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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Conclusion: Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes. (HEPATOLOGY 2009.)

Due to the difficulties of in vivo studies, primary cultures of hepatocytes are widely studied in vitro to examine the expression of liver-specific functions, notably regulation and activity of detoxifying pathways; this is a good method of predicting the hepatic elimination of xenobiotics. Nevertheless, the main obstacle in developing culture conditions that allow these investigations is the maintenance of cell survival and liver-specific functions that rapidly and markedly decrease after hepatocyte seeding.1, 2 Primary culture also represents an interesting tool for cell cycle studies. A growth factor restriction point has been located at two-thirds of G1 phase,3 and hepatocytes usually undergo only one round of division in conventional primary cultures. Many studies have shown that hepatocyte growth factor/scatter factor, epidermal growth factor (EGF), and transforming growth factor α are the primary mitogens for hepatocytes. In addition to their well-known proliferating functions, growth factors are also characterized by pleiotropic activities. EGF has indeed been associated with regulation of cell spreading4 and apoptosis5 through mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK, c-Jun N-terminal kinase, p38, and phosphoinositide-3 kinase/AKT pathways. Consequently, EGF appears to be a major and powerful factor of cell survival. In this report, our aim was to investigate the specific role of early or sequential EGF-dependent ERK1/ERK2 MAPK pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes.

The highly conserved ERK1/2 signaling pathway plays a key role in several physiological events, including the proliferative response of mammalian cells to mitogens. In most vertebrates studied, including mammals, two isoforms of ERK, ERK1 and ERK2, are expressed and activated. Both kinases are closely related (85% identity at the amino acid level) and are activated by MEK1/2 with similar efficiency, and no specific substrate for either enzyme has been identified to date. However, data emerging from the literature indicate that ERK1 and ERK2 MAPKs could have distinct functions in the regulation of proliferation and survival related to specific roles of each kinase and/or according to the ratio of their expression levels. ERK1 knockout mice are viable,6 whereas ERK2 invalidation induces embryonic lethality.7–9 In a model of cardiac ischemic injury, loss of a single allele of Erk2 increases apoptosis of myocardial cells, whereas Erk1 total knockout had no effect.10 ERK2 silencing in C2C12 myoblasts disrupts their terminal differentiation, whereas ERK1 knockdown has little effect.11 Interestingly, Lefloch et al.12 showed in a recent study that when ERK2 expression was reduced using RNA interference (RNAi), proliferation became dependent on ERK1, supporting the hypothesis of shared functions for ERK1 and ERK2 in the regulation of proliferation. Inversely, Vantaggiato et al.13 reported that ERK1 knockout or silencing enhance ERK2 signaling and fibroblast proliferation, whereas ERK2 knockdown abolishes cell cycle progression. In hepatocytes, activation of the ERK1/2 cascade was shown to play a critical role in inducing cyclin D1 at the restriction point in G1 and G1/S transition.14 More recently, we precisely revealed that ERK2 isoform was the key protein for crossing this restriction point.15

Long-term survival and control of multiple cell cycles of hepatocytes is a major challenge. In the present study, we investigated the effects of early and sustained stimulation of rat hepatocytes by EGF after seeding in a serum-free context. We ascertained the effects of such stimulation on the levels of proliferation and long-term survival of cells. We then investigated the ability of hepatocytes cultured in this model to perform multiple rounds of proliferation in association with MEK/ERK pathway activation. Finally, we determined the consequences of specific ERK1 and ERK2 silencing on apoptosis progression using both ERK1−/− mice and RNA interference.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Reagents.

MEK inhibitor U0126 and recombinant human EGF were from Promega (Charbonnières, France). Insulin I-5500, dimethyl sulfoxide (DMSO), colcemid, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and fluorimetric substrate of caspase (Asp-Glu-Val-Asp-7-amino-4-methylcoumarin [DEVD-AMC]) were purchased from Sigma (St. Quentin Fallavier, France). Etoposide was obtained from Merck (Fontenay sous Bois, France). Bromodeoxyuridine (BrdU) was from Amersham Corporation (Buckinghamshire, England). Phospho-ERK1/2 were investigated using a mouse monoclonal antibody directed to a synthetic phospho-peptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAPK (Cell Signaling, Ozyme, France). Polyclonal antibodies against ERK1 (sc-94), ERK2 (sc-154), cyclin A2 (sc-596), cyclin E (sc-481), and cyclin-dependent kinase (Cdk) 2 (sc-163) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–cyclin D1 (DCS-6) and p21WAF1 (CP74) antibodies were supplied from Neomarkers (Fremont, CA). Anti-Cdk1 is a polyclonal antiserum specifically directed against the C-terminal part of human p34.3 Anti–proliferating cell nuclear antigen (PC12) was obtained from Dako (Trappes, France). Anti–myosin light chain antibody was from Sigma. Secondary antibodies conjugated to horseradish peroxidase were obtained from Dako.

Animals.

Wild-type mice (weighing around 20 g) and male Sprague-Dawley rats (weighing around 200 g) were from Charles Rivers (L'Arbresle, France). ERK1−/− mice were obtained from Gilles Pagès (Nice, France). Animals were given food and water ad libitum, and experiments were performed in accordance with French laws and regulations.

Isolation and Primary Culture of Hepatocytes.

Hepatocytes were isolated and purified from mouse and rat liver by in situ perfusion and cultured as described.16 For standard culture conditions, the plating medium was supplemented with 10% fetal bovine serum (FBS). After plating for 4 hours, the medium was removed and cultures were retained in basal medium deprived of FBS and supplemented with 1.4 × 10−7 M hydrocortisone hemisuccinate. EGF stimulations (50 ng/mL) were performed 48 hours after plating. In the EGF model, hepatocytes were cultured in medium deprived of FBS and supplemented with EGF (50 ng/mL) and 1.4 × 10−7 M hydrocortisone hemisuccinate as soon as seeding and were maintained in this medium for the entire culture time.

Small Interfering RNA Transfection.

Small interfering RNA (siRNA) oligonucleotides (Eurogentec, Belgium) were designed, and transfections were performed as described.15 The following sequences with two 3′ deoxythymidine overhands were used: siERK2 (duplex 1), 5′-GUG CUG UGU CUU CAA GAG C-3′; siERK2 (duplex 2), 5′-UCA CAA GAG GAU UGA AGU U-3′; siERK-1 (duplex 1), 5′-UGA CCA CAU CUG CUA CUU C-3′; siERK1 (duplex 2), 5′-CUG GCU UUC UGA CCG AGU A-3′; sip21 (duplex 1), 5′-CAC GUG GCC UUG UCG CUG U-3′; sip21 (duplex 2), 5′-GCC GAU UGG UCU UCU GCA A-3′; and a control siRNA differing from the siERK2 (duplex 1) by three nucleotides.

Immunoblotting Analysis.

Aliquots of 30 μg of proteins were resolved on 7.5% to 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes using a Trans-Blot TM Cell apparatus (Biorad) for 1 hour at 400 mA in Tris buffer 25 mM, glycine 192 mM, ethanol 20% as described.6

Hepatocyte DNA Synthesis and Mitotic Index.

Incorporation of the thymidine analog BrdU was used as an index of cell proliferation. After fixation, BrdU-positive cells were detected using a cell proliferation kit (Amersham, Orsay, France). Mitotic index was determined after treatment with 1 μM colcemid for 12 hours. Cells were fixed and nuclear-stained with Hoechst. Percentages of BrdU-labeled and mitotic hepatocytes were determined in each condition (more than 300 cells per experiment).

Caspase Activity Assay.

Hepatocytes were lysed in the caspase activity buffer containing 20 mM piperazine-N,N′-bis-(2-ethanesulfonic acid) (pH 7.2), 100 mM NaCl, 10 mM dithiothreitol, 1 mM ethylene diamine tetraacetic acid, 0.1% 3-[3-cholamidopropyl-dimethylammonio]-2-hydroxy-1-propanesulfonic acid, and 10% sucrose as described.17 Fifty micrograms of crude cell lysate were incubated with 80 μM DEVD-AMC at 37°C for 1 hour, and substrate AMC was measured (Vmax) via spectrofluorometry (Molecular Devices, Wokingham, England) at 380/440 nm (ex/em).

Cell Viability Measurement.

Cell viability was determined via MTT assay. Briefly, hepatocytes were incubated for 4 hours at 37°C with MTT (0.25 g/L) and dissolved in DMSO. Absorbance (OD value) was measured at 570 nm. The lactate dehydrogenase (LDH) leakage was measured using a Cytotoxicity Detection Kit (Roche). The LDH leakage was expressed as a percentage of the total LDH (extracellular LDH + intracellular LDH). Iron-nitrilotriacetic acid 50 μM was used as a positive control of cytotoxicity.

Statistical Analysis.

Results are expressed as the mean ± standard deviation. Data were analyzed with Student t test. The level of significance was P < 0.05 or P < 0.01. All experiments were performed at least three times.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Highly Proliferating Hepatocytes Depend on ERK2 for Cell Cycle Progression and Replication.

We hypothesized that early and sustained MAPK activation could greatly enhance the cell proliferation capability of normal hepatocytes.

First, DNA synthesis was analyzed via BrdU incorporation (Fig. 1A). In standard culture conditions (FBS at seeding and EGF at 48 hours), only 25 (±5%) of hepatocytes incorporated BrdU from day 3 to day 5. Interestingly, EGF-seeded hepatocytes showed a very high and rapid BrdU incorporation: 70% (±8%) at days 2 and 3 decreasing thereafter. By performing a three-day BrdU incorporation, we determined that 57% (±5%) of hepatocytes from standard cultures entered into S phase, whereas almost all EGF-seeded hepatocytes incorporated BrdU 97% (±2%) (Supplementary Fig. 1A). Hepatocytes accomplished a complete cell cycle as the peak of mitosis was measured between 42 and 66h (Fig. 1B). Time-lapse micro cinematography revealed that both mononuclear (i) and binuclear (ii) hepatocytes underwent mitosis (Fig. 1C). Moreover, early EGF stimulation contributed to maintain hepatocytes in good conditions of survival since apoptotic progression was lower compared to standard cultures (Supplementary Fig. 1B).

