PDGF-induced glycosaminoglycan synthesis is mediated via
phosphatidylinositol 3-kinase
Jason
Liu,
Dora
Fitzli,
Mingyao
Liu,
Irene
Tseu,
Isabella
Caniggia,
Daniela
Rotin, and
Martin
Post
Medical Research Council Group in Lung Development and Lung Biology
Program, Department of Pediatrics, Hospital for Sick Children
Research Institute, University of Toronto, Toronto, Ontario, Canada
M5G 1X8
 |
ABSTRACT |
Platelet-derived
growth factor (PDGF)-BB has been shown previously to increase
glycosaminoglycan (GAG) synthesis but not DNA synthesis in freshly
isolated fetal lung fibroblasts. In the present study, we found that
PDGF-BB also enhanced
35SO4
incorporation into the small, soluble proteoglycan biglycan without
affecting biglycan's core protein mRNA expression, suggesting that
PDGF-BB mainly affects GAG chain elongation and/or sulfation. PDGF-BB-stimulated GAG synthesis was abrogated by tyrphostin 9, a PDGF
receptor-associated tyrosine kinase inhibitor, implying that the
stimulatory effect is mediated via the PDGF
-receptor (PDGFR). The
intracellular signal transduction pathways that mediate PDGF-BB-stimulated GAG synthesis in fetal lung fibroblasts were investigated. On ligand-induced tyrosine phosphorylation, PDGFR associated with phospholipase C (PLC)-
1, Ras GTPase
activating protein (RasGAP), and phosphatidylinositol 3-kinase (PI3K)
but not with the Syp-growth factor receptor-bound protein 2-Son of Sevenless complex. Association of PDGFR with PLC-
1 and
RasGAP followed by their tyrosine phosphorylation failed, however, to activate PLC-
1, protein kinase C (PKC), and Ras. Neither a PLC-
inhibitor, U-73122; a PKC inhibitor, calphostin C; nor a
mitogen-activated protein kinase kinase inhibitor, PD-98059, inhibited
PDGF-BB-induced GAG synthesis. In contrast, PDGF-BB stimulation
triggered PDGFR-associated PI3K activity. Both PDGF-BB-induced PI3K
activation and GAG synthesis were abolished by the PI3K
inhibitors wortmannin and LY-294002. The results suggest that PI3K is a
downstream mediator of PDGF-BB-stimulated GAG synthesis in fetal rat
lung fibroblasts.
platelet-derived growth factor; deoxyribonucleic acid synthesis; fetal lung fibroblasts
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INTRODUCTION |
PLATELET-DERIVED GROWTH FACTOR (PDGF) is a dimer of two
distinct but related polypeptide chains (A and B) that assemble as a
heterodimer, PDGF-AB, or as a homodimer, PDGF-AA or PDGF-BB (20). PDGF
exerts its biological effect via specific high-affinity cell-surface
receptors. There are two PDGF receptor subunits,
and
, that
dimerize after PDGF binding. The
-receptor binds only
PDGF-BB with high affinity, whereas the
-receptor binds all three
isoforms of PDGF (10). We have previously reported that both homodimers
of PDGF (AA and BB) and both PDGF receptors (
and
) are present
in early embryonic rat lung (18, 19). Also, we reported that rat lung
fibroblasts at late fetal gestation express both PDGF-AA and PDGF-BB
(4). Rat lung epithelial cells at late fetal gestation have PDGF
-
and
-receptors and respond mitogenically to both PDGF isoforms (3,
5). Although fetal lung fibroblasts have PDGF
-receptors (4, 5),
PDGF-BB does not stimulate fibroblast proliferation (5). However, fetal lung fibroblasts respond to PDGF-BB with increased glycosaminoglycan (GAG) synthesis (7). Thus the PDGF-BB signal is transduced in different
physiological responses depending on the lung cell type. These
observations heightened our interest in understanding how the
biological signals of PDGF-BB are intracellularly relayed in both cell
types.
In general, PDGF effects are relayed through a variety of intracellular
signal transduction pathways that are initiated by ligand-induced
receptor dimerization and autophosphorylation. On activation, the
receptor associates with a number of downstream signaling molecules,
including phospholipase C (PLC)-
1 (48), phosphatidylinositol
3-kinase (PI3K) (24, 48), Ras GTPase activating protein
(RasGAP) (25), and the tyrosine phosphatase Syp/SH-PTP2 (14, 30).
Further downstream, protein kinase C (PKC) and Ras are believed to be
important signaling intermediates in PDGF-initiated pathways leading to
biological responses (9, 15, 33).
PDGF-induced signal transduction in mitogenesis has been widely studied
in transformed and immortalized cell lines as well as in PDGF
-receptor (PDGFR)-overexpressing cells, but less is known about the intracellular signaling pathways leading to other biological functions in cells expressing PDGFR at physiological levels.
In the present study, we investigated the signaling pathway by which
PDGF-BB-induced GAG synthesis is relayed in primary cultures of fetal
lung fibroblasts. First, we determined PDGFR activation and subsequent
association of signaling proteins (PLC-
1, RasGAP, PI3K, Syp). To
further study the role of these signaling proteins in PDGF-mediated GAG
synthesis, we then measured their activities after PDGF-BB stimulation
and tested whether specific inhibitors of these proteins could block
PDGF-BB-stimulated GAG synthesis. Our results suggest that PI3K
functions as a downstream mediator of PDGF-BB-induced GAG
synthesis.
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MATERIALS AND METHODS |
Materials.
Female (200-250 g) and male (250-300 g) Wistar rats were
purchased from Charles River (St. Constant, PQ, Canada) and bred in our
animal facility. The sources of all cell culture material have been
described elsewhere (8).
