Liver X receptor β regulates bile volume and the expression of aquaporins and cystic fibrosis transmembrane conductance regulator in the gallbladder

Abstract

The gallbladder is considered an important organ in maintaining digestive and metabolic homeostasis. Given that therapeutic options for gallbladder diseases are often limited to cholecystectomy, understanding gallbladder pathophysiology is essential in developing novel therapeutic strategies. Since liver X receptor β (LXRβ), an oxysterol-activated transcription factor, is strongly expressed in gallbladder cholangiocytes, the aim was to investigate LXRβ physiological function in the gallbladder. Thus, we studied the gallbladders of WT and LXRβ^-/- male mice using immunohistochemistry, electron microscopy, qRT-PCR, bile duct cannulation, bile and blood biochemistry, and duodenal pH measurements. LXRβ^-/- mice presented a large gallbladder bile volume with high duodenal mRNA levels of the vasoactive intestinal polypeptide (VIP), a strong mediator of gallbladder relaxation. LXRβ^-/- gallbladders showed low mRNA and protein expression of Aquaporin-1, Aquaporin-8, and cystic fibrosis transmembrane conductance regulator (CFTR). A cystic fibrosis-resembling phenotype was evident in the liver showing high serum cholestatic markers and the presence of reactive cholangiocytes. For LXRβ being a transcription factor, we identified eight putative binding sites of LXR on the promoter and enhancer of the Cftr gene, suggesting Cftr as a novel LXRβ regulated gene. In conclusion, LXRβ was recognized as a regulator of gallbladder bile volume through multiple mechanisms involving CFTR and aquaporins.NEW & NOTEWORTHY This report reveals a novel and specific role of the nuclear receptor liver X receptor β (LXRβ) in controlling biliary tree pathophysiology. LXRβ^-/- mice have high gallbladder bile volume and are affected by a cholangiopathy that resembles cystic fibrosis. We found LXRβ to regulate the expression of both aquaporins water channels and the cystic fibrosis transmembrane conductance regulator. This opens a new field in biliary tree pathophysiology, enlightening a possible transcription factor controlling CFTR expression.

Keywords: CFTR; aquaporins; cholangiocytes; gallbladder; nuclear receptors.

Graphical abstract

Figure 1.

High gallbladder bile volume and reduced AQP1, AQP8 expression in LXRβ^−/− male gallbladders from fasted mice. A: gross gallbladder pictures show an increased volume in LXRβ^−/− mice, confirmed by a significant (*P < 0.05) difference in bile volume measurements. LXRα^−/− gallbladder bile volume did not differ from the one of wild-type (WT) mice. Data are expressed as means ± SE; n = 5 for each mouse strain. B: shows qRT-PCR results. Relative mRNA expression of AQP1 was significantly (*P < 0.05) reduced in LXRβ^−/− mice (n = 5) compared with WT littermates (n = 5). Data are expressed as means ± SD. C: immunohistochemistry demonstrates a reduced immunoreactivity of AQP1 in male LXRβ^−/− gallbladders (n = 5) compared with WT (n = 5) where AQP1 is expressed on the luminal and basolateral plasma membranes as well as in intracellular subapical vesicles. No differences are detectable between LXRα^−/− (n = 5) and WT gallbladders. Scale bar 50 μm. D: quantification of immunogold electron microscopy labeling for AQP1 shows a reduced labeling in LXRβ^−/− apical membrane of cholangiocytes. The immunogold particles were counted in 15 randomly taken images and a significant difference (*P < 0.05) was evident between WT (6.0 ± 1.7) and LXRβ^−/− (0.8 ± 0.7) mice. Data are expressed as means ± SD. E: shows qRT-PCR results. Relative mRNA expression of AQP8 was significantly (*P < 0.05) reduced in LXRβ^−/− mice (n = 5) compared with WT littermates (n = 5). Data are expressed as means ± SD. F: AQP8 immunoreactivity is evident on the plasma membrane of WT and LXRα^−/− gallbladder cholangiocytes and in intracellular vesicles. In LXRβ^−/− gallbladders, the staining appears reduced in both compartments. No differences are detectable between LXRα^−/− (n = 5) and WT gallbladders. Scale bar 25 μm. n, number of mice.

Figure 2.

Preserved response to feeding and high duodenal vasoactive intestinal polypeptide (VIP) levels in LXRβ^−/− male mice. A: hepatic bile flow measured over a time of 30 min. No significant differences were detected in LXRβ^−/− mice (n = 8) compared with wild type (WT) (n = 8). Data are expressed as means ± SE. B: shows gallbladder bile volume in LXRβ^−/− mice 12 h fasted (n = 5) and fed (n = 5). A significant reduction in the volume is seen in fed mice. *P < 0.05. Data are expressed as means ± SE. C: shows qRT-PCR results from male WT (n = 4) and LXRβ^−/− (n = 4) gallbladder, duodenum, and ileum. In the gallbladder, no differences in the expression of fibroblast growth factor receptor (FGFR)3, FGFR4, cholecystokinin-receptor (CCK-R1), VIP-R1 were detected between LXRβ^−/− and WT mice (top). VIP relative mRNA expression is higher in LXRβ^−/− duodenum compared with WT while the CCK expression did not differ (bottom left). In the ileum (bottom right), the relative mRNA expression of FGF-15 was not different in LXRβ^−/− mice compared with WT. *P < 0.05. Data are expressed as means ± SD; n, number of mice.

Figure 3.

Reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression in LXRβ^−/− male gallbladders and detection of liver X receptor elements (LXRE) in Cftr genomic locus. A: shows qRT-PCR results from male wild type (WT) (n = 4) and LXRβ^−/− (n = 4) gallbladders. Cftr relative mRNA expression is significantly reduced in LXRβ^−/− gallbladder compared with WT. *P < 0.05. Data are expressed as means ± SD. B: shows CFTR immunoreactivity in subapical vesicles of WT cholangiocytes. A similar pattern is detected in LXRβ^−/− gallbladders but with a much weaker immunoreactivity. Representative pictures from the body of the gallbladder are shown. Scale bar 25 μm. C: shows a schematic representation of the Cftr genomic locus, displaying the exon/intron structure of the gene and peaks of enrichment in H3K27Ac histone mark in a panel of seven cell lines of different origin and in A549 lung adenocarcinoma cell line. The genomic regions corresponding to the promoter and to putative enhancers are underlined and LXR binding sites in each region are marked by a green dot. Modified from Genome Browser (http://genome.ucsc.edu/).

Figure 4.

Cystic fibrosis (CF) resembling cholangiopathy in LXRβ^−/− male livers. A: CK-19 immunohistochemistry shows the presence of reactive, dysmorphic cholangiocytes in the livers from LXRβ^−/− male mice 14-mo old. Scale bar 50 μm. A, bottom: the number of CK-19-positive cells is significantly increased in LXRβ^−/− livers compared with wild type (WT) (*P < 0.01). Data are expressed as means ± SE. B: PCNA immunohistochemistry in WT and LXRβ^−/− male mice 14-mo old. Scale bar 50 μm. B, bottom: higher number of PCNA-positive nuclei are detected in LXRβ^−/− livers compared with WT (*P < 0.01). Data are expressed as means ± SE.

Figure 5.

Proposed model of impaired liver X receptor (LXR)β signaling. In LXRβ^−/− male gallbladders there is a downregulation of the water channels Aquaporins (AQP)-1, AQP-8 and the chloride-bicarbonate channel cystic fibrosis transmembrane conductance regulator (CFTR). At the same time, in the duodenum there are higher mRNA levels of vasoactive intestinal polypeptide (VIP), an important relaxing factor for the gallbladder, known to be upregulated also in CFTR^−/− animals. The additive effect of the described changes results in a markedly increased gallbladder bile volume in the presence of a normal bile flow from the liver and a preserved contraction signaling regulated by cholecystokinin (CCK).