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Figure 1. Hepatocyte replication is dependent on ERK2. (A) Hepatocytes were cultured in standard conditions or with EGF at seeding. Percentages of BrdU-labeled hepatocytes were determined via immuno-cytochemistry after 24-hour BrdU incorporation from day 1 to day 9. Representative pictures are shown at day 4. Bar = 60 μm. (B) Mitotic index was determined in EGF-seeded hepatocytes at the indicated times after a 12-hour colcemid treatment (1 μM). Percentages of dividing cells were determined; values are expressed as the mean ± standard deviation. (C) Phase contrast micrographs of EGF-cultured hepatocytes obtained via time-lapse micro-cinematography performed between 24 and 48 hours of culture represent the chronological events of mitosis in (i) mononuclear and (ii) binuclear hepatocytes at the indicated times (in minutes). Black arrows indicate cells under division progression. Bar = 20 μm. (D) Expression levels and/or activation were analyzed in EGF-seeded hepatocytes at the indicated times of culture. Myosin light chain (MLC) was used as a loading control. (E) Time course of [methyl-3H] thymidine incorporation into DNA in hepatocytes seeded in the presence or absence of EGF and U0126 (50 μM). Expression and activation levels of ERK1/2 were analyzed at the indicated times. (F) Time course of [methyl-3H] thymidine incorporation in EGF-seeded hepatocytes transfected with siERK2, siERK1, siERK2 and siERK1, or control siRNA and analyzed 48 hours after transfection.

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We characterized the expression profiles of different cell cycle markers in early EGF-seeded hepatocytes (Fig. 1D). Cyclin D1 (late G1 phase), Cdk1 (G1/S transition), and proliferating cell nuclear antigen (S phase) showed maximal expression levels at day 2 and 3. Cyclin B2 (M phase) expression was maximal at day 4 and 5. Our data confirmed a sustained ERK1/2 activation/phosporylation in the presence of EGF.

To further understand the role of ERK1/2, we first blocked MAPK activation using the specific inhibitor U0126. We confirmed that DNA replication was almost totally inhibited in the presence of U0126 (Fig. 1E). Control experiments showed that ERK1/2 phosphorylations were completely abolished by the MEK inhibitor. Second, specific extinction of ERK1 and ERK2 expressions were performed. As described previously in conventional culture conditions,15 silencing ERK2 in early EGF-stimulated hepatocytes induced an important decrease in DNA synthesis, whereas inhibition of ERK1 did not affect the progression in S phase (Fig. 1F). Dual knockdown of ERK1 and ERK2 decreased DNA replication in the same way as in ERK2 knockdown cells.

Transient MEK/ERK Inhibition Allows Multiple Cell Cycles.

After their first cell cycle, EGF-stimulated hepatocytes failed to perform a second one, despite the elevated viability of the cells (Fig. 2A). Some reports have suggested that sustained activation of ERK inhibits hepatocyte DNA replication, whereas transient activation of this pathway could stimulate DNA synthesis.18–20 We hypothesized that maintained EGF stimulation of hepatocytes that leads to sustained activation of ERK would be responsible for the blockage of progression into a second cell cycle. We first depleted the growth factor from culture for 48 hours and DNA synthesis was then quantified after restimulation (Fig. 2B). EGF depletion allowed a significant part of hepatocytes (11 ± 2%) to re-enter into S phase, but led to a high mortality and rapid apoptotic engagement (data not shown). Then, we transiently inhibited the MAPK pathway for 48 hours with the MEK inhibitor U0126 (Fig. 2C). Interestingly, 1 and 2 days after U0126 removal, 45% ± 5% and 18% ± 4% of hepatocytes could replicate their DNA respectively. Cumulative BrdU incorporation during the 3 days following inhibitor removal showed that 60% ± 5% of the cells replicated their DNA (data not shown).

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Figure 2. Blockage of the MEK/ERK pathway allows engagement of hepatocytes in a new cell cycle. (A) Percentages of BrdU-labeled hepatocytes were determined from day 1 to day 14. (B, C) Percentages of BrdU-labeled hepatocytes were determined in 7-day-old EGF-seeded hepatocytes (B) depleted for 2 days of EGF or (C) treated for 2 days with the MEK inhibitor U0126 or its control solvent DMSO from day 7 and 9. Representative pictures are shown. (D) Hepatocytes were treated with U0126 or DMSO from day 7 to day 9 and from day 13 to day 15. Percentages of BrdU-labeled hepatocytes were determined at the indicated times. Values represent the mean ± standard deviation. *P < 0.05 and **P < 0.01 versus controls.

