Cell-Cell and Cell-Extracellular Matrix Communication Pathways Identified in the Polarized Surface Proteome of Retinal Pigment Epithelial Cells

Background Polarized epithelial cells express distinct sets of surface proteins at the apical and basolateral membranes. This surface proteome is crucial for epithelial physiology as it includes channels and transporters, surface receptors and adhesion molecules among other proteins. We recently developed a quantitative approach to obtain polarized surface proteomes of epithelial cells in culture. This information could benefit efforts to understand epithelial cell/extracellular medium interactions as mechanisms to maintain higher order multi-tissue structures. Such interactions could represent organ-specific pathways of homeostasis and disease. In order to exploit the potential of our polarized epithelial surface proteomics approach, we pursued a detailed analysis of members of the Plexin/Semaphorin/Integrin (PSI) superfamily we found in the proteome of retinal pigment epithelial cells. In other cells, plexins are known receptors for semaphorins and mediate cell-cell communication, while integrins bind components of the extracellular matrix. In epithelial cells, some PSI isoforms have well known roles in cell adhesion, cytoskeletal remodeling and homotypic cell-cell junctions. However, the wide range of isoforms identified by surface proteomics provides a unique opportunity to analyze less-known isoforms as well as cell-type specific pathways. Objective To identify semaphorins and integrin isoforms in retinal pigment epithelium that mediate interactions with the extracellular matrix and other cell types. Methods We identified possible targets of interests based on their surface expression in retinal pigment epithelium and absence in the generic epithelial cell line MDCK. Next, we utilized gene knockdown via CRISPR or shRNA to analyze their role in cell-adhesion, migration and communication with endothelial cells. To study interaction with endothelial cells in vivo, we measured angiogenesis in a mouse model of matrigel-induced neovascularization. Results We determined that retinal pigment epithelial cells expressed semaphorin-4A (Sema4A) and Beta-8 integrin (ItgB8) at the surface, but they were absent in MDCK epithelial cells. Knockdown of ItgB8 significantly decreased retinal pigment epithelial cell migration in wound healing assays and cell adhesion to uncoated and collagen IV-coated substrate. Knockdown of Sema4A in retinal pigment epithelial cells enhanced angiogenesis in co-cultured choroidal explants (contains native endothelial cells that supply the retinal pigment epithelium in vivo). This enhanced angiogenesis suggests a baseline inhibitory role of secreted Sema4A. To test this possibility in vivo, we injected recombinant Sema4A intraocularly in mice, and we observed that this reduced matrigel-induced neovascularization. Conclusion Analysis of the surface proteome of epithelial cells led to identification of ItgB8 as mediator of retinal pigment epithelial cell adhesion and migration, and Sema4A as an anti-angiogenic cell-cell communication signal. Support or Funding Information NIH EY008538 and EY008538-28S1