Multiple roles of mesenchymal beta-catenin during murine limb patterning

Recently canonical Wnt signaling in the ectoderm has been shown to be required for maintenance of the apical ectodermal ridge (AER) and for dorsoventral signaling. Using conditional gain- and loss-of-function beta-catenin alleles, we have studied the role of mesenchymal beta-catenin activity during limb development. Here, we show that loss of beta-catenin results in limb truncations due to a defect in AER maintenance. Stabilization of beta-catenin also results in truncated limbs, caused by a premature regression of the AER. Concomitantly, in these limbs, the expression of Bmp2, Bmp4 and Bmp7, and of the Bmp target genes Msx1, Msx2 and gremlin, is expanded in the mesenchyme. Furthermore, we found that the expression of Lmx1b, a gene exclusively expressed in the dorsal limb mesenchyme and involved in dorsoventral patterning, is reduced upon loss of beta-catenin activity and is expanded ventrally in gain-of-function limbs. However, the known ectodermal regulators Wnt7a and engrailed 1 are expressed normally. This suggests that Lmx1b is also regulated, in part, by a beta-catenin-mediated Wnt signal, independent of the non-canoncial Wnt7a signaling pathway. In addition, loss of beta-catenin results in a severe agenesis of the scapula. Concurrently, the expression of two genes, Pax1 and Emx2, which have been implicated in scapula development, is lost in beta-catenin loss-of-function limbs; however, only Emx2 is upregulated in gain-of-function limbs. Mesenchymal beta-catenin activity is therefore required for AER maintenance, and for normal expression of Lmx1b and Emx2.     

Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.     

Regulation of the Wnt signaling pathway by disabled-2 (Dab2)

The adaptor molecule Disabled-2 (Dab2) has been shown to link cell surface receptors to downstream signaling pathways. Using a small-pool cDNA screening strategy, we identify that the N-terminal domain of Dab2 interacts with Dishevelled-3 (Dvl-3), a signaling mediator of the Wnt pathway. Ectopic expression of Dab2 in NIH-3T3 mouse fibroblasts attenuates canonical Wnt/beta-catenin-mediated signaling, including accumulation of beta-catenin, activation of beta-catenin/T-cell-specific factor/lymphoid enhancer-binding factor 1-dependent reporter constructs, and endogenous cyclin D1 induction. Wnt stimulation leads to a time-dependent dissociation of endogenous Dab2-Dvl-3 and Dvl-3-axin interactions in NIH-3T3 cells, while Dab2 overexpression leads to maintenance of Dab2-Dvl-3 association and subsequent loss of Dvl-3-axin interactions. In addition, we find that Dab2 can associate with axin in vitro and stabilize axin expression in vivo. Mouse embryo fibroblasts which lack Dab2 exhibit constitutive Wnt signaling as evidenced by increased levels of nuclear beta-catenin and cyclin D1 protein levels. Based on these results, we propose that Dab2 functions as a negative regulator of canonical Wnt signaling by stabilizing the beta-catenin degradation complex, which may contribute to its proposed role as a tumor suppressor.     

Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/beta-catenin activity in kidney epithelial cells

The Wnt signaling pathway is central to the development of all animals and to cancer progression, yet largely unknown are the pairings of secreted Wnt ligands to their respective Frizzled transmembrane receptors or, in many cases, the relative contributions of canonical (beta-catenin/LEF/TCF) versus noncanonical Wnt signals. Specifically, in the kidney where Wnt-4 is essential for the mesenchymal to epithelial transition that generates the tissue's collecting tubules, the corresponding Frizzled receptor(s) and downstream signaling mechanism(s) are unclear. In this report, we addressed these issues using Madin-Darby Canine Kidney (MDCK) cells, which are competent to form tubules in vitro. Employing established reporter constructs of canonical Wnt/beta-catenin pathway activity, we have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions, precisely reflecting functional findings from Wnt-4 null kidney mesenchyme ex vivo rescue studies. We have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/beta-catenin pathway using beta-Engrailed and dnTCF-4 constructs that suppress this pathway. We have further found that MDCK cells express the Frizzled-6 receptor and that Wnt-4 forms a biochemical complex with the Frizzled-6 CRD. Since Frizzled-6 did not appear to transduce Wnt-4's canonical signal, data supported recently by Golan et al., there presumably exists another as yet unknown Frizzled receptor(s) mediating Wnt-4 activation of beta-catenin/LEF/TCF. Finally, we report that canonical Wnt/beta-catenin signals cells help maintain cell growth and survival in MDCK cells but do not contribute to standard HGF-induced (nonphysiologic) tubule formation. Our results in combination with work from Xenopus laevis (not shown) lead us to believe that Wnt-4 binds both canonical and noncanonical Frizzled receptors, thereby activating Wnt signaling pathways that may each contribute to kidney tubulogenesis.     

