Volume 129, Issue 1, Pages 259-268 (July 2005) Cell Type-Specific Intervention of Transforming Growth Factor β/Smad Signaling Suppresses Collagen Gene Expression and Hepatic Fibrosis in Mice  Yutaka Inagaki, Miwa Kushida, Kiyoshi Higashi, Johbu Itoh, Reiichi Higashiyama, Yun Yu Hong, Norifumi Kawada, Kazuhiko Namikawa, Hiroshi Kiyama, George Bou-Gharios, Tetsu Watanabe, Isao Okazaki, Kazuo Ikeda  Gastroenterology  Volume 129, Issue 1, Pages 259-268 (July 2005) DOI: 10.1053/j.gastro.2005.03.088 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Schematic representation of recombinant adenoviruses used in this study. In the Ax17COL-NCre adenovirus, Cre recombinase was expressed by a strong tissue-specific COL1A2 enhancer (−17.0 to −15.5 kilobases) linked to the −350 minimal promoter sequence. The potent CAG expression unit, consisting of the cytomegalovirus immediate early enhancer, modified chicken β-actin promoter, and rabbit β-globin polyadenylation signal, was used to drive Cre in a positive control, AxCANCre. AxCALNLYB1 and AxCALNLGFP adenoviruses were also generated to express YB-1 and GFP, respectively, by the CAG unit after the loxP-flanked stuffer sequence had been excised by Cre recombinase. Gastroenterology 2005 129, 259-268DOI: (10.1053/j.gastro.2005.03.088) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Adenovirus-mediated YB-1 expression in activated hepatic stellate cells. CFSC-2G cells were infected for 1 hour with AxCALNLYB1 recombinant adenovirus alone (−) or together with either Ax17COL-NCre (COL) or AxCANCre (CAG) vector in the absence (UNTR) or the presence of 2 ng/mL TGF-β. (A) Whole-cell lysates were prepared and immunoblotted with anti-YB-1 antibodies. (B) The steady-state levels of endogenous COL1A2 mRNA were determined by real-time reverse-transcription polymerase chain reaction. Relative expression levels of COL1A2 mRNA were normalized against those of glyceraldehyde phosphate dehydrogenase mRNA. The values are mean ± SD obtained from 3 independent RNA preparations and are expressed relative to the levels in untreated cells infected with AxCALNLYB1 virus alone. *Significant difference between groups. Gastroenterology 2005 129, 259-268DOI: (10.1053/j.gastro.2005.03.088) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Adenovirus-mediated GFP expression in normal and carbon tetrachloride-treated liver. Mice were injected via the tail vein with saline (A and D) or 5 × 108 50% tissue culture infectious dose each of AxCALNLGFP adenovirus together with either AxCANCre (B and E) or Ax17COL-NCre (C and F). GFP fluorescence was viewed and analyzed by using a confocal laser scanning microscope in normal (A–C; bar = 200 μm) and carbon tetrachloride-treated (D–F; bar= 250 μm) liver tissues. Gastroenterology 2005 129, 259-268DOI: (10.1053/j.gastro.2005.03.088) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Characterization of GFP-expressing cells in carbon tetrachloride-treated liver. (A) GFP fluorescence expressed by the COL1A2 enhancer was observed in nonparenchymal cells within and around the centrilobular necrotic areas (arrows; bar = 25 μm). (B) Immunofluorescence study using anti-α-SMA antibodies indicated the localization of α-SMA (red) in the GFP (green)-expressing activated HSCs (arrows). An arrowhead shows a GFP-expressing parenchymal hepatocyte that is negative for α-SMA staining (bar = 20 μm). Gastroenterology 2005 129, 259-268DOI: (10.1053/j.gastro.2005.03.088) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Cell type-specific GFP expression in carbon tetrachloride-treated fibrotic liver. Mice were chronically treated with repeated carbon tetrachloride injections for 9 weeks. They were injected via the tail vein with 1 × 109 50% tissue culture infectious dose each of AxCALNLGFP adenovirus together with either AxCANCre (A) or Ax17COL-NCre (B and C) after 7 weeks of carbon tetrachloride intoxication and were continuously treated with carbon tetrachloride for a further 2 weeks. The expression of GFP (green) and the localization of α-SMA (red) were analyzed by using a confocal laser scanning microscope. Arrows indicate the GFP (green)-expressing activated HSCs that are positive for α-SMA (red) staining (C) (bar = 100 μm in A and B; bar = 25 μm in C). Gastroenterology 2005 129, 259-268DOI: (10.1053/j.gastro.2005.03.088) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Effects of YB-1 expression on the activation of the COL1A2 promoter. Transgenic reporter mice harboring the COL1A2 upstream sequence linked to a firefly luciferase gene were injected with control saline, 5 × 108 50% tissue culture infectious dose (TCID50) of Ax17COL-NCre adenovirus alone (COL), or 5 × 108 TCID50 each of Ax17COL-NCre and AxCALNLYB-1 (COL/YB-1). Twenty-four hours later, they were treated with a single injection of 1 mL/kg body weight of carbon tetrachloride. Livers were excised 72 hours after carbon tetrachloride injection and subjected to luciferase assays. The values are mean ± SD obtained from 7 mice in each group. Values in the control mice without carbon tetrachloride injection (UNTR) are also presented. *Significant difference between groups. NS, not significant; RLU, relative luminescence units. Gastroenterology 2005 129, 259-268DOI: (10.1053/j.gastro.2005.03.088) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Suppressive effects of YB-1 on the progression of hepatic fibrosis. Mice were chronically treated with 1 mL/kg body weight carbon tetrachloride twice a week for 9 weeks. They were injected with either control saline or 5 × 108 50% tissue culture infectious dose each of Ax17COL-NCre and AxCALNLYB1 (COL/YB-1) after 7 weeks of carbon tetrachloride intoxication and continuously treated with carbon tetrachloride for a further 2 weeks. Some mice were treated simultaneously with 10,000 U/day of recombinant murine IFN-γ every day during the last 2 weeks. They were killed 72 hours after the last carbon tetrachloride injection and subjected to (A) real-time reverse-transcription polymerase chain reaction assays quantifying the steady-state levels of COL1A2 and YB-1 mRNA, (B) Western blot analyses to detect type I collagen expression, (C) measurement of serum hyaluronic acid concentration, and (D) Azan-Mallory staining (original magnification, 100×). In the real-time reverse-transcription polymerase chain reaction assays, relative expression levels of COL1A2 and YB-1 mRNA were normalized against those of glyceraldehyde phosphate dehydrogenase mRNA. After Azan-Mallory staining, the degree of hepatic fibrosis was semiquantified by measuring the mean relative areas of fibrosis with the aid of computer software. The values are mean ± SD obtained from 7 mice in each group. *Significant difference between groups. NS, not significant. Gastroenterology 2005 129, 259-268DOI: (10.1053/j.gastro.2005.03.088) Copyright © 2005 American Gastroenterological Association Terms and Conditions