Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression

Abstract

Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ-binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism.

Keywords: GSK5182; bile acid; cannabinoid receptors; cholesterol 7α-hydroxylase (CYP7A1); oestrogen-related receptor γ (ERRγ); orphan nuclear receptor; small heterodimer partner (SHP).

Figure 1. 2-AGE induces ERRγ and CYP7A1 gene expression

(A and B) AML12 cells were treated with 2-AGE (10 µM) for the indicated time period. (C and D) Rat primary hepatocytes were treated with 2-AGE (10 µM) for 12 h. ***P < 0.001. (E and F) HepG2 cells were treated with 2-AGE (10 µM) for 12 h. ***P < 0.001. ERRγ and CYP7A1 expression in (A–F) were analysed by qPCR and Western blot analysis. (G) Mouse primary hepatocytes isolated from WT and CB1^−/− mice were treated with 2-AGE (10 µM) for 12 h. CYP7A1, ERRγ and CB1R gene expression were measured by qPCR analysis and normalized to actin expression. ***P < 0.001. All data are representative of at least three independent experiments. Error bars show ± S.E.M.

Figure 2. ERRγ is involved in 2-AGE-mediated induction of CYP7A1 gene expression

(A and B) AML12 cells were infected with adenovirus US (Ad-US) or adenovirus sh-ERRγ (Ad-shERRγ) for 48 h followed by treatment with 2-AGE (10 µM). Cell culture media were collected to determine bile acid levels (A, right panel). ***P < 0.001. (C and D) AML12 cells were infected with Ad-GFP (control), Ad-ERRα and Ad-ERRγ . ***P < 0.001. (E and F) HepG2 cells were infected with Ad-GFP (control), Ad-ERRα and Ad-ERRγ. Total RNA and protein in (A–F) were isolated and used for qPCR and Western blot analysis. ***P < 0.001. All data are representative of at least three independent experiments. Error bars show ± S.E.M.

Figure 3. ERRγ activates the CYP7A1 gene promoter

(A) 293T cells were transfected with hCYP7A1-luc (−1887 bp) and ERRγ expression vector. (B) 293T cells were transfected with mCYP7A1-luc (−3.2 kb to +234 bp) and different doses of the ERR subfamily cDNA expression vectors: 1:100 ng, 2:200 ng, 4:400 ng. (C) AML12 cells were infected with Ad-US or Ad-shERRγ for 48 h then transfected with mCYP7A1-luc (−3.2 kb to +234 bp) and treated with 2-AGE (10 µM). (D) 293T cells were transfected with deletion constructs of mCYP7A1-luc and ERRγ . (E) An ERRγ -binding site point mutation was made in mCYP7A1 (−1.5 kb)-luc as described in the ‘Materials and Methods’ section. 293T cells were transiently transfected with pCDNA3 – FLAG–ERRγ, mCYP7A1 (−1.5 kb to +234 bp)-luc (WT), mCYP7A1–mtERRE2-Luc, mCYP7A1–mtERRE1-Luc and mCYP7A1–mtERRE1 and 2-Luc. The cell lysates in (A–E) were utilized for luciferase and β-galactosidase assays. Experiments in (A–E) were conducted in triplicate and data are expressed as fold activation relative to the control. (F) ChIP assay. AML12 cells were infected with Ad-GFP or Ad-ERRγ for 48 h. Input represents 10% of purified DNA in each sample. Cell extracts were immunoprecipitated with anti-ERRγ antibody and purified DNA samples were employed for PCR with primers binding to ERRE1 (−1.4 kb to −1.2 kb) and distal site (−2.3 kb to −2.1 kb) on the CYP7A1 gene promoter.

Figure 4. ERRγ-mediated induction of CYP7A1 gene expression is inhibited by SHP

(A) AML12 cells were transfected with mCYP7A1-luc (−3.2 kb to +234 bp). At 36 h post transfection, cells were treated with 2-AGE (10 µM) in the presence or absence of CDCA (25 µM). (B) AML12 cells were treated with 2-AGE (10 µM) in the presence or absence of CDCA (25 µM) and CYP7A1 expression was analysed by qPCR. **P < 0.01; ***P < 0.001. (C) ChIP assay. AML12 cells were treated with 2-AGE (10 µM) in the presence or absence of CDCA (25 µM). Input represents 10% of purified DNA in each sample. Cell extracts were immunoprecipitated with anti-ERRγ antibody and purified DNA samples were employed for PCR with primers binding to ERRE1 (−1.4 kb to −1.2 kb) and distal site (−2.3 kb to −2.1 kb) on the CYP7A1 gene promoter. (D) AML12 cells were transfected with mCYP7A1-luc (−3.2 kb to +234 bp) and co-transfected with ERRγ or SHP expression vectors. (E) AML12 cells were infected with indicated adenoviruses and CYP7A1 expression was analysed by qPCR analysis. (F and G) AML12 cells were transfected with the si-SHP expression vector and after 24 h, the cells were co-transfected with mCYP7A1-luc (−3.2 kb to +234 bp) and ERRγ expression vector in the presence or absence of CDCA (25 µM). SHP mRNA level analysed by qPCR analysis. (H) AML12 cells were infected with Ad-ERRγ and Ad-shSHP in the presence or absence of CDCA (25 µM). CYP7A1 expression was analysed by qPCR analysis. *P < 0.05; ***P < 0.001. The cell lysates in (A, D and G) were utilized for luciferase and β-galactosidase assays.

Figure 5. GSK5182 inhibits CYP7A1 gene expression and bile acid synthesis

(A) AML12 cells were transfected with mCYP7A1-luc (−3.2 kb to +234 bp) then treated with 2-AGE (10 µM) and GSK5182 (10 µM). Cell lysates were utilized for luciferase and β-galactosidase assays. (B and C) AML12 cells were treated with 2-AGE (10 µM) for 12 h. Then, the cell culture medium was changed and GSK5182 (10 µM) was added for the final 24 h. Total mRNAs and protein were extracted for qPCR (B) and Western blot analysis (C). ***P < 0.001. (D–F) AML12 cells were infected with Ad-GFP and Ad-ERRγ and then treated with GSK5182 (10 µM) for 24 h. CYP7A1 mRNA level was analysed by qPCR (D) and the ERRγ and CYP7A1 protein levels were measured by Western blot analysis (E), ***P < 0.001. Cell culture media were collected to determine bile acid levels (F), ***P < 0.001.

Figure 6. Hepatic CB1 receptor signalling regulates CYP7A1 gene expression via induction of ERRγ in mice

(A) Ad-GFP or Ad-ERRγ were injected via the tail-vein into male C57BL/6J mice (n = 3–4 per group) and mice were killed at day 3. qPCR analysis and Western blot analysis were performed to measure the mRNA and protein levels in liver. ***P < 0.001. (B–D) Mice (n = 3–4) were treated with alcohol for 1 day in the presence of GSK5182. Total RNA was extracted for qPCR analyses (B). Liver tissue (C) and serum (D) were used for bile acid analysis; **P < 0.01; ***P < 0.001. (E) Proposed model for CB1 receptor-mediated induction of CYP7A1 gene expression via ERRγ. Activation of hepatic CB1 receptor increases ERRγ gene expression, which in turn increases the promoter activity and mRNA level of CYP7A1. Induction of CYP7A1 gene expression promotes bile acid synthesis and induces SHP expression. SHP inhibits ERRγ -mediated induction of CYP7A1 gene expression and promoter activity. GSK5182, an ERRγ inverse agonist, inhibits ERRγ -mediated CYP7A1 gene expression and bile acid (BA) synthesis.