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C Blackwell Wissenschafts-Verlag 2001

Differentiation (2000) 67:50–58

ORIGINAL ARTICLE

Joy Roy ¡ Monsur Kazi ¡ Ulf Hedin ¡ Johan Thyberg

Phenotypic modulation of arterial smooth muscle cells is associated with prolonged activation of ERK1/2

Accepted in revised form: 25 October 2000

Abstract Arterial smooth muscle cells grown in primary culture on a substrate of fibronectin in serum-free medium are converted from a contractile to a synthetic phenotype. This process is dependent on integrin signaling and includes a major structural reorganization with loss of myofilaments and formation of a large secretory apparatus. Functionally, the cells lose their contractility and become competent to migrate, secrete extracellular matrix components, and proliferate in response to growth factor stimulation. Here, it is demonstrated that the mitogen-activated protein kinases ERK1/2 play a vital role in the fibronectin-mediated modification of rat aortic smooth muscle cells. Immunoblotting showed that phosphorylated ERK1/2 (p44/p42) were expressed throughout the period when the change in phenotypic properties of the cells took place. Moreover, phosphorylated ERK1/2 accumulated in the nucleus as revealed by immunocytochemical staining. Additional support for an active role of ERK1/2 in the shift in smooth muscle phenotype was obtained by the finding that PD98059, an inhibitor of the upstream kinase MEK1, potently suppressed both the expression of phosphorylated ERK1/2 and the fine structural rebuilding of the cells. In conclusion, the observations point to an important and multifaceted role of ERK1/2 in the regulation of differentiated properties and growth of vascular smooth muscle cells.

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J. Roy ( ) ¡ M. Kazi ¡ U. Hedin Department of Surgical Sciences, Karolinska Hospital, S-171 76 Stockholm, Sweden e-mail: Joy.Roy/kirurgi.ki.se Tel: π 46 851 773 561, Fax: π 46 8 33 93 09 J. ThybergDepartment of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden U. S. Copyright Clearance Center Code Statement:

Key words smooth muscle cells ¡ MAP kinase ¡ differentiation ¡ phenotypic modulation

Introduction Smooth muscle cells (SMCs) build up the arterial media and constitute a major cell type in atherosclerotic and restenotic lesions. In the normal media, the SMCs are in a differentiated, quiescent, and contractile phenotype distinguished by the abundance of myofilaments [33]. On the other hand, the SMCs in developing intimal lesions express an immature, synthetic phenotype resembling that seen during vasculogenesis in the fetus [36]. As a part of this phenotypic modification, myofilaments disassemble and are replaced by a widespread endoplasmic reticulum (ER) and a prominent Golgi complex. In parallel, the cells become competent to migrate, proliferate, and secrete extracellular matrix components and so contribute to the increase in mass of the intimal thickenings. A similar change in differentiated properties occurs when SMCs are established in culture, and the in vitro system has been used extensively to study this process [41]. The mechanisms behind the transition of the SMCs from a contractile to a synthetic phenotype are still incompletely known. Earlier studies in our laboratories have suggested that this process is regulated by interactions between extracellular matrix components and integrins [20]. We thus proposed that the basement membrane (a network of laminin, collagen type IV, and heparan sulfate proteoglycans) which usually surrounds the SMCs favors the expression of a contractile phenotype. After its destruction, e.g. by matrix metalloproteases [40], the plasma and extracellular matrix protein fibronectin (FN) is able to interact with the SMCs and promote their conversion into a synthetic phenotype. In support of this model, immunoelectron microscopic

