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(Circulation Research. 2004;95:380.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Glucose-Potentiated Chemotaxis in Human Vascular Smooth Muscle Is Dependent on Cross-Talk Between the PI3K and MAPK Signaling Pathways

Malcolm Campbell, William E. Allen, Carol Sawyer, Bart Vanhaesebroeck, Elisabeth R. Trimble

From the Department of Clinical Biochemistry and Metabolic Medicine (M.C., W.E.A., E.R.T.), Queen’s University Belfast, Institute of Clinical Science, Royal Victoria Hospital, Belfast; and Ludwig Institute for Cancer Research (C.S., B.V.), London, UK.

Correspondence to Prof E.R. Trimble, MD, Department of Clinical Biochemistry and Metabolic Medicine, Queen’s University Belfast, Institute of Clinical Science, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BJ, UK. E-mail e.trimble{at}qub.ac.uk

	Abstract

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Atheroma formation involves the movement of vascular smooth muscle cells (VSMC) into the subendothelial space. The aim of this study was to determine the involvement of PI3K and MAPK pathways and the importance of cross-talk between these pathways, in glucose-potentiated VSMC chemotaxis to serum factors. VSMC chemotaxis occurred in a serum gradient in 25 mmol/L glucose (but not in 5 mmol/L glucose) in association with increased phosphorylation (activation) of Akt and ERK1/2 in PI3K and MAPK pathways, respectively. Inhibitors of these pathways blocked chemotaxis, as did an mTOR inhibitor. VSMC expressed all class IA PI3K isoforms, but microinjection experiments demonstrated that only the p110ß isoform was involved in chemotaxis. ERK1/2 phosphorylation was reduced not only by MAPK pathway inhibitors but also by PI3K and mTOR inhibitors; when PI3K was inhibited, ERK phosphorylation could be induced by microinjected activated Akt, indicating important cross-talk between the PI3K and ERK1/2 pathways. Glucose-potentiated phosphorylation of molecules in the p38 and JNK MAPK pathways inhibited these pathways but did not affect chemotaxis. The statin, mevinolin, blocked chemotaxis through its effects on the MAPK pathway. Mevinolin-inhibited chemotaxis was restored by farnesylpyrophosphate but not by geranylgeranylpyrophosphate; in the absence of mevinolin, inhibition of farnesyltransferase reduced ERK phosphorylation and blocked chemotaxis, indicating a role for the Ras family of GTPases (MAPK pathway) under these conditions. In conclusion, glucose sensitizes VSMC to serum, inducing chemotaxis via pathways involving p110ß-PI3K, Akt, mTOR, and ERK1/2 MAPK. Cross-talk between the PI3K and MAPK pathways is necessary for VSMC chemotaxis under these conditions.

Key Words: diabetes • atherosclerosis • Akt • ERK • statin

	Introduction

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Cardiovascular disease is responsible for most of the complications associated with diabetes. Impairment of endothelium and muscle contributes to abnormalities of vasodilation.¹ Expansion of the subendothelial space occurs with the development of atheromatous plaques, which contain cellular components that have migrated from the endothelium and the tunica media, in addition to invading inflammatory cells and macrophages.² Both PI3K and MAPK have been implicated in chemotaxis.

PI3K enzymes regulate many cellular responses, including proliferation, protection from apoptosis, intracellular vesicular transport, cytoskeletal rearrangements, and cell migration.³ PI3Ks signal via the production of 3-phosphoinositides, lipid second messengers that bind to lipid-binding domains in a wide variety of proteins, including protein kinases (such as Akt/PKB) and regulators of small GTPases. The plectrin homology lipid–binding domain of Akt binds to the lipid products of PI3K; Akt is then recruited to the plasma membrane and phosphorylated at T308 and S473 to yield a fully activated kinase.⁴ PI3Ks are heterodimers consisting of a 110-kDa catalytic subunit (p110, p110ß, or p110) in complex with a regulatory adapter molecule (p85 and its splice variants, p85ß and p55), which contains Src-homology-2 (SH2) domains.⁴ Whereas p110 and p110ß are ubiquitously expressed, p110 was previously thought to be restricted to leukocytes⁵ but has recently been found in other cell types.⁶ Many studies implicating PI3K activity have not distinguished between the 3 p110 isoforms, and little is known about their distinct signaling roles. All isoforms have the same lipid substrate, but the protein kinase substrates are isoform-specific.^4,7 Functional inactivation of individual PI3K isoforms (microinjection of isoform-specific antibodies) revealed isoform-specific roles in mediating cellular responses to specific extracellular signals.^3,6,8–10 In this study, we aimed to identify the isoform(s) of PI3K involved in glucose-potentiated vascular smooth muscle cell (VSMC) chemotaxis.

Many molecules with chemotactic properties activate the MAPK pathways, in addition to the PI3K pathway.¹¹ The 3 main human MAPK pathway cascades terminate in ERK1/2, p38, and JNK, respectively. The ERK1/2 MAPK pathway, which involves sequential activation of Ras, Raf, MEK1/2, and ERK1/2, is involved in platelet-derived growth factor (PDGF)–induced^8,12,13 and tumor necrosis factor--induced¹⁴ VSMC chemotaxis. By contrast, p38 has been implicated in sphingosine-1–phosphate-stimulated chemotaxis of rat VSMC.¹⁵ Glucose activates the ERK1/2 pathway in VSMC^16,17 and the p38 pathway in mesothelial cells,¹⁸ Schwann cells,¹⁹ and endothelial cells.¹⁷ In addition, glucopenia is also associated with activation of ERK1/2 and JNK.²⁰

Cross-talk occurs among the different MAPK pathways, the degree of which depends on both the conditions and the tissues involved;²¹ however, in many publications only 1 of the pathways has been investigated. Cross-talk between the PI3K and MAPK pathways has been implicated in phagocytic cell chemotaxis.^22,23 Furthermore, glucose can alter the cross-talk between signaling systems, as has been shown for the insulin and ß-adrenergic signaling systems in VSMC.²⁴

The aims of the present study were to investigate the involvement of the PI3K and MAPK in glucose-potentiated chemotaxis of VSMC to serum factors and to determine whether cross-talk between these pathways played an important role in chemotaxis under these conditions.

	Materials and Methods

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Chemicals and Antibodies
The following chemicals were purchased: D-glucose and L-glucose (BDH, Poole, England); Wortmannin and LY294002 (PI3K inhibitors), rapamycin (mTOR inhibitor), mevinolin (lovastatin), geranylgeranylpyrophosphate, and farnesylpyrophosphate (Sigma); PD98059 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor) (Calbiochem, La Jolla, Calif); the farnesyltransferase inhibitor H-D-Trp-D-Met-p-chloro-D-Phe-Gla-NH₂ (Bachem, St. Helens, UK); PDGF-ß receptor antibody (R&D System, Minneapolis, Minn); antibodies to p110ß (sc-602), p85, total and phosphorylated ERK, and total and phosphorylated JNK (Santa Cruz Biotechnologies, Calif); antibodies to total and Ser473-phosphorylated Akt/PKB (Cell Signaling Technology); antibodies to total and phosphorylated p38 MAPK (Calbiochem); HRP-conjugated antimouse or antirabbit IgG (DAKO, Ely, UK); and rabbit or mouse IgG (Sigma). C-terminal–directed, affinity-purified antibodies to p110, p110ß, and p110, used for microinjection and Western blotting, have been described elsewhere.^1–3

For an expanded Materials and Methods section, see the online data supplement available at http://circres.ahajournals.org.

	Results

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Glucose-Potentiated Chemotaxis Is Dependent on the p110ß Isoform of PI3K
We have previously shown that VSMC will show chemotaxis in fetal calf serum (FCS) gradient without previous starvation in the presence of 9 to 25 mmol/L glucose¹⁶ (see online Figure A). PI3K inhibitors, wortmannin (10 nmol/L) or LY294002 (10 µmol/L), blocked chemotaxis of VSMC in a gradient of FCS in 25 mmol/L glucose, with the Rayleigh test demonstrating no significant chemotaxis; the solvent, DMSO 0.1% (v/v), did not affect chemotaxis (Table 1). The present results show that VSMC express all class IA PI3K isoforms (see online Figure B). To determine the roles of these isoforms in VSMC chemotaxis, isoform-specific neutralizing antibodies were microinjected. After recovery (2 hours), cells were placed in a chemotaxis chamber with 25 mmol/L glucose and FCS gradient; for controls, nonspecific IgG was injected. As additional controls, each antibody was pre-absorbed with x30 molar excess of its cognate peptide before injection. Chemotaxis was blocked only in cells injected with antibody to p110ß, and preabsorption of this antibody with excess p110ß abrogated its effect on chemotaxis (Table 1).

