Abstract
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.
Footnotes
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This work was supported by Canadian Institute for Health Research grant (MT13747, to S.I.); by University Grants Committee of the Hong Kong Special Administrative Region Areas of Excellence Scheme grant AoE/P-10/01, and by Research Grant Council grants HKU 7427/04M (to B.C.B.K); by the Fellowship award from the Japanese Society for Clinical Pharmacology and Therapeutics (to K.K.); by the Pfizer fellowship (to S.U.); by the Ontario Graduate Scholarship (to A.I.C.); and by the RSTRACOMP studentship, Research Institute, Hospital for Sick Children (to A.I.C. and K.P.T).
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K.K. and A.I.C. contributed equally to this work.
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This work was presented in part at the annual meetings of the Japanese Society for Clinical Pharmacology and Therapeutics (December 2005, Oita, Japan) and the American Society for Clinical Pharmacology and Therapeutics (March 2006, Baltimore, MD).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.034504.
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ABBREVIATIONS: PXR, pregnane X receptor; CAR, constitutive androstane receptor; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NFAT5, the nuclear factor of activated T-cells 5; PCR, polymerase chain reaction; siRNA, small interfering RNA; SMIT, sodium/myoinositol cotransporter; SV, simian virus; TonE, tonicity-responsive enhancer; VDR, vitamin D receptor; bp, base pair(s); kb, kilobase(s); MEM, minimal essential medium; PBS, phosphate-buffered saline; dn, dominant negative.
- Received January 25, 2007.
- Accepted June 28, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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