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Manumycin A, Inhibitor of ras Farnesyltransferase, Inhibits Proliferation and Migration of Rat Vascular Smooth Muscle Cells Hirosuke Kouchi,* ,1 Kazufumi Nakamura,* Kazuo Fushimi,² Masakiyo Sakaguchi,² Masahiro Miyazaki,² Tohru Ohe,* and Masayoshi Namba² *Department of Cardiovascular Medicine and ²Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, Okayama, Japan Received September 18, 1999 Restenosis after angioplasty is thought to be caused by proliferation and migration of vascular smooth muscle cells (VSMCs), and it is a most serious problem in medical treatment. A low dose (50 ng/ml) of manu- mycin A, an inhibitor of p21 ras (ras) farnesylation, sig- nificantly inhibited proliferation of rat VSMCs stimu- lated by the platelet-derived growth factor (PDGF). The mitoinhibitory effect of manumycin A was dose- and time-dependent but was independent of cell den- sity. Western blot analysis showed that manumycin A reduced the amount of functional ras localized at the cytoplasmic membrane and inhibited the phosphory- lation of p42/44 mitogen-activated protein kinase (MAPK). Manumycin A also inhibited VSMC migration and disorganized a actin fibers, as shown by immnofluorecence staining. These results indicate that the interruption of the ras/MAPK signal transduc- tion pathway and the disorganization of a actin fibers are the main cause of manumycin A inhibition of VSMC proliferation and migration induced by PDGF. © 1999 Academic Press Despite great efforts to find ways to prevent reste- nosis, it occurs within a year in 40% of patients who undergo angioplasty (1). The operation entails injury to the media of the atrial wall by a PTCA balloon in order to obtain a larger lumen area and a longer effective period (2, 3, 4). However, a too-deep injury in the atrial wall results in exposure of vascular smooth muscle cells (VSMCs) to several cytokines and an excessive proliferation and migration of VSMCs (5, 6), which is thought to be a cause of restenosis after angio- plasty (7). Platelet-derived growth factor (PDGF) is one of the most powerful promoters of the proliferation and mi- gration of VSMCs (8, 9). The binding of PDGF to its receptor results in phosphorylation of the receptor ty- rosine residue (10). The phosphorylated PDGF recep- tor activates p21 ras (ras), and the activated ras triggers a phosphorylation cascade of p42/44 mitogen-activated protein kinase (MAPK), followed by proliferation and migration of VSMCs (7, 11). Signal transduction medi- ated by ras requires localization of the protein at the plasma membrane after farnesylation of cystine in the CAAX consensus sequence at the COOH terminus of the protein (12, 13). Therefore, inhibition of ras farne- sylation may block this cascade and inhibit the prolif- eration and migration of VSMCs (14). In this study, we examined the cytological effects of manumycin A, a potent new peptidomimetic inhibitor of ras farnesylation (15), on VSMCs in vitro. At a low concentration (50 ng/ml), manumycin A significantly inhibited the proliferation and migration of VSMCs, reduced the amount of ras protein localized at the cytoplasmic membrane, inhibited the phosphorylation of MAPK, and disorganized a actin fibers. MATERIALS AND METHODS Chemicals. Manumycin A was purchased from Sigma Chemical Co. (St. Louis, MO). It was dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium when used (16). The final concen- tration of DMSO was 0.1%, which is not cytotoxic to quiescent and growth-stimulated VSMCs, as reported previously (17). Recombi- nant human PDGF-BB, purchased from Pepro Tech (Rocky Hill, NJ), was dissolved in 4 3 10 23 N HCl containing 0.2% bovine serum albumin (BSA; Sigma). Cell culture. VSMCs were isolated from the thoracic aorta of adult Wistar-Kyoto rats (weighing 240 –320 g) by the explant culture method as previously described (18). The cells were grown in Dul- becco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) and 0.1 mg/ml kanamycin (high-serum medium) in a humidified 5% CO 2 atmo- sphere at 37°C and were used at passages 3–13. They were seeded at 1 To whom correspondence should be addressed at Department of Cardiovascular Medicine, Okayama University Medical School, 2-5-1 Shikata, Okayama 700-8558, Japan. Fax: 181-86-235-7353. E-mail: [email protected]. Biochemical and Biophysical Research Communications 264, 915–920 (1999) Article ID bbrc.1999.1546, available online at http://www.idealibrary.com on 915 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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Page 1: Manumycin A, Inhibitor of ras Farnesyltransferase, Inhibits Proliferation and Migration of Rat Vascular Smooth Muscle Cells

