Coordinate Control of Proliferation and Migration by the p27Kip1/Cyclin-Dependent Kinase/Retinoblastoma Pathway in Vascular Smooth Muscle Cells and Fibroblasts
Previous studies have demonstrated a protective effect of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 against atherosclerosis and restenosis, two disorders characterized by abundant proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts. These therapeutic effects might result from p27Kip1-dependent suppression of both cell proliferation and migration. However, the interplay between cell growth and locomotion remains obscure. We show here that p27Kip1 inhibits cellular changes that normally occur during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and focal adhesions). Importantly, a p27Kip1 mutant lacking CDK inhibitory activity failed to inhibit vascular smooth muscle cell and fibroblast proliferation and migration. Moreover, a constitutively active mutant of the retinoblastoma protein (pRb) insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration. In contrast, inactivation of pRb by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Collectively, these results suggest that cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/CDK/pRb pathway. These findings might have important implications for the development of novel therapeutic strategies targeting the fibroproliferative/migratory component of vascular occlusive disorders.
Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts play an important role in the pathobiology of vascular occlusive disease (eg, atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel bypass graft failure).1,2 Thus, understanding the molecular mechanisms that control hyperplastic growth and the locomotion of vascular cells should aid in the development of novel therapeutic strategies to reduce neointimal thickening.
Cell cycle progression is controlled by several cyclin-dependent kinases (CDKs) that associate with regulatory cyclins.3 Mitogenic stimuli activate CDK/cyclin holoenzymes, thus causing hyperphosphorylation of the retinoblastoma protein (pRb) and related “pocket” proteins from mid G1 to mitosis. CDK-dependent pRb hyperphosphorylation releases E2F transcription factors, thus contributing to the expression of several growth and cell cycle–regulatory genes with functional E2F-binding sites in their promoters.4
Interaction of CDKs/cyclins with CDK inhibitory proteins (CKIs) attenuates CDK activity and promotes growth arrest.5 CKIs of the Cip/Kip family (p21Cip1, p27Kip1, and p57Kip2) bind to and inhibit a wide spectrum of CDK/cyclin holoenzymes, whereas members of the Ink4 family (p16Ink4a, p15Ink4b, p18Ink4c, and p19Ink4d) are specific for cyclin D–associated CDKs. Mitogenic and antimitogenic stimuli affect the rates of synthesis and degradation of CKIs as well as their redistribution among different CDK/cyclin pairs.5 For example, p27Kip1 promotes the assembly of CDK4/cyclin D complexes, thus facilitating CDK2/cyclin E activation through the G1/S phase. Moreover, the proto-oncogene c-myc plays a key role in p27 sequestration through modulation of the level of cyclin D and E proteins.
Upregulation of p21Cip1 and p27Kip1 expression at late time points after experimental balloon angioplasty might limit neointimal hyperplasia after the initial proliferative wave.6,7 Consistent with this notion, ectopic expression of p21Cip1 and p27Kip1, but not p16Ink4a, has been shown to significantly reduce neointimal thickening after angioplasty6,8–12 and vein grafting.13 The protective role of p27Kip1 against neointimal thickening has also been demonstrated in hypercholesterolemic apolipoprotein-deficient mice, in which genetic inactivation of one or two p27Kip1 alleles progressively accelerated atherogenesis.14 However, neointimal hyperplasia after mechanical damage of the arterial wall was similar in wild-type and p27Kip1-null mice.15 Redundant roles between p21Cip1 and p27Kip1 or compensatory increase in p21Cip1 expression (or other CKIs) might account for the lack of phenotype of p27Kip1-null mice in the setting of mechanical arterial injury. It is also noteworthy to consider several studies with rapamycin, a bacterial macrolide that attenuates experimental restenosis16,17 and may prevent human in-stent restenosis.18 Rapamycin-dependent growth arrest in cultured cells depends on the stabilization of p27Kip1.19,20 However, rapamycin failed to prevent the downregulation of p27Kip1 seen 24 hours after balloon injury,21 and rapamycin-dependent inhibition of neointimal formation after mechanical injury was similar in wild-type and p27Kip1-null mice.15 Collectively, these studies suggest that the protective effect of rapamycin against neointimal thickening is not mediated by p27Kip1.
Regarding the role of CKIs during human atherosclerosis, more frequent expression of p27Kip1 and p21Cip1 has been reported within regions of human coronary atheromas not undergoing proliferation.7 Concordant expression of transforming growth factor-β receptors I and II in virtually all cells positive for p27Kip1 within human atherosclerotic plaques indicates that the transforming growth factor-β1 present in these lesions may contribute to p27Kip1 upregulation.22 Moreover, coexpression of p53 and p21Cip1 in human carotid atheromatous plaque cells that revealed a lack of proliferation markers suggests that an induction of p21Cip1 may occur via transcriptional activation by p53.23
Recent studies have implicated p21Cip1 and p27Kip1 as negative regulators of vascular cell migration in vitro. First, the sensitivity of VSMCs to the antimigratory activity of rapamycin depends on p27Kip1.24 Second, overexpression of p27Kip1 and p21Cip1 reduced human vein endothelial cell (HUVEC) and VSMC migration in vitro.25–27 Thus, protection against neointimal thickening by these CKIs might result from the combination of growth suppression and migration blockade. Although the mechanisms responsible for p27Kip1- and p21Cip1-dependent growth arrest have been extensively investigated, how these CKIs exert their antimigratory function remains unknown. In the present study, we examined the molecular mechanisms underlying the antimigratory function of p27Kip1 in VSMCs and fibroblasts and investigated the interplay between cell proliferation and migration. Our findings suggest that the p27Kip1/CDK/pRb pathway coordinately regulates cell proliferation and migration.
Materials and Methods
Cell Culture, Retroviral Infection, and Proliferation Assays
Primary VSMCs were obtained from explant cultures of aortic tissue from adult Sprague-Dawley rats (Harlan Interfauna Iberica, Barcelona, Spain). E19P cells (gift from C. Shanahan, Addenbrooke’s Hospital, Cambridge, UK) have been described previously.27a These cells expressed smooth muscle 22α, calponin, and smooth muscle α-actin mRNAs (C. Shanahan, PhD, personal communication, 2001). NIH-3T3 cells were obtained from the American Type Culture Collection. Cells were incubated at 37°C in a humidified 5% CO2/95% O2 atmosphere in DMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mmol/L l-glutamine, and either 10% FBS (primary VSMCs, E19P) or 10% NCS (NIH-3T3 cells). Unless otherwise stated, experiments were performed with cells harvested at subconfluent densities.
Infection with retroviruses was as described.28 Briefly, cells were seeded 24 hours before infection at 70% to 80% confluence (100-mm dishes). Viral supernatants were mixed with Polybrene (8 μg/mL, Sigma) and added to cells. After 4 hours at 37°C, viral supernatants were removed, and cells were incubated with normal growth medium for 8 hours. This process was repeated twice to improve infection. Infected cells were selected at least for 36 to 48 hours with 2.5 mg/L puromycin (Sigma). For E1A rescue experiments, cells were first infected with retroviral vector (Rev) encoding for E1A (Rev-E1A) or control Rev-hygro, selected with 100 mg/L hygromycin (Invitrogen), and infected again with Rev-LacZ or Rev-p27. Doubly infected cells were selected with both hygromycin and puromycin. The generation of clones from individual cells requires multiple cell divisions over a long period of time, an approach that is not possible when dealing with growth suppressor proteins (eg, p27 and pRb). Thus, in the present study, we used uncloned pooled populations of antibiotic-resistant cells.
[3H]Thymidine incorporation assays were performed in triplicate as previously described25 in cultures plated on 24-well culture dishes at a density of 5×103 cells per well and incubated for 4 hours with [3H]thymidine (1 μCi/mL, Amersham).
Western Blot Analysis and CDK Assay
Whole-cell extracts and Western blot analysis were performed as described6 with the use of anti-p27Kip1 (1:250) and anti-CDK2 (1:500) antibodies (sc-1641 and sc-163, respectively; Santa Cruz Biotechnology). Densitometric analysis of the blots was performed with Labimage version 2.6 software.
CDK activity in cell lysates was determined as described,6 except that CDK/cyclin holoenzymes were immunoprecipitated with 0.2 μg of each anti-cyclin E (sc-481) and anti-cyclin A (sc-751) antibody.
Migration of cells labeled with calcein-AM (Molecular Probes) was assessed using the FALCON HTS FluoroBlock transwell system (Becton Dickinson) as described.25 Cells were placed in the inserts (5×104 per insert) in serum-free medium. The lower chamber contained either serum-free medium supplemented with 0.1% BSA (unstimulated cells) or the chemotactic agent (10% FBS and 10% NCS for VSMCs and NIH-3T3 cells, respectively). The extent of cell migration was estimated by the fluorescence in the lower chamber, which was measured using a Victor 4120 multilabel counter (Wallac). Results represent the average fluorescence of induced cells (n=3) after subtracting the fluorescence of unstimulated cells (n=2).
Cells were seeded on glass coverslips and serum-starved in 0.2% FBS for 2 days. When indicated, cells were challenged with a 0.8% agarose plug in PBS containing 50% FBS after serum starvation. The coverslips were washed with PBS, fixed in 4% paraformaldehyde/PBS, and permeabilized with 0.5% Triton X-100/PBS. After blocking with 1% BSA/PBS, cells were incubated for 90 minutes with TRITC-conjugated phalloidin (1:500, Sigma) to label actin filaments and/or anti-paxillin antibody (1:200, clone 349, Transduction Laboratories). A mouse monoclonal antibody was used for p27 expression (DSC-72.F6, Master Diagnostica). Immunocomplexes were detected with biotinylated anti-mouse antibody (1:600, E0413, DAKO) followed by streptavidin-FITC (1:600, F0422, DAKO) or streptavidin–Texas Red (1:600, 5-872, Molecular Probes). Microscopic images were digitally recorded on an Axioscope II microscope (Zeiss). Confocal microscopy was performed at the SCSIE (University of Valencia) using a TCS SP LEICA microscope and VISILOG.5 software.
Results are reported as mean±SEM. Unless otherwise stated, differences were evaluated by ANOVA and Fisher post hoc test. Statistically significant differences were considered at P<0.05.
Effect of p27Kip1 on Focal Adhesions and Cytoskeletal Architecture
Using transwell migration assays, we have previously demonstrated the antimigratory activity of p27Kip1 in primary cultures of HUVECs25 and rabbit VSMCs.27 Because cell locomotion is a complex dynamic process that requires changes in the distribution of focal adhesions (FAs) and reorganization of the actin cytoskeleton,29 we investigated the effect of p27 overexpression on these processes by infecting cells with Rev-p27. Control cultures were infected with Rev-LacZ. Preliminary Western blot analysis revealed a 2.6- to 4.2-fold increase in p27Kip1 expression in rat VSMCs (E19P) and murine fibroblasts (NIH-3T3) infected with Rev-p27 (Figures 1A and 1B). In uninfected E19P cells, the endogenous p27Kip1 protein level was 3.1-fold higher in highly confluent versus subconfluent cultures (Figure 1A), and NIH-3T3 cells maintained in 0.5% FBS increased p27Kip1 expression by 3.8-fold compared with cultures fed 10% FBS (data not shown). Thus, retrovirally expressed p27Kip1 accumulated to physiologically high levels. Indirect immunofluorescence experiments revealed consistent and uniform overexpression of p27Kip1 in Rev-p27–infected cells (Figure 1C).
