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Ectopic expression of Smad7 inhibits transforming growth factor-β responses in vascular smooth muscle cells
Life Sciences 69 (2001) 2641–2652
Ectopic expression of Smad7 inhibits transforming growth factor-b responses in vascular smooth muscle cells Seiya Katoa,* Seiji Uedab, Kiyoshi Tamakib, Makiko Fujiic, Kohei Miyazonoc, Peter ten Dijked, Minoru Morimatsua, Seiya Okudab a
Department of Pathology, Kurume University, School of Medicine, Kurume, Japan Department of Pathology, Kurume University, School of Medicine, Kurume, Japan c Department of Biochemistry, The Cancer Institute, Tokyo, Japan d Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands b
Received 20 December 2000; accepted 8 February 2001
Abstract Vascular injury stimulates the cytokine-growth factor network in the vascular wall, including transforming growth factor-b (TGF-b). Reportedly, the intracellular signaling of TGF-b is mediated by Smad proteins. We tested the effects of the ectopic expression of inhibitory Smads in cultured rat smooth muscle cells (SMC) to identify the role of TGF-b/Smad signaling on the phenotypic modulation of SMC. The cells exposed to human recombinant TGF-b1 (10 ng/ml) were stimulated Smad2 phosphorylation. Infection with the replication-deficient adenovirus vector expressing Smad7, but not bacterial b-galactosidase or Smad6, was found to inhibit TGF-b-induced Smad2 phosphorylation in a dose-dependent manner. TGF-b suppressed the serum-induced proliferation of SMC from 36.3% to 51.0% (p,0.01), as measured by hand-counting, and this inhibition was attenuated by the ectopic expression of Smad7 (from 30.7% to 74.8% of the reduction of TGF-b-response, p,0.05), but not Smad6. A BrdU incorporation assay also showed that TGF-b-mediated growth inhibition was attenuated by exogenous Smad7 and that this inhibition can be reversed by an additional expression of exogenous Smad2. TGF-b increased the expression of a-smooth muscle actin and myosin heavy chain by 1.3-fold and 1.6-fold in comparison to the control, respectively, and these increases were attenuated by exogenous Smad7, but not Smad6. Our data indicate that Smads mediate TGF-b responses on SMC phenotypes. Smad7, but not Smad6, may specifically act as an inhibitor of TGF-b responses. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Vascular smooth muscle cells; Phenotypes; Transforming growth factor-b (TGF-b); Smad; Atherosclerosis
* Corresponding author. Department of Pathology, Kurume University, School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Tel: 181-(0)942-31-7547; fax: 181-(0)942-31-0342. E-mail address: [email protected] (S. Kato) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 3 5 0 -9
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Introduction In response to the injury process, vascular smooth muscle cells (SMC) undergo a transition from a contractile (quiescent) to a synthetic (proliferative) phenotype, which is characteristic of an early event in human atherogenesis [1,2]. A variety of growth factors, including transforming growth factor-b (TGF-b), have been shown to play pivotal roles in the regulation of SMC phenotypes [3]. It has been reported that TGF-b inhibits growth factor- or serum-stimulated SMC proliferation in vitro [4–6]. Regarding differentiation, TGF-b induces the expression of contractile proteins such as a-smooth muscle actin (SMA) and myosin heavy chain (MHC) in SMC [7,8]. The production of matrices and the balance between matrix-degrading enzymes and their inhibitors are also regulated by TGF-b [9,10]. At the site of vascular injury, TGF-b is released from aggregated platelets [11], activated macrophages [12], and SMC themselves [13]. The production of TGF-b is up-regulated in the rat balloon arterial injury model [9,13], and the neutralizing antibody against TGF-b suppresses the formation of neointimal hyperplasia [14]. The upregulated expression of TGF-b has also been demonstrated in human coronary restenotic lesions [15]. Taken together, the above findings indicate a significant role of TGF-b signaling in the phenotypic modulation of SMC and atherogenesis, which could be a potential target for molecular-based approaches to the control of disease processes. Smad proteins have been identified as intercellular signaling transducers of the TGF-b superfamily, which includes TGF-b, activin, and bone morphogenic protein (BMP) [16–18]. Signaling of the TGF-b superfamily occurs via ligand-induced dimerization of distinct type I and II serine/threonine kinase receptors [19]. Formation of the heteromer with a constitutive active type II receptor results in the transactivation of type I receptor kinase and propagates the downstream signaling [20,21]. Three types of Smad proteins have been isolated in mammals and classified by structure and function. Pathway-restricted Smad (Smad1, 2, 3, 5, 8) is directly phosphorylated by type I receptor kinase and subsequently binds with commonmediator Smad (Smad4/DPC4) [22]. Smad2 and Smad3 mediate TGF-b/activin signaling, and Smad1, Smad5, and Smad8 mediate BMP signaling [23,24]. The heterometric complex of Smad4/DPC4 and either TGF-b/activin-specific or BMP-specific Smads translocates into the nucleus, leading to TGF-b-dependent transcriptional responses [25]. In contrast, the last group of Smad proteins, inhibitory Smads (Smad6, 7), prevents cellular responses against TGF-b family members. Due to the lack of a C-terminal phosphorylation site, which is required for the formation of an active heterometric complex with Smad4/DPC4, inhibitory Smads bind with the activated type I receptor without mediating downstream signaling and therefore acts as a competitor with pathway-restricted Smads [26]. It has been reported that Smad6 and Smad7 show differential inhibitory effects on BMP- and activin/TGF-b-mediated responses, respectively [27, 28]. The expression of inhibitory Smad is induced by TGF-b itself or bone morphologic protein (BMP), possibly forming an autoregulatory negative feedback loop [29]. Mice lacking Smad proteins have been probed in relation to the significance of TGF-b/ Smad signaling in pathogenesis [30]. Share stress has been found to induce the expression of both Smad6 and Smad7 in cultured endothelial cells [31]. Transient expression of Smad7 prevents bleomycin-induced lung fibrosis in the mouse model [32]. These findings suggest
