RESUMEN
Pharmacological modulation of the Wnt/ß-catenin signaling pathway holds promises for both basic research and therapeutic applications. In this issue of Cell Chemical Biology, Kschonsak et al.1 engineered knotted peptides that promote Wnt signaling by targeting ZNRF3 and serve as pharmacological tools for studying Wnt biology and supporting organoid growth.
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Vía de Señalización Wnt , Vía de Señalización Wnt/efectos de los fármacos , Humanos , Proteínas Wnt/metabolismo , Proteínas Wnt/agonistas , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismo , beta Catenina/metabolismo , Animales , Receptores Wnt/metabolismoRESUMEN
Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically prevents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RWd) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RWd in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RWd with enhanced target selectivity and limited side effects.
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Receptores de Proteínas Morfogenéticas Óseas , Dendrímeros , Leucemia Mieloide Aguda , Proteínas Wnt , Adulto , Animales , Receptores de Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas , Dendrímeros/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fragmentos de Péptidos , Proteínas/farmacología , Proteínas Wnt/agonistas , Vía de Señalización Wnt , Xenopus laevisRESUMEN
Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper.
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Activinas/metabolismo , Blastocisto/metabolismo , Células Madre Embrionarias/metabolismo , Proteína Homeótica Nanog/metabolismo , Factores de Transcripción SOXB1/metabolismo , Proteínas Wnt/agonistas , Animales , Bovinos , Línea Celular , Desarrollo Embrionario/genética , Estratos Germinativos , Piridinas/metabolismo , Pirimidinas/metabolismo , Porcinos , Vía de Señalización Wnt/genéticaRESUMEN
The Wnt signaling pathway is intricately connected with bone mass regulation in humans and rodent models. We designed an antibody-based platform that generates potent and selective Wnt mimetics. Using this platform, we engineer bi-specific Wnt mimetics that target Frizzled and low-density lipoprotein receptor-related proteins and evaluate their effects on bone accrual in murine models. These synthetic Wnt agonists induce rapid and robust bone building effects, and correct bone mass deficiency and bone defects in various disease models, including osteoporosis, aging, and long bone fracture. Furthermore, when these Wnt agonists are combined with antiresorptive bisphosphonates or anti-sclerostin antibody therapies, additional bone accrual/maintenance effects are observed compared to monotherapy, which could benefit individuals with severe and/or acute bone-building deficiencies. Our data support the continued development of Wnt mimetics for the treatment of diseases of low bone mineral density, including osteoporosis.
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Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/tratamiento farmacológico , Fracturas del Fémur/tratamiento farmacológico , Osteoporosis Posmenopáusica/tratamiento farmacológico , Proteínas Wnt/agonistas , Anciano , Envejecimiento/fisiología , Animales , Densidad Ósea/efectos de los fármacos , Densidad Ósea/fisiología , Conservadores de la Densidad Ósea/uso terapéutico , Resorción Ósea/fisiopatología , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Femenino , Fracturas del Fémur/patología , Fémur/efectos de los fármacos , Fémur/lesiones , Fémur/patología , Humanos , Ratones , Osteoporosis Posmenopáusica/fisiopatología , Vía de Señalización Wnt/efectos de los fármacos , Adulto JovenRESUMEN
Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.
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Pulmón/efectos de los fármacos , Pulmón/fisiología , Receptor beta de Linfotoxina/antagonistas & inhibidores , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/agonistas , Inmunidad Adaptativa , Envejecimiento/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Animales , Apoptosis/efectos de los fármacos , Enfisema/metabolismo , Femenino , Humanos , Inmunidad Innata , Pulmón/metabolismo , Receptor beta de Linfotoxina/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Humo/efectos adversos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Acute liver failure (ALF) is known for extremely high mortality rate, the result of widespread damage of hepatocytes. Orthotopic liver transplantation is the only effective therapy but its application is limited by the scarcity of donor organs. Given the importance in the liver biology of Wnt/beta-catenin signaling pathway, we hypothesized that its stimulation could enhance hepatocyte regeneration and attenuate the course of thioacetamide (TAA)-induced ALF in Lewis rats. Chronic treatment with Wnt agonist was started either immediately after hepatotoxic insult ("early treatment") or when signs of ALF had developed ("late treatment"). Only 23 % of untreated Lewis rats survived till the end of experiment. They showed marked increases in plasma alanine aminotransferase (ALT) activity and bilirubin and ammonia (NH3) levels; plasma albumin decreased significantly. "Early" and "late" Wnt agonist treatment raised the final survival rate to 69 % and 63 %, respectively, and normalized ALT, NH3, bilirubin and albumin levels. In conclusion, the results show that treatment with Wnt agonist attenuates the course of TAA-induced ALF in Lewis rats, both with treatment initiated immediately after hepatotoxic insult and in the phase when ALF has already developed. Thus, the pharmacological stimulation of Wnt/beta-catenin signaling pathway can present a new approach to ALF treatment.
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Fallo Hepático Agudo/tratamiento farmacológico , Hígado/efectos de los fármacos , Proteínas Wnt/agonistas , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismo , Animales , Evaluación Preclínica de Medicamentos , Hígado/metabolismo , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/metabolismo , Masculino , Ratas Endogámicas Lew , TioacetamidaRESUMEN
The present study aimed to investigate the role and mechanisms of microRNA (miR)33b in endometriosis (Ems). Reverse transcriptionquantitative polymerase chain reaction (RTqPCR), MTT assays, flow cytometry, caspase3/9 activity assays and western blotting were performed in the present study. Initially, miR33b expression in an Ems rat model was investigated by RTqPCR and was demonstrated to be upregulated in Ems tissue samples of rats compared with the control group. In addition, miR33b upregulation inhibited cell growth and enhanced apoptosis in an Ems model (primary cell cultures) compared with the control group. In addition, miR33b upregulation reduced Wnt/ßcatenin signaling pathway and suppressed zinc finger Eboxbinding homeobox 1 (ZEB1) protein expression in the in vitro Ems model (primary cell cultures) compared with the control group. Furthermore, small interferingZEB1 ameliorated the effects of miR33b downregulation on Ems cell growth in the in vitro Ems model. Additionally, a Wnt agonist reduced the effects of miR33b upregulation on Ems cell growth in the in vitro Ems model. In conclusion, the present study demonstrated that upregulation of miR33b may promote Ems through Wnt/ßcatenin by ZEB1 expression.
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Endometriosis/genética , MicroARNs/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , beta Catenina/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Modelos Animales de Enfermedad , Endometriosis/tratamiento farmacológico , Endometriosis/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Ratas , Activación Transcripcional/efectos de los fármacos , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Inhibitors of Apoptosis Protein (IAP) genes participate in processes like apoptosis, proliferation, innate immunity, inflammation, cell motility, differentiation and in malignancies. Here we reveal 25 IAP genes in the tunicate Botryllus schlosseri's genome and their functions in two developmental biology phenomena, a new mode of whole body regeneration (WBR) induced by budectomy, and blastogenesis, the four-staged cycles of botryllid ascidian astogeny. IAP genes that were specifically upregulated during these developmental phenomena were identified, and protein expression patterns of one of these genes, IAP28, were followed. Most of the IAP genes upregulation recorded at blastogenetic stages C/D was in concert with the upregulation at 100⯵M H2O2 apoptotic-induced treatment and in parallel to expressions of AIF1, Bax, Mcl1, caspase 2 and two orthologues of caspase 7. Wnt agonist altered the takeover duration along with reduced IAP expressions, and displacement of IAP28+ phagocytes. WBR was initiated solely at blastogenetic stage D, where zooidal absorption was attenuated and regeneration centers were formed either from remains of partially absorbed zooids or from deformed ampullae. Subsequently, bud-bearing zooids developed, in concert with a massive IAP28-dependent phagocytic wave that eliminated the old zooids, then proceeded with the establishment of morphologically normal-looking colonies. IAP4, IAP14 and IAP28 were also involved in WBR, in conjunction with the expression of the pro-survival PI3K-Akt pathway. IAPs function deregulation by Smac mimetics resulted in severe morphological damages, attenuation in bud growth and differentiation, and in destabilization of colonial coordination. Longtime knockdown of IAP functions prior to the budectomy, resulted in colonial death.
