Published online before print November 22, 2006, doi: 10.1161/01.RES.0000253533.65446.33
© 2006 American Heart Association, Inc.
Molecular Medicine |
Regulation of Smooth Muscle Cell Proliferation by ß-Catenin/T-Cell Factor Signaling Involves Modulation of Cyclin D1 and p21 Expression
From the Bristol Heart Institute (H.Q., S.C.S., C.A.B., G.B.S.-N., S.J.G.), Level 7, Bristol Royal Infirmary, Upper Maudlin St, BRISTOL, BS2 8HW, UK, Cardiovascular Branch (M.B.), National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland.
Correspondence to Dr Sarah Jane George, Bristol Heart Institute, Level 7, Bristol Royal Infirmary, Upper Maudlin St BRISTOL, BS2 8HW. E-mail s.j.george{at}bris.ac.uk
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Abstract |
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We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of ß-catenin. In this study we demonstrate directly that growth factors stimulate ß-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether ß-catenin/TCF signaling regulates VSMC proliferation via modulation of the ß-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited ß-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of ß-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by ß-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting ß-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and ß-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 ß-catenin/TCF responsive genes.
Key Words: smooth muscle • proliferation • cell cycle
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Introduction |
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Vascular smooth muscle cell (VSMC) proliferation is a key event in the pathogenesis of vascular diseases characterized by intimal thickening, such as atherosclerosis, vascular rejection and restenosis after angioplasty. In the healthy vasculature, VSMCs display a low proliferative rate and are maintained in a quiescent and contractile phenotype, despite the presence of endogenous growth stimuli. Furthermore, exposure of intact blood vessels to exogenous growth factors in vitro or in vivo does not lead to proliferation.1,2 Intimal thickening results from migration of VSMCs from the medial layer into the intimal layer, accompanied by a switch to a proliferative synthetic phenotype whereby they become responsive to growth factor stimulation. The mechanism by which VSMCs break their proliferative constraint is not fully understood. However studies by our group and others have implicated the cadherin family of cell adhesion molecules as having an important role.3–6
Cadherins are a family of transmembrane proteins that mediate Ca2+-dependant homophilic cell-cell contacts and form adherens junctions through interaction with the actin cytoskeleton via adapter molecules known as catenins. ß-catenin binds the cytoplasmic tail of cadherins and regulates the formation of adherens junctions. In addition to its structural role complexed with cadherins, ß-catenin is an important signaling molecule and a potent regulator of cell behavior.
Dismantling of adherens junctions frees ß-catenin from the membrane and in the presence of Wnt signaling, ß-catenin escapes proteosomal degradation by the adenomatous polyposis coli (APC), Axin, and glycogen synthase kinase-3ß (GSK-3ß) complex, translocates to the nucleus and binds members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor family. The ß-catenin/TCF complex subsequently regulates the transcription of many genes involved in cell function, including survival, migration, differentiation and proliferation. cyclin D1 and p21 are 2 key cell cycle proteins, whose expression are regulated by ß-catenin/TCF signaling in colon carcinoma cells7,8 and human embryonic kidney cells,9 respectively. Cyclin D1 expression is increased7,8 and p21 repressed9 by ß-catenin/TCF signaling. Cyclin D1 is a major promoter of the cell cycle and its expression is induced soon after mitogen stimulation. In contrast, p21 is a member of the Cip/Kip family of cyclin-dependant kinase inhibitors (CKIs) it has growth permissive effects in VSMCs at low levels, by promoting cdk:cyclin complex formation, but growth inhibitory effects at higher levels.10–12
Recent studies have indicated that cadherin:catenin signaling is involved in the regulation of VSMC proliferation. First, upregulation of ß-catenin after induction of VSMC proliferation by injury of the rat carotid artery has been observed by several groups.4,6,13 Furthermore, we have previously shown that a rapid reduction in cadherin levels accompanied by an increase in nuclear ß-catenin and VSMC proliferation occurs after arterial injury.4 We have also demonstrated that growth factor stimulation leads to dismantling of cadherin junctions, in vitro, which is associated with increased VSMC proliferation, increased nuclear ß-catenin and increased ß-catenin complexed with LEF-1.3 In addition, cyclin D1 promoter activity is regulated by over-expression of constitutively active ß-catenin in a rat smooth muscle cell line.13,14 However, it is unknown whether growth factors modulate VSMC proliferation via induction of endogenous ß-catenin/TCF signaling and modulation cyclin D1 or p21 in VSMCs. Consequently, in this study we tested the hypothesis that ß-catenin/TCF signaling modulates VSMC proliferation in response to growth factors via the regulation of cyclin D1 and p21 expression.
