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Manish Mishra, Renu A Kowluru; A novel regulator of cytosolic oxidative stress in the development of Diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4031.
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In diabetes, increased retinal activity of Rac1, a small molecular weight GTPase, increases NADPH-oxidase 2 mediated cytosolic reactive oxygen species (ROS) that damages retinal mitochondria and accelerates capillary cell death. Activity of Rac1, in turn, is stimulated by p66Shc, an adapter protein that promotes oxidative stress. Regulation of p66Shc is controlled by histone acetylation, which facilitates the binding of p53 transcription factor at its promoter. In diabetes, expression of p66Shc and p53 are increased in the retina, activity of Sirt1, a histone deacetylase, is decreased, and induction of Sirt1 prevents increase in cellular ROS. Our aim is to examine the role of p66Shc in Rac1 activation and its regulation in the development of diabetic retinopathy.
To investigate the interrelationship between p66Shc and the Rac1, human retinal endothelial cells, incubated in high glucose for 24-96 hours were employed and tested for p66Shc expression and Rac1 activity using qPCR and G-LISA colorimetric assay respectively. Cellular localization of p66Shc and Rac1 was determined by immunofluorescence imaging. Role of p53 and the effect of histone acetylation on its binding at p66Shc promoter was determined in the cells overexpressing Sirt1 using chromatin immunoprecipitation assay.
High glucose increased p66Shc expression as early as 24 hours of its exposure, and it continued with the increased duration of glucose exposure. P66Shc expression and Rac1 activation presented a strong correlation, and this was confirmed by cytosolic co-localization of Rac1 and p66Shc. The binding of p53 at p66Shc promoter was increased by ~40%, which was prevented in the cells overexpressing Sirt1. In the same Sirt1 overexpressing cells, glucose-induced Rac1 activation was also ameliorated.
In diabetes, p66Shc plays an important role in retinal Rac1 activation, and Sirt1 regulates p66Shc via modulating p53 binding at p66Shc promoter. Thus, understanding the relationship between Rac1, p66Shc and Sirt1 will provide a novel mechanism of regulating early oxidative response to maintain cellular homeostasis and prevent the development of diabetic retinopathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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