RESUMO
Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. Small interfering RNA knockdown or retroviral-mediated expression of wild-type human or constitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a function shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene Wwtr1-/- ) knockout mice, there were no overt effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapfl °x/fl °x :Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some genes independently of YAP, including myogenic genes such as Pax7, Myf5, and Myod1 (ArrayExpress-E-MTAB-5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in myogenesis and aspects of cytoskeleton organization (ProteomeXchange-PXD005751). Neither TAZ nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to enhance myogenic differentiation. Stem Cells 2017;35:1958-1972.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Músculo Esquelético/citologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Fusão Celular , Proliferação de Células , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Via de Sinalização Hippo , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células-Tronco/citologia , Transativadores , Via de Sinalização Wnt/genética , Proteínas de Sinalização YAPRESUMO
Estrogen-related receptor alpha (ERRalpha) and proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) play central roles in the transcriptional control of energy homeostasis, but little is known about factors regulating their activity. Here we identified the homeobox protein prospero-related homeobox 1 (Prox1) as one such factor. Prox1 interacts with ERRalpha and PGC-1alpha, occupies promoters of metabolic genes on a genome-wide scale, and inhibits the activity of the ERRalpha/PGC-1alpha complex. DNA motif analysis suggests that Prox1 interacts with the genome through tethering to ERRalpha and other factors. Importantly, ablation of Prox1 and ERRalpha have opposite effects on the respiratory capacity of liver cells, revealing an unexpected role for Prox1 in the control of energy homeostasis.
Assuntos
Metabolismo Energético , Proteínas de Homeodomínio/metabolismo , Receptores de Estrogênio/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Células Hep G2 , Proteínas de Homeodomínio/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores de Estrogênio/genética , Regulon/genética , Transativadores/genética , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Sarcomas are rare cancers (≈1% of all solid tumours) usually of mesenchymal origin. Here, we review evidence implicating the Hippo pathway in soft tissue sarcomas. Several transgenic mouse models of Hippo pathway members (Nf2, Mob1, LATS1 and YAP1 mutants) develop various types of sarcoma. Despite that, Hippo member genes are rarely point mutated in human sarcomas. Instead, WWTR1-CAMTA1 and YAP1-TFE3 fusion genes are found in almost all cases of epithelioid haemangioendothelioma. Also copy number gains of YAP1 and other Hippo members occur at low frequencies but the most likely cause of perturbed Hippo signalling in sarcoma is the cross-talk with commonly mutated cancer genes such as KRAS, PIK3CA, CTNNB1 or FBXW7. Current Hippo pathway-targeting drugs include compounds that target the interaction between YAP and TEAD G protein-coupled receptors (GPCR) and the mevalonate pathway (e.g. statins). Given that many Hippo pathway-modulating drugs are already used in patients, this could lead to early clinical trials testing their efficacy in different types of sarcoma.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Sarcoma/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Via de Sinalização Hippo , Humanos , Camundongos , Sarcoma/genética , Sarcoma/patologiaRESUMO
Over the past decade, the Hippo signaling cascade has been linked to organ size regulation in mammals. Indeed, modulation of the Hippo pathway can have potent effects on cellular proliferation and/or apoptosis and a deregulation of the pathway often leads to tumor development. Importantly, emerging evidence indicates that the Hippo pathway can modulate its effects on tissue size by the regulation of stem and progenitor cell activity. This role has recently been associated with the central position of the pathway in sensing spatiotemporal or mechanical cues, and translating them into specific cellular outputs. These results provide an attractive model for how the Hippo cascade might sense and transduce cellular 'neighborhood' cues into activation of tissue-specific stem or progenitors cells. A further understanding of this process could allow the development of new therapies for various degenerative diseases and cancers. Here, we review current and emerging data linking Hippo signaling to progenitor cell function.
Assuntos
Transdução de Sinais , Células-Tronco/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mamíferos , Especificidade de Órgãos , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Satellite cells are the resident stem cells of skeletal muscle. Mitotically quiescent in mature muscle, they can be activated to proliferate and generate myoblasts to supply further myonuclei to hypertrophying or regenerating muscle fibres, or self-renew to maintain the resident stem cell pool. Here, we identify the transcriptional co-factor Yap as a novel regulator of satellite cell fate decisions. Yap expression increases during satellite cell activation and Yap remains highly expressed until after the differentiation versus self-renewal decision is made. Constitutive expression of Yap maintains Pax7(+) and MyoD(+) satellite cells and satellite cell-derived myoblasts, promotes proliferation but prevents differentiation. In contrast, Yap knockdown reduces the proliferation of satellite cell-derived myoblasts by ≈40%. Consistent with the cellular phenotype, microarrays show that Yap increases expression of genes associated with Yap inhibition, the cell cycle, ribosome biogenesis and that it represses several genes associated with angiotensin signalling. We also identify known regulators of satellite cell function such as BMP4, CD34 and Myf6 (Mrf4) as genes whose expression is dependent on Yap activity. Finally, we confirm in myoblasts that Yap binds to Tead transcription factors and co-activates MCAT elements which are enriched in the proximal promoters of Yap-responsive genes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Processos de Crescimento Celular/fisiologia , Núcleo Celular/metabolismo , Embrião de Galinha , Via de Sinalização Hippo , Cavalos , Camundongos , Fosfoproteínas/genética , Transdução de Sinais , Transfecção , Proteínas de Sinalização YAPRESUMO
Interplay between different posttranslational modifications of transcription factors is an important mechanism to achieve an integrated regulation of gene expression. For the estrogen-related receptors (ERRs) alpha and gamma, regulation by posttranslational modifications is still poorly documented. Here we show that transcriptional repression associated with the ERR amino-terminal domains is mediated through sumoylation at a conserved phospho-sumoyl switch, psiKxEPxSP, that exists within a larger synergy control motif. Arginine substitution of the sumoylatable lysine residue or alanine substitution of a nearby phosphorylatable serine residue (serine 19 in ERRalpha) increased the transcriptional activity of both ERRalpha and -gamma. In addition, phospho-mimetic substitution of the serine residue with aspartate restored the sumoylation and transcriptional repression activity. The increased transcriptional activity of the sumoylation-deficient mutants was more pronounced in the presence of multiple adjacent ERR response elements. We also identified protein inhibitor of activated signal transducer and activator of transcription y as an interacting partner and a small ubiquitin-related modifier E3 ligase for ERRalpha. Importantly, analysis with a phospho-specific antibody revealed that sumoylation of ERRalpha in mouse liver requires phosphorylation of serine 19. Taken together, these results show that the interplay of phosphorylation and sumoylation in the amino-terminal domain provides an additional mechanism to regulate the transcriptional activity of ERRalpha and -gamma.
Assuntos
Regulação da Expressão Gênica/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Estrogênio/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , DNA/química , DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Inibidoras de STAT Ativados/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Transcrição Gênica , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
The ubiquitous transcriptional coactivators Yap (gene symbol Yap1) and Taz (gene symbol Wwtr1) regulate gene expression mainly by coactivating the Tead transcription factors. Being at the center of the Hippo signaling network, Yap and Taz are regulated by the Hippo kinase cassette and additionally by a plethora of exercise-associated signals and signaling modules. These include mechanotransduction, the AKT-mTORC1 network, the SMAD transcription factors, hypoxia, glucose homeostasis, AMPK, adrenaline/epinephrine and angiotensin II through G protein-coupled receptors, and IL-6. Consequently, exercise should alter Hippo signaling in several organs to mediate at least some aspects of the organ-specific adaptations to exercise. Indeed, Tead1 overexpression in muscle fibers has been shown to promote a fast-to-slow fiber type switch, whereas Yap in muscle fibers and cardiomyocytes promotes skeletal muscle hypertrophy and cardiomyocyte adaptations, respectively. Finally, genome-wide association studies in humans have linked the Hippo pathway members LATS2, TEAD1, YAP1, VGLL2, VGLL3, and VGLL4 to body height, which is a key factor in sports.
Assuntos
Exercício Físico/fisiologia , Condicionamento Físico Animal/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Fatores de Transcrição/metabolismoRESUMO
The role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transformação Celular Neoplásica/metabolismo , Neoplasias Musculares/metabolismo , Fosfoproteínas/fisiologia , Rabdomiossarcoma Embrionário/metabolismo , Células Satélites de Músculo Esquelético/patologia , Animais , Diferenciação Celular/genética , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Dosagem de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias Musculares/mortalidade , Neoplasias Musculares/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Proteína MyoD , Transplante de Neoplasias , Proteínas Nucleares/metabolismo , Oncogenes , Rabdomiossarcoma Embrionário/mortalidade , Rabdomiossarcoma Embrionário/patologia , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor highly expressed in the kidney, an organ playing a central role in blood pressure regulation through electrolyte homeostasis and the renin-angiotensin system. Physiological analysis revealed that, relative to wild-type mice, ERRalpha null mice are hypotensive despite significant hypernatremia, hypokalemia, and slight hyperreninemia. Using a combination of genome-wide location analysis and expression profiling, we demonstrate that ERRalpha regulates the expression of channels involved in renal Na(+) and K(+) handling (Scnn1a, Atp1a1, Atp1b1) and altered in Bartter syndrome (Bsnd, Kcnq1). In addition, ERRalpha regulates the expression of receptors implicated in the systemic regulation of blood pressure (Ghr, Gcgr, Lepr, Npy1r) and of genes within the renin-angiotensin pathway (Ren1, Agt, Ace2). Our study thus identifies ERRalpha as a pleiotropic regulator of renal control of blood pressure, renal Na(+)/K(+) homeostasis, and renin-angiotensin pathway and suggests that modulation of ERRalpha activity could represent a potential avenue for the management of hypertension.
Assuntos
Pressão Sanguínea , Regulação da Expressão Gênica , Rim/metabolismo , Receptores de Estrogênio/fisiologia , Sistema Renina-Angiotensina/genética , Equilíbrio Hidroeletrolítico , Animais , Síndrome de Bartter/fisiopatologia , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Genômica/métodos , Hipotensão/genética , Hipotensão/metabolismo , Hipotensão/fisiopatologia , Bombas de Íon/genética , Bombas de Íon/metabolismo , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Receptores de Estrogênio/deficiência , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Sódio na Dieta/efeitos adversos , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Posttranslational modifications are instrumental to achieve gene- and tissue-specific regulatory outcomes by transcription factors. Nuclear receptors are dynamically modulated by several types of posttranslational modifications including phosphorylation, methylation, acetylation, ubiquitination, and sumoylation. The estrogen-related receptor alpha (ERRalpha, NR3B1) is phosphorylated on multiple sites, and sumoylated in the amino-terminal region in a phosphorylation-dependent manner. Here we demonstrate that ERRalpha interacts with and is acetylated by p300 coactivator associated factor (PCAF) in vitro and in mouse liver. Purified PCAF acetylated the DNA-binding domain of ERRalpha on four highly-conserved lysines. In addition, coexpression of PCAF reduced the transcriptional activity of ERRalpha and, reciprocally, a deacetylase screen identified histone deacetylase 8 (HDAC8) and sirtuin 1 homolog (Sirt1) as independent enhancers of ERRalpha transcriptional function. HDAC8 and Sirt1 were also demonstrated to interact directly with ERRalpha in vivo and to deacetylate and increase the DNA binding affinity of ERRalpha in vitro. The removal of PCAF increases the DNA binding of ERRalpha in vivo, whereas the removal of Sirt1 and HDAC8 decreases it as assessed by chromatin immunoprecipitation assay. Altogether, our results show that ERRalpha is an acetylated protein and imply the existence of a dynamic acetylation/deacetylation switch involved in the control of ERRalpha transcriptional activity.
Assuntos
Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transcrição Gênica/genética , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Camundongos , Ligação Proteica , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fatores de Transcrição de p300-CBP/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Members of the NR3B group of the nuclear receptor superfamily, known as the estrogen-related receptors (ERRs), were the first orphan receptors to be identified two decades ago. Despite the fact that a natural ligand has yet to be associated with the ERRs, considerable knowledge about their mode of action and biological functions has emerged through extensive biochemical, genetic and functional genomics studies. This review describes our current understanding of how the ERRs work as transcription factors and as such, how they control diverse developmental and physiological programs.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/fisiologiaRESUMO
A eukaryotic protein is often subject to regulation by multiple modifications like phosphorylation, acetylation, ubiquitination, and sumoylation. How these modifications are coordinated in vivo is an important issue that is poorly understood but is relevant to many biological processes. We recently showed that human MEF2D (myocyte enhancer factor 2D) is sumoylated on Lys-439. Adjacent to the sumoylation motif is Ser-444, which like Lys-439 is highly conserved among MEF2 proteins from diverse species. Here we present [corrected] several lines of evidence to demonstrate that Ser-444 of MEF2D is required for sumoylation of Lys-439. Histone deacetylase 4 (HDAC4) stimulated this modification by acting through Ser-444. In addition, phosphorylation of Ser-444 by Cdk5, a cyclin-dependent kinase known to inhibit MEF2 transcriptional activity, stimulated sumoylation. Opposing the actions of HDAC4 and Cdk5, calcineurin (also known as protein phosphatase 2B) dephosphorylated Ser-444 and inhibited sumoylation of Lys-439. This phosphatase, however, exerted minimal effects on the phosphorylation catalyzed by ERK5, an extracellular signal-regulated kinase known to activate MEF2D. These results identify [corrected] an essential role for Ser-444 in MEF2D sumoylation and reveal [corrected] a novel mechanism by which calcineurin selectively "edits" phosphorylation at different sites, thereby reiterating that interplay between different modifications represents a general mechanism for coordinated regulation of eukaryotic protein functions in vivo.