RESUMO
Notch signaling plays crucial roles in the control of cell fate and physiology through local cell-cell interactions. The core processes of Notch signal transduction are well established, but the mechanisms that fine-tune the pathway in various developmental and post-developmental contexts are less clear. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. In this study, we examined the role of almondex in Notch signaling during early Drosophila embryogenesis. We found that in addition to being required for lateral inhibition in the neuroectoderm, almondex is also partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, we found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, we speculate that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Neurogênese , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Receptores Notch/genéticaRESUMO
Mitral cells (MCs) of the mammalian olfactory bulb have a single primary dendrite extending into a single glomerulus, where they receive odor information from olfactory sensory neurons (OSNs). Molecular mechanisms for controlling dendritic arbors of MCs, which dynamically change during development, are largely unknown. Here we found that MCs displayed more complex dendritic morphologies in mouse mutants of Maml1, a crucial gene in Notch signaling. Similar phenotypes were observed by conditionally misexpressing a dominant negative form of MAML1 (dnMAML1) in MCs after their migration. Conversely, conditional misexpression of a constitutively active form of Notch reduced their dendritic complexity. Furthermore, the intracellular domain of Notch1 (NICD1) was localized to nuclei of MCs. These findings suggest that Notch signaling at embryonic stages is involved in the dendritic complexity of MCs. After the embryonic misexpression of dnMAML1, many MCs aberrantly extended dendrites to more than one glomerulus at postnatal stages, suggesting that Notch signaling is essential for proper formation of olfactory circuits. Moreover, dendrites in cultured MCs were shortened by Jag1-expressing cells. Finally, blocking the activity of Notch ligands in OSNs led to an increase in dendritic complexity as well as a decrease in NICD1 signals in MCs. These results demonstrate that the dendritic complexity of MCs is controlled by their presynaptic partners, OSNs.
Assuntos
Proteínas Nucleares/genética , Bulbo Olfatório/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptor Notch1/genética , Fatores de Transcrição/genética , Animais , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Jagged-1/biossíntese , Proteína Jagged-1/genética , Camundongos , Proteínas Nucleares/biossíntese , Bulbo Olfatório/crescimento & desenvolvimento , Neurônios Receptores Olfatórios/citologia , Receptor Notch1/biossíntese , Transdução de Sinais/genética , Fatores de Transcrição/biossínteseRESUMO
Mastermind-like 1 (MAML1) is a transcriptional co-activator in the Notch signaling pathway. Recently, however, several reports revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. We found that MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2), a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2) from the Runx2-regulated osteocalcin gene promoter. Interestingly, a deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Moreover, inhibition of Notch signaling did not affect the action of MAML1 on Runx2, suggesting that the activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1(-/-) embryos at embryonic day 16.5 (E16.5) had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1) and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1) expression, a marker of hypertrophic chondrocyte, were observed. These observations suggest that chondrocyte maturation was impaired in MAML1(-/-) mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.
Assuntos
Desenvolvimento Ósseo/genética , Condrócitos , Subunidade alfa 1 de Fator de Ligação ao Core , Desenvolvimento Embrionário/genética , Proteínas Nucleares , Fatores de Transcrição , Animais , Diferenciação Celular , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocalcina/genética , Osteocalcina/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The rostro-caudal polarity within a somite is primarily determined by the on/off state of Notch signaling, but the mechanism by which Notch is repressed has remained elusive. Here, we present genetic and biochemical evidence that the suppression of Notch signaling is essential for the establishment of rostro-caudal polarity within a somite and that Mesp2 acts as a novel negative regulator of the Notch signaling pathway. We generated a knock-in mouse in which a dominant-negative form of Rbpj is introduced into the Mesp2 locus. Intriguingly, this resulted in an almost complete rescue of the segmental defects in the Mesp2-null mouse. Furthermore, we demonstrate that Mesp2 potently represses Notch signaling by inducing the destabilization of mastermind-like 1, a core regulator of this pathway. Surprisingly, this function of Mesp2 is found to be independent of its function as a transcription factor. Together, these data demonstrate that Mesp2 is a novel component involved in the suppression of Notch target genes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Somitos/embriologia , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Nucleares/genética , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Mastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, ß-catenin and NF-κB. We have demonstrated previously that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. Here, we report the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Southern Blotting , Western Blotting , Primers do DNA/genética , Fibroblastos , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicosiltransferases/metabolismo , Hibridização In Situ , Luciferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Nucleares/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
Notch signaling is an evolutionarily conserved mechanism required for numerous types of cell fate decisions in metazoans. It mediates short-range communication between cells with receptors and ligands, both of which are expressed on the cell surfaces. In response to the ligand-receptor interaction, the ligand and the extracellular domain of the Notch receptor (NECD) in the complex are internalized into ligand-expressing cells by endocytosis, a prerequisite process for the conformational change of the membrane proximal region of Notch to induce critical proteolytic cleavages for its activation. Here we report that overexpression of transmembrane 2 (TM2) domain containing 3 (TM2D3), a mammalian homologue of Drosophila melanogaster Almondex (Amx), activates Notch1. This activation requires the ligand-binding domain in Notch1 and the C-terminal region containing TM2 domain in TM2D3. TM2D3 physically associates with Notch1 at the region distinct from the ligand-binding domain and enhances expression of Notch1 on the cell surface. Furthermore, cell surface expression of Notch1 and Notch2 is reduced in Tm2d3-deficient cells. Finally, amx-deficient Drosophila early embryos exhibit impaired endocytosis of NECD and Delta ligand, for which surface presentation of Notch is required. These results indicate that TM2D3 is an element involved in Notch signaling through the surface presentation.
Assuntos
Proteínas de Drosophila , Receptores Notch , Animais , Receptores Notch/genética , Receptores Notch/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ligantes , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Mamíferos/metabolismoRESUMO
Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis.
RESUMO
Delta-like (Dll) 1 and Dll4 differently function as Notch ligands in a context-dependent manner. As these ligands share structural properties, the molecular basis for their functional difference is poorly understood. Here, we investigated the superiority of Dll4 over Dll1 with respect to induction of T cell development using a domain-swapping approach in mice. The DOS motif, shared by Notch ligands-except Dll4-contributes to enhancing the activity of Dll for signal transduction. The module at the N-terminus of Notch ligand (MNNL) of Dll4 is inherently advantageous over Dll1. Molecular dynamic simulation revealed that the loop structure in MNNL domain of Dll1 contains unique proline residues with limited range of motion. The Dll4 mutant with Dll1-derived proline residues showed reduced activity. These results suggest that the loop structure-present within the MNNL domain-with a wide range of motion ensures the superiority of Dll4 and uniquely contributes to the triggering of Notch signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Linfopoese , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Mutação/genética , Domínios Proteicos , Relação Estrutura-Atividade , Linfócitos T/metabolismoRESUMO
Notch signalling plays pivotal roles in development and homeostasis of all metazoan species. Notch is a receptor molecule that directly translates information of cell-cell contact to gene expression in the nucleus. Mastermind is a conserved and essential nuclear factor that supports the activity of Notch. Here, the past and current studies of the interplay between these factors are reviewed.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Receptores Notch/genética , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
We have developed a method that we call 'drug-sensitivity pattern analysis', or DSPA, for analysis of protein function. Cells are transfected with cDNA of the test molecule, followed by analysis of the sensitivity of the transfected cells to multiple growth-inhibitory drugs. If two cDNA products have similar functions, their transfected cells should show similar drug-sensitivity patterns. The cDNAs of some signaling molecules were transfected into NIH3T3 or Ha-ras-transformed NIH3T3 (ras-NIH) cells and stable transfectants, which expressed high amounts of the gene product, were isolated. Chemosensitivity of the transfected clone was compared with the parental cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method using more than 40 drugs. The chemosensitivity changes caused by the transfected gene were calculated and expressed numerically as 'drug chemosensitivity index' (DCI). When the DCI values were analyzed by regression analysis, a significant positive relationship between IkappaBalpha superrepressor and dominant-negative IKKbeta and an inverse relationship between p53 and Mdm2 were consistent with previous reports. Thus, the DSPA method is useful for identifying functional similarities between gene products.
Assuntos
Resistência a Medicamentos , Proteínas/genética , Proteínas/fisiologia , Células 3T3 , Animais , DNA Complementar/genética , Quinase I-kappa B , Proteínas I-kappa B/genética , Proteínas I-kappa B/fisiologia , Técnicas In Vitro , Camundongos , Inibidor de NF-kappaB alfa , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sais de Tetrazólio , Tiazóis , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Adhesion molecules can initiate intracellular signaling. Engagement of CD44 either by its natural ligand hyaluronan or a specific antibody on a cell line induced tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which then associated with phosphatidylinositol 3-kinase (PI3K) and activated mitogen-activated protein kinase at its downstream. However, the introduction of dominant negative Rho into the cells inhibited the CD44-stimulated FAK phosphorylation. Cells expressing CD44 were significantly resistant to etoposide-induced apoptosis. This anti-apoptotic effect was cancelled by the inhibition of either Rho, FAK or PI3K. These results may indicate a signaling pathway from CD44 to mediate the resistance against drug-induced apoptosis in cancer cells.
