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Circulation Research. 2000;86:15-23

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(Circulation Research. 2000;86:15.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Phosphatidylinositol 3-Kinase Is Required for Insulin-Like Growth Factor-I–Induced Vascular Smooth Muscle Cell Proliferation and Migration

Cunming Duan, Jeanette R. Bauchat, Tzefu Hsieh

From the Department of Biology, University of Michigan, Ann Arbor, Mich.

Correspondence to Cunming Duan, PhD, Department of Biology, The University of Michigan, Natural Science Building, Ann Arbor, MI 48109-1048. E-mail cduan{at}umich.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Insulin-like growth factor–I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. The mitogenic and chemotactic actions of IGF-I are mediated through the IGF-I receptor, but how the activation of the IGF-I receptor leads to these biological responses is poorly understood. In this study, we examined the role of phosphatidylinositol 3-kinase (PI3 kinase) in mediating the mitogenic and chemotactic signals of IGF-I. IGF-I treatment resulted in a significant increase in phosphotyrosine-associated PI3 kinase activity in cultured primary VSMCs. To determine whether insulin receptor substrate (IRS)–1, -2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti-IRS-1 or an anti-IRS-2 antibody, and the associated PI3 kinase activity was determined. IGF-I stimulation resulted in a significant increase in IRS-1– but not IRS-2–associated PI3 kinase activity, suggesting that IGF-I primarily utilizes IRS-1 to transmit its signal in VSMCs. The IGF-I–induced increase in IRS-I–associated PI3 kinase activity was concentration dependent. At the maximum concentration (50 ng/mL), IGF-I induced a 60-fold increase. This activation occurred within 5 minutes and was sustained at high levels for at least 6 hours. IGF-I also caused a concentration-dependent and long-lasting activation of protein kinase B (PKB/Akt). Inhibition of PI3 kinase activation by LY294002 or wortmannin abolished IGF-I–stimulated VSMC proliferation and reduced IGF-I–directed VSMC migration by {approx}60%. These results indicate that activation of PI3 kinase is required for both IGF-I–induced VSMC proliferation and migration.


Key Words: insulin-like growth factor-I • phosphatidylinositol 3-kinase • proliferation • migration • vascular smooth muscle cell


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abnormal vascular smooth muscle cell (VSMC) proliferation and directed migration from media into intima play major roles in the pathogenesis of atherosclerotic lesions, the formation of restenosis after angioplasty, and the accelerated arteriopathy after cardiac transplantation.1 Studies using human, porcine, bovine, rabbit, and rat VSMCs have shown that insulin-like growth factor–I (IGF-I) is a mitogen and a potent chemoattractant for VSMCs.2 3 The mitogenic and chemotactic actions of IGF-I are mediated through the IGF-I receptor (IGF-IR), a transmembrane tyrosine kinase that is abundantly expressed in VSMCs. Selective blockage of the IGF-IR with a specific antibody inhibits IGF-I–stimulated cell proliferation and migration.4 5 6 Likewise, reducing the IGF-IR numbers using antisense DNA approaches suppresses the proliferative responses of VSMCs to IGF-I.7 8

While it has become evident that IGF-I actions in VSMC proliferation and migration are mediated through the IGF-IR, the intracellular signaling mechanisms that IGF-I utilizes to elicit these biological actions are poorly understood in this cell type. Because the signaling pathways initiated from the IGF-IR are very complex, previous studies on the IGF signal transduction pathways have been carried predominantly in immortalized cell lines, such as 3T3 cells. Studies using these "model" systems indicate that one of the earliest steps in signal transduction initiated by the IGF-IR is the phosphorylation of adaptor/docking proteins such as insulin receptor substrate (IRS)–1 or –2, Shc, Grab2, and Grab10.9 10 These molecules then interact with downstream signal transducers and effectors, resulting in activation of the mitogen-activated protein kinase (MAPK, also known as ERK, extracellular signal–regulated kinase) pathway and phosphatidylinositol 3-kinase (PI3 kinase) signaling pathways. Activation of the MAPK pathway is considered to be critical for cell proliferation, whereas the PI3 kinase pathway is important for mediating the metabolic and antiapoptotic signals of IGF-I. Although these "model" systems are ideal for demonstrating protein-protein interactions, they are less suited for elucidating the physiological outcomes of the activation of these signaling pathways. Furthermore, intracellular signaling pathways induced by the IGF-IR are highly cell-type specific. Diploid, normal smooth muscle cells (SMCs) in culture, which are untransformed, may respond differently from 3T3 cells or other immortalized cell lines often used for signal transduction studies.11 Indeed, previous studies using human, bovine, and rat SMCs indicate that IGF-I stimulation either did not activate or only weakly activated MAPK in these cells.12 13 14 15 The inability or meager ability of IGF-I in activating MAPK implies that this signaling pathway may play an insignificant role in IGF signaling in VSMCs.11 The alternative intracellular signaling pathway or pathways involved in transmitting the mitogenic signal of IGF-I have not been determined. Recent studies have indicated that PI3 kinase rather than MAPK activity correlated with IGF-I–induced proliferation in early passages of cultured normal human fibroblasts and mouse C2C12 myoblasts.16 17 Although one previous study indicated that IGF-I stimulation increases PI3 kinase activity in rat VSMCs,18 the functional significance of this activation has not been determined. Equally poorly understood are the intracellular signaling pathways involved in IGF-I–regulated VSMC chemotaxis. Bornfeldt et al5 11 suggested that the MAPK signaling pathway is unlikely to be involved in chemotaxis induced by IGF-I or by platelet-derived growth factor (PDGF)–BB, because (1) although both IGF-I and PDGF-BB stimulate phosphatidylinositol-4,5-bisphosphate hydrolysis, diacylglycerol formation, calcium mobilization, and chemotaxis in cultured human VSMCs, only PDGF-BB but not IGF-I activates MAPK activation in these cells, and (2) PDGF-BB stimulation of MAPK correlates well with stimulation of VSMC proliferation but not with chemotaxis. It is unknown at present whether the PI3 kinase signaling pathway is involved in transducing the chemotactic signal of IGF-I.

