Distinct effects of glutathione disulphide on the nuclear transcription factors κB and the activator protein‐1

Abstract

Oxidative conditions potentiate the activation of the nuclear transcription factor KB (NFKB) and the activator protein-1 (AP-1) in intact cells, but inhibit their DNA binding activity in vitro. We now show that both the activation of NFKB and the inhibition of its DNA binding activity is modulated in intact cells by the physiological oxidant glutathione disulphide (GSSG). NFKB activation in human T lineage cells (Molt-4) by 12-0-tetradecanoyl-phorbol 13-acetate was inhibited by dithiothreitol, and this was partly reversed by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1 -nitrosourea (BCNU) or by hydrogen peroxide, indicating that GSSG may be required for NFKB activation. These effects of BCNU and hydrogen peroxide were not seen in glutathionedepleted cells. However, NFKB and AP-1 activation were potentiated by dithiothreitol if added to cell cultures 1 h after the phorbol ester, indicating that a shift of redox conditions may support optimal oxidative activation with minimal inhibition of DNA binding. The elevation of intracellular GSSG levels by BCNU before stimulation suppressed the chloramphenicol acetyltransferase expression dependent on NFKB but increased that dependent on AP-1. This selective suppression of NFKB was also demonstrable by electrophoretic mobility shift assays. In vitro, GSSG inhibited the DNA binding activity of NFKB more effectively than that of AP-1, while AP-3 was inhibited more effectively by oxidized thioredoxin.The nuclear transcription factor KB (NFKB) is involved in the inducible transcription of immunologically relevant genes including the genes of the interleukin-2 receptor achain, tumor necrosis factor-a, major histocompatibility complex antigens and c-fos (Leung and Nabel, 1988;Cross et al., 1989; reviewed by Ullmann et al., 1990). The NFKB protein is found in many different cell types including B and T lymphocytes, macrophages and monocytes (reviewed by Baeuerle and Baltimore, 1991). The transcriptional activation of NFKB-dependent genes is usually induced by dissociation of the inhibitory protein IKB from the NFKB heterodimer in the cytoplasm and the subsequent translocation of active NFKB into the nucleus. The Fos and Jun proteins are components of another family of nuclear transcription factors that have been implicated in the regulation of growth, differentiation, neuronal excitation and cellular stress in a number of different cell types (reviewed by Angel and Karin, 1991;. Fos and Jun form a heterodimeric complex that interacts with a DNA sequence known as the activator protein-1 (AP-1) binding site or TPA-responsive element (TRE; TPA = 12-0-tetradecanoyl-phorbol 13-acetate). NFKB and other related factors of the v-and c-Re1 oncoprotein family share a characteristic sequence motif with a cysteine and three arginine residues in the DNA binding region (Ghosh et al., 1990;Kieran et al., 1990;Kumar et al., 1992). The DNA binding region of Fos, Jun and several related transcription factors also contains a characteristic sequence with a cysteine and up to seven ar...

Overexpression of γ-glutamylcysteine synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappa B and activator protein-1

“…Though AP-1 activation was blocked by transfection with g-GCS gene, published reports indicate that antioxidants including NAC, PDTC and thioredoxin, will suppress NF-kB activation (Meyer et al, 1993;Schenk et al, 1994;Galter et al, 1994), themselves activate AP-1. This suggests the two transcription factors are activated by distinct mechanism.…”

“…Several reports indicate that the requirements for AP-1 activation di er from that of NF-kB activation (Meyer et al, 1993;Shenk et al, 1994;Galter et al, 1994). Therefore, we investigated the e ect of g-GCS on TNF-induced AP-1 activation.…”

Oxidative stress and regulation of glutathione in lung inflammation

“…This may be due to conservation of the cysteine (sulphydryl) residue required for the nuclear AP-1 DNA binding. The DNA-binding of AP-1 can be enhanced by thioredoxin as well as the nuclear redox protein, Ref-1, and inhibited by GSSG in many cell types, suggesting that disulphide bond formation by cysteine residues inhibits AP-1 DNA-binding [58,59]. By contrast, oxidative stress imposed by H 2 O 2 treatment, depletion of intracellular GSH using DL-buthionine-(S,R)-sulphoximine (BSO) or an increase in the ratio GSH/GSSG by diamide treatment of the liver cell line HepG2 also stimulates AP-1 binding [60].…”

NF-κB Inhibition Ameliorates Angiotensin II–Induced Inflammatory Damage in Rats

“…19 However, in some biological systems, PDTC has been shown to exert pro-oxidative effects. 20,21 This could mean that the inhibitory effect of redox-active substances is related to reductive as well as oxidative processes, most likely depending on the step in the signaling cascade with which they interfere. Other transcription factors, such as AP-1, are influenced by the redox status of the cell.…”