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Ablation of 4E-BP1/2 prevents hyperglycemia-mediated induction of VEGF expression in the rodent retina and in Muller cells in culture.

Schrufer TL, Antonetti DA, Sonenberg N, Kimball SR, Gardner TW, Jefferson LS - Diabetes (2010)

Bottom Line: Whereas VEGF mRNA in the retina remained constant, VEGF expression was increased as early as 2 weeks after the onset of diabetes.Similar results were observed after 10 h of exposure of cells in culture to hyperglycemic conditions.Because the VEGF mRNA contains two internal ribosome entry sites, the increased expression is likely a consequence of ribosome loading at these sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.

ABSTRACT

Objective: Vascular endothelial growth factor (VEGF) contributes to diabetic retinopathy, but control of its expression is not well understood. Here, we tested the hypothesis that hyperglycemia mediates induction of VEGF expression in a eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) 1 and 2 dependent manner.

Research design and methods: The retina was harvested from control and type 1 diabetic rats and mice and analyzed for VEGF mRNA and protein expression as well as biomarkers of translational control mechanisms. Similar analyses were performed in Müller cell cultures exposed to hyperglycemic conditions. The effect of 4E-BP1 and 4E-BP2 gene deletion on VEGF expression was examined in mice and in mouse embryo fibroblasts (MEFs).

Results: Whereas VEGF mRNA in the retina remained constant, VEGF expression was increased as early as 2 weeks after the onset of diabetes. Increases in expression of 4E-BP1 protein mirrored those of VEGF and expression of 4E-BP1 mRNA was unchanged. Similar results were observed after 10 h of exposure of cells in culture to hyperglycemic conditions. Importantly, the diabetes-induced increase in VEGF expression was not observed in mice deficient in 4E-BP1 and 4E-BP2, nor in MEFs lacking the two proteins.

Conclusions: Hyperglycemia induces VEGF expression through cap-independent mRNA translation mediated by increased expression of 4E-BP1. Because the VEGF mRNA contains two internal ribosome entry sites, the increased expression is likely a consequence of ribosome loading at these sites. These findings provide new insights into potential targets for treatment of diabetic retinopathy.

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Related in: MedlinePlus

The relative phosphorylation of a number of biomarkers of mRNA translational control is unchanged in the retina after induction of diabetes. Samples from retinal homogenates prepared as described in research design and methods were assessed for the relative phosphorylation of (A) eIF2α on Ser51, (B) eIF4G on Ser1108, and (C) eIF4E on Ser209. Values for phosphorylation status were normalized to the amount of the respective total protein. Values represent the mean ± SEM of at least three independent sets of animals at each time point (n = 2–4 within each set), except that the control value for eIF2α at 6 weeks represents two animals. Control (white bars) and diabetic (black bars) animals.
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Figure 2: The relative phosphorylation of a number of biomarkers of mRNA translational control is unchanged in the retina after induction of diabetes. Samples from retinal homogenates prepared as described in research design and methods were assessed for the relative phosphorylation of (A) eIF2α on Ser51, (B) eIF4G on Ser1108, and (C) eIF4E on Ser209. Values for phosphorylation status were normalized to the amount of the respective total protein. Values represent the mean ± SEM of at least three independent sets of animals at each time point (n = 2–4 within each set), except that the control value for eIF2α at 6 weeks represents two animals. Control (white bars) and diabetic (black bars) animals.

Mentions: Because the expression of the VEGF mRNA did not change over the time course used above, mechanisms involved in the regulation of mRNA translation were explored that might explain increased expression of the protein. Such mechanisms include changes in phosphorylation of eIF4E, eIF4G, and the α-subunit of eIF2. Therefore, the phosphorylation state of each of these proteins was assessed at various times after STZ treatment. No significant difference in phosphorylation of eIF2α on Ser51 (Fig. 2A), eIF4G on Ser1108 (Fig. 2B), or eIF4E on Ser209 (Fig. 2C) was observed in the retina of diabetic compared with control rats at 1, 2, 4, or 6 weeks of diabetes.


Ablation of 4E-BP1/2 prevents hyperglycemia-mediated induction of VEGF expression in the rodent retina and in Muller cells in culture.

Schrufer TL, Antonetti DA, Sonenberg N, Kimball SR, Gardner TW, Jefferson LS - Diabetes (2010)

The relative phosphorylation of a number of biomarkers of mRNA translational control is unchanged in the retina after induction of diabetes. Samples from retinal homogenates prepared as described in research design and methods were assessed for the relative phosphorylation of (A) eIF2α on Ser51, (B) eIF4G on Ser1108, and (C) eIF4E on Ser209. Values for phosphorylation status were normalized to the amount of the respective total protein. Values represent the mean ± SEM of at least three independent sets of animals at each time point (n = 2–4 within each set), except that the control value for eIF2α at 6 weeks represents two animals. Control (white bars) and diabetic (black bars) animals.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927931&req=5

Figure 2: The relative phosphorylation of a number of biomarkers of mRNA translational control is unchanged in the retina after induction of diabetes. Samples from retinal homogenates prepared as described in research design and methods were assessed for the relative phosphorylation of (A) eIF2α on Ser51, (B) eIF4G on Ser1108, and (C) eIF4E on Ser209. Values for phosphorylation status were normalized to the amount of the respective total protein. Values represent the mean ± SEM of at least three independent sets of animals at each time point (n = 2–4 within each set), except that the control value for eIF2α at 6 weeks represents two animals. Control (white bars) and diabetic (black bars) animals.
Mentions: Because the expression of the VEGF mRNA did not change over the time course used above, mechanisms involved in the regulation of mRNA translation were explored that might explain increased expression of the protein. Such mechanisms include changes in phosphorylation of eIF4E, eIF4G, and the α-subunit of eIF2. Therefore, the phosphorylation state of each of these proteins was assessed at various times after STZ treatment. No significant difference in phosphorylation of eIF2α on Ser51 (Fig. 2A), eIF4G on Ser1108 (Fig. 2B), or eIF4E on Ser209 (Fig. 2C) was observed in the retina of diabetic compared with control rats at 1, 2, 4, or 6 weeks of diabetes.

Bottom Line: Whereas VEGF mRNA in the retina remained constant, VEGF expression was increased as early as 2 weeks after the onset of diabetes.Similar results were observed after 10 h of exposure of cells in culture to hyperglycemic conditions.Because the VEGF mRNA contains two internal ribosome entry sites, the increased expression is likely a consequence of ribosome loading at these sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.

ABSTRACT

Objective: Vascular endothelial growth factor (VEGF) contributes to diabetic retinopathy, but control of its expression is not well understood. Here, we tested the hypothesis that hyperglycemia mediates induction of VEGF expression in a eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) 1 and 2 dependent manner.

Research design and methods: The retina was harvested from control and type 1 diabetic rats and mice and analyzed for VEGF mRNA and protein expression as well as biomarkers of translational control mechanisms. Similar analyses were performed in Müller cell cultures exposed to hyperglycemic conditions. The effect of 4E-BP1 and 4E-BP2 gene deletion on VEGF expression was examined in mice and in mouse embryo fibroblasts (MEFs).

Results: Whereas VEGF mRNA in the retina remained constant, VEGF expression was increased as early as 2 weeks after the onset of diabetes. Increases in expression of 4E-BP1 protein mirrored those of VEGF and expression of 4E-BP1 mRNA was unchanged. Similar results were observed after 10 h of exposure of cells in culture to hyperglycemic conditions. Importantly, the diabetes-induced increase in VEGF expression was not observed in mice deficient in 4E-BP1 and 4E-BP2, nor in MEFs lacking the two proteins.

Conclusions: Hyperglycemia induces VEGF expression through cap-independent mRNA translation mediated by increased expression of 4E-BP1. Because the VEGF mRNA contains two internal ribosome entry sites, the increased expression is likely a consequence of ribosome loading at these sites. These findings provide new insights into potential targets for treatment of diabetic retinopathy.

Show MeSH
Related in: MedlinePlus