In this study, we examined the ability of IGF-I in activating PI3 kinase and protein kinase B (PKB/Akt) activation and investigated the role of this signaling pathway in mediating the mitogenic and chemotactic signals of IGF-I using cultured newborn porcine VSMCs. We found that IGF-I exposure strongly activated the PI3 kinase signaling cascade in these primary cells. Furthermore, specific inhibition of PI3 kinase negated IGF-I–dependent DNA synthesis and partially blocked IGF-I–induced VSMC migration, indicating that activation of PI3 kinase is required for both IGF-I–stimulated VSMC proliferation and migration.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
All chemicals and reagents were purchased from Sigma unless noted otherwise. IGF-IR ß-subunit antibody was purchased from Santa Cruz Biotechnology, Inc. The IRS-1 and -2 antibodies were a gift from Joslin Diabetes Center, Harvard Medical School (Boston, Mass). 4G10 anti-phosphotyrosine antibody was purchased from Upstate Biotechnology Inc. Phosphospecific (activated) and control antibodies for PKB/Akt and MAPK kinase (MEK) 1 inhibitor PD98059 were purchased from New England Biolabs. Horseradish peroxidase–linked anti-rabbit and anti-mouse antibodies and the rainbow molecular weight markers [32P]ATP and [32P]dCTP were obtained from Amersham Life Science. Human IGF-I was purchased from GroPep. LY294002 was purchased from BIOMOL (Plymouth Meeting, PA). FBS, DMEM with high glucose, and penicillin-streptomycin were purchased from GIBCO-BRL. Trypsin was obtained from Boehringer Mannheim.

Cell Culture
Porcine VSMCs were isolated from thoracic aorta of 3-week-old piglets.19 The cells were grown in 10-cm dishes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) in DMEM supplemented with 4 mmol/L glutamine, penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% FBS. The medium was changed every fourth day until the cells became confluent. Before stimulation experiments, medium was changed to serum-free DMEM (SFM) for 18 to 24 hours. This SFM was then replaced with fresh SFM plus indicated growth factors for various times.

Western Immunoblotting Analysis
The cell lysates were separated by SDS-PAGE. After transfer to filters (Immunobilon P, 0.45-µm pore size, Millipore), the membranes were blocked in 3% BSA (Fisher Scientific) in Tris-buffered saline–Tween 20 (TBST). For anti-PKB/Akt blotting, membranes were incubated overnight with a 1:1000 dilution in TBST-BSA buffer at 4°C and then washed five times with TBST. All other blots were incubated with a 1:1000 to 1:5000 dilution of the indicated antibody in blocking buffer for 1 to 2 hours at room temperature. Blots were then washed with TBST and incubated with a 1:3000 dilution of horseradish peroxidase–linked anti-rabbit secondary antibody in blocking buffer for 2 to 3 hours, followed by further washing. Enhanced chemiluminescence was performed according to the manufacturer’s instructions (Amersham). Densitometry was performed by scanning the autoradiographs (ScanJet IIcx, Hewlett-Packard) and the intensity of each band analyzed using Scion Image software.

