Wnt/beta-catenin pathway activation and myogenic differentiation are induced by cholesterol depletion

Myogenic differentiation is a multistep process that begins with the commitment of mononucleated precursors that withdraw from cell cycle. These myoblasts elongate while aligning to each other, guided by the recognition between their membranes. This step is followed by cell fusion and the formation of long and striated multinucleated myotubes. We have recently shown that cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) induces myogenic differentiation by enhancing myoblast recognition and fusion. Here, we further studied the signaling pathways responsible for early steps of myogenesis. As it is known that Wnt plays a role in muscle differentiation, we used the chemical MbetaCD to deplete membrane cholesterol and investigate the involvement of the Wnt/beta-catenin pathway during myogenesis. We show that cholesterol depletion promoted a significant increase in expression of beta-catenin, its nuclear translocation and activation of the Wnt pathway. Moreover, we show that the activation of the Wnt pathway after cholesterol depletion can be inhibited by the soluble protein Frzb-1. Our data suggest that membrane cholesterol is involved in Wnt/beta-catenin signaling in the early steps of myogenic differentiation.     

Nodal signaling in Xenopus gastrulae is cell-autonomous and patterned by beta-catenin

The classical three-signal model of amphibian mesoderm induction and more recent modifications together propose that an activin-like signaling activity is uniformly distributed across the vegetal half of the Xenopus blastula and that this activity contributes to mesoderm induction. In support of this, we have previously shown that the activin-response element (DE) of the goosecoid promoter is uniformly activated across the vegetal half of midgastrula-stage embryos. Here, we further examine the nature of this activity by measuring DE activation by endogenous signals over time. We find that the spatiotemporal pattern of DE activation is much more dynamic than was previously appreciated and also conclude that DE(6X)Luc activity reflects endogenous nodal signaling in the embryo. Using both the DE(6X)Luc construct and endogenous Xbra and Xgsc expression as read-outs for nodal activity, and the cleavage-mutant version of Xnr2 (CmXnr2) to regionally suppress endogenous nodal activity, we demonstrate that nodal signals act cell-autonomously in Xenopus gastrulae. Nodal-expressing cells are unable to rescue either reporter gene activation or target gene expression in distant nodal-deficient cells, suggesting that nodals function at short range in this context. Finally, we show that DE activation by endogenous signals occurs in the absence of dorsal beta-catenin-mediated signaling, but that the timing of dorsal initiation is altered. We conclude that nodal signals in Xenopus gastrulae function cell autonomously at short ranges and that the spatiotemporal pattern of this signaling along the dorsoventral axis is regulated by maternal Wnt-like signaling.     

Novel genes involved in canonical Wnt/beta-catenin signaling pathway in early Ciona intestinalis embryos

We report here characterization of five genes for novel components of the canonical Wnt/ β -catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss-of-function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci-PGAP1 , a Ciona orthologue of human PGAP1 , which encodes GPI (glycosylphosphatidylinositol) inositol-deacylase, Ci-ZF278 , a gene encoding a C2H2 zinc-finger protein, Ci-C10orf11 , a Ciona orthologue of human C10orf11 that encodes a protein with leucine-rich repeats, Ci-Spatial/C4orf17 , a single counterpart for two human genes Spatial and C4orf17 , and Ci-FLJ10634 , a Ciona orthologue of human FLJ10634 that encodes a member of the J-protein family. Knockdown of each of the genes mimicked β -catenin knockdown and resulted in suppression of the expression of β -catenin downstream genes ( Ci-FoxD , Ci-Lhx3 , Ci-Otx and Ci-Fgf9/16/20 ) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild-type, of Ci- β -catenin. Dosage-sensitive interactions were found between Ci-β-catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci- β -catenin in the Wnt/ β -catenin signaling pathway in early Ciona embryos.     

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.     

Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung

Branching morphogenesis in the lung serves as a model for the complex patterning that is reiterated in multiple organs throughout development. Beta-catenin and Wnt signaling mediate critical functions in cell fate specification and differentiation, but specific functions during branching morphogenesis have remained unclear. Here, we show that Wnt/beta-catenin signaling regulates proximal-distal differentiation of airway epithelium. Inhibition of Wnt/beta-catenin signaling, either by expression of Dkk1 or by tissue-specific deletion of beta-catenin, results in disruption of distal airway development and expansion of proximal airways. Wnt/beta-catenin functions upstream of BMP4, FGF signaling, and N-myc. Moreover, we show that beta-catenin and LEF/TCF activate the promoters of BMP4 and N-myc. Thus, Wnt/beta-catenin signaling is a critical upstream regulator of proximal-distal patterning in the lung, in part, through regulation of N-myc, BMP4, and FGF signaling.     

