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PubMed1997

Activation of the Wnt signaling pathway: a molecular mechanism for lithium action.

Glycogen synthase kinase-3 beta (GSK-3 beta/zeste-white-3/shaggy) is a negative regulator of the wnt signaling pathway which plays a central role in the development of invertebrates and vertebrates; loss of function and dominant negative mutations in GSK-3 beta lead to activation of the wnt pathway in Drosophila and Xenopus. We now provide evidence that lithium activates downstream components of the wnt signaling pathway in vivo, leading to accumulation of beta-catenin protein. Our data indicate that this activation of the wnt pathway is a consequence of inhibition of GSK-3 beta by lithium. Using a novel assay for GSK-3 beta in oocytes, we show that lithium inhibits GSK-3 beta from species as diverse as Dictyostelium discoideum and Xenopus laevis, providing a biochemical mechanism for the action of lithium on the development of these organisms. Lithium treatment also leads to activation of an AP-1-luciferase reporter in Xenopus embryos, consistent with previous observations that GSK-3 beta inhibits c-jun activity. Activation of the wnt pathway with a dominant negative form of GSK-3 beta is inhibited by myo-inositol, similar to the previously described effect of coinjecting myo-inositol with lithium. The mechanism by which myo-inositol inhibits both dominant negative GSK-3 beta and lithium remains uncertain.

Animals↗

PubMed2004

Cross-talk between Rac1 GTPase and dysregulated Wnt signaling pathway leads to cellular redistribution of beta-catenin and TCF/LEF-mediated transcriptional activation.

Aberrant activation of the Wnt pathway is observed in numerous cancers, and is particularly important in colon cancer. We demonstrate that Rac1 GTPase can significantly increase the signaling activity of beta-catenin in cells with inherent dysregulation of the canonical Wnt signaling pathway. Expression of dominant-negative (N17)Rac1 mutant in colon cancer cells caused a marked inhibition of Wnt signaling, as determined by the TCF/LEF-responsive (TOPFLASH) transcription assay. Expression of a constitutively active (V12)Rac1 mutant caused up to 40-fold induction from the TOPFLASH promoter, and this was dependent on the presence of stabilized beta-catenin. This induction was completely blocked by the expression of dominant-negative TCF-4, suggesting that beta-catenin and TCF-4 complex formation is required for Rac1-mediated transcription. Furthermore, we show that Cyclin D1, an important biological Wnt target gene, is regulated by Rac1 in a beta-catenin/TCF-dependent manner. We observed that Rac1 co-immunoprecipitates with beta-catenin and TCF-4 only in its active GTP-bound form. Both cell fractionation studies and fluorescence microscopy indicate that overexpression of V12Rac1 results in increased cytosolic and nuclear expression of beta-catenin. Interestingly, mutation of the polybasic region of Rac1, which prevents its nuclear localization, also caused an appreciable decrease in nuclear localization of beta-catenin, and effectively abolished its beta-catenin-dependent transcription co-activator function. Taken together, our data demonstrate a novel mechanism of Wnt pathway regulation whereby activation of Rac1 amplifies the signaling activity of stabilized/mutated beta-catenin by promoting its accumulation in the nucleus, and synergizing with beta-catenin to augment TCF/LEF-dependent gene transcription.

Cell Line, Tumor↗

PubMed2002

Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC.

