GNMT Expression Increases Hepatic Folate Contents and Folate-Dependent Methionine Synthase-Mediated Homocysteine Remethylation
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GNMT Expression Increases Hepatic Folate Contents and Folate-Dependent Methionine Synthase-Mediated Homocysteine Remethylation Yi-Cheng Wang,1 Yi-Ming Chen,2* Yan-Jun Lin,1 Shih-Ping Liu,2 and En-Pei Isabel Chiang1 1Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan, R.O.C; 2Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, R.O.C. Glycine N-methyltransferase (GNMT) is a major hepatic enzyme that converts S-adenosylmethionine to S-adenosylhomocys- teine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. GNMT is also a major hepatic folate binding protein that binds to, and, subsequently, may be inhibited by 5-methyltetrafolate. GNMT is commonly diminished in human hepatoma; yet its role in cellular folate metabolism, in tumorigenesis and antifolate ther- apies, is not understood completely. In the present study, we investigated the impacts of GNMT expression on cell growth, folate status, methylfolate-dependent reactions and antifolate cytotoxicity. GNMT–diminished hepatoma cell lines transfected with GNMT were cultured under folate abundance or restriction. Folate-dependent homocysteine remethylation fluxes were investi- gated using stable isotopic tracers and gas chromatography/mass spectrometry. Folate status was compared between wild-type (WT), GNMT transgenic (GNMTtg ) and GNMT knockout (GNMTko ) mice. In the cell model, GNMT expression increased folate con- centration, induced folate-dependent homocysteine remethylation, and reduced antifolate methotrexate cytotoxicity. In the mouse models, GNMTtg had increased hepatic folate significantly, whereas GNMTko had reduced folate. Liver folate levels corre- lated well with GNMT expressions (r = 0.53, P = 0.002); and methionine synthase expression was reduced significantly in GNMTko, demonstrating impaired methylfolate-dependent metabolism by GNMT deletion. In conclusion, we demonstrated novel findings that restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT ex- pression in vivo improves folate retention and bioavailability in the liver. Studies on how GNMT expression impacts the distribution of different folate cofactors and the regulation of specific folate dependent reactions are underway. © 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2010.00243 INTRODUCTION ating methionine from homocysteine hepatic folate-binding protein (2,3) that Different forms of folate serve as carri- remethylation (1). binds to, and, subsequently, may be in- ers of one-carbon units in DNA synthesis Glycine N-methyltransferase (GNMT, hibited by 5-methyl-THF (2). The expres- and biological methylation in mammals. EC2.1.1.20) is an abundant liver protein sion and function of GNMT have been in- The 10-formyl tetrahydrofolate is re- that converts S-adenosylmethionine to vestigated in human diseases (4–7) and quired for purine synthesis whereas the S-adenosylhomocysteine while generat- mouse models (8–10). Downregulation of 5,10-methylenetetrahydrofolate is essen- ing sarcosine from glycine. The GNMT GNMT has also been reported in human tial for pyrimidine synthesis. Among all reaction serves as an alternative pathway hepatocellular carcinoma. Loss of het- forms of folates, the 5-methyl tetrahydro- to regulate the S-adenosylmethionine to erozygosity within the GNMT gene in the folate (5-methyl-THF) is the most abun- S-adenosylhomocysteine balance and liver tissues of hepatocellular carcinoma dant that transfers the methyl group to availability of methyl group in mam- patients has been reported, and GNMT the enzyme methionine synthase, gener- malian cells (2). GNMT is also a major alteration appears to be an early event in human hepatocellular carcinoma (11). GNMT is commonly diminished in *Y-MC is a co–first author. human hepatoma and hepatoma cell lines Address correspondence and reprint requests to En-Pei Chiang, National Chung Hsing (4–10); hence it is believed to be a suscep- University, Taiwan, 250 Kuo-Kuang Road, Taichung, Taiwan 402, R.O.C. Phone: +886-4- tibility gene and a potential tumor sup- 22840385 ext 2190; Fax: +886-4-22876211; E-mail: [email protected]. pressor for human hepatoma (11). Indi- Submitted November 30, 2010; Accepted for publication December 30, 2010; Epub viduals with mutant GNMT and GNMT (www.molmed.org) ahead of print January 3, 2011. knockout mice showed that inactivation 486 | WANG ET AL. | MOL MED 17(5-6)486-494, MAY-JUNE 2011 RESEARCH ARTICLE of GNMT had significant impacts on enzyme that catalyzes the irreversible con- restoring GNMT protein on folate metab- methyl donor S-adenosylmethionine sup- version of 5,10-methylene- tetrahydrofolate olism. Establishing stable clones with and plies as well as the S-adenosylmethionine to 5-methyl-THF, is inhibited by without GNMT was done in the human to S-adenosylhomocysteine balance S-adenosylmethionine (22–23). In mam- hepatoblastoma cell line HepG2 by trans- (8–10). Deletion of GNMT increased the mals, the only known reaction that requires fection and hygromycin (300 μg/mL) se- susceptibility of liver cancer in mice (10). 