Dapper1 is a nucleocytoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus

Wnt signaling, via the activation of the canonical beta-catenin and lymphoid enhancer factor (LEF)/T-cell factor pathway, plays an important role in embryogenesis and cancer development by regulating the expression of genes involved in cell proliferation, differentiation, and survival. Dapper (Dpr), as a Dishevelled interactor, has been suggested to modulate Wnt signaling by promoting Dishevelled degradation. Here, we provide evidence that Dpr1 shuttles between the cytoplasm and the nucleus. Although overexpressed Dpr1 was mainly found in the cytoplasm, endogenous Dpr1 was localized over the cell, and Wnt1 induced its nuclear export. Treatment with leptomycin B induced nuclear accumulation of both endogenous and overexpressed Dpr1. We further identified the nuclear localization signal and the nuclear export signal within Dpr1. Using reporter assay and in vivo zebrafish embryo assay, we demonstrated that the forced nuclearly localized Dpr1 possessed the ability to antagonize Wnt signaling. Dpr1 interacted with beta-catenin and LEF1 and disrupted their complex formation. Furthermore, Dpr1 could associate with histone deacetylase 1 (HDAC1) and enhance the LEF1-HDAC1 interaction. Together, our findings suggest that Dpr1 negatively modulates the basal activity of Wnt/beta-catenin signaling in the nucleus by keeping LEF1 in the repressive state. Thus, Dpr1 controls Wnt/beta-catenin signaling in both the cytoplasm and the nucleus.     

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.     

Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway

The effect of a noncanonical Wnt, Wnt11, on canonical Wnt signaling stimulated by Wnt1 and activated forms of LRP5 (low density lipoprotein receptor-related protein-5), Dishevelled1 (Dvl1), and beta-catenin was examined in NIH3T3 cells and P19 embryonic carcinoma cells. Wnt11 repressed Wnt1-mediated activation of LEF-1 reporter activity in both cell lines. However, Wnt11 was unable to inhibit canonical signaling activated by LRP5, Dvl1, or beta-catenin in NIH3T3 cells, although it could in P19 cells. In addition, Wnt11-mediated inhibition of canonical signaling in NIH3T3 cells is ligand-specific; Wnt11 could effectively repress canonical signaling activated by Wnt1, Wnt3, or Wnt3a but not by Wnt7a or Wnt7b. Co-culture experiments with NIH3T3 cells showed that the co-expression of Wnt11 with Wnt1 was not an essential requirement for the inhibition, suggesting receptor competition as a possible mechanism. Moreover, in both cell types, elevation of intracellular Ca(2+) levels, which can result from Wnt11 treatment, led to the inhibition of canonical signaling. This result suggests that Wnt11 might not be able to signal in NIH3T3. Furthermore, P19 cells were found to express both endogenous canonical Wnts and Wnt11. Knockdown of Wnt11 expression using siRNA resulted in increased LEF-1 reporter activity, thus indicating that Wnt11-mediated suppression of canonical signaling exists in vivo.     

Wnt-mediated regulation of chondrocyte maturation: modulation by TGF-beta

Wnt proteins are expressed during limb morphogenesis, yet their role and mechanism of action remains unclear during long bone growth. Wnt expression, effects and modulation of signaling events by BMP and transforming growth factor-beta (TGF-beta) were evaluated in chick embryonic chondrocytes. Chondrocyte cell cultures underwent spontaneous maturation with increased expression of colX and this was associated with an increase in the expression of multiple Wnts, including Wnt 4, 5a, 8c, and 9a. Both parathyroid hormone related peptide (PTHrP) and TGF-beta inhibited colX, but had disparate effects on Wnt expression. While TGF-beta strongly inhibited all Wnts, PTHrP did not inhibit either Wnt8c or Wnt9a and had lesser effects on the expression of the other Wnts. BMP-2 induced colX expression, and also markedly increased Wnt8c expression. Overexpression of beta-catenin and/or T cell factor (TCF)-4 also induced the type X collagen promoter. Overexpression of Wnt8c induced maturation, as did overexpression of beta-catenin. The Wnt8c/beta-catenin maturational effects were enhanced by BMP-2 and inhibited by TGF-beta. TGF-beta also inhibited activation of the Topflash reporter by beta-catenin, suggesting a direct inhibitory effect since the Topflash reporter contains only beta-catenin binding sequences. In turn beta-catenin inhibited activation of the p3TP-Luc reporter by TGF-beta, although the effect was partial. Thus, Wnt/beta-catenin signaling is a critical regulator of the rate of chondrocyte differentiation. Moreover, this pathway is modulated by members of the TGF-beta family and demonstrates the highly integrated nature of signals controlling endochondral ossification.     

Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis

Wnt/beta-catenin signaling plays key roles in tooth development, but how this pathway intersects with the complex interplay of signaling factors regulating dental morphogenesis has been unclear. We demonstrate that Wnt/beta-catenin signaling is active at multiple stages of tooth development. Mutation of beta-catenin to a constitutively active form in oral epithelium causes formation of large, misshapen tooth buds and ectopic teeth, and expanded expression of signaling molecules important for tooth development. Conversely, expression of key morphogenetic regulators including Bmp4, Msx1, and Msx2 is downregulated in embryos expressing the secreted Wnt inhibitor Dkk1 which blocks signaling in epithelial and underlying mesenchymal cells. Similar phenotypes are observed in embryos lacking epithelial beta-catenin, demonstrating a requirement for Wnt signaling within the epithelium. Inducible Dkk1 expression after the bud stage causes formation of blunted molar cusps, downregulation of the enamel knot marker p21, and loss of restricted ectodin expression, revealing requirements for Wnt activity in maintaining secondary enamel knots. These data place Wnt/beta-catenin signaling upstream of key morphogenetic signaling pathways at multiple stages of tooth development and indicate that tight regulation of this pathway is essential both for patterning tooth development in the dental lamina, and for controlling the shape of individual teeth.     

Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors, transformed cell lines, and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves, as has been seen in a number of human breast cancers, or through mutation of intermediates in the Wnt pathway, such as adenomatous polyposis coli or beta-catenin, as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development, but overexpression of certain Wnts, such as Wnt-1, leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG, morphological changes and increased proliferation are accompanied by increased levels of CK2, as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins, which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin, Dvl-2, and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells, abrogates phosphorylation of beta-catenin, and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells.     

Stabilization of beta-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast

Many components of the Wnt/beta-catenin signaling pathway are expressed during mouse pre-implantation embryo development, suggesting that this pathway may control cell proliferation and differentiation at this time. We find no evidence for a functional activity of this pathway in cleavage-stage embryos using the Wnt-reporter line, BAT-gal. To further probe the activity of this pathway, we activated beta-catenin signaling by mating a zona pellucida3-cre (Zp3-cre) transgenic mouse line with a mouse line containing an exon3-floxed beta-catenin allele. The result is expression of a stabilized form of beta-catenin, resistant to degradation by the GSK3beta-mediated proteasome pathway, expressed in the developing oocyte and in each cell of the resulting embryos. Nuclear localization and signaling function of beta-catenin were not observed in cleavage-stage embryos derived from these oocytes. These results indicate that in pre-implantation embryos, molecular mechanisms independent of the GSK3beta-mediated ubiquitination and proteasome degradation pathway inhibit the nuclear function of beta-catenin. Although the mutant blastocysts initially developed normally, they then exhibited a specific phenotype in the embryonic ectoderm layer of early post-implantation embryos. We show a nuclear function of beta-catenin in the mutant epiblast that leads to activation of Wnt/beta-catenin target genes. As a consequence, cells of the embryonic ectoderm change their fate, resulting in a premature epithelial-mesenchymal transition.