View this table: [in this window] [in a new window]   Table 1. Glucose-Potentiated Chemotaxis is PI3K-Dependent (p110ß), Akt-Dependent, mTOR-Dependent, and ERK1/2-Dependent

Glucose-Potentiated Chemotaxis Is Dependent on Akt and mTOR
To test the effect of 25 mmol/L glucose on pathways mediated by PI3K, the activity of Akt a downstream target for PI3K, was investigated. No specific Akt inhibitors are available; therefore, we microinjected an antibody directed against the N terminus of Akt, which was previously used to demonstrate Akt involvement in insulin-stimulated glucose transport in adipocytes.²⁵ Cells injected with the anti-Akt antibody showed no chemotaxis in 25 mmol/L glucose and FCS gradient, whereas those injected with a nonspecific IgG control antibody did show chemotaxis (Table 1).

A pathway closely linked to the PI3K pathway is that of mTOR, leading to activation of p70-S6 kinase.²⁶ The mTOR inhibitor, rapamycin, also blocked chemotaxis (Table 1).

Glucose Increases the Expression and Phosphorylation Levels of MAPK Proteins
We tested the impact of 24-hour incubation in 25 mmol/L D-glucose on the expression levels and phosphorylation states of p38, JNK, and ERK1/2 members of the MAPK family by immunoprecipitation and Western blotting of lysates using total and phospho-specific antibodies and densitometric evaluation (Table 2). Incubation in 25 mmol/L glucose caused a significant increase in total protein level of all 3 proteins (P<0.05 in all cases). Both p38 and JNK showed significant increases (21% and 28%, respectively) in their phosphorylation level (P<0.05), but these did not differ significantly from the increases seen in total protein level (27% and 22%, respectively). A significant increase in ERK1/2 phosphorylation was seen in 25 mmol/L glucose (P<0.01 versus 5 mmol/L glucose), which was greater than 3-fold the increase seen in ERK1/2 total protein level (95% versus 30%, respectively).

View this table: [in this window] [in a new window]   Table 2. Glucose Effects on MAPK Proteins

Which MAPK Pathway Is Required for Glucose-Potentiated Chemotaxis?
To determine the role of the 3 main MAPK signaling pathways in D-glucose–mediated chemotaxis, VSMC were placed in a gradient of FCS in 25 mmol/L glucose in the presence of 600 nmol/L SB203580 (a p38 inhibitor), 90 nmol/L SP600125 (a JNK inhibitor), or 10 µmol/L PD98059 (a MEK1/2 inhibitor). VSMC treated with SB203580 or SP600125 still showed chemotaxis, but PD98059 blocked chemotaxis (Table 1), suggesting that only the MEK/ERK is involved in glucose-potentiated chemotaxis. To investigate this further, an anti-ERK antibody (directed to the C-terminus) was microinjected into VSMC. Anti-ERK antibody blocked chemotaxis whereas nonspecific IgG did not (Table 1).

Glucose-Potentiated Akt Activation Is PI3K-Dependent but not MAPK-Dependent
We next investigated how the PI3K and MAPK pathways interact to control chemotaxis in VSMC. Total and Ser473-phosphorylated Akt were immunoprecipitated from lysates of cells treated with 25 mmol/L D-glucose with or without the PI3K inhibitor LY294002, the mTOR inhibitor rapamycin, the MEK1/2 inhibitor PD98059, or the Ras farnesylation inhibitor, mevinolin. None of these inhibitors and/or L-glucose controls (latter not shown) caused a significant change in total Akt protein (Figure 1A and 1D). The level of Ser473-phosphorylated Akt (Figure 1B and 1E) increased significantly in 25 mmol/L glucose (versus 5 mmol/L glucose, P<0.05). Akt Ser473 phosphorylation was reduced by the PI3K inhibitors LY294002 (P<0.05) and wortmannin (not shown, P<0.05) but not by rapamycin, PD98059, or mevinolin (Figure 1B and 1E), indicating that the effects of glucose on the PI3K to Akt pathway are independent of mTOR and MAPK. We next tested the activity of immunoprecipitated Ser473-phosphorylated Akt by assessing its ability to phosphorylate GSK-3, a downstream target of Akt. The results were similar to those found for phosphorylation of Akt. Akt activity increased (80%) in cells cultured in 25 mmol/L glucose for 24 hours (Figure 1C and 1F); this was inhibited by co-incubation with the PI3K inhibitor, LY294002, further confirming that glucose-stimulated Akt activation is PI3K-dependent.

