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J. Biol. Chem., Vol. 277, Issue 19, 17016-17022, May 10, 2002
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From the
Received for publication, August 16, 2001, and in revised form, January 22, 2002
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. 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)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 anti-receptor 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 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),
likely a complete loss-of-function, or null, allele. Mertk was an appropriate candidate for rdy in light of evidence
that a signaling defect might underlie the RCS RPE phagocytic phenotype (19-21). 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 (22). Subsequently, in vivo genetic complementation
of the RCS phenotype by viral mediated gene transfer conclusively
demonstrated that Mertk is the gene for rdy
(23).
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 of
Mertk was indeed the RPE, as suggested by genetic chimera
experiments (24) 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.
Primary RPE Culture and OS Isolation--
All tissue culture
reagents were purchased from Invitrogen. Primary RPE cells were
isolated essentially as described (25) from pigmented
RCS-p+ and wild-type congenic
RCS-rdy+p+ rats at P6 to
P9. The RPE cells were plated on 4-well Lab-Tek chamber slides (Nunc,
Naperville, IL) for phagocytic assays or on cell culture dishes
(Corning Glass) for protein studies. The cultures were maintained in
low glucose Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal bovine serum and used after the cells grew confluent. OS were
isolated from P30 Sprague-Dawley rats (Simonsen Laboratories, Gilroy,
CA) as described (15), and the purified OS were stored in 1× BSS (10 mM HEPES, 137 mM NaCl, 5.36 mM KCl,
0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM
MgSO4, 1.27 mM CaCl2, pH 7.4)
containing 5% sucrose at RPE-J and NRK-49F Cell Culture--
The rat RPE cell line RPE-J
and the rat kidney fibroblast cell line NRK-49F were obtained from
American Type Culture Collection. RPE-J cells were cultured as
described (26), and NRK-49F cells were maintained as recommended by the company.
Generation of a Polyclonal Antibody against Rat Mertk--
A DNA
fragment encoding the 103 C-terminal amino acids of rat Mertk was
inserted into pGEX-1 (AMRAD Corp. Ltd., Australia) and transformed into
bacterial strain BL21(DE3)pLysS (Invitrogen, Carlsbad, CA) to produce a
glutathione S-transferase fusion protein. The fusion protein
was purified from crude bacterial lysate by affinity chromatography on
immobilized glutathione (Sigma) and injected into rabbits to generate
polyclonal antibodies.
Recombinant Adenovirus Infection of Primary RPE, RPE-J, and
NRK-49F Cells--
A recombinant adenovirus containing the complete
open reading frame of rat Mertk driven by a cytomegalovirus promoter
was constructed as described (23). To deliver Mertk into RPE cells, Ad-Mertk was added to confluent RPE cultures at various
multiplicities of infection (m.o.i.) and incubated for 42 h at
37 °C. A recombinant Ad-GFP (a gift from Dr. Yongjian Wu,
Stanford University) was used as a control. RPE-J cells were grown on
Matrigel (Fisher) for 6 days and then infected with Ad-Mertk
at an m.o.i. of 2 for 42 h at 32 °C. NRK-49F cells were
incubated with Ad-Mertk or Ad-GFP at an m.o.i. of
10 for 42 h at 37 °C.
Immunoblotting--
Wheat germ agglutinin (WGA)-enriched
samples, deglycosylated samples, immunoprecipitation samples, and whole
cell lysates were separated by 6 or 7.5% SDS-PAGE and transferred to a
nitrocellulose membrane (Fisher). Mertk protein was detected by the
C-terminal polyclonal antibody (1:1,000 dilution) followed by
horseradish peroxidase-conjugated secondary antibody and the Super
Signal West Pico Chemiluminescent Substrate system (Pierce). Pre-immune serum was used as a negative control. A polyclonal antibody directed against the ectodomain of murine Mertk (R & D Systems, Minneapolis, MN), which cross-reacts weakly with rat Mertk, was also used for immunoblotting (1:100 dilution). Tyrosine-phosphorylated Mertk was
detected in immunoprecipitation samples by immunoblot with an
anti-phosphotyrosine monoclonal antibody (P-Tyr-100, Cell Signaling, Beverly, MA).
Glycoprotein Enrichment by WGA-Sepharose Beads--
Neural
retina, RPE/sclera, brain, and kidney were dissected from
RCS-p+ and wild-type
RCS-rdy+p+ rats at
P23-P26 and homogenized in 1% Nonidet P-40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM
EDTA, 1% Nonidet P-40, pH 8.0) containing a protease inhibitor mixture
(Roche Molecular Biochemicals). Equivalent growth areas of wild-type
and mutant rat primary RPE cultures were washed three times with
Dulbecco's phosphate-buffered saline and treated with 1% Nonidet P-40
lysis buffer on ice for 20 min before cell lysates were collected.
Tissue homogenates and cell lysates were centrifuged at 14,000 rpm in a
Sorvall Microspin 24S centrifuge for 15 min at 4 °C. Supernatants
were collected and incubated with WGA-Sepharose beads (Sigma) for
3 h at 4 °C with rotation. Beads were washed five times with
1% Nonidet P-40 lysis buffer, and glycoproteins were dissociated from
the beads by boiling for 5 min in 2× loading buffer (0.125 M Tris-HCl, 4% SDS, 20% glycerol, 0.2 M
dithiothreitol, 0.02% bromphenol blue, pH 6.8).
Protein Deglycosylation--
Glycoproteins enriched with WGA
beads were dissociated from the beads in 1× denaturing buffer (New
England Biolabs, Beverly, MA) and treated according to the
manufacturer's instructions with either PNGase F (New England
Biolabs), to remove all forms of the N-linked
oligosaccharides, or with Endo H (New England Biolabs), to cleave the
chitobiose core of high mannose and hybrid form of N-linked oligosaccharides.
Phalliodin Labeling for F-actin in RPE Culture--
Confluent
wild-type primary RPE cells were washed with DMEM and fixed in 4%
paraformaldehyde for 10 min at room temperature. Cell membranes were
permeabilized with 0.5% Triton X-100, and cells were incubated for 30 min with fluorescein phalloidin (Molecular Probes, Eugene, OR) to
detect F-actin by fluorescence microscopy.
Phagocytic Assay--
The ability of RPE cells to phagocytize OS
was measured as reported previously (27). OS were suspended in DMEM
containing 5% fetal bovine serum and 5% sucrose at a concentration of
1 × 107 OS per ml. One ml of OS was added to each
well of a 4-well chamber slide and incubated with adenovirus-infected
or uninfected cells for 4 h at 37 °C. Then unbound OS were
washed away with DMEM, and cells were fixed for 15 min with 4%
paraformaldehyde in phosphate-buffered saline (137 mM NaCl,
3 mM KCl, 5 mM Na2HPO4,
2 mM,KH2PO4, pH 7.4). To
distinguish total and bound OS, samples were divided into two groups.
Each group contained 2 wells of cells. Group 1 was permeabilized with
0.5% Triton X-100 and group 2 remained unpermeabilized. OS were
immunolabeled with anti-rhodopsin monoclonal antibody Rho 4D2 (8)
(kindly provided by Dr. Robert S. Molday), followed by a Texas
Red-conjugated anti-mouse IgG (Molecular Probes). Fluorescent labeling
was observed under a Zeiss fluorescence microscope, and images were
taken from 10-15 random fields (0.036 mm2 per field) for
each group. OS with an estimated diameter of 1 µm or larger were
manually counted. The total number of OS (bound plus ingested) was
obtained from group 1 samples, and the number of bound OS was obtained
from group 2 samples. The number of ingested OS was obtained by
subtracting bound OS from total OS. For each experimental condition,
the assay was repeated at least three times. Results for each condition
were presented as a means ± S.E. A Student's t test
was used for statistical evaluation.
Double Immunolabeling for OS and Mertk--
Confluent wild-type
primary RPE cells were incubated with OS for 1-3 h, and unbound OS
were removed by washing with DMEM. Cells were fixed in 100% ethanol
for 5 min at Immunoprecipitation--
RPE-J cells were incubated with OS
(3 × 107 per ml) or control medium for 1-3 h at
32 °C. The cells were washed twice with cold phosphate-buffered
saline containing 1 mM sodium orthovanadate (Sigma) and
lysed in RIPA buffer (50 mM Tris, 150 mM NaCl,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0)
containing 10 mM sodium orthovanadate and the protease
inhibitor mixture. The cell lysate was centrifuged at 16,000 × g for 10 min at 4 °C, and the supernatant was incubated
with 20 µl of the Mertk C-terminal antiserum overnight at 4 °C.
The antigen-antibody complex was precipitated by protein A-Sepharose
beads (Amersham Biosciences AB), and proteins were dissociated from the
beads by boiling for 5 min in 2× loading buffer.
Mertk Protein Is Absent from the RCS Rat--
Our molecular
genetic data predict that only a 20-amino acid N-terminal peptide of
Mertk can be synthesized in the RCS rat (18). It is therefore likely
that most or all of the protein is missing from RCS tissues. To test
this prediction, we generated a polyclonal antibody directed against
the C terminus of rat Mertk and assessed its specificity by
immunoblotting. As shown in Fig. 1A, anti-Mertk recognized two
bands of ~200 and 150 kDa from rat kidney fibroblasts infected with a
recombinant adenovirus, Ad-Mertk, which expresses rat
Mertk (23). These bands were absent from Ad-GFP-infected or uninfected cells, indicating that the
antibody specifically recognizes Mertk. Pre-immune serum did not detect bands in any of the samples (data not shown).
The C-terminal antibody was used to assess Mertk expression in primary
RPE cultures from RCS or a wild-type, congenic strain, RCS-rdy+. As expected, anti-Mertk recognized a
single band of about 200 kDa from wild-type RPE cells but not from RCS
RPE cells (Fig. 1B). We next used the C-terminal antibody
and a polyclonal antiserum that reacts with the Mertk ectodomain to
examine various RCS and wild-type RCS-rdy+
tissues including neural retina, RPE/sclera, brain, and kidney (Fig.
2). The C-terminal antibody (Fig.
2A) recognized an ~190-kDa band from each of the four
wild-type tissues tested, as well as smaller bands from wild-type
neural retina (~160 kDa) and brain (~150 kDa). None of these bands
was present in any of the RCS samples. The ectodomain antibody (Fig.
2B) recognized the same Mertk bands as the C-terminal
antibody in wild-type tissues, although with lower avidity.
Mertk Glycosylation--
The primary amino acid sequence of Mertk
predicts a protein of about 107 kDa, significantly smaller than the
proteins we detected. There are 14 putative N-linked
glycosylation sites in the ectodomain of rat Mertk
(GenBankTM accession number P57097), and evidence indicates
that human MERTK from embryonic kidney cells is about 200 kDa in size
(28), consistent with our data. To determine whether the different
protein bands we detected with anti-Mertk are due to differential
glycosylation, WGA-enriched protein samples were digested with the
deglycosylase, PNGase F. After PNGase F treatment, the 150-200-kDa
bands disappeared, and a new band of about 120 kDa was detected by the
C-terminal antibody (Fig. 3A).
The size of the new band is slightly larger than the predicted size of
rat Mertk. These results indicate that rat Mertk exists in at least two
glycosylated forms. To determine which of these forms are mature
proteins, WGA-enriched proteins from brain were treated with Endo H. In
the early steps of N-linked oligosaccharide processing in
the endoplasmic reticulum and Golgi, oligosaccharides are Endo
H-sensitive until Golgi mannosidase II removes the last two mannose
residues from the final core of three residues present in a complex
oligosaccharide (29). As shown in Fig. 3B (lanes
1 and 2), the 190-kDa band remained intact after
digestion with Endo H, indicating that the band corresponds to a mature
form of Mertk. By contrast, Endo H treatment resulted in disappearance
of the 150-kDa band and appearance of a smaller band (lane
2). The size of the new band was greater than that of the band
produced by PNGase F treatment (compare lanes 2 and 3). Extended Endo H treatment did not further
reduce the size of the new band. The partial Endo H sensitivity of
the 150-kDa protein indicates that it contains both complex and high
mannose forms of N-linked oligosaccharides.
