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Discovery of Osmosensitive Transcriptional Regulation of Human Cytochrome P450 3As by the Tonicity-Responsive Enhancer Binding Protein (Nuclear Factor of Activated T Cells 5)

Division of Clinical Pharmacology & Toxicology, Department of Paediatrics, Physiology and Experimental Medicine Program, Research Institute, Hospital for Sick Children, Toronito, Ontario, Canada (K.K., A.I.C., S.U., K.P.T., S.I.); Department of Anatomical and Cellular Pathology, Sir Y.K. Pao Centre for Cancer, The Chinese University of Hong Kong, and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, the University of Hong Kong, Hong Kong, People's Republic of China (B.C.B.K.); and Department of Clinical Pharmacology and Therapeutics, Oita University Faculty of Medicine, Oita-shi, Japan (K.O.)

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 10- to 20-fold in human-intestinal C₂bbe1 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 (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.

Address correspondence to: Dr. Shinya Ito, Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8 Canada. E-mail: shinya.ito{at}sickkids.ca