Gene Expression In Human Hepatocytes Research Articles

Heterocyclic amines reduce insulin-induced AKT phosphorylation and induce gluconeogenic gene expression in human hepatocytes.

Heterocyclic amines (HCAs) are well-known for their mutagenic properties. One of the major routes of human exposure is through consumption of cooked meat, as certain cooking methods favor formation of HCAs. Recent epidemiological studies reported significant associations between dietary HCA exposure and insulin resistance and type II diabetes. However, no previous studies have examined if HCAs, independent of meat consumption, contributes to pathogenesis of insulin resistance or metabolic disease. In the present study, we have assessed the effect of three HCAs commonly found in cooked meat (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [MeIQ], 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [PhIP]) on insulin signaling and glucose production. HepG2 or cryopreserved human hepatocytes were treated with 0-50μM of MeIQ, MeIQx, or PhIP for 3days. Treatment of HepG2 cells and hepatocytes with MeIQ and MeIQx resulted in a significant reduction in insulin-induced AKT phosphorylation, suggesting that HCA exposure decreases hepatic insulin signaling. HCA treatment also led to significant increases in expression of gluconeogenic genes, G6PC and PCK1, in both HepG2 and cryopreserved human hepatocytes. Additionally, the level of phosphorylated FOXO1, a transcriptional regulator of gluconeogenesis, was significantly reduced by HCA treatment in hepatocytes. Importantly, HCA treatment of human hepatocytes led to increases in extracellular glucose level in the presence of gluconeogenic substrates, suggesting that HCAs induce hepatic glucose production. The current findings suggest that HCAs induce insulin resistance and promote hepatic glucose production in human hepatocytes. This implicates that exposure to HCAs may lead to the development of type II diabetes or metabolic syndrome.

Gefitinib Inhibits Rifampicin-Induced CYP3A4 Gene Expression in Human Hepatocytes.

During multidrug combination chemotherapy, activation of the nuclear receptor and the transcription factor human pregnane xenobiotic receptor (hPXR) has been shown to play a role in the development of chemoresistance. Mechanistically, this could occur due to the cancer drug activation of hPXR and the subsequent upregulation of hPXR target genes such as the drug metabolism enzyme, cytochrome P450 3A4 (CYP3A4). In the context of hPXR-mediated drug resistance, hPXR antagonists would be useful adjuncts to PXR-activating chemotherapy. However, there are currently no clinically approved hPXR antagonists in the market. Gefitinib (GEF), a tyrosine kinase inhibitor used for the treatment of advanced non-small-cell lung cancer and effectively used in combinational chemotherapy treatments, is a promising candidate owing to its hPXR ligand-like features. We, therefore, investigated whether GEF would act as an hPXR antagonist when combined with a known hPXR agonist, rifampicin (RIF). At therapeutically relevant concentrations, GEF successfully inhibited the RIF-induced upregulation of endogenous CYP3A4 gene expression in human primary hepatocytes and human hepatocells. Additionally, GEF inhibited the RIF induction of hPXR-mediated CYP3A4 promoter activity in HepG2 human liver carcinoma cells. The computational modeling of molecular docking predicted that GEF could bind to multiple sites on hPXR including the ligand-binding pocket, allowing for potential as a direct antagonist as well as an allosteric inhibitor. Indeed, GEF bound to the ligand-binding domain of the hPXR in cell-free assays, suggesting that GEF directly interacts with the hPXR. Taken together, our results suggest that GEF, at its clinically relevant therapeutic concentration, can antagonize the hPXR agonist-induced CYP3A4 gene expression in human hepatocytes. Thus, GEF could be a potential candidate for use in combinational chemotherapies to combat hPXR agonist-induced chemoresistance. Further studies are warranted to determine whether GEF has sufficient hPXR inhibitor abilities to overcome the hPXR agonist-induced chemoresistance.

