Frat is dispensable for canonical Wnt signaling in mammals

Wnt-signal transduction through beta-catenin is thought to require the inhibition of GSK3 by Frat/GBP. To investigate the role of Frat in mammalian development, we have generated mice with targeted mutations in all three murine Frat homologs. We show that Frat is normally expressed at sites of active Wnt signaling. Surprisingly, Frat-deficient mice do not display gross abnormalities. Moreover, canonical Wnt signaling in primary cells is unaffected by the loss of Frat. These studies show that Frat is not an essential component of the canonical Wnt pathway in higher organisms, despite the strict requirement of Frat/GBP for maternal Wnt signaling in Xenopus.     

Wnt10b Promotes Differentiation of Mouse Hair Follicle Melanocytes

Previous research has revealed that Wnt10b activates canonical Wnt signaling, which is integral to melanocyte differentiation in hair follicles (HFs). However, the function of Wnt10b in HF melanocytes remains poorly understood. We determined using Dct-LacZ transgenic mice that Wnt10b is mainly expressed near and within melanocytes of the hair bulbs during the anagen stage of the hair cycle. We also found that Wnt10b promotes an increase in melanocyte maturation and pigmentation in the hair bulbs of the mouse HF. To further explore the potential functions of Wnt10b in mouse HF melanocytes, we infected iMC23 cells with Ad-Wnt10b to overexpress Wnt10b. We demonstrated that Wnt10b promotes the differentiation of melanocytes by activating canonical Wnt signaling in melanocytes.     

Identification and Characterization of Wnt Signaling Pathway in Keloid Pathogenesis

Keloid is characterized by fibroblastic cell proliferation and abundant collagen synthesis. Numerous studies have shown that the Wingless type (Wnt) signaling pathways play key roles in various cellular functions including proliferation, differentiation, survival, apoptosis and migration. The aim of this study was to clarify the role of Wnt signaling pathway in keloid pathogenesis. Primary fibroblast cultures and tissue samples from keloid and normal appearing dermis were used. The expression of Wnt family members, frizzled (FZD)4 receptor, receptor tyrosine kinase-like orphan receptor (ROR)2 and the Wnt signaling downstream targets, glycogen synthase kinase (GSK)3-β and β-catenin were assessed using semi-quantitative RT-PCR, Western blot, or immunohistochemical methods. Of the Wnt family members, Wnt5a mRNA and protein levels were elevated in keloid fibroblasts (KF) as compared to normal fibroblasts (NF). A higher expression of β-catenin protein was also found in KF. No detectable levels of FZD4 receptor and ROR2 proteins were observed in both NF and KF. Functional analysis showed that treatment of NF and KF with recombinant Wnt5a peptide resulted in an increase in protein levels of total β-catenin and phosphorylated β-catenin at Ser33/37/Thr 41 but no significant change in phosphorylated β-catenin at Ser45/Thr 41 positions. In addition, the expression of total GSK3-β protein was not affected but its phosphorylated/inactivated form was increased in NF and KF. Our findings highlight a potential role for a Wnt/β-catenin canonical signaling pathway triggered by Wnt5a in keloid pathogenesis thereby providing a new molecular target for therapeutic modulations.     

Tankyrase is necessary for canonical Wnt signaling during kidney development

Recent studies using small molecule antagonists have revealed that the poly(ADP‐ribose) polymerases (PARPs) Tankyrase 1 and 2 are critical regulators of canonical Wnt signaling in some cellular contexts. However, the absence of any activity during zebrafish embryogenesis suggested that the tankyrases may not be general/core components of the Wnt pathway. Here, we show that Tnks1 and 2 are broadly expressed during mouse development and are essential during kidney and lung development. In the kidney, blockage of tankyrase activity phenocopies the effect of blocking production of all Wnt ligands. Tankyrase inhibition can be rescued by activation of β‐catenin demonstrating its specificity for the Wnt pathway. In addition, treatment with tankyrase inhibitors appears to be completely reversible in some cell types. These studies suggest that the tankyrases are core components of the canonical Wnt pathway and their inhibitors should enjoy broad usage as antagonists of Wnt signaling. Developmental Dynamics 239:2014–2023, 2010 © 2010 Wiley‐Liss, Inc.     

PX-RICS mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex

Cadherins mediate Ca2+-dependent cell-cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with beta-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a beta-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/beta-catenin complex in the ER and ER exit site, resulting in a decrease in cell-cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with beta-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner gamma-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/beta-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling.     

Active Wnt/beta-catenin signaling is required for embryonic thymic epithelial development and functionality ex vivo

The Wnt/beta-catenin signaling pathway plays an important role in the commitment and development of thymic epithelial precursors. Here we document similarities of thymic epithelial development during embryogenesis in human and mouse. We stained for thymic epithelial surface markers (EpCAM1, Ly51, K8) and ligand/receptor pair (Wnt4, Fz4). Our results confirm the relevance of using murine test systems to model human embryonic thymic epithelial cell development.     

β-catenin expression in benign and malignant pleural disorders

Benign and malignant pleural processes display a large and overlapping spectrum of morphological appearances, and can be difficult to distinguish, histologically, from each other. β-catenin, a participant in the wingless-type (Wnt) transduction pathway, is involved in the pathogenesis of malignant mesothelioma and has received limited evaluation for its ability to serve as a diagnostic aid for distinguishing between individual pleural disorders. We performed immunohistochemistry for β-catenin on 10 pleural malignant mesotheliomas, 10 examples of mesothelial hyperplasia and 18 cases of organizing pleuritis. Although differences were noted in staining intensity between the mesothelioma and mesothelial hyperplasia groups, extensiveness and cellular location were similar. Staining intensity (mean +/- s.d.) in mesotheliomas (2.00 +/- 0.67) was significantly less intense than in mesothelial hyperplasia cases (3.00 +/- 0.00) (p=0.0005). Stromal cell staining was cytoplasmic in all cases, and endothelial cell staining was membranous, submembranous and cytoplasmic. Nuclear expression of β-catenin was not observed in any of the cases studied. This lack of nuclear staining in the stromal cells of organizing pleuritis differs markedly from the previously reported high frequencies of nuclear β-catenin expression in other pleural spindle cell proliferations (desmoid tumors and solitary fibrous tumors). In summary, the current study adds to previous work indicating a role for β-catenin in the genesis of pleural conditions including organizing pleuritis, mesothelial hyperplasia and malignant mesothelioma. Although IHC for β-catenin does not appear to be conclusive for separating benign from malignant mesothelial proliferations, it may be valuable for assisting in the differential diagnosis of mesothelial and spindle cell proliferations in the pleura.     

CTNNB1, AXIN1 and APC expression analysis of different medulloblastoma variants

OBJECTIVES:

We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatric brain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor.

METHODS:

Sixty-one medulloblastoma cases were analyzed for β-catenin gene (CTNNB1) mutations, β-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. All data were correlated with histological subtypes and patient clinical information.

RESULTS:

CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3β phosphorylation sites, which participate in β-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear β-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1) were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants.

CONCLUSIONS:

A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of β-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularity medulloblastoma variants but not in large-cell medulloblastomas.     

基于Wnt/β-catenin信号通路治疗骨关节炎研究热点及应用前景

文题释义：Wnt信号通路：Wnt是Wingless/Integrated的缩写,由配体蛋白质Wnt和膜蛋白受体结合,激发的一组多下游通道的信号转导的途径,该信号通路在不同的动物物种间极为相似,遗传学上具有高度保守性。通过该途径,细胞表面受体胞内段将活化,将细胞外的信号传递到细胞内。表现为两种信号传导方式,分别是胞间交流(旁分泌)和自体细胞交流(自分泌),经典代表有：Wnt/β-catenin信号通路、平面细胞极性通路(Wnt/PCP通路)和Wnt/Ca²⁺通路。 骨硬化蛋白：由Sost基因编码,是一种分泌型半胱氨酸结蛋白,是Wnt共受体和低蛋白脂蛋白相关受体蛋白5 /低蛋白脂蛋白相关受体蛋白6相关受体的拮抗剂,作用于通路的上游,抑制Wnt 信号通路,从而起到软骨保护作用。背景：Wnt/β-catenin信号通路在骨关节炎的发展中起着重要的作用。 目的：基于Wnt/β-catenin信号通路,对骨关节炎的研究进展做一综述。方法：查阅PubMed、中国知网和万方数据库,以“catenin;wnt;osteoarthritis;arthritis;degenerative;arthritides;deformans;pathway;wnt signaling;signaling pathway;wnt signaling pathways;wnt beta catenin signaling pathway;canonical wnt pathway;canonical wnt;骨关节炎”为检索词,分别组合检索,查找Wnt/β-catenin信号通路治疗骨关节炎的文献,最终纳入74篇文献进行分析。结果与结论：基于Wnt/β-catenin信号通路治疗骨关节炎,主要途径有天然的拮抗剂、小分子抑制剂、激动剂、中草药和药物重新定位等方面。药物作用途径通过激活或者抑制Wnt/β-catenin信号通路,对软骨起到保护作用。通过Wnt/β-catenin信号通路治疗骨关节炎,目前处于实验性研究阶段,但具有较好应用前景。如何精准调控通路,更好的转化应用,有望成为未来研究的热点。ORCID: 0000-0002-9553-7171(贝涛) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Parathyroid hormone signaling through low-density lipoprotein-related protein 6

Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of β-catenin. Activation of PKA is essential for PTH-induced β-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of β-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity.     

Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells

Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cortex differentiate into neurons and its possible molecular mechanism is also not clear. Wnt signaling is implicated in the control of cell growth and differentiation during CNS development in animal model, but its action at the cellular level has been poorly understood. In this experiment, we examined neuronal differentiation of NSCs induced by VPA culture media using vitro immunochemistry assay. The neuronal differentiation of NSCs was examined after treated with 0.75 mM VPA for three, seven and ten days. RT-PCR assay was employed to examine the level of Wnt-3α and β-catenin. The results indicated that there were more β-tublin III positive cells in NSCs treated with VPA medium compared to the control group. The expression of Wnt-3α and β-catenin in NSCs treated with VPA medium was significantly greater compared to that of control media. In conclusion, these findings indicated that VPA could induce neuronal differentiation of NSCs by activating Wnt signal pathway.     

Wnt/β-catenin信号通路在骨肉瘤与骨重建及血管生成中的多重话题

文题释义：肿瘤微环境：是指肿瘤的发生、生长及转移与肿瘤细胞所处的内外环境有着密切关系,它不仅包括肿瘤所在组织的结构、功能和代谢,而且亦与肿瘤细胞自身的(核和胞质)内在环境有关。肿瘤细胞可以通过自分泌和旁分泌,改变和维持自身生存和发展的条件,促进肿瘤的生长和发展。上皮间质转化：指上皮到间质细胞的转化,它赋予细胞转移和入侵的能力,包括干细胞特征、减少凋亡与衰老,和促进免疫抑制,不仅在发育过程中起着关键的作用,而且还参与组织愈合、器官纤维化和癌症发生等过程。 背景：Wnt/β-catenin信号通路在骨肉瘤的肿瘤发生和骨重建中起的关键作用,及其在血管生成和免疫监视中的意义,是转移性播散的2个关键机制。 目的：文章就Wnt/β-catenin通路在骨肉瘤发生发展中的作用及其对特定微环境的调控进行综述。方法：检索2000年至2020年为止的PubMed数据库,以“Wnt/β-catenin;bone tumors”为英文检索词,得到Wnt/β-catenin通路与骨肿瘤关系的相关研究。结果与结论：典型的Wnt/β-catenin信号通路通过直接作用于骨肉瘤细胞和间接调节骨肉瘤细胞活性2种方式在骨肉瘤生长和转移过程中起着重要作用。Wnt/β-catenin通路也参与了骨肉瘤细胞对骨肿瘤微环境的劫持,促进细胞外基质的调节,使肿瘤细胞具有侵袭性,诱导肿瘤细胞的免疫耐受和血管生成,最终导致转移扩散的增加。另外,骨肉瘤细胞中典型Wnt信号通路的异常激活似乎与细胞增殖、上皮间质转化样过程的诱导、骨肉瘤促肿瘤细胞干细胞特性的获得以及转移扩散密切相关。ORCID: 0000-0003-0353-4895(刘畅) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

独活寄生汤拆方通过Wnt/β-catenin信号通路抑制软骨细胞炎症反应

文题释义：Wnt/β-catenin信号通路：是一条在生物进化中极为保守的通路。在正常细胞中,β-catenin只是作为一种细胞骨架蛋白在胞膜处与E-cadherin形成复合体对维持同型细胞的黏附、防止细胞的移动发挥作用。只有当细胞外Wnt信号分子与细胞膜上特异性受体Frizzled蛋白结合激活胞内散乱的蛋白导致GSK-3β失活,进而使β-catenin磷酸化降解,当解离的β-catenin达到一定浓度时细胞功能发生异常,表现为病理状态。独活寄生汤拆方：由中药独活、桑槲寄生、防风、秦艽、细辛、肉桂心组成具有祛风湿、止痹痛的祛湿止痛方,处方比例为3︰2︰2︰2︰2︰2。背景：Wnt/β-catenin信号通路在骨关节炎的炎症反应病理过程中扮演着重要角色,而独活寄生汤拆方可有效抑制软骨细胞的炎症反应,但其具体作用机制尚不明确。有待进一步深入研究。目的：探讨独活寄生汤拆方是否可以通过调控Wnt/β-catenin信号通路抑制由脂多糖诱导的软骨细胞炎症反应。方法：①取SD雄性大鼠,采用机械Ⅱ型胶原酶消化法获取膝关节软骨细胞,倒置相差显微镜观察软骨细胞的形态结构,Ⅱ型胶原免疫组化鉴定软骨细胞。②用不同质量浓度的独活寄生汤拆方(300,400,500 mg/L)干预由脂多糖(10 μg/L)诱导的软骨细胞炎症模型,干预8 h后,采用酶联免疫吸附(ELISA)法测定各组软骨细胞培养液中白细胞介素1β、肿瘤坏子因子α水平,确定独活寄生汤拆方干预浓度。③将第2代软骨细胞分为4组：空白组含正常培养液;模型组培养液中含10 μg/L脂多糖;独活寄生汤拆方组培养液中含10 μg/L脂多糖和400 mg/L独活寄生汤拆方;抑制剂组培养液中含10 μg/L脂多糖和10 mg/L Dickkopf-1。干预8 h后,采用Western blot法检测β-catenin、Wnt-4、Frizzled-2、GSK-3β、CKI-ε的蛋白表达量。结果与结论：①软骨细胞鉴定：获取的软骨细胞Ⅱ型胶原免疫组化染色胞浆呈棕黄色,具备软骨细胞典型特征;②ELISA结果显示：模型组培养液中白细胞介素1β和肿瘤坏子因子α水平均高于空白组,400 mg/L独活寄生汤拆方干预后两种炎症因子水平较模型组显著降低,所以确定独活寄生汤拆方干预浓度为     400 mg/L;③Western blot结果显示：模型组β-catenin、Wnt-4、Frizzled-2、CKI-ε的蛋白表达量均高于空白组,而GSK-3β蛋白表达量低于空白组;独活寄生汤拆方组和抑制剂组β-catenin、Wnt-4、Frizzled-2、CKI-ε蛋白表达量低于模型组,而GSK-3β蛋白表达量高于模型组。④结果表明：独活寄生汤拆方可以通过调控Wnt/β-catenin信号通路抑制由脂多糖诱导的软骨细胞炎症反应。ORCID: 0000-0003-3530-5683(李慧)中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Wnt Signaling as a Possible Promoting Factor of Cell Differentiation in Pleomorphic Adenomas

There are well known that Wnt signaling was some roles of cell differentiation at the development tissues, especially the oral and maxillofacial regions of some developmental stages. Therefore, to determine Wnt signaling in the pleomorphic adenoma tissues, we examined. The expression of Wnt1 and β-catenin as well as the distribution of various cytoskeletal proteins CK7 and CK13 was examined in 30 cases of pleomorphic adenoma by immunohistochemistry. Wnt1 was detected in almost all tumor cells. The peripheral columnar cells in squamous metaplasia and small cuboidal cells in duct-like structures were strongly positive to Wnt1. Although β-catenin was clearly localized on the cell membrane of tumor cells, nuclear translocation was observed in small cuboidal cells and in some basaloid cells. The immunofluorescent staining pattern of Wnt1 and CK7 as well as Wnt1 and CK13 was consistent with IHC results. Thus, in pleomorphic adenoma, Wnt is involved in tumor cell differentiation of peripheral columnar cells forming solid nests and small peripheral columnar cells forming duct-like structures. Moreover, among the three currently known Wnt pathways, β-catenin is the suggested pathway working during cell differentiation. Furthermore, peripheral columnar cells in solid tumor nests and in squamous metaplasia are governed by another Wnt pathway other than β-catenin. Therefore, Wnt signaling through β-catenin pathway may be involved in the ''mixed'' differentiation characteristic of pleomorphic adenoma although another pathway may also be possibly working in other parts of the tumor tissue.