Title: S-Adenosylmethionine Blocks Collagen I Production by Preventing Transforming Growth Factor-β Induction of the COL1A2 Promoter
Abstract: To study the antifibrogenic mechanisms of S-adenosylmethionine (AdoMet), transgenic mice harboring the -17 kb to +54 bp of the collagen α2 (I) promoter (COL1A2) cloned upstream from the β-gal reporter gene were injected with carbon tetrachloride (CCl4) to induce fibrosis and coadministered either AdoMet or saline. Control groups received AdoMet or mineral oil. AdoMet lowered the pathology in CCl4-treated mice as shown by transaminase levels, hematoxylin and eosin, Masson's trichrome staining, and collagen I expression. β-Galactosidase activity indicated activation of the COL1A2 promoter in stellate cells from CCl4-treated mice and repression of such activation by AdoMet. Lipid peroxidation, transforming growth factor-β (TGFβ) expression, and decreases in glutathione levels were prevented by AdoMet. Incubation of primary stellate cells with AdoMet down-regulated basal and TGFβ-induced collagen I and α-smooth muscle actin proteins. AdoMet metabolites down-regulated collagen I protein and mRNA levels. AdoMet repressed basal and TGFβ-induced reporter activity in stellate cells transfected with COL1A2 promoter deletion constructs. AdoMet blocked TGFβ induction of the -378 bp region of the COL1A2 promoter and prevented the phosphorylation of extracellular signal-regulated kinase 1/2 and the binding of Sp1 to the TGFβ-responsive element. These observations unveil a novel mechanism by which AdoMet could ameliorate liver fibrosis. To study the antifibrogenic mechanisms of S-adenosylmethionine (AdoMet), transgenic mice harboring the -17 kb to +54 bp of the collagen α2 (I) promoter (COL1A2) cloned upstream from the β-gal reporter gene were injected with carbon tetrachloride (CCl4) to induce fibrosis and coadministered either AdoMet or saline. Control groups received AdoMet or mineral oil. AdoMet lowered the pathology in CCl4-treated mice as shown by transaminase levels, hematoxylin and eosin, Masson's trichrome staining, and collagen I expression. β-Galactosidase activity indicated activation of the COL1A2 promoter in stellate cells from CCl4-treated mice and repression of such activation by AdoMet. Lipid peroxidation, transforming growth factor-β (TGFβ) expression, and decreases in glutathione levels were prevented by AdoMet. Incubation of primary stellate cells with AdoMet down-regulated basal and TGFβ-induced collagen I and α-smooth muscle actin proteins. AdoMet metabolites down-regulated collagen I protein and mRNA levels. AdoMet repressed basal and TGFβ-induced reporter activity in stellate cells transfected with COL1A2 promoter deletion constructs. AdoMet blocked TGFβ induction of the -378 bp region of the COL1A2 promoter and prevented the phosphorylation of extracellular signal-regulated kinase 1/2 and the binding of Sp1 to the TGFβ-responsive element. These observations unveil a novel mechanism by which AdoMet could ameliorate liver fibrosis. S-Adenosylmethionine (AdoMet) 1The abbreviations used are: AdoMet, S-adenosylmethionine; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AU, arbitrary units; CAT, chloramphenicol acetyltransferase; CCl4, carbon tetrachloride; C/EBP, CAAT/enhancer-binding protein; COL1A1, collagen α1 (I) promoter; COL1A2, collogen α2 (I) promoter; CYP2E1, cytochrome P450 2E1; ERK1/2, extracellular signal-regulated kinase 1/2;β-gal, β-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; HSC, hepatic stellate cells; MO, mineral oil; MTA, 5′-deoxy-5′-(methylthio)adenosine; pCα, procollagen α; pERK1/2, phosphorylated ERK1/2; ROS, reactive oxygen species; SAH, S-adenosylhomocysteine; α-Sma, α-smooth muscle actin; TGFβ, transforming growth factor-β; TNFα, tumor necrosis factor-α. is the principal biologic methyl donor, the precursor of aminopropyl groups used in polyamine biosynthesis in liver, and the precursor of reduced glutathione (GSH) through its conversion to cysteine by means of the transsulfuration pathway (1Finkelstein J.D. Am. J. Clin. Nutr. 2003; 77: 1094-1095Crossref PubMed Scopus (59) Google Scholar). Under normal conditions, most of the AdoMet generated is used in transmethylation reactions, in which methyl groups are added to compounds and AdoMet is converted to S-adenosylhomocysteine (SAH) (2Mato J.M. Alvarez L. Ortiz P. Pajares M.A. Pharmacol. Ther. 1997; 73: 265-280Crossref PubMed Scopus (412) Google Scholar). In alcoholic liver disease, many of the enzymatic steps in methionine metabolism are affected (3Lu S.C. Tsukamoto H. Mato J.M. Alcohol. 2002; 27: 155-162Crossref PubMed Scopus (80) Google Scholar). In the intragastric infusion model of ethanol feeding, hepatic levels of methionine, AdoMet, and DNA methylation decrease by ∼40% (4Huang Z.Z. Mao Z. Cai J. Lu S.C. Am. J. Physiol. 1998; 275: G14-G21PubMed Google Scholar). The hepatic methionine level depends on the stage of liver injury in rats fed ethanol (5Barak A.J. Beckenhauer H.C. Junnila M. Tuma D.J. Alcohol Clin. Exp. Res. 1993; 17: 552-555Crossref PubMed Scopus (167) Google Scholar). Reduced AdoMet levels and methylation can affect gene expression, membrane fluidity, and GSH levels in liver (2Mato J.M. Alvarez L. Ortiz P. Pajares M.A. Pharmacol. Ther. 1997; 73: 265-280Crossref PubMed Scopus (412) Google Scholar, 6Lieber C.S. Casini A. DeCarli L.M. Kim C.I. Lowe N. Sasaki R. Leo M.A. Hepatology. 1990; 11: 165-172Crossref PubMed Scopus (314) Google Scholar, 7Colell A. Garcia-Ruiz C. Morales A. Ballesta A. Ookhtens M. Rodes J. Kaplowitz N. Fernandez-Checa J.C. Hepatology. 1997; 26: 699-708PubMed Google Scholar). Rats with decreased hepatic levels of AdoMet are predisposed to liver injury caused by lipopolysaccharide, and this effect is prevented by exogenous AdoMet treatment (8Chawla R.K. Watson W.H. Eastin C.E. Lee E.Y. Schmidt J. McClain C.J. Am. J. Physiol. 1998; 275: G125-G129PubMed Google Scholar). AdoMet has been used increasingly for the treatment of liver diseases, although the protective mechanisms remain unclear and are likely to be multiple. Impaired mitochondrial uptake of GSH has been postulated to be an important pathogenic factor in alcoholic liver injury. AdoMet administration restores GSH uptake into mitochondria in rats fed ethanol for 4 weeks by preventing changes in mitochondrial membrane fluidity (7Colell A. Garcia-Ruiz C. Morales A. Ballesta A. Ookhtens M. Rodes J. Kaplowitz N. Fernandez-Checa J.C. Hepatology. 1997; 26: 699-708PubMed Google Scholar). AdoMet inhibits tumor necrosis factor-α (TNFα) release from macrophages (8Chawla R.K. Watson W.H. Eastin C.E. Lee E.Y. Schmidt J. McClain C.J. Am. J. Physiol. 1998; 275: G125-G129PubMed Google Scholar), and it has also been used to prevent the development of hepatocarcinogenesis (9Pascale R.M. Marras V. Simile M.M. Daino L. Pinna G. Bennati S. Carta M. Seddaiu M.A. Massarelli G. Feo F. Cancer Res. 1992; 52: 4979-4986PubMed Google Scholar) and to increase survival in patients with alcoholic liver disease (10Mato J.M. Camara J. Fernandez de Paz J. Caballeria L. Coll S. Caballero A. Garcia-Buey L. Beltran J. Benita V. Caballeria J. Sola R. Moreno-Otero R. Barrao F. Martin-Duce A. Correa J.A. Pares A. Barrao E. Garcia-Magaz I. Puerta J.L. Moreno J. Boissard G. Ortiz P. Rodes J. J. Hepatol. 1999; 30: 1081-1089Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Excessive collagen I accumulation is the histopathologic hallmark of liver fibrosis. Central to the development and progression of fibrosis are cytokines that are normally involved in matrix remodeling; among them, transforming growth factor-β (TGFβ) enhances collagen I production and inhibits the synthesis of proteolytic enzymes that catalyze extracellular matrix degradation while enhancing the expression of protease inhibitors (11Inagaki Y. Truter S. Ramirez F. J. Biol. Chem. 1994; 269: 14828-14834Abstract Full Text PDF PubMed Google Scholar, 12Inagaki Y. Truter S. Tanaka S. Di Liberto M. Ramirez F. J. Biol. Chem. 1995; 270: 3353-3358Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). As a result, TGFβ is believed to play a critical role in liver fibrosis. There are no data as to whether administration of AdoMet may affect levels of TGFβ itself and/or the actions of TGFβ on collagen I expression and fibrosis. The current study focused on analyzing potential mechanisms by which administration of AdoMet could decrease collagen I deposition under fibrogenic stimuli. Treatment with AdoMet was found to have a repressive effect on the COL1A2 promoter both in vivo and in vitro. We now propose that these antifibrogenic effects of AdoMet appear to be mediated, at least in part, by lowering TGFβ levels and by inhibiting TGFβ binding to the COL1A2 promoter through a Sp1-pERK1/2 coupled mechanism. Most reagents unless otherwise stated were purchased from Sigma. Protein concentration was determined by the method of Lowry et al. (13Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using the DC-20 protein assay kit (Bio-Rad). Animal Study Design—Transgenic mice harboring the -17 kb to +54 bp of the proximal promoter of the mouse COL1A2 gene cloned upstream from the Escherichia coli β-gal reporter gene (LacZ) were used. These transgenic mice were obtained from Dr. Benoit de Crombrugghe (Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston) (14Antoniv T.T. De Val S. Wells D. Denton C.P. Rabe C. de Crombrugghe B. Ramirez F. Bou-Gharios G. J. Biol. Chem. 2001; 276: 21754-21764Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Bou-Gharios G. Garrett L.A. Rossert J. Niederreither K. Eberspaecher H. Smith C. Black C. Crombrugghe B. J. Cell Biol. 1996; 134: 1333-1344Crossref PubMed Scopus (135) Google Scholar, 16Kinbara T. Shirasaki F. Kawara S. Inagaki Y. de Crombrugghe B. Takehara K. J. Cell. Physiol. 2002; 190: 375-381Crossref PubMed Scopus (21) Google Scholar, 17Inagaki Y. Nemoto T. Kushida M. Sheng Y. Higashi K. Ikeda K. Kawada N. Shirasaki F. Takehara K. Sugiyama K. Fujii M. Yamauchi H. Nakao A. de Crombrugghe B. Watanabe T. Okazaki I. Hepatology. 2003; 38: 890-899Crossref PubMed Google Scholar, 18De Val S. Ponticos M. Antoniv T.T. Wells D.J. Abraham D. Partridge T. Bou-Gharios G. J. Biol. Chem. 2002; 277: 9286-9292Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Mice were bred in our institution and received humane care in compliance with the guidelines of the National Institutes of Health and the Animal Care Committee of Mount Sinai School of Medicine. Chronic liver injury was induced by injecting intraperitoneally 5 ml of CCl4/kg of body weight (25% v/v in mineral oil) three times a week for 4 weeks. AdoMet was administered intraperitoneally at a dose of 10 mg/kg body weight every day for 4 weeks and was always given 2 h before CCl4. Control groups were injected with mineral oil or mineral oil plus AdoMet alone. Mice were maintained at 22 °C with a 12 h light/dark cycle, were fed standard chow diet, had free access to water, and were sacrificed under pentobarbital anesthesia 48 h after the last dose of CCl4. Serum Transaminases—Blood was collected from the retro-orbital vein