Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

Retained Cell�CCell Adhesion in Serrated Neoplastic Pathway as Opposed to Conventional Colorectal Adenomas

The molecular features of serrated polyps of colorectum remain to be elucidated. The expression pattern of adhesive molecules (E-cadherin, α-catenin, and β-catenin) has not been examined in serrated neoplastic pathway. The expression of E-cadherin, α-catenin, and β-catenin were analyzed by immunohistochemistry in 32 hyperplastic polyps (HPs), 28 sessile serrated adenomas (SSAs), 37 traditional serrated adenomas (TSAs), 51 traditional adenomas (TAs), and 10 normal colonic tissues (NCs). Retained membranous expression for E-cadherin, α-catenin, and β-catenin was more frequent in HPs, SSAs, and TSAs than that in TAs (p < 0.001). Nuclear labeling of β-catenin was detected in 19.6% of TAs, but in none of HPs, SSAs, and TSAs (p < 0.001). Cytoplasmic accumulation of β-catenin was found in 3.1% of HPs, 3.6% of SSAs, and 21.6% of TSAs, significantly lower than that in TAs (60.8%, p < 0.001). The membranous co-expression of E-cadherin, α-catenin, and β-catenin was more frequent in HPs (68.8%), SSAs (60.7%), and TSAs (37.8%) than that in TAs (7.8%, p < 0.001). Cell adhesion function is retained in serrated neoplastic pathway. Wnt signaling pathway plays a less active role in the development of colorectal serrated polys than in TAs.     

Beta-catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions

Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/beta-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as beta-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A(1) (PLA(1)), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of beta-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA(1) gene, cortical beta-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of beta-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of beta-catenin.     

Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

Cantharidin inhibits osteosarcoma proliferation and metastasis by directly targeting miR-214-3p/DKK3 axis to inactivate β-catenin nuclear translocation and LEF1 translation

Background: As the leading primary bone cancer in adolescents and children, osteosarcoma patients with metastasis show a five-year-survival-rate of 20-30%, without improvement over the past 30 years. Wnt/β-catenin is important in promoting osteosarcoma development. DKK3 is a Wnt/β-catenin antagonist and predicted to have the specific binding site in 3′-UTR with miR-214-3p.Methods: miR-214-3p and DKK3 levels were investigated in human osteosarcoma tissues and cells by RT-qPCR; the prognostic importance of DKK3 level in osteosarcoma patients was determined with Log-rank test; direct binding between DKK3 with miR-214-3p was identified with targetscan; anti-osteosarcoma mechanism of cantharidin was investigated by miR-214-3p silence/over-expression with or without cantharidin treatment, and nuclear/cytoplasmic protein assay in osteosarcoma cells.Results: Down-regulated DKK3 indicated poor prognosis of osteosarcoma patients. Up-regulated miR-214-3p promoted proliferation and migration, while suppressed apoptosis of osteosarcoma cells by increasing β-catenin nuclear translocation and LEF1 translation via degradation of DKK3. Cantharidin suppressed viabilities, migration and invasion, while promoted cell cycle arrest and apoptosis in 143B and U-2 OS cells via down-regulating miR-214-3p to up-regulate DKK3, thus inhibited p-GSK-3β expression, β-catenin nuclear translocation and LEF1 translation. Meanwhile, cantharidin inhibited tumor growth in xenograft-bearing mice with 143B cell injection in tibia.Conclusion: miR-214-3p mediated Wnt/β-catenin/LEF1 signaling activation by targeting DKK3 to promote oncogenesis of osteosarcoma; cantharidin inhibited proliferation and metastasis of osteosarcoma cells via down-regulating miR-214-3p to up-regulate DKK3 and decrease β-catenin nuclear translocation, indicating that cantharidin may be a prospective candidate for osteosarcoma treatment by targeting miR-214-3p/DKK3/β-catenin signaling.     

FMR1/FXR1 and the miRNA pathway are required for eye and neural crest development

FMR1 and FXR1 are RNA binding proteins interacting with the miRNA-induced silencing complex, RISC. Here we describe for the first time the function of these proteins during eye and neural crest (NC) development in Xenopus laevis. A loss of FMR1 or FXR1 results in abnormal eye development as well as defects in cranial cartilage derived from cranial NC cells. We further investigated the possible mechanism of these phenotypes by showing that a depletion of Dicer, an important enzyme for generating all mature miRNAs, in the anterior neural tissue also leads to eye and cranial cartilage defects. Furthermore, we examined the function of 12 miRNAs during anterior neural development. We show a specific requirement of six selected miRNAs during eye and cranial cartilage development. Mir-130a, -219, and -23b are involved in eye formation only whereas loss of miR-200b, miR-96 and miR-196a results in strong defects during eye as well as cranial cartilage development. Our results suggest an essential role for FMR1 and FXR1 for eye and NC development in X.laevis likely through an interaction with the miRNA pathway.     

Cardiomyocyte-like cells differentiation from non β-catenin expression mesenchymal stem cells

Recent studies have shown that block wnt/β-catenin signaling pathway is integrant for cardiomyocytes differentiation from bone marrow mesenchymal stem cells (MSCs). By transducing the MSCs with lentivirus which contain β-catenin interference RNA, we screened out the non β-catenin expression clone. In the establishment of knockdown β-catenin in MSCs, we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (salB), and cardiomyocytes lysis medium (CLM) in inducing MSCs to differentiate into cardiomyocyte-like cells. A method for culturing MSCs and cardiomyocytes was established. Purified MSCs were investigated by flow cytometry. The MSCs were positive for CD90 and CD29, but negative for CD34 and CD45. Meanwhile, the cardiomyocytes contracted spontaneously after 24 h of seeding into the plates. The fourth-passage non-β-catenin expression MSCs were divided into eight groups: control group, 5-aza, salB, CLM, 5-aza + salB, 5-aza + CLM, salB + CLM, and 5-aza + salB + CLM. The gene and protein expression of cTnT, α-actin, β-myosin, β-catenin, and GSK-3β were detected by quantitative real-time PCR and Western blotting. Our results showed that cTnT expression in 5-aza + salB + CLM group was ninefold higher than in the control group in the non-β-catenin MSCs model, implying that cardiomyocytes differentiation from MSCs is an extremely complicated process and it is necessary to consider the internal and external environmental conditions, such as suitable pharmaceutical inducers, cardiomyocytes microenvironments, inhibition of the negative signaling pathway and so on.     

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.     

Ras mutation cooperates with ��-catenin activation to drive bladder tumourigenesis

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras^Q61L or K-Ras^G12D) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.     

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.     

Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis.

The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector beta-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

Plx1 is required for chromosomal DNA replication under stressful conditions

Polo-like kinase (Plk)1 is required for mitosis progression. However, although Plk1 is expressed throughout the cell cycle, its function during S-phase is unknown. Using Xenopus laevis egg extracts, we demonstrate that Plx1, the Xenopus orthologue of Plk1, is required for DNA replication in the presence of stalled replication forks induced by aphidicolin, etoposide or reduced levels of DNA-bound Mcm complexes. Plx1 binds to chromatin and suppresses the ATM/ATR-dependent intra-S-phase checkpoint that inhibits origin firing. This allows Cdc45 loading and derepression of DNA replication initiation. Checkpoint activation increases Plx1 binding to the Mcm complex through its Polo box domain. Plx1 recruitment to chromatin is independent of checkpoint mediators Tipin and Claspin. Instead, ATR-dependent phosphorylation of serine 92 of Mcm2 is required for the recruitment of Plx1 to chromatin and for the recovery of DNA replication under stress. Depletion of Plx1 leads to accumulation of chromosomal breakage that is prevented by the addition of recombinant Plx1. These data suggest that Plx1 promotes genome stability by regulating DNA replication under stressful conditions.