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1.
Proc Natl Acad Sci U S A ; 117(14): 8064-8073, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198200

RESUMO

Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, PLoS One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen Clostridioides difficile, we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Infecções por Clostridium/patologia , Clostridium difficile/patogenicidade , Colo/patologia , Mucosa Intestinal/patologia , Células-Tronco/patologia , Animais , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Células Cultivadas , Infecções por Clostridium/microbiologia , Clostridium difficile/metabolismo , Colo/citologia , Colo/microbiologia , Modelos Animais de Doenças , Feminino , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Camundongos , Organoides , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células-Tronco/microbiologia
2.
Gastroenterol. hepatol. (Ed. impr.) ; 43(3): 107-116, mar. 2020. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-190783

RESUMO

Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca2+ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca2+ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis


Frizzled-2 desempeña un papel importante en el mantenimiento de la funcionalidad normal de los hepatocitos. Este estudio tiene como objetivo analizar el papel de la inhibición de Frizzled-2 en la protección de los hepatocitos BRL-3A de rata de la hipoxia/reoxigenación (H/R). El modelo de hepatocitos H/R in vitro se demostró con el cultivo de células BRL-3A en condiciones de H/R. El ARNip de Frizzled-2 se transinfectó en células BRL-3A para inhibir la señalización de Frizzled-2. Wnt5a y Frizzled-2 aumentaron considerablemente en las células BRL-3A tras el tratamiento con H/R. El tratamiento con H/R provocó citotoxicidad celular, una tasa de apoptosis temprana y el nivel de Ca2+ intracelular en células BRL-3A mientras que el gen frizzled-2 silenciado redujo la citotoxicidad celular inducida por H/R, la apoptosis y el nivel de Ca2+ intracelular. El estudio in vivo con ratones mostró, además, la regulación al alza de la vía de Frizzled-2/Wnt 5 y la expresión de caspasa 3 escindida en tejidos hepáticos con lesión por isquemia y reperfusión (LIR). En resumen, la inhibición de Frizzled-2 por su ARNip puede proteger a las células BRL-3A al atenuar la citotoxicidad celular y la apoptosis inducida por H/R


Assuntos
Animais , Camundongos , Ratos , Receptores Frizzled/antagonistas & inibidores , RNA/isolamento & purificação , Hipóxia/metabolismo , Apoptose/fisiologia , Receptores Frizzled/genética , Reação em Cadeia da Polimerase , Western Blotting , Caspase 3
3.
Am J Physiol Cell Physiol ; 318(1): C48-C62, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618077

RESUMO

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of ß-catenin. The treatment also activated ß-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or ß-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-ß-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of ß-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.


Assuntos
Centrossomo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteínas 14-3-3/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Glicemia/metabolismo , Centrossomo/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Exorribonucleases/metabolismo , Feminino , Receptores Frizzled/genética , Produtos Finais de Glicação Avançada/farmacologia , Células HCT116 , Humanos , Insulina/sangue , Camundongos Endogâmicos ICR , Ácido Palmítico/farmacologia , Ligação Proteica , Ratos , Proteínas Wnt/genética , Quinases Associadas a rho/metabolismo
4.
Med Sci Monit ; 25: 8637-8644, 2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31733054

RESUMO

BACKGROUND Although the promoting roles of Frizzled-7 (Fzd7) have been shown before, its effects in gastric cancer (GC) cell stemness are still unclear. The present study assessed the effects of Fzd7 on GC cell stemness and chemoresistance. MATERIAL AND METHODS Clinical samples were used to detect Fzd7 expression and online datasets were used to analyze the correlation between Fzd7 expression and GC patient prognosis. Quantitative real-time PCR (qPCR), Western blot, and spheroid formation were used to detect the stemness of cells and Fzd7-mediated effects on GC cell stemness. Cell viability was assessed to evaluate the role of Fzd7 in chemoresistance of GC cells. RESULTS We found that the expression of Frizzled-7 (Fzd7), a Wnt receptor, was increased in gastric cancer (GC) cells and tissues. Additionally, Fzd7 expression was correlated with shorter overall survival of GC patients. Knockdown of Fzd7 or using inhibitors of Wnt/Fzd (OMP-18R5/Vantictumad) decreased GC cell stemness, characterized as a decrease of spheroid formation ability and expression of stemness regulators. Notably, Fzd7 knockdown or inhibitors of Wnt/Fzd attenuated the chemoresistance of GC cells. Furthermore, elevation of Myc expression rescued the effects of Fzd7 inhibition on GC cell stemness and chemoresistance. CONCLUSIONS Our results suggest that inhibition of Fzd7 decreases the stemness and chemotherapeutic resistance of GC cells.


Assuntos
Receptores Frizzled/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , China , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Genes myc/genética , Genes myc/fisiologia , Humanos , Estimativa de Kaplan-Meier , Neoplasias Gástricas/genética , Via de Sinalização Wnt/genética , beta Catenina/metabolismo
5.
Int J Biol Sci ; 15(11): 2330-2339, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31595151

RESUMO

Many studies have shown that FZD2 is significantly associated with tumor development and tumor metastasis. The purpose of the present study was to gain insight into the role of FZD2 in the cell proliferation and invasion of tongue squamous cell carcinoma. According to TCGA-HNSC dataset, among the 10 Frizzled receptors, FZD2 exhibited the highest degree of differential expression between cancer tissues and normal tissues, and the overall survival of patients with higher FZD2 levels was shown to be significantly shorter compared with those with lower FZD2 levels. The upregulation of FZD2 in clinical tongue cancer tissues was validated by real-time PCR. Knockdown of FZD2 inhibited the proliferation, migration and invasion of CAL-27 and TCA-8113 cells, whereas overexpression of FZD2 led to the opposite results. Further analysis revealed that FZD2 is positively correlated with WNT3A, WNT5B, WNT7A and WNT2 and is negatively correlated with WNT4. These results indicated that FZD2 may act as an oncogene in tongue squamous cell carcinoma. Therefore, FZD2 may be a target for the diagnosis, prognosis and gene therapy of tongue cancer.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Receptores Frizzled/fisiologia , Neoplasias da Língua/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Movimento Celular , Proliferação de Células , Feminino , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Neoplasias da Língua/genética , Neoplasias da Língua/mortalidade , Neoplasias da Língua/patologia , Proteínas Wnt/metabolismo
6.
J Exp Clin Cancer Res ; 38(1): 358, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31419987

RESUMO

BACKGROUND: MicroRNAs (miRNAs) play crucial roles in tumor initiation and development. Previously, we indicated that miR-504 is downregulated and suppresses tumor proliferation in glioblastoma (GBM). However, the regulation and relevant mechanism of miR-504 in GBM mesenchymal (ME) transition remain unclear. METHODS: Transcriptome and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. The potential functions of miR-504 were predicted using gene ontology analysis. GBM cell migration and invasion were examined using wound healing and Transwell assays. Epithelial-mesenchymal transition (EMT) progression in GBM cell lines was detected with immunofluorescence and western blotting. The stemness activity of glioma stem-like cells (GSCs) was assessed by sphere formation assay and tumor xenograft model. miR-504 binding to the FZD7 (frizzled class receptor 7) 3' untranslated region (3'UTR) was validated using dual luciferase reporter assay. TOP/FOP Flash assays were conducted to determine the effects of miR-504 on Wnt/ß-catenin signaling. RESULTS: Analysis of TCGA transcriptomic data showed that low miR-504 expression correlated with ME subtype transition and poor survival in patients with GBM. Functional experiments showed that miR-504 overexpression suppressed malignant behaviors of GBM cells, such as migration, invasion, EMT, and stemness activity. Furthermore, miR-504 was a negative regulator of the Wnt-ß-catenin pathway by directly repressing FZD7 expression, and FZD7 overexpression reversed the EMT inhibition caused by miR-504. Moreover, the low miR-504/FZD7 expression ratio was a ME subtype marker and could serve as a significant prognostic indicator and predict the clinical outcome of chemotherapy and radiotherapy for patients with GBM in TCGA dataset. CONCLUSIONS: Our results suggest that miR-504 suppresses the aggressive biological processes associated with the ME phenotype of GBM and could be a potential candidate for therapeutic applications in these malignant brain tumors.


Assuntos
Neoplasias Encefálicas/patologia , Transição Epitelial-Mesenquimal , Receptores Frizzled/metabolismo , Glioblastoma/patologia , MicroRNAs/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Estudos de Coortes , Feminino , Seguimentos , Receptores Frizzled/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
7.
Exp Eye Res ; 186: 107720, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299183

RESUMO

Familial exudative vitreoretinopathy (FEVR) is a hereditary retinal vascular disorder. Among the various clinical phenotypes of this disease, retinal folds are the primary and typical feature of FEVR. However, little is known about the clinical characteristics, genetic spectrum, or potential phenotype-genotype correlation of retinal folds. Herein, we describe and analyze the clinical and genetic characteristics of retinal folds in FEVR. Eighty-nine patients with unilateral or bilateral retinal folds were included in this study. Clinical examinations showed that the retinal folds were bilateral in 37 patients (41.6%). Various retinal abnormalities were noted in the fellow eyes in the remaining 52 patients with unilateral folds. Most of the folds were located temporally (98.4%, 124/126), and were complete (97.6%, 123/126). 67.5% (60/89) probands were genetic confirmed FEVR. 25 novel pathogenic mutations (7 in FZD4, 7 in LRP5, 1 in NDP, 4 in TSPAN12, and 6 in KIF11) were identified in 25 families. Overall, 87.5% (14/16) and 73.7%(14/19) patients with LRP5 and FZD4 mutations were with unilateral folds, respectively.Nevertheless, only 25% (2/8), 36.4%(4/11) and 16.7%(1/6) patients with NDP, TSPAN12, and KIF11 mutations were with unilateral folds. Moreover, 85.7%(12/14),100% (6/6) and 100%(8/8) of the patients with mutated TSPAN12, KIF11, and NDP genes presented with symmetry in disease staging, while 55% and 64.7% of patients with FZD4 and LRP5 mutation displayed symmetry in staging. In conclusion, the majority of retinal folds extended completely and radially in the temporal peripheral retina. Patients with causative mutations in NDP, TSPAN12, or KIF11 were more likely to have bilaterally symmetrical severe retinopathy. In contrast, patients with LRP5 and FZD4 mutations displayed a relatively milder but broader spectrum of phenotypes and a higher frequency of asymmetry.


Assuntos
Vitreorretinopatias Exsudativas Familiares , Descolamento Retiniano , Adolescente , Criança , Pré-Escolar , Proteínas do Olho/genética , Vitreorretinopatias Exsudativas Familiares/genética , Vitreorretinopatias Exsudativas Familiares/patologia , Feminino , Receptores Frizzled/genética , Genótipo , Humanos , Lactente , Recém-Nascido , Cinesina/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Proteínas do Tecido Nervoso/genética , Fenótipo , Descolamento Retiniano/genética , Descolamento Retiniano/patologia , Tetraspaninas/genética
8.
Invest Ophthalmol Vis Sci ; 60(7): 2659-2666, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31237656

RESUMO

Purpose: The purpose of this study was to investigate the genetic mutation spectrum in Chinese patients with familial exudative vitreoretinopathy-associated rhegmatogenous retinal detachment (FEVR-RRD) and to analyze the preliminary genotype-phenotype association. Methods: In this consecutive, cross-sectional study, 54 patients with FEVR-RRD were studied. Comprehensive ophthalmic examinations and targeted next-generation sequencing were performed in all patients. The genotype-phenotype association was also analyzed. Results: Causative mutations were identified in 38.9% (21/54) of patients (14/54 in LRP5, 4/54 in FDZ4, and 3/54 in TSPAN12). The study identified 22 potentially pathogenic mutations in 21 unrelated FEVR probands, and 14 were novel (10/15 in LRP5, 1/4 in FZD4, and 3/3 in TSPAN12). Furthermore, to explore the genotype-phenotype association, late-phase angiographic posterior and peripheral leakage (LAPPEL) was identified in 100% (4/4) of patients with FZD4 mutations and 100% (3/3) of patients with TSPAN12 mutations but only in 42.9% (6/14) of patients with LRP5 mutations. Extraretinal neovascularization (ERNV) was found in 100% (4/4) of patients with FZD4 mutations and in 66.7% (2/3) of patients with TSPAN12 mutations, but only in 21.4% (3/14) of patients with LRP5 mutations. Conclusions: The positive rate for pathogenic mutations in the known FEVR-associated genes was 38.9% (21/54). Among the mutations, LRP5 mutation was the predominant, accounting for 66.7% (14/21) of genetic positive patients. Patients with FEVR-RRD due to LRP5 mutations have less retinal vascular leakage or neovasculization than do patients with FEVR-RRD due to TSPAN12/FZD4 mutations. Moreover, 14 novel variants were found, which provided a deeper understanding of this disease.


Assuntos
Vitreorretinopatias Exsudativas Familiares/genética , Receptores Frizzled/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mutação , Descolamento Retiniano/genética , Tetraspaninas/genética , Adolescente , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Criança , Pré-Escolar , Estudos Transversais , Análise Mutacional de DNA , Vitreorretinopatias Exsudativas Familiares/diagnóstico , Feminino , Angiofluoresceinografia , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Descolamento Retiniano/diagnóstico , Adulto Jovem
9.
Pathol Res Pract ; 215(8): 152478, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31176574

RESUMO

BACKGROUND: Overexpression of Frizzled-7 (FZD7) has been associated with tumor invasion and distant metastases, but little is known about the relationship between FZD7 expression and prognosis in colon cancer. PATIENTS AND METHODS: A total of 114 patients with colon cancer between June 2010 and December 2010 were enrolled in this study. The expression of FZD7 in cancerous and adjacent non-cancerous tissues was determined by immunohistochemistry, and the association between FZD7 expression and patient's clinicopathological characteristics was explored. The correlation between FZD7 expression and prognosis of colon cancer patients was analyzed using the Oncomine database and R2. RESULTS: FZD7 expression levels were significantly higher in colon cancer tissues compared with adjacent non-cancerous tissues (P < 0.001). High expression of FZD7 was significantly associated with metastatic or recurrent disease in colon cancer (P = 0.010). Kaplan-Meier survival analysis demonstrated that colon cancer patients with high expression of FZD7 had a significantly poorer OS (P = 0.013) and DFS (P = 0.010). Cox regression demonstrated that the expression of FZD7 was an independent prognostic factor for DFS (HR = 6.647, P = 0.023). A meta-analysis from the Oncomine database demonstrated that FZD7 mRNA levels were significantly higher in colorectal cancer tissues than in normal colorectal tissues, and FZD7 high expression was associated with a significantly poorer event and relapse-free survival time by analyzing the data from the R2: Genomics Analysis and Visualization Platform. CONCLUSIONS: Overexpression of FZD7 was associated with poor survival in patients with colon cancer. Our data suggest that FZD7 expression could be an effective prognostic biomarker for colon cancer.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/mortalidade , Receptores Frizzled/genética , Recidiva Local de Neoplasia/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , RNA Interferente Pequeno/genética
10.
Development ; 146(8)2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036548

RESUMO

Zebrafish kidneys use resident kidney stem cells to replace damaged tubules with new nephrons: the filtration units of the kidney. What stimulates kidney progenitor cells to form new nephrons is not known. Here, we show that wnt9a and wnt9b are induced in the injured kidney at sites where frizzled9b- and lef1-expressing progenitor cells form new nephrons. New nephron aggregates are patterned by Wnt signaling, with high canonical Wnt-signaling cells forming a single cell thick rosette that demarcates: domains of cell proliferation in the elongating nephron; and tubule fusion where the new nephron plumbs into the distal tubule and establishes blood filtrate drainage. Pharmacological blockade of canonical Wnt signaling inhibited new nephron formation after injury by inhibiting cell proliferation, and resulted in loss of polarized rosette structures in the aggregates. Mutation in frizzled9b reduced total kidney nephron number, caused defects in tubule morphology and reduced regeneration of new nephrons after injury. Our results demonstrate an essential role for Wnt/frizzled signaling in adult zebrafish kidney development and regeneration, highlighting conserved mechanisms underlying both mammalian kidney development and kidney stem cell-directed neonephrogenesis in zebrafish.


Assuntos
Rim/citologia , Rim/metabolismo , Néfrons/citologia , Néfrons/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regeneração/fisiologia , Via de Sinalização Wnt/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
11.
Mol Cells ; 42(4): 345-355, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31082802

RESUMO

Eosinophilic chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most challenging problems in clinical rhinology. FZD5 is a receptor for Wnt5A, and its complex with Wnt5A contributes to activating inflammation and tissue modification. Nasal polyps and eosinophil/non-eosinophil counts are reported to be directly correlated. This study investigated the expression and distribution of FZD5, and the role of eosinophil infiltration and FZD5 in eosinophilic CRSwNP pathogenesis. The prognostic role of eosinophil levels was evaluated in seven patients with CRSwNP. Fifteen patients with CRS were classified based on the percentage of eosinophils in nasal polyp tissue. Methylated genes were detected using methyl-CpG-binding domain sequencing, and qRT-PCR and immunohistochemistry were used to detect FZD5 expression in nasal polyp tissue samples. The results showed that mRNA expression of FZD5 was upregulated in nasal polyps. FZD5 expression was significantly higher in nasal polyp samples from patients with eosinophilic CRSwNP than in those from patients with non-eosinophilic CRSwNP, as indicated by immunohistochemistry. Furthermore, inflammatory cytokine levels were higher in eosinophilic CRSwNP-derived epithelial cells than in normal tissues. In conclusion, FZD5 expression in nasal mucosal epithelial cells is correlated with inflammatory cells and might play a role in the pathogenesis of eosinophilic CRSwNP.


Assuntos
Metilação de DNA , Eosinófilos/metabolismo , Receptores Frizzled/genética , Pólipos Nasais/genética , Rinite/genética , Sinusite/genética , Adulto , Idoso , Citocinas/metabolismo , Epigênese Genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Receptores Frizzled/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/citologia , Mucosa Nasal/metabolismo , Pólipos Nasais/imunologia , Prognóstico , Regiões Promotoras Genéticas , Rinite/imunologia , Sinusite/imunologia , Regulação para Cima
12.
Cancer Lett ; 457: 60-73, 2019 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-31078732

RESUMO

tRNA-derived fragments offer a recently identified group of non-coding single-stranded RNAs that are often as abundant as microRNAs in cancer cells and play important roles in carcinogenesis. However, the biological functions of them in breast cancer are still unclear. Hence, we focused on investigating whether tiRNAs could play a key role in the progression of breast cancer. We have identified 5'-tiRNAVal with significantly low expression in breast cancer tissues. The down-regulation of serum 5'-tiRNAVal was positively correlated with stage progression and lymph node metastasis. Overexpression of 5'-tiRNAVal suppressed cells malignant activities. FZD3 was confirmed to be a direct target of 5'-tiRNAVal in breast cancer. In addition, FZD3, ß-Catenin, c-myc and cyclinD1 levels in 5'-tiRNAVal overexpressing cells were downregulated while APC was inversely upregulated. Moreover, 5'-tiRNAVal inhibited the FZD3-mediated Wnt/ß-Catenin signaling pathway in breast cancer cells. Finally, 5'-tiRNAVal levels differentiated breast cancer from healthy controls with a sensitivity of 90.0% and specificity of 62.7%. This is the first study to show that 5'-tiRNAVal as a new tumor-suppressor through inhibition of FZD3/Wnt/ß-Catenin signaling pathway, which could be as a potential diagnostic biomarker for breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Receptores Frizzled/metabolismo , RNA de Transferência de Valina/metabolismo , Via de Sinalização Wnt , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação para Baixo , Feminino , Receptores Frizzled/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Células MCF-7 , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA de Transferência de Valina/genética , beta Catenina/genética , beta Catenina/metabolismo
13.
Cancer Res ; 79(11): 2812-2820, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30967398

RESUMO

Despite the high initial response rates to PARP inhibitors (PARPi) in BRCA-mutated epithelial ovarian cancers (EOC), PARPi resistance remains a major challenge. Chemical modifications of RNAs have emerged as a new layer of epigenetic gene regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of mRNA, yet the role of m6A modification in PARPi resistance has not previously been explored. Here, we show that m6A modification of FZD10 mRNA contributes to PARPi resistance by upregulating the Wnt/ß-catenin pathway in BRCA-mutated EOC cells. Global m6A profile revealed a significant increase in m6A modification in FZD10 mRNA, which correlated with increased FZD10 mRNA stability and an upregulation of the Wnt/ß-catenin pathway. Depletion of FZD10 or inhibition of the Wnt/ß-catenin sensitizes resistant cells to PARPi. Mechanistically, downregulation of m6A demethylases FTO and ALKBH5 was sufficient to increase FZD10 mRNA m6A modification and reduce PARPi sensitivity, which correlated with an increase in homologous recombination activity. Moreover, combined inhibition of PARP and Wnt/ß-catenin showed synergistic suppression of PARPi-resistant cells in vitro and in vivo in a xenograft EOC mouse model. Overall, our results show that m6A contributes to PARPi resistance in BRCA-deficient EOC cells by upregulating the Wnt/ß-catenin pathway via stabilization of FZD10. They also suggest that inhibition of the Wnt/ß-catenin pathway represents a potential strategy to overcome PARPi resistance. SIGNIFICANCE: These findings elucidate a novel regulatory mechanism of PARPi resistance in EOC by showing that m6A modification of FZD10 mRNA contributes to PARPi resistance in BRCA-deficient EOC cells via upregulation of Wnt/ß-catenin pathway.


Assuntos
Adenosina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores Frizzled/genética , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Homólogo AlkB 5 da RNA Desmetilase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Proteína BRCA2/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Receptores Frizzled/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Metilação , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , RNA Mensageiro/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , beta Catenina/metabolismo
14.
Development ; 146(9)2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30952667

RESUMO

Arl/ARF GTPases regulate ciliary trafficking, but their tissue-specific functions are unclear. Here, we demonstrate that ciliary GTPase Arl3 is required for mitotic spindle orientation of mouse basal stem cells during skin development. Arl3 loss diminished cell divisions within the plane of the epithelium, leading to increased perpendicular divisions, expansion of progenitor cells and loss of epithelial integrity. These observations suggest that an Arl3-dependent mechanism maintains cell division polarity along the tissue axis, and disruption of planar spindle orientation has detrimental consequences for epidermal architecture. Defects in planar cell polarity (PCP) can disrupt spindle positioning during tissue morphogenesis. Upon Arl3 loss, the PCP signaling molecules Celsr1 and Vangl2 failed to maintain planar polarized distributions, resulting in defective hair follicle angling, a hallmark of disrupted PCP. In the absence of Celsr1 polarity, frizzled 6 lost its asymmetrical distribution and abnormally segregated to the apical cortex of basal cells. We propose that Arl3 regulates polarized endosomal trafficking of PCP components to compartmentalized membrane domains. Cell-cell communication via ciliary GTPase signaling directs mitotic spindle orientation and PCP signaling, processes that are crucial for the maintenance of epithelial architecture.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Fuso Acromático/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Cultivadas , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Técnicas In Vitro , Queratinócitos/metabolismo , Lentivirus/genética , Camundongos , Mitose/genética , Mitose/fisiologia , Morfogênese/genética , Morfogênese/fisiologia , Reação em Cadeia da Polimerase , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
15.
Cell Prolif ; 52(3): e12607, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30955216

RESUMO

OBJECTIVES: Tumour-targeted gene therapy is a promising approach for effective control of gastric cancer cell proliferation. Our study aims to develop a cancer therapy which combines tumour-targeting promoters with cytotoxins. METHODS: The expression of globotriaosylceramide (Gb3), which is a Shiga-like toxin I (Stx1) receptor, was verified in gastric cancer compared with normal stomach tissues as assessed by flow cytometry and immunohistochemical analysis. We therefore constructed the recombinant pFZD7-Stx1 plasmid vectors with tumour-preferential Frizzled-7 promoter and Stx1. pFZD7-Stx1 was used to treat gastric cancer in vitro and in vivo. The gastric cancer cell proliferation and tumour growth were identified after the transfection with the pFZD7-Stx1. RESULTS: Globotriaosylceramide was obviously increased in gastric cancer compared with normal stomach. The gastric cancer cell proliferation and tumour growth decreased significantly after the transfection with the pFZD7-Stx1. CONCLUSION: Frizzled-7 promoter is preferentially active, and Gb3 is abundant in gastric cancer cells. Frizzled-7 promoter and Stx1 may be used to determine a novel and relatively specific and potent gastric cancer therapeutic strategy.


Assuntos
Receptores Frizzled/genética , Terapia Genética/métodos , Toxina Shiga I/genética , Toxina Shiga I/uso terapêutico , Neoplasias Gástricas/terapia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Receptores Frizzled/metabolismo , Humanos , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Toxina Shiga I/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transfecção , Triexosilceramidas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Proc Natl Acad Sci U S A ; 116(14): 6812-6817, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894493

RESUMO

Aberrant activation of Wnt/ß-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.


Assuntos
Especificidade de Anticorpos , Antineoplásicos Imunológicos , Receptores Frizzled/antagonistas & inibidores , Neoplasias Pancreáticas , Engenharia de Proteínas , Animais , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Feminino , Receptores Frizzled/genética , Receptores Frizzled/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Medicine (Baltimore) ; 98(11): e14803, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30882657

RESUMO

RATIONALE: The case with congenital macular coloboma and cataract was rarely reported, and the pathogenic gene of the disease is still not clear. Moreover, it is difficult to improve the visual acuity of the eye with this disease. PATIENT CONCERNS: An 11-year-old boy presented low visual acuity and horizontal nystagmus in both eyes. Ophthalmologic examination showed the patient with bilateral congenital coloboma and cataract. The visual acuity of the patient improved slightly after cataract surgery. Heterozygous mutations of frizzled-4 (FZD4) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) were identified by next-generation sequencing in this case. DIAGNOSIS: Congenital macular coloboma and cataract of both eyes. INTERVENTIONS: We performed the standard phacoemulsification and intraocular lens implantation on both eyes of the patient for the treatment of congenital cataract, and then followed up the fundus lesions regularly. OUTCOMES: Cataract surgery may improve the visual acuity of the eyes with congenital macular coloboma and cataract at some degree, but the vision of this patient was still very poor postoperatively. Furthermore, the heterozygous mutations of FZD4 and NOD2 were found in this patient. LESSONS: Cataract surgery may improve the visual acuity of the eyes with congenital macular coloboma and cataract at some degree, and heterozygous mutations of FZD4 and NOD2 may be involved in the occurrence of congenital macular coloboma and cataract.


Assuntos
Catarata , Coloboma , Receptores Frizzled/genética , Implante de Lente Intraocular/métodos , Macula Lutea/anormalidades , Proteína Adaptadora de Sinalização NOD2/genética , Facoemulsificação/métodos , Catarata/congênito , Catarata/diagnóstico , Catarata/genética , Catarata/fisiopatologia , Criança , Coloboma/diagnóstico , Coloboma/genética , Coloboma/fisiopatologia , Coloboma/cirurgia , Técnicas de Diagnóstico Oftalmológico , Humanos , Macula Lutea/fisiopatologia , Macula Lutea/cirurgia , Masculino , Nistagmo Patológico/diagnóstico , Nistagmo Patológico/etiologia , Resultado do Tratamento , Acuidade Visual
18.
Mol Vis ; 25: 60-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820142

RESUMO

Purpose: To identify novel mutations in FZD4 and to investigate their pathogenicity in a cohort of Chinese patients with familial exudative vitreoretinopathy (FEVR). Methods: Next-generation sequencing was performed in patients with a clinical diagnosis of FEVR. Wide-field angiography was performed in probands and family members if available. Clinical data were collected from patient charts. The effect of the mutations in FZD4 on its biologic activity in the Norrin/ß-catenin signaling pathway was analyzed with the luciferase reporter assay. Results: Four novel mutations in FZD4 (c.1188_1192del/p.F396fs, c.1220delC/p.A407Vfs*24, c.905G>A/p.C302Y, c.1325T>A/p.V442E) were identified in four unrelated families. The mutations were not detected in 200 healthy individuals. The variability of the ocular phenotypes was not only observed in the probands and parents harboring the same mutation but also between two eyes in one individual. All four novel mutations introduced reduction in luciferase activity. Compared with the wild-type, the FZD4 level of the four mutants also decreased variably. Conclusions: Four novel mutations in FZD4 were identified in Chinese patients with FEVR. No correlation in the reduced luciferase activity and the ocular phenotype was observed in this study. This study further emphasized the complexity of the FEVR-causing machinery.


Assuntos
Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Receptores Frizzled/genética , Mutação , Proteínas do Tecido Nervoso/genética , Doenças Retinianas/genética , beta Catenina/genética , Adulto , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , Estudos de Casos e Controles , Pré-Escolar , Oftalmopatias Hereditárias/diagnóstico por imagem , Oftalmopatias Hereditárias/etnologia , Oftalmopatias Hereditárias/patologia , Proteínas do Olho/metabolismo , Vitreorretinopatias Exsudativas Familiares , Feminino , Angiofluoresceinografia , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Fenótipo , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/etnologia , Doenças Retinianas/patologia , Transdução de Sinais , beta Catenina/metabolismo
19.
Gene Expr Patterns ; 32: 44-52, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30851426

RESUMO

Wnts are secreted signaling molecules that are implicated in a variety of growth-related processes. Frizzled proteins have been identified as receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for dioecious flatworm Frizzleds has not been determined. To evaluate the endogenous role of Frizzled proteins during development, we have identified and characterized a Schistosoma japonicum frizzled gene (Sjfz7). We found that Sjfz7 encodes a 698 amino acid protein with typical characteristics of Frizzled proteins. The immunohistochemical localization pattern showed that Sjfz7 protein was extensively distributed in almost all tissues of S. japonicum, including subtegumental muscle cells, parenchymal cells, intestinal epithelial cells and male and female germ cells. This indicated that Sjfz7-mediated Wnt signaling might be associated with the development of musculature, intestinal tract and reproductive organs in schistosome. Comparing mRNA levels between frizzled family members showed that Sjfz7 mRNA was consistently higher in the developmental stages analyzed, suggesting that Sjfz7 may be responsible for more functional tasks than other frizzled family members. Comparing frizzled mRNA levels between not fully developed and normal worms suggested that Wnt signaling might be abnormal in not fully developed worms.


Assuntos
Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Schistosoma japonicum/genética , Sequência de Aminoácidos/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Schistosoma japonicum/metabolismo , Esquistossomose Japônica/genética , Esquistossomose Japônica/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Transcriptoma/genética
20.
BMC Evol Biol ; 19(1): 72, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30849938

RESUMO

BACKGROUND: Frizzled family members belong to G-protein coupled receptors and encode proteins accountable for cell signal transduction, cell proliferation and cell death. Members of Frizzled receptor family are considered to have critical roles in causing various forms of cancer, cardiac hypertrophy, familial exudative vitreoretinopathy (FEVR) and schizophrenia. RESULTS: This study investigates the evolutionary and structural aspects of Frizzled receptors, with particular focus on FEVR associated FZD4 gene. The phylogenetic tree topology suggests the diversification of Frizzled receptors at the root of metazoans history. Moreover, comparative structural data reveals that FEVR associated missense mutations in FZD4 effect the common protein region (amino acids 495-537) through a well-known phenomenon called epistasis. This critical protein region is present at the carboxyl-terminal domain and encompasses the K-T/S-XXX-W, a PDZ binding motif and S/T-X-V PDZ recognition motif. CONCLUSION: Taken together these results demonstrate that during the course of evolution, FZD4 has acquired new functions or epistasis via complex patter of gene duplications, sequence divergence and conformational remodeling. In particular, amino acids 495-537 at the C-terminus region of FZD4 protein might be crucial in its normal function and/or pathophysiology. This critical region of FZD4 protein may offer opportunities for the development of novel therapeutics approaches for human retinal vascular disease.


Assuntos
Evolução Molecular , Oftalmopatias Hereditárias/genética , Receptores Frizzled/química , Receptores Frizzled/genética , Doenças Retinianas/genética , Vitreorretinopatias Exsudativas Familiares , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação de Sentido Incorreto/genética , Filogenia , Domínios Proteicos
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