Inhibition of canonical Wnt signaling promotes gliogenesis in P0-NSCs

Wnt signaling plays an essential role in the development of mammalian central nervous system. We investigated the impact of activation/inhibition of the Wnt signaling pathway on neuronal/glial differentiation in neurospheres derived from neonatal mouse forebrains. For short term alterations, neurospheres were stimulated with recombinant Wnt-3a, Wnt-5a and the Wnt inhibitor Dickkopf-1 (Dkk1). Furthermore, neurospheres were transduced with retroviral vectors encoding Wnt-3a, Wnt-7a and their inhibitors Dkk1 and soluble Frizzled related protein-5 (sFRP5). Long-term activation of Wnt pathway by Wnt-7a or by treatment with GSK3 inhibitors promoted a moderate increase of the neuronal differentiation and blocked gliogenesis. In contrast, Wnt pathway inhibition in neurospheres, induced by retroviral overexpression of either Dkk1 or sFRP5, robustly increased the gliogenesis at the expense of neurogenesis. In summary, our data demonstrate that activation or inhibition of Wnt/β-catenin signaling in neurospheres regulates neuronal and glial differentiation, respectively. Thus, our results suggest that Wnt signaling may also contribute to regulate these processes in the neonatal brain.     

Isorhamnetin-induced anti-adipogenesis is mediated by stabilization of β-catenin protein

AimsPrevious studies have shown that isorhamnetin has anti-adipogenic effects in mouse 3T3-L1 cells. This study was conducted to elucidate the inhibitory mechanisms of isorhamnetin during adipogenic differentiation of human adipose tissue-derived stem cells (hAMSCs).Main methodsThe effect of isorhamnetin on adipogenic differentiation of hAMSCs was quantified by Oil Red O staining and a triglyceride assay. In addition, real-time PCR and Western blot were used to determine the expression of adipogenesis-related genes.Key findingsIsorhamnetin inhibited the adipocyte differentiation of hAMSCs. Additionally, when the effects of Wnt antagonists that promote adipogenesis were evaluated, isorhamnetin was found to down-regulate the mRNA levels of sFRP1 and Dkk1, but had no effect on the mRNA levels of sFRP2, sFRP3, sFRP4 and Dkk3. Isorhamnetin also inhibited the expression of Wnt receptor and co-receptor genes. Furthermore, isorhamnetin increased the protein levels of β-catenin, an effector molecule of Wnt signaling, but had no effect on the mRNA levels of β-catenin. The phosphorylation level of GSK 3β was also increased by isorhamnetin. These results were confirmed by the fact that the expression of c-myc, cyclin D1 and PPARδ, which are target genes of β-catenin, was upregulated by isorhamnetin. Moreover, isorhamnetin reduced the mRNA expression levels of C/EBPα and PPARγ, which are known to be inhibited by c-myc or by cyclin D1 and PPARδ, respectively.SignificanceOur results indicate that isorhamnetin inhibits the adipogenic differentiation of hAMSCs and that its mechanisms are mediated by the stabilization of β-catenin.     

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/β-catenin signalling

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise β-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3], [4], [5] and [6], the effect of other Dkks on the Wnt/β-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/β-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/β-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.     

The roles of Wnt antagonists Dkk1 and sFRP4 during adipogenesis of human adipose tissue‐derived mesenchymal stem cells

Abstract. Objectives: The canonical Wnt signalling pathway performs an important function in the control of adipogenesis. However, the mechanisms and mediators underlying these interactions have yet to be defined in detail. Thus, this study was performed in order to elucidate the roles of the Wnt family during adipogenic differentiation of human adipose tissue‐derived mesenchymal stem cells (hAMSCs). Materials and methods: We assessed several members of the Frizzled (FZD) family, the receptors of Wnts, inhibitors including the secreted frizzled‐related protein (sFRP) family and Dickkopfs (Dkks), and the downstream factor, β‐catenin. Expressional levels of adipogenic markers regulated by the small interfering RNA of Dkk1 (siDkk1) and sFRP4 (sisFRP4) were assessed using real‐time quantitative PCR and Western blot analysis. Results: The mRNA level of Dkk1 was expressed abundantly in the early stages of adipogenesis and decreased rapidly during the late stages of adipogenesis. However, sFRP4 mRNA was up‐regulated gradually during adipogenic differentiation in hAMSCs. Expression of FZD1, FZD7 and β‐catenin were reduced during adipogenic differentiation. Transfection of hAMSCs with siDkk1 or sisFRP4 partially inhibited differentiation of hAMSCs into adipocytes and restored levels of β‐catenin. Conclusions: We determined that Dkk1 was up‐regulated transiently in the early stages of adipogenesis, and that sFRP4 levels increased gradually during adipogeneis via inhibition of Wnt signalling. Collectively, these results show that Dkk1 and sFRP4 perform an important function in adipogenesis in hAMSCs.     

Wnt3a binds to several sFRPs in the nanomolar range

Secreted Frizzled-related proteins (sFRPs) are modulators of the Wnt signaling pathway that plays important roles in both embryogenesis and oncogenesis. sFRPs have been proposed to antagonize Wnt activity by binding to Wnts. However, the affinity of this binding is unknown. Here we show, using surface plasmon resonance and purified proteins, that sFRP1, sFRP2, sFRP4, and Frzb bind directly to Wnt3a with affinities in the nanomolar range. However, only sFRP1 and sFRP2 antagonize Wnt3a activity by blocking Wnt3a induced β-catenin accumulation in L cells. Furthermore, sFRP2, but not Frzb, antagonizes Wnt3a signaling in an ES cell model of mesoderm differentiation. These results provide the first measurement of binding affinity of sFRPs for a Wnt, which together with the measurement of antagonistic activity of sFRPs could help understand how sFRPs regulate Wnt signaling.     

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.     

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/beta-catenin signalling

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise beta-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3-6], the effect of other Dkks on the Wnt/beta-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/beta-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/beta-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.     

The COP9 Signalosome Mediates β-Catenin Degradation by Deneddylation and Blocks Adenomatous Polyposis coli Destruction via USP15

The Wnt/β-catenin signalling pathway has important roles in normal cellular proliferation, development and angiogenesis. Many malignant transformations, including sporadic colorectal tumours, are caused by constitutive activation of the Wnt route due to mutations in the tumour suppressor protein adenomatous polyposis coli (APC) or the β-catenin oncogene, ultimately resulting in reduced β-catenin degradation by the ubiquitin (Ub) proteasome system (UPS). The COP9 signalosome (CSN) regulates the UPS by controlling cullin-RING Ub ligases (CRLs). We show here that the CSN and the β-catenin destruction complex cooperate in targeting β-catenin for degradation by the UPS. Together with the CRL that ubiquitinates β-catenin, they form a supercomplex responsible for β-catenin degradation. Wnt3A, glycogen synthase kinase 3β inhibitors or mutation of CSN-mediated deneddylation induce the disassembly of the supercomplex and the accumulation of β-catenin. Likewise, downregulation of the CSN in HeLa cells leads to retarded degradation of β-catenin. Additionally, we found that the knockdown of the CSN causes accelerated proteolysis of APC, an essential component of the β-catenin destruction complex, which is degraded by the UPS as β-catenin. We show here that APC is stabilised by the Ub-specific protease 15 (USP15) associated with the CSN. This is demonstrated by over-expression of siRNA oligonucleotides against USP15 or by over-expression of an USP15 mutant, which is unable to degrade poly-Ub chains. Thus, the CSN controls the Wnt/β-catenin signalling by assisting the assembly of β-catenin-degrading supercomplexes by deneddylation and, simultaneously, by stabilising APC via CSN-associated USP15. The CSN regulates the balance between β-catenin and APC. Disturbance of this balance can cause cancer by driving cell transformation, tumour angiogenesis and metastasis. A model is provided that proposes a role of CSN-mediated deneddylation in the formation of the β-catenin-degrading supercomplex and the protection of complex-bound APC via CSN-associated USP15.     

WIF-1研究进展

WIF-1是Wnt信号通路上的拮抗物之一,可以阻断Wnt的经典通路和非经典通路。目前在人类多种肿瘤的研究发现WIF-1表达异常。WIF-1（Wnt inhibitory factor-1）,sFRP（Frizzled related protein）和CER（Cerberus）属于Wnt拮抗物家族,通过直接与Wnt蛋白相连从而阻止Wnt与受体蛋白复合物相连,使细胞质中的β-catenin由于磷酸化而不能积累,进而阻断了经典通路和非经典通路。WIF-1可能与中胚层的发生以及肿瘤细胞的生长分化有关。Wnt家族其他成员已经被证实与早期冠心病、Ⅱ型糖尿病、肥胖症、骨质疏松症等相关。因此了解WIF-1在通路上的更多信息,解释WIF-1调节Wnt信号通路的机理和过程,为疾病治疗和预防以及药物开发提供新的方法。     

Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/β-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and β-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.     

Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed

Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin. In normal development, Wnt5a is secreted and directs the migration of target cells along concentration gradients. The effect of Wnt5a on target cells is regulated by many factors, including the expression level of inhibitors and receptors. Dysregulated Wnt5a signalling facilitates invasion of multiple tumor types into adjacent tissue. However, the expression and distribution of Wnt5a in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as the effect of Wnt5a on keratinocyte migration has not been studied in detail to date. We here report that Wnt5a is upregulated in SCC and BCC and localised to the leading edge of tumors, as well as tumor-associated fibroblasts. The Wnt5a-triggered bundling of its receptor Fzd3 provides evidence of Wnt5a concentration gradients projecting into the tumor. In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient. Expression profiling of the Wnt pathway shows that the upregulation of Wnt5a in SCC is coupled to repression of canonical Wnt signalling. This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2. Since both types of Wnt signalling act mutually antogonistically at multiple levels, the concurrent repression of canonical Wnt signalling suggests hyper-active Wnt5a signal transduction. Significantly, this combination of gene dysregulation is not observed in the benign hyperproliferative inflammatory skin disease psoriasis. Collectively, our data strongly suggest that Wnt5a signalling contributes to tissue invasion by non-melanoma skin cancer.     

A genetic study of the role of the Wnt/beta-catenin signalling in Paneth cell differentiation

Wnt/β-catenin signalling plays a key role in the homeostasis of the intestinal epithelium. Whereas its role in the maintenance of the stem cell compartment has been clearly demonstrated, its role in the Paneth cell fate remains unclear. We performed genetic studies to elucidate the functions of the Wnt/β-catenin pathway in Paneth cell differentiation. We analysed mice with inducible gain-of-function mutations in the Wnt/β-catenin pathway and mice with a hypomorphic β-catenin allele that have not been previously described. We demonstrated that acute activation of Wnt/β-catenin signalling induces de novo specification of Paneth cells in both the small intestine and colon and that colon cancers resulting from Apc mutations expressed many genes involved in Paneth cell differentiation. This suggests a key role for the Wnt/β-catenin pathway in Paneth cell differentiation. We also showed that a slight decrease in β-catenin gene dosage induced a major defect in Paneth cell differentiation, but only a modest effect on crypt morphogenesis. Overall, our findings show that a high level of β-catenin activation is required to determine Paneth cell fate and that fine tuning of β-catenin signalling is critical for correct Paneth cell lineage.     

Secreted Frizzled-related protein potentiation versus inhibition of Wnt3a/β-catenin signaling

Wnt signaling regulates a variety of cellular processes during embryonic development and in the adult. Many of these activities are mediated by the Frizzled family of seven-pass transmembrane receptors, which bind Wnts via a conserved cysteine-rich domain (CRD). Secreted Frizzled-related proteins (sFRPs) contain an amino-terminal, Frizzled-like CRD and a carboxyl-terminal, heparin-binding netrin-like domain. Previous studies identified sFRPs as soluble Wnt antagonists that bind directly to Wnts and prevent their interaction with Frizzleds. However, subsequent observations suggested that sFRPs and Frizzleds form homodimers and heterodimers via their respective CRDs, and that sFRPs can stimulate signal transduction. Here, we present evidence that sFRP1 either inhibits or enhances signaling in the Wnt3a/β-catenin pathway, depending on its concentration and the cellular context. Nanomolar concentrations of sFRP1 increased Wnt3a signaling, while higher concentrations blocked it in HEK293 cells expressing a SuperTopFlash reporter. sFRP1 primarily augmented Wnt3a/β-catenin signaling in C57MG cells, but it behaved as an antagonist in L929 fibroblasts. sFRP1 enhanced reporter activity in L cells that were engineered to stably express Frizzled 5, though not Frizzled 2. This implied that the Frizzled expression pattern could determine the response to sFRP1. Similar results were obtained with sFRP2 in HEK293, C57MG and L cell reporter assays. CRD_sFRP1 mimicked the potentiating effect of sFRP1 in multiple settings, contradicting initial expectations that this domain would inhibit Wnt signaling. Moreover, CRD_sFRP1 showed little avidity for Wnt3a compared to sFRP1, implying that the mechanism for potentiation by CRD_sFRP1 probably does not require an interaction with Wnt protein. Together, these findings demonstrate that sFRPs can either promote or suppress Wnt/β-catenin signaling, depending on cellular context, concentration and most likely the expression pattern of Fzd receptors.     

Dkk1 regulates ventral midbrain dopaminergic differentiation and morphogenesis

Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1(+/-) embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1(-/-) embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis.