Direct protein-protein interaction between Npas4 and IPAS mutually inhibits their critical roles in neuronal cell survival and death.

Inhibitory PAS domain protein (IPAS) is a bifunctional protein that acts as a transcriptional repressor in hypoxia and as a pro-apoptotic protein involved in neuronal cell death. Npas4 (NXF or LE-PAS) is a transcriptional factor that protects nerve cells from endogenous and foreign neurotoxins. Here we show that IPAS and Npas4 antagonize each other through their direct interaction. Coimmunoprecipitation experiments revealed that multiple binding sites on each protein were involved in the interaction. CoCl treatment of PC12 cells that induces IPAS repressed the transactivation activity of Npas4, and IPAS siRNA treatment reduced the CoCl-induced repression. CoCl-induced apoptosis was suppressed by the addition of KCl that induces Npas4. The protective effect of KCl was attenuated by siRNA-mediated gene silencing of Npas4. Npas4 and IPAS proteins were induced and localized in the cytoplasm of the dopaminergic neurons in the substantia nigra pars compacta after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Npas4 mice exhibited greater sensitivity to MPTP in nigral dopaminergic neurons. Together, these results strongly suggest that neuroprotective activity of Npas4 was, at least partly, exerted by inhibiting the pro-apoptotic activity of IPAS through direct interaction.