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Furthermore, we tested the ability of these EGF-seeded hepatocytes to perform a third peak of DNA synthesis by inducing a second 2-day break (between day 13 and day 15) of MEK/ERK activity after the second peak of proliferation (Fig. 2D). In these experimental conditions, we clearly demonstrated that 30% ± 5% and 20% ± 5% of hepatocytes incorporated BrdU 24 hours and 48 hours, respectively, after the second transient MAPK inhibition, showing that EGF-seeded hepatocytes are able to perform at least three successive waves of replication after two sequential MEK/ERK pathway inhibitions.

In parallel, we looked at ERK1/2 activation and cell cycle marker expression during and after U0126 treatment (Fig. 3A). As expected, MEK inhibitor completely abolished ERK1/2 phosphorylation, and the reversion experiment revealed rapid and sustained ERK1/2 activation. Interestingly, all the cell cycle markers dropped during U0126 treatment. After transient MEK inhibition, a progressive induction of these markers could be observed, confirming the re-entry into a second cell cycle.

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Figure 3. Cell cycle re-engagement following MEK inhibition involves down-regulation of p21. (A) ERK1/2, cyclins (D1, E, A2), proliferating cell nuclear antigen, Cdk1, Cdk2, p21 expression, and ERK1/2 activation (P-ERK) were analyzed at the indicated times in EGF-seeded hepatocytes treated with U0126 (U) or control DMSO (D) from day 7 to day 9. (B) ERK1/2, cyclin D1, cyclin E, p21 expression, and ERK1/2 activation were investigated in hepatocytes transfected at seeding with siERK2, siERK1, both siERK1 and siERK2, or control siRNA. Expression levels were analyzed 2 and 4 days after transfection. Myosin light chain (MLC) was used as a loading control. (C) p21 expression in hepatocytes transfected with sip21 (duplex 1 and 2) or control siRNA 24 hours after seeding and analyzed at the indicated time of culture. (D) Percentages of BrdU-labeled hepatocytes were determined from day 1 to day 7 in hepatocytes transfected with sip21 (duplex 1 and 2) or control siRNA. **P < 0.01 versus controls.

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In order to further analyze the specific involvement of ERK1 and ERK2 in these multiple cell cycle regulation, we then silenced both kinases in EGF-seeded hepatocytes via RNAi and looked at expression level of markers of G1 and S phase progression (Fig. 3B). ERK1 and ERK2 expression/phosphorylation were greatly to totally inhibited 2 and 4 days after transfection. Our results established that cyclin D1, cyclin E, and p21 were under an ERK2-dependent mechanism as silencing of the kinase alone or in association with ERK1 induced the drop of their expression. On the other hand, ERK1 silencing did not affect these expression levels. As already shown, ERK1 and ERK2 silencing were accompanied by an increase of ERK2 or ERK1 phosphorylation, respectively.21

Finally, we demonstrated that cell cycle repotentialization was supported in part by a MAPK-dependent p21 expression. We performed a silencing of p21 by RNA interference in EGF-stimulated cells once the first cell cycle was well engaged. Two different p21 siRNAs highly reduced the amount of p21 3 days and 7 days after transfection (Fig. 3C). These p21 inhibitions induced a significant rise of BrdU incorporation at days 6 and 7 as compared with control siRNA (Fig. 3D). These results attest that p21-deficient hepatocytes can progress into S phase of a second cell cycle.

Long-Term MEK Inhibition and ERK1 Knockdown or Knockout Increase Cell Survival.

Interestingly, U0126 treatment of EGF-seeded hepatocytes seemed to emphasize their survival. Consequently, we hypothesized that MEK/ERK blockage could increase long-term survival in MEK-inhibited cultures. Percent of apoptotic cells was significantly lower in U0126 conditions compared with control at 12 and 14 days (Fig. 4A) and U0126-treated hepatocytes showed a lower apoptosis engagement (Fig. 4B). As ascertained by LDH leakage (Fig. 4C) and MTT assays (Fig. 4D), cell viability was increased in U0126 cultures. Finally, we tested the sensitivity of U0126-treated hepatocytes to the apoptotic inducer etoposide (Fig. 4E). Interestingly, while a dose-dependent apoptosis progression was observed in both DMSO- and U0126-treated hepatocytes, U0126 pretreatment conferred a higher resistance to cell death as compared with control noninhibited cultures.

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Figure 4. Permanent MEK inhibition improves hepatocyte survival. (A) Percentage of apoptotic cells was determined using Hoechst staining in hepatocytes treated with U0126 or solvent control DMSO from day 7 to day 14. Representative pictures were taken at day 14. (B) DEVD-AMC caspase activities were investigated at the indicated times. Cells were treated with U0126 or DMSO from day 7 to day 14. (C) Efflux of LDH and (D) cell viability monitored by MTT assay from hepatocytes treated at day 7 with U0126 or DMSO for 48 hours. Treatment with iron-nitrilotriacetic acid (50 μM) for 12 hours was used as a positive control of cytotoxicity. Results are expressed as the mean ± standard deviation. Results are expressed as the mean ± standard deviation. *P < 0.05 and **P < 0.01 versus DMSO-treated hepatocytes. (E) Induction of apoptosis by etoposide (VP16) in U0126- or DMSO-treated hepatocytes. Hepatocytes were first treated at day 7 with U0126 or DMSO for 48 hours; apoptosis was then induced with growing concentrations of VP16 for 7 hours. Results are expressed as the mean ± standard deviation. **P < 0.01 versus control-treated hepatocytes for the same concentrations of VP16.

