Abstract
Purpose :
Characterization of the surface proteome of the retinal pigment epithelium (RPE) holds great potential for identifying targets to treat retinopathies associated with defective RPE physiology, like age-related macular degeneration and proliferative vitreoretinopathy. We analyzed the polarized distribution of hundreds of surface proteins in RPE primary cultures in order to identify proteins of previously unknown function in the RPE. Among them, we focused on Beta-8 Integrin (ItgB8) as a potential mediator of RPE adhesion by testing the hypothesis that basolateral distribution of ItgB8 in cultured RPE cells mediates cell adhesion to substrate and migration.
Methods :
Quantitative surface proteomics was performed via TMT-labeling in RPE primary cultures grown in permeable support. Surface proteins were isolated by surface biotinylation at 4°C for 30 mins. Knockdown of ItgB8 was performed via shRNAs (Sigma-Mission) delivered via lentiviruses to RPE cell line ARPE-19. Adhesion to plastic and collagen IV-coated substrates was tested over 1 hour at 37°C and measured with MTT-based assay. Cell migration was measured via wound healing assay. Controls consisted in non-targeting shRNAs. Data was analyzed as mean ± SEM and statistical analysis performed with GraphPad Prism.
Results :
ItgB8 was detected by proteomics and confirmed by western blot with a predominant basolateral distribution at the RPE cell surface (77±4% basolateral, n=4). Knockdown of ItgB8 delayed RPE cell migration from 11.7±1.1 μm/h to 6.6±0.3 μm/h (n=4; p<0.01), it decreased cell adhesion by 60±15% (n=3; p<0.01) and completely prevented adhesion to collagen-IV (n=3; p<0.01). Finally, we tested whether knockdown of the endosomal trafficking protein AP-1A prevented delivery of ItgB8 to the plasma membrane. We found that AP-1A shRNA attenuated the polarized distribution of ItgB8 (53±8% basolateral, n=4; p<0.01 vs. control-shRNA) and it decreased cell migration by 48±3% (n=4; p<0.01).
Conclusions :
Analysis of the surface proteome of cultured RPE cells led us to identify ItgB8 as a novel mediator of RPE cell adhesion, migration and attachment to collagen IV, most likely via basolateral trafficking mediated by AP-1A.
This is a 2020 ARVO Annual Meeting abstract.