Hepatocytes Of Transgenic Mice Research Articles

Mitochondrial rupture after photodamage initiates parkin-independent mitophagy in mouse hepatocytes.

Involvement of parkin in mitophagy is well studied. Translocation of parkin from cytosol to depolarized mitochondria triggers ubiquitination-dependent mitophagy. In this study, we used Airyscan super-resolution microscopy and correlative light-electron microscopy (CLEM) to characterize parkin association during photodamage-induced mitophagy, a form of phosphoinositide 3-kinase-independent or Type 2 mitophagy. After photodamage with 488-nm light to small groups of mitochondria within GFP-LC3 transgenic mouse hepatocytes, U-shaped crescents of GFP-LC3 fluorescence formed around damaged, depolarized mitochondria beginning after 31 ± 18 min. Fluorescence of crescents progressively intensified, but their length remained unchanged. Subsequently, LC3-containing puncta merged with damaged mitochondria containing crescents to form a continuous ring. Adenovirally expressed mCherry-TOMM20 (outer membrane marker) labeled the surface of all mitochondria. After photodamage, gaps of mCherry-TOMM20 surface labeling appeared as a result of mitochondrial swelling and outer membrane rupture. Crescents of GFP-LC3 fluorescence formed over these gaps not containing mCherry-TOMM20. Brightfield microscopy confirmed mitochondrial swelling after photodamage, leading to outer membrane rupture and herniation of the inner membrane. CLEM then revealed that GFP-LC3 labeling occurred at the bare surfaces of herniated inner membranes at early stages of photodamage-induced mitophagy. Adenoviral expression of mCherry-parkin showed that parkin did not associate with photodamaged mitochondria at any stage after up to 2 h. In parkin KO mouse hepatocytes adenovirally expressing GFP-LC3 and mCherry-TOMM20, the same mitophagy pattern was observed. Thus, mitochondrial swelling, outer membrane rupture, and inner membrane herniation after photodamage exteriorizes bare surfaces of mitochondrial inner membranes that become the proximate site for LC3 but not parkin association and the initiation of mitophagy (AA021191, AA025379, DK073336).

Hypothermia Advocates Functional Mitochondria and Alleviates Oxidative Stress to Combat Acetaminophen-Induced Hepatotoxicity.

For years, moderate hypothermia (32 °C) has been proposed as an unorthodox therapy for liver injuries, with proven hepatoprotective potential. Yet, limited mechanistic understanding has largely denied its acceptance over conventional pharmaceuticals for hepatoprotection. Today, facing a high prevalence of acetaminophen-induced liver injury (AILI) which accounts for the highest incidence of acute liver failure, hypothermia was evaluated as a potential therapy to combat AILI. For which, transforming growth factor-α transgenic mouse hepatocytes (TAMH) were subjected to concomitant 5 mM acetaminophen toxicity and moderate hypothermic conditioning for 24 h. Thereafter, its impact on mitophagy, mitochondrial biogenesis, glutathione homeostasis and c-Jun N-terminal kinase (JNK) signaling pathways were investigated. In the presence of AILI, hypothermia displayed simultaneous mitophagy and mitochondrial biogenesis to conserve functional mitochondria. Furthermore, antioxidant response was apparent with higher glutathione recycling and repressed JNK activation. These effects were, however, unremarkable with hypothermia alone without liver injury. This may suggest an adaptive response of hypothermia only to the injured sites, rendering it favorable as a potential targeted therapy. In fact, its cytoprotective effects were displayed in other DILI of similar pathology as acetaminophen i.e., valproate- and diclofenac-induced liver injury and this further corroborates the mechanistic findings of hypothermic actions on AILI.

Moderate Hypothermia Effectively Alleviates Acetaminophen-Induced Liver Injury With Prolonged Action Beyond Cooling.

