CHARACTERIZATION OF THE EXPRESSION AND REGULATION OF VEGF AND 4E-BP1 IN THE RETINA DURING DIABETES AND UNDER HYPERGLYCEMIC CONDITIONS

Open Access
- Author:
- Schrufer, Tabitha Lee
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 08, 2009
- Committee Members:
- Leonard Shelton Jefferson Jr., Dissertation Advisor/Co-Advisor
Leonard Shelton Jefferson Jr., Committee Chair/Co-Chair
Scot R Kimball, Committee Member
David A Antonetti, Committee Member
Yuguang Shi, Committee Member
Williard M Freeman, Committee Member - Keywords:
- VEGF
4E-BP1
diabetic retinopathy
diabetes
retina
translational control - Abstract:
- Diabetic retinopathy is the leading cause of blindness among working-age adults, occurring in the majority of individuals who have had diabetes for twenty or more years. Among the causes implicated in the development of diabetic retinopathy, increased expression of vascular endothelial growth factor (VEGF) has been implicated in the development of both abnormal neovascularization increased vascular permeability of nascent retinal vessels. Currently, the only treatment for diabetic retinopathy is laser-mediated ablation of the peripheral retina. Because this treatment results in permanent laser scars and is only effective in completely relieving the neovascularization in half of the patients undergoing the procedure, subsequent treatments are often required. This leads to a reduction in peripheral vision, night vision, and color detection. Additionally, this procedure only relieves the symptoms, but does not approach the molecular basis behind the disease. Therefore alternate molecular approaches to treatment are required. The first aim of this project was to define the time course of VEGF protein upregulation in the retina of diabetic rats to better understand the early progression of the disease. Retinas were isolated from rats 1, 2, 4, 6, and 12 weeks after streptozotocin (STZ) treatment and assessed for VEGF mRNA and protein expression. VEGF protein, but not mRNA expression was upregulated after 2 weeks of diabetes, and was returned to control levels after 12 weeks. The absence of a change in the expression of VEGF mRNA suggested a post-transcriptional mechanism for upregulated protein expression. The VEGF mRNA is complex and contains multiple elements that allow for translational control, including a uORF, 5 initiation codons, and 2 internal ribosome entry sites (IRESs). VEGF IRES utilization in response to cell stress has been well documented, but the factors involved in its utilization have not been identified. Therefore, the expression of proteins involved in translational control was evaluated. Eukaryotic initiation factor-4E-binding protein 1 (4E-BP1), a key repressor of translation initiation, was upregulated following the same time course as VEGF protein expression. Altered expression of 4E-BP1 correlated with increased sequestration of eIF4E, the mRNA cap-binding protein, an event that would be expected to repress global rates of mRNA translation. Ablation of 4E-BP1/2 in mice prevented the diabetes-induced increase in VEGF, suggesting that 4E-BP1 is required in this process. The second aim was designed to delineate the mechanism by which 4E-BP1 expression is increased in Müller cells exposed to hyperglycemic conditions. 4E-BP1 expression was maximally increased in the absence of a change in its mRNA after exposure to hyperglycemic conditions for 10 hours. Phosphorylation of 4E-BP1 by mammalian target of rapamycin complex 1 (mTORC1) results in release from eIF4E and targets the protein for ubiquitination and subsequent degradation. Analysis of the retinal lysates revealed a decrease in the relative phosphorylation of 4E-BP1 on T37/46. O-GlcNAcylation of 4E-BP1 was increased, which, unlike phosphorylation, does not interfere with the ability of 4E-BP1 to bind to eIF4E. O-GlcNAcylation of 4E-BP1 did, however, interfere with its ubiquitination, which would be expected to contribute to the stabilization and increased expression of the protein under hyperglycemic conditions. Overall the data are consistent with a model in which hyperglycemia-induced O-GlcNAcylation decreases the phosphorylation and ubiquitination of 4E-BP1. This leads to increased expression of 4E-BP1 and sequestration of eIF4E, which would be expected to repress cap-dependent translation, making complex mRNAs and those that can be translated cap-independently more competitive for translation, including the mRNA encoding VEGF.