Abstract
Purpose:
Daily clearance of aged photoreceptor outer segment (POS) tips by retinal pigment epithelial (RPE) cells is crucial for retinal health and function. 2 main extracellular ligands for the internalization receptor MerTK, Gas6 and Protein S, have been shown to stimulate POS phagocytosis in vitro. In vivo, both ligands are expressed and necessary for retinal survival, however their exact role for MerTK regulation remains unclear. We showed previously that Protein S expression follows a circadian rhythm peaking at maximum phagocytosis time, whereas Gas6 expression is stable overtime. Thus, we set out to analyze how stimulation of RPE cells by ligand/s or ligand/s production by RPE cells themselves could regulate POS phagocytosis. We also aimed at identifying MerTK extracellular residues required for ligand binding and POS uptake.
Methods:
We incubated RPE-J cells with various doses of ligands, either alone or in combination. We inhibited ligand expression, alone or in combination, using siRNA in RPE-J cells. We introduced mutations in the 2 putative binding sites of mouse MerTK extracellular domain by site-directed mutagenesis and constructs were transfected into rat NRK-49F fibroblasts devoid of endogenous MerTK and in RPE-J cells. We then analyzed the phagocytic capabilities of the cells following these various treatments using in vitro phagocytosis assays. Treatments and results were verified using immunoblots.
Results:
Exogenous recombinant Gas6 inhibits phagocytosis with increasing doses while Protein S is stimulatory. When combined to a 1:1 ratio, Gas6 has a dominant effect. Inhibiting endogenous Protein S has not effect while Gas6 or Gas6/Protein S combined down-regulates phagocytosis. Gas6 effect is more pronounced for the binding step whereas Protein S seems to be more implicated in the internalization step of phagocytosis. Mutagenesis of either MerTK minor or major binding site appears to stimulate phagocytosis, altough to various extents.
Conclusions:
These results indicate an opposite role of Gas6 and Protein S for the regulation of daily MerTK function, Gas6 inhibiting and Protein S stimulating MerTK. Associated with our previous in vivo data, Gas6 seems to sustain a continuous inhibitory role on MerTK function, whereas Protein S appears to help stimulate MerTK at the peak phagocytic time, thus participating in the fine-tuning of this intricate function.
Keywords: 701 retinal pigment epithelium •
645 phagocytosis and killing •
695 retinal degenerations: cell biology