From the Department of Gastroenterology, Hepatology, and Endocrinology, Medizinische Hochschule Hannover, 30625 Hannover, Germany and § Gesellschaft für Biotechnologische Forschung mbH, 38124 Braunschweig, Germany
Received for publication, August 19, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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After tissue loss the liver has the unique
capacity to restore its mass by hepatocyte proliferation. Interleukin-6
(IL6)-deficient mice show a lack in DNA synthesis after partial
hepatectomy (PH). To define better the role of IL6 and its family
members for liver regeneration after PH, we used conditional knockout
mice for glycoprotein 130 (gp130), the common signal transducer of all
IL6 family members. We show that gp130-dependent pathways
control Stat3 activation after PH. By using gene array analysis, we
demonstrate that c-jun, NF- Interleukin 6 (IL6)1
belongs to a family comprising of IL6, IL11, leukemia inhibitory
factor, oncostatin M, ciliary neurotropic factor, and cardiotropin 1. They all need the gp130 molecule for signal transduction (1, 2).
Knockout mice with deletion of individual cytokines of the IL6 family
have a rather mild phenotype (3). Experiments elucidating the role of
one specific cytokine of the IL6 family during different
pathophysiological conditions are thus hampered by a potential
redundancy in activity by another family member. Because all known IL6
family members use gp130 for signal transduction, mice lacking
functional gp130 are an attractive model to study the role of
IL6-related pathways in vivo.
gp130 knockout mice die in uteri or directly after birth due
to myocardial hypoplasia and reduced hemopoiesis in the fetal liver (4,
5). In order to circumvent this problem, conditional gp130 knockout
mice using the Cre/loxP system were generated (6). In these mice exon
16 coding for the gp130 transmembrane domain can be deleted as two loxP
sites flank it. In animals with the Cre recombinase under the control
of the Mx promoter, expression is induced by interferon- In the liver, IL6 is a major regulator of the acute phase response. It
has also been shown that IL6 is essential for liver regeneration after
partial hepatectomy (PH) (8, 9). First experiments indicated that there
is an elevation of TNF However, other reports indicated the possibility that the role of IL6
on cell cycle progression during liver regeneration after PH is less
essential as hypothesized previously (12). These results suggest the
possibility that other factors, e.g. the expression of other
IL6 family members, may modulate the outcome after PH and raise the
question of the ultimate role of IL6 during liver regeneration. In
order to circumvent a possible redundancy of different IL6 family
members, we used conditional gp130 knockout mice to study liver
regeneration after PH. By using this approach, we define more precisely
the role of IL6/gp130-dependent signaling, and we show that
these pathways may determine survival after PH but not the expression
of genes directly involved in cell cycle control.
Mice Strains, pIpC Stimulation, and Southern Blot
Experiments--
gp130 loxP (+/+) Mx-Cre (
Two IL6 knockout strains and respective controls were used in this
analysis (13, 14). The IL6 IL6 Stimulation Experiments and Preparation of Nuclear
Extracts--
Gp130 loxP/Mx-Cre-positive (+) and -negative ( Determination of IL6 Serum Levels--
Enzyme-linked
immunosorbent assay using the OptEIA mouse IL6 kit (Pharmingen)
measured IL6 serum levels.
Two-thirds Hepatectomy and Preparation of Nuclear
Extracts--
6-8-Week-old male gp130 loxP/Mx-Cre + and
For each time point indicated at least 4 mice were used in parallel.
Blood was drawn from the animals at each time point. The remaining
livers were removed, pooled, and rinsed in ice-cold phosphate-buffered
saline, and part of the livers were frozen immediately in liquid
nitrogen or tissue-tek (Sakura Europe, Netherlands). The remaining
liver was used to prepare nuclear extracts (15).
SDS-PAGE and Western Blot Analysis--
Nuclear extracts were
separated on a 10% SDS-polyacrylamide gel and blotted onto a
nitrocellulose membrane (Millipore Corp., Bedford, MA) as described
previously (15), and Western blot analysis was performed. Membranes
were probed with anti-cyclin E and anti-cyclin A as primary antibodies
(SC-481, SC-751; Santa Cruz Biotechnology, Santa Cruz, CA). As a
secondary antibody, peroxidase-conjugated donkey anti-rabbit IgG
(Jackson ImmunoResearch, number 711-035-152) was used. The
antigen-antibody complexes were visualized using the ECL detection
system as recommended by the manufacturer (Amersham Biosciences).
Western blot analysis was performed for each protein of interest
derived least at three times from different animals.
Gel Retardation Assays--
Gel shift experiments (EMSA) were
performed as described previously (15). Liver nuclear extracts were
incubated with a 32P-labeled consensus Stat3 (5'-GTG CAT
TTC CCG TAA ATC TTG TCT ACA-3') site, AP-1 site (5'-CGC TTG ATG ACT CAG
CAG CCG GAA-3'), and NF- BrdUrd Labeling--
For in vivo pulse-labeling
experiments, 5'-bromo-2'-deoxyuridine (BrdUrd, Amersham Biosciences),
30 µg/g mice, was injected intraperitoneally 2 h before killing.
Liver tissue was frozen immediately in liquid nitrogen. 5-µm
cryosections were fixed in ice-cold acetone/methanol and stained
according to the cell proliferation kit manual (Amersham
Biosciences).
Northern Blot Analysis--
Northern blot analysis was performed
as described previously, according to standard procedures (17). The
serum amyloid A, haptoglobin, hemopexin, albumin, c-myc, and
Clontech Gene Array--
For sample
preparation for cDNA expression arrays, three livers from each
group were homogenized in RNA Clean Solution (HYPAID-AGS) for isolation
and purification of total RNA. The RNA was pooled and treated with
DNase according to the manufacturer's instructions (Clontech Laboratories, Palo Alto, CA), and the
quality of 10 µg of RNA was evaluated by denaturing MOPS gel electrophoresis.
The cDNA probes prepared from DNase-treated RNAs obtained from each
of the experimental groups were hybridized simultaneously to four
membranes containing Atlas mouse cDNA expression arrays (Clontech). 5 µg of DNase-treated RNA was labeled
with [
The PhosphorImaging results were interpreted by using OptiQuant image
analysis software (PerkinElmer Life Sciences). Quantification of each
detectable band was performed from the digital light units generated by
OptiQuant. The background was subtracted.
TUNEL Assay--
The TUNEL Test (TdT-mediated dUTP nick end
labeling) was performed using the "In Situ Cell Death
Detection Kit, POD" from Roche Diagnostics according to the
instructions of the manufacturer. Sections were analyzed with a
fluorescence microscope (Olympus, Hamburg, Germany).
RT-PCR--
RNA was isolated as reported previously (17). First
strand synthesis for RT-PCR was performed using oligo(dT)15
primer (Promega) and the Omniscript RT kit (Qiagen) in combination with
1 µg of total RNA per reaction. Amplification of Bcl-xL and GAPDH
cDNA was carried out in one reaction using the specific primers
(Bcl-xL: sense, 5'-cgacccagccaccacctcctc-3', and antisense, 5'-tgg ggc ctc agt cct att ctc-3', cDNA length 316 bp; GAPDH: sense, 5'-tga tga cat caa gaa ggt ggt gaa g-3', and antisense, 5'-tcc ttg gag gcc atg
tag gcc at-3', cDNA length 249 bp) and 1 µl of the RT reaction.
Briefly, PCR was performed for 10 cycles with first strand DNA and
Bcl-xL primers at a Tanneal of 58 °C. Then
the reaction was interrupted; primers for GAPDH were added, and the reaction was continued for another 25 cycles. PCR products were documented and quantitated using a Gel Doc 1000 apparatus (Bio-Rad) and
Molecular Analyst software (Bio-Rad). For direct comparison the
GAPDH:Bcl-xL ratio was determined.
Quantification--
Quantification of results was performed with
a Fuji imager and Pcbas as described earlier (16).
pIpC Results in Exon 16 Deletion in gp130loxP/Mx-Cre
Animals--
pIpC induces interferon-
Next, we tested the functional consequences of a deletion higher than
95% of gp130 exon 16 after 80 µg of pIpC injection in the liver.
gp130 loxP (+/+/)/Mx-Cre (+) animals were stimulated with 10 µg of IL6/mouse for 3 h at different days after pIpC treatment. Gel shift analysis with a 32P-labeled Stat3 consensus
oligonucleotide showed that IL6-dependent Stat3 complex
formation was completely blocked 10 days after pIpC administration
(data not shown).
Stat3 Activation Is Blocked in gp130 loxP/Mx-Cre Animals
after PH--
Earlier results using IL6 knockout mice implied that IL6
is involved in cell cycle progression after PH (9). We performed partial hepatectomies in conditional gp130 knockout mice. gp130 loxP
(+/+)/Mx-Cre (+) (also referred to as gp130-deleted) and gp130 loxP (+/+)/Mx-Cre (
We first measured IL6 serum levels. In both groups IL6 serum levels
increased 3 h after PH (Fig. 1B). In controls peak IL6 expression was found after 6 h and in the gp130-deleted group after 12 h. Maximal IL6 levels were more than 2-fold higher in the
gp130-deleted group, and at later time points the decrease was slower
compared with controls (Fig. 1B). Between the two groups there was no difference in mortality after PH.
EMSA experiments revealed strong Stat3 complex formation 1 h after
PH in the control group. At later time points complex formation decreased and returned to background levels (Fig. 1C). In
gp130-deleted animals Stat3 activation after PH was completely blocked.
