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.

• Klíčová slova
• CAR, NR1I3, cytochrome P450, diazepam, drug interaction, gene regulation,
• MeSH
• buněčné linie MeSH
• diazepam farmakologie   MeSH
• dospělí MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• konstitutivní androstanový receptor MeSH
• lidé středního věku MeSH
• lidé MeSH
• ligandy MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• proteinové domény účinky léků   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• reportérové geny účinky léků   genetika   MeSH
• transport proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diazepam MeSH
• konstitutivní androstanový receptor MeSH
• ligandy MeSH
• NR1I3 protein, human MeSH Prohlížeč
• Nr1i3 protein, mouse MeSH Prohlížeč
• receptory cytoplazmatické a nukleární MeSH

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.

• Klíčová slova
• 3′-UTR, 3′-untranslated region, CAR, constitutive androstane receptor, CYP3A4, cytochrome P450 3A4, Cytochrome P450 3A4, DEX, dexamethasone, DMEs, drug metabolizing enzymes, DMSO, dimethyl sulfoxide, ER, estrogen receptor, GRα, glucocorticoid receptor α, Gene expression, Gluc, Gaussia luciferase, Glucocorticoid, LBD, ligand binding domain, MRE, miRNA-response element, MicroRNA, NR, nuclear receptor, PB, phenobarbital, PCN, pregnenolone 16α-carbonitrile, PHHs, primary human hepatocytes, PPARα, peroxisome proliferator-activated receptor α, PXR, pregnane X receptor, Pregnane X receptor, RXRα, retinoid X receptor α, Regulation, Rif, rifampicin, SEAP, secreted alkaline phosphatase, miRNA, microRNA,
• Publikační typ
• časopisecké články MeSH