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VASCULAR BIOLOGY

Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 14 June 2005 ; accepted in final form 5 September 2005

	ABSTRACT

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To understand the mechanisms by which thrombin induces vascular smooth muscle cell (VSMC) DNA synthesis and motility, we have studied the role of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-S6K1 signaling. Thrombin stimulated the phosphorylation of Akt and S6K1 in VSMC in a sustained manner. Blockade of PI3K-Akt-mTOR-S6K1 signaling by LY-294002, and rapamycin suppressed both thrombin-induced VSMC DNA synthesis and migration. Adenovirus-mediated expression of dominant-negative Akt also inhibited thrombin-induced VSMC DNA synthesis and migration. Furthermore, thrombin induced the expression of Fra-1 in a sustained PI3K-Akt-dependent and mTOR-independent manner in VSMC. Suppression of Fra-1 by its small interfering RNA attenuated both thrombin-induced VSMC DNA synthesis and migration. Thrombin also induced the expression of FGF-2 in a PI3K-Akt-Fra-1-dependent and mTOR-independent manner, and neutralizing anti-FGF-2 antibodies inhibited thrombin-stimulated VSMC DNA synthesis and motility. In addition, thrombin stimulated the tyrosine phosphorylation of EGF receptor (EGFR), and inhibition of its kinase activity significantly blocked Akt and S6K1 phosphorylation, Fra-1 and FGF-2 expression, DNA synthesis, and motility induced by thrombin in VSMC. Together these observations suggest that thrombin induces both VSMC DNA synthesis and motility via EGFR-dependent stimulation of PI3K/Akt signaling targeting in parallel the Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation.

Phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-S6K1 signaling plays an essential role in cell survival (40, 45, 46). Akt, an effector of PI3K via phosphorylation of a number of molecules, mediates antiapoptotic, mitogenic, motogenic, and thrombotic effects in a variety of target cells in response to the respective external cues (1, 5, 10, 13, 15, 19, 40, 45, 46). Despite the involvement of Akt in mediating antiapoptotic, mitogenic, motogenic, and thrombotic signaling events, its role in the nonthrombotic effects of thrombin was either less understood or disputed. For example, it was reported that thrombin causes a transient and weak activation of Akt in VSMC, and this mode of Akt stimulation was attributed to modulation of the regulation of expression of genes such as smooth muscle-specific myosin heavy chain gene enduring redifferentiation of VSMC (35). In contrast, in other cell types such as Chinese hamster fibroblasts, thrombin activated Akt in a sustained and robust manner, and this mode of Akt stimulation was shown to be associated with the proliferative capacity of the cell (17). In fact, Akt has been shown to be involved in the regulation of Fra-1, a protooncogene that plays an important role in the mediation of cell motility and proliferation (2, 39). The inability of thrombin to stimulate Akt in a sustained manner in VSMC was even thought to be a cause for its lack of effect as a mitogen to this cell type (35). However, work from various laboratories, including ours (30), has shown that thrombin elicits both mitogenic and motogenic effects in VSMC (4, 17, 20, 26, 33). To understand the signaling events of its nonthrombotic effects in VSMC, we have studied the role of PI3K-Akt-mTOR-S6K1 signaling. We found that thrombin activates Akt and one of its effectors, S6K1, in a sustained and PI3K-dependent manner in VSMC. Inhibition of Akt and S6K1 activation by pharmacological and genetic approaches suppressed both the mitogenic and motogenic effects of thrombin in VSMC. Thrombin induced the expression of Fra-1 in a sustained PI3K-Akt-dependent and mTOR-independent manner in VSMC. Inhibition of expression of Fra-1 by its small interfering RNA (siRNA) attenuated both thrombin-induced VSMC DNA synthesis and motility. Thrombin also induced the expression of FGF-2 in a PI3K-Akt-dependent and mTOR-independent manner in VSMC. Neutralizing anti-FGF-2 antibodies suppressed both thrombin-induced VSMC DNA synthesis and motility. In addition, thrombin stimulated tyrosine phosphorylation of EGF receptor (EGFR), and inhibition of its kinase activity reduced Akt and S6K1 phosphorylation, Fra-1 and FGF-2 expression, DNA synthesis, and motility induced by thrombin in VSMC. Together these observations suggest that thrombin elicits both mitogenic and motogenic effects in VSMC, and these responses require EGFR-dependent stimulation of PI3K-Akt signaling targeting in parallel the Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation.

