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Inhibition of smooth muscle cell migration by the p21 cyclin-dependent kinase inhibitor (Cip1)
R. Fukui et al. / Atherosclerosis 132 (1997) 53–59
(1/200). Alkaline phosphatase-conjugated second antibodies were used in the detection system.
2.9. Adhesion assay Adhesion studies were performed as described previously [19]. Briefly, a 96-well plate was coated with 60 mg/ml human fibronectin (Sigma, USA), 100 mg/ml bovine collagen type I (Chemicon, USA), 20 mg/ml human vitronectin (Takara, Japan), or 1 mg/ml bovine elastin (Elastin Products, USA), and blocked with 10 mg/ml bovine serum albumin (BSA). The transfected cells were trypsinized, and approximately 2.0× 104 cells were added to each well. After 1 h of incubation at 37°C, the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, stained with 0.5% toluidine blue and rinsed in water. Cells were solubilized by the addition of 100 ml of 1% SDS and quantified using a microtiter plate reader at 595 nm. The experiments described were performed in quadruplicate and were repeated a minimum of 3 times. Means of the data from experiments are shown.
55
transfected SM3 cells with a Cip1 encoding plasmid. Imunocytochemistry showed that the efficiency of Cip1 transfection was approximately 30% (Fig. 1A). Furthermore, we assessed the overexpression of the Cip1 protein by Western Blotting. Lysates from the Cip1transfected cells had a large amount of a 23–24 kDa protein, which was consistent with the Cip1 molecule as previously reported (Fig. 1B) [17]. In contrast, lysates from the vector-transfected cells did not give any obvious bands. Thus we used these transiently transfected cells to analyze the actions of Cip1. These cells were all still viable (\ 97%) as assesed by trypan blue exclusion as previously reported [22]. To investigate the regulating effect of Cip1 on SMC growth, we assessed the incorporation of BrdU of SM3 cells transfected with or without Cip1. The total number of BrdU-labeled cells was significantly decreased in Cip1 transfected cells when compared to vector transfected cells (Fig. 2). Thus Cip1 inhibited SMC proliferation as previously described [13,22].
2.10. Migration assay Migration was assayed by a modification of the Boyden’s chamber method using microchemotaxis chambers (Neuro Probe, USA) and polycarbonate filters (Necleopore, USA) with a pore size of 5.0 mm [20,21]. The filters were coated with 20 mg/ml fibronectin and placed between the chambers. Cells were trypsinized and suspended at a concentration of 5.0 ×105 cells/ml in MEM supplemented with 10% CS. The SMC suspension (50 ml) was placed in the upper chamber, and 25 ml of MEM containing human recombinant PDGF-BB (Sigma, USA) was placed in the lower chamber. The chamber was incubated at 37°C in 5% CO2 in air for 3 h. The filter was removed and the SMCs which had migrated to the upper side of the filter were scraped off. The SMC which migrated to the lower side of the filter were fixed in methanol, stained with Diff-Quick staining solution, and counted under a microscope ( × 100) to quantify SMC migration. Migration activity was expressed as the mean number of cells that had migrated per high-power field.
3. Results To study the effects of Cip1 on SMCs, especially, on adhesion and migration, we constructed an expression plasmid harboring the human Cip1 cDNA and transfected it into SM3 cells, a strain of cultured rabbit arterial SMCs, but we could not isolate colonies of stable transformants because of the effect Cip1 has on cell cycle arrest. Thus we decided to use transiently
Fig. 1. A, Cip1 expression of the transfected SM3 cells using immunocytochemistry. Magnification × 200. The round cell expressed the Cip1, on the other hand, spread cells did not express it. B, lysates from the cells were blotted with antibody. Lane 1, lysate of Cip1 transfected SM3 cells; lane 2, lysate of vector transfected SM3 cells.
