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Cardiovascular Research 2007 74(1):159-168; doi:10.1016/j.cardiores.2007.01.012
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Copyright © 2007, European Society of Cardiology

Syk contributes to PDGF-BB-mediated migration of rat aortic smooth muscle cells via MAPK pathways

Chang-Kwon Leea,1, Hwan Myung Leea,1, Hyo Jin Kima, Hyo-Jun Parka, Kyung-Jong Wona, Hui Yul Roha, Wahn Soo Choia, Byeong Hwa Jeonc, Tae-Kyu Parkb and Bokyung Kima,*

aDepartments of Physiology and Immunology, College of Medicine, Konkuk University, Danwol-dong 322, Chungju 380-701, Republic of Korea
bDivision of Life Science, Konkuk University, Danwol-dong 322, Chungju 380-701, Republic of Korea
cDepartment of Physiology, Chungnam National University, Taejeon 301-131, Republic of Korea

* Corresponding author. Tel.: +82 43 8403726; fax: +82 43 8519329. Email address: bkkim2{at}kku.ac.kr

Received 25 October 2006; revised 26 December 2006; accepted 11 January 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Here we investigated the role of spleen tyrosine kinase (Syk) in the migration induced by platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells (RASMC).

Methods: Cell migration was determined using a Boyden chamber, by wound-healing, and by aortic ring assays. Activity of Syk, mitogen-activated protein kinase (MAPK), and heat shock protein 27 (HSP27) were tested using immunoblotting with kinase inhibitors and small interference RNAs.

Results: PDGF-BB induced binding of Syk to the PDGFβ receptor and increased the phosphorylation of Syk and migration in RASMC. These effects of PDGF-BB were inhibited by piceatannol, an inhibitor of Syk. PDGF-BB increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and HSP27, which were significantly inhibited by piceatannol and in Syk-knockdown cells. The p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD98059 inhibited the migration, which was further inhibited by the combination of these inhibitors. SB203580, but not PD98059, inhibited the phosphorylation of HSP27 induced by PDGF-BB in RASMC. PDGF-BB-induced migration was attenuated in HSP27-knockdown cells. Kinase inhibitors and Syk-knockdown diminished PDGF-BB-induced sprout outgrowth in the aortic ring assay.

Conclusions: These results imply that Syk is an upstream signal of the p38 MAPK/HSP27 and ERK1/2 pathways that contributes to PDGF-BB-mediated migration in RASMC.

KEYWORDS Smooth muscle; Growth factor; MAP kinase; Tyrosine protein kinase; Remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Platelet-derived growth factor (PDGF) regulates vascular cell proliferation and migration [1–4]. PDGF binds to protein tyrosine kinase receptor on the cell surface and this leads to autophosphorylation of the receptor and the binding of Src homology 2 (SH2) domain-containing signaling molecules [5]. A number of different signaling molecules that contain the SH2 domain, including phospholipase C (PLC) {gamma}, phosphatidylinositol 3-kinase (PI3K), and ras/raf-1, are initiated by PDGF, and these are coupled to the activation of mitogen-activated protein kinases (MAPK) [6]. It has been established that ras, PI3K, and PLC{gamma} play important roles in actin reorganization, growth, and migration in vascular smooth muscle cells [7–9].

Moreover, the PDGF-induced signaling commonly requires the activation of MAPK, a family of serine/threonine-specific protein kinases, consisting of three isoforms: extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (JNK) [6]. It is now clear from numerous studies that activation of MAPK is important for PDGF-induced cell migration [10–14]. It has been reported that ERK1/2 can be involved in cell proliferation and migration [13,15]. Moreover, a small heat shock protein (HSP), HSP27, is a physiological substrate for p38 MAPK, and phosphorylation of HSP27 can modulate the polymerization of actin and migration [16,17].

Recently, it has been shown that signals from extracellular stimuli reach the intracellular space via spleen tyrosine kinase (Syk), a 70 kDa nonreceptor protein tyrosine kinase [18]. Syk is composed of a C-terminal kinase domain and two SH2 domains that bind to the phosphorylated immunoreceptor tyrosine-based activating motif of the immune receptor [19,20]. Syk is ubiquitously expressed by hematopoietic cells and is also expressed in epithelial and endothelial cells [21,22]. These results imply that Syk family may be involved in a wide variety of functions, as well as immune functions [23]. Syk is a major upstream effector of the PI3K/Akt pathway and participates in PI3K activation, which plays a significant role in the regulation of cancer cell motility [24,25]. It has been suggested that Syk plays a critical role in cell morphogenesis, growth, migration, and survival [22]. These effects of Syk are followed by downstream signaling events such as activation of PLC{gamma}, PI3K, and MAPK, which have been demonstrated to be effectors of PDGF signaling.

It has been reported that the Syk inhibitor attenuates the growth factor-mediated ERK activity in rat carotid arterial smooth muscle [26], suggesting that Syk can be involved in the regulation of vascular function. However, the role of Syk has not been directly determined in vascular smooth muscle. In this study, therefore, we examined the expression and activation of Syk on PDGF-induced migration and its signaling in rat aortic smooth muscle cells (RASMC).


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Materials
Piceatannol, SB203580, and PD98059 were purchased from Tocris Bioscience (Bristol, UK). PDGF-BB, TNF{alpha}, and IgG were obtained from R&D Systems. Angiotensin II (AngII), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and other culture ware and chemical reagents were obtained from Hyclone and Sigma. The following antibodies were used: monoclonal anti-Syk (Abcam, Cambridge, UK), polyclonal anti-Syk (Santa Cruz), anti-phospho tyrosine (Upstate), anti-PDGFβ receptor (PDGFβR), anti-p38 MAPK, anti-phospho p38 MAPK, anti-ERK1/2, anti-phospho ERK1/2 (Cell Signaling), anti-HSP27, anti-phospho HSP27 (Affinity BioReagents), and anti-β-actin (Sigma) antibodies.

2.2 Cell culture and immunoblotting
All experiments were performed in accordance with the institutional guidelines of Konkuk University, Korea. RASMC (in passages 5–11) were enzymatically isolated from male Sprague Dawley rats (6 weeks old, 190 g, n=6), and were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 200 mM glutamine. For all experiments, RASMC were grown to 70–80% confluence and starved in DMEM without FBS for 24 h. After treatment with stimulants, cells were lysed with cold extraction buffer (20 mM HEPES, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 2.5 mM 4-nitrophenylphosphate, 0.5 mM PMSF, and 1 tablet of complete proteinase inhibitor cocktail [Roche]). Cell lysates were centrifuged (13,000 xg, 15 min, 4 °C), and the supernatants were collected as protein samples. Protein concentrations were determined using Bio-Rad DC protein assay reagents. The protein homogenates were diluted 1:1 (v/v) with SDS sample buffer containing 40 mM Tris–HCl (pH 6.8), 8 mM EGTA, 4% 2-mercaptoethanol, 40% glycerol, 0.01% bromophenol blue, and 4% SDS, and then boiled for 5 min. Proteins (30–50 µg/lane) were separated using 12–14% polyacrylamide SDS gels, and then transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore). The membrane was then blocked for 2 h at room temperature with PBS containing 0.05% Tween 20 and 5% fat-free dried milk. The membranes were incubated with antibodies diluted 1:1000–2000 overnight at 4 °C. Immune complexes were detected with horseradish peroxidase-conjugated antibodies (Amersham-Pharmacia) diluted 1:1000 and incubated for 1 h at room temperature. After application of the secondary antibody, blots were incubated in enhanced chemiluminescence kits (Amersham-Pharmacia) and exposed to photographic film. Band intensity was measured by computer analysis using Quantitation software (Bio-Rad).

2.3 Immunoprecipitation
Cells were lysed in extraction buffer and lysates containing 200 µg of proteins were incubated with 4 µg/ml antibodies for 5 h at room temperature. The immunocomplex was precipitated using protein A-agarose beads (Roche) overnight at 4 °C. Beads were washed with phosphate buffered saline containing 0.1% Tween 20 and resuspended in SDS sample buffer, and boiled for 5 min. Protein samples collected were immunoblotted with antibodies as described above.

