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Sandra Suarez, John S Penn; GAPDH/Siah1 Signaling Mediates Apoptosis in High Glucose-treated Human Retinal Pericytes. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):924.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetic Retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive and are of limited effectiveness. Retinal pericyte death is an early pathologic
feature of DR. Though it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in non-ocular tissues. Subsequently, we showed that high glucose causes Siah1 upregulation, GAPDH nuclear translocation and human retinal pericyte (HRP) apoptosis. The goal of the present study was to determine if GAPDH and Siah1 interact in the presence of high glucose and if inhibition of Siah1 expression or GAPDH/Siah1 interaction prevents high glucose-induced HRP apoptosis.
HRPs were cultured in 25mM L- or D-glucose for 48hrs. Siah1 siRNA was used to downregulate Siah1 expression. TAT-FLAG GAPDH (Pep 1) and/or Siah1 (Pep 2) peptides were used to block GAPDH and Siah1 interaction, respectively. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on GAPDH/Siah1 interaction. Apoptosis was measured by caspase-3 enzymatic activity assay.
Co-immunoprecipitation of GAPDH and Siah1 was significantly increased as a result of high glucose treatment (p=0.0292). The inhibition of GAPDH/Siah1 association with blocking peptides significantly reduced high glucose-induced GAPDH/Siah1 co-immunoprecipitation (Pep 1, p=0.0194; Pep 2, p=0.00066). Furthermore, both high glucose-induced GAPDH nuclear translocation and apoptosis, as measured by caspase 3-enzymatic activity, were significantly inhibited by Siah1 siRNA (nuclear translocation, p=0.0469; apoptosis, p=0.0419), Pep 1 (nuclear translocation, p=0.0142; apoptosis, p=0.0013) and Pep 2 (nuclear translocation, p=0.0221; apoptosis, p=0.005).
Our findings demonstrate that inhibition of the GAPDH/Siah1 pro-apoptotic complex blocks high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Future experiments will examine the role of GAPDH nuclear translocation and HRP apoptosis in an animal model of DR.
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