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 anti-apoptotic 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.