Volume 127, Issue 5, Pages 1497-1512 (November 2004) The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis  Stefano Fiorucci, Elisabetta Antonelli, Giovanni Rizzo, Barbara Renga, Andrea Mencarelli, Luisa Riccardi, Stefano Orlandi, Roberto Pellicciari, Antonio Morelli  Gastroenterology  Volume 127, Issue 5, Pages 1497-1512 (November 2004) DOI: 10.1053/j.gastro.2004.08.001 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 In vivo delivery of 6-ECDCA protects against development of hepatic fibrogenesis induced by PS administration to rats. (A) Structure of the FXR ligand: 6-ECDCA. (B) Sirius red staining of a liver section obtained from a control rat (original magnification ×40). (C) Liver section from a control rat, showing Sirius red-positive fibrils around the central vein (original magnification ×100). (D and E) Sirius red staining of collagen in rats administered PS alone for 12 weeks (original magnification ×40 and ×100, respectively). (F) Sirius red staining of collagen in a rat administered PS in combination with 1 mg/kg 6-ECDCA (1–12 weeks) (original magnification ×40). (G) Sirius red staining of collagen in a rat administered PS in combination with 3 mg/kg 6-ECDCA (1–12 weeks) (original magnification ×40). (H and I) Reduction of liver fibrosis in rats administered PS in combination with 5 mg/kg 6-ECDCA (1–12 weeks) (original magnification ×40 and ×100, respectively). (J) Sirius red staining of collagen in a rat administered PS in combination with 10 mg/kg 6-ECDCA (1–12 weeks) (original magnification ×40). Representative micrographs of 6 specimens per rat are shown. (K) Attenuation of liver fibrosis in rats administered PS alone or in combination with 6-ECDCA. Morphometric analysis was carried out as described in the Materials and Methods section. Data are the mean ± SE of 6–12 rats per group. *P < .001 in comparison with control rats. **P < .01 in comparison with PS-treated rats. (L and M) FXR agonism reduces liver hydroxyproline content and urinary excretion of hydroxyproline in PS-treated rats. Data are mean ± SE of 6–12 rats. *P < .001 in comparison with control rats. **P < .01 in comparison with PS-treated rats. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 6-ECDCA protects from liver fibrosis induced by PS administration to rats. (A, B, and D–I) Quantitative RT-PCR of FXR and FXR-regulated genes was carried out as described in the Materials and Methods section on RNA purified from liver homogenates. Data are mean ± SE of 6–12 rats. *P < .001 in comparison with control rats. **P < .01 in comparison with PS treated rats. (C) Western blot analysis of α-SMA expression on liver homogenates. Lanes: 1, liver sample from a control rat; 2, liver sample from a rat administered PS alone; 3, liver sample from a rat administered PS and 1 mg/kg 6-ECDCA; 4, liver sample from a rat administered PS and 3 mg/kg 6-ECDCA; 5, liver sample from a rat administered PS and 5 mg/kg 6-ECDCA; and 6, liver sample from a rat administered PS and 10 mg/kg 6-ECDCA. The blot is representative of 4 other experiments that show a nearly identical pattern. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 The FXR ligand 6-ECDCA prevents liver fibrosis in BDL rats. Because control rats and BDL did not differ in any antropometric and biochemical parameters (see Tables 1 and 2), they were considered together. (A) Liver section from a control rat showing Sirius red-positive fibrils around the central vein (original magnification ×40). (B) Sirius red staining of collagen in BDL rats 2 weeks after ligation (original magnification ×40). (C) Sirius red staining of collagen in a liver section from a BDL rat administered CDCA 3 mg/kg (7–14 days) (original magnification ×40). (D) Sirius red staining of collagen in a liver section from a BDL rat administered 6-ECDCA 3 mg/kg (7–14 days). (E) Sirius red staining of collagen in a liver section from a BDL rat administered UDCA 15 mg/kg (7–14 days). (F) Morphometric assessment of liver fibrosis in BDL rats. Data are mean ± SE of 6–12 rats per group. *P < .001 vs. control rats. **P < .01 vs. BDL rats. (G and H) FXR ligand reduces liver hydroxyproline content and urinary excretion of hydroxyproline in BDL rats. Data are mean ± SE of 6–12 rats. *P < .001 vs. control rats. **P < .01 vs. BDL rats. (I) 6-ECDCA increases the liver expression of SHP mRNA as assessed by qRT-PCR in BDL rats. **P < .01 vs. BDL rats. (J–M) 6-ECDCA reduces liver expression of α1 (I) collagen, α-SMA, TIMP-1, and TIMP-2 mRNA as assessed by qRT-PCR in BDL rats. No effect was detected on MMP-2 mRNA (N). Data are mean ± SE of 6–12 rats. *P < .001 vs. control rats. **P < .01 vs. BDL rats. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 FXR is expressed in primary cultures of rat HSCs (A) and HSC-T6 (C). The RT-PCR shown is representative of 5 other experiments. (B and D) Quantitative RT-PCR analysis of FXR mRNA expression in freshly isolated HSC (day 0) and HSCs grown in 10% FCS for 7 days. The expression of FXR in HSC-T6 is shown by comparison. Data are mean ± SE of 6 separate experiments. *P < .05 vs. day 0. (E) Expression of SHP, NTCP, BSEP, and CYP7A1 in HSCs and HSC-T6. RT-PCR was carried out on HSCs cultured for 7 days in 10% FCS. The RT-PCR shown was confirmed by 5 other experiments. (F) FXR ligands induce SHP mRNA in HSCs. Cells were cultured for 7 days in 10% FCS and then stimulated with 20 μmol/L CDCA, 1 μmol/L 6-ECDCA, or 100 nmol/L GW4064 for 18 hours, and SHP expression was measured by quantitative RT-PCR as described in the Material and Methods section. Data are mean ± SE of 6 experiments. *P < .001 vs. control cells. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 FXR ligands inhibit α1(I) collagen gene expression in HSCs and HSC-T6. (A and B) Cells were cultured for 7 days in 10% FCS (lane 1) and then stimulated with 20 μmol/L CDCA (lane 2), 1 μmol/L 6-ECDCA (lane 3), or 100 nmol/L GW4064 (lane 4) for 18 hours. The symbol ”-“ indicates addition of water. The RT-PCR was representative of 4 other experiments. (C and D) Quantitative RT-PCR analysis of α1(I) collagen gene expression in HSCs and HSC-T6. Experimental conditions were the same as in panels A and B. Data are mean ± SE of 6 experiments. *P < .01 vs. control cells. (E) FXR ligands abrogate induction of α1(I) collagen gene in response to growth factors. HSCs were cultured for 7 days in 10% FCS alone (control) or 10% FCS medium containing thrombin (10 U), PDGF, or TGF-β1 (100 ng/mL) in the presence of 20 μmol/L CDCA, 1 μmol/L 6-ECDCA, or 100 nmol/L GW4064. *P < .001 vs. control cells. Data are mean ± SE of 6 experiments. **P < .01 vs. growth factors alone. (F) FXR ligands inhibit collagen synthesis induced by growth factors. HSCs were cultured for 7 days in 10% FCS alone (control) or 10% FCS medium containing thrombin (10 U), PDGF, or TGF-β1 (100 ng/mL) in the presence of 20 μmol/L CDCA, 1 μmol/L 6-ECDCA, or 100 nmol/L GW4064. Data are mean ± SE of 6 experiments. *P < .001 vs. control cells. **P < .01 vs. growth factors alone. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 FXR ligands do not inhibit DNA synthesis or HSC proliferation induced by thrombin and growth factors nor induce apoptosis. DNA synthesis was measured by assessing H3-thymidine incorporation (A), whereas cell proliferation was evaluated by cell counting (B). Apoptosis (C) was measured by assessing propidium iodide incorporation. HSCs were cultured for 7 days in 10% FCS alone (control) or 10% FCS medium containing thrombin (10 U), PDGF, or TGF-β1 (100 ng/mL) in the presence of 20 μmol/L CDCA or 1 μmol/L 6-ECDCA. Data are mean ± SE of 6 experiments. *P < .01 vs. control cells. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 SHP overexpression is sufficient to down-regulate α1(I) collagen mRNA in HSC-T6. (A) Western blot analysis of cellular extracts obtained from HSCT6 infected with empty vector (WT) and vector carrying the HA-SHP chimera. The relative expression of SHP protein in HSC-T6 infected with HA-SHP is visualized using an anti-HA antibody. (B) SHP overexpression in HSC-T6 down-regulates α1(I) collagen mRNA. Data are mean ± SE of 4 experiments. P < .01 vs. control cells (WT). α1(I) Collagen mRNA expression was measured by QRT-PCR. (C) SHP overexpression in HSC-T6 (SPH) abrogates α1(I) collagen mRNA induction caused by growth factors. HA-SHP transfected HSC-T6s were cultured for 24 hours in 10% FCS alone (control) or 10% FCS medium containing thrombin (10 U), PDGF, or TGF-β1 (100 ng/mL), and α1(I) collagen mRNA expression was measured by QRT-PCR. Data are mean ± SE of 6 experiments. P < .01 vs. WT cells. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 8 Abrogation of SHP expression by anti-SHP siRNA up-regulates α1(I) collagen mRNA expression in HSC-T6 cells. Anti-SHP siRNA was carried out as described in the Materials and Methods section. (A and B) HSC-T6 were incubated with 1–15 nmol/L SHP siRNA, and SHP expression was assessed by RT-PCR and Western blot analysis. *P < .01 vs. control cells. (C) Lack of inhibition of α1(I) collagen mRNA expression by FXR ligands in SHP-defective HSC-T6. HSC-T6 were cultured for 18 hours in 10% FCS alone (control) or 10% FCS medium containing CDCA (20 μmol/L), 6-6-ECDCA (1 μmol/L) or GW4064 (100 nmol/L) and α1(I) collagen mRNA expression assessed by QRT-PCR. Data are mean ± SE of 6 experiments. P < .01 vs. WT cells. (D) Enhanced expression of α1(I) collagen mRNA in SHP defective HSC-T6. HSC-T6 were cultured for 18 hours in 10% FCS alone (control) or 10% FCS medium containing thrombin (10 U) and TGF-β1 (100 ng/mL), and α1(I) collagen mRNA expression was assessed by RT-PCR. Data are the mean ± SE of 6 independent experiments. P < .01 vs. WT cells. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 9 SHP binds and inactivates AP-1 in HSCs. (A and B) Western blot analysis of JunD and c-Jun in anti-HA-SHP immunoprecipitates obtained from HSC-T6 infected with HA-SHP fusion protein. In both panels, the densitometric analysis of Western blot with anti-JunD and anti-c-Jun antibodies in HA-SHP immunoprecipitates is shown. The 10% input shown represents the amount of JunD and c-Jun that was expressed by HA-SHP cells exposed to 10 U thrombin for 18 hours. Densitometric analysis indicates that 30%–40% of HA-SHP imunoprecipitates bind to JunD or c-Jun. Data are mean ± SE of 4 experiments. (C) SHP overexpression inhibits the binding of AP-1 to DNA. EMSA was performed with nuclear extracts obtained from HSC-T6 cells infected with empty vector (lanes 1–4, 8, and 10) or HA-SHP chimera (lanes 5–7 and 9) and then incubated with thrombin with or without FXR 1 μmol/L 6-ECDCA or 100 nmol/L GW-4064. The nuclear extracts were preincubated with anti-JunD antibody at room temperature for 20 minutes before the addition of the radiolabeled probe. Although thrombin induces AP-1 binding in WT (lane 4 vs. lane 8), 6-ECDCA and GW-4064 (lanes 3 and 2 vs. lane 4) reduced the binding of AP-1 to DNA. SHP overexpression inhibits AP-1 DNA binding in all treatments (lanes 5–7 vs. lanes 2–4 and lane 9 vs. lane 8). The specificity of AP-1 DNA binding was demonstrated both by using anti-JunD antibody, which reduces AP-1 binding (lane 1), and an excess of cold probe, which abrogates AP to DNA binding (lane 10). P = free probe. The absence of the band of supershift depends on the interaction between the anti-JunD antibody and the protein that blocks the binding of JunD to response element. The EMSA shown is representative of 3 other experiments showing the same pattern. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 10 In vivo administration of FXR ligand increases SHP expression and SHP/JunD interaction in HSCs. HSCs were isolated from BDL rats after treatment with 6-ECDCA, 3 mg/kg, for 7 days as described in the Materials and Methods section. An equal amount of lysates (10 μg per lane) obtained from day 0 HSCs was used for these experiments. HSCs were lysed, and Western blot analysis was performed as described in the Materials and Methods section. The blot shown is representative of at least 3 other experiments showing the same pattern. Gastroenterology 2004 127, 1497-1512DOI: (10.1053/j.gastro.2004.08.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions