Departments of Medicine and Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27707
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ABSTRACT |
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Transforming growth factor- (TGF-
)-activated kinase 1 (TAK1), a serine/threonine kinase, is reported to function in the signaling pathways of TGF-
, interleukin 1, and ceramide.
However, the physiological role of TAK1 in vivo is largely unknown. To assess the function of TAK1 in vivo, dominant-negative TAK1 (dnTAK1) was expressed in the rat liver by adenoviral gene transfer. dnTAK1 expression abrogated c-Jun NH2-terminal kinase and c-Jun
but not nuclear factor (NF)-
B or SMAD activation after partial
hepatectomy (PH). Expression of dnTAK1 or TAM-67, a dominant-negative
c-Jun, induced G0 exit in quiescent liver and accelerated
cell cycle progression after PH. Finally, dnTAK1 and TAM-67 induced
c-myc expression in the liver before and after PH,
suggesting that G0 exit induced by dnTAK1 and TAM-67 is
mediated by c-myc induction.
partial hepatectomy; proliferation; c-Jun NH2-terminal
kinase; transforming growth factor-
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INTRODUCTION |
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ALTHOUGH HEPATOCYTES ARE NORMALLY in a quiescent state, the liver has a remarkable capacity for regeneration. Failure of the regenerative response in the face of chronic toxic injury results in liver scarring and cirrhosis (63). Regeneration is also impaired in fatty livers, which may be a forerunner of more serious disease (22). Liver regeneration is required after surgical resection of tumors, which frequently arise in patients with underlying hepatitis or cirrhosis. Thus strategies to improve the liver's regenerative response could improve the surgical outcome for diseased livers and may also be beneficial for the prevention and treatment of cirrhosis.
Liver regeneration after 70% partial hepatectomy (PH) in rats is a
well-characterized in vivo model of proliferation (33, 49). After PH, hepatocytes undergo a synchronous round of cell division, with peak DNA synthesis at 18-24 h and cell division at
~30 h. Several signaling molecules are rapidly induced after 70% PH,
including the transcription factors c-myc, nuclear factor (NF)-B, and activator protein-1 (AP-1) and the signaling kinase c-Jun NH2-terminal kinase (JNK) (3, 28, 46,
84). c-myc function is required for cell cycle
progression after PH (17), whereas NF-
B plays
protective and pro-proliferative roles after PH (37). The
roles of JNK and AP-1 activation after PH are unknown. Tumor necrosis
factor-
(TNF-
) signaling is required for liver regeneration, as
shown by treatment with anti-TNF-
antibodies (1) and by
targeted disruption of tumor necrosis factor receptor 1 (TNFR1)
(86). In mice with TNFR1 disruption, regeneration is restored by exogenous interleukin (IL)-6, which restores AP-1 but
not NF-
B activation (86).
Transforming growth factor- (TGF-
) is a pleiotropic cytokine that
has potent antiproliferative effects in the liver and other tissues
(60). TGF-
inhibits cell cycle progression induced during liver regeneration (13, 64) and promotes fibrosis
in both liver and lung (67, 71). Exogenous hepatocyte
growth factor inhibits TGF-
production and resolves experimental
liver fibrosis (79), demonstrating the importance of
hepatocyte proliferation in combating this disease state.
TGF- signals by binding to serine/threonine kinase receptors, which
phosphorylate and activate SMAD family transcription factors (60,
32). In addition, TGF-
signals through TGF-
-activated kinase 1 (TAK1), a mitogen-activated protein kinase (MAPK) kinase kinase family member that has been associated with the differentiation and antiproliferation functions of TGF-
(51, 77, 87).
Although TAK1 is expressed in the liver (87), its function
is unknown. The purpose of this study was to assess the role of TAK1 in
the regenerating liver. The results show that adenovirus-mediated expression of kinase-inactive TAK1 induced G0 exit in the
quiescent liver and accelerated hepatic cell cycle progression during
regeneration. In addition, the results suggest that these effects
result from induction of c-myc in the quiescent liver. Thus
dominant-negative TAK1 (dnTAK1) may be an attractive candidate for
pro-proliferative hepatic gene therapy.
