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(Circulation Research. 2000;87:92.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Heparin Blockade of Thrombin-Induced Smooth Muscle Cell Migration Involves Inhibition of Epidermal Growth Factor (EGF) Receptor Transactivation by Heparin-Binding EGF-Like Growth Factor

Presented in part at the Experimental Biology ‘99 meeting, Washington DC, April 17–21, 1999, and published in abstract form (FASEB J. 1999;13:A35, A136).

Andreas Kalmes, Beatrice R. Vesti, Günter Daum, Judith A. Abraham, Alexander W. Clowes

From the Department of Surgery (A.K., B.R.V., G.D., A.W.C.), University of Washington (Seattle); and Scios Inc (J.A.A.), Sunnyvale, Calif.

Correspondence to Andreas Kalmes, PhD, University of Washington School of Medicine, Department of Surgery, Box 356410, 1959 NE Pacific St, Seattle, WA 98195-6410. E-mail kalmes{at}u.washington.edu

	Abstract

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Abstract—Agonists of G protein–coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein–coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF–dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF–mediated EGFR transactivation. (Circ Res. 2000;87:92-98.)

Key Words: heparin • growth factors • muscle, smooth • migration

	Introduction

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The accumulation of vascular smooth muscle cells (SMCs) in the arterial intima contributes to pathological disorders such as atherosclerosis and the development of stenotic lesions after arterial injury (reviewed in Ross¹ and Schwartz et al² ). Heparin, a glycosaminoglycan, inhibits SMC proliferation and migration in vivo and in vitro.^{3 4 5 6 7} It is not known for certain how heparin blocks SMC function. The effects of heparin on SMCs include the inhibition of the expression of early genes,⁸ as well as matrix-degrading proteases^{9 10 11} and certain matrix molecules.^{12 13} We and others have previously shown that heparin suppresses activation of the extracellular signal-regulated kinases, ERK1 and ERK2, in response to serum and thrombin and other agonists of G protein-coupled receptors (GPCRs).¹⁴

Signaling through ERK1 and ERK2 in response to various GPCR agonists relies on a transactivation of the epidermal growth factor (EGF) receptor (EGFR) (reviewed in Zwick et al¹⁵ ). Thrombin and the thrombin receptor agonist peptide SFLLRN (thrombin receptor agonist peptide) have been shown to transactivate the EGFR in Rat-1 fibroblasts, keratinocytes, primary astrocytes, and COS-7 cells.^{16 17} EGFR transactivation had first been suggested to be independent of EGFR ligands, based on the rapid onset of EGFR tyrosine phosphorylation and the failure to detect EGFR ligands, in particular EGF, in conditioned medium of stimulated cells.^{16 17 18 19} Recently, it has been shown that EGFR transactivation can be mediated by heparin-binding EGF-like growth factor (HB-EGF).²⁰ HB-EGF is a member of the EGF family and is synthesized as a transmembrane precursor that is proteolytically processed into the mature, soluble growth factor (reviewed in Raab and Klagsbrun²¹ ). HB-EGF is a mitogen as well as a stimulator of cell migration for fibroblasts and SMCs.²¹ We report here that in rat and baboon SMCs, thrombin-stimulated SMC migration relies on HB-EGF–dependent EGFR transactivation, a mechanism that is blocked with heparin.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Antibodies against phosphorylated ERK1/2 were obtained from New England Biolabs. ERK1/2 antiserum (7884) was a generous gift from Rony Seger. EGFR antibodies (Abs) were from Santa Cruz Biotechnology and Upstate Biotechnology, respectively. Rat-specific EGFR blocking monoclonal antibody (mAb) (mAb151-IgG,²² ) was obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Human-specific EGFR blocking Ab (mAb 225²³ ) was a generous gift from Stephen Prescott. Neutralizing HB-EGF Abs^{24 25 26} were raised by injecting goats with recombinant human HB-EGF (Ab 197) or rat HB-EGF (Ab 19). Batimastat (BB94) was from British Biotech. Plasmid pGEX-RBD was a generous gift from David Shalloway. Antibodies against Shc and H-Ras were from Transduction Laboratories. Phosphotyrosine Ab (clone 4G10) was from Upstate Biotechnology. EGF and the tyrphostins AG1478 and AG1296 were from Calbiochem. Protein A– and protein G–agarose were from Roche Molecular Biochemicals. Human -thrombin was from American Diagnostica. Platelet-derived growth factor (PDGF)-BB was a generous gift from Zymogenetics. Heparin (porcine intestinal mucosa) was from Sigma Chemical Co.

