Published online before print December 29, 2004, doi: 10.1161/01.RES.0000154595.87608.db
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© 2005 American Heart Association, Inc.
Molecular Medicine |
Platelet-Derived Growth Factor-BB–Induced Human Smooth Muscle Cell Proliferation Depends on Basic FGF Release and FGFR-1 Activation
From the University of Washington School of Medicine, Department of Surgery, Seattle, Wash.
Correspondence to Esther Millette, PhD, University of Washington School of Medicine, Department of Surgery, Box 356410, 1959 NE Pacific St, Seattle, WA 98195-6410. E-mail millette{at}u.washington.edu
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Abstract |
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We have shown that the G protein–coupled receptor (GPCR) agonists, thrombin and Factor Xa, stimulate smooth muscle cell (SMC) proliferation through transactivation of the EGF receptor (EGFR) or the FGF receptor (FGFR), both of which are tyrosine kinase receptors. In the present study, we investigated whether platelet-derived growth factor (PDGF), a tyrosine kinase receptor agonist, might transactivate another tyrosine kinase receptor to induce SMC proliferation. Because heparin inhibits PDGF-mediated proliferation in human SMCs, we investigated whether the heparin-binding growth factor basic fibroblast growth factor (bFGF) and one of its receptors, FGFR-1, play a role in the response of human arterial SMCs to PDGF-BB. PDGF-BB induced the release of bFGF and sustained phosphorylation of FGFR-1 (30 minutes to 6 hours). A bFGF-neutralizing antibody inhibited PDGF-BB–mediated phosphorylation of FGFR-1, DNA synthesis, and cell proliferation. In the presence of bFGF antibody, PDGF-BB–induced early activation of ERK (0 to 60 minutes) was not affected, whereas late ERK activation (2 to 4 hours) was reduced. When FGFR-1 expression was suppressed using small interfering RNA (siRNA), ERK activation was reduced at late, but not early, time points after PDGF-BB stimulation. Addition of bFGF antibody to cells treated with siRNA to FGFR-1 had no further effect on ERK activation. Our results provide support for a novel mechanism by which PDGF-BB induces the release of bFGF and activation of FGFR-1 followed by the sustained activation of ERK and proliferation of human SMCs.
Key Words: platelet-derived growth factor • bFGF release • FGFR-1 • ERK • Akt
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Introduction |
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Activation of the ERK pathway is critical for cell proliferation.1 Although mitogens appear to activate maximally ERK within 15 minutes, prolonged activity of ERK is necessary for cell cycle progression through G1/S.2–4 Several authors have described two waves of signaling in response to mitogens: the first leads cells into G1, whereas the second is responsible for progression from G1 to S phase.3,5,6 For mitogens that bind to G protein–coupled receptors (GPCRs), the second wave of signaling depends on a receptor-linked tyrosine kinase.3,7,8 For example, in rat smooth muscle cells (SMCs), sustained ERK activity in response to thrombin and angiotensin II involves the release of heparin-binding EGF-like growth factor (HB-EGF), which in turn, activates the EGF receptor (EGFR).7,9,10 Heparin may inhibit thrombin-induced migration and proliferation of rat SMCs, in part, by preventing the binding of HB-EGF to heparan sulfate proteoglycans (HSPGs),7,11 because heparin binding growth factors (eg, HB-EGF, basic fibroblast growth factor [bFGF]) require HSPGs as coreceptors for biological activity.12
Like GPCR ligands, platelet-derived growth factor (PDGF)-BB induces two waves of signaling, and the second peak of ERK and phosphatidylinositol 3 (PI3)-kinase activity is required for progression through G1.6 However, EGFR transactivation is not involved.7 Of significant interest is the observation that ERK activity induced by PDGF-BB is inhibited at late time points by heparin.13 This suggests that heparin binding growth factors, other than EGFR ligands, may be involved in the response to PDGF-BB. In this regard, we recently found that thrombin- and factor Xa–induced proliferation of human SMCs depends on endogenous bFGF.14 In addition, lysophosphatidylcholine was reported to induce migration and angiotensin II to induce proliferation of human SMCs through the release of bFGF.15,16 For these reasons, we hypothesized that endogenous bFGF mediates the proliferative activity of PDGF-BB, a tyrosine kinase receptor agonist. In this study, we report a novel role for endogenous bFGF and FGF receptor (FGFR)-1 in PDGF-BB–mediated human SMC proliferation. In addition, prolonged activity of ERK is required for cell cycle progression and is dependent on bFGF-FGFR-1.
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Materials and Methods |
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Materials
PDGF-AA and -BB were a gift from Zymogenetics (Seattle, Wash). PDGF-CC, PDGF-DD, and the bFGF ELISA kit were purchased from R&D Systems. Anti–smooth muscle-
-actin, recombinant bFGF, actinomycin D, and heparin (porcine intestinal mucosa) were from Sigma-Aldrich. AG1296, PD98059, LY294002, and GF109203X (bisindolylmaleimide) were from Calbiochem. Neutralizing Ab against human basic fibroblast growth factor (bFGF) and ERK1/2 antiserum were generous gifts from Drs Michael Reidy17 and Karen Bornfeld (University of Washington, Seattle, Wash), respectively. The phosphotyrosine Ab (clone 4G10) was from Upstate Biotechnology, and the Ab against FGF receptor-1 was from Santa Cruz Biotechnology. Ab against phosphorylated ERK1/2 and Akt were obtained from Cell Signaling Technology. Ab against phosphorylated MEK 1/2 was obtained from New England Biolabs. Protein A-agarose was from Roche Diagnostics. All cell culture solutions were from Gibco.
Cell Culture
Human SMCs were prepared from abdominal aortas of organ donors with the approval of the Human Subject Review Board of the University of Washington. After removing endothelial cells, the inner part of the media was dissected and minced into explants, which were maintained in DMEM with 10% serum. After passage, the cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 200 U/mL penicillin, and 200 µg/mL streptomycin. Cell lines were used from passage 5 to 12.
Basic FGF Immunoassay
SMCs in 6-well plates were serum-deprived for 72 hours. At different times after stimulation, media was removed and cells were incubated with heparin (10 µg/mL) in PBS for 30 minutes at room temperature on a shaker. The heparin solution was removed and cells were detached with trypsin/EDTA. Cells were pelleted in a microcentrifuge and lysed in 1 mL HEB (25 mmol/L HEPES-NaOH, pH 7.5, 1 mmol/L EDTA, 150 mmol/L NaCl, 10 mmol/L NaF, 2 mmol/L sodium vanadate, 1 mmol/L benzamidine, 1% Nonidet P-40, 0.1% 2-mercaptoethanol, 1 mmol/L pepstatin A, 2 µg/mL leupeptin, and 20 kallikrein inhibitor units/mL aprotinin) and 20 µmol/L of phenylmethylsulfonyl fluoride. Levels of bFGF in media, heparin-wash, and cell lysates were determined by ELISA according to the manufacturer’s instructions.
3H-Thymidine Incorporation
Cells at 60% to 80% confluence were serum-deprived for 48 to 72 hours. Cells were stimulated with PDGF (10 ng/mL) and [3H]-thymidine (1 µCi/mL) was added 18 hours later. After another 8 hours, [3H]-thymidine incorporation was determined as previously described.14
Migration Assay
Microchemotaxis assays were performed as described18 for 5 hours at 37°C with 48-well chambers (Neuro Probe) and polycarbonate filters (10-µm pores; Nucleopore Corp) coated with monomeric collagen (100 µg/mL Vitrogen 100 in 0.1 mol/L acetic acid; Collagen Corp). Cells (35 000/well) were added to the upper chamber and chemoattractants or DMEM were added to the lower chamber. The migrating cells per high-power field were counted.
Growth Curve
SMCs were seeded (1200 cells/cm2) with media containing 10% FBS. The following day, media was changed to 0.5% FBS, and cells were stimulated 4 hours later. The media was changed every 48 hours. Cells were counted by Coulter counter (Beckman).
Immunoprecipitation of FGFR-1 and Western Blot Analysis
Immunoprecipitation of FGFR-1 was performed as previously described.14 Samples were subjected to SDS-PAGE (7% for FGFR-1 and 10% for ERK, Akt, MEK) and transferred to nitrocellulose membranes. Membranes were probed with antibodies at 4°C overnight. Immunodetection was performed by enhanced chemiluminescence (Amersham). Densitometric analysis of bands was performed using Image Quant (Molecular Dynamics).
Preparation of Small Interfering RNAs Targeting FGFR-1
Small interfering RNAs (siRNAs) targeting human FGFR-1 were designed using a siRNA construction kit, (Ambion) and were constructed from sense and anti-sense DNA oligonucleotides (Integrated DNA Technologies) using Basic Local Alignment Search Tool (BLAST) to avoid homology with other mRNA. The sense sequence for FGFR-1 siRNA was 5'-AAGTCGGACGCAACAGAGAAA-3'. The sequence for the scrambled siRNA (control) was 5'-AACAGAGAAAGTCGGACGCAA-3'.
Cells were transfected in 100-mm dishes with siRNAs (10 nmol/L) by calcium phosphate-precipitation for 15 hours, as described.19 Cells were washed three times with PBS, twice with media containing 15% FBS, and allowed to recover for at least 9 hours. For DNA synthesis and Western blot analysis for phosphoERK, cells were trypsinized and seeded into 12-well plates.
Statistical Analysis
All experiments were repeated at least four times. Statistical analyses were performed using one-way ANOVA followed by Bonferroni multiple comparison test or by a paired two-tailed t test as indicated. Statistical significance was accepted at P
0.05.
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Results |
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Endogenous bFGF Contributes to PDGF-BB–Induced Cell Proliferation, but Not Migration
In human SMCs, we found a strong inhibitory effect of heparin (100 µg/mL) on PDGF (AA, BB, CC, or DD)-induced DNA synthesis (Figure 1A). To determine whether an HSPG-sensitive EGFR ligand was involved, we blocked EGFR kinase activity with the selective inhibitor AG1478 (150 nmol/L). Because this treatment did not reduce PDGF-BB–induced DNA synthesis (unpublished data, 2002), we tested the involvement of bFGF. Preincubation of SMCs with bFGF antibody (30 µg/mL) inhibited DNA synthesis induced by the PDGF ligands (Figure 1A). In addition, cell proliferation induced by PDGF-BB was inhibited by 90±15% by the bFGF antibody and was completely abolished by heparin (Figure 1B).
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In contrast to proliferation, the bFGF antibody did not inhibit PDGF-BB-induced migration of human SMCs (Figure 1C). In control experiments, bFGF antibody inhibited migration in response to bFGF (Figure 1C).
PDGF-BB Activates FGFR-1
To investigate whether PDGF-BB activates FGFR-1, cells were stimulated with PDGF-BB for 30 minutes to 6 hours. Phosphorylation of FGFR-1 was increased within 30 minutes, lasted at least 6 hours, was comparable to receptor phosphorylation caused by bFGF (Figure 2A) and was inhibited by the bFGF antibody and heparin (Figure 2B).
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PDGF-BB Releases bFGF
The effect of the bFGF antibody suggests that bFGF was released by SMCs in response to PDGF. To test this hypothesis, levels of bFGF in the media, in the cell/matrix layer (released by heparin), and in intracellular stores were measured. After stimulation with PDGF-BB, bFGF increased slightly in the culture media at 1 hour (Table ). In the cell/matrix layer, the levels of bFGF increased by 2-fold within 30 minutes and 8-fold after 4 hours (Figure 3). Basic FGF in the cell/matrix layer remained elevated at 24 hours (Figure 3). In contrast, there was no difference in the levels of intracellular bFGF between PDGF-BB–stimulated cells and unstimulated control cells (Table).
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Time Window of Stimulatory Effect of bFGF
To determine when bFGF is required for PDGF-BB–induced DNA synthesis, cells were incubated with bFGF antibody 30 minutes before or 2 to 16 hours after PDGF-BB stimulation. As shown in Figure 4, treatment of cells with bFGF antibody could be delayed up to 4 hours without significantly reducing the inhibitory effect, whereas further delay resulted in a loss of inhibition. This suggests that the stimulatory effect of bFGF on PDGF-BB–induced DNA synthesis occurs within the first 4 to 6 hours.
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Basic FGF Contributes to PDGF-BB–Induced MEK and ERK Activation
To determine whether the activation of the ERK signal transduction pathway by PDGF-BB depends on endogenous bFGF, quiescent cells were stimulated with PDGF-BB, in the absence or the presence of bFGF antibody, nonspecific IgG, or heparin. PDGF-BB induced two peaks of ERK activation: the first was observed within 15 to 30 minutes, whereas the second was observed between 2 to 4 hours (Figure 5A and 5B). Preincubation of SMCs with bFGF antibody or heparin did not affect early ERK activation (0 to 60 minutes), but inhibited PDGF-BB–induced ERK activation at 2 and 4 hours (P<0.05) and at 8 hours (P=0.06) (Figure 5A and 5B). Consistent with this observation, PDGF-BB–induced phosphorylation of the ERK activator, MEK 1/2, was not altered by bFGF antibody or heparin at 15 to 30 minutes, but was inhibited by both after 2 and 4 hours (Figure 5A).
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Because PI3-kinase is known to contribute to PDGF-BB–induced mitogenesis, we investigated whether bFGF is required for the activation of Akt, a downstream protein kinase in the PI3-kinase signaling pathway. Preincubation of SMCs with bFGF antibody or heparin did not affect phosphorylation of Akt in response to PDGF-BB (Figure 5A), suggesting a specific effect of bFGF on MEK/ERK activity.
FGFR-1 Contributes to PDGF-BB–Induced ERK Activation and DNA Synthesis
FGFR-1 is reported to be the main receptor contributing to bFGF-induced mitogenesis in rat SMCs.20 To investigate the role of FGFR-1 in PDGF-BB–induced mitogenesis in human SMCs, we used siRNA to downregulate FGFR-1. As shown in Figure 6A, treatment of cells with FGFR-1 siRNA downregulated FGFR-1 expression from day 2 to 4 after transfection, while not affecting levels of smooth muscle--actin. In addition, there was no difference in cell number between cells transfected with FGFR-1 or scrambled siRNA (6978±623 and 7079±665 cells at day 4, respectively, n=4). Downregulation of FGFR-1 inhibited DNA synthesis in response to PDGF-BB (Figure 6B) and bFGF antibody did not further reduce PDGF-BB–induced [3H]-thymidine incorporation in FGFR-1 downregulated cells. In contrast, heparin was still inhibitory, suggesting additional mechanisms for heparin-mediated inhibition of PDGF-BB–induced DNA synthesis.
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To confirm a role for FGFR-1 in PDGF-BB signaling, we investigated whether FGFR-1 downregulation also affects late ERK phosphorylation (Figure 6C). Although bFGF-mediated ERK activation at 30 minutes was completely blocked in FGFR-1 downregulated cells, PDGF-BB–induced ERK activation was similar in FGFR-1 and scrambled siRNA-treated cells. In contrast, at 4 hours PDGF-BB–mediated ERK activation was reduced in FGFR-1 downregulated cells. In addition, bFGF antibody and heparin had no further inhibitory effect on ERK phosphorylation at 4 hours in FGFR-1 downregulated cells (Figure 6C). Depletion of FGFR-1 did not affect Akt phosphorylation by PDGF-BB (Figure 6C).
We investigated whether downregulation of FGFR-1 affects PDGF-BB–induced migration in human SMCs. Treatment of cells with FGFR-1 siRNA did not alter PDGF-BB–induced migration (unpublished data, 2004). This is consistent with the lack of an inhibitory effect of the bFGF antibody on PDGF-BB–induced migration in control cells (Figure 1C).
Signaling Proteins Involved in PDGF-BB–Induced bFGF Release
To investigate the pathways signaling in PDGF-BB–mediated bFGF release, quiescent cells were stimulated for 4 hours with PDGF-BB, in the absence or presence of 20 µmol/L AG1296 (AG, an inhibitor of PDGF receptor kinase), 10 µmol/L LY294002 (LY, a PI3-kinase inhibitor), 40 µmol/L PD98059 (PD, a MAPK inhibitor), or 10 µmol/L GF109203X (GFX, a PKC inhibitor). After 4 hours of stimulation with PDGF-BB, bFGF increased in the pericellular layer (heparin releasable; Figure 7A). Pretreatment of the cells with AG, LY, or GFX, but not PD, reduced PDGF-BB–induced bFGF levels in the pericellular layer (Figure 7A), suggesting that PI-3 kinase and PKC are involved in PDGF-BB–induced bFGF release and that PDGF receptors mediate this effect.
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To determine the effect of these inhibitors on phosphorylation of FGFR-1, cells were stimulated with PDGF-BB for 4 hours in the absence or the presence of AG, PD, LY, or GFX. Consistent with the previous data showing an inhibitory effect of AG, LY, and GFX on bFGF release, all but PD reduced PDGF-BB–induced FGFR-1 phosphorylation (Figure 7B).
Finally, we investigated the role of PDGF receptor kinase, PKC, and PI3-kinase on the sustained, bFGF-dependent, activity of ERK. Preincubation of SMCs with AG, LY, and GFX reduced the phosphorylation of ERK 4 hours after PDGF-BB stimulation (Figure 7C). Thus, PDGF-BB–induced bFGF release, FGFR-1 phosphorylation, and sustained ERK activity depend on PDGF receptor kinase, PKC and Akt activation. In addition, PDGF-BB–induced Akt activity, which is not affected by bFGF antibody (Figure 5), was unaltered by the MAPK inhibitor (Figure 7C), but was abolished by the PDGF receptor kinase inhibitor (Figure 7C), confirming that PDGF-BB activates Akt independently of bFGF and sustained MAPK activity.
We investigated whether bFGF transcription is required for PDGF-BB to induce bFGF release. We found that release of bFGF after 4 hours of stimulation with PDGF-BB was not altered by actinomycin D starting 1 hour before treatment (6.94±0.56 and 5.54±0.63 ng/106 cells without or with actinomycin D, respectively; mean±SEM; n=5; P=0.175), indicating that transcription is not required for PDGF-BB–mediated bFGF release.
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Discussion |
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The novel finding in the present study is that sustained ERK activation and subsequent proliferation in response to PDGF-BB is partially mediated by endogenous basic FGF through activation of FGFR-1. It is known that entry into S phase requires ERK activity1 and that sustained ERK activity is correlated with mitogenic activity.6 We demonstrate that early activation of ERK by PDGF-BB is independent of bFGF, whereas sustained ERK activation depends on bFGF and FGFR-1. Treatment with bFGF antibody could be delayed for 4 hours without reducing its inhibitory effect, suggesting that bFGF is an autocrine "progression factor."21 Our data further suggest that endogenous bFGF does not contribute to PI3-kinase activation by PDGF-BB because phosphorylation of the PI3-kinase substrate, Akt, is not affected by either bFGF blockade or FGFR-1 suppression.
To our knowledge, this is the first report showing that PDGF-BB induces activation of FGFR-1 and that FGFR-1 is required for a full mitogenic response to PDGF-BB. In rat aortic SMCs, FGFR-1 was shown to play an essential role in bFGF-induced effects.20 Although our observation suggests a crucial role for FGFR-1 in bFGF-dependent, PDGF-BB–induced proliferation, we cannot rule out the contribution of additional members of the FGFR family.22 FGFR-1 phosphorylation was observed as early as 30 minutes after PDGF-BB stimulation and the receptor remained phosphorylated after 4 hours. Because neutralizing bFGF antibody reduced PDGF-BB–induced mitogenesis and FGFR-1 phosphorylation, we conclude that PDGF-BB induces bFGF release.
We found that PDGF-BB significantly increases bFGF on the cell surface and the extracellular matrix, whereas the amount of bFGF in the media increases only slightly (Table). This result is in agreement with other studies showing that bFGF is bound to HSPGs on the cell surface and in the matrix.14,23,24 Some of the HSPGs protect bFGF from degradation, whereas others on the cell surface are required for bFGF signaling.25 Although syndecan-4 has been shown to increase bFGF-FGFR-1 signaling,26,27 downregulation of syndecan-4 by siRNA does not interfere with PDGF-BB–induced proliferation in human SMCs (unpublished data, 2004). Because the inhibitory effect of bFGF antibody is lost when administration is delayed by 6 hours (Figure 4), we conclude that bFGF is required within the first 4 to 6 hours for prolonged ERK activity, which allows entry to S phase. However, because the largest increase of bFGF in the pericellular pool (from 2 to 4 hours; Figure 3) coincides with the late peak of bFGF-dependent ERK activation (from 2 to 8 hours; Figure 5), it is possible that, whereas late FGFR-1 activation depends on bFGF release, early FGFR-1 activation may depend on another mechanism, such as transactivation by the PDGF receptor.
Basic FGF lacks a traditional signal peptide for secretion and different mechanisms for its release have been proposed, including an integrin-dependent process, mechanical strain, membrane disruption, and the Na+/K+-ATPase.23,28 Heparinase24 and protease activity29 can also release bFGF from the extracellular matrix. Our data demonstrate that PI3-kinase and protein kinase C, but not MAPK, are involved in bFGF release. This is, to our knowledge, the first observation of the requirement for PI3-kinase activity in bFGF release. FGFR-1 phosphorylation and prolonged ERK activity in response to PDGF-BB also requires active PI3-kinase and protein kinase C. Interestingly, PI3-kinase mediates translocation of exogenous bFGF into the cell.30 Although PDGF-BB is known to induce bFGF mRNA in rat SMCs,31 PDGF-BB reduces bFGF mRNA in bovine SMCs.32 We found in human SMCs that transcription is not necessary for PDGF-BB to increase proliferation through bFGF release. Further work will be required to elucidate whether PI3-kinase and PKC induce bFGF release through increased membrane permeability, Na+/K+-ATPase activity, FGF-binding protein availability,33–36 or other mechanisms.
Whether bFGF contributes to PDGF-BB–induced migration of rat SMCs is uncertain.31,37 Our experiments suggest that bFGF and FGFR-1 do not contribute to PDGF-BB–induced migration in human SMCs. This result is in accordance with our data showing that bFGF contributes to late, but not early, ERK activation in response to PDGF-BB.
Heparin suppresses rat SMC proliferation in vitro and in vivo,38 in part by displacing bFGF from sites of injury.39 In the present study, heparin inhibited PDGF-BB–induced ERK activation at 4 hours in normal and scrambled siRNA-treated cells but not in FGFR-1 downregulated cells. This result also suggests that heparin displaces bFGF from the cell surface, thus preventing binding to FGFR-1. However, heparin may also inhibit PDGF-BB–induced SMC proliferation by mechanisms unrelated to its interaction with bFGF. The variability of heparin responsiveness of human SMCs40–42 may be of great clinical significance, because it has been reported that resistance to the inhibitory effect of heparin is related to increased risk of restenosis.43 Whether heparin resistance is related to variations in bFGF signaling is an intriguing possibility.
In conclusion, the present study demonstrates that in human SMCs, maximal stimulation of proliferation by the tyrosine kinase receptor agonist PDGF-BB requires the sustained activation of ERK by another tyrosine kinase receptor, FGFR-1, which is activated by endogenous bFGF. This represents a novel mechanism that supports an important autocrine function of bFGF in human SMC proliferation.
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Acknowledgments |
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This study was supported by a grant from the NIH (HL-18645), a fellowship from the Heart and Stroke Foundation of Canada (to E.M.), and a fellowship from the German Academy of Nature Scientists Leopoldina (to B.H.R.). We thank Dr Michael A. Reidy (University of Washington) for providing the bFGF antibody and Dr Karen Bornfeld for the ERK antibody.
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Footnotes |
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Original received May 11, 2004; resubmission received November 18, 2004; revised resubmission received December 16, 2004; accepted December 16, 2004.
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