MicroRNA-30c-1-3p is a silencer of the pregnane X receptor by targeting the 3′-untranslated region and alters the expression of its target gene cytochrome P450 3A4

The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. Recently, activation of PXR has also been linked to the development of many disease conditions such as metabolic disorders and malignancies. MicroRNAs (miRs) emerge as important molecular species involved in these conditions. This study was undertaken to test a large number of miRs for their ability to regulate PXR expression. As many as 58 miRs were tested and miR-30c-1-3p was identified to suppress PXR expression. The suppression was achieved by targeting the 3′-untranslated region, 438 nucleotides from the stop codon. The suppression was detected in multiple cell lines from different organ origins. In addition, miR-30c-1-3p altered basal and induced expression of cytochrome P450 3A4 (CYP3A4), a prototypical target gene of PXR. The alteration varied depending on the time and amounts of miR-30c-1-3p. CYP3A4 is responsible for the metabolism of more than 50% medicines. The interconnection between miR-30c-1-3p and PXR signifies a role of miRs in drug–drug interactions and chemosensitivity. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie

Dexamethasone transcriptionally increases the expression of the pregnane X receptor and synergistically enhances pyrethroid esfenvalerate in the induction of cytochrome P450 3A23

The pregnane X receptor (PXR) is recognized as a key regulator for the induction of a large number of genes in drug metabolism and transport. The transactivation of PXR is enhanced by the glucocorticoid dexamethasone and the enhancement is linked to the induction of PXR in humans and rats. The present study was undertaken to determine the mechanism for the induction and ascertain the synergistic effect on the expression of CYP3A23, a rat PXR target. In primary hepatocytes, significant induction of PXR was detected as early as 2 h after the treatment and the maximal induction occurred at 1 μM dexamethasone. Similar induction kinetics was observed in the hepatoma line H4-II-E-C3. The induction was abolished by actinomycin D and dexamethasone efficaciously stimulated the rat PXR promoter. In addition, dexamethasone synergized esfenvalerate (an insecticide and a PXR activator) in inducing CYP3A23 and stimulating the CYP3A23 promoter. The full promoter of CYP3A23 (−1445/+74) was activated in a similar pattern as the changes in PXR mRNA in response to dexamethasone, esfenvalerate and co-treatment. In contrast, different responding patterns were detected on the stimulation of the CYP3A23 proximal promoter. Synergistic stimulation was also observed on the CYP3A4-DP-Luc reporter, the human counterpart of CYP3A23. These findings establish that transactivation is responsible for the induction of rat PXR and the induction presents potential interactions with insecticides in a species-conserved manner. The different responding patterns among CYP3A23 reporters point to an involvement of multiple transcriptional events in the regulation of CYP3A23 expression by dexamethasone, esfenvalerate and both. [Refer to PDF for graphical abstract

DEC1, a Basic Helix-Loop-Helix Transcription Factor and a Novel Target Gene of the p53 Family, Mediates p53-dependent Premature Senescence

Cellular senescence plays an important role in tumor suppression. p53 tumor suppressor has been reported to be crucial in cellular senescence. However, the underlying mechanism is poorly understood. In this regard, a cDNA microarray assay was performed to identify p53 targets involved in senescence. Among the many candidates is DEC1, a basic helix-loop-helix transcription factor that has been recently shown to be up-regulated in K-ras-induced premature senescence. However, it is not clear whether DEC1 is capable of inducing senescence. Here, we found that DEC1 is a novel target gene of the p53 family and mediates p53-dependent premature senescence. Specifically, we showed that DEC1 is induced by the p53 family and DNA damage in a p53-dependent manner. We also found that the p53 family proteins bind to, and activate, the promoter of the DEC1 gene. In addition, we showed that overexpression of DEC1 induces G1 arrest and promotes senescence. Moreover, we found that targeting endogenous DEC1 attenuates p53-mediated premature senescence in response to DNA damage. Furthermore, overexpression of DEC1 induces cellular senescence in p53-knockdown cells, albeit to a lesser extent. Finally, we showed that DEC1-induced senescence is p21-independent. Taken together, our data provided strong evidence that DEC1 is one of the effectors downstream of p53 to promote premature senescence

Dexamethasone suppresses the expression of multiple rat carboxylesterases through transcriptional repression: Evidence for an involvement of the glucocorticoid receptor

