Switch from Mnt-Max to Myc-Max induces p53 and cyclin D1 expression and apoptosis during cholestasis in mouse and human hepatocytes

Authors

  • Heping Yang,

    Corresponding author
    1. Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA
    • Division of Gastrointestinal and Liver Diseases, HMR Building 414, Department of Medicine, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90033
    Search for more papers by this author
    • fax: 323-442-3234

  • Tony W. H. Li,

    1. Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA
    Search for more papers by this author
  • Kwang Suk Ko,

    1. Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA
    Search for more papers by this author
  • Meng Xia,

    1. Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA
    Search for more papers by this author
  • Shelly C. Lu

    Corresponding author
    1. Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA
    • Division of Gastrointestinal and Liver Diseases, HMR Building 415, Department of Medicine, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90033
    Search for more papers by this author
    • fax: 323-442-3234


  • Potential conflict of interest: Nothing to report.

Abstract

Toxic bile acids induce hepatocyte apoptosis, for which p53 and cyclin D1 have been implicated as underlying mediators. Both p53 and cyclin D1 are targets of c-Myc, which is also up-regulated in cholestasis. Myc and Mnt use Max as a cofactor for DNA binding. Myc-Max typically activates transcription via E-box binding. Mnt-Max also binds the E-box sequence but serves as a repressor and inhibits the enhancer activity of Myc-Max. The current work tested the hypothesis that the switch from Mnt-Max to Myc-Max is responsible for p53 and cyclin D1 up-regulation and apoptosis during cholestasis. Following common bile duct ligation or left hepatic bile duct ligation, the expression of p53, c-Myc, and cyclin D1 increased markedly, whereas Mnt expression decreased. Nuclear binding activity of Myc to the E-box element of p53 and cyclin D1 increased, whereas that of Mnt decreased in a time-dependent fashion. Lithocholic acid (LCA) treatment of primary human hepatocytes and HuH-7 cells induced a similar switch from Mnt to Myc and increased p53 and cyclin D1 promoter activity and endogenous p53 and cyclin D1 expression and apoptosis. Blocking c-Myc induction in HuH-7 cells prevented the LCA-mediated increase in p53 and cyclin D1 expression and reduced apoptosis. Lowering Mnt expression further enhanced LCA's inductive effect on p53 and cyclin D1. Bile duct–ligated mice treated with a lentivirus harboring c-myc small interfering RNA were protected from hepatic induction of p53 and cyclin D1, a switch from Mnt to Myc nuclear binding to E-box, and hepatocyte apoptosis. Conclusion: The switch from Mnt to Myc during bile duct ligation and in hepatocytes treated with LCA is responsible for the induction in p53 and cyclin D1 expression and contributes to apoptosis. (HEPATOLOGY 2008.)

Retention of toxic bile acids is the main feature in a variety of cholestatic liver diseases, including biliary atresia, primary sclerosing cholangitis, and primary biliary cirrhosis.1 Cholestasis contributes to hepatocellular injury, progressive fibrosis, cirrhosis, and death from liver failure.2 Thus, the mechanisms by which toxic bile acids modulate liver damage in cholestasis are of major interest.

c-Myc is a basic helix-loop-helix-leucine zipper transcription factor that binds to E-box sequences as part of a heterodimeric complex with another basic helix-loop-helix-leucine zipper protein, Max, to activate transcription.3 c-Myc is commonly known to stimulate cell proliferation, but it has also been shown to sensitize cells to apoptosis.4 However, the mechanism by which c-Myc does this remains unclear. Because c-Myc is a transcriptional factor, it has been suggested that c-Myc may induce apoptosis by affecting other genes, such as p53.5 The promoter of the p53 gene has been noted to contain an E-box resembling the c-Myc binding site and can be directly transactivated by c-Myc/Max heterodimers.6 Aberrant expression of p53 is associated with hepatocyte apoptosis in cholestasis.7 c-Myc also can either positively or negatively regulate the expression of cyclin D1.8 Although cyclin D1 promotes cell growth, overexpression of cyclin D1 can lead to premature G1-S phase transition and cause serum-starved cells to go into apoptosis.9 In addition, cyclin D1 overexpression has been shown to increase toxic bile acid–induced Bax translocation, cytochrome c release, and apoptosis of primary hepatocytes.10

Mnt was identified as a Max-interacting transcriptional repressor.11 Deletion of Mnt can predispose a cell to apoptosis.12 Induction of c-Myc during cell cycle entry results in a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc–Max on shared target genes. Indeed, the ratio of Mnt-Max to c-Myc–Max modulates cell cycle entry.11 This ratio may also be important for the binding to the E-box sequences at target genes such as p53 and cyclin D1, leading to their altered expression and contributing to hepatocyte apoptosis induced by toxic bile acids.

On the basis of the functions of Myc-Max and Mnt-Max, we hypothesized that the altered ratio of Mnt-Max to Myc-Max may be an important determinant for cholestasis-induced apoptosis. Using a combination of in vivo and in vitro models, we provide evidence that the switch from Myc-Max to Mnt-Max is largely responsible for the induction of p53 and cyclin D1 expression and cell death during cholestasis, and we suggest that targeting this pathway may be an effective therapeutic strategy against cholestatic liver injury.

