Altered S-Phase Kinase-Associated Protein-2 Levels Are a Major Mediator of Cyclic Nucleotide–Induced Inhibition of Vascular Smooth
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Yih-Jer Wu, Mark Bond, Graciela B. Sala-
参见附件。
the Bristol Heart Institute, University of Bristol, United Kingdom.
Abstract
Cyclic nucleotides inhibit vascular smooth muscle cell (VSMC) proliferation but the underlying molecular mechanisms are incompletely understood. We studied the role of S-phase kinase-associated protein-2 (Skp2), an F-box protein of SCFSkp2 ubiquitin ligase responsible for polyubiquitylation of and subsequent proteolysis of p27Kip1, a key step leading to cell cycle progression. Skp2 mRNA and protein were upregulated in mitogen-stimulated VSMCs and after balloon injury in rat carotid arteries, where the time course and location of Skp2 expression closely paralleled that of proliferating cell nuclear antigen. Skp2 small interference RNA (siRNA) reduced Skp2 expression, increased p27Kip1 levels, and inhibited VSMC proliferation in vitro. cAMP-elevating agents prominently inhibited VSMC proliferation and Skp2 expression through inhibiting Skp2 transcription as well as decreasing Skp2 protein stability. Consistent with this, activation of protein kinase A, a downstream target of cAMP, was shown to negatively regulate focal adhesion kinase (FAK) phosphorylation and Skp2 expression. Adenovirus-mediated Skp2 expression reversed cAMP-induced p27Kip1 upregulation and rescued cAMP-related S-phase entry inhibition up to 50%. 8-Bromo-cGMP also moderately reduced Skp2 and cell proliferation when VSMCs were incubated with low serum concentration. Interestingly, we showed that 8-bromo-cGMP inhibited Skp2 expression also through activation of protein kinase A, not protein kinase G, which conversely enhanced FAKY397 phosphorylation and Skp2 expression. After balloon injury of rat carotid arteries, local forskolin treatment significantly reduced FAKY397 phosphorylation, Skp2 expression, VSMC proliferation, and subsequent neointimal thickening. These data demonstrate for the first time that Skp2 is an important factor in VSMC proliferation and its inhibition by cyclic nucleotides.
Key Words: Skp2 cyclic nucleotides smooth muscle cell proliferation focal adhesion kinase
Introduction
Vascular smooth muscle cell (VSMC) proliferation contributes to intima formation after balloon injury with and without stenting, venous graft failure, transplant vasculopathy, and atherosclerosis. This motivates efforts to understand the regulation of VSMC proliferation and in particular to identify pathways that could mediate inhibition of proliferation. In healthy, uninjured vessels, VSMCs are quiescent and exhibit extremely low rates of proliferation, even in the presence of mitogenic stimuli.1 The mechanisms responsible for overcoming VSMC quiescence and initiating VSMC proliferation in response to vessel injury include release of growth factors2,3 and remodeling of the vascular extracellular matrix.4–6 This stimulates signaling pathways downstream of growth factor receptors or focal adhesions, respectively.7–9 Signaling pathways related to cell–cell adhesion through cadherins may also be involved.10
Cell-type specific antiproliferative effects of cyclic nucleotides (cAMP and cGMP) are well documented.11 In particular, increased levels of cAMP and cGMP were shown to inhibit VSMC proliferation both in vitro in response to mitogens12,13 and in vivo after vascular injury.14,15 However, the mechanism of growth inhibition by cyclic nucleotides remains unclear. Early studies pointed to effects on mitogen-activating protein kinase (MAPK) signaling16,17 and the expression of cyclin D1.13,18,19 However, experiments showing that delaying addition of cyclic nucleotides for up to 16 hours after growth factors still results in inhibition, suggesting additional major effects on later events in the cell cycle. Consistent with this, several studies demonstrated that inhibition of proliferation by cAMP is associated with upregulation of the cyclin-dependent kinase inhibitor p27Kip1.19 Furthermore, prostacyclin mimetics, cicaprost20 or beraprost,21 which increase intracellular cAMP, also inhibit cyclin E-cyclin–dependent kinase (cdk) 2 activity via upregulation of p27Kip1. cGMP has also been demonstrated to inhibit VSMC proliferation through a transient increase of p27Kip1.19 Besides, VSMCs overexpress endothelial nitric oxide (NO) synthase, which increase NO production and thereby intracellular cGMP levels, is also demonstrated to upregulate p27Kip1.22 However, it is not known how cyclic nucleotides regulate p27Kip1 levels and whether this mechanism is essential for their growth inhibitory effects.
S-phase kinase-associated protein 2 (Skp2), an F-box protein component of the SCFskp2 ubiquitin ligase, is responsible for polyubiquitylation of many important cell-cycle regulators, including cdk inhibitor p27Kip1.23,24 p27Kip1 has been thought to be the major physiological target of Skp2, based on evidence that deletion of the p27Kip1 gene almost completely rescues the abnormal phenotypes of Skp2 knockout mice.25 Ectopic Skp2 expression in various cell types, including VSMC, has been shown to decrease p27Kip1 levels and force cells into S phase.9,23,26 Skp2, therefore, could play a key role in VSMC proliferation and related vascular pathologies.
The proposed role of Skp2 in regulation of p27Kip1 levels in VSMCs9 and the effects of cyclic nucleotides on p27Kip1 prompted us to test the hypothesis that Skp2 is an important factor in VSMC proliferation and neointimal thickening and that the growth-inhibitory effects of cyclic nucleotides in VSMCs are mediated via changes in the levels of Skp2 protein.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Cell Culture
VSMCs were obtained by the explant method from thoracic aortas of male Sprague–Dawley rats as described previously.1 VSMC proliferation was quantified by bromodeoxyuridine (BrdUrd) labeling.
Balloon Injury of Rat Carotid Arteries and Local Administration of Forskolin
Balloon injury of left common carotid artery was performed in male Sprague–Dawley rats as described in detail in the online data supplement. Where indicated, pluronic gel containing either forskolin (200 μmol/L) or 0.1% dimethyl sulfoxide (control) was applied around the injured common carotid arteries (1 cm in length) before closure of the wound.
