Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) phosphorylate caldesmon in vivo, but the function of caldesmon phosphorylation in smooth muscle physiology is controversial. We hypothesized that ERK MAPKs and caldesmon modulate chemotactic migration of cultured canine pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF; 10 ng/ml) and endothelin-1 (ET-1; 100 nM) transiently activated ERK MAPKs: PDGF produced higher maximal and more potent activation of ERK MAPKs over 5 h. While both PDGF and ET-1 increased caldesmon phosphorylation, only PDGF stimulated migration of cultured cells (13 times over basal migration). At concentrations from 0.01 to 10 nM, ET-1 failed to enhance migration; 100 nM ET-1 produced only a slight increase (1.31 ± 0.18 times basal migration). ET-1 (100 nM) did not potentiate migration triggered by 0.5 or 3 ng/ml PDGF. The MEK1 inhibitor PD-98059 (50 µM) abolished the PDGF-stimulated phosphorylation of ERK MAPKs and caldesmon and reduced cell migration by 50%. We conclude that while ERK MAPK activity is not required to initiate migration, an ERK MAPK-caldesmon pathway may modulate later events necessary for PDGF-stimulated migration of cultured PASMCs.
endothelin-1; PD-98059; extracellular signal-regulated kinase; mitogen-activated protein kinase; platelet-derived growth factor; smooth muscle cells
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INTRODUCTION |
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ANGIOGENESIS, ATHEROSCLEROSIS, AND VASCULAR HYPERTENSION are associated with accumulation of platelets and macrophages in the vicinity of wall lesions (1). Released growth factors, hormones, and extracellular matrix constituents stimulate cell membrane receptors and activate intracellular signal transduction pathways that include the mitogen-activated protein kinases (MAPKs) (3). Growth factors also stimulate migration of vascular smooth muscle cells (VSMCs), but it is controversial whether activation of extracellular signal-regulated (ERK) MAPKs and cell migration are associated. Studies with human vascular (21), rat aortic, and human umbilical smooth muscle cells (11) have shown that platelet-derived growth factor (PDGF) triggers a chemotactic response that is sensitive to the ERK MAPK inhibitor PD-98059. Other laboratories have confirmed the inhibitory effect of PD-98059 on the migration of different cell types (13, 14). Downregulation of ERK1 and ERK2 MAPKs with antisense oligodeoxynucleotides also results in reduction of migration of VSMCs (15). While these studies support a role for ERK MAPKs in some cell types, in rat fibroblasts PDGF-stimulated migration was reduced neither by chemical inhibition of the ERK MAPKs nor by expression of dominant negative MEK1, the kinase that phosphorylates and activates the ERK MAPKs (4). These results suggest that the role of ERK MAPKs in chemotactic migration is cell type specific and/or depends on differences in the experimental approach.
Caldesmon is a recognized effector of the ERK MAPKs and the actin binding protein that stabilizes actin structures, and it may be involved in regulation of the actomyosin ATPase (19). Caldesmon has been extensively studied as a modulator of smooth muscle contraction, and various laboratories have reported that activation of the ERK MAPKs and phosphorylation of caldesmon in intact smooth muscle increase in parallel with isometric force (10, 25, 27, 30). Development of phosphospecific anti-caldesmon antibodies has allowed others to determine that caldesmon phosphorylation on the ERK MAPK consensus sites increases during smooth muscle stimulation with contractile agonists (7, 8, 12). The necessity for caldesmon phosphorylation in development of Ca2+-independent contraction, however, has been challenged by studies that dissociate caldesmon phosphorylation from isometric force (2, 22). Meanwhile, evidence that caldesmon is involved in cellular functions other than the smooth muscle contraction has been produced. Caldesmon was localized at leading edges and membrane ruffles (5, 24), and caldesmon overexpression has been shown to increase the total number and stability of actin bundles in fibroblasts (24). One could predict that by stabilizing actin filaments, caldesmon decreases the rate and magnitude of actin rearrangement and attenuates processes depending on actin turnover. However, Warren et al. (26) showed that overexpression of a COOH-terminal fragment that contains the major actin binding affinity of human caldesmon increases adhesion and spreading of fibroblasts but moderately changes the velocity of cell translocation (26). The COOH terminus also harbors some of the major phosphorylation sites of mammalian caldesmon (19). This advances the possibility that an increased COOH-terminal phosphorylation may facilitate dissociation from actin, allow more rapid actin turnover, and accelerate cell motility. Whether phosphorylation of caldesmon occurs during cell attachment and migration, however, has not yet been established. Therefore, predictions such as whether caldesmon phosphorylation facilitates actin rearrangement at the leading edges and membrane ruffles, and thereby affects cell motility, are speculative.
