Dataset Information

Regulation of Na,K-ATPase ?1-subunit in TGF-?2-mediated epithelial-to-mesenchymal transition in human retinal pigmented epithelial cells.

ABSTRACT: Proliferative vitreo retinopathy (PVR) is associated with extracellular matrix membrane (ECM) formation on the neural retina and disruption of the multilayered retinal architecture leading to distorted vision and blindness. During disease progression in PVR, retinal pigmented epithelial cells (RPE) lose cell-cell adhesion, undergo epithelial-to-mesenchymal transition (EMT), and deposit ECM leading to tissue fibrosis. The EMT process is mediated via exposure to vitreous cytokines and growth factors such as TGF-?2. Previous studies have shown that Na,K-ATPase is required for maintaining a normal polarized epithelial phenotype and that decreased Na,K-ATPase function and subunit levels are associated with TGF-?1-mediated EMT in kidney cells. In contrast to the basolateral localization of Na,K-ATPase in most epithelia, including kidney, Na,K-ATPase is found on the apical membrane in RPE cells. We now show that EMT is also associated with altered Na,K-ATPase expression in RPE cells. TGF-?2 treatment of ARPE-19 cells resulted in a time-dependent decrease in Na,K-ATPase ?1 mRNA and protein levels while Na,K-ATPase ?1 levels, Na,K-ATPase activity, and intracellular sodium levels remained largely unchanged. In TGF-?2-treated cells reduced Na,K-ATPase ?1 mRNA inversely correlated with HIF-1? levels and analysis of the Na,K-ATPase ?1 promoter revealed a putative hypoxia response element (HRE). HIF-1? bound to the Na,K-ATPase ?1 promoter and inhibiting the activity of HIF-1? blocked the TGF-?2 mediated Na,K-ATPase ?1 decrease suggesting that HIF-1? plays a potential role in Na,K-ATPase ?1 regulation during EMT in RPE cells. Furthermore, knockdown of Na,K-ATPase ?1 in ARPE-19 cells was associated with a change in cell morphology from epithelial to mesenchymal and induction of EMT markers such as ?-smooth muscle actin and fibronectin, suggesting that loss of Na,K-ATPase ?1 is a potential contributor to TGF-?2-mediated EMT in RPE cells.

SUBMITTER: Mony S

PROVIDER: S-EPMC3796007 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Regulation of Na,K-ATPase β1-subunit in TGF-β2-mediated epithelial-to-mesenchymal transition in human retinal pigmented epithelial cells.

Mony Sridevi S Lee Seung Joon SJ Harper Jeffrey F JF Barwe Sonali P SP Langhans Sigrid A SA

Experimental eye research 20130628

Proliferative vitreo retinopathy (PVR) is associated with extracellular matrix membrane (ECM) formation on the neural retina and disruption of the multilayered retinal architecture leading to distorted vision and blindness. During disease progression in PVR, retinal pigmented epithelial cells (RPE) lose cell-cell adhesion, undergo epithelial-to-mesenchymal transition (EMT), and deposit ECM leading to tissue fibrosis. The EMT process is mediated via exposure to vitreous cytokines and growth facto ...[more]

PMID: 23810808

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Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD.Transcriptome wide expression profiles of human fetal RPE cell cultures as a function of passage and time post-plating were determined using Agilent 44 K whole genome microarrays and RNA-Seq. Using a systems level analysis, differentially expressed genes and pathways of interest were identified and their role in the establishment of a persistent mesenchymal state was assessed using pharmacological-based experiments.Using a human fetal RPE cell culture model that considers monolayer disruption and subconfluent culture as a proxy for wound stimulus, we show that prolonged wound stimulus leads to terminal acquisition of a mesenchymal phenotype post-confluence and altered expression of more than 40 % of the transcriptome. In contrast, at subconfluence fewer than 5 % of expressed transcripts have two-fold or greater expression differences after repeated passage. Protein-protein and pathway interaction analysis of the genes with passage-dependent expression levels in subconfluent cultures reveals a 158-node interactome comprised of two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGF? pathway activators: TGFB1, TGFB2, INHBA, INHBB, GDF6, CTGF, and THBS1. Significantly, inhibition of TGFBR1/ACVR1B mediated signaling using receptor kinase inhibitors both forestalls and largely reverses the passage-dependent loss of epithelial potential; thus extending the effective lifespan by at least four passages. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD, including key members of the TGF? pathway.In RPE cells the switch to a persistent mesenchymal state following prolonged wound stimulus is driven by lasting activation of the TGF? pathway. Targeted inhibition of TGF? signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell-based therapies.

Dataset Information

Regulation of Na,K-ATPase ?1-subunit in TGF-?2-mediated epithelial-to-mesenchymal transition in human retinal pigmented epithelial cells.

Publications

Regulation of Na,K-ATPase β1-subunit in TGF-β2-mediated epithelial-to-mesenchymal transition in human retinal pigmented epithelial cells.

Similar Datasets

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