C/EBP β isoforms LIP and LAP modulate progression of the cell cycle in the regenerating mouse liver

Authors


Abstract

The CCAAT enhancer-binding protein (C/EBP) β gene can produce several N-terminally truncated isoforms. Liver-enriched activator protein (LAP) is a transcriptional activator in many systems, whereas liver-enriched inhibitory protein (LIP) is regarded as a functional LAP antagonist. In this study, we examined the impact of these two proteins on cell cycle progression in the regenerating liver. Adenoviral overexpression of LAP, in addition to its role as a transactivator of liver-specific genes, led to a delayed S-phase entry of hepatocytes after partial hepatectomy (PH) in vivo. This delay was accompanied by decreased expression of cyclin A and E as well as proliferating cell nuclear antigen and decreased cyclin-dependent kinase 2 activity at the G1/S boundary. This observation is not explained by increased p21CIP1/Waf1 expression or lack of phosphorylation of external LAP, but LAP overexpression triggered a decreased C/EBP-α/C/EBP-α-30 ratio and a reduced basal c-jun level in the liver. In contrast, adenoviral overexpression of LIP resulted in a stronger and earlier induction of cyclin A and E after PH, but did not change the timing and extent of cyclin-dependent kinase 2 activity or the amount of hepatocytes that entered S phase in this model. In the LIP expressing group, both C/EBP-α isoforms and c-jun were more strongly induced after PH. In conclusion, the LAP/LIP ratio is an important modulator of cell cycle progression during liver regeneration. In the context of previous studies, our results demonstrate that LAP, through a dose-dependent effect, withholds a dual activating and inhibiting role on hepatocyte proliferation in vivo. (HEPATOLOGY 2004;40:356–365.)

The liver stands out in its capacity to regenerate lost parenchymal mass.1, 2 A well-established model for studying liver regeneration and cell cycle regulation in vivo is that of partial (two-thirds) hepatectomy in rodents. The proposed sequence of events involves the induction of cytokines. Tumor necrosis factor α rises early after partial hepatectomy (PH) and induces interleukin 6 expression in Kupffer cells. Interleukin 6 then leads to activation of STAT proteins, mainly STAT-3, and also the Ras/mitogen-activated protein kinase pathways, driving hepatocytes sensitive to the action of growth factors.3 Signal transduction pathways couple these extracellular factors to the key proteins regulating cell cycle progression, for example, kinase complexes consisting of the cyclin-dependent kinases (CDKs) and their regulatory subunits, the cyclins.4 A modification of these complexes by CDK inhibitors, (e.g., from the CIP/KIP family [p21CIP/WAF1, p27KIP1, p57KIP2]) has been demonstrated as an important factor.4, 5

The liver must maintain metabolic and synthetic homeostasis throughout regeneration.6 We and others have provided evidence for the importance of the CCAAT enhancer-binding proteins (C/EBP) α and β in this process.7–9 The C/EBPs are transcription factors belonging to the bZIP family of proteins, which are characterized by the presence of a basic region of amino acids involved in DNA binding followed by a leucine zipper motif.10 High expression of C/EBP-α is restricted to liver, adipose tissue, and lung, while C/EBP-β is expressed in many other tissues and plays an important role in the regulation of liver development or acute phase response to inflammation.11, 12 The C/EBP-β messenger RNA directs production of four isoforms: full-length C/EBP-β (38-kDa), 34-kDa liver-enriched activating protein (LAP), 21-kDa liver-enriched inhibitory protein (LIP), and a smaller 14-kDa isoform13 through alternative translation from different AUG codons (see also Fig. 1). Because the low molecular weight isoform LIP lacks most of the transactivation domain but contains the DNA-binding and dimerization domains, it is proposed to function as a dominant negative regulator of full-length C/EBP protein and LAP.13

Figure 1.

