Alrp,a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats

Abstract

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of ‘Alrp homologous proteins’ in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.     

Pro-apoptotic Sorafenib signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo

The multi-kinase inhibitor Sorafenib increases the survival of patients with advanced hepatocellular carcinoma (HCC). Current data suggest that Sorafenib inhibits cellular proliferation and angiogenesis and promotes apoptosis. However, the underlying pro-apoptotic molecular mechanisms are incompletely understood. Here we compared the pro-apoptotic and anti-proliferative properties of Sorafenib in murine hepatoma cells and syngeneic healthy hepatocytes in vitro and in animal models of HCC and liver regeneration in vivo. In vitro, we demonstrate that cell cycle activity and expression of anti-apoptotic Bcl-2 like proteins are similarly downregulated by Sorafenib in Hepa1-6 hepatoma cells and in syngeneic primary hepatocytes. However, Sorafenib-mediated activation of caspase-3 and induction of apoptosis were exclusively found in hepatoma cells, but not in matching primary hepatocytes. We validated these findings in vivo by applying an isograft HCC transplantation model and partial hepatectomy (PH) in C57BL/6 mice. Sorafenib treatment activated caspase-3 and thus apoptosis selectively in small tumor foci that originated from implanted Hepa1-6 cells but not in surrounding healthy hepatocytes. Similarly, Sorafenib did not induce apoptosis after PH. However, Sorafenib treatment transiently inhibited cell cycle progression and resulted in mitotic catastrophe and enhanced non-apoptotic liver injury during regeneration. Importantly, Sorafenib-mediated apoptosis in hepatoma cells was associated with the expression of p53-upregulated-modulator-of-apoptosis (PUMA). In contrast, regenerating livers after PH revealed downregulation of PUMA and were completely protected from Sorafenib-mediated apoptosis. We conclude that Sorafenib induces apoptosis selectively in hepatoma cells but not in healthy hepatocytes and can additionally increase non-apoptotic hepatocyte injury in the regenerating liver.     

Bezafibrate decreases growth stimulatory action of the sphingomyelin signaling pathway in regenerating rat liver

Liver regeneration after partial hepatectomy (PH) is achieved through proliferation of hepatocytes and non-parenchymal cells. The nuclear peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in regulation of lipid metabolism and proliferation of hepatic cells. The sphingomyelin signal transduction pathway is involved in the regulation of the cell cycle in eukaryotic organisms. Sphingosine-1-phosphate (S1P) and ceramide (CER)-- the intermediates of the pathway--are known to stimulate and to inhibit cellular proliferation. The aim of the present study was to investigate the effect of PPARalpha activation by bezafibrate on the sphingomyelin signaling pathway during the first 24h of liver regeneration after PH in the rat. The content of sphingomyelin, ceramide, sphingosine, sphinganine, sphingosine-1-phosphate and the activity of sphingomyelinases and ceramidases were determined at various time points after PH. It has been found that the activity of neutral Mg(2+)-dependent sphingomyelinase (nSMase) increased, whereas the activity of acidic sphingomyelinase (aSMase) decreased in the regenerating liver. Activation of PPARalpha by bezafibrate lower the activity of nSMase and increased the activity of aSMase in the regenerating rat liver. The content of ceramide was higher in bezafibrate-treated rats, whereas the content of sphingosine-1-phosphate was markedly lower as compared to the untreated rats. Therefore, it is concluded that activation of PPARalpha by bezafibrate decreases the growth-stimulatory activity of the sphingomyelin pathway in regenerating rat liver.     

Cell proliferation, apoptosis, NF-kappaB expression, enzyme, protein, and weight changes in livers of burned rats

Thermal injury has been shown to alter gut epithelium and heart myocyte homeostasis by inducing programmed cell death. The effect of thermal injury on hepatocyte apoptosis and proliferation, however, has not been established. The purpose of this study was to determine whether a large thermal injury increases liver cell apoptosis and proliferation and whether these changes were associated with alterations in hepatic nuclear factor kappaB (NF-kappaB) expression and changes in liver enzymes and amount of protein. Sprague-Dawley rats received a 40% total body surface area scald burn or sham burn. Rats were killed and livers were harvested at 1, 2, 5, and 7 days after burn. Liver cell apoptosis was determined by terminal deoxyuridine nick end labeling (TUNEL) assay and cell proliferation by immunohistochemistry for proliferating cell nuclear antigen. Hepatic NF-kappaB expression was determined by Western blot, and total hepatic protein content was determined by protein assay. Protein concentration decreased after burn compared with sham controls (P < 0.05). Liver cell apoptosis, proliferation, and NF-kappaB expression in hepatocytes increased in burned rats compared with controls (P < 0.05). It was concluded that thermal injury induces hepatic cell apoptosis and proliferation associated with an increase in hepatic NF-kappaB expression and a decrease in hepatic protein concentration.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

Serum ethanolamine and hepatocyte proliferation in perinatal and partially hepatectomized rats

It has been shown that the administration of ethanolamine (Etn) to partially hepatectomized rats enhances stimulation of DNA synthesis in regenerating hepatocytes. The present study aimed to test the hypothesis that the level of serum Etn in vivo may be regulated to control the growth of hepatocytes. Concentrations of serum Etn were determined in rats 1) of varying ages (from embryonic-19 (E-19) to 7-week-old), and 2) during regeneration following two-thirds hepatectomy (PH), to investigate whether serum Etn concentration correlates with the rate of proliferation of hepatocytes in growing animals or during regeneration. Serum Etn levels were 3 fold higher in E-19 fetuses and newborns than in adults, and were increased 2 fold 4 h after PH and remained high for at least 24 h. Results in both systems indicated a significant positive correlation between the rate of hepatocyte proliferation and serum Etn levels. Furthermore, Etn supplementation of 0.1 to 1 mmol immediately after PH promoted a significant weight gain and stimulated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) synthesis in the regenerating liver. We also observed that whenever serum Etn levels were elevated, the metabolism of PE and PC in the liver changed dynamically, first by elevating the net synthesis of PE. Taken together, these results suggested that the levels of serum Etn might be regulated based on the physiological state of an animal, which consequently regulates the proliferation of hepatocytes.     

Kinetics of DNA synthesis in feeding-dependent and independent hepatocyte populations of rats after partial hepatectomy

The effects of food consumption on the kinetics of hepatic DNA synthesis after partial hepatectomy (PH) have been studied in rats. Short-term (4-24 hr) fasting before or after PH resulted in depression and/or delay of DNA synthesis on days 1, 2 and 3 of regeneration. This depression was found in hepatocytes and, to a lesser extent, in littoral cells. Re-feeding resulted in an increase of DNA synthesis within 3-8 hr. The results suggest that two different hepatocyte subpopulations exist in regenerating rat liver: one which proceeds to DNA synthesis without apparent exogenous signals, and another one which needs, in addition to the specific mitogenic action of PH, food intake as a secondary permissive signal in order to initiate DNA synthesis. In the latter population food consumption appears to be required at two different stages: (1) in G0 or the early pre-replicative phase (PRP); (2) in the late PRP 3-8 hr before initiation of DNA synthesis. In the latter stage dietary protein is needed, but no so in the former. The dependence on feeding in the late PRP increases relatively with time after PH. No evidence was found to suggest a different distribution of the two cell populations throughout the liver acinus. The findings support the hypothesis that the known effects of the light-dark rhythm on the timing of DNA synthesis after PH are mediated by the natural feeding rhythm of rats fed ad libitum. In addition they offer a means for improving the synchrony of hepatocyte proliferation in regenerating rat liver.     

Transforming growth factor-β signaling confers hepatic stellate cells progenitor features after partial hepatectomy

Liver regeneration involves not only hepatocyte replication but progenitor aggregation and scarring. Partial hepatectomy (PH), an established model for liver regeneration, reactivates transforming growth factor-β (TGF-β) signaling. Hepatic stellate cells (HSCs) are primarily responding cells for TGF-β and resident in stem cell niche. In the current study, PH mice were treated with SB-431542, an inhibitor of TGF-β Type I receptor, aiming to address the role of TGF-β signaling on the fate determination of HSCs during liver regeneration. After PH, control mice exhibited HSCs activation, progenitor cells accumulation, and a fraction of HSCs acquired the phenotype of hepatocyte or cholangiocyte. Blocking TGF-β signaling delayed proliferation, impaired progenitor response, and scarring repair. In SB-431542 group, merely no HSCs were found coexpressed progenitor makers, such as SOX9 and AFP. Inhibition of TGF-β pathway disturbed the epithelial-mesenchymal transitions and diminished the nuclear accumulation of β-catenin as well as the expression of cytochrome P450 2E1 in HSC during liver regeneration. We identify a key role of TGF-β signaling on promoting HSC transition, which subsequently becomes progenitor for generating liver epithelial cells after PH. This process might interact with an acknowledged stem cell function signaling, Wnt/β-catenin.     