Serum-starved NIH-3T3 fibroblasts infected with either Rev-p27 or Rev-LacZ displayed a flat nonpolarized shape with a similar organization of actin filaments (Figure 2A) and widespread distribution of the FA-associated protein paxillin (Figure 2B). We next examined well-defined characteristics of migrating cells in cultures challenged with a chemotactic stimulus, including cell polarization with the formation of lamellipodia, redistribution of FAs toward these protrusions, and reorganization of the actin cytoskeleton.29 Analysis of stimulated cultures infected with control Rev-LacZ revealed the appearance of polarized cells that accumulated FAs within the lamellipodial protrusions (Figure 2E). Moreover, confocal microscopy of polarized cells revealed the characteristic organization of actin filaments in migrating cells, with long actin filaments oriented parallel to the long axis and a network of diagonally linked actin filaments within the lamellipodia (Figure 2D, left), in contrast to the pattern of relatively short actin filaments arranged in multiple orientations seen in nonpolarized cells (Figure 2D, right). Cells with an unambiguous migratory phenotype were also observed in stimulated cultures infected with Rev-p27; however, their frequency was relatively low compared with Rev-LacZ–infected NIH-3T3 cells (17±4.6% versus 46±1.4%, respectively) (Figure 2C).
Although polarization in stimulated E19P cells was not as evident as in NIH-3T3 cells, we also noted redistribution of FAs in E19P cultures (Figure 2F; compare left panel showing accumulation of paxillin expression in opposite poles versus widespread expression in the right panel). Moreover, in agreement with the results with NIH-3T3 cells, E19P cells displaying redistribution of FAs were more frequent in cultures infected with Rev-LacZ compared with Rev-p27 (59±3.6% versus 29±2.2%, respectively) (Figure 2F).
Augmented cell size was noticeable on infection with Rev-p27, an effect that was exacerbated in mitogen-stimulated cultures (Figure 2; compare Rev-LacZ– and Rev-p27–infected cultures in the absence and presence of chemotactic stimulus). We scored the number of cells that visibly exceeded the average population size in stimulated cultures. This analysis disclosed 24±1.2% and 8±1.5% of hypertrophic Rev-p27– and Rev-LacZ–infected NIH-3T3 cells, respectively (Figure 2C). Likewise, 40±1.4% of E19P-stimulated cells infected with Rev-p27 were clearly hypertrophic compared with 12±0.8% of hypertrophic Rev-LacZ–infected cells (Figure 2F). These results are in agreement with previous studies showing that p27Kip1 overexpression induced hypertrophy in mitogen-stimulated VSMCs and that angiotensin II–mediated VSMC hypertrophy required a high level of p27Kip1 expression.30,31
The above-mentioned experiments extend our previous studies illustrating the antimigratory activity of p27Kip1 in transwell migration assays25,27 by demonstrating that p27Kip1 can alter the phenotypic modulation that normally occurs during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and FAs). In the presence of serum, adhesion of NIH-3T3 and E19P cells to culture dishes was similar when uninfected cultures were compared with either Rev-p27– or Rev-LacZ–infected cells (Figure 3). Likewise, Rev-p27 did not affect the adhesion of rat primary VSMCs compared with cells infected with Rev-LacZ. Thus, defective cell adhesion does not seem to account for p27Kip1-dependent migration blockade in cells stimulated with serum (Figure 2).
p27Kip1-Dependent Growth Arrest and Migration Blockade Require Inhibition of CDK Activity
The p27CK− mutant protein displays impaired CDK inhibitory activity and lack of growth-suppressive function.28 To investigate whether growth arrest and migration blockade are separate functions of p27Kip1, we compared in E19P and NIH-3T3 cells the effect of Rev-p27 and Rev-p27CK− on growth factor–stimulated proliferation and locomotion. The level of overexpression achieved on infection with Rev-p27CK− was similar to that obtained with Rev-p27 (Figure 1B and data not shown). As expected, Rev-p27 but not Rev-p27CK− reduced CDK activity (Figure 4A). Consistent with these findings, infection of NIH-3T3 and E19P cells with Rev-p27 inhibited [3H]thymidine incorporation compared with that in uninfected cells or in cultures infected with control Rev-LacZ (Figure 4B). In contrast, Rev-p27CK− slightly increased de novo DNA synthesis.
