Inhibition of IKB-a Phosphorylation and Degradation and Subsequent NF-KB Activation by Glutathione Peroxidase Overexpression

We report here that both KB-dependent transactivation of a reporter gene and NF-KB activation in response to tumor necrosis factor (TNFc 0 or H202 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNFot treatment. Decreased ROS levels and NF-KB activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-KB subunits (p65 and p50) and of the inhibitory subunit IKB-a were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of NF-KB. Nuclear translocation of NF-KB as well as IKB-ot degradation were inhibited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNFa, a time correlation was observed between elevated ROS levels and IKB-ot degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of IKB-et, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNFe~-mediated transient accumulation of the acidic and highly phosphorylated IKB-et isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of IKB~, a phenomenon that precedes and controls the degradation of this protein, and then NF-KB activation. T rtE transcription factor NF-KB plays a pivotal role in the regulation of a wide variety of cellular genes, particularly those involved in immune and inflammatory responses, and also participates in the regulation of viral promoters, including the human immunodeficiency virus long terminal repeat (HIV-1 LTR) (3, 7, 29, 51, 66). Five different subunits of NF-KB have been described that can homoand heterodimerize (21, 53). These polypeptides belong to the rel family of transcription factors, and the more frequent and therefore prototypical form of NF-KB is a heterodimer complex containing the p50 and p65/RelA subunits (6, 11, 28, 59, 67). Unlike most transcription factors, these proteins reside in the cytoplasm in a latent form and must therefore translocate into the nucleus to function (5). In unstimulated cells, the nuclear import of the NF-KB DNA-binding dimer p65/RelAp50 is prevented by high-affinity association of the p65/ RelA subunit with a cytoplasmic inhibitor called IKB (4, 13, 57, 71). The inhibitory subunit IKB belongs also to a Address all correspondence to Andrr-Patrick Arrigo, Laboratoire du Stress Cellulaire, Centre de Grn~tique Molreulaire et Cellulaire, CNRSUMR 106, Universit6 Claude Bernard Lyon-I, 43 Boulevard du 11 Novembre, 69622 Villeurbanne Cedex, France. Tel.: (33) 72 44 85 95. Fax: (33) 72 44 05 55. e-mail: [email protected]. family of distinct proteins (62) and interacts, through its ankyrin-like repeats, with the nuclear localization signals of p50 and p65/RelA (9, 34). The prototypical IKB protein involved in cytoplasmic retention of NF-KB dimers is IKB-a, encoded by the MAD-3 gene (32). The inactive NF-KBIKB-ot complexes are dissociated in response to a variety of extracellular stimuli, thereby allowing free NF-KB dimers to translocate to the nucleus and activate transcription of genes containing KB regulatory elements. Phosphorylation of IKB-ot is required for NF-KB activation (10, 27, 35) but does not induce IKB dissociation from the inactive NF-KB-IKB-ot complex (20, 25, 39). This complex is more probably disrupted because of the selective degradation of phosphorylated IKB-a in response to extracellular signals (14, 33, 44). Recently, it has been shown that IKB-a turnover was regulated by phosphorylation at serine residues 32 and 36 (12, 14, 74). A multiprotease complex, the proteasome (2, 22), as well as I KB-a ubiquitination, is involved in this process (16, 37, 56, 74, 75). Stimulation results in a rapid loss of IKB-t~ and the rapid nuclear translocation of NF-KB. Transactivation by this factor, in turn, induces high levels of IKB-a synthesis that probably restore the unstimulated inhibited state (1, 13, 17, 71). The proteasome also appears involved in the proteolytic processing of p50 from a 105-kD precursor protein (p105) (24, 44, 45). © The Rockefeller University Press, 0021-9525/96/06/1083/11 $2.00 The Journal of Cell Biology, Volume 133, Number 5, June 1996 1083-1093 1083 on July 1, 2017 jcb.rress.org D ow nladed fom Transcription factors are usually activated by a restricted number of specific extracellular stimuli. In contrast, NF-KB is activated by an extraordinarily large number of