Abstract
Purpose::
Our previous work has shown that oxidative stress contributes to ceramide-induced apoptosis in RPE. The aim of the present study was to determine the effect of ceramide on the expression of key redoxin proteins and to examine the effect of hepatocyte growth factor (HGF).
Methods::
Confluent RPE cells in early (2-4) passages were exposed to 25 uM C2- Ceramide for 24 h with and with out preincubation with HGF (20 ng/ml). Gene and protein expression of Trx1, Trx2, Grx1, Grx2, PrxV, TR and Thioredoxin interacting protein (Txnip) were determined by RT-PCR and immunoblot analysis. Nuclear translocation of Trx1 in control and ceramide-induced RPE was examined by confocal microscopy.
Results::
RPE cells exposed to C2 ceramide for 24 h showed increased gene expression of cytosolic and mitochondrial thioredoxins (Trx1 and Trx2), glutaredoxins (Grx1 and Grx2) and PrxV. HGF preincubation followed by ceramide treatment further increased expression of only Trx1, Grx2 and PrxV while Trx2 and Grx1 were unaltered. Thioredoxin reductase was downregulated with ceramide treatment and with HGF preincubation followed by ceramide treatment. None of the redoxins showed a significant change in protein expression upon ceramide exposure vs. controls. Translocation of Trx1 from cytosol to nucleus was observed after ceramide treatment of RPE. Gene and protein expression of Txnip increased significantly (p<0.05 vs. controls) with ceramide treatment and HGF caused a further significant increase of Txnip.
Conclusions::
Our data suggest that genes of the redoxin family are upregulated in RPE under oxidative stress. HGF pretreatment 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 • protective mechanisms • retinal pigment epithelium