Abstract
Purpose :
RPE cells have multiple functions to protect the neural retina from oxidative stresses. It has been reported that the inhibition of de novo ceramide synthesis protects rat retina from light-induced degeneration. Ceramide is a key metabolic cellular factor that acts as 2nd messenger for cell death. To determine the role of ceramide metabolism in RPE cell death, we induced oxidative stress in RPE cells and studied the role of ceramide by modulating its level with N-Acylsphingosine Amidohydrolase (ASAH1).
Methods :
ARPE-19 cells were plated and after 24hrs of the cultivation, medium was replaced with serum free medium. To induce the oxidative stress, 4-hydroxy-2-nonenal (4-HNE), H2O2 or C2-ceramide was added. The expression of ASAH1 in ARPE-19 cells was investigated by RT-PCR. Two isoforms of ASAH1s were cloned from ARPE-19 cells and inserted into the mammalian expression vector with the fusion to Venus gene. Vectors were linearized and electroporated into ARPE cells. Following the isolation of venus positive cells by a cell sorter, 2 types of stable cells that expressed ASAH1 or its transcript variant were established. The localization of the ASAH1s were examined with Cell Navigator Lysosome Staining. The protective effect of ASAH1s against oxidative stresses were studied by cell viability assay.
Results :
The expression of ASAH1 was not detected in non-oxidative condition, however, up-regulated by 4-HNE and H2O2. Two isoforms of ASAH1s were detected in ARPE cells. One was expressed in the cytoplasm (cy-ASAH1, NM_004315) and the other was in lysosome (ly-ASAH1, BC_016481). The Cy-ASAH1 was the transcript variant, however, the function has not been elucidated. Cy-ASAH1 and Ly-ASAH1 was extremely increased by 4-HNE and H2O2, respectively. Both of stably ASAH1- expressing cells (ARPE-cy-ASAH1 and ARPE-Ly-ASAH1) survived more than that of native ARPE cells under oxidative stresses.
Conclusions :
ASAH1 is the key enzyme that hydrolyzes cytotoxic ceramide and generates sphingosine and cytoprotective sphingosine 1-phosphate. We conclude, the stably ASAH1 expressing ARPE-19 cells protected these cells from oxidative stress by reducing cellular ceramides. We found two types of ASAH1s were up-regulated in ARPE cells during the oxidative stress that might be dependent on the type of oxidative agents and may indicate different ceramide generation pathways in different form of stresses.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.