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that TGF-b/Smad signaling is involved in many types of disease processes. However, the role of TGF-b/Smad signaling in the phenotypic modulation of SMC and atherogenesis has not been fully explored. In the present study, we tested the hypothesis that TGF-b/Smad signaling regulates SMC phenotypes and investigated the ectopic expression of inhibitory Smads (Smad6, 7) in cultured SMC. Materials and methods Cultured rat vascular smooth muscle cells Rat aortic smooth muscle cells were isolated by enzymatic digestion from the thoracic aorta of 6 week old Sprague-Dawley rats (Charles River, Tokyo Japan) as previously described [33]. All surgical interventions and anesthesia were performed in accordance with the guidelines of the Animal Experiments Committee in our institute. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical Co., Tokyo, Japan) containing 10% fetal calf serum (FCS) (Filtron Pty Ltd., Brooklyn, Australia) together with antibiotics (100 U/ml penicillin and 100 mg/ml of streptomycin) (Life Technologies, Grand Island, NY) at 378C in a humidified 5% CO2-95% air atmosphere, and SMC of less than 10 passages were subjected to the following experiments. SMC were maintained at a subconfluent density, and human recombinant TGF-b1 (PeproTech, Rocky Hill, NJ) was administered at a concentration of 10 ng/ml. Upon infection with adenovirus vectors, the cells were incubated for 12 hours in DMEM containing 2% FCS and vectors at the indicated MOI (multiplicity of infection). Recombinant adenoviruses Replication-deficient human adenovirus vectors expressing mouse Smad2, Smad6, and Smad7 under the control of CMV promoter (AdvCMVSmad2, AdvCMVSmad6, and AdvCMVSmad7, respectively) were generated as previously described [32]. Expression of the transgene in SMC was confirmed by Western blotting with an anti-Flag antibody (Eastman Kodak Co. Scientific Imaging System, Rochester, NY) via expression of the fused Flag-epitope sequence. AdvCMVLacZ encoding bacterial b-galactosidase was used as a control virus in each experiment. The transfection efficiencies were tested by in situ X-Gal staining and more than 90% of cells were positive in the infection with 200 MOI viruses. Negligible cellular toxicity was also observed under this condition based on the measurement of lactate dehydrogenase in the conditioned media (data not shown). Proliferation assay In order to prepare the cells at defined densities and to measure cell growth, the cells were counted using a hemocytometer after trypan blue exclusion. Alternatively, SMC were inoculated on a 96-well plate and 5-bromo-29deoxyuridine (BrdU) incorporation assay [34] was performed using a cell proliferation ELISA colorimetric kit (Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s protocol. Absorbance at 450 nm and 690 nm were measured using a scanning multiwell spectrophotometer (Eimax A-4, Fujirebio, Japan).
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Western blotting The whole-cell lysate was directly dissolved in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (50 mM Tris-HCl [pH6.8], 2% SDS, 10% Glycerol, 0.1% bromophenol blue, and 100 mM dithiothreitol), and sheared three times through a 23-gauge needle. Equal amounts of the samples normalized by the cell number were subsequently resolved by SDS-PAGE. For the detection of phosphorylated Smad2 protein and the expression of endogenous Smad7, polyclonal antibodies generated previously were used [28,32]. For the detection of contractile proteins, membranes were probed with a monoclonal mouse antibody (mAb) against a-smooth muscle actin (Sigma, St. Louis, MO) or a myosin heavy chain (Santa-Cruz Biotechnology, Santa Cruz, CA). The membranes were then incubated with a 1 : 2000 dilution of alkaline phosphatase-conjugated secondary antibodies and visualized with nitroblue tetrazolium (Boehringer Mannheim) and 5-bromo-4-chloro3-indolyl phosphate p-toluidine salt (Boehringer Mannheim) in a carbonic acid buffer (pH 9.8). Some membranes were re-probed with a mAb for b-actin (Sigma) to confirm that an equal amount of sample was loaded in every lane. Densitometric analysis was performed with N.I.H. image-analyzing software. Immunohistochemical examination The cells were grown on a glass cover-slip and cultured as described above. To examine the expression of a contractile protein, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (pH7.4) for 10 minutes, then permealized with 0.2% Triton X-100. The cells were incubated with a 1 : 250 dilution of mAb for a-smooth muscle actin (Dako Japan, Kyoto, Japan) for 1 hour, followed by incubation with a 1 : 1,000 dilution of FITCconjugated rabbit anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA). After counterstaining with 0.1 mg/ml of propidium iodide (Sigma), the specimens were observed with an Olympus fluorescence microscope. Statistical analysis The data are presented as a mean6SD. Statistical analysis was performed by the Student’s t-test. A level of p,0.05 was accepted as statistically significant.
Results TGF-b induced phosphorylation of endogenous Smad2 and exogenous Smad7, but not Smad6, blocked Smad2 activation Culture conditions are considered to profoundly affect SMC phenotypes [35]; cells were therefore constantly seeded at sub-confluent density with media containing 10% FCS to maintain the proliferating state. Western blotting revealed that human recombinant TGF-b1 (10 ng/ml) transiently upregulated the level of phosphorylated Smad2 from 0.5 to 6 hours after administration. The expression of b-actin was not altered during this process (Fig. 1A). Although the expression level of total Smad2 protein was not specifically changed during
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Fig. 1. TGF-b-induced Smad2 phosphorylation and the expression of endogenous Smad7 in SMC. The cells were stimulated with 10 ng/ml of TGF-b1, and the cell lysate was harvested after the indicated incubation period. Samples containing 13105 cells were analyzed by Western blotting, and the membranes were probed with antiphosphorylated Smad2 (A; upper panel), anti-b-actin (A; lower panel), or anti-Smad7 (B) antibodies.
the above time-course (data not shown), endogenous Smad7 expression was transiently induced by TGF-b (Fig. 1B). Next, an ectopic expression of Smad proteins was induced in an adenovirus-mediated system. The infection with AdvCMVSmad7 (10–200 MOI) attenuated TGF-b-induced phosphorylation of Smad2 in a dose-dependent manner (Fig. 2A). However, a vector encoding neither bacterial b-galactosidase (AdvCMVLacZ) nor Smad6
Fig. 2. Ectopic expression of Smad7-inhibited Smad2 phosphorylation in SMC. The cells were infected with AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), or AdvCMVSmad7 (Smad7) at the indicated MOI, or were mock-infected (NV). Two hours after the stimulation with 10 ng/ml of TGF-b1, the cell lysate containing equal numbers of the cells (13105 cells) was analyzed by Western blotting. The membranes were probed with anti-Flag (A&B; upper panel) or anti-phosphorylated Smad2 antibodies (A&B; lower panel). A; The dose-dependent effect of the ectopic expression of Smad7, B; Comparing the effects of the ectopic expression of Smad6 and Smad7.
