RESUMEN
PURPOSE: WNT5A/ROR2 signaling pathway has been involved in many human cancers. Its role in pancreatic ductal adenocarcinoma (PDAC) has not been clarified yet. The purpose of this study was to determine the prognostic value of WNT5A expression in conjunction with the ROR2 expression in the same PDAC human tissues. METHODS: We retrospectively analyzed by immunohistochemistry the WNT5A and ROR2 expression in117 paraffin-embedded PDAC specimens following surgical pancreatic resection. The prognostic value of WNT5A and ROR2 was assessed using Kaplan-Meier survival curves and multivariate Cox regression models. RESULTS: High ROR2 expression was detected in 65.8% (77/117) of PDAC tumors, in 28.2% (33/117) in tumor-stroma, and in 71.1% (65/90) of normal pancreatic tissue. High WNT5A expression was found in 76.9% (90/117) of tumors, in 59.0% (69/117) of tumor-stroma, and in 83.0% (73/88) of normal pancreatic tissue. Spearman's correlation coefficiency demonstrated weak association between ROR2 and WNT5A expression in tumor (r=0.184; p=0.047), and no association in stroma (r=0.036; p=0.699). Multivariate analysis showed that regional lymph node invasion and differentiation were independent prognostic factors of survival, while ROR2- and WNT5A expression were not. CONCLUSIONS: Variable expression patterns for ROR2 and WNT5A were demonstrated in PDAC and normal pancreatic tissues suggesting a role for WNT5A/ROR2 signalling pathway, not only in PDAC but also in the normal pancreatic tissue during inflammation. The lack of prognostic significance for ROR2 and WNT5A expression in our cohort, either alone or in subgroup analysis, underlines the complexity of their role in PDAC, which is highly dependent on the different molecular receptor-ligand tissue contexts.
Asunto(s)
Adenocarcinoma/etiología , Carcinoma Ductal Pancreático/etiología , Neoplasias Pancreáticas/etiología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/fisiología , Transducción de Señal , Proteína Wnt-5a/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios RetrospectivosRESUMEN
In recent decades, the prevalence of obesity has risen dramatically worldwide among all age groups. Obesity is characterized by excess fat accumulation and chronic low-grade inflammation. The adipose tissue functions as a metabolically active endocrine organ secreting adipokines. A novel duo of adipokines, the anti-inflammatory secreted frizzled-related protein 5 (Sfrp5) and the proinflammatory wingless type mouse mammary tumor virus (MMTV) integration site family member 5A (Wnt5a), signal via the non-canonical Wnt pathway. Recent evidence suggests that Sfpr5 and Wnt5a play a key role in the pathogenesis of obesity and its metabolic complications. This review summarizes the current knowledge on the novel regulatory system of anti-inflammatory Sfrp5 and pro-inflammatory Wnt5a, and their relation to obesity and obesity-related complications. Future studies are required to investigate the potential role of Sfrp5 and Wnt5a as biomarkers for monitoring the response to lifestyle interventions and for predicting the development of cardiometabolic risk factors. These adipokines may also serve as novel therapeutic targets for obesity-related disorders.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Obesidad/etiología , Proteína Wnt-5a/fisiología , Tejido Adiposo/fisiopatología , Adolescente , Adulto , Animales , Factores de Riesgo Cardiometabólico , Niño , Dieta Saludable , Humanos , Inflamación/fisiopatología , Resistencia a la Insulina , Estilo de Vida , Enfermedad del Hígado Graso no Alcohólico , Obesidad/fisiopatología , Obesidad/terapiaRESUMEN
During digit development, the correct balance of chondrogenic signals ensures the recruitment of undifferentiated cells into the cartilage lineage or the maintenance of cells at the undifferentiated stage. WNT/ß catenin maintains the pool of progenitor cells, whereas TGFß signalling promotes cartilage differentiation by inducing Sox9 expression. Moreover, WNT5A promotes the degradation of ß catenin during mouse limb development. Although these mechanisms are well established, it is still unknown whether the signalling pathway downstream WNT5A is also involved in early chondrogenesis during digit formation. Thus, the aim of this study was to determine the role of WNT5A during the recruitment of progenitor cells during digit development. Our results showed that WNT5A activated calcium (Ca2+) release in the undifferentiated region during digit development. Further, the blockade of Ca2+ release or calcineurin (CaN) or nuclear factor of activated T-cells (NFAT) functions resulted in an inhibition of cartilage differentiation. Together, our results demonstrate that non canonical WNT5A-Ca2+-CaN-NFAT signalling plays a key role during embryonic digit development in vivo promoting the competence for chondrogenic signals and also acts as a permissive factor for chondrogenesis independently of cell death mechanisms.
Asunto(s)
Señalización del Calcio , Condrogénesis , Factores de Transcripción NFATC/metabolismo , Dedos del Pie/embriología , Proteína Wnt-5a/fisiología , Animales , Calcineurina/metabolismo , Calcio/metabolismo , Embrión de Pollo , Extremidades/embriología , Factor de Transcripción SOX9/metabolismoRESUMEN
The ovarian surface epithelium (OSE) is a monolayer that covers the ovarian surface and is involved in ovulation by rupturing and enabling release of a mature oocyte and by repairing the wound after ovulation. Epithelial-to-mesenchymal transition (EMT) is a mechanism that may promote wound healing after ovulation. While this process is poorly understood in the OSE, in other tissues wound repair is known to be under the control of the local microenvironment and different growth factors such as the WNT signaling pathway. Among WNT family members, WNT4 and WNT5a are expressed in the OSE and are critical for the ovulatory process. The objective of this study was to determine the potential roles of WNT4 and WNT5a in regulating the OSE layer. Using primary cultures of mouse OSE cells, we found WNT5a, but not WNT4, promotes EMT through a non-canonical Ca2+-dependent pathway, up-regulating the expression of Vimentin and CD44, enhancing cell migration, and inhibiting the CTNNB1 pathway and proliferation. We conclude that WNT5a is a stimulator of the EMT in OSE cells, and acts by suppressing canonical WNT signaling activity and inducing the non-canonical Ca2+ pathway.
Asunto(s)
Transición Epitelial-Mesenquimal , Epitelio/metabolismo , Ovario/metabolismo , Proteína Wnt-5a/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Immunoblotting , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba , Vía de Señalización Wnt , Proteína Wnt-5a/metabolismo , Proteína Wnt4/metabolismo , Proteína Wnt4/fisiologíaRESUMEN
BACKGROUND: Tumor-associated macrophages (TAMs) in the tumor microenvironment influence tumor initiation, invasion and metastasis. Several studies have shown that Wnt5a is mainly expressed in the tumor stroma, especially in TAMs. However, whether Wnt5a regulates the polarization and biological function of TAMs in colorectal cancer (CRC) is incompletely understood. METHODS: Immunofluorescence staining was performed to detect CD68 and Wnt5a expression in colorectal tissues from patients (63 CRC specimens VS 20 normal tissues). RT-qPCR, flow cytometry, ELISA and inhibitors were carried out to explore the role of Wnt5a in the polarization of TAMs. Clone formation and transwell assays were performed to determine the effects of Wnt5a-treated macrophages on tumor proliferation, migration and invasion in vitro. Finally, a xenograft model was applied to confirm the effects of Wnt5a+ TAMs on CRC tumorigenesis. RESULTS: We found that high Wnt5a+CD68+/CD68+ TAMs ratio was significantly associated with poor prognosis in CRC patients and Wnt5a+ TAM was an M2-like TAM subtype. Subsequently, we found that Wnt5a induced macrophages to secrete IL-10, which then acted as an autocrine cytokine to induce M2 polarization of these macrophages. IL-10 neutralizing antibody completely reversed the pro-M2 effect of Wnt5a. Mechanistically, the CaKMII-ERK1/2-STAT3 pathway was required for Wnt5a-mediated IL-10 expression in macrophages. Furthermore, Wnt5a-induced M2 macrophages promoted CRC cells proliferation, migration and invasion; knockdown of Wnt5a in TAMs significantly impaired the pro-tumor functions of TAMs. CONCLUSIONS: Our data indicate that Wnt5a could induce M2 polarization of TAMs by regulating CaKMII-ERK1/2-STAT3 pathway-mediated IL-10 secretion, ultimately promoting tumor growth and metastasis of CRC.
Asunto(s)
Carcinogénesis/metabolismo , Neoplasias Colorrectales/metabolismo , Interleucina-10/inmunología , Microambiente Tumoral , Macrófagos Asociados a Tumores/inmunología , Proteína Wnt-5a/fisiología , Animales , Línea Celular Tumoral , Femenino , Humanos , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Macrófagos Asociados a Tumores/citologíaRESUMEN
LncRNAs play critical roles in various pathophysiological and biological processes, such as protein translation, RNA splicing, and epigenetic modification. Indeed, abundant evidences demonstrated that lncRNA act as competing endogenous RNAs (ceRNAs) to participate in tumorigenesis. However, little is known about the underlying function of lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1) in pediatric and adolescent acute myeloid leukemia (AML). The expression of LINP1 was examined in AML patient samples by qRT-PCR. Cell proliferation was examined by CCK-8 and Edu assays. ß-Galactosidase senescence assay, mGlucose uptake assay, lactate production assay, and Gene Ontology (GO) analysis were performed for functional analysis. We found that LINP1 was significantly overexpressed in AML patients at diagnosis, whereas downregulated after complete remission (CR). Furthermore, knockdown of LINP1 expression remarkably suppressed glucose uptake and AML cell maintenance. Mechanistically, LINP1 was found to inhibit the glucose metabolism by suppressing the expression of HNF4a. Both LINP1 and HNF4a knockdown reduced the expression levels of AMPK phosphorylation and WNT5A, indicating for the first time that LINP1 strengthened the HNF4a-AMPK/WNT5A signaling pathway involved in cell glucose metabolism modulation and AML cell survival. Taken together, our results indicated that LINP1 promotes the malignant phenotype of AML cells and stimulates glucose metabolism, which can be regarded as a potential prognostic marker and therapeutic target for AML.
Asunto(s)
Adenilato Quinasa/fisiología , Factor Nuclear 4 del Hepatocito/fisiología , Leucemia Mieloide Aguda/genética , ARN Largo no Codificante/fisiología , ARN Neoplásico/fisiología , Transducción de Señal/fisiología , Proteína Wnt-5a/fisiología , Adolescente , Animales , Médula Ósea/patología , División Celular , Niño , Regulación Leucémica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ontología de Genes , Glucosa/metabolismo , Factor Nuclear 4 del Hepatocito/biosíntesis , Factor Nuclear 4 del Hepatocito/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Púrpura Trombocitopénica Idiopática/metabolismo , Interferencia de ARN , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , Distribución Aleatoria , Inducción de Remisión , Transducción de Señal/genética , Células THP-1RESUMEN
BACKGROUND: Although the complex roles of macrophages in myocardial injury are widely appreciated, the function of neutrophils in nonischemic cardiac pathology has received relatively little attention. METHODS: To examine the regulation and function of neutrophils in pressure overload-induced cardiac hypertrophy, mice underwent treatment with Ly6G antibody to deplete neutrophils and then were subjected to transverse aortic constriction. RESULTS: Neutrophil depletion diminished transverse aortic constriction-induced hypertrophy and inflammation and preserved cardiac function. Myeloid deficiency of Wnt5a, a noncanonical Wnt, suppressed neutrophil infiltration to the hearts of transverse aortic constriction-treated mice and produced a phenotype that was similar to the neutropenic conditions. Conversely, mice overexpressing Wnt5a in myeloid cells displayed greater hypertrophic growth, inflammation, and cardiac dysfunction. Neutrophil depletion reversed the Wnt5a overexpression-induced cardiac pathology and eliminated differences in cardiac parameters between wild-type and myeloid-specific Wnt5a transgenic mice. CONCLUSIONS: These findings reveal that Wnt5a-regulated neutrophil infiltration has a critical role in pressure overload-induced heart failure.
Asunto(s)
Hipertrofia Ventricular Izquierda/fisiopatología , Neutrófilos/fisiología , Proteína Wnt-5a/fisiología , Animales , Aorta Torácica , Quimiotaxis de Leucocito , Constricción , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/inmunología , Inflamación , Procedimientos de Reducción del Leucocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infarto del Miocardio/complicaciones , Infarto del Miocardio/fisiopatología , Presión , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , Estrés Mecánico , Remodelación Ventricular/genética , Proteína Wnt-5a/biosíntesis , Proteína Wnt-5a/deficiencia , Proteína Wnt-5a/genéticaRESUMEN
Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarity and cytoskeletal oscillation. These processes diminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.
Asunto(s)
Polaridad Celular , Citoesqueleto/fisiología , Mandíbula/embriología , Mandíbula/fisiología , Proteína Wnt-5a/fisiología , Citoesqueleto de Actina , Actomiosina/metabolismo , Animales , Calcio/metabolismo , Ciclo Celular , Citosol/metabolismo , Elasticidad , Células Epiteliales/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Mutación , Oscilometría , Transducción de Señal , Estrés Mecánico , Vinculina/metabolismo , ViscosidadRESUMEN
Fungal keratitis causes devastating corneal ulcers which can result in significant visual impairment and even blindness. As a ligand that activates the non-canonical Wnt signaling pathways, Wnt5a triggers the production of important inflammatory chemokines and the chemotactic migration of neutrophils. In this study we aimed to characterize the role of Wnt5a production, in situ, in vivo and in vitro in response to fungal keratitis. Wnt5a expression in corneas of Aspergillus fumigatus (A. fumigatus) keratitis patients was determined by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. In vivo and in vitro experiments were then performed in mouse models and THP-1 macrophages cell cultures infected with A. fumigatus, respectively. C57BL/6 mice were pretreated with siRNAs or neutralizing antibodies for dectin-1, LOX-1 and Wnt5a, or inhibitors of erk1/2 and JNK. Changes in Wnt5a expression were assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blot and bioluminescence imaging system image acquisition. We confirmed that corneal Wnt5a expression increased with A. fumigatus keratitis in patients and a murine model. Wnt5a production was dependent on dectin-1 and LOX-1 expression with contributions by Erk1/2 and JNK pathways. Additionally, Wnt5a knockdown revealed decreased levels of MPO, lower neutrophil recruitment, and a higher fungal load in mouse models. Compared with controls, Wnt5a knockdown impaired pro-inflammatory cytokine IL-1ß production in response to A. fumigatus exposure. Wnt5a also produces dectin-1 and LOX-1 induced inflammatory signature via effective neutrophil recruitment and inflammatory cytokine production in response to A. fumigatus keratitis. These findings demonstrate that Wnt5a is a critical component of the antifungal immune response.
Asunto(s)
Aspergilosis/inmunología , Aspergillus fumigatus/inmunología , Resistencia a la Enfermedad/inmunología , Infecciones Fúngicas del Ojo/inmunología , Queratitis/inmunología , Proteína Wnt-5a/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-1beta/inmunología , Lectinas Tipo C/inmunología , Sistema de Señalización de MAP Quinasas , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores Depuradores de Clase E/inmunología , Células THP-1 , Vía de Señalización Wnt , Proteína Wnt-5a/genética , Proteína Wnt-5a/inmunologíaRESUMEN
Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-ß in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.
Asunto(s)
Vesículas Extracelulares , Fibrosis Pulmonar Idiopática/etiología , Transducción de Señal , Proteína Wnt-5a/fisiología , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The early midgut undergoes intensive elongation, but the underlying cellular and molecular mechanisms are unknown. The early midgut epithelium is pseudostratified, and its nuclei travel between apical and basal surfaces in concert with cell cycle. Using 3D confocal imaging and 2D live imaging, we profiled behaviors of individual dividing cells. As nuclei migrate apically for mitosis, cells maintain a basal process (BP), which splits but is inherited by only one daughter. After mitosis, some daughters directly use the inherited BP as a "conduit" to transport the nucleus basally, while >50% of daughters generate a new basal filopodium and use it as a path to return the nucleus. Post-mitotic filopodial "pathfinding" is guided by mesenchymal WNT5A. Without WNT5A, some cells fail to tether basally and undergo apoptosis, leading to a shortened midgut. Thus, these studies reveal previously unrecognized strategies for efficient post-mitotic nuclear trafficking, which is critical for early midgut elongation.
