Role of AP-1 in the Coordinate Induction of Rat Glutamate-cysteine Ligase and Glutathione Synthetase bytert-Butylhydroquinone

Abstract

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...

Induction of Human Methionine Adenosyltransferase 2A Expression by Tumor Necrosis Factor α

“…At the end of the TNF␣ treatment, total RNA was extracted, and Northern hybridization analysis was performed using specific MAT2A cDNA probe as described previously (18). Northern hybridization analysis was also performed using a specific c-Jun cDNA probe as we described (22), and a p65 cDNA probe that corresponds to nucleotides 481-1032 of the published human p65 sequence (23). The p65 cDNA probe was obtained by reverse transcription and PCR using a one step RT-PCR kit (Clontech).…”

“…Representative DNase I footprinting is shown. using the SuperFect Transfection Reagent (Qiagen, Valencia, CA) as we described previously (22). To control for transfection efficiency, cells were co-transfected with the Renilla phRL-TK vector (Promega, Madison, WI), which is a plasmid containing Renilla luciferase gene driven by HSV-TK promoter.…”

“…Electrophoretic Mobility Shift and Supershift Assays-EMSAs for the putative binding sites were done as described previously (18,22). The probes are 32 P-end-labeled double-stranded DNA fragments (Ϫ354 to Ϫ326, which is the probe used in our previous study (18)), NFB site at Ϫ326: 5Ј-AGTTGAAAGGGAGGTGCCAGGC-3Ј, AP-1 site at Ϫ318: 5Ј-GGCTTTGCCATGTCACCGGAA-3Ј, where the MAT2A sequence is underlined, and mutant probes of these two putative binding sites.…”

“…Mutated probes from these two sites are: NF-B wild type, 5Ј-GGGAGGTGC-3Ј; 1-bp mutation, 5Ј-GGGTGGTGC-3Ј; 2-bp mutation, 5Ј-GGGTAGTGC-3Ј; 3-bp mutation, 5Ј-GGATAGTGC-3Ј and 4-bp mutation, 5Ј-GGATAATGC-3Ј; AP-1 wild type, 5Ј-TGCCATGTCAC-3Ј; 1-bp mutation, 5Ј-TGCCACGTCAC-3Ј; 2-bp mutation, 5Ј-TGCCACTTCAC-3Ј; 3-bp mutation, 5Ј-TGCCTCTTCAC-3Ј; and 4-bp mutation, 5Ј-TGC-CTCTACAC-3Ј. Further confirmation of the identity of the binding proteins was done by antibody supershift assays for NF-1, Nrf-2, p65, and c-Jun (Biotechnology, Lake Placid, NY, or Santa Cruz Biotechnology Inc., Santa Cruz, CA) as we described (18,22).…”

Nrf2-Keap1 Signaling Pathway Regulates Human UGT1A1 Expression in Vitro and in Transgenic UGT1 Mice

“…When oxidative or xenobiotic stimuli occur, Nrf2 and Keap1 dissociate, and Nrf2 migrates to the nucleus where it functions as a strong transcriptional activator of selected target genes (20). Therefore, agents that inhibit the interaction between Keap1 and Nrf2 lead to an accumulation of the Nrf2 transcription factor in the nucleus followed by enhanced expression of target genes (16,21,22).…”