Acetaminophen (APAP) overdose accounts for the highest incidence of acute liver failure, despite the availability of an antidote i.e. N-acetylcysteine. This calls for alternative strategies to manage APAP-induced liver injury (AILI). Therapeutic hypothermia has been explored in past studies for hepatoprotection, but these phenomenal reports lack clarification of its optimal window for application, and mechanistic effects in specific AILI. Hence, we conducted an in vitro study with transforming growth factor-α transgenic mouse hepatocytes cell line, TAMH, and human liver hepatocytes cell line, L-02, where cells were conditioned with deep (25°C) or moderate (32°C) hypothermia before, during or after APAP toxicity. Cell viability was evaluated as a hallmark of cytoprotection, along with cell death. Simultaneously, cold shock proteins (CSPs) and heat shock proteins expressions were monitored; key liver functions including drug-metabolizing ability and hepatic clearance were also investigated. Herein, we demonstrated significant hepatoprotection with 24-hour moderate hypothermic conditioning during AILI and this effect sustained for at least 24 hours of rewarming. Such liver preservation was associated with a CSP—RNA-binding motif protein 3 (RBM3) as its knockdown promptly abolished the cytoprotective effects of hypothermia. With mild and reversible liver perturbations, hypothermic therapy appears promising and its RBM3 involvement deserves future exploration.

Evaluation of In Vitro Models for Pyrrolizidine Alkaloid‐Induced Hepatocellular Injury

ObjectivePyrrolizidine alkaloids (PAs) are toxic chemicals found in many herbal medicines, dietary supplements and even food sources such as honey, bread, and tea. Cases of drug‐induced liver injury due to herbal and dietary supplements are relatively common in Asia and are increasing in the United States. The literature suggests that PAs containing macrocyclic structure are more toxic than those with smaller sidechains. The goal of this project was to evaluate two PAs with macrocyclic structure (monocrotaline and retrorsine) and one PA will a smaller sidechain (retronecine) for their potential to elicit toxicity across two in vitro model systems.MethodsPA toxicity was measured by determining the concentration at which 50% of the cell population was killed (LC50). PAs were dissolved in DMSO and PA‐induced toxicity was measured after 48 hours in two in vitro models: TAMH (transforming growth factor‐α transgenic mouse hepatocytes) and THLE‐2 (transformed human liver epithelial‐2 cells). Statistical analysis of cell viability data was performed using SPSS (Probit regression) to obtain LC50 values.ResultsA decrease in cell viability was observed as PA concentration increased. Interestingly, both cell lines were relatively resistant to PA‐induced toxicity. In many cases only predicted LC50 values for each compound could be generated and are indicated by an asterisk (7.0mM*, 11mM*, and 30.mM* in TAMH for monocrotaline, retrorsine and retronecine, respectively; and 2.2mM, 3.2mM, and >100mM* in THLE‐2 for monocrotaline, retrorsine and retronecine, respectively. Based on the toxicologic parameters, monocrotaline and retrorsine were more toxic (lower LC50 values) than retronecine.ConclusionsWhile PAs have been associated with hepatotoxicity, limited toxicity was seen in both the TAMH and THLE‐2 cell lines. Predicted LC50 values were higher than expected, suggesting TAMH and THLE‐2 cells are not good models for studying PA‐induced liver toxicity. Retronecine, which contains a smaller sidechain, was found to be less toxic than the larger compounds. Future work is currently underway to identify biomarkers responsible for the observed resistance to PA‐induced toxicity as potential targets for preventing hepatocellular injury.Support or Funding InformationThis work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.

