RESUMEN
The Wnt/ß-catenin signalling pathway regulates multiple cellular processes during development and many diseases, including cell proliferation, migration, and differentiation. Despite their hydrophobic nature, Wnt proteins exert their function over long distances to induce paracrine signalling. Recent studies have identified several factors involved in Wnt secretion; however, our understanding of how Wnt ligands are transported between cells to interact with their cognate receptors is still debated. Here, we demonstrate that gastric cancer cells utilise cytonemes to transport Wnt3 intercellularly to promote proliferation and cell survival. Furthermore, we identify the membrane-bound scaffolding protein Flotillin-2 (Flot2), frequently overexpressed in gastric cancer, as a modulator of these cytonemes. Together with the Wnt co-receptor and cytoneme initiator Ror2, Flot2 determines the number and length of Wnt3 cytonemes in gastric cancer. Finally, we show that Flotillins are also necessary for Wnt8a cytonemes during zebrafish embryogenesis, suggesting a conserved mechanism for Flotillin-mediated Wnt transport on cytonemes in development and disease.
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Neoplasias Gástricas , Pez Cebra , Animales , Desarrollo Embrionario , Proteínas Wnt/fisiología , Vía de Señalización WntRESUMEN
Wnt/beta-catenin signaling may play an essential role in endometrial decidualization, placentation, and the establishment of pregnancy. We investigate here the possible roles, immunolocalizations, and synthesis of the Wnt3, Wnt7a, and beta-catenin proteins in the rat endometrium during the estrous cycle and early postimplantation period. Wnt3 and Wnt7a had a similar localization and dynamic expression relative to the endometrial stages. Wnt7a immunostaining was not limited only to the luminal epithelial cells, but also to strong stainings in the stromal and endothelial cells. Wnt3, Wnt7a, and beta-catenin were highly synthesized and colocalized at the trophoblast-decidual interface; and were more obvious in the primary decidual zone, the GTCs, and the ectoplacental cone. Beta-catenin was strongly localized at the borders of the mature decidual cells; however, Wnt3 and Wnt7a immunolocalizations were decreased in those cells. As such, the immunolocalization of Wnt3, Wnt7a, and beta-catenin shifted with decidualization and placentation. The expression level of Wnt3, Wnt7a, and beta-catenin messenger RNAs increased in early pregnancy, and especially between Days 8.5 and 9.5. The dramatic changes in the expression of Wnt3, Wnt7a, and beta-catenin observed during the early days of pregnancy and the estrous cycle may indicate their roles in decidualization, stromal cell proliferation, and trophoblast invasion.
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Endometrio/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Wnt/fisiología , Vía de Señalización Wnt , Proteína Wnt3/fisiología , beta Catenina/fisiología , Animales , Decidua/citología , Embrión de Mamíferos/metabolismo , Ciclo Estral/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Embarazo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Células del Estroma/metabolismo , Trofoblastos/citología , Proteínas Wnt/biosíntesis , Proteínas Wnt/genética , Proteína Wnt3/biosíntesis , Proteína Wnt3/genética , beta Catenina/biosíntesis , beta Catenina/genéticaRESUMEN
During embryogenesis, morphogens form a concentration gradient in responsive tissue, which is then translated into a spatial cellular pattern. The mechanisms by which morphogens spread through a tissue to establish such a morphogenetic field remain elusive. Here, we investigate by mutually complementary simulations and in vivo experiments how Wnt morphogen transport by cytonemes differs from typically assumed diffusion-based transport for patterning of highly dynamic tissue such as the neural plate in zebrafish. Stochasticity strongly influences fate acquisition at the single cell level and results in fluctuating boundaries between pattern regions. Stable patterning can be achieved by sorting through concentration dependent cell migration and apoptosis, independent of the morphogen transport mechanism. We show that Wnt transport by cytonemes achieves distinct Wnt thresholds for the brain primordia earlier compared with diffusion-based transport. We conclude that a cytoneme-mediated morphogen transport together with directed cell sorting is a potentially favored mechanism to establish morphogen gradients in rapidly expanding developmental systems.
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Tipificación del Cuerpo/fisiología , Regulación del Desarrollo de la Expresión Génica , Vertebrados/embriología , Proteínas Wnt/fisiología , Animales , Apoptosis , Encéfalo/embriología , Linaje de la Célula , Movimiento Celular , Biología Computacional , Simulación por Computador , Desarrollo Embrionario , Cresta Neural/embriología , Placa Neural/embriología , Transporte de Proteínas , Transducción de Señal , Programas Informáticos , Procesos Estocásticos , Pez Cebra/embriología , beta Catenina/fisiologíaRESUMEN
To spatially co-exist and differentially specify fates within developing tissues, morphogenetic cues must be correctly positioned and interpreted. Here, we investigate mouse hair follicle development to understand how morphogens operate within closely spaced, fate-diverging progenitors. Coupling transcriptomics with genetics, we show that emerging hair progenitors produce both WNTs and WNT inhibitors. Surprisingly, however, instead of generating a negative feedback loop, the signals oppositely polarize, establishing sharp boundaries and consequently a short-range morphogen gradient that we show is essential for three-dimensional pattern formation. By establishing a morphogen gradient at the cellular level, signals become constrained. The progenitor preserves its WNT signaling identity and maintains WNT signaling with underlying mesenchymal neighbors, while its overlying epithelial cells become WNT-restricted. The outcome guarantees emergence of adjacent distinct cell types to pattern the tissue.
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Folículo Piloso/embriología , Células Madre/fisiología , Proteínas Wnt/antagonistas & inhibidores , Vía de Señalización Wnt/fisiología , Animales , Polaridad Celular , Ratones , Morfogénesis/fisiología , Proteínas Wnt/fisiologíaRESUMEN
Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.
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Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Fosfohidrolasa PTEN/fisiología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Ratones Noqueados , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Wnt/ß-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components.
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División Celular Asimétrica/genética , Caenorhabditis elegans/embriología , Células-Madre Neurales/citología , Proteínas Wnt/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , División Celular/genética , Polaridad Celular , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/fisiología , Ligandos , Células-Madre Neurales/fisiología , Neuronas/citología , Neuronas/fisiología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismoRESUMEN
The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/ß-catenin pathway in IPF. The potential impact of WNT/ß-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/ß-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16-24â¯months) versus young (3â¯months) mice had relatively less epithelial (EpCAM+) but more inflammatory (CD45+) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/ß-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/ß-catenin activation for up to 7â¯days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/ß-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/ß-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/ß-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.
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Células Epiteliales Alveolares , Senescencia Celular , Fibrosis Pulmonar Idiopática/metabolismo , Proteínas Wnt/fisiología , Vía de Señalización Wnt , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Línea Celular , Humanos , Pulmón/citología , Pulmón/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
A Wnt signaling network governs early anterior-posterior (AP) specification and patterning of the deuterostome sea urchin embryo. We have previously shown that non-canonical Fzl1/2/7 signaling antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by canonical Wnt/ß-catenin and non-canonical Wnt1/Wnt8-Fzl5/8-JNK signaling. This study focuses on the non-canonical function of the Wnt16 ligand during early AP specification and patterning. Maternally supplied wnt16 is expressed ubiquitously during cleavage and zygotic wnt16 expression is concentrated in the endoderm/mesoderm beginning at mid-blastula stage. Wnt16 antagonizes the ANE restriction mechanism and this activity depends on a functional Fzl1/2/7 receptor. Our results also show that zygotic wnt16 expression depends on both Fzl5/8 and Wnt/ß-catenin signaling. Furthermore, Wnt16 is necessary for the activation and/or maintenance of key regulatory endoderm/mesoderm genes and is essential for gastrulation. Together, our data show that Wnt16 has two functions during early AP specification and patterning: (1) an initial role activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism; and (2) a subsequent function in activating key endoderm GRN factors and the morphogenetic movements of gastrulation.
