Abstract
Purpose::
Müller cell dysfunction has been suggested to contribute to the development of diabetic retinopathy. Previously, we have demonstrated that high glucose induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear accumulation in retinal Müller cells in vitro and in vivo, a process which is dependent on the activation of the caspase-1/interleukin-1ß (IL-1ß) signaling pathway subsequently leading to apoptosis of these cells. The mechanism of GAPDH translocation to and accumulation in the nucleus is unclear because GAPDH lacks a nuclear localization signal (NLS). Recent studies done in macrophages demonstrated that complex formation of GAPDH with seven in absentia homolog-1 (siah-1), which has a NLS, is necessary for GAPDH translocation to the nucleus. Therefore, this study was focused on identifying (1) whether high glucose leads to siah-1 up-regulation and (2) whether GAPDH forms a complex with siah-1.
Methods::
Transformed rat retinal Müller (rMC-1) cells were incubated with normal (5mM) glucose medium or high (25mM) glucose medium for up to 96 hours. Following high glucose incubation cytosolic siah-1 amounts were determined by Western Blot analysis. To examine GAPDH/siah-1 complex formation, co-immunoprecipitation experiments were performed.
Results::
High glucose induced a two-fold increase in siah-1 protein levels starting at 5 hours of high glucose treatment. Elevated siah-1 protein levels were sustained under hyperglycemic conditions for up to 96 hours. Further, GAPDH/siah-1complex formation was detected at 12 hours of high glucose treatment. This interaction was inhibited by a caspase-1 inhibitor (YVAD-fmk).
Conclusions::
High glucose-induced siah-1 up-regulation and subsequent complex formation with GAPDH presents a possible mechanism by which GAPDH translocates from the cytosol into the nucleus to induce apoptosis. Inhibition of caspase-1 prevented GADPH/siah-1 complex formation indicating that a caspase-1/IL-1ß signaling driven mechanism contributes to this process subsequently leading to Müller cell dysfunction and the development of diabetic retinopathy.
Keywords: diabetic retinopathy • Muller cells • apoptosis/cell death