1. Volume 143, Issue 6, Pages 1630-1640.e8 (December 2012)
Glucocorticoids Promote Hepatic Cholestasis in Mice by Inhibiting the Transcriptional Activity of the Farnesoid X Receptor 
Yan Lu, Zhijian Zhang, Xuelian Xiong, Xiaolin Wang, Jin Li, Guojun Shi, Jian Yang, Xianfeng Zhang, Huijie Zhang, Jie Hong, Xuefeng Xia, Guang Ning, Xiaoying Li 
Gastroenterology 
Volume 143, Issue 6, Pages e8 (December 2012)
DOI: /j.gastro
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2. Figure 1
Serum BA levels in patients with Cushing syndrome and obesity. (A) Serum cortisol and (B) BA levels in patients with Cushing syndrome. (C) Serum GC and (D) BA levels in obese subjects. (E) Pearson correlation of serum cortisol and BA levels in obese and nonobese subjects. ***P < .001.
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3. Figure 2
Overproduction of BA and hepatobiliary dysfunction caused by DEX. (A) Liver weights (left), and gallbladder volume (right), (B) serum BA levels (left), and hepatic BA contents (right), (C) serum TC levels (left), and hepatic TC contents (right), and (D) serum ALT levels (left), and bilirubin (right) levels in C57/BL6 mice treated with DEX and vehicle (Ctrl) (n = 5). (E) Histologic examination of gallbladder mucosal epithelium by H&E staining. Stromal granulocyte infiltration is indicated by black arrows. (F) Immunohistology of macrophage infiltration in gallbladder epithelium stained with anti-CD68 antibody is indicated by black arrows in DEX-treated and control mice (left). CD68-positive cells in gallbladder epithelium were counted (right). *P < .05; **P < .01; ***P < .001.
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4. Figure 3
Gene expression profiles related to BA homeostasis. (A and B) Real-time polymerase chain reaction for gene expression related to (A) BA synthesis (left), BA and phosphatidylcholine (PC) efflux into bile ducts (middle), BA efflux into circulation (right), and (B) cholesterol metabolism (left), and cholesterol efflux into bile ducts (right) in DEX-treated and control mice (n = 5). (C) Critical transcription factors (TFs) related to BA homeostasis regulation were analyzed in 2 groups of mice. (D) Representative protein levels of hepatic SHP and Cyp7A1 are shown. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P < .05; **P < .01; ***P < .001.
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5. Figure 4
BA overproduction attenuated by GR antagonists or silence in obese mice. (A) Liver BA contents (left) and serum BA levels (right) in db/db mice treated with RU486 or vehicle (Ctrl) (n = 7). (B) Real-time PCR for BA synthesis gene expression in db/db mice treated with RU486. (C and D) Hepatic GR expression silenced by adenoviral shRNA shown by (C) real-time polymerase chain reaction and (D) Western blotting in db/db mice (n = 8). (E) Liver BA contents (left) and serum BA levels (right) in db/db mice with GR silenced. (F) Real-time polymerase chain reaction for BA synthesis gene expression in db/db mice with GR silenced. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **P < .01; ***P < .001.
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6. Figure 5
GR down-regulates SHP expression through repression of FXR activity. (A) Luciferase assay for SHP transcription activity using SHP promoter (SHP-Luc) in HEK293T cells treated with DEX (100 nmol/L) or GW4064 (1 μmol/L). (B and C) Luciferase assay for (B) SHP-Luc in HEK293T cells transfected with wild type (WT) and ligand binding domain (LBD) deleted (left), E558A and L584D mutants (right), and (C) DNA binding domain (DBD) deleted GR in the presence of DEX. (D) HEK293T cells were transfected with the indicated plasmids, treated with DEX for 1 or 2 hours, and subjected to immunoprecipitation (IP) with Flag-M2 beads. The immunoprecipitates were immunoblotted (IB) with specific antibodies as indicated. (E) GST pull-down assays with full-length (left) or truncated FXR (right). Input lanes represent 10% of the input. (F) DEX induced GR binding to the FXRE region of SHP promoter in HepG2 cells. For ChIP analyses, soluble chromatin prepared from HepG2 cells treated with DEX for 1 hour was immunoprecipitated with GR antibody or IgG as a control. **P < .01; ***P < .001.
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7. Figure 6
Overproduction of BA induced by glucocorticoids unobserved in FXR null mice. (A) Liver BA contents (left) and serum BA levels (right) in FXR wild-type (WT) and knock-out (KO) mice treated with DEX or vehicle control (n = 6–9). (B and C) Real-time polymerase chain reaction for BA synthesis gene expression from livers of FXR WT and KO mice. (D) Primary hepatocytes were seeded on 6-well plates and treated with vehicle (Ctrl) or DEX (100 nmol/L) for 24 hours. Total RNA was extracted and analyzed for SHP gene expression by real-time polymerase chain reaction. (E) ChIP assays for GR binding to the promoter of SHP in FXR WT and KO primary hepatocytes. *P < .05; **P < .01; ***P < .001.
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8. Figure 7
CtBP recruited to GR as co-repressor to reduce FXR transactivation activity. (A) ChIP assay shows that CtBP binds to SHP promoter in HepG2 cells treated with DEX (100 nmol/L) or vehicle. (B) Real-time polymerase chain reaction for CtBP (left) and SHP expression (right) in HepG2 with CtBP silenced. (C) Co-immunoprecipitation assays for CtBP binding to GR in HEK293T cells (left) or HepG2 cells (right) treated with DEX for 1 hour. HEK293T cells were transfected with the indicated plasmids subjected to immunoprecipitation (IP) with Flag-M2 beads. The lysates of HepG2 cells were subjected to IP with IgG or GR antibodies. (D) Hepatic CtBP silenced by adenoviral shRNA shown by real-time polymerase chain reaction (left) and Western blotting (right) (n = 5–6). (E) Liver BA contents in mice with CtBP silenced treated with DEX or vehicle (Ctrl) (n = 5–6). (F) Hepatic BA synthesis gene expression in mice as in panel E. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P < .05; **P < .01; ***P < .001.
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9. Supplementary Figure 1
(A and B) Blood biochemical characteristics in patients with (A) Cushing syndrome and (B) obese subjects. FBG, fasting blood glucose; TG, triglyceride; TC, cholesterol; AST, aspartate aminotransferase.
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10. Supplementary Figure 2
(A) Fasting blood glucose, (B) serum triglyceride (TG) levels, and (C) hepatic TG contents in C57/BL6 mice treated with DEX or vehicle control (Ctrl) (n = 5). *P < .05.
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11. Supplementary Figure 3
(A) Biliary BA, (B) cholesterol, and (C) phospholipid levels were analyzed in mice treated with vehicle or DEX (n = 5). (D) Representative images of gallbladder bile from 2 groups of mice. *P < .05; **P < .01.
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12. Supplementary Figure 4
(A) Serum corticosterone levels, (B) liver BA contents, and (C) serum BA levels in obese db/db and lean control mice (n = 6). ***P < .001.
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13. Supplementary Figure 5
(A) Fasting blood glucose and (B) hepatic TG contents in db/db mice treated with GR-specific or nonspecific adenoviral shRNA (n = 8). *P < .05.
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14. Supplementary Figure 6
(A) Circadian alteration of hepatic GR and FXR expression at the indicated time points in C57BL/6 mice (n = 5–7). (B) Circadian alteration of serum corticosterone levels and hepatic SHP expression at the indicated time points in C57/BL6 mice (n = 5–7). (C) Circadian alteration of serum corticosterone levels and hepatic SHP mRNA expression at the indicated time points in C57/BL6 mice with GR silenced by adenoviral shRNA (n = 5–6). **P < .01; ##P < .01; ***P < .001or ###P < .001.
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15. Supplementary Figure 7
(A) Real-time polymerase chain reaction (PCR) for SHP expression in HepG2 cells treated with DEX (100 nmol/L), GW4064 (2 μmol/L), or combined DEX and GW4064 for 24 hours. (B and C) Luciferase assay for GRE activity (GRE-Luc) for various mutants and truncated GR in HEK293T cells treated with or without DEX for 8 hours (100 nmol/L). (D) Co-immunoprecipitation assay for GR and FXR interaction in cytoplasm (C) and nucleus (N) fractionations in the presence or absence of DEX. (E) Co-localization of GR with FXR. HepG2 cells incubated with DEX for 1 hour were immunostained with the indicated antibodies. **P < .01.
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16. Supplementary Figure 8
(A) Serum adrenocorticotropic hormone and (B) corticosterone levels in FXR wild-type (WT) and knockout (KO) mice treated with vehicle (Ctrl) or DEX (n = 6–9). ***P < .001.
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17. Supplementary Figure 9
Real-time polymerase chain reaction analysis for Abcb11 gene expression in livers from FXR wild-type (WT) and knockout (KO) mice treated with dexamethasone (DEX) or vehicle (Ctrl) (n = 6–9). **P < .01.
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18. Supplementary Figure 10
(A) Pyruvate tolerance test (PTT) in FXR wild-type (WT) or knock-out (KO) mice treated with DEX or vehicle. Mice were fasted overnight before intraperitoneal administration of 1.5 g/kg pyruvate (n = 6–9). (B) Serum and (C) hepatic triglyceride contents in FXR WT and KO mice treated with DEX or vehicle. (D and E) Real-time polymerase chain reaction analysis of (D) phosphoenolpyruvate carboxykinase (PEPCK) and (E) glucose-6-phosphatase (G6Pase) in mice. *P < .05; **P < .01; ***P < .001.
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19. Supplementary Figure 11
(A) ChIP assays of SMRT or NcoR on the SHP promoter in HepG2 cells. (B) Occupancy of the SHP promoter by GR and CtBP at different times after DEX treatments in HepG2 cells. (C) ChIP assays for CtBP binding to the SHP promoter in HepG2 cells after short interfering RNA (siRNA) transfection. (D and E) Real-time polymerase chain reaction and Western blotting analysis of GR and CtBP in HepG2 cells transfected with control (Ctrl) or GR-specific siRNAs. *P < .05; ***P < .001.
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20. Supplementary Figure 12
(A) Fasting glucose and (B) serum TG levels in C57/BL6 mice with CtBP silenced by adenoviral shRNA (n = 5–6). **P < .01; ***P < .001.
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21. Supplementary Figure 13
(A) GR bound to the promoter region of Abcb4 gene in HepG2 cells. For ChIP analyses, soluble chromatin prepared from HepG2 cells treated with DEX (100 nmol/L) or vehicle control (Ctrl) for 1 hour was immunoprecipitated with GR antibody or IgG as a control. (B) ChIP assays for GR binding to the promoter of Abcb4 in FXR wild-type (+/+) and knockout (–/–) primary hepatocytes. (C) ChIP assays for the CtBP recruitment to Abcb4 gene promoter in HepG2 cells treated with DEX (100 nmol/L) or vehicle control (Ctrl). *P < .05; **P < .01; ***P < .001.
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22. Supplementary Figure 14
Working model: GR activated by GCs binds to FXR and suppresses FXR transactivation activity through recruiting CtBP. We propose that the reduction of FXR function adds an additional mechanism in GC-induced hepatic cholestasis and BA overproduction.
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23. Supplementary Figure 15
(A) Schematic diagram of rat CYP7A1 promoter containing wild-type and mutant (underlined) GR response elements (GRE) (left). Luciferase reporter assays in HepG2 cells transfected with wild-type and mutated Cyp7A1 promoters (right). After transfection for 24 hours, cells were treated with DEX (100 nmol/L) or vehicle control (Ctrl) for another 12 hours. (B) Primary hepatocytes from FXR wild-type (+/+) and knockout (–/–) mice were seeded on 6-well plates and treated with DEX (100 nmol/L) or vehicle control (Ctrl) for 24 hours. Total RNA was extracted and analyzed for Cyp7A1 expression by real-time polymerase chain reaction. **P < .01.
Gastroenterology  , e8DOI: ( /j.gastro )
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