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Tipificación del Cuerpo/genética , Morfogénesis/genética , Erizos de Mar , Proteínas Wnt/fisiología , Animales , Embrión no Mamífero , Receptores Frizzled/genética , Receptores Frizzled/fisiología , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Mesodermo/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Erizos de Mar/embriología , Erizos de Mar/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
The link between single-cell variation and population-level fate choices lacks a mechanistic explanation despite extensive observations of gene expression and epigenetic variation among individual cells. Here, we found that single human embryonic stem cells (hESCs) have different and biased differentiation potentials toward either neuroectoderm or mesendoderm depending on their G1 lengths before the onset of differentiation. Single-cell variation in G1 length operates in a dynamic equilibrium that establishes a G1 length probability distribution for a population of hESCs and predicts differentiation outcome toward neuroectoderm or mesendoderm lineages. Although sister stem cells generally share G1 lengths, a variable proportion of cells have asymmetric G1 lengths, which maintains the population dispersion. Environmental Wingless-INT (WNT) levels can control the G1 length distribution, apparently as a means of priming the fate of hESC populations once they undergo differentiation. As a downstream mechanism, global 5-hydroxymethylcytosine levels are regulated by G1 length and thereby link G1 length to differentiation outcomes of hESCs. Overall, our findings suggest that intrapopulation heterogeneity in G1 length underlies the pluripotent differentiation potential of stem cell populations.
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Diferenciación Celular , Células Madre Embrionarias/fisiología , Fase G1 , Proteínas Wnt/fisiología , Línea Celular , HumanosRESUMEN
There are 19 Wnt genes in mammals that belong to 12 subfamilies. Wnt signaling pathways participate in regulating numerous homeostatic and developmental processes in animals. However, the function of Wnt10b in fatty acid synthesis remains unclear in fish species. In the present study, we uncovered the role of the Wnt10b signaling pathway in the regulation of fatty acid synthesis in the muscle of zebrafish. The gene of Wnt10b was overexpressed in the muscle of zebrafish using pEGFP-N1-Wnt10b vector injection, which significantly decreased the expression of glycogen synthase kinase 3ß (GSK-3ß), but increased the expression of ß-catenin, peroxisome proliferators-activated receptor γ (PPARγ), and CCAAT/enhancer binding protein α (C/EBPα). Moreover, the activity and mRNA expression of key lipogenic enzymes ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC) and fatty acid synthetase (FAS), and the content of non-esterified fatty acids (NEFA), total cholesterol (TC), and triglyceride (TG) were also significantly decreased. Furthermore, interference of the Wnt10b gene significantly inhibited the expression of ß-catenin, PPARγ, and C/EBPα, but significantly induced the expression of GSK-3ß, FAS, ACC, and ACL. The content of NEFA, TC, and TG as well as the activity of FAS, ACC, and ACL significantly increased. Thus, our results showed that Wnt10b participates in regulating fatty acid synthesis via ß-catenin, C/EBPα and PPARγ in the muscle of zebrafish.
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Ácidos Grasos/metabolismo , Músculos/metabolismo , Proteínas Wnt/fisiología , Vía de Señalización Wnt/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , PPAR gamma/metabolismo , Proteínas Wnt/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. METHODS: Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. RESULTS: Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. CONCLUSIONS: Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.
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Próstata/crecimiento & desarrollo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Wnt/fisiología , Animales , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/patología , Trasplante de Neoplasias , Técnicas de Cultivo de Órganos , Células PC-3 , Neoplasia Intraepitelial Prostática/patología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Wnt/análisis , Proteínas Wnt/genéticaRESUMEN
The central regulator of the Wnt/ß-catenin pathway is the Axin/APC/GSK3ß destruction complex (DC), which, under unstimulated conditions, targets cytoplasmic ß-catenin for degradation. How Wnt activation inhibits the DC to permit ß-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using bimolecular fluorescence complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3ß interactions that prevent ß-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control the degradation of ß-catenin and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signal transduction in vivo.
