NGF Activates Similar Intracellular Signaling Pathways in Vascular Smooth Muscle Cells as PDGF-BB...

Rosemary Kraemer, Hiep Nguyen, Keith L. March and Barbara Hempsteadas PDGF-BB But Elicits Different Biological Responses

NGF Activates Similar Intracellular Signaling Pathways in Vascular Smooth Muscle Cells

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NGF Activates Similar Intracellular Signaling Pathways inVascular Smooth Muscle Cells as PDGF-BB But Elicits

Different Biological ResponsesRosemary Kraemer, Hiep Nguyen, Keith L. March, Barbara Hempstead

Abstract—The signaling pathways that regulate smooth muscle cell migration and proliferation are incompletelyunderstood. 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 factor1 receptor. The neurotrophin, nerve growth factor (NGF), and insulin-like growth factor 1 induce the migration but notthe proliferation of smooth muscle cells, whereas PDGF-BB stimulates both responses. To determine whether distinctsignaling pathways downstream of receptor tyrosine kinases specifically mediate smooth muscle cell migration orproliferation, the ligand-induced activation of different signaling pathways in smooth muscle cells was examined. NGFinduces prolonged activation of the Shc/MAP kinase pathway and phospholipase Cg compared with PDGF-BB. Theactivation 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 ofphosphatidylinositol-3 kinase, Wortmannin and LY294002, inhibit PDGF-BB and NGF-induced migration, whereas aninhibitor of MAP kinase kinase, PD98059, has no effect. Our results suggest that (1) different receptor tyrosine kinasesuse similar patterns of activation of signaling pathways to mediate distinct biological outcomes of cell migration andproliferation, (2) NGF activates signaling proteins in smooth muscle cells similar to those activated during NGF-inducedneuronal differentiation, and (3) the combinatorial effects of different signaling pathways are important for theregulation of smooth muscle cell migration and proliferation. Further studies using mutant trk receptors will help todefine the signal transduction pathways mediating NGF-induced smooth muscle cell migration.(Arterioscler ThrombVasc Biol. 1999;19:1041-1050.)

Key Words: trk n smooth muscle cellsn migrationn proliferationn signal transduction

A rterial injury, as occurs in atherosclerosis and balloonangioplasty, results in the migration of medial smooth

muscle cells into the intima. This is followed by a period ofproliferation, resulting in the formation of a neointimallesion.1 Ligand-induced activation of several receptor ty-rosine kinases has been implicated in the migration andproliferation of smooth muscle cells in the neointima aftervascular injury. Growth factors, such as platelet-derivedgrowth factor (PDGF) and insulin-like growth factor 1(IGF-1), and their receptors show increased expression in thearterial wall after balloon angioplasty.2–6 However, the bio-logical responses elicited by these 2 agonists are different inthat PDGF mediates both smooth muscle cell proliferationand migration whereas IGF-1 specifically induces onlysmooth muscle cell migration.7

Ligand binding to the PDGF and IGF-1 receptor tyrosinekinases induces autophosphorylation of tyrosines within thecytoplasmic domain of the receptors, resulting in the recruit-ment and activation of specific signaling molecules that may

mediate the migration and proliferation of vascular smoothmuscle cells in response to vascular injury. One of these is theadapter protein Shc, which binds either directly (PDGF-R) orindirectly (IGF-1 receptor through the insulin receptor sub-strate) to activated receptor tyrosine kinases, and initiatessignaling via the ras/MAP kinase pathway. The enzymesphospholipase C (PLC) and phosphatidylinositol-3 (PI-3)kinase also bind to activated receptor tyrosine kinases,localizing these normally cytoplasmic proteins to their mem-brane lipid substrates.

In human and rat vascular smooth muscle cells, PDGFactivates the ras/MAP kinase pathway and increases phos-phatidylinositol (PI) turnover, presumably through activationof PLCg and PI-3 kinase.7–10 In contrast, treatment of humansmooth muscle cells with IGF-1 increases PI turnover, butdoes not activate the ras/MAP kinase pathway.7 These results,coupled with experiments performed with epithelial cellsexpressing mutant PDGF receptors, have implicated theactivation of the ras/MAP kinase pathway in cell prolifera-

Received February 11, 1998; revision accepted October 5, 1998.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.Address correspondence to Barbara Hempstead, MD, PhD, Cornell University Medical College, 1300 York Ave, Room C-606, New York, NY 10021.

E-mail [email protected]© 1999 American Heart Association, Inc.

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tion, whereas activation of PI-3 kinase and PLCg has corre-lated with smooth muscle cell migration.7,11 However, inother studies, inhibition of PI-3 kinase does not reducePDGF-induced migration of vascular smooth muscle cells,12

and PLCg activation does not appear to be required for basicfibroblast growth factor (bFGF)-induced migration of myofi-broblasts.13 Thus, it is unclear whether there is a uniquesignaling pathway in vascular smooth muscle cells thatregulates cell migration.

To resolve these issues, we have used recent results thatindicate that the trk family of receptor tyrosine kinases andtheir ligands, the neurotrophins, are expressed by the neoin-timal smooth muscle cells.14 The neurotrophins (nerve growthfactor, NGF; brain-derived neurotrophic factor, BDNF; neu-rotrophin 3, NT-3; and neurotrophin 4/5, NT-4/5) are a classof polypeptide growth factors that were originally describedas differentiation and survival factors for neurons (reviewedin References 15 and 16). The trk family consists of trkA, trkB, and trkC, each sharing approximately 50% sequencehomology in the extracellular domain and 85% homology inthe cytoplasmic domain. TrkA binds NGF, trk B binds BDNFand NT-4/5, and trkC binds predominantly NT-3 (reviewed inReferences 17 and 18). After ligand-induced trkA phosphor-ylation, signaling proteins including PLCg and Shc bind toactivated trk receptors and participate in NGF-induced neu-ronal differentiation.19–22

Previous studies demonstrated increased expression oftrkA and trkB as well as their ligands, NGF and BDNF, in thelesion that develops after balloon deendothelialization of therat aorta.14 Moreover, trkB, trkC, and the neurotrophins areexpressed in primary and restenotic human atheroscleroticlesions. Finally, rat and human cultured aortic smooth musclecells, at low passage, express mRNA for the neurotrophinsand low levels of trkA, trkB, and trkC protein, although thepredominant trkB and trkC receptors are truncated isoformsthat lack kinase activity.14 Biologically, NGF is a potentchemotactic agent for human aortic smooth muscle cells,eliciting a response comparable to PDGF-BB.14

Although primary cultured smooth muscle cells expressneurotrophins and trk receptors, the use of these cells islimited because of the inability to culture these cells forprolonged periods without a significant change in pheno-type,23 precluding generation of stable clones expressingreceptors by gene transfer. Thus, to define the intracellularsignaling pathways activated by NGF in vascular smoothmuscle cells, conditionally immortalized mouse smooth mus-cle cells were used to generate smooth muscle cell lines thatstably express trkA. Vascular smooth muscle cells are derivedfrom thoracic aortic explants of transgenic mice expressing atemperature-sensitive SV40-T antigen, under the control ofthe smooth muscle cell–specifica-actin promoter (Fan et al,unpublished data, 1999). These cells exhibit an immortalizedphenotype at 33°C, when T antigen is expressed. At thenonpermissive 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 at33°C, then shifted to 39.5°C, for 3 to 4 days, to acquire anontransformed state, for functional and biochemical assays.

The signaling pathways that mediate NGF-induced smoothmuscle cell migration are not known. Because these cellsexpress native PDGF-BB (Fan et al, unpublished data, 1999)

and IGF-1 receptors (see below), we have tested whetherunique signaling pathways mediate migration or proliferationof vascular smooth muscle cells. Therefore, the presentstudies were undertaken to determine (1) whether NGFactivates signaling pathways in trkA-expressing smooth mus-cle 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 indifferent cell types, such as migration of smooth muscle cellsand neuronal differentiation, are mediated by similar ordivergent signaling cascades.

MethodsCell CultureTemperature-sensitive mouse smooth muscle cells (TsTmSMC)were grown from aortic explants of a transgenic mouse line express-ing a temperature-sensitive SV40-T antigen, under the control of thepromoter for smooth muscle cella 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(100mg/mL; Gibco), and glutamine (2 mmol/L; Gibco). TsTmSMCgrown at 33°C were transfected, using CaPO4, with the pMEXvector25 containing the cDNA for human trkA and encoding neomy-cin (G418) resistance. After selection in G418 (1 mg/mL; Gibco),colonies were subcloned, expanded, and tested for stable expressionof full-length trk receptors by Western blot analysis (see below).Human aortic smooth muscle cells (HASMC) were obtained fromATCC at passage 12 and were cultured at 37°C in M199 and 20%FCS, penicillin, streptomycin, glutamine, and endothelial cell growthfactor (30mg/mL; Biotechnology Research Institute).

RT-PCR TechniquesTotal RNA was isolated from TsTmSMC grown at either 33°C or39.5°C by extraction in guanidinium isothiocyanate and purifiedover a cesium chloride cushion by ultracentrifugation. One micro-gram of total RNA was subjected to reverse transcription usingmurine leukemia virus transcriptase (Perkin-Elmer). Total RNA notincubated with reverse transcriptase was used as a negative control.cDNA products were amplified by incubation with AmpliTaqpolymerase (Perkin-Elmer) and primers specific for each neurotro-phin, as previously described.14 Products were resolved by electro-phoresis in 7% polyacrylamide gels, followed by visualization withethidium bromide.

Western Blot AnalysisNative TsTmSMC and TsTmSMC expressing trkA were lysed inRIPA buffer containing the protease inhibitors phenylmethylsulfonylfluoride (1 mmol/L), aprotinin (1 mg/mL), and leupeptin (10mg/mL).25 After incubation on ice for 15 minutes, lysates wereclarified by centrifugation at 14 000g (Beckman microfuge) at 4°C,and the protein content of the supernatant determined by a Bio-Radprotein assay (Bio-Rad Laboratories) using bovine serum albumin asa standard. Detergent lysates containing equivalent amounts ofprotein were separated by 9% SDS-PAGE and blotted onto nitrocel-lulose. Western blot analysis was performed using a polyclonalantisera that recognizes all full-length trk isoforms (203 antisera26).Immunoreactive proteins were detected using enhanced chemilumi-nescence (Amersham) with anti-rabbit or anti-murine IgG-horseradish peroxidase (Boehringer Mannheim).

Migration AssayTrkA-expressing clones (designated mtrkA4 and mtrkA48) or nativeTsTmSMC cultured for 3 days at 39.5°C were rinsed with PBS andcultured in 0.5% FCS for 18 hours. Migration assays were performedusing a transwell filter 48-well chamber microchemotaxis apparatus(Neuroprobe Inc) as previously described,14 with slight modifica-tions. A cell suspension was obtained by incubating the cells inPBS/EDTA solution for 5 minutes. After centrifugation, the cellswere resuspended in DMEM containing 0.5% FCS at a concentration

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of 200 000 to 400 000 cells/mL. NGF (1 to 50 ng/mL; murine;Harlan Bioproducts for Science), PDGF-BB (10 ng/mL; recombinanthuman; R&D) or IGF-1 (35 ng/mL; recombinant human; R&D) wereresuspended in 0.5% FCS and placed in the lower chamber. Cells (50mL) were added to the upper chamber, over a polycarbonatepolyvinylpyrrolidone-free 8-mm pore membrane (Poretics Corp)coated with nondenatured rat tail collagen (Biomedical Technolo-gies), and incubated for 4 hours at 37°C in 95% air/5% CO2. Cellsattaching to the upper surface were removed by scraping, and cellsmigrating through the pores were fixed and visualized by stainingwith Dif-quik (Baxter Scientific) and migrating cells counted man-ually. Results are expressed as the fold increase in migration of cellsin the presence of the chemoattractant over cells in the absence of thechemoattractant, with 3 to 4 replicates per experimental group. Inexperiments in which specific enzyme inhibitors were used, the cellswere preincubated with either Wortmannin (10 to 50 nmol/L; SigmaChemical Co), LY294002 (10 to 20mmol/L; Sigma), PD98059 (7 to15 mmol/L; Cal Biochem), or vehicle for 30 minutes at 37°C beforeaddition to the upper chamber. The inhibitors were also added to thebottom wells at the same concentrations. Statistical differencesbetween the groups were determined by Student’st test, andstatistical significance was determined at aP level of ,0.05.

Cell Counting AssayHASMC (passage 14 to 16) were seeded in a 96-well microtiter plateat 5000 cells/well and cultured at 37°C. At 12 hours, the cells weretreated with either 5% FCS alone or 5% FCS containing NGF (10 or50 ng/mL) or PDGF-BB (30 ng/mL) every other day. After 7 days,the cells were resuspended and an aliquot was counted using ahemocytometer. The results are expressed as the number of cells/well6SE of 3 replicates per experimental group.

TrkA-expressing or native TsTmSMC were seeded onto 6-wellcluster plates and coated with rat tail collagen at a density of 30 000cells/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 for48 hours. The cells were then treated in 3% FCS alone or 3% FCScontaining 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 describedabove and results are presented as the mean6SE of 3 replicates perexperimental group.

Mitogenesis Assay ([3H]ThymidineIncorporation Assay)Cell proliferation assessed as [3H]thymidine incorporation in re-sponse to NGF and PDGF-BB was performed as previously de-scribed.27 Briefly, mtrkA48 were grown on collagen-coated 96-wellmicrotiter plates (10 000 cells/well) for 2 days at 39.5°C in 10% FCSmedia followed by 18 hours in 0.5% FCS. The cells were then treatedwith either 0.5% FCS or 0.5% FCS containing PDGF-BB (10ng/mL) or NGF (10 or 50 ng/mL). After 27 hours, [3H]thymidinewas added at 1mCi/well, for 5 hours. The cells were then harvestedonto glass fiber filters using an automated cell harvester (PharmaciaLKB Biotechnology, Inc) and quantified by liquid scintillationcounting. The data are expressed as counts per minute per well.

Assays Detecting Activation of Signaling ProteinsNative or trkA-expressing TsTmSMC were cultured on collagen-coated dishes 3 to 4 days at 39.5°C in 10% FCS media, followed by18 hours in 0.5% FCS. The cells were then treated with NGF (10 or50 ng/mL), PDGF-BB (10 ng/mL), or IGF-1 (35 ng/mL) for theindicated times and lysed with RIPA buffer containing proteaseinhibitors and sodium vanadate (1 mmol/L). Proteins in the celllysates were separated by SDS-PAGE and transferred to nitrocellu-lose. Western blot analysis was performed using an anti-phosphotyrosine antisera (monoclonal 4G10; Upstate Biotechnolo-gy25,28). In some experiments, 300 to 400mg of cell lysate were firstimmunoprecipitated with 203 antisera, followed by proteinA-Sepharose to immunoprecipitate trkA, or with an anti-PLCg1

antibody (mixed monoclonal; Upstate Biotechnology) using proteinG-Sepharose; Shc proteins were immunoprecipitated using an anti-Shc antibody (rabbit polyclonal; Upstate Biotechnology) conjugated

to protein A-Sepharose; immunoprecipitates were separated bySDS-PAGE, followed by Western blot analysis with 4G10.

MAP kinase activation was assessed using an immunoprecipitation-kinase assay.29,30Briefly, 200mg of protein from total cell lysates wereimmunoprecipitated using antisera specific for the MAP extracellularsignal regulated kinase (ERK)-1 or ERK-2 (Santa Cruz Biotechnology)and incubated with [g-32P]ATP using myelin basic protein (Sigma) as asubstrate.30 Kinase activity was quantified using a phosphoimager and isexpressed as relative units, with the activity after 5 minutes of treatmentwith NGF expressed as 1.0 U.

PI-3 Kinase ActivityCells were cultured and treated with growth factors as describedabove and PI-3 kinase assays were performed as previously de-scribed.31,32 Briefly, cells were rinsed with ice-cold PBS, then with137 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°Cfor 20 minutes. After clarification by centrifugation, 1500mg of cellprotein was immunoprecipitated using 4G1020,25 and proteinA-Sepharose. Immunoprecipitates were incubated with sonicatedphosphatidylinositol (final concentration of 0.2 mg/mL; Avanti PolarLipids) and 20mCi [g-32P]ATP in 40mL kinase buffer (30 mmol/LHEPES, pH 7.4, 30 mmol/L MgCl2, 50mmol/L ATP, and 40mmol/Ladenosine) for 10 minutes at room temperature. After termination ofthe reaction and extraction of the phospholipids, the products werespotted onto precoated silica gel 60 thin-layer chromatography plates(Whatman Chromatography) and developed in chloroform/metha-nol/ammonium hydroxide/H2O (45:35:2.5:7.5; vol/vol/vol/vol). Ra-diolabeled phospholipids were visualized by autoradiography, andlabeled phosphatidylinositol phosphate (PIP) migration was com-pared with cold PIP standard (Sigma). Kinase activity was quantifiedusing a phosphoimager and is expressed as relative units, with theactivity after 5 minutes of treatment with NGF expressed as 1.0 U.

ResultsExpression of Neurotrophins and trk Receptorsby TsTmSMCThe expression of the neurotrophins and trk receptors wasassessed by RT-PCR and Western blot analysis.14 TsTmSMC

Figure 1. RT-PCR for detection of mRNA of the neurotrophinsin TsTmSMC. mRNA purified from native TsTmSMC grown at33°C or 39.5°C was subjected to reverse transcription (RT) fol-lowed by PCR using primers specific for each of the neurotro-phins. The PCR product size is indicated by arrows. mRNA puri-fied from NIH3T3 cells was used as a positive control14; mRNAthat was not subjected to reverse transcription (no RT) wasused as a negative control. The marker sizes are in descendingorder 1.4, 1.1, 0.9, 0.6, 0.3, 0.28/0.27, 0.23, 0.19, 0.12, and0.072 kb. This represents 1 of 2 identical experiments.

Kraemer et al April 1999 1043



grown at 33°C and 39.5°C express mRNA for NGF, BDNF,and NT 4/5, but not NT3 (Figure 1). However, immunohis-tochemical analysis using an antibody that recognizes all 4neurotrophins14 indicates that these cells express negligiblelevels of neurotrophin protein when grown at 33°C or 39.5°C(data not shown). No expression of full-length or truncatedtrk proteins could be detected as assessed by Western blotanalysis in TsTmSMC grown at 33°C (Figure 2) or 39.5°C(data not shown) using an antibody that recognizes allfull-length trk isoforms (Figure 2) or antibodies that recog-nize truncated trkB or trkC (data not shown). The lack of trkexpression by native TsTmSMC is confirmed in functionalanalysis of neurotrophin action; TsTmSMC do not migrate inresponse 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).

Derivation of trkA-Expressing TsTmSMCTo study trk receptor signaling in smooth muscle cells,TsTmSMC were transfected with pMEX vector containingthe cDNA for human trkA. Six clones stably expressingsignificant levels of trkA were isolated, and 2 clones wereexamined in detail (Figure 2; designated mtrkA4 andmtrkA48). In comparison with cell lines expressing a definednumber of receptors determined by equilibrium bindinganalysis,25 the trkA-TsTmSMC express approximately10 000 and 40 000 trkA receptors per cell, respectively

(Figure 2). These clones were chosen as they reflect theexpression of trkA in neointimal smooth muscle cells.14

Cell Migration and Proliferation oftrkA-Expressing TsTmSMCThe ability of mtrkA4 and mtrkA48 cells to functionallyrespond to NGF was assessed using chemotaxis (Figure 3)and cell proliferation assays (Figure 4). NGF induces migra-tion of mtrkA4 and mtrkA48 cells at doses ranging from 1 to50 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 10ng/mL of NGF was comparable to that observed withPDGF-BB in both clones. An additional 4 clones of cellsexpressing significant levels of trkA responded similarly toNGF, with maximal migration occurring at approximately 5to 10 ng/mL of NGF (results not shown). Both PDGF-BB andIGF-1 induce migration of trkA-expressing TsTmSMC aseffectively as with native TsTmSMC, ranging from 5- to10-fold for PDGF-BB and 2- to 3-fold for IGF-1.

In contrast, NGF does not induce proliferation of trkA-expressing TsTmSMC (Figure 4). Treatment of mtrkA4 ormtrkA48 as well as native TsTmSMC with PDGF-BB resultsin a 2- to 4-fold increase in cell number, whereas neither NGFnor IGF-1 has an effect (Figure 4A). This was confirmed in a[3H]thymidine incorporation assay (Figure 4B), in whichPDGF-BB induced a 2-fold increase in [3H]thymidine incor-poration in mtrkA48, whereas NGF had no effect. Finally,these results are comparable to those using HASMC, in whichNGF (50 ng/mL) does not stimulate the proliferation ofHASMC (results not shown), whereas PDGF-BB (30 ng/mL)induces a 2- to 4-fold increase in cell number after 7 days oftreatment (4.860.23103 versus 9.460.33103 versus4.9.60.43103 cells/well; control versus PDGF-BB versusNGF). Comparable results using PDGF-BB-treated culturedhuman aortic smooth muscle cells have been reported.9

Therefore, NGF behaves similarly to IGF-1, as a chemotacticbut not a mitogenic agent for human vascular smooth mus-cle.7 Moreover, native TsTmSMC, which do not expressfull-length or truncated isoforms of trk, fail to respond toNGF in either chemotaxis (Figure 3) or proliferation assays(Figure 4). Thus, similar to early passage HASMC, trkA-

Figure 2. Expression of trkA in native and stably transfectedTsTmSMC. Expression of trkA in native TsTmSMC and 2 stablytransfected clones (mtrkA48 and mtrkA4) was detected byWestern blot analysis using an antisera detecting full-lengthtrkA, trkB, or trkC (203 antisera26). PC12 cells expressing highlevels of trkA25 were used as a positive control.

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, 35ng/mL), and NGF (1 to 50 ng/mL as indicated). Cell numbers for wells treated with no ligand (control, C) were TsTmSMC (8765.4);mtrkA48 (11262.4); mtrkA4 (6868.2). This represents 1 of 4 or 5 similar experiments for each cell group. Numbers are mean6SE of 4replicates per experimental group.




expressing TsTmSMC demonstrate chemotactic but not mi-togenic responses to NGF.

Activation of Signal Transduction PathwaysOur previous studies demonstrated that TsTmSMC and cul-tured 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 notinduce the tyrosine phosphorylation of cellular proteins inTsTmSMC cells (Figure 5A), consistent with their lack oftrkA expression. However, trkA-expressing TsTmSMC re-spond to NGF with a dose-dependent increase in the phos-phorylation of the trkA receptor (Figure 5B) as well asincreases in the tyrosine phosphorylation of several proteins,in a pattern similar to that of PDGF-BB (Figure 5A).

To further compare the signaling pathways activated byNGF, PDGF-BB, and IGF-1, mtrkA4 and mtrkA48 cellsgrown 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 kinasepathway, PLCg, and PI-3 kinase was assessed. The resultsusing mtrkA48 cells are shown in Figures 6, 7, and 8 andsimilar results were obtained with mtrkA4 cells (data notshown).

NGF treatment of mtrkA48 at both 10 and 50 ng/mLinduces prolonged phosphorylation of Shc and the MAPkinase enzymes, ERK-1 and ERK-2 (Figure 6). Anti-phosphotyrosine Western blot analysis of immunoprecipi-tated Shc proteins demonstrates increased phosphorylation ofproteins migrating at 46, 52, and 68 kDa (Figure 6A),confirmed by Western blot analysis to be the 46-, 52-, and68-kDa isoforms of Shc (results not shown). Shc proteins arephosphorylated within 5 minutes of NGF treatment ofmtrkA48, and remained phosphorylated for up to 1 hour aftertreatment (Figure 6A).

NGF also induces the tyrosine phosphorylation of 43- and42-kDa proteins (Figure 6B), confirmed by Western blotanalysis to be ERK-1 and ERK-2 (results not shown). ERK-1and ERK-2 remain phosphorylated for 1 (Figure 6B) to 4hours (not shown) after NGF treatment. The phosphorylationof MAP kinase correlates with its activation (Figure 6B), such

that 50 ng/mL of NGF treatment induces a 6- to 8-foldincrease in MAP kinase activity within 5 minutes of treatmentand the activity remains elevated up to 4 hours (Figure 6C anddata not shown). Similar results were obtained using 10ng/mL of NGF.

In contrast, PDGF-BB induces transient phosphorylation ofShc and MAP kinase in mtrkA48 cells. Peak phosphorylationof the 46- and 56-kDa isoforms of Shc and ERK-1 and ERK-2

Figure 4. Proliferation of TsTmSMC, mtrkA4,and mtrkA48. A, Cell counting assay of native ortrkA-expressing TsTmSMC. Native TsTmSMC,mtrkA48, or mtrkA4 were cultured at 39.5°C for3 days and then treated for 10 days in either 3%FCS alone (C), or 3% FCS containing eitherPDGF-BB (P, 10 ng/mL), IGF-1 (I, 10 ng/mL), orthe indicated doses of NGF. The dose of NGF(N) used in the experiments with TsTmSMC andmtrkA48 was 10 ng/mL. Values are mean6SE of3 replicates per group. Similar results wereobtained in 2 additional experiments. B, [3H]Thy-midine incorporation in mtrkA48. mtrkA48 wereseeded on collagen-coated 96-well microtiterplates and cultured at 39.5°C for 3 days, thentreated 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 asdescribed in the Methods. Values are mean6SEof 6 replicates per group. Similar results wereobtained in 3 additional experiments.

Figure 5. Tyrosine phosphorylation in native TsTmSMC andtrkA-expressing TsTmSMC in response to PDGF-BB and NGF.A, Anti-phosphotyrosine Western blot analysis of cell lysatesobtained from native TsTmSMC or mtrkA48 grown for 3 days at39.5°C then treated with either PDGF-BB (10 ng/mL) or NGF (50ng/mL) for 5 minutes. Similar results were obtained with mtrkA4.B, Dose-dependent phosphorylation of trkA receptor in mtrkA4cells. mtrkA4 cells grown for 3 days at 39.5°C were treated withthe indicated doses of NGF for 5 minutes. Whole cell lysateswere immunoprecipitated with the anti-trk antibody (a203) fol-lowed by Western blot analysis using an anti-phosphotyrosineantibody. Similar results were obtained within 1 other experi-ment. Similar results were obtained with mtrkA48 cells.




occurs at 5 minutes in response to PDGF-BB and begins toreturn to control levels within 20 to 60 minutes (Figure 6A).Interestingly, PDGF-BB did not induce the phosphorylationof the 68-kDa isoform of Shc. Peak MAP kinase activity alsooccurs within 5 minutes of treatment with PDGF-BB andreturns to a level approximately 2-fold above control by 1

hour (Figure 6B). This pattern of PDGF-induced MAP kinaseactivation is similar to that observed using cultured humansmooth muscle cells.7,8 IGF-1 does not significantly activateMAP kinase in trkA-expressing TsTmSMC (Figure 6B) nordoes it phosphorylate Shc (Figure 6A), consistent with resultsobtained with human neonatal vascular smooth muscle cells.7

Thus, in smooth muscle cells expressing trkA receptors, NGFinduces a prolonged activation of the Shc/MAP kinasepathway whereas PDGF-BB induces a transient activationand IGF-1 treatment is without effect.

Activation of PLC enzymes and PI-3 kinase has beenimplicated in the migration of human smooth muscle cells inresponse to PDGF and IGF-1.7,9 In mtrkA48 cells, both NGF(50 ng/mL) and PDGF-BB induce a rapid phosphorylation ofPLCg1, within 5 minutes of treatment (Figure 7), whereasIGF-1 has no effect. However, PLCg1 remains phosphorylat-ed in response to NGF, whereas the response to PDGF-BB istransient, returning to control levels within 20 minutes. Thus,as is observed with MAP kinase activation, NGF inducesprolonged phosphorylation of PLCg1 in smooth muscle cells.

NGF, PDGF-BB, or IGF-1 treatment of mtrkA48 cellsincreases PI-3 kinase activity, but to variable degrees, asassessed by immunoprecipitation kinase assays (Figure 8).NGF and IGF-1 induce a 2- to 3-fold increase in PI-3 kinaseactivity, and the activity remains increased relative to controlcells for up to 1 hour. In contrast, PDGF-BB markedlyinduces PI-3 kinase activity, with a greater than 20-foldincrease in activity within 5 minutes of treatment, and theactivity remains elevated by approximately 2- to 3-fold overcontrol cells at 1 hour. Thus, NGF, IGF-1, and PDGF-BBhave different effects on the level of activation of PI-3 kinasein smooth muscle cells. Table 1 summarizes the biologicalresponsiveness and the signal transduction pathways acti-vated by NGF, IGF-1, and PDGF-BB in trkA-expressingsmooth muscle cells.

To determine which of the above signaling pathwaysmediated migration and proliferation in response to NGF and

Figure 6. Activation of Shc/MAP kinase signal transduction path-way in trkA-expressing TsTmSMC. A, Anti-phosphotyrosine West-ern blot analysis of Shc proteins immunoprecipitated from mtrkA48treated 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 ligandaddition; Whole cell lysates were immunoprecipitated with an anti-Shc antibody conjugated to protein A-Sepharose followed byWestern blot analysis using an anti-phosphotyrosine antibody. B,ERK-1 and ERK-2 activation. Cells were treated with PDGF-BB (10ng/mL), NGF (10 or 50 ng/mL), IGF-1 (35 ng/mL), or no ligand (Ctl)at the indicated time points. Anti-phosphotyrosine Western blotanalysis (top) demonstrates increased phosphorylation of ERK-1and ERK-2. MAP kinase activity (bottom) in ligand-treated mtrkA48cells was measured from detergent lysates of cells treated withPDGF (E, 10 ng/mL), NGF (F, 50 ng/mL), or IGF-1 (M, 35 ng/mL)at the indicated time points. Data were normalized so that 1.0equals the volume data obtained by phosphoimager analysis after5 minutes of treatment with NGF. Data are presented as themean6SE of 4 experiments.

Figure 7. Phosphorylation of PLCg in mtrkA48 in response toNGF. 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-PLCgantibody followed by Western blot analysis using an anti-phosphotyrosine antibody. Similar results were obtained in 4experiments using mtrkA48 cells or mtrkA4 cells.

Figure 8. Activation of PI-3 kinase in mtrkA48 in response toNGF. mtrkA48 cells were treated with either PDGF-BB (E, 10ng/mL), NGF (F, 50 ng/mL), or IGF-1 (M, 35 ng/mL) for the indi-cated time and PI-3 kinase assays performed as described.Data were normalized so that 1.0 equals the volume analysisafter 5 minutes of treatment with NGF. Data are presented asthe mean6SE of 3 to 5 experiments.




PDGF-BB, pharmacological inhibitors of either PI-3 kinase,Wortmannin and LY294002, or MAP kinase kinase, PD98059, were used in the migration assay (Figure 9). Onlyresponses to PDGF-BB and NGF were assessed, as they aremore potent chemoattractants compared with IGF-1. BothWortmannin and LY294002 inhibited migration in responseto PDGF-BB and NGF in a dose-dependent manner (Figure9), similar to their inhibitory activity on PI-3 kinase-dependent responses in other cell systems.33–35 Wortmannin(10 nmol/L) resulted in a 50% reduction in migration, andWortmannin (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 LY294002similarly inhibited migration at doses ranging from 10 to20 mmol/L (Figure 9A). In contrast, PD 98059 had no effecton PDGF-BB- or NGF-induced migration at doses rangingfrom 7 to 15mmol/L (Figure 9B), concentrations that com-pletely inhibit phosphorylation of the MAP kinases, ERK-1and ERK-2, as assessed by phosphotyrosine Western blotanalysis (results not shown; also see Reference 36).

DiscussionMigration of medial smooth muscle cells and their subse-quent proliferation are the primary causes of smooth musclecell accumulation in the neointima after vascular injury.1

Growth factors expressed in neointimal lesions have distinc-

tive effects on smooth muscle cells, with PDGF inducing bothproliferation and migration, and IGF-1 and NGF promotingonly chemotaxis. It has been suggested that different signal-ing pathways regulate the proliferative compared with themigratory responses of PDGF on smooth muscle cells, withMAP kinase regulating proliferation and PLCg and PI-3-kinase regulating migration.7,11 This hypothesis has remaineduntested with regard to other receptor tyrosine kinases ex-pressed by vascular smooth muscle cells.

To address these issues, we have established a modelsystem using TsTmSMC grown at 39.5°C to examine theability of different growth factors to mediate directed migra-tion of smooth muscle cells using the transwell microchamberchemotaxis assay. These cells migrate in response toPDGF-BB or IGF-1 comparably to that observed with adultand fetal human7,9 and fetal bovine aortic smooth musclecells.37 Moreover, similar signal transduction pathways areactivated in TsTmSMC in response to PDGF and IGF-1 asthose observed in human smooth muscle cells and other cellsystems.7–9,38,39Finally, when trkA receptors are expressed inTsTmSMC, NGF is chemotactic, but not mitogenic, similar towhat is observed for HASMC (present paper and Reference14). Thus, this model system allows for direct comparison ofsignaling pathways activated by different receptor tyrosinekinases to determine whether activation of specific pathwayscorrelates with their ability to mediate either directed migra-tion or cell proliferation.

Figure 9. Pharmacological inhibition ofligand-induced migration of smooth musclecells. Microchamber chemotaxis assays wereperformed as described above. mtrka48 cellswere grown for 3 days at 39.5°C, then prein-cubated with Wortmannin (A, left), LY294002(A, right), or PD 98059 (B) at the indicateddoses, or in vehicle (DMSO or ethanol[EtOH]), for 30 minutes at 37°C before addi-tion to the upper well. Similar concentrationsof the inhibitors were added to the bottomwells. PDGF-BB and NGF were added to thebottom wells as before. Cell numbers forwells treated with no ligand (control, C) wereas follows: A, DMSO, 8968; Wortmannin (10nmol/L), 7565; Wortmannin (50 nmol/L),3665; B, EtOH, 162618; LY294002(10 mmol/L), 87.5611; LY294002 (20 mmol/L),4564; C, DMSO, 2468; PD 98059(15 mmol/L), 4565; PD 98059 (30 mmol/L),9367.

Comparison of Biological Activity and Time Course of Activation of Signal TransductionPathways Activated by NGF, IGF-1, and PDGF-BB in TsTmSMC Expressing trkA

Ligand Migration Proliferation Shc/MAP Kinase PLCg PI-3 Kinase

NGF 11 2 Prolonged, high levelof activation

Prolonged, high levelof activation

Prolonged, low levelof activation

PDGF-BB 11 11 Transient, high levelof activation

Transient, high levelof activation

Prolonged, highlevel of activation

IGF-1 1 2 Minimal activation Minimal activation Prolonged, low levelof activation

11 indicates 5- to 8-fold increase over control; 1, 2-fold increase over control; and 2, no increase over control.




The results of the current study suggest that ligand-inducedactivation of different receptor tyrosine kinases results in adistinct pattern of signal transduction pathways and that theultimate biological responses to each growth factor areregulated by the integration of these signals. Although bothNGF and IGF-1 are strictly chemotactic for smooth musclecells, NGF is a potent activator of MAP kinase, PLCg, and,to a lesser extent, PI-3 kinase, whereas only PI-3 kinaseactivation could be detected in response to IGF-1. Con-versely, similar signaling pathways are activated byPDGF-BB and NGF, although PDGF-BB is both chemotacticand mitogenic, whereas NGF is solely chemotactic. There aredifferences, however, in the extent and duration of activationof the signaling molecules. (1) PI-3 kinase activation is10-fold higher in response to PDGF-BB compared with NGF.However, PI-3 kinase inhibitors dose-dependently reducedboth PDGF-BB- and NGF-induced migration, suggesting thatthis signaling pathway mediates migration to both growthfactors despite differences in the level and duration of PI-3kinase activation by the 2 growth factors. Our results differfrom Higaki et al,12 who found no inhibition of PDGF-induced smooth muscle cell migration by either Wortmanninor LY294002. This can be reconciled by the fact that in theprevious studies, the cells were not preincubated with theinhibitors before the migration assay, and under these condi-tions, the enzyme may not have been completely inhibited.(2) NGF induces a more prolonged activation of MAP kinaseand PLCg than that which occurs in response to PDGF-BB.In prior studies, prolonged activation of MAP kinase hascorrelated with smooth muscle cell proliferation, becausecontractile agents, such as angiotensin II, that do not inducesmooth muscle cell proliferation only cause a transientincrease in MAP kinase activity.8 However, the more pro-longed activation of MAP kinase by NGF in trkA-expressingTsTmSMC compared with PDGF-BB argues against theduration of MAP kinase activation controlling proliferation.

How can the present results be reconciled with prior resultsin which the ras/MAP kinase pathway mediates distinctdownstream responses depending on the duration of itsactivation? Both the proliferation and migration of cells aredependent on coupling cytoskeletal reorganization to alter-ations in gene transcription.40–44Given the inability to detectthe activation of a single pathway controlling proliferation, itis likely that the combinatorial effects of several upstreamsignaling pathways may converge to induce a particularresponse. For example, activation of the Shc/MAP kinasecascade regulates the transcription and translation of certainmRNAs thought to be important for proliferation.11 However,ras can activate raf, MEK-1, and MAP kinase; it can alsoactivate rac and rho, events that are associated with cytoskel-etal rearrangement.45–49 Activation of PLCg can induce theformation of inositol 1,4,5-trisphosphate, an important medi-ator of calcium mobilization from intracellular stores, whichcan modulate actin reorganization.11 Lipid products of PI3-kinase activate the serine/threonine kinase Akt, whosedownstream targets include p70 S6 kinase, a protein thatregulates cell proliferation (reviewed in References 50 and51). Alternatively, inhibition of PI 3-kinase prevents PDGF-induced activation of rac and may also influence receptortyrosine kinase-mediated integrin activation (reviewed inReference 52). Thus, the coordinate activation of MAP kinase

with either PLCg or PI-3 kinase, including the appropriatelevel and duration of activation, may be necessary to inducethe proper cytoskeletal and gene transcription events thatcontrol proliferation. This is supported by the fact thatinhibitors of PI-3 kinase or microinjection of antibodiesdirected against PI-3 kinase or PLCg can prevent bFGF- orPDGF-induced proliferation of smooth muscle cells or fibro-blasts, whereas a PDGF receptor mutated at the PI-3 kinasebinding site prevents PDGF-induced DNA synthesis infibroblasts.53–56

Alternatively, common signaling pathways may mediatedistinctive biological outcomes by activating different effec-tor proteins. As mentioned above, in addition to MEK-1 andMAP kinase, ras can also activate rac and rho. Prolongedactivation of Shc and ras may favor activation of rac and rhoor other, as yet unidentified, cytoskeletal components and, incombination with prolonged PLCg activation, may mediateNGF-induced migration.

An additional possibility is that the differential intracellularlocation of activated signaling molecules can result in differ-ent functions. Thus, prolonged activation of MAP kinase mayinduce translocation to the nucleus in response to an agonist,and regulate the gene-induction profile. In quiescent smoothmuscle cells, MAP kinase is predominantly located in thecytosol.8,57 After PDGF and bFGF treatment, MAP kinasetranslocates to the nucleus, where it may induce transcriptionof genes important for smooth muscle cell proliferation.8,58 Incontrast, contractile agents stimulate a transient translocationof MAP kinase to the surface membrane and a later, moresustained redistribution to the contractile filaments.57,58 Thepotential for redistribution of MAP kinase by NGF intrkA-expressing TsTmSMC will require further investigation.Finally, it is possible that novel, or as yet untested, signalingpathways are activated, resulting in solely a migratory orproliferative stimulus.

Using TsTmSMC, we have been unable to detect thephosphorylation of PLCg in response to IGF-1. Our resultsare consistent with data that PLCg does not bind to thetyrosine-phosphorylated IGF-1 receptor nor is it activated byIGF-1.59,60 Previous studies of IGF-1 treatment of humansmooth muscle cells have detected the formation of diacyl-glycerol in response to IGF-1 as an index of PLC activa-tion.7,61,62 These results can be reconciled with the currentstudy as there are 3 families of PLC enzymes,b, g, and disoforms, each of which has multiple members. AlthoughIGF-1 does not activate PLCg, it can stimulate the activity ofPLCb1 in Swiss 3T3 cells.59 Thus, an increase in PI turnoverin response to IGF-1 may be caused by the activation of adifferent PLC isoform, which would not be detected with theisoform-specific antibody used in the present study.

Trk activation leads to distinctive biological responses ofsurvival and differentiation in neural crest-derived cells,15,16

and migration in vascular smooth muscle cells (Reference 14and present paper). However, the profiles of activation of the3 signaling pathways examined are remarkably similar. NGF-induced trkA activation in neuronal cells results in phosphor-ylation of Shc, MAP kinase, and PLCg, and the activation ofPI-3 kinase.20,25The MAP kinase kinase inhibitor, PD 98059,inhibits NGF-induced differentiation of the rat pheochromo-cytoma cell line, PC12,63 but does not inhibit NGF-inducedsmooth muscle cell migration (Figure 9), suggesting that the




cell type-specific biological responses elicited by NGF insmooth muscle cells, compared with neuronal cells, maydepend on the intracellular environment and the specificisoforms of the different signaling molecules that are acti-vated in a specific cell type. For example, there are at least 2isoforms of the p85 subunit of the PI-3 kinase and 2 isoformsof the p110 subunit.64–67Similarly, different protein kinase Cisoforms promote different responses depending on the intra-cellular molecules with which they interact.68,69 Moreover,the cytoskeletal components with which the signaling mole-cules interact in differentiated smooth muscle cells versusneuronal cells may also contribute to the different biologicalresponses observed.

Thus, NGF induces the migration, but not the proliferation,of smooth muscle cells that express trkA receptors, yetactivates similar signaling pathways as PDGF-BB, includingShc/MAP kinases, PLCg, and PI-3 kinases. Studies usingmutant trk receptors that can no longer bind or activate eachof these specific pathways will help in assessing which arerequired for the chemotactic response of smooth muscle cellsto NGF.

AcknowledgmentsThis 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 ReviewGrant (K.L.M.). Special thanks to Dominick Falcone for thoughtfuldiscussion, and Cherie Wieland for secretarial assistance.

References1. Ross R. Pathogenesis of atherosclerosis: a perspective for the 1990s.

Nature. 1993;362:801–809.2. Majack RA, Grieshaber NA, Cook CL, Weiser MCM, McFall RC,

Grieshaber SS, Reidy MA, Reilly CF. Smooth muscle cells isolated fromthe neointima after vascular injury exhibit altered responses to platelet-derived growth factor and other stimuli.J Cell Physiol. 1996;167:106–112.

3. Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox JN,Schwartz SM. PDGF ligand and receptor gene expression during repair ofarterial injury.J Cell Biol. 1990;111:2149–2158.

4. Ross R, Masuda J, Raines E, Gown A, Kutsuda S, Sasahara M, MaldenL, Masuko H, Sato H. Localization of PDGF-B protein in macrophages inall phases of atherogenesis.Science. 1990;248:1009–1012.

5. Cercek B, Fishbein MC, Forrester JS, Helfant RH, Fagin JA. Induction ofinsulin-like growth factor I messenger RNA in rat aorta after balloondenudation.Circ Res. 1990;66:1755–1760.

6. Khorsandi MJ, Fagin JA, Giannella-Neto D, Forrester JS, Cercek B.Regulation of insulin-like growth factor-I and its receptor in rat aorta afterballoon denudation.J Clin Invest. 1992;90:1926–1931.

7. Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, Ross R.Insulin-like growth factor-I and platelet-derived growth factor-BB inducedirected migration of human arterial smooth muscle cells via signalingpathways that are distinct from those of proliferation.J Clin Invest.1994;93:1266–1274.

8. Mii S, Khalil RA, Morgan KG, Ware JA, Kent KC. Mitogen-activatedprotein kinase and proliferation of human vascular smooth muscle cells.Am J Physiol. 1996;39:H142–H150.

9. Bornfeldt KE, Graves LM, Raines EW, Igarashi Y, Wayman G,Yamamura S, Yatomi Y, Sidhu JS, Krebs EG, Hakomori S, Ross R.Sphingosine-1-phosphate inhibits PDGF-induced chemotaxis of humanarterial smooth muscle cells: spatial and temporal modulation of PDGFchemotactic signal transduction.J Cell Biol. 1995;130:193–206.

10. Ranganna K, Joshi T, Yatsu FM. Sodium butyrate inhibits platelet-derived growth factor-induced proliferation of vascular smooth musclecells.Arterioscler Thromb Vasc Biol. 1995;15:2273–2283.

11. Bornfeldt KE. Intracellular signaling in arterial smooth muscle migrationversus proliferation.Trends Cardiovasc Med. 1996;6:143–151.

12. Higaki M, Sakaue H, Ogawa W, Kasuga M, Shimokado K. Phosphati-dylinositol 3 kinase-independent signal transduction pathway for plate-

let-derived growth factor-induced chemotaxis.J Biol Chem. 1996;271:29342–29346.

13. Clyman RI, Peters KG, Chen YQ, Escobedo J, Williams LT, Ives HE,Wilson E. Phospholipase-C gamma activation, phosphatidylinositolhydrolysis, and calcium mobilization are not required for FGF receptor-mediated chemotaxis.Cell Adhes Commun. 1994;1:333–342.

14. Donovan MJ, Miranda RC, Kraemer R, Mccaffrey TA, Tessarollo L,Mahadeo D, Sharif S, Kaplan DR, Tsoulfas P, Parada L, Toran-AllerandD, Hajjar DP, Hempstead BL. Neurotrophin and neurotrophin receptors invascular smooth muscle cells: regulation of expression in response toinjury. Am J Pathol. 1995;147:309–324.

15. Barde Y-A. Trophic factors and neuronal survival.Neuron. 1989;2:1525–1534.

16. Levi-Montalcini R. The nerve growth factor: 35 years later.Science.1987;237:1154–1162.

17. Chao M. Neurotrophin receptors: a window into neuronal differentiation.Neuron. 1992;9:583–593.

18. Chao MV, Hempstead BL. p75 and trk: a two-receptor system.TrendsNeurosci. 1995;18:321–326.

19. Soltoff S, Rabin S, J., Cantley L, Kaplan DR. Nerve growth factorpromotes the activation of phosphatidylinositol 3-kinase and its asso-ciation with the trk tyrosine kinase.J Biol Chem. 1992;267:17472–17477.

20. Stephens RM, Loeb DM, Copeland TD, Pawson T, Greene LA, KaplanDR. Trk receptors use redundant signal transduction pathways involvingSHC and PLC-gamma 1 to mediate NGF responses.Neuron. 1994;12:691–705.

21. Segal RA, Bhattacharyya A, Rua LA, Alberta JA, Stephens RM, KaplanDR, Stiles CD. Differential utilization of Trk autophosphorylation sites.J Biol Chem. 1996;271:20175–20181.

22. Greene LA, Kaplan DR. Early events in neurotrophin signalling via Trkand p75 receptors.Curr Opin Neurobiol. 1995;5:579–587.

23. Pauly RR, Bilato C, Sollott SJ, Monticone R, Kelly PT, Lakatta EG, CrowMT. Role of calcium/calmodulin-dependent protein kinase II in the reg-ulation of vascular smooth muscle cell migration.Circulation. 1995;91:1107–1115.

24. Deleted in proof.25. Hempstead BL, Rabin SJ, Kaplan L, Reid S, Parada LF, Kaplan DR.

Overexpression of the trk tyrosine kinase rapidly accelerates nervegrowth factor-induced differentiation.Neuron. 1992;9:883–896.

26. Huber LJ, Chao MV. A potential interaction of p75 and trk A NGFreceptors revealed by affinity crosslinking and immunoprecipitation.J Neurosci Res. 1995;40:557–563.

27. Kraemer R, Pomerantz K, Joseph-Silverstein J, Hajjar D. Induction ofbasic FGF mRNA and protein synthesis in smooth muscle cells bycholesterol-enrichment and 25-hydroxy-cholesterol.J Biol Chem. 1993;268:8040–8045.

28. Hempstead BL, Birge RB, Fajardo JE, Glassman R, Mahadeo D, KraemerR, Hanafusa H. Expression of the v-crk oncogene product in PC12 cellsresults in rapid differentiation by both nerve growth factor and epidermalgrowth factor pathways.Mol Cell Biol. 1994;14:1964–1971.

29. Loeb DM, Tsao H, Cobb MH, Green LA. NGF and other growth factorsinduce an association between ERK1 and the NGF receptor, gp140prototrk.Neuron. 1992;9:1053–1065.

30. Teng KK, Landers H, Mahadeo D, Hanafusa H, Hempstead BL, BirgeRB. Sustained activation of a Ras/MAP kinase cascade in PC12 mediatedby v-Crk in response to EGF and NGF.J Biol Chem. 1995;270:20677–20685.

31. Whitman M, Kaplan DR, Schaffhausen B, Cantly L, Roberts TM. Asso-ciation of phosphatidylinositol kinase activity with polyoma middle-Tcompetent for transformation.Nature. 1985;315:239–242.

32. Kaplan DR, Morrison DK, Wong G, McCormick F, Williams LT. PDGFb-receptor stimulates tyrosine phosphorylation of GAP and association ofGAP with a signaling complex.Cell. 1990;61:125–133.

33. Arcaro A, Wymann MP. Wotmannin is a potent phosphatidylinositol3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-triphosphate inneutrophil responses.Biochem J. 1993;296:297–301.

34. Ui M, Okada T, Hazeki K, Hazeki O. Wortmannin as a unique probe foran intracellular signalling protein, phosphoinositide 3-kinase.TrendsBiochem Sci. 1995;20:303–307.

35. Yano H, Agatsuma T, Nakanishi S, Saitoh Y, Nonomura Y, Matsuda Y.Biochemical and pharmacological studies with KT7692 andLY294002onthe role of phosphatidylinositol 3-kinase in Fc epsilon RI-mediated signaltransduction.Biochem J. 1995;312:145–150.

36. Bornfeldt KE, Campbell JS, Koyama H, Argast GM, Leslie CC, RainesEW, Krebs EG, Ross R. The mitogen-activated protein kinase pathwaycan mediate growth inhibition and proliferation in smooth muscle cells.J Clin Invest. 1997;100:875–885.




37. Grotendorst GR, Igarashi A, Larson R, Soma Y, Charette M. Differentialbinding, biological and biochemical actions of recombinant PDGF AA,AB, and BB molecules on connective tissue cells.J Cell Physiol. 1991;149:235–243.

38. Fantl WJ, Johnson DE, Williams LT. Signalling by receptor tyrosinekinases.Annu Rev Biochem. 1993;62:453–481.

39. Pazin MJ, Williams LT. Triggering signaling cascades by receptortyrosine kinases.Trends Biochem Sci. 1992;17:374–378.

40. Wary KK, Mainiero F, Isakoff SJ, Marcanotonio EE, Giancotti FG,Marcantonio EE. The adaptor protein Shc couples a class of integrins tothe control of cell cycle progression.Cell. 1996;87:733–743.

41. Folkman J, Moscona A. Role of cell shape in growth control.Nature.1978;273:345–349.

42. Hansen LK, Mooney DJ, Vacanti JP, Ingber DE. Integrin binding and cellspreading on extracellular matrix act at different points in the cell cycleto promote hepatocyte growth.Mol Biol Cell. 1994;5:967–975.

43. Fang F, Orend G, Watanabe N, Hunter T, Ruoslahti E. Dependence ofcyclinE-CDK2 kinase activity on cell anchorage.Science. 1996;271:499–502.

44. Koyama H, Raines EW, Bornfeldt KE, Roberts JM, Ross R. Fibrillarcollagen inhibits arterial smooth muscle proliferation through regulationof Cdk2 inhibitors.Cell. 1996;87:1069–1078.

45. Joneson T, White MA, Wigler MH, Bar-Sagi D. Stimulation ofmembrane ruffling and MAP kinase activation by distinct effectors ofRAS. Science. 1996;271:810–812.

46. Davis RJ. The mitogen-activated protein kinase signal transductionpathway.J Biol Chem. 1993;268:14553–14556.

47. Nobes CD, Hall A. Rho, Rac, and Cdc42 GTPases regulate the assemblyof multimolecular focal complexes associated with actin stress fibers,lamellipodia and filopodia.Cell. 1995;81:53–62.

48. Symons M. Rho family GTPases: the cytoskeleton and beyond.TrendsBiochem Sci. 1996;21:178–181.

49. Ridley AJ, Hall A. The small GTP-binding protein rho regulates theassembly of focal adhesions and actin stress fibers in response to growthfactors.Cell. 1992;70:389–399.

50. Toker A, Cantley LC. Signalling through the lipid products of phospho-inositide-3-OH kinase.Nature. 1997;387:673–676.

51. Chou MM, Blenis J. The 70 kDa S6 kinase: regulation of a kinase withmultiple roles in mitogenic signalling.Curr Opin Cell Biol. 1995;7:806–814.

52. Carpenter CL, Cantley LC. Phosphoinositide kinases.Curr Opin CellBiol. 1996;8:153–158.

53. Fantl WJ, Escobedo JA, Martin GA, Turck CW, Del Rosario M,McCormick F, Williams LT. Distinct phosphotyrosines on a growthfactor receptor bind to specific molecules that mediate different signalingpathways.Cell. 1992;69:413–423.

54. Roche S, Koegl M, Courtneidge SA. The phosphatidylinositol 3 kinasealpha is required for DNA synthesis induced by some, but not all growthfactors.Proc Natl Acad Sci U S A. 1994;91:9185–9189.

55. Roche S, McGlade J, Jones M, Gish GD, Pawson T, Courtneidge SA.Requirement of phospholipase C gamma, the tyrosine phosphatase Sypand the adaptor proteins Sch and Nck for PDGF-induced DNA synthesis:evidence for the existence of Ras-dependent and Ras-independentpathways.EMBO J. 1996;15:4940–4948.

56. Weiss RH, Yabes AP. Mitogenic inhibition by phorbol esters is asso-ciated with decreased phosphatidylinositol-3 kinase activation.Am JPhysiol. 1996;39:C619–C627.

57. Khalil RA, Morgan KG. PKC-mediated redistribution of mitogen-acti-vated protein kinase during smooth muscle cell activation.Am J Physiol.1993;265:C406–C411.

58. Lenormand P, Sardet C, Pages G, L’Allemain G, Brunet A, PouyssegurJ. Growth factors induce nuclear translocation of MAP kinases (p42MAPK and p44 MAPK) but not of their activator MAP kinase kinase(p45 MAPK) in fibroblasts.J Cell Biol. 1993;122:1079–1088.

59. Maraldi NM, Zini N, Ognibene A, Martelli AM, Barbieri M, Mazzotti G,Manzoli FA. Immunocytochemical detection of the intracellular vari-ations of phosphatidylinositol 4,5-biphosphate amount associated withchanges of activity and amount of phospholipase C beta 1 in cells exposedto mitogenic or differentiating agonists.Biol Cell. 1995;83:201–210.

60. Seely BL, Reichart DR, Staubs PA, Jhun BH, Hsu D, Maegawa H,Milarski KL, Saltiel AR, Olefsky JM. Localization of the insulin-likegrowth factor I receptor binding sites for the SH2 domain proteins p85,Syp, and GTPase activating protein.J Biol Chem.1995;270:19151–19157.

61. Lee SB, Rhee SG. Significance of PIP2 hydrolysis and regulation ofphospholipase C isozymes.Curr Opin Cell Biol. 1995;7:183–189.

62. Spiegal S, Foster D, Kolesnick R. Signal transduction through lipidsecond messengers.Curr Opin Cell Biol. 1996;8:159–167.

63. Pang L, Sawada T, Decker SJ, Saltiel AR. Inhibition of MAP kinasekinase blocks the differentiation of PC-12 cells induced by nerve growthfactor.J Biol Chem. 1995;270:13585–13588.

64. Baltensperger K, Kozma LM, Jaspers SR, Czech MP. Regulation byinsulin of phosphatidylinositol 39-kinase bound to alpha- and beta-isoforms of p85 regulatory subunit.J Biol Chem. 1994;269:28937–28946.

65. Reif K, Gout I, Waterfield MD, Cantrell DA. Divergent regulation ofphosphatidylinositol 3-kinase alpha and p85 beta isoforms upon T cellactivation.J Biol Chem. 1993;268:10780–10788.

66. Hu P, Schlessinger J. Direct association of the p110 beta phosphatidyl-inositol 3 kinase with p85 is mediated by an N terminal fragment of p110beta.Mol Cell Biol. 1994;14:2577–2583.

67. Volinia S, Hiles I, Ormondroyd E, Nizetic D, Antonacci R, Rocchi M,Waterfield MD. Molecular cloning, cDNA sequence, and chromosomallocalization of the human phosphatidylinositol 3-kinase p110 alpha(PI3CA) gene.Genomics. 1994;24:472–477.

68. Andrea JE, Walsh MP. Protein kinase C of smooth muscle.Hypertension.1992;20:585–595.

69. Leng L, Du B, Consigli S, Mccaffrey TA. Translocation of protein kinaseC-delta by PDGF in vascular smooth muscle cells: inhibition by TGF-beta1.Artery. 1996;22:140–154.




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