Beta-catenin-dependent Wnt signaling in apical ectodermal ridge induction and FGF8 expression in normal and limbless mutant chick limbs

The fibroblast growth factor (FGF) and beta-catenin-dependent Wnt signaling pathways are key regulators of vertebrate limb development. FGF10 induces expression of Wnt3a, which regulates the formation and FGF8 expression of the apical ectodermal ridge (AER). In amelic limbless limbs, an AER fails to form and FGF8 is not expressed, despite expression of FGF10. It has been found that Wnt3a is initially expressed in limbless ectoderm, although subsequently is drastically reduced. In addition, changes in the expression pattern or level of several Frizzled receptors, Axin, Lef1/Tcf1 and beta-catenin have been found in limbless limbs. Notably, while normal wing buds respond to LiCl-stimulated activation of beta-catenin-dependent signaling by forming ectopic, FGF8-expressing AER, LiCl was unable to induce an AER in limbless wing buds. The results of this study suggest that the limbless gene is required for beta-catenin-dependent Wnt signaling in limb ectoderm leading to FGF8 expression and AER formation.     

N-glycosylation at Asn residues 554 and 566 of E-cadherin affects cell cycle progression through extracellular signal-regulated protein kinase signaling pathway

E-cadherin, which has a widely acknowledged role in mediating calcium-dependent cell-cell adhesion between epithelial cells, also functions as a tumor suppressor. The ectodomain of human E-cadherin contains four potential Nglycosylation sites at Asn residues 554, 566, 618, and 633. We investigated the role of E-cadherin N-glycosylation in cell cycle progression by site-directed mutagenesis. We showed previously that all four potential N-glycosylation sites of E-cadherin w ere N-gly cosylated in human breast carcinoma MDA-MB-435 cells. Removal of N-glycan at Asn633 dramatically affected E-cadherin stability. In this study we showed that E-cadherin mutant missing N-glycans at AsnS54, Asn566 and Asn618 failed to induce cell cycle arrest in Gt phase and to suppress cell proliferation in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn554 and Asn566, but not at Asn618, seemed to be indispensable for E-cadherin-mediated suppression of cell cycle progression. Removal of N-glycans at either Asn554 or Asn566 of E-cadherin was accompanied with the activation of the extracellular signal-regulated protein kinase signaling pathway. After treatment with PD98059, an inhibitor of the extracellular signal-regulated protein kinase signaling pathway, wild-type E-cadherin transfected MDA-MB-435 and E-cadherin N-glycosylation-deficient mutant transfected MDA-MB-435 cells had equivalent numbers of cells in G1 phase. These rmdings implied that N-glycosylation might be crucial for E-cadherin-mediated suppression of cell cycle progression.     

Activation of the canonical Wnt pathway by the antipsychotics haloperidol and clozapine involves dishevelled-3

Protein kinase B (Akt), glycogen synthase kinase-3 (GSK-3) and members of the Wnt signal transduction pathway were recently found to be altered in schizophrenia and targeted by antipsychotic drugs. In the current study, selected Wnt signalling proteins were investigated to determine if they are altered by the antipsychotics clozapine or haloperidol in the rat prefrontal cortex. Pheochromocytoma (PC12) and neuroblastoma (SH-SY5Y) cells were also used to elucidate how antipsychotics generated the pattern of changes observed in vivo . Western blotting (WB) revealed that treatment with haloperidol or clozapine caused an up-regulation of Wnt-5a, dishevelled-3, Axin, total and phosphorylated GSK-3 and β-catenin protein levels. Treatment of PC12 and SH-SY5Y cells with a variety of pharmacological agents as well as the over-expression of several Wnt related proteins failed to mimic the pattern observed in vivo following antipsychotic treatment. However, the over-expression of dishevelled-3 nearly perfectly duplicated the changes observed in vivo . Immunoprecipitations (IP) conducted using protein isolated from the rat prefrontal cortex indicated that dishevelled-3 is associated with the D₂ dopamine receptor thereby suggesting that antipsychotics may act on dishevelled-3 via D₂ dopamine receptors to initiate a cascade of downstream changes involving Axin, GSK-3 and β-catenin that may help to alleviate psychosis in schizophrenic patients.     

Effects of rat Axin domains on axis formation in Xenopus embryos

Wnt signaling plays an important role in axis formation in early vertebrate development. Axin is one Wnt signaling regulator that inhibits this pathway. The effects of the injection of mRNA of several rat Axin (rAxin) mutants on axis formation in Xenopus embryos were examined. It was found that rAxin mutants containing only a regulation of G-protein signaling (RGS) domain fragment or with deletion of the RGS domain induced axis formation. Because the RGS domain is a major adenomatous polyposis coli gene product (APC)-binding domain, APC association with glycogen synthase kinase 3beta (GSK3beta) on the Axin molecule may be important in inhibition of axis formation. The ventralizing activities of wild-type rAxin and a mutant in which the Dishevelled and Axin (DIX) domain was deleted (deltaDIX mutant) were examined. Histological examination and gene expression revealed that the ventralizing activity of the deltaDIX mutant was weaker than that of wild-type rAxin. This finding suggests that the C-terminus of rAxin contributes to the inhibition of Wnt signaling in Xenopus embryos. Furthermore, an rAxin mutant that contained both the RGS and GSK3beta-binding domains affected both the dorsal and ventral sides of blastomeres, mediated ectodermal fate and induced expansion of notochord and/or endoderm, but did not induce axis formation.     

The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

To better understand the role of the canonical Wnt signaling pathway in cartilage development, we adenovirally expressed a constitutively active (ca) or a dominant negative (dn) form of lymphoid enhancer factor-1 (LEF-1), the main nuclear effector of the pathway, in undifferentiated mesenchymal cells, chondrogenic cells, and primary chondrocytes, and examined the expression of markers for chondrogenic differentiation and hypertrophy. caLEF-1 and LiCl, an activator of the canonical pathway, promoted both chondrogenic differentiation and hypertrophy, whereas dnLEF-1 and the gene silencing of beta-catenin suppressed LiCl-promoted effects. To investigate whether these effects were dependent on Sox9, a master regulator of cartilage development, we stimulated Sox9-deficient ES cells with the pathway. caLEF-1 and LiCl promoted both chondrogenic differentiation and hypertrophy in wild-type, but not in Sox9-deficient, cells. The response of Sox9-deficient cells was restored by the adenoviral expression of Sox9. Thus, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner.     

The Wnt signaling pathway has tumor suppressor properties in retinoblastoma

Retinoblastoma is a pediatric retinal tumor caused by mutational inactivation of the tumor suppressor pRb. Additional genetic changes, as yet unidentified, are believed to be required for tumor initiation. Mutations in the Wnt signaling pathway have been implicated in the pathogenesis of many cancers. Multiple Wnt pathway genes are expressed in the retina and the pRb and Wnt pathways interact biochemically, raising the possibility that alterations in the Wnt pathway contribute to retinoblastoma. Our studies showed that Wnt signaling activation significantly decreased the viability of retinoblastoma cell lines by inducing cell cycle arrest, which was associated with upregulated p53. Furthermore, immunolocalization of the Wnt signaling mediator beta-catenin in human and mouse retinoblastoma tissue indicated that canonical Wnt signaling is suppressed in tumors in vivo. These studies are consistent with the Wnt pathway acting as a tumor suppressor in retinoblastoma and suggest that loss of Wnt signaling is tumorigenic in the retina.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

Negative regulation of beta-catenin/Tcf signaling by naringenin in AGS gastric cancer cell

Functional activation of beta-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against beta-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism was related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the beta-catenin distribution and the levels of nuclear beta-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits beta-catenin/Tcf signaling in gastric cancer with unknown mechanisms.     

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.     

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.     

The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm

In the current analysis, we have investigated both the cytoskeletal and signaling roles of beta-catenin during the early phases of lens development using conditional loss- and gain-of-function strategies. Conditional loss of beta-catenin in the presumptive lens does not perturb the normal sequential appearance of lens fate markers but results in a dramatic failure of the coordinated epithelial cell behavior that constitutes lens morphogenesis. Similarly, loss-of-function for Lrp6, the Wnt pathway coreceptor expressed in the eye primordium, does not prevent expression of lens induction markers. Surprisingly, conditional deletion of beta-catenin in periocular ectoderm results in the formation of Prox-1 and beta-crystallin-positive ectopic lentoid bodies. Combined with the observation that the Wnt pathway reporter TOPGAL is expressed in nasal periocular ectoderm, these data suggest that, in this location, the canonical Wnt signaling pathway normally suppresses lens fate in favor of other structures. Consistent with this proposal, a dominant-active form of beta-catenin causes a loss of lens fate and a complete absence of lens development when expressed in the presumptive lens ectoderm.     

The Akt signaling pathway contributes to postconditioning's protection against stroke; the protection is associated with the MAPK and PKC pathways

We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c- Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning's protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning's protection.