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Am J Physiol Heart Circ Physiol 288: H2861-H2868, 2005. First published February 11, 2005; doi:10.1152/ajpheart.00561.2004
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Rapamycin inhibits fibronectin-induced migration of the human arterial smooth muscle line (E47) through the mammalian target of rapamycin

Kenji Sakakibara,* Bo Liu,* Scott Hollenbeck, and K. Craig Kent

Division of Vascular Surgery, Department of Surgery, New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, New York

Submitted 9 June 2004 ; accepted in final form 4 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The matrix protein fibronectin (FN) is a potent agoinst of vascular smooth muscle cell (SMC) migration. The role of rapamycin and the mammalian target of rapamycin (mTOR) in matrix protein-induced migration has not yet been defined. In these studies, we found that rapamycin (10 nM) markedly diminished chemotaxis of E47 cells (a cell line derived from human atherosclerotic plaques) and rat aortic SMCs toward FN as well as type I collagen and laminin; however, a period of preincubation >20 h was required. Subsequently, we showed that treatment with FN induced a rapid activation of mTOR as well as its downstream effector, S6 kinase (S6K). Moreover, FN-induced activation of both proteins was inhibited by preincubation with rapamycin for only 30 min. We then explored the upstream signaling pathway through which FN might mediate mTOR activation. A blocking antibody to {alpha}v{beta}3 inhibited FN-induced mTOR/S6K activation as well as E47 cell chemotaxis, implicating {alpha}v{beta}3 as the integrin receptor responsible for initiating FN-induced migration. Moreover, preincubation of E47 cells with wortmannin or LY-294002 blocked FN-induced mTOR/S6K activation, demonstrating that phosphatidylinositol 3-kinase (PI3K) plays a critical role in this rapamycin-sensitive signaling pathway. It has been previously suggested that rapamycin's effect on migration maybe related to enhancement of p27kip1. However, treatment of E47 cells with rapamycin did not alter the level of p27kip1 in the presence or absence of FN. Taken together, our data demonstrate that rapamycin inhibits FN-induced SMC migration through a pathway that involves at least {alpha}v{beta}3-integrin, PI3K, mTOR, and S6K.

S6 kinase; phosphatidylinositol 3-kinase; integrin; p27kip1; extracellular matrix proteins

RAPAMYCIN (sirolimus), initially developed as antibiotic and then as an immunosuppressant, has recently been used in drug-eluting stents to prevent restenosis. In several clinical trials, these stents have reduced the rate of significant restenosis from ~30 to <5% (25, 33, 35). The mechanism that underlies rapamycin's potent effect on intimal hyperplasia is not yet completely understood; however, it is believed rapamycin's action may be in part mediated through a direct effect on vascular smooth muscle cells (SMCs) (20).

The inhibitory effect of rapamycin on vascular SMC proliferation has been well established. Both in vitro and in vivo, rapamycin inhibits cell cycle progression of vascular SMCs from G1 to S (6, 13, 22); this effect may be related to an induction of p27kip1, a cyclin-dependent kinase (CDK) inhibitor (20). Rapamycin has also been found to inhibit migration of cultured SMCs. Treatment of vascular SMCs with rapamycin diminishes the ability of SMCs to migrate toward the potent chemokine platelet-derived growth factor (PDGF) (29).

Rapamycin's cellular effects appear to be mediated through the intracellular protein target of rapamycin (TOR). TORs were first identified in Saccharomyces cerevisiae during a genetic screening for mutants resistant to growth inhibition by rapamycin (10). TOR and its mammalian homolog (mTOR) are members of a unique family of protein kinases that contain domains similar to lipid kinases such as phosphatidylinositol 3-kinase (PI3K) (14). Several key protein translation regulators, including p70 ribosomal S6 kinase (S6K), are postulated as downstream effectors of mTOR. S6K is a mitogen-activated Ser/Thr kinase that controls translation of a unique family of mRNAs, presumably by inducing multiple phosphorylations of the 40 S ribosomal protein S6 (4). Selective inhibition of mitogen-induced mTOR-dependent activation of S6K by rapamycin impedes cell growth (4). However, the precise link among mTOR, S6K, and cell cycle regulators remains unclear.

