Wnt1 and Wnt2b immunodetection of the regenerating tail and comparative ultrastructure of tail spinal cord in the Scincella tsinlingensis / Inmunodetección Wnt1 y Wnt2b de la cola en regeneración y ultraestructura comparativa de la médula espinal de la cola en Scincella tsinlingensis

SUMMARY: The Wnt pathway is essential for the initiation of lizard tail regeneration. The regenerated lizard tails exhibit obvious morphological differences compared to the original ones. The expression of Wnt1 and Wnt2b proteins in the regenerating tail of Scincella tsinlingensis was detected by immunohistochemistry and then comparatively analyzed for ultrastructural changes in the original and regenerated spinal cord. The ependymal layer of the original spinal cord was pseudostratified with multiciliated cells and primary monociliated cells, while the cells of the ependymal layer of the regenerated spinal cord were organized in a monolayer with a few biciliated cells. Immunolocalization indicated that Wnt1 and Wnt2b were mainly distributed in the dermis near the original tail stump, spinal cord, and clot-positive migratory cells during Stage I, 0-1 days post-amputation (dpa). Wnt1 and Wnt2b were predominantly detected in the epaxial and hypaxial musculature near the original tail stump, wound epithelium, and spinal cord in the original tail during Stage II, 1-7 dpa. Mesenchymal cells and wound epithelium showed immunostaining during Stage III and IV, 7-15 dpa. The ependymal tubes contained these signaling proteins during Stage V and VI, 20- 30 dpa. Labeling was mainly observed in nearby regenerative blood vessels, ependymal cells, epaxial and hypaxial musculature in the apical epithelial layer (AEC) after 45-160 dpa. These findings indicated that Wnt1 and Wnt2b proteins presented primarily in regenerating epidermis and nerve tissues were a critical signal for tail regeneration in S. tsinlingensis.

RESUMEN: La vía Wnt es esencial para el inicio de la regeneración de la cola del lagarto. Las colas de lagarto regeneradas exhiben diferencias morfológicas obvias en comparación con las originales. La expresión de las proteínas Wnt1 y Wnt2b en la cola en regeneración de Scincella tsinlingensis se detectó mediante inmunohistoquímica y luego se analizaron comparativamente los cambios ultraestructurales en la médula espinal original y regenerada. La capa ependimaria de la médula espinal original se pseudoestratificó con células multiciliadas y células monociliadas primarias, mientras que las células de la capa ependimaria de la médula espinal regenerada se organizaron en monocapa con algunas células bicilicadas. La inmunolocalización indicó que Wnt1 y Wnt2b se distribuyeron principalmente en la dermis cerca del muñón de la cola original, la médula espinal y las células migratorias positivas en el coágulo durante la Etapa I, 0-1 días después de la amputación (dpa). Wnt1 y Wnt2b se detectaron predominantemente en la musculatura epaxial e hipaxial cerca del muñón de la cola original, el epitelio de la herida y la médula espinal en la cola original durante la Etapa II, 1-7 dpa. Las células mesenquimales y el epitelio de la herida mostraron inmunomarcaje durante la Etapa III y IV, 7- 15 dpa. Los tubos ependimarios contenían estas proteínas de señalización durante la Etapa V y VI, 20-30 dpa. El marcaje se observó principalmente en vasos sanguíneos regenerativos cercanos, células ependimarias, musculatura epaxial e hipaxial en la capa epitelial apical (AEC) después de 45-160 dpa. Estos hallazgos indicaron que las proteínas Wnt1 y Wnt2b están presentes principalmente en la epidermis en regeneración y en los tejidos nerviosos y eran una señal crítica para la regeneración de la cola en S. tsinlingensis.

Expression of Wnt and beta-catenin in mouse tooth germs and chick facial primordia: a comparison / Expresión de Wnt y beta-catenina en gérmenes de dientes de ratón y primordios faciales de polluelos: una comparación

In order to compare Wnt/ beta-catenin expression in mouse and chick facial primordia during development, we performed an immunohistochemical analysis of both protein expressions in E12 to E17 mouse embryos and in E3 and E10 chick embryos. During odontogenesis, from bud to bell stage, both proteins exhibit similar fixation patterns, with epithelial and mesenchymal immunoreactivity, consistant with literature data. Double labelling demonstrates that the same cells express both antigens, even in undifferentiated mesenchyme. The enamel knot, and the ameloblastic and odontoblastic layers are stained at the same manner. In the chick, Wnt and beta-catenin are diffusely present on craniofacial mesenchyme. In both species, premuscular blastemata express Wnt and b-catenin, but Wnt is specifically expressed on the perichondrium and ossification centers, suggesting a role independent from beta-catenin pathway.

Propofol inhibits proliferation, migration, and invasion but promotes apoptosis by regulation of Sox4 in endometrial cancer cells

Propofol is an intravenous sedative hypnotic agent of which the growth-inhibitory effect has been reported on various cancers. However, the roles of propofol in endometrial cancer (EC) remain unclear. This study aimed to explore the effects of propofol on EC in vitro and in vivo. Different concentrations of propofol were used to treat Ishikawa cells. Colony number, cell viability, cell cycle, apoptosis, migration, and invasion were analyzed by colony formation, MTT, flow cytometry, and Transwell assays. In addition, the pcDNA3.1-Sox4 and Sox4 siRNA plasmids were transfected into Ishikawa cells to explore the relationship between propofol and Sox4 in EC cell proliferation. Tumor weight in vivo was measured by xenograft tumor model assay. Protein levels of cell cycle-related factors, apoptosis-related factors, matrix metalloproteinases 9 (MMP9), matrix metalloproteinases 2 (MMP2) and Wnt/β-catenin pathway were examined by western blot. Results showed that propofol significantly decreased colony numbers, inhibited cell viability, migration, and invasion but promoted apoptosis in a dose-dependent manner in Ishikawa cells. Moreover, propofol reduced the expression of Sox4 in a dose-dependent manner. Additionally, propofol significantly suppressed the proportions of Ki67+ cells, but Sox4 overexpression reversed the results. Furthermore, in vivo assay results showed that propofol inhibited tumor growth; however, the inhibitory effect was abolished by Sox4 overexpression. Moreover, propofol inhibited Sox4 expression via inactivation of Wnt/β-catenin signal pathway. Our study demonstrated that propofol inhibited cell proliferation, migration, and invasion but promoted apoptosis by regulation of Sox4 in EC cells. These findings might indicate a novel treatment strategy for EC.

Rspo1 promotes osteoblast differentiation via Wnt signaling pathway.

R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of skeletal system. Recent studies in various murine osteoblast models have proposed that Rspo1 may interact with Wnt signaling pathway to induce differentiation in osteoblasts. Though findings in murine osteoblasts implicate a synergestic role of Rspo1 with Wnt signaling, still no study has addressed the similar role in more clinically applicable osteoblast models i.e., human cell lines or primary cells. Therefore, in the present study, we investigated the possible role of Rspo1 during differentiation process of human in vitro osteoblast cell models like primary osteoblasts or human osteoprogenitor cell line hFOB1.19 along with murine preosteoblast cell line MC3T3 E-1. Our results showed increase in Rspo1 at transcript level during differentiating phase of human primary osteoblasts and human FOB1.19 cells. We also found that Rspo1 (100 ng/mL) acts additively with Wnt3a to activate Wnt signaling, as confirmed by luciferase activity after transfection of TOPFLASH construct to hFOB1.19 cells. Similar additive role of Rspo1 and Wnt3a was apparent in alkaline phosphatase (ALP) activity analysis of human primary cells. Moreover, a reduction in ALP activity was observed with knock-down of Rspo1 by transfected shRNA in hFOB1.19 cells. These results suggested the possibility of autocrine regulation by Rspo1 on the osteogenic activities in human in vitro osteoblast models. Furthermore, these results were corroborated in MC3T3-E1, murine osteoblast cell model. Osteoblastic differentiation was induced by transfection of Rspo1 which was confirmed by increased ALP staining and qRT-PCR analysis of osteogenic markers, such as Runx2 and osteocalcin. In conclusion, present study highlights the role of Rspo1 in bone remodeling where it activates Wnt signaling to induce differentiation, as shown in human as well murine in vitro osteoblast cell models.

Canonical and noncanonical Wnt pathways: a comparison between endometrial cancer type I and atrophic endometrium in Brazil / Vias Wnt canônica e não canônica: uma comparação entre o câncer endometrial tipo I e endométrio atrófico no Brasil

CONTEXT AND OBJECTIVE: The Wnt pathway is involved in tumorigenesis of several tissues. For this reason, we proposed to evaluate Wnt gene expression in endometrial cancer type I. DESIGN AND SETTING: Cross-sectional study on materials gathered from the tissue bank of the Department of Pathology, Universidade Federal de São Paulo. METHODS: Endometrial specimens were obtained from surgeries performed between 1995 and 2005 at São Paulo Hospital, Universidade Federal de São Paulo. The material was divided into two groups according to tissue type: Group A, atrophic endometrium (n = 15); and Group B, endometrial adenocarcinoma (n = 45). We compared the immunohistochemical expression of Wnt1, Frizzled-1 (FZD1), Wnt5a, Frizzled-5 (FZD5) and beta-catenin between endometrial cancer type I and atrophic endometrium. RESULTS: Regarding Wnt1, FZD1 and Wnt5a expression, no significant association was observed between the groups. A significant association was observed between the groups in relation to FZD5 expression (P = 0.001). The proportion of FZD5-positive samples was significantly higher in group A (80.0 percent) than in group B (31.1 percent). Regarding the survival curve for FZD5 in group B, we did not find any significant association between atrophic endometrium and endometrial adenocarcinoma. We also did not find any significant association regarding beta-catenin expression (P = 1.000). CONCLUSION: FZD5 is downregulated in endometrial adenocarcinoma, in comparison with atrophic endometrium.

CONTEXTO E OBJETIVO: A via Wnt está envolvida na tumorigênese de diversos tipos de tecidos. Por essa razão, propusemo-nos a avaliar a expressão de genes da família Wnt no câncer endometrial tipo I. TIPO DE ESTUDO E LOCAL: Estudo transversal com coleta de materiais do banco de tecidos do Departamento de Patologia da Universidade Federal de São Paulo. MÉTODOS: Amostras endometriais foram obtidas de cirurgias que ocorreram entre 1995 e 2005 no Hospital São Paulo, Universidade Federal de São Paulo. Foram separados dois grupos segundo o tipo de tecido obtido: grupo A, com endométrio atrófico (n = 15); e grupo B, com adenocarcinoma endometrial (n = 45). Comparamos a expressão imunoistoquímica de Wnt 1, Frizzled-1 (FZD1), Wnt 5a, Frizzled-5 (FZD 5) e beta-catenina entre câncer endometrial tipo I e endométrio atrófico. RESULTADOS: Na expressão do Wnt1, FZD1 e Wnt5a, não observamos associação significante entre os grupos. Na expressão do FZD5, encontramos associação significante entre os grupos (P = 0,001). A proporção de positividade do FZD5 foi significantemente maior no grupo A comparado ao grupo B (31,1 por cento). Em relação à curva de sobrevida para o FZD5 no grupo B, não tivemos associação significante entre endométrio atrófico e adenocarcinoma do endométrio. Também não observamos associação significante na expressão da beta-catenina (P = 1,000). CONCLUSÃO: FZD5 é downregulated no adenocarcinoma endometrial quando comparado ao endométrio atrófico.

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80 percent of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38 percent of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64 percent, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

TSH signalling and cancer

Thyroid cancers are the most frequent endocrine neoplasms and mutations in the thyrotropin receptor (TSHR) are unusually frequent. Here we present the state-of-the-art concerning the role of TSHR in thyroid cancer and discuss it in light of the cancer stem cell theory or the classical view. We briefly review the gene and protein structure updating the cancer related TSHR mutations database. Intriguingly, hyperfunctioning TSHR mutants characterise differentiated cancers in contrast to undifferentiated thyroid cancers which very often bear silenced TSHR. It remains unclear whether TSHR alterations in thyroid cancers play a role in the onset or they appear as a consequence of genetic instability during evolution, but the presence of functional TSHR is exploited in therapy. We outline the signalling network build up in the thyrocyte between TSHR/PKA and other proliferative pathways such as Wnt, PI3K and MAPK. This networks integrity surely plays a role in the onset/evolution of thyroid cancer and needs further research. Lastly, future investigation of epigenetic events occurring at the TSHR and other loci may give better clues for molecular based therapy of undifferentiated thyroid carcinomas. Targeted demethylating agents, histone deacetylase inhibitors combined with retinoids and specific RNAis may help treatment in the future.

Os cânceres de tiróide são as neoplasias endócrinas mais frequentes e as mutações no receptor de tirotrofina (TSHR) são incomumente frequentes. Nesta revisão nós apresentamos o "estado da arte" com relação ao papel do TSHR no câncer de tiróide e o discutimos à luz da teoria da célula matriz do câncer ou a visão clássica. Revisamos brevemente a estrutura do gene e da proteína, atualizando a base de dados das mutações do TSHR relacionadas ao câncer. Curiosamente, mutações do TSHR com hiperfunção caracterizam cânceres diferenciados, em contraste com os cânceres de tiróide indiferenciados, os quais muito comumente mostram TSHR silenciados. Permanece obscuro se as alterações do TSHR em cânceres de tiróide têm algum papel no surgimento ou se elas aparecem como conseqüência da instabilidade genética durante seu desenvolvimento, mas a presença de TSHR funcional é explorada na terapia. Nós delineamos a rede de sinalizacão desenvolvida no tirócito entre TSHR/PKA e outras vias proliferativas como a Wnt, PI3k e MAPK. A integridade desta rede certamente tem um papel no surgimento/evolução do câncer de tiróide e necessita de novas pesquisas. Finalmente, novas investigacões sobre os eventos epigenéticos que ocorrem no TSHR e outros locais poderão trazer novas informações para uma terapia de base molecular nos carcinomas indiferenciados de tiróide. Agentes demetilantes direcionados, inibidores da histona-deacetilase, combinados com retinóides e RNAs específicos poderão auxiliar no tratamento futuro.