Asunto(s)
Seudópodos/fisiología , Proteína Wnt-5a/fisiología , Animales , Ciclo Celular , Movimiento Celular , Núcleo Celular/metabolismo , Sistema Digestivo/metabolismo , Fenómenos Fisiológicos del Sistema Digestivo , Endodermo , Epitelio/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Mitosis/fisiología , Proteína Wnt-5a/metabolismoRESUMEN
Embryonic morphogenesis of a complex organism requires proper regulation of patterning and directional growth. Planar cell polarity (PCP) signaling is emerging as a crucial evolutionarily conserved mechanism whereby directional information is conveyed. PCP is thought to be established by global cues, and recent studies have revealed an instructive role of a Wnt signaling gradient in epithelial tissues of both invertebrates and vertebrates. However, it remains unclear whether Wnt/PCP signaling is regulated in a coordinated manner with embryonic patterning during morphogenesis. Here, in mouse developing limbs, we find that apical ectoderm ridge-derived Fgfs required for limb patterning regulate PCP along the proximal-distal axis in a Wnt5a-dependent manner. We demonstrate with genetic evidence that the Wnt5a gradient acts as a global cue that is instructive in establishing PCP in the limb mesenchyme, and that Wnt5a also plays a permissive role to allow Fgf signaling to orient PCP. Our results indicate that limb morphogenesis is regulated by coordination of directional growth and patterning through integration of Wnt5a and Fgf signaling.
Asunto(s)
Tipificación del Cuerpo/fisiología , Polaridad Celular/fisiología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Factor 8 de Crecimiento de Fibroblastos/fisiología , Proteína Wnt-5a/fisiología , Animales , Tipificación del Cuerpo/genética , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Extremidades/embriología , Factor 4 de Crecimiento de Fibroblastos/deficiencia , Factor 4 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/deficiencia , Factor 8 de Crecimiento de Fibroblastos/genética , Mesodermo/embriología , Ratones , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal , Proteína Wnt-5a/deficiencia , Proteína Wnt-5a/genéticaRESUMEN
BACKGROUND: Skeletal muscle atrophy induced by either aging (sarcopenia) or mechanical unloading is associated with serious health consequences. Long non-coding RNAs (lncRNAs) are implicated as important regulators in numerous physiological and pathological processes. METHODS: Microarray analysis was performed to identify the differentially expressed lncRNAs in skeletal muscle between adult and aged mice. The most decreased lncRNA in aged skeletal muscle was identified. The C2C12 mouse myoblast cells were used to assess the biological function of the lncRNA in vitro. The target microRNA of lncRNA and the target protein of microRNA were predicted by bioinformatics analysis and validated in vitro. Furthermore, the biology function of the lncRNA in vivo was investigated by local overexpression or knockdown the lncRNA in skeletal muscle. The therapeutic effect of the lncRNA overexpression in age-related or mechanical unloading-induced muscle atrophy was also evaluated. RESULTS: We identified a novel lncRNA (muscle anabolic regulator 1, MAR1) which was highly expressed in mice skeletal muscle and positively correlated with muscle differentiation and growth in vitro and in vivo. We predicted and validated that microRNA-487b (miR-487b) was a direct target of MAR1. We also predicted and validated that Wnt5a, an important regulator during myogenesis, was a target of miR-487b in C2C12 cells. Our findings further demonstrated that enforced MAR1 expression in myoblasts led to derepression of Wnt5a. Moreover, MAR1 promoted skeletal muscle mass/strength and Wnt5a protein level in mice. Enforced MAR1 expression in mice attenuated muscle atrophy induced by either aging or unloading. CONCLUSIONS: The newly identified lncRNA MAR1 acts as a miR-487b sponge to regulate Wnt5a protein, resulting in promoting muscle differentiation and regeneration. MAR1 could be a novel therapeutic target for treating muscle atrophy induced by either aging or mechanical unloading.
Asunto(s)
MicroARNs/fisiología , Músculo Esquelético/fisiología , ARN Largo no Codificante/fisiología , Proteína Wnt-5a/fisiología , Envejecimiento/fisiología , Animales , Diferenciación Celular , Línea Celular , Ratones Endogámicos C57BL , Desarrollo de Músculos , Mioblastos/metabolismo , RegeneraciónRESUMEN
Alterations in ERBB family members have been associated with many tumor malignancies. EGFR and ERBB2 have been extensively explored in clinical oncology and several drugs currently target them therapeutically. However, the significance of ERBB4 as a potential therapeutic target remains mostly unexplored, even though ERBB4 is overexpressed or mutated in many solid tumors. Using a unique functional protein microarray platform, we found that ibrutinib inhibits ERBB4 activity in the same nM range as its canonical target, BTK. Cell-based assays revealed that ibrutinib treatment inhibited cell growth and decreased phosphorylation of ERBB4 and downstream targets MEK and ERK in cancer cell lines with high levels of endogenous ERBB4. In vivo, ibrutinib-responsive mouse xenograft tumors showed decreased tumor volumes with ibrutinib treatment. Interestingly, global gene expression comparisons between responsive and non-responsive cells identified a signature featuring the WNT pathway that predicts growth responsiveness to ibrutinib. Non-responsive ERBB4-expressing cell lines featured elevated activity of the WNT pathway, through the overexpression of WNT5A. Moreover, inhibition of WNT5A expression led to an ibrutinib response in non-responsive cell lines. Our data show that inhibiting ERBB4 reduces cell growth in cells that have low WNT5A expression and reveal a link between the ERBB4 and WNT pathways.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias/patología , Pirazoles/farmacología , Pirimidinas/farmacología , Receptor ErbB-4/antagonistas & inhibidores , Proteína Wnt-5a/fisiología , Células A549 , Adenina/análogos & derivados , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Piperidinas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
WNT5A, a representative ligand of activating several non-canonical WNT signal pathways, plays significant roles in oncogenesis and tumor inhibition. It has been shown that the non-receptor tyrosine kinase SRC is required for WNT5A-induced invasion of osteosarcoma cells. However, the precise molecular mechanism underlying WNT5A/SRC-mediated osteosarcoma cells invasion remains poorly defined. The study was designed to explore the role of ERK1/2 in WNT5A/SRC-induced osteosarcoma cells invasion and the downstream target of the SRC/ERK1/2 signalings. We found that WNT5A (100 ng/mL) remarkably stimulated migration and invasion of human osteosarcoma MG-63 cells, whereas inhibiting either SRC kinase activity by siRNA-mediated SRC silence or ERK1/2 phosphorylation by PD98059 treatment suppressed these effects, which suggested that the activation of SRC and ERK1/2 is essential for WNT5A-induced MG-63 cells migration and invasion. Furthermore, ERK1/2 phosphorylation induced by WNT5A was dramatically blocked by SRC siRNA. Additionally, our study further demonstrated that MMP-14 was upregulated after exposure to WNT5A in MG-63 cells, and the increased expression was blocked by SRC siRNA or PD98059. Collectively, these results indicate that WNT5A activates SRC/ERK1/2 signal pathway, leading to the upregulation of MMP-14 expression and MG-63 cells migration and invasion.
Asunto(s)
Neoplasias Óseas/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Metaloproteinasa 14 de la Matriz/biosíntesis , Osteosarcoma/enzimología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteína Wnt-5a/fisiología , Transporte Activo de Núcleo Celular , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular , Núcleo Celular/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Invasividad Neoplásica , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Proto-Oncogénicas pp60(c-src)/fisiologíaRESUMEN
Lineage-committed differentiation is an essential biological program during odontogenesis, which is tightly regulated by lineage-specific genes. Some of these genes are modified by colocalization of H3K4me3 and H3K27me3 marks at promoter regions in progenitors. These modifications, named "bivalent domains," maintain genes in a poised state and then resolve for later activation or repression during differentiation. Wnt5a has been reported to promote odontogenic differentiation in dental mesenchyme. However, relatively little is known about the epigenetic modulations on Wnt5a activation during tooth development. Here, we investigated the spatiotemporal patterns of H3K4me3 and H3K27me3 marks in developing mouse molars. Associated H3K4me3 methylases (mixed-lineage leukemia [MLL] complex) and H3K27me3 demethylases (JMJD3 and UTX) were dynamically expressed between early and late bell stage of human tooth germs and in cultured human dental papilla cells (hDPCs) during odontogenic induction. Poised WNT5A gene was marked by bivalent domains containing repressive marks (H3K27me3) and active marks (H3K4me3) on promoters. The bivalent domains tended to resolve during inducted differentiation, with removal of the H3K27me3 mark in a JMJD3-dependent manner. When JMJD3 was knocked down in cultured hDPCs, odontogenic differentiation was suppressed. The depletion of JMJD3 epigenetically repressed WNT5A activation by increased H3K27me3 marks. In addition, JMJD3 could physically interact with ASH2L, a component of the MLL complex, to form a coactivator complex, cooperatively modulating H3K4me3 marks on WNT5A promoters. Overall, our study reveals that transcription activities of WNT5A were epigenetically regulated by the negotiated balance between H3K27me3 and H3K4me3 marks and tightly mediated by JMJD3 and MLL coactivator complex, ultimately modulating odontogenic commitment during dental mesenchymal cell differentiation.