Gene Expression Analysis
Gene expression was quantified by RT-PCR and Western blotting, as described in detail in the online data supplement.
Results
Skp2 Regulates VSMC Proliferation In Vitro
Serum stimulation increased Skp2 protein levels 10±2-fold (n=6, P<0.05). Skp2 mRNA was also significantly upregulated by mitogen stimulation (Figure 1A) but to a lesser extent than the protein (1.8±0.4-fold; n=6, P<0.05). To determine the importance of this increase in Skp2 expression for S-phase entry and proliferation in VSMC, we designed small interfering RNA (siRNA) to specifically silence Skp2 expression. Transfection of VSMCs with siRNA27 targeting Skp2 but not siRNA targeting luciferase inhibited Skp2 mRNA and protein expression (by 79±4% and 93±3%, respectively; n=3, P<0.05) and increased the levels of p27Kip1 3 days post transfection. Importantly, total protein levels of GAPDH and focal adhesion kinase (FAK) were not affected by Skp2 siRNA, further demonstrating specificity. Skp2 siRNA significantly reduced VSMC proliferation measured by BrdUrd incorporation (from 24±1% to 12±2%; n=4; P<0.05) and cell counts per microscopic field (795±79 to 240±31 cells/field; n=4, P<0.05) (Figure 1B). Interestingly, VSMCs treated with Skp2 siRNA demonstrated the giant nuclei with increased size variation (Y.-J.W. and M.B., unpublished data, 2005), consistent with the phenotype of cells derived from Skp2-deficient mice.25
Skp2 Is Upregulated In Vivo in the Proliferating Arterial VSMCs Induced by Balloon Injury
As a first step to investigate the role of Skp2 in VSMC proliferation in vivo, we compared the distribution of Skp2 protein and proliferating cells (using proliferating cell nuclear antigen [PCNA]) in rat carotid arteries after balloon injury. As shown in Figure 2A and 2B (n=3 for each time point), Skp2 protein and PCNA were almost undetectable in the uninjured arteries. However, Skp2 protein and PCNA increased in response to balloon injury, with medial expression of both being maximal 2 days after injury. Medial Skp2 staining waned after day 7; however, VSMCs were highly proliferative and strongly Skp2 positive in the neointima. Intimal Skp2 and PCNA expression was maximal on day 10 and then gradually returned to almost baseline levels on day 28. Quantitative results confirmed that the expression of PCNA was temporally associated with expression of Skp2 (Figure 2B). From Figure 2A, the distribution of Skp2 appears broader than that of PCNA, and this finding was confirmed by dual-labeling studies (supplemental Figure IA). This result was to be expected because Skp2 expression occurs in cells from late G1 phase onward, whereas PCNA is confined to S-phase cells. Neither rabbit immunoglobulin fraction nor mouse IgG as the substitute for primary antibody produces any staining on the sections (supplemental Figure IB). To confirm upregulation of Skp2 following injury, mRNA and protein expression were quantified after 7 days using real-time RT-PCR and Western blotting. Skp2 mRNA was upregulated 8.4±2.7-fold, whereas Skp2 protein was increased 12±3-fold, versus uninjured (3 experiments, 3 rats for each experiment) (Figure 2C).
Regulation of Skp2 Transcription and Stability by cAMP and cGMP
Treatment of VSMCs with dibutyryl-cAMP (db-cAMP) (0.1 to 1 mmol/L) or forskolin (adenylate cyclase activator, 10 to 100 μmol/L), or isobutylmethylxanthine (IBMX) (nonspecific phosphodiesterase inhibitor, 0.1 to 0.5 mmol/L), but not 8-bromo-cGMP (Br-cGMP, up to 1 mmol/L), resulted in a dose-dependent inhibition of Skp2 protein (supplemental Figure II). At the maximal dose used, Skp2 remaining was 16±4%, 25±11%, and 20±8% of control, respectively (Figure 3A). The same cAMP-elevating agents elevated p27Kip1 levels (to 279±43%, 233±31%, and 226±34% of control, respectively) and inhibited hyperphosphorylation of retinoblastoma (Rb) protein, a marker of G1–S-phase progression (to 7.0±4.4%, 14±4%, and 18±5% of control, respectively) (Figure 3A). The same agents also similarly, but to a lesser extent, decreased Skp2 mRNA expression (to 48±8%, 53±9%, and 36±4% of control, respectively) (Figure 3B). To further elucidate whether cAMP inhibits Skp2 mRNA expression through regulation of promoter activity, we performed luciferase reporter assays on VSMCs following transfection of reporter plasmid containing Skp2 promoter. Treatment with db-cAMP significantly decreased Skp2 promoter activity to an extent similar to that seen in mRNA expression (to 50±5% of control, P<0.05) (Figure 3C). The failure of db-cAMP to reduce the SV40 and CMV promoter activities indicates a specific effect on the Skp2 promoter rather than a global inhibition of transcriptional activity (Figure 3C). Intriguingly, Br-cGMP treatment, which did not show any effect on Skp2 protein expression, did result in a moderate inhibition of Skp2 mRNA levels (to 79±9% of control, P<0.05) (Figure 3B) and Skp2 promoter activity (to 71±3% of control, P<0.05) (Figure 3C). The disproportionate regulation between Skp2 protein and mRNA levels by cyclic nucleotides made us consider the possibility that the cyclic nucleotides may also have their distinct posttranscriptional effects on Skp2 protein expression. We demonstrated that db-cAMP treatment but not Br-cGMP treatment results in a significant reduction in Skp2 protein stability (half-life of Skp2: db-cAMP=3.1±1.2 hours; control: 8.4±1.3 hours; Br-cGMP: 11±1 hours; ANOVA, P<0.01) (Figure 3D). The opposite effects of cAMP and cGMP on Skp2 stability help to explain their divergent effects on steady-state Skp2 protein and mRNA levels. Consistent with a decrease in levels of Skp2, which targets p27Kip1 for proteasomal degradation, we observed an increase in the stability of p27Kip1 after db-cAMP treatment (Figure 3D).
Inhibition of VSMC Proliferation and Skp2 Expression by cGMP Is Observed at Low Serum Concentrations
Although the growth inhibitory effects of cAMP were consistently observed in previous studies, the effects of cGMP were variable.13,28,29 Either treatment with high concentration of cGMP13 or stimulation with relatively low concentrations of mitogens29,30 appears to be required for the growth inhibitory effects of cGMP. To further clarify this controversial issue and determine whether Skp2 regulation is also a common underlying mechanism in inhibition of proliferation by cGMP, we treated quiescent VSMCs with Br-cGMP (1 mmol/L) in presence of different FCS concentrations. Unlike db-cAMP (1 mmol/L), which potently inhibited BrdUrd incorporation from 69±8% to 8.4±3.1% (P<0.05) in response to 15% serum mitogens, Br-cGMP did not show any significant effect on proliferation in the presence of 15% or even 10% of serum mitogens. However, at lower concentration of FCS (ie, 5%, 2%, and 0%), Br-cGMP treatment resulted in a significant reduction in BrdUrd incorporation (by 30%, P=0.06; 45% and 65%, both P<0.05, respectively) (Figure 4A). Similar to the pattern of BrdUrd incorporation, Skp2 expression (Figure 4B) was potently inhibited by db-cAMP by 84% (P<0.05) in response to 15% FCS. However, Skp2 expression was only inhibited by Br-cGMP at lower FCS concentration (ANOVA, P<0.01); the inhibition was concentration-dependent (supplemental Figure II). Taken together, these data suggest that regulation of Skp2 expression is a common mechanism underlying the antiproliferative effects of both cAMP and cGMP.
Adenovirus-Mediated Expression of Skp2 Reverses Forskolin-Related p27Kip1 Upregulation and Rescues VSMC Proliferation
To investigate whether Skp2 downregulation is necessary for antiproliferative effects of cAMP in VSMCs, we attempted to rescue VSMC proliferation in cells treated with forskolin using adenovirus-mediated expression of exogenous wild-type Skp2. Infection with adenoviral vector expressing Skp2 (AdSkp2) resulted in increased expression of Skp2 protein and completely prevented p27Kip1 upregulation by forskolin (from 166±21% to 66±6% of control, P<0.05). Furthermore, AdSkp2 infection also significantly, although partially, rescued Rb hyperphosphorylation inhibited by forskolin treatment (from 5.1±1.8% to 54±9% of control, P<0.05) (Figure 5A). More importantly, exogenous Skp2 expression reversed, at least partially, the forskolin-induced inhibition of BrdUrd incorporation (from 11±2% to 52±2% of control, P<0.05, Figure 5B).
cAMP and cGMP Have Opposite Effects on Focal Adhesion Kinase Phosphorylation at High Serum Concentration
Because we previously demonstrated that Skp2 expression is absolutely dependent on FAK activity,9 we investigated whether inhibition of Skp2 expression by cyclic nucleotides and cyclic nucleotide–elevating agents was associated with a change of FAK signaling. Treatment of cultured VSMCs with db-cAMP, forskolin or IBMX in the presence of 15% FCS significantly inhibited FAKY397 phosphorylation (Figure 6A; n=3, all P<0.05), an autophosphorylation site required for FAK activation. Levels of phospho-FAKY397 were significantly increased by Br-cGMP (to 154±7% of control; n=3; P<0.05) in the presence of 15% FCS but inhibited in serum-free conditions (Figure 6B), when Skp2 was decreased also (Figure 4B). Levels of GAPDH protein did not change after treatment. Expression of a constitutively active mutant of FAK (cd2-FAK) reversed forskolin induced downregulation of FAKY397 phosphorylation, partially but significantly rescued Skp2 expression, reversed the upregulation of p27Kip1 levels, and increased Rb hyperphosphorylation, a marker of G1–S-phase progression (Figure 6C), confirming that changes in FAK activity are, at least in part, responsible for regulating Skp2 expression.
Protein Kinases A and G Have Opposite Effects on Skp2 Expression
We investigated the role of protein kinase A (PKA) and G (PKG) signaling in the regulation of FAK phosphorylation and downregulation of Skp2 by cyclic nucleotides. Treatment of VSMC with a cell-permeable peptide inhibitor of PKA (PKAI) significantly rescued FAKY397 phosphorylation and Skp2 expression after treatment with db-cAMP (500 μmol/L) in presence of 10% serum (Figure 7A). Conversely, treatment with a PKGI resulted in a further dose-dependent inhibition of Skp2 expression and FAKY397 phosphorylation after Br-cGMP treatment in 2% serum (Figure 7B). Interestingly, PKAI treatment was also able to completely rescue Skp2 expression after Br-cGMP treatment in the presence of 2% serum (Figure 7C), which implies that the inhibitory effect of Br-cGMP on Skp2 levels is mediated by PKA.
Forskolin Inhibits FAK Phosphorylation, Skp2 Expression, and BrdUrd Incorporation After Balloon Injury of Rat Carotid Artery
We observed a significant increase in phospho-FAKY397–positive VSMCs in vivo 2 days after balloon injury to the rat carotid artery compared with uninjured control arteries (Figure 8A). Consistent with a role of FAK as a downstream effector of cyclic nucleotides, treatment with 30% pluronic gel containing 200 μmol/L forskolin significantly inhibited medial phospho-FAKY397 expression (from 6.6±1.2% to 1.4±0.4%; P<0.05) (Figure 8A). Forskolin treatment also inhibited Skp2 expression (from 7.5±2.7% to 1.0±0.2%, P<0.05) and BrdUrd incorporation (from 12±2% to 3.8±1.2%, P<0.05) (Figure 8B) 2 days after balloon injury. Consistent with previous studies, forskolin treatment significantly reduced the intima/media ratio 7 days after balloon injury, from 0.33±0.05 to 0.14±0.02 (P<0.05).
Discussion
In this study, we show for the first time that Skp2, an F-box component of the SCFSkp2 ubiquitin ligase that targets p27Kip1 for proteasome-mediated degradation,23,24 is an essential intermediate in the inhibitory effects of cAMP on VSMC proliferation in vitro. We show, moreover, that Skp2 expression closely parallels VSMC proliferation after injury to the rat carotid artery in vivo and that the cAMP-elevating agent forskolin suppresses both Skp2 expression and proliferation. Previously, work in a number of cell types demonstrated that growth inhibition by cAMP or cAMP-elevating agents is associated with increases in expression of p27Kip1,19–21 but these studies did not establish a causal relationship or define any upstream regulators. Our studies now show that Skp2 overexpression can reverse the increase in p27Kip1 and partially rescue VSMC proliferation from inhibition by cAMP. These experiments not only demonstrate that Skp2 is an essential upstream regulator of p27Kip1 degradation but that both Skp2 downregulation and p27Kip1 upregulation are essential for the full antiproliferative effects of cAMP. Interestingly, we found that the relative effects of cAMP and cGMP on Skp2 protein expression closely mirror their inhibitory effects on VSMC proliferation, which further supports our conclusion that Skp2 is a major effector of cyclic nucleotide–mediated growth arrest in VSMC. Previous data showing that 8-Br-cAMP has no effect on Skp2 expression in mouse embryonic fibroblasts20 suggest an interesting species or cell-type difference between VSMCs and fibroblasts. Skp2 overexpression completely reversed the effect of cyclic nucleotides on p27Kip1 but only partially rescued VSMC proliferation. Hence our results complement rather than contradict previous studies showing important effects of cAMP on MAPK activation,16 cyclin D1,13,18,19 and cyclin A expression.31,32 Notwithstanding, our results demonstrate that Skp2 is a major effector of the growth inhibitory effects of cyclic nucleotides.
Consistent with our previous work,9 we demonstrated that Skp2 mRNA and protein levels are both elevated in serum-stimulated isolated VSMCs. Silencing Skp2 expression by siRNA inhibited proliferation, confirming our previous results with adenovirus-mediated overexpression of dominant negative F-box–deleted Skp2.9 Although these results firmly establish a role for Skp2 in VSMC proliferation in vitro, no previous studies have addressed its role in vivo. As a first step, we showed that Skp2 mRNA and protein levels were also upregulated after balloon injury to the rat carotid in vivo. Secondly, the time course and localization of Skp2 protein expression closely paralleled that of the proliferation marker PCNA. Interestingly, the expression pattern of Skp2 reported here appears to be the reciprocal of that previously reported for p27Kip1 by Tanner et al.33 In that study, p27 Kip1 protein levels are high in uninjured vessels and decline shortly after injury. Subsequently, p27Kip1 rises to very high levels 14 to 21 days after injury, coinciding with the fall in Skp2 expression we report here, and VSMC proliferation levels returned toward baseline values. Thirdly, and most importantly, we showed that Skp2 upregulation and VSMC proliferation were inhibited in parallel by the cAMP-elevating agent forskolin, which also inhibited neointima formation.
To elucidate the mechanisms by which cyclic nucleotides regulate Skp2 expression, we first compared the effects of serum and cyclic nucleotides on steady-state Skp2 mRNA and protein levels. Skp2 mRNA levels were less responsive to serum than protein levels, which implied both transcriptional and posttranscriptional controls. This was confirmed by studies of promoter activity and protein stability, both of which were inhibited by cAMP-related interventions and only transcription by cGMP analogs. We demonstrated that inhibition of Skp2 protein by cAMP and cAMP-elevating agents both in vitro and in vivo was associated with a reduction in the phosphorylation of FAK, which regulates Skp2 protein stability in VSMCs.9 Consistent with this, the cGMP-induced increase in Skp2 protein stability in 15% FCS was found to be associated with an increase in FAK phosphorylation and the cGMP-induced inhibition of Skp2 protein expression in serum-free environment was also associated with a decrease of FAK phosphorylation. Exogenous expression of a constitutively active mutant of FAK was also able to rescue Skp2 expression and markers of G1–S-phase progression after forskolin treatment, confirming that FAK is an important downstream mediator of cyclic nucleotide inhibition of Skp2 expression and VSMC proliferation. The inhibitory effects of both cAMP and cGMP on Skp2 expression and the cAMP-mediated inhibition of FAK phosphorylation were mediated via PKA signaling and could be rescued by PKA inhibition. This is consistent with previous observations showing that cGMP can act through PKA.34 Interestingly, signaling through PKG appears to represent a positive growth regulatory signal, as PKG-inhibition blocked Skp2 and FAK phosphorylation. Importantly, the net outcome of elevated cGMP on Skp2 expression is dependent on serum mitogen levels. cGMP is only able to inhibit Skp2 expression in low serum concentrations, which probably reflects the unmasking of cGMP/PKA signaling, and thereby inhibit FAK phosphorylation in these conditions. In the presence of high serum, cGMP stimulates PKG-dependent increase of FAK phosphorylation, which we previously demonstrated promotes Skp2 protein stability.9 Presumably, this mechanism counters the negative effects of cGMP/PKA signaling on Skp2 and, at least in part, explains the relatively poor effects of cGMP on VSMC proliferation compared with cAMP.
The cyclic nucleotides cAMP and cGMP are important intracellular secondary messengers for prostanoids and NO, respectively, which regulate several functions of VSMC, including contraction as well as proliferation.11,35,36 The ability of cells to synthesize cAMP and cGMP is high in healthy uninjured vessels,37 and this probably contributes toward the maintenance of VSMC quiescence. Furthermore, reduced synthesis of cyclic nucleotides after vessel injury37 is likely to be an important mechanism in removing the "brake" on proliferation.
Taken together, our data demonstrate that cyclic nucleotide–dependent regulation of Skp2 represents an important pathway for controlling VSMC proliferation. Hence, Skp2 may emerge as a promising target of therapy for vasculoproliferative disorders.
Acknowledgments
This work was supported by the British Heart Foundation. Y.-J.W. was supported by Mackay Memorial Hospital (Taipei, Taiwan). We thank Dr R. C. Bush and Dr C. L. Jackson for expert advice. We also thank Dr J. L. Johnson for suggestions and help with immunohistochemistry and J. Tarlton for preparation of adenoviral vectors.
Footnotes
Both authors contributed equally to this study.
Original received October 24, 2005; resubmission received February 6, 2006; revised resubmission received March 10, 2006; accepted March 22, 2006.
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Jeremy JY, Dashwood MR, Timm M, Izzat MB, Mehta D, Bryan AJ, Angelini GD. Nitric oxide synthase and adenylyl and guanylyl cyclase activity in porcine interposition vein grafts. Ann Thorac Surg. 1997; 63: 470–476.
the Bristol Heart Institute, University of Bristol, United Kingdom.
Abstract
Cyclic nucleotides inhibit vascular smooth muscle cell (VSMC) proliferation but the underlying molecular mechanisms are incompletely understood. We studied the role of S-phase kinase-associated protein-2 (Skp2), an F-box protein of SCFSkp2 ubiquitin ligase responsible for polyubiquitylation of and subsequent proteolysis of p27Kip1, a key step leading to cell cycle progression. Skp2 mRNA and protein were upregulated in mitogen-stimulated VSMCs and after balloon injury in rat carotid arteries, where the time course and location of Skp2 expression closely paralleled that of proliferating cell nuclear antigen. Skp2 small interference RNA (siRNA) reduced Skp2 expression, increased p27Kip1 levels, and inhibited VSMC proliferation in vitro. cAMP-elevating agents prominently inhibited VSMC proliferation and Skp2 expression through inhibiting Skp2 transcription as well as decreasing Skp2 protein stability. Consistent with this, activation of protein kinase A, a downstream target of cAMP, was shown to negatively regulate focal adhesion kinase (FAK) phosphorylation and Skp2 expression. Adenovirus-mediated Skp2 expression reversed cAMP-induced p27Kip1 upregulation and rescued cAMP-related S-phase entry inhibition up to 50%. 8-Bromo-cGMP also moderately reduced Skp2 and cell proliferation when VSMCs were incubated with low serum concentration. Interestingly, we showed that 8-bromo-cGMP inhibited Skp2 expression also through activation of protein kinase A, not protein kinase G, which conversely enhanced FAKY397 phosphorylation and Skp2 expression. After balloon injury of rat carotid arteries, local forskolin treatment significantly reduced FAKY397 phosphorylation, Skp2 expression, VSMC proliferation, and subsequent neointimal thickening. These data demonstrate for the first time that Skp2 is an important factor in VSMC proliferation and its inhibition by cyclic nucleotides.
Key Words: Skp2 cyclic nucleotides smooth muscle cell proliferation focal adhesion kinase
Introduction
Vascular smooth muscle cell (VSMC) proliferation contributes to intima formation after balloon injury with and without stenting, venous graft failure, transplant vasculopathy, and atherosclerosis. This motivates efforts to understand the regulation of VSMC proliferation and in particular to identify pathways that could mediate inhibition of proliferation. In healthy, uninjured vessels, VSMCs are quiescent and exhibit extremely low rates of proliferation, even in the presence of mitogenic stimuli.1 The mechanisms responsible for overcoming VSMC quiescence and initiating VSMC proliferation in response to vessel injury include release of growth factors2,3 and remodeling of the vascular extracellular matrix.4–6 This stimulates signaling pathways downstream of growth factor receptors or focal adhesions, respectively.7–9 Signaling pathways related to cell–cell adhesion through cadherins may also be involved.10
Cell-type specific antiproliferative effects of cyclic nucleotides (cAMP and cGMP) are well documented.11 In particular, increased levels of cAMP and cGMP were shown to inhibit VSMC proliferation both in vitro in response to mitogens12,13 and in vivo after vascular injury.14,15 However, the mechanism of growth inhibition by cyclic nucleotides remains unclear. Early studies pointed to effects on mitogen-activating protein kinase (MAPK) signaling16,17 and the expression of cyclin D1.13,18,19 However, experiments showing that delaying addition of cyclic nucleotides for up to 16 hours after growth factors still results in inhibition, suggesting additional major effects on later events in the cell cycle. Consistent with this, several studies demonstrated that inhibition of proliferation by cAMP is associated with upregulation of the cyclin-dependent kinase inhibitor p27Kip1.19 Furthermore, prostacyclin mimetics, cicaprost20 or beraprost,21 which increase intracellular cAMP, also inhibit cyclin E-cyclin–dependent kinase (cdk) 2 activity via upregulation of p27Kip1. cGMP has also been demonstrated to inhibit VSMC proliferation through a transient increase of p27Kip1.19 Besides, VSMCs overexpress endothelial nitric oxide (NO) synthase, which increase NO production and thereby intracellular cGMP levels, is also demonstrated to upregulate p27Kip1.22 However, it is not known how cyclic nucleotides regulate p27Kip1 levels and whether this mechanism is essential for their growth inhibitory effects.
S-phase kinase-associated protein 2 (Skp2), an F-box protein component of the SCFskp2 ubiquitin ligase, is responsible for polyubiquitylation of many important cell-cycle regulators, including cdk inhibitor p27Kip1.23,24 p27Kip1 has been thought to be the major physiological target of Skp2, based on evidence that deletion of the p27Kip1 gene almost completely rescues the abnormal phenotypes of Skp2 knockout mice.25 Ectopic Skp2 expression in various cell types, including VSMC, has been shown to decrease p27Kip1 levels and force cells into S phase.9,23,26 Skp2, therefore, could play a key role in VSMC proliferation and related vascular pathologies.
The proposed role of Skp2 in regulation of p27Kip1 levels in VSMCs9 and the effects of cyclic nucleotides on p27Kip1 prompted us to test the hypothesis that Skp2 is an important factor in VSMC proliferation and neointimal thickening and that the growth-inhibitory effects of cyclic nucleotides in VSMCs are mediated via changes in the levels of Skp2 protein.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Cell Culture
VSMCs were obtained by the explant method from thoracic aortas of male Sprague–Dawley rats as described previously.1 VSMC proliferation was quantified by bromodeoxyuridine (BrdUrd) labeling.
Balloon Injury of Rat Carotid Arteries and Local Administration of Forskolin
Balloon injury of left common carotid artery was performed in male Sprague–Dawley rats as described in detail in the online data supplement. Where indicated, pluronic gel containing either forskolin (200 μmol/L) or 0.1% dimethyl sulfoxide (control) was applied around the injured common carotid arteries (1 cm in length) before closure of the wound.
Gene Expression Analysis
Gene expression was quantified by RT-PCR and Western blotting, as described in detail in the online data supplement.
Results
Skp2 Regulates VSMC Proliferation In Vitro
Serum stimulation increased Skp2 protein levels 10±2-fold (n=6, P<0.05). Skp2 mRNA was also significantly upregulated by mitogen stimulation (Figure 1A) but to a lesser extent than the protein (1.8±0.4-fold; n=6, P<0.05). To determine the importance of this increase in Skp2 expression for S-phase entry and proliferation in VSMC, we designed small interfering RNA (siRNA) to specifically silence Skp2 expression. Transfection of VSMCs with siRNA27 targeting Skp2 but not siRNA targeting luciferase inhibited Skp2 mRNA and protein expression (by 79±4% and 93±3%, respectively; n=3, P<0.05) and increased the levels of p27Kip1 3 days post transfection. Importantly, total protein levels of GAPDH and focal adhesion kinase (FAK) were not affected by Skp2 siRNA, further demonstrating specificity. Skp2 siRNA significantly reduced VSMC proliferation measured by BrdUrd incorporation (from 24±1% to 12±2%; n=4; P<0.05) and cell counts per microscopic field (795±79 to 240±31 cells/field; n=4, P<0.05) (Figure 1B). Interestingly, VSMCs treated with Skp2 siRNA demonstrated the giant nuclei with increased size variation (Y.-J.W. and M.B., unpublished data, 2005), consistent with the phenotype of cells derived from Skp2-deficient mice.25
Skp2 Is Upregulated In Vivo in the Proliferating Arterial VSMCs Induced by Balloon Injury
As a first step to investigate the role of Skp2 in VSMC proliferation in vivo, we compared the distribution of Skp2 protein and proliferating cells (using proliferating cell nuclear antigen [PCNA]) in rat carotid arteries after balloon injury. As shown in Figure 2A and 2B (n=3 for each time point), Skp2 protein and PCNA were almost undetectable in the uninjured arteries. However, Skp2 protein and PCNA increased in response to balloon injury, with medial expression of both being maximal 2 days after injury. Medial Skp2 staining waned after day 7; however, VSMCs were highly proliferative and strongly Skp2 positive in the neointima. Intimal Skp2 and PCNA expression was maximal on day 10 and then gradually returned to almost baseline levels on day 28. Quantitative results confirmed that the expression of PCNA was temporally associated with expression of Skp2 (Figure 2B). From Figure 2A, the distribution of Skp2 appears broader than that of PCNA, and this finding was confirmed by dual-labeling studies (supplemental Figure IA). This result was to be expected because Skp2 expression occurs in cells from late G1 phase onward, whereas PCNA is confined to S-phase cells. Neither rabbit immunoglobulin fraction nor mouse IgG as the substitute for primary antibody produces any staining on the sections (supplemental Figure IB). To confirm upregulation of Skp2 following injury, mRNA and protein expression were quantified after 7 days using real-time RT-PCR and Western blotting. Skp2 mRNA was upregulated 8.4±2.7-fold, whereas Skp2 protein was increased 12±3-fold, versus uninjured (3 experiments, 3 rats for each experiment) (Figure 2C).
Regulation of Skp2 Transcription and Stability by cAMP and cGMP
Treatment of VSMCs with dibutyryl-cAMP (db-cAMP) (0.1 to 1 mmol/L) or forskolin (adenylate cyclase activator, 10 to 100 μmol/L), or isobutylmethylxanthine (IBMX) (nonspecific phosphodiesterase inhibitor, 0.1 to 0.5 mmol/L), but not 8-bromo-cGMP (Br-cGMP, up to 1 mmol/L), resulted in a dose-dependent inhibition of Skp2 protein (supplemental Figure II). At the maximal dose used, Skp2 remaining was 16±4%, 25±11%, and 20±8% of control, respectively (Figure 3A). The same cAMP-elevating agents elevated p27Kip1 levels (to 279±43%, 233±31%, and 226±34% of control, respectively) and inhibited hyperphosphorylation of retinoblastoma (Rb) protein, a marker of G1–S-phase progression (to 7.0±4.4%, 14±4%, and 18±5% of control, respectively) (Figure 3A). The same agents also similarly, but to a lesser extent, decreased Skp2 mRNA expression (to 48±8%, 53±9%, and 36±4% of control, respectively) (Figure 3B). To further elucidate whether cAMP inhibits Skp2 mRNA expression through regulation of promoter activity, we performed luciferase reporter assays on VSMCs following transfection of reporter plasmid containing Skp2 promoter. Treatment with db-cAMP significantly decreased Skp2 promoter activity to an extent similar to that seen in mRNA expression (to 50±5% of control, P<0.05) (Figure 3C). The failure of db-cAMP to reduce the SV40 and CMV promoter activities indicates a specific effect on the Skp2 promoter rather than a global inhibition of transcriptional activity (Figure 3C). Intriguingly, Br-cGMP treatment, which did not show any effect on Skp2 protein expression, did result in a moderate inhibition of Skp2 mRNA levels (to 79±9% of control, P<0.05) (Figure 3B) and Skp2 promoter activity (to 71±3% of control, P<0.05) (Figure 3C). The disproportionate regulation between Skp2 protein and mRNA levels by cyclic nucleotides made us consider the possibility that the cyclic nucleotides may also have their distinct posttranscriptional effects on Skp2 protein expression. We demonstrated that db-cAMP treatment but not Br-cGMP treatment results in a significant reduction in Skp2 protein stability (half-life of Skp2: db-cAMP=3.1±1.2 hours; control: 8.4±1.3 hours; Br-cGMP: 11±1 hours; ANOVA, P<0.01) (Figure 3D). The opposite effects of cAMP and cGMP on Skp2 stability help to explain their divergent effects on steady-state Skp2 protein and mRNA levels. Consistent with a decrease in levels of Skp2, which targets p27Kip1 for proteasomal degradation, we observed an increase in the stability of p27Kip1 after db-cAMP treatment (Figure 3D).
Inhibition of VSMC Proliferation and Skp2 Expression by cGMP Is Observed at Low Serum Concentrations
Although the growth inhibitory effects of cAMP were consistently observed in previous studies, the effects of cGMP were variable.13,28,29 Either treatment with high concentration of cGMP13 or stimulation with relatively low concentrations of mitogens29,30 appears to be required for the growth inhibitory effects of cGMP. To further clarify this controversial issue and determine whether Skp2 regulation is also a common underlying mechanism in inhibition of proliferation by cGMP, we treated quiescent VSMCs with Br-cGMP (1 mmol/L) in presence of different FCS concentrations. Unlike db-cAMP (1 mmol/L), which potently inhibited BrdUrd incorporation from 69±8% to 8.4±3.1% (P<0.05) in response to 15% serum mitogens, Br-cGMP did not show any significant effect on proliferation in the presence of 15% or even 10% of serum mitogens. However, at lower concentration of FCS (ie, 5%, 2%, and 0%), Br-cGMP treatment resulted in a significant reduction in BrdUrd incorporation (by 30%, P=0.06; 45% and 65%, both P<0.05, respectively) (Figure 4A). Similar to the pattern of BrdUrd incorporation, Skp2 expression (Figure 4B) was potently inhibited by db-cAMP by 84% (P<0.05) in response to 15% FCS. However, Skp2 expression was only inhibited by Br-cGMP at lower FCS concentration (ANOVA, P<0.01); the inhibition was concentration-dependent (supplemental Figure II). Taken together, these data suggest that regulation of Skp2 expression is a common mechanism underlying the antiproliferative effects of both cAMP and cGMP.
Adenovirus-Mediated Expression of Skp2 Reverses Forskolin-Related p27Kip1 Upregulation and Rescues VSMC Proliferation
To investigate whether Skp2 downregulation is necessary for antiproliferative effects of cAMP in VSMCs, we attempted to rescue VSMC proliferation in cells treated with forskolin using adenovirus-mediated expression of exogenous wild-type Skp2. Infection with adenoviral vector expressing Skp2 (AdSkp2) resulted in increased expression of Skp2 protein and completely prevented p27Kip1 upregulation by forskolin (from 166±21% to 66±6% of control, P<0.05). Furthermore, AdSkp2 infection also significantly, although partially, rescued Rb hyperphosphorylation inhibited by forskolin treatment (from 5.1±1.8% to 54±9% of control, P<0.05) (Figure 5A). More importantly, exogenous Skp2 expression reversed, at least partially, the forskolin-induced inhibition of BrdUrd incorporation (from 11±2% to 52±2% of control, P<0.05, Figure 5B).
cAMP and cGMP Have Opposite Effects on Focal Adhesion Kinase Phosphorylation at High Serum Concentration
Because we previously demonstrated that Skp2 expression is absolutely dependent on FAK activity,9 we investigated whether inhibition of Skp2 expression by cyclic nucleotides and cyclic nucleotide–elevating agents was associated with a change of FAK signaling. Treatment of cultured VSMCs with db-cAMP, forskolin or IBMX in the presence of 15% FCS significantly inhibited FAKY397 phosphorylation (Figure 6A; n=3, all P<0.05), an autophosphorylation site required for FAK activation. Levels of phospho-FAKY397 were significantly increased by Br-cGMP (to 154±7% of control; n=3; P<0.05) in the presence of 15% FCS but inhibited in serum-free conditions (Figure 6B), when Skp2 was decreased also (Figure 4B). Levels of GAPDH protein did not change after treatment. Expression of a constitutively active mutant of FAK (cd2-FAK) reversed forskolin induced downregulation of FAKY397 phosphorylation, partially but significantly rescued Skp2 expression, reversed the upregulation of p27Kip1 levels, and increased Rb hyperphosphorylation, a marker of G1–S-phase progression (Figure 6C), confirming that changes in FAK activity are, at least in part, responsible for regulating Skp2 expression.
Protein Kinases A and G Have Opposite Effects on Skp2 Expression
We investigated the role of protein kinase A (PKA) and G (PKG) signaling in the regulation of FAK phosphorylation and downregulation of Skp2 by cyclic nucleotides. Treatment of VSMC with a cell-permeable peptide inhibitor of PKA (PKAI) significantly rescued FAKY397 phosphorylation and Skp2 expression after treatment with db-cAMP (500 μmol/L) in presence of 10% serum (Figure 7A). Conversely, treatment with a PKGI resulted in a further dose-dependent inhibition of Skp2 expression and FAKY397 phosphorylation after Br-cGMP treatment in 2% serum (Figure 7B). Interestingly, PKAI treatment was also able to completely rescue Skp2 expression after Br-cGMP treatment in the presence of 2% serum (Figure 7C), which implies that the inhibitory effect of Br-cGMP on Skp2 levels is mediated by PKA.
Forskolin Inhibits FAK Phosphorylation, Skp2 Expression, and BrdUrd Incorporation After Balloon Injury of Rat Carotid Artery
We observed a significant increase in phospho-FAKY397–positive VSMCs in vivo 2 days after balloon injury to the rat carotid artery compared with uninjured control arteries (Figure 8A). Consistent with a role of FAK as a downstream effector of cyclic nucleotides, treatment with 30% pluronic gel containing 200 μmol/L forskolin significantly inhibited medial phospho-FAKY397 expression (from 6.6±1.2% to 1.4±0.4%; P<0.05) (Figure 8A). Forskolin treatment also inhibited Skp2 expression (from 7.5±2.7% to 1.0±0.2%, P<0.05) and BrdUrd incorporation (from 12±2% to 3.8±1.2%, P<0.05) (Figure 8B) 2 days after balloon injury. Consistent with previous studies, forskolin treatment significantly reduced the intima/media ratio 7 days after balloon injury, from 0.33±0.05 to 0.14±0.02 (P<0.05).
Discussion
In this study, we show for the first time that Skp2, an F-box component of the SCFSkp2 ubiquitin ligase that targets p27Kip1 for proteasome-mediated degradation,23,24 is an essential intermediate in the inhibitory effects of cAMP on VSMC proliferation in vitro. We show, moreover, that Skp2 expression closely parallels VSMC proliferation after injury to the rat carotid artery in vivo and that the cAMP-elevating agent forskolin suppresses both Skp2 expression and proliferation. Previously, work in a number of cell types demonstrated that growth inhibition by cAMP or cAMP-elevating agents is associated with increases in expression of p27Kip1,19–21 but these studies did not establish a causal relationship or define any upstream regulators. Our studies now show that Skp2 overexpression can reverse the increase in p27Kip1 and partially rescue VSMC proliferation from inhibition by cAMP. These experiments not only demonstrate that Skp2 is an essential upstream regulator of p27Kip1 degradation but that both Skp2 downregulation and p27Kip1 upregulation are essential for the full antiproliferative effects of cAMP. Interestingly, we found that the relative effects of cAMP and cGMP on Skp2 protein expression closely mirror their inhibitory effects on VSMC proliferation, which further supports our conclusion that Skp2 is a major effector of cyclic nucleotide–mediated growth arrest in VSMC. Previous data showing that 8-Br-cAMP has no effect on Skp2 expression in mouse embryonic fibroblasts20 suggest an interesting species or cell-type difference between VSMCs and fibroblasts. Skp2 overexpression completely reversed the effect of cyclic nucleotides on p27Kip1 but only partially rescued VSMC proliferation. Hence our results complement rather than contradict previous studies showing important effects of cAMP on MAPK activation,16 cyclin D1,13,18,19 and cyclin A expression.31,32 Notwithstanding, our results demonstrate that Skp2 is a major effector of the growth inhibitory effects of cyclic nucleotides.
Consistent with our previous work,9 we demonstrated that Skp2 mRNA and protein levels are both elevated in serum-stimulated isolated VSMCs. Silencing Skp2 expression by siRNA inhibited proliferation, confirming our previous results with adenovirus-mediated overexpression of dominant negative F-box–deleted Skp2.9 Although these results firmly establish a role for Skp2 in VSMC proliferation in vitro, no previous studies have addressed its role in vivo. As a first step, we showed that Skp2 mRNA and protein levels were also upregulated after balloon injury to the rat carotid in vivo. Secondly, the time course and localization of Skp2 protein expression closely paralleled that of the proliferation marker PCNA. Interestingly, the expression pattern of Skp2 reported here appears to be the reciprocal of that previously reported for p27Kip1 by Tanner et al.33 In that study, p27 Kip1 protein levels are high in uninjured vessels and decline shortly after injury. Subsequently, p27Kip1 rises to very high levels 14 to 21 days after injury, coinciding with the fall in Skp2 expression we report here, and VSMC proliferation levels returned toward baseline values. Thirdly, and most importantly, we showed that Skp2 upregulation and VSMC proliferation were inhibited in parallel by the cAMP-elevating agent forskolin, which also inhibited neointima formation.
To elucidate the mechanisms by which cyclic nucleotides regulate Skp2 expression, we first compared the effects of serum and cyclic nucleotides on steady-state Skp2 mRNA and protein levels. Skp2 mRNA levels were less responsive to serum than protein levels, which implied both transcriptional and posttranscriptional controls. This was confirmed by studies of promoter activity and protein stability, both of which were inhibited by cAMP-related interventions and only transcription by cGMP analogs. We demonstrated that inhibition of Skp2 protein by cAMP and cAMP-elevating agents both in vitro and in vivo was associated with a reduction in the phosphorylation of FAK, which regulates Skp2 protein stability in VSMCs.9 Consistent with this, the cGMP-induced increase in Skp2 protein stability in 15% FCS was found to be associated with an increase in FAK phosphorylation and the cGMP-induced inhibition of Skp2 protein expression in serum-free environment was also associated with a decrease of FAK phosphorylation. Exogenous expression of a constitutively active mutant of FAK was also able to rescue Skp2 expression and markers of G1–S-phase progression after forskolin treatment, confirming that FAK is an important downstream mediator of cyclic nucleotide inhibition of Skp2 expression and VSMC proliferation. The inhibitory effects of both cAMP and cGMP on Skp2 expression and the cAMP-mediated inhibition of FAK phosphorylation were mediated via PKA signaling and could be rescued by PKA inhibition. This is consistent with previous observations showing that cGMP can act through PKA.34 Interestingly, signaling through PKG appears to represent a positive growth regulatory signal, as PKG-inhibition blocked Skp2 and FAK phosphorylation. Importantly, the net outcome of elevated cGMP on Skp2 expression is dependent on serum mitogen levels. cGMP is only able to inhibit Skp2 expression in low serum concentrations, which probably reflects the unmasking of cGMP/PKA signaling, and thereby inhibit FAK phosphorylation in these conditions. In the presence of high serum, cGMP stimulates PKG-dependent increase of FAK phosphorylation, which we previously demonstrated promotes Skp2 protein stability.9 Presumably, this mechanism counters the negative effects of cGMP/PKA signaling on Skp2 and, at least in part, explains the relatively poor effects of cGMP on VSMC proliferation compared with cAMP.
The cyclic nucleotides cAMP and cGMP are important intracellular secondary messengers for prostanoids and NO, respectively, which regulate several functions of VSMC, including contraction as well as proliferation.11,35,36 The ability of cells to synthesize cAMP and cGMP is high in healthy uninjured vessels,37 and this probably contributes toward the maintenance of VSMC quiescence. Furthermore, reduced synthesis of cyclic nucleotides after vessel injury37 is likely to be an important mechanism in removing the "brake" on proliferation.
Taken together, our data demonstrate that cyclic nucleotide–dependent regulation of Skp2 represents an important pathway for controlling VSMC proliferation. Hence, Skp2 may emerge as a promising target of therapy for vasculoproliferative disorders.
Acknowledgments
This work was supported by the British Heart Foundation. Y.-J.W. was supported by Mackay Memorial Hospital (Taipei, Taiwan). We thank Dr R. C. Bush and Dr C. L. Jackson for expert advice. We also thank Dr J. L. Johnson for suggestions and help with immunohistochemistry and J. Tarlton for preparation of adenoviral vectors.
Footnotes
Both authors contributed equally to this study.
Original received October 24, 2005; resubmission received February 6, 2006; revised resubmission received March 10, 2006; accepted March 22, 2006.
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