RESUMO
Mammalian NUMB is alternatively spliced generating four isoforms NUMB1-NUMB4 that can function as tumor suppressors. NUMB1-NUMB4 proteins, which normally determine how different cell types develop, are reduced in 21% of primary breast tumors. Our previous work has, however, indicated that two novel NUMB isoforms, NUMB5 and NUMB6 have the pro-oncogenic functions. Herein, we address a novel function of human NUMB isoform 6 (NUMB6) in promoting cancer cell migration and invasion. We found that NUMB6 induced expression of embryonic transcription factor Slug, which in turn actively repressed E-cadherin, prompting cells to undergo epithelial-mesenchymal transition (EMT). Low-metastatic breast cancer cells DB-7 stably expressing NUMB6, lost their epithelial phenotype, exhibited migratory and pro-invasive behavior, and ultimately elevated expression of mesenchymal markers. Among these markers, increased vimentin, ß-catenin, and fibronectin expression elicited metalloproteinase 9 (MMP9) production. Our results revealed that NUMB6-DB-7 cells have significantly increased level of Akt1 and Akt2 phosphorylation. Therefore, antagonizing Akt signaling using a chemical inhibitor LY294002, we found that NUMB6-induced Slug expression was reduced, and ultimately accompanied with decreased cell migration and invasion. In summary, this study identified a novel molecular determinant of breast cancer progression, uncovering a potential oncogenic role for the NUMB6 protein in cancer cell migration and invasion, coupled to the maintenance of mesenchymal-like cells. J. Cell. Biochem. 118: 237-251, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Membrana/genética , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, beta-arrestin Ral-GDS protein complexes dissociate and Ral-GDS translocates with beta-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of beta-arrestin Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, beta-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.
Assuntos
Arrestinas/metabolismo , Citoesqueleto/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Animais , Arrestinas/química , Transporte Biológico Ativo , Células COS , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Biológicos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ratos , Receptores de Formil Peptídeo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , beta-Arrestinas , Proteínas ral de Ligação ao GTP/química , Fator ral de Troca do Nucleotídeo Guanina/químicaRESUMO
BACKGROUND: Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-kappaB has yet to be explored. RESULTS: Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -alpha/beta and subsequent transcriptional activation of the p65 subunit of NF-kappaB. Ablation of endogenous NRAGE by siRNA inhibited NF-kappaB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-kappaB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor. CONCLUSION: Modulation of NRAGE expression revealed novel roles in regulating NF-kappaB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Animais , Antígenos de Neoplasias/genética , Western Blotting , Proteína Morfogenética Óssea 4/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Quinase I-kappa B/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Rim/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Transdução de Sinais/genética , Fator de Transcrição RelA/genéticaRESUMO
Bone morphogenetic signaling (BMP) is a key pathway during neurogenesis and depends on many downstream intermediators to carry out its signaling. One such signaling pathway utilizes neurotrophin receptor-interacting MAGE protein (NRAGE), a member of the melanoma-associated antigen (MAGE) family, to upregulate p38 mitogen activated protein kinase (p38(MAPK)) in response to cellular stress and activate caspases which are critical in leading cells to death. NRAGE consists of two conserved MAGE homology domains separated by a unique hexapeptide repeat domain. Although we have previously implicated NRAGE in inducing apoptosis in neural progenitors and P19 cells, a model system for neural progenitors, its domains have yet to be explored in determining which one may be responsible for setting up the signaling for apoptosis. Here, we overexpressed a series of deletion mutations in P19 cells to show that only those with at least half of the repeat domain, activated p38(MAPK) and underwent apoptosis offering intriguing incite into NRAGE's contribution in BMP apoptotic signaling.
Assuntos
Apoptose , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Neoplasias/química , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Sobrevivência Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estrutura Terciária de Proteína , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cellular interactions in development of the kidney are used as a model of reciprocal inductive events between epithelium and mesenchyme. Time- and labor-intensive methods have been developed to study this phenomenon. For example, in mice, the targeted disruption of genes in vivo has been used to modify the genetic program directing kidney development. However, gene targeting is a resource-intensive approach and alternative strategies for gene and protein modification in the kidney need to be developed. Herein, we have developed an efficient system for the delivery of antisense morpholino to alter normal protein expression. We describe the use of Endo-Porter to effectively deliver morpholinos to all parts and regions of the kidney explant. Also, we definitively show via confocal microscopy and Western blot analysis that the use of Endo-Porter in delivering antisense morpholinos is robust throughout the entire kidney explant, providing efficient suppression of protein expression. This method saves time and cost when compared with targeted disruption and is an improvement upon previous kidney organ culture methods.
Assuntos
Sistemas de Liberação de Medicamentos , Rim/citologia , Peptídeos , Animais , Fluoresceína/metabolismo , Corantes Fluorescentes/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Proteínas de Neoplasias/antagonistas & inibidores , Oligonucleotídeos Antissenso/química , Técnicas de Cultura de Órgãos , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
The activation of Notch signaling in neural crest stem cells (NCSCs) results in the rapid loss of neurogenic potential and differentiation into glia. We now show that the attenuation of endogenous Notch signaling within expanding NCSC clones by the Notch ligand soluble Jagged1 (sJ1), maintains NCSCs in a clonal self-renewing state in vitro without affecting their sensitivity to instructive differentiation signals observed previously during NCSC self-renewal. sJ1 functions as a competitive inhibitor of Notch signaling to modulate endogenous cell-cell communication to levels sufficient to inhibit neural differentiation but insufficient to instruct gliogenic differentiation. Attenuated Notch signaling promotes the induction and nonclassic release of fibroblast growth factor 1 (FGF1). The functions of sJ1 and FGF1 signaling are complementary, as abrogation of FGF signaling diminishes the ability of sJ1 to promote NCSC expansion, yet the secondary NCSCs maintain the dosage sensitivity of the founder. These results validate and build upon previous studies on the role of Notch signaling in stem cell self-renewal and suggest that the differentiation bias or self-renewal potential of NCSCs is intrinsically linked to the level of endogenous Notch signaling. This should provide a unique opportunity for the expansion of NCSCs ex vivo without altering their differentiation bias for clinical cell replacement or transplant strategies in tissue repair. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Diferenciação Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Crista Neural/citologia , Crista Neural/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Células Clonais/citologia , Células Clonais/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Inibição Neural/genética , Inibição Neural/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Ratos , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Receptor Notch1/fisiologia , Proteínas Serrate-Jagged , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Solubilidade , Células-Tronco/metabolismoRESUMO
Understanding the molecular events that govern neural progenitor lineage commitment, mitotic arrest, and differentiation into functional progeny are germane to our understanding of neocortical development. Members of the family of bone morphogenetic proteins (BMPs) play pivotal roles in regulating neural differentiation and apoptosis during neurogenesis through combined actions involving Smad and TAK1 activation. We demonstrate that BMP signaling is required for the induction of apoptosis of neural progenitors and that NRAGE is a mandatory component of the signaling cascade. NRAGE possesses the ability to bind and function with the TAK1-TAB1-XIAP complex facilitating the activation of p38. Disruption of NRAGE or any other member of the noncanonical signaling cascaded is sufficient to block p38 activation and thus the proapoptotic signals generated through BMP exposure. The function of NRAGE is independent of Smad signaling, but the introduction of a dominant-negative Smad5 also rescues neural progenitor apoptosis, suggesting that both canonical and noncanonical pathways can converge and regulate BMP-mediated apoptosis. Collectively, these results establish NRAGE as an integral component in BMP signaling and clarify its role during neural progenitor development.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas de Neoplasias/metabolismo , Neurônios/citologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Proteína Morfogenética Óssea 4 , Caspase 3 , Caspases/metabolismo , Ciclo Celular , Diferenciação Celular , Células Cultivadas , Ativação Enzimática , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Neurônios/metabolismo , Células-Tronco/metabolismo , Tretinoína/farmacologiaRESUMO
