PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs

Ilia A. Yamboliev, Jennifer Chen, and William T. Gerthoffer

Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pulmonary artery smooth muscle cell (PASMC) adhesion, spreading, and migration depend on matrix-stimulated reorganization of focal adhesions. Platelet-derived growth factor (PDGF) activates intracellular signal transduction cascades that also regulate adhesion, spreading, and migration, but the signaling molecules involved in these events are poorly defined. We hypothesized that phosphatidylinositol (PI) 3-kinases and Src tyrosine kinases translate matrix and PDGF-initiated signals into cell motility. In experiments with cultured canine PASMCs, inhibition of PI 3-kinases with wortmannin (0.3 µM) and LY-294002 (50 µM) and of Src kinase with PP1 (30 µM) did not decrease spontaneous (nonstimulated) or PDGF-stimulated (10 ng/ml) adhesion onto collagen. PI 3-kinase and Src kinase activities, however, were necessary for cell spreading: PP1 inhibited cell spreading and Src Tyr-418 phosphorylation in a concentration-dependent manner. Inhibition of PI 3-kinase and Src partially reduced cell migration, while at 10 and 30 µM, PP1 eliminated migration, likely due to inhibition of PDGF receptors. In conclusion, both PI 3-kinases and Src tyrosine kinases are components of pathways that mediate spreading and migration of cultured PASMCs on collagen.

phosphatidylinositol 3-kinases; cell adhesion; vascular smooth muscle cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DYNAMIC INTERACTIONS between cell surface receptors and components of the extracellular matrix play a key role for the control of cell behavior, which includes cell adhesion, spreading, and migration (18). Integrin receptor binding initiates signals, termed outside-in signaling, that stimulate tyrosine phosphorylation of several intracellular proteins including members of the Src family and the focal adhesion kinase (FAK) (4, 7, 23). Integrin receptors thus provide linkage between extracellular matrix proteins and focal adhesions, cytoskeleton, and other components of the intracellular space. Src family tyrosine kinases have been proposed to play an active role in processes localized at the focal adhesion structures. It has been shown that during cell adhesion, members of the Src family kinases translocate to focal adhesions and regulate rearrangement of actin structures. Overexpression of kinase-inactive Src reduces the rate of vascular smooth muscle cell (VSMC) spreading on collagen, while wild-type Src kinase enhances spreading (12). These observations suggest that Src kinase activity is required for cytoskeletal reorganization and signal transduction at the focal adhesions. However, Src kinase activity may be insufficient to elicit these effects: there is growing evidence to suggest that association between Src and FAK is necessary for full activation of both tyrosine kinases (reviewed in Ref. 6). It has been shown that Src activation is regulated by factors such as the COOH-terminal Src kinase (Csk), which interferes with the association of Src and FAK (25). In turn, inhibition of Src activity leads to decreased phosphorylation of FAK (29). Association of Src and FAK is not only necessary for their mutual activation but also may recruit other signaling molecules to the focal adhesions (8, 10), mediate phosphorylation of focal adhesion proteins (6), and ultimately cause reorganization of the focal adhesion structure (13). These observations identify Src tyrosine kinases as key components of the integrin-activated signaling pathways.

Smooth muscle cell adhesion, spreading, and migration also depend on growth factors, hormones, and cytokines (28). Activation of tyrosine kinase or G protein-coupled receptors mediates downstream activation of Src and leads to increased phosphorylation of FAK and focal adhesion proteins (10). This is another mechanism, referred to as inside-out signaling, that triggers reorganization of focal adhesions and affects cell motility (15). Thus Src tyrosine kinases are components of both the outside-in and inside-out signaling pathways.

Platelet-derived growth factor (PDGF)-stimulated cell migration is also associated with increased phosphatidylinositol (PI) 3-kinase activity (14) and phosphatidylinositol turnover (3). Activated PI 3-kinases also associate with FAK and increase FAK phosphorylation (1, 19). Thus PI 3-kinase activity may be required for focal adhesion reorganization and cell-matrix interactions. Although cell adhesion (13) and migration (2) may depend on PI 3-kinase activity, the importance of PI 3-kinases and Src tyrosine kinases in mediation of PDGF-stimulated inside-out signaling is poorly characterized. In the present study we hypothesized that PI 3-kinases and Src tyrosine kinase activity is required for adhesion, spreading, and migration of cultured VSMCs on collagen-coated surfaces. We show that while PI 3-kinases appear to mediate PDGF receptor-initiated effects, Src tyrosine kinases are involved in both PDGF receptor- and matrix protein-initiated events and are important regulators of cell adhesion, spreading, and migration.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. PDGF-AB and wortmannin were purchased from Sigma (St. Louis, MO), LY-294002 was purchased from Calbiochem (La Jolla, CA), and 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP1) was from Alexis (San Diego, CA). Transwell cell migration plates were purchased from Corning Costar (Corning, NY), cell culture medium M199 and newborn calf serum (NCS) were from GIBCO (Gaithersburg, MD), and Diff-Quik staining solutions I and II were from Baxter Diagnostics (McGaw Park, IL). Polyclonal phosphospecific anti-Akt/protein kinase B (PKB) antibody was purchased from New England Biolabs (Beverly, MA), polyclonal phosphospecific anti-Src (pY418) antibody was from BioSource International (Camarillo, CA), polyclonal antiPDGF receptor (PDGFR) type B antibody was from Upstate Biotechnology (Lake Placid, NY), mouse monoclonal anti-phosphotyrosine (pY99) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and secondary anti-rabbit and anti-mouse antibodies conjugated to alkaline phosphatase were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All commonly used reagents were from commercial sources.

Cell culture. Canine pulmonary artery smooth muscle tissue was obtained from adult mongrel dogs of either sex, euthanized by barbiturate overdose. 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 digested with 1 mg/ml type II collagenase, 0.1 mg/ml protease (P5147; Sigma), 2 mg/ml bovine serum albumin (BSA), 2 mg/ml trypsin inhibitor, and 0.3 mg/ml Na2ATP at 37°C for 1.5 h. Cells were then recovered by three washes of the partially digested tissue with Ca2+-free Hanks' solution at 37°C. Dispersed cells were sedimented by centrifugation and resuspended in M199 cell culture medium supplemented with 5% NCS, 0.2 mM glutamine, and antibiotics. Primary cultures were passaged onto 75-cm2 cell culture flasks coated with type I rat-tail collagen and grown to 80% confluence. Cells were starved for 24 h before each assay in M199 cell culture 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. For adhesion and spreading experiments, cells were detached from the culture flasks by a 1-min incubation with 3 ml of 0.1% trypsin/EGTA/M199 at 37°C and harvested with 20 ml of 0.3% BSA/M199. After a 5-min centrifugation at 5,000 rpm, the growth medium was discarded, cells were resuspended in 3 ml of fresh 0.3% BSA/M199 medium and counted to obtain the number of cells per 1 ml of suspension.

Cell adhesion assay. These experiments were carried out in 24-well culture plates previously coated with 0.3 ml of rat-tail collagen solution (15 µg of collagen per 1 ml of 0.1% acetic acid). Plates were kept in a laminar flow hood until wells were completely dry. The acetic acid was neutralized by extensive rinsing with sterile Milli-Q water, and plates were allowed to dry and were stored at 4°C until use. The experiments were started with transfer of cell suspension containing 4 × 104 cells in 0.5 ml of 0.3% BSA/M199 medium into the wells. In some adhesion experiments, PASMCs were allowed to attach to the surfaces without stimulant (spontaneous adhesion) but in the presence of kinase inhibitors such as the PI 3-kinase inhibitors wortmannin (0.3 µM) or LY-294002 (50 µM) or different concentrations of the Src inhibitor PP1. In other experiments, cell attachment was stimulated with 10 ng/ml PDGF, without (controls) or with the same concentrations of wortmannin, LY-294002, or PP1. After 1 h at 37°C, cell adhesion was stopped by removal of the growth medium and a twofold wash 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 then fixed with 0.5 ml of 3.7% formaldehyde/PBS for 10 min. After two washes with 0.5 ml of PBS, cells were stained with Diff-Quik solutions I and II as recommended by the manufacturer (Baxter Diagnostics). The excess stain was rinsed with PBS, and the residual cell-bound stain was recovered in 100 µl of 0.1 N HCl. Ninety microliters of this solution were transferred into 96-well plates, and absorbance was read at 595 nm. To determine the cell number that allows reliable quantification of cell adhesion, we tested the cell number-absorbance relationship in preliminary experiments. This relationship was linear within a range from 1 × 104 to 1 × 105 cells. Forty thousand cells provide reliable sensitivity of absorbance readings and were used in each adhesion experiments. To ensure reproducibility, we carried out all cell treatments in triplicate, and the average of the three absorbances represented the result of the experiment.

Cell spreading. These experiments were carried out in 24-well culture plates previously coated with rat-tail collagen as described in Cell adhesion assay. Forty thousand cells in 0.5 ml of 0.3% BSA/M199 medium were transferred into the wells and allowed to attach for 1 h at 37°C in the absence (controls) or presence of PDGF (10 ng/ml), with or without the kinase inhibitors wortmannin, LY-294002, or PP1. Media were then discarded, and unattached cells were removed by two washes with 0.5 ml of PBS prewarmed to 37°C. Fresh media (0.5 ml) with all constituents, including PDGF and kinase inhibitors, were added to the wells, and bright-field images of marked microscope fields were collected at ×40 magnification. Pictures of the same fields were also taken at 5 h. Analysis of cell spreading was based on cell morphology: cells that assumed long and thin morphology were considered spread cells, whereas non-spread cells retained a round shape with uneven outline. We measured both the long and short axes of each cell in the image, using tools of the Mocha Image Analysis software (Jandel, San Rafael, CA), and calculated the cell axes ratio as the ratio of the long to short cell axes. Cell axes ratios were used to construct frequency distribution histograms and to calculate the medians for each treatment group. Frequency distribution plots were fitted to polynomials to determine the maximal cell axes ratio for the treatment group. The effect of kinase inhibitors on cell spreading was evaluated by the leftward or rightward shift of the distribution curve and the change of median values compared with cell controls.

Cell migration assay. First-passage pulmonary artery cells at 90% confluence were growth-arrested for 24 h and used in the 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 (6.5-mm internal diameter, 8-µm pore size). The polycarbonate membranes used in all migration experiments were coated with 3 mg/ml rat-tail collagen solution as described in Cell adhesion assay. The wells were filled with 0.6 ml of M199 cell culture medium containing 0.3% BSA. One hundred microliters of cell suspension (~7.5 × 105 cells) in 0.3% BSA/M199 were transferred into the insert, and the inserts were transferred into the wells. Cell migration was stimulated by PDGF (10 ng/ml) present in the bottom solution. Spontaneous cell migration was evaluated in experiments without chemoattractant. For some experiments, cells were preincubated with different concentrations of the PI 3-kinase inhibitors wortmannin and LY-294002 or the Src inhibitor PP1 for 15 min before and throughout the experiment. Migration was carried out in a humidified CO2 incubator at 37°C for 5 h. At the end, the top solutions were removed, the cells on the top membrane surface were gently scraped with cotton swabs, and the cells on the bottom surface were fixed and stained with the Diff-Quik solutions I and II. 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 number of spontaneously migrated cells (without PDGF).

Protein phosphorylation assay. Changes in protein phosphorylation during cell adhesion experiments were assayed by Western blotting analysis of total cell lysates. To obtain a sufficient amount of total cell protein required for immunoblotting, we carried out these experiments in collagen-coated six-well cell culture plates using 2.4 × 105 cells in 2.4-ml cell suspension. The treatment groups and conditions were the same as in the adhesion experiments. Growth media were removed after 1-h incubation at 37°C, and cells were washed twice with ice-cold PBS buffer and lysed with 100 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 (AEBSF), and 10% glycerol]. An equal amount of total cell protein (15 µg for immunoblotting of Akt/PKB and PDGFR and 30 µg for Src phosphorylation assay) was resolved by SDS-PAGE (10% acrylamide) and transferred onto nitrocellulose membranes for 1.5 h at 24 V, 4°C (Genie blotter; Idea Scientific, Minneapolis, MN), and the membranes were blocked for 2 h with 0.5% gelatin in TNT buffer (100 mM Tris, pH 7.5, 0.1% Tween 20, and 150 mM NaCl). Akt/PKB phosphorylation was assayed with a phosphospecific rabbit polyclonal antibody raised against a peptide sequence containing phosphorylated Ser-473 of Akt/PKB (New England Biolabs). The phosphospecific anti-Src antibody recognized phosphorylated Tyr-418 amino acid (BioSource International). The membranes were incubated for 1 h with the primary antibody diluted 1:1,000 dilution in 0.1% gelatin/TNT. Excess primary antibody was removed by three 5-min washes with TNT, followed by a 1-h incubation with secondary alkaline phosphatase-conjugated antibodies diluted 1:10,000 with 0.1% gelatin/TNT. Color was developed as appropriate, blots were scanned with a UMAX Powerlook flatbed scanner, and immunoreactive band volumes were analyzed by scanning densitometry with the Molecular Analyst software (Bio-Rad, Hercules, CA). The relative increase in protein phosphorylation was calculated by dividing the density of immunoreactive bands of cells incubated with PDGF and the kinase inhibitors wortmannin, LY-294002, or PP1 by the band density of control cells.

Statistical methods. Results of cell adhesion, migration, and kinase phosphorylation are presented as the mean relative increase above the basal level (not stimulated with PDGF; ±SE). The n value when given refers to the number of parallel experiments. Three or more parallel experiments were usually conducted for each experimental point. Student's t-test for paired and unpaired data or one-way ANOVA for multiple group comparisons to a single control (Dunnett's test) were applied to test for differences between treatment means by using the Sigma Stat software (Jandel). Values of P < 0.05 were considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PI 3-kinases and Src tyrosine kinases are signaling molecules involved in reorganization of focal adhesions and actin cytoskeleton, but less is known about their role in PDGF-stimulated cell motility. To better understand this role, we hypothesized that members of the PI 3-kinases and Src tyrosine kinases are necessary for regulation of adhesion, spreading, and migration of vascular myocytes.

PI 3-kinases and Src kinases may be unnecessary for cell adhesion. To investigate how PI 3-kinases and Src kinases regulate cell adhesion, we carried out adhesion experiments using selective inhibitors of these kinases. The fungal metabolite wortmannin is an established inhibitor of PI 3-kinases and has been extensively used in studying roles of PI 3-kinases in various cellular processes (5, 16). In low nanomolar concentrations, wortmannin is a selective PI 3-kinase inhibitor, whereas inhibition of other kinases was shown at higher concentrations (17). Although less potent, LY-294002 exhibits better selectivity for PI 3-kinases and is a preferred alternative (26). We used PP1, the selective inhibitor of Src family tyrosine kinases, to inhibit signaling via Src-mediated pathways (9).

Adhesion onto the matrix is an early event in regulation of cell migration toward a gradient of chemoattractant. To address the question of how PI 3-kinases and Src kinases regulate adhesion of PASMCs onto collagen, we allowed 4 × 104 cells to adhere onto collagen-coated 24-well cell culture plates. After 1 h at 37°C, the attached cells were stained, the dye was extracted, and cell adhesion was quantified from dye absorbances. PDGF was not present in some of the wells: the adhesion of these cells will be further referred to as the spontaneous adhesion. Even without PDGF, a significant number of PASMCs adhered onto the collagen-coated surface. Cell adhesion remained virtually unchanged in the presence of the PI 3-kinase inhibitors LY-294002 (50 µM) and wortmannin (0.3 µM) and the Src tyrosine kinase inhibitor PP1 (30 µM) (Fig. 1A). Different reasons could account for this result: PI 3-kinase and Src kinase activities are not suppressed by the inhibitors, or the activity of these kinases is not necessary for cell adhesion onto collagen. Because PDGF was not present, activation of PI 3-kinases and Src kinases by ligand-receptor association can be ruled out. The presence of PI 3-kinase and Src kinase inhibitors, therefore, could be expected to affect either the basal activity of these kinases or activation initiated by integrin receptors during adhesion. To test whether wortmannin and LY-294002 affected the basal activity of PI 3-kinases, we determined phosphorylation of Akt/PKB, a kinase that is activated by PI 3-kinases in different cell types (11). These measurements were made with total lysates obtained from cells that underwent an experimental protocol similar to the standard cell adhesion protocol described in MATERIALS AND METHODS. We used the basal phosphorylation of Akt/PKB (Fig. 1B, control) as a reference value to determine the relative change of response. In cells that adhered in the presence of LY-294002 and wortmannin, phosphorylation of Akt/PKB was significantly reduced to about 40 and 30% of the basal phosphorylation, respectively. PP1 (30 µM) did not decrease cell attachment onto collagen and did not alter the basal phosphorylation of Akt/PKB (Fig. 1B). Our present results therefore suggest that spontaneous adhesion of PASMCs onto collagen proceeded in a manner apparently independent of PI 3-kinases or Src kinases.


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Fig. 1.   Phosphatidylinositol (PI) 3-kinase and Src kinase activities are not required for spontaneous (nonstimulated; left) and platelet-derived growth factor (PDGF)-stimulated (right) adhesion of pulmonary artery smooth muscle cells (PASMCs) onto collagen. Forty thousand cells in 0.5-ml suspension were allowed to adhere onto collagen-coated 24-well cell culture plates in the absence (control, C) or in the presence of the PI 3-kinase inhibitors LY-294002 (LY; 50 µM) and wortmannin (Wm; 0.3 µM) or the Src tyrosine kinase inhibitor PP1 (30 µM). After 1 h at 37°C, the attached cells were stained with Diff-Quik solutions I and II, and adhesion was quantified by absorbances at 595 nm and presented as the relative change compared with cell controls (A and C; n = 4). In parallel wells, attached cells were scraped and lysed, and extracts were used for assay of Akt/protein kinase B (PKB) phosphorylation by immunoblotting. The immunoreactive bands of phosphorylated Akt/PKB in controls and inhibitor-treated cells were assayed in duplicate (B, blot). Band densities were used to calculate the relative change of Akt/PKB phosphorylation relative to controls (B and D, bar graphs; n = 4). *P < 0.05 compared with controls; **P < 0.05 compared with PDGF-stimulated cells without kinase inhibitors (Student's t-test). Dotted lines in bar graphs project adhesion of cell controls.

Similar results were obtained when cell adhesion was stimulated with 10 ng/ml PDGF: the net absorbances of Diff-Quik-stained cells were comparable to these of spontaneously adhering control cells, suggesting that PDGF did not significantly change the net cell adhesion. Presented in relative units of change, cell adhesion was neither increased by PDGF nor decreased by the PI 3-kinase or Src kinase inhibitors (Fig. 1C). On the other hand, PDGF potently increased phosphorylation of Akt/PKB to about nine times basal level, and this increase was eliminated by 50 µM LY-294002 and 0.3 µM wortmannin and partially decreased by 30 µM PP1 (Fig. 1D). Failure to increase cell adhesion to collagen was not specifically associated with PDGF, since 5% NCS also failed to stimulate adhesion while producing phosphorylation of Akt/PKB similar to that stimulated by PDGF (not shown). These results therefore suggest that while PDGF and NCS activate multiple intracellular pathways, activation of PI 3-kinases and Src did not translate into an increase of PASMC adhesion onto collagen.

Results from other laboratories have shown that Src activity may more closely control cell adhesion and spreading on fibronectin than on collagen (13, 25). To test this possibility, we conducted adhesion experiments by following the same general approach but using fibronectin-coated culture plates. Under our experimental conditions, however, both the spontaneous and PDGF-stimulated adhesion of PASMCs onto fibronectin was very poor compared with the adhesion onto collagen. Because these experiments did not produce reliable results, detailed comparative studies between fibronectin and collagen with respect to cell adhesion, spreading, and migration were not carried out.

PI 3-kinase and Src kinase activities are required for cell spreading on collagen. To evaluate the role of PI 3-kinases and Src kinases in spreading of PASMCs on collagen, we inhibited kinase activities and monitored the changes in cell morphology during spreading on collagen. Using phase-contrast microscopy, we generated images of one selected field at 1 and 5 h after the initiation of the assay and used these images to quantitatively evaluate cell spreading. One hour after initiation of the experiment, PASMCs were usually in the initial phase of attachment to the matrix and appeared as either dark dots surrounded by bright rings or round-shaped bodies with uneven outline. At later times the cells assumed longer and thinner morphology, which is characteristic of cultured smooth muscle cells. To quantify cell spreading, we collected images after 5 h and calculated the ratio of the long to short axes of all cells in the image. Using these ratios, we calculated the median of the axes ratio distributions within the treatment groups and visualized the results using frequency distribution histograms. Figure 2 depicts images of cells allowed to spontaneously attach and spread on collagen-coated surfaces. Most cells within the monitored populations appeared as balls and were not spread after 1 h (Fig. 2, A-C). The progress of spontaneous cell spreading was more obvious at 3 h (not shown) and was clear at 5 h. The frequency distribution plots showed that most of the cells in the control group had an axes ratio of 2-3 and a median of 3.43 (Fig. 3A). Inhibition of PI 3-kinases with 0.3 µM wortmannin did not significantly change the axes ratio (range 2-3) of the spontaneously spread cells but significantly decreased the median cell distribution from 3.43 in controls to 2.55 in this treatment group (Fig. 3B). Inhibition of Src kinases with 30 µM PP1 did not notably alter the total number of cells per field; however, PP1 treatment dramatically changed cell morphology toward more round, less spread cells (Fig. 2F). These Src kinase-dependent morphological changes resulted in a leftward shift of the cell axes ratio to a range of 1-2 and a decrease of the median distribution to 2.12 (Fig. 3C). Because cell spreading in these experiments was not stimulated by PDGF, the results suggest that both PI 3-kinases and Src kinases are activated by events localized at focal adhesions. While both kinase activities mediate spreading, catalytic activity of Src tyrosine kinases seems crucial during spontaneous spreading of PASMCs on collagen.


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Fig. 2.   Spontaneous (nonstimulated) PASMC spreading on collagen depends on PI 3-kinase and Src kinase activity. Bright-field images of the same microscope field were generated at 1 and 5 h of cell spreading assays. The progress of spreading of control cells (A and D), cells incubated with the PI 3-kinase inhibitor wortmannin (0.3 µM; B and E) and the Src kinase inhibitor PP1 (30 µM; C and F) is presented with images taken at 1 (left) and 5 h (right) of the experiment. The 5-h images were used to measure the long and short cell axes of all cells in the field (Mocha Image Analysis software; Jandel). These data were used to calculate the cell axes ratios (long/short axes) and the median of the cell distribution and to construct the frequency distribution plots (shown in Figs. 3 and 4). The cell spreading experiment was repeated with 3 different batches of cells.



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Fig. 3.   Frequency distribution histograms depicting the spontaneous (left) and 10 ng/ml PDGF-stimulated (right) cell spreading of PASMCs on collagen. The PI 3-kinase inhibitor wortmannin (0.3 µM) and the Src kinase inhibitor PP1 (30 µM) reduced the spontaneous (B and C) and PDGF-stimulated (E and F) cell spreading, compared with controls (A and D). Phase-microscopy images were taken 5 h after the initiation of PASMC spreading assay, and the cell axes ratios of all cells in the images were used to construct the frequency distribution histogram for each treatment group and to calculate the medians. The histograms were fitted to polynomials to determine the maximum cell axes ratio for the group. Results are averages of 3 parallel experiments.

Furthermore, we tested how activation of intracellular pathways by PDGF affects cell spreading on collagen and what role PI 3-kinases and Src kinases play in this event. The PDGF-stimulated cell spreading of control group of cells (without kinase inhibitors) produced longer and thinner cells compared with spontaneously spread controls. As a result, there was a net rightward shift of the cell axes ratio to the range of 3-4 and an increase of the median to 4.47 (Fig. 3D). Compared with PDGF-stimulated controls, inhibition of PI 3-kinases with 0.3 µM wortmannin delayed cell spreading, caused a leftward shift of the axes ratio to 2-3, and decreased the median to 2.28 (Fig. 3E). The median and the cell axes ratio distribution in this treatment group were similar to these of spontaneously spread cells treated with wortmannin (Fig. 3B), suggesting that wortmannin abolished the PDGF-evoked increase of cell spreading. Inhibition of Src kinases by 30 µM PP1 in the PDGF-treated cells resulted in a dramatic reduction of cell spreading to a cell axes ratio range of 1-2 and a median of 2.06 (Fig. 3F). Because Src inhibition by PP1 effectively reduced cell spreading in both spontaneous and PDGF-stimulated groups, Src kinases are key enzymes not only in local focal adhesion events but also in regulation of PDGF receptor-initiated mechanisms of cell spreading on collagen.

The concentration of PP1 used in our cell adhesion and spreading experiments (30 µM) is about two orders of magnitude higher than the reported IC50 of PP1 in other cell types, including human coronary artery smooth muscle cells (27). To our knowledge, relevant information in canine PASMCs is not available. To test the inhibitory potency of PP1 for Src during spreading of canine pulmonary artery myocytes on collagen, we stimulated cell spreading with PDGF and, after 1 h, lysed cells and assayed phosphorylation of Src in the presence of PP1 at concentrations ranging from 0.3 to 30 µM. For these experiments we measured phosphorylation of Src at Tyr-418, which is necessary for activation of the kinase (21). PDGF (10 ng/ml) significantly increased cell spreading compared with PDGF-nonstimulated controls (Fig. 4, A and B). PDGF-stimulated cell spreading was slightly decreased by 0.3 µM PP1 (Fig. 4C), significantly retarded by 1 and 3 µM PP1, and dramatically affected by 10 and 30 µM PP1 (Fig. 4, D and E). Src phosphorylation at Tyr-418 was also concentration-dependently decreased by PP1: 0.3 µM PP1 significantly decreased PDGF-stimulated Src phosphorylation, 1 µM PP1 caused almost complete inhibition, and 3 and 10 µM PP1 abolished Src phosphorylation (Fig. 5, A and B). In these experiments PP1 did not cause obvious changes of Src protein levels (Fig. 5A, bottom). After normalization of the Tyr-418 phosphorylation bands to the respective Src protein band densities, we calculated an apparent IC50 of PP1 for Src of 0.88 µM (n = 2), a value consistent with previously reported data by Waltenberger et al. (27). Because these investigators also showed that, in addition to Src, PP1 could inhibit other signaling molecules, we further tested whether PDGFR phosphorylation was affected by the utilized concentration of PP1. This was a necessary assay since inhibitory effects of PP1 on PDGFR could obscure the role of PDGF-stimulated Src in regulation of cell spreading. Cell stimulation with 10 ng/ml PDGF did not significantly alter PDGFR expression (Fig. 5C, bottom) but produced a significant increase in tyrosine phosphorylation of PDGFR type B (Fig. 5C, top). Normalized data revealed that PP1 decreased PDGFR phosphorylation in a concentration-dependent fashion, with an IC50 = 9.7 µM (Fig. 5D). In comparison, however, the inhibitory affinity of PP1 for PDGFR was about 10 times lower than for Src kinase. These results allow us to distinguish effects mediated by Src kinase from effects mediated by PDGFR. For example, 3 µM PP1 completely inhibited tyrosine phosphorylation of Src, while PDGFR was only slightly affected. At this concentration of PP1, therefore, decrease of cell spreading is predominantly due to inhibition of Src kinase activity, rather than to inhibition of PDGFR. As shown with Fig. 4, Src activity is necessary for cell spreading, although inhibition of Src with 3 µM PP1 did not abolish cell adhesion and spreading on collagen.


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Fig. 4.   PDGF (10 ng/ml)-stimulated spreading of PASMCs on collagen (B) compared with PDGF-nonstimulated cell controls (A). The Src tyrosine kinase inhibitor PP1 (0.3-10 µM) reduced PDGF-stimulated spreading in a concentration-dependent manner (C-F). Frequency distribution histograms were generated from the cell axes ratios of all cells in phase-microscopy images, taken 5 h after the initiation of cell spreading assay. Results are averages of 2 parallel experiments.



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Fig. 5.   PP1 inhibits Src kinases and PDGF receptor (PDGFR) type B in a concentration-dependent fashion. Src and PDGFR protein levels and Src (Tyr-418) and PDGFR tyrosine phosphorylation were assayed in cell lysates obtained at 1 h of PDGF-stimulated spreading of PASMCs on collagen. Cell lysates were subjected to Western blotting analysis with regular and phosphospecific antibodies against Src and PDGFR, followed by densitometry of immunoreactive bands. Immunoreactive bands of phosphorylated proteins (A and C, top) were normalized to the protein levels of Src and PDGFR (A and C, bottom), and protein phosphorylation was then presented in relative units compared with PDGF-stimulated controls (B and D, solid bars). Results are averages of 2 parallel experiments. Dotted lines in bar graphs project phosphorylation of Src and PDGFR in cell controls.

PI 3-kinases and Src kinases are required for PDGF-stimulated cell migration. Cell adhesion and spreading may occur independently of a PDGF gradient. Cell crawling, however, is a directional process toward the increasing gradient of a chemoattractant. Because PI 3-kinases and Src tyrosine kinases are necessary for cell spreading, we next tested the hypothesis that PI 3-kinases and Src kinase activities are required for PDGF-stimulated directional migration. PDGF (10 ng/ml) was used to stimulate migration of control PASMCs or cells incubated with PI 3-kinase and Src inhibitors before and throughout the migration assay. As a result of spontaneous cell migration (without PDGF), 20-25 cells per microscope field migrated from the top to the bottom membrane surface of the migration inserts. PDGF stimulated about an eightfold increase of this number (Fig. 6A). Cell migration was insignificantly decreased by 0.03 and 0.1 µM wortmannin, while at 0.3 µM and higher concentrations, wortmannin significantly inhibited migration. Because 0.3 µM wortmannin is sufficient to completely inhibit PI 3-kinases, decrease of cell migration at the higher concentrations could be attributed to nonspecific effects of wortmannin on other signaling molecules. A dose-dependent decrease of cell migration was also obtained when PI 3-kinases were inhibited with LY-294002, but complete inhibition was again not achieved (Fig. 6A). These results suggest that, while important, PI 3-kinases are not crucial for chemotactic migration of cultured PASMCs.


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Fig. 6.   PI 3-kinase and Src tyrosine kinase mediated PDGF-stimulated chemotactic migration of PASMCs. Migration was carried out with 7.5 × 105 cells in Transwell cell migration plates with 6-mm internal insert diameter and 8-µm pore size. Cells were incubated with kinase inhibitors for 10 min before and throughout the assay. Migration was stimulated with 10 ng/ml PDGF in the bottom solutions and was carried out for 5 h at 37°C. Cells were then fixed and stained with Diff-Quik solutions I and II. Cells were removed from the top membrane surface and counted in 5 contiguous fields on the bottom surface, and the average cell number was used to calculate the change of migration relative to the count of the spontaneously migrated cells (open bars). The PI 3-kinase inhibitors wortmannin and LY-294002 (A, n = 6) and the Src kinase inhibitor PP1 (B, n = 8) dose-dependently reduced PDGF-stimulated migration. Data were evaluated using one-way ANOVA for multiple group comparisons to a single control (Dunnett's test). *P < 0.05 compared with spontaneously migrating cells; **P < 0.05 compared with PDGF-stimulated migration without kinase inhibitors (solid bars). Dotted lines in bar graphs project spontaneous migration of cell controls.

Similar experiments were carried out to test the role of Src tyrosine kinases for migration of cultured PASMC. In these experiments PDGF (10 ng/ml) stimulated a 6.71 ± 1.49-fold increase of the number of migrated cells compared with controls (Fig. 6B). At 1 µM, PP1 slightly decreased cell migration to 6.22 ± 0.38 times basal level and 3 µM PP1 significantly reduced migration to 3.71 ± 0.76 times basal level, while 10 µM PP1 abrogated the PDGF-stimulated increase of cell migration (Fig. 6B). These results suggest that both PI 3-kinases and Src tyrosine kinases regulate migration of cultured PASMCs toward PDGF.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sheer stress and vascular wall damage activate multiple intracellular signal transduction pathways in smooth muscle cells and induce responses such as adhesion onto matrix proteins, spreading, migration, and growth. Some major mechanisms involved in these processes include an increase of diacylglycerol and phosphatidylinositol 1,4,5-trisphosphate concentrations and Ca2+ mobilization (8), activation and translocation of protein kinase C's (PKCs) (20), and activation of mitogen-activated protein kinases and FAK (22). In this study we evaluated the role of PI 3-kinases and Src tyrosine kinases in the discrete steps of smooth muscle cell migration: adhesion, spreading, and crawling. One major observation was that cell adhesion onto collagen was apparently independent of PI 3-kinase and Src kinase activity but may instead depend on adhesion-activated integrin receptors and subsequent reorganization of focal adhesion structures. It has been suggested that weak associations between integrin receptors and components of the extracellular matrix characterize the early stages of cell adhesion. Cell tethering during this phase may be mediated by protein phosphorylation and/or activation of kinases localized at the focal adhesions. Cell adhesion therefore might be independent of signals initiated by cell membrane receptors and propagated through intracellular signaling cascades (7, 24). Such a model is consistent with our observations and can explain why activation of PI 3-kinases and Src by PDGF did not translate into enhanced cell adhesion. It can also explain why inhibition of PI 3-kinases and Src kinases did not attenuate attachment of canine PASMCs to collagen. It should be pointed out, however, that while this model is consistent with our results, it may have limited applicability to other smooth muscle cell types and may depend on the individual experimental protocols. For example, adhesion studies of other laboratories have produced a prominent increase of human VSMC attachment onto collagen I upon stimulation with PDGF (13). With respect to cell spreading on collagen, the results of that laboratory are similar to our observations: in both studies, PDGF produced an increase of cell spreading (12). These observations imply that human and canine vascular smooth muscle cells may differ in their timing or magnitude of responses to PDGF, in the orchestration of mechanisms that couple upstream signals to focal adhesion effectors, or in the organization of the focal adhesions and regulation of cell-matrix interactions.

Regulation of cell spreading may include different intracellular signaling pathways that function in a cooperative manner. Significant cell spreading occurred even when cells were not stimulated with PDGF. Clearly, spontaneous adhesion and spreading are initiated by physical interactions between cell structures at the basement cell membrane and matrix components, and extracellular stimuli may not be required to accelerate these processes. Unlike adhesion to collagen, however, Src kinase activity was required for cell spreading: inhibition of Src kinases dramatically decreased cell spreading. Integrin-stimulated Src kinase activity may be important for tyrosine phosphorylation and regulation of FAK (6), which is then necessary for reorganization of focal adhesions. PI 3-kinase activity appeared less important for spontaneous spreading, suggesting that PI 3-kinases were not components of the outside-in signaling during spontaneous spreading of canine PASMCs on collagen.

The role of PI 3-kinases and Src may be quite different in the presence of exogenous stimulants of cell spreading such as PDGF. In our experiments, inhibition of PI 3-kinases decreased, although did not abolish, cell spreading, suggesting a moderate role of PI 3-kinase in the ligand-activated intracellular signaling to focal adhesions. Although blockade of Src activation by PP1 more dramatically decreased cell spreading, even complete inhibition of Src did not abrogate cell spreading. Similar results were obtained with respect to cell migration: even complete inhibition of PI 3-kinases and Src kinases only partially blocked cell migration. The remaining cell spreading and migration likely reflects the contribution of other mechanisms, which operate in a PDGF-dependent manner, since more thorough inhibition of PDGFR by higher concentrations of PP1 more prominently decreased cell motility. Therefore, simultaneous removal of both the Src-dependent outside-in signaling by lower concentrations of PP1 and the PDGFR-dependent signal transduction at higher concentrations of PP1 compromises crucial mechanisms that regulate focal adhesions and, ultimately, eliminates cell migration.

In conclusion, in this study we showed that PI 3-kinase and Src kinase activity may not be required for adhesion onto collagen, but these activities were necessary for spreading and directional migration of cultured canine PASMCs toward PDGF. These results suggest that local events initiated by interaction of integrins or other proteins at the basement cell membrane with matrix proteins mediate cell adhesion to collagen. Outside-in signaling requires Src activation to trigger moderate spreading and migration. However, stimulation of surface membrane receptors is required for full activation of PI 3-kinases, Src, and other intracellular signal transduction systems that mediate extensive cell spreading and migration. Because lesions of the vascular wall are associated with local release and accumulation of growth factors, our results point to PI 3-kinases and Src tyrosine kinases as important enzymes in processes such as wound healing/restenosis and possibly neovascularization of pulmonary circulation.


    ACKNOWLEDGEMENTS

We acknowledge Shanti Rawat and Michelle Deetken for excellent technical assistance.


    FOOTNOTES

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 2 April 2001.


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