To address some of these issues, in the present study we hypothesized that ERK MAPKs are activated by growth factors and hormones and, via phosphorylation of caldesmon, modulate chemotactic migration of VSMCs. Our results, obtained by using cultured canine pulmonary artery smooth muscle cells (PASMCs) as a model system, show that PDGF and endothelin-1 (ET-1) activate ERK MAPKs and increase caldesmon phosphorylation and that PDGF, but not ET-1, stimulates chemotactic cell migration. Inhibition of the ERK MAPKs abolishes the PDGF-stimulated phosphorylation of caldesmon and partially decreases cell migration. These observations support a model in which ERK MAPKs play a modulatory role in chemotactic migration of cultured PASMCs. Caldesmon may be the link that translates activation of the ERK MAPKs to actin remodeling and changes in migratory responses of cultured smooth muscle cells.
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MATERIALS AND METHODS |
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Materials. ET-1 and PDGF antibodies were purchased from Sigma (St. Louis, MO). PD-98059 was from Calbiochem (La Jolla, CA). Transwell cell migration plates were purchased from Costar (Corning, NY). Cell culture medium M199 was from GIBCO (GIBCO BRL, Gaithersburg, MD), and Diff-Quik staining solutions I and II were from Baxter Diagnostics (McGraw Park, IL). Phosphospecific anti-ERK MAPK rabbit polyclonal antibody was purchased from New England Biolabs (Beverly, MA). Phosphospecific rabbit polyclonal anti-caldesmon antibodies were kindly provided by Dr. L. P. Adam. Secondary goat anti-rabbit antibodies conjugated to alkaline phosphatase were from Promega (Madison WI). All other reagents came from commercial sources.
Cell culture. Canine pulmonary artery smooth muscle tissue was obtained from adult mongrel dogs of either sex euthanized by barbiturate overdose. The tissue was minced and placed in Ca2+-free Hanks' solution, which contained 125 mM NaCl, 5.36 mM KCl, 15.5 mM NaHCO3, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 10 mM glucose, 2.9 mM sucrose, and 10 mM HEPES, pH 7.4, at 37°C. Minced tissue was then digested with 1 mg/ml type II collagenase, 0.1 mg/ml protease (Sigma; P5147), 2 mg/ml bovine serum albumin (BSA), 2 mg/ml trypsin inhibitor, and 0.3 mg/ml Na2ATP for 1.5 h at 37°C. Cells were recovered after three washes of the partially digested tissue with Ca2+-free Hanks' solution. Dispersed cells were sedimented by centrifugation at 100 g and resuspended in M199 supplemented with 10% newborn calf serum, 0.2 mM glutamine, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. Primary cultures were passaged onto 75-cm2 flasks, and cells were grown to 90% confluence. All culture flasks, as well as six-well culture plates and Transwell inserts, were previously coated with collagen extracted from rat tails and diluted to a working concentration of 15 µg/ml of 0.1% acetic acid. Culture vessels were incubated with 3, 0.3, and 0.1 ml of collagen solution, respectively, in a laminar flow hood until they were completely dry. Extensive rinsing with sterile Milli-Q water neutralized the acetic acid, and vessels were again air-dried, sterilized under ultraviolet light for 30 min, and stored at 4°C until use. Before stimulation or migration assays, cells were starved for 24 h in Ham's F-12 serum-free medium supplemented with 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenious acid, and 5.35 µg/ml linoleic acid. Cells were detached from culture flasks after a 1-min incubation with 3 ml of 0.1% trypsin-M199 at 37°C and harvested with 20 ml of 0.3% BSA-M199. After a 5-min centrifugation at 5,000 rpm, the medium was removed, and cells were resuspended in 1 ml of 0.3% BSA-M199 and counted. For the migration or protein phosphorylation assays, cells were transferred onto Transwell cell-migration inserts or six-well plates, previously coated with rat tail collagen as described above.
