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Endocrinology Vol. 140, No. 9 4228-4235
Copyright © 1999 by The Endocrine Society


ARTICLES

Roles of Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase Pathways in Stimulation of Vascular Smooth Muscle Cell Migration and Deoxyriboncleic Acid Synthesis by Insulin-Like Growth Factor-I1

Yumi Imai and David R. Clemmons

Division of Endocrinology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7170

Address all correspondence and requests for reprints to: David R. Clemmons Division of Endocrinology, University of North Carolina, Chapel Hill, North Carolina 27599-7170. E-mail: dpm{at}med.unc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor-I (IGF-I) is a potent stimulator of vascular smooth muscle cell (SMC) migration, a process that contributes to the accumulation of SMC within atherosclerotic lesions. Our previous studies have shown that IGF-I increases the affinity of the {alpha}Vß3 integrin toward ligands and that occupancy of this integrin is indispensable for IGF-I to stimulate cell migration. In this study, the role of phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) pathways in IGF-I induced cell motility and integrin activation was studied using porcine aortic smooth muscle cells (pSMC). Two structurally different inhibitors of PI 3-kinase decreased IGF-I-stimulated pSMC migration in a dose-dependent manner. The IC50 of wortmannin for inhibiting migration was 10 nM, and that of LY294002 was 0.3 µM. These inhibitors also suppressed IGF-I-induced phosphorylation of protein kinase B PKB/Akt at Ser437 using concentrations that also inhibited cell motility. PD98059, an inhibitor of the MAPK pathway, was somewhat less potent than PI 3-kinase inhibitors in blocking cell migration that had been stimulated by IGF-I. When IGF-I increased migration of pSMC 2.1-fold above control, 100 nM wortmannin inhibited this response by 79%, 1 µM LY294002 inhibited it by 58%, and 50 µM PD98059 caused a 34% reduction. In comparison, 100 nM wortmannin inhibited IGF-I stimulated DNA synthesis by 57%, 1 µM LY294002 inhibited it by 59%, whereas 50 µM PD98059 suppressed it completely. Thus, activation of PI 3-kinase plays the major role in IGF-I-stimulated migration and proliferation of pSMC. While the activation of the MAPK pathway seems to be necessary for stimulation of mitogenesis by IGF-I, the contribution of this pathway in IGF-I-induced cell migration is limited in pSMC. Interestingly, neither PI 3-kinase inhibitors nor PD98059 blocked the increase in {alpha}Vß3 integrin affinity that followed IGF-I treatment. Therefore, although both the PI 3-kinase and MAPK pathways were used by IGF-I to increase migration of pSMC, {alpha}Vß3 integrin activation did not depend on either PI 3-kinase or MAPK activation, suggesting the possible importance of some other signal transduction pathway to account for its full actions on pSMC.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ACCUMULATION OF VASCULAR smooth muscle cells in neointima is the pathological hallmark of atherosclerosis. Smooth muscle cells (SMC) that are normally quiescent in the tunica media proliferate and migrate into the intima after vascular injury and contribute to the narrowing of vessels. This process is triggered and regulated by multiple factors, including cytokines, peptide growth factors, integrins, and specific components of the extracellular matrix (ECM) (1). Insulin-like growth factor-I (IGF-I) is one factor that may play an important role in the progression of atherosclerosis because it has been shown to be increased at the site of vascular injury (2) and is a potent stimulator of proliferation and migration of vascular SMC in culture (3, 4, 5). One of the mechanisms by which IGF-I promotes cellular migration is by regulating integrins, heterodimeric transmembrane proteins that control cell-matrix and cell surface-cytoskeletal interactions (6). IGF-I treatment of SMC has been shown to increase the affinity of the {alpha}Vß3 integrin toward ligands, and specific inhibitors of ligand occupancy of the {alpha}Vß3 integrin block IGF-I-stimulated SMC migration (7).

IGF-I elicits its actions on cells by binding to the insulin-like growth factor receptor and activating its intrinsic receptor tyrosine kinase (8). Tyrosine kinase activity of the receptor is indispensable for IGF-I actions on the target cells (9). When activated, the IGF-I receptor phosphorylates docking proteins, including the insulin receptor substrates (IRS 1–4), Shc, and Crk, and these molecules activate downstream signaling proteins that lead to biological responses, such as cell migration and proliferation (8, 10). Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) are two key enzymes that are activated by IGF-I, and each of them represents a distinct cascade of activation steps that result in the biological functions of IGF-I. PI 3-kinase can be activated through IRS-1, IRS-2, and Shc, whereas MAPK can be activated through IRS-1 or IRS-2, Shc, and Crk following IGF-I stimulation (8, 10). Both the PI 3-kinase and MAPK kinase pathways have been shown to be involved in many aspects of IGF-I action, including cell proliferation, protection of cells from apoptosis, and cell differentiation (11, 12, 13, 14). Evidence in other cell systems implies that activation of PI-3 kinase and MAPK play roles in cell motility. Expression of a constituitively activated form of PI 3-kinase increases the motility of mammary gland epithelial cells (15). PI 3-kinase is indispensable for platelet-derived growth factor (PDGF) to induce chemotaxis in NIH3T3 cells, TRMP cells, and CHO cells (16). PDGF-induced migration of mesangial cells depends on PI 3-kinase and MAPK activation (17). However, there is only limited information about the roles of PI 3-kinase and MAPK activation in IGF-I-stimulated migration of vascular SMC, and it is not known if either pathway is involved in controlling integrin activation.

The purpose of the present study was to determine the contribution of the PI 3-kinase and MAPK pathways in IGF-I-stimulated cell migration, proliferation, and {alpha}Vß3 integrin activation.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Materials
Porcine aortic smooth muscle cells (pSMC) were prepared and maintained as previously described (5). IGF-I was a gift from Genentech, Inc. (South San Fransisco, CA). Wortmannin, LY294002, and PD98059 were purchased from Sigma (St. Louis, MO). Stock solutions of 10 mM wortmannin, 10 mM LY294002, and 50 mM PD98059 were prepared in 100% dimethylsulfoxide (DMSO) and stored at -70 C. Each inhibitor was diluted in DMEM so that the final concentration of DMSO in test solutions would be 0.1%. An antibody to Akt that has been phosphorylated at Ser473 (Phospho-Akt (Ser473)) and an antibody to p44/p42 MAPK that has been phosphorylated at Thr202 and Tyr204 (phospho-p44/p42 MAP kinase antibody) were obtained from New England Biolabs, Inc. (Beverly, MA). An antibody that recognizes both phosphorylated and nonphosphorylated forms of Akt and an antibody that recognizes both phosphorylated and nonphosphorylated forms of p44/p42 MAPK were obtained from New England Biolabs, Inc. Kistrin was a gift from Dr. Robert Lazarus (Genentech, Inc.).

Wounding assay
PSMC that had been grown to confluence in six-well culture plates (Falcon no. 3046, Falcon Labware Division of Becton Dickinson and Co., Fairbanks, NJ) were subjected to wounding as previously described (7). Cell layers were scraped with a single-edged razor blade and rinsed with serum-free medium. The edges of the wound were then observed under microscope to select straight and sharp edges. For each treatment, 6–17 1-mm regions along the edge of the wound were chosen and marked. Cells were incubated in DMEM containing 0.2% FBS plus 0.1% DMSO with the addition of various concentrations of wortmannin, LY294002, or PD98059 for 30 min. IGF-I (0 or 100 ng/ml) was then added to the cultures. Following a 48 h incubation at 37 C, the number of cells that migrated across the regions of the wound edge that had been marked before the treatment was quantified by direct counting of total cell number. Our previous analysis of the wounded monolayer by [3H]thymidine autoradiography demonstrated that the labeling index of pSMC at the wound edge was 7 ± 4% at the basal level and 18 ± 7% after exposure to IGF-I (7). Therefore, less than 10% of cells present in the denuded region at the end of migration assay are considered to result from cell division rather than cell migration. To measure the effect of the inhibitors on cell detachment, confluent pSMC in six-well culture plates were also incubated with DMEM containing 0.2% FBS plus 0.1% DMSO or the inhibitors in the absence or presence of 100 ng/ml IGF-I for 48 h. At the end of the incubation, the number of cells that remained attached to the plate was counted by releasing the cells from the plate with 0.02% EDTA plus 0.125% trypsin in PBS.

Detection of Akt phosphorylation at Ser473 and p44/p42 MAPK phosphorylation at Thr202 and Tyr204
pSMC were seeded using a density of 1 x 106/well onto six-well culture plates in DMEM with 1% FBS, incubated for 24 h, and serum-starved in serum-free DMEM for 2 h. The medium was changed to DMEM with 0.1% DMSO plus the indicated concentration of inhibitors, and the cells were incubated for 30 min at 37 C followed by stimulation with 100 ng/ml IGF-I for 30 min at 37 C. The cells were solubilized in 100 µl of SDS sample buffer, sonicated for 15 sec, and boiled for 10 min. Forty microliters of the resulting lysate were loaded onto an 8% polyacrylamide gel, separated, and transferred onto a polyvinylidine difluoride (PVDF) membrane, as described previously (18). The membrane was blocked with 3% nonfat milk in Tris-buffered saline (TBS), incubated with a 1:500 dilution of Phospho-Akt (Ser473) antibody or a 1:1000 dilution of phospho-p44/p42 MAPK (Thr202 and Tyr204) antibody in TBS containing 3% BSA plus 0.2% Nonidet P40 at 4 C overnight, washed, and treated with a 1:10,000 dilution of peroxidase conjugated antirabbit IgG antibody. To determine the protein amount of Akt or p44/p42 MAPK on the membranes, they were also probed with a 1:1000 dilution of Akt antibody or a 1:1000 dilution of p44/p42 MAPK antibody.The bands were visualized with enhanced chemiluminescence, as described previously (19). Densitometric analysis of the bands was performed by scanning x-ray films with Scan Maker IV from Microtek (Redondo Beach, CA) and analyzing the band density using NIH Image from Scion Corp. (Frederick, MD).

[125I]kistrin binding
Kistrin is a small peptide, termed a disintegrin, with a high affinity for {alpha}Vß3. [125I]kistrin was prepared by radiolabeling kistrin with [125I]NaI as described previously (7). pSMC were grown to subconfluence on 48-well culture plates. The cultures were washed three times with serum-free DMEM and preincubated with DMEM containing 0.01% BSA plus 0.1% DMSO in the presence or absence of inhibitors for 30 min at 37 C. IGF-I (0 or 100 ng/ml) was then added to the incubation medium that contained DMSO and the inhibitors, and the plates were incubated for an additional 16 h at 37 C. The plates were placed on ice, washed twice with DMEM containing 20 mM HEPES (pH 7.3), and then incubated with [125I]kistrin (1 x 106 cpm/ml) in DMEM containing 0.1% BSA plus 20 mM HEPES (pH 7.3) at 4 C for 5 h. The cell monolayers were washed three times with DMEM containing 20 mM HEPES (pH 7.3) and solubilized in 0.1% SDS plus 0.1 N NaOH. The bound radioactivity was determined by {gamma} counting.

Measurement of [3H]thymidine incorporation into pSMC
pSMC were plated at a density of 2.5 x 104/cm2 in 96-well tissue culture plates and grown for 5 days without a medium change. They were rinsed once with serum-free DMEM and serum starved by incubating with DMEM plus 0.2% platelet poor plasma (PPP) for 24 h (20). The medium was changed to DMEM with 0.2% PPP plus 0.1% DMSO that contained varying concentrations of inhibitors and IGF-I plus 0.5 µCi/well of [3H]thymidine (specific activity 35 Ci/mmol). The cells were incubated at 37 C for 24 h, and the amount of [3H]thymidine incorporated into DNA was determined as described previously (18).

Statistical analysis
Alternate Welch’s t test and the Mann Whitney test were used to compare the differences between control and test groups. P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both wortmannin and LY294002 inhibited IGF-I stimulated migration of pSMC in a dose-dependent manner
To determine whether the activation of PI 3-kinase is involved in IGF-I stimulation of SMC migration, pSMC were treated with IGF-I and increasing concentrations of two different types of PI 3-kinase inhibitors, wortmannin and LY294002 (21, 22). IGF-I (100 ng/ml) increased migration of pSMC to 377 ± 33% (mean ± SEM, n = 12) when the number of cells migrating in the absence of IGF-I is expressed as 100%. Wortmannin, a nonreversible inhibitor of PI 3-kinase, decreased the IGF-I-stimulated cell migration in a dose-dependent manner, and 50 nM wortmannin resulted in complete inhibition (Fig. 1aGo). LY294002, a reversible inhibitor of PI 3-kinase, also suppressed IGF-I-stimulated migration of pSMC in a dose-dependent manner. While IGF-I increased the cell migration to 228 ± 38% (mean ± SEM, n = 12), addition of 5 µM LY294002 resulted in 98% inhibition of IGF-I-stimulated cell migration (Fig. 1bGo). Therefore, the activation of PI 3-kinase is required for IGF-I to increase the migration of pSMC following wounding. The basal migration of the cells treated with 50 nM wortmannin was 158 ± 21% compared with migration of the cells treated with 0.1% DMSO carrier expressed as 100% (mean ± SEM, n = 17, NS, Fig. 1aGo).



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Figure 1. Concentration-dependent decrease in IGF-I-stimulated migration of pSMC by wortmannin and LY294002. pSMC were grown to confluence on six-well culture plates. The cultures were wounded with a single razor blade and then treated with IGF-I (0 or 100 ng/ml) and increasing concentrations of wortmannin (WMN, panel a) or LY294002 (LY, panel b) for 48 h. The number of cells that migrated across the wound was counted as described in Materials and Methods. The results are shown as the mean ± SEM (n = 8–20 replicates per experiment). The representative experiment that is shown was repeated three times with similar results. *, P < 0.05 when treatment without IGF-I is compared with treatment with IGF-I by the Mann Whitney test. {ddagger}, P < 0.05 when IGF-I (100 ng/ml) in the absence of an inhibitor is compared with IGF-I (100 ng/ml) in the presence of an inhibitor using the Mann Whitney test.

 
The effect of PD98059, which is an inhibitor of mitogen-activated protein kinase kinase (MAPKK), was compared with that of PI 3-kinase inhibitors (23). When IGF-I increased the cell migration to 214 ± 10% (mean ± SEM, n = 15), 100 nM wortmannin suppressed the IGF-I stimulated migration by 79%, 1 µM LY294002 suppressed it by 58%, and 50 µM PD 98059 caused a 34% decrease (Fig. 2Go). Therefore, MAPK also participates in the increase of pSMC migration after IGF-I treatment, but its contribution is less than that of PI 3-kinase because 50 µM PD98059 was a high enough concentration to give significant suppression of MAPK activity, as is shown below (Fig. 3Go). Treatment of cells with 100 nM wortmannin plus 50 µM PD98059 did not significantly decrease the IGF-I-stimulated cell migration compared with the effect of 100 nM wortmannin alone (Fig. 2Go). Thus, wortmannin and PD98059 do not have an additive effect in inhibiting the promotion of cell migration by IGF-I.



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Figure 2. Comparison of the effect of PD98059, wortmannin, and LY294002 on IGF-I-stimulated migration of pSMC. pSMC that were grown to confluence on six-well culture plates were wounded, and cell migration was determined after 48 h in the presence or absence of IGF-I (100 ng/ml), WMN (100 nM), LY (1 µM), and PD (50 µM). The results are the mean ± SEM (n = 9–19 determinations per experiment). The figure is a representative result from three separate experiments that gave similar results. *, P < 0.05 when treatment without IGF-I is compared with treatment with IGF-I by the Mann Whitney test. {ddagger}, P < 0.05 when 100 ng/ml IGF-I in the absence of an inhibitor is compared with 100 ng/ml IGF-I in the presence of an inhibitor using the Mann-Whitney test.

 


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Figure 3. Effects of wortmannin, LY294002, and PD98059 on IGF-I-stimulated phosphorylation of p44/p42 MAPK. pSMC were plated at a density of 1 x 106 cells per six-well culture plate in DMEM containing 1% FBS, incubated for 24 h, and then serum starved for 2 h. The cells were treated with 100 nM wortmannin, 1 µM LY294002, or 50 µM PD98059 for 30 min, stimulated with 100 ng/ml of IGF-I for 30 min, solubilized in Laemmli sample buffer, and analyzed for the dual phosphorylation of p44/p42 MAPK at Thr202 and Tyr204 (phospho-p44/p42 MAPK) or the amount of p44/p42 MAPK (p44/p42 MAPK) by immunoblotting (8% polyacrylamide gel). Lane 1, no treatment; lane 2, 100 ng/ml IGF-I; lane 3, 100 ng/ml IGF-I and PD98059; lane 4, 100 ng/ml IGF-I and LY294002; lane 5, 100 ng/ml IGF-I and wortmannin. The figure shows a representative blot of three independent experiments that gave similar results.

 
To rule out the possibility that cellular detachment caused by inhibitors had decreased the number of cells measured at the end of wounding assays, the loss of cells from culture dishes after 48 h of exposure to the inhibitors plus IGF-I was determined. When the cells were incubated in the presence of both IGF-I (100 ng/ml) and the inhibitors for 48 h, the number of cells did not decrease significantly compared with the cells that were incubated with IGF-I alone. The number of cells were 97 ± 7%, 95 ± 9%, and 91 ± 8% of control after 100 nM wortmannin, 1 µM LY294002, and 50 µM PD98059, respectively, when the number of cells in the cultures treated with IGF-I alone is expressed as 100% (mean ± SEM, n = 3). Therefore, it is unlikely that the loss of cells significantly affected the wounding assay results.

PI 3-kinase inhibitors inhibit phosphorylation of Akt after IGF-I stimulation
The finding that both wortmannin and LY294002 inhibited the effect of IGF-I on cell migration strongly supports the hypothesis that both inhibitors work by suppressing PI 3-kinase activity. To further strengthen this hypothesis, we tested whether similar concentrations of these inhibitors decreased the phosphorylation of Akt. After a 30-min preincubation with or without inhibitors, pSMC were stimulated with 100 ng/ml of IGF-I for 30 min, and the extent of serine phosphorylation was determined by immunoblotting the cell lysate using an antibody that specifically recognizes Akt that has been phosphorylated at Ser473. The intensity of immunoreactive bands was considered to reflect the change in the phosphorylation status of Akt because the treatment did not change the amount of Akt protein in pSMC when it was analyzed by immunoblotting of cell lysates with an Akt antibody that recognizes both phosphorylated and nonphosphorylated forms of Akt (data not shown). While phosphorylation of Akt was undetectable in the basal state, IGF-I stimulated phosphorylation of Akt. 100 nM wortmannin completely inhibited the Akt phosphorylation, and 1 µM LY294002 significantly decreased the extent of Akt phosphorylation. On the other hand, 50 µM PD98059 did not affect Akt phosphorylation after IGF-I stimulation (Fig. 4Go). Therefore, the doses of wortmannin and LY294002 that were shown to suppress the cell migration were sufficient to prevent PI 3-kinase dependent phosphorylation of Akt after IGF-I stimulation. In contrast, the concentration of PD98059 that inhibited cell migration did not suppress PI 3-kinase activity in pSMC.



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Figure 4. Effects of wortmannin, LY294002, and PD98059 on IGF-I-stimulated phosphorylation of Akt. pSMC were plated at a density of 1 x 106 cells per six-well culture in DMEM containing 1% FBS, incubated for 24 h, and then serum starved for 2 h. The cells were treated with 100 nM wortmannin, 1 µM LY294002, or 50 µM PD98059 for 30 min, stimulated with 100 ng/ml of IGF-I for 30 min, solubilized in Laemmli sample buffer, and analyzed for the phosphorylation of Akt by immunoblotting (8% polyacrylamide gel). Immunoblotting of the cell lysates with an Akt antibody that recognizes both phosphorylated and nonphosphorylated forms of Akt showed that the total amount of Akt was not significantly different after the treatments with IGF-I and inhibitors (data not shown). Lane 1, no treatment; lane 2, 100 ng/ml IGF-I; lane 3, 100 ng/ml IGF-I and wortmannin; lane 4, 100 ng/ml IGF-I and LY294002; lane 5, 100 ng/ml IGF-I and PD98059. The figure shows a representative blot of three independent experiments that gave similar results.

