Abstract
Purpose :
Coloboma is a congenital disorder caused by failure of optic fissure closure (OFC) resulting in the incomplete formation of eye tissues. Our studies previously showed the evolutionarily-conserved role of FAT1 in mediating OFC in human, mouse, and zebrafish eyes and revealed its localization in the leading edges of retinal pigment epithelial (RPE) cells, which participate in the early steps of OFC. FAT1 may modulate Hippo signaling, a tumor suppressor pathway associated with coloboma. This study further investigated the role of FAT1 and its downstream proteins in the Hippo signaling pathway in human RPE cells.
Methods :
Three different induced pluripotent stem cell lines were differentiated to RPE cells. FAT1, Hippo signaling proteins (MST, LATS, MOB, YAP/TAZ), and their phosphorylated forms were studied via Western blots and immunofluorescence staining followed by confocal microscopy. To determine cellular localization of proteins, we calculated membrane:cytoplasm protein fluorescence intensity ratios using tight junction protein ZO1 as a control. Retrospective transcript analysis for FAT1 and Hippo pathway genes was carried out with the National Eye Institute’s eyeIntegration database.
Results :
Gene expression analysis showed presence of transcripts for FAT1 and Hippo pathway genes, and Western blots confirmed the presence of FAT1 and MST, LATS, MOB, and YAP/TAZ in the RPE. FAT1 was found to localize at the apical side of RPE membranes and demonstrated a membrane:cytoplasm ratio of 0.96 [95% CI 0.86,1.06]. pYAPS127 expression was consistently observed in RPE cells but not in HeLa cell controls, suggesting active Hippo signaling resulting in phosphorylation of YAP/TAZ localized predominantly at the apical surfaces and in the cytoplasm (0.75 [0.56,0.95]); unphosphorylated YAP/TAZ revealed lower relative cytoplasmic expression (1.42 [1.15,1.68], p<0.01).
Conclusions :
FAT1 and key Hippo signaling proteins were expressed in RPE cells at the RNA and protein levels. Preliminary protein phosphorylation studies suggest the Hippo pathway may be active in the RPE based on the presence of phosphorylated YAP/TAZ. Concurrent with strong FAT1 expression levels, these results suggest a potential upstream role for FAT1 in regulating Hippo signaling, indicating the need to study this pathway in RPE junction formation and as a participant in OFC.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.