Studies On Circadian Clock Regulation Of Flavin-containing Monooxygenase 5

ObjectivesCircadian clock genes play an important regulatory role in circadian variations of physiological processes?e.g.,temperature,blood pressure,the metabolism of lipid,glucose and cholesterol?.It has been reported that many phase I drug-metabolizing enzymes?DMEs?including a number of CYPs,such as CYP1A2,CYP2A4/5,CYP2B10 and CYP3A11,display circadian rhythms in their expression.Flavin-containing monooxygenases?FMOs?,a family of phase I enzymes,are involved in metabolism of numerous drugs?e.g.,albendazole,benzydamine and methimazole?and environmental toxicants?e.g.,aldicarb and insecticides?.However,the diurnal rhythmicities of FMOs remain unknown.In this study,we aimed to characterize circadian rhythms of FMO5 expression and activity in mouse liver,and to investigate the potential roles of circadian clock genes?Bmal1,Rev-erb?and E4bp4/Dbp?in generation of diurnal rhythms of FMO5.Methods1.qPCR and Western blotting were used to detect the expression of FMO5 in wild-type mice at six circadian time points?i.e.,ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22?.Serum shock experiments were used to determine the rhythmic expression of Fmo5 in vitro.In addition,FMO5 activity assay was determined using hepatic microsomal incubation assays.2.Pharmacokinetic studies were performed with wild-type mice after intraperitoneal injection of PTX?50 mg/kg?at two different circadian points?i.e.,ZT2 and ZT14?.Blood and liver samples were collected at predetermined time points.The plasma and liver concentrations of PTX and its metabolite PTX-M were determined by UPLC-QTOF/MS.Non-compartmental model was used to estimate the time-varying PTX and PTX-M pharmacokinetics and liver distribution in mice.3.qPCR and Western blotting were used to detect the expression of FMO5 in Bmal1^-/-,Rev-erb?^-/-,E4bp4^-/-and their littermates(Bmal1^+/+,Rev-erb?^+/+and E4bp4^+/+)at six circadian time points?i.e.,ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22?.4.The regulatory effects of circadian clock genes?Bmal1,Rev-erb?and E4bp4?on the rhythmic expression of FMO5 were determined by overexpression and knockdown of gene?Bmal1,Rev-erb?and E4bp4?expression in primary mouse hepatocytes and hepatoma cells.5.The regulation mechanism of circadian clock genes on Fmo5 was elucidated by combining luciferase reporter gene assay and Ch IP assay.Results1.Circadian rhythms of FMO5 expression and activity in mouse liverFMO5 mRNA and protein in mouse liver oscillated in a circadian time-dependent fashion that peaked at ZT14.We confirmed that PTX is specific metabolized by mouse FMO5 to form PTX-M.Consistent with the enzyme's rhythmic expression,in vitro hepatic microsomal metabolism assays with PTX revealed a robust diurnal variation in Fmo5 activity.We also observed the rhythmic expression of Fmo5 in synchronized Hepa1-6 cells.These data demonstrated Fmo5 as a circadian gene.2.The activity of FMO5 in vivo displays a circadian time-dependent manner.A marked dosing time effect was observed in PTX metabolism?i.e.,formation of the metanolite PTX-M?.Plasma and hepatic levels of PTX-M were higher at dosing time of ZT14than at ZT2.3.Bmal1,Rev-erb?and E4bp4 ablation led to disrupted Fmo5 rhythms.We investigated the roles of the three cis-elements in regulation of rhythmic FMO5expression using Bmal1^-/-,E4bp4^-/-,and Rev-erb?^-/-mice in this study.Bmal1,Rev-erb?and E4bp4 knockdown led to disrupted FMO5 rhythms.FMO5 levels were decreased in Bmal1^-/-and Rev-erb?^-/-,but increased in E4bp4^-/-mice.4.FMO5 is positively regulated by Bmal1 in cells.Overexpression of Bmal1 led to significant increases in FMO5 mRNA and protein in primary mouse hepatocytes and Hepa1-6 cells.By contrast,knockdown of Bmal1 by si RNA caused decreases in FMO5 mRNA and protein.5 FMO5 is regulated by E4bp4 and Rev-erb?in primary mouse hepatocytes.E4bp4 overexpression led to significant decreases in FMO5 mRNA and protein,whereas E4bp4 knockdown resulted in an increase in FMO5 expression,indicating E4bp4 as a negative regulator of Fmo5.We observed positive regulatory effects of Rev-erb?on Fmo5.It is impossible that Rev-erb?directly regulate FMO5 expression due to Rev-erb?is a well-known transcriptional repressor.Instead,a negative mediator are essential for the positive regulation of Fmo5 by Rev-erb?.Interestingly,the regulatory effects of Rev-erb?on Fmo5 were attenuated in E4bp4-deficient cells.6 Circadian clock genes regulate Fmo5 expression.Bmal1 directly activated Fmo5 through binding to an E-box?-1822/-1816 bp?in the gene promoter.E4bp4,a repressor,directly inhibit Fmo5 through binding to both D-box1 and D-box3in gene promoter.Dbp activated Fmo5 via direct binding to D-box1 in the gene promoter.ConclusionThis study showed diurnal rhythms of hepatic FMO5 at the levels of mRNA,protein and enzymatic activity.The rhythmic expression of FMO5 was generated through transcriptional actions of circadian clock genes?including Bmal1,E4bp4 and Dbp?on E-box and D-box cis-elements.Our study has implications for understanding of clock-controlled drug metabolism and for facilitating the practice of chronotherapeutics.