Hepatocyte-Specific Inhibitor-of-kappaB-Kinase Deletion
Triggers the Innate Immune Response and Promotes
Earlier Cell Proliferation During Liver Regeneration
Yann Malato, Leif E. Sander, Christian Liedtke, Malika Al-Masaoudi, Frank Tacke,
Christian Trautwein,* and Naiara Beraza*
after partial hepatectomy (PH). It is activated upon I?B phosphorylation by the I?B kinase
(IKK) complex comprising inhibitor of kappaB kinase 1 (IKK1), inhibitor of kappaB kinase 2
specific IKK2 deletion during liver regeneration. A 70% PH was performed on IKK2f/f(wild-
type) and IKK2?LPCmice (hepatocyte-specific IKK2 knockout mice). PH in IKK2?LPC
compared with IKK2f/fmice resulted in weaker and delayed NF-?B activation in hepatocytes,
while nonparenchymal liver cells showed earlier NF-?B activation and higher tumor necrosis
liver. These results correlated with earlier Jun kinase activity, c-myc expression, and matrix
preceded a more rapid cell cycle progression and earlier hepatocyte proliferation as evidenced
through cyclin and 5-bromo-2-deoxyuridine analysis. Interestingly, despite faster G1/S progres-
sion, IKK2?LPC mice exhibited an enduring mitosis phase, because mitotic bodies were still
observed at later stages after PH. Conclusion: We demonstrate that PH in IKK2?LPC mice
triggers a more rapid and pronounced inflammatory response in nonparenchymal liver cells,
which triggers earlier hepatocyte proliferation. (HEPATOLOGY 2008;47:2036-2050.)
liver can restore its mass and cell function upon hepato-
he healthy liver is a quiescent organ with low cell
turnover. However, after injury caused by surgi-
cal resection, chemicals, or viral infections, the
liver regeneration in rodents is partial hepatectomy (PH)
of two-thirds (70%) of the liver. Liver regeneration after
PH is a compensatory hyperplasic response and not a
complete restoration of the resected lobes.1PH causes a
significant stress to the liver as it dramatically disrupts
organ homeostasis, and the proinflammatory response,
triggered by surgery, primes the remnant hepatocytes to
proliferate and recover liver mass.1
Activation of the innate immune system through
Toll-like receptor (TLR) and other pattern-recogni-
tion molecules plays a key role in host defense against
invading pathogens.2Recent studies have highlighted
the crucial contribution of these mechanisms to the
inflammatory response after PH.3-5During the prim-
ing phase of liver regeneration, Kupffer cells are acti-
vated and secrete proinflammatory cytokines, most
prominently tumor necrosis factor (TNF)-?, interleu-
kin (IL)-6, and IL-1?, which can initiate the acute
phase response in hepatocytes.1,6,7Serum amyloid A
(SAA), C-reactive protein, and the complement system
are proteins involved in this physiologic response to
Abbreviations: BrdU, 5-bromo-2-deoxyuridine; CXC, chemotactic cytokines;
DAPI, 4?,6?-diamidino-2-phenylindole; ECM, extracellular matrix; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; JNK, c-Jun N-terminal
kinase; MMP-9, matrix metalloproteinase-9; mRNA, messenger RNA; NF-?B,
nuclear factor ?B; NPC, nonparenchymal cell; PCR, polymerase chain reaction;
PH, partial hepatectomy; PMNC, polymorphonuclear cell; SAA, serum amyloid A.
From the Medical Clinic III, University Hospital Aachen (RWTH), Aachen,
Received August 10, 2007; accepted January 29, 2008.
Supported by a grant of the Deutsche Krebshilfe. Yann Malato was supported by
the Sheila Sherlock entry level research fellowship of the EASL. Naiara Beraza was
supported in part by a postdoctoral fellowship from the Program of Researchers’
*These authors contributed equally to this study.
ical Clinic III, University Hospital Aachen (RWTH), Pauwelsstrasse 30, 52074
Aachen, Germany. E-mail: firstname.lastname@example.org; fax: (49)-241-80-82455
Copyright © 2008 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Nothing to report.
restore liver homeostasis.6-8Proinflammatory cytokine
secretion from Kupffer cells during liver injury requires
nuclear factor ?B (NF-?B) activation and triggers a
cascade leading to influx of inflammatory cells.9
Lipopolysaccharide, IL-1?, or TNF can induce pro-
duction of chemotactic cytokines (chemokines [CXC])
such as CXC ligand-1 (CXCL-1 or KC). These chemo-
kines belong to the Glu-Leu-Arg positive (ELR?) CXC
family, which differ from ELR?CXC chemokines both
structurally (due to the presence of a three–amino acid
sequence [Glu-Leu-Arg]) and functionally, because they
can exert antagonistic effects.10ELR?CXC chemokines
contribute to wound healing and resolution of inflamma-
tory events by stimulating polymorphonuclear cell
they have protective and proregenerative properties as
shown during acetaminophen-induced liver injury.11
Earlier work defined the proinflammatory cytokine
TNF as an essential molecule involved in liver regenera-
patocyte proliferation.12These results indicate that
intracellular pathways of the TNF/TNF-R1 system are
involved in mediating hepatocyte proliferation in vivo.
Shortly after PH, TNF is rapidly secreted from Kupffer
cells, inducing NF-?B activation.1NF-?B is held in the
cytoplasm in its inactive form when bound to I?B. Upon
tor-B essential modifier (NEMO)) phosphorylates I?B,
leading to its degradation and resulting in NF-?B nuclear
translocation and target gene transcription.13
The role of NF-?B during liver regeneration has been
previously investigated although it remains controver-
sial.14-16Moreover, the implication of IKK2-driven
NF-?B signaling in hepatocytes during liver regeneration
remains to be elucidated. Because IKK?/IKK2 knockout
mice die in utero due to massive liver apoptosis, we used
hepatocyte-specific IKK2 knockout mice (IKK2?LPC).17
Our previous work has shown that hepatocyte-specific
IKK2 deletion exerts different outcomes depending on
IKK2?LPC hepatocytes after exogenous TNF adminis-
tration, NF-?B activation is attenuated during ischemia/
reperfusion-induced liver injury.17Therefore, we used
our IKK2?LPC mice to elucidate the impact of hepato-
and its implication during liver regeneration.
Materials and Methods
Generation of Conditional IKK2 Knockout Mice.
(IKK2f/f) were generated as described previously.17
IKK2f/fmice were then crossed with Alfp-cre transgenic
albumin promoter.18Crossings rendered Cre-positive
(IKK2?LPC) and Cre-negative (IKK2f/f) mice that were
genotyped via polymerase chain reaction (PCR) analysis
as described previously.17All mice were backcrossed to a
pure Bl6/C57 background, and 8-week-old mice were
used for the experiments. Animals were held according to
the human care criteria prepared by the National Acad-
emy of Sciences (NIH publication 86-23, revised 1985).
Systemic NF-?B inhibition. To inhibit IKK2 in all
cell compartments, we administrated 10 ?g/g body
weight of the specific pharmaceutical IKK2 inhibitor
AS60286817(kindly provided by Merck-Serono Interna-
tional SA) or vehicle (0.5% carboxymethylcellulose/
Tween 20 0.25%). Treatment was given orally 24 hours
after liver resection.
Partial Hepatectomy. Pathogen-free 7-week-old to
9-week-old male mice were used for PH. PH operations
were performed under ketamin/xylazine anesthesia as de-
scribed previously.19Each time point provides at least four
liquid nitrogen and Tissue Tek (Sakura Europe).
RNA Isolation and Quantitative Real-Time PCR.
Livers were harvested and snap frozen in liquid nitrogen.
RNA was isolated with PeqGold-RNA pure kit (Peqlab).
ers and reverse transcription with M-MLV Reverse Tran-
scriptase (Invitrogen). Quantitative real-time PCR was
performed using SYBR Green reagent (Invitrogen) in an
ABI-Prism 7300 real-time PCR system (Applied Biosys-
tems). Reactions were performed twice in triplicate, and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
values were used to normalize gene expression which was
expressed in times versus control basal expression and
quantified via delta-delta CT calculation. Primers can be
provided under request.
Quantification of Liver Cytokine Levels. Frozen liv-
ers were homogenized in ice-cold lysis buffer containing 10
mM [4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid],
2 mM ethylene diamine tetraacetic acid (pH 8.0), 5 mM
dithiothreitol, 1 mM Pefabloc, and 1 tablet of cocktail of
proteinase inhibitors (Roche). TNF and IL-6 liver expres-
immunosorbent assay that was performed following the
manufacturer’s instructions (R&D Systems).
c-Jun N-Terminal Kinase Activity Assay. c-Jun N-
terminal kinase (JNK) activity was assessed as described
previously.20As a substrate, we used a purified recombi-
HEPATOLOGY, Vol. 47, No. 6, 2008 MALATO ET AL.2037
nant protein containing the N-terminal amino acids
1-223 of the human c-Jun fused to glutathion-S-trans-
ferase. For immobilization, 15 ?g recombinant glutathion-
beads (Sigma) in buffer containing 1.0% Triton X-100 and
1 mM dithiothreitol followed by incubation with 50 ?g of
total liver protein extract at 4°C overnight. After extensive
washing in 20 mM [4-(2-hydroxyethyl)-1-piperazine eth-
anesulfonic acid] (pH 7.5); 20 mM MgCl2; 20 mM glycer-
ophosphate; and 2 mM dithiothreitol, the bead-protein
complexes were incubated with [32P]?–adenosine triphos-
phate and 40 pM adenosine triphosphate for 20 minutes at
30°C. After kinase reaction, the proteins were fraction-
ated on a 10% sodium dodecylsulfate–polyacrylamidegel
and visualized on X-ray film (Amersham). A single promi-
Western Blot Analysis. Protein extracts were resolved
in a 10% sodium dodecyl sulfate–polyacrylamide gel and
transferred to nitrocellulose membranes (Whatman).
Membranes were blocked with 5% milk in Tris-buffered
saline and probed with the following primary antibodies:
p-IKK1/2 (Ser176/180), p-I?B-?, p-STAT-3, p-cdc2
(Cell Signaling Technology), cyclin D1, and cyclin A
(Santa Cruz Biotechnology). As a loading control, we
used GAPDH antibody (Biogenesis) and ?-tubulin
(Sigma). As secondary antibodies, we used anti-rabbit
IgG–HRP-linked (Cell Signaling Technology) and anti-
mouse IgG–HRP-linked (Santa Cruz Biotechnology).
Immunofluorescence. All immunofluorescence de-
scribed in this study was performed on liver cryosections.
anol–30% acetone 10 minutes at room temperature for
p65 (sc-109; Santa Cruz Biotechnology) and F4/80 (Se-
rotec) and in 4% paraformaldehyde for CD11b (Becton
for p65, goat anti-rat with fluorescein isothiocyanate for
Hepatocyte proliferation was detected via immunoflu-
orescence through 5-bromo-2-deoxyuridine (Amersham
Pharmacia) incorporation to the nuclei of hepatocytes
undergoing DNA synthesis and anti-phospho-Histone
H3 antibody (Cell Signaling Technology) was used to
assess the presence of mitotic bodies after PH on liver
sections. As secondary antibodies we used goat-anti-
mouse Cy-3 labeled and goat anti-rabbit fluorescein iso-
thiocyanate labeled respectively (Jackson Research).
Quantification of proliferating and mitotic hepatocytes
was performed by counting 5-bromo-2-deoxyuridine
(BrdU) or phospho-Histone H3–positive nuclei relative
to the total nuclei per power field (?200) stained with
Zymography. Whole liver proteins, extracted with an
Nonidet P-40 buffer, were subjected to electrophoresis in
an 8% sodium dodecyl sulfate–polyacrylamide gel sup-
plemented with 10 mg/mL of Gelatinase A (Sigma). Gel
2.5% in a rocking surface. Next, the gel was incubated at
37 °C for 24 hours in a buffer (pH 7.4) containing 50
mM Tris HCl, 10 mM CaCl2? 2H2O, 100 mM NaCl
and 0.05% Brij 35 (Sigma). Gel was fixed and stained
with 0.5% Coomassie blue in 10% acetic acid, 30% iso-
matrix metalloproteinase-9 (MMP-9) activity was de-
tected as a nonstained band. This experiment was re-
peated three times in all samples (n ? 4).
Cell Isolation and Flow Cytometry. Single-cell sus-
pensions were yielded through mechanical enzymatic di-
whole liver samples were minced with scissors and subse-
quently digested at 37°C for 30 minutes in 0.3225 U/mL
collagenase D (Roche Applied Science) in RPMI me-
dium. After digestion, the extract was filtered twice
though a 12-?m filter (Becton Dickinson). The suspen-
conglomerates. The supernatant was washed twice in
Hank’s balanced salt solution supplemented with 1% bo-
tic acid. Cells were stained for CD45, F4/80, Gr1 (BD
Biosciences), and CD11b (Becton Dickinson), as well as
NK1.1 and CD3 (eBioscience) and analyzed via flow cy-
tometry (FACSCanto II; Becton Dickinson).
For analysis of blood leukocytes, blood was taken via
the retro-orbital venous plexus. Red blood cell lysis was
performed using 1X Pharmlyse buffer (Becton Dickin-
son) following the manufacturer’s instructions. Leuko-
cytes were then stained for CD11b, Gr1, CD19 (Becton
Dickinson), CD115, and CD3 (eBioscience) and sub-
jected to flow cytometric analysis. White blood cells were
counted with an automat in the animal facility of the
cells per microliter.
Statistical Analysis. Data are expressed as the
mean ? standard deviation. Statistical significance was
determined via 2-way analysis of variance followed by
Student t test.
IKK2 Hepatocyte-Specific Deletion Modifies I-?B?
Phosphorylation and NF-?B Activation After PH. Af-
ter stimulation, the IKK complex gets activated by phos-
2038 MALATO ET AL. HEPATOLOGY, June 2008
phorylation of the catalytic subunits IKK1 and IKK2.
These kinases phosphorylate I-?B, eventually triggering
NF-?B nuclear translocation and target gene activation.
We previously generated hepatocyte-specific IKK2 ko
mice (IKK2?LPC), which show effective IKK2 dele-
tion in hepatocytes.17PH was performed in these
knockout mice and wild-type controls (IKK2f/f), and
we first studied the impact of IKK2 deletion in the IKK
complex using an IKK1/2 phospho-specific antibody
(Ser176/180). In IKK2f/f, we observed transient
IKK1/2 phosphorylation 1 hour after PH (Fig. 1A),
whereas strong phospho-IKK1/2 expression was found
in IKK2?LPC 1, 3, and 6 hours after PH (Fig. 1A). No
significant differences in IKK1 protein level were evi-
dent between the 2 mice strains at different time points
after PH (Fig. 1A).
