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From the The Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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METHODS. Polymerase chain reaction–based site-directed mutagenesis was used to
generate point mutations in the human PDGF 
receptor (PDGFR)
cDNA, which resulted in single amino acid substitutions. These changes
were based on naturally occurring point mutations in the c-kit receptor
tyrosine kinase, which suppresses the function of wild-type c-kit. A
truncated PDGFR was also made, in which the receptor ended just
after the juxtamembrane domain. As with the point mutants, truncated
receptors have been shown to block the action of wild-type receptors.
All the PDGFR mutants were introduced into cells that naturally
express the wild-type receptor, and the PDGF-dependent output of the
resultant cell lines was determined. In addition, the PVR potential of
cell lines expressing the mutant receptors was tested in a PVR rabbit
model.
RESULTS. Although the mutants differed in their ability to suppress PDGF-dependent signaling of the wild-type receptor, each mutant effectively blocked cell cycle progression. When expressed in rabbit conjunctival fibroblasts, a cell line that effectively induces PVR, the mutant receptors blocked PVR to various degrees. The most effective receptor was the truncated mutant.
CONCLUSIONS. These data suggest that the PDGFR plays an important role in
PVR. In addition, these mutant receptors appear to have therapeutic
potential for prevention of this blinding
disease.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Growth factors such as transforming growth factor (TGF)-ß3 4 and platelet-derived growth factor (PDGF)5 6 7 8 9 are believed to play an important role in promoting the events that contribute to PVR. Other growth factors such as hepatocyte growth factor,10 basic fibroblast growth factor, or interleukin-64 8 have also been implicated. We have recently reported that cells unable to respond to PDGF induce PVR poorly and that the PVR potential increases substantially when they are made responsive to PDGF by expression of the PDGF receptor.11 This finding strongly suggests that PDGF is an important growth factor in at least an experimental model of PVR.
PDGF is a potent mitogen for fibroblasts and induces DNA synthesis and
chemotaxis and sometimes serves as a survival factor. Two PDGF genes
have been identified, and they encode the PDGF-A and PDGF-B chains.
Biologically active PDGF is either a homo- or heterodimer; therefore,
there are three kinds of combinations, PDGF-AA, -AB, and -BB. The
receptor for PDGF is a homo- or heterodimer of the and ß
subunits. The receptor subunits differ in their affinity for ligand,
and, hence, the composition of receptor subunits is in part dependent
on the isoform of PDGF. For instance, PDGF-AA only binds to
homodimer, -AB to homo- or ß heterodimer, and –BB to any
subunit combination. In the studies described herein, we focus on the
PDGF receptor (PDGFR), which is a homodimer of the subunits
and can be assembled by any of the three PDGF isoforms. PDGF dimerizes
the PDGFR, leading to activation of the receptor’s tyrosine
activity, which is encoded in the intracellular domain of the receptor.
Activation of the receptor’s kinase is a prerequisite for subsequent
signal relay and biological responses.
The c-kit receptor belongs to the same family of tyrosine
receptor kinases as the PDGFR; and like the PDGFR it has an
extracellular domain, transmembrane domain, juxtamembrane domain, and
tyrosine kinase that is interrupted by a kinase
insert.12
13
14
Single point mutations in c-kit are
responsible for the deficits of W37,
Wv, W42, and
W41 strains of mice. The abnormalities include
white spotting on the skin, infertility, stem cell deficiency, and
anemia. W37 has a substitution of Glu to Lys at
position 582 (juxtamembrane domain); Wv has Met
instead of Thr at 660 (first half of the kinase domain); Asp replaces
Asn at 790 (second half of the kinase domain) in
W42; and W41 has a Val to
Met substitution at 831 (second half of the kinase
domain).15
16
The affected mice were heterozygous for the
mutations, suggesting that the mutant form of c-kit was dominant to the
normal copy of c-kit, which was also expressed. In fact, all the c-kit
receptor mutants have been shown to function as dominant negatives in a
mast cell proliferation assay.15
Mice do not survive when
both c-kit alleles harbor either the W37 or
W42 point mutant.17
The point mutants in c-kit lie in regions of the intracellular domain
that are highly conserved within this class of receptor tyrosine
kinases. When the single amino acid substitution corresponding to
W37 was introduced into Xenopus
PDGFR, the resultant mutant blocked PDGFR-dependent events during
development. Furthermore, this mutant prevented PDGF-dependent tyrosine
phosphorylation of the wild-type (WT) PDGFR when the mutant and WT
receptor were coexpressed.18
These findings suggest that
making the W mutations in the PDGFR would yield a panel of mutants
able to block activation of the WT PDGFR.
A widely used approach to make dominant negative receptor tyrosine
kinase is to truncate the kinase domain. Such a truncated receptor
heterodimerizes with a WT receptor and prevents activation of the WT
receptor. An important distinction between the truncated receptors and
point mutants in the W series is the level of expression needed to
effectively block signaling of the WT receptor. For the PDGFR, the WT
receptor was silenced by a 90-fold overexpression of the truncated
receptor.19
In contrast, a 4-fold overexpression of the
PDGFR point mutant corresponding to W37 was
sufficient to block activation of the WT receptor.18
In
the W mice, even a single copy of the mutant was sufficient to have an
effect, because the W phenotype was seen in heterozygotes. Hence, the W
panel of the receptor mutants may be more potent than truncated
mutants.
Here we generated a panel of PDGFR mutants that either had a single
amino acid substitution, which corresponded to the
W37, Wv,
W42, or W41 c-kit mutants,
or was truncated to eliminate the kinase domain and carboxyl terminus.
Each of the mutants was expressed in cells that naturally express the
PDGFR and proved to block PDGF-dependent entry into S phase. We also
tested the effect of expressing these mutants in rabbit conjunctival
fibroblasts (RCFs), which can induce PVR in rabbit eyes when coinjected
with platelet-rich plasma (PRP).20
Importantly, the cells
with these mutants were impaired to induce PVR.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Polymerase Chain Reaction Mutagenesis
The truncated receptor was generated as follows. The 4.3-kb
NotI/XbaI cDNA fragment containing the /ß
chimeric receptor21
was subcloned into pBlueScript SK
(Stratagene, La Jolla, CA). This construct was cut with
SacII and XbaI, and the liberated cDNA fragment
(which contains all of the ßPDGFR sequence) was discarded. The
remaining fragment was treated with Klenow to blunt-end the DNA and
then religated. The 2.0-kb NotI/SalI PDGFR
fragment was subcloned into
pLXSN2 21
that had also been cut
with this enzyme pair. The protein encoded by this portion of the
PDGFR cDNA includes all of the extracellular, transmembrane, and
juxtamembrane domains and has a stop codon at nucleotide 1972, with no
change in the predicted amino acid sequence. The last amino acid of the
truncated receptor is proline 589, in the sequence "DSRWEFP," and
is near or at the juxtamembrane/kinase domain junction.
Because the protein sequence surrounding the mutated amino acid in the
W37, Wv,
W42, and W41 in c-kit
receptors was highly conserved, we made the corresponding substitution
in the PDGFR. More specifically, Glu to Lys at the position 587 for
W37, Thr to Met at 665 for
Wv, Asp to Asn at 818 for
W42, Val to Met at 858 for
W41 (Fig. 1A
). The mutants were generated using a polymerase chain reaction
(PCR)–based strategy and the template was 18G generated from
18F. The 18F was made by subcloning the 3.5-kb wild-type human PDGFR
cDNA into pBlueScript II SK+ (Stratagene) using
NotI/BamHI site.22
We generated 18G
from 18F by introducing SacII site at 1975, which is a
unique site for this construct.21
The PCR-generated
mutants were subcloned into 18G as an NcoI/SacII
fragment (E587K), as a SacII/StuI fragment
(T665M), or as a StuI/SphI fragment (D818N and
V858M). The sequence of the point mutants and the truncated receptor
was confirmed by sequencing DNA.
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PDGFR cDNAs were subcloned into
pLHDCX2 retroviral vector using the
NotI/SalI site. This vector has a modified
multiple cloning site containing
NotI-BglII-SalI-HindIII
driven by cytomegalovirus promoter. The vector also encodes a
histidinol-resistant gene, which is driven by the long terminal repeat
(LTR) promoter. Purified DNA (25 µg) was transfected into 293 GPG
replication-incompetent retrovirus-producing cells23
using
lipofectamine (GIBCO–BRL) according to the manufacturer’s
instruction, and virus in the supernatant was collected from days 3
through 8. The virus was concentrated by centrifugation at
30,000g at 4°C for 90 minutes and resuspended in TNE
solution (50 mM Tris–HCl, pH 7.8, 130 mM NaCl, 1 mM EDTA), and it was
stored at -70°C until use. To express the mutant receptors, NIH 3T3
cells and RCFs were incubated overnight with the virus harboring
mutated PDGFR or empty vector in the presence of 4 µg/ml of
polybrene in DMEM with 10% FBS. The infected cells were passaged into
new dishes and cultured in DMEM with 10% FBS supplemented with 5 mM
histidinol (Sigma, St. Louis, MO). Mass population of drug-resistant
cells was used in the experiments. Fluorescent based cell
sorting (FACS) analysis of the E587K, T665M, D818N, and V858M mutant
receptor–expressing RCFs indicated a single population of
receptor-expressing cells. Two populations of truncated
receptor–expressing cells were detected by FACS, and the higher
expressing population was sorted and collected, and these cells were
used for the PVR studies shown in Figure 6
.
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Receptors were immunoprecipitated from the soluble fraction with the 27P or 292 antibody. Immune complex was bound to formalin-fixed membranes of Staphylococcus aureus, spun through an EB sucrose gradient, and washed twice with EB, then with PAN (10 mM Pipes, pH 7.0, 100 mM NaCl, 1% aprotinin) + 0.5% NP-40, and, finally, with PAN. The samples were resuspended in PAN before using for kinase assay or Western blot analysis.
Total cell lysates containing 20 µg of protein or receptor immunoprecipitates from 1.0 x 106 cells were resolved in 7.5% SDS—polyacrylamide gel electrophoresis (PAGE) gel under reducing conditions. Proteins were transferred onto Immobilon (Millipore, Bedford, MA). Membranes were blocked using Block (10 mg/ml BSA, 10 mg/ml ovalbumin, 0.05% Tween-20, dissolved in Western Rinse; 8 mM Tris-HCl, 2 mM Tris-base, pH 7.5, 150 mM NaCl) for anti-phosphotyrosine blotting. The membranes were blocked in Blotto (10 mg/ml nonfat dry milk, 0.05% Tween-20 in Western Rinse) for other blotting. Membranes were incubated with primary antibodies for 1 hour at room temperature and washed 5 times with Western Rinse. Consequently, they were incubated with secondary antibody for 1 hour at room temperature and washed 5 times in Western Rinse as well. Finally, all blots were visualized using ECL (Amersham Pharmacia Biotech, Piscataway, NJ).
Antibodies
The 27P antibody is a crude polyclonal rabbit antibody raised
against a glutathione S-transferase (GST) fusion protein, including the
human PDGFR carboxyl terminus (amino acids 951–1089). The 80.8
antibody was raised against a GST-fusion protein, including a portion
of the first immunoglobulin domain (amino acids 52–94) of human
PDGFR. These antibodies recognize the human and mouse PDGFR. The
292 antibody is a mouse monoclonal antibody that specifically
recognizes primate PDGFR; therefore, this antibody does not
recognize the endogenous receptor in NIH 3T3 cells. The Ras
GTP–activating protein (RasGAP) antibody is a crude rabbit antisera
against the SH2-SH3-SH2 of the human RasGAP (69.3). 4G10 and PY20 are
mouse monoclonal anti-phosphotyrosine antibodies, purchased from
Upstate Biotechnology (Lake Placid, NY) or Transduction Laboratories
(San Diego, CA). The phospho-extracellular signal related kinase (Erk)
rabbit polyclonal antibody was purchased from New England Biolabs
(Beverly, MA). For Western blot analysis the following dilutions were
used for each primary antibody: PDGFR, 27P:80.8 (1:1), 1:1000;
anti-phosphotyrosine, 4G10:PY20 (1:1), 1:5000; 69.3, 1:4000;
anti–phospho-Erk, 1:1000. Secondary antibodies were horseradish
peroxidase–conjugated goat anti-rabbit or anti-mouse antibodies
(Amersham Pharmacia Biotech) diluted 1:5000.
In Vitro Kinase Assay
Mutant PDGFRs were selectively immunoprecipitated with the 292
antibody, and samples representing 2 x 105
cells were subjected to an in vitro kinase assay. Immunoprecipitates
were preincubated with 2 µg of GST protein for 10 minutes at 0°C,
then 2 µg of GST–phospholipase C (PLC)-
, 10 µCi of
-[32P] ATP (DuPont–NEN Research Products,
Boston, MA), and universal kinase buffer (20 mM Pipes, pH 7.0, 10 mM
MnCl2, 20 µg/ml aprotinin) were added. The
samples were incubated at 30°C for 5 minutes, the proteins were
separated by 7.5% SDS–PAGE, the gel was dried, and the radiolabeled
protein was detected by autoradiography.
DNA Synthesis Assay
NIH 3T3 cells were trypsinized, resuspended in DMEM with 10% FBS,
and plated at a density of 5 x 105
cells/well in a 24-well tissue culture plate and cultured overnight.
The cells were rinsed twice with phosphate-buffered saline (PBS), 0.5
ml of DMEM with 0.1% FBS and 0.4 mg/ml of BSA (Sigma) was added, and
the cells were incubated for 48 hours. The cells were then exposed to
50 ng/ml of PDGF-AA, 10% FBS (vol/vol), or buffer for 22 hours, after
which time the cells were pulsed for 4 hours in DMEM with 10% FBS
containing 0.8 µCi/ml of [3H]-thymidine
(DuPont–NEN Research Products). Finally, the cells were washed twice
with PBS, washed once with 5% trichloroacetic acid, and lysed in 250
µl of 0.25 N NaOH. The lysates were transferred into scintillation
tubes containing 50 µl of 6 N HCl, and then 3 ml of
scintillation fluid (ICN Biochemical) was added. The
incorporated radioactivity was quantitated in a scintillation counter
(Packard, Meriden, CT). The data were expressed as fold induction,
which was calculated by dividing stimulated samples by the buffer
control. Each condition was assayed in triplicate, and the mean ±
SD was obtained.
Rabbit Model for PVR
PVR was induced in the rabbit eyes as previously
described.20
Briefly, gas vitrectomy was performed by
injecting 0.4 ml of perfluoropropane
(C3F8) into the vitreous
cavity 4 mm posterior to the corneal limbus under anesthetic
conditions. Three days later, the rabbits were anesthetized and the
pupils were dilated. Then 0.1 ml DMEM containing 1 x
105 of RCFs expressing empty vector or PDGFR
mutant was injected into the vitreous cavity together with 0.1 ml of
PRP using a 30-gauge needle. Ten rabbits underwent surgery for each
group, with the cells expressing the empty vector or the truncated,
E587K, T665M, D818N, V858M PDGFR mutant. One rabbit in the D818N
group died just after the surgery; therefore, the total number was 9
for this group. The retinal status was evaluated with an indirect
ophthalmoscope fitted with a +30 D fundus lens at days 1, 4, 7, 14, 21,
and 28 after the surgery. The PVR was graded according to the
Fastenberg score from 0 through 5.25
All surgeries were
performed under aseptic conditions and pursuant to the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research. Only the left
eye of each rabbit was used for the experiments.
Statistical Analysis
To determine whether the differences among groups of rabbits were
statistically significant, we performed the Mann–Whitney U test for
nonparametric ordinal data. The response of rabbits injected with empty
vector–expressing cells were compared with the response of those
injected with mutant receptor–expressing cells. In all cases,
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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PDGFR MutantsPDGFR
mutants capable of blocking the output of the WT PDGFR. To this end,
we made the five mutants shown in Figure 1A
. All five were expressed in
NIH 3T3 cells using the replication-incompetent retroviral approach
described in the Methods section. NIH 3T3 cells were chosen as the cell
line for the characterization studies because it is a
well-characterized cell line that responds mitogenically to PDGF-AA
(see Fig. 4
below). A Western blot analysis of total cell lysates using
a combination of PDGFR antibodies that recognizes both endogenous
and introduced receptors indicated that each of the mutants were
expressed two- to fourfold above the level of the endogenous WT
PDGFR (Fig. 1B)
. The smaller size of the truncated receptor is
expected, because it lacks the kinase domain and carboxyl terminus.
Because only one of the antibodies recognizes the truncated receptor
(it lacks the 27P epitope), the level of expression of this receptor
may be underestimated. We conclude that all five of the mutant
receptors were successfully constructed and stably expressed.
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PDGFR, which is an early required event in the signaling
cascade. Cells expressing an empty vector or the PDGFR mutants were
arrested by serum deprivation and then left resting or stimulated with
PDGF-AA for 5 minutes. The cells were lysed and the receptors were
immunoprecipitated using 27P, an antibody that recognizes both the
introduced mutant and endogenous WT receptors, except for the truncated
receptor which is lacking the epitope seen by this antibody.
Anti-phosphotyrosine Western blot analysis of these samples indicated
that PDGF triggered the expected increase in the phosphorylation
content of the WT receptor (Fig. 2A
, lane "EMP"). In contrast, the WT receptor was poorly
phosphorylated in cells expressing the truncated receptor, even though
there were comparable amounts of WT receptor recovered in the
"TRUNC" and "EMP" samples (Fig. 2A)
. The phosphotyrosine signal
of the immunoprecipitated receptor was not inhibited in any of the
other cells. This is probably at least in part because some of these
mutants retain kinase activity (see Fig. 3 ) and were immunoprecipitated with the 27P antibody.
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PDGFR, as well as many other
receptors. Indeed, stimulation of the control cells (EMP) resulted in
enhanced phosphorylation of Erk (Fig. 2B)
, which is a commonly used
indicator of activation. Similar to the effect on tyrosine
phosphorylation of the receptor, expression of the truncated receptor
greatly diminished PDGF-dependent activation of Erk (Fig. 2B)
. Cells
expressing the D818N mutant also failed to fully activate Erk in
response to PDGF, whereas Erk activation in cells expressing the other
point mutants was unaffected or even enhanced in some experiments (Fig. 2B
and data not shown). We conclude that the truncated receptor
efficiently blocks PDGF-dependent signaling events, whereas the point
mutants either had no effect or effects were only partially inhibited.
To better characterize the kinase activity of the point mutants we
immunoprecipitated them using the 292 monoclonal antibody, which
selectively recognizes an extracellular epitope of the introduced
receptor and recognizes all the mutants used in this study. Although
PDGF-stimulation is expected to dimerize mutant and WT receptors,
lysing cells in RIPA buffer breaks receptor dimers.26
Consequently, 292 immunoprecipitates are not expected to contain a
coimmunoprecipitating WT receptor. NIH 3T3 cells expressing the empty
vector or receptor mutants were left resting or stimulated with 50
ng/ml of PDGF-AA for 5 minutes, the cells were lysed in RIPA buffer,
and the resultant samples were subjected to anti-phosphotyrosine and
anti-PDGFR Western blot analyses. In this series of experiments, we
included the previously described F cell line, which is an NIH
3T3–like cell line that expresses the introduced human
PDGFR.11
This cell line was included as a positive
control, because the WT receptor in NIH 3T3 cells is mouse, and not
recognized by the 292 antibody. As expected, the PDGFR was
immunoprecipitated from the F cells, but not the NIH 3T3 cells
expressing the empty vector, and PDGF stimulation increased the
phosphotyrosine content of the receptor (Fig. 3A) . PDGF promoted
tyrosine phosphorylation of three of the four point mutants. There was
no detectable basal or PDGF-stimulated tyrosine phosphorylation of the
truncated and D818N PDGFR receptor mutant.
We also tested the ability of immunoprecipitated receptors to
phosphorylate an exogenous substrate. The WT PDGFR phosphorylated
the exogenous substrate and there was a modest enhancement of this
activity when the receptor was immunoprecipitated from PDGF-stimulated
cells (Fig. 3B)
. In contrast, the substrate was not phosphorylated by
the truncated or D818N receptor. The kinase activity of the E587K
mutant was comparable to the WT receptor, whereas the T665M and V858M
mutants were more active than the WT receptor. We conclude that the
truncated and D818N mutants appear to be kinase dead and that the E587K
is comparable to WT, whereas T665M and V858M are activated as kinases.
Note that the behavior of these PDGFR mutants is similar, although
not identical, to that of the analogous c-kit receptor mutants, in
which kinase activity was lowest in W42
(corresponds to D818N) and W37 (E587K),
Wv (T665M) was intermediate, and
W41 (V858M) was the best, although still below
the WT levels.15
PDGFR Mutants Inhibit PDGF-AA–Dependent DNA Synthesis
The next question we addressed is whether these mutants inhibit
PDGF-dependent biological responses. We focused on cell cycle
progression, which can readily be monitored in NIH 3T3 cells in a DNA
synthesis assay. Cells were plated in 24-well dishes, arrested by serum
deprivation, then tested for their ability to enter the S phase after
exposure to PDGF. As shown in Figure 4
, cells expressing an empty vector responded to PDGF-AA, and the
magnitude of the response was typically at least 50% of the response
seen when cells were stimulated with 10% FBS. In all the other cell
lines PDGF-AA failed to induce a robust response, whereas each of the
cell lines did respond normally to serum. The somewhat elevated
response to serum in the V858M cells was not routinely observed. We
conclude that expression of the mutant receptors selectively blocks
PDGF-dependent cell cycle progression, whereas it has little effect on
the response of these cells to serum.
PDGFR Mutants Attenuated Experimental PVR in Rabbits
Data presented thus far indicated that the mutant receptors were
capable of blocking PDGF-dependent cellular responses such as cell
cycle progression. Consequently, we wanted to test whether they could
prevent PVR, which we have recently found is dependent on PDGF in a
rabbit model of the disease.11
To avoid species variables,
we switched from the mouse NIH 3T3 cell line to primary RCF, which has
previously been used extensively with this PVR model.
An empty vector or each of the PDGFR mutants was introduced into
fourth passage RCF, and mass populations of drug-resistant cells were
obtained. Western blot analysis of the resultant cell lines indicated
that the cells do indeed express endogenous PDGFR, and the
introduced receptor was expressed at a 6- to 10-fold higher level (Fig. 5)
. The truncated receptor was expressed at least 30-fold over the
endogenous receptor. Because mass populations of the cells were used,
we also tested the heterogenicity of the population with respect to
receptor expression. FACS analysis, using the 292 monoclonal antibody,
indicated that there was a single population of receptor-expressing
cells for all the samples, except the truncated receptor. In this case,
there were two populations, and we sorted the cells to obtain a single
population of high expressors, which were used in the analysis shown in
Figure 5 . These experiments show that the PDGFR mutants were
expressed in RCF and that the cells used in our experiments were
homogenous with respect to receptor expression.
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PDGFR mutants we performed the following
experiments. For each of the six experimental groups, 9 to 10 rabbits
were first subjected to gas compression vitrectomy and then 1 x
105 cells were coinjected with 0.1 ml of PRP. The
PVR grade was evaluated on days 1, 4, 7, 14, 21, and 28. Under these
experimental conditions PVR is induced rapidly, such that 30% of the
rabbits injected with cells expressing the empty vector underwent total
retinal detachment (stage 5) by day 4. By day 7, 100% of the rabbits
in the control group had reached the most severe form of the disease.
In contrast, PVR was less severe in all the experimental groups (Fig. 6
, top panel) at day 7. At this time point the truncated receptor
appeared to be the best in preventing PVR. The experiment was extended
for 3 more weeks, during which time PVR worsened in all experimental
groups. However, in all cases there was a statistically significant
difference in severity of the disease between the animals injected with
cells expressing PDGFR mutants versus cells of the control group. As
with the earlier time point, the truncated receptor was the best in
preventing PVR. None of the rabbits in this group achieved stage 5, and
80% remained at stage 1 or below. We conclude that the mutant
PDGFRs are capable of significantly attenuating experimental PVR. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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PDGFR
mutants. Although the mutants differed in their intrinsic kinase
activity and potential to prevent PDGF-dependent signaling, they were
all effective in blocking PDGF-stimulated cell cycle progression.
Furthermore, these receptor mutants were able to prevent PVR, and the
truncated receptor was the most effective. One reason why the truncated receptor was more effective in blocking PVR might be because it was expressed to a higher level than the other receptors. To investigate this possibility we compared PDGF-dependent signaling in cells expressing high or low levels of the point mutants. Unlike the truncated receptor, increasing the expression level of the point mutants did not further block PDGF-dependent responses (authors’ unpublished observations, July 1999). These findings suggest that the higher level of expression of the truncated receptor may not be the reason it was the most effective in blocking PVR. The key difference may relate to the step in signaling cascade at which the mutant receptors inhibit. The truncated receptor seems to block at a very early step, whereas the point mutants appear to have an effect further downstream in the signaling cascade.
The mechanism by which the truncated PDGFR inhibits DNA synthesis is
thought to be by competitive inhibition of ligand binding to the WT
receptor.27
To be effective, this type of dominant
negative receptor typically needs to be expressed at levels that
greatly exceed the level of the WT receptor. In contrast, mice
heterozygous for the W mutants of c-kit display a
phenotype,15
16
17
indicating that these mutants are
effective when expressed at levels comparable to those of the WT
receptor. This suggests that the point mutants block receptor function
by a mechanism that is different from that of the truncated receptor.
The idea that the truncated and point mutants used in this study
function by different mechanism is further supported by the finding
that some of the point mutants are kinase active, whereas the truncated
receptor is not (Fig. 3) .The mechanism by which the point mutants block
PDGF-dependent cellular responses, as well as the possibility that
there may be more than one mechanism of action within the group of 4
point mutants, is actively under investigation.
Given that a key step in activation of the WT receptor is engaging its
kinase activity, it is a bit puzzling how a kinase active receptor
could have a negative influence on the overall response of a cell to
the growth factor. However, other investigators have also shown that
kinase active receptor tyrosine kinase mutants are dominant negative.
The Y845F mutant of epidermal growth factor receptor (EGFR) has full
kinase activation but inhibits EGF- and serum-dependent
mitogenesis.28
Another example is the thanatophoric
dysplasia (TD) II mutant of fibroblast growth factor receptor (FGFR).
The TDII mutant is constitutively active, and it causes cell cycle
arrest by activating Stat 1 and consequent upregulation of cell cycle
inhibitor p21waf1/cip1.29
The T665M
and V858M PDGFR mutants, which have elevated kinase activity (Fig. 3)
, may be mimicking these ways to inhibit the endogenous
PDGFR-dependent cell cycle progression.
Our data that inhibition of PDGFR can reduce the PVR score strongly
suggest that PDGFR is a critical contributor in this experimental
model of PVR. This is somewhat surprising granted that the RCFs are
likely to have receptors for many growth factors. Furthermore, the PRP
that is coinjected with the RCFs is a rich source of serum growth
factors. The idea that the PDGFR is important for experimental PVR
is consistent with our previous findings, in which a different approach
was used to address this question.11
In this study we
found that the PVR potential of cells that lack PDGFR is low and that
expressing the PDGFR significantly enhances the ability of such
cells to induce PVR. Taken together, our findings make a strong case
for the idea that the PDGFR is contributing to experimental PVR.
One possible mechanism by which the PDGFR promotes the events
culminating in PVR is by driving cell proliferation. However, the
observation that the panel of mutants were equipotent in blocking
PDGF-dependent DNA synthesis (Fig. 4)
, but differed in their ability to
prevent PVR (Fig. 6)
, suggests that there are processes in addition to
cell proliferation that are required for PVR. In fact, other
investigators have found that even irradiated cells, which fail to
proliferate, can induce PVR in animal eyes, provided that a sufficient
number of cells is injected.25
Our studies support those
previous findings that PVR requires more than just cell proliferation.
Further characterization of the panel of PDGFR mutants may help in
identifying the other cellular processes required for PVR.
Other than cell proliferation, what are other cellular processes that
are likely to be contributing to PVR? In humans, it seems that cell
migration is an important component in PVR, because the cells of the
epiretinal membranes have arrived from distant locations. In the
commonly used PVR rabbit that was used in this series of experiments,
cell migration may not be an important issue because the cells are
injected into the final anatomic location. An additional contributing
factor to PVR is likely to be secretion of extracellular matrix (ECM),
which constitutes a major portion of the membrane in experimental and
clinical PVR. Whether the PDGFR is particularly capable of
triggering ECM production is an area of active investigation. In
addition, the PDGFR may be part of a more elaborate network of
growth factors. For instance, TGF-ß is able to promote
PVR,3
4
and TGF-ß upregulates PDGF-AA secretion and
PDGFR activation.30
31
PDGF-AA also has a synergistic
effect on TGF-ß–associated collagen synthesis.32
33
It
may be interesting to investigate the relationship between PDGFR and
TGF-ß, especially in terms of collagen and/or ECM synthesis. Finally,
contraction of the epiretinal membrane or vitreous makes a major
contribution to retinal detachment in later stages of PVR, and PDGF
enhances the contraction of fibroblasts,34
and RPE
cells.35
36
37
The establishment of experimental PVR that is dependent on the PDGFR
and the availability of PDGFR signaling mutants provide an
opportunity to begin to define the signaling enzymes that are involved
with PVR. For instance, an PDGFR mutant that is unable to bind or
activate the Src family of kinases is better than the WT receptor in
the early phase of experimental PVR.38
In the present
study, we found a positive correlation between activation of Erk
signals (Fig. 2)
and PVR scores at 7 days (Fig. 6
; P =
0.035, by Spearman rank correlation test). Thus, in addition to
identifying important cellular responses that contribute to PVR, we are
also actively investigating the signaling enzymes that participate in
these cellular responses and in PVR. Finally, PVR may be a simple model
for other fibrotic diseases such as atherosclerosis and kidney
fibrosis,39
40
and so our PVR findings may shed light on
the cellular process that contribute to other diseases as well.
The present study points to the PDGFR as a target for the prevention
of PVR. Several approaches have been developed to block PDGFR
activation. These include a variety of ways to prevent the ligand from
interacting with the receptor: antibodies to the ligand41
or receptor42
and peptides that compete with the ligand
for binding to the receptor.43
44
Others have focused on
ways to inhibit activation of the receptor’s kinase activity, and
developed antibodies that block receptor dimerization,45
or drugs that inhibit the receptor’s enzymatic activity
(AG1296).46
Elucidation of the signaling enzymes that the
PDGFR uses to promote PVR will further expand the targets and
available drugs to treat PVR. Finally, we believe that gene therapy is
also viable approach to prevent PVR, and our success with the ex vivo
approach described in this article encourages us to try to develop an
in vivo, gene therapy–based treatment for PVR.
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Acknowledgements |
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Footnotes |
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Submitted for publication January 26, 2000; revised April 24, 2000; accepted May 22, 2000.
Commercial relationships policy: N.
Corresponding author: Andrius Kazlauskas, The Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. kazlauskas{at}vision.eri.harvard.edu
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or phosphatidylinositol 3 kinase J Biol Chem 272,9011-9018This article has been cited by other articles:
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