Mertk Complements the Phagocytic Defect of RCS RPE--
After demonstrating that RCS RPE lacked Mertk protein, we sought to
determine whether transfer of the gene to RCS RPE would complement the
well described OS phagocytic defect in primary cell culture. Rat RPE
cells in culture form a monolayer with polygonal cell morphology (Fig.
4A). We chose
Ad-Mertk as the means of delivering the gene to these cells
because earlier studies had demonstrated that adenovirus efficiently
infects primary RCS RPE (30). As expected, when RCS RPE cells were
incubated with Ad-GFP at a low m.o.i. of 2, almost all were
found to be GFP-positive after 42 h (Fig. 4B).
Similarly, Mertk was delivered into RCS RPE cells with high
efficiency by Ad-Mertk; cells infected at increasing m.o.i.
values expressed proportionally more Mertk protein (Fig. 4C). Even at an m.o.i. of 2, infected cells produced more
Mertk than wild-type RPE.
We assessed the effect of Mertk expression on the phagocytic ability of
RCS RPE by a cell culture assay. RPE monolayers were incubated with
purified rat OS for 4 h. The cells were then washed, fixed, and
either permeabilized with Triton X-100 or not, and OS were detected
with a monoclonal antibody directed against rhodopsin to determine the
number of bound and ingested OS. Bound and ingested OS in RCS RPE,
virus-infected RCS RPE, and wild-type RPE cells were quantified (Fig.
5A). Consistent with the
results of Chaitin and Hall (15), RCS RPE cells bound similar numbers
of OS as did wild-type RPE (p = 0.70) but could not
ingest any of the bound OS. By contrast, wild-type RPE cells ingested
73 ± 8% of total OS. Introduction of Mertk into RCS
RPE dramatically altered the difference in OS ingestion between these
two types of RPE cells (Fig. 5A). Infection of RCS RPE by
Ad-Mertk at an m.o.i. of 2 caused the infected cells to
ingest as many OS as wild-type RPE (p = 0.84). The
number of bound OS was not altered in comparison to uninfected RCS
(p = 0.47) or wild-type RCS-rdy+
cells (p = 0.70). The functional rescue that we
observed is specifically due to Mertk because infection with
Ad-GFP did not result in an increase in OS ingestion
(p = 0.86).
Additional Mertk did not increase OS ingestion in wild-type RPE (Fig.
5A). Cells infected with Ad-Mertk at an m.o.i. of
2 ingested an equal amount of OS as compared with uninfected cells (p = 0.88), despite the higher level of Mertk in
virus-infected cells (Fig. 5B). The slight increase in the
number of bound OS (Fig. 5A) was not significant
(p = 0.34).
Mertk Co-localizes with OS--
The preceding data demonstrate
that the RPE is indeed the site of action of Mertk with
respect to OS phagocytosis. To assess whether Mertk is directly
involved in OS ingestion, we examined the subcellular localization of
the protein during a time course of RPE phagocytosis. Double
immunolabeling of uninfected wild-type RPE cells during the first
3 h of phagocytosis revealed progressive co-localization of Mertk
with OS (Fig. 6). A small number of OS co-localized with punctate Mertk signals after 1 h of incubation (Fig. 6, A-C, arrowheads). The number OS with accompanying
Mertk increased by 2 h (Fig. 6, D-F), and
by 3 h, the patterns of OS staining and punctate Mertk staining
were almost identical (Fig. 6, G-I). These data indicate
that Mertk gradually accumulates around OS with a time course that is
similar to the progressive ingestion of OS (7, 15).
Mertk Phosphorylation during RPE Phagocytosis--
If Mertk is
directly involved in OS ingestion, as suggested by the co-localization
data, then the receptor should be increasingly activated after addition
of OS. To test this hypothesis, we examined the activation state of the
receptor by monitoring the extent of phosphorylated tyrosine residues
in Mertk during the first 3 h of OS phagocytosis. To obtain a
sufficient number of cells, we used RPE-J, a well characterized rat RPE
cell line with the ability to phagocytize OS (12, 26), and infected
them with Ad-Mertk (m.o.i. = 2) to enhance detection of
tyrosine-phosphorylated receptor molecules. After 1 h of OS
incubation, we did not detect activated Mertk by immunoprecipitation
with anti-Mertk and immunoblotting with an anti-phosphotyrosine
monoclonal antibody (Fig. 7). However, after 3 h, two tyrosine-phosphorylated forms of Mertk were readily detected (Fig. 7). Activated Mertk was not observed in the absence of
added OS (Fig. 7). A parallel experiment with uninfected RPE-J cells
yielded similar results, except the signals were much weaker (data not
shown).
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 (24). 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 (31), 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
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 (7, 12,
15). By 3 h of incubation, however, substantial OS ingestion has
occurred (7), and about 70% of total OS are ingested by 4 h (Fig.
5A). 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 (32), 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 (33). Moreover, apoptotic cell uptake by
professional and non-professional phagocytes requires a tyrosine kinase
signaling pathway to activate CrkII and Rac (33, 34). The
Caenorhabditis 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 (35). 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 (36). It is interesting that annexin V
binds avidly to purified rat OS, indicating that phosphatidylserine is
exposed on the outside.3 A
secreted ligand of Mertk, Gas6 (28, 37), binds phosphatidylserine (38)
and may serve as a bridge between OS and Mertk at the RPE plasma
membrane during phagocytosis, as we suggested previously (18).
Consistent with this model, Hall and colleagues (39) 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 cells
in 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.
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.
*
This work was supported by a postdoctoral fellowship from
the Fight for Sight research division of Prevent Blindness America (to
W. F.), by grants from the Ruth and Milton Steinbach Fund and the Karl
Kirchgessner Foundation (to D. V.), by National Institutes of Health
Grants EY01919, EY02162, and EY06842, the Foundation Fighting
Blindness, and the Macula Vision Research Foundation (to M. M. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Research to Prevent Blindness Senior Scientist Investigator.
Published, JBC Papers in Press, February 22, 2002, DOI 10.1074/jbc.M107876200
2
J. Duncan, X. Huang, M. M. LaVail,
D. Sheppard, and D. Vollrath, unpublished observations.
3
W. Feng and D. Vollrath, unpublished observations.
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-
Mertk Triggers Uptake of Photoreceptor Outer Segments during
Phagocytosis by Cultured Retinal Pigment Epithelial Cells*
,
Department of Genetics, Stanford University
School of Medicine, Stanford, California 94305-5120 and the
§ Departments of Anatomy and Ophthalmology, University of
California, San Francisco School of Medicine, San
Francisco, California 94143-0730
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
5 integrin (12-14).
Inhibition of v5 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 (15), indicating that the protein encoded by the
rdy locus is critical, directly or indirectly, for OS uptake.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. All animal procedures adhered to
the Association for Research in Vision and Ophthalmology Resolution on
the Use of Animals in Research.
20 °C. Localization of the remaining OS and Mertk was
examined by immunolabeling with the Rho 4D2 (1:100) and the C-terminal
antibody (1:100) followed by Texas Red-conjugated anti-mouse IgG and
Oregon Green-conjugated anti-rabbit IgG (Molecular Probes).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
An antiserum detects rat Mertk. A,
equal amounts of whole cell lysate from NRK-49F cells uninfected
(lane 1), infected by Ad-GFP (m.o.i. = 10, lane 2), or infected by Ad-Mertk (m.o.i. = 10, lane 3) were resolved by 6% SDS-PAGE and analyzed by
immunoblot with a polyclonal rabbit antiserum directed against the C
terminus of Mertk. B, equal volumes of WGA-enriched
glycoproteins from Nonidet P-40 cell lysates of cultured wild-type
RCS-rdy+ (lane 1) and RCS (lane
2) RPE cells were resolved by 7.5% SDS-PAGE and analyzed as in
A.
View larger version (65K):
[in a new window]
Fig. 2.
Full-length Mertk protein is absent from RCS
tissues. Equal amounts of WGA-enriched glycoproteins isolated from
various wild-type RCS-rdy+ (odd numbered
lanes) and RCS (even numbered lanes) tissues were
resolved by 6% SDS-PAGE and analyzed by immunoblot with the C-terminal
antibody (A) or a polyclonal antiserum directed against the
ectodomain of murine Mertk (B). A single membrane was probed
twice. B, lane 1, two bands are clearly, but
faintly, visible on the original film. The band in lane 6 is
probably an artifact because it is not detected by the C-terminal
antibody and is too large to be the predicted truncated protein.
View larger version (36K):
[in a new window]
Fig. 3.
Two differentially glycosylated forms of
Mertk. A, untreated ( ) or PNGase F-treated (+)
glycoproteins from wild-type RCS-rdy+ neural
retina, RPE/sclera, brain, and kidney were resolved by 7.5% SDS-PAGE
and analyzed by immunoblot with the C-terminal antibody. B,
untreated (lane 1), Endo H-treated (lane 2), and
PNGase F-treated (lane 3) glycoproteins from wild-type
RCS-rdy+ brain tissue were analyzed as in
A.
View larger version (33K):
[in a new window]
Fig. 4.
High efficiency gene delivery to rat
RPE primary cultures by recombinant adenovirus. A,
morphology of rat primary RPE culture highlighted by fluorescein
phalloidin. Bar, 50 µm. B, GFP expression in
binucleate RCS RPE cells infected by Ad-GFP at an m.o.i. of
2. Bar, 50 µm. C, equal amounts of whole cell
lysate from uninfected RCS RPE (lane 1), RCS RPE infected by
Ad-Mertk at an m.o.i. of 2 (lane 2), 5 (lane 3), and 10 (lane 4), and wild-type
RCS-rdy+ RPE (lane 5) were resolved
by 6% SDS-PAGE and analyzed by immunoblot with the C-terminal
antibody.
View larger version (15K):
[in a new window]
Fig. 5.
Mertk complements the OS ingestion defect of
RCS RPE. A, binding and ingestion of OS by uninfected RCS
RPE, Ad-GFP (m.o.i. = 2) or Ad-Mertk (m.o.i. = 2)-infected RCS RPE, and uninfected and Ad-Mertk (m.o.i. = 2)-infected wild-type RCS-rdy+ RPE.
B, equal amounts of whole cell lysates from uninfected ( )
and Ad-Mertk (m.o.i. = 2)-infected (+) wild-type
RCS-rdy+ RPE were resolved by 6% SDS-PAGE and
analyzed by immunoblot with the C-terminal antibody.
View larger version (52K):
[in a new window]
Fig. 6.
Mertk co-localizes with OS during
phagocytosis. Primary wild-type RPE cells were incubated with OS
for 1-3 h. An anti-rhodopsin monoclonal antibody combined with Texas
Red-conjugated anti-mouse IgG was used to label OS (A,
D, G, and J). An anti-Mertk polyclonal
antibody combined with Oregon Green-conjugated anti-rabbit IgG was used
to label Mertk (B, E, and H).
Pre-immune serum was used as a negative control to demonstrate the
specificity of anti-Mertk labeling (K). Single color images
from the same field were merged (C, F,
I, and L). Arrowheads indicate
selected examples of co-localization sites A-F.
Bar, 10 µm.
View larger version (85K):
[in a new window]
Fig. 7.
Outer segments activate Mertk. RPE-J
cells were infected with Ad-Mertk, and 2 days later cells
were incubated with or without exogenous rat OS for 1-3 h. Equal
amounts of immunoprecipitated samples were resolved by 6% SDS-PAGE and
analyzed by immunoblotting with an anti-phosphotyrosine monoclonal
antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v5 integrin is a major OS binding
receptor for RPE in cell culture (12, 14). However,
v5 integrin cannot be essential
for retinal structure and function because mice with a targeted
disruption of the 5 gene have normal retinal anatomy and
electroretinography at 1 and 4 months of
age,2 despite the fact that
disc shedding and phagocytosis begin around P12. The apparent
discrepancy between the role of v5
integrin 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 normal
in vivo OS phagocytic process. By contrast, Mertk is
required for RPE phagocytosis of OS both in vivo (23) and in
cell culture (the present study).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
650-723-3290; Fax: 650-723-7016; E-mail:
vollrath@genome.stanford.edu.
