Effects of Extracellular Matrix on Phenotype Modulation and MAPK Transduction of Rat Aortic Smooth Muscle Cells in Vitro

Experimental and Molecular Pathology 69, 79–90 (2000) doi:10.1006/exmp.2000.2321, available online at http://www.idealibrary.com on

Effects of Extracellular Matrix on Phenotype Modulation and MAPK Transduction of Rat Aortic Smooth Muscle Cells in Vitro

Hanjuan Qin, Toshiyuki Ishiwata, Roujiao Wang, Mitsuhiro Kudo, Munehiro Yokoyama, Zenya Naito, and Goro Asano Department of Pathology, Nippon Medical School, Tokyo, Japan

Received February 5, 2000

The transition of arterial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype may play an essential role in the formation of atherosclerotic and restenotic lesions. This process includes a prominent structural reorganization and allows cells to acquire the ability to migrate, proliferate, and secrete extracellular matrix components. According to Western blotting analysis and immunohistochemical and morphological observations, laminin not only retains SMCs in a contractile state but also possibly stimulates cells to transform a synthetic to a contractile phenotype at an early stage, mediated by P38 MAPK signal transduction. However, fibronectin promotes SMCs to transform from a contractile to a synthetic phenotype, mediated by the ERK MAPK signal pathway. The localization of smooth muscle a -actin, myosin heavy chain isoform SM2, and vimentin in explant-isolated rat SMCs was affected by a substrate of fibronectin and laminin and also by ERK MAP kinase inhibitor (PD098059) and P38 MAPK inhibitor (SB203580). Furthermore, vimentin may play a much more important role in differentiation than desmin in phenotype modulation in rat aortic smooth muscle cells. q 2000 Academic Press Key Words: extracellular marix (ECM); smooth muscle cells (SMCs); phenotype modulation; MAPK transduction.

Ross, 1986; Schwartz et al., 1986) and may play an integral role in the restenotic change of an artery (Ross, 1986; Schwartz et al., 1986; Thyberg, 1996). SMCs reveal a marked decrease in the number of microfilaments in association with an extensive rough endoplasmic reticulum (RER) and a large Golgi complex in the cytoplasm. Similarly, the cells undergo a prominent reorganization of filament proteins, such as actin, myosin, and vimentin (Palmberg et al., 1985; Gabbiani et al., 1984; Owens et al., 1986; Chamley et al., 1977; Skalli and Bloom, 1986; Travo and Weber, 1982; Zhihe and Heping, 1997). As a result of these changes, cells lose their contractility and exhibit the competence to migrate and proliferate in response to mitogen stimuli (Chamley et al., 1977; Chamley-Campbell et al., 1979), as well as to secrete large numbers of extracellular matrix components (Ang et al., 1990; Merrilees et al., 1990; Sjolund et al., 1986). Moreover, once they have entered the cellular cycle, these cells start to produce growth factors themselves and acquire the ability to replicate in the absence of exogenous stimuli (Thyberg, 1996). Extracellular martix components take part in the control of SMC phenotype (Thyberg, 1996), and cell proliferation in vascular disease. Earlier studies have indicated that blood and fibronectin promote modulation of SMC from a contractile to a synthetic phenotype. However, laminin maintains them in a contractile phenotype (Thyberg, 1996). The different effects of fibronectin and laminin suggest that fibronectin

INTRODUCTION

The phenotype modulation of arterial smooth muscle cells (SMCs) from contractile to synthetic phenotype is an early event in atherosclerosis (Campbell and Campbell, 1985;

0014-4800/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.

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80 and laminin may be mediated by different signal pathways. Mitogen-activiated protein kinase (MAPK) cascades are major signal transducers from which cells transduce extracellular stimuli into an intracellular response. MAPKs translocate into the nucleus, where they phosphorylate and regulate transcription factors. MAPKs include at least three groups: the extracellular signal-regulated kinase (ERK); the stressactivated protein kinase (SAPK)/N-terminal c-jun kinase (JNKs), and P38 (Robinson and Cobb, 1997). Wary et al. reported that there are several mechanisms by which integrins lead to MAP kinase activation (Schlaepfer et al., 1994; Clark and Hynes, 1996; Chen et al., 1996; Renshaw et al., 1996; Wary et al., 1996). We used PD098059 (which blocks ERK by preventing the activation of its immmediate activator) and SB203580 (which directly inhibits P38 MAPK activation) inhibitors and observed the roles of ERK and P38 MAPK signal transduction pathways on fibronectin and laminin. Fibronectin/laminin mediating contractile proteins have been implicated as differentiation markers for SMCs in vivo and in vitro (Owens and Thompson, 1986). This study was designed to clarify a synthetic phenotype of the explantisolated rat SMCs affected by a substrate of fibronectin and laminin, as well as its MAPK signal transduction.

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Preparation of Substrates Substrate proteins were dissolved in phosphate-buffered saline (PBS) at 20 mg/ml and used to coat the bottom of the culture dishes or chamber slides and then absorbed overnight at 378C in an atmosphere of 5% CO2 in air. The dishes or chamber slides were rinsed twice with PBS and cells were incubated with medium DF-12/0.1% BSA for 30 min to block nonspecific binding. MAP Kinase Inhibitor PD098059, a specific ERK inhibitor, was purchased from Biomol Research Labs, U.S.A., and SB203580, a specific P38 inhibitor, was purchased from Yamasa Corporation Japan. Then 10 M/L stock solution was prepared in DMSO and stored a 2208C. Final dilutions of the drug were made with medium DF-12/0.1% BSA. Controls were made by 0.05% DMSO/medium DF-12/0.1% BSA that did not affect the cells adversely. Immunological Reagents Mouse monoclonal antibodies against smooth muscle aactin (Dakopatts A/S, Denmark), mouse monoclonal antibodies against vimentin (Dakopatts A/S), and antibodies

MATERIALS AND METHODS

Cell Culture Rat aortic SMCs were isolated from the thoracic aorta of 250-g male Wistar rats using the explant method described by Yoshida (1990). The aorta was carefully opened longitudinally, and the tissue was cut into small pieces and placed into a six-well dish with DF-12 containing 10% FBS, 2.5 mg/ml amphotericin B, and 100 U/ml penicillin. Cells were used between passages 2 and 4. Before all experiments, the subconfluent cells were seeded and cultured with DF-12 without serum for 48 h, and subsequently, the cells were detached with 0.05% trypsin and rinsed twice in medium DF-12/0.1% BSA with 40 mg/ml trypsin inhibitor and suspended in the same medium. However, cells were pretreated for 15 min by the different dose inhibitors. They were then seeded on freshly prepared substrates at a density of 4 3 104 cells/cm2 and incubated at 378C in an atmosphere of 5% CO2 in air until the selected days (medium was changed after 1 day).

FIG. 1. Time course of smooth muscle cell growth cultured with fibronectin and laminin. The number of SMCs expresses as mean 6SEM from three independent experiments is not changed under the two conditions.

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against smooth muscle myosin 2 heavy chain (Biomedical Technology Inc., U.S.A.) were used. Immunofluorescence Microscopic Observations SMCs were seeded on the chamber slide coated with fibronectin or laminin in serum-free medium DF-12/0.1% BSA and cultured for 1, 3, and 5 days. Otherwise, after treatment with different inhibitors for 15 min, they were seeded on chamber slides coated with fibronectin or laminin in serum-deprived medium DF-12/0.1% BSA and cultured for 24 h. The cells were fixed in methanol: acetone (1:1 vol/ vol) for 10 min at 48C. After rinsing with PBS, the cells were blocked for nonspecific reaction using normal animal serum for 30 min at room temperature and exposed to several

primary antibodies, the monoclonal anti-smooth muscle aactin, myosin 2, and vimentin (diluted 1:100, 1:20, 1:50 in 0.1% BSA/PBS seperatly) for 2 h at room temperature, followed by streptavidin-conjugated secondary antibody (diluted 1:50 in PBS/0.1% BSA) for 1 h at room temperature. Subsequently, the immunostained cells were labeled with FITC-conjugated biotin and examined using on Olympus microscope with epifluorescence optics. Protein Extraction and Immunoblotting Analysis Smooth muscle a -actin, smooth muscle myosin heavy chain isoform SM2, and vimentin were quantified using the modified method of Laemmli et al. Cells were counted using a hemocytometer and were lysed in SDS sample buffer,

FIG. 2. Ultrastructure of aortic SMC cultured with fibronectin (a,c) and laminin (b,d) for day 1 (a,b) and day 5 (c,d). On fibronectin, fewer filaments are located under the cytomembrane than on laminin at day 1 and the number gradually decreased with time, but there is no change with time on laminin. However, rough endoplasmic reticulum in the cytoplasm is much more abundant at day 1 than at day 5 on fibronectin. Bars, 3 mm.

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FIG. 3. Ultrastructure of aortic SMC cultured with the inhibitors PD098059 (c,d) and SB203580 (e,f) on fibronectin (a,c,e) and laminin (b,d,f) for 24 h. The filaments located under the cytomembrane are inhibited by PD098059 (c) and SB203580 (f) over controls (a,b), and rough endoplasmic reticulum in the cytoplasm is increased compared with controls. Bars, 3 mm.

