PI-3-kinase and MAPK regulate mesangial cell proliferation and
migration in response to PDGF
Goutam Ghosh
Choudhury,
C.
Karamitsos,
James
Hernandez,
Alessandra
Gentilini,
John
Bardgette, and
Hanna E.
Abboud
Division of Nephrology, Department of Medicine, University of Texas
Health Science Center, and Audie L. Murphy Memorial Veterans Affairs
Medical Center, San Antonio, Texas 78284-7882
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ABSTRACT |
Proliferation and
migration are important biological responses of mesangial cells to
injury. Platelet-derived growth factor (PDGF) is a prime candidate to
mediate these responses in glomerular disease. PDGF and its receptor
(PDGFR) are upregulated in the mesangium during glomerular injury. We
have recently shown that PDGF activates phosphatidylinositol 3-kinase
(PI-3-kinase) in cultured mesangial cells. The role of
this enzyme and other more distal signaling pathways in regulating
migration and proliferation of mesangial cells has not yet been
addressed. In this study, we used two inhibitors of PI-3-kinase,
wortmannin (WMN) and LY-294002, to investigate the role of this enzyme
in these processes. Pretreatment of mesangial cells with WMN and
LY-294002 dose-dependently inhibited PDGF-induced PI-3-kinase activity
assayed in antiphosphotyrosine immunoprecipitates. WMN pretreatment
also inhibited the PI-3-kinase activity associated with anti-PDGFR
immunoprecipitates prepared from mesangial cells treated with PDGF.
Pretreatment of the cells with different concentrations of WMN resulted
in a dose-dependent inhibition of PDGF-induced DNA synthesis. Both WMN
and LY-294002 inhibited PDGF-stimulated migration of mesangial cells in
a dose-dependent manner. It has recently been shown that PI-3-kinase
physically interacts with Ras protein. Because Ras is an upstream
regulator of the kinase cascade leading to the activation of
mitogen-activated protein kinase (MAPK), we determined whether
activation of PI-3-kinase is necessary for activation of MAPK.
Pretreatment of mesangial cells with WMN and LY-294002 significantly
inhibited PDGF-induced MAPK activity as measured by immune complex
kinase assay of MAPK immunoprecipitates. Furthermore, PD-098059, an
inhibitor of MAPK-activating kinase inhibited PDGF-induced MAPK
activity and resulted in significant reduction of mesangial cell
migration in response to PDGF. These data indicate that MAPK is a
downstream target of PI-3-kinase and that both these enzymes are
involved in regulating proliferation and migration of mesangial cells.
phosphatidylinositol 3-kinase; mesangial cells; migration; mitogenesis; mitogen-activated protein kinase; platelet-derived growth
factor
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INTRODUCTION |
CELL MIGRATION and cell proliferation are fundamental
responses of mesangial cells to glomerular injury and contribute to hypercellularity observed in a number of glomerular
diseases. Mesangial cell migration may also contribute to
repopulation of glomerular cells that follows cytolytic lesions as
observed in experimental and human forms of glomerulonephritis.
Platelet-derived growth factor (PDGF), secreted by glomerular cells as
well as activated platelets and macrophages, is the most potent mitogen for mesangial cells in vitro and in vivo (1). PDGF also induces directed migration during inflammatory glomerular disease (3). In
immune-mediated human glomerulonephritis and in experimental models of
glomerular injury, mesangial cell proliferation and migration are
accompanied by increased expression of PDGF and its receptor (PDGFR).
Binding of PDGF causes dimerization of its cognate receptor and induces
its intrinsic protein tyrosine kinase activity leading to
autophosphorylation of the receptor and to the phosphorylation of
target substrates on tyrosine residues (2). Tyrosine
autophosphorylation of the receptor creates binding sites for a set of
proteins characterized by the presence of ~100 amino acid residue
sequence motifs known as src homology 2 (SH2) domain. Some of the
proteins that associate with PDGFR include phospholipase C
1,
guanosinetriphosphatase activating protein, phosphotyrosine phosphatase
(PTP) 1D, and phosphatidylinositol 3-kinase (PI-3-kinase)
(2). Tyrosine phosphorylation and association of these enzymes with
PDGFR stimulate their enzymatic activity. PI-3-kinase is activated by
several growth factors and cytokines, including different tyrosine
kinase oncogenes (14). This enzyme is a heterodimer of 110-kDa
catalytic and 85-kDa regulatory subunits (2, 6). Activation of this
enzyme results in the production of D-3 phosphorylated inositides, the
precise functions of which are not yet clear. Several investigators
reported that PI-3-kinase lipid products, the D-3 phosphorylated
inositides, are necessary for cell proliferation. Also, activation of
this enzyme is necessary for cell migration and PDGFR internalization (13, 16). The 85-kDa regulatory subunit contains two SH2 domains through which it can associate with tyrosine-phosphorylated PDGFR on
the plasma membrane, thus stimulating the enzymatic activity of its
110-kDa catalytic subunit.
Another signal transduction pathway utilized by PDGFR is the Ras-Raf
mitogen-activated protein kinase (MAPK or ERK) (17). Activated PDGFR
binds the SH2 domain-containing adaptor protein Grb-2, which brings the
guanine nucleotide exchange factor, son of sevenless
(SOS), to the plasma membrane to replace GDP with GTP in
Ras. GTP-bound Ras interacts with Raf serine threonine kinase,
localizing it in the plasma membrane to activate its serine threonine
kinase. Raf thus initiates the kinase cascade to finally stimulate
MAPK, which phosphorylates downstream target proteins including
transcription factors (17). Modulation of any component of this kinase
cascade including the upstream regulator Ras may have an impact on
PDGF-stimulated signals and their biological consequences. With the
discovery of the drugs wortmannin and LY-294002 as potent inhibitors of
PI-3-kinase (28, 29), it is now possible to study the biological role
of PI-3-kinase in different cellular responses by directly inhibiting
its enzymatic activity after PDGF stimulation of cells. In this study,
we demonstrate that wortmannin and LY-294002 dose-dependently inhibit
PDGF-induced PI-3-kinase activity in mesangial cells. Inhibition of
PI-3-kinase activity leads to inhibition of PDGF-induced chemotaxis and
DNA synthesis. In addition, we demonstrate that inhibition of
PI-3-kinase blocks activation of MAPK in response to PDGF. These data
indicate that PI-3-kinase regulates mesangial cell proliferation and
chemotaxis in a MAPK-dependent manner.
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MATERIALS AND METHODS |
Materials. Tissue culture materials
were obtained from GIBCO-BRL. Nonidet P-40 (NP-40),
phenylmethylsulfonyl fluoride (PMSF), Na3VO4,
phosphatidylinositol, and wortmannin were purchased from Sigma.
LY-294002 was obtained from Calbiochem. PD-098059 was provided by
Parke-Davis Pharmaceutical Research Division. Aprotinin was obtained
from Miles Laboratories. Human recombinant PDGF BB was obtained from
Amgen. Human PDGFR
monoclonal antibody was obtained from Genzyme.
Antiphosphotyrosine and PI-3-kinase antibodies were obtained from
Upstate Biotechnology. MAPK antibody was from Santa Cruz Biotechnology.
Protein measurement and polyacrylamide gel reagents were purchased from
Bio-Rad. Protein A-Sepharose CL4B was obtained from Pharmacia.
[
-32P]ATP was from
New England Nuclear. All other reagents were of analytical grade.
Cell culture. Human mesangial cells
were propagated in Waymouth's medium in the presence of 17% fetal
calf serum as described (6). Cells were made quiescent by serum
starvation for 48 h in the same medium. Cells were treated with
wortmannin for 1 h before addition of PDGF. In these experiments, the
cells were used between passages 6 and
10.
Preparation of membrane and cytoplasmic
fractions. Solubilization buffer (0.5 ml) [20 mM
tris(hydroxymethyl)aminomethane hydrochloride (Tris · HCl), pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM
Na3VO4,
1 mM PMSF, and 0.1% aprotinin] is added to the cell monolayer.
The cells are collected by scrapping and lysed by 20 brisk strokes in a
Dounce homogenizer. The nuclear pellet is removed by centrifugation at
403 g for 5 min at 4°C. The
postnuclear supernatant is centrifuged at 242,000 g for 30 min at 4°C to separate
the cytosolic and membrane fraction. The cytosolic supernatant is
adjusted to 1% NP-40 in solubilization buffer. The membrane pellet is
resuspended in RIPA buffer (solubilization buffer with 1% NP-40) and
lysed for 30 min at 4°C. The soluble proteins are separated by
centrifugation at 10,000 g for 30 min
and used as the membrane fraction.
Immunoprecipitation and PI-3-kinase
assay. Cells are lysed in RIPA buffer at 4°C for 30 min. The debris is separated by centrifugation at 10,000 g for 30 min at 4°C. Protein is
estimated in the cleared supernatant, and an equal amount of protein is
used for immunoprecipitation with different antibodies as described
(7). Antiphosphotyrosine or PDGFR
immunoprecipitates are used for
PI-3-kinase assay as described (6). Briefly, the immunobeads are
resuspended in PI-3-kinase assay buffer [20 mM
Tris · HCl, pH 7.5, 0.1 M NaCl, and 0.5 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid]; 0.5 µl of phosphatidylinositol was added and incubated at
25°C for 10 min. One microliter of 1 M
MgCl2 and 10 µCi of [
-32P]ATP are added
simultaneously to the reaction mixture and incubated at 25°C for
another 10 min. A mixture of chloroform-methanol and 11.6 N HCl (150 µl, at a ratio of 50:100:1) is added to stop the reaction. The
reaction is then extracted with 100 µl of chloroform. The organic
layer is washed with 80 µl of methanol and 1 N HCl (1:1). The
reaction product is dried under a stream of nitrogen and resuspended in
10 µl of chloroform, separated by thin-layer chromatography, and
developed with CHCl3/methanol/28%
NH4OH/H2O (129:114:15:21). The spots are visualized by autoradiography.