	MATERIALS AND METHODS

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Reagents. Aprotinin, dithiothreitol, HEPES, hydroxyurea, PMSF, sodium orthovanadate, sodium deoxycholate, leupeptin, and thrombin were purchased from Sigma (St. Louis, MO). AG-1478, LY-294002, and rapamycin were obtained from Calbiochem (San Diego, CA). Anti-Akt (catalog no. 9272), anti-phospho-Akt (catalog no. 9271), and anti-phospho-S6K1 (catalog no. 9204) antibodies were procured from Cell Signaling Technology (Beverly, MA). Anti-EGFR (catalog no. SC-03) and anti-FGF-2 (catalog no. SC-079) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibodies (catalog no. 05-321, clone 4G10) were obtained from Upstate (Charlottesville, VA). Neutralizing anti-FGF-2 (AB-33-NA) antibodies were bought from R & D Systems (Minneapolis, MN). Lipofectamine 2000 was bought from Invitrogen (Carlsbad, CA). [³H]thymidine (specific activity 78 or 20 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA). Fra-1 siRNA sense (5'-AAG UUC CAC CUU GUG GCA AGC dTdT-3') and antisense (5'-GCU UGG CAC AAG GUG GAA CUU dTdT-3') oligonucleotides were made by Dharmacon (Lafayette, CO). Adenoviral vector harboring dominant-negative (dn)Akt (ad-dnAkt) was constructed by Fujio et al. (12) and was generously provided to us by Dr. Kenneth Walsh (Boston University School of Medicine, Boston, MA) for use in our experiments. Adenoviral vector harboring green fluorescent protein (ad-GFP) was constructed as described previously (25).

Cell culture. VSMC were isolated from the thoracic aortas of 120- to 150-g male Sprague-Dawley rats by enzymatic dissociation as described previously (9). Cells were grown in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37°C in a humidified 95% air-5% CO₂ atmosphere. Cells were quiesced by incubating in DMEM containing 0.1% calf serum for 72 h and used to perform the experiments unless otherwise stated.

Cell motility. VSMC motility was measured by cell wounding assay (9). Quiescent confluent monolayers of VSMC were wounded with a sterile pipette tip to generate a cell-free gap of 1-mm width, and the wound location in the culture dish was marked. Cells were washed, and fresh serum-free DMEM was added and photographed to record the wound width at 0 h. To prevent replicative DNA synthesis, hydroxyurea was added to the medium to a final concentration of 5 mM just before the addition of agonist. Twenty-four hours after the appropriate treatments, photographs were taken again at the marked wound location. Cell migration was measured with the NIH Image 1.62 program, and cell motility was expressed as distance migrated in micrometers.

DNA synthesis. VSMC with and without appropriate treatments were labeled with 1 µCi/ml [³H]thymidine for the last 12 h of the 24-h incubation period. After being labeled, cells were washed with cold PBS, trypsinized, and collected by centrifugation. The cell pellet was suspended in cold 10% (wt/vol) TCA and vortexed vigorously to lyse cells. After standing on ice for 20 min, the cell lysate mixture was passed through a glass fiber filter (GF/C, Whatman). The filter was washed once with cold 5% TCA and once with cold 70% (vol/vol) ethanol. The filter was dried and placed in a liquid scintillation vial containing the scintillant fluid, and radioactivity was measured in a Beckman Coulter liquid scintillation counter (LS 6500).

Immunoprecipitation. After appropriate treatments, VSMC were rinsed with cold PBS and lysed in 500 µl of lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 100 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate) on ice for 20 min. The cell extracts were scraped into 1.5-ml Eppendorf tubes and cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Cell extracts containing 500 µg of protein from control and each treatment were immunoprecipitated with 3 µg of anti-EGFR antibodies overnight at 4°C. The immunocomplexes were collected by incubation with 40 µl of 50% (wt/vol) protein A/G Sepharose beads, followed by serial washings with lysis buffer and PBS. The immunocomplexed protein A/G Sepharose beads were suspended in 50 µl of the Laemmli sample buffer, heated for 10 min in boiling water, resolved by 0.1% SDS-10% PAGE, and subjected to Western blot analysis using anti-phosphotyrosine antibodies.

Western blot analysis. After appropriate treatments, VSMC were rinsed with cold PBS and lysed in 250 µl of lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 100 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate) on ice for 20 min. The cell extracts were scraped into 1.5-ml Eppendorf tubes and cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Cell extracts containing an equal amount of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond; Amersham Biosciences, Piscataway, NJ). After blocking in 10 mM Tris·HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (wt/vol) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected with a chemiluminescence reagent kit (Amersham Biosciences).

Statistics. All experiments were performed three times with reproducible results. Data are presented as means ± SD, and the treatment effects were analyzed by one-way ANOVA, followed by Bonferroni's multiple comparison test. Comparisons between two groups were performed using Student's t-test. P values <0.05 were considered to be statistically significant. In the case of Western blot analysis, one representative set of data is presented.

	RESULTS

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To understand the mechanisms by which thrombin influences its nonthrombotic effects in VSMC, we have studied the role of Akt. Quiescent VSMC were treated with and without thrombin (0.5 U/ml) for the indicated times, and cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for phosphorylation of Akt and one of its effectors, S6K1, using their phosphospecific antibodies. Thrombin stimulated phosphorylation of both Akt (Ser473) and S6K1 (Thr421/Ser424) in a time-dependent manner, with a maximum of fivefold increase at 1 h of treatment and a return to near-basal levels by 8 h (Fig. 1, A and B). Akt and S6K1 are downstream effector molecules of PI3K (11, 22, 42). Therefore, to find out whether thrombin-stimulated Akt and S6K1 phosphorylation depend on activation of PI3K, we tested the effect of its specific inhibitor, LY-294002 (41). As shown in Fig. 1, C and D, LY-294002 (25 µM) completely inhibited thrombin-induced phosphorylation of Akt and S6K1. This finding suggests that thrombin stimulates the phosphorylation of both Akt and S6K1 in a PI3K-dependent manner in VSMC. mTOR is a downstream effector of Akt and an upstream regulator of S6K1 (11, 22, 42). Therefore, to understand the order in which Akt and S6K1 phosphorylation occur, we next tested the effect of rapamycin, a potent inhibitor of mTOR (3). Rapamycin (50 ng/ml) also completely inhibited S6K1 phosphorylation induced by thrombin (Fig. 1, C and D). Rapamycin, however, alone increased Akt phosphorylation compared with control, and this response may reflect a compensatory loop in the activation of Akt, perhaps to override mTOR inhibition. Together these results indicate that thrombin activates PI3K-Akt-mTOR-S6K1 signaling in this order in VSMC.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Thrombin stimulates sustained phosphorylation of Akt and S6K1 in vascular smooth muscle cells (VSMC). Quiescent VSMC were treated with and without thrombin (0.5 U/ml) for the indicated times (A) or with and without thrombin (0.5 U/ml) in the presence and absence of LY-294002 (25 µM) or rapamycin (50 ng/ml) for 1 h (C), and cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for phosphorylated (p) Akt and S6K1 using their phosphospecific antibodies. As a loading control, the same blot was reprobed with anti-Akt antibodies. Bar graphs in B and D show quantitative analysis of 3 independent experiments on the time course and the effect of LY-294002 and rapamycin on thrombin-induced phosphorylation of Akt and S6K1, respectively. *P < 0.01 vs. control; **P < 0.01 vs. thrombin treatment alone.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Inhibition of phosphatidylinositol 3-kinase (PI3K) or mammalian target of rapamycin (mTOR) suppresses thrombin-induced VSMC DNA synthesis and motility. A: quiescent VSMC were treated with and without thrombin (0.5 U/ml) in the presence and absence of LY-294002 (25 µM) or rapamycin (50 ng/ml) for 24 h, and DNA synthesis was measured by [3H]thymidine incorporation. B: a cell-free gap was produced in the quiescent VSMC monolayer, treated with and without thrombin (0.5 U/ml) in the presence and absence of LY-294002 (25 µM) or rapamycin (50 ng/ml) for 24 h, and cell migration was measured. *P < 0.01 vs. control; **P < 0.01 vs. thrombin treatment alone.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Thrombin induces Fra-1 expression in PI3K-dependent and mTOR-independent manner in VSMC. Quiescent VSMC were treated with and without thrombin (0.5 U/ml) for the indicated times (A) or with and without thrombin (0.5 U/ml) in the presence and absence of LY-294002 (25 µM) or rapamycin (50 ng/ml) for 8 h (C), and cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for Fra-1 levels using its specific antibodies. Bar graphs in B and D show quantitative analysis of 3 independent experiments on the time course and the effect of LY-294002 and rapamycin on thrombin-induced expression of Fra-1, respectively. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.05 vs. thrombin treatment alone.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Suppression of Fra-1 by small interfering RNA (siRNA) prevents thrombin-induced VSMC DNA synthesis and motility. A: VSMC that were transfected with mock or Fra-1 siRNA were quiesced and treated with and without thrombin (0.5 U/ml) for 8 h, and cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for Fra-1 levels using its specific antibodies. B: quantitative analysis of 3 independent experiments on the effect of Fra-1 siRNA on thrombin-induced Fra-1 expression. C and D: VSMC that were transfected with mock or Fra-1 siRNA were quiesced, and DNA synthesis (C) and motility (D) in response to thrombin (0.5 U/ml) were measured as described in Fig. 2. *P < 0.01 vs. control; **P < 0.05 vs. thrombin treatment alone.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Adenovirus-mediated expression of dominant-negative (dn)Akt but not GFP suppresses thrombin-induced S6K1 phosphorylation, Fra-1 expression, DNA synthesis, and motility. A: VSMC were infected with adenovirus expressing either GFP or dnAkt, quiesced, and treated with and without thrombin (0.5 U/ml) for 1 h or 8 h, and cell extracts were prepared. S6K1 phosphorylation and Fra-1 levels were measured by Western blot analysis using their specific antibodies. B: quantitative analysis of 3 independent experiments on the effect of dnAkt on thrombin-induced S6K1 phosphorylation and Fra-1 expression. C: VSMC that were infected with ad-GFP or ad-dnAkt and quiesced were treated with and without thrombin (0.5 U/ml) for 24 h, and DNA synthesis was measured by [3H]thymidine incorporation. D: VSMC that were infected with ad-GFP or ad-dnAkt were quiesced, and a cell-free gap was generated and treated with and without thrombin (0.5 U/ml) for 24 h. At the end of 24-h thrombin treatment, VSMC migration was measured as described in Fig. 2. *P < 0.01 vs. control; **P 0.01 vs. thrombin treatment alone; ***P < 0.05 vs. thrombin treatment alone.

