RESUMO
The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the ß-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for ß-TrCP recruitment to the complex and ß-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/ß-catenin signaling. In line, the ß-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Aciltransferases , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Proteínas de Sinalização YAPRESUMO
The assembly of the Smad complex is critical for TGFbeta signaling, yet the mechanisms that inactivate or empower nuclear Smad complexes are less understood. By means of siRNA screen we identified FAM (USP9x), a deubiquitinase acting as essential and evolutionarily conserved component in TGFbeta and bone morphogenetic protein signaling. Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits Smad4 by impeding association with phospho-Smad2. FAM reverts this negative modification, re-empowering Smad4 function. FAM opposes the activity of Ectodermin/Tif1gamma (Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 monoubiquitin ligase. Our study points to Smad4 monoubiquitination and deubiquitination as a way for cells to set their TGFbeta responsiveness: loss of FAM disables Smad4-dependent responses in several model systems, with Ecto being epistatic to FAM. This defines a regulative ubiquitination step controlling Smads that is parallel to those impinging on R-Smad phosphorylation.
Assuntos
Proteína Smad4/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular Tumoral , Embrião não Mamífero/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação , XenopusRESUMO
The Spemann organizer stands out from other signaling centers of the embryo because of its broad patterning effects. It defines development along the anteroposterior and dorsoventral axes of the vertebrate body, mainly by secreting antagonists of growth factors. Qualitative models proposed more than a decade ago explain the organizer's region-specific inductions (i.e., head and trunk) as the result of different combinations of antagonists. For example, head induction is mediated by extracellular inhibition of Wnt, BMP, and Nodal ligands. However, little is known about how the levels of these antagonists become harmonized with those of their targets and with the factors initially responsible for germ layers and organizer formation, including Nodal itself. Here we show that key ingredients of the head-organizer development, namely Nodal ligands, Nodal antagonists, and ADMP ligands reciprocally adjust each other's strength and range of activity by a self-regulating network of interlocked feedback and feedforward loops. A key element in this cross-talk is the limited availability of ACVR2a, for which Nodal and ADMP must compete. By trapping Nodal extracellularly, the Nodal antagonists Cerberus and Lefty are permissive for ADMP activity. The system self-regulates because ADMP/ACVR2a/Smad1 signaling in turn represses the expression of the Nodal antagonists, reestablishing the equilibrium. In sum, this work reveals an unprecedented set of interactions operating within the organizer that is critical for embryonic patterning.
Assuntos
Organizadores Embrionários/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animais , Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Ligantes , Modelos Biológicos , Transdução de Sinais , Fatores de Tempo , Transfecção , Xenopus laevis/metabolismoRESUMO
The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGFbeta signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known about the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, we show that the Smad4 inhibitor ectodermin (Ecto, also known as Trim33 or Tif1gamma) is required to limit Nodal responsiveness in vivo. New phenotypes, which are linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extra-embryonic territories. In extra-embryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the anterior visceral endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates towards node/organizer fates, revealing the requirement of Smad inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular negative control of Smad function by ectodermin/Tif1gamma is a crucial element in the cellular response to TGFbeta signals in mammalian tissues.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Smad/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Padronização Corporal , Diferenciação Celular , Cruzamentos Genéticos , Ectoderma/metabolismo , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Fenótipo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
MicroRNAs are crucial modulators of gene expression, yet their involvement as effectors of growth factor signalling is largely unknown. Ligands of the transforming growth factor-beta superfamily are essential for development and adult tissue homeostasis. In early Xenopus embryos, signalling by the transforming growth factor-beta ligand Nodal is crucial for the dorsal induction of the Spemann's organizer. Here we report that Xenopus laevis microRNAs miR-15 and miR-16 restrict the size of the organizer by targeting the Nodal type II receptor Acvr2a. Endogenous miR-15 and miR-16 are ventrally enriched as they are negatively regulated by the dorsal Wnt/beta-catenin pathway. These findings exemplify the relevance of microRNAs as regulators of early embryonic patterning acting at the crossroads of fundamental signalling cascades.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Receptores de Activinas Tipo II/biossíntese , Receptores de Activinas Tipo II/genética , Ativinas/metabolismo , Animais , Padronização Corporal , MicroRNAs/genética , Proteína Nodal , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , Proteínas Wnt/metabolismo , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMO
The ability to induce autologous tissue-specific stem cells in culture could have a variety of applications in regenerative medicine and disease modeling. Here we show that transient expression of exogenous YAP or its closely related paralogue TAZ in primary differentiated mouse cells can induce conversion to a tissue-specific stem/progenitor cell state. Differentiated mammary gland, neuronal, and pancreatic exocrine cells, identified using a combination of cell sorting and lineage tracing approaches, efficiently convert to proliferating cells with properties of stem/progenitor cells of their respective tissues after YAP induction. YAP-induced mammary stem/progenitor cells show molecular and functional properties similar to endogenous MaSCs, including organoid formation and mammary gland reconstitution after transplantation. Because YAP/TAZ function is also important for self-renewal of endogenous stem cells in culture, our findings have implications for understanding the molecular determinants of the somatic stem cell state.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glândulas Mamárias Animais/citologia , Especificidade de Órgãos , Fosfoproteínas/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Aciltransferases , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Feminino , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Organoides/citologia , Pâncreas Exócrino/citologia , Regeneração , Reprodutibilidade dos Testes , Proteínas de Sinalização YAPRESUMO
p53 is a protein with many talents. One of the most fundamental is the ability to act as essential growth checkpoint that protects cells against cellular transformation. p53 does so through the induction of genes leading to growth arrest or apoptosis. Most of the studies focusing on the mechanisms of p53 activity have been performed in cultured cells upon treatment with well-established p53-activating inputs, such as high doses of radiations, DNA-damaging drugs and activated oncogenes. However, how the tumor suppressive functions of p53 become concerted with the extracellular cues arriving at the cell surface during tissue homeostasis, remains largely unknown. Intriguingly, two recent papers have shed new light into this unexplored field, indicating that p53 plays a key role in TGF-beta-induced growth arrest and, unexpectedly, in the developmental effects of TGF-beta in early embryos. Here we review and comment on these findings and on their implications for cancer biology.
Assuntos
Transformação Celular Neoplásica/genética , Dano ao DNA , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Proteína Supressora de Tumor p53/farmacologia , Divisão Celular , Desenvolvimento Embrionário e Fetal , Crescimento/genética , Homeostase , Humanos , Fator de Crescimento Transformador beta/biossínteseRESUMO
The TGFß pathway is critical for embryonic development and adult tissue homeostasis. On ligand stimulation, TGFß and BMP receptors phosphorylate receptor-activated SMADs (R-SMADs), which then associate with SMAD4 to form a transcriptional complex that regulates gene expression through specific DNA recognition. Several ubiquitin ligases serve as inhibitors of R-SMADs, yet no deubiquitylating enzyme (DUB) for these molecules has so far been identified. This has left unexplored the possibility that ubiquitylation of R-SMADs is reversible and engaged in regulating SMAD function, in addition to degradation. Here we identify USP15 as a DUB for R-SMADs. USP15 is required for TGFß and BMP responses in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes R-SMAD monoubiquitylation, which targets the DNA-binding domains of R-SMADs and prevents promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters by the SMAD complex. These data identify an additional layer of control by which the ubiquitin system regulates TGFß biology.
Assuntos
Endopeptidases/genética , Processamento de Proteína Pós-Traducional , Proteína Smad3/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Proteína Morfogenética Óssea 2/metabolismo , DNA/metabolismo , Endopeptidases/metabolismo , Células HCT116 , Células HEK293 , Humanos , Oócitos , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Proteína Smad3/genética , Proteína Smad4/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Proteases Específicas de Ubiquitina , Ubiquitinação , XenopusRESUMO
During development and tissue homeostasis, cells must integrate different signals. We investigated how cell behavior is controlled by the combined activity of transforming growth factor-beta (TGF-beta) and receptor tyrosine kinase (RTK) signaling, whose integration mechanism is unknown. We find that RTK/Ras/MAPK (mitogen-activated protein kinase) activity induces p53 N-terminal phosphorylation, enabling the interaction of p53 with the TGF-beta-activated Smads. This mechanism confines mesoderm specification in Xenopus embryos and promotes TGF-beta cytostasis in human cells. These data indicate a mechanism to allow extracellular cues to specify the TGF-beta gene-expression program.
Assuntos
Proliferação de Células , Embrião não Mamífero/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/metabolismo , Substituição de Aminoácidos , Animais , Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Idelta/metabolismo , Linhagem Celular Tumoral , Desenvolvimento Embrionário , Indução Embrionária , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesoderma/metabolismo , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Smad/metabolismo , XenopusRESUMO
TGF-beta proteins are main regulators of blood vessel development and maintenance. Here, we report an unprecedented link between TGF-beta signaling and arterial hypertension based on the analysis of mice mutant for Emilin1, a cysteine-rich secreted glycoprotein expressed in the vascular tree. Emilin1 knockout animals display increased blood pressure, increased peripheral vascular resistance, and reduced vessel size. Mechanistically, we found that Emilin1 inhibits TGF-beta signaling by binding specifically to the proTGF-beta precursor and preventing its maturation by furin convertases in the extracellular space. In support of these findings, genetic inactivation of Emilin1 causes increased TGF-beta signaling in the vascular wall. Strikingly, high blood pressure observed in Emilin1 mutants is rescued to normal levels upon inactivation of a single TGF-beta1 allele. This study highlights the importance of modulation of TGF-beta availability in the pathogenesis of hypertension.
Assuntos
Pressão Sanguínea/fisiologia , Homeostase , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Artérias/citologia , Artérias/metabolismo , Furina/metabolismo , Dosagem de Genes , Genes Reporter , Humanos , Hipertensão/etiologia , Hipertensão/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína Nodal , Fenótipo , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
TGF-beta signaling is essential for development and proliferative homeostasis. During embryogenesis, maternal determinants act in concert with TGF-beta signals to form mesoderm and endoderm. In contrast, ectoderm specification requires the TGF-beta response to be attenuated, although the mechanisms by which this is achieved remain unknown. In a functional screen for ectoderm determinants, we have identified Ectodermin (Ecto). In Xenopus embryos, Ecto is essential for the specification of the ectoderm and acts by restricting the mesoderm-inducing activity of TGF-beta signals to the mesoderm and favoring neural induction. Ecto is a RING-type ubiquitin ligase for Smad4, a TGF-beta signal transducer. Depletion of Ecto in human cells enforces TGF-beta-induced cytostasis and, moreover, plays a causal role in limiting the antimitogenic effects of Smad4 in tumor cells. We propose that Ectodermin is a key switch in the control of TGF-beta gene responses during early embryonic development and cell proliferation.