Abbreviations

BDL, bile duct ligation; CBDL, common bile duct ligation; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; H & E, hematoxylin and eosin; LCA, lithocholic acid; LHBDL, left hepatic bile duct ligation; mRNA, messenger RNA; SEM, standard error of the mean; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling.

Materials and Methods

Materials.

Cell culture media and fetal bovine serum were obtained from Mediatech (Herndon, VA) and Omega Scientific (Tarzana, CA), respectively. a-32P-dCTP and γ-32P-ATP (3000 Ci/mmol) was purchased from PerkinElmer (Boston, MA). All other reagents were analytical-grade and were obtained from commercial sources.

Cell Culture.

HuH-7 cells, 293A cells, and mouse hepatocytes were obtained from the Cell Culture Core of the USC Research Center for Liver Diseases. Primary human hepatocytes were obtained in a suspension culture in a cold preservation medium (24 hours after the livers were harvested) from CellzDirect (Pittsboro, NC). Cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin-streptomycin. Mouse hepatocytes were centrifuged and purified through Percoll as described.13 Hepatocyte viability was detected by trypan blue exclusion and was 75 to 95% in common bile duct ligation (CBDL) mice.

CBDL and Left Hepatic Bile Duct Ligation (LHBDL) in Mice.

The use and care of the animals were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Southern California. Three-month-old male C57/B6 mice were subjected to CBDL as described14 and sacrificed on day 0, 1, 3, 7, 10, 14, 21, or 28 post-surgery. In separate experiments, the left hepatic bile duct was selectively ligated immediately before the common bile duct was entered. The livers were excised 3 days post-operation. Liver tissue samples were obtained from CBDL mice and ligated left lobes and nonligated right lobes of LHBDL mice and were processed for the various studies described next.

Necrosis and Apoptosis Determination.

Replicate 4-μm-thick sections of formalin-fixed liver tissues embedded in paraffin were cut and stained with hematoxylin and eosin for the evaluation of necrosis. The percentage of necrosis was estimated in these sections by the evaluation of the number of microscopic fields with necrosis in comparison with the entire histological section. Fifteen fields were examined at 100× magnification. Tissue sections were stained with terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) according to the manufacturer's suggested protocol (in situ cell death detection kit, Roche). Five random fields containing an average of 250 nuclei were counted for each TUNEL-stained tissue sample. The apoptotic index (percentage of apoptotic nuclei) of hepatocytes was calculated as follows: (Apoptotic nuclei/Total nuclei) × 100%. Samples from at least three independent experiments were scored. Primary human hepatocytes and HuH-7 apoptotic cells were assessed by Hoechst staining as previously described.15 All histological evaluations were done in a blinded fashion.

Northern Blot of Liver Tissues and Hepatocytes Isolated from Bile Duct Ligation (BDL) Mice.

Total RNA was isolated with the TRIzol reagent (Invitrogen). Northern blot analysis was done as previous described.15 Specific c-myc, p53, cyclin D1, Mnt, Max, and β-actin complementary DNA probes (see Supporting Table 1) were labeled with [32P]dCTP with a DECAprime II kit (Ambion, Austin, TX). Autoradiography and densitometry were used to quantitate RNA expression as we described.15 Results of Northern blot analyses were normalized to β-actin.

Table 1. Hepatic Apoptosis and Necrosis During Common Bile Duct Ligation (%)
 ShamDay 1Day 3Day 7Day 10Day 14Day 21Day 28
  • Results are expressed as the mean ± standard deviation from four mice for each group. Necrosis and apoptosis were determined as described in the Materials and Methods section.

  • *

    P < 0.05 versus the sham group.

Necrosis05 ± 1*15 ± 3*19 ± 5*21 ± 4*27 ± 6*31 ± 5*34 ± 6*
Apoptosis0.8 ± 0.213.5 ± 3.1*35.9 ± 4.7*30.3 ± 5.0*27.6 ± 6.3*21.5 ± 5.7*16.0 ± 4.0*14.7 ± 4.6*

Western Blot Analysis of Liver Tissues and Hepatocytes from BDL Mice.

Western blot analyses were done as previously described.15 Membranes were probed with antibodies to c-Myc, Mnt, Max, p53, or cyclin D1 (Novus Biologicals, Littleton, CO). To ensure equal loading, membranes were stripped and reprobed with anti-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

Small Interfering RNA (siRNA) Transfection and Lithocholic Acid (LCA) Treatment.

Double-stranded c-myc siRNA and scrambled siRNA were purchased from Ambion (Silencer c-Myc siRNA control, catalog number 4604). SignalSilence p53 siRNA (human) was purchased from Cell Signaling Technology (catalog number 6231), and Smartpool cyclin D1 siRNA was obtained from Millipore Corp. (catalog number M-003210). Mnt siRNA was obtained from Santa Cruz (catalog number sc-38083). HuH-7 cells were transfected with c-myc, p53, cyclin D1, Mnt, or scrambled siRNA (10 nM per 1 × 105 cells) with the Lipofectamine RNAiMAX transfection reagent (Invitrogen) in six-well plates at 30% confluence for 0, 8, 16, 24, or 48 hours and then treated with 100 μM LCA (Sigma) for 8 hours for the evaluation of knockdown efficiency. For LCA-mediated cell apoptosis, HuH-7 cells were cultured in six-well plates and treated with siRNA for 24 hours and then with LCA for another 24 hours. In experiments looking at the effect of c-myc, p53, and cyclin D1 siRNAs on LCA-mediated promoter activities, HuH-7 cells were treated with c-myc siRNA or scrambled siRNA for 24 hours and then transfected with either the p53 or cyclin D1 promoter constructs for another 10 hours. LCA was added during the last 8 hours of the p53 or cyclin D1 promoter transfection.

Construction of Lentivirus Vectors and Gene Delivery In Vivo.

The empty lentivirus vector pLEN (H1GFP) was a gift from Dr. Stuart A. Berger (University Health Network). Mouse c-myc siRNA (target sequence: 5′-GAACATCATCATCCAGGAC-3′)16 was synthesized from the USC Norris Comprehensive Cancer Center DNA Core Facility. The annealed mixture was ligated into pLEN vector that had been digested with PacI and XbaI. Lentiviral vectors containing human c-myc short hairpin RNA were produced in 293A cells.17 In brief, the pLEN plasmid vector (10 μg) was mixed with the accessory plasmids VSVG (3.5 μg), pRRE (6.5 μg), and pREV (2.5 μg) and transfected into 293A cells with SuperFect (Qiagen). The culture medium was replaced with fresh Iscove's minimal essential medium containing 10% fetal bovine serum at 24 hours, and the virus was harvested 48 hours post-transfection. A total of 1 × 105 HuH-7 cells were infected at a multiplicity of 20 plaque-forming units/cell for 24 hours. Transducing units (1×109; final volume: 0.1 mL) were injected into the spleens of CBDL, sham, and LHBDL operated mice immediately after the BDL was performed under the same anesthesia.

P53 and Cyclin D1 Promoter and E-Box Mutational Analysis.

The p53 promoter and the 3.3-kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, MD) and Dr. R. G. Pestell (Thomas Jefferson University, Philadelphia, PA), respectively. The E-box element of p53 [5′-(CTCCCATGTGCTCA)4-3′], its mutant [5′-(CTCCAATGTTCTCA)4-3′], cyclin D1 [5′-(TTTACACGTGTTGA)4-3′], and its mutant [5′-(TTTAAACGTTTTGA)4-3′] were cloned into the HindIII and Bgl II sites of pLuc-MCS vector (Stratagene, Cedar Creek, TX). HuH-7 cells were transfected with Superfect (Qiagen, Texas), the media were changed 2 hours post-transfection, and LCA (100 μM) was added. Luciferase activity was determined 8 hours post–LCA treatment. For siRNA treatment and promoter cotransfection, HuH-7 cells were transfected with c-myc siRNA for 24 hours and then transfected with p53 or cyclin D1 promoters or native or mutated E-box elements of these promoters for an additional 2 hours. The medium was changed and treated with 100 μM LCA or vehicle for 8 hours.

Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay.

EMSAs were done as described previously.18 The probes were32P-end-labeled double-stranded p53 DNA fragments (−20 to −40 of the human E-box region and +82 to +62 of the mouse E-box region) and cyclin D1 DNA fragments (−446 to −467 of the human E-box region and −425 to −446 of the mouse E-box region). Supershift assays were done with antibodies for c-Myc or Mnt (Biotechnology, Lake Placid, NY, or Santa Cruz Biotechnology) as we described.18

Statistical Analysis.

Data are given as the mean ± standard error of the mean. Statistical analysis was performed with analysis of variance followed by Fisher's test for multiple comparisons. For changes in messenger RNA (mRNA) and protein levels, ratios of c-Myc, p53, cyclin D1, and Mnt to actin densitometric values were compared by analysis of variance. Significance was defined by P < 0.05.

Results

CBDL-Mediated and LHBDL-Mediated Necrosis and Apoptosis.

Following CBDL, both necrosis (Fig. 1A) and apoptosis (Fig. 1B) occurred. However, although apoptosis peaked around day 3 following CBDL, necrotic areas continued to expand with time (see Fig. 1 and Table 1 for quantitation).

Figure 1.

(A) Representative liver sections stained with H&E from CBDL and sham-operated mice from day 1 to day 28. The liver was histologically normal in sham-operated mice. Liver tissues from day 1 onward had increased necrosis in the CBDL group. Stars indicate areas of hepatic necrosis (H&E staining, 200×). (B) Representative liver sections stained with TUNEL from CBDL and sham-operated mice from day 1 to day 28. Cells negative for apoptosis exhibited no nuclear staining, whereas apoptotic cells exhibited brown nuclear staining. Hepatocyte apoptosis was not detected in sham-operated mice. Apoptotic cells were observed on day 1, peaked at day 3, maintained a persistently high level of apoptosis up to day 10, and decreased from day 14 to day 28 (TUNEL staining, 200×). Abbreviations: CBDL, common bile duct ligation; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling.

