Background & Aim: We evaluated the full range effects of FOXO3a, one of the main forkhead transcription factors expressed in endothelial cells (ECs), by microarray analysis and investigated the role of FOXO3a regulating TNF receptor signaling pathway.
Methods and Results: Human umbilical vein endothelial cells (HUVECs) were transfected with adenoviral vectors expressing constitutively active FOXO3a (Ad-TM-FOXO3a), and oligonucleotide microarray was performed. Gene transfer of TM-FOXO3a to HUVECs caused remarkable apoptosis, which were accompanied with up-regulation of several genes related with TNF receptor signaling, such as TNF-a, TANK (TRAF-associated NF-kB activator), and TTRAP (TRAF and TNF receptor-associated protein). Furthermore, kB-Ras1 (IkB-interacting Ras-like protein-1) which is known to block IkB degradation was found increased, and intranuclear translocation of NF-kB was inhibited. Simultaneously, GADD45B and XIAP, negative regulators of c-Jun N-terminal kinase (JNK), were suppressed and JNK activity was found increased. Co-transfection of Ad-TAM67 (dominant negative c-Jun) with Ad-TM-FOXO3a significantly reduced TNF-a expression, suggesting that the increased c-Jun contributes to FOXO3a-induced TNF-a expression. Attenuation of TNF signaling pathway either by blocking antibody for TNF receptor or by blocking JNK with DMAP (6-dimethylaminopurine) or Ad-TAM67 co-transfection, significantly reduced FOXO3a-induced apoptosis of ECs. Finally, treatment of vasculature with heat shock, an activator of endogenous FOXO3a, resulted in endothelial damage, which was completely rescued by Ad-TAM67.
Conclusion: FOXO3a promotes apoptosis of ECs, through activation of JNK and suppression of NF-kB. These data identify a novel role of FOXO3a to turn TNF receptor signaling to a pro-apoptotic JNK-dependent pathway.