- Email: [email protected]
c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration
c-Jun-N-Terminal Kinase Drives Cyclin D1 Expression and Proliferation During Liver Regeneration Robert F. Schwabe,1,2 Cynthia A. Bradham,1,2 Tetsuya Uehara,1,2 Etsuro Hatano,1,2 Brydon L. Bennett,3 Robert Schoonhoven,4 and David A. Brenner1,2 The c-Jun-N-terminal kinase (JNK) pathway is strongly activated after partial hepatectomy (PH), but its role in hepatocyte proliferation is not known. In this study, JNK activity was blocked with the small molecule inhibitor JNK SP600125 in vivo and in vitro as shown by a reduction of c-Jun phosphorylation, AP-1 DNA binding activity, and c-jun messenger RNA (mRNA) expression. SP600125 inhibited proliferating cell nuclear antigen (PCNA) expression, cyclin D1 mRNA and protein expression and reduced mitotic figures after PH. Survival was reduced significantly 3 days after PH in SP600125-treated versus vehicletreated rats (3 of 11 vs. 8 of 9, P < .01). In epidermal growth factor (EGF)-treated primary cultures of rat hepatocytes, SP600125 decreased 3H-thymidine uptake, cyclin D1 mRNA and protein expression, and inhibited the EGF-induced transcription of a cyclin D1 promoter-driven reporter gene. The defective regeneration and the decreased survival in SP600125-treated rats did not result from a major increase in apoptosis as shown by normal levels of caspase 3 activity and only slight increases in apoptotic figures. In conclusion, our data show that JNK drives G0 to G1 transition in hepatocytes and that cyclin D1 is a downstream target of the JNK pathway during liver regeneration. (HEPATOLOGY 2003;37: 824-832.)
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wo-thirds partial hepatectomy (PH) of the liver results in the rapid and potent activation of multiple cellular signaling pathways inducing a proliferative response of hepatocytes, which allows the liver to regain its original size and cell mass within 7 to 10 days.1,2 Although c-Jun-N-terminal kinase (JNK) is strongly activated within minutes after PH,3,4 the function of JNK activation in liver regeneration has not yet been determined. The JNK/c-Jun pathway is a critical component of the proliferative response and induces G0 to G1 cell cycle progression in many cell types.5 Fibroblasts derived Abbreviations: PH, partial hepatectomy; JNK, c-Jun-N-terminal kinase; AP-1, activating protein 1; mRNA, messenger RNA; TNF, tumor necrosis factor; EGF, epidermal growth factor; HGF, hepatocyte growth factor; NF-B, nuclear factor B; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; IL, interleukin; DMSO, dimethyl sulfoxide. From the 1Departments of Medicine, 2Biochemistry and Biophysics, 4Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC; and 3Signal Research Division, Celgene Corporation, San Diego, CA. Received August 8, 2002; accepted January 6, 2003. Supported in part by grants DK-34987 and GM 41804 from the National Institutes of Health. Address reprint requests to: David A. Brenner, M.D., University of North Carolina, Department of Medicine, CB #7038, Chapel Hill, NC 27599. E-mail: [email protected]; fax: 919-966-7468. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3704-0016$30.00/0 doi:10.1053/jhep.2003.50135 824
from c-Jun null mice have an impaired proliferation and reduced activity of cyclin D1– dependent kinases.6 Fibroblasts derived from JNK 1⫺/⫺ JNK 2⫺/⫺ mice have a defect in proliferation that resembles the defect of c-Jun ⫺/⫺ fibroblast and fibroblasts with a mutated c-Jun.6-8 The cyclin D1 gene has emerged as an important target for the JNK/c-Jun pathway in driving proliferation. The cyclin D1 promoter contains an activating protein 1 (AP-1) site and ectopic expression of either c-fos or c-Jun induces cyclin D1 messenger RNA (mRNA),9 which in turn is critical in driving G0 to G1 cell cycle progression in many cell types including hepatocytes.10 During liver regeneration, the activation of JNK is preceded by the release of tumor necrosis factor ␣ (TNF-␣). Inhibition of TNF-␣ or inactivation of the TNF receptor (TNFR)1 lead to defects in liver regeneration and increased mortality after PH and is associated with a reduced JNK activation.3,11 TNF-␣ does not act as a strong mitogen itself, but may prime hepatocytes for the proliferative action of growth factors such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor ␣ through mechanisms that potentially involve AP-1, STAT3, C/EBP, and nuclear factor B (NF-B).2,12 In this study, we determined the role of JNK in hepatocyte proliferation during liver regeneration and in EGF-
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treated primary hepatocytes by blocking JNK with the small molecule inhibitor SP600125.13 Inhibition of JNK resulted in a diminished proliferative response as shown by a decrease of proliferating cell nuclear antigen (PCNA) expression and mitotic figures. Cyclin D1 was a transcriptional target of JNK and may mediate its pro-proliferative effects in hepatocytes.
Materials and Methods SP600125 Treatment in Animals Undergoing PH. All animal studies were approved by the University of North Carolina Ethics Board. Male Sprague-Dawley rats (200-225 g) were subcutaneously injected with either SP600125 (a gift from Celgene Corporation, San Diego, CA) at a dose of 6 mg/kg or the corresponding volume (6 mL/kg) of PPCES vehicle (30% PEG-400/20% polypropylene glycol/15% Cremophor EL/5% ethanol/30% saline). One hour later, animals were anesthetized with ketamine and two-thirds PH was performed according to Anderson and Higgins as previously described.14 Animals were killed at various time points after PH and the remnant liver was snap frozen or fixed in 4% paraformaldehyde. For all 24- and 60-hour PH and survival experiments, treatment groups (i.e., vehicle or SP600125 treated) consisted of at least 4 rats. Hepatocyte Isolation and SP600125 Treatment of Cultured Hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats by collagen perfusion as described previously.15 Hepatocytes were plated in 6-well plates (0.5 ⫻ 106 cells/well) coated with rat type I collagen in Waymouth’s medium containing 10% fetal bovine serum, 0.1 mmol/L insulin, and 0.1 mmol/L dexamethasone in a humidified atmosphere at 37°C and 5% CO2. After 3 hours, the culture was washed with phosphatebuffered saline and changed to hormonally defined medium containing 0.1 mmol/L insulin, 2 mmol/L L-glutamine, 5 mg/mL transferrin, 1 nmol/L selenium, and 10 nmol/L free fatty acids in RPMI basal medium. For cyclin D1 expression and 3H-thymidine incorporation studies, hepatocytes were plated in Williams E media containing 5 mmol/L pyruvate and 0.1 mmol/L insulin. Two hours before EGF (20 ng/mL) treatment, hepatocytes were pretreated with SP600125 at a concentration of 20 mol/L or 0.1% dimethyl sulfoxide (DMSO). Western Blot Analysis. Snap frozen liver samples or cultured hepatocytes were lysed as previously described.16 Acrylamide gels were loaded with 50 to 100 g protein. Protein transfer onto nitrocellulose membranes was checked by Ponceau S. Blots were incubated with either anti– cyclin D1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) anti–phospho-c-Jun (Santa Cruz Bio-
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technology), anti–phospho-STAT3 (Cell Signaling, Beverly, MA), or ␣-phospho Erk antibody (New England Biolabs, Beverly, MA) at a dilution of 1:1,000 for 1 to 3 hours followed by incubation with goat-anti-mouse secondary antibody at 1:1,000 for 1 hour and chemiluminescent detection. For the confirmation of equal loading, blots were reprobed with anti-tubulin primary antibody (Oncogene Science, Cambridge, MA) or anti-actin primary antibody (ICN, Costa Mesa, CA). Northern Blot Analysis. Twenty micrograms of RNA was separated by gel electrophoresis on 2.2 mol/L formaldehyde 1% agarose gels, transferred to nylon membranes, and hybridized with 32P-labeled probes for c-jun as previously described.17 Reverse-Transcription Polymerase Chain Reaction. RNA was prepared from frozen liver tissue by the Trizol method. One microgram of RNA was reverse transcribed as previously described.16 One microliter of the reversetranscription reaction was subjected to polymerase chain reaction (PCR) for either cyclin D1, -actin, or interleukin 6 (IL-6). Cyclin D1 was amplified for 28 cycles using 5⬘-GCG AAG TGG AGA CCA TCC G-3⬘ sense and 5⬘-GTC CAC ATC TCG GAC GTC G-3⬘ antisense primer at a concentration of 1 mol/L in 10 mmol/L Tris (pH 9.2), 35 mmol/L MgCl2, 75 mmol/L KCl, and 100 g/mL bovine serum albumin. IL-6 was amplified for 40 cycles in 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, and 1.5 mmol/L MgCl2 using previously described primers.18 -actin was amplified for 28 cycles as previously described.16 Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared as described.16 Five micrograms of nuclear protein were incubated with 100 pg of 32P labeled probe containing the AP-1 consensus site in buffer containing 10 mmol/L HEPES pH 7.8, 2 mmol/L MgCl2, 50 mmol/L KCl, 1 mmol/L dithiothreitol, 0.1 mmol/L ethylenediaminetetraacetic acid, 20% glycerol, singlestranded oligonucleotide (25 g/mL), and poly dI/dC (25 g/mL) for 15 minutes on ice. Immunohistochemistry. Liver tissue was fixed in 4% paraformaldehyde for 20 hours. PCNA expression was detected by immunostaining using monoclonal antiPCNA primary antibody (DAKO, Carpinteria, CA) at a concentration of 50 g/mL for 10 minutes, the DAKO Envision system (DAKO), and 3.3-diaminobenzidine substrate as previously described.19 PCNA expression was quantified using Bioquant TCW 98 software (Biometrics, Nashville, TN) and an Olympus microscope (Melville, NY). 3H-Thymidine Incorporation Assay. Two hours after plating, hepatocytes were pretreated with 20 mol/L SP600125 or DMSO for 2 hours and then treated with
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20 ng/mL EGF (R&D Systems, Minneapolis, MN). Two Ci 3H-thymidine were added 18 hours before subjecting the cells to precipitation with 10% trichloroacetic acid. Cells were lysed in 0.2 N NaOH and counted in a scintillation counter (Beckman-Coulter, Fullerton, CA). TNF-␣ Enzyme-Linked Immunosorbent Assay. Whole-liver tissue was lysed in 25 mmol/L HEPES (pH 7.4) containing 0.1% CHAPS (Sigma), 5 mmol/L MgCl2, 1.3 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L EGTA, protease and phosphatase inhibitors. Cleared lysates were measured by enzyme-linked immunosorbent assay (R&D Systems) at a 1:5 dilution according to the manufacturer’s description and adjusted to total protein concentration. Detection of Apoptosis. Apoptosis was quantified by counting nuclei with an apoptotic morphology as previously described.14 Caspase 3–like activity was determined by incubating whole-cell extracts with amino-4-trifluoromethyl coumarin-DEVD as previously described.16 Reporter Gene Assays. To assess the effects of SP600125 on cyclin D1 transcription, hepatocytes were transfected with either ⫺964CD1Luc, or ⫺964mtCD1luc.9 For transfection, 2.5 g of plasmid DNA, 0.5 g of Renilla-luc Tk plasmid, and 2.5 L transfect F1 reagent (Targeting Systems, San Diego, CA) were added to the cells in OptiMEM media for 2 hours. After 12 hours, the cells were treated with EGF for 16 hours. Luciferase activity was determined on a 2010 luminometer (Analytical Luminescence, San Diego, CA) and adjusted to the internal Renilla-luc control. Statistics. A paired Student’s t test was performed using Microsoft Excel (Redmond, WA) and a P level less than .01 was considered statistically significant. For survival studies after PH, the Wilcoxon test was performed and a P value of .01 was used as criterion of statistical significance.
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Results
Fig. 1. SP600125 inhibits JNK activity in the regenerating liver and in cultured hepatocytes. (A) The effect of SP600125 on JNK activity after PH was determined by Western blot analysis of the JNK target c-Jun in vehicle- and SP600125-treated rats (upper panel). To show the specificity of SP600125, STAT3 phosphorylation was analyzed after PH in vehicle- and SP600125-treated rats (lower panel). (B) The induction of c-jun mRNA and AP-1 DNA binding activity was examined by Northern blot and electrophoretic mobility shift assays, respectively, 1 hour after 70% PH in untreated, vehicle-treated, and SP600125-treated rats. (C) Hepatocytes were pretreated with SP600125 (20 mol/L) for 2 hours followed by EGF (20 nmol/L) treatment for 20 minutes. Phosphorylation of c-Jun and Erk were determined by Western blot analysis. (D) IL-6 mRNA was determined by reverse-transcription PCR in livers from vehicleor SP600125-treated rats before or after PH and compared with levels of -actin. RNA from Rat1 fibroblasts served as a positive control for IL-6. (E) TNF-␣ levels from whole-liver extracts from vehicle- or SP600125treated rats were determined by enzyme-linked immunosorbent assays before PH, 30 minutes, and 90 minutes after PH.
SP600125 Inhibits JNK Activity After PH and in Cultured Hepatocytes. To analyze the effects of SP600125 on JNK activity, rats were treated with SP600125 or vehicle 1 hour before PH and killed 1 hour after PH, which corresponded to the peak of JNK activity after PH.4 Because SP600125 acts as a reversible inhibitor of JNK and may be washed out in JNK activity assays, we analyzed signaling events downstream of JNK activation. SP600125 specifically inhibited c-Jun phosphorylation (Fig. 1A) and c-Jun mRNA induction and blunted the induction of AP-1 DNA binding after PH (Fig. 1B), but did not affect signal transducer and activator of (STAT) 3 phosphorylation (Fig. 1A). In cultured hepatocytes, EGF
rapidly induced the phosphorylation of c-Jun, which was almost completely blocked by SP600125 (Fig. 1C). SP600125 specifically blocked JNK and did not influence TNF-␣–induced IB degradation, EGF-induced Akt activation (unpublished data), and EGF-induced Erk phosphorylation (Fig. 1C), which is consistent with previous studies in other cell types.13,20 To exclude that the effects of SP600125 after PH were caused mainly by blocking the release of the cytokines that are required for normal liver regeneration, we determined the effects of SP600125 on IL-6 and TNF-␣ after PH. Hepatic levels of IL-6 mRNA and TNF-␣ protein were not influenced by
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Fig. 2. SP600125 inhibits PCNA expression and mitosis in the regenerating liver. (A) PCNA expression in vehicle- and SP600125-treated rats was examined 24 and 60 hours after PH by immunohistochemistry as described in the Materials and Methods section and quantified using image analysis software. Statistical significance was determined by the t test. (B) Mitotic figures are indicated by arrows in hematoxylineosin–stained section 60 hours after PH. Mitotic figures were counted in 20 fields of animals killed after 24 and 60 hours after PH.
SP600125 (Fig. 1D and E). The decrease of intrahepatic TNF-␣ seen after PH most likely was caused by the release of preformed TNF-␣ and loss of intracellular storages. SP600125 Inhibits Proliferation After PH. To address the specific role of JNK in liver regeneration, we analyzed the proliferative response after PH in the presence or absence of SP600125. As expected, vehicletreated rats showed a strong increase in the number of PCNA-positive nuclei 24 hours after PH (12.3% ⫾ 7%, n ⫽ 4) compared with rats before PH (0.15% ⫾ 0.12%, Fig. 2A). In contrast, SP6-treated rats showed a marked decrease in the number of PCNA-positive nuclei (1.14% ⫾ 0.22%, n ⫽ 4) that significantly differed from vehicletreated rats (Student’s t test, P value ⬍ .01) and was nearly as low as those of livers before PH. After 60 hours there was a further increase in PCNA expression in vehicletreated animals (31.7% ⫾ 2.9%) and at this time point, SP600125-treated animals also showed a significant percentage of PCNA-positive nuclei (21.0% ⫾ 7.1%) that was reduced only slightly in comparison with the vehicle control (Fig. 2A). Mitotic figures were low in both treatment groups 24 hours after PH. Sixty hours after PH there was a high percentage of mitotic hepatocytes in vehicle-treated animals, but almost no mitotic figures in SP600125-treated hepatocytes (Fig. 2B). These findings
suggest that JNK exerts an important role in promoting G0 exit and cell cycle progression in hepatocytes after PH. SP600125 Inhibits Cyclin D1 Expression After PH. To further investigate the functional links between JNK activation and proliferation, we analyzed the expression of the G1 cell cycle regulator cyclin D1 that contains an AP-1 site in its promoter.9 Cyclin D1 expression is sufficient to drive hepatocytes to enter the cell cycle.10 In vehicle-treated animals, cyclin D1 protein expression was strongly up-regulated 24 hours after PH. In contrast, SP600125-pretreated animals showed a marked reduction in cyclin D1 protein expression after 24 hours (Fig. 3A). To determine whether JNK exerted its effect on cyclin D1 through a transcriptionally regulated mechanism as suggested by the AP-1 site in its promoter, we analyzed cyclin D1 mRNA levels by reverse-transcription PCR. Cyclin D1 mRNA was induced 24 hours after PH in vehicletreated animals (Fig. 3B). This induction was reduced in SP600125-treated animals in comparison with vehicle treated animals (Fig. 3B), but to a slightly lesser degree than cyclin D1 protein levels, whereas -actin mRNA levels were not affected, suggesting that JNK controls cyclin D1 expression mainly through a transcriptionally regulated pathway.
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in cells transfected with ⫺964mtCD1Luc, which lacks the AP-1 binding site in its promoter (Fig. 4B). To further confirm this data, we analyzed cyclin D1 mRNA and protein expression in EGF-treated hepatocytes in the presence or absence of SP600125. Both cyclin D1 mRNA and protein levels were induced after 48 hours of EGF treatment, and pretreatment with SP600125 strongly reduced the levels of cyclin D1 mRNA and protein (Fig. 4C and D). Inhibition of JNK Decreases Survival After PH. Previous studies have shown that the absence of critical signaling molecules such as c-Jun or TNF-receptor 1 inhibits liver regeneration and decreases survival after PH and is associated with increased microvesicular steatosis.11,22 In the present study, JNK inhibition decreased Fig. 3. SP600125 inhibits cyclin D1 expression after PH. (A) Cyclin D1 protein expression was determined by Western blot analysis in SP600125- or vehicle-treated rats 24 hours after PH. Cyclin D1 expression was quantified using NIH Image software (Bethesda, MD) and normalized to a band from the Coomassie stained blot. (B) Cyclin D1 mRNA and -actin mRNA were detected by reverse-transcription PCR in either SP600125- or vehicle-treated rats 24 hours after PH. Cyclin D1 and -actin bands were quantified by densitometry. Shown is the average of the ratio between cyclin D1 and -actin mRNA in each treatment group.
