Albert Braeuning

Differential gene expression in periportal and perivenous mouse hepatocytes

Hepatocytes located in the periportal and perivenous zones of the liver lobule show remarkable differences in the levels and activities of various enzymes and other proteins. To analyze global gene expression patterns of periportal and perivenous hepatocytes, enriched populations of the two cell types were isolated by combined collagenase/digitonin perfusion from mouse liver and used for microarray analysis. In total, 198 genes and expressed sequences were identified that demonstrated a ≥ 2‐fold difference in expression between hepatocytes from the two different zones of the liver. A subset of 20 genes was additionally analyzed by real‐time RT‐PCR, validating the results obtained by the microarray analysis. Several of the differentially expressed genes encoded key enzymes of intermediary metabolism, including those involved in glycolysis and gluconeogenesis, fatty acid degradation, cholesterol and bile acid metabolism, amino acid degradation and ammonia utilization. In addition, several enzymes of phase I and phase II of xenobiotic metabolism were differentially expressed in periportal and perivenous hepatocytes. Our results confirm previous findings on metabolic zonation in liver, and extend our knowledge of the regulatory mechanisms at the transcriptional level.

Zonal gene expression in murine liver: Lessons from tumors

Gene expression in hepatocytes within the liver lobule is differentially regulated along the portal to central axis; however, the mechanisms governing the processes of zonation within the lobule are unknown. A model for zonal heterogeneity in normal liver is proposed, based on observations of differential expression of genes in liver tumors from mice that harbor activating mutations in either Catnb (which codes for β‐catenin) or Ha‐ras. According to the model, the regulatory control consists of two opposing signals, one delivered by endothelial cells of the central veins activating a β‐catenin–dependent pathway (retrograde signal), the other by blood‐borne molecules activating Ras‐dependent downstream cascades (anterograde signal). In conclusion, gradients of opposing signaling molecules along the portocentral axis determine the pattern of enzymes and other proteins expressed in hepatocytes of the periportal and pericentral domains of the liver lobule. (HEPATOLOGY 2006;43:407–414.)

Inducibility of Drug-metabolizing Enzymes by Xenobiotics in Mice with Liver-specific Knockout of Ctnnb1

Basal as well as xenobiotic-induced expression of the main enzymes from phase I and phase II of drug metabolism is confined to the perivenous areas of the mammalian liver lobule. Whereas signal transduction pathways that govern xenobiotic-induced expression of these enzymes via ligand-activated transcription factors such as constitutive androstane receptor (CAR) or the aryl hydrocarbon receptor (AhR) have been intensively studied, the mechanisms regulating zone-specific basal expression of genes related to drug metabolism and preferential response of perivenous hepatocytes to xenobiotic inducers are still largely unknown. Recent publications by our and other groups point to an important role for the Wnt/β-catenin pathway in the maintenance of the perivenous hepatocyte gene expression profile including the main hepatic detoxification enzymes, and β-catenin signaling was recently implicated in the expression of several cytochrome P450 isoenzymes. To analyze, whether the β-catenin pathway would also affect inducible expression of drug-metabolizing enzymes, mice with liver-specific knockout of the Ctnnb1 gene (encoding β-catenin) were treated with different model inducers of xenobiotic metabolism. Knockout of β-catenin led to alterations in basal expression of most drug metabolism-related genes analyzed and resulted in strongly diminished responses to agonists of CAR-, AhR-, and nuclear factor erythroid-related factor 2-dependent transcription. Taken together, the data presented in this study indicate that β-catenin not only regulates basal expression of drug-metabolizing enzymes but also determines the magnitude and hepatic localization of response to xenobiotic inducers in vivo.

T‐cell factor 4 and β‐catenin chromatin occupancies pattern zonal liver metabolism in mice

β‐catenin signaling can be both a physiological and oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20%‐40% of hepatocellular carcinomas (HCCs) with specific metabolic features. We decipher the molecular determinants of β‐catenin‐dependent zonal transcription using mice with β‐catenin‐activated or ‐inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T‐cell factor (Tcf)−4 and β‐catenin, transcriptome, and metabolome. We find that Tcf‐4 DNA bindings depend on β‐catenin. Tcf‐4/β‐catenin binds Wnt‐responsive elements preferentially around β‐catenin‐induced genes. In contrast, genes repressed by β‐catenin bind Tcf‐4 on hepatocyte nuclear factor 4 (Hnf‐4)‐responsive elements. β‐Catenin, Tcf‐4, and Hnf‐4α interact, dictating β‐catenin transcription, which is antagonistic to that elicited by Hnf‐4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by β‐catenin, partly through xenobiotic nuclear receptors. Conclusions: β‐catenin patterns the zonal liver together with Tcf‐4, Hnf‐4α, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with β‐catenin mutational activation. (Hepatology 2014;59:2344–2357)

Identification of Dlk1-Dio3 imprinted gene cluster noncoding RNAs as novel candidate biomarkers for liver tumor promotion.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells

Abstract Because of the rising application of nanoparticles in food and food-related products, we investigated the influence of the digestion process on the toxicity and cellular uptake of silver nanoparticles for intestinal cells. The main food components – carbohydrates, proteins and fatty acids – were implemented in an in vitro digestion process to simulate realistic conditions. Digested and undigested silver nanoparticle suspensions were used for uptake studies in the well-established Caco-2 model. Small-angle X-ray scattering was used to estimate particle core size, size distribution and stability in cell culture medium. Particles proved to be stable and showed radii from 3.6 to 16.0 nm. Undigested particles and particles digested in the presence of food components were comparably taken up by Caco-2 cells, whereas the uptake of particles digested without food components was decreased by 60%. Overall, these findings suggest that in vivo ingested poly (acrylic acid)-coated silver nanoparticles may reach the intestine in a nanoscaled form even if enclosed in a food matrix. While appropriate for studies on the uptake into intestinal cells, the Caco-2 model might be less suited for translocation studies. Moreover, we show that nanoparticle digestion protocols lacking food components may lead to misinterpretation of uptake studies and inconclusive results.

Coordinate Regulation of Cytochrome P450 1A1 Expression in Mouse Liver by the Aryl Hydrocarbon Receptor and the β-Catenin Pathway

The expression of cytochrome P450 (CYP) 1a1 and other drug-metabolizing enzymes is controlled by the aryl hydrocarbon receptor (AhR), which is activated by dioxin-type inducers leading to transcriptional induction of target genes. Here, we show that a second level of transcriptional control exists in hepatocytes, which is tightly linked to the Wnt/β-catenin/T-cell factor (TCF) signaling pathway. In transgenic mice, hepatic expression of CYP1A (and other CYP isoforms) is stimulated by the expression of mutationally activated β-catenin(S33Y) in the absence of AhR-activating compounds but repressed after knockout of β-catenin. These effects were further analyzed in vitro, and the stimulatory role of β-catenin was ascribed to a TCF-binding site within the CYP1A1 promoter. Moreover, β-catenin signaling acted cooperatively with AhR agonists via AhR-binding sites on the DNA during the induction of Cyp1a1 in vivo and in vitro. Activation of β-catenin enhanced the transactivation potential of ligand-activated AhR at its DNA-binding sites without altering the total amount of DNA-bound AhR. Coimmunoprecipitation demonstrated a physical interaction between AhR and β-catenin. Furthermore, the present results suggest that transcriptional induction of the AhR by β-catenin does not play a major role in β-catenin-dependent regulation of Cyp1a1 expression and that inhibition of β-catenin signaling by ligand-activated AhR, as recently observed in the intestine does not occur in mouse liver. In conclusion, signaling through β-catenin activates basal CYP1A1 expression and augments CYP1A1 induction by AhR ligands through enhancement of the transactivation potential of the AhR.

Tumor formation in liver of conditional β-catenin-deficient mice exposed to a diethylnitrosamine/phenobarbital tumor promotion regimen

The antiepileptic drug phenobarbital (PB) is a potent tumor promoter in mouse liver, where it stimulates the selective outgrowth of tumor populations harboring activating mutations in Ctnnb1, encoding β-catenin. A tumor initiation-promotion study was conducted in mice with conditional hepatocyte-specific knockout (KO) of Ctnnb1 and in Ctnnb1 wild-type controls. Mice received a single injection of N-nitrosodiethylamine (DEN) at the age of 6 weeks followed by continuous administration of PB given in the diet (0.05%) for 27 weeks. Metabolic activation of DEN in hepatocytes from both Ctnnb1 wild-type and KO mice was demonstrated. PB strongly enhanced liver tumor formation in Ctnnb1 wild-type mice, and 90% of the PB-promoted tumors were Ctnnb1-mutated. A similar increase in carcinogenic response was seen when using glucose-6-phosphatase and glutamine synthetase as tumor markers. The prevalence of tumors in Ctnnb1 KO mice was ∼7-fold higher than in wild-type mice, suggesting an enhancing effect of the gene KO on liver tumor development. However, in strong contrast to wild-type mice, PB did not promote tumor formation in the Ctnnb1 KO mice. Livers of KO mice, particularly from the PB treatment group, demonstrated fibrosis and massive infiltration of immune cells, an effect not seen in wild-type mice. In summary, our data demonstrate that (i) liver tumor promotion by PB requires functional β-catenin signaling and (ii) absence of β-catenin enhances carcinogen-induced hepatocarcinogenesis and induces a pre-cirrhotic phenotype in mouse liver.

Phenobarbital Induces Cell Cycle Transcriptional Responses in Mouse Liver Humanized for Constitutive Androstane and Pregnane X Receptors

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

Phenobarbital-mediated tumor promotion in transgenic mice with humanized CAR and PXR.

The nuclear receptors CAR (constitutive androstane receptor) and possibly PXR (pregnane X receptor) mediate the hepatic effects of phenobarbital (PB) and similar-acting compounds. Although PB is a potent nongenotoxic tumor promoter in rodent liver, epidemiological data from epilepsy patients treated with phenobarbital do not show a specific role of PB in human liver cancer risk. That points to species differences in the susceptibility to tumor promotion by PB, which might be attributed to divergent functions of the PB receptors CAR and PXR in mice and humans. In the present study, male transgenic mice expressing human CAR and PXR were used to detect possible differences between wild-type (WT) and humanized mice in their response to CAR activation in a tumor initiation/promotion experiment with a single injection of the tumor initiator N-nitrosodiethylamine preceding chronic PB treatment for 10 months. Analysis of liver tumor burden revealed that PB strongly promoted the outgrowth of hepatocellular adenoma driven by activated β-catenin in WT mice, whereas the tumor-promoting effect of PB was much less pronounced in the humanized group. In conclusion, the present findings demonstrate that human CAR and PXR support tumor promotion by PB in mouse liver, but to a significantly lesser extent than the WT murine receptors.