We then tested if lack of hepatocytic IKK2 expression
and strong IKK activation alters I-?B? phosphorylation
after PH. In IKK2f/fmice, high p-I-?B? expression was
already found 1 hour after PH, and lower I-?B? phos-
phorylation was evident 6 hours after PH (Fig. 1B). In
IKK2?LPC mice, p-I-?B? expression was detected start-
ing 3 hours after PH, indicating delayed NF-?B activa-
tion (Fig. 1B). These data suggest that IKK2 deletion and
PH trigger increased IKK1 activation leading to delayed
activate the NF-?B noncanonical pathway.21
Because these results demonstrated altered IKK com-
plex activity in IKK2?LPC mice, we now investigated
NF-?B nuclear translocation in both animal strains via
immunofluorescence using a p65 antibody. In order to
dissect the cellular distribution of NF-?B activation we
costained with a F4/80 antibody, which labels Kupffer
cells. In IKK2f/fmice, p65 staining was positive 1 hour
after liver resection in hepatocytes (full arrows pointing
down), whereas 3 hours after PH, nonparenchymal cells
detected in hepatocytes 1 hour after resection, whereas
positive signals were detected in nonparenchymal cells—
(Fig. 1C). Three hours after resection, NF-?B positive
hepatocytes were detected in IKK2?LPC (Fig. 1C). The
p65 antibody used in this study binds to the N-terminal
lated cells remain p65-negative.22
In summary, lack of IKK2 expression in hepatocytes
alters the NF-?B activation pattern in both parenchymal
and nonparenchymal liver cells after PH.
Lack of Hepatocyte IKK2 Promotes Earlier Cell
Cycle Entry After PH. Kupffer cell–derived TNF pro-
duction is induced after PH triggering NF-?B activa-
tion.1As expected, liver TNF expression increased in
IKK2f/fanimals up to 6 hours after PH, whereas in
IKK2?LPC mice it was already increased 1 hour after
resection, remaining elevated 3 and 6 hours after PH
TNF-dependent JNK activation is involved in trigger-
ing cell cycle progression after PH.12,23,24Kinase assays of
IKK2f/fmice whole-liver extracts showed transient JNK
activation 1 hour after PH, whereas IKK2?LPC mice
exhibited sustained JNK activity during the initial time
points after liver resection (Fig. 2B).
Because c-myc is shortly activated after PH as part of
the immediate-early gene response, it mediates early cell
cycle progression of hepatocytes.25In IKK2f/fmice c-myc
messenger RNA (mRNA) expression increased already 3
Unexpectedly, faster c-myc expression was evident in
IKK2?LPC animals, because levels were found a maxi-
mum 3 hours after PH.
In the early phase of liver regeneration, extracellular
matrix (ECM) degradation allows release of growth fac-
produced in response to TNF and mediates ECM degra-
dation.28Zymography analysis showed that in IKK2f/f
animals MMP-9 proteinase activity was transiently acti-
vated 6 hours after PH, while in IKK2?LPC mice earlier
and prolonged MMP-9 activity was observed (Fig. 2D).
hepatocytes to leave earlier the quiescent (G0) state.
Earlier S-Phase Progression and Hepatocyte Pro-
liferation in IKK2?LPC Mice After PH. Next, we
analyzed G1/S progression of hepatocytes in IKK2?LPC
IKK2f/fand IKK2?LPC mice between 24 and 72 hours
after PH. Between 24 and 40 hours after liver resection,
significantly more BrdU-positive hepatocytes were found
in IKK2?LPC mice and maximum DNA synthesis was
evident 48 hours after PH (Fig. 3A-B). In IKK2f/fmice a
single and pronounced peak of DNA synthesis was found
48 hours after PH, and the number of proliferating cells
was higher than IKK2?LPC mice at this time point.
D1 expression was found between mouse strains, because
protein levels in IKK2?LPC peaked 24 hours after PH,
whereas in wild-type littermates it was found a maximum
of 40 hours after resection (Fig. 4A). Cyclin E mRNA
analysis showed maximum up-regulation in IKK2?LPC
40 hours after PH that was delayed to 48 hours in wild-
type mice (Fig. 4B). Cyclin A mRNA expression was first
elevated 40 hours after PH in IKK2?LPC mice, while in
HEPATOLOGY, Vol. 47, No. 6, 2008MALATO ET AL.2039
Fig. 1. Hepatocyte-specific IKK2 deletion attenuates parenchymal NF-?B activation after PH. (A) Western blot analysis of p-IKK1/2, total IKK1,
and p-I-?B? before and 1, 3, and 6 hours after PH. GAPDH and ?-tubulin (lower panel) were used as loading controls. (B) Immunohistochemistry
using p65?secondary Cy3-labeled and F4/80?secondary fluorescein isothiocyanate antibodies in livers of IKK2f/f and IKK2?LPC mice before and
1 and 3 hours after PH (left panel). DAPI staining was performed to detect nuclei. (C) Merged picture (right panel) of p65 (left panel) and F4/80
stainings (middle panel). Thin arrows pointing up show Kupffer cell p65 staining; full arrows pointing down show hepatocyte p65 staining.
2040MALATO ET AL. HEPATOLOGY, June 2008
wild-type mice no significant increase could be observed
at this time point (Fig. 4C). At 48 hours, cyclin A expres-
sion peaked in both groups but was higher in IKK2f/f
cyclin A activation in IKK2?LPC mice (Fig. 4D). These
results demonstrate earlier G1/S-phase progression and
DNA synthesis in IKK2?LPC mice after PH.
Prolonged Mitotic Phase in IKK2?LPC Mice after
PH. Cells remain arrested in G2phase when cyclin B1
forms a complex with cdc2 (also known as Cdk1) that is
inactive upon Tyr-15 and Thr-14 phosphorylation.29
G2/M phase progression occurs after dephosphorylation
of cdc2 by Cdc25C phosphatase.30Cyclin B1 mRNA
expression was not different between IKK2?LPC and
IKK2f/fmice (Fig. 5A). However, a different kinetic in
cdc2 activation was observed. In IKK2?LPC, faster pro-
gression into the mitotic phase occurred, becuase we
found earlier phosphorylation of cdc2 that was dephos-
phorylated (and thus activated) 60 hours after PH,
whereas in IKK2f/fmice a certain degree of phosphory-
lated cdc-2 (inactive form) could still be detected at this
time point (Fig. 5B).
Phosphorylation of Histone H3 is essential for chro-
mosome condensation and progression from G2to mito-
sis.31Immunohistochemistry using a phospho-Histone
mitosis in both mouse strains 60 hours after PH (Fig.
5C-D). However, although the mitotic rate remained at
the same level in IKK2f/fmice 72 hours after PH, we
observed a significant increase in IKK2?LPC mice. After
96 hours, few phospho-Histone H3–positive cells could
be detected, whereas no mitosis could be detected in
ECM modulation is important in order to shift hepa-
tocytes in a differentiated and quiescent state.26Differ-
ences in ECM degradation were evident between the 2
mouse strains at late time points after PH. In IKK2f/f
hours (Fig. 5E), while in IKK2?LPC mice it was found
later (48-60 hours after PH). Together, these results sug-
gest that IKK2?LPC mice require a prolonged mitotic
phase in order to reach a quiescent state after PH.
Stronger CXCL1 and Acute Phase Gene Expression
in IKK2?LPC Mice After PH. Recent studies high-
lighted the role of the innate immune response during
liver regeneration3-5where proinflammatory cytokines
and acute phase genes play a main role. Our first results
accompanied by high TNF expression in IKK2?LPC
mice leading to earlier priming. In addition to TNF, IL-6
f/fanimals at this time point.
Fig. 2. (A) IKK2?LPC mice exhibit earlier cell cycle priming after PH. TNF protein expression detected by enzyme-linked immunosorbent assay in
livers of IKK2f/fand IKK2?LPC mice. Solid lines represent IKK2f/f; dashed lines represent IKK2?LPC (n ? 4 animals/time point; ***P ? 0.001,
**P ? 0.01 [IKK2f/fversus IKK2?LPC]). (B) JNK activity was detected in whole liver extracts showing stronger and sustained kinase activity in
IKK2?LPC mice after PH. (C) Real-time PCR for c-myc liver mRNA expression in IKK2?LPC and IKK2f/fmice before and after PH. (D) Zymography
to determine MMP-9 gelatinase activity in IKK2?LPC mice and IKK2f/fmice before and after PH. Two representative samples from each time point
and genotype are shown. The position of the digested band is shown by an arrow.
HEPATOLOGY, Vol. 47, No. 6, 2008 MALATO ET AL.2041
is involved in triggering hepatocytes to leave their quies-
cent state. Liver IL-6 expression increased in both IKK2f/f
and IKK2?LPC mice during the first 6 hours after PH;
however, no significant differences were found between
the two mouse strains (Fig. 6A). This result was further
confirmed after no significant difference in nuclear phos-
pho-STAT-3 expression was found between both groups
after PH (Fig. 6B).
SAA is one of the main acute phase response proteins
and is part of the unspecific defense in mice after liver
injury.7,26Maximal SAA levels were found in IKK2f/fand
IKK2?LPC mice 24 hours after PH. However, earlier
and more intense acute phase response was detected in
IKK2?LPC livers as evidenced by the 200-fold increase
in SAA expression found already 6 hours after liver resec-
tion (Fig. 6C).
Fig. 3. IKK2 deletion in hepatocytes accelerates hepatocyte proliferation after PH. (A) Immunohistochemistry using a BrdU antibody followed by
a Cy3-labeled secondary antibody in livers of IKK2f/fand IKK2?LPC mice before and between 24 and 72 hours after PH (upper panel). Merged DAPI
and BrdU staining is shown in the lower panels. (B) Quantification of BrdU-positive cells relative to total nuclei (DAPI) per ?200 power field. Solid
lines represent IKK2f/f; dashed lines represent IKK2?LPC (n ? 4 animals/time point; **P ? 0.01, ***P ? 0.001 [IKK2f/fversus IKK2?LPC]).