under anesthesia, centrifuged at 3,000 rpm for 3 min, and serum was separated to assay for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using kits from Sigma (Infinity ALT and AST). Liver Histology and Immunohistochemistry—Liver samples were fixed in 10% buffered formalin and embedded in paraffin. Five-μm sections were dehydrated and stained with hematoxylin and eosin (H&E) or Masson's trichrome and evaluated by a liver pathologist who was blinded from the experimental conditions. The Sirius red/fast green staining was carried out as according to López-De León and Rojkind (19Lopez-De León A. Rojkind M. J. Histochem. Cytochem. 1985; 33: 737-743Crossref PubMed Scopus (565) Google Scholar) as described previously (20Carmiel-Haggai M. Cederbaum A.I. Nieto N. FASEB J. 2005; 19: 136-138Crossref PubMed Scopus (266) Google Scholar). Immunofluorescence was carried out using a rabbit IgG fraction to β-gal (ICN Biomedical), the reporter protein for the COL1A2 promoter activation, and a rabbit IgG to α-smooth muscle actin (α-Sma), a specific marker for stellate cell activation. Alexa Fluor 488 dye-conjugated goat anti-rabbit antibody and Alexa Fluor 568 dye-conjugated goat anti-mouse antibody (Molecular Probes) were used for immunofluorescent staining of β-gal (corresponding to the active COL1A2 promoter) and α-Sma, respectively, and colocalization by overlaying both stainings using Adobe Photoshop computer software. TGFβ immunostaining was carried out using a TGFβ monoclonal antibody (Sigma) and a ImmunoCruz staining system. Immunoblotting and Western Blot Analysis—Immunoblotting and Western blot analysis were performed as described previously (21Nieto N. Friedman S.L. Greenwel P. Cederbaum A.I. Hepatology. 1999; 30: 987-996Crossref PubMed Scopus (161) Google Scholar). Anti-collagen type I antibody (1/5,000) was kindly provided by Dr. Detlef Schuppan (Harvard Medical School) (22Rombouts K. Niki T. Greenwel P. Vandermonde A. Wielant A. Hellemans K. De Bleser P. Yoshida M. Schuppan D. Rojkind M. Geerts A. Exp. Cell Res. 2002; 278: 184-197Crossref PubMed Scopus (109) Google Scholar, 23Niki T. Rombouts K. De Bleser P. De Smet K. Rogiers V. Schuppan D. Yoshida M. Gabbiani G. Geerts A. Hepatology. 1999; 29: 858-867Crossref PubMed Scopus (177) Google Scholar). Procollagen type I was detected as several bands, i.e. high molecular mass chains of procollagen α1(I) and α2(I) and the N-terminally processed pCα1(I) and pCα2(I). In blots where the intracellular collagen expression was analyzed, the band labeled as collagen I corresponds to the pCα1(I) and pCα2(I), which overlap at ∼200 kDa. In the culture media, intact procollagen predominated, and only little fully processed collagen was present (22Rombouts K. Niki T. Greenwel P. Vandermonde A. Wielant A. Hellemans K. De Bleser P. Yoshida M. Schuppan D. Rojkind M. Geerts A. Exp. Cell Res. 2002; 278: 184-197Crossref PubMed Scopus (109) Google Scholar, 23Niki T. Rombouts K. De Bleser P. De Smet K. Rogiers V. Schuppan D. Yoshida M. Gabbiani G. Geerts A. Hepatology. 1999; 29: 858-867Crossref PubMed Scopus (177) Google Scholar). Anti-α-Sma and anti-TGFβ were obtained from Sigma. Anti-cytochrome P450 2E1 (CYP2E1) antibody was a gift from Dr. Jerome Lasker (Hackensack Biomedical Research Institute, Hackensack, NJ). Sp1, CAAT/enhancer-binding protein γ (C/EBPγ), C/EBPδ, Smad3, Smad4, ERK1/2, pERK1/2, p38, and pp38 antibodies were from Santa Cruz Biotechnologies. Goat anti-rabbit IgG and goat anti-mouse IgG (both at 1/10,000) were used as secondary antibodies (Chemicon). For the immunoblotting, pERK1/2 was immunoprecipitated with anti pERK1/2 IgG-protein G-agarose, resolved on a 10% SDS-PAGE, and immunoblotted for Sp1. The