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Myocardin and smooth muscle differentiation
Archives of Biochemistry and Biophysics 543 (2014) 48–56
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Invited Review
Myocardin and smooth muscle differentiation Xi-Long Zheng ⇑ Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, Libin Cardiovascular Institute of Alberta, Faculty of Medicine, University of Calgary, Alberta, Canada
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Article history: Received 15 October 2013 and in revised form 15 December 2013 Available online 25 December 2013 Keywords: Myocardin Smooth muscle differentiation Smooth muscle cell proliferation
a b s t r a c t Myocardin (MYOCD), a co-transcriptional activator of serum response factor (SRF), stimulates the expression of smooth muscle (SM) genes and inhibits the cell cycle. In addition to its roles in the development, MYOCD may be critically involved in the pathogenesis of proliferative vascular diseases. This review mainly focuses on how MYOCD activity is regulated and how it inhibits cell proliferation. Ó 2013 Elsevier Inc. All rights reserved.
Introduction SM contractility is critical to maintaining the physiological functions of the internal organs, such as the blood vessel wall, gastrointestinal tract, airway, uterus and bladder. Notably, deregulation of vascular contractility, for example, contributes to the pathogenesis of hypertension. In addition, abnormal proliferation of SMCs is involved in neointima formation in arteries, uterine leiomyoma (a benign tumor within the uterus), renal leiomyolipoma in tuberous sclerosis complex (TSC)1 patients, and lung lymphangioleiomyomatosis (LAM) in female patients. The methodology for the culture of smooth muscle cells (SMCs) was established about 30 years ago, but it remained unknown as to why SMCs could be cultured and what controlled SM differentiation until MYOCD was discovered in 2001. In order to proliferate, SMCs are believed to undergo phenotypic conversion from contractile differentiation to synthetic dedifferentiation status. This phenotypic conversion model has been ⇑ Corresponding author. Address: 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada. Fax: +1 (403) 270 0737. E-mail address: [email protected] URL: http://webapps2.ucalgary.ca/~zhenglab/index.html 1 Abbreviations used: TSC, tuberous sclerosis complex; LAM, lymphangioleiomyomatosis; SMCs, smooth muscle cells; MRTF-A, MYOCD-related transcription factorA; TADs, transcription activation domains; Foxo, forkhead transcription factor; CRM, chromosomal region maintenance protein; PECAM-1, platelet endothelial cell adhesion molecule 1; MHC, myosin heavy chain; MLCK, myosin light chain kinase; SBE, Smad-binding element; HRT, hairy-related transcription factor; uPAR, urokinase-type plasminogen activator receptor; TDG, thymine DNA glycosylase; KLF, Kruppel-like transcription factor; ER, estrogen receptor; SRC3; steroid receptor coactivator 3; STRAP, SRF-dependent transcription regulation-associated protein; Cdc7, cell division cycle 7; Ang II, Angiotensin II; IGF, insulin-like growth factor; NKE, Nkx2.5 responsive element; HSC, hepatic stellate cells; ATV, atorvastatin; CBP, CREB-binding protein; CHIP, C-terminus of Hsc70-interacting protein; ERa, estrogen receptor a; TFs, transcription factors; PDGF, platelet-derived growth factor. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.12.015
challenged by a recent study showing proliferating SMCs in culture resulting from vascular stem cells, instead of dedifferentiated SMCs [1]. Nevertheless, the critical roles of MYOCD have well been established in SMC differentiation. This review will focus on the following questions: how is MYOCD activity regulated, and how does MYOCD regulate differentiation and the cell cycle of SMCs? MYOCD and its expression MYOCD (935 aa) was discovered in a research screening for novel cardiac-specific genes in silico through a BLAST search using ESTs from mouse embryonic heart cDNA libraries in the database [2]. Two other family members, MYOCD-related transcription factor-A (MRTF-A, 929 aa, also called MAL, MKL-1, and BSAC), and MRTF-B (1080 aa, also called MKL-2), were identified through searching the NCBI databases using the mouse Myocd cDNA sequence [3]. None of the members bind to DNA, but rather they initiate SRF-dependent gene transcription [2,3]. MYOCD forms a stable ternary complex with SRF and is defined as a co-transcriptional activator [2]. SRF binds to CArG [CC(A/T)6GG] boxes and transactivates transcription of many genes, including SM-specific ones (Fig. 1). MYOCD does not have transcriptional activity in Srf/ cells [3]. Notably, MYOCD may exert its effects independently of SRF resulting from its binding to other signaling proteins. MYOCD is expressed in the heart and in most developing and adult SMC compartments including the dorsal aorta, bladder, stomach, intestine, and uterus [3,4]. Evidence suggests that MYOCD may be expressed in other cells or tissues. For example, lineage tracing studies have shown that MYOCD is transiently expressed in skeletal muscle during development and plays a suppressor role in the skeletal muscle differentiation program [5]. In endothelial cells,
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Fig. 1. Myocardin (MYOCD) stimulation of gene expression through serum response factor (SRF). MYOCD activity is indicated by the expression levels of MYOCD target genes, such as SM a-actin and SM22.
MYOCD mediates hypoxia-induced transdifferentiation into SMlike cells [6]. In peripheral blood mononuclear cells, thrombin stimulates MYOCD and SM myosin heavy chain expression [7]. In addition, distinct MYOCD splice variants have been reported [8,9], which may confer differential tissue expression and functions. In contrast, MRTF-A and -B are expressed in a broad range of embryonic and adult tissues, including ES cells [10].
The features of domain structures First, there has been no DNA binding domain revealed in any member of the MYOCD family. Their C-terminal regions contain transcription activation domains (TADs, Fig. 2), which are required for transcriptional activity of MRTFs, but have no target gene specificity [3]. Deletion of this region results in a dominant-negative mutant. Second, their interaction with SRF is through a short peptide sequence containing a basic and glutamine-rich region (Fig. 2) [2,3]. The binding region of Smad1 includes a basic domain and a stretch of glutamine residues [11]. The forkhead transcription factor (Foxo) 4 (amino acid residues 89–325) directly interacts with the 129–510 aa fragment of myocardin [12]. GATA4 directly binds two regions (326–438 and 439–713 aa) of myocardin [13]. In addition, several other domain structures have been identified in the MYOCD family. (1) RPEL motifs: Two are present in the MYOCD N-terminal region (Fig. 2). Three are in MRTF-A/B, which mediate their association with G-actin [14]. (2) SAP domain (SAF-A/B, Acinus, and PIAS): It contains 35 amino acids (residues 380–414) and may participate in chromosomal dynamics, nuclear breakdown, and apoptotic DNA fragmentation as described for other SAP-containing proteins [15]. (3) Coiled-coil motif: It resembles a leucine zipper and mediates homo- and heterodimerization of MYOCD members, which stabilizes their binding with SRF on CArG boxes of target genes (Fig. 2) [2,3,10,14].
Subcellular localization MYOCD is in the nucleus, but MRTF-A/B are in the cytosol. This is possibly because MRTF-A/B have three RPEL motifs, but there are only two in MYOCD. The three RPEL motifs can mediate MRTF-A/B binding to cytosolic G-actin [16], but the two RPEL domains in MYOCD have relatively weak binding to actin [16]. It is also possibly due to: (1) the higher affinity of MYOCD to importin/1 than MRTF-A/B, and (2) G-actin inhibition of importin/1 binding with MRTF-A/B [17]. In addition to importin, recent studies have shown that difference in subcellular localization is also determined by their differential interaction with chromosomal region maintenance protein (CRM)1, SRF, and G-actin [18]. Differential localization may be explained by MYOCD’s weak binding to CRM1, but strong binding to SRF, which are opposite to MRTF-A [18]. Notably, the subcellular localization of all members may change in response to various stresses. Hyperosmotic stress, for example, was reported to regulate the distribution and stability of MRTF in kidney tubular cells [19]. The phenotypes of knockout mice Myocd knockout Mice with a homozygous null mutation of Myocd died at embryonic day 10.5 (E10.5) [20]. This study showed no staining for either SM a-actin or SM22 in the dorsal aortae in transverse section of Myocd/ embryos, suggesting the lack of SMC differentiation. However, the staining for PECAM-1 (Platelet Endothelial Cell Adhesion Molecule 1), an endothelial marker, at E8.5 suggested a normal vascular patterning in mutant embryos [20]. It is unclear what causes the embryonic death, but the lethality may result from abnormal vasculature of the yolk sac and the pericardial effusion observed by E10.5 [20]. Selective knockout of Myocd in neural crest-derived SMCs results in the lack of the contractile phenotype
Fig. 2. Domain structure of myocardin (MYOCD). The functions of the various domains have been described in the text. SAP: SAF-A/B, Acinus, and PIAS; TAD: transactivation domain; SRF: serum response factor; ++: basic domain.
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of SMCs and patent ductus arteriosus, in which SMCs demonstrate the synthetic phenotype [21]. However, Myocd-null SMCs can still differentiate and also be incorporated into vascular tissues with normal morphology [22]. In chimeric knockout mice, it was reported that MYOCD is dispensable for development of vascular, but not visceral, SMCs [23]. Nevertheless, it remains interesting to address whether the death of Myocd-null embryos results from loss of MYOCD in vascular SMCs or in other cells. Phenotype of MRTF-A/B knockout mice Knockout of MRTF-A is not lethal, but female mice are not able to release milk during lactation [24]. However, knockout of MRTFB is lethal as demonstrated by several independent laboratories. First, homozygous MRTF-B gene-trap mice die between E17.5 and postnatal day 1 due to a defect in the cardiac outflow tract. Although migration and early patterning of cardiac neural crest cells appear normal, there is a significant reduction of SMC differentiation in the arch arteries and the aorticopulmonary septum at E11.5 [25]. Other studies show that mice die from several cardiovascular defects including abnormal patterning of the branchial arch arteries, double-outlet right ventricle, ventricular septal defects, and thin-walled myocardium [26]. A failure in SMC differentiation within the branchial arch arteries is also observed [26]. In addition, other defects in this knockout may contribute to the lethality. It has been reported that mice containing an insertional mutation of MRTF-B not only have vascular defects, but also liver hemorrhage. These mice usually die in late gestation with defective expression of SM genes in the liver sinusoids, vitelline veins, and yolk sac [27]. Mechanisms underlying the cellular effects of MYOCD First, MYOCD induces SRF-dependent gene expression. Many genes contain one or multiple CArG boxes, which is specific for SRF binding, in their promoters (Fig. 1) [28,29]. However, the expression of only a portion of these SRF-responsive genes are driven by MYOCD. The potential mechanism(s) underlying MYOCD specificity in gene transcription is discussed in the next section. Most SM genes contain CArG boxes and are stimulated by MYOCD, including SM a-actin (ACTA2), SM c-actin, SM myosin heavy chain (MHC), calponin, SM22, h-caldesmon and SM myosin light chain kinase (MLCK) [4,30–33]. Transforming growth factor-beta1 (TGFb1)-induced transcript 1 (TGFB1I1, also known as Hic-5), which was recently recognized as a novel marker for the SM contractile phenotype, is also regulated by SRF/MYOCD [34]. Expression of smoothelin-A, but not -B, is controlled by SRF/MYOCD [35]. Notably, MYOCD also induces the expression of SM genes in a variety of non-muscle cell types [31,32], such as peripheral blood mononuclear cells [7]. Besides SM-related genes, MYOCD stimulates the expression of many other genes through the SRF-CArG box mechanism, such as p21, a cell cycle inhibitory protein [36], cysteine-rich protein 2, a SRF co-activator [37], large conductance calcium-activated potassium (MaxiK) channel subunit b, a SM-restricted ion channel [38], and lipoma preferred partner, a nucleocytoplasmic shuttling adaptor protein [39]. In addition to proteins, MYOCD induces the expression of microRNAs, such as miRNA1 [40,41] and miRNA143/145 [42–45]. Second, MYOCD also has SRF-independent effects through the following mechanisms. (1) MEF2A: MYOCD binds to the MEF2A transcription factor, and potently stimulates MEF2-dependent gene expression. It binds MEF2 at the MADS-box/MEF2 domain through a short amphipathic helix [46,47]. (2) NF-jB: MYOCD
directly interacts with p65 and decreases p65-mediated target gene activation by interfering with p65 DNA binding [48]. (3) Smad3: MYOCD interacts with Smad3, which binds to the Smadbinding element (SBE) through the C-terminal TADs to activate SM gene expression through a CArG box-independent manner [49]. Specificity of MYOCD induction of gene expression Several laboratories have been addressing why MYOCD specifically induces the expression of SM genes, instead of all CArGcontaining genes. To date, the published studies suggest a complexity of the underlying mechanism with several possibilities proposed. (1) Uniqueness of CArG boxes: The evidence comes from the studies of the human ACTG2 promoter, in which there are four conserved CArG boxes. Substitution of CArG2 with other CArG sequences results in loss of promoter inducibility by MYOCD [33]. (2) Non-CArG sequence: Evidence suggests the sequences adjacent to the CArG boxes are critical in determining MYOCD specificity. For example, mutation of an adjacent binding site for NKX3.1 results in reduction of MYOCD-dependent transactivation of the ACTG2 promoter [33]. Supportively, MYOCD stimulates the expression of telokin, which has one CArG box, but not c-fos. In the c-fos promoter, the ets binding site, together with elements 30 of the CArG box, inhibits transactivation by MYOCD [50]. (3) Chromatin structure: MYOCD recruits chromatin-remodeling enzymes to SRF target genes with involvement of the p300 histone acetyltransferase [51]. SRF/MYOCD complexes are associated with a specific variant of histone H3 on SMC gene loci in vivo [52]. (4) Other determinants: SWI/SNF complexes are required for MYOCD to increase SRF binding to CArG boxes in MYOCD target genes [53]. Recruitment of Jmjd1a, the histone 3 lysine 9 (H3K9)-specific demethylase, is required for MYOCD-directed gene transcription [54]. Regulation of MYOCD activity Protein–protein interaction regulates MYOCD activity First, interaction of MYOCD with other proteins inhibits MYOCD transcriptional activity, because of the reduction of MYOCD availability for SRF. For example, interaction of Foxo4 with MYOCD prevents MYOCD transcriptional activation of SM genes. However, when Foxo4 is phosphorylated through the Akt pathway, it translocates to the cytoplasm, leading to the release of MYOCD [12]. SOX9 also interacts with MYOCD and suppresses its transcriptional activity [55]. In addition, the hairy-related transcription factor (HRT)-2, which is expressed in developing vasculature [56], associates with MYOCD and represses its activity through a mechanism as yet to be defined [57]. In addition, the urokinase-type plasminogen activator receptor (uPAR) can interact with MYOCD in the nucleus, resulting in displacement of MYOCD from SRF and the reduction of MYOCD-induced gene expression [58]. Second, proteins may compete with MYOCD for the binding site on SRF so as to reduce MYOCD activity. These proteins include Runx2, a key osteogenic transcription factor [59], thymine DNA glycosylase (TDG) [60], HERP1 [61] and GATA factors [13,62]. Additionally, some proteins bind to the MYOCD–SRF complex and reduce its binding to DNA, including Msx transcription factors (Msx1 and Msx2) [63], Yap1 [64], and the Kruppel-like transcription factor (KLF) 4 [52,65]. Notably, the actin cytoskeleton can also regulate the activity of MRTFs [66,67]. MRTFs directly bind actin
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monomers and are sequestered in the cytoplasm. Actin polymerization results in MRTF translocation to the nucleus, where it coactivates high levels of CArG-dependent gene transcription [14,68]. Finally, MYOCD activity can be enhanced by interaction with other proteins. For example, the N terminus of estrogen receptor (ER) coactivator steroid receptor coactivator 3 (SRC3) contains a basic helix-loop-helix/Per-ARNT-Sim protein–protein interaction domain. Through this domain, SRC3 interacts with MYOCD TAD to enhance its transcriptional activity [69]. Therefore, SRC3 is defined as a coactivator for MYOCD. In addition, GATA4, which does not necessarily bind to DNA, directly interacts with MYOCD to stimulate MYOCD transcriptional activity in some target genes [13]. Regulation of MyoCD gene expression directly relates to its activity Stimulation of MyoCD gene expression First, an enhancer has been identified to control Myocd gene expression, which requires Mef2, Foxo and TEAD transcription factors for its activity [70]. Interestingly, MYOCD was found to regulate its own enhancer through an SRF-independent mechanism [70]. However, other studies have suggested that SRF may stimulate the expression of Myocd gene since loss of SRF results in decrease of Myocd mRNA [71,72] and MYOCD promoter contains a CArG binding site [73]. In addition to MYOCD itself, other SRF-cofactor, such as Nkx2.5 and p49/STRAP (SRF-dependent transcription regulation-associated protein), may be involved in SRF-induced MYOCD gene expression [74]. Second, MYOCD gene expression is stimulated by the Rho-ROCK (Rho-associated kinase) pathway as demonstrated in cultured SMCs derived from human aorta, porcine coronary and mouse aorta [75–77]. The RhoA inhibitor Y27632 inhibits thrombin-induced MYOCD gene expression in peripheral blood mononuclear cells [7]. Several other mechanisms have also been proposed to stimulate MYOCD gene expression. (1) TGFb1: a recent study has revealed that TGFb1 induces the mRNA expression and promoter activity of MYOCD through cell division cycle 7 (Cdc7), a non-transcription factor, and its interaction with Nkx2.5 [78]. (2) Angiotensin II (Ang II): Ang II is a well-known inducer of SMC hypertrophy. It also increases MYOCD expression through up-regulation of Prx1, a homeodomain protein [79]. (3) Hypoxia: MYOCD expression is increased by hypoxia in cardiomyocytes through the induction of Ang II and activation of the ERK pathway [80]. In contrast, in rat pulmonary arterial SMCs, hypoxia reduces MYOCD gene expression through up-regulation of PDGF-BB [81] or inhibition of cGMP-dependent protein kinase [82]. (4) Insulin-like growth factor (IGF)-1: IGF-1 stimulates the activity and expression of MYOCD through Foxo4 [12]. It was reported that homeobox protein Nkx2.5 binding to a Nkx2.5 responsive element (NKE) within the MYOCD promoter promotes MYOCD gene expression [83]. Notably, MYOCD is up-regulated in non-muscle cells, such as rat primary trans-differentiated hepatic stellate cells (HSC) [84], suggesting non-vascular pathophysiological roles of MYOCD. Inhibition of MYOCD gene expression MYOCD gene expression is inhibited by overexpression of Smad3, but not Smad2, due to its interaction with Nkx2.5 and subsequent inhibition of MYOCD gene promoter [85]. Resveratrol suppresses MYOCD gene expression through induction of p53 [86]. In addition, methylation of the MYOCD gene prevents its expression in nasopharyngeal carcinoma cell lines [87]. Interestingly, recent studies have revealed that atorvastatin (ATV), the most widely prescribed lipid-lowering drug [88],