No increase in complex formation was found compared with the
pretreatment level (Fig. 1C).
Suppressor of cytokine signaling 3 (SOCS-3) is a potent inhibitor of
Stat3-dependent gene activation. A recent study by Campbell et al. (19) showed a strong regulation of SOCS-3
during liver regeneration. Therefore, we investigated SOCS-3
mRNA expression in both groups. SOCS-3 transcripts were
strongly increased 3 and 6 h (more than 30-fold) after PH in the
control group, whereas no regulation was found in gp130-deleted animals
(Fig. 1D). No significant regulation was found for
SOCS-1 and -2 in both groups after PH (data not shown).
DNA Synthesis after Partial Hepatectomy in gp130-deleted
Mice--
gp130-dependent signaling was blocked in
gp130-deleted mice after PH. In further experiments we determined the
effect on DNA synthesis after PH by BrdUrd pulse-labeling experiments
in both groups.
In the control group a maximum of BrdUrd-positive cells was evident
48 h after PH. 39% ± 5 of all hepatocytes were BrdUrd-positive (Fig. 2, A and B).
At later time points BrdUrd-positive cells decreased.
In gp130-deleted animals the maximum of BrdUrd-positive cells was
observed 48 h after PH and was lower compared with controls. 31% ± 4 nuclei of all hepatocytes were BrdUrd-positive in gp130-deleted animals. At later time points the amount of BrdUrd-positive hepatocytes decreased (Fig. 2, A and B).
Delayed Induction of Cyclins in gp130-deleted Animals--
In
further experiments we studied the impact of gp130 deletion on cyclin
expression after PH. Cyclin A and E expression was studied by Western
blot experiments.
In control animals cyclin E expression was first positive
12 h after PH. There was a continuous increase in cyclin E
expression for up to 48 h. At later time points cyclin E
expression decreased (Fig.
3A). In gp130-deleted animals,
cyclin E was only detected 24 h after PH, and a strong increase
was observed up to 48 h after PH. At 72 h cyclin E expression
declined (Fig. 3B).
Cyclin A expression in controls was detected 40 h after PH and
decreased thereafter, whereas in gp130-deleted animals cyclin A
expression was first positive at the 48-h time point (Fig. 3, C and D).
Gene Array Analysis in gp130-deleted Mice after IL6
Stimulation--
Earlier experiments showed even more pronounced
differences in cell cycle progression after hepatectomy in IL6 knockout
animals compared with controls (9, 12). Therefore we searched for specific pathways to understand how IL6 may influence cell cycle progression in the liver. We stimulated gp130-deleted and control mice
with IL6 for 6 h and prepared mRNA. The mRNAs of untreated and IL6-stimulated animals were used for gene array analysis. The focus
of this analysis was on genes that are involved in cell cycle control.
This analysis revealed two genes, c-jun and
c-myc, that might have an impact on
G0/G1 phase transition and are regulated in an
IL6-dependent manner in the liver (Fig.
4A). No impact on genes
directly involved in cell cycle control-like cyclins was found.
The gene array analysis showed also a strong effect on the expression
pattern of the TNF-R1 and -R2 (Fig.
4B). The gp130-deleted animals are blocked in regulating the
TNF-R expression.
Activation of Immediate Early Genes Is Inhibited in gp130-deleted
Animals--
In further experiments we tried to specify our gene array
data by studying c-myc RNA expression by Northern blot
analysis before and after PH. Basal expression and the induction after PH as found in controls were reduced in gp130-deleted animals. Thus
gp130-dependent pathways contribute to the basal and
inducible c-myc expression after PH (data not shown).
c-Jun is a member of the AP1 complex. Activation after PH was studied
by gel shift experiments using a 32P-labeled AP1 consensus
oligonucleotide. Strong complex formation was evident 1-36 h after PH
in controls. At later time points complex formation decreased and was
reduced to pretreatment levels (Fig. 4C). In the
gp130-deleted animals specific complex formation was first evident only
36 h after hepatectomy and remained high 48 h after PH (Fig.
4C).
In addition to c-Jun, it has been shown also for NF- Activation of DNA Synthesis after Partial Hepatectomy in IL6
Knockout Animals--
Our experiments in the gp130-deleted animals
indicated that gp130-dependent pathways are essential for
liver cells in contributing to the activation of distinct immediate
early genes as shown previously (9). Because in our experiments the
impact on DNA synthesis was less prominent after PH as in IL6
As strain-specific differences may account for changes in hepatocyte
proliferation, we additionally performed PHs in a second IL6 Impaired Acute Phase Gene Regulation in gp130-deleted
Animals--
IL6 contributes to acute phase gene expression and
represents a first line of defense after bacterial infections (22).
