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doi: 10.1242/10.1242/jcs.00151

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Temporal pattern of NFB activation influences apoptotic cell fate in a stimuli-dependent fashion

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

View larger version (33K): [in a new window]   Fig. 1. TNF- and UV treatment lead to serine phosphorylation of IB, whereas pervanadate and H/R induce tyrosine phosphorylation of IB. HeLa cells were treated with UV (50 J/m2), TNF- (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 IB at S32/36 was evaluated by a phosphospecific antibody. (B) 200 µg of total cytoplasmic protein was immunoprecipitated with anti-IB antibody followed by western blotting with an anti-phosphotyrosine antibody to detect tyrosine phosphorylation of IB. Samples analyzed were identical to those evaluated in A.

View larger version (19K): [in a new window]   Fig. 2. IB(S32/36A) inhibits NFB activation following UV and TNF- treatment. (A) HeLa cells were co-infected with Ad.IB(S32/36A) or Ad.BglII together with Ad.NFBLuc 24 hours before treatment. Cells were harvested 6 hours after UV (50 J/m2), TNF- (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. NFB activity was determined by the relative luciferase activity (as light units). The relative NFB activation was calculated by deducting the mean NFB 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 NFB activation is plotted for each individual stimulus (±s.e.m., n=6). (B) Percent NFB inhibition by IB(S32/36A) for each experimental point was calculated using the following formula: percent inhibition=1—(relative activation of each Ad.IB(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.

View larger version (19K): [in a new window]   Fig. 3. IB(Y42F) inhibits NFB activation following pervanadate and H/R treatment. (A) HeLa cells were co-infected with Ad.IB(Y42F) or Ad.BglII together with Ad.NFBLuc 24 hours before treatment. Cells were treated with UV (50 J/m2), TNF-(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. NFB activity was determined by the relative luciferase activity (as light units). The relative NFB activation was calculated by deducting the mean NFB 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 NFB activation is plotted for each individual stimulus (±s.e.m., n=6). (B) Percent NFB inhibition by IB(Y42F) for each experimental point was calculated using the following formula: percent inhibition=1—(relative activation of each Ad.IB(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.

View larger version (20K): [in a new window]   Fig. 4. Inhibition of either tyrosine or serine IB phosphorylation stimulates apoptosis in an injury-context specific fashion. HeLa cells were infected with Ad.BglII, Ad.IB(S32/36A) or Ad.IB(Y42F) at an moi of 1000 particles/cell for 24 hours. Cells were treated with UV (50 J/m2), TNF- (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.IB(S32/36A) more significantly increased apoptosis following UV or TNF- treatment, whereas Ad.IB(Y42F) preferentially increased apoptosis following pervanadate or H/R treatment. Paired t-test analysis was performed between Ad.BglII infected and Ad.IB(S32/36A) or Ad.IB(Y42F) infected samples for each stimulus, and a statistically significant difference is denoted by *(P<0.05) or (P<0.001). P-values for paired t-test analysis comparing Ad.IB(S32/36A) and Ad.IB(Y42F) infected samples for each stimulus are denoted above brackets.

View larger version (37K): [in a new window]   Fig. 5. Ad.IBAS inhibits IB protein expression and elevates baseline levels of NFB in the nucleus. HeLa cells were infected at an moi of 1000 particles/cell with Ad.IBAS 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-IB antibody, anti-IBß antibody or anti-actin antibody. Ad.IBAS infection led to a reduction in IB protein expression at all time points (lane 2-4) and an increase in IBß expression compared with Ad.GFP-infected controls (lane 1). Changes in IB expression were referenced to actin protein levels, which did not change. (B) 5 µg of nuclear protein was evaluated by EMSA to determine NFB DNA-binding activity. The induced p65-p50 heterodimer of NFB, 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.IBAS (same sample as from lane 5 Panel B) were evaluated by EMSA supershift to determine the subunit composition of the NFB 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.

View larger version (63K): [in a new window]   Fig. 6. IBAS enhances NFB binding activity following UV, TNF-, pervanadate or H/R treatment. HeLa cells were infected with either Ad.BglII or Ad.IBAS at an moi of 1000 particles/cell for 24 hours. Cells were treated with (A) UV (50 J/m2), (B) TNF- (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.IBAS-infected cells demonstrated a higher baseline of NFB DNA-binding activity (lanes 1 versus lane 4) and enhanced NFB 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.

View larger version (24K): [in a new window]   Fig. 7. IBAS enhances NFB transcriptional activity following UV, TNF-, pervanadate or H/R treatment. HeLa cells were co-infected with Ad.NFBluc (500 particles/cell) together with either Ad.BglII or Ad.IBAS at an moi of 1000 particles/cell for 24 hours. Cells were treated with (A) UV (50 J/m2), (B) TNF-(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). NFB transcriptional activity was significantly enhanced (P<0.01) in the Ad.IBAS infected groups, as compared to the groups infected by Ad.BglII, following UV, TNF-, pervanadate or H/R treatment.

View larger version (22K): [in a new window]   Fig. 8. NFB activation is pro-apoptotic following UV and TNF- treatments, while anti-apoptotic following pervanadate and H/R treatments. HeLa cells were infected with either Ad.BglII or Ad.IBAS at an MOI of 1000 particles/cell for 24 hours. Cells were treated with (A) UV (50 J/m2), (B) TNF- (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- treatment, IBAS expression significantly (P<0.01) increased apoptosis compared with the Ad.BglII-infected control group. By contrast, following pervanadate and H/R treatments, IBAS expression significantly decreased (P<0.01) the percentage of apoptotic cells as compared to the Ad.BglII-infected control group.