Aryl hydrocarbon receptor is a transcriptional activator of the human breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP/ABCG2) is a membrane-bound 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 -194/-190 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.

Ambient tonicity and intestinal cytochrome CYP3A.

IMPORTANCE OF THE FIELD: Recently-discovered tonicity-dependence of human CYP3A expression in vitro may be a novel mechanism of CYP3A regulation in the intestinal epithelia, which exists in a dynamic osmotic environment influenced by food intake. AREAS COVERED IN THIS REVIEW: A combination of focused and comprehensive literature searches to identify any relevant reports using Medline (from 1950 to 7 November 2009) through the OVID system. WHAT THE READER WILL GAIN: An update on current knowledge on osmotic environment in the gastrointestinal (GI) tract and its impact on intestinal CYP3A expression and function with special emphasis on the tonicity-sensitive transcription factor nuclear factor of activated T cells 5 (NFAT5). TAKE HOME MESSAGE: 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.

Discovery of osmosensitive transcriptional regulation of human cytochrome P450 3As by the tonicity-responsive enhancer binding protein (nuclear factor of activated T cells 5).

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 approximately 10- 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 +/-10 kilobases (kb) from the transcription start sites of CYP3A showed that only the CYP3A7 intron 2 region ( approximately 5 kb downstream from the transcription start site), which contains two TonE motifs (+5076/+5086 and + 5417/+5427), 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 (+5417/+5427) 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.