Immunoprecipitation
Equal amounts of cell lysates were incubated with the indicated antibodies overnight at 4°C according to the manufacturer’s instructions. Protein A–Sepharose (50 µL) was then added for 4 hours or overnight at 4°C and followed by 3 washes. Beads were resuspended in 30 µL Laemmli loading buffer containing 30 mg/mL DTT, boiled, and separated by SDS-PAGE followed by Western blotting.

PI3 Kinase Assay
After growth factor treatment, cell cultures were washed, lysed, and incubated with primary antibody overnight followed by further incubation with protein A–Sepharose for 2 hours. After washing 3 times, PI3 kinase assay was performed as described previously.20 Briefly, samples were resuspended in 30 µL of PI3 kinase buffer (in mmol/L, Tris [pH 7.5] 20, NaCl 100, and EGTA 0.5), and 20 µg of phosphatidylinositol was added. After 5 minutes at room temperature, 10 µCi of [32P]ATP was added. After 10 minutes at room temperature, lipids were extracted with 80 µL of MeOH:1N HCl (1:1). Samples were spotted on 1% potassium oxalate–treated TLC plates (Analtech) and developed in CHCl3:MeOH:NH4OH (129:114:15). The highest migrating spots on the TLC plate, representing phosphatidylinositol phosphate, were quantified by densitometry, as described above.

[3H]Thymidine Incorporation Assay
To determine the rate of DNA synthesis, porcine VSMCs were plated onto 96-well plates (Falcon) at 15 000 cells/well in DMEM supplemented with 10% FBS and incubated for 3 to 5 days without a medium change. After being rinsed three times with DMEM, the cultures were exposed to DMEM containing 1 µCi [3H]thymidine (ICN Biochemicals, Inc) and the desired concentrations of IGF-I and/or inhibitors in a final volume of 200 µL. Each treatment was added to triplicate cultures. After {approx}48 hours, cells were washed twice with PBS, twice with cold 5% trichloroacetic acid for 10 minutes at 4°C, and solubilized in 200 µL of 0.1 mol/L NaOH/1% SDS at room temperature. The solubilized DNA was harvested for liquid scintillation counting. The results are expressed as the percentage change from the controls.

5-Bromo-2-Deoxyuridine (BrdU) Staining
Immunocytochemical analysis of BrdU incorporation into DNA was used to examine the effect of IGF-I on cell proliferation. VSMCs were growth arrested for 24 hours in SFM. BrdU (20 µmol/L), IGF-I, and/or inhibitors were added directly to cell cultures, and the cells were further incubated for 20 hours. Cells were fixed in 2% paraformaldehyde, processed to expose incorporated BrdU, and then incubated with a 1:2000 dilution of a mouse monoclonal anti-BrdU primary antibody (Sigma), 0.5 µg/mL TRITC-conjugated goal anti-mouse secondary antibody. The cells were visualized under a fluorescence microscope. The BrdU-positive cells were counted, and the results are expressed as percentage of BrdU-labeled cells in the cell population.

Migration Assay
Migration assays were performed using 24-well cell culture inserts with 8.0 µm polyethylene terephthalate Cyclopore membranes (Falcon). The inserts were coated with 0.1% gelatin before each experiment. Porcine VSMCs at {approx}70% confluence were incubated in 0.2% BSA in SFM-DMEM for 3 hours and trypsinized. After trypsinization, the cells were washed once in 1x PBS and resuspended. Growth factors diluted in DMEM were loaded into the lower wells of the inserts and cells (50 000 cells in 200 µL) were subsequently loaded into the upper wells. The chambers were incubated for 8 hours at 37°C. After the incubation, cells were removed from the upper side of the membranes using cotton swabs, and the membranes with the migratory cells on the underside were fixed and stained in toluidine blue. The inserts were then examined under the microscope, and the total number of migratory cells was counted.

Statistical Analysis
Values are mean±SE. Differences among groups were analyzed by 1-way ANOVA followed by the Fisher protected least significance difference test using Statview (Abacus Concepts, Inc).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
The effect of IGF-I on DNA synthesis in cultured newborn porcine VSMCs was examined by [3H]thymidine incorporation assay and BrdU immunostaining. As reported previously,21 exposure of quiescent porcine VSMCs to IGF-I resulted in a dose-dependent increase in [3H]thymidine incorporation (Figure 1ADown). At 50 ng/mL, IGF-I induced a maximum increase of 348±100.7% (P<0.01, n=4). No further increase was seen at concentrations higher than 50 ng/mL. The mitogenic activity of IGF-I was further studied by immunocytochemical detection of BrdU incorporation. For this, VSMCs were grown to confluence, and growth was arrested by serum starvation for 24 hours. As shown in Figure 1CDown, BrdU staining was not detected in serum-starved VSMCs, with the exception of rare, lightly stained nuclei. IGF-I (50 ng/mL) treatment of growth-arrested VSMCs resulted in a 440±58% (n=3, P<0.05) increase in the number of BrdU-positive cells over the control (Figure 1CDown). The chemotactic activity of IGF-I was determined by a transwell migration assay. This assay measures the movements of cells across a porous membrane in response to a concentration gradient of a chemoattractant and has previously been used to provide quantitative measurements of human VSMC motility in response to PDGF-BB and IGF-I.5 As shown in Figure 1BDown, IGF-I strongly stimulated directed migration of porcine VSMCs with a bell-shaped dose-response curve often seen in classical chemotactic responses.22 The maximal response, being 1736±658% (n=3) over the control, was seen at the concentration of 50 ng/mL (P<0.05). At 100 ng/mL, the levels decreased to 793±57% over the control (n=3). Checkerboard analysis in which the amount of IGF-I was varied in both the top and bottom wells indicated that porcine VSMCs displayed predominantly directed motility (chemotaxis) toward an IGF-I gradient ({approx}80%), with a minor component attributed to random motility.



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  		Figure 1. IGF-I stimulates porcine VSMC proliferation and directional migration through an IGF-IR–mediated mechanism. A, Concentration-dependent stimulation of DNA synthesis by IGF-I. Confluent cells were exposed to SFM in the presence or absence of various concentrations of IGF-I or the IGF-I analog [Leu24]IGF-I. The [3H]thymidine incorporation was determined as described in Materials and Methods. Values are mean±SE of 4 separate experiments, each of which was performed in triplicate. B, Concentration-dependent stimulation of cell migration by IGF-I. Migration assays were performed using 24-well cell culture inserts as described in Materials and Methods in the presence or absence of the indicated concentrations of IGF-I or [Leu24]IGF-I. Values are mean±SE of 3 separate experiments. C, Effect of IGF-I on BrdU incorporation. Growth-arrested VSMCs were treated with (c and d) or without (a and b) IGF-I (50 ng/mL) in the presence of BrdU (20 µmol/L) for 20 hours. Cells were fixed and stained for BrdU. a and c, Phase-contrast views of control and IGF-I–treated cells. b and d, Fluorescent view of the same cells. Magnification x100.

To determine the involvement of IGF-IR, the growth and chemotactic effects of [Leu24]IGF-I, an IGF-I analog with greatly reduced affinity for the IGF-IR but normal affinity for IGF binding proteins,23 were examined. [Leu24]IGF-I did not cause any significant increase in either DNA synthesis or migration (Figures 1AUp, 1BUp, and 7CDown). These results suggest that IGF-I stimulates porcine VSMC proliferation, motility, and directed migration through the IGF-IR–mediated mechanism(s).



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  		Figure 7. Inhibition of PI3 kinase activity by LY294002 abolishes IGF-I–induced DNA synthesis in cultured porcine VSMCs. A, Inhibition of PI3 kinase by LY294002 abolished IGF-I–stimulated thymidine incorporation. Confluent cells were exposed to various concentrations of IGF-I in the presence and absence of LY294002 (20 µmol/L). Values are mean±SE of 3 separate experiments, each of which was performed in triplicate. B, Dose-dependent effect of LY294002. Confluent cells were exposed to varying concentrations of LY294002 in the presence and absence of IGF-I (50 ng/mL). Values are mean±SE of 3 separate experiments, each of which was performed in triplicate. C, Inhibition of PI3 kinase by LY294002 or wortmannin abolished IGF-I–stimulated cell proliferation. Growth-arrested VSMCs were treated with or without IGF-I (100 ng/mL), wortmannin (20 µmol/L), or LY294002 (10 µmol/L) in the presence of BrdU (20 µmol/L) for 20 hours. Cells were fixed and immunocytochemically stained for BrdU. Cells were visualized under a fluorescence microscope, and BrdU-positive cells were analyzed. Values are mean±SE of 4 to 8 replications.

To examine the effect of IGF-I stimulation in tyrosine phosphorylation of IGF-IR and other endogenous proteins, confluent porcine VSMCs were treated with various concentrations of IGF-I for 10 minutes after serum starvation for 24 hours. IGF-I caused tyrosine phosphorylation of several major proteins, including two proteins with the apparent molecular sizes of 185 and 96 kDa (Figure 2ADown). Immunoblotting analysis using specific antibodies indicated that the 96-kDa protein is the IGF-IR ß-subunit, and the 185-kDa protein is IRS-1. The tyrosine phosphorylation of IGF-IR and IRS-1 and their association with each other were further examined by immunoprecipitation with either an IGF-IR or IRS-1 antibody followed with immunoblotting analysis. As shown in Figure 2BDown, receptor autophosphorylation was undetectable in 24-hour serum-starved cells. Incubation of VSMCs with IGF-I (100 ng/mL) for 10 minutes resulted in the tyrosine phosphorylation of IGF-IR and IRS-1 and their physical association.



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  		Figure 2. IGF-I stimulation leads to tyrosine phosphorylation of IGF-IR and its downstream proteins in cultured porcine VSMCs. A, Stimulation of tyrosine phosphorylation by IGF-I. Serum-starved cells were treated with or without IGF-I at various concentrations for 10 minutes. Cells were lysed and subjected to SDS-PAGE on a 10% gel. Proteins containing phosphotyrosine were detected with Western immunoblotting as described in Materials and Methods with a monoclonal anti-phosphotyrosine antibody. Arrows indicate proteins with increased tyrosine phosphorylation. B, IGF-I stimulates IGF-IR and IRS-I association and phosphorylation. Serum-starved cells were treated with or without IGF-I (100 ng/mL) for 10 minutes. The cells were lysed, immunoprecipitated (IP) with an anti–IGF-IRß (left) or an anti–IRS-1 antibody (right), and subjected to Western blotting (IB) with the antibodies indicated. Cont indicates control.

We next examined the effect of IGF-I stimulation in activating the PI3 kinase. First, the IGF-I–induced change in tyrosine-phosphorylated protein-associated PI3 kinase activity was analyzed. Binding of the p85 regulatory subunit of PI3 kinase to tyrosine-phosphorylated proteins is a major mechanism of PI3 kinase activation.24 Serum-starved porcine VSMCs were treated with IGF-I (50 ng/mL) for 10 minutes. The cell lysates were immunoprecipitated using an anti-phosphotyrosine antibody (4G10), and the PI3 kinase activity was analyzed. As shown in Figure 3ADown, IGF-I treatment resulted in a significant increase in PI3 kinase activity. To determine whether IRS-1, IRS-2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti IRS-1 or anti IRS-2 antibody, and the associated PI3 kinase activity was determined. IGF-I stimulation resulted in a significant increase in IRS-1–associated PI3 kinase activity (Figure 3BDown). In contrast, there was no change in the IRS-2–associated PI3 kinase activity after IGF-I stimulation, suggesting that IGF-I primarily utilizes IRS-1 to transmit its signal downstream in cultured porcine VSMCs. The IGF-I–induced increase in IRS-I–associated PI3 kinase activity was dose dependent at concentrations ranging from 1 to 50 ng/mL. At the maximum concentration (50 ng/mL), IGF-I induced a {approx}54–61-fold increase (Figure 4ADown). This activation occurred within 5 minutes of IGF-I stimulation and was sustained at high levels for 6 hours (Figure 4BDown). The activity began to decline after 6 hours but was nonetheless higher than the basal level even after 24 hours. To further examine the effect of IGF-I in activating the PI3 kinase signaling cascade, the phosphorylation of PKB/Akt was examined using a phosphospecific and a control antibody. The results indicate that IGF-I induced a concentration-dependent increase in the serine phosphorylation of PKB/Akt at concentrations ranging from 1 to 50 ng/mL (Figure 5ADown). Consistent with the PI3 kinase results, the IGF-I–induced PKB/Akt phosphorylation was observed within 5 minutes and lasted for at least 8 hours (Figure 5BDown). These results suggest that IGF-I has a strong effect in activating the PI3 kinase-PKB/Akt signaling pathway in porcine VSMCs and that this activation is primarily mediated through IRS-1.



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  		Figure 3. IGF-I–induced PI3 kinase activation is mediated by IRS-1 but not IRS-2 in cultured porcine VSMCs. A, Effect of IGF-I on the tyrosine phosphorylated protein–associated PI3 kinase activity. Serum-starved cells were treated with or without IGF-I (50 ng/mL) for 10 minutes, immunoprecipitated with the anti-phosphotyrosine antibody 4G10, and assayed for PI3 kinase activity in vitro. B, Effect of IGF-I on the IRS-1– or IRS-2–associated PI3 kinase activity. Serum-starved cells were treated with or without IGF-I (5 or 50 ng/mL) for 10 minutes. Cells were lysed, immunoprecipitated with either an anti–IRS-1 or anti–IRS-2 antibody, and assayed for PI3 kinase activity in vitro.



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  		Figure 4. IGF-I stimulation leads to a strong and sustained activation of PI3 kinase in cultured porcine VSMCs. A, Dose-dependent effect of IGF-I on IRS-1–associated PI3 kinase activity. Serum-starved cells were treated with or without IGF-I at various concentrations for 10 minutes, immunoprecipitated with an IRS-1 antibody, and assayed for PI3 kinase activity in vitro. Radioactivity in the spots corresponding to phosphatidylinositol (PIP) were measured, and results are shown in the right panel (n=2). B, Time-course effect of IGF-I on total PI3 kinase activity. Serum-starved cells were treated with or without IGF-I (50 ng/mL) for various periods of time. Cells were lysed, immunoprecipitated, and assayed for PI3 kinase activity in vitro. Radioactivity in the spots corresponding to phosphatidylinositol (PIP) were measured, and results are shown in the right panel (n=2).



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  		Figure 5. IGF-I stimulation leads to a sustained activation of PKB/Akt in cultured porcine VSMCs. A, Dose-dependent effect of IGF-I. Serum-starved cells were treated with or without IGF-I at various concentrations for 10 minutes and were subjected to Western blotting with phosphospecific and control antibodies. B, Time-dependent phosphorylation of PKB/Akt induced by IGF-I. Serum-starved cells were treated with or without IGF-I (50 ng/mL) for various periods of time, and lysates were subjected to Western blotting with phosphospecific and control antibodies.

To determine whether the mitogenic and/or chemotactic effects of IGF-I involve the activation of PI3 kinase in VSMCs, two structurally distinct PI3 kinase inhibitors, wortmannin and LY294002, were used to block PI3 kinase activation. The effectiveness of these compounds in porcine VSMCs was examined by directly monitoring the IRS-1–associated PI3 kinase activity as well as the phosphorylation status of PKB/Akt. As shown in Figure 6ADown, pretreatment with wortmannin (10 µmol/L) resulted in a 95% inhibition in the IGF-I–induced PI3 kinase activation. LY294002 at 10 µmol/L also significantly inhibited the activation (70% inhibition). In comparison, the MEK inhibitor PD98059 (40 µmol/L) had no such effect. Likewise, wortmannin and LY294002, but not PD98059, blocked the IGF-I–stimulated PKB/Akt phosphorylation (Figure 6BDown). At 10 µmol/L, LY294002 completely inhibited IGF-I–stimulated PKB/Akt phosphorylation (Figure 6CDown). The involvement of PI3 kinase in IGF-I–stimulated VSMC proliferation was determined by incubating cells with LY294002 or wortmannin in the presence or absence of IGF-I. LY294002 inhibited IGF-I–stimulated thymidine incorporation in a dose-dependent manner (Figure 7Up). At 10 µmol/L, it completely inhibited the IGF-I–stimulated DNA synthesis. LY294002 alone lowered the basal level to 56% of the SFM control group. Because wortmannin has a very short half-life (3 to 4 hours) in cell cultures,25 it was not used in the long-term thymidine incorporation experiments. To further investigate the role of PI3 kinase in mediating the mitogenic effect of IGF-I, BrdU staining experiments were carried out using growth-arrested VSMCs. As shown in Figure 7CUp, both wortmannin (20 µmol/L) and LY294002 (10 µmol/L) treatment nearly completely inhibited IGF-I–stimulated VSMC proliferation (89% and 100% inhibition). Blocking PI3 kinase activation by LY294002 (20 µmol/L), on the other hand, resulted in a significant but partial decrease (58%) in the number of cells migrated toward IGF-I (Figure 8Down). Likewise, wortmannin treatment also reduced IGF-I–induced cell migration by 57%. These results indicate that activation of PI3 kinase is required for the mitogenic and chemotactic actions of IGF-I in porcine VSMCs.



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  		Figure 6. Inhibition of IGF-I–stimulated PI3 kinase and PKB/Akt activation in cultured porcine VSMCs. A, Inhibition of PI3 kinase activity. Serum-starved cells were pretreated with and without wortmannin (10 µmol/L), LY294002 (10 µmol/L), or PD98059 (40 µmol/L) for 2 hours before IGF-I (50 ng/mL) stimulation. Lysates were immunoprecipitated with an anti–IRS-1 antibody and assayed for PI3 kinase activity in vitro. Radioactivity in phosphatidylinositol (PIP) was measured, and means of 2 separate experiments are shown as fold change over control. B, Inhibition of PKB/Akt phosphorylation. Serum-starved cells were pretreated with PD98059 (40 µmol/L), LY294002 (10 µmol/L), or wortmannin (10 µmol/L) for 2 hours before IGF-I (50 ng/mL) stimulation. C, Dose-dependent inhibition by LY294002. Serum-starved cells were pretreated for 2 hours with varying concentrations of LY294002 before IGF-I (50 ng/mL) stimulation.



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  		Figure 8. Activation of PI3 kinase is required for IGF-I–directed VSMC migration. Cells were pretreated with and without wortmannin (20 µmol/L) or LY294002 (10 µmol/L) for 2 hours before migration assay. Migration assays were performed using 24-well cell culture inserts, as described in Materials and Methods, in the presence or absence of IGF-I (50 ng/mL). Values are mean±SE of 4 separate experiments.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The effects of IGF-I on VSMC proliferation and directional migration are widely recognized across mammalian species. Although it is evident that the mitogenic and chemotactic actions of IGF-I are mediated through the same IGF-IR, the intracellular signaling mechanisms that IGF-I utilizes to elicit these biological actions are poorly understood. In the present study, we demonstrated that IGF-I strongly activates PI3 kinase in cultured porcine VSMCs. This activation occurs within minutes and is sustained for hours. Although the activation of PI3 kinase by IGF-I has recently been documented in rat VSMCs,18 the relative importance of IRS-1 or IRS-2 has not been determined. In this study, we have demonstrated that IGF-I activates the PI3 kinase signaling pathway primarily through IRS-1. IRS-2 plays an insignificant role in IGF signaling in VSMCs. In addition to induction of PI3 kinase, IGF-I stimulation also leads to a dose-dependent and sustained activation of PKB/Akt, indicating that IGF-I is a strong activator of the PI3 kinase signaling cascade in cultured VSMCs.

Previous studies using human, bovine, and rat SMCs indicate that IGF-I stimulation either did not activate or only weakly activated MAPK in these cells.12 13 14 15 Although IGF-I is a weaker mitogen compared with PDGF-BB, it nonetheless induces a significant increase in VSMC proliferation and DNA synthesis (References 4, 15, 21, and 264 15 21 26 , and the present study). The inability or meager ability of IGF-I to activate the MAPK signaling pathway implied that an alternative intracellular signaling pathway or pathways may mediate the mitogenic signal initiated from the IGF-IR in VSMCs. Recent studies have indicated that the PI3 kinase signaling pathway may be involved in IGF-I–induced proliferation in certain cell types.27 Milansincic et al17 reported that IGF-I elicited a strong mitogenic response whereas it only had minimal effect on MAPK activity in mouse C2C12 myoblasts. In these cells, IGF-I strongly activated PI3 kinase. Likewise, it has been shown that PI3 kinase rather than MAPK activity is correlated with IGF-I–induced mitogenesis in early passages of cultured human fibroblasts.16 In this study, we have found that the mitogenic signal of IGF-I is primarily mediated through the PI3 kinase signaling pathway in cultured porcine VSMCs. Support for this conclusion came from experiments using two PI3 kinase–specific inhibitors, LY294002 and wortmannin. Wortmannin and LY294002 are two potent and specific inhibitors of PI3 kinase, the use of which has established a role for PI3 kinase in transducing numerous effects of IGF-I in regulating the metabolism, differentiation, and inhibition of apoptosis of IGF-I.9 10 LY294002, a synthetic inhibitor of PI3 kinase, strongly inhibited PI3 kinase activity and PKB/Akt activation in porcine VSMCs. Wortmannin binds irreversibly to the catalytic subunit (p110) of PI3 kinase, thereby inhibiting PI3 kinase activation with a high degree of specificity.28 In porcine VSMCs, it completely blocked the IGF-I–induced PI3 kinase activation and PKB/Akt phosphorylation. Both wortmannin and LY294002 inhibited IGF-I–stimulated VSMC proliferation, respectively. In addition, pretreatment of cells with either of these compounds blocked the IGF-I–stimulated gene expression (C. Duan, M.B. Liimatta, O.L. Bottum, unpublished observation).

Another novel finding made in this study is that activation of PI3 kinase is required for optimal chemotactic response of VSMCs to IGF-I. Although initiation of cellular motility has been demonstrated with a number of growth factors, including PDGF, IGF-I, hepatocyte growth factor (HGF), and fibroblast growth factor (FGF) in a number of cell types,22 5 29 30 31 the intracellular signaling pathways involved in cellular motility and chemotaxis in general are less well understood compared with those of mitogenesis. PDGF-BB is the most extensively studied growth factor in the context of chemotaxis. It has been shown that PDGF-BB–stimulated chemotaxis relies on activation of both phospholipase C (PLC)–{gamma} and PI3 kinase in Chinese hamster ovary cells.29 HGF also activates both PLC-{gamma} and PI3 kinase, and both signaling pathways are required for HGF-induced chemotaxis in murine renal epithelial cells.31 Not all chemoattractants fall into this paradigm. For example, FGF mediates chemotaxis without activating PLC-{gamma}.30 In rat VSMCs, activation of calcium/calmodulin–dependent protein kinase II has been implicated in FGF-stimulated chemotaxis.32 Likewise, IGF-I does not activate PLC-{gamma} but is a potent chemoattractant for human VSMCs.5 Therefore, the signal transduction mechanism utilized among different chemoattractants is not universal. Although IGF-I is a potent chemoattractant for VSMCs, very little is known about the intracellular signaling mechanisms underlying the chemotactic action of IGF-I in VSMCs or any other cell types. In this study, we have shown that two PI3 kinase–specific inhibitors, LY294002 and wortmannin, both significantly inhibited IGF-I–induced chemotaxis of VSMCs. These findings support a role for PI3 kinase in chemotactic response to IGF-I. This finding is in good agreement with those of 2 previous studies using rat VSMCs showing inhibition of PI3 kinase activation by wortmannin-attenuated PDGF-BB and 12-myristate 13-acetate–induced VSMC migration,33 34 suggesting a general role of PI3 kinase in the chemotactic responses of VSMCs to various chemoattractants. It is of interest to note that, although inhibition of PI3 kinase activity by either LY294002 or wortmannin abolished IGF-I–induced DNA synthesis, the IGF-I–induced VSMC migration was only partially inhibited ({approx}57–58%). Because LY294002 and wortmannin at the concentrations used suppressed PI3 kinase activity and PKB/Akt phosphorylation by >90%, it is possible that another PI3 kinase-independent signaling pathway(s) is also involved in IGF-I–induced VSMC migration. Indeed, a previous study in rat VSMCs indicating that there may be PI3 kinase–independent chemotactic signaling pathways involved in PDGF-BB–induced cell migration.35 Further studies are needed to elucidate the PI3 kinase-independent signaling pathway(s) that mediates the chemotactic signal of IGF-I in VSMCs.

Although the results of this study indicated that activation of PI3 kinase is required for both IGF-I–regulated VSMC proliferation and migration, how the mitogenic and chemotactic signals diverge further downstream of PI3 kinase is not yet clear. One of the well-characterized downstream pathways is the PKB/Akt-p70 s6 kinase (p70s6k) pathway. PKB/Akt is a serine-threonine kinase immediately downstream of PI3 kinase.36 In this study, we have found that treatment of porcine VSMCs with IGF-I results in a profound and long-lasting activation of PKB/Akt. PKB/Akt has been shown to be a major mediator for IGF-I actions in promoting neuronal survival.37 38 Recent studies in Dictyostelium discoideum suggest that PKB/Akt is involved in directional sensing of chemoattractant and plays an important role in chemotaxis in these amoeba cells.39 40 Acting further downstream is p70s6k, a serine/threonine kinase that has been shown to play a role in regulating cell cycle progression and protein synthesis, but not apoptosis.36 41 Higaki et al35 reported that rapamycin, which inhibits p70s6k along with other signaling events, did not inhibited PDGF-BB–induced rat VSMC chemotaxis. In contrast, Poon et al42 showed that pretreatment of SMCs with rapamycin ({approx}2–100 ng/mL) for 48 hours inhibited PDGF-BB–induced VSMC migration. Therefore, it remains to be determined as to whether p70s6k is involved in the IGF-I–regulated VSMC migration. Two other proteins downstream of PI3 kinase are Rho and Rac. These small G proteins are well known for their involvement in PDGF-stimulated cytoskeletal reorganization and membrane ruffling.43 44 There is, however, no direct evidence for a role for Rho and Rac in mediating the chemotactic responses of VSMCs to IGF-I to date. A third group of possible downstream signaling intermediates of PI3 kinase comprises several protein kinase C (PKC) isoforms. It has long been established that the products of PI3 kinase, including phosphatidylinositol-3,4,5-trisphosphate, are capable of activating PKC-{zeta} and PKC-{epsilon}.45 46 47 Recently, we have shown that several PKC isoenzymes, including PKC-{epsilon} and -{zeta}, are expressed in porcine VSMCs and are under the regulation of IGF-I.20 In these cells, down-regulation or inhibition of PKC activity by high doses of 12-myristate 13-acetate or a specific PKC inhibitor (GF109203X) abolished IGF-I–induced DNA synthesis and migration. Therefore, these PKC isoforms may be essential signaling intermediates that act downstream of PI3 kinase in the IGF signaling network in VSMCs.20 Experiments are currently underway to determine the role(s) of these PKC isoforms in mediating the growth and chemotactic signals of IGF-I and their relationship with PI3 kinase and PKB/Akt in cultured porcine VSMCs.


*    Acknowledgments
 
This study was supported in part by National Science Foundation Grant IBN-9728911 and by National Institute of Diabetes and Digestive and Kidney Diseases (NIH) Grant 5P60DK-20572 through a Pilot/Feasibility Grant from Michigan Diabetes Research and Training Center, University of Michigan. We thank Kasiani Pozios for her help with the BrdU staining experiments.

Received August 16, 1999; accepted October 8, 1999.


*    References
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 *References
 

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