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We then determined the involvement of ERK1 and ERK2 in cell survival by performing RNAi silencing of each kinase. First, we ascertained a high inhibition of ERK1 and ERK2 in knockdown cells. As already observed,22 dual inhibition led to a strong decrease of both protein expressions. Whereas caspase activity was not significantly modified in hepatocytes silenced for ERK2, ERK1-silenced hepatocytes showed a 50% decrease of apoptosis progression in comparison with control transfected cells. A similar phenotype was observed in hepatocytes dually inhibited for ERK1 and ERK2, confirming the results previously obtained with the MEK inhibitor U0126 (Fig. 4).

In order to confirm the involvement of ERK1 in apoptosis, we investigated apoptotic activity in hepatocytes isolated from wild-type and ERK1 knockout mice (Fig. 5B). Primary hepatocyte cultures from wild-type mice massively performed programmed cell death 4 and 5 days after seeding, whereas ERK1−/− hepatocytes showed a low apoptotic engagement. As expected, ERK1 was not found in hepatocytes from knockout mice (Fig. 5C). Finally, we induced apoptosis in ERK1 knockout and wild-type hepatocytes using etoposide (Fig. 5D). As previously observed in U0126-treated cells, ERK1−/− knockout hepatocytes exhibited an enhanced resistance to apoptosis compared with wild-type hepatocytes.

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Figure 5. ERK1 negatively regulates cell survival. (A) DEVD-AMC caspase activities were investigated in hepatocytes seeded with FBS and stimulated with EGF at 48 hours. Cells were transfected with siERK2 (duplex 1 and 2), siERK1 (duplex 1 and 2), siERK1 and siERK2, and control siRNA 24 hours after seeding. ERK1/2 silencing and caspase activities were investigated 5 days after seeding. Results are expressed as the mean ± standard deviation. *P < 0.05 versus control siRNA. (B) DEVD-AMC caspase assay in EGF-seeded hepatocytes coming from ERK1−/− and wild-type mice at the indicated times of culture. Data represent the mean ± standard deviation. **P < 0.01 versus hepatocytes from wild-type mice. (C) ERK1/2 expressions and activations were analyzed at the indicated times of culture. Myosin light chain (MLC) was used as a loading control. (D) Induction of apoptosis by etoposide (VP16) in hepatocytes coming from ERK1−/− and wild-type mice. Hepatocytes were treated at day 2 with growing concentrations of VP16 for 24 hours. Data represent the mean ± standard deviation. *P < 0.05 and **P < 0.01 versus wild-type mice hepatocytes for the same concentrations of VP16.

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Taken together, our results strongly argue for a critical and specific role of ERK1 in the regulation of apoptosis engagement.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we attest that early EGF-dependent MAPK activation greatly enhanced hepatocyte proliferation capacity. Interestingly, we demonstrated an antagonist effect of sustained MAPK activation. Temporal activation/inhibition are key determinants in generating precise biological responses. Indeed, a transient inhibition of the MEK/ERK pathway after the first cycle results in the reinitialization of new cell cycles. Using siRNA silencing, we show that this process is ERK2-dependent and is supported in part by ERK2-dependent p21 expression. Moreover, sustained blockage of the MEK/ERK pathway is associated with a great reduction of apoptosis engagement of up to 3 weeks, specifically involving ERK1 as demonstrated by survival of ERK1-deficient hepatocytes isolated from ERK1 knockout mice and ERK1 knockdown cells.

Antagonist Effect of Temporal ERK2 MAPK Activation on Hepatocyte Cell Cycle Progression.

Stimulating hepatocytes with EGF at seeding increases their motility leading, to cell–cell contacts and establishment of compact colonies. These morphological effects of EGF have been proved to be MEK/ERK-dependent.4, 23 In this study, we demonstrated that EGF-seeded hepatocytes exhibited a high ability to proliferate, because almost all cells responded to the growth factor and progressed through the S phase, as opposed to commonly used serum-seeded hepatocytes. We established that DNA replication requires growth factor–dependent ERK2 activation. Indeed, early inhibition of ERK2 expression/phosphorylation by RNAi greatly inhibited DNA replication, whereas ERK1 inhibition had no effect on S phase progression. We also showed recently that, in serum-seeded hepatocytes, crossing the growth factor restriction point requires an ERK2 activation leading to accumulation of cyclin D1 and progression to S phase in rodent hepatocytes.15