Construction of adenoviral vectors for overexpression of C/EBP-β isoforms LIP and LAP. (A) The C/EBP-β gene contains several AUG codons that serve as start sites of translation. Depicted is the 38-kd full-length isoform with the activation domain, the basic region, and the leucin zipper domain. We constructed adenoviral vectors to overexpress two N-terminally truncated isoforms, LAP (34 kd) and LIP (21 kd), which lacks the activation domain. (B) Overexpression of LAP/LIP by adenoviral constructs in cell culture. 293 cells were infected with advLAP and advLIP at a multiplicity of infection of 30 and harvested 24 hours later. Twenty micrograms of cellular protein extracts were subjected to a Western blot analysis using an antibody detecting the C-terminus to test for expression of C/EBP-β isoforms. The arrows indicate the position of signals for LAP and LIP. (C) Mice were injected with 1.6 × 109 Pfu of the respective virus or a LacZ control virus. The mice were sacrificed 24 hours later. Whole liver protein extracts were prepared, and 50 μg were subjected to Western blotting with the respective antibody. C/EBP-β, CCAAT enhancer-binding protein β; mRNA, messenger RNA; FL, full-length; AD, activation domain; BR, basic region; LZ, leucin zipper; LAP, liver-enriched activator protein; LIP, liver-enriched inhibitory protein.

In the regenerating liver, it has been demonstrated that C/EBP-β increases during G1 phase of the hepatocyte cell cycle, while levels of C/EBP-α messenger RNA decrease transiently.14, 15 C/EBP-α−/− mice display increased hepatocyte proliferation at birth, while C/EBP-β knockout mice exhibit impaired liver regeneration after PH.16–18 It has been demonstrated that low molecular weight isoforms of C/EBP-β are induced in response to PH,19–21 but the direct contribution of the particular C/EBP-β isoforms to cell cycle progression after PH is not clear. In this study, we show that adenoviral overexpression of LAP leads to a delayed cell cycle progression after PH in vivo, while LIP overexpression results in earlier induction of key cell cycle markers.

Abbreviations

C/EBP, CCAAT enhancer-binding protein; LAP, liver-enriched activator protein; LIP, liver-enriched inhibitory protein; PH, partial hepatectomy; CDK, cyclin-dependent kinase; CMV, cytomegalovirus; PCNA, proliferating cell nuclear antigen; BrdU, 5-bromo-2′-deoxyuridine.

Materials and Methods

Construction of Adenoviral Vectors.

Construction of advLacZ has been previously described.22 Recombinant adenoviruses were constructed using the AdEasy system.23 Viral vectors expressing LAP or LIP were generated by inserting either LAP complementary DNA containing a HindIII/EcoRV fragment derived from cytomegalovirus (CMV)-LAP wt or the respective fragment from CMV-LIP124 into the shuttle vector AdTrack-CMV. The infective adenoviral vectors were generated by homologous recombination of the shuttle vectors with AdEasy-1 in Escherichia coli BJ5183. Preparation of high-titer viral stocks has been described previously.22

Cell Culture and Infection Experiments.

293 cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum. Cells were grown on 100-mm dishes to approximately 80% confluence and then infected with advLIP or advLAP at a multiplicity of infection of 30 for 24 hours. Cells were then washed twice in phosphate-buffered saline, harvested, and lysed in an NP-40 lysis buffer (150 mmol/L NaCl, 10 mmol/L Tris/HCl, pH 7.4, 1 mmol/L ethylenediaminetetraacetic acid, 1% NP-40).

In Vivo Infection and PH.

Pathogen-free male balb/c mice (age 7–8 weeks) were obtained from the animal facility of the Hannover Medical School. Animals received humane care according to the criteria prepared by the National Academy of Sciences (National Institutes of Health publication no. 86-23, revised 1985). Viruses advLacZ, advLIP, and advLAP were prepared, purified, and titered as described above. In vivo infection of mice, PH procedure, and generation of nuclear protein extracts were performed as previously described.22 Alanine aminotransferase activities were measured using standard procedures.

Preparation of Cellular Extracts.

Mouse liver was homogenized in an NP-40 buffer containing protease and phosphatase inhibitors (1 mmol/L Na3VO4, 1 mmol/L dithiothreitol, 1 mmol/L phenylsmethylsulfonyl-fluoride and 1× protease-inhibitor minitablets [Roche, Mannheim, Germany]). The homogenates were sonicated and clarified by centrifugation for 10 minutes at 15,000 rpm. Supernatants were aliquoted and stored at −80°C. Protein concentration was measured using the BioRad DC protein reagent (BioRad, Hercules, CA).