Asunto(s)
Código de Histonas/fisiología , Odontogénesis/genética , Proteína Wnt-5a/genética , Animales , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Papila Dental/citología , Papila Dental/crecimiento & desarrollo , Epigénesis Genética/genética , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos BALB C , Diente Molar/crecimiento & desarrollo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Wnt-5a/fisiologíaRESUMEN
A recent study indicated that high Wnt5a expression is associated with poor prognosis in non-small-cell lung cancer (NSCLC) patients; however, the underlying mechanism was not clear yet. Immunohistochemistry and Western blotting were performed to examine the protein expression level in NSCLC tissues and cell lines. The role of Wnt5a in clone formation, invasiveness, migration, and epithelial-to-mesenchymal transition (EMT) of NSCLC cells was studied. Luciferase reporter assay was used to evaluate the Tcf/Lef transcriptional activity. For assessing the effects of Wnt5a on tumor growth and metastasis in vivo, A549 cells transfected with sh-Wnt5a were subcutaneously or orthotopically injected into nude mice. In NSCLC tissues, higher expression levels of Wnt5a and ROR2 were found, ß-Catenin was expressed exceptionally, and EMT was prompted. Wnt5a overexpression increased clone formation, migration, and invasion, as well as prompted EMT of NSCLC cell in vitro, whereas Wnt5a knockdown showed the absolutely reversed results. Wnt5a overexpression enhanced the Tcf/Lef transcriptional activity and elevated the nuclear ß-catenin level in NSCLC cells, without altering the ROR2 expression. We also demonstrated that si-ß-catenin antagonized Wnt5a overexpression nduced EMT and invasiveness. Besides, in vivo experiment showed that sh-Wnt5a significantly increased tumor volume and tumor weight, and prompted EMT in A549 tumor-bearing mice as compared with the control. No metastasis was found in the liver tissue after sh-Wnt5a-transfected cells were orthotopically injected into nude mice as compared with the control. In conclusion, Wnt5a promotes EMT and metastasis in NSCLC, which is involved in the activation of ß-catenin-dependent canonical Wnt signaling.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Proteína Wnt-5a/fisiología , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/secundario , Movimiento Celular , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Carga Tumoral , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
OBJECTIVE: Aberrant Wnt signaling may contribute to osteoarthritis (OA) but the Wnt family members involved have not been fully identified. The purpose of this study was to investigate the role of Wnt5a as a potential mediator of cartilage destruction in OA. DESIGN: Immunohistochemistry to detect Wnt5a was performed using normal and OA human articular cartilage. Cultured normal human chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus or recombinant Wnt5a protein with or without pretreatment using a panel of signaling inhibitors. Expression of Wnt5a, anabolic genes and catabolic genes were determined by quantitative real-time PCR. Production of Wnt5a protein and matrix metalloproteinases (MMPs) as well as activation of signaling proteins were analyzed by immunoblotting. RESULTS: Wnt5a was present in human articular cartilage with OA changes and its expression and secretion were increased in FN-f stimulated chondrocytes. FN-f stimulated Wnt5a production through the c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) pathways. Wnt5a reduced aggrecan gene expression after 48 h of treatment. Wnt5a seemed to promote MMP1, -3, and -13 expression as well as MMP1 and MMP13 protein production in normal human chondrocytes. Wnt5a inhibitor peptides did not affect FN-f induced MMP production. Wnt5a activated ß-catenin independent signaling including calmodulin-dependent protein kinase II (CaMKII), JNK, p38, ERK1/2, p65 and Akt. Inhibition of JNK, p38, ERK, PI-3 kinase and CaMKII by specific signaling inhibitors suppressed Wnt5a mediated MMP1 and MMP13 production. CONCLUSIONS: Wnt5a is present in human OA cartilage and can promote chondrocyte catabolic activity through non-canonical Wnt signaling, which suggests a potential role in OA.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Metaloproteinasas de la Matriz/biosíntesis , Osteoartritis/metabolismo , Proteína Wnt-5a/fisiología , Adulto , Anciano , Agrecanos/biosíntesis , Agrecanos/genética , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo/fisiología , Persona de Mediana Edad , Osteoartritis/patología , Proteínas Recombinantes/farmacología , Vía de Señalización Wnt/fisiología , Proteína Wnt-5a/farmacología , Adulto JovenRESUMEN
OBJECTIVE: Our previous study showed that WNT5A, a member of the noncanonical WNT pathway, is involved in interleukin-1beta induced matrix metalloproteinase expression in temporomandibular joint (TMJ) condylar chondrocytes. The purpose of this study is to further explore the roles of WNT5A in cartilage biology of the TMJ. METHODS: An early TMJ osteoarthritis-like rat model was constructed by a mechanical method (steady mouth-opening). The gene and protein levels of WNT5A during the condylar cartilage changes were measured. Effects of WNT5A on chondrocyte proliferation, hypertrophy and migration were analyzed after WNT5A gain or loss of function in vitro. A c-Jun N-terminal kinase (JNK) inhibitor SP600125 was used to evaluate the involvement of JNK pathway in these effects of WNT5A. The expression and transcription activity of cell cycle regulators c-MYC and Cyclin D1 were examined to determine the mechanism behind WNT5A regulation of chondrocyte proliferation. RESULTS: WNT5A was significantly upregulated in the condylar cartilage of rats in the early TMJ osteoarthritis-like model. Activating WNT5A facilitated condylar chondrocyte proliferation, hypertrophy and migration. Conversely, inhibiting WNT5A activity in chondrocytes decreased their proliferation, hypertrophy and migration. Blockage of the JNK pathway by its inhibitor, SP600125, impaired these effects of WNT5A on chondrocytes. WNT5A regulated both the expression and transcriptional activity of c-MYC and Cyclin D1 in chondrocytes, both of which were upregulated in condylar cartilage of the rat early TMJ osteoarthritis. CONCLUSION: WNT5A regulates condylar chondrocyte proliferation, hypertrophy and migration. These findings provide new insights into the role of WNT5A signaling in TMJ cartilage biology and its potential in future therapy for TMJ degenerative diseases.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular/fisiología , Condrocitos/metabolismo , Cóndilo Mandibular/citología , Trastornos de la Articulación Temporomandibular/metabolismo , Proteína Wnt-5a/fisiología , Animales , Condrocitos/citología , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Hipertrofia , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Regulación hacia ArribaRESUMEN
The signaling mechanisms that choreograph the assembly of the highly asymmetric pre- and postsynaptic structures are still poorly defined. Using synaptosome fractionation, immunostaining, and coimmunoprecipitation, we found that Celsr3 and Vangl2, core components of the planar cell polarity (PCP) pathway, are localized at developing glutamatergic synapses and interact with key synaptic proteins. Pyramidal neurons from the hippocampus of Celsr3 knockout mice exhibit loss of â¼50% of glutamatergic synapses, but not inhibitory synapses, in culture. Wnts are known regulators of synapse formation, and our data reveal that Wnt5a inhibits glutamatergic synapses formed via Celsr3. To avoid affecting earlier developmental processes, such as axon guidance, we conditionally knocked out Celsr3 in the hippocampus 1 week after birth. The CA1 neurons that lost Celsr3 also showed a loss of â¼50% of glutamatergic synapses in vivo without affecting the inhibitory synapses assessed by miniature excitatory postsynaptic current (mEPSC) and electron microscopy. These animals displayed deficits in hippocampus-dependent behaviors in adulthood, including spatial learning and memory and fear conditioning. In contrast to Celsr3 conditional knockouts, we found that the conditional knockout of Vangl2 in the hippocampus 1 week after birth led to a large increase in synaptic density, as evaluated by mEPSC frequency and spine density. PCP signaling is mediated by multiple core components with antagonizing functions. Our results document the opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.