Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin-proteasome-mediated degradation of FLICE inhibitory protein

Stimulation of cell surface Fas ( CD95) results in recruitment of cytoplasmic proteins and activation of caspase-8, which in turn activates downstream effector caspases leading to programmed cell death. Nitric oxide ( NO) plays a key role in the regulation of apoptosis, but its role in Fas-induced cell death and the underlying mechanism are largely unknown. Here we show that stimulation of the Fas receptor by its ligand ( FasL) results in rapid generation of NO and concomitant decrease in cellular FLICE inhibitory protein ( FLIP) expression without significant effect on Fas and Fas-associated death domain ( FADD) adapter protein levels. FLIP down-regulation as well as caspase-8 activation and apoptosis induced by FasL were all inhibited by the NO-liberating agent sodium nitroprusside and dipropylenetriamine NONOate, whereas the NO synthase inhibitor aminoguanidine and NO scavenger 2-(4-carboxyphenyl)4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide ( PTIO) had opposite effects, indicating an anti-apoptotic role of NO in the Fas signaling process. FasL-induced down-regulation of FLIP is mediated by a ubiquitin-proteasome pathway that is negatively regulated by NO. S-nitrosylation of FLIP is an important mechanism rendering FLIP resistant to ubiquitination and proteasomal degradation by FasL. Deletion analysis shows that the caspase-like domain of FLIP is a key target for S-nitrosylation by NO, and mutations of its cysteine 254 and cysteine 259 residues completely inhibit S-nitrosylation, leading to increased ubiquitination and proteasomal degradation of FLIP. These findings indicate a novel pathway for NO regulation of FLIP that provides a key mechanism for apoptosis regulation and a potential new target for intervention in death receptor-associated diseases.