Migration assay. First-passage pulmonary artery cells growth arrested for 24 h were used in all migration experiments. Transwell cell-migration plates, a modification of the Boyden chamber method, were used for this assay. The plates were equipped with inserts whose bottoms were sealed with polycarbonate membranes with a 6.5-mm internal diameter and 8-µm pore size. The wells of Transwell plates were filled with 0.6 ml of bottom solution (M199 cell culture medium containing 0.3% BSA). Cell suspension (100 µl) in M199-0.3% BSA (top solution) was pipetted into the insert, and the insert was transferred into the well. Cell migration was stimulated by PDGF, present in the bottom solution, and with different concentrations of ET-1, placed in the bottom, top, or both solutions. Spontaneous (chemokinetic) migration was evaluated in experiments without chemoattractant. In some inserts, cells were preincubated with PD-98059 for 15 min before and throughout the experiment. The Transwell plates were incubated in a humidified CO2 incubator at 37°C during the migration (5 h). The top solution was then removed, the cells on the top membrane surface were gently scraped with a cotton swab, and the cells on the bottom surface were fixed and stained with Diff-Quik solutions I and II, as recommended by the manufacturer. Cells from five adjacent microscope fields for each membrane were counted at ×40 magnification to obtain the average number of cells per field. The change in chemotactic cell migration was calculated relative to the chemokinetic migration, established as the control in all migration experiments.
ERK MAPK and caldesmon phosphorylation assay. Cultured VSMCs in collagen-coated six-well plates were stimulated with 100 nM ET-1 for 20 min or with 10 ng/ml PDGF for 30 min, unless otherwise specified. Growth medium was withdrawn, and cells were washed twice with ice-cold PBS buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 2.6 mM KCl, and 137 mM NaCl, pH 7.4). Cells were lysed with 150 µl of RIPA buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM Na2EDTA, 0.5% (vol/vol) Nonidet P-40, 0.5% Triton X-100, 1 mM NaF, 1 µM leupeptin, 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 10% glycerol]. Equal amounts of total cell protein (15 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes at 24 V and 4°C for 1.5 h (Genie blotter; Idea Scientific, Minneapolis, MN). For ERK MAPK phosphorylation, membranes were probed for 2 h with a phosphospecific rabbit polyclonal antibody diluted 1:1,000 in 0.1% gelatin-TNT buffer (100 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20). Two polyclonal phosphospecific antibodies were used for assay of caldesmon phosphorylation. They were raised against peptide sequences surrounding the phosphorylated MAPK consensus domains Ser-759 (B1 antibody) and Ser-789 (B3 antibody) of mammalian smooth muscle caldesmon. Antibodies were affinity-purified and characterized as previously described (7). Caldesmon blots were probed for 1.5 h at dilution 1:2,000 in 0.1% gelatin-TNT. After being washed three times (5 min each), membranes were incubated for 1 h in secondary goat anti-rabbit alkaline phosphatase conjugate diluted 1:10,000 in 0.1% gelatin-TNT. Color was developed as appropriate, blots were scanned with a UMAX Powerlook flat-bed scanner (Bio-Rad, Hercules, CA), and immunoreactive band volumes were analyzed by scanning densitometry with Molecular Analyst software. By using dilution series, we have established for ERK MAPKs and caldesmon in preliminary experiments that the immunoreactive signal obtained from 15 µg of total sample protein falls within the linear range of detection (5-25 µg). The increase in protein phosphorylation is expressed relative to bands of control samples.
Time courses of ERK MAPK and caldesmon phosphorylation. First-passage pulmonary artery cells were starved for 24 h and harvested as described in Cell culture. To obtain sufficient cell number and total protein for immunoblotting at the early time points, we transferred 1 × 105 cells in 300 µl of suspension into inserts with a 12-mm diameter and 8-µm pore size. To investigate the dynamics of cell attachment, we allowed cells in six parallel Transwell migration chambers to spontaneously attach and migrate (without PDGF, controls). In a second set of chambers, cell migration was stimulated with 10 ng/ml PDGF. Migration in one chamber of both sets was terminated at 0.5, 1, 2, 3, and 5 h, respectively. Cells that did not attach to the membranes were rinsed with PBS buffer. Cells attached to the top and bottom membrane surfaces were stained with Diff-Quik solutions I and II. Cell number in five contiguous microscope fields was counted twice. With the first count we obtained the total cell number on top and bottom membrane surfaces, which represents total cell attachment. Cells from the top membrane surface were then scraped with a cotton swab, and the cells on the bottom surface were counted to obtain the number of migrated cells.
A similar approach was taken to study phosphorylation of ERK MAPKs and caldesmon during cell migration. Two 12-mm inserts with 1 × 105 cells each were used for all migration times. Cells in one insert were allowed to attach and migrate without attractant (controls), and cells in the other insert were attracted with 10 ng/ml PDGF. At each time point, the polycarbonate membranes were detached from the insert bottom and sprayed with ice-cold PBS buffer to remove all unattached cells. Membranes were then immersed into 50 µl of SDS-containing extraction buffer [25 mM Tris, pH 7.4, 2% SDS, 10% glycerol, 1 mM dithiothreitol (DTT), 1 µM leupeptin, 10 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride] to lyse the attached cells, followed by sonication to extract total protein. These protein homogenates were then mixed with 4× SDS sample buffer to produce final contractions of 0.06 M Tris · HCl (pH 7.8), 2% SDS, 10% glycerol, 1 mM DTT, and 0.03% bromphenol blue. After being boiled for 5 min, proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with phosphospecific anti-ERK and anti-caldesmon antibodies, and the immunoreactive bands were analyzed by densitometry. The PDGF-stimulated protein phosphorylation was corrected for the increase of cell number during the migration experiments, obtained as described in the legend to Fig. 3.Statistical methods. Results are presented as means ± SE. Student's t-test for paired and unpaired data was applied to test for differences between treatment means, as appropriate. A probability of P < 0.05 was accepted as a statistically significant difference.
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RESULTS |
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PDGF and ET-1 stimulate transient phosphorylation of ERK MAPKs and caldesmon in cultured VSMCs. MAPKs phosphorylate mammalian caldesmon at two major sites, equivalent to Ser-759 and Ser-789 of human caldesmon. Data of other laboratories (7) and our results (12) show that while both sites are phosphorylated in vitro, the preferred phosphorylation site in vivo is Ser-789. To test whether phosphorylation of these sites occurs during cell migration, we used antibodies raised against peptide sequences surrounding phosphorylated Ser-759 and Ser-789 of human caldesmon (7). For these experiments, we treated cultured canine PASMCs in collagen-coated six-well plates with 10 ng/ml PDGF and 100 nM ET-1. The time course of phosphorylation of the target proteins (ERK MAPKs and caldesmon) was monitored for 5 h, the duration of a routine cell migration experiment. Protein phosphorylation was assayed in total cell lysates after SDS-PAGE separation and immune detection with phosphospecific antibodies: the anti-ERK MAPK phosphospecific antibodies recognized only the dual-phosphorylated enzymes, while the anti-caldesmon antibodies reacted with caldesmon phosphorylated at Ser-759 (B1 antibody) or Ser-789 (B3 antibody).
