The Molecular Mechanism Regulating Diurnal Rhythm of Flavin-Containing Monooxygenase 5 in Mouse Liver

Flavin-containing monooxygenase 5 (FMO5) is a phase I enzyme that plays an important role in xenobiotic metabolism. Here, we aimed to characterize diurnal rhythms of Fmo5 expression and activity in mouse liver and to investigate the potential roles of clock genes (Bmal1, Rev-erbα, and E4bp4) in the generation of diurnal rhythms. Fmo5 mRNA and protein showed robust diurnal rhythms, with peak values at zeitgeber time (ZT) 10/14 and trough values at ZT2/22 in mouse liver. Consistently, a diurnal rhythm was observed for in vitro microsomal Baeyer-Villiger oxidation of pentoxifylline (PTX), a specific reaction catalyzed by Fmo5. Pharmacokinetic studies revealed a more extensive Baeyer-Villiger oxidation of PTX at dosing time of ZT14 than at ZT2, consistent with the diurnal pattern of Fmo5 protein. Fmo5 expression was downregulated and its rhythm was blunted in Bmal1^−/− and Rev-erbα^−/− mice. Positive regulation of Fmo5 by Bmal1 and Rev-erbα was confirmed in primary mouse hepatocytes and/or Hepa1-6 cells. Furthermore, Fmo5 expression was upregulated and its rhythm was attenuated in E4bp4^−/− mice. Negative regulation of Fmo5 by E4bp4 was validated using primary mouse hepatocytes. Combined luciferase reporter and chromatin immunoprecipitation assays demonstrated that Bmal1 (a known Rev-erbα activator) activated Fmo5 transcription via direct binding to an E-box (−1822/−1816 bp) in the promoter, whereas E4bp4 (a known Rev-erbα target gene) inhibited Fmo5 transcription by binding to two D-boxes (−1726/−1718 and −804/−796 bp). In conclusion, circadian clock genes control diurnal expression of Fmo5 through transcriptional actions on E-box and D-box cis-elements.

SIGNIFICANCE STATEMENT

Hepatic Fmo5 displayed diurnal rhythmicities in expression and activity in mice. We uncovered the molecular mechanism by which the rhythmic Fmo5 expression was generated. Fmo5 promoter presents E-box and D-box binding elements for transcriptional actions from circadian clock proteins such as Bmal1, E4bp4, and Dbp. These findings have implications for understanding clock-controlled drug metabolism and for facilitating the practice of chronotherapeutics.