Structure‐Based Assessment of the Toxicity and Cytoprotective Effects of a Series of Small Molecule Polyphenolic Compounds

ObjectivePolyphenols are widely known to possess antioxidant and anti‐inflammatory properties, but are also capable of eliciting toxicity at higher doses. This goal of this project was to investigate whether a structure‐toxicity relationship exists among five polyphenolic compounds: trans‐cinnamic acid (tCA), p‐coumaric acid (COU), caffeic acid (CAF), resveratrol (RES), and phloretin (PHL). Each compound is relatively similar in structure, but varies in the number of phenolic groups it contains (tCA=0, COU=1, CAF=2, RES=3, PHL=4).MethodsPolyphenol toxicity was measured by determining the median lethal dose (LC50) and toxicant threshold concentration (LC1) for each compound using an in vitro mouse liver model (TAMH; transforming growth factor‐α transgenic mouse hepatocytes). TAMH cells were cultured and exposed to varying concentrations of each compound for 24‐hours followed by a test for cell viability using resazurin dye. Subsequent hepatoprotective studies based on LC1 values were then conducted by incubating TAMH cells with each polyphenol for 24 h prior to a hepatotoxic dose of diquat (18μM). Changes in cell viability were again measured using the resazurin‐based assay. Data analysis was performed using SPSS to calculate the LC50 and LC1 values for each compound. Significance was determined using one‐way analysis of variance (ANOVA) and post‐hoc Least Significant Difference tests with a fold cut‐off of p < 0.05.ResultstCA and COU were relatively non‐toxic (LC50 > 5mM) compared to the compounds with multiple phenolic groups. In fact, addition of phenolic groups directly correlated with a decrease in the LC50 value indicating that as the number of phenolic groups increased so did the toxicity for this series of compounds. For the hepatoprotection studies, significant increases in cell viability were observed across only some of the polyphenolic compounds; however, this protection did not correlate with phenolic group number.ConclusionsWhile greater toxicity was associated with an increase in the number of phenolic groups based on the LC50 determinations, no common trend was observed relating phenolic group number and hepatoprotective ability using an LC1‐based dosing regimen. Perhaps this suggests that subtle differences in the carbon skeleton among the polyphenols tested influences protective ability. Future work is currently underway to investigate the mechanistic features differentiating the degree of cytoprotection between the polyphenolic compounds.Support or Funding InformationSource of Research Support: This work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellow (SURF) Individual Award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

S1P Stimulates Proliferation by Upregulating CTGF Expression through S1PR2-Mediated YAP Activation.

Dysregulation of the Hippo pathway in the liver results in overgrowth and eventually tumorigenesis. To date, several upstream mechanisms have been identified that affect the Hippo pathway, which ultimately regulate YAP, the major downstream effector of the pathway. However, upstream regulators of the Hippo pathway in the liver remain poorly defined. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been shown to stimulate hepatocellular carcinoma (HCC) cell proliferation, but whether the Hippo pathway is involved in S1P-stimulated HCC cell proliferation remains to be determined. Here it is demonstrated that S1P activates YAP and that the S1P receptor 2 (S1PR2/S1P2) mediates S1P-induced YAP activation in both human and mouse HCC cells. S1P promotes YAP-mediated upregulation of cysteine-rich protein 61 and connective tissue growth factor (CTGF), and stimulates HCC cell proliferation. By using siRNA-mediated knockdown approaches, only CTGF was required for S1P-stimulated cell proliferation. Of note, S1P activates YAP in a MST1/2-independent manner suggesting that the canonical Hippo kinase is not required for S1P-mediated proliferation in liver. The upregulation of CTGF and S1P2 were also observed in liver-specific YAP overexpression transgenic mouse hepatocytes. Moreover, YAP regulated liver differentiation-dependent gene expression by influencing the chromatin binding of HNF4α based on ChIP-seq analysis. Finally, results using gain- and loss-of-function approaches demonstrate that HNF4α negatively regulated S1P-induced CTGF expression.Implications: These findings reveal a role for S1P in stimulating HCC cell proliferation by upregulating CTGF expression through S1P2-mediated YAP activation. Mol Cancer Res; 16(10); 1543-55. ©2018 AACR.

The Mechanistic Effects of JNK and p53 Knockdown on Hepatocyte Viability Following Dipyridyl Herbicide Exposure

ObjectiveThe objective of this study is to characterize key pathways that contribute to the hepatotoxic effects of two dipyridyl herbicides, paraquat and diquat.MethodsDose‐dependent toxicity assays were performed in an in vitro liver model (TGF‐alpha transgenic mouse hepatocytes; TAMH) to determine LC50 values for paraquat and diquat. Transient knockdown of JNK and p53 was performed using siRNA. Target knockdowns were confirmed using protein immunoblotting. Cell viability assays were then conducted to determine how each transfection affected cell survival following exposure to either paraquat or diquat.ResultsLC50 values were determined to be 57μM and 18μM for paraquat and diquat, respectively. siRNA‐based knockdown decreased JNK protein expression by 31%. This correlated with significant increases in cell viability for transfected cells exposed to either paraquat (21%) or diquat (15%). siRNA‐based knockdown decreased p53 protein expression by 66%. This correlated with a significant but small decrease in the viability of transfected cells exposed to paraquat only (6%).ConclusionExperimental LC50 values revealed diquat to be more hepatotoxic than paraquat in the TAMH line. The siRNA‐based knockdown of JNK provides evidence that JNK plays a crucial role in propagating toxicity caused by these herbicides. In contrast, effects from the p53 knockdown experiments had either no effect (diquat exposure) or propagated toxicity to a small but significant extent (paraquat). These observations suggest affecting targets that precede p53 during apoptotic signaling are crucial to rescuing hepatocytes from the toxic effects of dipyridyl herbicides.Support or Funding InformationThis work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellow (SURF) Individual Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leukocyte Specific Protein‐1 (LSP1) Functions as a Suppressor of Proliferation During Liver Regeneration After Partial Hepatectomy (PHx) and in Primary Hepatocytes

Leukocyte specific protein‐1 (LSP1), an f‐actin binding protein expressed in hematopoietic cells and hepatocytes, is the gene with the highest copy number variation in human hepatocellular carcinoma. Our previous work has demonstrated that loss of LSP1 expression leads to increased proliferation and migration of rat hepatocellular carcinoma cells. We have also demonstrated that overexpression of LSP1 in mouse liver through hydrodynamic tail vein injection of LSP1 plasmid DNA results in a significant decrease in proliferation on day 2 after partial hepatectomy (PHx). To further characterize the role of LSP1 in liver regeneration, we performed 2/3 PHx on both a global LSP1 knockout mouse as well as a hepatocyte specific LSP1 transgenic mouse model and analyzed proliferation using ki67 immunohistochemistry as well as western blot analysis of ERK signaling. Since expression of LSP1 leads to decreased proliferation, we hypothesized that the knockout mouse model would exhibit increased proliferation and activation of ERK whereas the transgenic mouse model would display decreased proliferation and pERK expression. LSP1 knockout mice displayed an increase in Ki67 labeling on day 4 after PHx and western blot analysis shows increased pERK expression after PHx in comparison to controls. In the transgenic mouse model, a significant decrease in Ki67 positive hepatocytes was observed on day 4 after PHx as well as decreased pERK expression. Transgenic mice also displayed decreased liver to body weight ratios on day 7 following PHx. Hepatocytes isolated from knockout mice exhibited increased proliferation in the absence of growth factors compared to controls whereas transgenic mouse hepatocytes displayed decreased BrdU incorporation in both the presence and absence of growth factors. LSP1 knockout hepatocytes and transgenic hepatocytes also displayed increased and decreased pERK expression, respectively. Our results demonstrate that LSP1 functions as an inhibitor of proliferation in primary hepatocytes as well as during liver regeneration following PHx. Future studies aim to address the signaling pathways downstream of LSP1 that effect proliferation during liver regeneration as well as how LSP1 functions in hepatocarcinogenesis mechanisms and sensitivity to sorafenib.Support or Funding InformationMenten Endowment, University of Pittsburgh

Oxidation potentials of N-modified derivatives of the analgesic flupirtine linked to potassium KV 7 channel opening activity but not hepatocyte toxicity.

