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L-type amino acid transporter 1 (LAT1) transfers essential amino acids across cell membranes. Owing to its predominant expression in the blood–brain barrier and tumor cells, LAT1 has been exploited for drug delivery and targeting to the central nervous system (CNS) and various cancers. Although the interactions of amino acids and their mimicking compounds with LAT1 have been extensively investigated, the specific structural features for an optimal drug scaffold have not yet been determined. Here, we evaluated a series of LAT1-targeted drug-phenylalanine conjugates (ligands) by determining their uptake rates by in vitro studies and investigating their interaction with LAT1 via induced-fit docking. Combining the experimental and computational data, we concluded that although LAT1 can accommodate various types of structures, smaller compounds are preferred. As the ligand size increased, its flexibility became more crucial in determining the compound’s transportability and interactions. Compounds with linear or planar structures exhibited reduced uptake; those with rigid lipophilic structures lacked interactions and likely utilized other transport mechanisms for cellular entry. Introducing polar groups between aromatic structures enhanced interactions. Interestingly, compounds with a carbamate bond in the aromatic ring’s para-position displayed very good transport efficiencies for the larger compounds. Compared to the ester bond, the corresponding amide bond had superior hydrogen bond acceptor properties and increased interactions. A reverse amide bond was less favorable than a direct amide bond for interactions with LAT1. The present information can be applied broadly to design appropriate CNS or antineoplastic drug candidates with a prodrug strategy and to discover novel LAT1 inhibitors used either as direct or adjuvant cancer therapy.

Frontotemporal lobar degeneration (FTLD) comprises a heterogenous group of fatal neurodegenerative diseases and, to date, no validated diagnostic or prognostic biomarkers or effective disease-modifying therapies exist for the different clinical or genetic subtypes of FTLD. Current treatment strategies rely on the off-label use of medications for symptomatic treatment. Changes in several neurotransmitter systems including the glutamatergic, GABAergic, dopaminergic, and serotonergic systems have been reported in FTLD spectrum disease patients. Many FTLD-related clinical and neuropsychiatric symptoms such as aggressive and compulsive behaviour, agitation, as well as altered eating habits and hyperorality can be explained by disturbances in these neurotransmitter systems, suggesting that their targeting might possibly offer new therapeutic options for treating patients with FTLD. This review summarizes the present knowledge on neurotransmitter system deficits and synaptic dysfunction in model systems and patients harbouring the most common genetic causes of FTLD, the hexanucleotide repeat expansion in C9orf72and mutations in the granulin (GRN)and microtubule-associated protein tau (MAPT)genes. We also describe the current pharmacological treatment options for FLTD that target different neurotransmitter systems.

The constitutive androstane receptor (CAR; NR1I3) has been established as one of the main drug- and xenobiotic-responsive transcriptional regulators, collectively called xenosensors. CAR activates the expression of several oxidative, hydrolytic, and conjugative drug-metabolizing enzymes and drug transporters, and therefore, it contributes to drug and xenobiotic elimination, drug interactions, and toxicological processes. This minireview introduces mechanisms that modulate CAR activity and focuses on the recent approaches used to search and characterize CAR agonists, inverse agonists, and indirect activators. This minireview is dedicated to Dr. Masahiko Negishi to celebrate his scientific achievements during his long service at the National Institutes of Health.SIGNIFICANCE STATEMENTDiscovery and characterization of human constitutive androstane receptor (CAR) modulators is important for drug development, toxicity studies, and in generation of chemical tools to dissect biological functions of CAR. This minireview focuses on the main methods used to search for these compounds and discusses their essential features.

As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that play a major role in drug metabolism and bioactivation of prodrugs have been detected in ocular tissues, but data on their quantitative expression is scarce. Also, many ophthalmic drugs are prone to hydrolysis. Metabolic characterization of individual ocular tissues is useful for the drug development process, and therefore, seven individual ocular tissues from human eyes were analyzed for the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC). Generic and selective human esterase substrates 4-nitrophenyl acetate (most esterases), D-luciferin methyl ester (CES1), fluorescein diacetate and procaine (CES2), and phenacetin (AADAC) were applied to determine the enzymes’ specific activities. Enzyme kinetics and inhibition studies were performed with isoform-selective inhibitors digitonin (CES1) and verapamil and diltiazem (CES2). Enzyme contents were determined using quantitative targeted proteomics, and CES2 expression was confirmed by western blotting. The expression and activity of human CES1 among ocular tissues varied by >10-fold, with the highest levels found in the retina and iris-ciliary body. In contrast, human CES2 expression appeared lower and more similar between tissues, whereas AADAC could not be detected. Inhibition studies showed that hydrolysis of fluorescein diacetate is also catalyzed by enzymes other than CES2. This study provides, for the first time, quantitative information on the tissue-dependent expression of human ocular esterases, which can be useful for the development of ocular drugs, prodrugs, and in pharmacokinetic modeling of the eye.SIGNIFICANCE STATEMENTNovel and comprehensive data on the protein expression and activities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical species, this study will improve the understanding of interspecies differences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, viahydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC (d-luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.

Organic solute transporter α/β (OSTα/β) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/βplays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/Bhave been associated with clinical cholestasis. OSTα/βalso transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/β, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTαthat are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTαsubunit to generate OSTα/βmutant cell lines. Out of the 10 OSTα/βmutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/βwild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/βmutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/βWT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/βmutants demonstrated altered expression and cellular localization when compared with OSTα/βWT. In summary, we identified OSTαresidues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/βand may influence OSTα/β-mediated bile acid transport. These data advance our understanding of OSTα/βstructure/function and can inform future studies designed to gain further insight into OSTα/βstructure or to identify additional OSTα/βsubstrates and inhibitors.SIGNIFICANCE STATEMENTOSTα/βis a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTαamino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/βand may influence OSTα/β-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/βstructure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/β.

Drug interactions with the organic solute transporter alpha/beta (OSTα/β) are understudied even though OSTα/β is an important transporter that is expressed in multiple human tissues including the intestine, kidneys, and liver. In this study, an in vitromethod to identify novel OSTα/β inhibitors was first developed using OSTα/β-overexpressing Flp-In 293 cells. Incubation conditions were optimized using previously reported OSTα/β inhibitors. A method including a 10 min preincubation step with the test compound was used to screen for OSTα/β inhibition by 77 structurally diverse compounds and fixed-dose combinations. Seven compounds and one fixed-dose combination (100 μM final concentration) inhibited OSTα/β-mediated dehydroepiandrosterone sulfate (DHEAS) uptake by >25%. Concentration-dependent OSTα/β inhibition was evaluated for all putative inhibitors (atorvastatin, ethinylestradiol, fidaxomicin, glycochenodeoxycholate, norgestimate, troglitazone, and troglitazone sulfate). Ethinylestradiol, fidaxomicin, and troglitazone sulfate yielded a clear concentration–inhibition response with IC50values <200 μM. Among all tested compounds, there was no clear association between physicochemical properties, the severity of hepatotoxicity, and the degree of OSTα/β inhibition. This study utilized a novel in vitromethod to identify OSTα/β inhibitors and, for the first time, provided IC50values for OSTα/β inhibition. These data provide evidence that several drugs, some of which are associated with cholestatic drug-induced liver injury, may impair the function of the OSTα/β transporter.

4-Nonylphenol (4-NP), a para-substituted phenolic compound with a straight or branched carbon chain, is a ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor (ED) with potent activities on estrogen receptors. Constitutive Androstane Receptor (CAR) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in the ED mechanism of action. An NP mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by 4-NP are enigmatic, and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here, we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR variants using molecular dynamics (MD) simulations, cellular experiments with various CAR expression constructs, recombinant CAR LBD in a TR-FRET assay, or a differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type CAR1 and CAR3 variant LBDs in MD simulations. Consistently, branched 4-NPs activated CAR3 and CAR1 LBD more efficiently than linear 4-NP. Furthermore, in HepaRG cells, we observed that all 4-NPs upregulated CYP2B6 mRNA, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and human CAR-LBD, as well as its dominant variant CAR3. The work could contribute to the safer use of individual 4-NPs in many areas of industry.

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions.SIGNIFICANCE STATEMENTWe report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.

Aldehyde hydrogenases (ALDHs) belong to a large gene family involved in oxidation of both endogenous and exogenous compounds in mammalian tissues. Among ALDHs, the rat ALDH1A7gene displays a curious strain dependence in phenobarbital (PB)-induced hepatic expression: the responsive RRstrains exhibit induction of both ALDH1A7 and CYP2B mRNAs and activities, whereas the nonresponsive rrstrains show induction of CYP2B only. Here, we investigated the responsiveness of ALDH1A1, ALDH1A7, CYP2B1, and CYP3A23genes to prototypical P450 inducers, expression of nuclear receptors CAR and pregnane X receptor, and structure of the ALDH1A7promoter in both rat strains. ALDH1A7 mRNA, associated protein and activity were strongly induced by PB and modestly induced by pregnenolone 16α-carbonitrile in the RRstrain but negligibly in the rrstrain, whereas induction of ALDH1A1 and P450 mRNAs was similar between the strains. Reporter gene and chromatin immunoprecipitation assays indicated that the loss of ALDH1A7inducibility in the rrstrain is profoundly linked with a 16-base pair deletion in the proximal promoter and inability of the upstream DNA sequences to recruit constitutive androstane receptor-retinoid X receptor heterodimers.SIGNIFICANCE STATEMENTGenetic variation in rat ALDH1A7promoter sequences underlie the large strain-dependent differences in expression and inducibility by phenobarbital of the aldehyde dehydrogenase activity. This finding has implications for the design and interpretation of pharmacological and toxicological studies on the effects and disposition of aldehydes.

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes are anticipated as important surrogates for primary human hepatocytes in applications ranging from basic research to drug discovery and regenerative medicine. Although methods for differentiating hepatocyte-like cells (HLCs) from hiPSCs have developed remarkably, the limited yield of fully functional HLCs is still a major obstacle to their utility. A three-dimensional (3D) culture environment could improve the in vitrohepatic maturation of HLCs. Here we compare 3D hydrogel models of hiPSC-derived HLCs in agarose microwells (3D Petri Dish; 3DPD), nanofibrillar cellulose hydrogels (Growdex; 3DNFC), or animal extracellular matrix-based hydrogels (3D Matrigel; 3DMG). In all the tested 3D biomaterial systems, HLCs formed aggregates. In comparison with two-dimensional monolayer culture, 3DPD and 3DMG models showed both phenotypic and functional enhancement in HLCs over 2.5 weeks of 3D culture. Specifically, we found higher hepatocyte-specific gene expression levels and enhanced cytochrome P450 functions. Our work suggests that transferring HLCs into 3D hydrogel systems can expedite the hepatic maturation of HLCs irrespective of the biochemical nature of the 3D hydrogel. Both plant-based nonembedding and animal-based embedding 3D hydrogel models enhanced the maturation.

Induction of cytochrome P450 (CYP) enzymes is commonly analyzed in cultured human primary hepatocytes (HPHs) by measuring CYP1A2, CYP2B6 and CYP3A4/3A5 activities after exposure to test and reference compounds. Because chemicals can both inhibit and induce CYP enzymes, this traditional approach fails to distinguish such simultaneous effects. Regulatory authorities have therefore suggested that measurement of CYP expression levels should complement activity measurements. We aimed to compare a hybridization and bead-based assay termed transcript analysis with the aid of affinity capture (TRAC) with the routinely used quantitative real-time PCR (qRT-PCR) assay and to study its suitability for CYP induction studies on mRNA level. HPHs from three donors were treated with vehicle, reference substances omeprazole, phenobarbital and rifampicin and six test compounds on 48-well plates. The mRNA expression of ten CYP isoforms important for drug metabolism was determined by TRAC and qRT-PCR methods in order to validate the novel TRAC method. The fold-increases of CYP mRNA levels showed a good correlation between the assays. With TRAC, the marker CYP mRNAs for induction could be easily detected from about 10 000 hepatocytes per sample, with a coefficient of variation below 10% between triplicates. Time spent for TRAC analysis was significantly shorter. Thus, TRAC is a sensitive and reproducible high-throughput assay, which enables accurate and direct detection of multiple mRNA targets simultaneously from large number of samples without enzymatic reactions inherent to qRT-PCR. It is a valuable method to study CYP induction and expandable to other genes relevant for drug metabolism and toxicity.

AbstractThe constitutive androstane receptor (CAR; NR1I3) has emerged as one of the main drug- and xenobiotic-sensitive transcriptional regulators. It has a major effect on the expression of several oxidative and conjugative enzymes and transporters, and hence, CAR can contribute to drug/drug interactions. Novel functions for CAR are also emerging: it is able to modulate the metabolic fate of glucose, lipids, and bile acids, and it is also involved in cell-cell communication, regulation of the cell cycle, and chemical carcinogenesis. Here, we will review the recent information available on CAR and its target gene expression, its interactions with partner proteins and mechanisms of action, interindividual and species variation, and current advances in CAR ligand selectivity and methods used in interrogation of its ligands.

The human constitutive androstane receptor (CAR, NR1I3) is one of the key regulators of xenobiotic and endobiotic metabolism. The unique properties of human CAR, such as the high constitutive activity and the complexity of signaling, as well as the lack of functional and predictive cell-based assays to study the properties of the receptor, have hindered the discovery of selective human CAR ligands. Here we report a novel human CAR inverse agonist, 1-[(2-methylbenzofuran-3-yl)methyl]-3-(thiophen-2-ylmethyl) urea (S07662), which suppresses human CAR activity, recruits the corepressor NCoR in cell-based assays, and attenuates the phenytoin- and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO)-induced expression of CYP2B6 mRNA in human primary hepatocytes. The properties of S07662 are also compared with those of known human CAR inverse agonists by using an array of different in vitroand in silicoassays. The identified compound S07662 can be used as a chemical tool to study the biological functions of human CAR and also as a starting point for the development of new drugs for various conditions involving the receptor.

To characterise in detail the patterns of expression and functional activities of CYP and efflux pump genes in Caco-2 cells stably transfected with human Pregnane X Receptor or murine Constitutive Androstane Receptor.Cell lines transfected with nuclear receptors were treated with established ligands, and gene expression of CYP and efflux pump genes were quantified by qRT-PCR and Western blot. P-glycoprotein activity was assessed by measuring calcein-AM accumulation and bidirectional permeability coefficients of digoxin and quinidine. CYP activities were measured with both fluorescent and non-fluorescent substrates.hPXR and mCAR upregulated some CYP and efflux pump genes ligand dependently. P-glycoprotein level was increased, but CYP3A4 protein remained below the limit of detection. P-glycoprotein activity was markedly elevated in Caco/mCAR cells and more modestly in Caco/hPXR cells. CYP3A4 activity remained lower than that in vitamin D-treated Caco-2 cells.Nuclear receptors can modulate the expression of metabolic genes in Caco-2 cells, but the overall level of metabolism could not be efficiently controlled. P-glycoprotein activity increased, but CYP activities remained very low.

Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are the principal regulators of drug/xenobiotic disposition and toxicity. These nuclear receptors display considerable cross-regulation of their target genes, and species-specific, yet promiscuous activation by a large number of structurally dissimilar ligands. Activation of PXR and/or CAR will frequently result in enhanced drug metabolism, disturbances in homeostasis of endogenous substances, and increased toxicity. Thus, understanding, measurement and prediction of ligand-elicited activation of PXR and CAR receptors is of utmost importance for the drug development process. In this mini-review, we will review the recent elucidation of structural properties of PXR and CAR, the molecular determinants of their ligand and species specificities and progress made in in silico models for identification of PXR and CAR activators.

The constitutive androstane receptor (CAR) belongs to the superfamily of nuclear-hormone receptors that function as ligand-activated transcription factors. CAR plays an essential role in the metabolism of xenobiotics and shows—in contrast to related receptors—constitutive activity. However, the molecular basis for the constitutive activity remains unclear. In the present study, homology models of the ligand binding domain (LBD) were generated based on the crystal structures of the related pregnane X (PXR) and the vitamin D receptor (VDR). The models were used to investigate the basal activity of CAR and the effect of coactivator binding. Molecular dynamics (MD) simulations of complexed and uncomplexed receptor revealed a hypothesis for the activation mechanism. The suggested mechanism is supported by experimental results from site-directed mutagenesis. The basal activity of CAR can be explained by specific van-der-Waals interactions between amino acids on the LBD and its C-terminal activation domain (AF-2). Docking studies with the GOLD program yielded the interaction modes of structurally diverse agonists, giving insight into mechanisms by which ligands enhance CAR activity. Figure The constitutive activity. Favorable regions of interactions between the GRID methyl probe and the AF-2 truncated LBD (colored magenta, contour level -2.5 kcal mol 1). Only the MOLCAD surface of the LBD is shown, colored according to the lipophilic potential (blue polar, brown lipophilic). The position of the two hydrophobic residues Leu343 and Ile346 from the AF-2 helix (colored cyan) is in close agreement with the GRID results.

The absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of a candidate drug influence its final clinical success. These properties have traditionally been evaluated by using various in vivoanimal approaches, but recently, a number of in vitroand in silicomethods have been introduced to determine key ADMET features. Basic events, such as absorption through the gut wall, binding to plasma proteins, active and passive transfer through the blood–brain barrier, and various metabolic parameters, can now be screened with rapid in vitroand computer modelling methods. The focus in this short review is on the basic in vitroand in silicomethods that are used for studying the metabolism properties of new drug molecules.

mCAR (mouse constitutive androstane receptor; NR1I3) controls the expression of cytochrome P450 as well as other enzymes involved in drug and steroid metabolism. The high basal activity of mCAR can be modulated by inhibitory steroids related to androstenol and by activating xenobiotic chemicals such as 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene and chlorpromazine. The ability of oestrogens and some other xenobiotics to activate mCAR is not clear. In the present study, co-transfection assays in HEK-293 cells indicated that oestrogens varied in their efficacy to activate mCAR, depending on variation at the steroid D-ring and position of hydroxy groups. In general, oestrogens were weaker activators of mCAR than 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene and chlorpromazine. Also, the induction of CYP2B10 mRNA by oestrogens was less pronounced in mouse primary hepatocytes. Yeast two-hybrid assays indicated that, unlike androstenol and the established activators, oestrogens attracted both nuclear receptor co-repressors and co-activators to the mCAR ligand-binding domain, thus limiting the extent of mCAR activation. This novel dual action is not limited to oestrogens, but is shared by some xenobiotic CYP2B inducers such as clotrimazole and methoxychlor. These findings offer an alternative explanation for the recently suggested nuclear activation step of mCAR.

It has been shown that extracellular glycosaminoglycans (GAGs) limit the gene transfer by cationic lipids and polymers. The purpose of this study was to clarify how interactions with anionic GAGs (hyaluronic acid and heparan sulfate) modify the cellular uptake and distribution of lipoplexes and polyplexes. Experiments on cellular DNA uptake and GFP reporter gene expression showed that decreased gene expression can rarely be explained by lower cellular uptake. In most cases, the cellular uptake is not changed by GAG binding to the lipoplexes or polyplexes. Reporter gene expression is decreased or blocked by heparan sulfate, but it is increased by hyaluronic acid; this suggests that intracellular factors are involved. Confocal microscopy experiments demonstrated that extracellular heparan sulfate and hyaluronic acid are taken into cells both with free and DNA-associated carriers. We conclude that extracellular GAGs may alter both the cellular uptake and the intracellular behavior of the DNA complexes.

Purpose. To develop and characterize a new drug-regulated gene expression system based on the nuclear receptor constitutive androstane receptor (CAR). Methods. Both transient and stable transfection into HEK293 cells of luciferase plasmids under the control of either drug- and steroid-responsive nuclear receptor CAR or the tetracycline-sensitive transactivator tTA were used in development of stable cell lines. Results. A stable first-generation cell line that expresses luciferase gene under the control of nuclear receptor CAR was developed. The luciferase expression in CAR-producing cells could be suppressed by androstanes and reactivated by structurally unrelated drugs chlorpromazine, metyrapone, phenobarbital, and clotrimazole. The kinetics of luciferase expression in CAR-producing cells and the tTA system were comparable. The overall regulation of CAR system was improved by modifications to the DNA binding domain and site. Conclusions. Because of its wide ligand selectivity and transferable ligand binding domain, CAR expands the repertoire of regulated gene expression systems.

Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks.

This article reviews recent progress in characterizing cis-acting DNA elements of the phenobarbital-inducible CYP2B genes. Whereas proximal DNA elements such as the C/EBP binding site regulate basal transcription activity, phenobarbital-responsive enhancer activity is governed by the distal element (designated phenobarbital-responsive enhancer module, PBREM) residing about -2.3 kbp upstream from the transcription start site. Proximal elements are not required to enhance the phenobarbital-inducible transcription, since the PBREM can confer the inducibility to several heterologous promoters. Repression of the basal transcription by a negative element upstream of the -0.8 kbp region, however, may be necessary for the proper regulation of the CYP2B genes. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 3–9, 1998

This article reviews recent progress in characterizing cis‐acting DNA elements of the phenobarbital‐inducible CYP2B genes. Whereas proximal DNA elements such as the C/EBP binding site regulate basal transcription activity, phenobarbital‐responsive enhancer activity is governed by the distal element (designated phenobarbital‐responsive enhancer module, PBREM) residing about ‐2.3 kbp upstream from the transcription start site. Proximal elements are not required to enhance the phenobarbital‐inducible transcription, since the PBREM can confer the inducibility to several heterologous promoters. Repression of the basal transcription by a negative element upstream of the ‐0.8 kbp region, however, may be necessary for the proper regulation of the CYP2B genes. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12:3–9, 1998

We cloned cDNAs which encode a mouse liver nuclear protein with an apparent molecular mass of 51 kDa, using sequences derived from a purified protein as the basis for designing specific primers. The deduced amino acid sequences revealed that the 51-kDa protein contains characteristic subdomain structures of a protein kinase. The bacterially expressed recombinant 51-kDa protein catalyzed phosphorylation of general substrates such as casein and was autophosphorylated at serine residue(s). This 51-kDa protein kinase, designated 51PK, is 40% identical to the 34-kDa protein kinase encoded by the vaccinia virus B1 gene and 25% identical to the casein kinase I isoforms, including yeast HRR25. The 51PK mRNA was expressed as two splice variants and the 51PK protein was exclusively localized in nuclei. Northern hybridization showed that 51PK mRNA was expressed in various tissues, with highest levels in testis, spleen, lung, and liver. These results, therefore, indicate that 51PK is a nuclear serine/threonine kinase and a novel distinct member of the protein kinase superfamily.

ABSTRACTPBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as atrans-acting factor for the phenobarbital-inducible Cyp2b10gene.

Using a primary hepatocyte culture in which the mouse Cyp2b10 gene transcription is activated by phenobarbital (PB)-type inducers, we examined the cellular signalling mechanisms associated with PB induction. Low nanomolar concentrations of protein serine/threonine phosphatase inhibitors okadaic acid (OA) and calyculin A blocked the induction of CYP2B10 mRNA. Nuclear run-on assays indicated that OA suppressed Cyp2b10 gene transcription. Pretreatment of the cells with an inhibitor of Ca2+/calmodulin-dependent protein kinases {1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62)} or with a flavonoid, naringin, were completely or partly protective respectively against the OA-mediated suppression of CYP2B10 mRNA. Several other established modulators of protein kinase activities did not greatly affect the induction of CYP2B10 mRNA, nor could they prevent the suppressive effect of OA. Our results indicate that specific protein phosphorylation-dephosphorylation is required for the induction of Cyp2b10 gene expression, which is modulated through multiple endogenous and exogenous signals.

The mouse phenobarbital (PB)-inducible Cyp2b10gene promoter has been isolated and sequenced, and control of its expression has been characterized. The 1405-base pair (bp) Cyp2b10promoter sequence is 83% identical to the corresponding region from the rat CYP2B2 gene. In addition to the lack of CA repeats, differences include insertion of 42 base pairs (−123/-82 bp) into the middle of a consensus sequence to the so-called “Barbie box.” In this report, we have developed a primary mouse hepatocyte culture system in which endogenous 2B10 mRNA as well as Cyp2b10-driven CAT activity were induced by PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), but not by the 3-chloro derivative of TCPOBOP. Deletion analysis of the Cyp2b10promoter identified a basal transcription element at −64/-34 bp and a negative element at −971/-775 bp. Sequences contained within the −1404/-971 bp region are responsible for the induced CAT activity. DNase I protection and gel shift assays detected five major protein binding sites within the −1404/-971 bp fragment, one of which shared high sequence identity with a portion of a regulatory element in CYP2B2 gene (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A.(1995) Gene158, 263-268). Our results indicate that sequences important for PB-induced transcription of Cyp2b10gene are located in the distal promoter.

ABSTRACTPBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as atrans-acting factor for the phenobarbital-inducibleCyp2b10gene.