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Article ID bbrc.1999.1546, available online at http://www.idealibrary.com on

anumycin A, Inhibitor of ras Farnesyltransferase,nhibits Proliferation and Migrationf Rat Vascular Smooth Muscle Cells

irosuke Kouchi,*,1 Kazufumi Nakamura,* Kazuo Fushimi,† Masakiyo Sakaguchi,†asahiro Miyazaki,† Tohru Ohe,* and Masayoshi Namba†

Department of Cardiovascular Medicine and †Department of Cell Biology, Institute of Molecular and Cellular Biology,kayama University Medical School, Okayama, Japan

eceived September 18, 1999

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Restenosis after angioplasty is thought to be causedy proliferation and migration of vascular smoothuscle cells (VSMCs), and it is a most serious problem

n medical treatment. A low dose (50 ng/ml) of manu-ycin A, an inhibitor of p21ras (ras) farnesylation, sig-ificantly inhibited proliferation of rat VSMCs stimu-

ated by the platelet-derived growth factor (PDGF).he mitoinhibitory effect of manumycin A was dose-nd time-dependent but was independent of cell den-ity. Western blot analysis showed that manumycin Aeduced the amount of functional ras localized at theytoplasmic membrane and inhibited the phosphory-ation of p42/44 mitogen-activated protein kinaseMAPK). Manumycin A also inhibited VSMC migrationnd disorganized a actin fibers, as shown bymmnofluorecence staining. These results indicatehat the interruption of the ras/MAPK signal transduc-ion pathway and the disorganization of a actin fibersre the main cause of manumycin A inhibition ofSMC proliferation and migration induced by PDGF.1999 Academic Press

Despite great efforts to find ways to prevent reste-osis, it occurs within a year in 40% of patients whondergo angioplasty (1). The operation entails injury tohe media of the atrial wall by a PTCA balloon in ordero obtain a larger lumen area and a longer effectiveeriod (2, 3, 4). However, a too-deep injury in the atrialall results in exposure of vascular smooth muscle

ells (VSMCs) to several cytokines and an excessiveroliferation and migration of VSMCs (5, 6), whichs thought to be a cause of restenosis after angio-lasty (7).

1 To whom correspondence should be addressed at Department ofardiovascular Medicine, Okayama University Medical School, 2-5-1hikata, Okayama 700-8558, Japan. Fax: 181-86-235-7353. E-mail:

[email protected].

915

Platelet-derived growth factor (PDGF) is one of theost powerful promoters of the proliferation and mi-

ration of VSMCs (8, 9). The binding of PDGF to itseceptor results in phosphorylation of the receptor ty-osine residue (10). The phosphorylated PDGF recep-or activates p21ras (ras), and the activated ras triggersphosphorylation cascade of p42/44 mitogen-activatedrotein kinase (MAPK), followed by proliferation andigration of VSMCs (7, 11). Signal transduction medi-

ted by ras requires localization of the protein at thelasma membrane after farnesylation of cystine in theAAX consensus sequence at the COOH terminus of

he protein (12, 13). Therefore, inhibition of ras farne-ylation may block this cascade and inhibit the prolif-ration and migration of VSMCs (14).In this study, we examined the cytological effects ofanumycin A, a potent new peptidomimetic inhibitor

f ras farnesylation (15), on VSMCs in vitro. At a lowoncentration (50 ng/ml), manumycin A significantlynhibited the proliferation and migration of VSMCs,educed the amount of ras protein localized at theytoplasmic membrane, inhibited the phosphorylationf MAPK, and disorganized a actin fibers.

ATERIALS AND METHODS

Chemicals. Manumycin A was purchased from Sigma Chemicalo. (St. Louis, MO). It was dissolved in dimethyl sulfoxide (DMSO)nd diluted with culture medium when used (16). The final concen-ration of DMSO was 0.1%, which is not cytotoxic to quiescent androwth-stimulated VSMCs, as reported previously (17). Recombi-ant human PDGF-BB, purchased from Pepro Tech (Rocky Hill, NJ),as dissolved in 4 3 1023 N HCl containing 0.2% bovine serumlbumin (BSA; Sigma).

