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doi: 10.1242/10.1242/jcs.00151

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Temporal pattern of NF{kappa}B activation influences apoptotic cell fate in a stimuli-dependent fashion

Chenguang Fan1,2,3, Jusan Yang2,3 and John F. Engelhardt1,2,3,*

1 Molecular Biology Graduate Program, University of Iowa College of Medicine, Iowa City, Iowa, 52242 USA
2 The Center for Gene Therapy, University of Iowa College of Medicine, Iowa City, Iowa, 52242 USA
3 Department of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, Iowa, 52242 USA



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  		Fig. 1. TNF-{alpha} and UV treatment lead to serine phosphorylation of I{kappa}B{alpha}, whereas pervanadate and H/R induce tyrosine phosphorylation of I{kappa}B{alpha}. HeLa cells were treated with UV (50 J/m2), TNF-{alpha} (10 ng/ml), pervanadate (50 µM) or H/R (5 hours hypoxia, 15 and 30 minutes reoxygenation) for the indicated times. Both untreated and treated samples were harvested for cytoplasmic extracts. (A) 5 µg of total protein was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Phosphorylation of I{kappa}B{alpha} at S32/36 was evaluated by a phosphospecific antibody. (B) 200 µg of total cytoplasmic protein was immunoprecipitated with anti-I{kappa}B{alpha} antibody followed by western blotting with an anti-phosphotyrosine antibody to detect tyrosine phosphorylation of I{kappa}B{alpha}. Samples analyzed were identical to those evaluated in A.

 


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  		Fig. 2. I{kappa}B{alpha}(S32/36A) inhibits NF{kappa}B activation following UV and TNF-{alpha} treatment. (A) HeLa cells were co-infected with Ad.I{kappa}B{alpha}(S32/36A) or Ad.BglII together with Ad.NF{kappa}BLuc 24 hours before treatment. Cells were harvested 6 hours after UV (50 J/m2), TNF-{alpha} (10 ng/ml), pervanadate (50 µM) and H/R (5 hours hypoxia, 6 hours reoxygenation) treatments, and whole cell extracts were normalized by total protein content and subjected to luciferase assays. NF{kappa}B activity was determined by the relative luciferase activity (as light units). The relative NF{kappa}B activation was calculated by deducting the mean NF{kappa}B baseline activation for each vector group (in the absence of stimulation) from the corresponding individual values from the same vector group in the presence of stimulus. The relative NF{kappa}B activation is plotted for each individual stimulus (±s.e.m., n=6). (B) Percent NF{kappa}B inhibition by I{kappa}B{alpha}(S32/36A) for each experimental point was calculated using the following formula: percent inhibition=1—(relative activation of each Ad.I{kappa}B{alpha}(S32/36A) infected sample/mean relative activation of the Ad.BglII infected group). Results depict the mean (±s.e.m.) for n=6 independent data points in each group.

 


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  		Fig. 3. I{kappa}B{alpha}(Y42F) inhibits NF{kappa}B activation following pervanadate and H/R treatment. (A) HeLa cells were co-infected with Ad.I{kappa}B{alpha}(Y42F) or Ad.BglII together with Ad.NF{kappa}BLuc 24 hours before treatment. Cells were treated with UV (50 J/m2), TNF-{alpha}(10 ng/ml), pervanadate (50 µM) and H/R (5 hours hypoxia, 6 hours reoxygenation) then harvested 6 hours after treatment. Whole cell extracts were normalized by total protein content and subjected to luciferase assay. NF{kappa}B activity was determined by the relative luciferase activity (as light units). The relative NF{kappa}B activation was calculated by deducting the mean NF{kappa}B baseline activation for each vector group (in the absence of stimulation) from the corresponding individual values from the same vector group in the presence of stimulus. The relative NF{kappa}B activation is plotted for each individual stimulus (±s.e.m., n=6). (B) Percent NF{kappa}B inhibition by I{kappa}B{alpha}(Y42F) for each experimental point was calculated using the following formula: percent inhibition=1—(relative activation of each Ad.I{kappa}B{alpha}(Y42F) infected sample/mean relative activation of the Ad.BglII infected group). Results depict the mean (±s.e.m.) for n=6 independent data points in each group.

 


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  		Fig. 4. Inhibition of either tyrosine or serine I{kappa}B{alpha} phosphorylation stimulates apoptosis in an injury-context specific fashion. HeLa cells were infected with Ad.BglII, Ad.I{kappa}B{alpha}(S32/36A) or Ad.I{kappa}B{alpha}(Y42F) at an moi of 1000 particles/cell for 24 hours. Cells were treated with UV (50 J/m2), TNF-{alpha} (10 ng/ml), pervanadate (50 µM) or H/R (5 hours hypoxia, 18 hours reoxygenation) and then harvested 18 hours after treatment. Cells were then stained with annexin-V—FITC and propidium iodine. Results depict the mean (±s.e.m., n=3) percent of apoptotic cells as shown by FACS analysis. Ad.I{kappa}B{alpha}(S32/36A) more significantly increased apoptosis following UV or TNF-{alpha} treatment, whereas Ad.I{kappa}B{alpha}(Y42F) preferentially increased apoptosis following pervanadate or H/R treatment. Paired t-test analysis was performed between Ad.BglII infected and Ad.I{kappa}B{alpha}(S32/36A) or Ad.I{kappa}B{alpha}(Y42F) infected samples for each stimulus, and a statistically significant difference is denoted by *(P<0.05) or {dagger}(P<0.001). P-values for paired t-test analysis comparing Ad.I{kappa}B{alpha}(S32/36A) and Ad.I{kappa}B{alpha}(Y42F) infected samples for each stimulus are denoted above brackets.

 


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  		Fig. 5. Ad.I{kappa}B{alpha}AS inhibits I{kappa}B{alpha} protein expression and elevates baseline levels of NF{kappa}B in the nucleus. HeLa cells were infected at an moi of 1000 particles/cell with Ad.I{kappa}B{alpha}AS or Ad.GFP. Cytoplasmic and nuclear extracts were prepared from cells collected at 24, 48 and 72 hours after infection. (A) 5 µg of cytoplasmic protein was evaluated by western blotting using anti-I{kappa}B{alpha} antibody, anti-I{kappa}Bß antibody or anti-actin antibody. Ad.I{kappa}B{alpha}AS infection led to a reduction in I{kappa}B{alpha} protein expression at all time points (lane 2-4) and an increase in I{kappa}Bß expression compared with Ad.GFP-infected controls (lane 1). Changes in I{kappa}B expression were referenced to actin protein levels, which did not change. (B) 5 µg of nuclear protein was evaluated by EMSA to determine NF{kappa}B DNA-binding activity. The induced p65-p50 heterodimer of NF{kappa}B, identified by supershift assays, is marked by an arrow. Results in A and B are derived from the same experimental samples, and experimental conditions are marked above each lane. (C) 5 µg of nuclear protein from 72 hour post-infection time points with Ad.GFP (same sample as from lane 2 Panel B) and Ad.I{kappa}B{alpha}AS (same sample as from lane 5 Panel B) were evaluated by EMSA supershift to determine the subunit composition of the NF{kappa}B DNA binding complex. Antibodies used for supershift were against p50, p52, C-Rel, Rel B and p65. The active DNA-binding complex is identified as p65-p50 heterodimers, and supershifted bands (in lanes 2, 6, 8 and 12) are marked by an asterisk to the left of the gel.

 


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  		Fig. 6. I{kappa}B{alpha}AS enhances NF{kappa}B binding activity following UV, TNF-{alpha}, pervanadate or H/R treatment. HeLa cells were infected with either Ad.BglII or Ad.I{kappa}B{alpha}AS at an moi of 1000 particles/cell for 24 hours. Cells were treated with (A) UV (50 J/m2), (B) TNF-{alpha} (10 ng/ml), (C) pervanadate (50 µM) or (D) H/R (5 hours hypoxia, 6 hours reoxygenation). Both treated and untreated cells were collected for nuclear extract preparation and EMSA analysis. Ad.I{kappa}B{alpha}AS-infected cells demonstrated a higher baseline of NF{kappa}B DNA-binding activity (lanes 1 versus lane 4) and enhanced NF{kappa}B activation following all treatments compared with Ad BglII-infected cells (lanes 2,3 versus lanes 5,6). The conditions for various experimental groups are marked above each panel.

 


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  		Fig. 7. I{kappa}B{alpha}AS enhances NF{kappa}B transcriptional activity following UV, TNF-{alpha}, pervanadate or H/R treatment. HeLa cells were co-infected with Ad.NF{kappa}Bluc (500 particles/cell) together with either Ad.BglII or Ad.I{kappa}B{alpha}AS at an moi of 1000 particles/cell for 24 hours. Cells were treated with (A) UV (50 J/m2), (B) TNF-{alpha}(10 ng/ml), (C) pervanadate (50 µM) and (D) H/R (5 hours hypoxia, 6 hours reoxygenation). Luciferase assays were performed on samples harvested 6 hours after treatment. Results depict the mean (±s.e.m., n=6) raw unadjusted relative light units (RLU). NF{kappa}B transcriptional activity was significantly enhanced (P<0.01) in the Ad.I{kappa}B{alpha}AS infected groups, as compared to the groups infected by Ad.BglII, following UV, TNF-{alpha}, pervanadate or H/R treatment.

 


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  		Fig. 8. NF{kappa}B activation is pro-apoptotic following UV and TNF-{alpha} treatments, while anti-apoptotic following pervanadate and H/R treatments. HeLa cells were infected with either Ad.BglII or Ad.I{kappa}B{alpha}AS at an MOI of 1000 particles/cell for 24 hours. Cells were treated with (A) UV (50 J/m2), (B) TNF-{alpha} (10 ng/ml), (C) pervanadate (50 µM) and (D) H/R (5 hours hypoxia, 18 hours reoxygenation) then harvested 18 hours after treatment. Cells are then trypsinized and stained with annexin-V-FITC and propidium iodine. Results depict the mean (±s.e.m., n=3) percent of apoptotic cells as shown by FACS analysis. Following UV and TNF-{alpha} treatment, I{kappa}B{alpha}AS expression significantly (P<0.01) increased apoptosis compared with the Ad.BglII-infected control group. By contrast, following pervanadate and H/R treatments, I{kappa}B{alpha}AS expression significantly decreased (P<0.01) the percentage of apoptotic cells as compared to the Ad.BglII-infected control group.

 

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