Myocardin Is Sufficient for a Smooth Muscle-Like Contractile Phenotype
Background— Myocardin (Myocd) is a strong coactivator that binds the serum response factor (SRF) transcription factor over CArG elements embedded within smooth muscle cell (SMC) and cardiac muscle cyto-contractile genes. Here, we sought to ascertain whether Myocd-mediated gene expression confers a structural and physiological cardiac or SMC phenotype.
Methods and Results— Adenoviral-mediated expression of Myocd in the BC3H1 cell line induces cardiac and SMC genes while suppressing both skeletal muscle markers and cell growth. Immunofluorescence microscopy shows that SRF and a SMC-like cyto-contractile apparatus are elevated with Myocd overexpression. A short hairpin RNA to Srf impairs BC3H1 cyto-architecture; however, cotransduction with Myocd results in complete restoration of the cyto-architecture. Electron microscopic studies demonstrate a SMC ultrastructural phenotype with no evidence for cardiac sarcomerogenesis. Biochemical and time-lapsed videomicroscopy assays reveal clear evidence for Myocd-induced SMC-like contraction.
Conclusion— Myocd is sufficient for the establishment of a SMC-like contractile phenotype.
Differentiated vascular smooth muscle cells (SMCs) have 2 important phenotypic characteristics. First, they replicate infrequently within the normal vessel wall.1 Second, they express a unique cyto-contractile gene program encoding a subproteome necessary for the principal function of these cells, namely contraction. After physical or chemical injury to the vessel wall however, vascular SMCs increase their replication rate and reduce the expression of many cyto-contractile genes including the smooth muscle isoforms of alpha actin, myosin heavy chain, and calponin.2 Such changes in SMC-restricted genes are thought to contribute to the pathogenesis of atherosclerosis, transplant arteriopathy, hypertension, bypass-graft failure, and the malignant phenotype.2,3⇓ Although the majority of vascular diseases correlate with reductions in SMC contractile proteins, at least one example exists in which increases in SMC contractile proteins are associated with a disorder.4 Elucidating the intrinsic and extrinsic cues that specify one vascular SMC phenotype over another has therefore been the subject of intense study, with myriad proteins and signal transduction pathways identified.2
See accompanying article on page 1416
The differentiated phenotype of a muscle cell is largely determined by the expression of both ubiquitous and cell-specific transcription factors (TFs). The latter are exemplified by members of the MyoD family of basic helix-loop-helix TF which can convert a variety of cells, including cultured SMCs, into skeletal muscle cells.5 Widely expressed TFs such as serum response factor (SRF) are critical for normal skeletal muscle, cardiomyocyte, and SMC differentiated phenotypes.6 Ubiquitous TFs such as SRF orchestrate specific programs of gene expression through combinatorial associations with coregulators, some of which display cell-specific patterns of expression.7 Among SRF coregulators, myocardin (Myocd) has emerged as one of central importance for the establishment of SMC identity. First cloned in a bioinformatic screen for unknown cardiac-specific genes, Myocd was shown initially to stimulate a battery of SRF target genes associated with cardiac muscle differentiation.8 A subsequent series of complementary reports demonstrated the pivotal role of Myocd in directing a genetic program of SMC differentiation.9–13⇓⇓⇓⇓ Levels of Myocd mRNA are reduced concurrently with SMC contractile genes on SMC phenotypic change in culture9 and in vivo after arterial injury.14,15⇓ These results and the growing number of studies showing Myocd induces SMC contractile gene expression in nonmuscle cell types suggest that, similar to MyoD in skeletal muscle, Myocd is a nodal point for the specification of SMC. Interestingly, Myocd can repress MyoD family members and redirect cells fated for skeletal muscle to adopt a SMC-like phenotype, suggesting Myocd is dominant over the MyoD program of skeletal muscle cell differentiation.16
Although a growing number of studies have documented the ability of Myocd to stimulate SMC-restricted gene expression, to what extent this program of gene expression recapitulates aspects of a structural and functional vascular SMC phenotype is unclear.17 Here, we show that despite the activation of both cardiac and SMC genes, Myocd confers an ultrastructural and contractile phenotype that most closely resembles that seen in SMCs; no evidence for structural or physiological signs of cardiac muscle differentiation are manifest. These data demonstrate that Myocd overexpression is sufficient for structural and functional attributes of mature SMC, implicating it as a potential master regulator for the SMC contractile phenotype.
Materials and Methods
An expanded Materials and Methods section is available in the online Data Supplement (available at http://atvb.ahajournals.org).
Adenoviral transductions were carried out as described4 using multiplicity of infections (moi) from 10 to 100.
Bright field, immunofluorescence, and electron microscopic analyses of Myocd versus control expressing BC3H1 cells were performed without knowledge of the experimental condition.
Contractile Competence Assays
Contractile competence was carried out in control and Myocd-transduced cells (BC3H1 and human airway SMC) using a standard cell shortening assay and a 3-dimensional collagen gel system.
Myocd Inhibits Cell Growth and Cyclin D1 Expression
A hallmark of mature, differentiated vascular SMCs is a low replication rate.1 To assess the effects of Myocd on BC3H1 cell proliferation, cells were transduced with either a control adenovirus or one carrying the short form of Myocd (amino acids 128 to 935). Overexpression of Myocd changes BC3H1 cell morphology from a polygonal to more spindle-like shape, reminiscent of mature SMCs (Figure 1A). Data in Figure 1A further demonstrate a reduction in cell density with Myocd overexpression. A temporal study of cell proliferation reveals decreases in Myocd-transduced cell number beginning 2 days after adenoviral transduction and persisting over 5 days (Figure 1B). Consistent with growth attenuation, Myocd transduction leads to lower level expression of cyclin D1 (Figure 1C). Transient promoter transfection studies suggest that Myocd directly represses cyclin D1 transcription (Figure 1D). These results suggest that Myocd promotes a more SMC-like morphology and suppresses growth in BC3H1 cells, in part, through direct repression of a key cell cycle-associated gene.
Reciprocal Changes in SMC Versus Skeletal Muscle Markers in BC3H1 Cells
BC3H1 cells can adopt a skeletal muscle fate on serum withdrawal.18 Consistent with this concept, BC3H1 cells induced to differentiate to skeletal muscle with low serum exhibit robust expression of myogenin (Myog) mRNA, which encodes for 1 of 4 myogenic regulatory factors that orchestrate skeletal muscle cell differentiation (Figure 2A, compare WT lanes). In contrast, endogenous Myocd mRNA levels are seen to diminish on BC3H1 cell differentiation as are levels of the Myocd target gene, SM calponin (Cnn1, Figure 2A, compare WT lanes). Interestingly, we observe a similar decrease in Srf mRNA with BC3H1 cell differentiation (Figure 2B). To determine whether the changes in mouse Cnn1 expression apply to the human ortholog as well, we generated 3 BC3H1 cell lines stably-expressing the human CNN1 gene harbored within a bacterial artificial chromosome19 (lanes labeled BAC in Figure 2A). As with endogenous mouse Cnn1, human CNN1 mRNA levels are higher in growing versus differentiated BC3H1 cells (Figure 2A), and immunocytochemistry confirms that similar changes apply to human CNN1 protein as well (Figure 2C). Taken together, results reveal reciprocal expression profiles between key SMC and skeletal muscle markers in cells that transition from a SMC- to skeletal muscle cell-like phenotype in vitro.
Disparate Effects of Myocd on the 3 Muscle Cell Programs of Differentiation
Myocd is a potent inducer of SMC and cardiac muscle genes that contain SRF-binding CArG elements.20 To determine the effects of Myocd on these muscle cell programs as well as skeletal myogenic regulatory factors, we transduced BC3H1 cells that had undergone initial differentiation to the skeletal muscle phenotype with control or Myocd adenovirus. Myocd induces endogenous SMC markers such as Acta2, Cnn1, Myh11, and Tagln (Figure 3A) as well as human CNN1 mRNA (data not shown). Similarly, several cardiac muscle transcripts are elevated on Myocd overexpression (Figure 3A). Consistent with a recent report in C2C12 myoblasts,16 skeletal myogenic regulatory factors are attenuated with Myocd expression in BC3H1 cells (Figure 3A). We see similar trends with respect to SMC and skeletal muscle proteins (Figure 3B). These results show that Myocd induces SMC and cardiac muscle target genes, but represses skeletal muscle markers of differentiation in the BC3H1 cell line.