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(AdvCMVSmad6) was found to inhibit TGF-b-induced Smad2 phosphorylation in SMC (Fig. 2B). Exogenous Smad7, but not Smad6, suppressed TGF-b-mediated growth inhibition in SMC TGF-b1 (10 ng/ml) inhibited serum-induced proliferation of SMC from 36.3% to 51.0% (p,0.01), as measured by cell-counting. TGF-b-mediated growth inhibition was partially inhibited by the infection of AdvCMVSmad7 (from 30.7% to 74.8% of the reduction of TGF-b-mediated growth inhibition, p,0.05), however AdvCMVLacZ and AdvCMVSmad6 had no effect (Fig. 3). The changes in SMC proliferation in response to TGF-b1 (10 ng/ml) were also tested by a BrdU incorporation assay. Under serum-free conditions, TGF-b in this dosage did not appear to exert significant influence on BrdU incorporation. In the culture with 10% FCS, TGF-b reduced BrdU incorporation. The infection with 200 MOI of both AdvCMVLacZ and AdvCMVSmad6 did not alter the BrdU incorporation levels under either TGF-b-untreated or -treated conditions; however, the infection with AdvCMVSmad7 (50– 200 MOI) attenuated TGF-b-mediated growth inhibition in a dose-dependent manner (Fig. 4A). We next tested the up-regulation of Smad2 availability with the infection of AdvCMVSmad2. The infection with AdvCMVSmad2 itself did not alter the BrdU incorporation levels under either TGF-b-untreated or -treated conditions. However, the infection with AdvCMVSmad2 reversed TGF-b-mediated growth inhibition in AdvCMVSmad7-infected cells (Fig. 4B).
Fig. 3. Ectopic expression of Smad7 suppressed TGF-b-mediated growth inhibition in SMC. The cells were seeded at a density of 2.53104 cells/cm2 on a 6-well culture plate at day 0 and were infected with 200 MOI of AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), or AdvCMVSmad7 (Smad7), or were mock-infected (NV) at day 1. The cells were then cultured under serum (10% FCS) with or without TGF-b1 (10 ng/ml), or under serumfree conditions, and cell counting was carried out each day. The data shown are mean6SD in triplicated wells.
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Fig. 4. Ectopic expression of Smad7 suppressed TGF-b-mediated growth inhibition in SMC; this suppression was then reversed by additional expression of exogenous Smad2. The cells were inoculated on a 96-well plate and were then infected with AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), AdvCMVSmad7 (Smad7), or AdvCMVSmad2 (Smad2) at the indicated MOI, or were mock-infected (NV). After 24 hours of incubation under serum (10% FCS) or serum-free conditions with or without TGF-b1 (10 ng/ml), a BrdU incorporation assay was performed. The data shown are mean6SD (n56). # p,0.05, * p,0.01, or NS (not statistically significant) were indicated. A; The dose-dependent effect of ectopic expression of Smad7. B; The effect of exogenous Smad2 on the exogenous Smad7-expressing cells.
Ectopic expression of Smad7 inhibited TGF-b-mediated contractile protein expression in SMC To test the potentiality of TGF-b/Smad signaling in regulating SMC differentiation, we measured the expression of a-smooth muscle actin and myosin heavy chain by Western blotting. Under serum condition, TGF-b1 (10 ng/ml) stimulated both a-smooth muscle actin and myosin heavy chain expression (1.3 and 1.6 fold, respectively), of which stimulation was inhibited by the ectopic expression of Smad7, but not Smad6 (Fig. 5). Immunohistochemistry also revealed that TGF-b-induced a-smooth muscle actin expression and its assembly were suppressed by exogenous Smad7, but not Smad6 (Fig. 6). Discussion In SMC, TGF-b was found to transiently upregulate Smad2 phosphorylation, which was detectable within 6 hours by Western blotting (Fig. 1A). Thus, the phosphorylation of pathway-restricted Smads such as Smad2 may participate in the early response to TGF-b, which is consistent with previous studies using other cell types [16–18]. In this experiment, detectable phosphorylation of Smad2 in the untreated cells may be dependent on endogenous TGF-b. As the Smad2 phosphorylation stimulated by TGF-b1 was weakly observed, it could be an-
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Fig. 5. TGF-b stimulated contractile protein expression of SMC, which was attenuated by exogenous Smad7. The cells were infected with 200 MOI of AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), or AdvCMVSmad7 (Smad7), or were mock-infected (NV). After 48 hours of incubation in serum (10% FCS) with or without TGF-b (10 ng/ml), or under serum-free conditions, the expression of a-smooth muscle actin (A) and myosin heavy chain (B) were measured by Western blotting. The results were also expressed in relative densitometric units with the value for the serum condition without TGF-b in mock-infected cells being 1.0 (mean6SD, n53). # p,0.05, * p,0.01, or NS (not statistically significant) were indicated.
ticipated that Smad3, another member of pathway-restricted Smad, also plays a role in TGF-b signaling in SMC. Further studies should be required for identifying the role of Smad3 in this context. The endogenous expression of an inhibitory Smad, Smad7, and its induction by TGF-b were also detected in SMC (Fig. 1B). There may be an intracrine negative feedback loop in SMC regulating TGF-b responses. Our data may suggest that the ectopic expression of Smad7 effectively blocks Smad2 phosphorylation (Fig. 2) and TGF-b responses (Fig. 3–6) in SMC. In other mammalian cells such as mink lung epithelial cells, Smad2 and Smad7 have been reported to interact at the TGF-b type I receptor level [16–18]. In SMC, we assume that Smad7 also binds to activated Smad2, and thus competes with Smad4/DPC4 (a common-mediator Smad) for heteromer formation with Smad2. On the other hand, exogenous Smad6 did not exhibit any inhibitory effect on TGF-b responses in SMC (Fig. 2–6). In the present study, we did not show the exogenous Smad6 transfected with an adenovirus vector was functional, however we have reported that the transfection using this vector effectively inhibited the signaling mediated by TGF-b superfamily. For example, the exogenous Smad6 has been shown to inhibit osteoblast differentiation of BMP6-treated C2C12 cells [36]. It was shown that Smad7 is more potent in inhibiting TGF-b signaling than Smad6 [27, 28] and Smad6 preferentially attenuates signaling of BMPs, but not TGF-b [37]. Thus, it is considered that Smad7, but not Smad6, specifically inhibits TGF-b-mediated signaling in SMC.
