Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells
PurposeTo 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.MethodsPrimary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to expres...
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Frontiers Media S.A.
2020-06-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fcell.2020.00469/full |
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| author | Mi Zhou Sarah R. Weber Yuanjun Zhao Han Chen Alistair J. Barber Stephanie L. Grillo Carson A. Wills Hong Gang Wang John D. Hulleman Jeffrey M. Sundstrom |
| author_facet | Mi Zhou Sarah R. Weber Yuanjun Zhao Han Chen Alistair J. Barber Stephanie L. Grillo Carson A. Wills Hong Gang Wang John D. Hulleman Jeffrey M. Sundstrom |
| author_sort | Mi Zhou |
| collection | DOAJ |
| description | PurposeTo 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.MethodsPrimary 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.ResultsCompared 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.ConclusionsThe results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells. |
| first_indexed | 2024-12-12T05:59:15Z |
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| language | English |
| last_indexed | 2024-12-12T05:59:15Z |
| publishDate | 2020-06-01 |
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| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-23db40c4680440e7ab5dafdc5ecb170f2022-12-22T00:35:28ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2020-06-01810.3389/fcell.2020.00469545698Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial CellsMi Zhou0Sarah R. Weber1Yuanjun Zhao2Han Chen3Alistair J. Barber4Stephanie L. Grillo5Carson A. Wills6Hong Gang Wang7John D. Hulleman8Jeffrey M. Sundstrom9Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, United StatesDepartment of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, United StatesDepartment of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, United StatesTEM Facility, Penn State Hershey College of Medicine, Hershey, PA, United StatesDepartment of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, United StatesDepartment of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, United StatesDivision of Hematology and Oncology, Department of Pediatrics, Penn State Hershey College of Medicine, Hershey, PA, United StatesDivision of Hematology and Oncology, Department of Pediatrics, Penn State Hershey College of Medicine, Hershey, PA, United StatesDepartment of Ophthalmology, UT Southwestern Medical Center, Dallas, TX, United StatesDepartment of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, United StatesPurposeTo 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.MethodsPrimary 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.ResultsCompared 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.ConclusionsThe results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.https://www.frontiersin.org/article/10.3389/fcell.2020.00469/fullRPEdedifferentiationEMTprotein misfoldingmacular degeneration |
| spellingShingle | Mi Zhou Sarah R. Weber Yuanjun Zhao Han Chen Alistair J. Barber Stephanie L. Grillo Carson A. Wills Hong Gang Wang John D. Hulleman Jeffrey M. Sundstrom Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells Frontiers in Cell and Developmental Biology RPE dedifferentiation EMT protein misfolding macular degeneration |
| title | Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells |
| title_full | Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells |
| title_fullStr | Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells |
| title_full_unstemmed | Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells |
| title_short | Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells |
| title_sort | expression of r345w fibulin 3 induces epithelial mesenchymal transition in retinal pigment epithelial cells |
| topic | RPE dedifferentiation EMT protein misfolding macular degeneration |
| url | https://www.frontiersin.org/article/10.3389/fcell.2020.00469/full |
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