NF-κB potentiates caspase independent hydrogen peroxide induced cell death

Abstract

Background: The pro-survival activity of NF-κB in response to a variety of stimuli has been extensively characterized. Although there have been a few reports addressing the pro-cell death role of NF-κB, the precise mechanism of NF-κB's pro-cell death function still remains elusive.

Methodology/principal findings: In the present study, we investigated the role of NF-κB in cell death induced by chronic insult with hydrogen peroxide (H(2)O(2)). Here, we show that NF-κB promotes H(2)O(2) induced caspase independent but PARP dependent fibroblast cell death. The pro-death activity of NF-κB is due to the DNA binding activity of RelA, which is induced through IKK- mediated IκBα degradation. NF-κB dependent pro-survival genes, Bcl-2 and XIAP, were significantly repressed, while NF-κB dependent pro-death genes, TNFα and Fas Ligand, were induced in response to H(2)O(2).

Conclusions/significance: We discovered an unexpected function of NF-κB, in that it potentiates chronic H(2)O(2) exposure induced cell death, and suggest that NF-κB mediates cell death through the repression of pro-survival genes and induction of pro-death genes. Since unremitting exposure of tissues to H(2)O(2) and other reactive oxygen species can lead to several degenerative disorders and diseases, our results have important implications for the use of NF-κB inhibitors in therapeutic drug design.

Figure 1. Continuous H₂O₂ exposure (via GO) to fibroblasts induces a caspase independent but PARP dependent cell death.

(A) Wild type (wt) MEFs were either untreated (-) or treated with 25 mU/ml GO for the indicated periods of time. H₂O₂ readings for the H₂O₂ standard curve were taken 10 min after addition. All H₂O₂ levels are shown as normalized to untreated samples and are given in arbitrary units (a.b.u). (B) Following pretreatment for 1 hr in the presence of 100 µM z-VAD-fmk or DMSO, MEFs were subsequently treated with 25 mU/ml GO or 200 J/m² UV and cell death was analyzed at the indicated time points. (C) Cell lysate was analyzed by western blotting against pro-caspase3, caspase3, and α-tubulin. The cell lysates were also stained by Coomassie Brilliant Blue to demonstrate equal loading. (D) Following pretreatment for 1 hr in the presence of 60 µM of DPQ or DMSO, MEFs were subsequently treated with 25 mU/ml GO or 2 mM H₂O₂ and cell death was analyzed at the indicated time points. (E) Cell lysate was analyzed by western blotting against PAR (poly (ADP-ribose) polymer), PARP, and α-tubulin. Both z-VAD-fmk and DPQ were kept in the media following GO or UV stimulation. Cell death was determined as the normalized value of propidium iodide incorporation (see Methods). All results are presented as the average of triplicate experiments. Error bars signify ±s.e.m (standard error of mean).

Figure 2. NF-κB augments cell death in H₂O₂ induced cell death.

Cell death assays were performed on wt, nfkb^−/− MEFs (A), and nfkb^−/− cells reconstituted with empty vector (pBabe), rela transgene (Tg), or rela mutant (R35AY36A Tg) (B) treated with 25 mU/ml GO for the indicated periods of time. RelA was verified by western blot. Results are presented as the average of 3 independent experiments. Error bars signify ±s.e.m. * denotes p<0.05. (C–D) wt, relA Tg, R35AY36A Tg, and pBabe nfkb^−/− cells were analyzed for cell viability in terms of AnnexinV positive cells following 16 hrs of treatment with 10 ng/ml TNFα (C) and for nuclear localization and DNA binding by EMSA analysis following treatment with 1 ng/ml TNFα (D). Supershift analysis was performed by adding RelA antibody to the EMSA reaction.

Figure 3. The canonical activation pathway is required for the pro-cell death function of NF-κB.

(A) Cell viability assays were performed on ikba^−/− MEFs reconstituted with either wt ikba Tg, or with aa ikba Tg treated with 50 mU/ml GO for the indicated periods. (B) wt ikba Tg and aa ikba Tg MEFs were treated with 1 ng/ml TNFα for 0.5 hr for EMSA analysis. (C) wt MEFS were treated with either 1.0 ng/ml TNFα or 25 mU/ml GO for the indicated times, for EMSA (C) IKK activity (D) or IκBα degradation analysis (E). Quantification of EMSA and IKK kinase assay experiments are shown. All results are presented as the average of 3 independent experiments. Error bars signify ±s.e.m.

Figure 4. NF-κB dependent survival genes, Bcl-2 and XIAP, are repressed, while cell death genes, TNFα and FasL, are induced.

Total RNA was isolated from wt and pBabe or rela reconstituted nfkb^−/− MEFs without treatment (black bars) or following 4 hrs of treatment with 25 mU/ml GO (grey bars). Relative gene expression (Rel. Gene. Expr.) levels were determined using real time RT-PCR and are presented as compared to untreated samples. Results are presented as the average of three triplicate experiments. Error bars signify ± standard deviation. * denotes p<0.05.