Role of MAP kinase signalling pathways in the mode of action of peroxisome proliferators


Peroxisome proliferators (PPs) are a class of non-genotoxic chemicals that cause rodent liver enlargement and hepatocarcinogenesis. In primary rat hepatocytes, PPs cause cell proliferation, suppression of apoptosis and peroxisome proliferation. We have investigated the role of different families of mitogen-activated protein (MAP) kinases in the mode of action of PPs. Addition of 50 μM nafenopin to primary rat hepatocyte cultures caused weak activation of extracellular signal regulated kinases and p38 MAP kinase. However, incubation of primary hepatocytes with the p38 MAP kinase inhibitor SB203580 or the MAP kinase kinase (MEK) inhibitor PD098059 prevented the induction of DNA synthesis and the suppression of transforming growth factor β1-induced apoptosis by the PP nafenopin. In contrast, in the presence of these MAP kinase inhibitors, nafenopin still induced palmitoyl CoA oxidation, a measure of peroxisome proliferation. We have shown previously that PPs such as nafenopin require tumour necrosis factor α (TNF-α) to exert their effects on cellular proliferation and apoptosis. Here we show that treatment of primary rat hepatocyte cultures with nafenopin causes an increase in bioactive TNF-α and that this process requires p38 MAP kinase activity.



Peroxisome proliferators (PPs) are a class of non-genotoxic carcinogens that cause peroxisome proliferation and tumours in rodent liver. The induction of DNA synthesis and suppression of apoptosis are implicated in the mechanism of carcinogenicity for these structurally diverse compounds (14). The action of PPs in rodents is mediated via activation of the peroxisome proliferator activated receptor α (PPARα), a ligand-activated transcription factor that can cause transcriptional regulation of genes associated with peroxisome proliferation and fatty acid β-oxidation (5,6).

The mechanisms by which PPs perturb liver growth are unknown but may involve the pro-inflammatory cytokine tumour necrosis factor α (TNF-α) (7,8). TNF-α is produced mainly by activated macrophages and in smaller amounts by other cell types (9,10). Kupffer cells, the resident hepatic macrophages, are a rich source of cytokines in the liver (11,12) and have been suggested to play a key role in the induction of hepatocyte proliferation caused by PPs (13,14).

Mitogen-activated protein (MAP) kinase pathways contribute to the transmission of extracellular signals that result in the direct or indirect phosphorylation of transcription factors and alterations in gene expression (15). The MEK (MAP kinase kinase) and extracellular signal regulated kinases (ERK) pathway is primarily responsible for responding to cellular proliferation signals, while the p38 MAP kinases and c-Jun N-terminal kinases (JNK) respond to cellular stress signals (1517). p38 MAP kinases are key mediators of the inflammatory response and are activated by cytokines, growth factors and a variety of cellular stresses, including heat shock, ionizing radiation, UV radiation and hyperosmolarity (18,19). Activation of both the p38 and the ERK families of MAP kinases has been shown to be involved in signalling pathways leading to TNF-α release and IL-6 production by macrophages and other cell types (20,21).

To address the mechanism of hepatic growth perturbation by PPs, we have investigated the role of different families of MAP kinases. Using specific inhibitors, we demonstrate that although MAP kinases are only weakly activated by the PP nafenopin, these pathways are required for growth regulation and suppression of apoptosis by PPs. However, neither family of MAP kinases are involved in PP-induced peroxisome proliferation.

Materials and methods


MAP kinase inhibitors PD098059 (22), SB203580 (23) and Ac-DEVD-AMC were purchased from Calbiochem. SB203580 was also obtained from AstraZeneca Pharmaceuticals. Recombinant rat TNF-α was purchased from Insight Biotechnologies Ltd, transforming growth factor β1 (TGF-β1), 7-amino-4-methyl-coumarin (AMC), actinomycin D and epidermal growth factor (EGF) from Sigma, UK. Bromodeoxyuridine (BrdU)-labelling reagents were purchased from Sigma, UK and the anti-BrdU antibody from Roche, UK. All other tissue culture reagents were purchased from Life Technologies, UK.

Cell culture and treatment

Primary rat hepatocyte cultures were prepared as follows: at termination, rats were subject to terminal anaesthesia with ether and hepatocytes isolated by a two-step collagenase perfusion (24) using 0.05% (w/v) collagenase. Hepatocytes (2×106 viable cells) were inoculated into tissue culture flasks in Williams medium E supplemented with 10% fetal calf serum, 10 μg/ml insulin, 0.1 mM hydrocortisone, 2 mM l-glutamine, 100 U/ml penicillin, 0.01% BSA and 100 μg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere and medium was changed after 4 h. MAP kinase inhibitors SB203580 (10 μM) and PD098059 (20 μM) were added 2 h prior to addition of nafenopin which was used at a concentration of 50 μM in order to avoid toxicity. In apoptosis experiments, 5 ng/ml TGF-β1 was added 8 h after addition of nafenopin.

Western blotting

Cells were lysed in Buffer A (50 mM Tris–HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-mercaptoethanol, 1% Triton X-100, 50 mM sodium fluoride, 5 mM sodim pyrophosphate, 10 mM sodium β-glycerophosphate, 0.1 mM PMSF, 1 μg/ml aprotinin, leupeptin, pepstatin). Hepatocyte lysates (50 μg protein/sample) were mixed with NuPAGE sample buffer (Novex) and run on NuPAGE Bis–Tris polyacrylamide gels (Novex) according to the manufacturer's instructions. Equal loading was verified by Ponceau S solution staining (Sigma). Western blotting was carried out using PVDF membranes and anti-phospho-p44/42 MAP kinase antibodies (New England Biolabs).

Measurement of apoptosis and S-phase


Sixteen hours after addition of TGF-β1, the monolayers were fixed with ice-cold methanol for 5 min and then stained with Hoechst 33258 (5 ng/ml) as described previously (25). Cells were then washed in distilled water and mounted in a solution of 20 mM citric acid, 50 mM disodium orthophosphate and 50% glycerol. Apoptotic cells were scored by nuclear morphology.


Eight hours after treatment with nafenopin, cells were incubated with BrdU for a further 16 h. Monolayers were then washed, fixed and incubated in the presence of anti-BrdU antibody. Incorporated BrdU was localized using peroxidase-linked secondary antibody and a diaminobenzidine (DAB) substrate as described previously (25). DNA synthesis was measured by scoring the number of DAB stained nuclei. More than 1000 cells were counted for each flask.

p38 activity assays

p38 MAP kinase activity assays were carried out using an Upstate Biotechnology MAPKAP kinase 2 immunoprecipitation–kinase assay kit, according to the manufacturer's instructions and using [33P]dATP (Amersham, UK). After transfer on P81 phosphocellulose paper, samples were read on a scintillation counter. MAPKAP kinase 2 substrate peptide was also obtained from AstraZeneca Pharmaceuticals. Cells were treated with 25 ng/ml TNF-α for 5 min prior to assaying MAPKAP kinase 2 activity.

Assay for cyanide-insensitive palmitoyl CoA oxidation (CIPCO)

Cells were washed in PBS, scraped in ice-cold PBS and pelleted by centrifugation at 2000 r.p.m. for 2 min. The cell pellet was then resuspended in TES (20 mM Tris–HCl pH 7.4, 5 mM EDTA, 250 mM sucrose), disrupted by sonication and stored at –70°C for CIPCO and protein assays. CIPCO assays were carried out as described previously (26) with some modifications. The assay medium contained 60 mM Tris–HCl pH 8.3, 50 μM Co enzyme A, 370 μM NAD+, 94 mM nicotinamide, 2.8 mM dithiothreitol, 2 mM KCN, 12.5 μg/ml bovine serum albumin (fatty acid free), 100 μg/ml flavin adenosine dinucleotide, 50 μg/ml palmitoyl CoA. Protein concentration in the CIPCO assay aliquot of each sample was assessed using Bio-Rad Bradford protein assay reagent following the manufacturer's instructions. CIPCO activity was expressed as nmol NAD+ reduced/min/mg protein.