Openers of neuronal voltage-gated potassium channels (KV ) are of interest as therapeutic agents for treating pain (flupirtine) and epilepsy (retigabine). In an effort to better understand the mechanisms of action and toxicity of flupirtine, we synthesized nine novel analogues with varying redox behavior. Flupirtine can be oxidatively metabolized into azaquinone diimines; thus, the oxidation potentials of flupirtine and its analogues were measured by cyclic voltammetry. KV 7.2/3 (KCNQ2/3) opening activity was determined by an established assay with HEK293 cells overexpressing these channels. A link was found between the oxidation potentials of the compounds and their EC50 values for potassium channel opening activity. On the other hand, no correlation was observed between oxidation potentials and cytotoxicity in cultures of transgenic mouse hepatocytes (TAMH). These results support the idea that oxidative metabolites of flupirtine contribute to the mechanism of action, similar to what was recently proposed for acetaminophen (paracetamol), but not to hepatotoxicity.

Differential Regulation of Mitogen-Activated Protein Kinase Pathways by Acetaminophen and Its Nonhepatotoxic Regioisomer 3′-Hydroxyacetanilide in TAMH Cells

Acetaminophen (APAP), a widely used analgesic and antipyretic that is considered to be relatively safe at recommended doses, is the leading cause of drug-induced liver failure in the United States. 3'-Hydroxyacetanilide (AMAP), a regioisomer of APAP, is useful as a comparative tool for studying APAP-induced toxicity because it is nontoxic relative to APAP. Transforming growth factor-alpha transgenic mouse hepatocytes were treated with both isomers to investigate mitogen-activated protein kinase (MAPK) cascades in order to differentiate their toxicological outcomes. Posttranslational modifications of MAPK signaling were assessed using immunoblotting and Bioplex technology, whereas gene expression changes were measured using Affymetrix Mouse Gene 1.0 ST arrays. APAP treatment led to higher levels of glutathione depletion at 6 and 24 h compared with AMAP in mitochondria. Glutathione depletion was preceded by increased levels of c-Jun N-terminal kinase (JNK) phosphorylation at 2 and 6 h after APAP treatment compared with AMAP, whereas AMAP treatment led to increased extracellular signal-regulated protein kinase (ERK) phosphorylation at 2 and 6 h compared with APAP. Furthermore, APAP treatment significantly upregulated jun oncogene (c-Jun) gene expression, which was confirmed by Western blotting for both the phosphorylated and the nonphosphorylated forms of c-Jun protein. Transfection with JNK siRNA attenuated APAP toxicity after 24 h, suggesting that higher levels of APAP-induced activation of JNK were related to higher rates of cell death. In summary, genomic regulation of MAPK-related transcription factors coupled with posttranslational activation of their upstream kinases is critical in differentiating the toxicities of APAP and AMAP.

Hepatitis B Virus (HBV) Surface Antigen Interacts with and Promotes Cyclophilin A Secretion: Possible Link to Pathogenesis of HBV Infection

Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.

Susceptibility to liver fibrosis in mice expressing a connective tissue growth factor transgene in hepatocytes