 
Effect of PI 3-kinase inhibitors and PD98059 on phosphorylation of MAPK after IGF-I stimulation
Because it has been reported that activation of PI 3-kinase results in the secondary activation of the MAPK pathway in certain cell types, effects of PI 3-kinase inhibitors and PD98059 on activation of MAPK after IGF-I stimulation was studied by analyzing phosphorylation of MAPK, a substrate for activated MAPKK (24). After a 30-min preincubation with or without inhibitors, pSMC were stimulated with 100 ng/ml of IGF-I for 30 min and the extent of p44/p42 MAPK phosphorylation was determined by immunoblotting the cell lysate with an antibody that recognizes MAPK phosphorylated at both Thr202 and Tyr204. The treatment did not affect the total amount of p44/p42 MAPK in pSMC, as was demonstrated by immunoblotting of the cell lysates with a p44/p42 MAPK antibody, which recognizes both phosphorylated and nonphosphorylated forms of MAPK (Fig. 3Go). IGF-I stimulation clearly increased phosphorylation of MAPK (Fig. 3Go). 50 µM PD98059 significantly reduced phosphorylation of MAPK after IGF-I stimulation, indicating that it effectively inhibited activation of MAPKK. Densitometric analysis of the bands that correspond to p42 MAPK demonstrated that 50 µM PD98059 treatment decreased band intensity by 70% compared with that obtained with cells treated with IGF-I alone (Fig. 3Go, lanes 2 and 3). 1 µM LY294002 did not reduce MAPK phosphorylation after IGF-I treatment, whereas 100 nM wortmannin seemed to decrease it slightly in repeated experiments. Density of the immunoreactive band corresponding to p42 MAPK after 100 nM wortmannin treatment was reduced by 40% compared with that after IGF-I treatment alone (Fig. 3Go, lanes 2 and 5). Therefore, the majority of MAPK activation after IGF-I treatment seems to occur independently of PI 3-kinase activation in pSMC. However, the partial reduction of MAPK phosphorylation after 100 nM wortmannin treatment indicates that the activation of PI 3-kinase may play a small role in the full activation of MAPK pathway upon IGF-I stimulation. The failure of LY294002 to suppress MAPK activation after IGF-I treatment may be due to the fact that 1 µM of LY294002 is less potent than 100 nM wortmannin in inhibiting PI 3-kinase activity, as was demonstrated by their potency to inhibit Akt phosphorylation (Fig. 4Go). Alternatively, it is possible that wortmannin inhibits enzymes other than PI 3-kinase that activate MAPK pathway after IGF-I treatment.

Neither wortmannin nor PD98059 inhibited activation of the {alpha}Vß3 integrin by IGF-I in pSMC
Our previous studies have demonstrated that IGF-I increases affinity of the {alpha}Vß3 integrin toward ligands and that ligand occupancy of the {alpha}Vß3 integrin is required for IGF-I to stimulate migration of pSMC (7). To determine whether the PI 3-kinase or MAPK pathways are required for {alpha}Vß3 integrin activation by IGF-I, we determined the effect of these inhibitors on {alpha}Vß3 activation. The binding of [125I]kistrin, which is a specific ligand for the {alpha}Vß3 integrin, to pSMC surfaces was measured after cells were treated with IGF-I and the indicated amounts of inhibitors. 100 ng/ml IGF-I increased the [125I]kistrin binding by 42 ± 4% (mean ± SEM, n = 3) over control cultures not exposed to IGF-I. Neither 100 nM wortmannin nor 50 µM PD98059 decreased the [125I]kistrin binding after IGF-I stimulation significantly (Fig. 5Go). The treatment of cells with 100 nM wortmannin plus 50 µM PD98059 also did not decrease [125I]kistrin binding after IGF-I stimulation (data not shown). Therefore, the inhibition of PI 3-kinase and MAPK diminished the IGF-I effect on cell migration by mechanisms other than inhibition of {alpha}Vß3 integrin activation, and {alpha}Vß3 integrin activation after IGF-I stimulation most likely involves pathways other than PI 3-kinase or MAPK activation.



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Figure 5. Effects of wortmannin (WMN) and PD98059 (PD) on IGF-I-stimulated increase in [125I]kistrin binding to pSMC. pSMC were grown to subconfluence on 48-well plates, then incubated in the presence or absence of IGF-I 100 ng/ml, wortmannin (100 nM), or PD98059 (50 µM) for 16 h. Activation of {alpha}Vß3 integrin was measured as the increase in [125I]kistrin binding to pSMC as described in Materials and Methods. The data are mean ± SEM (n = 3 per experiment) and is a representative result of three separate experiments that gave similar results.

 
IGF-I-stimulated DNA synthesis was suppressed completely by the dose of a MAPK inhibitor that only partially reduced IGF-I-stimulated migration in pSMC
MAPK inhibition was less effective than PI 3-kinase inhibition in blocking IGF-I-stimulated cell migration. Therefore, we compared the effect of MAPK inhibition to PI 3-kinase inhibition on IGF-I-stimulated cellular replication. In other systems, stimulation of cell replication by IGF-I has also been shown to involve both PI 3-kinase and MAPK activation (12). [3H]thymidine incorporation was measured in cells treated with the combination of various concentrations of IGF-I and the inhibitors for 24 h. IGF-I increased [3H]thymidine incorporation into pSMC in a dose-dependent manner: 20 ng/ml IGF-I increased incorporation to 211 ± 24% (Fig. 6Go, b and c) to 421 ± 62% (Fig. 6aGo) (mean ± SEM, n = 3) when the value without IGF-I was expressed as 100%. Increasing concentrations of wortmannin inhibited the cellular response to IGF-I without changing the basal level of [3H]thymidine incorporation significantly. 1.0 µM wortmannin was required to completely diminish the effect of IGF-I on DNA synthesis, and 100 nM wortmannin decreased [3H]thymidine incorporation by 57% (Fig. 6aGo). 1 µM LY294002 suppressed the maximum response to IGF-I (20 ng/ml) by 59%, and 10 µM of LY294002 was required to completely inhibit [3H]thymidine incorporation after IGF-I stimulation (Fig. 6bGo). On the other hand, 50 µM PD98059, which decreased IGF-I-stimulated cell migration by only 34%, inhibited IGF-I-stimulated DNA synthesis completely (Fig. 6cGo). Therefore, although MAPK plays a significant role in the cell replication response to IGF-I, its contribution to IGF-I-stimulated cell migration is not as important as PI 3-kinase pathway activation.



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Figure 6. Dose-dependent inhibition of IGF-I-stimulated DNA synthesis by wortmannin, LY294002, and PD98059. pSMC were treated with increasing concentrations of wortmannin (nM), LY294002 (µM), or PD98059 (µM). The change in [3H]thymidine incorporation into DNA after IGF-I stimulation was measured. The data are the mean ± SEM (n = 3 per experiment) and include the results from three separate experiments: panel a, wortmannin; panel b, LY294002; panel c, PD98059. *, P < 0.05 when treatment without IGF-I was compared with treatment with IGF-I using the alternate Welch t test. {ddagger}, P < 0.05 when treatment with 100 ng/ml IGF-I in the absence of an inhibitor was compared to 100 ng/ml IGF-I in the presence of an inhibitor using the alternate Welch t test.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activation of PI 3-kinase was required for IGF-I to increase migration of pSMC in our assays. Two unrelated inhibitors of PI 3-kinase, wortmannin and LY294002, completely blocked IGF-I-stimulated cell migration at the doses that are known to suppress PI 3-kinase activity in many other cell types (21, 22). The IC50 of wortmannin that was required to inhibit cell migration was 10 nM, and that of LY294002 was 0.3 µM. Thus, the abrogation of IGF-I-stimulated cell migration by both inhibitors is most likely due to blockade of PI 3-kinase. The decrease in IGF-I-stimulated Akt phosphorylation by these inhibitors further supports the conclusion that they strongly suppressed PI 3-kinase activity in our system. Phosphorylation of Akt at Ser437 is downstream of PI 3-kinase and is dependent upon prior PI 3-kinase activation (25). Therefore, pSMC seem to be reasonably sensitive to these inhibitors when PI 3-kinase activation is examined.

Although both the PI 3-kinase and MAPK pathways have been described as distinctive signal transduction pathways, cross-talk between the two pathways has been documented. This includes the activation of MAPK by PI 3-kinase, especially by the PI 3 {gamma} isoform (26, 27). The analysis of IGF-I induced Akt phosphorylation in the presence of PD98059 indicated that the suppression of MAPK had little effect on Akt phosphorylation after IGF-I treatment in our system. The IGF-I induced MAPK phosphorylation was only partially suppressed by 100 nM wortmannin and was not suppressed at all by 1 µM LY294002. Therefore, the activation of PI 3 and MAPK pathways seems to occur independently of each other after IGF-I stimulation in pSMC. The discrepancy between wortmannin and LY294002 in their effects on MAPK phosphorylation could be due to differences in their potency, or to suppression of enzymes other than PI 3-kinase by wortmannin. In either case, PI 3-kinase inhibitors effectively reduced IGF-I induced cell migration at the doses that have little effect on MAPK activation, indicating that the secondary activation of MAPK by PI 3-kinase is not the major mechanism responsible for IGF-I-dependent cell migration in pSMC.

PD98059, an inhibitor of MAPKK, was only partially effective in suppressing IGF-I-stimulated migration of pSMC even at the dose that completely blocked IGF-I-stimulated DNA synthesis in the same cells. Because PD98059 decreased IGF-I-induced cell migration by 34% (P < 0.05), IGF-I apparently utilizes the MAPK pathway in stimulating the migration of pSMC. However, the contribution of the MAPK pathway for this IGF-I action is limited compared with that of PI 3-kinase, which is required for IGF-I to up-regulate cell motility. Several studies have analyzed the contribution of PI 3-kinase and MAPK pathways in the vascular SMC migration that occurs in response to PDGF (1). The results reported to date have placed variable relative importance on each pathway, depending on the source of SMC and on the conditions of the experiments (28, 29, 30, 31). To our knowledge, there has been only one other study that analyzed signal transduction pathways responsible for IGF-I-stimulated migration of vascular SMC. Pukac et al. (31) compared effects of signal transduction inhibitors on the migration of rat vascular SMC after IGF-I, PDGF, and phorbol 12-myristate 13-acetate (PMA) treatment. Our observation that the PI 3-kinase pathway is more involved than the MAPK pathway in IGF-I-stimulated pSMC migration is similar to their conclusion. However, unlike their speculation that the MAPK pathway was not activated after IGF-I treatment in their system, phosphorylation of MAPK was increased after IGF-I stimulation in pSMC, suggesting that, although the MAPK pathway is activated by IGF-I, this plays a minor role in IGF-I stimulation of pSMC migration.

Because occupancy of the {alpha}Vß3 integrin is required for IGF-I to increase migration of SMC, we hypothesized that activation of the PI 3 and MAP kinase pathways might be involved in increasing the affinity of the {alpha}Vß3 integrin after IGF-I treatment. The role of PI 3-kinase activation in integrin functions has been reported in several other systems. Wortmannin effectively inhibited maintenance of the active state in the platelet specific integrin, {alpha}IIbß3, which is highly homologous to {alpha}Vß3 (32). In lymphocytes, CD2-induced activation of ß1 integrin, measured as adhesion to fibronectin, was suppressed by wortmannin (33). However, neither PI 3-kinase inhibitors nor a MAPKK inhibitor affected the change in {alpha}Vß3 integrin affinity upon IGF-I treatment of pSMC. Although MAPK activation has been suggested to inactivate constituitively activated integrins in CHO cells (34), the inhibition of MAPK did not increase {alpha}Vß3 integrin affinity upon IGF-I treatment. We conclude that IGF-I probably activates pathways other than PI 3-kinase and MAPK to induce activation of the {alpha}Vß3 integrin in pSMC. The possible roles of pathways other than PI 3-kinase and MAPK in modulating IGF-I actions have been documented. Expression of mutant IGF-I receptors with amino acid substitutions for specific tyrosine phosphorylation sites results in a form of receptor that fails to elucidate the full actions of IGF-I, even though these forms of the receptor can activate maximal PI 3-kinase and MAPK responses (35, 36). The activation of protein kinase C (PKC) is one possible pathway that is responsible for the change in {alpha}Vß3 integrin affinity, since it has been known to be activated by IGF-I and is involved in integrin function in mast and other cell types (37, 38).

There are several possible mechanisms by which activation of PI 3-kinase and MAPK increase cell migration. Although activation of MAPK played a limited role in stimulation of pSMC migration by IGF-I, it has been shown to play an important role for the increase in cell migration after PDGF treatment and H-Ras activation (17, 39). Phosphorylation of the myosin light chain by MAPK has been documented and may be one of the substrates used by this enzyme to regulate cell motility (40). As for PI 3-kinase, it has been well documented that its activation alters the cytoskeletal organization that changes dynamically when cells increase their motility. In neutrophils, PI 3-kinase is required for the cells to increase cytoskeletal actin and respond to chemoattractants (41). PI 3-kinase has been shown to mediate IGF-I and PDGF-stimulated membrane ruffling and lamellipodia formation, which are considered to be an important component of a set of responses that leads to cell migration (42, 43, 44). Cdc42 and Rac1 activation disrupt actin organization and increase cell motility through activation of PI 3-kinase in mammary epithelial cells (15). It will require further analysis to determine whether PI 3-kinase inhibitors change actin organization in pSMC and if this leads to a block in IGF-I-stimulated migration.

Both PI 3-kinase and MAPK pathways are involved in proliferation and migration of pSMC after IGF-I treatment, with PI 3-kinase bieng more involved in migration and MAPK being more important for proliferation. Considering the well established role of vascular SMC in neointima formation, knowledge about intracellular signaling pathways governing proliferation and migration of these cells may facilitate formulation of effective strategies against atherosclerosis.


    Acknowledgments
 
The authors wish to thank Mr. George Mosley for his help in preparing the manuscript. We thank Ms. Gayle Horvitz for her technical support.