View larger version (38K): [in this window] [in a new window]   Figure 1. The effect of PI3K and MAPK pathway inhibitors on total protein level and activation of the downstream target of the PI3K pathway, Akt. This figure shows a representative immunoblot of (A) total Akt, (B) Ser473-phosphorylated Akt, and (C) phosphorylated GSK3-/ß (a measure of Akt activation) under the conditions of 5 or 25 mmol/L glucose, with or without the addition of inhibitors. Graphs (D), (E), and (F) show results for the same measurements in 4 (n=4) independent experiments. The effects of glucose and inhibitors on the activation of Akt were investigated using a kinase assay that tests the ability of isolated Akt to phosphorylate its substrate GSK-3/ß. Inhibitors: LY (PI3K inhibitor, LY294002, 10 µmol/L), Rap (mTOR inhibitor, rapamycin, 1 nmol/L), PD9 (MEK1/2 inhibitor, PD98059, 10 µmol/L), and Mev (a statin, Ras inhibitor, mevinolin, 10 µmol/L).

Glucose-Potentiated ERK1/2 Activation Is Affected by MEK/MAPK Pathway Inhibitors and by Inhibitors of the PI3K and mTOR Pathways
To determine whether ERK activation in VSMC depends on PI3K activation, total and phosphorylated ERK1/2 were immunoprecipitated from lysates of cells treated with 25 mmol/L D-glucose with or without wortmannin and LY294002. Additionally, rapamycin, PD98059, or mevinolin were also used. The small but significant increase in total ERK1/2 protein level in 25 mmol/L glucose (P<0.05 versus 5 mmol/L glucose alone) was not altered by rapamycin or mevinolin (Figure 2A and 2D) but was reduced by LY294002 and PD98059 (P<0.05 versus 25 mmol/L glucose alone). The increased level of ERK phosphorylation (Figure 2B and 2E) in 25 mmol/L glucose (compared with 5 mmol/L glucose, P<0.05) was blocked by LY294002, wortmannin (data not shown), rapamycin, PD98059, and mevinolin (P<0.05 for all), indicating that the effect of glucose on the MAPK pathway leading to ERK1/2 is dependent on PI3K, mTOR, and, as expected, on Ras and MEK1/2 (Figure 2E). For dose-response to mevinolin, see online Figure C. Furthermore, an inhibitor of farnesyltransferase (FTI; 2 nmol/L), H-D-Trp-D-Met-p-chloro-D-Phe-Gla-NH_2, also reduced glucose-induced ERK phosphorylation (online Figure D). To investigate the relationship between ERK phosphorylation under these experimental conditions, a MAPK activity assay was undertaken; phosphorylated ERK1/2 was isolated from cell lysates and assessed for its ability to phosphorylate its target, ELK1. The results were similar to those found for phosphorylation of ERK1/2 (Figure 2C and 2F) with 300% increase in ERK1/2 activity after culture in 25 mmol/L glucose for 24 hours compared with 5 mmol/L glucose conditions. The PI3K inhibitor (LY294002), rapamycin, PD98059, and mevinolin all significantly reduced the activity of ERK1/2 in 25 mmol/L glucose conditions (P<0.05 for all).

View larger version (36K): [in this window] [in a new window]   Figure 2. The effect of PI3K and MAPK pathway inhibitors on total protein level and activation of the downstream target of the MAPK pathway, ERK1/2. This figure shows a representative immunoblot of (A) total ERK1/2, (B) Tyr204-phosphorylated ERK1/2, and (C) phosphorylated ELK-1 (a measure of ERK1/2 activation) under conditions of 5 or 25 mmol/L glucose, with or without the addition of inhibitors. Graphs (D), (E), and (F) give results for the same measurements for 4 to 7 (n=4 to 7) independent experiments. The ability of isolated ERK1/2 to phosphorylate its substrate ELK1 under the indicated conditions was assessed using a MAPK assay (F). Inhibitors: LY (LY294002, 10 µmol/L), Rap (rapamycin, 1 nmol/L), PD9 (PD98059, 10 µmol/L), and Mev (mevinolin, 10 µmol/L).

