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Haoshen Shi, Jena J Steinle, Elizabeth A Berger; Site specific phosphorylation of NF-κB p65 in human retinal cells exposed to pro- versus anti-inflammatory cytokines under high glucose conditions. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5212.
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Inflammation has more recently been considered an important contributor to the pathogenesis of diabetic retinopathy (DR). Site-specific phosphorylation of NF-κB p65 subunit leads to selective activation and transcription of downstream pro-inflammatory genes. IL-1β and TNF-α are direct activators of NF-κB and known to contribute to the overall inflammatory response in DR; while anti-inflammatory cytokine IL-4 suppresses inflammation. As such, the current study is designed to characterize site specific NF-κB p65 phosphorylation and downstream activation of inflammatory mediators following high glucose exposure associated with DR.
Primary human retinal endothelial cells (HREC) and MIO-M1 Müller cells were exposed to either normal glucose (5 mM, NG) or high glucose (25 mM, HG) for four days. Cells were serum starved for 24h, then treated with IL-1β, TNF-α or IL-4 in HG for 10 min, 30 min, 2 hours or 24 hours. Total protein was collected for Western blot analysis of p65 phosphorylation sites – s276, s311, s468, s529 and s536. Total RNA was collected to confirm NF-κB-induced inflammatory molecules that are known to be altered in DR.
Exposure to HG conditions resulted in increased phosphorylation of NF-κB p65 subunits s311 and s276 in HREC; while s468 was elevated in MIO-M1. Treatment with pro-inflammatory cytokines IL-1β and TNF-α demonstrated up-regulated phosphorylation of NF-κB p65 subunits s311 and s536 in HREC; subunits s536, s529 and s468 were elevated in MIO-M1 cells. Anti-inflammatory IL-4 reduced phosphorylation of subunit s536 in HREC; while s529 and s468 were decreased in MIO-M1 cells.
These data suggest that NF-κB p65 phosphorylation sites are differentially phosphorylated in HREC versus MIO-M1 in response to HG; further exposure to cytokines such as IL-1β and TNF-α leads to differential activation of pathogenic molecules known to be associated with DR. In contrast, IL-4 can selectively inhibit phosphorylation of NF-κB p65 at different sites, potentially suppressing NF-κB’s activity as it relates to DR. The current study suggests potential therapeutic intervention points of NF-κB p65 phosphorylation sites that may improve disease pathogenesis as it relates to DR.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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