Na2O435S,
[
-32P]ATP, and
[
-32P]GTP were from
ICN Biomedicals (St. Laurent, PQ). Human recombinant PDGF-BB and
antibodies to PDGFR, PLC-
1, RasGAP, PI3K regulatory subunit p85,
growth factor receptor-bound protein 2 (GRB2), the mammalian Son of
Sevenless (Sos) 1, p21ras (Ras),
and phosphotyrosine were purchased from Upstate Biotechnology (Lake
Placid, NY). Antibodies to Syp (SH-PTP2) were from Santa Cruz
Biotechnology (Santa Cruz, CA). Chondroitinase ABC was from Seikagaku
America (Rockville, MD). The 1.7-kb human biglycan cDNA was from Dr. L. W. Fisher (National Institutes of Health, Bethesda, MD). The inositol
trisphosphate (IP3) assay kit
and enhanced chemiluminescence detection reagents were from Amersham
(Oakville, ON, Canada). Phorbol 12-myristate 13-acetate (PMA),
phosphatidylinositol (PI), calphostin C, and streptolysin O were from
Sigma (St. Louis, MO). Wortmannin was purchased from Calbiochem (La
Jolla, CA). Tyrphostin 1, tyrphostin 9, U-73122, U-73343, and
LY-294002 were from Biomol (Plymouth Meeting, PA). PD-98059 was from
New England Biolabs (Beverly, MA). Polyethylenimine cellulose TLC
plates were obtained from Macherey-Nagel (Duren, Germany). Silica
gel-60 plates were from Fisher Scientific (Toronto, ON). NIH/3T3 cells
were from American Type Culture Collection (Rockville, MD).
Cell culture.
Pregnant rats were killed on day 19 of
fetal gestation (term = 22 days) by diethyl ether excess. The fetuses
were aseptically removed from the mothers, and the fetal lungs were
dissected out in cold Hanks' balanced salt solution without calcium
and magnesium [HBSS(
)] and cleared of major airways
and vessels. The lungs were washed twice in HBSS(
), minced, and
suspended in HBSS(
). Fibroblasts were isolated from the fetal
lungs as previously described in detail (8). At subconfluency, fetal
lung fibroblasts, cultured in
75-cm2 tissue culture flasks, were
washed three times with serum-free minimal essential medium (MEM) and
serum starved for 24 h in MEM. Cells were rinsed once with serum-free
MEM and incubated in either MEM or MEM supplemented with PDGF-BB.
Incubation was stopped by removing the medium and washing the cells
three times with ice-cold PBS. For DNA and GAG measurements, fetal lung
fibroblasts were diluted in MEM + 5% (vol/vol) fetal bovine serum
(FBS) to a concentration of 1 × 106 cells/ml. One milliliter of
this cell suspension was seeded in wells of 24-well tissue culture
plates. At subconfluency, fibroblasts were serum starved for 24 h and
then used for DNA synthesis measurements. GAG synthesis was measured in
confluent cultures of fibroblasts that were serum starved for 24 h.
Effect of PDGF-BB on DNA synthesis.
Fetal rat lung fibroblasts were incubated for 24 h with 1 µCi/ml of
[3H]thymidine in
either serum-free MEM or MEM containing 20-50 ng/ml of PDGF-BB.
DNA synthesis was determined by the incorporation of
[3H]thymidine into DNA
as previously described (5, 8).
Effect of PDGF-BB on biglycan
synthesis.
Fetal lung fibroblasts were incubated with 50 µCi/ml of
35SO4
in the absence or presence of 20-100 ng/ml of PDGF-BB. After a 24-h incubation, culture media were collected and dialyzed extensively against distilled water (<3,500) in the presence of 1 mM
phenylmethylsulfonyl fluoride (PMSF). The dialyzed media were then
lyophilized, dissolved in PBS, and incubated for 2 h with or without
chondroitinase ABC (100 mU/ml) at 37°C. After addition of SDS
sample buffer [10% (vol/vol) glycerol, 2%
(wt /vol) SDS, 5% (vol/vol)
-mercaptoethanol, 0.0025% (wt /vol) bromphenol blue, and 0.06 M Tris, pH 8.0],
samples were boiled and radiolabeled proteoglycans were
separated by 5% (wt /vol) SDS-PAGE. Gels were fixed in 10%
(vol/vol) acetic acid in 40% (vol/vol) methanol, prepared for
fluorography by soaking in
EN3HANCE (DuPont), dried, and
exposed to Kodak XAR-5 film using DuPont Cronex intensifying screens.
The films were quantified using an Ultroscan XL laser densitometer
(LKB, Bromma, Sweden).
Effect of PDGF-BB on biglycan mRNA
expression.
After a 24-h incubation of fetal lung fibroblasts with or without 20 ng/ml of PDGF-BB, total RNA was isolated by lysing the cells in 4 M
guanidinium thiocyanate followed by centrifugation on a 5.7 M cesium
chloride cushion to pellet RNA. Total RNA (15 µg) was size
fractionated on 1% (wt /vol) agarose gels containing 3% (vol/vol)
formaldehyde, transferred to Hybond
N+ membranes (Amersham), and
immobilized by ultraviolet cross-linking. The human biglycan (1.7 kb)
cDNA probe was labeled with
D-[
-32P]CTP
using a random-primed labeling system (Amersham). Prehybridization and
hybridization were performed in 50% (wt /vol) formamide, 5× SSPE (1× SSPE is 0.15 M NaCl, 0.01 M
NaHPO4, and 0.001 M EDTA, pH 7.4),
0.5% (wt /vol) SDS, 5× Denhardt's solution, and 100 µg/ml of denatured salmon sperm DNA at 42°C. After hybridization, the blots were washed with 5× SSC (1× SSC is 0.15 M NaCl and
0.015 M sodium citrate, pH 7.0) and 0.2% (wt /vol) SDS at 42°C
for 20 min followed by 0.5× SSC and 0.2% (wt /vol) SDS at
42°C for 10 min and then exposed for 24 h to Kodak XAR-5 film using
DuPont Cronex intensifying screens. The blots were then stripped and, for normalization, hybridized with a rat
-actin cDNA probe.
Inhibition of PDGF-BB-induced GAG
synthesis.