conditions and agents (3, 7, 29, 73). Of great interest was the discovery that most inducers of NF-KB seem to rely on the production of intracellular reactive oxygen species (ROS) 1 as evidenced by the inhibitory effect of several antioxidants, including N-acetylcysteine (63, 70) and the activation induced by hydrogen peroxide (63). ROS include superoxide radicals (O2"-/.O2H), hydrogen peroxide (H202), organic hydroperoxides, and hydroxyl radical (OH-). Eukaryotic cells produce ROS continuously as side products of the mitochondrial electron transfer chain reactions (31), but also upon exposure to different stimuli that can activate NF-KB, including UV light, hydrogen peroxide, and inflammatory cytokines, such as tumor necrosis factor t~ (TNFet) and interleukin 1 (18, 19, 43, 64). The intracellular balance between ROS formation and detoxification is regulated by nonenzymatic as well as enzymatic defenses. In mammalian cells, major antioxidant enzymes include superoxide dismutases (SOD), catalase, and a family of selenium-dependent glutathione peroxidases (68). In addition to the classical seleno-glutathione peroxidase (GSHPx) (26), which can reduce H202 and a variety of organic hydroperoxides in the presence of glutathione, this family of enzymes includes a plasmatic, a gastrointestinal, and a phospholipid hydroperoxide glutathione peroxidase. In the cytoplasm and in the mitochondria, H202 is mainly detoxified by GSHPx; catalase, which has a much higher Michaelis Menten constant (Km) for H202 than GSHPx, is found predominantly in peroxisomes. GSHPx is a homotetramer, and each subunit contains one selenocysteine residue in its active site (40). In higher eukaryotes, selenium (Se) is a trace element that is essential for the activity of these glutathione peroxidases. Lowering Se cellular contents, either in vivo by dietary manipulations or in vitro by using selenium-deprived growth media, decreased the selenoperoxidase-mediated cytoprotection against oxidative stress (26, 69, 72). On the other hand, cells grown in selenium-supplemented growth media showed increased GSHPx activity that resulted in a decreased NF-KB activation by oxidative stress and reduced HIV-1 reactivation in HIV-1 latently infected T lymphocytes exposed to oxidative stress (60). Moreover, Se deficiency that results in reduced glutathione peroxidase activities has been detected in HIV-infected patients (23, 54), suggesting that, in vivo, Se levels may be an important determinant of the progression and pathology of AIDS. Until recently, most studies aimed to demonstrate a link between NF-KB activation and ROS were performed with chemicals and antioxidants that were often used at very high inhibitory doses and not devoid of possible side effects. To overcome this problem, Schmidt et al. (61) used cell lines that overexpress SOD or catalase to modulate intracellular ROS levels. These authors pointed out the essential role of ROS, probably H 2 0 2 , in NF-KB activation. 1. Abbreviat ions used in this paper: DOC, sodium desoxycholate; EB, ethidium bromide; ECL, enhanced chemiluminescence; GSHPx, selenoglutathione peroxidase; HE, hydroethidine; ROS, reactive oxygen species; SOD, superoxide dismutase; TNFa, tumor necrosis factor a. In the present study, the overexpression of glutathione peroxidase was used to further demonstrate the implication of ROS in NF-KB activation by hydrogen peroxide and TNFa. To this end, human breast carcinoma T47D cells, which are characterized by low endogenous GSHPx levels, were stably transfected with a cDNA gene encoding human GSHPx (49, 50). GSHPx overexpression induced oxidoresistance status and decreased intracellular ROS levels. Here, we show that the overexpression of GSHPx abolished NF-KB-IKB-et activation by TNFot or hydrogen peroxide. This phenomenon was characterized, in vitro, by an inhibition of NF-~B DNA-binding activity and, in live cells, by a strong decrease in NF-KB nuclear translocation, IKB-c~ phosphorylation and subsequent degradation, and KB-dependent transcription. Moreover, a time correlation was observed between the TNFa-mediated intracellular burst of ROS and I~B-a degradation. The data presented suggest that IKB-ot phosphorylation and subsequent degradation are controlled by intracellular ROS levels. Materials and Methods