Abstract
Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-to-mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy, diabetic retinopathy, and age-related macular degeneration. Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human-induced pluripotent stem cell–based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag labeling followed by high-resolution tandem MS for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantified 7937 protein groups in our tandem mass tag–based MS analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Furthermore, we integrated our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label-free single-shot data-independent acquisition MS study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple age-related macular degeneration–associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease.
Highlights
Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-tomesenchymal transition (EMT)
To obtain insight into the proteomic pathways involved in RPEEMT, we utilized our previously described system for generating RPE monolayers from human-induced pluripotent stem cell (hiPSC) and human-embryonic stem cell (hESC) [35, 36] and induced EMT by proteocollagenolytic treatment [46]
We confirmed upregulation of key EMT transcriptional factors (SNAI1 7-fold, SNAI2 8-fold, TWIST1 8-fold, ZEB1 3-fold, and HMGA2 8-fold) by Quantitative PCR (qPCR) at 3 to 12 h postdissociation (Fig. 1D). These changes were accompanied by decreased expression of both epithelial cell morphology–related genes (CDH1 [Ecadherin] fourfold) and RPE-specific genes (LRAT 84-fold, BEST1 26-fold, OTX2 15-fold, PMEL17 12-fold, CRX 12-fold, RLBP1 9-fold, TYR 7-fold, and MITF 6-fold) (Fig. 1, E and F)
Summary
We present a comprehensive proteomic analysis aimed at defining the temporal protein expression changes associated with EMT of stem cell–derived retinal pigment epithelial cells. Temporal Proteomic Profiling of RPE-EMT the realization that EMT plays an essential role in many ocular degenerative diseases, including proliferative vitreoretinopathy [11,12,13], neovascular (“wet”) age-related macular degeneration (AMD) [14, 15], atrophic (“dry”) AMD [16, 17], and diabetic retinopathy [18], there has been increasing interest in mechanisms and regulation of EMT of the retinal pigment epithelium (RPE). In addition to presenting a comprehensive temporal proteomic analysis of hRPE monolayers and enzymatically dissociated single cells by high-resolution MS, we describe an integrative proteogenomic analysis to identify the proteins and associated biological pathways that drive EMT in RPE. We hope that our dataset and analysis will provide the vision research community a comprehensive resource for the increasing understanding of RPE-EMT progression mechanisms and point toward potential targets for therapeutic intervention
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