Fetal lung fibroblasts were preincubated for 1 h in serum-free MEM with
or without either 5 µM U-73122, 5 µM U-73343 (32), 0.1 µM
calphostin C (26), 1 µM tyrphostins 1 and 9 (2), 100 µM PD-98059
(37), or the indicated concentrations of wortmannin (47) or LY-294002.
We first assessed the cytotoxicity of these agents for fetal lung
fibroblasts in 24-h culture. Release of [14C]adenine was used
as an indicator of cell injury. The above-mentioned concentrations of
agents had no cytotoxic effect on lung fibroblasts in 24-h culture
experiments. Cells were then incubated for 24 h in serum-free MEM
supplemented with inhibitors and 10 µCi/ml of
35SO4
in the presence or absence of 20 ng/ml of PDGF-BB. Total radiolabeled GAGs (medium+cell layer fraction) were measured as described previously (7).
Immunoprecipitation and Western
blotting.
After incubation with or without PDGF-BB, fibroblasts were scraped in
lysis buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM
MgCl2, 1 mM EGTA, 10% (vol/vol)
glycerol, 1% (vol/vol) Triton X-100, 100 mM sodium fluoride, 10 mM
pyrophosphate, 200 µM sodium orthovanadate, 10 µg/ml of aprotinin,
10 µg/ml of leupeptin, and 1 mM PMSF], sonicated, and
centrifugated at 10,000 g for 15 min at 4°C. Aliquots of cell lysates equalized to 300 µg of protein were precleared by incubation with nonimmune rabbit IgG for 30 min at
4°C, followed by incubation with 10% (vol/vol) Formalin-fixed Staphylococcus aureus Cowan strain A
(Zysorbin) in PBS for another 30 min at 4°C. Specific primary
antibodies were then added to the cleared supernatants for overnight
incubation on an end-to-end rotator at 4°C. Zysorbin was used to
collect the immune complexes, which were extensively washed with lysis
buffer, dissociated by boiling in sample buffer, and subjected to 10%
SDS-PAGE. After electrophoresis, proteins were transferred to a
nitrocellulose membrane. Nonspecific binding was blocked by incubation
with 3% (wt /vol) nonfat milk powder in PBS at 4°C for 60 min.
The membrane was then incubated with designated primary antibodies.
After overnight incubation at 4°C, the membrane was washed three
times with PBS, followed by incubation with horseradish
peroxidase-conjugated goat anti-mouse IgG (1:20,000) or goat
anti-rabbit IgG (1:30,000). After washes with PBS, blots were developed
with an enhanced chemiluminescence detection kit. The films were
quantified with the use of an Ultroscan XL laser densitometer.
IP3 measurement.
Fetal lung fibroblasts treated with or without 20 ng/ml of PDGF-BB were
lysed in 15% (wt /vol) ice-cold trichloroacetic acid, which was
removed by three sequential extractions with diethyl ether. The final
extract was neutralized with 1 M
NaHCO3. Cellular IP3 content was determined using
an IP3 assay kit (Amersham).
PKC activity measurement.
After incubation with or without 20 ng/ml of PDGF-BB, fetal lung
fibroblasts were homogenized by sonication for 15 s in ice-cold 0.2 M
Tris-0.5 mM EDTA-0.5 mM EGTA-25 µg/ml of leupeptin-25 µg/ml of
aprotinin-0.1 M
-mercaptoethanol, pH 7.5. Cytosolic and membrane fractions were isolated by ultracentrifugation, and PKC activity was
measured (31).
Ras (p21ras) assay.
Fetal lung fibroblasts were incubated with or without 20 ng/ml of
PDGF-BB. After 10 min of incubation, Ras activity was assayed as
described by Naberg and Westermark (36). The flasks were rapidly
transferred to ice, and fibroblasts were washed with a salt solution
containing 5.2 mM MgCl2, 131 mM
KCl, 20 mM NaCl, 0.3 µM CaCl2,
1.3 mM EGTA, and 12.5 mM PIPES, pH 7.0. Fibroblasts were permeabilized
with 1 ml of salt solution supplemented with 0.5 U streptolysin O, 100 µM ATP, and 10 µCi/ml of
[
-32P]GTP
on ice for 30 min. The cells were then washed three times with salt
buffer; lysed in 400 µl of 50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM
MgCl2, 1% (vol/vol) Triton X-100,
10 µg/ml of leupeptin, 10 µg/ml of aprotinin, and 1 mM PMSF; and
centrifuged for 3 min at 10,000 g. The
supernatants were transferred to Eppendorf tubes containing 50 µl of
4 M NaCl, 4% (wt /vol) sodium deoxycholate, and 0.4% (wt /vol)
SDS. Monoclonal anti-Ras antibody (1 µg) was added to the samples,
which were then incubated overnight at 4°C. Immunocomplexes were
collected by adding 100 µl of protein G-Sepharose to the samples. The
Ras-bound guanosine nucleotides were eluted from the immunoprecipitates
by incubation with 15 µl of 0.5 mM GTP, 0.5 mM GDP, 2 mM EDTA, 2 mM
dithiothreitol, and 0.2% (wt /vol) SDS for 20 min at 68°C. The
eluates were spotted on polyethylenimine cellulose TLC plates, and
nucleotides were separated using 1.2 M ammonium formate in 0.8 M HCl as
eluent. The TLC plates were exposed to Kodak-XAR film, and GTP and GDP
spots were quantified using an Ultroscan XL laser densitometer (LKB).
PI3K assay.
Fetal lung fibroblasts were incubated for 10 min with or without 20 ng/ml of PDGF-BB. Cells were lysed, and PI3K was immunoprecipitated with either p85, phosphotyrosine, or PDGFR antibodies. PI3K activity was measured as described by Whitman et al. (53). Five microliters of
immunoprecipitates, resuspended in 30 µl of 40 mM HEPES, pH 7.4, were
mixed with 5 µl of 40 mM HEPES and 5 µl of PI liposomes (0.8 mg/ml
in water). The reaction was initiated by adding 5 µl containing 5 µCi of [
-32P]ATP
in 40 mM HEPES, 200 µM ATP, and 20 mM
MgCl2. After a 15-min incubation
at 30°C, the reaction was terminated by adding 100 µl of 1 M HCl.
PI-containing lipids were extracted and separated by TLC on silica gel
G plates impregnated with 60 mM EDTA, 2% (wt /vol) sodium tartrate,
and 50% (vol/vol) ethanol using chloroform-methanol-4 M
NH4OH (9:7:2, vol/vol) as
developing solvent. PI-containing lipid standards
[phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2)]
were visualized with iodine vapor, and radiolabeled lipids were
detected by exposing the TLC plates to Kodak-XAR film.
For PI3K inhibition studies, fetal lung fibroblasts were preincubated
for 1 h with the indicated concentrations of PI3K inhibitor wortmannin
(47). Cells were then stimulated for 10 min with 20 ng/ml of PDGF-BB,
and PI3K activity was assayed as described above. After
autoradiography, radiolabeled PIP spots were scraped from the plates,
transferred to scintillation vials, and counted in a scintillation
counter. Alternatively, PIP spots were quantified using an Ultroscan XL
laser densitometer (LKB).
Statistical analysis.
Unless stated differently, experiments were carried out for a minimum
of three times with materials collected from separate cell
cultures. All values are shown as means ± SE. Statistical analysis
was by Student's t-test or, for
comparison of more than two groups, by one-way analysis of variance
followed by Duncan's multiple range comparison test, with significance
defined as P < 0.05.
 |
RESULTS |
Effect of PDGF-BB on DNA and proteoglycan
synthesis.
To investigate the effects of PDGF-BB on fetal lung fibroblast
proliferation, fibroblasts were first serum starved for 24 h in MEM.
Although these quiescent cells responded to mitogenic stimuli such as
FBS, a 24-h exposure to 0-50 ng/ml of PDGF-BB failed to increase
the [3H]thymidine
incorporation into DNA (Table 1). Under
similar experimental conditions, NIH/3T3 fibroblasts responded
mitogenically to PDGF-BB (Table 1). Because PDGF-BB has been shown to
stimulate GAG synthesis of fetal rat lung fibroblasts without affecting
the composition of individual GAG molecules (7), we then investigated
the effect of PDGF-BB on proteoglycan synthesis. Fetal lung fibroblasts
were radiolabeled with
35SO4
in the presence of various concentrations of PDGF-BB. Media were then
collected and analyzed by SDS-PAGE to identify soluble proteoglycans
(Fig.
1A).
A concentration of 50 ng/ml of PDGF-BB maximally (~4-fold) augmented
the synthesis of the soluble proteoglycan with a relative molecular
mass of 200-250 kDa (Fig.
1A). PDGF-BB also
increased the
35SO4
incorporation into large macromolecules, which remained on top of the
gel. These larger
35SO4-labeled
macromolecules most likely represent the large chondroitin/dermatan (CS/DS) proteoglycan versican, whereas the sulfated small CS/DS proteoglycan (~250 kDa) corresponds to biglycan (6). Both
35SO4-labeled
macromolecules were sensitive to chondroitinase ABC digestion,
consistent with our previous studies (6). Northern analysis revealed
that core protein mRNA expression for biglycan in fetal lung
fibroblasts was not altered by 50 ng/ml of PDGF-BB (Fig.
1B), suggesting that the increase in
35SO4
incorporation into biglycan (Fig.
1A) was not due to increased core
protein gene expression. GAG formation was further used as a biological
marker for studying PDGF-BB-induced intracellular signaling in fetal
rat lung fibroblasts.

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Fig. 1.
Effect of platelet-derived growth factor (PDGF)-BB on biglycan
synthesis by fetal rat lung fibroblasts.
A: cells were incubated for 24 h with
35SO4
in presence of various concentrations of PDGF-BB. Media samples were
dialyzed against distilled water, lyophilized, reconstituted in PBS,
and incubated with chondroitinase ABC (ABC) or left untreated (C).
Proteoglycans were separated on 5% SDS-polyacrylamide gels. A
representative autoradiogram is shown. Position of undigested biglycan
is shown at left. Molecular-mass
marker positions are at right.
B: cells were exposed to 50 ng/ml of
PDGF-BB for 24 h. Total cellular RNA (10 g) was analyzed by Northern
hybridizations using a biglycan cDNA probe. A representative
autoradiogram is shown.
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PDGFR activation.
We first determined whether the increase in GAG production by PDGF-BB
requires tyrosine-phosphorylated PDGF receptors. Fetal lung fibroblasts
were incubated with 1 µM tyrphostin 9, a specific blocker of
intrinsic tyrosine kinase activity of the PDGFR (2), before exposure to
PDGF-BB. Tyrphostin 9 abolished the stimulatory effect of PDGF-BB on
GAG synthesis (see Fig. 2B). Also,
tyrphostin 9 completely blocked PDGF-BB-induced tyrosine
phosphorylation of the PDGF receptors (Fig.
2A). In
contrast, its inactive analog tyrphostin 1 had no such effects. Because
previous studies have shown that fetal lung fibroblasts do not express
the PDGF
-receptor (3, 5), these results indicate that PDGF-BB
exerts its stimulatory effect on GAG synthesis via the
-receptor
(PDGFR). Activation of PDGFR on PDGF-BB stimulation is shown in Fig.
3. We also noticed that the amount of
PDGFR was significantly decreased in PDGF-BB-stimulated cells (Fig.
4A),
implying a rapid internalization and degradation of the ligand-receptor
complex.

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Fig. 2.
Effect of tyrphostin (Tyr) 9 on PDGF-BB-stimulated -receptor ( R)
autophosphorylation and glycosaminoglycan (GAG) formation.
A: fetal rat lung fibroblasts grown to
subconfluence and serum starved for 24 h were stimulated with 20 ng/ml
of PDGF-BB for 5 min in presence (+) or absence ( ) of 1 µM Tyr
9. Cell lysates were prepared and immunoblotted with phosphotyrosine
antibody (anti-PY). Position of R is shown at
left. Molecular-mass standards are at
right.
B: serum-starved cells were incubated
with
35SO4
and 20 ng/ml of PDGF-BB in presence or absence of Tyr 9 or its inactive
analog Tyr 1. After a 24-h incubation, incorporation of
35SO4
into GAGs was determined. * P < 0.05 compared with control value.
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Fig. 3.
Autophosphorylation of PDGF R by PDGF-BB.
A: serum-starved fetal rat lung
fibroblasts were stimulated with various concentrations of PDGF-BB for
5 min and then lysed. Cell lysates, normalized for protein content,
were analyzed by SDS-PAGE followed by immunoblotting with anti-PY and
detection with 125I-labeled
protein A. Position of R is shown at
left. Molecular-mass standards are at
right.
B: serum-starved cells were stimulated
with PDGF (20 ng/ml) for indicated times (5 and 10", 5 and 10 s,
respectively; 1, 10, and 30', 1, 10, and 30 min, respectively).
Time 0 represents unstimulated cells.
Cell lysates were prepared and immunoblotted with anti-PY as in
A.
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Fig. 4.
Phosphorylation and binding of phospholipase C (PLC)- 1, Ras GTPase
activating protein [RasGAP (GAP)], and phosphatidylinositol
3-kinase (PI3K) to autophosphorylated PDGF R. Serum-starved cells
were stimulated with (+) or without ( ) 20 ng/ml of PDGF-BB for 5 min and then lysed. Cell lysates, normalized for protein content, were
immunoprecipitated (Ip) with antibodies against either PDGF R
(anti- R), PLC- , PI3K regulatory subunit p85 (anti-p85), or RasGAP
(anti-GAP). Resulting immune complexes were subjected to immunoblot
analysis (Blot) with anti- R (A)
or anti-PY (B). Phosphorylation of
PLC- was determined by immunoprecipitation with anti-PY followed by
immunoblotting with anti-PLC-
(B). Positions of R, RasGAP, and
PLC- are shown at left.
Molecular-mass standards are at
right.
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Proteins that associate with the PDGF receptor in
fetal lung fibroblasts.
To determine whether PDGF-BB induces an association of PDGFR with known
receptor-binding proteins in primary fetal lung fibroblast cultures,
extracts of resting and PDGF-stimulated fibroblasts were
immunoprecipitated with antibodies against either PLC-
1, RasGAP, or
PI3K regulatory subunit p85. The immunoprecipitates were analyzed by
SDS-PAGE, followed by immunoblotting with anti-PDGFR. PDGF-BB
stimulation promoted the binding of PLC-
1, RasGAP, and p85
to PDGFR (Fig. 4A). Although Syp was
present in the cells, we were unable to detect any binding of Syp to
the activated receptor (Fig. 5). We
confirmed these results by first immunoprecipitating PDGFR with
anti-PDGFR and then immunoblotting with antibodies against either
PLC-
1, RasGAP, p85, or Syp (data not shown). In a control experiment
using NIH/3T3 fibroblasts, which responded mitogenically to PDGF-BB
(Table 1), Syp associated with the activated PDGF receptor, consistent
with previous reports (14). In addition, we investigated
PDGF-BB-induced tyrosine phosphorylation of these receptor-binding
proteins. PDGF-BB stimulation resulted in an increase in tyrosine
phosphorylation of PLC-
1 (Fig.
4B). RasGAP tyrosine phosphorylation
was slightly increased by PDGF-BB (Fig. 4B), whereas PDGF-BB did not trigger
any significant tyrosine phosphorylation of the PI3K regulatory subunit
p85 (Fig. 4B).

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Fig. 5.
Lack of binding of Syp, growth factor receptor-bound protein 2 (GRB2),
and Son of Sevenless (Sos) to autophosphorylated PDGF R.
Serum-starved fetal lung fibroblasts were stimulated with or without
PDGF-BB (20 ng/ml) for 5 min. Cell lysates were prepared and
immunoprecipitated with anti- R. Cell lysates and resulting immune
complexes were subjected to immunoblot analysis with antibodies to Syp,
GRB2, and Sos. Positions of Syp (65 kDa), GRB2 (25 kDa), and Sos (170 kDa) in lysates of unstimulated cells are shown at
left.
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PDGF-BB does not activate the
PLC-
1-PKC pathway in fetal rat lung
fibroblasts.
Although PDGF-BB stimulated tyrosine phosphorylation and binding of
PLC-
1 to the receptor, these events did not trigger any IP3 production in fetal rat lung
fibroblasts (Table 2). PDGF-BB did not
activate PKC either (Table 2). Neither the PLC-
1 inhibitor U-73122
nor the PKC inhibitor calphostin C blocked PDGF-BB-enhanced GAG
synthesis (Fig. 6). A potent activator of
PKC, the phorbol ester PMA also did not stimulate GAG synthesis (data
not shown). These results suggest that neither PLC-
1 nor PKC is
involved in PDGF-stimulated GAG synthesis in fetal lung fibroblasts.

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Fig. 6.
Lack of effect of U-73122 and calphostin on PDGF-BB-induced GAG
synthesis. Serum-starved fetal rat lung fibroblasts were preincubated
for 1 h with or without 5 µM U-73122, U-73343 (an inactive analog of
U-73122), or calphostin before stimulation with 20 ng/ml of PDGF-BB.
GAG synthesis was measured as described in MATERIALS
AND METHODS. Data are means ± SE of 4 separate
experiments carried out in quadruplicate. Open bars, unstimulated;
solid bars, PDGF-BB stimulated.
* P < 0.05 compared with
unstimulated cells (1-way analysis of variance followed by Duncan's
multiple range test).
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PDGF-BB does not activate Ras in fetal lung
fibroblasts.
We examined the binding of the GRB2-Sos complex to PDGFR in fetal lung
fibroblasts. Although both components were present in the cells, we
were unable to detect any binding of GRB2 or Sos to the activated
receptor (Fig. 5). As mentioned in Proteins that
associate with the PDGF receptor in fetal lung
fibroblasts, no association of Syp with
PDGFR was observed after PDGF-BB stimulation (Fig. 5). Under similar
experimental conditions, however, both GRB2 and Sos associated with the
PDGF receptor after PDGF-BB exposure in NIH/3T3 fibroblasts (not
shown). To examine whether Ras is along the signal transduction pathway
for PDGF-BB-mediated GAG synthesis, we tested the activity of Ras on
PDGF-BB stimulation (36). Fetal lung fibroblasts were incubated with or
without 20 ng/ml of PDGF-BB and subsequently permeabilized in the
presence of
[
-32P]GTP. Cell
extracts were immunoprecipitated with a monoclonal anti-p21ras antibody, and the
Ras-bound guanosine nucleotides were analyzed by TLC. In preliminary
experiments, we tested the specificity of the Ras antibody and found
that the antibody recognized a single protein of 21 kDa (data not
shown). A 10-min preincubation with PDGF-BB did not increase the amount
of labeled GTP and GDP in the anti-Ras immunoprecipitates [1.08 ± 0.37-fold increase in Ras-bound nucleotides (GTP+GDP) over
control; n = 3 separate experiments in
duplicate], suggesting that Ras activity was not increased on
PDGF-BB stimulation
(Fig.7A,
lanes 1 and
2 vs. lanes
3 and 4). Under
similar experimental conditions, PDGF-BB increased Ras-bound guanosine
nucleotide binding [2.05 ± 0.18-fold increase in Ras-bound nucleotides (GTP+GDP) over control; n = 3 separate experiments in duplicate] in NIH/3T3 fibroblasts
(Fig. 7B). PDGF-BB caused a parallel
increase in both Ras-GTP and Ras-GDP and no relative increase in
Ras-GTP. This response is consistent with PDGF-activated nucleotide
exchange via Sos. When we analyzed the radiolabeled lysates of fetal
lung fibroblasts after the 30 min of incubation at 4°C, it appeared
that >50% of the added radioactive GTP was hydrolyzed to GDP without
any detectable formation of GMP. The hydrolysis of
[
-32P]GTP
was not significantly affected by PDGF-BB (Fig.
7A, lanes 5 and
6 vs. lanes 7 and
8). Similar hydrolysis results were obtained with
NIH/3T3 cells. Although we did not determine whether the formed Ras-GDP
in the 3T3 cells originated from binding of GDP to Ras or from
hydrolysis of bound GTP, previous studies with Swiss 3T3 cells have
shown that PDGF activates an unidirectional exchange mechanism (36). To
confirm that PDGF-BB stimulation of fetal lung fibroblasts did not
activate Ras, we tested the phosphorylation of the Raf-1
serine/threonine kinase (53), previously reported to be a downstream
effector of Ras (51). PDGF-BB has been shown to phosphorylate Raf-1
(41), and Raf-1 has been linked to Ras-GTP and mitogen-activated
protein (MAP) kinase complexes (34). In keeping with our negative Ras
activity results, we were unable to detect a molecular weight shift by
Western analysis using an anti-Raf-1 antibody (not shown). Although a
mobility assay is rather insensitive, this result suggests that PDGF-BB did not stimulate the phosphorylation of Raf-1 in fetal lung
fibroblasts. To further exclude the MAP kinase pathway in
PDGF-BB-induced GAG synthesis, fetal lung fibroblasts were incubated
with PDGF-BB with and without PD-98059, an inhibitor of MAP kinase
kinase (37). In agreement with the observed absence of
Ras and Raf-1 activation by PDGF-BB, PD-98059 (100 µM) did not block
PDGF-BB-induced GAG synthesis (relative increase in GAG synthesis
over control: 194 ± 15 vs. 170 ± 10%, PDGF-BB vs.
PDGF-BB+PD-98059, respectively, n = 4).

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Fig. 7.
Lack of PDGF-BB effect on p21ras
activity. A: serum-starved fetal lung
fibroblasts were stimulated with or without PDGF-BB (20 ng/ml) for 10 min. Cells were transferred to 4°C and permeabilized with 0.5 U of
streptolysin O in an intracellular buffer in presence of 10 µCi/ml of
[ -32P]GTP. After 30 min of incubation, cell lysates were prepared and precipitated with
monoclonal anti-p21ras antibody.
Immune complexes were collected with protein G-Sepharose, and
p21ras-bound guanosine
nucleotides were analyzed (lanes
1-4). To determine stability of
[32P]GTP in
permeabilized cells, lysates were also analyzed before
immunoprecipitation (lanes
5-8). B:
serum-starved NIH/3T3 fibroblasts were also treated with and without
PDGF-BB, and p21ras-bound
guanosine nucleotides were analyzed. Data show autoradiographs after 1 wk of exposure at 70°C.
|
|
PDGF-BB-stimulated GAG synthesis is mediated via
PI3K.
To investigate whether PDGF-BB activated the PI3K pathway in fetal lung
fibroblasts, we first performed PI3K activity measurements. Fetal rat
lung fibroblasts were stimulated with 20 ng/ml of PDGF-BB for 10 min at
37°C and lysed; lysates were immunoprecipitated with either p85,
phosphotyrosine, or PDGFR antibodies; and the immunoprecipitates were
assayed for PI3K activity. PI3K activity in p85 precipitate was
increased 2.4 ± 0.5-fold (PIP densitometry, mean ± range,
n = 2 separate experiments in
duplicate) in PDGF-BB-stimulated cells (Fig.
8A),
likely because of the association of PI3K with the
-receptor (Fig.
4). Indeed, PI3K activity was readily detected in
-receptor
immunoprecipitates of PDGF-BB-stimulated cells (Fig. 8B).
-Receptor-associated PI3K
activity increased 2.1 ± 0.4-fold (PIP densitometry, mean ± range, n = 2 separate experiments in triplicate) on PDGF-BB stimulation. Also, PI3K activity in
phosphotyrosine immunoprecipitates was slightly increased in
PDGF-BB-stimulated cells (Fig. 8A),
in agreement with the undetectable tyrosine phosphorylation of p85
(Fig. 4). To evaluate the importance of the PI3K pathway in mediating
PDGF-BB-induced GAG synthesis, fetal rat lung fibroblasts were
preincubated for 1 h at 37°C with various concentrations of the
PI3K inhibitor wortmannin (47) before PDGF-BB stimulation. Wortmannin reduced PDGF-BB-stimulated GAG
synthesis in a dose-dependent manner (Fig.
9). PDGF-BB-induced GAG synthesis was
completely abolished by 500 nM wortmannin. Such a concentration had no
inhibitory effect on GAG synthesis in unstimulated fibroblasts (not
shown). PDGF-BB-induced GAG synthesis was also abrogated by another
PI3K inhibitor, LY-294002 (50), at a concentration of 5 µM (Fig. 9).
Equal amounts of DMSO, the solvent in which wortmannin and LY-294002
were dissolved, did not influence PDGF-BB-induced GAG synthesis. To
confirm that the inhibitory effect of wortmannin on PDGF-BB-stimulated
GAG synthesis was mediated via PI3K, we measured PDGF-BB-induced PI3K
activity in cells exposed to the different concentrations of
wortmannin. A similar dose-dependent inhibitory effect of wortmannin on
PDGF-BB-triggered PI3K activation was observed (Fig.
10). As anticipated, 500 nM wortmannin
completely abrogated the stimulatory effect of PDGF-BB on PI3K
activity. A similar concentration of wortmannin did not affect the
PDGFR tyrosine phosphorylation (Fig. 10). These results suggest that PI3K is a downstream mediator of PDGF-BB-triggered GAG synthesis in
fetal lung fibroblasts.

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Fig. 8.
PDGF-BB induces PI3K activity in fetal lung fibroblasts. Serum-starved
cells were stimulated with and without PDGF-BB (20 ng/ml) for 10 min
and then lysed. Cell lysates were immunoprecipitated with either
anti-p85 (A), anti-PY
(A), or anti- R
(B) followed by protein A-Sepharose.
Resulting immune complexes were analyzed for PI3K activity as described
in MATERIALS AND METHODS. PI3K
reaction products were separated by TLC. Positions of
phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol
4,5-bisphosphate (PIP2), and
origin are shown at right. Experiments
were repeated twice with duplicate
(A) and triplicate
(B) samples.
|
|

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Fig. 9.
Wortmannin and LY-294002 inhibit PDGF-BB-stimulated GAG synthesis.
Serum-starved fetal lung fibroblasts were preincubated for 1 h with
various concentrations of wortmannin
(left) or LY-294002
(right) before a 24-h incubation
with or without 20 ng/ml of PDGF-BB. GAG synthesis was measured as
described in MATERIALS AND METHODS.
Data are %stimulation per GAG synthesis by control fibroblasts
incubated without PDGF-BB.
|
|

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Fig. 10.
Wortmannin inhibits PDGF-BB-induced PI3K activity without affecting
autophosphorylation of R. Serum-starved fetal lung fibroblasts were
preincubated for 1 h with 500 nM wortmannin before a 5-min incubation
with or without 20 ng/ml of PDGF-BB.
A: PI3K activity was measured by
isolating PIP from reaction mixture by TLC and assaying for
radioactivity. Data are %stimulation per PI3K activity by control
fibroblasts incubated without PDGF-BB. Values are representative of 2 experiments carried out in triplicate.
B: in parallel experiments, cell
lysates were prepared and immunoblotted with anti-PY. Molecular-mass
standards are at right.
|
|
 |
DISCUSSION |
Although PDGF is considered to be a major mitogen for mesenchymal
tissues, it regulates a variety of other biological processes. Previously, it was reported that quiescent fetal rat lung fibroblasts did not respond mitogenically to either isoform of PDGF (5). However,
PDGF-BB but not PDGF-AA increased GAG synthesis without changing GAG
composition (7). PDGF-BB has also been shown to markedly augment the
synthesis and deposition of GAGs during skin injury and dermal wound
healing (39, 40). Exposure of monkey arterial smooth muscle cells to
PDGF-BB resulted in an increased sulfation of biglycan (43). Herein, we
found that PDGF-BB increased the
35SO4
incorporation into biglycan and larger sulfated proteoglycans by fetal
lung fibroblasts. However, PDGF-BB did not affect the gene expression
of biglycan's core protein, consistent with the previous finding that
actinomycin D and cycloheximide did not abrogate PDGF-BB-stimulated GAG
synthesis (7). Also, the proteoglycan inhibitor
D-
-xyloside does not inhibit
the stimulatory effect of PDGF-BB on GAG formation (7). These results
are compatible with PDGF-BB stimulating GAG synthesis at a
posttranslational level, most likely via activation of enzymes involved
in GAG chain elongation and/or sulfation.
In the present study, we found that PDGF-BB-induced GAG synthesis was
relayed by PI3K. In fetal lung fibroblasts, PDGF-BB-induced activation
of the intrinsic kinase activity of the
-receptor led to the binding
of PLC-
1, PI3K, and RasGAP to the receptor. The binding was
accompanied by tyrosine phosphorylation of PLC-
1 and RasGAP.
Although tyrosine phosphorylation of PDGFR-associated PI3K was hardly
detectable, PDGF-BB stimulated PI3K activity but not that of PLC-
1,
PKC, or Ras. Inhibition of PDGF-BB-induced PI3K activation with
wortmannin abrogated PDGF-induced GAG synthesis in fetal lung
fibroblasts, suggesting that phosphatidylinositol 3,4,5-trisphosphate
(PIP3) accumulation is essential
for this cell response. PDGF-BB has also been shown to stimulate
hyaluronan synthesis and secretion in the human foreskin fibroblast
cell line AG-1523 (46) and vascular smooth muscle cells (37),
respectively. In contrast to our findings, the stimulatory effect of
PDGF-BB on hyaluronan synthesis was not inhibited by wortmannin but by calphostin C (46). The dosage of wortmannin to inhibit PI3K activity in
fetal lung fibroblasts was greater than that required for PI3K
inhibition in AG-1523 cells (46), neutrophils (47), and rat adipocytes
(42). However, PDGFR tyrosine phosphorylation was not affected by the
higher concentration of wortmannin. Furthermore, LY-294002, another
PI3K inhibitor, also inhibited PDGF-BB-induced GAG synthesis at a
dosage compatible with previous studies (50).