Tyrosine kinase assay. Tyrosine kinase
activity was measured directly on the immunobeads as described
previously (6, 7). Briefly, the immunoprecipitates are resuspended in
kinase buffer [50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), pH 7.4, and 10 mM
MnCl2].
[
-32P]ATP (20 µCi) is added, and the reaction is incubated at 30°C for 15 min.
At the end of the reaction, 2× sample buffer is added, and the
labeled proteins are separated on sodium dodecyl sulfate (SDS) gel.
Measurement of DNA synthesis. DNA
synthesis is measured as incorporation of
[3H]thymidine into trichloroacetic acid-insoluble material as described previously (6).
Cell migration assay. Cell migration
in response to PDGF is determined using a modified method of Boyden
chamber assay (8). Briefly, confluent mesangial cells are serum starved
for 48 h. One hour before harvest, cells are incubated with
wortmannin. The cells are then trypsinized, washed with
Hanks' solution without Ca2+, and
finally resuspended in serum-free medium in the presence of 1% human
serum albumin in a 50-ml Falcon tube. The tube is kept on a rotating
apparatus until use to avoid the attachment of the cells to the plastic
tube wall. PDGF is added to the bottom chamber of the Boyden apparatus.
A polycarbonate membrane filter coated with 0.02 mg/ml collagen type I
is placed in the middle of the chamber. Cell suspension is applied to
the top chamber and incubated for 8 h at 37°C. After the
incubation, the filter is inverted on a glass slide, fixed with
methanol, and stained with Giemsa. The dried filter is mounted on a
slide with Permount, and cells are counted in 10 high-power fields in
the center of each filter (magnification, ×450). Each assay was
performed in triplicate. The data are presented as number of cells per
high-power field. When PDGF is added to the top and bottom chambers, no
cell migration was observed. Similarly, the addition of wortmannin to
the bottom chamber in concentrations up to 1 µM had no effect on
chemotaxis of mesangial cells (data not shown).
MAPK assay. MAPK assay was performed
using a modified method of Kribben et. al. (15). Briefly, cleared cell
lysate was immunoprecipitated with MAPK-specific antibody, and the
immunobeads were resuspended in MAPK assay buffer (10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.5 mM dithiothreitol, and 0.5 mM
Na3VO4)
in the presence of 0.5 mg/ml myelin basic protein (MBP), 0.5 µM
protein kinase A inhibitor, and 25 µM cold ATP plus 1 µCi
[
-32P]ATP. The
reaction was incubated at 30°C for 30 min followed by a 10-min
incubation on ice. The reaction mixture was then separated on 15% SDS
polyacrylamide gel. Phosphorylated MBP was visualized by
autoradiography.
Data analysis. Significance of the
data was determined by unpaired Student's
t-test.
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RESULTS |
Inhibition of PI-3-kinase by wortmannin
in mesangial cells. PDGF stimulates PI-3-kinase
activity in antiphosphotyrosine-associated protein fraction from
mesangial cells (6). We tested the effect of wortmannin on PDGF-induced
PI-3-kinase activity in these cells. Cleared cell lysate from
PDGF-stimulated mesangial cells pretreated with different concentration
of wortmannin was immunoprecipitated with antiphosphotyrosine
monoclonal antibody. The washed immunobeads were assayed for
PI-3-kinase activity using phosphatidylinositol as substrate in the
presence of
[
-32P]ATP. As shown
in Fig. 1, PDGF-induced PI-3-kinase
activity that associates with antiphosphotyrosine immunoprecipitates
was inhibited by wortmannin in a dose-dependent manner. At
100 nM wortmannin, >90% of the activity was inhibited.

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Fig. 1.
Effect of wortmannin (WMN) on platelet-derived growth factor
(PDGF)-induced phosphatidylinositol 3-kinase (PI-3-kinase) activity in
antiphosphotyrosine immunoprecipitates. Quiescent mesangial cells were
preincubated with different concentrations of wortmannin or with
vehicle dimethyl sulfoxide (DMSO) for 1 h and stimulated with 10 ng/ml
PDGF BB for 10 min. Cleared cell lysate was immunoprecipitated with
antiphosphotyrosine antibody, and immunobeads were assayed for
PI-3-kinase activity in the presence of PI and
[ -32P]ATP. Organic
extract of reaction product was separated by thin-layer chromatography.
Concentrations of wortmannin (in nM) are indicated. Arrow, position of
phosphatidylinositol phosphate (PI-3-P).
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PI-3-kinase activation requires translocation of this enzyme to the
plasma membrane and its association with tyrosine-phosphorylated proteins that include tyrosine-phosphorylated growth factor receptors. We have recently demonstrated that, in mesangial cells, PDGF stimulates association of PI-3-kinase with PDGFR, confirming translocation of this
enzyme to the plasma membrane in response to PDGF (6). To confirm
direct translocation of PI-3-kinase, we isolated membrane and
cytoplasmic fraction from mesangial cells treated with PDGF. Both these
fractions were immunoprecipitated with antiphosphotyrosine antibody,
and the immunoprecipitates were used in PI-3-kinase assay. The data
show that PDGF-stimulated PI-3-kinase activity is associated with the
membrane fraction (Fig. 2). Next we tested the effect of wortmannin on PDGFR-associated PI-3-kinase activity, which is also a measure of membrane-associated PI-3-kinase activity. Lysates from PDGF-treated mesangial cells preincubated with wortmannin were immunoprecipitated with PDGFR monoclonal antibody. The
immunoprecipitates were assayed for PI-3-kinase activity. The data show
that wortmannin inhibits the PDGFR-associated PI-3-kinase activity
(Fig. 3).

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Fig. 2.
Activation of PI-3-kinase in plasma membrane of PDGF-stimulated
mesangial cells. Quiescent mesangial cells were treated with 10 ng/ml
of PDGF BB. Cytoplasmic and membrane fractions were isolated from cell
lysate as described in MATERIALS AND
METHODS. Antiphosphotyrosine immunoprecipitates from
each fraction were assayed for PI-3-kinase activity. Arrow, PI-3-P
spot.
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Fig. 3.
Effect of wortmannin (WMN) on PDGF receptor (PDGFR)-associated
PI-3-kinase activity. Quiescent mesangial cells were pretreated with
wortmannin or DMSO. Cells were treated with 10 ng/ml of PDGF BB for 10 min. Cell lysate was immunoprecipitated with human PDGFR -specific
monoclonal antibody, and immunobeads were assayed for PI-3-kinase
activity. Arrow, position of PI-3-P.
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Role of PI-3-kinase in PDGF-induced DNA synthesis in
mesangial cells. We and others have previously shown
that PDGF is a potent mitogen for mesangial cells in culture (1, 6).
However, the requirement of PI-3-kinase in PDGF-mediated DNA synthesis in mesangial cells has not yet been investigated. To explore the potential involvement of PI-3-kinase in PDGF mitogenic signaling in
mesangial cells, we measured PDGF-induced DNA synthesis in the presence
of the PI-3-kinase inhibitor wortmannin. The data in Fig.
4 show that wortmannin inhibits
PDGF-induced DNA synthesis in a dose-dependent manner. These data
indicate that inhibition of PI-3-kinase completely blocks mesangial
cell DNA synthesis in response to PDGF.

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Fig. 4.
Effect of wortmannin (WMN) on PDGF-induced DNA synthesis in mesangial
cells. Quiescent mesangial cells were pretreated with different
concentrations of wortmannin for 1 h and followed by 10 ng/ml PDGF BB.
[3H]thymidine incorporation was
measured as an index of DNA synthesis. Results are means ± SE of 4 independent experiments each done in quadruplicate.
* P < 0.05 vs. untreated
cells. + P < 0.05 vs. PDGF alone.
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Effect of PI-3-kinase inhibition on PDGF-mediated
mesangial cell migration. Mesangial cell migration is
an important biological response during glomerular injury. We tested
the involvement of PI-3-kinase in PDGF-induced mesangial cell
migration. Quiescent cells were treated with different concentrations
of wortmannin and subsequently used in chemotaxis assay in the presence
of PDGF. The results show that wortmannin inhibits PDGF-induced
mesangial cell migration in a concentration-dependent manner similar to that observed for PI-3-kinase inhibition and DNA synthesis inhibition (Fig. 5). These data suggest that mesangial
cell migration involves PI-3-kinase activation.

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Fig. 5.
Effect of wortmannin on PDGF-induced chemotaxis of mesangial cells.
Quiescent mesangial cells were preincubated with different
concentrations of wortmannin for 1 h. Cells were then tested for
directed migration in the presence of 10 ng/ml PDGF BB in a modified
Boyden chamber using polycarbonate filters. After staining, cells that
migrated to underside of filter were quantitated by light microscopy
(×450). Results are means ± SE of 3 independent experiments
each done in triplicate. * P < 0.05 vs. untreated cells.
+ P < 0.05 vs.
PDGF alone.
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Although wortmannin has been extensively used as an inhibitor of
PI-3-kinase, other enzymes are also inhibited by this fungal metabolite
in different cell types. For example, wortmannin inhibits bombesin-induced phospholipase A2
in Swiss 3T3 cells and anti-CD3-stimulated phospholipase D in Jurkat T
cells (4, 18). This drug also inhibits myosin light chain kinase and
the biological effect mediated by this kinase (20). To address the
involvement of PI-3-kinase in mesangial cells, we used the chromone
derivative, LY-294002, which is known to block the activity of this
enzyme in different cells (28). Lysates from PDGF-treated mesangial
cells preincubated with LY-294002 were immunoprecipitated with
antiphosphotyrosine antibody followed by measurement of PI-3-kinase
activity in these immunoprecipitates. The data show that 50 µM and
100 µM of LY-294002 significantly inhibit the PDGF-stimulated
PI-3-kinase activity (Fig. 6).

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Fig. 6.
Effect of LY-294002 (LY) on PDGF-stimulated PI-3-kinase activity in
mesangial cells. Quiescent mesangial cells were incubated with
indicated concentrations of LY-294002 for 40 min prior to stimulation
with 10 ng/ml of PDGF BB for 10 min. Cleared cell lysate was
immunoprecipitated with antiphosphotyrosine antibody. Immunobeads were
assayed for PI-3-kinase activity. Arrow, PI-3-P (PIP) spot.
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To test whether this chromone derivative inhibits PDGF-induced
chemotaxis, mesangial cells were incubated with LY-294002 and then used
in chemotaxis assay in the presence of PDGF. The results show that 50 µM and 100 µM LY-294002 significantly inhibit PDGF-induced chemotaxis of mesangial cells (Fig. 7).

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Fig. 7.
Effect of LY-294002 on PDGF-induced chemotaxis of mesangial cells.
Quiescent mesangial cells were pretreated with different concentration
of LY-294002 for 40 min. Cells were then tested for directed migration
in response to 10 ng/ml of PDGF as described in Fig. 5.
* P < 0.05 vs. PDGF alone.
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PI-3-kinase regulates PDGF-induced MAPK activity in
mesangial cells. It is well established that PDGF
activates Ras, which induces translocation of Raf kinase to the plasma
membrane to bind physically with Ras protein (9, 17). This
translocation of Raf increases its intrinsic kinase activity to
initiate the kinase cascade to finally stimulate MAPK. Although
PI-3-kinase can physically associate with Ras protein, the upstream
regulator of MAPK (24), it is not clear whether PI-3-kinase can
regulate MAPK activation. To address this issue, we measured kinase
activity in MAPK immunoprecipitates of PDGF-stimulated mesangial cells pretreated with wortmannin and LY-294002. As shown in Fig.
8, both these compounds significantly
inhibited PDGF-induced MAPK activity in mesangial cells. These data
indicate that in mesangial cells, PI-3-kinase activity stimulated by
PDGF regulates MAPK activity.

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Fig. 8.
Effect of wortmannin (WMN) and LY-294002 (Ly) on PDGF-stimulated
mitogen-activated protein kinase (MAPK) activity. Quiescent mesangial
cells were treated with 100 nM wortmannin for 1 h or 100 µM LY-294002
for 40 min followed by 10 ng/ml PDGF BB. Cleared cell lysate (100 µg)
was immunoprecipitated with a MAPK-specific antibody.
Immunoprecipitates were used in an in vitro immunocomplex kinase assay
in the presence of myelin basic protein (MBP) and
[ -32P]ATP.
Phosphorylated MBP was separated on a 15% SDS polyacrylamide gel.
Molecular mass markers are shown (in kDa) at
left.
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MAPK regulates PDGF-induced mesangial cell
chemotaxis. To study the role of MAPK in PDGF-induced
mesangial cell chemotaxis, we used the MEK inhibitor PD-098059.
Mesangial cells were preincubated with this compound followed by
treatment with PDGF. The cell lysates were immunoprecipitated with MAPK
antibody and used in an in vitro immunocomplex kinase assay to
determine MAPK activity. As shown in Fig.
9, the MEK inhibitor abolished
PDGF-stimulated MAPK activity. Next we treated mesangial cells with
PD-098059, and the cells were used in chemotaxis assay in response to
PDGF. The data show that inhibition of MAPK activity significantly
inhibits PDGF-induced chemotaxis of mesangial cells (Fig.
10). Note that, despite complete inhibition of MAPK activity by PD-098059 (Fig. 9), PDGF-stimulated chemotaxis was inhibited by only 41%.

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Fig. 9.
Effect of MEK inhibitor on PDGF-induced MAPK activity in mesangial
cells. Quiescent mesangial cells were treated with 20 µM PD-098059
(PD) for 45 min followed by 10 ng/ml of PDGF BB. Lysate (100 µg) was
immunoprecipitated with MAPK antibody. The MAPK activity was measured
in the immunoprecipitates as described in Fig. 8. Molecular
mass markers are shown in kDa at
left.
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Fig. 10.
Effect of MEK inhibitor on PDGF-induced chemotaxis of mesangial cells.
Quiescent mesangial cells were pretreated with 20 µM PD-098059 for 45 min. Cells were then tested for directed migration in response to 10 ng/ml of PDGF as described in Fig. 5.
+ P < 0.05 vs. untreated cells. * P < 0.05 vs. PDGF alone.
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DISCUSSION |
Studies in human and experimental animals suggest that PDGF plays a key
role in proliferative and inflammatory glomerular disease (1, 2). PDGF
stimulates pleotrophic effects in cells of mesenchymal origin including
glomerular mesangial cells. Addition of PDGF to cultured mesangial
cells stimulates early signal transduction pathways leading to DNA
synthesis and PDGF A- and B-chain gene induction (1). We have recently
shown that PDGF activates PI-3-kinase as one of the early signaling
pathways (6). Activation of PI-3-kinase has been implicated in an array
of biological responses in different cell types (14). These include DNA
synthesis, activation of p70S6 kinase, insulin and PDGF-stimulated
membrane localization of GLUT4, receptor downregulation, adipocyte
differentiation, glycogen synthesis, PDGF-induced membrane ruffling,
activation of integrins in platelets, and histamine release from
RBL-2H3 cells (see Ref. 21, references therein). In the present study, we used two structurally different PI-3-kinase inhibitors wortmannin and LY-294002 to examine the role of PI-3-kinase in mesangial cell
migration and DNA synthesis in response to PDGF. Pretreatment of these
cells with wortmannin or LY-294002 prior to addition of PDGF inhibited
the antiphosphotyrosine-associated PI-3-kinase activity in a
dose-dependent manner (Figs. 1 and 6).
PI-3-kinase is a heterodimeric cytoplasmic enzyme. However, in tumor
cells, activated PI-3-kinase is localized to the plasma membrane (19).
In addition, this enzyme physically associates with
tyrosine-phosphorylated membrane-bound cellular proteins via the SH2
domain of its 85-kDa regulatory subunit at the vicinity of its
phosphoinositide substrates (14). These observations lead to the
hypothesis that translocation and membrane localization of PI-3-kinase
is necessary for its activation in vivo. Our data show that in
mesangial cells, PDGF stimulates translocation of activated PI-3-kinase
to the plasma membrane (Fig. 2). These data confirm the hypothesis that
membrane bound PI-3-kinase is active. In support of this notion, we
reported earlier that PI-3-kinase associates with PDGFR in mesangial
cells in response to PDGF (6). This also demonstrated the presence of
PI-3-kinase in the plasma membrane. Of interest is our observation that
wortmannin inhibits PDGFR-associated PI-3-kinase activity (Fig. 3),
PDGF-induced DNA synthesis (Fig. 4) and chemotaxis (Fig. 5). These data
indicate that activated PI-3-kinase is necessary for PDGF-stimulated
mesangial cell proliferation and migration.
Mesangial cell migration has been implicated in the pathology of
different glomerular diseases (1, 2). Cytokines and growth factors are
the principal mediators of mesangial cell migration during
inflammation. PI-3-kinase has recently been implicated in regulated on
activation normal T-expressed and presumably secreted (RANTES)-mediated
lymphocyte migration (26). Also hepatocyte growth factor (HGF)-mediated
mitogenesis, measured by chemotaxis, of renal inner medullary
collecting duct (IMCD) cells was inhibited by wortmannin suggesting the
involvement of PI-3-kinase in this process (5). However, unlike the
effect of wortmannin on PDGF-mediated mitogenesis in mesangial cells,
in IMCD cells, HGF-stimulated mitogenesis was inhibited to a lesser
extent by wortmannin (5). These data indicate that PI-3-kinase
regulates growth factor-induced mitogenesis in a cell type-specific
manner. By mutagenesis studies of PDGFR, the role of PI-3-kinase in
PDGF-mediated cell migration is controversial (16, 30). Using
wortmannin to inhibit PI-3-kinase enzymatic activity, we now show
complete inhibition of mesangial cell migration (Fig. 5). Because
wortmannin inhibits other enzymes such as phospholipase
A2, phospholipase D, and myosin
light chain kinase (4, 18, 20), we confirmed the role of PI-3-kinase in
mesangial cell migration using another PI-3-kinase inhibitor LY-294002.
This chromogen inhibited PDGF-induced PI-3-kinase activity assayed in
the antiphosphotyrosine immunoprecipitates (Fig. 6). The same
concentration of LY-294002 also significantly inhibited PDGF-induced
mesangial cell chemotaxis (Fig. 7). These data taken together
with the results obtained with wortmannin indicate that activation of
PI-3-kinase in PDGF-stimulated mesangial cells is an essential
enzymatic pathway that mediates cell migration. Of interest is the
recent observation that PI-3-kinase can physically bind to Rac1, which
is a member of Rho family of small GTP binding proteins (25). It has
been shown that these proteins play important role in
cytoskeletal organization during formation of focal adhesion and cell
migration (10, 22, 23).
It has recently been shown that the SH3 domain of Grb2 can bind the
proline-rich region of the 85-kDa subunit of PI-3-kinase, thus bringing
this enzyme in the vicinity of Ras (31). In another study, it has been
reported that PI-3-kinase binds Ras protein directly suggesting that
this lipid kinase can modulate Ras function (24). These observations
provide two independent mechanisms for PI-3-kinase translocation to the
plasma membrane away from its binding to the activated PDGFR, which is
also a means of translocation to the plasma membrane. It is known that
this translocation is required for PI-3-kinase activity. We have also
shown that PDGF-stimulated PI-3-kinase activity resides in the membrane
fraction of mesangial cells (Fig. 2). In the PDGF signaling pathway,
Ras is the upstream regulator of the kinase cascade that ultimately
stimulates MAPK (17). In the present study, we have shown that
inhibition of PI-3-kinase activity by wortmannin reduced MAPK activity
(Fig. 8). Another PI-3-kinase inhibitor, LY-294002, also significantly inhibited PDGF-induced MAPK activity in mesangial cells. Neither wortmannin nor LY-294002 inhibits the enzymatic activity of MAPK in an
in vitro MAPK assay (data not shown), suggesting that the inhibition of
MAPK activity we observed in the intact cells is secondary to their
effect on PI-3-kinase activation. These data also suggest that
PI-3-kinase modulates Ras function, and hence inhibition of PI-3-kinase
may inhibit downstream MAPK activity. Alternatively, the D-3
phosphorylated products produced by activated PI-3-kinase may directly
or indirectly modulate MAPK activity. These observations of regulatory
role of PI-3-kinase in activation of MAPK in mesangial cell migration
indicate that MAPK is also involved in this PDGF-induced biological
response. Our data showing that indirect inhibition of MAPK activity by
PD-098059 (Fig. 9), a potent inhibitor of the MAPK-activating kinase
MEK, is associated with significant inhibition of PDGF-induced
mesangial cell chemotaxis provide the first evidence that MAPK
modulates PDGF-mediated mesangial cell migration (Fig. 10). However, it
is important to emphasize that, although treatment of mesangial cells
with the MEK inhibitor PD-098059 caused complete inhibition of MAPK
activity in these cells, PDGF-induced chemotaxis was inhibited by only
41%. These data suggest that additional signaling pathway(s) are
involved in PDGF-induced chemotaxis in mesangial cells.
Migration of mesangial cells in the glomerulus contributes to
structural remodeling in proliferative glomerulonephritis. PI-3-kinase is a central downstream signaling enzyme for many growth factor and
cytokine receptors including PDGF. Neutralization of PDGFR or PDGF
directly by injection of antibodies attenuates the pathological lesion
during the course of anti Thy-1-induced glomerulonephritis (11, 12).
However, during glomerular injury, several inflammatory cytokines and
growth factors besides PDGF may be expressed. PI-3-kinase targeting may
provide a convenient mechanism to inhibit signals transduced
simultaneously or in an overlapping fashion by several inflammatory
cytokines.
 |
ACKNOWLEDGEMENTS |
We thank Sergio Garcia for help with the cell culture.
 |
FOOTNOTES |
This study was supported in part by the Dept. of Veterans Affairs
Medical Research Service and National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-50190 (to G. Ghosh Choudhury).
H. E. Abboud is supported by a Dept. of Veterans Affairs Medical
Research Service grant and National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-43988 and DK-33665.
Address for reprint requests: G. Ghosh Choudhury, Div. of Nephrology,
Dept. of Medicine, Univ. of Texas Health Science Center, San Antonio,
TX 78284-7882.
Received 13 November 1996; accepted in final form 29 July 1997.
 |
REFERENCES |
1.
Abboud, H. E.
Role of platelet-derived growth factor in renal injury.
Annu. Rev. Physiol.
57:
297-309,
1995[Medline].
2.
Abboud, H. E.,
B. Bhandari,
and
G. Ghosh Choudhury.
Cell biology of platelet-derived growth factor.
In: Molecular Nephrology. Kidney Function in Health and Disease, edited by J. Bonventre,
and D. Schlondorf. New York: Dekker, 1995, p. 573-590.
3.
Barnes, J. L.,
and
K. A. Hevey.
Glomerular mesangial cell migration in response to platelet-derived growth factor.
Lab. Invest.
62:
379-382,
1990[Medline].
4.
Cross, M. J.,
A. Stewart,
M. N. Hodgkin,
D. J. Kerr,
and
M. J. Wakelam.
Wortmannin and its structural analogue demethoxyviriin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase.
J. Biol. Chem.
270:
25352-25355,
1995[Abstract/Free Full Text].
5.
Derman, M. P.,
M. J. Cunha,
E. J. Barros,
S. K. Nigam,
and
L. G. Cantley.
HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3 kinase.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F1211-F1217,
1995[Abstract/Free Full Text].
6.
Ghosh Choudhury, G.,
P. Biswas,
G. Grandaliano,
B. Fouqueray,
S. A. Harvey,
and
H. E. Abboud.
PDGF-mediated activation of phosphatidylinositol 3 kinase in human mesangial cells.
Kidney Int.
46:
37-47,
1994[Medline].
7.
Ghosh Choudhury, G.,
L.-M. Wang,
J. Pierce,
S. A. Harvey,
and
A. Y. Sakaguchi.
A mutational analysis of phosphatidylinositol 3 kinase activation of colony stimulating factor 1 receptor.
J. Biol. Chem.
266:
8068-8072,
1991[Abstract/Free Full Text].
8.
Grandaliano, G.,
A. J. Valente,
M. M. Rozek,
and
H. E. Abboud.
Gamma interferon stimulates monocyte chemotactic protein (MCP-1) in human mesangial cells.
J. Lab. Clin. Med.
123:
282-289,
1994[Medline].
9.
Hall, A.
A biochemical function for Ras-At last.
Science
264:
1413-1414,
1995.
10.
Huttenlocher, A.,
R. R. Sandborg,
and
A. L. Horwitz.
Adhesion in cell migration.
Curr. Opin. Cell Biol.
7:
697-706,
1995[Medline].
11.
Johnson, R. J.
Cytokine networks and the pathogenesis of glomerulonephritis.
J. Lab. Clin. Med.
121:
190-192,
1993[Medline].
12.
Johnson, R. J.,
E. W. Raines,
J. Floege,
A. Yoshimura,
P. Pritzl,
C. Alpers,
and
R. Ross.
Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor.