Changes in Insulin Signaling and Gluconeogenic Gene Expression in Human Hepatocytes Following Exposure to Heterocyclic Amines

Humans are exposed to heterocyclic amine (HCA) mutagens produced in meats cooked at high temperatures or until well-done. A recent epidemiological study documented associations of high HCA intake with increased prevalence of insulin resistance, which is one of the hallmarks of type II diabetes mellitus and metabolic syndrome. However, it is unknown if HCAs directly induce insulin resistance. To investigate the effects of HCA exposure on insulin sensitivity, we subjected HepG2 (hepatocellular carcinoma) cells and primary human hepatocytes to 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and measured changes in insulin signaling, gluconeogenic gene expression, and glucose production. HepG2 cells were treated with varying concentrations of MeIQ (0-100 μM), then treated with insulin and analyzed for changes in insulin signaling via p-AKT induction on Western blot. MeIQ exposure decreased insulin-induced p-AKT levels in low glucose conditions, suggesting that the cells become insulin resistant in the presence of MeIQ. Additionally, the MeIQ treatment significantly upregulated the transcripts of genes involved in gluconeogenesis (e.g., G6PC and PCK1) in HepG2 cells. Similarly, primary human hepatocytes exposed to MeIQ also showed significant increases in gluconeogenic genes, compared to the vehicle treated cells. These results suggest that MeIQ impairs insulin signaling and potentially induces glucose production in hepatocytes. We then tested if the gene expression changes induced by MeIQ leads to increases in glucose production in primary hepatocytes. Despite upregulation of gluconeogenic genes, there were no significant changes in glucose production between control and MeIQ-treated cells. Future studies will examine whether MeIQ exposure alters the suppressive effects of insulin on gluconeogenic gene expression and glucose production and test additional HCAs. The alterations in insulin signaling and gluconeogenic gene expression following MeIQ exposure in hepatocytes indicate that MeIQ may directly alter insulin sensitivity.

Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA

We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.

Gefitinib, at Its Clinically Relevant Concentrations, Inhibits Rifampicin‐Induced CYP3A4 Gene Expression in Human Hepatocytes

Pregnane xenobiotic receptor (PXR) has been shown to induce chemoresistance during multidrug chemotherapy via upregulation of PXR target genes such as cytochrome P450 3A4 (CYP3A4). CYP3A4 contributes to the metabolism of over 50% of clinical drugs. Therefore, during multidrug chemotherapy, drug induction of hPXR‐mediated CYP3A4, can reduce the therapeutic concentration of coadministered drugs, leading to chemoresistance. It is possible to overcome a PXR agonist‐induced chemoresistance with a PXR antagonist. However, proposed antagonists lack appropriate pharmacological characteristics that allow them to be active in the clinic. We propose that a PXR antagonist would be a cancer drug itself that is part of a “cancer drug cocktail”, and effective as PXR antagonist at therapeutic concentrations. Gefitinib (GEF), a tyrosine kinase inhibitor approved for the treatment of advanced non‐small cell lung cancer, and shown to be effective in combinational chemotherapy treatment, is a promising candidate due to its PXR ligand‐like features. We therefore sought to determine whether GEF would behave as a PXR antagonist at its clinically relevant concentrations in human primary hepatocytes, human hepatocells, and HepG2 human hepatocellular carcinoma cells. Rifampicin, an agonist of PXR, was used to activate PXR‐mediated CYP3A expression. Reporter gene assays were conducted to determine PXR transactivation of CYP3A4 promoter activity. Cell viability and qRT‐PCR assays were performed to assess the cytotoxicity and CYP3A4 gene expression, respectively. PXR molecular docking studies were carried out to predict binding affinity of GEF. Finally, competitive ligand binding and steroid receptor coactivator‐1 (SRC‐1) recruitment assays were performed to examine GEF ability to bind to PXR and alter SRC‐1 recruitment. GEF, at its therapeutic concentrations, repressed rifampicin‐induced endogenous CYP3A4 gene expression. Additionally, GEF inhibited rifampicin induction of PXR‐mediated CYP3A4 promoter activity. These findings indicate that GEF could inhibit PXR agonists‐induced CYP3A4 gene expression. Molecular docking studies predicted that GEF can bind to multiple sites on PXR, including the ligand binding pocket, AF‐2 region and alpha‐8 pocket. Indeed, GEF bound to PXR and attenuated the PXR agonist‐induced SRC‐1 interaction, suggesting that GEF directly interacts with multiple sites on the PXR. Taken together, these results suggest that GEF, at its clinically relevant therapeutic concentration, can antagonize PXR agonists‐induced CYP3A4 gene expression. Thus, GEF could be a potential candidate for use in combinational chemotherapies to combat PXR‐mediated chemoresistance. Future studies are required to examine GEF selectivity towards PXR, and to determine GEF suppression of PXR agonists‐induced chemoresistance.Support or Funding InformationThe authors thank Drs. Coleman, Mansour, Schwartz &amp; Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant and Animal Health and Disease Research Grant.