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R. Fukui et al. / Atherosclerosis 132 (1997) 53–59
Fig. 2. The percentage of BrdU-labeled cells in Cip1 transfected and vector transfected. Cells were incubated with or without Cip1 for 24 h and then maintained in 10%CS–MEM plus 10 mM BrdU for 24 h. The cells were plated on fibronectin coated 8 mm square Lab-Tek chamber slides and stained with anti-BrdU antibody. Total BrdU-labeled cells were quantitated by counting BrdU-labeled cells. * P B 0.05.
In order to investigate the assembly of the actin stress fiber network and focal adhesions in response to fibronectin, transfected cells were plated onto fibronectin-coated coverslips for 30, 60, 90 and 180 min. Vector-transfected SM3 cells were attached to fibronectin but were round conformation after 30 min of incubation; began to spread after about 60 min; and become highly spread after about 90 min of incubation, forming a monolayer. In contrast, the shape of Cip1transfected SM3 cells kept round conformation on fibronectin even after 180 min. These results led us to examine the protein expression involved in cytoskeletal machinery, specifically, integrin, vinculin, and a-actin. Cytoskeletal organization was assessed immunocytochemically with an anti-aactin antibody, an anti-integrin a5b1 antibody and an anti-vinculin antibody. After 60 min, vector-transfected cells were spread out on fibronectin, the actin filaments were detectable and the integrin a5b1 and vinculin were localized on the cell periphery (Fig. 3). However, Cip1-transfected cells had not spread after 60 min and even after 180 min, the actin, integrin a5b1 and vinculin staining showed faint perinuclear localization and an absence of filaments (Fig. 3). Furthermore, we tested whether Cip1 influenced the total amounts of a-actin, integrin a5b1, and vinculin using Western Blotting or Immunoprecipitation. There was no significant difference of these amounts between them (Fig. 4). Thus Cip1 altered the localization of a-actin, vinculin, and integrin a5b1, but did not alter the whole amounts of these molecules significantly in these partially transfected cells. order to measure the binding ability of Cip1-transfected cells to ECM, cell adhesion assays were per-
formed. Fibronectin and collagen type I have been described as molecules that promote the adhesion and spreading of SMCs. Thus experiments were designed to test the adhesion of transfected cells to fibronectin, collagen type I, vitronectin and elastin substrates. Cells attaching to these substrates were counted via the absorbance, 60 min after delivering the cells suspension. The number of Cip1-transfected cells attached to all the substrates was significantly less than in the vector transfected cells (Fig. 5). Cells plated in control wells with only BSA did not adhere (not shown). Furthermore, we collected both the detached floating cells and the attached cells, and assessed their Cip1 expression by Western Blotting. The detached floating cells showed strong Cip1 expression compared to the attached cells (Fig. 6). Both of the cells were still viable ( \ 97%) as assessed by trypan blue exclusions (not shown). In addition, we functionally characterized Cip1 by testing for its effects on SMC migration using a modified Boyden’s chamber assay. SM3 cells were induced to migrate in a dose dependent manner at concentrations of up to 30.0 ng/ml on fibronectin-coated membrane toward platelet-derived growth factor-BB (PDGF-BB). The basal level of the migratory activity of Cip1-transfected cells was about half that compared with the vector-transfected cells. The PDGF-induced migration activity was significantly lower in the Cip1transfected cells than in the vector-transfected cells at every concentration (Fig. 7). The elevation of the migratory activity induced by PDGF in Cip1-transfected cells was also lower than in vector-transfected cells.
