Vladimir O. Pustylnyak

Constitutive androstane receptor (CAR) is a xenosensor and target for therapy

Constitutive androstane receptor (CAR, NR1I3), which is under consideration in this review, is a member of the superfamily of nuclear receptors. However, certain features distinguish CAR from the variety of nuclear receptors. First, this receptor has structural features that allow it to display constitutive activity in the absence of a ligand and to interact in a species-specific manner with a huge number of ligands diverse in chemical structure and origin. Second, recently many researchers are focused on CAR because the significance is increasingly shown of its influence on a variety of physiological functions, such as gluconeogenesis, metabolism of xenobiotics, fatty acids, bilirubin, and bile acids, hormonal regulation, etc. In addition to the fundamental scientific interest, the study of CAR is of practical importance because changes in CAR activity can lead to disorders in physiological processes, which finally can result in changes in pathological states. However, despite intensive studies, many mechanisms are still unclear, which makes it difficult to understand the role of CAR in the overall picture of molecular regulation of physiological processes. This review analyzes the features and diversity of the functions of CAR.

The constitutive androstane receptor activator 4‐[(4R,6R)‐4,6‐diphenyl‐1,3‐dioxan‐2‐yl]‐N,N‐dimethylaniline inhibits the gluconeogenic genes PEPCK and G6Pase through the suppression of HNF4α and FOXO1 transcriptional activity

The dual role of the constitutive androstane receptor (CAR) as both a xenosensor and a regulator of endogenous energy metabolism (lipogenesis and gluconeogenesis) has recently gained acceptance. Here, we investigated the effects of 4‐[(4R,6R)‐4,6‐diphenyl‐1,3‐dioxan‐2‐yl]‐N,N‐dimethylaniline (transpDMA), an effective CAR activator, on the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose‐6‐phosphatase (G6Pase) in rat livers.

Genetic polymorphism of estrogen metabolizing enzymes in Siberian women with breast cancer.

Allelic variants of cytochrome P450: CYP1A1, CYP1A2, CYP19 (aromatase), and the II-phase enzyme sulfotransferase 1A1 (SULT1A1) genes are associated with a high risk of hormone-dependent cancers. We estimated the frequency of these allelic variants in the female population of the Novosibirsk district and their association with the elevated risk of breast cancer. A DNA bank of patients with gynecologic oncology, patients with breast cancer (n = 335), and healthy women (n = 530) was created, and the following single-nucleotide polymorphisms were examined: CYP1A1 M1 polymorphism, that is, T264-C transition in the 30-noncoding region; CYP1A2*1F polymorphism, that is, C734-A transversion in the CYP1A2 gene; C-T transition (Arg264Cys) in exon 7 of CYP19; and SULT1A1*2 polymorphism, that is, G638-A transition (Arg213His) in the SULT1A1 gene. The results of our study indicate that women with mutant allele C and genotypes C/T, C/C of the CYP1A1 gene, wild-allele C, and genotype C/C of the CYP1A2 gene, mutant allele A and genotypes A/G, A/A of the SULT1A1 gene have an increased risk of development of breast cancer. Women with body mass index ≥ 30 and the heterozygous genotype C/T of the CYP19 gene have an increased risk of breast cancer. The CYP1A2 heterozygous variant genotype C/A is associated with an increased risk of an estrogen receptor (ER(+)) tumor.

Species-specific induction of CYP2B by 2,4,6-tryphenyldioxane-1,3 (TPD)

AIM The aim of the current study was to investigate the species-specific induction of CYP2B by 2,4,6-tryphenyldioxane-1,3 (TPD) in relation to activation of CAR. MAIN METHODS 7-Pentoxyresorufin O-dealkylase (PROD) activity, RT-PCR, Western blot, Electrophoretic mobility shift assays (EMSA). KEY FINDINGS Phenobarbital-like inducer administration significantly up-regulated CYP2B activity in rat and mouse liver in a species-specific manner, in contrast to the effects on CYP2B in lungs, kidneys and brains. In parallel, Western blot analysis showed that the species-specific increase of PROD in liver is related to the high content of CYP2B: phenobarbital (PB) and TPD increased CYP2B in rat liver, PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) - in mouse liver. The CYP2B protein level was unchanged in the lungs of rats and mice after inducer treatment, whereas it was not detected in the kidney and brain of control and treated animals. The hepatic CYP2B activity in both species paralleled the increase of CYP2B mRNA. A detectable CYP2B mRNA level was measured in the lungs of untreated mice and rats, though it was unchanged during induction. Noninducibility of CYP2B in extrahepatic tissues accompanied an absence of constitutive androstane receptor (CAR) gene expression in these tissues. In liver CYP2B induction paralleled the high level of CAR expression detected by RT-PCR. Moreover, PB, TPD and TCPOBOP treatment stimulated nuclear accumulation of CAR and increased CAR receptor NR1-binding activity in animal liver in a species-specific manner. SIGNIFICANCE We have shown that the increased nuclear accumulation and binding activity of CAR are associated with the species-specific up-regulation of CYP2B by TPD in rat liver.

Constitutive androstane receptor activation by 2,4,6-triphenyldioxane-1,3 suppresses the expression of the gluconeogenic genes

The constitutive androstane receptor (CAR, NR1I3) has a central role in detoxification processes, regulating the expression of a set of genes involved in metabolism. The dual role of NR1I3 as both a xenosensor and as a regulator of endogenous energy metabolism has recently been accepted. Here, we investigated the mechanism of transcriptional regulation of the glucose metabolising genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) by the cis isomer of 2,4,6-triphenyldioxane-1,3 (cisTPD), a highly effective NR1I3 activator in rat liver. It was shown that expression of the gluconeogenic genes PEPCK and G6Pase was repressed by cisTPD treatment under fasting conditions. Western-blot analysis demonstrated a clear reduction in the intensity of PEPCK and G6Pase immunobands from the livers of cisTPD-treated animals relative to bands from the livers of control animals. Chromatin immunoprecipitation assays demonstrated that cisTPD prevents the binding of FOXO1 to the insulin response sequences in the PEPCK and G6Pase gene promoters in rat liver. Moreover, cisTPD-activated NR1I3 inhibited NR2A1 (HNF-4) transactivation by competing with NR2A1 for binding to the NR2A1-binding element (DR1-site) in the gluconeogenic gene promoters. Thus, our results are consistent with the hypothesis that the cisTPD-activated NR1I3 participates in the regulation of the gluconeogenic genes PEPCK and G6Pase.

A Comparison of the Dynamics of S100B, S100A1, and S100A6 mRNA Expression in Hippocampal CA1 Area of Rats during Long-Term Potentiation and after Low-Frequency Stimulation

The interest in tissue- and cell-specific S100 proteins physiological roles in the brain remains high. However, necessary experimental data for the assessment of their dynamics in one of the most important brain activities, its plasticity, is not sufficient. We studied the expression of S100B, S100A1, and S100A6 mRNA in the subfield CA1 of rat hippocampal slices after tetanic and low-frequency stimulation by real-time PCR. Within 30 min after tetanization, a 2–4 fold increase of the S100B mRNA level was observed as compared to the control (intact slices) or to low-frequency stimulation. Subsequently, the S100B mRNA content gradually returned to baseline. The amount of S100A1 mRNA gradually increased during first hour and maintained at the achieved level in the course of second hour after tetanization. The level of S100A6 mRNA did not change following tetanization or low-frequency stimulation.

Regulation of S100B gene in rat hippocampal CA1 area during long term potentiation.

In the present study we investigated the regulation of S100B expression during tetanization-induced hippocampal long term potentiation, one of the best characterized forms of synaptic plasticity. Tetanization resulted in time-dependent change in S100B gene expression and protein content in hippocampal CA1 area. We analyzed the promoter region of the rat S100B gene and identified response elements for the tumor suppressor p53. ChIP assay revealed that p53 could bind to putative p53-binding sites of the S100B promoter. The time-dependent recruitment of p53 to its putative binding sites in the S100B gene promoter paralleled the time-course change of S100B mRNA and protein levels. Thus, these results strongly support the view that S100B gene may be a target of p53. Moreover, we demonstrated that the increase of S100B protein content was accompanied with the decrease of p53 protein content, and it seems that the decrease is regulated on post-translational level. Thus, our results may help to understand the physiological function of the p53-S100B-p53 loop in the process of synaptic plasticity.

CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments.

Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on CYP2B induction in mouse liver

2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective inducer of CYP2В in rats, but not in mice. Several analogs of TPD were synthesized. All substituents were entered into the same position of TPD (R=H, cisTPD and transTPD; R=N(CH(3))(2), transpDMA; R=NO(2), transpNO(2); R=F, transpF; R=OCH(3), transpMeO). The purpose of the present study was to investigate the effect of TPD analogs on CYP2B induction in mouse livers. Among the six test compounds, four (R=-N(CH(3))(2), -NO(2), -F, -OCH(3)) demonstrated a dose-dependent induction of mouse CYP2B. To further characterize the compounds, we determined ED50s using sigmoidal dose-response curves. The dose-response study has shown that all active compounds have similar potencies to induce CYP2B in mouse livers. Western-blot analysis and multiplex RT-PCR have shown that the increase of CYP2B activity in mouse liver is related to the high content of CYP2B proteins and paralleled the increase of cyp2b10 mRNA level. ChIP results have demonstrated that the transcriptional enhancement of cyp2b10 gene in response to compounds is accompanied by the increased recruitment of the constitutive androstane receptor (CAR) to its specific binding site (PBREM) on the target gene. Thus, minor structural changes in TPD cause dramatic changes in its ability to induce mouse CYP2B, and it is likely several TPD analogs act by activation of mouse CAR.

Role of Nuclear Constitutive Androstane Receptor in Regulation of Hepatocyte Proliferation and Hepatocarcinogenesis.

Activation of the constitutive androstane receptor (CAR) in hepatocytes occurs as a body adaptation in response to a number of external influences, and its functional activity is primarily related to induction of enzymes detoxifying xenobiotics. However, special attention was recently given to CAR due to the fact that its key role becomes unveiled in various physiological and pathophysiological processes occurring in the liver: gluconeogenesis, metabolism of fatty acids and bilirubin, hormonal regulation, proliferation of hepatocytes, and hepatocarcinogenesis. Here we review the main pathways and mechanisms that elevate hepatocyte proliferative activity related to CAR and whose disturbance may be a pivotal factor in hepatocarcinogenesis.