ABBREVIATIONS
-N-acetylglucosaminidase H;
PNGase F, peptide N-glycosidase F;
GFP, green fluorescent
protein.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Bok, D.,
and Young, R. W.
(1979)
in
The Retinal Pigment Epithelium
(Zinn, K. E.
, and Marmor, M. F., eds)
, pp. 148-174, Harvard University Press, Cambridge, MA
2.
Young, R. W.,
and Bok, D.
(1969)
J. Cell Biol.
42,
392-403 3.
Young, R. W.
(1978)
Investig. Ophthalmol. Vis. Sci.
17,
105-116 4.
Bok, D.,
and Hall, M. O.
(1971)
J. Cell Biol.
49,
664-682 5.
Dowling, J. E.,
and Sidman, R. L.
(1962)
J. Cell Biol.
14,
73-109 6.
Mayerson, P. L.,
and Hall, M. O.
(1986)
J. Cell Biol.
103,
299-308 7.
Hall, M. O.,
and Abrams, T.
(1987)
Exp. Eye Res.
45,
907-922[CrossRef][Medline]
[Order article via Infotrieve] 8.
Laird, D. W.,
and Molday, R. S.
(1988)
Investig. Ophthalmol. Vis. Sci.
29,
419-428 9.
Boyle, D.,
Tien, L. F.,
Cooper, N. G,
Shepherd, V.,
and McLaughlin, B. J.
(1991)
Investig. Ophthalmol. Vis. Sci.
32,
1464-1470 10.
Shepherd, V. L.,
Tarnowski, B. I.,
and McLaughlin, B. J.
(1991)
Investig. Ophthalmol. Vis. Sci.
32,
1779-1784 11.
Ryeom, S. W.,
Sparrow, J. R.,
and Silverstein, R. L.
(1996)
J. Cell Sci.
109,
387-395[Abstract] 12.
Finnemann, S. C.,
Bonilha, V. L.,
Marmorstein, A. D.,
and Rodriguez-Boulan, E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12932-12937 13.
Miceli, M. V.,
Newsome, D. A.,
and Tate, D. J., Jr.
(1997)
Investig. Ophthalmol. Vis. Sci.
38,
1588-1597 14.
Lin, H.,
and Clegg, D. O.
(1998)
Investig. Ophthalmol. Vis. Sci.
39,
1703-1712 15.
Chaitin, M. H.,
and Hall, M. O.
(1983)
Investig. Ophthalmol. Vis. Sci.
24,
812-820 16.
Wilt, S. D.,
Greaton, C. J.,
Lutz, D. A.,
and McLaughlin, B. J.
(1999)
Exp. Eye Res.
69,
405-411[CrossRef][Medline]
[Order article via Infotrieve] 17.
Silverstein, R. L.,
Sparrow, J. R.,
and Ryeom, S. W.
(1998)
Exp. Eye Res.
67,
534 18.
D'Cruz, P. M.,
Yasumura, D.,
Weir, J.,
Matthes, M. T.,
Abderrahim, H.,
LaVail, M. M.,
and Vollrath, D.
(2000)
Hum. Mol. Genet.
9,
645-652 19.
Chaitin, M. H.,
and Hall, M. O.
(1983)
Investig. Ophthalmol. Vis. Sci.
24,
821-831 20.
Heth, C. A.,
and Schmidt, S. Y.
(1992)
Investig. Ophthalmol. Vis. Sci.
33,
2839-2847 21.
Heth, C. A.,
and Marescalchi, P. A.
(1994)
Investig. Ophthalmol. Vis. Sci.
35,
409-416 22.
Gal, A., Li, Y.,
Thompson, D. A.,
Weir, J.,
Orth, U.,
Jacobson, S. G.,
Apfelstedt-Sylla, E.,
and Vollrath, D.
(2000)
Nat. Genet.
26,
270-271[CrossRef][Medline]
[Order article via Infotrieve] 23.
Vollrath, D.,
Feng, W.,
Duncan, J.,
Yasumura, D.,
D'Cruz, P. M.,
Chappelow, A.,
Matthes, M. T.,
Kay, M. A.,
and LaVail, M. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
12584-12589 24.
Mullen, R. J.,
and LaVail, M. M.
(1976)
Science
192,
799-801 25.
Chang, C. W.,
Roque, R. S.,
Defoe, D. M.,
and Caldwell, R. B.
(1991)
Curr. Eye Res.
10,
1081-1086[Medline]
[Order article via Infotrieve] 26.
Nabi, I. R.,
Mathews, A. P.,
Cohen-Gould, L.,
Gundersen, D.,
and Rodriguez-Boulan, E.
(1993)
J. Cell Sci.
104,
37-49[Abstract] 27.
Feng, W.,
Lutz, D. A.,
and McLaughlin, B. J.
(1996)
Investig. Ophthalmol. Vis. Sci.
37,
378 28.
Chen, J.,
Carey, K.,
and Godowski, P. J.
(1997)
Oncogene
14,
2033-2039[CrossRef][Medline]
[Order article via Infotrieve] 29.
Alberts, B.,
Bray, D.,
Lewis, J.,
Raff, M.,
Roberts, K.,
and Watson, J. D.
(1994)
Molecular Biology of the Cell
, 3rd Ed.
, pp. 604-605, Garland Publishing, Inc., New York
30.
da Cruz, L.,
Robertson, T.,
Hall, M. O.,
Constable, I. J.,
and Rakoczy, P. E.
(1998)
Curr. Eye Res.
17,
668-672[CrossRef][Medline]
[Order article via Infotrieve] 31.
Strauss, O.,
Stumpff, F.,
Mergler, S.,
Wienrich, M.,
and Wiederholt, M.
(1998)
Acta Anat.
162,
101-111[CrossRef][Medline]
[Order article via Infotrieve] 32.
Scott, R. S.,
McMahon, E. J.,
Pop, S. M.,
Reap, E. A.,
Caricchio, R.,
Cohen, P. L.,
Earp, H. S.,
and Matsushima, G. K.
(2001)
Nature
411,
207-211[CrossRef][Medline]
[Order article via Infotrieve] 33.
Leverrier, Y.,
and Ridley, A. J.
(2001)
Curr. Biol.
11,
195-199[CrossRef][Medline]
[Order article via Infotrieve] 34.
Albert, M. L.,
Kim, J. I.,
and Birge, R. B.
(2000)
Nat. Cell Biol.
2,
899-905[CrossRef][Medline]
[Order article via Infotrieve] 35.
Reddien, P. W.,
and Horvitz, H. R.
(2000)
Nat. Cell Biol.
2,
131-136[CrossRef][Medline]
[Order article via Infotrieve] 36.
Fadok, V. A.,
Voelker, D. R.,
Campbell, P. A.,
Cohen, J. J.,
Bratton, D. L.,
and Henson, P. M.
(1992)
J. Immunol.
148,
2207-2216[Abstract] 37.
Nagata, K.,
Ohashi, K.,
Nakano, T.,
Arita, H.,
Zong, C.,
Hanafusa, H.,
and Mizuno, K.
(1996)
J. Biol. Chem.
271,
30022-30027 38.
Nakano, T.,
Ishimoto, Y.,
Kishino, J.,
Umeda, M.,
Inoue, K.,
Nagata, K.,
Ohashi, K.,
Mizuno, K.,
and Arita, H.
(1997)
J. Biol. Chem.
272,
29411-29414 39.
Hall, M. O.,
Prieto, A. L.,
Obin, M. S.,
Abrams, T. A.,
Burgess, B. L.,
Heeb, M. J.,
and Agnew, B. J.
(2001)
Exp. Eye Res.
73,
509-520[CrossRef][Medline]
[Order article via Infotrieve]
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