Several studies reported antagonist effects of MEK/ERK activation on cell proliferation. In PC12 cells, transient induction of the pathway by EGF mediates proliferation, whereas sustained activation by nerve growth factor causes cell cycle arrest and differentiation.21 Multiple hepatocyte cell cycle induction is a challenge for future biotherapy, and some studies indicated that cells could need cyclic stimulations to allow further DNA replication. For instance, multiple rounds of replication could be observed by alternating addition and removal of the solvent in DMSO culture conditions.22, 24 In coculture with liver biliary cells, EGF alone prolonged cell progression up to late G1, whereas tumor necrosis factor α–mediated extracellular matrix remodeling is required for multiple division cycles.25

Our study demonstrated, at a mechanistic level, that sustained activation of the pathway in hepatocytes is responsible for the inhibition of new cell cycles, and the results attest that transient MAPK inhibition after the first cell cycle resensitizes the cells to the mitogen. At least 60 ± 5% of the hepatocytes progressed into S phase after transient MAPK inhibition. Furthermore, cells were able to undergo a third cell cycle after a second transient U0126 treatment. Thus, we clearly demonstrated that it is possible to induce multiple cycles of DNA synthesis by performing cycles of activation/repression of the ERK pathway. Our results clearly emphasize the role of an ERK2-dependent p21 expression in the reinduction of DNA replication. p21 inhibition by RNAi clearly induced a second S phase entry. Indeed, hepatocytes enter S phase more quickly during liver regeneration in p21−/− mice,26–28 and inhibition of p21 expression accelerates S phase entry.28 Recent data from Albrecht et al.27 strongly suggested that liver overgrowth is associated with an antiproliferative response involving p21 and may involve liver size homeostatic responses.

MAPK via ERK1 as a Negative Regulator of Hepatocyte Survival.

Few data concerning the specific functions of ERK1 and ERK2 are reported in the literature. Recent gene disruption experiments have provided demonstrations that ERK1 and ERK2 control specific functions. ERK2 knockout induces embryonic lethality with death of the animals in utero around embryonic day 7.5 due to defects in mesoderm and placental development.7–9 Inversely, ERK1−/− mice are viable, fertile, and ostensibly normal,6 showing that ERK1 is dispensable and could be compensated by ERK2. Additionally, using ERK1 knockout mice, we recently showed that ERK2 was the key form involved in the regulation of hepatocyte replication both in vitro and in vivo, while ERK1 targeting had no effect.15 Proliferation of thymocytes was also ascertained to be specifically dependent on ERK2 signaling through the DN4 stage of development.29 In HeLa cells, however, both ERK2 and ERK1 silencing led to a similar attenuation of proliferation.30 The ERK1 isoform has been shown to be specifically required for in vitro and in vivo adipogenesis.31 In human hepatocellular carcinoma cells, we have underlined that down-regulation of ERK2 by RNAi strongly reduced cell motility and invasiveness, whereas ERK1 inhibition had no impact on hepatoma cell motility.32

In the present study, MEK inhibition by U0126 reduced apoptotic and necrotic progression and extended survival up to 3 weeks. By performing specific RNAi-mediated targeting of ERK1 and ERK2, we did observe an apoptotic protection in rat hepatocytes silenced for ERK1 alone or in combination with ERK2. On the contrary, ERK2 silencing never induced survival or apoptotic engagement. Inversely, in a model of myocardial infarction, loss of one of the two ERK2 alleles is associated with an increase of cardiac cell death, whereas ERK1-null mice showed a level of injury equivalent to that in wild-type mice.10 Using ERK1 knockout mice, we ascertained that apoptotic engagement was slower in ERK1−/− hepatocytes compared to wild type animals, both in basal and apoptosis-inducing conditions. A similar result was also obtained in keratinocytes obtained from these same ERK1 knockout mice, which were shown to be more resistant to apoptotic signals such as UV light, staurosporine, growth factor deprivation, or cycloheximide plus tumor necrosis factor α treatment.33 Our study demonstrates that ERK1 but not ERK2 specifically regulates survival and apoptotic progression. Conversely, silencing ERK1 expression alone was shown to be sufficient to significantly decrease ovarian cancer cells viability.34

Recent data suggests that ERK1 could act as a negative regulator of cell proliferation in fibroblasts by competing ERK2-dependent signaling.13 Indeed, inhibition of ERK1 using RNAi or genetic disruption was accompanied with an increase of ERK2 phosphorylation, probably due to loss of competition between ERK1 and ERK2 for their binding and activation by MEKs. Similar observations were obtained in brain and skin derived from ERK1−/− mice and, as we previously reported, in hepatocytes silenced for one of both kinases.15, 33, 35 Vantaggiato et al.13 associated this inactivation of ERK1 with an increase of the proliferation rate of fibroblasts. We and others have never observed any differences in the proliferation level of ERK1 deficient hepatocytes or fibroblasts.6, 15 However, these findings are discrepant with observations showing no difference in the proliferation rate of ERK1-deficient embryonic fibroblasts,6 whereas RNAi silencing of ERK1 inhibited the proliferation of HeLa cells to the same extent as ERK2 silencing.30 Recent studies emphasize this controversy.36 Using B cell–specific targeted mice, Sanjo et al.36 showed the absolute normal proliferation of B cells lacking ERK2, and proposed that ERK1 compensate for the loss of ERK2. Lefloch et al.12 reported that ERK1 and ERK2 kinase activities are indistinguishable in NIH3T3 cells and proposed that ERK gene dosage is essential and drives their apparent biological differences.

