RESUMO
Peptic ulcer disease is a frequent clinical problem with potentially serious complications such as bleeding or perforation. A decisive factor in the pathogenesis of peptic ulcers is gastric acid, the secretion of which is controlled by the hormone gastrin released from gastric G cells. However, the molecular mechanisms regulating gastrin plasma concentrations are poorly understood. Here, we identified a semaphorin-plexin signaling pathway that operates in gastric G cells to inhibit gastrin expression on a transcriptional level, thereby limiting food-stimulated gastrin release and gastric acid secretion. Using a systematic siRNA screening approach combined with biochemical, cell biology, and in vivo mouse experiments, we found that the RasGAP protein Rasal1 is a central mediator of plexin signal transduction, which suppresses gastrin expression through inactivation of the small GTPase R-Ras. Moreover, we show that Rasal1 is pathophysiologically relevant for the pathogenesis of peptic ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs), a main risk factor of peptic ulcers in humans. Last, we show that application of recombinant semaphorin 4D alleviates peptic ulcer disease in mice in vivo, demonstrating that this signaling pathway can be harnessed pharmacologically. This study unravels a mode of G cell regulation that is functionally important in gastric homeostasis and disease.
Assuntos
Úlcera Péptica , Semaforinas , Animais , Moléculas de Adesão Celular , Proteínas Ativadoras de GTPase , Gastrinas/efeitos adversos , Gastrinas/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso , Úlcera Péptica/induzido quimicamente , Transdução de SinaisRESUMO
Plexins comprise a family of transmembrane receptors for semaphorins. Plexins of the B- and D-subfamily interact with the receptor tyrosine kinase ErbB-2, and this interaction has been shown to be functionally relevant for various biological processes including tumor metastasis and bone formation. Binding of semaphorins to B- and D-subfamily plexins results in the activation of ErbB-2, which in turn phosphorylates these plexins. This phosphorylation triggers the activation of the small GTPases RhoA and RhoC downstream of B-subfamily plexins. Here we describe a methodology that allows the analysis of ErbB-2-mediated plexin phosphorylation and signaling.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor ErbB-2/metabolismo , Tirosina/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Células MCF-7 , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor ßPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration.
Assuntos
Movimento Celular , Células Dendríticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Semaforinas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Genótipo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Espectrometria de Massas , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Interferência de RNA , Receptores de Superfície Celular/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Semaforinas/deficiência , Semaforinas/genética , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
AIMS: VEGF A (VEGF-A) is a central regulator of pre- and postnatal vascular development. In vitro studies suggested that heterotrimeric G-proteins of the Gq/11 family contribute to VEGF receptor 2 (VEGFR2) signalling, but the mechanism and physiological relevance of this finding is unknown. The aim of this study is to understand the role of endothelial Gαq/11 in VEGF-dependent regulation of vascular permeability and angiogenesis. METHODS AND RESULTS: We show here that VEGF-A-induced signalling events, such as VEGFR2 autophosphorylation, calcium mobilization, or phosphorylation of Src and Cdh5, were reduced in Gαq/11-deficient endothelial cells (ECs), resulting in impaired VEGF-dependent barrier opening, tube formation, and proliferation. Agonists at Gq/11-coupled receptors facilitated VEGF-A-induced VEGFR2 autophosphorylation in a Gαq/11-dependent manner, thereby enhancing downstream VEGFR2 signalling. In vivo, EC-specific Gαq/11- and Gαq-deficient mice showed reduced VEGF-induced fluid extravasation, and retinal angiogenesis was significantly impaired. Gαq-deficient ECs showed reduced proliferation, Cdh5 phosphorylation, and fluid extravasation, whereas apoptosis was increased. CONCLUSION: Gαq/11 critically contributes to VEGF-A-dependent permeability control and angiogenic behaviour in vitro and in vivo.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Permeabilidade Capilar/fisiologia , Células Cultivadas , Humanos , Camundongos , Neovascularização Fisiológica/fisiologia , Fosforilação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane.
Assuntos
Moléculas de Adesão Celular/metabolismo , Divisão Celular/fisiologia , Rim/metabolismo , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Semaforinas/metabolismo , Transdução de Sinais , Fuso Acromático/metabolismo , Animais , Moléculas de Adesão Celular/genética , Comunicação Celular/genética , Polaridade Celular/fisiologia , Células Epiteliais/citologia , Epitélio/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Rim/embriologia , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Semaforinas/genética , Transdução de Sinais/fisiologia , Fuso Acromático/genética , Cicatrização/genéticaRESUMO
The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKß-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which â¼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKß substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKß on serine 298. We provide evidence that IKKß-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.
Assuntos
Moléculas de Adesão Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Quinase I-kappa B/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Western Blotting , Moléculas de Adesão Celular/metabolismo , Imunoprecipitação da Cromatina , Cromatografia Líquida , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B , Imunoprecipitação , Células MCF-7 , Espectrometria de Massas , Proteínas de Membrana , Inibidor de NF-kappaB alfa , NF-kappa B , Fosfoproteínas , Proteínas de Ligação a RNA , Serina , Ensaio Tumoral de Célula-Tronco , Regulação para CimaRESUMO
Plexins are widely expressed transmembrane proteins that mediate the cellular effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still poorly understood. Here we show that signalling via B-family plexins leading to the activation of the small GTPase RhoA requires activation of the IκB kinase (IKK)-complex. In contrast, plexin-B-dependent regulation of R-Ras activity is not affected by IKK activity. This regulation of plexin signalling depends on the kinase activity of the IKK-complex, but is independent of NF-κB activation. We confirm that the IKK-complex is active in tumour cells and osteoblasts, and we demonstrate that plexin-B-dependent tumour cell invasiveness and regulation of osteoblast differentiation require an active IKK-complex. This study identifies a novel, NF-κB-independent function of the IKK-complex and shows that IKK directs plexin-B signalling to the activation of RhoA.
Assuntos
Moléculas de Adesão Celular/metabolismo , Quinase I-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Transdução de Sinais/genéticaRESUMO
Mammalian plexins constitute a family of transmembrane receptors for semaphorins and represent critical regulators of various processes during development of the nervous, cardiovascular, skeletal, and renal system. In vitro studies have shown that plexins exert their effects via an intracellular R-Ras/M-Ras GTPase-activating protein (GAP) domain or by activation of RhoA through interaction with Rho guanine nucleotide exchange factor proteins. However, which of these signaling pathways are relevant for plexin functions in vivo is largely unknown. Using an allelic series of transgenic mice, we show that the GAP domain of plexins constitutes their key signaling module during development. Mice in which endogenous Plexin-B2 or Plexin-D1 is replaced by transgenic versions harboring mutations in the GAP domain recapitulate the phenotypes of the respective null mutants in the developing nervous, vascular, and skeletal system. We further provide genetic evidence that, unexpectedly, the GAP domain-mediated developmental functions of plexins are not brought about via R-Ras and M-Ras inactivation. In contrast to the GAP domain mutants, Plexin-B2 transgenic mice defective in Rho guanine nucleotide exchange factor binding are viable and fertile but exhibit abnormal development of the liver vasculature. Our genetic analyses uncover the in vivo context-dependence and functional specificity of individual plexin-mediated signaling pathways during development.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/genética , Animais , Camundongos , Camundongos TransgênicosRESUMO
The vertebrate central nervous system develops from an epithelium where cells are polarized along the apicobasal axis. Loss of this polarity results in abnormal organ architecture, morphology and proliferation. We found that mutations of the guanine nucleotide exchange factor ArhGEF18 affect apicobasal polarity of the retinal neuroepithelium in medaka fish. We show that ArhGEF18-mediated activation of the small GTPase RhoA is required to maintain apicobasal polarity at the onset of retinal differentiation and to control the ratio of neurogenic to proliferative cell divisions. RhoA signals through Rock2 to regulate apicobasal polarity, tight junction localization and the cortical actin cytoskeleton. The human ArhGEF18 homologue can rescue the mutant phenotype, suggesting a conserved function in vertebrate neuroepithelia. Our analysis identifies ArhGEF18 as a key regulator of tissue architecture and function, controlling apicobasal polarity and proliferation through RhoA activation. We thus identify the control of neuroepithelial apicobasal polarity as a novel role for RhoA signaling in vertebrate development.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Oryzias/embriologia , Oryzias/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Pulmonary arterial hypertension (PAH) is a fatal disease for which no cure is yet available. The leading cause of death in PAH is right ventricular (RV) failure. Previously, the TNF receptor superfamily member fibroblast growth factor-inducible molecule 14 (Fn14) has been associated with different fibrotic diseases. However, so far there is no study demonstrating a causal role for endogenous Fn14 signaling in RV or LV heart disease. The purpose of this study was to determine whether global ablation of Fn14 prevents RV fibrosis and remodeling improving heart function. Here, we provide evidence for a causative role of Fn14 in pulmonary artery banding (PAB)-induced RV fibrosis and dysfunction in mice. Fn14 expression was increased in the RV after PAB. Mice lacking Fn14 (Fn14(-/-)) displayed substantially reduced RV fibrosis and dysfunction following PAB compared to wild-type littermates. Cell culture experiments demonstrated that activation of Fn14 induces collagen expression via RhoA-dependent nuclear translocation of myocardin-related transcription factor-A (MRTF-A)/MAL. Furthermore, activation of Fn14 in vitro caused fibroblast proliferation and myofibroblast differentiation, which corresponds to suppression of PAB-induced RV fibrosis in Fn14(-/-) mice. Moreover, our findings suggest that Fn14 expression is regulated by endothelin-1 (ET-1) in cardiac fibroblasts. We conclude that Fn14 is an endogenous key regulator in cardiac fibrosis and suggest this receptor as potential new target for therapeutic interventions in heart failure.
Assuntos
Hipertrofia Ventricular Direita/prevenção & controle , Miocárdio/patologia , Receptores do Fator de Necrose Tumoral/fisiologia , Disfunção Ventricular Direita/prevenção & controle , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Diferenciação Celular , Proliferação de Células , Colágeno/metabolismo , Citocina TWEAK , Endotelina-1/fisiologia , Hipertensão Pulmonar Primária Familiar , Fibrose/prevenção & controle , Imunofluorescência , Hipertensão Pulmonar/complicações , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Miofibroblastos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Receptor de TWEAK , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismo , Regulação para Cima , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/fisiopatologiaRESUMO
Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.
Assuntos
Antígenos CD/metabolismo , Proteína Adaptadora GRB2/metabolismo , Semaforinas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Ativação Enzimática , Proteína Adaptadora GRB2/genética , Células HEK293 , Humanos , Células MCF-7 , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2-dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2-overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2-overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2-positive breast cancer.
Assuntos
Neoplasias da Mama/patologia , Invasividade Neoplásica/fisiopatologia , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Processamento de Proteína Pós-Traducional , Receptor ErbB-2/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologia , Adulto , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Genes erbB-2 , Células HEK293 , Humanos , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Invasividade Neoplásica/genética , Metástase Neoplásica , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fosforilação , Prognóstico , Receptores de Superfície Celular/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
Semaphorin 3E (Sema3E) is a secreted molecule implicated in axonal path finding and inhibition of developmental and postischemic angiogenesis. Sema3E is also highly expressed in metastatic cancer cells, but its mechanistic role in tumor progression was not understood. Here we show that expression of Sema3E and its receptor Plexin D1 correlates with the metastatic progression of human tumors. Consistent with the clinical data, knocking down endogenous expression of either Sema3E or Plexin D1 in human metastatic carcinoma cells hampered their metastatic potential when injected into mice, while tumor growth was not markedly affected. Conversely, overexpression of exogenous Sema3E in cancer cells increased their invasiveness, transendothelial migration, and metastatic spreading, although it inhibited tumor vessel formation, resulting in reduced tumor growth in mice. The proinvasive and metastatic activity of Sema3E in tumor cells was dependent on transactivation of the Plexin D1-associated ErbB2/Neu oncogenic kinase. In sum, Sema3E-Plexin D1 signaling in cancer cells is crucially implicated in their metastatic behavior and may therefore be a promising target for strategies aimed at blocking tumor metastasis.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Metástase Neoplásica , Semaforinas/fisiologia , Transdução de Sinais/fisiologia , Animais , Células COS , Linhagem Celular Tumoral , Movimento Celular , Chlorocebus aethiops , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Receptor ErbB-2/fisiologia , Semaforinas/análise , Proteínas ras/genética , Proteínas rho de Ligação ao GTP/análiseRESUMO
Semaphorins and their receptors, plexins, have emerged as key regulators of various aspects of neuronal development. In contrast to the Plexin-A family, the cellular functions of Plexin-B family proteins in developing neurons are only poorly understood. An activation of Plexin-B1 via its ligand, semaphorin 4D (Sema4D), produces an acute collapse of axonal growth cones in hippocampal and retinal neurons over the early stages of neurite outgrowth. However, the functional role of Sema4D-Plexin-B interactions over subsequent stages of neurite development, differentiation and maturation has not been characterized. Here we addressed this question using morphogenetic assays and time-lapse imaging on developing rat hippocampal neurons as a model system. Interestingly, Sema4D treatment over several hours was observed to promote branching and complexity in hippocampal neurons via the activation of Plexin-B1. The activation of receptor tyrosine kinases and the Rho kinase following Sema4D treatment was found to control dendritic and axonal morphogenesis by differentially regulating branching and extension. Phosphoinositide-3-kinase, but not extracellular signal-regulated kinase 1/2, was observed to be important for the stimulatory effects of Sema4D on dendritic branching. Furthermore, we observed that the mammalian target of rapamycin is activated downstream of Plexin-B1 and contributes to Sema4D-induced effects on dendritic branching. In contrast, glycogen synthase kinase-3 beta, another effector of phosphoinositide-3-kinase signalling, was not involved. Thus, our results show that Sema4D-Plexin-B interactions modulate dendritic and axonal arborizations of developing neurons by co-ordinated and concerted activation of diverse signalling pathways.