2

View larger version (35K): [in this window] [in a new window]   Fig. 6. Thrombin induces FGF-2 expression in a PI3K-Akt-Fra-1-dependent and mTOR-independent manner in VSMC. Quiescent VSMC were treated with and without thrombin (0.5 U/ml) for the indicated times (A) or with and without thrombin (0.5 U/ml) in the presence and absence of LY-294002 (25 µM) or rapamycin (50 ng/ml) for 16 h (B), and cell extracts were prepared. C: VSMC that were infected with either ad-GFP or ad-dnAkt were quiesced and treated with and without thrombin (0.5 U/ml) for 16 h, and cell extracts were prepared. D: conditions were the same as in C, except that cells were transfected with mock or Fra-1 siRNA before being subjected to quiescence followed by treatment with and without thrombin. Equal amounts of protein from control and each treatment were analyzed by Western blotting for FGF-2 levels using its specific antibodies.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Neutralizing anti-FGF-2 antibodies suppress thrombin-induced VSMC DNA synthesis and motility. Quiescent VSMC were treated with and without thrombin (0.5 U/ml) in the presence and absence of neutralizing anti-FGF-2 antibodies (30 µg/ml) for 24 h, and DNA synthesis (A) and motility (B) were determined as described in Fig. 2. *P < 0.01 vs. control; **P < 0.01 vs. thrombin treatment alone.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Inhibition of EGF receptor (EGFR) tyrosine kinase activity suppresses thrombin-induced Akt and S6K1 phosphorylation, Fra-1 and FGF-2 expression, DNA synthesis, and motility. Quiescent VSMC were treated with and without thrombin (0.5 U/ml) in the presence or absence of AG-1478 (1 µM) for the indicated times (A and C), 1 h (B), or 16 h (D), and cell extracts were prepared. Equal amounts of protein from control and each treatment were either immunoprecipitated (IP) with anti-EGFR antibodies followed by immunoblotting (IB) with anti-PY20 antibodies (A) or directly analyzed by Western blotting for phosphorylated Akt and S6K1 (B) and total Fra-1 (C) and FGF-2 (D) levels using the respective antibodies. E: quiescent VSMC were treated with and without thrombin (0.5 U/ml) in the presence and absence of AG-1478 (1 µM) for 24 h, and DNA synthesis was measured by [3H]thymidine incorporation. F: a cell-free gap was produced in the quiescent VSMC monolayer and treated with and without thrombin (0.5 U/ml) in the presence and absence of AG-1478 (1 µM) for 24 h, and cell migration was measured. *P < 0.01 vs. control; **P < 0.01 vs. thrombin treatment alone.

	DISCUSSION

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The present results also reveal that stimulation of EGFR-PI3K-Akt signaling targeting in parallel the Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation is needed for thrombin-induced VSMC migration. Several studies have reported that activation of PI3K-Akt-mTOR-S6K1 signaling is required for mediating cell motility in response to various stimulants (21, 38). However, the mechanisms by which this signaling contributes to cell motility are largely unclear. In this regard, it was recently reported that group 1B secretory phospholipase A₂ mediates NIH3T3 fibroblast migration via PI3K-Akt-dependent induction of expression of matrix metalloproteinase-2 (5). Matrix metalloproteinases such as matrix metalloproteinase-2 and -9, by catalyzing the degradation of the extracellular matrix proteins, play an important role in cell migration (5, 44). Furthermore, it was shown that PI3K mediates the expression of matrix metalloproteinases such as matrix metalloproteinase-12 via the involvement of Fra-1 in human aortic smooth muscle cells derived from atherosclerotic lesions (43). A role for PI3K-Akt in the regulation of AP-1 activity has also been reported (24). In addition, the present study shows that PI3K-Akt-Fra-1 plays a role in thrombin-induced expression of FGF-2, a growth factor that has been shown to be a very potent chemoattractant for VSMC (34). On the basis of these observations, it is quite possible that EGFR-PI3K-Akt signaling via the involvement of Fra-1 may lead to induction of expression of a variety of molecules such as FGF-2 and matrix metalloproteinases and thereby mediate thrombin-induced VSMC migration. Because inhibition of mTOR-S6K1 axis also prevents thrombin-induced VSMC migration, it is likely that, similar to their involvement in DNA synthesis, both Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation subsequent to stimulation of EGFR-PI3K-Akt signaling may be necessary for mediation of VSMC motility in response to thrombin.

In summary, our results demonstrate for the first time that thrombin-induced VSMC DNA synthesis and migration require activation of EGFR-PI3K-Akt signaling targeting in parallel Fra-1-mediated FGF-2 expression and mTOR-S6K1 stimulation.

	GRANTS

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This work was supported by National Heart, Lung, and Blood Institute Grant HL-64165 (to G. N. Rao).

	ACKNOWLEDGMENTS

 
We are thankful to Dr. Kenneth Walsh for providing ad-dnAkt.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. N. Rao, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (e-mail: grao{at}physio1.utmem.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.

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