Expression of c-Myc, Mnt, Max, p53, and Cyclin D1 in Liver Tissues and Hepatocytes During CBDL.

We first examined changes in expression of c-Myc, Mnt, Max, p53, and cyclin D1 during CBDL. Hepatic protein levels of c-Myc, p53, and cyclin D1 increased by day 1 after CBDL, peaked around day 3, and remained elevated up to day 28 (Fig. 2A). On the other hand, the Mnt protein level fell quickly and remained at about 50% of baseline from day 3 onward. Max expression was unchanged. These changes were largely due to a change in the mRNA levels (Fig. 2B). Hepatocytes isolated from CBDL and sham-operated mice exhibited similar changes (Fig. 2C; see Supporting Fig. 1 for densitometry). Thus, expression of genes in the whole liver largely reflects hepatocyte expression.

Figure 2.

(A) Effect of CBDL on the protein levels of c-Myc, Mnt, Max, p53, and cyclin D1 in liver tissues. Protein samples from liver tissues of CBDL mice from 0 to 28 days were analyzed by Western blot analysis. To ensure equal loading, membranes were stripped and reprobed with anti-actin antibodies. Total protein (15 μg/lane) was loaded for protein expression analysis. Representative blots from at least three mice for each time point are shown, and densitometric values are shown in Supporting Fig. 1A. (B) Effects of CBDL on mRNA levels of c-myc, Mnt, Max, p53, and cyclin D1 in liver tissues. RNA was isolated from liver tissues of CBDL mice from day 0 to day 28 for Northern blot analysis (15 μg/lane). Membranes were stripped and reprobed with b-actin to ensure equal loading. Representative blots from at least three mice for each time point are shown, and densitometric values are shown in Supporting Fig. 1B. (C) Effects of CBDL on mRNA levels of c-myc, Mnt, Max, p53, and cyclin D1 in hepatocytes isolated from CBDL mice from day 0 to 28 post-operation. Representative blots from at least three mice for each time point are shown, and densitometric values are shown in Supporting Fig. 1C. Abbreviations: CBDL, common bile duct ligation; mRNA, messenger RNA.

Switch of Nuclear Binding Activity to E-Box from Mnt-Max to Myc-Max During CBDL.

The ability of c-Myc to potentiate apoptosis is well documented.4 In contrast, the role of Mnt in apoptosis during cholestasis is unknown. EMSAs were performed to test the binding activity of c-Myc and Mnt to the E-box elements of p53 and cyclin D1. The E-box sequences of mouse and human cyclin D1 and p53 are highly conserved (Fig. 3A). Liver nuclear protein extracts from different stages of CBDL mice shifted two major bands using probes to the E-box region of either p53 or cyclin D1. The top band decreased whereas the bottom band increased in intensity from day 0 to day 14 (Fig. 3B). Supershift assays using c-Myc antibody generated a strong supershift band derived from the lower band, whereas Mnt antibody supershifted the top band (Fig. 3C).

Figure 3.

(A) E-box sequences of mouse and human cyclin D1 and p53. The E-box sequence of mouse and human cyclin D1 is completely conserved, and the E-box sequence of mouse p53 has only one mismatch in comparison with the human p53. (B) EMSA analysis using the mouse p53 E-box element showed that the top band (Mnt) was decreased and the bottom band (c-Myc) was increased from day 0 to day 14 after CBDL. (C) Supershift analysis using the mouse p53 E-box element identified bound proteins to include c-Myc and Mnt. The c-Myc antibody generated a strong supershift band derived from the lower band; whereas the Mnt antibody supershifted the top band. Similar results were obtained with the mouse cyclin D1 E-box element (not shown). Abbreviations: CBDL, common bile duct ligation; EMSA, electrophoretic mobility shift assay.

Effect of LCA on Expression of c-Myc, p53, Cyclin D1, and Mnt in Primary Human Hepatocytes and HuH-7.

LCA is the most toxic bile acid with genotoxic and mutagenesis-enhancing properties.19 In rodents, LCA leads to intrahepatic cholestasis-like hepatotoxicity and apoptosis of hepatocytes.20 To ascertain whether LCA-mediated hepatocyte apoptosis is associated with aberrant expression of c-Myc, p53, cyclin D1, and Mnt, HuH-7 cells and primary human hepatocytes were treated with LCA. LCA increased the protein expression of c-Myc, p53, and cyclin D1 in a time-dependent manner, whereas Mnt protein expression decreased in both HuH-7 cells (Fig. 4A) and primary human hepatocytes (Fig. 4B; see Supporting Fig. 2 for densitometry). In HuH-7 cells after 24 hours of treatment, 50μM LCA induced apoptosis, whereas 25μM LCA induced gene expression changes (Fig. 2C,D). Compared to HuH-7 cells, 100μM LCA induced apoptosis in primary human hepatocytes at an earlier time point (Fig. 4E).

Figure 4.