SP600125 Inhibits Hepatocyte Proliferation and Cyclin D1 Expression in Cultured Hepatocytes. The complete mitogen EGF significantly increases c-Jun phosphorylation in cultured primary rat hepatocytes (Fig. 1C) and drives cell cycle progression in cultured hepatocytes, thereby mimicking some of the pro-proliferative signaling occurring during PH.4,21 To determine whether JNK is critical for EGF-induced proliferation in cultured hepatocytes, we examined the effect of SP600125 on 3Hthymidine incorporation and cyclin D1 expression. EGF induced a 2-fold increase in 3H-thymidine incorporation after 24 hours, and a 6-fold increase after 48 hours (Fig. 4A). When preincubated with SP600125, 3H-thymidine uptake decreased by 48% (24-hour time point, P ⬍ .01) and 67% (48-hour time point, P ⬍ .01) in the EGFtreated hepatocytes and also was lower in the absence of EGF. To test whether cyclin D1 transcription was inhibited by SP600125 in vitro, we transfected hepatocytes with a reporter gene containing the ⫺964 cyclin D1 promoter linked to firefly luciferase (⫺964CD1Luc) or the same reporter gene with a mutated AP-1 site at ⫺954 (⫺964mtCD1Luc).9 EGF induced the expression of this reporter gene 2-fold (Fig. 4B). Pretreatment with SP600125 reduced the transcription of this reporter gene in EGF-treated hepatocytes by 40% but did not affect the internal control Tk-driven renilla luciferase. The reduced luciferase activity was comparable with the level observed
Fig. 4. SP600125 inhibits 3H-thymidine incorporation and cyclin D1 transcription in cultured hepatocytes. (A) 3H-thymidine incorporation was measured in hepatocytes in the presence or absence of EGF (20 nmol/L) in either SP600125-treated (20 mol/L) or DMSO-treated (0.1%) hepatocytes after 24 and 48 hours of treatment. Statistical significance was determined by the t test. (B) Cyclin D1– dependent transcription was determined by reporter gene assay. Hepatocytes were transfected with ⫺964CD1luc or ⫺964mutCD1luc and treated with EGF (20 nmol/L) after SP600125 (20 mol/L) or DMSO (0.1%) pretreatment as described in the Materials and Methods section. Statistical significance was determined by the t test. (C) Cyclin D1 and -actin mRNA levels were determined by PCR 48 hours after EGF (20 nmol/L) stimulation in the presence or absence of SP600125 (20 mol/L). (D) Cyclin D1 and actin protein levels were determined by Western blot analysis 48 hours after EGF (20 nmol/L) stimulation in the presence or absence of SP600125 (20 mol/L).
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Fig. 5. JNK inhibition increases mortality after PH. (A) Rats were pretreated with SP600125 or vehicle 1 hour before PH. Survival was monitored over the following 3 days and is shown as a Kaplan-Meier graph. Statistical significance was determined by the Wilcoxon test. (B) Rats were pretreated with SP600125 or vehicle 1 hour before PH. The levels of aspartate and alanine aminotransferases were determined 60 hours after PH. Statistical significance was determined by the t test. (C) Sections from SP600125- or vehicle-treated animals 60 hours after PH were stained with hematoxylineosin. (D) Rats were pretreated with SP600125 or vehicle 1 hour before PH and livers were harvested 24 or 60 hours after PH. Cells with typical apoptotic morphology were counted in 20 ⫻ 200 fields in 2 different animals per treatment group. The number of apoptotic cells was divided by the total number of parenchymal cells and is expressed as percentage. (E) Caspase 3 activity was determined by incubating cell extracts from livers harvested 24 and 60 hours after PH with the caspase 3 substrate AFC-DEVD for 2 hours. Mouse hepatocytes treated with the agonistic Jo2 antibody served as positive control. Caspase 3 activity is shown as AFC release (pmol) per g protein extract after 2 hours of incubation.
survival 3 days after PH (Fig. 5A, vehicle 89% vs. SP600125 27%, P ⫽ .01, Wilcoxon test) and SP600125treated animals had elevated transaminase levels 60 hours after PH (Fig. 5B, P ⬍ .01). SP600125-treated animals showed a high degree of microvesicular steatosis 2 to 3 days after PH (Fig. 5C). However, vehicle-treated animals also showed microvesicular steatosis, albeit to a lesser degree. To determine whether JNK blockade induced an increase in mortality due to higher rates of apoptosis as shown in animals with defective NF-B and inducible nitric oxide synthase in previous studies,14,23 we quantified apoptotic figures in hematoxylin-eosin–stained liver sections of SP600125- and vehicle-treated animals. In addition, caspase 3–like activity was measured in extracts from these livers. In both vehicle- and SP600125-treated animals, the numbers of apoptotic hepatocytes were low. Twenty-four hours after PH, apoptotic figures were elevated slightly in SP600125-treated animals, but at all other time points the number of apoptotic figures was
similar (Fig. 5D). These findings were confirmed by the low caspase 3–like activity after PH in both SP600125and vehicle-treated animals after 24 and 60 hours, whereas caspase 3–like activity was highly elevated in hepatocytes treated with the agonistic Jo2 antibody (Fig. 5E). Thus, the reduced hepatocyte proliferation in SP600125-treated animals after PH did not appear to be caused by an increase in hepatocyte apoptosis.
Discussion In the adult liver, hepatocytes are long-lived and rarely undergo proliferation, yet they retain a remarkable ability to proliferate.1 This allows the liver to regain its original size within 7 to 10 days after 70% PH and quickly restore function. This regenerative response is initiated by a series of signaling events that allow the hepatocyte to enter the cell cycle and undergo several rounds of proliferation. JNK activation is one of the earliest signals to be detected
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after PH, but the function of JNK in liver regeneration remains largely unknown.4 Several agonists may induce JNK during liver regeneration: (1) TNF-␣ is an important mediator of early-phase JNK activation after PH as shown by the reduced JNK activation and AP-1 binding activity in rats injected with antibodies to TNF-␣ and mice with inactivated TNFR1.3,11 TNF-␣ acts as a comitogen that primes hepatocytes for the pro-proliferative effects of hepatic mitogens including EGF and HGF through the activation of NF-B, AP-1, STAT3, and the release of IL-6, but alone is incapable to induce hepatocyte proliferation.3,24,25 However, the relative role of these pathways in driving proliferation after PH is unclear and some factors such as NF-B may only play a minor role .26 (2) EGF and HGF are believed to play a key role in liver regeneration and both induce JNK activation and AP-1– dependent gene transcription in cultured hepatocytes.4,27 The merging of 2 crucial pro-proliferative signaling cascades (i.e., cytokines and growth factors) at the level of JNK points toward a role for JNK in driving proliferation during liver regeneration. This hypothesis is supported by studies showing JNKs to be an important regulator of cell proliferation in other cell types.8,28 JNKs phosphorylate the N-terminal domain of c-Jun, increase its transactivation, and thereby up-regulate AP-1– dependent transcription. Additionally, JNKs phosphorylate the transcription factors ATF2 and JunD. These factors homodimerize and heterodimerize with other AP-1 components to induce AP-1– dependent transcription and the up-regulation of many genes that are critically involved in cell proliferation and survival.5 JNK Activity Is Required for Hepatocyte Proliferation. The liver contains JNK1 and JNK2, which are believed to fulfill similar functions, but it does not contain the neuronal form JNK3. Because JNK1 ⫺/⫺/JNK2 ⫺/⫺ mice are not viable,29,30 our study used a pharmacologic approach to study the role of JNKs in liver regeneration. The small molecule JNK inhibitor SP600125 blocks both JNK1 and JNK2 in a highly specific manner13 and efficiently inhibited JNK activity in the liver and cultured hepatocytes in our study. Blocking JNK activity with SP600125 inhibited the normal proliferative response after PH, indicating that JNKs are essential components of the cellular machinery that drives hepatocytes to enter the cell cycle after PH. SP600125-treated animals showed a blocked G1/S transition, as apparent by the decreased expression of PCNA and by the reduction of the levels of cyclin D1, which is predominantly expressed during G1. SP600125 additionally reduced mitotic figures after PH and 3H-thymidine incorporation in cultured hepatocytes. At later time points, SP600125-treated animals had similar expression levels of PCNA, indicating the absence of
