Volume 134, Issue 4, Pages 1180-1190 (April 2008) Hepatocyte Growth Factor Suppresses Profibrogenic Signal Transduction via Nuclear Export of Smad3 With Galectin-7  Yutaka Inagaki, Kiyoshi Higashi, Miwa Kushida, Yun Yu Hong, Sachie Nakao, Reiichi Higashiyama, Tadashi Moro, Johbu Itoh, Toshiyuki Mikami, Toru Kimura, Goshi Shiota, Ichiro Kuwabara, Isao Okazaki  Gastroenterology  Volume 134, Issue 4, Pages 1180-1190 (April 2008) DOI: 10.1053/j.gastro.2008.01.014 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Antagonistic effects of HGF over expression on TGF-β-stimulated COL1A2 transcription. (A) Schematic representation of the −2900COL1A2 promoter region with the TGF-β-responsive element (TbRE). Sp1 and phosphorylated Smad3 bind to the TbRE and interact with each other to mediate the stimulatory effect of TGF-β on gene transcription. (B) CFSC-2G cells were cotransfected with the different lengths of COL1A2 promoter/luciferase constructs and either HGF expression plasmid or empty control vector then left untreated (UNTR) or treated with 2 ng/mL TGF-β for 36 hours. Transcriptional activity was normalized against that of cotransfected pRL-CMV. The values are mean ± SD obtained from 5 independent tests and expressed relative to the activity in untreated −2900COL1A2/LUC control transfectants. The asterisk indicates that the difference between the groups is significant. RLU, relative luminescence units; NS, not significant; +1, transcription start site. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Effects of HGF on subcellular localization of Smad3. (A) CFSC-2G cells were untreated (UNTR) or treated with 50 ng/mL HGF for 1 hour. In some cases, cells were preincubated with 10 μmol/L PD98059 for 1 hour followed by a further incubation in the absence or presence of HGF. Subcellular localization of Smad3 (green) was determined by using rabbit anti-Smad3 antibodies and Alexa Fluor 488-conjugated anti-rabbit IgG. All images were viewed and analyzed using a confocal laser-scanning microscopy with the same conditions of identical excitation strength and detection gain. Scale bar, 20 μm. (B) Fluorescence signals present in the nucleus and the cytoplasm of CFSC-2G cells were quantified by using 3-dimensional confocal laser-scanning microscopic pictures. After being projected onto a single 2-dimensional image, the localization of cytoplasmic and nuclear fluorescence signals was determined by a transparent image and 7-amino-actinomycin D nuclear staining, respectively. In the left panel are shown representative pictures of nuclear and cytoplasmic fluorescence signals present in an HGF-treated cell. The vertical arrow means that the fluorescence on a confocal microscopic picture (upper panel) is projected onto a 2-dimensional picture (lower panel). The smaller arrow means that the fluorescence signals present in the nucleus are separated from those in the cytoplasm to semi-quantify the signals. The right histograms summarize a result of statistical analyses using 20 randomly selected cells for each group. The asterisk indicates that the difference between the groups is significant. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Identification of nuclear proteins interacting with phosphorylated Smad3 following HGF treatment. (A) Nuclear extracts were prepared from CFSC-2G cells treated with 50 ng/mL HGF for the indicated lengths of time. After immunoprecipitation using antiphospho-Smad2/3 antibodies, they were separated on a 10%–20% gradient SDS-polyacrylamide gel and visualized by silver staining. (B) In some experiments, cells were preincubated with 10 μmol/L PD98059 for 1 hour before HGF treatment. Arrows 1 to 3 indicate the bands corresponding to galectin-7, MRP 14, and MRP 8, respectively, which became evident following HGF treatment. Heavy (H) and light (L) chains of IgG are also shown. M, molecular weight markers. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Physical interaction between Smad3 and galectin-7. (A) Whole cell lysates were prepared from CFSC-2G cells treated with 50 ng/mL HGF for the indicated lengths of time. They were separated on a 12.5% SDS-polyacrylamide gel, electroblotted, and incubated with antibodies against Smad3, galectin-7, or β-actin as a control. (B) COS-7 cells were cotransfected with a Myc-tagged Smad3 expression vector and Flag-tagged expression plasmids encoding galectin-7 (G7), galectin-1 (G1), or galectin-3 (G3). After 48 hours of incubation, whole cell lysates were prepared from transfected cells, immunoprecipitated (IP) with anti-Flag antibodies, and immunoblotted (IB) with anti-Myc antibodies. They were also immunoblotted directly with anti-Myc or anti-Flag antibodies to confirm the expression of each protein in the transfected cells. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Subcellular localization of Smad3 and galectin-7 following HGF treatment. (A) Colocalization of Smad3 (green) and galectin-7 (red) was examined in CFSC-2G cells treated with 50 ng/mL HGF for the indicated lengths of time. Nuclei were stained with 7-amino-actinomycin D (blue). In the middle and lower panels are shown the images of only Smad3 and galectin-7, respectively. Scale bar, 20 μm. (B) Nuclear and cytoplasmic proteins were prepared from CFSC-2G cells treated with 50 ng/mL HGF for the indicated lengths of time. They were separated on a 12.5% SDS-polyacrylamide gel, electroblotted, and incubated with antibodies against phosphorylated Smad3 (pSmad3), galectin-7, lamin A/C, or β-actin. The right histograms summarize the results of statistical analyses using 3 independent preparations of nuclear and cytoplasmic extracts. Relative expression levels of phosphorylated Smad3 and galectin-7 were normalized against those of lamin A/C or β-actin. The asterisk indicates that the value is significantly different from that with untreated cell extracts. (C) Translocation of Smad3 (green) and galectin-7 (red) was also examined in primary cultures of rat HSC at day 7 after plating. Cells were either untreated (UNTR) or treated with 100 ng/mL HGF for 1 hour. In the middle and lower panels are shown the images of only Smad3 and galectin-7, respectively. Scale bar, 20 μm. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Effects of transfection of galectin-7 siRNA on subcellular localization of Smad3 and TGF-β-stimulated gene expression. (A) CFSC-2G cells were transfected with 0.5 μg double-stranded galectin-7 or control siRNA and incubated for 48 hours. After additional transfection with 0.5 μg siRNA for 48 hours, whole cell lysates were separated by an SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies against galecin-7, phosphorylated Smad3 (pSmad3), or β-actin. (B) Following transfection with the indicated siRNA, subcellular localization of Smad3 and galectin-7 was examined in CFSC-2G cells untreated (UNTR) or treated with 50 ng/mL HGF for 1 hour. All images were viewed and analyzed using a confocal laser-scanning microscopy with the same conditions of identical excitation strength and detection gain. Scale bar, 20 μm. (C) CFSC-2G cells were transfected with the indicated siRNA then untreated (UNTR) or treated with 5 ng/mL TGF-β and/or 50 ng/mL HGF for 24 hours. Total RNA was extracted, and the steady state levels of endogenous COL1A2 and PAI-1 mRNA were measured by real-time RT-PCR. The values are mean ± SD obtained from 6 different RNA preparations, and relative expression levels of COL1A2 and PAI-1 mRNA were normalized against those of GAPDH mRNA. The asterisk indicates that the difference between the groups is significant. NS, not significant. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Effects of over expression of galectin-7 on TGF-β-stimulated gene expression. (A) CFSC-2G cells were cotransfected with 0.2 μg (SBE)4-luciferase construct, 0.2 μg pRL-TK, and increasing amounts (0, 0.25, 0.5, 1, or 2 μg) of galectin-7 expression plasmid. After 6 hours of incubation, cells were either untreated (UNTR) or treated with 5 ng/mL TGF-β for 24 hours. Transcriptional activity was normalized against that of cotransfected pRL-TK. The values are mean ± SD obtained from 6 independent tests and expressed relative to the activity in untreated cells transfected with an empty control vector. RLU, relative luminescence units. (B) CFSC-2G cells were transfected with increasing amounts (0, 0.25, 0.5, 1, or 2 μg) of galectin-7 expression plasmid. After 6 hours of incubation, cells were either untreated (UNTR) or treated with 5 ng/mL TGF-β for 24 hours. Total RNA was extracted and subjected to real-time RT-PCR to determine the steady state levels of endogenous COL1A2 and PAI-1 mRNA. The values are mean ± SD obtained from 6 different RNA preparations, and relative expression levels of COL1A2 and PAI-1 mRNA were normalized against those of GAPDH mRNA. In all cases, an empty control vector was used to ensure an equal amount of DNA in each sample. The asterisk indicates that the value is significantly lower than that in control transfectants with only an empty vector. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Effects of HGF over expression on activation of COL1A2 promoter. Double transgenic mice over expressing HGF driven by the mouse albumin enhancer/promoter and harboring a COL1A2 upstream sequence linked to a firefly luciferase gene (HGF Tg), as well as control reporter mice (WT), were untreated (UNTR) or treated with a single subcutaneous injection of 1 mL/kg body weight of carbon tetrachloride (CCl4). Mice were killed 72 hours after the CCl4 injection and subjected to (A) luciferase assays determining COL1A2 promoter activity in liver tissue and (B) real-time RT-PCR measuring the steady state levels of endogenous COL1A2 expression. Luciferase activity was normalized against the protein concentration of tissue homogenates, whereas relative expression levels of COL1A2 mRNA were normalized against those of GAPDH mRNA. The values are mean ± SD obtained from 7 mice in each group. The asterisk indicates that the difference between the groups is significant. RLU, relative luminescence units; NS, not significant. (C) HSC were isolated from wild-type or HGF-over expressing transgenic mice before and 24 hours after a single CCl4 injection. HSC were subjected to primary culture for 24 hours, and immunofluorescence studies were performed to determine subcellular localization of Smad3 (green) and galectin-7 (red). Nuclei were stained with 7-amino-actinomycin D (blue). In the middle and lower panels are shown the images of only Smad3 and galectin-7, respectively. Scale bar, 10 μm. Gastroenterology 2008 134, 1180-1190DOI: (10.1053/j.gastro.2008.01.014) Copyright © 2008 AGA Institute Terms and Conditions