Abstract
Purpose: :
Iduna is a neuroprotective protein against glutamate NMDA receptor-mediated excitotoxicity both in vitro and in vivo through interfering with PAR polymer-induced cell death. Mutation at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. NPD1, a docosahexanoic acid-derived mediator, induced cell survival through the upregulation of Bcl2 class of survival proteins under oxidative-stress (OS) in RPE cells. The goal of this study is to examine if NPD1 survival bioactivity engages Iduna expression in RPE cells undergoing OS.
Methods: :
72h grown ARPE-19 cells were serum starved overnight, oxidative-stress was introduced, and then challenged with various concentrations (1nM-2µM) of NPD1 for 3, 4, 6, 8, 10, and 12h. Cell lysates were made, and Western blot analysis was performed. Iduna protein was detected by either anti-Iduna antibody (RNF146) or clone N201/35 containing anti-iduna (UC Davis/NIH Neiro Mab Facility, CA). Immunocytochemical studies were performed on ARPE-19 cells after 6h treatment with 50nM NPD1 under OS, and probed with Iduna-specific antibody.
Results: :
Our results indicated that NPD1 and DHA at 50 and 100 nM concentrations enhanced expression of Iduna in ARPE-19 cells undergoing OS. However, OS or NPD1 alone did not have any effect on the Iduna expression. Moreover, the NPD1-mediated expression of Iduna is time and concentration-dependent. The enhanced expression of Iduna by NPD1 started at least 4h after initiation of OS. NPD1 treatment, maximized at 6h, continued until 10h, and declined at 12h in ARPE-19 cells. Concentration kinetic of NPD1 (1 nM to 2 µM)-mediated expression of Iduna in OS ARPE-19 cells indicated that NPD1 as low as 10 nM was able to enhance Iduna expression and maximized at 50 nM, after which it started to decline. Additionally, human primary retinal culture (hRPE) also followed the same path. Immunocytochemical analysis of the Iduna expression in ARPE-19 cells under oxidative stress by NPD1 corroborated the above observations.
Conclusions: :
Here we identify that NPD1 exerted enhanced expression of Iduna in RPE cells undergoing OS. To our knowledge, this is the first report that a bioactive lipid molecule NPD1 mediates enhanced expression of Iduna in RPE cells. This NPD1 mediated expression of Iduna may be a key regulator of cell survival in OS mediated cell death.
Keywords: cell survival • gene/expression • retinal pigment epithelium