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1.
Cell Chem Biol ; 31(6): 1044-1046, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38906109

RESUMO

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.


Assuntos
Via de Sinalização Wnt , Via de Sinalização Wnt/efeitos dos fármacos , Humanos , Proteínas Wnt/metabolismo , Proteínas Wnt/agonistas , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , beta Catenina/metabolismo , Animais , Receptores Wnt/metabolismo
2.
J Biol Chem ; 298(2): 101586, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35032551

RESUMO

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.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas , Dendrímeros , Leucemia Mieloide Aguda , Proteínas Wnt , Adulto , Animais , Receptores de Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas , Dendrímeros/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fragmentos de Peptídeos , Proteínas/farmacologia , Proteínas Wnt/agonistas , Via de Sinalização Wnt , Xenopus laevis
3.
Biol Open ; 10(11)2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34643229

RESUMO

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.


Assuntos
Ativinas/metabolismo , Blastocisto/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Proteínas Wnt/agonistas , Animais , Bovinos , Linhagem Celular , Desenvolvimento Embrionário/genética , Camadas Germinativas , Piridinas/metabolismo , Pirimidinas/metabolismo , Suínos , Via de Sinalização Wnt/genética
4.
Nat Commun ; 12(1): 3247, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059688

RESUMO

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.


Assuntos
Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/tratamento farmacológico , Fraturas do Fêmur/tratamento farmacológico , Osteoporose Pós-Menopausa/tratamento farmacológico , Proteínas Wnt/agonistas , Idoso , Envelhecimento/fisiologia , Animais , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Conservadores da Densidade Óssea/uso terapêutico , Reabsorção Óssea/fisiopatologia , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Feminino , Fraturas do Fêmur/patologia , Fêmur/efeitos dos fármacos , Fêmur/lesões , Fêmur/patologia , Humanos , Camundongos , Osteoporose Pós-Menopausa/fisiopatologia , Via de Sinalização Wnt/efeitos dos fármacos , Adulto Jovem
5.
Nature ; 588(7836): 151-156, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33149305

RESUMO

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.


Assuntos
Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/agonistas , Imunidade Adaptativa , Envelhecimento/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Enfisema/metabolismo , Feminino , Humanos , Imunidade Inata , Pulmão/metabolismo , Receptor beta de Linfotoxina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça/efeitos adversos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
6.
Physiol Res ; 69(1): 113-126, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31852203

RESUMO

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.


Assuntos
Falência Hepática Aguda/tratamento farmacológico , Fígado/efeitos dos fármacos , Proteínas Wnt/agonistas , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Fígado/metabolismo , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/metabolismo , Masculino , Ratos Endogâmicos Lew , Tioacetamida
7.
Mol Med Rep ; 19(3): 2144-2152, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664209

RESUMO

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.


Assuntos
Endometriose/genética , MicroRNAs/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , beta Catenina/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Endometriose/tratamento farmacológico , Endometriose/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ativação Transcricional/efeitos dos fármacos , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Via de Sinalização Wnt/efeitos dos fármacos
8.
Dev Biol ; 448(2): 320-341, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30385275

RESUMO

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 µM 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.


Assuntos
Proteínas Inibidoras de Apoptose/genética , Regeneração/genética , Urocordados/genética , Urocordados/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Peróxido de Hidrogênio/administração & dosagem , Peróxido de Hidrogênio/toxicidade , Proteínas Inibidoras de Apoptose/metabolismo , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/genética , Família Multigênica , Regeneração/efeitos dos fármacos , Urocordados/efeitos dos fármacos , Urocordados/embriologia , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismo
9.
Cell Physiol Biochem ; 51(2): 961-978, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30466106

RESUMO

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.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Via de Sinalização Wnt , Idoso , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Metilação de DNA , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Fatores Reguladores de Interferon/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Fator 1 de Ligação ao Facilitador Linfoide/química , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
10.
Eur J Med Chem ; 157: 1491-1499, 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30282321

RESUMO

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.


Assuntos
Fenantridinas/síntese química , Fenantridinas/farmacologia , Proteínas Wnt/agonistas , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/agonistas , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Fenantridinas/química , Relação Estrutura-Atividade , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
11.
PLoS One ; 13(2): e0192654, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29444187

RESUMO

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.


Assuntos
Corantes Fluorescentes/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Fator Regulador Miogênico 5/genética , Fator de Crescimento Transformador alfa/metabolismo , Proteínas Wnt/agonistas
12.
J Nutr Biochem ; 47: 94-105, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28570944

RESUMO

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.


Assuntos
Transplante de Medula Óssea/efeitos adversos , Suplementos Nutricionais , Ácidos Graxos Ômega-3/uso terapêutico , Ácidos Graxos Ômega-6/uso terapêutico , Hematopoese , Trombopoese , Regulação para Cima , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Suplementos Nutricionais/efeitos adversos , Ácidos Graxos Ômega-3/efeitos adversos , Ácidos Graxos Ômega-6/efeitos adversos , Feminino , Regulação da Expressão Gênica , Sobrevivência de Enxerto , Hematínicos/uso terapêutico , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Receptor Notch1/agonistas , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Condicionamento Pré-Transplante/efeitos adversos , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
13.
Nature ; 545(7653): 234-237, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28467818

RESUMO

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.


Assuntos
Transdução de Sinais , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Receptores Frizzled/metabolismo , Células HEK293 , Hepatócitos/citologia , Hepatomegalia/metabolismo , Hepatomegalia/patologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Intestinos/citologia , Ligantes , Fígado/metabolismo , Fígado/patologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Modelos Moleculares , Organoides/citologia , Organoides/metabolismo , Multimerização Proteica , Solubilidade , Técnicas de Cultura de Tecidos
14.
Mol Cell Endocrinol ; 439: 26-34, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27769713

RESUMO

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.


Assuntos
Alopecia/patologia , Androgênios/farmacologia , Diferenciação Celular/efeitos dos fármacos , Folículo Piloso/patologia , Células-Tronco/patologia , Proteínas Wnt/agonistas , Proteínas Wnt/antagonistas & inibidores , Alopecia/genética , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Ligantes , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
15.
J Nutr Biochem ; 37: 101-108, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27697743

RESUMO

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.


Assuntos
Proteína Axina/agonistas , Diferenciação Celular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/agonistas , Fator de Crescimento Transformador beta2/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Biologia Computacional , Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Células HEK293 , Humanos , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Elementos de Resposta , Transdução de Sinais , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
16.
J Neurochem ; 139(6): 1175-1191, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27778356

RESUMO

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.


Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/biossíntese , Fragmentos de Peptídeos/biossíntese , Agregados Proteicos/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Diterpenos/farmacologia , Humanos , Camundongos , Agregados Proteicos/efeitos dos fármacos , Proteínas Wnt/agonistas , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/biossíntese , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/agonistas , Proteína Wnt-5a/antagonistas & inibidores , Proteína Wnt-5a/biossíntese
17.
BMC Biochem ; 17(1): 9, 2016 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-27207629

RESUMO

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.


Assuntos
Benzodioxóis/química , Pirimidinas/química , Tubulina (Proteína)/química , Proteínas Wnt/agonistas , Benzodioxóis/metabolismo , Benzodioxóis/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Análise por Conglomerados , Bases de Dados Factuais , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Microscopia de Fluorescência , Ligação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologia , Proteínas Wnt/metabolismo
18.
Mol Cell Biol ; 36(5): 731-41, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26711256

RESUMO

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.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteína Quinase C/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ativação Enzimática/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/análise , Glicogênio Sintase Quinase 3 beta , Humanos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/análise , Proteínas Wnt/agonistas
20.
Stem Cell Reports ; 5(5): 895-907, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26455412

RESUMO

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.


Assuntos
Células-Tronco Adultas/metabolismo , Terapia Genética/métodos , Hepatócitos/metabolismo , Degeneração Hepatolenticular/terapia , Proteínas Adaptadoras de Transdução de Sinal/genética , Células-Tronco Adultas/citologia , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Cães , Hepatócitos/citologia , Degeneração Hepatolenticular/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
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