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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

p38α mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor β, tumor necrosis factor-α, interleukin-1β, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α-deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver-specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life-span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α-deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α-deficient mice during chronic cholestasis through down-regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down-regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α-deficient mice. p38α-knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α-deficient mice. Conclusion: Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis. (HEPATOLOGY 2013)

Mitogen-activated protein kinases (MAPKs) are essential for the cellular response against injury and for regulation of cell death and tissue homeostasis. p38 MAPKs are a family of serine/threonine protein kinases activated by environmental and genotoxic stress that have key roles in the control of cell proliferation, differentiation, and survival, as well as in the regulation of the inflammatory response.1

p38α is the most abundant kinase within the p38 MAPK family and displays relevant biological roles in pathophysiology. Increased proliferation and impaired differentiation have been considered hallmarks of p38α-deficient cells.2 Mice with liver-specific deletion of p38α exhibited enhanced hepatocyte proliferation after partial hepatectomy2 and developed more liver tumors with increased numbers of proliferative tumor cells.3 p38α may repress cell proliferation by antagonizing the c-Jun N-terminal kinase (JNK)/c-Jun pathway.1, 3 Thus, up-regulation of cyclin D1 and Cdc2, two cell cycle regulators that are targets of the JNK/c-Jun pathway, may account for the increased cell proliferation found in liver tumor cells lacking p38α.3 p38α can also regulate cell division at the level of G1/S and G2/M cell cycle checkpoints.1

p38 MAPK activity exhibited an inverse relationship with hepatocyte proliferation during the perinatal and postnatal transitions.4 Accordingly, p38 MAPK activation resulted in hepatocyte growth arrest and inhibition of DNA synthesis in cultured fetal rat hepatocytes.4 Furthermore, inhibition of p38 MAPK in vivo was sufficient to markedly increase the number of proliferating cell nuclear antigen (PCNA)-positive hepatocytes.4

p38α is widely expressed in endothelial and inflammatory cells and it has been involved in the up-regulation of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β.5 Several pathophysiological mediators activate p38α in vitro including transforming growth factor beta (TGF-β), TNF-α, IL-1β, and oxidative stress.4, 6, 7 TNF-α-induced regulated upon activation, normal T-cell expressed, and secreted (RANTES) production in hepatocytes seems to require p38α activation.8 Thus, p38α is often overactivated within inflamed tissues and inhibitors for p38 MAPKs exhibit antiinflammatory effects primarily through repression of inflammatory mediators.5, 9 Accordingly, p38α is a potential target for the treatment of chronic inflammatory disorders and phase 2 clinical trials using p38α inhibitors have been performed during the last two decades.5 However, these clinical studies show disappointing results and a number of side effects such as transaminase elevations.5

Therefore, assessment of the role of long-term p38α deficiency in the progression of chronic inflammation may be of interest on the one hand to understand the pathophysiology of chronic liver diseases and on the other hand to explain potential hepatic side effects upon chronic treatment with p38α inhibitors. Chronic cholestasis induced by bile duct ligation (BDL) causes hepatocyte and cholangiocyte proliferation together with cell death, leukocyte infiltration, oxidative stress, and eventually fibrosis.10-12 p38 MAPK is activated in cholestatic liver, but its role in the onset and development of this disease has not been established. Hence, our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis, particularly to dissect its role in compensatory proliferation of parenchymal cells, inflammation, and fibrogenesis.

Materials and Methods

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

Animals.

To delete p38α specifically in the hepatocytes, we generated mice carrying p38α floxed alleles13 and the Afp-Cre transgene, expressing Cre under the control of the alpha-fetoprotein promoter, which is active during embryonic hepatic development. The liver-specific p38α knockout (KO) mice were kept in a C57BL/6 genetic background. Adult male mice weighing 20 ± 3 g were held in individual cages. Animals were distributed into four groups: two groups that underwent BDL (BDL wildtype [WT] and BDL p38α KO), and two sham-operated group animals (sham WT and sham p38α KO). The total number of mice used was 36: eight BDL mice after 12 days cholestasis, 12 BDL mice after 28 days cholestasis, and 16 control mice. Animals were euthanized under anesthesia at 12 and 28 days postsurgery. All mice received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23 revised 1985). The study was approved by the Ethics Committee of Animal Experimentation and Welfare of the University of Valencia (Valencia, Spain).