    Footnotes
 
1 This work was supported by Grant HL-56850 from the National Institutes of Health. Back

Received December 9, 1998.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ross R 1993 The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809[CrossRef][Medline]
  2. Miano JM, Vlasic N, Tota RR, Stemerman MB 1993 Smooth muscle cell immediate-early gene and growth factor activation follows vascular injury. A putative in vivo mechanism for autocrine growth. Arterioscler Thromb 13:211–219[Abstract/Free Full Text]
  3. Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, Ross R 1994 Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest 93:1266–1274
  4. Khorsandi MJ, Fagin JA, Giannella-Neto D, Forrester JS, Cercek B 1992 Regulation of insulin-like growth factor-I and its receptor in rat aorta after balloon denudation. Evidence for local bioactivity. J Clin Invest 90:1926–1931
  5. Gockerman A, Prevette T, Jones JI, Clemmons DR 1995 Insulin-like growth factor (IGF)-binding proteins inhibit the smooth muscle cell migration responses to IGF-I and IGF-II. Endocrinology 136:4168–4173[Abstract]
  6. Hynes RO 1992 Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11–25[CrossRef][Medline]
  7. Jones JI, Prevette T, Gockerman A, Clemmons DR 1996 Ligand occupancy of the {alpha}-V-ß3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor. Proc Natl Acad Sci USA 93:2482–2487[Abstract/Free Full Text]
  8. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[CrossRef][Medline]
  9. Kato H, Faria TN, Stannard B, Roberts Jr CT, LeRoith D 1993 Role of tyrosine kinase activity in signal transduction by the insulin- like growth factor-I (IGF-I) receptor. Characterization of kinase-deficient IGF-I receptors and the action of an IGFI-mimetic antibody ({alpha} IR-3). J Biol Chem 268:2655–2661[Abstract/Free Full Text]
  10. Beitner-Johnson D, LeRoith D 1995 Insulin-like growth factor-I stimulates tyrosine phosphorylation of endogenous c-Crk. J Biol Chem 270:5187–5190[Abstract/Free Full Text]
  11. Miller TM, Tansey MG, Johnson Jr EM, Creedon DJ 1997 Inhibition of phosphatidylinositol 3-kinase activity blocks depolarization- and insulin-like growth factor I-mediated survival of cerebellar granule cells. J Biol Chem 272:9847–9853[Abstract/Free Full Text]
  12. Kuemmerle JF, Bushman TL 1998 IGF-I stimulates intestinal muscle cell growth by activating distinct PI 3-kinase and MAP kinase pathways. Am J Physiol 275:G151–G158
  13. Parrizas, M 1997 Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J Biol Chem 272:154–161[Abstract/Free Full Text]
  14. Liu Q, Ning W, Dantzer R, Freund GG, Kelley KW 1998 Activation of protein kinase C-zeta and phosphatidylinositol 3'-kinase and promotion of macrophage differentiation by insulin-like growth factor-I. J Immunol 160:1393–1401[Abstract/Free Full Text]
  15. Keely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV 1997 Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 390:632–636[CrossRef][Medline]
  16. Kundra V, Escobedo JA, Kazlauskas A, Kim HK, Rhee HG, Williams LT, Zetter BR 1994 Regulation of chemotaxis by the platelet-derived growth factor receptor-ß. Nature 367:474–476[CrossRef][Medline]
  17. Choudhury GG, Karamitsos C, Hernandez J, Gentilini A, Bardgette J, Abboud HE 1997 PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF. Am J Physiol 273:F931–F938
  18. Imai Y, Busby Jr WH, Smith CE, Clarke JB, Garmong AJ, Horvitz GB, Rees CR, Clemmons DR 1997 Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture. J Clin Invest 100:2596–2605[Medline]
  19. Imai Y, Philippe N, Sesti G, Accili D, Taylor SI 1997 Expression of variant forms of insulin receptor substrate-1 identified in patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 82:4201–4207[Abstract/Free Full Text]
  20. Clemmons DR 1985 Exposure to platelet-derived growth factor modulates the porcine aortic smooth muscle cell response to somatomedin-C. Endocrinology 117:77–83[Abstract]
  21. Arcaro A, Wymann MP 1993 Wortmannin is a potent phosphatidylinositol 3kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J 296:297–301
  22. Vlahos CJ, Matter WF, Hui KY, Brown RF 1994 A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269:5241–5248[Abstract/Free Full Text]
  23. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR 1995 A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92:7686–7689[Abstract/Free Full Text]
  24. Cross DAE, Alessi DR, Vanderheede JR, McDowell HE, Hundal HS, Cohen P 1994 The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor-1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J 303:21–26
  25. Franke TF, Kaplan DR, Cantley LC 1997 PI3K: downstream AKTion blocks apoptosis. Cell 88:435–437[CrossRef][Medline]
  26. Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, Wetzker R 1997 Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase {gamma}. Science 275:394–397[Abstract/Free Full Text]
  27. Bondeva T, Pirola L, Bulgarelli-Leva G, Rubio I, Wetzker R, Wymann MP 1998 Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science 282:293–296[Abstract/Free Full Text]
  28. Graf K, Xi XP, Yang D, Fleck E, Hsueh WA, Law RE 1997 Mitogen-activated protein kinase activation is involved in platelet-derived growth factor-directed migration by vascular smooth muscle cells. Hypertension 29:334–339[Abstract/Free Full Text]
  29. Jiang B, Yamamura S, Nelson PR, Mureebe L, Kent KC 1996 Differential effects of platelet-derived growth factor isotypes on human smooth muscle cell proliferation and migration are mediated by distinct signaling pathways. Surgery 120:427–431[CrossRef][Medline]
  30. Higaki M, Sakaue H, Ogawa W, Kasuga M, Shimokado K 1996 Phosphatidylinositol 3-kinase-independent signal transduction pathway for platelet-derived growth factor-induced chemotaxis. J Biol Chem 271:29342–29346[Abstract/Free Full Text]
  31. Pukac L, Huangpu J, Karnovsky MJ 1998 Platelet-derived growth factor-BB, insulin-like growth factor-I, and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration. Exp Cell Res 242:548–560[CrossRef][Medline]
  32. Kovacsovics TJ, Bachelot C, Toker A, Vlahos CJ, Duckworth B, Cantley, LC, Hartwig JH 1995 Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem 270:11358–11366[Abstract/Free Full Text]
  33. Shimizu Y, Mobley JL, Finkelstein LD, Chan AS 1995 A role for phosphatidylinositol 3-kinase in the regulation of ß 1 integrin activity by the CD2 antigen. J Cell Biol 131:1867–1880[Abstract/Free Full Text]
  34. Hughes PE, Renshaw MW, Pfaff M, Forsyth J, Keivens VM, Schwartz HA, Ginsberg MH 1997 Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway. Cell 88:521–530[CrossRef][Medline]
  35. Esposito DL 1997 Tyrosine residues in the C-terminal domain of the insulin-like growth factor-I receptor mediate mitogenic and tumorigenic signals. Endocrinology 138:2979–2988[Abstract/Free Full Text]
  36. Scrimgeour AG, Blakesley VA, Stannard BS, LeRoith D 1997 Mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways are not sufficient for insulin-like growth factor I-induced mitogenesis and tumorigenesis. Endocrinology 138:2552–2558[Abstract/Free Full Text]
  37. Tranque PA, Calle R, Naftolin F, Robbins R 1992 Involvement of protein kinase-C in the mitogenic effect of insulin-like growth factor-I on rat astrocytes. Endocrinology 131:1948–1954[Abstract]
  38. Vosseller K, Stella G, Yee NS, Besmer P 1997 c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling. Mol Biol Cell 8:909–922[Abstract]
  39. Khosravi-Far R, Solski PA, Clark GJ, Kinch MS, Der CJ 1995 Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. Mol Cell Biol 15:6443–6453[Abstract]
  40. Ni H, Wang XS, Diener K, Yao Z 1998 MAPKAPK5, a novel mitogen-activated protein kinase (MAPK)-activated protein kinase, is a substrate of the extracellular regulated kinase (ERK) and p38 kinase. Biochem Biophys Res Commun 243:492–496[CrossRef][Medline]
  41. Niggli V, Keller H 1997 The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptide-induced locomotion and increases in cytoskeletal actin in human neutrophils. Eur J Pharmacol 335:43–52[CrossRef][Medline]
  42. Kadowaki T, Koyasu S, Nishida E, Sakai H, Takaku F, Yahara I, Kasuga M 1986 Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors. J Biol Chem 261:16141–16147[Abstract/Free Full Text]
  43. Leventhal PS, Shelden EA, Kim B, Feldman EL 1997 Tyrosine phosphorylation of paxillin and focal adhesion kinase during insulin-like growth factor-I-stimulated lamellipodial advance. J Biol Chem 272:5214–5218[Abstract/Free Full Text]
  44. Nobes CD, Hall A 1995 Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53–62[CrossRef][Medline]



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