Effect of platelet-derived growth factor isoforms in rat
metanephric mesenchymal cells
Jill M.
Ricono1,2,
Mazen
Arar3,
Goutam Ghosh
Choudhury4,5, and
Hanna E.
Abboud1,2,5
Departments of 1 Medicine and 3 Pediatrics, and
2 Institute of Biotechnology, Department of Molecular Medicine,
4 The University of Texas Health Science Center and Geriatric
Research, Education and Clinical Center, 5 South Texas Veterans
Health Care System, San Antonio, Texas 78229-3900
 |
ABSTRACT |
Platelet-derived growth factor
(PDGF) B-chain or PDGF
-receptor-deficient mice lack mesangial
cells. To explore potential mechanisms for failure of PDGF A-chain to
rescue mesangial cell phenotype, we investigated the biological effects
and signaling pathways of PDGF AA and PDGF BB in metanephric
mesenchymal (MM) cells isolated from rat kidney. PDGF AA caused modest
cell migration but had no effect on DNA synthesis, unlike PDGF BB,
which potently stimulated migration and DNA synthesis. PDGF AA and PDGF
BB significantly increased the activities of phosphatidylinositol
3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK). PDGF BB
was more potent than PDGF AA in activating PI 3-K or MAPK in these cells. Pretreatment of MM cells with the MAPK kinase (MEK) inhibitor PD-098059 abrogated PDGF BB-induced DNA synthesis, whereas the PI 3-K
inhibitor wortmannin had a very modest inhibitory effect on DNA
synthesis (approximately
20%). On the other hand, wortmannin completely blocked PDGF AA- and PDGF BB-induced migration, whereas PD-098059 had a modest inhibitory effect on cell migration. These data
demonstrate that activation of MAPK is necessary for the mitogenic
effect of PDGF BB, whereas PI 3-K is required for the chemotactic
effect of PDGF AA and PDGF BB. Although PDGF AA stimulates PI 3-K and
MAPK activity, it is not mitogenic and only modestly chemotactic.
Collectively, the data may have implications related to the failure of
PDGF AA to rescue mesangial cell phenotype in PDGF B-chain or
PDGF-
-receptor deficiency.
kidney; development; mesangial cell; signal transduction
 |
INTRODUCTION |
THERE IS EVIDENCE THAT
GLOMERULAR microvascular cells arise from metanephric mesenchyme
(14). The spatial and temporal distribution of
platelet-derived growth factor (PDGF) and its receptors (PDGFR) suggest
a role for this growth factor in the development of mesangial cells (18) and have been conclusively demonstrated in
two studies where PDGF B-chain or PDGFR-
-deficient mice
lack mesangial cells (16, 21). PDGF is widely expressed in
a variety of mesenchymal cells during development. Siefert et al.
(18) mapped the expression patterns of PDGF ligands and
receptors in the developing and adult murine kidney using in situ
hybridization (Fig. 1). During glomerular development, as the renal vesicle epithelium progresses through the
comma- and S-shape stages, PDGF A-chain and B-chains are expressed in
epithelial cells. PDGF A-chain is expressed earlier and is seen even at
the renal vesicle stage, whereas PDGF B-chain expression occurs at
later stages and, at the earliest, is seen in the S-shaped bodies of
the developing glomerular structures. PDGFR-
and PDGFR-
are
expressed by mesenchymal cells in the metanephric blastema. At later
stages, PDGFR-
is expressed in cells surrounding the glomerulus, and
PDGFR-
transcripts are present in the mesenchymal/interstitial cells
that are recruited into the glomerular cleft, which will form the
vascular tuft of the mature glomerulus (18). These cells
express PDGFR-
and PDGF B-chain transcripts at high levels during
the later stages of glomerular development when microvasular (capillary) cells begin to fill the glomerular tuft. At this stage, PDGFR-
transcripts are barely detectable and PDGF A-chain
transcripts are undetectable. Utilizing both immunohistochemistry and
in situ hybridization, Arar et al. (2) showed similar
findings in the rat, with PDGFR-
localizing to metanephric
mesenchymal (MM) cells at early stages of development, cells within the
cleft of the S-shaped bodies of the maturing glomerulus, and, at later
stages, in the mature glomerulus. This study also demonstrated that
activation of PDGFR-
by PDGF BB isoform mediates MM cell migration
and DNA synthesis, providing one mechanism by which a subpopulation of these cells potentially develop into mesangial cells. However, the
failure of PDGFR-
to compensate for the lack of
-receptor in the
PDGFR-
-deficient mice remains unexplained, considering that the
-receptor, similar to the
-receptor, localizes to mesenchymal cells in the metanephric blastema, and PDGF A-chain, similar to the
B-chain, is also expressed in epithelial cells of the maturing glomerulus.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of the
localization of platelet-derived growth factor (PDGF) ligands and
receptors at various stages of the developing kidney
(A-E) based on data from Siefert et al.
