Mitogen-Activated Protein Kinase Activation Is Involved in Platelet-Derived Growth Factor-Directed Migration by Vascular Smooth Muscle Cells
Migration of vascular smooth muscle cells (VSMCs) is a crucial response to vascular injury resulting in neointima formation and atherosclerosis. Platelet-derived growth factor (PDGF-BB) functions as a potent chemoattractant for VSMCs and enhances these pathologies in the vasculature. However, little is known about the intracellular pathways that mediate VSMC migration. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) activation in this function, since PDGF-BB as well as other growth factors activate this pathway. Using an in-gel kinase assay, we observed that PD 98059, an inhibitor of MEK that activates MAP kinase, inhibited PDGF-BB-induced activation of ERK-1 and ERK-2 in cultured rat aortic smooth muscle cells in a concentration-dependent manner. In contrast, PDGF-mediated activation of intracellular calcium release was not affected by PD 98059. The chemotactic response of both rat aortic smooth muscle cells (RASMCs) and human umbilical vein smooth muscle cells (HUSMCs) toward PDGF-BB (10 ng/mL) was significantly reduced by PD 98059 (10 μmol/L) to 41.7±7.1% in RASMCs (P>.01) and to 47.2 ±5.3% in HUSMCs (P>.01). Similar inhibition was seen at 30 μmol/L, less at 1 μmol/L. To further confirm the specificity of these results implicating the MAPK pathway, an antisense oligodeoxynucleotide (ODN) directed against the initiation translation site of rat ERK-1 and ERK-2 mRNA was used to suppress MAP kinase synthesis and function in rat VSMCs. Liposomal transfection with 0.4 μmol/L antisense ODN reduced ERK-1 and ERK-2 protein by 65% (P>.01) after 48 hours. The chemotactic response to PDGF-BB (10 ng/mL) was reduced by 75% (P>.01) in rat VSMCs transfected with the same antisense ODN concentration. Sense and scrambled control ODNs (0.4 μmol/L) did not affect ERK-1 and ERK-2 protein concentrations or chemotaxis of VSMCs induced by PDGF-BB. These experiments provide the first evidence that activation of MAPK is a critical event in PDGF-mediated signal transduction regulating VSMC migration.
- FBS = fetal bovine serum
- HBKM = HEPES-buffered Krebs' medium
- IGF-1 = insulin-like growth factor
- MAP = mitogen-activated protein
- MAPK = mitogen-activated protein kinase
- MEK = mitogen-activated protein kinase kinase
- ODN = oligodeoxynucleotide
- PDGF = platelet-derived growth factor
- RAF = MEKK, MEK kinase
- RAS = guanine-nucleotide binding protein
- VSMC = vascular smooth muscle cell
Accumulation of VSMCs and formation of connective tissue matrix are critical events in the development of both restenosis and atherosclerosis. The accumulation of VSMCs in the intima results from both cell proliferation and directed migration of VSMCs from the media into the intima.1,2⇓ These processes are regulated by several growth factors that may be released by cells within the injured vessel or from circulating cells. PDGF is a potent chemoattractant inducing directed migration of VSMCs.3,4⇓ The importance of PDGF is underscored by studies in the rat balloon injury model resulting in arterial intimal hyperplasia, in which PDGF has been shown to be required for accumulation of VSMCs in the intima due to migration.5,6⇓
Intracellular signaling pathways controlling cell migration are poorly understood in comparison to our extensive knowledge of mitogenic signal transduction. Signaling proteins and second messengers implicated in directed migration of VSMCs to growth factors include: (1) increased intracellular calcium and the activation of calcium/calmodulin-dependent protein kinase II7; (2) increased phosphatidylinositol turnover linked to the activation of phospholipase Cγ or phospholipase Cβ3; (3) activation of phosphatidylinositol 3-kinase4; and (4) activation of RAS and RAF.8 The mitogenic signaling pathway for PDGF involves the activation of RAS, which activates the serine threonine kinase RAF, which triggers a protein kinase cascade.9 The signal for proliferation is transmitted into the nucleus though the activation of MAPK, which phosphorylates transcription factors that induce expression of c-fos and other early growth response genes. The involvement of the RAS-RAF-MAPK cascade in cell migration is controversial. In fibroblasts expressing a dominant negative RAS, directed migration toward PDGF was suppressed,8 suggesting this pathway contributed to cell migration. In contrast, IGF-I and PDGF-BB were shown to equally induce VSMC migration, but IGF-1 only weakly induced MAPK in contrast to PDGF.3 The authors concluded that activation of MAPK may not required for VSMC migration. The availability of the MEK inhibitor PD 98059, which is widely used as a pharmacological inhibitor of the MAPK pathway, and antisense ODN against MAPK mRNA now provides critical tools to determine the specific role of MAPK activation in cell functions such as migration.
