1. Volume 136, Issue 4, Pages 1435-1443 (April 2009)
Bile Salts Control the Antimicrobial Peptide Cathelicidin Through Nuclear Receptors in the Human Biliary Epithelium 
Emilie D'Aldebert, Marie–Jeanne Biyeyeme Bi Mve, Martine Mergey, Dominique Wendum, Delphine Firrincieli, Audrey Coilly, Laura Fouassier, Christophe Corpechot, Raoul Poupon, Chantal Housset, Nicolas Chignard 
Gastroenterology 
Volume 136, Issue 4, Pages (April 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
Cathelicidin expression in the human biliary epithelium. Representative immunostaining of cathelicidin in the biliary epithelium (A) within normal human liver and (B) in the liver of a patient with suppurative cholangitis. Immunostaining is localized in biliary epithelial cells (arrows and higher magnification within insets in the lower left corners) and in infiltrating inflammatory cells (arrowheads). Inset in the upper right corner of panel B illustrates positive staining in the biliary tract lumen.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
1α,25(OH)2D3 (VD3) induces cathelicidin expression through VDR in biliary epithelial cells. (A) Representative immunostaining of VDR in the biliary epithelium within normal human liver. Immunostaining is intense in biliary epithelial cells and not different from background in hepatocytes. Arrowhead in the higher magnification inset in the lower left corner indicates VDR nuclear staining. (B) Biliary epithelial cells (Mz-ChA-1) were treated with VD3 (closed circles) or maintained in control conditions (open circles) for the indicated time and subjected to cathelicidin messenger RNA (mRNA) detection by reverse-transcription quantitative PCR. Data represent means ± SEM of 4 experiments performed in duplicate. Values at all time points were significantly higher in VD3-treated cells when compared with basal values (P < .05 by the Student t test for paired samples). (C) The cells were transfected either with scramble (sc) or VDR small interfering RNA (si) and subjected to detection of VDR mRNA by reverse-transcription quantitative PCR (data represent means ± SEM of 3 experiments performed in duplicate; *P < .05 vs scramble by the Student t test for paired samples). (D) Detection of VDR and β-actin by immunoblot–enhanced chemiluminescence analyses (representative gels of 3 different experiments are shown). (E) Cells transfected either with scramble (sc) or VDR small interfering RNA (si) were incubated with VD3 for 24 hours before cathelicidin expression was analyzed by reverse-transcription quantitative PCR (data represent means ± SEM of 3 experiments performed in duplicate; *P < .05 vs control; **P < .05 vs scramble by the Student t test for paired samples).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
Bile salts control VDR expression in biliary epithelial cells. Biliary epithelial cells were incubated with CDCA or UDCA (A) at the indicated concentration or (B) with 100 μmol/L for various time periods. Whole-cell protein extracts then were submitted to VDR and β-actin immunoblot–enhanced chemiluminescence analyses. Representative gels of 3 and 5 different experiments are shown, respectively. (C and D) Biliary epithelial cells were incubated for 2 hours with CDCA (100 μmol/L) or UDCA (100 μmol/L) with or without PD98059 (50 μmol/L). Whole-cell protein extracts then were submitted to (C) p-ERK 1/2 and ERK 1/2 immunoblot–enhanced chemiluminescence analyses or (D) VDR and β-actin immunoblot–enhanced chemiluminescence analyses. Representative gels of 3 different experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
Bile salts control VDR activity in biliary epithelial cells. (A) Biliary epithelial cells were incubated for 24 hours with CDCA (100 μmol/L), UDCA (100 μmol/L), or VD3 (0.1 μmol/L). Cytosolic (20 μg) and nuclear proteins (10 μg) then were submitted to VDR, tubulin, lamin A/C, and β-actin immunoblot–enhanced chemiluminescence analyses. Representative gels of 3 different experiments are shown. (B) Nuclear proteins from biliary epithelial cells incubated with CDCA (100 μmol/L), UDCA (100 μmol/L), or VD3 (0.1 μmol/L) were subjected to electrophoretic mobility shift assay using a biotin-labeled VDRE consensus sequence in the absence (−) or presence (+) of excess unlabeled VDRE consensus sequence. Representative gels of 3 different experiments are shown. (C) Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with CDCA (100 μmol/L) or UDCA (100 μmol/L). Data represent means ± SEM of 4 experiments. *P < .05 vs untreated cells. **P < .05 vs VD3 or bile salts alone by the Student t test for paired samples. (D) Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with CDCA (100 μmol/L) or UDCA (100 μmol/L) in the presence or absence of PD (50 μmol/L). Data represent means ± SEM of 4 experiments. *P < .05 vs UDCA alone by the Student t test for paired samples. (E) Nuclear proteins from biliary epithelial cells incubated with GW4064 (1 μmol/L) were subjected to electrophoretic mobility shift assay using a biotin-labeled VDRE consensus sequence in the absence (−) or presence (+) of excess unlabeled VDRE consensus sequence. Representative gels of 3 different experiments are shown. (F) Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with the FXR agonist, GW4064 (1 μmol/L). Data represent means ± SEM of 3 experiments. *P < .05 vs control by the Student t test for paired samples.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
Bile salts induce cathelicidin expression in biliary epithelial cells. (A) Total RNA from biliary epithelial cells treated with CDCA (100 μmol/L), UDCA (100 μmol/L), VD3 (0.1 μmol/L), or the combination of bile salts with VD3 was subjected to reverse-transcription quantitative PCR with primers designated to amplify cathelicidin. Data represent means ± SEM of 4 experiments performed in duplicate. *P < .05 vs untreated cells. **P < .05 vs VD3 or bile salts alone by the Student t test for paired samples. (B) Proteins from biliary epithelial cells transfected with either scramble (sc) or VDR small interfering RNA (si) were incubated with CDCA (100 μmol/L), UDCA (100 μmol/L), or the combination of bile salts with VD3 (0.1 μmol/L) were submitted to VDR and β-actin immunoblot–enhanced chemiluminescence analyses. Representative gels of 3 different experiments are shown. (C and D) Biliary epithelial cells transfected either with scramble or VDR small interfering RNA were incubated with CDCA (100 μmol/L), UDCA (100 μmol/L), or the combination of bile salts with VD3 (0.1 μmol/L) and subjected to cathelicidin mRNA detection by real-time reverse-transcription PCR. Data represent means ± SEM of 3 experiments performed in duplicate. *P < .05 vs scramble by the Student t test for paired samples. (E) Biliary epithelial cells were incubated with CDCA (100 μmol/L) or GW4064 (1 μmol/L) in the presence of either a control plasmid or a FXR dominant-negative plasmid (DN hFXR) and subjected to real-time reverse-transcription PCR with primers designated to amplify cathelicidin. Data represent means ± SEM of 3 experiments performed in duplicate. *P < .05 vs respective control condition by the Student t test for paired samples.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
UDCA treatment causes increased expressions of VDR and cathelicidin in the human liver. Total RNA from the liver of PBC patients before or after the onset of UDCA treatment was subjected to reverse-transcription quantitative PCR with primers designated to amplify either VDR or cathelicidin. Results are expressed as mRNA levels relative to the mean value of PBC patients before UDCA treatment. (A) Correlation between VDR expression and cathelicidin expressions in all human liver samples. R = ; P < .05. (B and C) Comparison between PBC patients before and after the onset of UDCA treatment. *P < .05 by the Mann–Whitney rank test for unpaired data.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

8. Figure 7
Representation of vitamin D and bile salt action on cathelicidin expression in biliary epithelial cells. (A) Vitamin D (VD3) induces cathelicidin expression through VDR in biliary epithelial cells. (B) UDCA, by increasing VDR protein expression through ERK activation, induces cathelicidin expression in biliary epithelial cells. When UDCA and vitamin D are used in combination, the induction of cathelicidin expression is potentiated. (C) CDCA is able to increase VDR protein expression. However, CDCA induces cathelicidin expression through FXR. When vitamin D is combined with CDCA, a synergistic effect on cathelicidin expression is observed.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions