RESUMO
Wnt signaling plays an important role in adult brain function, and its dysregulation has been implicated in the loss of neuronal homeostasis. Despite the existence of many studies on the participation of the Wnt pathway in adult neurons, its regulation in astrocytes has been scarcely explored. Several reports point to the presence of Wnt ligands in astrocytes and their possible impact on neuronal plasticity or neuronal death. We aimed to analyze the effect of the neurotransmitter glutamate and the inflammatory cytokine TNFα on the mRNA and protein levels of the canonical Wnt agonist Wnt7a and the antagonist Dkk1 in cultured astrocytes. Primary astrocyte cultures from rat cerebral cortices were exposed to glutamate or TNFα. Wnt7a and Dkk1 expression was analyzed by RT-qPCR and its protein abundance and distribution was assessed by immunofluorescence. We found high basal expression and protein levels of Wnt7a and Dkk1 in unstimulated astrocytes and overproduction of Dkk1 mRNA induced by the two stimuli. These results reveal the astrocytic source of the canonical Wnt ligands Wnt7a and Dkk1, whose levels are differentially regulated by glutamate and TNFα. Astrocytes are a significant source of Wnt ligands, the production of which can be differentially regulated under excitatory or proinflammatory conditions, thereby impacting neuronal function.
Assuntos
Astrócitos , Ácido Glutâmico , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Proto-Oncogênicas , Fator de Necrose Tumoral alfa , Proteínas Wnt , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Animais , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Wnt/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Ratos , RNA Mensageiro/metabolismo , Ratos Wistar , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/citologiaRESUMO
The aim of this study is to evaluate if single nucleotide polymorphisms (SNPs) in WNT6 and WNT10A are associated with the risk of dental pulp calcification in orthodontic patients. This cross-sectional study followed the "Strengthening the Reporting of Genetic Association Studies" (STREGA) guidelines. Panoramic radiographs (pre- and post-orthodontic treatment) and genomic DNA from 132 orthodontic patients were studied. Dental pulp calcification (pulp stones and/or pulp space narrowing) was recorded in upper and lower first molars. The SNPs in WNT6 and WNT10A (rs7349332, rs3806557, rs10177996, and rs6754599) were assessed through genotyping analysis using DNA extracted from buccal epithelial cells. The association between pulp calcification and SNPs were analyzed using allelic and genotypic distributions and haplotype frequencies (p<0.05). Prevalence of dental pulp calcification was 42.4% in the 490 studied molars. In the genotypic analysis, the SNPs in WNT10A showed a statistically significant value for molar calcification (p = 0.027 for rs1017799), upper molar calcification (p = 0.040 for rs1017799) (recessive model), and molar calcification (p = 0.046 for rs3806557) (recessive model). In the allelic distribution, the allele C of the SNP rs10177996 in WNT10A was associated with molar calcifications (p = 0.042) and with upper first molar calcification (p = 0.035). Nine combinations of haplotypes showed statistically significant value (p<0.05). The findings of this study indicates that SNPs in WNT10A and WNT6 are associated with dental pulp calcification in molars after orthodontic treatment and may be considered as biomarkers for dental pulp calcification.
Assuntos
Calcificações da Polpa Dentária , Polimorfismo de Nucleotídeo Único , Humanos , Estudos Transversais , Radiografia Panorâmica , Dente Molar , Polpa Dentária , Proteínas Wnt/genéticaRESUMO
The receptor tyrosine kinase orphan receptor 1 (ROR1) is a receptor for WNT5A and related Wnt proteins, that play an important role during embryonic development by regulating cell migration, cell polarity, neural patterning, and organogenesis. ROR1 exerts these functions by transducing signals from the Wnt secreted glycoproteins to the intracellular Wnt/PCP and Wnt/Ca++ pathways. Investigations in adult human cells, particularly cancer cells, have demonstrated that besides these two pathways, the WNT5A/ROR1 axis can activate a number of signaling pathways, including the PI3K/AKT, MAPK, NF-κB, STAT3, and Hippo pathways. Moreover, ROR1 is aberrantly expressed in cancer and was associated with tumor progression and poor survival by promoting cell proliferation, survival, invasion, epithelial to mesenchymal transition, and metastasis. Consequently, numerous therapeutic tools to target ROR1 are currently being evaluated in cancer patients. In this review, we will provide a detailed description of the signaling pathways regulated by ROR1 in cancer and their impact in tumor progression.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias , Gravidez , Feminino , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Neoplasias/genética , Proteínas Wnt/metabolismo , Linhagem Celular Tumoral , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismoRESUMO
BACKGROUND: Ewing's sarcoma is the second most common bone and soft tissue malignancy in children and adolescents. Tumor necrosis factor-α-induced protein 8-like 1 (TIPE1) functions as a tumor suppressor in several cancers. Activation of Wnt/ß-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing's sarcoma. The exact role of TIPE1 in Ewing's sarcoma remains to be elucidated. PURPOSE: This study aimed to assess the expression and function of TIPE1 in Ewing's sarcoma. METHODS: TIPE1 expression in Ewing's sarcoma cells was determined by qPCR and western blotting. Furthermore, the Ewing's sarcoma cell line RD-ES was transfected with a lentivirus-based TIPE1 expression system to upregulate the expression of TIPE1. The Cell Counting Kit 8 was used to assess the effect of TIPE1 on cell proliferation. The effects of TIPE1 on cell migration and invasion was detected by Transwell assay. Flow cytometry was performed to detect apoptosis. RESULTS: Our results suggested lower TIPE1 expression in Ewing's sarcoma cell lines compared with normal osseous cells. TIPE1 remarkably inhibited the growth and proliferation of Ewing's sarcoma cell; TIPE1 also induced apoptosis and inhibited invasion in vitro. TIPE1 inhibited Ewing's sarcoma growth, motility, and survival through regulation of Wnt/ß-catenin signaling. CONCLUSIONS: Our results demonstrated the anti-tumor function of TIPE1 in Ewing's sarcoma and reveal a novel therapeutic target.
Assuntos
Neoplasias Ósseas , Sarcoma de Ewing , Adolescente , Criança , Humanos , Apoptose , beta Catenina/genética , beta Catenina/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Proliferação de Células , Perfilação da Expressão Gênica , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
The Wnt/ß-catenin signaling pathway is frequently activated in hepatocellular carcinoma (HCC). A number of studies have focused on the aberrant hypermethylation of the DKK family proteins and its role in regulating the activation of specific signaling pathways. However, the exact way by which DKK regulates the signaling pathway caused by Core protein of HCV has not been reported. In the present study, we evaluated the expression level of DKK and its aberrant promoter methylation to investigate the involvement of epigenetic regulation in hepatoma cell lines. The transcription and protein expression of DKK1 was significantly increased, whereas the transcription and protein expression levels of DKK2, DKK3, and DKK4 were significantly decreased following overexpression of Core protein. Pyrosequencing indicated that hypermethylation of DKK3 was increased. This was associated with increased expression of Dnmt1. The investigation of the molecular mechanism indicated that HCV Core protein interacted with Dnmt1, which combined with the promoter of DKK3, leading to methylation of DKK3. Functional studies indicated that Core protein promoted the growth, migration and invasion of cancer cells. However, upregulation of the expression of DKK3 and/or the knockdown of the expression of Dnmt1 inhibited the growth, migration and invasion of cancer cells. Taken together, the data indicated that epigenetic silencing of DKK3 caused by Dnmt1 activated the Wnt/ß-catenin pathway in HCV Core-mediated HCC. Therefore, DKK3 may be a potential diagnostic and therapeutic target for HCC.
Assuntos
Carcinoma Hepatocelular , Hepatite C , Neoplasias Hepáticas , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Proliferação de Células , Epigênese Genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Wnt , Via de Sinalização Wnt , beta CateninaRESUMO
Oncostatin M (OSM), which belongs to the IL-6 family of cytokines, is the most potent and effective stimulator of osteoclast formation in this family, as assessed by different in vitro assays. Osteoclastogenesis induced by the IL-6 type of cytokines is mediated by the induction and paracrine stimulation of the osteoclastogenic cytokine receptor activator of nuclear factor κ-B ligand (RANKL), expressed on osteoblast cell membranes and targeting the receptor activator of nuclear factor κ-B (RANK) on osteoclast progenitor cells. The potent effect of OSM on osteoclastogenesis is due to an unusually robust induction of RANKL in osteoblasts through the OSM receptor (OSMR), mediated by a JAK-STAT/MAPK signaling pathway and by unique recruitment of the adapter protein Shc1 to the OSMR. Gene deletion of Osmr in mice results in decreased numbers of osteoclasts and enhanced trabecular bone caused by increased trabecular thickness, indicating that OSM may play a role in physiological regulation of bone remodeling. However, increased amounts of OSM, either through administration of recombinant protein or of adenoviral vectors expressing Osm, results in enhanced bone mass due to increased bone formation without any clear sign of increased osteoclast numbers, a finding which can be reconciled by cell culture experiments demonstrating that OSM can induce osteoblast differentiation and stimulate mineralization of bone nodules in such cultures. Thus, in vitro studies and gene deletion experiments show that OSM is a stimulator of osteoclast formation, whereas administration of OSM to mice shows that OSM is not a strong stimulator of osteoclastogenesis in vivo when administered to adult animals. These observations could be explained by our recent finding showing that OSM is a potent stimulator of the osteoclastogenesis inhibitor WNT16, acting in a negative feedback loop to reduce OSM-induced osteoclast formation.
Assuntos
Oncostatina M/metabolismo , Osteoclastos , Ligante RANK , Animais , Diferenciação Celular , Retroalimentação , Interleucina-6/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Proteínas Wnt/metabolismoRESUMO
SUMMARY: The Wnt pathway is essential for the initiation of lizard tail regeneration. The regenerated lizard tails exhibit obvious morphological differences compared to the original ones. The expression of Wnt1 and Wnt2b proteins in the regenerating tail of Scincella tsinlingensis was detected by immunohistochemistry and then comparatively analyzed for ultrastructural changes in the original and regenerated spinal cord. The ependymal layer of the original spinal cord was pseudostratified with multiciliated cells and primary monociliated cells, while the cells of the ependymal layer of the regenerated spinal cord were organized in a monolayer with a few biciliated cells. Immunolocalization indicated that Wnt1 and Wnt2b were mainly distributed in the dermis near the original tail stump, spinal cord, and clot-positive migratory cells during Stage I, 0-1 days post-amputation (dpa). Wnt1 and Wnt2b were predominantly detected in the epaxial and hypaxial musculature near the original tail stump, wound epithelium, and spinal cord in the original tail during Stage II, 1-7 dpa. Mesenchymal cells and wound epithelium showed immunostaining during Stage III and IV, 7-15 dpa. The ependymal tubes contained these signaling proteins during Stage V and VI, 20- 30 dpa. Labeling was mainly observed in nearby regenerative blood vessels, ependymal cells, epaxial and hypaxial musculature in the apical epithelial layer (AEC) after 45-160 dpa. These findings indicated that Wnt1 and Wnt2b proteins presented primarily in regenerating epidermis and nerve tissues were a critical signal for tail regeneration in S. tsinlingensis.
RESUMEN: La vía Wnt es esencial para el inicio de la regeneración de la cola del lagarto. Las colas de lagarto regeneradas exhiben diferencias morfológicas obvias en comparación con las originales. La expresión de las proteínas Wnt1 y Wnt2b en la cola en regeneración de Scincella tsinlingensis se detectó mediante inmunohistoquímica y luego se analizaron comparativamente los cambios ultraestructurales en la médula espinal original y regenerada. La capa ependimaria de la médula espinal original se pseudoestratificó con células multiciliadas y células monociliadas primarias, mientras que las células de la capa ependimaria de la médula espinal regenerada se organizaron en monocapa con algunas células bicilicadas. La inmunolocalización indicó que Wnt1 y Wnt2b se distribuyeron principalmente en la dermis cerca del muñón de la cola original, la médula espinal y las células migratorias positivas en el coágulo durante la Etapa I, 0-1 días después de la amputación (dpa). Wnt1 y Wnt2b se detectaron predominantemente en la musculatura epaxial e hipaxial cerca del muñón de la cola original, el epitelio de la herida y la médula espinal en la cola original durante la Etapa II, 1-7 dpa. Las células mesenquimales y el epitelio de la herida mostraron inmunomarcaje durante la Etapa III y IV, 7- 15 dpa. Los tubos ependimarios contenían estas proteínas de señalización durante la Etapa V y VI, 20-30 dpa. El marcaje se observó principalmente en vasos sanguíneos regenerativos cercanos, células ependimarias, musculatura epaxial e hipaxial en la capa epitelial apical (AEC) después de 45-160 dpa. Estos hallazgos indicaron que las proteínas Wnt1 y Wnt2b están presentes principalmente en la epidermis en regeneración y en los tejidos nerviosos y eran una señal crítica para la regeneración de la cola en S. tsinlingensis.
Assuntos
Animais , Cauda/metabolismo , Cauda/ultraestrutura , Via de Sinalização Wnt , Lagartos/anatomia & histologia , Imuno-Histoquímica , Proteínas Wnt/metabolismo , Regeneração da Medula EspinalRESUMO
Antecedentes: Las células madres intestinales generan las distintas estirpes celulares a dicho nivel. Estas se regulan por interacciones entre el epitelio y las células del nicho celular anexo. Estas se pueden ver dañadas en tratamientos con radiación, generando el síndrome gastrointestinal inducido por radiación. Se ha visto que células madre mesenquimales (MSC) y macrófagos de médula ósea (BMM) tienen propiedades de regeneración tisular. Objetivos: Evaluar la expresión génica de IL-4, Wnt6, VEGF y bFGF, a partir de cultivos celulares primarios independientes de MSC derivadas de tejido adiposo y BMM de ratones C57BL/6, por medio de PCR en tiempo real (qRT-PCR). Diseño experimental: A partir de un análisis in silico, se confeccionaron primers para evaluar la expresión génica de las moléculas propuestas, en los cultivos primarios por medio de qRT-PCR y electroforesis. Resultados y proyecciones: IL-4 y Wnt6 no son expresadas en las muestras de BMM y MSC. VEGF y bFGF son expresadas por diferentes células, dando expresión diferenciada. A futuro, se deben evaluar las mismas estirpes celulares en un ambiente inflamatorio y su efecto en la expresión génica, en especial VEGF y bFGF. Limitaciones: El número de moléculas en estudio es limitado y la expresión se evalúo solo a nivel genético.
Background: Intestinal stem cell generates diferents cellular types in their niche. They're regulated by interactions between epithelium and niche's cells, and can be damaged by medical radiation treatments causing radiation-induced gastrointestinal syndrome. It has seen that mesenchymal stem cells (MSC) d and bone marrow-derived macrophages (BMM) have propierties of tissular regeneration. Objectives: Determinated genetic expression of IL-4, Wnt6, VEGF and bFGF, in primary cellular cultures of MSC derivated of adipose tissue and BMM of C57BL/6 mice, through real time PCR (qRT-PCR). Methods: By an in silico analysis, we created primers to evaluate the proposed molecules in the primary cellular cultives, with qRT-PCR and electrophoresis. Results and projections: IL-4 and Wnt6 were not expressed in the MSC and BMM samples. VEGF and bFGF were expressed by different cells, giving differential expression. In the future, the same samples should be analyzed in an inflammatory environment, especially VEGF and bFGF. Limitations: The number of molecules are limited and the expression of them is only in a genetic level.
Assuntos
Animais , Camundongos , Lesões por Radiação , Fatores Biológicos/genética , Interleucina-4/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas Wnt/genética , Células-Tronco Mesenquimais/efeitos da radiação , Células-Tronco/efeitos da radiaçãoRESUMO
BACKGROUND: Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/ß-catenin-dependent or ß-catenin-independent or "non-canonical", both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in embryonic hippocampal neurons (DIV 4). Under these conditions, the activity of exogenous Wnt ligands on the complexity of the dendritic tree and axonal polarity were evaluated METHODS: Cultured primary embryonic hippocampal neurons obtained from Sprague-Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24 h, Wnt ligands were added to the cultures on DIV 3 for 24 h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry was carried out to evaluate neuronal polarity. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, ß-catenin and GSK-3ß (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin. RESULTS: We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of embryonic hippocampal neurons, with decreases ß-catenin and Wnt3a and an apparent increase in GSK-3ß (p-Ser9) levels. No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative to Wnt concentrations. CONCLUSIONS: Results indicated that Wnt ligands, added exogenously, restored dendritic arbor complexity in embryonic hippocampal neurons, previously treated with a high affinity specific Porcupine inhibitor. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat Wnt-related diseases. Video Abstract.
Assuntos
Glicogênio Sintase Quinase 3 beta/genética , Neurônios/metabolismo , Proteína Wnt3A/genética , beta Catenina/genética , Animais , Axônios/metabolismo , Benzenoacetamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Polaridade Celular/genética , Proliferação de Células/efeitos dos fármacos , Feto , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Ligantes , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Piridinas/farmacologia , Ratos , Proteínas Wnt/genética , Proteína Wnt-5a/genéticaRESUMO
Despite advances in the understanding of the molecular mechanisms underlying the basic biology and pathogenesis of pediatric central nervous system (CNS) malignancies, patients still have an extremely unfavorable prognosis. Over the years, a plethora of natural and synthetic compounds has emerged for the pharmacologic intervention of the NF-kB pathway, one of the most frequently dysregulated signaling cascades in human cancer with key roles in cell growth, survival, and therapy resistance. Here, we provide a review about the state-of-the-art concerning the dysregulation of this hub transcription factor in the most prevalent pediatric CNS tumors: glioma, medulloblastoma, and ependymoma. Moreover, we compile the available literature on the anti-proliferative effects of varied NF-kB inhibitors acting alone or in combination with other therapies in vitro, in vivo, and clinical trials. As the wealth of basic research data continues to accumulate, recognizing NF-kB as a therapeutic target may provide important insights to treat these diseases, hopefully contributing to increase cure rates and lower side effects related to therapy.