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Proteínas Inhibidoras de la Apoptosis/genética , Regeneración/genética , Urocordados/genética , Urocordados/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/administración & dosificación , Peróxido de Hidrógeno/toxicidad , Proteínas Inhibidoras de la Apoptosis/metabolismo , Estadios del Ciclo de Vida/efectos de los fármacos , Estadios del Ciclo de Vida/genética , Familia de Multigenes , Regeneración/efectos de los fármacos , Urocordados/efectos de los fármacos , Urocordados/embriología , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismoRESUMEN
BACKGROUND/AIMS: Interferon consensus sequence-binding protein 8 (IRF8) belongs to a family of interferon (IFN) regulatory factors that modulates various important physiological processes including carcinogenesis. As reported by others and our group, IRF8 expression is silenced by DNA methylation in both human solid tumors and hematological malignancies. However, the role of IRF8 in lung carcinoma remains elusive. In this study, we determined IRF8 epigenetic regulation, biological functions, and the signaling pathway involved in non-small cell lung cancer (NSCLC). METHODS: IRF8 expression were determined by Q- PCR. MSP and A+T determined promotor methylation. MTS, clonogenic, Transwell assay, Flow cytometry, three-dimensional culture and AO/EB stain verified cell function. In vivo tumorigenesis examed the in vivo effects. By Chip-QPCR, RT-PCR, Western blot and Immunofluorescence staining, the mechanisms were studied. RESULTS: IRF8 was significantly downregulated in lung tumor tissues compared with adjacent non-cancerous tissues. Furthermore, methylation-specific PCR analyses revealed that IRF8 methylation in NSCLC was a common event, and demethylation reagent treatment proved that downregulation of IRF8 was due to its promoter CpG hypermethylation. Clinical data showed that the IRF8 methylation was associated with tumor stage, lymph node metastasis status, patient outcome, and tumor histology. Exogenous expression of IRF8 in the silenced or downregulated lung cancer cell lines A549 and H1299 at least partially restored the sensitivity of lung cancer cells to apoptosis, and arrested cells at the G0/G1 phase. Cell viability, clonogenicity, and cell migration and invasive abilities were strongly inhibited by restored expression of IRF8. A three-dimensional culture system demonstrated that IRF8 changed the cells to a more spherical phenotype. Moreover, ectopic expression of IRF8 enhanced NSCLC chemosensitivity to cisplatin. Furthermore, as verified by Chip-qPCR, immunofluorescence staining, and western blotting, IRF8 bound to the T-cell factor/lymphoid enhancer factor (TCF /LEF) promoter, thus repressing ß-catenin nuclear translocation and its activation. IRF8 significantly disrupted the effects of Wnt agonist, bml284, further suggesting its involvement in the Wnt/ß-catenin pathway. CONCLUSION: IRF8 acted as a tumor suppressor gene through the transcriptional repression of ß-catenin-TCF/LEF in NSCLC. IRF8 methylation may serve as a potential biomarker in NSCLC prognosis.
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Factores Reguladores del Interferón/metabolismo , Vía de Señalización Wnt , Anciano , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular/efectos de los fármacos , Cisplatino/farmacología , Metilación de ADN , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Factores Reguladores del Interferón/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Factor de Unión 1 al Potenciador Linfoide/química , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Estadificación de Neoplasias , Regiones Promotoras Genéticas , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
Previously, HLY78, a lycorine derivative, was identified as the first Wnt/ß-catenin signaling agonist through binding to the DAX domain of Axin, a scaffold of Wnt/ß-catenin complex. In this study, to obtain more potent Wnt/ß-catenin agonist, the structure optimization of HLY78 was carried out by design and synthesis of six phenanthridine derivatives, which afforded five active ones. In particular, 8,9-bis((1,3-dimethyl-1H-pyrazol)methoxy)-5-ethyl-4-methyl-5,6-dihydrophenanthridine showed the most potent activity (0.15/µM) that was increased nearly 30 times as that of the lead HLY78. These compounds may be valuable in future pharmacological or biological studies.
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Fenantridinas/síntesis química , Fenantridinas/farmacología , Proteínas Wnt/agonistas , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/agonistas , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Fenantridinas/química , Relación Estructura-Actividad , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.
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Colorantes Fluorescentes/metabolismo , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Animales , Diferenciación Celular , Línea Celular , Ratones , Músculo Esquelético/citología , Músculo Esquelético/crecimiento & desarrollo , Factor 5 Regulador Miogénico/genética , Factor de Crecimiento Transformador alfa/metabolismo , Proteínas Wnt/agonistasRESUMEN
Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1+ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure.
