Dominant-negative TAK1 induces c-Myc and G0 exit in liver

Cynthia A. Bradham, Etsuro Hatano, and David A. Brenner

Departments of Medicine and Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27707


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta )-activated kinase 1 (TAK1), a serine/threonine kinase, is reported to function in the signaling pathways of TGF-beta , 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)-kappa 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-beta


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-kappa 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-kappa 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-alpha (TNF-alpha ) signaling is required for liver regeneration, as shown by treatment with anti-TNF-alpha 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-kappa B activation (86).

Transforming growth factor-beta (TGF-beta ) is a pleiotropic cytokine that has potent antiproliferative effects in the liver and other tissues (60). TGF-beta 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-beta production and resolves experimental liver fibrosis (79), demonstrating the importance of hepatocyte proliferation in combating this disease state.

TGF-beta signals by binding to serine/threonine kinase receptors, which phosphorylate and activate SMAD family transcription factors (60, 32). In addition, TGF-beta signals through TGF-beta -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-beta (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.


    METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   A: transforming growth factor-beta (TGF-beta )-activated kinase 1 (TAK1) expression in hepatocytes. Primary hepatocyte cultures were uninfected or infected with Ad5TAK1, and then whole cell extracts were immunoprecipitated (IP) with anti-TAK1. Immunoprecipitates were then immunoblotted (IB) with either anti-TAK1 or anti-hemagglutinin (HA) antibodies. B: expression of viral proteins in primary hepatocyte cultures. Hepatocytes were uninfected (U) or infected with AD51kappa B or Ad5TAK, and then whole cell extracts (50 µg) were immunoblotted with anti-HA primary antibodies.

Because TAK1 can mediate signals to JNK from TGF-beta , as well as TNF-alpha , IL-1, and ceramide (57, 70, 90, 93), JNK activation was assessed after PH (Fig. 2). Animals were injected with PBS or adenoviruses expressing either dnTAK1 (Ad5TAK) or luciferase control (Ad5Luc) and then subjected to 70% PH. JNK activation was compared in extracts prepared from the originally resected liver lobes and from the remnant liver after PH (Fig. 2A). 70% PH induced hepatic JNK activation in PBS- and Ad5Luc-injected animals as expected (84). JNK activation after PH was abrogated in Ad5TAK-injected animals (Fig. 2A). Because JNK phosphorylates c-Jun on serines 63 and 73 to activate c-Jun-mediated transcription (24), c-Jun serine 63 phosphorylation was assessed by Western blotting using a phospho-specific antibody (Fig. 2B). c-Jun phosphorylation was induced at 1 h after PH in livers from PBS- and Ad5Luc-, but not Ad5TAK-, injected animals (Fig. 2B). Activated c-Jun induces transcription of a variety of genes, including the c-jun gene itself in an autofeedback loop (66). To assess c-Jun functional activity after PH, steady-state c-jun mRNA levels were determined by Northern blotting (Fig. 2C). Hepatic c-jun was induced at 1 h after PH in PBS- and Ad5Luc-, but not Ad5TAK-, injected animals (Fig. 2C). Together, these results show that JNK and c-Jun activities are induced after PH and that expression of dnTAK1 completely blocks activation of both JNK and c-Jun.


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Fig. 2.   Dominant-negative (dn)TAK1 inhibits JNK activation after partial hepatectomy (PH). Rats were injected with PBS, Ad5Luc, or Ad5TAK and then subjected to 70% PH. A: whole cell extracts were prepared from liver samples from original resected lobes (time 0) or 1 h after PH and then assessed for c-Jun NH2-terminal kinase (JNK) activity, using GST-jun as a substrate. B: whole cell extracts from A were assessed for phospho-c-Jun expression using Western blotting. C: total RNA was prepared from liver samples as in A, and c-jun mRNA levels were determined by Northern blotting. 18S and 28S ribosomal bands are shown as an RNA loading control.

dnTAK1 does not effect p38, NF-kappa 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-kappa B (57, 65, 66). Therefore, the effect of dnTAK1 expression on p38 and NF-kappa 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-beta signaling to SMADs and TAK1 (35). NF-kappa 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-kappa B activation in this setting. TNF-alpha signaling is responsible for NF-kappa B induction after PH (86). We have similarly observed that dnTAK1 does not inhibit TNF-alpha -mediated NF-kappa B activation in primary hepatocyte cultures or COS cells (data not shown). In contrast, dnTAK1 strongly inhibits IL-1-mediated NF-kappa B activation (57). This differential result may be explained by the observation that TAK1 signals to NF-kappa B via interaction with Traf6 (57, 74), which is involved in IL-1 but not TNF-alpha signaling (18).


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Fig. 3.   dnTAK1 does not block nuclear factor-kappa B (NF-kappa B) or SMAD activation after PH. Rats were injected with PBS or Ad5TAK and then subjected to 70% PH. Whole cell extracts were prepared from liver samples from original resected lobes (time 0) or 1 h after PH. A: phospho-p38 (P-p38) was assessed by Western blotting. C, untreated control liver. B: NF-kappa B DNA binding activity was assessed by electrophoretic mobility shift assay (EMSA) using a probe containing a consensus kappa B binding site. C: SMAD DNA binding activity was assessed by EMSA using a probe containing a consensus SMAD3/4 binding site.

Thus, of the pathways examined, only the JNK pathway was blocked by kinase-inactive TAK1 in this model of PH, although effects on other unassessed pathways cannot be ruled out. To delineate the contribution of c-Jun activation to the overall impact of dnTAK1 expression, an adenovirus expressing HA-tagged TAM-67 (Ad5TAM) was generated. TAM-67 is a truncation mutant of c-Jun lacking amino acids 3-122, comprising the transactivation domain, whereas the DNA binding domain remains intact, permitting dimerization with other AP-1 family members and DNA binding activity (14). TAM-67 thus functions as a dominant-negative c-Jun, inhibiting AP-1-mediated transcription (15). The ability of adenovirally expressed TAM-67 to block expression from an AP-1 reporter was confirmed in MCF7 cells and primary hepatocyte cultures (data not shown).

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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Fig. 4.   dnTAK1 and TAM-67 accelerate proliferating cell nuclear antigen (PCNA) expression. Rats were injected with PBS (A and B), Ad5Luc (C and D), Ad5TAK (E and F), or Ad5TAM adenoviruses (G and H) and then subjected to 70% PH. Liver sections from original resected lobes (time 0, A, C, E, and G) or 24 h after PH (B, D, F, and H) were immunostained for PCNA. Insets, Western blots for HA-dnTAK1 (E) and HA-TAM-67 (G) expression in uninfected (U) and infected (I) livers.


                              
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Table 1.   PCNA-positive cells



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Fig. 5.   dnTAK1 and TAM-67 accelerate the appearance of mitotic figures. Rats were injected and treated as in Fig. 4, and liver sections were stained with hematoxylin and eosin. Sections are labeled as in Fig. 4. Arrows identify mitotic figures; arrowheads identify representative endothelial cell (e) nuclei.


                              
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Table 2.   Mitotic figures

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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Fig. 6.   dnTAK1 and TAM-67 accelerate cyclin mRNA accumulation. Rats were injected with PBS, Ad5TAK, or Ad5TAM and then subjected to 70% PH. Total mRNA was prepared from original resected livers (time 0) or from 1, 12, or 24 h after PH and then subjected to Northern blot analysis for cyclin mRNAs as indicated. 18S and 28S ribosomal bands are shown as a control for RNA loading.



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Fig. 7.   dnTAK1 and TAM-67 accelerate cyclin protein expression. Rats were injected with PBS, Ad5Luc, Ad5TAKn or Ad5TAM and then subjected to 70% PH. Whole cell lysates were prepared from original resected livers (time 0) or from 12 or 24 h after PH and then subjected to Western blot analysis for cyclins as indicated. In the cyclin E panel, the upper band is nonspecific (n.s.), while the lower band corresponds to cyclin E (cycE).

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-beta (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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Fig. 8.   dnTAK1 and TAM-67 accelerate expression of p21 and c-myc. Rats were injected with PBS, Ad5Luc, Ad5TAK, or Ad5TAM and then subjected to 70% PH. Whole cell lysates and RNA were prepared from original resected livers (time 0) or from 12 or 24 h after PH and then subjected to Western blot analysis for p21 (A) or p27 (B) or Northern blot analysis for c-myc (C).

dnTAK1 and TAM-67 induce c-myc. c-Myc expression is associated with G0 exit and is inhibited by TGF-beta (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.

In summary, dnTAK1 expression in vivo stimulated hepatocyte proliferation in the quiescent liver and accelerated cell cycle progression after PH. This effect was associated with inhibition of JNK and AP-1 but not NF-kappa B and was reproduced by expression of TAM-67, a dominant-negative form of the AP-1 component c-Jun. Expression of dnTAK1 or TAM-67 induced c-myc expression, consistent with G0 exit.


    DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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-beta is a potent inhibitor of proliferation and is strongly associated with the induction and maintenance of liver fibrosis (67, 79). TAK1 mediates some TGF-beta signals, including antiproliferative and prodifferentiation functions. For example, TAK1 mediates bone morphogenic protein (BMP, a TGF-beta family member)-induced differentiation of P19 cells (51). Exogenous active TAK1 expressed in the heart induces cardiac hypertrophy, a condition associated with TGF-beta expression (92). Additionally, in kidney cells, TAK1 inhibits cell cycle progression, whereas dnTAK1 expression reverses TGF-beta -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-beta -mediated growth arrest involves downregulation of c-myc (27, 47, 54, 61), whereas enforced myc expression overcomes TGF-beta -mediated growth arrest (80). c-myc transcription is induced by beta -catenin/TCF-4, whereas beta -catenin/TCF DNA binding is antagonized by TAK1 homologs in Caenorhabditis elegans (31, 48, 38). beta -Catenin is expressed in the normal liver (16, 36) and is upregulated during liver regeneration (36). Thus TAK1 may also inhibit c-myc via beta -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-beta to activate TNF-alpha expression (91), although TNF-alpha 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-alpha -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-beta -mediated growth suppression and apoptosis (78). TGF-beta is a tumor suppressor, and this suppressive effect, and TGF-beta -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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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