Lithocholic acid induction of the FGF19 promoter in intestinal cells is mediated by PXR

Abstract

Aim: To study the effect of the toxic secondary bile acid lithocholic acid (LCA) on the expression of fibroblast growth factor 19 (FGF19) in intestinal cells and to characterize the pregnane-X-receptor (PXR) response of the FGF19 promoter region.

Methods: The intestinal cell line LS174T was stimulated with various concentrations of chenodeoxy-cholic acid and lithocholic acid for several time points. FGF19 mRNA levels were determined with quantitative realtime RT-PCR. FGF19 deletion promoter constructs were generated and the LCA response was analzyed in reporter assays. Co-transfections with PXR and RXR were carried out to study FGF19 regulation by these factors.

Results: LCA and CDCA strongly up-regulate FGF19 mRNA expression in LS174T cells in a time and dose dependent manner. Using reporter gene assays with several deletion constructs we found that the LCA responsive element in the human FGF19 promoter maps to the proximal regulatory region containing two potential binding sites for PXR. Overexpression of PXR and its dimerization partner retinoid X receptor (RXR) and stimulation with LCA or the potent PXR ligand rifampicin leads to a significant induction of FGF19 promoter activity in intestinal cells.

Conclusion: LCA induced feedback inhibition of bile acid synthesis in the liver is likely to be regulated by PXR inducing intestinal FGF19 expression.

Figure 1

mRNA induction of FGF19 in LS174T cells by CDCA (A) and LCA (B) (mean ± SD, Student’s t-test). P < 0.05 between 3 h, 6 h and 24 h treatment groups and 0 h group in both A and B.

Figure 2

CYP3A4 and FGF19 promoter constructs and sequence alignment of the human and murine FGF19/Fgf15 regulatory regions. A: CYP3A4 and FGF19 promoter constructs used for transient transfection assays. The positions are calculated relative to the major transcription start site; B: Sequence comparison of homologous regions in the human FGF19 and murine Fgf15 promoters. The locations of the DR3 and ER6 elements identified in the human FGF19 promoter by NUBIScan analysis are indictated by bold letters.

Figure 3

Localization of the LCA/Rif/PXR responsive element in the human FGF19 promoter. A: Relative CYP3A4 and FGF19 promoter activities of LS174T cells treated with LCA (250 μmol/L) and Rif (10 μmol/L) compared to control treated cells for 24 h (P < 0.05 between LCA and Rif treatment groups and control treated groups); B: Reporter constructs were cotransfected with PXR/RXR or mock plasmids and stimulated with LCA (250 μmol/L) and Rif (10 μmol/L) as indicated for 24 h. Mock transfected and solely LCA/Rif stimulated cells were used for normalization and calculation of relative promoter induction (mean ± SD, Student’s t-test). The dotted line indicates the threshold for significant promoter induction. P < 0.05 between PXR/RXR cotransfected and LCA/Rif treated groups and mock transfected and LCA/Rif treated groups. The used reporter constructs are indicated on the x-axis.