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Circulation. 1995;91:1107-1115

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(Circulation. 1995;91:1107-1115.)
© 1995 American Heart Association, Inc.

Articles

Role of Calcium/Calmodulin-Dependent Protein Kinase II in the Regulation of Vascular Smooth Muscle Cell Migration

Rebecca R. Pauly, MD; Claudio Bilato, MD; Steven J. Sollott, MD; Robert Monticone, MS; Paul T. Kelly, PhD; Edward G. Lakatta, MD; Michael T. Crow, PhD

From the Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Md (R.R.P., C.B., S.J.S., R.M., E.G.L., M.T.C.), and the Department of Neurobiology and Anatomy, University of Texas Medical School, Houston (P.T.K.).

Correspondence to Michael T. Crow, PhD, Laboratory of Cardiovascular Science, Vascular Biology Group, NIH, National Institute on Aging, 4940 Eastern Ave, Baltimore, MD 21224.


*    Abstract
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 *Abstract
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Background The migration of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of many vascular diseases. We have previously shown that VSMC migration in response to platelet-derived growth factor (PDGF) is suppressed when cultured cells are growth-arrested and induced to differentiate. The present study was undertaken to elucidate the mechanism of this suppression.

Methods and Results While both proliferating and growth-arrested VSMCs upregulated expression of the immediate early response genes, c-fos and JE (monocyte chemoattractant protein 1), growth-arrested VSMCs exhibited much smaller changes in intracellular calcium in response to PDGF and failed to activate the calcium/calmodulin-dependent protein kinase II (CaM kinase II). Blocking calcium-calmodulin interactions (50 µmol/L W7) or the activation of CaM kinase II (10 µmol/L KN62) in proliferating cells blocked their migration by more than 90%, whereas inhibition of protein kinase C activation had no significant effect on migration. Pretreatment of growth-arrested VSMCs with the calcium ionophore ionomycin resulted in an approximately 2.5-fold activation of CaM kinase II and increased migration of growth-arrested cells to 84±6% that of proliferating cells. These effects of ionomycin were blocked by inhibitors of CaM kinase II. Constitutively activated (ie, calcium/calmodulin-independent) CaM kinase II introduced by gene transfection into growth-arrested cells significantly increased migration toward PDGF from <20% to >70% that of proliferating cells.

Conclusions These results demonstrate that activation of CaM kinase II is required for VSMC migration, that its activation in response to PDGF is suppressed in growth-arrested VSMCs, and that this suppression of CaM kinase II activation is responsible, in large part, for the failure of growth-arrested VSMCs to migrate toward PDGF.


Key Words: calcium • protein kinases • muscles, smooth • platelet-derived factors • cells


*    Introduction
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 up arrowAbstract
 *Introduction
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In normal blood vessels, the vast majority of vascular smooth muscle cells (VSMCs) reside in the tunica media, where these cells are relatively quiescent with respect to cell proliferation and where they express a host of differentiation-specific genes, many of which play an important role in the regulation of blood vessel tone.1 2 A hallmark of many vascular diseases and all vessel injury is the increased extramedial presence of VSMCs in the tunica intima.3 4 In the case of vessel injury involving endothelial denudation or destruction, the increase in intimal VSMCs often results in the formation of a fibrocellular mass known as the neointima.1 While part of the increase may arise from the proliferation of the small number of VSMCs normally present in the intima of some vessels, the vast majority of VSMCs present in the neointima after balloon catheter injury are thought to originate from medial VSMCs that have migrated across the internal elastic membrane.

VSMCs display at least two distinct phenotypic states in vivo—a "contractile" phenotype, in which the cells are quiescent and in which a relatively high percentage of the cell volume contains longitudinally organized microfilaments composed of smooth muscle–specific contractile proteins, and a "synthetic" phenotype, in which the microfilaments are replaced by extensive rough endoplasmic reticulum and a large Golgi complex.2 Contractile VSMCs compose most, if not all, the VSMCs found in the media of uninjured vessels, whereas VSMCs exhibiting the synthetic phenotype are typically observed in the neointima. A phenotype similar to the contractile state is seen in cell culture in postconfluent growth-arrested VSMCs. While such cells still express large amounts of nonmuscle contractile proteins, they do not proliferate and, depending on their time in culture, express a variety of differentiation-specific genes.2 5 6 VSMCs proliferating in cell culture resemble the "synthetic" phenotype in that they exhibit decreased contractile function, express low levels of smooth muscle–specific genes and increased amounts of interstitial matrix components, and secrete various cytokines and growth factors.5 6 7 The ability of VSMCs to modulate between phenotypic states raises the question of whether these states affect the ability of VSMCs to migrate in response to chemoattractants released at the sites of vessel injury, such as platelet-derived growth factor (PDGF), and whether differences in intracellular signaling in response to PDGF can account for this behavior. We report here that the migration of VSMCs is regulated by their phenotypic state. Proliferating VSMCs readily migrate toward PDGF, whereas the migration of growth-arrested VSMCs is substantially suppressed. This suppression is accompanied by the failure of growth-arrested VSMCs to activate the multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) in response to PDGF and is rapidly reversed by elevating intracellular calcium. By use of specific inhibitors CaM kinase II and gene transfection to express constitutively activated CaM kinase II, we show that activation of CaM kinase II is required for VSMC migration and that the restoration of this activity in growth-arrested cells results in the restoration of their ability to migrate in response to PDGF.


*    Methods
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Materials
Recombinant human PDGF BB was obtained from Collaborative Research. It was dissolved in Dulbecco's modified minimal essential medium (DMEM; high glucose) containing 0.1% BSA and stored in aliquots at -70°C. Each aliquot was used only once. Ionomycin and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical Co; KN-62, W7, and H7 from LC Laboratories; calmodulin from Upstate Biotechnology; and autocamtide-2 from Peninsula Laboratories. With the exception of calmodulin, these reagents were dissolved in dimethyl sulfoxide, divided into aliquots, stored at -70°C, and used only once after thawing.

Cell Culture and Migration Assays
VSMC cultures were established from 3- to 6-month-old Wistar rats as described previously.8 Cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mmol/L nonessential amino acids, 20 mmol L-glutamine, 50 mg/mL penicillin, 50 mg/mL streptomycin, and 10 mg/mL neomycin (GIBCO), in a humidified 5% CO2 atmosphere at 37°C, and were used between the 7th and 14th passages. Proliferating cells were harvested for migration assays or biochemical analyses between 50% and 80% confluency. Growth was arrested by switching to serum-free medium (SFM; DMEM, antibiotic [as above], and 1 mg/L insulin, 0.67 µg/L selenium, and 0.55 mg/L transferrin) for 5 to 7 days. Under these conditions, VSMCs failed to incorporate [3H]thymidine and, depending on cell passage, expressed some of the differentiation-specific markers of the intact vessel. Growth arrest was induced in either preconfluent cultures or in cultures in which the cells were maintained for 1 to 2 days postconfluence to produce the characteristic "hill and valley" morphology. Migration assays were performed with a modified Boyden chamber as previously described.8 9

Intracellular Calcium Measurements
VSMCs were cultured on glass coverslips and incubated in 25 µmol/L of the acetoxymethyl ester derivative of calcium indicator indo-1 (Molecular Bioprobes, Inc) for 45 minutes at 37°C. Ca2+-dependent indo-1 fluorescence was measured in a small number (10 to 20) of indo-1–loaded cells bathed in (mmol/L) HEPES 20, pH 7.4; glucose 15; NaC 137; KCl 5.4; CaCl2 1.8; and MgSO4 1.2. Fluorescence was excited by epi-illumination with 10-microsecond flashes of 350±5-nm light. Indo-1 emission, in spectral windows of 390 to 434 and 477 to 507 nm (corresponding to the Ca2+-bound and Ca2+-free forms of the indicator, respectively), was simultaneously collected by means of paired photomultipliers.10 The ratio of these two channels was used to estimate intracellular calcium levels ([Ca2+]i).11

Protein Kinase C and CaM Kinase II Activity Assays
Protein kinase C (PKC) activity was measured in situ in permeabilized cells according to the procedure of Sadoshima and Izumo12 by use of the PKC-specific substrate PLSRTLSVAAKK alone or in combination with 100 µmol/L PKC pseudosubstrate peptide (RFAKGALRQKNVHEVKN). After stimulation with PDGF or PMA, cells were rinsed once in PKC buffer, consisting of Dulbecco's PBS (DPBS) containing 1 mg/mL glucose, 20 mmol/L HEPES (pH 7.2), 25 mmol/L ß-glycerophosphate, 5 mmol/L EGTA, and 2.5 mmol/L CaCl2, and then permeabilized while attached to the plate in PKC buffer containing 0.01% saponin, 10 µmol/L ATP, 5 nmol/L {gamma}[32P]ATP (5 µCi per reaction: 6000 Ci/mol), and 200 µmol/L PKC-specific substrate peptide. After a 10-minute reaction at 37°C, the reaction was terminated by addition of trichloroacetic acid (TCA) to a final concentration of 8%. A 50-µL aliquot of the reaction mixture was removed, spotted onto Whatman P81 paper, then washed in five changes of 100 mmol/L phosphoric acid (10 minutes each) and once in 95% ethanol for 2 minutes. The filters were then dried and counted by liquid scintillation counting.

CaM kinase II activity was measured with a modification of the procedure described by Tansey and coworkers.13 Cell extracts were prepared by incubating the cells still attached to the culture plates for 7 minutes at 4°C in solution containing equal parts of Hanks' buffered saline solution and a mixture of 500 mmol/L MOPS, pH 7.5; 1% nonidet-P40 (NP-40); 20 mmol/L Na4P2O7; 250 mmol/L NaCl; 3 mmol/L EGTA; 1 mmol/L dithiothreitol; 20 µg/mL aprotinin; and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). The extract was then collected, and total and autonomous CaM kinase II activity was measured. Total CaM kinase II was measured in a reaction mixture (25 µL) containing 50 mmol/L MOPS, 10 mmol/L magnesium acetate, 15 mmol/L 2-mercaptoethanol, 5 mmol/L {gamma}[32P]ATP (5 µCi per reaction; 6000 Ci/mol), 0.2 mmol/L ATP, 3 mol/L EGTA, 4 mol/L CaCl2, 0.4 µmmol/L calmodulin, and 20 µmol/L autocamtide-2 at pH 7.5 at 30°C for 2 minutes. Autonomous activity was measured in a similar reaction mixture, except that both calcium and calmodulin were removed from the reaction buffer. Reaction mixtures were then processed as described for the PKC assay above. After the background was subtracted, autonomous activity was presented as percentage total activity [autonomous divided by (total multiplied by 100)].

CaM Kinase II Activation Assay
Autophosphorylation was used to assess activation of CaM kinase II and was measured by following the incorporation of radioactive phosphate into the molecule. Cells on a 60-mm plastic plate were incubated in 0.4 mCi/mL [32P]orthophosphoric acid (NEX-054, New England Nuclear) in phosphate-free SFM for 3 hours, stimulated with PDGF or other agents, then extracted in 1 mL RIPA buffer (150 mmol/L NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mmol/L Tris HCl at pH 8.0). Extracts were clarified by centrifugation, and aliquots containing equal amounts of TCA-precipitable counts were incubated overnight at 4°C with an antiserum to the {delta}-isoforms of CaM kinase II (generously provided by Hal Singer, Geisinger Clinic, Danville, Pa). To precipitate immune complexes, protein A-Sepharose (Sigma) was added for 1 hour. The precipitate was washed and prepared for electrophoresis as described previously.8 An aliquot was separated by 10% SDS-PAGE. Gels were dried and exposed to X-ray film. Quantification of results was performed on a phosphorimager (Betascope model 603, Betagen Corp).

RNA Analyses
Total RNA was isolated from VSMC cultures with the guanidine isothiocyanate procedure,14 and Northern blotting analyses were performed as described elsewhere.8 15 The c-fos mRNA probe has been described previously,13 whereas the probe used to detect JE (monocyte chemoattractant protein [MCP]-1) mRNA was an approximately 500-bp cDNA probe corresponding to the conserved protein coding region of the mouse JE cDNA. It was also generated by polymerase chain reaction using primers complementary to the published nucleotide sequence16 and cloned into the pCRII vector (InVitrogen Corp). The complete nucleotide sequence of the cDNA insert for both probes was verified by the dideoxy-mediated chain termination method.17

Gene Transfections and the Isolation and Characterization of Stable Transformants
Full-length wild-type and mutant cDNAs for rat brain CaM kinase II {alpha}-subunit were cloned into the eukaryotic expression vector pRC/CMV (InVitrogen Corp).18 VSMCs were then transfected with the expression vectors by the Ca2PO4 method.19 Cell lines containing stably integrated expression plasmids were generated by selection for resistance to 250 µg/mL geneticin (G418, GIBCO). Individual clones were then pooled for expansion and analyses. Expression of the transfected CaM kinase II {alpha}-subunit was determined by analyzing protein extracts of the cultures with an {alpha}-isoform–specific monoclonal antibody (generously provided by Dr H. Schulman, Stanford University). Samples (50 µg total protein) were separated by 10% SDS–PAGE and then transferred electrophoretically to polyvinylidine difluoride (PVDF) membranes (Amersham Corp) in a buffer consisting of 50 mmol/L Tris base, 157 mmol/L glycine, and 20% methanol at 12°C for 4 hours at 250 mA. The PVDF membrane was blocked to reduce nonspecific protein binding (Immunoblock, Amersham Corp) and stored with 10 µg/mL primary antibody overnight at 4°C. Membranes were then washed in DPBS containing 0.1% Tween 20 and incubated with 1 µg/mL rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase (Promega Biotech) at room temperature for 4 hours. Membranes were washed with DPBS–0.1% Tween 20 and developed using the enhanced chemiluminescent method according to the manufacturer's instructions (Amersham Corp).

Statistical Evaluations and Comparisons
All data are expressed as mean±SEM. Student's unpaired t test was used to compare the data presented in Figs 1Down and 2Down. A value of P<.001 is taken as statistically significant. For the CaM kinase II activity measurements in the TableDown, the mean values of the control and treated groups were compared by ANOVA, with P values corrected by the Bonferroni method.20 For these measurements, P<.01 was considered statistically significant.



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  		Figure 1. Bar graphs showing that migration is suppressed in growth-arrested vascular smooth muscle cells (VSMCs) and restored by pretreatment with ionomycin. A, Migration (cells/field) measured as chemotaxis of proliferating (filled bars) and growth-arrested (open bars) VSMCs. Cell migration is from the upper to the lower chamber. Additions to the upper and lower chambers used in the different sets of data are indicated below the bars. BSA (0.1%) in the upper chamber and 10 ng/mL platelet-derived growth factor (PDGF) BB in the lower chamber is the standard chemotaxis assay condition. When BSA or PDGF is in both chambers, no gradient of chemoattractant exists and migration is substantially reduced. Increasing the concentration of PDGF BB in the lower chamber to either 20 or 40 ng/mL did not affect the migration of growth-arrested cells. B, Migration measured as chemotaxis of ionomycin-pretreated proliferating (filled bars) and growth-arrested (open bars) VSMCs. Data in the first set of bars are under standard conditions, with 10 ng/mL PDGF BB as a chemoattractant in the lower chamber and 0.1% BSA in the upper chamber, and cells were pretreated for 20 minutes with 1 µmol/L ionomycin before being added to the upper chamber. The second set of data is with VSMCs not pretreated with ionomycin but where 1 µmol/L ionomycin alone was added in the lower chamber to test the ability of ionomycin as a chemoattractant.



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  		Figure 2. Tracings showing calcium responsiveness of proliferating and growth-arrested vascular smooth muscle cells (VSMCs). Changes in intracellular calcium levels ([Ca2+]i) as estimated from changes in intracellular indomethacin-1 fluorescence in proliferating (A) and growth-arrested (B) VSMCs (groups of 10 to 20 cells) exposed to 10 ng/mL platelet-derived growth factor (PDGF) BB or 1 µmol/L ionomycin (continuous exposure, initiated at the arrows).


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  		Table 1. Inhibition of Calcium/Calmodulin-Dependent Protein Kinase II Activity by KN62


*    Results
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*Results
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VSMC Migration Is Suppressed by Growth Arrest and Reversed by Pretreatment With Ionomycin
The migration of cultured VSMCs was measured in a Boyden chamber as chemotaxis toward PDGF BB. Cells were plated on filters that had been coated with collagen type I and fibronectin and placed into the upper chamber of the apparatus. The migration of cells to the underside of the filter in response to the chemoattractant PDGF BB in the bottom chamber was measured after 4 hours. Fig 1AUp shows that, during this period and under these conditions, the migration of VSMCs taken from growth-arrested cultures (open bar) was approximately fourfold less than that of VSMCs taken from proliferating cultures (filled bar). Migration always depended on a gradient of PDGF established across the filter. When PDGF in the lower chamber was replaced by BSA (BSA/BSA) or when PDGF was placed in both chambers (PDGF/PDGF), migration was sharply reduced (Fig 1AUp). Both proliferating and growth-arrested VSMCs adhered equally to the filter in the upper chamber of the Boyden apparatus (data not shown).

If growth-arrested VSMCs were pretreated for as little as 20 minutes with the calcium ionophore ionomycin (1 µmol/L) before exposure to the PDGF gradient, migration was restored to a level that was 85±4% of that observed for proliferating VSMCs (open bars in Fig 3BDown). Pretreatment of proliferating VSMCs with ionomycin did not increase the number of cells migrating to the underside of the filter, which suggests that ionomycin did not activate a different population of VSMCs from those activated by PDGF (Fig 1BUp). Ionomycin also was not a chemoattractant for either proliferating or growth-arrested cells (see "IONOMYCIN" in Fig 1BUp).



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  		Figure 3. Blots showing stimulation of Immediate-early gene expression in proliferating and growth-arrested vascular smooth muscle cells (VSMCs). Ten micrograms of total RNA from proliferating and growth-arrested (postconfluent/serum-starved) VSMC cultures treated with 10 ng/mL platelet-derived growth factor (PDGF) BB for 30 minutes, 1 hour, or 3 hours were probed with cDNA probes to rat c-fos (c-Fos) and mouse JE (monocyte chemoattractive protein-1; MCP-1) mRNA.

Early Responses of Proliferating and Growth-Arrested VSMCs to PDGF
On the basis of the response of growth-arrested cells to ionomycin (Fig 1BUp), we examined the changes in intracellular calcium that occurred in proliferating and growth-arrested VSMCs in response to PDGF (Fig 2Up). In proliferating VSMCs, PDGF caused a rapid rise in [Ca2+]i a sustained elevation (Fig 2AUp). In growth-arrested VSMCs, the initial response to PDGF was dramatically blunted and there was a lower sustained elevation (Fig 2BUp). Addition of ionomycin to growth-arrested cells, however, produced a rapid increase in [Ca2+]i followed by a sustained elevation, both of which were similar in magnitude to those of PDGF-stimulated proliferating cells.

Despite the dramatic difference in the response of proliferating and growth-arrested VSMCs to PDGF with respect to changes in [Ca2+]i, the activation of immediate early gene expression was, if anything, augmented in growth-arrested cells (Fig 3Up). PDGF activated c-fos and JE (MCP-1) expression in both proliferating and growth-arrested VSMCs. In both cell phenotypes, the expression of c-fos mRNA peaked at 30 minutes, whereas JE did not increase until 1 hour. These results indicated that growth-arrested cells could still respond to PDGF and retained at least some of the intracellular signaling pathways associated with PDGF signaling in proliferating cells but were specifically defective in their [Ca2+]i responsiveness.

Inhibiting CaM Kinase II but not PKC Activity Inhibits VSMC Migration
Given this difference in [Ca2+]i responsiveness, we looked for differences in the activation of the calcium-sensitive enzyme CaM kinase II. During activation by calcium and calmodulin, this kinase undergoes stoichiometric autophosphorylation, which produces an activated kinase that no longer requires calcium or calmodulin.21 Fig 4ADown and 4CDown shows that the addition of PDGF to proliferating cells caused a rapid increase in CaM kinase II activation as assessed by changes in its phosphorylation, which increased more than threefold within 1 minute of PDGF addition and remained elevated for at least 15 minutes. Ionomycin also increased CaM kinase II activation. In growth-arrested cells, however, PDGF failed to increase CaM kinase II phosphorylation (Fig 4BDown and 4CDown), whereas ionomycin could activate CaM kinase II phosphorylation to levels similar to that in proliferating cells (Fig 4CDown).



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  		Figure 4. Activation of calcium/calmodulin-dependent protein (CaM) kinase II in proliferating and growth-arrested vascular smooth muscle cells (VSMCs). A, Autogram of 32P incorporation into CaM kinase II in proliferating VSMCs after stimulation with 10 ng/mL platelet-derived growth factor (PDGF) BB or 1 µmol/L ionomycin. The positions of the 79- and 46-kD prestained markers are indicated to the left. B, Autoradiogram of 32P incorporation into CaM kinase II in growth-arrested VSMCs after stimulation with 10 ng/mL PDGF BB or 1 µmol/L ionomycin. Markings as in A. C, Bar graph of 32P incorporation into CaM kinase II in proliferating (filled bars) and growth-arrested (open bars) VSMCs expressed relative to control (unstimulated) incorporation. ' indicates time in minutes.

To examine the consequences of CaM kinase II phosphorylation and activation, the effects of CaM kinase II inhibitors on migration were examined in proliferating VSMCs (Fig 5Down). CaM kinase II activation in response to PDGF was inhibited by preventing either calcium-calmodulin interactions with W7 (50 µmol/L) or calmodulin–CaM kinase II interactions with KN62 (10 µmol/L). KN62 inhibited PDGF-stimulated CaM kinase activity (measured as percent autonomous activity) by >80% (see TableUp below). Both KN62 and W7 attenuated migration by >80%. In sharp contrast, inhibition of PKC had no effect on the extent of migration by proliferating VSMCs. PKC was either downregulated by chronic (24-hour) pretreatment with 10 µmol/L PMA22 or its activity inhibited with the highly specific inhibitor chelerythrine.23 The effectiveness of these reagents in inhibiting PKC activation in response to PDGF was verified by an in situ permeabilization assay to monitor PKC activation (data not shown).



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  		Figure 5. Bar graph showing effect of protein kinase C and calcium/calmodulin-dependent protein kinase II inhibitors and vascular smooth muscle cell (VSMC) migration. Ten nanograms per milliliter platelet-derived growth factor BB was used as chemoattractant. Except for long-term phorbol 12-myristate 13-acetate (PMA) stimulation, inhibitors (10 µmol/L KN62, 50 µmol/L W7, and 10 µmol/L chelerythrine) were added to the upper chamber of the Boyden apparatus at the same time that cells were added. PMA-treated cells were incubated in 10 µmol/L PMA for 24 hours before the migration assay.

We next tested whether the effect of ionomycin in stimulating the migration of growth-arrested cells was due to activation of CaM kinase II by examining their response to KN62. The migration of proliferating and growth-arrested VSMCs toward PDGF was measured after pretreatment with 1 µmol/L ionomycin and/or 10 µmol/L KN62 (Fig 6Down). The increase in the migration of growth-arrested VSMCs (IONO) was completely blocked by inhibiting CaM kinase II activation (IONO+KN62), which indicates that the increase in migration seen with ionomycin was due, in part, to its activation of CaM kinase II.



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  		Figure 6. Bar graph showing that Ionomycin (IONO) restores migration in growth-arrested vascular smooth muscle cells (VSMCs) through a calcium/calmodulin-dependent protein (CaM) kinase II–dependent mechanism. Ten nanograms per milliliter platelet-derived growth factor BB was used as the chemoattractant in the lower chamber. Ionomycin (1 µmol/L) and KN62 (10 µmol/L) were added to the upper chamber at the time of cell addition. Ionomycin was removed after 20 minutes, whereas KN62 remained in the chamber throughout the migration experiment. PROL indicates proliferating VSMCs; GROWTH-ARRESTED, growth arrested VSMCs. *Significant difference between KN62-treated and control VSMCs at the level of P<.001 (n=8); **significant difference at a level of P<.001 between ionomycin/KN62-treated and ionomycin-treated VSMCs that have been growth-arrested.

Effects of Constitutively Active CaM Kinase II in VSMCs
To determine whether activation of CaM kinase II alone was sufficient to restore PDGF-directed migration in growth-arrested cells, we created stable transfectants harboring wild-type and genetically engineered forms of the rat brain {alpha}-subunit of CaM kinase II. Mutations involved changing amino acid 286 from a threonine to an aspartic acid (T286D) or amino acids 286 and 287 to aspartic acid (D3) (Fig 7ADown) Thr-286 is the site of autophosphorylation that results in an active enzyme independent of calcium and calmodulin. The substitution of the threonine with a negatively charged amino acid, such as aspartic acid, is thought to mimic the negative charge introduced into the wild-type protein at this site by phosphorylation and is a strategy used to modify the activity of numerous transcription factors25 as well as with CaM kinase II.26



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  		Figure 7. Characterization of vascular smooth muscle cell (VSMC) calcium/calmodulin-dependent protein (CaM) kinase II stable transfectant. A, Schematic of CaM kinase II {alpha}-subunit cDNAs in the pRC/CMV expression vectors. Wild-type and mutant cDNAs expressing the rat {alpha}-subunit of CaM kinase II were subcloned into the pRC/CMV vector, in which the cytomegaloviral (CMV) early promoter drives expression of the cDNA. cDNAs are shown as consisting of three protein domains: an inhibitory domain that suppresses activity, a catalytic domain, and an association domain. The single-letter codes for amino acids 286, 287, and 288 are shown. In T286D, Thr-286 was replaced by a negatively charged amino acid (aspartic acid) resulting in constitutive activation of the protein. In D3, both Thr-286 and Val-287 were converted to aspartic acid, which together with aspartic acid-288 present in unmodified CaM kinase II resulted in three negatively charged amino acids in a row. B, Western blot analysis of stable transfectants of CaM kinase II {alpha}-subunit transfectants. Total cellular protein (50 µg) was separated by SDS-PAGE, electroblotted onto nitrocellulose, then incubated with an {alpha}-subunit–specific monoclonal antibody (gift of H. Shulman, Stanford University). Antibody binding was detected with an alkaline phosphatase–conjugated anti-rabbit immunoglobulin. Cytosolic extracts of brain and heart were used as positive and negative controls, respectively. Both heart and VSMCs contain mostly {delta}-isoform and very little, if any, {alpha}-isoforms of CaM kinase II.42 Transfx indicates transfectants.

Western blot analysis of pooled transfectants indicated that only in transfectants receiving the expression vectors containing the {alpha}-subunit CaM kinase II cDNAs was the {alpha}-subunit expressed (Fig 7BUp). The data indicated that the level of expression of the three different {alpha}-subunit expression vectors (wild-type, T286D, and D3) was similar. CaM kinase II activity was assayed in control, wild-type, and D3 transfected cells. Basal autonomous activity was low in both control and wild-type cells (5±2.2% total activatable CaM kinase II; n=4) but relatively high in D3 transfected cells (29±3.4%, n=4). There were no observable differences in cell shape, cell proliferation, or the ability of cells to adhere or form the characteristic hill and valley morphology between transfected or untransfected cells.

The migration of control and CaM kinase II stable transfectants was measured (Fig 8Down). Migration was measured in proliferating transfected VSMCs in the absence and presence of 10 µmol/L KN62 and in growth-arrested transfected VSMCs. As expected, migration toward PDGF BB in stable transfectants containing the vector alone or expressing wild-type cDNA was inhibited by KN-62 or suppressed when the cells were growth-arrested. In contrast, PDGF-directed cell migration in stable transfectants expressing either constitutively activated form of the {alpha}-subunit was resistant to inhibition by KN62 and only partially inhibited by growth arrest. The resistance of the D3 and T286D cells to KN62 was consistent with the facts that these cells express constitutively activated CaM kinase II and that KN62 cannot inhibit previously activated CaM kinase II.



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  		Figure 8. Bar graph showing effects of constitutively active calcium/calmodulin-dependent protein (CaM) kinase II on vascular smooth muscle cell (VSMC) migration. Standard assay conditions were used with 10 ng/mL platelet-derived growth factor BB as chemoattractant. The migration of proliferating cells (Prol) in the absence and presence of the inhibitor of CaM kinase II activation KN62 (10 µmol/L) was measured, as was the migration of the cells after they had been growth-arrested. Cells labeled Vector are stable transfectants containing the pRC/CMV expression vector without the CaM kinase II cDNAs, whereas those labeled as Wild Type, T286D, and D3 are stable transfectants expressing the wild-type and mutant forms of the {alpha}-subunit cDNA. n.s. indicates no statistically significant difference (P>.1) between control and KN62-treated cells. *Statistically significant difference at the level of P<.001 between growth-arrested T286D or D3 VSMC transfectants and growth-arrested vector or wild-type transfected VSMCs.


*    Discussion
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*Discussion
down arrowReferences
 
The present studies were undertaken to identify potential regulatory mechanisms controlling VSMC migration. We showed that migration toward the chemoattractant PDGF BB was suppressed in growth-arrested VSMCs compared with proliferating VSMCs and that suppression was removed by brief pretreatment of the cells with a calcium ionophore. PDGF stimulated expression of the early response genes c-fos and MCP-1 (JE) in both proliferating and growth-arrested VSMCs, which indicated that PDGF receptors and part of the intracellular signaling pathway present in proliferating VSMCs were intact and operative in growth-arrested VSMCs. Compared with proliferating cells, however, growth-arrested VSMCs exhibited a much-reduced calcium response to PDGF. Consistent with this hypothesis, we showed that the activation of CaM kinase II did not occur in growth-arrested VSMCs stimulated with PDGF. The functional importance of CaM kinase II activation was then established by showing (1) that inhibiting CaM kinase II in migrating VSMCs inhibited migration; (2) that treatment of growth-arrested VSMCs with the calcium ionophore ionomycin, which activated CaM kinase II in these cells, restored their ability to migrate toward PDGF; (3) that the ionomycin-induced restoration of migration in growth-arrested VSMCs was inhibited by blocking CaM kinase II activity; and (4) that stable VSMC transfectants expressing constitutively activated forms of CaM kinase II continued to migrate even when the cells were growth-arrested or treated with the specific inhibitor of activation KN62. Taken together, these data show not only that CaM kinase II activity is required for VSMC migration but also that the suppression of cell migration in quiescent growth-arrested VSMCs is due to the failure of these cells to activate CaM kinase II.

CaM kinase II is a multifunctional enzyme that phosphorylates a number of cellular proteins.27 The protein targets that CaM kinase II phosphorylates to facilitate or regulate VSMC migration are not known. CaM kinase II has been shown to phosphorylate the actin-binding protein calponin28 and myosin light-chain kinase (MLCK).13 The latter may play a role in the desensitization of contractile filaments to activation by Ca2+, whereas phosphorylation of calponin has been shown to release the inhibition that this molecule exerts on actin-activated myosin ATPases.13 28 It is not known, however, whether MLCK is present in these cells, although smooth muscle–specific contractile proteins are generally absent from proliferating late-passage VSMCs.29 On the other hand, it is known that calponin levels rapidly decrease with continued cell cultivation,29 and they are barely detectable in the VSMC cultures used in the present study.

A number of observations suggest that CaM kinase II may affect events immediately after PDGF addition that influence changes in the polymerization state of actin. The movement of cells orchestrated by chemoattractants is mediated by the coordinated changes in the polymerization and depolymerization of actin-containing microfilaments present in the cell cortex, cytoplasm, and at specialized membrane contact points with the extracellular matrix.30 With PDGF as the chemoattractant, multiple intracellular signaling pathways are activated by its receptor tyrosine kinase, leading to the increased activity of GTP-binding proteins, such as ras, increased polyphosphoinositide metabolism and turnover, and a transient elevation of intracellular calcium.31 Proteins that control the activity of the small, ras-like GTP-binding proteins involved in the regulation of actin assembly, such as rac and rho32 33 are particularly susceptible to changes in intracellular calcium and polyphosphoinositides, whereas actin-binding proteins, such as profilin, regulate the hydrolysis of polyphosphoinositides.30 Both calcium and polyphosphoinositides have been proposed to regulate actin reorganization in response to growth-factor stimulation.34

More directly, though, migrating cells form membrane ruffles at their leading edges.35 Such ruffles occur within minutes after growth-factor stimulation and are thought to play a fundamental role in cell movement. Inhibition of CaM kinase II activation in chicken fibroblasts using W7, which inhibits calcium binding to calmodulin and the subsequent activation of CaM kinase II, has been reported to inhibit growth-factor–stimulated membrane ruffling.36 Likewise, we have observed that membrane ruffling of VSMCs in response to PDGF is suppressed by KN62 in proliferating VSMCs expressing only endogenous or wild-type transfected CaM kinase II but not in VSMCs expressing constitutively activated CaM kinase II (J. Kinsella, R.R.P., and M.T.C., unpublished observations). These observations suggest that examining the role of CaM kinase II in regulating the function of the small GTP-binding proteins involved in actin stress fiber assembly and disassembly (ie, rac and rho) or the activity of enzymes that modify these ras-like proteins may provide new insights into the regulation of cell migration.

A direct pathway that would affect phosphoinositide metabolism, changes in [Ca2+]i, and CaM kinase II activity after PDGF receptor stimulation is the activation of phospholipase C{gamma} (PLC{gamma}). One recent study showed that PLC activity is a common intracellular pathway activated by growth factors or cytokines that serve as chemoattractants for human VSMCs,37 whereas a second study demonstrated that activation of PLC{gamma} is required for the migration of many different cell lines toward PDGF BB.38 Activated PLC{gamma} catalyzes the turnover of phosphatidylinositol biphosphate, leading to the production of diacylglycerol and inositol triphosphate (IP3). Diacylglycerol activates PKC, whereas IP3 causes the release of intracellular calcium–activated CaM kinase II. Whether activation of CaM kinase II is the critical downstream effect of PLC{gamma} activation with respect to the regulation of cell migration is unknown. While our results, as well as those of others,39 indicate that activation of PKC is not necessary for migration, note that increases in diacylglycerol can influence actin turnover and migration through a PKC-independent mechanism.39 40 In this regard, it will be of interest to examine the effects of constitutively activated CaM kinase II in cells in which PLC{gamma} activity has been abolished by a dominant negative mutant.38

VSMCs in vivo are usually quiescent and do not migrate from the confines of the medial space. The establishment of VSMC cultures from such tissues results in a transformation of these cells from a quiescent to a proliferative phenotype, mimicking in many results the phenotypic transformation of VSMCs that occurs when the vessel is injured. As we have shown, the transformation that occurs upon culturing is associated with an ability to respond to the chemoattractant PDGF. When these cells are then growth-arrested and become quiescent, they no longer respond to PDGF. The behavior of such cells is similar, in at least some respects, to that of normal medial cells in vivo. To reacquire the ability to migrate, additional factor(s) are needed to activate pathways similar to that activated in our in vitro experiments by ionomycin. Our preliminary data indicate that there are many factors of potential physiological importance that could cooperate with PDGF to activate growth-arrested cells to migrate. These are basic fibroblast growth factor, insulin-like growth factor-1 (IGF-1), and endothelin-1. None of these is a very potent chemoattractant, especially compared with PDGF. But the administration of any one of these reagents to growth-arrested VSMCs can partially or fully restore the ability to migrate toward PDGF (R.R.P., R.M., and M.T.C., unpublished results, 1994). Both endothelin41 and IGF-137 have been shown to increase [Ca2+]i and would be expected to active CaM kinase II even in growth-arrested cells.

There are, however, important differences between growth-arrested cultured VSMCs and medial VSMCs in vivo. The ability of growth-arrested VSMCs to express the smooth muscle–specific genes characteristic of in vivo differentiation is rapidly lost with cell passage,29 and many of these markers were not detected in the later-passage VSMCs used in the present study. The expression of these differentiation-specific genes may provide additional barriers to the activation of migration. This notion is supported by a preliminary report demonstrating that overexpression of one of these differentiation-specific markers, the actin-binding protein calponin, inhibits migration in late (greater than nine-passage VSMCs.42 Calponin is expressed by cultured VSMCs only during the first few passages after cell isolation.29 The marked effects of calponin reported in the preliminary communication42 suggest that it and possibly other actin-modulating proteins may provide a block to migration different from the one involving growth arrest and changes in CaM kinase II. Such results are not inconsistent with the findings reported here and indicate that multiple steps may be required for the phenotypic transformation of VSMCs. The requirement of multiple steps to activate the program for cell migration in VSMCs may explain why migration occurs only several days after injury to vessels by balloon catheter injury.


*    Acknowledgments
 
The authors acknowledge the generosity and assistance of Hal Singer (Weis Center for Research, Danville, Pa) for his advice on CaM kinase II assays and antibodies to {delta}-CaM kinase II and to Howard Schulmann (Stanford University) for antibodies to {alpha}-CaM kinase II.

Received May 18, 1994; revision received September 12, 1994; accepted October 5, 1994.


*    References
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 up arrowAbstract
 up arrowIntroduction
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*References
 

