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Abstract

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

The cytokine tumor necrosis factor alpha (TNF-α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF-κB) activation, apoptosis signaling by way of caspase-8 (Casp8), and activation of cJun N-terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF-dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor-interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF-κB and JNK/cJun related signals. In order to define the role of NF-κB in this setting we blocked NF-κB activation in Casp8Δhepa mice by concomitant inactivation of the NF-κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF-κB, and JNK activation. While loss of Casp8 triggers NF-κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Abbreviations
BrdU

bromodeoxyuridine

Casp8

caspase-8

EMSA

electrophoretic mobility shift assay

FADD

Fas-associated protein with death domain

JNK

c-Jun N-terminal protein kinase

NEMO

NF-κB-essential-modulator

NF-κB

nuclear factor kappa B

PH

partial hepatectomy

RIP1

receptor-interacting protein 1

TNF

tumor necrosis factor alpha

TNFR1

TNF receptor 1

TRADD

tumor necrosis factor receptor type 1-associated death domain protein

TRAF2

TNF receptor-associated factor 2

WT

wild-type

The cytokine tumor necrosis factor alpha (TNFα; TNF) mediates pleiotropic effects by triggering inflammation and cell proliferation by way of nuclear factor kappa B (NF-κB), apoptosis through caspase-8 (Casp8), or activation of cJun N-terminal kinases (JNK). It has been identified as a crucial mediator for the priming phase of liver regeneration. Genetic inactivation of TNF-receptor 1 (TNF-R1) results in decreased NF-κB and JNK signaling leading to impaired hepatocyte proliferation after 70% partial hepatectomy (PH).[1]

In the adult liver, hepatocytes are long-lived and rarely undergo proliferation, yet they retain a remarkable ability to proliferate.[2] This allows the liver to restore its original mass within 7 to 10 days after PH. The regenerative response is initiated by a series of signaling events that allow the quiescent hepatocytes to reenter the cell cycle and undergo several rounds of proliferation until the original liver mass is restored.[3]

Binding of TNF to TNF-R1 rapidly initiates assembly of a plasma membrane bound complex-I, composed of TNF-R1, the tumor necrosis factor receptor type 1-associated death domain protein (TRADD), the protein kinase RIP1, and the TNF receptor-associated factor 2 (TRAF2). Complex-I induces immediate downstream activation of both the JNK and NF-κB signaling pathways and prevents apoptosis in part by inducing antiapoptotic proteins such as FLIPL.[4] Upon inhibition of NF-κB signaling, a competing complex (complex-II) is formed immediately after TNF ligation. Complex-II includes the adapter proteins TRADD, FADD (Fas-associated protein with death domain), and the proapoptotic protease pro-caspase-8, which eventually initiates the apoptotic signal cascade.[5] Constitutive targeted disruption of Casp8 results in embryonic lethality presumably due to an abundance of developmental defects.[6] More recent studies revealed that Casp8 plays also an essential role for prevention of an alternative mode of programmed cell death, termed necroptosis.[7] We recently reported that loss of Casp8 in hepatocytes protects from acute Fas and lipopolysaccharide (LPS)-induced liver injury but also triggers increased nonapoptotic cell death in mice lacking the NF-κB essential modulator (NEMO) involving enhanced RIP1 kinase activity and necroptosis.[8]

The aim of the present study was to investigate the consequences of genetic Casp8 inactivation in hepatocytes for liver regeneration following PH. We demonstrate that loss of Casp8 leads to an accelerated onset of hepatocyte priming and DNA synthesis following PH without affecting proper termination of liver growth. We provide evidence that this protective effect is due to early NF-κB activation associated with premature expression of the upstream RIP1 kinase. Our findings may have an impact for the evaluation of human therapies using low-molecular caspase-inhibitors.

Materials and Methods

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

For our studies we used genetically modified mice of male gender carrying a hepatocyte-specific deletion (Δhepa) of Casp8 and/or Nemo genes (Casp8Δhepa; NEMOΔhepa;Casp8ΔhepaNEMOΔhepa) and cre-negative littermates (Casp8f/f; NEMOf/f;Casp8f/fNEMOf/f) as described recently.[8] Mice were maintained in the animal facility of the University Hospital Aachen in a temperature-controlled room with 12-hour light/dark cycle. Animal husbandry and procedures were approved by the authority for environment conservation and consumer protection of the state North Rhine-Westfalia (LANUV, Germany).

For PH, pathogen-free 7-9-week-old male mice were used as described.[9] For each experimental condition a minimum of five mice per group were included in the study. All mice received a single injection of the nucleoside analog bromodeoxyuridine (BrdU) (30 μg/g, intraperitoneally, Applichem, Cheshire, CT) 2 hours before sacrificing.

Messenger RNA (mRNA) Expression Analysis and Quantitative Real-Time Polymerase Chain Reaction (qPCR) and Immunoblotting

Isolation of total RNA from liver tissues and reverse-transcription reactions were performed as described recently.[8] Primer sequences are listed in Supporting Table 1. Target gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression as internal standard. Values were expressed as fold increase compared to untreated controls. Western blot analysis was performed under reducing conditions according to standard procedures using primary and secondary antibodies as listed in Supporting Table 2. As internal loading control, membranes were probed with antibodies against GAPDH or β-actin.

Measurement of Aminotransferase Activity

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured in serum according to standard methods (UV test at 37°C) using a Roche Modular preanalytics system (Roche, Grenzach, Germany).

Statistical Data Analysis

Data are expressed as the mean ± SD. Statistical significance was determined by 2-way analysis of variance followed by Student t test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
Hepatocyte-Specific Deletion of Casp8 Accelerates the Onset of Liver Regeneration in Mice After PH

We performed PH in Casp8Δhepa mice and Casp8f/f controls and analyzed cell cycle initiation and progression 24-96 hours after surgery. Surprisingly, we observed an accelerated and overall stronger DNA synthesis in Casp8-deficient hepatocytes between 30-48 hours after surgery as demonstrated by increased incorporation of the thymidine analog BrdU (Fig. 1A,B). In order to elucidate the aberrant signals in Casp8Δhepa livers resulting in early onset of liver regeneration, we systematically analyzed the regulation of G1- and S-phase cyclins (Supporting Fig. 1A). In agreement with our initial observation, Casp8Δhepa mice also revealed an earlier induction cyclin A2 (Fig. 1C,D), cyclin E1 (Fig. 1E), and the cyclin E/A inducing transcription factor E2F1 (Supporting Fig. 1B), further indicating premature onset of G1/S-phase transition.

image

Figure 1. Hepatocyte-specific deletion of Casp8 accelerates the onset of liver regeneration in mice after PH. Casp8Δhepa mice and Casp8f/f controls were subjected to PH. At the indicated timepoints after surgery, extracted livers were investigated for cell cycle progression. Representative immunoblots were performed on the same gel and aligned for better overview. (A) Measurement of BrdU incorporation. Green: nuclear BrdU incorporation; blue: nuclear staining with DAPI. (B) Quantification of BrdU incorporation. (C,D) Gene and protein expression of cyclin A2 was analyzed by qPCR (C) and immunoblots (D). (E). Gene expression analysis of cyclin E1. (F) Protein expression of cyclin D1. *P < 0.05, **P < 0.01; ***P < 0.001.

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Cyclin D1 is the apical cyclin for cell cycle activation and is predominantly regulated by growth factors and immediate early transcription factors.[10] Loss of Casp8 also resulted in accelerated onset of cyclin D1 gene and protein expression (Supporting Fig. 1C and Fig. 1F), which suggests that Casp8 controls an important upstream mediator of cyclin D1 gene regulation.

Progression and Termination of Liver Mass Restoration After PH Is Independent of Casp8 Function

We hypothesized that receptor-mediated apoptosis and Casp8 activation is important for terminating liver regeneration after PH following restoration of the original liver mass. Our previous data demonstrated that loss of Casp8 results in excessive DNA synthesis. From these results we expected deregulated liver regeneration and potentially hepatomegaly in Casp8Δhepa mice. Surprisingly, 1 week after PH Casp8Δhepa mice revealed normal liver size and liver morphology (Supporting Fig. 2A) and showed identical liver mass restoration compared to wild-type (WT) controls (Fig. 2A).

image

Figure 2. Progression and termination of hepatic regeneration after PH is independent of Casp8. (A) Liver weight index (percent of body weight) at indicated timepoints after PH. (B) Quantitative gene expression analysis of cyclin B1 in Casp8Δhepa mice and Casp8f/f controls. (C) Detection of Cyclin B1, phosphorylated Cyclin B1 (p-Cyclin B1) and phosphorylated histone H3 (p-histone H3) by immunoblot analysis. (D,E) Histological Analysis of mitosis. At timepoints after PH as indicated, liver sections were stained with hematoxylin/eosin (H&E) and analyzed for mitotic figures (arrows). (D) Representative H&E histologies. (E) Number of mitotic figures was determined from at least 40 independent high-power fields (HPF) per condition at a magnification of 400×. *P < 0.05; **P < 0.01; ***P < 0.001.

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In order to elucidate the apparent contradiction between excessive DNA synthesis and normal liver mass reconstitution in Casp8Δhepa mice, we analyzed hepatocyte mitosis by determining cyclin B1 expression and phosphorylation (indicating G1/M-phase transition), and phosphorylation of histone H3 at Ser10, which is required for chromosome condensation and thus is specific for mitosis progression. In agreement with earlier reports,[11] liver regeneration in WT mice was associated with two peaks of cyclin B1 mRNA expression 36 and 48 hours after PH, which correlated with a biphasic protein expression, phosphorylation, and nuclear translocation of cyclin B1 (Fig. 2B,C; Supporting Fig. 2B). Histone H3 phosphorylation in WT controls started 36 hours post-PH and was maximal after 48 hours (Fig. 2C). In contrast, Casp8Δhepa mice revealed deregulated and overall reduced cyclin B1 gene expression (Fig. 2B) and poor cyclin B1 phosphorylation, which correlated with marginal phosphorylation of histone H3 (Fig. 2C). This indicated that accelerated DNA synthesis in regenerating Casp8Δhepa liver is compensated by retarded mitosis, eventually resulting in normal liver mass reconstitution. In fact, histologic evaluation demonstrated substantial delay of hepatocyte mitosis in Casp8Δhepa mice (Fig. 2D,E).

We further evaluated a potential function of proapoptotic Casp8 protease activity for termination of the regenerating process after liver resection and analyzed livers of Casp8f/f and Casp8Δhepa mice for apoptosis between 0-96 hours after PH. However, at any timepoint investigated, enzymatic activities of Casp8 or Casp3 did not exceed baseline levels of untreated WT controls (Supporting Fig. 2C,D) in either group. These findings suggest that the proapoptotic function of Casp8 is not involved in terminating liver regeneration after PH.

Hepatocyte-Specific Casp8 Deletion Accelerates Activation of TNF-Induced Signaling Pathways After PH

Untreated Casp8Δhepa mice displayed signs of moderate basal liver inflammation as evidenced by frequent accumulation of infiltrating mononuclear cells (Fig. 3A). Consistently, basal hepatic TNF mRNA levels in Casp8Δhepa mice were 5-fold elevated and more strongly induced following PH compared to WT controls (Fig. 3B). Six hours after PH, Casp8Δhepa mice revealed significantly reduced AST levels (Fig. 3C), suggesting that loss of Casp8 and elevated TNF induction are mediating hepatoprotective effects in this setting.

image

Figure 3. Ablation of Casp8 results in accelerated onset of priming phase after PH. Casp8Δhepa mice and Casp8f/f controls were characterized at early timepoints after PH (0-6 hours). (A) H&E stainings of untreated livers. Arrows: spontaneous inflammatory liver infiltrates in Casp8Δhepa mice. (B) Determination of TNF mRNA expression by qPCR. *P < 0.05. (C) AST serum transaminase levels during priming phase of PH.