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METHODS |
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Adenoviruses.
Recombinant, replication-defective adenoviruses expressing dnTAK1 and
TAM-67 were constructed according to the methods of Graham and Prevec
(29). The plasmid pEF-HA-TAKKW, encoding a hemagglutinin
(HA)-tagged, kinase-inactive TAK1 mutant, K63W, was the kind gift of
Dr. K. Matsumoto (Nagoya University, Nagoya, Japan). HA-TAKKW
cDNA was released using EcoRI and DraI and then subcloned into EcoRI and blunted BamHI sites in
the adenoviral vector pACCMV.PLPASR (pACCMV). TAM-67 was
amplified by polymerase chain reaction from the plasmid CMV TAM 67 to
incorporate a 5' EcoRI site and HA tag and a 3'
BamHI site and then subcloned into the EcoRI and
BamHI sites in pACCMV. Both clones were confirmed by
sequence analysis. Adenoviruses were generated by cotransfection of 293 embryonic human kidney cells with pACCMV constructs and purified,
5'-ClaI-truncated human type 5 dl309 adenoviral
DNA. Resultant adenoviral plaques were screened by Western blotting for
HA tags. Recombinant adenoviruses were grown in 293 cells and purified
by CsCl density gradient centrifugation (29). Purified viruses were dialyzed against 10 mM Tris, pH 7.5, 1 mM EDTA, 150 mM
NaCl, and 10% glycerol to remove CsCl and then aliquoted and stored at
80°C. Viral titers were determined by plaque assays.
Animals and surgery. Male Sprague-Dawley rats (175-225 g) were injected with 1010 plaque-forming units of adenovirus or an equivalent volume of PBS via the tail vein (37). Forty-eight hours after viral injection, 70% PH was performed (33). The initially resected liver lobes were collected as time 0 samples. At 1, 12, or 24 h after PH, the animals were killed and the remnant liver was collected. At all time points, portions of the liver were fixed in neutral-buffered formalin for subsequent histological analysis, and the remaining liver was divided and snap-frozen in liquid nitrogen. Three to six rats were analyzed per condition.
Histology and immunohistochemistry. Paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E). For proliferating cell nuclear antigen (PCNA) staining, sections were deparaffinized (10) and stained using the DAKO Envision system and anti-PCNA antibody (DAKO) and then counterstained with hematoxylin. Ten to twelve 20× fields were digitally captured, and PCNA-positive nuclei were quantitated by automated counting with the density slice function in NIH Image. Mitotic figures were counted manually from digitized images of H&E-stained sections.
Western blotting. Total cellular extracts were prepared from frozen tissues (10). Proteins were separated on polyacrylamide gels and then transferred to nitrocellulose membranes. Primary antibodies directed against cyclins D, E, and A and p21 and p27 were the kind gift of Dr. Y. Xiong (University of North Carolina at Chapel Hill). Anti-cyclin B was from Neomarkers, anti-phospho-c-Jun and phospho-p38 were from New England Biolabs, and anti-HA antibody was from Babco. Horseradish peroxidase-tagged secondary antibodies were from Santa Cruz Biotechnologies, and membranes were developed using ECL plus (Amersham).
JNK assay. JNK activity was measured with an in vitro kinase assay using GST-cJun(1-79) as a substrate (10, 83).
Northern blotting. Total RNA was isolated from frozen tissue using TRIzol (GIBCO BRL). RNA (20 µg) was separated on 1% agarose-formaldehyde gels, then transferred to nylon membranes (MSI). cDNA probes were radiolabeled using PrimeIt II kit and NucTrap push columns (Stratagene). Membranes were hybridized in RapidHyb buffer (Amersham) at 60°C and then washed in 2× SSC-0.1% SDS. Probe hybridization was assessed by autoradiography and quantitated by phosphorimager analysis (Molecular Dynamics).