Carboxylesterases play important roles in the metabolism of xenobiotics and detoxication of insecticides. Without exception, all mammalian species studied express multiple forms of carboxylesterases. Several rat carboxylesterases are well-characterized including hydrolase A, B and S, and the expression of these enzymes is significantly suppressed by glucocorticoid dexamethasone. In this study, we used multiple experimental systems and presented a molecular mechanism for the suppression. Rats receiving one or more daily injections of dexamethasone consistently expressed lower HA, HB and HS. The suppression occurred at the levels of mRNA, protein and hydrolytic activity. In hepatoma cell line H4-II-E-C3, nanomolar dexamethasone caused significant decreases in HA, HB and HS mRNA, and the decreases were abolished by antiglucocorticoid RU486. Additionally, dexamethasone at nanomolar concentrations repressed the promoters of carboxylesterases, and the repression was reduced by glucocorticoid receptor-β, a dominant negative regulator of the glucocorticoid receptor (GR). In contrast, co-transfection of the pregnane X receptor (PXR) increased the reporter activities, but the increase occurred only at micromolar concentrations of dexamethasone. These findings establish that both GR and PXR are involved in the regulated expression of rat carboxylesterases by dexamethasone but their involvement depends on the concentrations

Liver receptor homolog 1 transcriptionally regulates human bile salt export pump expression

The metabolic conversion of cholesterol into bile acids in liver is initiated by the rate-limiting cholesterol 7α-hydroxylase (CYP7A1), whereas the bile salt export pump (BSEP) is responsible for the canalicular secretion of bile acids. Liver receptor homolog 1 (LRH-1) is a key transcriptional factor required for the hepatic expression of CYP7A1. We hypothesized that LRH-1 was also involved in the transcriptional regulation of BSEP. In support of our hypothesis, we found that overexpression of LRH-1 induced, whereas knockdown of LRH-1 decreased, BSEP expression. Consistent with its role in transcriptional regulation, LRH-1 dose-dependently transactivated the BSEP promoter. In addition, such transactivation by LRH-1 was required for maximal induction of BSEP expression through the bile acid/farnesoid X receptor (FXR) activation pathway. Bioinformatic and mutational analysis led to the identification of a functional liver receptor homolog 1-responsive element (LRHRE) in the BSEP promoter. Specific binding of LRH-1 to the LRHRE and recruitment of LRH-1 to the BSEP promoter were demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, respectively. In conclusion, LRH-1 transcriptionally activated the BSEP promoter and functioned as a modulator in bile acid/FXR-mediated BSEP regulation. These results suggest that LRH-1 plays a supporting role to FXR in maintaining hepatic bile acid levels by coordinately regulating CYP7A1 and BSEP for bile acid synthesis and elimination, respectively

Antioxidant sulforaphane and sensitizer trinitrobenzene sulfonate induce carboxylesterase-1 through a novel element transactivated by nuclear factor-E2 related factor-2

Carboxylesterase-1 (CES1), the most versatile human carboxylesterase, plays critical roles in drug metabolism and lipid mobilization. This enzyme is highly induced by antioxidants and sensitizers in various cell lines. These compounds are known to activate nuclear factor-E2 related factor-2 (Nrf2) by reacting to kelch-like ECH-associated protein-1 (Keap1). The aims of this study were to determine whether antioxidant sulforaphane (SFN) and sensitizer trinitrobenzene sulfonate (TNBS) target Keap1 similarly and whether they use the same element for CES1 induction. Cells over-expressing Keap1 were treated with TNBS or SFN and the formation of disulfide bonds among Keap1 molecules were determined. SFN promoted intramolecular disulfide formation whereas TNBS promoted intermolecular disulfide formation of Keap1. Two elements, sensitizing/antioxidant response element (S/ARE) and ARE4, were identified to support Nrf2 in the regulated expression of CES1A1. Both elements were bound by Nrf2, however, the S/ARE element supported, whereas the ARE4 element repressed Nrf2 transactivation. The repression required higher amounts of Nrf2, suggesting that the transactivation through the S/ARE element dominates the trans-repression through the ARE4 element under normal antioxidative condition. These findings conclude that compounds, although triggering the Keap1-Nrf2 pathway, may differ in the mode of reacting with Keap1. These findings also conclude that both positive and negative Nrf2 elements exist even within the same gene, and such opposing mechanisms provide fine-tuning in transcriptional regulation by the Keap1-Nrf2 pathway. High levels of CES1 are linked to lipid retention. Excessive induction of CES1 by antioxidants and sensitizers likely provides a mechanism for potential detrimental effect on human health

Cooperation between MEF2 and PPARγ in human intestinal β,β-carotene 15,15'-monooxygenase gene expression

BACKGROUND: Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. β, β-carotene 15,15'-monooxygenase 1 (BCMO1) catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression. RESULTS: We identified and functionally characterized the human BCMO1 promoter sequence and determined the transcriptional regulation of the BCMO1 gene in a BCMO1 expressing human intestinal cell line, TC-7. Several functional transcription factor-binding sites were identified in the human promoter that are absent in the mouse BCMO1 promoter. We demonstrate that the proximal promoter sequence, nt -190 to +35, confers basal transcriptional activity of the human BCMO1 gene. Site-directed mutagenesis of the myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) binding elements resulted in decreased basal promoter activity. Mutation of both promoter elements abrogated the expression of intestinal cell BCMO1. Electrophoretic mobility shift and supershift assays and transcription factor co-expression in TC-7 cells showed MEF2C and PPARγ bind to their respective DNA elements and synergistically transactivate BCMO1 expression. CONCLUSION: We demonstrate that human intestinal cell BCMO1 expression is dependent on the functional cooperation between PPARγ and MEF2 isoforms. The findings suggest that the interaction between MEF2 and PPAR factors may provide a molecular basis for interspecies differences in the transcriptional regulation of the BCMO1 gene

Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity

Surge in Expression of Carboxylesterase 1 During the Post-neonatal Stage Enables a Rapid Gain of the Capacity to Activate the Anti-influenza Prodrug Oseltamivir

Background. Oseltamivir, a widely used anti-influenza drug, is hydrolytically activated by carboxylesterase 1 (CES1). The expression of this carboxylesterase is developmentally regulated. This study was performed to determine when after birth infants acquire competence of activating this prodrug. Methods. Liver tissue samples were collected and divided into 5 age groups: group 1 (1-31 d old), group 2 (35-70 d old), group 3 (89-119 d old), group 4 (123-198 d old), and group 5 (>18 years of age). These samples were analyzed for oseltamivir hydrolysis and CES1 expression. Results. Liver samples in group 1 expressed the lowest level of CES1 with the lowest hydrolytic activity toward oseltamivir. A 4-7-fold increase between groups 1 and 2 (1-31 vs 35-70 d of age) was detected in the hydrolysis and expression analyses, respectively. Liver samples in the other 3 pediatric groups (35-198 d of age) exhibited similar expression and hydrolysis levels. Overall, liver samples in group 1 had CES1 expression and hydrolysis levels that were 10% of those of adults, whereas liver samples in the other 3 pediatric groups had levels that were ∼50% of adult levels. Conclusions. The post-neonatal surge in CES1 expression ensures the hydrolytic capacity to be gained rapidly after birth in infants, but the larger variability during this period suggests that caution should be exercised on the extrapolated dosing regimens of ester drugs from other age group

Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs

Orlistat has been the most used anti-obesity drug and the mechanism of its action is to reduce lipid absorption by inhibiting gastrointestinal lipases. These enzymes, like carboxylesterases (CESs), structurally belong to the α/β hydrolase fold superfamily. Lipases and CESs are functionally related as well. Some CESs (e.g., human CES1) have been shown to hydrolyze lipids. This study was designed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. Liver microsomes and recombinant CESs were tested for the inhibition of the hydrolysis of standard substrates and the anticancer prodrugs pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) and irinotecan. Contrary to the hypothesis, orlistat at 1 nM inhibited CES2 activity by 75% but no inhibition on CES1, placing CES2 one of the most sensitive targets of orlistat. The inhibition varied among some CES2 polymorphic variants. Pretreatment with orlistat reduced the cell killing activity of PPD. Certain mouse but not rat CESs were also highly sensitive. CES2 is responsible for the hydrolysis of many common drugs and abundantly expressed in the gastrointestinal track and liver. Inhibition of this carboxylesterase probably presents a major source for altered therapeutic activity of these medicines if co-administered with orlistat. In addition, orlistat has been linked to various types of organ toxicities, and this study provides an alternative target potentially involved in these toxicological responses