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.     

Overexpression of suppressor of cytokine signaling-3 in T cells exacerbates acetaminophen-induced hepatotoxicity

Cytokines have been implicated in the progression of acetaminophen (APAP)-induced acute liver injury. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT pathway, but their role in APAP hepatotoxicity is unknown. In this present study, we attempted to explore the role of SOCS3 in T cells in APAP-induced liver injury. Mice with a cell-specific overexpression of SOCS3 in T cells (SOCS3Tg, in which Tg is transgenic) exhibited exaggerated hepatic injury after APAP challenge, as evidenced by increased serum alanine aminotransferase levels, augmented hepatic necrosis, and decreased survival relative to the wild-type mice. Adaptive transfer of SOCS3Tg-CD4(+) T cells into T and B cell-deficient RAG-2(-/-) mice resulted in an exacerbated liver injury relative to the control. In SOCS3Tg mice, hepatocyte apoptosis was enhanced with decreased expression of antiapoptotic protein bcl-2, whereas hepatocyte proliferation was reduced with altered cell cycle-regulatory proteins. Levels of IFN-gamma and TNF-alpha in the circulation were augmented in SOCS3Tg mice relative to the control. Studies using neutralizing Abs indicated that elevated IFN-gamma and TNF-alpha were responsible for the exacerbated hepatotoxicity in SOCS3Tg mice. Activation of STAT1 that is harmful in liver injury was augmented in SOCS3Tg hepatocytes. Alternatively, hepatoprotective STAT3 activation was decreased in SOCS3Tg hepatocytes, an event that was associated with augmented SOCS3 expression in the hepatocytes. Altogether, these results suggest that forced expression of SOCS3 in T cells is deleterious in APAP hepatotoxicity by increasing STAT1 activation while decreasing STAT3 activation in hepatocytes, possibly through elevated IFN-gamma and TNF-alpha.     

LPS／D—GalN诱发ⅣF—KB转基因小鼠急性致死性肝损伤模型的建立

目的以NF—KB转基因BALB／c小鼠建立一个LPS／D—GaIN诱发的急性致死性肝损伤模型。方法采取腹腔注射高剂量的LPS／D-GalN建立急性致死性肝损伤小鼠模型,观察模型小鼠的促炎症细胞因子水平和NF—KB的活性改变,以及肝脏功能和病理改变情况。结果模型组小鼠生存时间为8—10h,模型建立后小鼠血清TNF—a、IL-6和MCP-1水平显著升高,在2—4h达到高峰;肝脏外观出现瘀血和出血,肝脏小叶被严重破坏,肝细胞严重坏死和出血;血清ALT／AST水平在模型诱发后持续迅速上升;整体成像显示胛-KB的活性在4～6h达到高峰。正常对照组小鼠以上指标无显著变化。结论成功建立LPS／D-GalN诱发的M-船转基因小鼠的急性致死性肝损伤模型。     

The effect of Z-FA.FMK on D-galactosamine/TNF-alpha-induced liver injury in mice

Cathepsin B is a cysteine proteinase, considered to have an important role in apoptosis, which is activated by D-galactosamine and tumor necrosis factor-alpha (D-GalN/TNF-alpha). Benzyloxycarbonyl-L-phenylalanine fluoromethyl ketone (Z-FA.FMK) is a cathepsin B inhibitor used in research on apoptotic pathways. The aim of this study was to investigate the role of Z-FA.FMK on apoptotic cell death, cell proliferation and liver damage induced by a D-GalN/TNF-alpha combination in mice. In the study, 1 h after administration of 8 mg/kg Z-FA.FMK by intravenous injection, D-GalN (700 mg/kg) and TNF-alpha (15 microg/kg) were administered by a single intraperitoneal injection. In the group given D-GalN/TNF-alpha, the following results were found: Degenerative changes in the liver tissue, significant increase in the number of both TUNEL and activated caspase-3-positive hepatocytes, a decrease in the number of PCNA-positive hepatocytes, an increase in lipid peroxidation (LPO) levels and a decrease in glutathione (GSH) and DNA levels in the liver tissue. In contrast, in the group given D-GalN/TNF-alpha and Z-FA.FMK, a decrease in the damage of the liver tissue, a significant decrease in TUNEL and activated caspase-3-positive hepatocytes, a significant increase in the number of PCNA-positive hepatocytes, a decrease in the LPO levels, an increase in GSH and DNA levels in the liver tissue were found. As a result, microscopic and biochemical evaluations indicate that Z-FA.FMK plays a protective role against liver injury induced by D-GalN/TNF-alpha and it has an inverse effect on hepatocyte apoptosis and proliferation in BALB/c mice.     

Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy.

Partial hepatectomy (PH) triggers a rapid regenerative response in the remaining tissue to reinstate the organ function and the cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 in the sera of rats with PH. Analysis of the cyclooxygenase (COX) isoenzymes in the remnant liver showed the preferential expression of COX-2 in hepatocytes. Cultured regenerating hepatocytes expressed significant levels of COX-2, a process that was not observed in the sham counterparts. Maximal expression of COX-2 was detected 16 h after PH with increased levels present even at 96 h. Pharmacological inhibition of COX-2 activity with NS398 shunted the up-regulation of cell proliferation after PH, which suggests a positive interaction of prostaglandins with the progression of the cell cycle. Similar results were obtained after PH of mice lacking the COX-2 gene. The expression of COX-2 in regenerating liver was concomitant with a decrease in CCAAT-enhancer binding protein (C/EBP-a) level and an increase in the expression of C/EBP-b and C/EBP-d. These results suggest a contribution of the enhanced synthesis of prostaglandins to liver regeneration observed after PH.     

Hepatocytes proteomic alteration and seroproteome analysis of HBV‐transgenic mice

Hepatitis B is the most common and serious liver disease, especially in developing countries. Although HBV pathogenesis has been extensively investigated, the proteomic alteration of hepatocytes during HBV chronic infection is still unclear. Using the purified hepatocytes, we compared the protein profiles by 2‐DE and LC‐MS between HBV‐transgenic (Tg) and corresponding background mice. Twenty‐seven altered proteins were identified in hepatocytes from HBV‐Tg mice, among which 13 proteins were involved in mitochondrion metabolism pathway including tricarboxylic acid (TCA) cycle and oxidative response; four proteins (SELENBP, SCP2, RGN and PRDX1) were also dramatically changed in liver samples from HBV‐infected patients. Important genes (gpx, sod, ogg et al.) correlated to oxidative damage were up‐regulated in the liver of HBV‐Tg mice. Reactive oxygen species production was significantly increased while ATP production was decreased in liver mitochondria from HBV‐Tg mice. Moreover, hepatocytes of HBV‐Tg mice were more sensitive to hydrogen peroxide‐induced cell death than that of wild‐type control. Using 2‐D Western blotting analysis, eight hepatocyte proteins were found to react with sera of HBV‐Tg mice but not with that of background mice. Interestingly, two (Etfa and Dmgdh) of the eight reactive proteins were overexpressed in HBV‐Tg mice. We believe this study is the first proteomic and seroproteome analysis of HBV‐infected mammalian hepatocyte and provides insightful links between HBV infection and HBV‐induced liver diseases.     

Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes

Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2‐Cre or VE‐cadherin‐Cre constructs to facilitate fate‐mapping of LSECs in liver regeneration. Some YFP‐positive LSECs were observed to convert into hepatocytes following a two‐thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non‐hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte‐like (iHep) cells may provide a new approach to tissue engineering.     

Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2-/- mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication, and survival. Cdk2-/- hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2-/- liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2-/- hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2-/- mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation, and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.     

CpG DNA-mediated induction of acute liver injury in D-galactosamine-sensitized mice: the mitochondrial apoptotic pathway-dependent death of hepatocytes

Unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce innate inflammatory responses, including rapid induction of proinflammatory cytokines. Although innate inflammatory responses induced by CpG DNA and other pathogen-associated molecular patterns are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is detrimental to the host. In this study, we demonstrate that CpG DNA, but not control non-CpG DNA, induces a fulminant liver failure with subsequent shock-mediated death by promoting massive apoptotic death of hepatocytes in D-galactosamine (D-GalN)-sensitized mice. Inhibition of mitochondrial membrane permeability transition pore opening or caspase 9 activity in vivo protects D-GalN-sensitized mice from the CpG DNA-mediated liver injury and death. CpG DNA enhanced production of proinflammatory cytokines in D-GalN-sensitized mice via a TLR9/MyD88-dependent pathway. In addition, CpG DNA failed to induce massive hepatocyte apoptosis and subsequent fulminant liver failure and death in D-GalN-sensitized mice that lack TLR9, MyD88, tumor necrosis factor (TNF)-alpha, or TNF receptor I but not interleukin-6 or -12p40. Taken together, our results provide direct evidence that CpG DNA induces a severe acute liver injury and shock-mediated death through the mitochondrial apoptotic pathway-dependent death of hepatocytes caused by an enhanced production of TNF-alpha through a TLR9/MyD88 signaling pathway in D-GalN-sensitized mice.     

High levels of Myc expression are required for the robust proliferation of hepatocytes,but not for the sustained weak proliferation

In contrast to the robust proliferation exhibited following acute liver injury, hepatocytes exhibit long-lasting proliferative activity in chronic liver injury. The mechanistic differences between these distinct modes of proliferation are unclear. Hepatocytes exhibited robust proliferation that peaked at 2 days following partial hepatectomy in mice, but this proliferation was completely inhibited by hepatocyte-specific expression of MadMyc, a Myc-suppressing chimeric protein. However, Myc suppression induced weak but continuous hepatocyte proliferation, thereby resulting in full restoration of liver mass despite an initial delay. Late-occurring proliferation was accompanied by prolonged suppression of proline dehydrogenase (PRODH) expression, and forced PRODH overexpression inhibited hepatocyte proliferation. In hepatocytes in chronic liver injury, Myc was not activated but PRODH expression was suppressed in regenerating hepatocytes. In liver tumors, PRODH expression was often suppressed, especially in the highly proliferative tumors with distinct Myc expression. Our results indicate that the robust proliferation of hepatocytes following acute liver injury requires high levels Myc expression and that there is a compensatory Myc-independent mode of hepatocyte proliferation with the regulation of proline metabolism, which might be relevant to liver regeneration in chronic injury.     

Kupffer cell-derived cyclooxygenase-2 regulates hepatocyte Bcl-2 expression in choledocho-venous fistula rats

We have previously demonstrated that after bile duct ligation hepatocytes express Bcl-2, although the mechanisms regulating Bcl-2 expression were not identified. Our aim was to determine if biliary constituents induce hepatocellular expression of Bcl-2 by a cyclooxygenase-2 (COX-2)-dependent mechanism. We used the choledocho-venous fistula (CVF) rat model for these studies and inhibited COX-2 by feeding the animals nimesulide, a selective inhibitor of COX-2 activity. Serum bile acids were 70-fold greater in CVF animals compared with controls, although liver histology and serum alanine aminotransferase values remained normal for the duration of the study. Neither Bcl-2 nor COX-2 was detected in sham-operated animals. However, Bcl-2 was expressed in hepatocytes but not in other liver cells in the CVF animals. In contrast, COX-2 protein was identified in Kupffer cells but not in hepatocytes of CVF animals. Hepatic Bcl-2 protein expression was fourfold lower in the livers from nimesulide-treated CVF rats. In conclusion, high circulating concentrations of biliary constituents are associated with stimulation of de novo hepatocyte expression of Bcl-2 and Kupffer cell expression of COX-2. These data suggest Kupffer cell-derived prostanoids may regulate Bcl-2 expression in the hepatocyte.     

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.     

Hepatoprotective principles from the flowers of Tilia argentea (linden): structure requirements of tiliroside and mechanisms of action

The methanolic extract from the flowers of Tilia argentea (linden) was found to show a hepatoprotective effect against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. By bioassay-guided separation using in vitro D-GalN-induced damage to hepatocytes, five flavonol glycosides were isolated as the hepatoprotective constituents of the methanolic extract. Tiliroside, the principal flavonol glycoside, strongly inhibited serum GPT and GOT elevations at doses of 25-100 mg/kg (p.o.) in D-GalN/LPS-treated mice. By comparing the inhibitory effects of tiliroside with those of its components alone, the kaempferol 3-O-beta-D-glucopyranoside moiety was found to be essential for the activity, and its effect was suggested to depend on the inhibition of tumor necrosis factor-alpha (TNF-alpha) production, decreased sensitivity of hepatocytes to TNF-alpha, and on the protection of hepatocytes against D-GalN.     

Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration

After tissue loss the liver has the unique capacity to restore its mass by hepatocyte proliferation. Interleukin-6 (IL6)-deficient mice show a lack in DNA synthesis after partial hepatectomy (PH). To define better the role of IL6 and its family members for liver regeneration after PH, we used conditional knockout mice for glycoprotein 130 (gp130), the common signal transducer of all IL6 family members. We show that gp130-dependent pathways control Stat3 activation after PH. By using gene array analysis, we demonstrate that c-jun, NF-kappa B, c-myc, and tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the acute phase response was completely abolished, we considered that other means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and also IL6 -/- animals after PH leads to a significant reduction in survival and DNA synthesis, which was associated with decreased Bcl-xL expression and higher apoptosis in the liver. These results indicate that the phenotype concerning the reduction in DNA synthesis might be linked to the degree of infection after PH. Thus our results suggest that the role of gp130-dependent signaling is not a direct influence on cell cycle progression after partial hepatectomy but is to activate protective pathways important to enable hepatocyte proliferation.     

CCl4-induced acute liver injury in mice is inhibited by hepatocyte growth factor overexpression but stimulated by NK2 overexpression

Hepatocyte growth factor (HGF) inhibits acute liver injury. NK2 acts as an antagonist to HGF in vitro, but its in vivo function has reached no consensus conclusions. We have investigated in vivo effects of HGF and NK2 on CCl4-induced acute liver injury. Elevation of the serum alanine aminotransferase level and extension of centrilobular necrosis were inhibited in HGF transgenic mice but were promoted in NK2 transgenic mice. Hepatocyte proliferation after liver injury was not inhibited in NK2 transgenic mice. Thus, this study indicates that HGF inhibits liver injury, and NK2 antagonizes HGF on liver injury, however, NK2 may not antagonize HGF on hepatocyte proliferation.     

LPS/D-GalN诱导小鼠急性肝损伤模型的建立

目的建立脂多糖(lipopolysaccharide,LPS)/D-氨基半乳糖(D-galactosamine,D-GalN)诱导小鼠急性肝损伤模型。方法 40只雌性C57BL/6小鼠用于观察8种不同LPS与D-GalN剂量配比联合刺激后小鼠存活时间,以确定模型建立的最佳剂量。使用腹腔注射最佳剂量染毒32只雌性C57BL/6小鼠,分别在0、1、4、8 h处死,每组8只,0 h注射相同剂量生理盐水作为对照。观察染毒后小鼠肝组织病理损伤,检测血清中ALT及炎症因子IL-6、MCP-1和TNF-α表达水平变化。结果通过观察小鼠存活时间,确定腹腔注射最佳染毒剂量为LPS(2.5 mg/kg)/D-GalN(0.3 g/kg);小鼠染毒后肝组织呈进程性病变,最终发展为肝脏弥漫性坏死,肝细胞核崩解。与对照组相比,血清ALT显著升高(P0.001),IL-6、MCP-1、TNF-α均在1 h后达到最高水平(P0.001),然后持续下降。结论成功建立LPS/D-GaIN诱导小鼠急性肝损伤模型,为探索急性肝损伤的致病机制以及药物干预治疗提供有效的动物模型。     

Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.