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FIG. 4. Immunofluorescence staining of SMC cultured with fibronectin on day 1 (A,C,E) and day 5 (B,D,F) in a -SM actin (A,B), SM2 (C,D), and vimentin (E,F). The distribution of filaments is much less on day 5 than day 1.

84 drawn repeatedly through a 23-gauge syringe, and heated at 948C for 10 min. The samples were loaded and run on a 7.5% SDS–polyacrylamide gel and electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore Bedford, MA). Blots were blocked for 2 h with 5% dried milk in PBS containing 0.1% NaN3 and subsequently incubated overnight at 48C with a 1:1000 dilution of smooth muscle a-actin and vimentin antibody or a 1:150 dilution of smooth muscle myosin SM2 antibody. After 20 min in washing buffer solution (1 M Tris–HCl; 5 M NaCl;10% Triton X-100), blots were incubated with a 1:3000 dilution of alkaline phosphatase-conjugated secondary antibody in TBST (0.2 M Tris–HCl, 0.14 M NaCl, 0.01% Tween 20) for 1 h at room temperature. Immunoreactive protein bands were visualized using nitroblue tetrazolium(NBT)– bromochloroindolyl phosphate (BCIP) and the resultant bands quantified using NIH image software.

Electron Microscopic Observations Cells were cultured on eight-well chamber slides coated with fibronectin and laminin for 1 and 5 days. The cells were fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (PB, pH 7.4). After rinsing in PB, the specimens were postfixed in 1% osmium tetroxide in PB for 1 h at 48C, dehydrated in graded ethanol, stained with 2% uranyl acetate in ethanol, and embeded in epoxy resin. The ultrathin sections were cut on an LKB ultramicrotome, stained with uranyl acetate and lead citrate, and observed in a Hitachi HT 7000 electron microscope. All cells were registered as being of either a contractile or a synthetic phenotype. Cells in intermediate stages accounted for a low percentage and were not assigned to one phenotype or the other.

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Ultrastructure Change In the fibronectin-coated wells, the filament bundles (myofilaments) of SMCs were localized under cytomembranes at day 1 and gradually decreased in a time-dependent manner at day 5. At this stage, SMCs contained a large amount of rough endoplasmic reticulum in the cytoplasm. In contrast, in the laminin-coated wells, the filament bundles were more abundant in the perinuclear zone than in fibronectin at day 1, and there was a small amount of rough endoplasmic reticulum in the cytoplasm up to day 5 (Fig. 2). However, in PD098059, the filament bundles were decreased in fibronectin and in SB203580; the same findings were seen in laminin (Fig. 3).

lmmunofluorescence Observation Using antibodies against a-actin, myosin 2, and vimentin, we observed the localization of microfilament bundles in the substrates using immunocytochemistry. In the fibronectincoated wells, cells which contained stress fibers such as antia-actin, myosin 2 and vimentin were strongly reactive at day 1 and gradually become reduced at day 5 (Fig. 4). In contrast, in the laminin-coated wells, the stress fibers were retained and/or increased with time (Fig. 5). However, the subconfluent cells were pretreated with inhibitor for 15 min and then seeded on the substrates of fibronectin and laminin in serum-free medium and fixed after 24 h of culture. In the PD098059 group, cells on fibronectin stained less strongly from the small dose (0.5 mM). And stress fibers disappeared from the perinucleus and were seen only on the submembrane. In contrast, there was no change in cells on laminin. In cells on laminin, of the SB230580 group the stress fibers became reduced in the cytoplasm, but there was no change in cells on fibronectin (Fig. 6). The immunocytochemical findings using SM2 and vimentin are not shown.

RESULTS Immunoblotting Analysis Cell Growth on Substrates SMCs were seeded on coated substrates of six-well plates and incubated with DF-12/0.1% BSA at 378C in an atmosphere of 5% CO2 in air for up to 5 days. The cells attached and spread out on both substrates and in less than 1 day a subconfluent layer of flattened cells was formed. The cell numbers were kept constant during the experiments using the hemocytometer. The cells did not proliferate throughout the observation period in either substrate (Fig. 1).

To study the differences in proteins present on both substrates, immunoblotting was performed after SDS–PAGE to semiquantify smooth muscle a-actin, myosin 2, and vimentin in freshly isolated SMCs. In the fibronectin-coated dish, the quantities of smooth muscle a-actin, vimentin, and myosin 2 clearly decreased with time. However, in the laminincoated dish, smooth muscle a-actin, myosin 2, and vimentin amounts remained unchanged with time. However, on the first day, the respective amounts of smooth muscle a-actin, myosin 2, and vimentin increased more than on fibronectin

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(Fig. 7). In the PD098059 group, the amount of protein decreased with concentrations from 0.5 mM in SMCs on fibronectin. Also, in the SB203580 group, the protein was reduced in the laminin-coated dish (Fig. 8).

DISCUSSION

Earlier studies have shown that SMCs in the intima of human arteries involved in atherogenesis express a reduced volume density of myofilaments (synthetic phenotype) (Mosse et al., 1985, 1986). In addition, similar phenotypic changes occurred in subconfluent cells, accompanied by distinct alterations in biochemical characteristics. However, the signaling events in this process have not yet been elucidated. Recently, several MAPK signal transduction pathways have been reported. Extensive studies have shown that there were multistep phosphorylation cascades by ligand–cell surface receptor bindings which then transmitted signals to cytosolic and nuclear targets (Seger and Krebs, 1995). ERK MAPK is activated by integrin-mediated adhesion. However, JNK and P38 MAP kinase are strongly activated by environmental stress factors, inducing alterations in cell growth, prostanoid production, and other cellular dysfunctions (Xia et al., 1995; Kramer et a1., 1996). P38 MAPK is activated by dual phosphorylation on tyrosine and threonine residues in cells subjected to various types of stress (Raingeaud et al., 1995). This finding supports the results that after SMCs were subcultured in 1 or 2 passages, the cells revealed a synthetic phenotype (Thyberg, 1996; Chamley-Campbell et al., 1979) and no proliferation was found when they were seeded on substrates in serum-free wells. The cells seeded on a laminin substrate contained a large number of filament bundles compared with cells seeded on a fibronectin substrate on day 1 using immunoblotting and ultrastructural analysis. Moreover, the amount of smooth muscle a-actin, myosin 2, and vimentin remained unchanged until 5 days of immunoblotting analysis. However, when SMCs were seeded on fibronectin substrate, smooth muscle a-actin, myosin 2, and vimentin amounts distinctly decreased with time, as indicated from immunoblotting analysis and ultrastructural and immunocytochemical observations. These findings suggest that the interaction between SMCs and the laminin substrate promotes the synthesis of smooth muscle a-actin (Thyberg, 1996), myosin 2, and vimentin mRNA at an early stage, followed by inhibition of the degeneration of these proteins. Thyburg et al. reported that laminin retained SMCs in a contractile phenotype, whereas fibronectin transfers SMCs

85 from a contractile to a synthetic phenotype (Thyberg, 1996). Hayashi et al. also reported that laminin only had the potency to delay this transition but was unable to maintain it for a long time (Hayashi, 1998). We demonstrated the contractile proteins as differentiation markers (Owens and Thompson, 1986) and showed the possibility that laminin were not only able to retain the SMCs in a contractile phenotype but also possibly stimulate cells from a synthetic to a contractile phenotype at an early stage. The intermediate filaments were vimentin and desmin with a 7- to 12-nm diameter (Bennett et al., 1979; Quinlan and Franke, 1982). Although the functional properties of vascular SMCs have not yet been completely elucidated, the role of maintaining the functional cytoskeleton as well as the phenotypic state has been well established. Earlier studies reported that vimentin is a growth regulator, while desmin is related to contractile activity (Rittling and Baseraga, 1987; Fujimoto et al., 1987). In the present study, vimentin played a key role in phenotype modulation, while desmin’s contribution was not observed (data not shown). These findings indicated that the substrates of extracellular matrix were able to modify the expression of smooth muscle a-actin, myosin 2, and intermediate filaments in cultured SMCs and to participate in phenotype modulation. This suggests that smooth muscle cells seeded on extracellular matrix substrates under serum-free conditions were not able to proliferate without DNA synthesis and fibronectin promoting the transformation of SMCs to a synthetic phenotype. By contrast, laminin not only retained SMCs in a contractile state but also possibly stimulated the cells’ transformation from a synthetic to a contractile phenotype. Since the effects were different, this suggests that there may be different signal transductions. In the present study, the molecular mechanisms by which fibronectin and laminin regulated SMC phenotypes in vitro were investigated by analyzing the changes in a-smooth muscle actin, SM2, and vimentin using MAP kinase inhibitors PD098059 (ERK MAPK) and SB203580 (P38 MAPK). We treated the SMCs in 0.1, 0.5, 1, and 5 mM densities, respectively. In fibronectin, using PD098059, smooth muscle a-actin, SM2, and vimentin were decreased and rearrangement was clearly observed at the 0.5 mM density using immunofluorence staining. The expression of these proteins decreased in a dose-dependent manner by immumoblotting, and the decreased filaments were observed by electron microscopy. However, in laminin, using SB203580, the present study showed similar findings by immunoblotting, immunofluorence staining, and electron microscopic observation. The organization of actin filaments and marked changes in cell morphology are related to functional transition from

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FIG. 5. Immunofluorescence staining of SMC cultured with laminin on day 1 (A,C,E) and day 5 (B,D,F) in a-SM actin (A,B), SM2 (C,D), and vimentin (E,F). The distribution of filaments shows no change with time, respectively.

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FIG. 6. Immunofluorescence staining of SMC cultured with the inhibitors PD098059 (C,E) and SB203580 (D,F) on fibronectin (A,C,E) and laminin (B,D,F) for 24 h. The content of filaments is decreased in fibronectin (C) and laminin (F) compared with controls (A,B).

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FIG. 7. Immunoblotting after SDS–PAGE of a -SM actin (42 kDa), SM2 (200 kDa), and vimentin (57 kDa) on fibronectin and laminin at 1, 3, and 5 days, respectively. The protein content decreases with time on fibronectin, but there is no change on laminin.

a contractile to a synthetic phenotype. It is known that the classic MAP kinase, ERK, is activated through Ras-dependent signal transduction by growth factors and hormones, inducing cellular proliferation and differentiation by stimulating transcription factors that lead to the expression of c-fos and other growth responsive genes (Kundra et al., 1994; Bilato et al., 1995). Integrin-dependent adhesion can lead to activation of the ERK-type MAP kinases (Mark et al., 1996). The small GTP-binding proteins play a key role in membrane receptors and signaling of cytoplasm involved primarily in the regulation of cytoskeletal organization in reponse to extracellular growth factors. Ras has been indicated via a connection to focal adhesion kinase through a Shr/ Grb2/SOS interaction and then via the Raf/MEK pathway to the ERK MAPK transduction cascade to transcriptional activation in the nucleus. Coso et al. (1995) and Minden

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et al. reported that Cdc42 and Rac stimulated a kinase cascade that leads to subsequent activation of the P38 MAPK and JNK/SAPK, but not the MAPK ERK1/2 in fibroblasts (Minden et al., 1995). Wary et al. pointed out that MAP kinase activation was found to be a subunit dependent, occurring with integrins containing a1, a5, or aV subunits but not with integrins containing a2 or a3 subunits (Wary et al., 1996). Using this model, the different effects on phenotype modulation when cultured on fibronectin and laminin are probably due to the different signal transduction pathways induced by the small GTP-binding proteins. It is known that Rac/Cdc42 small GTP-binding proteins link plasma membrane receptors to the assembly and organization of the actin cytoskeleton. Early studies reported that Rac was a key control element in the reorganization of the actin cytoskeleton induced by growth factors in fibroblasts (Ridley and Hall, 1992) and Cdc42 in actin remodeling has also been established. Expression of constitutively active mutants of Rac/Cdc42 in Hela, NIH-3T3, and Cos cells resulted in stimulation of P38 activity (Coso et al., 1995). There is evidence indicating that Rho(Rac/Cdc42) plays a role in integrin-mediated signaling events (Mark et al., 1996), which suggests that laminin should be mediated by laminin receptors to associate with and activate Rac/Cdc42 small GTP-binding proteins and then via the downstream signal cascade to P38 MAPK. Fibronectin should be mediated by a5b1 receptors to associate with and activate Ras small GTP-binding protein and then through the Raf/MEK pathway to ERK MAPK. These findings suggest that laminin promotes SMC transformation from a synthetic to a contractile phenotype at an

FIG. 8. Immunoblotting after SDS-PAGE of a -SM actin (42 kDa), SM2 (200 kDa), and vimentin (57 kDa) with the inhibitors PD098059 and SB203580 on fibronectin and laminin at 0, 0.5, and 5 mM doses for 24 h. On fibronectin and laminin, the protein content was inhibited by PD098059 and SB203580 dose dependently.

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early stage and then retains it in the contractile phenotype. This is mediated by the P38 MAP kinase signal cascade. Otherwise, fibronectin promotes SMCs from a contractile to a synthetic phenotype, which is mediated by the ERK MAPK signal pathway. In addition, vimentin plays a much more important role in differentiation than desmin in the phenotype modulation of rat aortic smooth muscle cells.

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