SDS–Polyacrylamide Gel Electrophoresis and Western Blot Analysis.

Nuclear protein extracts or whole cellular protein extracts were subjected to SDS–polyacrylamide gel electrophoresis and Western blot analysis as described previously.5 For primary antibody incubation, membranes were probed with anti-LAP,24 anti–proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Santa Cruz, CA, #sc-56), anti–cyclin A (Santa Cruz Biotechnology, #sc-751), anti–cyclin E (Santa Cruz Biotechnology, #sc-481), anti–C/EBP-α (Santa Cruz Biotechnology, #sc-61), anti–c-jun (Santa Cruz Biotechnology, #sc-1694), anti-p21 (BD Pharmingen, San Diego, CA, #556431), anti–α tubulin (Santa Cruz Biotechnology, #sc-8035), anti-CDK2 (Santa Cruz Biotechnology, #Sc-163) or anti–Phospho-C/EBP-β (Thr235) (Cell Signaling Technology, Beverly, MA, #3084). As a secondary antibody, anti–rabbit immunoglobulin G (Jackson Immuno Research Laboratories Inc., West Grove, PA, #711-035-152) or anti–mouse immunoglobulin G (Chemicon International, Temecula, CA, #AQ 127P) were used. The antigen–antibody complexes were visualized using the ECL Chemiluminescence Kit (Amersham, Arlington Heights, IL). Coomassie staining was used to demonstrate equal protein loading. Quantification of signal intensity was performed with phosphoimager analysis and correction against loading control.

CDK2 Kinase Assay.

For measuring CDK2 activity, immunoprecipitation was performed with 1 μL anti-CDK2 (Santa Cruz Biotechnology, #sc-163) in 300 μg of whole cell protein extracts for 1 hour on ice. Forty microliters of prewashed protein-A-agarose beads (Oncogene Science, Cambridge, MA) were added for 1 hour. After washing twice with NP-40 buffer, CDK2 activity was determined as described previously.5

5-Bromo-2′-deoxyuridine In Vivo Labeling.

Two hours before sacrificing, 30 μg/mouse of 5-bromo-2′-deoxyuridine (BrdU) (Amersham, Braunschweig, Germany) were injected intraperitoneally. Cryosections were fixed and stained according to the Amersham cell proliferation kit manual. At least 4 independent experiments were performed. Statistical analysis was performed using the SPSS statistical package (version 10.0.7; SPSS Inc., Chicago, IL).

Caspase Assay.

Caspase 3–like activity was measured using an enzymatic, fluorometric assay. Samples were lysed in assay buffer (50 mmol/L hydroxyethylpiperazine-N-2 ethanesulfonic acid, pH 7.4, 100 mmol/L NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, 10 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetraacetic acid, 10% glycerol). After centrifugation for 10 minutes at 14,000g, samples were incubated with a specific substrate (Ac-DEVD-AFC, Biomol, Plymouth Meeting, PA) for 3 hours. Fluorescence was measured (Fluostar, Biomedizintechnik, Offenburg, Germany), and readings were compared with values after incubation with a specific inhibitor (DEVD-CHO, Biomol) using an excitation filter for 400 nm and an emission filter for 505 nm. As a positive control, galactosamine (700 mg/kg) was administered intraperitoneally; 2 minutes later, tumor necrosis factor α (5 μg/mouse, kindly provided by Dr. G. R. Adolf, Bender GmbH, Vienna, Austria) was intravenously injected.

c-jun Immunohistochemistry.

Immunohistochemical staining of c-jun on cryosections was performed using a standard protocol.25 The c-jun–specific antibody was the same as that used for Western blot analysis (Santa Cruz Biotechnology). As a secondary antibody, goat anti–rabbit immunoglobulin G Cy3 (Jackson Immuno Research Laboratories Inc., cat. #111-166) was used. The nuclei were counterstained with a 4′,6-diamidino-2-phenylindole solution (1:15000 dilution of stock solution, Boehringer Mannheim, Mannheim, Germany) for 2 minutes.

Results

Expression of LIP and LAP With Recombinant Adenoviruses in Cell Culture and in Mouse Livers In Vivo.

The C/EBP-β gene can code for different proteins, mainly by alternative translation of its messenger RNA13 (Fig. 1A). To examine the role of LIP and LAP in liver regeneration, we constructed adenoviral vectors expressing either LIP (advLIP) or LAP (advLAP) under control of a CMV promotor (see Materials and Methods). As a control, we used a recombinant adenovirus that expressed the LacZ gene (advLacZ).

Expression of the vectors was first tested in vitro by infecting 293 cells at a multiplicity of infection of 30 and harvesting 24 hours later. For advLAP, Western blot analysis showed a strong expression of a 34-kd protein and, as expected, a slight signal at 21 kd representing LIP (Fig. 1B, lane 1). In contrast, cells infected with advLIP showed a single strong band of the expected size (Fig. 1B, lane 2). To examine expression of the constructs in vivo, we injected advLIP and advLAP as well as the control virus (Fig. 1C). Twenty-four hours after infection with advLAP, both LAP and LIP were expressed in the liver. Interestingly, the expression of LIP was relatively stronger than that in vitro. Animals injected with advLIP showed an isolated overexpression of the LIP isoform. Thus, the advLAP construct led to an efficient expression of both LAP and LIP in cell culture and in vivo, whereas advLIP infection resulted in strong overexpression of LIP in both systems.

LIP Overexpression Has a Minor Influence on S Phase Entry After PH, While LAP Overexpression Leads to a Delayed S Phase Entry.

To examine the influence of LIP and LAP in controlling cell cycle progression in the liver in vivo, we injected the advLIP and advLAP constructs as well as the advLacZ virus into mice, which then underwent PH 24 hours after infection. Two hours before sacrificing, mice were injected with BrdU to label cells in S phase. In the control group, DNA synthesis started at 24 hours after PH and peaked at 36 hours after PH, with approximately 20% of BrdU-positive hepatocytes (Fig. 2A and 2B). In the advLIP-infected mice, peak DNA synthesis was not shifted. In contrast, mice infected with advLAP showed a lower number of S phase–positive hepatocytes at 24 hours after PH and an even stronger difference to the control group at the 36-hour time point, with only about 7% of BrdU-positive hepatocytes. Therefore, advLIP has a minor effect on the timing of G1/S phase transition and the extent of S phase–positive hepatocytes after PH, whereas the advLAP construct leads to a delayed S phase entry of hepatocytes.

Figure 2.

Modulation of cell cycle progression by adenoviral overexpression of LAP and LIP. Mice were injected with 1.6 × 109 Pfu of the LacZ control virus and the advLIP and advLAP construct. PH was performed 24 hours later. (A) BrdU uptake was determined before PH and at 24 hours, 36 hours, and 48 hours after PH. (B) Quantification of the percentage of BrdU-positive hepatocyte nuclei at 0 hours, 24 hours, 36 hours, and 48 hours after PH in advLacZ control, advLIP, and advLAP mice. *Significant difference (P < .05) to the control group. (C) Determination of relative caspase 3–like activity using an enzymatic fluorometric assay. The relative level of AFC release reflecting caspase 3 activation is indicated for the advLacZ, advLIP and advLAP mice at different time points after PH. As control, the relative value for mice that were injected with galactosamine and tumor necrosis factor α are depicted. LIP, liver-enriched inhibitory protein; LAP, liver-enriched activator protein; PH, partial hepatectomy; BrdU, 5-bromo-2′-deoxyuridine.

Previous studies demonstrated an involvement of C/EBP-β in the regulation of apoptosis.26, 27 To exclude that the observed decreased proliferation in the advLAP group results from an increased apoptotic cell death at the G1/S phase transition, serum aminotransferase activities (aspartate aminotransferase/alanine aminotransferase) and hepatic caspase 3 activation were tested. No significant difference in the serum aminotransferase activities was measured (data not shown). Moreover, the course of caspase 3 activation after PH showed only minor changes between the different groups (Fig. 2C). All groups showed only moderate caspase 3 activation compared with a positive control with strong hepatic apoptosis after treatment with tumor necrosis factor α and galactosamine.

LAP Expression Leads to a Delayed Activation and Reduced Expression of CDK2.

CDK2 is activated during the G1/S phase transition.28 Therefore, we examined the influence of LIP or LAP overexpression on CDK2 activity (Fig. 3A). In the advLacZ control group, maximum CDK2 activation was first detected 36 hours after PH, and at the 48-hour time point a minor reduction in CDK2 activity was found. In the advLIP group, the maximum CDK2 activity was also seen 36 hours after PH with an intensity comparable to the control group. However, 48 hours after PH, CDK2 activity was significantly reduced, suggesting that peak activity was slightly shifted to an earlier time point. In contrast, at 36 hours after PH no CDK2 activity was detected in the advLAP group, whereas 48 hours after PH a signal of a similar intensity as seen at 36 hours in both other groups was found. Thus, LIP overexpression concentrates CDK2 activation to an earlier time point after PH, whereas LAP expression results in a clear delay of CDK2 activation after PH.

Figure 3.

Differences in CDK2 activity and expression in advLacZ control, advLIP, and advLAP mice. (A) A total of 300 μg of protein extracts from the different mice harvested at time points from 0–48 hours after PH were subjected to immunoprecipitation using an anti-CDK2 antibody. Immune complex kinase assays were performed. The position of the labeled substrate Histone H1 is indicated. (B) One hundred micrograms of protein extracts were loaded and subjected to Western blot analysis using the same CDK2 antibody. The membrane was stripped and reincubated with an antibody directed against α tubulin to ensure equal loading (bottom panel). PH, partial hepatectomy; LIP, liver-enriched inhibitory protein; LAP, liver-enriched activator protein; CDK2, cyclin-dependent kinase 2.

We also tested for an influence of the viral constructs on the expression levels of CDK2 (Fig. 3B). Whereas no difference was detected between the advLIP group and the control group, infection with advLAP resulted in lower CDK2 levels at 24 hours and 36 hours after PH. Therefore, besides the effect on the activation of the CDK2 complex, LAP inhibits the up-regulation of CDK2 at the G1/S phase transition.

Influence of LIP/LAP Expression on Key Cell Cycle Markers After PH.

To examine the effect of LIP or LAP on early steps of cell cycle progression, we performed Western blot analysis of key proteins that are involved in cell cycle progression. First, expression of PCNA, which is an essential component for eukaryotic chromosomal DNA replication,28 was examined.

In the advLacZ control group, only low levels of PCNA were seen during early time points after PH, but expression increased strongly at 36 hours and 48 hours after PH (Fig. 4A). Animals infected with advLIP showed stronger PCNA expression during early time points with a gradual increase until 48 hours, but maximum intensity was less pronounced at 36 hours and 48 hours compared with control. In the advLAP group, PCNA expression was similar during early time points after PH, but the maximum intensity at 36 hours and 48 hours was lower than in both other groups (see Fig. 4A).

Figure 4.

Expression of key cell cycle markers is influenced by LIP/LAP overexpression. Nuclear protein extracts were gained at the indicated time points from livers of mice infected with the advLacZ, advLIP, and advLAP viruses. Six micrograms of nuclear proteins were loaded onto an SDS gel and subjected to Western blot analysis. The respective antibodies were used to examine expression of (A) PCNA, (B) cyclin A, and (C) cyclin E. The intensities of the signals were quantified using phosphoimager analysis and corrected against the respective loading control. (D) The relative intensity is indicated compared with the 36 hours time point in the advLacZ group, which is set as 100%. PH, partial hepatectomy; PCNA, proliferating cell nuclear antigen; LIP, liver-enriched inhibitory protein; LAP, liver-enriched activator protein.

Expression of cyclin A, the key cyclin assembling with CDK2 during S phase, showed kinetics similar to PCNA (Fig. 4B). In the control group, cyclin A was maximally expressed at 36 hours and slightly lower at 48 hours after PH. Overexpression of LIP led to a slight basal signal of cyclin A even before PH and a strong gradual increase until 48 hours. In the advLAP group, cyclin A expression could be detected at a low level 24 hours after PH, but no strong increase at 36 hours as seen in the control group could be detected.

Cyclin E was strongly induced between 24 hours and 48 hours in the control group (Fig. 4C). AdvLIP infection before surgery resulted in a strong basal induction of cyclin E and a gradual increase after PH. In contrast, cyclin E expression was significantly reduced in the advLAP group. Equal loading was ensured by Coomassie-Blue staining (Fig. 4D). Thus, LIP-overexpression led to a stronger basal expression of PCNA, cyclin A, and cyclin E and a gradual increase of these proteins after PH, while overexpression of LAP instead resulted in a reduction in their expression.

Influence of LIP and LAP on p21WAF1/Cip1 expression.

We next were interested to identify other cofactors that might mediate the modulating effect of C/EBP-β isoforms on proliferation. Previous studies have suggested a role of C/EBP family members in the transcriptional control of the cell cycle inhibitor p21WAF1/Cip1.29–31 We performed a p21 Western blot analysis (Fig. 5A). In the advLacZ control group and the advLIP group, a slight p21 signal could be detected even before PH, which gradually increased until 48 hours after PH. In the advLAP group, there was no basic p21 expression detectable. Instead, a signal was only detected 24 hours after PH, and the maximum intensity at 48 hours was lower than in both other groups. Thus, the delay in cell cycle progression in the advLAP group is not mediated by up-regulation of p21, but instead is accompanied by a delayed and weaker p21 induction.

Figure 5.

Modulation of p21Cip1/Waf1/C/EBP-β expression and LAP phosphorylation in response to LIP and LAP overexpression. Whole cell protein extracts were gained at the indicated time points from livers of mice that had been infected with the advLacZ, advLIP, and advLAP virus. 100μg of protein extracts were loaded onto an SDS gel and subjected to Western blot analysis using antibodies specific for (A) p21Cip1/Waf1, (B) C/EBP-α, and (C) phospho-C/EBP-β. In (A) and (B), the intensities of the signals were quantified via phosphoimager analysis and corrected against the respective loading control (α tubulin, lower panel). The relative intensity is indicated compared with (A) the 36 hours time point or (B) the 0 hours time point in the advLacZ group, which are set as 100%. PH, partial hepatectomy; PCNA, proliferating cell nuclear antigen; LIP, liver-enriched inhibitory protein; LAP, liver-enriched activator protein; C/EBP-β, CCAAT enhancer-binding protein β.

Changes in the Expression of C/EBP-α Isoforms and LAP Phosphorylation.

C/EBP-α is a known inhibitor of cell growth in some systems,32 and previous studies have suggested an influence of C/EBP-β isoforms on C/EBP-α expression.33 We therefore performed Western Blot analysis to examine C/EBP-α expression around the G1/S phase boundary (Fig. 5B). As described for C/EBP-β, the translation of the C/EBP-α messenger RNA can result in different protein isoforms depending on the five AUG transcription starts.34 In all groups, 2 specific bands could be detected at 43 kd and 30 kd, reflecting the two most prominent C/EBP-α isoforms. In the advLacZ control group, a gradual down-regulation at 24 and 36 hours after PH of both isoforms could be detected. In the advLIP group, the pattern of regulation was the same, but a stronger signal of both isoforms was detected at all time points compared with control. In contrast, in the advLAP group the signal for the 43-kd isoform was comparable to the control group, whereas the amount of the 30-kd isoform was higher than in the control group. Thus, overexpression of LIP results in an up-regulation of both C/EBP-α isoforms, whereas LAP overexpression results in an attenuation of the C/EBP-α-30/C/EBP-α ratio.

It has been shown previously that phosphorylation of LAP at specific residues is an essential step in its transcriptional activation.35, 36 To exclude that the observed effect of LAP expression on cell cycle progression might be caused by a lack of phosphorylation of external LAP, we performed Western blot analysis with a specific antibody detecting phosphorylated LAP isoforms (Fig. 5C). Compared with the control and the advLIP group, a strong signal for phoshorylated LAP could be detected in the advLAP group. Therefore, LAP expressed via adenoviral gene transfer is efficiently phosphorylated by intracellular kinases.

Effect of LAP and LIP on c-jun Expression.

Phosphorylation of c-jun by upstream kinases is important in mediating signals that regulate proliferation and cell death.37 In Fig. 6A, the expression of c-jun was examined. In the control group, c-jun was minimally induced at 24 hours and declined below baseline level at 36 hours after PH. In the advLIP group, basal c-jun level was equal to the control group, but the induction at 24 hours was stronger. In contrast, almost no c-jun was detectable before PH in the advLAP group, and the induction at 24 hours and 36 hours after PH was slightly reduced compared with the control group.

Figure 6.

Modulation of c-jun expression by LIP and LAP. Whole cell protein extracts were gained at the indicated time points from livers of mice that had been infected with the advLacZ, advLIP, and advLAP virus. (A) One hundred micrograms of protein extracts were loaded onto an SDS gel and subjected to Western blot analysis using an antibody specific for c-jun. The intensities of the signals were quantified via phosphoimager analysis and corrected against the loading control (α tubulin, lower panel). The relative intensity is indicated compared with the 0 hours time point in the advLacZ group, which is set as 100%. (B) Immunohistochemical stainings on liver sections from mice from the advLacZ, advLIP, and advLAP groups at 36 hours after PH. Immunofluorescence for c-jun (top panel) and 4′,6-diamidino-2-phenylindole counterstaining (lower panel). PH, partial hepatectomy; LIP, liver-enriched inhibitory protein; LAP, liver-enriched activator protein; DAPI, 4′,6-diamidino-2-phenylindole.

To confirm up-regulation of c-jun by advLIP, we analyzed c-jun expression via immunohistochemistry (Fig. 6B). At 36 hours after PH, c-jun was detectable in the nuclei of animals infected with advLIP, whereas almost no signal could be detected in the adcLacZ and advLAP group. Thus, LIP expression emphasizes c-jun expression after PH, while LAP overexpression decreases basal c-jun expression in the liver.

Discussion

In our study, we show that LAP—in addition to its regulatory function on metabolic events after PH—also withholds a role as a negative regulator of cell cycle progression of hepatocytes in the model of liver regeneration after PH. This effect is mediated through decreased expression of cyclins A and E, PCNA, and CDK2 and subsequently delays CDK2 activation. In contrast, LIP overexpression leads to an earlier expression of the respective cyclin. In fact, advLIP-treated mice already had elevated baseline levels of the respective cell cycle markers, a finding that supports the activating function of LIP on hepatocyte proliferation.

This finding is in line with previous reports that suggest a negative effect of LAP on cell proliferation in various systems. In intestinal epithelial cells, overexpression of LAP has resulted in an inhibition of cell growth.38 In the liver during postnatal development, LAP expression is minimal when hepatocyte proliferation occurs, and actively dividing cells specifically exclude the expression of LAP. Expression of LAP arrests the cell cycle before the G1/S boundary in hepatoma cells.24 Our data indicate that this effect is not only observed in a tumor cell line but is also relevant in vivo during liver regeneration.

In contrast, other studies have demonstrated an essential function of LAP in mediating proproliferative signals. Mice with a targeted deletion of C/EBP-β are resistant to the induction of skin tumors by Ras, suggesting that C/EBP-β is indispensable for the stimulation of cell proliferation by this mitogen-activated protein kinase pathway.39 Moreover, mice lacking the C/EBP-β gene show a reduced regenerative response after PH. Interestingly, these animals showed a normal expression of cyclin D1, but reduced expression of cyclin E—and to some extent cyclin A—at the G1/S boundary.18 In our study, we enhanced LAP expression as an alternative approach to the knockout studies. However, we also found down-regulation of the respective cyclins in our mice. Moreover, impaired LAP phosphorylation could be excluded as a major reason for this observation. Therefore, LAP withholds a dual pro- and antiproliferative role in the regenerating liver that seems to be dose-dependent. Although lack of as well as high expression of LAP inhibit hepatocyte proliferation, physiological doses are required for cell proliferation. This is in line with our earlier studies, in which an increase in LAP expression was found in G1, while a dramatic reduction was found exactly at the G1/S phase boundary after PH.7

