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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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Ablation of 4E-BP1/2 in mice attenuates the diabetes-induced increase in VEGF expression. A: Western blot analysis of 4E-BP1/2 protein expression in the retina of wild-type (WT) and Eif4ebp1;Eif4ebp2 double knockout (DKO) control mice. Four-week-old mice were injected with STZ as described in research design and methods, and 5 weeks later protein (60 μg) was analyzed from six WT and six DKO mice. Representative blots are shown. All samples were run on the same gel, but not in contiguous lanes. B: Western blot analysis of VEGF expression in the retina of WT and DKO control and diabetic mice. Protein (60 μg) was analyzed from 12 control and 12 diabetic wild-type mice, and 7 control and 8 diabetic DKO mice; control (white bars) and diabetic (black bars) animals. Values represent the mean ± SEM. *P < 0.05 versus corresponding control value.
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Figure 5: Ablation of 4E-BP1/2 in mice attenuates the diabetes-induced increase in VEGF expression. A: Western blot analysis of 4E-BP1/2 protein expression in the retina of wild-type (WT) and Eif4ebp1;Eif4ebp2 double knockout (DKO) control mice. Four-week-old mice were injected with STZ as described in research design and methods, and 5 weeks later protein (60 μg) was analyzed from six WT and six DKO mice. Representative blots are shown. All samples were run on the same gel, but not in contiguous lanes. B: Western blot analysis of VEGF expression in the retina of WT and DKO control and diabetic mice. Protein (60 μg) was analyzed from 12 control and 12 diabetic wild-type mice, and 7 control and 8 diabetic DKO mice; control (white bars) and diabetic (black bars) animals. Values represent the mean ± SEM. *P < 0.05 versus corresponding control value.

Mentions: If an increase in 4E-BP1 availability is necessary for the increased expression of VEGF, then the absence of 4E-BP1 would be expected to prevent the diabetes-induced change in VEGF expression. To test this hypothesis, retinas from control and STZ-induced diabetic, wild-type, and 4E-BP1 and 4E-BP2 double knockout (Eif4ebp1;Eif4ebp2) mice were analyzed. While retinal 4E-BP1 protein was detected in Eif4ebp1;Eif4ebp2 wild-type mice, the protein was undetectable in the retinal extracts from Eif4ebp1;Eif4ebp2 double knockout mice, confirming lack of 4E-BP1 expression in these mice (Fig. 5A). After 5 weeks of STZ-induced diabetes, VEGF expression was significantly higher in the retina of the diabetic compared with the control Eif4ebp1;Eif4ebp2 wild-type mice. In contrast, diabetes had no effect on VEGF expression in the retina of Eif4ebp1;Eif4ebp2 double knockout mice (Fig. 5B), despite severe hyperglycemia (supplementary Fig. 1C). This result demonstrates that an increase in 4E-BP1 availability is necessary for the diabetes-induced increase in VEGF expression.


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)

Ablation of 4E-BP1/2 in mice attenuates the diabetes-induced increase in VEGF expression. A: Western blot analysis of 4E-BP1/2 protein expression in the retina of wild-type (WT) and Eif4ebp1;Eif4ebp2 double knockout (DKO) control mice. Four-week-old mice were injected with STZ as described in research design and methods, and 5 weeks later protein (60 μg) was analyzed from six WT and six DKO mice. Representative blots are shown. All samples were run on the same gel, but not in contiguous lanes. B: Western blot analysis of VEGF expression in the retina of WT and DKO control and diabetic mice. Protein (60 μg) was analyzed from 12 control and 12 diabetic wild-type mice, and 7 control and 8 diabetic DKO mice; control (white bars) and diabetic (black bars) animals. Values represent the mean ± SEM. *P < 0.05 versus corresponding control value.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Ablation of 4E-BP1/2 in mice attenuates the diabetes-induced increase in VEGF expression. A: Western blot analysis of 4E-BP1/2 protein expression in the retina of wild-type (WT) and Eif4ebp1;Eif4ebp2 double knockout (DKO) control mice. Four-week-old mice were injected with STZ as described in research design and methods, and 5 weeks later protein (60 μg) was analyzed from six WT and six DKO mice. Representative blots are shown. All samples were run on the same gel, but not in contiguous lanes. B: Western blot analysis of VEGF expression in the retina of WT and DKO control and diabetic mice. Protein (60 μg) was analyzed from 12 control and 12 diabetic wild-type mice, and 7 control and 8 diabetic DKO mice; control (white bars) and diabetic (black bars) animals. Values represent the mean ± SEM. *P < 0.05 versus corresponding control value.
Mentions: If an increase in 4E-BP1 availability is necessary for the increased expression of VEGF, then the absence of 4E-BP1 would be expected to prevent the diabetes-induced change in VEGF expression. To test this hypothesis, retinas from control and STZ-induced diabetic, wild-type, and 4E-BP1 and 4E-BP2 double knockout (Eif4ebp1;Eif4ebp2) mice were analyzed. While retinal 4E-BP1 protein was detected in Eif4ebp1;Eif4ebp2 wild-type mice, the protein was undetectable in the retinal extracts from Eif4ebp1;Eif4ebp2 double knockout mice, confirming lack of 4E-BP1 expression in these mice (Fig. 5A). After 5 weeks of STZ-induced diabetes, VEGF expression was significantly higher in the retina of the diabetic compared with the control Eif4ebp1;Eif4ebp2 wild-type mice. In contrast, diabetes had no effect on VEGF expression in the retina of Eif4ebp1;Eif4ebp2 double knockout mice (Fig. 5B), despite severe hyperglycemia (supplementary Fig. 1C). This result demonstrates that an increase in 4E-BP1 availability is necessary for the diabetes-induced increase in VEGF expression.

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