Published online before print June 7, 2007, doi: 10.1161/CIRCRESAHA.106.145235
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Yin Yang-1 Inhibits Vascular Smooth Muscle Cell Growth and Intimal Thickening by Repressing p21WAF1/Cip1 Transcription and p21WAF1/Cip1-Cdk4-Cyclin D1 Assembly
From the Centre for Vascular Research, Department of Pathology, University of New South Wales, and Department of Haematology, The Prince of Wales Hospital (F.S.S., H.I., R.B. C.N.C., L.M.K.), Sydney, NSW, Australia; Centre for Cardiovascular Biology and Medicine, BHF Laboratories, Department of Medicine, University College London (S.S., R.K., M.J.R., J.F.M., I.C.Z.), United Kingdom; Cell Biology Laboratory (P.K., A.B.), Baker Heart Research Institute, Australia.
Correspondence to Levon M. Khachigian, PhD, DSc, Centre for Vascular Research, Department of Pathology, The University of New South Wales, Sydney NSW 2052 Australia. E-mail L.Khachigian{at}unsw.edu.au
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Abstract |
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Vascular injury initiates a cascade of phenotype-altering molecular events. Transcription factor function in this process, particularly that of negative regulators, is poorly understood. We demonstrate here that the forced expression of the injury-inducible GLI-Krüppel zinc finger protein Yin Yang-1 (YY1) inhibits neointima formation in human, rabbit and rat blood vessels. YY1 inhibits p21WAF1/Cip1 transcription, prevents assembly of a p21WAF1/Cip1-cdk4-cyclin D1 complex, and blocks downstream pRbSer249/Thr252 phosphorylation and expression of PCNA and TK-1. Conversely, suppression of endogenous YY1 elevates levels of p21WAF1/Cip1, PCNA, pRbSer249/Thr252 and TK-1, and increases intimal thickening. YY1 binds Sp1 and prevents its occupancy of a distinct element in the p21WAF1/Cip1 promoter without YY1 itself binding the promoter. Additionally, YY1 induces ubiquitination and proteasome-dependent degradation of p53, decreasing p53 immunoreactivity in the artery wall. These findings define a new role for YY1 as both an inducer of p53 instability in smooth muscle cells, and an indirect repressor of p21WAF1/Cip1 transcription, p21WAF1/Cip1-cdk4-cyclin D1 assembly and intimal thickening.
Key Words: gene expression • arterial injury • intimal thickening • vascular biology
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Introduction |
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Injury to the blood vessel wall initiates a cascade of molecular events that includes the induction of transcriptional activators and repressors. The net balance in the activities of these regulators in the reparative response to injury governs the phenotypic outcome, which includes vascular disorders such as atherosclerosis, bypass graft failure, hemodialysis access graft failure, restenosis after percutaneous coronary angioplasty and stenting, the pathogenesis of each of which involves the formation of a smooth muscle cell (SMC)-rich intima. The roles played by transcriptional regulators controlling these processes are only partly defined. Greater mechanistic insights would provide an invaluable basis for novel targeted interventional approaches.
Yin Yang-1 (YY1 [also known as 
], NF-E1, UCRBP and CF1])1,2 was first identified on the basis of its capacity to negatively regulate the adeno-associated virus P5 promoter,3 the immunoglobulin kappa 3' enhancer4 and the upstream conserved long terminal repeat region of Moloney murine leukemia virus.5 This GLI-Krüppel zinc finger protein plays a regulatory role in a variety of fundamental biological processes, such as growth, apoptosis and differentiation. This is, at least in part, because of its capacity to influence gene expression via its ability to initiate, activate or repress transcription depending on the context in which it binds. YY1 regulates transcription by direct or indirect activation or repression via cofactor recruitment or disruption of binding sites causing conformational changes in DNA.1,2,6
El Affar et al7 recently used genome-wide expression profiling to identify a plethora of YY1 target genes (primary and secondary) implicated in cell proliferation, cytokinesis, apoptosis, development, and differentiation. Levels of the cyclin-dependent kinase inhibitor, p21WAF1/Cip1 increased 3.3-fold in hypomorphic mice expressing 25% of normal YY1 levels,7 suggesting an inverse relationship between the transcription factor and the cell cycle regulator. This is notionally consistent with our previous demonstration in SMCs of YY1 inhibition of cell proliferation.8 Other studies, however, have linked YY1 with increased cell growth, prompting some to propose YY1 as a therapeutic target in cancer.6,9 In this study, we explored the relationship between YY1 and p21WAF1/Cip1 transcription in SMC growth and intimal thickening after arterial injury.
We demonstrate here that YY1 blocks p21WAF1/Cip1 transcription in SMCs by preventing Sp1 occupancy of a distinct element upstream in the p21WAF1/Cip1 promoter, thereby perturbing assembly of a p21WAF1/Cip1-cdk4-cyclin D1 complex, blocking pRbSer249/Thr252 phosphorylation, E2F-dependent gene expression and intimal thickening. In addition, we show that YY1 destabilizes p53, a positive regulator of p21WAF1/Cip1 transcription by inducing its ubiquitination and proteasomal degradation.
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Materials and Methods |
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Methodologies used for adenoviral YY1 construction, human saphenous vein culture and transfection, cell culture and plasmid transfection, proliferation assays, carotid artery injury and adenovirus delivery, rabbit collar model and periadventitial gene transfer, immunohistochemical staining, transient transfection and reporter gene analysis, Western blot analysis, immunoprecipitation analysis, RT-PCR, EMSA and ChIP are described in the online supplement available at http://circres.ahajournals.org.
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Results |
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YY1 Inhibits Intimal Thickening in Human, Rabbit and Rat Blood Vessels
YY1, like many other transcription factors, is inducibly expressed in vascular SMCs within hours of balloon injury.8 However, whether this zinc transcription factor influences intimal thickening or plays a gene regulatory role in injured vessels is unknown. We explored these questions given the apparent paradox between the growth-promoting properties of YY1 in non-SMC types6,9 and the inverse relationship between YY1 and the cyclin-dependent kinase inhibitor p21WAF1/Cip1 in hypomorphic mice.7 Adenoviral overexpression of YY1 (Ad-YY1), but not LacZ (Ad-LacZ), inhibited intimal thickening in explants of human saphenous veins, commonly used as autologous conduits for CABG10 within 14 days (Figure 1a & supplemental Figure I). Consistent with these observations, Ad-YY1 also blocked rabbit and rat primary SMC proliferation (Figure 1b), but intriguingly, had no effect on vascular endothelial cell growth (Figure 1b). YY1 inhibition of SMC proliferation in vitro or neointima formation was not associated with increased apoptosis, as determined by assessment of internucleosomal DNA fragmentation or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) immunostaining (data not shown).
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We next used 2 well-established animal models of SMC hyperplasia to determine the effect of YY1 on intimal thickening in intact arteries. YY1 inhibited neointima formation in rabbit carotid arteries 9 days after periadventitial collar placement11 (Figure 1c) and in rat carotid arteries 14 days after balloon catheter injury12 (Figure 1d), coincident with reduced SMC numbers in the neointima (Figure 1, c and d, right panels). We also observed no difference in intensity of immunostaining for fibronectin and collagen type I, or staining with Masson’s Trichrome or Alcian Blue per field of SMCs in the neointima between groups (data not shown), suggesting that YY1 negatively regulates intimal thickening primarily by reducing SMC proliferation rather than the matrix content as an independent variable.
YY1 Represses p21WAF1/Cip1 Transcription in SMCs
In efforts to define the molecular basis for YY1 inhibition, we investigated whether YY1 perturbs a principal component of the cell cycle machinery. Immunohistochemical analysis revealed that YY1 overexpression in the artery wall decreased levels of p21WAF1/Cip1 (Figure 2a and supplemental Figure II). YY1 also reduced levels of proliferating nuclear cell antigen (PCNA) and pRbSer249/Thr252 (Figure 2a and supplemental Figure II). Moreover, transient transfection analysis demonstrates dose-dependent inhibition of Firefly luciferase reporter activity driven by 1975 bp of the p21WAF1/Cip1 promoter (3*6) (Figure 2b, upper left panel). These findings are supported by Western blot analysis demonstrating that exogenous YY1 accumulates in SMC nuclei (Figure 2b, lower left panel) as cellular p21WAF1/Cip1 levels decrease (Figure 2b, right panel). We reasoned that if YY1 inhibition of SMC replication involved p21WAF1/Cip1 then the forced expression of p21WAF1/Cip1 may rescue the cells from growth suppression. Delivery of p21WAF1/Cip1 together with YY1 reversed YY1 inhibition (Figure 2c). Conversely, antisense (p21AS)-mediated depletion of p21WAF1/Cip113 not only reduced SMC proliferation, it rendered YY1 unable to further inhibit growth (Figure 2d).
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Serum induced the assembly of a cyclin D1-cdk4-p21WAF1/Cip1 complex (Figure 3a), which mediates pRbSer249/Thr252 phosphorylation.14 YY1 inhibited both formation of the complex and pRbSer249/Thr252 phosphorylation (Figure 3a). These data show for the first time that YY1 inhibits p21WAF1/Cip1 transcription and consequently, the formation of a cyclin D1-cdk4-p21WAF1/Cip1 complex. In contrast, cyclin D1-cdk4-p21WAF1/Cip1 assembly and pRbSer249/Thr252 levels were unaltered by YY1 in ECs (Figure 3a), consistent with the inability of YY1 to alter p21WAF1/Cip1 expression in this cell type (Figure 3b).
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YY1 Represses p21WAF1/Cip1 Transcription Indirectly by Preventing Sp1 Occupancy of the p21WAF1/Cip1 Promoter
Inspection of the p21WAF1/Cip1 promoter by Match or P-Match or AliBaba2.1 (80% minimal matrix conservation, www.gene-regulation.com) revealed that it does not contain consensus recognition elements for YY1 (5'NNNNNCCATNTWNNNWN3' or 5'NNNCGGCCATCTTGNCTSNW3'’, where it serves as a repressor or activator, respectively).15 5' deletion and transient transfection analysis determined that the region mediating YY1 repression of the p21WAF1/Cip1 promoter is located at bps –1975/–1270 (Figure 4a, upper panel). This region does, however bear an atypical motif (5'CCTCCC3') previously uncharacterized in this promoter that supports the interaction of Sp1 with the smooth muscle myosin heavy chain promoter16 and the lecithin:cholesterol acyltransferase promoter.17 Mutation of this element (Sp1–7: –1375CCTCCC–1370 to mSp1–7: –1375TTGTTT–1370) in construct 3*6 (bearing 2kb of the p21WAF1/Cip1 promoter) rendered it refractory to YY1 repression (Figure 4a, lower left panel). When element Sp1–7 was inserted in front of the SV40 promoter in plasmid pGL3-prom, this rendered the heterologous reporter construct Sp1 inducible (Figure 4a, lower right panel). In contrast, the –1375TTGTTT–1370 mutation did not support Sp1 induction by Sp1 (Figure 4a, lower right panel). Electrophoretic mobility shift analysis (EMSA) with SMC nuclear extracts (Figure 4b, left panel) and chromatin immunoprecipitation analysis (ChIP) (Figure 4c, middle panel, lanes 1 to 4) confirmed this region of the p21WAF1/Cip1 promoter interacts with Sp1. In contrast, YY1 did not bind the promoter (Figure 4, b and c). To exclude the possibility of a defective YY1 antibody or the lack of YY1 in the extracts, we obtained a positive supershift with probe 32P-MVYY1, bearing a consensus YY1 binding element8 (Figure 4b, right panel). This led us to hypothesize that YY1 represses p21WAF1/Cip1 transcription indirectly by interacting with Sp1.
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Immunoprecipitation analysis with GST antibodies demonstrated that GST-YY1 and Sp1 physically interact (Figure 5a, left panel). Immunoprecipitation studies confirmed the interaction of YY1 with Sp1 in SMCs, as observed in other cell types18,19 (Figure 5a, right panel); however, this interaction was not observed in ECs (Figure 5a, right panel). EMSA revealed that recombinant Sp1 but not YY1 binds the –1375CCTCCC–1370 element (Figure 5b, upper panel). Interestingly, YY1 prevents the interaction of Sp1 with the promoter fragment, an effect not observed with BSA (Figure 5b, upper panel). The integrity of the GST-YY1 was confirmed using Oligo-MVYY1 (Figure 5b, middle panel), and mutation of this element abolished Sp1 binding (Figure 5b, lower panel). Moreover, YY1 perturbs the interaction of Sp1’s C-terminal region (residues 620 to 778, spanning the zinc finger domain and activation domain D19) with the p21WAF1/Cip1 promoter (Figure 5c). ChIP analysis revealed that exogenous YY1 (as either the wild-type or GST fusion protein) displaced endogenous Sp1 from the authentic p21WAF1/Cip1 promoter (Figure 4c, middle and lower panels). In contrast, GST-YY1
ZNF lacking YY1’s zinc finger region (which binds to the zinc finger region of Sp118,19) failed to inhibit the interaction of Sp1 with the promoter (Figure 4c, lower panel). To further demonstrate specificity of YY1 inhibition, we performed ChIP analysis for the well-established Sp1-dependent gene, platelet-derived growth factor A-chain (PDGF-A)20 in SMCs transfected with YY1. YY1 did not affect Sp1’s interaction with a 777 bp region of the PDGF-A promoter containing Sp1-binding elements (SBE)20 (Figure 5d, upper panel), nor did it affect Sp1’s interaction with the proximal SBE of the p21WAF1/Cip1 promoter21 (Figure 5d, lower panel). These findings demonstrate that YY1 prevents Sp1 occupancy of a novel recognition element (–1375CCTCCC–1370) upstream in the p21WAF1/Cip1 promoter in a site-selective manner. This mechanism of YY1 repression involving Sp1 displacement has hitherto not been described.
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YY1 siRNA Stimulates SMC Growth and Intimal Thickening
Because YY1 overexpression inhibits SMC proliferation and neointima formation after vascular injury, we reasoned that strategies reducing the expression of endogenous YY1 in the vessel wall would, conversely, increase SMC growth and intimal thickening. siRNA targeting YY1 abrogated serum-inducible YY1 expression and increased p21WAF1/Cip1 expression without affecting levels of GAPDH (Figure 6a, right panel). The siRNA increased serum-inducible SMC proliferation within 3 days, whereas the nonsense (ns) siRNA counterpart had no effect (Figure 6a, left panel). Moreover, the siRNA increased in the rat carotid artery intimal thickening 14 days after balloon injury beyond that of the nonsense or vehicle groups (Figure 6b). Immunohistochemical analysis revealed that the siRNA suppressed YY1 expression in the artery wall while increasing levels of p21WAF1/Cip1, PCNA and pRbSer249/Thr252 (Figure 6c and supplemental Figure III).
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YY1 Regulates Thymidine Kinase-1 Expression
Rb phosphorylation triggers transcription factor E2F release14 which in turn activates thymidine kinase-1 (TK-1) stimulating salvage DNA synthesis and cell cycle progression.22 We examined the influence of YY1 on events downstream of pRbSer249/Thr252 phosphorylation and examined E2F-dependent TK-1 expression14,23 as a surrogate measure of pRbSer249/Thr252 phosphorylation. YY1 overexpression inhibited TK-1 expression in the injured vessel wall (Figure 7a and supplemental Figure IV). Moreover, YY1 inhibited both TK-1 promoter activity and protein expression (Figure 7b). Conversely, the YY1 siRNA, but not siRNAns increased TK-1 expression (Figure 7a and supplemental Figure IV). YY1 repression of TK-1 expression is blocked in the presence of E2F siRNA (Figure 7b) indicating therefore, that YY1 regulation of TK-1 is mediated through E2F.
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Sp1 displacement may not be the only mechanism with which YY1 controls p21WAF1/Cip1. Previous studies have demonstrated that p53 positively regulates p21WAF1/Cip1.24 We reasoned, therefore, that as well as causing Sp1 displacement, YY1 may reduce p21WAF1/Cip1 expression via p53. p53 stability is regulated by Mdm2-mediated ubiquitination and degradation via the proteasome.25,26 We found that YY1 overexpression reduces p53 immunoreactivity in SMCs, both in culture and in the artery wall (Figure 7c, left panel), consistent with our previous observation of an inverse relationship between p53 and YY1 expression in SMCs in the artery wall.8 Immunoprecipitation analysis revealed that YY1 increased p53 ubiquitination (Figure 7c, top right panel), as observed by Sui and coworkers in an osteosarcoma cell line.27 This was accompanied by reduced p21WAF1/Cip1 levels (Figure 7c, top right panel). Experiments further showed that p53 accumulates in SMCs treated with the proteasome inhibitor MG132, and that blockade of the proteasome prevents YY1 suppression of p53 protein levels (Figure 7c, bottom right panel). In contrast, p53 mRNA was not altered by YY1, either in the absence or presence of MG132 (Figure 7c, bottom right panel). These data thus demonstrate that YY1 suppression of p21WAF1/Cip1 in SMCs also involves p53 ubiquitination and proteasomal degradation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We demonstrate here that YY1 inhibits intimal thickening in human, rabbit and rat blood vessels, and that YY1 inhibits p21WAF1/Cip1 transcription in SMCs by preventing Sp1 occupancy of an element upstream in the p21WAF1/Cip1 promoter, perturbing assembly of a p21WAF1/Cip1-cdk4-cyclin D1 complex, thereby blocking Rb phosphorylation. This study challenges the general dogma that p21WAF1/Cip1 plays a negative regulatory role in cell cycle progression and provides a mechanism.28–34 The notion that p21WAF1/Cip1 may function as a positive cell cycle regulator has been described before.14,35,36 Weiss et al have shown that p21WAF1/Cip1 is an assembly factor required for PDGF-induced vascular SMC proliferation.37 Moreover, p21 WAF1/Cip1 expression is proatherogenic in apolipoprotein E-null mice, possibly as a consequence of cell cycle-independent activities of p21WAF1/Cip1.38,39 Using both gain- and loss-of-function approaches, we provide evidence for YY1 regulation of p21WAF1/Cip1 and SMC growth via perturbed assembly of the p21WAF1/Cip1-cdk4-cyclin D1 complex, thereby extending our understanding of the mechanisms of YY1-dependent repression.1,6 Moreover, we provide evidence that YY1 suppression of p21WAF1/Cip1 in SMCs also involves p53 ubiquitination and proteasomal degradation (Figure 8).
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YY1 had no effect on vascular endothelial proliferation nor did it influence p21WAF1/Cip1-cdk4-cyclin D1 complex assembly or pRbSer249/Thr252 phosphorylation in this cell type. Consistent with our model, YY1 failed to alter p21WAF1/Cip1 levels in ECs, unlike SMCs. The molecular basis for the inability of YY1 to repress p21WAF1/Cip1 expression in ECs is not clear but may involve cell-specific differences in YY1-dependent cofactor interactions. For example, YY1 collaborates with a large number of transcription factors including RYBP (Ring1- and YY1-binding protein),40 AP-2,41 c-Myc,42 YEAF1/RYBP, YAF-2,43 as well as the general factors TBP, TAFII55, and TFIIB.44,45 These and other collaborative factors may be preferentially expressed in SMCs but not ECs.46 Different CArG elements in the SM 
-actin promoter confer or repress TGF-ß1 responsiveness depending on the cell type.47 Alternatively, differences in chromatin structure and methylation state of the promoter may influence YY1’s accessibility and capacity to alter transcription. YY1 has been found to bind to and recruit the histone H4 (Arg 3)-specific methyltransferase, PRMT1, to the c-myc promoter.48 YY1 is also known to repress the human immunodeficiency virus type-1 5' long terminal repeat via recruitment of histone deacetylase-1 cooperatively with LSF.49
In this study we used siRNA strategies to knock down YY1 in vivo and in vitro rather than using genetically-modified mice, complementing the YY1 overexpression data and strengthening the physiological relevance of our conclusions. Mice in which YY1 is selectively ablated in vascular SMCs may be useful in response-to-injury studies of intimal thickening. However, given the critical role played by YY1 in key genes regulating SMC development such as (SM22- and SM-actin)50 the successful generation of such mice would seem unlikely. Alternatively, YY1 might be forcibly expressed in p21WAF1/Cip1-deficent mice, although again there is some evidence of compensatory changes by substitute factors such as p27Kip1 and p57Kip2. Studies by Sata et al have shown that intimal thickening in p21WAF1/Cip1-null mice does not differ from that in wild-type mice.51
Although our data demonstrate the role of p21WAF1/Cip1 in YY1-suppression of SMC growth and arterial wound repair via Sp1 and p53, it is still possible that p21WAF1/Cip1 may not be the sole YY1-dependent gene responsible because YY1 is a transcription factor with multiple targets7 and is involved in many cell regulatory systems.2,6 The role of other cyclins and cdks also requires exploration. This study will nonetheless facilitate further investigation into YY1-dependent processes mediating SMC growth and intimal hyperplasia. From a therapeutic standpoint, the resistance of arterial ECs to YY1-mediated growth inhibition would make YY1-based gene therapy potentially attractive in pathophysiologic situations, such as restenosis and vein grafting, where re-endothelialization is desirable and currently-used therapies, such as stent-delivered drugs52–54 (eg, rapamycin and taxol) lack cell specificity. Moreover, recent studies demonstrate the safety of using locally-delivered adenovirus in vascular applications. Trinam EG004, which locally generates VEGF-D via an adenovirus impregnated, bio-degradable collagen-based collar akin to that used in our rabbit model (Figure 1c), has been successfully trialed in hemodialysis patients to increase access graft patency (http://www.arktherapeutics.com/).
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Acknowledgments |
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We thank Dr Ryan Peden (Centre for Vascular Research, Sydney) for in vitro apoptosis assays, Dr Wafik El-Deiry (University of Pennsylvania) for pCEP-WAF1, Dr Mary Kavurma (Centre for Vascular Research, Sydney, Australia) for construct 3*6-Sp1m7, Dr Michael Atchison (University of Pennsylvania) for pCB6-YY1, Dr Matthew Horton (St George Hospital, Sydney, Australia) for saphenous veins, and Dr. Yang Shi (Harvard Medical School) for Sp1 and YY1 constructs.
Sources of Funding
This work was supported by grants from the National Health and Medical Research Council (N.H.M.R.C. to L.M.K., C.N.C., A.B.), National Heart Foundation (to L.M.K., R.B.), N.S.W. State Government Department of Health (to L.M.K., C.N.C.), British Heart Foundation (to I.C.Z., J.F.M., R.K.) and the Japan Research Foundation for Clinical Pharmacology (to H.I.). L.M.K. is a Senior Principal Research Fellow of the N.H.M.R.C.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received November 22, 2006; revision received May 17, 2007; accepted May 29, 2007.
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References |
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