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Trasplante de Médula Ósea/efectos adversos , Suplementos Dietéticos , Ácidos Grasos Omega-3/uso terapéutico , Ácidos Grasos Omega-6/uso terapéutico , Hematopoyesis , Trombopoyesis , Regulación hacia Arriba , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Suplementos Dietéticos/efectos adversos , Ácidos Grasos Omega-3/efectos adversos , Ácidos Grasos Omega-6/efectos adversos , Femenino , Regulación de la Expresión Génica , Supervivencia de Injerto , Hematínicos/uso terapéutico , Ratones Congénicos , Ratones Endogámicos C57BL , Receptor Notch1/agonistas , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Acondicionamiento Pretrasplante/efectos adversos , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing ß-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic ß-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.
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Transducción de Señal , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Receptores Frizzled/metabolismo , Células HEK293 , Hepatocitos/citología , Hepatomegalia/metabolismo , Hepatomegalia/patología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Intestinos/citología , Ligandos , Hígado/metabolismo , Hígado/patología , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Modelos Moleculares , Organoides/citología , Organoides/metabolismo , Multimerización de Proteína , Solubilidad , Técnicas de Cultivo de TejidosRESUMEN
In androgenetic alopecia, androgens impair dermal papilla-induced hair follicle stem cell (HFSC) differentiation inhibiting Wnt signaling. Wnt agonists/antagonists balance was analyzed after dihydrotestosterone (DHT) stimulation in androgen-sensitive dermal papilla cells (DPC) cultured as spheroids or monolayer. In both culture conditions, DHT stimulation downregulated Wnt5a and Wnt10b mRNA while the Wnt antagonist Dkk-1 was upregulated. Notably, tissue architecture of DPC-spheroids lowers Dkk-1 and enhances Wnt agonists' basal expression; probably contributing to DPC inductivity. The role of Wnt agonists/antagonists as mediators of androgen inhibition of DPC-induced HFSC differentiation was evaluated. Inductive DPC-conditioned medium supplemented with DKK-1 impaired HFSC differentiation mimicking androgens' action. This effect was associated with inactivation of Wnt/ß-catenin pathway in differentiating HFSC by both DPC-conditioned media. Moreover, addition of WNT10b to DPC-medium conditioned with DHT, overcame androgen inhibition of HFSC differentiation. Our results identify DKK1 and WNT10b as paracrine factors which modulate the HFSC differentiation inhibition involved in androgen-driven balding.
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Alopecia/patología , Andrógenos/farmacología , Diferenciación Celular/efectos de los fármacos , Folículo Piloso/patología , Células Madre/patología , Proteínas Wnt/agonistas , Proteínas Wnt/antagonistas & inhibidores , Alopecia/genética , Línea Celular , Medios de Cultivo Condicionados/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Humanos , Ligandos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
Human milk contains growth factors that maintain intestinal mucosal homeostasis, but the molecular mechanisms behind how these growth factors regulate gene transcription are largely unknown. In this study, IEC-6 (rat intestinal epithelial cells) cells were used as a model to study cell differentiation mediated by transforming growth factor-ß2 (TGF-ß2), the most abundant growth factor in human milk. We focused on the transcription factor early growth response-1 (EGR-1), as we found a robust and rapid response in our initial transcription factor screen. Immunoblotting and immunofluorescent assays confirmed the phenotype change upon TGF-ß2 treatment and EGR-1 stimulation in the nucleus, with maximum expression occurring at 1 h. Chromatin immunoprecipitation sequencing was performed to map genome-wide EGR-1 binding sites on more than 1800 genes, widely involved in processes such as gene expression, transcription, membrane invagination and metabolism. In particular, more than 15 Wnt signaling pathway genes have EGR-1 binding sites; among them, Axin1 was the limiting factor, ensuring proper ß-catenin accumulation in the cytoplasm. We further used chromatin immunoprecipitation quantitative PCR to validate that EGR-1 binds to the region of -636/-454 bp and -454/-200 bp of the Axin1 promoter and functionally activates gene expression. The effect of TGF-ß2 on maintaining small intestinal cell homeostasis was partially explained by Axin1 activation through EGR-1.
