Ceramide–induced Upregulation of Redoxin Proteins in RPE and Effect of Hepatocyte Growth Factor

Abstract

Abstract: : Purpose: We have shown that the lipid second messenger ceramide produces oxidant stress and apoptosis in RPE which can be partially attenuated by pretreatment with hepatocyte growth factor (HGF) or GSH ester. The aim of this study was to determine the role, regulation and mechanism of action of endogenous redoxins in ceramide–induced oxidative injury and protection by HGF. Methods: Confluent RPE in early (2–4) passages were exposed to 25µM C₂ ceramide overnight with or without pretreatment with HGF (20 ng/ml). Gene expression of cytosolic and mitochondrial thioredoxins (TRX1and TRX2), glutaredoxins (GRX1 and GRX2) and periredoxin (PRX5) was quantitated by real time PCR. TRX1 and PRX5 protein expression was determined by western blot analyses. The gene expression of thioredoxin–interacting protein (Txnip), which inhibits TRX by forming a complex with TRX, was also studied. Results: All redoxins showed an increase in gene expression (p<0.05 vs controls) with C₂ ceramide treatment. HGF pretreatment showed a further significant increase (p<0.01 vs ceramide) in TRX1 and GRX2 while TRX2 and PRX5 remained unchanged. HGF treatment alone did not cause significant changes in gene expression as compared to controls. TRX1 and PRX5 protein expression showed a mild, non–significant elevation with ceramide treatment but a significant (p<0.05) increase with HGF pretreatment. Txnip decreased significantly with ceramide and HGF partially reversed this effect. Conclusions: Redoxin genes are elevated in RPE under oxidative stress from ceramide exposure. HGF protection from ceramide–induced cell death may involve improvement of redox status of RPE by synergistic upregulation of several redoxin genes.

Keywords: oxidation/oxidative or free radical damage • antioxidants • apoptosis/cell death