Assuntos
Apoptose/fisiologia , Receptores de Hialuronatos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoAssuntos
Doença de Fabry/patologia , Fibrose/patologia , Ventrículos do Coração/patologia , Idoso , Biópsia , Ecocardiografia/métodos , Eletrocardiografia , Terapia de Reposição de Enzimas/métodos , Doença de Fabry/sangue , Doença de Fabry/fisiopatologia , Coração/fisiopatologia , Ventrículos do Coração/anatomia & histologia , Ventrículos do Coração/fisiopatologia , Humanos , Espectroscopia de Ressonância Magnética/métodos , Masculino , Tomografia Computadorizada Multidetectores/métodos , Peptídeo Natriurético Encefálico/sangue , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/fisiopatologia , alfa-Galactosidase/sangue , alfa-Galactosidase/genéticaRESUMO
The human ortholog of mammalian enabled (hMena), a member of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family, is an actin regulatory protein involved in the regulation of cell motility. Increasing evidence suggests that hMena overexpression is involved in human cancers, but the upstream events that influence the expression of hMena remain to be elucidated. In this study, we performed immunohistochemical analysis of the expression of hMena protein in paraffin-embedded archival tissues of infiltrating ductal carcinomas (IDCs) obtained from 52 cases. We found that elevated hMena expression is associated with larger tumor size (>2.5 cm, p<0.01), HER2 expression (p<0.05), p53 index (p<0.03) and Ki67 index (p<0.01), suggesting that hMena is a predictor of poor prognosis in IDCs. The histological characteristics of each specimen showed that hMena was overexpressed in the tumor cells at the invasive front of IDCs, indicating that hMena expression is at least partly mediated by tumor cell-matrix interactions. To explore the role of the absence of p53 function in hMena overexpression of IDCs, wild-type p53 cDNA was introduced into SW620 cells, which originally express mutant p53. In wild-type p53-transfected cells, hMena mRNA expression was decreased to 70% of the levels in mock transfected cells (p<0.01). In conclusion, our study indicates that hMena overexpression is involved in the progression of IDCs, and raises the possibility that wild-type p53 may suppress hMena expression.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal/metabolismo , Proteínas dos Microfilamentos/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal/genética , Carcinoma Ductal/patologia , DNA Complementar/genética , Progressão da Doença , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Genes p53 , Células HeLa , Humanos , Imuno-Histoquímica , Antígeno Ki-67/biossíntese , Antígeno Ki-67/genética , Proteínas dos Microfilamentos/genética , Prognóstico , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genéticaRESUMO
Hyaluronan (HA) is synthesized by HA synthase (HAS) 1, HAS2 and HAS3, and degraded by hyaluronidase (HYAL) 1 and HYAL2 in a CD44-dependent manner. HA and HYALs are intricately involved in tumor growth and metastasis. Random cell movement is generally described as chemokinesis, and represents an important step at the beginning of tumor cell liberation from the primary site. To investigate the roles of HAS2 and HYAL2/CD44 in cell motility, we examined HeLa-S3 cells showing spontaneous chemokinesis. HeLa-S3 cells expressed HAS2 and HAS3. siRNA-mediated knockdown of HAS2 decreased spontaneous chemokinesis of HeLa-S3 cells. Although HeLa-S3 cells secreted 50 ng/ml of high molecular weight (HMW)-HA (peak: 990 kDa) into the culture supernatant after 6 h of culture, exogenously added HMW-HA did not enhance spontaneous chemokinesis of the cells. These observations suggested that HeLa-S3 cells may have a self-degrading system for HA to regulate their spontaneous chemokinesis. To examine this possibility, we investigated the effects of siRNA-mediated knockdown of HYAL2 or CD44 on the spontaneous chemokinesis of HeLa-S3 cells. Knockdown of either molecule decreased the spontaneous chemokinesis of the cells. Low molecular weight (LMW)-HA (23 kDa) reversed the HYAL2 siRNA-mediated reduction in spontaneous chemokinesis of HeLa-S3 cells to the level in control cells stimulated with the same HA. These findings indicate that the HAS2-HYAL2/CD44 system may support spontaneous chemokinesis of human cancer cells through self-degradation of HMW-HA to produce LMW-HA by an autocrine mechanism. Consequently, our study may further expand our understanding of HA functions in cancer.
Assuntos
Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Glucuronosiltransferase/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Receptores de Hialuronatos/genética , Hialuronan Sintases , Ácido Hialurônico/farmacologia , Hialuronoglucosaminidase/genética , Peso MolecularRESUMO
The Notch signalling pathway has a crucial function in determining cell fates in multiple tissues within metazoan organisms. On binding to ligands, the Notch receptor is cleaved proteolytically and releases its intracellular domain (NotchICD). The NotchICD enters the nucleus and acts cooperatively with other factors to stimulate the transcription of target genes. High levels of Notch-mediated transcriptional activation require the formation of a ternary complex consisting of NotchICD, CSL (CBF-1, suppressor of hairless, LAG-1) and a Mastermind family member. However, it is still not clear how the formation of the ternary complex is regulated. Here we show that Nemo-like kinase (NLK) negatively regulates Notch-dependent transcriptional activation by decreasing the formation of this ternary complex. Using a biochemical screen, we identified Notch as a new substrate of NLK. NLK-phosphorylated Notch1ICD is impaired in its ability to form a transcriptionally active ternary complex. Furthermore, knockdown of NLK leads to hyperactivation of Notch signalling and consequently decreases neurogenesis in zebrafish. Our results both define a new function for NLK and reveal a previously unidentified mode of regulation in the Notch signalling pathway.
Assuntos
Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Substituição de Aminoácidos/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , DNA/metabolismo , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 3 , Embrião não Mamífero/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/genética , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores Notch/genética , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Xenopus , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mammarian enabled (Mena), a member of the Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) family of proteins, has been implicated in cell motility through regulation of the actin cytoskeleton assembly, including lamellipodial protrusion. Rac1, a member of the Rho family GTPases, also plays a pivotal role in the formation of lamellipodia. Here we report that human Mena (hMena) colocalizes with Rac1 in lamellipodia, and using an unmixing assisted acceptor depletion fluorescence resonance energy transfer (u-adFRET) analysis that hMena associates with Rac1 in vivo in the glioblastoma cell line U251MG. Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia. This cellular phenotype is canceled by introduction of a dominant negative form of Rac1. A Rac activity assay and FRET analysis showed that hMena knock-down cells increased the activation of Rac1 at the lamellipodia. These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Proteínas dos Microfilamentos/fisiologia , Proteínas de Neoplasias/fisiologia , Pseudópodes/química , Proteínas rac1 de Ligação ao GTP/fisiologia , Neoplasias Encefálicas/química , Linhagem Celular Tumoral/química , Linhagem Celular Tumoral/ultraestrutura , Movimento Celular , Forma Celular , Transferência Ressonante de Energia de Fluorescência , Glioblastoma/química , Células HeLa/química , Células HeLa/ultraestrutura , Humanos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Mapeamento de Interação de Proteínas , Pseudópodes/ultraestrutura , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/químicaRESUMO
Fibronectin (FN) is thought to play an important role in various aspects of hematopoiesis through binding to very late antigen (VLA)-4 and VLA-5. Little is known, however, about the effects of FN on the proliferation of B cell progenitors. In this study, we investigated the effects of immobilized FN on the proliferation of the pre-B cell line, Nalm-6, which expresses both VLA-4 and VLA-5. Immobilized FN significantly promoted the proliferation of Nalm-6 cells through the synergistic effects of VLA-4 and VLA-5. Furthermore, FN induced the phosphorylation of mitogen-activated protein kinases (MAPKs) of Nalm-6 cells. The MAPK kinase 1 (MEK1) inhibitor, PD98059, and Src family tyrosine kinase inhibitor, herbimycin A, inhibited the FN-promoted proliferation of Nalm-6 cells. These results demonstrate that the interactions of FN and VLA-4/VLA-5 transmit the growth signals that are mediated through Src family tyrosine kinases and the MAPK cascade in Nalm-6 cells. The precise mechanism of synergistic effect of VLA-4 and VLA-5 on FN-promoted proliferation of Nalm-6 cells should be further investigated.
Assuntos
Fibronectinas/farmacologia , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Células Precursoras de Linfócitos B/citologia , Benzoquinonas/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Fibronectinas/antagonistas & inibidores , Fibronectinas/metabolismo , Flavonoides/farmacologia , Citometria de Fluxo , Humanos , Lactamas Macrocíclicas/farmacologia , Leucemia de Células B , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/enzimologia , Células Precursoras de Linfócitos B/metabolismo , Rifabutina/análogos & derivados , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
The cell adhesion molecule CD44, which is the major hyaluronan receptor, has been implicated in the binding, endocytosis, and metabolism of hyaluronan. Previous studies have revealed that CD44 plays crucial roles in a variety of inflammatory diseases. In recent years, TLRs, which are ancient microbial pattern recognition receptors, have been shown to initiate an innate immune response and have been linked to a variety of inflammatory diseases. The present study shows that CD44 negatively regulates in vivo inflammation mediated by TLRs via NF-kappaB activation, which leads to proinflammatory cytokine production. Furthermore, our results show that CD44 directly associates with TLR2 when stimulated by the TLR2 ligand zymosan and that the cytoplasmic domain of CD44 is crucial for its regulatory effect on TLR signaling. This study indicates that CD44 plays a protective role in TLR-mediated inflammation and is the first to demonstrate a direct association between CD44 and a TLR.
Assuntos
Regulação para Baixo/imunologia , Receptores de Hialuronatos/fisiologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/fisiologia , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/fisiologia , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Experimental/prevenção & controle , Linhagem Celular , Células Cultivadas , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo , Zimosan/metabolismo , Zimosan/toxicidadeRESUMO
Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wild-type CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.