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.