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

Open AccessPublished:February 22, 2002DOI:https://doi.org/10.1074/jbc.M107876200
      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 andin vivo genetic complementation to demonstrate thatMertk 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. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.
      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)
      The abbreviations used are: RPE
      retinal pigment epithelium
      OS
      outer segment
      P
      postnatal day
      m.o.i.
      multiplicity of infection
      WGA
      wheat germ agglutinin
      DMEM
      Dulbecco's modified Eagle's medium
      Endo H
      endo-β-N-acetylglucosaminidase H
      PNGase F
      peptide N-glycosidase F
      GFP
      green fluorescent protein
      1The abbreviations used are: RPE
      retinal pigment epithelium
      OS
      outer segment
      P
      postnatal day
      m.o.i.
      multiplicity of infection
      WGA
      wheat germ agglutinin
      DMEM
      Dulbecco's modified Eagle's medium
      Endo H
      endo-β-N-acetylglucosaminidase H
      PNGase F
      peptide N-glycosidase F
      GFP
      green fluorescent protein
      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 (
      • Bok D.
      • Young R.W.
      ). The RPE phagocytizes photoreceptor outer segment (OS) membranes (
      • Young R.W.
      • Bok D.
      ) that are shed as part of the normal ongoing process of photoreceptor OS renewal (
      • Young R.W.
      ). Failure of OS membrane uptake leads to photoreceptor cell death (
      • Bok D.
      • Hall M.O.
      ), 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 (
      • Bok D.
      • Hall M.O.
      ). This defect leads to an accumulation of shed OS membranes in the subretinal space (
      • Bok D.
      • Hall M.O.
      ) and a rapid and progressive degeneration of photoreceptor cells (
      • Dowling J.E.
      • Sidman R.L.
      ).
      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 (
      • Mayerson P.L.
      • Hall M.O.
      ,
      • Hall M.O.
      • Abrams T.
      ,
      • Laird D.W.
      • Molday R.S.
      ). Inhibition of the RPE cell culture phagocytic assay by anti-receptor antibodies or competitive ligands suggested several specific proteins that might play a role in the process, including the mannose receptor (
      • Boyle D.
      • Tien L.F.
      • Cooper N.G.
      • Shepherd V.
      • McLaughlin B.J.
      ,
      • Shepherd V.L.
      • Tarnowski B.I.
      • McLaughlin B.J.
      ), CD36 (
      • Ryeom S.W.
      • Sparrow J.R.
      • Silverstein R.L.
      ), and αvβ5 integrin (
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      ,
      • Miceli M.V.
      • Newsome D.A.
      • Tate Jr., D.J.
      ,
      • Lin H.
      • Clegg D.O.
      ). Inhibition of αvβ5 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 wild-type levels. However, only a small percentage of bound OS are ingested by RCS RPE cells (
      • Chaitin M.H.
      • Hall M.O.
      ), indicating that the protein encoded by therdy 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 (
      • Wilt S.D.
      • Greaton C.J.
      • Lutz D.A.
      • McLaughlin B.J.
      ). CD36 null mice have been reported to have normal electroretinography and retinal histology (
      • Silverstein R.L.
      • Sparrow J.R.
      • Ryeom S.W.
      ). 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 Mertktranscript with a translation termination signal after codon 20 (18), likely a complete loss-of-function, or null, allele. Mertkwas an appropriate candidate for rdy in light of evidence that a signaling defect might underlie the RCS RPE phagocytic phenotype (
      • Chaitin M.H.
      • Hall M.O.
      ,
      • Heth C.A.
      • Schmidt S.Y.
      ,
      • Heth C.A.
      • Marescalchi P.A.
      ). The discovery of mutations in the human ortholog,MERTK, in individuals with retinitis pigmentosa indicated that Mertk is essential for maintenance of the mammalian retina (
      • Gal A.
      • Li Y.
      • Thompson D.A.
      • Weir J.
      • Orth U.
      • Jacobson S.G.
      • Apfelstedt-Sylla E.
      • Vollrath D.
      ). Subsequently, in vivo genetic complementation of the RCS phenotype by viral mediated gene transfer conclusively demonstrated that Mertk is the gene for rdy(
      • Vollrath D.
      • Feng W.
      • Duncan J.
      • Yasumura D.
      • D'Cruz P.M.
      • Chappelow A.
      • Matthes M.T.
      • Kay M.A.
      • LaVail M.M.
      ).
      The identification of Mertk provides an initial focus for elucidating molecular mechanisms of RPE phagocytosis. In the present study, we sought to determine whether the site of action ofMertk was indeed the RPE, as suggested by genetic chimera experiments (
      • Mullen R.J.
      • LaVail M.M.
      ) and, if so, whether Mertk protein was directly involved in the ingestion step of OS phagocytosis. We tested whether viral mediated gene transfer of Mertk to cultured RCS RPE cells could complement their ingestion defect. We also generated a polyclonal antibody directed against rat Mertk and used it to examine the activation state and subcellular localization of the receptor over a time course of OS phagocytosis by cultured RPE cells.

      DISCUSSION

      We have transferred wild-type Mertk to RCS RPE cells, which we showed lacked Mertk protein, and completely corrected the phagocytic defect of the cells. These results definitively establish the RPE as the site of action of Mertk with respect to OS phagocytosis, as suggested previously (
      • Mullen R.J.
      • LaVail M.M.
      ). Moreover, the fact that Ad-Mertk-infected RCS cells bound and ingested OS at wild-type levels demonstrates that the rest of the phagocytic machinery in RCS RPE is normal and that RCS RPE cells have the same potential as wild-type RPE for OS internalization. Reported biochemical abnormalities of RCS RPE, such as increased calcium membrane conductance and altered cAMP and inositol phosphate second messenger metabolism (
      • Strauss O.
      • Stumpff F.
      • Mergler S.
      • Wienrich M.
      • Wiederholt M.
      ), are likely secondary to the loss of Mertk function.
      Mertk did not affect the binding phase of phagocytosis in primary RPE cell culture; modest overexpression of the protein (m.o.i. = 2) in RCS RPE and wild-type RPE cells did not significantly increase OS binding. These results are consistent with previous reports indicating that αvβ5 integrin is a major OS binding receptor for RPE in cell culture (
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      ,
      • Lin H.
      • Clegg D.O.
      ). However, αvβ5 integrin cannot be essential for retinal structure and function because mice with a targeted disruption of the β5 gene have normal retinal anatomy and electroretinography at 1 and 4 months of age,
      J. Duncan, X. Huang, M. M. LaVail, D. Sheppard, and D. Vollrath, unpublished observations.
      despite the fact that disc shedding and phagocytosis begin around P12. The apparent discrepancy between the role of αvβ5integrin in cell culture and in vivo may result from physical differences in the process of RPE phagocytosis in these two settings. In vivo, OS are closely apposed to RPE microvilli, whereas in cell culture, purified OS are suspended in culture medium and added to cells. Thus, OS binding in cell culture may not be relevant to, or may be substantially different from, the normalin vivo OS phagocytic process. By contrast, Mertk is required for RPE phagocytosis of OS both in vivo (
      • Vollrath D.
      • Feng W.
      • Duncan J.
      • Yasumura D.
      • D'Cruz P.M.
      • Chappelow A.
      • Matthes M.T.
      • Kay M.A.
      • LaVail M.M.
      ) and in cell culture (the present study).
      We generated a polyclonal antibody suitable for immunoblotting and demonstrated that the antibody specifically recognizes 190–200- and 150–160-kDa forms of Mertk in the RPE and assorted other tissues. These sizes are significantly larger than the molecular weight predicted on the basis of the rat primary amino acid sequence. We found that a large majority of the excess molecular weight is due to the presence of N-linked oligosaccharides and that the two forms arise from differential glycosylation. Both forms of the receptor present in RPE-J cells can be activated by OS (Fig. 7). It therefore appears that both forms are functional and probably localize to the plasma membrane.
      Mertk appears to be an integral component of the phagocytic machinery. During the early stages of RPE phagocytosis, Mertk progressively co-localized with OS. The time course of co-localization matched that of the activation of Mertk, as measured by tyrosine phosphorylation, suggesting that a close association with OS may be required to activate the receptor. Because Mertk only stimulated OS internalization and not binding, the protein must be critical for the ingestion phase. The delayed activation of Mertk is consistent with the observed initial delay in the kinetics of OS ingestion by cultured RPE cells (
      • Hall M.O.
      • Abrams T.
      ,
      • Finnemann S.C.
      • Bonilha V.L.
      • Marmorstein A.D.
      • Rodriguez-Boulan E.
      ,
      • Chaitin M.H.
      • Hall M.O.
      ). By 3 h of incubation, however, substantial OS ingestion has occurred (
      • Hall M.O.
      • Abrams T.
      ), and about 70% of total OS are ingested by 4 h (Fig. 5 A). The fact that nearly all OS were accompanied by punctate Mertk signals at 3 h (Fig. 6) suggests that the receptor becomes internalized with OS as part of the phagosome. Further studies are required to address the turnover of Mertk.
      The requirement for Mertk in both RPE phagocytosis of OS in the RCS rat and macrophage phagocytosis of apoptotic cells in the Merkd mouse (
      • Scott R.S.
      • McMahon E.J.
      • Pop S.M.
      • Reap E.A.
      • Caricchio R.
      • Cohen P.L.
      • Earp H.S.
      • Matsushima G.K.
      ), combined with the general similarities between RPE phagocytosis and the uptake of apoptotic cells by macrophages and other professional phagocytes, suggests that the two processes may share mechanistic features. Activation of Mertk could trigger an intracellular signaling pathway that controls rearrangement of cytoskeletal components necessary for OS or apoptotic cell ingestion. Phosphotyrosine accumulates within actin cups that form immediately beneath the site of apoptotic cell ingestion during macrophage phagocytosis (
      • Leverrier Y.
      • Ridley A.J.
      ). Moreover, apoptotic cell uptake by professional and non-professional phagocytes requires a tyrosine kinase signaling pathway to activate CrkII and Rac (
      • Leverrier Y.
      • Ridley A.J.
      ,
      • Albert M.L.
      • Kim J.I.
      • Birge R.B.
      ). TheCaenorhabditis elegans homologs CED-2 (CrkII) and CED-10 (Rac) are required for engulfment of apoptotic cells, demonstrating an ancient origin to at least part of this pathway (
      • Reddien P.W.
      • Horvitz H.R.
      ). This signaling pathway may also be activated during RPE phagocytosis of OS.
      It is not yet known whether phosphatidylserine plays a key role in the recognition of OS by RPE cells, as it does in the recognition of apoptotic cells by macrophages (
      • Fadok V.A.
      • Voelker D.R.
      • Campbell P.A.
      • Cohen J.J.
      • Bratton D.L.
      • Henson P.M.
      ). It is interesting that annexin V binds avidly to purified rat OS, indicating that phosphatidylserine is exposed on the outside.
      W. Feng and D. Vollrath, unpublished observations.
      A secreted ligand of Mertk, Gas6 (
      • Chen J.
      • Carey K.
      • Godowski P.J.
      ,
      • Nagata K.
      • Ohashi K.
      • Nakano T.
      • Arita H.
      • Zong C.
      • Hanafusa H.
      • Mizuno K.
      ), binds phosphatidylserine (
      • Nakano T.
      • Ishimoto Y.
      • Kishino J.
      • Umeda M.
      • Inoue K.
      • Nagata K.
      • Ohashi K.
      • Mizuno K.
      • Arita H.
      ) and may serve as a bridge between OS and Mertk at the RPE plasma membrane during phagocytosis, as we suggested previously (
      • D'Cruz P.M.
      • Yasumura D.
      • Weir J.
      • Matthes M.T.
      • Abderrahim H.
      • LaVail M.M.
      • Vollrath D.
      ). Consistent with this model, Hall and colleagues (
      • Hall M.O.
      • Prieto A.L.
      • Obin M.S.
      • Abrams T.A.
      • Burgess B.L.
      • Heeb M.J.
      • Agnew B.J.
      ) recently reported that Gas6 stimulates phagocytosis of exogenous OS by rat primary RPE cells. It will be of great interest to determine whether Gas6 plays a key role in RPE phagocytosis and/or internalization of apoptotic cellsin vivo.
      In summary, we have demonstrated that Mertk is an integral component of the RPE phagocytic process in cell culture, in which it probably functions to trigger ingestion of bound OS. Future studies on the interaction of Mertk with upstream and downstream proteins will help to elaborate the molecular mechanism of RPE phagocytosis. The common requirement for Mertk in uptake of apoptotic cells by professional phagocytes and OS phagocytosis by RPE indicates that elucidation of this mechanism may have general implications.

      Acknowledgments

      We thank Jessica Weir for DNA sequencing and Nancy Lawson and Dean Cruz for help with the animals. We also thank Dr. Silvia Finnemann for detailed instructions for RPE-J culture.

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