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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2133-2140

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2133-2140.)
© 1999 American Heart Association, Inc.

Vascular Biology

Phosphatidylinositol 3-Kinase and Focal Adhesion Kinase Are Early Signals in the Growth Factor–Like Responses to Thrombospondin-1 Seen in Human Vascular Smooth Muscle

Joanne S. Lymn; Sarafina J. Rao; Gerard F. Clunn; Karen L. Gallagher; Clive O'Neil; Neil T. Thompson; Alun D. Hughes

From Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology & Medicine, St. Mary's Hospital, London (J.S.L., S.J.R., G.F.C., K.L.G., A.D.H.), and the Immunology Unit, Glaxo-Wellcome, Medicines Research Centre, Stevenage (C.N., N.T.T.), England.

Correspondence to Joanne S. Lymn, Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology & Medicine, QEQM Wing, St. Mary's Hospital, Paddington, London W2 1NY, England. E-mail j.lymn{at}ic.ac.uk


*    Abstract
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Abstract—Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1–stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1–induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure.


Key Words: thrombospondin-1 • focal adhesion kinase • phosphatidylinositol 3-kinase • human vascular smooth muscle


*    Introduction
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 *Introduction
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Thrombospondin-1 (TSP-1), a large, homotrimeric glycoprotein, is a member of the TSP family of matricellular proteins. This family currently consists of 5 isoforms: TSP-1 through TSP-4 and TSP-5/cartilage oligomeric matrix protein,1 which, although implicated in a wide variety of biological processes,2 show differential tissue expression both temporally and spatially.3 4 TSP-1 was originally described as a product of platelet {alpha}-granules5 but has since been shown to be synthesized by a number of different cell types, including vascular endothelial and smooth muscle cells.6 7 TSP-1 is present in negligible amounts in normal human blood vessels, but the expression of this protein is markedly upregulated in injured and diseased vasculature,8 9 10 suggesting a possible role for TSP-1 in the abnormal cellular response to vascular injury.

Literature evidence would suggest that TSP-1 levels are regulated by growth factors. Both platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß) have been shown to induce TSP-1 secretion from vascular smooth muscle cells (VSMCs).11 12 TSP-1 mRNA levels have been shown to be similarly regulated. Basic fibroblast growth factor upregulates TSP-1 mRNA in 3T3 cells,13 and PDGF induces upregulation in a manner similar to that of c-myc and c-fos, with TSP-1 being classified as an immediate early response gene.14 These data, in conjunction with the role of TSP-1 in potentiating the chemotactic response of VSMCs to PDGF15 and the proliferative response to epidermal growth factor,16 have led to the belief that TSP-1 plays a modulatory role in the cellular response to vascular injury.

Recent work from our laboratory, however, demonstrates that TSP-1 can in fact act as an independent growth factor for human (H) VSMCs. We have shown that TSP-1 is capable of inducing not only migration but also proliferation of these cells to levels similar to those seen with PDGF, although we have shown that PDGF does not contribute to these mitogenic effects. Indeed, TSP-1 in combination with PDGF induced a synergistic effect on DNA synthesis.17 18 This direct effect of TSP-1 on HVSMCs suggests a growth factor–like role for TSP-1 in the cellular response to injury. Although the functional consequences of TSP-1 stimulation have been demonstrated, the mechanisms by which these signals are transmitted intracellularly remain obscure.

Our previous data have shown that TSP-1 acts as a pleiotropic growth regulator of HVSMCs in much the same way as growth factors like PDGF. In keeping with this observation, recent data from our laboratory have shown that TSP-1–induced DNA synthesis in HVSMCs is completely inhibited by the tyrosine kinase inhibitors genistein and tyrphostin A23. Cellular migration in response to TSP-1 is also substantially reduced by these tyrosine kinase inhibitors.18 This suggests that tyrosine phosphorylation of certain key proteins plays an important role in the intracellular transmission of signals induced by TSP-1. Interestingly TSP-1–enhanced cell spreading of C32 human melanoma cells on sparse vitronectin coatings has been correlated with increased tyrosine phosphorylation of focal adhesion kinase (FAK).19 Although TSP-induced disassembly of focal adhesion plaques in aortic endothelial cells has recently been reported to occur via a pathway involving stimulation of the lipid kinase, phosphatidylinositol 3-kinase (PI 3-K), tyrosine phosphorylation of the p85 regulatory subunit of this enzyme has not been reported.20

We report here that soluble TSP-1 induces tyrosine phosphorylation of a number of cellular proteins in quiescent HVSMCs, with a complex temporal pattern of activation. The primary phosphotyrosine proteins have been identified as PI 3-K (the p85 regulatory subunit) and FAK. PI 3-K is upstream from FAK in the signaling process, in that FAK phosphorylation is dependent on PI 3-K activation.


*    Methods
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 *Methods
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HVSMC Culture
HVSM was obtained from saphenous veins from patients undergoing cardiovascular surgery. Tissues were surplus to requirements, and their use conformed to the guidelines of our local ethics committee. VSMCs were cultured by an explant technique as previously described.21 VSMCs were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) buffered with 25 mmol/L HEPES and supplemented with 15% (vol/vol) FCS, 4 mmol/L L-alanyl-L-glutamine (Glutamax-I), penicillin (100 U/mL), streptomycin (100 µg/mL), and gentamicin (25 µg/mL). Cell cultures were maintained in a humidified atmosphere of 5% CO2 (vol/vol) in air at 37°C. VSMCs were routinely used at third passage unless otherwise stated. VSMC purity was routinely checked by immunocytochemical studies for {alpha}-actin staining.

Cell Stimulation
Passage 2 HVSMCs were subcultured and plated onto Petri dishes at a known density (106 cells per dish) and allowed to attach for 24 hours in DMEM supplemented with 15% (vol/vol) FCS. Cells were then washed twice with PBS and maintained in serum-free DMEM for a period of 7 days. Quiescent cells were then washed in serum-free DMEM and stimulated with 1 µg/mL TSP-1 in DMEM for a period of 30 minutes at 37°C, unless otherwise stated. Cells were then washed twice with ice-cold PBS, scraped into 1 mL of ice-cold lysis buffer (50 mmol/L Tris [pH 7.4]; 150 mmol/L NaCl; 1 mmol/L EGTA; 1% [vol/vol] NP-40; 0.25% sodium deoxycholate, 1 mmol/L NaF, sodium orthovanadate, and PMSF; and 1 µg/mL aprotinin, pepstatin, and leupeptin), and allowed to stand on ice for 10 minutes before centrifugation (14 000 rpm, 15 minutes, 4°C). The resulting cell lysates were used for protein measurement.

Immunoprecipitation
A known concentration of cellular protein ({approx}250 µg) was removed from all samples and precleared for 1 hour with albumin-agarose before incubation with primary antibody (1 µg of antibody per 100 µg of protein) for 3 hours at 4°C. Immunoprecipitates were captured on protein A–agarose in the manner described.22

Immunoblotting
Proteins were separated on 10% SDS-polyacrylamide gels before being transferred to nitrocellulose by using a Bio-Rad wet gel transfer system. After successful transfer, the nitrocellulose blots were blocked in 5% BSA for 1 hour and washed 3 times in Tris-buffered saline containing 0.05% Tween-20 before being probed with the primary antibody for 1 hour. Blots were washed well in Tris-buffered saline/Tween-20 before being probed with the appropriate secondary antibody and developed by enhanced chemiluminescence.

PI 3-K Activity Assay
Passage 4 cells were washed twice with PBS and maintained in serum-free DMEM for a period of 7 days before stimulation with TSP-1. Cells were stimulated and scraped into lysis buffer as described above and then incubated with anti–rat PI 3-K, rabbit whole antiserum, and protein A–agarose overnight at 4°C. The immunoprecipitate was washed twice with 0.1 mol/L Tris-HCl, 0.5 mol/L LiCl (pH 7.5) and twice with kinase assay buffer (20 mmol/L HEPES, 10 mmol/L MgCl2, and 25 µmol/L ATP, pH 7.4). Samples were resuspended in 40 µL of kinase buffer with 10 µL of [33P]ATP and 10 µL of PI and incubated at room temperature for 15 minutes. The reaction was stopped by the addition of 50 µL of 1 mol/L HCl and 100 µL of chloroform/methanol (1:1, vol/vol), and the samples were centrifuged at 14 000 rpm for 5 minutes to give 2 separate phases. The chloroform layer was spotted onto potassium oxalate–coated thin-layer chromatography plates and developed in a mixture of chloroform/acetone/methanol/acetic acid/water (40:15:13:12:7, vol/vol/vol/vol/vol) with a running time of {approx}2 hours. Plates were dried thoroughly, wrapped in Clingo-rap, and exposed to a PhosphorImager cassette for 2 days.

Cell Migration Assays
These assays were performed with blind-well chemotaxis chambers as previously described.23 In brief, the upper and lower compartments of the blind-well chambers were separated by gelatin-coated polycarbonate filters. TSP-1 (10 µg/mL) was added to the lower chamber and acted as the chemoattractant. The cell suspension (2.25x105 cells/mL), with or without PI 3-K inhibitors, was added to the upper chamber, and migration was allowed to proceed for 5 hours. The filters were then removed, fixed in ethanol, and stained in toluidine blue (1%, vol/vol). The migrated cells were counted under a light microscope.

Materials
Cell-culture plasticware, media, supplements, and TSP-1 (purified from human platelets) were obtained from Life Technologies. FCS was obtained from M.B. Meldrum Ltd. BSA fraction V was purchased from Boehringer Mannheim. The bicinchoninic acid protein assay kits were obtained from Pierce Warriner. Hybond C nitrocellulose, enhanced chemiluminescence reagents, and streptavidin complex were obtained from Amersham. Protein A–agarose was obtained from Cambridge Bioscience. Anti-phosphotyrosine antibody PY20 and anti-rabbit IgG (horseradish peroxidase linked) were obtained from Affiniti. All other antibodies were obtained from TCS, and all other reagents were obtained from Sigma.

Statistical Analysis of Data
Control data were defined as 100%, and experimental data were expressed as mean±SEM in relation to the control. Statistical differences between means in terms of analysis of temporal effects were determined with a Freidman nonparametric ANOVA for multiple repeated measures. Other data were compared by a Wilcoxon matched-pairs signed rank test. A value of P<=0.05 was considered significant.


*    Results
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TSP-1 Stimulates Tyrosine Phosphorylation of Novel Protein Bands
HVSMCs were incubated with or without TSP-1 (1 µg/mL) for 60 minutes, and the tyrosine phosphorylation profiles of crude cell lysates were examined and compared. Control cells demonstrated a small number of constitutively phosphorylated protein bands, 3 of which (approximate molecular weights of 97, 75, and 63 kDa) exhibited significantly enhanced tyrosine phosphorylation after stimulation with TSP-1. More important, TSP-1 stimulation of HVSMCs resulted in the appearance of a number of tyrosine-phosphorylated protein bands that were not observed in control, unstimulated cells. Densitometric profile analysis of the TSP-1–stimulated cell lysates compared with biotinylated molecular weight markers attributed molecular weights of {approx}170, 118, and 88 kDa to these novel tyrosine-phosphorylated bands (data not shown). The stimulation of novel tyrosine-phosphorylated proteins by TSP-1 correlates well with previous data from our laboratory that suggest that TSP-1 acts as an independent growth factor for HVSMCs.18

Temporal Nature of TSP-1–Induced Tyrosine Phosphorylation
Further investigation of the effect of TSP-1 stimulation on the tyrosine phosphorylation profile of cell lysates demonstrated a complex temporal pattern of tyrosine phosphorylation in response to cell stimulation. Protein bands that showed some constitutive tyrosine phosphorylation appeared to demonstrate increasing tyrosine phosphorylation in response to TSP-1 in a time-dependent manner. The peak tyrosine phosphorylation levels of these particular protein bands were generally observed between 60 and 90 minutes after initiation of TSP-1 stimulation. Other proteins, however, appeared to be tyrosine phosphorylated early in response to TSP-1, between 20 and 30 minutes. These proteins then appeared to undergo a certain amount of subsequent dephosphorylation on prolonged exposure to TSP-1, although they remained phosphorylated above control levels at 60 minutes. Protein bands falling into this category of rapidly phosphorylated signals included tyrosine-phosphorylated proteins of {approx}118 and 88 kDa (Figure 1Down).



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  		Figure 1. Temporal characteristics of cellular tyrosine phosphorylation after TSP-1 stimulation. A, Western blot of whole-cell lysate, showing the temporal pattern of tyrosine phosphorylation in response to TSP-1 (1 µg/mL) stimulation. Cells were stimulated with TSP-1 for between 10 and 120 minutes before lysis. Fifteen micrograms of total cell protein was separated by 10% polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose before being probed with an anti-phosphotyrosine antibody (PY20) at 1:1000. Gel shown is a representative example of 5 separate experiments. B, Densitometric scanning of the main bands of tyrosine phosphorylation was performed, and the mean±SEM of all experiments was plotted. Tyrosine phosphorylation in control cells was expressed as 100%, and all values for stimulated cells were calculated with respect to this value. 63-kDa band, black box; 75-kDa band, white box; 88-kDa band, dark gray box; and 118-kDa band, light gray box.

Identification of Early Signals in Response to TSP-1
Owing to the complex temporal nature of the phosphotyrosyl protein profile and because the nature of the primary signaling process in response to TSP-1 in HVSMCs is obscure, we concentrated on identifying and understanding the early phosphotyrosine signals. The growth factor–like nature of TSP-1 and the estimated molecular weights of the early tyrosine-phosphorylated proteins led us to investigate the possibility that these protein bands might represent FAK and the p85 regulatory subunit of PI 3-K. Quiescent cells were exposed to TSP-1 (1 µg/mL), and the temporal pattern of tyrosine phosphorylation of p85 PI 3-K and FAK was identified by immunoprecipitation followed by Western blotting techniques. Initially, phosphotyrosine immunoprecipitates were collected from 200 µg of crude cell lysate and probed with antibodies to specific cellular proteins. Parallel studies in which the specific protein was immunoprecipitated followed by immunoblotting with an anti-phosphotyrosine antibody yielded results similar to those in which phosphotyrosine proteins were immunoprecipitated. These results demonstrated a single peak of FAK phosphorylation in response to TSP-1 stimulation at {approx}20 to 30 minutes (Figure 2ADown and 2BDown). In contrast, the p85 regulatory subunit of PI 3-K exhibited a biphasic tyrosine phosphorylation in response to TSP-1 stimulation. Tyrosine phosphorylation levels were markedly increased within the first 10 minutes of TSP-1 stimulation, decreased transiently, and then exhibited a second peak in tyrosine phosphorylation at {approx}20 to 30 minutes (Figure 2CDown). Prolonged exposure to TSP-1 was generally associated with reduced phosphorylation of the p85 regulatory subunit of PI 3-K to near control levels. Intriguingly, the peak in FAK phosphorylation coincided exactly with the later peak in the biphasic PI 3-K response. This behavior was a consistent trend in all of the human cell strains tested, although the time of peak phosphorylation and the degree of phosphorylation above control levels varied from strain to strain (Figure 3ADown).



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  		Figure 2. Identification of early tyrosine-phosphorylated cellular proteins. A, Western blot showing the temporal effect of TSP-1 stimulation on the association of FAK with phosphotyrosine proteins. Tyrosine-phosphorylated proteins were immunoprecipitated from 200 µg of whole-cell lysate after stimulation with TSP-1. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to FAK. Gel is a representative example of 3 separate cell strains. B, Histogram shows the densitometric analysis of TSP-1–induced tyrosine phosphorylation of FAK. FAK phosphorylation in control cells was taken as 100%, and all other data were calculated with respect to this value. Data represent mean±SEM of 3 separate cell strains. C, Western blot showing the biphasic nature of the tyrosine phosphorylation of the p85 regulatory subunit of PI 3-K after stimulation with 1 µg/mL TSP-1. Immunoprecipitated phosphotyrosine proteins were separated by 10% PAGE and transferred to nitrocellulose before being probed with a monoclonal antibody to p85 (primary, 1:1000, 1 hour; secondary, rabbit anti-mouse horseradish peroxidase conjugate, 1:1000). Blot shown is a representative example of 4 separate cell strains.



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  		Figure 3. Time course of TSP-1 stimulation on PI 3-K activity. A, Histogram represents the mean tyrosine phosphorylation of the p85 regulatory subunit as determined by densitometric analysis. Tyrosine phosphorylation in control cells is taken to be 100%, and all other data are calculated with respect to this value. Data represent the mean±SEM of n=5 to 15 separate cell strains. Statistical analysis of data by a Friedman nonparametric ANOVA for multiple repeated measures demonstrated significantly increased tyrosine phosphorylation above control levels (*P<0.05) at 5, 20, 30, and 60 minutes. B, Passage 4 HVSMCs were stimulated with TSP-1 for between 5 and 90 minutes. Cells were lysed and the total cell protein determined. PI 3-K was immunoprecipitated from an equal quantity of cell protein from each lysate and its activity determined as described in Methods. Activity of control, unstimulated cells was expressed as 100%, and the activity of all stimulated samples was calculated with respect to this value. Graph represents the mean±SEM of 4 to 7 separate experiments.

PI 3-K Activity
Although FAK phosphorylation can be taken as an indicator of activation,24 the same is not necessarily true of PI 3-K. With PI 3-K, phosphorylation occurs in the p85 regulatory subunit whereas catalytic activity is located in the p110 subunit, and although phosphorylation and activity are generally linked, activation of PI 3-K is not concomitant with tyrosine phosphorylation of the p85 subunit. Consequently, modulation of PI 3-K activity in response to TSP-1 was measured and compared with the p85 tyrosine phosphorylation results. Temporal analysis of the PI 3-K activity in immunoprecipitates from stimulated cells (5- to 90-minute time course) demonstrated that this lipid kinase activity was indeed stimulated by TSP-1 in a biphasic manner (Figure 3BUp). This increase in activity, which was statistically significant (P<0.05) at 5 minutes, correlates well with the temporal nature of the increased tyrosine phosphorylation of the p85 regulatory subunit of PI 3-K in response to TSP-1, which was statistically significant at both 5 and 20 minutes (Figure 3AUp). This finding suggests that TSP-1 stimulates a biphasic increase in PI 3-K enzyme activity, with the primary, transient stimulation occurring within 5 to 10 minutes after exposure to TSP-1, and the second prolonged peak of activity occurring at {approx}30 minutes. These peaks in PI 3-K activity appear to be coupled to increases in the tyrosine phosphorylation levels of the p85 regulatory subunit of this enzyme.

Effect of PI 3-K Inhibition on FAK Phosphorylation
The biphasic nature of the PI 3-K response to TSP-1 compared with that of FAK led us to investigate possible interactions between PI 3-K and FAK in the signaling pathway. Wortmannin at nanomolar concentrations has been shown to be a highly selective inhibitor of class 1 PI3-K,25 as has LY294002, a structurally dissimilar but equally effective PI 3-K inhibitor.26 Pretreatment of HVSMCs with either of these membrane-permeable inhibitors before a 30-minute exposure to TSP-1 resulted in a substantial decrease in the level of TSP-1–stimulated tyrosine phosphorylation of FAK. This inhibition was apparent in samples wherein immunoprecipitated FAK was probed with an anti-phosphotyrosine antibody (Figure 4ADown) and in phosphotyrosine immunoprecipitates probed with anti-FAK (Figure 4BDown). These data suggest that not only is PI 3-K activity stimulated by TSP-1 but also that this activation occurs upstream from FAK activation in the signaling cascade and that FAK activation is dependent on PI 3-K activity.



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  		Figure 4. Effect of PI 3-K inhibitors on TSP-1–induced FAK phosphorylation. Third-passage HVSMCs were pretreated for 15 minutes with or without the PI 3-k inhibitors wortmannin (50 nmol/L) and LY294002 (1 µmol/L). Cells were then stimulated with TSP-1 (1 µg/mL) for 30 minutes in the continued presence of these inhibitors. Control cells were unstimulated. Total cell protein was measured and equal amounts removed from each lysate for immunoprecipitation. A, Immunoprecipitated FAK was analyzed by SDS-PAGE and Western blotting with a monoclonal anti-phosphotyrosine antibody (PY20). B, Anti-phosphotyrosine immunoprecipitates were analyzed by SDS-PAGE and Western blotting with an antibody to FAK. C, TSP-1–stimulated tyrosine phosphorylation of FAK was measured after treatment with 50 nmol/L wortmannin and 1 µmol/L LY294002. Results are expressed as percent of the TSP-1–stimulated response, which was taken to be 100%. Data shown represent mean±SEM of n=6 to 8 separate cell strains. Statistical analysis of data by the Wilcoxon matched-pairs signed rank test demonstrated that the effects of both wortmannin and LY294002 on TSP-1–induced FAK phosphorylation were highly significant. **P<0.02, ***P<0.008.

Neither wortmannin nor LY294002 had a significant effect on the ability of the p85 regulatory subunit to associate with tyrosine-phosphorylated proteins or on tyrosine phosphorylation of the subunit itself (densitometric analysis of p85 tyrosine phosphorylation in arbitrary units: TSP-1, -4.5±1.5, n=8; wortmannin, -3.8±1.5, n=8; and LY294002, -2.4±1.3, n=6). Both wortmannin and LY294002 interact with the p110 catalytic subunit of PI 3-K and would not necessarily be expected to affect tyrosine phosphorylation of the p85 subunit.

The effect of wortmannin and LY294002 on cell viability was determined by measuring lactic dehydrogenase release from the cells by using a nonradioactive cytotoxicity assay. Lactic dehydrogenase released from control cells was expressed as 100%, and all other data were compared with this value (lactic dehydrogenase positive control for these cells was 3136±1380%; TSP-1, 105±27%; wortmannin, 296±150%; and LY294002, 102±15%). These data indicated that PI 3-K inhibitor treatment did not significantly affect cell viability.

Nature of the FAK–PI 3-K Interaction
Western blotting techniques were used to determine the nature of the interaction between FAK and PI 3-K and the temporal effect of TSP-1 stimulation on this interaction. Lysates from agonist-stimulated cells were immunoprecipitated with FAK, and these immunoprecipitates were immunoblotted for PI 3-K. At no stage after TSP-1 stimulation was the p85 regulatory subunit of PI 3-K detected in these immunoprecipitates. Conversely, FAK protein expression was not detected in p85 PI3-K immunoprecipitates from agonist-stimulated cells (data not shown). These data suggest that the TSP-1–stimulated interaction between FAK and PI 3-K may not constitute a direct protein-protein interaction between the cytosolic tyrosine kinase and the p85 regulatory subunit of the lipid kinase.

TSP-1–Induced Cellular Migration
TSP-1 (10 µg/mL) has been shown to induce levels of HVSMC chemotaxis similar to those seen with PDGF-BB. These are significantly reduced by tyrosine kinase inhibitors, suggesting that tyrosine phosphorylation of cell proteins plays a key role in this process.18 FAK has long been recognized as a constituent of focal adhesions,27 and the assembly of focal adhesions accompanies cell migration.28 Activation of FAK in focal adhesions appears to occur downstream from their assembly and to be a key factor in the regulation of cell motility.29 30 31 32 If FAK is associated with the ability of cells to migrate, then inhibition of signals upstream from FAK in the signaling process should also have the ability to inhibit cell migration. Consequently, we set out to examine the effect of inhibiting PI 3-K activity on cell migration in response to TSP-1.

The addition of both PI 3-K inhibitors, wortmannin and LY294002, to cells before their exposure to a directed gradient of TSP-1 resulted in a concentration dependent decrease in the ability of these cells to migrate in response to TSP-1 (Figure 5Down). The PI 3-K inhibitor wortmannin has been shown in some cell lines to affect phospholipase A2 (PLA2), which may in turn affect cellular responses such as chemotaxis. Consequently, the effect of the PLA2 inhibitor 7,7-dimethyleicosadienoic acid on TSP-1–induced chemotaxis was investigated. PLA2 inhibition had no effect on TSP-1–induced chemotaxis (TSP-1–induced chemotaxis was taken to represent 100%; the TSP-1+100 µmol/L 7,7-dimethyleicosadienoic acid value was -114±22%, n=3), suggesting that inhibition of PLA2 did not contribute to the action of wortmannin. This result suggests that PI 3-K plays a key role in the regulation of cellular migration. Wortmannin (25 to 100 nmol/L) inhibited the ability of cells to migrate to TSP-1 in a concentration-dependent manner. LY294002 (0.1 to 10 µmol/L) similarly inhibited TSP-1–induced migration.



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  		Figure 5. Effect of PI 3-K inhibitors on TSP-1–induced cellular chemotaxis. A, The numbers of cells migrating to TSP-1 (10 µg/mL) in the presence of increasing concentrations of wortmannin were counted and the mean value of duplicate filters calculated. Histogram shows the mean±SEM from experiments with 4 separate cell strains. Effect of wortmannin was statistically significant (*) at both 75 and 100 nmol/L. B, The effect of LY294002 on the cellular migration induced by TSP-1. The number of cells migrating to TSP-1 (10 µg/mL) in the presence of increasing concentrations of LY294002 were counted. Histogram shows the mean±SEM of experiments with 5 separate cell strains. Effect of LY294002 was statistically significant (*) at 1 and 10 µmol/L.


*    Discussion
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 *Discussion
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Previous data from our laboratory have shown that TSP-1 may act as an independent growth factor for HVSMCs.18 We have previously shown that TSP-1–induced DNA synthesis in these HVSMCs is unaffected by the presence of neutralizing antibodies to both PDGF and TGF-ß1, although the response to these factors is completely inhibited in the presence of the appropriate antibody. Similarly, we have been unable to detect the presence of either PDGF or TGF-ß1 in the commercially available TSP-1 used for these experiments. Indeed, we have demonstrated both DNA synthesis and migration of HVSMCs in response to recombinant TSP-1.18 These results suggest that the responses seen to TSP-1 are unlikely to be due to contamination of this protein with either PDGF or TGF-ß1.

The data presented here argue strongly in favor of TSP-1 inducing its functional effects through the temporally regulated tyrosine phosphorylation of a series of cellular proteins. The early tyrosine-phosphorylated signals seen in response to TSP-1 include FAK and PI 3-K, the activity of both enzymes being significantly enhanced by a 20- to 30-minute stimulation with 1 µg/mL TSP-1. Indeed, preliminary data from this laboratory have recently demonstrated tyrosine phosphorylation of both FAK and the PI 3-K p85 regulatory subunit by baculovirus-derived recombinant TSP-1 (a generous gift from Dr J. Adams, University College, London). Although stimulation of FAK activity by TSP-1 has been previously shown in human C32 melanoma cells,19 our data represent the first direct demonstration that soluble TSP-1 induces tyrosine phosphorylation of FAK in VSMCs. Similarly, these data represent the first demonstration of PI 3-K activation, accompanied by a corresponding tyrosine phosphorylation of the p85 regulatory subunit, induced by TSP-1 in any cells to date.

TSP belongs to a family of matricellular proteins that bind to other extracellular matrix proteins as well as to cell-surface receptors and other molecules such as serine proteases. Although signaling through TSP-1 is obscure, there is evidence that another member of this matricellular family, osteopontin, produces rapid activation of PI 3-K.33 Recent evidence from Greenwood and colleagues20 have demonstrated that TSP-1 signals for focal adhesion disassembly in cultured bovine aortic endothelial cells operate through a pathway involving stimulation of PI 3-K. These authors showed a 2.5-fold increase in PI 3-K activity with Hep-I (a synthetic peptide containing amino acids 17 to 35 of TSP-1), although these authors failed to detect a corresponding increase in tyrosine phosphorylation of the p85 regulatory subunit of the lipid kinase. This endothelial cell activation data correlate well with the HVSMC data reported here, which show a similar increase in PI 3-K activity ({approx}2-fold) at 20 to 30 minutes after TSP-1 stimulation.

Although both FAK and PI 3-K are early tyrosine-phosphorylated signals in response to TSP-1, immunoprecipitation data show a biphasic tyrosine phosphorylation response for PI 3-K that is correlated with a biphasic activation of the lipid kinase. This biphasic phosphorylation was not detected initially when we examined tyrosine phosphorylation profiles, primarily because of the time course used for assessment and the somewhat crude picture that cell lysate analysis gives. The single peak of FAK phosphorylation correlates well with the latter peak of PI 3-K phosphorylation and activation, suggesting that PI 3-K activation may precede FAK activation. In fact, both of the PI 3-K inhibitors, wortmannin and LY294002, though structurally different, substantially reduced the level of TSP-1–stimulated FAK phosphorylation, suggesting that FAK phosphorylation occurs downstream from PI 3-K and that activation of the former is dependent on the activation of PI 3-K.

There is evidence to suggest that the p110 subunit of PI 3-K functions as a dual kinase. Aside from its ability to phosphorylate inositol lipids at the D3 position of the inositol ring, PI 3-K has been implicated as a serine/threonine protein kinase. Although no protein kinase substrates have yet been identified in vivo,34 a number of serine/threonine kinases have been placed downstream from PI 3-K in receptor-stimulated signaling. These downstream targets of PI 3-K include protein kinase B,35 protein kinase C,36 and p70s6K.37 Data presented here, however, suggest that the cytosolic tyrosine kinase FAK can also be placed downstream from PI 3-K in TSP-1–stimulated cells. Such a link has previously been suggested by other groups with regard to PDGF-stimulated phosphorylation of FAK.38 39 The mechanism by which PI 3-K exerts its effects on FAK phosphorylation remains obscure, although a direct and stable association between FAK and the p85 subunit of PI 3-K has previously been reported in vitro.40 41 42 Data presented here, however, provide no evidence for such a direct protein-protein association between FAK and PI 3-K in HVSMCs. FAK was consistently absent from our immunoprecipitates of PI 3-K, and similarly, PI 3-K was consistently absent from immunoprecipitates of FAK, suggesting an indirect mechanism of FAK activation by PI 3-K. In this respect, the p85 subunit of PI 3-K has been demonstrated to contain a breakpoint cluster region–homology domain, which has been suggested to contain a G protein–binding site and may function as a GTPase-activating protein for monomeric GTP-binding proteins.43 Similarly, both rac-mediated lamellipodia and rho-mediated stress fiber and focal adhesion formation can be induced by membrane targeting of the p110 catalytic subunit of PI 3-K,44 suggesting that PI 3-K can induce small GTPase-mediated cellular responses. Interestingly, an increasing number of reports now link activated rho to the tyrosine phosphorylation of a number of cellular proteins, including FAK.45 A possibility then is that tyrosine phosphorylation and the concomitant activation of FAK by PI 3-K presented in this report may occur indirectly through the activation of a small GTPase of the rho family. In keeping with this concept, the lipid products of PI 3-K have recently been shown to bind selectively to a number of small G protein exchange factors.46 Alternatively, there is evidence to suggest that the products of PI 3-K activity initiate downstream signaling by recruiting and/or activating target proteins. PI 3,4,5-trisphosphate, a product of PI 3-K activity, has been demonstrated to compete with tyrosine-phosphorylated proteins for binding to SH2 domains. Thus, PI 3,4,5-trisphosphate can act to recruit SH2 domain–containing proteins to the cell membrane and has been shown to bind to src47 and to activate phospholipase C{gamma}.48 It is possible then that PI 3-K exerts its effects on FAK indirectly through the cytosolic tyrosine kinase src. Such an interaction between src and FAK has been seen in a number of cell types.33 49

TSP-1 can induce levels of chemotaxis similar to those seen with PDGF-BB in HVSMCs.18 This suggests that the upregulation in TSP-1 levels noted after platelet degranulation or in injured and diseased blood vessels may act as a powerful chemoattractant, inducing cells to migrate into the intima of the vessel. In atherosclerosis and saphenous vein graft occlusion, lesions are found in the vascular intima, and the SMCs that compose these lesions are clonal. This suggests that migration of SMCs from the media is a key event in the early stages of atherosclerosis and restenosis.50 51 Understanding the cellular signals involved in the TSP-1–induced migration of HVSMCs may therefore enable us to target specific signaling molecules in a therapeutic approach for prevention of these vascular occlusions.

In conclusion, TSP-1 stimulates the phosphorylation and activation of both PI 3-K and FAK. PI 3-K lies upstream from FAK in the signaling cascade that leads to cell chemotaxis. Inhibition of PI 3-K activity leads to both a decrease in FAK phosphorylation and a decrease in the ability of cells to migrate in response to a directed gradient of TSP-1. The mechanism by which PI 3-K exerts its effects on FAK, however, remains to be defined.


*    Acknowledgments
 
This work was supported by the British Heart Foundation (PG/98141) (to J.S.L. and A.D.H.). We thank the surgeons and theater staff of St Mary's Hospital for supplying samples of saphenous vein.

Received August 7, 1998; accepted January 25, 1999.


*    References
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