BDL was performed as described.10 Liver injury and function were assessed as indicated in the Supporting Material and Supporting Figs. S1 and S2. For biochemical assays, western blotting, and real-time reverse-transcription polymerase chain reaction (RT-PCR), see the Supporting Material. For histological analysis and immunofluorescence, see the Supporting Material.

For binucleation rate and number of nuclei per field, 50-60 slides from all different animals were blindly scored. In BDL animals, foci with high inflammatory infiltrate were avoided.

Statistical Analysis.

All results are given as mean ± standard deviation (SD). Significant differences were assessed by one-way analysis of variance (ANOVA) followed by a Tukey's post-hoc test. Survival curves were constructed by the Kaplan-Meyer method and analyzed for differences using the score test of Cox proportional hazards model for grouped data.

Results

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

Survival of Cholestatic Mice Is Reduced by Liver-Specific p38α Deficiency.

A survival curve was performed in order to assess if there were differences between the WT group and p38α KO mice. The mean life-span was significantly less in mice with a deficiency of p38α after BDL than in WT mice. The Kaplan-Meier curve (Fig. 1) showed that WT mice had a favorable response to BDL in comparison with KO mice (P < 0.01). BDL p38α KO mice had a 50% decrease in mean life-span (30.16 ± 4.06 days versus 59.2 ± 7.2 days) and a significant difference in maximum lifespan (76 days versus 106 days) versus WT BDL mice. The presence of p38α was also associated with a delay in the onset of mortality.

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Figure 1. Survival curve after cholestasis induction in WT and liver-specific p38α KO mice. Both groups, WT (n = 20) and p38α KO (n = 18) of mice underwent BDL. Statistical differences in animal survival were found between groups following a Kaplan-Meier test. P < 0.001 was obtained using the score test of the Mantel-Cox proportional hazards model for grouped data.

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p38α Signaling Pathway Is Acting Through MK2 in Liver Cholestasis.

p38α phosphorylation was increased in WT BDL mice upon chronic cholestasis (Fig. 2). This activation of p38α led to a significant increase in MAPK-activated kinase 2 (MK2) phosphorylation on threonine 334. Indeed, only in WT BDL mice there was a significant increase in phosphorylation of MK2 and, therefore, activation of MK2, when compared with WT sham mice and KO mice. It has been reported that MK2 can phosphorylate Akt on serine 473.14

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Figure 2. p38α downstream signaling pathway (I) (A) Signaling proteins related to the p38α pathway. (B) Representative western blot images from total liver homogenates WTS, sham (n = 8); KOS, sham (n = 8); WTB, BDL (n = 6); KOB, BDL (n = 6). Samples were analyzed after 28 days of cholestasis induction. α-Tubulin was used as loading control. Densitometries are shown in Supporting Fig. S3.

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We also tested two other regulators of Akt, phosphoinositide-dependent kinase-1 (PDK1) and phosphatase and tensin homolog (PTEN), but no differences were found in their phosphorylation and protein levels among groups. Similar results were obtained 12 days after BDL (data not shown). Quantification of the western blots is shown in Supporting Fig. S3.

p38α Deficiency Causes a Decrease in AKT Phosphorylation.

The p38α downstream pathway was assessed starting with one of its major targets, MK2. As shown in Fig. 2B, phosphorylation of MK2 on threonine 334 was strongly regulated by this pathway. However, neither PDK-1 nor PTEN levels and phosphorylation were modified upon p38α deficiency. Akt may be phosphorylated on serine 473 by p-MK2 and this phosphorylation was markedly reduced upon p38 deficiency, whereas phosphorylation on threonine 308 remained unaffected (Fig. 3A). Other downstream targets such as mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) 3β were phosphorylated after BDL in a p38α-dependent manner (Fig. 3B). GSK3β phosphorylation only increased markedly in WT BDL mice, which would inactivate the enzyme. One of the major targets of GSK3β is β-catenin, which exhibited an increase only in BDL WT mice. The same western blots were performed with mice after 12 days of BDL (results not shown).