Assuntos
Antígenos CD/metabolismo , Axônios/fisiologia , Dendritos/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , Animais , Células Cultivadas , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/genética , Transdução de SinaisRESUMO
The semaphorin 4D (Sema4D) receptor plexin-B1 constitutively interacts with particular Rho guanine nucleotide exchange factors (RhoGEFs) and thereby mediates Sema4D-induced RhoA activation, a process which involves the tyrosine phosphorylation of plexin-B1 by ErbB-2. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGEF activity. We show here that activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation creates docking sites for the SH2 domains of phospholipase Cgamma (PLCgamma). PLCgamma is thereby recruited into the plexin-B1 receptor complex and via its SH3 domain activates the Rho guanine nucleotide exchange factor PDZ-RhoGEF. PLCgamma-dependent RhoGEF activation is independent of its lipase activity. The recruitment of PLCgamma has no effect on the R-Ras GTPase-activating protein activity of plexin-B1 but is required for Sema4D-induced axonal growth cone collapse as well as for the promigratory effects of Sema4D on cancer cells. These data demonstrate a novel nonenzymatic function of PLCgamma as an important mechanism of plexin-mediated signaling which links tyrosine phosphorylation of plexin-B1 to the regulation of a RhoGEF protein and downstream cellular processes.
Assuntos
Antígenos CD/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfolipase C gama/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Linhagem Celular , Movimento Celular , Ativação Enzimática , Cones de Crescimento/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Fosfotirosina/metabolismo , Ligação Proteica , Receptores de Superfície Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Proteínas ras/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Semaphorins and their receptors, plexins, carry out important functions during development and disease. In contrast to the well-characterized plexin A family, however, very little is known about the functional relevance of B-type plexins in organogenesis, particularly outside the nervous system. Here, we demonstrate that plexin B1 and its ligand Sema4d are selectively expressed in epithelial and mesenchymal compartments during key steps in the genesis of some organs. This selective expression suggests a role in epithelial-mesenchymal interactions. Importantly, using the developing metanephros as a model system, we have observed that endogenously expressed and exogenously supplemented Sema4d inhibits branching morphogenesis during early stages of development of the ureteric collecting duct system. Our results further suggest that the RhoA-ROCK pathway, which is activated downstream of plexin B1, mediates these inhibitory morphogenetic effects of Sema4d and suppresses branch-promoting signalling effectors of the plexin B1 signalling complex. Finally, mice that lack plexin B1 show early anomalies in kidney development in vivo. These results identify a novel function for plexin B1 as a negative regulator of branching morphogenesis during kidney development, and suggest that the Sema4d-plexin B1 ligand-receptor pair contributes to epithelial-mesenchymal interactions during organogenesis via modulation of RhoA signalling.
Assuntos
Antígenos CD/metabolismo , Epitélio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organogênese/genética , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , Animais , Antígenos CD/genética , Imuno-Histoquímica , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Morfogênese/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Semaforinas/genética , Transdução de Sinais/genéticaRESUMO
Sema4D-induced activation of plexin-B1 has been reported to evoke different and sometimes opposing cellular responses. The mechanisms underlying the versatility of plexin-B1-mediated effects are not clear. Plexin-B1 can associate with the receptor tyrosine kinases ErbB-2 and Met. Here we show that Sema4D-induced activation and inactivation of RhoA require ErbB-2 and Met, respectively. In breast carcinoma cells, Sema4D can have pro- and anti-migratory effects depending on the presence of ErbB-2 and Met, and the exchange of the two receptor tyrosine kinases is sufficient to convert the cellular response to Sema4D from pro- to anti-migratory and vice versa. This work identifies a novel mechanism by which plexin-mediated signaling can be regulated and explains how Sema4D can exert different biological activities through the differential association of its receptor with ErbB-2 and Met.
Assuntos
Movimento Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Antígenos CD/genética , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met , Receptor ErbB-2/genética , Receptores de Superfície Celular/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Neural alterations and aberrantly expressed nerve-specific factors promoting tumor progression are known to contribute to pancreatic cancer's extremely poor prognosis. Despite hints that axon guidance factor semaphorin 3A (SEMA3A) may function as a tumor inhibitor, its clinical importance and therapeutic potential have not yet been explored. The present study investigated the role of SEMA3A and its receptors-plexins A1-A4 (PLXNA1-A4) and neuropilin-1 (NRP1)-in pancreatic cancer. QRT-PCR and immunohistochemical analyses revealed overexpression of SEMA3A, NRP1 and PLXNA1 in metaplastic ducts, malignant cells and nerves of cancerous specimens, and showed that elevated levels of corresponding mRNA (6.8-fold, 2.0-fold and 1.5-fold, respectively) clearly correlated with negative clinicopathological manifestations such as shorter survival (SEMA3A and PLXNA1) and a lesser degree of tumor differentiation (NRP1) in Stages I-III patients. High SEMA3A expression in pancreata of Stage IV M1 patients and in peritoneal metastases, and consequent functional studies indicated that poor clinical outcome might be related to the ability of SEMA3A to promote dissemination and invasiveness of pancreatic cancer cells through activation of multiple pathways involving Rac1, GSK3b or p42/p44 MAPK, but not E- to N-cadherin switch, MMP-9 or VEGF induction. Thus, this study is the first to quantify expression of the SEMA3A system in human malignancy and to show that overexpression of SEMA3A by nerves and transformed cells leads to a SEMA3A-rich environment which may favor malignant activities of tumor cells. Furthermore, negative clinicopathological correlations suggest that SEMA3A might represent a novel intervention target but not a treatment option for pancreatic cancer patients.
Assuntos
Adenocarcinoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforina-3A/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Diferenciação Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Alemanha/epidemiologia , Humanos , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Prognóstico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Células Tumorais Cultivadas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Semaphorins and their receptors, plexins, have emerged as important cellular cues regulating key developmental processes. B-type plexins directly regulate the actin cytoskeleton in a variety of cell types. Recently, B-type plexins have been shown to be expressed in striking patterns in the nervous system over critical developmental windows. However, in contrast to the well characterized plexin-A family, the functional role of plexin-B proteins in neural development and organogenesis in vertebrates in vivo is not known. Here, we have elucidated the functional contribution of the two neuronally expressed plexin-B proteins, Plexin-B1 or Plexin-B2, toward the development of the peripheral nervous system and the CNS by generating and analyzing constitutive knock-out mice. The development of the nervous system was found to be normal in mice lacking Plexin-B1, whereas mice lacking Plexin-B2 demonstrated defects in closure of the neural tube and a conspicuous disorganization of the embryonic brain. After analyzing mutant mice, which bypassed neural tube defects, we observed a key requirement for Plexin-B2 in proliferation and migration of granule cell precursors in the developing dentate gyrus, olfactory bulb, and cerebellum. Furthermore, we identified semaphorin 4C as a high-affinity ligand for Plexin-B2 in binding and functional assays. Semaphorin 4C stimulated activation of ErbB-2 and RhoA via Plexin-B2 and enhanced proliferation and migration of granule cell precursors. Semaphorin 4C-induced proliferation of ventricular zone neuroblasts was abrogated in mice lacking Plexin-B2. These genetic and functional analyses reveal a key requirement for Plexin-B2, but not Plexin-B1, in patterning of the vertebrate nervous system in vivo.
Assuntos
Movimento Celular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sistema Nervoso/crescimento & desenvolvimento , Receptores de Superfície Celular/fisiologia , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Células COS , Movimento Celular/genética , Proliferação de Células , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Chlorocebus aethiops , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Organogênese/genética , Prosencéfalo/citologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genéticaRESUMO
Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2-mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse.