Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4alpha - PubMed (original) (raw)

Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4alpha

Yusuke Inoue et al. J Lipid Res. 2006 Jan.

Abstract

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

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Figures

Fig. 1.

BA homeostasis in HNF4αΔL and HNF4αF/F mice. (A) Serum total BA levels in 14, 28, and 45-day-old mice were measured. (B) Total volume of biofluid in gallbladder. (C) Total BA amount in gallbladder. (D) Total BA pool. (E) Fecal and (F) Urinary BA excretion rate. (B-D) All mice were 45-days-old. Data are mean ± S.E. (HNF4αF/F mice [_n_=4-6], HNF4αΔL mice, [_n_=4-8]). Significant differences compared to HNF4αF/F mice: *, p< 0.05; **, p<0.01; ***, p<0.001.

Fig. 2.

Expression of the genes involved in BA biosynthesis pathways. (A) Northern blot analysis of liver RNA from 14, 28, and 45-day-old mice killed at 10 am. Real-time PCR of liver mRNA from 45-day-old mice killed at 10 am for CYP27A1 (B), CYP39A1 (C), ACOX2 (D), and D-PBE (E). Data are mean ± S.E. (_n_=8 for each group). Significant difference compared to HNF4αF/F mice: *, p< 0.001.

Fig. 2.

Fig. 3.

Circadian rhythm of expression of CYP7A1 and CYP8B1 mRNAs in the livers of HNF4αΔL and HNF4αF/F mice. Northern blot analysis of mRNA in livers from 45-day-old mice at 10 am (A) and 10 pm (B). (C) Real-time PCR of liver mRNA for CYP7A1 at 10 am (left) and 10 pm (right). Data are mean ± S.E. (_n_=8 for each group). Significant differences compared to the other three groups: *, p< 0.01. Western blots of total liver proteins (100 μg) at 10 am (D) and 10 pm (E). CYP7A1 and β-actin polyclonal antibodies were used to assess protein expression.

Fig. 4.

Composition of BA in HNF4αΔL and HNF4αF/F mice. Amounts of individual BAs from 45-day-old mice were analyzed by GC/MS and are indicated as a percentage of the entire pool. The position and stereochemistry of hydroxyl groups on the ring structure of each BA are indicated in parentheses.

Fig. 5.

Specific activities and expression of CYP8B1 in HNF4αΔL and HNF4αF/F mice. (A) Specific activities of CYP8B1 were measured using liver microsomal protein from HNF4αΔL and HNF4αF/F mice. Data are mean ± S.E. (_n_=5). Significant differences between HNF4αΔL and HNF4αF/F mice: *, p< 0.001; **, p<0.005, respectively. (B) Specific activities of CYP8B1 were measured using liver microsomal protein from _Cyp8b1_-null and wild-type mice. Data are mean ± S.E. (_n_=2).

Fig. 6.

Promoter analysis of the mouse Cyp8b1 gene. (A) Luciferase reporter plasmids containing the mouse Cyp8b1 promoter were transfected into HepG2 cells. The normalized activity ± S.E. (_n_=4) of each construct is presented as arbitrary units. (B) CV-1 cells were co-transfected with the HNF4α expression vector, as indicated. The normalized activity ± S.E. (_n_=4) of each construct is presented as arbitrary units. (C) Nuclear extracts from liver of HNF4αF/F mice (left panel) and HNF4αΔL mice (right panel) mice were incubated with labeled HNF4α binding site of the mouse Cyp8b1 promoter in the absence (lanes 1 and 8) or presence of unlabeled each oligonucleotide for the HNF4α binding site of the mouse Cyp8b1 promoter (lanes 2 and 9), consensus HNF4α binding site (lanes 3 and 10), and consensus PPARα binding site (PPRE, lanes 4 and 11). For the supershift assay, nuclear extracts were incubated with labeledoligonucleotide probe in the presence of the anti-HNF4α (lanes 5 and 12), anti-PPARα (lanes 6 and 13), and anti-RXRα antibodies (lanes 7 and 14). The HNF4α-DNA complex and its supershifted complex, caused by the HNF4α-specific antibody, are indicated by the lower and upper arrows, respectively.

Fig. 7.

Effects of mutations of the HNF4α binding site in the mouse Cyp8b1 promoter. (A) Schematic representation of the wild-type (WT) and mutated (Mut) HNF4α binding site of the mouse Cyp8b1 promoter. Mutations in the HNF4α binding site are represented in bold type. Plasmids were transfected into HepG2 (B) and CV-1 (C) cells and the normalized activity ± S.E. (_n_=4) of each construct is presented as relative activity.

TABLE 1

Analysis of free bile acids in serum HNF4αΔL and HNF4αF/F mice.

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Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4alpha - PubMed (original) (raw)