Peroxisome proliferator-activated receptor γ activation inhibits liver growth through miR-122-mediated downregulation of cMyc.

Although NR1C3 agonists inhibit cell growth, the molecular mechanism of their action has not been thoroughly characterized to date. A recent study demonstrated that NR1C3 can regulate miR-122 by binding to its promoter. Given that miR-122 can indirectly regulate cMyc-mediated promitogenic signaling by targeting E2f1, we hypothesized that NR1C3 activation inhibits hepatocyte proliferation through miR-122-mediated cMyc downregulation. In the present study, we examined if liver hyperplasia induced by a strong chemical mitogen for the liver, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is an agonist of NR1I3, can be repressed by NR1C3 activation through miR-122 upregulation. Acute TCPOBOP treatment caused a significant increase in liver-to-body weight ratio. The liver mass increase was accompanied with miR-122 downregulation. ChIP assays demonstrated that TCPOBOP-activated NR1I3 accumulated on the DR1 site in the pri-miR-122 promoter; and the NR1I3 accumulation is accompanied by a decrease in miR-122 and an increase in E2f1 and its transcription target cMyc. Rosiglitazone (Ros) treatment, which is an agonist of NR1C3, caused an opposite effect on liver-to-body weight ratio. When Ros was given with TCPOBOP, it attenuated the inhibitory effect of TCPOBOP on miR-122. Moreover, Ros treatment inhibited the NR1I3 binding with the DR1 site in the pri-miR-122 promoter. Furthermore, the increase of miR-122 produced by Ros was correlated with the downregulation of its targets, E2f1 and cMyc. Thus, our finding demonstrated that the liver growth inhibitory effect of NR1C3 activation was at least partly related to the decrease of cMyc though the activation of miR-122 and the downregulation of E2f1.

Constitutive androstane receptor activation evokes the expression of glycolytic genes.

It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in a mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation.

Xenosensor CAR mediates down-regulation of miR-122 and up-regulation of miR-122 targets in the liver.

MiR-122 is a major hepatic microRNA, accounting for more than 70% of the total liver miRNA population. It has been shown that miR-122 is associated with liver diseases, including hepatocellular carcinoma. Mir-122 is an intergenic miRNA with its own promoter. Pri-miR-122 expression is regulated by liver-enriched transcription factors, mainly by HNF4α, which mediates the expression via the interaction with a specific DR1 site. It has been shown that phenobarbital-mediated activation of constitutive androstane receptor (CAR), xenobiotic nuclear receptor, is associated with a decrease in miR-122 in the liver. In the present study, we investigated HNF4α-CAR cross-talk in the regulation of miR-122 levels and promitogenic signalling in mouse livers. The level of miR-122 was significantly repressed by treatment with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is an agonist of mouse CAR. ChIP assays demonstrated that TCPOBOP-activated CAR inhibited HNF4α transactivation by competing with HNF4α for binding to the DR1 site in the pri-miR-122 promoter. Such transcription factor replacement was strongly correlated with miR-122 down-regulation. Additionally, the decrease in miR-122 levels produced by CAR activation is accompanied by an increase in mRNA and cellular protein levels of E2f1 and its accumulation on the target cMyc gene promoter. The increase in accumulation of E2f1 on the target cMyc gene promoter is accompanied by an increase in cMyc levels and transcriptional activity. Thus, our results provide evidence to support the conclusion that CAR activation decreases miR-122 levels through suppression of HNF4α transcriptional activity and indirectly regulates the promitogenic protein cMyc. HNF4α-CAR cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments.

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.

Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers.

Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p'-DDT (85%) and o,p'-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CAR and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45ß, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45ß. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the ß-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers.