The Hippo signal transduction network in skeletal and cardiac muscle.

REVIEW CELL BIOLOGY

The Hippo signal transduction network in skeletal and cardiac muscle Henning Wackerhage,1* Dominic P. Del Re,2 Robert N. Judson,1,3 Marius Sudol,4,5 Junichi Sadoshima2 The discovery of the Hippo pathway can be traced back to two areas of research. Genetic screens in fruit flies led to the identification of the Hippo pathway kinases and scaffolding proteins that function together to suppress cell proliferation and tumor growth. Independent research, often in the context of muscle biology, described Tead (TEA domain) transcription factors, which bind CATTCC DNA motifs to regulate gene expression. These two research areas were joined by the finding that the Hippo pathway regulates the activity of Tead transcription factors mainly through phosphorylation of the transcriptional coactivators Yap and Taz, which bind to and activate Teads. Additionally, many other signal transduction proteins crosstalk to members of the Hippo pathway forming a Hippo signal transduction network. We discuss evidence that the Hippo signal transduction network plays important roles in myogenesis, regeneration, muscular dystrophy, and rhabdomyosarcoma in skeletal muscle, as well as in myogenesis, organ size control, and regeneration of the heart. Understanding the role of Hippo kinases in skeletal and heart muscle physiology could have important implications for translational research.

Introduction

Skeletal and cardiac muscle function to generate body movement and blood flow. Both skeletal and cardiac muscle cells have sarcomeric motor proteins that convert the chemical energy of nutrients into work and heat. Skeletal and cardiac muscles develop from differentiation of the mesoderm, but express different genes and vary in their morphological and physiological properties. In addition, skeletal muscle has the ability to regenerate after chemical or mechanical damage (1): a property conferred by the activation of resident stem cells known as satellite cells (2–5). In contrast, adult mammalian cardiac muscle does not effectively regenerate after cardiac injury, such as a heart attack in humans (6). However, neonatal mammalian (7) and adult zebrafish hearts (8) can regenerate, suggesting that the underlying molecular program responsible for regeneration exists in the heart and may be activated for therapeutic purposes. Both heart and skeletal muscle are subject to progressive degeneration, as seen in aging (9) and muscular dystrophy (10), and skeletal, but not cardiac, muscle can undergo oncogenic transformation, for example, in patients with rhabdomyosarcoma (11–13). Several signal transduction pathways have been linked to muscle development, regeneration, and disease. Here, we review the evidence for the emerging role of the Hippo signal transduction network in these processes. Discovery of the Hippo signal transduction network

The Hippo pathway is an important signal transduction pathway involved in development, stem cell function, regeneration, and organ size in multiple tissues in various species, and underlies several human pathologies, including cancer 1

School of Medical Sciences, University of Aberdeen, Health Sciences Building, Foresterhill, AB25 2ZD Aberdeen, Scotland, UK. 2Department of Cell Biology and Molecular Medicine, New Jersey Medical School, Rutgers University, 185 South Orange Avenue, Newark, NJ 07103, USA. 3Biomedical Research Centre, University of British Columbia, 317-2194 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. 4Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Republic of Singapore. 5Department of Medicine, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA. *Corresponding author. E-mail: [email protected]

(14–18). Genetic screens designed to identify tumor suppressors in the fruit fly Drosophila melanogaster led to the discovery of the Hippo pathway, including the kinases Hippo and Warts, the kinase-binding proteins Salvador and Mob1, and several upstream kinase activators. Consistent with a role as tumor suppressors, loss-of-function mutations in the genes encoding these proteins typically increase cell proliferation and decrease apoptosis (19, 20). Several genetic epistasis and protein interaction studies indicate that these proteins constitute a distinct, integrated signal transduction pathway (21). As early as the late 1980s, transcriptional regulators, which are now known to be part of the Hippo pathway, were studied independently from the work in flies, often in the context of muscle research. The transcription factor Tead1 (TEA domain containing 1, also referred to as Tef-1 in early studies) binds to a CATTCC DNA binding motif (22), which is variously referred to as a muscle CAT (MCAT) motif (23), GTIIC motif (22), or Hippo response element (24). A conserved 66– to 68–amino acid region known as the TEA/ ATTS (TEA) domain in Teads (Tead1 to Tead4) binds to MCAT elements (Fig. 1, A and B) (25). The activity of Teads requires binding to transcriptional coactivators (26), including Yap (yes-associated protein, encoded by the gene Yap1) (27, 28), Taz (transcription coactivator with PDZ binding motif, encoded by the gene Wwtr1) (29–31), and Vglls (Vestigial-like proteins) (26, 32, 33). The discovery that the Hippo pathway inhibits the transcriptional cofactor Yorkie (34), which is the fly homolog of Yap and Taz, connected the Hippo pathway to the transcriptional regulators Yap, Taz, and Teads. In recent years, the list of genes and proteins linked to the Hippo pathway has expanded greatly. Research demonstrates that the Hippo pathway is only one of several signal transduction modules that target the Hippo transcriptional regulators Yap, Taz, Teads, and Vglls. Additionally, the Hippo transcriptional regulators interact with many downstream signaling proteins (35, 36). Therefore, to most appropriately describe the structure of the entire signaling system, we refer to it as the “Hippo signal transduction network.” Hippo pathway

The central Hippo pathway comprises the mammalian STE20-like protein kinases 1 and 2 (Mst1 and Mst2, encoded by the genes Stk4 and Stk3, orthologs of Hippo in flies) and the large tumor suppressor kinases

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REVIEW motifs, all of which are phosphorylated by Lats1/2 (49). Phosphorylation of the best-characterized phosUpstream phosite of YAP (Ser127) leads to sequestration in the elements PPxY/F cytosol by 14-3-3 proteins (50). Mutating YAP Ser127 P Tondu to alanine prevents phosphorylation and promotes TEA constitutive activation of its transcriptional function Sav1 Mst1/2 (47, 51). Except in intestinal stem cells (52), expression P of YAP S127A increases cell proliferation and inhibits apoptosis (47, 51, 53, 54). Ser89 of TAZ is analogous to C Mob1 Lats1/2 Ser127 of YAP, and the TAZ S89A mutant is also a P constitutive transcriptional activator (30). Yap can be activated independently of Lats1/2 by phosphorylation Vgll1–3 Yap/Taz by the tyrosine kinase Yes (27, 55). Moreover, the PPxY motif of the nonreceptor tyrosine phosphatase Ptpn14 Vgll4 binds to the WW domain of Yap and inhibits nuclear Interface 1 Vgll1 D Tead1–4 localization of Yap (56–58). The serine and threonine phosphatase PP1 activates Taz by dephosphorylation Interface 2 Tead4 of Ser89 and Ser311, which induces nuclear localization CATTCC and stabilization of Taz (59). Monomethylation of Yap (MCAT) at Lys494 by Setd7 (SET domain containing lysine Interface 3 YAP methyltransferase 7) leads to cytoplasmic localization and inhibition of Yap (60). Moreover, murine Yap phosFig. 1. Protein interactions in the Hippo pathway. (A) The Hippo pathway comprises a cassette phorylated at Ser112, which is homologous to Ser127 in of serine and threonine kinases, including Mst1 or Mst2 (Mst1/2) and Lats1 or Lats2 (Lats1/2), human YAP, localizes to both the cytoplasm and the nuthat phosphorylates the functionally redundant transcriptional coactivators Yap and Taz (Yap/ cleus of cells grown at low density in culture (61). Thus, Taz). The key domains and motifs that enable protein-protein and protein-DNA interactions are Yap and Taz are inhibited and activated by proteins other WW, Tondu, and Tea domains and PPxY or PPxF motifs (PPxY/F). P, phosphorylation; Yap/Taz, than Lats kinases, suggesting that the phosphorylation Yap or Taz. (B) Structural model of the TEA domain of Tead (red, yellow) binding to DNA (white). state of YAP Ser127 and TAZ Ser89 may not be sufficient Image adapted with permission from (210). (C) Structural model of the b strands in the WW to indicate inhibition of their transcriptional activity. domain (green) of YAP in complex with a LATS1 peptide (yellow) containing the PPxY motif. In the absence of coactivators, Teads repress exThe side-chain moieties of amino acids within the WW domain (red) and the consensus residues pression of target genes (25). Yap and Taz bind to within the PPxY motif (blue) (assigned P0, P + 1, and Y + 3) are engaged in intermolecular three interfaces on Teads (32) (Fig. 1D) and thereby contacts. Credit: A. Farooq/University of Miami. (D) Structural model of the transactivation domain relieve repression and promote transcription in a manof Tead4 (red) binding to Yap (orange) at three interfaces or Vgll1 (green) at two interfaces. Image ner analogous to transcription coactivators in other reproduced with permission from (32). developmental signaling pathways. Chromatin immunoprecipitation studies in breast epithelial cells show 1 and 2 (Lats1 and Lats2, paralogous to Warts in flies) of the NDR (nuthat Yap and Tead occupy about 80% of the same genomic loci (62). Howclear dbf2-related) family (Fig. 1A) (37). Multiple proteins in the Hippo ever, Tead-dependent transcription can also occur independently of coactisignal transduction network interact using WW domains, which are vation by Yap or Taz. Like Yap and Taz, binding of some Vgll proteins can defined by two highly conserved tryptophans located 20 to 22 amino acids activate Tead-based transcription (32, 33). In contrast, Vgll4 is a repressor of apart that bind to proline-rich motifs [PPxYor PPxF (PPxY/F)] (38). Mst1 Teads (26, 63). The Tondu domains of Vgll proteins interact with Teads or Mst2 (Mst1/2) binds to the auxiliary protein Sav1 (Salvador homolog 1), (32, 33) and may compete for common binding interfaces with Yap and presumably through interactions of WW domains in Sav1 and noncanon- Taz (26) (Fig. 1D). Vgll4 has two Tondu domains, whereas other Vgll paraical PPxF motifs in Mst1/2 (39, 40), and this complex directly phosphoryl- logs have only one. ates a threonine in the kinase domain of Lats1 or Lats2 (Lats1/2) (Thr1041 of Lats1 or Thr1079 of Lats2) (41). Moreover, Mst1/2 bound to Sav1 binds Crosstalk with the Hippo pathway to and phosphorylates Mob1a or Mob1b (Mob kinase activator 1), which then binds to the autoinhibitory loop of Lats1/2 to promote autophosphor- The Hippo pathway extends beyond a simple kinase cascade, leading to ylation (Ser909 of Lats1 or Ser872 of Lats2) (41, 42). The PPxY motif of inhibition of Yap and Taz. Several proteins have been discovered to either Lats1/2 binds to the WW domains of Yap or Taz (43, 44) (Fig. 1C). Yap completely or partially bypass the Hippo pathway to target Yap and Taz. is alternatively spliced to produce variants with either one or two WW do- Likewise, Yap and Taz can influence the activity of DNA binding transcripmains (45), and Taz has one WW domain (30). Phosphorylation and action factors other than Teads (Fig. 2). tivation of Lats1/2 by Mst1/2 promote the ability of Lats1/2 to phosphorylate Cells grown on stiff substrates or at low density form stress fibers Yap or Taz (30, 34, 46, 47). Many cell adhesion and cell junction proteins can (64, 65) composed of filamentous (F)–actin, myosin II, and a-actin (66). affect the activity of Mst1/2 and Lats1/2; however, these proteins primarily Actin polymerization is required for Yap and Taz nuclear localization and have been studied in epithelial cells (14, 48), and it is unclear whether they activation by mechanical signals or low-density cell culture (61, 64, 65). have similar functions in skeletal and cardiac muscle cells. Several angiomotin proteins, which are present in skeletal muscle and the Phosphorylation of Yap or Taz by Lats1/2 occurs on HXRXXS amino heart in addition to other tissues (67), bind to F-actin and Yap and are acid motifs (46). Human YAP has five (Ser61, Ser109, Ser127, Ser164, and required for inhibition of Yap by mechanical stimuli (68). Disruption Ser381) and human TAZ has four (Ser66, Ser89, Ser117, and Ser311) HXRXXS of actin polymerization can activate Lats (69), and expression of a kinaseB

A

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REVIEW in orthotopic xenografts in mice (55). Additionally, members of the b-catenin destruction Mechanotransduction Epinephrine complex can promote the cytosolic sequestration of Yap and Taz and the ubiquitin-dependent degradation of Taz and thereby inhibit Yap- and TazFrz Lrp dependent gene expression (71, 72). G Gs PKA 12/13 Yap and Taz bind and coactivate transcripGq/11 tion factors other than Teads (36). Of these, Smads (sma gene mothers against decapentaRho F-Actin plegic peptide) and Tbx5 are especially releAxin vant to skeletal and cardiac muscle biology. APC GSK3 Cytoplasm The WW domains of Yap bind to the PPxY motifs of the phosphorylated linker region of the Hippo Sav1 Mst1/2 bone morphogenetic protein (BMP) signaling– pathway associated receptor Smad, Smad1, to promote P Smad1-dependent transcription (73). The WW domains of Yap can also bind to the PPxY motif Mob1 Lats1/2 -Cat of Smad7, an inhibitor of transforming growth P Set7 P factor–b (TGFb) signaling (74). The C termiM Yes nus of Taz binds to complexes of the TGFb Vgll1–3 -Cat Taz Yap Yap/Taz Taz Yap signaling–associated Smads, Smad2 and Smad3, and the DNA binding Smad, Smad4, in the nuVgll4 cleus and promotes the expression of Smad2Tbx5 Smad TCF Tead1–4 and Smad3-dependent genes (75). Moreover, Yap and Taz can bind to Tbx5 and promote Tbx5-dependent gene expression (55, 76). CATTCC (MCAT) Nucleus Hippo signaling can influence mTOR (mamFig. 2. Schematic of key signaling modules within the Hippo signal transduction network. As detailed in malian target of rapamycin) kinase signaling. Yap the main text, evidence suggests that crosstalk between the Hippo pathway and other signaling mod- drives the expression of miR-29, which promotes ules occurs in parallel to the core kinase cassette. P, phosphorylation; M, methylation; LPA, lysopho- the degradation of the phosphatase Pten. Pten inhibits Akt, and Akt indirectly activates mTOR. sphatidic acid; S1P, sphingosine 1-phosphate. Knockdown of Lats1 and Lats2 in cells or overexpression of Yap in the skin of mice increases dead form of Lats2 can prevent the ability of the actin depolymerizing mTOR activity (77). Mst1/2 can bind in a complex with Akt1, and knockagents to promote the cytosolic localization of Yap (69). However, knockdown of Mst1/2 reduces the activating phosphorylation of Akt1 at Ser473 down of Lats1 and Lats2 does not rescue Yap and Taz inhibition in cells grown and reduces phosphorylation of Akt substrates (78). Moreover, Akt phoson a soft extracellular matrix (65), suggesting that inhibition of Yap and Taz phorylates Mst1 at Thr120 and Thr387, leading to inhibition of Mst1 activby mechanical stimuli can function in parallel with or independent of Lats. ity (79–81). Because Yap and Taz promote cell proliferation and because G protein (heterotrimeric guanine nucleotide–binding protein)–coupled mTOR signaling is required for protein synthesis during cell proliferation, receptors (GPCRs) are a large family of seven-transmembrane receptor the mechanistic connection between Hippo and mTOR signaling suggests proteins (70), which can regulate the nuclear translocation of Yap and that cell proliferation and protein synthesis could be coordinated through Taz. Exposing cultured cells to fetal bovine serum or GPCR ligands, in- this crosstalk. cluding lysophosphatidic acid or S1P, which activates Ga12/13-, Gaq/11-, There is evidence that other signaling pathways regulate Hippo sigand Gai/d-coupled receptors, inhibits phosphorylation of Lats, Yap, and naling by altering the transcription of genes encoding proteins in the Taz, and promotes nuclear localization of Yap (69). Disruption of the actin Hippo pathway. For example, exposing cerebellar neuronal precursors to cytoskeleton by exposing cells to latrunculin B prevents the ability of ly- the secreted glycoprotein Shh (sonic hedgehog) increases Yap1 expression sophosphatidic acid or fetal bovine serum to activate Yap and Taz (69), (82). Moreover, the Notch-associated transcription factor Rbpj (recombisuggesting that the cytoskeleton may integrate mechanotransduction and nation signal binding protein for immunoglobulin kappa J) directly induces GPCR signaling. In contrast to Ga12/13-, Gaq/11-, and Gai/d-coupled receptors, transcription of Yap1 and Tead2 in mouse cortical neural stem cells (83). Sox2 the activation of GaS-coupled receptors by epinephrine or other ligands (sex determining region box 2) directly binds to the Yap1 promoter and inincreases inhibitory phosphorylation and cytosolic localization of Yap (69). creases its expression in mesenchymal stem cells (84). Moreover, the GaS increases the production of cyclic AMP (adenosine 5′-monophosphate), promoter of Yap1 contains several binding sites for the transcription factor which activates protein kinase A (PKA), and exposing cells to forskolin, GA-binding protein, which also promotes Yap1 expression (85). which increases cyclic AMP, increases phosphorylation of Lats1/2 and Yap The abundance of Yap and Taz protein is controlled by phosphorylationand promotes cytosolic localization of Yap (69). dependent degradation. Phosphorylation of Ser381 by Lats1 or Lats2 primes Multiple studies provide evidence for crosstalk between the Wnt–b- YAP for phosphorylation by casein kinases CK1d or CK1e, which creates catenin and Hippo signaling pathways (35). For example, Yap can form a a phosphodegron motif that leads to Yap degradation by the proteasome complex with b-catenin, Yes, and Tbx5 (T-box transcription factor 5) and (49). Similarly, glycogen synthase kinase 3 of the b-catenin destruction is required in colorectal cancer cell lines with high b-catenin activity for complex phosphorylates Taz, creating a phosphodegron motif and targetproliferation and colony formation in culture and tumor-forming potential ing it for degradation (86, 87).

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Wnt

S1P LPA Thrombin

Extracellular


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REVIEW Several other signaling proteins crosstalk with the Hippo pathway. For example, the kinase Lkb1 (also known as serine and threonine protein kinase 11) (88, 89) and integrin-linked protein kinase (90) interact with Hippo transcriptional regulators and may be relevant to skeletal and cardiac muscle biology. Moreover, recent proteomic studies in several cell types have identified previously uncharacterized protein interactions with members of the Hippo signal transduction network. For example, Yorkie can interact with proteins of lysosomal pathway, and knockdown of these proteins or neutralization of lysosomal pH increases the activity of a Yorkie transcriptional reporter (91). Likewise, interaction with coiled-coil domaincontaining protein 85C regulates the localization of Yap (92). Striatin-interacting phosphatase and kinase (STRIPAK) complexes bind Mst1 and Mst2, but the functional implications of this interaction are poorly understood (93, 94). Collectively, this diversity of crosstalk supports the notion that the Hippo pathway does not function in isolation but participates in various other pathways as part of a wider Hippo signal transduction network. Hippo signaling and skeletal muscle myogenesis

of Hippo signaling in activating ECR111, overexpression of YAP S127A in C2C12 myoblasts increases Myf5 expression (107). It is unknown whether Yap1 is required for Myf5 expression during developmental myogenesis in vivo because mice with global knockout of Yap1 die around E8.5 (111). Global knockout of Wwtr1, the gene encoding Taz (112), does not result in an obvious skeletal muscle phenotype, and in-depth analysis of this tissue was not the focus of that study. Additional circumstantial evidence suggests that inhibition of Yap is essential for myoblast differentiation into multinucleated myotubes. Cell culture conditions optimal for differentiating myoblasts into myotubes are similar to those that inhibit Yap activity in other cell types. Myoblasts grown to a high degree of confluence maximize cell-cell contact, which is known to inhibit Yap in other cell types (46, 65) and enhances myoblast differentiation (113). Similarly, myoblast differentiation is enhanced when cells are cultured on soft hydrogel substrates with a stiffness of about 12 kPa similar to the stiffness of muscle, rather than directly on plastic, which is much stiffer (114). Reducing the concentration of serum in the medium, which should inhibit Yap by reducing activation of GPCR signaling (69), also promotes terminal differentiation of C2C12 myoblasts (113). Together, these results imply that inhibition of Yap may be a key event for the terminal differentiation of myoblasts into myotubes. In contrast to the observation that active Yap prevents the differentiation in myoblasts (107) and activated satellite cells (108), some genes with MCAT motifs are expressed in differentiated muscle. In zebrafish, activation of a transgenic reporter for Tead-dependent transcription is highly abundant in differentiated trunk muscles at 2 and 3 days after fertilization (Fig. 3A) (115). Similarly, the expression of genes that encode proteins characteristic of differentiated muscle, such as cardiac troponin T (Tnnt2) (116) and a-actin (117) in mouse, relies at least partially on MCAT response elements (118). One possible explanation is that different Tead, Yap, Taz, and Vgll complexes may selectively target myoblast or differentiated muscle genes, leading to differential expression. Similar target specificity occurs in flies where the homologs of Vgll and Teads homologs form complexes that target different genes than complexes of Yorkie and Tead homologs (26, 119). In mammals, Yap1 is highly expressed in myoblasts and inhibits differentiation (107, 108), whereas Wwtr1 [Taz (120–122)], Vgll2 (123–126), and Tead4 (127) are more highly expressed in differentiated muscle and promote differentiation. A second possibility is that different Tead, Yap, Taz, and

Several studies implicate the Hippo signal transduction network in muscle development, regeneration, and disease. Skeletal muscle myogenesis begins around embryonic day 8 (E8) in mice, when Pax3 (paired box protein-3)–positive cells in the dermomyotome begin to undergo epithelialto-mesenchymal transitions, delaminate, and migrate (95). Migratory Pax3positive cells begin to express the myogenic regulatory transcription factors Myf5 and MyoD (MyoD is expressed later than Myf5) (96–98), which promote the specification of Pax3-positive progenitor cells into mononucleated myoblasts (98). Overexpression of Myf5 or MyoD in non-muscle cells is sufficient to initiate myogenesis in vivo (99), suggesting that these factors function redundantly (100, 101). MyoD induces the expression of the transcription factors myogenin and Mrf4, causing myoblasts to undergo terminal differentiation, in which they fuse into multinucleated myotubes that mature into striated muscle fibers (98). MyoD has been reported to bind more than 20,000 DNA loci in myoblasts and multinucleated myotubes (102). Although some of these loci might be detected due to nonspecific MyoD binding (103), the large number of sites is consistent with the idea that MyoD acts as a “pioneer” transcription factor. Pioneer transcription factors stably bind and “preselect” a set of genes to be expressed in a given cell lineage (104). Consistent with this model, MyoD recruits histone methyltransferases and aceC tyltransferases to the enhancers and promoters of myoblast-associated genes, which prepares the A chromatin for active transcription (105, 106). There is both direct and indirect evidence that the Hippo signal transduction network is involved in the regulation of myogenic differentiation in cultured myoblasts in vitro and during embryonic B myogenesis in vivo. Studies suggest that Yap is Basal lamina active in myoblasts and inhibits terminal differentiation into myotubes (107, 108). In Xenopus laevis embryos, overexpression of constitutively Plasmalemma active Yap increases proliferation of neural progenitor cells and reduces the expression of markers of somatic muscle differentiation, including Fig. 3. The Hippo signal transduction network and skeletal muscle. (A) Green fluorescent protein MyoD (109). The evolutionarily conserved ECR111 (GFP) fluorescence in a 4xGTIIC:dGFP (MCAT reporter) zebrafish at 2 days after fertilization. Note enhancer, which contains an MCAT motif that the intense GFP signal in skeletal muscle (arrowheads). Image reproduced with permission from binds Teads, is located ~111 kilo–base pairs (115). (B) Transmission electron micrograph of a satellite cell (sc) between the plasmalemma and upstream of the Myf5 gene and is required for basal lamina. Image reproduced with permission from (4). (C) Immunofluorescence of ex vivo the expression of Myf5 in ventral somatic com- muscle fibers showing that Yap abundance (red) is higher in activated (48 hours, MyoD+) than in partments (110). Consistent with a potential role quiescent (0 hours, Pax7+) satellite cells. Image reproduced with permission from (108).


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REVIEW Vgll complexes target the same genes but exert different effects. During neurogenesis, the transcription factors Sox2, Sox3, and Sox11 are expressed at different stages of neuronal differentiation and bind to identical DNA loci; however, the function of the different isoforms can be either to preselect genes for expression or to actively promote their expression (128). A third possibility is that Yap or Taz could not only coactivate MCAT elements but also bind and coactivate additional transcription factors such as Smads (75, 129) or Tbx5 (55, 76) to drive myoblast or myotube and differentiated muscle-specific gene expression. The Hippo pathway in satellite cells and skeletal muscle regeneration

In skeletal muscle, nuclei within differentiated muscle fibers do not divide and, thus, do not contribute to regeneration. Resident Pax7-expressing stem cells, called satellite cells (4), proliferate and differentiate in response to injury to give rise to new muscle (130). Satellite cells reside between the basal lamina and the plasma membrane of differentiated muscle cells (4, 5, 131) (Fig. 3B). The nuclei of satellite cells account for between 1.4 and 7.3% of all nuclei within an adult human muscle (132). Satellite cells undergo selfrenewing cell divisions and, when stimulated, can differentiate into muscle fibers (2). Satellite cells are essential for regeneration (3), but not for shortterm hypertrophy after overload (133). Hippo signaling is likely to play a role in the proliferation and differentiation of satellite cells. The expression of Yap1 increases about threefold during activation of mouse satellite cell grown in mitogen-rich medium (108) (Fig. 3C), and overexpression of human YAP S127A in activated satellite cells increases proliferation and inhibits differentiation (108), consistent with the activation and function of Yap in other stem and progenitor cells (51, 53, 54). Additional evidence suggests that Yap activity in satellite cells may be regulated by pathways that crosstalk to the Hippo pathway. S1P, which activates Yap through GPCR signaling (69, 134), promotes the proliferation of satellite cells (135). Notch, which can increase the expression of Yap1 in cortical neural stem cells (83), promotes satellite cell proliferation (136). Recent reports suggest that Notch can also promote satellite cell self-renewal (137) and niche colonization (138). Wnt signaling regulates myogenesis during development and differentiation of satellite cells (139). Yap activates the expression of Bmp4 in satellite cells (108), and Bmp4 protein promotes proliferation and inhibits differentiation of satellite cells (140). Moreover, overexpression of YAP S127A in muscle satellite cells increases the expression of genes encoding proteins angiomotin-like 2 and Frmd6 (also known as Willin) and decreases the expression of genes that encode GPCRs (107). Because these changes in gene expression should result in inhibition of Yap activity (68, 141, 142), this observation suggests that negative feedback mechanisms may serve to limit Yap-dependent cell proliferation. Thus, these data suggest that Yap may be an important regulator of the proliferation of activated satellite cells, and future studies should test the role of Yap on skeletal muscle regeneration in vivo. The Hippo pathway in terminally differentiated skeletal muscle

In adult humans, muscle fibers can be up to ~20 cm long (143), and a single fiber may contain several tens of thousands of nuclei (144). The human vastus lateralis of young males comprises ~400,000 to 900,000 muscle fibers, and the number of fibers decreases during aging (145). Fibers can be distinguished into slow type I, intermediate type IIa, and fast type IIx and IIb fibers based on the presence of myosin heavy chain isoforms and on the abundance and isoforms of other motor, metabolic, and mitochondrial proteins (146, 147). The number of fi-

bers and the percentage of different fiber types vary greatly both within and between differing muscles of the body and among individuals (145, 148, 149). Muscle fibers hypertrophy in response to overload (for example, as a result of resistance training), and increase mitochondrial biogenesis and change the concentrations and isoforms of motor and metabolic proteins in response to endurance training (147). Thus, differentiated skeletal muscle has a high degree of plasticity, enabling it to change its force production and metabolic capacity in response to various types of stimuli. Hippo signaling may regulate gene expression in differentiated skeletal muscle. Genes and reporter genes with MCAT elements are actively expressed in differentiated skeletal muscle (115–117, 150). Acute resistance exercise increases expression of the genes encoding cysteine-rich angiogenic inducer 61 and connective tissue growth factor (Ctgf) (151), the latter of which has three MCAT elements in its proximal promoter (62). These genes are frequently used as marker genes for Yap and/or Taz activity [for example (152)], suggesting that Yap or Taz may be activated by acute resistance exercise. Teads have been shown to regulate a-actin promoter activity in a model of stretch overload–induced hypertrophy in chicken (117). Overexpression of Tead1 in muscle fibers in mice causes a fast-to-slow fiber type transition, but not hypertrophy (153). Moreover, denervation of fast, but not slow, skeletal muscle induces atrophy and increases the expression of Mst1 (154). Mst1 can phosphorylate Ser207 of the forkhead transcription factor Foxo3a, promoting muscle atrophy in mice (154) and thereby increasing the expression of genes encoding skeletal muscle atrophy–regulating factors known as atrogins (155). Future studies are needed to clarify the role of the Hippo pathway in overload-induced hypertrophy, the regulation of muscle fiber type–specific gene expression, muscle atrophy, and other related phenomena. Hippo pathway and myopathies

Perturbation of Hippo signaling may contribute to the pathology of different myopathies, including muscular dystrophies (10). We recently found that the expression of human YAP S127A in muscle fibers in adult mice causes a “fulminant” myopathy characterized by atrophy, signs of centronuclear myopathy, deterioration, and, after several weeks, death of the mice (156). The WW domain–containing protein Bag3 (Bag family molecular chaperone regulator 3) interacts directly with the Hippo pathway proteins angiomotin 1, angiomotin 2, and Lats1, and is a positive regulator of Yap and Taz and of Ctgf expression (157). Bag3 knockout causes a fulminant muscular dystrophy in mice (158), and in humans, loss-of-function mutations of Bag3 are associated with severe childhood muscular dystrophy (159). Loss-of-function mutations in the WW domain–containing protein dystrophin (DMD) causes either Duchenne’s (severe) or Becker’s (mild) muscular dystrophy in humans (10, 160). It is unknown whether DMD, or the related protein utrophin, uses WW domains to interact with the Hippo pathway and whether mutations in these proteins perturb Hippo signaling and thereby contribute to the pathology of muscular dystrophies. However, considering the relatively small number of human proteins with WW domains (39), it is intriguing that mutations in these proteins cause human muscular dystrophy. Other studies further support a role for perturbation of Hippo signaling in myopathies. The Hippo pathway target gene CTGF is highly expressed in muscles of patients with muscular dystrophy (161). Overexpression of Ctgf in mouse skeletal muscle is sufficient to cause muscular dystrophy (162), whereas knockout of Ctgf in a mouse model of muscular dystrophy partially ameliorates the pathology (163). Laminopathies, which can result in muscular dystrophy, are diseases that result from mutation in LMNA, which encodes the nuclear lamina protein lamin A/C (164). Human


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REVIEW myoblasts with LMNA mutations have impaired ability to produce appropriate cytoskeletal rearrangements in response to mechanical stimuli, such as the changes in substrate stiffness (165). Consistent with the role of Hippo in mechanotransduction (64, 65), YAP is more active, and CTGF expression is higher in LMNA mutant compared to wild-type myoblasts cultured on substrates with the same stiffness (165). Collectively, these results suggest that dysregulation of Hippo signaling in skeletal muscle due to the mutations in DMD, BAG3, or LMNA may result in changes in expression of Hippo target genes such as CTGF and thereby contribute to pathology. Myopathies resulting from nongenetic causes may also involve Hippo signaling. Statins are widely prescribed drugs that act on the mevalonate pathway, the activation of which increases Yap and Taz transcriptional activity (166, 167). Statins induce myopathies with symptoms ranging from muscle weakness to rhabdomyolysis in as many as 1.5 million patients per year (168). Thus, investigating whether the inhibition of Yap and Taz contributes to statin-induced myopathies may have important clinical ramifications. Hippo and rhabdomyosarcoma

Rhabdomyosarcomas, diagnosed on the basis of the presence of rhabdomyoblasts, are the most common soft tissue sarcomas in children and adolescents (11). Rhabdomyosarcomas are classified as embryonal (ERMS), alveolar (ARMS), and pleomorphic (anaplastic). ERMS occur in infants and young children, ARMS occur in adolescents and young adults and have a poorer prognosis than ERMS, and pleomorphic rhabdomyosarcomas occur in adults and are relatively rare (11). About 70 to 80% of ARMS express PAX3-FOXO1 or PAX7-FOXO1 fusion genes, which indicates a poorer prognosis than other ARMS (169). Expression of PAX3-FOXO1 in mice with homozygous loss of the gene encoding tumor protein 53 (p53) or homozygous loss of the gene encoding cyclin-dependent kinase inhibitor 2A (CDKN2A, also known as p16/Ink4A) gives rise to tumors that resemble human ARMS (170), supporting the tissue-specific, oncogenic potential of this fusion gene. Given that YAP S127A overexpression drives proliferation and inhibits differentiation of C2C12 myoblasts (107) and activated satellite cells (108), deregulation of Hippo signaling may contribute to the pathogenesis of rhabdomyosarcoma. The Ras association (RalGDS/AF-6) domain family (Rassf ) of proteins contain SARAH (Salvador, Rassf, and Hippo) domains that bind to and inhibit Mst1 and Mst2 (171). The expression of the tumor suppressor RASSF4 is increased in PAX3-FOXO1–positive ARMS (172). The PAX3FOXO1 fusion protein directly binds to the 5′ enhancer of RASSF4, and knockdown of RASSF4 in cultured ARMS cell lines reduces cell proliferation and the survival of mice with ARMS xenotransplants (172). Similar to other Rassf proteins, RASSF4 binds and inhibits MST1 in human myoblast and ARMS cell lines (172); however, a functional connection between RASSF4 and YAP in ARMS has not been identified. Hippo signaling may also be important in the development of ERMS. YAP is more abundant in the nucleus of ERMS compared to ARMS patient samples, which, in some cases, may be explained by increased copy number of the YAP1 locus (173). Overexpression of YAP S127A in activated, but not quiescent, satellite cells causes ERMS-like tumors in mice with high penetrance and a short latency to tumor onset. Cessation of YAP S127A transgene expression in YAP S127A–driven ERMS-like tumors in mice or a knockdown of YAP in human ERMS cells causes differentiation of tumor cells into myosin heavy chain–expressing muscle fibers. Knockdown of YAP reduces proliferation and anchorage-independent growth in human ERMS cells in culture and decreases the tumor burden in mice with human ERMS xenotransplants. Combined analyses of Yap and Tead1 genome-wide chromatin immunoprecipitation and quantitative reverse transcription polymerase chain reaction (RT-qPCR) studies and cDNA microarrays of YAP S127A–driven ERMS-like tumors suggest that in mouse

ERMS myoblasts, Yap and Tead1 bind to and increase the expression of genes that regulate cell proliferation (Ccnd1 and Cdc6), as well as oncogenes and cancer-related genes (Met, Myc, and Birc5), and conversely repress the expression of genes typically expressed in terminally differentiated skeletal muscle (Myl4, Myh2, Mybph, and Tnnc2) (173). Other studies provide additional evidence for a connection between Hippo signaling and rhabdomyosarcoma. One case of a spindle cell variant of ERMS contained a TEAD1-NCOA2 fusion gene (174), and knockdown of the tyrosine kinase Yes, which binds and phosphorylates Yap (27, 55), reduces proliferation of ARMS and ERMS cell lines (175). Collectively, these studies suggest that Hippo pathway dysregulation causes or contributes to rhabdomyosarcoma, identifying the Hippo pathway as a treatment target for these cancers. The Hippo pathway in heart development

The heart is a heterogeneous organ comprising cardiomyocytes, endocardial cells, valvular components, connective tissues, cells of the electrical conduction system, as well as the smooth muscle and endothelial cells of the coronary arteries and veins (176). It develops from mesodermal progenitor cells located in the anterior region of the primitive streak and the proepicardium (176). These cardiac progenitor populations migrate away from the primitive streak and give rise to the primary and secondary heart fields. Cells in the primary heart field differentiate and form a linear heart tube that begins to beat. The heart tube grows unevenly, using cells from the primary heart field to form the lower bulk of the heart. Cells from the secondary heart field migrate to the heart tube to form the outflow tract. Heart looping enables the developing heart tube to fold within the pericardial cavity (6, 176). The growth of the heart during embryonic development is driven by cardiomyocyte and precursor cell proliferation, but just after birth, cardiomyocyte proliferation stops and heart growth occurs primarily by cardiomyocyte hypertrophy (176). These processes are regulated through complex intrinsic and extrinsic signaling events among cells in both heart fields. Several lines of evidence suggest that Hippo signaling is involved in heart development. Pathways that crosstalk with the Hippo pathway, including BMP, Wnt, Notch, and Shh (51, 54, 71, 177, 178), regulate transcription factors critical for heart development. The genes encoding the transcription factors Gata4 and Nkx2.5 are expressed in the primary and secondary heart fields, along with the gene encoding Tbx5, which is expressed only in the primary heart field, and these proteins work in concert to promote cardiomyocyte differentiation and heart maturation (179). Tbx5, which can be coactivated by both Yap and Taz (55, 76), is a critical mediator of embryonic heart development (180), as mutations in TBX5 cause Holt-Oram syndrome, which is characterized by heart and limb abnormalities (181). Conditional deletion of Sav1, Lats2, or Mst1 and Mst2 in Nkx2.5-positive cardiomyocytes in mice increases proliferation, leading to cardiomegaly and perinatal lethality (Fig. 4A) (182). Crossing Sav1 conditional knockout mice to those with heterozygous deletion of Ctnnb1, which encodes b-catenin, rescues cardiomyocyte hyperproliferation, indicating that Wnt–b-catenin signaling acts downstream of Hippo signaling (182). Cardiomyocytes from Sav1 conditional knockout mice have increased expression of Wnt–b-catenin target genes, including the transcription factors Sox2 and Snail2. Because Sox2 increases the expression of Yap (84), this could represent a positive feedback mechanism. Moreover, using either Nkx2.5- or Tnnt2-Cre–mediated excision to create conditional deletion of Yap1 in cardiomyocytes during embryonic development results in lethality between E10.5 and E16.5 (183, 184). Hearts of these mice have normal cardiac looping and chamber formation, but reduced cardiomyocyte proliferation and smaller, thinner ventricles, indicating that Yap is an important regulator of embryonic cardiac growth.


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REVIEW Whether Hippo signaling also plays a role in cardiac hypertrophy is less clear. Conditional deletion of Yap1 in cardiomyocytes in Tnnt2-Cre mice during development does not affect the size of cardiomyocytes (184). Likewise, postnatal retroorbital injection (192) of adenovirus encoding GFP and Tnnt2-Cre into mice with heterozygous floxed alleles of Yap1 results in targeted deletion of Yap1 3 mM 3 mM in a small percentage of cardiomyocytes. GFPpositive (Yap1-deleted) cardiomyocytes have normal size at baseline and after pressure overload stress (184). Likewise, aMHC promoter–driven YAP S127A or S112A in the heart of postnatal mice does not alter cardiomyocyte size (184, 189). In contrast, conditional overexpression of wild-type Yap in neonatal cardiomyocytes increases size and induces expression of genetic markers of the hypertrophic fetal cardiomyocytes, including genes that encode 3000 µM 3000 µM atrial natriuretic factor, brain natriuretic peptide, and b-myosin heavy chain (190). Exposing cultured Fig. 4. The Hippo signal transduction network and the heart. (A) Conditional cardiomyocytespecific knockout of Sav1 (Salv) results in cardiac hypertrophy (cardiomegaly) associated with cardiomyocytes to the GPCR agonist phenylephrine increased cardiomyocyte proliferation. ra, right atrium; la, left atrium; rv, right ventricle; lv, left induces hypertrophy and increases expression of ventricle. Image reproduced with permission from (182). (B) Serial sections of Masson’s trichrome– atrial natriuretic factor mRNA, and expression of short hairpin RNAs targeting endogenous Yap1 atstained wild-type (WT) and aMHC promoter–driven Yap S112A transgenic (Tg1 and Tg2) hearts showing scar tissue (blue) 21 days after transient ligation of the left anterior descending artery at tenuates the effects of phenylephrine in other cell types (69), suggesting that phenylephrine may inpostnatal day 7. Image reproduced with permission from (189). hibit Hippo signaling in these cells. Moreover, adult mice with conditional deletion of Yap1 with aMHCThe Hippo pathway in cardiac regeneration and growth Cre have reduced cardiomyocyte hypertrophy in response to myocardial ischemia (190). Thus, Yap can either promote or inhibit cardiomyocyte The potential for cardiomyocyte proliferation in adult mammals is low hypertrophy, a discrepancy that may be explained by the method of gene and declines with age (185–187). Therefore, after injury, the adult mam- targeting, the timing or duration of Yap depletion, the type of myocardial malian myocardium replaces lost cardiomyocytes with fibrotic scar tissue, stress, or the mutational status of Yap1. Nevertheless, these studies reveal and the functional output of the heart is reduced. In contrast, the neonatal that Yap, and potentially Hippo signaling, can influence cardiomyocyte mouse heart can regenerate after partial resection or ischemia within the hypertrophy. first week after birth (7). In these animals, mature cardiomyocytes, rather Mst1 is activated by oxidative stress and promotes cardiomyocyte than distinct cardiac stem or progenitor cells, undergo proliferation to pro- death (193, 194). Overexpression of Mst1 promotes apoptosis of cultured mote regeneration (188). In neonatal mice, conditional deletion of Yap1 in cardiomyocytes. In contrast, the inhibition of endogenous Mst1 by exprescardiomyocytes, using aMHC-Cre, impairs heart regeneration after ische- sion of a kinase-inactive (K59R) variant, which functions as a dominant mia (189). In contrast, cardiomyocyte-specific overexpression of constitu- negative, reduces cardiomyocyte apoptosis induced by pharmacological tively active (S112A) murine Yap (homologous to human YAP S127A) inhibition of protein kinase C or protein phosphatases (195). Furthermore, reduces fibrosis and promotes cardiac regeneration beyond postnatal cardiomyocyte-specific overexpression of Mst1 causes a dose-dependent day 7 (Fig. 4B) (189). This is consistent with the observation that over- increase in cardiomyocyte apoptosis: mice with low amounts of Mst1 expression of active Yap promotes cardiomyocyte proliferation both in vivo overexpression have a modest increase in basal apoptosis, whereas mice and in cultured cardiomyocytes (183, 184, 190). In response to myocardial with higher expression have a robust increase in apoptosis and rapidly infarction, Yap can be found in the nuclei of cardiomyocytes surrounding progress to dilated cardiomyopathy, heart failure, and premature death the site of injury (190), suggesting increased activation of Yap at the border (195). The SARAH domain of Rassf1a binds to Mst1 and promotes of the infarcted area. Moreover, adult mice with conditional Yap1 knockout its activation in response to pressure overload in mouse hearts (196). using aMHC-Cre display greater injury, increased apoptosis, and reduced Cardiomyocyte-specific overexpression of Rassf1a increases Mst1 activaproliferation of cardiomyocytes after myocardial infarction (190). However, tion and exacerbates cardiac dysfunction induced by pressure overload. In it is unknown whether Yap is involved in cardiac remodeling and myocar- contrast, overexpression of Rassf1a with a mutation in the SARAH dodial regeneration after myocardial infarction, and if so, which cells express main (L308P), which cannot bind Mst1, acts as a dominant negative Yap1, how are they activated, and how do they contribute to the functional and protects against cardiac injury in this context. Furthermore, cardiomyorecovery of the heart. Conditional deletion of Sav1, or Lats1 and Lats2, in cyte-specific deletion of Rassf1A attenuates Mst1 activation and is protective cardiomyocytes using Nkx2.5-Cre stimulates proliferation in uninjured against injury and heart failure due to pressure overload. Knockout of hearts and increases proliferation, reduces fibrosis, and improves cardiac Rassf1A in mice does not protect against fibrosis and cardiac hypertrophy function in response to partial heart resection at postnatal day 8 in mice, induced by tumor necrosis factor–a (196), indicating cell type–specific effects as well as myocardial infarction in adult mice (191). Thus, inhibition of of Rassf1A in heart. Hippo signaling promotes heart regeneration through increased cardioYap is also important for heart homeostasis in adult mice. Homozygous myocyte proliferation. deletion of Yap1 in cardiomyocytes of postnatal mice using aMHC-Cre A

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REVIEW results in a rapidly developing dilated cardiomyopathy, and these mice die of heart failure by 12 weeks of age (190). Conditional knockout of Yap1 in the heart leads to robust increases in cardiomyocyte apoptosis and fibrosis with suppressed cardiac function, possibly due to suppression of the prosurvival kinase Akt. Activation of Hippo signaling may contribute to arrhythmogenic cardiomyopathy. Neurofibromin 2 (Nf2), a cytoskeletal protein that modulates Yap activity through physical interaction with the Hippo pathway members Ww45 and Kibra (197), and Mst1 are activated in a mouse model of arrhythmogenic cardiomyopathy and in human patients (198). The inactivation of Yap in these contexts may contribute to increased adipogenesis, which is a hallmark and contributing factor in arrhythmogenic cardiomyopathy. Thus, Yap is likely a mediator of cardiomyocyte survival, proliferation, and signal transduction in the adult mammalian heart. The myocardin family of coactivators bind to the transcription factor Srf (serum response factor) and may affect both Yap and Srf signaling in muscle cells (199). Myocardins inhibit skeletal muscle differentiation (200) and drive the expression of contractile genes in smooth muscle and cardiac cells (201). Yap physically interacts with myocardin and inhibits its ability to elicit expression of contractile genes through interaction with Srf (199). The presence of a PPxY motif in myocardin suggests that it could bind to the WW domains of Yap, although this has not been empirically tested. The Hippo signal transduction network as a drug target

Given the importance of Hippo signaling in skeletal and cardiac muscle biology, drugs that target this pathway may be relevant for therapeutic purposes. Verteporfin, which is used as a photosensitizer in photodynamic therapy in the eye (202), can disrupt the interaction between Yap and Tead in human embryonic kidney (HEK) 293 cells, inhibit activation of Tead target genes, and reverse Yap-induced hepatomegaly in vivo (203). Therefore, verteporfin may be useful in treating hyperproliferative muscle disorders, such as rhabdomyosarcoma. In addition, drugs that target GPCRs, such as b-blockers (69, 204–206), or the mevalonate pathway, such as statins (166, 167), potentially could be used to target the Hippo pathway in muscle disease or to promote regeneration. Statins and GPCR-targeting drugs are among the most widely prescribed drugs, and it has been estimated that 30 to 50% of all medications exert their effect via GPCRs (207). Although not all statins and GPCR-targeted drugs necessarily affect Hippo signaling, this suggests that the effects and side effects of these drugs should be considered in light of potential impact on Hippo signaling. The availability of approved drugs that may target the Hippo pathway provides an exciting opportunity to test whether they can be used to treat skeletal muscle or heart diseases. Conclusions and outlook

Since the late 1980s, proteins in the Hippo signal transduction network have been identified as regulators of skeletal and cardiac muscle gene expression, development, organ growth, stem cell function, regeneration, and disease. Additionally, in nonmuscle cells, proteins in the Hippo pathway interact with proteins involved in skeletal and cardiac muscle biology, suggesting that these mechanisms may exist in muscle and the heart. However, our knowledge of the Hippo signal transduction network in skeletal and cardiac muscle is modest compared to that of other signal transduction pathways in these tissues. Thus, there are still many unanswered questions in this field, for example: • Can information from the ENCODE project (208) and other genomewide analyses that have fundamentally changed our understanding of chromatin and gene regulation lead to better understanding of the regulation of gene expression by the Hippo pathway? Genome-wide chromatin immunoprecipitation analysis shows that Yorkie DNA binding correlates

with the activating chromatin mark, trimethylation of Lys4 of histone H3. The same study also shows that Yorkie directly binds to chromatin-remodeling complexes (209). Thus, do Yap and Taz have similar functions to Yorkie? How do the Hippo transcriptional regulators interact with chromatin and chromatin-remodeling proteins throughout the mammalian genome, especially in skeletal muscle and heart cells, and during tumorigenesis in rhabdomyosarcoma? • Given that Yap and Taz typically promote cell proliferation, which is associated with progressive shortening of telomeres, is there a relationship between the Hippo pathway and aging in skeletal muscle and the heart? • Is the Hippo pathway involved in regulating the skeletal and heart muscle adaptation to endurance and resistance exercise training or in mediating other forms of muscle plasticity? • Given that several approved drugs target Hippo signaling, can these drugs be used to treat skeletal and heart muscle diseases, including muscular dystrophy, cellular damage after myocardial infarction, and rhabdomyosarcoma? Are there other molecules that can target Hippo signaling specifically through Hippo pathway proteins that are only present in muscle? REFERENCES AND NOTES 1. A. Studitsky, Free auto- and homografts of muscle tissue in experiments on animals. Ann. N.Y. Acad. Sci. 120, 789–801 (1964). 2. C. A. Collins, I. Olsen, P. S. Zammit, L. Heslop, A. Petrie, T. A. Partridge, J. E. Morgan, Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289–301 (2005). 3. C. Lepper, T. A. Partridge, C. M. Fan, An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development 138, 3639–3646 (2011). 4. A. Mauro, Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, 493–495 (1961). 5. J. Scharner, P. S. Zammit, The muscle satellite cell at 50: The formative years. Skelet. Muscle 1, 28 (2011). 6. M. Xin, E. N. Olson, R. Bassel-Duby, Mending broken hearts: Cardiac development as a basis for adult heart regeneration and repair. Nat. Rev. Mol. Cell Biol. 14, 529–541 (2013). 7. E. R. Porrello, A. I. Mahmoud, E. Simpson, J. A. Hill, J. A. Richardson, E. N. Olson, H. A. Sadek, Transient regenerative potential of the neonatal mouse heart. Science 331, 1078–1080 (2011). 8. K. D. Poss, L. G. Wilson, M. T. Keating, Heart regeneration in zebrafish. Science 298, 2188–2190 (2002). 9. M. V. Narici, N. Maffulli, Sarcopenia: Characteristics, mechanisms and functional significance. Br. Med. Bull. 95, 139–159 (2010). 10. J. C. Kaplan, The 2012 version of the gene table of monogenic neuromuscular disorders. Neuromuscul. Disord. 21, 833–861 (2011). 11. D. M. Parham, D. A. Ellison, Rhabdomyosarcomas in adults and children: An update. Arch. Pathol. Lab. Med. 130, 1454–1465 (2006). 12. C. Keller, D. C. Guttridge, Mechanisms of impaired differentiation in rhabdomyosarcoma. FEBS J. 280, 4323–4334 (2013). 13. S. Hettmer, A. J. Wagers, Muscling in: Uncovering the origins of rhabdomyosarcoma. Nat. Med. 16, 171–173 (2010). 14. F. X. Yu, K. L. Guan, The Hippo pathway: Regulators and regulations. Genes Dev. 27, 355–371 (2013). 15. A. M. Tremblay, F. D. Camargo, Hippo signaling in mammalian stem cells. Semin. Cell Dev. Biol. 23, 818–826 (2012). 16. J. S. Mo, H. W. Park, K. L. Guan, The Hippo signaling pathway in stem cell biology and cancer. EMBO Rep. 15, 642–656 (2014). 17. D. Pan, Hippo signaling in organ size control. Genes Dev. 21, 886–897 (2007). 18. K. F. Harvey, X. Zhang, D. M. Thomas, The Hippo pathway and human cancer. Nat. Rev. Cancer 13, 246–257 (2013). 19. I. K. Hariharan, D. Bilder, Regulation of imaginal disc growth by tumor-suppressor genes in Drosophila. Annu. Rev. Genet. 40, 335–361 (2006). 20. N. Tapon, K. F. Harvey, D. W. Bell, D. C. Wahrer, T. A. Schiripo, D. A. Haber, I. K. Hariharan, salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines. Cell 110, 467–478 (2002). 21. D. Pan, The Hippo signaling pathway in development and cancer. Dev.Cell 19, 491–505 (2010). 22. I. Davidson, J. H. Xiao, R. Rosales, A. Staub, P. Chambon, The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence. Cell 54, 931–942 (1988).


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Veeraraghavan, Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain. Proc. Natl. Acad. Sci. U.S.A. 103, 17225–17230 (2006). Acknowledgments: We would like to thank A. Farooq, B. Link, J. Miesfeld, W. J. Hong, A. Pobbati, S. Veeraraghavan, T. Heallen, and J. F. Martin for sending high-resolution figures of their work. Funding: Research in the Aberdeen lab has been supported by a Medical Research Council project grant (99477; H.W., P. S. Zammit, and C. De Bari), Sarcoma UK grant (H.W., G. Murray, R. Urcia, and M. Jaspars), Friends of Anchor grant (H.W. and C. De Bari), and a grant by Tenovus Scotland (G11/05; C. De Bari and H.W.). Research in the Sudol lab was supported by the Mechanobiology Institute of the National University of Singapore. Research in the Sadoshima lab has been supported by NIH grants HL112330, HL102738 (J.S.), and HL122669 (D.P.D.R.) and American Heart Association Scientist Development grant 11SDG7240066 (D.P.D.R.). Competing interests: The authors declare that they have no competing financial interests. Submitted 22 January 2014 Accepted 2 July 2014 Final Publication 5 August 2014 10.1126/scisignal.2005096 Citation: H. Wackerhage, D. P. Del Re, R. N. Judson, M. Sudol, J. Sadoshima, The Hippo signal transduction network in skeletal and cardiac muscle. Sci. Signal. 7, re4 (2014).


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Exercise-induced signal transduction and gene regulation in skeletal muscle.

The Hippo signal transduction pathway in soft tissue sarcomas.

Charge movement and the nature of signal transduction in skeletal muscle excitation-contraction coupling.

Acute acidosis attenuates leucine stimulated signal transduction and protein synthesis in rat skeletal muscle.

Angiogenesis in skeletal and cardiac muscle.

An integrated signal transduction network of macrophage migration inhibitory factor.

Assessment of signal transduction mechanisms regulating airway smooth muscle contractility.

Signal transduction.

Signal transduction.

Signal transduction in atherosclerosis: integration of cytokines and the eicosanoid network.

Oncogenes and signal transduction.

Signal transduction in erythropoiesis.

Signal transduction in bacteria.

Signal transduction in cancer.

Hippo Pathway and Skeletal Muscle Mass Regulation in Mammals: A Controversial Relationship.

Signal transduction: the nuclear target.

Alcohol intoxication following muscle contraction in mice decreases muscle protein synthesis but not mTOR signal transduction.

NF-κB signal transduction network: a long lasting relationship in Philadelphia positive Leukemias.

microRNA and thyroid hormone signaling in cardiac and skeletal muscle.

The PtdIns-PLC superfamily and signal transduction.

Signal, transduction, and the hematopoietic stem cell.

Signal transduction and gene control.

In utero Undernutrition Programs Skeletal and Cardiac Muscle Metabolism.

Signal transduction mechanisms in cancer.