Temporal pattern of NFκB activation influences apoptotic cell fate in a stimuli-dependent fashion

The transcription factor NFkappaB is a critical immediate early response gene involved in modulating cellular responses and apoptosis following diverse environmental injuries. The activation of NFkappaB is widely accepted to play an antiapoptotic role in cellular responses to injury. Hence, enhancing NFkappaB activation in the setting of injury has been proposed as one potential therapeutic approach to environmental injuries. To this end, we constructed a recombinant adenoviral vector (Ad.IkappaBalphaAS) expressing antisense IkappaBalpha mRNA that is capable of augmenting NFkappaB activation prior to and following four types of cellular injury [TNF-alpha, UV, hypoxia/reoxygenation (H/R) or pervanadate treatment]. Biochemical and functional analyses of NFkappaB activation pathways for these injuries demonstrated two categories involving either serine (S32/36) phosphorylation (TNF-alpha, UV) or tyrosine (Y42) phosphorylation (H/R or PV) of IkappaBalpha. We hypothesized that activation of NFkappaB prior to injury using antisense IkappaBalpha mRNA would reduce apoptosis. As anticipated, recombinant adenoviral IkappaBalpha phosphorylation mutants (Ad.IkappaBalphaS32/36A or Ad.IkappaBalphaY42F) preferentially reduced NFkappaB activation and enhanced apoptosis following injuries associated with either serine or tyrosine phosphorylation of IkappaBalpha, respectively. These studies demonstrate for the first time that an IkappaBalphaY42F mutant can effectively modulate NFkappaB-mediated apoptosis in an injury-context-dependent manner. Interestingly, constitutive activation of NFkappaB following Ad.IkappaBalphaAS infection reduced apoptosis only following injuries associated with IkappaBalpha Y42, but not S32/36, phosphorylation. These findings demonstrate that the temporal regulation of NFkappaB and the apoptotic consequences of this activation are differentially influenced by the pathway mediating NFkappaB activation. They also provide new insight into the therapeutic potential and limitations of modulating NFkappaB for environmental injuries such as ischemia/reperfusion and pro-inflammatory diseases.