2042 MALATO ET AL.HEPATOLOGY, June 2008
CXC chemokines like CXCL-1 attract neutrophils to
facilitating tissue repair.10We found that basal CXCL-1
mRNA expression was higher in livers of IKK2?LPC
compared with IKK2f/fanimals (Fig. 6D). After PH,
CXCL-1 expression in IKK2f/fmice increased over time
during the first 24 hours, whereas a dramatically faster
and stronger up-regulation was evident in IKK2?LPC
IKK2?LPC after PH.
IKK2?LPC Mice Show Enhanced Recruitment of
Inflammatory Cells After PH. To study if the increased
chemoattractant response observed in IKK2?LPC mice
formed FACS analysis and white blood cell counts col-
lected from IKK2f/fand IKK2?LPC mice before and 12
hours after PH. In untreated animals from both strains, a
comparable percentage of CD11b?/Gr1? cells, mainly
PMNCs, were detected in peripheral blood (Fig. 7A).
This cell population increased 12 hours after PH in
IKK2?LPC, while no regulation was evident in IKK2f/f
mice (Fig. 7A).
We then investigated if increased PMNC mobiliza-
tion in IKK2?LPC mice after PH correlated with a
higher recruitment to the liver (Fig. 7B). Similar num-
bers of liver resident CD45?/CD11b?/Gr1?/F4/
80? cells—mainly PMNCs—could be detected in
both strains before PH. Twelve hours after PH, the
amount of these cells did not increase significantly in
IKK2f/fanimals, whereas livers of IKK2?LPC mice
already revealed a 3.5-fold increase of CD45?/
CD11b?/Gr1?/F4/80? cells compared with the
basal level and control wild-type littermates. These re-
sults suggest that the earlier inflammatory and che-
moattractant response in IKK2?LPC after PH had a
direct impact on mobilization of neutrophils to the
blood stream and recruitment to the liver. This finding
was further confirmed by immunofluorescence of liver
sections using a CD11b antibody, supporting a more
pronounced infiltration of CD11b?cells after PH in
IKK2?LPC mice (Fig. 7C).
Fig. 4. Lack of hepatocyte IKK2 expression triggers earlier G1/S phase transition after PH. (A) Western blot analysis for cyclin D1 expression of
whole protein liver extracts of IKK2f/fand IKK2?LPC mice at time points before and after PH. (B) Real-time PCR for liver cyclin E mRNA expression
of IKK2f/fand IKK2?LPC animals at time points before and after PH. (C) Liver cyclin A mRNA and (D) protein expression in IKK2f/fand IKK2?LPC
mice before and after PH. Solid lines represent IKK2f/f; dashed lines represent IKK2?LPC [n ? 4 animals/time point; *P ? 0.05, **P ? 0.01 (IKK2f/f
HEPATOLOGY, Vol. 47, No. 6, 2008 MALATO ET AL.2043
Fig. 5. Misregulation of mitosis in hepatocyte-specific IKK2-deleted mice after PH. (A) Real-time PCR for liver cyclin B1 mRNA expression in IKK2f/f
and IKK2?LPC mice before and after PH. (B) Phospho-cdc2 expression as evidenced via Western blot analysis in IKK2f/fand IKK2?LPC mice. (C)
Immunofluorescence with a phospho-Histone H3 antibody before and between 60 and 96 hours after PH in IKK2f/fand IKK2?LPC mice (upper panel).
Merged DAPI and anti-phospho-Histone H3 staining is shown in the lower panel. (D) Quantification of anti-phospho-Histone H3 positive cells in livers
of IKK2f/fand IKK2?LPC mice. Solid lines represent IKK2f/f; dashed lines represent IKK2?LPC (n ? 4 animals/time point; ***P ? 0.001 [IKK2f/f
versus IKK2?LPC]). (E) Zymography for MMP-9 gelatinase activity in IKK2?LPC mice occurred later than in IKK2f/fmice after PH. Two representative
samples from each time point and genotype are shown. The position of the digested band is shown by an arrow.
2044 MALATO ET AL. HEPATOLOGY, June 2008
Systemic NF-?B Inhibition with AS602868 Does
Not Affect Hepatocyte Proliferation After PH. Our
data show that hepatocyte-specific IKK2 deletion is asso-
ciated with a proliferative advantage after PH. There-
fore, we tested if systemic inhibition is also associated
with earlier proliferation after PH. We thus applied an
IKK2 inhibitor AS602868 in a dose we used in previ-
ous experiments to block IKK2 after ischemia/reperfu-
sion injury17and to block progression of NASH.32
We administered AS602868 24 hours and 1 hour be-
fore PH as well as 24 hours after PH. AS602868 treat-
ment compared with controls (vehicle) had no impact on
the increase and the maximum of BrdU synthesis 40 and
48 hours after PH (Fig. 8A-B). These results indicate that
systemic, in contrast to hepatocytic IKK2 inhibition, has
no impact on cell cycle progression after PH.
NF-?B is an essential transcription factor to maintain
liver homeostasis.13,33Additionally, knockout mice of
members of the IKK complex controlling NF-?B activa-
tion die from hepatocyte apoptosis during embryonal de-
different factors involved in this pathway is of central
relevance for the liver. In the present study, we used hep-
atocyte-specific IKK2 knockout mice to further elucidate
the role of the IKK complex during liver regeneration.
After PH in IKK2?LPC mice, major differences in
NF-?B activation were found compared to wild-type
IKK2f/fanimals. In wild-type mice NF-?B activation was
first found in hepatocytes, while in IKK2?LPC animals
p65 was first positive in nonparenchymal cells (NPCs),
resulting in a stronger inflammatory response. In our pre-
vious study using IKK2?LPC animals the inflammatory
response after I/R injury was attenuated compared with
sion in hepatocytes is essential to direct the crosstalk be-
tween hepatocytes and NPCs in a stimulus-dependent
crosstalk between IKK2-deficient hepatocytes and NPCs
during liver regeneration is not fully understood.
Fig. 6. Earlier and stronger inflammatory response in hepatocyte-specific IKK2 knockout mice. (A) IL-6 protein expression detected via
enzyme-linked immunosorbent assay at time points before and after PH in livers of IKK2f/fand IKK2?LPC mice. (B) Western blot analysis of whole
liver extracts of IKK2f/fand IKK2?LPC animals for phospho-STAT-3 expression before and after PH. GAPDH was used as a loading control. Real-time
PCR to analyze liver mRNA levels in IKK2f/fand IKK2?LPC mice for (C) SAA and (D) CXCL-1 before and after PH. Solid lines represent IKK2f/f; dashed
lines represent IKK2?LPC (n ? 4 animals/time point; *P ? 0.05, **P ? 0.01, ***P ? 0.001 [IKK2f/fversus IKK2?LPC]).
HEPATOLOGY, Vol. 47, No. 6, 2008 MALATO ET AL.2045
Fig. 7. Mobilization and recruitment of inflammatory cells after PH. (A) FACS analysis of peripheral blood leukocytes before (left panel) and 12
horus after PH (right panel). Fraction of CD11b? cells is given in percentage of total leukocytes. Diagram shows total number of CD11b?/Gr1?/
CD115? cells (PMNCs) in the peripheral blood at the given time points. (B) Fluorescence-activated cell sorting analysis of immune cells isolated
from hepatic tissue before and 12 hours after PH. Dot plots show CD11b and F4/80 expression of cells pregated for CD45? respectively. Bar chart
shows the absolute frequency of CD11b?/Gr1?/F4/80? cells (mainly PMNCs) displayed as the percentage of total liver cells (normalized to
CD45? cells). (C) Immunofluorescent staining of frozen liver sections for CD11b? confirmed the results obtained via fluorescence-activated cell
sorting (red, CD11b-positive cells; blue, DAPI-stained nuclei). Black bars represent IKK2f/f; grey bars represent IKK2?LPC (n ? 4 animals/time point;
*P ? 0.05 [IKK2f/fversus IKK2?LPC]).
2046MALATO ET AL. HEPATOLOGY, June 2008
Analysis of the IKK complex activity revealed that
IKK2?LPC mice showed stronger IKK1 activation after
PH despite later p-I?B? expression that correlated with
delayed kinetics of p65 activation in hepatocytes. A po-
tential explanation for the stronger IKK1 activation ob-
served could be that lack of IKK2 protein expression
results in increased activation (phosphorylation) of IKK1
and consequently, enhanced signaling via the noncanoni-
role of IKK1 during liver regeneration has not been stud-
Fig. 8. Systemic pharmaceutical IKK2 inhibition with AS602868 had no impact on hepatocyte proliferation after PH. (A) Immunohistochemistry using
BrdU and a Cy3-labeled antibody in livers orally treated with AS602868 or vehicle showed no significant differences between treatment groups (AS602868
versus vehicle). (B) Solid lines represent quantification of BrdU-positive cells relative to vehicle; dashed lines represent AS602868 (n ? 5
HEPATOLOGY, Vol. 47, No. 6, 2008MALATO ET AL. 2047
such as lymphotoxin-? (LT?) and its receptor (LT?R),
show impaired liver regeneration after PH36pointing to
the relevance of the noncanonical pathway during liver
via the noncanonical pathway in IKK2?LPC mice may
cooperate in the production of ligands that activate
NPCs, contributing to the crosstalk between cell com-
partments after PH.37
In order to further test the relevance of IKK2 for the
crosstalk between hepatocytes and NPCs, we used the
IKK2 inhibitor AS602868 to systemically block IKK2
activation during liver regeneration. Interestingly, inhibi-
alter DNA synthesis after PH. Earlier results demon-
strated that IKK2 inhibition in myeloid cells blocks
NF-?B activation38and that by using AS602868 the in-
flammatory response is also blocked in NPCs in the liv-
er.32This, together with our present data suggests that
AS602868 administration during PH also reduces the
inflammatory response in NPCs which reverts the prolif-
erative advantage shown in IKK2?LPC mice.
Our results using IKK2?LPC mice and AS602868
cannot be compared to previous experiments using either
an adenoviral I?B superrepressor or transgenic mice car-
rying an inducible I?B mutant in hepatocytes.15,16Both
approaches blocked NF-?B activation downstream of the
IKK complex; however, the outcome of the studies was
liferation were found when the adenoviral vector was
applied,15whereas no significant effects on liver regener-
ation were observed using transgenic mice carrying the
inducible I?B? mutant.16A major problem using the
apoptosis and DNA synthesis already before PH.15In the
Chaisson et al.16study, NF-?B was completely blocked
and not delayed, as found in our study. This difference
between the 2 studies might also contribute to the obser-
vation why the imbalance in NF-?B activation as found
in IKK2?LPC mice triggers the strong inflammatory re-
sponse in the NPC compartment.
We show that earlier NF-?B activation in the NPC
compartment observed in mice lacking IKK2 correlated
with a rapid and strong inflammatory response. PMNCs
(neutrophils) have differential roles depending on the
or display beneficial effects by mediating resolution of
wound healing, tissue repair, and cell proliferation.11,39
Targeting immune cells to the damaged tissue relies on
the production of chemoattractants. ELR?CXC chemo-
kines mediate chemotaxis of neutrophils. After PH,
higher expression of CXCL1 in IKK2?LPC mice corre-
ment to the liver that may contribute to the earlier
ELR?CXC chemokines contribute to liver regeneration
after acetaminophen-induced injury.11
In order to elucidate the molecular mechanism by
liver of IKK2?LPC mice we studied the activation of
in wild-type and IKK2?LPC mice after PH. We have
previously demonstrated that hepatocytes are a potent
source of CXCL1, which is strongly induced in response
and pSTAT3 were equal in the 2 groups. However, there
communicate41,42in an IKK2-dependent fashion.43We
therefore propose that increased CXCL-1 levels observed
in IKK2?LPC mice after PH are a result of disrupted
inhibition of STAT-3 by IKK2. This is further supported
by enhanced SAA transcription in IKK2?LPC mice as
SAA gene expression is tightly controlled by gp130/
both synergistic and inhibitory consequences during
acute phase gene transcription, because they share bind-
ing sites in the promoters of these genes, rendering en-
hanced or competitive overlapping interactions.41-43
Thus, it is very likely that despite comparable IL-6 pro-
duction and STAT-3 phosphorylation after PH,
IKK2?LPC mice exhibit earlier, stronger, and sustained
SAA production than wild-type littermates due to the
lower presence of competitive NF-?B, allowing more ef-
ficient STAT-3 binding to the SAA promoter in hepato-
Additionally, we investigated the degree of ECM deg-
radation in the liver, because this promotes the bioavail-
ability of growth factors such as hepatocyte growth
inflammatory response.44MMP-9 is a TNF-inducible
molecule45that plays a key role in ECM degradation and
deficient mice exhibit slower and delayed hepatocyte pro-
expression in livers of hepatocyte-IKK2 deleted mice cor-
contribute to earlier priming as observed in IKK2?LPC
These results correlated with prolonged JNK activa-
tion, a known target of TNF that can be negatively regu-
IKK2?LPC mice after PH. Both pathways are directly
stimuli into earlier cell cycle progression. Because cyclin
D is a direct target gene of JNK/AP-1 signaling, these
in IKK2?LPC miceas
2048MALATO ET AL. HEPATOLOGY, June 2008
results provide the molecular link between the stronger
inflammatory response and earlier G1/S-phase transition
in IKK2?LPC mice.24
During mitosis, differences were found between
els peaked at the same time point, exit of mitosis was
prolonged in IKK2?LPC animals as evidenced by longer
Histone H3 phosphorylation and delayed ECM degrada-
tion. At present, we have no obvious explanation for this
observation. However, these differences could either be a
direct effect of lack of IKK2 expression in hepatocytes or
lier time point of hepatocyte proliferation in IKK2?LPC
The present work contributes essentially to the under-
standing of the role of IKK2 and consequently NF-?B
signaling in injury induced liver regeneration. Addition-
ally, it highlights the relevance of NPCs and their
crosstalk with hepatocytes during this process and sup-
ports our previous work depicting the importance of pre-
serving the balance of NF-?B activity between the liver
cell compartments in order to maintain liver homeostasis
and efficiently counteract injury.17,32,47Our results sug-
future target for regenerative or anti-inflammatory treat-
ment strategies in liver diseases.
Guyot for her expert help in establishing the zymography
analysis. We are very grateful to Dr. Michel Dreano for
promptly providing us the AS602868. We are also very
thankful to Prof. Dr. Manolis Pasparakis (University of
Cologne, Germany) for kindly providing us with the
We want to thank Dr Christelle
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