Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver

Abstract

Here, we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a-knockout (Cyp3a(-/-)) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a(-/-) mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element-binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a(-/-) mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a(-/-) mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a(-/-) mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7α-hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a(-/-) mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a(-/-) mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo.

Keywords: 25-hydroxy-cholesterol; HMG-CoA synthase 1; cytochrome P450 3A; squalene epoxidase; sterol regulatory element-binding protein-2.

Fig. 1.

Expression levels of HMGCS1 (A), HMGCR (B), and SQLE (C) mRNAs in the livers of WT and Cyp3a^−/− (KO) mice. Expression levels were normalized by expression levels of GAPDH and are shown as means ± SEM of six mice in each group. cDNAs prepared from each mouse were used for triplicate determination. **P < 0.01 versus WT mice.

Fig. 2.

Expression levels of SREBP-2 in the livers of WT and KO mice. A: Expression levels of SREBF2 mRNAs were normalized by expression levels of GAPDH and are shown as means ± SEM of six mice in each group. cDNAs prepared from each mouse were used for triplicate determination. ***P < 0.001 versus WT mice. B: SREBP-2 protein was assessed using Western blotting of isolated hepatic nuclear protein. TBP was used as a loading control.

Fig. 3.

SREBP-2 binding to Hmgcs1 promoter (−348 to −208 bp) in the livers of WT and KO mice. ChIP assay was performed using anti-SREBP-2 antibody. Liver tissues from two mice in each group were pooled and subjected to the assay. The immunoprecipitated DNA, along with the DNA isolated before immunoprecipitation (Input), were analyzed by semi-quantitative PCR (A) and quantitative PCR (B). Signals were normalized to input DNA. Data are means ± SD of three independent determinations, each performed in duplicate. **P < 0.01 versus WT mice.

Fig. 4.

Expression levels of ABCG5, ABCG8, ABCA1, LDLR, and NPC1L1 mRNAs in the livers (A) and intestines (B) of WT and KO mice. Expression levels were normalized by expression levels of GAPDH and are shown as means ± SEM of six mice in each group. cDNAs prepared from each mouse were used for triplicate determination. *P < 0.05 versus WT mice.

Fig. 5.

Expression levels of CYP7A1 (A), CYP8B1 (B), CYP27A1 (C), CYP7B1 (D), and SHP (E) mRNAs in the livers of WT and KO mice. Expression levels were normalized by expression levels of GAPDH and are shown as means ± SEM of six mice in each group. cDNAs prepared from each mouse were used for triplicate determination. **P < 0.01 and ***P < 0.001 versus WT mice.

Fig. 6.

Concentration of C4 in plasma (A), hepatic bile acid (B), and bile acid pool size (C) of WT and KO mice. Plasma C4 concentrations are expressed as a mean ± SD of triplicate determinations, using pooled plasma from six mice. Hepatic bile acid levels were expressed as means ± SEM of three mice in each group. Bile acid pool size is determined as total bile acid from liver, gallbladder, and intestine. The values of bile acid pool size were shown as means ± SEM of three mice in each group. *P < 0.05 versus WT mice.