Asunto(s)
Proteína Axina/agonistas , Diferenciación Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/agonistas , Factor de Crecimiento Transformador beta2/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteína Axina/genética , Proteína Axina/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Biología Computacional , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Mucosa Intestinal/citología , Intestino Delgado/citología , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Ratas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Elementos de Respuesta , Transducción de Señal , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disorder and the most frequent cause of dementia in the aged population. According to the amyloid hypothesis, the amyloid-ß (Aß) peptide plays a key role in the pathogenesis of AD. Aß is generated from the amyloidogenic processing of amyloid precursor protein and can aggregate to form oligomers, which have been described as a major synaptotoxic agent in neurons. Dysfunction of Wnt signaling has been linked to increased Aß formation; however, several other studies have argued against this possibility. Herein, we use multiple experimental approaches to confirm that the inhibition of Wnt signaling promoted the amyloidogenic proteolytic processing of amyloid precursor protein. We also demonstrate that inhibiting Wnt signaling increases the production of the Aß42 peptide, the Aß42 /Aß40 ratio, and the levels of Aß oligomers such as trimers and tetramers. Moreover, we show that activating Wnt signaling reduces the levels of Aß42 and its aggregates, increases Aß40 levels, and reduces the Aß42 /Aß40 ratio. Finally, we show that the protective effects observed in response to activation of the Wnt pathway rely on ß-catenin-dependent transcription, which is demonstrated experimentally via the expression of various 'mutant forms of ß-catenin'. Together, our findings indicate that loss of the Wnt signaling pathway may contribute to the pathogenesis of AD.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/biosíntesis , Fragmentos de Péptidos/biosíntesis , Agregado de Proteínas/fisiología , Vía de Señalización Wnt/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Diterpenos/farmacología , Humanos , Ratones , Agregado de Proteínas/efectos de los fármacos , Proteínas Wnt/agonistas , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/biosíntesis , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt-5a/agonistas , Proteína Wnt-5a/antagonistas & inhibidores , Proteína Wnt-5a/biosíntesisRESUMEN
BACKGROUND: In drug discovery research, cell-based phenotypic screening is an essential method for obtaining potential drug candidates. Revealing the mechanism of action is a key step on the path to drug discovery. However, elucidating the target molecules of hit compounds from phenotypic screening campaigns remains a difficult and troublesome process. Simple and efficient methods for identifying the target molecules are essential. RESULTS: 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) was identified as a senescence inducer from a phenotypic screening campaign. The compound is widely used as a Wnt agonist, although its target molecules remain to be clarified. To identify its target proteins, we compared a series of cellular assay results for the compound with our pathway profiling database. The database comprises the activities of compounds from simple assays of cellular reporter genes and cellular proliferations. In this database, compounds were classified on the basis of statistical analysis of their activities, which corresponded to a mechanism of action by the representative compounds. In addition, the mechanisms of action of the compounds of interest could be predicted using the database. Based on our database analysis, the compound was anticipated to be a tubulin disruptor, which was subsequently confirmed by its inhibitory activity of tubulin polymerization. CONCLUSION: These results demonstrate that tubulin is identified for the first time as a target molecule of the Wnt-activating small molecule and that this might have misled the conclusions of some previous studies. Moreover, the present study also emphasizes that our pathway profiling database is a simple and potent tool for revealing the mechanisms of action of hit compounds obtained from phenotypic screenings and off targets of chemical probes.
Asunto(s)
Benzodioxoles/química , Pirimidinas/química , Tubulina (Proteína)/química , Proteínas Wnt/agonistas , Benzodioxoles/metabolismo , Benzodioxoles/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Análisis por Conglomerados , Bases de Datos Factuales , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Humanos , Redes y Vías Metabólicas/efectos de los fármacos , Microscopía Fluorescente , Unión Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacología , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacología , Proteínas Wnt/metabolismoRESUMEN
The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3ß (GSK-3ß) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3ß interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3ß redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3ß at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3ß in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3ß. In vitro activity assays demonstrated that GSK-3ß phosphorylation mediated by PKCζ enhanced GSK-3ß activity. We mapped Ser147 of GSK-3ß as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3ß activity, resulting in ß-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3ß basal activity. Thus, we found that PKCζ phosphorylates GSK-3ß at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3ß activity in opposite manners in normal and malignant colon cells.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Proteína Quinasa C/metabolismo , Línea Celular , Línea Celular Tumoral , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Activación Enzimática/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/análisis , Glucógeno Sintasa Quinasa 3 beta , Humanos , Fosforilación/efectos de los fármacos , Proteína Quinasa C/análisis , Proteínas Wnt/agonistasRESUMEN
The recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease.