From the Department of Gastroenterology and Hepatology,
Medizinische Hochschule, Hannover, Federal Republic of Germany and
I. Medical Clinic, Section of Pathophysiology, University
of Mainz, Federal Republic of Germany
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ABSTRACT |
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Interleukin-6 (IL-6) triggers pivotal pathways
in vivo. The designer protein hyper-IL-6 (H-IL-6) fuses the
soluble IL-6 receptor (sIL-6R) through an intermediate linker with
IL-6. The intracellular pathways that are triggered by H-IL-6 are not
defined yet. Therefore, we studied the molecular mechanisms leading to
H-IL-6-dependent gene activation. H-IL-6 stimulates
haptoglobin mRNA expression in HepG2 cells, which is
transcriptionally mediated as assessed by run-off experiments. The
increase in haptoglobin gene transcription correlates with higher
nuclear translocation of tyrosine-phosphorylated STAT3 and its DNA
binding. As H-IL-6 stimulates STAT3-dependent gene
transcription, we compared the molecular mechanism between IL-6 and
H-IL-6. Transfection experiments were performed with a
STAT3-dependent luciferase construct. The same amount of
H-IL-6 stimulated luciferase activity faster, stronger, and for a
longer period of time. Dose response experiments showed that a 10-fold lower dose of H-IL-6 stimulated STAT3-dependent gene
transcription comparable with the higher amount of IL-6. Cotransfection
with the gp80 and/or gp130 receptor revealed that the effect of H-IL-6 on STAT3-dependent gene transcription is restricted to the
gp80/gp130 receptor ratio. High amounts of gp130 increased and high
amounts of gp80 decreased the effect on H-IL-6-dependent
gene transcription. To investigate the in vivo effect of
H-IL-6 on gene transcription in the liver, H-IL-6 and IL-6 were
injected into C3H mice. H-IL-6 was at least 10-fold more effective in
stimulating the DNA binding and nuclear translocation of STAT3, which
enhances haptoglobin mRNA and protein expression. Thus H-IL-6
stimulates STAT3-dependent gene transcription in liver
cells in vitro and in vivo at least 10-fold
more effectively than IL-6. Our results provide evidence that H-IL-6 is
a promising designer protein for therapeutic intervention during
different pathophysiological conditions also in humans.
Cytokines are important to maintain several physiological and
pathophysiological functions in vivo. Binding to a specific membrane receptor activates intracellular signaling pathways, which
trigger a variety of intracellular events. IL-6 is a cytokine with
pleiotropic functions in the immune system, hematopoietic cells,
hepatocytes, and the nervous system. It belongs to a family consisting
of IL-6,1 CNTF, LIF, OSM,
IL-11, and CT-1. All these family members interact with a ligand
binding domain that confers specificity. The different ligand binding
domains are all linked to the signal transducer gp130 (1, 2). Three
members of the Janus tyrosine kinase family, Jak1, Jak2, and Tyk, are
constitutively associated with the intracellular domain of gp130 (3,
4).
IL-6 associates with the IL-6 receptor (IL-6R, gp80). Binding of IL-6
to its receptor induces homodimerization of gp130 resulting in
autophosphorylation of the associated Jak kinases, which in turn
phosphorylate gp130. Phosphorylation of gp130 creates docking sites for
SH2 domains containing signaling molecules (5). At least two signaling
cascades have been characterized that either trigger STAT3 or
mitogen-activated protein kinase activation (6). After association with
the gp130 receptor, STAT3 becomes phosphorylated at tyrosine 705 through Jak kinases (7). Tyrosine phosphorylation of STAT3 results in
homo- or heterodimerization of STAT3 and its nuclear translocation (8).
In the nucleus STAT3 binds to target sequences in different promoters
and enhances gene transcription (8). In pro-B cells, STAT3 is required
for bcl-2 induction, and thus has an anti-apoptotic effect. The
mitogen-activated protein kinase pathway, which also diverges at the
intracellular domain of gp130, involves the activation of ras
and is essential for cell cycle progression and induction of
DNA synthesis (6).
In hepatocytes, IL-6 induces the synthesis of acute phase plasma
proteins, which play a protective role during the acute phase response
(9). Binding sites for STAT3 are located in most of the promoters of
the acute phase genes, and originally STAT3 was also called acute phase
response factor (APRF) (10). Besides the role of IL-6 in inducing the
acute phase response, there is evidence that through IL-6, STAT3 is
also activated during liver regeneration (11, 12). Additional, recent
experiments in IL-6 and tumor necrosis factor receptor 1 knockout mice
show that IL-6 is essential to induce cell cycle progression during
liver regeneration. In both mice there is a lack of DNA synthesis after
hepatectomy that can be prevented by IL-6 injection (13, 14).
Therefore, IL-6 and, thus, the activation of the
intracellular-signaling cascades that diverge at the gp130 transducer
molecule are essential to maintain and restore liver function during
different conditions.
Assembly of IL-6 with the IL-6 receptor and the gp130 molecule requires
the association of three different molecules (15, 16). The on-off rate
on the cell membrane and the availability of each partner might be a
rate-limiting step. A designer protein (H-IL-6), which fuses IL-6 to
the soluble IL-6 receptor through an intermediate linker, might also be
of therapeutic benefit in humans, for example during infection or liver
injury (17). Therefore, we were interested in studying the effect of
H-IL-6 on gene transcription in vivo and in vitro
in liver cells. We show that H-IL-6 is severalfold more potent in
vivo and in vitro in activating
STAT3-dependent gene transcription. The effect on gene
transcription is dependent on the gp80/gp130 ratio on a given cell.
Therefore, H-IL-6 could be of therapeutic benefit during different
pathophysiological conditions also in vivo.
Recombinant H-IL-6 and IL-6--
Recombinant H-IL-6 was
synthesized in yeast cells as described earlier (17). The H-IL-6
protein was purified from yeast supernatants by anion-exchange
chromatography and gel filtration. The purified H-IL-6 fraction was
visualized by running on a SDS-polyacrylamide gel electrophoresis
followed by silver staining. Human IL-6 was produced in
Escherichia coli and purified as described previously (18).
IL-6 Determination--
IL-6 serum concentrations obtained from
the liver vein were measured essentially as described previously
(12).
Stimulation of Mice and Preparation of Liver Nuclear
Extracts--
C3H mice were either stimulated with H-IL-6 or IL-6
intraperitoneally by the doses indicated. Tissue for Northern blot
analysis or preparation of liver nuclear extracts was perfomed at
different time points after injection. At each time point at least
three animals were used in parallel.
For preparation of nuclear extracts the pooled livers were rinsed in
freezing phosphate-buffered sulfate, and liver nuclear proteins were
prepared as described previously (19). All the steps were performed at
4 °C. Nuclear proteins were aliquoted and frozen immediately in
liquid nitrogen.
Cell Culture, Transfection Experiments, and Luciferase
Assays--
HepG2 cells (ATCC) were cultured in Dulbecco's modified
essential medium supplemented with 10% fetal calf serum. DNA
transfection into HepG2 cells was performed as described previously
(19). The reporter construct used in these experiments represents the promoter of the SDS-polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Liver nuclear proteins or 20 µg of serum were
separated on a 10% SDS-polyacrylamide gel (19) and blotted onto a
nitrocellulose membrane (Schleicher & Schuell) in 1% SDS, 20%
methanol, 400 mM glycine, 50 mM Tris-HCl, pH
8.3, at 4 °C for 2 h at 200 mA. STAT3 and phospho-STAT3 was
visualized using polyclonal antibodies supplied by Santa Cruz
Biotechnology (Santa Cruz, CA) and haptoglobin by using a polyclonal
rabbit antibody supplied from DAKO (Hamburg, Germany). The
antigen-antibody complexes were visualized using the ECL detection
system as recommended by the manufacturer (Amersham, Braunschweig, Germany).
Gel Retardation Assays--
For gel retardation assays, liver
nuclear extracts were used as indicated. The binding reaction was
performed for 20 min on ice (22). For binding assays, an
oligonucleotide spanning the STAT3 site in the Northern Blot Analysis--
Total RNA was isolated by the
guanidium isothiocyanate method from the liver of the mice or from
HepG2 cells at the time points indicated. Northern blot analysis was
performed as described before, according to standard procedures (19).
15 µg of total RNA was analyzed through a 1% agarose formaldehyde
gel, followed by transfer to Hybond N membranes (Amersham). The
haptoglobin and GAPDH cDNA probes were labeled with
[ Run-off Assays--
Run-off experiments were performed
essentially as described before (12). 5 × 107
isolated nuclei were incubated in reaction buffer (5 mM
Tris-Cl, pH 8.0, 2.5 mM MgCl2, and 0.15 M KCl) containing 10 ml of 100 mM ATP, 10 ml of
100 mM CTP, 10 ml of 100 mM GTP, and 10 ml of 10 mCi/ml [
For hybridization, 6 µg of cDNA was denatured and fixed on nylon
membrane in a total dilution volume of 100 ml TES buffer. DNA was fixed
for 1 h at 80 °C.
Prehybridization was performed for 1 h, 65 °C in hybridization
buffer (20 mM TES, pH 7.4, 10 mM EDTA, 400 mM NaCl, 0.2% SDS, and 2× Denhardt's solution).
Hybridization was performed by adding the RNA-equivalent of 2 × 106 counts/ml in hybridization buffer in a 5-ml
polypropylene tube at 65 °C overnight. After extensive washing in
2× SSC and 0.2% SDS, a final RNase digestion (8 ml of 2× SSC and
8-10 mg/ml RNase A) was performed.
For quantification, exposure was performed on a Fuji image plate. After
subtraction of the vector control signal, counts of the haptoglobin
signal were distributed through the GAPDH value and set to 1. The
values at different time points after IL-6 or H-IL-6 treatment were
shown as fold activation compared with the pretreatment level.
Quantification--
Quantification of results was performed with
a Fuji imager as described before (19).
H-IL-6 Stimulates Transcription of the Haptoglobin
Gene--
Several intracellular pathways are located downstream of the
intracellular domain of the gp130 molecule (6). We were interested in
studying whether H-IL-6 can stimulate gene expression on a transcriptional level in liver cells. Therefore HepG2 cells were stimulated with H-IL-6, and the mRNA expression was studied after different time points. Northern blot analysis showed that after 10 ng/ml H-IL-6 haptoglobin mRNA expression was increased for up to
96 h (Fig. 1A).
Quantitative analysis was performed by comparing the haptoglobin signal
with the GAPDH signal. These results showed that 1 h after H-IL-6
stimulation, haptoglobin mRNA expression was already more than
5-fold enhanced to the pretreatment level. The most prominent increase
was found 15 h after stimulation exceeding the pretreatment level
more than 120-fold (Fig. 1B).
In further experiments, run-off analysis was performed to demonstrate
that the effect on haptoglobin mRNA expression is transcriptionally mediated. As shown in Fig. 1, C and D, the
transcription rate of the haptoglobin gene was already more than
16-fold enhanced 45 min after H-IL-6 stimulation. At later time points,
the transcription rate of the haptoglobin gene decreased. However
15 h after stimulation, the transcription rate was still
more than 6-fold higher compared with the pretreatment level. Thus
these results showed that H-IL-6 stimulates haptoglobin mRNA
expression via higher gene transcription.
H-IL-6 Induces the Nuclear Translocation, Tyrosine Phosphorylation,
and DNA Binding of STAT3--
H-IL-6 induces higher transcription of
the haptoglobin gene. The transcription rate of the haptoglobin gene
during the acute phase response in the liver is controlled via STAT3,
and therefore its nuclear expression was studied following H-IL-6
stimulation (Fig. 2A). Higher
STAT3 expression was immediately found after H-IL-6 stimulation, and
the effect lasted for up to 9 h. Increased nuclear translocation
of STAT3 was associated with an increase in tyrosine phosphorylation of
STAT3 (Fig. 2B). Before stimulation, no
tyrosine-phosphorylated STAT3 was detected in the nucleus and a clear
increase in intensity of the STAT3 tyrosine-phosphorylated band was
obvious 5 min after stimulation. Strong STAT3 tyrosine phosphorylation
was observed for up to 1 h after H-IL-6 stimulation. A weak
signal, higher than the pretreatment level, was still detected for up
to 12 h.
H-IL-6 stimulation of HepG2 cells also increased the DNA-binding of
STAT3 versus its cognate DNA. As shown in Fig. 2C
by gel shift experiments, new complex formation was detected 5 min
after stimulation and no DNA binding was detected again 6 h
after treatment with H-IL-6. STAT3 binding to the cognate DNA was
confirmed by supershift experiments (Fig. 2D).
The results shown in Figs. 1 and 2 indicate that H-IL-6 triggers STAT3
translocation and haptoglobin gene transcription to maximal levels
during the first hour after stimulation. However, because of the
accumulation of haptoglobin mRNA levels, maximal haptoglobin
mRNA expression was found only 15 h after stimulation. These
results indicate that up to this time point the increase in haptoglobin
mRNA levels is higher than its subsequent degradation.
Stimulation of STAT3-dependent Gene Transcription via
H-IL-6 and IL-6--
H-IL-6 stimulates nuclear STAT3 translocation,
and this increase is associated with higher haptoglobin mRNA
transcription. We performed further experiments to investigate whether
H-IL-6 and IL-6 have comparable effects on STAT3-dependent
gene transcription. Thus we transfected HepG2 cells with a
STAT3-dependent reporter gene construct and stimulated the
cells with 10 ng/ml of IL-6 or H-IL-6. Cells were harvested at
different time points after stimulation (Fig.
3A). 1 h after
stimulation the reporter activity in the cells activated with H-IL-6
was more than 10-fold higher compared with the pretreatment level and
at least 2-fold higher compared with the level found in the
IL-6-stimulated cells at the same time point. At all later time points
H-IL-6 leads to at least 2-fold higher luciferase activity compared
with the cells stimulated with IL-6. Because the half-life of the
luciferase protein is around 3 h, the high luciferase activity
found 24 and 48 h after H-IL-6 administration indicates that there
is still an increased transcription rate after these intervals.
Additionally, when the luciferase activity of the H-IL-6 treated cells
was compared with those treated with IL-6, these results showed that at
later time points the difference increased even more. Therefore, the absolute luciferase activity after H-IL-6 treatment at the different time points was set at 100%. The activity after IL-6 stimulation was
expressed as percentage of the H-IL-6-treated cells at the same points.
As shown in Fig. 3B, the relative percentage of the luciferase activity of IL-6-treated cells decreased with time (51%
after 4 h versus 38% after 48 h), which indicates
that after H-IL-6 stimulation the effect on gene transcription was not
only stronger but also more prolonged compared with the IL-6
treatment.
In further experiments the dose-dependent effect of H-IL-6
on STAT3-dependent gene transcription was assessed. The
cells were harvested 4 h after stimulation. As shown in Fig.
3C in the range between 0.1 and 10 ng, a 10-fold higher dose
of IL-6 compared with H-IL-6 leads to similar effect on gene
transcription. When 100 ng/ml of each protein were used there was
no further increase in gene transcription compared with 10 ng/ml of
IL-6 or H-IL-6. However the difference on gene transcription between
IL-6 and H-IL-6 remained the same (Fig. 3C).
STAT3-dependent Gene Transcription Activated Through
H-IL-6 or IL-6 Depends on the gp130 and gp80/IL-6R Status--
To
further understand the mechanism that is responsible for the increased
effect of H-IL-6 compared with IL-6 on gene transcription, we performed
cotransfection experiments with either the gp130, gp80/IL-6R or both
receptors (Fig. 4, A and
B).
First, the cells were treated with H-IL-6 when increasing amounts of
the gp130 receptor was cotransfected with the
STAT3-dependent reporter gene construct (Fig.
4A). These experiments revealed that by increasing the
amount of gp130, the luciferase activity could be further stimulated. A
30% increase in luciferase activity was found when 1 µg of the gp130
plasmid was used. When the highest amount (4 µg) of the gp130 plasmid
was cotransfected the maximal luciferase activity dropped again but was
still 15% higher compared with the level when no gp130 was
cotransfected. In contrast to the gp130 experiments, cotransfection of
the gp-80/IL-6R construct with the STAT3-dependent reporter
plasmid leads to a direct reduction of luciferase activity after H-IL-6
stimulation compared with the level when no gp80/IL-6R was added. When
gp130 and gp80/IL-6R were cotransfected and stimulated with H-IL-6, low
amounts of the plasmids led to a reduction in luciferase activity
compared with the cells that were not transfected with both receptors.
The same cotransfection experiments with the different receptor
combinations were performed when the cells were stimulated with IL-6
(Fig. 4B). Cotransfection of increasing amounts of the gp130
receptor with the STAT3-dependent reporter gene showed the following results compared with the initial activity when no receptor was added (control). 500 ng of gp130 receptor resulted in a 19% increase compared with control. In contrast, when 100, 1,000, or 4,000 ng were transfected, luciferase activity was
Cotransfection experiments of lower amounts (100, 500, and 1,000 ng) of
the gp80/IL-6R plasmid led to an increase in luciferase activity that
was maximal with 500 ng (+77%). However, when 4,000 ng of gp80/IL-6R
were cotransfected, the activity of the STAT3-dependent reporter construct decreased again. A similar kinetic was found when
both receptors were cotransfected. Low amounts of gp80 and gp130
increased, and high amounts decreased luciferase activity (Fig.
4B).
H-IL-6 Is more Active than IL-6 in Stimulating Acute Phase Gene
Expression in Vivo--
The in vivo relevance of H-IL-6 was
still unclear. As our results in cell culture experiments showed,
mainly that gp130 is essential for the interaction with H-IL-6,
additional cells may bind the molecule. Therefore it seemed possible
that in vivo activation of STAT3-dependent gene
transcription in liver cells might be less effective. 4 µg of H-IL-6
or 40 µg of IL-6 were injected intraperitoneally into C3H mice
according to the in vitro results where H-IL-6 was 10-fold
more active. At different time points after injection IL-6 serum levels
were determined in the liver vein, and haptoglobin mRNA expression
was measured in the liver.
As shown in Fig. 5, A and
B, haptoglobin mRNA increased to similar levels when
10-fold higher amounts of IL-6 were injected compared with H-IL-6.
After normalization with the GAPDH signals, some of the time points (1, 6, and 12 h) determined after IL-6 stimulation were higher
compared with the corresponding time points after H-IL-6 injection
(Fig. 5C).
As H-IL-6 might have a different half-life in vivo compared
with IL-6, the IL-6 serum concentration was measured in the liver vein
(Fig. 6, A and B).
The IL-6 serum concentration after H-IL-6 injection was more than
10-fold lower compared with the levels found after IL-6 administration
0.5 h after injection. At later time points, the difference
between the H-IL-6- and IL-6-treated animals was even more dramatic.
For example, after 3 h the IL-6 concentration in the
H-IL-6-treated animals was more than 20-fold lower compared with the
IL-6 treated mice.
The haptoglobin protein expression in the serum of the mice was
determined by Western blot analysis. Time and dose kinetic experiments
were performed with 20 µg of serum. Four different doses of either
IL-6 or H-IL-6 were used. As H-IL-6 was 10-fold more active in inducing
haptoglobin mRNA expression, the dose kinetics were started with a
10-fold lower amount of H-IL-6 compared with IL-6. As shown in Fig.
7, A and B, the
mice were stimulated for 4 h with 0.04, 0.4, 4, or 40 µg of IL-6
and 0.004, 0.04, 0.4, or 4 µg of H-IL-6. These results showed that a
10-fold lower amount of H-IL-6 had a similar effect on haptoglobin
serum expression as did the higher IL-6 concentration (see Fig. 7,
A and B).
The dose kinetic experiments indicated that H-IL-6 was 10-fold more
active than IL-6, therefore time kinetic experiments were performed
with either 40 µg of IL-6 or 4 µg of H-IL-6. Fig. 7, C
and D, shows that in vivo the 10-fold lower
amount of H-IL-6 induced at least the same strength in haptoglobin
expression in the time kinetic experiment as the higher amount of
IL-6. Denistometric evaluation revealed that after 6 h,
haptoglobin expression was nearly 2-fold after H-IL-6 compared with
IL-6 stimulation. At later time points, the kinetic of 40 µg of IL-6
and 4 µg of H-IL-6 was not significantly different. Loading of
the gels was checked by Coomassie staining (data not shown).
Therefore, the experiments as shown in Figs. 5-7 demonstrated that
in vivo H-IL-6 was at least 10-fold more active in inducing haptoglobin mRNA and protein levels.
In Vivo H-IL-6 Induces Stronger Nuclear Translocation of STAT3
Compared With IL-6--
In further experiments, we investigated
whether mainly STAT3 is involved in regulating higher haptoglobin
mRNA levels. Thus, the nuclear translocation and tyrosine
phosphorylation of STAT3 was studied in liver nuclei. Liver nuclear
extracts were prepared from the mice that were either treated with 4 µg of H-IL-6 or 40 µg of IL-6.
Western blot analysis was performed using anti-STAT3 and
anti-phospho-STAT3 antibodies. H-IL-6 and IL-6 induced an increase in
nuclear STAT3 expression and tyrosine-phosphorylated STAT3 30 min after
injection (Fig. 8). In the H-IL-6 treated
animals at later time points, the nuclear concentration of STAT3
continuously fell and reached pretreatment levels 6 h after
injection. After IL-6 injection, the nuclear concentration of
phospho-STAT3 remained high for the first 2 h. At later time
points the nuclear concentration decreased, and no nuclear
tyrosine-phosphorylated STAT3 was found after 12 h again. The time
kinetic of the nuclear expression of tyrosine-phosphorylated STAT3 was,
therefore, closely linked to the kinetic found for the IL-6 serum
levels (Fig. 6).
The nuclear translocation of STAT3 is associated with an increased
affinity toward its cognate DNA motif. Perhaps additionally posttranscriptional modifications might be involved in mediating DNA
binding of STAT3 and target gene transcription (23, 24). Therefore, gel
shift experiments were performed with nuclear extracts to study whether
H-IL-6 also triggers higher DNA binding. New complex formation was
found 30 min after H-IL-6 and IL-6 injection (Fig.
9, A and B). As
shown by supershift experiments, the complex was completely
supershifted by an anti-STAT3 antibody (Fig. 9C). Increased
DNA binding of STAT3 after IL-6 and H-IL-6 injection closely followed
the kinetic of the nuclear translocation of tyrosine-phosphorylated STAT3. After H-IL-6 injection STAT3 showed its strongest affinity toward its cognate DNA after 30 min, and a decrease in DNA-binding was
found at later time points. No complex formation was detected at the
6 h time point. After IL-6 treatment, a different time kinetic was
found. The intensity of the STAT3 complex was lower compared with
H-IL-6 stimulation after 1 h and remained high for up to 4 h.
The DNA-binding of STAT3 slightly decreased only after this time point,
and no complex formation was detected after 12 h. Additionally,
after H-IL-6 stimulation a second faster migrating complex was found
1 h after injection. As IL-6 can also activate STAT1 (25),
DNA-binding competition experiments were performed with antibodies that
either detect STAT1- IL-6 is a pleiotropic cytokine with several essential functions
involved in the induction of immunoglobulin secretion of B cells, the
maturation of megakaryocytes, colony formation of hematopoietic stem
cells, and growth and differentiation of T cells (9, 17, 26). IL-6 is
essential for the activation of the acute phase response in the liver
and for triggering liver regeneration (13, 27). Therefore, designer
molecules that activate IL-6-dependent intracellular
signals would offer the possibility of therapeutic intervention during
different pathophysiological conditions.
In this study, we investigated whether H-IL-6 might activate
STAT3-dependent gene transcription in vitro and
in vivo and whether the gp130 to gp80/IL-6R status on the
cell surface might influence STAT3-dependent gene
transcription. H-IL-6 enhances transcription of the haptoglobin gene in
HepG2 cells, which is associated with higher nuclear translocation,
tyrosine phosphorylation, and DNA binding of STAT3. These results show
that the designer protein H-IL-6 activates homodimerization of two
gp130 molecules and phosphorylation of the intracellular tyrosines,
which trigger STAT3 activation. In run-off experiments and by using a
STAT3-dependent reporter gene construct, we show that the
same amount of H-IL-6 induces 2-3-fold higher gene transcription
compared with IL-6. When the cells were transfected with the
STAT3-dependent reporter construct and stimulated with
increasing amounts of H-IL-6 or IL-6, a 10-fold lower amount of H-IL-6
led to the same luciferase activity as the higher IL-6 concentration.
Therefore H-IL-6 leads to 10-fold stronger activation of
STAT3-dependent gene transcription. Additionally, differences were found in the time kinetic. 1 h after stimulation the luciferase activity in H-IL-6-treated cells was 3-fold higher compared with IL-6. After 4 h the difference was 2-fold and at later time points the difference between the two molecules increased again. These differences in the kinetic of STAT3-dependent
gene transcription after H-IL-6 and IL-6 stimulation is most likely explained on the receptor level.
