Breast cancer resistance protein (BCRP/ABCG2) is a membranebound efflux transporter important in cellular detoxification and multidrug resistance. Some aryl hydrocarbon receptor (AHR) agonists were reported to induce BCRP expression in human colon carcinoma cells. However, a direct involvement of AHR transcriptional regulation remains unexplored. In this study, we show that BCRP induction by AHR ligands occurs in human intestinal, liver, and mammary carcinoma cells and in primary colonocytes and hepatocytes. Increased BCRP transporter activity consistent with gene induction was also evident in the Caco2 subclone C2bbe1 cells. Using RNA interference and ectopic expression techniques to manipulate cellular AHR status, we confirmed AHR dependence of ABCG2 gene regulation. By gene promoter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays, an active, proximal dioxin-response element at Ϫ194/ Ϫ190 base pairs upstream of the transcription start site of the human ABCG2 gene was identified. Despite a common observation in human-derived cells, our in vitro and in vivo studies supported by phylogenetic footprinting analysis did not find that mouse Abcg2 is subject to AHR regulation. We conclude that AHR is a direct transcriptional regulator of human BCRP and provide an unprecedented role of AHR in cellular adaptive response and cytoprotection by up-regulating an important ATP-binding cassette efflux transporter.Breast cancer resistance protein (BCRP) is an ATP-binding cassette (ABC) transporter encoded by the ABCG2 gene, which attracts growing research efforts directed at its involvement in toxicity and elimination of various drugs and xenobiotics (Doyle et al., 1998;Ishikawa, 2009). BCRP is a promiscuous cellular efflux pump of a broad spectrum of drugs (e.g., topotecan, acyclovir, mitoxantrone), carcinogens [e.g., aflatoxin, benzo(a)pyrene, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine], nutrients and phytochemicals (e.g., riboflavin, folate, daidzein, flavonoids), and metabolites (e.g., sulfated estrogens, eicosanoids). The apical domain of intestinal mucosa, mammary ductal epithelia, liver, kidney, and sanctuary barrier tissues (blood-brain barrier, blood-testis barrier, and blood-placental barrier) is an important site of BCRP expression (Ishikawa, 2009;Robey et al., 2009). Thus, BCRP has important pharmacological and toxicological implications in systemic pharmacokinetics, tissue distribution,

We report the discovery of an osmosensitive transcriptional control of human CYP3A4, CYP3A7, and CYP3A5. Ambient hypertonicity (350 -450 mOsmol/kg) increased mRNA expressions of the CYP3A by ϳ10-to 20-fold in human-intestinal C 2 bbe1 cells, followed by an increase of CYP3A protein. Hypotonicity, on the other hand, suppressed CYP3A mRNA levels, indicating that physiological isotonic conditions may regulate the basal expression of CYP3A. Similar responses to ambient tonicity were observed in other human-derived cell lines (intestinal LS180 and hepatic HepG2) and human primary colonic cells. The 11-base pair tonicity-responsive enhancer (TonE) is an osmosensitive regulator that is activated by the transcription factor, the nuclear factor of activated T-cells 5 (NFAT5). Luciferase-based reporter assays of 13 consensus TonE motifs within Ϯ10 kilobases (kb) from the transcription start sites of CYP3A showed that only the CYP3A7 intron 2 region (ϳ5 kb downstream from the transcription start site), which contains two TonE motifs (ϩ5076/ϩ5086 and ϩ 5417/ ϩ5427), was responsive to hypertonicity stimuli. This observation was confirmed upon cotransfection with an NFAT5 expression vector, small interfering RNA, or dominant-negative NFAT5. Deletion and mutation analyses suggested that the TonE (ϩ5417/ ϩ5427) is indispensable for the enhancer activity. NFAT5 binding to the CYP3A7 intron 2 TonE motif was demonstrated with electrophoretic mobility shift assay and in a native cell context by chromatin immunoprecipitation. We conclude that transcription of human CYP3A is influenced by ambient tonicity. The physiological significance of the tonic regulation of CYP3A enzymes remains to be determined.The human cytochrome P450 3A (CYP3A) subfamily represents the most abundant cytochrome P450 drug-metabolizing enzymes in the liver and intestine. Together with membrane-bound transporters, they constitute a crucial component for drug elimination and excretion. Of the three major isoforms (CYP3A4, CYP3A5, and CYP3A7), CYP3A4 is the most abundant adult form, whereas CYP3A7 is the main fetal form. These isozymes collectively metabolize nearly half of all currently used medications.The human CYP3A genes reside in a cluster on chromosome 7 (Nelson et al., 2004), and their expression is characterized by wide interindividual variations. Significant coex-

In vitro hypertonicity of ambient osmotic environment in cultured human cells increases expression of CYP3A through transcriptional enhancement by osmosensitive NFAT5. Although post-prandial osmolality in the GI lumen in vivo is substantially increased, NFAT5 activation has not been reported. Similarly, high-salt diet increases intestinal CYP3A function in humans, but it is not known whether these changes are mediated directly by NFAT5.

We previously showed that human CYP3A expression in vitro is under the transcriptional control mediated by NFAT5, which is a tonicity responsive transcriptional factor. Interestingly, hypertonicity increases CYP3A expression, while hypotonicity decreases it, suggesting constitutive regulation roles of the NFAT5‐CYP3A pathway. In contrast, mouse Cyp3a was unresponsive to ambient hypertonicity in vitro. In the present study, our objective was to show if human CYP3A expression can be induced by hypertonic stimuli in vivo. The humanized CYP3A4–3A7 transgenic mice were subjected to 4 cycles of 24h water deprivation every other day, or to a 7‐day high‐salt diet (8% salt content, HSD), that mimics the dehydration and hypertonic luminal content in the intestine, respectively. In the kidney of mice undergoing the cyclic dehydration, NFAT5‐regulated BGT1 mRNA was significantly increased compared to control (5.9±1.1 fold: M±SEM; p=0.003). Fold‐changes of the CYP3A4 and 3A7 were 2.5±0.5 (p=0.02) and 0.7±0.2 (p=0.13), respectively. In mice after HSD, fold‐induction of the duodenal BGT1, CYP3A4 and 3A7 expressions were 25.2±8.2 (p=0.02), 4.7±0.9 (p=0.01) and 2.0±0.3 (p=0.08), respectively. Our findings suggest that human CYP3A transcription is under the influence of ambient tonicity. Direct involvement of NFAT5 in vivo awaits further studies.CIHR supported