(18) and Arar et al. (2).
|
|
Binding of PDGF dimers to the extracellular domain of the receptor
induces the receptor to dimerize and transautophosphorylate. Autophosphorylation of the receptor increases its intrinsic tyrosine kinase activity and provides docking sites for downstream signal transduction proteins (1, 9). Many of these tyrosines
interact directly or indirectly with the SH2 domains of signaling
molecules and are present in homologous positions with respect to
PDGFR-
and PDGFR-
. Activation of PDGFR initiates several major
signal transduction cascades, which include activation of
phosphoinositiol 3-kinase (PI 3-K), phospholipase C
(PLC)
1, and Ras-Raf-mitogen-activated protein kinase
(MAPK) kinase (MEK)-MAPK- [extracellular signal-activated kinase
(ERK)1/2] pathways (3, 5, 8). Expression of mutant PDGFR-
, the tyrosine residues of which are replaced with
phenylalanine, demonstrated an essential role for PI 3-K and
PLC
1 in proliferation and chemotaxis in different
cell types (6, 20, 22, 23). Additional studies
demonstrated that these responses are cell-specific (12). Because PDGF B-chain and PDGFR-
appear to be
essential for mesangial cell development, and signaling through the
PDGFR-
does not seem to compensate for the loss of PDGFR-
signaling, we examined the biological effects of PDGF AA and PDGF BB on
MM cells and the role of ERK1/2 and PI 3-K.
 |
MATERIALS AND METHODS |
Materials.
Tissue culture materials were purchased from GIBCO BRL (Rockville, MD).
Recombinant PDGF-AA and PDGF-BB were obtained from R&D Systems
(Minneapolis, MN). Wortmannin and PD-098059 were purchased from Alexis
(San Diego, CA). Myelin basic protein (MBP), phosphatidylinositol (PI),
collagenase, and collagen type I were obtained from Sigma (St. Louis,
MO). Primary antibodies to PDGFR-
(A-3) and PDGFR-
(951) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Secondary antibodies conjugated to Cy3 or FITC were
obtained from Chemicon (Temecula, CA). Protein measurement and
polyacrylamide gel reagents were purchased from Bio-Rad (Hercules, CA).
Anti-phosphotyrosine and Erk-1 polyclonal antibodies were also
purchased from Santa Cruz Biotechnology. Protein A-Sepharose was
obtained from Pierce (Rockford, IL). All other reagents were
high-quality analytic grade.
Cultured cells.
Primary MM cell cultures were prepared as previously described
(2, 10). Briefly, pregnant Sprague-Dawley rats were
purchased at 10-11 days of gestation. At gestational day
13, mothers were anesthetized by intramuscular injection of rat
mixture (60% ketamine, 40% xylazine), and embryos were collected. The
age of the embryo was counted from the day of the vaginal plug
(day 0). Embryos were dissected in 1× phosphate-buffered
saline under a zoom model SZH Olympus stereomicroscope. Embryonic
kidneys were collected, and cells were propagated in Dulbecco's
modified Eagle medium (GIBCO) including 10% fetal calf serum and grown
at 37°C, 5% carbon dioxide.
MAPK assay.
MM cells were plated 7.5 × 105 cells/60-mm dish,
grown to confluency, and serum starved for 48 h. Respective cells
were pretreated with 50 µM PD-098059, an MEK inhibitor, for 45 min
before being stimulated with PDGF-AA or PDGF-BB. Cells were lysed with
RIPA buffer (20 mM Tris · HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA,
1% Nonidet P-40, 1 mM Na3VO4 , 1 mM
polymethylsulfonyl fluoride, and 0.1% aprotinin) for 30 min at 4°C
and centrifuged at 10,000 g for 20 min at 4°C. Protein
concentrations were measured in the cell lysates. Equal amounts of
protein (100 µg) were incubated with ERK-1 polyclonal antibody for 30 min on ice. Fifteen microliters of protein A-Sepharose beads (50%
vol/vol slurry) were added and incubated at 4°C on a rocking platform
for 2 h. The immunobeads were washed and resuspended in MAPK assay
buffer (in mM: 10 HEPES, pH 7.4, 10 MgCl2, 0.5 dithiothreitol, and 0.5 Na3VO4) in the presence
of 0.5 mg/ml MBP and 25 µM cold ATP plus 1 µCi
[
-32P]ATP. The mixture was incubated at 30°C for 30 min, followed by a 10-min incubation on ice. Protein-loading buffer was
added, and reactions were boiled. Samples were then loaded on a 12.5% SDS-PAGE, and phosphorylated MBP was visualized by autoradiography (5).
Western blotting.
Equal amounts of protein from cell lysates were separated on a 12.5%
SDS-PAGE gel and electrophoretically transferred to polyvinyl membrane.
The membrane was blocked with 5% nonfat milk prepared in Tris-buffered
saline with Tween 20 (TBST) buffer, washed with TBST, and
incubated with ERK-1 primary antibody (1:200 dilution; Santa Cruz
Biotechnology). The membrane was then washed and incubated with
horseradish peroxidase-conjugated goat anti-rabbit IgG. The blot was
developed with enhanced chemiluminesence reagent.
PI 3-K assay.
MM cells were plated as above. Respective cells were pretreated with
250 nM wortmannin, a PI 3-K inhibitor, for 3 h before treatment
with PDGF. Cells were lysed and protein was analyzed as previously
mentioned. One hundred micrograms of protein were incubated with
anti-phoshotyrosine antibody for 30 min on ice. Fifteen microliters
protein A-Sepharose beads (50% vol/vol slurry) were added and
incubated at 4°C on a rocking platform for 2 h. The immunobeads
were washed three times with RIPA, once with PBS, once with
buffer A (0.5 mM LiCl, 0.1 M Tris · HCl, pH 7.5, and 1 mM Na3VO4), once with doubly distilled water,
and once with buffer B (0.1 M NaCl, 0.5 mM EDTA, 20 mM
Tris · HCl, pH 7.5). The immunobeads were then resuspended in
50 µl of PI 3-K assay buffer (20 mM Tris · HCl, pH 7.5, 0.1 M
NaCl, and 0.5 mM EGTA). PI (0.5 µl of 20 mg/ml) was added and
incubated at 25°C for 10 min. A cocktail of 1 µl of 1 M
MgCl2 and 10 µCi [
-32P]ATP was added and
incubated at room temperature for 10 min. A mixture of chloroform,
methanol, and 11.6 N HCl (150 µl, 50:100:1) was added to stop the
reaction, and an additional 100 µl of chloroform were added. The
organic layer is extracted and washed with methanol and 1 N HCl (1:1).
The reaction was dried overnight and resuspended in 10 µl of
chloroform. The samples were separated by thin-layer chromatography and
developed with CHCl3/MeOH/28%
NH4OH/H2O (129:114:15:21). The spots were
visualized by autoradiography (5).
DNA synthesis.
MM cells were plated at 7.5 × 104 cells/24-well dish,
grown to confluency, and serum starved for 48 h. Cells were either
pretreated with PD-098059 or wortmannin as previously described before
stimulation with PDGF isoforms. [3H]TdR (1 µCi/25 ml)
was added to each well. DNA synthesis is measured as the incorporation
of [3H]thymidine into trichoroactic acid-insoluble
material (7).
Cell migration assay.
Cell migration in response to PDGF was determined using blind well
chamber assays. Confluent MM cells were serum starved for 48 h and
then pretreated with PD-098059, wortmannin, or LY-294002. The monolayer
of cells were trypsinized and resuspended in serum-free media. The cell
suspension was added to the top chamber, while the PDGF was added to
the bottom chamber of the apparatus. A polycarbonate membrane filter
coated with collagen I separated the chambers. After 4 h at
37°C, the cells on the upper surface of the filter were removed with
a cotton-tipped applicator, and migratory cells on the lower surface of
the filter were fixed in methanol and stained with Giemsa. With the use
of high magnification (×450), the migration of cells was analyzed by
counting the number of cells that had migrated through the
polycarbonate filter (7).
Immunofluorescence.
For PDGFR-
and -
double immunofluorescent staining, cells were
grown to near confluency on eight-well coverslips. Cells were fixed in
methanol and washed in 1× PBS with 0.1% BSA. The primary antibody
(PDGFR-
) 1:20 was added, and coverslips were incubated in a
humidifier for 30 min at room temperature. Cells were washed three
times for 5 min each. The respective secondary antibody (donkey
anti-mouse, Cy3 conjugated) 1:30 was added, and coverslips were
incubated in a humidifier for an additional 30 min at room temperature.
For the PDGFR-
, cells were washed three times for 5 min each, the
primary antibody (PDGFR-
) 1:20 was added, and coverslips were
incubated in a humidifier for 30 min at room temperature. The
respective secondary antibody (donkey anti-rabbit, FITC conjugated)
1:20 was added, and coverslips were incubated in a humidifier for an
additional 30 min at room temperature. Cells were washed three times
for 5 min each and mounted with crystal mounts. Cells were viewed with
respective fluorescent filters with ultraviolet light.
 |
RESULTS |
Effect of PDGF isoforms on MM cell DNA synthesis and migration.
PDGF AA and PDGF BB were examined for their ability to stimulate DNA
synthesis as measured by [3H]thymidine incorporation into
DNA of quiescent MM cells. When cells were treated with PDGF BB for
24 h, [3H]thymidine incorporation increased nearly
fivefold above basal levels at concentrations of 10 and 100 ng/ml
of PDGF BB. However, similar concentrations of PDGF AA did not increase
DNA synthesis above basal levels (Fig.
2A). In addition to
proliferation, migration is an important biological response during
organ development. MM cells from the same passage used for the
[3H]thymidine incorporation were used for the migration
assays. PDGF BB induced migration of MM cells four- to fivefold above baseline, with a maximal effect seen at a dose of 10 ng/ml. PDGF AA
also induced migration in the MM cells about twofold above basal levels
at a dose of both 10 and 100 ng/ml; however, the response was much
weaker than that of PDGF BB (Fig. 2B).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of PDGF isoforms on DNA synthesis and migration.
A: [3H]thymidine incorporation was measured as
an index of DNA synthesis in response to treatment of 10 or 100 ng/ml
of PDGF AA or PDGF BB in quiescent metanephric mesenchymal (MM) cells.
Values are means ± SE of 4 independent experiments.
**P < 0.01 vs. untreated control. B:
serum-deprived quiescent MM cells were used in cell migration assay in
the presence of 10 or 100 ng/ml of PDGF AA or PDGF BB as described in
MATERIALS AND METHODS. Values are means ± SE of 3 independent experiments. **P < 0.01 vs.
untreated control.
|
|
PDGF activation of MAPK in MM cells.
Activated PDGFR-
is known to associate with Grb2/sos, which lies
upstream of the Ras-MEK-ERK signaling pathway. In contrast, activated
PDGFR-
has been reported to associate with Crk adaptor protein,
which may also lie upstream of the Ras-MEK-ERK signaling pathway. To
determine whether MAPK (ERK1/2) is activated in MM cells, we measured
the kinase activity in ERK1/2 immunoprecipitates of PDGF-stimulated MM
cells. MM cells were stimulated with PDGF AA or PDGF BB for 5, 10, and
15 min. Both PDGF AA and PDGF BB stimulated ERK1/2 activity in MM cells
(Fig. 3). Maximal activation was observed
at 15 min, and therefore in all subsequent experiments cells were
treated for 15 min with the PDGF isoforms. Dose-responses of
PDGF-induced ERK1/2 activity showed that PDGF AA induced maximal ERK1/2
activity at 50 and 100 ng/ml, whereas PDGF BB induced maximum activity
at 10 ng/ml (Fig. 4). These
concentrations correspond to those required to stimulate
proliferation and migration (Fig. 2, A and
B). In some experiments, cells were pretreated with an MEK
inhibitor, PD-098059, and assayed for ERK1/2 activity (Fig. 5). PD-098059 reduced PDGF-induced ERK1/2
activity to near basal levels, indicating that ERK1/2 activation in MM
cells is mediated by the Ras-Raf-MEK-MAPK pathway. Western blot
analysis of the ERK1/2 protein was performed on the same cell lysates
as for loading controls.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 3.
Time course of activation of mitogen-activated protein
kinase (MAPK) in PDGF-treated MM cells. Serum-deprived quiescent MM
cells were treated with 100 ng/ml of PDGF AA or 10 ng/ml of PDGF BB for
various time points, i.e., 5, 10, and 15 min. Cleared cell lysates were
immunoprecipitated with extracellular signal-regulated kinase (ERK)-1
polyclonal antibody. The immunoprecipitates were then used in an in
vitro immunecomplex kinase assay in the presence of myelin basic
protein (MBP) and [ -32P]ATP as described in
MATERIALS AND METHODS. Phosphorylated MBP was separated on
a 12.5% SDS-polyacrylamide gel. Western blot analysis of ERK 1/2 was
done on the same cell lysates to determine the loading control. Each
barogram represents the ratio of the radioactivity incorporated into
the phosphorylated MBP quantified by PhosphorImager analysis factored
by the densitometric measurement of the ERK1/2 band. Values are
means ± SE of 3 independent experiments expressed as the
percentage of control, where the ratio in the untreated cells was
defined as 100%. **P < 0.01 vs. control.
|
|

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of different doses of PDGF AA and BB on MAPK
activity in MM cells. Quiescent MM cells were treated with varying
concentrations of PDGF AA and BB. Cells were stimulated for 15 min with
10, 25, 50, or 100 ng/ml of PDGF AA or BB. Cleared cell lysates were
immunoprecipitated with ERK-1 polyclonal antibody. The
immunoprecipitates were then used in an in vitro immunecomplex kinase
assay in the presence of MBP and [ -32P]ATP as
described in MATERIALS AND METHODS. Phosphorylated MBP was
separated on a polyacrylamide gel. Western blot analysis of ERK 1/2 was
done on the same cell lysates, which were used as loading controls.
Each barogram represents the ratio of the radioactivity
incorporated into the phosphorylated MBP, quantified by PhosphorImager
analysis factored by the densitometric measurement of ERK1/2 band.
Values are mean ± SE of 3 independent experiments and are
expressed as the percentage of control, where the ratio in the
untreated cells was defined as 100%. **P < 0.01 vs.
control. *P < 0.05.
|
|

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of MEK inhibitor on the Ras-Raf-MAPK kinase
(MEK)-ERK pathway. Quiescent MM cells were pretreated with 50 µM
PD-098059 for 45 min before treatment with 100 ng/ml of PDGF AA or 10 ng/ml of PDGF BB. Cleared cell lysates were immunoprecipitated with
ERK-1 polyclonal antibody. The immunoprecipitates were then used in an
in vitro immunecomplex kinase assay in the presence of MBP and
[ -32P]ATP as described in MATERIALS AND
METHODS. Phosphorylated MBP was separated on a 12.5%
SDS-polyacrylamide gel. Western blot analysis of ERK1/2 was done on the
same cell lysates and used as loading controls. Each barogram
represents the ratio of the radioactivity incorporated into the
phosphorylated myelin basic protein quantified by PhosphorImager
analysis factored by the densitometric measurement of ERK1/2 band.
Values are means ± SE of 3 independent experiments and are
expressed as the percentage of control, where the ratio in the
untreated cells was defined as 100%. **P < 0.01 vs.
control.
|
|
Effect of MAPK inhibitor on PDGF-induced DNA synthesis and
migration in MM cells.
To assess the involvement of MAPK signaling pathways in DNA synthesis
and migration of the MM cells, cells were pretreated with 50 µM
PD-098095 for 45 min before the addition of PDGF AA or PDGF BB.
Pretreatment of cells with the MEK inhibitor abolished PDGF-induced DNA
synthesis (Fig. 6A). This
suggests that the Ras-Raf-MEK-MAPK pathway is a major contributor of
PDGF-induced DNA synthesis in MM cells. Pretreatment of the MM cells
with PD-098095 significantly decreased migration induced by PDGF BB
(Fig. 6B). However, PD-098095 did not inhibit migration to
basal levels as in PDGF-induced DNA synthesis (Fig. 6A). The
same concentration of PD-098095 that completely blocked PDGF AA-induced
ERK1/2 activity (Fig. 5) did not significantly reduce PDGF AA-induced
migration. These data suggest that other pathways are involved in
PDGF-induced migration.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of MEK inhibitor on PDGF-induced DNA synthesis and
migration. Quiescent MM cells were pretreated with 50 µM PD-098059
for 45 min before treatment with PDGF AA or PDGF BB. A:
[3H]thymidine incorporation was measured as an index of
DNA synthesis in response to treatment of 10 or 100 ng/ml of PDGF AA or
BB in quiescent MM cells. Values are means ± SE of 4 independent
experiments. **P < 0.01 vs. untreated and treated with
PD-098059. B: serum-deprived quiescent MM cells were used in
cell migration assay in the presence of 10 or 100 ng/ml of PDGF AA or
BB, as described in MATERIALS AND METHODS. Values are
means ± SE of 3 independent experiments. **P < 0.01 vs. untreated and treated with PD-098059.
|
|
PDGF activation of PI 3-K.
PI 3-K has previously been shown to associate with
tyrosine-phosphorylated PDGF receptors. However, it has not been
established whether signaling by both PDGF isoforms through their
respective receptors can activate PI 3-K in MM cells. PI 3-K activity
was determined in anti-phosphotyrosine immunoprecipitates of lysate from PDGF AA- or PDGF BB-stimulated cells. The immunoprecipitates were
assayed for PI 3-K activity as described in MATERIALS AND METHODS. As shown in Fig. 7, both
isoforms of PDGF activate PI 3-K activity. PDGF BB showed a significant
effect at 10 ng/ml, whereas 100 ng/ml of PDGF AA was necessary to
induce significant activation. When cells were pretreated with
wortmaninn, a PI 3-K inhibitor, the PDGFR-associated PI 3-K activity in
MM cells was significantly reduced (Fig.
8).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 7.
Activation of phosphatidylinositol 3-kinase (PI 3-K) in
PDGF-treated MM cells. A: serum-deprived MM cells were
treated with 10 or 100 ng/ml PDGF AA or PDGF BB for 15 min and cell
lysates were immunoprecipitated with anti-phosphotyrosine antibody. The
immunoprecipitates were then assayed for PI 3-kinase activity in the
presence of phosphotidyl inositol (PI) and [ -32P]ATP
as described in MATERIALS AND METHODS. The arrow indicates
the position of PI 3-P spot. B: each barogram represents the
radioactivity incorporated into PI 3-P by PhosphorImager analysis.
Values are means ± SE of 3 independent experiments and are
expressed as the percentage of control, where the untreated cells were
defined as 100%. **P < 0.01 vs. untreated control.
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of PI 3-K inhibitor on PDGF-activated PI 3-K in MM
cells. A: quiescent MM cells were pretreated with 250 nM
wortmannin for 3 h before treatment with PDGF AA or PDGF BB.
Cleared cell lysates were immunoprecipitated with anti-phosphotyrosine
antibody and the immunoprecipitates were then assayed for PI 3-K
activity in the presence of PI and [ -32P]ATP as
described in MATERIALS AND METHODS. Arrow, position of
PI 3-P spot. B: each barogram represents the radioactivity
incorporated into PI 3-P by PhosphorImager analysis. Values are
means ± SE of 3 independent experiments and are expressed as the
percentage of control, where the untreated cells were defined as 100%.
**P < 0.01 vs. untreated and treated with
wortmannin.
|
|
Effect of PI 3-K inhibitor on PDGF-induced DNA synthesis and
migration in MM cells.
We have recently shown that activation of PI 3-K is necessary for
PDGF-induced DNA synthesis and migration in mesangial cells (4). To determine the importance of PI 3-K in MM cells,
cells were pretreated with 250 nM wortmannin for 3 h before
stimulation with PDGF isoforms. Wortmannin decreased PDGF-induced
[3H]thymidine incorporation by ~20% (Fig.
9A), unlike the complete abolition of activity when cells were pretreated with PD-098059 (Fig.
6A). These data indicate that PI 3-K plays a lesser role in
PDGF-induced DNA synthesis than MAPK in MM cells. Two structurally dissimilar PI 3-K inhibitors, wortmannin and LY-294002, completely blocked PDGF-induced migration in MM cells (Fig. 9, B and
C), whereas the MEK inhibitor decreased PDGF BB-induced
migration by ~30% and PDGF AA-induced migration by ~15% (Fig.
6B). These data suggest that PI 3-K, rather than MAPK, plays
a predominant role in PDGF-induced migration.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of PI 3-K inhibitor on PDGF-induced DNA
synthesis and migration. Quiescent MM cells were pretreated with 250 nM
wortmannin for 3 h or 25 µM LY-294002 for 1 h before
treatment with PDGF AA or PDGF BB. A:
[3H]thymidine incorporation was measured as an index of
DNA synthesis in response to treatment of 10 or 100 ng/ml of PDGF AA or
BB in quiescent MM cells. Results are means ± SE of 4 independent
experiments. *P < 0.05 vs. untreated and treated with
wortmannin. B: serum-deprived quiescent MM cells were used
in cell migration assay in the presence of 10 or 100 ng/ml of PDGF AA
or PDGF BB, as described in MATERIALS AND METHODS. The data
represent means ± SE of 3 independent experiments.
**P < 0.01 vs. untreated and treated with
wortmannin. C: serum-deprived quiescent MM cells were used
in cell migration assay in the presence of 100 ng/ml of PDGF AA or 10 ng/ml of PDGF BB, as described in MATERIALS AND METHODS.
The data represent means ± SE of 3 independent experiments.
**P < 0.01 vs. untreated and treated with LY-294002.
|
|
Expression of PDGF receptors in MM cells.
To study the expression of PDGFR-
and -
in MM cells,
immunofluorescence was performed using PDGFR-
- and
PDGFR-
-specific antibodies. Figure
10 shows abundant expression of
both PDGFR-
and PDGFR-
in MM cells. This suggests
that these cells have the potential to respond to both PDGF isoforms.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 10.
Expression of PDGFR- and - in MM cells. A:
immunofluorescent localization of PDGFR- . B:
immunofluorescent localization of PDGFR- . All MM cells stained for
both PDGFR- and - . Magnification: ×40.
|
|
 |
DISCUSSION |
There are several potential mechanisms by which PDGF, acting on MM
cells, leads to the development of a subset of these cells into mature
differentiated mesangial cell phenotype. Two mechanisms pertinent
to development include cell migration and cell proliferation. This study explored the effects of PDGF AA and BB isoforms on DNA
synthesis and migration of MM cells isolated at the earliest stage of
the developing rat metanephric blastema, embryonic day 13.
Arar et al. (2) have recently demonstrated that PDGF BB stimulates DNA synthesis and migration of these cells. Stimulation of
cell migration by PDGF BB was associated with activation of PI 3-K, and
inhibition of PI 3-K blocked PDGF BB-induced migration. However, the
role of other signal transduction pathways activated by PDGF BB was not
explored. More importantly, the role of the PDGF A-chain in activating
MM cells is not known. This information is pertinent because
PDGFR-
does not appear to compensate for PDGFR-
in rescuing
mesangial cell phenotype in PDGFR-
deficiency. Moreover, during
early stages of development and maturation of the glomerular capillary
bed, the PDGF A-chain and PDGFR-
have a spatial and temporal
distribution similar to that of the PDGF B-chain and PDGFR-
,
respectively (18).
PDGF BB, as reported previously (2), stimulates DNA
synthesis and robust migration of MM cells. We now demonstrate that PDGF AA, even at a concentration as high as 100 ng/ml, was not mitogenic for these cells and had a modest effect on cell migration. Therefore, the lack of a mitogenic effect of the PDGF A-chain and its
inability to stimulate robust migration are potential mechanisms for
failure of PDGFR-
to compensate for PDGFR-
in rescuing mesangial
cell phenotype. Mouse chimeras composed of PDGFR-
+/+ and PDGFR-
/
cells demonstrated that PDGFR
/
cells fail to
populate the glomerular mesangium, whereas PDGFR
+/+ cells do,
suggesting a direct permissive role of PDGF BB in mesangial cell
development and maturation (17). Studies utilizing bromodeoxyuridine labeling demonstrated active proliferation of mesangial cell progenitors in cup-shaped and S-shaped glomeruli of
wild-type, but not mutant, mice. These studies suggested that proliferation of mesangial cell progenitors is a critical step for
mesangial cell development (17). Our finding of the
failure of PDGF AA to induce proliferation of MM cells supports
this contention. We next examined the activation of ERK1/2 by PDGF
AA or PDGF BB and its involvement in PDGF-induced DNA synthesis and
migration of MM cells. PDGF AA and PDGF BB increased ERK1/2 activity in a dose- and time-dependent manner (Figs. 3 and 4), with PDGF BB being
slightly more potent than PDGF AA. Pretreatment of MM cells with the
MEK inhibitor PD-098059 at a concentration that abolished MAPK activity
resulted in complete inhibition of DNA synthesis. However, the MEK
inhibitor only partially blocked PDGF BB-induced cell migration and
exerted a small but insignificant effect on PDGF AA-induced cell
migration. These data indicate that the Ras-Raf-MEK-MAPK pathway is
essential for PDGF BB-induced DNA synthesis in MM cells.
Cells expressing a PDGFR-
mutant devoid of the binding sites for PI
3-K, i.e., lacking Tyr740 and Tyr751, show no
chemotactic responses to PDGF (15, 24), suggesting a role
for PI 3-K in cell migration. However, PI 3-K regulates growth
factor-induced migration in a cell type-specific manner. For example,
there is evidence that PI 3-K does not mediate cell migration in smooth
muscle cells activated by PDGF BB (12). PDGFR-
-mediated
migration also appears to be cell type specific. In lung fibroblasts,
Swiss 3T3 and hematopoeitic 32D cells, activation of PDGFR-
induces
migration (13, 19, 25). However, in other cell types, such
as aortic endothelial cells and vascular smooth muscle cells, PDGF AA
inhibits the chemotactic response. PDGF BB as well as PDGF AA induce PI
3-K activity in MM cells, with the AA isoform resulting in somewhat
lesser induction of enzyme activity. Wortmannin, at concentrations that
decreased PI 3-K enzymatic activity, markedly inhibited PDGF-induced
migration of MM cells. In contrast to its potent effect on cell
migration, pretreatment of MM cells with wortmannin reduced PDGF
BB-induced DNA synthesis by ~20%. The data indicate that migration
of MM cells in response to both PDGF isoforms is mediated via PI 3-K. It is very unlikely that the differential effect of PDGF isoforms is
due to differential expression of PDGFR-
and -
in the cells, because both receptors were homogenously distributed in MM cells. The
data also demonstrate that the lack of biological response to PDGF AA
is not due to a low number of PDGFR-
or poor coupling of the AA
ligand with the receptor, because PDGF AA was almost as potent as PDGF
BB in activating ERK1/2 at a wide range of concentrations. Furthermore, the PDGF AA isoform potently activated PI 3-K to a degree
almost similar to that for the PDGF BB isoform. However, activation of
these pathways, PI 3-K and MAPK, by PDGF AA is not sufficient to induce
DNA synthesis or robust migration in these cells. These data, taken
together with the mesangial cell phenotype in the
PDGFR-
-deficient mouse, suggest that, in the absence of significant
DNA synthesis, activation of PI 3-K and subsequent migration is
insufficient for PDGFR-
, which can be activated by PDGF AA or PDGF
BB, to compensate for the loss of PDGFR-
. Of interest is the recent
observation that mice with a PDGFR-
mutant for PI 3-K binding sites
develop normally and do not exhibit an overt phenotype in the mesangium
(11), suggesting that
-receptor-mediated signaling
through activated PI 3-K is only of minor importance during mesangial
cell development. Alternatively, other signaling molecules activated by
the mutant
-receptor are able to compensate for the loss of PI 3-K signaling.
In conclusion, in this study we have shown that PDGF AA and PDGF BB
activate PI 3-K and MAPK enzymatic signaling pathways in MM cells. We
have also shown that PDGF BB induces DNA synthesis primarily through
the MAPK pathway and migration through the PI 3-K pathway. The finding
that PDGF AA had no effect on DNA synthesis, whereas it stimulated
modest migration of the cells, suggests that the failure of PDGFR-
to compensate for loss of PDGFR-
may be due to its inability to
mediate these fundamental biological responses of MM cells. It is
tempting to speculate that mesangial cell progenitors that may be
stimulated to migrate eventually undergo apoptosis in the
absence of the PDGF B-chain or PDGFR-
or fail to sustain their
proliferation or even their survival. A more comprehensive examination
of molecules activated by PDGFR-
is required to understand the
mechanism by which PDGF BB and PDGFR-
activation result in mesangial
cell development and maturation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Yves Gorin for critically reading the manuscript and
for valuable discussions.
 |
FOOTNOTES |
This study was supported in part by a Department of Veterans Affairs
Medical Research Service Merit Review Award and a Research Excellence
Area Program (REAP) Award (to G. G. Choudhury and H. E. Abboud), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33665 (to H. E. Abboud) and DK-55815 (to
G. G. Choudhury), American Heart Association, Texas Affiliate,
Grant-in-Aid 97G-439, and a Clinical Scientist Award from the National
Kidney Foundation (to M. Arar).
Address for reprint requests and other correspondence: H. E. Abboud, Dept. of Medicine, The Univ. of Texas at San Antonio Health
Science Center, Div. of Nephrology-MC 7882, 7703 Floyd Curl Dr.,
San Antonio, TX 78229-3900 (E-mail: abboud{at}uthscsa.edu).
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.
First published August 8, 2001; 10.1152/ajprenal.00323.2000
Received 27 November 2000; accepted in final form 20 September 2001.
 |
REFERENCES |
1.
Abboud, H,
Bhandari B,
and
Choudhury GG.
Cell biology of platelet-derived growth factor.
In: Molecular Nephrology. Kidney Function in Health and Disease, edited by Bonventre J,
and Schlondorf D.. New York: Dekker, 1995, p. 573-590.
2.
Arar, M,
Xu YC,
Elshihabi I,
Barnes JL,
Choudhury GG,
and
Abboud HE.
Platelet-derived growth factor receptor beta regulates migration and DNA synthesis in metanephric mesenchymal cells.
J Biol Chem
275:
9527-9533,
2000[Abstract/Free Full Text].
3.
Bazenet, CE,
and
Kazlauskas A.
The PDGF receptor alpha subunit activates p21ras and triggers DNA synthesis without interacting with rasGAP.
Oncogene
9:
517-525,
1994[ISI][Medline].
4.
Choudhury, GG,
Grandaliano G,
Jin DC,
Katz MS,
and
Abboud HE.
Activation of PLC and PI 3 kinase by PDGF receptor alpha is not sufficient for mitogenesis and migration in mesangial cells.
Kidney Int
57:
908-917,
2000[ISI][Medline].
5.