Electrophoretic mobility shift assays.
Protein extracts (5 µg) were incubated with radiolabeled
double-stranded DNA probes containing the consensus binding sites for
NF-B or SMAD3/4 and then subjected to electrophoresis on 6%
acrylamide gels as described previously (10).
Immunoprecipitation of TAK1. Primary rat hepatocytes were isolated as described previously (11). Rat hepatocytes were either left uninfected or infected with Ad5TAK and then incubated for 12 h. Hepatocytes (3.2 × 107) were washed with PBS, lysed in 300 µl of extraction buffer, and immunoprecipitated with 2 µg of rabbit anti-TAK1 antibody (a kind gift from Dr. Jun Ninomiya-Tsuji, Nagoya University, Nagoya, Japan) (40). No kinase activity was retained by immunoprecipitated TAK1. For immunoblotting, anti-TAK1 antibody and mouse anti-HA antibody (Boehringer Mannheim) were used at 1:500 and 1:1,000 dilution, respectively.
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RESULTS |
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dnTAK1 expression inhibits JNK and c-Jun activation after PH.
To determine the role of TAK1 in hepatocyte proliferation in vivo, an
adenovirus expressing an HA-tagged kinase-inactive dominant-negative mutant of TAK1, TAK1 K63W(20) (dnTAK1), was generated.
Although TAK1 mRNA is detectable in the liver (87), TAK1
protein expression was assessed in hepatocyte cultures by
immunoprecipitation and Western blotting (Fig.
1). The results confirm that both
endogenous and virally expressed TAK1 protein can be detected in
quiescent hepatocytes. Although overall protein levels appear to be
similar in control and Ad5TAK-infected cells, the relative amount of
exogenous protein is strongly increased, suggesting that the
immunoprecipitation antibody may be saturated during the precipitation
even in the control cells. Direct Western blotting with anti-HA
antibody reveals much higher levels of viral protein in infected cells
than is indicated by the immunoprecipitation (Fig. 1B).
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dnTAK1 does not effect p38, NF-B, or SMAD activation.
dnTAK1 has been reported to block activation of the MAPK family member
p38 (29) as well as the transcription factor NF-
B (57, 65, 66). Therefore, the effect of dnTAK1 expression on p38 and NF-
B activation after PH was assessed. p38 activity was
assessed by Western blotting using a phospho-specific antibody. Phospho-p38 was detected in the initially resected liver lobes, although little or no phospho-p38 was seen after PH (Fig.
3A). Phospho-p38 was also
detected in untreated control livers (Fig. 3A), indicating
that the p38 activation observed at the time of hepatectomy reflects
basal activity and not surgical stress. No decrease in phospho-p38
levels was observed in Ad5TAK-injected animals compared with
PBS-injected animals. Thus p38 is inhibited during liver regeneration,
and p38 activity before PH was unaffected by dnTAK1 expression. Hepatic
SMAD3/4 DNA binding activity was increased after PH and was unaffected
by dnTAK1 expression (Fig. 3C), consistent with possibly
divergent TGF-
signaling to SMADs and TAK1 (35).
NF-
B binding activity was induced after PH in PBS-injected animals
as expected (28) and was unaffected by expression of
dnTAK1 (Fig. 3B), indicating that TAK1 is not required for
NF-
B activation in this setting. TNF-
signaling is responsible for NF-
B induction after PH (86). We have similarly
observed that dnTAK1 does not inhibit TNF-
-mediated NF-
B
activation in primary hepatocyte cultures or COS cells (data not
shown). In contrast, dnTAK1 strongly inhibits IL-1-mediated NF-
B
activation (57). This differential result may be explained
by the observation that TAK1 signals to NF-
B via interaction with
Traf6 (57, 74), which is involved in IL-1 but not TNF-
signaling (18).
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dnTAK1 and TAM-67 induce G0 exit.
Hepatocyte proliferation after PH was assessed by immunostaining liver
sections for the G1 marker PCNA, before and after PH (Fig.
4 and Table
1). As expected, PCNA expression was
not induced until 24 h after PH in livers from PBS- and
Ad5Luc-injected animals (62, 68). In contrast, hepatic
PCNA expression was detected at all time points in Ad5TAK- and
Ad5TAM-injected animals (Fig. 4 and Table 1). The increased levels of
PCNA staining were statistically significant for both Ad5TAK and Ad5TAM
compared with PBS (P < 0.01 at all time points). M
phase was assessed by counting mitotic figures in H&E-stained sections
from livers before and after PH (Fig. 5 and Table
2). In PBS- and Ad5Luc-injected
animals, mitotic figures were only detected at 24 h after PH, as
expected (68). In contrast, mitotic figures were detected
before and after PH in dnTAK1- and TAM-67-expressing livers (Fig.
5 and Table 2). TAM-67-expressing livers
consistently showed approximately two-thirds as many mitotic figures as
dnTAK1-expressing livers at all time points examined. The increased
numbers of mitotic figures were statistically significant for both
dnTAK1 and TAM-67 compared with PBS (P < 0.01 for all
time points, except P < 0.025 for Ad5TAM compared with
PBS at 24 h).
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dnTAK1 and TAM-67 accelerate cyclin expression.
Cyclin mRNA levels were assessed before and after PH by Northern blot
analysis (Fig. 6). The results indicate
an earlier and stronger accumulation of cyclin mRNAs in Ad5TAK- and
Ad5TAM-injected animals compared with PBS-injected animals. These
results were confirmed by assessing cyclin protein levels before PH and
at 12 and 24 h after PH with Western blotting (Fig.
7). Consistent with the Northern blot
results, cyclin protein expression occurred early in Ad5TAK- and
Ad5TAM-injected animals compared with PBS- and Ad5Luc-injected animals.
The kinetics of cyclin expression are consistent with previous reports
(44, 45). With one exception, increased cyclin mRNAs and
proteins were not observed before PH in dnTAK- and TAM-67-expressing
livers, despite the ongoing cell cycle progression detected
histologically. One possible explanation for this apparant
inconsistency is that because the hepatocytes are not undergoing
synchronous cell division, increased cyclin levels fall below the
limits of detection by Western blotting. Cyclin E protein was observed
before PH in dnTAK1- and TAM-67-expressing livers, although cyclin E
mRNA levels were not yet increased, suggesting that the initial
induction of cyclin E expression may be controlled
posttranscriptionally. Together, these results show that dnTAK1
expression in the liver enhanced proliferation and that this effect was
mimicked by expression of the c-Jun dominant-negative TAM-67.
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dnTAK1 and TAM accelerate p21 expression.
Cyclin-dependent kinases are negatively regulated by the cyclin kinase
inhibitors p21 and p27, both of which are positively regulated by
TGF- (56). Targeted disruption of p21 in mice accelerates liver regeneration (2), whereas hepatic p21
overexpression blocks regeneration (85). However, p21
protein expression was induced more rapidly in dnTAK1- and
TAM-76-expressing livers compared with Luc and PBS livers (Fig.
8A). Similar to the cyclins,
p21 expression at 24 h after PH was more robust in Ad5TAK- and
Ad5TAM- compared with PBS- and Ad5Luc-injected animals (Fig.
8A). Hepatic p27 protein expression remained unchanged in
all animals at all time points (Fig. 8B), consistent with
previous reports (2, 73). The lack of inhibition of p21
expression by dnTAK1 is not surprising, because p21 expression is
regulated by SMADs (53), which were not affected by
dnTAK1 (Fig. 3). Because p21 is normally expressed at 24 h after
PH as an apparently normal aspect of cell cycle progression
(26), perhaps the accelerated p21 expression induced by dnTAK1 and TAM-67 reflects the overall acceleration of the
cell cycle.
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dnTAK1 and TAM-67 induce c-myc.
c-Myc expression is associated with G0 exit and is
inhibited by TGF- (8, 47). c-myc mRNA was
not detected by Northern blotting at 12 or 24 h after PH or before
PH in livers from PBS- or Ad5Luc-injected animals. This was not
surprising, because c-Myc is normally expressed during the initial
"priming" phase, lasting until 8 h after PH (46).
In contrast, c-myc expression was detected before and after
hepatectomy in dnTAK- and TAM-67-expressing livers. c-myc
levels were initially higher in dnTAK-expressing livers and increased
more slowly in TAM-67-expressing livers. Thus hepatic expression of
either dnTAK1 or TAM-67 resulted in abnormal induction of
c-myc expression before PH.
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DISCUSSION |
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Hepatocytes possess an amazing replicative capacity. Serial transplantation and repopulation studies in transgenic mice have shown that hepatocytes have the capacity to undergo >85 rounds of cell division in vivo with no indications of either senescence or transformation (13). After 70% PH, the remaining hepatocytes need only divide an average of 1.5 times to completely restore the liver mass (13). Liver regeneration is required after surgical resection of diseased liver or after PH for transplantation to living related donors. Hepatocyte proliferation is crucial for recovery from liver injury induced by alcohol, adverse drug reactions, viruses, or other toxins. Hepatocyte proliferation is impaired in fatty livers (6, 68) as well as cirrhotic livers (23), in which inhibition of proliferation may be causal (63).
TGF- is a potent inhibitor of proliferation and is strongly
associated with the induction and maintenance of liver fibrosis (67, 79). TAK1 mediates some TGF-
signals, including
antiproliferative and prodifferentiation functions. For example, TAK1
mediates bone morphogenic protein (BMP, a TGF-
family
member)-induced differentiation of P19 cells (51).
Exogenous active TAK1 expressed in the heart induces cardiac
hypertrophy, a condition associated with TGF-
expression
(92). Additionally, in kidney cells, TAK1 inhibits cell
cycle progression, whereas dnTAK1 expression reverses TGF-
-mediated inhibition of 3H incorporation and increases cyclin
expression levels above normal (77), similar to the
findings reported here.
Hepatocyte proliferation in vivo requires an initial priming stimulus,
such as one-third hepatectomy, which alone is insufficient to induce
proliferation but causes normally nonresponsive hepatocytes to become
permissive to growth factor stimulation (81).
Priming probably reflects G0/G1 transition and
is marked by induction of c-myc (46), which is
an established regulator of the G0/G1 transition in hepatocytes (81) and other cells (7,
8, 30, 69). Fibroblasts containing only one c-myc
allele display delayed cell cycle entry and reduced cyclin levels
(30, 69), and blockade of c-myc transcription
correlates with an inhibition of proliferation after PH
(17). Our results suggest that dnTAK1 induces hepatocyte
proliferation through upregulation of c-myc mRNA, consistent
with previous studies demonstrating that TGF--mediated growth arrest
involves downregulation of c-myc (27, 47, 54, 61), whereas enforced myc expression overcomes TGF-
-mediated growth arrest (80). c-myc transcription is
induced by
-catenin/TCF-4, whereas
-catenin/TCF DNA binding is
antagonized by TAK1 homologs in Caenorhabditis
elegans (31, 48, 38).
-Catenin is expressed in the normal liver (16, 36) and is upregulated during
liver regeneration (36). Thus TAK1 may also inhibit
c-myc via
-catenin in the liver.
AP-1 binds to a negative element in c-myc promoter
(75), potentially explaining the similar induction of
c-myc mRNA observed in livers expressing TAM-67 and dnTAK1.
However, TAM-67 induced c-myc expression in the quiescent
liver, in which there is no AP-1 expression (Fig. 2), making this
simple explanation unlikely. Targeted disruption of c-jun is
embryonic lethal as a result of a failure in liver development
(34). A recent study unexpectedly showed that targeted
replacement of c-jun with an inactive mutant containing
serine-to-alanine substitutions at 63 and 73 restores normal
development (5), suggesting that c-Jun protein plays a
necessary yet passive role during liver development, perhaps by
blocking transcription of a proapoptotic protein. Because hepatic c-myc mRNA is constitutively transcribed and regulated
posttranscriptionally by a labile destabilizing protein (46, 8,
52), one might speculate that TAM-67 expression passively
inhibits transcription of this destabilizing protein, thereby inducing
c-myc expression. This hypothesis is consistent with the
observed correlation of c-jun and c-myc
expression in various in vivo models of hepatic proliferation
(21). An alternative possibility is that TAM-67 synergizes
with the pro-proliferative factor C/EBP- to activate TNF-
expression (91), although TNF-
is not required for
liver mitogenesis in vivo (43).
An unexpected result of this study is the finding that inhibition of
JNK and AP-1 correlates with the induction of proliferation. Indeed, a
pro-proliferative role for JNK has been described in various cells
(4, 9, 50, 72). However, several counterexamples of
antiproliferative, prodifferentiation roles for JNK and AP-1 have also
been described. JNK activity occurs independently of or correlates
inversely with proliferation induced by insulin-like growth factor I,
adrenomedullin, or Rac-1/cdc42 (42, 58, 59). Epo-1-mediated erythroid differentiation of SKT6 cells requires JNK
(83), and vitamin D-mediated Caco-2 cell differentiation requires both JNK and AP-1 (19). Jnk1 knockout T cells
show increased proliferation and decreased differentiation
(25), and Jnk2 knockouts display defective T cell
differentiation (89). Interestingly, studies in primary
hepatocytes have reported that JNK and AP-1 are required for hepatocyte
growth factor-, glucose- and TNF--mediated hepatocyte proliferation
in vitro (4), although our preliminary data showed that
neither dnTAK1 or TAM-67 had a specific effect on EGF-mediated
hepatocyte proliferation in vitro (data not shown). A differential role
for cAMP in hepatocyte proliferation has been observed in vitro and in
vivo (82), similar to the differential findings regarding
the proliferative role of JNK and AP-1 in hepatocytes in vitro
(4) and in vivo (this study). This may possibly reflect
the strongly reduced requirement for priming hepatocyte proliferation
in vitro vs. in vivo. Direct inhibition of JNK in vivo will ultimately
be necessary to clearly define the role of JNK and AP-1 activation
during liver regeneration.
Potentiation of the liver's regenerative response would have great
clinical utility in patients with liver cirrhosis. However, caution
regarding the potential induction of hepatocellular transformation is
an important caveat here and in other pro-proliferative gene therapy
approaches (63, 79). The onset of hepatocellular carcinoma is marked by a loss of sensitivity to TGF--mediated growth
suppression and apoptosis (78). TGF-
is a tumor
suppressor, and this suppressive effect, and TGF-
-induced
apoptosis, appear to be mediated by SMADs (20, 76, 88,
94). However, although mutations in TAK1 were not detected in
lung cancer (41), TAK1 has been reported to mediate
BMP-induced apoptosis (39). Gene therapy
approaches with transient expression may alleviate concerns regarding
cellular transformation; thus, for situations in which a temporary
induction of hepatic proliferation would be beneficial, adenoviral gene therapy with dnTAK1 may prove to be useful.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. A. Brenner, 154 Glaxo CB 7038, UNC Chapel Hill, Chapel Hill, NC 27707 (E-mail dab{at}med.unc.edu).
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.
Received 5 March 2001; accepted in final form 3 July 2001.
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