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Figure 3. p38α downstream signaling pathway (II). Representative western blot images from total liver homogenates WTS, sham (n = 8); KOS, sham (n = 8); WTB, BDL (n = 6); KOB, BDL (n = 6). Samples were analyzed after 28 days of cholestasis induction. α-Tubulin or Ponceau was used as loading control. (A) Phosphorylation of Akt and mTOR. (B) GSK3β phosphorylation is increased BDL WT compared with sham (P < 0.01). Western blot densitometry revealed higher GSK3β phosphorylation and β-catenin levels in WT BDL compared to KO BDL (P < 0.05). Data are shown as mean and SD. Black, WT; white, KO. Statistical significance was analyzed by one-way ANOVA. *P < 0.05 WT versus KO; **P < 0.01 WT versus KO; #P < 0.05 BDL versus sham; ##P < 0.01 WT versus KO BDL.

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Inflammation, Apoptosis, and Fibrosis Profiles Associated with p38α Deficiency in Chronic Cholestasis.

The inflammatory and profibrogenic profiles were assessed in WT and p38α KO mice (Fig. 4). p38α KO mice had higher messenger RNA (mRNA) levels of some proinflammatory cytokines, such as RANTES under basal conditions (Fig. 4B), and the BDL group had higher mRNA levels of adhesion factor Icam-1 (Fig. 4C). Although TNF-α expression was not affected by the absence of p38α, the mRNA levels of receptor 1 for TNF-α increased in p38α KO mice, making these animals likely more sensitive to this cytokine (Fig. 4A). On the other hand, the antiinflammatory cytokine IL-10 mRNA level markedly increased in p38α KO BDL mice after 12 days of BDL (Fig. 4B), probably to restrain the inflammatory response. STAT3 phosphorylation was increased after BDL similarly in both WT and KO mice (Supporting Fig. S4). However, no significant changes in phosphorylation of p65 were found upon BDL (Supporting Fig. S4).

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Figure 4. Inflammation and fibrosis after BDL (12 and 28 days). (A) Real-time PCR analysis of the mRNA for TNF-α and its receptors 1(tnfsr1a) and 2(tnfsr1b); (B) interleukin 6 and interleukin 10, RANTES and its receptor Ccr5; (C) adhesion molecules CCl2 and Icam1 (D) profibrotic gene Timp1 and collagen a1. Data are shown as fold increase in mRNA level compared to the control liver tissue and were normalized by TATA-binding protein mRNA. Data are the mean and SD of n(wts) = 8; n(kos) = 8; n(wtb 12 days) = 4; (wtb 28 days) = 6 (kob 12 days) = 4; n(kob 28 days) = 6. Statistical significance was analyzed by one-way ANOVA. *P < 0.05 WT versus KO; **P < 0.01 WT versus KO; #P < 0.05 BDL versus sham; ##P < 0.01 WT versus KO BDL; $P < 0.05 BDL 28 days versus BDL 12 days; $$P < 0.01 BDL 28 days versus BDL 12 days. (E) Representative images of Sirius Red staining in liver tissue slides, WT mice after 28 days of BDL; (F) KO mice after 28 days of BDL. At least 20 fields per animal were examined. A minimum of four experiments were performed for each group of animals. Scale bars = 100 μm.

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Liver-specific p38α-deficient mice did not show a higher degree of apoptosis upon chronic cholestasis compared with WT mice (Fig. S5). Indeed, the cleavage of caspase 3 (Fig. S5) showed no further increase in apoptosis upon p38α deficiency.

We have previously shown that p38α KO cardiomyocytes produce increased levels of collagen.15 Therefore, we also studied fibrogenesis by measuring mRNA expression of collagen and metalloprotease inhibitor (Timp 1), which decreased in p38α KO after they underwent BDL (Fig. 4D), showing that these KO mice exhibited a lower profibrotic stage. Moreover, collagen deposition in the liver by the Sirius Red staining method showed that p38α KO mice did not exhibit more fibrosis than WT mice in chronic cholestasis (Fig. 4E,F). Consequently, liver fibrosis does not seem to account for the reduced life span of p38α KO BDL mice.

p38α Deficiency Decreased Albumin and Increased HSP27 Levels.

Albumin is considered a marker of hepatic function, and hence a decrease in its synthesis could indicate impairment of the liver capacity for protein synthesis. p38α KO sham mice already showed decreased albumin mRNA levels before inducing the illness (Fig. 5A). In WT mice, the decrease in albumin mRNA expression began when BDL was performed, so the reduction in the albumin mRNA levels was seen at 12 days of cholestasis, and kept on descending until 28 days (Fig. 5A). In the p38α KO group, mRNA expression of albumin remained at the same low level during evolution of the illness, showing also no significant differences with the p38α sham group at 12 and 28 days (Fig. 5A). Albumin immunohistochemical staining showed the same profile (Fig. 5B). Since activation of p70 S6 kinase and subsequent phosphorylation of S6 ribosomal protein are involved in protein synthesis and are downstream MAPK, we assessed activation of this pathway. Figure 6 and Supporting Fig. S6 show significantly reduced phosphorylation of p70 S6 kinase and S6 only in p38-deficient mice after BDL. Since endoplasmic reticulum stress might also contribute to the reduced albumin synthesis, we measured GADD 153 and phosphorylation of eIF2a. Supporting Fig. S7 shows that liver-specific p38-deficient mice did not exhibit more endoplasmic reticulum stress than WT mice.

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Figure 5. Hepatic expression of albumin and HSP27. (A) Real-time PCR analysis of the expressions of mRNA for liver albumin. Data are shown as fold increase in mRNA level compared to the control and were normalized by TATA-binding protein mRNA. Data are the mean and SD of n(wts) = 8; n(kos) = 8; n(wtb 12 days) = 4; (wtb 28 days) = 6 (kob 12 days) = 4; n(kob 28 days) = 6. Statistical significance was analyzed by one-way ANOVA. *P < 0.05 WT versus KO; **P < 0.01 WT versus KO; #P < 0.05 BDL versus sham; ##P < 0.01 WT versus KO BDL; $P < 0.05 BDL 28 days versus BDL 12 days; $$P < 0.01 BDL 28 days versus BDL 12 days. (B) Representative images of albumin staining in liver tissues in the indicated liver tissues after sham or 28 days BDL. A minimum of 4 experiments were performed for each group of animals. Scale bars = 100 μm. (C) Representative western blot images from total liver homogenates n(wts) = 8; n(kos) = 8; n(wtb) = 6; n(kob) = 6. Protein extracts from sham and BDL animals were taken after 28 days of cholestasis induction. α-Tubulin was used as loading control. (D,E) Immunofluorescence analysis of HSP27 in WT (D) or KO mice (E) after 28 days of BDL. A minimum of four experiments were performed for each groups of animals. Scale bars = 50 μm.

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Figure 6. Effect of p38α on hepatocyte size. (A) Number of hepatocytes per field in sham and BDL animals after 12 and 28 days of BDL. At least 20 fields per animal were examined. A minimum of four experiments were performed for each groups of animals. Black: WT; white: p38α KO. Data are shown as mean and SD. Statistical significance was analyzed by one-way ANOVA. **P < 0.01 WT versus KO; ##P < 0.01 BDL versus sham; $$P < 0.01 BDL 28 days versus BDL 12 days. Representative images of hematoxylin-eosin histological staining in liver tissue of WT sham (B), KOS (C); WT BDL after 28 days (D); and KO BDL after 28 days (E). Experiments were performed four times. Scale bars = 100 μm. (F) Representative western blot images of phospho-p70 S6 kinase (phosphorylated on T389), p70 S6 kinase, phospho-S6 protein (phosphorylated on S235), S6 protein, and α-tubulin [n(wts) = 8; n(kos) = 8; n(wtb 12 days) = 4; (wtb 28 days) = 6 (kob 12 days) = 4; n(kob 28 days) = 6]. α-Tubulin was used as loading control. Corresponding densitometries are shown in Supporting Fig. S6.

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On the other hand, looking for cellular structure alterations we investigated HSP27, a phosphorylation target of MK2, and found a significant increase (P < 0.01) in phospho-HSP27 by western blotting only in BDL WT mice but not in p38α KO mice (Fig. 5C; Supporting Fig. S3). Interestingly, p38α KO BDL mice had higher expression of HSP27 in comparison with WT BDL mice (Fig. 5C). This result was confirmed by confocal image analysis (Fig. 5D,E).