Choudhury, GG,
Karamitsos C,
Hernandez J,
Gentilini A,
Bardgette J,
and
Abboud HE.
PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF.
Am J Physiol Renal Physiol
273:
F931-F938,
1997[ISI][Medline].
6.
Cospedal, R,
Abedi H,
and
Zachary I.
Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases.
Cardiovasc Res
41:
708-721,
1999[ISI][Medline].
7.
Grandaliano, G,
Valente AJ,
Rozek MM,
and
Abboud HE.
Gamma interferon stimulates monocyte chemotactic protein (MCP-1) in human mesangial cells.
J Lab Clin Med
123:
282-289,
1994[ISI][Medline].
8.
Heldin, CH.
Structural and functional studies on platelet-derived growth factor.
Embo J
11:
4251-4259,
1992[ISI][Medline].
9.
Heldin, CH,
Ostman A,
and
Ronnstrand L.
Signal transduction via platelet-derived growth factor receptors.
Biochim Biophys Acta
1378:
79-113,
1998.
10.
Herzlinger, D,
Koseki C,
Mikawa T,
and
al-Awqati Q.
Metanephric mesenchyme contains multipotent stem cells whose fate is restricted after induction.
Development
114:
565-572,
1992[Abstract].
11.
Heuchel, R,
Berg A,
Tallquist M,
Ahlen K,
Reed RK,
Rubin K,
Claesson-Welsh L,
Heldin CH,
and
Soriano P.
Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling.
Proc Natl Acad Sci USA
96:
11410-11415,
1999[Abstract/Free Full Text].
12.
Higaki, M,
Sakaue H,
Ogawa W,
Kasuga M,
and
Shimokado K.
Phosphatidylinositol 3-kinase-independent signal transduction pathway for platelet-derived growth factor-induced chemotaxis.
J Biol Chem
271:
29342-29346,
1996[Abstract/Free Full Text].
13.
Hosang, M,
Rouge M,
Wipf B,
Eggimann B,
Kaufmann F,
and
Hunziker W.
Both homodimeric isoforms of PDGF (AA and BB) have mitogenic and chemotactic activity and stimulate phosphoinositol turnover.
J Cell Physiol
140:
558-564,
1989[ISI][Medline].
14.
Hyink, DP,
Tucker DC,
St John PL,
Leardkamolkarn V,
Accavitti MA,
Abrass CK,
and
Abrahamson DR.
Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F886-F899,
1996[Abstract/Free Full Text].
15.
Kundra, V,
Escobedo JA,
Kazlauskas A,
Kim HK,
Rhee SG,
Williams LT,
and
Zetter BR.
Regulation of chemotaxis by the platelet-derived growth factor receptor-beta.
Nature
367:
474-476,
1994[ISI][Medline].
16.
Leveen, P,
Pekny M,
Gebre-Medhin S,
Swolin B,
Larsson E,
and
Betsholtz C.
Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities.
Genes Dev
8:
1875-1887,
1994[Abstract].
17.
Lindahl, P,
Hellstrom M,
Kalen M,
Karlsson L,
Pekny M,
Pekna M,
Soriano P,
and
Betsholtz C.
Paracrine PDGF-B/PDGF-Rbeta signaling controls mesangial cell development in kidney glomeruli.
Development
125:
3313-3322,
1998[Abstract/Free Full Text].
18.
Seifert, RA,
Alpers CE,
and
Bowen-Pope DF.
Expression of platelet-derived growth factor and its receptors in the developing and adult mouse kidney.
Kidney Int
54:
731-746,
1998[ISI][Medline].
19.
Siegbahn, A,
Hammacher A,
Westermark B,
and
Heldin CH.
Differential effects of the various isoforms of platelet-derived growth factor on chemotaxis of fibroblasts, monocytes, and granulocytes.
J Clin Invest
85:
916-920,
1990[ISI][Medline].
20.
Smith, RJ,
Sam LM,
Justen JM,
Bundy GL,
Bala GA,
and
Bleasdale JE.
Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness.
J Pharmacol Exp Ther
253:
688-697,
1990[Abstract].
21.
Soriano, P.
Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.
Genes Dev
8:
1888-1896,
1994[Abstract].
22.
Valius, M,
Bazenet C,
and
Kazlauskas A.
Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively.
Mol Cell Biol
13:
133-143,
1993[Abstract].
23.
Valius, M,
Secrist JP,
and
Kazlauskas A.
The GTPase-activating protein of Ras suppresses platelet-derived growth factor beta receptor signaling by silencing phospholipase C-gamma 1.
Mol Cell Biol
15:
3058-3071,
1995[Abstract].
24.
Wennstrom, S,
Siegbahn A,
Yokote K,
Arvidsson AK,
Heldin CH,
Mori S,
and
Claesson-Welsh L.
Membrane ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3' kinase.
Oncogene
9:
651-660,
1994[ISI][Medline].
25.
Yu, JC,
Heidaran MA,
Pierce JH,
Gutkind JS,
Lombardi D,
Ruggiero M,
and
Aaronson SA.
Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol 3-kinase activity without affecting mitogenic or chemotactic signal transduction.
Mol Cell Biol
11:
3780-3785,
1991[ISI][Medline].
Am J Physiol Renal Fluid Electrolyte Physiol 282(2):F211-F219