外源性Wnt3a持续作用对小鼠胚胎干细胞Wnt／β-catenin信号通路的调控作用

目的观察Wnt/β-catenin信号通路是否在体外以外源性Wnt3a持续作用小鼠胚胎干细胞后被激活,并进一步调控该通路下游基因的表达。方法应用外源性Wnt3a持续作用ES-E14TG2a小鼠胚胎干细胞21d,通过细胞免疫荧光及Western Blotting检测细胞内β-catenin蛋白,以观察该蛋白的胞内积聚情况;同时QRT-PCR检测WNT下游靶标基因的表达量,采用完全随机F检验并用LSD法进行两两比较,来确定经典WNT／β-catenin信号通路是否被激活。结果ES-E14TG2a小鼠胚胎干细胞经Wnt3a连续培养21d后,β-catenin蛋白的细胞荧光明显较强,而对照组中的荧光强度较弱,说明细胞内β-catenin蛋白没有被降解而是在胞内大量积累;Western Blotting检测结果显示Wnt3a连续培养21d后ES-E14TG2a细胞内β-catenin蛋白条带明显比空白对照的蛋白条带粗;ES—E14TG2a细胞经wnt3a培养后Pitx2、Frizzled、Sox17的表达量均持续上升,Pitx2在培养7d、14d、21d分别为4．17±0．20、7．27±0．35、8．59±0．21（F=222．757,P=0．000）;Frizzled在培养7d、14d、21d分别为1．01±0．06、2．93±0．22、5．44±0．30（F=302．703,P=0．000）;Sox17在培养7d、14d、21d分别为8．45±0．41、18．35±0．17、34．93±0．16（F=7217．083,P=0．000）;Oct4培养到7d、14d的表达量持续增加分别为1．22±0．21、1．56±0．04,而连续培养21d后Oct4基因的表达量下降为1．15±0．07（F=8．827,P=0．016）。结论Wnt3a持续作用可激活Wnt／β-catenin信号通路,并调控下游基因的表达。     

Lithium chloride promotes proliferation of neural stem cells in vitro,possibly by triggering the Wnt signaling pathway

The objective of this study was to clarify the relationship between the effect and associated mechanisms of lithium chloride on neural stem cells (NSCs) and the Wnt signaling pathway. The expression of key molecules proteins related to the Wnt signaling pathway in the proliferation and differentiation of control NSCs and lithium chloride-treated NSCs was detected by Western blot analysis. Flow cytometry analysis was applied to study the cell cycle dynamics of control NSCs and NSCs treated with lithium chloride. The therapeutic concentrations of lithium chloride stimulated NSC proliferation. β-catenin expression gradually decreased, while Gsk-3β expression gradually increased (P?P?in vitro and preventing the cells from differentiating, which is potentially mediated by activation of the Wnt signaling pathway.     

Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.     

ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled

Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/β-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with β-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/β-catenin signaling pathway by phosphorylating Dsh.     

Inhibition of ClC-5 suppresses proliferation and induces apoptosis in cholangiocarcinoma cells through the Wnt/β-catenin signaling pathway

Chloride channel-5 (ClC-5), an important branch of the ClC family, is involved in the regulation of the proliferation and cell-fate of a variety of cells, including tumor cells. However, its function in cholangiocarcinoma (CCA) cells remains enigmatic. Here, we discovered that ClC-5 was up-regulated in CCA tissues and CCA cell lines, while ClC-5 silencing inhibited CCA cell proliferation and induced apoptosis. Further mechanism studies revealed that ClC-5 inhibition could inhibit Wnt/β-catenin signaling activity and further activate the mitochondria apoptotic pathway in CCA cells. Furthermore, rescuing Wnt/β-catenin signaling activation eliminated the anti-tumor function of ClC-5 knockdown. Together, our research findings illustrated that ClC-5 inhibition plays an anti-tumor role in CCA cells via inhibiting the activity of the Wnt/β-catenin pathway, which in turn activates the mitochondrial apoptotic pathway.     

Resveratrol targeting the Wnt signaling pathway: A focus on therapeutic activities

Wingless-type MMTV integration site (Wnt) signaling pathway is considered as an important pathway regulating a variety of biological processes such as tissue formation and homeostasis, cell proliferation, cell migration, cell differentiation, and embryogenesis. Impairment in the Wnt signaling pathway is associated with pathological conditions, particularly cancer. So, modulation of this pathway can be considered as a promising strategy and several drugs have been developed in line with this strategy. Resveratrol (Res) is a naturally occurring nutraceutical compound exclusively found in different fruits and nuts such as grape, peanut, and pistachio. This compound has favorable biological and therapeutic activities such as antioxidant, anti-inflammatory, antitumor, hepatoprotective, cardioprotective, and antidiabetic. At the present review, we demonstrate how Res modulates Wnt signaling pathway to exert its pharmacological effects.     

Down-regulation of SLC35C1 induces colon cancer through over-activating Wnt pathway

The canonical Wnt signalling pathway is a critical pathway involved in the proliferation of cells. It has been well-established that it plays the central role during colorectal carcinogenesis and development. Yet the exact molecular mechanism of how the canonical Wnt pathway is fine-tuned remains elusive. We found that SLC35C1, a GDP-fucose transporter, negatively regulates the Wnt signalling pathway. We show here that SLC35C1 is reduced in all colon cancer by both immunohistochemistry images and TCGA data, whereas β-catenin is increased. Down-regulation of SLC35C1 is also detected by real-time PCR in stage 3 and stage 4 colorectal cancer tissues. Moreover, analysing the TCGA database with cBioPortal reveals the negative correlation of SLC35C1 mRNA level to the expression of β-catenin. Reduced SLC35C1 significantly promotes cell proliferation and colony formation of HEK293 cells. Meanwhile, in HEK293 cells silencing SLC35C1 activates canonical Wnt pathway, whereas overexpressing SLC35C1 inhibits it. Consistently, the reduction of SLC35C1 in HEK293 cells also elevated the mRNA level of Wnt target genes C-myc, Axin2 and Cyclin D1, as well as the secretion of Wnt3a. In conclusion, we identified SLC35C1 as a negative regulator of the Wnt signalling pathway in colon cancer. Decreased SLC35C1 may cause over-activation of Wnt signalling in colorectal cancer.     

Wnt signaling associated small molecules improve the viability of pPSCs in a PI3K/Akt pathway dependent way

Although we have obtained porcine pluripotent stem cell lines (pPSCs) from blastocysts, the cells exhibit flat clonal morphology and do not support single-cell passage. There is massive cell death after cell dissociation, and the efficiency of single-cell colony is generally ≤10%. In a recent study, we got a new pPSCs using two Wnt signaling pathway regulators CHIR99021 and XAV939. This cell had strong biological viability, small-domed morphology, and its cloning efficiency after dissociation was 80–90%. The CH/XAV-treated cells expressed elevated levels of pluripotent genes, and possessed differentiation abilities both in vitro and in vivo, proven by the formation of embryonic bodies and teratomas with three germ layers. Furthermore, we found that the combinative use of CHIR99021 and XAV939 resulted in β-catenin-maintained expression in the cytoplasm but not translocation to the nuclei for WNT/TCF activation. In the meanwhile, E-cadherin located on the cell membrane, thereby activated the PI3K/Akt signaling pathway to enhance the pluripotency of the cells. Our study obtained new pPSCs, which were even closer to the naïve state with only two small molecule inhibitors, and the improved pluripotency of pPSCs could facilitate transgenic manipulation and regenerative medicine research. Besides, our study casted a light on the understanding of pPSCs and the derivation of authentic porcine embryonic stem cells.     

Role of Wnt signaling in fracture healing

The Wnt signaling pathway is well known to play major roles in skeletal development and homeostasis. In certain aspects, fracture repair mimics the process of bone embryonic development. Thus, the importance of Wnt signaling in fracture healing has become more apparent in recent years. Here, we summarize recent research progress in the area, which may be conducive to the development of Wnt-based therapeutic strategies for bone repair. [BMB Reports 2014; 47(12): 666-672]     

Berberine ameliorates DSS-induced intestinal mucosal barrier dysfunction through microbiota-dependence and Wnt/β-catenin pathway

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colon, and it has become one of the world-recognized medical problems as it is recurrent and refractory. Berberine (BBR) is an effective drug for UC treatment. However, the underlying mechanism and targets remain obscure. In this study, we systematically investigated the therapeutic effect and its mechanism of BBR in ameliorating DSS-induced mouse colitis. Expectedly, the colon inflammation was significantly relieved by BBR, and microbiota depletion by antibiotic cocktail significantly reversed the therapeutic effect. Further studies showed that BBR can regulate the abundance and component of bacteria, reestablish the broken chemical and epithelial barriers. Meanwhile, BBR administration dramatically decreased ILC1 and Th17 cells, and increased Tregs as well as ILC3 in colonic tissue of DSS-induced mice, and it was able to regulate the expression of various immune factors at the mRNA level. Moreover, a proteomic study revealed that Wnt/β-catenin pathway was remarkably enhanced in colonic tissue of BBR-treated mice, and the therapeutic effect of BBR was disappeared after the intervention of Wnt pathway inhibitor FH535. These results substantially revealed that BBR restores DSS-induced colon inflammation in a microbiota-dependent manner, and BBR performs its protective roles in colon by maintaining the structure and function of the intestinal mucosal barrier, regulating the intestinal mucosal immune homeostasis and it works through the Wnt/β-catenin pathway. Importantly, these findings also provided the proof that BBR serves as a potential gut microbiota modulator and mucosal barrier protector for UC prevention and therapy.     

IDKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.