At this point, considering all the parameters that were indicating that p38α KO BDL mice exhibited worse liver conditions and could suffer from the illness more than the WT BDL mice, we decided to study the evolution of hepatomegaly and cell size in the progress of the illness.

p38α KO Mice Have Smaller Hepatocytes than WT Ones.

Comparing the hepatocyte size at the very beginning, we found differences between WT sham and p38α KO sham (Fig. 6A). In addition, when mice underwent BDL a different phenomenon was observed. In a WT liver, cell growth tried to compensate for liver injury and loss of function. This process can be observed in the hematoxylin and eosin images, where WT BDL cells tend to be bigger (Fig. 6). On the other hand, p38α KO mice hepatocytes kept the same smaller size after BDL and, therefore, remained small after impairment of liver function. We assessed this alteration in two ways: by measuring the hepatocyte area and by counting the number of nuclei in each field (Fig. 6A). Both parameters showed the same profile as markers of cell growth. We also assessed the involvement of the p70S6 kinase pathway, downstream of Akt/mTOR, in the reduction of hepatocyte growth. Figure 6C shows increased phosphorylation of both p70S6 kinase and S6 protein in WT mice upon BDL, whereas this phosphorylation was much less intense in p38α-deficient mice (Supporting Fig. S7).

p38α KO Mice Exhibit Less Hepatocyte Proliferation and Liver Mass.

When WT mice underwent BDL, an adaptive response was found in order to compensate for the injury-induced loss of liver function at 12 days postinduction, which consisted of an increase of liver weight (Fig. 7A).

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Figure 7. Effect of p38α on hepatocyte proliferation. (A) Liver weight evolution after BDL [n(wts) = 8; n(kos) = 8; n(wtb 12 days) = 4; (wtb 28 days) = 6 (kob 12 days) = 4; n(kob 28 days) = 6]. Data are the mean and SD. Statistical significance was analyzed by one-way ANOVA. *P < 0.05 WT versus KO; **P < 0.01 WT versus KO; #P < 0.05 BDL versus sham; ##P < 0.01 WT versus KO BDL; $P < 0.05 BDL 28 days versus BDL 12 days; $$P < 0.01 BDL 28 days versus BDL 12 days. (B) Representative western blot images from liver nuclei homogenates using the indicated antibodies [n(wts) = 8; n(kos) = 8; n(wtb 12 days) = 4; (wtb 28 days) = 6 (kob 12 days) = 4; n(kob 28 days) = 6]. H3 was used as loading control. (C) Mitotic index was measured by the ratio between phosphorylated Histone 3 and PCNA. This ratio shows the number of cells in the mitosis phase, which are also proliferating. (D) Ratio PCNA (versus H3) / alanine amino transferase (ALT): Measurement of hepatic proliferative response after liver injury34 shows reduced proliferation in p38α KO livers after stress stimuli. Black, WT; white, KO. Data are shown as mean and SD. (E) Immunofluorescence at 12 days after BDL: WT BDL mice after 12 days of BDL; KO BDL mice after 12 days of BDL. ki67 as a proliferative marker (red), pH3 (S10) as a mitotic marker (green). Representative images of liver sections: n(wts) = 8; n(kos) = 8; n(wtb 12 days) = 4; (kob 12 days) = 4. A minimum of four experiments were performed for each groups of animals. Scale bars = 50 μm.

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At 12 days after BDL WT animals had larger livers than the p38α KO ones, although liver size decreased at 28 days after cholestasis induction. This phenomenon partially fits with the observation that WT mice had larger hepatocytes than the p38α KO mice. Nevertheless, it was necessary to check if cell proliferation was also related to the enlargement of the liver. We assessed cell proliferation by performing western blotting of nuclei fraction to detect PCNA (Fig. 7B; Supporting Fig. S6). Only WT BDL mice at 12 days had higher levels of PCNA. Similar findings were observed by confocal image analysis using ki-67 as a marker of proliferation (Fig. 7E). As described previously, the absence of p38α may produce a delay in mitotic exit, which means that cells remain longer in mitosis than expected.16 This delay may be estimated by measuring the mitotic index (pH3/PCNA), which indicates the ratio between mitotic cells and proliferating cells.16 Calculating this index with our data we found that p38α KO BDL mice livers had high mitotic index and low proliferating response, suggesting that proliferating cells may suffer from a delay in mitosis (Fig.7C).

After 12 days of cholestasis, p38α KO mice responded with less cell proliferation than the WT ones, and similar results were observed after 28 days of BDL (Fig. 7D).

Since p38α may antagonize the JNK pathway1 which may also affect cell proliferation, we measured phospho-JNK but it did not increase significantly in liver of p38-deficient mice (Supporting Fig. S8).

p38α Deficiency Increases Cyclin Levels but Induces Mitosis Blockade and Cytokinesis Failure.

First we wanted to check, using cholestasis as a stimulus, if the role of p38α in cell cycle checkpoints was conserved. Sham mice did not show any changes in cyclin levels, but in the BDL groups an increase of cyclin levels occurred when p38α was knocked out. These mice exhibited more cyclin D1 and B1 expression after cholestasis induction, and hence, interphase could be taking place faster than in the BDL WT animals. The increase in cyclin B1 and D1 levels was much more intense in p38α KO animals after 12 days of BDL (Fig. 8A). We also assessed the expression of p21 by western blot, and found a progressive increase during cholestasis only in p38α KO mice (Fig. 8A). This could be related to the cell cycle alterations observed upon p38α down-regulation. The mitotic delay could be reflected in an increase in polyploid nuclei (nonmitotic nuclei division) or binucleated cells (nonmitotic cytoplasm division). By identifying nuclei with 4,6-diamidino-2-phenylindole (DAPI) and cellular membranes with anti-β-catenin immunostaining, we found that p38α KO mice exhibited a higher rate of binucleated hepatocytes than WT mice both before and after BDL, whereas no differences were observed between BDL and sham groups in p38α KO animals (Fig. 8). In contrast, the percentage of binucleated cells in WT sham livers was markedly reduced when they start proliferating after induction of cholestasis.

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Figure 8. Effect of p38α on hepatocyte cell cycle and cytokinesis. (A) Representative western blot images from liver nuclei homogenates [n(wts) = 8; n(kos) = 8; n(wtb 12 days) = 4; (wtb 28 days) = 6 (kob 12 days) = 4; n(kob 28 days) = 6] for p21, cyclin D1, and cyclin B1. H3 was used as loading control. Western blot densitometry showed significant differences between groups. Data are the mean and SD. Black, WT; white, KO. Statistical significance was analyzed by one-way ANOVA. *P < 0.05 WT versus KO **P < 0.01 WT versus KO; #P < 0.05 BDL versus sham; ##P < 0.01 WT versus KO BDL; $P < 0.05 BDL 28 days versus BDL 12 days; $$P < 0.01 BDL 28 days versus BDL 12 days. (B) Representative DAPI staining image of WT sham, (C) KO sham, (D) WT after 28 days of BDL, (E) KO after 28 days of BDL. At least 80 nuclei per field were examined. A minimum of four experiments were performed for each groups of animals. Scale bars = 100 μm. (F) Ratio of binucleated hepatocytes per field in sham and BDL animals after 12 and 28 days of cholestasis induction both in WT and KO groups. At least 20 fields per animal were examined.

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Discussion

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

p38α Deficiency Reduces Hepatocyte Growth Through AKT/mTOR Down-regulation.

The two major groups of proteins that are regulated by p38 MAPK-mediated phosphorylation are protein kinases, such as MK2, and transcription factors, such as p53.1 p38α may negatively regulate AKT activity independently of PI3K, by regulating its interaction with PP2A or through the activation of MK2 (Fig. 2A). Indeed, MK2 mediates HSP27-dependent activation of AKT by way of phosphorylation on Ser473.13 Accordingly, phosphorylation of MK2 on Thr334 and of AKT on Ser473 were markedly reduced in liver of p38α-deficient mice upon chronic cholestasis (Figs. 2B, 3A). Activation of AKT triggers a key antiapoptotic signaling pathway in the liver. However, in our model of chronic cholestasis the lack of AKT activation did not lead to increased apoptosis (Supporting Fig. S5).

