A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium

Abstract

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.

Keywords: TGFB1; age-related macular degeneration; gene set enrichment; proteome; retinal pigment epithelium; secretome; transcriptome; zinc.

Figure 1

Differences in characteristics between cultures from three donors. There were differences between the three batches of cells in their pigmentation (A) and maximal transepithelial electrical resistance (TEER) (B); each point is an average +/-SEM from triplicate measurements from each of three donors.). Apical and basal zinc supplementation for 4 weeks significantly increased TEER values (C); the samples from the three donors represent replicates, plotted as values normalised to 100% at day 4; control (●), zinc apical supplementation (■), and zinc basal supplementation (▲). Scale bar represents 100 µm. Experimental workflow for downstream analysis (D). * represents p-value < 0.05, two-way ANOVA.

Figure 2

Changes in transcriptome and proteome following zinc supplementation. Comparison of cellular gene expressional after apical (A), or basal (B) zinc supplementation compared to untreated controls. Red dots represent significantly upregulated while blue dot represents significantly downregulated genes (adjusted p-value <0.05). Blue line indicates log2 fold change: 1 and -1. Analysis of the cellular proteome following apical (C) or basal (D) zinc supplementation. When changes in secreted proteins were analysed we found significant changes in both the apical (E,F) and basal (G,H) secretomes following apical (E,G) or basal (F,H) supplementation with zinc. In C–H, open black circles represent no change while red dots show significant significance B changes (adjusted p-value < 0.05).

Figure 3

Overlapping canonical pathways upon zinc apical (A) or basal (B) supplementation. Each pathway is a single “node” colored according to significance (the brighter red, the more significant the pathway). Blue lines connect any two pathways which have at least three dataset molecules in common. GROUP #1: adhesion to polarity; GROUP #2: protein export/import; GROUP #3: extracellular matrix organization, GROUP #4: protein processing, GROUP #5: oxidative stress response, GROUP #6: embryonic cell development, GROUP #7: immune response, and GROUP #8: lipid metabolism.

Figure 4

Influence of zinc supplementation culturing condition in retinal pigment epithelial cells. Measured and predicted upstream and downstream regulatory effect. Orange lines and objects: predicted activation; grey lines: not predicted effect; yellow lines: inconsistency with state of downstream molecule; light red objects: measured increase; green object: measured decrease; blue object: predicted inhibition. Note, this regulatory effect of TGFB1 was only detected upon zinc apical supplementation.