Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK.

Silvia C. Finnemann,Yingyu Mao

doi:10.3390/cells10081927

Abstract

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE.

Highlights

In the mammalian retina, photoreceptor neurons continuously renew their lightsensitive outer segments to maintain retinal function for life
retinal pigment epithelium (RPE) cells in the mutant Royal College of Surgeons (RCS) rats lacking functional Mer tyrosine kinase (MerTK) still activate Rac1 during photoreceptor outer segment fragments (POS) binding like wt RPE cells, but fail to engulf surface-tethered POS
To assess if MerTK activity is required for F-actin assembly beneath bound POS, we used a synchronized POS phagocytosis assay testing unpassaged, differentiated primary rat RPE cells in culture

Summary

Introduction

Photoreceptor neurons continuously renew their lightsensitive outer segments to maintain retinal function for life. Extracellular phosphatidylserine-binding proteins MFG-E8, Gas, and Protein S in the subretinal space between RPE cells and photoreceptor outer segments serve to activate at least two phagocytic receptors in the RPE cells: apical αvβ integrin receptors are stimulated by MFG-E8 to signal towards the. RCS RPE cells fail to engulf POS, leading to the accumulation of outer segment debris in the subretinal space, photoreceptor death and retinal degeneration [22]. Downstream signaling stimulated by MerTK ligation is complex and may include Src family kinase and phospholipase involvement [25,26] It remains poorly understood which specific aspects of the MerTK signaling response in the RPE are required for POS engulfment. We show that RPE cells cannot form phagocytic cups beneath surface-tethered POS in the absence of MerTK signaling, which implies roles for F-actin-regulating mechanisms in phagocytosis other than and in addition to Rac activation. POS by MerTK-deficient RPE, suggesting that RhoA/ROCK pathway inhibition is both sufficient and necessary to overcome MerTK deficiency

Animals

Primary RPE Cell Culture

POS Phagocytosis Assays

RhoA Pathway Manipulations in RPE in Culture and RPE Ex Vivo

SDS-PAGE and Immunoblotting

Statistical Analysis

Results

Inhibition of the RhoA-ROCK Pathway Restores Phagocytic Cup Formation and POS

Discussion

Methods