5-methyl-THF is the synthesis of methion- lection (25). A stable cell line, cotrans- The presence of GNMT in the liver and ine from homocysteine (16). On the other fected with pGNMT and pTK-Hyg kidney implies that this protein could hand, 5-methyl-THF is tightly bound to (Clontech, Palo Alto, CA, USA) plasmid participate in gluconeogenesis (12). The GNMT, and such binding inhibits GNMT DNAs, was used to represent cells with function of GNMT in extra hepatic tis- (24). Both we and others have shown that normal GNMT function (GNMT+). The sues is less clear. GNMT also is present in GNMT deletion led to abnormally high other stable cell line that cotransfected pancreas, prostate, intestinal mucosa, elevations in S-adenosylmethionine and with pFLAG-CMV-5 and pTK-Hyg plas- plasma and semen (12–17). The localiza- altered methylation status in vivo (8–10). mids was used to represent cells with di- tion of GNMT in the exocrine cells in Via its regulation in intracellular minished GNMT (GNMT–). GNMT ex- the above tissues suggests a potential role S-adenosyl methionine homeostasis pressions were confirmed by Western in secretion (12). GNMT serves as an al- and/or its tight binding to folate enzymes, blot analyses in both cell lines. ternative mechanism for utilizing it is plausible that GNMT can alter the To investigate the impacts of GNMT S-adenosylmethionine that does not nec- availability of cellular folate cofactors and expression on folate status, intracellular essarily involve the methylation of physi- further affect folate- dependent reactions. folate contents and folate-dependent ologically important acceptors (12). The In the present study, we hypothesized homocysteine remethylation fluxes were crystal structure of rat GNMT demon- that deletion of GNMT will result in low compared between GNMT+ and GNMT– strated two 5-methyl-THF binding sites intracellular folate levels owing to de- cells under folate-adequate (regular located in the intersubunit areas of the creased folate retention in the liver; and αMEM media containing 2.2 μmol/L tetramer (18). Each folate binding site the loss of GNMT can cause folate defi- folic acid) and folate-restricted consists of two 1–7 N-terminal regions of ciency specifically in those tissues origi- (10 nmol/L folinate) conditions. Cells one pair of subunits and two 205–218 re- nally expressing this folate binding pro- were grown in α-MEM containing 10% gions of the other pair of subunits, thus, tein. Alternatively, the tight binding (v/v) FCS, 0.12% NaHCO3, penicillin each GNMT tetramer binds two folate between GNMT and 5-methyl-THF might (100,000 units/L), streptomycin molecules. The N-terminal fragments of reduce the availability or bioactivity of (100 mg/L), amphotericin (0.25 mg/mL) GNMT need significant conformational the enzyme-bound 5-methyl-THF, result- and 5% CO2 in an incubator at 37°C. The freedom to provide access to the active ing in a decrease in the 5-methyl-THF- medium was replaced every 72 h. The sites for folate binding as well as for the dependent homocysteine remethylation treatments, including adequate folate, inhibition by 5-methyl-THF (18). Although and methionine synthase expression. Im- and low folate (10 nmol/L folinate), were the binding between GNMT and 5-methyl- pacts of GNMT expression on folate sta- treated for 144 h. Cellular folate, THF has been well characterized, the sig- tus were investigated both in vitro and in S-adenocylmethionine and S-adenosyl - nificance of maintaining optical folate sta- vivo using cell lines with and without homocysteine concentrations were deter- tus in human pathological conditions is GNMT transfection and by using genetic mined as described previously (21). less clear, and the specific impact of mouse models with GNMT transgene or GNMT expression on hepatic methyl- GNMT disruption. The 5-methyl-THF de- Stable Isotope Tracer Studies folate-dependent reactions has not been pendent methionine synthesis metabolic GNMT-expressing HepG2 cells elucidated. In recent years, stable isotope fluxes were investigated in cell models (GNMT+) and the negative control tracers have been widely utilized to eluci- with and without GNMT expression. The (GNMT–) cells were cultured under con- date how specific folate enzymes mediate