RESUMO
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
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
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
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.