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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1041-1050.)
© 1999 American Heart Association, Inc.

Original Contributions

NGF Activates Similar Intracellular Signaling Pathways in Vascular Smooth Muscle Cells as PDGF-BB But Elicits Different Biological Responses

Rosemary Kraemer; Hiep Nguyen; Keith L. March; Barbara Hempstead

From the Departments of Pathology (R.K., H.N.) and Medicine (B.H.), Cornell University Medical College, New York, NY, and Krannert Institute of Cardiology and Richard L. Roudebush Veterans Administration Medical Center (K.L.M.), Indiana University School of Medicine, Indianapolis, Ind.

	Abstract

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Abstract—The signaling pathways that regulate smooth muscle cell migration and proliferation are incompletely understood. Smooth muscle cells express at least 3 families of receptor tyrosine kinases that mediate cell migration: platelet-derived growth factor (PDGF) receptors, the trk family of neurotrophin receptors, and insulin-like growth factor 1 receptor. The neurotrophin, nerve growth factor (NGF), and insulin-like growth factor 1 induce the migration but not the proliferation of smooth muscle cells, whereas PDGF-BB stimulates both responses. To determine whether distinct signaling pathways downstream of receptor tyrosine kinases specifically mediate smooth muscle cell migration or proliferation, the ligand-induced activation of different signaling pathways in smooth muscle cells was examined. NGF induces prolonged activation of the Shc/MAP kinase pathway and phospholipase C compared with PDGF-BB. The activation of phosphatidylinositol-3 kinase, however, was 10-fold greater in response to PDGF-BB compared with NGF. Insulin-like growth factor 1 activates only phosphatidylinositol-3 kinase. Pharmacological inhibitors of phosphatidylinositol-3 kinase, Wortmannin and LY294002, inhibit PDGF-BB and NGF-induced migration, whereas an inhibitor of MAP kinase kinase, PD98059, has no effect. Our results suggest that (1) different receptor tyrosine kinases use similar patterns of activation of signaling pathways to mediate distinct biological outcomes of cell migration and proliferation, (2) NGF activates signaling proteins in smooth muscle cells similar to those activated during NGF-induced neuronal differentiation, and (3) the combinatorial effects of different signaling pathways are important for the regulation of smooth muscle cell migration and proliferation. Further studies using mutant trk receptors will help to define the signal transduction pathways mediating NGF-induced smooth muscle cell migration.

Key Words: trk • smooth muscle cells • migration • proliferation • signal transduction

	Introduction

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Arterial injury, as occurs in atherosclerosis and balloon angioplasty, results in the migration of medial smooth muscle cells into the intima. This is followed by a period of proliferation, resulting in the formation of a neointimal lesion.¹ Ligand-induced activation of several receptor tyrosine kinases has been implicated in the migration and proliferation of smooth muscle cells in the neointima after vascular injury. Growth factors, such as platelet-derived growth factor (PDGF) and insulin-like growth factor 1 (IGF-1), and their receptors show increased expression in the arterial wall after balloon angioplasty.^{2 3 4 5 6} However, the biological responses elicited by these 2 agonists are different in that PDGF mediates both smooth muscle cell proliferation and migration whereas IGF-1 specifically induces only smooth muscle cell migration.⁷

Ligand binding to the PDGF and IGF-1 receptor tyrosine kinases induces autophosphorylation of tyrosines within the cytoplasmic domain of the receptors, resulting in the recruitment and activation of specific signaling molecules that may mediate the migration and proliferation of vascular smooth muscle cells in response to vascular injury. One of these is the adapter protein Shc, which binds either directly (PDGF-R) or indirectly (IGF-1 receptor through the insulin receptor substrate) to activated receptor tyrosine kinases, and initiates signaling via the ras/MAP kinase pathway. The enzymes phospholipase C (PLC) and phosphatidylinositol-3 (PI-3) kinase also bind to activated receptor tyrosine kinases, localizing these normally cytoplasmic proteins to their membrane lipid substrates.

In human and rat vascular smooth muscle cells, PDGF activates the ras/MAP kinase pathway and increases phosphatidylinositol (PI) turnover, presumably through activation of PLC and PI-3 kinase.^{7 8 9 10} In contrast, treatment of human smooth muscle cells with IGF-1 increases PI turnover, but does not activate the ras/MAP kinase pathway.⁷ These results, coupled with experiments performed with epithelial cells expressing mutant PDGF receptors, have implicated the activation of the ras/MAP kinase pathway in cell proliferation, whereas activation of PI-3 kinase and PLC has correlated with smooth muscle cell migration.^{7 11} However, in other studies, inhibition of PI-3 kinase does not reduce PDGF-induced migration of vascular smooth muscle cells,¹² and PLC activation does not appear to be required for basic fibroblast growth factor (bFGF)-induced migration of myofibroblasts.¹³ Thus, it is unclear whether there is a unique signaling pathway in vascular smooth muscle cells that regulates cell migration.

To resolve these issues, we have used recent results that indicate that the trk family of receptor tyrosine kinases and their ligands, the neurotrophins, are expressed by the neointimal smooth muscle cells.¹⁴ The neurotrophins (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT-3; and neurotrophin 4/5, NT-4/5) are a class of polypeptide growth factors that were originally described as differentiation and survival factors for neurons (reviewed in References 15 and 16^{15 16} ). The trk family consists of trkA, trk B, and trkC, each sharing approximately 50% sequence homology in the extracellular domain and 85% homology in the cytoplasmic domain. TrkA binds NGF, trk B binds BDNF and NT-4/5, and trkC binds predominantly NT-3 (reviewed in References 17 and 18^{17 18} ). After ligand-induced trkA phosphorylation, signaling proteins including PLC and Shc bind to activated trk receptors and participate in NGF-induced neuronal differentiation.^{19 20 21 22}

Previous studies demonstrated increased expression of trkA and trkB as well as their ligands, NGF and BDNF, in the lesion that develops after balloon deendothelialization of the rat aorta.¹⁴ Moreover, trkB, trkC, and the neurotrophins are expressed in primary and restenotic human atherosclerotic lesions. Finally, rat and human cultured aortic smooth muscle cells, at low passage, express mRNA for the neurotrophins and low levels of trkA, trkB, and trkC protein, although the predominant trkB and trkC receptors are truncated isoforms that lack kinase activity.¹⁴ Biologically, NGF is a potent chemotactic agent for human aortic smooth muscle cells, eliciting a response comparable to PDGF-BB.¹⁴

Although primary cultured smooth muscle cells express neurotrophins and trk receptors, the use of these cells is limited because of the inability to culture these cells for prolonged periods without a significant change in phenotype,²³ precluding generation of stable clones expressing receptors by gene transfer. Thus, to define the intracellular signaling pathways activated by NGF in vascular smooth muscle cells, conditionally immortalized mouse smooth muscle cells were used to generate smooth muscle cell lines that stably express trkA. Vascular smooth muscle cells are derived from thoracic aortic explants of transgenic mice expressing a temperature-sensitive SV40-T antigen, under the control of the smooth muscle cell–specific -actin promoter (Fan et al, unpublished data, 1999). These cells exhibit an immortalized phenotype at 33°C, when T antigen is expressed. At the nonpermissive temperature of 39.5°C, T antigen is degraded, and the cells exhibit a more differentiated phenotype.²⁴ Thus, stably transfected cells can be generated and propagated at 33°C, then shifted to 39.5°C, for 3 to 4 days, to acquire a nontransformed state, for functional and biochemical assays.

The signaling pathways that mediate NGF-induced smooth muscle cell migration are not known. Because these cells express native PDGF-BB (Fan et al, unpublished data, 1999) and IGF-1 receptors (see below), we have tested whether unique signaling pathways mediate migration or proliferation of vascular smooth muscle cells. Therefore, the present studies were undertaken to determine (1) whether NGF activates signaling pathways in trkA-expressing smooth muscle cells that have been implicated in PDGF- or IGF-1-induced vascular smooth muscle cell migration, and (2) whether distinct biological responses to trk activation in different cell types, such as migration of smooth muscle cells and neuronal differentiation, are mediated by similar or divergent signaling cascades.

	Methods

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Cell Culture
Temperature-sensitive mouse smooth muscle cells (TsTmSMC) were grown from aortic explants of a transgenic mouse line expressing a temperature-sensitive SV40-T antigen, under the control of the promoter for smooth muscle cell actin (Fan et al, unpublished data, 1999). The cells were cultured in DMEM with 4500 mg glucose/L, 10% FCS (GibcoBRL), penicillin (1000 U/mL; Gibco), streptomycin (100 µg/mL; Gibco), and glutamine (2 mmol/L; Gibco). TsTmSMC grown at 33°C were transfected, using CaPO₄, with the pMEX vector²⁵ containing the cDNA for human trkA and encoding neomycin (G418) resistance. After selection in G418 (1 mg/mL; Gibco), colonies were subcloned, expanded, and tested for stable expression of full-length trk receptors by Western blot analysis (see below). Human aortic smooth muscle cells (HASMC) were obtained from ATCC at passage 12 and were cultured at 37°C in M199 and 20% FCS, penicillin, streptomycin, glutamine, and endothelial cell growth factor (30 µg/mL; Biotechnology Research Institute).

RT-PCR Techniques
Total RNA was isolated from TsTmSMC grown at either 33°C or 39.5°C by extraction in guanidinium isothiocyanate and purified over a cesium chloride cushion by ultracentrifugation. One microgram of total RNA was subjected to reverse transcription using murine leukemia virus transcriptase (Perkin-Elmer). Total RNA not incubated with reverse transcriptase was used as a negative control. cDNA products were amplified by incubation with AmpliTaq polymerase (Perkin-Elmer) and primers specific for each neurotrophin, as previously described.¹⁴ Products were resolved by electrophoresis in 7% polyacrylamide gels, followed by visualization with ethidium bromide.

Western Blot Analysis
Native TsTmSMC and TsTmSMC expressing trkA were lysed in RIPA buffer containing the protease inhibitors phenylmethylsulfonyl fluoride (1 mmol/L), aprotinin (1 µg/mL), and leupeptin (10 µg/mL).²⁵ After incubation on ice for 15 minutes, lysates were clarified by centrifugation at 14 000g (Beckman microfuge) at 4°C, and the protein content of the supernatant determined by a Bio-Rad protein assay (Bio-Rad Laboratories) using bovine serum albumin as a standard. Detergent lysates containing equivalent amounts of protein were separated by 9% SDS-PAGE and blotted onto nitrocellulose. Western blot analysis was performed using a polyclonal antisera that recognizes all full-length trk isoforms (203 antisera²⁶ ). Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham) with anti-rabbit or anti-murine IgG-horseradish peroxidase (Boehringer Mannheim).

Migration Assay
TrkA-expressing clones (designated mtrkA4 and mtrkA48) or native TsTmSMC cultured for 3 days at 39.5°C were rinsed with PBS and cultured in 0.5% FCS for 18 hours. Migration assays were performed using a transwell filter 48-well chamber microchemotaxis apparatus (Neuroprobe Inc) as previously described,¹⁴ with slight modifications. A cell suspension was obtained by incubating the cells in PBS/EDTA solution for 5 minutes. After centrifugation, the cells were resuspended in DMEM containing 0.5% FCS at a concentration of 200 000 to 400 000 cells/mL. NGF (1 to 50 ng/mL; murine; Harlan Bioproducts for Science), PDGF-BB (10 ng/mL; recombinant human; R&D) or IGF-1 (35 ng/mL; recombinant human; R&D) were resuspended in 0.5% FCS and placed in the lower chamber. Cells (50 µL) were added to the upper chamber, over a polycarbonate polyvinylpyrrolidone-free 8-µm pore membrane (Poretics Corp) coated with nondenatured rat tail collagen (Biomedical Technologies), and incubated for 4 hours at 37°C in 95% air/5% CO₂. Cells attaching to the upper surface were removed by scraping, and cells migrating through the pores were fixed and visualized by staining with Dif-quik (Baxter Scientific) and migrating cells counted manually. Results are expressed as the fold increase in migration of cells in the presence of the chemoattractant over cells in the absence of the chemoattractant, with 3 to 4 replicates per experimental group. In experiments in which specific enzyme inhibitors were used, the cells were preincubated with either Wortmannin (10 to 50 nmol/L; Sigma Chemical Co), LY294002 (10 to 20 µmol/L; Sigma), PD98059 (7 to 15 µmol/L; Cal Biochem), or vehicle for 30 minutes at 37°C before addition to the upper chamber. The inhibitors were also added to the bottom wells at the same concentrations. Statistical differences between the groups were determined by Student's t test, and statistical significance was determined at a P level of <0.05.

Cell Counting Assay
HASMC (passage 14 to 16) were seeded in a 96-well microtiter plate at 5000 cells/well and cultured at 37°C. At 12 hours, the cells were treated with either 5% FCS alone or 5% FCS containing NGF (10 or 50 ng/mL) or PDGF-BB (30 ng/mL) every other day. After 7 days, the cells were resuspended and an aliquot was counted using a hemocytometer. The results are expressed as the number of cells/well±SE of 3 replicates per experimental group.

TrkA-expressing or native TsTmSMC were seeded onto 6-well cluster plates and coated with rat tail collagen at a density of 30 000 cells/well in 10% FCS media and cultured at 39.5°C. After 2 days, the cells were rinsed with PBS, then cultured in 3% FCS media for 48 hours. The cells were then treated in 3% FCS alone or 3% FCS containing NGF (10 or 50 ng/mL), PDGF-BB (10 ng/mL), or IGF-1 (10 ng/mL) every day for 10 days. Cells were counted as described above and results are presented as the mean±SE of 3 replicates per experimental group.

Mitogenesis Assay ([³H]Thymidine Incorporation Assay)
Cell proliferation assessed as [³H]thymidine incorporation in response to NGF and PDGF-BB was performed as previously described.²⁷ Briefly, mtrkA48 were grown on collagen-coated 96-well microtiter plates (10 000 cells/well) for 2 days at 39.5°C in 10% FCS media followed by 18 hours in 0.5% FCS. The cells were then treated with either 0.5% FCS or 0.5% FCS containing PDGF-BB (10 ng/mL) or NGF (10 or 50 ng/mL). After 27 hours, [³H]thymidine was added at 1 µCi/well, for 5 hours. The cells were then harvested onto glass fiber filters using an automated cell harvester (Pharmacia LKB Biotechnology, Inc) and quantified by liquid scintillation counting. The data are expressed as counts per minute per well.

Assays Detecting Activation of Signaling Proteins
Native or trkA-expressing TsTmSMC were cultured on collagen-coated dishes 3 to 4 days at 39.5°C in 10% FCS media, followed by 18 hours in 0.5% FCS. The cells were then treated with NGF (10 or 50 ng/mL), PDGF-BB (10 ng/mL), or IGF-1 (35 ng/mL) for the indicated times and lysed with RIPA buffer containing protease inhibitors and sodium vanadate (1 mmol/L). Proteins in the cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using an anti-phosphotyrosine antisera (monoclonal 4G10; Upstate Biotechnology^{25 28} ). In some experiments, 300 to 400 µg of cell lysate were first immunoprecipitated with 203 antisera, followed by protein A-Sepharose to immunoprecipitate trkA, or with an anti-PLC₁ antibody (mixed monoclonal; Upstate Biotechnology) using protein G-Sepharose; Shc proteins were immunoprecipitated using an anti-Shc antibody (rabbit polyclonal; Upstate Biotechnology) conjugated to protein A-Sepharose; immunoprecipitates were separated by SDS-PAGE, followed by Western blot analysis with 4G10.

MAP kinase activation was assessed using an immunoprecipitation-kinase assay.^{29 30} Briefly, 200 µg of protein from total cell lysates were immunoprecipitated using antisera specific for the MAP extracellular signal regulated kinase (ERK)-1 or ERK-2 (Santa Cruz Biotechnology) and incubated with [-³²P]ATP using myelin basic protein (Sigma) as a substrate.³⁰ Kinase activity was quantified using a phosphoimager and is expressed as relative units, with the activity after 5 minutes of treatment with NGF expressed as 1.0 U.

PI-3 Kinase Activity
Cells were cultured and treated with growth factors as described above and PI-3 kinase assays were performed as previously described.^{31 32} Briefly, cells were rinsed with ice-cold PBS, then with 137 mmol/L NaCl, 20 mmol/L Tris, pH 8.0, 1 mmol/L MgCl, 1 mmol/L CaCl₂ (buffer A), and lysed in buffer A containing 10% glycerol, 1% NP40, protease inhibitors, and sodium vanadate at 4°C for 20 minutes. After clarification by centrifugation, 1500 µg of cell protein was immunoprecipitated using 4G10^{20 25} and protein A-Sepharose. Immunoprecipitates were incubated with sonicated phosphatidylinositol (final concentration of 0.2 mg/mL; Avanti Polar Lipids) and 20 µCi [-³²P]ATP in 40 µL kinase buffer (30 mmol/L HEPES, pH 7.4, 30 mmol/L MgCl₂, 50 µmol/L ATP, and 40 µmol/L adenosine) for 10 minutes at room temperature. After termination of the reaction and extraction of the phospholipids, the products were spotted onto precoated silica gel 60 thin-layer chromatography plates (Whatman Chromatography) and developed in chloroform/methanol/ammonium hydroxide/H₂O (45:35:2.5:7.5; vol/vol/vol/vol). Radiolabeled phospholipids were visualized by autoradiography, and labeled phosphatidylinositol phosphate (PIP) migration was compared with cold PIP standard (Sigma). Kinase activity was quantified using a phosphoimager and is expressed as relative units, with the activity after 5 minutes of treatment with NGF expressed as 1.0 U.

	Results

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Expression of Neurotrophins and trk Receptors by TsTmSMC
The expression of the neurotrophins and trk receptors was assessed by RT-PCR and Western blot analysis.¹⁴ TsTmSMC grown at 33°C and 39.5°C express mRNA for NGF, BDNF, and NT 4/5, but not NT3 (Figure 1). However, immunohistochemical analysis using an antibody that recognizes all 4 neurotrophins¹⁴ indicates that these cells express negligible levels of neurotrophin protein when grown at 33°C or 39.5°C (data not shown). No expression of full-length or truncated trk proteins could be detected as assessed by Western blot analysis in TsTmSMC grown at 33°C (Figure 2) or 39.5°C (data not shown) using an antibody that recognizes all full-length trk isoforms (Figure 2) or antibodies that recognize truncated trkB or trkC (data not shown). The lack of trk expression by native TsTmSMC is confirmed in functional analysis of neurotrophin action; TsTmSMC do not migrate in response to NGF, in doses ranging from 10 to 50 ng/mL, whereas the cells migrate in response to PDGF-BB and IGF-1 (Figure 3).

View larger version (61K): [in this window] [in a new window]   Figure 1. RT-PCR for detection of mRNA of the neurotrophins in TsTmSMC. mRNA purified from native TsTmSMC grown at 33°C or 39.5°C was subjected to reverse transcription (RT) followed by PCR using primers specific for each of the neurotrophins. The PCR product size is indicated by arrows. mRNA purified from NIH3T3 cells was used as a positive control14 ; mRNA that was not subjected to reverse transcription (no RT) was used as a negative control. The marker sizes are in descending order 1.4, 1.1, 0.9, 0.6, 0.3, 0.28/0.27, 0.23, 0.19, 0.12, and 0.072 kb. This represents 1 of 2 identical experiments.

View larger version (50K): [in this window] [in a new window]   Figure 2. Expression of trkA in native and stably transfected TsTmSMC. Expression of trkA in native TsTmSMC and 2 stably transfected clones (mtrkA48 and mtrkA4) was detected by Western blot analysis using an antisera detecting full-length trkA, trkB, or trkC (203 antisera26 ). PC12 cells expressing high levels of trkA25 were used as a positive control.

View larger version (19K): [in this window] [in a new window]   Figure 3. Migration of native TsTmSMC, mtrkA48, and mtrkA. Chemotaxis was measured by a microchamber chemotaxis assay. Native TsTmSMC, mtrkA48, and mtrkA4 were grown for 3 days at 39.5°C before treatment with PDGF-BB (P, 10 ng/mL), IGF-1 (I, 35 ng/mL), and NGF (1 to 50 ng/mL as indicated). Cell numbers for wells treated with no ligand (control, C) were TsTmSMC (87±5.4); mtrkA48 (112±2.4); mtrkA4 (68±8.2). This represents 1 of 4 or 5 similar experiments for each cell group. Numbers are mean±SE of 4 replicates per experimental group.

Derivation of trkA-Expressing TsTmSMC
To study trk receptor signaling in smooth muscle cells, TsTmSMC were transfected with pMEX vector containing the cDNA for human trkA. Six clones stably expressing significant levels of trkA were isolated, and 2 clones were examined in detail (Figure 2; designated mtrkA4 and mtrkA48). In comparison with cell lines expressing a defined number of receptors determined by equilibrium binding analysis,²⁵ the trkA-TsTmSMC express approximately 10 000 and 40 000 trkA receptors per cell, respectively (Figure 2). These clones were chosen as they reflect the expression of trkA in neointimal smooth muscle cells.¹⁴

Cell Migration and Proliferation of trkA-Expressing TsTmSMC
The ability of mtrkA4 and mtrkA48 cells to functionally respond to NGF was assessed using chemotaxis (Figure 3) and cell proliferation assays (Figure 4). NGF induces migration of mtrkA4 and mtrkA48 cells at doses ranging from 1 to 50 ng/mL, with maximal migration at a dose of 10 ng/mL (Figure 3; 8.8- and 5-fold increase in migration over control, mtrkA4 and mtrkA48, respectively). The response to 10 ng/mL of NGF was comparable to that observed with PDGF-BB in both clones. An additional 4 clones of cells expressing significant levels of trkA responded similarly to NGF, with maximal migration occurring at approximately 5 to 10 ng/mL of NGF (results not shown). Both PDGF-BB and IGF-1 induce migration of trkA-expressing TsTmSMC as effectively as with native TsTmSMC, ranging from 5- to 10-fold for PDGF-BB and 2- to 3-fold for IGF-1.

View larger version (19K): [in this window] [in a new window]   Figure 4. Proliferation of TsTmSMC, mtrkA4, and mtrkA48. A, Cell counting assay of native or trkA-expressing TsTmSMC. Native TsTmSMC, mtrkA48, or mtrkA4 were cultured at 39.5°C for 3 days and then treated for 10 days in either 3% FCS alone (C), or 3% FCS containing either PDGF-BB (P, 10 ng/mL), IGF-1 (I, 10 ng/mL), or the indicated doses of NGF. The dose of NGF (N) used in the experiments with TsTmSMC and mtrkA48 was 10 ng/mL. Values are mean±SE of 3 replicates per group. Similar results were obtained in 2 additional experiments. B, [3H]Thymidine incorporation in mtrkA48. mtrkA48 were seeded on collagen-coated 96-well microtiter plates and cultured at 39.5°C for 3 days, then treated for 32 hours in either 0.5% FCS alone (C) or 0.5% FCS containing either PDGF-BB (P, 10 ng/mL) or NGF at the indicated doses. [3H]Thymidine incorporation was performed as described in the Methods. Values are mean±SE of 6 replicates per group. Similar results were obtained in 3 additional experiments.

In contrast, NGF does not induce proliferation of trkA-expressing TsTmSMC (Figure 4). Treatment of mtrkA4 or mtrkA48 as well as native TsTmSMC with PDGF-BB results in a 2- to 4-fold increase in cell number, whereas neither NGF nor IGF-1 has an effect (Figure 4A). This was confirmed in a [³H]thymidine incorporation assay (Figure 4B), in which PDGF-BB induced a 2-fold increase in [³H]thymidine incorporation in mtrkA48, whereas NGF had no effect. Finally, these results are comparable to those using HASMC, in which NGF (50 ng/mL) does not stimulate the proliferation of HASMC (results not shown), whereas PDGF-BB (30 ng/mL) induces a 2- to 4-fold increase in cell number after 7 days of treatment (4.8±0.2x10³ versus 9.4±0.3x10³ versus 4.9.±0.4x10³ cells/well; control versus PDGF-BB versus NGF). Comparable results using PDGF-BB-treated cultured human aortic smooth muscle cells have been reported.⁹ Therefore, NGF behaves similarly to IGF-1, as a chemotactic but not a mitogenic agent for human vascular smooth muscle.⁷ Moreover, native TsTmSMC, which do not express full-length or truncated isoforms of trk, fail to respond to NGF in either chemotaxis (Figure 3) or proliferation assays (Figure 4). Thus, similar to early passage HASMC, trkA-expressing TsTmSMC demonstrate chemotactic but not mitogenic responses to NGF.

Activation of Signal Transduction Pathways
Our previous studies demonstrated that TsTmSMC and cultured rat smooth muscle cells respond comparably to PDGF-BB, with increased phosphorylation of the PDGF-B receptor, Shc, and the MAP kinases, ERK-1 and ERK-2 (Fan et al, unpublished observations, 1999). In contrast, NGF does not induce the tyrosine phosphorylation of cellular proteins in TsTmSMC cells (Figure 5A), consistent with their lack of trkA expression. However, trkA-expressing TsTmSMC respond to NGF with a dose-dependent increase in the phosphorylation of the trkA receptor (Figure 5B) as well as increases in the tyrosine phosphorylation of several proteins, in a pattern similar to that of PDGF-BB (Figure 5A).

View larger version (36K): [in this window] [in a new window]   Figure 5. Tyrosine phosphorylation in native TsTmSMC and trkA-expressing TsTmSMC in response to PDGF-BB and NGF. A, Anti-phosphotyrosine Western blot analysis of cell lysates obtained from native TsTmSMC or mtrkA48 grown for 3 days at 39.5°C then treated with either PDGF-BB (10 ng/mL) or NGF (50 ng/mL) for 5 minutes. Similar results were obtained with mtrkA4. B, Dose-dependent phosphorylation of trkA receptor in mtrkA4 cells. mtrkA4 cells grown for 3 days at 39.5°C were treated with the indicated doses of NGF for 5 minutes. Whole cell lysates were immunoprecipitated with the anti-trk antibody (203) followed by Western blot analysis using an anti-phosphotyrosine antibody. Similar results were obtained within 1 other experiment. Similar results were obtained with mtrkA48 cells.

To further compare the signaling pathways activated by NGF, PDGF-BB, and IGF-1, mtrkA4 and mtrkA48 cells grown at 39.5°C for 3 to 4 days were treated with either NGF, PDGF-BB, or IGF-1 and activation of the Shc/MAP kinase pathway, PLC, and PI-3 kinase was assessed. The results using mtrkA48 cells are shown in Figures 6, 7, and 8 and similar results were obtained with mtrkA4 cells (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 6. Activation of Shc/MAP kinase signal transduction pathway in trkA-expressing TsTmSMC. A, Anti-phosphotyrosine Western blot analysis of Shc proteins immunoprecipitated from mtrkA48 treated with PDGF-BB (10 ng/mL), NGF (10 or 50 ng/mL), or IGF-1 (35 ng/mL). Cells were lysed at the indicated times after ligand addition; Whole cell lysates were immunoprecipitated with an anti-Shc antibody conjugated to protein A-Sepharose followed by Western blot analysis using an anti-phosphotyrosine antibody. B, ERK-1 and ERK-2 activation. Cells were treated with PDGF-BB (10 ng/mL), NGF (10 or 50 ng/mL), IGF-1 (35 ng/mL), or no ligand (Ctl) at the indicated time points. Anti-phosphotyrosine Western blot analysis (top) demonstrates increased phosphorylation of ERK-1 and ERK-2. MAP kinase activity (bottom) in ligand-treated mtrkA48 cells was measured from detergent lysates of cells treated with PDGF (, 10 ng/mL), NGF (, 50 ng/mL), or IGF-1 (, 35 ng/mL) at the indicated time points. Data were normalized so that 1.0 equals the volume data obtained by phosphoimager analysis after 5 minutes of treatment with NGF. Data are presented as the mean±SE of 4 experiments.

View larger version (23K): [in this window] [in a new window]   Figure 7. Phosphorylation of PLC in mtrkA48 in response to NGF. mtrkA48 cells were treated with PDGF-BB (10 ng/mL), NGF (50 ng/mL), or IGF-1 (35 ng/mL) for the indicated time. Whole cell lysates were immunoprecipitated with an anti-PLC antibody followed by Western blot analysis using an anti-phosphotyrosine antibody. Similar results were obtained in 4 experiments using mtrkA48 cells or mtrkA4 cells.

View larger version (22K): [in this window] [in a new window]   Figure 8. Activation of PI-3 kinase in mtrkA48 in response to NGF. mtrkA48 cells were treated with either PDGF-BB (, 10 ng/mL), NGF (, 50 ng/mL), or IGF-1 (, 35 ng/mL) for the indicated time and PI-3 kinase assays performed as described. Data were normalized so that 1.0 equals the volume analysis after 5 minutes of treatment with NGF. Data are presented as the mean±SE of 3 to 5 experiments.

NGF treatment of mtrkA48 at both 10 and 50 ng/mL induces prolonged phosphorylation of Shc and the MAP kinase enzymes, ERK-1 and ERK-2 (Figure 6). Anti-phosphotyrosine Western blot analysis of immunoprecipitated Shc proteins demonstrates increased phosphorylation of proteins migrating at 46, 52, and 68 kDa (Figure 6A), confirmed by Western blot analysis to be the 46-, 52-, and 68-kDa isoforms of Shc (results not shown). Shc proteins are phosphorylated within 5 minutes of NGF treatment of mtrkA48, and remained phosphorylated for up to 1 hour after treatment (Figure 6A).

NGF also induces the tyrosine phosphorylation of 43- and 42-kDa proteins (Figure 6B), confirmed by Western blot analysis to be ERK-1 and ERK-2 (results not shown). ERK-1 and ERK-2 remain phosphorylated for 1 (Figure 6B) to 4 hours (not shown) after NGF treatment. The phosphorylation of MAP kinase correlates with its activation (Figure 6B), such that 50 ng/mL of NGF treatment induces a 6- to 8-fold increase in MAP kinase activity within 5 minutes of treatment and the activity remains elevated up to 4 hours (Figure 6C and data not shown). Similar results were obtained using 10 ng/mL of NGF.

In contrast, PDGF-BB induces transient phosphorylation of Shc and MAP kinase in mtrkA48 cells. Peak phosphorylation of the 46- and 56-kDa isoforms of Shc and ERK-1 and ERK-2 occurs at 5 minutes in response to PDGF-BB and begins to return to control levels within 20 to 60 minutes (Figure 6A). Interestingly, PDGF-BB did not induce the phosphorylation of the 68-kDa isoform of Shc. Peak MAP kinase activity also occurs within 5 minutes of treatment with PDGF-BB and returns to a level approximately 2-fold above control by 1 hour (Figure 6B). This pattern of PDGF-induced MAP kinase activation is similar to that observed using cultured human smooth muscle cells.^{7 8} IGF-1 does not significantly activate MAP kinase in trkA-expressing TsTmSMC (Figure 6B) nor does it phosphorylate Shc (Figure 6A), consistent with results obtained with human neonatal vascular smooth muscle cells.⁷ Thus, in smooth muscle cells expressing trkA receptors, NGF induces a prolonged activation of the Shc/MAP kinase pathway whereas PDGF-BB induces a transient activation and IGF-1 treatment is without effect.

Activation of PLC enzymes and PI-3 kinase has been implicated in the migration of human smooth muscle cells in response to PDGF and IGF-1.^{7 9} In mtrkA48 cells, both NGF (50 ng/mL) and PDGF-BB induce a rapid phosphorylation of PLC₁, within 5 minutes of treatment (Figure 7), whereas IGF-1 has no effect. However, PLC₁ remains phosphorylated in response to NGF, whereas the response to PDGF-BB is transient, returning to control levels within 20 minutes. Thus, as is observed with MAP kinase activation, NGF induces prolonged phosphorylation of PLC₁ in smooth muscle cells.

NGF, PDGF-BB, or IGF-1 treatment of mtrkA48 cells increases PI-3 kinase activity, but to variable degrees, as assessed by immunoprecipitation kinase assays (Figure 8). NGF and IGF-1 induce a 2- to 3-fold increase in PI-3 kinase activity, and the activity remains increased relative to control cells for up to 1 hour. In contrast, PDGF-BB markedly induces PI-3 kinase activity, with a greater than 20-fold increase in activity within 5 minutes of treatment, and the activity remains elevated by approximately 2- to 3-fold over control cells at 1 hour. Thus, NGF, IGF-1, and PDGF-BB have different effects on the level of activation of PI-3 kinase in smooth muscle cells. Table 1 summarizes the biological responsiveness and the signal transduction pathways activated by NGF, IGF-1, and PDGF-BB in trkA-expressing smooth muscle cells.