Four waves of hepatocyte proliferation linked with three waves of hepatic fat accumulation during partial hepatectomy-induced liver regeneration

Partial hepatectomy (PH) triggers hepatocyte proliferation-mediated liver repair and is widely used to study the mechanisms governing liver regeneration in mice. However, the dynamics of the hepatocyte proliferative response to PH remain unclear. We found that PH-induced mouse liver regrowth was driven by four consecutive waves of hepatocyte replication. The first wave exhibited the highest magnitude followed by two moderate waves and one minor wave. Underlying this continuous hepatocyte replication was persistent activation of cell cycle components throughout the period of liver regeneration. Hepatocyte mitotic activity in the first three proliferative cycles showed a circadian rhythm manifested by three corresponding mitosis peaks, which were always observed at Zeitgeber time 0. The Bmal1-Clock/Wee1/Cdc2 pathway has been proposed by others to govern the circadian rhythm of hepatocyte mitosis during liver regeneration. However, we did not observe the correlations in the expression or phosphorylation of these proteins in regenerating livers. Notably, Bmal1 protein displayed frequent changes in hepatic distribution and cellular localization as the liver regrowth progressed. Further, three waves of hepatic fat accumulation occurred during hepatic regeneration. The first started before and lasted through the first round of hepatocyte proliferation, whereas the second and third occurred concomitantly with the second and third mitotic peaks, respectively. CONCLUSION: PH-induced liver regeneration consists of four continuous waves of hepatocyte proliferation coupled with three waves of hepatic fat accumulation. Bmal1, Wee1, and Cdc2 may not form a pathway regulating the circadian rhythm of hepatocyte mitosis during liver regeneration.     

Prolonged Jun N-terminal kinase (JNK) activation and the upregulation of p53 and p21(WAF1/CIP1) preceded apoptosis in hepatocytes after partial hepatectomy and cisplatin

Cisplatin induced apoptosis in regenerating liver after partial hepatectomy (PH). Apoptosis was determined by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and peaked at 8 h after PH. The activity of Jun N-terminal kinase (JNK) transiently increased at 1 h after PH. However, in cisplatin-injected rats, the JNK activity increased at 30 min and the increased level was maintained up to 4 h after PH. The in vivo activation of JNK was confirmed by the increased level of the phosphorylated c-Jun protein. Western blot analysis showed that the phosphorylated c-Jun level increased at 1 h and reached more than 30-fold the control level at 2 h after PH with cisplatin. The c-jun mRNA levels also markedly increased at 1 h after PH with cisplatin. The protein level of p53 increased after 1 h on cisplatin injection, but no significant change in the mRNA level was observed. The rise in the p53 protein level was followed by the upregulation of p21(WAF1/CIP1) mRNA and protein levels. These results suggested that the enhanced and sustained JNK activation and the upregulation of p53 and p21(WAF1/CIP1) were involved in hepatocyte apoptosis induced by PH with cisplatin.     

Autocrine expression of activated transforming growth factor-beta(1) induces apoptosis in normal rat liver

The aim of this study was to determine the differential effects of latent and activated transforming growth factor (TGF)-beta(1) in growth control of normal and proliferating hepatocytes in vivo. Rats were injected with adenoviruses expressing control transgenes (Ctrl), latent TGF-beta(1) [TGF-beta(L)], or activated TGF-beta(1) [TGF-beta(A)]. Additional animals underwent two-thirds partial hepatectomy (PH) 24 h after injection. Increased hepatocyte apoptosis was observed in TGF-beta(A)-injected but not TGF-beta(L)-injected animals 24 h postinjection (10.5%) compared with Ctrl animals (0.37%). The percent of apoptotic cells increased to 32.1% in TGF-beta(A)-injected animals 48 h after injection. Furthermore, TGF-beta(A)-injected rats did not survive 24 h after PH. Four hours after PH, 0.25 and 14.1% apoptotic hepatocytes were seen in Ctrl- and TGF-beta(A)-injected rats, respectively. TGF-beta(A)-induced apoptosis in primary rat hepatocytes was blocked with a pancaspase inhibitor. Thus autocrine expression of TGF-beta(A) but not TGF-beta(L) induces hepatocyte apoptosis in the normal rat liver. Rats overexpressing TGF-beta(A) do not survive two-thirds PH due to hepatic apoptosis. Thus activation of TGF-beta(1) may be a critical step in the growth control of normal and proliferating rat hepatocytes.     

A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice

The ability of hepatocytes to enter the cell cycle and regenerate the liver after tissue loss provides an in vivo model to study the regulation of proliferation and organ regeneration. The extent of hepatocyte proliferation is directly proportional to the amount of resected liver tissue, and 2/3 partial hepatectomy (2/3 PH) leads to highly synchronized hepatocyte cell-cycle entry and progression. This surgical technique was first described in rats and requires modification for application in mice. Lack of standardization of 2/3 PH in mice has caused discrepancies in the results obtained in different laboratories. Here, we provide a protocol and a movie describing a straightforward surgical technique, which takes 15-20 min, to consistently remove two-thirds of the liver in mice. As this protocol is not associated with mortality and gives highly reproducible results, we hope that it will be widely used and serve to standardize 2/3 PH in mice.     

Molecular dissection of gp130-dependent pathways in hepatocytes during liver regeneration

Interleukin-6 (IL-6) via its signal transducer gp130 is an important mediator of liver regeneration involved in protecting from lipopolysaccharide (LPS)-induced liver injury after partial hepatectomy (PH). Here we generated mice either defective (Delta) in hepatocyte-specific gp130-dependent Ras or STAT activation to define their role during liver regeneration. Deletion of gp130-dependent signaling had major impact on acute phase gene (APG) regulation after PH. APG expression was blocked in gp130-DeltaSTAT animals, whereas gp130-DeltaRas mice showed an enhanced APG response and stronger SOCS3 regulation correlating with delayed hepatocyte proliferation. To define the role of SOCS3 during hepatocyte proliferation, primary hepatocytes were co-stimulated with IL-6 and hepatocyte growth factor. Higher SOCS3 expression in gp130-DeltaRas hepatocytes correlated with delayed hepatocyte proliferation. Next, we tested the impact of LPS, mimicking bacterial infection, on liver regeneration. LPS and PH induced SOCS3 and APG in all animal strains and delayed cell cycle progression. Additionally, IL-6/gp130-dependent STAT3 activation in hepatocytes was essential in mediating protection and thus required for maximal proliferation. Unexpectedly, oncostatin M was most strongly induced in gp130-DeltaSTAT animals after PH/LPS-induced stress and was associated with hepatocyte proliferation in this strain. In summary, gp130-dependent STAT3 activation and concomitant SOCS3 during liver regeneration is involved in timing of DNA synthesis and protects hepatocyte proliferation during stress conditions.     

Expression and localization of regenerating gene I in a rat liver regeneration model

Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.     

Accelerated proliferation of hepatocytes in rats with iron overload after partial hepatectomy

Although iron overload is implicated in hepatocarcinogenesis, the precise mechanism was not known yet. In the present study, we investigated the effect of iron overload upon the induction of hepatocyte proliferation after 70?% partial hepatectomy (PH) in rats fed with rat chow with 3?% carbonyl iron for 3?months. In normal-diet rats, the increase in Ki-67 labeling index (LI) commenced at 24?h post-PH and the LIs of proliferating cell nuclear antigen (PCNA) incorporated 5-bromo-2′-deoxyuridine (BrdU) and phospho-histone H3 reached maximum values at 36 and 48?h after PH, respectively. In iron-overload rats, the above parameters occurred 12?h earlier compared to that of normal-diet rats, shortening the G0–G1 transition. Interestingly, nuclear staining for metallothionein (MT), which is essential for hepatocyte proliferation, was noted even at 0?h in iron-overload rats, while MT expression occurred at 6?h in the normal rats. Moreover, nuclear factor kappa B (NF-κB) expression, which is an essential early event leading to liver regeneration, was detected in Kupffer cells at 0?h in iron-overload rats. These results may indicate that overloaded iron, maybe through the induction of MT and NF-κB, may keep liver as a state ready to regenerate in response to PH, by bypassing signal transduction cascades involved in the initiation of liver regeneration.