Dataset Information

Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

ABSTRACT: Purpose:To investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied. Methods:Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial-mesenchymal transition (EMT) phenotype was assessed by a migration assay. Results:Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT. Conclusions:The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.

SUBMITTER: Zhou M

PROVIDER: S-EPMC7317295 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

Zhou Mi M Weber Sarah R SR Zhao Yuanjun Y Chen Han H Barber Alistair J AJ Grillo Stephanie L SL Wills Carson A CA Wang Hong Gang HG Hulleman John D JD Sundstrom Jeffrey M JM

Frontiers in cell and developmental biology 20200619

<h4>Purpose</h4>To investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied.<h4>Methods</h4>Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized s ...[more]

PMID: 32637411

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Project description:We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFβ1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFβ was determined by pre-incubation of EVs with a pan-TGFβ blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFβ1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFβ-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFβ dependent manner. These results suggest that EV-bound TGFβ plays a critical role in the induction of EMT in RPE cells.

Dataset Information

Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

Publications

Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

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