The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 205, No. 1, January 21, 2008 91-103 www.jem.org/cgi/doi/
Cytokines are secreted proteins that regulate
multiple processes, including growth and dif-
ferentiation, cell survival, hematopoiesis, and
immunological functions. Many cytokine ef-
fects are transduced through the JAK – STAT
pathway. JAK proteins, when bound to cyto-
kine receptors, assemble in phosphorylated recep-
tor complexes that create docking sites for proteins
such as the STATs, which contain Src homol-
ogy 2 domains. STATs are activated through
phosphorylation by JAKs, and the activated
STATs can dimerize and bind to DNA to acti-
vate transcription of target genes. Important
STAT targets include the suppressor of cytokine
signaling ( Socs ) genes, which encode eight pro-
teins that inhibit a variety of signaling pathways
( 1 ). SOCS proteins appear to inhibit cytokine
signaling by targeting diff erent components of
the signaling complex either (a) by directly bind-
ing to activated, tyrosine phosphorylated cyto-
kine receptors or JAKs via their Src homology 2
domains, or (b) by targeting receptor complexes
for proteosomal degradation via the SOCS box
( 1 ). Thus, stimulation of Socs transcription by
STATs establishes a negative feedback loop that
inhibits ongoing activation of cytokine signaling.
SOCS proteins have been shown to play an im-
portant role in regulating cytokine activity at
several levels, including modulating cytokine
production and by inhibiting downstream sig-
naling cascades ( 1, 2 ).
Both in the hematopoietic system and in
the liver, SOCS3 is a critical inhibitor of IL-6
signal ing mediated through the gp130 recep-
tor ( 2 ). Mice defi cient in the gp130 receptor
in the liver do not produce STAT3 in response
to IL-6 ( 3 ), and Yang et al. showed that IL-6
has a crucial role in the expression of Socs3 dur-
ing infl ammatory responses ( 4 ). Inhibition of
Abbreviations used: cDNA,
complementary DNA; DAVID,
database for annotation, visualiza-
tion, and integrated discovery;
DEN, N -nitrosodiethylamine;
EGF, epidermal growth factor;
ERK1/2, extracellular signal-
regulated kinase 1/2; h-KO,
hepatocyte-specifi c knockout;
HCC, hepatocellular carcinoma;
HIF1 ? , hypoxia-inducible factor
1 ? ; MAPK, mitogen-activated
protein kinase; MEK, MAPK/
ERK kinase; mTOR, mamma-
lian target of rapamycin; NPC,
nonparenchymal cell; PAINT,
promoter analysis and interaction
network tool; PDGF, platelet-
derived growth factor; PH,
two-thirds partial hepatectomy;
SOCS, suppressor of cytokine
signaling; TLR, Toll-like receptor;
TRE, transcriptional regulatory
element; TRNA, transcriptional
regulatory network analysis. The online version of this article contains supplemental material.
Regulation of liver regeneration
and hepatocarcinogenesis by suppressor
of cytokine signaling 3
Kimberly J. Riehle , 1,2 Jean S. Campbell , 1 Ryan S. McMahan , 1
Melissa M. Johnson , 1 Richard P. Beyer , 3 Theo K. Bammler , 3
and Nelson Fausto 1
1 Department of Pathology, 2 Department of Surgery, and 3 Department of Environmental and Occupational Health Sciences,
University of Washington School of Medicine, Seattle, WA 98195
Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in
multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation
at the early stages of liver regeneration (Campbell, J.S., L. Prichard, F. Schaper, J. Schmitz,
A. Stephenson-Famy, M.E. Rosenfeld, G.M. Argast, P.C. Heinrich, and N. Fausto. 2001.
J. Clin. Invest. 107:1285–1292). We developed Socs3 hepatocyte-specifi c knockout ( Socs3
h-KO) mice to directly study the role of SOCS3 during liver regeneration after a two-thirds
partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA
replication and liver weight restoration after PH in comparison with littermate controls.
Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation
is prolonged, and activation of the mitogenic extracellular signal-regulated kinase 1/2
(ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 defi ciency enhances hepato-
cyte proliferation in association with enhanced STAT3 and ERK activation after epidermal
growth factor or interleukin 6 stimulation. Microarray analyses show that SOCS3 modu-
lates a distinct set of genes, which fall into diverse physiological categories, after PH.
Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop
hepatocellular carcinoma at an accelerated rate. By acting on cytokines and multiple
proliferative pathways, SOCS3 modulates both physiological and neoplastic proliferative
processes in the liver and may act as a tumor suppressor.
ROLE OF SOCS3 IN LIVER REGENERATION AND HCC | Riehle et al.
Liver regeneration is enhanced in Socs3 h-KO mice
Socs3 is robustly induced during the fi rst few hours after PH
( 9 ), suggesting that SOCS3 might act as a negative regulator
of liver regeneration. To determine whether Socs3 defi ciency
altered regeneration, we generated Socs3 h-KO mice ( 2 ). In the
absence of an operative or chemical stimulus, Socs3 h-KO liv-
ers were identical to those of control littermates in weight,
histology, and proliferative and apoptotic indices (unpublished
data). To determine whether the lack of SOCS3 would have
an eff ect on DNA replication after PH, we evaluated the pro-
liferative response to PH in Socs3 h-KO mice compared with
that of littermates with intact Socs3 . Surprisingly, Socs3 h-KO
mice display marked enhancement of hepatocyte DNA repli-
cation, as shown by increased BrdU incorporation ( Fig. 1 A ).
Nuclear hepatocyte BrdU labeling in Socs3 h-KO mice is sig-
nifi cantly higher than in control littermates from 32 to 48 h
after PH, and is 90 – 160% higher at the peak of DNA replica-
tion between 36 and 40 h after PH. Additionally, the number
of hepatocyte mitoses is 85 and 89% higher in Socs3 h-KO
mice than that of controls at 48 and 72 h, respectively ( Fig. 1 B ).
As a consequence of the increased hepatocyte replication
in Socs3 h-KO mice, these animals restore their liver weights
after PH 2 d earlier than do controls ( Fig. 1 C ). To further
demonstrate negative regulation by SOCS3 of the progression
of hepatocytes through the cell cycle in the regenerating liver,
we performed immunoblotting for the cell-cycle proteins cy-
clin A and p107, which are known to be up-regulated during
liver regeneration ( 10 ). Lysates harvested between 24 and 48 h
after PH showed that Socs3 h-KO mice had both earlier and
increased expression of these proteins during liver regenera-
tion, particularly p107, which is strongly expressed in Socs3
h-KO mice from 32 to 48 h after PH ( Fig. 1 D ).