4. Discussion This is the first time that an endogenous cell cycle regulated factor such as Cip1 has been associated with cytoskeletal organization and adhesion and in the inhibition of adhesion and migration in this transient system. Actin filament disassembly and assembly play an important role in the leading edge of cells migrating in a PDGF-BB gradient and these processes enable cytoplasmic flow and protrusion of new leading lamellae [23]. Focal adhesion sites are sites where actin filaments attach to the plasma membrane and actin nucleation sites [24]. We showed that Cip1-transfection altered SM3 cell shape; inhibited the spreading of a-actin, integrin a5b1 and vinculin; and kept their round conformation on fibronectin. But the total amount of these molecules in Cip1-transfected cells was not reduced significantly. Although we did not analyze whether this Cip1transfected round cell was apoptotic or not, Cip1 did not induce cell death as estimated by trypan blue staining in our study. In other kinds of SMCs, Cip1 does not induce apoptosis, but was shown to induce G1
58
R. Fukui et al. / Atherosclerosis 132 (1997) 53–59
Fig. 6. Cip1 expression of both detached and attached cells by Western Blotting. The transfected cells were adhesion assayed on fibronectin and the detached cells were separated from the attached cells by two PBS rinses. Equal amounts of proteins from the cells were immunoblotted with anti-Cip1 antibody. Lane 1, lysate of Cip1 transfected detached cells; lane 2, lysate of Cip1 transfected attached cells.
adhesion molecules by Cip1 may lead to inhibition of integrin-transmembrane signaling. As migration is also critically affected by its adhesive property [28], the inhibitory effect of Cip1 on the adhesion of SMC is thought to be important for migratory activity. DiMilla et al. [28] reported that maximal migration of SMCs on fibronectin and collagen type VI occurred at an inter-
mediate attachment strength. Appropriate strength, therefore, is important for SMC migration. Thus, the inhibition of SMCs migration on fibronectin by Cip1 is considered to be due to its decreasing adhesion to fibronectin. Yang et al. [22] reported that endogenous Cip1 protein was induced in porcine arteries following balloon catheter injury. They also reported that endogenous Cip1 was detected in the neointima and correlated inversely with the location and kinetics of intimal cell proliferation. Taken together, these findings suggest that Cip1 may normally regulate cellular proliferation and migration following arterial injury, and strategies to increase its expression may be therapeutically beneficial in vascular diseases. The signal transduction pathways and the molecular mechanisms associated with cell cycle, cell adhesion and cell migration are not well understood. Integrins are ab heterodimers that mediate adhesion of cells to ECMs and to other cells, and have been suggested to form the basis of the molecular signal transduction cascade to the nucleus [29]. Microinjection of integrin b1c cDNA is reported to inhibit DNA synthesis [30]. These results suggest that adhesion molecules directly associate with the cell cycle. We therefore asked whether a cell cycle inhibitor Cip1 may be involved in the adhesion and the migration. Various cellular functions are regulated by small GTP- binding proteins of the Ras superfamily. In particular, the Rho family, which includes rho, rac and cdc42 has been shown to regulate cell shape [31,32], ruffling [32,33], motility [34,35], proliferation [36], and adhesion [37–39]. Furthermore, inhibition of rho by C3 exoenzyme is thought to induce G1 arrest in fibroblast with rounding of cell bodies [40]. These results may imply that Cip1 is associated with these proteins. Further studies are required to clarify the mechanism of Cip1.
Acknowledgements We are grateful for the comments and encouragement given to us by Dr N. Koyama (Esai Corporation, Japan). We thank Dr M. Seto (Asahikasei Corporation, Japan) for providing SM3 cells, a strained arterial cultured SMC, and Dr J. Miyazaki (Institute of Development, Aging and Cancer, Tohoku University) for providing the eukaryotic expression vector pCAGGS.
References Fig. 7. Modified Boyden’s chamber assay. Migration activity was expressed as the mean number of cells that had migrated per highpower field. **, Vs. vector transfected (PB 0.01).
[1] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801. [2] Sanders M. Molecular and cellular concepts in atherosclerosis. Pharmacol Ther 1994;61:109.