Accompanying vascular injury are significant changes in the composition of the extracellular matrix (ECM) of the arterial wall. Levels of fibronectin (FN) are increased; FN, in turn, is a potent activator of SMCs (28). We and others (26, 40) have previously demonstrated that ECM proteins, including FN, are potent stimulants of vascular SMC chemotaxis. The effect of these matrix proteins can be at least equivalent to that of the prototypical chemokine PDGF-BB (26). Stimulation of cellular migration by ECM proteins is mediated by a diverse class of heterodimeric {alpha}{beta}-membrane receptors known as integrins (7, 11, 12). Although many growth factors and ECM proteins induce SMC migration, the signaling pathways used by these two classes of agonists can be distinct. For example, PDGF-induced chemotaxis requires the signaling protein phospholipase C (PLC) but not PI3K, whereas ECM protein-induced chemotaxis requires PI3K but not PLC (17). The finding that rapamycin inhibits PDGF-induced SMC chemotaxis implies mTOR as an essential signaling step in PDGF-induced chemotaxis. Whether a rapamycin-sensitive pathway is necessary for ECM protein-induced chemotaxis is not known. In this study, we first assessed the effect of rapamycin on FN-induced SMC migration. We then define the rapamycin-sensitive signaling pathway through which FN affects migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
General materials. Rapamycin was obtained from Calbiochem (La Jolla, CA). PDGF-BB was obtained from Upstate Biotechnologies (Lake Placid, NY). Cell culture reagents were obtained from GIBCO-BRL Life Technologies (Gaithersburg, MD). Bovine FN and other chemicals if not otherwise specified were purchased from Sigma (St. Louis, MO).

Antibodies. Mouse antihuman {alpha}2{beta}1 and {alpha}v{beta}3 blocking monoclonal antibodies were purchased from Chemicon (Temecula, CA). Rabbit antihuman mTOR, phospho-mTOR (Ser2448), S6K, and phospho-S6K (Thr389) were obtained from Cell Signaling (Beverly, MA). p27kip1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

SMC culture. E47 cells, a generous gift from Dr. T. McCaffrey (The George Washington University Medical Center), are a spontaneous cell line originally isolated from a human carotid endarterectory specimen. In addition to expressing {alpha}-actin and calponin, E47 cells behave physiologically similarly to human saphenous vein SMCs in terms of proliferation and migration (B. Liu and K. C. Kent, unpublished observations). E47 cells were maintained in199 Earle's minimal essential medium (medium 199) supplemented with 10% FBS at 37°C with 5% CO2.

Rat aortic SMCs were isolated from the thoracic aorta of male Sprague-Dawley rats based on a protocol described by Clowes et al. (2) and maintained in DMEM containing 10% fetal FBS at 37°C with 5% CO2. The use of animals for this research was approved by the Institutional Review Board at Weill Cornell Medical.

Chemotaxis and attachment assays. Cells were made quiescent by incubation in media containing 0.5% FBS for 48 h. Cells were harvested using 0.05% trypsin-EDTA and resuspended in media supplemented with 0.5% FBS. Chemotaxis assays were performed at 37°C using a 24-well transwell containing a polycarbonate filter with 5-µm (E47 cells) or 8-µm (rat aortic SMCs) pores (Poretics; Livermore, CA). Cells, control or pretreated with an inhibitor, were seeded in the upper well at a density of 40,000 cells/well. FN or other matrix proteins, diluted in 0.5% FBS media, were added to the lower well. After a 2-h (E47 cells) or 6-h (rat aortic SMC) incubation, membranes were removed, fixed in 70% ethanol at –20°C for 30 min, and stained with hematoxylin at room temperature for 30 min. The upper side of the membrane was scraped using a cotton swab to remove cells that had attached but not migrated. The membrane was then mounted onto a microscope slide. Chemotaxis was assessed by counting the number of cells that migrated in five independent high-power fields (x200 magnification). For the cell attachment assay, E47 cells were seeded to top wells at a density of 10,000 cells/well. Fewer cells than the migration assay were used to allow more accurate cell counting. After a 30-min incubation, membranes were removed, fixed, and stained as noted above. The lower side of the membrane was scraped to remove any cells that might have migrated. Cell attachment was evaluated by counting the number of cells that attached to the upper surface of the membrane.

Immunoblotting. Cells were starved with or without rapamycin (10 nM) for 48 h and then stimulated with FN or PDGF-BB for the indicated times. Cells were lysed in radioimmunoprecipitation assay buffer [25 mM Tris·HCl (pH 7.4), 25 mM NaCl, 1 mM sodium orthovanadate,10 mM sodium pyrophosphate, 10 mM NaF, 0.5 mM EGTA, 1.0% Triton X-100, 1 mM PMSF, and 10 mM okadaic acid] for 60 min at 4°C. Samples were then centrifuged for 20 min at 4°C. The total protein concentration of the resulting supernatant was determined by a modification of the method of Lowry. Equal amount of protein extracts (50–75 µg) were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and blotted with antibodies. Labeled proteins are visualized with an ECL system (Amersham Biosciences; Piscataway, NJ). Band intensity was determined using densitometric analysis (NIH Image software). The degree of mTOR or S6K activation was expressed as the ratio of phospho-mTOR to total mTOR or phospho-S6K to total S6K. The level of p27kip was expressed as its ratio to {beta}-actin.

Statistical analysis. Values are expressed as means ± SE. An unpaired Student's t-test was used to evaluate the statistical differences between the control and treated groups. Values of P < 0.05 were considered significant. All experiments were repeated at least three times.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Rapamycin inhibits FN-induced SMC migration. To investigate whether rapamycin affects vascular SMC migration induced by matrix proteins, we used FN as our prototype. An elevated level of FN is found in hyperplastic plaques, and we and others (26, 40) have demonstrated that FN is a potent agonist of SMC migration. E47 cells, a human arterial SMC line derived from a carotid plaque, were made quiescent by incubation in 0.5% serum for 48 h. Chemotaxis of E47 cells toward soluble FN was determined using a transwell assay. In the absence of FN, only a few E47 cells migrated through the porous membrane. The addition of FN (20 µg/ml) produced a two- to fivefold increase in SMC chemotaxis, which is comparable to the fold induction in chemotaxis that we have previously observed with PDGF (data not shown). To evaluate the effect of rapamycin on chemotaxis, we preincubated E47 cells with rapamycin (10 nM for 48 h), a concentration that has preciously been shown to inhibit PDGF-induced chemotaxis. Rapamycin produced no significant effect on basal migration. However, rapamycin completely eliminated FN-induced chemotaxis (Fig. 1A).



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  		Fig. 1. Rapamycin inhibits fibronectin (FN)-induced smooth muscle cell (SMC) chemotaxis. A: E47 cells were preincubated with rapamycin (10 nM) for 48 h, and SMC chemotaxis toward FN (20 µg/ml) was determined using a transwell assay (*P < 0.05, n = 4). B: FN-induced chemotaxis of E47 cells preincubated with rapamycin for the indicated times (*P < 0.05, n = 3). C: rat aortic SMCs were incubated with rapamycin (10 nM) for 48 h. Chemotaxis toward FN (20 µg/ml), type I collagen (1 µg/ml), or laminin (20 µg/ml) was determined using a transwell assay (*P < 0.05, n = 3). HPF, high-powered fields.

 
After establishing that preincubation with rapmycin for 48 h inhibited E47 cell chemotaxis, we then investigated whether shorter periods of incubation might yield a similar effect. In a second series of experiments, chemotaxis was evaluated in E47 cells preincubated with rapamycin (10 nM) for 0.5, 6, 12, 20, 24, or 48 h. FN-induced chemotaxis was unaffected by pretreatment of cells with rapamycin for 20 h or less, whereas inhibition occurred when cells were preincubated for 24 and 48 h (Fig. 1B). These results demonstrate that rapamycin's effect on migration requires a prolonged period of pretreatment. To exclude the possibility that the inhibition of chemotaxis was related to cell death, we assessed cell viability with trypan blue. There were no differences in cell viability between vehicle-treated control and rapamycin-treated cells at any time point. We also evaluated whether prolonged rapamycin treatment might affect cell attachment. Control or rapamycin-treated (10 nM, 48 h) E47 cells were seeded to transwells. After 30 min of incubation, cell attachment was assayed. There were no significant differences in attachment between control and rapamycin-treated cells (control 349 ± 32 cells/well vs. rapamycin 359 ± 56 cells/well). These observations demonstrate that findings from chemotaxis assays were solely dependent on cell migration and not cell attachment.

