Identification of O-GlcNAc modification targets in mouse retinal pericytes: implication of p53 in pathogenesis of diabetic retinopathy
Zafer Gurel1, Balyn W Zaro2, Matthew R Pratt2
1Department of Ophthalmology and Visual Sciences, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin, United States of America; McPherson Eye Research Institute, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin, United States of America.
Abstract:
Hyperglycemia is the primary cause of the majority of diabetes complications, including diabetic retinopathy (DR). Hyperglycemic conditions have a detrimental effect on many tissues and cell types, especially the retinal vascular cells including early loss of pericytes (PC). However, the mechanisms behind this selective sensitivity of retinal PC to hyperglycemia are undefined. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is elevated under hyperglycemic condition, and thus, may present an important molecular modification impacting the hyperglycemia-driven complications of diabetes. We have recently demonstrated that the level of O-GlcNAc modification in response to high glucose is variable in various retinal vascular cells. Retinal PC responded with the highest increase in O-GlcNAc modification compared to retinal endothelial cells and astrocytes. Here we show that these differences translated into functional changes, with an increase in apoptosis of retinal PC, not just under high glucose but also under treatment with O-GlcNAc modification inducers, PUGNAc and Thiamet-G. To gain insight into the molecular mechanisms involved, we have used click-It chemistry and LC-MS analysis and identified 431 target proteins of O-GlcNAc modification in retinal PC using an alkynyl-modified GlcNAc analog (GlcNAlk). Among the O-GlcNAc target proteins identified here 115 of them were not previously reported to be target of O-GlcNAc modification. We have identified at least 34 of these proteins with important roles in various aspects of cell death processes. Our results indicated that increased O-GlcNAc modification of p53 was associated with an increase in its protein levels in retinal PC. Together our results suggest that post-translational O-GlcNAc modification of p53 and its increased levels may contribute to selective early loss of PC during diabetes. Thus, modulation of O-GlcNAc modification may provide a novel treatment strategy to prevent the initiation and progression of DR.
Insights
High glucose increases O-linked β-N-acetylglucosamine (O-GlcNAc) modification in retinal pericytes (PC), leading to increased apoptosis. This suggests O-GlcNAc modification of p53 may drive pericyte loss in diabetic retinopathy.
Area of Science:
- Biochemistry
- Cell Biology
- Ophthalmology
Background:
- Diabetic retinopathy (DR) is a major complication of hyperglycemia, characterized by early loss of retinal pericytes (PC).
- The precise mechanisms underlying the selective vulnerability of retinal PCs to hyperglycemia remain unclear.
- O-linked β-N-acetylglucosamine (O-GlcNAc) modification is elevated in hyperglycemia and may contribute to diabetes complications.
Purpose of the Study:
- To investigate the role of O-GlcNAc modification in the selective loss of retinal pericytes (PCs) under hyperglycemic conditions.
- To identify O-GlcNAc-modified proteins in retinal PCs and elucidate their involvement in cell death pathways.
Main Methods:
- Cultured retinal pericytes (PCs) were exposed to high glucose and O-GlcNAc inducers (PUGNAc, Thiamet-G).
- Apoptosis was assessed, and O-GlcNAc-modified proteins were identified using click-It chemistry and LC-MS analysis with an alkynyl-modified GlcNAc analog (GlcNAlk).
Main Results:
- Retinal PCs exhibited a significantly higher increase in O-GlcNAc modification compared to endothelial cells and astrocytes under high glucose.
- Increased O-GlcNAc modification correlated with enhanced apoptosis in PCs, both under high glucose and with O-GlcNAc inducers.
- LC-MS identified 431 O-GlcNAc target proteins in PCs, including 115 novel targets, with 34 implicated in cell death.
- Increased O-GlcNAc modification of p53 was observed, along with elevated p53 protein levels.
Conclusions:
- Elevated O-GlcNAc modification in retinal PCs under hyperglycemic conditions contributes to increased apoptosis.
- The O-GlcNAc modification of p53 and its subsequent increase in levels may play a critical role in the selective loss of PCs during diabetes.
- Modulating O-GlcNAc modification presents a potential therapeutic strategy for preventing diabetic retinopathy progression.
