1. Volume 126, Issue 3, Pages 756-764 (March 2004)
Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity 
Frank Chen, M. Ananthanarayanan, Sukru Emre, Ezequiel Neimark, Laura N. Bull, A.S. Knisely, Sandra S. Strautnieks, Richard J. Thompson, Margret S. Magid, Ronald Gordon, N. Balasubramanian, Frederick J. Suchy, Benjamin L. Shneider 
Gastroenterology 
Volume 126, Issue 3, Pages (March 2004)
DOI: /j.gastro

2. Figure 1
(A) Northern analysis of ileal mRNA. Lane 1, cholestatic patient with high γ-glutamyl transpeptidase; lanes 2–4, PFIC1 disease patients; lane 5, PFIC2 patient. The mRNA levels were measured by PhosphorImager analysis of a Northern blot of 20 μg of total RNA. ASBT mRNA levels were increased and ILBP and FXR mRNA levels were decreased when FIC1 mRNA was absent (namely, in the patients with PFIC1 disease). Loading controls for total RNA (28S) were similar in the 5 patients. (B) Quantitation of steady-state mRNA levels. PhosphorImager analysis of the Northern blot in (A) was performed, and data were analyzed for FIC1-positive (lanes 1 and 5) vs. FIC1-negative (lanes 2–4) samples. Statistically significant (P < 0.01) increases in ASBT mRNA levels were observed in the FIC1-negative patients relative to the FIC1-positive patients. Statistically significant (P < 0.01) decreases in FXR, SHP, and ILBP mRNA levels were observed in the FIC1-negative patients relative to the FIC1-positive patients. 28S RNA levels were similar between groups (P > 0.05).
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Northern analysis of FIC1 antisense-treated Caco-2 cells. Three separate sets of cells were treated with FIC1 antisense RNA (lanes 2, 4, and 6). The mRNA levels were measured by PhosphorImager analysis of a Northern blot of 20 μg of total RNA. FIC1 mRNA was undetectable in the FIC1 antisense-treated cells. FIC1-negative cells (lanes 2, 4, and 6) had enhanced ASBT and diminished ILBP, FXR, and SHP mRNA expression. Similar RNA loading is shown by equivalent 28S RNA.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Promoter activity in FIC1 antisense-treated Caco-2 cells. Caco-2 cells were pretreated with FIC1 antisense RNA (FIC1 negative) and then transfected with luciferase reporter constructs for human ASBT, human FXR, or human BSEP (C, Caco-2; H, HepG2). Luciferase activity was normalized to simultaneously co-transfected Renilla luciferase. The pGL3-basic vector activity was 0.3 ± 0.1 (data not shown). Human ASBT promoter activity was enhanced in FIC1-negative cells, whereas FXR and BSEP promoter activities were diminished (differences between wild-type and FIC1 antisense-treated cells were significant at P < 0.01 for all promoters tested).
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Effect of RAR/RXR mutation on the FIC1-mediated response of ASBT. A 3′ RAR/RXR cis-element in human ASBT was mutated by site-directed mutagenesis to yield the mutant luciferase reporter construct HASBTμ. Caco-2 cells were pretreated with FIC1 antisense RNA and then transfected with the ASBT or HASBTμ luciferase reporter constructs. Luciferase activity was normalized to simultaneously co-transfected Renilla luciferase. The y-axis is logarithmic. HASBTμ promoter activity was reduced in wild-type Caco-2 cells compared with the human ASBT promoter construct (P < 0.01). FIC1 antisense treatment activated ASBT activity (P < 0.01) but not HASBTμ activity (P > 0.05).
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Effect of FXR and SHP overexpression on the FIC1-mediated response of ASBT. The effect of overexpression of exogenous FXR or SHP on the FIC1 response was assessed. Caco-2 cells were transfected with either an FXR or SHP expression vector (HASBT + FXR or HASBT + SHP) to assess the ability to overcome the predicted effect of FIC1 knock-down (+FIC1−, FIC antisense oligonucleotide treatment; +sFIC1−, scrambled oligonucleotide treatment) on FXR expression. Overexpression of FXR had no effect on the FIC1 antisense treatment on HASBT promoter activity (P > 0.05). Overexpression of SHP reduced the activity of the HASBT promoter (P < 0.01) and abrogated the FIC1 knock-down effect (P < 0.01).
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
FXR analysis in Caco-2 FIC-1 knock-down studies. (A) Electrophoretic mobility gel shift assay of the effect of FIC1 antisense treatment. Cytoplasmic and nuclear extracts were prepared from Caco-2 cells that were untreated (wt) or treated with FIC1 antisense (neg) or scrambled antisense (scr) oligonucleotides. Gel shift assays were performed with an oligonucleotide for the human FXR binding element. Binding was observed (arrow) with the cytoplasmic and nuclear extracts from the untreated and scrambled antisense-treated Caco-2 cells. In contrast, FIC1 antisense treatment had no effect on binding by cytoplasmic extracts, but nuclear extracts were unable to bind the FXR DNA element. (B) Supershift analysis. Cytoplasmic and nuclear extracts were prepared from wild-type Caco-2 cells. Electrophoretic mobility shift assays were performed with an FXR cis-element oligonucleotide. An FXR antibody (Ab = F) supershifted the DNA/protein complex compared with no antibody (N) or an irrelevant anti-histone antibody (H). (C) Competition assays. Cytoplasmic and nuclear extracts were prepared from wild-type Caco-2 cells. Electrophoretic mobility shift assays were performed with an FXR cis-element oligonucleotide. Specific and nonspecific unlabeled competitor oligonucleotides were used at the concentrations listed in the figure. The specific oligonucleotide competed for protein binding as shown by the diminished intensity of the band at the arrow. In contrast, nonspecific DNA did not compete for the protein binding. (D) Western blot analysis. Cytoplasmic and nuclear extracts were prepared from Caco-2 cells that were untreated (wt) or treated with FIC1 antisense (neg) or scrambled antisense (scr) oligonucleotides. FXR protein expression was reduced in nuclear extracts of Caco-2 cells treated with the FIC1 antisense oligonucleotide. Equivalent loading is shown by the signals for actin (cytoplasmic protein marker) and histone H1 (nuclear protein marker).
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Autoactivation of FXR. The human FXR promoter was electroporated into Caco-2 cells. Promoter activity was markedly enhanced when the cells were treated with chenodeoxycholic acid (CDCA) 100 μmol/L (P < 0.01, Caco-2 vs. CDCA). Promoter activity was reduced by overexpression of a mutant FXR protein (FXRμ) that binds bile acids but does not transactivate (P < 0.01, Caco-2 vs. FXRμ). Overexpression of FXRμ abrogated the bile acid response of the FXR promoter (P < 0.01, + CDCA vs. FXRμ; CDCA).
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Hypothetical model of the effect of FIC1. FIC1 is a membrane protein that alters membrane aminophospholipid asymmetry and transduces an unknown signaling pathway (curved arrows). This leads to a posttranslational modification of FXR (indicated by shading and attached star). The posttranslational modification is necessary for nuclear translocation of FXR. FXR in the nucleus then activates itself (FXR), the ileal lipid-binding protein (ILBP), the short heterodimer protein (SHP), and the bile salt excretory pump (BSEP). FXR, via the effect of SHP, inhibits the expression of the apical sodium-dependent bile acid transporter (ASBT) and cholesterol 7-α hydroxylase (C7AH).
Gastroenterology  , DOI: ( /j.gastro )