Abstract
Cytokine-activated inhibitor of κB kinase β (IKKβ) is a key mediator of immune and inflammatory responses, but recent studies suggest that IKKβ is also required for tissue homeostasis in physiopathological processes. Here we report a novel role for IKKβ in maintenance of constitutive levels of the redox scavenger GSH. Inactivation of IKKβ by genetic or pharmacological means results in low cellular GSH content and marked reduction of redox potential. Similar to Ikkβ(−/−) cells, Tnfr1(−/−) and p65(−/−) cells are also GSH-deficient. As a consequence, cells deficient in IKKβ signaling are extremely susceptible to toxicity caused by environmental and pharmacological agents, including oxidants, genotoxic agents, microtubule toxins, and arsenic. GSH biosynthesis depends on the activity of the rate-limiting enzyme glutamate-cysteine ligase (GCL), consisting of a catalytic subunit (GCLC) and a modifier subunit (GCLM). We found that loss of IKKβ signaling significantly reduces basal NF-κB activity and decreases binding of NF-κB to the promoters of Gclc and Gclm, leading to reduction of GCLC and GCLM expression. Conversely, overexpression of GCLC and GCLM in IKKβ-null cells partially restores GSH content and prevents stress-induced cytotoxicity. We suggest that maintenance of GSH is a novel physiological role of the IKKβ-NF-κB signaling cascade to prevent oxidative damage and preserve the functional integrity of the cells.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported in part by the National Institutes of Health National Institute of Environmental Health Sciences [Grants ES11798, ES10708, ES06096, T32-ES007250]; by the National Institutes of Health National Eye Institute [Grant EY15227]; and by Intramural Research Program of the National Institutes of Health National Cancer Institute.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.061424.
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ABBREVIATIONS:
- NF-κB
- nuclear factor-κB
- IκBα
- inhibitor of κB
- TNFα
- tumor necrosis factor
- TNFR
- tumor necrosis factor receptor
- TRAF
- TNFR-associated factor
- IKK
- IκB kinase
- ROS
- reactive oxygen species
- GCL
- glutamate cysteine ligase
- GCLC
- glutamine cysteine ligase catalytic subunit
- GCLM
- glutamine cysteine ligase modifier subunit
- AP-1
- activator protein-1
- NRF2
- NF-E2-related factor-2
- JSH23
- 4-methyl-N1-(3-phenylpropyl)benzene-1,2-diamine
- BMS-345541
- 4-(2′-aminoethyl)amino-1,8-dimethylimidazo[1,2-a]quinoxaline
- TPCA-1
- [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide
- luminol
- 5-amino-2,3-dihydro-1,4-phthalazinedione
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- DTPA
- diethylenetriaminepenta-acetic acid
- DCFDA
- 2′,7′-dichlorofluorescin-diacetate
- PBS
- phosphate-buffered saline
- DAPI
- 4,6-diamidino-2-phenylindole
- KRB
- KCl-respiratory buffer
- TUNEL
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PCR
- polymerase chain reaction
- siRNA
- small interfering RNA
- ChIP
- chromatin immunoprecipitation
- kbp
- kilobase pair(s)
- RT-PCR
- reverse transcription-polymerase chain reaction
- IgG
- immunoglobulin G.
- Received October 1, 2009.
- Accepted February 12, 2010.
- U.S. Government work not protected by U.S. copyright
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