View this table: [in this window] [in a new window]   Table 1. Comparison of Biological Activity and Time Course of Activation of Signal Transduction Pathways Activated by NGF, IGF-1, and PDGF-BB in TsTmSMC Expressing trkA

To determine which of the above signaling pathways mediated migration and proliferation in response to NGF and PDGF-BB, pharmacological inhibitors of either PI-3 kinase, Wortmannin and LY294002, or MAP kinase kinase, PD 98059, were used in the migration assay (Figure 9). Only responses to PDGF-BB and NGF were assessed, as they are more potent chemoattractants compared with IGF-1. Both Wortmannin and LY294002 inhibited migration in response to PDGF-BB and NGF in a dose-dependent manner (Figure 9), similar to their inhibitory activity on PI-3 kinase-dependent responses in other cell systems.^{33 34 35} Wortmannin (10 nmol/L) resulted in a 50% reduction in migration, and Wortmannin (50 nmol/L) reduced migration to control levels (Figure 9A), similar to its inhibitory activity on PI-3 kinase-dependent responses in other cell systems.^{33 34} LY294002 similarly inhibited migration at doses ranging from 10 to 20 µmol/L (Figure 9A). In contrast, PD 98059 had no effect on PDGF-BB- or NGF-induced migration at doses ranging from 7 to 15 µmol/L (Figure 9B), concentrations that completely inhibit phosphorylation of the MAP kinases, ERK-1 and ERK-2, as assessed by phosphotyrosine Western blot analysis (results not shown; also see Reference 36³⁶ ).

View larger version (32K): [in this window] [in a new window]   Figure 9. Pharmacological inhibition of ligand-induced migration of smooth muscle cells. Microchamber chemotaxis assays were performed as described above. mtrka48 cells were grown for 3 days at 39.5°C, then preincubated with Wortmannin (A, left), LY294002 (A, right), or PD 98059 (B) at the indicated doses, or in vehicle (DMSO or ethanol [EtOH]), for 30 minutes at 37°C before addition to the upper well. Similar concentrations of the inhibitors were added to the bottom wells. PDGF-BB and NGF were added to the bottom wells as before. Cell numbers for wells treated with no ligand (control, C) were as follows: A, DMSO, 89±8; Wortmannin (10 nmol/L), 75±5; Wortmannin (50 nmol/L), 36±5; B, EtOH, 162±18; LY294002 (10 µmol/L), 87.5±11; LY294002 (20 µmol/L), 45±4; C, DMSO, 24±8; PD 98059 (15 µmol/L), 45±5; PD 98059 (30 µmol/L), 93±7.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Migration of medial smooth muscle cells and their subsequent proliferation are the primary causes of smooth muscle cell accumulation in the neointima after vascular injury.¹ Growth factors expressed in neointimal lesions have distinctive effects on smooth muscle cells, with PDGF inducing both proliferation and migration, and IGF-1 and NGF promoting only chemotaxis. It has been suggested that different signaling pathways regulate the proliferative compared with the migratory responses of PDGF on smooth muscle cells, with MAP kinase regulating proliferation and PLC and PI-3-kinase regulating migration.^{7 11} This hypothesis has remained untested with regard to other receptor tyrosine kinases expressed by vascular smooth muscle cells.

To address these issues, we have established a model system using TsTmSMC grown at 39.5°C to examine the ability of different growth factors to mediate directed migration of smooth muscle cells using the transwell microchamber chemotaxis assay. These cells migrate in response to PDGF-BB or IGF-1 comparably to that observed with adult and fetal human^{7 9} and fetal bovine aortic smooth muscle cells.³⁷ Moreover, similar signal transduction pathways are activated in TsTmSMC in response to PDGF and IGF-1 as those observed in human smooth muscle cells and other cell systems.^{7 8 9 38 39} Finally, when trkA receptors are expressed in TsTmSMC, NGF is chemotactic, but not mitogenic, similar to what is observed for HASMC (present paper and Reference 14¹⁴ ). Thus, this model system allows for direct comparison of signaling pathways activated by different receptor tyrosine kinases to determine whether activation of specific pathways correlates with their ability to mediate either directed migration or cell proliferation.

The results of the current study suggest that ligand-induced activation of different receptor tyrosine kinases results in a distinct pattern of signal transduction pathways and that the ultimate biological responses to each growth factor are regulated by the integration of these signals. Although both NGF and IGF-1 are strictly chemotactic for smooth muscle cells, NGF is a potent activator of MAP kinase, PLC, and, to a lesser extent, PI-3 kinase, whereas only PI-3 kinase activation could be detected in response to IGF-1. Conversely, similar signaling pathways are activated by PDGF-BB and NGF, although PDGF-BB is both chemotactic and mitogenic, whereas NGF is solely chemotactic. There are differences, however, in the extent and duration of activation of the signaling molecules. (1) PI-3 kinase activation is 10-fold higher in response to PDGF-BB compared with NGF. However, PI-3 kinase inhibitors dose-dependently reduced both PDGF-BB- and NGF-induced migration, suggesting that this signaling pathway mediates migration to both growth factors despite differences in the level and duration of PI-3 kinase activation by the 2 growth factors. Our results differ from Higaki et al,¹² who found no inhibition of PDGF-induced smooth muscle cell migration by either Wortmannin or LY294002. This can be reconciled by the fact that in the previous studies, the cells were not preincubated with the inhibitors before the migration assay, and under these conditions, the enzyme may not have been completely inhibited. (2) NGF induces a more prolonged activation of MAP kinase and PLC than that which occurs in response to PDGF-BB. In prior studies, prolonged activation of MAP kinase has correlated with smooth muscle cell proliferation, because contractile agents, such as angiotensin II, that do not induce smooth muscle cell proliferation only cause a transient increase in MAP kinase activity.⁸ However, the more prolonged activation of MAP kinase by NGF in trkA-expressing TsTmSMC compared with PDGF-BB argues against the duration of MAP kinase activation controlling proliferation.

How can the present results be reconciled with prior results in which the ras/MAP kinase pathway mediates distinct downstream responses depending on the duration of its activation? Both the proliferation and migration of cells are dependent on coupling cytoskeletal reorganization to alterations in gene transcription.^{40 41 42 43 44} Given the inability to detect the activation of a single pathway controlling proliferation, it is likely that the combinatorial effects of several upstream signaling pathways may converge to induce a particular response. For example, activation of the Shc/MAP kinase cascade regulates the transcription and translation of certain mRNAs thought to be important for proliferation.¹¹ However, ras can activate raf, MEK-1, and MAP kinase; it can also activate rac and rho, events that are associated with cytoskeletal rearrangement.^{45 46 47 48 49} Activation of PLC can induce the formation of inositol 1,4,5-trisphosphate, an important mediator of calcium mobilization from intracellular stores, which can modulate actin reorganization.¹¹ Lipid products of PI 3-kinase activate the serine/threonine kinase Akt, whose downstream targets include p70 S6 kinase, a protein that regulates cell proliferation (reviewed in References 50 and 51^{50 51} ). Alternatively, inhibition of PI 3-kinase prevents PDGF-induced activation of rac and may also influence receptor tyrosine kinase-mediated integrin activation (reviewed in Reference 52⁵² ). Thus, the coordinate activation of MAP kinase with either PLC or PI-3 kinase, including the appropriate level and duration of activation, may be necessary to induce the proper cytoskeletal and gene transcription events that control proliferation. This is supported by the fact that inhibitors of PI-3 kinase or microinjection of antibodies directed against PI-3 kinase or PLC can prevent bFGF- or PDGF-induced proliferation of smooth muscle cells or fibroblasts, whereas a PDGF receptor mutated at the PI-3 kinase binding site prevents PDGF-induced DNA synthesis in fibroblasts.^{53 54 55 56}

Alternatively, common signaling pathways may mediate distinctive biological outcomes by activating different effector proteins. As mentioned above, in addition to MEK-1 and MAP kinase, ras can also activate rac and rho. Prolonged activation of Shc and ras may favor activation of rac and rho or other, as yet unidentified, cytoskeletal components and, in combination with prolonged PLC activation, may mediate NGF-induced migration.

An additional possibility is that the differential intracellular location of activated signaling molecules can result in different functions. Thus, prolonged activation of MAP kinase may induce translocation to the nucleus in response to an agonist, and regulate the gene-induction profile. In quiescent smooth muscle cells, MAP kinase is predominantly located in the cytosol.^{8 57} After PDGF and bFGF treatment, MAP kinase translocates to the nucleus, where it may induce transcription of genes important for smooth muscle cell proliferation.^{8 58} In contrast, contractile agents stimulate a transient translocation of MAP kinase to the surface membrane and a later, more sustained redistribution to the contractile filaments.^{57 58} The potential for redistribution of MAP kinase by NGF in trkA-expressing TsTmSMC will require further investigation. Finally, it is possible that novel, or as yet untested, signaling pathways are activated, resulting in solely a migratory or proliferative stimulus.

Using TsTmSMC, we have been unable to detect the phosphorylation of PLC in response to IGF-1. Our results are consistent with data that PLC does not bind to the tyrosine-phosphorylated IGF-1 receptor nor is it activated by IGF-1.^{59 60} Previous studies of IGF-1 treatment of human smooth muscle cells have detected the formation of diacylglycerol in response to IGF-1 as an index of PLC activation.^{7 61 62} These results can be reconciled with the current study as there are 3 families of PLC enzymes, ß, , and isoforms, each of which has multiple members. Although IGF-1 does not activate PLC, it can stimulate the activity of PLCß1 in Swiss 3T3 cells.⁵⁹ Thus, an increase in PI turnover in response to IGF-1 may be caused by the activation of a different PLC isoform, which would not be detected with the isoform-specific antibody used in the present study.

Trk activation leads to distinctive biological responses of survival and differentiation in neural crest-derived cells,^{15 16} and migration in vascular smooth muscle cells (Reference 14¹⁴ and present paper). However, the profiles of activation of the 3 signaling pathways examined are remarkably similar. NGF-induced trkA activation in neuronal cells results in phosphorylation of Shc, MAP kinase, and PLC , and the activation of PI-3 kinase.^{20 25} The MAP kinase kinase inhibitor, PD 98059, inhibits NGF-induced differentiation of the rat pheochromocytoma cell line, PC12,⁶³ but does not inhibit NGF-induced smooth muscle cell migration (Figure 9), suggesting that the cell type-specific biological responses elicited by NGF in smooth muscle cells, compared with neuronal cells, may depend on the intracellular environment and the specific isoforms of the different signaling molecules that are activated in a specific cell type. For example, there are at least 2 isoforms of the p85 subunit of the PI-3 kinase and 2 isoforms of the p110 subunit.^{64 65 66 67} Similarly, different protein kinase C isoforms promote different responses depending on the intracellular molecules with which they interact.^{68 69} Moreover, the cytoskeletal components with which the signaling molecules interact in differentiated smooth muscle cells versus neuronal cells may also contribute to the different biological responses observed.

Thus, NGF induces the migration, but not the proliferation, of smooth muscle cells that express trkA receptors, yet activates similar signaling pathways as PDGF-BB, including Shc/MAP kinases, PLC, and PI-3 kinases. Studies using mutant trk receptors that can no longer bind or activate each of these specific pathways will help in assessing which are required for the chemotactic response of smooth muscle cells to NGF.

	Acknowledgments

 
This work was supported by American Heart Association Grant-in-Aid 95015150 (R.K.), public health service grant, P01 HL46403-06, Hirschl/Caulliers-Weill Foundation (B.H.), and a VA Merit Review Grant (K.L.M.). Special thanks to Dominick Falcone for thoughtful discussion, and Cherie Wieland for secretarial assistance.

	Footnotes

 
Address correspondence to Barbara Hempstead, MD, PhD, Cornell University Medical College, 1300 York Ave, Room C-606, New York, NY 10021.

Received February 11, 1998; accepted October 5, 1998.

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