Myocd Induces Structural Attributes of a Contractile SMC Phenotype
A structural hallmark of mature differentiated SMCs is the presence of a rich array of myofilaments. Immunofluorescence microscopy shows Myocd-mediated increases in cyto-contractile fibers and SRF expression in BC3H1 (Figure 4, panels a versus c). Further studies demonstrate that the increase in cyto-contractile fibers is attributable to elevations in SM α-actin filaments (supplemental Figure I, please see http://atvb.ahajournals.com). The cyto-contractile apparatus in BC3H1 cells is disrupted with a short hairpin RNA to Srf (Figure 4, panels a versus b). Cotransduction with Myocd results in complete restoration of the cyto-contractile phenotype (Figure 4, panels b versus d). Western blotting results indicate that cotransduction of Myocd and shSRF normalizes SRF levels (supplemental Figure II) though we cannot rule out an SRF-independent effect of Myocd21,22⇓ in rescuing the cyto-contractile phenotype. Importantly, staining with an antibody to cardiac alpha actinin in Myocd-transduced BC3H1 cells does not show the typical periodic staining indicative of cardiac sarcomeres (supplemental Figure II).
To probe the structure of Myocd-transduced cells deeper, we performed transmission electron microscopy. Mature SMCs of the adult mouse aorta show characteristic myofilaments throughout the cytosol, punctuated with focal densities (Figure 5A). Control-transduced BC3H1 cells show little, if any, indication of myofilament arrays (Figure 5B). Myocd-transduced BC3H1 cells, however, exhibit bands of myofilaments that appear similar to those in mature vascular SMC in vivo (Figure 5C and supplemental Figure III). In agreement with the cardiac alpha actinin staining, we found no evidence for cardiac myofilaments organized as repeating sarcomeres. A blind, quantitative analysis of more than 80 cells each from control- and Myocd-transduced cultures reveals, respectively, 2.3% and 58.8% cells exhibiting ultrastructural evidence of smooth myofilaments (Figure 5D). These results establish Myocd as a mediator of the SMC ultrastructure phenotype.
Myocd Induces SMC-Like Contractile Competence in 2 Distinct Cell Types
The preceding structural data suggest that Myocd facilitates bonafide SMC contractions in BC3H1 cells. To explore this novel concept, we first assessed expression of additional proteins necessary for SMC contractile competence. As shown in Figure 6A, Myocd dose-dependently increases the SM isoform of myosin light chain kinase (SM-MLCK), a known SRF target gene.23 We also see Myocd-induced increases in phosphorylated myosin light chain 20 (Figure 6B), an essential mediator of SMC contractile activity.24 No spontaneous contractions are seen in Myocd-transduced BC3H1 cells. However, time-lapsed videomicroscopy shows slow SMC-like contractions in Myocd-transduced BC3H1 cells stimulated with 75 mmol/L KCl (Figure 6C and supplemental Movies). Quantitative measures of percent cell shortening indicate that Myocd-transduced cells display >3-fold increases over control cells (Figure 6D). Human bronchial SMC transduced with Myocd also show a dramatic contractile response following histamine stimulation, suggesting Myocd evokes contractile competence in visceral SMC with inherent deficits in contractile activity in vitro (supplemental Figure IV). Taken in aggregate, the results of this report support the notion that Myocd is sufficient for a SMC-like contractile phenotype.
