Alterations in hepatic mRNA expression of phase II enzymes and xenobiotic transporters after targeted disruption of hepatocyte nuclear factor 4 alpha

Abstract

Hepatocyte nuclear factor 4 alpha (HNF4a) is a liver-enriched master regulator of liver function. HNF4a is important in regulating hepatic expression of certain cytochrome P450s. The purpose of this study was to use mice lacking HNF4a expression in liver (HNF4a-HNull) to elucidate the role of HNF4a in regulating hepatic expression of phase II enzymes and transporters in mice. Compared with male wild-type mice, HNF4a-HNull male mouse livers had (1) markedly lower messenger RNAs (mRNAs) encoding the uptake transporters sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide (Oatp) 1a1, Oatp2b1, organic anion transporter 2, sodium phosphate cotransporter type 1, sulfate anion transporter 1, sodium-dependent vitamin C transporter 1, the phase II enzymes Uridine 5'-diphospho (UDP)-glucuronosyltransferase (Ugt) 2a3, Ugt2b1, Ugt3a1, Ugt3a2, sulfotransferase (Sult) 1a1, Sult1b1, Sult5a1, the efflux transporters multidrug resistance-associated protein (Mrp) 6, and multidrug and toxin extrusion 1; (2) moderately lower mRNAs encoding Oatp1b2, organic cation transporter (Oct) 1, Ugt1a5, Ugt1a9, glutathione S-transferase (Gst) m4, Gstm6, and breast cancer resistance protein; but (3) higher mRNAs encoding Oatp1a4, Octn2, Ugt1a1, Sult1e1, Sult2a2, Gsta4, Gstm1-m3, multidrug resistance protein (Mdr) 1a, Mrp3, and Mrp4. Hepatic signaling of nuclear factor E2-related factor 2 and pregnane X receptor appear to be activated in HNF4a-HNull mice. In conclusion, HNF4a deficiency markedly alters hepatic mRNA expression of a large number of phase II enzymes and transporters, probably because of the loss of HNF4a, which is a transactivator and a determinant of gender-specific expression and/or adaptive activation of signaling pathways important in hepatic regulation of these phase II enzymes and transporters.

FIG. 1.

Hepatic mRNAs encoding uptake transporters in adult male mice with liver-specific deletion (HNull) of HNF4a. The y-axis represents relative mRNA expression with values of WT controls set at 1.0. Data without error bar represent transporter mRNAs in pooled total RNA from livers of HNF4a-HNull and WT control mice (N = 5–6) determined by the Quantigene Plex assay. Data with error bar represent transporter mRNAs in individual samples of total RNA from livers of HNF4a-HNull and WT control mice (N = 5–6) determined by the bDNA assay. Mean ± SE. *p < 0.05 compared with WT control.

FIG. 2.

Hepatic mRNAs encoding Ugts in adult male mice with liver-specific deletion (HNull) of HNF4a. Ugt mRNAs in total RNA from livers of HNF4a-HNull and WT control mice (N = 5–6) were determined by the Quantigene Plex assay. The y-axis represents relative mRNA expression of Ugts with values of WT controls set at 1.0. Mean ± SE. *p < 0.05 compared with WT control.

FIG. 3.

Hepatic mRNAs encoding Sults in adult male mice with liver-specific deletion (HNull) of HNF4a. Sult mRNAs in total RNA from livers of HNF4a-HNull and WT control mice (N = 5–6) were determined by the Quantigene Plex assay. The y-axis represents relative mRNA expression of Sults with values of WT controls set at 1.0. Mean ± SE. *p < 0.05 compared with WT control.

FIG. 4.

Hepatic mRNAs encoding Gsts in adult male mice with liver-specific deletion (HNull) of HNF4a. Gst mRNAs in total RNA from livers of HNF4a-HNull and WT control mice (N = 5–6) were determined by the Quantigene Plex assay. The y-axis represents relative mRNA expression of Gsts with values of WT controls set at 1.0. Mean ± SE. *p < 0.05 compared with WT control.

FIG. 5.

Hepatic mRNAs encoding efflux transporters in adult male mice with liver-specific deletion (HNull) of HNF4a. The y-axis represents relative mRNA expression with values of WT controls set at 1.0. Data without error bar represent transporter mRNAs in pooled total RNA from livers of HNF4a-HNull and WT control mice (N = 5–6) determined by the Quantigene Plex assay. Data with error bar represent transporter mRNAs in individual samples of total RNA from livers of HNF4a-HNull and WT control mice (N = 5–6) determined by the bDNA assay. Mean ± SE. *p < 0.05 compared with WT control.

FIG. 6.

Renal mRNAs encoding transporters in adult male and female mice with liver-specific deletion (HNull) of HNF4a. The y-axis represents relative mRNA expression with values of male WT controls set at 1.0. Data with error bar represent transporter mRNAs in individual samples of total RNA, whereas data without error bar represent transporter mRNAs in pooled total RNA, from kidneys of HNF4a-HNull and WT control mice (N = 5–6) determined by the bDNA assay. Mean ± SE. *p < 0.05 compared with WT male mice; ^†p < 0.05 compared with WT female mice.

FIG. 7.

(A–B) Hepatic mRNA expression of nuclear receptors/TFs important in the regulation of drug-processing genes in adult male and/or female mice with liver-specific deletion (HNull) of HNF4a. The y-axis represents relative mRNA expression with values of male WT controls set at 1.0. Data with error bar represent mRNAs in individual samples of total RNA, whereas data without error bar represent mRNAs in pooled total RNA, from livers of HNF4a-HNull and WT control mice (N = 5–6) determined by the bDNA assay. (C) Binding of TFs to their consensus cis-response elements. Nuclear extracts from livers of male HNF4a-Hnull and WT mice (N = 5) were used to determine in vitro DNA binding of TFs with Procarta TF binding assays. Mean ± SE. *p < 0.05 compared with WT control.

FIG. 8.

Thematic model illustrating the hypothesized direct and indirect roles of HNF4a in regulating hepatic expression of phase II enzymes and transporters in mice. BA, bile acids; FA, fatty acids; HNF4-RE, HNF4a response element; ROS, reactive oxygen species.