We next examined cell migration using a transwell assay. Preliminary control experiments in NIH-3T3 cells and rat primary VSMCs demonstrated similar migratory capacities of uninfected and Rev-LacZ–infected cultures over a stimulation period of 8 hours (Figure 5A). Under these conditions, infection of E19P cells, NIH-3T3 cells, and rat primary VSMCs with Rev-p27, but not Rev-p27CK−, markedly inhibited serum-induced cell migration (Figure 5B). Of note, p27Kip1 also inhibited platelet-derived growth factor (PDGF)-BB–dependent migration of human vein endothelial cells25 and VSMCs (please see the online data supplement, available at http://www.circresaha.org) in transwell assays.
pRb Status Coordinately Regulates Cell Proliferation and Migration
The above-described results demonstrate that CDK inhibition is essential for the dual function of p27Kip1 as a growth suppressor and migration blocker. Thus, we reasoned that downstream targets of CDK activity might contribute to coordinately regulate cell cycle progression and locomotion. Because CDK-dependent hyperphosphorylation of pRb is critical for cell cycle progression,4 we investigated whether pRb can affect cell migration. We first examined the effect of Rb-HAΔp34, a phosphorylation-deficient pRb mutant insensitive to CDK-dependent inactivation.32 As expected, cultures of asynchronously growing NIH-3T3 cells infected with Rev-Rb-HAΔp34 disclosed a dramatic reduction in [3H]thymidine incorporation versus Rev-LacZ–infected cells (Figure 6A). The antiproliferative effect of Rb-HAΔp34 was markedly correlated with a diminished migratory capacity (Figure 6B).
If inhibition of CDK-mediated pRb hyperphosphorylation contributes to the antimigratory function of p27Kip1, as suggested by the above results, one would expect high proliferative and migratory activity by inactivating pRb function even in the presence of high level of p27 expression and reduced CDK activity. To test this possibility, NIH-3T3 cells were doubly infected with Rev-p27 and Rev-E1A, which encodes for the adenovirus E1A oncoprotein. In previous studies with rodent fibroblasts and cardiac myocytes, E1A has been shown to associate with the growth suppressor pRb, thus alleviating pRb-mediated repression of E2F transcriptional activity and evoking cell cycle progression.33,34 These studies also demonstrated the ability of E1A in preventing growth arrest by p21Cip1 and p27Kip1 without increasing CDK activity. Consistent with these findings, Rev-E1A overcame the proliferative blockade by high level of p27Kip1 expression (Figure 7A) without restoring CDK activity (Figure 7B). Importantly, Rev-E1A restored cell migration in cultures coinfected with Rev-p27 (Figure 7C).
In the present study, we have identified molecular mechanisms that regulate cell proliferation and migration in a coordinated manner (Figure 8). Our results suggest that changes in p27Kip1 expression within the physiological range might concomitantly regulate VSMC and fibroblast growth and locomotion. A low level of p27Kip1 expression was correlated with high proliferative and migratory capacity, whereas nuclear accumulation of this CKI was associated with a quiescent and static phenotype. The observation that blockade of CDK activity was essential for this dual inhibitory function of p27Kip1 (Figure 5B) suggests that modulation of CDK/cyclin activity may coordinately control cell proliferation and locomotion. Indeed, migration induced by PDGF-BB was maximal in VSMCs that were predominantly in late G1,35 a phase of the cell cycle associated with high CDK activity. Moreover, CDK5 and CDK6 appear to regulate several processes that are relevant for cell migration.36–39
McAllister et al40 have recently reported that efficient transduction of a misfolded TAT-p27Kip1 fusion protein into HepG2 human hepatocellular carcinoma cells induces cell scattering, a finding that seemingly disagrees with the present study. However, it is important to note that this activity of TAT-p27Kip1 occurred without causing cell cycle arrest because of the cytoplasmic accumulation of TAT-p27Kip1 and the lack of association with CDK/cyclin complexes. Importantly, a mutant TAT-p27Kip1 protein that accumulated in the nucleus failed to induce cell migration. In the present study, Rev-p27 led to nuclear accumulation of p27Kip1 (Figure 1C), and p27Kip1-dependent migration blockade required inactivation of CDK/cyclin complexes and growth arrest.