Materials were obtained from the following suppliers: PDGF-BB, DMEM, glutamine, antibiotics, HEPES, DMSO, FBS, myelin basic protein, and monoclonal antibody against smooth muscle α-actin from Sigma; culture plasticware from Becton Dickinson; the transwell chambers from Costar; and [γ32P]ATP from ICN. Antibody against rat ERK-1 was obtained from Transduction Laboratories. The MEK inhibitor PD 98059 was kindly provided by Dr Alan R. Saltiel (Park-Davies, Ann Arbor, Mich) and was dissolved in DMSO 0.1% at 100 mol/L.
Rat aortic smooth muscle cells were prepared from thoracic aorta of 2- to 3-month-old Sprague-Dawley rats using the explant technique.10 Rats were handled as described previously.11 The cells were cultured in DMEM containing 10% FBS, 150 mmol/L HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, and 200 mmol/L glutamine. The purity and identity of the smooth muscle cell cultures were verified by using a monoclonal antibody against smooth muscle α-actin. For all experiments, earlypassaged (≤4) rat VSMCs were grown to 60% to 70% confluence and made quiescent by serum starvation (0.4%) for at least 16 hours, when MAPK activity was assayed. When used, PD 98059 was added 30 minutes before the addition of growth factors. For all data shown, each individual experiment represented by the n value was performed using an independent preparation of VSMCs.
Human smooth muscle cells were prepared from human umbilical cords by preparation of the umbilical vein using the explant technique.10 Human material was obtained with the administrative approval of the institutional review board. When required, informed consent was obtained from patients who provided material. Cells were cultured in DMEM culture medium as described above. Cells from passages 4 to 7 were used for chemotaxis experiments.
MAPK In-Gel Kinase Assay
MAPK activity was measured by the in-gel kinase assay.11 Cells were lysed in a buffer containing 50 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 μmol/L Na3 VO4, 10 mmol/L HEPES, pH 7.4, 0.1% Triton X-100, 500 μmol/L PMSF, and 10 μg/mL leupeptin, then flash-frozen on a dry ice/ethanol bath. After the cells were allowed to thaw, they were scraped off the dish and centrifuged at 14 000 rpm (4°C for 30 minutes), and protein concentrations were determined using the Bradford protein assay (BioRad). Equal amounts of proteins (5 to 10 μg) were separated by SDS-PAGE through a gel containing 0.4 mg/mL myelin basic protein. The gel was then incubated twice in buffer A (50 mmol/L HEPES, pH 7.4, and 5 mmol/L β-mercaptoethanol) containing 20% isopropyl alcohol for 30 minutes; once in buffer A for 1 hour; twice in buffer A containing 6 mol/L guanidine HCl for 30 minutes; twice in buffer A containing 0.04% Tween 20 at 4°C for 16 and 2 hours; and once in buffer A containing 100 μmol/L Na3 VO4, 50 μmol/L ATP, and 50 μCi [g32P]ATP for 1 hour at 30°C. The reaction was terminated by washing the gel five to eight times in fixative solution containing 10 mmol/L sodium pyrophosphate and 5% trichloroacetic acid for 15 minutes. The gel was dried and subjected to autoradiography.
Chemotaxis experiments were performed as described previously.11 VSMC migration was examined in transwell cell-culture chambers using a gelatin-coated polycarbonate membrane with 8-μm pores. Preconfluent smooth muscle cells were suspended in DMEM/0.4% FBS to a concentration of 5.0×105 cells/mL. Cells were pretreated with PD 98059 (1 to 30 μmol/L) or vehicle for 30 minutes at 20°C. DMEM/0.4% FCS (0.6 mL) was added to the lower compartment. A 0.1-mL cell suspension (final concentration: 50 000 cells/well, diameter 6.5 mm) was added to the upper compartment, and cells were then incubated at 37°C (95% air/5% CO2). Chemotaxis was induced by addition of PDGF-BB in a final concentration of 10 ng/mL to the lower compartment. After 4 hours, the filters were fixed with methanol (10 minutes at 4°C), followed by counterstaining with hematoxylin. The number of VSMCs per 320× high-power field that migrated to the lower surface of the filters was determined microscopically. Four to six randomly chosen high-power fields were counted per filter. Experiments were performed in duplicate or triplicate and were repeated at least three times.
Liposomal Transfection With Antisense ODN
The antisense phosphorothioate-modified ODN was a 17-mer (5′-GCCGCCGCCGCCGCCAT-3′) directed against the initiation of translation site of rat ERK-1 and ERK-2, which have the identical sequence at this site.12 This ODN has been used successfully to downregulate MAPK expression in rat cardiac myocytes13 and in 3T3 cells.12 Sense (5′-ATGGCGGCGGCGG CGGC-3′) and scrambled controls (5′-CGCGCGCTCGCG CACCC-3′) were used. All ODNs were synthesized at the Microsequencing Core facility of the University of Southern California using an automated DNA synthesizer (Applied Biosystems, Perkin Elmer). The ODNs were purified on oligopurification cartridges (Applied Biosystems), dried, and re-suspended in sterile water.
Transfection with ODNs was performed in DMEM/serum-free medium with lipofectin at a final concentration of 10 μg/mL for 6 hours at 37°C. Medium was then replaced with lipofectin-free DMEM/2% FBS containing the original ODN concentration, and incubation was allowed to proceed further for 42 hours at 37°C. For migration experiments, 100 000 cells were placed into the upper compartment of gelatin-coated transwell chambers in DMEM/10% FBS 20 hours before transfection. Transfection was performed for 48 hours and migration was induced by addition of PDGF-BB to the lower compartment.
Cells were washed twice with ice-cold PBS and lysed with the same buffer as used in the in-gel kinase assay. Equal amounts of proteins (15 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes using a Bio-Rad transblotter. Nonspecific binding was blocked by using 5% fat-free milk powder. The membrane was incubated with a mouse monoclonal anti-ERK1 antibody (1 μg/mL), which also recognizes ERK-2, for 1 hour in blocking solution. Blots were washed and incubated for another hour with a goat anti-rabbit horseradish-conjugated antibody (1:500, Amersham Life Science Inc) before final development using the ECL Detection System (Amersham).
Cell Ca2+ Measurement
Confluent VSMC monolayers were starved in serum-free medium for 24 hours at 37°C. Cells were trypsinized (0.25% trypsin, 1 mmol/L EDTA; Gibco) briefly and cell suspensions were washed once with HBKM (107 mmol/L NaCl, 6 mmol/L KCl, 1.2 mmol/L MgSO4, 1 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 11.5 mmol/L glucose, 20 mmol/L HEPES, pH 7.4). Cells were incubated with 5 mmol/L Fura-2-Am (Molecular Probes Inc) in HBKM containing 1% BSA for 30 minutes. Then cells were washed twice with HBKM to remove excess dye. Before measurement of cell Ca2+, cells were pretreated with PD 98059 for 45 minutes. One milliliter of cell suspension was transferred to cuvettes and stirred continuously with a magnetic stir bar with constant temperature of 37°C. Fluorescence was detected with a fluorescence spectrophotometer (Hitachi F-200) at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. PDGF-BB was added to induce intracellular Ca2+ release after starting the experiment. Intracellular Ca2+ concentrations were estimated by calibration of the intracellular Ca2+ signal. Cells were lysed with 0.02% Triton-X100 to achieve 100% saturation of the dye, whereas minimal fluorescence was determined with 4 mmol/L EGTA, 30 mmol/L Tris, Kd=224 nm, as described previously.14
ANOVA and paired or unpaired t tests were performed for statistical analysis as appropriate. A value of P>.05% was considered to be statistically significant. Data are expressed as mean±SEM.
PD 98059 Inhibits MAPK Activation by PDGF-BB
Rat aortic VSMCs were made quiescent by 16-hour treatment in 0.4% serum. Quiescent VSMCs exhibited low MAPK activity, as evidenced by the faint signals produced by the phosphorylation of myelin basic protein by p44 (ERK 1) and p42 (ERK 2) in the in-gel kinase assay (Fig 1A, lane 1). Strong induction of ERK 1 (≈18-fold) and ERK 2 activity (≈5-fold) was observed after 10-minute treatment with PDGF-BB, a potent chemoattractant for VSMCs (Fig 1A and 1B). PDGF-BB-induced MAPK activation was inhibited in a concentration-dependent manner by PD 98059, a synthetic inhibitor of MEK, a dual specific kinase that activates both ERK-1 and ERK-2 by phosphorylation at specific threonine and tyrosine residues (Fig 1A). PD 98059 at 1 μmol/L inhibited the PDGF-induced activation of ERK-1 by 62.4±7.9% and ERK-2 by 45.3±9.7% in rat VSMCs (n=4; P>.01 versus PDGF alone). At the maximal tested concentration of 30 μmol/L PD 98059, PDGF-induced ERK-1 and ERK-2 activity declined nearly to the baseline MAPK activity seen in quiescent cells (Fig 1B). Half-maximal inhibition was observed at 0.7 μmol/L for ERK-1 and at 4 μmol/L for ERK-2 in rat VSMCs.
PD 98059 Inhibits PDGF-BB-Mediated Migration in Rat and Human VSMCs
Migration experiments with VSMCs were performed using two different protocols. In the first protocol, cells were treated with PD 98059 or vehicle for 30 minutes and were added afterward to the upper compartment of the modified Boyden chamber. Chemotaxis was induced by addition of PDGF-BB to the lower compartment. PDGF-BB (10 ng/mL) induced a 5.0±0.6-fold increase of migrated cells (P>.01 versus control, n = 6) compared with control. This effect was significantly reduced to a 2.3±0.3-fold increase by 1 μmol/L PD 98059 (P>.05 versus PDGF alone), to a 1.8±0.4-fold increase by 10 μmol/L PD 98059 (P>.01 versus PDGF alone), and to a 1.2±0.3-fold increase by 30 μmol/L PD 98059 (P>.01 versus PDGF alone).
To distinguish between a possible effect of PD 98059 on adhesion of VSMCs to the gelatin-coated membranes and a subsequent inhibition of chemotaxis, cells were added to the upper compartment and treatment with PD 98059 was initiated after a delay of 60 minutes in the second protocol. In adhesion experiments using gelatin-coated membranes, we have observed that the adhesion by rat aortic smooth muscle cells is maximally achieved after 40 to 45 minutes at 37°C (data not shown). After 45 minutes' incubation with the MEK inhibitor, PDGF-BB was added to the lower compartment and migration was stopped 4 hours later. PDGF-BB (10 ng/mL) induced a 4.5±0.5-fold increase in migrated cells (P>.01 versus control), which was already diminished by 1 μmol/L PD compound to 2.1±0.2-fold (P>.01 versus PDGF alone). At higher concentrations, the MEK inhibitor completely abolished the PDGF-induced migration (Fig 2A). PD 98059 (30 μmol/L) alone had no effect on unstimulated cells.
We performed parallel experiments with VSMCs obtained from human umbilical veins. In human vascular smooth muscle cells, PD 98059 was also able to block PDGF-directed migration nearly completely at 10 and 30 μmol/L (Fig 2B). PD 98059 (1 μmol/L) was less effective in diminishing PDGF-mediated migration in human VSMCs than in rat VSMCs. The MEK inhibitor PD 98059 was effective in preventing PDGF-mediated migration with a half-maximal inhibition observed at 0.9 μmol/L for rat VSMCs and 4.8 μmol/L for human VSMCs.
PD 98059 Does Not Affect Intracellular Ca2+ Release by PDGF-BB
PDGF-BB (10 ng/mL) induced a rapid increase in intracellular calcium concentrations that was not significantly altered by pretreatment with PD 98059 for 45 minutes. The absorbances were as follows: PDGF 10 ng/mL, 285.4± 48.6 nm; PDGF plus 1 μmol/L PD 98059, 265.6±25.7 nm; PDGF plus 10 μmol/L PD 98059, 265.7±19.0 nm; and PDGF plus 30 μmol/L PD 98059, 236±13.1 nm (the latter was nonsignificant versus PDGF alone; results shown are mean±SD from three different experiments).
Antisense ODN Against MAPK mRNA Downregulates MAPK Protein and Inhibits PDGF-Mediated Migration
To further establish the crucial role of MAPK activation in PDGF-BB-directed migration, we performed antisense experiments using a 17-mer ODN targeting the initiation site for ERK 1 and ERK 2 mRNA. Liposomal transfection with the antisense ODN for 48 hours induced a concentration-dependent suppression of ERK-1 and ERK-2 protein levels (Fig 3). At a concentration of 0.4 μmol/L, MAPK protein was reduced by 65% (P>.01). Lipofectin alone or control ODNs, both sense and scrambled at identical concentrations to antisense, did not affect ERK-1 and ERK-2 protein concentrations (Fig 3).
Liposomal transfection of rat VSMCs with the MAPK antisense ODNs also attenuated PDGF-mediated migration. After 48 hours of transfection, the migratory response of rat VSMCs was reduced by 75% with 0.4 μmol/L antisense ODN and by 40% in the presence of 0.1 μmol/L antisense ODN (both P>.05 versus control and lipofectin). Liposomal transfection alone and with control ODNs did not significantly alter the migratory response of rat VSMCs to PDGF-BB (Fig 4).
The present investigation provides strong evidence for the involvement of the MAPK signaling pathway in PDGF-mediated migration of VSMCs. PDGF-BB is a potent activator of the MAPK pathway by triggering RAS-RAF activation, which activates MEK, which then phosphorylates ERK-1 and ERK-2 in VSMCs. In the present study, PDGF-BB induced a 5-fold increase of ERK-1 and an 18-fold increase of ERK-2 activity after 10 minutes. A synthetic inhibitor of the ERK pathway, PD 98059, demonstrated a concentration-dependent inhibitory effect on PDGF-induced MAPK activation in VSMCs. The half-maximal inhibitory concentrations measured in our system were similar to those observed to inhibit MAPK activation and growth in 3T3 fibroblasts15 and in VSMCs.16 PD 98059 also demonstrated a concentration-dependent inhibition on PDGF-BB-mediated migration in both rat and human VSMCs. The half-maximal inhibitory concentrations to inhibit migration were in the same range that inhibited MAPK activity in our in-gel kinase assay. The specific inhibition of MAPK activation using antisense ODNs against ERK-1 and ERK-2 mRNA suppressed MAPK protein expression and inhibited PDGF-mediated migration of VSMCs toward PDGF-BB. The sense and scrambled control ODNs had no effect on MAPK expression or PDGFmediated migration. These data provide more conclusive proof that the MAPK pathway plays an important role in PDGF-induced migration of human and rat VSMCs.
Transfection of VSMCs with plasmid DNA occurs with low efficiency. In contrast, ODNs complexed with liposomes transfect VSMCs at high efficiency. A recent study by Pickering et al17 using fluorescein-tagged ODNs revealed that virtually all VSMCs treated with as little as 0.2 μmol/L for 1 hour incorporated the ODNs into both the nucleus and the cytoplasmic compartment. A similar high efficiency of transfection can be inferred from our results showing that MAPK protein levels were specifically depleted to 35% of the levels in VSMCs treated with sense and scrambled ODNs (Fig 3).