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Fig. 6. TGF-b stimulated a-smooth muscle actin expression of SMC, which was attenuated by exogenous Smad7. The cells seeded on a glass coverslip were infected with adenovirus vectors (200 MOI) and further cultured for 48 hours as shown in Fig. 5. Immunohistochemical analysis was carried out for the expression of asmooth muscle actin. A&B; mock infected, C&D; AdvCMVSmad6 infected. E&F; AdvCMVSmad7 infected. A,C&E; 10% FCS, B,D&F; 10% FCS1TGF-b1 (10 ng/ml).
In the present study, a relatively high dose (10 ng/ml) of TGF-b1 inhibited serum-stimulated SMC proliferation when the cells were seeded at a sub-confluent density (Fig. 3,4), which is consistent with previous investigations [4–6,38]. TGF-b-mediated growth inhibition of SMC was attenuated by exogenous Smad7, the effect of which was reversed by additional administration of exogenous Smad2 (Fig. 4B). These results may indicate the significance of Smad-mediated signaling in TGF-b-mediated growth control of SMC. The effect of TGF-b on SMC growth is generally considered to be bifunctional. It has been reported that TGF-b stimulates SMC proliferation when cells are seeded at a post-confluent density [39]. When the cells are exposed to the lowest concentration of TGF-b, such as 0.25 ng/ml, TGF-b shows a stimulus effect on SMC proliferation [39]. Thus, our observations should be limited to the sub-confluent proliferating SMC exposed to a highdose of TGF-b. The availability of TGF-b/Smad signaling and its biological significance under each cell condition or different TGF-b dosages remains to be elucidated. TGF-b has been shown to induce specific contractile protein expression and hypertrophy in SMC [5]. We tested the expression of a-smooth muscle actin as an early differentiation marker and myosin heavy chain as a late differentiation marker [40], and the expression of both was found to be increased in TGF-b-treated cells (Fig. 5, 6). In the present study, the exogenous Smad7 resulted in both recovery of proliferation and the reduction of contractile expressions in TGF-b-stimulated SMC. It has been reported that TGF-b-induced SMC hypertrophy was not dependent on withdrawal of cells from cell cycle [6]. Thus, we assume that TGF-b/Smad signaling may regulate SMC differentiation, which is independent of its growth regulatory mechanism. Exogenous Smad7 inhibited TGF-b-induced contractile expression
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not only in TGF-b-treated cells but also in TGF-b-untreated cells (data not shown). We therefore assume that endogenous TGF-b also plays a role in the expression of contractile proteins and in the SMC differentiation. In conclusion, our data indicate that TGF-b/Smad signaling plays a role in the regulation of vascular smooth muscle phenotype and ectopic expression of an inhibitory Smad, Smad7, appears to inhibit TGF-b responses. Although a little information has been available on the target genes of TGF-b/Smad signaling in SMC, Kanai et al. recently reported that CARP (Cardiac ankyrin repeat protein), a nuclear factor, is a downstream target of TGF-b/Smad signaling and plays a role in TGF-b-mediated growth regulation in SMC [41]. Modification of the signaling loop of TGF-b may allow for the development of molecular approaches for preventing vascular diseases. References 1. Chamley-Campbell JH, Champbell GR, Ross R. Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. Journal of Cell Biology 1981; 89 (2): 379–383. 2. Thyberg J, Hedin, U, Sjölund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 1990; 10 (6): 966–990. 3. Chabrier PE. Growth factors and vascular wall. International Angiology 1996; 15 (2): 100–103. 4. Ouchi Y, Hirosumi J, Watanabe M, Hattori A, Nakamura T, Orimo H. Inhibitory effect of transforming growth factor-beta on epidermal growth factor-induced proliferation of cultured rat aortic smooth muscle cells. Biochemical and Biophysical Research Communication 1989; 157 (1): 301–307. 5. Owens GK, Geisterfer AA, Yang YW, Koyama A. Transforming growth factor-b-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. Journal of Cell Biology 1988; 107 (2): 771–780. 6. Morisaki N, Kawano M, Koyama N, Koshikawa T, Umemiya K, Saito Y, Yoshida S. Effects of transforming growth factor-beta 1 on growth of aortic smooth muscle cells. Influences of interaction with growth factors, cell state, cell phenotype, and cell cycle. Atherosclerosis 1991; 88 (2–3): 227–2343. 7. Hautmann MB, Madsen CS, Owens GK. A transforming growth factor b (TGFb) control element drives TGFb-induced stimulation of smooth muscle a-actin gene expression in concert with two CArG elements. Journal of Biological Chemistry 1997; 272 (16): 10948–10956. 8. Orlandi A, Ropraz P, Gabbiani G. Proliferative activity and a-smooth muscle actin expression in cultured rat aortic smooth muscle cells are differently modulated by transforming growth factor-b1 and heparin. Experimental Cell Research 1994; 214 (2): 528–536. 9. Rasmussen LM, Wolf YG, Ruoslahti E. Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor-b activity. American Journal of Pathology 1995; 147 (4): 1041–1048. 10. Fabunmi RP, Baker AH, Murray EJ, Booth RF, Newby AC. Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells. Biochemical Journal 1996; 315 (Pt1): 335–42. 11. Associan RK, Sporn MB. Type beta transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells. Journal of Cell Biology 1986; 102 (4): 1217–1223. 12. Associan RK, Fleurdelys BE, Stevenson HC, Miller PJ, Madtes DK, Raines EW, Ross R, Sporn MB. Expression and secretion of type beta transforming growth factor by activated human macrophages. Proceedings of the National Academy of Sciences of the United States of America 1987; 84 (17):6020–6024. 13. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor beta 1 during repair of arterial injury. Journal of Clinical Investigation 1991; 88 (3):904–910. 14. Nikol S, Isner JM, Pickering JG, Kearney M, Leclerc G, Weir L. Expression of transforming growth factor-beta is increased in human vascular restenosis lesions. Journal of Clinical Investigation 1992; 90 (4):1582–1592.