To investigate further the effects of glucose activation of the PI3K pathway on the MAPK pathway, VSMC were treated with the PI3K inhibitor and then microinjected with activated Akt to see whether phosphorylated Akt could induce phosphorylation of ERK. At the inhibitor concentration used, the glucose-induced increase in PI3K activity was totally inhibited (online Figure E). Microinjection of activated Akt into VSMC pretreated with LY294002 resulted in a 4-fold increase in phosphorylated ERK in cells incubated in either 5 mmol/L or 25 mmol/L glucose (Figure 3).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of activated Akt on ERK activation in the presence of PI3K inhibition. ERK phosphorylation was measured by image analysis of immunoflourescently stained VSMC treated with 5 mmol/L glucose or 25 mmol/L glucose with and without treatment with the PI3K inhibitor LY294002. To determine whether Akt, a major downstream target of PI3K, is involved in regulating the glucose effect on ERK, an activated version of Akt was microinjected into cells pretreated with LY294002. Graph shows results from image analysis of confocal micrographs. As shown in Figure 2, by immunoprecipitation, the increased phosphorylation of ERK associated with 25 mmol/L glucose was inhibited by treatment with LY294002, indicating that glucose upregulated ERK via PI3K. Cells injected with activated Akt showed a 4-fold increase in phosphorylated ERK, indicating that Akt activity can mimic the effect of the active PI3K pathway. All results shown are for cells injected with the rabbit IgG and show that injection of this IgG in the absence of activated Akt has no effect on ERK phosphorylation.

Glucose-Potentiated Chemotaxis Is Ras-Dependent
Statins, in addition to inhibition of cholesterol synthesis, also cause impaired farnesylation of Ras and impaired geranylation of Rho family proteins.²⁷ Mevinolin acts as a MAPK inhibitor because of its ability to prevent Ras farnesylation and therefore subsequent recruitment to the membrane and activation. To investigate the role of Ras and Rho family members in glucose-mediated chemotaxis, VSMC were placed in a gradient of FCS in 25 mmol/L glucose in the presence of various concentrations of mevinolin (0.1 nmol/L to 10 µmol/L); concentrations as low as 1 nmol/L inhibited chemotaxis (not shown). To investigate whether mevinolin was acting through the Ras or Rho family of proteins, geranylgeranylpyrophosphate (10 µmol/L), the activated substrate for Rho geranylation, or farnesylpyrophosphate (10 µmol/L), required for Ras farnesylation, was placed in the chemotaxis chamber with mevinolin-treated cells; farnesylpyrophosphate, but not geranylpyrophosphate, restored chemotaxis (Table 3). This suggests that under the conditions of these experiments, it is the interference of mevinolin with farnesylation of Ras that blocked activation of the ERK1/2 MAPK pathway and prevented chemotaxis. Furthermore, the inhibitor of FTI, in the absence of mevinolin, prevented chemotaxis to FCS in 25 mmol/L glucose (Table 3).

View this table: [in this window] [in a new window]   Table 3. Ras Farnesylation Is Necessary for Glucose-Potentiated Chemotaxis to FCS

Morphological Changes Associated With Glucose-Potentiated Chemotaxis Are Ras-Dependent
Confocal microscopy was used to assess the morphological alterations associated with Ras inhibition in 25 mmol/L glucose conditions. In the sample micrographs shown in Figure 4, a novel technique was used where the pre-etched coverslip from the chemotaxis experiment was gently removed after identifying the direction of the FCS gradient. Cells were stained with TRITC-phalloidin. The images have been rotated so that the FCS source (chemoattractant) is located at the top.

View larger version (75K): [in this window] [in a new window]   Figure 4. Morphological alterations associated with glucose-potentiated chemotaxis in VSMC depend on Ras. Confocal micrographs of VSMC migrating in a gradient of FCS in a chemotaxis chamber. Cells were stained with TRITC-phalloidin to reveal the actin cytoskeleton. The chemoattractant source is at the top of each image. Scale bars represent 10 µm. A, 5 mmol/L glucose: cells showed no filopodia. B, 25 mmol/L glucose: resulted in the formation of filopodia along the lamellipodium (see high-power view in insert). The lamellipodium has formed along the leading edge of the migrating cell in the direction of the FCS gradient. C, 25 mmol/L glucose plus 10 µmol/L mevinolin: prevents filopodia formation along the lamellipodium. D, 25 mmol/L glucose plus 10 µmol/L mevinolin and 10 µmol/L geranylgeranylpyrophosphate: filopodia are not detected along the lamellipodia. E, 25 mmol/L glucose plus 10 µmol/L mevinolin and 10 µmol/L farnesylpyrophosphate: filopodia are restored.

Figure 4A shows a cell in 5 mmol/L glucose in an FCS gradient. Lamellipodia are present without an obvious leading edge or filopodia (insert). In Figure 4B, a cell in 25 mmol/L glucose in FCS gradient shows that direction-sensing filopodia are present on the lamellipodia at the leading edge of the cell. In Figure 4C, a cell in 25 mmol/L glucose treated with mevinolin in a FCS gradient shows actin cables but lacks filopodia (insert) along the extensive lamellipodium, which is not restricted to the leading edge. In Figure 4D, a cell exposed to 25 mmol/L glucose in FCS gradient and treated with mevinolin and geranylgeranylpyrophosphate is shown; filopodia are absent along the lamellipodium. In Figure 4E, a cell exposed to 25 mmol/L glucose and treated with mevinolin and farnesylpyrophosphate in a FCS gradient is shown. The cell has a distinct leading edge in the direction of the chemoattractant (FCS), with filopodia (insert) along the lamellipodium. It would therefore appear that Ras farnesylation is a prerequisite for formation of filopodia in VSMC under these experimental conditions.

	Discussion

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The results clearly show that both the p110ß-PI3K and MEK1/2 MAPK pathways are involved in glucose-potentiated VSMC chemotaxis to serum and that there is significant cross-talk between the 2 signaling pathways with PI3K influencing the MAPK pathway, but not vice versa. There was no evidence that the p38 and JNK pathways were involved in chemotaxis in these conditions.

The p110ß isoform of PI3K (but not p110 or p110) was required for glucose-potentiated VSMC chemotaxis to serum. The expression of p110, previously thought to be confined to a few cell types,^5,6,8 has been found here for the first time to our knowledge in human aortic VSMC. Our observation that p110ß, but not p110 or p110, mediates glucose-potentiated VSMC chemotaxis to serum indicates that these isoforms are not functionally redundant, and it is confirmed by previous studies of the roles of the p110 isoforms.^6,8–10 However, although p110 isoforms play distinct roles, these vary with cell type and stimuli. For example, in porcine aortic endothelial cells, PDGF-stimulated actin reorganization is p110-dependent, whereas insulin-induced actin reorganization is p110ß-dependent.¹⁰

PI3K inhibitors also reduced glucose-induced phosphorylation of ERK1/2 and MAPK activity, showing that there was significant cross-talk between these 2 pathways. An important site of the cross-talk centered on Akt. This was shown in 2 sets of experiments: microinjection of Akt antibodies blocked chemotaxis, and microinjection of activated Akt into PI3K-inhibited cells resulted in phosphorylation of ERK. Conversely, inhibition of MEK1/2 did not affect Akt phosphorylation or activity.

Microinjection of antibodies to ERK1/2 prevented chemotaxis. Additionally, inhibitors of both MEK1/2 and PI3K resulted in reduced phosphorylation of ERK and reduced MAPK activity. Mevinolin, a member of the statin group of drugs that inhibits Ras, also inhibited chemotaxis and ERK1/2 phosphorylation, but not Akt phosphorylation. Similar observations were made with rapamycin, an mTOR inhibitor. Although mTOR is known to be PI3K-dependent,²⁸ it is not clear how mTOR relates to the PI3K–Akt pathway with respect to VSMC chemotaxis. The effect of rapamycin on VSMC migration has been shown, in part, to be p27^Kip1-dependent.²⁹ However, the complete signaling pathway that links mTOR to VSMC migration is not yet fully delineated.³⁰ PI3K inhibitors and rapamycin inhibited ERK activation to a similar degree.

There have been conflicting reports in the literature of the importance of PI3K and MAPK in VSMC chemotaxis. Cospedal et al³¹ found that PDGF-mediated chemotaxis of VSMC was dependent on MAPK but not PI3K. This was unexpected because PI3K has been implicated in cell migration in numerous cell types and with various stimuli, whereas MAPK has often been associated with proliferation but not chemotaxis.³¹ A few studies have investigated both effects under the same experimental conditions. Nelson et al³² found that when VSMC were stimulated with PDGF, MAPK-dependent migration occurred much more rapidly than did MAPK-dependent proliferation, suggesting distinct phases of enzyme activation for these different cellular phenomena, a point of view supported by other work showing a long lag phase for glucose effects on VSMC, such as thymidine uptake^33,34 or cell multiplication.³⁵ We also have found that VSMC replication does not significantly change until after 8 to 10 days in 25 mmol/L glucose,³⁵ and thymidine uptakes in 5 or 25 mmol/L glucose conditions are similar over the course of 72 hours (unpublished results). Chemotaxis induced by thrombospondin-1 has also been found to be both PI3K-dependent³⁶ and MAPK-dependent.³⁷ It is of interest that in the other important cell of the vascular wall, the endothelial cell, glucose activates JNK but not ERK1/2 or p38, and it is associated with a reduction in endothelial cell proliferation.³⁸ As further evidence of the diversity of glucose-related chemotactic responses, it has been shown that neural crest cell migration in rat embryos is inhibited by elevated glucose concentrations.³⁹

The inhibitory effect of mevinolin on chemotaxis was of particular interest. Statin drugs are in widespread use for the reduction of cholesterol levels; their primary action is to inhibit the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase). It has become clear that this group of drugs has additional effects that can be ascribed to the reduction of intermediates in the cholesterol synthesis pathway downstream of HMG CoA reductase,^40–42 which are required for prenylation of small GTPase proteins. A significant proportion of subjects with diabetes have dyslipidemia that is treated with a drug of the statin group. It seems probable that in those patients, statins may help to prevent the development of atherosclerosis by a lipid-lowering action and by interruption of the signaling pathway involved in glucose-enhanced chemotaxis of VSMC from the tunica media to the subendothelial space of the arterial wall. In this study, the statin influence on chemotaxis could be detected at concentrations as low as 1 nmol/L and was mediated via Ras rather than via the Rho family of GTPases. This was deduced from the fact that addition of farnesylpyrophosphate, required for the prenylation of Ras, and not geranylgeranylpyrophosphate, required for prenylation of Rho A, restored chemotaxis in VSMC treated with mevinolin. Furthermore, the use of a farnesyltransferase inhibitor in the absence of a statin reduced ERK phosphorylation and prevented chemotaxis. By contrast, in neonatal rat cardiomyocytes, statins have been shown to downregulate Rac1 and Rho A.⁴³ In addition to these mechanisms, a previous study⁴⁰ suggested that statins may also inhibit chemotaxis through an associated inhibition of oxidative stress. Another area of variability in statin action relevant to the present study is the effects on the PI3K pathway. It is clear that mevinolin neither activated nor suppressed PI3K activity under the conditions of the present investigations. Different statins and conditions are associated with different activation profiles. Atorvastatin treatment during oxidative stress associated with reperfusion injury in mouse myocardium is associated with increased phosphorylation of Akt.⁴⁴ Simvastatin treatment reduces PDGF-induced Akt phosphorylation in the aortas of low-density lipoprotein receptor-deficient mice subjected to coronary angioplasty.⁴⁵ Pravastatin (100 µmol/L) has been shown to inhibit PI3K and phosphorylation of Akt in the A10 aortic VSMC cell line.⁴⁶ We have observed a significant reduction in glucose-induced Akt phosphorylation in primary VSMC with concentrations of pravastatin as low as 10 nmol/L (ERK phosphorylation is reduced at 1 nmol/L), and pravastatin blocks glucose-potentiated chemotaxis to serum (unpublished). Thus, there are differences in the inhibitory action of statins on the PI3K pathway, even under the same experimental conditions of glucose-potentiated chemotaxis to serum.

In conclusion, this study has shown that glucose-potentiated VSMC chemotaxis to serum involves the p110ß catalytic subunit of PI3K and the ERK1/2 MAPK pathway, and that mTOR and Ras are involved. The results reveal significant cross-talk between the PI3K and MAPK pathways, with PI3K/Akt having a major influence on the extent of activation within the ERK1/2 pathway in VSMC. Statins, a widely used group of drugs in diabetic subjects with dyslipidemias, block glucose-dependent chemotaxis of VSMC.

	Acknowledgments

 
This work was supported by the R&D Office, Department of Health, Social Services and Public Safety, Northern Ireland, by the Irish Endocrine Society (E.R.T. and W.E.A.), and by Diabetes UK (grant RD01:0002179 to B.V.).

	Footnotes

 
Original received February 26, 2004; revision received June 18, 2004; accepted June 24, 2004.

	References

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