  1. Chamley-Campbell JH, Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res. 1981;40:347-357.
  2. Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res. 1986;58:427-444.[Abstract/Free Full Text]
  3. Liu MW, Roubin GS, King SB. Restenosis after coronary angioplasty. Circulation. 1989;79:1374-1387. [Abstract/Free Full Text]
  4. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488-500. [Medline] [Order article via Infotrieve]
  5. Blank RS, Thompson MM, Owens GK. Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells. J Cell Biol. 1988;107:299-306. [Abstract/Free Full Text]
  6. Pauly RR, Passaniti A, Crow M, Kinsella JL, Papadopoulos N, Monticone R, Lakatta EG, Martin GR. Experimental models that mimic the differentiation and dedifferentiation of vascular cells. Circulation. 1992;86(suppl III):II-68-III-73.
  7. Ross R. Polypeptide growth factors and atherosclerosis. Trends Cardiovasc Med. 1991;1:277-282.
  8. Pauly RR, Passaniti A, Bilato C, Monicone R, Cheng L, Papadopoulos N, Gluzband YA, Smith L, Weinstein C, Lakatta E, Crow MT. The migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation. Circ Res. 1994;75:41-54. [Abstract/Free Full Text]
  9. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987;47:3239-3245. [Abstract/Free Full Text]
  10. Spurgeon HA, Stern MD, Baartz G, Raffaeli S, Hansford RG, Talo A, Lakatta EG, Capogrossi MC. Simultaneous measurement of Ca2+, contraction, and potential in cardiac myocytes. Am J Physiol. 1990;258:H574-H586. [Abstract/Free Full Text]
  11. Ziegelstein RC, Cheng L, Blank PS, Spurgeon HA, Lakatta EGF, Hansford RG, Capogrossi MC. Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification. Am J Physiol. 1993;265:H1424-H1433. [Abstract/Free Full Text]
  12. Sadoshima J-I, Izumo S. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro. Circ Res. 1993;73:424-438. [Abstract/Free Full Text]
  13. Tansey MG, Word RA, Hidaka H, Singer HA, Schworer CM, Kamm KE, Stull JT. Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells. J Biol Chem. 1992;267:12511-12516. [Abstract/Free Full Text]
  14. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294-5299. [Medline] [Order article via Infotrieve]
  15. Zheng J-S, Boluty MO, O'Neill LO, Crow MT, Lakatta EG. Extracellular ATP induces immediate-early gene expression but not cellular hypertrophy in neonatal cardiac myocytes. Circ Res. 1994;74:1034-1041. [Abstract/Free Full Text]
  16. Rollins, RJ, Morrison ED, Stiles CD. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc Natl Acad Sci U S A.1988;85:3738-3742.
  17. Sanger F, Niclen S, Coulsen AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A.. 1977;74:5463-5467. [Abstract/Free Full Text]
  18. Waxham MN, Aronowski J, Westgate SA, Kelly PR. Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-independent protein kinase II eliminates Ca2+/calmodulin-independent activity. Proc Natl Acad Sci U S A. 1990;87:1273-1277. [Abstract/Free Full Text]
  19. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.
  20. Devore J. Probability and Statistics for Engineering and the Sciences. Monterey, Calif: Brooks/Cole Publishing Co; 1982.
  21. Miller SG, Kennedy MB. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+ triggered molecular switch. Cell. 1986;44:861-870. [Medline] [Order article via Infotrieve]
  22. Ballester R, Rosen OM. Fate of immunoprecipitable protein kinase C in G3H cells treated with phorbol 12-myristate 13-acetate. J Biol Chem. 1985;260:15194-15199. [Abstract/Free Full Text]
  23. Herbert JM, Augereau JM, Gleye J, Maffrand JM. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990;172:993-999. [Medline] [Order article via Infotrieve]
  24. Schworer CM, Rothblum LI, Thekkumkara TJ, Singer HA. Identification of novel isoforms of the {delta} subunit of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem. 1993;268:14443-14449. [Abstract/Free Full Text]
  25. Hunter T, Karin M. The regulation of transcription by phosphorylation. Cell. 1992;70:375-387. [Medline] [Order article via Infotrieve]
  26. Waldmann R, Hanson PI, Schulman H. Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2+-independent for functional studies. Biochemistry. 1990;29:1679-1684. [Medline] [Order article via Infotrieve]
  27. Hanson PI, Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinases. Annu Rev Biochem. 1992;61:559-601. [Medline] [Order article via Infotrieve]
  28. Wills FL, McCubbin WD, Kay CM. Characterization of the smooth muscle calponin and calmodulin complex. Biochemistry. 1993;32:2321-2328. [Medline] [Order article via Infotrieve]
  29. Shanahan CM, Weissberg PL, Metcalfe JC. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ Res. 1993;73:193-204. [Abstract]
  30. Stossel T. From signal to pseudopod. J Biol Chem. 1989;264:18621-18624.
  31. Pazin MJ, Williams LT. Triggering signalling cascades by receptor tyrosine kinases. Trends Biochem Sci. 1992;17:374-378. [Medline] [Order article via Infotrieve]
  32. Settleman J, Albright LC, Weinberg RA. Association between GTPase activators for rho and ras families. Nature. 1992;359:153-154. [Medline] [Order article via Infotrieve]
  33. Tsai H-H, Hall A, Stacey DW. Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins. Mol Cell Biol. 1989;9:5260-5264. [Abstract/Free Full Text]
  34. Rijken PJ, Hage WJ, van Begen en Henegouwen PMP, Verkleij AJ, Boonstra J. Epidermal growth factor induces actin organization of the actin microfilament system in human A431 cells. J Cell Sci. 1991;100:491-499. [Abstract/Free Full Text]
  35. Mellstrom K, Heldin CH, Westermark B. Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor. Exp Cell Res. 1988;177:347-359. [Medline] [Order article via Infotrieve]
  36. Hasegawa T. Production of a unique antibody specific for membrane ruffles and its use to characterize the behavior of two distinct types of ruffles. J Cell Biol. 1993;6:1439-1448.
  37. Bornefeldt KE, Raines EW, Makano T, Graves LM, Krebs EG, Ross R: Insulin-like growth factor-1 and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signalling pathways that are distinct from those of proliferation. J Clin Invest. 1994;93:1266-1274.
  38. Kundra V, Escobedo JA, Kazlauskas A, Kim HK, Rhee SAG, Williams LT, Zetter BR. Regulation of chemotaxis by the platelet-derived growth factor receptor-ß. Nature. 1994;367:474-476. [Medline] [Order article via Infotrieve]
  39. Shariff A, Luna EJ. Diacylglycerol-stimulated formation of actin nucleation sites at plasma membranes. Science. 1992;256:245-247. [Abstract/Free Full Text]
  40. Omann GM, Porasik MM, Sklar LA. Oscillating actin polymerization/depolymerization responses in human polymorphonuclear leukocytes. J Biol Chem. 1989;264:16355-16358. [Abstract/Free Full Text]
  41. Griendling KK, Tsuda T, Alexander RW. Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells. J Biol Chem. 1989;264:8237-8240. [Abstract/Free Full Text]
  42. Takahashi K, Takagi M, Ohgami K, Nakai M, Kojima A, Nadal-Ginard B. Inhibition of smooth muscle cell migration and proliferation caused by transfection of the human calponin gene is associated with enhanced cell-matrix adhesion and reduced PDGF responsiveness. Circulation. 1993;88:I-174. Abstract.



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