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35. Kato S, Shanlay JR, Fox JC. Serum Stimulation, cell-cell interactions, and extracellular matrix independently influence smooth muscle cell phenotype in vitro. American Journal of Pathology 1996; 149 (2): 687–697. 36. Fujii M, Takeda K, Imamura T, Aoki H, Sampath TK, Enomoto S, Kawabata M, Kato M, Ichjo H, Miyazono K. Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation. Molecular Biology of the Cell 1999; 10 (11): 3801–3813. 37. Zhao J, shi W, Chen H, Warburton D. Smad7 and Smad6 differntially modulate transforming growth factor b-induced inhibition of embryonic lung morphogenesis. Journal of Biological Chemistry 2000; 275(31): 23992–23997. 38. Majack, RA, Majesky MW, Goodman LV. Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-beta. Journal of Cell Biology 1990; 111 (1): 239–247. 39. Björkerud S. Effects of transforming growth factor-b1 on human arterial smooth muscle cells in vitro. Arteriosclerosis and Thrombosis 1991; 11 (4):892–902. 40. Christen T, Bochaton-Piallat ML, Neuville P, Rensen S, Redard M, van Eys G, Gabbiani G. Cultured porcine coronary artery smooth muscle cells. A new model with advanced differentiation. Circulation Research 1999; 85 (1):99–107. 41. Kanai H, Tanaka T, Aihara Y, Takeda S, Kawabata M, Miyazono K, Nagai R, Kurabayashi M. Transforming growth factor-b/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CACA motif in vascular smooth muscle cells. Circulation Research 2001; 88(1):30–36.
Ectopic expression of Smad7 inhibits transforming growth factor-b responses in vascular smooth muscle cells Seiya Katoa,* Seiji Uedab, Kiyoshi Tamakib, Makiko Fujiic, Kohei Miyazonoc, Peter ten Dijked, Minoru Morimatsua, Seiya Okudab a
Department of Pathology, Kurume University, School of Medicine, Kurume, Japan Department of Pathology, Kurume University, School of Medicine, Kurume, Japan c Department of Biochemistry, The Cancer Institute, Tokyo, Japan d Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands b
Received 20 December 2000; accepted 8 February 2001
Abstract Vascular injury stimulates the cytokine-growth factor network in the vascular wall, including transforming growth factor-b (TGF-b). Reportedly, the intracellular signaling of TGF-b is mediated by Smad proteins. We tested the effects of the ectopic expression of inhibitory Smads in cultured rat smooth muscle cells (SMC) to identify the role of TGF-b/Smad signaling on the phenotypic modulation of SMC. The cells exposed to human recombinant TGF-b1 (10 ng/ml) were stimulated Smad2 phosphorylation. Infection with the replication-deficient adenovirus vector expressing Smad7, but not bacterial b-galactosidase or Smad6, was found to inhibit TGF-b-induced Smad2 phosphorylation in a dose-dependent manner. TGF-b suppressed the serum-induced proliferation of SMC from 36.3% to 51.0% (p,0.01), as measured by hand-counting, and this inhibition was attenuated by the ectopic expression of Smad7 (from 30.7% to 74.8% of the reduction of TGF-b-response, p,0.05), but not Smad6. A BrdU incorporation assay also showed that TGF-b-mediated growth inhibition was attenuated by exogenous Smad7 and that this inhibition can be reversed by an additional expression of exogenous Smad2. TGF-b increased the expression of a-smooth muscle actin and myosin heavy chain by 1.3-fold and 1.6-fold in comparison to the control, respectively, and these increases were attenuated by exogenous Smad7, but not Smad6. Our data indicate that Smads mediate TGF-b responses on SMC phenotypes. Smad7, but not Smad6, may specifically act as an inhibitor of TGF-b responses. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Vascular smooth muscle cells; Phenotypes; Transforming growth factor-b (TGF-b); Smad; Atherosclerosis
* Corresponding author. Department of Pathology, Kurume University, School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Tel: 181-(0)942-31-7547; fax: 181-(0)942-31-0342. E-mail address: [email protected] (S. Kato) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 3 5 0 -9
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Introduction In response to the injury process, vascular smooth muscle cells (SMC) undergo a transition from a contractile (quiescent) to a synthetic (proliferative) phenotype, which is characteristic of an early event in human atherogenesis [1,2]. A variety of growth factors, including transforming growth factor-b (TGF-b), have been shown to play pivotal roles in the regulation of SMC phenotypes [3]. It has been reported that TGF-b inhibits growth factor- or serum-stimulated SMC proliferation in vitro [4–6]. Regarding differentiation, TGF-b induces the expression of contractile proteins such as a-smooth muscle actin (SMA) and myosin heavy chain (MHC) in SMC [7,8]. The production of matrices and the balance between matrix-degrading enzymes and their inhibitors are also regulated by TGF-b [9,10]. At the site of vascular injury, TGF-b is released from aggregated platelets [11], activated macrophages [12], and SMC themselves [13]. The production of TGF-b is up-regulated in the rat balloon arterial injury model [9,13], and the neutralizing antibody against TGF-b suppresses the formation of neointimal hyperplasia [14]. The upregulated expression of TGF-b has also been demonstrated in human coronary restenotic lesions [15]. Taken together, the above findings indicate a significant role of TGF-b signaling in the phenotypic modulation of SMC and atherogenesis, which could be a potential target for molecular-based approaches to the control of disease processes. Smad proteins have been identified as intercellular signaling transducers of the TGF-b superfamily, which includes TGF-b, activin, and bone morphogenic protein (BMP) [16–18]. Signaling of the TGF-b superfamily occurs via ligand-induced dimerization of distinct type I and II serine/threonine kinase receptors [19]. Formation of the heteromer with a constitutive active type II receptor results in the transactivation of type I receptor kinase and propagates the downstream signaling [20,21]. Three types of Smad proteins have been isolated in mammals and classified by structure and function. Pathway-restricted Smad (Smad1, 2, 3, 5, 8) is directly phosphorylated by type I receptor kinase and subsequently binds with commonmediator Smad (Smad4/DPC4) [22]. Smad2 and Smad3 mediate TGF-b/activin signaling, and Smad1, Smad5, and Smad8 mediate BMP signaling [23,24]. The heterometric complex of Smad4/DPC4 and either TGF-b/activin-specific or BMP-specific Smads translocates into the nucleus, leading to TGF-b-dependent transcriptional responses [25]. In contrast, the last group of Smad proteins, inhibitory Smads (Smad6, 7), prevents cellular responses against TGF-b family members. Due to the lack of a C-terminal phosphorylation site, which is required for the formation of an active heterometric complex with Smad4/DPC4, inhibitory Smads bind with the activated type I receptor without mediating downstream signaling and therefore acts as a competitor with pathway-restricted Smads [26]. It has been reported that Smad6 and Smad7 show differential inhibitory effects on BMP- and activin/TGF-b-mediated responses, respectively [27, 28]. The expression of inhibitory Smad is induced by TGF-b itself or bone morphologic protein (BMP), possibly forming an autoregulatory negative feedback loop [29]. Mice lacking Smad proteins have been probed in relation to the significance of TGF-b/ Smad signaling in pathogenesis [30]. Share stress has been found to induce the expression of both Smad6 and Smad7 in cultured endothelial cells [31]. Transient expression of Smad7 prevents bleomycin-induced lung fibrosis in the mouse model [32]. These findings suggest