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inhibits MYOCD gene expression in cultured SMCs through inhibition of the Rho-ROCK pathway [77]. Injection of ATV in mice significantly downregulates MYOCD expression, contractile proteins and contractility in mouse carotid arteries and aorta [77]. In addition, ATV was reported to inhibit hypoxia-induced MYOCD expression in cardiomyocytes [89] and attenuate the expression of MYOCD and ACTA2 in human fetal penile SMCs [90]. Therefore, inhibition of MYOCD gene expression may be a therapeutic approach for cardiovascular disease. Post-translation modifications and MYOCD activity MYOCD activity is also regulated by post-translational modifications, such as phosphorylation, acetylation, sumoylation and ubiquitination. First, MAPK p44/42 phosphorylates MYOCD at four sites (Ser812, Ser859, Ser866, and Thr893) in its TAD and increases its transcriptional activity. Mutation of all four sites with aspartate (4xD), but not alanine (4xA), reduces its transcriptional activity, likely through its reduced interaction with the cAMP response element binding protein (CREB)-binding protein (CBP) acetyltransferase [91]. GSK-3b phosphorylates MYOCD, but inhibits its transcriptional activity in cardiomyocytes [92]. How GSK-3b phosphorylation regulates MYOCD activity in vascular SMCs remains to be investigated. Second, MYOCD is acetylated, which is required for its transcriptional activity. Acetylation increases the association of MYOCD and SRF. Lysine residues locate at the N terminus are acetylated by chromatin-modifying histone acetylase p300 [93]. Third, SUMO-1 modification of a lysine residue at position 445 of MYOCD increases its transcriptional activity. E3 SMO-protein ligase 3, a protein inhibitor of activated STAT (PIAS)1 interacts with MYOCD, and controls MYOCD activity through its E3 ligase activity, which supposedly stimulates MYOCD sumoylation on an atypical sumoylation site(s) [94]. Finally, MYOCD protein undergoes ubiquitination-degradation, resulting in either decreased or increased transcriptional activities, which will be detailed in the next section. MYOCD is regulated by ubiquitination and proteasomal degradation Inhibition of MYOCD activity MYOCD, when phosphorylated by GSK-3b, can be ubiquitinated by C-terminus of Hsc70-interacting protein (CHIP), a cytosolic E3 ligase, resulting in proteasomal degradation and reduction of MYOCD transcriptional activity (Fig. 3) [95]. Note that CHIP-mediated ubiquitination-degradation is mainly responsible for protein quality control. Polyubiquitinated proteins are rapidly destroyed by the 26S proteasome, a large complex with multiple proteolytic activities [96,97]. The ubiquitin proteasome system (UPS) accounts for the degradation of 80–90% of all intracellular proteins [96,97]. A few studies suggest that regulation of MYOCD activity is through a decrease or increase of MYOCD degradation. Four and a half LIM domain protein 2 (FHL2) interacts with MYOCD and stimulates its transcriptional activity. This protein–protein interaction reduces proteasome-mediated degradation of MYOCD [98]. In addition, UBR5 (ubiquitin protein ligase E3 component n-recognin 5) binds to MYOCD and stimulates its transcriptional activity through reduction of MYOCD degradation, but independent of the E3 ligase activity of UBR5 [99]. Stimulation of MYOCD activity Evidence also indicates that inhibition of MYOCD degradation blocks MYOCD activity [100]. First, treatment with proteasomal inhibitors, such as MG132, results in a significant increase in MYOCD
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Fig. 3. Regulation of myocardin (MYOCD) by the ubiquitin proteasomal degradation system. CHIP: C-terminus of Hsc70-interacting protein with a U-box domain, an E3 ubiquitin-ligase.
protein [100]. Paradoxically, increased MYOCD due to decreased degradation is accompanied by decreased expression of SM target genes, including ACTA2, microRNA (miRNA)1 and 143/145, suggesting that accumulated MYOCD does not have transcriptional activity. This has also been supported by an earlier study showing that proteasome inhibitors decreased SRF-dependent expression of SM a-actin [101]. Second, a chromatin immunoprecipitation assay revealed that proteasome inhibition increases the occupancy of MYOCD and SRF on the acta2 gene promoter, but abolishes MYOCD-dependent recruitment of RNA polymerase II and its serine5-phosphorylated species (p-Pol II) [100]. Proteasome inhibition did not prevent Pol II recruitment in non-MYOCD target genes. This degradation-induced transactivation model is also supported by the following in vivo studies: (1) Ubiquitin, E2, E3 and proteasome subunits are downregulated in anastomotic intimal hyperplasia in a canine carotid model [102], and (2) MYOCD accumulation occurs in balloon injury-induced neointima of rat aorta [61] and neointimal lesion of mouse carotid arteries in response to ligation with a reduced expression of MyoCD gene [100]. Given that SMCs are de-differentiated and synthetic in neointimal lesions, these findings suggest that accumulation of MYOCD due to inhibition of proteasomal degradation results in inhibition of MYOCD activity. Notably, several other studies have also shown a reduced expression of MyoCD gene in proliferative vascular lesions, such as those in mouse carotid arteries in response to ligation [41,103,104] or wire injury [105] and in femoral artery via wire injury [106,107], but downregulation of MYOCD protein was also reported in neointimal lesions in femoral arteries [107]. Nevertheless, This speculation has been validated by a recent finding showing that stabilization of MYOCD protein in neointimal lesions is accompanied by downregulation of transcripts for ubiquitin and proteasome subunits [100]. In the SMC heterogeneity model, MYOCD in more differentiated spindle-shaped SMCs is more quickly degraded and has a shorter half-life than in epithelioid SMCs [100]. Taken together, these evidence suggests that MYOCD with increased stability due to reduced degradation is likely inactive and unable to transactivate its target genes. This proposed model may also explain how dysfunction of the UPS is involved in the initiation, progression and complications of atherosclerosis [108].
‘‘Licensing’’ model Above features described for MYOCD perfectly fit the ‘‘Licensing’’ model described for many other transcription factors (Fig. 4), such as Gcn4 [109], Gal4 [109,110] and Ino2/4 [109] in yeast and estrogen receptor a (ERa) [111–113], progesterone receptor [114], retinoic acid receptor [115], p53 [116] and c-myc [117] in mammalian cells. Their transcriptional activities require them to be degraded via the proteasome. Additionally, their transactivation potencies are positively correlated with the rate of proteasomal degradation [111,118–120]. Inhibition of their degradation through either inhibition of 26S proteasome activity by MG132, a specific, potent, reversible, and cell-permeable proteasome inhibitor, or downregulation of E3 ligases abolishes their transcriptional activities [109,112,117,121]. Deshaies and Lipford proposed a ‘‘Licensing’’ model to explain this Degradation-Activation mechanism (Fig. 4) [122], which was further developed by the Tansey et al. [110,117,121] and Cannon et al. [112]. Essentially, transcription factors (TFs) recruit not only the core components of the transcriptional machinery, but also E3 ligase(s) and proteasomes to the promoter. Upon initiation, TFs undergo ubiquitin-proteasomal degradation, which results in disassembly of the pre-initiation complex and subsequent transactivation [112]. This clearance, triggered by proteasomal degradation, immediately restores promoter responsiveness to further binding of TFs. If TFs are accumulated at the promoter, for example due to proteasome inhibition, it will prevent further binding of TFs. Accumulated TFs are incapable of Pol II to the promoter [109,112,121], and subsequently repress the transcription of target genes [112,123]. Therefore, it is speculated that promoter-bound MYOCD for transactivation may have to be degraded by the proteasome so as to clear the promoter for the next-round of transcriptional activation, as described for ERa (Fig. 4) [111–113]. Notably, all these previously described transcription factors have a degradation signal or degron, a sequence containing a proteasome-binding tag and a proteasome initiation region [124], in their TADs. MYOCD likely has a degron in its TAD as well, since MYOCD lacking the C-terminal region does not undergo proteasomal degradation and lacks transcriptional activity. In
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Fig. 4. The licensing/degradation-activation model. The mechanism proposed for stimulation of gene transcription by those transcription factors including myocardin (MYOCD) involves three stages. (1) Initiation of transcription: MYOCD, for example, binds to serum response factor (SRF) to form a complex, which is subsequently recruited to the promoters of MYOCD target genes (A). The MYOCD–SRF complex results in further recruitment of a specific E3 ligase (E3), proteasomes and the core components of the transcriptional machinery, including RNA polymerase II (Pol II), to initiate the transcription of MYOCD target genes (B). (2) Degradation of the original transcriptional activator: The complex formation at the promoter results in ubiquitination-degradation of transcriptional activator(s), such as MYOCD (B). (3) Restoration of promoter responsiveness: Proteasomal degradation of MYOCD due to ubiquitination by an unknown E3 will clear the accumulation of MYOCD and allow the formation of a new complex to ensure continuous transactivation of MYOCD target genes (B).
addition, MYOCD was reported to exhibit auto-inhibition, because the K259R mutation at the amino terminus impairs MYOCD activity [125]. Whether the K259R mutation reduces MYOCD degradation remains to be tested. Evidence supports that MYOCD transcriptional activity requires its degradation [100]. Therefore, the critical questions are: Which E3 ligase mediates degradation and how does it happen at the promoter(s) of its target genes?
MYOCD regulation of SM contractility MYOCD induction of contractility MYOCD increases SM contractility, although it does not stimulate the expression of SM proteins that lack efficacious CArG elements, such as smoothelin-B, aortic carboxypeptidase-like protein, and focal adhesion kinase-related non-kinase. In cultured SMCs, overexpression of MYOCD induces the expression of multiple contractile proteins, such as SM a-actin, SM-myosin heavy chain
(MHC), SM22, calponin, and desmin [2,4,31,32,126,127]. MYOCD is sufficient to promote the development of a mature SMC-like phenotype from human embryonic stem (ES) cells with up-regulated contractility [128]. Platelet-derived growth factor (PDGF) modulates the SRF/MYOCD complex to enhance ES cell differentiation into SMCs through up-regulation of HDAC7 splicing [129]. As mentioned earlier, MYOCD induces SM gene expression in a number of non-SM cell types including mouse ES cells, 10T1/2 cells, NIH 3T3 fibroblasts, L6 myoblasts, and cardiac fibroblasts [4,30–32]. In the BC3H1 cell line, for example, MYOCD expression induces SMC-like contraction [126]. Notably, evidence also suggests that MYOCD induces SMC differentiation through induction of a SM-specific miRNA gene, miRNA143/145, mediating MYOCDinduced expression of SMC differentiation marker genes [43] and the contractile phenotype of vascular SMCs [42–45,130]. However, it remains unknown how to coordinate these two mechanisms in the same cells. Notably, more detailed studies are required to illustrate how MYOCD directly regulates SM contractility. MYOCD results in increased expression of contractile proteins, but the question
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is: Does the upregulation of contractile proteins increase sensitivity or maximal contractility? Another question is: Does this increase in contractile proteins contribute to SMC hypertrophy or increased muscle mass? It is well known that SM contraction results from a series of intracellular signaling events, such as phosphorylation of regulatory myosin light chain [131]. Therefore, it will be interesting to investigate how myocardin regulates the components in the contractile machinery, for example, increases phosphorylation of regulatory myosin light chain [126,131].
Auto-inhibition of SM contractility Forced expression of MYOCD increases SM contractile proteins and contributes to the development of SM contractility, but forced expression of MYOCD also induces the expression of skeletal and cardiac CArG-dependent genes in cultured SMCs [132]. Notably, MYOCD, which inhibits differentiation of the L6 myoblasts [133], is only transiently expressed in embryonic skeletal muscle during mouse development [5]. In SM, therefore, a critical question is: Does MYOCD continue increasing contractility in SMCs? Or, how is MYOCD expression or activity suppressed to maintain proper contractility in SMCs? There are three possibilities. Studies from our group using human aortic SMCs have revealed that MYOCD induces expression of miRNA1, which subsequently inhibits MYOCDinduced expression and SM contractility as illustrated in a collagen lattice contraction assay [40]. Exogenous miRNA1, which does not affect MYOCD or SRF expression, not only suppresses the expression of contractile proteins, such as SM a-actin and SM22, but also impairs actin cytoskeletal organization [40]. These results have suggested a ‘‘buffer’’ role of miRNA1 in the negative feedback loop in the regulation of contractility induced by MYOCD, representing a self-limiting mechanism of MYOCD to avoid a super-contractile phenotype. In addition, too much MYOCD may be degraded through CHIP-triggered ubiquitination proteasomal degradation. Interestingly, it was also reported that MYOCD represses its own expression in human fibroblasts [134].
MYOCD inhibition of cell proliferation MYOCD inhibits proliferation of SMCs and other cells as well. At least three independent mechanisms have been proposed. First, MYOCD directly binds to the NF-jB (p65) subunit, and decreases p65-mediated target gene activation by interfering with p65 DNA binding, leading to inhibition of cell proliferation (Fig. 5) [48]. Second, MYOCD induces the expression of miRNA1, which also inhibits SMC proliferation, likely through downregulation of Pim-1, an oncogenic Ser/Thr kinase, with a role in SMC proliferation in vitro and in vivo (Fig. 5) [41,135]. There has been a low basal level of miRNA1 in vascular SMCs and tissues [41]. MYOCD also induces the expression of miRNA1 in cultured rat bladder SMC and in vivo bladders to down-regulate connexin 43 (GJA1) expression [136]. Notably, miRNA1 also inhibits proliferation of cardiomyocytes, skeletal myoblasts, lung cancer cells and hepatocellular carcinogenesis [137–140]. Third, MYOCD also inhibits proliferation of human leiomyosarcoma cells through up-regulation of p21, a cell cycle inhibitory protein [36], which has been defined as a tumor suppressor [141]. MYOCD has also been used in the classification of mesenchymal tumors of the uterus [142]. There might be some other mechanisms involved in MYOCD-induced inhibition of the cell cycle. For example, calponin, whose expression is also induced by MYOCD, is well known to inhibit proliferation of SMCs [143,144]. Perspectives The development of SM-containing organs involves SM differentiation and cell proliferation, but the critical question is: how are these two events coordinated? Or, how does MYOCD play a role in the coordination of these two processes? During vascular development, increased SMC cell number may result from proliferation of stem cells. At a certain point, SMCs start to differentiate in response to MYOCD activation and/or expression. When MYOCD activity reaches maximal levels, the cell cycle will be arrested also due to MYOCD expression. These cells represent a group of mature
Fig. 5. Myocardin (MYOCD) inhibition of cell proliferation. MYOCD directly binds to the NF-jB (p65) subunit and suppresses the expression of p65-dependent growth genes, such as c-Myc. MYOCD also up-regulates the expression of microRNA-1 (miR-1), which subsequently down-regulates the Pim-1 transcription factor, which is involved in SMC proliferation. SRF: serum response factor.
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SMCs with contractility. If these cells are sufficient to maintain vascular functions, other stem cells remain quiescent within the blood vessel wall, but will be re-activated and proliferate again in response to environmental alterations. Downregulation of MYOCD in those ‘‘mature’’ SMCs may not be sufficient to induce so-called ‘‘phenotype-conversion and subsequent proliferation.’’ However, those ‘‘mature’’ SMCs may continue synthesizing DNA without mitosis due to the restriction of extracellular matrix proteins and mechanical stress. In this case, polyploidization may occur, which is well documented in hypertension [145,146]. To date, the exact role for MYOCD in the pathogenesis of cardiovascular disease remains largely unknown. If up-regulation and downregulation of MYOCD activity results in vascular dysfunction or malfunction in other SM-containing organ systems, MYOCD may become a therapeutic target for various diseases. Deregulation of MYOCD protein and/or its activity is present in vascular lesions. In cerebral amyloid angiopathy, for example, MYOCD is proposed to mediate arterial hyper-contractility and cerebral blood flow deregulation in an Alzheimer’s phenotype [147]. MYOCD also regulates the low-density lipoprotein receptor-related protein 1 (LRP)-mediated amyloid-b clearance in brain vascular cells [148]. In addition, ATV, although well-known to lower blood cholesterol, significantly reduces MYOCD expression in the cardiovascular system [77], which may contribute to its protective effects. Therefore, statins improve Alzheimer’s disease likely through inhibition of MyoCD gene expression. More studies are warranted to validate this speculation. Another research direction may be to investigate the roles of MYOCD in both muscle and non-muscle cells in development and/or disease conditions. Acknowledgments This research was supported by the Canadian Institutes of Health Research (CIHR MOP-119511 to X.-L.Z.). X.-L. Zheng is the recipient of a Senior Scholar Award of Alberta Innovates—Health Solutions (AIHS). References [1] Z. Tang, A. Wang, F. Yuan, Z. Yan, B. Liu, J.S. Chu, J.A. Helms, S. Li, Nat. Commun. 3 (2012) 875. [2] D.-Z. Wang, P. Chang, Z. Wang, L. Sutherland, J. Richardson, E. Small, P. Krieg, E.N. Olson, Cell 105 (2001) 851–862. [3] D.-Z. Wang, S. Li, D. Hockemeyer, L. Sutherland, Z. Wang, G. Schratt, J.A. Richardson, A. Nordheim, E.N. Olson, Proc. Natl. Acad. Sci. USA 99 (2002) 14855–14860. [4] J. Chen, C.M. Kitchen, J.W. Streb, J.M. Miano, J. Mol. Cell. Cardiol. 34 (2002) 1345–1356. [5] X. Long, E.E. Creemers, D.Z. Wang, E.N. Olson, J.M. Miano, Proc. Natl. Acad. Sci. US A 104 (2007) 16570–16575. [6] P. Zhu, L. Huang, X. Ge, F. Yan, R. Wu, Q. Ao, Int. J. Exp. Pathol. 87 (2006) 463– 474. [7] K. Martin, S. Weiss, P. Metharom, J. Schmeckpeper, B. Hynes, J. O’Sullivan, N. Caplice, Circ. Res. 105 (2009) 214–218. [8] M. Imamura, X. Long, V. Nanda, J.M. Miano, Gene 464 (2010) 1–10. [9] M. Saha, S.E. Ingraham, A. Carpenter, M. Robinson, K.E. McHugh, S. Singh, M.L. Robinson, K.M. McHugh, J. Urol. 182 (2009) 766–775. [10] K.L. Du, M. Chen, J. Li, J.J. Lepore, P. Mericko, M.S. Parmacek, J. Biol. Chem. 279 (2004) 17578–17586. [11] T.E. Callis, D. Cao, D.-Z. Wang, Circ. Res. 97 (2005) 992–1000. [12] Z. Liu, Z. Wang, H. Yanagisawa, E.N. Olson, Dev. Cell 9 (2005) 261–270. [13] J. Oh, Z. Wang, D.-Z. Wang, C.-L. Lien, W. Xing, E.N. Olson, Mol. Cell. Biol. 24 (2004) 8519–8528. [14] F. Miralles, G. Posern, A.-I. Zaromytidou, R. Treisman, Cell 113 (2003) 329– 342. [15] L. Aravind, E.V. Koonin, Trends Biochem. Sci. 25 (2000) 112–114. [16] S. Guettler, M.K. Vartiainen, F. Miralles, B. Larijani, R. Treisman, Mol. Cell. Biol. 28 (2008) 732–742. [17] S. Nakamura, K. Hayashi, K. Iwasaki, T. Fujioka, H. Egusa, H. Yatani, K. Sobue, J. Biol. Chem. 285 (2010) 37314–37323. [18] K. Hayashi, T. Morita, J. Biol. Chem. 288 (2013) 5743–5755. [19] D.L. Ly, F. Waheed, M. Lodyga, P. Speight, A. Masszi, H. Nakano, M. Hersom, S.F. Pedersen, K. Szaszi, A. Kapus, Am. J. Physiol. Cell Physiol. 304 (2013) C115–C127.
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Invited Review
Myocardin and smooth muscle differentiation Xi-Long Zheng ⇑ Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, Libin Cardiovascular Institute of Alberta, Faculty of Medicine, University of Calgary, Alberta, Canada
a r t i c l e
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Article history: Received 15 October 2013 and in revised form 15 December 2013 Available online 25 December 2013 Keywords: Myocardin Smooth muscle differentiation Smooth muscle cell proliferation
a b s t r a c t Myocardin (MYOCD), a co-transcriptional activator of serum response factor (SRF), stimulates the expression of smooth muscle (SM) genes and inhibits the cell cycle. In addition to its roles in the development, MYOCD may be critically involved in the pathogenesis of proliferative vascular diseases. This review mainly focuses on how MYOCD activity is regulated and how it inhibits cell proliferation. Ó 2013 Elsevier Inc. All rights reserved.
Introduction SM contractility is critical to maintaining the physiological functions of the internal organs, such as the blood vessel wall, gastrointestinal tract, airway, uterus and bladder. Notably, deregulation of vascular contractility, for example, contributes to the pathogenesis of hypertension. In addition, abnormal proliferation of SMCs is involved in neointima formation in arteries, uterine leiomyoma (a benign tumor within the uterus), renal leiomyolipoma in tuberous sclerosis complex (TSC)1 patients, and lung lymphangioleiomyomatosis (LAM) in female patients. The methodology for the culture of smooth muscle cells (SMCs) was established about 30 years ago, but it remained unknown as to why SMCs could be cultured and what controlled SM differentiation until MYOCD was discovered in 2001. In order to proliferate, SMCs are believed to undergo phenotypic conversion from contractile differentiation to synthetic dedifferentiation status. This phenotypic conversion model has been ⇑ Corresponding author. Address: 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada. Fax: +1 (403) 270 0737. E-mail address: [email protected] URL: http://webapps2.ucalgary.ca/~zhenglab/index.html 1 Abbreviations used: TSC, tuberous sclerosis complex; LAM, lymphangioleiomyomatosis; SMCs, smooth muscle cells; MRTF-A, MYOCD-related transcription factorA; TADs, transcription activation domains; Foxo, forkhead transcription factor; CRM, chromosomal region maintenance protein; PECAM-1, platelet endothelial cell adhesion molecule 1; MHC, myosin heavy chain; MLCK, myosin light chain kinase; SBE, Smad-binding element; HRT, hairy-related transcription factor; uPAR, urokinase-type plasminogen activator receptor; TDG, thymine DNA glycosylase; KLF, Kruppel-like transcription factor; ER, estrogen receptor; SRC3; steroid receptor coactivator 3; STRAP, SRF-dependent transcription regulation-associated protein; Cdc7, cell division cycle 7; Ang II, Angiotensin II; IGF, insulin-like growth factor; NKE, Nkx2.5 responsive element; HSC, hepatic stellate cells; ATV, atorvastatin; CBP, CREB-binding protein; CHIP, C-terminus of Hsc70-interacting protein; ERa, estrogen receptor a; TFs, transcription factors; PDGF, platelet-derived growth factor. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.12.015
challenged by a recent study showing proliferating SMCs in culture resulting from vascular stem cells, instead of dedifferentiated SMCs [1]. Nevertheless, the critical roles of MYOCD have well been established in SMC differentiation. This review will focus on the following questions: how is MYOCD activity regulated, and how does MYOCD regulate differentiation and the cell cycle of SMCs? MYOCD and its expression MYOCD (935 aa) was discovered in a research screening for novel cardiac-specific genes in silico through a BLAST search using ESTs from mouse embryonic heart cDNA libraries in the database [2]. Two other family members, MYOCD-related transcription factor-A (MRTF-A, 929 aa, also called MAL, MKL-1, and BSAC), and MRTF-B (1080 aa, also called MKL-2), were identified through searching the NCBI databases using the mouse Myocd cDNA sequence [3]. None of the members bind to DNA, but rather they initiate SRF-dependent gene transcription [2,3]. MYOCD forms a stable ternary complex with SRF and is defined as a co-transcriptional activator [2]. SRF binds to CArG [CC(A/T)6GG] boxes and transactivates transcription of many genes, including SM-specific ones (Fig. 1). MYOCD does not have transcriptional activity in Srf/ cells [3]. Notably, MYOCD may exert its effects independently of SRF resulting from its binding to other signaling proteins. MYOCD is expressed in the heart and in most developing and adult SMC compartments including the dorsal aorta, bladder, stomach, intestine, and uterus [3,4]. Evidence suggests that MYOCD may be expressed in other cells or tissues. For example, lineage tracing studies have shown that MYOCD is transiently expressed in skeletal muscle during development and plays a suppressor role in the skeletal muscle differentiation program [5]. In endothelial cells,
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Fig. 1. Myocardin (MYOCD) stimulation of gene expression through serum response factor (SRF). MYOCD activity is indicated by the expression levels of MYOCD target genes, such as SM a-actin and SM22.
MYOCD mediates hypoxia-induced transdifferentiation into SMlike cells [6]. In peripheral blood mononuclear cells, thrombin stimulates MYOCD and SM myosin heavy chain expression [7]. In addition, distinct MYOCD splice variants have been reported [8,9], which may confer differential tissue expression and functions. In contrast, MRTF-A and -B are expressed in a broad range of embryonic and adult tissues, including ES cells [10].
The features of domain structures First, there has been no DNA binding domain revealed in any member of the MYOCD family. Their C-terminal regions contain transcription activation domains (TADs, Fig. 2), which are required for transcriptional activity of MRTFs, but have no target gene specificity [3]. Deletion of this region results in a dominant-negative mutant. Second, their interaction with SRF is through a short peptide sequence containing a basic and glutamine-rich region (Fig. 2) [2,3]. The binding region of Smad1 includes a basic domain and a stretch of glutamine residues [11]. The forkhead transcription factor (Foxo) 4 (amino acid residues 89–325) directly interacts with the 129–510 aa fragment of myocardin [12]. GATA4 directly binds two regions (326–438 and 439–713 aa) of myocardin [13]. In addition, several other domain structures have been identified in the MYOCD family. (1) RPEL motifs: Two are present in the MYOCD N-terminal region (Fig. 2). Three are in MRTF-A/B, which mediate their association with G-actin [14]. (2) SAP domain (SAF-A/B, Acinus, and PIAS): It contains 35 amino acids (residues 380–414) and may participate in chromosomal dynamics, nuclear breakdown, and apoptotic DNA fragmentation as described for other SAP-containing proteins [15]. (3) Coiled-coil motif: It resembles a leucine zipper and mediates homo- and heterodimerization of MYOCD members, which stabilizes their binding with SRF on CArG boxes of target genes (Fig. 2) [2,3,10,14].
Subcellular localization MYOCD is in the nucleus, but MRTF-A/B are in the cytosol. This is possibly because MRTF-A/B have three RPEL motifs, but there are only two in MYOCD. The three RPEL motifs can mediate MRTF-A/B binding to cytosolic G-actin [16], but the two RPEL domains in MYOCD have relatively weak binding to actin [16]. It is also possibly due to: (1) the higher affinity of MYOCD to importin/1 than MRTF-A/B, and (2) G-actin inhibition of importin/1 binding with MRTF-A/B [17]. In addition to importin, recent studies have shown that difference in subcellular localization is also determined by their differential interaction with chromosomal region maintenance protein (CRM)1, SRF, and G-actin [18]. Differential localization may be explained by MYOCD’s weak binding to CRM1, but strong binding to SRF, which are opposite to MRTF-A [18]. Notably, the subcellular localization of all members may change in response to various stresses. Hyperosmotic stress, for example, was reported to regulate the distribution and stability of MRTF in kidney tubular cells [19]. The phenotypes of knockout mice Myocd knockout Mice with a homozygous null mutation of Myocd died at embryonic day 10.5 (E10.5) [20]. This study showed no staining for either SM a-actin or SM22 in the dorsal aortae in transverse section of Myocd/ embryos, suggesting the lack of SMC differentiation. However, the staining for PECAM-1 (Platelet Endothelial Cell Adhesion Molecule 1), an endothelial marker, at E8.5 suggested a normal vascular patterning in mutant embryos [20]. It is unclear what causes the embryonic death, but the lethality may result from abnormal vasculature of the yolk sac and the pericardial effusion observed by E10.5 [20]. Selective knockout of Myocd in neural crest-derived SMCs results in the lack of the contractile phenotype
Fig. 2. Domain structure of myocardin (MYOCD). The functions of the various domains have been described in the text. SAP: SAF-A/B, Acinus, and PIAS; TAD: transactivation domain; SRF: serum response factor; ++: basic domain.
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of SMCs and patent ductus arteriosus, in which SMCs demonstrate the synthetic phenotype [21]. However, Myocd-null SMCs can still differentiate and also be incorporated into vascular tissues with normal morphology [22]. In chimeric knockout mice, it was reported that MYOCD is dispensable for development of vascular, but not visceral, SMCs [23]. Nevertheless, it remains interesting to address whether the death of Myocd-null embryos results from loss of MYOCD in vascular SMCs or in other cells. Phenotype of MRTF-A/B knockout mice Knockout of MRTF-A is not lethal, but female mice are not able to release milk during lactation [24]. However, knockout of MRTFB is lethal as demonstrated by several independent laboratories. First, homozygous MRTF-B gene-trap mice die between E17.5 and postnatal day 1 due to a defect in the cardiac outflow tract. Although migration and early patterning of cardiac neural crest cells appear normal, there is a significant reduction of SMC differentiation in the arch arteries and the aorticopulmonary septum at E11.5 [25]. Other studies show that mice die from several cardiovascular defects including abnormal patterning of the branchial arch arteries, double-outlet right ventricle, ventricular septal defects, and thin-walled myocardium [26]. A failure in SMC differentiation within the branchial arch arteries is also observed [26]. In addition, other defects in this knockout may contribute to the lethality. It has been reported that mice containing an insertional mutation of MRTF-B not only have vascular defects, but also liver hemorrhage. These mice usually die in late gestation with defective expression of SM genes in the liver sinusoids, vitelline veins, and yolk sac [27]. Mechanisms underlying the cellular effects of MYOCD First, MYOCD induces SRF-dependent gene expression. Many genes contain one or multiple CArG boxes, which is specific for SRF binding, in their promoters (Fig. 1) [28,29]. However, the expression of only a portion of these SRF-responsive genes are driven by MYOCD. The potential mechanism(s) underlying MYOCD specificity in gene transcription is discussed in the next section. Most SM genes contain CArG boxes and are stimulated by MYOCD, including SM a-actin (ACTA2), SM c-actin, SM myosin heavy chain (MHC), calponin, SM22, h-caldesmon and SM myosin light chain kinase (MLCK) [4,30–33]. Transforming growth factor-beta1 (TGFb1)-induced transcript 1 (TGFB1I1, also known as Hic-5), which was recently recognized as a novel marker for the SM contractile phenotype, is also regulated by SRF/MYOCD [34]. Expression of smoothelin-A, but not -B, is controlled by SRF/MYOCD [35]. Notably, MYOCD also induces the expression of SM genes in a variety of non-muscle cell types [31,32], such as peripheral blood mononuclear cells [7]. Besides SM-related genes, MYOCD stimulates the expression of many other genes through the SRF-CArG box mechanism, such as p21, a cell cycle inhibitory protein [36], cysteine-rich protein 2, a SRF co-activator [37], large conductance calcium-activated potassium (MaxiK) channel subunit b, a SM-restricted ion channel [38], and lipoma preferred partner, a nucleocytoplasmic shuttling adaptor protein [39]. In addition to proteins, MYOCD induces the expression of microRNAs, such as miRNA1 [40,41] and miRNA143/145 [42–45]. Second, MYOCD also has SRF-independent effects through the following mechanisms. (1) MEF2A: MYOCD binds to the MEF2A transcription factor, and potently stimulates MEF2-dependent gene expression. It binds MEF2 at the MADS-box/MEF2 domain through a short amphipathic helix [46,47]. (2) NF-jB: MYOCD
directly interacts with p65 and decreases p65-mediated target gene activation by interfering with p65 DNA binding [48]. (3) Smad3: MYOCD interacts with Smad3, which binds to the Smadbinding element (SBE) through the C-terminal TADs to activate SM gene expression through a CArG box-independent manner [49]. Specificity of MYOCD induction of gene expression Several laboratories have been addressing why MYOCD specifically induces the expression of SM genes, instead of all CArGcontaining genes. To date, the published studies suggest a complexity of the underlying mechanism with several possibilities proposed. (1) Uniqueness of CArG boxes: The evidence comes from the studies of the human ACTG2 promoter, in which there are four conserved CArG boxes. Substitution of CArG2 with other CArG sequences results in loss of promoter inducibility by MYOCD [33]. (2) Non-CArG sequence: Evidence suggests the sequences adjacent to the CArG boxes are critical in determining MYOCD specificity. For example, mutation of an adjacent binding site for NKX3.1 results in reduction of MYOCD-dependent transactivation of the ACTG2 promoter [33]. Supportively, MYOCD stimulates the expression of telokin, which has one CArG box, but not c-fos. In the c-fos promoter, the ets binding site, together with elements 30 of the CArG box, inhibits transactivation by MYOCD [50]. (3) Chromatin structure: MYOCD recruits chromatin-remodeling enzymes to SRF target genes with involvement of the p300 histone acetyltransferase [51]. SRF/MYOCD complexes are associated with a specific variant of histone H3 on SMC gene loci in vivo [52]. (4) Other determinants: SWI/SNF complexes are required for MYOCD to increase SRF binding to CArG boxes in MYOCD target genes [53]. Recruitment of Jmjd1a, the histone 3 lysine 9 (H3K9)-specific demethylase, is required for MYOCD-directed gene transcription [54]. Regulation of MYOCD activity Protein–protein interaction regulates MYOCD activity First, interaction of MYOCD with other proteins inhibits MYOCD transcriptional activity, because of the reduction of MYOCD availability for SRF. For example, interaction of Foxo4 with MYOCD prevents MYOCD transcriptional activation of SM genes. However, when Foxo4 is phosphorylated through the Akt pathway, it translocates to the cytoplasm, leading to the release of MYOCD [12]. SOX9 also interacts with MYOCD and suppresses its transcriptional activity [55]. In addition, the hairy-related transcription factor (HRT)-2, which is expressed in developing vasculature [56], associates with MYOCD and represses its activity through a mechanism as yet to be defined [57]. In addition, the urokinase-type plasminogen activator receptor (uPAR) can interact with MYOCD in the nucleus, resulting in displacement of MYOCD from SRF and the reduction of MYOCD-induced gene expression [58]. Second, proteins may compete with MYOCD for the binding site on SRF so as to reduce MYOCD activity. These proteins include Runx2, a key osteogenic transcription factor [59], thymine DNA glycosylase (TDG) [60], HERP1 [61] and GATA factors [13,62]. Additionally, some proteins bind to the MYOCD–SRF complex and reduce its binding to DNA, including Msx transcription factors (Msx1 and Msx2) [63], Yap1 [64], and the Kruppel-like transcription factor (KLF) 4 [52,65]. Notably, the actin cytoskeleton can also regulate the activity of MRTFs [66,67]. MRTFs directly bind actin
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monomers and are sequestered in the cytoplasm. Actin polymerization results in MRTF translocation to the nucleus, where it coactivates high levels of CArG-dependent gene transcription [14,68]. Finally, MYOCD activity can be enhanced by interaction with other proteins. For example, the N terminus of estrogen receptor (ER) coactivator steroid receptor coactivator 3 (SRC3) contains a basic helix-loop-helix/Per-ARNT-Sim protein–protein interaction domain. Through this domain, SRC3 interacts with MYOCD TAD to enhance its transcriptional activity [69]. Therefore, SRC3 is defined as a coactivator for MYOCD. In addition, GATA4, which does not necessarily bind to DNA, directly interacts with MYOCD to stimulate MYOCD transcriptional activity in some target genes [13]. Regulation of MyoCD gene expression directly relates to its activity Stimulation of MyoCD gene expression First, an enhancer has been identified to control Myocd gene expression, which requires Mef2, Foxo and TEAD transcription factors for its activity [70]. Interestingly, MYOCD was found to regulate its own enhancer through an SRF-independent mechanism [70]. However, other studies have suggested that SRF may stimulate the expression of Myocd gene since loss of SRF results in decrease of Myocd mRNA [71,72] and MYOCD promoter contains a CArG binding site [73]. In addition to MYOCD itself, other SRF-cofactor, such as Nkx2.5 and p49/STRAP (SRF-dependent transcription regulation-associated protein), may be involved in SRF-induced MYOCD gene expression [74]. Second, MYOCD gene expression is stimulated by the Rho-ROCK (Rho-associated kinase) pathway as demonstrated in cultured SMCs derived from human aorta, porcine coronary and mouse aorta [75–77]. The RhoA inhibitor Y27632 inhibits thrombin-induced MYOCD gene expression in peripheral blood mononuclear cells [7]. Several other mechanisms have also been proposed to stimulate MYOCD gene expression. (1) TGFb1: a recent study has revealed that TGFb1 induces the mRNA expression and promoter activity of MYOCD through cell division cycle 7 (Cdc7), a non-transcription factor, and its interaction with Nkx2.5 [78]. (2) Angiotensin II (Ang II): Ang II is a well-known inducer of SMC hypertrophy. It also increases MYOCD expression through up-regulation of Prx1, a homeodomain protein [79]. (3) Hypoxia: MYOCD expression is increased by hypoxia in cardiomyocytes through the induction of Ang II and activation of the ERK pathway [80]. In contrast, in rat pulmonary arterial SMCs, hypoxia reduces MYOCD gene expression through up-regulation of PDGF-BB [81] or inhibition of cGMP-dependent protein kinase [82]. (4) Insulin-like growth factor (IGF)-1: IGF-1 stimulates the activity and expression of MYOCD through Foxo4 [12]. It was reported that homeobox protein Nkx2.5 binding to a Nkx2.5 responsive element (NKE) within the MYOCD promoter promotes MYOCD gene expression [83]. Notably, MYOCD is up-regulated in non-muscle cells, such as rat primary trans-differentiated hepatic stellate cells (HSC) [84], suggesting non-vascular pathophysiological roles of MYOCD. Inhibition of MYOCD gene expression MYOCD gene expression is inhibited by overexpression of Smad3, but not Smad2, due to its interaction with Nkx2.5 and subsequent inhibition of MYOCD gene promoter [85]. Resveratrol suppresses MYOCD gene expression through induction of p53 [86]. In addition, methylation of the MYOCD gene prevents its expression in nasopharyngeal carcinoma cell lines [87]. Interestingly, recent studies have revealed that atorvastatin (ATV), the most widely prescribed lipid-lowering drug [88],
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inhibits MYOCD gene expression in cultured SMCs through inhibition of the Rho-ROCK pathway [77]. Injection of ATV in mice significantly downregulates MYOCD expression, contractile proteins and contractility in mouse carotid arteries and aorta [77]. In addition, ATV was reported to inhibit hypoxia-induced MYOCD expression in cardiomyocytes [89] and attenuate the expression of MYOCD and ACTA2 in human fetal penile SMCs [90]. Therefore, inhibition of MYOCD gene expression may be a therapeutic approach for cardiovascular disease. Post-translation modifications and MYOCD activity MYOCD activity is also regulated by post-translational modifications, such as phosphorylation, acetylation, sumoylation and ubiquitination. First, MAPK p44/42 phosphorylates MYOCD at four sites (Ser812, Ser859, Ser866, and Thr893) in its TAD and increases its transcriptional activity. Mutation of all four sites with aspartate (4xD), but not alanine (4xA), reduces its transcriptional activity, likely through its reduced interaction with the cAMP response element binding protein (CREB)-binding protein (CBP) acetyltransferase [91]. GSK-3b phosphorylates MYOCD, but inhibits its transcriptional activity in cardiomyocytes [92]. How GSK-3b phosphorylation regulates MYOCD activity in vascular SMCs remains to be investigated. Second, MYOCD is acetylated, which is required for its transcriptional activity. Acetylation increases the association of MYOCD and SRF. Lysine residues locate at the N terminus are acetylated by chromatin-modifying histone acetylase p300 [93]. Third, SUMO-1 modification of a lysine residue at position 445 of MYOCD increases its transcriptional activity. E3 SMO-protein ligase 3, a protein inhibitor of activated STAT (PIAS)1 interacts with MYOCD, and controls MYOCD activity through its E3 ligase activity, which supposedly stimulates MYOCD sumoylation on an atypical sumoylation site(s) [94]. Finally, MYOCD protein undergoes ubiquitination-degradation, resulting in either decreased or increased transcriptional activities, which will be detailed in the next section. MYOCD is regulated by ubiquitination and proteasomal degradation Inhibition of MYOCD activity MYOCD, when phosphorylated by GSK-3b, can be ubiquitinated by C-terminus of Hsc70-interacting protein (CHIP), a cytosolic E3 ligase, resulting in proteasomal degradation and reduction of MYOCD transcriptional activity (Fig. 3) [95]. Note that CHIP-mediated ubiquitination-degradation is mainly responsible for protein quality control. Polyubiquitinated proteins are rapidly destroyed by the 26S proteasome, a large complex with multiple proteolytic activities [96,97]. The ubiquitin proteasome system (UPS) accounts for the degradation of 80–90% of all intracellular proteins [96,97]. A few studies suggest that regulation of MYOCD activity is through a decrease or increase of MYOCD degradation. Four and a half LIM domain protein 2 (FHL2) interacts with MYOCD and stimulates its transcriptional activity. This protein–protein interaction reduces proteasome-mediated degradation of MYOCD [98]. In addition, UBR5 (ubiquitin protein ligase E3 component n-recognin 5) binds to MYOCD and stimulates its transcriptional activity through reduction of MYOCD degradation, but independent of the E3 ligase activity of UBR5 [99]. Stimulation of MYOCD activity Evidence also indicates that inhibition of MYOCD degradation blocks MYOCD activity [100]. First, treatment with proteasomal inhibitors, such as MG132, results in a significant increase in MYOCD
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Fig. 3. Regulation of myocardin (MYOCD) by the ubiquitin proteasomal degradation system. CHIP: C-terminus of Hsc70-interacting protein with a U-box domain, an E3 ubiquitin-ligase.
protein [100]. Paradoxically, increased MYOCD due to decreased degradation is accompanied by decreased expression of SM target genes, including ACTA2, microRNA (miRNA)1 and 143/145, suggesting that accumulated MYOCD does not have transcriptional activity. This has also been supported by an earlier study showing that proteasome inhibitors decreased SRF-dependent expression of SM a-actin [101]. Second, a chromatin immunoprecipitation assay revealed that proteasome inhibition increases the occupancy of MYOCD and SRF on the acta2 gene promoter, but abolishes MYOCD-dependent recruitment of RNA polymerase II and its serine5-phosphorylated species (p-Pol II) [100]. Proteasome inhibition did not prevent Pol II recruitment in non-MYOCD target genes. This degradation-induced transactivation model is also supported by the following in vivo studies: (1) Ubiquitin, E2, E3 and proteasome subunits are downregulated in anastomotic intimal hyperplasia in a canine carotid model [102], and (2) MYOCD accumulation occurs in balloon injury-induced neointima of rat aorta [61] and neointimal lesion of mouse carotid arteries in response to ligation with a reduced expression of MyoCD gene [100]. Given that SMCs are de-differentiated and synthetic in neointimal lesions, these findings suggest that accumulation of MYOCD due to inhibition of proteasomal degradation results in inhibition of MYOCD activity. Notably, several other studies have also shown a reduced expression of MyoCD gene in proliferative vascular lesions, such as those in mouse carotid arteries in response to ligation [41,103,104] or wire injury [105] and in femoral artery via wire injury [106,107], but downregulation of MYOCD protein was also reported in neointimal lesions in femoral arteries [107]. Nevertheless, This speculation has been validated by a recent finding showing that stabilization of MYOCD protein in neointimal lesions is accompanied by downregulation of transcripts for ubiquitin and proteasome subunits [100]. In the SMC heterogeneity model, MYOCD in more differentiated spindle-shaped SMCs is more quickly degraded and has a shorter half-life than in epithelioid SMCs [100]. Taken together, these evidence suggests that MYOCD with increased stability due to reduced degradation is likely inactive and unable to transactivate its target genes. This proposed model may also explain how dysfunction of the UPS is involved in the initiation, progression and complications of atherosclerosis [108].
‘‘Licensing’’ model Above features described for MYOCD perfectly fit the ‘‘Licensing’’ model described for many other transcription factors (Fig. 4), such as Gcn4 [109], Gal4 [109,110] and Ino2/4 [109] in yeast and estrogen receptor a (ERa) [111–113], progesterone receptor [114], retinoic acid receptor [115], p53 [116] and c-myc [117] in mammalian cells. Their transcriptional activities require them to be degraded via the proteasome. Additionally, their transactivation potencies are positively correlated with the rate of proteasomal degradation [111,118–120]. Inhibition of their degradation through either inhibition of 26S proteasome activity by MG132, a specific, potent, reversible, and cell-permeable proteasome inhibitor, or downregulation of E3 ligases abolishes their transcriptional activities [109,112,117,121]. Deshaies and Lipford proposed a ‘‘Licensing’’ model to explain this Degradation-Activation mechanism (Fig. 4) [122], which was further developed by the Tansey et al. [110,117,121] and Cannon et al. [112]. Essentially, transcription factors (TFs) recruit not only the core components of the transcriptional machinery, but also E3 ligase(s) and proteasomes to the promoter. Upon initiation, TFs undergo ubiquitin-proteasomal degradation, which results in disassembly of the pre-initiation complex and subsequent transactivation [112]. This clearance, triggered by proteasomal degradation, immediately restores promoter responsiveness to further binding of TFs. If TFs are accumulated at the promoter, for example due to proteasome inhibition, it will prevent further binding of TFs. Accumulated TFs are incapable of Pol II to the promoter [109,112,121], and subsequently repress the transcription of target genes [112,123]. Therefore, it is speculated that promoter-bound MYOCD for transactivation may have to be degraded by the proteasome so as to clear the promoter for the next-round of transcriptional activation, as described for ERa (Fig. 4) [111–113]. Notably, all these previously described transcription factors have a degradation signal or degron, a sequence containing a proteasome-binding tag and a proteasome initiation region [124], in their TADs. MYOCD likely has a degron in its TAD as well, since MYOCD lacking the C-terminal region does not undergo proteasomal degradation and lacks transcriptional activity. In
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Fig. 4. The licensing/degradation-activation model. The mechanism proposed for stimulation of gene transcription by those transcription factors including myocardin (MYOCD) involves three stages. (1) Initiation of transcription: MYOCD, for example, binds to serum response factor (SRF) to form a complex, which is subsequently recruited to the promoters of MYOCD target genes (A). The MYOCD–SRF complex results in further recruitment of a specific E3 ligase (E3), proteasomes and the core components of the transcriptional machinery, including RNA polymerase II (Pol II), to initiate the transcription of MYOCD target genes (B). (2) Degradation of the original transcriptional activator: The complex formation at the promoter results in ubiquitination-degradation of transcriptional activator(s), such as MYOCD (B). (3) Restoration of promoter responsiveness: Proteasomal degradation of MYOCD due to ubiquitination by an unknown E3 will clear the accumulation of MYOCD and allow the formation of a new complex to ensure continuous transactivation of MYOCD target genes (B).
addition, MYOCD was reported to exhibit auto-inhibition, because the K259R mutation at the amino terminus impairs MYOCD activity [125]. Whether the K259R mutation reduces MYOCD degradation remains to be tested. Evidence supports that MYOCD transcriptional activity requires its degradation [100]. Therefore, the critical questions are: Which E3 ligase mediates degradation and how does it happen at the promoter(s) of its target genes?
MYOCD regulation of SM contractility MYOCD induction of contractility MYOCD increases SM contractility, although it does not stimulate the expression of SM proteins that lack efficacious CArG elements, such as smoothelin-B, aortic carboxypeptidase-like protein, and focal adhesion kinase-related non-kinase. In cultured SMCs, overexpression of MYOCD induces the expression of multiple contractile proteins, such as SM a-actin, SM-myosin heavy chain
(MHC), SM22, calponin, and desmin [2,4,31,32,126,127]. MYOCD is sufficient to promote the development of a mature SMC-like phenotype from human embryonic stem (ES) cells with up-regulated contractility [128]. Platelet-derived growth factor (PDGF) modulates the SRF/MYOCD complex to enhance ES cell differentiation into SMCs through up-regulation of HDAC7 splicing [129]. As mentioned earlier, MYOCD induces SM gene expression in a number of non-SM cell types including mouse ES cells, 10T1/2 cells, NIH 3T3 fibroblasts, L6 myoblasts, and cardiac fibroblasts [4,30–32]. In the BC3H1 cell line, for example, MYOCD expression induces SMC-like contraction [126]. Notably, evidence also suggests that MYOCD induces SMC differentiation through induction of a SM-specific miRNA gene, miRNA143/145, mediating MYOCDinduced expression of SMC differentiation marker genes [43] and the contractile phenotype of vascular SMCs [42–45,130]. However, it remains unknown how to coordinate these two mechanisms in the same cells. Notably, more detailed studies are required to illustrate how MYOCD directly regulates SM contractility. MYOCD results in increased expression of contractile proteins, but the question
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is: Does the upregulation of contractile proteins increase sensitivity or maximal contractility? Another question is: Does this increase in contractile proteins contribute to SMC hypertrophy or increased muscle mass? It is well known that SM contraction results from a series of intracellular signaling events, such as phosphorylation of regulatory myosin light chain [131]. Therefore, it will be interesting to investigate how myocardin regulates the components in the contractile machinery, for example, increases phosphorylation of regulatory myosin light chain [126,131].
Auto-inhibition of SM contractility Forced expression of MYOCD increases SM contractile proteins and contributes to the development of SM contractility, but forced expression of MYOCD also induces the expression of skeletal and cardiac CArG-dependent genes in cultured SMCs [132]. Notably, MYOCD, which inhibits differentiation of the L6 myoblasts [133], is only transiently expressed in embryonic skeletal muscle during mouse development [5]. In SM, therefore, a critical question is: Does MYOCD continue increasing contractility in SMCs? Or, how is MYOCD expression or activity suppressed to maintain proper contractility in SMCs? There are three possibilities. Studies from our group using human aortic SMCs have revealed that MYOCD induces expression of miRNA1, which subsequently inhibits MYOCDinduced expression and SM contractility as illustrated in a collagen lattice contraction assay [40]. Exogenous miRNA1, which does not affect MYOCD or SRF expression, not only suppresses the expression of contractile proteins, such as SM a-actin and SM22, but also impairs actin cytoskeletal organization [40]. These results have suggested a ‘‘buffer’’ role of miRNA1 in the negative feedback loop in the regulation of contractility induced by MYOCD, representing a self-limiting mechanism of MYOCD to avoid a super-contractile phenotype. In addition, too much MYOCD may be degraded through CHIP-triggered ubiquitination proteasomal degradation. Interestingly, it was also reported that MYOCD represses its own expression in human fibroblasts [134].
MYOCD inhibition of cell proliferation MYOCD inhibits proliferation of SMCs and other cells as well. At least three independent mechanisms have been proposed. First, MYOCD directly binds to the NF-jB (p65) subunit, and decreases p65-mediated target gene activation by interfering with p65 DNA binding, leading to inhibition of cell proliferation (Fig. 5) [48]. Second, MYOCD induces the expression of miRNA1, which also inhibits SMC proliferation, likely through downregulation of Pim-1, an oncogenic Ser/Thr kinase, with a role in SMC proliferation in vitro and in vivo (Fig. 5) [41,135]. There has been a low basal level of miRNA1 in vascular SMCs and tissues [41]. MYOCD also induces the expression of miRNA1 in cultured rat bladder SMC and in vivo bladders to down-regulate connexin 43 (GJA1) expression [136]. Notably, miRNA1 also inhibits proliferation of cardiomyocytes, skeletal myoblasts, lung cancer cells and hepatocellular carcinogenesis [137–140]. Third, MYOCD also inhibits proliferation of human leiomyosarcoma cells through up-regulation of p21, a cell cycle inhibitory protein [36], which has been defined as a tumor suppressor [141]. MYOCD has also been used in the classification of mesenchymal tumors of the uterus [142]. There might be some other mechanisms involved in MYOCD-induced inhibition of the cell cycle. For example, calponin, whose expression is also induced by MYOCD, is well known to inhibit proliferation of SMCs [143,144]. Perspectives The development of SM-containing organs involves SM differentiation and cell proliferation, but the critical question is: how are these two events coordinated? Or, how does MYOCD play a role in the coordination of these two processes? During vascular development, increased SMC cell number may result from proliferation of stem cells. At a certain point, SMCs start to differentiate in response to MYOCD activation and/or expression. When MYOCD activity reaches maximal levels, the cell cycle will be arrested also due to MYOCD expression. These cells represent a group of mature
Fig. 5. Myocardin (MYOCD) inhibition of cell proliferation. MYOCD directly binds to the NF-jB (p65) subunit and suppresses the expression of p65-dependent growth genes, such as c-Myc. MYOCD also up-regulates the expression of microRNA-1 (miR-1), which subsequently down-regulates the Pim-1 transcription factor, which is involved in SMC proliferation. SRF: serum response factor.
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SMCs with contractility. If these cells are sufficient to maintain vascular functions, other stem cells remain quiescent within the blood vessel wall, but will be re-activated and proliferate again in response to environmental alterations. Downregulation of MYOCD in those ‘‘mature’’ SMCs may not be sufficient to induce so-called ‘‘phenotype-conversion and subsequent proliferation.’’ However, those ‘‘mature’’ SMCs may continue synthesizing DNA without mitosis due to the restriction of extracellular matrix proteins and mechanical stress. In this case, polyploidization may occur, which is well documented in hypertension [145,146]. To date, the exact role for MYOCD in the pathogenesis of cardiovascular disease remains largely unknown. If up-regulation and downregulation of MYOCD activity results in vascular dysfunction or malfunction in other SM-containing organ systems, MYOCD may become a therapeutic target for various diseases. Deregulation of MYOCD protein and/or its activity is present in vascular lesions. In cerebral amyloid angiopathy, for example, MYOCD is proposed to mediate arterial hyper-contractility and cerebral blood flow deregulation in an Alzheimer’s phenotype [147]. MYOCD also regulates the low-density lipoprotein receptor-related protein 1 (LRP)-mediated amyloid-b clearance in brain vascular cells [148]. In addition, ATV, although well-known to lower blood cholesterol, significantly reduces MYOCD expression in the cardiovascular system [77], which may contribute to its protective effects. Therefore, statins improve Alzheimer’s disease likely through inhibition of MyoCD gene expression. More studies are warranted to validate this speculation. Another research direction may be to investigate the roles of MYOCD in both muscle and non-muscle cells in development and/or disease conditions. Acknowledgments This research was supported by the Canadian Institutes of Health Research (CIHR MOP-119511 to X.-L.Z.). X.-L. Zheng is the recipient of a Senior Scholar Award of Alberta Innovates—Health Solutions (AIHS). References [1] Z. Tang, A. Wang, F. Yuan, Z. Yan, B. Liu, J.S. Chu, J.A. Helms, S. Li, Nat. Commun. 3 (2012) 875. [2] D.-Z. Wang, P. Chang, Z. Wang, L. Sutherland, J. Richardson, E. Small, P. Krieg, E.N. Olson, Cell 105 (2001) 851–862. [3] D.-Z. Wang, S. Li, D. Hockemeyer, L. Sutherland, Z. Wang, G. Schratt, J.A. Richardson, A. Nordheim, E.N. Olson, Proc. Natl. Acad. Sci. USA 99 (2002) 14855–14860. [4] J. Chen, C.M. Kitchen, J.W. Streb, J.M. Miano, J. Mol. Cell. Cardiol. 34 (2002) 1345–1356. [5] X. Long, E.E. Creemers, D.Z. Wang, E.N. Olson, J.M. Miano, Proc. Natl. Acad. Sci. US A 104 (2007) 16570–16575. [6] P. Zhu, L. Huang, X. Ge, F. Yan, R. Wu, Q. Ao, Int. J. Exp. Pathol. 87 (2006) 463– 474. [7] K. Martin, S. Weiss, P. Metharom, J. Schmeckpeper, B. Hynes, J. O’Sullivan, N. Caplice, Circ. Res. 105 (2009) 214–218. [8] M. Imamura, X. Long, V. Nanda, J.M. Miano, Gene 464 (2010) 1–10. [9] M. Saha, S.E. Ingraham, A. Carpenter, M. Robinson, K.E. McHugh, S. Singh, M.L. Robinson, K.M. McHugh, J. Urol. 182 (2009) 766–775. [10] K.L. Du, M. Chen, J. Li, J.J. Lepore, P. Mericko, M.S. Parmacek, J. Biol. Chem. 279 (2004) 17578–17586. [11] T.E. Callis, D. Cao, D.-Z. Wang, Circ. Res. 97 (2005) 992–1000. [12] Z. Liu, Z. Wang, H. Yanagisawa, E.N. Olson, Dev. Cell 9 (2005) 261–270. [13] J. Oh, Z. Wang, D.-Z. Wang, C.-L. Lien, W. Xing, E.N. Olson, Mol. Cell. Biol. 24 (2004) 8519–8528. [14] F. Miralles, G. Posern, A.-I. Zaromytidou, R. Treisman, Cell 113 (2003) 329– 342. [15] L. Aravind, E.V. Koonin, Trends Biochem. Sci. 25 (2000) 112–114. [16] S. Guettler, M.K. Vartiainen, F. Miralles, B. Larijani, R. Treisman, Mol. Cell. Biol. 28 (2008) 732–742. [17] S. Nakamura, K. Hayashi, K. Iwasaki, T. Fujioka, H. Egusa, H. Yatani, K. Sobue, J. Biol. Chem. 285 (2010) 37314–37323. [18] K. Hayashi, T. Morita, J. Biol. Chem. 288 (2013) 5743–5755. [19] D.L. Ly, F. Waheed, M. Lodyga, P. Speight, A. Masszi, H. Nakano, M. Hersom, S.F. Pedersen, K. Szaszi, A. Kapus, Am. J. Physiol. Cell Physiol. 304 (2013) C115–C127.
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