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Materials and Methods |
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Further details regarding the experimental protocols can be found in the online data supplement available at http://circres.ahajournals.org.
Cell Culture
Primary VSMCs were grown from human saphenous vein segments and aortas from cyclin D1–/–, cyclin D1+/+, p21–/–, p21+/+ and TOPGAL mice as previously described.3 Immortalized human aortic intimal VSMCs, ISS10s were cultured as previously described.15 Quiesced cells were stimulated with fetal calf serum (FCS) or 20 ng/mL platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF).
Adenoviral Infection
Human VSMCs were incubated with 300 plaque forming units (pfu) per cell of RAd ICAT, RAd dnTCF-4, RAd mutant ß-catenin or RAd LacZ control, or 1000pfu/cell of RAd FL-N-cadherin or RAd LacZ control for 18 hours. TOPGAL mouse VSMCs were infected with 300pfu/cell of RAd ICAT, RAd dnTCF-4, RAd FL-N-cadherin or RAd 66 control. Medial layer segments were then incubated for 18 hours with 5 x108pfu of RAd LacZ, RAd ICAT or RAd dnTCF-4. Over-expression was predominantly in luminal cells but also deeper into the medial layer (see supplemental Figure I in the online data supplement).
Cationic Lipid Delivery of Antibodies
1.25 µg of anti-ß-catenin antibody or nonimmune IgG was delivered to human VSMCs with 2.5 µL of BioPORTER reagent (Genlantis, UK) per 2x104 cells, and by following the manufacturer’s instructions. After quiescing for 24 hours, VSMCs were stimulated with PDGF+bFGF for 6 or 12 hours, or with 10% FCS for 24 hours. Intracellular antibody was detected by immunocytochemistry (data not shown) and Western blotting (supplemental Figure II). Proliferation was assessed by using the WST-1 assay (Roche) and lysates were pre-incubated with Protein G-coated agarose (Pierce, Northumberland, UK) to remove delivered antibody before Western blotting.
Assessment of ß-catenin Reporter Activity
TOPGAL VSMCs were quiesced for 36 hours and then stimulated with 10% FCS or PDGF+bFGF for 12 hours. ß-galactosidase activity was determined using the Galacto-Light Plus kit (Tropix, Bedford, UK).
Assessment of Proliferation
Proliferation in the presence of 10% FCS was assessed by bromodeoxyuridine (BrdU) incorporation by immunocytochemistry16 and immunohistochemistry.17
Expression of Cell Cycle Proteins
Quiescent VSMCs were stimulated with PDGF+bFGF for 6 or 12 hours for analysis of p21 and cyclin D1, respectively. Equal protein concentrations were subjected to Western blotting as previously described.3 Densitometric analysis results were expressed as a percentage of RAd LacZ infected cells.
Expression of Cell Cycle Messenger RNA
Quiesced VSMCs were incubated with PDGF+bFGF for 6 hours and total RNA extracted. 18s, p21, and cyclin D1 messenger RNA (mRNA) were analyzed by semi-quantitative RT-PCR. Optical densities were normalized by 18s ribosomal mRNA and expressed as a percentage of RAd LacZ control.
Transfection
The reporter plasmids (TOPFlash, CD1LUC or -2400p21LUC) or their corresponding controls (TKLUC, 
LEFCD1LUC or -94p21LUC, respectively) were transiently cotransfected with LacZ or dnTCF4 constructs into ISS10 VSMCs using the Lipofectamine Plus (Invitrogen). Quiesced cells were stimulated with either 10% FCS, PDGF or PDGF+bFGF for 24 hours (TOPFlash or CD1LUC) or 48 hours (p21LUC). Firefly luciferase activity was determined using a luciferase assay system (Promega) and normalized by total cell protein.
Immunohistochemistry
Cyclin D1 and MYC-tag were detected in paraffin-wax embedded sections by fluorescent immunohistochemistry and p21 was detected with color immunohistochemistry.
Statistical Analysis
Experiments were performed at least 3 times with VSMCs grown from different vein segments or mouse aortas. Data from 3 or more groups were analyzed by ANOVA and Student Newman Keuls post-test, data from 2 groups were analyzed by Student t tests, data expressed as a percentage of the control were analyzed by One sample t tests; significant differences were taken if P<0.05.