0301–4681/2000/6701–50$ 15.00/0

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analyses demonstrated that the phenotypic modification of the SMCs following arterial injury is accompanied by disappearance of pericellular laminin and that the modified cells move into a FN-rich matrix in the intima [46]. The interaction between FN and SMCs is mediated by the RGD sequence in FN and the integrin a5b1 [3, 16, 17]. This promotes formation of focal adhesions and activation of focal adhesion kinase (FAK) in primary cultures. Moreover, suppression of FAK using the tyrosine kinase inhibitor genistein was shown to prevent the change in phenotype of the cells [18]. The mitogen-activated protein kinase (MAPK) pathway represents another signal transduction system that has been associated with integrin activation. This system is usually organized as a chain of three kinases: a MAPK (entering the nucleus and activating transcription factors), a MAPK activator (MEK or MAPK kinase), and a MEK activator (MEK kinase or MAPK kinase kinase). In the case of growth factor and extracellular matrix signaling, the components in this kinase chain are typically ERK1/2, MEK1/2, and Raf [10, 37]. Integrin-mediated activation of ERK is dependent on receptor clustering, recruitment of the adaptor protein Shc, and an intact cytoskeletal organization. In this manner, extracellular matrix components provide for a sustained ERK activity through the mid G1 phase and so cooperate with growth factors in cell cycle progression [5, 31, 38, 48, 51]. Here, we have studied the role of ERK activation in the phenotypic modulation of arterial SMCs induced by primary culture on a substrate of FN in serum-free medium. The expression and localization of phosphorylated (active) ERK1/2 in the cells and the effect of the synthetic MEK1 inhibitor PD98059 on the structural reorganization of the cells was examined by immunoblotting, immunocytochemical stainings, and electron microscopy. Our findings show that the SMCs maintained a substantial level of ERK1/2 phosphorylation throughout six days of culture on FN in the absence of exogenous growth factors and that inhibition of ERK1/2 activation using PD98059 prevented the transition of the cells from a contractile to a synthetic phenotype.

Methods Materials Ham’s medium F12, fetal calf serum (FCS), collagenase, EHS laminin and the synthetic peptides GRGDSP and GRGESP were obtained from Gibco BRL (Paisley, Scotland), bovine serum albumin (BSA) and bovine plasma FN from Sigma Chemical Company (St. Louis, MO, USA), and cell-culture plasticware from Nunc (Roskilde, Denmark). The medium was supplemented with 10 mM Hepes/10 mM Tes (pH 7.3), 50 mg/ml L-ascorbic acid, and 50 mg/ml gentamycin sulfate (medium F12). GRGDSP and GRGESP peptides were diluted to a final concentration of 250 mg/ml in cell culture medium and added 12 hours after cell

seeding. To prepare substrates for cell culture, FN and laminin were diluted to 10 mg/ml and 20 mg/ml respectively in Dulbecco’s phosphate-buffered saline (PBS, pH 7.3) and allowed to adsorb to the bottom of plastic petri dishes or glass coverslips for 15–20 hours at 20 æC. Before seeding of cells, the dishes/coverslips were rinsed twice with PBS and incubated with medium F12/0.1 % BSA for 30 minutes to block unspecific binding. MEK1 inhibitor (PD98059) was obtained from New England Biolabs (Beverly, MA, USA). Stock solutions were prepared in dimethylsulfoxide (DMSO) at 20 mM and diluted in culture medium to a final concentration of 40 mM (controls received equivalent amounts of DMSO). Mouse monoclonal antibodies against smooth muscle a-actin were from Sigma, mouse monoclonal antibodies against cyclin D1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and rabbit polyclonal antibodies against phosphorylated (active) ERK1/2 from New England Biolabs. Rabbit polyclonal antibodies against ERK1/2 [39] were a generous gift from Dr. R. Seger (University of Washington, Seattle, WA, USA) and rabbit polyclonal antibodies against Golgi a-mannosidase II [30] were kindly provided by Dr. K. W. Moremen (University of Georgia , Athens, GA, USA) and Dr. M. G. Farquhar (University of California, San Diego, CA, USA). Alkaline phosphatase-conjugated goat anti-rabbit IgG, rhodamine-labeled rabbit anti-mouse IgG and rhodamine-labeled swine anti-rabbit IgG were from Dako (Glostrup, Denmark), HRP-labeled goat anti-rabbit IgG from Amersham Pharmacia Biotech (Uppsala, Sweden), and 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) from Boehringer Mannheim (Mannheim, Germany). Cell culture SMCs were isolated from the aortic media of 350–400 g male Sprague-Dawley rats (B&K Universal, Sollentuna, Sweden) by digestion with 0.1 % collagenase in medium F12/0.1 % BSA [44]. After rinsing, the cells were seeded on a substrate of FN in medium F12/0.1 % BSA (40,000 cells/cm2). The cultures were incubated at 37 æC in a humid atmosphere of 5 % CO2 in air, and medium was changed daily. Immunoblotting Freshly isolated SMCs were seeded in FN-coated petri dishes and grown for various periods. The cells were rinsed with cold PBS and lysed in 200 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2 % SDS, 0.5 % sodium deoxycholate, 0.5 % Triton X-100, 0.2 mM sodium vanadate, 100 mM sodium fluoride, 1 mM EDTA) with protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride). The cells were scraped off the dishes and insoluble material was removed by centrifugation. The lysates were mixed with 5 ¿ SDS sample buffer and boiled for 5 minutes. Proteins were separated by SDS-PAGE in 10 % gels followed by transfer to presoaked nitrocellulose membranes (Hybond-C pure; Amersham Pharmacia Biotech) for 70 minutes at 100 V in a Mini Protean II Trans-Blot Apparatus with cooling (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5 % nonfat dry milk in TTBS (25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05 % Tween-80) with agitation at 20 æC for 60 minutes and then incubated for 15 hours at 4 æC with primary antibodies. After washing with TTBS 3 ¿ 15 minutes, the membranes were incubated with either alkaline phosphatase-conjugated goat anti rabbit IgG (1:500) or HRP-labeled goat anti-rabbit 1gG (1:3000). The alkaline phosphatase activity was detected using an alkaline phoshatase conjugate substrate kit (Bio-Rad Laboratories) and HRP activity by enhanced chemiluminescence (ECL, Amersham). Immunofluorescence microscopy Cytospins of freshly isolated SMCs and cells grown on glass coverslips were fixed in 2 % formaldehyde in PBS (pH 7.3) for 60 min-

52 utes, rinsed with PBS, and stored in PBS with 0.02 % sodium azide at 4 æC before use. For immunostaining of smooth muscle a-actin and mannosidase II, the cells were treated with 50 mM ammonium chloride in PBS for 15 minutes and permeabilized with 0.2 % Triton X-100 in PBS for 2–3 minutes. They were then exposed to primary and secondary antibodies for 90 minutes each (diluted in PBS with 0.2 % BSA). For immunostaining of phosphorylated ERK1/2, the cells were permeabilized with 0.2 % Triton X-100 in PBS for 5 minutes and treated with 0.15 M glycine for 2 ∫ 15 minutes. After blocking with 1 % BSA, 0.05 % Tween-20 in PBS for 30 minutes, the coverslips were incubated with primary antibodies (diluted in PBS with 0.1 % BSA) for 15 hours at 4 æC and then with secondary antibodies for 60 minutes at 20 æC. The coverslips were mounted in a glycerol-based medium and the specimens were examined in a Nikon Labophot microscope with epifluorescence optics. Controls incubated without primary antibodies or with unspecific mouse or rabbit IgG were negative. Nuclear staining was performed by incubating coverslips with 1 mg/ml DAPI in PBS for 1 minute just prior to mounting. Electron microscopy The cells were fixed in 3 % glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer (pH 7.3) with 0.05 M sucrose for at least 2 hours, scraped off the dishes with a plastic spatula, transferred to small plastic tubes, and pelleted by centrifugation. After rinsing, they were postfixed in 1.5 % osmium tetroxide in 0.1 M cacodylate buffer (pH 7.3) with 0.5 % potassium ferrocyanate for 2 hours at 4 æC, dehydrated in ethanol (70, 95, 100 %), stained with 2 % uranyl acetate in ethanol, and embedded in Spurr low viscosity epoxy resin. Sections of uniform thickness were cut with a diamond knife on an LKB Ultrotome IV, picked up on carbon-coated formvar films, stained with alkaline lead citrate, and examined in a Philips CM120TWIN electron microscope at 80 kV (Philips, Eindhoven, The Netherlands). For quantitative evaluation of cell fine structure, two non-overlapping sections from each cell pellet were scanned and mid-sagittal sections through the central parts of the cells (extending from the nucleus toward the periphery) were photographed at a magnification of 15,000 ∫ using a Megaplus CCD camera (Eastman Kodak Company, San Diego, CA, USA). The pictures were subsequently examined using the analySIS system (Soft Imaging Software, Münster, Germany). A square lattice with test points 0.5 mm apart was superimposed on the micrographs and the volume density of the main cytoplasmic organelles was determined by point counting [50]. In addition, the number of caveolae on the cell surface was counted and the length of the plasma membrane within the plane of section was measured.

Fig. 1 Lysates prepared from rat aorta and cultured rat aortic SMCs were analyzed by immunoblotting using primary antibodies that detect phosphorylated ERK1/2. (A) Phosphorylated ERK1/2 in aorta (A), freshly isolated SMCs (F), and SMCs grown in primary culture on a substrate of FN in medium F12/0.1 % BSA for up to six days. (B) ERK1/2 phosphorylation in SMCs incubated in secondary culture on a substrate of FN in medium F12/0.1 % BSA for up to 4 hours. (C) ERK1/2 phosphorylation in SMCs grown for three days in primary culture on a substrate of FN in medium F12/0.1 % BSA and then treated with 10 % FCS for 15 or 30 minutes. The experiments were performed at least three times with similar results.

Results Sustained ERK1/2 phosphorylation in SMCs in primary culture To study a possible role of ERK activation in the FN-mediated change in phenotypic properties of SMCs, we have analyzed the expression of phosphorylated ERK1/2 in rat aorta and in SMCs grown in primary culture on a substrate of FN in serum-free medium. Phosphorylated ERK1/2 could not be detected in the intact aorta by immunoblotting. On the other hand, phosphorylated ERK1/2 was found in freshly isolated SMCs, directly after adhesion to the substrate, and throughout six days of primary culture (Fig. 1A). The

total levels of enzyme remained unchanged during this period as revealed by blotting with the general ERK1/2 antibodies (see Fig. 4A). We also studied the subcellular localization of ERK using indirect immunofluorescence microscopy. In freshly isolated SMCs a diffuse staining was detected in the cytoplasm (Fig. 2A). The same glass slides were costained with DAPI to confirm the extranuclear location of the phosphorlyated ERK1/2 (Fig. 2B). In primary culture, a faint nuclear staining for phosphorylated ERK1/2 could be detected after 1 day (Fig. 2C) and a strong nuclear staining at later time points (Fig. 2D–F). In contrast to the sustained ERK activation seen during primary culture, adhesion of subcul-

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tured SMCs to FN, i.e. cells already converted into a synthetic phenotype, was followed by the appearance of phosphorylated ERK1/2 30 minutes after adhesion, but little or no active enzyme remained after 4 hours (Fig. 1B). SMCs grown on substrates of FN or laminin for 2 days in the presence of the GRGDSP peptide (added to inhibit the integrin-mediated interaction of the cells with exogenous and/or endogenous FN; see [16]) stained weakly for phosphorylated ERK1/2 (Fig. 3B, C) as compared to SMCs grown on FN in the presence of the control GRGESP peptide (Fig 3A). In addition, no nuclear phosphorylated ERK1/2 was found after treatment of SMCs grown on FN with the MEK1 inhibitor PD98059 for two hours (Fig. 3D). These findings verify the exceptional ability of the SMCs to activate ERK1/2 during a

Fig. 2 Immunofluorescence microscopy of freshly isolated SMCs and SMCs grown in primary culture on a substrate of FN in medium F12/0.1 % BSA for one to six days and stained with primary antibodies against phosphorylated ERK1/2 (A, C–F). Freshly isolated SMCs were also costained with DAPI to identify the nuclei (B). Bars, 50 mm. Similar results were obtained in three separate experiments.

Fig. 3 Immunofluorescence microscopy of SMCs grown in primary culture on substrates of FN (A, B and D) or laminin (C) for 2 days. SMCs were either incubated with 250 mg/ml of the GRGESP (A) or GRGDSP (B and C) peptides (added 12 hours after seeding) or treated with 40 mM PD98059 for 2 hours (D). Bar, 35 mm.

Fig. 4 (A) Immunoblot analysis of phosphorylated ERK1/2 and total ERK1/2 in freshly isolated SMCs and SMCs grown in primary culture on a substrate of FN in medium F12/0.1 % BSA for one or six days either without or with 40 mM PD98059. (B) Immunoblot analysis of cyclin D1 in freshly isolated SMCs and SMCs grown in primary culture on a substrate of FN in medium F12/0.1 % BSA for three days either with or without 40 mM PD98059. The experiments were performed three times with similar results.

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prolonged period in association with the shift in phenotype and in the absence of exogenous mitogens. Stimulation of three-day-old primary cultures with 10 % FCS for 15 or 30 minutes was followed by a further increase

in ERK1/2 phosphorylation (Fig. 1C), indicating that the phosphorylation level induced by binding of the SMCs to FN did not represent a maximal level of ERK1/2 activation.

Effects of the MEK1 inhibitor PD98059 on the change in phenotype of SMCs in primary culture

Fig. 5 Light microscopic appearance of SMCs grown in primary culture on a substrate of FN in medium F12/0.1 % BSA for six days either without (A) or with 40 mM PD98059 (B). Bar, 50 mm.

To explore the role of ERK activation in the phenotypic modulation process, we studied the effects of the MEK1 inhibitor PD98059 on the morphological reorganization of the SMCs in serum-free primary cultures. At 40 mM, PD98059 strongly inhibited ERK1/2 phosphorylation without affecting the total levels of enzyme (Fig. 4A). In addition, PD98059 suppressed the expression of cyclin D1 as analyzed in three-day-old cultures (Fig. 4B). Examination in an inverted microscope showed that the cells attached to the FN substrate and within one day assumed an elongated shape. In control cultures, they subsequently spread out and after six days were still flattened in shape (Fig. 5A). On the other hand, PD98059 prevented cell spreading and the cells remained elongated throughout the observation period (Fig. 5B). Immunofluorescence microscopy demonstrated that the cells were strongly positive for the differentiation marker smooth muscle a-actin early in culture (Fig. 6A). The staining became weaker with time in the controls (Fig. 6B), but remained high in cells exposed to PD98059 (Fig. 6C). In a corresponding manner, staining with antibodies against the Golgi marker mannosidase II re-

Table 1 Effects of MEK1 inhibitor on the structural reorganization of arterial smooth muscle cells in primary culture Organelles

Freshly isolated cells (n Ω 3)

Vv ( %) Filaments 55.3 (4.5) ER 5.2 (3.6) Golgi complex 0.5 (0.4) Lysosomes 3.0 (1.0) Mitochondria 5.9 (1.9) Cytoplasm 30.1 (1.2) Caveolae/mm

Fig. 6 Immunofluorescence microscopy of SMCs grown in primary culture on a substrate of FN in medium F12/0.1 % BSA for one (A) or six days (B–E) either without (A, B and D) or with 40 mM PD98059 (C and E). Stainings were made with primary antibodies against smooth muscle a-actin (A–C) or the Golgi marker enzyme mannosidase II (D and E). Bars, 20 mm.

number/mm 0.5 (0.2)

Control 6 days (n Ω 5)

PD98059 6 days (n Ω 5)

Vv (%) 18.4 (4.9) 23.2 (4.5) 11.8 (4.4) 2.8 (0.4) 7.3 (1.9) 36.5 (4.2)

Vv (%) 37.7 (0.7) 9.5 (2.1) 4.8 (0.8) 3.2 (0.9) 8.7 (1.2) 36.2 (3.9)

⬍ 0.001 ⬍ 0.001 ⬍ 0.05 ⬎ 0.1 ⬎ 0.1 ⬎ 0.1

number/mm 1.1 (0.1)

number/mm 2.2 (0.4)

⬍ 0.01

P

Rat aortic smooth muscle cells were grown on a substrate of plasma FN in medium F12/0.1 % BSA with or without 40 mM PD98059 for 6 days (medium changed daily). The cells were then processed for electron microscopy and determination of the cytoplasmic volume density (Vv) of the main organelles and the number of caveolae per micron plasma membrane length in sections. Results are given as means of five separate experiments (20–30 cells per group and experiment) with standard deviation in parentheses. The statistical significance of the differences of the means was calculated using Student’s t test (control versus PD98059)

55 Fig. 7 Electron microscopy of SMCs grown in primary culture on a substrate of FN in medium F12/0.1 % BSA for six days either without (A) or with 40 mM PD98059 (B). ER, endoplasmic reticulum; F, myofilaments; G, Golgi stacks; M, mitochondria; N, nucleus. Bars, 0.5 mm.

vealed that the Golgi complex grew into a large juxtanuclear network in control cells (Fig. 6D), but stayed small in drug-treated cells (Fig. 6E). Electron microscopic analysis confirmed that the freshly isolated cells were all in a contractile phenotype. Like the situation in vivo [45, 46], more than half of the cytoplasmic volume was occupied by myofilaments, whereas the ER and the Golgi complex were small in size (Table 1). In contrast, caveolae were less apparent than in vivo due to the formation of deep cell surface invaginations during the collagenase digestion. However, as soon as the cells had attached to the substrate and acquired an elongated shape, caveolae were again numerous. After 6 days culture on a substrate of FN in serum-free medium, a marked reorganization of the cells had taken place as previously described [16, 43]. The volume density of myofilaments in the cytoplasm had decreased from ⬎ 50 % to ⬍20 %. At the same time, both the ER and the Golgi complex had increased severalfold in size (Fig. 7A; Table 1). Moreover, cell surface caveolae were few as compared to the situation during the first 1–2 days [47]. In cultures treated with PD98059 for 6

days, most of the cells retained a morphology similar to that seen directly after isolation and during the first 1–2 days in the controls (Fig. 7B). The cytoplasmic volume density of myofilaments was thus more than twofold larger and the volume densities of the ER and the Golgi complex less than half of that in the six-day-old controls. In addition, caveolae were more numerous in the drug-treated cells (Table 1).

Discussion The results of this study indicate that ERK1/2 play an essential role in the FN-mediated transition of vascular SMCs from a contractile to a synthetic phenotype. Phosphorylated ERK1/2 was expressed during the change in phenotypic properties of the cells, and the MEK1 inhibitor PD98059 potently suppressed both the expression of phosphorylated ERK1/2 and the structural reorganization of the cells. In accord with previous findings [23, 24], no phosphorylated ERK1/2 was detected in the intact artery by immunoblotting with phosphospecific

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p42/p44 antibodies. On the other hand, freshly isolated SMCs showed a distinct level of ERK1/2 phosphorylation that was subsequently maintained after attachment and spreading of the cells on FN and throughout six days of culture. Since the ERK activity was extinguished by PD98059, an inhibitor of the upstream kinase MEK1 [1, 9], it is concluded that a continually ongoing phosphorylation of ERK occurred during culture of the SMCs on FN. It should be pointed out that the MEK1 inhibitors PD98059 and U0126 were recently shown to inhibit phosphorylation of another MAPK, ERK5 [22]. Therefore, it cannot be excluded that this kinase may also be involved in the phenotypic modulation of the SMCs. As a further indication of the role of FN in activation of the MAPK pathway in the SMCs, only a weak reaction for phosphorylated ERK1/2 was detected if an RGD-containing peptide was added to the medium in order to inhibit the interaction of FN in the substrate with FN receptors on the cell surface. In a similar manner, only a weak reaction for phosphorylated ERK1/2 was noted in cells grown on a substrate of laminin, a basement membrane protein that in contrast to FN tends to maintain the SMCs in a contractile phenotype [16]. Activation of ERK in response to adhesion has been described in several cell types (for reviews, see [10, 37]). It was recently also reported that ligation of the FN receptor a5b1 is sufficient to activate ERK in fibroblasts [35]. Nevertheless, sustained ERK activation and cell cycle progression usually require cooperation between integrins and growth factor receptors [2, 34, 35, 49]. Accordingly, we observed but a transient ERK phosphorylation when subcultured SMCs were seeded on FN in serum-free medium. In contrast, a constant level of active ERK was noticed for six days in primary culture under identical conditions, despite the fact that more than 95 % of the cells remain in the G0/G1 phase of the cell cycle in this case ([15], Roy et al., unpublished observations). These findings support the idea that the observed ERK activation was induced and maintained by signaling via integrins rather than growth factor receptors. Upon activation, the MEK-ERK complexes dissociate, the phosphorylated ERKs dimerize, and the dimers are actively translocated into the nucleus [6, 8, 11, 26, 37]. Here, we observed a diffuse extranuclear immunostaining for phosphorylated ERK in freshly isolated cells, followed by a weak nuclear staining after one day of culture, and an increased nuclear reactivity over the next few days. In contrast, immunoblotting revealed that a maximum level of ERK phosphorylation had been reached already after one day. A possible explanation could be that the newly activated ERK was dispersed in the cells and did not stain well. Moreover, it has been demonstrated that nuclear retention of MAPK requires de novo synthesis of short-lived proteins that act as nuclear anchors [27]. In this context, it should be

stressed that both the nucleic acid and protein synthetic activities of the SMCs are low during the first days of primary culture [16]. In cycling CHO cells, it was likewise found that nuclear translocation occurred a few hours after the initial detection of phosphorylated ERK2 and coincided with the G1/S phase transition [21]. Such a delayed accumulation could allow the activated kinase to phosphorylate substrates in other parts of the cell than the nucleus [29, 32]. It has been proposed that a sustained ERK activation is necessary [35, 49] but not sufficient for progression through the G1 phase of the cell cycle [7, 25]. In a recent investigation, we saw induction of cyclin D1 in primary cultures of SMCs seeded on a substrate of FN in serumfree medium but no entrance into S-phase (Roy et al., unpublished observations). It is here important to recall that the level of ERK phosphorylation induced by FN was clearly lower than that obtained after serum stimulation. On the basis of these findings, it is suggested that the quantity of active ERK generated by interaction of the SMCs with FN is sufficient to support the transition from a contractile to a synthetic phenotype but not to drive the cell cycle and induce mitosis. This is in agreement with earlier conclusions that the change in SMC phenotype is a necessary but not sufficient requirement for induction of cell growth [4, 15, 43]. The proteins that act as substrates for and mediate the effects of ERK1/2 in the SMCs are not known. Likewise, only little is known about the gene products that are responsible for the shift in SMC phenotype. In both cases, transcription factors that control expression of specific genes as well as regulatory molecules that function in the cytoplasm are likely to be involved. Interestingly, ERK was recently observed to participate in the control of nucleotide synthesis, i.e. the initial step in nucleic acid production. It does so by regulating the activity of carbamoyl phosphate synthetase, an enzyme that catalyzes the first and rate-limiting step in pyrimidine nucleotide synthesis [12]. The conversion of the SMCs from a contractile to a synthetic phenotype includes a burst of synthetic activities, initially the construction of numerous ribosomes and later the manufacturing of a multitude of cellular and extracellular matrix proteins [41]. An overall inhibitory effect of PD98059 on RNA synthesis could thus be expected to strongly suppress the modification of the SMCs. Cyclin D1 is another possible target for ERK in the SMCs and its expression was found to be distinctly inhibited by PD98059. Activation of ERK during cell-matrix interactions is believed to be dependent on cell spreading and focal adhesion formation rather than attachment alone [5, 10, 31]. Accordingly, it has been found that newly isolated SMCs seeded on a basement membrane substrate remain more elongated in shape and show a weaker induction of ERK activity than cells seeded on a FN substrate in serum-free medium or in plain dishes in serum-containing medium [16, 28]. Inhibition of SMC spreading

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on FN using antibodies against integrin b1 or the tyrosine kinase inhibitor genistein restrained the shift in phenotype in a similar manner [17–19]. Notably, integrins are able to activate not only ERK but also JNK and p38, two other members of the MAPK family [37]. However, the ability of the specific MEK1 inhibitor PD98059 to potently interfere with the spreading and phenotypic modification of the SMCs suggests that JNK and p38 are of minor significance in this process as compared to ERK. Another intracellular signaling system that has been proposed to take part in integrin-mediated cell activation is the phosphoinositide 3-kinase (PI3K) pathway [10]. We recently observed that the PI3K inhibitor wortmannin blocked the serum-stimulated proliferation of adult rat aortic SMCs in primary culture, but had little or no effect on the preceding change in overall phenotypic properties of the cells [42]. In contrast, IGF-Istimulated expression of a contractile phenotype in chick embryo gizzard SMCs grown on a substrate of laminin was reported to depend on PI3K activity [13]. Moreover, the PDGF-BB-induced dedifferentiation of these cells was found to involve both the ERK and p38 MAPK pathways [14]. Because of the differences in cell system and experimental setup, a direct comparison between these studies is difficult. A possible interpretation could be that the growth factor- and extracellular matrix-induced changes in SMC phenotype are mediated via different signaling pathways. Acknowledgements The authors thank Karin Blomgren and Mariette Lengquist for expert technical assistance. Financial support was obtained from the Swedish Medical Research Council (06537 and 12233), the Swedish Heart Lung Foundation, the King Gustaf V 80th Birthday Fund, the Swedish Society for Medical Research, the Magn. Bergvall Foundation, the Åke Wiberg Foundation, and Karolinska Institutet.

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Erratum Differentiation 66: 189–196 Adriano Ceccarelli, Natasha Zhukovskaya, Takefumi Kawata, Salvatore Bozzaro, Jeffrey Williams In issue 67:4π5 in the article entitled ‘‘Characterisation of a DNA sequence element that directs Dictyostelium stalk cell–specific gene expression’’ by Adriano Ceccarelli, Natasha Zhukovskaya, Takefumi Kawata, Salvatore Bozzaro, and Jeffrey Williams, the first two authors should have appeared as ‘‘joint first authors’’. The Publisher regrets this omission and apologizes for any misrepresentation it may have caused for the authors.