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Wan Jin Jahng, Megan Frost, Weilue He, Ji-Yeon Um, Rissa McDonough, Sydney Bruestle, Trevor Moser, Srinivas R. Sripathi; Transition in Oxygen Stoichiometry Determines Vimentin/PP2A Nitration-Phosphorylation Signaling Through Nitric Oxide. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1596.
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How much nitric oxide (NO) is produced and what duration is needed for protein nitration in the RPE? We directly measured NO production from RPE cells exposed to different stresses, including light/dark cycles, continuous illumination, different light intensities (200-7000 lux) and oxidative stress. We aimed to establish therapeutic levels of NO that induces up-regulation of protecting molecules by delivering precisely controlled surface fluxes of NO to cultured cells using a novel NO-generating polymer. This will establish an in vitro model to allow the systematic study of RPE damage caused by hypoxia, hyperoxia, bright light and constant light and both the protective and pathological roles that NO plays in retinal degeneration.
The levels of NO produced by RPE cells were directly measured under different levels of light or oxygen concentration (1, 5, 10, 21, 50% O2). NO damage to RPE cells was examined by delivering precisely controlled surface fluxes of NO to the cells in vitro. We have quantitatively established what level and duration of NO account for its neuroprotective or cytotoxic properties and at what level NO becomes pathological in RPE cell death. These measurements were made using a Sievers’ 280i Nitric Oxide Analyzer and BODIPY compound.
Nitric oxide was generated oxygen-dependent manner in RPE cells under stress conditions. Inducible and endothelial nitric oxide synthase (iNOS, eNOS) were confirmed by Western blot. Excess nitric oxide synthesized peroxynitrite and accelerated PP2A nitration. PP2A modulates cell proliferation directly or indirectly through MAPK, p-53, NF-ΚB by phosphorylation levels. Accumulation of phosphorylated vimentin by inactive PP2A was positively correlated with an apoptotic marker, BAK. Elevated levels of iNOS under oxidative stress at 12, 36, 48 hrs were observed. PP2A dephosphorylated vimentin at Ser38 site whereas phosphorylation of PP2A-Cα/β at Tyr307 decreased under oxidative stress. Increased p-vimentin and decreased BAK were confirmed by okadaic acid. Bcl-xL signaling was regulated by substrate (Arg) and a competitive inhibitor (L-NAME) of NOS.
Upregulated phosphorylation of Bcl-xL and PP2A inhibition may suggest that the apoptotic connection between PP2A activity and RPE cell death. Combinatorial network of modifications, including phosphorylation, nitration, and methylation of PP2A is the determinant of the activity and turnover rate. Our data suggests that nitric oxide plays a key role in dephosphorylation signaling through PP2A phosphorylation-nitration. We propose that nitric oxide is a neuroprotective or cytotoxic second messenger based on its local concentration.
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