Received for publication January 29, 2007.
Revised June 28, 2007.
Accepted for publication June 28, 2007.

Discovery of Osmo-sensitive Transcriptional Regulation of Human Cytochrome P450 3As (CYP3As) by the Tonicity-Responsive Enhancer Binding Protein (TonEBP/ NFAT5)

Abstract

We report the discovery of an osmo-sensitive transcriptional control of human CYP3A4, CYP3A7, and CYP3A5. Ambient hypertonicity (350 - 450 mOsm/kg) increased mRNA expressions of the CYP3As by ~10-20-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-bp tonicity-responsive enhancer (TonE) is an osmo-sensitive 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 ±10kb from the transcription start sites of CYP3As showed that only the CYP3A7 intron 2 region (~5kb 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, siRNA 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 the human CYP3As is influenced by ambient tonicity. The physiological significance of the tonic regulation of CYP3A enzymes remains to be determined.

Key words: NFAT, DNA binding sites, MDR/p-Glycoprotein, Regulation of gene expression, Cytochrome P450, Regulation - transcriptional