PDK1 and AKT are required for normal cell growth and liver regeneration after partial hepatectomy.17 Taking into account the absence of significant PDK1-mediated AKT phosphorylation on Thr308 upon chronic cholestasis (Fig. 3A), it seems that p38α/MK2-dependent AKT activation is essential for liver regeneration and hepatomegaly in this chronic disorder. In vitro in rat hepatoma cells, AKT activation increased cell size through mTOR-dependent and mTOR-independent pathways and the latter also involved inhibition of protein degradation.18 Accordingly, p38α deficiency may reduce hepatocyte growth during chronic cholestasis through down-regulation of both AKT and mTOR (Fig. 3A).

Similar to yeast, in mammals two distinct protein kinase mTOR complexes have been characterized. mTORC1 is rapamycin-sensitive and controls protein synthesis, whereas mTORC2 is rapamycin-insensitive and controls the actin cytoskeleton.19 Hence, down-regulation of mTOR may contribute to reduce albumin levels in liver of p38α-deficient mice. The Akt/mTOR pathway may lead to activation of the p70 S6 kinase/S6 pathway. Our findings suggest that blockade of this pathway seems to be involved in the lack of hepatocyte proliferation and growth that occurs upon p38α deficiency during chronic cholestasis. Endoplasmic reticulum stress could also contribute to reduced albumin synthesis but this idea was discarded as GADD 153 and eIF2α phosphorylation did not increase in liver-specific p38α-deficient mice upon cholestasis (Supporting Fig. S7).

p38α may inactivate GSK3β by direct phosphorylation of Ser389 or indirectly through phosphorylation of Thr9 by Akt, leading to β-catenin accumulation.20 Thus, p38 modulates canonical Wnt-β-catenin signaling, which is critical for normal cell proliferation and homeostasis.21

Inactivation of GSK3β produces embryonic lethality caused by severe liver degeneration associated with hypersensitivity to TNF-α and reduced NF-κB function.22 Inhibition of GSK3β may sensitize rat hepatocytes to apoptosis by reducing p65 phosphorylation and down-regulating NF-κB transactivation.23 In p38α-deficient livers, activation of GSK3β due to reduced phosphorylation (Fig. 3B) does not seem to be associated with changes in apoptosis or p65 phosphorylation upon BDL (Fig. S4).

p38α Deficiency Enhances the Long-Term Inflammatory Response in the Liver.

The p38α pathway is also involved in the up-regulation of inflammatory cytokines. p38 may positively regulate NF-κB activity by different mechanisms, including chromatin remodeling through Ser10 phosphorylation of histone H3 at NF-κB-dependent promoters or by impinging on IκB kinase (IKK) or the p65 subunit.20 However, in chronic cholestasis p38α deficiency did not significantly affect NF-κB activation (Supporting Fig. S4) or the expression of TNF-α and interleukin-6 (see Fig. 4). Nevertheless, RANTES and receptor 1 of TNF-α were up-regulated in the liver of p38α-deficient mice under basal conditions and remained high during the first 12 days after cholestasis (Fig. 4B). RANTES is one of the major adjacent cysteines motif (CC) chemokines that is produced by T-lymphocytes, monocytes, endothelial cells, and fibroblasts. It is worth noting that expression of antiinflammatory IL-10 was markedly up-regulated at 12 days after cholestasis induction only in p38α KO mice, which should provide protection restraining the inflammatory response, but this protection was lost in the long term (i.e., at 28 days) leading to up-regulation of Icam-1 and chemokine (C-C motif) ligand 2 (Ccl2) (Fig. 4B).

Although previous reports have associated p38α with the regulation of apoptosis and fibrogenesis, liver-specific p38α-deficient mice did not show a higher degree of apoptosis or fibrosis upon chronic cholestasis compared with WT mice (see Fig. 4 and Supporting Fig. S5). Hence, neither apoptosis nor fibrosis would contribute to the increased mortality of these animals.

p38α Deficiency Induces Mitosis Blockade and Cytokinesis Failure in Hepatocytes.

p38α controls the differentiation and proliferation of many cell types, including hepatocytes.4, 24, 25 p38α may negatively regulate cell cycle progression at the G1/S and the G2/M transitions triggering cell cycle arrest by down-regulation of cyclins, up-regulation of cyclin-dependent kinase inhibitors, and by inducing p53 phosphorylation and the up-regulation of p16.1, 24 p38α-deficient myogenic cells exhibited delayed cell-cycle exit and continued proliferation under conditions that normally induce cell-cycle withdrawal and differentiation.26 p38α controls myoblast proliferation by antagonizing the proliferation-promoting function of JNK, and this effect is at least in part mediated by up-regulation of the phosphatase MAPK phosphase-1 (MKP-1).26 Hence, p38α and JNK MAPKs may exert antagonistic effects on cell proliferation and survival.1 However, phospho-JNK did not increase upon cholestasis in the liver of p38α-deficient mice (Fig. S8) and therefore the JNK pathway would not contribute to the reduced cell proliferation in our chronic model.

PCNA is expressed in replicating cells during S phase, thus allowing detection of dividing cells. The number of PCNA-expressing cells was higher in skeletal muscle from mice deficient in p38α than in WTs.26 Continuous myoblast proliferation and reduced myofiber growth were attributed to the persistence of cyclin D1.26 Indeed, down-regulation of cyclin D1 by p38α has been reported in different cell types.26 Accordingly, inhibition of p38α in vivo was sufficient to stimulate hepatocyte cell cycle activity, whereas p38α activation resulted in hepatocyte growth arrest and decreased cyclin D1 in cultured fetal rat hepatocytes.4 Accordingly, cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis (see Fig. 8). However, PCNA was surprisingly down-regulated at 12 days after cholestasis induction and the mitotic index was extremely high in long-term cholestasis in p38α-deficient mice (i.e., at 28 days) (see Fig. 7). Hence, unexpectedly p38α deficiency blockades progression of mitosis towards the S phase in hepatocytes during the initial course of chronic cholestasis. The increased death rate that occurs in liver-specific p38α KO mice could be due to the blockade of hepatocyte growth with impaired protein synthesis and lack of proliferative adaptive response in the liver.

Cardiac-specific p38α-KO mice exhibited an increase in neonatal cardiomyocyte mitoses and inhibition of p38α in adult cardiomyocytes promotes karyokinesis and cytokinesis.25 However, liver-specific p38α-KO mice exhibit cytokinesis failure evidenced by enhanced binucleation rate (see Fig. 8). Moreover, as chronic cholestasis evolves, the binucleation rate decreases in WT animals, whereas it remains high in p38α-deficient mice.

Incomplete cytokinesis may be associated with developmental or pathological cell division programs leading to polyploid progenies.27, 28 AKT activity regulates cytoskeleton organization and its down-regulation might be involved in cytokinesis failure.29 Indeed, during postnatal development binucleated tetraploid cells arise in the liver due to AKT-mediated failure in cytokinesis.29 Down-regulation of mTOR might also contribute to the p38α-dependent AKT-mediated cytokinesis failure since complex mTORC2 also controls the actin cytoskeleton.19

AKT and GSK3β cooperate in spindle formation.29 AKT phosphorylates GSK3β decreasing its activity. By limiting the activity of GSK3β at the cell cortex and at the centrosomes, AKT allows for the selective stabilization of microtubules. GSK3β is crucial for the regulation of microtubule organization and dynamics, particularly for mitotic spindle organization.29 p38α deficiency alters the balance between AKT and GSK3β leading to AKT down-regulation and GSK3β activation (Fig. 3), which seems to impair normal cytokinesis completion.

MK2 also plays a significant role downstream of p38α in remodeling the actin cytoskeleton.30 Particularly, MK2 triggers phosphorylation of HSP27 inducing its release from F-actin.30 HSP27 protects against apoptosis and actin fragmentation, promoting resistance against cell death.31 We found an increase in HSP27 levels, which may be an adaptive response against liver injury, with significant changes in phosphorylation.

Mnk1 and Polo-like kinase 1 (Plk1), two potential downstream targets of p38α signaling, may contribute to cytokinesis failure in p38α-deficient liver. Inhibition of Mnk1, a kinase target for MAPK pathways, causes cytokinesis failure inducing the formation of multinucleated cells.32 In addition, MK2 directly phosphorylates Plk1, and down-regulation of p38α or MK2 induces mitotic defects that can be rescued by Plk1.33

In conclusion, the present work shows that liver-specific p38α deficiency leads to reduced hepatocyte size, blockade of mitosis, cytokinesis failure, and eventually shorter life span upon chronic cholestasis induced by BDL. These results highlight the key role of p38α in cell proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis.

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

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

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