On the Possible Effect of Phytic Acid (Myo-Inositol Hexaphosphoric Acid, IP6) on Cytochromes P450 and Systems of Xenobiotic Metabolism in Different Hepatic Models.

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug-drug interactions involving IP6 are low.

Epidermal Growth Factor Represses Constitutive Androstane Receptor Expression in Primary Human Hepatocytes and Favors Regulation by Pregnane X Receptor.

Growth factors have key roles in liver physiology and pathology, particularly by promoting cell proliferation and growth. Recently, it has been shown that in mouse hepatocytes, epidermal growth factor receptor (EGFR) plays a crucial role in the activation of the xenosensor constitutive androstane receptor (CAR) by the antiepileptic drug phenobarbital. Due to the species selectivity of CAR signaling, here we investigated epidermal growth factor (EGF) role in CAR signaling in primary human hepatocytes. Primary human hepatocytes were incubated with CITCO, a human CAR agonist, or with phenobarbital, an indirect CAR activator, in the presence or absence of EGF. CAR-dependent gene expression modulation and PXR involvement in these responses were assessed upon siRNA-based silencing of the genes that encode CAR and PXR. EGF significantly reduced CAR expression and prevented gene induction by CITCO and, to a lower extent, by phenobarbital. In the absence of EGF, phenobarbital and CITCO modulated the expression of 144 and 111 genes, respectively, in primary human hepatocytes. Among these genes, only 15 were regulated by CITCO and one by phenobarbital in a CAR-dependent manner. Conversely, in the presence of EGF, CITCO and phenobarbital modulated gene expression only in a CAR-independent and PXR-dependent manner. Overall, our findings suggest that in primary human hepatocytes, EGF suppresses specifically CAR signaling mainly through transcriptional regulation and drives the xenobiotic response toward a pregnane X receptor (PXR)-mediated mechanism.

Novel role for carbohydrate responsive element binding protein in the control of ethanol metabolism and susceptibility to binge drinking.

UNLABELLED: Carbohydrate responsive element binding protein (ChREBP) is central for de novo fatty acid synthesis under physiological conditions and in the context of nonalcoholic fatty liver disease. We explored its contribution to alcohol-induced steatosis in a mouse model of binge drinking as acute ethanol (EtOH) intoxication has become an alarming health problem. Within 6 hours, ChREBP acetylation and its recruitment onto target gene promoters were increased in liver of EtOH-fed mice. Acetylation of ChREBP was dependent on alcohol metabolism because inhibition of alcohol dehydrogenase (ADH) activity blunted ChREBP EtOH-induced acetylation in mouse hepatocytes. Transfection of an acetylation-defective mutant of ChREBP (ChREBP(K672A) ) in HepG2 cells impaired the stimulatory effect of EtOH on ChREBP activity. Importantly, ChREBP silencing in the liver of EtOH-fed mice prevented alcohol-induced triglyceride accumulation through an inhibition of the lipogenic pathway but also led, unexpectedly, to hypothermia, increased blood acetaldehyde concentrations, and enhanced lethality. This phenotype was associated with impaired hepatic EtOH metabolism as a consequence of reduced ADH activity. While the expression and activity of the NAD(+) dependent deacetylase sirtuin 1, a ChREBP-negative target, were down-regulated in the liver of alcohol-fed mice, they were restored to control levels upon ChREBP silencing. In turn, ADH acetylation was reduced, suggesting that ChREBP regulates EtOH metabolism and ADH activity through its direct control of sirtuin 1 expression. Indeed, when sirtuin 1 activity was rescued by resveratrol pretreatment in EtOH-treated hepatocytes, a significant decrease in ADH protein content and/or acetylation was observed. CONCLUSION: our study describes a novel role for ChREBP in EtOH metabolism and unravels its protective effect against severe intoxication in response to binge drinking.

Activation of the Constitutive Androstane Receptor induces hepatic lipogenesis and regulates Pnpla3 gene expression in a LXR-independent way.

The Constitutive Androstane Receptor (CAR, NR1I3) has been newly described as a regulator of energy metabolism. A relevant number of studies using animal models of obesity suggest that CAR activation could be beneficial on the metabolic balance. However, this remains controversial and the underlying mechanisms are still unknown. This work aimed to investigate the effect of CAR activation on hepatic energy metabolism during physiological conditions, i.e. in mouse models not subjected to metabolic/nutritional stress. Gene expression profiling in the liver of CAR knockout and control mice on chow diet and treated with a CAR agonist highlighted CAR-mediated up-regulations of lipogenic genes, concomitant with neutral lipid accumulation. A strong CAR-mediated up-regulation of the patatin-like phospholipase domain-containing protein 3 (Pnpla3) was demonstrated. Pnpla3 is a gene whose polymorphism is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD) development. This observation was confirmed in human hepatocytes treated with the antiepileptic drug and CAR activator, phenobarbital and in immortalized human hepatocytes treated with CITCO. Studying the molecular mechanisms controlling Pnpla3 gene expression, we demonstrated that CAR does not act by a direct regulation of Pnpla3 transcription or via the Liver X Receptor but may rather involve the transcription factor Carbohydrate Responsive Element-binding protein. These data provide new insights into the regulation by CAR of glycolytic and lipogenic genes and on pathogenesis of steatosis. This also raises the question concerning the impact of drugs and environmental contaminants in lipid-associated metabolic diseases.

Human induced pluripotent stem cells in hepatology: beyond the proof of concept.

The discovery of the wide plasticity of most cell types means that it is now possible to produce virtually any cell type in vitro. This concept, developed because of the possibility of reprogramming somatic cells toward induced pluripotent stem cells, provides the opportunity to produce specialized cells that harbor multiple phenotypical traits, thus integrating genetic interindividual variability. The field of hepatology has exploited this concept, and hepatocyte-like cells can now be differentiated from induced pluripotent stem cells. This review discusses the choice of somatic cells to be reprogrammed by emergent new and nonintegrative strategies, as well as the application of differentiated human induced pluripotent stem cells in hepatology, including liver development, disease modeling, host-pathogen interactions, and drug metabolism and toxicity. The actual consensus is that hepatocyte-like cells generated in vitro present an immature phenotype. Currently, developed strategies used to resolve this problem, such as overexpression of transcription factors, mimicking liver neonatal and postnatal modifications, and re-creating the three-dimensional hepatocyte environment in vitro and in vivo, are also discussed.

PBPK modeling of irbesartan: incorporation of hepatic uptake.

Physiological based pharmacokinetic (PBPK) modeling is now commonly used in drug development to integrate human or animal physiological data in order to predict pharmacokinetic profiles. The aim of this work was to construct and refine a PBPK model of irbesartan taking into account its active uptake via OATP1B1/B3 in order to predict more accurately its pharmacokinetic profile using Simcyp(®). The activity and expression of the human hepatocyte transporters OATP1B1 and OATP1B3 were studied. The relative activity factors (RAFs) for OATP1B1 and OATP1B3 transporters were calculated from intrinsic clearances obtained by concentration dependent uptake experiments in human hepatocytes and HEK overexpressing cells: RAF1B1 using estrone-3-sulfate and pitavastatine clearances, and RAF1B3 using cholecystokinine octapeptide (CCK-8) clearances. The relative expression factor (REF) was calculated by comparing immunoblotting of hepatocytes (REFHH ) or tissues (REFtissue) with those of overexpressing HEK cells for each transporter. These scaling factors were applied in a PBPK model of irbesartan using the Simcyp® simulator. Pharmacokinetic simulation using REFHH (1.82 for OATP1B1, 8.03 for OATP1B3) as an extrapolation factor was the closest to the human clinical pharmacokinetic profile of irbesartan. These investigations show the importance of integrating the contribution of the active uptake of a drug in the liver to improve PBPK modeling.

The WNT/ß-catenin pathway is a transcriptional regulator of CYP2E1, CYP1A2, and aryl hydrocarbon receptor gene expression in primary human hepatocytes.

The wingless-type MMTV integration site family (WNT)/ß-catenin/adenomatous polyposis coli (CTNNB1/APC) pathway has been identified as a regulator of drug-metabolizing enzymes in the rodent liver. Conversely, little is known about the role of this pathway in drug metabolism regulation in human liver. Primary human hepatocytes (PHHs), which are the most physiologically relevant culture system to study drug metabolism in vitro, were used to investigate this issue. This study assessed the link between cytochrome P450 expression and WNT/ß-catenin pathway activity in PHHs by modulating its activity with recombinant mouse Wnt3a (the canonical activator), inhibitors of glycogen synthase kinase 3ß, and small-interfering RNA to invalidate CTNNB1 or its repressor APC, used separately or in combination. We found that the WNT/ß-catenin pathway can be activated in PHHs, as assessed by universal ß-catenin target gene expression, leucine-rich repeat containing G protein-coupled receptor 5. Moreover, WNT/ß-catenin pathway activation induces the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor, but not of CYP3A4, hepatocyte nuclear factor-4α, or pregnane X receptor (PXR) in PHHs. Specifically, we show for the first time that CYP2E1 is transcriptionally regulated by the WNT/ß-catenin pathway. Moreover, CYP2E1 induction was accompanied by an increase in its metabolic activity, as indicated by the increased production of 6-OH-chlorzoxazone and by glutathione depletion after incubation with high doses of acetaminophen. In conclusion, the WNT/ß-catenin pathway is functional in PHHs, and its induction in PHHs represents a powerful tool to evaluate the hepatotoxicity of drugs that are metabolized by CYP2E1.

Nuclear receptors in the cross-talk of drug metabolism and inflammation.

Inflammation and infection have long been known to affect the activity and expression of enzymes involved in hepatic and extrahepatic drug clearance. Significant advances have been made to elucidate the molecular mechanisms underlying the complex cross-talk between inflammation and drug-metabolism alterations. The emergent role of ligand-activated transcriptional regulators, belonging to the nuclear receptor (NR) superfamily, is now well established. The NRs, pregnane X receptor, constitutive androstane receptor, retinoic X receptor, glucocorticoid receptor, and hepatocyte nuclear factor 4, and the basic helix-loop-helix/Per-ARNT-Sim family member, aryl hydrocarbon receptor, are the main regulators of the detoxification function. According to the panel of mediators secreted during inflammation, a cascade of numerous signaling pathways is activated, including nuclear factor kappa B, mitogen-activated protein kinase, and the Janus kinase/signal transducer and activator of transcription pathways. Complex cross-talk is established between these signaling pathways regulating either constitutive or induced gene expression. In most cases, a mutual antagonism between xenosensor and inflammation signaling occurs. This review focuses on the molecular and cellular mechanisms implicated in this cross-talk.

Mitochondria Transfer from Mesenchymal Stem Cells Confers Chemoresistance to Glioblastoma Stem Cells through Metabolic Rewiring.

Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ. Significance: Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.

Chromatin dynamics of gene activation and repression in response to interferon alpha (IFN(alpha)) reveal new roles for phosphorylated and unphosphorylated forms of the transcription factor STAT2.

Signal transducer and activator of transcription 2 (STAT2), the critical component of type I interferons signaling, is a prototype latent cytoplasmic signal-dependent transcription factor. Activated tyrosine-phosphorylated STAT2 associates with STAT1 and IRF9 to bind the ISRE elements in the promoters of a subset of IFN-inducible genes (ISGs). In addition to activate hundreds of ISGs, IFNα also represses numerous target genes but the mechanistic basis for this dual effect and transcriptional repression is largely unknown. We investigated by ChIP-chip the binding dynamics of STAT2 and "active" phospho(P)-STAT2 on 113 putative IFNα direct target promoters before and after IFNα induction in Huh7 cells and primary human hepatocytes (PHH). STAT2 is already bound to 62% of our target promoters, including most "classical" ISGs, before IFNα treatment. 31% of STAT2 basally bound promoters also show P-STAT2 positivity. By correlating in vivo promoter occupancy with gene expression and changes in histone methylation marks we found that: 1) STAT2 plays a role in regulating ISGs expression, independently from its phosphorylation; 2) P-STAT2 is involved in ISGs repression; 3) "activated" ISGs are marked by H3K4me1 and H3K4me3 before IFNα; 4) "repressed" genes are marked by H3K27me3 and histone methylation plays a dominant role in driving IFNα-mediated ISGs repression.

Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal.

Pluripotent stem cells (PSC) are functionally characterized by their capacity to differentiate into all the cell types from the three germ layers. A wide range of markers, the expression of which is associated with pluripotency, has been used as surrogate evidence of PSC pluripotency, but their respective relevance is poorly documented. Here, we compared by polychromatic flow cytometry the kinetics of loss of expression of eight widely used pluripotency markers (SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24, OCT4, NANOG, and alkaline phosphatase [AP]) at days 0, 5, 7, and 9 after induction of PSC differentiation into cells representative of the three germ layers. Strikingly, each marker showed a different and specific kinetics of disappearance that was similar in all the PSC lines used and for all the induced differentiation pathways. OCT4, SSEA3, and TRA-1-60 were repeatedly the first markers to be downregulated, and their expression was completely lost at day 9. By contrast, AP activity, CD24, and NANOG proteins were still detectable at day 9. In addition, we show that differentiation markers are coexpressed with pluripotency markers before the latter begin to disappear. These results suggest that OCT4, SSEA3, and TRA-1-60 might be better to trace in vitro the emergence of pluripotent cells during reprogramming.

Butyrate, a typical product of gut microbiome, affects function of the AhR gene, being a possible agent of crosstalk between gut microbiome, and hepatic drug metabolism.

Modulation of gut microbiome composition seems to be a promising therapeutic strategy for a wide range of pathologic states. However, these microbiota-targeted interventions may affect production of microbial metabolites, circulating factors in the gut-liver axis influencing hepatic drug metabolism with possible clinical relevance. Butyrate, a short-chain fatty acid produced through microbial fermentation of dietary fibers in the colon, has well established anti-inflammatory role in the intestine, while the effect of butyrate on the liver is unknown. In this study, we have evaluated the effect of butyrate on hepatic AhR activity and AhR-regulated gene expression. We have showed that AhR and its target genes were upregulated by butyrate in dose-dependent manner in HepG2-C3 as well as in primary human hepatocytes. The involvement of AhR has been proved using specific AhR antagonists and siRNA-mediated AhR silencing. Experiments with AhR reporter cells have shown that butyrate regulates the expression of AhR target genes by modulating the AhR activity. Our results suggest also epigenetic action by butyrate on AhR and its repressor (AHRR) presumably through mechanisms based on HDAC inhibition in the liver. Our results demonstrate that butyrate may influence the drug-metabolizing ability of liver enzymes e.g., through the interaction with AhR-dependent pathways.

Glucose Starvation or Pyruvate Dehydrogenase Activation Induce a Broad, ERK5-Mediated, Metabolic Remodeling Leading to Fatty Acid Oxidation.

Cells have metabolic flexibility that allows them to adapt to changes in substrate availability. Two highly relevant metabolites are glucose and fatty acids (FA), and hence, glycolysis and fatty acid oxidation (FAO) are key metabolic pathways leading to energy production. Both pathways affect each other, and in the absence of one substrate, metabolic flexibility allows cells to maintain sufficient energy production. Here, we show that glucose starvation or sustained pyruvate dehydrogenase (PDH) activation by dichloroacetate (DCA) induce large genetic remodeling to propel FAO. The extracellular signal-regulated kinase 5 (ERK5) is a key effector of this multistep metabolic remodeling. First, there is an increase in the lipid transport by expression of low-density lipoprotein receptor-related proteins (LRP), e.g., CD36, LRP1 and others. Second, an increase in the expression of members of the acyl-CoA synthetase long-chain (ACSL) family activates FA. Finally, the expression of the enzymes that catalyze the initial step in each cycle of FAO, i.e., the acyl-CoA dehydrogenases (ACADs), is induced. All of these pathways lead to enhanced cellular FAO. In summary, we show here that different families of enzymes, which are essential to perform FAO, are regulated by the signaling pathway, i.e., MEK5/ERK5, which transduces changes from the environment to genetic adaptations.

Primary hepatocytes isolated from human and porcine donors display similar patterns of cytochrome p450 expression following exposure to prototypical activators of AhR, CAR and PXR.

The hepatic cytochrome p450's (CYP) are of major importance for the metabolism of xenobiotics and knowledge about their regulation is crucial. This knowledge often originates from cell models; primary human hepatocytes (PHH) being the gold standard. However, due to limited availability of high-quality human donor organs, basic knowledge on alternative models are needed. Primary porcine hepatocytes (PPH) have been suggested as an alternative to PHH. Unfortunately, data comparing the response in gene-transcription to standard CYP inducers between PHH and PPH are missing. In the present study we, cultured PHH and PPH under the same conditions, treated them with standard inducers of the CYP1-3 and determined the response in gene and protein expression. The results demonstrated that in both species TCDD and omeprazole caused an increase in CYP1A/B expression. In PPH, CITCO increased the content of CYP1A/B. For the CYP2B/C/D's, phenobarbital and rifampicin caused increases in expression. For the CYP2D's, TCDD and omeprazole caused increased gene expression in PPH, which were not the case for PHH. Both phenobarbital, rifampicin and omeprazole increased CYP3A expression in PHH and PPH. Moreover, TCDD increased the gene expression of CYP3A in PPH; this was not the case for PHH. Multivariate data analysis found no difference in gene expression between PHH and PPH for phenobarbital, rifampicin and CITCO. However, differential clustering was observed for TCDD and omeprazole. In conclusion, despite model specificity, there are a high number of similar responses, and experiments investigating mRNA regulation made in PPH permits for a reliable translation into human setting.

Comparative effects of microtubules disruption on glucocorticoid receptor functions in proliferating and quiescent cells.

We have recently demonstrated that the alkaloid colchicine (COL) inhibits glucocorticoid receptor (GR) transcriptional activity. In addition, we described proteasome-mediated degradation of GR in COL-treated HeLa cells. While these effects were previously attributed to cell cycle arrest in G2/M phase, this explanation is not applicable for nonproliferating cells such as human hepatocytes (HH). In the current study, we compared COL-mediated microtubule disruption and cell cycle arrest with selected GR functions in HeLa cells and HH as models of proliferating and quiescent cells, respectively. Microtubule disruption led to irreversible decrease in GR binding capacity and protein level in HeLa cells. None of the parameters was restored 24 hours after COL withdrawal. In contrast, dexamethasone (DEX) binding was increased in HH at the beginning of the treatment, with following transient activation of extracellular signal-regulated kinase (ERK). The findings of these investigations emphasize the GR-signaling differences between primary and transformed cells.

Regulation of CAR and PXR Expression in Health and Disease.

Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are members of the nuclear receptor superfamily that mainly act as ligand-activated transcription factors. Their functions have long been associated with the regulation of drug metabolism and disposition, and it is now well established that they are implicated in physiological and pathological conditions. Considerable efforts have been made to understand the regulation of their activity by their cognate ligand; however, additional regulatory mechanisms, among which the regulation of their expression, modulate their pleiotropic effects. This review summarizes the current knowledge on CAR and PXR expression during development and adult life; tissue distribution; spatial, temporal, and metabolic regulations; as well as in pathological situations, including chronic diseases and cancers. The expression of CAR and PXR is modulated by complex regulatory mechanisms that involve the interplay of transcription factors and also post-transcriptional and epigenetic modifications. Moreover, many environmental stimuli affect CAR and PXR expression through mechanisms that have not been elucidated.

Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain.

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

The 3'-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids.

Pregnane X receptor (PXR) is the major regulator of xenobiotic metabolism. PXR itself is controlled by various signaling molecules including glucocorticoids. Moreover, negative feed-back regulation has been proposed at the transcriptional level. We examined the involvement of the 3'-untranslated region (3'-UTR) of NR1I2 mRNA and microRNAs in PXR- and glucocorticoid receptor (GR)-mediated regulation of NR1I2 gene expression. PXR ligands were found to significantly downregulate NR1I2 mRNA expression in a set of 14 human hepatocyte cultures. Similarly, PXR was downregulated by PCN in the C57/BL6 mice liver. In mechanistic studies with the full-length 3'-UTR cloned into luciferase reporter or expression vectors, we showed that the 3'-UTR reduces PXR expression. From the miRNAs tested, miR-18a-5p inhibited both NR1I2 expression and CYP3A4 gene induction. Importantly, we observed significant upregulation of miR-18a-5p expression 6 h after treatment with the PXR ligand rifampicin, which indicates a putative mechanism underlying NR1I2 negative feed-back regulation in hepatic cells. Additionally, glucocorticoids upregulated NR1I2 expression not only through the promoter region but also via 3'-UTR regulation, which likely involves downregulation of miR-18a-5p. We conclude that miR-18a-5p is involved in the down-regulation of NR1I2 expression by its ligands and in the upregulation of NR1I2 mRNA expression by glucocorticoids in hepatic cells.

The Anti-Cancer Drug Dabrafenib Is a Potent Activator of the Human Pregnane X Receptor.

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in both the development of multidrug resistance and enhanced cancer cells aggressiveness. Moreover, its unintentional activation by pharmaceutical drugs can mediate drug-drug interactions and cause severe adverse events. In that context, the potential of the anticancer BRAF inhibitor dabrafenib suspected to activate hPXR and the human constitutive androstane receptor (hCAR) has not been thoroughly investigated yet. Using different reporter cellular assays, we demonstrate that dabrafenib can activate hPXR as efficiently as its reference agonist SR12813, whereas it does not activate mouse or zebrafish PXR nor hCAR. We also showed that dabrafenib binds to recombinant hPXR, induces the expression of hPXR responsive genes in colon LS174T-hPXR cancer cells and human hepatocytes and finally increases the proliferation in LS174T-hPXR cells. Our study reveals that by using a panel of different cellular techniques it is possible to improve the assessment of hPXR agonist activity for new developed drugs.

ARID1A loss in adult hepatocytes activates ß-catenin-mediated erythropoietin transcription.

Erythropoietin (EPO) is a key regulator of erythropoiesis. The embryonic liver is the main site of erythropoietin synthesis, after which the kidney takes over. The adult liver retains the ability to express EPO, and we discovered here new players of this transcription, distinct from the classical hypoxia-inducible factor pathway. In mice, genetically invalidated in hepatocytes for the chromatin remodeler Arid1a, and for Apc, the major silencer of Wnt pathway, chromatin was more accessible and histone marks turned into active ones at the Epo downstream enhancer. Activating ß-catenin signaling increased binding of Tcf4/ß-catenin complex and upregulated its enhancer function. The loss of Arid1a together with ß-catenin signaling, resulted in cell-autonomous EPO transcription in mouse and human hepatocytes. In mice with Apc-Arid1a gene invalidations in single hepatocytes, Epo de novo synthesis led to its secretion, to splenic erythropoiesis and to dramatic erythrocytosis. Thus, we identified new hepatic EPO regulation mechanism stimulating erythropoiesis.