Designating a transcription factor a master regulator of differentiation implies an intrinsic ability of the factor to autoregulate its expression while eliciting biochemical and physiological attributes of the differentiated cell. MyoD was among the first master regulators of differentiation defined by virtue of its capacity to induce contractile proteins unique to skeletal muscle, organizing proteins into repeating sarcomeres, and eliciting contractile activity25 as well as auto-regulating its expression.26 Might we similarly consider Myocd a master regulator of SMC differentiation? Clearly, Myocd can activate SMC genes in a variety of cell contexts, and there is some evidence for Myocd auto-regulating its own expression.27 However, until now, nothing was known about the ability of Myocd to orchestrate both ultrastructural and physiological attributes of the SMC differentiated phenotype. Here, we show expected changes in SMC, cardiac muscle, and skeletal muscle marker expression on Myocd overexpression in BC3H1 cells. Despite the activation of some cardiac-restricted genes, Myocd stimulates smooth myofilaments with no evidence for cardiac sarcomerogenesis. Consistent with ultrastructure studies, Myocd provokes SMC-like contractions in 2 distinct cell types that otherwise are weakly responsive to contractile agonists. We conclude, therefore, that although Myocd may not stimulate every SMC-associated gene,28 the ability to autoregulate its own expression27 combined with the structural and functional data reported here, support the designation of Myocd as a master regulator of the SMC differentiated phenotype.
Despite a literature replete with studies showing Myocd-mediated SMC contractile gene/protein expression, little is known as to the ability of this cofactor to mediate contractile competence. Wamhoff and colleagues showed that voltage-gated calcium channel activation stimulated Myocd mRNA expression in rat SMCs.29 The same study used embryoid bodies to show spontaneous SMC-like contractions were dependent on voltage-gated channel activity. Husain and colleagues recently demonstrated defective Myocd expression and reduced SMC-like contractions in embryoid bodies null for the c-Myb transcription factor.30 It will be important to determine whether this model of SMC contractile competence is dependent on Myocd expression and whether Myocd is a direct target of c-Myb. Moreover, effects of Myocd modulation on SMC-restricted ion channel expression and activity should be assessed to gain further insight into the role of Myocd in mediating SMC contractile competence. While the present work was underway, parallel studies uncovered Myocd as a marker for Alzheimer angiopathy, and gain-of-function studies demonstrated increases in human cerebral SMC shortening.4 Thus, there may be a number of SMC-associated diseases where exaggerated Myocd expression influences disease progression (eg, Alzheimer disease, asthma, intestinal pseudo-obstruction).
It is important to point out that because Myocd levels are low in cultured SMCs where contractile competence is rarely seen and most SMC markers are severely downregulated, we were unable to address effects of loss of Myocd on SMC ultrastructure and contractile activity. However, ultrastructural analysis of the mouse ductus arteriosis where Myocd is conditionally ablated reveals loss in SMC myofilaments and an abundance of rough endoplasmic reticulum indicative of a SMC synthetic phenotype.31 These results are congruent with a Myocd pan-knockout study where aortic SM alpha actin expression was essentially absent in day 10.5 embryos.13 In both cases, loss in SMC Myocd is apparently uncompensated for by the myocardin-related transcription factors.32
The results of this study and others clearly show that cardiac and SMC genes are coactivated with ectopic Myocd expression. Indeed, we have yet to find a cell line that is not responsive to Myocd in terms of SMC gene activation (see supplemental Figure V for examples). The initial report of Myocd8 proposed it playing a critical role in cardiac muscle differentiation. Subsequent studies in Xenopus showed ectopic Myocd to activate cardiac muscle (and SMC) genes.33 Importantly, the latter article as well as a more recent study where Myocd was ectopically expressed in human cardiac fibroblasts34 found no evidence for striated structures or spontaneous contractions that would support a structural and functional cardiac state. Similarly, we have never seen any evidence for cardiac sarcomerogenesis in cells overexpressing Myocd. The absence of cardiac sarcomerogenesis with Myocd overexpression may not be surprising given the highly organized construction of the sarcomere involving dozens of proteins.35 Nevertheless, there is some evidence for Myocd-mediated cardiac channel induction and the restoration of cardiac electric conduction in vitro,34 suggesting some contexts exist where the milieu and signaling input facilitates the coexistence of both cardiac and SMC markers of differentiation. Indeed, a number of SMC restricted genes are known to be expressed in early cardiac muscle,36 and some of these are redeployed during pathological remodeling of cardiac muscle where Myocd levels are also elevated.37,38⇓
One of the hallmarks of a mature, differentiated SMC phenotype is growth cessation. We first reported that Myocd could reduce cell growth9; however, the mechanisms underlying such growth attenuation were not addressed. Here, we provide evidence for Myocd directly repressing cyclin D1 promoter activity. Downregulated Myocd and SMC contractile markers have been associated with human malignant transformation.3,39⇓ Thus, it is tempting to speculate that in addition to activating genes involved in SMC contraction, Myocd represses cell cycle activity thereby contributing to the quiescent phenotype of differentiated SMC in the vessel wall and perhaps other cell types as well. In this context, Myocd represses gene expression in skeletal muscle,16 and a more recent study40 demonstrated Myocd-mediated inhibition of NF-κB/p65 transcriptional activity and cell cycle protein levels in SMCs. Myocd therefore appears to be a multi-functional protein with transcriptional coactivator and repressor activities.