Neurotrophin receptor-interacting MAGE (NRAGE) is the most recently identified p75 neurotrophin receptor (p75(NTR)) intracellular binding protein. Previously, NRAGE over-expression was shown to mediate cell cycle arrest and facilitate nerve growth factor (NGF) dependent apoptosis of sympathetic neuroblasts in a p75(NTR) specific manner. Here we have examined the temporal and spatial expression patterns of NRAGE over the course of murine embryogenesis to determine whether NRAGE's expression is consistent with its proposed functions. We demonstrate that NRAGE mRNA and protein are expressed throughout embryonic and adult tissues. The mRNA is constitutively expressed within each tissue across development. However, expression of NRAGE protein displays a tight temporal tissue specific regulation. During early CNS development, NRAGE protein is expressed throughout the neural tube, but by later stages of neurogenesis, NRAGE protein is restricted within the ventricular zone, subplate and cortical plate. Moreover, NRAGE protein expression is limited to proliferative neural subpopulations as we fail to detect NRAGE expression co-localized with mature/differentiation associated neuronal markers. Interestingly, NRAGE's expression is not restricted solely to areas of p75(NTR) expression suggesting that NRAGE may mediate proliferation and/or apoptosis from other environmental signals in addition to NGF within the CNS. Our data support previously characterized roles for NRAGE as a mediator of precursor apoptosis and a repressor of cell cycle progression in neural development.
Assuntos
Apoptose/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Antígenos de Neoplasias , Apoptose/genética , Sequência de Bases , Ciclo Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Sistema Nervoso/embriologia , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor de Fator de Crescimento Neural , Transdução de Sinais , Especificidade da Espécie , Distribuição TecidualRESUMO
The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily. p75NTR signaling events have been implicated in both cell cycle arrest and apoptosis depending on which effector molecules are associated with its intracellular domain after ligand binding. Two such effector proteins, p75NTR-associated cell death executor (NADE) and neurotrophin receptor interacting factor (NRIF) promote p75NTR-mediated apoptosis, whereas Schwann cell factor-1 (SC-1) mediates neurotrophin-dependent withdrawal from the cell cycle. An understanding of the expression profiles of these three interacting proteins and p75NTR during embryogenesis is critical for addressing whether these effector proteins might function outside of p75NTR-mediated signaling events. The distribution of NADE, NRIF and SC-1 mRNAs during murine development suggests that the action of these genes is in fact not limited to regions of p75NTR expression. Specifically, a detailed comparison of the spatial and temporal expression domains of NADE, NRIF and SC-1 during brain development revealed regions of co-expression with p75NTR but also illustrates a distinct and discordant spatial and temporal expression. These results yield novel insights into the unique developmental characteristics of the three p75NTR-interacting proteins, thus revealing their diverse signaling potential during embryonic development.
Assuntos
Proteínas de Transporte/biossíntese , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Neurônios/metabolismo , Biossíntese de Proteínas , Envelhecimento , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Masculino , Camundongos , Gravidez , Proteínas/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Distribuição TecidualRESUMO
In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38(MAPK) and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38(MAPK) phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/ß phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/química , Peptídeos/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Compartimento Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Microscopia Confocal , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Sequências Repetitivas de Aminoácidos , Transdução de Sinais/efeitos dos fármacosRESUMO
We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.
