Inhibition Of Canonical Wnt Signaling Research Articles

Cross-talk between Wnt and Bone Morphogenetic Protein 2 (BMP-2) Signaling in Differentiation Pathway of C2C12 Myoblasts

Loss of function of the Wnt co-receptor, lipoprotein receptor-related protein 5, decreases bone formation, and a point mutation in this gene results in high bone mass, indicating the importance of this signaling pathway in bone formation. However, the exact mechanism is currently unknown. We examined a potential role for Wnt signaling and functional cross-talk of bone morphogenetic protein 2 (BMP-2) in osteoblast differentiation. To assess the contribution of Wnt, we generated C2C12 cells over-expressing Wnt3a or Wnt5a and treated these with BMP-2. We showed that expression of matrix extracellular phosphoglycoprotein was induced by BMP-2 in Wnt3a over-expressing C2C12 cells but not in Wnt5a over-expressing C2C12 cells. Over-expression of Wnt3a blocked BMP-2-induced inhibition of myotube formation in C2C12 cells when switched to low mitogen medium. In these cultures, expression of inhibitor of DNA binding/differentiation (Id) 1, a helix-loop-helix protein induced by BMP-2, decreased in stable Wnt3a- but not in Wnt5a-expressing cells. This suppression is mediated by a GC-rich region of the BMP-2-responsive element of the Id1 gene promoter, and interaction between Smad1/4 and beta-catenin is crucial for Wnt-mediated suppression of the BMP-2 response in C2C12 cells. Over-expression of the inhibitor of canonical Wnt signaling, Dickkopf, inhibits this suppression. In contrast, BMP-2 or Smad1/4 up-regulated Wnt3a or activated beta-catenin-induced lymphoid-enhancing factor 1/T cell factor-dependent transcriptional activity. These findings identify functional cross-talk of Id1 expression between Wnt and BMP signaling and demonstrate a novel mechanism for Wnt regulation of the BMP-2 response, linking Id1 expression to Wnt/beta-catenin signaling.

Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways

Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.

Global Inhibition of Lef1/Tcf-dependent Wnt Signaling at Its Nuclear End Point Abrogates Development in Transgenic Xenopus Embryos

Analysis of canonical Wnt signaling during vertebrate development by means of knock-out or transgenic approaches is often hampered by functional redundancy as well as pathway bifurcations downstream of the manipulated components. We report the design of an optimized chimera capable of blocking transcriptional activation of Lef1/Tcf-beta-catenin target genes, thus enabling intervention with the canonical Wnt pathway at its nuclear end point. This construct was made hormone-inducible, both functionally and transcriptionally, and was transgenically integrated in Xenopus embryos. Down-regulation of target genes was clearly observed upon treatment of these embryos with dexamethasone. In addition, exposure of variously aged transgenic embryos to dexamethasone caused complex phenotypes with many new but also several recognizable features stemming from inhibition of canonical Wnt signaling. At least in some tissues, a significant reduction in cell proliferation and an increase in programmed cell death appeared to underlie these phenotypes. Our inducible transgenic system can serve a broad range of experimental settings designed to unveil new functional aspects of Lef1/Tcf-beta-catenin signaling during vertebrate embryogenesis.

Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells.