Though previous work has demonstrated that 90 – 95% of
hepatocyte genomic Socs3 is excised in Socs3 h-KO mice ( 2 ),
we wanted to be certain that the strong physiological stimulus
of PH ( 9 ) would not lead to signifi cant expression from resid-
ual copies of the gene and that expression by nonparenchymal
cells (NPCs) was very low. We thus performed Northern
blotting for Socs3 on RNA isolated from Socs3 h-KO and
control littermates at various times after PH. We found virtu-
ally no induction of Socs3 after PH in the KO mice at any of
the times examined ( Fig. 1 E ) and, similarly, did not observe a
compensatory up-regulation of Socs1 ( Fig. 1 F ). In summary,
the data presented in this section clearly demonstrate that
SOCS3 defi ciency increases hepatocyte replication and accel-
erates liver regeneration after PH.
Activation of STAT3 and extracellular signal-regulated
kinase (ERK) signaling pathways in Socs3 h-KO mice
After PH, IL-6 is released by Kupff er cells ( 11 ) and subse-
quently binds its specifi c receptor on the surface of hepato-
cytes. Receptor binding activates JAK to phosphorylate and
activate STAT3, which then dimerizes and translocates to the
nucleus. As we have previously shown that Socs3 expression
this pathway involves the binding of SOCS3 to phosphoty-
rosine 759 of the activated gp130 receptor ( 5 ). IL-6 is one of
the main mediators of the acute-phase response, which is
induced by infl ammatory stimuli in the liver. By regulating
the activity of the IL-6 – gp130 pathway, SOCS3 could have
a major effect on the acute-phase response to liver injury
or infl ammation.
One of the most interesting fi ndings regarding the mech-
anisms that initiate liver regeneration after two-thirds partial
hepatectomy (PH) is the demonstration that several compo-
nents of the innate immune system may be involved in the
initiation process. These components, which include cyto-
kines such as IL-6, proteins of the complement system, and
lymphotoxins, are particularly active during the fi rst 12 – 18 h
after PH, a time period during which hepatocytes transition
from a quiescent state into the cell cycle. Through work with
animal models, we and others have shown that IL-6 is a com-
ponent of a cytokine pathway activated very shortly after PH
that involves signaling through TNFR1 and the activation
of the transcription factors NF- ? B and STAT3 ( 6 – 8 ). It ap-
pears that the components of this pathway are involved in
both cell survival and hepatocyte replication ( 6, 8 ). Data from
our laboratory showed that Socs3 is highly expressed between
2 and 8 h after PH, which partially overlaps with the time
frame of STAT3 expression, and that Socs3 expression in the
regenerating liver is IL-6 dependent ( 9 ). We concluded from
these experiments that in the initial stages of liver regenera-
tion, SOCS3 might shut off the early cytokine response, per-
haps to protect liver cells against the cytotoxic eff ects of
prolonged cytokine expression. However we did not have
direct evidence either to support this hypothesis or to deter-
mine whether SOCS3 might have other functions in the
regenerating liver. Because Socs3 KO mice die during em-
bryogenesis, we developed mice in which Socs3 was specifi -
cally deleted in hepatocytes ( Socs3 fl /fl , Alb-Cre + ; termed Socs3
hepatocyte-specifi c knockout [h-KO] mice) and used these
animals to directly investigate the role of SOCS3 during
Our main expectation was that Socs3 h-KO mice would
show a prolonged acute-phase response, and that excessive
cytokine signaling would lead to toxicity and a decrease in
cell proliferation after PH. Contrary to these expectations,
we demonstrate that in the absence of SOCS3, (a) hepato-
cyte DNA replication and progression through the cell cycle
are markedly enhanced after PH, leading to an acceleration
of liver regeneration; (b) hepatocytes isolated from Socs3
h-KO mice have an increased replication capacity; and (c)
Socs3 -defi cient mice develop hepatocellular carcinoma (HCC)
at an accelerated rate. These data suggest that, in addition to
its role in the control of cytokine expression in the regener-
ating liver, SOCS3 coordinates the responses of innate im-
mune system components with that of proliferative pathways.
Coordination between these systems may be required for the
precise regulation and synchronization of hepatocyte pro-
liferation during liver regeneration and for the prevention
of tumorigenesis in such a highly prolifera tive environment.
JEM VOL. 205, January 21, 2008
PH is prolonged in Socs3 h-KO mice compared with litter-
mates ( Fig. 2, C and D ). We measured IL-6 levels at 18 and
24 h after PH to confi rm that this cytokine is not expressed
later in Socs3 h-KO mice, thus contributing to prolonged ex-
pression of these genes, and did not observe elevated levels of
IL-6 in either Socs3 h-KO or control littermates at these times
(unpublished data). We also determined whether either STAT1
or STAT5 are abnormally activated in the absence of SOCS3
by immunoblotting for phospho-STAT1 and -STAT5 on
liver lysates prepared from Socs3 h-KO mice and littermates
from 0 to 12 h after PH. We did not observe any activation
of either STAT1 or STAT5 at any of the times examined
TNF and IL-6 participate in both the initiation of liver
regeneration and the induction of the acute-phase response,
which are very diff erent biological processes ( 12 ). We show
that in SOCS3-defi cient mice, both cell proliferation and
the expression of some acute-phase response genes are en-
hanced, suggesting that both processes may be negatively
regulated by SOCS3. Phosphorylation of the mitogen-activated
after PH is largely dependent on the IL-6 – STAT3 signaling
pathway ( 9 ), we examined the activation of this pathway
during liver regeneration in Socs3 h-KO mice. Serum IL-6
levels were determined by ELISA from 30 min to 12 h after
PH and do not signifi cantly diff er between Socs3 h-KO and
control littermates ( Fig. 2 A ). This result is not unexpected,
because the production of IL-6 by NPCs would not be al-
tered by SOCS3 defi ciency in hepatocytes. However, there
is a marked diff erence in the phosphorylation of STAT3 be-
tween Socs3 h-KO and control littermates. STAT3 phos-
phorylation is still present 12 h after PH in Socs3 h-KO mice,
whereas it is no longer detectable in control littermates, as
shown by immunoblotting ( Fig. 2 B ). These data confi rm the
observation that SOCS3 acts in a negative feedback loop to
arrest STAT3 activation in hepatocytes during liver regenera-
tion. To confi rm that this prolonged activation of STAT3
would lead to diff erences in gene expression, we examined
the induction Cd14 and Saa2 , which are two acute-phase
response genes and known STAT3 targets in the liver. North-
ern blot analysis shows that the expression of these genes after
Figure 1. Liver regeneration is enhanced in Socs3 h-KO mice. (A) Staining for BrdU indicates increased DNA synthesis in Socs3 h-KO mice.
