Abstract
Purpose :
The von Hippel Lindau protein (VHL) is a multifunctional adaptor with many antiangiogenic roles in the cell. In addition to oxygen-dependent degradation of hypoxia inducible factor 1 alpha (HIF1a), VHL also has roles in extracellular matrix organization and negative regulation of VEGF-A expression. Independent of HIF1a degradation, VHL also regulates VEGF-A expression by binding and sequestering transcription factor Sp1. VHL-mediated negative regulation of Sp1 represses VEGF-A expression. It was reported that PKC-zeta can outcompete VHL to phosphorylate Sp1. PKC-zeta is stimulated by hyperglycemia. We have previously shown that hyperglycemia drives Sp1-mediated VEGF-A upregulation. To further investigate how hyperglycemia impacts Sp1 regulation, we analyzed VHL-Sp1 interaction in these retinal cell types.
Methods :
ARPE-19, RPE-1, and MIO-M1 cell lines were used for this study. Sp1-VHL interactions were analyzed by co-immunoprecipitation and Western blotting. CRISPR/Cas9 was used for gene knockout. Cells were stably transduced with lentivirus containing cDNA for HA-VHL, and Flag-tagged wild-type or mutant Sp1. Inhibitors of O-GlcNAc transferase (OGT) or a protein kinase c zeta (PKCz) pseudosubstrate were used to probe the dynamics of their interaction.
Results :
Hyperglycemia significantly decreases interaction between Sp1 and VHL in RPE and Müller glia. The decrease in interaction was dependent on post-translational modification of Sp1 zinc finger residues. OGT inhibition revealed that this was O-GlcNAc-independent. PKC-zeta is known to phosphorylate Sp1 zinc fingers 1 and 2; analysis of Sp1 mutants revealed that modification of zinc finger 1 residues was important for interaction. Membrane sequestration of PKC-zeta by pseudosubstrate inhibitor rescued Sp1-VHL interaction in hyperglycemia.
Conclusions :
Here, we describe an antiangiogenic VHL function that may be perturbed by hyperglycemia. Although VHL depletion is used to model proliferative DR, to our knowledge VHL has not been implicated in DR. We have found that hyperglycemia significantly decreases Sp1-VHL interaction in RPE and Müller glia, consistent with increased Sp1 transcriptional activity. Post-translational modification of Sp1 zinc finger 1 residues is important for VHL interaction. Sp1-VHL interaction may be disrupted via PKC-zeta kinase. Further studies are required to assess additional VHL functions as well as the impact on VEGF-A regulation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.