Previous investigations aimed at identifying whether the MAPK pathway mediates cell migration are inconclusive. Chronically transfected cells expressing a dominant negative RAS demonstrated decreased migration toward PDGF-BB, and cells expressing a dominant negative RAS also demonstrated decreased chemotaxis toward lysophosphatidic acid but not fibronectin.8 However, fibroblasts expressing an excess of constitutive RAS activity failed to migrate toward PDGF-BB. Moreover, if the cells constitutively expressed RAF, the step immediately downstream from RAS, there was no effect on migration toward PDGF-BB. The authors proposed that an unknown alternate pathway distal to RAS, but not involving RAF, was responsible for cell migration and that too little or too much RAS activity could inhibit migration. These findings are not necessarily inconsistent with ours. All of their findings are compatible with a mechanism for chemotactic signaling in which cells can transition from a ground level, or unstimulated state, to an induced state as they respond to gradients of chemoattractants. Constitutive activation of chemotactic signal transduction by overexpression of signaling components or inhibition of the pathway by blocking the function of a signaling protein like MAPK would each result in an inhibition of migration. We investigated signaling events immediately downstream of RAF and found that inhibition of MEK or MAPK itself inhibited PDGF-directed migration.8 Whether alternate routes to activate MEK independent of RAF are involved is unknown.
Bornfeldt and colleagues3 investigated migration of human VSMCs toward IGF-1 and PDGF-BB and found that both growth factors increased phosphoinositol turnover and calcium flux. They found that only PDGF-BB induced MAPK activation and concluded that MAPK activation is not required for chemotaxis signaling. However, their study did not address whether inhibition of MAPK activation had any effect on PDGF-mediated migration. Furthermore, activation of the MAPK pathway by both insulin and IGF-1 has been observed in our laboratory (R.E.L., unpublished data, 1996) for VSMCs and reported by other groups for several other cell types.18,19⇓
Another essential step in the intracellular pathway mediating chemotaxis upstream of MEK is the activation of the calcium/calmodulin-dependent protein kinase II, which is normally activated by increased Ca2+ transients.7 MAPK can be activated through at least two different pathways, one that is dependent on calcium mobilization and one that is calcium independent.20 We therefore tested the effect of PD 98059 on PDGF-induced Ca2+ transients. PDGF induced a strong Ca2+ transient in rat VSMCs that was unaffected in the presence of the PD compound. This finding suggests that increased intracellular calcium levels without a subsequent activation of the MAPK are insufficient for chemotactic signaling.
PD 98059 appears to be a highly selective inhibitor of MEK; it has been shown to have no effect on p70 S6 kinase, phospholipase C, Raf-kinase, cAMP-dependent kinase, protein kinase C, v-Src, EGF receptor kinase, the PDGF receptor kinase, and the phosphatidylinositol 3-kinase.15,16,19,21⇓⇓⇓ However, the possibility that this synthetic inhibitor affects other cellular targets cannot be ruled out completely. Therefore, we employed antisense ODNs against MAPK to inhibit this pathway. Both approaches decreased MAPK activity and inhibited PDGF-directed migration of VSMCs.
In this study, we present the first evidence for a potential role of the MAPK pathway in the regulation of chemotaxis toward PDGF-BB gradients in rat and human VSMCs. Thus, two important actions of VSMC involved in restenosis and atherosclerosis, migration and proliferation, are dependent on the MAPK pathway. Approaches designed to inhibit this pathway, therefore, may be useful to prevent or inhibit the progression of this mechanisms in the vasculature. Indeed, we have recently reported that the insulin-sensitizing drug troglitazone inhibits basic fibroblast growth factor-induced VSMC growth and PDGF-induced migration, induction of c-fos transcription regulated via MAPK activation, and intimal hyperplasia resulting from balloon injury of the rat aorta.11 Whether antisense against MAPK will have a similar impact in vivo deserves further investigation.
This study was supported by a Specialized Center for Research Grant in hypertension from the National Heart, Lung, and Blood Institute (1P50-HL4404) to Willa Hsueh and a research grant from the American Diabetes Association (ADA 96) to Ronald Law. We thank Denise Edwards and Janie Teran for their secretarial contributions.
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