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Spermidine Attenuates Oxidative Stress-Induced Apoptosis via Blocking Ca²⁺ Overload in Retinal Pigment Epithelial Cells Independently of ROS.

ABSTRACT: Retinal pigment epithelial (RPE) cells occupy the outer layer of the retina and perform various biological functions. Oxidative damage to RPE cells is a major risk factor for retinal degeneration that ultimately leads to vision loss. In this study, we investigated the role of spermidine in a hydrogen peroxide (H₂O₂)-induced oxidative stress model using human RPE cells. Our findings showed that 300 ?M H₂O₂ increased cytotoxicity, apoptosis, and cell cycle arrest in the G2/M phase, whereas these effects were markedly suppressed by 10 ?M spermidine. Furthermore, spermidine significantly reduced H₂O₂-induced mitochondrial dysfunction including mitochondrial membrane potential and mitochondrial activity. Although spermidine displays antioxidant properties, the generation of intracellular reactive oxygen species (ROS) upon H₂O₂ insult was not regulated by spermidine. Spermidine did suppress the increase in cytosolic Ca²⁺ levels resulting from endoplasmic reticulum stress in H₂O₂-stimulated human RPE cells. Treatment with a cytosolic Ca²⁺ chelator markedly reversed H₂O₂-induced cellular dysfunction. Overall, spermidine protected against H₂O₂-induced cellular damage by blocking the increase of intracellular Ca²⁺ independently of ROS. These results suggest that spermidine protects RPE cells from oxidative stress, which could be a useful treatment for retinal diseases.

SUBMITTER: Kim DH

PROVIDER: S-EPMC7866386 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Spermidine Attenuates Oxidative Stress-Induced Apoptosis via Blocking Ca2+ Overload in Retinal Pigment Epithelial Cells Independently of ROS.

Kim Da Hye DH Kim Jeong-Hwan JH Hwangbo Hyun H Kim So Young SY Ji Seon Yeong SY Kim Min Yeong MY Cha Hee-Jae HJ Park Cheol C Hong Su Hyun SH Kim Gi-Young GY Park Seh-Kwang SK Jeong Ji-Won JW Kim Mi-Young MY Choi Yung Hyun YH Lee Hyesook H

International journal of molecular sciences 20210129 3

Retinal pigment epithelial (RPE) cells occupy the outer layer of the retina and perform various biological functions. Oxidative damage to RPE cells is a major risk factor for retinal degeneration that ultimately leads to vision loss. In this study, we investigated the role of spermidine in a hydrogen peroxide (H2O2)-induced oxidative stress model using human RPE cells. Our findings showed that 300 μM H2O2 increased cytotoxicity, apoptosis, and cell cyc ...[more]

PMID: 33572992

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Project description:PurposeMutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1.MethodsUsing adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp.ResultsBest1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling.ConclusionsThese data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.

Dataset Information

Spermidine Attenuates Oxidative Stress-Induced Apoptosis via Blocking Ca²⁺ Overload in Retinal Pigment Epithelial Cells Independently of ROS.

Publications

Spermidine Attenuates Oxidative Stress-Induced Apoptosis via Blocking Ca<sup>2+</sup> Overload in Retinal Pigment Epithelial Cells Independently of ROS.

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