Mertk Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells

Abstract

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. Phagocytosis is a process by which large particles are internalized by cells to form phagosomes. The process can be divided into three phases: binding, ingestion, and digestion. Retinal pigment epithelial (RPE) 1 cells, which form a polarized epithelium between the photoreceptor cells and the choroid in the outer retina, phagocytize more biomass than any other mammalian cell type (1). The RPE phagocytizes photoreceptor outer segment (OS) membranes (2) that are shed as part of the normal ongoing process of photoreceptor OS renewal (3). Failure of OS membrane uptake leads to photoreceptor cell death (4), as illustrated by the RCS rat, a widely used model for recessively inherited retinal degeneration. The RCS mutation rdy (retinal dystrophy) causes, either directly or indirectly, a defect in RPE phagocytosis (4). This defect leads to an accumulation of shed OS membranes in the subretinal space (4) and a rapid and progressive degeneration of photoreceptor cells (5).The molecular mechanisms of RPE phagocytosis are unclear. Studies of the internalization of exogenous OS by cultured primary RPE cells suggested a receptor-mediated process (6 -8). Inhibition of the RPE cell culture phagocytic assay by antireceptor antibodies or competitive ligands suggested several specific proteins that might play a role in the process, including the mannose receptor (9, 10), CD36 (11), and ␣ v ␤ 5 integrin (12-14). Inhibition of ␣ v ␤ 5 integrin function disrupts the OS binding phase of RPE phagocytosis, whereas the mannose receptor and CD36 have been implicated in both OS binding and ingestion. Cultured RCS RPE cells bind exogenous OS at wildtype levels. However, only a small percentage of bound OS are ingested by RCS RPE cells (15), indicating that the protein encoded by the rdy locus is critical, directly or indirectly, for OS uptake.The gene corresponding to rdy remained unknown until recently. The mannose receptor protein and messenger RNA are present in the RPE of both wild-type and RCS rats from postnatal day (P) 5 to adult (16). CD36 null mice have been reported to have normal electroretinography and retinal histology (17). These data suggest that neither the mannose receptor nor CD36 is the gene mutated in the RCS rat. We used positional cloning to identify a mutation in the receptor tyrosine kinase gene Mertk in the RCS rat. A deletion of RCS genomic DNA results in expression of an aberrant Mertk transcript with a translation termination signal after codon 20 (18),...

Bimodal in vivo imaging provides early assessment of stem-cell-based photoreceptor engraftment

“…This resulted from either RPE uptake of lone QT or phagocytosis of PPC-graft containing QT. The latter scenario was observed in vitro here (Figure 3f) and in others studies 21,22 and is expected given the RPE's phagocytic roles. 23 …”

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

“…It is established that binding of POS to RPE cells activates a hierarchy of tyrosine kinases that are essential for phagosome internalization, and that FAK and MerTK play prominent roles in this process (Feng et al, 2002;Finnemann, 2003). Because FAK and anx A2 are substrates for several tyrosine kinases, including Src (Bellagamba et al, 1997), and because we have recently shown that anx A2 is required for the plasma membrane targeting and activation of Src (Hayes and Moss, 2009), we next tested whether anx A2 and c-Src become tyrosine-phosphorylated during POS phagocytosis by ARPE19 cells.…”

“…Intriguingly, engagement of the ␣v␤5 integrin by its cognate ligand, milk fat globule-EGF8 (MFG-E8), is essential for the circadian synchronization of phagocytosis (Nandrot and Finnemann, 2006;Nandrot et al, 2007), and although phagocytosis of POS occurs in MFG-E8 Ϫ/Ϫ and ␤5 Ϫ/Ϫ mice, in both mutants the daily rhythm is absent. In contrast, focal adhesion kinase (FAK) and MerTK are not required for the initial binding of POS to the apical surface of the RPE, but they are sequentially activated by ␣v␤5 receptors and are essential for phagosome internalization (Feng et al, 2002;Finnemann, 2003).…”

“…In these experiments, we observed enrichment of anx A2-GFP on forming but not mature phagosomes ( Figure 2E), which when taken with the data mentioned above strongly suggests that a role for anx A2 in POS phagocytosis is most likely to be at the time of POS engagement and initial internalization. It is established that binding of POS to RPE cells activates a hierarchy of tyrosine kinases that are essential for phagosome internalization, and that FAK and MerTK play prominent roles in this process (Feng et al, 2002;Finnemann, 2003). Because FAK and anx A2 are substrates for several tyrosine kinases, including Src (Bellagamba et al, 1997), and because we have recently shown that anx A2 is required for the plasma membrane targeting and activation of Src (Hayes and Moss, 2009), we next tested whether anx A2 and c-Src become tyrosine-phosphorylated during POS phagocytosis by ARPE19 cells.…”

Mertk Activation During RPE Phagocytosis in Vivo Requires αVβ5 Integrin

“…Moreover, we still do not know which RPE proteins serve as substrates for MerTK's kinase activity during RPE phagocytosis. However, both endogenous and overexpressed MerTK reveal a striking redistribution to the sites of internalized POS in in vitro phagocytosis assays suggesting that MerTK receptors may be components of the phagocytic machinery of the RPE (Feng et al, 2002;Finnemann, 2003 residues of MerTK in RPE in culture (Feng et al, 2002;Finnemann, 2003). Although their mutual dependence has not been demonstrated directly, levels of MerTK tyrosine phosphorylation commonly serve to assess the extent of MerTK activity.…”