J. Exp. Med.
175:
1413-1416,
1992[Abstract].
13.
Joly, M.,
A. Kazlauskas,
F. S. Fay,
and
S. Covera.
Disruption of PDGF receptor trafficking by mutation of its PI 3 kinase binding sites.
Science
263:
684-687,
1994[Medline].
14.
Kapeller, R.,
and
L. C. Cantley.
Phosphatidylinositol 3 kinase.
Bioassays
16:
565-576,
1994[Medline].
15.
Kribben, A.,
E. D. Weider,
L. Xiaomel,
V. V. Putten,
Y. Granot,
R. W. Schrier,
and
R. A. Nemenoff.
AVP-induced activation of MAP kinase in vascular smooth muscle cells is mediated through protein kinase C.
Am. J. Physiol.
265 (Cell Physiol. 34):
C939-C945,
1993[Abstract/Free Full Text].
16.
Kundra, V.,
J. A. Escobedo,
A. Kazlauskas,
H. K. Kim,
S. G. Rhee,
L. T. Williams,
and
B. Zetter.
Regulation of chemotaxis by platelet-derived growth factor receptor-
.
Nature
367:
474-476,
1994[Medline].
17.
Margolis, B.,
and
E. Y. Skolnik.
Activation of Ras by receptor tyrosine kinases.
J. Am. Soc. Nephrol.
5:
1288-1299,
1994[Abstract].
18.
Mollinedo, F.,
C. Gajate,
and
I. Flores.
Involvement of phospholipase D in the activation of transcription factor AP-1 in human T lymphoid Jurkat cells.
J. Immunol.
153:
2457-2469,
1994[Abstract/Free Full Text].
19.
Narayanan, U.,
C. Keuker,
and
R. Hilf.
Membrane associated phosphatidylinositol kinase of R3230AC mammary tumors and normal mammary glands and effects of insulin on tumor enzyme activity.
Cancer Res.
48:
6727-6732,
1988[Abstract].
20.
Ozawa, K.,
T. Masujima,
K. Ikeda,
Y Kodama,
and
Y. Nonomura.
Different pathways of inhibitory effects of wortmannin on exocytosis are revealed by video-enhanced light microscope.
Biochem. Biophys. Res. Commun.
222:
243-248,
1996[Medline].
21.
Rameh, L. E.,
C-S. Chen,
and
L. C. Cantley.
Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3 kinase association with tyrosine phosphorylated proteins.
Cell
83:
821-830,
1995[Medline].
22.
Ridley, A. J.,
and
A. Hall.
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibres in response to growth factors.
Cell
70:
389-399,
1992[Medline].
23.
Ridley, A. J.,
H. F. Paterson,
C. L. Johnson,
D. Diekmann,
and
A. Hall.
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70:
401-410,
1992[Medline].
24.
Rodriguez-Viciana, P.,
P. H. Warne,
R. Dhand,
B. Vanhaesebroeck,
I. Gout,
M. J. Fry,
M. D. Waterfield,
and
J. Downward.
Phosphatidylinositol-3-OH kinase as a direct target of Ras.
Nature
370:
527-532,
1994[Medline].
25.
Tolias, L. F.,
L. C. Cantley,
and
C. L. Carpenter.
Rho family GTPases bind to phosphoinositide kinases.
J. Biol. Chem.
270:
17656-17659,
1995[Abstract/Free Full Text].
26.
Turner, L.,
S. G. Ward,
and
J. Westwick.
RANTES-activated human T-lymphocytes. A role for phosphoinositide 3 kinase.
J. Immunol.
155:
2437-2444,
1995[Abstract].
27.
Valius, M.,
and
A. Kazlauskas.
Phospholipase C
1 and phosphatidylinositol 3 kinase are the downstream mediators of PDGF receptor's mitogenic signaling.
Cell
73:
321-334,
1993[Medline].
28.
Vlahos, C. J.,
W. F. Matter,
K. Y. Hui,
and
R. F. Brown.
A specific inhibitor of phosphatidylinositol 3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J. Biol. Chem.
269:
5241-5248,
1994[Abstract/Free Full Text].
29.
Yano, H.,
S. Nakanishi,
K. Kimura,
N. Hanai,
Y. Fukui,
Y. Nonomura,
and
Y. Matsuda.
Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3 kinase in RBL-2H3 cells.
J. Biol. Chem.
268:
25846-25856,
1993[Abstract/Free Full Text].
30.
Yu, J. C.,
J. S. Gutkind,
D. Mahadevan,
W. Si,
K. A. Meyers,
J. H. Pierce,
and
M. A. Heidaran.
Biological function of PDGF-induced PI 3 kinase activity: its role in alpha PDGF receptor mediated mitogenic signaling.
J. Biol. Chem.
127:
479-487,
1994.
31.
Wang, J.,
K. R. Auger,
l. Jarvis,
Y. Shi,
and
T. M. Roberts.
Direct association of Grb2 with the p85 subunit of phosphatidylinositol 3 kianse.
J. Biol. Chem.
270:
12774-12780,
1995[Abstract/Free Full Text].
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