Home LiteratureArticle Details
PMID: 8655581 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression.

The Journal of cell biology ·Vol. 133 ·No. 5 ·1996-06-00 ·Pages 1083-93

Kretz-Remy C, Mehlen P, Mirault ME, Arrigo AP

Abstract

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 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 TNF alpha treatment. Decreased ROS levels and NF-kappa B 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-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B-alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF-kappa B activation.

MeSH Terms
Cell Line Cell Nucleus/metabolism DNA/metabolism DNA-Binding Proteins/chemistry,metabolism Gene Expression Genes, Reporter Glutathione Peroxidase/genetics,metabolism Humans Hydrogen Peroxide/pharmacology I-kappa B Proteins NF-KappaB Inhibitor alpha NF-kappa B/metabolism Oxidative Stress Phosphorylation Reactive Oxygen Species/metabolism Transcriptional Activation Transfection Tumor Necrosis Factor-alpha/pharmacology
Chemicals
DNA-Binding Proteins I-kappa B Proteins NF-kappa B NFKBIA protein, human Reactive Oxygen Species Tumor Necrosis Factor-alpha NF-KappaB Inhibitor alpha DNA Hydrogen Peroxide Glutathione Peroxidase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Kretz-Remy C
Centre National de la Recherche Scientifique Unité Mixte de Recherche Université Claude Bernard Lyon-I, France.
Mehlen P
Mirault M E
Arrigo A P
References (71)
71 references, click to expand
  1. Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus.
    Proc Natl Acad Sci U S A. 1990 Dec;87(24):9943-7 PMID: 2263644
  2. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review).
    Free Radic Res Commun. 1992;17(4):221-37 PMID: 1473734
  3. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway.
    Genes Dev. 1995 Jul 1;9(13):1586-97 PMID: 7628694
  4. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase.
    Biotechniques. 1989 Jun;7(6):576-9 PMID: 2517211
  5. Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide.
    Proc Natl Acad Sci U S A. 1990 Jul;87(13):5006-10 PMID: 2164216
  6. Constitutive expression of human hsp27, Drosophila hsp27, or human alpha B-crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts.
    J Immunol. 1995 Jan 1;154(1):363-74 PMID: 7995955
  7. Function and activation of NF-kappa B in the immune system.
    Annu Rev Immunol. 1994;12:141-79 PMID: 8011280
  8. Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal.
    Cell. 1990 Sep 7;62(5):1019-29 PMID: 2203532
  9. The kinetics of HIV-1 long terminal repeat transcriptional activation resemble those of hsp70 promoter in heat-shock treated HeLa cells.
    FEBS Lett. 1994 Oct 24;353(3):339-44 PMID: 7811329
  10. Importance of the glutathione redox cycle for the resistance of lung epithelial cells against a polymorphonuclear leukocyte-mediated oxidant attack.
    Biochem Pharmacol. 1986 Jul 1;35(13):2268-70 PMID: 3729983
  11. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome).
    Nature. 1988 Jan 14;331(6152):192-4 PMID: 3277060
  12. Strategies of antioxidant defense.
    Eur J Biochem. 1993 Jul 15;215(2):213-9 PMID: 7688300
  13. Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism.
    Cell. 1986 Dec 26;47(6):921-8 PMID: 3096580
  14. Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation.
    Mol Cell Biol. 1995 May;15(5):2809-18 PMID: 7739562
  15. NF-kappa B and Rel: participants in a multiform transcriptional regulatory system.
    Int Rev Cytol. 1993;143:1-62 PMID: 8449662
  16. Stimulation of free radical generation in human leukocytes by various agents including tumor necrosis factor is a calmodulin dependent process.
    Biochem Biophys Res Commun. 1990 Mar 30;167(3):1030-6 PMID: 2157420
  17. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B.
    Chem Biol. 1995 Jan;2(1):13-22 PMID: 9383399
  18. H2O2 and antioxidants have opposite effects on activation of NF-kappa B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor.
    EMBO J. 1993 May;12(5):2005-15 PMID: 8491191
  19. Abnormalities of blood selenium and glutathione peroxidase activity in patients with acquired immunodeficiency syndrome and aids-related complex.
    Biol Trace Elem Res. 1988 Jan-Apr;15:167-77 PMID: 2484515
  20. Cloning of a mitogen-inducible gene encoding a kappa B DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs.
    Nature. 1990 Nov 1;348(6296):76-80 PMID: 2234062
  21. Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B.
    Nature. 1993 Sep 9;365(6442):182-5 PMID: 8371761
  22. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation.
    Mol Cell Biol. 1993 Jun;13(6):3301-10 PMID: 8497253
  23. Generation of p50 subunit of NF-kappa B by processing of p105 through an ATP-dependent pathway.
    Nature. 1991 Dec 5;354(6352):395-8 PMID: 1956402
  24. Inactivation of NF-kappa B inhibitor I kappa B alpha: ubiquitin-dependent proteolysis and its degradation product.
    Biochem Biophys Res Commun. 1995 Oct 4;215(1):292-301 PMID: 7575604
  25. Activation of NF-kappa B requires proteolysis of the inhibitor I kappa B-alpha: signal-induced phosphorylation of I kappa B-alpha alone does not release active NF-kappa B.
    Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):552-6 PMID: 7831327
  26. The proteasome (multicatalytic protease) is a component of the 1500-kDa proteolytic complex which degrades ubiquitin-conjugated proteins.
    J Biol Chem. 1990 Mar 25;265(9):4789-92 PMID: 2180950
  27. I kappa B: a specific inhibitor of the NF-kappa B transcription factor.
    Science. 1988 Oct 28;242(4878):540-6 PMID: 3140380
  28. Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli.
    EMBO J. 1995 Jun 15;14(12):2876-83 PMID: 7796813
  29. Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B.
    Mol Cell Biol. 1995 Mar;15(3):1302-11 PMID: 7862124
  30. I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention.
    Genes Dev. 1992 Oct;6(10):1899-913 PMID: 1340770
  31. Structure, regulation and function of NF-kappa B.
    Annu Rev Cell Biol. 1994;10:405-55 PMID: 7888182
  32. A time-course study on superoxide generation and protein kinase C activation in human neutrophils.
    FEBS Lett. 1988 Nov 7;239(2):195-8 PMID: 2846355
  33. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin.
    J Leukoc Biol. 1990 May;47(5):440-8 PMID: 2159514
  34. The rel-associated pp40 protein prevents DNA binding of Rel and NF-kappa B: relationship with I kappa B beta and regulation by phosphorylation.
    Genes Dev. 1991 Aug;5(8):1464-76 PMID: 1907941
  35. Proteins controlling the nuclear uptake of NF-kappa B, Rel and dorsal.
    Trends Cell Biol. 1991 Nov;1(5):130-7 PMID: 14731544
  36. Autoregulation of I kappa B alpha activity.
    Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):28-32 PMID: 8278379
  37. A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB.
    Genes Dev. 1989 Nov;3(11):1689-98 PMID: 2691328
  38. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits.
    Biochim Biophys Acta. 1991 Apr 16;1072(1):63-80 PMID: 2018779
  39. Differential interactions of Rel-NF-kappa B complexes with I kappa B alpha determine pools of constitutive and inducible NF-kappa B activity.
    EMBO J. 1994 Oct 3;13(19):4608-16 PMID: 7925301
  40. DNA binding of purified transcription factor NF-kappa B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition.
    J Biol Chem. 1991 Jan 5;266(1):252-60 PMID: 1985897
  41. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells.
    J Leukoc Biol. 1994 Feb;55(2):253-8 PMID: 8301222
  42. Isozymes of superoxide dismutase from wheat germ.
    Biochim Biophys Acta. 1973 Jul 12;317(1):50-64 PMID: 4723247
  43. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF.
    EMBO J. 1993 Aug;12(8):3095-104 PMID: 8344250
  44. The inhibitory ankyrin and activator Rel proteins.
    Curr Opin Genet Dev. 1992 Apr;2(2):211-20 PMID: 1386268
  45. Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B.
    Science. 1991 Mar 22;251(5000):1490-3 PMID: 2006423
  46. An inducible transcription factor activates expression of human immunodeficiency virus in T cells.
    Nature. 1987 Apr 16-22;326(6114):711-3 PMID: 3031512
  47. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha.
    Biochem J. 1989 Oct 15;263(2):539-45 PMID: 2556998
  48. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1.
    EMBO J. 1991 Aug;10(8):2247-58 PMID: 2065663
  49. Overexpression of seleno-glutathione peroxidase by gene transfer enhances the resistance of T47D human breast cells to clastogenic oxidants.
    J Biol Chem. 1991 Nov 5;266(31):20752-60 PMID: 1939125
  50. Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha.
    Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2532-6 PMID: 8460169
  51. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B.
    Cell. 1994 Sep 9;78(5):773-85 PMID: 8087845
  52. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor.
    Cell. 1988 Apr 22;53(2):211-7 PMID: 3129195
  53. Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.
    Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11884-8 PMID: 7991551
  54. Glutathione peroxidase.
    Methods Enzymol. 1985;113:490-5 PMID: 4088069
  55. p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction.
    Genes Dev. 1993 Apr;7(4):705-18 PMID: 8458581
  56. The inducible transcription factor NF-kappa B: structure-function relationship of its protein subunits.
    Biochem J. 1993 Mar 1;290 ( Pt 2):297-308 PMID: 8452515
  57. Glutathione peroxidase activity and metabolism of arachidonic acid in peripheral blood mononuclear cells from elderly subjects.
    Clin Sci (Lond). 1993 Aug;85(2):203-11 PMID: 8403789
  58. Increased thermal aggregation of proteins in ATP-depleted mammalian cells.
    Eur J Biochem. 1994 Feb 15;220(1):239-46 PMID: 7907018
  59. Intracellular reactive oxygen species as apparent modulators of heat-shock protein 27 (hsp27) structural organization and phosphorylation in basal and tumour necrosis factor alpha-treated T47D human carcinoma cells.
    Biochem J. 1995 Dec 1;312 ( Pt 2):367-75 PMID: 8526844
  60. Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity.
    Cell. 1991 Jun 28;65(7):1281-9 PMID: 1829648
  61. Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-kappa B precursor p105 are obligatory steps in the activation of NF-kappa B.
    Nucleic Acids Res. 1993 Nov 11;21(22):5059-66 PMID: 8255759
  62. Relationships between in vitro selenium supply, glutathione peroxidase activity, and phagocytic function in the HL-60 human myeloid cell line.
    J Biol Chem. 1985 Jul 25;260(15):8951-5 PMID: 2991228
  63. A novel signal transduction pathway from the endoplasmic reticulum to the nucleus is mediated by transcription factor NF-kappa B.
    EMBO J. 1995 Jun 1;14(11):2580-8 PMID: 7781611
  64. Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65.
    Mol Cell Biol. 1995 Apr;15(4):2166-72 PMID: 7891711
  65. Stimulation of glutathione peroxidase activity decreases HIV type 1 activation after oxidative stress.
    AIDS Res Hum Retroviruses. 1994 Nov;10(11):1451-61 PMID: 7888200
  66. NF-kappa B: a lesson in family values.
    Cell. 1995 Feb 24;80(4):529-32 PMID: 7867060
  67. Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B.
    Mol Cell Biol. 1995 May;15(5):2689-96 PMID: 7739549
  68. Selenium supplementation of symptomatic human immunodeficiency virus infected patients.
    Biol Trace Elem Res. 1989 Apr-May;20(1-2):59-65 PMID: 2484402
  69. In vivo control of NF-kappa B activation by I kappa B alpha.
    EMBO J. 1993 Dec;12(12):4685-95 PMID: 8223478
  70. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation.
    Science. 1995 Mar 10;267(5203):1485-8 PMID: 7878466
  71. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway.
    Science. 1993 Mar 26;259(5103):1912-5 PMID: 8096091
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.133.5.1083
Published
1996-06-00
Pages
1083-93
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2120847
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.133.5.1083 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2