Connective tissue growth factor (CCN2) is a matricellular protein that is up-regulated in many fibrotic disorders and coexpressed with transforming growth factor beta. CCN2 promotes fibrogenesis and survival in activated hepatic stellate cells, and injured or fibrotic liver contains up-regulated levels of CCN2 that are produced by a variety of different cell types, including hepatocytes. To investigate CCN2 action in vivo, transgenic FVB mice were created in which the human CCN2 gene was placed under the control of the albumin enhancer promoter to elevate hepatocyte CCN2 levels. Production of human CCN2 (hCCN2) messenger RNA and elevated CCN2 protein levels was demonstrated in transgenic livers, whereas levels of endogenous mouse CCN2 were comparable between transgenic and wild-type mice. Liver histology and liver function tests were unaffected in transgenic animals. However, after chronic administration of CCl(4), alpha-smooth muscle actin (alpha-SMA)-expressing cells and collagen deposition were increased as a function of the dosage of the hCCN2 transgene (hccn2(+/+) > hccn2(+/-) > hccn2(-/-)). Moreover, CCl(4)-induced serum hyaluronic acid, hepatic tissue levels of alpha-SMA or acid-soluble collagen, and messenger RNA expression of alpha-SMA, collagen alpha1 (I), matrix metalloprotease-2, or tissue inhibitor of metalloprotease-1 were greater in transgenic mice than in wild-type mice. Transgenic mice also exhibited enhanced hepatic deposition of collagen 2 weeks after bile duct ligation. Production of elevated CCN2 levels in hepatocytes of transgenic mice in vivo does not cause hepatic injury or fibrosis per se but renders the livers more susceptible to the injurious actions of other fibrotic stimuli. These studies support a central role of CCN2 in hepatic fibrosis and demonstrate a role of the microenvironment in regulating the profibrotic action of CCN2.

PIK3IP1, a Negative Regulator of PI3K, Suppresses the Development of Hepatocellular Carcinoma

Phosphatidylinositol-3-kinase (PI3K) is a well-known regulator of cell division, motility, and survival in most cell types. Recently, we characterized a novel protein that we call PI3K Interacting Protein 1 (PIK3IP1), which binds to the p110 catalytic subunit of PI3K and reduces its activity in vitro. Little is known about the role of PIK3IP1 in normal and neoplastic growth in vivo. Proper liver function and development depend on intact PI3K signal transduction; when dysregulated, the PI3K pathway is linked to the development of liver cancer. To begin to dissect the contribution of PIK3IP1 to hepatic PI3K signaling in vivo and to liver tumorigenesis in particular, we formulated the following hypothesis: because PIK3IP1 down-regulates PI3K signaling and uncontrolled PI3K signaling is associated with liver cancer, then PIK3IP1-mediated down-regulation of the PI3K pathway should inhibit hepatocellular carcinoma (HCC) development. To test this idea, we generated transgenic mice overexpressing PIK3IP1 in hepatocytes in a mouse strain prone to develop HCC. Isolated PIK3IP1 transgenic mouse hepatocytes showed blunted PI3K signaling, DNA synthetic activity, motility, and survival compared with controls. In vivo, spontaneous liver tumorigenesis was significantly dampened in the transgenic animals. This was accompanied by decreased hepatic PI3K activity and reduced hepatocyte proliferation in the transgenics compared with controls. We also observed that human HCC expressed less PIK3IP1 protein than adjacent matched liver tissue. Our data show that PIK3IP1 is an important regulator of PI3K in vivo, and its dysregulation can contribute to liver carcinogenesis.

Ablation of gap junctional communication in hepatocytes of transgenic mice does not lead to disrupted cellular homeostasis or increased spontaneous tumourigenesis

Gap junctions between murine hepatocytes are composed of two subunit proteins, connexin26 (Cx26) and connexin32 (Cx32). Previously, we found increased formation of chemically induced liver tumours but no increase in spontaneous development of preneoplastic hepatic foci in mice that lacked Cx32 and expressed decreased amounts of Cx26. In order to clarify this tumour-suppressive effect and to overcome embryonic lethality of constitutive Cx26-deficient mice, cell type-specific targeting of the Cx26 gene was performed. Mice with loxP-flanked Cx26 coding DNA were crossed with mice expressing the Cre recombinase exclusively in hepatocytes. Progeny mice lacking Cx26 in the liver were viable and fertile with no obvious signs of phenotypic alterations. To generate mice that totally lack gap junctional intercellular coupling, these mice were crossed with constitutive Cx32-deficient mice. We found no increase in spontaneously induced liver tumour formation in Cx26 and double deficient Cx26/Cx32 mice. Occasionally, double deficient livers exhibited morphological alterations, like amyloidosis, and a slightly increased basal proliferation rate of hepatocytes. Although the absence of gap junction channels led to altered expression of adhesion-related proteins like E-cadherin and actin, microarray analyses of total liver transcripts yielded only few differences between Cx26-deficient and double deficient livers compared to control samples. Our results suggest that total lack of gap junctional communication due to hepatocytic ablation of Cx26 and Cx32 does not drastically alter basal hepatocytic function and does not lead to increased spontaneous liver tumour formation.

Gene Expression Analysis in HBV Transgenic Mouse Liver: A Model to Study Early Events Related to Hepatocarcinogenesis

Hepatitis B virus (HBV) is one of the major etiological factors responsible for the development of hepatocellular carcinoma (HCC). We used a transgenic mouse, containing HBV sequences, as a model system to unravel the molecular mechanisms of hepatocarcinogenesis induced by HBV. We chose this animal model because it consistently develops liver cancer after intermediate steps that mimic the natural history of HBV infection in humans. In this study, we focus our attention on the early events leading to liver cancer. We compared the gene expression profile of 3-month-old transgenic mice with that of 3-month-old wild-type (wt) animals. In the transgenic mouse, microarray data analysis showed a total of 45 significantly differentially expressed genes, 25 highly expressed (fold change > or =2; P = 0.0025), and 20 downregulated (fold change < or =0.5; P = 0.0025). These genes belong to several different functional categories such as the regulation of immunological response, transcription, intracellular calcium ion mobilization, regulation of cell cycle and proliferation, NF-kappab signal transduction cascades, and apoptosis. In particular, the upregulation of the antiapoptotic gene NuprI and the downregulation of the proapoptotic gene Bnip3 were found. This observation was supported by an in vitro apoptosis assay that showed downregulation of apoptosis in hepatocytes of HBV transgenic mouse compared with wt mice treated with staurosporine. In conclusion, our experimental approach allowed identification of new genes modulated by HBV and showed that the apoptotic process was deregulated in transgenic mouse hepatocytes. These data shed light on one possible mechanism by which HBV induces hepatocarcinogenesis.

Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A

Background & Aims: To establish a chronic infection, hepatitis C virus (HCV) has to evade the host defense. Expression of HCV proteins in cultured cells and in hepatocytes of transgenic mice inhibits interferon-α-induced intracellular signaling through the Jak-STAT (signal transducer and activator of transcription) pathway. It is not known if interferon-α signaling is inhibited in patients with chronic hepatitis C as well, and the molecular mechanisms are not well defined. Methods: Interferon-α-induced signaling was investigated in liver biopsies from patients with chronic hepatitis C. The molecular mechanisms of HCV interference with Jak-STAT signaling were analyzed in cultured cells, HCV transgenic mice, and liver biopsies. Results: Interferon-α-induced DNA binding of STAT1 was significantly impaired in liver biopsies from patients with chronic hepatitis C compared with controls. Tyrosine and serine phosphorylation of STAT1 were intact, but methylation of STAT1 on arginine 31 was reduced. Hypomethylated STAT1 associated with PIAS1, an inhibitor of STAT DNA binding. Increased expression levels of Protein Phosphatase 2A were found in liver extracts from HCV transgenic mice and in liver biopsies of patients with chronic hepatitis C. Overexpression of PP2Ac in Huh7 cells resulted in hypomethylation of STAT1, increased binding to PIAS1, and reduced interferon-α-induced DNA binding of STAT1. Conclusions: We conclude that HCV interferes with interferon-α signaling via up-regulation of PP2Ac, hypomethylation of STAT1, and increased STAT1-PIAS1 association, resulting in reduced transcriptional activation of interferon-α-stimulated genes.

Fibroblast growth factor receptor signalling is crucial for liver homeostasis and regeneration.