Cell Culture
Aortic SMCs were prepared from Fischer rats and baboons as described previously.^{27 28} Human embryonic kidney (HEK) 293 cells were from American Type Culture Collection. SMCs (passages 3 to 15) and HEK 293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 200 U/mL penicillin, and 200 µg/mL streptomycin. Before stimulation, cells were starved for 2 to 3 days in medium without fetal bovine serum (SMCs) or overnight in medium containing 0.3% (HEK 293 cells).

Ras Assay
Ras activity was determined as described previously²⁹ through affinity purification of Ras-GTP with a GST-fusion protein containing the Ras binding domain of Raf-1 (GST-RBD), followed by Western blot analysis with an H-Ras Ab.

Immunoprecipitations
Cells from 100-mm culture dishes were harvested in 1 mL ice-cold buffer HEB²⁸ (25 mmol/L HEPES-NaOH, pH 7.5, 10% glycerol, 150 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 1 mmol/L sodium vanadate, 1 mmol/L benzamidine, 0.1% 2-mercaptoethanol, 1% Triton X-100, 1 µmol/L pepstatin A, 2 µg/mL leupeptin, and 20 kallikrein inhibitor units/mL aprotinin) and lysed for 10 minutes on ice. The lysates were cleared with centrifugation at 10 000g for 10 minutes in a microfuge. One microgram of Abs and 10 µL protein A– or protein G–agarose slurry were added to the supernatants and incubated overnight at 4°C with constant agitation. The beads were washed 3 times in HEB and boiled in 20 µL Laemmli’s sample buffer.

Western Blots
Total protein from cell lysates or immunocomplexes were subjected to SDS-PAGE and transferred to nitrocellulose membranes with electroblotting. Immunodetection was performed with the enhanced chemiluminescence kit (Amersham) according to the manufacturer’s protocol.

Migration Assay for SMCs In Vitro
Modified Boyden chamber assays were performed as described³⁰ for 5 hours at 37°C under 5% CO₂ with 48-well microchemotaxis chambers (Neuro Probe) and polycarbonate filters (10-µm pores; Nucleopore Corp) coated with collagen (Vitrogen 100; Collagen Corp). Chemoattractants or serum-free DMEM as a control was added to the lower chamber.

Statistical Analysis
All experiments were repeated at least twice with similar results. Statistical analysis of the migration data shown in the Table was performed with the Wilcoxon signed rank test (SPSS/PC⁺). Statistical significance was accepted at P<0.05.

View this table: [in this window] [in a new window]   Table 1. Thrombin-Induced Migration of Rat SMCs Depends on EGFR Transactivation by HB-EGF

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heparin Inhibits EGFR Transactivation by Thrombin
We found previously that in SMCs, heparin blocks the activation of ERK and its activator MEK by thrombin and other agonists of GPCRs.¹⁴ Because thrombin has been reported to cause EGFR transactivation in other cell types, we investigated whether this pathway is also used in SMCs and whether it is affected by heparin.

Thrombin causes an increase in tyrosine phosphorylation of the EGFR (Figure 1) that is detectable within 2 minutes of the exposure of rat SMCs to thrombin (data not shown). As expected, thrombin-induced EGFR tyrosine phosphorylation in SMCs is blocked by an inhibitor of the intrinsic EGFR kinase activity, the tyrphostin AG1478 (Figure 1). EGFR transactivation is also inhibited by heparin (Figure 1). In addition, thrombin-induced signaling events upstream of ERK, such as tyrosine phosphorylation of the adapter protein Shc and GTP binding of the small GTPase Ras, as well as ERK phosphorylation, are inhibited by both AG1478 and heparin (Figure 2). Activation of the same signaling elements by EGF is inhibited by AG1478 but not by heparin (Figure 2 and data not shown). On the other hand, thrombin-induced Shc and ERK phosphorylations are not inhibited by a PDGF receptor inhibitor, the tyrphostin AG1296, at concentrations that completely abolish PDGF-induced signaling (Figures 2A and 2C). We conclude from these results that thrombin induces EGFR transactivation in rat SMCs and that the inhibitory effect of heparin on thrombin-induced ERK activation is due to a blockade of this transactivation.

View larger version (30K): [in this window] [in a new window]   Figure 1. Thrombin-induced EGFR transactivation requires the intrinsic EGFR kinase activity and is inhibited by heparin. Quiescent rat SMCs were stimulated with 10 nmol/L thrombin for 15 minutes in the presence or absence of heparin (100 µg/mL) or AG1478 (100 nmol/L, preincubated for 30 minutes). EGFR was immunoprecipitated and Western blots of the immunoprecipitated material were probed with anti-phosphotyrosine Ab (PY, top); blots were reprobed with anti-EGFR Ab (bottom). IB indicates immunoblot.

View larger version (44K): [in this window] [in a new window]   Figure 2. Thrombin-induced signaling through Shc, Ras, and ERK is EGFR dependent and is inhibited by heparin. Quiescent rat SMCs were stimulated with 10 nmol/L thrombin, 10 ng/mL EGF, or 10 ng/mL PDGF-BB for 15 minutes in the presence or absence of heparin (100 µg/mL) or after preincubation for 30 minutes with AG1478 (100 nmol/L) or AG1296 (25 µmol/L), as indicated. A, Shc proteins were immunoprecipitated and Western blots of the immunoprecipitated material were probed with anti-phosphotyrosine (PY) Ab (top) and reprobed with anti-Shc Ab (bottom). Arrows indicate the position of the 3 Shc isoforms. B, Activation of Ras was determined as described in Materials and Methods. C, ERK activation was analyzed by probing Western blots of total protein extracts with anti-phospho-ERK Ab (top) or anti-ERK Ab (bottom). IB indicates immunoblot.

EGFR Transactivation by Thrombin Is Mediated by HB-EGF
Because EGFR transactivation has been reported to be either ligand independent^{16 17 18 19} or dependent on HB-EGF,²⁰ we further investigated the mechanism involved in thrombin signaling by using an EGFR blocking Ab (mAb 151-IgG). This Ab binds to the extracellular portion of rat EGFR, thereby competing with ligand binding. The preincubation of SMCs with this Ab suppresses ERK activation by thrombin as well as by EGF. The Ab has no effect when PDGF-BB is used for stimulation (Figure 3), demonstrating its specificity. This result suggested that an EGFR ligand might be involved in EGFR transactivation. Because heparin blocks EGFR transactivation, we examined whether HB-EGF is involved. HB-EGF requires cell surface–associated heparan sulfate proteoglycans (HSPGs) as coreceptors for the EGFR,²¹ raising the possibility that exogenously added heparin could inhibit HB-EGF by competing with cell surface HSPGs for HB-EGF-binding. Consistent with this model, heparin at the concentration used here (100 µg/mL) blocks HB-EGF–induced EGFR activation (Figure 4). In addition, neutralizing Ab against rat HB-EGF (Ab 19) prevents EGFR phosphorylation and ERK activation by HB-EGF as well as by thrombin (Figure 4) but has no effect when EGF is used as the stimulant (data not shown). We conclude from these findings that HB-EGF mediates EGFR transactivation by thrombin in SMCs.

View larger version (29K): [in this window] [in a new window]   Figure 3. ERK activation by thrombin is inhibited by an EGFR blocking Ab. Quiescent rat SMCs were stimulated with 10 ng/mL EGF, 10 ng/mL PDGF-BB, or 10 nmol/L thrombin for 15 minutes with or without preincubation for 30 minutes with anti-EGFR blocking Ab (151-IgG, 15 µg/mL). Total protein extracts were analyzed on Western blots for ERK phosphorylation as described in Figure 2. IB indicates immunoblot.

View larger version (58K): [in this window] [in a new window]   Figure 4. EGFR transactivation by thrombin is blocked by heparin and by a neutralizing HB-EGF Ab. Quiescent rat SMCs were stimulated with 10 nmol/L thrombin or 1 ng/mL rat HB-EGF in the presence or absence of heparin (100 µg/mL) or neutralizing anti–HB-EGF (Ab 19, 100 µg/mL). EGFR tyrosine phosphorylation (A) and ERK activation (B) were analyzed as described in the legends for Figures 1 and 2, respectively. IB indicates immunoblot; PY, phosphotyrosine.

EGFR Transactivation by Thrombin Receptor Agonist Peptide in HEK 293 Cells Is Mediated by HB-EGF
Because thrombin is a protease, we wanted to exclude the possibility that it might act on the HB-EGF precursor to release the mature growth factor in a thrombin receptor–independent manner. The peptide SFLLRN (thrombin receptor agonist peptide [TRAP]) activates the proteinase-activated receptor (PAR)1 thrombin receptor in the absence of thrombin.³¹ In SMCs, however, TRAP is only a weak ERK activator¹⁴ and does not cause detectable EGFR transactivation (data not shown). HEK 293 cells express endogenous PAR1 thrombin receptor and, in contrast to SMCs, respond to TRAP with EGFR transactivation (Figure 5). Therefore, we examined whether TRAP causes EGFR activation in these cells in an HB-EGF–dependent manner. Both EGFR phosphorylation and ERK activation stimulated by TRAP are inhibited by the tyrphostin AG1478, by a human-specific EGFR blocking Ab (mAb 225), and by a human-specific neutralizing HB-EGF Ab (Ab 197; Figure 5). Because human pro-HB-EGF acts as the cellular receptor for diphtheria toxin, we used its nontoxic analog, CRM197, which has been shown to block HB-EGF signaling.³² In HEK 293 cells, CRM197 abolishes EGFR phosphorylation and ERK activation induced by TRAP (Figure 5), as well as by HB-EGF, but has no effect on ERK activation in response to EGF (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 5. In HEK 293 cells, EGFR transactivation by TRAP requires HB-EGF. Quiescent cells were stimulated with 30 µmol/L TRAP for 15 minutes after preincubation for 30 minutes with the indicated reagents: neutralizing anti–HB-EGF (Ab 197, 100 µg/mL), control IgG (normal goat IgG, 100 µg/mL), AG1478 (100 nmol/L), EGFR blocking Ab (mAb 225, 25 µg/mL), and CRM197 (10 µg/mL). EGFR tyrosine phosphorylation (A) and ERK activation (B) were analyzed as described in the legends for Figures 1 and 2. Data in B were obtained by scanning autoradiographs and are presented as a percentage of ERK activation achieved with TRAP stimulation alone (mean±SD, n=3 or 4). IB indicates immunoblot; PY, phosphotyrosine.

HB-EGF–Dependent EGFR Transactivation Requires Matrix Metalloproteinase Activity
HB-EGF shedding in different cell types has been reported to be matrix metalloproteinase (MMP) dependent.^{20 33 34 35} To examine whether MMP activity is involved in thrombin-induced EGFR transactivation, we used the MMP inhibitor batimastat (BB94). Pretreatment of cells with batimastat inhibits ERK activation induced by thrombin in rat SMCs and by TRAP in HEK 293 cells (Figure 6); in contrast, ERK activation by EGF or recombinant HB-EGF is not affected (Figure 6).

View larger version (35K): [in this window] [in a new window]   Figure 6. HB-EGF–dependent EGFR transactivation in response to thrombin and TRAP requires MMP activity. A, Quiescent rat SMCs were stimulated with 10 nmol/L thrombin, 1 ng/mL rat HB-EGF, or 10 ng/mL EGF for 15 minutes after preincubation with 5 µmol/L batimastat (BB94) for 30 minutes. B, Quiescent HEK 293 cells were stimulated with 30 µmol/L TRAP (SFLLRN), 1 ng/mL human HB-EGF, or 10 ng/mL EGF for 15 minutes after preincubation with 5 µmol/L (BB94) for 30 minutes. ERK activation was analyzed as described in the legend to Figure 2. IB indicates immunoblot.

SMC Migration Induced by Thrombin Requires HB-EGF
To address whether this novel signaling pathway plays a role in SMC migration, we determined the ability of SMCs to migrate toward thrombin in a modified Boyden chamber assay. In rat SMCs, thrombin causes an average 1.75-fold stimulation of migration. The inhibition of EGFR kinase activity by AG1478, the inhibition of ligand binding by EGFR blocking Ab, and the sequestration of HB-EGF by heparin or neutralizing HB-EGF Ab all completely block thrombin-induced migration (Table).

To examine whether the same pathway is used in primate cells, we used baboon SMCs. Under the conditions tested, we did not observe significant differences between the 2 species: migration in response to thrombin is activated to the same extent (Figure 7A) and is inhibited by blocking EGFR Abs as well as neutralizing HB-EGF Abs (Figure 7B). Because PDGF-BB–induced SMC migration is not inhibited by heparin,³⁶ HB-EGF should not be involved. PDGF-BB is an equally potent chemoattractant for rat and baboon SMCs (Figure 7A) and migration toward PDGF-BB is not affected by Abs against EGFR or HB-EGF (Figure 7B).

View larger version (20K): [in this window] [in a new window]   Figure 7. Migration of both rat and baboon SMCs in response to thrombin, but not PDGF-BB, depends on EGFR transactivation by HB-EGF. Migration of SMCs was induced in a Boyden chamber by the addition of 10 nmol/L thrombin or 10 ng/mL PDGF-BB to the lower chamber. Where indicated, anti-EGFR Abs (25 µg/mL) and anti–HB-EGF Abs (100 µg/mL) were added together with the cells to the upper chamber. Migration was measured as the number of cells per high-power field migrating across the membrane between the 2 chambers. Data are presented as mean±SD (corresponding numbers of independent experiments [n] are indicated in the figure). Filled columns represent rat SMCs; open columns, baboon SMCs. A, Induction of migration by PDGF-BB and thrombin is shown as percent migration of unstimulated controls. B, Effect of anti-EGFR and anti–HB-EGF Abs on migration in response to PDGF-BB and thrombin. Migration induced in the controls (PDGF-BB or thrombin alone, minus background of unstimulated cells) was set to 100%, and the activity in the Ab groups is shown as a percentage of control.