Recent experiments published by Somers et al. (28) suggested
that the first event in IL-6 signal transduction is the binding of IL-6
through site1 to gp80/IL-6R. The second event is the binding of this
heterodimer to the gp130 molecule, which results in the formation of a
trimeric complex on the surface of the cell. The third event is the
association of two trimeric to a hexameric complex, which results in
intracellular dimerization of two gp130 molecules and the activation of
intracellular-signaling cascades (28). The first step is characterized
by binding of two specific and low affinity partners. In the case of
H-IL-6, this first event is not necessary. This advantage may trigger
faster assembly and gp130 dimerization, which results in a more rapid
STAT3 phosphorylation and luciferase activity as shown in the cell
culture experiments. A second mechanism, which might explain the more
rapid effect on gene transcription after H-IL-6 compared with IL-6
stimulation, is the observation that IL-6 might form IL-6/IL-6 dimers.
The IL-6 homodimer has higher affinity toward gp80/IL-6R; however, a
complex consisting of two IL-6 and gp80/IL-6R molecules is less potent
in complex formation with gp130 and inducing STAT3 phosphorylation (29). Therefore, H-IL-6 might preferentially first lead to a monomeric
IL-6·gp80·gp130 complex, which more rapidly induces hexamer
formation, STAT3 phosphorylation, and thus gene activation.
Besides the faster early events, H-IL-6 has also a more pronounced
effect on gene transcription compared with IL-6 as assessed by
haptoglobin Northern blot analysis, run-off assays, and
STAT3-dependent luciferase reporter gene assays in HepG2
cells. The hexameric IL-6·gp80·gp130 complex at the surface of
hepatocytes is dissolved by internalization and degradation of IL-6
(30, 31). A di-leucine internalization motif in the cytoplasmic domain
of gp130 seems essential for controlling IL-6 internalization and
degradation (32-34). The internalization process of H-IL-6 is most
likely comparable with the one of IL-6 and soluble IL-6R/gp80. However
in H-IL-6 the linker peptide, which fuses IL-6 and soluble IL-6R/gp80,
could lead to less effective internalization that results in a more prolonged effect on gene transcription. The second mechanism that may
account for the longer effect on gene transcription is based on the
three-step model during receptor assembly. The first step with low
affinity binding is not necessary for H-IL-6. The second step is the
essential interaction for H-IL-6 and results in high affinity binding
between H-IL-6 and gp130. Because of the high affinity between the two
partners the on/off rate at the receptor level is shifted toward longer
assembly of an active complex on the cell membrane, which results in a
more prolonged effect on STAT3-dependent gene transcription.
The interaction of IL-6 with the soluble gp80/IL-6R is also relevant
in vivo (35, 36). The association of IL-6 and soluble gp80/IL-6R renders cells that express only gp130 sensitive for IL-6 and
this mechanism has been termed transignaling (37, 38). H-IL-6 closely
resembles the association between soluble gp80/IL-6R and IL-6;
therefore, the experiments where gp130 and/or the gp80/IL-6R molecule
were cotransfected with a STAT3-dependent reporter gene construct might also have direct implications for the physiological assembly of soluble gp80 and IL-6. These results show that the receptor
status of a specific cell influences the effect on gene transcription
induced by H-IL-6. When the STAT3-dependent reporter gene
construct was cotransfected with the gp80/IL-6R molecule, the
H-IL-6-dependent effect on gene transcription was reduced, whereas higher luciferase activity was found after IL-6 stimulation. In
contrast, cotransfecting the gp130 molecule had the opposite effect.
Therefore the availability of gp130 molecules on the cell surface is
the rate-limiting step for inducing STAT3-dependent gene
transcription. These results also suggest that in cells which only
express gp130 molecules, STAT3-dependent gene transcription can be stimulated, which might explain the observation that
hematopoietic progenitor cells strongly respond to H-IL-6 but not to
IL-6 stimulation (17). Thus our cotransfection experiments with the
gp80 and gp130 receptors clearly show that the target cells, which can be stimulated by IL-6 and H-IL-6, are not absolutely comparable. A
higher ratio toward the gp130 receptor would render the cell more
susceptible toward H-IL-6 but not IL-6, which results in higher
STAT3-dependent gene transcription. In contrast, cells with
a 1:1 gp130/gp80 ratio would be less ideal target cells for H-IL-6.
H-IL-6 triggers STAT3-dependent gene transcription also
in vivo. The IL-6 serum levels show that the decay of the
H-IL-6 protein is not dramatically reduced compared with IL-6.
Additionally, these experiments indicate that even without the
specificity of IL-6, which needs gp80 and gp130 for trimer formation,
most of the protein is still very active in the liver. A 10-fold lower dose and serum levels of H-IL-6 have a similar effect on activating STAT3 and haptoglobin mRNA and protein levels compared with IL-6. Earlier reports showed that injection of the gp80/IL-6R protein into
rats lead to an enrichment of the protein mainly in liver, muscle,
skin, and kidney (39). Because H-IL-6 consists of IL-6 and soluble
gp80/IL-6R it may have a similar tissue distribution as the gp80/IL-6R
molecule alone. This characteristic would help to specify potential
target tissues in vivo, where H-IL-6 might have a potential
therapeutic effect.
Because H-IL-6 is more active for inducing STAT3-dependent
gene transcription in the liver, several therapeutic implications are
evident. The induction of acute phase proteins contributes to a first
line of defense during infection (9, 26). H-IL-6 would induce these
defense mechanisms stronger and more rapidly, and therefore might help
to eliminate infectious agents, especially in immunosuppressed
patients. Besides mediating the activation of acute phase genes, IL-6
seems involved in activating pathways that are important to trigger
cell proliferation and anti-apoptotic mechanism (6, 40, 41).
Hepatocytes of IL-6-deficient and type I tumor necrosis factor
receptor- deficient mice are unable to proliferate after partial
hepatectomy and recombinant IL-6 is able to restore cell proliferation
and thus liver regeneration (13, 14). Additionally, administration of
IL-6 before Con A injection prevents hepatocytes from undergoing tumor
necrosis factor-induced apoptosis (42). Therefore, H-IL-6 seems a
promising designer protein to influence several pathophysiological
conditions of the liver in vivo and thus might also be
important for later therapy in humans.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-macroglobulin gene linked to a luciferase reporter gene (20). For H-IL-6 and IL-6 stimulation experiments, cells were
cultured with 1% fetal calf serum for 24 h after transfection. Cotransfection of the reporter gene construct with the gp130 and IL-6
receptor expression vector was performed with the amounts indicated.
Cells were stimulated with H-IL-6 or IL-6 in the amounts and for the
time points indicated. Luciferase activity was measured as described
earlier (21) using a Lumat LB 9501 (Berthold, Germany). The relative
luciferase activity was normalized by cotransfecting a Rous sarcoma
virus-
-galactosidase expression vector. The results, which are
shown, represent the normalized luciferase activity. Nuclear extracts
were prepared from HepG2 hepatoma cells using the Dignam C method as
described previously (22).
2-macroglobulin
promoter was used as a 32P-labeled probe. The
oligonucleotides (sense 5'-GATCCTTCTGGGAATTCCTA-3' and antisense
5'-GATCTAGGAATTCCCAGAAG-3') were purchased from Naps (Göttingen,
Germany). Free DNA and DNA-protein complexes were resolved on a 6%
polyacrylamide gel as described previously (22). "Supershift"
experiments were performed with nuclear extracts prepared at the time
points indicated. Complex formation for supershift experiments was
performed with either an antibody directed against STAT3 purchased from
Santa Cruz Biotechnology or antibodies directed against STAT1, which
were a generous gift from Thomas Decker (Vienna, Austria).
-32P]ATP according to random priming (Boehringer
Mannheim). The hybridization procedure was performed as described
previously (19).
-32P]UTP for 30 min at 30 °C. RNA was
extracted, precipitated, and dissolved in TES buffer exactly as
described. 2 µl of the labeled RNA was counted in a scintillation vial.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Increased haptoglobin gene transcription in
HepG2 cells after H-IL-6 stimulation. Northern blot analysis with
15 µg RNA at each time point was performed when HepG2 cells were
stimulated with 10 ng/ml H-IL-6 (panel A). As
controls, RNA of HepG2 cells was used before treatment (0,
lane 1) and 96 h after stimulation of the carrier
solution (96, lane 10). The carrier solution
consisted of 0.9% NaCl. The same membranes were hybridized with a
probe for haptoglobin (HAP) and GAPDH. In panel B
the relative changes in haptoglobin mRNA expression are shown in
comparison with the GAPDH signal. The haptoglobin to GAPDH ratio before
treatment was set to 1 and the relative changes compared with the
pretreatment level were calculated accordingly. The transcription rate
of the haptoglobin gene was studied by run-off experiments in HepG2
cells before and at different time points after 10 ng/ml H-IL-6
(panel C) stimulation. In panel D the relative
changes are shown as fold induction compared with the pretreatment
level.
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Fig. 2.
Nuclear STAT3 expression and DNA-binding in
HepG2 cells after H-IL-6 injection. Western blot analysis was
performed with 10 µg of nuclear extracts prepared before or at
different time points after treatment of HepG2 cells with 10 ng/ml
H-IL-6. Nuclear STAT3 (panel A) or tyrosine-phosphorylated
nuclear STAT3 (panel B) expression was detected with
antibodies directed against STAT3 or tyrosine-phosphorylated STAT3. Gel
shift experiments were performed with 1 µg of liver nuclear extracts
prepared from HepG2 cells at different time points after 10 ng/ml
H-IL-6 (panel C) stimulation. The nuclear extracts were
incubated with a probe spanning the STAT3 site of the
2-macroglobulin promoter. The position of the bound (panel
B) DNA is indicated. In panel D, supershift experiments
were performed using HepG2 cell nuclear extracts 30 min after H-IL-6
stimulation. In lane 1 no antibody was added, whereas, in
lanes 2 and 3, nuclear extracts were either incubated with 1 µl of an anti-STAT1 (1 µS1) or 1 µl of an anti-STAT3
(1 µS3) antibody.
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Fig. 3.
Time course and dose response of
STAT3-dependent gene transcription after IL-6 or H-IL-6
stimulation. 2 µg of a STAT3-dependent reporter gene
construct (20) were transfected into HepG2 cells and stimulated with
either 10 ng/ml IL-6 or H-IL-6 (panel A). The luciferase
activity before stimulation was set to 1. The activity at different
time points after stimulation is shown as fold induction compared with
the pretreatment level. In panel B the luciferase
activity after H-IL-6 stimulation at each time point was set to 100%.