Because E47 is a cell line derived from a human carotid plaque, we queried whether rapamycin's effect on FN-induced chemotaxis could be generalized to other types of SMCs. To accomplish this, we treated rat aortic SMCs (passage 3) with rapamycin (10 nM) for 48 h in the presence of 0.5% FBS. Chemotaxis was assessed as described. Rapamycin significantly inhibited chemotaxis of rat aortic SMCs toward FN (Fig. 1C). Interestingly, the degree of inhibition caused by rapamycin was less in primary rat aortic SMCs, which could perhaps reflect a different rapamycin sensitivity between these two types of SMCs or differences in the culture environment. Furthermore, to address whether the inhibitory effect of rapamycin is unique to FN versus other matrix proteins, we induced chemotaxis with type I collagen as well as laminin, two matrix proteins that we have previously shown to stimulate migration of human vascular SMCs (26). Type I collagen (1 µg/ml) stimulated chemotaxis of rat aortic SMCs in a fashion similar to that of FN, whereas laminin (20 µg/ml) also produced chemotaxis but to a lesser degree (Fig. 1C). Importantly, incubation with rapamycin (10 nM) for 48 h significantly inhibited chemotaxis induced by both type I collagen as well as laminin (Fig. 1C). Taken together, our data demonstrate that rapamycin affects SMC chemotaxis in response to a variety of matrix proteins.

FN activates mTOR and S6K in E47 cells. Because mTOR has been identified as the mammalian target of rapamycin, we postulated that mTOR might be a critical element of the signaling pathway that facilitates FN-induced chemotaxis. To test this hypothesis, we evaluated mTOR activity in E47 cells stimulated with FN. E47 cells were seeded at a density of 7.0 x 105 cells/100-mm dish (80% confluent) and made quiescent by incubation in media containing 0.5% FBS for 48 h. FN (0–40 µg/ml) was then added directly to the culture supernatant. After a 30-min incubation, cells were lysed. Activation of mTOR in E47 cell lysates was measured by Western blott analysis with an antibody that recognizes the phosphorylated form of mTOR (phosphorylation at Ser2448 is essential for mTOR activation). As shown in Fig. 2A, an increase in mTOR phosphorylation was evident in cells treated with 1 µg/ml FN, but more prominent induction was obtained with higher concentrations. In parallel experiments, we found that FN (1–40 µg/ml) stimulated E47 SMC chemotaxis in a similar dose-dependent fashion (data not shown). PDGF-BB, a potent stimulant of SMC chemotaxis, has been previously shown to activate mTOR in vascular SMCs. We therefore compared activation of mTOR by FN with that of PDGF. As shown in Fig. 2, B and C, 20 µg/ml FN produced a 1.94 ± 0.31-fold increase in phosphorylation of mTOR. This effect was similar to the 1.83 ± 0.25-fold increase in phosphorylated mTOR produced by PDGF-BB (25 ng/ml). Total mTOR protein levels were not altered by either FN or PDGF (Fig. 2, B and C). We next tested whether the concentration of rapamycin shown to inhibit FN-induced chemotaxis was able to affect FN-induced phosphorylation of mTOR. Pretreated E47 cells with rapamycin (10 nM) for 48 h completely eliminated phosphorylation of mTOR induced by FN (Fig. 2B).



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  		Fig. 2. Rapamycin inhibits FN-induced activation of mammalian target of rapamycin (mTOR). A: 80% confluent, growth-arrested E47 cells were stimulated with FN (0–40 µg) for 30 min. Cell lysates were then immunobloted with anti-phospho-mTOR or anti-mTOR antibodies. B and C: subconfleunt, growth-arrested E47 cells were preincubated with rapamycin (10 nM) for 48 h and then stimulated with FN (20 µg/ml) or PDGF-BB (25 ng/ml) for 30 min. Cell lysates were then immunobloted with anti-phospho-mTOR or anti-mTOR antibodies. B: representative blot; C: densitometry analysis (n = 4). *P < 0.05 compared with unstimulated, nonrapamycin-treated control; **P < 0.05 compared with FN- or PDGF-stimulated, nonrapamycin-treated cells.