Our results represent an advance in understanding long-term survival and multiple cell cycles regulation of hepatocytes in vitro. However, survival is limited to 2 or 3 weeks, and in vivo experiments would be necessary to improve the regenerating capability of the hepatocytes—a major challenge in biotherapy and in hepatic function recovery. Transient activations could be the key signal allowing S phase entry in vivo.14 Indeed, in regenerating liver, a transient mid-late G1 MAPK activation peak was located just before transient cyclin D1 induction. Events during progression through the initial G1 restriction point such as cyclin D1 and p21 expression must be recapitulated by inhibition/reactivation of ERK2 in subsequent cell cycles, as shown by our in vitro results.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Catherine Ribault for technical assistance and Carly Gamble for critical reading of the manuscript and the Platform Fluorescent Microscopy IFR 140, Rennes.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Guillouzo A. Liver cell models in in vitro toxicology. Environ Health Perspect 1998; 106(Suppl 2): 511532.
  • 2
    Clayton DF, Darnell JE Jr. Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol 1983; 3: 15521561.
  • 3
    Loyer P, Cariou S, Glaise D, Bilodeau M, Baffet G, Guguen-Guillouzo C. Growth factor dependence of progression through G1 and S phases of adult rat hepatocytes in vitro. Evidence of a mitogen restriction point in mid-late G1. J Biol Chem 1996; 271: 1148411492.
  • 4
    Rescan C, Coutant A, Talarmin H, Theret N, Glaise D, Guguen-Guillouzo C, et al. Mechanism in the sequential control of cell morphology and S phase entry by epidermal growth factor involves distinct MEK/ERK activations. Mol Biol Cell 2001; 12: 725738.
  • 5
    Danielsen AJ, Maihle NJ. The EGF/ErbB receptor family and apoptosis. Growth Factors 2002; 20: 115.
  • 6
    Pages G, Guerin S, Grall D, Bonino F, Smith A, Anjuere F, et al. Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 1999; 286: 13741377.
  • 7
    Saba-El-Leil MK, Vella FD, Vernay B, Voisin L, Chen L, Labrecque N, et al. An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development. EMBO Rep 2003; 4: 964968.
  • 8
    Hatano N, Mori Y, Oh-hora M, Kosugi A, Fujikawa T, Nakai N, et al. Essential role for ERK2 mitogen-activated protein kinase in placental development. Genes Cells 2003; 8: 847856.
  • 9
    Yao Y, Li W, Wu J, Germann UA, Su MS, Kuida K, et al. Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation. Proc Natl Acad Sci U S A 2003; 100: 1275912764.
  • 10
    Lips DJ, Bueno OF, Wilkins BJ, Purcell NH, Kaiser RA, Lorenz JN, et al. MEK1-ERK2 signaling pathway protects myocardium from ischemic injury in vivo. Circulation 2004; 109: 19381941.
  • 11
    Li J, Johnson SE. ERK2 is required for efficient terminal differentiation of skeletal myoblasts. Biochem Biophys Res Commun 2006; 345: 14251433.
  • 12
    Lefloch R, Pouyssegur J, Lenormand P. Single and combined silencing of ERK1 and ERK2 reveals their positive contribution to growth signaling depending on their expression levels. Mol Cell Biol 2008; 28: 511527.
  • 13
    Vantaggiato C, Formentini I, Bondanza A, Bonini C, Naldini L, Brambilla R. ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially. J Biol 2006; 5: 14.
  • 14
    Talarmin H, Rescan C, Cariou S, Glaise D, Zanninelli G, Bilodeau M, et al. The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes. Mol Cell Biol 1999; 19: 60036011.
  • 15
    Fremin C, Ezan F, Boisselier P, Bessard A, Pages G, Pouyssegur J, et al. ERK2 but not ERK1 plays a key role in hepatocyte replication: an RNAi-mediated ERK2 knockdown approach in wild-type and ERK1 null hepatocytes. HEPATOLOGY 2007; 45: 10351045.
  • 16
    Guguen Guillouzo C. Isolation and culture of animals and human hepatocytes. New York: John Wiley and Sons, Inc, 2002.
  • 17
    Coutant A, Rescan C, Gilot D, Loyer P, Guguen-Guillouzo C, Baffet G. PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival. HEPATOLOGY 2002; 36: 10791088.
  • 18
    Auer KL, Park JS, Seth P, Coffey RJ, Darlington G, Abo A, et al. Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice. Biochem J 1998; 336: 551560.
  • 19
    Tombes RM, Auer KL, Mikkelsen R, Valerie K, Wymann MP, Marshall CJ, McMahon M, et al. The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic. Biochem J 1998; 330: 14511460.
  • 20
    Park JS, Qiao L, Gilfor D, Yang MY, Hylemon PB, Benz C, et al. A role for both ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21 (Cip-1/WAF1/mda6) protein levels in primary hepatocytes. Mol Biol Cell 2000; 11: 29152932.
  • 21
    Bessard A, Coutant A, Rescan C, Ezan F, Fremin C, Courselaud B, et al. An MLCK-dependent window in late G1 controls S phase entry of proliferating rodent hepatocytes via ERK-p70S6K pathway. HEPATOLOGY 2006; 44: 152163.
  • 22
    Traverse S, Gomez N, Paterson H, Marshall C, Cohen P. Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J 1992; 288: 351355.
  • 23
    Cable EE, Isom HC. Exposure of primary rat hepatocytes in long-term DMSO culture to selected transition metals induces hepatocyte proliferation and formation of duct-like structures. HEPATOLOGY 1997; 26: 14441457.
  • 24
    Kost DP, Michalopoulos GK. Effect of 2% dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture. J Cell Physiol 1991; 147: 274280.
  • 25
    Serandour AL, Loyer P, Garnier D, Courselaud B, Theret N, Glaise D, et al. TNFalpha-mediated extracellular matrix remodeling is required for multiple division cycles in rat hepatocytes. HEPATOLOGY 2005; 41: 478486.
  • 26
    Mullany LK, Nelsen CJ, Hanse EA, Goggin MM, Anttila CK, Peterson M, et al. Akt-mediated liver growth promotes induction of cyclin E through a novel translational mechanism and a p21-mediated cell cycle arrest. J Biol Chem 2007; 282: 2124421252.
  • 27
    Jaime M, Pujol MJ, Serratosa J, Pantoja C, Canela N, Casanovas O, et al. The p21(Cip1) protein, a cyclin inhibitor, regulates the levels and the intracellular localization of CDC25A in mice regenerating livers. HEPATOLOGY 2002; 35: 10631071.
  • 28
    Mullany LK, Nelsen CJ, Hanse EA, Goggin MM, Anttila CK, Peterson M, et al. Akt-mediated liver growth promotes inducation of cyclin E through a novel translational mechanism and a p21-mediated cell cycle arrest. J Biol Chem 2007; 282: 2124421252.
  • 29
    Ilyin GP, Glaise D, Gilot D, Baffet G, Guguen-Guillouzo C. Regulation and role of p21 and p27 cyclin-dependent kinase inhibitors during hepatocyte differentiation and growth. Am J Physiol Gastrointest Liver Physiol 2003; 285: G115G127.
  • 30
    Fischer AM, Katayama CD, Pages G, Pouyssegur J, Hedrick SM. The role of erk1 and erk2 in multiple stages of T cell development. Immunity 2005; 23: 431443.
  • 31
    Liu X, Yan S, Zhou T, Terada Y, Erikson RL. The MAP kinase pathway is required for entry into mitosis and cell survival. Oncogene 2004; 23: 763776.
  • 32
    Bost F, Aouadi M, Caron L, Even P, Belmonte N, Prot M, et al. The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis. Diabetes 2005; 54: 402411.
  • 33
    Bessard A, Fremin C, Ezan F, Coutant A, Baffet G. MEK/ERK-dependent uPAR expression is required for motility via phosphorylation of P70S6K in human hepatocarcinoma cells. J Cell Physiol 2007; 212: 526536.
  • 34
    Bourcier C, Jacquel A, Hess J, Peyrottes I, Angel P, Hofman P, et al. p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1)-dependent signaling contributes to epithelial skin carcinogenesis. Cancer Res 2006; 66: 27002707.
  • 35
    Zeng P, Wagoner HA, Pescovitz OH, Steinmetz R. RNA interference (RNAi) for extracellular signal-regulated kinase 1 (ERK1) alone is sufficient to suppress cell viability in ovarian cancer cells. Cancer Biol Ther 2005; 4: 961967.
  • 36
    Mazzucchelli C, Vantaggiato C, Ciamei A, Fasano S, Pakhotin P, Krezel W, et al. Knockout of ERK1 MAP kinase enhances synaptic plasticity in the striatum and facilitates striatal-mediated learning and memory. Neuron 2002; 34: 807820.
  • 37
    Sanjo H, Hikida M, Aiba Y, Mori Y, Hatano N, Ogata M, et al. Extracellular signal-regulated protein kinase 2 is required for efficient generation of B cells bearing antigen-specific immunoglobulin G. Mol Cell Biol 2007; 27: 12361246.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
hep_22730_sm_SupFig1.tif95KFigure 1S. (A) A 3-day BrdU incorporation was done covering S phase (from day 2 to day 5 for standard culture + EGF, or from day 1 to day 4 for EGF-seeded cultures). Results represent means ± SD. ** P < 0.01 when compared with standard condition. (B) DEVD-AMC caspase activities were investigated in hepatocytes seeded with FCS or with EGF at the indicated times of culture.

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