Abstract
Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.
Highlights
The MerTK receptor is necessary for retinal phagocytosis and its daily rhythm
Consistent with a previous report using J774 macrophages, MerTK extracellular domain was cleaved and released in the conditioned media (CM) from J774 macrophages as a soluble protein of ϳ150 kDa [41]. soluble form of MerTK (sMerTK) release increased with time, and a concomitant significant decrease of full-length MerTK levels was observed in corresponding lysates
To J774 macrophages, a soluble form of the receptor was detected in retinal pigment epithelial (RPE)-J cells with a size around 120 kDa (Fig. 1B). sMerTK cleavage increased with time and was slightly more pronounced when cells were incubated with
Summary
The MerTK receptor is necessary for retinal phagocytosis and its daily rhythm. Results: MerTK is cleaved from the apical cell surface in vitro and in vivo, reducing the phagocytic capacity of RPE cells. The ␣v5 integrinMFG-E8 couple is essential for the daily burst of phagocytosis observed 1–2 h after light onset as shown by knock-out animal models [12, 14] Another series of soluble ligands able to bind PS for apoptotic cell elimination are Gas and protein S [15, 16], vitamin K-dependent proteins that are common ligands for the TAM (Tyro, Axl, and MerTK) family of tyrosine kinase receptors [17]. Soluble receptors can have more than one occupation [39]; they may function as binding proteins to stabilize ligands in the extracellular matrix and enhance interaction with the membrane-bound receptors, can down-regulate ligand-related activation and associated downstream signaling, or might act as decoy receptors by competing with full-length membrane receptors for ligand binding Both Axl and MerTK have been shown to be cleaved and released as soluble proteins by fibroblasts and macrophages, respectively [40, 41]. SMerTK release is rhythmic in vivo and may help limit the phagocytic peak duration
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