 
Next, we tested whether FN activates S6K, a major downstream effector of mTOR, using a monoclonal antibody that recognizes phospho-S6K (Thr389). We found that FN produced a similar dose-dependent induction of S6K phosphorylation (Fig. 3A). Specifically, FN (20 µg/ml for 30 min) induced an ~200% increase of phospho-S6K; this was comparable to the ~190% increase in phospho-S6K by PDGF-BB (Fig. 3, B and C). Preincuabation with rapamycin (10 nM for 48 h) completely eliminated this effect (Fig. 3, B and C), supporting the importance of S6K in the rapamycin-sensitive signaling pathway responsible for FN-induced migration.



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  		Fig. 3. Rapamycin inhibits FN-induced activation of S6 kinase (S6K). A: subconfluent, growth-arrested E47 cells were stimulated with FN (0–40 µg) for 30 min. Cell lysates were then immunobloted with anti-phospho-S6K or anti-S6K antibodies. B and C: subconfluent, growth-arrested E47 cells were preincubated with rapamycin (10 nM) or solvent for 48 h and then stimulated with FN (20 µg/ml) or PDGF-BB (25 ng/ml) for 30 min. Cell lysates were then immunobloted with anti-phospho-S6k or anti-S6k antibodies. B: representative blot; C: densitometry analysis (n = 4). *P < 0.05 compared with unstimulated, nonrapamycin-treated control; **P < 0.05 compared with FN- or PDGF-stimulated, nonrapamycin-treated cells.

 
In our initial studies, we found that inhibition of migration by rapamycin required pretreatment of E47 cells for periods >20 h. Consequently, we evaluated the length of preincubation required to inhibit mTOR and S6K. Interestingly, only 30 min of preincubation with rapamycin was sufficient to eliminate FN-induced activation of mTOR or S6K (Fig. 4). These data indicate that rapamycin has an immediate effect on the FN-induced mTOR/S6K activation but a delayed effect on migration. The combination of these findings suggests that the pathway beyond S6K may involve a delayed process such as protein synthesis or degradation.



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  		Fig. 4. Pretreatment with rapamycin for 30 min was sufficient to inhibit FN-induced activation of mTOR as well as S6K. Subconfluent, growth-arrested E47 cells were preincubated with rapamycin (10 nM) for 30 min and then stimulated with FN (20 µg/ml) or PDGF-BB (25 ng/ml) for 30 min. Cell lysates were then immunobloted with anti-phospho-mTOR/anti-mTOR antibodies (A) or anti-phospho-S6K/anti-S6k antibodies (B).

 
PI3K mediates mTOR/S6K activation. PI3K is a family of enzymes that phosphorylate the 3'-hydroxyl group of phosphatidylinositol, producing the second messengers phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. To determine whether PI3K is required for FN-induced mTOR and S6K activation, we evaluated the effect of inhibition of PI3K on activation of these two proteins. Serum-starved E47 cells were preincubated with specific inhibitors of PI3K, wortmannin (50 nM) and LY-294002 (25 µM), for 30 min before FN treatment. Both inhibitors, at these concentrations, have been shown to selectively inhibit PI3K activity (30, 39). We found that pretreatment with wortmannin or LY-294002 completely abolished activation of mTOR and S6K in response to FN, indicating that PI3K is necessary for mTOR/S6K activation (Fig. 5, A and B). We next evaluated the requirement of PI3K for FN-induced E47 cell migration. We found that inhibition of PI3K completely (LY-294002) or markedly (wortmannin) blocked FN-induced chemotaxis (Fig. 5C). These data suggest a critical role for PI3K in FN-induced migration as well as activation of mTOR and S6K.



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  		Fig. 5. Phosphatidylinositol 3-kinase (PI3K) is necessary for FN-induced mTOR/S6K activation as well as chemotaxis. Subconfluent, growth-arrested E47 cells were preincubated with LY-294002 (25 µM) or wortmannin (50 nM) for 30 min and then stimulated with FN (20 µg/ml) for 30 min. Cell lysates were immunobloted with anti-phospho-mTOR/anti-mTOR (A) or anti-phospho-S6K/anti-S6K (B). C: chemotaxis toward FN (20 µg/ml) in the presence or absence of LY-294002 or Wortmannin was determined using a transwell assay (*P < 0.05).

 
Activation of the mTOR/S6K pathway is dependent on {alpha}v{beta}3-integrin. Using Western blot analysis, we detected multiple FN-binding integrin subunits in E47 cells, including {alpha}2, {alpha}3, {alpha}v, {beta}1, and {beta}3, but not {alpha}5. On the basis of the availability of blocking antibodies, we chose to evaluate the role of {alpha}v{beta}3, an established receptor for FN expressed in both arterial and venous vascular SMCs (12, 23), in FN-induced activation of mTOR/S6K. We found that the {alpha}v{beta}3 blocking antibody, at a dilution of 1:100, significantly inhibited FN-induced phosphorylation and thus activation of mTOR (Fig. 6A). In contrast, neither control IgG nor a blocking antibody to {alpha}2{beta}1 (a collagen-binding integrin) mimicked this effect (Fig. 6A).



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  		Fig. 6. {alpha}v{beta}3-Integrin mediates FN-induced mTOR/S6K activation as well as chemotaxis. A: subconfleunt, growth-arrested E47 cells were preincubated with IgG or blocking antibodies to {alpha}2{beta}1 or {alpha}v{beta}3 for 1 h and then stimulated with FN (20 µg/ml) for 30 min. Cell lysates were analyzed for anti-phospho-mTOR or anti-total-mTOR. A blot representative of 3 experiments is shown. B: subconfluent, growth-arrested E47 cells were pretreated with IgG or blocking antibodies for 1 h. Chemotaxis toward FN (20 µg/ml) was determined using a transwell assay. *P < 0.05 compared with FN-stimulated control.

 
After establishing a role for {alpha}v{beta}3 in FN-induced activation of mTOR/S6K, we then evaluated the importance of this integrin in FN-induced migration. We found that a blocking antibody to {alpha}v{beta}3 produced a 63.5 ± 3.1% decrease in chemotaxis of E47 cells toward FN, whereas control IgG and a blocking antibody to {alpha}2{beta}1 had no effect (Fig. 6B). Taken together, our data demonstrated that {alpha}v{beta}3-integrin is necessary for activation of mTOR/S6K as well as migration of vascular E47 cells in response to FN.

p27kip1 is not involved in rapamycin's inhibition of FN-induced migration. p27kip1 is a CDK inhibitor that has been previously shown to block SMC proliferation (1, 37). p27kip1 has also been observed to be upregulated by rapamycin, and this mechanism may be in part responsible for rapamycin's effect on SMC proliferation (18). It has been postulated that p27kip1 might also mediate rapamycin's effect on SMC migration. Evidence for this was produced by Sun and colleagues (36), who found that rapamycin (10 nM) failed to inhibit PDGF-induced chemotaxis of vascular SMCs isolated from mice lacking the gene encoding p27kip1.

We postulated that p27kip1 might also play a role in rapamycin's effect on FN-induced SMC migration. We therefore hypothesized that rapamycin might increase levels of p27kip1 in vascular SMCs or, alternatively, that FN-induced mTOR activity might diminish levels of p27kip1. We first confirmed previous observations with PDGF by demonstrating that treatment of SMCs with PDGF-BB (25 ng/ml) resulted in an ~50% reduction of p27kip1 protein (data not shown). However, under similar circumstances, stimulation of E47 cells with FN did not alter levels of p27kip1 (Fig. 7). To test whether rapamycin enhanced levels of p27kip1, we treated cells with rapamycin (10 nM) for 48h before the addition of FN or solvent. Rapamycin did not alter the level of p27kip1 in either the presence or absence of FN (Fig. 7). These data suggest that p27kip1 does not play a role in the FN/mTOR/migration signaling pathway, nor is inhibition of FN-induced E47 cell migration by rapamycin mediated by the induction of p27kip1.



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  		Fig. 7. Neither rapamycin nor FN affect protein levels of p27kip. E47 cells were preincubated with rapamycin (10 nM) for 48 h and then stimulated with FN (20 µg/ml) for 30 min. Cell lysates were immunobloted with anti-p27 or {beta}-actin antibodies. A: representative blot; B: densitometry analysis of 3 separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although rapamycin-coated stents appear to be effective in the prevention of intimal hyperplasia after coronary angioplasty, the precise mechanism underlying rapamycin's action has not been fully elucidated. An effect of rapamycin on SMC migration was first described by Poon and colleagues (29). These investigators found that PDGF-induced chemotaxis was markedly inhibited by preincubation of SMCs with rapamycin. Our findings now further this observation in that rapamycin inhibits SMC chemotaxis in response to the matrix protein FN as well as type I collagen and laminin. FN is prevalent after vascular injury. The accumulation of FN in the neoinima is noted after balloon injury, especially in the early stages of intima development, i.e., before SMCs have moved through the internal elastic lamina (38). In studies of human vascular SMCs, we (26) have previously shown that chemotaxis in response to matrix proteins including FN is at least equivalent if not greater than that produced by PDGF. Thus the ability of rapamycin to inhibit SMC migration induced by matrix proteins provides yet another important mechanism through which this inhibitor produces its overall effect on the development of intimal hyperpalasia.

We then investigated the rapamycin-sensitive pathway used by FN to stimulate vascular SMC migration. We found that FN rapidly activates mTOR in the E47 human vascular SMC line as well as its downstream effector S6K. Moreover, activation of mTOR and S6K is dependent on {alpha}v{beta}3-integrin as well as the signaling intermediate PI3K. Our finding that rapamycin blocks FN-induced activation of mTOR and S6K as well as migration suggests that mTOR and S6K play important role in regulating ECM protein-induced SMC migration. To our knowledge, this is the first study that directly establishes mTOR and S6K as regulators of SMC migration induced by ECM proteins. A relationship between matrix proteins and S6K has been explored in other cell types. Malik and Parsons (19) showed that adhesion of REF 52 cells to FN increased the activity of S6K. More recently, Levy et al. (15) demonstrated that gelectin-8 (a mammalian lectin) as well as FN induced phosphorylation of S6K in Chinese hamster ovary cells.

Our findings regarding the kinetics of rapamycin's effect on FN-induced SMC migration are worthy of discussion. An effect of rapamycin on E47 cell chemotaxis was not evident unless cells were exposed to this inhibitor for >20 h. A similar period of preincubation has also been shown to be necessary for rapamycin's inhibitory effect on PDGF-induced SMC chemotaxis (29, 36). Rapamycin can rapidly penetrate the cell membrane (31). Moreover, we observed rapid inhibition of the mTOR-dependent phosphorylation of S6K by rapamycin. Nevertheless, despite inhibition of mTOR/S6K by 30 min, >20 h of continuous incubation with rapamycin was necessary to prevent FN-induced E47 cell SMC migration. This delayed effect of rapamycin on cell migration seems to be cell specific. Incubation of neutrophils with rapamycin at 10 nM (the same concentration used in our study) for only 30 min was sufficient to inhibit chemotaxis of these cells (8). One possible explanation for the delayed effect of rapamycin in SMCs is that rapamycin alters chemotaxis indirectly, either by inhibiting synthesis of a protein factor that is necessary for migration or by inducing a protein factor that negatively regulates migration (Fig. 8). Rapamycin is known to inhibit protein translation in many types of cells; the mechanism appears to be through inhibition of S6K as well as the eukaryotic initiation factor 4E (5). In bovine aortic SMCs, 48-h incubation with rapamycin has been reported to inhibit total protein synthesis by 30% (21). In separate experiments, we also observed a similar reduction in total protein levels after treating E47 cells with rapamycin (data not shown). This observation raises the possibility that among the proteins, the syntheses of which are diminished by rapamycin, is one or more key factors needed for SMC migration. Alternatively, rapamycin, through S6K, could affect SMC migration by inducing a protein factor that inhibits cell migration. Converse to its inhibitory effect on total protein synthesis, rapamycin has recently been reported to increase the expression of certain proteins such as the vascular SMC specific markers ({alpha}-actin, calponin, and SMC myosin heavy chain) (21).



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  		Fig. 8. Simplified schematic of FN signaling pathways in E47 cells.

 
Our data also suggest that this delayed rapamycin-sensitive FN/mTOR/S6K pathway is distinct from the more immediate and direct signaling sequence that initiates migration. Chemotaxis can be observed as early as 30 min after stimulation of E47 cells with FN. Our data suggest that {alpha}v{beta}3-integrin and PI3K are both necessary for this rapid E47 chemotaxis in response to FN. We did not evaluate the importance of additional integrin receptors, but it is possible that others are also needed. We were surprised to find the critical necessity of {alpha}v{beta}3 because in previous experiments we had found this integrin to not be important in saphenous vein SMC chemotaxis in response to FN. This difference in requirement for {alpha}v{beta}3 may reflect a physiological difference between arterial versus venous SMCs or a difference between cells isolated from normal vessel (saphenous vein) versus cells originated from a carotid plaque (E47). Although in the present study we did not specifically explore the downstream signaling proteins required for the more rapid or immediate pathway leading to SMC migration, we have previously shown that both ERK and the small G protein Rho are critical elements (16, 27) (see Fig. 8). Interestingly, {alpha}v{beta}3 and PI3K are also necessary for activation of the mTOR (rapamycin sensitive) pathway that also facilitates FN-induced migration. PI3K activates mTOR and S6K and perhaps eventually leads to manipulation of protein synthesis or degradation (Fig. 8). Although the rapamycin-sensitive signaling pathway that we have described does not appear to be prerequisite for the rapid initiation of chemotaxis, activation of this pathway may well be necessary to maintain cell movement. These two pathways may be interrelated in that a critical protein altered by S6K may be a necessary component of the more immediate signaling response. The importance of S6K/protein synthesis and cell migration has been preciously explored. Crouch et al. (3) postulated a role for S6K in the regulation of mobility of Swiss 3T3 cells based on the finding that S6K associates with stress fibers in these cells.

Using p27kip1-defecient mice, Sun and colleagues (36) found that the loss of p27kip1 diminished rapamycin's ability to inhibit PDGF-induced chemotaxis. These findings suggest that rapamycin's effect on cell migration may be mediated by the induction of p27kip1. Further supporting this hypothesis, overexpression of p27kip1 in endothelial cells has been shown to inhibit migration (9). With these provocative findings, we explored whether the induction of p27kip1 might mediate the inhibitory effect of rapamycin in FN-induced chemotaxis. We found that treatment of E47 cells with FN did not alter levels of p27kip1. Moreover, preincubation of E47 cells with 10 nM rapamycin, a concentration that inhibited chemotaxis, had no effect on the level of cellular p27kip1. Therefore, rapamycin's effect on FN-induced SMC chemotaxis appears to not be mediated by p27kip1. The data suggesting p27kip1 as an effector for rapamycin are not conclusive. Roque et al. (34) showed that deletion of the p27kip1 gene did not influence rapamycin's antiproliferative and antimigratory effects in a vascular model of intimal hyperplasia. Moreover, McAllister et al. (24) found that application of TAT-tagged p27kip1 protein to HepG2 cells increased rather than inhibited cell migration.

In addition to their function as chemokines, matrix proteins are known to support and modify many SMC cellular processes including growth, survival, and differentiation. Therefore, matrix proteins as well as the proteases that degrade them are thought to be critical in the pathogenesis of atherosclerosis and restenosis (32). FN, which is elevated in early atherosclerotic and restenotic lesions, produces signals that lead to migration as well as survival and proliferation (28) of vascular SMCs. By blocking mTOR signaling, rapamycin may inhibit the effects of both growth factors and matrix proteins on SMC migration and proliferation.

In summary, our results demonstrate that the matrix protein FN rapidly activates mTOR and S6K in E47 SMCs. This activation is mediated, at least in part, by {alpha}v{beta}3-integrin and by PI3K. Blocking by rapamycin, of a pathway that includes FN -> {alpha}v{beta}3 -> PI3K-> mTOR -> S6K and a manipulation of protein synthesis, leads to inhibition of FN induced migration. These data demonstrate yet another mechanism by which rapamycin can modify the cellular events that lead to intimal hyperplasia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grant HL-68673 (to K. Kent and B. Liu), NIH Training Grant T32 GM-08466 (to S. Hollenbeck), an American Association of Surgeons Resident Research Scholarship (to S. Hollenbeck), and the Atorvastatin Research Award (to B. Liu) sponsored by Pfizer Incorporated.


   ACKNOWLEDGMENTS
 
The authors thank Dr. T. McCaffrey for the generous gift of E47 cells and Sophia Chu for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. C. Kent, Dept. of Surgery, New York Presbyterian Hospital, 525 E. 68th St., Payson 707, New York, NY 10021 (E-mail: kckent{at}med.cornell.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K. Sakakibara and B. Liu contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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