Original ContributionImpairment of the Mitochondrial Electron Chain Transport Prevents NF-κB Activation by Hydrogen Peroxide
Introduction
NF-κB (Nuclear Factor-κB) is a rapidly inducible transcription factor involved in the response of various cell types to a number of external or internal stimuli. Following its intracellular activation, NF-κB regulates the expression of many genes that code for cytokines, growth factors, acute phase response proteins, or cellular receptors, and thus modulates the cellular response to the applied stimulus.1, 2, 3Several viruses such as the Human Immunodeficiency Virus type-1 (HIV-1) or the cytomegalovirus also depend on NF-κB for their intracellular replication.1, 2, 3
NF-κB complexes bind DNA as dimers constituted from a family of proteins designated as the Rel/NF-κB family. In mammals, this family contains the proteins p50, p52, p65 (RelA), RelB, and c-Rel (Rel).1, 2, 3These five proteins harbor a related, but nonidentical 300 amino acid-long Rel homology domain (RelHD) that is responsible for dimerization, nuclear translocation, and specific DNA-binding. In addition, RelA, RelB, and c-Rel, but not p50 or p52, contain one or two transactivating domains. p50 and p52 derive from cytoplasmic precursors named p105 and p100, respectively. NF-κB complexes are sequestered in the cytoplasm of most resting cells by inhibitory proteins belonging to the IκB family.4, 5, 6, 7, 8The members of the IκB family are IκB-α, IκB-β, IκB-ε, p100, and p105.4, 5, 6, 7, 8
Following various stimuli, including the interaction of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) with their receptors, IκB-α is first phosphorylated on serines 32 and 36, then ubiquitinated at lysines 21 and 22, and rapidly degraded by the proteasome, allowing NF-κB nuclear translocation and gene activation.9, 10, 11, 12, 13In the case of these two types of cytokines, the signal transduction pathways leading to the phosphorylation and degradation of IκB proteins has been recently clarified in HeLa and in L293 cells.14, 15, 16, 17, 18It is included into a 700–900 kDa complex called signalosome, whose important partners are proteins associated to the TNF-α or IL-1 receptors, NIK and IKK-1 and −2.14, 15, 16, 17, 18Proinflammatory cytokines such as TNF-α or IL-1β or the bacterial outer membrane component (LPS) are potent activators of NF-κB, which mediates several of their biological activities such as stimulation of the transcription in lymphocytes through the intracellular generation of an oxidative stress.19, 20, 21However, the assumption that a similar mechanism is effective in other cell lines such as lymphocytes has not yet been demonstrated. Up to now, the mechanism by which ROS activate NF-κB translocation or how ROS feed in the signalosome pathway is still unknown.
In this work, we have demonstrated that NF-κB activation in human transformed T lymphocytes (Jurkat JR cells) stimulated with 150 μM of H2O2 can be strongly inhibited when the mitochondrial respiration is impaired. NF-κB activation in similar conditions by other stimuli such as TNF-α or LPS was not affected by these respiration inhibitors. These data demonstrate that in T lymphocytes, NF-κB translocation mediated by H2O2 strongly relies on ATP generated by mitochondria and that H2O2 could promote NF-κB translocation by a mechanism involving other components than those present in the signalosome.
Section snippets
Cell Culture and Biological Reagents
The human T lymphoid cell line Jurkat JR and the murine pre-B 70Z/3 cells were grown in RPMI 1640 medium + ultraglutamine I + 25 mM Hepes (Bio-Whittaker, Belgium), 10% fetal bovine serum (Gibco BRL) and 50 μM β-mercaptoethanol.
NF-κB inducers were usually added in the following final concentrations: H2O2 (Merck, Germany) at 150 μM in RPMI medium, LPS (Sigma, St. Louis, Mo) from E. coli, serotype 0111:B4 at 5 mg/ml in sterile water and TNF-α (Boeringher Mannheim, Germany) at 100 U/ml in RPMI
NF-κB activation by H2O2 and TNF-α in Jurkat JR cells
Electrophoretic Mobility Shift Assays (EMSA) showed that stimulation of Jurkat JR cells with H2O2 at 150 μM induced a strong NF-κB DNA-binding activity, which reached its maximum between 150 and 240 min of treatment (Fig. 1A) but is sustained up to 300 min. Under our experimental conditions, one main band was visible on the autoradiographies. Supershifting experiments performed with antibodies directed toward NF-κB proteins demonstrated that the shifted band, which was displaced by both p50
Discussion
Because NF-κB can be activated by an extraordinarily large number of different signals, ranging from ultraviolet radiation, oxidative stress to cytokines, and T cell activation, the mechanism by which these signals converge on IκB-α is of the wide interest. Recently, many components from certain signaling pathways that lead to NF-κB activation have been described.14, 15, 16, 17, 18The recent identification of a high molecular weight IκB kinase complex and the identification of two unusual IκB
Acknowledgements
J.P. is a Research Director at the National Fund for Scientific Research (Belgium) and SL-P is scientific collaborator at the National Fund for Scientific Research (Belgium). C.J. and B.P. are supported by FRIA fellowships. This work has been supported by grants from the National Fund for Scientific Research (Belgium), Télévie (Belgium), and the Sidaction program (Paris, France).
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