title: O-GlcNAc modification of transcription factor Sp1 mediates hyperglycemia-induced VEGF-A upregulation in retinal cells.
abstract: OBJECTIVE
  Proangiogenic protein VEGF-A contributes significantly to retinal lesions and neovascularization in diabetic retinopathy (DR). In preclinical DR, hyperglycemia can upregulate VEGF-A in retinal cells. The VEGF-A promoter is responsive to the transcription factor specificity protein 1 (Sp1). The O-GlcNAc modification is driven by glucose concentration and has a profound effect on Sp1 activity. This study investigated the effects of hyperglycemia on Sp1-mediated expression of VEGF-A in the retinal endothelium and pigment epithelium.
  
  
  METHODS
  Hyperglycemia-exposed ARPE-19 (human retinal pigment epithelial cells) and TR-iBRB (rat retinal microendothelial cells) were assayed for levels of VEGF-A by qRT-PCR, Western blot, and ELISA. Small molecule inhibitors of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) were used to manipulate O-GlcNAc levels. Vascular endothelial growth factor-A protein and transcript were measured in cells depleted of OGT or Sp1 by shRNA. The proximal VEGF-A promoter was analyzed for glucose sensitivity by luciferase assay. Chromatin immunoprecipitation (ChIP) was used to assess Sp1 occupancy on the VEGF-A promoter.
  
  
  RESULTS
  Hyperglycemia increased VEGF-A promoter activity and upregulated VEGF-A transcript and protein. Elevation of O-GlcNAc by OGA inhibitors was sufficient to increase VEGF-A. O-GlcNAc transferase inhibition abrogated glucose-driven VEGF-A. Cellular depletion of OGT or Sp1 by shRNA significantly abrogated glucose-induced changes in VEGF-A. ChIP analysis showed that hyperglycemia significantly increased binding of Sp1 to the VEGF-A promoter.
  
  
  CONCLUSIONS
  Hyperglycemia-driven VEGF-A production is mediated by elevated O-GlcNAc modification of the Sp1 transcription factor. This mechanism may be significant in the pathogenesis of preclinical DR through VEGF-A upregulation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States.
authors.forename: Kelly
authors.initials: K
authors.lastname: Donovan
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States.
authors.forename: Oleg
authors.initials: O
authors.lastname: Alekseev
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States Department of Ophthalmology, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Qi
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States.
authors.forename: William
authors.initials: W
authors.lastname: Cho
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States.
authors.forename: Jane
authors.initials: J
authors.lastname: Azizkhan-Clifford
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D012160:Retina / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000502:physiology*; D015533:Transcriptional Activation; D015854:Up-Regulation / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D016329:Sp1 Transcription Factor; D042461:Vascular Endothelial Growth Factor A; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase
keywords: O-GlcNAc; OGT; Sp1; VEGF-A; diabetic retinopathy