Multipotential adult mesenchymal stem cells (MSCs) are able to differentiate along several known lineages, and lineage commitment is tightly regulated through specific cellular mediators and interactions. Recent observations of a low/high bone-mass phenotype in patients expressing a loss-/gain-of-function mutation in LRP5, a coreceptor of the Wnt family of signaling molecules, suggest the importance of Wnt signaling in bone formation, possibly involving MSCs. To analyze the role of Wnt signaling in mesenchymal osteogenesis, we have profiled the expression of WNTs and their receptors, FRIZZLEDs (FZDs), and several secreted Wnt inhibitors, such as SFRPs, and examined the effect of Wnt 3a, as a representative canonical Wnt member, during MSC osteogenesis in vitro. WNT11, FZD6, SFRP2, and SFRP3 are upregulated during MSC osteogenesis, while WNT9A and FZD7 are downregulated. MSCs also respond to exogenous Wnt 3a, based on increased beta-catenin nuclearization and activation of a Wnt-responsive promoter, and the magnitude of this response depends on the MSC differentiation state. Wnt 3a exposure inhibits MSC osteogenic differentiation, with decreased matrix mineralization and reduced alkaline phosphatase mRNA and activity. Wnt 3a treatment of fully osteogenically differentiated MSCs also suppresses osteoblastic marker gene expression. The Wnt 3a effect is accompanied by increased cell number, resulting from both increased proliferation and decreased apoptosis, particularly during expansion of undifferentiated MSCs. The osteo-suppressive effects of Wnt 3a are fully reversible, i.e., treatment prior to osteogenic induction does not compromise subsequent MSC osteogenesis. The results also showed that sFRP3 treatment attenuates some of the observed Wnt 3a effects on MSCs, and that inhibition of canonical Wnt signaling using a dominant negative TCF1 enhances MSC osteogenesis. Interestingly, expression of Wnt 5a, a non-canonical Wnt member, appeared to promote osteogenesis. Taken together, these findings suggest that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor MSC population, whereas non-canonical Wnts facilitate osteogenic differentiation. Release from canonical Wnt regulation is a prerequisite for MSC differentiation. Thus, loss-/gain-of-function mutations of LRP5 would perturb Wnt signaling and depress/promote bone formation by affecting the progenitor cell pool. Elucidating Wnt regulation of MSC differentiation is important for their potential application in tissue regeneration.

Regulating the Balance between Peroxisome Proliferator-activated Receptor γ and β-Catenin Signaling during Adipogenesis: A GLYCOGEN SYNTHASE KINASE 3β PHOSPHORYLATION-DEFECTIVE MUTANT OF β-CATENIN INHIBITS EXPRESSION OF A SUBSET OF ADIPOGENIC GENES

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of beta-catenin to the proteasome. In fact, sustained activation of beta-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. In this report, we investigated the mechanisms regulating the balance between beta-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes. Specifically, we show that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of beta-catenin, which depends on glycogen synthase kinase (GSK) 3beta activity. Mutation of serine 37 (a target of GSK3beta) to an alanine renders beta-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3beta phosphorylation-defective S37A-beta-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-beta-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant beta-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-beta-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKbeta is a key regulator of the balance between beta-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes.

Zygotic Wnt Activity Is Required for Brachyury Expression in the Early Xenopus laevis Embryo

The canonical, β-catenin-dependent Wnt pathway is a crucial player in the early events of Xenopus development. Dorsal axis formation and mesoderm patterning are accepted effects of this pathway, but the regulation of expression of genes involved in mesoderm specification is not. This conclusion is based largely on the inability of the Wnt pathway to induce mesoderm in animal cap explants. Using injections of inhibitors of canonical Wnt signaling, we demonstrate that expression of the general mesodermal marker Brachyury (Xbra) requires a zygotic, ligand-dependent Wnt activity throughout the marginal zone. Analysis of the Xbra promoter reveals that putative TCF-binding sites mediate Wnt activation, the first sites in this well-studied promoter to which an activation role can be ascribed. However, established mesoderm inducers like eFGF and activin can bypass the Wnt requirement for Xbra expression. Another mesoderm promoting factor, VegT, activates Xbra in a Wnt-dependent manner. We also show that the activin/nodal signaling is necessary for ectopic Xbra induction by the Wnt pathway, but not by VegT. Our data significantly change the understanding of Brachyury regulation in Xenopus, implying the existence of an unknown zygotic Wnt ligand in Spemann's organizer. Since Brachyury is considered to have a major role in mesoderm formation, it is possible that Wnts might play a role in mesoderm specification, in addition to patterning.