2.4 Transfection siRNA for RASMC
RASMC (1x105) were seeded in 60 mm plates. After 12 h incubation in antibiotic-free DMEM containing 10% FBS at 37 °C and 5% CO2, the medium was replaced with fresh antibiotics and FBS-free DMEM. The cells were subsequently added and incubated with the small interference RNA (siRNA) or nonsilencing control siRNA to a final concentration of 250 pM siRNA using transfection reagent (Welfect-QTM Gold, Welgene, Korea). After transfection for 48 h, cells were harvested with extraction buffer. The relative expression of Syk and HSP27 were determined by immunoblotting analysis using anti-Syk and -HSP27 antibodies, respectively. siRNAs were designed to target the sequences of rat Syk (5'-GAGGUAUAUGAGAGUCCUU-3', Accession Number: NM012758) and HSP27 (5'-GGAGCCAAGUAGAAGCCUU-3', Accession Number: NM031970). Two RNA of 19 nucleotides followed by TT were designed and chemically synthesized, purified, and annealed (Bioneer, Korea). Nonsilencing siRNA (5'-CCUACGCCACCAAUUUCGU-3') was provided by Bioneer.

2.5 Boyden chamber assay
Migration assays were performed in 48-well microchemotaxis Boyden chambers (Neuro Probe). Polycarbonate membranes with 8 µm pores (Neuro Probe) were coated with a 0.1 mg/ml of type I collagen from rat tail tendon (BD Bioscience) in double-distilled water and then dried for 60 min. RASMC were harvested using trypsin–EDTA (Life Technologies) and resuspended in DMEM containing 0.1% BSA and test inhibitors. In some experiment, to determine the PDGFβR-mediated pathway, cells were pretreated in DMEM containing 1 µg/ml of PDGFβR or isotype-matched control IgG for 60 min. The bottom chamber was loaded with 3x104 cells and the membrane was laid over the cells. The microchamber was then inverted and incubated at 37 °C for 120 min. The chamber was then returned to an upright position, and the upper wells were loaded with DMEM containing 0.1% BSA, PDGF-BB, and test inhibitors. The chamber was then incubated 37 °C for 90 min, and the membranes were fixed and stained using Diff-Quik (Baxter Healthcare). The number of cells migrating through the membrane was determined by counting four randomly chosen regions of each well under a microscope (x400). All the samples used in the experiments contained 0.1% DMSO, and the presence of DMSO did not affect RASMC migration.

2.6 Wound-healing assay
RASMC were grown in cell culture plates (60 mm) using cell growth media. After the cells had reached subconfluence, the medium was replaced with FBS-free media for 24 h. The monolayer cells were wounded with 200 µl pipette tips and the medium was replaced with FBS-free medium containing different concentrations of stimulants. After the cells had been cultured for 36 h, cell movement into the wound area was examined at approximately the same fields. The migration distances between the leading edge of the migrating cells and the edge of the wound were compared, and total wound areas were analyzed using Scion Image software.

2.7 Aortic ring assay
Ex vivo migrations of RASMC were measured by an aortic ring assay using Matrigel with some modifications [27]. The endothelium and adventitium of the aorta from Sprague Dawley rats (8 weeks, n=8) was removed enzymatically, the vessels were cut into rings (1 mm). The rings were placed and embedded in 48-well plates coated with Matrigel (BD Bioscience), and simultaneously added PDGF-BB and test inhibitors in FBS-free medium. In some experiments embedded rings were incubated in 10% FBS-medium for 3 days, and then transfected with 50 pM of siRNA under transfection conditions. The rings were stained with Diff-Quik and photographed and analyzed the length of sprouts using Scion Image software on day 5.

2.8 Statistical analysis
Data are expressed as means±SE of the mean. Statistical evaluation of the data was performed using Student's t tests for comparisons between pairs of groups and by ANOVA for multiple comparisons. P<0.05 was considered to indicate a significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 The Syk inhibitor piceatannol in PDGF-BB-induced migration
To determine the role of Syk in vascular cell migration, piceatannol, a selective inhibitor of Syk, was tested on PDGF-BB-induced migration of RASMC using a modified Boyden chamber assay. PDGF-BB (0.1–20 ng/ml) dose-dependently increased migration, which reached a submaximum at 10 ng/ml of PDGF-BB (Fig. 1A). The migration stimulated by 10 ng/ml PDGF-BB was 300.2±36.0% of nonstimulated controls (23.8±2.4 cells/field in control, n=8). Piceatannol (1–30 µM) inhibited migration induced by PDGF-BB (10 ng/ml) in a dose-dependent manner, and inhibition was complete with 30 µM of the inhibitor (n=8; Fig. 1B). In the linear wound-healing assay, PDGF-BB (10 ng/ml) treatment for 36 h repaired the scratch (4.3±1.1%, n=4, vs. 85.1±8.7%, n=4, of wound width; Fig. 1C and D). Piceatannol (30 µM) inhibited PDGF-BB-induced wound-healing (14.1±11.3%, n=4), which was similar to the wound obtained in the nonstimulated control cells.


Figure 1
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  		Fig. 1 Effects of PDGF-BB and piceatannol on migration and linear wound closure in RASMC. (A) Effect of PDGF-BB on RASMC migration. The cells were treated with or without PDGF-BB (0.1–20 ng/ml) for 90 min. (B) Effect of piceatannol on PDGF-BB-stimulated RASMC migration. Cells were preincubated with or without piceatannol (1–30 µM) for 120 min, and then treated with PDGF-BB (10 ng/ml) for 90 min (n=8). (C) and (D) Effect of PDGF-BB and piceatannol in linear wound-healing. The cells were subjected to injury by scratching with a plastic pipette tip. The cells were then treated for 36 h with or without PDGF-BB (10 ng/ml) or piceatannol (30 µM). The scratched wound width was expressed as 100% (n=4). (E) Effect of PDGF-BB (1 and 10 ng/ml) on migration in the PDGFβR-neutralized cells. Cells were pretreated with 1 µg/ml of anti-PDGFβR antibody or control IgG for 60 min, and measured the PDGF-BB-induced migration (n=8). (F) Effect of piceatannol (30 µM) on AngII (100 nM)- and TNF{alpha} (5 ng/ml)-induced migration (n=8). *p<0.01.

 
To confirm the PDGF-BB pathways that induce RASMC migration, the effects of PDGF-BB were determined in PDGFβR-neutralized cells. Cells were preincubated in media containing 1 µg/ml of anti-PDGFβR antibody for 60 min, and measured the PDGF-BB-induced migration. As shown in Fig. 1E, PDGF-BB (1 and 10 ng/ml)-induced RASMC migration was completely abolished by the neutralization of PDGFβR. There was no change in cells pretreated with an isotype-matched IgG (1 µg/ml, 60 min) as a control.

The effects of Syk inhibitor on the receptor agonist- and cytokine-mediated migration were analyzed. AngII (100 nM) increased RASMC migration to 211±16.2% of control, which was inhibited by treatment with 30 µM piceatannol (Fig. 1F). Similar effect was obtained in TNF{alpha} (5 ng/ml)-induced migration. There were no effects on RASMC viability by stimulators or piceatannol (data not shown).

3.2 PDGF-BB in phosphorylation of Syk
Given the migration finding, we next analyzed the effects of PDGF-BB on Syk activation. To determine the Syk activity, cell extracts were immunoprecipitated with anti-Syk antibody and then immunoblotted with anti-phospho tyrosine (4G10) antibody. As shown in Fig. 2A and C, PDGF-BB (10 ng/ml) elevated Syk phosphorylation in a time-dependent manner with a maximum at 300 s and decreased slightly thereafter. PDGF-BB (0.1–20 ng/ml; 10 min) increased Syk phosphorylation in a dose-dependent manner, with a maximum at 10 ng/ml of the stimulator (n=4; Fig. 2B and D). The total expression of Syk, determined using a nonphospho-Syk antibody, was not changed during treatment with PDGF-BB (n=4).


Figure 2
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  		Fig. 2 PDGF-induced phosphorylation of Syk in RASMC. (A) and (B) RASMC were stimulated with PDGF (10 ng/ml) for indicated times (A) and with PDGF (0.1–20 ng/ml) for 10 min (B). The lysates were immunoprecipitated with anti-Syk antibody, and then immunoblotted with anti-phospho tyrosine (4G10) antibody (upper panels). (C) and (D) The statistical results obtained from panels A and B, respectively. (E) and (F) The binding between Syk and PDGFβR in the PDGF-treatment. Cell extracts from RASMC stimulated with PDGF (10 ng/ml) for indicated times were immunoprecipitated with anti-Syk (E) or-PDGFβR (F) antibodies, and then immunoblotted with anti-PDGFβR or Syk antibodies, respectively. Total expressions of PDGFβR and Syk were determined using immunoblotting with an anti-Syk or -PDGFβR antibodies (lower panels). The ratio of phosphorylated/nonphosphorylated Syk in the basal state is expressed as 100% (n=4). *p<0.01. IB, immunoblotting. IP, immunoprecipitation. p-Syk, phosphorylated Syk. pY, phospho tyrosine.

 
PDGF-BB (10 ng/ml) increased the binding PDGFβR to Syk in RASMC (Fig. 2E). The maximum signal appeared at 30 s after the treatment with PDGF-BB (566.2±30.2% of controls, n=4), and then gradually declined close to the basal level within 10 min. To confirm this association, we performed a reciprocal experiment in which cell lysates were immunoprecipitated with anti-PDGFβR antibody, and then immunoblotted with anti-Syk antibody. PDGF-BB (10 ng/ml) increased the binding Syk to PDGFβR in RASMC, which reached at a maximum at 30 s (401.2±10.0% of controls, n=4; Fig. 2F). In contrast, no association between PDGFβR and Syk was obtained in the absence of PDGF-BB.

3.3 The Syk inhibitor piceatannol in PDGF-BB-induced Syk phosphorylation
To determine the role of Syk inhibitor on PDGF-BB-induced Syk activation, RASMC were preincubated with piceatannol at different concentrations and then stimulated with PDGF-BB (10 ng/ml) for 10 min. PDGF-BB-induced Syk phosphorylation was inhibited by treatment with piceatannol (1–30 µM) in a dose-dependent manner (n=4; Fig. 3A and B). However, piceatannol did not influence the total expression of Syk.


Figure 3
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  		Fig. 3 Effects of Syk inhibitor on PDGF-BB-induced Syk activity in RASMC. (A) RASMC were incubated with piceatannol (1–30 µM) for 30 min, and then treated with PDGF-BB (10 ng/ml) for 10 min. The lysates from cells were immunoprecipitated with anti-Syk and then immunoblotted with anti-phospho tyrosine (4G10) antibodies. (B) The statistical results obtained from panel A. The ratio of phosphorylated/nonphosphorylated Syk in the basal state is expressed as 100% (n=4). *p<0.01.

 
3.4 Role of Syk in MAPK-mediated responses
To evaluate the roles of Syk activation on MAPK-mediated responses, the effect of piceatannol on the phosphorylation of p38 MAPK, ERK1/2, and HSP27 by PDGF-BB was tested. SB203580 (30 µM), an inhibitor of p38 MAPK, inhibited the migration induced by 10 ng/ml of PDGF-BB in RASMC (159±9.4%, n=8, vs. 234.4±5.9%, n=8, in control; Fig. 4A). PD98059 (30 µM), an inhibitors of ERK1/2, inhibited the PDGF-BB (10 ng/ml)-induced migration to 182±9.8% of nonstimulated control (Fig. 4A). Treatment of cells with the combination of SB203580 (30 µM) and PD98059 (30 µM) further inhibited the migration to 97.2±7.1% of controls (Fig. 4A). PDGF-BB (10 ng/ml) elevated the phosphorylation of p38 MAPK (259.5±4.5% of controls, n=4; Fig. 4C) and ERK1/2 (269.2±28.8% of controls, n=4; Fig. 4B). Piceatannol (30 µM) inhibited the phosphorylation of p38 MAPK to 78.3±1.6% (n=4) and ERK1/2 to 120.1±4.9% (n=4) of nonstimulated controls. PDGF-BB (10 ng/ml) elevated the phosphorylation of HSP27, which was attenuated by piceatannol (30 µM) to 71.6±5.8% (n=4) of controls (Fig. 4D). SB203580 (30 µM), but not PD98059 (30 µM), inhibited the phosphorylation of p38 MAPK and HSP27 induced by 10 ng/ml of PDGF-BB (n=4). PDGF-BB-elevated the phosphorylation of ERK1/2 was inhibited by PD98059 (149.9±24.9% of control, n=4), but not by SB203580 (365.7±69.1% of control, n=4) (Fig. 4B).


