PII‐45

PURPOSE Expression of CYP3A is regulated by transcriptional factors such as PXR. However, an entire spectrum of its regulation remains elusive. We describe a novel regulatory pathway for CYP3A transcription, which is mediated by the tonicity‐responsive enhancer (TonE) and its binding protein (TonEBP), also known as NFAT5. METHODS Human intestinal C2bbe1 cells, a subclone of Caco2 cells, were exposed to various tonicity changes encountered in physiological conditions of the intestinal lumen. Real‐time RT‐PCR and western blotting were used to analyze gene and protein expression. TonEBP expression plasmid, siRNA, and dominant‐negative TonEBP were used for gain‐ and loss‐of‐function assays. Luciferase‐based reporter constructs with CYP3A promoters were used to identify the TonE element within the CYP3A gene cluster. RESULTS The C2bbe1 cells showed significant tonicity‐dependent increase in CYP 3A4, 7 and 5 mRNA (5‐10‐fold increase at 400 mOsm/kg) and protein expressions with no appreciable change in PXR. This was confirmed in the primary culture of human colon, and the other cell lines of human intestinal and hepatic origins. Screening of the CYP3A gene region revealed an active TonE sequence within a CYP3A7 intron. CONCLUSION Human CYP3A expression is under the influence of external tonicity changes. We propose that binding of the tonicity‐activated TonEBP to the TonE element in the CYP3A gene cluster is responsible for this phenomenon. Clinical Pharmacology & Therapeutics (2005) 79 , P48–P48; doi: 10.1016/j.clpt.2005.12.170