Hepatic flavin-containing monooxygenase gene regulation in different mouse inflammation models

Abstract

The objective of the study was to investigate the regulation of hepatic flavin-containing monooxygenases (Fmo) Fmo1, Fmo3, Fmo4, and Fmo5 in three different mouse models of inflammation, including treatment with Citrobacter rodentium, lipopolysaccharide (LPS), and dextran sulfate sodium (DSS). Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the steady-state mRNA levels for the various Fmo isoforms in these mouse models of inflammation during different treatment time courses. Fmo3 mRNA was most significantly down-regulated in C. rodentium-treated female mice. Fmo1, Fmo3, and Fmo5 mRNAs were also found to be down-regulated in LPS models of inflammation. The significant down-regulation of hepatic FMO3 protein during C. rodentium treatment was confirmed with Western blot analysis of liver microsomes from treated animals. Toll-like receptor (TLR) 4 is known to be responsible for LPS signaling in association with several proteins. To investigate whether TLR4 was responsible for regulation of Fmo genes in both LPS and C. rodentium animal models, Fmo mRNA levels in female wild-type (C3H/HeOuJ) and TLR4 mutant (C3H/HeJ) mice were compared in both inflammatory models by real-time RT-PCR. The results showed that Fmo3 down-regulation during C. rodentium infection is independent of TLR4. Whereas TLR4 is likely to play only a partial role in Fmo1 gene regulation in LPS-treated animals, our results show that the down-regulation of Fmo3 and Fmo5 in this model is TLR4-dependent. Unlike cytochrome P450 regulation measured in the same mouse strains, Fmo3 expression was largely refractory to down-regulation in the DSS model of inflammatory colitis.

Fig. 1.

Effect of C. rodentium treatment on hepatic Fmo mRNA regulation in C57BL/6 mice. Hepatic Fmo1 (A), Fmo3 (B), Fmo4 (C), and Fmo5 (D) mRNA levels in C. rodentium-infected female mice after 7, 10, and 15 days in comparison with the corresponding control mice groups. Day 24 post-treatment also available for Fmo3 (B). Number of mice for each group is as listed in Table 1, and mean levels are graphed. Error bars represent S.E.M. Statistically significant differences between the control and C. rodentium-infected mice are identified by *, P < 0.05 and ***, P < 0.001.

Fig. 2.

Effect of C. rodentium treatment on hepatic FMO protein levels in C57BL/6 mice. Microsomal FMO3 (A) and FMO1 (B) protein levels during C. rodentium infection were quantified based on Western blot analysis of FMO3 (C) and FMO1 (D), respectively. Protein expression of control mice (n = 4 for each group) and of mice at 7, 10, 15, and 24 days after C. rodentium infection (n = 5 for each group) are compared. Mean levels are graphed, and error bars represent S.E.M. Statistically significant differences between the control and C. rodentium-infected mice are identified by *, P < 0.05 and ***, P < 0.001.

Fig. 3.

Effect of C. rodentium treatment on hepatic Fmo3 mRNA expression in HeJ and HeOu mice. Hepatic Fmo3 mRNA levels were measured in C. rodentium-treated female HeJ and HeOu mice after 6 days of infection in comparison with the corresponding control mice groups. Number of mice for each group is as listed in Table 1. Mean levels are graphed, and error bars represent S.E.M. Statistically significant differences between the control and C. rodentium-infected mice are identified by ***, P < 0.001.

Fig. 4.

Effect of LPS treatment on hepatic Fmo mRNA regulation. Hepatic Fmo1 (A), Fmo3 (B), Fmo4 (C), and Fmo5 (D) mRNA levels were measured in female HeJ and HeOu mice 24 h after injection with 1 mg/kg LPS or saline (control groups). Number of mice for each group is as listed in Table 1. Mean levels are graphed, and error bars represent S.E.M. Statistically significant differences between the control and LPS-treated (LPS) mice are identified by *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

Fig. 5.

Effect of DSS treatment on hepatic Fmo mRNA regulation. Hepatic Fmo1 (A), Fmo3 (B), Fmo4 (C), and Fmo5 (D) mRNA levels in DSS-treated female HeJ and HeOu mice after 5 days are shown in comparison with the corresponding control mouse groups. Number of mice for each group is as listed in Table 1. Mean levels are graphed, and error bars represent S.E.M. Statistically significant differences between the control and DSS-treated (DSS) mice are identified by **, P < 0.01.