Basal MET phosphorylation is an indicator of hepatocyte dysregulation in liver disease.

Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, metabolic dysfunction-associated steatotic liver disease (MASLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in MASLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) an increased basal MET phosphorylation and a strong downregulation of the PI3K-AKT pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

Multilineage communication regulates human liver bud development from pluripotency.

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

PFASs-restriction proposal commentary on ECHA's Annex XV restriction report, proposal for a restriction, March 2023.

PFASs are defined as substances that contain at least one fully fluorinated methyl (CF3-) or methylene (-CF2-) carbon atom. The excellent technical properties of members of the PFAS group have led to their use in a wide range of applications. The substance group comprises more than 10,000 individual compounds. A variety of adverse effects has been described for single substances. For the majority of the PFASs, neither toxicokinetic data nor effect data is available. Hence, because of the small number of PFASs for which a full toxicological profile is available, grouping based on the existing data is not feasible. A critical problem of PFASs and their degradation products is the very high persistence, which clearly fulfils the criterion for the substance property Very Persistent (vP) according to Annex XIII of the REACH Regulation. Because of this property the European Commission is planning to take action. Defining suitable subgroups appears to be a scientifically based approach. However, to reach this goal, large data gaps would have to be closed which would take up to centuries, a time-frame, which is not defendable with respect to potential irreversible harm. Because of the time pressure resulting from the potential irreversible harm, the precautionary principle has been selected as an appropriate tool to handle PFASs and in the restriction proposal PFASs are treated as one group. This approach is justified in the view of the advisory committee of the German Society for Toxicology. ECHA's proposal received a lot of attention in the public. However, communication so far has obviously led to the misunderstanding of a proven health hazard for all PFASs. Communication should explain the justification of the broad inclusion of substances as being based on the precautionary principle. Data gaps versus current knowledge need to be clearly communicated; communication should also include the possibility for derogation of essential use. It should address the issue of suitable substitutes to avoid unintended health consequences; and it should mention that existing persistent environmental contamination calls for developing innovation in remediation techniques.

Identification of Interleukin1ß as an Amplifier of Interferon alpha-induced Antiviral Responses.

The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFNα)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFNα-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFNα-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFNα-induced antiviral genes can also be achieved by co-application of IFNα and interleukin1 beta (IL1ß). Consistently, we found that IL1ß enhances IFNα-mediated repression of viral replication. Conversely, we observed that in IL1ß receptor knock-out mice replication of viruses sensitive to IFNα is increased. Thus, IL1ß is capable to potentiate IFNα-induced antiviral responses and could be exploited to improve antiviral therapies.

Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.

BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1ß secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction.

Tightly interlinked feedback regulators control the dynamics of intracellular responses elicited by the activation of signal transduction pathways. Interferon alpha (IFNα) orchestrates antiviral responses in hepatocytes, yet mechanisms that define pathway sensitization in response to prestimulation with different IFNα doses remained unresolved. We establish, based on quantitative measurements obtained for the hepatoma cell line Huh7.5, an ordinary differential equation model for IFNα signal transduction that comprises the feedback regulators STAT1, STAT2, IRF9, USP18, SOCS1, SOCS3, and IRF2. The model-based analysis shows that, mediated by the signaling proteins STAT2 and IRF9, prestimulation with a low IFNα dose hypersensitizes the pathway. In contrast, prestimulation with a high dose of IFNα leads to a dose-dependent desensitization, mediated by the negative regulators USP18 and SOCS1 that act at the receptor. The analysis of basal protein abundance in primary human hepatocytes reveals high heterogeneity in patient-specific amounts of STAT1, STAT2, IRF9, and USP18. The mathematical modeling approach shows that the basal amount of USP18 determines patient-specific pathway desensitization, while the abundance of STAT2 predicts the patient-specific IFNα signal response.

Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels.

Long-term simulation of lead concentrations in agricultural soils in relation to human adverse health effects.

Lead (Pb) exposure of consumers and the environment has been reduced over the past decades. Despite all measures taken, immission of Pb onto agricultural soils still occurs, with fertilizer application, lead shot from hunting activities, and Pb from air deposition representing major sources. Little is known about the intermediate and long-term consequences of these emissions. To gain more insight, we established a mathematical model that considers input from fertilizer, ammunition, deposition from air, uptake of Pb by crops, and wash-out to simulate the resulting Pb concentrations in soil over extended periods. In a further step, human oral exposure by crop-based food was simulated and blood concentrations were derived to estimate the margin of exposure to Pb-induced toxic effects. Simulating current farming scenarios, a new equilibrium concentration of Pb in soil would be established after several centuries. Developmental neurotoxicity represents the most critical toxicological effect of Pb for humans. According to our model, a Pb concentration of ~ 5 mg/kg in agricultural soil leads to an intake of approximately 10 µg Pb per person per day by the consumption of agricultural products, the dose corresponding to the tolerable daily intake (TDI). Therefore, 5 mg Pb/kg represents a critical concentration in soil that should not be exceeded. Starting with a soil concentration of 0.1 mg/kg, the current control level for crop fields, our simulation predicts periods of ~ 50 and ~ 175 years for two Pb immission scenarios for mass of Pb per area and year [scenario 1: ~ 400 g Pb/(ha × a); scenario 2: ~ 175 g Pb/(ha × a)], until the critical concentration of ~ 5 mg/kg Pb in soil would be reached. The two scenarios, which differ in their Pb input via fertilizer, represent relatively high but not unrealistic Pb immissions. From these scenarios, we calculated that the annual deposition of Pb onto soil should remain below ~ 100 g/(ha × a) in order not to exceed the critical soil level of 5 mg/kg. We propose as efficient measures to reduce Pb input into agricultural soil to lower the Pb content of compost and to use alternatives to Pb ammunition for hunting.

Prolonged Lipid Accumulation in Cultured Primary Human Hepatocytes Rather Leads to ER Stress than Oxidative Stress.

Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Epigenetic Modifications of the Liver Tumor Cell Line HepG2 Increase Their Drug Metabolic Capacity.

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards 'normal' liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.

Genomewide comparison of the inducible transcriptomes of nuclear receptors CAR, PXR and PPARα in primary human hepatocytes.

The ligand-activated nuclear receptor pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3) are two master transcriptional regulators of many important drug metabolizing enzymes and transporter genes (DMET) in response to xenobiotics including many drugs. The peroxisome proliferator-activated receptor alpha (PPARα, NR1C1), the target of lipid lowering fibrate drugs, primarily regulates fatty acid catabolism and energy-homeostasis. Recent research has shown that there are substantial overlaps in the regulated genes of these receptors. For example, both CAR and PXR also modulate the transcription of key enzymes involved in lipid and glucose metabolism and PPARα also functions as a direct transcriptional regulator of important DMET genes including cytochrome P450s CYP3A4 and CYP2C8. Despite their important and widespread influence on liver metabolism, comparative data are scarce, particularly at a global level and in humans. The major objective of this study was to directly compare the genome-wide transcriptional changes elucidated by the activation of these three nuclear receptors in primary human hepatocytes. Cultures from six individual donors were treated with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARα (WY14,643) or DMSO as vehicle control. Genomewide mRNA profiles determined with Affymetrix microarrays were analyzed for differentially expressed genes and metabolic functions. The results confirmed known prototype target genes and revealed strongly overlapping sets of coregulated but also distinctly regulated and novel responsive genes and pathways. The results further specify the role of PPARα as a regulator of drug metabolism and the role of the xenosensors PXR and CAR in lipid metabolism and energy homeostasis. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

The Cell-Surface N-Glycome of Human Embryonic Stem Cells and Differentiated Hepatic Cells thereof.

Human embryonic stem cells (hESCs) are pluripotent stem cells that offer a wide range of applications in regenerative medicine. In addition, they have been proposed as an appropriate alternative source of hepatocytes. In this work, hESCs were differentiated into definitive endodermal cells (DECs), followed by maturation into hepatocyte-like cells (HLCs). Their cell-surface N-glycome was profiled and also compared with that of primary human hepatocytes (PHHs). Undifferentiated hESCs contained large amounts of high-mannose N-glycans. In contrast, complex-type N-glycans such as asialylated or monosialylated biantennary and triantennary N-glycans were dominant in HLCs, and fully galactosylated structures were significantly more abundant than in undifferentiated hESCs. The cell-surface N-glycosylation of PHHs was more biologically processed than that of HLCs, with bisialylated biantennary and trisialylated triantennary structures predominant. This is the first report of the cell surface N-glycome of PHHs and of HLCs being directly generated from hESCs without embryoid body formation.

Imatinib and spironolactone suppress hepcidin expression.

Disorders of iron metabolism are largely attributed to an excessive or insufficient expression of hepcidin, the master regulator of systemic iron homeostasis. Here, we investigated whether drugs targeting genetic regulators of hepcidin can affect iron homeostasis. We focused our efforts on drugs approved for clinical use to enable repositioning strategies and/or to reveal iron-related side effects of widely prescribed therapeutics. To identify hepcidin-modulating therapeutics, we re-evaluated data generated by a genome-wide RNAi screen for hepcidin regulators. We identified 'druggable' screening hits and validated those by applying RNAi of potential drug targets and small-molecule testing in a hepatocytic cell line, in primary murine and human hepatocytes and in mice. We initially identified spironolactone, diclofenac, imatinib and Suberoylanilide hydroxamic acid (SAHA) as hepcidin modulating drugs in cellular assays. Among these, imatinib and spironolactone further suppressed liver hepcidin expression in mice. Our results demonstrate that a commonly used anti-hypertensive drug, spironolactone, which is prescribed for the treatment of heart failure, acne and female hirsutism, as well as imatinib, a first-line, lifelong therapeutic option for some frequent cancer types suppress hepcidin expression in cultured cells and in mice. We expect these results to be of relevance for patient management, which needs to be addressed in prospective clinical studies.

Global Transcriptional Response of Human Liver Cells to Ethanol Stress of Different Strength Reveals Hormetic Behavior.

BACKGROUND: The liver is the major site for alcohol metabolism in the body and therefore the primary target organ for ethanol (EtOH)-induced toxicity. In this study, we investigated the in vitro response of human liver cells to different EtOH concentrations in a perfused bioartificial liver device that mimics the complex architecture of the natural organ. METHODS: Primary human liver cells were cultured in the bioartificial liver device and treated for 24 hours with medium containing 150 mM (low), 300 mM (medium), or 600 mM (high) EtOH, while a control culture was kept untreated. Gene expression patterns for each EtOH concentration were monitored using Affymetrix Human Gene 1.0 ST Gene chips. Scaled expression profiles of differentially expressed genes (DEGs) were clustered using Fuzzy c-means algorithm. In addition, functional classification methods, KEGG pathway mapping and also a machine learning approach (Random Forest) were utilized. RESULTS: A number of 966 (150 mM EtOH), 1,334 (300 mM EtOH), or 4,132 (600 mM EtOH) genes were found to be differentially expressed. Dose-response relationships of the identified clusters of co-expressed genes showed a monotonic, threshold, or nonmonotonic (hormetic) behavior. Functional classification of DEGs revealed that low or medium EtOH concentrations operate adaptation processes, while alterations observed for the high EtOH concentration reflect the response to cellular damage. The genes displaying a hormetic response were functionally characterized by overrepresented "cellular ketone metabolism" and "carboxylic acid metabolism." Altered expression of the genes BAHD1 and H3F3B was identified as sufficient to classify the samples according to the applied EtOH doses. CONCLUSIONS: Different pathways of metabolic and epigenetic regulation are affected by EtOH exposition and partly undergo hormetic regulation in the bioartificial liver device. Gene expression changes observed at high EtOH concentrations reflect in some aspects the situation of alcoholic hepatitis in humans.

Hemolysis after Oral Artemisinin Combination Therapy for Uncomplicated Plasmodium falciparum Malaria.

Episodes of delayed hemolysis 2-6 weeks after treatment of severe malaria with intravenous artesunate have been described. We performed a prospective observational study of patients with uncomplicated malaria to investigate whether posttreatment hemolysis also occurs after oral artemisinin-based combination therapy. Eight of 20 patients with uncomplicated malaria who were given oral artemisinin-based combination therapy met the definition of posttreatment hemolysis (low haptoglobin level and increased lactate dehydrogenase level on day 14). Five patients had hemolysis persisting for 1 month. Patients with posttreatment hemolysis had a median decrease in hemoglobin level of 1.3 g/dL (interquartile range 0.3-2.0 g/dL) in the posttreatment period, and patients without posttreatment hemolysis had a median increase of 0.3 g/dL (IQR -0.1 to 0.7 g/dL; p = 0.002). These findings indicate a need for increased vigilance for hemolytic events in malaria patients, particularly those with predisposing factors for anemia.

Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures.

Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis.

Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.

Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.