Abstract
Purpose :
Diabetic hyperglycemia increases protein modification by the glucose-derived nucleoside sugar, O-GlcNAc. Increased pan-cellular O-GlcNAcylation has been associated with pathological molecular consequences in the diabetic retina. The transcription factor Sp1 is heavily O-GlcNAc modified, which dysregulates its transcriptional activity at multiple target genes including vascular endothelial growth factor A (VEGF-A). We investigated the effect of Sp1 site-specific modification on Sp1 transactivity. We also evaluated the O-GlcNAc transferase (OGT) enzyme as a therapeutic target for early diabetic retinopathy.
Methods :
Human retinal pigment epithelial cell lines ARPE-19 and RPE-1 as well as the human Müller glial cell line MIO-M1 were used for these studies. The VEGF-A promoter was analyzed by luciferase reporter assay. The CRISPR/Cas9 system was used for gene knockout. Cells treated with 25mM glucose and OGT inhibitors were assayed for RNA by qRT-PCR, protein expression by Western blotting, and factor secretion by ELISA. Promoter occupancy was assessed by chromatin immunoprecipitation. Sp1 affinity for its consensus motif was assessed by electrophorectic mobility shift assay.
Results :
We assayed serial truncations of the VEGF-A promoter by luciferase assay; Sp1 binding sites in the proximal VEGF-A promoter are both necessary and sufficient for glucose stimulation. Pharmacologic OGT inhibition effectively prevented glucose-driven luciferase activity and upregulation of VEGF-A in an Sp1-dependent manner. OGT inhibition also prevented upregulation of TGFβ1 and PAI-1 transcript and protein by an Sp1-independent mechanism. Comparison of wild-type and an O-GlcNAc-blunted mutant Sp1 revealed that Sp1 PTM on zinc finger 3 serine residues is necessary for promoter stimulation and VEGF-A upregulation.
Conclusions :
Transcription factor Sp1 has been reported by many groups to be pathologically transcriptionally active in multiple diabetic tissues, which has been linked to its O-GlcNAc status. Here, we report that hyperglycemia-driven Sp1 O-GlcNAcylation of zinc finger 3 is required for VEGF-A upregulation in RPE and Müller cell lines. Inhibtion of O-GlcNAc transferase impacted multiple disease-relevant factors including VEGF-A, TGFβ1, and PAI-1, making OGT a promising therapeutic target.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.