Role of O-GlcNAcylation of Sp1 in the pathogenesis of diabetic retinopathy

    loading  Checking for direct PDF access through Ovid

Abstract

Purpose

Central to the pathogenesis of diabetic retinopathy (DR) is vascular endothelial growth factor A (VEGF-A). Gene expression of VEGF-A is regulated by several transcription factors, including specificity protein 1 (Sp1). Sp1 is known to participate in angiogenesis and co-localizes with VEGF-A in the epiretinal membranes of DR patients. Sp1 is heavily O-GlcNAcylated by the enzyme O-GlcNAc transferase (OGT). This glycosylation modification affects its transcriptional activity, localization, stability, and protein interactions. Since O-GlcNAcylation of proteins is elevated under high glucose conditions, we investigated the relationship between O-GlcNAcylation of Sp1 and increased VEGF-A transcription.

Methods

Hyperglycemia-exposed ARPE-19 (retinal pigment epithelial cells) and TR-iBRB (rat retinal microendothelial cells) were assayed for levels of VEGF-A by RT-qPCR and Western blot. VEGF-A protein and mRNA levels were measured in cells depleted of Sp1 or OGT by shRNA. Small molecule inhibitors of OGA and OGT were used to increase or decrease total O-GlcNAc. Chromatin immunoprecipitation was used to assess levels of Sp1 bound to the VEGF-A promoter.

Results

Hyperglycemia caused increased VEGF-A transcript and protein expression in ARPE-19 and TR-iBRB cells. OGT or Sp1 depletion significantly abrogated these glucose-induced changes in both cell types, while minimally affecting basal expression. ChIP analysis showed that glucose increased the amount of promoter-bound Sp1.

Conclusion

Glucose-induced increases in pan O-GlcNac in the RPE and vascular retina may be participating in the aberrant expression of VEGF-A in DR. The highly O-glcosylated Sp1 transcription factor may be responsible for early VEGF-A production in the retina.

Related Topics

    loading  Loading Related Articles