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Silvia C Finnemann, Yingyu Mao; Inhibiting cytosolic RhoA pathway signaling rescues the phagocytic deficiency of RCS RPE lacking the Mer tyrosine kinase receptor (MerTK). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2407.
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© ARVO (1962-2015); The Authors (2016-present)
Diurnal phagocytosis of photoreceptor outer segment tips (POS) by retinal pigment epithelial (RPE) cells is essential for vision. POS clearance is triggered by sequential engagement of αvβ5 integrin and Mer tyrosine kinase (MerTK) RPE surface receptors. Inactivating mutations of MerTK in RCS rats or mice diminish POS clearance causing rapid postnatal photoreceptor degeneration. Several MerTK mutations cause severe retinitis pigmentosa in human patients. Here, we tested whether and which manipulation of cytosolic signal transduction pathways may be sufficient to promote POS engulfment by RPE lacking MerTK.
Purified POS, receptor ligands, pharmacological agents, and adenoviruses encoding mutant signaling proteins were applied in combination to rat eyecup preparations and/or unpassaged, primary wild-type and RCS rat RPE cells. Synchronized POS uptake assays and microscopy were used to quantify F-actin phagocytic cup recruitment and internalization capacity of RPE in culture. Lysotracker live imaging verified POS engulfment. Activities of small GTPases were measured using G-lisa kits. Expression and activity of cytosolic kinases were quantified by immunoblotting.
A drug mini-screen revealed that inhibition of RhoA signaling promoted POS internalization into acidified phagosomes by RCS rat RPE tissue ex vivo and by RCS primary RPE. The daily peak of POS clearance coincided with a dip in endogenous RhoA activity in wild-type but not RCS rat RPE in vivo. Either constitutively active Rac1 or RhoA inhibition yielded F-actin phagocytic cup assembly beneath bound POS and POS engulfment in RPE lacking MerTK or in RPE with MerTK lacking MerTK ligand. Yet, RhoA pathway inhibition did not restore phagocytic cups or POS engulfment abolished by dominant-negative Rac1. Likewise, constitutively active Rac1 failed to restore phagocytic cups or POS engulfment inhibited by RhoA activation.
Decreasing RhoA pathway activity is sufficient to promote POS internalization by MerTK-deficient RPE ex vivo or in culture. RPE with wild-type MerTK briefly alters both endogenous Rac1 and RhoA activities during phagocytosis to allow recruitment of functional F-actin phagocytic cups and POS engulfment. Manipulating cytosolic signaling can replace the contribution of MerTK to phagocytic F-actin dynamics and restore phagocytic function to RPE lacking MerTK.
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