Figure 4
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  		Fig. 4 Effects of kinase inhibitors on PDGF-BB-induced responses in RASMC. (A) Effects of kinase inhibitors on PDGF-BB-induced RASMC migration. The cells were preincubated for 120 min with SB203580 (30 µM), piceatannol (30 µM), PD98059 (30 µM), or SB203580 (30 µM) plus PD98059 (30 µM), and then stimulated with 10 ng/ml PDGF-BB for 90 min. The migration in the quiescent states is expressed as 100% (n=8). (B) RASMC were treated with or without piceatannol (30 µM), PP2 (10 µM), PD98059 (30 µM), or SB203580 (30 µM) for 30 min, and then stimulated with PDGF-BB (10 ng/ml) for 10 min. (C) and (D) The statistical results obtained from panel B. The ratio of phosphorylated/nonphosphorylated p38 MAPK (C) and phosphorylated HSP27/actin (D) in the basal state is expressed as 100% (n=4). (E) The HSP27 expression in nontransfected control (Con), nonsilencing siRNA (non-siRNA), or siRNA-HSP27 transfected cells. Whole cell extracts were subjected to immunoblotting for protein expression using anti-HSP27 antibody (n=3). (F) Effects of transfection with siRNA-HSP27 on PDGF-BB-induced migration in RASMC (n=8). *p<0.01. PIC, piceatannol. SB, SB203580. PD, PD98059.

 
To clarify the roles of HSP27 on vascular cell migration, the expression of HSP27 was knocked down using an siRNA technique. As shown in Fig. 4E, transfection of siRNA-HSP27 reduced the expression of endogenous HSP27, but not in cells transfected with nonsilencing siRNA. The migration induced by PDGF-BB (10 ng/ml) was attenuated in cells transfected with siRNA-HSP27 (190.8±16.9% vs. 268.5±11.4% in nontransfected control, n=8, Fig. 4F). There was no inhibition of the migration in nonsilencing siRNA, which was similar to the levels from nontransfected controls. Similar effects of siRNA-HSP27 were obtained in AngII (100 nM)- and TNF{alpha} (5 ng/ml)-induced migration (Fig. 4F).

3.5 Changes of response to PDGF-BB in the Syk-knockdown cells
To confirm the effect of piceatannol and role of Syk on PDGF-BB-induced migration, Syk was knocked down using an siRNA-Syk. Transfection of siRNA-Syk drastically reduced the expression of endogenous Syk (31.7±1.0% of nontransfected control, n=4), but not in cells transfected with nonsilencing siRNA (90.8±2.7% of nontransfected control, n=4, Fig. 5A). Cell migration and detection of downstream signaling molecules were determined using nontransfected and transfected cells. PDGF-BB (10 ng/ml) increased RASMC migration in cells transfected with siRNA-Syk and nonsilencing siRNA. PDGF-BB-induced migration was attenuated in cells transfected with siRNA-Syk (264±25.8% vs. 158.6±5.6%, n=8). There was no inhibition of the migration in nonsilencing siRNA. Treatment with siRNA-Syk did not affect to the migration in resting state (Fig. 5A). Similar results were obtained in AngII (100 nM)- and TNF{alpha} (5 ng/ml)-induced responses (Fig. 5A). In Syk-knockdown cells, the phosphorylation of p38 MAPK and ERK1/2 by 10 ng/ml of PDGF-BB (10 min) was significantly inhibited to 92.9±1.2% and 88.1±34.9%, respectively, of control (n=4). Similarly, HSP27 phosphorylation induced by PDGF-BB (10 ng/ml; 10 min) was diminished by the siRNA-Syk (360.3±59.7%, n=4, vs. 648.4±104.5%, n=4; Fig. 5B). In contrast, transfection of nonsilencing siRNA failed to attenuate the responses by PDGF-BB, which showed a similarity to the results from nontransfected control cells (732.5±161.2%, n=4).


