Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP)

Abstract

Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids.

Fig. 1.

Relationship between small heterodimer partner (SHP) and secretin gene expression in human duodenal HuTu-80 cells. A: effects of overexpressing SHP on secretin gene promoter. p341 (2.0 μg), and various amounts of SHP/pcDNA3.1 (0, 0.5, 1.0, and 2.0 μg) were cotransfected into HuTu-80 cells. B: real-time quantitative PCR to show the relative mRNA levels of secretin and S14 in HuTu-80 cells transiently transfected with the indicated amount of SHP vector. C: effects of silencing endogenous SHP on secretin promoter activity. p341 (2.0 μg) was cotransfected with various amounts of siSHP-1 and siSHP-2 (1.0 and 2.0 μg), pSilencer, and siControl. D: Top: Western blot analysis of SHP protein in HuTu-80 cells transfected with pSilencer (1), siSHP-1 (2), siSHP-2 (3), and siControl (4). Bottom: Coomassie blue staining of a SDS-PAGE ran in parallel as a loading control; *P < 0.001; **P < 0.05 vs. p341 2.0 μg.

Fig. 2.

Functions of SHP and NeuroD on secretin promoter. A: coimmunoprecipitation demonstrating the physical interaction between SHP and NeuroD in HuTu-80 cells. SHP in the cell lysate immunoprecipitated with NeuroD antibody (NeuroD-IP) was detected by Western Blot. A mock immunoprecipitation using no antibody was conducted as a negative control. B: coexpression of SHP, NeuroD, and E47 in HuTu-80 cells. Different combinations of SHP/pcDNA3.1, NeuroD/pCMV, and E47/pCMX were cotransfected with 2.0 μg of p341 into HuTu-80 cells. *P < 0.001 vs. controls as indicated by the bars.

Fig. 3.

Studies of SHP function on mouse secretin promoter. A: effects of SHP overexpression on mouse secretin promoter (MSCTP). Various amount of SHP/pcDNA3.1 (0, 0.5, 1.0, and 2.0 μg) were cotransfected with 2.0 μg MSCTP into HuTu-80 cells. B: mouse secretin promoter activity under shRNA silencing of endogenous SHP. 2.0 μg siSHP-1 or siSHP-2 was cotransfected with 2.0 μg MSCTP. C: schematic representation of human and mouse secretin promoters. The boxes represent the NeuroD-binding E-box and both identified and potential GC-boxes as indicated in the diagram. The indicated positions of the boxes are relative to the deduced transcription start site. *P < 0.001; **P < 0.05 vs. MSCTP 2.0 μg.

Fig. 4.

Effects of chenodeoxycholic acid (CDCA) and fexaramine on secretin gene expression in HuTu-80 cells. A: Western blot analysis demonstrating the presence of farnesoid X receptor (FXR) protein in HuTu-80 cells. SDS PAGE resolved 100 μg of the protein extracts from normal cell (1) and cell transient transfected with FXR as a positive control (2). B: real-time quantitative PCR to show relative transcript levels of SHP in HuTu-80 cells with 24 h of CDCA or fexaramine treatment. C: chromatin immunoprecipitation assay showing the relative occupancy of the immunoprecipitated SHP and NeuroD at the human secretin promoter. Data were calculated using the equation: 2^(Ct^mock − Ct^specific) and normalized with Input, which was defined as 1.00, where Ct represents fractional cycle number at which the fluorescence signal reaches 10-fold standard deviation of the baseline. A mock immunoprecipitation without using antibody was carried out as negative control. D: secretin relative transcript level. E: luciferase-promoter activity after 24-h treatment of CDCA or fexaramine. F: shRNA silencing endogenous SHP with 2.0 μg siSHP-1 followed by 24-h treatment of 200 μM CDCA or 50 μM fexaramine; *P < 0.001; **P < 0.05 vs. no treatment control.

Fig. 5.

Double fluorescence immunohistochemistry of secretin and SHP in mouse duodenum. Secretin was detected by Alexa Fluor 594 green fluorescent secondary antibody, whereas SHP was stained by Alexa Fluor 488 red fluorescent secondary antibody. The merged image shows the colocalization of secretin and SHP, indicated by the arrow. In the control, PBS was used instead of the primary antibodies. Scale bar = 20 μm.

Fig. 6.

In vivo effects of bile acids on SHP and secretin gene expression in mouse duodenum. Mice were fasted for 24 h and then fed ad libitum with control or CA-enriched (1%) diet for 1 day. Duodenal transcript levels of SHP (A) and secretin (B) were measured by real-time quantitative PCR assay. The secretin (C) protein levels in the duodenum were assessed by enzyme immunoassay. An independent group of mice were treated with control or cholestyramine (CY)-enriched (5%) diet. After the 10-day treatment, mice were euthanized for the measurement of SHP transcript level (D), secretin transcript (E), and protein levels (F); **P < 0.05 vs. control.