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1.
Gene ; 761: 145038, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777532

RESUMO

Neuropathic pain, which results from impairment of the somatosensory system, has affected about 8% population around the world and leads to considerable burdens for patients and world health care system. However, its underlying mechanisms remain poorly understood. In this study, we hypothesized that miR-24-3p was involved in the progression of neuropathic pain in CCI rat models. By measuring miR-24-3p expression in CCI rats, we found that miR-24-3p expression was increased in CCI rats, suggesting miR-24-3p might participate in neuropathic pain progression. Next, by conducting a serial in vitro and vivo experiments, we found that miR-24-3p regulated Wnt5a/ß-Catenin Signaling levels to promote neuropathic pain progression via targeting LPAR3 in CCI rats. Furthermore, we explored the upstream regulator of miR-24-3p by conducting bioinformatics analysis, we found that circular RNA cZRANB1 might sponge to miR-24-3p. Then we applied biotinylated RNA pull-down and luciferase reporter assays to assess the association between cZRANB1 and miR-24-3p. It was found that cZRANB1 mediated LPAR3 expression via sponging miR-24-3p. Collectively, our study suggests that cZRNAB1 regulated Wnt5a/ß-Catenin Signaling expression via miR-24-3p/LPAR3 axis in CCI rat models.


Assuntos
MicroRNAs/genética , Neuralgia/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Constrição Patológica/genética , Progressão da Doença , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Inflamação/genética , Masculino , MicroRNAs/metabolismo , RNA Circular/genética , RNA Longo não Codificante/genética , Ratos , Ratos Sprague-Dawley , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Ubiquitina Tiolesterase/genética , Via de Sinalização Wnt/genética , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , beta Catenina/metabolismo
2.
Anticancer Res ; 40(5): 2725-2737, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366418

RESUMO

BACKGROUND/AIM: Glioblastoma (GB) is the most aggressive type of tumor in the central nervous system and is characterized by resistance to therapy and abundant vasculature. Tumor vessels contribute to the growth of GB, and the tumor microenvironment is thought to influence tumor vessels. We evaluated the molecular communication between human GB cells and human brain microvascular endothelial cells (HBMEC) in vitro. MATERIALS AND METHODS: We investigated whether GB-conditioned media (GB-CM) influenced HBMEC proliferation and migration, as well as the levels of MMP-9, CXCL12, CXCR4, CXCR7, VEGFs, VEGFR-2, and WNT5a in HBMEC. RESULTS: Although HBMEC proliferation was not modified, increased HBMEC migration was detected after GB-CM treatment. Furthermore, treatment of HBMEC with GB-CM resulted in increased levels of MMP-9 and CXCR4. The levels of WNT5a, VEGFs and VEGFR-2 were not affected. CONCLUSION: GB-secreted factors lead to increased endothelial cell migration and to increased levels of MMP-9 and CXCR4.


Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Movimento Celular , Células Endoteliais/patologia , Glioblastoma/patologia , Metaloproteinase 9 da Matriz/metabolismo , Receptores CXCR4/metabolismo , Neoplasias Encefálicas/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo
3.
Cell Prolif ; 53(6): e12825, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32391621

RESUMO

OBJECTIVES: Little is known about the roles of integral membrane proteins beyond channels, carriers or receptors in meiotic oocytes. The transmembrane protein Fam70A was previously identified as a likely "female fertility factor" in Fox3a-knockout mouse ovaries where almost all follicles underwent synchronous activation and the mice became infertile very early. However, whether Fam70A functions in oocyte meiosis remains unknown. Therefore, the present study aimed to address this question. MATERIALS AND METHODS: Co-immunoprecipitation, immunogold labelling-electron microscopy, co-localization and yeast two-hybrid assays were used to verify the interaction. Antibody or small interfering RNA transfection was used to deplete the proteins. Immunofluorescence, immunohistochemistry and live tracker staining were used to examine the localization or characterize phenotypes. Western blot was used to examine the protein level. RESULTS: Fam70A was enriched in oocyte membranes important for normal meiosis. Fam70A depletion remarkably disrupted spindle assembly, chromosome congression and first polar body extrusion, which subsequently increased aneuploidy and abnormal fertilization. Moreover, Fam70A directly bound Wnt5a, the most abundant Wnt member within oocytes. Depletion of either Fam70A or Wnt5a remarkably increased adenomatous polyposis coli (APC), which stabilizes active ß-catenin and microtubules. Consequently, depletion of either Fam70A or Wnt5a remarkably increased p-ß-catenin (inactive form) and acetylated tubulin, while APC knockdown remarkably decreased these two. Furthermore, Fam70A depletion remarkably reduced Akt phosphorylation. CONCLUSIONS: Fam70A regulates meiosis and quality of mouse oocytes through both canonical and non-canonical Wnt5a signalling pathways.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Meiose , Proteínas de Membrana/metabolismo , Oócitos/metabolismo , Proteína Wnt-5a/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Camundongos , Microtúbulos/metabolismo , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo
4.
Nat Commun ; 11(1): 1936, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321913

RESUMO

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.


Assuntos
Enterócitos/metabolismo , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Enterócitos/citologia , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas-G/genética , Células Estromais/metabolismo , Proteína Wnt-5a/metabolismo
5.
Dev Cell ; 52(5): 647-658.e6, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32155439

RESUMO

During development, intestinal epithelia undergo dramatic morphogenesis mediated by mesenchymal signaling to form villi, which are required for efficient nutrient absorption and host defense. Although both smooth-muscle-induced physical forces and mesenchymal cell clustering beneath emerging villi are implicated in epithelial folding, the underlying cellular mechanisms are unclear. Hedgehog (Hh) signaling can mediate both processes. We therefore analyzed its direct targetome and revealed GLI2 transcriptional activation of atypical cadherin and planar cell polarity (PCP) genes. By examining Fat4 and Dchs1 knockout mice, we demonstrate their critical roles in villus formation. Analyses of PCP-mutant mice and genetic interaction studies show that the Fat4-Dchs1 axis acts in parallel to the core-Vangl2 PCP axis to control mesenchymal cell clustering. Moreover, live light-sheet fluorescence microscopy and cultured PDGFRα+ cells reveal a requirement for PCP in their oriented cell migration guided by WNT5A. Therefore, mesenchymal PCP induced by Hh signaling drives cell clustering and subsequent epithelial remodeling.


Assuntos
Caderinas/metabolismo , Polaridade Celular , Proteínas Hedgehog/metabolismo , Mucosa Intestinal/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Microvilosidades/metabolismo , Animais , Caderinas/genética , Diferenciação Celular , Movimento Celular , Células Cultivadas , Feminino , Proteínas Hedgehog/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo
6.
Open Biol ; 10(2): 190273, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32097584

RESUMO

Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.


Assuntos
Quinase 1 de Adesão Focal/genética , Proteína Wnt-5a/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Movimento Celular , Quinase 1 de Adesão Focal/metabolismo , Gastrulação , Camundongos , Mutação , Transdução de Sinais , Proteína Wnt-5a/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética
7.
Am J Respir Crit Care Med ; 201(10): 1249-1262, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32023086

RESUMO

Rationale: Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth that affects infants born in the saccular stage of lung development at <32 weeks of gestation. Although the mechanisms driving BPD remain uncertain, exposure to hyperoxia is thought to contribute to disease pathogenesis.Objectives: To determine the effects of hyperoxia on epithelial-mesenchymal interactions and to define the mediators of activated Wnt/ß-catenin signaling after hyperoxia injury.Methods: Three hyperoxia models were used: A three-dimensional organotypic coculture using primary human lung cells, precision-cut lung slices (PCLS), and a murine in vivo hyperoxia model. Comparisons of normoxia- and hyperoxia-exposed samples were made by real-time quantitative PCR, RNA in situ hybridization, quantitative confocal microscopy, and lung morphometry.Measurements and Main Results: Examination of an array of Wnt ligands in the three-dimensional organotypic coculture revealed increased mesenchymal expression of WNT5A. Inhibition of Wnt5A abrogated the BPD transcriptomic phenotype induced by hyperoxia. In the PCLS model, Wnt5A inhibition improved alveolarization following hyperoxia exposure, and treatment with recombinant Wnt5a reproduced features of the BPD phenotype in PCLS cultured in normoxic conditions. Chemical inhibition of NF-κB with BAY11-7082 reduced Wnt5a expression in the PCLS hyperoxia model and in vivo mouse hyperoxia model, with improved alveolarization in the PCLS model.Conclusions: Increased mesenchymal Wnt5A during saccular-stage hyperoxia injury contributes to the impaired alveolarization and septal thickening observed in BPD. Precise targeting of Wnt5A may represent a potential therapeutic strategy for the treatment of BPD.


Assuntos
Células Epiteliais Alveolares/metabolismo , Fibroblastos/metabolismo , Hiperóxia/genética , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína Wnt-5a/genética , Animais , Displasia Broncopulmonar , Técnicas de Cocultura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hiperóxia/metabolismo , Hibridização In Situ , Pulmão/crescimento & desenvolvimento , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Microscopia Confocal , NF-kappa B/antagonistas & inibidores , Nitrilos/farmacologia , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Sulfonas/farmacologia , Proteína Wnt-5a/efeitos dos fármacos , Proteína Wnt-5a/metabolismo
8.
Nat Commun ; 11(1): 445, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974352

RESUMO

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.


Assuntos
Doenças dos Ductos Biliares/patologia , Colangite Esclerosante/patologia , Cicatriz/patologia , Via de Sinalização Wnt , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Doenças dos Ductos Biliares/induzido quimicamente , Doenças dos Ductos Biliares/metabolismo , Ductos Biliares/citologia , Polaridade Celular , Colangite Esclerosante/metabolismo , Cicatriz/metabolismo , Modelos Animais de Doenças , Células Epiteliais , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Piridinas/toxicidade , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/metabolismo
9.
Life Sci ; 245: 117338, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31981630

RESUMO

Secreted frizzled-related protein 5 (Sfrp5) primarily acts in combination with wingless-type family member 5a (Wnt5a), to inhibits chronic inflammation and repress atherosclerosis and other metabolic disorders. Epicardial adipose tissue (EAT), surrounding the heart and coronary arteries, has been found to be highly related to the progression of coronary artery disease through adipokines production. However, little is known about EAT-derived Sfrp5 and Wnt5a in humans. We aimed to investigate whether the EAT-derived Sfrp5/Wnt5a levels are altered in patients with CAD. Fifty-eight patients with CAD and 29 patients without CAD who underwent cardiac surgery were enrolled. Serum samples and paired adipose biopsies from EAT and subcutaneous adipose tissue (SAT) were collected, and Sfrp5 and Wnt5a levels were detected. Correlation and multivariate regression analyses were performed to determine the relationship between Sfrp5/Wnt5a expression and CAD and other clinical risk factors. According to the results, the CAD group had lower Sfrp5 and higher Wnt5a levels in EAT and serum (all p < 0.05). Serum Sfrp5 levels were significantly lower in CAD patients with impaired myocardial function. EAT Sfrp5 mRNA levels and serum Sfrp5 levels were both negatively associated with the presence of CAD, after adjustment for known biomarkers, EAT mRNA and serum Wnt5a levels correlated positively with the presence of CAD. Thus, we concluded that low Sfrp5 and high Wnt5a levels are associated with the presence of CAD, independent of other conventional risk factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tecido Adiposo/metabolismo , Doença da Artéria Coronariana/metabolismo , Pericárdio/metabolismo , Proteína Wnt-5a/metabolismo , Tecido Adiposo/patologia , Estudos de Casos e Controles , Doença da Artéria Coronariana/patologia , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pericárdio/patologia , Reação em Cadeia da Polimerase em Tempo Real
10.
Oncol Rep ; 43(2): 601-608, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894282

RESUMO

Receptor tyrosine kinase like orphan receptor 2 (ROR2) regulates Wnt5a-induced cell migration by phosphorylating PI3K/Akt and activating RhoA in osteosarcoma. However, the role of Wnt5a signaling and its corresponding receptors in the regulation of osteosarcoma metastasis remains poorly understood. ROR1 monoclonal antibody (mAb) and short hairpin (sh)RNA targeting ROR2 markedly inhibited the activity of dishevelled associated activator of morphogenesis 1 (DAAM1) and RhoA and retarded cell migration in osteosarcoma. ROR1 mAb and ROR2 shRNA destroyed the microfilament formation of osteosarcoma cells. Silencing of DAAM1 (with DAAM1 shRNA) downregulated RhoA activity and inhibited cell migration. The decrease of cell migration caused by DAAM1 shRNA was rescued by wild-type DAAM1 overexpression. DAAM1 and PI3Kα/Akt were parallel signaling pathways mediating osteosarcoma cell migration in response to Wnt5a. It was concluded that Wnt5a promotes osteosarcoma cell migration via ROR1/2 receptors, and then activates DAAM1 and RhoA.


Assuntos
Neoplasias Ósseas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Osteossarcoma/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Transdução de Sinais
11.
J Clin Invest ; 130(2): 853-862, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31929186

RESUMO

Oncogene-targeted and immune checkpoint therapies have revolutionized the clinical management of malignant melanoma and now offer hope to patients with advanced disease. Intimately connected to patients' overall clinical risk is whether the initial primary melanoma lesion will metastasize and cause advanced disease, but underlying mechanisms are not entirely understood. A subset of melanomas display heightened peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α) expression that maintains cell survival cues by promoting mitochondrial function, but also suppresses metastatic spread. Here, we show that PGC1α expression in melanoma cells was silenced by chromatin modifications that involve promoter H3K27 trimethylation. Pharmacological EZH2 inhibition diminished H3K27me3 histone markers, increased PGC1α expression, and functionally suppressed invasion within PGC1α-silenced melanoma cells. Mechanistically, PGC1α silencing activated transcription factor 12 (TCF12), to increase expression of WNT5A, which in turn stabilized YAP protein levels to promote melanoma migration and metastasis. Accordingly, inhibition of components of this transcription-signaling axis, including TCF12, WNT5A, or YAP, blocked melanoma migration in vitro and metastasis in vivo. These results indicate that epigenetic control of melanoma metastasis involved altered expression of PGC1α and an association with the inherent metabolic state of the tumor.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Melanoma Experimental/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Fatores de Transcrição/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular Tumoral , Células HEK293 , Histonas/genética , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fatores de Transcrição/genética , Proteína Wnt-5a/genética
12.
Cancer Res ; 80(5): 1156-1170, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932454

RESUMO

The noncanonical Wnt ligand Wnt5a is found in high concentrations in ascites of women with ovarian cancer. In this study, we elucidated the role of Wnt5a in ovarian cancer metastasis. Wnt5a promoted ovarian tumor cell adhesion to peritoneal mesothelial cells as well as migration and invasion, leading to colonization of peritoneal explants. Host components of the ovarian tumor microenvironment, notably peritoneal mesothelial cells and visceral adipose, secreted Wnt5a. Conditional knockout of host WNT5A significantly reduced peritoneal metastatic tumor burden. Tumors formed in WNT5A knockout mice had elevated cytotoxic T cells, increased M1 macrophages, and decreased M2 macrophages, indicating that host Wnt5a promotes an immunosuppressive microenvironment. The Src family kinase Fgr was identified as a downstream effector of Wnt5a. These results highlight a previously unreported role for host-expressed Wnt5a in ovarian cancer metastasis and suggest Fgr as a novel target for inhibition of ovarian cancer metastatic progression.Significance: This study establishes host-derived Wnt5a, expressed by peritoneal mesothelial cells and adipocytes, as a primary regulator of ovarian cancer intraperitoneal metastatic dissemination and identifies Fgr kinase as novel target for inhibition of metastasis.


Assuntos
Carcinoma Epitelial do Ovário/patologia , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/imunologia , Peritônio/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteína Wnt-5a/metabolismo , Quinases da Família src/metabolismo , Animais , Carcinoma Epitelial do Ovário/imunologia , Adesão Celular/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Metástase Neoplásica/imunologia , Metástase Neoplásica/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Peritoneais/secundário , Peritônio/citologia , Peritônio/imunologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Proteína Wnt-5a/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Mol Cell ; 77(3): 633-644.e5, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31836388

RESUMO

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.


Assuntos
Melanoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Wnt-5a/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , MAP Quinase Quinase Quinases/metabolismo , Melanoma/genética , Melanoma/patologia , Terapia de Alvo Molecular , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia
14.
Am J Physiol Gastrointest Liver Physiol ; 318(2): G352-G360, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31869240

RESUMO

Gastric cancer (GC) is the most prevalent human cancer around the globe. In GC, Wnt signaling is deregulated, and receptor-like tyrosine kinase (RYK) coreceptors have been identified to interact with noncanonical Wnt ligand Wnt5a. We, therefore, aimed to evaluate the role of RYK in GC development and metastasis. GC tumor samples were collected from 250 GC patients. Expressions of RYK, as well as markers for the epithelial-mesenchymal transition (EMT), such as N-cadherin and E-cadherin, were subjected to correlation analysis with clinicopathological features. Endogenous RYK expression levels were compared in GC cell lines with ascending metastatic potentials followed by stable RYK knockdown. Effect of RYK knockdown on GC cell migration, invasion, and EMT phenotype were assessed in vitro, and on GC tumor growth in vivo in a xenograft rodent model. Particularly, liver metastasis potential of tail vein-injected GC cells was also analyzed following RYK knockdown. RYK was highly correlated with liver metastasis of GC tumors and the expression profiles of EMT markers toward the mesenchymal tendency. RYK expression was also positively correlated with the metastasis potential of GC cells. RYK knockdown not only inhibited migration, invasion, and EMT of GC cells in vitro, but also suppressed tumorigenesis and liver metastasis of GC cells in vivo using the mouse xenograft model. RYK is highly correlated with GC tumorigenesis and potential of liver metastasis, suggesting it may be a novel oncogenic factor of the noncanonical Wnt signaling pathway contributing to GC.NEW & NOTEWORTHY RYK is highly correlated with gastric cancer tumorigenesis and the potential of liver metastasis, suggesting it may be a novel oncogenic factor of the noncanonical Wnt signaling pathway contributing to gastric cancer.


Assuntos
Neoplasias Hepáticas/secundário , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias Gástricas/metabolismo , Proteína Wnt-5a/metabolismo , Animais , Biomarcadores Tumorais/análise , Caderinas/análise , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica/genética , Receptores Proteína Tirosina Quinases/análise , Proteína Wnt-5a/análise , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cancer Res ; 80(3): 576-590, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31719098

RESUMO

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to ß-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of ß-catenin activation in prostate cancer progression and treatment resistance. ß-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with ß-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of ß-catenin activation, by mediating nuclear localization of NFκBp65 and ß-catenin. Overall, WNT/ß-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/ß-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. SIGNIFICANCE: Targeting of both AR and WNT/ß-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , PTEN Fosfo-Hidrolase/deficiência , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Proteína Wnt-5a/metabolismo , beta Catenina/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Masculino , Camundongos , Prognóstico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteína Wnt-5a/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
16.
J Periodontal Res ; 55(2): 199-208, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31593304

RESUMO

BACKGROUND AND OBJECTIVE: Peri-implantitis is a plaque-associated pathological condition occurring in tissues around dental implants, characterized by inflammation in the peri-implant mucosa and subsequent progressive loss of supporting bone. Wnt5a is the activating ligand of the non-canonical Wnt signaling pathways and plays important roles in leukocyte infiltration and cytokine/ chemokine production in inflammatory disorders. Previous studies showed that Wnt5a was significantly up-regulated in gingival tissues of chronic and aggressive periodontitis. However, the roles and the regulatory mechanisms of Wnt5a in peri-implantitis are not well known. METHODS: The expression of Wnt5a in gingival tissues collected from 8 healthy implant patients and 8 peri-implantitis patients was analyzed by Western blotting and immunofluorescence. Porphyromonas gingivalis infected macrophages isolated from the peripheral blood of healthy volunteers were used as an in vitro cellular model of peri-implantitis. Using neutralizing antibodies, inhibitors and siRNA, the production and roles of Wnt5a in peri-implantitis were assessed by immunofluorescence, quantitative polymerase chain reaction (RT-PCR) and Western blotting. Unpaired two-tailed Student's t test was used to compare qRT-PCR and Western blotting results. P ≤ .05 was considered statistically significant. RESULTS: Wnt5a was highly expressed in the gingival tissues of peri-implantitis patients. Compared to controls, Wnt5a increased in P gingivalis infected macrophages. Wnt5a production in response to P gingivalis infection was dependent on LOX-1 and TLR4. Compared to controls, Wnt5a knockdown impaired IL-1ß, MCP-1, and MMP2 production induced by P gingivalis infection. CONCLUSION: Our results indicate that Wnt5a is involved in LOX-1 and TLR4 induced inflammatory signature via inflammatory cytokines production in response to P gingivalis infection. These findings demonstrate that Wnt5a maybe an important component of the host immune response in peri-implantitis.


Assuntos
Implantes Dentários , Peri-Implantite/metabolismo , Receptores Depuradores Classe E/metabolismo , Receptor 4 Toll-Like/isolamento & purificação , Proteína Wnt-5a/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/microbiologia , Porphyromonas gingivalis
17.
J Diabetes Investig ; 11(2): 307-314, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31368666

RESUMO

AIMS/INTRODUCTION: Type 2 diabetes mellitus is a chronic metabolic disorder characterized by islet ß-cell dysfunction, which might result from the activation of islet stellate cells (ISCs). Our recent study showed that a specific population of ISCs is prone to be activated in type 2 diabetes mellitus accompanied by reduced secretion of insulin. The wingless-type MMTV integration site family member 5a (Wnt5a)/frizzled-5 signaling pathway might play an important role in this process. The present study aimed to explore the effects of Wnt5a on the activation of ISCs isolated from db/db mice. MATERIALS AND METHODS: ISCs were isolated from db/db mice and matched db/m mice. Immunohistochemistry and western blotting analysis were applied for the determination of Wnt5a expression. Exogenous Wnt5a and lentivirus containing the target gene Wnt5a short hairpin ribonucleic acid were used as a molecular intervention. The experiment of transwell and wound healing was used to evaluate the migration of the isolated ISCs. RESULTS: Our data showed that the expression of Wnt5a and frizzled-5 was decreased in the ISCs isolated from db/db mice compared with db/m mice. Both the exogenous Wnt5a and the overexpression of Wnt5a could inhibit the outgrowth rate of ISCs from islets, and its viability, migration and α smooth muscle actin expression. These changes were associated with the inactivation of the Smad2/3 signaling pathway in a frizzled-5-dependent manner. CONCLUSIONS: Our observations revealed a potential role of Wnt5a in preventing ISC activation. The maintenance of quiescent ISCs might be a desirable outcome of therapeutic strategies for diabetes mellitus.


Assuntos
Receptores Frizzled/metabolismo , Células Estreladas do Pâncreas/metabolismo , Proteína Wnt-5a/metabolismo , Animais , Apoptose , Células Cultivadas , Receptores Frizzled/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
18.
Cell Mol Life Sci ; 77(5): 919-935, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31312879

RESUMO

Wnt ligands signal through canonical or non-canonical signaling pathways. Although both routes share common elements, such as the Fz2 receptor, they differ in the co-receptor and in many of the final responses; for instance, whereas canonical Wnts increase ß-catenin stability, non-canonical ligands downregulate it. However, both types of ligands stimulate tumor cell invasion. We show here that both the canonical Wnt3a and the non-canonical Wnt5a stimulate Fz2 tyrosine phosphorylation, Fyn binding to Fz2, Fyn activation and Fyn-dependent Stat3 phosphorylation. Wnt3a and Wnt5a require Src for Fz2 tyrosine phosphorylation; Src binds to canonical and non-canonical co-receptors (LRP5/6 and Ror2, respectively) and is activated by Wnt3a and Wnt5a. This Fz2/Fyn/Stat3 branch is incompatible with the classical Fz2/Dvl2 pathway as shown by experiments of over-expression or depletion. Fyn is necessary for transcription of genes associated with invasiveness, such as Snail1, and for activation of cell invasion by both Wnt ligands. Our results extend the knowledge about canonical Wnt pathways, demonstrating additional roles for Fyn in this pathway and describing how this protein kinase is activated by both canonical and non-canonical Wnts.


Assuntos
Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteína Wnt-5a/metabolismo , Proteína Wnt3A/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular , Ativação Enzimática/genética , Células HEK293 , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Invasividade Neoplásica/genética , Neoplasias/patologia , Fosforilação/fisiologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Fator de Transcrição STAT3/metabolismo , Transcrição Genética/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-31678514

RESUMO

Atherosclerosis (AS) is characterized by lipids metabolism disorder and inflammatory response. Accumulating evidence has demonstrated that Wingless type 5a (Wnt5a) is implicated in cardiovascular diseases through non-canonical Wnt cascades. However, its precise role during the pathogenesis of AS is still unclear. Therefore, the present study aims to investigate the role and the underlying mechanism of Wnt5a/receptor tyrosine kinase-like orphan receptor 2 (Ror2) pathways in the promotion of AS process through affecting lipid accumulation and inflammation. In atherosclerotic clinical samples, Wnt5a levels were measured by using enzyme-linked immunosorbent assay (ELISA) assay. In vivo experiments were conducted by using apolipoprotein E knockout (apoE-/-) mice model. Vascular smooth muscle cells (VSMCs) were applied for in vitro studies. Wnt5a was highly expressed in both of atherosclerotic clinical samples and apoE-/- mice. The knockdown of Wnt5a significantly inhibited cholesterol accumulation and inflammatory response. Additionally, the lipopolysaccharide (LPS)-induced inflammation aggravated the cholesterol accumulation and decreased adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression in VSMCs. Depletion of intracellular cholesterol by ß-cyclodextrin (ß-CD) led to the upregulation of ABCA1 and the inhibition of inflammation. Conversely, the overexpression of Wnt5a inhibited ABCA1 expression, facilitated cholesterol accumulation, impared cholesterol efflux, promoted NF-κB nuclear translocation and the inflammatory cytokines secretion. Moreover, the knockdown of Ror2 increased ABCA1 expression and reduced Wnt5a-induced cholesterol accumulation and inflammatory responses. Furthermore, the knockdown of ABCA1 enhanced cholesterol accumulation and inflammatory response. Therefore, Wnt5a/Ror2 pathway was critical in regulating cholesterol homeostasis and inflammatory response, which might be a promising therapeutic target for AS therapy.


Assuntos
Aterosclerose/metabolismo , Colesterol/metabolismo , Inflamação/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteína Wnt-5a/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/imunologia , Estudos de Casos e Controles , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Inflamação/sangue , Inflamação/imunologia , Masculino , Camundongos , Camundongos Knockout para ApoE , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/sangue , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais/imunologia , Proteína Wnt-5a/sangue , Proteína Wnt-5a/genética
20.
Cell Mol Biol (Noisy-le-grand) ; 65(7): 138-145, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31880532

RESUMO

It was to study the influence of Wilms tumor suppressor gene (WT1) on ovarian granular cells (GCs) in mice, and the molecular mechanism involved. LV-WT1 short hairpin ribonucleic acid (shRNA) vector was used to downregulate WT1 expression in granular cells (GCs). The effects of WTI on proliferation and apoptosis of GCs were investigated. Western blot and qRT-PCR were used to assay the mRNA and protein expressions of Bax/bcl-2 in GCs transfected with LV-WT1-RNAi. The expression levels of SUZ12, Wnt5a, Wnt11, Wnt4, Wnt3a, Wnt2 mRNA in GCs were also determined. LV-WT1-RNAi significantly reduced WT1 expression, increased apoptosis and inhibited proliferation of GCs. The inhibition of WT1 had no significant effect on the expression of bcl-2 in GCs. The expressions of Wnt2, Wnt4 and Wnt5a were augmented in WT1-knockdown GCs, relative to non-transfected cells. WT1 activation is necessary for maintaining early survival of GCs in follicles via activation of the Wnt/ß-catenin signal pathway.


Assuntos
Células da Granulosa/metabolismo , Proteínas WT1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Imunofluorescência , Imuno-Histoquímica , Camundongos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas WT1/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , Proteína Wnt4/genética , Proteína Wnt4/metabolismo , beta Catenina/genética
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