Figure 5
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  		Fig. 5 Effects of transfection with siRNA-Syk on PDGF-BB-, AngII-, and TNF{alpha}-induced responses in RASMC. (A) Effect of siRNA-Syk on PDGF-BB (10 ng/ml)-, AngII (100 nM)-, and TNF{alpha} (5 ng/ml)-induced migration of RASMC. The basal activity in migration of RASMC is expressed as 100% (n=8). The inset indicates the Syk expression in nontransfected control (Con), nonsilencing siRNA (non-siRNA), or siRNA-Syk transfected cells. Whole cell extracts were subjected to immunoblotting for protein expression using anti-Syk antibody. (B) Effects of Syk-knockdown on PDGF-BB-induced phosphorylation of p38 MAPK, ERK1/2, and HSP27. Whole extracts from control or transfected cells treated with PDGF-BB (10 ng/ml) for 10 min were immunoblotted with the phospho p38 MAPK, ERK1/2, and HSP27 antibodies. *p<0.01.

 
3.6 Roles of Syk in the sprout growth of aortic rings
To investigate the role of Syk ex vivo, piceatannol was tested using the aortic ring assay with Matrigel. Rings were embedded in Matrigel and the outgrowth of sprouting was determined as cell migration and proliferation. As shown in Fig. 6, PDGF-BB (10 ng/ml) increased the sprout of aortic rings. Piceatannol (30 µM) diminished the aortic sprouts by PDGF-BB. Treatment with SB203580 (30 µM) or PD98059 (30 µM) inhibited PDGF-BB (10 ng/ml)-induced sprout of aortic rings. The outgrowth of sprout by 10 ng/ml of PDGF-BB was inhibited in cells transfected with siRNA-Syk (n=4).


Figure 6
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  		Fig. 6 Effects of PDGF-BB and kinase inhibitors on the sprout formation of aortic ring. Aortic rings (1 mm) were cultured and embedded in Matrigel, and then added PDGF-BB or piceatannol. The result was obtained on day 5. (A) Upper panel, Rings without any treatments (vehicle) and PDGF-BB (10 ng/ml)-treated rings. Lower panels, PDGF-BB-induced inhibition of outgrowth of sprout by piceatannol (30 µM), SB203580 (30 µM), and PD98059 (30 µM). (B) Effects of Syk-knockdown on PDGF-BB-induced outgrowth of sprout (n=4). (C) and (D) The statistical results obtained from panels A and B, respectively.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In this study, we identified that the protein tyrosine kinase Syk was expressed in vascular smooth muscle cells, as we previously determined in mast cells [28], and played an important role in the vascular cell migration elevated by PDGF-BB. Although it has been reported that the Syk inhibitor piceatannol inhibits the MAPK phosphorylation elicited by fibroblast growth factor in vascular smooth muscle [26], to the best of our knowledge, this study is the first to directly show the expression and activation of Syk by PDGF in vascular smooth muscle cells. We confirm the hypothesis that this kinase can be involved in the regulation of signal transduction in variety types of cells, including vascular smooth muscle cells [22,23].

In our study, PDGF-BB increased the phosphorylation of Syk and migration in RASMC, which were significantly inhibited by the Syk inhibitor and in the transfected cells with siRNA-Syk. The Syk inhibitor decreased the PDGF-BB-induced outgrowth of aortic sprout, known as ex vivo model for vascular cell proliferation and migration [27]. Moreover, this study showed that TNF{alpha} and AngII increased RASMC migration, which was inhibited by the Syk inhibitor and siRNA-Syk. It has been shown that the Syk pathway is involved in the cell migration induced by growth factors and cytokines in leukocytes [29]. Moreover, Syk participates in the migration of platelets [30]. Migration of vascular smooth muscle cells is commonly elevated by several factors, especially PDGF [2]. From these results, we suggest that the Syk pathway contributes to the vascular cell migration.

It is well known that MAPK isoforms are common signals contributing to a variety of cell functions [6,15]. PDGF increases both MAPK activation and migration in several cells, implying MAPK can be a crucial pathway in PDGF-mediated migration [11,14,19]. In this study, PDGF-BB increased phosphorylation of p38 MAPK in RASMC. p38 MAPK phosphorylates and activates downstream protein kinases, i.e. MAPK-activated protein kinase-2/3 and p38-regulated/activated protein kinase, which phosphorylate HSP27 in many cell types [17,31,32]. There are data indicating that the phosphorylation of HSP27 can modulate the polymerization of actin and cytoskeleton remodeling, which are thought to be a key step in cell migration [17,32]. In our study, PDGF-BB-activated migration was significantly inhibited in the transfected cells with siRNA-HSP27. Moreover, present and previous results have shown that p38 MAPK inhibitor attenuates the migration and HSP27 phosphorylation induced by PDGF-BB in smooth muscle cells [14,16]. These results imply that p38 MAPK contributes to PDGF-BB-induced migration through phosphorylation of HSP27, and this may be important in vascular smooth muscle cell migration. Moreover, the upper signal Syk also can contribute to the activation of p38 MAPK in cellular systems [33,34]. Our results showed that the phosphorylation of p38 MAPK and HSP27 induced by PDGF-BB was significantly inhibited by treatment with Syk inhibitor and in the siRNA-Syk transfected cells. Moreover, PDGF-BB-mediated migration was attenuated by inhibition of p38 MAPK, as previously described [12,35]. These results strongly suggest that PDGF-BB induces Syk activation and this subsequently stimulates the p38 MAPK/HSP27 pathway that leads to vascular cell migration. Moreover, this study showed that the inhibition of Syk inhibited the activation of ERK1/2 elevated by PDGF-BB and the ERK1/2 inhibitor diminished the PDGF-BB-induced migration and aortic sprouts. These results imply that ERK1/2, as well as p38 MAPK, is involved in the migration by PDGF-BB. Moreover, treatment with combination of ERK1/2 and p38 MAPK inhibitors further inhibited the migration induced by PDGF-BB. In contrast, ERK1/2 inhibitor did not affect to the phosphorylation of HSP27, which imply that ERK contributes to vascular cell migration through HSP27-independent manners. These results imply that activation of Syk also stimulates ERK1/2 activity and this may result in RASMC migration.

Our results also showed that PDGF-BB-induced migration was completely abolished by the neutralization of its receptor with anti-PDGFβR antibody and PDGF-BB induced the binding of Syk to PDGFβR, implying that Syk protein does associate with the PDGFβR in a PDGF-dependent manner. It has been reported that autophosphorylation of tyrosine kinase receptor by extracellular stimuli leads to the binding of a group of molecules containing SH2 domain and subsequently transmits its signals into the intracellular space [5]. Moreover, the finding that Syk binds to tyrosine receptor kinase has been reported previously [33,34]. Activation of receptor tyrosine kinases can result in Syk activation, and the Syk kinase pathway stimulates the elevation of MAPK phosphorylation [33]. These results suggest that the binding between PDGFβR and Syk is important in transmitting the signal into intracellular space in RASMC.

In summary, our study revealed that Syk was expressed in vascular smooth muscle and this kinase was significantly activated with PDGF-BB treatment. Cells treated with a Syk inhibitor and transfected with siRNA-Syk showed inhibition in PDGF-BB-induced migration and Syk phosphorylation. Moreover, PDGF-BB increased the phosphorylation of p38 MAPK, ERK1/2, and HSP27, which were also inhibited by the Syk inhibition. From these results, we suggest that Syk plays a crucial role in the regulation of PDGF-BB-induced migration in vascular smooth muscle, which is mediated by the p38 MAPK/HSP27 and ERK1/2 pathways.


   Acknowledgements
 
This work was supported by grant from the Bio-Food and Drug Research Center and the Second-Phase of BK (Brain Korea) 21 Project in 2006 at Konkuk University and by grant No. R01-2004-000-10045-0 from the Basic Research Program of the Korea Science and Engineering Foundation, Korea.


   Notes
 
1 Contributed equally to the manuscript. Back

Time for primary review 34 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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