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Results |
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Over-expression of ß-Catenin/TCF Signaling Modulators
The role of ß-catenin/TCF signaling in the regulation of VSMCs was investigated by over-expression of ICAT, and dnTCF-4. Both proteins inhibit the ß-catenin/TCF pathway by sequestering endogenous ß-catenin and preventing the activation of TCF/LEF transcription factors. Western blotting revealed over-expression of dnTCF-4 and ICAT with 300 pfu/cell (supplemental Figure IIIA) at 42 hours after infection in human primary VSMCs (supplemental Figure IIIB).
We also over-expressed full length N-cadherin (FL-N-cad), the predominant classical cadherin in human VSMCs3 to indirectly inhibit ß–catenin/TCF signaling by increasing cell-cell junctions and thereby lowering free cytosolic ß-catenin by binding it to the cell membrane. We previously showed that 1000pfu/cell of the RAd FL-N-cad increased the association of N-cadherin and ß-catenin suggesting elevated numbers of cell-cell contacts.18 To demonstrate this directly we performed adhesion assays and observed that cell-cell adhesion significantly increased from 13.9±3.6% in control cells to 24.3±1.2% in cells over-expressing N-cadherin (n=3, P<0.05)
To stimulate ß-catenin/TCF signaling in VSMCs we used adenoviral delivery of a constitutively active (mutant) form of ß-catenin. Western blotting for the V5 tag and active (nonphosphorylated on Ser37 and Thr41) ß-catenin revealed maximal expression of mutant (active) ß-catenin with 300pfu/cell (supplemental Figure IIIC).
ß-Catenin/TCF Signaling in Human VSMCs
To examine whether ß-catenin/TCF signaling is induced with FCS or growth factor stimulation in primary VSMCs, we used TOPGAL transgenic mice, which carry the TCF responsive LacZ transgene. We chose to stimulate VSMCs with PDGF+bFGF because these growth factors are known to regulate VSMC proliferation after vascular injury.19,20 Figure 1A shows that stimulation of quiesced primary VSMCs, isolated from TOPGAL mouse aortas, with FCS or PDGF+bFGF significantly increased LacZ expression and therefore ß-catenin/TCF signaling. Over-expression of N-cadherin, dnTCF-4 and ICAT in TOPGAL primary VSMCs, stimulated with FCS or PDGF+bFGF, significantly reduced LacZ expression, and therefore endogenous ß-catenin/TCF signaling, compared with RAd 66 controls (Figure 1B).
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ß-Catenin/TCF Signaling Promotes VSMC Proliferation
Figure 2A shows incorporation of BrdU was approximately 45%, 55% and 20% of the LacZ controls in primary human VSMCs infected with RAd FL-Ncad, RAd dnTCF-4 and RAd ICAT, respectively. Similarly, intracellular delivery of anti-ß-catenin antibody reduced proliferation by approximately 45% compared with the IgG control (Figure 2B). Figure 2C shows infection with RAd dnTCF-4 and RAd ICAT significantly reduced incorporation of BrdU by approximately 50% compared with LacZ controls in saphenous vein segments. In addition, proliferation was significantly higher (1.52±0.18-fold P<0.05, n=5) in serum-starved human VSMCs infected with RAd mutant-ß-catenin than in control VSMCs infected with RAd LacZ.
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To determine whether cyclin D1 and p21 are necessary to exert the proliferative effect of ß-catenin/TCF signaling we examined the effect of inhibition of this pathway in cyclin D1 deficient (CD1–/–) and p21 deficient (p21–/–) mouse aortic VSMCs. Figure 3 (A and B) show that stimulation with FCS elevated BrdU incorporation in both the wild type and CD1–/– and p21–/– VSMCs. Over-expression of dnTCF-4 and ICAT significantly lowered BrdU incorporation in the wild type mouse cells (P<0.05 and P<0.01 respectively), as seen in the human VSMCs. This attenuation, however, was not observed in the CD1–/– or p21–/– VSMCs. Indeed, BrdU incorporation was significantly lower in the wild type VSMCs infected with RAd ICAT and RAd dnTCF-4 compared with the CD1–/– and p21–/– VSMCs with the same treatment.