The underlying mechanism by which the PI3K product,
PIP3, relays the biological signal
of increased GAG synthesis remains unknown. As suggested above, PDGF-BB
may increase GAG synthesis by regulating GAG chain elongation and
sulfation, two processes occurring in the
trans-Golgi cisternae (13, 49). It is
possible that small GTP-binding proteins such as Rab play
a role in controlling GAG synthesis and secretion (17). Increasing
evidence suggests that Rab proteins, which are localized to different
subcellular compartments, exert a regulatory role in the transport of
newly synthesized proteins from the endoplasmic reticulum to secretory vesicles through the various stacks of the Golgi complex (1). Rab
proteins may also direct trafficking of secretory vesicles to the
plasma membrane (52). We speculate that
PIP3 may be involved in regulating
the transport of proteoglycans through the Golgi complex by activating
and stabilizing Rab proteins, thereby affecting the elongation
and/or sulfation of GAGs linked to proteoglycan core proteins.
Consistent with this possibility is the finding that the yeast homolog
of the catalytic subunit of PI3K, Vps34, is involved in the sorting of
proteins to the vacuole (21). Other intracellular membrane trafficking
events in which PI3K activities appear to be involved are PDGF
internalization (22), fluid phase endocytosis (12), and early endosome
fusion (23).
PDGF-BB did not trigger DNA synthesis in fetal lung fibroblasts. Three
major signaling pathways, PLC-
1-PKC, PI3K, and Ras, have been
implicated in mitogenic signaling of PDGF (10, 17, 48). Activation of
PLC-
1 increases intracellular calcium levels and activates the
serine/threonine-specific PKC (48). In the present study, the content
of IP3, a product of PLC-
1
activity, and PKC activity were not altered after PDGF-BB exposure. Ras is also an important mediator of PDGF-induced mitogenesis (33). Recent
studies have shown that PDGF-BB-stimulated chemotaxis of 3T3 cells is
mediated via Ras (28). Ras activity might be regulated by the
PDGFR-associated proteins RasGAP and Sos (10, 29). Ras is a guanine
nucleotide-binding protein that is active when bound to GTP and
inactive when bound to GDP (16). The nucleotide-exchange factor Sos
binds directly to Ras and is linked to the activated PDGFR complex via
GRB2 and Syp (14, 30). We found that RasGAP bound to the activated
PDGFR, but no Sos binding via GRB2 and Syp was observed. No stimulatory
effect of PDGF-BB on Ras activity was noted in fetal lung fibroblasts.
In addition, the observation that RasGAP binding to PDGFR did not lead
to an increased accumulation of Ras-GTP in the absence of
PDGF-activated nucleotide exchange indicates that RasGAP activity was
not altered by the PDGF-BB treatment. In contrast, PDGF-BB stimulated
DNA synthesis in NIH/3T3 fibroblasts, and PDGFR activation led to
binding of Syp, GRB2, and Sos as well as increased Ras activity,
supporting the view that Ras activation is critical for PDGF-BB-induced
proliferation. Also, PI3K has been implicated in triggering the
mitogenic response of PDGF (25, 48). Although receptor-associated PI3K
activity was stimulated by PDGF-BB, PDGFR failed to relay a mitogenic
signal in fetal rat lung fibroblasts. It is possible that some of the signaling proteins that associate with PDGFR, such as RasGAP, counteract the positive mitogenic signals originating from other PDGFR-associated proteins, PLC-
1 and PI3K, as has been suggested by
Valius and Kazlauskas (48). RasGAP binding to the receptors has also
been shown to inhibit migration of 3T3 fibroblasts toward PDGF-BB (27).
Alternatively, studies with PDGFR mutants have demonstrated that the
ligand-dependent receptor association of either RasGAP, PLC-
1, or
PI3K (25, 48) is dispensable for mitogenic signaling, suggesting that
none of these molecules is directly involved in mitogenesis. Another
explanation for PDGF-BB not being mitogenic for fetal rat lung
fibroblasts may be that an additional mitogen is required to trigger
the mitogenic pathway. It has recently been found that PDGF-AA is not
mitogenic in 3T3 cells unless transforming growth factor-
, which is
not mitogenic itself, is added simultaneously to the culture medium
(44). PDGF isoforms have been shown to be important mitogens for human fetal lung fibroblast cell lines IMR-90 and WI-38 (11). The difference
in the ability of human and rat fetal lung fibroblasts to respond to
PDGF isoforms appears to be due to the type of receptor subunit present
on the cell membrane. IMR-90 and WI-38 cells responded to both PDGF-AA
and PDGF-BB, indicating that these cells have both PDGF
-
and
-receptors. In contrast, fetal rat lung fibroblasts express only
-receptors (4, 5). Together, all these data suggest that the
mitogenic response of cells to PDGF depends on many variables,
including receptor density and receptor isoform on the cell surface and
the sum of intracellular signals arising from activated PDGFRs.
Recently, it has been found that embryonic rat lung fibroblasts
(day 13) respond mitogenically to
PDGF-BB (45), implying that the PDGF-BB signal in the same cell type is
transduced in different physiological responses depending on gestation
and/or differentiation.
 |
ACKNOWLEDGEMENTS |
These studies were supported by a group grant from the Medical
Research Council of Canada and an equipment grant from the Ontario
Thoracic Society. M. Liu and D. Rotin are recipients of a scholarship
from the Medical Research Council of Canada.
 |
FOOTNOTES |
Address for reprint requests: M. Post, Lung Biology Program, Hospital
for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G
1X8.
Received 1 October 1997; accepted in final form 23 January 1998.
 |
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