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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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Hyperglycemic conditions stimulate secretion of VEGF in retinal Müller cells, but not in Eif4ebp1;Eif4ebp2 double knockout (DKO) MEFs. A: Analysis of VEGF secretion. Medium was collected from TR-MUL cells exposed to control medium (white bar) or high-glucose medium (black bar) and subjected to ELISA analysis of VEGFA concentration. VEGF was normalized to protein content in the cell lysates. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. B: Quantitative RT-PCR of VEGF mRNA. RNA was extracted from TR-MUL cells exposed to control or high-glucose medium followed by RNA isolation as described in research design and methods. Values represent the mean of 4E-BP1 mRNA expression normalized to an internal β-actin mRNA control and are expressed as a percentage of control ± SEM of 6 dishes of cells per condition. C: Analysis of VEGF secretion in the human Müller cell line, MIO-M1. Medium was collected from TR-MUL cells exposed to control or high-glucose medium and subjected to ELISA for measurement of VEGFA concentration as described above. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. D: Analysis of VEGF secretion. Medium was collected from Eif4ebp1;Eif4ebp2 WT and DKO MEFs exposed to control or high-glucose medium and subjected to ELISA for VEGFA concentration. Values represent the mean expressed as a percentage of the control ± SEM of 9 dishes of cells of WT cells per condition and 6 dishes of double-knockout cells per condition. *P < 0.001, †P < 0.01 when compared with DKO cells maintained in control medium.
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Figure 8: Hyperglycemic conditions stimulate secretion of VEGF in retinal Müller cells, but not in Eif4ebp1;Eif4ebp2 double knockout (DKO) MEFs. A: Analysis of VEGF secretion. Medium was collected from TR-MUL cells exposed to control medium (white bar) or high-glucose medium (black bar) and subjected to ELISA analysis of VEGFA concentration. VEGF was normalized to protein content in the cell lysates. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. B: Quantitative RT-PCR of VEGF mRNA. RNA was extracted from TR-MUL cells exposed to control or high-glucose medium followed by RNA isolation as described in research design and methods. Values represent the mean of 4E-BP1 mRNA expression normalized to an internal β-actin mRNA control and are expressed as a percentage of control ± SEM of 6 dishes of cells per condition. C: Analysis of VEGF secretion in the human Müller cell line, MIO-M1. Medium was collected from TR-MUL cells exposed to control or high-glucose medium and subjected to ELISA for measurement of VEGFA concentration as described above. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. D: Analysis of VEGF secretion. Medium was collected from Eif4ebp1;Eif4ebp2 WT and DKO MEFs exposed to control or high-glucose medium and subjected to ELISA for VEGFA concentration. Values represent the mean expressed as a percentage of the control ± SEM of 9 dishes of cells of WT cells per condition and 6 dishes of double-knockout cells per condition. *P < 0.001, †P < 0.01 when compared with DKO cells maintained in control medium.

Mentions: To determine whether hyperglycemic conditions resulted in the induction of VEGF, medium was collected for measurement of VEGF secretion by ELISA. After 10 h of high-glucose treatment, the VEGF concentration in the cell culture medium was significantly elevated for VEGFA content (Fig. 8A). However, there was no change in the expression of VEGFA mRNA in the cell lysates (Fig. 8B), suggesting a posttranscriptional mechanism for the stimulated production of VEGF. VEGF secretion was also stimulated in MIO-M1 cells incubated in the high-glucose medium compared with the control medium (Fig. 8C).


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)

Hyperglycemic conditions stimulate secretion of VEGF in retinal Müller cells, but not in Eif4ebp1;Eif4ebp2 double knockout (DKO) MEFs. A: Analysis of VEGF secretion. Medium was collected from TR-MUL cells exposed to control medium (white bar) or high-glucose medium (black bar) and subjected to ELISA analysis of VEGFA concentration. VEGF was normalized to protein content in the cell lysates. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. B: Quantitative RT-PCR of VEGF mRNA. RNA was extracted from TR-MUL cells exposed to control or high-glucose medium followed by RNA isolation as described in research design and methods. Values represent the mean of 4E-BP1 mRNA expression normalized to an internal β-actin mRNA control and are expressed as a percentage of control ± SEM of 6 dishes of cells per condition. C: Analysis of VEGF secretion in the human Müller cell line, MIO-M1. Medium was collected from TR-MUL cells exposed to control or high-glucose medium and subjected to ELISA for measurement of VEGFA concentration as described above. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. D: Analysis of VEGF secretion. Medium was collected from Eif4ebp1;Eif4ebp2 WT and DKO MEFs exposed to control or high-glucose medium and subjected to ELISA for VEGFA concentration. Values represent the mean expressed as a percentage of the control ± SEM of 9 dishes of cells of WT cells per condition and 6 dishes of double-knockout cells per condition. *P < 0.001, †P < 0.01 when compared with DKO cells maintained in control medium.
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Figure 8: Hyperglycemic conditions stimulate secretion of VEGF in retinal Müller cells, but not in Eif4ebp1;Eif4ebp2 double knockout (DKO) MEFs. A: Analysis of VEGF secretion. Medium was collected from TR-MUL cells exposed to control medium (white bar) or high-glucose medium (black bar) and subjected to ELISA analysis of VEGFA concentration. VEGF was normalized to protein content in the cell lysates. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. B: Quantitative RT-PCR of VEGF mRNA. RNA was extracted from TR-MUL cells exposed to control or high-glucose medium followed by RNA isolation as described in research design and methods. Values represent the mean of 4E-BP1 mRNA expression normalized to an internal β-actin mRNA control and are expressed as a percentage of control ± SEM of 6 dishes of cells per condition. C: Analysis of VEGF secretion in the human Müller cell line, MIO-M1. Medium was collected from TR-MUL cells exposed to control or high-glucose medium and subjected to ELISA for measurement of VEGFA concentration as described above. Values represent the mean expressed as a percentage of the control ± SEM of 6 dishes of cells per condition. D: Analysis of VEGF secretion. Medium was collected from Eif4ebp1;Eif4ebp2 WT and DKO MEFs exposed to control or high-glucose medium and subjected to ELISA for VEGFA concentration. Values represent the mean expressed as a percentage of the control ± SEM of 9 dishes of cells of WT cells per condition and 6 dishes of double-knockout cells per condition. *P < 0.001, †P < 0.01 when compared with DKO cells maintained in control medium.
Mentions: To determine whether hyperglycemic conditions resulted in the induction of VEGF, medium was collected for measurement of VEGF secretion by ELISA. After 10 h of high-glucose treatment, the VEGF concentration in the cell culture medium was significantly elevated for VEGFA content (Fig. 8A). However, there was no change in the expression of VEGFA mRNA in the cell lysates (Fig. 8B), suggesting a posttranscriptional mechanism for the stimulated production of VEGF. VEGF secretion was also stimulated in MIO-M1 cells incubated in the high-glucose medium compared with the control medium (Fig. 8C).

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