Dataset Information

Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina.

ABSTRACT: Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.

SUBMITTER: Ananth S

PROVIDER: S-EPMC7823377 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina.

Ananth Sudha S Miyauchi Seiji S Thangaraju Muthusamy M Jadeja Ravirajsinh N RN Bartoli Manuela M Ganapathy Vadivel V Martin Pamela M PM

Antioxidants (Basel, Switzerland) 20201224 1

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a m ...[more]

PMID: 33374239

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Project description:The cystine/glutamate exchanger (xCT, SLC7A11) is a component of the system Xc amino-acid antiporter that is able to export glutamate and import cysteine into cells. The xCT amino acid exchanger has received a lot of attention, due to the fact that cysteine is an essential substrate for the synthesis of glutathione (GSH), an endogenous antioxidant in cells. The objective of this research was to clone the full-length cDNA of chicken xCT, and to investigate the gene expression of xCT in different tissues, including intestinal segments of broiler chickens during development. The full-length cDNA of chicken xCT (2,703 bp) was obtained from the jejunum by reverse transcription-PCR and sequenced. Homology tests showed that chicken xCT had 80.4%, 80.2%, and 71.2% homology at the nucleotide level with humans, cattle, and rats, respectively. Likewise, amino acid sequence analysis showed that chicken xCT protein is 86.4%, 79.3%, and 75.6% homologous with humans, cattle, and rats, respectively. Additionally, phylogenetic analysis indicated that chicken xCT genes share a closer genetic relationship with humans and cattle, than with rats. The chicken xCT protein has 12 transmembrane helixes, 6 extracellular loops, and 5 intracellular loops. The mRNA of xCT was detected in all tissues, including intestinal segments, in which the mRNA expression of xCT was significantly higher (P < 0.05) within the colon, compared to the jejunum and ileum. During development, a linear pattern of changes regarding the levels of the xCT mRNA was found, indicating that there was an abundance of xCT within the duodenum (P < 0.05). Furthermore, there were changes of the xCT mRNA abundance in the colon during development, which displayed linear and cubic patterns (P < 0.05). These results indicated that xCT is widely expressed both in intestinal segments, as well as other organs that are not associated with nutrient absorption. Further investigation is needed to characterize the functional relevance of xCT activity in oxidative stress and inflammation in the small intestine of broiler chickens.

Project description:Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Keywords: Tissue interactions, retina, retinal pigment epithelium, blood-retinal barrier, tight junctions

Dataset Information

Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina.

Publications

Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina.

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