ET-1 increased ERK1 and ERK2 MAPK phosphorylation to 3.02 ± 0.51 and 1.87 ± 0.13 times basal migration at 30 min. This phosphorylation then transiently decreased and was indistinguishable from the basal level after 2 h (Fig. 1A). PDGF-stimulated ERK MAPK phosphorylation was also transient but was more potent than ET-1-stimulated phosphorylation, with maximum activation of 13.40 ± 1.16 times basal migration at 15 min (Fig. 1C). PDGF-stimulated ERK MAPK phosphorylation was also higher at later times, remaining five times higher than the basal level even at 5 h (Fig. 1C).
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PDGF, but not ET-1, stimulates chemotactic migration of cultured
PASMCs.
To test the potency of PDGF to stimulate migration of cultured PASMCs,
we carried out migration experiments with different concentrations of
PDGF in the range from 1 to 30 ng/ml. As expected, PDGF triggered a
dose-dependent increase of cell migration: the relationship was almost
linear up to 5 ng/ml PDGF and leveled off at higher concentrations
(Fig. 2A). On the basis of
these observations, we used 10 ng/ml PDGF in the following experiments; this concentration stimulates significant and consistent migration for
routine migration studies.
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ERK MAPKs and caldesmon are phosphorylated during PDGF-stimulated cell migration. In the experiments presented with Fig. 1, cells were uniformly exposed to a constant concentration of PDGF (10 ng/ml) for 5 h in six-well culture plates. Unlike this protocol, cell migration in Transwell plates is driven by a gradient of chemoattractant, which diffuses from the bottom to the top solution through the pores of the separating membrane. In addition, during migration, cells continuously detach and reattach to the matrix, and these processes probably activate transmembrane protein receptors and intracellular protein kinase pathways (1). Therefore, chemotactic cell migration is the response of coordinated ligand-membrane receptor and matrix-transmembrane receptor-initiated intracellular signaling. To test how the gradient of the chemoattractant affects phosphorylation of ERK MAPKs and caldesmon, we carried out cell migration experiments in Transwell plates. Migration was stimulated with 10 ng/ml PDGF, and in separate inserts the process was stopped at different times for 5 h. Time courses of protein phosphorylation in control (chemokinetic migration) and PDGF-stimulated experiments were monitored by immunoblotting. The success of these measurements crucially depends on whether a sufficient amount of total protein is available for immunoblotting, especially at the early times of the experiment. Because the total protein is a function of the cell number on the membrane, it was necessary to first establish the number of cells required for the experiment. The concentration of PDGF used in these experiments (10 ng/ml) was based on our preliminary results: at this concentration PDGF stimulates near maximal cell migration and, hence, allows highly reliable quantitation (Fig. 2A). Migration experiments were then carried out with a different number of cells in the top solution in the range from 2.5 × 104 to 2 × 105 cells. PDGF stimulated significant migration of cells from the top to the bottom membrane surface: ~50 cells per microscope field were counted at 5 h when 2.5 × 104 cells were used for the experiment. This number increased to >200 cells per field with 2 × 105 cells. Under the same conditions, the chemokinetic cell motility accounted for 16% with 5 × 104 cells and decreased to ~10% of the total migration with 2 × 105 cells. For our experiments, an initial number of 1 × 105 cells was determined to provide an optimal ratio of chemotactic vs. chemokinetic migration and a sufficient total number of cells on the bottom membrane surface for reliable protein phosphorylation assay.
The time course of protein phosphorylation during cell migration in Transwell plates depends not only on activation of intracellular signal transduction pathways by PDGF and matrix stimuli but also on changes of the total number of cells on the membrane at the selected times during the experiment. To obtain this information, we investigated the binding of cells to the upper membrane and the rate of appearance of cells on the lower surface of the membrane during migration without PDGF or in the presence of 10 ng/ml PDGF in the bottom solution. The polycarbonate membranes were immersed in Diff-Quik solution at different times during these experiments, and the stained cells on the top and bottom membrane surfaces were counted. Overall, cell attachment was a rapid process. At 30 min, attachment was almost completed, and the total cell number changed little at later times (Fig. 3, E), although tightly bound cells occupied only ~40% of the top membrane surface (Fig. 3, A and C). Also, at early times, cell attachment was apparently independent of PDGF (Fig. 3E, solid triangles and circles). The chemokinetic cell migration was trivial, and only six or seven cells per microscope field were found on the bottom membrane surface even at 5 h (Fig. 3E, open triangles). In contrast, in the presence of PDGF, the number of cells on the bottom membrane surface significantly increased as a result of directional migration. Although at 30 min and 1 h many cells were attached to the top membrane surface, cell migration to the bottom surface was relatively modest (Fig. 3, B and E, open circles). A significant number of cells on the bottom membrane surface were first observed at 2 h, and a more rapid increase was registered between 2 and 5 h (Fig. 3E, open circles). These results suggest that the top membrane surface is saturated with attached cells after ~30 min of the experiment regardless of PDGF. It appears that cell migration from the top to the bottom surface is necessary to permit attachment of new cells and that the increase in the number of attached cells between 0.5 and 5 h reflects net cell migration. Figure 3 allows accurate calculation of the increase of total cell number as a function of the migration time. The average increase of the total cell number in the spontaneous (Fig. 3E, solid triangles) and PDGF-stimulated (Fig. 3E, solid circles) cell migration groups were calculated relative to the total number of cells at 0.5 h. As predicted, PDGF-stimulated cell migration resulted in a total cell number increase of ~40% at 5 h. The determined relative increase of cell number was used to normalize protein phosphorylation and calculate the net increase of ERK MAPK and caldesmon activity during cell migration.
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ERK MAPKs modulate migration of cultured PASMCs.
Because activation of ERK MAPKs and caldesmon occurs simultaneously
with cell migration, we tested the hypothesis that these events are
functionally connected. Migration of cultured PASMCs stimulated
with 10 ng/ml PDGF was used as a functional test, and PDGF-stimulated ERK MAPK activation was inhibited with the
selective MEK1 inhibitor PD-98059. Cell migration was carried out at
different concentrations of PD-98059 ranging from 3 to 50 µM and was
quantified by cell count, as detailed in MATERIALS AND
METHODS. In control experiments, PDGF stimulated a 14.38 ± 2.31-fold increase of chemotactic migration (Fig.
6, solid bar) relative to the
chemokinetic cell movement (Fig. 6, open bar). The lowest concentration
of PD-98059 used (3 µM) did not affect PDGF-stimulated cell
migration, and only a modest reduction was observed at 10 µM PD-98059
(Fig. 6, shaded bars). At a concentration of 50 µM, however, PD-98059
significantly decreased cell migration to ~50% of the maximal level.
As shown in Fig. 5, at the same concentration of PD-98059 (50 µM),
the PDGF-stimulated ERK MAPK phosphorylation is essentially eliminated, and so is the phosphorylation of caldesmon. These results imply that
activation of the ERK MAP kinase-caldesmon pathway may participate in
processes that modulate chemotactic migration.
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DISCUSSION |
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PDGF and ET-1 are simultaneously released at the sites of vascular
wall lesion and stimulate cell proliferation and growth (18). Although the underlying mechanisms are not quite
well understood, PDGF and ET-1 appear to activate similar intracellular events that include calcium mobilization, activation of
phosphatidylinositol 3-kinases, phospholipase C-, and monomeric G
proteins of the Ras, Rac, and Rho family (3). However, the
functional effects of PDGF and ET-1 may be different. With respect to
cell migration, for example, PDGF is considered one of the most potent
chemoattractants, while the features of ET-1 as a chemoattractant are
disputed. In some studies ET-1 has been shown to stimulate cell
migration (9) or potentiate migration initiated by PDGF
(16). In cultured canine PASMCs, PDGF elicited a potent
cell migratory response, while ET-1 failed to stimulate chemotactic
migration in a broad range of concentrations (10 pM to 100 nM). Also,
ET-1 failed to potentiate chemotactic migration triggered by various
concentrations of PDGF. It was recently proposed that a fast activation
of MAPKs by PDGF is required to initiate migration of cultured human
VSMCs (21). Indeed, our experiments with PDGF support this
observation: along with the increase of chemotactic migration, PDGF
stimulated an early peak of ERK MAPK activation. With respect to ET-1,
however, the early activation of ERK MAPKs did not correlate with
increased cell migration. It is likely, therefore, that activation of
the ERK MAPKs is a secondary event and is not required for initiation of chemotactic migration of cultured canine PASMCs. Nonetheless, the
ERK MAPK pathway may still have a role in PDGF-stimulated cell
migration. In studies of other laboratories, as well as in the present
study, inhibition of the ERK MAPKs resulted in decreased migration.
This strongly suggests that MAPKs modulate cell migration. Because the
ERK MAPKs are one of the multiple pathways that are activated by PDGF
(255), it is not surprising that inhibition of ERK MAPKs
does not abolish cell motility.
Mechanisms employed by ERK MAPKs in modulation of cell migration are largely unexplored. Development of force is necessary for cell migration, and ERK MAPKs have been shown to mediate activation of myosin light chain kinase and phosphorylation of regulatory myosin light chains in fibroblasts (15). Actin remodeling is also important for cell migration, and ERK MAPKs have been proposed to participate in actin cytoskeleton remodeling (17). However, the proteins that translate MAPK activation into changes of actin assembly have not been identified. Caldesmon is a plausible candidate, because by binding to several actin monomers, caldesmon stabilizes the filaments and delays actin disassembly and turnover (6). Phosphorylation of l-caldesmon at cyclin-dependent kinase recognition sites has been suggested to cause dissociation from actin filaments during cytokinesis (28). Caldesmon phosphorylation is increased during PDGF-stimulated migration of cultured PASMCs, and it is tempting to speculate that this phosphorylation is necessary to facilitate actin reorganization during migration. Other factors may also be important for actin turnover. The phenotypic shift from h-caldesmon to l-caldesmon in dedifferentiated migrating cells, for example (23), may also be necessary to facilitate actin disassembly. These factors are favorably present in cultured PASMCs. There is a prevalence of the l-caldesmon isoform compared with h-caldesmon. Stimulation with PDGF increases caldesmon phosphorylation along with chemotactic cell migration, and migration is reduced upon inhibition of the MAPK-mediated caldesmon phosphorylation. Caldesmon, therefore, may be one of the ERK MAPK targets that translates activation of MAPKs to alterations of the actin assembly and modulation of PDGF-stimulated migration of cultured PASMCs. Different approaches including actin assembly analysis and/or wound healing models should be employed to establish relevance of caldesmon phosphorylation for VSMC physiology.
In conclusion, we provide evidence that PDGF- stimulated migration of cultured canine PASMCs is paralleled by enhanced phosphorylation of ERK MAPKs and their target protein, caldesmon. Activation of ERK MAPKs is insufficient to initiate chemotactic migration, but inhibition of ERK MAPKs completely blocks caldesmon phosphorylation and partially decreases the PDGF-stimulated migration. On the basis of these results, we propose that the ERK MAP kinase-caldesmon pathway be considered as a modulator of actin remodeling and chemotactic migration of cultured PASMCs.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Shanti Rawat and Michelle Deetken.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-48183 (to W. T. Gerthoffer).
Address for reprint requests and other correspondence: I. A. Yamboliev, Dept. of Pharmacology, MS 318, Univ. of Nevada School of Medicine, Reno, NV 89557-0046 (E-mail: yambo{at}med.unr.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.
Received 1 December 2000; accepted in final form 7 February 2001.
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