Abstract
Purpose :
Molecular mechanisms regulating diurnal phagocytosis by the retinal pigment epithelium (RPE), a process fundamental to life-long outer segment renewal, remain incompletely understood. The Hippo pathway, through yes-associated protein (YAP), influences retinal tissue development, yet how Hippo signaling functions in adult retina remains unknown. Here, we explore effects of Hippo pathway manipulation on phagocytosis by differentiated RPE in culture.
Methods :
Primary, polarized RPE cells from Sprague-Dawley rats were transfected with Yap-targeting or non-targeting siRNA, and cell lysates were probed for YAP via Western blot. Cells were challenged with Texas-Red pre-labeled POS particles isolated from porcine eyes. Effects of Hippo pathway drugs, XMU-MP-1 (an indirect YAP activator) or CA3 (a YAP inhibitor) were also tested. Opsin labeling after paraformaldehyde fixation was used to exclusively label surface-bound POS. Confocal microscopy imaging followed by ImageJ image analyses yielded POS quantification. Data sets of n≥3 were analyzed using two-tailed Student’s t-test for groups of two. Groups of three were evaluated using ANOVA and Tukey’s post hoc tests.
Results :
Transfecting primary RPE cells with Yap-targeting siRNA decreased YAP protein by 74% relative to control cells (p<0.05). Yap-silenced or CA3-treated primary RPE cells showed an increase in total and bound POS/cell, relative to control cells (p<0.05), suggesting inhibition of phagocytosis. XMU-MP-1-treated cells showed a decrease in total and bound POS/cell (p<0.05). CA3 treatment of Yap-silenced cells had no effect on POS uptake, confirming that silencing specifically of YAP affected phagocytosis. In discontinuous phagocytosis assays, administering drugs after POS binding but prior to internalization, CA3 increased total and bound POS/cell, while XMU-MP-1 decreased total and bound POS/cell (both p<0.05).
Conclusions :
Our data provide new insight into the cellular signaling pathways controlling RPE phagocytosis: the internalization phase of RPE phagocytosis relies on YAP/Hippo pathway signaling.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.