Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor activators

In this report we show that submicromolar concentrations of dexamethasone enhance pregnane X receptor (PXR) activator-mediated CYP3A4 gene expression in cultured human hepatocytes. Because this result is only observed after 24 h of cotreatment and is inhibited by pretreatment with cycloheximide, we further investigated which factor(s), induced by dexamethasone, might be responsible for this effect. We report that dexamethasone increases both retinoid X receptor-alpha (RXRalpha) and PXR mRNA expression in cultured human hepatocytes, whereas PXR activators such as rifampicin and clotrimazole do not. Accumulation of RXRalpha and PXR mRNA reaches a maximum at a concentration of 100 nM dexamethasone after treatment for 6 to 12 h and is greatly diminished by RU486. A similar pattern of expression is observed with tyrosine aminotransferase mRNA. Moreover, the effect of dexamethasone on PXR mRNA accumulation seems to be through direct action on the glucocorticoid receptor (GR) because the addition of cycloheximide has no effect, and dexamethasone does not affect the degradation of PXR mRNA. Furthermore, dexamethasone induces the accumulation of a RXRalpha-immunoreactive protein and increases the nuclear level of RXRalpha:PXR heterodimer as shown by gel shift assays with a CYP3A4 ER6 PXRE probe. This accumulation of latent PXR and RXRalpha in the nucleus of hepatocytes explains the synergistic effect observed with dexamethasone and PXR activators together on CYP3A4 induction. These results reveal the existence of functional cross talk between the GR and PXR, and may explain some controversial aspects of the role of the GR in CYP3A4 induction.     

Assessment of human pregnane X receptor involvement in pesticide-mediated activation of CYP3A4 gene.

Assessment of foreign chemical inducibility on CYP3A4 is necessary to optimize drug therapies. The properties of chemicals such as pesticides, however, are not well investigated. In the present study, properties of various pesticides on human CYP3A4 induction have been tested using HepG2-derived cells stably expressing the CYP3A4 promoter/enhancer (3-1-10 cells) and the human pregnane X receptor (hPXR)-small interfering RNA (siRNA) system. Among the examined pesticides, 13 pesticides were observed to activate the CYP3A4 gene. Surprisingly, pyributicarb was found to increase the CYP3A4 reporter activity at 0.1 to 1 microM more strongly than typical CYP3A4 inducer rifampicin. Expression of hPXR-siRNA clearly diminished the pyributicarb-stimulated CYP3A4 reporter activity in 3-1-10 cells and decreased the endogenous CYP3A4 mRNA levels in HepG2 cells. Pyributicarb caused enhancement of CYP3A4-derived reporter activity in mouse livers introduced with hPXR by adenovirus. These results indicate pyributicarb as a potent activator of CYP3A4 gene, suggesting the existence of pesticides leading to CYP3A4 induction in our environment.     

银杏内酯B通过激活孕烷X受体诱导CYP3A4的表达

目的研究银杏内酯B(ginkgolide B)对CYP3A4的诱导作用,进一步验证是否通过激活孕烷X受体实现对CYP3A4 mRNA和蛋白水平的诱导表达。方法用不同浓度银杏内酯B处理LS174T细胞,通过Q-PCR法检测CYP3A4 mRNA表达变化,进一步利用本实验室构建的PXRCYP3A4稳定转染HepG2工程细胞株,结合荧光素酶报告基因技术,显示检测银杏内酯B对PXR的转录激活活性的影响。蛋白质免疫印迹法检测CYP3A4蛋白水平表达的变化;利用siRNA技术敲低PXR的mRNA表达,检测在PXR低表达的条件下银杏内酯B对CYP3A4 mRNA和蛋白水平表达影响。结果银杏内酯B可以浓度依赖性的使CYP3A4基因及蛋白表达水平上调,报告基因结果显示,银杏内酯B能够浓度依赖性的增强PXR的转录激活活性,在PXR低表达的条件下,银杏内酯B对CYP3A4诱导作用明显低于正常表达组PXR。结论以上表明银杏内酯B通过激活PXR受体,促进其转录激活活性进而诱导CYP3A4表达上调,同时对PXR自身的表达无明显影响。     

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 µM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 µM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 µM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 µM and 10 µM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.     

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.     

Involvement of glucocorticoid receptor and pregnane X receptor in the regulation of mouse CYP3A44 female-predominant expression by glucocorticoid hormone.

The role of the glucocorticoid receptor (GR) and pregnane X receptor (PXR) in the regulation of female-predominant expression of mouse CYP3A44 by glucocorticoid hormones was evaluated using a primary culture of female mouse hepatocytes, as the expression was suppressed in adrenalectomized female mice, restored by dexamethasone (DEX) treatment and was not detected in male mouse livers. Glucocorticoid hormones, such as DEX, hydrocortisone, and corticosterone, 11beta-[4-dimethylamino] phenyl-17beta-hydroxy-17-[1-propynyl]estra-4,9-diene-3-one (RU486), antagonists for GR and an agonist for PXR, and rifampicin, an agonist for PXR, were chosen to investigate the relationship of GR/PXR activation and Cyp3a44 gene expression. Glucocorticoid-inducible expression of CYP3A44 was not suppressed but rather was increased by RU486. Treatment of GR expression plasmid-transfected hepatocytes with DEX concentration dependently enhanced the expression of PXR as well as CYP3A44 mRNAs. A synergistic effect of DEX at submicromolar concentrations and rifampicin is observed. Furthermore, transfection of PXR and retinoid X receptor-alpha (RXRalpha) also showed prominent induction of CYP3A44 mRNA by DEX. These results suggest that DEX plays a dual role in CYP3A44 expression: first, direct activation of the Cyp3a44 gene by the PXR-RXRalpha complex, and, second, indirect activation of the Cyp3a44 gene through the induction of PXR gene expression by the GR pathway.     

人参皂苷F1通过激活孕烷X受体诱导CYP3A4的表达

目的探索人参皂苷F1(ginsenoside F1)是否通过激活孕烷X受体(PXR)实现对CYP3A4基因表达及酶活性的诱导作用。方法利用本实验室构建的PXR-CYP3A4稳定转染Hep G2工程细胞株结合荧光素酶报告基因技术,检测人参皂苷F1对PXR的转录激活效应;用不同浓度的人参皂苷F1处理LS174T细胞,并通过Q-PCR和酶活性试剂盒检测CYP3A4的mRNA表达和酶活性变化。结果不同浓度人参皂苷F1作用于LS174T细胞以后,可以浓度依赖性地诱导CYP3A4 mRNA水平的表达,并且增强其酶活性;同时,PXR-CYP3A4稳定转染Hep G2工程细胞株结合荧光素酶报告基因检测结果亦表明,人参皂苷F1能够浓度依赖性地增强PXR的转录激活效应。结论本研究揭示了人参皂苷F1可诱导CYP3A4的基因表达并增强其酶活性,这一过程可能与人参皂苷F1对孕烷X受体的激活有关。     

孕烷X受体和CYP3A相关性的研究进展

CYP3A是生物体内化学物代谢的关键酶 ,孕烷X受体 (PXR)是CYP3A基因表达的转录活化因子。PXR分子结构的不同导致CYP3A的种属差异。化学物通过PXR调节CYP3A的表达可能是影响化学物体内代谢的一条重要途径。研究PXR和CYP3A的相互作用对于新药设计、指导临床合理用药、预测药物相互作用、减少药物不良反应都具有重要意义。     

Differences in transactivation between rat CYP3A1 and human CYP3A4 genes by human pregnane X receptor

In an assay system using a human CYP3A4 reporter constructed with the promoter (+11 nt to -362 nt) and enhancer (-7.2 knt to -7.8 knt) regions including everted repeat separated by six nucleotides (ER-6) and direct repeat separated by three nucleotides (DR-3) motifs, the CYP3A4 transactivation was detected without overexpression of any nuclear receptors in rifampicin-treated HepG2 cells. Overexpression of human pregnane X receptor (hPXR) enhanced the transactivation. Rat CYP3A1 reporter constructed with the promoter region (+31 nt to -171 nt) including both DR-3 and ER-6 motifs was, however, not transactivated in rifampicin-treated cells, even after overexpression of hPXR. Although overexpression of retinoid X receptor alpha (RXRalpha) had no clear effect for both CYP3A reporters, co-expression of apolipoprotein AI regulatory protein-1 (ARP-1) with hPXR resulted in the rifampicin-induced transactivation of the CYP3A1 reporter. A truncated CYP3A4 reporter retaining the both motifs showed the rifampicin-induced transactivation by overexpression of hPXR and ARP-1, while the transactivation in hPXR-overexpressed cells was not observed. These results support the idea that a nuclear receptor other than RXRalpha may play a role in the CYP3A transactivation together with hPXR. The present study also suggests the involvement of a novel cis-element in the hPXR-mediated CYP3A4 transactivation.     

Natural protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4.

Between 45 and 60% of all drugs currently used are metabolized by the CYP3A4 protein. CYP3A4 expression in liver varies up to 60-fold in the general population, which can lead to ineffective drug therapy (high CYP3A4) or, on the other hand, to harmful drug reactions (low CYP3A4). Most of this variability has been attributed to genetic factors, but to date their identity remains unknown. Recently, it was shown that CYP3A expression is largely controlled by the pregnane X receptor (PXR). We, therefore, hypothesized that polymorphisms in PXR may contribute to CYP3A4 variability. The presence of PXR variants was investigated in two ethnic groups, Caucasians and Africans. Six missense mutations leading to variant PXR proteins were identified, and their consequences on CYP3A4 expression were analyzed. Expressed in LS174T cells, three protein variants, V140M, D163G, and A370T, exhibited altered basal and/or induced transactivation of CYP3A promoter reporter genes. Thus, these natural PXR protein variants may play a role in the observed interindividual variability of CYP3A4 expression and may be involved in rare, atypical responses to drugs or altered sensitivities to carcinogens.     

PXR受体调控的CYP3A诱导及其在药物代谢中的重要意义

机体每日都要接触大量外源性化合物（xenobiotics），包括环境、饮食、药物中的各种成分，其中亲脂性化合物如果不能被及时代谢为极性化合物，就会在肝脏蓄积并影响机体正常生理功能，产生毒性甚至致癌。细胞色素P450（CYPS）属于血红素蛋白基因超家族，编码一系列代谢酶系统，参与各类不同结构亲脂性化合物的生物转化，增强代谢物水溶性，利于排出体外，     

Induction of CYP3A4 by 1 alpha,25-dihydroxyvitamin D3 is human cell line-specific and is unlikely to involve pregnane X receptor.

Under certain culture conditions, exposure of the human colon adenocarcinoma cell line Caco-2 to 1,25-(OH)(2)-D(3) induces expression of CYP3A4 to levels comparable to that in human small intestinal epithelium. To determine whether 1,25-(OH)(2)-D(3) could be used to restore CYP3A expression in other culture models, we examined several cell lines derived from malignancies of human tissues known to express CYP3A enzymes: Hep G2 (liver), LS180 (colon), HPAC (pancreas), Hs746T (stomach). Primary cultures of human hepatocytes from two donors were also examined. 1,25-(OH)(2)-D(3) increased CYP3A catalytic activity in LS180 (15-fold), HPAC (6-fold), and hepatocytes (2- to 3-fold); this was accompanied by induction of CYP3A4 mRNA and CYP3A immunoreactive protein. However, 1,25-(OH)(2)-D(3) had no effect on CYP3A expression in Hs746T or Hep G2. Known ligands for pregnane X receptor (PXR) (rifampin, dexamethasone, and dexamethasone t-butyl acetate) markedly induced CYP3A4 expression in human hepatocytes. In contrast, these ligands had little or no effect on CYP3A4 expression in Caco-2 cells, even at concentrations 1 to 2 orders of magnitude greater than effective concentrations of 1,25-(OH)(2)-D(3) or two other vitamin D receptor (VDR) ligands (25-OH-D(3) and 1-OH-D(3)). The retinoic acid receptor ligand all-trans-retinoic acid augmented the 1,25-(OH)(2)-D(3)-mediated induction of CYP3A4 catalytic activity up to 2-fold in Caco-2 cells, while having no demonstrable effect on levels of CYP3A4 mRNA or protein. The retinoid X receptor ligand 9-cis-retinoic acid appeared to slightly reduce CYP3A4 catalytic activity. We conclude that 1,25-(OH)(2)-D(3) can be used to increase CYP3A4 expression in some, but not all, human cell lines derived from tissues known to express CYP3A enzymes. The mechanisms involved in this induction are unlikely to involve PXR and may involve VDR.     

Induction of cytochrome P450 3A by paclitaxel in mice: pivotal role of the nuclear xenobiotic receptor, pregnane X receptor.

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.     

CYP3A induction by liver x receptor ligands in primary cultured rat and mouse hepatocytes is mediated by the pregnane X receptor.

The effects of oxysterol and drug ligands of the liver X receptor (LXR) on cytochrome P450 expression were evaluated in primary cultured rodent hepatocytes. Treatment of rat hepatocyte cultures with either 25-hydroxycholesterol or 24(S),25-epoxycholesterol (10(-5) to 5 x 10(-5) M) produced concentration-dependent elevations in CYP3A mRNA and immunoreactive protein levels but did not increase the amounts of CYP1A1, CYP2B, or CYP4A gene products. The effects of 24(S),25-epoxycholesterol on CYP3A content were much greater than were those of 25-hydroxycholesterol, consistent with the relative abilities of these sterols to bind and activate LXR. To understand the mechanistic basis of these observations, experiments were performed using primary cultured hepatocytes prepared from LXRalpha/beta- or pregnane X receptor (PXR)-null mice. CYP3A mRNA levels were increased after treatment with 24(S),25-epoxycholesterol in both wild-type and LXR-null mouse hepatocytes. In contrast, neither 24(S),25-epoxycholesterol nor either of two additional potent LXR ligands, 22(R)-hydroxycholesterol and N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)ethyl-]phenyl]-benzenesulfonamide (T0901317), altered CYP3A mRNA levels in hepatocytes prepared from PXR-null mice, although these agents induced CYP3A mRNA content in wild-type cultures. As evidence that the LXR ligands also activated PXR in rat hepatocytes, cotransfection of primary cultures with a dominant negative PXR abolished reporter gene induction after treatment with any of the test agents. These results indicate that selected LXR ligands are capable of activating PXR, probably as a defensive measure to prevent the accumulation of these potentially toxic endogenous molecules.     

