Abstract
Purpose: :
Previous studies in our laboratory demonstrated a role of intralysosomally-generated reactive oxygen species (ROS) in oxidative stress-induced damage in TM cells. Here we investigate the mechanisms by which lysosomal ROS induces cell death in TM cells.
Methods: :
Oxidative stress was applied by exposing TM cells with increasing concentrations of H2O2. Lysosomal ROS production was inhibited using desferrioxamine (DFO, 0.5 mM). Lysosomal permeabilization was monitored by flow cytometry using the Lysotracker (LTR) uptake and acridine orange (AO) relocation techniques. Translocation of cathepsins into the cytosol was visualized by confocal microscopy using pepstatin A BODIPY FL. Chemical inhibition of cathepsins was achieved with either leupeptin (0.1 mM) or with pepstatin A (0.1 mM). Silencing of cathepsin D (CTSD) and cathepsin B (CTSB) expression was accomplished by nucleofecting TM cells with plasmids containing miRNA sequences to knock-down the expression of porcine CTSD or porcine CSTB. Cytotoxicity was quantified by LDH assay.
Results: :
Exposure of TM cells to H2O2 significantly induced lysosomal labilization in a concentration-dependent manner as quantified by the appearance of pale cells using the LTR uptake method (282.4±100% increase, p=0.034), or increase green fluorescence using the AO relocation technique (162.3±37.6% increase, p=0.04). While control cells showed an expected pepstatin BODIPY FL-cathepsin D complexes localization in the perinuclear region, cells treated with H2O2 showed a diffused pattern distribution and decrease in green fluorescence, suggesting the loss of CTSD from the lysosomes in response to H2O2. Pre-incubation with DFO completely prevented both lysosomal permeabilization and relocation of CTSD. Chemical inhibition of CTSD significantly decreased the amount of secreted LDH (130.1 ± 25.7 % versus 427.5 ± 51.1 %, p=0.0008). Similar results were obtained when blocking CTSD expression with miRNA (0.41 ± 0.09 RFU versus 1.03 ± 0.1 RFU, p=0.001). Neither chemical inhibition of cysteine proteinases nor CTSB-miRNA showed any protective effect against H2O2-induced cell death.
Conclusions: :
Our results indicate that generation of intralysosomal ROS induces lysosomal membrane permeabilization and release of CTSD into the cytosol, leading to TM cell death. Here, we propose a mechanism by which oxidative stress might contribute to the loss of cellularity reported in the TM tissue with both aging and disease.
Keywords: trabecular meshwork • oxidation/oxidative or free radical damage • apoptosis/cell death