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early-phase JNK activity still allows hepatocytes to enter the cell cycle, albeit in a delayed manner. JNK Activation Drives Cyclin D1 Expression. Several of the AP-1 regulated genes are critical regulators of cell growth, among them cyclin D1 and PCNA.9,31 The importance of cyclin D1 as a critical downstream target of JNK is suggested by the impaired transcription of cyclin D1 and the reduced activity of cyclin D1– dependent kinases in c-jun ⫺/⫺ mouse embryonic fibroblasts.6,32 In hepatocytes, cyclin D1 plays a crucial role in driving proliferation as recently shown by a study in which cyclin D1 overexpression per se was sufficient to promote hepatocyte replication.10 Moreover, it has been shown that cyclin D1 is the most prominently up-regulated type D cyclin after PH and in response to mitogenic stimuli and plays a critical role in the driving cells through the G1 restriction point.33 Our study found a strong reduction of cyclin D1 protein and mRNA expression in SP600125treated animals after PH. Similar results were obtained in cultured hepatocytes in which cyclin D1 mRNA and protein levels as well as the transcription of a cyclin D1– promoter-driven reporter gene were decreased by SP600125 in EGF-treated hepatocytes. Cyclin D1 expression in the liver may be regulated at the posttranscriptional level.34,35 In our study, however, cyclin D1 was regulated mainly at the transcriptional level through a JNK-dependent pathway, suggesting that cyclin D1 is an important mediator of pro-proliferative effects of JNK. In conjunction with results from previous studies, our data indicates that the growth factor ⫹ TNF-␣ 3 JNK 3 AP-1 3 cyclin D1 pathway is crucial in driving proliferation during liver regeneration. JNK Blockade Decreases Survival After PH. Inactivation of important signaling pathways such as TNF-␣ and IL-6 impairs liver regeneration and decreases survival after PH.11,25 In our study, SP600125 significantly reduced survival 3 days after PH. SP600125-treated animals showed a higher degree of microvesicular steatosis similar to mice with inactivated TNFR1 or c-jun.11,22 However, vehicle-treated animals, also showed increased microvesicular steatosis in our study, presumably due to components in the drug vehicle that may exacerbate the low degree of microvesicular steatosis occurring normally after PH.11 Animals that survived day 3 after PH showed a slightly delayed but ultimately normal liver regeneration with a slightly decreased liver-to-body-weight ratio at day 3 and a normal liver-to-body-weight ratio at day 5 (data not shown). These data are consistent with other studies in which the blockade of single signaling pathways caused a significant delay of liver regeneration and increased mortality, but an ultimately normal regeneration in surviving animals, most likely due to compensatory effects of
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other pathways.25 This hypothesis is supported additionally by our finding that the expression of PCNA was almost as high in SP600125- as in vehicle-treated animals 60 hours after PH. JNK Blockade Does Not Significantly Increase Apoptosis After PH. Previous studies have shown that some signaling cascades after PH such as NF-B, inducible nitric oxide sythase, and Akt function to protect hepatocytes from undergoing apoptosis.14,23,36 JNK is involved in the regulation of cell death in many cell types and may have both pro- or antiapoptotic effects.37 JNK activity and c-Jun are required for normal liver development and appear to protect hepatocytes from apoptosis during development as seen in mice lacking either the JNK kinase SEK1 or c-Jun.38-42 Therefore, we wanted to exclude the possibility that the decreased survival and the decreased proliferation in SP600125-treated animals after PH was caused by an increased rate of apoptosis. Caspase 3–like activity was similar in the treatment groups and apoptotic bodies were only slightly more frequent in SP600125-treated animals 24 hours after PH, but not at other time points. These findings are further supported by data from our laboratory that JNK has proapoptotic effects in TNF-␣–induced apoptosis of hepatocytes43 and reperfusion injury.44 The Role of c-Jun Phosphorylation in Liver Regeneration. The AP-1 component c-Jun is a major contributor of AP-1 activity in quiescent liver and after PH.4 The occurrence of proliferative defects in both JNK 1⫺/⫺/ JNK 2 ⫺/⫺ MEFs and c-jun⫺/⫺ MEFs indicate that c-Jun serves as a major JNK target in proliferation.6,8,32 However, proliferative defects in c-jun⫺/⫺ MEFs are more severe than in MEFs containing a c-Jun with mutated phosphorylation sites 63 and 73,7 and N-terminal phosphorylation of c-Jun is dispensable for liver development and hepatocyte proliferation after PH.22,45 These results indicate that c-Jun has functions independent of its N-terminal phosphorylation. Our study indicates that JNK activity is required for hepatocyte proliferation after PH and in culture and seems to contradict previous studies.22,45 However, JNKs regulate AP-1– dependent transcription through the phosphorylation of several targets including c-Jun, ATF2, and JunD, indicating that inhibition of c-Jun phosphorylation through the use of a mutated c-Jun in previous studies may have been compensated for by other JNK targets. Evidence supporting this hypothesis comes from a recent study showing that the knock-in of a second JunB allele can reverse developmental hepatic abnormalities and embryonic lethality of c-Jun⫺/⫺ mice and up-regulate c-Jun targets including cyclin D1,46 indicating that Jun family members, and potentially other AP-1 components, have a
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higher degree of redundancy than previously appreciated. However, it appears that the presence of c-Jun in the AP-1 complex is absolutely required for cell cycle progression and protection from apoptosis after PH as seen in the c-jun⫺/⫺ mice.22 The striking difference between the complete absence of c-Jun and deletion of its N-terminal phosphorylation sites indicate that unphosphorylated c-Jun may contribute to AP-1– dependent transcription after PH.22 Potential explanations are that (1) heterodimerization of other AP-1 components with c-Jun induces AP-1 transcription independent of the phosphorylation status of c-Jun and/or that (2) JNK targets such as JunD, which lack a JNK docking site, use a JNK docking site containing partners (including mutated c-Jun) for JNK recruitment and their subsequent phosphorylation resulting in increased AP-1– dependent transcription.47 Future studies have to determine whether JNK has other substrates than c-Jun to drive hepatocyte proliferation.
References 1. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997; 276:60-66. 2. Fausto N. Liver regeneration. J Hepatol 2000;32:19-31. 3. Diehl AM, Yin M, Fleckenstein J, Yang SQ, Lin HZ, Brenner DA, Westwick J, et al. Tumor necrosis factor-alpha induces c-jun during the regenerative response to liver injury. Am J Physiol 1994;267:G552-G561. 4. Westwick JK, Weitzel C, Leffert HL, Brenner DA. Activation of Jun kinase is an early event in hepatic regeneration. J Clin Invest 1995;95:803-810. 5. Shaulian E, Karin M. AP-1 in cell proliferation and survival. Oncogene 2001;20:2390-2400. 6. Schreiber M, Kolbus A, Piu F, Szabowski A, Mohle-Steinlein U, Tian J, Karin M, et al. Control of cell cycle progression by c-Jun is p53 dependent. Genes Dev 1999;13:607-619. 7. Behrens A, Sibilia M, Wagner EF. Amino-terminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation. Nat Genet 1999;21:326-329. 8. Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, Bar-Sagi D, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000;288:870-874. 9. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, Pestell RG. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem 1995;270:2358923597. 10. Nelsen CJ, Rickheim DG, Timchenko NA, Stanley MW, Albrecht JH. Transient expression of cyclin D1 is sufficient to promote hepatocyte replication and liver growth in vivo. Cancer Res 2001;61:8564-8568. 11. Yamada Y, Kirillova I, Peschon JJ, Fausto N. Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc Natl Acad Sci U S A 1997;94:14411446. 12. Taub R. Liver regeneration 4: transcriptional control of liver regeneration. FASEB J 1996;10:413-427. 13. Bennett BL, Sasaki DT, Murray BW, O’Leary EC, Sakata ST, Xu W, Leisten JC, et al. SP600125, an anthrapyrazolone inhibitor of Jun Nterminal kinase. Proc Natl Acad Sci U S A 2001;98:13681-13686. 14. Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, Brenner DA. NFkappaB prevents apoptosis and liver dysfunction during liver regeneration [published erratum appears in J Clin Invest 1998;101:1541]. J Clin Invest 1998;101:802-811.