Polycyclic aromatic hydrocarbons activate CYP3A4 gene transcription through human pregnane X receptor

Aryl hydrocarbon receptor (AhR) activators have been shown to induce members of the cytochrome P450 (P450) 1 family. Here we demonstrate that the AhR activators induce CYP3A4 through human pregnane X receptor (PXR). AhR activators, polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased CYP3A4 reporter activity and CYP3A4 mRNA expression in HepG2 cells. The CYP3A4 reporter activity was also increased by treatment with cigarette tar. The increased CYP3A4 reporter activity was clearly knocked down by the introduction of human PXR-small interfering RNA, but not by that of human AhR-small interfering RNA. The CYP3A4 reporter activity enhanced by overexpression of human PXR was further increased by treatment with PAHs and TCDD as well as by treatment with rifampicin. These results suggest that PAHs contained in cigarette smoke induce CYP3A4 in human liver.     

Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of CYP3A4 gene

Metformin is widely used in the treatment of type-2 diabetes. The pleotropic effects of metformin on glucose and lipid metabolism have been proposed to be mediated by the activation of AMP-activated protein kinase (AMPK) and the subsequent up-regulation of small heterodimer partner (SHP). SHP suppresses the functions of several nuclear receptors involved in the regulation of hepatic metabolism, including pregnane X receptor (PXR), which is referred to as a “master regulator” of drug/xenobiotic metabolism.In this study, we hypothesize that metformin suppresses the expression of CYP3A4, a main detoxification enzyme and a target gene of PXR, due to SHP up-regulation.We employed various gene reporter assays in cell lines and qRT-PCR in human hepatocytes and in Pxr^−/− mice.We show that metformin dramatically suppresses PXR-mediated expression of CYP3A4 in hepatocytes. Consistently, metformin significantly suppressed the up-regulation of Cyp3a11 mRNA in the liver and intestine of wild-type mice, but not in Pxr^−/− mice. A mechanistic investigation of the phenomenon showed that metformin does not significantly up-regulate SHP in human hepatocytes. We further demonstrate that AMPK activation is not involved in this process. We show that metformin disrupts PXR's interaction with steroid receptor coactivator-1 (SRC1) in a two-hybrid assay independently of the PXR ligand binding pocket. Metformin also inhibited vitamin D receptor-, glucocorticoid receptor- and constitutive androstane receptor (CAR)-mediated induction of CYP3A4 mRNA in human hepatocytes.We show, therefore, a suppressive effect of metformin on PXR and other ligand-activated nuclear receptors in transactivation of the main detoxification enzyme CYP3A4 in human hepatocytes.     

Indirect activation of pregnane X receptor in the induction of hepatic CYP3A11 by high-dose rifampicin in mice

Rifampicin (RIF), a typical ligand of human pregnane X receptor (PXR), powerfully induces the expression of cytochrome P450 3A4 (CYP3A4) in humans. Although it is thought that RIF is not a ligand of rodent PXR, treatment with high-dose RIF (e.g. more than 20?mg/kg) increases the expression of CYP3A in the mouse liver. In this study, we investigated whether the induction of CYP3A by high-dose RIF in the mouse liver is mediated via indirect activation of mouse PXR (mPXR). The results showed that high-dose RIF increased the expression of CYP3A11 and other PXR-target genes in the liver of wild-type mice but not PXR-knockout mice. However, the results of reporter gene and ligand-dependent assembly assays showed that RIF does not activate mPXR in a ligand-dependent manner. In addition, high-dose RIF stimulated nuclear accumulation of mPXR in the mouse liver, and geldanamycin and okadaic acid attenuated the induction of Cyp3a11 and other PXR-target genes in primary hepatocytes, suggesting that high-dose RIF triggers nuclear translocation of mPXR. In conclusion, the present study suggests that high-dose RIF stimulates nuclear translocation of mPXR in the liver of mice by indirect activation, resulting in the transactivation of Cyp3a11 and other PXR-target genes.