Cell culture. VSMCs were isolated from the thoracic aorta ofdult Wistar-Kyoto rats (weighing 240–320 g) by the explant cultureethod as previously described (18). The cells were grown in Dul-

ecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan)upplemented with 10% fetal bovine serum (FBS) and 0.1 mg/mlanamycin (high-serum medium) in a humidified 5% CO2 atmo-phere at 37°C and were used at passages 3–13. They were seeded at

0006-291X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

Page 2: Manumycin A, Inhibitor of ras Farnesyltransferase, Inhibits Proliferation and Migration of Rat Vascular Smooth Muscle Cells

various densities into dishes or multiwell plates containing a high-svptdar

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erum medium and were allowed to attach themselves to the cultureessels for 12 hours. The cells were then incubated in DMEM sup-lemented with 0.1% FBS (low-serum medium) for 24 hours to makehem quiescent. They were then fed with the fresh low-serum me-ium and stimulated with 10 ng/ml of PDGF in the presence orbsence of manumycin A. All dishes and plates were coated withat-tail collagen before use (19), except in the transwell assay.

[3H]Thymidine incorporation. VSMCs plated in 24-well culturelates at densities of 1.0 3 105 or 1.0 3 106 cells per well were labeledith [methyl-3H]-thymidine (American Radiolabeled Chemicals, St.ouis, MO) at 1 mCi/ml for the last 2 hours in the presence or absencef manumycin A. After labeling was completed, the cells wereashed in situ twice each with ice-cold phosphate-buffered saline

PBS), 5% trichloroacetic acid and 95% ethanol. The cells were thenysed with 0.5 N NaOH. Aliquots of the cell lysates were neutralizedith HCl, and radioactivity was measured in a liquid scintillation

ounter.

Western blot analysis. VSMCs (1.0 3 105) were plated into00-mm culture dishes. After treatment with or without manumycin, the cells were scraped off for analysis of ras protein and phosphor-lated MAPK. For preparation of the membrane fractions for rasrotein detection, the cell pellets were lysed in lysis buffer [10 mMris (pH 7.6), 5 mM MgCl2, 1 mM dithiothreitol, and 1 mM phenyl-ethylsulfonyl fluoride] and sonicated for 5 minutes. The cell lysatesere centrifuged at 55,000 rpm (100,000 g) and 4°C for 30 minutes to

eparate membrane and cytosol fractions. For detection of phosphor-lated MAPK, the cell pellets were lysed in lysis buffer [50 mM TrispH 7.6), 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100,.1% sodium dodecyl sulfate, 0.2 mM Na3VO4, and 2 mM NaF]. Theell lysates were then centrifuged at 15,000 rpm (16,000 g) and 4°Cor 20 minutes, and the supernatants were used as whole cell ex-racts. The membrane fractions and whole cell extracts were frac-ionated by 12% SDS-polyacrylamide gel electrophoresis and thenransferred to nitrocellulose membranes (Amersham Life Science,uckinghamshire, United Kingdom). The membranes were immu-oblotted with anti-ras antibody (Transduction Laboratories, Lex-

ngton, KY) or with anti-phosphorylated MAPK antibody (New En-land Biolabs, Beverly, MA). Then the immunoblotted membranesere incubated with secondary antibodies (peroxidase-conjugatedouse IgG or rabbit IgG antibodies, MBL, Nagoya, Japan). The

ands were visualized using an ECL Plus detection kit (Amershamife Science).

Transwell migration assay. The motility assay was performedith transwell tissue culture inserts composed of polycarbonateembranes containing 12-mm pores (Millipore, Bedford, MA) (20).he basal (outer) chamber of each transwell contained 600 ml ofigh-serum medium that was supplemented with 10 ng/ml of PDGF

n the presence or absence of manumycin A (50 ng/ml). The apicalinner) chamber of each transwell contained VSMCs (4.0 3 104) in00 ml of high-serum medium. After 24-hour incubation, cells thatigrated from the apical to the basal chamber surface were stainedith 5% Giemsa solution after fixation with methanol and were

ounted under a light microscope.

Immunocytochemistry. For double stains of nuclei and a actin,.0 3 104 cells were plated on glass coverslips coated with rat-tailollagen. The cells were incubated for 12 hours in low-serum mediumontaining PDGF (10 ng/ml) in the presence or absence of manumy-in A (50 ng/ml), fixed with 4% paraformaldehyde, 0.1% glutalarde-yde and 5 mM MgCl2 in PBS, and stained with 1 mM Hoechst 33258

n PBS. After permeabilization in ice-cold ethanol, cells with staineduclei were incubated with anti-a actin mouse monoclonal antibody

Progen, Heidelberg, Germany) at a dilution of 1:100 with PBSontaining 1% BSA and 0.1% sodium azide, and they were thenncubated with FITC-conjugated anti-mouse IgG antibody (Santaruz Biotechnology, Santa Cruz, CA) as a secondary antibody.