(B) Increased mitotic fi gures in hepatocytes lacking Socs3. (C) More rapid return to preoperative liver weight in the absence of Socs3. (D) Increased
expression of cyclin A and p107 in Socs3 h-KO mice after PH, demonstrated by immunoblotting. ? -Actin and ? -tubulin are shown as loading controls.
(E) Confi rmation of lack of Socs3 induction after PH in Socs3 h-KO mice by Northern blotting, with cyclophilin as a loading control. (F) Northern blotting
demonstrates no compensatory up-regulation of Socs1 in the absence of Socs3 . Data shown are representative of three to six mice per genotype per time
point and are presented as mean ± SEM. *, P < 0.05.
ROLE OF SOCS3 IN LIVER REGENERATION AND HCC | Riehle et al.
hepatocytes lacking SOCS3 is approximately double of that
of hepatocytes with intact SOCS3 ( Fig. 3 A ). These data in-
dicate that SOCS3 defi ciency appears to result in autocrine
mechanisms that lead to enhanced hepatocyte replication.
The diff erence between the proliferative response of hepato-
cytes from Socs3 h-KO mice and their littermates in culture
was further accentuated by exposure of the cells to EGF ( Fig.
3 B ). After 62 and 86 h in culture, EGF-stimulated hepato-
cytes from SOCS3-defi cient mice incorporate [ 3 H]thymidine
at a rate that is almost fourfold higher than the hepatocytes
from control littermates. Similar to our fi ndings in the regen-
erating liver in vivo, SOCS3-defi cient hepatocytes in culture
displayed increased activation of STAT3 and ERK1/2 in
response to EGF ( Fig. 3, C and D ). Thus, the defi ciency in
SOCS3 not only increases the intrinsic replicative capacity of
hepatocytes but also makes them more responsive to the pro-
liferative eff ects of growth factors such as EGF.
JAK inhibition by AG490 and MAPK/ERK kinase (MEK)
inhibition by U0126 inhibit hepatocyte proliferation in vitro
The data presented suggest that enhanced signaling through
STAT3 and ERK1/2 may be responsible for the heightened
proliferative state of SOCS3-defi cient cells. We thus used small
molecule inhibitors of the upstream kinases JAK (upstream
of STAT3) and MEK (upstream of ERK1/2) to determine
protein kinases (MAPKs) ERK1/2 has been shown to be a
key event in the regenerative response to PH and is believed
to occur via activation of the epidermal growth factor recep-
tor (EGFR) by a variety of ligands ( 13 ). We found that the
increased proliferative activity in Socs3 h-KO mice after PH
is associated with a marked increase in phospho-ERK1/2,
particularly at 18 and 24 h after PH ( Fig. 2 E ). Thus, SOCS3
defi ciency after PH accelerates liver regeneration, prolongs
STAT3 activation and the acute-phase response, and enhance
Socs3 -defi cient hepatocytes exhibit enhanced proliferation
in vitro, associated with increased phosphorylation of
STAT3 and ERK1/2
After demonstrating the marked enhancement of both cyto-
kine signaling and hepatocyte proliferation during liver re-
generation, an in vivo growth process that restores liver mass
after removal of two thirds of the liver, we asked whether
primary hepatocytes isolated from Socs3 h-KO mice also dis-
play enhanced proliferative activity in culture. For these
experiments, we perfused the livers of Socs3 h-KO and con-
trol mice with collagenase, Percoll-purifi ed the hepatocytes,
plated them in media containing 5% serum for attachment,
and maintained the cells in the absence of serum or growth
factors. We found that the incorporation of [ 3 H]thymidine in
Figure 2. Activation of STAT3 and ERK1/2 are enhanced in Socs3 h-KO mice. (A) Serum IL-6 levels after PH are unchanged in Socs3 h-KO mice
(ELISA). (B) Lack of SOCS3 in hepatocytes leads to prolonged phosphorylation of STAT3 (Y705) after PH, demonstrated by immunoblotting. (C) Northern
blot analysis indicates prolonged expression of Cd14 Socs3 h-KO mice. (D) Saa2 expression is also prolonged in Socs3 h-KO mice. (E) Socs3 defi ciency
leads to enhanced phosphorylation of ERK1/2 after PH, demonstrated by immunoblotting. All demonstrated data are representative of three to six mice
per genotype per time point and are presented as mean ± SEM. *, P < 0.05.
JEM VOL. 205, January 21, 2008
Socs3 -defi cient hepatocytes exhibit enhanced activation
of multiple IL-6 – dependent signaling pathways
The observations that both STAT3 and ERK1/2 activation
are prolonged in vivo after PH in Socs3 -defi cient livers and in
vitro after EGF stimulation of Socs3 defi cient hepatocytes
suggested that SOCS3 may also inhibit signaling pathways
downstream of IL-6. To determine whether IL-6 stimulation
of Socs3 -defi cient hepatocytes in culture would alter the re-
sponse of downstream pathways, hepatocytes isolated from
Socs3 h-KO mice and control littermates were exposed to
IL-6. Protein lysates were prepared at the indicated times, and
phosphorylation of STAT3, ERK1/2, Akt, and S6 protein was
analyzed by phosphoimmunoblotting. IL-6 – induced STAT3
phosphorylation is prolonged in Socs3 h-KO hepatocytes
whether we could decrease the proliferation of Socs3 KO
cells to the level of control hepatocytes. As expected, AG490,
a JAK inhibitor, inhibits STAT3 phosphorylation in response
to IL-6 in cultured hepatocytes, whereas the vehicle control
(DMSO) has no eff ect on IL-6 – stimulated STAT3 phosphoryl-
ation ( Fig. 4 A ). When added to culture medium 1 h before the
addition of EGF, AG490 also inhibits hepatocyte prolifera-
tion, as measured by [ 3 H]thymidine incorporation ( Fig. 4 B ).
Addition of U0126, a selective MEK inhibitor, inhibits both
ERK1/2 activation ( Fig. 4 C ) and hepatocyte proliferation
( Fig. 4 D ) in response to EGF. These results indicate that
SOCS3 can modulate hepatocyte replication in vitro through
eff ects on both the STAT3 and MAPK signaling pathways,
similar to our in vivo observations.
Figure 3. SOCS3 down-regulates hepatocyte proliferation and growth factor signaling in vitro. (A) [ 3 H]Thymidine incorporation by primary
hepatocytes in the absence of growth factors 36 h after isolation and initiation of cell culture. (B) Hepatocyte proliferation in response to 20 ng/ml EGF.
(C) Pi-STAT3 Y705 is enhanced in response to EGF in Socs3 -defi cient hepatocytes. (D) Similar enhancement of Pi-ERK1/2 in response to EGF in Socs3 KO
hepatocytes. All data are representative of at least three separate experiments, with four to six replicates in each 3 H experiment, and are presented as
mean ± SEM. *, P < 0.05.
Figure 4. Inhibition of JAK or MEK in culture inhibits hepatocyte proliferation. (A) AG490 inhibits tyrosine phosphorylation of STAT3. (B) AG490
inhibits hepatocyte proliferation ([ 3 H]thymidine assay). (C) U0126 inhibits ERK activation in response to EGF. (D) U0126 inhibits hepatocyte proliferation
([ 3 H]thymidine assay). Data are representative of three independent experiments and are presented as mean ± SEM. *, P < 0.05.
ROLE OF SOCS3 IN LIVER REGENERATION AND HCC | Riehle et al.
the lack of SOCS3 has broad eff ects on gene expression dur-
ing liver regeneration, we performed complementary DNA
(cDNA) microarray analysis on RNA prepared from Socs3
h-KO mice and control littermates 18 h after PH. Three pooled
samples per genotype were applied to Aff ymetrix oligonucle-
otide arrays, and data were analyzed as described in Supple-
mental materials and methods (available at http://www.jem
.org/cgi/content/full/jem.20070820/DC1). The heatmap
shown in Fig. 6 A represents a global view of diff erential gene
expression patterns in Socs3 h-KO mice versus control litter-
mates at 18 h after PH. The map demonstrates uniformity of
expression among the pools for each genotype and reveals
striking diff erences in gene expression profi les between Socs3
h-KO mice and control littermates. The Aff ymetrix data were
subjected to National Institutes of Health (NIH) database for
annotation, visualization, and integrated discovery (DAVID)
analysis and Kyoto Encyclopedia of Genes and Genomes an-
notation. NIH DAVID analysis of diff erentially regulated
genes revealed that several pathways thought to be important in
liver regeneration are enhanced in Socs3 h-KO mice ( Fig. 6 B ).
In addition to the JAK – STAT and MAPK signaling pathways,
which we had already shown to be enhanced in the absence
of SOCS3, we found that Toll-like receptor (TLR) signaling
and cytokine – cytokine receptor interaction, focal adhesion,
and Wnt signaling pathways are similarly up-regulated. These
pathways have been shown by multiple investigators to be
critical to normal regeneration ( 13, 15, 16 ), and in some cases
may be involved in the development of HCC ( 8, 17, 18 ). Our
microarray data support the view that the enhancement of
multiple intracellular signaling pathways in Socs3 h-KO mice
allows them to regenerate more effi ciently than control litter-
mates. Interestingly, DAVID analysis revealed that bile acid
synthesis and fatty acid metabolism were down-regulated in
Socs3 h-KO mice in comparison with control littermates, sug-
gesting that SOCS3 may enhance rather than inhibit these
functions. Recent data suggest that these pathways are them-
selves necessary for optimal liver regeneration ( 19, 20 ). Our
results do not necessarily contradict these studies, as the multi-
ple changes created by SOCS3 defi ciency may alter the liver
metabolic requirements during regeneration.
To validate our microarray gene expression data, we per-
formed real-time RT-PCR on several genes that had been
shown to be up-regulated in Socs3 h-KO mice. Work by
several investigators has shown that c-jun, a subunit of the
transcription factor activator protein 1, is critical to the pro-
liferative response after PH ( 21 ), and data from our laboratory
suggest that AP-1 activity may be necessary for induction of
heparin-binding EGF (unpublished data; Mitchell, C., per-
sonal communication), a member of the EGF family that par-
ticipates in the linkage between cytokine activity and cell-cycle
progression during regeneration ( 22 ). In the present study,
we found that the expression of c-jun messenger RNA is ap-
proximately twofold higher in Socs3 h-KO mice compared
with littermates ( Fig. 6 C ).
We also observed increased expression of haptoglobin, an
acute-phase response protein, further supporting our observation
compared with controls ( Fig. 5 A ). ERK1/2 activation is bi-
phasic, peaking at 30 and 120 min after IL-6 treatment ( Fig.
5 B ), and although the initial activation of ERK1/2 appears
to be similar in hepatocytes from Socs3 h-KO mice and lit-
termates, Socs3 KO hepatocytes demonstrate prolonged acti-
vation of ERK1/2 compared with control cells.
The protein kinase target of rapamycin (TOR) complexes
1/2, which regulate mammalian TOR (mTOR), are in-
volved in multiple cellular processes, such as protein transla-
tion, nutrient sensing, and cell growth ( 14 ). To determine
whether Socs3 could regulate IL-6 – stimulated mTOR activ-
ity, we examined the levels of phospho-S6 ribosomal pro-
tein, a downstream target of mTOR. We observed enhanced
and prolonged phosphorylation of S6 protein in Socs3 KO
cells ( Fig. 5 C ), indicating that S6 kinase is activated to a
greater extent, and thus, the mTOR pathway may also be
enhanced. In contrast, Akt phosphorylation and activation, as
determined by phospho-S473 – Akt and a phospho-Akt sub-
strate antibody, is similar in Socs3 KO and control hepato-
cytes after IL-6 treatment ( Fig. 5 D ). These results confi rm
that IL-6 stimulates multiple signaling pathways, such as those
mediated by STAT3, ERK1/2, Akt, and S6 kinase in pri-
mary hepatocytes, and that SOCS3 defi ciency results in pro-
longed and enhanced activation of some of these pathways.
The absence of Socs3 results in profound changes in gene
expression after PH
Given the signifi cant enhancement of liver regeneration ob-
served in Socs3 h-KO mice and our fi ndings in vitro, we hy-
pothesized that diverse cellular pathways contribute to the
proliferative advantage of these cells. To determine whether
Figure 5. Activation of STAT3, ERK1/2, and Akt are prolonged
in Socs3 KO hepatocytes after IL-6 treatment. (A) Pi-STAT3 Y705 is
prolonged in response to IL-6 in Socs3 KO hepatocytes. (B) Activation of
ERK1/2 is enhanced in response to IL-6 in Socs3 KO hepatocytes. (C)
Prolonged activation of Pi-S6 S240/244 ribosomal protein in Socs3 KO versus
littermate hepatocytes. ? -Actin is shown as a loading control. (D) No
difference in activation of Akt in Socs3 versus control hepatocytes. Data are
representative of three to fi ve independent experiments.
JEM VOL. 205, January 21, 2008
of three angiogenic factors in the livers of Socs3 h-KO mice
suggests that initiation of liver architecture remodeling seen
after PH may occur earlier in these mice. Collectively, our
real-time RT-PCR results both validate our microarray data
and provide additional insight into potential mechanisms be-
hind the enhancement in liver regeneration seen in Socs3
Promoter analysis after PH in Socs3 h-KO mice
We were also interested in identifying the potential regula-
tory networks that might account for the changes in messen-
ger RNA expression identifi ed by the microarray and, thus,
used transcriptional regulatory network analysis (TRNA) us-
ing the promoter analysis and interaction network tool
(PAINT), as described in Materials and methods ( 27 ). TRNA
of extended STAT3 activation in Socs3 h-KO mice ( Fig. 2 ).
I ? B ? is rapidly resynthesized after it is phosphorylated and de-
graded, which results in the release and activation of NF- ? B
( 23, 24 ). We observed increased expression of I ? b ? in Socs3
h-KO mice, suggesting that NF- ? B was active at 18 h after PH
in these animals. Increased expression of I ? b ? is also consistent
with the enrichment of genes in the TLR pathway ( Fig. 6 B ).
Hypoxia-inducible factor 1 ? ( Hif1 ? ) is induced under hyp-
oxic conditions and transcribes factors that are important to
angiogenesis, and has been reported to increase after PH ( 25 ).
Hif1 ? expression was signifi cantly increased in Socs3 h-KO mice
compared with littermates after PH. Both platelet-derived
growth factor C ( Pdgfc ) and PDGF receptor ? ( Pdgfr ? ) tran-
scribe potent angiogenic factors ( 26 ), and were signifi cantly
up-regulated in Socs3 h-KO mice. The increased expression
Figure 6. Oligonucleotide microarrays demonstrate broad and diverse effects of SOCS3 on gene expression after PH. (A) Heatmap demon-
strating a global picture of differential gene expression in Socs3 h-KO mice versus control littermates at 18 h after PH. (B) NIH DAVID analysis of a
differentially regulated gene list reveals that multiple pathways are differentially regulated during regeneration in Socs3 h-KO mice. (C) Confi rmed up-
regulation of c-jun , Haptoglobin , Hif1 a , I k B a , Pdgf-c , and Pdgfr a in Socs3 h-KO mice versus control littermates at 18 h after PH, demonstrated by
real-time RT-PCR. Data are presented as fold change using the values from nonoperated mice as the denominator and are presented as mean ± SEM.
*, P < 0.05; ***, P < 0.001.
ROLE OF SOCS3 IN LIVER REGENERATION AND HCC | Riehle et al.
up-regulated in these cancers ( 17 ). Because we have shown
that SOCS3 is a critical negative regulator of these pathways
during the physiological regenerative response to PH, we
wondered whether a lack of SOCS3 would promote neoplas-
tic proliferative processes as well. To test this hypothesis, we
used a model of DEN-induced hepatocarcinogenesis, in
which Socs3 h-KO mice and control littermates were injected
with a single dose of DEN at 12 – 14 d of life. The mice re-
ceived no other treatment and were killed between 3 and 12 mo
of age. Foci of altered hepatocytes were observed by 6 mo of
age in both Socs3 h-KO mice and littermates but were larger
in size in Socs3 h-KO mice (1.2 vs. 0.5% of the area). Socs3
h-KO mice developed HCC signifi cantly earlier and in greater
numbers than did littermate controls ( Table II ). By 9 mo, four
out of six SOCS3-defi cient mice had developed tumors,
whereas none were detected in the control littermates; by 12
mo, all Socs3 h-KO mice had developed HCC. The tumors
were typical trabecular-type HCCs with a moderate degree of
diff erentiation (unpublished data). Using immunoblot analy-
sis, we observed increased levels of activated, phosphorylated
ERK1/2 in the tumor tissue from 9-mo Socs3 h-KO mice
compared with adjacent tissue, as well as from liver tissue
was performed for genes found to be up-regulated > 1.5-fold
in Socs3 h-KO mice to identify transcription factor binding
sites or transcriptional regulatory elements (TREs) in the 5 ?
fl anking regions. The most enriched TREs in the gene set are
listed by consensus sequence, along with their associated tran-
scription factors, categorized, and ranked by frequency of
presence in Table I . Most notably, TREs for several members
of the NF- ? B promoter family (p65, NF- ? B, and c-Rel) are
among the most overrepresented, consistent with the enrich-
ment of cytokine signaling and TLR pathways determined by
DAVID analysis ( 28 ). Additionally, the presence of the TRE
for Elk-1, a downstream target of the MAPK pathway, sup-
ports our fi ndings of extended and increased ERK1/2 activa-
tion in Socs3 h-KO mice ( Fig. 2 E ). Collectively, these results
corroborate our in vivo and in vitro data demonstrating the
increased proliferative capacity in Socs3 h-KO mice after PH,
in the context of enhanced signaling via multiple pathways.
Accelerated development of N -nitrosodiethylamine (DEN) –
induced HCC in Socs3 h-KO mice
Recent work on human HCCs demonstrated that the JAK –
STAT and/or Ras-Raf – MAPK pathways are virtually always
Table I. TREs enriched in Socs3 h-KO mice 18 h after PH
Candidate binding factor for TRE TRE consensus sequenceCountsp-value
NF- ? B superfamily
NF- ? B
NF- ? B
NF- ? B (p65)
Cell growth and proliferation
Differentiation and hepatocyte specifi city
Enriched TREs (biologically relevant transcription factor binding sites) were identifi ed by comparing promoter regions of genes up-regulated > 1.5-fold in Socs3 h-KO mice
to all M. musculus promoter regions in the PAINT database. To identify TREs overrepresented, 5 kb of upstream sequence of each gene from the list of signifi cantly up-
regulated genes was analyzed by PAINT. TRE consensus sequence is the consensus binding site for the indicated binding factor (known transcription factor), provided as the
International Union of Pure and Applied Chemistry 15-letter code. Multiple listings of candidate binding factors are based on differences in consensus binding sites. Count
refers to the total number of times the TRE was found in the gene set. p-values were calculated as described in Materials and methods.
JEM VOL. 205, January 21, 2008
cell factor participate in the replicative response of hepato-
cytes after PH ( 32, 34 ). Although it may appear that the cyto-
kine and growth factor pathways in the regenerating liver may
have distinct functions, these networks may overlap in some
cases. For instance, it has been demonstrated that STAT3 ac-
tivates multiple genes and contributes to hepatocyte replication
during liver regeneration ( 16 ). Moreover, IL-6 has multiple
functions, including cell survival, cell proliferation, and the
acute-phase response to infl ammation ( 35 – 37 ), and the role of
IL-6 in the regenerating liver remains a controversial issue.
Thus, it is unclear how cytokines and growth factors may
interact to produce the precisely orchestrated liver growth re-
sponse that occurs after PH.
SOCS proteins were originally described as negative regu-
lators of cytokine signaling. More recent studies using specifi -
cally targeted mouse models reveal that, in the hematopoietic
system, SOCS1 and SOCS3 inhibit myeloid signaling path-
ways and may infl uence conditions such as infl ammation, auto-
immunity, and malignancy ( 17, 38 – 41 ). In the liver, SOCS3
regulates the activity of the cytokine pathway stimulated by
from control littermates (unpublished data), suggesting that
signaling pathways that control proliferation may be activated
in Socs3 -defi cient tumors.
Previous work suggested that SOCS3 may decrease tu-
mor formation in the liver by blocking apoptosis ( 29 ), so we
examined the extent of apoptosis induced by DEN in our
mice using a caspase 3 activity assay ( 30 ). We found no diff er-
ences among caspase activities of Socs3 h-KO mice and con-
trol littermates 24 h after injection ( Fig. 7 A ), though caspase 3
is activated in both groups of mice 48 h after DEN injection.
To determine whether DEN injection induces expression of
the antiapoptotic protein Bcl-xL, we performed immunoblot
analysis of liver lysates. We observed a modest induction of
Bcl-xL at 24 and 48 h after DEN injection, but the levels
were not diff erent between Socs3 h-KO mice compared with
littermate controls (unpublished data), suggesting that dysreg-
ulated apoptotic pathways did not result in the earlier devel-
opment of HCC in the absence of SOCS3 in our model.
It has recently been demonstrated that IL-6 is rapidly re-
leased after DEN injection ( 31 ), and we found that serum
IL-6 levels were dramatically elevated in Socs3 h-KO mice
compared with littermates at 24 h after injection ( Fig. 7 B ).
Elevated levels of phospho-STAT3 are observed at both 24
and 48 h after DEN injection in Socs3 h-KO mice ( Fig. 7 C ).
These data support the hypothesis that SOCS3 may prevent
DEN-induced HCC formation by altering the response to
IL-6 rather than by inhibiting apoptotic pathways.
Liver regeneration after PH is a unique growth process in
which the hepatic mass is rapidly restored after surgical re-
moval of two thirds of the liver. The regenerative process af-
ter PH is dependent on the replication of hepatocytes, which
are completely diff erentiated and normally quiescent cells,
and does not rely on the activation of a compartment of liver
stem cells. Hepatocyte replication does require the involve-
ment of NPCs, the major source of hepatic cytokines and
growth factors, and the remodeling of extracellular matrix
( 6, 8, 32 ). A large number of components of the innate immune
system, including TNF, complement proteins, and the IL-6 –
STAT3 pathway, participate in the initiation of liver regener-
ation ( 6, 8, 33 ). Additionally, studies have shown that growth
factors such as HGF, members of the EGF family, and stem
Table II. Development of HCC in Socs3 h-KO and control
littermates after DEN injection
3 mo 6 mo9 mo 12 mo
Control 0/100/50/6 4/8
Socs3 h-KO0/4 0/34/6 4/4
12 – 14-d-old pups were injected with a single dose of 5 mg/kg DEN. At the indicated
times, male mice were killed and livers were examined for tumors. Macroscopic
HCCs were confi rmed by hematoxylin and eosin analysis, as described in Materials
and methods. Tumor development was signifi cantly different in Socs3 h-KO mice as
determined by a log-rank test, with a p-value of 0.0027.
Figure 7. Analysis of early response to DEN injection in Socs3
h-KO mice versus littermates. Mice were injected with 100 ? g DEN per
gram of body weight, and liver tissues were harvested 24 and 48 h later.
(A) DEN injection induces caspase 3 activity at 48 h but similarly in Socs3
h-KO (hatched bars) and littermate (black bars) mice. (B) Serum IL-6 levels
are elevated in Socs3 h-KO versus littermate mice 24 h after DEN injection.
(C) Enhanced activation of STAT3 after DEN injection in Socs3 h-KO mice.
Data presented are representative of six mice per genotype per time point
and are presented as mean ± SEM. *, P < 0.05.
ROLE OF SOCS3 IN LIVER REGENERATION AND HCC | Riehle et al.
activation of STAT3 and ERK1/2 after IL-6 or EGF stimu-
lation. These eff ects can be blocked by inhibitors of the JAK –
STAT (AG490) or MEK – ERK1/2 (U0126) pathways. Our
results are consistent with other work demonstrating that
SOCS3 (as well as other SOCS proteins) can regulate signal-
ing through the EGFR ( 47, 48 ).
Calvisi et al. reported that the JAK – STAT pathway is en-
hanced in human HCC compared with nonneoplastic liver
and is associated with the down-regulation of various suppres-
sors of this pathway ( 17 ). We wondered whether the enhanced
hepatocyte proliferation in the regenerating liver and the high
proliferative capacity of Socs3 KO hepatocytes in culture would
predispose these mice to liver carcinogenesis. We found that
tumor development is accelerated in Socs3 h-KO mice that are
injected with DEN, a known hepatocarcinogen. These data
are consistent with the observation that SOCS3 defi ciency
promotes cell growth in human HCC by enhancing the JAK –
STAT and focal adhesion kinase signaling pathways ( 18 ). Our
microarray analysis of post-PH liver RNA using DAVID and
the Kyoto Encyclopedia of Genes and Genomes annotation
identifi ed both of these pathways as being activated in Socs3
h-KO mice. Recently, Ogata et al. reported that Socs3 hepatic-
defi cient mice developed a greater number of hepatic tumors
that were larger than those of control mice when injected with
DEN for 6 wk or DEN in combination with 6 mo of a cho-
line-defi cient, l – amino acid diet ( 29 ). Ogata et al. concluded
that in the setting of infl ammation-induced tumorigenesis,
STAT3 was activated and induced expression of antiapoptotic
proteins such as Bcl-2 and Bcl- xL ( 29 ). Our fi ndings included
accelerated HCC development in Socs3 h-KO mice after a
single DEN injection ( Table II ), but we did not observe a dif-
ference in DEN-induced apoptosis 24 or 48 h after DEN in-
jection as measured by caspase 3 activation or Bcl- xL expression.
However we did observe increased release of IL-6 in Socs3 h-
KO mice and subsequent enhanced phosphorylation of STAT3.
It is possible that the elevated levels of IL-6 provide a cell pro-
liferative or survival advantage to tumor cells in Socs3 h-KO
mice ( 35 – 37, 49 ). Regardless of mechanisms, our results and
those of Ogata et al. demonstrate that SOCS3 defi ciency in-
creases the risk of HCC development.
Despite the fact that individual pathways involving vari-
ous cytokines and growth factors during liver regeneration
have been described in detail, there is little information re-
garding the mechanisms that coordinate these events and lead
to a precisely regulated and synchronized growth process.
Our work demonstrates that, in the regenerating liver, SOCS3
regulates not only cytokine expression through various path-
ways involving TLR receptors and the IL-6 – STAT3 pathway
but also controls the expression of multiple genes involved in
proliferative pathways that require ERK activation. We sug-
gest that SOCS3 expression at the early stages of liver regen-
eration is an essential element that coordinates the termination
of the main cytokine response with the activation of growth
factors that regulate cell-cycle progression. In the absence of
SOCS3, hepatocytes acquire an enhanced proliferative ca-
pacity, both in vivo and in culture. Thus, hepatic SOCS3 can
the IL-6 family, which signals through the IL-6R – gp130 to
activate the STAT3 transcription factor. IL-6 is a key mediator
of the acute-phase response to infl ammation, and by control-
ling the IL-6 – STAT3 pathway, SOCS3 functions as a regu-
lator of this response. Previous work from our laboratory
showed that Socs3 expression is greatly induced during the fi rst
12 h after PH ( 9 ). Our interpretation of these results was that
SOCS3 halts STAT3 activation and terminates the early phase
of liver regeneration in which cytokines are major partici-
pants, leading to the growth factor – regulated progression of
hepatocytes through the cell cycle and, ultimately, DNA rep-
lication. Thus, SOCS3 could function at the interface between
cytokine expression and growth factor activity during the re-
generative response. To directly analyze the role of SOCS3
during liver regeneration after PH, we studied a variety of
proliferative processes in Socs3 h-KO mice. The main fi ndings
of this work were that (a) Socs3 h-KO mice show an enhance-
ment of hepatocyte DNA synthesis and mitosis after PH; (b)
Socs3 KO hepatocytes are highly proliferative in primary cul-
ture, even in the absence of growth factors; (c) SOCS3 defi -
ciency enhances multiple pathways related to both cytokine
activity and cell proliferation; and (d) Socs3 h-KO mice de-
velop HCC at an accelerated rate.
The enhancement of liver regeneration caused by SOCS3
defi ciency was an unexpected result. Based on existing data
on the role of SOCS3 in blocking STAT3 expression, it was
expected that SOCS3 defi ciency after PH would lead to an
enhanced and prolonged acute-phase reaction that might in-
hibit the proliferative response. However, SOCS3 defi ciency
led to both an enhancement of the expression of acute-phase
response genes and the up-regulation of multiple pathways
related to cell proliferation. These data are consistent with
reports in other systems showing that SOCS3 has a broader
role than the control of cytokine activity, which include the
enhancement of the proliferation of myeloid cells in the he-
matopoietic system ( 38 ), islet cell hyperplasia ( 42 ), inhibition
of insulin receptor phosphorylation associated with the meta-
bolic syndrome in the liver ( 43, 44 ), and the promotion of
liver fi brosis through TGF- ? production ( 45 ). Sun et al. re-
ported that STAT3 activation was enhanced after PH in Socs3
heterozygous (+/ ? ) mice, but that hepatocytes isolated from
these mice and exposed to IL-6 in culture showed a decrease
in cell proliferation because of increased expression of p21
( 46 ). We suggest that the diff erence in our results from those
of Sun et al. may be explained by the use of Socs3 h-KO mice
in our experiments.
As expected, IL-6 levels after PH were not altered by he-
patocyte Socs3 defi ciency because IL-6 is produced by liver
NPCs, which were not genetically targeted in our mice. The
data we obtained with hepatocytes isolated from Socs3 h-KO
mice and placed in primary culture demonstrate that these
cells have a high proliferative activity even when maintained
in medium without growth factors and are highly sensitive to
EGF stimulation. Similar to the experiments performed in
regenerating livers, the increased proliferation of cultured he-
patocytes from Socs3 h-KO mice is associated with enhanced
JEM VOL. 205, January 21, 2008
Primary hepatocyte culture, [ 3 H]thymidine incorporation, and
kinase inhibitor treatment. Primary hepatocytes were isolated from Socs3
h-KO mouse and control livers by collagenase perfusion and Percoll gradi-
ents, as previously described ( 54 ). To measure DNA replication in cultured
hepatocytes, [ 3 H]thymidine was added to media at a fi nal concentration of
1 mCi/ml for 4 h, and incorporation was measured as previously described
( 55 ). Where indicated in the fi gures, 20 ng/ml IL-6 or 30 ng/ml EGF was
added to media. For kinase inhibitor experiments, 50 ? M AG490, 5 ? M
U0126, or DMSO (vehicle control) was added 1 h before IL-6 or EGF
stimulation. Total RNA and protein lysates were prepared from hepatocyte
cell cultures as described.
Microarray analysis of 18-h PH samples. To perform statistically rele-
vant gene expression profi ling at 18 h after PH, additional PHs were per-
formed so that there were 9 – 11 samples for each genotype at this time point.
Total RNA was isolated as described in Materials and methods and then pu-
rifi ed using the RNeasy kit (QIAGEN). Equal amounts of each sample were
pooled, resulting in three groups per genotype. All samples were of good
quality, as assessed by a bioanalyzer (model 2100; Agilent Technologies). A
detailed description of microarray methods can be found in Supplemental
materials and methods. Microarray data have been deposited in the Gene
Expression Omnibus under accession no. GSE9549 .
TRNA. Promoter analysis was performed using PAINT (version 3.5, avail-
able at http://www.dbi.tju.edu/dbi/tools/paint) ( 27 ). The input fi le con-
tained the Locuslink identifi ers for 583 Aff ymetrix probe sets determined by
microarray to be up-regulated > 1.5-fold in Socs3 h-KO mice 18 h after PH.
PAINT retrieved 5 kb of 5 ? fl anking sequence for 524 genes and identifi ed
473 unique promoters. Using a TRE core similarity threshold of 1, 132
TREs were found for known promoters within the input sequences.
PAINT tested for enrichment of specifi c TREs and transformed the results
into a candidate interaction matrix based on a p-value threshold of 0.05 and
a false discovery rate threshold of 0.3. For the group of genes analyzed, the
p-values for signifi cance of enrichment of each TRE were calculated using
hypergeometric distribution, with the abundance of each TRE compared
with the default PAINT reference set of randomly selected Mus musculus
promoters. For additional details, please refer to Vadigepalli et al. ( 27 ) and
Addya et al. ( 56 ).
Statistical analyses. For all in vivo experiments, three to six mice per gen-
otype per time point were used, except for the microarray analyses done on
18-h post-PH samples. All in vitro experiments were performed at least
three times. Data presented are either representative of the replicates, or
quantitation and statistical analysis of all replicates. Statistical analysis was
done by nonparametric analysis, using the Mann-Whitney test or an un-
paired t test with Welch ’ s correction. Data are presented as mean ± SEM,
with P < 0.05 considered statistically signifi cant. Prism software (GraphPad)
was used for all statistical analyses except for microarray data.
Online supplemental material. Supplemental materials and methods provides
detailed methods for the generation and analysis of microarray data ( Fig. 6,
A and B ), as performed by the Center for Ecogenetics and Environmental
Health at the University of Washington. Table S1 provides a complete list of
the genes and identifi ers presented in Fig. 6 A . Online supplemental material
is available at http://www.jem.org/cgi/content/full/jem.20070820/DC1.
The authors would like to thank Doug Hilton, Warren Alexander, and Ben Croker for
developing and providing the Socs3 fl /fl mice. The authors also wish to thank John
Brooling and Renay Bauer for their technical assistance.
This work was supported by NIH grants CA-23226 (to J.S. Campbell) and
CA-023226 and CA-074131 (to N. Fausto), an American College of Surgeons Resident
Research Scholarship (to K.J. Riehle), and University of Washington National
Institute of Environmental Health Sciences – sponsored Center for Ecogenetics and
Environmental Health grant P30ES07033.
The authors have no confl icting fi nancial interests.
function both as an antiinfl ammatory agent and a tumor sup-
pressor. SOCS3 may be a suitable target for the regulation of
acute-phase responses to infl ammation and for the prevention
or treatment of HCC.
MATERIALS AND METHODS
Animals and operative technique. The generation of mice in which the
Socs3 gene has been fl anked by loxP sequences ( Socs3 fl /fl mice) has been pre-
viously described ( 2 ). Hepatocyte-specifi c excision of the Socs3 gene was
achieved by breeding Socs3 fl /fl mice with mice expressing the Cre recombi-
nase transgene under control of the albumin promoter ( Alb-Cre + ), yielding
Socs3 h-KO mice. Socs3 fl /fl , Alb-Cre ? littermates were used as controls for all
experiments and are henceforth referred to as littermates. All mice (C57BL/6)
were free of Helicobacter species and were housed in a specifi c pathogen-free
facility with 12-h light/dark cycles with free access to standard food and
water. PH and sham operations were performed as previously described ( n =
three to six mice per genotype per time point) ( 15, 50 ). Liver remnants were
weighed after removal of necrotic stumps and sutures and compared with
postoperative body weight. For HCC experiments, a single i.p. injection of
5 mg DEN per kilogram (Sigma-Aldrich) was performed 12 – 14 d after
birth. For short time points, a single injection of 100 mg DEN per kilogram
( 31 ) was given to 4-wk-old mice. At the times indicated in the fi gures, mice
were killed by CO 2 inhalation. All animal studies were performed under ap-
proved institutional animal care and use committee protocols at the Univer-
sity of Washington.
Histology. For all mice harvested 24 h or later after PH, 50 ? g BrdU per
gram of body weight was injected i.p. 2 h before harvest. Liver lobes were
fi xed and stained as previously described ( 15, 22 ). For DEN-induced HCC
experiments, macroscopic tumors were counted at necropsy. Liver sections
were stained with hematoxylin and eosin and scored for foci of altered hepa-
tocytes and microscopic HCC using previously described criteria ( 51, 52 ).
ELISA. Serum IL-6 concentration was determined using an ELISA, accord-
ing to the manufacturer ’ s instructions (R & D Systems), as described previ-
ously ( 15, 22 ).
RNA isolation, Northern blotting, and real-time RT-PCR analysis.
RNA was isolated from whole liver or primary hepatocytes using TRI zol
(Invitrogen) as previously described ( 9, 15 ). Northern blot analyses were
done as previously described ( 9, 15 ) using 32 P-labeled cDNA probes for
Socs3 , Socs1 , Cd14 , or Saa2 ( 15 ), or a 32 P-labeled riboprobe for cyclophilin
(Ambion) as a loading control. Bands were quantifi ed using a phosphorim-
ager (Storm; Molecular Dynamics). For cDNA synthesis, 2 ? g RNA from
18-h PH samples were reverse transcribed using the Retroscript kit (Invitrogen),
and real-time RT-PCR was performed on the resultant cDNA using FAM-
labeled primers and reagents (Applied Biosystems). ? ? Ct values were cal-
culated by subtracting Ct values for the gene of interest from the Ct values
for 18S (a housekeeping gene) and then subtracting the ? Ct value obtained
for each gene from nonoperated control mice. Fold change was calculated by
normalizing all values to those of nonoperated wild-type mice.
Immunoblotting and caspase 3 activity assay. Whole liver or primary
hepatocyte protein lysates were prepared in a Triton X-100 lysis buff er and
quantifi ed as previously described ( 15 ). Caspase 3 activity was determined
using a fl uorogenic substrate as previously described ( 30 ). 20 or 50 ? g of
protein from each sample were separated using SDS-PAGE, and immuno-
blotting was performed using standard procedures with the following pri-
mary antibodies from Cell Signaling Technology (phosphotyrosine 705 STAT3,
total STAT3, phospho-ERK1/2, phospho-S473 – Akt, total Akt, phospho-
S/T Akt substrates, phospho-S6 S240/244 protein, phosphotyrosine 701
STAT1, total STAT1, and phosphotyrosine 649 STAT5), Santa Cruz Bio-
technology, Inc. (cyclin A and p107), Sigma-Aldrich ( ? -actin), and Zymed
(total STAT5a/b), and total ERK1/2 ( 53 ) as previously described ( 9, 22 ).
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Submitted: 24 April 2007
Accepted: 20 November 2007
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