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

Mitochondrial DNA depleted (ρ0) 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 ρ0 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 ρ0 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 ρ0 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.