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Proteína Axina/metabolismo , Complejo de Señalización de la Axina/fisiología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/fisiología , Animales , Animales Modificados Genéticamente , Proteína Axina/química , Complejo de Señalización de la Axina/química , Complejo de Señalización de la Axina/metabolismo , Tipificación del Cuerpo/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrión no Mamífero , Prueba de Complementación Genética , Glucógeno Sintasa Quinasa 3 beta/química , Imagen Óptica , Fosforilación/genética , Unión Proteica/genética , Conformación Proteica , Pliegue de Proteína , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiología , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
OBJECTIVES: This study investigates the role of Wnt7b in mouse dentin formation. DESIGN: C57BL/6 mouse tooth germs at different developmental stages were collected to measure the expression of Wnt7b by immunohistochemical staining. The morphology of mandibles of Dmp1-cre;ROSA26-Wnt7b transgenic mice and ROSA26-Wnt7b littermates was analyzed by Micro-CT and HE staining. The ultramicrostructure of dentin was scanned with an electron microscope. Primary mouse dental papillae cells (MDPCs) and odontoblastic cell line (A11) were cultured and infected with adenovirus to overexpress Wnt7b. Cell proliferation and cell apoptosis were evaluated using CCK-8 and flow cytometry. Osteogenic differentiation of MDPCs and A11 was assessed by Alizarin red staining, and qPCR detection of osteogenic gene expression. The activation of signaling pathways was measured by the use of western blot analysis. The ERK1/2 inhibitor was used to test the effect of Wnt7b regulated cell differentiation. RESULTS: Wnt7b was expressed principally in the mouse odontoblast layer after the early bell stage. In transgenic mice, Wnt7b was over-expressed in tooth mesenchyme, with a thinner predentin layer and thicker intertubular dentin. Both the micro-hardness value and the Ca/Pi ratio of dentin of transgenic mice were higher. Wnt7b promoted proliferation and mineralization of MDPCs and A11. The protein level of p-ERK1/2 was found to be higher in A11 infected with Ad-Wnt7b. The ERK signaling pathway inhibitor partly rescued the Wnt7b-induced differentiation of A11. CONCLUSIONS: Wnt7b enhances dentinogenesis by increasing the proliferation and differentiation of dental mesenchymal cells partly through ERK1/2 pathway.
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Dentinogénesis , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas , Proteínas Wnt , Animales , Diferenciación Celular , Pulpa Dental , Dentinogénesis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Odontoblastos , Osteogénesis , Proteínas Proto-Oncogénicas/fisiología , Proteínas Wnt/fisiologíaRESUMEN
Despite an extensive literature documenting the adaptive changes of bones and ligaments to mechanical forces, our understanding of how tissues actually mount a coordinated response to physical loading is astonishingly inadequate. Here, using finite element (FE) modeling and an in vivo murine model, we demonstrate the stress distributions within the periodontal ligament (PDL) caused by occlusal hyperloading. In direct response, a spatially restricted pattern of apoptosis is triggered in the stressed PDL, the temporal peak of which is coordinated with a spatially restricted burst in PDL cell proliferation. This culminates in increased collagen deposition and a thicker, stiffer PDL that is adapted to its new hyperloading status. Meanwhile, in the adjacent alveolar bone, hyperloading activates bone resorption, the peak of which is followed by a bone formation phase, leading ultimately to an accelerated rate of mineral apposition and an increase in alveolar bone density. All of these adaptive responses are orchestrated by a population of Wnt-responsive stem/progenitor cells residing in the PDL and bone, whose death and revival are ultimately responsible for directly giving rise to new PDL fibers and new bone.
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Ligamento Periodontal/fisiología , Periodoncio/fisiología , Estrés Mecánico , Proteínas Wnt/fisiología , Pérdida de Hueso Alveolar , Animales , Análisis de Elementos Finitos , RatonesRESUMEN
Wingless/integrated (Wnt) signaling has emerged as a major mechanism for promoting bone formation and a target pathway for developing bone anabolic agents against osteoporosis. However, the downstream events mediating the potential therapeutic effect of Wnt proteins are not fully understood. Previous studies have indicated that increased glycolysis is associated with osteoblast differentiation in response to Wnt signaling, but direct genetic evidence for the importance of glucose metabolism in Wnt-induced bone formation is lacking. Here, we have generated compound transgenic mice to overexpress Wnt family member 7B (Wnt7b) transiently in the osteoblast lineage of postnatal mice, with or without concurrent deletion of the glucose transporter 1 (Glut1), also known as solute carrier family 2, facilitated glucose transporter member 1. Overexpression of Wnt7b in 1-mo-old mice for 1 wk markedly stimulated bone formation, but the effect was essentially abolished without Glut1, even though transient deletion of Glut1 itself did not affect normal bone accrual. Consistent with the in vivo results, Wnt7b increased Glut1 expression and glucose consumption in the primary culture of osteoblast lineage cells, and deletion of Glut1 diminished osteoblast differentiation in vitro. Thus, Wnt7b promotes bone formation in part through stimulating glucose metabolism in osteoblast lineage cells.-Chen, H., Ji, X., Lee, W.-C., Shi, Y., Li, B., Abel, E. D., Jiang, D., Huang, W., Long, F. Increased glycolysis mediates Wnt7b-induced bone formation.
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Transportador de Glucosa de Tipo 1/fisiología , Glucosa/metabolismo , Glucólisis , Osteoblastos/metabolismo , Osteogénesis/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Wnt/fisiología , Animales , Linaje de la Célula , Células Cultivadas , Fémur/crecimiento & desarrollo , Fémur/ultraestructura , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Transportador de Glucosa de Tipo 1/deficiencia , Transportador de Glucosa de Tipo 1/genética , Ratones , Ratones Transgénicos , Osteogénesis/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/metabolismo , Tamoxifeno/farmacología , Tibia/crecimiento & desarrollo , Tibia/ultraestructura , Proteínas Wnt/genéticaRESUMEN
OBJECTIVES: Wnt16 is implicated in bone fracture and bone mass accrual both in animals and humans. However, its functional roles and molecular mechanism in chondrocyte differentiation and osteoarthritis (OA) pathophysiology remain largely undefined. In this study, we analysed its mechanistic association and functional relationship in OA progression in chondrocyte lineage. METHODS: The role of Wnt16 during skeletal development was examined by Col2a1-Wnt16 transgenic mice and Wnt16fl/fl;Col2a1-Cre (Wnt16-cKO) mice. OA progression was assessed by micro-CT analysis and Osteoarthritis Research Society International score after anterior cruciate ligament transection (ACLT) surgery with Wnt16 manipulation by adenovirus intra-articular injection. The molecular mechanism was investigated in vitro using 3D chondrocyte pellet culture and biochemical analyses. Histological analysis was performed in mouse joints and human cartilage specimens. RESULTS: Wnt16 overexpression in chondrocytes in mice significantly inhibited chondrocyte hypertrophy during skeletal development. Wnt16 deficiency exaggerated OA progression, whereas intra-articular injection of Ad-Wnt16 markedly attenuated ACLT-induced OA. Cellular and molecular analyses showed that, instead of ß-catenin and calcium pathways, Wnt16 activated the planar cell polarity (PCP) and JNK pathway by interacting mainly with AP2b1, and to a lesser extend Ror2 and CD146, and subsequently induced PTHrP expression through phosphor-Raptor mTORC1 pathway. CONCLUSIONS: Our findings indicate that Wnt16 activates PCP/JNK and crosstalks with mTORC1-PTHrP pathway to inhibit chondrocyte hypertrophy. Our preclinical study suggests that Wnt16 may be a potential therapeutic target for OA treatment.
Asunto(s)
Artritis Experimental/patología , Osteoartritis/patología , Proteínas Wnt/fisiología , Animales , Artritis Experimental/metabolismo , Artritis Experimental/fisiopatología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Diferenciación Celular/fisiología , Polaridad Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Condrocitos/patología , Condrocitos/fisiología , Progresión de la Enfermedad , Humanos , Hipertrofia/prevención & control , Sistema de Señalización de MAP Quinasas/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Ratones Transgénicos , Osteoartritis/metabolismo , Osteoartritis/fisiopatología , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Proteínas Wnt/deficiencia , Proteínas Wnt/metabolismoRESUMEN
Class III Semaphorin (Sema) secreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used in situ hybridization or immunohistochemistry to map the expression of Sema3D, Sema3F, Nrp1, Nrp2, and PlxnA1 in the chicken (Gallus gallus) inner ear from embryonic day (E)5-E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. Sema3D expression flanking the sensory tissue in vestibular organs suggests that it may repel Nrp2- and PlxnA1-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8-E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, Sema3D in the future tegmentum vasculosum opposes Nrp1 and PlxnA1 in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. Sema3D immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear.
Asunto(s)
Pollos/metabolismo , Oído Interno/metabolismo , Neuropilina-1/biosíntesis , Semaforinas/biosíntesis , Animales , Movimiento Celular , Embrión de Pollo , Oído Interno/embriología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Neurogénesis , Neuropilina-1/genética , Órgano Espiral/embriología , Órgano Espiral/metabolismo , Semaforinas/genética , Transducción de Señal , Organismos Libres de Patógenos Específicos , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismo , Proteínas Wnt/farmacología , Proteínas Wnt/fisiologíaRESUMEN
Crown shapes in mammalian teeth vary considerably from species to species, and morphological characters in crown shape have been used to identify species. Cusp pattern is one of the characters in crown shape. In the processes governing the formation of cusp pattern, the Shh pathway has been implicated as an important player. Suppression of Shh signaling activity in vitro in explant assays appears to induce supernumerary cusp formation in wild-type tooth germs. However, the in vivo role of Shh signaling in cusp pattern formation and the molecular mechanisms by which Shh regulates cusp patterning are not clear. Here, through in vivo phenotypic analyses of mice in which Shh activity was suppressed and compared with wild-type mice, we characterized differences in the location, number, incidence, and shape of supernumerary cusps in molars at embryonic day 15.5. We found that the distances between cusps were reduced in molars of Shh activity-suppressed mice in vivo. These findings confirm and extend the previous idea that Shh acts as an inhibitor in the reaction-diffusion model for cusp pattern formation by negatively regulating the intercuspal distance. We uncovered a significant reduction of expression level of Sostdc1, which encodes a secreted modulator of Wnt signaling, after suppression of Shh activity. The supernumerary cusp formation in Sostdc1-/- mice and compound Sostdc1 and Lrp mutant mice indicates a strong association between Wnt and Shh signaling pathways in cusp patterning. In further support of this idea, there is a high degree of similarity in the supernumerary cusp patterns of mice lacking Sostdc1 or Shh at embryonic day 15.5. These results suggest that Shh plays an inhibitory role in cusp pattern formation by modulating Wnt signaling through the positive regulation of Sostdc1.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/fisiología , Proteínas Hedgehog/fisiología , Diente/embriología , Proteínas Wnt/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Tipificación del Cuerpo/fisiología , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ratones , Diente Molar , Transducción de Señal , Diente/metabolismo , Corona del Diente , Germen Dentario , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización WntRESUMEN
BACKGROUND: The objective of this study was to determine acyl-CoA synthetase 5 (ACSL5) and Wnt2B expression patterns in human congenital pulmonary airway malformations (CPAMs) and to identify the possible roles of ACSL5 and Wnt2B in the pathogenesis of CPAM. METHODS: Expression of ACSL5 and Wnt2B was evaluated by immunohistochemical staining, Western blotting, and quantitative real-time polymerase chain reaction, which were performed on surgical specimens of CPAM and adjacent normal lung tissues as controls. RESULTS: Immunohistochemistry revealed that ACSL5 and Wnt2B immunopositive cells were predominantly detected in the mesenchymal cell nucleus, and there were lower expressions of ACSL5 and Wnt2B immunopositive cells in CPAM tissues than those in adjacent normal lung tissues. Western blotting and quantitative real-time polymerase chain reaction showed that ACSL5 and Wnt2B protein and mRNA expressions were significantly decreased in CPAM tissues as compared to the adjacent normal lung tissues (P < 0.05). In addition, there was a reduced level of ACSL5 relative to that of Wnt2B. CONCLUSIONS: The decreased ACSL5 and Wnt2B expressions correlated with aberrations in pulmonary development and in the pathogenesis of CPAM, so downregulation of ACSL5 and Wnt2B could play an important role in the development of bronchial-alveolar structures in CPAM.
Asunto(s)
Coenzima A Ligasas/genética , Malformación Adenomatoide Quística Congénita del Pulmón/etiología , Glicoproteínas/genética , Proteínas Wnt/genética , Coenzima A Ligasas/análisis , Coenzima A Ligasas/fisiología , Glicoproteínas/análisis , Glicoproteínas/fisiología , Humanos , Inmunohistoquímica , ARN Mensajero/análisis , Proteínas Wnt/análisis , Proteínas Wnt/fisiologíaRESUMEN
ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.