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ß-Catenin/TCF Signaling and Cyclin D1 in Isolated VSMCs
To establish whether ß-catenin/TCF signaling regulates cyclin D1 gene expression at the transcriptional level, we aimed to transfect a cyclin D1 reporter plasmid, CD1LUC, into the ISS10 VSMC line. However, before this, to confirm that ß-catenin/TCF signaling was induced with FCS stimulation in ISS10 cells, we transfected the TCF responsive reporter plasmid, TOPFlash, into the ISS10 VSMC line and stimulated them for 24 hours with FCS. TOPFlash was transiently cotransfected with either the LacZ (control) or dnTCF-4 construct to demonstrate the inhibitory effect of dnTCF-4. FCS significantly increased TOPFlash activity by 1.7-fold, but did not affect the negative control (TKLUC). Furthermore, expression of dnTCF-4 significantly reduced FCS stimulated TOPFlash activity by 6-fold (supplemental Figure IV), confirming the effect observed on ß-catenin/TCF signaling with dnTCF-4 over-expression in TOPGAL mouse cells. CD1LUC, or the control plasmid that lacks the TCF/LEF binding sequence (LEFCD1LUC), were transiently cotransfected into ISS10 VSMCs with either the LacZ or dnTCF-4 construct. Figure 4A shows that FCS or PDGF+bFGF, but not PDGF alone, significantly elevated CD1LUC activity, compared with unstimulated controls. Cotransfection with the dnTCF-4 construct significantly lowered CD1LUC activity in all conditions. Furthermore, the control reporter LEFCD1LUC exhibited significantly lower activity than CD1LUC after stimulation and was unaffected by cotransfection with dnTCF-4.
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Expression of cyclin D1 mRNA in primary human VSMCs was then determined. Figure 4B shows that cyclin D1 mRNA expression was significantly reduced in PDGF+bFGF stimulated VSMCs infected with RAd FL-Ncad, RAd ICAT or RAd dnTCF-4 (P<0.01, P<0.03 and P<0.03, respectively) compared with the RAd LacZ infected control.
We then investigated whether ß-catenin/TCF signaling regulated the expression of cyclin D1 protein in VSMCs. Cyclin D1 protein levels in VSMCs are consistently elevated 12 hours after stimulation with PDGF+bFGF and remain elevated thereafter (supplemental Figure V). We therefore determined the effect of ß-catenin/TCF inhibition after 12 hours of stimulation with PDGF+bFGF. Figure 5A shows that cyclin D1 protein expression was significantly lower in PDGF+bFGF stimulated primary human VSMCs infected with RAd FL-N-cad, RAd dnTCF-4 or RAd ICAT compared with the RAd LacZ infected control (P<0.05, P<0.05 and P<0.01, respectively). Similarly, intracellular delivery of anti-ß-catenin antibody significantly reduced cyclin D1 protein expression compared with the IgG control (Figure 5B, P<0.02). In contrast, cyclin D1 protein levels were significantly elevated in serum-starved human VSMCs infected with RAd mutant ß-catenin compared with the RAd LacZ infected control (Figure 5C, P<0.02).
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ß-Catenin/TCF Signaling and p21 in Isolated VSMCs
To establish whether ß-catenin/TCF signaling regulates p21 gene expression at the level of transcription, we transfected a p21 reporter plasmid, -2400p21LUC, which contains two ß-catenin/TCF sites, into ISS10 VSMCs. Figure 6A shows that neither FCS nor PDGF+bFGF, regulated -2400p21LUC activity. However cotransfection with the dnTCF-4 construct significantly increased –2400p21LUC activity in all conditions. The –94p21LUC control reporter however exhibited only basal activity in all conditions.
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The expression of p21 mRNA in primary human VSMCs was then determined. Figure 6B shows that p21 expression was significantly increased in PDGF+bFGF stimulated VSMCs infected with RAd ICAT, RAd dnTCF-4 or RAd FL-Ncad compared with the RAd LacZ infected control (P<0.04, P<0.04 and P<0.01 respectively).
We then investigated whether ß-catenin/TCF signaling regulated the expression of p21 protein in VSMCs. Levels of p21 protein in VSMCs are slightly elevated between 1 and 2 hours, reach a minimum at 6 hours and are then elevated 8 hours after stimulation with PDGF+bFGF and remain elevated thereafter (supplemental Figure V). We therefore determined the effect of ß-catenin/TCF signaling 6 hours after stimulation with PDGF+bFGF, when p21 levels are at a minimum. Figure 7A shows that p21 protein expression was significantly elevated in PDGF+bFGF stimulated primary human VSMCs infected with RAd FL-Ncad, RAd ICAT or RAd dnTCF-4, compared with the RAd LacZ infected control (P<0.02, P<0.05 and P<0.01 respectively). Similarly, intracellular delivery of anti-ß-catenin antibody significantly increased p21 protein expression compared with the IgG control (Figure 7B, P<0.02). In contrast, p21 protein levels were significantly decreased in serum-starved human VSMCs infected with RAd mutant ß-catenin compared with the RAd LacZ infected control (Figure 7C, P<0.02).
