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C. Bucolo, M. Amadio, F. Drago, S. Govoni, A. Pascale; Posttranscriptional Regulation of VEGF in Diabetic Retina: Role of the RNA-Binding Protein HuR. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5904.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether VEGF expression is regulated at posttranscriptional level in diabetic retinopathy through the mRNA-stabilizing protein HuR and via PKCβ activation.
Diabetes was induced in rats by streptozotocin (STZ) injection. Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Retinal tissues were processed to detect PKCβI, PKCβII, VEGF and HuR contents, as well as HuR phosphorylation. Immunoprecipitation coupled to western blotting or RT-PCR was employed to evaluate HuR phosphorylation and its binding to VEGF mRNA, respectively. Statistical analysis was performed by ANOVA followed by an appropriate post hoc comparison test.
Following experimental diabetes PKCβI and PKCβII levels were statistically (p<0.01) increased compared to sham; we also showed a PKC-mediated phosphorylation/activation of HuR. These effects were statistically (p<0.01) blunted by the co-administration of a PKCβ inhibitor (INH). A specific binding between HuR protein and VEGF mRNA was also evaluated in RiboNucleoProteic complexes from sham and diabetic animals indicating no significant difference in all retinal tissues. The PKCβ/HuR activation was accompanied by statistical (p<0.01) enhanced VEGF protein expression that was blunted by the PKCβ inhibitor. However, the total VEGF mRNA levels did not change following diabetes (mean ± S.E.M. of Ct values: sham = 28.8 ± 0.86; STZ = 28.5 ± 0.84; STZ+ INH = 28.7 ± 1.02; n=6).
The present findings indicate that, following diabetes, there is an higher phosphorylation of HuR, a nucleo-cytoplasmic shuttling protein, by PKCβ resulting in the activation of HuR itself that then targets VEGF mRNA, finally leading to an increased amount of the correspondent VEGF protein. These data, along with the observation that VEGF mRNA shows no changes following diabetes, suggest that VEGF may be regulated mainly at posttranscriptional level.
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