A Comprehensive Proteomic and Phosphoproteomic Analysis of Retinal Pigment Epithelium Reveals Multiple Pathway Alterations in Response to the Inflammatory Stimuli.

Juha Song,Joo-Youn Cho,Dohyun Han,Seung Hyeok Seok,Heonyi Lee,Da Jung Kim,Jae-Hak Park

doi:10.3390/ijms21093037

Abstract

Overwhelming and persistent inflammation of retinal pigment epithelium (RPE) induces destructive changes in the retinal environment. However, the precise mechanisms remain unclear. In this study, we aimed to investigate RPE-specific biological and metabolic responses against intense inflammation and identify the molecular characteristics determining pathological progression. We performed quantitative analyses of the proteome and phosphoproteome of the human-derived RPE cell line ARPE-19 after treatment with lipopolysaccharide (LPS) for 45 min or 24 h using the latest isobaric tandem-mass tags (TMTs) labeling approach. This approach led to the identification of 8984 proteins, of which 261 showed a 1.5-fold change in abundance after 24 h of treatment with LPS. A parallel phosphoproteome analysis identified 20,632 unique phosphopeptides from 3207 phosphoproteins with 3103 phosphorylation sites. Integrated proteomic and phosphoproteomic analyses showed significant downregulation of proteins related to mitochondrial respiration and cell cycle checkpoint, while proteins related to lipid metabolism, amino acid metabolism, cell-matrix adhesion, and endoplasmic reticulum (ER) stress were upregulated after LPS stimulation. Further, phosphorylation events in multiple pathways, including MAPKK and Wnt/β-catenin signalings, were identified as involved in LPS-triggered pathobiology. In essence, our findings reveal multiple integrated signals exerted by RPE under inflammation and are expected to give insight into the development of therapeutic interventions for RPE disorders.

Highlights

The retinal pigment epithelium (RPE) is a hexagonal pigmented cell layer located between the retina and choroid [1]
Low-grade parainflammation plays a cytoprotective role against local stress, overwhelming and persistent inflammation caused by complex biological disturbances, such as oxidative stress, complement abnormalities, and drusenoid accumulation, has been known to instigate the recruitment and activation of immune cells, exacerbating metabolic and pathobiological changes to the RPE [10]
The phosphorylation of β-catenin at Ser-552 residue was identified as increased by LPS challenge for 24 h (Figure S4B). These findings indicate that the tandem-mass tags (TMTs) labeling-based proteomic and phosphoproteomic analysis performed in this study possesses sufficient analytical power to confidently reveal changes in protein and phosphoprotein levels relevant to inflammation-driven pathology

Summary

Introduction

The retinal pigment epithelium (RPE) is a hexagonal pigmented cell layer located between the retina and choroid [1]. As the blood-ocular barrier component, the RPE helps maintain retinal homeostasis and visual function through mutualistic physical and metabolic interactions with the retina, and supports immune privilege by producing cell-surface and soluble inhibitory molecules such as CD86 and TGF-β [2,3,4,5]. Low-grade parainflammation plays a cytoprotective role against local stress, overwhelming and persistent inflammation caused by complex biological disturbances, such as oxidative stress, complement abnormalities, and drusenoid accumulation, has been known to instigate the recruitment and activation of immune cells, exacerbating metabolic and pathobiological changes to the RPE [10]. An improved understanding of RPE-specific inflammatory responses that determine disease progression is necessary

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