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

ABSTRACT: BACKGROUND:We aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress. METHODS: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 (NF?B1) gene expressions were evaluated by quantitative PCR analysis, while GRP 78, NF-?B 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. RESULTS: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, NF?B1 mRNA and NF-?B p65 protein levels in cells treated with TM compared to controls. Administration of TUDCA lead to a partial decrease in GRP78 expression, NF?B1 mRNA, NF-?B p65 protein, C22-C24 CERs and C1P levels along with a decrease in caspase-3 and -12 activity. CONCLUSIONS:The results of this study reveal the presence of increased long chain CERs, C1P and apoptotic markers in retinal cells undergoing ER stress.

SUBMITTER: Afsar E

PROVIDER: S-EPMC6976939 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Afşar Ebru E Kırımlıoglu Esma E Çeker Tuğçe T Yılmaz Çağatay Ç Demir Necdet N Aslan Mutay M

Redox biology 20200120

<h4>Background</h4>We aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress.<h4>Methods</h4>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 ...[more]

PMID: 31978676

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Project description:Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are the most potent known virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications such as acute renal failure, blindness and neurological abnormalities. Although numerous studies have defined apoptotic responses to Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) in a variety of cell types, the potential significance of Stx-induced apoptosis of photoreceptor and pigmented cells of the eye following intoxication is unknown. We explored the use of immortalized human retinal pigment epithelial (RPE) cells as an in vitro model of Stx-induced retinal damage. To the best of our knowledge, this study is the first report that intoxication of RPE cells with Stxs activates both apoptotic cell death signaling and the endoplasmic reticulum (ER) stress response. Using live-cell imaging analysis, fluorescently labeled Stx1 or Stx2 were internalized and routed to the RPE cell endoplasmic reticulum. RPE cells were significantly sensitive to wild type Stxs by 72 h, while the cells survived challenge with enzymatically deficient mutant toxins (Stx1A- or Stx2A-). Upon exposure to purified Stxs, RPE cells showed activation of a caspase-dependent apoptotic program involving a reduction of mitochondrial transmembrane potential (Δψm), increased activation of ER stress sensors IRE1, PERK and ATF6, and overexpression CHOP and DR5. Finally, we demonstrated that treatment of RPE cells with Stxs resulted in the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), suggesting that the ribotoxic stress response may be triggered. Collectively, these data support the involvement of Stx-induced apoptosis in ocular complications of intoxication. The evaluation of apoptotic responses to Stxs by cells isolated from multiple organs may reveal unique functional patterns of the cytotoxic actions of these toxins in the systemic complications that follow ingestion of toxin-producing bacteria.

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Dataset Information

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

Publications

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

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