 1;
IFN-, interferon
;
IFN-
R, IFN-
receptor;
MAP, mitogen-activate protein;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
EpoR, erythropoietin receptor;
CMV, cytomegalovirus;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl fluoride;
DOC, desoxycholic acid;
kb, kilobase(s);
PIAS3, protein inhibitor of
activated STAT3;
CRE, cAMP-response element;
CREB, CRE-binding protein;
SIE, sis-inducible element;
STAT1, -3, signal transducer and activator
of transcription-1, -3;
JAK, Janus kinase.
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REFERENCES |
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