RESUMO
TGF-ß signaling is a therapeutic target in advanced cancers. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a key component mediating pro-oncogenic TGF-ß-induced SMAD and non-SMAD signaling. Upon TGF-ß stimulation, TRAF4 is recruited to the active TGF-ß receptor complex, where it antagonizes E3 ligase SMURF2 and facilitates the recruitment of deubiquitinase USP15 to the TGF-ß type I receptor (TßRI). Both processes contribute to TßRI stabilization on the plasma membrane and thereby enhance TGF-ß signaling. In addition, the TGF-ß receptor-TRAF4 interaction triggers Lys 63-linked TRAF4 polyubiquitylation and subsequent activation of the TGF-ß-activated kinase (TAK)1. TRAF4 is required for efficient TGF-ß-induced migration, epithelial-to-mesenchymal transition, and breast cancer metastasis. Elevated TRAF4 expression correlated with increased levels of phosphorylated SMAD2 and phosphorylated TAK1 as well as poor prognosis among breast cancer patients. Our results demonstrate that TRAF4 can regulate the TGF-ß pathway and is a key determinant in breast cancer pathogenesis.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator 4 Associado a Receptor de TNF/genética , Fator 4 Associado a Receptor de TNF/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Fosforilação , Poliubiquitina/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.
Assuntos
Embrião não Mamífero/citologia , Células-Tronco Embrionárias/citologia , Proteína Smad7/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Diferenciação Celular/genética , Embrião não Mamífero/metabolismo , Células-Tronco Embrionárias/metabolismo , Gastrulação/genética , Humanos , Células Jurkat , Camundongos , Proteólise , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The expression of the bone morphogenetic protein antagonist, Gremlin 1, was recently shown to be increased in the lungs of pulmonary arterial hypertension patients, and in response to hypoxia. Gremlin 1 released from the vascular endothelium may inhibit endogenous bone morphogenetic protein signaling and contribute to the development of pulmonary arterial hypertension. Here, we investigate the impact of Gremlin 1 inhibition in disease after exposure to chronic hypoxia/SU5416 in mice. We investigated the effects of an anti-Gremlin 1 monoclonal antibody in the chronic hypoxia/SU5416 murine model of pulmonary arterial hypertension. Chronic hypoxic/SU5416 exposure of mice induced upregulation of Gremlin 1 mRNA in lung and right ventricle tissue compared with normoxic controls. Prophylactic treatment with an anti-Gremlin 1 neutralizing mAb reduced the hypoxic/SU5416-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy. Importantly, therapeutic treatment with an anti-Gremlin 1 antibody also reduced pulmonary vascular remodeling and right ventricular hypertrophy indicating a role for Gremlin 1 in the progression of the disease. We conclude that Gremlin 1 plays a role in the development and progression of pulmonary arterial hypertension in the murine hypoxia/SU5416 model, and that Gremlin 1 is a potential therapeutic target for pulmonary arterial hypertension.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/prevenção & controle , Hipóxia/complicações , Indóis/efeitos adversos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Pirróis/efeitos adversos , Animais , Anticorpos Monoclonais/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Doença Crônica , Hipertensão Pulmonar Primária Familiar , Células HEK293 , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/patologia , Hipóxia/fisiopatologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
Engagement of the immunoinhibitory receptor, programmed death-1 (PD-1) attenuates T-cell receptor (TCR)-mediated activation of IL-2 production and T-cell proliferation. Here, we demonstrate that PD-1 modulation of T-cell function involves inhibition of TCR-mediated phosphorylation of ZAP70 and association with CD3zeta. In addition, PD-1 signaling attenuates PKCtheta activation loop phosphorylation in a cognate TCR signal. PKCtheta has been shown to be required for T-cell IL-2 production. A phosphorylated PD-1 peptide, corresponding to the C-terminal immunoreceptor tyrosine-switch motif (ITSM), acts as a docking site in vitro for both SHP-2 and SHP-1, while the phosphorylated peptide containing the N-terminal PD-1 immunoreceptor tyrosine based inhibitory motif (ITIM) associates only with SHP-2.
Assuntos
Antígenos de Superfície/fisiologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Antígenos CD , Antígenos de Superfície/química , Proteínas Reguladoras de Apoptose , Humanos , Células Jurkat , Dados de Sequência Molecular , Fosforilação , Receptor de Morte Celular Programada 1 , Proteína Quinase C-theta , Homologia de Sequência de Aminoácidos , Proteína-Tirosina Quinase ZAP-70RESUMO
The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings.
Assuntos
Receptores de Activinas Tipo II/imunologia , Ativinas/metabolismo , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Hipertrofia/metabolismo , Mioblastos Esqueléticos/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Atrofia/imunologia , Atrofia/metabolismo , Diferenciação Celular/fisiologia , Humanos , Hipertrofia/patologia , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos Esqueléticos/imunologia , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
In advanced cancers, the TGF-ß pathway acts as an oncogenic factor and is considered to be a therapeutic target. Here using a genome-wide cDNA screen, we identify nuclear receptor NR4A1 as a strong activator of TGF-ß signalling. NR4A1 promotes TGF-ß/SMAD signalling by facilitating AXIN2-RNF12/ARKADIA-induced SMAD7 degradation. NR4A1 interacts with SMAD7 and AXIN2, and potently and directly induces AXIN2 expression. Whereas loss of NR4A1 inhibits TGF-ß-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-ß-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-ß-mediated breast cancer cell migration, invasion and metastasis in vitro and in vivo. Notably, NR4A1 expression is elevated in breast cancer patients with high immune infiltration and its expression weakly correlates with phosphorylated SMAD2 levels, and is an indicator of poor prognosis. Our results uncover inflammation-induced NR4A1 as an important determinant for hyperactivation of pro-oncogenic TGF-ß signalling in breast cancer.
Assuntos
Neoplasias Mamárias Animais/metabolismo , Metástase Neoplásica/fisiopatologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Feminino , Imuno-Histoquímica , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Knockout , Metástase Neoplásica/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/genética , Ubiquitinação/genética , Ubiquitinação/fisiologia , Peixe-ZebraRESUMO
The stability and membrane localization of the transforming growth factor-ß (TGF-ß) type I receptor (TßRI) determines the levels of TGF-ß signalling. TßRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-ß signalling. USP4 was found to directly interact with TßRI and act as a deubiquitylating enzyme, thereby controlling TßRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-ß-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TßRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-ß and AKT signalling pathways.