Abstract
Purpose: :
The E3 ubiquitin ligase seven in absentia homolog-1 (siah-1) participates in various cellular events including cell death. Siah-1 initiates cell death by binding and transporting the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the cytosol to the nucleus leading to activation of nuclear p53 by GAPDH. We have previously demonstrated that elevated glucose levels lead to GAPDH nuclear accumulation in Müller cells in vitro and in vivo. Therefore, this study examined the importance of siah-1 in the process of high glucose-induced GAPDH nuclear accumulation and cell death in Müller cells.
Methods: :
Müller cells were treated with either normal (5mM) or high (25mM) glucose medium for up to 96 hours. Co-immunoprecipitation assays were used to determine GAPDH-siah-1 complex formation. To assess the importance of siah-1 in the process of GAPDH nuclear accumulation and cell death, Müller cells were transfected with 50nM siah-1 siRNA (or scrambled siRNA for control) prior to treatment with normal and high glucose medium. GAPDH immunofluoresence analysis was used to determine GAPDH sub-cellular location. Trypan blue assays were performed to determine cell viability.
Results: :
High glucose treatment of retinal Müller cells led to a 2 fold increase in GAPDH-siah-1 complex formation in the nucleus at 24 hours. At concentrations of up to 50nM siah-1 siRNA, siah-1 protein levels were reduced by 52 ± 17.5%. More importantly, high glucose-induced GAPDH nuclear accumulation was diminished by 53±8.4% at these conditions. In addition, siah-1 knock down reduced high glucose-induced death of Müller cells.
Conclusions: :
The activation of siah-1 by high glucose is a critical event in the induction and execution of Müller cell death. Therefore, therapies that target the regulation of siah-1 might represent potential strategies to correct or preserve proper Müller cell function in a hyperglycemic environment.
Keywords: Muller cells • apoptosis/cell death • diabetic retinopathy