R. Fukui et al. / Atherosclerosis 132 (1997) 53–59 [3] Matsushime H, Quelle DE, Shurtleff SA, Shibuya M, Sherr CJ, Kato JY. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol 1994;14:2066. [4] Matsushime H, Ewen ME, Strom DK, et al. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell 1992;71:323. [5] Sherr CJ. Growth factor-regulated G1 cyclins. Stem Cells Dayt 1994;12(Suppl 1):47. [6] Simons M, Edelman ER, DeKeyser JL, Langer R, Rosenberg RD. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature 1992;359:67. [7] Morishita R, Gibbons GH, Ellison KE, et al. Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 1993;90:8474. [8] Morishita R, Gibbons GH, Ellison KE, et al. Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J Clin Invest 1994;93:1458. [9] Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 1993;75:805. [10] Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993;366:704. [11] Polyak K, Lee MH, Erdjument Bromage H, et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 1994;78:59. [12] Toyoshima H, Hunter T. p27, A novel inhibitor of G1 cyclinCdk protein kinase activity, is related to p21. Cell 1994;78:67. [13] Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest 1995;96:2260. [14] Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991;108:193. [15] Sasaki Y, Uchida T, Sasaki Y. A variant derived from rabbit aortic smooth muscle: phenotype modulation and restoration of smooth muscle characteristics in cells in culture. J Biochem Tokyo 1989;106:1009. [16] Sullian TR, Karas RH, Aronovitz M, et al. Estrogen inhibits the response-to-injury in a mouse carotid artery model. J Clin Invest 1995;96:2482. [17] Fredersdorf S, Milne AW, Hall PA, Lu X. Characterization of a panel of novel anti-p21Waf1/Cip1 monoclonal antibodies and immunochemical analysis of p21Waf1/Cip1 expression in normal human tissus. Am J Pathol 1996;148:825. [18] Warnke R, Levy R. Detection of T and B cell antigens hybridoma monoclonal antibodies: a biotin–avidin–horseradish peroxidase method. J Histochem Cytochem 1980;28:771. [19] Fujio Y, Yamada F, Takahashi K, Shibata N. Altered fibronectin-dependent cell adhesion by PDGF accompanies phenotypic modulation of vascular smooth muscle cells. Biochem Biophys Res Commun 1993;196:997. [20] Koyama N, Hart CE, Clowes AW. Different functions of the platelet-derived growth factor-alpha and -beta receptors for the migration and proliferation of cultured baboon smooth muscle cells. Circ Res 1994;75:682.
.
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[21] Koyama N, Harada K, Yamamoto A, Morisaki N, Saito Y, Yoshida S. Purification and characterization of an autocrine migration factor for vascular smooth muscle cells (SMC), SMCderived migration factor. J Biol Chem 1993;268:13301. [22] Yang ZY, Simari RD, Perkins ND, et al. Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc Natl Acad Sci USA 1996;93:7905. [23] Katayose D, Wersto R, Cowan K, Seth P. Consequences of p53 gene expression by adenovirus vector on cell cycle arrest and apoptosis in human aortic vascular smooth muscle cells. Biochem Biophys Res Commun 1995;215:446. [24] Stossel TP. On the crawling of animal cells. Science 1993;260:1086. [25] Mitchison TJ, Cramer LP. Actin-baced cell motility and cell locomotion. Cell 1996;84:371. [26] Wang J, Walsh K. Resistance to apoptosis conferred by cdk inhibitors during myocyte differentiation. Science 1996;273:359. [27] Zhang Z, Morla AO, Vuori K, Bauer JS, Juliano RL, Ruoslahti E. The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin. J Cell Biol 1993;122:235. [28] DiMilla PA, Stone JA, Quinn JA, Albelda SM, Lauffenburger DA. Maximal migration of human smooth mucle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength. J Cell Biol 1993;122:729 – 37. [29] Miyamoto S, Teramoto H, Coso OA, et al. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol 1995;131:791. [30] Meredith J, Takada Y, Fornaro M, Languino LR, Schwartz MA. Inhibition of cell cycle progression by the alternatively spliced integrin b1c. Science 1995;269:1570. [31] Bussey H. Cell shape determination: a pivotal role for Rho. Science 1996;272:224. [32] Miura Y, Kikuchi A, Musha T, et al. Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in Swiss 3T3 cells. J Biol Chem 1993;268:510. [33] Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992;70:401. [34] Takaishi K, Kikuchi A, Kuroda S, Kotani K, Sasaki T, Takai Y. Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility. Mol Cell Biol 1993;13:72. [35] Stasia MJ, Jouan A, Bourmeyster N, Boquet P, Vignais PV. ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility. Biochem Biophys Res Commun 1991;180:615. [36] Self AJ, Paterson HF, Hall A. Different structural organization of Ras and Rho effector domains. Oncogene 1993;8:655. [37] Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992;70:389. [38] Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol 1994;10:31. [39] Takai Y, Sasaki T, Tanaka K, Nakanishi H. Rho as a regulator of the cytoskeleton. Trends Biochem Sci 1995;20:227. [40] Yamamoto M, Marui N, Sakai T, et al. ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells accumulate in the G1 phase of the cell cycle. Oncogene 1993;8:1449.
(1/200). Alkaline phosphatase-conjugated second antibodies were used in the detection system.
2.9. Adhesion assay Adhesion studies were performed as described previously [19]. Briefly, a 96-well plate was coated with 60 mg/ml human fibronectin (Sigma, USA), 100 mg/ml bovine collagen type I (Chemicon, USA), 20 mg/ml human vitronectin (Takara, Japan), or 1 mg/ml bovine elastin (Elastin Products, USA), and blocked with 10 mg/ml bovine serum albumin (BSA). The transfected cells were trypsinized, and approximately 2.0× 104 cells were added to each well. After 1 h of incubation at 37°C, the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, stained with 0.5% toluidine blue and rinsed in water. Cells were solubilized by the addition of 100 ml of 1% SDS and quantified using a microtiter plate reader at 595 nm. The experiments described were performed in quadruplicate and were repeated a minimum of 3 times. Means of the data from experiments are shown.
55
transfected SM3 cells with a Cip1 encoding plasmid. Imunocytochemistry showed that the efficiency of Cip1 transfection was approximately 30% (Fig. 1A). Furthermore, we assessed the overexpression of the Cip1 protein by Western Blotting. Lysates from the Cip1transfected cells had a large amount of a 23–24 kDa protein, which was consistent with the Cip1 molecule as previously reported (Fig. 1B) [17]. In contrast, lysates from the vector-transfected cells did not give any obvious bands. Thus we used these transiently transfected cells to analyze the actions of Cip1. These cells were all still viable (\ 97%) as assesed by trypan blue exclusion as previously reported [22]. To investigate the regulating effect of Cip1 on SMC growth, we assessed the incorporation of BrdU of SM3 cells transfected with or without Cip1. The total number of BrdU-labeled cells was significantly decreased in Cip1 transfected cells when compared to vector transfected cells (Fig. 2). Thus Cip1 inhibited SMC proliferation as previously described [13,22].
2.10. Migration assay Migration was assayed by a modification of the Boyden’s chamber method using microchemotaxis chambers (Neuro Probe, USA) and polycarbonate filters (Necleopore, USA) with a pore size of 5.0 mm [20,21]. The filters were coated with 20 mg/ml fibronectin and placed between the chambers. Cells were trypsinized and suspended at a concentration of 5.0 ×105 cells/ml in MEM supplemented with 10% CS. The SMC suspension (50 ml) was placed in the upper chamber, and 25 ml of MEM containing human recombinant PDGF-BB (Sigma, USA) was placed in the lower chamber. The chamber was incubated at 37°C in 5% CO2 in air for 3 h. The filter was removed and the SMCs which had migrated to the upper side of the filter were scraped off. The SMC which migrated to the lower side of the filter were fixed in methanol, stained with Diff-Quick staining solution, and counted under a microscope ( × 100) to quantify SMC migration. Migration activity was expressed as the mean number of cells that had migrated per high-power field.
3. Results To study the effects of Cip1 on SMCs, especially, on adhesion and migration, we constructed an expression plasmid harboring the human Cip1 cDNA and transfected it into SM3 cells, a strain of cultured rabbit arterial SMCs, but we could not isolate colonies of stable transformants because of the effect Cip1 has on cell cycle arrest. Thus we decided to use transiently
Fig. 1. A, Cip1 expression of the transfected SM3 cells using immunocytochemistry. Magnification × 200. The round cell expressed the Cip1, on the other hand, spread cells did not express it. B, lysates from the cells were blotted with antibody. Lane 1, lysate of Cip1 transfected SM3 cells; lane 2, lysate of vector transfected SM3 cells.
