Volume 127, Issue 4, Pages 1174-1188 (October 2004) Regulation of hepatic stellate cell activation and growth by transcription factor myocyte enhancer factor 2  Xuemin Wang, Xiaoli Tang, Xiaoming Gong, Efsevia Albanis, Scott L. Friedman, Zixu Mao  Gastroenterology  Volume 127, Issue 4, Pages 1174-1188 (October 2004) DOI: 10.1053/j.gastro.2004.07.007 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Induction of MEF2 expression and activity during culture-induced activation of primary HSCs. (A) Relative levels of MEF2A, MEF2C, and MEF2D during HSC activation. The levels of MEF2 mRNA (MEF2A, top panel; MEF2C, middle panel; MEF2D, bottom panel) were determined by reverse-transcription PCR using total RNA prepared from HSCs. 18S RNA was used as a quantity control of RNA preparations. Day indicates the number of days in culture (day 0 means freshly isolated HSCs never attached to dishes). (B) Expression of MEF2 protein during HSC activation. The levels of MEF2A, MEF2C, and MEF2D protein were determined by Western blot using isoform-specific antibodies. The expression of activation marker α-SMA and loading control GAPDH was determined by Western blot analysis as indicated. Day indicates the number of days in culture. (C) Increase in MEF2 DNA binding activity during HSC activation. MEF2 DNA binding activity was determined by electrophoretic mobility shift assay using nuclear extracts prepared from cultured HSCs at the date indicated. Isoform-specific anti-MEF2A (lanes 9 and 10) and MEF2D (lanes 11 and 12) antibodies were used for supershift assays. An irrelevant antibody was used as control (lane 13). mt probe, a probe with a mutated MEF2 binding site; SSC, supershifted complex of MEF2 and DNA; NS, nonspecific complex. The experiment was repeated 5 times. (D) Increase in MEF2-dependent gene transactivation activity during HSC activation. MEF2-dependent luciferase reporter gene assay was determined at different days as indicated 48 hours after transfection. Values shown are relative fold of luciferase activity after adjusting for transfection efficiency. wt, wild-type MEF2 luciferase reporter; mt, the reporter with MEF2 binding sites mutated (n = 3; **P < 0.01 vs. wt reporter at day 3). Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Reduction of MEF2 expression and activity during Matrigel-induced HSC-T6 deactivation. (A) Matrigel-induced reduction of MEF2 protein expression. The expression of MEF2A and MEF2C as well as MEF2D (top panel), α-SMA, and control GAPDH (bottom panel) was analyzed as described in the legend to Figure 1. Time indicates the length of HSC-T6 cells plated on Matrigel. (B) Matrigel-induced inhibition of MEF2 DNA binding activity. MEF2 DNA binding activity was determined as described in the legend to Figure 1C following Matrigel treatment as described in A. The experiment was repeated 3 times. Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Transcriptional regulation of HSC activation by MEF2. (A) Diagram of the MEF2 region targeted by RNAi. The nucleotide sequence between positions 242 and 262 is shown and targeted in RNAi. (B) Quantitative real-time reverse-transcription PCR analysis of MEF2 mRNA. HSCs were transfected as described in Materials and Methods at day 5 with or without cotransfection of RNAi and analyzed for MEF2 mRNA levels (n = 3; *P < 0.05 vs. vector control without RNAi). (C) Inhibition of MEF2-dependent gene expression by MEF2 RNAi in HSCs. HSCs were transfected with MEF2 reporter and RNAi. Relative fold of luciferase activity is shown (n = 3; **P < 0.01 vs. wild-type MEF2 reporter control without RNAi). (D) Quantitative real-time reverse-transcription PCR analysis of mRNA levels for α-SMA, collagen α1(I), and PCNA in HSCs. Experiments were performed as described in B. (E) Quantitative real-time reverse-transcription PCR analysis of mRNA levels for interferon responsive gene OAS1. Experiments were performed as described in B. Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Regulation of α-SMA expression by MEF2. (A) HSCs transfected with vectors as indicated at day 5 were separated from untransfected cells by GFP signal-based FACS. The levels of α-SMA and GAPDH following transfection were determined by Western blot as described in the legend to Figure 1D. (B) Inhibition of α-SMA expression by MEF2 RNAi in HSCs. HSCs were cotransfected with GFP with or without RNAi cotransfection. Transfected cells were separated from untransfected cells by FACS, and the levels of α-SMA and GAPDH were determined by Western blot. Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 MEF2-induced increase in HSC proliferation and cell cycle progression by MEF2. (A) MEF2 enhanced HSC proliferation. HSCs transduced with Ad-GFP or Ad-MEF2C-VP16 were assayed for cellular proliferation by either measuring the levels of dehydrogenase (WST-8 kit) (left panel) or direct cell number counting (right panel). Data are shown as mean ± SD (n = 3; **P < 0.01 vs. controls). (B) MEF2 enhanced the cell cycle progression of HSCs. Cells cultured in medium with 10% fetal calf serum (left panel) or in serum-deprived medium (0%) for 24 hours (right panel) were transduced as described in the legend to Figure 4A and then kept in media with 10% serum for 48 hours (left panel) or 1% serum for 24 hours (right panel). Cell cycle analysis was performed as described in Materials and Methods. Data show the percentage of cells in each phase of the cell cycle of one representative experiment (the experiment was repeated 3 times). Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 MEF2-induced increase in collagen α1(I) promoter activity and mRNA expression. (A) Increase in collagen α1(I) promoter by MEF2. Cultured primary HSCs were transfected with collagen reporter along with vectors for pcDNA3 control, MEF2D, or MEF2C-VP16 at day 5 in culture. After 24 hours, luciferase activity was determined (n = 3; **P < 0.01 vs. pcDNA3). (B) Increase in collagen α1(I) mRNA expression. Primary HSCs were transduced with Ad-GFP or Ad-MEF2C-VP16 at day 5 in culture. After 2 days, total RNA was extracted and the level of collagen α1(I) mRNA was determined by real-time PCR. Values shown were relative fold of mRNA expression compared with Ad-GFP (n = 3; **P < 0.01). Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 Regulation of MEF2 activity and α-SMA expression by p38 MAPK in HSCs. (A) Inhibition of p38 MAPK but not ERK reduced MEF2 transactivation activity. Primary HSCs on day 5 in culture were transfected with a vector for MEF2 luciferase reporter for 12 hours and then treated with vehicle (dimethyl sulfoxide), PD98059 (10 μmol/L), SB203580 (10 μmol/L, 20 μmol/L), or SB202190 (25 μmol/L). Luciferase assay was performed 36 hours later (left panel) (n = 3; **P < 0.01 vs. wild-type reporter treated with dimethyl sulfoxide). The effect of PD98059 on the inhibition of MEK1/2 was determined by Western blot using anti-phospho-ERK antibody (top, right panel). The bottom of the right panel shows the amount of ERK determined by anti-ERK antibody. (B) Inhibition of p38 MAPK reduced α-SMA expression. For long-term treatment, HSCs after day 1 in culture were treated with dimethyl sulfoxide, SB203580 (10 μmol/L), SB202190 (25 μmol/L), PD98059 (10 μmol/L) (left panel), or SB203580 at different concentrations (middle panel) for 24 hours. Medium was changed every other day, and at day 7 whole cell lysate was prepared and analyzed for α-SMA and GAPDH by Western blot. For transient treatment, HSCs at day 4 in culture were treated with dimethyl sulfoxide or SB203580 at a different dose for 48 hours (right panel). Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 8 Increase in MEF2 expression in rat liver following TAA-induced fibrosis. Rat liver fibrosis was induced by TAA following the procedure described in Materials and Methods. The whole cell lysates were prepared from liver tissues of 3 groups of TAA-treated and normal control rats. The levels of MEF2A, MEF2C, and MEF2D protein were determined by Western blot analysis. Numbers 1, 2, and 3 indicate the individual rat used. Gastroenterology 2004 127, 1174-1188DOI: (10.1053/j.gastro.2004.07.007) Copyright © 2004 American Gastroenterological Association Terms and Conditions