(A) Western blot analysis of c-Myc, Mnt, Max, p53, and cyclin D1 in LCA-treated (100 μM) HuH-7 cells from 1 to 48 hours. To ensure equal loading, membranes were stripped and reprobed with anti-actin antibodies. Total protein (15 μg/lane) was used for the assay. Representative blots from at least three independent experiments for each time point are shown, and densitometric values are shown in Supporting Fig. 2A. (B) Effects of LCA on protein levels of c-Myc, Mnt, p53, and cyclin D1 in primary human hepatocytes treated for various times. Total protein (15 μg/lane) was subjected to Western blot analysis. Membranes were stripped and reprobed with actin for housekeeping control. Representative blots from at least three independent experiments for each time point are shown, and densitometric values are shown in Supporting Fig. 2B. (C) Dose response of LCA on apoptosis in HuH-7 cells. HuH-7 cells were treated with LCA (0-125 μM) for 24 hours, and apoptosis was assessed as described in the Materials and Methods section. Results represent the mean ± SEM from three experiments. *P < 0.05, **P < 0.005 versus 0. (D) Dose response of LCA on p53, cyclin D1, c-Myc, and Mnt protein levels in HuH-7 cells. HuH-7 cells were treated with LCA (0-100 μM) for 24 hours, and protein levels were determined by Western blot analysis as described previously. (E) Effect of LCA on HuH-7 and primary human hepatocyte apoptosis. After LCA treatment (100 μM), apoptosis increased from 24 to 48 hours in HuH-7 and from 12 to 36 hours in primary hepatocytes. Results represent the mean ± SEM from three independent experiments. *P < 0.05 versus respective controls for the different time points. Abbreviations: LCA, lithocholic acid; SEM, standard error of the mean.

Effect of In Vitro siRNA Knockdown and LCA-Mediated Gene Expression and Apoptosis in HuH-7 Cells.

To test whether c-Myc, p53, and cyclin D1 up-regulation is responsible for LCA-induced apoptosis, an siRNA strategy was used to prevent the increase in expression of c-Myc, p53, and cyclin D1 in HuH-7 cells treated with LCA. The siRNA knockdown efficiency of c-myc, p53, and cyclin D1 mRNA expression was 86%, 88%, and 82% in HuH-7 cells after 36 hours of transfection, respectively (Fig. 5A). To see if knockdown of c-Myc, p53, or cyclin D1 can prevent LCA-mediated induction of these genes, HuH-7 cells were transfected with siRNA for these genes for various time periods and then treated with LCA for 8 hours. Figure 5B shows that there is a time-dependent decrease in the protein levels of c-Myc, p53, and cyclin D1 following siRNA treatment of each respective gene, and LCA was unable to overcome this inhibitory effect. Although the decrease in protein level was maximum for each respective siRNA (70% for c-Myc, 65% for p53, and 75% for cyclin D1 at 48 hours), cells treated with c-myc siRNA also had a nearly 50% fall in p53 and a 40% fall in cyclin D1, which supported an important role of c-Myc in their up-regulation. Cells treated with p53 siRNA had no change in the cyclin D1 protein level but also had a slight fall in the c-Myc level. Cells treated with cyclin D1 siRNA had no effect on c-Myc or p53 protein levels. Scrambled siRNA had no effect on c-Myc, p53, and cyclin D1 expression (data not shown). These siRNA treatments significantly attenuated the LCA-induced apoptosis, with c-Myc siRNA offering the greatest protection and cyclin D1 siRNA offering the least protection (Fig. 5C). c-Myc knockdown in HuH-7 cells led to increased Mnt nuclear binding activity (Fig. 5D). Mnt knockdown led to increased c-Myc nuclear binding activity and p53 and cyclin D1 expression at baseline and further enhanced LCA's inductive effect on c-Myc nuclear binding and the expression of these genes (Fig. 5E,F).

Figure 5.

(A) Efficiency of c-myc, p53, and cyclin D1 knockdown in HuH-7 cells. Cells were treated with siRNA for 36 hours, and messenger RNA levels were determined by Northern blot analysis. Scrambled siRNA had no effect (not shown). (B) Effect of siRNA against c-myc, p53, and cyclin D1 on protein levels of c-Myc, p53, and cyclin D1 in LCA-treated HuH-7 cells. HuH-7 cells were transfected with siRNA for these genes for 0, 8, 16, 24, or 48 hours and then treated with LCA for 8 hours for evaluation of c-Myc, p53, and cyclin D1 knockdown efficiency. Numbers below each blot refer to densitometric values as percentages of 0 hours (LCA treatment alone). (C) Effect of c-myc, p53, and cyclin D1 siRNA on LCA-mediated HuH-7 apoptosis. HuH-7 cells were transfected with siRNA for 24 hours, and this was followed by LCA treatment for another 24 hours. *P < 0.05 versus respective controls. †P < 0.05 versus LCA plus scrambled siRNA. (D) Effect of c-myc siRNA on Mnt and Myc nuclear binding activity to the E box of p53 promoter in HuH-7 cells. HuH-7 cells were treated with c-myc siRNA for 48 hours, and EMSA for p53 promoter E-box binding was performed as described in the Materials and Methods section. For specificity, a 50× cold probe was added. (E) Effect of Mnt knockdown on baseline and LCA-mediated nuclear binding to E-box. HuH-7 cells were treated with Mnt siRNA for 48 hours, and this was followed by LCA (100 μM) for another 8 hours. EMSA for p53 promoter E-box binding activity was performed as previously described. (F) Effect of Mnt knockdown on baseline and LCA-mediated induction of p53 and cyclin D1 protein levels. HuH-7 cells were treated with Mnt siRNA for 48 hours, and this was followed by LCA (100 μM) for another 8 hours; Western blot analysis was performed as described in the Materials and Methods section. Abbreviations: EMSA, electrophoretic mobility shift assay; LCA, lithocholic acid; siRNA, small interfering RNA.

Role of c-Myc and E-Box in the Activity of p53 and Cyclin D1 Promoters.

To see whether LCA-mediated p53 and cyclin D1 induction required c-Myc binding to the E-box elements in the upstream region of the p53 and cyclin D1 genes, we examined the promoter activity of these genes as well as constructs containing either the native E-box element or mutated E-box element derived from these genes. The effect of c-Myc was assessed by the lowering of its expression with siRNA treatment. The luciferase activity of the p53 and cyclin D1 promoter was doubled by LCA treatment in comparison with the control. This activity was repressed by the lowering of c-Myc expression with c-myc siRNA (Fig. 6A). LCA also doubled whereas c-myc siRNA reduced luciferase activity driven by the E-box element alone, but mutations of the E-box element in the p53 and cyclin D1 promoter region completely obliterated the inductive effect of LCA as well as the repressive effect of c-myc siRNA (Fig. 6B).

Figure 6.

Effects of LCA and c-myc siRNA on the promoter activity of p53 and cyclin D1. (A) HuH-7 cells were treated with c-myc siRNA or scrambled siRNA for 24 hours and then transfected with either the p53 or cyclin D1 promoter for another 10 hours. LCA (100 μM) was added during the last 8 hours of the p53 or cyclin D1 promoter transfection. Results represent the mean ± SEM from three independent experiments performed in triplicate. *P < 0.05 versus control or scrambled siRNA. †P < 0.05 versus LCA. (B) The protocol was the same as that in part A, except that the promoter constructs consisted of only the E-box elements of these promoters in native or mutated sequences as described in the Materials and Methods section. Results represent the mean ± SEM from three independent experiments performed in triplicate. *P < 0.05 versus respective control. Abbreviations: LCA, lithocholic acid; SEM, standard error of the mean; siRNA, small interfering RNA.

Effect of In Vivo c-myc siRNA Knockdown on CBDL-Mediated and LHBDL-Mediated Liver Injury, Nuclear Binding Activity to E-Box, and Expression of p53 and Cyclin D1.

To confirm the importance of the switch from Mnt to Myc in the induction of p53 and cyclin D1 as well as cell death, we blocked c-Myc induction in the CBDL and LHBDL mouse models using siRNA. Figure 7A shows that c-myc siRNA treatment decreased the basal expression of c-myc by 47% in liver tissues of sham-operated mice and decreased the c-myc expression in the CBDL group by 57%. Importantly, c-myc siRNA treatment also decreased both basal and CBDL-induced p53 and cyclin D1 mRNA levels. Similarly, c-myc siRNA inhibited basal expression of c-myc, p53, and cyclin D1 in the right lobe of LHBDL mice and largely prevented the increase in LHBDL-induced expression of c-myc, p53, and cyclin D1 in the left lobe (Fig. 7B; see Supporting Fig. 3 for densitometry). To further test our hypothesis that c-Myc knockdown in vivo can interfere with c-Myc–Max binding to the E-box site of p53 and cyclin D1, we isolated nuclear protein from liver tissues of left or right lobes of LHBDL mice on day 3 for EMSA analysis. c-Myc knockdown reduced c-Myc binding and increased Mnt binding in the E-box region of both p53 (Fig. 7C) and cyclin D1 (data not shown) in left lobes of LHBDL mice.

Figure 7.

(A) Northern blot analysis of c-myc, p53, and cyclin D1 in livers of CBDL and sham-operated mice treated with a lentivirus harboring c-myc siRNA or empty vector injection. See Supporting Fig. 3A for densitometric analysis. (B) Northern blot analysis of c-myc, p53, and cyclin D1 in left lobes and right lobes from LHBDL mice treated with a lentivirus harboring c-myc siRNA or empty vector injection. See Supporting Fig. 3B for densitometric analysis. (C) Effect of c-myc siRNA on Myc and Mnt nuclear binding activity in LHBDL mice. Mice were treated with c-myc siRNA or an empty vector followed by LHBDL as described in the Materials and Methods section. EMSA for E-box binding of p53 was performed with nuclear protein extracts from the left and right lobes 3 days later. Abbreviations: CBDL, common bile duct ligation; EMSA, electrophoretic mobility shift assay; LHBDL, left hepatic bile duct ligation; siRNA, small interfering RNA.

In order to determine whether c-Myc knockdown can protect the liver from injury during cholestasis, we examined for necrosis and apoptosis after c-myc siRNA treatment in CBDL and LHBDL mice. At day 3, the liver tissues and hepatocytes were collected and analyzed for infection efficiency. Eighty percent of the hepatocytes were infected, as detected by direct green fluorescent protein fluorescence (Fig. 8A). c-Myc knockdown decreased hepatic necrosis and apoptosis in the entire liver of CBDL mice and the left lobe of LHBDL mice (Fig. 8B,C and Table 2).

Figure 8.

(A) Transduction efficiency of the lentivirus infection. Hepatocytes were isolated on day 3 from mice treated with CBDL, LHBDL (left lobe), or sham operation. Transduction efficiency was measured by positive GFP expression in hepatocytes. The corresponding phase image shows more than 80% of the hepatocytes were infected in sham-operated and bile duct–ligated groups. (B) H&E staining shows c-myc siRNA infection reduced hepatic necrosis in CBDL and LHBDL mice on day 3. The entire liver in sham-operated controls and right lobe in the LHBDL group by c-myc siRNA infection did not exhibit any necrosis. Arrowheads indicate hepatic necrosis (H&E staining, 200×). (C) TUNEL staining (200×) shows that c-myc siRNA infection also inhibited hepatocyte apoptosis in CBDL and LHBDL mice on day 3. The entire liver in sham-operated controls of CBDL and the right lobe of LHBDL mice did not exhibit any apoptosis. Abbreviations: CBDL, common bile duct ligation; GFP, green fluorescent protein; H&E, hematoxylin and eosin; LHBDL, left hepatic bile duct ligation; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling.

Table 2. Effect of c-myc siRNA on Hepatic Apoptosis and Necrosis in CBDL and LHBDL (%)
 ShamCBDLLHBDL Right LobeLHBDL Left Lobe
ControlsiRNAControlsiRNAControlsiRNAControlsiRNA
  • Data are expressed as the mean ± standard deviation from at least three mice per group. Necrosis and apoptosis were determined as described in the Materials and Methods section.

  • Abbreviations: CBDL, common bile duct ligation; LHBDL, left hepatic bile duct ligation; siRNA, small interfering RNA.

  • *

    P < 0.05 versus the sham group in CBDL mice and the right lobes of LHBDL mice.

  • P < 0.05 versus respective controls (empty vector for the CBDL and LHBDL mice).

Necrosis0016.4 ± 5.0*11.3 ± 3.20013.0 ± 3.8*7.2 ± 2.9
Apoptosis0.9 ± 0.11.1 ± 0.437.6 ± 4.5*19.0 ± 3.31.2 ± 0.41.1 ± 0.323.4 ± 4.0*14.34 ± 3.7

Discussion

Toxic bile acids induce apoptosis in vitro and in vivo,21 and Miyoshi et al.22 showed that the Fas signaling pathway is involved in apoptosis during the early time course of BDL. Later, in BDL, both Fas-dependent and Fas-independent pathways participated.22 However, Gujral et al.14 showed that necrosis but not apoptosis occurred during BDL. The explanations for these discrepant results are unclear. Recent work also revealed that Kupffer cells and the innate immune system also participate in liver injury during BDL.23 Clearly, the intense interest in studying the molecular mechanisms of liver injury during cholestasis is to uncover novel therapeutic strategies. Currently, only ursodeoxycholic acid is used in the treatment of chronic cholestatic disorders such as primary biliary cirrhosis, and a key molecular target of ursodeoxycholic acid is p53.24 Because p53 is pro-apoptotic and a downstream target of c-Myc,6 which is also induced during cholestasis,25 we hypothesized that the increase in c-Myc may lead to a switch from Mnt-Max to Myc-Max and induction of pro-apoptotic genes. The aims of this study were to test this hypothesis and to see if p53 and cyclin D1, both downstream targets of c-Myc, which has been implicated in toxic bile acid–induced apoptosis, play any role in liver injury in cholestasis.

CBDL has been used as an animal model of chronic liver injury because it duplicates the retention of toxic bile acids during human cholestatic liver disease. The LHBDL model is used to study prolonged liver fibrogenesis independent of liver failure.27 This model of LHBDL allows a comparison of the injured ligated left lobe and the nonligated right lobe. Using both models, we found that liver injury induced by CBDL and LHBDL up to day 3 occurs in a similar manner by both necrosis and apoptosis. However, although apoptosis peaked early at day 3, necrosis progressed with time. Our results indicate that both forms of liver injury occur following BDL, with apoptosis predominating early on, but necrosis is the main form of liver cell death at later stages of cholestasis.

The c-Myc oncogene, which is induced during BDL,25 functions as a positive regulator of cell proliferation and growth. Paradoxically, c-Myc can also result in the sensitization of cells to apoptosis under conditions of hepatocyte injury.27 Max serves as an obligate heterodimerization partner for Myc, allowing it to bind E-box consensus sequences to activate transcription.11 Max also interacts with the Myc antagonist Mnt.11 Excessive Myc levels result in the sequestration of Max to decrease its availability to other dimerization partners such as Mnt. Decreased Mnt expression increases the basal pool levels of Max available for dimerization with Myc. Both will alter the ratio between Myc-Max and Mnt-Max complexes and disrupt the balance of binding to E boxes and regulation of Myc target genes.11 Although one previous study showed that c-Myc is induced during BDL, whether Mnt expression is altered has not been examined. Our results show that there is a rapid induction of c-Myc expression with a concomitant fall in Mnt expression, both occurring at the mRNA level and in hepatocytes of mice subjected to either CBDL or LHBDL. Consistently, both p53 and cyclin D1, downstream targets of c-Myc, are up-regulated. This switch from Mnt to Myc resulted in a change in E-box binding from Mnt-Max to Myc-Max. We also confirmed that treatment of human primary hepatocytes and HuH-7 cells with the toxic bile acid LCA induced the same changes. Use of both in vitro and in vivo models was intended to facilitate mechanistic studies. The LHBDL model allowed us to dissociate cholestatic liver injury from that due to overwhelming liver failure that can occur in CBDL.

Using the in vitro model system, we demonstrated that LCA induced apoptosis in a time-dependent manner and the promoter activity of both p53 and cyclin D1, which required an intact E-box. To test the hypothesis that this induction in c-Myc with a subsequent increase in p53 and cyclin D1 is responsible for cell death, we employed the siRNA technology. Consistent with the fact that c-Myc regulates p53 and cyclin D1, knockdown of c-Myc resulted in lower expression of both and largely prevented the ability of LCA to induce either p53 or cyclin D1. This conclusively demonstrates that LCA induces c-Myc, which then induces p53 and cyclin D1 via E-box transactivation. Although siRNA knockdown of cyclin D1 had no effect on c-Myc or p53 expression at 48 hours, p53 knockdown was able to also reduce c-Myc expression in LCA-treated HuH-7 cells. HuH-7 cells express a mutant gain of function p53,28 and mutant p53 can up-regulate c-Myc expression29; hence, siRNA knockdown of p53 can reduce the LCA-mediated activation of c-Myc in HuH-7 cells. Importantly, blocking c-Myc up-regulation by LCA abolished significantly (but not entirely) apoptosis. This supports an important role of c-Myc in LCA-mediated apoptosis. Blocking p53 induction also protected, but less well than blocking c-Myc induction. Blocking cyclin D1 induction had the least protective effect but was still significantly different from the scrambled control.

Cyclin D1 is important in cell cycle progression, so it seems paradoxical that its induction would be involved in apoptosis. Although overexpression of cyclin D1 provides a growth advantage to tumor cells, overexpression of cyclin D1 in normal cells can trigger apoptosis. The induction of the apoptotic program by cyclin D1 overexpression can be attributed to a variety of cell cycle–dependent and cell cycle–independent mechanisms in normal cells.9 Toxic bile acid–induced apoptosis in hepatocytes is associated with cyclin D1–dependent Bax translocation.10 Still, given the small degree of protection of about 25%, cyclin D1 overexpression does not play a dominant role in LCA-induced apoptosis.

To confirm our in vitro results, we employed the CBDL and LHBDL models. Our results confirm that c-Myc induction is required for the up-regulation of p53 and cyclin D1 in vivo and c-Myc knockdown prevented the switch from Mnt-Max to Myc-Max E-box binding. Interestingly, the introduction of c-myc siRNA increased Mnt-Max binding to the E-box element in p53 and cyclin D1 in livers of CBDL mice, left lobes of LHBDL mice, and HuH-7 cells. This is likely due to lower levels of Myc-Max complexes, which make more Max available to complex with Mnt. Thus, the reduced expression of p53 and cyclin D1 is due to both lower Myc-Max and higher Mnt-Max (acting as a dominant-negative) binding to their corresponding E-boxes. The reverse is also true, so reduced Mnt expression alone led to higher baseline c-Myc nuclear binding and expression of p53 and cyclin D1and potentiated LCA's effect on these parameters. Most importantly, c-Myc knockdown significantly reduced both apoptosis and necrosis. However, the protection is not complete (about 40%-50% less apoptosis and 35%-45% less necrosis) at day 3, and this suggests that c-Myc–independent pathways are also important. The fact that both apoptosis and necrosis are protected supports the notion that c-Myc participates in one or more of the shared biochemical pathways that regulate them.

In conclusion, our study revealed a novel switch from Mnt expression to Myc expression during cholestasis in vivo and treatment of hepatocytes with a toxic bile acid. This has important pathological consequences as it leads to the induction of p53 and cyclin D1, which participate in the pro-apoptotic effect of toxic bile acid. Why the expression of Mnt falls and Myc increases during cholestasis remains unclear and will be the subject of a future study. Our findings support that this switch is an important target for designing therapy against cholestatic liver injury as preventing this switch significantly reduced liver cell death.

Acknowledgements

HuH-7 cells, 293A cells, and primary mouse and human hepatocytes were provided by the Cell Culture Core, and pathological sections and staining were performed by the Imaging Core of the USC Research Center for Liver Diseases (P30DK48522).

Ancillary