signal was detected using the ECL system (Amersham Biosciences). β-Gal Staining and Quantification—Samples were fixed in 10% formalin for 1 h, rinsed, and permeabilized in 0.1 m, pH 7.3, sodium phosphate, 2 mm MgCl2, 0.1% sodium deoxycholate, and 0.2% Nonidet P-40, incubated overnight with 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside plus 20 mm potassium ferri- and ferrocyanide in rinsing solution at 37 °C. Samples were washed in 1 × phosphate-buffered saline three times, followed by paraffin embedding and sectioning. Finally, 12-μm liver slices were deparaffinized, rehydrated, and counterstained with nuclear fast red for 1 min, rinsed, and rehydrated. Each β-gal staining was also performed using frozen sections from the same tissues that were embedded in optimal cutting temperature compound and immediately frozen using methylbutane on dry ice. Proteins were extracted from individual livers, and β-gal activity was measured by means of a chemiluminescent reporter assay (Galactolight Plus, Tropix). β-Gal activity was expressed as units/mg of protein. GSH Levels, Lipid Peroxidation, and CYP2E1 Activity—50 μg of liver was homogenized in 5% trichloroacetic acid at a ratio of 1:10 (w/v) and centrifuged for 5 min at 8,000 rpm and 4 °C. GSH levels were determined in the protein free extract by the enzymatic method of Tietze (24Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5554) Google Scholar). Levels of malondialdehyde were measured in liver homogenates using a lipid peroxidation colorimetric assay kit (Calbiochem). Catalytic activity of CYP2E1 was determined as the rate of production of p-nitrocatechol from p-nitrophenol (25Reinke L.A. Moyer M.J. Drug Metab. Dispos. 1985; 13: 548-552PubMed Google Scholar). Northern Blot Analysis—Northern blot analysis was performed as described previously (21Nieto N. Friedman S.L. Greenwel P. Cederbaum A.I. Hepatology. 1999; 30: 987-996Crossref PubMed Scopus (161) Google Scholar, 26Nieto N. Dominguez-Rosales J.A. Fontana L. Salazar A. Armendariz-Borunda J. Greenwel P. Rojkind M. Hepatology. 2001; 33: 597-607Crossref PubMed Scopus (45) Google Scholar) using cDNA probes for COL1A1 (provided by Dr. Scott L. Friedman, Mount Sinai School of Medicine) and COL1A2 (provided by Dr. Francesco Ramírez, New York Hospital for Special Surgery) and a cDNA for GAPDH, a housekeeping gene, from the ATCC. Cell Culture and Transfection Experiments—All cell culture experiments were carried out using primary hepatic stellate cells (HSC) isolated by in situ liver perfusion and Histodenz gradient centrifugation (27Nieto N. Friedman S.L. Cederbaum A.I. Hepatology. 2002; 35: 62-73Crossref PubMed Scopus (229) Google Scholar). HSC were cultured in Dulbecco's modified Eagle's medium without serum from the beginning of each treatment. The doses of AdoMet, MTA, SAH, and methionine were either 10 or 30 μm. TGFβ was used at 10 ng/ml. Reporter DNA constructs containing upstream sequences of the human COL1A2 promoter linked to the chloramphenicol acetyltransferase (CAT) gene were provided by Dr. Francesco Ramírez (New York Hospital for Special Surgery) (28Boast S. Su M.W. Ramirez F. Sanchez M. Avvedimento E.V. J. Biol. Chem. 1990; 265: 13351-13356Abstract Full Text PDF PubMed Google Scholar). In these constructs, human COL1A2 sequences span from -3500 to +58 bp (-3500COL1A2-CAT), from -772 to +58 bp (-772COL1A2-CAT), and from -378 to +58 bp (-378COL1A2-CAT) (28Boast S. Su M.W. Ramirez F. Sanchez M. Avvedimento E.V. J. Biol. Chem. 1990; 265: 13351-13356Abstract Full Text PDF PubMed Google Scholar). Parallel transfection of the corresponding empty vector pEMBL8-CAT at equivalent concentrations were performed. The total amount of plasmid DNA was equalized using pBlue-script SK- (Stratagene). Cells were plated at a density of 105/well in 6-well plates. Complexes containing FuGENE 6 (Roche Applied Science) plus plasmid DNA were prepared according to the manufacturer's instructions with a final concentration of plasmid DNA for each of the chimeric COL1A2 DNA constructs of 1 μg/ml. Parallel cotransfection with 25 ng/ml of the control pRL-null (Promega) containing the cDNA encoding for Renilla luciferase was performed to normalize for transfection efficiency. Cells were incubated in the presence of the transfection mix for 24 h, after which the media were replaced and the cells treated with 30 μm AdoMet for 24 h. Samples for the CAT activity were collected, and the reaction was run using a kit from Promega as described previously (29Nieto N. Greenwel P. Friedman S.L. Zhang F. Dannenberg A.J. Cederbaum A.I. J. Biol. Chem. 2000; 275: 20136-20145Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). TGFβ—TGFβ was measured by enzyme-linked immunosorbent assay using a kit from BIOSOURCE. Gel Mobility Shift Assays—Nuclear extracts were prepared according to the method of Dignam et al. (30Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9163) Google Scholar). For the gel mobility shift assays, a probe was generated by digestion with BglII and BstXI (-378 to -183 bp) of the -378COL1A2-CAT plasmid, which contains the TGFβ-responsive element (11Inagaki Y. Truter S. Ramirez F. J. Biol. Chem. 1994; 269: 14828-14834Abstract Full Text PDF PubMed Google Scholar, 12Inagaki Y. Truter S. Tanaka S. Di Liberto M. Ramirez F. J. Biol. Chem. 1995; 270: 3353-3358Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 31Inagaki Y. Truter S. Greenwel P. Rojkind M. Unoura M. Kobayashi K. Ramirez F. Hepatology. 1995; 22: 573-579PubMed Google Scholar, 32Zhang W. Ou J. Inagaki Y. Greenwel P. Ramirez F. J. Biol. Chem. 2000; 275: 39237-39245Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). This probe was end labeled with [γ-32P]ATP and T4 kinase (Promega). Binding reactions in a total volume of 10 μl contained 5 μg of the nuclear protein extract from HSC, 1 μl of 10 × binding buffer, 1 μg/μl poly[d(I-C)], 150 mm KCl, 0.05 mm ZnCl2, and 20,000 cpm of labeled probe at room temperature for 30 min. For competition studies, 200-fold of cold probe was added along with labeled probe. PAGE (6%) was performed at 150 V for 2 h in 0.25 × TBE buffer, the gels were dried under vacuum and exposed overnight using Kodak films. Southwestern Blot Analysis—Southwestern blot analysis was performed by the method of Singh et al. (33Singh H. LeBowitz J.H. Baldwin Jr., A.S. Sharp P.A. Cell. 1988; 52: 415-423Abstract Full Text PDF PubMed Scopus (419) Google Scholar). The membrane containing the immunoprecipitated pERK1/2 bound to the immunoblotted Sp1 was blocked in 10% nonfat dry milk in TNE buffer (50 mm Tris, pH 7.5, 40 mm NaCl, 1 mm EDTA). DNA binding was carried out for 3 h with TNE buffer containing 5 μg/ml poly[d(I-C)] and 2 × 105 cpm/ml [γ-32P]dCTP multiprime-labeled BglII and BstXI-digested fragment of the -378COL1A2-CAT plasmid containing the TGFβ-responsive element. Membranes were washed three times for 5 min each with TNE at room temperature and exposed in the PhosphorImager screen. Statistical Analysis—Analyses of variance were performed for all experiments except for those in Fig. 4B, which were analyzed by an unpaired Student's t test. Values are expressed as the means ± S.E. and are the average values of three and six experiments for the in vitro and for the in vivo studies, respectively. Induction of Liver Fibrosis in Mouse COL1A2 Promoter Transgenic Mice and Protection by AdoMet—Administration of CCl4 increased the liver to body weight ratio ∼15% over the non-CCl4-treated mice with no amelioration by AdoMet. Hepatocellular damage and protection by AdoMet were assayed by measuring serum levels of transaminases and by H&E staining. The CCl4 treatment elevated ALT and AST serum levels 7- and 3.5-fold, respectively. AdoMet lowered the elevated ALT levels in the CCl4-treated mice to control levels (Fig. 1A). H&E staining in the CCl4-treated mice revealed the presence of Mallory bodies, lymphocyte infiltration, centrilobular steatosis, and perivenular and pericellular fibrosis; AdoMet prevented these changes and minimized the pathology (Fig. 1B). Similarly, Masson's trichrome staining showed less endogenous total collagen deposition in mice treated with CCl4 plus AdoMet than in the CCl4-treated mice (Fig. 1C). Quantitative analysis of total collagen in Sirius red- and fast green-stained liver sections showed a 2.5-fold increase by CCl4, which was prevented by AdoMet treatment (Fig. 1E). Collagen I expression assessed by Western blot analysis was elevated 3-fold by the CCl4 treatment and reduced by about 50% by coadministration of AdoMet (Fig. 1D). AdoMet Prevents the Activation of the Mouse COL1A2 Promoter in Vivo—Transgenic mice harboring the -17 kb to +54 bp of the proximal promoter of the mouse COL1A2 gene cloned upstream from the E. coli β-gal LacZ reporter gene have been shown to be valuable in studies of activation of the COL1A2 gene in vivo (14Antoniv T.T. De Val S. Wells D. Denton C.P. Rabe C. de Crombrugghe B. Ramirez F. Bou-Gharios G. J. Biol. Chem. 2001; 276: 21754-21764Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Kinbara T. Shirasaki F. Kawara S. Inagaki Y. de Crombrugghe B. Takehara K. J. Cell. Physiol. 2002; 190: 375-381Crossref PubMed Scopus (21) Google Scholar, 18De Val S. Ponticos M. Antoniv T.T. Wells D.J. Abraham D. Partridge T. Bou-Gharios G. J. Biol. Chem. 2002; 277: 9286-9292Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). CCl4 activated the COL1A2 promoter as shown by the β-gal-positive blue staining (Fig. 2A). This activation was repressed in mice cotreated with AdoMet (Fig. 2A). Quantification of the β-gal activity by chemiluminescence showed an approximate 27-fold increase in β-gal activity by CCl4 which was reduced to a 5-fold increase in the presence of AdoMet (Fig. 2B). Immunofluorescence analysis of mice injected with CCl4 revealed colocalization of α-Sma, a marker for stellate cell activation, and β-gal-positive cells, indicating that the activation of the COL1A2 promoter occurred in HSC (Fig. 2D). AdoMet increased total GSH about 17% in control mice. When CCl4 was injected, a 15% decrease in GSH levels was observed compared with the mineral oil-injected mice; however, cotreatment with AdoMet restored GSH to above control levels (Fig. 3A). AdoMet decreased lipid peroxidation by-products generated under CCl4 treatment to the levels found in control mice (Fig. 3B). Immunostaining for 4-hydroxynonenal showed an extensive but diffuse pattern of positive staining, indicating development of oxidant stress in all liver cells (not shown). CYP2E1 expression and activity were lower in the CCl4-treated mice compared with the control mice because CCl4 is known to lower CYP2E1 levels via radical inactivation and lipid peroxidation (34Sohn D.H. Yun Y.P. Park K.S. Veech R.L. Song B.J. Biochem. Biophys. Res. Commun. 1991; 179: 449-454Crossref PubMed Scopus (51) Google Scholar, 35Tierney D.J. Haas A.L. Koop D.R. Arch. Biochem. Biophys. 1992; 293: 9-16Crossref PubMed Scopus (138) Google Scholar). AdoMet further decreased CYP2E1 activity and content in the presence of CCl4 (Fig. 3D); this would decrease the potential for reactive oxygen species (ROS) production. AdoMet Down-regulates Collagen I in Primary HSC—We analyzed the effect of AdoMet on the expression of collagen I protein in primary HSC. Neither cell viability (95%) nor cell proliferation assessed by [methyl-3H]thymidine incorporation was affected by AdoMet treatment (data not shown). HSC were incubated for 1, 3, 5, and 7 days in the presence or absence of 10 or 30 μm AdoMet added daily. Western blot analysis revealed a time-dependent up-regulation of collagen I production (Fig. 4A). AdoMet was very effective in lowering collagen I protein levels in cultured HSC. Part of the generated collagen I is usually secreted into the medium. AdoMet lowered basal intracellular as well as secreted collagen I protein (Fig. 4, A and B). The ability of AdoMet to prevent HSC activation in culture is shown by the 60% decrease in α-Sma levels at 7 days (Fig. 4B). Metabolites of AdoMet, such as SAH and MTA, and methionine, a precursor of AdoMet, were used at 30 μm and found to decrease collagen I protein and COL1A1 and COL1A2 mRNA levels in HSC (Fig. 4, C and D); however, the effects mediated by methionine were lower compared with those of the other treatments perhaps because of the extremely low expression of methionine adenosyltransferase 1A in HSC (36Shimizu-Saito K. Horikawa S. Kojima N. Shiga J. Senoo H. Tsukada K. Hepatology. 1997; 26: 424-431Crossref PubMed Scopus (25) Google Scholar). Transient transfection experiments with chimeric constructs harboring progressive 5′-deletions of the human COL1A2 promoter linked to the CAT reporter gene (see scheme in Fig. 5) were performed to identify the promoter regions required for the AdoMet-mediated repressive effect on collagen I production. Primary HSC were transfected with the constructs described in Refs. 28Boast S. Su M.W. Ramirez F. Sanchez M. Avvedimento E.V. J. Biol. Chem. 1990; 265: 13351-13356Abstract Full Text PDF PubMed Google Scholar and 29Nieto N. Greenwel P. Friedman S.L. Zhang F. Dannenberg A.J. Cederbaum A.I. J. Biol. Chem. 2000; 275: 20136-20145Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar or with the parental empty vector pEMBL8-CAT. As shown in Fig. 5, the basal acetylation of chloramphenicol in HSC transfected with the -3500COL1A2-CAT and the -378COL1A2-CAT plasmids was similar. On the other hand, the activity of the -772COL1A2-CAT vector was significantly lower. The -772 to -378 region of the human or mouse COL1A2 gene contains a silencer element (28Boast S. Su M.W. Ramirez F. Sanchez M. Avvedimento E.V. J. Biol. Chem. 1990; 265: 13351-13356Abstract Full Text PDF PubMed Google Scholar, 29Nieto N. Greenwel P. Friedman S.L. Zhang F. Dannenberg A.J. Cederbaum A.I. J. Biol. Chem. 2000; 275: 20136-20145Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 31Inagaki Y. Truter S. Greenwel P. Rojkind M. Unoura M. Kobayashi K. Ramirez F. Hepatology. 1995; 22: 573-579PubMed Google Scholar). Addition of 30 μm AdoMet for 24 h after transfection decreased the basal activity of both the -3500COL1A2-CAT and the -378COL1A2-CAT reporter vectors. It is interesting that AdoMet lowered CAT activity in cells transfected with the -378COL1A2-CAT construct, which contains the -378 to +58 bp region that is essential for increased basal COL1A2 expression and responsiveness to oxidative stress and cytokines such as TNFα and TGFβ (11Inagaki Y. Truter S. Ramirez F. J. Biol. Chem. 1994; 269: 14828-14834Abstract Full Text PDF PubMed Google Scholar, 12Inagaki Y. Truter S. Tanaka S. Di Liberto M. Ramirez F. J. Biol. Chem. 1995; 270: 3353-3358Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 21Nieto N. Friedman S.L. Greenwel P. Cederbaum A.I. Hepatology. 1999; 30: 987-996Crossref PubMed