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We thus investigated TNF-dependent pathways in Casp8Δhepa mice for up to 6 hours after PH. Induction of the receptor-interacting protein 1 (RIP1) through TNF is a prerequisite for NF-κB and JNK activation.[12, 13] In Casp8f/f livers, RIP1 expression was first detectable after 0.5 hours and peaked 2 hours after PH. In contrast, Casp8Δhepa livers already revealed slight basal RIP1 expression, which was almost immediately induced to maximal levels 0.5 hours after PH (Fig. 4A). Of note, we localized premature RIP1 in hepatocyte nuclei but could not detect pronecrotic RIP1/RIP3 complexes in Casp8Δhepa liver as determined by coimmunofluorescence analysis (Supporting Fig. 3A).

image

Figure 4. Casp8-deficiency triggers premature JNK and NF-κB signaling. Examination of immediate TNF signaling targets in Casp8Δhepa liver and Casp8f/f controls 0-6 hours after PH by immunoblot analysis. (A) RIP1 protein expression. (B) Measurement of JNK phosphorylation (p-JNK). JNK1 and JNK2 isoforms are highlighted by arrows. Total JNK1/JNK2 expression was used as internal control. (C) cJun phosphorylation. (D) Phosphorylation of NF-κB p65 at Ser536. (E) EMSA analysis using nuclear extracts from Casp8Δhepa and Casp8f/f livers at distinct timepoints after PH. Mobility shift signals indicate NF-κB activation (arrows). Signal specificity was confirmed by supershift analysis with p50 and p65 antibodies and nuclear extracts from Casp8Δhepa mice 2 hours post-PH. (F) Primary hepatocytes were isolated from Casp8Δhepa mice and Casp8f/f controls and treated with increasing concentrations (0-50 ng/mL) of recombinant TNF for 0.5 hours. Hepatocyte proteins were subjected to immunoblotting and probed for RIP1, phospho-p65 (p-p65), and phospho-JNK1/JNK2 (p-JNK). cl. RIP1: cleaved RIP1.

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We next addressed activation of the JNK/cJun pathway. WT controls predominantly displayed phosphorylation of JNK1, which was maximal 2 hours after PH, while Casp8Δhepa mice revealed accelerated and enhanced hepatic activation of both JNK1 and JNK2 already 0.5 hours after surgery (Fig. 4B; Supporting Fig. 3B). Consistently, Casp8Δhepa livers revealed an earlier and prolonged phosphorylation of the prototypical JNK-target cJun after PH (Fig. 4C).

Phosphorylation of the NF-κB subunit p65 at Ser536 is believed to enhance p65 transactivation potential.[14] In WT mice, p65 was first phosphorylated 2 hours after treatment (Fig. 4D), which resulted in robust transactivation 4 hours after surgery as evidenced by electrophoretic mobility shift assay (EMSA) (Fig. 4E). In contrast, Casp8Δhepa livers revealed constitutive p65 phosphorylation and accelerated nuclear NF-κB activation (Fig. 4D,E). To further support this finding, we analyzed expression of the Casp8-inhibitory protein FLIP, which is an immediate NF-κB downstream target.[15] In WT controls, FLIP was only transiently induced 1-2 hours after PH, while ablation of Casp8 resulted in constitutive FLIP expression 0-6 hours post-PH (Supporting Fig. 3C). In summary, these results demonstrate that loss of Casp8 triggers accelerated and prolonged NF-κB and JNK-dependent signaling after PH.

To further elucidate the mechanistic link between Casp8-deficiency and modulated TNF signaling, we investigated primary hepatocytes from Casp8Δhepa mice and WT controls after stimulation with different TNF concentrations mimicking low versus high TNF expression as found in Casp8f/f and Casp8Δhepa mice, respectively. Treatment with 10 ng/mL TNF resulted in time-dependent RIP1 activation in WT hepatocytes, while RIP1 was constitutively expressed in Casp8Δhepa cells, thereby completely reflecting our in vivo findings (Supporting Fig. 3D). Lower TNF concentrations were not sufficient to induce RIP1 or p65 phosphorylation in WT cells and only marginally activated JNK1, but not JNK2 (Fig. 4F). However, Casp8-deficient hepatocytes exhibited increased sensitivity towards TNF, resulting in improved activation of RIP1, p65, and both JNK1 and JNK2. Importantly, we detected cleaved RIP1 in WT hepatocytes without treatment and after high TNF dosage (≥10ng/mL, Fig. 4F), but not in Casp8-deficient cells. Thus, loss of Casp8 caused increased sensitivity towards TNF and enhanced stability of RIP1.

Spontaneous Liver Necrosis and Cholestasis in Casp8ΔhepaNEMOΔhepa Mice Is Largely Reverted by PH

We aimed to assess whether changes in NF-κB or JNK signaling explain accelerated cell cycle entry in Casp8Δhepa livers after PH. In vivo inhibition of NEMO in hepatocytes completely prevents NF-κB activation and results in a spontaneous liver phenotype including basal inflammation and apoptosis.[16, 17] We therefore blocked NF-κB activation in Casp8Δhepa mice by simultaneous genetic inactivation of NEMO.

We recently reported that Casp8ΔhepaNEMOΔhepa double deficient mice display basal necrotic liver injury with varying severity and thus classified these mice into three categories (type I, II, III) reflecting the grade of liver disease.[8] We performed PH in Casp8ΔhepaNEMOΔhepa mice of all subtypes and used the explanted liver lobes as reference. Type I livers appear mostly normal, whereas type II and type III livers display strong liver necrosis and cholestasis. Interestingly, 2 weeks after surgery all mice displayed substantially improved liver histology (Fig. 5A) and normal liver morphology (Fig. 5B) in comparison to their presurgical state. However, ALT levels in these mice were still elevated (Fig. 5C), indicating residual liver injury.

image

Figure 5. Spontaneous liver necrosis and cholestasis in Casp8ΔhepaNEMOΔhepa mice is largely reverted by PH. (A) Casp8ΔhepaNEMOΔhepa mice spontaneously develop liver necrosis and cholestasis with broad variations (type I-III). Type I: moderate liver necrosis and normal macroscopic appearance; type II: strong liver necrosis; type III: excessive tissue necrosis. Casp8ΔhepaNEMOΔhepa mice of all subtypes and Casp8f/fNEMOf/f controls were subjected to PH. prePH: Tissue sections from liver explants were used as pretreatment control and stained with H&E. Localization and size of necrotic liver lesions is highlighted by arrows. The same mice were sacrificed 2 weeks after PH and the regenerated livers were histologically analyzed. (B) Representative macroscopic appearance of a type II Casp8ΔhepaNEMOΔhepa liver before (prePH) and 2 weeks after PH. (C) ALT serum values from Casp8ΔhepaNEMOΔhepa mice and controls before and 2 weeks after PH. ***P < 0.001. (D) Forty-eight-hour survival of hepatectomized Casp8ΔhepaNEMOΔhepa mice. Survival rates were calculated for total Casp8ΔhepaNEMOΔhepa mice or differentiated by subtypes I, II, and III, respectively.

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The overall survival of these mice following PH was ∼75% after 48 hours (Fig. 5D). Interestingly, type I mice had a 90% survival rate, whereas type III mice demonstrated poor survival (<40%). These data are remarkable as a recent study demonstrates that only 50% of mice with genetic inactivation of NEMO survive PH.[18] Thus, inhibition of Casp8 improves the poor liver regeneration and survival of mice lacking NEMO after PH.

Combined Loss of Casp8 and NEMO Results in Constitutive cJun Phosphorylation and Increased Liver Injury During the Priming Phase After PH

We next investigated the immediate response of Casp8ΔhepaNEMOΔhepa mice within the first 6 hours after PH. Casp8ΔhepaNEMOΔhepa livers revealed a constitutive up-regulation of TNF, FLIP, and cJun mRNA, which was not significantly different between subtypes I-III (Fig. 6A-C). At the protein level, we found strong basal phosphorylation of p65 in NEMOΔhepa and Casp8ΔhepaNEMOΔhepa livers, reflecting strong inflammation and NF-κB activation of nonparenchymal liver cells (Fig. 6D). FLIP protein was also slightly up-regulated in both NEMOΔhepa and Casp8ΔhepaNEMOΔhepa livers, but less pronounced compared to Casp8Δhepa mice.

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Figure 6. Constitutive cJun activation and increased liver injury in Casp8ΔhepaNEMOΔhepa mice during the priming phase of liver regeneration. (A-C) Basal gene expression of TNF, cJun, and FLIP was determined by qPCR. Values were calculated for all Casp8ΔhepaNEMOΔhepa mice or differentiated by subtypes I, II, and III, respectively. (D) Basal hepatic protein levels of phospho-p65 (p-p65), phospho-cJun (p-cJun), and FLIP in Casp8ΔhepaNEMOΔhepa mice (types I-III) in comparison to single Casp8Δhepa and NEMOΔhepa mice. (E) Casp8ΔhepaNEMOΔhepa mice and Casp8f/fNEMOf/f controls were subjected to PH. Nuclear translocation and phosphorylation of cJun (stained in red) was visualized by immunofluorescence microscopy. (F) Measurement of ALT serum levels. *P < 0.05, **P < 0.01; ***P < 0.001.

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Importantly, we found constitutive cJun phosphorylation in untreated Casp8ΔhepaNEMOΔhepa livers (Fig. 6D), which further accumulated within 2 hours after PH (Fig. 6E). Overall, Casp8ΔhepaNEMOΔhepa mice revealed significantly elevated ALT levels compared to WT controls 0-6 hours after PH (Fig. 6F), suggesting that the protective effect of Casp8 inactivation in the priming phase of liver regeneration (compare Fig. 3D) is completely reverted by concomitant inhibition of NEMO.

Accelerated Onset of Liver Regeneration in Casp8Δhepa Mice Is Mediated by Way of NEMO/NF-κB Signaling

To elucidate the role of NF-κB for the accelerated onset of DNA synthesis in Casp8Δhepa mice, we performed PH on type II Casp8ΔhepaNemoΔhepa mice and compared initiation and progression of S-phase with Casp8f/fNEMOf/f controls. Both groups showed comparable initiation of DNA synthesis 30 hours after PH, while Casp8ΔhepaNemoΔhepa livers had reduced number of hepatocytes in S-phase exclusively 40 hours after surgery (Fig. 7A,B). This correlated with slightly impaired cyclin A2 mRNA and protein expression (Fig. 7C,D). However, cyclin D1 expression in Casp8ΔhepaNemoΔhepa mice and control livers was identical within the first 48 hours after PH (Fig. 7E), indicating that accelerated G1/S-phase transition and onset of DNA synthesis in regenerating Casp8Δhepa livers depends on aberrant NF-κB induction.

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Figure 7. Accelerated onset of liver regeneration in Casp8Δhepa mice is mediated through the NEMO/NF-κB signaling axis. Hepatocyte proliferation and liver mass restoration in Casp8Δhepa NEMOΔhepa mice and Casp8f/fNEMOf/f controls after PH was investigated for up to 6 weeks after surgery. (A) Determination of BrdU incorporation (green) indicating S-phase progression. (B) Quantification of BrdU incorporation. (C) Gene expression analysis of cyclin A2 by qPCR. (D) Determination of cyclin A2 and cyclin D1 protein expression after PH. (E) Cyclin D1 gene expression profile after PH. (F) Liver weight index was calculated as percent of body weight at indicated timepoints after PH. *P < 0.05, **P < 0.01; ***P < 0.001.

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Remarkably, the slightly delayed DNA synthesis in Casp8ΔhepaNemoΔhepa mice did not impair liver mass restoration during the first 14 days after PH (Fig. 7F). However, untreated Casp8ΔhepaNemoΔhepa mice exhibited hepatomegaly at baseline and 6 weeks after PH Casp8ΔhepaNemoΔhepa mice again revealed significantly increased liver mass (Fig. 7F) which was associated with slightly increased cyclin A and D levels after completion of liver mass restoration (336 hours post-PH, Fig. 7C,E). Accordingly, proper liver regeneration requires balanced expression of Casp8 and NEMO.

Discussion

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

Casp8 is the most apical caspase during extrinsic apoptosis mediated by death-receptors. In addition, Casp8 may have nonapoptotic functions, e.g., by regulating NF-κB transcriptional activity.[19] In the present study we characterized the consequences of hepatocyte-specific Casp8 deletion for liver regeneration in mice. We tested the hypothesis that loss of Casp8 would either prevent proper termination of liver growth or affect the early events of TNF-dependent signaling after PH.

The present study revealed that termination of liver regeneration is independent of Casp8. However, loss of Casp8 resulted in deregulation of all interphase cyclins and in accelerated onset of DNA synthesis after PH. These unexpected effects could be linked to premature RIP1 kinase activation affecting the downstream NF-κB and JNK/cJun pathways, respectively. Concomitant NEMO deletion restored normal onset of hepatocyte proliferation in Casp8-deficient livers but eventually induced hepatomegaly.

Previous work from Ben Moshe et al.[20] revealed completely opposite results compared to our own study, including high mortality postsurgery, impaired early liver regeneration, and delayed expression of interphase cyclins. However, that study used a different experimental setting (33% hepatectomy) and a different Casp8 knockout allele (ΔExon 1-2). We thus conclude that the effects of Casp8 depletion on liver regeneration could be allele-specific and may depend on the strength of the regeneration stimulus.

Casp8Δhepa mice showed normal liver regeneration after PH despite excessive DNA synthesis. This was unexpected, as aberrant DNA replication could result in enhanced cell division and in augmented liver growth. However, even 6 months after PH we did not detect any signs of hepatomegaly or precarcinogenic lesions in Casp8Δhepa mice (data not shown). These results suggest that termination of liver regeneration is predominantly controlled by nonapoptotic, Casp8-independent mechanisms. We further conclude that Casp8-deficient hepatocytes undergo delayed G1/M transition and slow progression through mitosis, as evidenced by impaired induction, phosphorylation, and nuclear translocation of cyclin B (indicative of late M-phase transition) and poor phosphorylation of histone H3 demonstrating low prophase activity.[21] Thus, accelerated DNA synthesis is most likely compensated by delayed mitosis progression eventually resulting in normal liver mass restoration.

Importantly, accelerated onset of DNA synthesis in Casp8Δhepa mice was also associated with earlier induction of cyclin D gene expression. Several experimental data demonstrated that the cyclin D gene promoter is regulated by NF-κB and by way of cJun and cFos in a JNK-dependent manner.[22-25] Thus, our data suggest that the early start of DNA synthesis in Casp8Δhepa liver is best explained by premature NF-κB or JNK/cJun activation.

However, our experiments using Casp8ΔhepaNEMOΔhepa double-deficient mice clearly demonstrated that accelerated onset of DNA replication in Casp8Δhepa livers is dependent on the NEMO/NF-κB axis and not due to aberrant JNK/cJun activation. Additional ablation of NEMO in Casp8Δhepa mice completely rescued the kinetics of liver regeneration, although it resulted in constitutive cJun activation.

In addition, Casp8ΔhepaNEMOΔhepa mice revealed improved survival after PH (75% total survival, 90% survival in type I) in comparison to single NEMOΔhepa mice, which showed 50% mortality due to excessive liver apoptosis and strong oxidative stress.[18] Interestingly, liver resection even improved the spontaneous necrotic liver injury in Casp8ΔhepaNEMOΔhepa mice. Therefore, loss of Casp8—and thus accumulation of RIP1—seems to predispose to liver necrosis in a purely inflammatory setting, while it appears highly protective in the setting of surgical liver injury. Additionally, our data demonstrate that NEMO and Casp8 expression are of major relevance to tightly balance the precise timing of liver regeneration by synergistically controlling NF-κB and cJun activation and thus cyclin D expression.

Ultimately, our data indicate that all observations in Casp8Δhepa mice can be attributed to increased sensitivity towards exocrine TNF and accelerated induction of RIP1 in Casp8-deficient hepatocytes. RIP1 is proteolytically degraded by Casp8[26] and we provided direct evidence that loss of Casp8 prevented RIP1 cleavage in primary hepatocytes. Instead, even low doses of external TNF enabled accelerated RIP1 induction in Casp8-deficient cells. We recently demonstrated that elevated expression of RIP1 in Casp8Δhepa mice can result in RIP1/RIP3 complex formation and nonapoptotic liver injury resembling features of necroptosis in the Concanavalin A model of acute hepatitis.[8] However, our present data strongly suggest that after PH premature RIP1 induction is rather protective. Following PH, we could not detect pronecrotic RIP1-RIP3 colocalization in Casp8Δhepa liver tissue, but identified excessive RIP1 in hepatocyte nuclei. In line with our findings, a recent study demonstrated that RIP1 is directly involved in TNF gene transcription under certain conditions.[27] Thus, it is tempting to speculate that improved RIP1 stability in Casp8-deficient cells triggers autocrine TNF gene expression in hepatocytes, which would also explain elevated TNF gene expression in Casp8Δhepa mice. However, our data from primary hepatocytes using different dosages of TNF indicate that increased sensitivity of Casp8-deficient hepatocytes towards low-dose TNF is of greater relevance to explain our findings, as this was sufficient to trigger enhanced activation of all downstream signals including RIP1, NF-κB, JNK1, and JNK2, which pushes these cells towards cell cycle entry.

Upon TNF stimulation, RIP1 is recruited to the TNF receptor complex and contributes to activation of NF-κB by way of binding to NEMO, which is the regulatory subunit of the IKK complex.[12] Previous data demonstrated that phosphorylation of p65 at Ser536, which was constitutively found in Casp8-deficient hepatocytes, is performed by IKK kinase,[28] further highlighting the importance of the RIP1-NEMO-NF-κB axis for accelerated onset of liver regeneration in Casp8Δhepa mice. In addition, overexpression of RIP1 also induces JNK activation.[13] However, by analyzing Casp8ΔhepaNEMOΔhepa mice we provided indirect evidence that enhanced JNK/cJun activation is not involved in premature cyclin D induction after PH. Thus, hepatoprotection and accelerated liver regeneration in Casp8Δhepa mice is best explained by aberrant high RIP1 expression and improved NF-κB activation. Our conclusions are illustrated in Supporting Fig. 4.

In summary, our study demonstrates that loss of Casp8 is protective in the priming phase of liver regeneration in a nonapoptotic manner as it triggers the RIP1/NF-κB axis. These findings could be clinically and potentially therapeutically relevant in patients undergoing extended surgical liver resection.

References

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

Supporting Information

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

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

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