Several growth factors have been suggested to play a crucial role in liver regeneration, but a functional proof is still missing. Since fibroblast growth factors are important for the initiation of mammalian liver development, we determined the roles of these mitogens in liver repair by targeted expression of a dominant-negative fibroblast growth factor receptor (FGFR) in hepatocytes of transgenic mice. The liver of young animals appeared histologically normal, and liver function was not obviously impaired. In aged transgenic mice, the frequency of fatty liver development was strongly increased compared to control animals. Following partial hepatectomy, transgenic mice showed markedly reduced hepatocyte proliferation because of an arrest in the late G(1) phase of the cell cycle. These data demonstrate a key role of FGFR signalling in repair after liver injury.

Transgenic mouse models of hepatitis B virus-associated hepatocellular carcinoma.

The multi-factorial and multi-step nature of cancer development makes analysis difficult in cell culture and non-genetic animal models. Recent progress in technology has allowed the development of several transgenic animal models addressing various aspects of liver diseases caused by hepatitis B virus in human patients. The experimental data from these studies in vivo highlight the importance of HBV gene products that alone or in conjunction with other host cellular protein(s) can deregulate the cell cycle control checkpoints in the hepatocytes of transgenic mice leading to the development of hepatocellular carcinoma. Moreover, these models are extremely useful in analysing and ascertaining the stages of malignant transformation linked to multiple genetic and non-genetic events of cancer development and in developing novel strategies of intervention.

Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes.

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and IFN-gamma inhibit hepatitis B virus (HBV) replication noncytopathically in the livers of HBV transgenic mice and in hepatocyte cell lines derived from these mice. The present study was designed to identify transcriptionally controlled hepatocellular genes that are tightly associated with the inhibition of HBV replication and that might, therefore, mediate the antiviral effect of these cytokines. Twenty-nine genes were identified, many of which have known or potential antiviral activity. Notably, multiple components of the immunoproteasome and ubiquitin-like proteins were strongly induced by both IFN-alpha/beta and IFN-gamma, as were a number of GTP-binding proteins, including GTPases with known antiviral activity, chemokines, signaling molecules, and miscellaneous genes associated with antigen processing, DNA-binding, or cochaperone activity and several expressed sequence tags. The results suggest that one or more members of this relatively small subset of genes may mediate the antiviral effect of IFN-alpha/beta and IFN-gamma against HBV. We have already exploited this information by demonstrating that the antiviral activity of IFN-alpha/beta and IFN-gamma is proteasome dependent.

STAT3 Down-regulates the Expression of Cyclin D during Liver Development

As the expression of cyclin D1 is induced during liver regeneration and also in hepatic tumor cells, cyclin D1 is likely to play an important role in the proliferation and transformation of hepatocytes. However, the role of cyclin D1 in liver development remains unknown. Here we show that the expression of D-type cyclins including cyclin D1, D2, and D3 is down-regulated along with liver development. In addition, oncostatin M (OSM), an interleukin-6 family cytokine, down-regulated the expression of cyclin D1 and D2 in a primary culture of fetal hepatocytes in which OSM induces hepatic differentiation. Ectopic expression of receptor mutants defective in the activation of either STAT3 or SHP-2/Ras indicated that the down-regulation of D1 and D2 cyclins by OSM was mediated by STAT3 but not by SHP-2/Ras. Consistently, expression of dominant negative STAT3 but not Ras relieved OSM-induced suppression of cyclin D expression. Activation of STAT3 in fetal hepatocytes of transgenic mice expressing the STAT3-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in the suppression of cyclin D1 and D2 expression. These results indicate that STAT3 activation is necessary and sufficient for down-regulation of D1 and D2 cyclins in fetal hepatocytes. Furthermore, STAT3-C, a constitutively active form of STAT3, suppressed transcription of the cyclin D1 promoter in fetal hepatocytes, whereas it activated the transcription in hepatic tumor cells, huH7 and HepG2. Thus, STAT3-mediated down-regulation of cyclin D expression is rather specific to fetal hepatocytes that are undergoing maturation processes including a reduction of their proliferation potential.