Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-κB and AP-1

Abstract

Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tertButylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidantresponsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-B family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-B binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-B family members.Glutathione (GSH) is the main nonprotein thiol in mammalian cells that participates in many critical cellular functions, including antioxidant defense and cell growth (14,24,28). The synthesis of GSH from its constituent amino acids involves two ATP-requiring enzymatic steps: the formation of ␥-glutamylcysteine from glutamate and cysteine and the formation of GSH from ␥-glutamylcysteine and glycine. The first step of GSH biosynthesis is rate limiting and catalyzed by glutamatecysteine ligase (GCL, also known as ␥-glutamylcysteine synthetase), while the second step is catalyzed by GSH synthetase (14). The GCL enzyme is composed of a catalytic (GCLC, M r of ϳ73,000) and a modifier (GCLM, M r of ϳ30,000) subunit which are encoded by different genes and dissociate under reducing conditions (7,27,35). The catalytic subunit exhibits all of the catalytic activity of the isolated enzyme as well as feedback inhibition by GSH (27). The modifier subunit is enzymatically inactive but plays an important regulatory function by lowering the K m of GCL for glutamate and raising the K i for GSH (7,8). GCL is a major determinant of the overall GSH synthesis capacity, and changes in GCL activity can result from regulation at multiple levels affecting only the catalytic or modifier subunit or both (14). Both human GCLC and GCLM promoters have been cloned (4,5,16,18,34). Antioxidantresponse element (ARE, also known as electrophile response element, EpRE) and activator protein 1 (AP-1) are two cisacting elements present in the promoter of both human GCL subunits that have been implicated in their transcriptional regulation by oxidants and ␤-naphthoflavone (5, 14, 16, 18).Nrf1 and Nrf2, members of the cap 'n' collar-basic leu...

S-glutathionylation: relevance in diabetes and potential role as a biomarker

“…Thyroid hormone and cysteine deprivation induce GCLm expression (Dasgupta et al, 2007). Nrf1, Nrf2, AP-1, NFκB and c-Myc seem to be involved in the regulation of the promoter activity of the GCLm gene (Wild et al, 1999;Yang et al, 2005;Benassi et al, 2006). Interestingly, it has been shown that insulin was unable to change the levels of GCLm mRNA (Okouchi et al, 2006), while GCLc levels were affected by this stimulus in the same report.…”

“…These and other considerations point to a major regulatory function for GCLm in glutathione synthesis. Some of the above-mentioned stimuli could induce both, GCLc and GCLm (Wild et al, 1999;Yang et al, 2005). However, there is some evidence supporting the specific up-regulation of GCLm.…”

“…NFκB (indirectly through AP-1), Nrf1 and Nrf2 Yang et al, 2005). Although GCL has been described as the rate-limiting enzyme in GSH generation, this second reaction can become limiting in some situations, such as when GCL reaction is under optimal conditions and GS levels are not sufficient to transform all the γ-glutamylcysteine produced by GCL (for example, when only GS is down-regulated or absent).…”

JunB and JunD Regulate Human Heme Oxygenase-1 Gene Expression in Renal Epithelial Cells

“…Third, mutations in multiple regulatory sequences, including the ARE, CRE/AP-1, and E box, were required to abolish HO-1 promoter responsiveness. It is possible that Nrf2 activates the human HO-1 promoter indirectly through AP-1 family members as shown recently for the rat glutamate-cysteine ligase catalytic subunit gene (55). A comparison of the HS-2 region with the previously described E1 enhancer region located at Ϫ4.0 kb of the mouse Ho-1 gene (7,56) reveals that the three StRE sequences of the mouse E1 region (Fig.…”

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“…Interplay between Nrf2 and NF-jB signaling is well documented. 45,48 Additionally, small molecule Nrf2 activators have been shown to be effective in inhibiting activation of NF-jB. Xu et al 49 demonstrated that sulforaphane exposure to a human prostate cancer cell line resulted in decreased UVCinduced NF-jB activation.…”