Aberrant activation of the Wnt signaling pathway has been reported in different human tumor types, including malignant melanomas. We investigated 37 malignant melanomas (15 primary tumors and 22 metastases) for alterations of 4 genes encoding members of this pathway, i.e., CTNNB1 (beta-catenin gene, 3p22.1), APC (adenomatous polyposis coli gene, 5q22.2), BTRC (beta-transducin repeat-containing protein gene, 10q24.3) and ICAT (inhibitor of beta-catenin and Tcf-4, 1p36.2). Mutational analysis of CTNNB1 identified somatic mutations in 1 primary melanoma and 1 melanoma metastasis from 2 different patients (5%). Both mutations affected the N-terminal degradation box of beta-catenin, which is important for the regulation of beta-catenin homeostasis. Another primary melanoma carried a somatic APC missense mutation within the known mutation cluster region in exon 15. Fourteen tumors (40%) showed LOH at microsatellite markers on 1p36. None of the tumors had lost both copies of the ICAT gene, but 1 melanoma metastasis carried a somatic point mutation altering the translation start codon of ICAT. Real-time RT-PCR showed markedly reduced ICAT transcript levels (<or=20% relative to normal skin and benign melanocytic nevi) in 28/36 malignant melanomas (78%), including 13/14 tumors with LOH on 1p36. Allelic loss on 10q was detected in 15 tumors (44%). We found neither mutations nor complete loss of expression of the BTRC gene in our melanoma series. Taken together, our results indicate that the Wnt pathway may be altered in malignant melanomas by different mechanisms, including rare somatic mutations in CTNNB1, APC or ICAT, as well as low or absent expression of ICAT transcripts.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Inhibition of the canonical Wnt signaling pathway in cytoplasm: a novel property of the carboxyl terminal domains of two Xenopus ELL genes.

The Wnt signaling pathways are important in many developmental events. The canonical Wnt pathway is one of the three major Wnt-mediated intracellular signaling pathways and is thought to activate Dvl followed by the stabilization of beta-catenin. In Xenopus, this pathway is involved in dorsal determination, anterior-posterior patterning during gastrulation, and neural induction. Here we describe a role for the Xenopus ELL (Eleven-nineteen Lysine-rich Leukemia) gene product in canonical Wnt signaling. Translocation of ELL has been associated with acute myeloid leukemia and the protein possesses three functional domains. We identified rELL-C from a rat brain cDNA library as a binding factor for Dishevelled (Dvl); it represents a partial sequence of rat ELL lacking the pol II elongation domain and has been shown to suppress canonical Wnt signaling. Next, we isolated two Xenopus homologs of ELL, xELL1 and xELL2. No obvious phenotypes were observed with microinjection of full-length xELL1 or xELL2 mRNA, however, microinjection with their occludin homology domain inhibited Wnt signaling at the level of Dvl and upstream of beta-catenin. Intracellular localization of microinjected xELL1- and xELL2-GFP mRNAs showed localization of the full-length products in the nucleus and the occludin-homology domain products in cytoplasm. These results raise the possibility that ELL, which is thought to function as a transcription factor in nuclei, can serve other, novel roles to suppress canonical Wnt signaling in the cytoplasm.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A Wnt signaling pathway controls hox gene expression and neuroblast migration in C. elegans.

The specification of body pattern along the anteroposterior (A/P) body axis is achieved largely by the actions of conserved clusters of Hox genes. Limiting expression of these genes to localized regional domains and controlling the precise patterns of expression within those domains is critically important for normal patterning. Here we report that egl-20, a C. elegans gene required to activate expression of the Hox gene mab-5 in the migratory neuroblast QL, encodes a member of the Wnt family of secreted glycoproteins. We have found that a second Wnt pathway gene, bar-1, which encodes a beta-catenin/Armadillo-like protein, is also required for activation of mab-5 expression in QL. In addition, we describe the gene pry-1, which is required to limit expression of the Hox genes lin-39, mab-5 and egl-5 to their correct local domains. We find that egl-20, pry-1 and bar-1 all function in a linear genetic pathway with conserved Wnt signaling components, suggesting that a conserved Wnt pathway activates expression of mab-5 in the migratory neuroblast QL. Moreover, we find that members of this Wnt signaling system play a major role in both the general and fine-scale control of Hox gene expression in other cell types along the A/P axis.

Amino Acid Sequence↗

PubMed2005

Changes in the Wnt signalling pathway in gastrointestinal cancers and their prognostic significance.

Many steps in the Wnt signalling pathway may be altered during the process of carcinogenesis. This Review focuses on the changes observed in gastrointestinal cancers. A literature search was undertaken and the currently available data summarised. Understanding the alterations to this signalling pathway may help to reveal future targets for therapeutic agents. In addition, since in some tumours, levels of components of the Wnt pathway have been found to correlate with clinical stage, their potential use as prognostic indicators is highlighted.

Axin Protein↗

PubMed2006

Molecular bases of the regulation of bone remodeling by the canonical Wnt signaling pathway.

Osteoporosis is a common, prevalent, and debilitating condition, particularly in postmenopausal women. Genetics play a major role in determining peak bone mass and fracture risk, but few genes have been demonstrated conclusively to be involved, much less the signaling pathways with which they are affiliated. The identification of mutations in the gene Lrp5, a Wnt coreceptor, as the cause for both osteoporotic and high-bone mass disorders implicated the canonical Wnt signaling pathway in bone mass regulation. Since Lrp5, other Wnt components have been identified as being regulators of bone mass, and Wnt target genes affecting bone homeostasis have begun to be elucidated. This chapter looks at the various components of the canonical Wnt signaling pathway and the data indicating that this pathway plays a major role in the control of both bone formation and bone resorption, the two key aspects of bone remodeling.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Expression of Wnt-signaling pathway proteins in intraductal papillary mucinous neoplasms of the pancreas: a tissue microarray analysis.

Abrogation of the Wnt-signaling pathway is implicated in the carcinogenesis of several malignancies, especially colorectal cancer where up to 90% of cases are thought to have impaired Wnt signaling. It is less frequently involved in conventional ductal pancreatic adenocarcinoma. This pathway has not been explored in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas previously and formed the basis of this study. A tissue microarray of 18 cases of IPMN was stained for proteins involved in the Wnt pathway: adenomatous polyposis coli (APC), pan-beta-catenin, axin 2, glycogen synthase 3alphabeta and 3beta, c-myc, E-cadherin, and cyclin D1. The IPMNs were classified as 8 adenomas, 3 borderline, and 7 cases with carcinoma in situ and/or invasive carcinoma, occurring in 13 females, and the overall age range was 45 to 73 years. Immunohistochemical analysis showed nuclear beta-catenin staining in 7 (39%) of the 18 cases. The cases with nuclear beta-catenin localization included 1 adenoma, 2 borderline IPMN, and 4 carcinomas in situ and/or invasive carcinomas. Seven cases showed absence of APC immunostaining and these included 4 cases with nuclear beta-catenin localization. Fourteen cases displayed marked diffuse up-regulation of c-myc protein, and 12 cases also showed diffuse cyclin D1 protein overexpression. E-cadherin expression was intense and membrane in location (comparable to normal tissue) in 6 of 8 adenomas (no tissue was available in 1 case). Decreased E-cadherin staining was noted in 8 cases where tissue was available for assessment. There was progressive decrease in membrane staining of E-cadherin in 2 of 3 borderline lesions, 1 of 2 carcinomas in situ, and 4 of 5 invasive carcinomas. All other immunostains were either normal in distribution or did not show any correlation with beta-catenin or clinicopathologic parameters. In conclusion, 7 (39%) of 18 cases of IPMN in this study demonstrated abnormal localization of beta-catenin, 4 of which also lacked APC expression. Of 5 carcinomas arising in IPMN, 4 displayed a decrease in E-cadherin expression. There was also a trend for the higher grades of IPMN to show nuclear localization of beta-catenin. These findings suggest that a proportion of cases of IPMN may show abnormalities in the Wnt-signaling pathway with consequent altered expression of downstream related proteins.

Adenoma↗

PubMed2026

Studies on functional differentiation of xpr1a and xpr1b genes in zebrafish.