In summary, we demonstrate that whereas ectopic Myocd expression induces both cardiac and SMC genes, structural and physiological data support its role in directing a SMC-like contractile phenotype. In no instance have we ever observed evidence for the manifestation of cardiac muscle structure or function. The results of the present study therefore support the designation of Myocd as a master regulator of SMC differentiation. The fact that Myocd levels decrease in parallel with contractile genes both in vitro and in vivo after vascular injury implies that the nearly 40-year-old phenomenon of SMC phenotypic modulation41 likely stems from defective Myocd expression. A critical goal for future research will be elucidating the transcriptional regulation of Myocd under normal and pathological conditions.
We thank Dr Burns C. Blaxall for the generous gift of neonatal cardiomyocytes.
Sources of Funding
This work was supported by grants from the National Institutes of Health (HL62572 and AG026950 to J.M.M.; AG023084 to B.V.Z.; and HL077726 to W.T.G.) and the American Heart Association (0625938T to X.L.).
Original received March 1, 2008; final version accepted April 17, 2008.
- ↵Spaet TH, Lejnieks I. Mitotic activity of rabbit blood vessels. Proc Soc Exp Biol Med. 1967; 125: 1197–1201.
- ↵Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84: 767–801.
- ↵Chow N, Bell RD, Deane R, Streb JW, Chen J, Brooks A, Van Nostrand W, Miano JM, Zlokovic BV. Serum response factor and myocardin mediate arterial hypercontractility and cerebral blood flow dysregulation in Alzheimer’s phenotype. Proc Natl Acad Sci USA. 2007; 104: 823–828.
- ↵Weintraub H, Tapscott SJ, Davis RL, Thayer MJ, Adam MA, Lassar AB, Miller AD. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci USA. 1989; 86: 5434–5438.
- ↵Miano JM, Long X, Fujiwara K. Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus. Am J Physiol Cell Physiol. 2007; 292: C70–C81.
- ↵Du K, Ip HS, Li J, Chen M, Dandre F, Yu W, Lu MM, Owens GK, Parmacek MS. Myocardin is a critical serum response factor cofactor in the transcriptional program regulating smooth muscle cell differentiation. Mol Cell Biol. 2003; 23: 2425–2437.
- ↵Yoshida T, Sinha S, Dandre F, Wamhoff BR, Hoofnagle MH, Kremer BE, Wang D-Z, Olson EN, Owens GK. Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes. Circ Res. 2003; 92: 856–864.
- ↵Wang Z, Wang D-Z, Pipes GCT, Olson EN. Myocardin is a master regulator of smooth muscle gene expression. Proc Natl Acad Sci USA. 2003; 100: 7129–7134.
- ↵Li S, Wang D-Z, Richardson JA, Olson EN. The serum response factor coactivator myocardin is required for vascular smooth muscle development. Proc Natl Acad Sci USA. 2003; 100: 9366–9370.
- ↵Tharp DL, Wamhoff BR, Turk JR, Bowles DK. Upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle. Am J Physiol Heart Circ Physiol. 2006; 291: H2493–H2503.
- ↵Long X, Creemers EE, Wang D-Z, Olson EN, Miano JM. Myocardin is a bifunctional switch for smooth versus skeletal muscle differentiation. Proc Natl Acad Sci USA. 2007; 104: 16570–16575.
- ↵Miano JM. Channeling to myocardin. Circ Res. 2004; 95: 340–342.
- ↵Olson EN, Caldwell KL, Gordon JI, Glaser L. Regulation of creatine phosphokinase expression during differentiation of BC3H1 cells. J Biol Chem. 1983; 258: 2644–2652.
- ↵Miano JM, Kitchen CM, Chen J, Maltby KM, Kelly LA, Weiler H, Krahe R, Ashworth LK, Garcia E. Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome. Am J Physiol Heart Circ Physiol. 2002; 282: H1793–H1803.
- ↵Pipes GCT, Creemers EE, Olson EN. The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes Dev. 2006; 20: 1545–1556.
- ↵Qiu P, Ritchie RP, Fu Z, Cao D, Cumming J, Miano JM, Wang D-Z, Li HJ, Li L. Myocardin enhances Smad3-mediated transforming growth factor-β1 signaling in a CArG box-independent manner: Smad-binding element is an important cis element for SM22α transcription in vivo. Circ Res. 2005; 97: 983–991.
- ↵Yin F, Hoggatt AM, Zhou J, Herring BP. The 130kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse MYLK gene. Am J Physiol Cell Physiol. 2006; 290: C1599–C1609.
- ↵Lattanzi L, Salvatori G, Coletta M, Sonnino C, Cusella De Angelis MG, Gioglio L, Murry CE, Kelly R, Ferrari G, Molinaro M, Crescenzi M, Mavilio F, Cossu G. High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies. J Clin Invest. 1998; 101: 2119–2128.
- ↵Creemers EE, Sutherland LB, McNally J, Richardson JA, Olson EN. Myocardin is a direct transcriptional target of Mef2, Tead and Foxo proteins during cardiovascular development. Development. 2006; 133: 4245–4256.
- ↵Yoshida T, Kawai-Kowase K, Owens GK. Forced expression of myocardin is not sufficient for induction of smooth muscle differentiation in multipotential cells. Arterioscler Thromb Vasc Biol. 2004; 24: 1596–1601.
- ↵Wamhoff BR, Bowles DK, McDonald OG, Sinha S, Somlyo AP, Somlyo AV, Owens GK. L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a Rho kinase/myocardin/SRF-dependent mechanism. Circ Res. 2004; 95: 406–414.
- ↵Kolodziejska KM, Ashraf HN, Nagy A, Bacon A, Frampton J, Xin H-B, Kotlikoff MI, Husain M. c-Myb-dependent smooth muscle cell differentiation. Circ Res. 2008; 102: 554–561.
- ↵Wang DZ, Li S, Hockemeyer D, Sutherland L, Wang Z, Schratt G, Richardson JA, Nordheim A, Olson EN. Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proc Natl Acad Sci USA. 2002; 99: 14855–14860.
- ↵Small EM, Warkman AS, Wang D-Z, Sutherland LB, Olson EN, Krieg PA. Myocardin is sufficient and necessary for cardiac gene expression in Xenopus. Development. 2005; 132: 987–997.
- ↵van Tuyn J, Pijnappels DA, de Vries AAF, de Vries I, van der Velde-van Dijke I, Knaan-Shanzer S, van der Laarse A, Schalij MJ, Atsma DE. Fibroblasts from human postmyocardial infarction scars acquire properties of cardiomyocytes after transduction with a recombinant myocardin gene. FASEB J. 2007; 21: 3369–3379.
- ↵Hoshijima M. Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures. Am J Physiol Heart Circ Physiol. 2006; 290: H1313–H1325.
- ↵Milyavsky M, Shats I, Cholostoy A, Brosh R, Buganim Y, Weisz L, Kogan I, Cohen M, Shatz M, Madar S, Kalo E, Goldfinger N, Yuan J, Ron S, Mackenzie K, Eden A, Rotter V. Inactivation of myocardin and p16 during malignant transformation contributes to a differentiation defect. Cancer Cell. 2007; 11: 133–146.
- ↵Tang R, Zheng X-L, Callis TE, Stansfield WE, He J, Baldwin AS, Wang D-Z, Selzman CH. Myocardin inhibits cellular proliferation by inhibiting NF-κB(p65)-dependent cell cycle progression. Proc Natl Acad Sci USA. 2008; 105: 3362–3367.