RESUMEN
In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.
Asunto(s)
Micronutrientes , Proteoma , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma , Factor de Crecimiento Transformador beta1/fisiología , Zinc/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Polaridad Celular/efectos de los fármacos , Polaridad Celular/genética , Células Cultivadas , Impedancia Eléctrica , Matriz Extracelular/metabolismo , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/prevención & control , Microvellosidades/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Pigmentación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/fisiología , Zinc/metabolismoRESUMEN
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that requires an active metabolism to maintain outer retinal homeostasis and compensate for oxidative stress. Using 13C metabolic flux analysis in human RPE cells, we found that RPE has an exceptionally high capacity for reductive carboxylation, a metabolic pathway that has recently garnered significant interest because of its role in cancer cell survival. The capacity for reductive carboxylation in RPE exceeds that of all other cells tested, including retina, neural tissue, glial cells, and a cancer cell line. Loss of reductive carboxylation disrupts redox balance and increases RPE sensitivity to oxidative damage, suggesting that deficiencies of reductive carboxylation may contribute to RPE cell death. Supporting reductive carboxylation by supplementation with an NAD+ precursor or its substrate α-ketoglutarate or treatment with a poly(ADP ribose) polymerase inhibitor protects reductive carboxylation and RPE viability from excessive oxidative stress. The ability of these treatments to rescue RPE could be the basis for an effective strategy to treat blinding diseases caused by RPE dysfunction.