Xenotropic and polytropic retrovirus receptor 1 (XPR1) is known to be involved in various biological processes, including phosphate homeostasis, cellular signaling, brain and vascular mineralization, whereas its specific contribution to bone development remains incompletely characterized. Due to genome duplication in teleosts, zebrafish Danio rerio possess two paralogous genes of XPR1 namely xpr1a and xpr1b, whose functional divergence remains unclear. The amino acid sequence similarity between zebrafish xpr1a and xpr1b was 83.26%. In situ hybridization demonstrated overlapping localization in the head and spinal cord at 24-48 hpf, while diverged by 72 hpf, with xpr1a becoming restricted to the head while xpr1b persisted in both regions. CRISPR/Cas9 was used to generate xpr1a and xpr1b mutants. The xpr1a mutants are comparatively healthy, viable but with mild growth reduction, whereas the xpr1b mutants display high mortality, reduced body length and severe vertebral deformities. Interestingly, all the double mutants died at the embryonic stage. Moreover, to further investigate the molecular and regulatory mechanisms, we conducted comparative transcriptome analysis on bone and brain tissues from xpr1b+/+ and xpr1b-/- zebrafish. In bone tissue, 6749 DEGs were identified, comprising 3846 upregulated and 2903 downregulated genes. These DEGs were mainly enriched in the MAPK signaling pathway, Wnt signaling pathway, cysteine and methionine metabolism, and ECM-receptor interaction. RT-qPCR validated results showed that seven osteogenesis-related genes (col1a1a, sp7, runx2b, col1a2, col1a1b, alp1 and entpd5), and two phosphate homeostasis related genes (slc20a2 and pdgfba), which are essential for skeletal mineralization and phosphate homeostasis, exhibited significantly downregulated expression in bone tissue of xpr1b mutant zebrafish. These results highlight the pivotal role of xpr1b in regulating skeletal mineralization and phosphate metabolism, thereby elucidating the functional specialization of XPR1 paralogs while providing a theoretical basis for understanding bone developmental mechanism in teleost vertebrates.

Animals↗

PubMed2006

Gene expression patterns of the liver in response to alcohol: in vivo and in vitro models compared.

Two basic models of alcoholic liver disease pathogenesis exist, one in vivo and one in vitro. To justify the in vitro model, evidence is needed to show that it stimulates the in vivo model. Therefore, changes in gene expression caused by high ethanol level were compared using the two models. Many functional pathways were upregulated in both models. These included the insulin signaling pathway, TGFbeta signaling pathway, apoptosis, MAPK signaling pathway, wnt signaling pathway and apoptosis. Differences were found in the fatty acids synthesis pathway, which was upregulated in vivo; and glycosylation enzymes which were downregulated in vivo. Also, downregulated in vitro were beta oxidation by mitochondria and translation factors. Catalase and superoxide dismutase in mitochondria were upregulated in vitro. These two enzymes have antioxidant effects. In summary, remarkably similar responses to high alcohol levels in the form of changes in gene expression pathways were found in the in vivo and in vitro models tested.

Animals↗

PubMed2006

Neuronal production and precursor proliferation defects in the neocortex of mice with loss of function in the canonical Wnt signaling pathway.

To better understand the function of the Wnt pathway in the developing telencephalon, we analyzed neocortical development in low density lipoprotein receptor-related protein (LRP) 6 mutants. LRP6 mutant mice are hypomorphic for the canonical Wnt signaling pathway and have hypoplasia of the developing neocortex. While early telencephalic morphogenesis is largely intact in these mice, probably due to compensation by LRP5, the mutant mice develop a dramatically thinner cortical plate. There is a prominent reduction of neurogenesis leading to a thin cortical plate. Reduced proliferation late in gestation probably also contributes to the hypoplasia. Although there are marked decreases in the numbers of layer 6 and layers 2-4 neurons all laminar identities are generated and there is no evidence of compensatory increases in layer 5 neurons. In addition, LRP6 mutants have partial penetrance of a complex of cortical dysmorphologies resembling those found in patients with developmental forms of epilepsy and mental retardation. These include ventricular and marginal zone heterotopias and cobblestone lissencephaly. This analysis demonstrates that canonical Wnt signaling is required for a diverse array of developmental processes in the neocortex in addition to the previously known roles in regulating precursor proliferation and patterning.

Animals↗

PubMed2006

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo.

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Amino Acid Sequence↗

PubMed2006

Alterations of the Wnt signaling pathway during the neoplastic progression of Barrett's esophagus.

Aberrant activation of the Wnt signaling pathway has been reported during neoplastic progression in Barrett's esophagus (BE). However, mutations in APC and CTNNB1 genes were rarely observed. In this study, expression pattern of Wnt ligands, Frizzled receptors and APC, as well as the methylation status of the APC, SFRP1 and SFRP2 promoter genes were investigated in normal esophageal mucosa and in preneoplastic and neoplastic lesions of BE patients. Promoter methylation of APC was found in all BE samples and in 95% of esophageal adenocarcinomas (EAC). Full methylation of APC correlated with lack of expression. In EAC, nuclear translocation of beta-catenin was observed regardless of the expression of APC. WNT2 expression was higher in dysplasia and EAC than in BE, with 20/26 (77%) of the EAC showing high expression of WNT2. SFRP1 methylation occurred in all BE samples and in 96% of EAC, while SFRP2 was methylated in 73% of the normal squamous esophageal mucosa samples. In conclusion, (1) alterations of key regulators of the Wnt signaling are frequent in the pathogenesis of BE; (2) the APC and SFRP1 genes are inactivated by promoter methylation in BE; (3) the WNT2 gene is upregulated along the progression from low-grade dysplasia to EAC.

Adenocarcinoma↗

PubMed2025

MEIS1 knockdown upregulates WNT signaling pathway genes in esophageal squamous cell carcinoma.

BACKGROUND: The transcription factor MEIS1 belongs to the 3-amino acid loop extension (TALE) family of homeodomain proteins which plays various functions in normal and tumor cell progression. The canonical WNT/β-catenin pathway governs a plethora of biological processes including cell proliferation, differentiation, and tumor development. In the present study, the effect of MEIS1 gene silencing was assessed on WNT pathway genes in esophageal squamous cell carcinoma (ESCC) cells. MATERIALS AND METHODS: Along with the packaging plasmids, the pLKO.1-MEIS1 plasmid was cotransfected into HEK293T to generate lentiviral particles, followed by transduction of a semi-confluent KYSE-30 cell culture. After total RNA extraction and cDNA synthesis, comparative real-time PCR was applied to assess the efficiency of MEIS1 knockdown and the expression of genes related to the WNT signaling pathway. RESULTS: The results revealed effective downregulation of MEIS1 in KYSE-30 cells. Interestingly, MEIS1 silencing led to a substantial overexpression of WNT pathway key components while the expression of negative regulators of this pathway was substantially decreased. CONCLUSIONS: Our data suggest that MEIS1 gene probably induces WNT/β-catenin pathway deactivation in ESCC cells. Consequently, the inverse correlation of MEIS1 expression and WNT signaling pathway activation may introduce a new molecular linkage through ESCC progression and aggressiveness.

Humans↗

PubMed2005

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.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells.

Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing beta-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast.

Animals↗

PubMed2002

Aberrant nuclear localization and gene mutation of beta-catenin in low-grade adenocarcinoma of fetal lung type: up-regulation of the Wnt signaling pathway may be a common denominator for the development of tumors that form morules.