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15. Herman B, Nieminen AL, Gores GJ, Lemasters JJ. Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability. FASEB J 1988;2:146-151. 16. Schwabe RF, Bennett BL, Manning AM, Brenner DA. Differential role of I kappa B kinase 1 and 2 in primary rat hepatocytes. HEPATOLOGY 2001; 33:81-90. 17. Bradham CA, Stachlewitz RF, Gao W, Qian T, Jayadev S, Jenkins G, Hannun Y, et al. Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases. HEPATOLOGY 1997; 25:1128-1135. 18. Stefanovic B, Schnabl B, Brenner DA. Inhibition of collagen alpha 1(I) expression by the 5⬘ stem-loop as a molecular decoy. J Biol Chem 2002; 277:18229-18237. 19. Schwabe RF, Schnabl B, Kweon YO, Brenner DA. CD40 activates NFkappa B and c-Jun N-terminal kinase and enhances chemokine secretion on activated human hepatic stellate cells. J Immunol 2001;166:68126819. 20. Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, Manning AM, et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 2001;108:7381. 21. Loyer P, Ilyin G, Cariou S, Glaise D, Corlu A, Guguen-Guillouzo C. Progression through G1 and S phases of adult rat hepatocytes. Prog Cell Cycle Res 1996;2:37-47. 22. Behrens A, Sibilia M, David JP, Mohle-Steinlein U, Tronche F, Schutz G, Wagner EF. Impaired postnatal hepatocyte proliferation and liver regeneration in mice lacking c-jun in the liver. EMBO J 2002;21:1782-1790. 23. Rai RM, Lee FY, Rosen A, Yang SQ, Lin HZ, Koteish A, Liew FY, et al. Impaired liver regeneration in inducible nitric oxide synthase deficient mice. Proc Natl Acad Sci U S A 1998;95:13829-13834. 24. Kirillova I, Chaisson M, Fausto N. Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation. Cell Growth Differ 1999;10:819-828. 25. Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, Taub R. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 1996;274:1379-1383. 26. Chaisson ML, Brooling JT, Ladiges W, Tsai S, Fausto N. Hepatocytespecific inhibition of NF-kappaB leads to apoptosis after TNF treatment, but not after partial hepatectomy. J Clin Invest 2002;110:193-202. 27. Auer KL, Contessa J, Brenz-Verca S, Pirola L, Rusconi S, Cooper G, Abo A, et al. The Ras/Rac1/Cdc42/SEK/JNK/c-Jun cascade is a key pathway by which agonists stimulate DNA synthesis in primary cultures of rat hepatocytes. Mol Biol Cell 1998;9:561-573. 28. Schnabl B, Bradham CA, Bennett BL, Manning AM, Stefanovic B, Brenner DA. TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells. HEPATOLOGY 2001;34:953-963. 29. Kuan CY, Yang DD, Samanta Roy DR, Davis RJ, Rakic P, Flavell RA. The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development. Neuron 1999;22:667-676.
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30. Sabapathy K, Jochum W, Hochedlinger K, Chang L, Karin M, Wagner EF. Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2. Mech Dev 1999;89:115-124. 31. Liu YC, Chang HW, Lai YC, Ding ST, Ho JL. Serum responsiveness of the rat PCNA promoter involves the proximal ATF and AP-1 sites. FEBS Lett 1998;441:200-204. 32. Wisdom R, Johnson RS, Moore C. c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J 1999;18:188-197. 33. Rickheim DG, Nelsen CJ, Fassett JT, Timchenko NA, Hansen LK, Albrecht JH. Differential regulation of cyclins D1 and D3 in hepatocyte proliferation. HEPATOLOGY 2002;36:30-38. 34. Albrecht JH, Hoffman JS, Kren BT, Steer CJ. Cyclin and cyclin-dependent kinase 1 mRNA expression in models of regenerating liver and human liver diseases. Am J Physiol 1993;265:G857-G864. 35. Awad MM, Gruppuso PA. Cell cycle control during liver development in the rat: evidence indicating a role for cyclin D1 posttranscriptional regulation [in process citation]. Cell Growth Differ 2000;11:325-334. 36. Hong F, Nguyen VA, Shen X, Kunos G, Gao B. Rapid activation of protein kinase B/Akt has a key role in antiapoptotic signaling during liver regeneration. Biochem Biophys Res Commun 2000;279:974-979. 37. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 2000;103:239-252. 38. Hilberg F, Aguzzi A, Howells N, Wagner EF. c-jun is essential for normal mouse development and hepatogenesis. Nature 1993;365:179-181. 39. Johnson RS, van Lingen B, Papaioannou VE, Spiegelman BM. A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture. Genes Dev 1993;7:1309-1317. 40. Ganiatsas S, Kwee L, Fujiwara Y, Perkins A, Ikeda T, Labow MA, Zon LI. SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis. Proc Natl Acad Sci U S A 1998;95:6881-6886. 41. Nishina H, Vaz C, Billia P, Nghiem M, Sasaki T, De la Pompa JL, Furlonger K, et al. Defective liver formation and liver cell apoptosis in mice lacking the stress signaling kinase SEK1/MKK4. Development 1999;126: 505-516. 42. Eferl R, Sibilia M, Hilberg F, Fuchsbichler A, Kufferath I, Guertl B, Zenz R, et al. Functions of c-Jun in liver and heart development. J Cell Biol 1999;145:1049-1061. 43. Schwabe RF, Bennett BL, Purbeck C, Brenner DA. JNK enhances TNF␣induced apoptosis in hepatocytes in a transcriptionally independent manner. HEPATOLOGY 2002;36:299A. 44. Uehara T, Bennett BL, Sakata ST, Satoh Y, Brenner DA. JNK inhibitors protect from hepatic ischemia/reperfusion. HEPATOLOGY 2001;34:348A. 45. Bradham CA, Hatano E, Brenner DA. Dominant-negative TAK1 induces c-Myc and G(0) exit in liver. Am J Physiol 2001;281:G1279-G1289. 46. Passegue E, Jochum W, Behrens A, Ricci R, Wagner EF. JunB can substitute for Jun in mouse development and cell proliferation. Nat Genet 2002; 30:158-166. 47. Kallunki T, Deng T, Hibi M, Karin M. c-Jun can recruit JNK to phosphorylate dimerization partners via specific docking interactions. Cell 1996;87:929-939.
T
wo-thirds partial hepatectomy (PH) of the liver results in the rapid and potent activation of multiple cellular signaling pathways inducing a proliferative response of hepatocytes, which allows the liver to regain its original size and cell mass within 7 to 10 days.1,2 Although c-Jun-N-terminal kinase (JNK) is strongly activated within minutes after PH,3,4 the function of JNK activation in liver regeneration has not yet been determined. The JNK/c-Jun pathway is a critical component of the proliferative response and induces G0 to G1 cell cycle progression in many cell types.5 Fibroblasts derived Abbreviations: PH, partial hepatectomy; JNK, c-Jun-N-terminal kinase; AP-1, activating protein 1; mRNA, messenger RNA; TNF, tumor necrosis factor; EGF, epidermal growth factor; HGF, hepatocyte growth factor; NF-B, nuclear factor B; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; IL, interleukin; DMSO, dimethyl sulfoxide. From the 1Departments of Medicine, 2Biochemistry and Biophysics, 4Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC; and 3Signal Research Division, Celgene Corporation, San Diego, CA. Received August 8, 2002; accepted January 6, 2003. Supported in part by grants DK-34987 and GM 41804 from the National Institutes of Health. Address reprint requests to: David A. Brenner, M.D., University of North Carolina, Department of Medicine, CB #7038, Chapel Hill, NC 27599. E-mail: [email protected]; fax: 919-966-7468. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3704-0016$30.00/0 doi:10.1053/jhep.2003.50135 824
from c-Jun null mice have an impaired proliferation and reduced activity of cyclin D1– dependent kinases.6 Fibroblasts derived from JNK 1⫺/⫺ JNK 2⫺/⫺ mice have a defect in proliferation that resembles the defect of c-Jun ⫺/⫺ fibroblast and fibroblasts with a mutated c-Jun.6-8 The cyclin D1 gene has emerged as an important target for the JNK/c-Jun pathway in driving proliferation. The cyclin D1 promoter contains an activating protein 1 (AP-1) site and ectopic expression of either c-fos or c-Jun induces cyclin D1 messenger RNA (mRNA),9 which in turn is critical in driving G0 to G1 cell cycle progression in many cell types including hepatocytes.10 During liver regeneration, the activation of JNK is preceded by the release of tumor necrosis factor ␣ (TNF-␣). Inhibition of TNF-␣ or inactivation of the TNF receptor (TNFR)1 lead to defects in liver regeneration and increased mortality after PH and is associated with a reduced JNK activation.3,11 TNF-␣ does not act as a strong mitogen itself, but may prime hepatocytes for the proliferative action of growth factors such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor ␣ through mechanisms that potentially involve AP-1, STAT3, C/EBP, and nuclear factor B (NF-B).2,12 In this study, we determined the role of JNK in hepatocyte proliferation during liver regeneration and in EGF-
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treated primary hepatocytes by blocking JNK with the small molecule inhibitor SP600125.13 Inhibition of JNK resulted in a diminished proliferative response as shown by a decrease of proliferating cell nuclear antigen (PCNA) expression and mitotic figures. Cyclin D1 was a transcriptional target of JNK and may mediate its pro-proliferative effects in hepatocytes.