The relative activity after IL-6 stimulation was calculated in percent
compared with the activity at each time point after H-IL-6 stimulation.
In panel C increasing amounts of either H-IL-6 or IL-6 were
administered and luciferase activity was measured 4 h after
stimulation. The changes are shown as fold induction in comparison with
the pretreatment level, which was set to 1.
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Fig. 4.
The gp130 to gp80/IL-6R ratio determines
STAT3-dependent gene transcription after IL-6 or H-IL-6
treatment. HepG2 cells were transfected with 2 µg of the
STAT3-dependent reporter gene construct. Cotransfection of
the gp80/IL-6R, the gp130, or both receptors were performed in the
concentrations indicated. When both receptors were cotransfected, the
same amount of each receptor was used. Stimulation with 10 ng/ml H-IL-6
(panel A) or IL-6 (panel B) was performed 24 h after transfection for 4 h. The luciferase activity when only
the STAT3-dependent reporter gene construct was transfected
was set to 100%. Changes after cotransfecting the different receptor
construct combinations are shown in percent.
14%,
4%, and
28%, respectively.
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Fig. 5.
Regulation of haptoglobin mRNA expression
through IL-6 and H-IL-6 in vivo. Northern blot analysis with
15 µg RNA at each time point was performed when C3H mice were either
stimulated with 40 µg of IL-6 (panel A) or 4 µg of
H-IL-6 (panel B). The same membranes were hybridized with a
probe for haptoglobin (HAP) and GAPDH. In panel C
the relative changes in haptoglobin mRNA expression are shown in
comparison with the GAPDH signal. The haptoglobin to GAPDH ratio before
treatment was set to 1, and the relative changes compared with the
pretreatment level were calculated accordingly.
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Fig. 6.
IL-6 serum levels after IL-6 and H-IL-6
injection. The IL-6 concentration was measured in the liver vein
before and at different time points after injecting either with 40 µg
of IL-6 (A) or 4 µg of H-IL-6 (B).
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Fig. 7.
Increase of haptoglobin protein expression in
serum following IL-6 and H-IL-6 stimulation in vivo. Western
blot analysis for dose (A and B) and time kinetic
(C and D) experiments with 20 µg of serum of
mice treated with IL-6 or H-IL-6 were performed to study haptoglobin
protein expression. For dose kinetic experiments increasing doses of
either IL-6 (0.04, 0.4, 4, or 40 µg), H-IL-6 (0.004, 0.04, 0.4, or 4 µg) or the buffer control (0) were injected for 4 h
(A and B). For time kinetic studies, serum was
used before treatment (0) or at different time points (6, 12, 24, and
48 h) after stimulation with either IL-6 (C) or H-IL-6
(D). The position of the specific haptoglobin
(HAP) signal is shown.
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Fig. 8.
Detection of nuclear STAT3 and nuclear
tyrosine phosphorylated STAT3 after IL-6 and H-IL-6 injection in
vivo. Western blot analysis was performed with 10 µg of liver
nuclear extracts prepared before and at different time points after
treatment of C3H mice with 40 µg of IL-6 (A) or 4 of µg
H-IL-6 (B). Nuclear STAT3 or tyrosine-phosphorylated nuclear
STAT3 expression was detected with antibodies directed against STAT3 or
tyrosine-phosphorylated STAT3.
or both STAT1 isoforms (
and
) (Fig.
9D). The STAT1 antibody which detects both STAT1 isoforms
completely competed the new complex, which was found 1 h after
H-IL-6 treatment. In contrast, the anti-STAT1-
antibody had only a
minor influence on the intensity of the new complex. Therefore, these
results indicated that in vivo H-IL-6 also stimulated nuclear translocation and DNA-binding of STAT1-
.
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Fig. 9.
DNA binding of STAT3 after IL-6 or H-IL-6
stimulation in vivo. Gel shift experiments were performed
with 1 µg of liver nuclear extracts prepared from animals before and
at different time points after 40 µg of IL-6 (A) or 4 µg
of H-IL-6 (B) stimulation. The nuclear extracts were
incubated with a probe spanning the STAT3 site of the
2-macroglobulin promoter. The position of the free (F)
and bound (B) DNA is indicated. For supershift experiments,
nuclear extracts were used from animals either treated for 1 h
with IL-6 (C) or H-IL-6 (D). Nuclear extracts
were incubated with increasing amounts of a STAT3 (S3)
antibody and antibodies that either detect STAT1-
(S1-
) or the C-terminal domain of STAT1-
+
(S1).
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ACKNOWLEDGEMENT |
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We thank Malte Peters for helpful discussions.
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FOOTNOTES |
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* This work was supported by the DFG Grant Tr 285 4-3.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.
Dedicated to Professor Dr. K.-H. Meyer zum Büschenfelde on the occasion of his 70th birthday.
§ To whom correspondence should be addressed: Dept. of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: 49-511-532-3489; Fax: 49-511-532-4896.
The abbreviations used are: IL-6, interleukin-6; H-IL-6, hyper IL-6; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid.
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REFERENCES |
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