Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7alpha-hydroxylase gene expression

Abstract

Mouse fibroblast growth factor 15 (FGF15) and human ortholog FGF19 have been identified as the bile acid-induced intestinal factors that mediate bile acid feedback inhibition of cholesterol 7alpha-hydroxylase gene (C YP7A1) transcription in mouse liver. The mechanism underlying FGF15/FGF19 inhibition of bile acid synthesis in hepatocytes remains unclear. Chenodeoxycholic acid (CDCA) and the farnesoid X receptor (FXR)-specific agonist GW4064 strongly induced FGF19 but inhibited CYP7A1 messenger RNA (mRNA) levels in primary human hepatocytes. FGF19 strongly and rapidly repressed CYP7A1 but not small heterodimer partner (SHP) mRNA levels. Kinase inhibition and phosphorylation assays revealed that the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) pathway played a major role in mediating FGF19 inhibition of CYP7A1. However, small interfering RNA (siRNA) knockdown of SHP did not affect FGF19 inhibition of CYP7A1. Interestingly, CDCA stimulated tyrosine phosphorylation of the FGF receptor 4 (FGFR4) in hepatocytes. FGF19 antibody and siRNA specific to FGFR4 abrogated GW4064 inhibition of CYP7A1. These results suggest that bile acid-activated FXR is able to induce FGF19 in hepatocytes to inhibit CYP7A1 by an autocrine/paracrine mechanism.

Conclusion: The hepatic FGF19/FGFR4/Erk1/2 pathway may inhibit CYP7A1 independent of SHP. In addition to inducing FGF19 in the intestine, bile acids in hepatocytes may activate the liver FGF19/FGFR4 signaling pathway to inhibit bile acid synthesis and prevent accumulation of toxic bile acid in human livers.

Fig. 1

CDCA and GW4064 increased FGF19 and repressed CYP7A1 mRNA expression in primary human hepatocytes. Primary human hepatocytes were treated with CDCA (50 μM) or GW4064 (1 μM) for a period of time indicated. Total RNA was isolated for real-time quantitative PCR analysis of relative mRNA expression of FGF19 (A), CYP7A1 (B), SHP (C) and HNF4α (D) in hepatocytes. Data represent the mean ± SD of at least three different donor human primary hepatocytes.

Fig. 2

CDCA induced FGF19 protein expression and secretion in primary human hepatocytes. (A) Western Blot analysis of FGF19 protein expression in primary human hepatocytes by CDCA. Primary human hepatocytes were treated with CDCA (50 μM) for a period of time indicated and whole cell extracts (50 μg/lane) were then analyzed by immunoblotting with FGF19 and actin antibodies. Data are representative of two independently performed experiments. An asterisk indicates non-specific binding. (B) Effects of CDCA on FGF19 secretion to culture media. Primary human hepatocytes were treated with CDCA for a period of time indicated and collected for analysis by ELISA assay. (C) CDCA dose-responses on FGF19 secretion. Primary human hepatocytes were treated with CDCA for 24 h in the concentrations indicated. FGF19 expression was determined by ELISA assay.

Fig. 3

FGF19 repressed CYP7A1 mRNA expression in primary human hepatocytes. Primary human hepatocytes were treated with FGF19 (40 ng/ml) for a period of time or treated for 24 h with different concentrations of FGF19. Total RNAs were isolated for real-time quantitative PCR analysis of relative mRNA expression levels of CYP7A1 (A and B) and SHP (C and D). Data represent the mean ± SD of at least three donors primary human hepatocytes.

Fig. 4

siRNA knockdown of SHP did not abolish FGF19 repression of CYP7A1 expression. (A) The effects of siRNA-SHP on the SHP protein expression were measured by Western blot analysis Actin expression was used as a loading control. Data represent one of three separate experiments. (B) HepG2 cells were transfected with the SMART Pool of siRNA-SHP (200 pmol) and control siRNA (cyclophilin B, 200 pmol) and treated with or without FGF19 (40 ng/ml) for 6 h. Total RNA was isolated for Q-PCR analysis of SHP ( A B) and CYP7A1 (C) mRNA levels. Data show relative mRNA expression of siRNA-SHP (200 pmol) treated to the control siRNA (200 pmol) treated samples. Data represent the mean ± SD of at least three individual experiments.

Fig. 5

CDCA, FGF19, and MAP kinase inhibitors stimulate CYP7A1 mRNA expression and activate MAP kinases in primary human hepatocytes. (A) Primary human hepatocytes were treated with specific MAP kinase inhibitors: ERK1/2:U0126 (10 μM) and/or JNK: SP600125 (SP, 25 μM) for 1 h, and then with FGF19 (40 ng/ml) for 6 h. Total RNAs were isolated for real-time quantitative PCR analysis of relative mRNA expression levels of CYP7A1 in primary human hepatocytes. Data represent the mean ± SD of at least three donor hepatocytes independently performed. (B) Primary human hepatocytes were treated with FGF19 (40 ng/ml) for a period of time indicated and whole cell extracts (50 μg/lane) were then analyzed by immunoblotting with antibodies against ERK1/2, phospho-ERK1/2, JNK, phospho-JNK, p38, or phospho-p38 as indicated. (C) Primary human hepatocytes were treated with specific MAP kinase inhibitors: ERK1/2:U0126 (U, 10 μM), PD98059 (PD, 25 μM) and/or JNK: SP600125 (SP, 25 μM) for 1 h, and then with FGF19 (40 ng/ml) for 30 min. Whole cell extracts (50 μg/lane) were then analyzed by immunoblotting with ERK1/2 or phospho-ERK1/2 antibodies. Data are representative of two donor hepatocytes independently performed. (D) Primary human hepatocytes were treated with CDCA (50 μM) for a period of time indicated. Cell lysates were isolated for western blots using antibodies against ERK or phospho-ERK. (E) Primary human hepatocytes were treated with CDCA (50 μM) for a period of time indicated and cell extracts were immunoprecipitated with anti-FGFR4 antibody. Western blot analysis was performed using anti-phosphor-tyrosine antibody. Western blot analysis for FGFR4 using goat anti-FGFR4 antibody was performed to confirm the equal amounts of FGFR4 precipitant.

Fig. 6

Inhibition of FGF19 and FGFR4 activity abrogates inhibition of CYP7A1 by FXR agonist GW4064 in human hepatocytes. (A) Primary human hepatocytes were treated with anti-FGF19 antibody (closed bar) with or without GW4064. Total RNAs were isolated for Q-PCR analysis of relative CYP7A1 mRNA expression levels in hepatocytes. (B) Western blot analysis of the effect of siRNA-FGFR4 on the FGFR4 protein expression. Actin expression was used as a loading control. Data represent one of three separate experiments. (C) HepG2 cells were transfected with the SMART Pool of siRNA to FGFR4 (siFGFR4, 200 pmol, closed bar) or control siRNA (siCon, 200 pmol, open bar) and treated with GW4064 for 24 h. Total RNAs were isolated for Q-PCR analysis of relative mRNA expression levels of FGFR4. (D) mRNAs isolated in (C) were used to assay relative mRNA expression levels of CYP7A1.