We conclude from these data that EGFR transactivation by HB-EGF is required for rat and baboon SMC migration stimulated by thrombin and that the inhibitory effect of heparin is based, at least in part, on a blockade of HB-EGF.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
EGFR transactivation is necessary for intracellular signaling by a variety of GPCR agonists, including thrombin.^{16 17 18 19 37 38} We show here that SMC migration in response to thrombin depends on EGFR activation and that this mechanism is blocked by heparin. This is to our knowledge the first report that describes a requirement of EGFR transactivation by a GPCR agonist for cell migration. In addition, our data demonstrate that EGFR transactivation by thrombin relies on a novel mechanism that has recently been described in nonvascular cells that overexpress various receptor molecules.²⁰ These investigators have shown that EGFR activation by GPCR agonists in genetically engineered Rat-1 fibroblasts, COS-7 cells, and HEK 293 cells requires MMP-mediated cleavage of the pro form of HB-EGF. Our data demonstrate that this mechanism is used in normal vascular SMCs to regulate migration. Because SMC migration is a crucial process in the development of pathological disorders of the vessel wall that involve neointima formation, our data indicate an important physiological role for this novel signaling pathway. Consistent with this hypothesis, HB-EGF expression has been shown to be upregulated in human arteriosclerotic plaques^{39 40 41} and in neointimal cells of rat carotid arteries in response to balloon injury.⁴²

HB-EGF requires cell surface HSPGs as coreceptors (reviewed in Raab and Klagsbrun²¹ ). HB-EGF binding to EGFR is antagonized in a dose-dependent manner by heparin.⁴³ In our experiments, heparin blocked EGFR activation by HB-EGF as well as EGFR transactivation and SMC migration induced by thrombin. Thus, the inhibitory effect of heparin on thrombin-induced ERK activation and SMC migration may be accounted for by a blockade of soluble HB-EGF.

Interestingly, the PAR1 thrombin receptor agonist peptide TRAP causes HB-EGF–dependent EGFR transactivation in HEK 293 cells but not in rat SMCs. Consistent with this failure to activate a pathway that, as shown here, is necessary for migration, TRAP does not act as a chemoattractant for rat SMCs (data not shown). In SMCs, TRAP is also a weak ERK activator that is not inhibited by heparin.¹⁴ Although we cannot completely rule out that TRAP-activated PAR1 does not fully duplicate the signaling induced by the proteolytically processed receptor, it may also be possible that thrombin-induced EGFR transactivation in SMCs is mediated by a different receptor. Recently, 2 novel members of the family of protease-activated receptors, PAR3 and PAR4, were cloned as additional thrombin receptors from human and mouse cells.^{44 45} The presence of PAR4 as a second thrombin receptor in vascular and gastric SMCs has been suggested.⁴⁶

Ectodomain shedding provides an important mechanism for HB-EGF signaling, although the pro form may also have biological activity (reviewed in Raab and Klagsbrun²¹ ). Because the MMP inhibitor batimastat inhibits thrombin receptor–induced EGFR transactivation in both SMCs and HEK 293 cells, we conclude that an MMP is involved and that the EGFR is activated by soluble HB-EGF. These data are consistent with the recent observation that batimastat inhibits signaling by lysophosphatidic acid, carbachol, and phorbol-12-myristate-13-acetate in COS-7 cells and by bombesin and phorbol-12-myristate-13-acetate in PC-3 prostate carcinoma cells.²⁰ The identity of the protease or proteases that mediate GPCR-induced HB-EGF shedding in SMCs remains to be determined.

Taken together, our data demonstrate that EGFR transactivation is an essential step in thrombin-induced SMC migration. Furthermore, they reveal that a novel mechanism of EGFR transactivation, which involves HB-EGF as an EGFR ligand, is used in normal vascular SMCs. This mechanism appears to account, at least in part, for the inhibitory effects of heparin on signaling by GPCR agonists.

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (HL-18645 and HL-30946) and a fellowship of the Swiss National Foundation (Dr Vesti). We thank Jessie Deou for technical help; Debby Damm, Brett Garrick, and Peter Strathis for the production of recombinant HB-EGF and HB-EGF Abs; and Michael Klagsbrun and Zeev Gechtman for helpful discussions. We also thank David Shalloway for the GST-RBD construct, Stephen Prescott for EGFR mAb 225, Rony Seger for ERK Ab 7884, and Zymogenetics for PDGF-BB.

Received April 28, 2000; accepted May 25, 2000.

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