Assuntos
Desenvolvimento Fetal/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Transdução de Sinais/fisiologia , Animais , Western Blotting , Movimento Celular/genética , Polaridade Celular/genética , Embrião de Galinha , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais , Neurogênese/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Branching morphogenesis is a developmental process characteristic of many organ systems. Specifically, during renal branching morphogenesis, its been postulated that the final number of nephrons formed is one key clinical factor in the development of hypertension in adulthood. As it has been established that BMPs regulate, in part, renal activity of p38 MAP kinase (p38(MAPK)) and it has demonstrated that the cytoplasmic protein Neurotrophin Receptor MAGE homologue (NRAGE) augments p38(MAPK) activation, it was hypothesized that a decrease in the expression of NRAGE during renal branching would result in decreased branching of the UB that correlated with changes in p38(MAPK) activation. To verify this, the expression of NRAGE was reduced in ex vivo kidney explants cultures using antisense morpholino. Morpholino treated ex vivo kidney explants expression were severely stunted in branching, a trait that was rescued with the addition of exogenous GDNF. Renal explants also demonstrated a precipitous drop in p38(MAPK) activation that too was reversed in the presence of recombinant GDNF. RNA profiling of NRAGE diminished ex vivo kidney explants resulted in altered expression of GDNF, Ret, BMP7 and BMPRIb mRNAs. Our results suggested that in early kidney development NRAGE might have multiple roles during renal branching morphogenesis through association with both the BMP and GDNF signaling pathways.
Assuntos
Rim/embriologia , Morfogênese , Proteínas de Neoplasias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Proteínas de Homeodomínio/metabolismo , Imunoprecipitação , Rim/citologia , Rim/efeitos dos fármacos , Rim/enzimologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Técnicas de Cultura de Órgãos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ureter/efeitos dos fármacos , Ureter/embriologia , Ureter/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Loss of numb function suggests that numb maintains progenitors in an undifferentiated state. Herein, we demonstrate that numb1 and numb3 are expressed in undifferentiated cortical progenitors, whereas numb2 and numb4 become prominent throughout differentiation. To further assess the role of different numb isoforms in cortical neural development, we first created a Numb-null state with antisense morpholino, followed by the re-expression of specific numb isoforms. The re-expression of numb1 or numb3 resulted in a significant reduction of neural differentiation, correlating with an expansion of the cortical progenitor pool. In contrast, the expression of numb2 or numb4 resulted in a reduction of proliferating progenitors and a corresponding increase in mammalian achete-scute homologue (MASH1) expression, concurrent with the appearance of the microtubule[corrected]-associated [corrected] protein-2-positive neurons. Of interest, the effect of numb isoforms on neural differentiation could not be directly related to Notch, because classic canonical Notch signaling assays failed to uncover any differences in the four isoforms to inhibit the Notch downstream events. This finding suggests that numb may have other signaling properties during neuronal differentiation in addition to augmenting notch signal strength.
Assuntos
Desenvolvimento Embrionário/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Oligonucleotídeos Antissenso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Genetic studies show that TGFbeta signaling is essential for vascular development, although the mechanism through which this pathway operates is incompletely understood. Here we demonstrate that the TGFbeta auxiliary coreceptor endoglin (eng, CD105) is expressed in a subset of neural crest stem cells (NCSCs) in vivo and is required for their myogenic differentiation. Overexpression of endoglin in the neural crest caused pericardial hemorrhaging, correlating with altered vascular smooth muscle cell investment in the walls of major vessels and upregulation of smooth muscle alpha-actin protein levels. Clonogenic differentiation assay of NCSCs derived from neural tube explants demonstrated that only NCSC expressing high levels of endoglin (NCSC(CD105+)) had myogenic differentiation potential. Furthermore, myogenic potential was deficient in NCSCs obtained from endoglin null embryos. Expression of endoglin in NCSCs declined with age, coinciding with a reduction in both smooth muscle differentiation potential and TGFbeta1 responsiveness. These findings demonstrate a cell autonomous role for endoglin in smooth muscle cell specification contributing to vascular integrity.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Desenvolvimento Muscular/fisiologia , Crista Neural/citologia , Crista Neural/metabolismo , Animais , Sequência de Bases , Movimento Celular , Células Cultivadas , Primers do DNA/genética , Endoglina , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Desenvolvimento Muscular/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/metabolismo , Ratos , Transdução de Sinais , Proteínas Smad/metabolismoRESUMO
RNA-binding proteins regulate cell fate decisions during nervous system development. The Msi family of RNA-binding proteins is expressed in multipotential neural progenitors, and is required for maintaining cells in a proliferative state. We demonstrate that Msi-1's ability to regulate progenitor maintenance is through the translational inhibition of the cyclin-dependent kinase inhibitor p21WAF-1. Msi-1 ectopic expression increases the proliferation rate and the capacity to regulate p21WAF-1 protein expression, independent of p53. The 3' untranslated region (UTR) of the native p21(WAF-1) mRNA contains a Msi-1 consensus-binding site that permits Msi-1 to directly repress the translation of p21WAF-1 protein. Reduction of Msi-1 through antisense leads to aberrant p21WAF-1 expression, which significantly impairs neural differentiation. A double knockdown for p21WAF-1 and Msi-1 rescues the production of mature MAP+ neurons. Our results further elucidate the symbiotic relationship between cell cycle withdrawal and the onset of differentiation in the developing nervous system, as well as increasing the understanding of the influence that RNA-binding proteins serve in regulating these processes.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Ligação a RNA/fisiologia , Ciclo Celular , Divisão Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/genética , Quinases Ciclina-Dependentes/metabolismo , Regulação da Expressão Gênica , Humanos , Rim , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
During muscle differentiation, a population of quiescent undifferentiated myoblasts (reserve cells) emerges among mature muscle cells. However, the molecular mechanisms underlying such cell segregation and the characterization of this subpopulation of myoblasts remain to be determined. Notch is known to control the behavior and fate of murine muscle stem cells. In this study, we examined the role of Notch in myoblast segregation. We showed that inhibition of Notch activity by either overexpressing Numb or by using a pharmacological gamma-secretase inhibitor (DAPT) enhanced differentiation of murine and human myoblasts. This effect was not restricted to in vitro culture systems since DAPT-treated zebrafish embryos also showed increased differentiation. Using C2.7 myoblasts as a model, we showed that inhibition of Notch induced myotube hypertrophy by recruiting reserve cells that do not normally fuse. We further showed that endogenous Notch-signaling components were differentially expressed and activated in reserve cells with respect to Notch 1 and CD34 expression. We identified CD34 negative reserve cells as the subpopulation of myoblasts recruited to fuse into myotubes during differentiation in response to Notch inhibition. Therefore, we showed here that the activation of Notch 1 is important to maintain a subpopulation of CD34 negative reserve cells in an undifferentiated state.
Assuntos
Fibras Musculares Esqueléticas/citologia , Receptores Notch/antagonistas & inibidores , Animais , Antígenos CD34/análise , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Fusão Celular , Linhagem Celular , Humanos , Hipertrofia , Cinética , Luciferases/análise , Luciferases/genética , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/citologia , Transdução de Sinais , Transfecção , Triglicerídeos/farmacologia , Peixe-Zebra , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/farmacologiaRESUMO
A numerical and functional deficiency in invariant NKT (iNKT) cells detectable by 3 wk of age in the thymus and spleen mediates the pathogenesis of type 1 diabetes in NOD mice, but the stage of T cell development at which this deficiency first occurs is unknown. We report in this study that this deficiency develops after the CD4(+)CD8(+) double-positive stage of thymic T cell development and is due to a lineage-specific depletion of CD4(-)CD8(-) double-negative alphabeta T cells and iNKT cells from the thymus between embryonic day 18 and day 1 after birth. Thus, an inheritable defect in a lineage fate decision that elicits a deficiency in fetal thymic iNKT cell development may predispose to susceptibility to type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Feto/imunologia , Predisposição Genética para Doença , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Diabetes Mellitus Tipo 1/genética , Feminino , Feto/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Linfopenia/genética , Linfopenia/imunologia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Técnicas de Cultura de Órgãos , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Timo/metabolismo , Timo/patologiaRESUMO
The signaling adapter proteins FRS2 and FRS3 are implicated in the transmission of extracellular signals from nerve growth factor (NGF) or fibroblast growth factor (FGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. This study presents the genomic sequence and exon-intron organization of the mouse FRS2 and FRS3 loci as well as their evolutionary conservation with their human counterparts. Both FRS2 and FRS3 contain 5 coding exons spanning over 7 kb of genomic sequence with similar exon sizes and organization. Comparative genomic sequence analyses show a highly conserved genomic organization between mouse and human in both FRS2 and FRS3 genes. Non-coding sequences, highly conserved between mouse and human, were identified in the FRS3 introns that may potentially function as regulatory elements. To assay potential differences in their patterns of expression, RT-PCR analysis was used to assay FRS2 and FRS3 expression in the developing embryo and neural tube (NT) during the time of neurogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas/genética , Proteínas de Membrana/genética , Fosfoproteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Humanos , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The development of the nervous system is regulated by trophic signals that control cell proliferation, differentiation, and survival. Numb is an evolutionarily conserved protein identified by its ability to control cell fate in the nervous system of Drosophila. Mammals express four isoforms of Numb that differ in the length of a phosphotyrosine-binding (PTB) domain and a proline-rich region (PRR). Using PC12 cells stably expressing each of the human isoforms, we show that Numb regulates sensitivity of the cells to neurotrophic factor-induced differentiation and neurotrophic factor withdrawal-induced death in an isoform-specific manner. Numb isoforms containing a short PTB domain enhance the differentiation response to NGF and enhance apoptosis upon NGF withdrawal; Numb isoforms containing a long PTB domain exhibit the same sensitivity to NGF as vector-transfected cells. These effects of Numb were found to be independent of the length of the PRR. In undifferentiated conditions, the levels of full-length TrkA and of phosphorylated p44/p42 mitogen-activated protein kinase (MAPK) are increased in cells expressing Numb isoforms with a short PTB domain, indicating an up-regulation of NGF signaling pathways. Furthermore, we provide evidence that the mechanism whereby short PTB domain Numb isoforms sensitize cells to trophic factor deprivation-induced apoptosis involves elevations in intracellular calcium concentrations. Our results suggest that Numb sensitizes cells to neurotrophin responses in an isoform-specific manner, an effect that may play an important role in the development and plasticity of the nervous system.
Assuntos
Proteínas de Membrana/farmacologia , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Feocromocitoma/metabolismo , Animais , Cálcio/metabolismo , Morte Celular , Diferenciação Celular/efeitos dos fármacos , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Estresse Oxidativo , Células PC12 , Feocromocitoma/tratamento farmacológico , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Espécies Reativas de Oxigênio , Receptor trkA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Neural stem cells become progressively less neurogenic and more gliogenic with development. Here, we show that between E10.5 and E14.5, neural crest stem cells (NCSCs) become increasingly sensitive to the Notch ligand Delta-Fc, a progliogenic and anti-neurogenic signal. This transition is correlated with a 20- to 30-fold increase in the relative ratio of expression of Notch and Numb (a putative inhibitor of Notch signaling). Misexpression experiments suggest that these changes contribute causally to increased Delta sensitivity. Moreover, such changes can occur in NCSCs cultured at clonal density in the absence of other cell types. However, they require local cell-cell interactions within developing clones. Delta-Fc mimics the effect of such cell-cell interactions to increase Notch and decrease Numb expression in isolated NCSCs. Thus, Delta-mediated feedback interactions between NCSCs, coupled with positive feedback control of Notch sensitivity within individual cells, may underlie developmental changes in the ligand-sensitivity of these cells.