Because pRb is a critical target of CDKs from mid G1 through S phase, we examined whether this growth suppressor can regulate cell migration. Forced expression of Rb-HAΔp34, a pRb mutant refractory to CDK-dependent hyperphosphorylation that prevents transcriptional activation of promoters containing the E2F-binding motif,32,41 evoked a quiescent and static phenotype (Figure 6). Conversely, the oncoprotein E1A, which can trigger cell proliferation by alleviating pRb-mediated repression of E2F transcriptional activity in a CDK-independent manner,33,34 overcame p27Kip1-dependent growth arrest and migration blockade without restoring CDK activity (Figure 7). Thus, p27Kip1-dependent growth suppression and inhibition of migration may be mediated by the accumulation of hypophosphorylated pRb and repression of E2F target genes. Of note in consideration of this model, E2F-1–null keratinocytes exhibited a delay in transit through both G1 and S phases of the cell cycle and substantially impaired migration.42
The interrelationships between cell cycle and cell locomotion appear to be a general phenomenon. For example, the growth suppressors p27Kip1, p16INK4a, and p21Cip1 are potent inhibitors of cell spreading and migration in a variety of cell types, including HUVECs, CS-1 β3 melanoma cells, VSMCs, and NIH-3T3 fibroblasts25–27,36 (also shown in the present study). Moreover, several cytostatic agents (eg, quercetin, mimosine, suramin, rapamycin, and troglitazone) reduced the migratory potential of VSMCs and tumor cells.43–48 Likewise, 17β-estradiol concurrently regulated cell growth and locomotion, leading to increased endothelial cell proliferation and migration and blockade of the chemotactic and mitogenic effects of PDGF-BB in VSMCs.49 Inactivation of activator protein-1 and c-myc transcription factors also inhibited both proliferation and migration of cultured VSMCs,50,51 and p53-deficient VSMCs displayed a higher rate of proliferation and migration than wild-type counterparts.52 It is noteworthy that NBT-II rat bladder carcinoma cells synchronized in G1 migrated simultaneously after fibroblast growth factor-1 stimulation.53 Moreover, cell movement was observed in all phases of the cell cycle, except G2/M, in which cells did not respond to stimulation by fibroblast growth factor-1.53 Fukui et al35 demonstrated maximal migration of PDGF-BB–stimulated VSMCs in late G1. Moreover, transcriptional profiling of human fibroblasts has identified diverse cytoskeletal reorganization genes and many genes involved in cell motility and remodeling of the extracellular matrix that exhibit cell cycle–dependent regulation.54 Thus, the position in the cell cycle appears to be a major determinant of the competence of a cell for migration.
In conclusion, we propose that cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/CDK/pRb/E2F pathway. Further clarification of the molecular networks underlying the interrelationships between cell cycle–regulatory factors and the chemotactic machinery should shed significant mechanistic insight into the pathobiology of neointimal lesion development, thus facilitating the development of novel therapies for the treatment of vascular occlusive disorders.
Work in the laboratory of Dr Andrés is currently supported by the Spanish Ministerio de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (grants SAF2001-2358 and SAF2002-1143) and by Generalitat Valenciana (grants GV01-488 and CTGCA/2002/04). Dr Díez-Juan received salary support from the Spanish Government and Fondo Europeo de Desarrollo Regional (grant 1FD97-1035-C02-02) and from Fondo Social Europeo (CSIC-Programa I3P). We thank B. Amati for providing retrovirus, C. Shanahan for providing the E19P cells, C. Guerri for expert advice on immunofluorescence, and E. Navarro-Raga for confocal microscopy.
Original received August 13, 2002; resubmission received December 27, 2002; revised resubmission received January 17, 2003; accepted January 21, 2003.
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