Dual effect of oxidative stress on NF-κB activation in HeLa cells

Abstract

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H(2)O(2) leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)- induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF- kappakB activation, HeLa cells were incubated with H(2)O(2) or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H(2)O(2) and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H(2)O(2) alone, nuclear NF-kappaB was induced after 2 h without detectable degradation of cytosolic IkappaBalphaa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappakB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.

Biphasic Role of Nuclear Factor-k B on Cell Survival and COX-2 Expression in SOD1 Tg Astrocytes after Oxygen Glucose Deprivation

“…regulation of IKK and NF-kB is cell-type restricted, but the molecular mechanisms for the astrocytespecific NF-kB pathway are not known (Schreck et al, 1991;Meyer et al, 1993;Anderson et al, 1994;Manna et al, 1998;Li and Karin, 1999;Byun et al, 2002). In the present study, we show that OGD injury activates the transcription factor NF-kB through phosphorylation of IkBa, which finally promotes cell death and COX-2 expression in mouse primary astrocytes.…”

NF-κB Signaling Pathway, Inflammation and Colorectal Cancer

“…Both pathways regulate cell survival and death (33), and now have been implicated in carcinogenesis (34,35). In addition to the classical IKKmediated NF-B activation, it has been shown that certain novel pathway can activate NF-B by IKK-independent such as UV and hydrogen peroxide (H 2 O 2 ) induced NF-B activation (36)(37)(38).…”

Cysteine-179 of IκB kinase β plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

“…Substitution of Cys-179 with alanine was carried out by site-directed mutagenesis as described previously (Jeon et al, 2003 Cell culture, IKK assay and reporter assay HEK-293 and HeLa cells were obtained from the American Type Culture Collection (Manassa, VA), and maintained in DMEM supplemented with 10% heat-inactivated FBS, and antibiotics. Cells were transfected with expression vectors for IKKβ and their mutants using Fugene 6 (Roche Molecular Biochemicals, Mannheim, Germany) and incubated for 48 h. Preparation of cytoplasmic extracts and immunoprecipitation were performed as described (Byun et al, 2002). Kinase activity was measured in reaction mixtures containing 10 µM ATP, [γ-32 P]ATP (2-5 µCi) and GST-IκBα (1 µg) (Jeon et al, 2000).…”

“…Phosphorylated GST-IκBα was visualized by autoradiography and quantitated in a phosphor image analyzer (Fujifilm, Tokyo, Japan). Proteins in the cell extracts were analyzed by immunoblotting using ECL system (Amersham Biosciences, Buckinghamshire, U.K.) (Byun et al, 2002). NF-κB reporter gene assay was performed in HeLa cells as described previously (Byun et al, 2002).…”

“…Other thiol-reactive agents such as cyclopentenone PGs [PGA1 and 15-deoxy-∆ 12-14 -PGJ2 (15dPGJ2)] (Rossi et al, 2000;Straus et al, 2000), arsenite anion (AsO3 3-) (Kapahi et al, 2000), parthenolide (Kwok et al, 2001), and epoxyquinone A (Liang et al, 2006) were also shown to inhibit NF-κB and IKK activation in cells stimulated with TNF, IL-1, and phorbol esters. It was reported that exposure of cells to oxidants such as hydrogen peroxide (H2O2) and diamide suppressed TNF-induced NF-κB and IKK activation (Korn et al, 2001;Byun et al, 2002). These results suggest that a cysteine sulfhydryl group, which is easily modified by thiol-reactive or oxidizing agents, is critically involved in IKK activation or regulation of IKK activity.…”