In alcoholic hepatitis, abnormal hepatic gene expression in methionine and GSH metabolism occurs and often contributes to decreased hepatic methionine, S-adenosylmethionine, cysteine, and GSH levels. It may be important to replenish these thiols in patients hospitalized with alcoholic hepatitis.

Background & Aims-Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2β) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5′-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2.

GSH synthesis occurs via two enzymatic steps catalyzed by glutamate-cysteine ligase (GCL, made up of two subunits) and GSH synthetase (GS). Recently, we described coordinate induction of GCL subunits and GS. To study GS transcriptional regulation, we have cloned and characterized a 2.2-kb 5-flanking region of the rat GS (GenBank TM accession number AF333982). One transcriptional start site is located at 51 nucleotides upstream of the translational start site. The rat GS promoter drove efficiently luciferase expression in H4IIE cells. Sequential deletion analysis revealed DNA regions that are involved in positive and negative regulation. One repressor identified was NF1. tert-Butylhydroquinone (TBH) exerted a dose-and time-dependent increase in the mRNA level and promoter activity of both GCL subunits and GS. TBH increased protein binding to several regions of the GS promoter, c-jun expression, and activator protein 1 (AP-1) binding activity to several of the putative AP-1-binding sites of the GS promoter. Blocking AP-1 binding with dominant-negative c-jun led to decreased basal expression and significantly blocked the TBH-induced increase in promoter activity and mRNA level of all three genes. In conclusion, AP-1 is required for basal expression of GCL and GS; while NF1 serves as a repressor of GS, increased AP-1 transactivation is the predominant mechanism for coordinate induction of GCL and GS expression by TBH.GSH is the main non-protein thiol in mammalian cells that participates in many critical cellular functions including antioxidant defense and cell growth (1-3). The synthesis of GSH from its constituent amino acids involves two ATP-requiring enzymatic steps: the formation of ␥-glutamylcysteine from glutamate and cysteine, and formation of GSH from ␥-glutamylcysteine and glycine. The first step of GSH biosynthesis is generally regarded as rate-limiting and catalyzed by glutamate-cysteine ligase (GCL, 1 also known as ␥-glutamylcysteine synthetase), whereas the second step is catalyzed by GSH synthetase (GS) (1). The GCL enzyme is composed of a catalytic (GCLC, M r ϳ73,000) and a modifier (GCLM, M r ϳ30,000) subunit that are encoded by different genes and dissociate under reducing conditions (4 -6). The catalytic subunit exhibits all of the catalytic activity of the isolated enzyme as well as feedback inhibition by GSH (6). The modifier subunit is enzymatically inactive but plays an important regulatory function by lowering the K m values of GCL for glutamate and raising the K i value for GSH (5, 7). Because GCL is a major determinant of the overall GSH synthesis capacity, regulation of GCL subunits has been a topic of extensive research (1). Changes in GCL activity can result from regulation at multiple levels affecting only the catalytic or modifier subunit or both. Both human and rat GCL promoters have been cloned (8 -12). Antioxidant response element (ARE, also known as electrophile-response element) and activator protein 1 (AP-1) are two cis-acting elements present in the promoter of both human GCL sub...

Two genes (MAT1A and MAT2A) encode for methionine adenosyltransferase (MAT), an essential cellular enzyme responsible for S-adenosylmethionine biosynthesis. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. We showed a switch from MAT1A to MAT2A expression in human hepatocellular carcinoma (HCC), which facilitates cancer cell growth. Using DNase I footprinting analysis, we previously identified a region in the MAT2A promoter protected from DNase I digestion in HCC. This region contains NF-B and AP-1 elements, and the present study examined whether they regulate MAT2A promoter activity. We Methionine adenosyltransferase (MAT)1 is an essential cellular enzyme that catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor and the ultimate source of the propylamine moiety used in polyamine biosynthesis (1, 2). In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, ␣1 and ␣2 (3-5). MAT1A is expressed mostly in liver, and it encodes the ␣1 subunit found in two native MAT isozymes, which are either a dimer (MAT III) or tetramer (MAT I) of this single subunit (5). MAT2A encodes for a catalytic subunit (␣2) found in a native MAT isozyme (MAT II), which is associated with a catalytically inactive regulatory subunit (␤) in lymphocytes encoded by yet a third gene (5, 6). MAT2A is widely distributed (3-5). MAT2A also predominates in the fetal liver and is progressively replaced by MAT1A during liver development (7,8). In adult liver, increased expression of MAT2A is associated with rapid growth or de-differentiation of the liver (9 -11). Using a cell line model that differs only in the type of MAT expressed, we demonstrated that a switch in MAT expression in liver cancer (from MAT1A to MAT2A) plays an important pathogenetic role by facilitating liver cancer growth (12). The influence of MAT expression on liver growth and injury was further demonstrated using a MAT1A knockout mouse model (13,14). In this model, absence of hepatic MAT1A is compensated by induction of MAT2A. These animals exhibit chronic hepatic SAMe deficiency, are prone to liver injury and develop spontaneous hepatocellular carcinoma (HCC) (13,14).Given the importance of MAT expression in liver disease and cancer, we have been interested in understanding transcriptional regulation of MAT genes. We characterized the promoter region of both human MAT genes (15, 16) and showed previously that in human HCC, both promoter hypomethylation (17) and increased expression of c-Myb and Sp1 with subsequent trans-activation of the MAT2A promoter contribute to transcriptional up-regulation of MAT2A in HCC (18). In the latter work we described increased protein binding to the MAT2A promoter region (Ϫ354 to Ϫ312) in HCC as compared with normal liver (18). Two other consensus elements in this DNase I protected region are nuclear factor kappa B (NF-B) and activator protein 1 (AP-1). We had previously observed that nuclear binding of NF-B and AP-1 to the promoter of huma...