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Proteomic analysis of retinal pigment epithelium cells after exposure to UVA radiation.

ABSTRACT: BACKGROUND:Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression. METHODS:ARPE-19 cells were irradiated with a UVA lamp at different doses (5, 10, 20, 30 and 40?J/cm2) from 10?cm. Cell viability was determined by MTT assay. Visual inspection was first achieved with inverted light microscopy and then the DeadEnd™ Fluorometric TUNEL System was used to observe nuclear DNA fragmentation. Flow cytometry based-Annexin V-FITC/PI double-staining was used to further quantify cellular viability. Mitochondrial membrane potential was assessed with JC-1 staining. 2D electrophoresis maps of exposed cells were compared to nonexposed cells and gel images analyzed with PDQuest 2-D Analysis Software. Spots with greater than a 1.5-fold difference were selected for LC-MS/MS analysis and some confirmed by western blot. We further investigated whether caspase activation, apoptotic-related mitochondrial proteins, and regulators of ER stress sensors were involved in UVA-induced apoptosis. RESULTS:We detected 29 differentially expressed proteins (9 up-regulated and 20 down-regulated) in the exposed cells. Some of these proteins such as CALR, GRP78, NPM, Hsp27, PDI, ATP synthase subunit alpha, PRDX1, and GAPDH are associated with anti-proliferation, induction of apoptosis, and oxidative-stress protection. We also detected altered protein expression levels among caspases (caspase 3 and 9) and in the mitochondrial (cytosolic cytochrome C, AIF, Mcl-1, Bcl-2, Bcl-xl, Bax, Bad, and p-Bad) and ER stress-related (p-PERK, p-eIF2?, ATF4 and CHOP) apoptotic pathways. CONCLUSIONS:UVA irradiation suppressed the proliferation of ARPE-19 cells in a dose-dependent manner, caused quantitative loses in transmembrane potential (??m), and induced both early and late apoptosis.

SUBMITTER: Chen JL

PROVIDER: S-EPMC6679551 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Proteomic analysis of retinal pigment epithelium cells after exposure to UVA radiation.

Chen Jiunn-Liang JL Hung Chun-Tzu CT Keller Joseph Jordan JJ Lin Hsien-Chung HC Wu Yu-Jen YJ

BMC ophthalmology 20190802 1

<h4>Background</h4>Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression.<h4>Methods</h4>ARPE-19 cells were irradiated ...[more]

PMID: 31375076

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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Project description:The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or 2 h after the onset of light has been reported for several diurnal and nocturnal species. This peak in phagocytic activity also persists under constant lighting conditions (i.e., constant light or dark) thus demonstrating that the timing of this peak is driven by a circadian clock. The aim of this study was to investigate the change in RPE whole transcriptome at two different circadian times (CT; 1 h before (CT23) and 1 h after (CT1) subjective light onset). C57BL/6J male mice were maintained in constant dark conditions for three days and euthanized under red light (<1 lux) at CT23 and CT1. RPE was isolated from whole eyes for RNA library preparation and sequencing on an Illumina HiSeq4000 platform. 14,083 mouse RPE transcripts were detected in common between CT23 and CT1. 12,005 were protein coding transcripts and 2078 were non-protein coding transcripts. 2421 protein coding transcripts were significantly upregulated whereas only 3 transcripts were significantly downregulated and 12 non-protein coding transcripts were significantly upregulated and 31 non-protein coding transcripts were significantly downregulated at CT1 when compared to CT23 (p < 0.05, fold change ≥ ±2.0). Of the protein coding transcripts, most of them were characterized as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included biotypes such as long-non coding RNAs and pseudogenes. Gene ontology analysis and ingenuity pathway analysis revealed that differentially expressed transcripts were associated with integrin signaling, oxidative phosphorylation, protein phosphorylation, and actin cytoskeleton remodeling suggesting that these previously identified phagocytic pathways are under circadian control. Our analysis identified new pathways (e.g., increased mitochondrial respiration via increased oxidative phosphorylation) that may be involved in the circadian control of phagocytic activity. In addition, our dataset suggests a possible regulatory role for the identified non-protein coding transcripts in mediating the complex function of RPE phagocytosis. Finally, our results also indicate, as seen in other tissues, about 20% of the whole RPE transcriptome may be under circadian clock regulation.

Project description:PURPOSE:The retinal pigment epithelium (RPE) and underlying Bruch's membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell-substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch's membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this "aged" substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. METHODS:Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. RESULTS:Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. CONCLUSIONS:This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch"s membrane could play a significant role in age-related dysfunction of the RPE.

Dataset Information

Proteomic analysis of retinal pigment epithelium cells after exposure to UVA radiation.

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Proteomic analysis of retinal pigment epithelium cells after exposure to UVA radiation.

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