© 1999 American Heart Association, Inc.
Original Contribution |
A Dual Inhibitor of Platelet-Derived Growth Factor ß-Receptor and Src Kinase Activity Potently Interferes With Motogenic and Mitogenic Responses to PDGF in Vascular Smooth Muscle Cells
A Novel Candidate for Prevention of Vascular Remodeling
From the Department of Internal Medicine II (J.W., J.K., H.F., U.M., V.H.), Ulm University Medical Center, Ulm, Germany; Research Unit "Molecular Cell Biology" (A.U., F-D.B.), Medical Faculty, Friedrich Schiller University, Jena, Germany; Cancer Biology Research Group (J.D.B., D.F.), University of Calgary Medical Centre, Calgary, Alberta, Canada; and Department of Biological Chemistry (A.G., A.L.), Institute of Life Sciences, and Department of Organic Chemistry (A.G.), Institute of Chemistry, The Hebrew University, Jerusalem, Israel.
Correspondence regarding biochemical aspects of this study to Frank-D. Böhmer, Research Unit "Molecular Cell Biology," Medical Faculty, Friedrich Schiller University, Drackendorfer Str 1, D-07747 Jena, Germany. E-mail i5frbo{at}rz.uni-jena.de
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Abstract—PP1 has previously been described as an inhibitor of the Src-family kinases p56Lck and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)–induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60c-src in vitro and Src-dependent tyrosine phosphorylation in p60c-srcF572-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor–stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF
-receptor was
susceptible, albeit to a lesser extent than PDGF ß-receptor. These
data suggest that the previously described tyrosine kinase
inhibitor PP1/AGL1872 is not selective for the
Src family of tyrosine kinases. It is also a potent
inhibitor of the PDGF ß-receptor kinase but is not a
ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits
migration and proliferation of HCASMCs probably by interference with 2
distinct tyrosine phosphorylation events, creating a
novel and potent inhibitory principle with possible
relevance for the treatment of pathological HCASMC activity, such as
vascular remodeling and restenosis.
Key Words: platelet-derived growth factor ß-receptor • coronary artery smooth muscle cell • atherosclerosis • restenosis • tyrphostins • PP1/AGL1872
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Protein tyrosine phosphorylation has been established as a crucial step in the regulation of normal cell proliferation, migration, differentiation, and survival. Specific tyrosine phosphorylation events are triggered by extracellular stimuli such as growth factors and processed by intracellular signal transducers.1 2 Aberrant tyrosine phosphorylation has been associated with numerous pathological states. Therefore, the development of tyrosine phosphorylation inhibitors (tyrphostins) for specific protein tyrosine kinases appears to be promising in the framework of the general concept of "signal transduction therapy."3 4 Indeed, in the last few years it has become apparent that tyrphostins with a high degree of selectivity and potency can be identified.4 5 6 For example, the tyrphostins AG1295 and AG1296, 2 closely related quinoxaline derivatives, selectively inhibit the platelet-derived growth factor (PDGF) ß-receptor tyrosine kinase, PDGF-stimulated proliferation, and cell transformation driven by autocrine PDGF receptor activation.7 8 The kinase inhibition is direct and involves interaction of the compounds with the ATP binding domain.9 Interestingly, KDR, a functional receptor for vascular endothelial growth factor (VEGF), which is structurally related to the PDGF ß-receptor, is not affected at all by AG1295 and AG1296, as are other tyrosine kinases such as the epidermal growth factor (EGF) receptor and Src.7
Inhibition of PDGF ß-receptor signaling has important potential clinical applications. Besides treatment of malignancies, which involve PDGF ß-receptor activation,10 11 12 the treatment of pathological vascular smooth muscle cell migration and proliferation in the context of atherosclerotic and restenotic processes appears as a promising application for PDGF ß-receptor kinase blockers.13 14 15 16 17 18 19 Because AG1295 and AG1296 do not inhibit the VEGF receptor KDR, and because endothelial cells of large arteries do not express PDGF receptors, it was postulated that AG1295 and AG1296 would not inhibit endothelial cell activation but only smooth muscle cell activity.9 20 This prediction turned out to be correct indeed, as AG1295 was shown to inhibit neointima formation in pigs by blocking proliferation and migration of smooth muscle cells without blocking endothelial cells.21
PDGF ß-receptor signaling involves interaction of the autophosphorylated receptor kinase with multiple SH2-domain–containing downstream effector molecules.22 Among other signaling events induced by PDGF, activation of Src-family kinases has been shown to be critical for the generation of the mitogenic signal.23 24 Activation occurs by the binding of Src to the autophosphorylated PDGF ß-receptor at phosphotyrosine residues 579 and 581.25 Src has also been reported to phosphorylate the PDGF ß-receptor at several sites, but its biological significance has not been fully established.26 To further evaluate the involvement of Src in PDGF signaling in vascular smooth muscle cells, we have used an Src-family kinase blocker, PP1, which was described recently.27 In the absence of a published synthesis protocol, we synthesized the compound, named it PP1/AGL1872, and tested its potential as a PDGF ß-receptor signaling antagonist. PP1/AGL1872 inhibits recombinant Src kinase and Src-dependent tyrosine phosphorylation. It potently inhibits human coronary artery smooth muscle cell (HCASMC) migration and also proliferation, 2 critical steps in the restenotic process.13 18 28 Surprisingly, however, we find that in addition to Src kinase inhibition, PP1/AGL1872 directly inhibits the PDGF ß-receptor tyrosine kinase, but it does not inhibit the kinase of the VEGF receptor KDR, the EGF receptor kinase, the fibroblast growth factor (FGF) receptor-1 kinase, or the insulin-like growth factor-1 (IGF-I) receptor kinase. In this article we describe in detail the dual Src and PDGF ß-receptor kinase inhibitor PP1/AGL1872.
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General
"Workup" refers to the process of adding water to the reaction mixture and extracting it with CH2Cl2 or ethyl acetate (EtAc); washing the organic phase; drying, filtering, and evaporating to dryness. All compounds were characterized spectroscopically and had the correct elemental analysis. Nuclear magnetic resonance (NMR) was recorded in CDCl3, with shifts in ppm downfield to tetramethyl silane. Compounds 1a, 1b, 2a, and 2b were prepared according to Reference 2929 . Compounds 4a (PP1/AGL1872) and 4b (PP2/AGL1879) were reported previously, but no synthesis or chemical data were described.27 Figure 1
presents the synthesis of
PP1/AGL1872 and PP2/AGL1879. Compounds 1a through 4b are described
below; their numbers correspond to those appearing in Figure 1.
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1. Acyl Malononitriles
Compound 1a is a light yellow solid, yield 60%, with melting
point 180°C (from CCl4). NMR (acetone
d6) measures 7.69, 7.40
(4H, ABq, JAB=8.0 Hz), and
2.43 (3H, s,
CH3).
Compound 1b is a light yellow solid, yield 55%, with melting point 187°C. NMR 7.50, 4H, narrow ABq.
2. Benzylidene Malononitriles
Compound 2a is a white solid, 40% yield, with melting point
95°C (from chromatography and trituration with
methanol). NMR 7.40 (4H, AB,
JAB=8.8 Hz), 3.95 (3H, s,
methoxy), and 2.46 (3H, s, methyl).
Compound 2b is a white solid (chromatography and trituration with hexane), yield 51%, with melting point 120°C. NMR 7.50 (4H, JAB=8.5 Hz) and 3.95 (3H, s, methoxy).
3. 1–Tertiary Butyl (t-Bu)–3 Aryl-4 Cyano-5 Amino
Pyrazoles
Compounds 3a (0.3 g, 1.5 mmol/L) and 2a (0.22 g, 1.8
mmol/L), t-Bu hydrazine HCl, and 0.09 g KOH in 20 mL ethanol were
refluxed for 1.5 hours. Workup (EtAc) and trituration with
benzene-hexane gave a 0.134 g white solid, 35% yield, with melting
point 157°C. NMR 7.82, 7.23 (4H, ABq,
JAB=8.6Hz), 4.37 (2H, br.s,
NH2), 2.39 (3H, s, methyl),
and 1.69 (9H, s, t-Bu).
Compounds 3b (1.5 g, 6.8 mmol/L) and 2b (1 g, 8 mmol/L), t-Bu hydrazine HCl, and 0.5 g KOH in 40 mL ethanol were refluxed for 3 hours. Workup (EtAc) and recrystallization from cyclohexane gave a 0.58-g white solid, 31% yield, with melting point 153°C. NMR 7.86, 7.38 (4H, ABq, JAB=8.8Hz), 4.36 (2H, br.s, NH2), and 1.68 (9H, s, t-Bu).
4. 1-t-Bu-3-Aryl-4 Amino Pyrazolo[3.4-d] Pyrimidines
Compound 4a (PP1/AGL1872 [0.13 g]). Compound 3a and 3 mL
formamide were heated at 180°C for 3 hours. To the cooled reaction
water was added the precipitated solid that was filtered and
recrystallized from benzene-hexane to give a 0.12 g white solid, 83%
yield, with melting point 175°C. NMR 8.35 (1H,
s, H2), 7.58, 7.32 (4H,
ABq, JAB=8.2 Hz), 5.36
(2H, br.s, NH2), 2.43
(3H, s, methyl), and 1.84
(9H, s).
Compound 4b (PP2/AGL1879 [0.25 g]). Compound 3b and 4 mL formamide were heated at 180°C for 3 hours. Workup (CH2Cl2) and chromatography gave a 0.16 g white solid, 58% yield, with melting point 203°C. NMR 8.35 (1H, s, H2), 7.65, 7.50 (4H, ABq, JAB=8.2 Hz), 5.54 (2H, br.s, NH2), and 1.82 (9H, s).
Cells and Reagents
Swiss 3T3 cells (American Type Culture Collection No. 92), NIH
3T3 cells stably transfected with a constitutively active mutant (F527)
of chicken c-src gene,7 and TRMP cells
overexpressing human PDGF ß-receptor9 were grown in
DMEM, supplemented with 4.5 g/L glucose, glutamine, antibiotics,
and 10% FCS (Life Technologies). PAE cells, stably transfected with
PDGF ß-receptor (PAE/PDGFß-R cells), PDGF -receptor
(PAE/PDGF-R cells), or FGF receptor-1 (PAE/FGFR-1 cells), were
kindly provided by Dr Lena Claesson-Welsh (Department of Medical
Chemistry, Uppsala University, Uppsala, Sweden) and were grown in
DMEM/Ham's F12 1:1 medium, supplemented as above. VEGF receptor
KDR-expressing cells (PAE/KDR cells) were previously
described.30 31 Human coronary artery
endothelial cells (HCAECs) and HCASMCs were purchased
from Clonetics and grown in EBM medium (supplemented with EGM-MV
SingleQuots) or in SmBM medium (supplemented with SmGM-2 SingleQuots),
respectively (Clonetics).
Recombinant human Src kinase
(p60c-src) was purified from overexpressing Sf9
cells as described earlier.32 The monoclonal
anti-Src antibody (OPO 7L) and the agarose-coupled
anti-Src antibody (OPO 7A) were obtained from Dianova.
Antibodies against phosphotyrosine (RC20H, horseradish peroxidase
conjugated) were from Transduction Laboratories. For detection of PDGF
ß-receptors by immunoblotting, antibody
DIG-17 or antibody 06-498 from Upstate Biotechnology was
used. The same antibody, 06-498, was used for immunoprecipitation of
PDGF ß-receptor33 and NEF antiserum for
immunoprecipitation of the VEGF receptor KDR,30
respectively. Wheat germ agglutinin coupled to agarose was obtained
from Pharmacia, and [
-32P]ATP was purchased
from NEN-DuPont. The "optimal Src substrate" peptide
AEEEIYGEFEAKKKK34 was kindly provided by U. Engström
(Ludwig Institute for Cancer Research, Uppsala, Sweden). Human
recombinant PDGF-BB, PDGF-AA, basic FGF (bFGF), acidic FGF (aFGF), and
IGF-I were obtained from TEBU, and human recombinant EGF was from
Biomol. Human recombinant VEGF165 was a kind gift
from D. Gospodarowicz (Chiron, Emeryville, Calif).
In Vitro Phosphorylation Assay of
Src
For in vitro assays of Src kinase activity, 0.2 to
0.4 U of p60c-src were incubated in the absence
or presence of the inhibitors or vehicle (DMSO, final
concentration 1%) and with the optimal Src substrate peptide (30
µmol/L) in assay buffer containing (in mmol/L) HEPES (pH 7.4)
50, MnCl2 10, and DTT 2 (final concentrations)
for 15 minutes on ice. Then, [-32P]ATP was
added (0.25 µCi, 10 µmol/L final concentration), and the
mixture (25 µL final volume) was transferred to 30°C and incubated
for 30 minutes. Thereafter, the reaction was quenched by adding 5 µL
of 120 mmol/L EDTA, and 10 µL of the reaction mixture was
spotted on Whatman P81 phosphocellulose paper, dried for 30 minutes,
washed 4 times for 10 minutes with 75 mmol/L phosphoric acid, and
dried. Radioactivity in the spots was quantified with a phosphor imager
(Molecular Imager GS250, Bio-Rad). Background correction was made using
reactions without peptide substrate. This assay was also applied to
PDGF ß-receptor preparations (see below) to reveal possible Src-like
kinase activity in the preparation. Preparations (5 µL) were
analyzed as above. IC50 values were
obtained by fitting the data points using a nonlinear regression
program (Graph III).
In Vitro Phosphorylation Assays of the PDGF
ß-Receptor, KDR, IGF-I Receptor, and FGF Receptor-1
In vitro autophosphorylation of PDGF
ß-receptor with purified Swiss 3T3 cell membranes was carried out as
described.7 To test the effect of inhibitors
on partially purified PDGF ß-receptor from TRMP cell membranes,
aliquots of a receptor preparation obtained as in Reference 99 were
preincubated with the compounds or vehicle (DMSO, final concentration
1%) for 15 minutes on ice in the presence of 50 mmol/L HEPES (pH
7.4), 5 mmol/L MnCl2, and 100 µmol/L
sodium orthovanadate (final concentrations). Then, PDGF-BB (or the
respective solvent containing 0.5 mol/L NaCl, 20 mmol/L HEPES [pH
7.4], and 5 mg/mL BSA) was added for 20 minutes on ice, and
subsequently, autophosphorylation was allowed in the
presence of [-32P]ATP (3.5 µCi, 10
µmol/L final concentration, final volume 25 µL) for 10 minutes. The
reaction was quenched by addition of SDS-PAGE sample buffer, and
receptor phosphorylation was evaluated by SDS-PAGE
followed by phosphor imager analysis of the fixed, stained, and
dried gels.
For in vitro phosphorylation of PDGF ß-receptor, FGF receptor-1 and KDR expressed in PAE cells, subconfluent PAE/PDGFß-R cells, PAE/FGFR1 cells, and PAE/KDR cells were used as previously described.30 In addition, primary HCASMCs and HCAECs were used in the same way to analyze the PDGF ß-receptor, FGF receptor-1, and IGF-I receptor. After a 10-minute preincubation with different concentrations of PP1/AGL1872, 100 µmol/L Na3VO4 was added 5 minutes before stimulation of cells at 37°C with 50 ng/mL PDGF-BB (5 minutes), 50 ng/mL bFGF (10 minutes), 50 ng/mL IGF-I (2 minutes), or 50 ng/mL VEGF for 5 minutes, respectively. Immunoprecipitation was performed with a PDGF ß-receptor–specific antiserum (06–498), a monoclonal chicken anti-FGF receptor antiserum (kindly provided by Sabine Werner, Max-Planck-Institute for Biochemistry, Martinsried/Munich, Germany), an anti–IGF-I receptor-specific antiserum (Santa Cruz Biotechnology, catalog No. sc-713), or an antiserum recognizing KDR (NEF). Immunoprecipitates immobilized on protein A–Sepharose CL 4B (Pharmacia) were used for an immune complex–kinase assay, which was carried out for 7 minutes at room temperature in 25 µL using the previously described buffer. After SDS-PAGE, gels were incubated for 30 minutes in 2.5% glutaraldehyde, washed twice for 15 minutes in 10% acetic acid/40% methanol, treated for 1 hour at 55°C in 1 mol/L KOH, washed 3 times for 20 minutes in 10% acetic acid/40% methanol, dried, and exposed to Hyperfilm MP (Amersham).
Protein Phosphorylation in Intact Cells
To analyze Src-dependent tyrosine
phosphorylation in intact cells, NIH3T3 fibroblasts
transfected with a cDNA for chicken p60srcF527
were treated in 24-well plates for 2 hours under serum-free conditions
with the inhibitors or corresponding vehicle (DMSO, final
concentration 0.3% to 1%). Thereafter, the cells were washed 3 times
with PBS and extracted with lysis buffer as
described.7 Lysate protein (10 µg) was subjected to
SDS-PAGE, immunoblotting using RC20H
anti-phosphotyrosine antibodies (1:2500, Transduction Laboratories),
and detection with the enhanced chemiluminescence reagent (Amersham).
To quantify inhibitory effects, major phosphotyrosine bands
at 
65 or 120 kDa were analyzed by scanning the films with a
flatbed office scanner and evaluating intensity using the program NIH
Image 1.57. To detect the Src
phosphorylation level, lysates of respectively treated
Src-transformed cells (1 nearly confluent well of a 6-well
plate per point) were subjected to immunoprecipitation with
agarose-coupled anti-Src OPO 7A antibodies (Dianova). The
immunoprecipitates were washed 3 times with lysis buffer and subjected
to SDS-PAGE and immunoblotting with
anti-phosphotyrosine antibodies or anti-Src antibodies.
For demonstration of tyrosine phosphorylation of PDGF ß-receptor or KDR in transfected PAE cells and in HCASMCs or HCAECs, respectively, cells were starved overnight in serum-free medium and incubated for 10 minutes at 37°C with different concentrations of PP1/AGL1872 and for another 5 minutes with 100 µmol/L Na3VO4 before stimulation of cells with 50 ng/mL PDGF-BB or 50 ng/mL VEGF for 5 minutes at 37°C. After a wash with ice-cold PBS containing 100 µmol/L Na3VO4, cells were solubilized in lysis buffer as previously described.35 The cell lysates were centrifuged at 10 000g for 15 minutes, and the supernatants were used for immunoprecipitation with antisera recognizing the PDGF ß-receptor (R3) or KDR (NEF). Immunoprecipitates were bound on protein A–Sepharose CL 4B, subjected to SDS-PAGE, and blotted onto a nitrocellulose membrane (Hybond C Extra, Amersham). Phosphorylated proteins were detected by immunoblotting using the horseradish peroxidase–conjugated anti-phosphotyrosine antibody RC20H (Transduction Laboratories) followed by a chemoluminescence-based detection system (enhanced chemiluminescence, Amersham).
PDGF ß-receptor and EGF receptor autophosphorylation
in intact Swiss 3T3 cells was estimated as described.7
Ligand-dependent receptor phosphorylation in PAE cells
overexpressing PDGF ß-receptor, PDGF -receptor, or FGF receptor-1
was estimated similarly, by starving confluent cultures for 6 to 16
hours in serum-free medium, treating for 2 hours with
inhibitors or vehicle, and stimulating for 10 minutes with
50 ng/mL PDGF-BB, 10 minutes with 50 ng/mL PDGF-AA, and 5 minutes with
100 ng/mL aFGF, respectively. Cell lysates (usually the lysates of 1
well of a 6-well plate per point) were incubated with 30 µL of lysis
buffer–equilibrated wheat germ agarose beads for 1 hour with
end-over-end rotation. The beads were washed 3 times with lysis buffer,
and bound proteins were extracted by boiling with SDS-PAGE sample
buffer and analyzed by immunoblotting.
Quantitative evaluation of the immunoblots was carried out by scanning appropriately exposed films with a flatbed scanner and analyzing the data with the program NIH Image 1.57.
Assessment of Cell Migration (Chemotaxis Assay)
The migratory response of HCASMCs and PAE/KDR cells was assessed
using the modified Boyden chamber (Neuro Probe, Inc) and
collagen-coated polycarbonate filters (Nuclepore) with pore diameters
of 8 µm, as previously described.36 In brief,
HCASMCs were starved for 24 hours in DMEM (Biochrom) containing 0.1%
BSA before the assay. To detach cells from the culture flasks, an
ice-cold 0.02% EDTA solution was added to the cells for 5 minutes.
Then, cells were collected by centrifugation and
resuspended in DMEM containing 0.1% BSA. PAE/KDR cells were cultivated
and assayed in Hamm F12 medium containing 10% FCS. To the medium in
the lower part of the Boyden chamber, PDGF-BB, VEGF, bFGF, or IGF-I
(each 10 ng/mL respectively), or 20% FCS, was added. PP1/AGL1872 was
added to both the upper and the lower chambers. Suspended cells were
given 4 hours for migration after a preincubation period of 30 minutes
in PP1/AGL1872. The number of cells that migrated without specific
stimulation was referred to as 100% baseline migration
(chemokinesis). The assay was performed in triplicate, and 5
medium-power fields per well were counted using a light microscope
(Jenalab). Data were reproduced in at least 2 independent
experiments.
Assessment of Cell Proliferation
Cell proliferation was assessed using a DNA synthesis assay
based on measurement of [3H]thymidine
incorporation. Assays with Swiss 3T3 cells were performed exactly as
previously described, and HCAEC and HCASMC proliferation was assessed
similarly.7 In brief, HCAECs or HCASMCs were seeded
sparsely in 12-well culture dishes. After 24 hours, cells were washed 2
times with endothelial basal medium containing 1% FCS (HCAECs) or
smooth muscle basal medium containing 1% FCS (HCASMCs),
respectively, and incubated for an additional 48 hours with 1 renewal
of medium. Cells were incubated for 15 minutes with different
concentrations of PP1/AGL1872 (0.1, 1, and 10 µmol/L) or with
the solvent DMSO alone and were stimulated with 3 ng/mL VEGF or with 15
ng/mL PDGF-BB for 20 hours, followed by addition of 0.25 µCi of
[3H]thymidine per milliliter (Amersham) for 2
hours. Finally, precipitated DNA was quantitated by liquid
scintillation counting.
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PP1/AGL1872 Is a Potent Inhibitor of p60c-src and Src Activity in Intact Cells
Because the synthetic protocols for PP1 and PP2 are not available,27 both compounds were synthesized by us and named PP1/AGL1872 and PP2/AGL1879, respectively. We assayed PP1/AGL1872 and PP2/AGL1879 for their activity on recombinant p60c-src using the optimal Src peptide substrate, AEEEIYGEFEAKKKK.34 PP1/AGL1872 inhibits p60c-src in vitro with an IC50 of 0.2 µmol/L (Table 1
); ie, we found an
IC50 50 times higher than that reported for
inhibition of p56Lck in
vitro.27 PP2/AGL1879 had a somewhat
lower activity in this assay with an IC50
of 0.3 µmol/L (Table 1). Therefore, further analysis
was carried out primarily with PP1/AGL1872; however, in all types of
assays described below, PP2/AGL1879 yielded similar results (data not
shown). In NIH3T3 cells transformed by expression of the constitutively
active Src variant p60c-srcF527,
highly elevated tyrosine phosphorylation of multiple
proteins is detectable as compared with untransfected NIH3T3 cells (see
also Reference 3737 . When Src-transformed NIH3T3 cells were
exposed to PP1/AGL1872, tyrosine phosphorylation was
inhibited in a dose-dependent manner (Figure 2). Also,
p60c-srcF527 autophosphorylation
detected in anti-Src immunoprecipitates from these cells was
inhibited in a dose-dependent manner (Figure 2). Quantification
revealed IC50 values of 0.7 and 0.9 µmol/L
for both effects, respectively (Table 1), ie, close to the value
reported for inhibition of p56Lck by PP1 in
intact T cells.27 Taken together, PP1/AGL1872
inhibits p60c-src in vitro and in intact
cells.
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PP1/AGL1872 Potently Inhibits PDGF-BB–Induced Migration of Human
Vascular Smooth Muscle Cells but Not VEGF-Induced Migration of Human
Endothelial Cells
To study the role of Src-family kinases in growth
factor–induced cell migration, we used a modified Boyden chamber assay
to measure PDGF-stimulated migration of HCASMCs. PDGF-BB (10 ng/mL)
stimulates HCASMC migration to about twice the baseline value (Figure 3A). PP1/AGL1872 exerts a strong and
dose-dependent inhibitory effect on this process. At a
concentration of 2 µmol/L, the PDGF-BB–stimulated value is
reduced to 69% of the stimulated control value
(IC50=3 µmol/L), and PP1/AGL1872 at
10 µmol/L abolishes the majority of the stimulatory effect of
PDGF-BB. PP1/AGL1872 does not affect the baseline value of HCASMC
migration in the absence of PDGF-BB. As compared with the selective
PDGF ß-receptor blocker AG1295 (IC50=7
µmol/L; J. Waltenberger et al, unpublished data), PP1/AGL1872
is more potent in this assay. In contrast, PP1/AGL1872 has only a weak
effect on VEGF-induced migration of endothelial cells
(HCAECs). Even at a concentration of 10 µmol/L, the VEGF-induced
migration of these cells was inhibited by 30% (Figure 3B).
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In addition, we have compared the inhibitory effect of
PP1/AGL1872 on PDGF-BB–stimulated migration of HCASMCs with its effect
on migration stimulated by IGF-I, bFGF, and FCS. The latter 2 agents
had little effect on HCASMC migration in our assays (not shown),
whereas IGF-I treatment resulted in a small but reproducible migratory
response (Figure 3C
). Given the small stimulation by IGF-I,
sensitivity to PP1/AGL1872 is difficult to evaluate. Within these
limits, the available data suggest that PP1/AGL1872 exerts only very
weak if any inhibitory action on IGF-I–stimulated
migration of HCASMCs (IC50>>10 µmol/L,
Figure 3C).
PP1/AGL1872 Inhibits PDGF ß-Receptor Kinase
We tested the effect of PP1/AGL1872 on PDGF ß-receptor
kinase autophosphorylation. Interestingly, we found
PP1/AGL1872 to potently inhibit PDGF ß-receptor autokinase activity
in Swiss 3T3 cell membranes (IC50=0.1
µmol/L, Figure 4A, upper panel, Table 2), in preparations of partially purified
human PDGF ß-receptor (IC50=0.1 µmol/L,
Figure 4, lower panel, Table 2) and in intact Swiss 3T3
cells (IC50=0.5 µmol/L, Figure 4B).
Because heterologous phosphorylation of PDGF
ß-receptor by Src-family kinases has been previously
described,26 we wondered whether this observed
inhibition could be secondary to Src-family kinase
inhibition. Therefore, we tested the possible presence of
Src-family kinases in the semipurified PDGF ß-receptor
fraction from TRMP cells using the Src kinase in vitro
assay with the optimal Src peptide substrate peptide (see
Materials and Methods). Only a very small activity toward the Src
substrate peptide could be detected in these preparations, and the
activity was completely suppressed by 10 µmol/L AG1296, the
selective PDGF receptor blocker (Figure 5). In contrast, as described
earlier,7 recombinant p60c-src
is not affected by AG1296 (Figure 5). Thus, the small activity
toward the Src substrate peptide in the PDGF ß-receptor
preparation represents some minor activity of PDGF receptor
toward this substrate. We conclude that the PDGF ß-receptor
preparations used in our assays are devoid of Src-family kinase
activity and, therefore, the inhibition of PDGF ß-receptor autokinase
activity by PP1/AGL1872 is not mediated by Src kinase inhibition, but
is a direct effect.
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We further investigated whether inhibition of the PDGF ß-receptor
kinase by PP1/AGL1872 would occur with some specificity or whether
other receptor tyrosine kinases would be affected similarly. EGF
receptor autophosphorylation in intact Swiss 3T3 cells
was completely refractory to inhibition by PP1/AGL1872 (Figure 4B). Also, EGF receptor autophosphorylation in
Swiss 3T3 cell membranes was inhibited with an
IC50 of >3 µmol/L (ie, at least 30 times
higher than that for inhibition of PDGF ß-receptor
autophosphorylation in the same type of assay [data
not shown]).
Using an in vitro kinase assay, PP1/AGL1872 inhibits
PDGF-BB–stimulated phosphorylation of the PDGF
ß-receptor in receptor immunoprecipitates from PAE/PDGFß-R cells in
a dose-dependent manner (IC50<1 µmol/L,
Figure 6A, Table 2). In contrast,
VEGF-induced phosphorylation of KDR in PAE/KDR cells
remained essentially unaffected by PP1/AGL1872 (Figure 6B).
Importantly, VEGF-stimulated autophosphorylation of KDR
in intact HCAECs is also resistant to PP1/AGL1872, whereas
PDGF-BB–stimulated PDGF receptor phosphorylation in
HCASMCs is potently inhibited by PP1/AGL1872 (Figure 6C and 6D).
Subjecting PAE cells overexpressing human PDGF ß-receptor, PDGF
-receptor, or FGF receptor-1 to PP1/AGL1872 treatment revealed
resistance of FGF receptor-1 to the compound (Figure 7A); rather than inhibition we
reproducibly observed enhancement of FGF receptor-1
phosphorylation in the presence of PP1/AGL1872. The
PDGF ß-receptor was similarly susceptible to inhibition in this
background (Table 2), as in Swiss 3T3 cells or HCASMCs, and PDGF
-receptor was inhibited at high concentrations of PP1/AGL1872 but
was clearly less susceptible than the PDGF ß-receptor (Figure 7A, Table 2).
|
|
Unlike the PDGF ß-receptor, the FGF receptor-1 showed a high baseline
level of tyrosine phosphorylation in HCASMCs.
Stimulation with bFGF (50 ng/mL) had only a very weak effect on FGF
receptor-1 phosphorylation. In the presence of
PP1/AGL1872, the phosphorylation pattern of the FGF
receptor-1 was not affected, and there was definitely no reduction
below the unstimulated baseline level (Figure 7B). In contrast,
IGF-I (50 ng/mL) strongly stimulated the
autophosphorylation of the IGF-I receptor, and the
presence of PP1/AGL1872 (>1 µmol/L) led not to inhibition, but
to a further enhancement of tyrosine phosphorylation
(Figure 7C).
In summary, PP1/AGL1872 inhibits PDGF ß-receptor kinase with efficacy
similar to that of Src-family kinase activity in vitro and in intact
cells, whereas PDGF -receptor is less sensitive and the agent has
little effect on EGF receptor kinase, VEGF receptor kinase KDR, FGF
receptor-1, and IGF-I receptor kinase.
PP1/AGL1872 Differentially Affects Cell Proliferation Driven by
Different Growth Factors
Given the putative role of Src-family kinases for downstream
signaling of PDGF ß-receptor and the susceptibility of both
Src-family kinases and PDGF ß-receptor to PP1/AGL1872, we expected
potent inhibition of PDGF-stimulated proliferation by the compound.
PDGF-BB (15 ng/mL) stimulated [3H]thymidine
incorporation of HCASMCs strongly (Figure 8A) and PP1/AGL1872 inhibited DNA
synthesis potently, with only a minor effect also on basal DNA
synthesis (Figure 8A) (mean IC50=1
µmol/L in multiple independent experiments). VEGF-induced stimulation
of DNA synthesis in HCAECs was comparable with that of PDGF-BB in
HCASMCs, and PP1/AGL1872 inhibited this stimulatory effect with an
IC50 of 3 µmol/L (Figure 8B).
PP1/AGL1872 did not affect cell viability within the studied
concentration range of up to 30 µmol/L. Also, adhesion of
endothelial cells and smooth muscle cells to culture
flasks was not affected by PP1/AGL1872. Cells neither detached nor
changed in shape. When DNA synthesis of HCASMCs was stimulated with
IGF-I (10 ng/mL), stimulation was only weak and the
inhibitory effect of PP1/AGL1872 was difficult to judge but
apparently less pronounced than that toward PDGF stimulation
(IC50 of 4 µmol/L). Likewise, FCS
(20%)–stimulated proliferation of HCASMCs was inhibited by
PP1/AGL1872, with a somewhat higher IC50 of
3 µmol/L (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PP1/AGL1872 Is a Dual Inhibitor of Src Kinase and PDGF ß-Receptor Kinase Activity
Because activation of Src-family kinases is an important downstream mediator of PDGF ß-receptor signaling, we wondered whether selective Src inhibitors might be used to interfere with PDGF-mediated mitogenesis and migration of HCASMCs in the context of atherosclerosis and restenosis. Also, the utilization of selective Src kinase inhibitors is expected to yield important information as to the extent to which Src is involved in the signaling of different receptor tyrosine kinases implicated in the proliferation of vascular cells as PDGF ß-receptor and the VEGF receptor KDR. The compound PP1 has been previously described as a selective inhibitor for the Src-family kinases p56Lck and FynT27 and was likely also to inhibit p60c-src. We therefore examined its inhibitory activity on this kinase. As expected, we found PP1/AGL1872 to potently inhibit p60c-src in vitro and Src-dependent tyrosine phosphorylation in intact cells (Figure 2
).
Because Src has been demonstrated to be essential for PDGF
ß-receptor signaling,26 we examined the ability of
PP1/AGL1872 to inhibit PDGF-dependent proliferation and migration of
HCASMCs. We found PP1/AGL1872 to be a potent inhibitor of
both processes. On detailed examination we found, to our surprise, that
PP1/AGL1872 potently inhibits the PDGF ß-receptor kinase itself but
has no inhibitory activity toward KDR, FGF receptor-1,
IGF-I receptor, or, at least in intact cells, EGF receptor. Also,
PP1/AGL1872 is only moderately effective on the PDGF -receptor.
These observations reveal that PP1/AGL1872 is not a selective Src
kinase inhibitor but interacts with Src kinase and
PDGFß-R kinase domain with similar efficacy. The specificity profile
of the compound, which is expected to reflect differential affinity to
the ATP-binding pockets of the various kinases,9 gives
rise to the interesting speculation that the PDGF ß-receptor kinase
ATP-binding pocket may be more related to the Src kinase
ATP-binding pocket than to that of the other receptors, including the
PDGF -receptor. This point requires further attention, including
future experiments to obtain detailed kinetic data on
inhibitor kinase interaction.
Dual Inhibition of PDGF ß-Receptor Kinase and Src-Family Kinases
as a Novel Concept to Prevent HCASMC Activation in Atherogenesis
and Restenosis
In line with the insensitivity of KDR to PP1/AGL1872, the compound
had little effect on VEGF-induced migration of
endothelial cells. Also, we observed little effect of
PP1/AGL1872 on the small increase of HCASMC migration in the presence
of IGF-I, whereas PDGF-stimulated migration was effectively blocked.
Thus, the inhibitory efficacy of PP1/AGL1872 on migration
induced by various motogens seems to be related to the susceptibility
of the receptor kinases to inhibition by the compound. Interestingly,
we even observed an enhanced phosphorylation of the FGF
receptor-1 and IGF-I receptor in the presence of PP1/AGL1872. One could
speculate that negative regulators of receptor
phosphorylation as protein-tyrosine phosphatases are
positively regulated by Src-family kinases, leading to elevated
receptor phosphorylation secondary to Src-family kinase
inhibition. Activation of protein-tyrosine phosphatases by
tyrosine-specific phosphorylation has been
reported.38 39 40
VEGF-stimulated DNA synthesis of HCAECs was susceptible to inhibition by PP1/AGL1872 (IC50=3 µmol/L). Likewise, DNA synthesis induced by FCS or IGF-I in HCASMCs was inhibited by PP1/AGL1872, albeit to a somewhat lesser extent than PDGF-stimulated DNA synthesis in HCASMCs. Similar data have been obtained for DNA synthesis stimulated by various mitogens in Swiss 3T3 fibroblasts (not shown). It seems tempting to speculate that the PP1/AGL1872 susceptibility of DNA synthesis induced by growth factors the receptor kinases of which are resistant to the agent reflects the involvement of Src-family kinases in the downstream mitogenic signaling of these receptors. In line with this assumption, PDGF-stimulated DNA synthesis of HCASMCs is rather sensitive to PP1/AGL1872 (IC50=1 µmol/L), possibly as the result of dual inhibition of the PDGF ß-receptor kinase itself and of downstream Src-family kinases. This interpretation of the data would imply that Src-family kinases are to some extent involved in mitogenic signaling of various growth factor receptors, including KDR, but not, or to a lesser extent, in the signaling events leading to cell migration/chemotaxis. Indeed, for the PDGF ß-receptor, abrogation of receptor interaction with Src by mutating a Src phosphorylation site of the receptor leads to a shift of receptor signaling activity in that the mitogenic response is attenuated and the motogenic response is enhanced.26 These data are also in line with the hypothesis that Src is primarily involved in mitogenic signaling of the PDGF ß-receptor and that the chemotactic response is not mediated by Src. Thus, the inhibitory effect of PP1/AGL1872 on migration observed by us is more likely to result from the direct effect of the compound on the receptor and not as a result of its anti-Src activity.
PDGF signaling inhibitors acting by different mechanisms have been shown to combat successfully PDGF-dependent pathological processes in restenosis.13 14 17 19 41 42 According to our data, PP1/AGL1872 potently interferes with PDGF-driven migration and proliferation of HCASMCs, including basal DNA synthesis, and differentially affects KDR signaling. It has little effect on VEGF-stimulated migration of PAE/KDR cells and no effect on basal DNA synthesis of HCAECs. The inhibitory effect on VEGF-stimulated DNA synthesis of HCAECs occurs at 10-fold higher concentrations than that on PDGF-stimulated DNA synthesis of HCASMCs. VSMC migration and proliferation, shown here to be both inhibited by PP1/AGL1872, are crucial events underlying neointima formation.43 It would, therefore, be worthwhile testing PP1/AGL1872 in different in vivo models to investigate the functional significance of a combined Src/PDGF ß-receptor inhibition on neointima formation (ie, in the rat and the pig). It will be interesting to examine whether the dual inhibition is more potent than just PDGF ß-receptor inhibition as achieved by using AG129516 to prevent vascular remodeling and restenosis.
|
Acknowledgments |
|---|
This work was supported in part by a joint German-Israeli research project (DISMED87) grant (to A.L., F.-D.B., and J.W.), a grant from The Israel Science Foundation (The Israel Academy of Sciences and Humanities) (to A.L.), a grant from the Max-Planck-Society (to F.-D.B.), and a grant from the Land Baden-Württemberg (Landesforschungsschwerpunkt "Wachstumsfaktor-Modulation als Therapieprinzip") (to J.W.). We are grateful to Ulla Engström for synthesis of the optimal Src peptide and to Dr L. Claesson-Welsh for kind provision of various cell lines.
Received June 12, 1998; accepted April 15, 1999.
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J. Graff, U. Klinkhardt, V. B. Schini-Kerth, S. Harder, N. Franz, S. Bassus, and C. M. Kirchmaier Close Relationship between the Platelet Activation Marker CD62 and the Granular Release of Platelet-Derived Growth Factor J. Pharmacol. Exp. Ther., March 1, 2002; 300(3): 952 - 957. [Abstract] [Full Text] [PDF]
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 F. Carlomagno, D. Vitagliano, T. Guida, M. Napolitano, G. Vecchio, A. Fusco, A. Gazit, A. Levitzki, and M. Santoro The Kinase Inhibitor PP1 Blocks Tumorigenesis Induced by RET Oncogenes Cancer Res., February 1, 2002; 62(4): 1077 - 1082. [Abstract] [Full Text] [PDF]
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 Y. Saito, J. Haendeler, Y. Hojo, K. Yamamoto, and B. C. Berk Receptor Heterodimerization: Essential Mechanism for Platelet-Derived Growth Factor-Induced Epidermal Growth Factor Receptor Transactivation Mol. Cell. Biol., October 1, 2001; 21(19): 6387 - 6394. [Abstract] [Full Text] [PDF]
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 E. Schneider, A. Schmid-Kotsas, J. Zhao, H. Weidenbach, R. M. Schmid, A. Menke, G. Adler, J. Waltenberger, A. Grunert, and M. G. Bachem Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells Am J Physiol Cell Physiol, August 1, 2001; 281(2): C532 - C543. [Abstract] [Full Text] [PDF]
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I. A. Yamboliev, J. Chen, and W. T. Gerthoffer PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs Am J Physiol Cell Physiol, August 1, 2001; 281(2): C709 - C718. [Abstract] [Full Text] [PDF]
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 J. N. Oak, N. Lavine, and H. H. M. Van Tol Dopamine D4 and D2L Receptor Stimulation of the Mitogen-Activated Protein Kinase Pathway Is Dependent on trans-Activation of the Platelet-Derived Growth Factor Receptor Mol. Pharmacol., July 1, 2001; 60(1): 92 - 103. [Abstract] [Full Text]
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 J. P. M. Wesselman, A. D. Dobrian, S. D. Schriver, and R. L. Prewitt Src Tyrosine Kinases and Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinases Mediate Pressure-Induced C-Fos Expression in Cannulated Rat Mesenteric Small Arteries Hypertension, March 1, 2001; 37(3): 955 - 960. [Abstract] [Full Text] [PDF]
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R. A. Blake, M. A. Broome, X. Liu, J. Wu, M. Gishizky, L. Sun, and S. A. Courtneidge SU6656, a Selective Src Family Kinase Inhibitor, Used To Probe Growth Factor Signaling Mol. Cell. Biol., December 1, 2000; 20(23): 9018 - 9027. [Abstract] [Full Text]
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S. Rosenkranz, Y. Ikuno, F. L. Leong, R. A. Klinghoffer, S. Miyake, H. Band, and A. Kazlauskas Src Family Kinases Negatively Regulate Platelet-derived Growth Factor alpha Receptor-dependent Signaling and Disease Progression J. Biol. Chem., March 24, 2000; 275(13): 9620 - 9627. [Abstract] [Full Text] [PDF]
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S. Gro{beta}, A. Knebel, T. Tenev, A. Neininger, M. Gaestel, P. Herrlich, and F. D. Bohmer Inactivation of Protein-tyrosine Phosphatases as Mechanism of UV-induced Signal Transduction J. Biol. Chem., September 10, 1999; 274(37): 26378 - 26386. [Abstract] [Full Text] [PDF]
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C. R. Hauck, D. A. Hsia, and D. D. Schlaepfer Focal Adhesion Kinase Facilitates Platelet-derived Growth Factor-BB-stimulated ERK2 Activation Required for Chemotaxis Migration of Vascular Smooth Muscle Cells J. Biol. Chem., December 22, 2000; 275(52): 41092 - 41099. [Abstract] [Full Text] [PDF]
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S. Heeneman, J. Haendeler, Y. Saito, M. Ishida, and B. C. Berk Angiotensin II Induces Transactivation of Two Different Populations of the Platelet-derived Growth Factor beta Receptor. KEY ROLE FOR THE p66 ADAPTOR PROTEIN Shc J. Biol. Chem., May 19, 2000; 275(21): 15926 - 15932. [Abstract] [Full Text] [PDF]
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