MERTK ACTIVATION DURING RPE PHAGOCYTOSIS IN VIVO REQUIRES αVβ5 INTEGRIN

1. INTRODUCTION

Daily phagocytosis of shed photoreceptor outer segment fragments (POS) is a key task of the retinal pigment epithelium (RPE) in the retina. Lack or inefficiency of daily POS clearance causes early onset, rapid, and complete retinal degeneration in experimental animals and likely contributes to human blinding diseases such as retinitis pigmentosa and age-related macular degeneration (Dowling and Sidman, 1962, Gal et al., 2000). The phagocytic mechanism of the RPE belongs to a group of conserved non-inflammatory clearance pathways that mediate recognition and engulfment of apoptotic cells in both non-professional and professional phagocytic cells, such as fibroblasts and macrophages, respectively (Finnemann and Rodriguez-Boulan, 1999). These pathways share the use of phagocyte cell surface receptors such as the lipid scavenger receptor CD36 (Ryeom et al., 1996), the integrin adhesion receptor αvβ5 (Finnemann et al., 1997; Miceli et al., 1997; Lin and Clegg, 1998) and the receptor tyrosine kinase Mer (MerTK) (D'Cruz et al., 2000; Nandrot et al., 2000). In vitro phagocytosis assays studying primary or permanent RPE in culture fed with isolated POS suggest that CD36 and MerTK participate in the engulfment step of the phagocytic process (Chaitin and Hall, 1983; Finnemann and Silverstein, 2001), while αvβ5 integrin promotes POS recognition/binding and initiates a downstream cytoplasmic signaling cascade in the RPE (Finnemann et al., 1997). However, the precise function of these receptors and their roles in the intact retina are so far only poorly understood. Most recently, we have begun to study phagocytosis and receptor activity in animal models that lack αvβ5 integrin or MerTK to determine how these different plasma membrane receptors of the RPE functionally interact to coordinate particle uptake.

2. ROLE OF MERTK ACTIVATION IN RPE PHAGOCYTOSIS

Activity of the Mer tyrosine kinase receptor MerTK is essential for efficient engulfment of POS by RPE in vivo and in vitro (Mullen and LaVail, 1976; Edwards and Szamier, 1977) Despite its importance, mechanisms of MerTK activation and MerTK downstream signaling target proteins in RPE are still largely obscure. Retinal ligands of MerTK have not yet been conclusively identified. 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). Furthermore, challenge with isolated POS causes increased phosphorylation at tyrosine 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.

3. FOCAL ADHESION KINASE SIGNALING ACTIVATES MERTK DURING RPE PHAGOCYTOSIS

Our previous studies on phagocytic signaling in RPE suggest an important role for focal adhesion kinase (FAK) in MerTK activation. FAK is a cytoplasmic non-receptor tyrosine kinase that colocalizes with integrin receptors at focal contacts where it commonly transduces signaling pathways downstream of activated integrins (for a recent review on FAK see (Parsons, 2003)). Reversible activation of FAK is critical for integrin functions that involve cytoskeletal reorganization (Ilic et al., 1995). We expressed a C-terminal fragment of FAK that competes with full-length endogenous FAK for cytoskeletal anchorage. This fragment has been shown to act as a dominant-negative inhibitor of endogenous FAK abrogating FAK downstream signal transduction. We showed that the rat derived RPE-J cell line, like primary wild-type rat RPE, utilizes endogenous MerTK to engulf POS. Importantly, expression of the dominant-negative FAK C-terminal fragment in RPE-J cells inhibited POS engulfment (Finnemann, 2003). Furthermore, it eliminated the increase in MerTK tyrosine phosphorylation that is elicited by phagocytic challenge of RPE cells in culture (Finnemann, 2003). On the contrary, RPE cultures derived from RCS rats retained similar FAK activation as wild-type Long Evans rat RPE cultures in response to OS challenge. These results identify a novel signal transduction pathway in which FAK acts upstream of MerTK to stimulate the internalization machinery of the RPE.

4. αvβ5 SIGNALING VIA FOCAL ADHESION KINASE ACTIVATES MERTK DURING RPE PHAGOCYTOSIS IN VIVO AND IN VITRO

Previous studies have shown that phosphorylation of FAK at tyrosine residue 861 promotes direct binding of FAK to the cytoplasmic face of αvβ5 integrin receptors (Eliceiri et al., 2002). In our studies, we observed increased levels of FAK in αvβ5 protein complexes isolated by immunoprecipitation from RPE-J cells during the early POS binding phase but loss of FAK from the integrin complex during the later POS internalization phase (Finnemann, 2003). Residence of FAK in the complex correlated well with elevated phosphorylation of tyrosine 861, while phosphorylation of other tyrosine residues that indicate FAK enzymatic activity persisted beyond the time of FAK in the integrin complex. These results suggest that RPE cells activate FAK recruited to its apical αvβ5 surface receptors in response to POS phagocytic challenge in vitro.

To directly determine whether αvβ5 integrin receptors were required for FAK and MerTK activation in RPE, we tested FAK and MerTK activation during RPE phagocytosis by RPE cells of β5 integrin knockout mice that lack all αvβ5 integrin receptors. β5 null RPE cells in culture largely fail to phagocytose isolated POS (Nandrot et al., 2004). β5 null retina lacks the synchronized burst of RPE phagocytosis that characteristically follows early morning rod shedding in rodent retina (Nandrot et al., 2004). The detrimental effects of this abnormal timing of phagocytosis on retinal function in β5 integrin null mice of age are discussed in more detail in the chapter by Nandrot and Finnemann in this volume.

When we fed isolated POS to wild-type mouse RPE, we found robust FAK and MerTK activation confirming our earlier results using stable and primary rat RPE (shown for MerTK in Figure 1a, β5+/+). In contrast, β5 null RPE cells in primary culture did not increase tyrosine phosphorylation of either FAK or MerTK in response to POS, although they expressed both proteins at normal levels (shown for MerTK in Figure 1a, β5−/−).Furthermore, in vivo phagocytic signaling via FAK and MerTK was strongly and transiently stimulated following light onset in wild-type mouse retina but was absent in β5 knockout mouse retina (shown for MerTK in Figure 1b). These data provide conclusive evidence that αvβ5 integrin signaling regulates rhythmic activation of FAK and MerTK during RPE phagocytosis in the intact retina.

Figure 1.

MerTK activation requires αvβ5 integrin. a. Primary RPE in culture from β5+/+ or β5−/− mice received isolated POS (POS) or assay medium alone (m) for 1.5 hours before lysis of cells. b. Eyecups were harvested from 3 week old strain-matched β5+/+ and β5−/− mice at different times of day as indicated and protein lysates prepared immediately. a and b. Lysates were analyzed by SDS-PAGE and immunoblotting for MerTK protein and tyrosine-phosphorylated MerTK (PY-MerTK). Band intensities were quantified to calculate relative levels of MerTK phosphorylation (= activation). Bars represent means ± SD, n=3. Significant differences between equivalent β5+/+ and β5−/− values were determined by Student’s t-test and are indicated by asterisks (P < 0.001 for a, P < 0.05 for b). Modified from Nandrot et al., 2004, with permission from Rockefeller University Press.

5. PERSPECTIVE

The results discussed here are the first to describe signaling activities by phagocytic receptors in intact retina that precisely correlate temporally with POS shedding and uptake by RPE cells. The late onset retinal dysfunction of the β5 knockout mouse as a consequence of lack of such phagocytic signaling emphasizes the importance of precise temporal regulation of POS phagocytosis by the RPE. In future studies, we will use similar experimental approaches exploring in vivo signaling in normal and mutant animal models to identify further components of the RPE phagocytic mechanism and to unravel their functional interactions.