Caspase-3 activity assays

Caspase-3 activity in cytosols was prepared using the method outlined in Swanton et al. (27). Cytosols (20 μg) were then incubated with 50 μM Ac-DEVD-AMC for 10 min at room temperature. Using these conditions, AMC release was found to be within the linear range. Samples were diluted to 2 ml in water and the generation of fluorescent product determined using an SLM Aminco fluorimeter.

Measurement of TNF-α levels

L929 cells were seeded in 96-well plates (2.5×104/well) and grown at 37°C/5% CO2. Twenty-four hours later 6 μg/ml actinomycin D was added, 1 h before the addition of 100 μl of medium from hepatocyte cultures. Plates were further incubated at 37°C/5% CO2 for 18 h. After incubation, the medium was removed before addition of 100 μl crystal violet solution (0.2% in 2% ethanol). Plates were washed in water, dried and incubated in 1% SDS before measuring OD at 620 nm. Standard concentrations of TNF-α were used on each individual plate to generate a standard curve.


The PP nafenopin weakly activates ERK MAP kinase and p38 MAP kinase

To establish whether nafenopin activated different MAP kinases directly in primary rat hepatocyte cultures, we analysed the phosphorylated forms of ERK and p38 MAP kinases. Western blotting using antibodies specific for the phosphorylated form of ERK MAP kinases showed strong activation of ERK MAP kinase in primary rat hepatocytes upon treatment with EGF (Figure 1A). However, weak activation of ERK MAP kinase was observed upon treatment with 50 μM nafenopin (Figure 1A). Treatment of primary rat hepatocyte cultures with TNF-α resulted in a 2–3-fold increase in p38 activity (measured by activation of MAPKAP-K2 kinase) (Figure 1B). Under these conditions, however, nafenopin only weakly activated p38 MAP kinase (Figure 1B). In agreement with previous studies (17), the basal levels of p38 MAP kinase were found to be high in primary rat hepatocytes compared with other cell lines (not shown).

Inhibition of MAP kinases prevents the suppression of apoptosis by the PP nafenopin

As described previously (25), addition of the PP nafenopin to primary rat hepatocyte cultures results in the suppression of spontaneous apoptosis and that induced by TGF-β1. Inhibition of TGF-β1-induced apoptosis by nafenopin was measured by scoring changes in nuclear morphology (Figure 2A and B) and by measuring activation of caspases using cytosolic extracts from hepatocyte cultures (Figure 2C). In both cases, nafenopin suppressed TGF-β1-induced apoptosis by ~50%.

To investigate the role of MAP kinase signalling pathways in the suppression of apoptosis by PPs, we evaluated the effects of SB203580 and PD098059 on the response of primary rat hepatocytes to the PP nafenopin. Addition of the p38 MAP kinase inhibitor SB203580 or the MEK inhibitor PD098059 did not affect the levels of spontaneous apoptosis (not shown) or TGF-β1-induced apoptosis (Figure 2A and B). However, pre-treatment of primary rat hepatocyte cultures with SB203580 or PD098059 prevented the suppression of TGF-β1-induced apoptosis by nafenopin (Figure 2A and B).

MAP kinase inhibitors prevent the induction of S-phase by the PP nafenopin

PPs cause cell replication in isolated rat hepatocytes. To determine whether induction of S-phase by the PP nafenopin was dependent on MAP kinases, we treated primary rat hepatocytes with SB203580 or PD098059. Stimulation of primary rat hepatocytes with nafenopin in the presence of SB203580 or PD098059, prevented the PP-induced increase in S-phase (Figure 3). This suggests that MAP kinase activity is required for primary hepatocytes to undergo S-phase in response to PPs.

Inhibition of MAP kinases does not affect CIPCO

Because MAP kinase inhibitors affected the regulation of cell growth by PPs, we examined whether MAP kinases were also involved in peroxisome proliferation. CIPCO measurements were used as an indicator of fatty acid β-oxidation associated with peroxisome proliferation. As expected, nafenopin gave a strong induction of β-oxidation of palmitoyl CoA in this system, but this was unaffected by the presence of MAP kinase inhibitors (Figure 4). Therefore, inhibition of MAP kinases did not affect the induction of fatty acid β-oxidation by nafenopin.

SB203580 prevents TNF-α production in response to PPs

p38 MAP kinase has been implicated in the regulation of TNF-α production in monocytes and other cell types (28). We therefore evaluated the effects of SB203580 on TNF-α production in hepatocyte cultures in response to PP treatment. Levels of bioactive TNF-α were found to increase in primary rat hepatocytes in response to treatment with nafenopin (Figure 5). This increase was inhibited when cultures were pre-treated with SB203580 (Figure 5), indicating that p38 MAP kinase activity is required for the release of bioactive TNF-α in response to PPs.


The mechanisms by which PPs regulate cellular proliferation and survival are still not known. In this study, we have investigated the role of intracellular signalling via different MAP kinases in the mode of action of PPs. Using specific inhibitors, we show that inhibition of different families of MAP kinases prevents the induction of S-phase and suppression of apoptosis by the PP nafenopin but does not prevent peroxisome proliferation. As TNF-α is thought to be a key mediator of the response to PPs, we show that one of the functions of MAP kinase signalling pathways may be to regulate the levels of this cytokine in the liver. Therefore, activation of distinct families of MAP kinases by different hepatic factors may play a permissive role in growth perturbation by PPs.

It has previously been shown that treatment of immortalized liver cell lines and rat hepatocytes with the PP Wy-14,643 causes activation of MAP kinases and induction of immediate early genes (29). The data presented here confirm and extend these observations, to establish a causative link between activation of MAP kinases and effects on cell growth and survival. However, it is yet to be established whether activation of MAP kinase pathways by PPs induces cellular proliferation and suppression of apoptosis by a direct mechanism. In this context, it is worth noting that under the conditions used in this study, PPs are weak mitogens compared with other factors, such as EGF in vitro (30). This may partly explain the weak activation of ERK MAP kinase and p38 MAP kinase observed.

The lack of effect of MAP kinase inhibitors on the levels of fatty acid β-oxidation in primary hepatocytes suggests that the mechanism by which PPs regulate cell growth and survival is distinct from the mechanism of induction of peroxisome proliferation. However, both effects are dependent on the receptor PPARα (31). The diverse effects of PPs in the liver may therefore be mediated by activation of different signalling pathways or by modulation of transcription of different genes. PPARα has previously been shown to be phosphorylated in response to insulin, resulting in enhanced transcriptional response (32). Recently, a ligand-independent transcriptional activation domain in PPARα has been shown to contain MAP kinase sites which are required for transcriptional activation by insulin (33). In contrast, phosphorylation of PPARγ has been shown to inhibit its transcriptional activity (34). Although we have established that MAP kinase activity is not required for PPARα-mediated transcriptional activation of genes required for peroxisome proliferation, we cannot exclude a role for MAP kinases in PPARα-mediated transcription of genes involved in growth regulation.

The inhibition of PP-mediated growth effects by MAP kinase inhibitors shown here, may be the result of indirect effects on signalling pathways triggered by cytokines and growth factors. The p38 MAP kinase inhibitor SB203580 has been shown to have anti-inflammatory properties and to inhibit the production of cytokines, including TNF-α, IL-1 and IL-6 (20,27,35). Indeed, in primary hepatocyte cultures, SB203580 prevented the increase in bioactive TNF-α levels induced by treatment with the PP nafenopin. These findings support a role for TNF-α and perhaps other hepatic cytokines, as mediators of the growth regulatory effects of PPs. In addition, our results support an indirect role for MAP kinases in the induction of cell growth and suppression of apoptosis by PPs. Activation of MAP kinase signalling pathways by hepatic cytokines or growth factors may play a permissive role in PP-induced growth regulation. Activation of ERK MAP kinase and p38 MAP kinase by mitogens or cytokines such as TNF-α may be required for the growth changes to occur. Induction of oxidative stress by PPs (14,36) may also play a role in the activation MAP kinase pathways. In particular, p38 MAP kinase has been associated with oxidative stress (18,37) and has been reported to be constitutively active in mouse liver (19).

Although the ability of the PP class of rodent hepatocarcinogens to cause hepatic cell proliferation and survival have been described extensively, the molecular mechanisms responsible for this perturbation are still unclear. Here we demonstrate that the PP-induced regulation of cell growth and apoptosis involves different molecular mechanisms from the induction of peroxisome proliferation.