Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: a role for NF-κB-STAT3-directed gene expression

Abstract

Mitochondrial DNA depleted (ρ(0)) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ(+) HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ(0) cells compared to ρ(+) HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ(+) HSF, but this response was substantially decreased in ρ(0) HSF. Suppression of the IKK-NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ(+) HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ(0) HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6-JAK2-STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.

Fig. 1. Mitochondrial (mt)DNA-depleted human skin fibroblasts (HSF ρ⁰) and their parental cells (HSF ρ⁺)

A. PCR amplification of the mtDNA fragment (251 bp) from HSF ρ⁺ using mtDNA primer set. B. Cytochrome-c oxidase activity (oxidized cytochrome-c, nmol/min/mg protein) in total cell extracts from ρ⁺ and ρ⁰ cells was determined as described in “Methods”. C. Immunofluorescence of mitochondrial pyruvate dehydrogenase (green) with PI-stained red nuclei in ρ⁺ and ρ⁰ cells. Mitochondrial membrane potential was detected using JC-1 probe based on change of green to red fluorescence. D. Rates of oxygen consumption (Femtomole/cell/min) for ρ⁺ and ρ⁰ cells were determined using an oxygen electrode unit.

Fig. 2. Differentially expressed genes in mtDNA-depleted human skin fibroblasts (HSF ρ⁰) compared with their parental cells (HSF ρ⁺)

A. Comparison of gene expression was performed using microarrays. The log transformed fold change in relative gene expression is plotted on the x-axis and the log transformed p-value is plotted on the y-axis. Each point in the graph represents an individual gene. Genes towards the top of the graph have a lower p-value and the genes further to the left and the right of the x-axis have greater fold change between classes. From paired class comparison of HSF ρ⁰ and HSF ρ⁺ gene expression, there were 2100 genes that were differentially expressed between the two conditions, FDR<0.04. B. Gene ontology analysis using PANTHER showing functional enrichment of genes, number of genes in each protein class is indicated out of the 2100 differentially expressed genes. Categories are ordered by significance (p-values).

Fig. 3. Hierarchical clustering of gene expression after alpha irradiation of HSF ρ⁰ compared with HSF ρ⁺

A. HSF gene expression responses at 4 hours after irradiation with 0.5 Gy alpha-particles; the heatmap of differentially expressed 265 genes was made using BRB tools (http://linus.nci.nih.gov/BRB-ArrayTools.html). The inset displays a subset of the differentially expressed genes showing MMP12 and PTGS2 gene expression. The scale bar indicates induced (red) and repressed (green) expression ratios. B. Gene ontology analysis using PANTHER biological processes comparing HSF ρ⁰ to ρ⁺ gene expression response after irradiation with 0.5 Gy α-particles. Functionally enriched categories are shown, p-values indicate statistical significance, p-value cut off 0.05 and NS is not significant. C. Expression levels of selected genes in HSF ρ⁰ compared with HSF ρ⁺ 4 hours after irradiation.

Fig. 4. Validation of differential gene expression in mtDNA-depleted human skin fibroblasts, (HSF ρ⁰), and their parental cells (HSF ρ⁺) after α-irradiation using qRT-PCR

A. Heatmap of gene expression levels in the low density array panel. Gene expression was determined by quantitative real time PCR (qRT-PCR). 33 genes were analyzed across the time course of 0.5, 1, 4 and 24 h in non-treated and α-irradiated (0.5 Gy) cells. Heatmap for up-regulated and down-regulated genes was generated using BRB Array-Tools software. B. Relative gene expression changes in IL6, IL8, PTGS2/COX2 and TNFRSF1 in HSF in the time course. Gene expression was determined qRT-PCR. Points are the mean of three independent replicates. C. Relative increase in IL6 secretion in ρ⁺ and ρ⁰ HSF 48 h after exposure to ionizing irradiation (0.5 Gy).

Fig. 5. Activation of the main signaling pathways and levels of target proteins after α-irradiation of HSF

A, B. Western blot analysis of indicated proteins 4 h and 24 h after α-irradiation (0.5 Gy) of ρ⁺ and ρ⁰ HSF. C. NF-κB-dependent luciferase reporter activity (2×NF-κB-Luc) was determined in transiently transfected ρ⁺ and ρ⁰ HSF before and 8 h after α-irradiation (0.5 Gy) or 8 h after TNFα (20 ng/ml) treatment. D. STAT-dependent luciferase reporter activity (3×STAT-Luc) was determined in transiently transfected ρ⁺ and ρ⁰ HSF before and 8 h after α-irradiation (0.5 Gy) or 8 h after IL6 (10 ng/ml) treatment. Error bars represent mean ± S.D. (Student's t test, p< 0.05). E. Surface expression of TRAIL-R2/DR5 in HSF before and 24 h after α-irradiation was determined using immunostaining and FACS analysis.

Fig. 6. Cell cycle and apoptotic analysis of mtDNA-depleted human skin fibroblasts (HSF ρ⁰) and their parental cells (HSF ρ⁺)

A. Cell cycle analysis of ρ⁺ and ρ⁰ HSF after α-irradiation (0.5 Gy) was performed using PI staining of DNA and FACS analysis. B. Clonogenic survival fraction (SF) of control and treated cells 12 days after irradiation is indicated in brackets. C. Western blot analysis of indicated proteins 24 h after α-irradiation (0.5 Gy) of ρ⁺ and ρ⁰ HSF. D. Apoptosis was induced by α-irradiation (0.5 Gy) alone or in combination with LY294002 (50 μM), sodium arsenite (As, 4 μM) alone or in combination with LY294002 (50 μM), in the presence or the absence of caspase inhibitors, IETD or LEHD (50 μM), in ρ⁺ and ρ⁰ HSF.

Fig. 7. Surface expression of receptors and death ligand/receptor-induced apoptosis in HSF

A. Surface expression of TRAIL-R2/DR5, TRAIL-R1/DR4, FAS, TNFR1, NGFR1 and IL6Rα was determined using immunostaining with the corresponding PE-labeled mAbs and the subsequent FACS analysis of ρ⁺ and ρ⁰ HSF. Medium fluorescent intensity (MFI) is indicated in brackets. B. Induction of apoptosis by ligands, FasL (25 ng/ml), TNFα (20 ng/ml) and NGF (50 ng/ml) in the presence CHX (1 μg/ml).

Fig. 8. TRAIL and CHX induced apoptosis in HSF

A. Dose-dependent response of ρ⁺ and ρ⁰ HSF to TRAIL (10-100 ng/ml) in the presence of cycloheximide (CHX, 1 μg/ml). Apoptotic levels (% cells in pre-G1/G0) were determined 48 h after treatment using PI staining and FACS analysis. Error bars represent mean ± S.D. (Student's t test, p< 0.05. B. Apoptosis induced by TRAIL (50 ng/ml) and CHX (1 μg/ml) in the presence or the absence of IETD or LEHD (50 μM) in ρ⁺ and ρ⁰ HSF. An additional effect of α-irradiation (IR at 0.5 Gy) on (TRAIL+CHX)-induced apoptosis was also determined. Apoptotic levels (% cells in pre-G1/G0) were determined 48 h after treatment using PI staining and FACS analysis. Error bars represent mean ± S.D. (Student's t test, p< 0.05). C. Western blot analysis of caspase-8-dependent pathway following TRAIL+CHX treatment of HSF. Decreased levels of procaspase-8 (determined with mAb to p55) reflect its cleavage and activation. The subsequent activation of caspase-3 results in PARP cleavage that was more advanced in ρ⁰ HSF. D. Relative specific activity of capase-8, caspase-9 and caspase-3 in total cell extracts of ρ⁺ and ρ⁰ HSF 6 h after TRAIL+CHX treatment.

Fig. 9. Modulation of TRAIL-induced apoptosis in HSF using inhibitory mAbs and low molecular inhibitors of cell signaling pathways

A, B. Effects of suppression IKKβ-NF-κB by BMS-345541 (10 μM) and JAK2-STAT3 by AG490 (50 μM) on 2×NF-κB-Luc and 3×STAT3-Luc reporter activities, respectively, in transiently transfected HSF. C. Effects of suppression IKKβ-NF-κB by BMS-345541 (10 μM) and JAK2-STAT3 by AG490 (50 μM) on TRAIL-induced apoptosis. Apoptotic levels (% cells in pre-G1/G0) were determined 48 h after treatment using PI staining and FACS analysis. Error bars represent mean ± S.D. (Student's t test, p< 0.05). D. Effect of IKKβ-NF-κB inhibitor, BMS-345541 (10 μM), on relative increase in IL6 secretion in ρ⁺ and ρ⁰ HSF 48 h after exposure to ionizing irradiation. E. Effects of blockage of IL6- or IL8-dependent signaling with anti-IL6 mAb (5 μg/ml) or anti-IL8 mAb (5 μg/ml), respectively, added into the cell media on TRAIL-induced apoptosis in ρ⁺ and ρ⁰ HSF. Cells were additionally pretreated with 0.5 Gy α-irradiation (the right panel). Error bars represent mean ± S.D. (Student's t test, p< 0.05).