The nuclear receptor Rev-erbα participates in circadian regulation of Ugt2b enzymes in mice

Circadian clock is known to modulate phase I metabolism, however whether and how the phase II enzymes UDP-glucuronosyltransferases (UGTs) are regulated by circadian clock are largely unknown. In this study, we aimed to investigate a potential role of the clock gene Rev-erb alpha in regulation of Ugt2b enzymes. Ugt2b mRNA and protein expression in mouse livers were determined at a 4-h interval around the clock. Ugt2b activity was probed using morphine as a specific substrate. Regulation of Ugt2b by Rev-erb alpha was investigated using mouse hepatoma Hepa-1c1c7 cells and Rev-erb alpha knock-out (Rev-erb alpha(-/-)) mice. Luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays were performed to identify the Rev-erb alpha binding site in Ugt2b36 promoter. Circadian variations in hepatic mRNA expression were observed for six Ugt2b genes (Ugt2b1, Ugt2b5, Ugt2b35, Ugt2b36, Ugt2b37, and Ugt2b38) in mice. Likewise, the total Ugt2b protein showed a circadian fluctuation. Glucuronidation of morphine (an Ugt2b substrate) both in vitro and in vivo was dosing-time dependent. Morphine glucuronidation was more extensive at the dosing time of ZT2 than at ZT14 consistent with the Ugt2b protein levels. Furthermore, Rev-erb alpha knockdown significantly increased Ugt2b mRNA and protein in Hepa-1c1c7 cells, whereas Rev-erb alpha overexpression or activation down-regulated Ugt2b expression. Moreover, Rev-erb alpha ablation in mice up-regulated the mRNA and protein expression of Ugt2b and blunted Ugt2b rhythmicity in the liver. In addition, Rev-erb alpha repressed the transcription of Ugt2b36 through specific binding to the -30 to -18 bp of promoter region based on a combination of luciferase reporter, mobility shift and ChIP assays. In summary, the clock gene Rev-erb alpha negatively regulates the expressions of Ugt2b genes, contributing to their circadian variations.