Materials and Methods SP600125 Treatment in Animals Undergoing PH. All animal studies were approved by the University of North Carolina Ethics Board. Male Sprague-Dawley rats (200-225 g) were subcutaneously injected with either SP600125 (a gift from Celgene Corporation, San Diego, CA) at a dose of 6 mg/kg or the corresponding volume (6 mL/kg) of PPCES vehicle (30% PEG-400/20% polypropylene glycol/15% Cremophor EL/5% ethanol/30% saline). One hour later, animals were anesthetized with ketamine and two-thirds PH was performed according to Anderson and Higgins as previously described.14 Animals were killed at various time points after PH and the remnant liver was snap frozen or fixed in 4% paraformaldehyde. For all 24- and 60-hour PH and survival experiments, treatment groups (i.e., vehicle or SP600125 treated) consisted of at least 4 rats. Hepatocyte Isolation and SP600125 Treatment of Cultured Hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats by collagen perfusion as described previously.15 Hepatocytes were plated in 6-well plates (0.5 ⫻ 106 cells/well) coated with rat type I collagen in Waymouth’s medium containing 10% fetal bovine serum, 0.1 mmol/L insulin, and 0.1 mmol/L dexamethasone in a humidified atmosphere at 37°C and 5% CO2. After 3 hours, the culture was washed with phosphatebuffered saline and changed to hormonally defined medium containing 0.1 mmol/L insulin, 2 mmol/L L-glutamine, 5 mg/mL transferrin, 1 nmol/L selenium, and 10 nmol/L free fatty acids in RPMI basal medium. For cyclin D1 expression and 3H-thymidine incorporation studies, hepatocytes were plated in Williams E media containing 5 mmol/L pyruvate and 0.1 mmol/L insulin. Two hours before EGF (20 ng/mL) treatment, hepatocytes were pretreated with SP600125 at a concentration of 20 mol/L or 0.1% dimethyl sulfoxide (DMSO). Western Blot Analysis. Snap frozen liver samples or cultured hepatocytes were lysed as previously described.16 Acrylamide gels were loaded with 50 to 100 g protein. Protein transfer onto nitrocellulose membranes was checked by Ponceau S. Blots were incubated with either anti– cyclin D1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) anti–phospho-c-Jun (Santa Cruz Bio-
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technology), anti–phospho-STAT3 (Cell Signaling, Beverly, MA), or ␣-phospho Erk antibody (New England Biolabs, Beverly, MA) at a dilution of 1:1,000 for 1 to 3 hours followed by incubation with goat-anti-mouse secondary antibody at 1:1,000 for 1 hour and chemiluminescent detection. For the confirmation of equal loading, blots were reprobed with anti-tubulin primary antibody (Oncogene Science, Cambridge, MA) or anti-actin primary antibody (ICN, Costa Mesa, CA). Northern Blot Analysis. Twenty micrograms of RNA was separated by gel electrophoresis on 2.2 mol/L formaldehyde 1% agarose gels, transferred to nylon membranes, and hybridized with 32P-labeled probes for c-jun as previously described.17 Reverse-Transcription Polymerase Chain Reaction. RNA was prepared from frozen liver tissue by the Trizol method. One microgram of RNA was reverse transcribed as previously described.16 One microliter of the reversetranscription reaction was subjected to polymerase chain reaction (PCR) for either cyclin D1, -actin, or interleukin 6 (IL-6). Cyclin D1 was amplified for 28 cycles using 5⬘-GCG AAG TGG AGA CCA TCC G-3⬘ sense and 5⬘-GTC CAC ATC TCG GAC GTC G-3⬘ antisense primer at a concentration of 1 mol/L in 10 mmol/L Tris (pH 9.2), 35 mmol/L MgCl2, 75 mmol/L KCl, and 100 g/mL bovine serum albumin. IL-6 was amplified for 40 cycles in 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, and 1.5 mmol/L MgCl2 using previously described primers.18 -actin was amplified for 28 cycles as previously described.16 Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared as described.16 Five micrograms of nuclear protein were incubated with 100 pg of 32P labeled probe containing the AP-1 consensus site in buffer containing 10 mmol/L HEPES pH 7.8, 2 mmol/L MgCl2, 50 mmol/L KCl, 1 mmol/L dithiothreitol, 0.1 mmol/L ethylenediaminetetraacetic acid, 20% glycerol, singlestranded oligonucleotide (25 g/mL), and poly dI/dC (25 g/mL) for 15 minutes on ice. Immunohistochemistry. Liver tissue was fixed in 4% paraformaldehyde for 20 hours. PCNA expression was detected by immunostaining using monoclonal antiPCNA primary antibody (DAKO, Carpinteria, CA) at a concentration of 50 g/mL for 10 minutes, the DAKO Envision system (DAKO), and 3.3-diaminobenzidine substrate as previously described.19 PCNA expression was quantified using Bioquant TCW 98 software (Biometrics, Nashville, TN) and an Olympus microscope (Melville, NY). 3H-Thymidine Incorporation Assay. Two hours after plating, hepatocytes were pretreated with 20 mol/L SP600125 or DMSO for 2 hours and then treated with
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20 ng/mL EGF (R&D Systems, Minneapolis, MN). Two Ci 3H-thymidine were added 18 hours before subjecting the cells to precipitation with 10% trichloroacetic acid. Cells were lysed in 0.2 N NaOH and counted in a scintillation counter (Beckman-Coulter, Fullerton, CA). TNF-␣ Enzyme-Linked Immunosorbent Assay. Whole-liver tissue was lysed in 25 mmol/L HEPES (pH 7.4) containing 0.1% CHAPS (Sigma), 5 mmol/L MgCl2, 1.3 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L EGTA, protease and phosphatase inhibitors. Cleared lysates were measured by enzyme-linked immunosorbent assay (R&D Systems) at a 1:5 dilution according to the manufacturer’s description and adjusted to total protein concentration. Detection of Apoptosis. Apoptosis was quantified by counting nuclei with an apoptotic morphology as previously described.14 Caspase 3–like activity was determined by incubating whole-cell extracts with amino-4-trifluoromethyl coumarin-DEVD as previously described.16 Reporter Gene Assays. To assess the effects of SP600125 on cyclin D1 transcription, hepatocytes were transfected with either ⫺964CD1Luc, or ⫺964mtCD1luc.9 For transfection, 2.5 g of plasmid DNA, 0.5 g of Renilla-luc Tk plasmid, and 2.5 L transfect F1 reagent (Targeting Systems, San Diego, CA) were added to the cells in OptiMEM media for 2 hours. After 12 hours, the cells were treated with EGF for 16 hours. Luciferase activity was determined on a 2010 luminometer (Analytical Luminescence, San Diego, CA) and adjusted to the internal Renilla-luc control. Statistics. A paired Student’s t test was performed using Microsoft Excel (Redmond, WA) and a P level less than .01 was considered statistically significant. For survival studies after PH, the Wilcoxon test was performed and a P value of .01 was used as criterion of statistical significance.
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Results
Fig. 1. SP600125 inhibits JNK activity in the regenerating liver and in cultured hepatocytes. (A) The effect of SP600125 on JNK activity after PH was determined by Western blot analysis of the JNK target c-Jun in vehicle- and SP600125-treated rats (upper panel). To show the specificity of SP600125, STAT3 phosphorylation was analyzed after PH in vehicle- and SP600125-treated rats (lower panel). (B) The induction of c-jun mRNA and AP-1 DNA binding activity was examined by Northern blot and electrophoretic mobility shift assays, respectively, 1 hour after 70% PH in untreated, vehicle-treated, and SP600125-treated rats. (C) Hepatocytes were pretreated with SP600125 (20 mol/L) for 2 hours followed by EGF (20 nmol/L) treatment for 20 minutes. Phosphorylation of c-Jun and Erk were determined by Western blot analysis. (D) IL-6 mRNA was determined by reverse-transcription PCR in livers from vehicleor SP600125-treated rats before or after PH and compared with levels of -actin. RNA from Rat1 fibroblasts served as a positive control for IL-6. (E) TNF-␣ levels from whole-liver extracts from vehicle- or SP600125treated rats were determined by enzyme-linked immunosorbent assays before PH, 30 minutes, and 90 minutes after PH.
SP600125 Inhibits JNK Activity After PH and in Cultured Hepatocytes. To analyze the effects of SP600125 on JNK activity, rats were treated with SP600125 or vehicle 1 hour before PH and killed 1 hour after PH, which corresponded to the peak of JNK activity after PH.4 Because SP600125 acts as a reversible inhibitor of JNK and may be washed out in JNK activity assays, we analyzed signaling events downstream of JNK activation. SP600125 specifically inhibited c-Jun phosphorylation (Fig. 1A) and c-Jun mRNA induction and blunted the induction of AP-1 DNA binding after PH (Fig. 1B), but did not affect signal transducer and activator of (STAT) 3 phosphorylation (Fig. 1A). In cultured hepatocytes, EGF
rapidly induced the phosphorylation of c-Jun, which was almost completely blocked by SP600125 (Fig. 1C). SP600125 specifically blocked JNK and did not influence TNF-␣–induced IB degradation, EGF-induced Akt activation (unpublished data), and EGF-induced Erk phosphorylation (Fig. 1C), which is consistent with previous studies in other cell types.13,20 To exclude that the effects of SP600125 after PH were caused mainly by blocking the release of the cytokines that are required for normal liver regeneration, we determined the effects of SP600125 on IL-6 and TNF-␣ after PH. Hepatic levels of IL-6 mRNA and TNF-␣ protein were not influenced by
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Fig. 2. SP600125 inhibits PCNA expression and mitosis in the regenerating liver. (A) PCNA expression in vehicle- and SP600125-treated rats was examined 24 and 60 hours after PH by immunohistochemistry as described in the Materials and Methods section and quantified using image analysis software. Statistical significance was determined by the t test. (B) Mitotic figures are indicated by arrows in hematoxylineosin–stained section 60 hours after PH. Mitotic figures were counted in 20 fields of animals killed after 24 and 60 hours after PH.
SP600125 (Fig. 1D and E). The decrease of intrahepatic TNF-␣ seen after PH most likely was caused by the release of preformed TNF-␣ and loss of intracellular storages. SP600125 Inhibits Proliferation After PH. To address the specific role of JNK in liver regeneration, we analyzed the proliferative response after PH in the presence or absence of SP600125. As expected, vehicletreated rats showed a strong increase in the number of PCNA-positive nuclei 24 hours after PH (12.3% ⫾ 7%, n ⫽ 4) compared with rats before PH (0.15% ⫾ 0.12%, Fig. 2A). In contrast, SP6-treated rats showed a marked decrease in the number of PCNA-positive nuclei (1.14% ⫾ 0.22%, n ⫽ 4) that significantly differed from vehicletreated rats (Student’s t test, P value ⬍ .01) and was nearly as low as those of livers before PH. After 60 hours there was a further increase in PCNA expression in vehicletreated animals (31.7% ⫾ 2.9%) and at this time point, SP600125-treated animals also showed a significant percentage of PCNA-positive nuclei (21.0% ⫾ 7.1%) that was reduced only slightly in comparison with the vehicle control (Fig. 2A). Mitotic figures were low in both treatment groups 24 hours after PH. Sixty hours after PH there was a high percentage of mitotic hepatocytes in vehicle-treated animals, but almost no mitotic figures in SP600125-treated hepatocytes (Fig. 2B). These findings
suggest that JNK exerts an important role in promoting G0 exit and cell cycle progression in hepatocytes after PH. SP600125 Inhibits Cyclin D1 Expression After PH. To further investigate the functional links between JNK activation and proliferation, we analyzed the expression of the G1 cell cycle regulator cyclin D1 that contains an AP-1 site in its promoter.9 Cyclin D1 expression is sufficient to drive hepatocytes to enter the cell cycle.10 In vehicle-treated animals, cyclin D1 protein expression was strongly up-regulated 24 hours after PH. In contrast, SP600125-pretreated animals showed a marked reduction in cyclin D1 protein expression after 24 hours (Fig. 3A). To determine whether JNK exerted its effect on cyclin D1 through a transcriptionally regulated mechanism as suggested by the AP-1 site in its promoter, we analyzed cyclin D1 mRNA levels by reverse-transcription PCR. Cyclin D1 mRNA was induced 24 hours after PH in vehicletreated animals (Fig. 3B). This induction was reduced in SP600125-treated animals in comparison with vehicle treated animals (Fig. 3B), but to a slightly lesser degree than cyclin D1 protein levels, whereas -actin mRNA levels were not affected, suggesting that JNK controls cyclin D1 expression mainly through a transcriptionally regulated pathway.
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in cells transfected with ⫺964mtCD1Luc, which lacks the AP-1 binding site in its promoter (Fig. 4B). To further confirm this data, we analyzed cyclin D1 mRNA and protein expression in EGF-treated hepatocytes in the presence or absence of SP600125. Both cyclin D1 mRNA and protein levels were induced after 48 hours of EGF treatment, and pretreatment with SP600125 strongly reduced the levels of cyclin D1 mRNA and protein (Fig. 4C and D). Inhibition of JNK Decreases Survival After PH. Previous studies have shown that the absence of critical signaling molecules such as c-Jun or TNF-receptor 1 inhibits liver regeneration and decreases survival after PH and is associated with increased microvesicular steatosis.11,22 In the present study, JNK inhibition decreased Fig. 3. SP600125 inhibits cyclin D1 expression after PH. (A) Cyclin D1 protein expression was determined by Western blot analysis in SP600125- or vehicle-treated rats 24 hours after PH. Cyclin D1 expression was quantified using NIH Image software (Bethesda, MD) and normalized to a band from the Coomassie stained blot. (B) Cyclin D1 mRNA and -actin mRNA were detected by reverse-transcription PCR in either SP600125- or vehicle-treated rats 24 hours after PH. Cyclin D1 and -actin bands were quantified by densitometry. Shown is the average of the ratio between cyclin D1 and -actin mRNA in each treatment group.
SP600125 Inhibits Hepatocyte Proliferation and Cyclin D1 Expression in Cultured Hepatocytes. The complete mitogen EGF significantly increases c-Jun phosphorylation in cultured primary rat hepatocytes (Fig. 1C) and drives cell cycle progression in cultured hepatocytes, thereby mimicking some of the pro-proliferative signaling occurring during PH.4,21 To determine whether JNK is critical for EGF-induced proliferation in cultured hepatocytes, we examined the effect of SP600125 on 3Hthymidine incorporation and cyclin D1 expression. EGF induced a 2-fold increase in 3H-thymidine incorporation after 24 hours, and a 6-fold increase after 48 hours (Fig. 4A). When preincubated with SP600125, 3H-thymidine uptake decreased by 48% (24-hour time point, P ⬍ .01) and 67% (48-hour time point, P ⬍ .01) in the EGFtreated hepatocytes and also was lower in the absence of EGF. To test whether cyclin D1 transcription was inhibited by SP600125 in vitro, we transfected hepatocytes with a reporter gene containing the ⫺964 cyclin D1 promoter linked to firefly luciferase (⫺964CD1Luc) or the same reporter gene with a mutated AP-1 site at ⫺954 (⫺964mtCD1Luc).9 EGF induced the expression of this reporter gene 2-fold (Fig. 4B). Pretreatment with SP600125 reduced the transcription of this reporter gene in EGF-treated hepatocytes by 40% but did not affect the internal control Tk-driven renilla luciferase. The reduced luciferase activity was comparable with the level observed
Fig. 4. SP600125 inhibits 3H-thymidine incorporation and cyclin D1 transcription in cultured hepatocytes. (A) 3H-thymidine incorporation was measured in hepatocytes in the presence or absence of EGF (20 nmol/L) in either SP600125-treated (20 mol/L) or DMSO-treated (0.1%) hepatocytes after 24 and 48 hours of treatment. Statistical significance was determined by the t test. (B) Cyclin D1– dependent transcription was determined by reporter gene assay. Hepatocytes were transfected with ⫺964CD1luc or ⫺964mutCD1luc and treated with EGF (20 nmol/L) after SP600125 (20 mol/L) or DMSO (0.1%) pretreatment as described in the Materials and Methods section. Statistical significance was determined by the t test. (C) Cyclin D1 and -actin mRNA levels were determined by PCR 48 hours after EGF (20 nmol/L) stimulation in the presence or absence of SP600125 (20 mol/L). (D) Cyclin D1 and actin protein levels were determined by Western blot analysis 48 hours after EGF (20 nmol/L) stimulation in the presence or absence of SP600125 (20 mol/L).
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Fig. 5. JNK inhibition increases mortality after PH. (A) Rats were pretreated with SP600125 or vehicle 1 hour before PH. Survival was monitored over the following 3 days and is shown as a Kaplan-Meier graph. Statistical significance was determined by the Wilcoxon test. (B) Rats were pretreated with SP600125 or vehicle 1 hour before PH. The levels of aspartate and alanine aminotransferases were determined 60 hours after PH. Statistical significance was determined by the t test. (C) Sections from SP600125- or vehicle-treated animals 60 hours after PH were stained with hematoxylineosin. (D) Rats were pretreated with SP600125 or vehicle 1 hour before PH and livers were harvested 24 or 60 hours after PH. Cells with typical apoptotic morphology were counted in 20 ⫻ 200 fields in 2 different animals per treatment group. The number of apoptotic cells was divided by the total number of parenchymal cells and is expressed as percentage. (E) Caspase 3 activity was determined by incubating cell extracts from livers harvested 24 and 60 hours after PH with the caspase 3 substrate AFC-DEVD for 2 hours. Mouse hepatocytes treated with the agonistic Jo2 antibody served as positive control. Caspase 3 activity is shown as AFC release (pmol) per g protein extract after 2 hours of incubation.
survival 3 days after PH (Fig. 5A, vehicle 89% vs. SP600125 27%, P ⫽ .01, Wilcoxon test) and SP600125treated animals had elevated transaminase levels 60 hours after PH (Fig. 5B, P ⬍ .01). SP600125-treated animals showed a high degree of microvesicular steatosis 2 to 3 days after PH (Fig. 5C). However, vehicle-treated animals also showed microvesicular steatosis, albeit to a lesser degree. To determine whether JNK blockade induced an increase in mortality due to higher rates of apoptosis as shown in animals with defective NF-B and inducible nitric oxide synthase in previous studies,14,23 we quantified apoptotic figures in hematoxylin-eosin–stained liver sections of SP600125- and vehicle-treated animals. In addition, caspase 3–like activity was measured in extracts from these livers. In both vehicle- and SP600125-treated animals, the numbers of apoptotic hepatocytes were low. Twenty-four hours after PH, apoptotic figures were elevated slightly in SP600125-treated animals, but at all other time points the number of apoptotic figures was
similar (Fig. 5D). These findings were confirmed by the low caspase 3–like activity after PH in both SP600125and vehicle-treated animals after 24 and 60 hours, whereas caspase 3–like activity was highly elevated in hepatocytes treated with the agonistic Jo2 antibody (Fig. 5E). Thus, the reduced hepatocyte proliferation in SP600125-treated animals after PH did not appear to be caused by an increase in hepatocyte apoptosis.
Discussion In the adult liver, hepatocytes are long-lived and rarely undergo proliferation, yet they retain a remarkable ability to proliferate.1 This allows the liver to regain its original size within 7 to 10 days after 70% PH and quickly restore function. This regenerative response is initiated by a series of signaling events that allow the hepatocyte to enter the cell cycle and undergo several rounds of proliferation. JNK activation is one of the earliest signals to be detected
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after PH, but the function of JNK in liver regeneration remains largely unknown.4 Several agonists may induce JNK during liver regeneration: (1) TNF-␣ is an important mediator of early-phase JNK activation after PH as shown by the reduced JNK activation and AP-1 binding activity in rats injected with antibodies to TNF-␣ and mice with inactivated TNFR1.3,11 TNF-␣ acts as a comitogen that primes hepatocytes for the pro-proliferative effects of hepatic mitogens including EGF and HGF through the activation of NF-B, AP-1, STAT3, and the release of IL-6, but alone is incapable to induce hepatocyte proliferation.3,24,25 However, the relative role of these pathways in driving proliferation after PH is unclear and some factors such as NF-B may only play a minor role .26 (2) EGF and HGF are believed to play a key role in liver regeneration and both induce JNK activation and AP-1– dependent gene transcription in cultured hepatocytes.4,27 The merging of 2 crucial pro-proliferative signaling cascades (i.e., cytokines and growth factors) at the level of JNK points toward a role for JNK in driving proliferation during liver regeneration. This hypothesis is supported by studies showing JNKs to be an important regulator of cell proliferation in other cell types.8,28 JNKs phosphorylate the N-terminal domain of c-Jun, increase its transactivation, and thereby up-regulate AP-1– dependent transcription. Additionally, JNKs phosphorylate the transcription factors ATF2 and JunD. These factors homodimerize and heterodimerize with other AP-1 components to induce AP-1– dependent transcription and the up-regulation of many genes that are critically involved in cell proliferation and survival.5 JNK Activity Is Required for Hepatocyte Proliferation. The liver contains JNK1 and JNK2, which are believed to fulfill similar functions, but it does not contain the neuronal form JNK3. Because JNK1 ⫺/⫺/JNK2 ⫺/⫺ mice are not viable,29,30 our study used a pharmacologic approach to study the role of JNKs in liver regeneration. The small molecule JNK inhibitor SP600125 blocks both JNK1 and JNK2 in a highly specific manner13 and efficiently inhibited JNK activity in the liver and cultured hepatocytes in our study. Blocking JNK activity with SP600125 inhibited the normal proliferative response after PH, indicating that JNKs are essential components of the cellular machinery that drives hepatocytes to enter the cell cycle after PH. SP600125-treated animals showed a blocked G1/S transition, as apparent by the decreased expression of PCNA and by the reduction of the levels of cyclin D1, which is predominantly expressed during G1. SP600125 additionally reduced mitotic figures after PH and 3H-thymidine incorporation in cultured hepatocytes. At later time points, SP600125-treated animals had similar expression levels of PCNA, indicating the absence of
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early-phase JNK activity still allows hepatocytes to enter the cell cycle, albeit in a delayed manner. JNK Activation Drives Cyclin D1 Expression. Several of the AP-1 regulated genes are critical regulators of cell growth, among them cyclin D1 and PCNA.9,31 The importance of cyclin D1 as a critical downstream target of JNK is suggested by the impaired transcription of cyclin D1 and the reduced activity of cyclin D1– dependent kinases in c-jun ⫺/⫺ mouse embryonic fibroblasts.6,32 In hepatocytes, cyclin D1 plays a crucial role in driving proliferation as recently shown by a study in which cyclin D1 overexpression per se was sufficient to promote hepatocyte replication.10 Moreover, it has been shown that cyclin D1 is the most prominently up-regulated type D cyclin after PH and in response to mitogenic stimuli and plays a critical role in the driving cells through the G1 restriction point.33 Our study found a strong reduction of cyclin D1 protein and mRNA expression in SP600125treated animals after PH. Similar results were obtained in cultured hepatocytes in which cyclin D1 mRNA and protein levels as well as the transcription of a cyclin D1– promoter-driven reporter gene were decreased by SP600125 in EGF-treated hepatocytes. Cyclin D1 expression in the liver may be regulated at the posttranscriptional level.34,35 In our study, however, cyclin D1 was regulated mainly at the transcriptional level through a JNK-dependent pathway, suggesting that cyclin D1 is an important mediator of pro-proliferative effects of JNK. In conjunction with results from previous studies, our data indicates that the growth factor ⫹ TNF-␣ 3 JNK 3 AP-1 3 cyclin D1 pathway is crucial in driving proliferation during liver regeneration. JNK Blockade Decreases Survival After PH. Inactivation of important signaling pathways such as TNF-␣ and IL-6 impairs liver regeneration and decreases survival after PH.11,25 In our study, SP600125 significantly reduced survival 3 days after PH. SP600125-treated animals showed a higher degree of microvesicular steatosis similar to mice with inactivated TNFR1 or c-jun.11,22 However, vehicle-treated animals, also showed increased microvesicular steatosis in our study, presumably due to components in the drug vehicle that may exacerbate the low degree of microvesicular steatosis occurring normally after PH.11 Animals that survived day 3 after PH showed a slightly delayed but ultimately normal liver regeneration with a slightly decreased liver-to-body-weight ratio at day 3 and a normal liver-to-body-weight ratio at day 5 (data not shown). These data are consistent with other studies in which the blockade of single signaling pathways caused a significant delay of liver regeneration and increased mortality, but an ultimately normal regeneration in surviving animals, most likely due to compensatory effects of
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other pathways.25 This hypothesis is supported additionally by our finding that the expression of PCNA was almost as high in SP600125- as in vehicle-treated animals 60 hours after PH. JNK Blockade Does Not Significantly Increase Apoptosis After PH. Previous studies have shown that some signaling cascades after PH such as NF-B, inducible nitric oxide sythase, and Akt function to protect hepatocytes from undergoing apoptosis.14,23,36 JNK is involved in the regulation of cell death in many cell types and may have both pro- or antiapoptotic effects.37 JNK activity and c-Jun are required for normal liver development and appear to protect hepatocytes from apoptosis during development as seen in mice lacking either the JNK kinase SEK1 or c-Jun.38-42 Therefore, we wanted to exclude the possibility that the decreased survival and the decreased proliferation in SP600125-treated animals after PH was caused by an increased rate of apoptosis. Caspase 3–like activity was similar in the treatment groups and apoptotic bodies were only slightly more frequent in SP600125-treated animals 24 hours after PH, but not at other time points. These findings are further supported by data from our laboratory that JNK has proapoptotic effects in TNF-␣–induced apoptosis of hepatocytes43 and reperfusion injury.44 The Role of c-Jun Phosphorylation in Liver Regeneration. The AP-1 component c-Jun is a major contributor of AP-1 activity in quiescent liver and after PH.4 The occurrence of proliferative defects in both JNK 1⫺/⫺/ JNK 2 ⫺/⫺ MEFs and c-jun⫺/⫺ MEFs indicate that c-Jun serves as a major JNK target in proliferation.6,8,32 However, proliferative defects in c-jun⫺/⫺ MEFs are more severe than in MEFs containing a c-Jun with mutated phosphorylation sites 63 and 73,7 and N-terminal phosphorylation of c-Jun is dispensable for liver development and hepatocyte proliferation after PH.22,45 These results indicate that c-Jun has functions independent of its N-terminal phosphorylation. Our study indicates that JNK activity is required for hepatocyte proliferation after PH and in culture and seems to contradict previous studies.22,45 However, JNKs regulate AP-1– dependent transcription through the phosphorylation of several targets including c-Jun, ATF2, and JunD, indicating that inhibition of c-Jun phosphorylation through the use of a mutated c-Jun in previous studies may have been compensated for by other JNK targets. Evidence supporting this hypothesis comes from a recent study showing that the knock-in of a second JunB allele can reverse developmental hepatic abnormalities and embryonic lethality of c-Jun⫺/⫺ mice and up-regulate c-Jun targets including cyclin D1,46 indicating that Jun family members, and potentially other AP-1 components, have a
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higher degree of redundancy than previously appreciated. However, it appears that the presence of c-Jun in the AP-1 complex is absolutely required for cell cycle progression and protection from apoptosis after PH as seen in the c-jun⫺/⫺ mice.22 The striking difference between the complete absence of c-Jun and deletion of its N-terminal phosphorylation sites indicate that unphosphorylated c-Jun may contribute to AP-1– dependent transcription after PH.22 Potential explanations are that (1) heterodimerization of other AP-1 components with c-Jun induces AP-1 transcription independent of the phosphorylation status of c-Jun and/or that (2) JNK targets such as JunD, which lack a JNK docking site, use a JNK docking site containing partners (including mutated c-Jun) for JNK recruitment and their subsequent phosphorylation resulting in increased AP-1– dependent transcription.47 Future studies have to determine whether JNK has other substrates than c-Jun to drive hepatocyte proliferation.
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