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ß-Catenin/TCF Signaling, Cyclin D1 and p21 in Vein Segments
We then determined whether these effects could be seen in VSMCs in vein segments. Immunohistochemical analysis of medial layer sections showed that over-expression of ICAT and dnTCF-4 significantly reduced cyclin D1 expression compared with the RAd LacZ infected control (Figure 8A and B, P<0.05 and P<0.01, respectively). Conversely, infection with RAd dnTCF-4 or RAd ICAT significantly elevated the number of p21 positive VSMCs compared with the RAd LacZ control (Figure 8A and 8C, P<0.05 and P<0.01, respectively).
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Discussion |
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We have previously shown that growth factor-dependent VSMC proliferation is accompanied by dismantling of N-cadherin cell-cell junctions and a subsequent increase in nuclear ß-catenin and ß-catenin/TCF complexes.3 In this study we have demonstrated that growth factors induce ß-catenin/TCF signaling in VSMCs and attenuation of ß-catenin/TCF signaling inhibits VSMC proliferation. Furthermore, inhibition of ß-catenin/TCF signaling reduces cyclin D1 expression and elevates p21 expression. We therefore conclude that VSMC proliferation is regulated by ß-catenin signaling, in part via the modulation of cyclin D1 and p21.
To our knowledge this is the first study to show that growth factors drive endogenous ß-catenin/TCF signaling in primary VSMCs. Interestingly, stimulation with PDGF+bFGF, growth factors that modulate VSMC proliferation and migration during remodelling after injury,1 was sufficient to induce ß-catenin/TCF signaling. To directly demonstrate the involvement of ß-catenin/TCF signaling in growth factor-dependent VSMC proliferation we used four inhibitory approaches; over-expression of ICAT and dnTCF-4 to inhibit the interaction of ß-catenin with TCF-4, over-expression of N-cadherin to increase localization of ß-catenin in adherens junctions, and intracellular delivery of ß-catenin antibodies. All approaches reduced proliferation by approximately 50% to levels comparable to quiescent cells, indicating that the N-cadherin and ß-catenin/TCF signaling pathway is a major regulator of VSMC proliferation. This is supported by a previous study which showed that transfection of degradation resistant (active) form of ß-catenin increased proliferation in a rat VSMC cell line.13 Importantly, we also observed that ß-catenin/TCF signaling regulates proliferation of human VSMCs embedded in native extracellular matrix of saphenous vein segments, indicating that this pathway is not restricted to isolated VSMCs.
We have directly shown that endogenous ß-catenin/TCF signaling induced cyclin D1 transcription and increased mRNA and protein levels in primary VSMCs. Importantly, we observed that ß-catenin/TCF-dependent regulation of cyclin D1 also occurs in saphenous vein segments. Because over-expression of N-cadherin reduced cyclin D1 expression we suggest that induction of ß-catenin/TCF signaling as a result of growth factor-dependent dismantling of adherens junctions, is an important pathway in VSMC proliferation via increased cyclin D1 expression. This is corroborated by our previous observation that cyclin D1 protein was induced after balloon injury in the rat carotid, concomitant with increased VSMC proliferation and reduced cadherin expression.4 Furthermore, it has been shown that degradation-resistant ß-catenin elevated cyclin D1 expression,13 over-expression of N-cadherin lowered exogenous ß-catenin-dependent cyclin D1 reporter activity,14 and Wnt signaling modulated cyclin D1 expression21 in a rat smooth muscle cell line.
In addition to regulation of cyclin D1, we also observed that ß-catenin/TCF signaling repressed p21 mRNA and protein expression in primary VSMCs, as seen in HEK293 cells.9 Importantly, inhibition of ß-catenin/TCF signaling also significantly increased p21 protein in VSMCs embedded in saphenous vein segments. Previous studies showed that N-cadherin plays a role in p21 regulation in other cell types,22,23 however our study has shown for the first time that endogenous ß-catenin/TCF signaling represses expression of p21 mRNA and protein in primary VSMCs. It is possible however, that other cell cycle genes including c-myc and c-jun, are also regulated by ß-catenin/TCF signaling. Although beyond the scope of this report, identification of other ß-catenin responsive genes involved in VSMC proliferation is a focus of our current studies.
Taken together our findings provide evidence for the involvement of N-cadherin and ß-catenin/TCF signaling in the regulation of VSMC proliferation and indicate that increased ß-catenin levels, previously detected after balloon injury in the rat carotid,4,13 may contribute to VSMC proliferation by modulating the expression of both cyclin D1 and p21. The importance of this pathway is further supported by our observation that elevated levels of active ß-catenin in the absence of growth factors promoted VSMC proliferation and modulation of cyclin D1 and p21 levels.
The inability of ICAT and dnTCF-4 to reduce proliferation in cyclin D1–/– or p21–/– VSMCs substantiates our evidence that cyclin D1 and p21 are essential for the regulation of ß-catenin-dependent proliferation. Because both cyclin D1 and p21 are regulated by ß-catenin/TCF signaling, one would have predicted that proliferation of cyclin D1–/– VSMCs would be partially inhibited because of elevated p21 levels and vice versa. However, inhibition of ß-catenin/TCF signaling was unable to attenuate proliferation in cyclin D1–/– or p21–/– VSMCs. Cyclin D1–/– cells are able to enter the cell cycle as elevated cyclin E levels may compensate for the lack of cyclin D1.24 An increase in cyclin D1 early in G1 is thought to allow progression through to S phase because of titration of p21 into nascent cyclin D1/cdk4 complexes, which progressively relieves cyclin E/cdk2 from its main inhibitory constraint.25 Because cyclin E is elevated in cyclin D–/– mouse cells, we suggest that inhibition of ß-catenin/TCF signaling and the subsequent elevation of p21 does not lead to cell cycle arrest in these VSMCs because there is sufficient p21-free cyclin E/cdk 2 complexes to allow S phase progression. Similarly, in p21–/–VSMCs a ß-catenin/TCF-dependent elevation of cyclin D1 in early G1 is redundant because cyclin E/cdk2 complexes are uninhibited by p21.
p21 is necessary for G1/S transition because it promotes cyclin D/cdk complex formation and lack of p21 leads to cell cycle arrest.10,25 Our study and others, have demonstrated that growth factor-dependent elevation of p21 is not seen until late in the cell cycle.10,26,27 Before that p21 is expressed at relatively low levels, presumably sufficient to allow the formation of cyclin D/cdk complexes but at low enough levels to permit kinase activity. PDGF-dependent induction of p21 occurs via the ras/MAPK pathway in VSMCs, and ras and ERK1/2 are activated as early as 15 minutes after stimulation.27,28 Because ß-catenin is active and present in the nucleus at 4 hours after growth factor stimulation in VSMCs,3 ß-catenin may antagonize ras/MAPK-dependant induction of p21, resulting in optimal levels of p21 to allow G1 progression. Interestingly, at 24 hours after stimulation, p21 levels are maximal and nuclear ß-catenin is diminished compared with 4 hours.3
Although inhibition of ß-catenin/TCF signaling had comparable effects on cyclin D1 mRNA and protein, a far greater effect was observed on p21 protein than mRNA, particularly with ICAT and dnTCF-4. This suggests the involvement of ß-catenin/TCF-dependent post-translational regulation of p21 in VSMCs; perhaps via regulation of CCAAT enhancer binding protein 
(C/EBP), since upregulation of ß-catenin was associated with downregulation of C/EBP29 and C/EBP increases p21 protein stability.30
Accumulation of free cytosolic ß-catenin is regulated by several pathways including expression of cadherins, Wnt signaling and other pathways which modulate GSK-3ß activation eg, integrin-linked kinase activity. The reduced effect of N-cadherin over-expression on ß-catenin/TCF signaling, cyclin D1 and p21 expression, compared with ICAT and dnTCF-4, may be because ICAT and dnTCF-4 inhibit ß-catenin released as a result of all pathways but over-expression of N-cadherin affects only one pathway. Furthermore, N-cadherin and ß-catenin must be correctly phosphorylated for adhesion to occur (see review by31). In addition, growth factors induce N-cadherin shedding from the cell surface3 which may counter-balance the over-expression of N-cadherin and thereby reduce it’s inhibitory capacity.
Over-expression of N-cadherin, however, had a comparable inhibitory effect on VSMC proliferation to ICAT and dnTCF-4. ICAT and dnTCF-4 only prevent TCF activation, whereas increasing the number of adherens junctions by N-cadherin over-expression could potentially have other downstream effects that may contribute to inhibition of proliferation. For example, adherens junction formation may reduce Rho activity which could reduce proliferation via regulation of p27Kip1 levels or ERK1/2, see review by.32
In summary we have shown that N-cadherin and ß-catenin/TCF signaling plays a major role in the regulation of VSMC proliferation in response to stimulation by growth factors via the regulation of the cell cycle genes cyclin D1 and p21. We suggest that this pathway may therefore be involved in the regulation of VSMC proliferation in vascular diseases involving intimal thickening.
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Acknowledgments |
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We thank Jill Tarlton for excellent technical assistance. We thank Drs Thilo Hagen, Avri Ben-Ze’ev, Bert Vogelstein, Mien-Chie Hung, and Tetsu Akiyama for supplying plasmids and adenoviruses. We are very grateful to Dr Vera Fantl and Prof Clive Dickson for providing cyclin D1 knockout mice, Professor Yingzi Yang for providing TOPGAL mice, and Dr Yasuyuki Sasaguri for providing ISS10 cells.
Sources of Funding
This work was supported by the British Heart Foundation.
Disclosures
None.
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Footnotes |
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Original received June 15, 2006; revision received October 9, 2006; accepted October 24, 2006.
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References |
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- Newby AC, George SJ. Proliferation, migration, matrix turnover and death of smooth muscle cells in native coronary and vein graft atherosclerosis. Curr Opin Cardiol. 1996; 11: 574–582.[Medline] [Order article via Infotrieve]
- Reidy M, Fingerle J, Lindner V. Factors controlling the development of arterial lesions after injury. Circulation. 1993; 86: III-43–III-46.
- Uglow EB, Slater S, Sala-Newby GB, Aguilera-Garcia CM, Angelini GD, Newby AC, George SJ. Dismantling of cadherin-mediated cell-cell contacts modulates smooth muscle proliferation. Circ Res. 2003; 92: 1314–1321.
[Abstract/Free Full Text] - Slater SC, Koutsouki E, Jackson CL, Bush RC, Angelini GD, Newby AC, George SJ. R-cadherin:ß-catenin complex and its association with vascular smooth muscle cell proliferation. Arterioscler Thromb Vasc Biol. 2004; 24: 1204–1210.
[Abstract/Free Full Text] - Blindt R, Bosserhoff A-K, Dammers J, Krott N, Demircan L, Hoffmann R, Hanrath P, Weber C, Vogt F. Downregulation of N-cadherin in the neointima stimulates migration of smooth muscle cells by RhoA deactivation. Cardiovasc Res. 2004; 62: 212–222.
[Abstract/Free Full Text] - Jones M, Sabatini PJ, Lee FS, Bendeck MP, Langille BL. N-cadherin upregulation and function in response of smooth muscle cells to arterial injury. Arterioscler Thromb Vasc Biol. 2002; 22: 1972–1977.
[Abstract/Free Full Text] - Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico M, Pestell R, Ben-Ze’ev A. The cyclin D1 gene is a target of the ß-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A. 1999; 96: 5522–5527.
[Abstract/Free Full Text] - Tetsu O, McCormick F. ß-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 1999; 398: 422–426.[CrossRef][Medline] [Order article via Infotrieve]
- Kamei J, Toyofuku T, Hori M. Negative regulation of p21 by ß-catenin/TCF signaling: a novel mechanism by which cell adhesion molecules regulate cell proliferation. Biochem Biophys Res Comm. 2003; 312: 380–387.[CrossRef][Medline] [Order article via Infotrieve]
- Bond M, Sala-Newby GB, Wu Y-J, Newby AC. Biphasic effect of p21Cip1 on smooth muscle cell proliferation: role of PI3-kinase and Skp-2-mediated degradation. Cardiovasc Res. 2006; 69: 198–206.
[Abstract/Free Full Text] - Dong Y, Chi SL, Borowsky AD, Fan Y, Weiss RH. Cytosolic p21Waf1/Cip1 increases cell cycle transit in vascular smooth muscle cells. Cell Signal. 2004; 16: 263–269.[CrossRef][Medline] [Order article via Infotrieve]
- Weiss RH, Randour CJ. The permissive effect of p21Waf1/Cip1 on DNA synthesis is dependent on cell type: effect is absent in p53-inactive cells. Cell Signal. 2000; 12: 413–418.[CrossRef][Medline] [Order article via Infotrieve]
- Wang X, Xiao Y, Mou Y, Zhao Y, Blankesteijn M, Hall JL. A role for the ß-catenin/T-cell factor signaling cascade in vascular remodeling. Circ Res. 2002; 90: 340–347.
[Abstract/Free Full Text] - Hou R, Liu S, Anees S, Hiroyasu S, Sibinga NES. The Fat1 cadherin integrates vascular smooth muscle cell growth and migration signals. J Cell Biol. 2006; 173: 417–429.
[Abstract/Free Full Text] - Murahashi N, Sasaguri Y, Ohuchida M, Kakita N, Morimatsu M. Immortatlization of human aortic smooth muscle cells with origin-minus simian virus 40 DNA. Biotechnol Appl Biochem. 1992; 16: 152–160.[Medline] [Order article via Infotrieve]
- Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis. J Clin Invest. 1998; 101: 1478–1487.[Medline] [Order article via Infotrieve]
- George SJ, Baker AH, Angelini GD, Newby AC. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther. 1998; 5: 1552–1560.[CrossRef][Medline] [Order article via Infotrieve]
- Koutsouki E, Beeching CA, Slater SC, Blaschuk OW, Sala-Newby GB, George SJ. N-cadherin-dependent cell-cell contacts promote human saphenous vein smooth muscle cell survival. Arterioscler Thromb Vasc Biol. 2005; 25: 982–988.
[Abstract/Free Full Text] - Walker LN, Bowen-Pope DF, Ross R, Reidy MA. Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Proc Natl Acad Sci U S A. 1986; 83: 7311–7315.
[Abstract/Free Full Text] - Lindner V, Lappi DA, Baird A, Majack RA, Reidy MA. Role of basic fibroblast growth factor in vascular lesion formation. Circ Res. 1991; 68: 106–113.
[Abstract/Free Full Text] - Ezan J, Leroux L, Barandon L, Dufourcq P, Jaspard B, Moreau C, Allieres C, Daret D, Couffinhal T, Duplaa C. FrzA/sFRP-1, a secreted antagonist of the Wnt-Frizzled pathway, controls vascular cell proliferation in vitro and in vivo. Cardiovasc Res. 2004; 63: 731–738.
[Abstract/Free Full Text] - Levenberg S, Yarden A, Kam Z, Geiger B. p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry. Oncogene. 1999; 18: 869–876.[CrossRef][Medline] [Order article via Infotrieve]
- Gavard J, Marthiens V, Monnet C, Lambert M, Mege R-M. N-cadherin activation substitutes for the cell contact control in cell cycle arrest and myogenic differentiation: involvement of p120 and ß-catenin. J Biol Chem. 2004; 279: 36795–36802.
[Abstract/Free Full Text] - Ledda-Columbano GM, Pibiri M, Concas D, Cossu C, Tripodi M, Columbano A. Loss of cyclin D1 does not inhibit the proliferative response of mouse liver to mitogenic stimuli. Hepatology. 2002; 36: 1098–1105.[CrossRef][Medline] [Order article via Infotrieve]
- Coqueret O. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 2003; 13: 65–70.[CrossRef][Medline] [Order article via Infotrieve]
- Moon S-K, Cha B-Y, Kim C-H. In vitro cellular aging is associated with enhanced proliferative capacity, G1 cell cycle modulation, and matrix metalloproteinase-9 regulation in mouse aortic smooth muscle cells. Arch Biochem Biophys. 2003; 418: 39–48.[CrossRef][Medline] [Order article via Infotrieve]
- Lee B, Moon S-K. Ras/ERK signaling pathway mediates activation of the p21WAF1 gene promoter in vascular smooth muscle cells by platelet-derived growth factor. Arch Biochem Biophys. 2005; 443: 113–119.[CrossRef][Medline] [Order article via Infotrieve]
- Izzard TD, Taylor C, Birkett SD, Jackson CL, Newby AC. Mechanisms underlying maintenance of smooth muscle cell quiescence in rat aorta: role of the cyclin dependent kinases and their inhibitors. Cardiovasc Res. 2002; 53: 242–252.
[Abstract/Free Full Text] - Her GM, Cheng CH, Hong JR, Sundaram GS, Wu JL. Imbalance in liver homeostasis leading to hyperplasia by overexpressing either one of the Bcl-2-related genes, zfBLP1 and zfMcl-1a. Dev Dyn. 2006; 235: 515–523.[CrossRef][Medline] [Order article via Infotrieve]
- Park JS, Qiao L, Gilfor D, Yang MY, Hylemon PB, Benz C, Darlington G, Firestone G, Fisher PB, Dent P. A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21 (Cip-1/WAF1/mda6) protein levels in primary hepatocytes. Mol Biol Cell. 2000; 11: 2915–2932.
[Abstract/Free Full Text] - Lilien J, Balsamo J. The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of ß-catenin. Curr Opin Cell Biol. 2005; 17: 459–465.[CrossRef][Medline] [Order article via Infotrieve]
- Loriand G, Guerin P, Pacaud P. Rho kinases in cardiovascular physiology and pathophysiology. Circ Res. 2006; 98: 322–334.
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