Thus after abdominal surgery the outcome may depend upon the ability of
the liver to control unspecific defense mechanisms. In further experiments the impact on acute phase gene expression was studied in
gp130-deleted mice. As shown in Fig.
6A, serum amyloid A,
haptoglobin and hemopexin gene expression were increased starting 3-6
h, and maximal levels were evident 24 h after PH in controls. In
contrast, this increase in acute phase gene expression after PH was not found in gp130-deleted mice (Fig. 6A).
Albumin mRNA expression decreased after PH in the control group
(Fig. 6A). In gp130-deleted animals only minor variations in
albumin mRNA levels were detected in the first 12 h post-PH (Fig. 6A). These results demonstrate that after PH
regulation of the acute phase response is blocked in gp130-deleted animals.
gp130-deleted and IL6 Knockout Animals Are More
Vulnerable to LPS after Partial Hepatectomy--
As regulation of the
acute phase response was impaired in gp130-deleted animals, we
investigated the impact of a bacterial infection, mimicked by LPS
injection, on liver regeneration. LPS was injected 3 h after PH,
and the impact on survival and DNA synthesis was studied.
LPS-treated, gp130-deleted animals had a significantly worse outcome
compared with controls. In the gp130-deleted group 40% of the animals
died during the first 48 h after PH. In contrast, the survival
rate in controls was 85% after LPS injection (p < 0.01, Fig. 6B).
Higher mortality correlated with a decrease in DNA synthesis in
gp130-deleted animals who survived. BrdUrd staining after 48 h
revealed an 80% lower rate of BrdUrd-positive hepatocytes in
gp130-deleted animals compared with controls (Fig. 6C).
As LPS had a significant effect on survival and DNA synthesis of
gp130-deleted animals, we also tested the impact of LPS on survival in
IL6 knockout animals compared with controls (Fig. 7A). IL6
These differences were also reflected when BrdUrd staining was
performed in the liver of these animals. DNA synthesis 48 h after
PH in LPS-treated IL6 Increased Apoptosis in gp130-deleted Animals after Partial
Hepatectomy and LPS Stimulation--
The LPS/PH experiments indicated
that protective mechanism might be impaired in gp130-deleted animals.
Additionally, earlier experiments showed that IL6 might stimulate
anti-apoptotic pathways by inducing higher Bcl-xL expression
(23). Therefore we studied Bcl-xL expression through an
RT-PCR approach early after PH. In control animals we found an increase
in Bcl-xL expression 3 h after PH which was not found in
gp130-deleted animals (Fig. 8, A and B).
As differences in Bcl-xL regulation might be one of the
gp130-dependent mechanisms protecting from apoptosis after
LPS stimulation, we now studied if there is a difference in the degree
of apoptosis between controls and gp130-deleted animals. At the 3-h
LPS/6-h PH time point we found a significant increase in apoptosis in the gp130-deleted animals compared with controls (Fig. 8C).
In gp130-deleted mice 15-fold more liver cells were TUNEL-positive compared with controls (Fig. 8D).
The molecular mechanisms essential to restore liver mass after
injury are complex, and the intra- and extracellular pathways that
orchestrate liver regeneration have recently become clearer on a
molecular level (11, 24, 25). Earlier experiments showed that after
partial hepatectomy there is activation of Stat3 in the first hours
after surgery (8, 26). Further experiments using IL6 knockout mice
demonstrated that this cytokine is involved in regulating the
regenerative response after PH (9). Additionally, TNF-R1 knockout mice
showed a lack in liver regeneration that could be rescued by injecting
IL6 (10). Based on these data a model has been deducted where first
TNF In the present study we were interested to examine further the role of
IL6 for liver regeneration after partial hepatectomy using conditional
gp130 knockout mice. After birth Cre expression in gp130loxP/Mx-Cre
animals was induced by pIpC injection. Earlier experiments demonstrated
that in these animals efficient deletion of gp130 exon 16 was achieved
in the liver and other tissue contributing to the immune response
(spleen, bone marrow, thymus) (9). In the liver more than 95% of exon
16 in the gp130 locus was deleted, and IL6-dependent Stat3
activation was inhibited, in contrast to controls. All of the IL6
family members need gp130 for signal transduction, and thus the use of
gp130 loxP/Mx-Cre animals circumvents the possibility that other
cytokines of this family may compensate for the loss of each other (1,
2).
After PH in hepatocytes of the control group strong Stat3 activation
was restricted to the first hours after PH, whereas maximal IL6 serum
levels were found after 6 h. Recent experiments published by
Fausto and co-workers (19) have revealed a molecular explanation for
this observation. They demonstrated that only 1 h after partial hepatectomy SOCS-3 is induced on the mRNA level and
reached maximum expression after 2 h. In the present analysis we
further characterized this mechanism and showed that SOCS-3
is controlled via gp130-dependent pathways in hepatocytes.
Thus, through increasing SOCS-3 expression via gp130, Stat3 activation
is strongly restricted after PH.
In our study there is a discrepancy between the relatively early
activation of Stat3 after partial hepatectomy and the late peak of IL6
serum expression. As in the gp130-deleted animals no Stat3 DNA binding
was found, and activation is likely to be mediated through a
gp130-dependent cytokine. Additionally in IL6 Liver regeneration in gp130-deleted animals showed marked differences
compared with controls. Stat3 activation was completely blocked, and
gene array analysis defined additional gp130-dependent downstream targets, c-myc and c-jun, after
PH. Additionally, our experiments suggest that NF- The explanation for diminished NF- Our work is in agreement and expands earlier reports of Taub and
co-workers (9) and Fausto and co-workers (10) indicating that
activation of distinct immediate early genes is dependent on IL6 after
PH (9, 10). However, DNA synthesis was reduced in conditional gp130
knockout animals but less severely impaired as described previously in
IL6 This result was unexpected. However, further evaluation of the IL6
In our earlier reports we used a transgenic mouse that overexpressed
the soluble IL6 receptor under the control of a hepatocyte-specific control element. In these animals IL6 induces severalfold stronger gp130-dependent signaling in hepatocytes. In contrast to
our initial hypothesis, PH and IL6 stimulation did not result in an
increase of hepatocyte proliferation but to a delay in cell cycle
progression. Thus hyperstimulation of gp130-dependent
pathways in hepatocytes was not associated with increased DNA synthesis.
The discrepancy in the results of different groups in the correlation
between IL6 and hepatocyte proliferation was obvious. Thus it was
difficult to ultimately define the role of IL6 and thus of
gp130-dependent signals for liver regeneration. Therefore, we considered other means to explain the role of IL6 for cell cycle
progression after PH. Recent studies (23, 28, 29) demonstrated that IL6
induces protective, e.g. anti-apoptotic, pathways in
hepatocytes. These results led us to consider the possibility that the
role of IL6 in the liver after PH is not mainly to trigger cell cycle
progression through immediate early genes but instead is to protect the
liver from injury during this complex step. We show in our study that
in gp130-deleted animals acute phase gene regulation and thus early
defense mechanisms are blocked after PH.
Therefore, to mimic a bacterial infection, we injected LPS in
gp130-deleted and control animals after PH. These experiments revealed
striking differences in the outcome of these animals similar to the
results obtained in the initial PH experiments reported in the IL6
In further experiments we investigated the possible mechanism that
might account for higher LPS sensitivity. As in earlier experiments
Kovalovich et al. (23) showed that IL6 induces Bcl-xL expression, and we investigated its expression by RT-PCR and found lower expression in the gp130-deleted animals. After PH the finding correlated with a higher rate of apoptosis in the gp130-deleted animals
after LPS stimulation indicating that this is most likely an important
mechanism that contributes to the worse outcome in these animals after
partial hepatectomy and LPS stimulation.
Based on these results we define more specifically the role of the
IL6/gp130-dependent pathway during liver regeneration. Higher IL6 expression after PH is important to activate protective pathways that render hepatocytes more resistant toward stress-inducing signals, e.g. bacterial infections after PH. Our results
suggest that the phenotype of gp130-deleted and IL6 knockout animals
after PH is related to the degree of additional injury. These results would nicely explain why there are differences in the literature upon
the strength of the phenotype in IL6 B, c-myc, and
tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor
effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the
acute phase response was completely abolished, we considered that other
means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and
also IL6
/
animals after PH leads to a significant reduction in
survival and DNA synthesis, which was associated with decreased Bcl-xL
expression and higher apoptosis in the liver. These results indicate
that the phenotype concerning the reduction in DNA synthesis might be
linked to the degree of infection after PH. Thus our results suggest
that the role of gp130-dependent signaling is not a direct
influence on cell cycle progression after partial hepatectomy but is to
activate protective pathways important to enable hepatocyte proliferation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or agents
triggering internal interferon-
expression, e.g. pIpC (6,
7).
followed by IL6 in the serum of animals after
PH (8). Further studies using IL6 and TNF receptor 1 (TNF-R1) knockout
mice (9, 10) demonstrated that in both knockout animals S phase
progression is impaired and some of the animals die after surgery. In
both IL6 and TNF-R1 knockout mice, the defect in hepatocyte
proliferation can be rescued by treating the animals with IL6. These
results imply that the increase in TNF
levels is important to induce IL6, which is ultimately involved in triggering liver regeneration (9,
11).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) mice were
bred with gp130 loxP (+/+) Mx-Cre (+) animals. By using this
approach, littermates were always homozygote for gp130 loxP and had a
50% chance for the Mx-Cre transgene. These animals
with identical backgrounds were then used for further experiments. Cre+
and Cre
(controls) animals were always treated in parallel with pIpC
(Sigma) as indicated. 30 µg of isolated genomic DNA derived from
mouse livers was digested with EcoRI overnight, separated on
a 1% agarose gel, and transferred to Hybond N+ membranes.
Membranes were hybridized with a 32P-labeled gp130 probe as
described before (6). The deletion efficiency in other organs has
already been described (6).
/
strain was a gift of Uli Rüther
(University of Düsseldorf, Germany) and was back-crossed to a U. S. Naval Medical Research Institute background. The
second IL6
/
strain (C57BL/6) (13) was received from Benhard
Schieffer (Department of Cardiology and Angiology, Medizinische
Hochschule Hannover, Hannover, Germany).
)
animals were stimulated with 80 µg of pIpC. At different days after
stimulation these animals were treated with 10 µg of IL6 per mouse.
3 h after IL6 injection, the liver was harvested, and liver
nuclear extracts were prepared as described before (15).
(controls) animals were treated with 80 µg of pIpC per mouse. 10 days
after stimulation and 12 h before surgery, food was withdrawn from
the animals. Two-thirds (partial) hepatectomy was performed as
described earlier (16). The removed median, right, and caudate liver
lobes were routinely tested for the efficiency of exon 16 deletion by
Southern blot analysis. In all mice the efficiency in the liver was
>95%. For LPS stimulation experiments 30 µg of LPS/20 g mouse
(Sigma) was injected intraperitoneally 3 h after partial hepatectomy.
B site (5'-AGT TGA GGG GAC TTT CCC AGG G-3')
in 1× binding buffer (25 mM HEPES, pH 7.6, 5 mM MgCl2, 34 mM KCl, 2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, 1 mg/ml poly(dI-dC), 2 mg/ml bovine serum albumin). 3.5 µg
of liver nuclear extracts was incubated for 30 min on ice. Free DNA and
DNA-protein complexes were resolved on a 4% TBE-polyacrylamide
gel. Supershift experiments were performed using anti-Stat1 and
anti-Stat3 antibodies (Santa Cruz Biotechnology).
-actin (GAPDH) cDNA probes were labeled with
[
-32P]CTP according to the instructions for Rediprime
(Amersham Biosciences). The hybridization procedure was performed as
described previously (17). Blots were exposed for autoradiography and
exposed to an imaging plate (Fuji) for quantification. The specific
signals were normalized to the
-actin signals and set to 1 for
untreated animals.
-32P]dCTP and reverse-transcribed to
cDNA, according to the manufacturer's protocol. The radioactively
labeled cDNA probes were hybridized overnight to cDNA
expression arrays using ExpressHyb hybridization solution with
continuous agitation at 68 °C. After two high stringency washes, the
hybridized membranes were exposed to a PhosphorImaging screen overnight.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
expression in vivo
and activates the Mx promoter (7, 18). We injected increasing amounts
of pIpC in gp130loxP (+/+)/Mx-Cre (+) animals and gp130loxP
(+/+)/Mx-Cre (
) to analyze deletion of gp130 exon 16 in
the liver. 72 h after pIpC injection, the liver of the animals was
harvested, and DNA was prepared. As evidenced by Southern blot
analysis, 20 µg of pIpC resulted in a more than 90% deletion of exon
16 in the gp130 locus in Mx-Cre (+) and not in
Mx-Cre (
) animals (data not shown). With 80 µg (Fig.
1A) and higher amounts (160 and 320 µg, data not shown) of pIpC more than 95% of exon 16 was
deleted in gp130loxP (+/+)/Mx-Cre (+) animals.
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Fig. 1.
Lack of Stat3 activation in
gp130-deleted animals after partial hepatectomy. A,
gp130loxP/Mx-Cre + and animals were stimulated with 80 µg of
pIpC/mouse. 10 days after stimulation liver DNA was prepared, and
Southern blot analysis was performed. The position of the gp130 loxP
(gp130 loxP) and the-deleted gp130 loxP (gp130
loxP) signal is indicated. B, IL6 serum levels were
determined in gp130 loxP/Mx-Cre + and
animals at the time
points (n >4 animals per time point) indicated before and
after PH. C, gel shift experiments were performed with liver
nuclear extracts (n >4 animals per time point) before and
after PH with a 32P-labeled Stat3 consensus
oligonucleotide. Gels for gp130 loxP/Mx-Cre + and
animals were
run in parallel. The position of Stat3 complex formation is indicated.
D, Northern blot analysis was performed including time
points before and after PH of gp130 loxP/Mx-Cre
and + mice.
Filters were probed with a 32P-labeled cDNA for SOCS-3
and
-actin (control). The positions of the SOCS-3 and
-actin
signals are indicated.
) (controls) animals were both
treated with 80 µg of pIpC. After 10 days PH was performed. For each
time point indicated at least 4 animals were included, and the
efficiency of gp130 exon 16 deletion was monitored by Southern blot
analysis in the resected liver lobes. For further analysis only mice
were included with a deletion efficiency >95%.
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Fig. 2.
DNA synthesis in gp130-deleted animals after
partial hepatectomy. A, BrdUrd staining of liver sections
was performed at different time points (n >4 animals per
time point) before and after PH in gp130 loxP/Mx-Cre and + animals. Distinct time points are shown. B, for statistical
analysis the amounts of BrdUrd-positive hepatocytes for each point of
time are shown per 100 hepatocytes.
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Fig. 3.
Cyclin E and A expression in gp130-deleted
mice after partial hepatectomy. A-D, cyclin E (A
and B) and A (C and D) expression was
determined in gp130 loxP/Mx-Cre (A and C)
and + (B and D) animals at time points
(n >4 animals per time point) before and after PH. An
arrowhead shows the position of cyclin E and A. PK, pyruvate kinase.
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Fig. 4.
Delayed activation of immediate early genes
in gp130-deleted mice after partial hepatectomy. A, gp130
loxP/Mx-Cre and + mice were included. Animals were stimulated
for 6 h (+) with IL6. cDNA array analysis was performed. The
impact on cell cycle-related genes is shown. B, basal and
IL-6-inducible expression of TNF-R1 and TNF-R2 in control and
gp130-deleted animals as evidenced by gene array analysis. C
and D, gel shift experiments were performed using liver
nuclear extracts derived from gp130 loxP/Mx-Cre
and + before
and after PH. 32P-Labeled consensus oligonucleotides for
AP1 (C) or NF-
B (D) were used in this
analysis. Specific complex formation is indicated. EMSA experiments for
both groups were performed in parallel.
B that its
activation correlates with liver regeneration after PH (20, 21). In
controls, biphasic NF-
B activation was evident 1-6 and 24-36 h
after PH (Fig. 4D). In gp130-deleted mice the first peak of
NF-
B activation was strongly reduced, whereas the second peak could
be detected equally to controls (Fig. 4D).
/
mice (9), we performed partial hepatectomies in IL6 knockout
animals (13) and monitored BrdUrd incorporation. DNA synthesis was
increased in controls 24 h after PH. Maximal levels were found
48 h after PH. 72 h after PH the amount of BrdUrd-positive
cells decreased (Fig. 5, A and B). Compared with controls in IL6
/
animals the increase
and maximum in DNA synthesis was moderately reduced (Fig. 5,
A and B).
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Fig. 5.
DNA synthesis in IL6 knockout animals after
partial hepatectomy. A, BrdUrd staining of liver sections
was performed at different time points (n = 4 animals
per time point) before and after PH in wild type (wt) and
IL6 /
mice. Distinct time points are shown. B, for
statistical analysis the amount of BrdUrd-positive hepatocytes for each
time point are shown per 100 hepatocytes.
/
strain (14) and stained for BrdUrd-positive cells. These results were
consistent with the data obtained in the first IL6
/
strain (data
not shown).
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Fig. 6.
Impaired acute phase gene regulation and LPS
injection reduces survival and DNA synthesis in gp130-deleted mice
after partial hepatectomy. A, RNA was prepared from mouse
liver before and after PH of gp130 loxP/Mx-Cre + and mice for
Northern blot analysis. Filters were probed with
32P-labeled cDNAs for serum amyloid A (SAA),
haptoglobin, hemopexin and albumin. As a loading control
-actin was
included in this analysis. B, PH was performed in gp130
loxP/Mx-Cre + mice and controls (n = 13/group). 3 h after PH 30 µg LPS/mouse was injected. Survival of the animals
after PH is indicated. C, BrdUrd (BrdU) staining
of liver sections was performed 48 h after PH of the gp130
loxP/Mx-Cre
and + mice treated with LPS. For statistical
analysis the amount of BrdUrd-positive hepatocytes is shown before and
48 h after PH as positive cells per 100 hepatocytes.
/
animals and
controls were treated with LPS 3 h after PH. Survival was
evaluated in the first 48 h after PH. In the IL6
/
animals
significantly more animals died after PH (p < 0.05)
when they were treated with LPS compared with controls and untreated
animals. Thus as found in the gp130-deleted animals, IL6
/
mice
were more susceptible to LPS after PH (Fig. 7A).
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Fig. 7.
LPS injection leads to reduced DNA synthesis
and survival after partial hepatectomy in IL6 knockout mice.
A and B, 3 h after PH 30 µg of LPS were
injected into wild type controls and IL6 /
animals
(n = 15/group). A, survival of the animals
was observed up to 48 h after PH and B, BrdUrd
(BrdU)-positive hepatocytes/100 cells were determined at the
time points indicated.
/
mice was significantly reduced compared
with controls (Fig. 7B).
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Fig. 8.
Lower Bcl-xL expression and higher apoptosis
in gp130-deleted animals after partial hepatectomy and LPS stimulation.
A, 1 µg of RNA at time points before and after PH of gp130
loxP/Mx-Cre + and mice were used for cDNA synthesis.
Primers for Bcl-xL and GAPDH were used as described under "Materials
and Methods" for semi-quantitative duplex PCR. The specific signals
of Bcl-xL and GAPDH are indicated. B, the relative
Bcl-xL:GAPDH ratio was calculated. The Bcl-xL:GAPDH ratio of untreated
animals was set to 1. C, TUNEL assays were performed of
liver sections derived from animals 3 h after PH and 6 h
after PH stimulated for 3 h with LPS. D, quantitative
analysis of TUNEL-positive cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and then IL6 serum levels are increased, and the intracellular
pathways that are activated through these two cytokines are essential
to trigger cell cycle progression after PH.
/
animals
no Stat3 activation was found after PH (9). These two observations
clearly indicate that IL6 induces Stat3 after PH. Therefore it is
likely that increased IL6 levels are first present in the liver after
PH as the cytokine at this stage may mainly bind to membrane-bound
gp80. At later time points, gp80 receptors in the liver are saturated,
and increased IL6 levels are present in the serum of the animals. In
the serum IL6 is bound to soluble gp80 and soluble gp130
receptors that limit the activation of gp130-dependent
pathways also in liver cells. As the pool of soluble gp130 is increased
in the gp130-deleted animals, IL6 has a longer half-life in the serum
of these animals.
B activation at
the early time points during liver regeneration is
gp130-dependent.
B and AP1 activation after partial
hepatectomy might be caused on different levels. However, our gene
array analysis also included TNF-R1 and TNF-R2.
Interestingly, the expression of both receptors is controlled via IL6
as in gp130-deleted animals the basal and the inducible expression of
TNF-R1 and the inducible expression of TNF-R2 are significantly reduced
(Fig. 4B). As NF-
B and AP1 are controlled via TNF after
PH, these results would explain in part a reduction in the
activation of both transcription factors in the gp130-deleted animals
after PH.
/
animals. The increase in cyclin A and E expression was
delayed in gp130-deleted animals, which was associated with a minor
reduction in DNA synthesis 48 h after PH. Gene array analysis
revealed no significant impact of IL6 on genes directly involved in
cell cycle control. Similar results were also reported in IL6
/
mice using gene array analysis after PH (27). Therefore, the
gp130-deleted animals could eventually restore liver mass and had no
increased mortality compared with controls. Obviously, the observed
reduction of immediate early gene activation in gp130-deleted mice
implies that in the liver compensatory pathways do exist to induce
hepatocyte proliferation.
/
experiments of Cressmann et al. (9) revealed that they
quoted those IL6
/
animals that survived the follow up after
hepatectomy had a normal liver weight 5 days after hepatectomy. These
results imply that IL6 is important but not absolutely necessary in
order to restore liver mass after PH. Otherwise there would be no
obvious explanation that some of the IL6
/
animals survived and
completely restored their liver mass. This hypothesis is further strengthened by the report of Demitris and co-workers (12) using IL6
/
mice. They demonstrated that in IL6
/
mice the reduction in
DNA synthesis after PH is less severe as originally observed by
Cressmann et al. (9) and was comparable with the degree of
reduction in DNA synthesis in our present experiments.
/
animals (9). After PH DNA synthesis was reduced in the surviving
LPS-injected gp130-deleted animals, and the same phenotype was found
when LPS was injected into IL6
/
mice after PH. Consistent with
this observation, hepatocyte-specific gp130 knockout animals are also
hypersensitive to LPS indicating that gp130-dependent
pathways in hepatocytes are essential for this observation (30).
/
mice after partial hepatectomy.
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FOOTNOTES |
---|
* This work was supported by 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.
Both authors contributed equally to this work.
¶ Present address: Bayer AG, Pharma Research Europe, G. 402, Apratherweg, D-42096 Wuppertal.
To whom correspondence should be addressed: Dept. of
Gastroenterology, Hepatology, and Endocrinology, Medizinische
Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
Tel.: 49-511-532-3865; Fax: 49-511-532-4896; E-mail:
trautwein.christian@mh-hannover.de.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M208470200
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ABBREVIATIONS |
---|
The abbreviations used are: IL6, interleukin-6; PH, partial hepatectomy; gp130, glycoprotein 130; TNF, tumor necrosis factor; TNF-R, TNF receptor 1; BrdUrd, bromodeoxyuridine; TUNEL, TdT-mediated dUTP nick end labeling; LPS, lipopolysaccharide; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; MOPS, 4-morpholinepropanesulfonic acid.
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