The salient histopathologic features of low-grade adenocarcinoma of the fetal lung type (L-FLAC)/well-differentiated fetal adenocarcinoma (WDFA) include complex glandular structures and morules with biotin-rich optically clear nuclei. Interestingly, these characteristic features are shared by the cribriform-morular variant of papillary thyroid carcinoma, whose morphology is identical to that of familial adenomatous polyposis (FAP)-associated thyroid carcinoma. Furthermore, the single reported case of lung cancer associated with FAP was L-FLAC/WDFA. These observations lead us to hypothesize that up-regulation of the Wnt signaling pathway underlies the development of L-FLAC/WDFA. To verify this hypothesis, 11 cases of L-FLAC/WDFA, including the one FAP-associated case, eight cases of high-grade adenocarcinoma of the fetal lung type (H-FLAC), 24 cases of conventional pulmonary adenocarcinoma (CAC), and 13 fetal lungs were immunostained for beta-catenin. All cases of L-FLAC/WDFA showed predominantly aberrant nuclear/cytoplasmic expression, especially in budding glands and morules, whereas six of eight cases (75%) of H-FLAC and all but one case (96%) of CAC showed predominantly membranous expression. Fetal lungs showed nuclear/cytoplasmic expression restricted to the distal branching airway epithelium. Mutational analysis of exon 3 of the beta-catenin gene in five sporadic cases of L-FLAC/WDFA showed a point mutation at codon 34 and codon 37 in two cases, respectively. The present study indicates that up-regulating disturbances in the Wnt signaling pathway, including mutation of the beta-catenin gene, underlie tumorigenesis of L-FLAC/WDFA. The expression pattern of beta-catenin in L-FLAC/WDFA resembles that of the developing fetal lung airway. With the expression pattern of beta-catenin as a marker, most cases of H-FLAC as well as CAC appear to have different oncogenic pathways from cases of L-FLAC/WDFA. The present study together with other available data also suggests that abnormal up-regulation of the Wnt signaling pathway may be a common denominator for the development of tumors with morular formation from a variety of anatomic sites.

Adenocarcinoma↗

PubMed2005

TOPGAL mice show that the canonical Wnt signaling pathway is active during bone development and growth and is activated by mechanical loading in vitro.

UNLABELLED: We identified cellular targets of canonical Wnt signaling within the skeleton, which included chondrocytes, osteoblasts, and osteocytes in growing bone, but only osteocytes and chondrocytes in the mature skeleton. Mechanical deformation induced Wnt signaling in osteoblasts in vitro. INTRODUCTION: Genetic evidence in mice and humans has implicated the canonical Wnt signaling pathway in the control of skeletal development and bone mass. However, little is known of the details of Wnt signaling in the skeleton in vivo. We used Wnt indicator TOPGAL mice to identify which cells activated this pathway during bone development and in the mature skeleton. MATERIALS AND METHODS: We examined canonical Wnt signaling during embryonic and neonatal bone development in TOPGAL mice. The TOPGAL transgene consists of a beta-galactosidase gene driven by a T cell factor (TCF)beta-catenin responsive promoter so that canonical Wnt activity can be detected by X-gal staining. Expression of Wnt signaling components was examined in primary calvarial cell cultures by RT-PCR. The effect of mechanical deformation on Wnt signaling was examined in primary calvarial cells grown on collagen I and stretched using Flexercell Tension Plus System FX-4000T. Immunohistochemistry was used to examine the localization of beta-catenin in cartilage, bone, and cultured calvarial cells exposed to physical deformation. RESULTS AND CONCLUSIONS: Canonical Wnt signaling was active in several cell types in the fetal and neonatal skeleton, including chondrocytes, osteoblasts, and osteocytes. With age, activation of Wnt signaling became less prominent but persisted in chondrocytes and osteocytes. Although osteoblasts in culture expressed many different individual Wnt's and Wnt receptors, the TOPGAL transgene was not active in these cells at baseline. However, Wnt signaling was activated in these cells by physical deformation. Together with the activation of canonical Wnt signaling in osteocytes seen in vivo, these data suggest that Wnt signaling may be involved in the coupling of mechanical force to anabolic activity in the skeleton.

Animals↗