Comparison of metabolic and mitogenic response in vitro of the rapid-acting insulin lispro product SAR342434, and US- and EU-approved Humalog®

SAR342434 is a biosimilar of insulin lispro (Humalog® U-100). Batches of SAR342434 were compared with Humalog® batches of either EU or US origin in a panel of in vitro biological assays that included insulin binding to insulin receptor (IR) isoforms A (IR-A) and B (IR-B) and IR-A/IR-B autophosphorylation. A surface plasmon resonance biosensor-based assay was developed to characterize the kinetics of insulin binding to solubilized full-length IR-A or IR-B. Insulin-dependent metabolic activity assays included inhibition of lipolysis in in vitro differentiated human adipocytes, glucose uptake in L6-myocytes, and repression of glucose-6-phosphatase gene expression in human hepatocytes. Mitogenic activity assays included insulin binding to insulin-like growth factor-1 receptor (IGF1R), IGF1R autophosphorylation, and cell proliferation in MCF-7 cells. Weighted geometric means and their respective 95% confidence intervals (CI) were calculated for all 50% inhibitory or effective concentration values and kinetic binding constants for IR-A and IR-B. Statistical evaluation of the data demonstrated that the 90% CIs of the ratio of geometric means between SAR342434 and Humalog® EU or Humalog® US were within the predefined acceptance limits for each assay. Insulin lispro as SAR342434 solution demonstrated similarity to both US- and EU-approved Humalog® based on a side-by-side biological similarity assessment.

Endoplasmic reticulum-mediated induction of interleukin-8 occurs by hepatitis B virus infection and contributes to suppression of interferon responsiveness in human hepatocytes

The events in the immune response to hepatitis B virus (HBV) remain unclear. We analyzed the direct influence of HBV on gene expression in human hepatocytes under immunodeficient conditions using a human hepatocyte chimeric mouse model. HBV-infected or non-infected chimeric mouse livers were collected, and gene expression profiles were compared. Since IL-8 was the most significantly up-regulated gene at 8 weeks after HBV infection, we focused on IL-8 and found that HBx and the large HBs (L-HBs) protein induce transcription of IL-8 via endoplasmic reticulum stress. This stress induces IL-8 transcription via NFAT activation and contributes to suppression of interferon responsiveness in HBV-infected human hepatocytes. In the present study, we identified a novel regulatory mechanism in which the L-HBs protein activates IL-8 via endoplasmic reticulum stress, suggesting a key role for IL-8 in the immune response to HBV and a potential new target for antiviral treatments of HBV infection.

Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy.

Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. The results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes.

Vinorelbine Inhibits Pregnane X Receptor‐Mediated Induction of Cytochrome P450 3A4 Gene Expression in Human Hepatocytes

Pregnane X receptor (PXR), a ligand‐dependent nuclear receptor, is involved in regulating the gene expression of enzymes and proteins involved in endobiotic and xenobiotic metabolism. In particular, PXR plays a crucial role in regulating the gene expression of drug‐metabolizing enzymes, including cytochrome p450 3A4 (CYP3A4). In cancer patients, upregulation of the PXR target genes, including CYP3A4, can lead to not only adverse drug interactions but also chemoresistance. It is possible to inhibit the activity of PXR in order to overcome PXR‐mediated adverse drug interactions and chemoresistance. In this regard, a few PXR inhibitors or antagonists have been identified and characterized. However, these compounds fall short of reaching their clinical utility as they are non‐selective and/or highly‐toxic. Therefore, there is a practical need to develop a selective and less‐toxic PXR antagonist to overcome PXR‐mediated adverse drug interactions and chemoresistance. We sought to determine whether vinorelbine (VNB), a clinically‐used antimicrotubule agent, inhibits the transcriptional activity of PXR in human hepatocytes. PXR transactivation and cell viability assays were performed to determine CYP3A4 promoter activity and cytotoxicity, respectively, in HepG2 human liver carcinoma cells. Quantitative RT‐PCR assays were conducted to study gene expression of CYP3A4 in human primary hepatocytes. VNB, at its non‐cytotoxic and therapeutically‐relevant concentrations, inhibited PXR transactivation of CYP3A4 promoter activity. Additionally, VNB attenuated the PXR agonist‐induced gene expression of CYP3A4, suggesting that VNB downregulates CYP3A4 gene expression by inhibiting the activity of PXR. These results support our conclusion that VNB inhibits the transcriptional activity of PXR at its non‐cytotoxic and therapeutically relevant concentrations. Future studies will be directed to study the selectivity of VNB to PXR as well as the mechanisms of VNB inhibition of PXR‐mediated CYP3A4 induction.Support or Funding InformationThis work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant, and Auburn University Startup Funds to Pondugula SR. The authors would like to thank Drs. Coleman, Tao, and Schwartz for sharing their research facilities.

Dose-response analysis of the effects of persistent organic pollutants (POPs) on gene expression in human hepatocytes

Determination of gene expression profile alterations following dose-dependent exposure to toxicants will greatly improve the utility of arrays in identifying biomarkers and elucidating modes of toxicity. In the present study, the effects of increasing doses of persistent organic pollutants (POPs) on the expression of toxicity-related genes were evaluated. Human hepatocellular carcinoma (HepG2) cells were exposed to nontoxic (NT) and 20% inhibitory concentrations (IC20) of the well-known toxic PoP compounds, toxaphene (TOX) and chlordane (CD), for 48 h. Transcriptomic profiling showed differential gene expression patterns and a profound effect on gene expression changes in the IC20 exposure group. Through clustering analysis, 98 NT- and 336 IC20-specific genes and 73 genes expressed dose-dependently were identified. Gene Ontology (GO) analysis identified several key pathways related to the adverse health effects of POPs. In conclusion, this report describes changes in gene expression profiles in hepatocytes in response to different doses of POPs and relates these changes to hepatotoxicity-related pathways. Moreover, novel genes and pathways that may potentially play a role in the prevention of liver disease due to POP exposure were identified.

Environmental pollutants parathion, paraquat and bisphenol A show distinct effects towards nuclear receptors-mediated induction of xenobiotics-metabolizing cytochromes P450 in human hepatocytes

Environmental pollutants parathion, bisphenol A and paraquat were not systematically studied towards the effects on the expression of phase I xenobiotics-metabolizing cytochromes P450 (CYPs). We monitored their effects on the expression of selected CYPs in primary cultures of human hepatocytes. Moreover, we investigated their effects on the receptors regulating these CYPs, particularly arylhydrocarbon receptor (AhR), pregnane X receptor (PXR) and glucocorticoid receptor (GR) by gene reporter assays. We found that parathion and bisphenol A are the activators of AhR. Moreover, they are the inducers of CYP1A1 mRNA in hepatoma cells HepG2 as well as in human hepatocytes by AhR-dependent mechanism via formation of AhR-DNA-binding complex, as revealed by gel shift assay. All three compounds possessed anti-glucocorticoid action as revealed by GR-dependent gene reporter assay and a decline in tyrosine aminotransferase (TAT) gene expression in human hepatocytes. Moreover, parathion and bisphenol A are the activators of PXR and inducers of CYP3A4 mRNA and protein in the primary cultures of human hepatocytes.In conclusion, the studied compounds displayed distinct activities towards nuclear receptors involved in many biological processes and these findings may help us to better understand their adverse actions in pathological states followed after their exposure.

Dose-dependent induction of signaling pathways by the flavonoid quercetin in human primary hepatocytes: A transcriptomic study.

Quercetin is widespread in plant kingdom and consumed regularly with human diet (16 mg/day). Due to reported positive effects on health, quercetin supplements with recommended doses up to 2 g/day are offered. However, molecular effects of such high doses on human liver have not been assessed yet. Therefore, molecular effects on human hepatocytes were analyzed to help assessing the risk of quercetin supplementation. Molecular effects of three different quercetin concentrations on gene expression in human hepatocytes were investigated by microarray analysis. Possible new signaling pathways were investigated using reporter gene assays. Quercetin concentrations representing the normal intake showed weak effects on mRNA expression in liver cells. In contrast, supplemental doses affect immune response and p53 signaling and might be associated with cancer. Additionally, quercetin showed inhibition of transcriptional activation and mRNA-expression of HNF4α and its target genes. Inhibitory effects were also found for FXR, LXRα, and PXR. Normal intake of quercetin seems to play a minor regulatory role, while supplement doses may have great effects on gene expression in hepatocytes. However, since it is not clarified whether such high doses of quercetin exert positive or negative effects, a careful handling of quercetin supplements is advised.

AAV shuffles to the liver: commentary on Lisowski et al.

AAV shuffles to the liver: commentary on Lisowski et al.

Effect of N,N-dimethylformamide on calcium homeostasis and the calpain gene expression in human hepatocytes

To investigate the effect of N,N-dimethylformamide (DMF) on calcium homeostasis and calpain I and II gene expression in human hepatocytes (HL-7702). HL-7702 cells were exposed to different concentrations of DMF (10, 25, 50, 100, or 200 mmol/L); other HL-7702 cells, which were used as a control group, were exposed to the equal volume of DMEM; the intracellular Ca(2+) concentration was monitored using the calcium fluorescent probe (fluo-3/AM). After 24-h exposure to DMF (10, 25, 50, 100, 150, or 200 mmol/L), the morphology of hepatocytes was observed under an inverted phase contrast microscope, and the cell viability was measured by MTT assay. After 24-h exposure to DMF (10, 25, 50, 100, or 150 mmol/L), the mRNA expression levels of calpain I and II in hepatocytes were measured by real-time quantitative PCR. There were significant differences in cell viability among different exposure groups (P < 0.01); the 50, 100, 150, and 200 mmol/L DMF exposure groups had a significantly lower cell viability than the control group (P < 0.05). Under the inverted phase contrast microscope, HL-7702 cells gradually lost the original shape, with swelling and shrinking, as the dose of DMF increased, and those treated with 150 mmol/L DMF even became round and floated. The fluorescence density of fluo-3 in hepatocytes increased as the dose of DMF rose, demonstrating a dose-response relationship, and there were significant differences among these exposure groups (P < 0.05). There were significant differences in mRNA expression levels of calpain I and II among these exposure groups (P < 0.01), and the expression increased as the dose of DMF rose; but DMF did not promote the mRNA expression of calpain I at a concentration of 150 mmol/L. DMF can cause damage to hepatocytes, which is related to intracellular calcium increase and calpain mRNA increase.

Cytokine gene expression in human hepatocytes infected with dengue virus serotype 3 (strain-16562)

Liver is a site of viral replication and liver dysfunction is a characteristic of severe dengue infection. To understand these mechanisms, we analyzed the response of a hepatic cell linage, HepG2 to infection with dengue 3 virus (strain 16562). Steady state levels of mRNA accumulation were assessed for 14 genes involved in modulation of the host immune responses, at 6, 24 and 48 hpi, by quantitative reverse transcription real-time PCR (qRT-PCR). Fourteen genes showed altered expression upon infection with D3V including; cytokines/chemokines (IL-1β, IL-6, IL-8, RANTES, MCP-2, IL-2Rα and TGF-βIIIR), type I interferon (IFN-α and IFN-β), and pattern-recognition receptors (TLR3, TLR8, RIG-1, MDA5 and MyD88). Although these genes are associated with mechanism of innate immune response and anti-viral activity, their altered expression does not inhibit D3V (strain 16562) growth kinetics and virus yield in HepG2 cells. Gene expression in liver may explain pathological changes associated with dengue virus infection.

Interferon-lambda (IFN-λ) induces signal transduction and gene expression in human hepatocytes, but not in lymphocytes or monocytes

This study compared the ability of IFN-α and IFN-λ to induce signal transduction and gene expression in primary human hepatocytes, PBLs, and monocytes. IFN-α drug products are widely used to treat chronic HCV infection; however, IFN-α therapy often induces hematologic toxicities as a result of the broad expression of IFNARs on many cell types, including most leukocytes. rIFN-λ1 is currently being tested as a potential alternative to IFN-α for treating chronic HCV. Although IFN-λ has been shown to be active on hepatoma cell lines, such as HepG2 and Huh-7, its ability to induce responses in primary human hepatocytes or leukocytes has not been examined. We found that IFN-λ induces activation of Jak/STAT signaling in mouse and human hepatocytes, and the ability of IFN-λ to induce STAT activation correlates with induction of numerous ISGs. Although the magnitude of ISG expression induced by IFN-λ in hepatocytes was generally lower than that induced by IFN-α, the repertoire of regulated genes was quite similar. Our findings demonstrate that although IFN-α and IFN-λ signal through distinct receptors, they induce expression of a common set of ISGs in hepatocytes. However, unlike IFN-α, IFN-λ did not induce STAT activation or ISG expression by purified lymphocytes or monocytes. This important functional difference may provide a clinical advantage for IFN-λ as a treatment for chronic HCV infection, as it is less likely to induce the leukopenias that are often associated with IFN-α therapy.

MiRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes.

Human inducible nitric oxide synthase (hiNOS) gene expression is regulated by transcriptional and posttranscriptional mechanisms. The purpose of this study was to determine whether specific microRNA (miRNA) directly regulate hiNOS gene expression. Sequence analysis of the 496-bp hiNOS 3'-untranslated region (3'-UTR) revealed five putative miR-939 binding sites. The hiNOS 3'-UTR conferred significant posttranscriptional blockade of luciferase activity in human A549, HCT8, and HeLa cells. The hiNOS 3'-UTR also exerted basal and cytokine-stimulated posttranscriptional repression in an orientation-dependent manner. Functional studies demonstrated that transfection of miR-939 into primary human hepatocytes (HCs) significantly inhibited cytokine-induced NO synthesis in a dose-dependent manner that was abrogated by a specific miR-939 inhibitor. MiR-939 (but not other miRNAs) abolished cytokine-stimulated hiNOS protein in human HC, but had no effect on hiNOS mRNA levels. Site-directed mutagenesis of miR-939 bindings sites at +99 or +112 bp in the hiNOS 3'-UTR increased reporter gene expression. Furthermore, intact miR-939 binding sites at +99 or +112 positions were required for posttranscriptional suppression by miR-939. Cytokine stimulation directly increased miR-939 levels in human HC. Transfection of miR-939 inhibitor (antisense miR-939) enhanced cytokine-induced hiNOS protein and increased NO synthesis in vitro in human HC. Finally, cytokine or LPS injection in vivo in mice increased hepatic miR-939 levels. Taken together, these data identify that miR-939 directly regulates hiNOS gene expression by binding in the 3'-UTR to produce a translational blockade. These findings suggest dual regulation of iNOS gene expression where cytokines induce iNOS transcription and also increase miR-939, leading to translational inhibition in a check-and-balance system.

Effects of Oxygen Concentration on the Expression of an Antioxidant Gene in HepG2

To clarify the effects of O(2) concentration (cO(2)) on antioxidant gene expression in human hepatocytes, mRNA expression of HepG2 cells cultured at 1, 3, and 5% cO(2) and atmospheric gas-phase, was measured. The expression of some genes fluctuated depending on the cO(2) in the incubator. This indicates that cO(2) is a critically important factor in the investigation of human biological mechanisms.

Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Investigation of the CYP2C9 Induction Profile in Human Hepatocytes by Combining Experimental and Modelling Approaches

The goal of the present review is to characterise the induction profile of CYP2C9, a member of the cytochrome P450 superfamily. Since the mechanism of CYP2C9 induction is fairly complex, with parallel processes triggered by various inducers, an evaluation of the experimental results is often a great challenge. At least three nuclear receptors, the glucocrticoid receptor (GR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), are known to mediate the CYP2C9 gene induction in man. However, mathematical modelling and simulation can provide an appropriate tool for the interpretation of CYP2C9 regulatory mechanisms. As an example, we present modelling and simulation approaches of the CYP2C9 gene expression in human hepatocytes treated with well-known CYP2C9 inducers: the steroid hormone precursor dehydroepiandrosterone (DHEA) and the synthetic glucocorticoid dexamethasone (DXM). The results of the analysis suggest that in addition to the potent function of GR and the further involvement of PXR and CAR activated by DXM or DHEA, an additional factor might play a role in CYP2C9 regulation by DHEA. The novel potential candidate for DHEA action in CYP2C9 induction is likely to be the estrogen receptor. Additionally, the balance of DHEA sulphation-desulphation processes should also be considered in any description of DHEA-induced CYP2C9 profiles.