Published online before print January 19, 2009, doi: 10.1161/CIRCULATIONAHA.108.822056
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(Circulation. 2009;119:577-586.)
© 2009 American Heart Association, Inc.
Vascular Medicine |
Deficiency of the NR4A Neuron-Derived Orphan Receptor-1 Attenuates Neointima Formation After Vascular Injury
From the Division of Endocrinology and Molecular Medicine, University of Kentucky, Lexington (T.N., Y.Z., F.G., H.M.F., E.H., K.J., D.B.), and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Tex (O.M.C.).
Correspondence to Dennis Bruemmer, MD, 575 Wethington Bldg, 900 S Limestone St, Lexington, KY 40536–0200. E-mail Dennis.Bruemmer{at}uky.edu
Received March 13, 2008; accepted November 19, 2008.
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Abstract |
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Background— The neuron-derived orphan receptor-1 (NOR1) belongs to the evolutionary highly conserved and most ancient NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily function as early-response genes regulating key cellular processes, including proliferation, differentiation, and survival. Although NOR1 has previously been demonstrated to be required for smooth muscle cell proliferation in vitro, the role of this nuclear receptor for the proliferative response underlying neointima formation and target genes trans-activated by NOR1 remain to be defined.
Methods and Results— Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in NOR1–/– mice compared with wild-type mice. In vitro, NOR1-deficient smooth muscle cells exhibit decreased proliferation as a result of a G1
S phase arrest of the cell cycle and increased apoptosis in response to serum deprivation. NOR1 deficiency alters phosphorylation of the retinoblastoma protein by preventing mitogen-induced cyclin D1 and D2 expression. Conversely, overexpression of NOR1 induces cyclin D1 expression and the transcriptional activity of the cyclin D1 promoter in transient reporter assays. Gel shift and chromatin immunoprecipitation assays identified a putative response element for NR4A receptors in the cyclin D1 promoter, to which NOR1 is recruited in response to mitogenic stimulation. Finally, we provide evidence that these observations are applicable in vivo by demonstrating decreased cyclin D1 expression during neointima formation in NOR1-deficient mice.
Conclusions— These experiments characterize cyclin D1 as an NOR1-regulated target gene in smooth muscle cells and demonstrate that NOR1 deficiency decreases neointima formation in response to vascular injury.
Key Words: cell cycle • receptors • muscle, smooth • survival
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Introduction |
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In an era marked by the increasing prevalence of obesity, diabetes, and cardiovascular disease, members of the nuclear hormone receptor superfamily have emerged as transcription factors that regulate diverse aspects of metabolism.1 In addition to their function of acting as molecular sensors of lipid and carbohydrate homeostasis, many nuclear receptors exert pleiotropic effects to control inflammatory and proliferative responses during vascular remodeling. This ability to integrate metabolic and vascular signaling networks has been well described for the ligand-activated peroxisome proliferator-activated receptors and liver X receptors.2,3 However, the nuclear receptor superfamily comprises a large number of receptors, for which ligands have not been identified, and they remain classified as orphan nuclear receptors.1 For many of these orphan receptors, the physiological functions and their target genes remain unknown, yet the high degree of conservation points to an important role in the control of gene expression.
Clinical Perspective p 586
The NR4A subfamily of orphan nuclear receptors consists of the members Nur77, Nurr1, and neuron-derived orphan receptor-1 (NOR1).4,5 These receptors bind as monomers to the nerve growth factor-induced clone B response element (NBRE) and as homodimers to the Nurr1 response element in the promoter of their target genes.4,5 In contrast to other members of the nuclear receptor superfamily, the ligand-binding pocket of these NR4A receptors is covered by hydrophobic residues, which has resulted in the characterization of NR4A receptors as ligand-independent and constitutively active transcription factors.6 Consistent with this concept, these receptors function as early-response genes, which are induced rapidly in response to various extracellular cues.4,5
In the vascular system, NOR1 is expressed in atherosclerotic lesions and in the neointima after vascular injury.7–9 At the cellular level, NOR1 expression is induced rapidly by inflammatory stimuli in macrophages10 and by growth factors in endothelial cells.11 Finally, NOR1 is highly expressed in smooth muscle cells (SMCs) after mitogenic stimulation,7,9 and recent studies,7 including our experiments using NOR1-deficient SMCs,9 pointed to a key role of this receptor in the transcriptional control of SMC proliferation. In the present study, we extend these observations by demonstrating that NOR1-deficient mice are protected from neointima formation because of altered cyclin D1 expression. These results indicate that NOR1 may constitute a potential novel target for the intervention for proliferative vascular diseases.
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Animals
All experiments were performed with littermate NOR1–/– and NOR1+/+ mice as recently described.9,12 The institutional animal care and use committee at the University of Kentucky approved all animal procedures.
Cell Culture
Rat and mouse aortic SMCs were cultured as described.9 Human coronary artery SMCs were commercially obtained (Lonza Inc, Allendale, NJ). Cells were grown to 60% to 70% confluence, serum deprived in 0.1% FBS for at least 24 hours, and subjected to stimulation with FBS at a final concentration of 10%. For all data shown, cells were used between passages 3 and 7, and individual experiments were repeated at least 3 times with different cell preparations.
Cell Cycle Distribution and Apoptosis Assays
After mitogenic stimulation, cells were collected by trypsinization, washed twice in PBS, and resuspended in staining buffer containing 20 µg/mL RNase A and 100 µg/mL propidium iodide. Cells were stained for 30 minutes at 4°C, and 1x106 cells were analyzed for cell cycle distribution with a FACSCalibur sorting system (Becton Dickinson, Franklin Lakes, NJ). Apoptosis was assessed with a fluorescence-based FragEL DNA fragmentation kit (QIA39, Calbiochem, San Diego, Calif).
Western Blot Analysis
Western blotting was performed as described9 with the following antibodies: Ser807/811 Phospho-Rb (9308, Cell Signaling Technology, Danvers, Mass), NOR1 (PP-H7833, R&D Systems, Minneapolis, Minn), Nurr1 (AF2156, R&D Systems), Nur77 (LS-B114, Lifespan Biosciences, Seattle, Wash), cyclin D1 (05–815, Millipore, Billerica, Mass), cyclin D2 (M-20), and GAPDH (FL 335) (both Santa Cruz Biotechnology, Santa Cruz, Calif).
Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
RNA was isolated and reverse transcribed (RT) as described.9 Polymerase chain reactions (PCRs) were performed with the iCycler and SYBR Green I system (Bio-Rad, Hercules, Calif). Each sample was analyzed in triplicate and normalized to values for transcription factor IIB mRNA expression. All primer sequences are provided in Table I of the online Data Supplement.
Plasmids, Transient Transfections, and Luciferase Assay
The human and murine cyclin D1 promoter constructs were kindly provided by Drs Richard G. Pestell13 and Nicholas H. Heintz,14 respectively. The NBRE sites in the murine and human cyclin D1 promoter located between –2197 and –2190 and between –1061 and –1054 were mutated from AAAGGTCA to AAAGAACA and from AAAGGTGA to AAAGAAGA, respectively, with the QuickChange II site-directed mutagenesis kit (Stratagene, La Jolla, Calif). The NOR1 expression vector was used as previously described.9 The E2F-Luc reporter construct was commercially obtained (Stratagene). SMCs were transfected with 1.5 µg reporter DNA with LipofectAMINE 2000 (Invitrogen, Carlsbad, Calif). After transfection, cells were serum deprived in 0.1% FBS for 12 hours and stimulated with 10% FBS for the indicated times. Luciferase activity was assayed with a Dual Luciferase Reporter Assay (Promega, Madison, Wis). Transfection efficiency was normalized to renilla luciferase activities generated by cotransfection of 5 ng pRLCMV (Promega).
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assays were performed with 5 µg nuclear extract as described15 and the oligonucleotides described in supplemental Table I. Supershift experiments were performed by incubating 5 µg nuclear extract with 2 µg NOR1 antibody (PP-H7833-00, R&D Systems) for 20 minutes before the addition of the radiolabeled probe.
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) assays were performed with the EZ-ChIP kit (Millipore) as described.9 Chromatin was immunoprecipitated with 5 µg antibody directed against NOR1 (PP-H7833-00, R&D Systems). Final DNA extractions were amplified by real-time PCR with primer pairs covering the NBRE cognate sequences in the human or mouse cyclin D1 promoter (supplemental Table I).
Endovascular Femoral Artery Guidewire Injury
Guidewire endothelial denudation injuries were performed on the left femoral artery of littermate NOR1+/+ (n=9) and NOR1–/– (n=8) mice at 8 weeks of age with a 0.25-mm SilverSpeed-10 hydrophilic guidewire (Micro Therapeutics Inc, Irvine, Calif) as described.16 The denudation injury was accomplished on 4 passages of the wire. Sham surgery without injury was performed on the right side. Mice were euthanized at the indicated time points, and femoral arteries were isolated for tissue analysis.
Tissue Preparation and Morphometry
After euthanasia, mice were perfused with PBS for 5 minutes, followed by 4% paraformaldehyde for 30 minutes at 100 cm H2O via cannulation of the left ventricle. Femoral arteries were embedded in paraffin and cut into 10-µm sections. Serial sections 2 mm proximal from the incision site of the wire insertion were evaluated by staining with an elastic Verhoeff-van Gieson staining kit (Sigma-Aldrich, St Louis, Mo) to visualize the internal and external elastic lamina. The intimal and medial areas were measured by computerized morphometry with the Image-Pro Plus 4.0 software (MediaCybernetics, Bethesda, Md). Intimal hyperplasia was defined as the formation of a neointimal layer medial to the internal elastic lamina. Media area was defined as the area encircled by the external elastic lamina and internal elastic lamina. The intima-to-media ratio was calculated as the intimal area divided by the media area.
Immunohistochemistry and Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling Staining
Statistical Analysis
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
NOR1 Deficiency Attenuates Neointima Formation After Vascular Injury
NOR1 Is Required for Cell Cycle Progression and Survival of SMCs
NOR1 Deficiency Increases Apoptosis in Neointimal SMC
NOR1 Deficiency Alters Phosphorylation of the Retinoblastoma Protein and E2F Activity
NOR1 Regulates Cyclin D1 and D2 Expression in Response to Mitogenic Stimulation
NOR1 Activates the Cyclin D1 Promoter
Electrophoretic mobility shift assays and ChIP assays were performed next to confirm that NOR1 directly binds to these NBRE sites. As depicted in Figure 7B, electrophoretic mobility shift assay experiments revealed a specific complex at the murine NBRE site that was induced by FBS and completely abolished by competition with an unlabeled NOR1 oligonucleotide but not by an AP-1 oligonucleotide. Diminished binding and supershift with an antibody raised against NOR1 further verified specificity of this complex. ChIP assays corroborated occupancy of the endogenous NBRE sites in the murine and human cyclin D1 promoter by NOR1 using murine wild-type SMCs and human coronary artery SMCs. As shown in Figure 7C, quantitative analysis by real-time PCR revealed that mitogenic stimulation results in a profound increase of NOR1 binding to the NBRE site located between –2197 and –2190 in the murine cyclin D1 promoter. Similarly, FBS induced NOR1 binding to the NBRE site located between –1061 and –1054 in the human cyclin D1 promoter (Figure 7D). This binding of NOR1 to the human cyclin D1 promoter was prevented by PD98059, an inhibitor of extracellular signal-regulating kinase/mitogen-activated protein kinase signaling, consistent with this signaling cascade being a key pathway in the control of SMC proliferation. In concert, these experiments demonstrate that NOR1 binds to an NBRE motif in the murine and human cyclin D1 promoter, resulting in the activation of the promoter and cyclin D1 transcription.
Cyclin D1 Expression During Neointima Formation Is Attenuated in NOR1-Deficient Mice
Consistent with the mitogenic activity of NOR1 in vitro,7,9 our data establish that NOR1 is required for cell cycle progression and cell survival. The main gatekeeper of G1
Compared with the well-studied early-response genes encoding proteins of the AP-1 complex, little is known about the endogenous binding activity of NOR1 to consensus sites in target promoters. A single recent study identified that NOR1 binds to an atypical NBRE with 1 base substitution in the uncoupling protein-1 promoter.20 In the present study, we identified a canonical NBRE site in the murine cyclin D1 promoter at –2197 to –2190 and a putative NBRE site with 1 base substitution in the human cyclin D1 promoter located at –1061 to –1054. Using gel shift and ChIP assays, we confirmed that NOR1 is recruited to these NBRE sites in response to mitogenic signals. The functional relevance of NOR1 binding to these NBRE sites was provided by the observation that overexpression of NOR1 induces cyclin D1 promoter activity, whereas mutation of the NBRE sites resulted in a complete loss of both FBS- and NOR1-induced cyclin D1 promoter activity. Although it is possible, if not likely, that NOR1 regulates additional genes involved in the regulation of cell proliferation, these studies characterize NOR1-regulated cyclin D1 expression as a novel transcriptional cascade promoting cell cycle progression.
Considering that all 3 members of the NR4A receptor subfamily bind to an NBRE site, functional redundancy between Nur77 and NOR1 has been suggested.21 However, 2 lines of evidence suggest that a possible redundancy is likely context and tissue dependent. First, Nur77–/– mice exhibit no overt phenotype,22 whereas NOR1–/– mice develop an obvious phenotype with inner ear defects and bidirectional circling behavior.12 Considering these differences between Nur77–/– and NOR1–/– mice, one might hypothesize that the Nur77 function can be replaced by NOR1 but not vice versa. Consistent with this notion would be the superinduction of NOR1 in Nur77–/– mice21 and the lack of increased Nur77 expression in NOR1–/– SMCs in our studies. Furthermore, because the maximal induction of Nur77 and Nurr1 mRNA during neointima formation was delayed in NOR1-deficient mice, it is possible that NOR1 is required for maximal expression of Nur77 and Nurr1, particularly because a positive autoregulation of NR4A receptors has recently been confirmed.23 Second, in the context of neointima formation, it appears that Nur77 and NOR1 exert distinct effects on SMC proliferation. Arkenbout et al17 recently reported that overexpression of Nur77 protects against neointima formation. This observation, along with our data, suggests that Nur77 and NOR1 exhibit opposite roles in the control of SMC proliferation. An obvious question for future investigation is how to reconcile the distinct biological effects of NOR1 and Nur77. Although the transcriptional target gene mediating the inhibition of SMC proliferation by Nur77 remains unknown, a possibility is that its biological effect may occur as a result of its heterodimeric partner retinoid X receptor (RXR). Nurr77, unlike NOR1, can heterodimerize with RXR and mediate efficient trans-activation in response to RXR-specific agonists from canonical DR5 sites.24 Ligand-induced RXR activation has been well established to inhibit mitogen-induced SMC proliferation,25 and interaction between Nur77 and RXR may at least need to be considered as an explanation for the distinct effect of Nur77 and NOR1 on SMC proliferation.
These studies were supported by the National Institutes of Health (RO1 HL084611
Disclosures
None. 2. Gervois P, Fruchart JC, Staels B. Drug insight: mechanisms of action and therapeutic applications for agonists of peroxisome proliferator-activated receptors. Nat Clin Pract Endocrinol Metab. 2007; 3: 145–156. 3. Zelcer N, Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling. J Clin Invest. 2006; 116: 607–614. 4. Maxwell MA, Muscat GE. The NR4A subgroup: immediate early response genes with pleiotropic physiological roles. Nucl Recept Signal. 2006; 4: e002. 5. Pols TW, Bonta PI, de Vries CJ. NR4A nuclear orphan receptors: protective in vascular disease? Curr Opin Lipidol. 2007; 18: 515–520. 6. Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker NPC, Perlmann T. Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors. Nature. 2003; 423: 555–560. 7. Martinez-Gonzalez J, Rius J, Castello A, Cases-Langhoff C, Badimon L. Neuron-derived orphan receptor-1 (NOR-1) modulates vascular smooth muscle cell proliferation. Circ Res. 2003; 92: 96–103. 8. Bonta PI, van Tiel CM, Vos M, van Thienen JV, Ferreira V, Arkenbout EK, Seppen J, Spek CA, van der Poll T, Pannekoek H, de Vries CJM. Nuclear receptors Nur77, Nurr1, and NOR-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses. Arterioscler Thromb Vasc Biol. 2006; 26: 2288–2294. 9. Nomiyama T, Nakamachi T, Gizard F, Heywood EB, Jones KL, Ohkura N, Kawamori R, Conneely OM, Bruemmer D. The NR4A orphan nuclear receptor NOR1 is induced by platelet-derived growth factor and mediates vascular smooth muscle cell proliferation. J Biol Chem. 2006; 281: 33467–33476. 10. Pei L, Castrillo A, Chen M, Hoffmann A, Tontonoz P. Induction of NR4A orphan nuclear receptor expression in macrophages in response to inflammatory stimuli. J Biol Chem. 2005; 280: 29256–29262. 11. Rius J, Martinez-Gonzalez J, Crespo J, Badimon L. NOR-1 is involved in VEGF-induced endothelial cell growth. Atherosclerosis. 2006; 184: 276–282. 12. Ponnio T, Burton Q, Pereira FA, Wu DK, Conneely OM. The nuclear receptor Nor-1 is essential for proliferation of the semicircular canals of the mouse inner ear. Mol Cell Biol. 2002; 22: 935–945. 13. Albanese C, D'Amico M, Reutens AT, Fu M, Watanabe G, Lee RJ, Kitsis RN, Henglein B, Avantaggiati M, Somasundaram K, Thimmapaya B, Pestell RG. Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis. J Biol Chem. 1999; 274: 34186–34195. 14. Ranjan P, Anathy V, Burch PM, Weirather K, Lambeth JD, Heintz NH. Redox-dependent expression of cyclin D1 and cell proliferation by Nox1 in mouse lung epithelial cells. Antioxid Redox Signal. 2006; 8: 1447–1459. 15. Ogawa D, Stone JF, Takata Y, Blaschke F, Chu VH, Towler DA, Law RE, Hsueh WA, Bruemmer D. Liver X receptor agonists inhibit cytokine-induced osteopontin expression in macrophages through interference with activator protein-1 signaling pathways. Circ Res. 2005; 96: e59–e67. 16. Ogawa D, Nomiyama T, Nakamachi T, Heywood EB, Stone JF, Berger JP, Law RE, Bruemmer D. Activation of peroxisome proliferator-activated receptor gamma suppresses telomerase activity in vascular smooth muscle cells. Circ Res. 2006; 98: e50–e59. 17. Arkenbout EK, de Waard V, van Bragt M, van Achterberg TAE, Grimbergen JM, Pichon B, Pannekoek H, de Vries CJM. Protective function of transcription factor TR3 orphan receptor in atherogenesis: decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. Circulation. 2002; 106: 1530–1535. 18. Sherr CJ. G1 phase progression: cycling on cue. Cell. 1994; 79: 551–555. 19. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 1993; 7: 812–821. 20. Kumar N, Liu D, Wang H, Robidoux J, Collins S. Orphan nuclear receptor NOR-1 enhances cAMP-dependent uncoupling protein-1 gene transcription. Mol Endocrinol. 2008; 22: 1057–1064. 21. Cheng LE, Chan FK, Cado D, Winoto A. Functional redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell apoptosis. EMBO J. 1997; 16: 1865–1875. 22. Lee SL, Wesselschmidt RL, Linette GP, Kanagawa O, Russell JH, Milbrandt J. Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77). Science. 1995; 269: 532–535. 23. Zhan Y, Du X, Chen H, Liu J, Zhao B, Huang D, Li G, Xu Q, Zhang M, Weimer BC, Chen D, Cheng Z, Zhang L, Li Q, Li S, Zheng Z, Song S, Huang Y, Ye Z, Su W, Lin SC, Shen Y, Wu Q. Cytosporone B is an agonist for nuclear orphan receptor Nur77. Nat Chem Biol. 2008; 4: 548–556. 24. Perlmann T, Jansson L. A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev. 1995; 9: 769–782. 25. Wakino S, Kintscher U, Kim S, Jackson S, Yin F, Nagpal S, Chandraratna RA, Hsueh WA, Law RE. Retinoids inhibit proliferation of human coronary smooth muscle cells by modulating cell cycle regulators. Arterioscler Thromb Vasc Biol. 2001; 21: 746–751.
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Paraffin sections were incubated with a Cy3-conjugated 
-smooth muscle actin antibody (C6198, Sigma Aldrich), a macrophage antiserum (AIA31240
To compare wild-type and NOR1-deficient mice on a single variable, we used an unpaired Student t test. One-way ANOVA and 2-way ANOVA were used to compare multiple groups as appropriate. Values of P<0.05 were considered statistically significant. Results are expressed as mean±SEM.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
Conclusions
References
NR4A Receptors Are Expressed During Neointima Formation
Previous studies demonstrated that NOR1, Nur77, and Nurr1 are expressed in atherosclerotic lesions8,17 and that NOR1 mRNA is induced after balloon angioplasty.7 To complement these studies, we analyzed their expression profiles during neointima formation in the mouse femoral artery using endothelial denudation injuries. As depicted in Figure 1A, all 3 members of the NR4A subfamily were expressed in the neointima of NOR1+/+ mice. In contrast, NOR1 protein was undetectable in mice deficient for the locus, confirming the phenotype of the mouse strain. In response to vascular injury of wild-type mice, transcript levels of all 3 receptors were induced rapidly within 3 to 6 hours (Figure 1B). Although Nur77 and Nurr1 were induced to a comparable extent in wild-type and NOR1-deficient mice, their induction was delayed in NOR1-deficient mice. In vitro, NOR1 mRNA expression was induced by >600-fold on mitogenic stimulation of wild-type SMCs, which is consistent with previous studies7,9 (Figure 1C). Although Nur77 mRNA expression increased by >20-fold in wild-type and NOR1–/– SMC, Nurr1 transcript levels were induced to only a modest extent in both genotypes. Finally, complementary results were obtained in Western blotting experiments analyzing protein levels of all 3 receptors in response to mitogenic stimulation (Figure 1D).
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Figure 1. NOR1, Nur77, and Nurr1 expression during neointima formation and in response to mitogenic stimulation of SMCs. A and B, Endothelial denudation injuries were performed in littermate NOR1+/+ and NOR1–/– mice. A, Tissues were harvested after 28 days, and NOR1, Nur77, and Nurr1 expression was analyzed by immunohistochemistry (scale bar=10 µm/L). B, mRNA was harvested from sham-operated and injured vessels at the indicated time points and analyzed for NOR1, Nur77, and Nurr1 expression with real-time RT-PCR. Data are normalized to transcription factor IIB mRNA expression levels and presented as mean±SEM-fold increase relative to sham-operated vessels (*P<0.05 vs time point 0 of the same genotype, 2-way ANOVA). C and D, SMCs were isolated from NOR1+/+ and NOR1–/– mice, serum deprived for 48 hours, and stimulated with 10% FBS. C, mRNA was harvested at the indicated time points and analyzed for NOR1, Nur77, and Nurr1 mRNA expression with real-time RT-PCR. Data are normalized to transcription factor IIB mRNA expression levels and presented as fold increase relative to quiescent cells (*P<0.05 vs quiescent, 2-way ANOVA). D, NOR1, Nur77, and Nurr1 protein expression was analyzed by Western blotting at the indicated time point. Cohybridization for GAPDH was performed to assess equal loading. The autoradiograms are representative of 3 independently performed experiments.
The profound increase in NOR1 expression during vascular remodeling and the reduction of cell proliferation in NOR1-deficient SMCs reported in our previous in vitro assays9 prompted us to examine neointima formation in NOR1-deficient mice. As depicted in Figure 2A, neointima formation was substantially decreased in NOR1-deficient mice compared with wild-type mice. Quantitative morphometry confirmed a significant 56.7% reduction in the intima-to-media ratio and a concomitant increase in lumen size in NOR1–/– mice (the Table). Cells present in the neointima were primarily SMCs staining positive for -smooth muscle actin (Figure 2B). Staining for von Willebrand factor identified an intact endothelial layer above the neointima, whereas no endothelial cells were detected within the neointima. Macrophages were largely absent within the neointimal layer and sparsely detected within the adventitial layer.
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Figure 2. Neointima formation is decreased in NOR1-deficient mice. Endothelial denudation injury was performed in NOR1+/+ (n=9) and NOR1–/– (n=8) mice. A, Tissues were harvested after 28 days, and elastic Verhoeff-van Gieson stain was performed to visualize the internal and external elastic lamina. Top, Magnification x200; scale bar=50 µm/L. Bottom, Magnification x400; scale bar=25 µm/L. B, Representative immunofluorescence image of injured femoral arteries from NOR1+/+ and NOR1–/– mice showing -smooth muscle actin (-SMA)-positive SMCs (red), macrophages (green), and endothelial cells stained for von Willebrand factor (vWF; yellow). Sections were counterstained with DAPI to visualize nuclei (blue) and merged to show the composite staining.
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Table. Morphological Analysis of Mouse Femoral Arteries
To determine the mechanisms underlying decreased neointima formation and altered SMC proliferation in NOR1–/– mice,9 we analyzed cell cycle distribution of wild-type and NOR1–/– SMCs. On mitogenic stimulation with 10% FBS, NOR1 wild-type SMCs progressed into the S phase, whereas NOR1–/– SMCs remained arrested in the G0/G1 phase (29.95±0.57% versus 4.18±1.23% of cells in the S phase, respectively; P<0.01, unpaired Student t test; Figure 3A and 3B). Analysis of cell cycle distribution further revealed that serum deprivation increased apoptosis of NOR1–/– SMCs compared with wild-type mice (9.3±0.19% versus 1.66±1.28%; P<0.05, unpaired Student t test; Figure 3A and 3B), an observation confirmed by an increased number of NOR1-deficient SMCs revealing DNA fragmentation (Figure 3C). In concert, these data point to an increased propensity for apoptosis in NOR1–/– SMCs and corroborate previous observations using NOR1 antisense to demonstrate that this receptor is required for S phase entry of the cell cycle.7
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Figure 3. NOR1 expression is required for cell cycle progression and survival of SMCs. Quiescent aortic SMCs isolated from NOR1+/+ and NOR1–/– were stimulated to enter the S phase by treatment with 10% FBS for 24 hours. A, Representative cell cycle distribution assessed by DNA staining with propidium iodide and subsequent flow cytometry. B, DNA histograms for cell cycle distribution of quiescent and mitogen-stimulated NOR1+/+ and NOR1–/– SMCs. C, NOR1+/+ and NOR1–/– SMCs were serum deprived for 48 hours and assayed by FACS for fluorescence-based DNA fragmentation. A representative histogram is depicted, and the percentage of apoptotic cells is indicated as mean±SEM in the top right corner.
To determine whether reduced neointima formation in NOR1–/– mice may be due in part to increased apoptosis, we next quantified SMC apoptosis in neointimal lesions. As shown in the representative Figure 4A and quantified in Figure 4B, <10% of the total number of neointimal SMCs in wild-type mice demonstrated the presence of DNA fragmentation. In contrast, NOR1 deficiency increased the number of apoptotic SMCs in the neointima by 
3-fold compared with wild-type mice (P<0.05). These results confirm the above-described in vitro observations and indicate that increased SMC apoptosis may be responsible in part for the decreased neointima formation in NOR1-deficient mice.
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Figure 4. NOR1 deficiency increases neointimal SMC apoptosis. A, Arterial tissues from NOR1+/+ and NOR1–/– mice were harvested 28 days after injury and analyzed for DNA fragmentation (green). Sections were counterstained with DAPI to visualize nuclei (blue) and merged to show the composite staining. B, Apoptotic cells with DNA fragmentation were quantified in the neointima and expressed as percent of DAPI-stained nuclei (*P<0.05 vs wild-type, unpaired Student t test).
Cell cycle transition through the G1 checkpoint is regulated by the retinoblastoma tumor suppressor protein (Rb) and requires its phosphorylation.18 Therefore, we next examined whether Rb phosphorylation is altered in NOR1-deficienct SMCs. As depicted in Figure 5A, quiescent NOR1+/+ SMCs exhibited low levels of Rb phosphorylated at Ser807/811, which increased after mitogenic stimulation. In marked contrast, deficiency of NOR1 resulted in significantly decreased Rb phosphorylation. Hypophosphorylated Rb sequesters the E2F transcription factor, restricting the expression of E2F-regulated target genes, the protein products of which are required for DNA replication.18 To further determine whether NOR1-regulated Rb phosphorylation affects downstream E2F activity, we performed trans-activation assays using a reporter construct driven by multiple E2F consensus sites. As demonstrated in Figure 5B, overexpression of NOR1 resulted in a profound 5.03±0.95-fold induction of E2F activity compared with cells transfected with the empty control vector. Taken together, these studies demonstrate that NOR1 is required for mitogen-induced Rb phosphorylation and is sufficient to increase the transcriptional activity of E2F.
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Figure 5. NOR1 deficiency alters Rb phosphorylation and E2F activity. A, Aortic SMCs isolated from NOR1+/+ or NOR1–/– mice were serum deprived and stimulated with 10% FBS. Cells were harvested at the indicated time points and analyzed for Rb (ser807/811) phosphorylation and GAPDH to assess equal loading. Top, Mean±SEM of Rb phosphorylation/GAPDH protein levels quantified by densitometry (n=3; *P<0.05 vs time point 0, #P<0.05 vs wild type, 2-way ANOVA). B, Rat SMCs were cotransfected with either an NOR1 expression vector or empty control vector and a luciferase reporter construct driven by multiple E2F binding sites. After transfection, cells were harvested after 24 hours, and luciferase activities were analyzed as described in Methods. Data are presented as mean±SEM from 3 independently performed experiments (*P<0.01 vs control vector, unpaired Student t test).
The phosphorylation of Rb is regulated by cyclin/cyclin-dependent kinase complexes, and cyclin D1 expression is limiting for cell proliferation and survival.13,19 In addition, recent studies identified cyclin D2 as a target gene for Nur77 in monocytes,10 and we have previously demonstrated that NOR1 expression is required for cyclin D1 and D2 expression in response to platelet-derived growth factor.9 Consistent with these prior studies, cyclin D1 and D2 protein expression was completely abolished in NOR1–/– cells stimulated with FBS compared with wild-type SMCs (Figure 6A). Conversely, overexpression of NOR1 in quiescent human coronary artery SMCs with an eukaryotic expression vector increased cyclin D1 protein expression and downstream Rb phosphorylation in the absence of mitogenic stimulation (Figure 6B). The mechanisms by which NOR1 induces cyclin D1 and D2 expression occur at the transcriptional level because mitogen-induced mRNA expression of cyclin D1 (Figure 6C) and D2 (Figure 6D) was completely repressed in NOR1-deficient SMC. In concert, these data demonstrate that NOR1 is necessary and sufficient to induce cyclin D1 protein expression, providing a potential mechanism by which NOR1 regulates Rb phosphorylation, cell cycle progression, and survival.
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Figure 6. NOR1 regulates mitogen-induced cyclin D expression. A, Serum-deprived SMCs isolated from NOR1+/+ or NOR1–/– mice were stimulated with 10% FBS. Cyclin D1 and D2 protein expression was analyzed at the indicated time points. B, Quiescent human coronary artery SMCs were transfected with either an NOR1 expression vector or a green fluorescent protein expression vector. Forty-eight hours after transfection, Rb phosphorylation, NOR1, cyclin D1, and GAPDH protein expression was analyzed. The autoradiograms shown are representative of 3 independently performed experiments. Cyclin D1 (C) and cyclin D2 (D) mRNA expression was analyzed by real-time RT-PCR in NOR1+/+ and NOR1–/– SMCs before (white bars) and after (black bars) stimulation with 10% FBS for 24 hours. Data are normalized to transcription factor IIB mRNA expression levels and presented as mean±SEM relative to quiescent SMCs (*P<0.05 vs quiescence, 2-way ANOVA).
To further investigate whether NOR1 induces cyclin D1 promoter activity, we performed trans-activation assays using an NOR1 expression vector cotransfected with reporter constructs driven by a murine 3.6-kb or human 1.7-kb cyclin D1 promoter fragment (Figure 7A). Compared with cells transfected with the control vector, overexpression of NOR1 resulted in a significant induction of basal and mitogen-induced activity of the cyclin D1 promoter. To characterize the transcriptional mechanisms governing this regulation, we screened both cyclin D1 promoter sequences for putative NBRE consensus sites. Using this approach, we identified a canonical consensus NBRE site within the murine cyclin D1 promoter (–2197 to –2190, AAAGGTCA) and a putative NBRE site with 1 base substitution in the human cyclin D1 promoter (–1061 to –1054, AAAGGTGA). To establish the functional relevance of these NBRE sites for the regulation of the cyclin D1 promoter by NOR1, site-directed mutations were next introduced into these NBRE sequences. In marked contrast to the regulation of the wild-type promoters, both murine and human cyclin D1 promoter constructs bearing mutations of the predicted NBRE sites exhibited low basal activity and were not inducible by FBS.
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Figure 7. NOR1 activates the cyclin D1 promoter. A, Rat SMCs were cotransfected with either an NOR1 or green fluorescent protein expression vector and a luciferase reporter construct driven by the wild-type or NBRE-mutated murine or human cyclin D1 promoter. After transfection, cells were starved for 12 hours, stimulated with 10% FBS for 48 hours, and analyzed for luciferase activities. Data are presented as mean±SEM from 3 independently performed experiments (*P<0.05 vs quiescence, #P<0.05 vs green fluorescent protein, 2-way ANOVA). B, Serum-deprived SMCs isolated from NOR1+/+ mice were stimulated with 10% FBS for 12 hours. DNA binding activities in nuclear extracts recognizing a radiolabeled murine cyclin D1 promoter region containing the NBRE site between –2197 and –2190 were detected by electrophoretic mobility shift assays. Specificity was confirmed by competition with cold cyclin D1 or AP-1 oligonucleotides and by supershift using an NOR1 antibody. The autoradiogram shown is representative of 3 independently performed experiments. C, Quiescent wild-type SMCs were stimulated with 10% FBS and harvested at the indicated time points for ChIP assays. After chromatin immunoprecipitation with an NOR1 antibody or control IgG, quantitative real-time PCR analysis was performed with primer pairs that cover the NBRE site between –2197 and –2190 in the murine cyclin D1 promoter. D, Quiescent human coronary artery SMCs were pretreated with vehicle (dimethyl sulfoxide) or the MEK1 inhibitor PD98059 (10 µmol/L) for 30 minutes before stimulation with 10% FBS for 24 hours. After chromatin immunoprecipitation with an NOR1 antibody or control IgG, quantitative real-time PCR analysis was performed with primer pairs that cover the NBRE site between –1061 and –1054 in the human cyclin D1 promoter. Data in C and D are normalized to total extract (input) and presented as fold±SEM increase relative to quiescent SMCs (*P<0.001 vs quiescent SMCs, 1-way ANOVA [C] and 2-way ANOVA [D]).
Finally, to investigate whether cyclin D1 expression during neointimal SMC proliferation is decreased in NOR1-deficient mice in vivo, we performed cyclin D1 immunohistochemistry of guidewire-injured femoral arteries isolated from NOR1+/+ and NOR1–/– mice. As depicted in Figure 8A, neointimal SMCs of wild-type mice revealed typical nuclear localization of cyclin D1 expression, which was decreased in the neointima of NOR1–/– mice. Quantification of neointimal cyclin D1 expression as a means of cyclin D1-positive nuclei divided by the total number of nuclei confirmed a significant decrease in cyclin D1 expression in the neointima of NOR1–/– mice compared with wild-type mice (11.5±3.49% versus 40.7±5.27%, respectively; P<0.001; Figure 8B).
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Figure 8. NOR1 deficiency attenuates cyclin D1 expression during neointima formation. Femoral artery sections were isolated 28 days after injury of NOR1+/+ and NOR1–/– mice. A, Cyclin D1 immunohistochemistry was performed on tissues obtained from NOR1+/+ and NOR1–/– mice. B, Cyclin D1-positive cells were quantified by analyzing the fraction of cyclin D1-stained nuclei in the neointima relative to the total number of nuclei in the neointimal area (n=8 to 9 per group). Values are expressed as mean±SEM (*P<0.001 vs NOR1+/+, unpaired Student t test).
Discussion
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Abstract
Introduction
Methods
Results
Discussion
Conclusions
References
Previous studies by Martínez-González et al7 using NOR1 silencing and our experiments in NOR1-deficient SMCs9 provided evidence that this receptor promotes SMC proliferation in vitro. Although these studies point to an important role for NOR1 in the regulation of SMC proliferation, the contribution of NOR1 to the proliferative response underlying neointima formation and the molecular mechanisms responsible for the mitogenic activity of NOR1 remain unknown. In the present study, we extend these earlier observations and demonstrate that NOR1 deficiency attenuates neointima formation, suggesting an important role of NOR1 signaling for proliferative vascular remodeling. S phase progression is the Rb tumor suppressor protein,18 and we observed altered Rb phosphorylation in NOR1-deficient SMCs. Conversely, overexpression of NOR1 in SMCs induced Rb phosphorylation and downstream E2F-dependent promoter trans-activation. Among the cyclin/cyclin-dependent kinase complexes that phosphorylate Rb, mammalian D-type cyclins drive cells into the S phase and prevent apoptosis,13,19 and mitogen-induced cyclin D1 and D2 expression was completely abolished as a consequence of NOR1 deficiency. Furthermore, we demonstrate that cyclin D1 expression is decreased in neointimal SMCs of NOR1–/– mice, suggesting that the regulation of cyclin D1 by NOR1 also is applicable in vivo. Interestingly, Pei et al10 previously demonstrated that NOR1 overexpression in macrophages induces cyclin D2 mRNA. In addition, we have recently observed that cyclin D1 and D2 protein levels were decreased in NOR1-deficient cells.9 These studies, combined with our present data, indicate that cyclin D1 may constitute a key target gene responsible for the mitogenic activity of NOR1.
Conclusions
Top
Abstract
Introduction
Methods
Results
Discussion
Conclusions
References
Our results demonstrate that NOR1 induces cyclin D1 promoter activity, allowing subsequent gene transcription. NOR1-induced cyclin D1 expression results in the phosphorylation of Rb and subsequent G1S phase transition, an effect that will ultimately act mitogenically on SMCs. Finally, NOR1 deficiency limits the proliferative response underlying neointima formation by compromising cyclin D1 expression. These findings identify NOR1 as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that NOR1 might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.
Acknowledgments
Source of Funding
References
Top
Abstract
Introduction
Methods
Results
Discussion
Conclusions
References
1. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science. 2001; 294: 1866–1870.
Footnotes
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.822056/DC1.
Circulation 2009 119: 487-488.
L. Martorell, M. Gentile, J. Rius, C. Rodriguez, J. Crespo, L. Badimon, and J. Martinez-Gonzalez
The Hypoxia-Inducible Factor 1/NOR-1 Axis Regulates the Survival Response of Endothelial Cells to Hypoxia
Mol. Cell. Biol.,
November 1, 2009;
29(21):
5828 - 5842.
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