LIP, the shorter translation product of the C/EBP-β gene initiated at the third AUG codon, behaves as a LAP transcription antagonist because it shares with LAP the DNA-binding and leucine zipper domains but lacks the transactivation domain.13 After PH, there is an increase in the nuclear expression of LAP and LIP, underlining a probable physiological function of both isoforms in this system.20 In our study, we demonstrate that overexpression of LIP leads to increased expression of cyclin A and cyclin E at early time points after PH but does not have a major impact on the rate of proliferation. Thus, a physiological function of LAP, which is up-regulated soon after PH,20 might be the inhibition of an early cyclin A and E expression prior to the G1/S boundary (e.g., to support the hepatocytes' synchronization of cell cycle progression after PH). This function is specifically mediated by its transactivation domain and not the basic and bZIP domain, which are conserved between LAP and LIP.

We intended to identify additional factors that might mediate the effect of LAP/LIP expression on the cell cycle machinery. From the known CDK inhibitors, p21Cip1/Waf1 has the most prominent effect on cell cycle progression after PH.4, 5 Moreover, C/EBP-α and C/EBP-β can inhibit proliferation in adipocytes and colorectal cancer cells, respectively, through a p21-dependent mechanism.29, 32 We demonstrated that the inhibitory effect of LAP at the G1/S boundary is not accompanied by a p21 up-regulation. In contrast, the p21 level was lower in the livers of advLAP mice. This lower p21 level is probably not directly caused by LAP but instead might reflect the dependence of p21 expression on the strength of the proliferative stimulus, underlining the role of p21 as a cell cycle inhibitor acting in a negative feedback loop in the regenerating liver.4

Another way that LAP and LIP could mediate their influence on proliferation might be a modulation of C/EBP-α expression. LIP overexpression led to a stronger expression of C/EBP-α in the regenerating liver. Because LIP animals had an increased expression of proteins that trigger G1/S phase transition, enhanced C/EBP-α expression was unexpected, because C/EBP-α has been characterized as an inhibitor of hepatocyte proliferation.32 However, we found alterations in the ratio of C/EBP-α isoforms, which may change their pro- and/or antiproliferative properties. Animals infected with the advLAP construct showed a shift of the proportion of C/EBP-α isoforms toward the shorter 30-kd form. Interestingly, in adipocytes the longer C/EBP-α wt form and the C/EBP-α-30 form fulfill an antagonistic function on proliferation and differentiation. Moreover, the ratio of C/EBP-α/C/EBP-α-30 changes during liver development,40 suggesting a possible modulating effect of the C/EBP-α/C/EBP-α-30 ratio on hepatocyte proliferation.

Recent results have demonstrated that c-jun expression is crucial in mediating hepatocyte proliferation, because c-jun conditional knockout animals have a severe phenotype in hepatocyte proliferation.41 Additionally, previous studies in HepG2 cells have suggested that LAP might indirectly inhibit c-jun expression.24 We show that LIP and LAP have an antagonistic function on c-jun and its expression after PH. Reduced c-jun expression was found in advLAP-infected mice compared with advLIP- and advLacZ-infected mice before PH. At later time points, advLAP-infected animals increase expression of c-jun. However, this increase was lower compared with advLIP-infected mice and slightly reduced compared with advLacZ-infected mice. Therefore c-jun could be one of the target genes and/or proteins that are modulated through LAP and LIP and that might mediate the effect on cell cycle progression after PH.

Taken together, our results show that overexpression of LAP results in decreased hepatocyte proliferation at the G1/S boundary after PH. This effect of LAP is mediated through its transactivation domain. The mechanisms on hepatocyte proliferation that mediate the effect likely involve the interaction with other C/EBP proteins—especially C/EBP-α—and the modulation of c-jun expression. Therefore, our results show that the fine tuning of LAP expression after PH is essential to mediate the timing of hepatocyte proliferation and represents a potential therapeutic target of intervention.

Ancillary