Abstract
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.
Highlights
Retinal pigment epithelial (RPE) cells, photoreceptors, and the choroid form a functional unit required for healthy vision
Compared to RPE cells expressing WT-Fibulin-3, zonula occludens-1 (ZO-1) distribution was disrupted and trans-epithelial electrical resistance (TER) values were significantly lower in RPE cells expressing R345WFibulin-3
Retinal pigment epithelial signature genes were downregulated and multiple genes associated with epithelial– mesenchymal transition (EMT) were upregulated in the mutant group
Summary
Retinal pigment epithelial (RPE) cells, photoreceptors, and the choroid form a functional unit required for healthy vision. Atrophy and dysfunction of the RPE with subsequent loss of photoreceptors plays a fundamental role in numerous retinal degenerations. RPE dysfunction manifests as a loss of barrier function, disrupted polarization, and downregulated expression of RPE signature genes and microRNA-204/211 (Wang et al, 2010; Adijanto et al, 2012). Recent evidence suggests that RPE cells lose terminal differentiation and acquire a mesenchymal cell phenotype in several retinal degenerations (Ghosh et al, 2018; Goldberg et al, 2018; Wu et al, 2019). Misfolded proteins accumulate in RPE cells with age, but the relationship between misfolded protein accumulation and RPE epithelial– mesenchymal transition (EMT) remains unclear
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