Activated natural killer T cells induce liver injury by Fas and tumor necrosis factor-alpha during alcohol consumption

BACKGROUND & AIMS: Chronic alcohol abuse induces liver injury and increases the severity of viral hepatitis, but the precise mechanisms responsible are not well understood. In particular, little is known about the role of natural killer T cells in alcohol-induced liver injury. Natural killer T cells are mediators of important regulator and effector functions making use of Fas and tumor necrosis factor (TNF)-alpha in apoptosis induction. This report analyzes the role of natural killer T cells, Fas, and TNF-alpha in a model of chronic alcohol consumption. METHODS: Mice fed alcohol by intragastric tube were assayed for serum alanine aminotransferase values, liver histology, and liver mononuclear cells before and after activation of natural killer T cells by ligand alpha-galactosylceramide. RESULTS: In alcohol-consuming animals, liver natural killer T cells increase, and further activation by alpha-galactosylceramide causes lethal liver injury. This is explained by alcohol-induced hepatocyte sensitization to cell-mediated lysis, which develops concomitant to increased cytolytic activity of natural killer T cells. Natural killer T cell-mediated apoptosis proceeds by the Fas pathway, and Fas is essential for alcohol-associated liver injury. TNF-alpha plays an additional role as a defect in TNF receptor-1 inhibits alcohol-associated liver injury. Alcohol-fed natural killer T cell-deficient Jalpha281(-/-) mice express a delay in alcohol-induced liver injury. CONCLUSIONS: Alcohol consumption induces an increase of natural killer T cells in the liver and a high sensitivity of hepatocytes to cell-mediated lysis. Stimulation of natural killer T cells during alcohol consumption induces serious liver injury by a mechanism that involves concomitant signals by Fas and tumor necrosis factor receptor-1 on alcohol-stressed hepatocytes.     

Neutralization of tumor necrosis factor abrogates hepatic failure induced by alpha-galactosylceramide without attenuating its antitumor effect in aged mice

BACKGROUND/AIMS: The functions of mouse liver NK1.1+ T (NKT) cells stimulated with alpha-galactosylceramide (alpha-GalCer) are enhanced age dependently, and the antitumor and anti-metastatic effect in the liver is dependent on IFN-gamma. However, hepatic injury is independent of IFN-gamma and Fas/Fas-ligand dependent. The aim of this study is to investigate how tumor necrosis factor is involved in the alpha-GalCer-mediated immune phenomena. METHODS: C57BL/6 mice were intraperitoneally treated with anti-TNF antibody 1 h before alpha-GalCer injection, and Fas-ligand expression of NKT cells, the serum ALT levels and histopathological findings of the liver, kidney and lung and mortality after alpha-GalCer injection were evaluated. IFN-gamma production and antitumor immunity in the liver after the intravenous injection of EL-4 cells were also assessed. RESULTS: Serum TNF levels after alpha-GalCer injection increased age dependently in mice. Anti-TNF Ab reduced Fas-ligand (Fas-L) expression of NKT cells while it completely inhibited organ injuries induced by alpha-GalCer and thereby reduced the mortality of old mice, whereas it did not affect the IFN-gamma production from NKT cells, the antitumor immunity in the liver nor the mouse survival after EL-4 injection. CONCLUSIONS: NKT cells activated by alpha-galactosylceramide participated in either antitumor immunity or hepatic injury using IFN-gamma and TNF/Fas-L, respectively.     

Impairment of liver regeneration correlates with activated hepatic NKT cells in HBV transgenic mice

A fraction of HBV carriers have a risk to develop liver cancer. Because liver possesses a strong regeneration capability, surgical resection of cancerous liver or transplantation with healthy liver is an alternate choice for HBV-caused hepatocarcinoma therapy. How HBV infection affects the regeneration of hepatectomized or transplanted liver remains elusive. We report that partial hepatectomy (PHx)-induced liver regeneration was reduced in HBV transgenic (HBV-tg) mice, a model of human HBV infection. PHx markedly triggered natural killer T (NKT) cell accumulation in the hepatectomized livers of HBV-tg mice, simultaneously with enhanced interferon gamma (IFN-gamma) production and CD69 expression on hepatic NKT cells at the early stage of liver regeneration. The impairment of liver regeneration in HBV-tg mice was largely ameliorated by NKT cell depletion, but not by natural killer (NK) cell depletion. Blockage of CD1d-NKT cell interaction considerably alleviated NKT cell activation and their inhibitory effect on regenerating hepatocytes. Neutralization of IFN-gamma enhanced bromodeoxyuridine incorporation in HBV-tg mice after PHx, and IFN-gamma mainly induced hepatocyte cell cycle arrest. Adoptive transfer of NKT cells from regenerating HBV-tg liver, but not from normal mice, could inhibit liver regeneration in recipient mice. CONCLUSION: Activated NKT cells negatively regulate liver regeneration of HBV-tg mice in the PHx model.     

Selective elimination of hepatic natural killer T cells with concanavalin A improves liver regeneration in mice.

BACKGROUND: Although concanavalin A (Con A) as a T cell stimulant can cause natural killer T (NKT) cell-mediated liver injury in mice and a nonhepatotoxic dose of Con A can trigger innate immune cells including NKT cells to prevent tumor metastasis in the liver, little is known about the role of Con A-primed NKT cells in liver repair. In this study, we aimed to investigate the effect of pretreatment with a nontoxic dose of Con A on subsequent liver regeneration in mice. METHODS: A nontoxic dose of Con A was injected intravenously 24 h before partial hepatectomy (PHx), which was used as a model of liver regeneration. Ratios of remnant liver mass to body weight, bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) labeling were used to assess liver regeneration. RESULTS: Hepatic mononuclear cells were isolated and analyzed by flow cytometry. After PHx, the ratios of liver weight to body weight, PCNA-positive hepatocytes and BrdU-positive hepatocytes in Con A-pretreated mice were significantly higher than that of phosphate-buffered saline-treated mice, indicating that Con A pretreatment can accelerate liver regeneration. Flow cytometric analysis showed that NKT cells were significantly activated and selectively eliminated after the Con A administration. Moreover, NKT cells expressed more apoptosis-related molecules, Fas and Annexin V. CONCLUSIONS: Taken together, Con A accelerates liver regeneration in mice by eliminating hepatic NKT cells via activation-induced cell death.     

Intensive expansion of natural killer T cells in the early phase of hepatocyte regeneration after partial hepatectomy in mice and its association with sympathetic nerve activation

When C57BL/6 mice were partially hepatectomized (PHx), severe lymphocytosis was induced in the liver in the early phase of hepatocyte regeneration (4 to 12 hours after PHx). A major lymphocyte subset expanding in this organ was estimated to be natural killer 1.1(+) (NK1.1(+)) intermediate CD3 (CD3(int)) cells (i.e., NKT cells). CD3(int) cells are extrathymic T cells generated in situ in the liver. These changes were suppressed when mice with PHx were pretreated with a beta-adrenergicD antagonist (i.e., beta-blocker), propranolol (PPL). This might have been caused by sympathetic nerve stimulation during hepatocyte regeneration. An alpha-blocker showed a similar effect, although the magnitude of suppression was lower than that of the beta-blocker. We previously showed that NK and NKT cells express surface beta-adrenergic receptors and are activated in number by sympathetic nerve stimulation. In the present study, NK cytotoxicity mediated by liver lymphocytes obtained from mice with PHx decreased, whereas NKT cytotoxicity against syngeneic thymocytes increased. Purified CD3(int) cells were also found to be able to mediate NKT cytotoxicity against regenerating hepatocytes. These results suggest that sympathetic nerve stimulation after PHx results in subsequent activation of NKT cells and that these NKT cells might be associated with immunologic surveillance during hepatocyte regeneration.     

Negative regulation of liver regeneration by innate immunity (natural killer cells/interferon-gamma)

BACKGROUND AND AIMS: Hepatic lymphocytes are composed mainly of natural killer (NK) cells and NKT cells, which play key roles in innate immune responses against pathogens and tumors in the liver. This report analyzes the effects of activation of innate immunity by viral infection or the toll-like receptor 3 (TLR3) ligand on liver regeneration. METHODS: The partial hepatectomy (PHx) method was used as a model of liver regeneration. Murine cytomegalovirus (MCMV) infection and the TLR3 ligand polyinosinic-polycytidylic acid [poly(I:C)] were used to activate innate immunity. RESULTS: NK cells are activated after PHx, as evidenced by producing interferon (IFN)-gamma. Infection with MCMV or injection of poly(I:C) further activates NK cells to produce IFN-gamma and attenuates liver regeneration in the PHx model. Depletion of NK cells or disruption of either the IFN-gamma gene or the IFN-gamma receptor gene enhances liver regeneration and partially abolishes the negative effects of MCMV and polyI:C on liver regeneration, whereas NKT cells may only play a minor role in suppression of liver regeneration. Adoptive transfer of IFN-gamma +/+ NK cells, but not IFN-gamma -/- NK cells, restores the ability of polyI:C to attenuate liver regeneration in NK-depleted mice. Finally, administration of polyI:C or IFN-gamma enhances expression of several antiproliferative proteins, including STAT1, IRF-1, and p21cip1/waf1 in the livers of partially hepatectomized mice. CONCLUSIONS: Our findings suggest that viral infection and the TLR3 ligand negatively regulate liver regeneration via activation of innate immunity (NK/IFN-gamma), which may play an important role in the pathogenesis of viral hepatitis.     

Role of Valpha 14 NKT cells in the development of impaired liver regeneration in vivo

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.     

Functional alterations of liver innate immunity of mice with aging in response to CpG-oligodeoxynucleotide

Immune functions of liver natural killer T (NKT) cells induced by the synthetic ligand alpha-galactosylceramide enhanced age-dependently; hepatic injury and multiorgan dysfunction syndrome (MODS) induced by ligand-activated NKT cells were also enhanced. This study investigated how aging affects liver innate immunity after common bacteria DNA stimulation. Young (6 weeks) and old (50-60 weeks) C57BL/6 mice were injected with CpG oligodeoxynucleotides (CpG-ODN), and the functions of liver leukocytes were assessed. A CpG-ODN injection into the old mice remarkably increased tumor necrosis factor (TNF) production in Kupffer cells, and MODS and lethal shock were induced, both of which are rarely seen in young mice. Old Kupffer cells showed increased Toll-like receptor-9 expression, and CpG-ODN challenge augmented TNF receptor and Fas-L expression in liver NKT cells. Experiments using mice depleted of natural killer (NK) cells by anti-asialoGM1 antibody (Ab), perforin knockout mice, and mice pretreated with neutralizing interferon (IFN)-gamma Ab demonstrated the important role of liver NK cells in antitumor immunity. The production capacities of old mice for IFN-gamma, IFN-alpha, and perforin were much lower than those of young mice, and the CpG-induced antitumor cytotoxicity of liver NK cells lessened. Lethal shock and MODS greatly decreased in old mice depleted/deficient in TNF, FasL, or NKT cells. However, depletion of NK cells also decreased serum TNF levels and FasL expression of NKT cells, which resulted in improved hepatic injury and survival, suggesting that NK cells are indirectly involved in MODS/lethal shock induced by NKT cells. Neutralization of TNF did not reduce the CpG-induced antitumor effect in the liver. CONCLUSION: Hepatic injury and MODS mediated by NKT cells via the TNF and FasL-mediated pathway after CpG injection increased, but the antitumor activity of liver NK cells decreased with aging.     

Anti-transforming growth factor-β1 antibody transiently enhances DNA synthesis during liver regeneration after partial hepatectomy in rats

The regulation of liver regeneration after partial hepatectomy (PHx) is complex and involves many different cytokines. We investigated the role of one of these, transforming growth factor-β1 (TGF-β1), an inhibitor of liver regeneration, in a Wistar male rat model, in which anti-TGF-β1 antibody was injected immediately or 24 h after 70% PHx. Livers from treated animals contained an increased number of cells in S phase, according to 5-bromo-2′-deoxyuridine (BrdU) labeling 36 h after PHx. Antibody administration 24 h after PHx resulted in the highest peak of proliferation; moreover, peak MIB-5 labeling was also observed at that time. However, neither residual liver-weight-to-body-weight ratios nor regeneration rates differed significantly between any of the animals. Therefore, we also measured levels of serum TGF-β1 and hepatocyte growth factor (HGF; an activator). With antibody administration at 0 or 24 h, TGF-β1 levels were diminished at 24 or 36 h as compared with levels in control rats, but then rebounded, reaching a delayed peak at 48 or 72 h after PHx, respectively. Interestingly, there were also similar trends in HGF levels. These results indicate that TGF-β1 may inhibit the G1 checkpoint, and serum TGF-β1 concentration may influence HGF to regulate liver regeneration and to maintain homeostasis of proliferation after PHx.     

Focal liver necrosis appears early after partial hepatectomy and is dependent on T cells and antigen delivery from the gut

Introduction: Progressive liver failure may develop following removal of a large part of the liver or transplantation of a small for size liver graft. The pathophysiology of this clinical syndrome is only partially understood. Methods: We assessed liver damage and hepatocyte 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation following partial hepatectomy (PH) in C57BL/6, BALB/C and immune‐deficient mice. Hepatic lymphocyte subpopulations were characterized. Lipopolysaccharide (LPS) treatment and bowel decontamination determined the role of gut antigens. Results: Discrete, round necrotic lesions were observed as early as 2 h following 70%, but not 30% PH. In immune competent mice the extent of hepatocyte necrosis inversely correlated with BrdU incorporation. T, natural killer and natural killer T cells were recruited to the liver early after PH; however, only T‐cell depletion abrogated hepatic necrosis. Hepatic injury was significantly reduced in non‐obese diabetic/severe combined immunodeficient mice undergoing PH, while BrdU incorporation was not affected. Liver injury was augmented by LPS injection and reduced by gut decontamination. Conclusions: A distinct pattern of early focal hepatic necrosis is observed following extensive PH in mice. T cells infiltrating the liver immediately after PH and gut‐derived antigens are indispensable for the observed liver necrosis and may thus provide therapeutic targets to ameliorate liver damage following PH.     

Lack of endothelial autophagy does not impair liver regeneration after partial hepatectomy in mice

Liver sinusoidal endothelial cells (LSEC) are key elements in regulating the liver response to injury and regeneration. While endothelial autophagy is essential to protect endothelial cells from injury-induced oxidative stress and fibrosis, its role in liver regeneration has not been elucidated. This study was intended to investigate the role of endothelial autophagy in liver regeneration in the context of partial hepatectomy (PHx). Analysis of autophagy levels in rat LSEC after PHx indicated a tendency to decrease activity the first 2 days after surgery. PHx performed in mice with impaired endothelial autophagy (Atg7^flox/flox;VE-Cadherin-Cre⁺) and their littermate controls showed no differences neither in liver-to-body weight ratio, histological analysis, hepatocyte proliferation nor vascular integrity during the first 7 days after PH and liver regeneration was completely achieved. Our results indicate that endothelial autophagy does not play an essential role in the coordination of the liver regeneration process after PHx.     

Hepatocyte growth factor gene therapy accelerates regeneration in cirrhotic mouse livers after hepatectomy

BACKGROUND: Impaired regeneration and dysfunction of the cirrhotic liver following partial hepatectomy (PHx) are the most serious risk factors for postoperative liver failure. AIMS: Using naked hepatocyte growth factor (HGF) plasmid by the electroporation (EP) in vivo method, we investigated HGF for its role and mechanism of proliferation and restoration of liver mass in cirrhotic mice following PHx. ANIMALS: Eight week old female mice were used. METHODS: HGF plasmid 50 micro g was injected intramuscularly and transferred by EP in vivo once a week for three weeks. After establishment of carbon tetrachloride induced cirrhosis, mice underwent PHx. The HGF treated group was given naked HGF plasmid four days before PHx, and additional HGF was given once a week until they were killed, while a control group was given only empty plasmid. Mice were killed 2, 4, 10, and 14 days after PHx. Morphological and functional restoration of the liver were examined, as well as activation of mitogen activated protein kinase (MAPK) and mRNA levels of HGF activator (HGFA). RESULTS: The HGF treated group demonstrated a continuous threefold increase in HGF levels in plasma. Therapy with HGF in cirrhotic PHx resulted in effective liver regeneration via restoration of HGFA and activation of MAPK p44/p42, accelerated normalisation of liver function, and increased collagen degradation. CONCLUSIONS: HGF gene therapy by in vivo EP may be useful for hepatic resection in cirrhotic livers by stimulating liver proliferative and collagenolytic capacities, as well as accelerating functional recovery.     

Effect of cytokines on liver necrosis

AIM To investigate the effect of cytokines on the liver necrosis.METHODS rIL (interleukin)-1, rIL-6, rIFN (interferon), rTNF (tumor necrosis factor) α with or without D-galactosamine (D-GAL) were injected into the abdominal cavity of mice separately. ALT, TBIL (total bilirubin) and histological changes were observed.RESULTS There was no effect on hepatocyte of normal mice after injection of rIL-1, rIL-6, rIFN alone or together. The serum total bilirubin (TBIL) and liver necrosis of mice increased after rTNFα, rIL-6 or rIFN were used separately with D-GAL. The TBIL level (μmol/L) was 46.19±10.62, 44.55±12.9 and 41.94±14.9, higher than that caused by D-GAL alone (TBIL, 26.67μmol/L±11.14μmol/L). The serum TBIL of mice and the degree of liver necrosis increased after injection of IL-1, IL-6 with D-GAL and rTNFα.CONCLUSION Cytokines, like IL-1,IL-6, IFN and TNFα joined in the process of hepatocyte necrosis. They can enhance the degree of liver necrosis induced by D-GAL.     

Mild hypothermia does not affect liver regeneration after partial hepatectomy in mice

Background: The use of mild hypothermia has been suggested to be therapeutically useful in treating acute liver failure. It is not known if hypothermia influences liver regeneration. Aim: To assess the effect of hypothermia on liver regeneration in mice. Methods: After partial (70%) hepatectomy (PHx), C57BL6/J mice were randomly assigned to either a hypothermic group or a normothermic group. Controlled mild hypothermia was maintained for up to 3 h after surgery. In addition, assessment of liver mass restitution was examined by studying the induction of key cell cycle proteins (cyclin A, D1 and E) and hepatocyte proliferation [assessment of proliferating cell nuclear antigen (PCNA) protein expression] by Western blotting and DNA synthesis by measuring 5‐bromo‐2‐deoxyuridine (BrdU) incorporation by immunohistochemical techniques 45 h after PHx. Results: Partial hepatectomy induced a vigorous proliferative response in the remnant livers of both groups of mice (normothermic and hypothermic groups), as evidenced by the induction of key cyclins, PCNA and incorporation of BrdU after PHx. The liver/body weight ratio and both cyclin and PCNA protein expression as well as BrdU incorporation did not differ between the regenerating livers of hypothermic and normothermic groups. Conclusion: Mild hypothermia does not influence liver regeneration in mice.     

Cellular and molecular mechanisms of liver injury

Derangements in apoptosis of liver cells are mechanistically important in the pathogenesis of end-stage liver disease. Vulnerable hepatocytes can undergo apoptosis via an extrinsic, death receptor-mediated pathway, or alternatively intracellular stress can activate the intrinsic pathway of apoptosis. Both pathways converge on mitochondria, and mitochondrial dysfunction is a prerequisite for hepatocyte apoptosis. Persistent apoptosis is a feature of chronic liver diseases, and massive apoptosis is a feature of acute liver diseases. Fibrogenesis is stimulated by ongoing hepatocyte apoptosis, eventually resulting in cirrhosis of the liver in chronic liver diseases. Endothelial cell apoptosis occurs in ischemia-reperfusion injury. Natural killer and natural killer T cells remove virus-infected hepatocytes by death receptor-mediated fibrosis. Lastly, activated stellate cell apoptosis leads to slowing and resolution of apoptosis. This review summarizes recent cellular and molecular advances in the understanding of the injury mechanisms leading to end-stage liver disease.     

Depletion of Thymus-Derived CD4+CD25+ T Cells Abrogates the Suppressive Effects of alpha-galactosylceramide Treatment on Experimental Allergic Conjunctivitis.

Background: We showed previously that alpha-galactosylceramide (alpha-GalCer) treatment elevated splenic CD4+CD25+Foxp3+ T-cell numbers and suppressed the development of experimental allergic conjunctivitis (EC). Here, we investigated whether CD4+CD25+Foxp3+ T cells mediate the suppressive effects of alpha-GalCer treatment on EC. Methods: To deplete CD4+CD25+Foxp3+ T cells, neonatal mice were thymectomized and intraperitoneally injected with anti-CD25 Ab. At 6 weeks of age, these mice were immunized with ragweed (RW) in aluminum hydroxide. Ten days later, the mice were challenged with RW in eye drops and 24 hours later, the conjunctivas and spleens were harvested for histological and flow cytometric analyses, respectively. alpha-GalCer or vehicle was injected 2 hours prior to RW challenge. In addition, alpha-GalCer was injected into thymus-intact EC-developing mice that had not been treated with anti-CD25 Ab. Results: alpha-GalCer treatment significantly suppressed EC in the thymus-intact mice that had not been treated with anti-CD25 Ab. In contrast, alpha-GalCer treatment of thymectomized and anti-CD25 Ab-treated mice did not affect the severity of EC or splenic CD4+CD25+Foxp3+ T-cell numbers. However, alpha-GalCer treatment did significantly increase splenic CD4+CD25+Foxp3+ T-cell numbers in thymectomized mice that had not received anti-CD25 Ab. Conclusions: alpha-GalCer treatment during the effector phase of EC increased CD4+CD25+Foxp3+ T-cell numbers, which in turn suppressed the development of EC.     

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.     

Effect of cytokines on liver necrosis

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Conditional deletion of beta-catenin reveals its role in liver growth and regeneration

BACKGROUND & AIMS: The Wnt/beta-catenin pathway plays a role in liver growth and development. To address this conclusively, we used a conditional knockout approach to delete beta-catenin in the liver. METHODS: Floxed beta-catenin (exons 2-6) mice were intercrossed with Albumin-Cre recombinase transgenic mice; considerable beta-catenin deletion was evident 15 days after birth by Western blot and immunohistochemistry analyses. RESULTS: Although these mice were viable, there was a significant decrease in their liver weight/body weight ratio by 14% at 1 month and 28%-35% by 2-6 months of age, which was sustained throughout their normal life span. There was an accompanying decrease in basal hepatocyte proliferation showed by Ki-67 staining. Additional analysis revealed several known and novel genes to be down-regulated in these mice that play a role in normal liver homeostasis. When subjected to two-thirds partial hepatectomy, the Ctnnb1(loxp/loxp); Alb-Cre(+/-) mice were sick and lethargic, especially during the first 2-3 days only. These mice display a 2-fold decrease in the number of Ki-67- or PCNA-positive cells at the time of peak hepatocyte proliferation at 40 hours, which coincided with decreased cyclin A, D, and E expression. However, a rebound increase in hepatocyte proliferation was evident in the knockout mice at 3 days. Also, increased apoptosis was observed in the knockout livers during regeneration at all stages. CONCLUSIONS: Thus, beta-catenin is essential for normal liver growth and development. Also, although regeneration is delayed in the absence of beta-catenin, it does occur suboptimally, showing its redundancy in the liver.