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R. Fukui et al. / Atherosclerosis 132 (1997) 53–59
Fig. 2. The percentage of BrdU-labeled cells in Cip1 transfected and vector transfected. Cells were incubated with or without Cip1 for 24 h and then maintained in 10%CS–MEM plus 10 mM BrdU for 24 h. The cells were plated on fibronectin coated 8 mm square Lab-Tek chamber slides and stained with anti-BrdU antibody. Total BrdU-labeled cells were quantitated by counting BrdU-labeled cells. * P B 0.05.
In order to investigate the assembly of the actin stress fiber network and focal adhesions in response to fibronectin, transfected cells were plated onto fibronectin-coated coverslips for 30, 60, 90 and 180 min. Vector-transfected SM3 cells were attached to fibronectin but were round conformation after 30 min of incubation; began to spread after about 60 min; and become highly spread after about 90 min of incubation, forming a monolayer. In contrast, the shape of Cip1transfected SM3 cells kept round conformation on fibronectin even after 180 min. These results led us to examine the protein expression involved in cytoskeletal machinery, specifically, integrin, vinculin, and a-actin. Cytoskeletal organization was assessed immunocytochemically with an anti-aactin antibody, an anti-integrin a5b1 antibody and an anti-vinculin antibody. After 60 min, vector-transfected cells were spread out on fibronectin, the actin filaments were detectable and the integrin a5b1 and vinculin were localized on the cell periphery (Fig. 3). However, Cip1-transfected cells had not spread after 60 min and even after 180 min, the actin, integrin a5b1 and vinculin staining showed faint perinuclear localization and an absence of filaments (Fig. 3). Furthermore, we tested whether Cip1 influenced the total amounts of a-actin, integrin a5b1, and vinculin using Western Blotting or Immunoprecipitation. There was no significant difference of these amounts between them (Fig. 4). Thus Cip1 altered the localization of a-actin, vinculin, and integrin a5b1, but did not alter the whole amounts of these molecules significantly in these partially transfected cells. order to measure the binding ability of Cip1-transfected cells to ECM, cell adhesion assays were per-
formed. Fibronectin and collagen type I have been described as molecules that promote the adhesion and spreading of SMCs. Thus experiments were designed to test the adhesion of transfected cells to fibronectin, collagen type I, vitronectin and elastin substrates. Cells attaching to these substrates were counted via the absorbance, 60 min after delivering the cells suspension. The number of Cip1-transfected cells attached to all the substrates was significantly less than in the vector transfected cells (Fig. 5). Cells plated in control wells with only BSA did not adhere (not shown). Furthermore, we collected both the detached floating cells and the attached cells, and assessed their Cip1 expression by Western Blotting. The detached floating cells showed strong Cip1 expression compared to the attached cells (Fig. 6). Both of the cells were still viable ( \ 97%) as assessed by trypan blue exclusions (not shown). In addition, we functionally characterized Cip1 by testing for its effects on SMC migration using a modified Boyden’s chamber assay. SM3 cells were induced to migrate in a dose dependent manner at concentrations of up to 30.0 ng/ml on fibronectin-coated membrane toward platelet-derived growth factor-BB (PDGF-BB). The basal level of the migratory activity of Cip1-transfected cells was about half that compared with the vector-transfected cells. The PDGF-induced migration activity was significantly lower in the Cip1transfected cells than in the vector-transfected cells at every concentration (Fig. 7). The elevation of the migratory activity induced by PDGF in Cip1-transfected cells was also lower than in vector-transfected cells.
4. Discussion This is the first time that an endogenous cell cycle regulated factor such as Cip1 has been associated with cytoskeletal organization and adhesion and in the inhibition of adhesion and migration in this transient system. Actin filament disassembly and assembly play an important role in the leading edge of cells migrating in a PDGF-BB gradient and these processes enable cytoplasmic flow and protrusion of new leading lamellae [23]. Focal adhesion sites are sites where actin filaments attach to the plasma membrane and actin nucleation sites [24]. We showed that Cip1-transfection altered SM3 cell shape; inhibited the spreading of a-actin, integrin a5b1 and vinculin; and kept their round conformation on fibronectin. But the total amount of these molecules in Cip1-transfected cells was not reduced significantly. Although we did not analyze whether this Cip1transfected round cell was apoptotic or not, Cip1 did not induce cell death as estimated by trypan blue staining in our study. In other kinds of SMCs, Cip1 does not induce apoptosis, but was shown to induce G1
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R. Fukui et al. / Atherosclerosis 132 (1997) 53–59
Fig. 6. Cip1 expression of both detached and attached cells by Western Blotting. The transfected cells were adhesion assayed on fibronectin and the detached cells were separated from the attached cells by two PBS rinses. Equal amounts of proteins from the cells were immunoblotted with anti-Cip1 antibody. Lane 1, lysate of Cip1 transfected detached cells; lane 2, lysate of Cip1 transfected attached cells.
adhesion molecules by Cip1 may lead to inhibition of integrin-transmembrane signaling. As migration is also critically affected by its adhesive property [28], the inhibitory effect of Cip1 on the adhesion of SMC is thought to be important for migratory activity. DiMilla et al. [28] reported that maximal migration of SMCs on fibronectin and collagen type VI occurred at an inter-
mediate attachment strength. Appropriate strength, therefore, is important for SMC migration. Thus, the inhibition of SMCs migration on fibronectin by Cip1 is considered to be due to its decreasing adhesion to fibronectin. Yang et al. [22] reported that endogenous Cip1 protein was induced in porcine arteries following balloon catheter injury. They also reported that endogenous Cip1 was detected in the neointima and correlated inversely with the location and kinetics of intimal cell proliferation. Taken together, these findings suggest that Cip1 may normally regulate cellular proliferation and migration following arterial injury, and strategies to increase its expression may be therapeutically beneficial in vascular diseases. The signal transduction pathways and the molecular mechanisms associated with cell cycle, cell adhesion and cell migration are not well understood. Integrins are ab heterodimers that mediate adhesion of cells to ECMs and to other cells, and have been suggested to form the basis of the molecular signal transduction cascade to the nucleus [29]. Microinjection of integrin b1c cDNA is reported to inhibit DNA synthesis [30]. These results suggest that adhesion molecules directly associate with the cell cycle. We therefore asked whether a cell cycle inhibitor Cip1 may be involved in the adhesion and the migration. Various cellular functions are regulated by small GTP- binding proteins of the Ras superfamily. In particular, the Rho family, which includes rho, rac and cdc42 has been shown to regulate cell shape [31,32], ruffling [32,33], motility [34,35], proliferation [36], and adhesion [37–39]. Furthermore, inhibition of rho by C3 exoenzyme is thought to induce G1 arrest in fibroblast with rounding of cell bodies [40]. These results may imply that Cip1 is associated with these proteins. Further studies are required to clarify the mechanism of Cip1.
Acknowledgements We are grateful for the comments and encouragement given to us by Dr N. Koyama (Esai Corporation, Japan). We thank Dr M. Seto (Asahikasei Corporation, Japan) for providing SM3 cells, a strained arterial cultured SMC, and Dr J. Miyazaki (Institute of Development, Aging and Cancer, Tohoku University) for providing the eukaryotic expression vector pCAGGS.
References Fig. 7. Modified Boyden’s chamber assay. Migration activity was expressed as the mean number of cells that had migrated per highpower field. **, Vs. vector transfected (PB 0.01).
[1] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801. [2] Sanders M. Molecular and cellular concepts in atherosclerosis. Pharmacol Ther 1994;61:109.
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