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Effect of ER stress on sphingolipid levels and apoptotic pathways in retinal pigment epithelial cells.

Redox biology (2020-01-25)
Ebru Afşar, Esma Kırımlıoglu, Tuğçe Çeker, Çağatay Yılmaz, Necdet Demir, Mutay Aslan
ABSTRACT

We aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress. Cells were treated with tunicamycin (TM) to induce ER stress and tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, was administered to decrease cytotoxicity. Cell viability was measured by MTT assay. Levels of C16-C24 sphingomyelins (SM) and C16-C24 ceramides (CERs) were determined by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78) and nuclear factor kappa-b subunit 1 (NFκB1) gene expressions were evaluated by quantitative PCR analysis, while GRP 78, NF-κB p65, cleaved caspase-3 and caspase-12 protein levels were assesed by immunofluorescence. Ceramide-1-phosphate (C1P) levels were determined by immunoassay, while caspase -3 and -12 activity in cell lysates were measured via a fluorometric method. Induction of ER stress in TM treated groups were confirmed by significantly increased mRNA and protein levels of GRP78. TM significantly decreased cell viability compared to controls. Treatment with TUDCA along with TM significantly increased cell viability compared to the TM group. A significant increase was observed in C22-C24 CERs, C1P, caspase-3, caspase-12, NFκB1 mRNA and NF-κB p65 protein levels in cells treated with TM compared to controls. Administration of TUDCA lead to a partial decrease in GRP78 expression, NFκB1 mRNA, NF-κB p65 protein, C22-C24 CERs and C1P levels along with a decrease in caspase-3 and -12 activity. The results of this study reveal the presence of increased long chain CERs, C1P and apoptotic markers in retinal cells undergoing ER stress.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Stearoyl-D-sphingosine, ≥98.0% (TLC)
Sigma-Aldrich
N-Palmitoyl-D-sphingosine, ≥98.0% (TLC)
Sigma-Aldrich
N-Palmitoyl-D-sphingomyelin, ≥96.0% (TLC)