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that TGF-b/Smad signaling is involved in many types of disease processes. However, the role of TGF-b/Smad signaling in the phenotypic modulation of SMC and atherogenesis has not been fully explored. In the present study, we tested the hypothesis that TGF-b/Smad signaling regulates SMC phenotypes and investigated the ectopic expression of inhibitory Smads (Smad6, 7) in cultured SMC. Materials and methods Cultured rat vascular smooth muscle cells Rat aortic smooth muscle cells were isolated by enzymatic digestion from the thoracic aorta of 6 week old Sprague-Dawley rats (Charles River, Tokyo Japan) as previously described [33]. All surgical interventions and anesthesia were performed in accordance with the guidelines of the Animal Experiments Committee in our institute. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical Co., Tokyo, Japan) containing 10% fetal calf serum (FCS) (Filtron Pty Ltd., Brooklyn, Australia) together with antibiotics (100 U/ml penicillin and 100 mg/ml of streptomycin) (Life Technologies, Grand Island, NY) at 378C in a humidified 5% CO2-95% air atmosphere, and SMC of less than 10 passages were subjected to the following experiments. SMC were maintained at a subconfluent density, and human recombinant TGF-b1 (PeproTech, Rocky Hill, NJ) was administered at a concentration of 10 ng/ml. Upon infection with adenovirus vectors, the cells were incubated for 12 hours in DMEM containing 2% FCS and vectors at the indicated MOI (multiplicity of infection). Recombinant adenoviruses Replication-deficient human adenovirus vectors expressing mouse Smad2, Smad6, and Smad7 under the control of CMV promoter (AdvCMVSmad2, AdvCMVSmad6, and AdvCMVSmad7, respectively) were generated as previously described [32]. Expression of the transgene in SMC was confirmed by Western blotting with an anti-Flag antibody (Eastman Kodak Co. Scientific Imaging System, Rochester, NY) via expression of the fused Flag-epitope sequence. AdvCMVLacZ encoding bacterial b-galactosidase was used as a control virus in each experiment. The transfection efficiencies were tested by in situ X-Gal staining and more than 90% of cells were positive in the infection with 200 MOI viruses. Negligible cellular toxicity was also observed under this condition based on the measurement of lactate dehydrogenase in the conditioned media (data not shown). Proliferation assay In order to prepare the cells at defined densities and to measure cell growth, the cells were counted using a hemocytometer after trypan blue exclusion. Alternatively, SMC were inoculated on a 96-well plate and 5-bromo-29deoxyuridine (BrdU) incorporation assay [34] was performed using a cell proliferation ELISA colorimetric kit (Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s protocol. Absorbance at 450 nm and 690 nm were measured using a scanning multiwell spectrophotometer (Eimax A-4, Fujirebio, Japan).
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Western blotting The whole-cell lysate was directly dissolved in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (50 mM Tris-HCl [pH6.8], 2% SDS, 10% Glycerol, 0.1% bromophenol blue, and 100 mM dithiothreitol), and sheared three times through a 23-gauge needle. Equal amounts of the samples normalized by the cell number were subsequently resolved by SDS-PAGE. For the detection of phosphorylated Smad2 protein and the expression of endogenous Smad7, polyclonal antibodies generated previously were used [28,32]. For the detection of contractile proteins, membranes were probed with a monoclonal mouse antibody (mAb) against a-smooth muscle actin (Sigma, St. Louis, MO) or a myosin heavy chain (Santa-Cruz Biotechnology, Santa Cruz, CA). The membranes were then incubated with a 1 : 2000 dilution of alkaline phosphatase-conjugated secondary antibodies and visualized with nitroblue tetrazolium (Boehringer Mannheim) and 5-bromo-4-chloro3-indolyl phosphate p-toluidine salt (Boehringer Mannheim) in a carbonic acid buffer (pH 9.8). Some membranes were re-probed with a mAb for b-actin (Sigma) to confirm that an equal amount of sample was loaded in every lane. Densitometric analysis was performed with N.I.H. image-analyzing software. Immunohistochemical examination The cells were grown on a glass cover-slip and cultured as described above. To examine the expression of a contractile protein, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (pH7.4) for 10 minutes, then permealized with 0.2% Triton X-100. The cells were incubated with a 1 : 250 dilution of mAb for a-smooth muscle actin (Dako Japan, Kyoto, Japan) for 1 hour, followed by incubation with a 1 : 1,000 dilution of FITCconjugated rabbit anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA). After counterstaining with 0.1 mg/ml of propidium iodide (Sigma), the specimens were observed with an Olympus fluorescence microscope. Statistical analysis The data are presented as a mean6SD. Statistical analysis was performed by the Student’s t-test. A level of p,0.05 was accepted as statistically significant.
Results TGF-b induced phosphorylation of endogenous Smad2 and exogenous Smad7, but not Smad6, blocked Smad2 activation Culture conditions are considered to profoundly affect SMC phenotypes [35]; cells were therefore constantly seeded at sub-confluent density with media containing 10% FCS to maintain the proliferating state. Western blotting revealed that human recombinant TGF-b1 (10 ng/ml) transiently upregulated the level of phosphorylated Smad2 from 0.5 to 6 hours after administration. The expression of b-actin was not altered during this process (Fig. 1A). Although the expression level of total Smad2 protein was not specifically changed during
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Fig. 1. TGF-b-induced Smad2 phosphorylation and the expression of endogenous Smad7 in SMC. The cells were stimulated with 10 ng/ml of TGF-b1, and the cell lysate was harvested after the indicated incubation period. Samples containing 13105 cells were analyzed by Western blotting, and the membranes were probed with antiphosphorylated Smad2 (A; upper panel), anti-b-actin (A; lower panel), or anti-Smad7 (B) antibodies.
the above time-course (data not shown), endogenous Smad7 expression was transiently induced by TGF-b (Fig. 1B). Next, an ectopic expression of Smad proteins was induced in an adenovirus-mediated system. The infection with AdvCMVSmad7 (10–200 MOI) attenuated TGF-b-induced phosphorylation of Smad2 in a dose-dependent manner (Fig. 2A). However, a vector encoding neither bacterial b-galactosidase (AdvCMVLacZ) nor Smad6
Fig. 2. Ectopic expression of Smad7-inhibited Smad2 phosphorylation in SMC. The cells were infected with AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), or AdvCMVSmad7 (Smad7) at the indicated MOI, or were mock-infected (NV). Two hours after the stimulation with 10 ng/ml of TGF-b1, the cell lysate containing equal numbers of the cells (13105 cells) was analyzed by Western blotting. The membranes were probed with anti-Flag (A&B; upper panel) or anti-phosphorylated Smad2 antibodies (A&B; lower panel). A; The dose-dependent effect of the ectopic expression of Smad7, B; Comparing the effects of the ectopic expression of Smad6 and Smad7.
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(AdvCMVSmad6) was found to inhibit TGF-b-induced Smad2 phosphorylation in SMC (Fig. 2B). Exogenous Smad7, but not Smad6, suppressed TGF-b-mediated growth inhibition in SMC TGF-b1 (10 ng/ml) inhibited serum-induced proliferation of SMC from 36.3% to 51.0% (p,0.01), as measured by cell-counting. TGF-b-mediated growth inhibition was partially inhibited by the infection of AdvCMVSmad7 (from 30.7% to 74.8% of the reduction of TGF-b-mediated growth inhibition, p,0.05), however AdvCMVLacZ and AdvCMVSmad6 had no effect (Fig. 3). The changes in SMC proliferation in response to TGF-b1 (10 ng/ml) were also tested by a BrdU incorporation assay. Under serum-free conditions, TGF-b in this dosage did not appear to exert significant influence on BrdU incorporation. In the culture with 10% FCS, TGF-b reduced BrdU incorporation. The infection with 200 MOI of both AdvCMVLacZ and AdvCMVSmad6 did not alter the BrdU incorporation levels under either TGF-b-untreated or -treated conditions; however, the infection with AdvCMVSmad7 (50– 200 MOI) attenuated TGF-b-mediated growth inhibition in a dose-dependent manner (Fig. 4A). We next tested the up-regulation of Smad2 availability with the infection of AdvCMVSmad2. The infection with AdvCMVSmad2 itself did not alter the BrdU incorporation levels under either TGF-b-untreated or -treated conditions. However, the infection with AdvCMVSmad2 reversed TGF-b-mediated growth inhibition in AdvCMVSmad7-infected cells (Fig. 4B).
Fig. 3. Ectopic expression of Smad7 suppressed TGF-b-mediated growth inhibition in SMC. The cells were seeded at a density of 2.53104 cells/cm2 on a 6-well culture plate at day 0 and were infected with 200 MOI of AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), or AdvCMVSmad7 (Smad7), or were mock-infected (NV) at day 1. The cells were then cultured under serum (10% FCS) with or without TGF-b1 (10 ng/ml), or under serumfree conditions, and cell counting was carried out each day. The data shown are mean6SD in triplicated wells.
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Fig. 4. Ectopic expression of Smad7 suppressed TGF-b-mediated growth inhibition in SMC; this suppression was then reversed by additional expression of exogenous Smad2. The cells were inoculated on a 96-well plate and were then infected with AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), AdvCMVSmad7 (Smad7), or AdvCMVSmad2 (Smad2) at the indicated MOI, or were mock-infected (NV). After 24 hours of incubation under serum (10% FCS) or serum-free conditions with or without TGF-b1 (10 ng/ml), a BrdU incorporation assay was performed. The data shown are mean6SD (n56). # p,0.05, * p,0.01, or NS (not statistically significant) were indicated. A; The dose-dependent effect of ectopic expression of Smad7. B; The effect of exogenous Smad2 on the exogenous Smad7-expressing cells.
Ectopic expression of Smad7 inhibited TGF-b-mediated contractile protein expression in SMC To test the potentiality of TGF-b/Smad signaling in regulating SMC differentiation, we measured the expression of a-smooth muscle actin and myosin heavy chain by Western blotting. Under serum condition, TGF-b1 (10 ng/ml) stimulated both a-smooth muscle actin and myosin heavy chain expression (1.3 and 1.6 fold, respectively), of which stimulation was inhibited by the ectopic expression of Smad7, but not Smad6 (Fig. 5). Immunohistochemistry also revealed that TGF-b-induced a-smooth muscle actin expression and its assembly were suppressed by exogenous Smad7, but not Smad6 (Fig. 6). Discussion In SMC, TGF-b was found to transiently upregulate Smad2 phosphorylation, which was detectable within 6 hours by Western blotting (Fig. 1A). Thus, the phosphorylation of pathway-restricted Smads such as Smad2 may participate in the early response to TGF-b, which is consistent with previous studies using other cell types [16–18]. In this experiment, detectable phosphorylation of Smad2 in the untreated cells may be dependent on endogenous TGF-b. As the Smad2 phosphorylation stimulated by TGF-b1 was weakly observed, it could be an-
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Fig. 5. TGF-b stimulated contractile protein expression of SMC, which was attenuated by exogenous Smad7. The cells were infected with 200 MOI of AdvCMVLacZ (LZ), AdvCMVSmad6 (Smad6), or AdvCMVSmad7 (Smad7), or were mock-infected (NV). After 48 hours of incubation in serum (10% FCS) with or without TGF-b (10 ng/ml), or under serum-free conditions, the expression of a-smooth muscle actin (A) and myosin heavy chain (B) were measured by Western blotting. The results were also expressed in relative densitometric units with the value for the serum condition without TGF-b in mock-infected cells being 1.0 (mean6SD, n53). # p,0.05, * p,0.01, or NS (not statistically significant) were indicated.
ticipated that Smad3, another member of pathway-restricted Smad, also plays a role in TGF-b signaling in SMC. Further studies should be required for identifying the role of Smad3 in this context. The endogenous expression of an inhibitory Smad, Smad7, and its induction by TGF-b were also detected in SMC (Fig. 1B). There may be an intracrine negative feedback loop in SMC regulating TGF-b responses. Our data may suggest that the ectopic expression of Smad7 effectively blocks Smad2 phosphorylation (Fig. 2) and TGF-b responses (Fig. 3–6) in SMC. In other mammalian cells such as mink lung epithelial cells, Smad2 and Smad7 have been reported to interact at the TGF-b type I receptor level [16–18]. In SMC, we assume that Smad7 also binds to activated Smad2, and thus competes with Smad4/DPC4 (a common-mediator Smad) for heteromer formation with Smad2. On the other hand, exogenous Smad6 did not exhibit any inhibitory effect on TGF-b responses in SMC (Fig. 2–6). In the present study, we did not show the exogenous Smad6 transfected with an adenovirus vector was functional, however we have reported that the transfection using this vector effectively inhibited the signaling mediated by TGF-b superfamily. For example, the exogenous Smad6 has been shown to inhibit osteoblast differentiation of BMP6-treated C2C12 cells [36]. It was shown that Smad7 is more potent in inhibiting TGF-b signaling than Smad6 [27, 28] and Smad6 preferentially attenuates signaling of BMPs, but not TGF-b [37]. Thus, it is considered that Smad7, but not Smad6, specifically inhibits TGF-b-mediated signaling in SMC.
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Fig. 6. TGF-b stimulated a-smooth muscle actin expression of SMC, which was attenuated by exogenous Smad7. The cells seeded on a glass coverslip were infected with adenovirus vectors (200 MOI) and further cultured for 48 hours as shown in Fig. 5. Immunohistochemical analysis was carried out for the expression of asmooth muscle actin. A&B; mock infected, C&D; AdvCMVSmad6 infected. E&F; AdvCMVSmad7 infected. A,C&E; 10% FCS, B,D&F; 10% FCS1TGF-b1 (10 ng/ml).
In the present study, a relatively high dose (10 ng/ml) of TGF-b1 inhibited serum-stimulated SMC proliferation when the cells were seeded at a sub-confluent density (Fig. 3,4), which is consistent with previous investigations [4–6,38]. TGF-b-mediated growth inhibition of SMC was attenuated by exogenous Smad7, the effect of which was reversed by additional administration of exogenous Smad2 (Fig. 4B). These results may indicate the significance of Smad-mediated signaling in TGF-b-mediated growth control of SMC. The effect of TGF-b on SMC growth is generally considered to be bifunctional. It has been reported that TGF-b stimulates SMC proliferation when cells are seeded at a post-confluent density [39]. When the cells are exposed to the lowest concentration of TGF-b, such as 0.25 ng/ml, TGF-b shows a stimulus effect on SMC proliferation [39]. Thus, our observations should be limited to the sub-confluent proliferating SMC exposed to a highdose of TGF-b. The availability of TGF-b/Smad signaling and its biological significance under each cell condition or different TGF-b dosages remains to be elucidated. TGF-b has been shown to induce specific contractile protein expression and hypertrophy in SMC [5]. We tested the expression of a-smooth muscle actin as an early differentiation marker and myosin heavy chain as a late differentiation marker [40], and the expression of both was found to be increased in TGF-b-treated cells (Fig. 5, 6). In the present study, the exogenous Smad7 resulted in both recovery of proliferation and the reduction of contractile expressions in TGF-b-stimulated SMC. It has been reported that TGF-b-induced SMC hypertrophy was not dependent on withdrawal of cells from cell cycle [6]. Thus, we assume that TGF-b/Smad signaling may regulate SMC differentiation, which is independent of its growth regulatory mechanism. Exogenous Smad7 inhibited TGF-b-induced contractile expression
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not only in TGF-b-treated cells but also in TGF-b-untreated cells (data not shown). We therefore assume that endogenous TGF-b also plays a role in the expression of contractile proteins and in the SMC differentiation. In conclusion, our data indicate that TGF-b/Smad signaling plays a role in the regulation of vascular smooth muscle phenotype and ectopic expression of an inhibitory Smad, Smad7, appears to inhibit TGF-b responses. Although a little information has been available on the target genes of TGF-b/Smad signaling in SMC, Kanai et al. recently reported that CARP (Cardiac ankyrin repeat protein), a nuclear factor, is a downstream target of TGF-b/Smad signaling and plays a role in TGF-b-mediated growth regulation in SMC [41]. Modification of the signaling loop of TGF-b may allow for the development of molecular approaches for preventing vascular diseases. References 1. Chamley-Campbell JH, Champbell GR, Ross R. Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. Journal of Cell Biology 1981; 89 (2): 379–383. 2. Thyberg J, Hedin, U, Sjölund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 1990; 10 (6): 966–990. 3. Chabrier PE. Growth factors and vascular wall. International Angiology 1996; 15 (2): 100–103. 4. Ouchi Y, Hirosumi J, Watanabe M, Hattori A, Nakamura T, Orimo H. Inhibitory effect of transforming growth factor-beta on epidermal growth factor-induced proliferation of cultured rat aortic smooth muscle cells. Biochemical and Biophysical Research Communication 1989; 157 (1): 301–307. 5. Owens GK, Geisterfer AA, Yang YW, Koyama A. Transforming growth factor-b-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. Journal of Cell Biology 1988; 107 (2): 771–780. 6. Morisaki N, Kawano M, Koyama N, Koshikawa T, Umemiya K, Saito Y, Yoshida S. Effects of transforming growth factor-beta 1 on growth of aortic smooth muscle cells. Influences of interaction with growth factors, cell state, cell phenotype, and cell cycle. Atherosclerosis 1991; 88 (2–3): 227–2343. 7. Hautmann MB, Madsen CS, Owens GK. A transforming growth factor b (TGFb) control element drives TGFb-induced stimulation of smooth muscle a-actin gene expression in concert with two CArG elements. Journal of Biological Chemistry 1997; 272 (16): 10948–10956. 8. Orlandi A, Ropraz P, Gabbiani G. Proliferative activity and a-smooth muscle actin expression in cultured rat aortic smooth muscle cells are differently modulated by transforming growth factor-b1 and heparin. Experimental Cell Research 1994; 214 (2): 528–536. 9. Rasmussen LM, Wolf YG, Ruoslahti E. Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor-b activity. American Journal of Pathology 1995; 147 (4): 1041–1048. 10. Fabunmi RP, Baker AH, Murray EJ, Booth RF, Newby AC. Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells. Biochemical Journal 1996; 315 (Pt1): 335–42. 11. Associan RK, Sporn MB. Type beta transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells. Journal of Cell Biology 1986; 102 (4): 1217–1223. 12. Associan RK, Fleurdelys BE, Stevenson HC, Miller PJ, Madtes DK, Raines EW, Ross R, Sporn MB. Expression and secretion of type beta transforming growth factor by activated human macrophages. Proceedings of the National Academy of Sciences of the United States of America 1987; 84 (17):6020–6024. 13. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor beta 1 during repair of arterial injury. Journal of Clinical Investigation 1991; 88 (3):904–910. 14. Nikol S, Isner JM, Pickering JG, Kearney M, Leclerc G, Weir L. Expression of transforming growth factor-beta is increased in human vascular restenosis lesions. Journal of Clinical Investigation 1992; 90 (4):1582–1592.
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