FOXO1 functions as a master switch that regulates gene expression necessary for tumor necrosis factor-induced fibroblast apoptosis

Tumor necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory and pro-apoptotic mediator that plays an important role in several normal and disease processes. TNF-induced cell death is one of the principal mechanisms by which cells are removed. Although TNF-mediated apoptosis has been the subject of intense investigation, the transcriptional mechanisms through which it promotes apoptosis are not well understood and, paradoxically, the archetypal TNF-induced nuclear factor NF kappa B is anti-apoptotic. To identify a potential master transcriptional regulator of apoptosis, we examined an array of TNF-alpha-activated transcription factors. Forkhead box class-O 1 (FOXO1) was strongly activated, which was confirmed in vitro and in vivo by electrophoretic mobility shift assay. The central importance of FOXO1 was established in experiments with small inhibitory RNA (siRNA) that specifically silenced FOXO1. When FOXO1 was silenced, fibroblast apoptosis was reduced 76%. Other siRNAs that partially inhibited FOXO1 expression were proportionately effective in reducing apoptosis. Transcriptional profiling was then carried out in conjunction with siRNA to establish mechanisms by which FOXO1 modulated apoptosis. In the absence of FOXO1, TNF-alpha failed to up-regulate a large number of pro-apoptotic gene families including ligands, receptors, adapter molecules, mitochondrial proteins, and caspases. siRNA silencing also blocked down-regulation of anti-apoptotic genes. These results indicate that TNF induces activation of the FOXO1 transcription factor, which acts as a master switch to control apoptosis.