916

al analyses were performed with the ANOVA method, and P , 0.05as considered statistically significant.

ESULTS

A low dose (less than 50 ng/ml) of manumycin A wasot cytotoxic to VSMCs during the 12-hour treatmentFigs. 1a–1c), but a high concentration (500 ng/ml) of

anumycin A was severely cytotoxic (Fig. 1d). Thenti-proliferative effects of manumycin A on VSMCstimulated with PDGF were examined at various con-entrations of manumycin A (n 5 3). Manumycin Anhibited the incorporation of [3H]-thymidine in a dose-ependent manner (Fig. 2a), and inhibition was alsobserved in cell growth (Fig. 2b). Time-course experi-ents showed that manumycin A (50 ng/ml) effectively

nhibited [3H]-thymidine incorporation up to 12 hoursfter its addition to VSMC cultures (Fig. 2c).Next, we examined whether or not the growth inhib-

tory effect of manumycin A on VSMCs was dependentn cell density (n 5 3). The cells were inoculated at.0 3 105 or 1.0 3 106 cells per well into 24-well plates,nd they were treated for 12 hours with or withoutanumycin A (50 ng/ml) in the presence of PDGF (10

g/ml) after 24-hour serum starvation. Manumycin Aonsistently inhibited [3H]-thymidine incorporation inoth the high- and low-density cultures (Fig. 3). Inther words, the inhibition ratios in the high- andow-density cultures were nearly the same: 0.41 (1250/040 cpm) and 0.32 (2380/7370 cpm), respectively.To elucidate the mechanism responsible for the in-

ibition of VSMC growth by manumycin A, we exam-ned the effects of manumycin A on the signal trans-uction pathway mediated by the ras protein afterDGF stimulation. Signal transduction mediated byhe ras protein is dependent on the protein’s localiza-ion at the plasma membrane after farnesylation as aost-translational modification (13). Thus, we exam-ned the level of ras protein localized at the plasma

embrane. Western blot analysis of membrane frac-ions showed a reduced amount of ras protein, suggest-ng that manumycin A inhibited ras farnesylation atetween 60 and 120 seconds after the addition ofanumycin A (Fig. 4a). To determine whether manu-ycin A could also affect a ras downstream signal

ransducer, MAPK, phosphorylation of the kinase wasxamined in the presence or absence of manumycin Ay Western blot analysis. In the absence of manumycin, phosphorylated MAPK increased within 20 minutesfter PDGF stimulation, but in the presence of manu-ycin A, phosphorylated MAPK did not increase but

ather decreased and was hardly detectable at 20 min-tes (Fig. 4b).Then we examined whether manumycin A had the

bility to inhibit the migration of VSMCs. In a trans-ell assay, manumycin A (50 ng/ml) showed an inhib-

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FIG. 1. VSMC cultures with or without manumycin A (5–500 ng/ml) in the presence of PDGF (10 ng/ml). VSMCs were exposed for 12ours to only the vehicle (0.1% DMSO) as a control (a), or 5–500 ng/ml of manumycin A dissolved in DMSO (b–d). In all cultures, the finaloncentration of DMSO was 0.1%. Photomicrographs were taken with a phase-contrast microscope at a magnification of 3200. Data areepresentative of three similar results.

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Vol. 264, No. 3, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tory effect on VSMC migration in the presence of 10g/ml of PDGF (n 5 3) (Fig. 5).Actin filaments organized in stress fibers and their

ites of attachment at focal adhesion are thought tolay a critical role in the regulation of cell migration21, 22). To examine whether or not manumycin A hadn effect on the arrangement of a actin, we stained a

FIG. 2. Antiproliferative effect of manumycin A on PDGF-timulated VSMCs. Cells were treated for 12–24 hours with or with-ut manumycin A (5–500 ng/ml) in the presence of PDGF (10 ng/ml).anumycin A inhibited VSMC growth in a dose-dependent manner:

a) [3H]Thymidine incorporation; (b) cell growth. Manumycin A (50g/ml) also showed time-dependent inhibition of VSMC growth: (c)

3H]Thymidine incorporation. Numbers with asterisks show a signif-cant difference (P , 0.01) by ANOVA. Data are shown as means ofhe results of triplicate experiments 6 SE.

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ctin in the presence or absence of manumycin A.igure 6 shows that the untreated control cells had aormal a actin arrangement but that the manumycin-treated cells had disorganized actin.

ISCUSSION

In this study, we demonstrated that manumycin A atlow dose (less than 50 ng/ml) markedly inhibited the

FIG. 3. Antiproliferative effects of manumycin A on both high-nd low-density VSMC cultures. Cells were inoculated at densitiesf 1.0 3 105 or 1.0 3 106 cells per well in 24-well plates, inhich serum had been starved for 24 hours and then stimulated for2 hours with PDGF (10 ng/ml) in the presence or absence of manu-ycin A (50 ng/ml). The cells were labeled with [3H]thymidine (1Ci/well) for 2 hours between 10 and 12 hours after the addition ofanumycin A. Data are shown as means of the results of triplicate

xperiments 6 SE.

FIG. 4. Inhibitory effects of manumycin A on localization of rasrotein at the cytoplasmic membrane and on phosphorylation ofAPK in VSMCs stimulated by PDGF. Membrane fractions (a) and

otal cell extracts (b) were prepared from VSMCs at various timesfter the addition of manumycin A (50 ng/ml) in the presence ofDGF (10 ng/ml), and they were analyzed for ras protein and phos-horylated MAPK by Western blot analyses. Data are representativef three similar results.

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Vol. 264, No. 3, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

roliferation of VSMCs stimulated with PDGF but ex-ibited severe cytotoxicity at a higher dose (more than00 ng/ml). In the presence of PDGF, manumycin A

FIG. 5. Inhibitory effect of manumycin A (50 ng/ml) on the mi-ration of VSMCs stimulated with PDGF (10 ng/ml). Cell mobilityas assessed by the transwell migration assay as described underaterials and Methods. The asterisk indicates a significant differ-

nce (P , 0.01) by ANOVA. Data are represented as means of theesults of riplicate experiments 6 SE.

FIG. 6. Disassembly of a actin stress fibers in VSMCs caused byg/ml). The cells were doubly stained for visualization of nuclei andmagnification of 3400. Data are representative of three similar re

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educed the amount of ras protein localized at thelasma membrane within 60–120 seconds. Previouseports have shown that fully processed ras localizes tohe plasma membrane, whereas unprocessed ras, be-ause of the lack of prenyl anchor, is not membrane-ssociated but accumulates in the cytosol (12, 21). Thisuggests that manumycin A inhibits farnesylation ofhe ras protein, with the result that it can not localizet the plasma membrane. Furthermore, this post-ranslational modification of the ras protein is neces-ary for activation of the protein itself (13). Thus,anumycin A seems to inhibit farnesylation and acti-

ation of the ras protein. Phosphorylation of MAPK, aas downstream signal transducer, was completely in-ibited within 20 minutes in the presence of manumy-in A. Taken together, interception of the PDGF signalransduction at the point of ras protein activation maye responsible for the growth arrest of VSMCs in theresence of manumycin A.Manumycin A markedly inhibited the migration ofSMCs stimulated by PDGF and disorganized a actinbers. Assembly and disassembly of actin filamentslay an important role in cell migration (22, 23). Thus,isorganization of the fibers may be a main cause of thenhibition of VSMC migration by manumycin A.

atment with manumycin A (50 ng/ml) in the presence of PDGF (10ctin. Photomicrographs were taken with a fluorescent microscope atts.

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Proliferation and migration of VSMCs may be closelyelated to restenosis after angioplasty (7). Althoughany clinical trials on the prevention of restenosisave been conducted, the results are not sufficient (24).n the present study, manumycin A at 50 ng/ml effec-ively inhibited both proliferation and migration ofSMCs stimulated by PDGF in vitro. Furthermore, it

nhibited the PDGF-induced proliferation of VSMCsndependently of cell density. However, the effectiveose range of manumycin A was narrow: it had noffect at a one-tenth concentration (5 ng/ml), yet wasytotoxic to VSMCs at a 10-fold higher concentration500 ng/ml). Thus, it may be difficult to deliver thegent in vivo to the site of a balloon injury at the properose. Recently, new local drug delivery systems haveeen developed in an attempt to reduce restenosis bydministering pharmacologic agents directly to the le-ion. When manumycin A is properly delivered in vivoy such a local delivery system (25, 26), it will be quiteseful in preventing restenosis after angioplasty.

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