Abstract
Purpose:
Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived lipid mediator, induces cell survival in uncompensated oxidative stress conditions. NPD1 is a critical modulator of homeostatic alterations during rod outer segment phagocytosis (Bazan NG, Invest Ophthalmol Vis Sci. 2007;48:4866-81). NPD1 effects include the down-regulation of pro-apoptotic proteins and the up-regulation of pro-survival counterparts, however the molecular principles underlying this modulation remain unknown.
Methods:
To define the mechanisms by which NPD1 modulates cell survival, we used human retinal pigment epithelial (RPE) cells as a cellular model. We analyzed the promoter of BIRC3, a pro-survival protein that is up-regulated by NPD1, to find the transcription factors that are implicated in the NPD1-mediated modulation. Finally, we silenced BIRC3 to assess its importance in the NPD1 pathway.
Results:
We report here that, in RPE cells, NPD1 induces nuclear translocation and cREL synthesis that, in turn, mediates BIRC3 transcription. NPD1 activates NF-kappaB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-kappaB pathways. BIRC3 promoter contains two binding sites for NF-kappaB that scored highly for cREL and ChIP assay, showing that this binding is enhanced by NPD1. cREL and RelB expression follows a similar pattern of BIRC3, indicating that NPD1 also is required to activate cREL-mediated RelB expression. BIRC3 silencing prevents NPD1 induction of survival against oxidative stress, noticed as increased activation of effector caspases 3 and 7. BIRC3 may exert its effects by modulation of RIP1 activity, which participates in regulation of the formation of complexes II and III.
Conclusions:
These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation. Thus, NPD1 bioactivity governs key counter-regulatory gene transcription events decisive for RPE/photoreceptor cell integrity when confronted with potential disruptions of homeostasis.
Keywords: 615 neuroprotection •
701 retinal pigment epithelium