Transcription factor SP1 mediates hyperglycemia-induced upregulation of roundabout4 in retinal microvascular endothelial cells.

Roundabout4 (Robo4) is a gene that is expressed specifically in vasculature and is involved in the angiogenesis and integrity of blood vessels. The expression level of Robo4 increases gradually along with the development of diabetic retinopathy (DR). In this study, we explored the mechanism of transcriptional regulation of Robo4 in retinal endothelial cells, and investigated the effects of this regulation on cellular functions under hyperglycemic conditions. Human retinal endothelial cells (HREC) exposed to hyperglycemia were used to detect the expression levels of specificity protein 1 (SP1) and Robo4 by RT-qPCR and western blotting. Small interfering RNA (SiRNA) transfection technology was used to analyze the regulatory relationship between SP1 and Robo4. The effect of transcription factor SP1 on Robo4 promoter activity and the location of SP1 binding sites were investigated using chromatin immunoprecipitation (ChIP) and luciferase assay. Cell migration, monolayer permeability and tube formation assays were performed to demonstrate the role of SP1/Robo4 in regulating HREC functions in hyperglycemic conditions. The results showed that hyperglycemia upregulated the mRNA and protein levels of SP1 and Robo4 in HREC. Depletion of SP1 by siRNA transfection inhibited the hyperglycemia induced overexpression of Robo4. ChIP combined with luciferase assay showed that under hyperglycemic conditions, SP1 significantly increased the transcriptional level of Robo4 via an additional SP1 binding site at -1912/-1908 in the Robo4 promoter. Repressing the SP1/Robo4 pathway effectively mitigated the abnormity in HREC migration, permeability and angiogenesis induced by hyperglycemia. All these findings indicate that hyperglycemia-induced upregulation of Robo4 is mediated by enhanced transcription of SP1. The SP1/Robo4 signaling pathway can regulate the migratory ability, monolayer permeability and angiogenesis of HREC under hyperglycemic conditions, suggesting that it may play an important role in microvascular dysfunction during DR.