The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells.

Thyroid iodide accumulation via the sodium/iodide symporter (NIS; SLC5A5) has been the basis for the longtime use of radio-iodide in the diagnosis and treatment of thyroid cancers. NIS is also expressed, but poorly functional, in some non-thyroid human cancers. In particular, it is much more strongly expressed in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines than in primary human hepatocytes (PHH). The transcription factors and signaling pathways that control NIS overexpression in these cancers is largely unknown. We identified two putative regulatory clusters of p53-responsive elements (p53REs) in the NIS core promoter, and investigated the regulation of NIS transcription by p53-family members in liver cancer cells. NIS promoter activity and endogenous NIS mRNA expression are stimulated by exogenously expressed p53-family members and significantly reduced by member-specific siRNAs. Chromatin immunoprecipitation analysis shows that the p53-REs clusters in the NIS promoter are differentially occupied by the p53-family members to regulate basal and DNA damage-induced NIS transcription. Doxorubicin strongly induces p53 and p73 binding to the NIS promoter, leading to an increased expression of endogenous NIS mRNA and protein in HCC and CCA cells, but not in PHH. Silencing NIS expression reduced doxorubicin-induced apoptosis in HCC cells, pointing to a possible role of a p53-family-dependent expression of NIS in apoptotic cell death. Altogether, these results indicate that the NIS gene is a direct target of the p53 family and suggests that the modulation of NIS by DNA-damaging agents is potentially exploitable to boost NIS upregulation in vivo.

p53-paralog DNp73 oncogene is repressed by IFNα/STAT2 through the recruitment of the Ezh2 polycomb group transcriptional repressor.

The DNp73 proteins act as trans-repressors of p53 and p73-dependent transcription and exert both anti-apoptotic activity and pro-proliferative activity. DNp73s are frequently up-regulated in a variety of human cancers, including human hepatocellular carcinomas (HCCs). Increased levels of DNp73 proteins confer to HCC cells resistance to apoptosis and, irrespective to p53 status, a chemoresistant phenotype. Here, we show that interferon (IFN)α down-regulates DNp73 expression in primary human hepatocytes (PHHs) and HCC cell lines. IFNα has been used as pro-apoptotic agent in the treatment of malignancies and there is increasing evidence of IFNα effectiveness in HCC treatment and prevention of recurrence. The precise mechanisms by which class I IFNs exert their anti-proliferative and anti-tumor activity remain unclear. IFNα binding to its receptor activates multiple intracellular signaling cascades regulating the transcription of numerous direct target genes through the recruitment of a complex comprising of STAT1, STAT2 and IFN regulatory factor (IRF)9 to their promoters. We found that, in response to IFNα, the P2p73 promoter undergoes substantial chromatin remodeling. Histone deacetylases (HDACs) replace histone acetyl transferases. STAT2 is recruited onto the endogenous P2p73 promoter together with the polycomb group protein Ezh2, leading to increased H3K27 methylation and transcriptional repression. The reduction of DNp73 levels by IFNα is paralleled by an increased susceptibility to IFNα-triggered apoptosis of Huh7 hepatoma cells. Our results show, for the first time, that IFN-stimulated gene factor 3 recruitment may serve both in activating and repressing gene expression and identify the down-regulation of DNp73 as an additional mechanism to counteract the chemoresistance of liver cancer cells.

Cross-talk between xenobiotic detoxication and other signalling pathways: clinical and toxicological consequences.

1. Recent investigations on nuclear receptors and other transcription factors involved in the regulation of genes encoding xenobiotic metabolizing and transport systems reveal that xenobiotic-dependent signalling pathways are embedded in, and establish functional interactions with, a tangle of regulatory networks involving the glucocorticoid and oestrogen receptors, the hypoxia-inducible factor, the vitamin D receptor and other transcription factors/nuclear receptors controlling cholesterol/bile salt homeostasis and liver differentiation. 2. Such functional interferences provide new insight, first for understanding how xenobiotics might exert adverse effects, and second how physiopathological stimuli affect xenobiotic metabolism.

The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors.

Numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cytochromes P450 (CYPs), transferases and transporters. For example, natural and synthetic glucocorticoids (agonists and antagonists) as well as other clinically important drugs induce the hepatic CYP2B, CYP2C and CYP3A subfamilies in man, and these inductions might lead to clinically important drug-drug interactions. Only recently, the key cellular receptors that mediate such inductions have been identified. They include nuclear receptors, such as the constitutive androstane receptor (CAR, NR1I3), the retinoid X receptor (RXR, NR2B1), the pregnane X receptor (PXR, NR1I2), and the vitamin D receptor (VDR, NR1I1) and steroid receptors such as the glucocorticoid receptor (GR, NR3C1). There is a wide promiscuity of these receptors in the induction of CYPs in response to xenobiotics. Indeed, this adaptive system appears now as a tangle of networks, where receptors share partners, ligands, DNA response elements and target genes. Moreover, they influence mutually their relative expression. This review is focused on these different pathways controlling human CYP2B6, CYP2C9 and CYP3A4 gene expression, and the cross-talk between these pathways.

Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor.

Although CYP3A induction by dexamethasone has been extensively documented, its mechanism is still unclear because both the role of the glucocorticoid receptor and the ability of dexamethasone to activate the human pregnane X receptor have been questioned. In an attempt to resolve this problem, we investigated the response of CYP3A4 to dexamethasone (10 nm-100 microm) in primary human hepatocytes and HepG2 cells, using a variety of methods: kinetic analysis of CYP3A4 and tyrosine aminotransferase expression, effects of RU486 and cycloheximide, ligand binding assay, cotransfection of HepG2 cells with CYP3A4 reporter gene constructs and vectors expressing the glucocorticoid receptor, pregnane X receptor or constitutively activated receptor. In contrast to rifampicin (monophasic induction), dexamethasone produces a biphasic induction of CYP3A4 mRNA consisting of a low-dexamethasone component (nmol concentrations) of low amplitude (factor of 3-4) followed by a high-dexamethasone component (supramicromolar concentrations) of high amplitude (factor of 15-30). We show that the low-dexamethasone component results from the glucocorticoid receptor-mediated expression of pregnane X receptor and/or constitutively activated receptor which, in turn, are able to transactivate CYP3A4 in a xenobiotic-independent manner. At supramicromolar concentrations (>10 microm), dexamethasone binds to and activates pregnane X receptor thus producing the high-dexamethasone component of CYP3A4 induction. We conclude that, in contrast to the other xenobiotic inducers of CYP3A4, glucocorticoids play a dual role in CYP3A4 expression, first by controlling the expression of PXR and CAR under physiological conditions (submicromolar concentrations) through the classical glucocorticoid receptor pathway, and second by activating the pregnane X receptor under bolus or stress conditions (supramicromolar concentrations).

Induction of CYP2C genes in human hepatocytes in primary culture.

The expression and inducibility of four CYP2C genes, including CYP2C8, -2C9, -2C18, and -2C19, was investigated in primary cultures of human hepatocytes. By the use of RNase protection assay and specific antibodies, each CYP2C mRNA and protein were quantified unequivocally. The four CYP2C mRNAs were expressed in human livers and cultured primary hepatocytes, but only the CYP2C18 protein was not detected. Compounds known to activate the pregnane X receptor (PXR) such as rifampicin, or the constitutively activated receptor (CAR) such as phenobarbital, induced CYP2C8, CYP2C9, and to a lesser extent CYP2C19 mRNAs and proteins. CYP2C18 mRNA was expressed but not inducible. The concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to rifampicin and phenobarbital paralleled that of CYP3A4 and CYP2B6, the maximum accumulation being reached with 10 microM rifampicin and 100 microM phenobarbital. In contrast, dexamethasone produced maximum induction of CYP2C8 and CYP2C9 mRNAs at 0.1 microM while in these conditions neither CYP3A4 nor CYP2B6 was significantly induced. Moreover, the concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to dexamethasone paralleled that of tyrosine aminotransferase. Furthermore, dexamethasone, which has been recently shown to up-regulate PXR and CAR expression through the glucocorticoid receptor, potentiated CYP2C8 and CYP2C9 mRNA induction in response to rifampicin and phenobarbital. Collectively, these results suggest the possible implication of at least three receptors in the regulation of CYP2C8 and CYP2C9 expression, i.e., glucocorticoid receptor, PXR, and/or CAR.

Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation.

The barbiturate phenobarbital induces the transcription of cytochromes P450 (CYPs) 2B through the constitutive androstane receptor (CAR; NR1I3). CAR is a member of the nuclear receptor family (NR1) mostly expressed in the liver, which heterodimerizes with retinoid X receptor (RXR) and was shown to transactivate both the phenobarbital responsive element module of the human CYP2B6 gene and the CYP3A4 xenobiotic response element. Because previous studies in rodent hepatocyte cultures have shown that the phenobarbital-mediated induction of CYP2B genes is potentiated by glucocorticoids, we examined the role of activated glucocorticoid receptor in this process. We show that submicromolar concentrations of dexamethasone enhance phenobarbital-mediated induction of CYP3A4, CYP2B6, and CYP2C8 mRNA in cultured human hepatocytes. In parallel, we observed that glucocorticoid agonists, such as dexamethasone, prednisolone, or hydrocortisone, specifically increase human car (hCAR) mRNA expression. Accumulation of hCAR mRNA parallels that of tyrosine aminotransferase: both mRNAs reach a maximum at a concentration of 100 nM dexamethasone and are down-regulated by concomitant treatment with the glucocorticoid antagonist RU486. Moreover, the effect of dexamethasone on hCAR mRNA accumulation appears to be of transcriptional origin because the addition of protein synthesis inhibitor cycloheximide has no effect, and dexamethasone does not affect the degradation of hCAR mRNA. Furthermore, dexamethasone increases both basal and phenobarbital-mediated nuclear translocation of CAR immunoreactive protein in human hepatocytes. The up-regulation of CAR mRNA and protein in response to dexamethasone explains the synergistic effect of this glucocorticoid on phenobarbital-mediated induction of CYP2B genes and the controversial role of the glucocorticoid receptor on phenobarbital-mediated CYP gene inductions.

Interleukin-6 negatively regulates the expression of pregnane X receptor and constitutively activated receptor in primary human hepatocytes.

The marked impairment of hepatic drug metabolism during inflammation and infections has been known for many years and shown to result from down-regulation of cytochrome P450s (CYP) by cytokines. However, the mechanism of this repression is unknown. Using primary cultures of human hepatocytes, we show here that interleukin-6 (IL-6) rapidly and markedly decreases the expression of PXR (pregnane X receptor) and CAR (constitutively activated receptor) mRNAs, but does not affect the levels of dioxin receptor and glucocorticoid receptor mRNA. In parallel, IL-6 decreases both rifampicin- and phenobarbital-mediated induction of CYP2B6, CYP2C8, CYP2C9, and CYP3A4. As the transcriptional activity of PXR and CAR is not affected by IL-6 in cell-based reporter assays, our data suggest that the loss of CYP2 and CYP3 inducibility results from the negative regulation of PXR and CAR gene expression by this cytokine.