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Sample records for activates canonical wnt

  1. Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration.

    PubMed

    Han, Pingping; Ivanovski, Saso; Crawford, Ross; Xiao, Yin

    2015-07-01

    Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost because of disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (1) local injection of lithium chloride; (2) local injection of sclerostin antibody; and (3) local injection of a lentiviral construct overexpressing β-catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well-organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs. PMID:25556853

  2. Canonical Wnt Signaling Activity in Early Stages of Chick Lung Development

    PubMed Central

    daMota, Paulo; Correia-Pinto, Jorge

    2014-01-01

    Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good

  3. Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells

    PubMed Central

    Corbett, Laura; Mann, Jelena; Mann, Derek A.

    2015-01-01

    The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis. PMID:26566235

  4. KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification

    PubMed Central

    Tetreault, Marie-Pier; Weinblatt, Daniel; Shaverdashvili, Khvaramze; Yang, Yizeng; Katz, Jonathan P.

    2016-01-01

    Epithelial differentiation and stratification are essential for normal homeostasis, and disruption of these processes leads to both injury and cancer. The zinc-finger transciption factor KLF4 is a key driver of epithelial differentiation, yet the mechanisms and targets by which KLF4 controls differentiation are not well understood. Here, we define WNT5A, a non-canonical Wnt ligand implicated in epithelial differentiation, repair, and cancer, as a direct transcriptional target that is activated by KLF4 in squamous epithelial cells. Further, we demonstrate functionally that WNT5A mediates KLF4 control of epithelial differentiation and stratification, as treatment of keratinocytes with WNT5A rescues defective epithelial stratification resulting from KLF4 loss. Finally, we show that the small GTPase CDC42 is regulated by KLF4 in a WNT5A dependent manner. As such, we delineate a novel pathway for epithelial differentiation and stratification and define potential therapeutic targets for epithelial diseases. PMID:27184424

  5. Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways

    PubMed Central

    Weivoda, Megan M; Ruan, Ming; Hachfeld, Christine M; Pederson, Larry; Howe, Alan; Davey, Rachel A; Zajac, Jeffrey D; Kobayashi, Yasuhiro; Williams, Bart O; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear β-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of β-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (β-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that β-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data

  6. Ligand-Independent Canonical Wnt Activity in Canine Mammary Tumor Cell Lines Associated with Aberrant LEF1 Expression

    PubMed Central

    van Wolferen, Monique E.; Rao, Nagesha A. S.; Grizelj, Juraj; Vince, Silvijo; Hellmen, Eva; Mol, Jan A.

    2014-01-01

    Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand–independent mechanisms. PMID:24887235

  7. Activation of the canonical Wnt/{beta}-catenin pathway enhances monocyte adhesion to endothelial cells

    SciTech Connect

    Lee, Dong Kun . E-mail: leedk@memorialhealthsource.com; Nathan Grantham, R.; Trachte, Aaron L.; Mannion, John D.; Wilson, Colleen L.

    2006-08-18

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/{beta}-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3{beta} or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/{beta}-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/{beta}-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules.

  8. Glucose induced activation of canonical Wnt signaling pathway in hepatocellular carcinoma is regulated by DKK4.

    PubMed

    Chouhan, Surbhi; Singh, Snahlata; Athavale, Dipti; Ramteke, Pranay; Pandey, Vimal; Joseph, Jomon; Mohan, Rajashekar; Shetty, Praveen Kumar; Bhat, Manoj Kumar

    2016-01-01

    Elevated glycemic index, an important feature of diabetes is implicated in an increased risk of hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of this association are relatively less explored. Present study investigates the effect of hyperglycemia over HCC proliferation. We observed that high glucose culture condition (HG) specifically activates canonical Wnt signaling in HCC cells, which is mediated by suppression of DKK4 (a Wnt antagonist) expression and enhanced β-catenin level. Functional assays demonstrated that a normoglycemic culture condition (NG) maintains constitutive expression of DKK4, which controls HCC proliferation rate by suppressing canonical Wnt signaling pathway. HG diminishes DKK4 expression leading to loss of check at G0/G1/S phases of the cell cycle thereby enhancing HCC proliferation, in a β-catenin dependent manner. Interestingly, in NOD/SCID mice supplemented with high glucose, HepG2 xenografted tumors grew rapidly in which elevated levels of β-catenin, c-Myc and decreased levels of DKK4 were detected. Knockdown of DKK4 by shRNA promotes proliferation of HCC cells in NG, which is suppressed by treating cells exogenously with recombinant DKK4 protein. Our in vitro and in vivo results indicate an important functional role of DKK4 in glucose facilitated HCC proliferation. PMID:27272409

  9. Glucose induced activation of canonical Wnt signaling pathway in hepatocellular carcinoma is regulated by DKK4

    PubMed Central

    Chouhan, Surbhi; Singh, Snahlata; Athavale, Dipti; Ramteke, Pranay; Pandey, Vimal; Joseph, Jomon; Mohan, Rajashekar; Shetty, Praveen Kumar; Bhat, Manoj Kumar

    2016-01-01

    Elevated glycemic index, an important feature of diabetes is implicated in an increased risk of hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of this association are relatively less explored. Present study investigates the effect of hyperglycemia over HCC proliferation. We observed that high glucose culture condition (HG) specifically activates canonical Wnt signaling in HCC cells, which is mediated by suppression of DKK4 (a Wnt antagonist) expression and enhanced β-catenin level. Functional assays demonstrated that a normoglycemic culture condition (NG) maintains constitutive expression of DKK4, which controls HCC proliferation rate by suppressing canonical Wnt signaling pathway. HG diminishes DKK4 expression leading to loss of check at G0/G1/S phases of the cell cycle thereby enhancing HCC proliferation, in a β-catenin dependent manner. Interestingly, in NOD/SCID mice supplemented with high glucose, HepG2 xenografted tumors grew rapidly in which elevated levels of β-catenin, c-Myc and decreased levels of DKK4 were detected. Knockdown of DKK4 by shRNA promotes proliferation of HCC cells in NG, which is suppressed by treating cells exogenously with recombinant DKK4 protein. Our in vitro and in vivo results indicate an important functional role of DKK4 in glucose facilitated HCC proliferation. PMID:27272409

  10. FZD6, targeted by miR-21, represses gastric cancer cell proliferation and migration via activating non-canonical wnt pathway

    PubMed Central

    Yan, Jin; Liu, Tingyu; Zhou, Xiaoying; Dang, Yini; Yin, Chengqiang; Zhang, Guoxin

    2016-01-01

    FZD6 plays crucial roles in human tumorigenesis. However, its mechanism in regulating cancers has not been fully elucidated. In the study, we found that FZD6 repressed gastric cancer cell proliferation and migration via activating non-canonical wnt pathway. In addition, non-canonical wnt pathway ameliorated expression of canonical wnt pathway. We also demonstrated that the FZD6 was involved in miR-21-dependent effects in the canonical and non-canonical wnt pathways in gastric cancer. These findings provide a better understanding of the development and progression of gastric cancer and may be an important implication for future therapy. PMID:27347343

  11. The Novel Secreted Adipokine WNT1-inducible Signaling Pathway Protein 2 (WISP2) Is a Mesenchymal Cell Activator of Canonical WNT*

    PubMed Central

    Grünberg, John R.; Hammarstedt, Ann; Hedjazifar, Shahram; Smith, Ulf

    2014-01-01

    WNT1-inducible-signaling pathway protein 2 (WISP2) is primarily expressed in mesenchymal stem cells, fibroblasts, and adipogenic precursor cells. It is both a secreted and cytosolic protein, the latter regulating precursor cell adipogenic commitment and PPARγ induction by BMP4. To examine the effect of the secreted protein, we expressed a full-length and a truncated, non-secreted WISP2 in NIH3T3 fibroblasts. Secreted, but not truncated WISP2 activated the canonical WNT pathway with increased β-catenin levels, its nuclear targeting phosphorylation, and LRP5/6 phosphorylation. It also inhibited Pparg activation and the effect of secreted WISP2 was reversed by the WNT antagonist DICKKOPF-1. Differentiated 3T3-L1 adipose cells were also target cells where extracellular WISP2 activated the canonical WNT pathway, inhibited Pparg and associated adipose genes and, similar to WNT3a, promoted partial dedifferentiation of the cells and the induction of a myofibroblast phenotype with activation of markers of fibrosis. Thus, WISP2 exerts dual actions in mesenchymal precursor cells; secreted WISP2 activates canonical WNT and maintains the cells in an undifferentiated state, whereas cytosolic WISP2 regulates adipogenic commitment. PMID:24451367

  12. Sulf1 has ligand-dependent effects on canonical and non-canonical Wnt signalling

    PubMed Central

    Fellgett, Simon W.; Maguire, Richard J.; Pownall, Mary Elizabeth

    2015-01-01

    ABSTRACT Wnt signalling plays essential roles during embryonic development and is known to be mis-regulated in human disease. There are many molecular mechanisms that ensure tight regulation of Wnt activity. One such regulator is the heparan-sulfate-specific 6-O-endosulfatase Sulf1. Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bio-availability of Wnt ligands. Sulf1 could, therefore, influence the formation of Wnt signalling complexes to modulate the activation of both canonical and non-canonical pathways. In this study, we use well-established assays in Xenopus to investigate the ability of Sulf1 to modify canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants. We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands differently, and inhibits the activity of canonical Wnt8a but enhances the activity of non-canonical Wnt11b. PMID:25681501

  13. Canonical Wnt activity regulates trunk neural crest delamination linking BMP/noggin signaling with G1/S transition.

    PubMed

    Burstyn-Cohen, Tal; Stanleigh, Jonathan; Sela-Donenfeld, Dalit; Kalcheim, Chaya

    2004-11-01

    Delamination of premigratory neural crest cells depends on a balance between BMP/noggin and on successful G1/S transition. Here, we report that BMP regulates G1/S transition and consequent crest delamination through canonical Wnt signaling. Noggin overexpression inhibits G1/S transition and blocking G1/S abrogates BMP-induced delamination; moreover, transcription of Wnt1 is stimulated by BMP and by the developing somites, which concomitantly inhibit noggin production. Interfering with beta-catenin and LEF/TCF inhibits G1/S transition, neural crest delamination and transcription of various BMP-dependent genes, which include Cad6B, Pax3 and Msx1, but not that of Slug, Sox9 or FoxD3. Hence, we propose that developing somites inhibit noggin transcription in the dorsal tube, resulting in activation of BMP and consequent Wnt1 production. Canonical Wnt signaling in turn stimulates G1/S transition and generation of neural crest cell motility independently of its proposed role in earlier neural crest specification. PMID:15456730

  14. Renal Hypodysplasia Associates with a Wnt4 Variant that Causes Aberrant Canonical Wnt Signaling

    PubMed Central

    Vivante, Asaf; Mark-Danieli, Michal; Davidovits, Miriam; Harari-Steinberg, Orit; Omer, Dorit; Gnatek, Yehudit; Cleper, Roxana; Landau, Daniel; Kovalski, Yael; Weissman, Irit; Eisenstein, Israel; Soudack, Michalle; Wolf, Haike Reznik; Issler, Naomi; Lotan, Danny; Anikster, Yair

    2013-01-01

    Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well understood. In this multicenter study, we identified 20 Israeli pedigrees with isolated familial, nonsyndromic RHD and screened for mutations in candidate genes involved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, GDNF, WNT4, and WT1. In addition to previously reported RHD-causing genes, we found that two affected brothers were heterozygous for a missense variant in the WNT4 gene. Functional analysis of this variant revealed both antagonistic and agonistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibited WNT3A induced canonical activation, and the WNT4 variant significantly enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cultures of human fetal kidney cells, which maintain WNT activation and more closely represent WNT signaling in renal progenitors during nephrogenesis, this mutation caused significant loss of function, resulting in diminished canonical WNT/β-catenin signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia. PMID:23520208

  15. Malignant gliomas induce and exploit astrocytic mesenchymal-like transition by activating canonical Wnt/β-catenin signaling.

    PubMed

    Lu, Ping; Wang, Yajing; Liu, Xiuting; Wang, Hong; Zhang, Xin; Wang, Kequan; Wang, Qing; Hu, Rong

    2016-07-01

    The complex microenvironment of malignant gliomas plays a dynamic and usually cancer-promoting role in glioma progression. Astrocytes, the major stromal cells in the brain, can be activated by glioma microenvironment, resulting in a layer of reactive astrocytes surrounding the gliomas. Reactive astrocytes are universally characterized with the upregulation of glial fibrillary protein and glycoprotein podoplanin. In this work, we investigated the role of reactive astrocytes on malignant glioma microenvironment and the potential mechanism by which glioma cells activated the tumor-associated astrocytes (TAAs). The reactive astrocytes were observed around gliomas in the intracranial syngeneic implantation of rat C6 and mouse GL261 glioma cells in vivo, as well as primary astrocytes cultured with glioma cells condition medium in vitro. Besides, reactive astrocytes exhibited distinct epithelial-to-mesenchymal (-like) transition and enhanced migration and invasion activity, with the decrease of E-cadherin and concomitant increase of vimentin and matrix metalloproteinases. Furthermore, canonical Wnt/β-catenin signaling was activated in TAAs. The Wnt/β-catenin pathway inhibitor XAV939 and β-catenin plasmid were used to verify the regulation of Wnt/β-catenin signaling on TAAs and their invasion ability. Taken together, our findings established that glioma cells remarkably activated astrocytes via upregulating Wnt/β-catenin signaling, with obviously mesenchymal-like transition and increased migration and invasion ability, indicating that glioma cells may stimulate adjacent astrocytes to degrade extracellular matrix and thereby promoting tumor invasiveness. PMID:27236327

  16. Canonical Wnt Signaling Regulates Atrioventricular Junction Programming and Electrophysiological Properties

    PubMed Central

    Gillers, Benjamin S; Chiplunkar, Aditi; Aly, Haytham; Valenta, Tomas; Basler, Konrad; Christoffels, Vincent M.; Efimov, Igor R; Boukens, Bastiaan J; Rentschler, Stacey

    2014-01-01

    Rationale Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease. Objective To determine the role of canonical Wnt signaling in the myocardium during AVC development. Methods and Results We utilized a novel allele of β-catenin that preserves β-catenin’s cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiologic criteria. Aberrant AVC development can lead to ventricular preexcitation, a characteristic feature of Wolff-Parkinson-White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of β-catenin protein levels can rescue Notch-mediated ventricular preexcitation and dysregulated ion channel gene expression. Conclusions Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electrical programming upstream of Tbx3. Our data further suggests that ventricular preexcitation may require both morphologic patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue. PMID:25599332

  17. Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation

    SciTech Connect

    Yoshida, Go J. Saya, Hideyuki

    2014-01-10

    Highlights: •CD44 variant8–10 and c-Myc are inversely expressed in gastric cancer cells. •Redox-stress enhances c-Myc expression via canonical Wnt signal. •CD44v, but not CD44 standard, suppresses redox stress-induced Wnt activation. •CD44v expression promotes both transcription and proteasome degradation of c-Myc. •Inversed expression pattern between CD44v and c-Myc is often recognized in vivo. -- Abstract: Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells. That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup high}/ c-Myc {sup low} and proliferative cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup low}/ c-Myc {sup high}.

  18. Canonical Wnt signaling in the oligodendroglial lineage--puzzles remain.

    PubMed

    Guo, Fuzheng; Lang, Jordan; Sohn, Jiho; Hammond, Elizabeth; Chang, Marcello; Pleasure, David

    2015-10-01

    The straightforward concept that accentuated Wnt signaling via the Wnt-receptor-β-catenin-TCF/LEF cascade (also termed canonical Wnt signaling or Wnt/β-catenin signaling) delays or blocks oligodendrocyte differentiation is very appealing. According to this concept, canonical Wnt signaling is responsible for remyelination failure in multiple sclerosis and for persistent hypomyelination in periventricular leukomalacia. This has given rise to the hope that pharmacologically inhibiting this signaling will be of therapeutic potential in these disabling neurological disorders. But current studies suggest that Wnt/β-catenin signaling plays distinct roles in oligodendrogenesis, oligodendrocyte differentiation, and myelination in a context-dependent manner (central nervous system regions, developmental stages), and that Wnt/β-catenin signaling interplays with, and is subjected to regulation by, other central nervous system factors and signaling pathways. On this basis, we propose the more nuanced concept that endogenous Wnt/β-catenin activity is delicately and temporally regulated to ensure the seamless development of oligodendroglial lineage cells in different contexts. In this review, we discuss the role Wnt/β-catenin signaling in oligodendrocyte development, focusing on the interpretation of disparate results, and highlighting areas where important questions remain to be answered about oligodendroglial lineage Wnt/β-catenin signaling. PMID:25782433

  19. Frat is dispensable for canonical Wnt signaling in mammals

    PubMed Central

    van Amerongen, Renée; Nawijn, Martijn; Franca-Koh, Jonathan; Zevenhoven, John; van der Gulden, Hanneke; Jonkers, Jos; Berns, Anton

    2005-01-01

    Wnt-signal transduction through β-catenin is thought to require the inhibition of GSK3 by Frat/GBP. To investigate the role of Frat in mammalian development, we have generated mice with targeted mutations in all three murine Frat homologs. We show that Frat is normally expressed at sites of active Wnt signaling. Surprisingly, Frat-deficient mice do not display gross abnormalities. Moreover, canonical Wnt signaling in primary cells is unaffected by the loss of Frat. These studies show that Frat is not an essential component of the canonical Wnt pathway in higher organisms, despite the strict requirement of Frat/GBP for maternal Wnt signaling in Xenopus. PMID:15681612

  20. Medicarpin, a Natural Pterocarpan, Heals Cortical Bone Defect by Activation of Notch and Wnt Canonical Signaling Pathways

    PubMed Central

    Gupta, Chandra Prakash; Kureel, Jyoti; Mansoori, Mohd Nizam; Shukla, Priyanka; John, Aijaz A.; Singh, Kavita; Purohit, Dipak; Awasthi, Pallavi; Singh, Divya; Goel, Atul

    2015-01-01

    We evaluated the bone regeneration and healing effect of Medicarpin (med) in cortical bone defect model that heals by intramembranous ossification. For the study, female Sprague–Dawley rats were ovariectomized and rendered osteopenic. A drill hole injury was generated in mid femoral bones of all the animals. Med treatment was commenced the day after and continued for 15 days. PTH was taken as a reference standard. Fifteen days post-treatment, animals were sacrificed. Bones were collected for histomorphometry studies at the injury site by micro-computed tomography (μCT) and confocal microscopy. RNA and protein was harvested from newly generated bone. For immunohistochemistry, 5μm sections of decalcified femur bone adjoining the drill hole site were cut. By μCT analysis and calcein labeling of newly generated bone it was found that med promotes bone healing and new bone formation at the injury site and was comparable to PTH in many aspects. Med treatment led to increase in the Runx-2 and osteocalcin signals indicating expansion of osteoprogenitors at the injury site as evaluated by qPCR and immunohistochemical localization. It was observed that med promoted bone regeneration by activating canonical Wnt and notch signaling pathway. This was evident by increased transcript and protein levels of Wnt and notch signaling components in the defect region. Finally, we confirmed that med treatment leads to elevated bone healing in pre-osteoblasts by co localization of beta catenin with osteoblast marker alkaline phosphatase. In conclusion, med treatment promotes new bone regeneration and healing at the injury site by activating Wnt/canonical and notch signaling pathways. This study also forms a strong case for evaluation of med in delayed union and non-union fracture cases. PMID:26657206

  1. Canonical wnt signaling is required for commissural axon guidance

    PubMed Central

    Avilés, Evelyn C.

    2015-01-01

    ABSTRACT Morphogens have been identified as guidance cues for postcrossing commissural axons in the spinal cord. Shh has a dual effect on postcrossing commissural axons: a direct repellent effect mediated by Hhip as a receptor, and an indirect effect by shaping a Wnt activity gradient. Wnts were shown to be attractants for postcrossing commissural axons in both chicken and mouse embryos. In mouse, the effects of Wnts on axon guidance were concluded to depend on the planar cell polarity (PCP) pathway. Canonical Wnt signaling was excluded based on the absence of axon guidance defects in mice lacking Lrp6 which is an obligatory coreceptor for Fzd in canonical Wnt signaling. In the loss‐of‐function studies reported here, we confirmed a role for the PCP pathway in postcrossing commissural axon guidance also in the chicken embryo. However, taking advantage of the precise temporal control of gene silencing provided by in ovo RNAi, we demonstrate that canonical Wnt signaling is also required for proper guidance of postcrossing commissural axons in the developing spinal cord. Thus, axon guidance does not seem to depend on any one of the classical Wnt signaling pathways but rather involve a network of Wnt receptors and downstream components. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 190–208, 2016 PMID:26014644

  2. β-Catenin-dependent pathway activation by both promiscuous "canonical" WNT3a-, and specific "noncanonical" WNT4- and WNT5a-FZD receptor combinations with strong differences in LRP5 and LRP6 dependency.

    PubMed

    Ring, Larisa; Neth, Peter; Weber, Christian; Steffens, Sabine; Faussner, Alexander

    2014-02-01

    The WNT/β-catenin signalling cascade is the best-investigated frizzled receptor (FZD) pathway, however, whether and how specific combinations of WNT/FZD and co-receptors LRP5 and LRP6 differentially affect this pathway are not well understood. This is mostly due to the fact that there are 19 WNTs, 10 FZDs and at least two co-receptors. In our attempt to identify the signalling capabilities of specific WNT/FZD/LRP combinations we made use of our previously reported TCF/LEF Gaussia luciferase reporter gene HEK293 cell line (Ring et al., 2011). Generation of WNT/FZD fusion constructs - but not their separate transfection - without or with additional isogenic overexpression of LRP5 and LRP6 in our reporter cells permitted the investigation of specific WNT/FZD/LRP combinations. The canonical WNT3a in fusion to almost all FZDs was able to induce β-catenin-dependent signalling with strong dependency on LRP6 but not LRP5. Interestingly, noncanonical WNT ligands, WNT4 and WNT5a, were also able to act "canonically" but only in fusion with specific FZDs and with selective dependence on LRP5 or LRP6. These data and extension of this experimental setup to the poorly characterized other WNTs should facilitate deeper insight into the complex WNT/FZD signalling system and its function. PMID:24269653

  3. Controlled levels of canonical Wnt signaling are required for neural crest migration.

    PubMed

    Maj, Ewa; Künneke, Lutz; Loresch, Elisabeth; Grund, Anita; Melchert, Juliane; Pieler, Tomas; Aspelmeier, Timo; Borchers, Annette

    2016-09-01

    Canonical Wnt signaling plays a dominant role in the development of the neural crest (NC), a highly migratory cell population that generates a vast array of cell types. Canonical Wnt signaling is required for NC induction as well as differentiation, however its role in NC migration remains largely unknown. Analyzing nuclear localization of β-catenin as readout for canonical Wnt activity, we detect nuclear β-catenin in premigratory but not migratory Xenopus NC cells suggesting that canonical Wnt activity has to decrease to basal levels to enable NC migration. To define a possible function of canonical Wnt signaling in Xenopus NC migration, canonical Wnt signaling was modulated at different time points after NC induction. This was accomplished using either chemical modulators affecting β-catenin stability or inducible glucocorticoid fusion constructs of Lef/Tcf transcription factors. In vivo analysis of NC migration by whole mount in situ hybridization demonstrates that ectopic activation of canonical Wnt signaling inhibits cranial NC migration. Further, NC transplantation experiments confirm that this effect is tissue-autonomous. In addition, live-cell imaging in combination with biophysical data analysis of explanted NC cells confirms the in vivo findings and demonstrates that modulation of canonical Wnt signaling affects the ability of NC cells to perform single cell migration. Thus, our data support the hypothesis that canonical Wnt signaling needs to be tightly controlled to enable migration of NC cells. PMID:27341758

  4. Modulation of canonical Wnt signaling by the extracellular matrix component biglycan

    PubMed Central

    Berendsen, Agnes D.; Fisher, Larry W.; Kilts, Tina M.; Owens, Rick T.; Robey, Pamela G.; Gutkind, J. Silvio; Young, Marian F.

    2011-01-01

    Although extracellular control of canonical Wnt signaling is crucial for tissue homeostasis, the role of the extracellular microenvironment in modulating this signaling pathway is largely unknown. In the present study, we show that a member of the small leucine-rich proteoglycan family, biglycan, enhances canonical Wnt signaling by mediating Wnt function via its core protein. Immunoprecipitation analysis revealed that biglycan interacts with both the canonical Wnt ligand Wnt3a and the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), possibly via the formation of a trimeric complex. Biglycan-deficient cells treated with exogenous Wnt3a had less Wnt3a retained in cell layers compared with WT cells. Furthermore, the Wnt-induced levels of LRP6 phosphorylation and expression of several Wnt target genes were blunted in biglycan-deficient cells. Both recombinant biglycan proteoglycan and biglycan core protein increased Wnt-induced β-catenin/T cell-specific factor–mediated transcriptional activity, and this activity was completely inhibited by Dickkopf 1. Interestingly, recombinant biglycan was able to rescue impaired Wnt signaling caused by a previously described missense mutation in the extracellular domain of human LRP6 (R611C). Furthermore, biglycan's modulation of canonical Wnt signaling affected the functional activities of osteoprogenitor cells, including the RUNX2-mediated transcriptional activity and calcium deposition. Use of a transplant system and a fracture healing model revealed that expression of Wnt-induced secreted protein 1 was decreased in bone formed by biglycan-deficient cells, further suggesting reduced Wnt signaling in vivo. We propose that biglycan may serve as a reservoir for Wnt in the pericellular space and modulate Wnt availability for activation of the canonical Wnt pathway. PMID:21969569

  5. Structure-activity relationship and properties optimization of a series of quinazoline-2,4-diones as inhibitors of the canonical Wnt pathway.

    PubMed

    Nencini, Arianna; Pratelli, Carmela; Quinn, Joanna M; Salerno, Massimiliano; Tunici, Patrizia; De Robertis, Alessandra; Valensin, Silvia; Mennillo, Federica; Rossi, Marco; Bakker, Annette; Benicchi, Tiziana; Cappelli, Federico; Turlizzi, Elisa; Nibbio, Martina; Caradonna, Nicola P; Zanelli, Ugo; Andreini, Matteo; Magnani, Matteo; Varrone, Maurizio

    2015-05-01

    Wnt signaling pathway plays a critical role in numerous cellular processes, including tumor initiation, proliferation, invasion/infiltration, metastasis formation and resistance to chemotherapy. In a drug discovery project aimed at the identification of inhibitors of the canonical Wnt pathway, we selected a series of quinazoline 2,4-diones as starting point for the therapeutic treatment of glioblastoma multiforme. Despite of poor physico-chemical properties of hit compound 1, our medicinal chemistry effort allowed the discovery and characterization of lead compound 33 (SEN461), with improved ADME profile, good bioavailability and active in vitro and in vivo in glioblastoma, gastric and sarcoma tumors. PMID:25847770

  6. Glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts

    SciTech Connect

    Ohnaka, Keizo . E-mail: oonaka@geriat.med.kyushu-u.ac.jp; Tanabe, Mizuho; Kawate, Hisaya; Nawata, Hajime; Takayanagi, Ryoichi

    2005-04-01

    To explore the mechanism of glucocorticoid-induced osteoporosis, we investigated the effect of glucocorticoid on canonical Wnt signaling that emerged as a novel key pathway for promoting bone formation. Wnt3a increased the T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-dependent transcriptional activity in primary cultured human osteoblasts. Dexamethasone suppressed this transcriptional activity in a dose-dependent manner, while 1,25-dihydroxyvitamin D3 increased this transcriptional activity. LiCl, an inhibitor of glycogen synthase kinase-3{beta}, also enhanced the Tcf/Lef-dependent transcriptional activity, which was, however, not inhibited by dexamethasone. The addition of anti-dickkopf-1 antibody partially restored the transcriptional activity suppressed by dexamethasone. Dexamethasone decreased the cytosolic amount of {beta}-catenin accumulated by Wnt3a and also inhibited the nuclear translocation of {beta}-catenin induced by Wnt3a. These data suggest that glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts, partially through the enhancement of the dickkopf-1 production.

  7. Beta-catenin versus the other armadillo catenins: assessing our current view of canonical Wnt signaling.

    PubMed

    Miller, Rachel K; Hong, Ji Yeon; Muñoz, William A; McCrea, Pierre D

    2013-01-01

    The prevailing view of canonical Wnt signaling emphasizes the role of beta-catenin acting downstream of Wnt activation to regulate transcriptional activity. However, emerging evidence indicates that other armadillo catenins in vertebrates, such as members of the p120 subfamily, convey parallel signals to the nucleus downstream of canonical Wnt pathway activation. Their study is thus needed to appreciate the networked mechanisms of canonical Wnt pathway transduction, especially as they may assist in generating the diversity of Wnt effects observed in development and disease. In this chapter, we outline evidence of direct canonical Wnt effects on p120 subfamily members in vertebrates and speculate upon these catenins' roles in conjunction with or aside from beta-catenin. PMID:23481204

  8. Beta-catenin versus the other catenins: assessing our current view of canonical Wnt signaling

    PubMed Central

    Miller, Rachel K.; Hong, Ji Yeon; Muñoz, William A.; McCrea, Pierre D.

    2013-01-01

    The prevailing view of canonical Wnt signaling emphasizes the role of beta-catenin acting downstream of Wnt activation to regulate transcriptional activity. However, emerging evidence indicates that other catenins in vertebrates, such as members of the p120 subfamily, convey parallel signals to the nucleus downstream of canonical Wnt pathway activation. Their study is thus needed to appreciate the networked mechanisms of canonical Wnt pathway transduction, especially as they may assist in generating the diversity of Wnt effects observed in development and disease. In this chapter, we outline evidence of direct canonical Wnt effects on p120-subfamily members in vertebrates, and speculate upon these catenins’ roles in conjunction with or aside from beta-catenin. PMID:23481204

  9. Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition

    PubMed Central

    Aisagbonhi, Omonigho; Rai, Meena; Ryzhov, Sergey; Atria, Nick; Feoktistov, Igor; Hatzopoulos, Antonis K.

    2011-01-01

    SUMMARY Despite available therapies, myocardial infarction (MI) remains a leading cause of death worldwide. Better understanding of the molecular and cellular mechanisms that regulate cardiac repair should help to improve the clinical outcome of MI patients. Using the reporter mouse line TOPGAL, we show that canonical (β-catenin-dependent) Wnt signaling is induced 4 days after experimental MI in subepicardial endothelial cells and perivascular smooth muscle actin (SMA)-positive (SMA+) cells. At 1 week after ischemic injury, a large number of canonical-Wnt-positive cells accumulated in the infarct area during granulation tissue formation. Coincidently with canonical Wnt activation, endothelial-to-mesenchymal transition (EndMT) was also triggered after MI. Using cell lineage tracing, we show that a significant portion of the canonical-Wnt-marked SMA+ mesenchymal cells is derived from endothelial cells. Canonical Wnt signaling induces mesenchymal characteristics in cultured endothelial cells, suggesting a direct role in EndMT. In conclusion, our study demonstrates that canonical Wnt activation and EndMT are molecular and cellular responses to MI and that canonical Wnt signaling activity is a characteristic property of EndMT-derived mesenchymal cells that take part in cardiac tissue repair after MI. These findings could lead to new strategies to improve the course of cardiac repair by temporal and cell-type-specific manipulation of canonical Wnt signaling. PMID:21324930

  10. Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway

    PubMed Central

    Yang, Jing; Ye, Ling; Hui, Tian-Qian; Yang, Dong-Mei; Huang, Ding-Ming; Zhou, Xue-Dong; Mao, Jeremy J; Wang, Cheng-Lin

    2015-01-01

    Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/β-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/β-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/β-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of β-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced β-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of β-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation. PMID:26047580

  11. TCDD Inhibition of Canonical Wnt Signaling Disrupts Prostatic Bud Formation in Mouse Urogenital Sinus

    PubMed Central

    Peterson, Richard E.

    2013-01-01

    In mice, in utero exposure to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) reduces the number of dorsolateral prostatic buds resulting in a smaller dorsolateral prostate and prevents formation of ventral buds culminating in ventral prostate agenesis. The genes and signaling pathways affected by TCDD that are responsible for disrupting prostate development are largely unknown. Here we show that treatment of urogenital sinus (UGS) organ cultures with known inhibitors of canonical Wnt signaling also inhibits prostatic bud formation. In support of the hypothesis that TCDD decreases canonical Wnt signaling, we identify inhibitory effects of TCDD on multiple components of the canonical Wnt signaling pathway in the UGS that temporally coincide with the inhibitory effect of TCDD on prostatic bud formation: (1) expression of R-spondins (Rspo2 and Rspo3) that promote canonical Wnt signaling is reduced; (2) expression of Lef1, Tcf1, and Wif1, established canonical Wnt target genes, is decreased; (3) expression of Lgr5, a RSPO receptor that activates canonical Wnt signaling, is reduced; and (4) expression of Dickkopfs (Dkks), inhibitors of canonical Wnt signaling, is not increased by TCDD. Thus, the TCDD-induced reduction in canonical Wnt signaling is associated with a decrease in activators (Rspo2 and Rspo3) rather than an increase in inhibitors (Dkk1 and Dkk2) of the pathway. This study focuses on determining whether treatment of TCDD-exposed UGS organ cultures with RSPO2 and/or RSPO3 is capable of rescuing the inhibitory effects of TCDD on canonical Wnt signaling and prostatic bud formation. We discovered that each RSPO alone or in combination partially rescues TCDD inhibition of both canonical Wnt signaling and prostatic bud formation. PMID:23429912

  12. Canonical Wnt signaling transiently stimulates proliferation and enhances neurogenesis in neonatal neural progenitor cultures

    SciTech Connect

    Hirsch, Cordula; Campano, Louise M.; Woehrle, Simon; Hecht, Andreas . E-mail: andreas.hecht@mol-med.uni-freiburg.de

    2007-02-01

    Canonical Wnt signaling triggers the formation of heterodimeric transcription factor complexes consisting of {beta}-catenin and T cell factors, and thereby controls the execution of specific genetic programs. During the expansion and neurogenic phases of embryonic neural development canonical Wnt signaling initially controls proliferation of neural progenitor cells, and later neuronal differentiation. Whether Wnt growth factors affect neural progenitor cells postnatally is not known. Therefore, we have analyzed the impact of Wnt signaling on neural progenitors isolated from cerebral cortices of newborn mice. Expression profiling of pathway components revealed that these cells are fully equipped to respond to Wnt signals. However, Wnt pathway activation affected only a subset of neonatal progenitors and elicited a limited increase in proliferation and neuronal differentiation in distinct subsets of cells. Moreover, Wnt pathway activation only transiently stimulated S-phase entry but did not support long-term proliferation of progenitor cultures. The dampened nature of the Wnt response correlates with the predominant expression of inhibitory pathway components and the rapid actuation of negative feedback mechanisms. Interestingly, in differentiating cell cultures activation of canonical Wnt signaling reduced Hes1 and Hes5 expression suggesting that during postnatal neural development, Wnt/{beta}-catenin signaling enhances neurogenesis from progenitor cells by interfering with Notch pathway activity.

  13. Three-dimensional culture of sebaceous gland cells revealing the role of prostaglandin E{sub 2}-induced activation of canonical Wnt signaling

    SciTech Connect

    Yoshida, Go J. Saya, Hideyuki

    2013-09-06

    Highlights: •Three-dimensional culture generates “semi-vivo” sebaceous glands. •Xenograft model failed to mimic the biology of sebaceous glands in vivo. •Proinflammatory cytokine PGE{sub 2} enhances Wnt signal activity in the organoids. •PGE{sub 2} influences on the mitochondrial and lipid metabolism in the organoids. •Considering 3R agenda, “semi-vivo” sebaceous glands are useful for research. -- Abstract: Background: Prostaglandin E{sub 2} (PGE{sub 2}) is a proinflammatory mediator and activates the canonical Wnt–β-catenin signaling pathway in hematopoietic stem cells. The SZ95 cell line was established from human sebaceous gland cells and is studied as a model system for these cells. Given that 2D culture of SZ95 cells does not recapitulate the organization of sebaceous glands in situ, we developed a 3D culture system for these cells and examined the effects of PGE{sub 2} on cell morphology and function. Results: SZ95 cells maintained in 3D culture formed organoids that mimicked the organization of sebaceous glands in situ, including the establishment of a basement membrane. Organoids exposed to PGE{sub 2} were larger and adopted a more complex organization compared with control organoids. PGE{sub 2} activated the canonical Wnt signaling pathway as well as increased cell viability and proliferation, mitochondrial metabolism, and lipid synthesis in the organoids. Conclusions: Culture of SZ95 cells in 3D culture system recapitulates the structure and susceptibility to PGE{sub 2} of sebaceous glands in situ and should prove useful for studies of the response of these glands to inflammation and other environmental stressors. Our results also implicate PGE{sub 2}-induced activation of canonical Wnt signaling pathway in regulation of the morphology,proliferation, and function of “semi-vivo” sebaceous glands.

  14. Mechanotransduction activates canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

    PubMed Central

    Cha, Boksik; Geng, Xin; Mahamud, Md. Riaj; Fu, Jianxin; Mukherjee, Anish; Kim, Yeunhee; Jho, Eek-hoon; Kim, Tae Hoon; Kahn, Mark L.; Xia, Lijun; Dixon, J. Brandon; Chen, Hong; Srinivasan, R. Sathish

    2016-01-01

    Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2. PMID:27313318

  15. Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

    PubMed Central

    Peng, ChiehFu Jeff; Wikramanayake, Athula H.

    2013-01-01

    Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved

  16. Nucleoporin 62-Like Protein Activates Canonical Wnt Signaling through Facilitating the Nuclear Import of β-Catenin in Zebrafish

    PubMed Central

    Yang, Xiaojie; Gu, Qilin; Lin, Li; Li, Shaoyang; Zhong, Shan

    2015-01-01

    Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, the developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. The nup62l gene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx, and intestine of developing embryos. Activation of the Wnt/β-catenin pathway positively modulates nup62l transcription, while Bmp signaling acts downstream of Wnt/β-catenin signaling to negatively regulate nup62l expression. Overexpression of nup62l dorsalized embryos and enhanced gastrula convergence and extension (CE) movements. In contrast, knockdown of Nup62l led to ventralized embryos, an impediment to CE movements, and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/β-catenin signaling through interaction with and facilitation of nuclear import of β-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements, and proper specification of midline organ precursors through mediating the nuclear import of β-catenins in zebrafish. PMID:25605329

  17. Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

    PubMed Central

    Hardy, Katharine M.; Garriock, Robert J.; Yatskievych, Tatiana A.; D’Agostino, Susan L.; Antin, Parker B.; Krieg, Paul A.

    2008-01-01

    Knowledge of the molecular mechanisms regulating cell ingression, epithelial-mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm and in differentiated myocardial cells. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full length or dominant negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo. PMID:18602094

  18. Ilexonin A Promotes Neuronal Proliferation and Regeneration via Activation of the Canonical Wnt Signaling Pathway after Cerebral Ischemia Reperfusion in Rats

    PubMed Central

    Zhang, Bi-Qin; Zheng, Guan-Yi; Han, Yu; Chen, Xiao-Dong; Jiang, Qiong

    2016-01-01

    Aims. Ilexonin A (IA), a component of the Chinese medicine Ilex pubescens, has been shown to be neuroprotective during ischemic injury. However, the specific mechanism underlying this neuroprotective effect remains unclear. Methods. In this study, we employed a combination of immunofluorescence staining, western blotting, RT-PCR, and behavioral tests, to investigate the molecular mechanisms involved in IA regulation of neuronal proliferation and regeneration after cerebral ischemia and reperfusion in rodents. Results. Increases in β-catenin protein and LEF1 mRNA and decreases in GSK3β protein and Axin mRNA observed in IA-treated compared to control rodents implicated the canonical Wnt pathway as a key signaling mechanism activated by IA treatment. Furthermore, rodents in the IA treatment group showed less neurologic impairment and a corresponding increase in the number of Brdu/nestin and Brdu/NeuN double positive neurons in the parenchymal ischemia tissue following middle cerebral artery occlusion compared to matched controls. Conclusion. Altogether, our data indicate that IA can significantly diminish neurological deficits associated with cerebral ischemia reperfusion in rats as a result of increased neuronal survival via modulation of the canonical Wnt pathway. PMID:27057202

  19. A divergent canonical WNT-signaling pathway regulates microtubule dynamics

    PubMed Central

    Ciani, Lorenza; Krylova, Olga; Smalley, Matthew J.; Dale, Trevor C.; Salinas, Patricia C.

    2004-01-01

    Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability. PMID:14734535

  20. Opposite Interplay between PPAR Gamma and Canonical Wnt/Beta-Catenin Pathway in Amyotrophic Lateral Sclerosis.

    PubMed

    Lecarpentier, Yves; Vallée, Alexandre

    2016-01-01

    The opposite interplay between peroxisome proliferator-activated receptor gamma (PPAR gamma) and Wnt/beta-catenin signaling has led to the categorization of neurodegenerative diseases (NDs) as either NDs in which PPAR gamma is downregulated while the canonical Wnt/beta-catenin pathway is upregulated [amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, multiple sclerosis, Friedreich's ataxia] or NDs in which PPAR gamma is upregulated while the canonical Wnt/beta-catenin signaling is downregulated (bipolar disorder, schizophrenia, Alzheimer's disease). ALS, a common adult-onset debilitating ND, is characterized by a chronic and progressive degeneration of upper and lower motor neurons resulting in muscular atrophy, paralysis, and ultimately death. The intent of this review is to provide an analysis of the integration of these two opposed systems, i.e., canonical Wnt/beta-catenin and PPAR gamma, in ALS. Understanding this integration may aid in the development of novel ALS therapies. Although the canonical Wnt/beta-catenin pathway is upregulated in ALS, riluzole, an enhancer of the canonical Wnt signaling, is classically prescribed in this disease in humans. However, studies carried out on ALS transgenic mice have shown beneficial effects after treatment by PPAR gamma agonists partly due to their anti-inflammatory effects. PMID:27445967

  1. Opposite Interplay between PPAR Gamma and Canonical Wnt/Beta-Catenin Pathway in Amyotrophic Lateral Sclerosis

    PubMed Central

    Lecarpentier, Yves; Vallée, Alexandre

    2016-01-01

    The opposite interplay between peroxisome proliferator-activated receptor gamma (PPAR gamma) and Wnt/beta-catenin signaling has led to the categorization of neurodegenerative diseases (NDs) as either NDs in which PPAR gamma is downregulated while the canonical Wnt/beta-catenin pathway is upregulated [amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Huntington’s disease, multiple sclerosis, Friedreich’s ataxia] or NDs in which PPAR gamma is upregulated while the canonical Wnt/beta-catenin signaling is downregulated (bipolar disorder, schizophrenia, Alzheimer’s disease). ALS, a common adult-onset debilitating ND, is characterized by a chronic and progressive degeneration of upper and lower motor neurons resulting in muscular atrophy, paralysis, and ultimately death. The intent of this review is to provide an analysis of the integration of these two opposed systems, i.e., canonical Wnt/beta-catenin and PPAR gamma, in ALS. Understanding this integration may aid in the development of novel ALS therapies. Although the canonical Wnt/beta-catenin pathway is upregulated in ALS, riluzole, an enhancer of the canonical Wnt signaling, is classically prescribed in this disease in humans. However, studies carried out on ALS transgenic mice have shown beneficial effects after treatment by PPAR gamma agonists partly due to their anti-inflammatory effects. PMID:27445967

  2. Canonical WNT signaling pathway and human AREG.

    PubMed

    Katoh, Yuriko; Katoh, Masaru

    2006-06-01

    AREG (Amphiregulin), BTC (beta-cellulin), EGF, EPGN (Epigen), EREG (Epiregulin), HBEGF, NRG1, NRG2, NRG3, NRG4 and TGFA (TGFalpha) constitute EGF family ligands for ERBB family receptors. Cetuximab (Erbitux), Pertuzumab (Omnitarg) and Trastuzumab (Herceptin) are anti-cancer drugs targeted to EGF family ligands, while Gefitinib (Iressa), Erlotinib (Tarceva) and Lapatinib (GW572016) are anti-cancer drugs targeted to ERBB family receptors. AREG and TGFA are biomarkers for Gefitinib non-responders. The TCF/LEF binding sites within the promoter region of human EGF family members were searched for by using bioinformatics and human intelligence (Humint). Because three TCF/LEF-binding sites were identified within the 5'-promoter region of human AREG gene, comparative genomics analyses on AREG orthologs were further performed. The EPGN-EREG-AREG-BTC cluster at human chromosome 4q13.3 was linked to the PPBP-CXCL segmental duplicons. AREG was the paralog of HBEGF at human chromosome 5q31.2. Chimpanzee AREG gene, consisting of six exons, was located within NW_105918.1 genome sequence. Chimpanzee AREG was a type I transmembrane protein showing 98.0% and 71.4% total amino-acid identity with human AREG and mouse Areg, respectively. Three TCF/LEF-binding sites within human AREG promoter were conserved in chimpanzee AREG promoter, but not in rodent Areg promoters. Primate AREG promoters were significantly divergent from rodent Areg promoters. AREG mRNA was expressed in a variety of human tumors, such as colorectal cancer, liver cancer, gastric cancer, breast cancer, prostate cancer, esophageal cancer and myeloma. Because human AREG was characterized as potent target gene of WNT/beta-catenin signaling pathway, WNT signaling activation could lead to Gefitinib resistance through AREG upregulation. AREG is a target of systems medicine in the field of oncology. PMID:16685431

  3. Canonical Wnt/β-catenin signaling drives human schwann cell transformation, progression, and tumor maintenance.

    PubMed

    Watson, Adrienne L; Rahrmann, Eric P; Moriarity, Branden S; Choi, Kwangmin; Conboy, Caitlin B; Greeley, Andrew D; Halfond, Amanda L; Anderson, Leah K; Wahl, Brian R; Keng, Vincent W; Rizzardi, Anthony E; Forster, Colleen L; Collins, Margaret H; Sarver, Aaron L; Wallace, Margaret R; Schmechel, Stephen C; Ratner, Nancy; Largaespada, David A

    2013-06-01

    Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with downregulation of β-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties in immortalized human Schwann cells, and downregulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small-molecule inhibition of Wnt signaling effectively reduced the viability of MPNST cell lines and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors. PMID:23535903

  4. Wnt3a regulates proliferation and migration of HUVEC via canonical and non-canonical Wnt signaling pathways

    SciTech Connect

    Samarzija, Ivana; Sini, Patrizia; Schlange, Thomas; MacDonald, Gwen; Hynes, Nancy E.

    2009-08-28

    Untangling the signaling pathways involved in endothelial cell biology is of central interest for the development of antiangiogenesis based therapies. Here we report that Wnt3a induces the proliferation and migration of HUVECs, but does not affect their survival. Wnt3a-induced proliferation was VEGFR signaling independent, but reduced upon CamKII inhibition. In a search for the downstream mediators of Wnt3a's effects on HUVEC biology, we found that Wnt3a treatment leads to phosphorylation of DVL3 and stabilization of {beta}-catenin. Moreover, under the same conditions we observed an upregulation in c-MYC, TIE-2 and GJA1 mRNA transcripts. Although treatment of HUVECs with Wnt5a induced DVL3 phosphorylation, we did not observe any of the other effects seen upon Wnt3a stimulation. Taken together, our data indicate that Wnt3a induces canonical and non-canonical Wnt signaling in HUVECs, and stimulates their proliferation and migration.

  5. Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival

    PubMed Central

    Luo, Tian; Dunphy, Paige S.; Lina, Taslima T.

    2015-01-01

    Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, β-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca2+ and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival. PMID:26712203

  6. Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival.

    PubMed

    Luo, Tian; Dunphy, Paige S; Lina, Taslima T; McBride, Jere W

    2016-03-01

    Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, β-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca(2+) and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival. PMID:26712203

  7. NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation

    SciTech Connect

    Adachi, Atsuo; Takahashi, Tomosaburo; Ogata, Takehiro; Imoto-Tsubakimoto, Hiroko; Nakanishi, Naohiko; Ueyama, Tomomi; Matsubara, Hiroaki

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer NFAT5 protein expression is downregulated during cardiomyogenesis. Black-Right-Pointing-Pointer Inhibition of NFAT5 function suppresses canonical Wnt signaling. Black-Right-Pointing-Pointer Inhibition of NFAT5 function attenuates mesodermal induction. Black-Right-Pointing-Pointer NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2 Prime Z, 3 Prime E]-6-bromoindirubin-3 Prime -oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

  8. Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells.

    PubMed

    Perkins, Timothy N; Dentener, Mieke A; Stassen, Frank R; Rohde, Gernot G; Mossman, Brooke T; Wouters, Emiel F M; Reynaert, Niki L

    2016-06-15

    Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. PMID:27095093

  9. Regulation of CXCL12 expression by canonical Wnt signaling in bone marrow stromal cells.

    PubMed

    Tamura, Masato; Sato, Mari M; Nashimoto, Masayuki

    2011-05-01

    CXCL12 (stromal cell-derived factor-1, SDF-1), produced by stromal and endothelial cells including cells of the bone marrow, binds to its receptor CXCR4 and this axis regulates hematopoietic cell trafficking. Recently, osteoclast precursor cells were found to express CXCR4 and a potential role for the CXCL12-CXCR4 axis during osteoclast precursor cell recruitment/retention and development was proposed as a regulator of bone resorption. We examined the role of canonical Wnt signaling in regulating the expression of CXCL12 in bone marrow stromal cells. In mouse stromal ST2 cells, CXCL12 mRNA was expressed, while its expression was reduced in Wnt3a over-expressing ST2 (Wnt3a-ST2) cells or by treatment with lithium chloride (LiCl). Wnt3a decreased CXCL12 levels in culture supernatants from mouse bone marrow stromal cells. The culture supernatant from Wnt3a-ST2 cells also reduced migratory activity of bone marrow-derived cells in a Transwell migration assay. Silencing of glycogen synthase kinase-3β decreased CXCL12 expression, suggesting that the canonical Wnt signaling pathway regulates CXCL12 expression. In a transfection assay, LiCl down-regulated the activity of a reporter gene, a 1.8kb fragment of the 5'-flanking region of the CXCL12 gene. These results show that canonical Wnt signaling regulates CXCL12 gene expression at the transcriptional level, and this is the first study linking chemokine expression to canonical Wnt signaling. PMID:21296678

  10. Activation of canonical wnt pathway promotes differentiation of mouse bone marrow-derived MSCs into type II alveolar epithelial cells, confers resistance to oxidative stress, and promotes their migration to injured lung tissue in vitro.

    PubMed

    Liu, Ai-Ran; Liu, Le; Chen, Song; Yang, Yi; Zhao, Hong-Jie; Liu, Ling; Guo, Feng-Mei; Lu, Xiao-Min; Qiu, Hai-Bo

    2013-06-01

    The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells in vivo and in vitro, is critical for reepithelization and recovery in acute lung injury (ALI), but the mechanisms responsible for differentiation are unclear. In the present study, we investigated the role of the canonical wnt pathway in the differentiation of mouse bone marrow-derived MSCs (mMSCs) into AT II cells. Using a modified co-culture system with murine lung epithelial-12 (MLE-12) cells and small airway growth media (SAGM) to efficiently drive mMSCs differentiation, we found that GSK 3β and β-catenin in the canonical wnt pathway were up-regulated during differentiation. The levels of surfactant protein (SP) C, SPB, and SPD, the specific markers of AT II cells, correspondingly increased in mMSCs when Wnt3a or LiCl was added to the co-culture system to activate wnt/β-catenin signaling. The expression of these factors was depressed to some extent by inhibiting the pathway with the addition of DKK 1. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. Our results suggested that the activation of wnt/β-catenin signaling promoted mMSCs migration towards ALI mouse-derived lung tissue in a Transwell assay, and ameliorated the cell death and the reduction of Bcl-2/Bax induced by H(2) O(2), which simultaneously caused reduced GSK 3β and β-catenin in mMSCs. These data supports a potential mechanism for the differentiation of mMSCs into AT II cells involving canonical wnt pathway activation, which may be significant to their application in ALI. PMID:23154940

  11. Ptk7 and Mcc, Unfancied Components in Non-Canonical Wnt Signaling and Cancer.

    PubMed

    Dunn, Norris Ray; Tolwinski, Nicholas S

    2016-01-01

    Human development uses a remarkably small number of signal transduction pathways to organize vastly complicated tissues. These pathways are commonly associated with disease in adults if activated inappropriately. One such signaling pathway, Wnt, solves the too few pathways conundrum by having many alternate pathways within the Wnt network. The main or "canonical" Wnt pathway has been studied in great detail, and among its numerous downstream components, several have been identified as drug targets that have led to cancer treatments currently in clinical trials. In contrast, the non-canonical Wnt pathways are less well characterized, and few if any possible drug targets exist to tackle cancers caused by dysregulation of these Wnt offshoots. In this review, we focus on two molecules-Protein Tyrosine Kinase 7 (Ptk7) and Mutated in Colorectal Cancer (Mcc)-that do not fit perfectly into the non-canonical pathways described to date and whose roles in cancer are ill defined. We will summarize work from our laboratories as well as many others revealing unexpected links between these two proteins and Wnt signaling both in cancer progression and during vertebrate and invertebrate embryonic development. We propose that future studies focused on delineating the signaling machinery downstream of Ptk7 and Mcc will provide new, hitherto unanticipated drug targets to combat cancer metastasis. PMID:27438854

  12. Canonical Wnt signaling maintains the quiescent stage of hepatic stellate cells

    SciTech Connect

    Kordes, Claus Sawitza, Iris; Haeussinger, Dieter

    2008-02-29

    It is well known that hepatic stellate cells (HSC) develop into cells, which are thought to contribute to liver fibrogenesis. Recent data suggest that HSC are progenitor cells with the capacity to differentiate into cells of endothelial and hepatocyte lineages. The present study shows that {beta}-catenin-dependent canonical Wnt signaling is active in freshly isolated HSC of rats. Mimicking of the canonical Wnt pathway in cultured HSC by TWS119, an inhibitor of the glycogen synthase kinase 3{beta}, led to reduced {beta}-catenin phosphorylation, induced nuclear translocation of {beta}-catenin, elevated glutamine synthetase production, impeded synthesis of {alpha}-smooth muscle actin and Wnt5a, but promoted the expression of glial fibrillary acidic protein, Wnt10b, and paired-like homeodomain transcription factor 2c. In addition, canonical Wnt signaling lowered DNA synthesis and hindered HSC from entering the cell cycle. The findings demonstrate that {beta}-catenin-dependent Wnt signaling maintains the quiescent state of HSC and, similar to stem and progenitor cells, influences their developmental fate.

  13. Canonical and non-canonical Wnt proteins program dendritic cell responses for tolerance

    PubMed Central

    Oderup, Cecilia; LaJevic, Melissa; Butcher, Eugene C.

    2013-01-01

    Antigen presenting dendritic cells (DC) interpret environmental signals to orchestrate local and systemic immune responses. They govern the balance between tolerance and inflammation at epithelial surfaces, where the immune system must provide robust pathogen responses while maintaining tolerance to commensal flora and food antigens. The Wnt family of secreted proteins, which control epithelial as well as hematopoietic development and homeostasis, is emerging as an important regulator of inflammation. Here we show that canonical and non-canonical Wnts directly stimulate murine DC production of anti-inflammatory cytokines. Wnt3A triggers canonical β-catenin signaling and preferentially induces DC TGF-β and VEGF production, whereas Wnt5A induces IL-10 through alternative pathways. The Wnts also alter DC responses to microbe- or pathogen-associated molecular patterns (PAMPs), inhibiting pro-inflammatory cytokine induction in response to toll like receptor ligands and promoting DC generation of Foxp3+ regulatory T cells. Moreover, although both Wnts suppress pro-inflammatory responses to bacterial endotoxin and to TLR1/2, TLR7 and TLR9 ligands, Wnt5A but not Wnt3A inhibits IL-6 production in response to the viral mimic, polyinosinic:polycytidylic acid. Wnt family members thus directly and differentially regulate DC functions, an ability that may contribute to the balance between tolerance and inflammation at epithelial sites of exposure to microbes and environmental antigens. PMID:23677472

  14. Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation.

    PubMed

    Di Liddo, Rosa; Bertalot, Thomas; Schuster, Anne; Schrenk, Sandra; Müller, Oliver; Apfel, Johanna; Reischmann, Patricia; Rajendran, Senthilkumar; Sfriso, Riccardo; Gasparella, Marco; Parnigotto, Pier Paolo; Conconi, Maria Teresa; Schäfer, Karl Herbert

    2016-03-15

    In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of β-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote β-catenin release and signaling without any specific activation of the canonical Wnt pathway. In the present work, we took advantage of the ability of lipofectamine-like reagent to cause a synchronous dissociation of adherent junctions in cells isolated from the rat enteric nervous system (ENS) for obtaining an in vitro model of deregulated β-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen β-catenin stabilization at resting and primed conditions with exogenous Wnt3a or lipopolysaccharide (LPS). ΔTop_EGFP3a provided a reliable and strong fluorescent signal that was easily measurable and at the same time highly sensitive to modulations of Wnt signaling following Wnt3a and LPS stimulation. The reporter gene was useful to demonstrate that Wnt3a exerts a protective activity in the ENS from overstimulated Wnt signaling by promoting a downregulation of the total β-catenin level. Based on this evidence, the use of ΔTop_EGFP3a reporter plasmid could represent a more reliable tool for the investigation of Wnt and cross-talking pathways in ENS inflammation. PMID:26767983

  15. Agonists of epoxyeicosatrienoic acids reduce infarct size and ameliorate cardiac dysfunction via activation of HO-1 and Wnt1 canonical pathway.

    PubMed

    Cao, Jian; Tsenovoy, Peter L; Thompson, Ellen A; Falck, John R; Touchon, Robert; Sodhi, Komal; Rezzani, Rita; Shapiro, Joseph I; Abraham, Nader G

    2015-01-01

    Myocardial infarction (MI) is complicated by ventricular fibrosis and associated diastolic and systolic failure. Emerging studies implicate Wnt1 signaling in the formation of new blood vessels. Epoxyeicosatrienoic acids (EETs)-mediated up-regulation of heme oxygenase-1 (HO-1) protects against the detrimental consequences of MI in several animal models, however, the mechanism(s) by which this occurs remains unclear. The aim of this study was to examine these mechanisms in the LAD ligation animal model of post infarcted heart failure. Specifically, we sought to clarify the mechanistic basis of the interactions of the Wnt1 canonical pathway, HO-1 and associated angiogenesis. Human microvascular endothelial cells (HMECs) were exposed to anoxia and treated with the EET agonist, NUDSA, in the presence and absence of tin mesoporphyrin (SnMP). Increased capillary density, and Wnt1 and HO-1 expression occurred in cells treated with NUDSA. Anoxic HMECs treated with NUDSA and Wnt1 siRNA, exhibited decreased in the expression of β-catenin and the Wnt1 target gene, PPARδ (p<0.05 vs. NUDSA). Furthermore, blocking the Wnt 1 antagonist, Dickkopf 1, by siRNA increased β-catenin and PPARδ expression, and this effect was further enhanced by the concurrent administration of NUDSA. In in vivo experiments, C57B16 mice were divided into 4 groups: sham, mice with MI via LAD ligation and mice with MI treated with NUDSA, with and without SnMP. Increased fractional area change (FAC) and myocardial angiogenesis were observed in mice treated with NUDSA (p<0.05 vs. MI). Increased expression of HO-1, Wnt1, β-catenin, adiponectin, and phospho-endothelial nitric oxide synthetase (p-eNOS), and a decrease in the glycosylated subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91(phox) expression occurred in cardiac tissue of mice treated with NUDSA (p<0.05 vs. MI). SnMP reversed these effects. This novel study demonstrates that increasing the canonical Wnt1 signaling

  16. LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure.

    PubMed

    Gray, Jason D; Kholmanskikh, Stanislav; Castaldo, Bozena S; Hansler, Alex; Chung, Heekyung; Klotz, Brian; Singh, Shawn; Brown, Anthony M C; Ross, M Elizabeth

    2013-11-01

    Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulation were examined using gain (Crooked tail, Lrp6(Cd)) and loss (Lrp6(-)) of function mouse lines. Two features often associated with canonical Wnt signaling, dorsal-ventral patterning and proliferation, were no different from wild-type (WT) in the Lrp6(Cd/Cd) neural tube. Lrp6(-/-) embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6(Cd/Cd) and Lrp6(-/-) cranial folds were indicated by cell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6(Cd/Cd) or Lrp6(-/-) embryos exhibited elevated and decreased RhoA basal activity levels, respectively. While ligand-independent activation of canonical Wnt signaling, bypassing Lrp-Frizzled receptors, did not activate RhoA, non-canonical Wnt5a stimulation of RhoA activity was impaired in Lrp6(-/-) MEFs. RhoA inhibition exacerbated NTDs in cultured Lrp6 knockout embryos compared with WT littermates. In contrast, a ROCK inhibitor rescued Lrp6(Cd/Cd) embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity in Wnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6(Cd) protein where interaction with DAAM1 could account for observed elevated RhoA activity. Conversely, null mutation that eliminates Lrp6 interaction with DAAM1 led to lower basal RhoA activity in Lrp6(-/-) embryos. These results indicate that Lrp6 mediates not only canonical Wnt signaling, but can also modulate non-canonical pathways involving RhoA-dependent mechanisms to impact neurulation, possibly through intracellular complexes with DAAM1. PMID:23773994

  17. Ptk7 and Mcc, Unfancied Components in Non-Canonical Wnt Signaling and Cancer

    PubMed Central

    Dunn, Norris Ray; Tolwinski, Nicholas S.

    2016-01-01

    Human development uses a remarkably small number of signal transduction pathways to organize vastly complicated tissues. These pathways are commonly associated with disease in adults if activated inappropriately. One such signaling pathway, Wnt, solves the too few pathways conundrum by having many alternate pathways within the Wnt network. The main or “canonical” Wnt pathway has been studied in great detail, and among its numerous downstream components, several have been identified as drug targets that have led to cancer treatments currently in clinical trials. In contrast, the non-canonical Wnt pathways are less well characterized, and few if any possible drug targets exist to tackle cancers caused by dysregulation of these Wnt offshoots. In this review, we focus on two molecules—Protein Tyrosine Kinase 7 (Ptk7) and Mutated in Colorectal Cancer (Mcc)—that do not fit perfectly into the non-canonical pathways described to date and whose roles in cancer are ill defined. We will summarize work from our laboratories as well as many others revealing unexpected links between these two proteins and Wnt signaling both in cancer progression and during vertebrate and invertebrate embryonic development. We propose that future studies focused on delineating the signaling machinery downstream of Ptk7 and Mcc will provide new, hitherto unanticipated drug targets to combat cancer metastasis. PMID:27438854

  18. Wnt5a-mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration

    SciTech Connect

    Cheng Chingwen Yeh Juching; Fan Taiping; Smith, Stephen K.; Charnock-Jones, D. Stephen

    2008-01-11

    Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. Wnt signalling is mediated through cell-cell interaction and is involved in many developmental processes and cellular functions. In this study, we investigated the possible function of Wnt5a and the non-canonical Wnt pathway in human endothelial cells. We found that Wnt5a-mediated non-canonical Wnt signalling regulated endothelial cell proliferation. Blocking this pathway using antibody, siRNA or a down-stream inhibitor led to suppression of endothelial cell proliferation, migration, and monolayer wound closure. We also found that the mRNA level of Wnt5a is up-regulated when endothelial cells are treated with a cocktail of inflammatory cytokines. Our findings suggest non-canonical Wnt signalling plays a role in regulating endothelial cell growth and possibly in angiogenesis.

  19. Secreted Frizzled-related protein-2 (sFRP2) augments canonical Wnt3a-induced signaling

    SciTech Connect

    Marschall, Zofia von; Fisher, Larry W.

    2010-09-24

    Research highlights: {yields} sFRP2 enhances the Wnt3a-induced {beta}-catenin stabilization and its nuclear translocation. {yields} sFRP2 enhances LRP6 phosphorylation and Wnt3a/{beta}-catenin transcriptional reporter activity. {yields} Dickkopf-1 (DKK1) fully antagonizes both Wnt3a/sFRP2-induced LRP6 phosphorylation and transcriptional activity. {yields} sFRP2 enhances expression of genes known to be regulated by Wnt3a signaling. -- Abstract: Secreted Frizzled-related proteins (sFRP) are involved in embryonic development as well as pathological conditions including bone and myocardial disorders and cancer. Because of their sequence homology with the Wnt-binding domain of Frizzled, they have generally been considered antagonists of canonical Wnt signaling. However, additional activities of various sFRPs including both synergism and mimicry of Wnt signaling as well as functions other than modulation of Wnt signaling have been reported. Using human embryonic kidney cells (HEK293A), we found that sFRP2 enhanced Wnt3a-dependent phosphorylation of LRP6 as well as both cytosolic {beta}-catenin levels and its nuclear translocation. While addition of recombinant sFRP2 had no activity by itself, Top/Fop luciferase reporter assays showed a dose-dependent increase of Wnt3a-mediated transcriptional activity. sFRP2 enhancement of Wnt3a signaling was abolished by treatment with the Wnt antagonist, Dickkopf-1 (DKK1). Wnt-signaling pathway qPCR arrays showed that sFRP2 enhanced the Wnt3a-mediated transcriptional up-regulation of several genes regulated by Wnt3a including its antagonists, DKK1, and Naked cuticle-1 homolog (NKD1). These results support sFRP2's role as an enhancer of Wnt/{beta}-catenin signaling, a result with biological impact for both normal development and diverse pathologies such as tumorigenesis.

  20. Reck enables cerebrovascular development by promoting canonical Wnt signaling.

    PubMed

    Ulrich, Florian; Carretero-Ortega, Jorge; Menéndez, Javier; Narvaez, Carlos; Sun, Belinda; Lancaster, Eva; Pershad, Valerie; Trzaska, Sean; Véliz, Evelyn; Kamei, Makoto; Prendergast, Andrew; Kidd, Kameha R; Shaw, Kenna M; Castranova, Daniel A; Pham, Van N; Lo, Brigid D; Martin, Benjamin L; Raible, David W; Weinstein, Brant M; Torres-Vázquez, Jesús

    2016-01-01

    The cerebral vasculature provides the massive blood supply that the brain needs to grow and survive. By acquiring distinctive cellular and molecular characteristics it becomes the blood-brain barrier (BBB), a selectively permeable and protective interface between the brain and the peripheral circulation that maintains the extracellular milieu permissive for neuronal activity. Accordingly, there is great interest in uncovering the mechanisms that modulate the formation and differentiation of the brain vasculature. By performing a forward genetic screen in zebrafish we isolated no food for thought (nft (y72)), a recessive late-lethal mutant that lacks most of the intracerebral central arteries (CtAs), but not other brain blood vessels. We found that the cerebral vascularization deficit of nft (y72) mutants is caused by an inactivating lesion in reversion-inducing cysteine-rich protein with Kazal motifs [reck; also known as suppressor of tumorigenicity 15 protein (ST15)], which encodes a membrane-anchored tumor suppressor glycoprotein. Our findings highlight Reck as a novel and pivotal modulator of the canonical Wnt signaling pathway that acts in endothelial cells to enable intracerebral vascularization and proper expression of molecular markers associated with BBB formation. Additional studies with cultured endothelial cells suggest that, in other contexts, Reck impacts vascular biology via the vascular endothelial growth factor (VEGF) cascade. Together, our findings have broad implications for both vascular and cancer biology. PMID:26657775

  1. Identification of 2-aminopyrimidine derivatives as inhibitors of the canonical Wnt signaling pathway.

    PubMed

    Del Bello, Fabio; Farande, Aniket; Giannella, Mario; Piergentili, Alessandro; Quaglia, Wilma; Benicchi, Tiziana; Cappelli, Federico; Nencini, Arianna; Salerno, Massimiliano; Thomas, Russell J; Travagli, Massimilano; Varrone, Maurizio

    2015-09-01

    The canonical Wnt signaling pathway plays a fundamental role in embryonic as well as in adult development. Consequently, dysregulation of the pathway has been linked to a wide spectrum of pathological conditions. In a program aimed at the identification of small molecule inhibitors of the canonical Wnt pathway we identified a series of 2-aminopyrimidine derivatives which specifically inhibited the pathway with minimal or no sign of cellular toxicity. The hit molecules 1 and 2 showed promising inhibitory activity with IC50 values of approximately 10 μM, but low solubility and metabolic stability. During the early stage of the hit series exploration, the pyrimidine core was variously decorated to obtain active compounds with a better physico-chemical profile. In particular, compound 13 showed Wnt inhibition activity comparable to hit molecules 1 and 2, with improved physico-chemical properties. Therefore, this series of compounds may be considered a promising starting point for the design of novel small molecule inhibitors of the canonical Wnt pathway. PMID:26233797

  2. Cdo suppresses canonical Wnt signalling via interaction with Lrp6 thereby promoting neuronal differentiation

    PubMed Central

    Jeong, Myong-Ho; Ho, Seok-Man; Vuong, Tuan Anh; Jo, Shin-Bum; Liu, Guizhong; Aaronson, Stuart A.; Leem, Young-Eun; Kang, Jong-Sun

    2015-01-01

    Canonical Wnt signalling regulates expansion of neural progenitors and functions as a dorsalizing signal in the developing forebrain. In contrast, the multifunctional co-receptor Cdo promotes neuronal differentiation and is important for the function of the ventralizing signal, Shh. Here we show that Cdo negatively regulates Wnt signalling during neurogenesis. Wnt signalling is enhanced in Cdo-deficient cells, leading to impaired neuronal differentiation. The ectodomains of Cdo and Lrp6 interact via the Ig2 repeat of Cdo and the LDLR repeats of Lrp6, and the Cdo Ig2 repeat is necessary for Cdo-dependent Wnt inhibition. Furthermore, the Cdo-deficient dorsal forebrain displays stronger Wnt signalling activity, increased cell proliferation and enhanced expression of the dorsal markers and Wnt targets, Pax6, Gli3, Axin2. Therefore, in addition to promoting ventral central nervous system cell fates with Shh, Cdo promotes neuronal differentiation by suppression of Wnt signalling and provides a direct link between two major dorsoventral morphogenetic signalling pathways. PMID:25406935

  3. The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: Implications in targeted cancer therapies

    PubMed Central

    Yang, Ke; Wang, Xin; Zhang, Hongmei; Wang, Zhongliang; Nan, Guoxin; Li, Yasha; Zhang, Fugui; Mohammed, Maryam K.; Haydon, Rex C.; Luu, Hue H.; Bi, Yang; He, Tong-Chuan

    2015-01-01

    The canonical WNT/β-catenin signaling pathway governs a myriad of biological processes underlying development and maintenance of adult tissue homeostasis, including regulation of stem cell self-renewal, cell proliferation, differentiation, and apoptosis. WNTs are secreted lipid-modified glycoproteins that act as short-range ligands to activate receptor-mediated signaling pathways. The hallmark of the canonical pathway is the activation of β-catenin mediated transcriptional activity. Canonical WNTs control the β-catenin dynamics as the cytoplasmic level of β-catenin is tightly regulated via phosphorylation by the ‘destruction complex’, consisting of glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), the scaffold protein AXIN, and the tumor suppressor adenomatous polyposis coli (APC). Aberrant regulation of this signaling cascade is associated with varieties of human diseases, especially cancers. Over the past decade, significant progress has been made in understanding the mechanisms of canonical WNT signaling. In this review, we focus on the current understanding of WNT signaling at the extracellular, cytoplasmic membrane, and intracellular/nuclear levels, including the emerging knowledge of crosstalk with other pathways. Recent progresses in developing novel WNT pathway-targeted therapies will also be reviewed. Thus, this review is intended to serve as a refresher of the current understanding about the physiologic and pathogenic roles of WNT/β-catenin signaling pathway, and to outline potential therapeutic opportunities by targeting the canonical WNT pathway. PMID:26618721

  4. The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: implications in targeted cancer therapies.

    PubMed

    Yang, Ke; Wang, Xin; Zhang, Hongmei; Wang, Zhongliang; Nan, Guoxin; Li, Yasha; Zhang, Fugui; Mohammed, Maryam K; Haydon, Rex C; Luu, Hue H; Bi, Yang; He, Tong-Chuan

    2016-02-01

    The canonical WNT/β-catenin signaling pathway governs a myriad of biological processes underlying the development and maintenance of adult tissue homeostasis, including regulation of stem cell self-renewal, cell proliferation, differentiation, and apoptosis. WNTs are secreted lipid-modified glycoproteins that act as short-range ligands to activate receptor-mediated signaling pathways. The hallmark of the canonical pathway is the activation of β-catenin-mediated transcriptional activity. Canonical WNTs control the β-catenin dynamics as the cytoplasmic level of β-catenin is tightly regulated via phosphorylation by the 'destruction complex', consisting of glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), the scaffold protein AXIN, and the tumor suppressor adenomatous polyposis coli (APC). Aberrant regulation of this signaling cascade is associated with varieties of human diseases, especially cancers. Over the past decade, significant progress has been made in understanding the mechanisms of canonical WNT signaling. In this review, we focus on the current understanding of WNT signaling at the extracellular, cytoplasmic membrane, and intracellular/nuclear levels, including the emerging knowledge of cross-talk with other pathways. Recent progresses in developing novel WNT pathway-targeted therapies will also be reviewed. Thus, this review is intended to serve as a refresher of the current understanding about the physiologic and pathogenic roles of WNT/β-catenin signaling pathway, and to outline potential therapeutic opportunities by targeting the canonical WNT pathway. PMID:26618721

  5. β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

    PubMed Central

    Liu, Yilin; Hancock, Morgan; Workman, Aspen; Doster, Alan

    2016-01-01

    ABSTRACT Like many Alphaherpesvirinae subfamily members, bovine herpesvirus 1 (BoHV-1) expresses an abundant transcript in latently infected sensory neurons, the latency-related (LR)-RNA. LR-RNA encodes a protein (ORF2) that inhibits apoptosis, interacts with Notch family members, interferes with Notch-mediated transcription, and stimulates neurite formation in cells expressing Notch. An LR mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency or replicate efficiently in certain tissues, indicating that LR gene products are important. In this study, β-catenin, a transcription factor activated by the canonical Wnt signaling pathway, was frequently detected in ORF2-positive trigeminal ganglionic neurons of latently infected, but not mock-infected, calves. Conversely, the lytic cycle regulatory protein (BoHV-1 infected cell protein 0, or bICP0) was not frequently detected in β-catenin-positive neurons in latently infected calves. During dexamethasone-induced reactivation from latency, mRNA expression levels of two Wnt antagonists, Dickkopf-1 (DKK-1) and secreted Frizzled-related protein 2 (SFRP2), were induced in bovine trigeminal ganglia (TG), which correlated with reduced β-catenin protein expression in TG neurons 6 h after dexamethasone treatment. ORF2 and a coactivator of β-catenin, mastermind-like protein 1 (MAML1), stabilized β-catenin protein levels and stimulated β-catenin-dependent transcription in mouse neuroblastoma cells more effectively than MAML1 or ORF2 alone. Neuroblastoma cells expressing ORF2, MAML1, and β-catenin were highly resistant to cell death following serum withdrawal, whereas most cells transfected with only one of these genes died. The Wnt signaling pathway interferes with neurodegeneration but promotes neuronal differentiation, suggesting that stabilization of β-catenin expression by ORF2 promotes neuronal survival and differentiation. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an

  6. The Importance of the Canonical Wnt Signaling Pathway in the Porcine Endometrial Stromal Stem/Progenitor Cells: Implications for Regeneration

    PubMed Central

    Bukowska, Joanna; Ziecik, Adam Janusz; Laguna, Joanna; Gawronska-Kozak, Barbara

    2015-01-01

    The regenerative ability of the endometrium is strongly associated with the presence of adult stem/progenitor cells. Purposes of the present study were (1) to establish the presence of stem/progenitor cells in porcine endometrial stroma using a clonogenic assay and (2) to investigate whether the canonical Wnt pathway affects the potential of stem/progenitor cells to undergo self-renewal or differentiation. The utility of endometrial stromal clones as a model for stem/progenitor studies was evaluated based on these cells' increased expression of mesenchymal stem cell (MSC) marker genes, including CD29, CD73, CD90, and CD105, compared with primary cultured cells. Small molecules were introduced to activate (BIO) or inhibit (XAV939) the canonical Wnt pathway during stromal clone formation. Cloning efficiency assays revealed that activation of the Wnt/β-catenin pathway promoted formation of more differentiated small clones. Moreover, activation of the Wnt/β-catenin pathway decreased, whereas inhibition of the pathway increased MSC marker expression. Additionally, we confirmed the importance of canonical Wnt pathway stimulation in endometrial stromal cells through observing the appropriate changes in β-catenin cellular localization. These data indicate that modulation of the canonical Wnt pathway effects the process of regeneration in the porcine endometrium during the course of the estrous cycle. PMID:26414529

  7. Inhibition of adipocytogenesis by canonical WNT signaling in human mesenchymal stem cells

    SciTech Connect

    Shen, Longxiang; Glowacki, Julie; Zhou, Shuanhu

    2011-08-01

    The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPAR{gamma}2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of {beta}-catenin protein, inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast, knockdown of {beta}-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.

  8. LRP5 and plasma cholesterol levels modulate the canonical Wnt pathway in peripheral blood leukocytes.

    PubMed

    Borrell-Pages, Maria; Carolina Romero, July; Badimon, Lina

    2015-08-01

    Inflammation is triggered after invasion or injury to restore homeostasis. Although the activation of Wnt/β-catenin signaling is one of the first molecular responses to cellular damage, its role in inflammation is still unclear. It was our hypothesis that the low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and the canonical Wnt signaling pathway are modulators of inflammatory mechanisms. Wild-type (WT) and LRP5(-/-) mice were fed a hypercholesterolemic (HC) diet to trigger dislipidemia and chronic inflammation. Diets were supplemented with plant sterol esters (PSEs) to induce LDL cholesterol lowering and the reduction of inflammation. HC WT mice showed increased serum cholesterol levels that correlated with increased Lrp5 and Wnt/β-catenin gene expression while in the HC LRP5(-/-) mice Wnt/β-catenin pathway was shut down. Functionally, HC induced pro-inflammatory gene expression in LRP5(-/-) mice, suggesting an inhibitory role of the Wnt pathway in inflammation. Dietary PSE administration downregulated serum cholesterol levels in WT and LRP5(-/-) mice. Furthermore, in WT mice PSE increased anti-inflammatory genes expression and inhibited Wnt/β-catenin activation. Hepatic gene expression of Vldlr, Lrp2 and Lrp6 was increased after HC feeding in WT mice but not in LRP5(-/-) mice, suggesting a role for these receptors in the clearance of plasmatic lipoproteins. Finally, an antiatherogenic role for LRP5 was demonstrated as HC LRP5(-/-) mice developed larger aortic atherosclerotic lesions than WT mice. Our results show an anti-inflammatory, pro-survival role for LRP5 and the Wnt signaling pathway in peripheral blood leukocytes. PMID:25748163

  9. AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of {beta}-catenin at Ser 552

    SciTech Connect

    Zhao, Junxing; Yue, Wanfu; Zhu, Mei J.; Sreejayan, Nair; Du, Min

    2010-04-23

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/{beta}-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing {beta}-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/{beta}-catenin signaling through phosphorylation of {beta}-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of {beta}-catenin at Ser 552. The {beta}-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated {beta}-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [{gamma}-{sup 32}P]ATP autoradiography. In conclusion, AMPK phosphorylates {beta}-catenin at Ser 552, which stabilizes {beta}-catenin, enhances {beta}-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/{beta}-catenin signaling pathway.

  10. Gene regulatory networks mediating canonical Wnt signal directed control of pluripotency and differentiation in embryo stem cells

    PubMed Central

    Zhang, Xiaoxiao; Peterson, Kevin A.; Liu, X. Shirley; McMahon, Andrew P.; Ohba, Shinsuke

    2013-01-01

    Canonical Wnt signaling supports the pluripotency of embryonic stem cells (ESCs) but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Further, canonical Wnt signaling up-regulates the activity of the Pou5f1 distal enhancer via the Sox motif in ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a MEK/ERK inhibitor essential for ESC culture suggests MEK/ERK signaling and canonical Wnt signaling combine to promote ESC differentiation. PMID:23505158

  11. Can Wnt5a and Wnt non-canonical pathways really mediate adipocyte de-differentiation in a tumour microenvironment?

    PubMed

    Chirumbolo, Salvatore; Bjørklund, Geir

    2016-09-01

    Wnt5a has been recently reported as a possible triggering factor of adipocyte de-differentiation into an adipocyte-derived fibroblast in the tumour microenvironment of pancreas cancer. The Wnt/β-catenin pathway was described in processes involving de-differentiation and epithelial-mesenchymal transition but some Wnt family-belonging molecules exert an adipogenic role on adipocyte, while other ones, such as Wnt10b or Wnt3a, an anti-adipogenic role. Although this ability depends on the different tumoural microenvironments, it is intriguing to ascertain if some Wnt molecules, participating in the non-canonical pathway, may be targeted as fundamental factors able to trigger the desmoplastic reaction of peritumoural white adipose tissue. PMID:27391920

  12. The orphan GPCR, Gpr161, regulates the retinoic acid and canonical Wnt pathways during neurulation.

    PubMed

    Li, Bo I; Matteson, Paul G; Ababon, Myka F; Nato, Alejandro Q; Lin, Yong; Nanda, Vikas; Matise, Tara C; Millonig, James H

    2015-06-01

    The vacuolated lens (vl) mouse mutation arose on the C3H/HeSnJ background and results in lethality, neural tube defects (NTDs) and cataracts. The vl phenotypes are due to a deletion/frameshift mutation in the orphan GPCR, Gpr161. A recent study using a null allele demonstrated that Gpr161 functions in primary cilia and represses the Shh pathway. We show the hypomorphic Gpr161(vl) allele does not severely affect the Shh pathway. To identify additional pathways regulated by Gpr161 during neurulation, we took advantage of naturally occurring genetic variation in the mouse. Previously Gpr161(vl-C3H) was crossed to different inbred backgrounds including MOLF/EiJ and the Gpr161(vl) mutant phenotypes were rescued. Five modifiers were mapped (Modvl: Modifier of vl) including Modvl5(MOLF). In this study we demonstrate the Modvl5(MOLF) congenic rescues the Gpr161(vl)-associated lethality and NTDs but not cataracts. Bioinformatics determined the transcription factor, Cdx1, is the only annotated gene within the Modvl5 95% CI co-expressed with Gpr161 during neurulation and not expressed in the eye. Using Cdx1 as an entry point, we identified the retinoid acid (RA) and canonical Wnt pathways as downstream targets of Gpr161. QRT-PCR, ISH and IHC determined that expression of RA and Wnt genes are down-regulated in Gpr161(vl/vl) but rescued by the Modvl5(MOLF) congenic during neurulation. Intraperitoneal RA injection restores expression of canonical Wnt markers and rescues Gpr161(vl/vl) NTDs. These results establish the RA and canonical Wnt as pathways downstream of Gpr161 during neurulation, and suggest that Modvl5(MOLF) bypasses the Gpr161(vl) mutation by restoring the activity of these pathways. PMID:25753732

  13. The canonical Wnt signaling activator, R-spondin2, regulates craniofacial patterning and morphogenesis within the branchial arch through ectodermal-mesenchymal interaction

    PubMed Central

    Jin, Yong-Ri; Turcotte, Taryn J.; Crocker, Alison L.; Han, Xiang Hua; Yoon, Jeong Kyo

    2011-01-01

    R-spondins are a recently characterized family of secreted proteins that activate Wnt/β-catenin signaling. Herein, we determine R-spondin2 (Rspo2) function in craniofacial development in mice. Mice lacking a functional Rspo2 gene exhibit craniofacial abnormalities such as mandibular hypoplasia, maxillary and mandibular skeletal deformation, and cleft palate. We found that loss of the mouse Rspo2 gene significantly disrupted Wnt/β-catenin signaling and gene expression within the first branchial arch (BA1). Rspo2, which is normally expressed in BA1 mesenchymal cells, regulates gene expression through a unique ectoderm-mesenchyme interaction loop. The Rspo2 protein, potentially in combination with ectoderm-derived Wnt ligands, up-regulates Msx1 and Msx2 expression within mesenchymal cells. In contrast, Rspo2 regulates expression of the Dlx5, Dlx6, and Hand2 genes in mesenchymal cells via inducing expression of their upstream activator, Endothelin1 (Edn1), within ectodermal cells. Loss of Rspo2 also causes increased cell apoptosis, especially within the aboral (or caudal) domain of the BA1, resulting in hypoplasia of the BA1. Severely reduced expression of Fgf8, a survival factor for mesenchymal cells, in the ectoderm of Rspo2−/− embryos is likely responsible for increased cell apoptosis. Additionally, we found that cleft palate in Rspo2−/− mice is not associated with defects intrinsic to the palatal shelves. A possible cause of cleft palate is a delay of proper palatal shelf elevation that may result from the small mandible and a failure of lowering the tongue. Thus, our study identifies Rspo2 as a mesenchyme-derived factor that plays critical roles in regulating BA1 patterning and morphogenesis through ectodermal-mesenchymal interaction and a novel genetic factor for cleft palate. PMID:21237142

  14. A possible role for the canonical Wnt pathway in endocrine cell development in chicks

    SciTech Connect

    Pedersen, Anna Hauntoft; Heller, R. Scott . E-mail: shll@hagedorn.dk

    2005-08-05

    Wnt signalling is involved in many developmental processes such as proliferation, differentiation, cell fate decisions, and morphogenesis. However, little is known about Wnt signalling during pancreas development. Multiple Wnt ligands and Frizzled receptors are expressed in the embryonic mouse pancreas, the surrounding mesenchyme, and have also been detected in the chicken endoderm during development. The aim of this study was to investigate the role of canonical Wnt signalling on endocrine cell development by use of the in ovo electroporation of the chicken endoderm. Overexpression with a constitutive active form of {beta}-catenin in combination with Ngn3 resulted in reduced numbers of glucagon cells. dnLEF-1 or naked-1 did not alter endocrine cell differentiation when co-expressed with Ngn3, but dnLEF-1 appeared to have some potential for inhibiting delamination of Ngn3 cells. In addition, neuronal {beta}-III-tubulin, which had previously been considered a specific marker for neuronal cells, was observed in the pancreas and was upregulated in the electroporated Ngn3 cells and thus may be a new endocrine marker in the chicken.

  15. Metastases and Colon Cancer Tumor Growth Display Divergent Responses to Modulation of Canonical WNT Signaling

    PubMed Central

    Seth, Chandan; Ruiz i Altaba, Ariel

    2016-01-01

    Human colon cancers commonly harbor loss of function mutations in APC, a repressor of the canonical WNT pathway, thus leading to hyperactive WNT-TCF signaling. Re-establishment of Apc function in mice, engineered to conditionally repress Apc through RNAi, resolve the intestinal tumors formed due to hyperactivated Wnt-Tcf signaling. These and other results have prompted the search for specific WNT pathway antagonists as therapeutics for clinically problematic human colon cancers and associated metastases, which remain largely incurable. This widely accepted view seems at odds with a number of findings using patient-derived material: Canonical TCF targets are repressed, instead of being hyperactivated, in advanced colon cancers, and repression of TCF function does not generally result in tumor regression in xenografts. The results of a number of genetic mouse studies have also suggested that canonical WNT-TCF signaling drives metastases, but direct in vivo tests are lacking, and, surprisingly, TCF repression can enhance directly seeded metastatic growth. Here we have addressed the abilities of enhanced and blocked WNT-TCF signaling to alter tumor growth and distant metastases using xenografts of advanced human colon cancers in mice. We find that endogenous WNT-TCF signaling is mostly anti-metastatic since downregulation of TCF function with dnTCF generally enhances metastatic spread. Consistently, elevating the level of WNT signaling, by increasing the levels of WNT ligands, is not generally pro-metastatic. Our present and previous data reveal a heterogeneous response to modulating WNT-TCF signaling in human cancer cells. Nevertheless, the findings that a fraction of colon cancers tested require WNT-TCF signaling for tumor growth but all respond to repressed signaling by increasing metastases beg for a reevaluation of the goal of blocking WNT-TCF signaling to universally treat colon cancers. Our data suggest that WNT-TCF blockade may be effective in inhibiting tumor

  16. WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway.

    PubMed

    Goto, Toshiyasu; Matsuzawa, Junhei; Iemura, Shun-Ichiro; Natsume, Tohru; Shibuya, Hiroshi

    2016-05-01

    The stability of β-catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3β phosphorylates β-catenin to be degraded by ubiquitination with β-TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected β-catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of β-catenin. These results suggest that WDR26 plays a negative role in β-catenin degradation in the Wnt signaling pathway. PMID:27098453

  17. Neuroprotective Potential of Novel Multi-Targeted Isoalloxazine Derivatives in Rodent Models of Alzheimer's Disease Through Activation of Canonical Wnt/β-Catenin Signalling Pathway.

    PubMed

    Machhi, Jatin; Sinha, Anshuman; Patel, Pratik; Kanhed, Ashish M; Upadhyay, Pragnesh; Tripathi, Ashutosh; Parikh, Zalak S; Chruvattil, Ragitha; Pillai, Prakash P; Gupta, Sarita; Patel, Kirti; Giridhar, Rajani; Yadav, Mange Ram

    2016-05-01

    Previous reports suggest that Alzheimer's disease is protected by cholinesterase inhibitors. We synthesized some isoalloxazine derivatives and evaluated them using in vitro cholinesterase inhibition assay. Two of the compounds (7m and 7q) were figured out as potent cholinesterase inhibitors. They further showed anti-Aβ aggregatory activity in the in vitro assay. The current study deals with the evaluation of neuroprotective potentials of the potent compounds (7m and 7q) using different in vitro and in vivo experiments. The compounds were first assessed for their tendency to cross blood-brain barrier using in vitro permeation assay. They were evaluated using scopolamine-induced amnesic mice model. Additionally, ROS scavenging and anti-apoptotic properties of 7m and 7q were established against Aβ1-42-induced toxicity in rat hippocampal neuronal cells. 7m and 7q were also evaluated using Aβ1-42-induced Alzheimer's rat model. Lastly, their involvement in Wnt/β-catenin pathway was also demonstrated. The results indicated good CNS penetration for 7m and 7q. The neuroprotective effects of 7m and 7q were evidenced by improved cognitive ability in both scopolamine and Aβ1-42-induced Alzheimer's-like condition in rodents. The in vivo results also confirmed their anti-cholinesterase and anti-oxidant potential. Immunoblot results showed that treatment with 7m and 7q decreased Aβ1-42, p-tau, cleaved caspase-3, and cleaved PARP levels in Aβ1-42-induced Alzheimer's rat brain. Additionally, immunoblot results demonstrated that 7m and 7q activated the Wnt/β-catenin pathway as evidenced by increased p-GSK-3, β-catenin, and neuroD1 levels in Aβ1-42-induced Alzheimer's rat brain. These findings have shown that isoalloxazine derivatives (7m and 7q) could be the potential leads for developing effective drugs for the treatment of AD. PMID:26797524

  18. IGF-1R inhibition in mammary epithelia promotes canonical Wnt signaling and Wnt1-driven tumors

    PubMed Central

    Rota, Lauren M.; Albanito, Lidia; Shin, Marcus E.; Goyeneche, Corey L.; Shushanov, Sain; Gallagher, Emily J.; LeRoith, Derek; Lazzarino, Deborah A.; Wood, Teresa L.

    2014-01-01

    Triple-negative breast cancers (TNBC) are an aggressive disease subtype which unlike other subtypes lack an effective targeted therapy. Inhibitors of the insullin-like growth factor receptor (IGF-1R) have been considered for use in treating TNBC. Here we provide genetic evidence that IGF-1R inhibition promotes development of Wnt1-mediated murine mammary tumors that offer a model of TNBC. We found that in a double transgenic mouse model carrying activated Wnt-1 and mutant IGF-1R, a reduction in IGF-1R signaling reduced tumor latency and promoted more aggressive phenotypes. These tumors displayed a squamal cell phenotype with increased expression of keratins 5/6 and β-catenin. Notably, cell lineage analyses revealed an increase in basal (CD29hi/CD24+) and luminal (CD24+/CD61+/CD29lo) progenitor cell populations, along with increased Nanog expression and decreased Elf5 expression. In these doubly transgenic mice, lung metastases developed with characteristics of the primary tumors, unlike MMTV-Wnt1 mice. Mechanistic investigations showed that pharmacological inhibition of the IGF-1R in vitro was sufficient to increase the tumorsphere-forming efficiency of MMTV-Wnt1 tumor cells. Tumors from doubly transgenic mice also exhibited an increase in the expression ratio of the IGF-II-sensitive, A isoform of the insulin receptor vs the IR-B isoform, which in vitro resulted in enhanced expression of β-catenin. Overall, our results revealed that in Wnt-driven tumors an attenuation of IGF-1R signaling accelerates tumorigenesis and promotes more aggressive phenotypes, with potential implications for understanding TNBC pathobiology and treatment. PMID:25092896

  19. [Dual-role regulations of canonical Wnt/beta-catenin signaling pathway].

    PubMed

    Liu, Yang; Zhang, Chen-guang; Zhou, Chun-yan

    2010-04-18

    In recent years, Wnt/beta-catenin signaling has been identified as a key player in embryogenesis and human diseases. Canonical Wnt signaling pathway is controlled by a variety of classic molecules like Wnt, beta-catenin, Axin, APC, GSK-3beta and CK1, which interact and coordinate to regulate the expressions of cell signaling molecules. The latest evidences suggest that some components of the Wnt/beta-catenin signaling, like APC, GSK-3beta, CK1, Dkk2 and WISE, play dual roles different from what they have been thought previously. Here we reviewed some recent discoveries on the canonical Wnt/beta-catenin signaling pathway to provide some new ideas and principles for signaling transduction studies. PMID:20396373

  20. Gonad differentiation in zebrafish is regulated by the canonical Wnt signaling pathway.

    PubMed

    Sreenivasan, Rajini; Jiang, Junhui; Wang, Xingang; Bártfai, Richárd; Kwan, Hsiao Yuen; Christoffels, Alan; Orbán, László

    2014-02-01

    Zebrafish males undergo a "juvenile ovary-to-testis" gonadal transformation process. Several genes, including nuclear receptor subfamily 5, group A (nr5a) and anti-Müllerian hormone (amh), and pathways such as Tp53-mediated germ-cell apoptosis have been implicated in zebrafish testis formation. However, our knowledge of the regulation of this complex process is incomplete, and much remains to be investigated about the molecular pathways and network of genes that control it. Using a microarray-based analysis of transforming zebrafish male gonads, we demonstrated that their transcriptomes undergo transition from an ovary-like pattern to an ovotestis to a testis-like profile. Microarray results also validated the previous histological and immunohistochemical observation that there is high variation in the duration and extent of commitment to the juvenile ovary phase among individuals. Interestingly, global gene expression profiling of diverging zebrafish juvenile ovaries and transforming ovotestes revealed that some members of the canonical Wnt/beta-catenin signaling pathway were differentially expressed between these two phases. To investigate whether Wnt/beta-catenin signaling plays a role in zebrafish gonad differentiation, we used the Tg (hsp70l:dkk1b-GFP)w32 line to inhibit Wnt/beta-catenin signaling during gonad differentiation. Activation of dkk1b-GFP expression by heat shock resulted in an increased proportion of males and corresponding decrease in gonadal aromatase gene (cyp19a1a) expression. The Wnt target gene, lymphocyte enhancer binding factor 1 (lef1), was also down-regulated in the process. Together, these results provide the first functional evidence that, similarly to mammals, Wnt/beta-catenin signaling is a "pro-female" pathway that regulates gonad differentiation in zebrafish. PMID:24174574

  1. Canonical Wnts, specifically Wnt-10b, show ability to maintain dermal papilla cells

    SciTech Connect

    Ouji, Yukiteru Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2013-08-30

    Highlights: •First report on effects of various Wnts on DP cells. •Wnt-10b promoted trichogenesis, while Wnt-3a showed to a limited extent. •Canonical Wnts, specifically Wnt-10b, is important for DP cells maintenance. -- Abstract: Although Wnts are expressed in hair follicles (HFs) and considered to be crucial for maintaining dermal papilla (DP) cells, the functional differences among them remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, 5a, 10b, 11) on the proliferation of mouse-derived primary DP cells in vitro as well as their trichogenesis-promoting ability using an in vivo skin reconstitution protocol. Wnt-10b promoted cell proliferation and trichogenesis, while Wnt-3a showed those abilities to a limited extent, and Wnt-5a and 11 had no effects. Furthermore, we investigated the effects of these Wnts on cultured DP cells obtained from versican-GFP transgenic mice and found that Wnt-10b had a potent ability to sustain their GFP-positivity. These results suggest that canonical Wnts, specifically Wnt-10b, play important roles in the maintenance of DP cells and trichogenesis.

  2. Secreted and transmembrane wnt inhibitors and activators.

    PubMed

    Cruciat, Cristina-Maria; Niehrs, Christof

    2013-03-01

    Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand-receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-β and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease. PMID:23085770

  3. Structural Insights into the C1q Domain of Caprin-2 in Canonical Wnt Signaling*

    PubMed Central

    Miao, Haofei; Jia, Yingying; Xie, Sichun; Wang, Xin; Zhao, Jianfei; Chu, Youjun; Zhou, Zhilei; Shi, Zhubing; Song, Xiaomin; Li, Lin

    2014-01-01

    Previously, we have identified Caprin-2 as a new regulator in canonical Wnt signaling through a mechanism of facilitating LRP5/6 phosphorylation; moreover, we found that its C-terminal C1q-related domain (Cap2_CRD) is required for this process. Here, we determined the crystal structures of Cap2_CRD from human and zebrafish, which both associate as a homotrimer with calcium located at the symmetric center. Surprisingly, the calcium binding-deficient mutant exists as a more stable trimer than its wild-type counterpart. Further studies showed that this Caprin-2 mutant disabled in binding calcium maintains the activity of promoting LRP5/6 phosphorylation, whereas the mutations disrupting Cap2_CRD homotrimer did impair such activity. Together, our findings suggested that the C-terminal CRD domain of Caprin-2 forms a flexible homotrimer mediated by calcium and that such trimeric assembly is required for Caprin-2 to regulate canonical Wnt signaling. PMID:25331957

  4. PTH/cAMP/PKA signaling facilitates canonical Wnt signaling via inactivation of glycogen synthase kinase-3beta in osteoblastic Saos-2 cells.

    PubMed

    Suzuki, Akira; Ozono, Keiichi; Kubota, Takuo; Kondou, Hiroki; Tachikawa, Kanako; Michigami, Toshimi

    2008-05-01

    Although the intermittent administration of PTH is known to stimulate the bone formation, the underlying mechanisms are not fully understood. Here we investigated the crosstalk between PTH/cAMP signaling and canonical Wnt signaling using the human osteoblastic cell line Saos-2. Treatment with PTH or forskolin, an activator of adenylate cyclase, facilitated T-cell factor (TCF)-dependent transactivation in a dose-dependent manner, which was abolished by pre-treatment with a PKA inhibitor, H89. Wnt3a and forskolin synergistically increased the TCF-dependent transactivation. Interestingly, intermittent treatment with PTH enhanced the TCF-dependent transactivation more profoundly than continuous treatment. In addition to the effects on TCF-dependent reporter activity, treatment with PTH or forskolin resulted in the increased expression of endogenous targets of Wnts, Wnt-induced secreted protein 2 (WISP2) and naked cuticle 2 (NKD2). We then investigated the convergence point of PTH/cAMP signaling and the canonical Wnt pathway. Western blotting demonstrated that GSK-3beta was rapidly phosphorylated at Ser(9) on treatment with PTH or forskolin, leading to its inactivation. Moreover, overexpression of a constitutively active mutant of GSK-3beta abolished the TCF-dependent transactivation induced by forskolin. On the other hand, overexpression of the Wnt antagonist Dickkopf-1 (DKK1) failed to cancel the effects of forskolin on the canonical Wnt pathway. Interestingly, treatment with Wnt3a markedly reduced the forskolin-induced expression of receptor activator of NF-kappaB ligand (RANKL), a target gene of PTH/cAMP/PKA. These results suggest that cAMP/PKA signaling activates the canonical Wnt pathway through the inactivation of GSK-3beta, whereas Wnt signaling might inhibit bone resorption through a negative impact on RANKL expression in osteoblasts. PMID:17990294

  5. The low-density lipoprotein receptor-related protein 10 is a negative regulator of the canonical Wnt/{beta}-catenin signaling pathway

    SciTech Connect

    Jeong, Young-Hee; Sekiya, Manami; Hirata, Michiko; Ye, Mingjuan; Yamagishi, Azumi; Lee, Sang-Mi; Kang, Man-Jong; Hosoda, Akemi; Fukumura, Tomoe; Kim, Dong-Ho; Saeki, Shigeru

    2010-02-19

    Wnt signaling pathways play fundamental roles in the differentiation, proliferation and functions of many cells as well as developmental, growth, and homeostatic processes in animals. Low-density lipoprotein receptor (LDLR)-related protein (LRP) 5 and LRP6 serve as coreceptors of Wnt proteins together with Frizzled receptors, triggering activation of canonical Wnt/{beta}-catenin signaling. Here, we found that LRP10, a new member of the LDLR gene family, inhibits the canonical Wnt/{beta}-catenin signaling pathway. The {beta}-catenin/T cell factor (TCF) transcriptional activity in HEK293 cells was activated by transfection with Wnt3a or LRP6, which was then inhibited by co-transfection with LRP10. Deletion of the extracellular domain of LRP10 negated its inhibitory effect. The inhibitory effect of LRP10 was consistently conserved in HEK293 cells even when GSK3{beta} phosphorylation was inhibited by incubation with lithium chloride and co-transfection with constitutively active S33Y-mutated {beta}-catenin. Nuclear {beta}-catenin accumulation was unaffected by LRP10. The present studies suggest that LRP10 may interfere with the formation of the {beta}-catenin/TCF complex and/or its binding to target DNA in the nucleus, and that the extracellular domain of LRP10 is critical for inhibition of the canonical Wnt/{beta}-catenin signaling pathway.

  6. Reducing canonical Wingless/Wnt signaling pathway confers protection against mutant Huntingtin toxicity in Drosophila.

    PubMed

    Dupont, Pascale; Besson, Marie-Thérèse; Devaux, Jérôme; Liévens, Jean-Charles

    2012-08-01

    Huntington's disease (HD) is a genetic neurodegenerative disease characterized by movement disorders, cognitive decline and neuropsychiatric symptoms. HD is caused by expanded CAG tract within the coding region of Huntingtin protein. Despite major insights into the molecular mechanisms leading to HD, no effective cure is yet available. Mutant Huntingtin (mHtt) has been reported to alter the stability and levels of β-Catenin, a key molecule in cell adhesion and signal transduction in Wingless (Wg)/Wnt pathway. However it remains to establish whether manipulation of Wg/Wnt signaling can impact HD pathology. We here investigated the phenotypic interactions between mHtt and Wg/Wnt signaling by using the power of Drosophila genetics. We provide compelling evidence that reducing Armadillo/β-Catenin levels confers protection and that this beneficial effect is correlated with the inactivation of the canonical Wg/Wnt signaling pathway. Knockdowns of Wnt ligands or of the downstream transcription factor Pangolin/TCF both ameliorate the survival of HD flies. Similarly, overexpression of one Armadillo/β-Catenin destruction complex component (Axin, APC2 or Shaggy/GSK-3β) increases the lifespan of HD flies. Loss of functional Armadillo/β-Catenin not only abolishes neuronal intrinsic but also glia-induced alterations in HD flies. Our findings highlight that restoring canonical Wg/Wnt signaling may be of therapeutic value. PMID:22531500

  7. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.

    PubMed

    Abedini, Atefeh; Zamberlam, Gustavo; Lapointe, Evelyne; Tourigny, Catherine; Boyer, Alexandre; Paquet, Marilène; Hayashi, Kanako; Honda, Hiroaki; Kikuchi, Akira; Price, Christopher; Boerboom, Derek

    2016-04-01

    Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation ofWnt5a(but notWnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genesin vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signalingviathe WNT/Ca(2+)or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both β-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decreaseviaa glycogen synthase kinase-3β-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.-Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling. PMID:26667040

  8. Down-Regulation of Canonical and Up-Regulation of Non-Canonical Wnt Signalling in the Carcinogenic Process of Squamous Cell Lung Carcinoma

    PubMed Central

    Weich, Alexander; Kiss, Edit; Barko, Szilvia; Kovacs, Tamas; Avdicevic, Monika; D’Souza, Vijay K.; Rapp, Judit; Kvell, Krisztian; Jakab, Laszlo; Nyitrai, Miklos; Molnar, Tamas F.; Thickett, David R.; Laszlo, Terezia; Pongracz, Judit E.

    2013-01-01

    The majority of lung cancers (LC) belong to the non-small cell lung carcinoma (NSCLC) type. The two main NSCLC sub-types, namely adenocarcinoma (AC) and squamous cell carcinoma (SCC), respond differently to therapy. Whereas the link between cigarette smoke and lung cancer risk is well established, the relevance of non-canonical Wnt pathway up-regulation detected in SCC remains poorly understood. The present study was undertaken to investigate further the molecular events in canonical and non-canonical Wnt signalling during SCC development. A total of 20 SCC and AC samples with matched non-cancerous controls were obtained after surgery. TaqMan array analysis confirmed up-regulation of non-canonical Wnt5a and Wnt11 and identified down-regulation of canonical Wnt signalling in SCC samples. The molecular changes were tested in primary small airway epithelial cells (SAEC) and various lung cancer cell lines (e.g. A549, H157, etc). Our studies identified Wnt11 and Wnt5a as regulators of cadherin expression and potentiated relocation of β-catenin to the nucleus as an important step in decreased cellular adhesion. The presented data identifies additional details in the regulation of SCC that can aid identification of therapeutic drug targets in the future. PMID:23505429

  9. Andrographolide activates the canonical Wnt signalling pathway by a mechanism that implicates the non-ATP competitive inhibition of GSK-3β: autoregulation of GSK-3β in vivo.

    PubMed

    Tapia-Rojas, Cheril; Schüller, Andreas; Lindsay, Carolina B; Ureta, Roxana C; Mejías-Reyes, Cristóbal; Hancke, Juan; Melo, Francisco; Inestrosa, Nibaldo C

    2015-03-01

    Wnt/β-catenin signalling is an important pathway that regulates multiple biological processes, including cell adhesion and determination of cell fate during animal development; in the adult nervous system it regulates the structure and function of synapses. Wnt-signalling dysfunction is associated with several neurodegenerative diseases such as schizophrenia and Alzheimer's disease. The use of natural compounds is an interesting strategy in the search for drugs with the therapeutic potential to activate this signalling pathway. In the present study, we report that andrographolide (ANDRO), a component of Andrographis paniculata, is a potent activator of Wnt signalling. Our results indicate that ANDRO activates this pathway, inducing the transcription of Wnt target genes by a mechanism that bypasses Wnt ligand binding to its receptor. In vitro kinase assays demonstrate that ANDRO inhibits glycogen synthase kinase (GSK)-3β by a non-ATP-competitive, substrate-competitive mode of action. In silico analyses suggest that ANDRO interacts with the substrate-binding site of GSK-3β. Finally, we demonstrated that the increase seen in the levels of GSK-3β phosphorylated at Ser⁹ is the result of an autoregulatory mechanism of the kinase in vivo, although not through activation of protein phosphatase type 1. Our results suggest that ANDRO could be used as a potential therapeutic drug for disorders caused by Wnt-signalling dysfunction such as neurodegenerative diseases. PMID:25423492

  10. Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

    PubMed Central

    Tsukiyama, Tadasuke; Fukui, Akimasa; Terai, Sayuri; Fujioka, Yoichiro; Shinada, Keisuke; Takahashi, Hidehisa; Yamaguchi, Terry P.; Ohba, Yusuke

    2015-01-01

    Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis. PMID:25825523

  11. The Canonical Wnt Signal Restricts the Glycogen Synthase Kinase 3/Fbw7-Dependent Ubiquitination and Degradation of Eya1 Phosphatase

    PubMed Central

    Sun, Ye

    2014-01-01

    Haploinsufficiency of Eya1 causes the branchio-oto-renal (BOR) syndrome, and abnormally high levels of Eya1 are linked to breast cancer progression and poor prognosis. Therefore, regulation of Eya1 activity is key to its tissue-specific functions and oncogenic activities. Here, we show that Eya1 is posttranslationally modified by ubiquitin and that its ubiquitination level is self-limited to prevent premature degradation. Eya1 has an evolutionarily conserved CDC4 phosphodegron (CPD) signal, a target site of glycogen synthase kinase 3 (GSK3) kinase and Fbw7 ubiquitin ligase, which is required for Eya1 ubiquitination. Genetic deletion of Fbw7 and pharmacological inhibition of GSK3 significantly decrease Eya1 ubiquitination. Conversely, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the canonical Wnt signal suppresses Eya1 ubiquitination. Compound Eya1+/−; Wnt9b+/− mutants exhibit an increased penetrance of renal defect, indicating that they function in the same genetic pathway in vivo. Together, these findings reveal that the canonical Wnt and PI3K/Akt signal pathways restrain the GSK3/Fbw7-dependent Eya1 ubiquitination, and they further suggest that dysregulation of this novel axis contributes to tumorigenesis. PMID:24752894

  12. The γ-Protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane

    PubMed Central

    Mah, Kar Men; Houston, Douglas W.; Weiner, Joshua A.

    2016-01-01

    The 22 γ-Protocadherin (γ-Pcdh) adhesion molecules encoded by the Pcdhg gene cluster play critical roles in nervous system development, including regulation of dendrite arborisation, neuronal survival, and synaptogenesis. Recently, they have been implicated in suppression of tumour cell growth by inhibition of canonical Wnt signalling, though the mechanisms through which this occurs remain unknown. Here, we show differential regulation of Wnt signalling by individual γ-Pcdhs: The C3 isoform uniquely inhibits the pathway, whilst 13 other isoforms upregulate signalling. Focusing on the C3 isoform, we show that its unique variable cytoplasmic domain (VCD) is the critical one for Wnt pathway inhibition. γ-Pcdh-C3, but not other isoforms, physically interacts with Axin1, a key component of the canonical Wnt pathway. The C3 VCD competes with Dishevelled for binding to the DIX domain of Axin1, which stabilizes Axin1 at the membrane and leads to reduced phosphorylation of Wnt co-receptor Lrp6. Finally, we present evidence that Wnt pathway activity can be modulated up (by γ-Pcdh-A1) or down (by γ-Pcdh-C3) in the cerebral cortex in vivo, using conditional transgenic alleles. Together, these data delineate opposing roles for γ-Pcdh isoforms in regulating Wnt signalling and identify Axin1 as a novel protein interactor of the widely-expressed γ-Pcdh-C3 isoform. PMID:27530555

  13. PTH1-34 blocks radiation-induced osteoblast apoptosis by enhancing DNA repair through canonical Wnt pathway.

    PubMed

    Chandra, Abhishek; Lin, Tiao; Zhu, Ji; Tong, Wei; Huo, Yanying; Jia, Haoruo; Zhang, Yejia; Liu, X Sherry; Cengel, Keith; Xia, Bing; Qin, Ling

    2015-01-01

    Focal radiotherapy for cancer patients has detrimental effects on bones within the radiation field and the primary clinical signs of bone damage include the loss of functional osteoblasts. We reported previously that daily injection of parathyroid hormone (PTH, 1-34) alleviates radiation-induced osteopenia in a preclinical radiotherapy model by improving osteoblast survival. To elucidate the molecular mechanisms, we irradiated osteoblastic UMR 106-01 cells and calvarial organ culture and demonstrated an anti-apoptosis effect of PTH1-34 on these cultures. Inhibitor assay indicated that PTH exerts its radioprotective action mainly through protein kinase A/β-catenin pathway. γ-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DNA double strand breaks (DSBs) in irradiated osteoblasts via activating the β-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Moreover, down-regulation of Ku70 by siRNA abrogated the prosurvival effect of PTH and Wnt3a on irradiated osteoblasts. In summary, our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1-34 or Wnt agonist as possible therapy for radiation-induced osteoporosis. PMID:25336648

  14. The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway

    PubMed Central

    Abdelhamed, Zakia A.; Natarajan, Subaashini; Wheway, Gabrielle; Inglehearn, Christopher F.; Toomes, Carmel; Johnson, Colin A.; Jagger, Daniel J.

    2015-01-01

    ABSTRACT Ciliopathies are a group of developmental disorders that manifest with multi-organ anomalies. Mutations in TMEM67 (MKS3) cause a range of human ciliopathies, including Meckel-Gruber and Joubert syndromes. In this study we describe multi-organ developmental abnormalities in the Tmem67tm1Dgen/H1 knockout mouse that closely resemble those seen in Wnt5a and Ror2 knockout mice. These include pulmonary hypoplasia, ventricular septal defects, shortening of the body longitudinal axis, limb abnormalities, and cochlear hair cell stereociliary bundle orientation and basal body/kinocilium positioning defects. The basal body/kinocilium complex was often uncoupled from the hair bundle, suggesting aberrant basal body migration, although planar cell polarity and apical planar asymmetry in the organ of Corti were normal. TMEM67 (meckelin) is essential for phosphorylation of the non-canonical Wnt receptor ROR2 (receptor-tyrosine-kinase-like orphan receptor 2) upon stimulation with Wnt5a-conditioned medium. ROR2 also colocalises and interacts with TMEM67 at the ciliary transition zone. Additionally, the extracellular N-terminal domain of TMEM67 preferentially binds to Wnt5a in an in vitro binding assay. Cultured lungs of Tmem67 mutant mice failed to respond to stimulation of epithelial branching morphogenesis by Wnt5a. Wnt5a also inhibited both the Shh and canonical Wnt/β-catenin signalling pathways in wild-type embryonic lung. Pulmonary hypoplasia phenotypes, including loss of correct epithelial branching morphogenesis and cell polarity, were rescued by stimulating the non-canonical Wnt pathway downstream of the Wnt5a-TMEM67-ROR2 axis by activating RhoA. We propose that TMEM67 is a receptor that has a main role in non-canonical Wnt signalling, mediated by Wnt5a and ROR2, and normally represses Shh signalling. Downstream therapeutic targeting of the Wnt5a-TMEM67-ROR2 axis might, therefore, reduce or prevent pulmonary hypoplasia in ciliopathies and other congenital

  15. Characterizing the Activation of the Wnt Signaling Pathway in Hilar Cholangiocarcinoma Using a Tissue Microarray Approach

    PubMed Central

    Chen, W.; Huang, L.; Liang, J.; Cai, J.; Lei, Y.; Lai, J.; Liang, L.; Zhang, K.

    2016-01-01

    Hilar cholangiocarcinoma (HCCA) is an invasive hepatic malignancy that is difficult to biopsy; therefore, novel markers of HCCA prognosis are needed. Here, the level of canonical Wnt activation in patients with HCCA, intrahepatic cholangiocarcinoma (IHCC), and congenital choledochal cysts (CCC) was compared to understand the role of Wnt signaling in HCCA. Pathology specimens from HCCA (n=129), IHCC (n=31), and CCC (n=45) patients were used to construct tissue microarrays. Wnt2, Wnt3, β-catenin, TCF4, c-Myc, and cyclin D1 were detected by immunohistochemistry. Parallel correlation analysis was used to analyze differences in protein levels between the HCCA, IHCC, and CCC groups. Univariate and multivariate analyses were used to determine independent predictors of successful resection and prognosis in the HCCA group. The protein levels of Wnt2, β-catenin, TCF4, c-Myc, and cyclin D1 were significantly higher in HCCA compared to IHHC or CCC. Wnt signaling activation (Wnt2+, Wnt3+, nuclear β-catenin+, nuclear TCF4+) was significantly greater in HCCA tissues than CCC tissues. Univariable analyses indicated that expression of cyclin D1 as well as Wnt signaling activation, and partial Wnt activation (Wnt2+ or Wnt3+ and nuclear β-catenin+ or nuclear TCF4+) predicted successful resection, but only cyclin D1 expression remained significant in multivariable analyses. Only partial Wnt activation was an independent predictor of survival time. Proteins in the canonical Wnt signaling pathway were present at higher levels in HCCA and correlated with tumor resecility and patient prognosis. These results suggest that Wnt pathway analysis may be a useful marker for clinical outcome in HCCA. PMID:26972709

  16. Fibroblast growth factor and canonical WNT/β-catenin signaling cooperate in suppression of chondrocyte differentiation in experimental models of FGFR signaling in cartilage.

    PubMed

    Buchtova, Marcela; Oralova, Veronika; Aklian, Anie; Masek, Jan; Vesela, Iva; Ouyang, Zhufeng; Obadalova, Tereza; Konecna, Zaneta; Spoustova, Tereza; Pospisilova, Tereza; Matula, Petr; Varecha, Miroslav; Balek, Lukas; Gudernova, Iva; Jelinkova, Iva; Duran, Ivan; Cervenkova, Iveta; Murakami, Shunichi; Kozubik, Alois; Dvorak, Petr; Bryja, Vitezslav; Krejci, Pavel

    2015-05-01

    Aberrant fibroblast growth factor (FGF) signaling disturbs chondrocyte differentiation in skeletal dysplasia, but the mechanisms underlying this process remain unclear. Recently, FGF was found to activate canonical WNT/β-catenin pathway in chondrocytes via Erk MAP kinase-mediated phosphorylation of WNT co-receptor Lrp6. Here, we explore the cellular consequences of such a signaling interaction. WNT enhanced the FGF-mediated suppression of chondrocyte differentiation in mouse limb bud micromass and limb organ cultures, leading to inhibition of cartilage nodule formation in micromass cultures, and suppression of growth in cultured limbs. Simultaneous activation of the FGF and WNT/β-catenin pathways resulted in loss of chondrocyte extracellular matrix, expression of genes typical for mineralized tissues and alteration of cellular shape. WNT enhanced the FGF-mediated downregulation of chondrocyte proteoglycan and collagen extracellular matrix via inhibition of matrix synthesis and induction of proteinases involved in matrix degradation. Expression of genes regulating RhoA GTPase pathway was induced by FGF in cooperation with WNT, and inhibition of the RhoA signaling rescued the FGF/WNT-mediated changes in chondrocyte cellular shape. Our results suggest that aberrant FGF signaling cooperates with WNT/β-catenin in suppression of chondrocyte differentiation. PMID:25558817

  17. Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

    PubMed Central

    Chen, Eleanor Y.; DeRan, Michael T.; Ignatius, Myron S.; Grandinetti, Kathryn Brooke; Clagg, Ryan; McCarthy, Karin M.; Lobbardi, Riadh M.; Brockmann, Jillian; Keller, Charles; Wu, Xu; Langenau, David M.

    2014-01-01

    Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS. PMID:24706870

  18. Canonical and non-canonical Wnt signaling control the regeneration of amputated rodent vibrissae follicles.

    PubMed

    Yuan, Yan-Ping; Huang, Keng; Xu, Yan-Min; Chen, Xian-Cai; Li, Hai-Hong; Cai, Bo-Zhi; Liu, Yang; Zhang, Huan; Li, Yu; Lin, Chang-Min

    2016-02-01

    Although mammals are notoriously poor at regeneration compared with many lower-order species, the hair follicle, particular to mammals, is capable of regeneration following partial amputation. The detailed internal mechanism of this phenomenon is still unclear. Development and regrowth of the hair follicle depends on dermal-epidermal interaction within the hair follicle. Previous studies have shown that Wnt/β-catenin, Shh, Bmp, PDGF, TGF and Notch signals all take part in the development and growth of the hair follicle, and the Wnt/β-catenin signaling additionally plays an indispensable role in hair follicle morphogenesis and regrowth. In this study, we investigated the localization, as well as, protein levels of Wnt/β-catenin signaling molecules during amputated whisker follicle regeneration. PMID:26742765

  19. BMP‐9‐induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β‐catenin signalling

    PubMed Central

    Tang, Ni; Song, Wen‐Xin; Luo, Jinyong; Luo, Xiaoji; Chen, Jin; Sharff, Katie A.; Bi, Yang; He, Bai‐Cheng; Huang, Jia‐Yi; Zhu, Gao‐Hui; Su, Yu‐Xi; Jiang, Wei; Tang, Min; He, Yun; Wang, Yi; Chen, Liang; Zuo, Guo‐Wei; Shen, Jikun; Pan, Xiaochuan; Reid, Russell R.; Luu, Hue H.; Haydon, Rex C.

    2008-01-01

    Abstract Bone morphogenetic protein 9 (BMP‐9) is a member of the transforming growth factor (TGF)‐β/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/β‐catenin signalling plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. Wnt3A and BMP‐9 enhanced each other’s ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP‐9‐induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR‐5 or low‐density lipoprotein receptor‐related protein (LRP)‐6 exerted no inhibitory effect on BMP‐9‐induced ALP activity. β‐Catenin knockdown in MSCs and MEFs diminished BMP‐9‐induced ALP activity, and led to a decrease in BMP‐9‐induced osteocalcin reporter activity and BMP‐9‐induced expression of late osteogenic markers. Furthermore, β‐catenin knockdown or FrzB overexpression inhibited BMP‐9‐induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP‐9 induced recruitment of both Runx2 and β‐catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between β‐catenin and Runx2, plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. PMID:19175684

  20. Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling

    PubMed Central

    Franco, Claudio A; Jones, Martin L; Bernabeu, Miguel O; Vion, Anne-Clemence; Barbacena, Pedro; Fan, Jieqing; Mathivet, Thomas; Fonseca, Catarina G; Ragab, Anan; Yamaguchi, Terry P; Coveney, Peter V; Lang, Richard A; Gerhardt, Holger

    2016-01-01

    Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus. DOI: http://dx.doi.org/10.7554/eLife.07727.001 PMID:26845523

  1. SOXC proteins amplify canonical WNT signaling to secure nonchondrocytic fates in skeletogenesis.

    PubMed

    Bhattaram, Pallavi; Penzo-Méndez, Alfredo; Kato, Kenji; Bandyopadhyay, Kaustav; Gadi, Abhilash; Taketo, Makoto M; Lefebvre, Véronique

    2014-12-01

    Canonical WNT signaling stabilizes β-catenin to determine cell fate in many processes from development onwards. One of its main roles in skeletogenesis is to antagonize the chondrogenic transcription factor SOX9. We here identify the SOXC proteins as potent amplifiers of this pathway. The SOXC genes, i.e., Sox4, Sox11, and Sox12, are coexpressed in skeletogenic mesenchyme, including presumptive joints and perichondrium, but not in cartilage. Their inactivation in mouse embryo limb bud caused massive cartilage fusions, as joint and perichondrium cells underwent chondrogenesis. SOXC proteins govern these cells cell autonomously. They replace SOX9 in the adenomatous polyposis coli-Axin destruction complex and therein inhibit phosphorylation of β-catenin by GSK3. This inhibition, a crucial, limiting step in canonical WNT signaling, thus becomes a constitutive event. The resulting SOXC/canonical WNT-mediated synergistic stabilization of β-catenin contributes to efficient repression of Sox9 in presumptive joint and perichondrium cells and thereby ensures proper delineation and articulation of skeletal primordia. This synergy may determine cell fate in many processes besides skeletogenesis. PMID:25452386

  2. Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical Wnt signaling and causes an osteogenesis-imperfecta-like phenotype

    PubMed Central

    Salazar, Valerie S.; Zarkadis, Nicholas; Huang, Lisa; Norris, Jin; Grimston, Susan K.; Mbalaviele, Gabriel; Civitelli, Roberto

    2013-01-01

    Summary To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-β–BMP pathway, in Osx1+ cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of β-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt. PMID:24006258

  3. Role of L1CAM in the Regulation of the Canonical Wnt Pathway and Class I MAGE Genes.

    PubMed

    Shkurnikov, M Yu; Knyazev, E N; Wicklein, D; Schumacher, U; Samatov, T R; Tonevitskii, A G

    2016-04-01

    Molecule L1CAM is specific for nerve cells and tumors of various localizations. The expression of L1CAM is significantly higher in melanoma in comparison with benign nevi and correlates with the progress of melanoma and transition from radial to vertical growth. Monoclonal antibodies to L1CAM effectively and specifically attenuate melanoma growth, though stimulates the epithelial-mesenchymal transition. shRNA-mediated knock-down of L1CAM showed the involvement of L1CAM in regulation of activity of the canonical Wnt pathway and expression of genes of class I melanoma-associated antigens (MAGE). PMID:27165065

  4. Canonical Notch activation in osteocytes causes osteopetrosis.

    PubMed

    Canalis, Ernesto; Bridgewater, David; Schilling, Lauren; Zanotti, Stefano

    2016-01-15

    Activation of Notch1 in cells of the osteoblastic lineage inhibits osteoblast differentiation/function and causes osteopenia, whereas its activation in osteocytes causes a distinct osteopetrotic phenotype. To explore mechanisms responsible, we established the contributions of canonical Notch signaling (Rbpjκ dependent) to osteocyte function. Transgenics expressing Cre recombinase under the control of the dentin matrix protein-1 (Dmp1) promoter were crossed with Rbpjκ conditional mice to generate Dmp1-Cre(+/-);Rbpjκ(Δ/Δ) mice. These mice did not have a skeletal phenotype, indicating that Rbpjκ is dispensable for osteocyte function. To study the Rbpjκ contribution to Notch activation, Rosa(Notch) mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and the NICD coding sequence, were crossed with Dmp1-Cre transgenic mice and studied in the context (Dmp1-Cre(+/-);Rosa(Notch);Rbpjκ(Δ/Δ)) or not (Dmp1-Cre(+/-);Rosa(Notch)) of Rbpjκ inactivation. Dmp1-Cre(+/-);Rosa(Notch) mice exhibited increased femoral trabecular bone volume and decreased osteoclasts and bone resorption. The phenotype was reversed in the context of the Rbpjκ inactivation, demonstrating that Notch canonical signaling was accountable for the phenotype. Notch activation downregulated Sost and Dkk1 and upregulated Axin2, Tnfrsf11b, and Tnfsf11 mRNA expression, and these effects were not observed in the context of the Rbpjκ inactivation. In conclusion, Notch activation in osteocytes suppresses bone resorption and increases bone volume by utilization of canonical signals that also result in the inhibition of Sost and Dkk1 and upregulation of Wnt signaling. PMID:26578715

  5. MicroRNA-142-3p Negatively Regulates Canonical Wnt Signaling Pathway

    PubMed Central

    Hu, Tanyu; Phiwpan, Krung; Guo, Jitao; Zhang, Wei; Guo, Jie; Zhang, Zhongmei; Zou, Mangge; Zhang, Xuejie; Zhang, Jianhua

    2016-01-01

    Wnt/β-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs) are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/β-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/β-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC) to upregulate Wnt/β-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/β-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding β-catenin) through binding to its 3’ untranslated region (3’ UTR). Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/β-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/β-catenin signaling, which help to understand the complex regulation of Wnt/β-catenin signaling. PMID:27348426

  6. BMP and non-canonical Wnt signaling are required for inhibition of secondary tail formation in zebrafish.

    PubMed

    Yang, Yi; Thorpe, Chris

    2011-06-01

    The role of bone morphogenetic protein (BMP) signaling in specifying cell fate in the zebrafish tailbud has been well established. In addition to a loss of ventral tissues, such as ventral tailfin and cloaca, some embryos with compromised BMP signaling produce an additional phenotype: a ventrally located secondary tail containing both somitic muscle and notochord. This phenotype has been proposed to reflect a fate-patterning defect due to a change in a hypothesized BMP activity gradient. Here, we show that a defect in morphogenetic movements, not fate patterning, underlies the formation of secondary tails in BMP-inhibited embryos. Our data indicate that BMP signaling is activated in the ventroposterior tailbud to promote cell migration during tailbud protrusion, and that defective migration of these cells in BMP mutants ultimately leads to bifurcation of the caudal notochord. Additionally, we show that non-canonical Wnt signaling is also required for proper tail morphogenesis, possibly by maintaining cohesion of notochord progenitors by regulation of cadherin localization. We propose a model in which BMP and the non-canonical Wnt pathway regulate tail morphogenesis by controlling cell migration and cell adhesion within the tailbud. PMID:21610036

  7. Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation.

    PubMed

    Brinkmann, Eva-Maria; Mattes, Benjamin; Kumar, Rahul; Hagemann, Anja I H; Gradl, Dietmar; Scholpp, Steffen; Steinbeisser, Herbert; Kaufmann, Lilian T; Özbek, Suat

    2016-06-24

    Convergent extension movements during vertebrate gastrulation require a balanced activity of non-canonical Wnt signaling pathways, but the factors regulating this interplay on the molecular level are poorly characterized. Here we show that sFRP2, a member of the secreted frizzled-related protein (sFRP) family, is required for morphogenesis and papc expression during Xenopus gastrulation. We further provide evidence that sFRP2 redirects non-canonical Wnt signaling from Frizzled 7 (Fz7) to the receptor tyrosine kinase-like orphan receptor 2 (Ror2). During this process, sFRP2 promotes Ror2 signal transduction by stabilizing Wnt5a-Ror2 complexes at the membrane, whereas it inhibits Fz7 signaling, probably by blocking Fz7 receptor endocytosis. The cysteine-rich domain of sFRP2 is sufficient for Ror2 activation, and related sFRPs can substitute for this function. Notably, direct interaction of the two receptors via their cysteine-rich domains also promotes Ror2-mediated papc expression but inhibits Fz7 signaling. We propose that sFRPs can act as a molecular switch, channeling the signal input for different non-canonical Wnt pathways during vertebrate gastrulation. PMID:27129770

  8. Hepatic Progenitor Cells Contribute to the Progression of 2-Acetylaminofluorene/Carbon Tetrachloride-Induced Cirrhosis via the Non-Canonical Wnt Pathway.

    PubMed

    Chen, Jiamei; Zhang, Xiao; Xu, Ying; Li, Xuewei; Ren, Shuang; Zhou, Yaning; Duan, Yuyou; Zern, Mark; Zhang, Hua; Chen, Gaofeng; Liu, Chenghai; Mu, Yongping; Liu, Ping

    2015-01-01

    Hepatic progenitor cells (HPCs) appear to play an important role in chronic liver injury. In this study, cirrhosis was induced in F-344 rats (n = 32) via subcutaneous injection of 50% carbon tetrachloride (CCl4) twice a week for 8 weeks. Then, 30% CCl4 was administered in conjunction with intragastric 2-acetylaminofluorine (2-AAF) for 4 weeks to induce activation of HPCs. WB-F344 cells were used to provide direct evidence for differentiation of HPCs to myofibroblasts. The results showed that after administration of 2-AAF, the hydroxyproline content and the expressions of α-SMA, Col I, Col IV, TGF-β1, CD68, TNF-α, CK19 and OV6 were significantly increased. OV6 and α-SMA were largely co-expressed in fibrous septum and the expressions of Wnt5b, frizzled2, frizzled3 and frizzled6 were markedly increased, while β-catenin expression was not statistically different among the different groups. Consistent with the above results, WB-F344 cells, treated with TGF-β1 in vitro, differentiated into myofibroblasts and α-SMA, Col I, Col IV, Wnt5b and frizzled2 expressions were significantly increased, while β-catenin expression was decreased. After blocking the non-canonical Wnt pathway via WIF-1, the Wnt5b level was down regulated, and α-SMA and F-actin expressions were significantly decreased in the WIF-1-treated cells. In conclusion, these results indicate that HPCs appear to differentiate into myofibroblasts and exhibit a profibrotic effect in progressive cirrhosis via activation of the non-canonical Wnt pathway. Blocking the non-canonical Wnt pathway can inhibit the differentiation of HPCs into myofibroblasts, suggesting that blocking this pathway and changing the fate of differentiated HPCs may be a potential treatment for cirrhosis. PMID:26087010

  9. A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.

    PubMed

    Waaler, Jo; Machon, Ondrej; Tumova, Lucie; Dinh, Huyen; Korinek, Vladimir; Wilson, Steven Ray; Paulsen, Jan Erik; Pedersen, Nina Marie; Eide, Tor J; Machonova, Olga; Gradl, Dietmar; Voronkov, Andrey; von Kries, Jens Peter; Krauss, Stefan

    2012-06-01

    Increased nuclear accumulation of β-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/β-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the β-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the β-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the β-catenin destruction complex, followed by increased degradation of β-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of β-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/β-catenin signaling through inhibiting the PARP domain of TNKS1/2. PMID:22440753

  10. Resveratrol augments the canonical Wnt signaling pathway in promoting osteoblastic differentiation of multipotent mesenchymal cells

    SciTech Connect

    Zhou, Haibin; Shang, Linshan; Li, Xi; Zhang, Xiyu; Gao, Guimin; Guo, Chenhong; Chen, Bingxi; Liu, Qiji; Gong, Yaoqin; Shao, Changshun

    2009-10-15

    Resveratrol has been shown to possess many health-benefiting effects, including the promotion of bone formation. In this report we investigated the mechanism by which resveratrol promotes osteoblastic differentiation from pluripotent mesenchymal cells. Since Wnt signaling is well documented to induce osteoblastogenesis and bone formation, we characterized the factors involved in Wnt signaling in response to resveratrol treatment. Resveratrol treatment of mesenchymal cells led to an increase in stabilization and nuclear accumulation of {beta}-catenin dose-dependently and time-dependently. As a consequence of the increased nuclear accumulation of {beta}-catenin, the ability to activate transcription of {beta}-catenin-TCF/LEF target genes that are required for osteoblastic differentiation was upregulated. However, resveratrol did not affect the initial step of the Wnt signaling pathway, as resveratrol was as effective in upregulating the activity of {beta}-catenin in cells in which Lrp5 was knocked down as in control cells. In addition, while conditioned medium enriched in Wnt signaling antagonist Dkk1 was able to inhibit Wnt3a-induced {beta}-catenin upregulation, this inhibitory effect can be abolished in resveratrol-treated cells. Furthermore, we showed that the level of glycogen synthase kinase 3{beta} (GSK-3{beta}), which phosphorylates and destabilizes {beta}-catenin, was reduced in response to resveratrol treatment. The phosphorylation of GSK-3{beta} requires extracellular signal-regulated kinase (ERK)1/2. Together, our data indicate that resveratrol promotes osteoblastogenesis and bone formation by augmenting Wnt signaling.

  11. Rabconnectin-3a Regulates Vesicle Endocytosis and Canonical Wnt Signaling in Zebrafish Neural Crest Migration

    PubMed Central

    Tuttle, Adam M.; Hoffman, Trevor L.; Schilling, Thomas F.

    2014-01-01

    Cell migration requires dynamic regulation of cell–cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand–receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial–mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts. PMID:24802872

  12. Abnormal expression of key genes and proteins in the canonical Wnt/β-catenin pathway of articular cartilage in a rat model of exercise-induced osteoarthritis

    PubMed Central

    LIU, SHEN-SHEN; ZHOU, PU; ZHANG, YANQIU

    2016-01-01

    To investigate the molecular pathogenesis of the canonical Wnt/β-catenin pathway in exercise-induced osteoarthritis (OA), 30 male healthy Sprague Dawley rats were divided into three groups (control, normal exercise-induced OA and injured exercise-induced OA groups) in order to establish the exercise-induced OA rat model. The mRNA and protein expression levels of Runx-2, BMP-2, Ctnnb1, Sox-9, collagen II, Mmp-13, Wnt-3a and β-catenin in chon-drocytes were detected by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemical staining. The mRNA levels of Runx-2, BMP-2 and Ctnnb1 were upregulated in the normal exercise-induced OA and injured exercise-induced OA groups; while Runx-2 and BMP-2 were upregulated in the injured exercise-induced OA group when compared with the normal exercise-induced OA group. The protein levels of Mmp-13, Wnt-3a and β-catenin were increased and collagen II was reduced in the normal exercise-induced OA and injured exercise-induced OA groups. Ctnnb1, Wnt-3a and β-catenin, which are key genes and proteins in the canonical Wnt/β-catenin pathway, were abnormally expressed in chondrocytes of the exercise-induced OA rat model. Ctnnb1, β-catenin and Wnt-3a were suggested to participate in the pathogenesis of exercise-induced OA by abnormally activating the Wnt/β-catenin pathway during physical exercise due to excessive pressure. The results of the present study may provide an improved understanding of the pathogenesis of exercise-induced OA. PMID:26794964

  13. Hypoxia-Inducible Factors Modulate the Stemness and Malignancy of Colon Cancer Cells by Playing Opposite Roles in Canonical Wnt Signaling

    PubMed Central

    Santoyo-Ramos, Paula; Likhatcheva, María; García-Zepeda, Eduardo A.; Castañeda-Patlán, M. Cristina; Robles-Flores, Martha

    2014-01-01

    This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling. Under normoxia, we determined that both HIF-1α and HIF-2α are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1α or HIF-2α expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1α depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on β-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2α knockdown did not affect β-catenin levels, increasing the transcriptional activity of β-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of β-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1α depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2α silencing altered the expression of the stem cell markers CD44, Oct4, and CD24 and of the differentiation marker CK20 in the opposite direction as HIF-1α silencing. Remarkably, HIF-2α knockdown also enhanced β-catenin transcriptional activity under hypoxia in cells that displayed normal Wnt signaling, suggesting that the gene negatively modulates canonical Wnt signaling in colon cancer cells. Taken together, our results indicate that HIFs play opposing roles in canonical Wnt signaling and are essential for the stemness and malignancy maintenance of colon cancer cells. PMID:25396735

  14. Dickkopf-1 induced apoptosis in human placental choriocarcinoma is independent of canonical Wnt signaling

    SciTech Connect

    Peng Sha; Miao Chenglin; Li Jing; Fan Xiujun; Cao Yujing; Duan Enkui . E-mail: duane@ioz.ac.cn

    2006-11-24

    Placental choriocarcinoma, a reproductive system carcinoma in women, has about 0.81% occurrence frequency in China, which leads to over 90% lethality due to indistinct pathogenesis and the absence of efficient therapeutic treatment. In the present study, using immunostaining and reverse transcription PCR, we reported that Dickkopf-1 (Dkk-1) is prominently expressed in human cytotrophoblast (CTB) cell, but absent in the human placental choriocarcinoma cell line JAR and JEG3, implicating an unknown correlation between Dkk-1 and carcinogenesis of placental choriocarcinoma. Further, through exogenous introduction of Dkk-1, we found repressed proliferation in JAR and JEG3, induced apoptosis in JAR, and discovered significant tumor suppression effects of Dkk-1 in placental choriocarcinoma. Moreover we found that this function of Dkk-1 is achieved through c-Jun N-terminal kinase (JNK), whereas the canonical Wnt pathway may not have a great role. This discovery is not symphonic to previous functional understanding of Dkk-1, a canonical Wnt signaling antagonist. Together, our data indicate the possible correlation between Dkk-1 and human placental choriocarcinoma and suggest potential applications of Dkk-1 in treatment of human placental choriocarcinomas.

  15. Resveratrol inhibits canonical Wnt signaling in human MG-63 osteosarcoma cells.

    PubMed

    Zou, Yonggen; Yang, Jiexiang; Jiang, Dianming

    2015-11-01

    In the last 30 years, the 5-year-survival rate of patients with osteosarcoma has not improved as a result of the low prevalence and large tumor heterogeneity. Therefore, the development of novel drugs for the treatment of osteosarcoma is urgently required. The present study aimed to identify potential novel drugs for the treatment of osteosarcoma, thus used β‑catenin as a target and performed high content screening. In a total of 14 botanical extracts assessed, resveratrol markedly downregulated the expression of β‑catenin and significantly inhibited MG‑63 cell proliferation. CCK‑8 assay was used to confirm the anti‑osteosarcoma effect of resveratrol and flow cytometry and western blotting were performed to analyze the underlying mechanisms of the proapoptotic effects of resveratrol. β‑catenin is a vital member of the canonical Wnt signaling pathway and, therefore, the target genes of this pathway were further analyzed. The results of this analysis demonstrated that resveratrol suppressed the MG‑63 cells by inhibiting the canonical Wnt signaling pathway. PMID:26398440

  16. Wnt pathway activation by ADP-ribosylation

    PubMed Central

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P.; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S.; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)—known to target Axin for proteolysis—regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  17. Wnt pathway activation by ADP-ribosylation.

    PubMed

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  18. Wnt activation promotes neuronal differentiation of Glioblastoma

    PubMed Central

    Rampazzo, E; Persano, L; Pistollato, F; Moro, E; Frasson, C; Porazzi, P; Della Puppa, A; Bresolin, S; Battilana, G; Indraccolo, S; Te Kronnie, G; Argenton, F; Tiso, N; Basso, G

    2013-01-01

    One of the biggest challenges in tumour research is the possibility to reprogram cancer cells towards less aggressive phenotypes. In this study, we reprogrammed primary Glioblastoma multiforme (GBM)-derived cells towards a more differentiated and less oncogenic phenotype by activating the Wnt pathway in a hypoxic microenvironment. Hypoxia usually correlates with malignant behaviours in cancer cells, but it has been recently involved, together with Wnt signalling, in the differentiation of embryonic and neural stem cells. Here, we demonstrate that treatment with Wnt ligands, or overexpression of β-catenin, mediate neuronal differentiation and halt proliferation in primary GBM cells. An hypoxic environment cooperates with Wnt-induced differentiation, in line with our finding that hypoxia inducible factor-1α (HIF-1α) is instrumental and required to sustain the expression of β-catenin transcriptional partners TCF-1 and LEF-1. In addition, we also found that Wnt-induced GBM cell differentiation inhibits Notch signalling, and thus gain of Wnt and loss of Notch cooperate in the activation of a pro-neuronal differentiation program. Intriguingly, the GBM sub-population enriched of cancer stem cells (CD133+ fraction) is the primary target of the pro-differentiating effects mediated by the crosstalk between HIF-1α, Wnt, and Notch signalling. By using zebrafish transgenics and mutants as model systems to visualize and manipulate in vivo the Wnt pathway, we confirm that Wnt pathway activation is able to promote neuronal differentiation and inhibit Notch signalling of primary human GBM cells also in this in vivo set-up. In conclusion, these findings shed light on an unsuspected crosstalk between hypoxia, Wnt and Notch signalling in GBM, and suggest the potential to manipulate these microenvironmental signals to blunt GBM malignancy. PMID:23429286

  19. Components of Intraflagellar Transport Complex A Function Independently of the Cilium to Regulate Canonical Wnt Signaling in Drosophila.

    PubMed

    Balmer, Sophie; Dussert, Aurore; Collu, Giovanna M; Benitez, Elvira; Iomini, Carlo; Mlodzik, Marek

    2015-09-28

    The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, we conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. Our results show no effect on Hedgehog signaling. In contrast, our screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting β-catenin/Armadillo activity. PMID:26364750

  20. Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/β-Catenin Pathway in Rat Brain Endothelial Cells Requires Cross-talk with Notch Signaling.

    PubMed

    Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R

    2016-04-01

    The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways. PMID:26872974

  1. WNT antagonist, DKK2, is a Notch signaling target in intestinal stem cells: augmentation of a negative regulation system for canonical WNT signaling pathway by the Notch-DKK2 signaling loop in primates.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-01-01

    Notch and WNT signaling pathways are key components of the stem cell signaling network. Canonical WNT signaling to intestinal progenitor cells leads to transcriptional activation of the JAG1 gene, encoding Serrate-type Notch ligand. JAG1 then binds to the Notch receptor on adjacent stem cells to induce Notch receptor proteolyses for the release of Notch intracellular domain (NICD). NICD is associated with CSL/RBPSUH and Mastermind (MAML1, MAML2, or MAML3) to activate Notch target genes, such as HES1 and HES5. Although WNT-dependent Notch signaling activation in intestinal stem cells is clarified, the effects of Notch signaling activation on WNT signaling in progenitor cells remain unclear. We searched for Notch-response element (NRE) in the promoter region of genes encoding secreted WNT signaling inhibitors, including DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1. Double NREs were identified within human DKK2 promoter by bioinformatics and human intelligence (Humint). The human DKK2 gene was characterized as Notch signaling target in intestinal stem cells. Because DKK2 is a key player in the stem cell signaling network, the DKK2 gene at human chromosome 4q25 is a candidate tumor suppressor gene inactivated due to epigenetic silencing and/or deletion. The chimpanzee DKK2 gene was identified within the NW_105990.1 genome sequence, while the cow Dkk2 gene was identified within the AC156664.2 and AC158038.2 genome sequences. Chimpanzee DKK2 and cow Dkk2 showed 98.5% and 95.8% total-amino-acid identity with human DKK2, respectively. Double NREs in human DKK2 promoter were conserved in chimpanzee DKK2 promoter, partially in rat Dkk2 promoter, but not in cow and mouse Dkk2 promoters. The Notch-DKK2 signaling loop, created or potentiated in primates, was complementary to WNT-DKK1 and BMP-IHH-SFRP1 signaling loops for negative regulation of canonical WNT signaling pathway. Together, these facts indicate that DKK2 promoter evolution resulted in the

  2. Hit to lead studies on (hetero)arylpyrimidines--agonists of the canonical Wnt-beta-catenin cellular messaging system.

    PubMed

    Gilbert, Adam M; Bursavich, Matthew G; Alon, Nippa; Bhat, Bheem M; Bex, Frederick J; Cain, Michael; Coleburn, Valerie; Gironda, Virginia; Green, Paula; Hauze, Diane B; Kharode, Yogendra; Krishnamurthy, Girija; Kirisits, Matthew; Lam, Ho-Sun; Liu, Yao-Bin; Lombardi, Sabrina; Matteo, Jeanne; Murrills, Richard; Robinson, John A; Selim, Sally; Sharp, Michael; Unwalla, Raymond; Varadarajan, Usha; Zhao, Weiguang; Yaworsky, Paul J

    2010-01-01

    A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone. PMID:19897365

  3. Activation of intracellular calcium by multiple Wnt ligands and translocation of β-catenin into the nucleus: a convergent model of Wnt/Ca2+ and Wnt/β-catenin pathways.

    PubMed

    Thrasivoulou, Christopher; Millar, Michael; Ahmed, Aamir

    2013-12-13

    Ca(2+) and β-catenin, a 92-kDa negatively charged transcription factor, transduce Wnt signaling via the non-canonical, Wnt/Ca(2+) and canonical, Wnt/β-catenin pathways independently. The nuclear envelope is a barrier to large protein entry, and this process is regulated by intracellular calcium [Ca(2+)]i and trans-nuclear potential. How β-catenin traverses the nuclear envelope is not well known. We hypothesized that Wnt/Ca(2+) and Wnt/β-catenin pathways act in a coordinated manner and that [Ca(2+)]i release facilitates β-catenin entry into the nucleus in mammalian cells. In a live assay using calcium dyes in PC3 prostate cancer cells, six Wnt peptides (3A, 4, 5A, 7A, 9B, and 10B) mobilized [Ca(2+)]i but Wnt11 did not. Based upon dwell time (range = 15-30 s) of the calcium waveform, these Wnts could be classified into three classes: short, 3A and 5A; long, 7A and 10B; and very long, 4 and 9B. Wnt-activated [Ca(2+)]i release was followed by an increase in intranuclear calcium and the depolarization of both the cell and nuclear membranes, determined by using FM4-64. In cells treated with Wnts 5A, 9B, and 10B, paradigm substrates for each Wnt class, increased [Ca(2+)]i was followed by β-catenin translocation into the nucleus in PC3, MCF7, and 253J, prostate, breast, and bladder cancer cell lines; both the increase in Wnt 5A, 9B, and 10B induced [Ca(2+)]i release and β-catenin translocation are suppressed by thapsigargin in PC3 cell line. We propose a convergent model of Wnt signaling network where Ca(2+) and β-catenin pathways may act in a coordinated, interdependent, rather than independent, manner. PMID:24158438

  4. Comparative integromics on non-canonical WNT or planar cell polarity signaling molecules: transcriptional mechanism of PTK7 in colorectal cancer and that of SEMA6A in undifferentiated ES cells.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-09-01

    Non-canonical WNT and planar cell polarity (PCP) are overlapping but distinct signaling pathways, which control convergent extension, neural tube closure, orientation of cilia and sensory hair cells, axon guidance, and cell motility. Non-canonical WNT signals, regulated by the interaction of WNT, WNT antagonist, Frizzled and ROR2, are transduced to JNK, ROCK, PKC, MAP3K7, and NFAT signaling cascades. PCP signals, regulated by the interaction of VANGL-PRICKLE complex, CELSR and Frizzled-DVL complex, are transduced to JNK, ROCK, and other uncharacterized signaling cascades. PTK7 signaling, regulated by SEMA6 and Plexin-A family members, affects PCP pathway through VANGL. Here, integrative genomic analyses on WNT5A, WNT5B, WNT11, FZD3, FZD6, ROR1, ROR2, RYK, CELSR1, CELSR2, CELSR3, VANGL1, VANGL2, PRICKLE1, PRICKLE2, PTK7, SEMA6A, SEMA6B, SEMA6C and SEMA6D were carried out. PTK7 and SEMA6A were expressed in undifferentiated embryonic stem (ES) cells, SEMA6A in endodermal progenitors, CELSR1, VANGL1 and PTK7 in gastrointestinal tumors. CELSR2, PRICKLE2 and SEMA6C were expressed in fetal brain, CELSR2, PRICKLE1 and SEMA6A in adult brain, WNT5A and CELSR3 in adult brain tumors. These facts indicate class switches of non-canonical WNT or PCP signaling molecules during embryogenesis and carcinogenesis. TCF/LEF-, SP1-, and 5 bHLH-binding sites within human PTK7 promoter were conserved in chimpanzee, rhesus monkey, mouse, and rat PTK7 orthologs, which explained the mechanism of PTK7 upregulation in colorectal cancer. NANOG-, SOX2-, and POU5F1 (OCT3/OCT4)-binding sites within intron 1 of the human SEMA6A gene were conserved in chimpanzee, rhesus monkey, mouse, and rat SEMA6A orthologs, which explained the mechanism of SEMA6A upregulation in undifferentiated ES cells. Most of non-canonical WNT or PCP signaling molecules, except PTK7 and SEMA6A, were not frequently expressed in undifferentiated human ES cells. Non-canonical WNT or PCP signaling pathway, activated to orchestrate

  5. TCF3, a novel positive regulator of osteogenesis, plays a crucial role in miR-17 modulating the diverse effect of canonical Wnt signaling in different microenvironments

    PubMed Central

    Liu, W; Liu, Y; Guo, T; Hu, C; Luo, H; Zhang, L; Shi, S; Cai, T; Ding, Y; Jin, Y

    2013-01-01

    Wnt signaling pathways are a highly conserved pathway, which plays an important role from the embryonic development to bone formation. The effect of Wnt pathway on osteogenesis relies on their cellular environment and the expression of target genes. However, the molecular mechanism of that remains unclear. On the basis of the preliminary results, we observed the contrary effect of canonical Wnt signaling on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the different culture environment. Furthermore, we found that the expression level of miR-17 was also varied with the change in the culture environment. Therefore, we hypothesized that miR-17 and canonical Wnt signaling may have potential interactions, particularly the inner regulation relationship in different microenvironments. In this paper, we observed that canonical Wnt signaling promoted osteogenesis of PDLSCs in the fully culture medium, while inhibited it in the osteogenic differentiation medium. Interestingly, alteration in the expression level of endogenous miR-17 could partially reverse the different effect of canonical Wnt signaling. Furthermore, the role of miR-17 was because of its target gene TCF3 (transcription factor 3), a key transcription factor of canonical Wnt pathway. Overexpression of TCF3 attenuated the effect of miR-17 on modulating canonical Wnt signaling. Finally, we elucidated that TCF3 enhanced osteogenesis both in vitro and in vivo. In brief, the different level of miR-17 was the main cause of the different effect of canonical Wnt signaling, and TCF3 was the crucial node of miR-17–canonial Wnt signaling regulation loop. This understanding of microRNAs regulating signaling pathways in different microenvironments may pave the way for fine-tuning the process of osteogenesis in bone-related disorders. PMID:23492770

  6. Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

    PubMed Central

    Aznar, Nicolas; Midde, Krishna K; Dunkel, Ying; Lopez-Sanchez, Inmaculada; Pavlova, Yelena; Marivin, Arthur; Barbazán, Jorge; Murray, Fiona; Nitsche, Ulrich; Janssen, Klaus-Peter; Willert, Karl; Goel, Ajay; Abal, Miguel; Garcia-Marcos, Mikel; Ghosh, Pradipta

    2015-01-01

    Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the β-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis. DOI: http://dx.doi.org/10.7554/eLife.07091.001 PMID:26126266

  7. Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling.

    PubMed

    Aznar, Nicolas; Midde, Krishna K; Dunkel, Ying; Lopez-Sanchez, Inmaculada; Pavlova, Yelena; Marivin, Arthur; Barbazán, Jorge; Murray, Fiona; Nitsche, Ulrich; Janssen, Klaus-Peter; Willert, Karl; Goel, Ajay; Abal, Miguel; Garcia-Marcos, Mikel; Ghosh, Pradipta

    2015-01-01

    Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the β-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis. PMID:26126266

  8. Liposomal packaging generates Wnt protein with in vivo biological activity.

    PubMed

    Morrell, Nathan T; Leucht, Philipp; Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M; Helms, Jill A; Nusse, Roel

    2008-01-01

    Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context. PMID:18698373

  9. Bisphenol-A Mediated Inhibition of Hippocampal Neurogenesis Attenuated by Curcumin via Canonical Wnt Pathway.

    PubMed

    Tiwari, Shashi Kant; Agarwal, Swati; Tripathi, Anurag; Chaturvedi, Rajnish Kumar

    2016-07-01

    Bisphenol A (BPA) is an environmental xenoestrogenic endocrine disruptor, utilized for production of consumer products, and exerts adverse effects on the developing nervous system. Recently, we found that BPA impairs the finely tuned dynamic processes of neurogenesis (generation of new neurons) in the hippocampus of the developing rat brain. Curcumin is a natural polyphenolic compound, which provides neuroprotection against various environmental neurotoxicants and in the cellular and animal models of neurodegenerative disorders. Here, we have assessed the neuroprotective efficacy of curcumin against BPA-mediated reduced neurogenesis and the underlying cellular and molecular mechanism(s). Both in vitro and in vivo studies showed that curcumin protects against BPA-induced hippocampal neurotoxicity. Curcumin protects against BPA-mediated reduced neural stem cells (NSC) proliferation and neuronal differentiation and enhanced neurodegeneration. Curcumin also enhances the expression/levels of neurogenic and the Wnt pathway genes/proteins, which were reduced due to BPA exposure in the hippocampus. Curcumin-mediated neuroprotection against BPA-induced neurotoxicity involved activation of the Wnt/β-catenin signaling pathway, which was confirmed by the use of Wnt specific activators (LiCl and GSK-3β siRNA) and inhibitor (Dkk-1). BPA-mediated increased β-catenin phosphorylation, decreased GSK-3β levels, and β-catenin nuclear translocation were significantly reversed by curcumin, leading to enhanced neurogenesis. Curcumin-induced protective effects on neurogenesis were blocked by Dkk-1 in NSC culture treated with BPA. Curcumin-mediated enhanced neurogenesis was correlated well with improved learning and memory in BPA-treated rats. Overall, our results conclude that curcumin provides neuroprotection against BPA-mediated impaired neurogenesis via activation of the Wnt/β-catenin signaling pathway. PMID:25963729

  10. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    SciTech Connect

    Sakisaka, Yukihiko; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

    2015-08-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.

  11. Wnt/β-catenin signaling via Axin2 is required for myogenesis and, together with YAP/Taz and Tead1, active in IIa/IIx muscle fibers.

    PubMed

    Huraskin, Danyil; Eiber, Nane; Reichel, Martin; Zidek, Laura M; Kravic, Bojana; Bernkopf, Dominic; von Maltzahn, Julia; Behrens, Jürgen; Hashemolhosseini, Said

    2016-09-01

    Canonical Wnt/β-catenin signaling plays an important role in myogenic differentiation, but its physiological role in muscle fibers remains elusive. Here, we studied activation of Wnt/β-catenin signaling in adult muscle fibers and muscle stem cells in an Axin2 reporter mouse. Axin2 is a negative regulator and a target of Wnt/β-catenin signaling. In adult muscle fibers, Wnt/β-catenin signaling is only detectable in a subset of fast fibers that have a significantly smaller diameter than other fast fibers. In the same fibers, immunofluorescence staining for YAP/Taz and Tead1 was detected. Wnt/β-catenin signaling was absent in quiescent and activated satellite cells. Upon injury, Wnt/β-catenin signaling was detected in muscle fibers with centrally located nuclei. During differentiation of myoblasts expression of Axin2, but not of Axin1, increased together with Tead1 target gene expression. Furthermore, absence of Axin1 and Axin2 interfered with myoblast proliferation and myotube formation, respectively. Treatment with the canonical Wnt3a ligand also inhibited myotube formation. Wnt3a activated TOPflash and Tead1 reporter activity, whereas neither reporter was activated in the presence of Dkk1, an inhibitor of canonical Wnt signaling. We propose that Axin2-dependent Wnt/β-catenin signaling is involved in myotube formation and, together with YAP/Taz/Tead1, associated with reduced muscle fiber diameter of a subset of fast fibers. PMID:27578179

  12. Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

    PubMed

    Yun, Kangsun; Ajima, Rieko; Sharma, Nirmala; Costantini, Frank; Mackem, Susan; Lewandoski, Mark; Yamaguchi, Terry P; Perantoni, Alan O

    2014-12-20

    Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations. PMID:25082826

  13. Epigenetic inactivation of the canonical Wnt antagonist secreted frizzled-related protein 1 in hepatocellular carcinoma cells.

    PubMed

    Wu, Y; Li, J; Sun, C Y; Zhou, Y; Zhao, Y F; Zhang, S J

    2012-01-01

    Secreted Frizzled-related protein 1 (sFRP1), as one of most important Wnt antagonists, is frequently silenced by promoter hypermethylation in many types of tumor, including hepatocellular carcinoma (HCC). In this study, we aimed to investigate whether restoration of sFRP1 affected HCC metastatic behavior. sFRP1 mRNA expression and promoter methylation in HCC tissues and cell lines were examined using RT-PCR and methylation-specific PCR (MS-PCR), respectively. sFRP1 protein expression was assessed by Western Blot. We generated stable HCC cell line restoration of sFRP1 in HepG2 cells, which naturally do not express detectable sFRP1 mRNA. The effects of exogenous sFRP1 on HepG2 cell invasion were investigated using trans-well assay. Also the effects of sFRP1 re-expression on the β-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay.sFRP1 promoter methylation was frequently observed in HCC tissues (60%) and cell lines (75%). All samples with sFRP1 methylation showed down-regulation of sFRP1 expression in HCC cell lines. Demethylation treatment with 5-aza-20-deoxycytidine in HCC cells restored sFRP1 expression. Restoration of sFRP1 substantially impaired the invasive potentials of HepG2 cells. Moreover, exogenous sFRP1 caused significant decrease of β-catenin/T-cell factor-dependent transcription activity.These findings demonstrate that sFRP1 silencing due to promoter hypermethylation is a major event during tumorigenesis. sFRP1 is also a negative modulator of canonical Wnt signaling, which could contribute to metastasis in HCC progression, thus providing a possible therapeutic strategy against HCC. PMID:22296502

  14. Cross-regulation of signaling pathways: An example of nuclear hormone receptors and the canonical Wnt pathway

    SciTech Connect

    Beildeck, Marcy E.; Gelmann, Edward P.; Byers, Stephen W.

    2010-07-01

    Predicting the potential physiological outcome(s) of any given molecular pathway is complex because of cross-talk with other pathways. This is particularly evident in the case of the nuclear hormone receptor and canonical Wnt pathways, which regulate cell growth and proliferation, differentiation, apoptosis, and metastatic potential in numerous tissues. These pathways are known to intersect at many levels: in the intracellular space, at the membrane, in the cytoplasm, and within the nucleus. The outcomes of these interactions are important in the control of stem cell differentiation and maintenance, feedback loops, and regulating oncogenic potential. The aim of this review is to demonstrate the importance of considering pathway cross-talk when predicting functional outcomes of signaling, using nuclear hormone receptor/canonical Wnt pathway cross-talk as an example.

  15. A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling.

    PubMed

    Gebruers, Evelien; Cordero-Maldonado, María Lorena; Gray, Alexander I; Clements, Carol; Harvey, Alan L; Edrada-Ebel, Ruangelie; de Witte, Peter A M; Crawford, Alexander D; Esguerra, Camila V

    2013-01-01

    Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility. PMID:24349481

  16. Extending the Convergence of Canonical WNT Signaling and Classic Cancer Pathways for Treatment of Malignant Peripheral Nerve Sheath Tumors

    PubMed Central

    Reilly, Karlyne M.

    2016-01-01

    Summary Malignant peripheral nerve sheath tumors (MPNSTs) are incurable tumors of the Schwann cell lineage that progress unpredictably from benign plexiform neurofibromas (PNFs). In this issue of Cancer Discovery Watson and colleagues (1) use an insertional mutagenesis screen combined with network analysis to identify the canonical Wnt signaling pathway as an important potential biomarker of tumor progression and target for combination therapy in MPNSTs. PMID:23749527

  17. Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling

    PubMed Central

    Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

    2015-01-01

    AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed s

  18. Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

    SciTech Connect

    Briolay, A.; Lencel, P.; Caverzasio, J.; Buchet, R.; Magne, D.

    2013-01-18

    Highlights: ► Ankylosing spondylitis (AS) leads to bone fusions and ankylosis. ► TNF-α stimulates osteoblasts through growth factors in AS. ► We compare the involvement of canonical vs non-canonical Wnt signaling. ► Canonical Wnt signaling is not involved in TNF-α effects in differentiating hMSCs. ► TNF-α stimulates osteoblasts through Wnt5a autocrine secretion in hMSCs. -- Abstract: Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased

  19. WNT3 Inhibits Cerebellar Granule Neuron Progenitor Proliferation and Medulloblastoma Formation via MAPK Activation

    PubMed Central

    Ayrault, Olivier; Kim, Jee Hae; Zhu, Xiaodong; Murphy, David A.; Van Aelst, Linda; Roussel, Martine F.; Hatten, Mary E.

    2013-01-01

    During normal cerebellar development, the remarkable expansion of granule cell progenitors (GCPs) generates a population of granule neurons that outnumbers the total neuronal population of the cerebral cortex, and provides a model for identifying signaling pathways that may be defective in medulloblastoma. While many studies focus on identifying pathways that promote growth of GCPs, a critical unanswered question concerns the identification of signaling pathways that block mitogenic stimulation and induce early steps in differentiation. Here we identify WNT3 as a novel suppressor of GCP proliferation during cerebellar development and an inhibitor of medulloblastoma growth in mice. WNT3, produced in early postnatal cerebellum, inhibits GCP proliferation by down-regulating pro-proliferative target genes of the mitogen Sonic Hedgehog (SHH) and the bHLH transcription factor Atoh1. WNT3 suppresses GCP growth through a non-canonical Wnt signaling pathway, activating prototypic mitogen-activated protein kinases (MAPKs), the Ras-dependent extracellular-signal-regulated kinases 1/2 (ERK1/2) and ERK5, instead of the classical β-catenin pathway. Inhibition of MAPK activity using a MAPK kinase (MEK) inhibitor reversed the inhibitory effect of WNT3 on GCP proliferation. Importantly, WNT3 inhibits proliferation of medulloblastoma tumor growth in mouse models by a similar mechanism. Thus, the present study suggests a novel role for WNT3 as a regulator of neurogenesis and repressor of neural tumors. PMID:24303070

  20. Extracellular activation of Wnt signaling through epigenetic dysregulation of Wnt inhibitory factor-1 (Wif-1) is associated with pathogenesis of adrenocortical tumor.

    PubMed

    Mitsui, Yozo; Yasumoto, Hiroaki; Nagami, Taichi; Hiraki, Miho; Arichi, Naoko; Ishikawa, Noriyoshi; Araki, Asuka; Maruyama, Riruke; Tanaka, Yuichiro; Dahiya, Rajvir; Shiina, Hiroaki

    2014-04-30

    Wnt/β-catenin signaling is considered to be an essential regulator of adrenocortical oncogenesis. Wnt inhibitory factor-1 (Wif-1), an extracellular regulator of Wnt signaling, is frequently down-regulated by hypermethylation of the promoter CpG. We investigated epigenetic regulation of Wif-1 and its association with adrenocortical (AC) tumor pathogenesis in light of Wnt activation. The AC tumors showed a high prevalence of Wif-1 promoter methylation and low prevalence of Wif-1 mRNA transcription as compared to the normal adrenal (NA) samples. Furthermore, a significant correlation was found between Wif-1 promoter methylation and mRNA transcription in the tumors. Either intracellular β-catenin accumulation or β-catenin mRNA transcription was significantly elevated in the AC tumors, which also showed an inverse correlation with Wif-1 mRNA transcription. Cyclin D1, a target gene of Wnt signaling, was also up-regulated in the AC tumors as compared with the NA samples. In addition, down-regulation of Wif-1 was correlated with increased cyclin D1 at both mRNA and protein levels. However, despite the proposed activation of Wnt signaling in AC tumors, only 2 of 20 with intracellular β-catenin accumulation showed β-catenin mutations. Thus, genetic alterations of β-catenin and epigenetics-related Wif-1 promoter hypermethylation may be important mechanisms underlying AC tumor formation though aberrant canonical Wnt/β-catenin signaling activation. PMID:24755523

  1. In vivo transcriptional governance of hair follicle stem cells by canonical Wnt regulators

    PubMed Central

    Lien, Wen-Hui; Polak, Lisa; Lin, Mingyan; Lay, Kenneth; Zheng, Deyou; Fuchs, Elaine

    2014-01-01

    Hair follicle stem cells (HFSCs) regenerate hair in response to Wnt signalling. Here, we unfold genome-wide transcriptional and chromatin landscapes of β-catenin–TCF3/4–TLE circuitry, and genetically dissect their biological roles within the native HFSC niche. We show that during HFSC quiescence, TCF3, TCF4 and TLE (Groucho) bind coordinately and transcriptionally repress Wnt target genes. We also show that β-catenin is dispensable for HFSC viability, and that if TCF3/4 levels are sufficiently reduced, it is dispensable for proliferation. However, β-catenin is essential to activate genes that launch hair follicle fate and suppress sebocyte fate determination. TCF3/4 deficiency mimics Wnt–β-catenin-dependent activation of these hair follicle fate targets; TCF3 overexpression parallels their TLE4-dependent suppression. Our studies unveil TCF3/4–TLE histone deacetylases as a repressive rheostat, whose action can be relieved by Wnt–β-catenin signalling. When TCF3/4 and TLE levels are high, HFSCs can maintain stemness, but remain quiescent. When these levels drop or when Wnt–β-catenin levels rise, this balance is shifted and hair regeneration initiates. PMID:24463605

  2. Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

    SciTech Connect

    Jensen, Taylor J.; Wozniak, Ryan J.; Eblin, Kylee E.; Wnek, Sean M.; Gandolfi, A. Jay; Futscher, Bernard W.

    2009-02-15

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.

  3. Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation.

    PubMed

    Li, Zhenfei; Nie, Fen; Wang, Sheng; Li, Lin

    2011-02-22

    Histone methylation has an important role in transcriptional regulation. However, unlike H3K4 and H3K9 methylation, the role of H4K20 monomethylation (H4K20me-1) in transcriptional regulation remains unclear. Here, we show that Wnt3a specifically stimulates H4K20 monomethylation at the T cell factor (TCF)-binding element through the histone methylase SET8. Additionally, SET8 is crucial for activation of the Wnt reporter gene and target genes in both mammalian cells and zebrafish. Furthermore, SET8 interacts with lymphoid enhancing factor-1 (LEF1)/TCF4 directly, and this interaction is regulated by Wnt3a. Therefore, we conclude that SET8 is a Wnt signaling mediator and is recruited by LEF1/TCF4 to regulate the transcription of Wnt-activated genes, possibly through H4K20 monomethylation at the target gene promoters. Our findings also indicate that H4K20me-1 is a marker for gene transcription activation, at least in canonical Wnt signaling. PMID:21282610

  4. Nimbolide, a neem limonoid abrogates canonical NF-κB and Wnt signaling to induce caspase-dependent apoptosis in human hepatocarcinoma (HepG2) cells.

    PubMed

    Kavitha, Krishnamurthy; Vidya Priyadarsini, Ramamurthi; Anitha, Prabukumar; Ramalingam, Krishnan; Sakthivel, Ramasamy; Purushothaman, Gempuraj; Singh, Abhishek Kumar; Karunagaran, Devarajan; Nagini, Siddavaram

    2012-04-15

    Nuclear factor kappa B (NF-κB), an oncogenic signaling factor plays a critical role in the development and progression of various cancers. The objective of this study was to investigate the effect of nimbolide, a neem derived tetranortriterpenoid on NF-κB signaling and its downstream events - Wnt/β-catenin activation and apoptosis evasion in human hepatocarcinoma (HepG2) cells by evaluating NF-κB family members (NF-κB-p50, p65, IκB-α, p-IκB-α, and IKKβ), members of Wnt signaling (GSK-3β and β-catenin), and intrinsic apoptosis (Bcl-2, Bax, cytochrome c, Smac/DIABLO, caspase-3, and caspase-9). Our results demonstrate that nimbolide concurrently abrogates canonical NF-κB and Wnt signaling and induces intrinsic apoptosis in HepG2 cells. These data suggest that phytochemicals such as nimbolide that can target multiple steps along the NF-κB signaling circuit are promising candidates for future phytochemical-based mechanistic pathway targeted anticancer regimens. PMID:22327045

  5. The canonical Wnt pathway regulates the metastasis-promoting mucin MUC4 in pancreatic ductal adenocarcinoma.

    PubMed

    Pai, Priya; Rachagani, Satyanarayana; Lakshmanan, Imayavaramban; Macha, Muzafar A; Sheinin, Yuri; Smith, Lynette M; Ponnusamy, Moorthy P; Batra, Surinder K

    2016-02-01

    Aberrant Wnt signaling frequently occurs in pancreatic cancer (PC) and contributes to disease progression/metastases. Likewise, the transmembrane-mucin MUC4 is expressed de novo in early pancreatic intraepithelial neoplasia (PanINs) and incrementally increases with PC progression, contributing to metastasis. To determine the mechanism of MUC4 upregulation in PC, we examined factors deregulated in early PC progression, such as Wnt/β-catenin signaling. MUC4 promoter analysis revealed the presence of three putative TCF/LEF-binding sites, leading us to hypothesize that MUC4 can be regulated by β-catenin. Immunohistochemical (IHC) analysis of rapid autopsy PC tissues showed a correlation between MUC4 and cytosolic/nuclear β-catenin expression. Knock down (KD) of β-catenin in CD18/HPAF and T3M4 cell lines resulted in decreased MUC4 transcript and protein. Three MUC4 promoter luciferase constructs, p3778, p3000, and p2700, were generated. The construct p3778, encompassing the entire MUC4 promoter, elicited increased luciferase activity in the presence of stabilized β-catenin. Mutation of the TCF/LEF site closest to the transcription start site (i.e., -2629/-2612) and furthest from the start site (i.e., -3425/-3408) reduced MUC4 promoter luciferase activity. Transfection with dominant negative TCF4 decreased MUC4 transcript and protein levels. Chromatin immunoprecipitation confirmed enrichment of β-catenin on -2629/-2612 and -3425/-3408 of the MUC4 promoter in CD18/HPAF. Functionally, CD18/HPAF and T3M4 β-catenin KD cells showed decreased migration and decreased Vimentin, N-cadherin, and pERK1/2 expression. Tumorigenicity studies in athymic nude mice showed CD18/HPAF β-catenin KD cells significantly reduced primary tumor sizes and metastases compared to scrambled control cells. We show for the first time that β-catenin directly governs MUC4 in PC. PMID:26526617

  6. Time-Course Gene Expression Profiling Reveals a Novel Role of Non-Canonical WNT Signaling During Neural Induction.

    PubMed

    Huang, Cindy Tzu-Ling; Tao, Yunlong; Lu, Jianfeng; Jones, Jeffrey R; Fowler, Lucas; Weick, Jason P; Zhang, Su-Chun

    2016-01-01

    The process of neuroepithelial differentiation from human pluripotent stem cells (PSCs) resembles in vivo neuroectoderm induction in the temporal course, morphogenesis, and biochemical changes. This in vitro model is therefore well-suited to reveal previously unknown molecular mechanisms underlying neural induction in humans. By transcriptome analysis of cells along PSC differentiation to early neuroepithelia at day 6 and definitive neuroepithelia at day 10, we found downregulation of genes that are associated with TGF-β and canonical WNT/β-CATENIN signaling, confirming the roles of classical signaling in human neural induction. Interestingly, WNT/Ca(2+) signaling was upregulated. Pharmacological inhibition of the downstream effector of WNT/Ca(2+) pathway, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), led to an inhibition of the neural marker PAX6 and upregulation of epidermal marker K18, suggesting that Ca(2+)/CaMKII signaling promotes neural induction by preventing the alternative epidermal fate. In addition, our analyses revealed known and novel expression patterns of genes that are involved in DNA methylation, histone modification, as well as epithelial-mesenchymal transition, highlighting potential roles of those genes and signaling pathways during neural differentiation. PMID:27600186

  7. Time-Course Gene Expression Profiling Reveals a Novel Role of Non-Canonical WNT Signaling During Neural Induction

    PubMed Central

    Huang, Cindy Tzu-Ling; Tao, Yunlong; Lu, Jianfeng; Jones, Jeffrey R.; Fowler, Lucas; Weick, Jason P.; Zhang, Su-Chun

    2016-01-01

    The process of neuroepithelial differentiation from human pluripotent stem cells (PSCs) resembles in vivo neuroectoderm induction in the temporal course, morphogenesis, and biochemical changes. This in vitro model is therefore well-suited to reveal previously unknown molecular mechanisms underlying neural induction in humans. By transcriptome analysis of cells along PSC differentiation to early neuroepithelia at day 6 and definitive neuroepithelia at day 10, we found downregulation of genes that are associated with TGF-β and canonical WNT/β-CATENIN signaling, confirming the roles of classical signaling in human neural induction. Interestingly, WNT/Ca2+ signaling was upregulated. Pharmacological inhibition of the downstream effector of WNT/Ca2+ pathway, Ca2+/calmodulin-dependent protein kinase II (CaMKII), led to an inhibition of the neural marker PAX6 and upregulation of epidermal marker K18, suggesting that Ca2+/CaMKII signaling promotes neural induction by preventing the alternative epidermal fate. In addition, our analyses revealed known and novel expression patterns of genes that are involved in DNA methylation, histone modification, as well as epithelial-mesenchymal transition, highlighting potential roles of those genes and signaling pathways during neural differentiation. PMID:27600186

  8. Modulating effects of acyl-CoA synthetase 5-derived mitochondrial Wnt2B palmitoylation on intestinal Wnt activity

    PubMed Central

    Klaus, Christina; Schneider, Ursula; Hedberg, Christian; Schütz, Anke K; Bernhagen, Jürgen; Waldmann, Herbert; Gassler, Nikolaus; Kaemmerer, Elke

    2014-01-01

    AIM: To investigate the role of acyl-CoA synthetase 5 (ACSL5) activity in Wnt signaling in intestinal surface epithelia. METHODS: Several cell lines were used to investigate the ACSL5-dependent expression and synthesis of Wnt2B, a mitochondrially expressed protein of the Wnt signaling family. Wnt activity was functionally assessed with a luciferase reporter assay. ACSL5-related biochemical Wnt2B modifications were investigated with a modified acyl-exchange assay. The findings from the cell culture models were verified using an Apcmin/+ mouse model as well as normal and neoplastic diseased human intestinal tissues. RESULTS: In the presence of ACSL5, Wnt2B was unable to translocate into the nucleus and was enriched in mitochondria, which was paralleled by a significant decrease in Wnt activity. ACSL5-dependent S-palmitoylation of Wnt2B was identified as a molecular reason for mitochondrial Wnt2B accumulation. In cell culture systems, a strong relation of ACSL5 expression, Wnt2B palmitoylation, and degree of malignancy were found. Using normal mucosa, the association of ACSL5 and Wnt2B was seen, but in intestinal neoplasias the mechanism was only rudimentarily observed. CONCLUSION: ACSL5 mediates antiproliferative activities via Wnt2B palmitoylation with diminished Wnt activity. The molecular pathway is probably relevant for intestinal homeostasis, overwhelmed by other pathways in carcinogenesis. PMID:25356045

  9. Loss of Tc-arrow and canonical Wnt signaling alters posterior morphology and pair-rule gene expression in the short-germ insect, Tribolium castaneum.

    PubMed

    Bolognesi, Renata; Fischer, Tamara D; Brown, Susan J

    2009-07-01

    Wnt signaling has been implicated in posterior patterning in short-germ insects, including the red flour beetle Tribolium castaneum (Bolognesi et al. Curr Biol 18:1624-1629, 2008b; Angelini and Kaufman Dev Biol 283:409-423, 2005; Miyawaki et al. Mech Dev 121:119-130, 2004). Specifically, depletion of Wnt ligands Tc-Wnt1 and Tc-WntD/8 produces Tribolium embryos lacking abdominal segments. Similar phenotypes are produced by depletion of Tc-porcupine (Tc-porc) or Tc-pangolin (Tc-pan), indicating that the signal is transmitted through the canonical Wnt pathway (Bolognesi et al. Curr Biol 18:1624-1629, 2008b). Here we show that RNAi for the receptor Tc-arrow produced similar truncated phenotypes, providing additional evidence supporting canonical signal transduction. Furthermore, since in Tribolium segments are defined sequentially by a pair-rule gene circuit that, when interrupted, produces truncated phenotypes (Choe et al. Proc Natl Acad Sci U S A 103:6560-6564, 2006), we investigated the relationship between loss of Wnt signaling and this pair-rule gene circuit. After depletion of the receptor Tc-arrow, expression of Tc-Wnt1 was noticeably absent from the growth zone, while Tc-WntD/8 was restricted to a single spot of expression in what remained of the posterior growth zone. The primary pair-rule genes Tc-runt (Tc-run) and Tc-even-skipped (Tc-eve) were expressed normally in the anterior segments, but were reduced to a single spot in the remnants of the posterior growth zone. Thus, expression of pair-rule genes and Tc-WntD/8 are similarly affected by depletion of Wnt signal and disruption of the posterior growth zone. PMID:19705150

  10. Hear the Wnt Ror: How Melanoma Cells Adjust to Changes in Wnt

    PubMed Central

    O’Connell, Michael P.; Weeraratna, Ashani T.

    2009-01-01

    The interplay between canonical and non-canonical Wnt pathways in development and tumorigenesis is tightly regulated. In this review we will describe the yin and the yang of canonical and non-canonical Wnt signaling pathways during melanocyte development, and melanoma genesis. Canonical Wnt signaling, represented by Wnts such as Wnt1 and Wnt3A, signals via β-catenin to promote melanocyte differentiation and tumor development. Non-canonical Wnt signaling, specifically Wnt5A, regulates canonical pathways, and signals to induce melanoma metastasis. This review will focus on the role of Wnt5A during melanoma progression, and its relationship to canonical Wnt signaling. PMID:19708915

  11. The Use of Chick Embryos to Study Wnt Activity Gradients.

    PubMed

    Galli, Lisa M; Barnes, Tiffany; Burrus, Laura W

    2016-01-01

    The chick spinal cord provides a valuable model for assessing Wnt signaling activity. Loss or gain of function constructs that are transfected by electroporation can be directed to a single side of the spinal cord, thus leaving the contralateral side as an internal control. Here, we describe a method for measuring Wnt signaling via the use of BAT-Gal, a β-catenin dependent Wnt reporter. PMID:27590153

  12. R26-WntVis reporter mice showing graded response to Wnt signal levels.

    PubMed

    Takemoto, Tatsuya; Abe, Takaya; Kiyonari, Hiroshi; Nakao, Kazuki; Furuta, Yasuhide; Suzuki, Hitomi; Takada, Shinji; Fujimori, Toshihiko; Kondoh, Hisato

    2016-06-01

    The canonical Wnt signaling pathway plays a major role in the regulation of embryogenesis and organogenesis, where signal strength-dependent cellular responses are of particular importance. To assess Wnt signal levels in individual cells, and to circumvent the integration site-dependent bias shown in previous Wnt reporter lines, we constructed a new Wnt signal reporter mouse line R26-WntVis. Heptameric TCF/LEF1 binding sequences were combined with a viral minimal promoter to confer a graded response to the reporter depending on Wnt signal strengths. The histone H2B-EGFP fusion protein was chosen as the fluorescent reporter to facilitate single-cell resolution analyses. This WntVis reporter gene was then inserted into the ROSA26 locus in an orientation opposite to that of the endogenous gene. The R26-WntVis allele was introduced into Wnt3a(-/-) and Wnt3a(vt/-) mutant mouse embryos and compared with wild-type embryos to assess its performance. The R26-WntVis reporter was activated in known Wnt-dependent tissues and responded in a graded fashion to signal intensity. This analysis also indicated that the major Wnt activity early in embryogenesis switched from Wnt3 to Wnt3a around E7.5. The R26-WntVis mouse line will be widely useful for the study of Wnt signal-dependent processes. PMID:27030109

  13. Curcumin-loaded nanoparticles potently induce adult neurogenesis and reverse cognitive deficits in Alzheimer's disease model via canonical Wnt/β-catenin pathway.

    PubMed

    Tiwari, Shashi Kant; Agarwal, Swati; Seth, Brashket; Yadav, Anuradha; Nair, Saumya; Bhatnagar, Priyanka; Karmakar, Madhumita; Kumari, Manisha; Chauhan, Lalit Kumar Singh; Patel, Devendra Kumar; Srivastava, Vikas; Singh, Dhirendra; Gupta, Shailendra Kumar; Tripathi, Anurag; Chaturvedi, Rajnish Kumar; Gupta, Kailash Chand

    2014-01-28

    Neurogenesis, a process of generation of new neurons, is reported to be reduced in several neurodegenerative disorders including Alzheimer's disease (AD). Induction of neurogenesis by targeting endogenous neural stem cells (NSC) could be a promising therapeutic approach to such diseases by influencing the brain self-regenerative capacity. Curcumin, a neuroprotective agent, has poor brain bioavailability. Herein, we report that curcumin-encapsulated PLGA nanoparticles (Cur-PLGA-NPs) potently induce NSC proliferation and neuronal differentiation in vitro and in the hippocampus and subventricular zone of adult rats, as compared to uncoated bulk curcumin. Cur-PLGA-NPs induce neurogenesis by internalization into the hippocampal NSC. Cur-PLGA-NPs significantly increase expression of genes involved in cell proliferation (reelin, nestin, and Pax6) and neuronal differentiation (neurogenin, neuroD1, neuregulin, neuroligin, and Stat3). Curcumin nanoparticles increase neuronal differentiation by activating the Wnt/β-catenin pathway, involved in regulation of neurogenesis. These nanoparticles caused enhanced nuclear translocation of β-catenin, decreased GSK-3β levels, and increased promoter activity of the TCF/LEF and cyclin-D1. Pharmacological and siRNA-mediated genetic inhibition of the Wnt pathway blocked neurogenesis-stimulating effects of curcumin. These nanoparticles reverse learning and memory impairments in an amyloid beta induced rat model of AD-like phenotypes, by inducing neurogenesis. In silico molecular docking studies suggest that curcumin interacts with Wif-1, Dkk, and GSK-3β. These results suggest that curcumin nanoparticles induce adult neurogenesis through activation of the canonical Wnt/β-catenin pathway and may offer a therapeutic approach to treating neurodegenerative diseases such as AD, by enhancing a brain self-repair mechanism. PMID:24467380

  14. MicroRNA-124 suppresses the migration and invasion of osteosarcoma cells via targeting ROR2-mediated non-canonical Wnt signaling.

    PubMed

    Zhang, Can; Hu, Yihe; Wan, Jun; He, Hongbo

    2015-10-01

    MicroRNAs (miRs) have been implicated in tumorigenesis through inhibition of the expression of their target genes at post-transcriptional levels. miR-124 has been found to be downregulated in many malignant tumors including osteosarcoma (OS). However, the detailed mechanism of miR-124 in the regulation of OS malignant phenotypes remains largely unclear. Here we aimed to explore the role of miR-124 in mediating OS cell migration and invasion, as well as the underlying regulatory mechanisms. Real-time RT-PCR data showed that miR-124 was frequently downregulated in OS cell lines compared to normal human osteoblast cells. We further conducted bioinformatic analysis and a luciferase reporter assay, and identified receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a novel target of miR-124. Furthermore, we found that ROR2 was significantly upregulated in OS cell lines compared to normal human osteoblast cells, and miR-124 negatively mediated the protein level of ROR2 in U-2OS and Saos-2 cells. Moreover, transfection with miR-124 mimics significantly suppressed migration and invasion in the U-2OS and Saos-2 cells, while overexpression of ROR2 in the miR-124-transfected OS cells reversed the inhibitory effect of miR-124 upregulation on OS cell migration and invasion. In addition, we found that overexpression of miR-124 significantly suppressed the activity of non-canonical Wnt signaling, downstream of ROR2. Based on these findings, we suggest that miR-124 may inhibit OS metastasis, partly at least, via targeting ROR2 and thus suppressing the activity of ROR2-mediated non-canonical Wnt signaling. PMID:26259653

  15. Molecular Genetics of Intracranial Meningiomas with Emphasis on Canonical Wnt Signalling

    PubMed Central

    Pećina-Šlaus, Nives; Kafka, Anja; Lechpammer, Mirna

    2016-01-01

    Research over the last decade recognized the importance of novel molecular pathways in pathogenesis of intracranial meningiomas. In this review, we focus on human brain tumours meningiomas and the involvement of Wnt signalling pathway genes and proteins in this common brain tumour, describing their known functional effects. Meningiomas originate from the meningeal layers of the brain and the spinal cord. Most meningiomas have benign clinical behaviour and are classified as grade I by World Health Organization (WHO). However, up to 20% histologically classified as atypical (grade II) or anaplastic (grade III) are associated with higher recurrent rate and have overall less favourable clinical outcome. Recently, there is emerging evidence that multiple signalling pathways including Wnt pathway contribute to the formation and growth of meningiomas. In the review we present the synopsis on meningioma histopathology and genetics and discuss our research regarding Wnt in meningioma. Epithelial-to-mesenchymal transition, a process in which Wnt signalling plays an important role, is shortly discussed. PMID:27429002

  16. Molecular Genetics of Intracranial Meningiomas with Emphasis on Canonical Wnt Signalling.

    PubMed

    Pećina-Šlaus, Nives; Kafka, Anja; Lechpammer, Mirna

    2016-01-01

    Research over the last decade recognized the importance of novel molecular pathways in pathogenesis of intracranial meningiomas. In this review, we focus on human brain tumours meningiomas and the involvement of Wnt signalling pathway genes and proteins in this common brain tumour, describing their known functional effects. Meningiomas originate from the meningeal layers of the brain and the spinal cord. Most meningiomas have benign clinical behaviour and are classified as grade I by World Health Organization (WHO). However, up to 20% histologically classified as atypical (grade II) or anaplastic (grade III) are associated with higher recurrent rate and have overall less favourable clinical outcome. Recently, there is emerging evidence that multiple signalling pathways including Wnt pathway contribute to the formation and growth of meningiomas. In the review we present the synopsis on meningioma histopathology and genetics and discuss our research regarding Wnt in meningioma. Epithelial-to-mesenchymal transition, a process in which Wnt signalling plays an important role, is shortly discussed. PMID:27429002

  17. Wnt signaling regulates multipolar-to-bipolar transition of migrating neurons in the cerebral cortex.

    PubMed

    Boitard, Michael; Bocchi, Riccardo; Egervari, Kristof; Petrenko, Volodymyr; Viale, Beatrice; Gremaud, Stéphane; Zgraggen, Eloisa; Salmon, Patrick; Kiss, Jozsef Z

    2015-03-01

    The precise timing of pyramidal cell migration from the ventricular germinal zone to the cortical plate is essential for establishing cortical layers, and migration errors can lead to neurodevelopmental disorders underlying psychiatric and neurological diseases. Here, we report that Wnt canonical as well as non-canonical signaling is active in pyramidal precursors during radial migration. We demonstrate using constitutive and conditional genetic strategies that transient downregulation of canonical Wnt/β-catenin signaling during the multipolar stage plays a critical role in polarizing and orienting cells for radial migration. In addition, we show that reduced canonical Wnt signaling is triggered cell autonomously by time-dependent expression of Wnt5A and activation of non-canonical signaling. We identify ephrin-B1 as a canonical Wnt-signaling-regulated target in control of the multipolar-to-bipolar switch. These findings highlight the critical role of Wnt signaling activity in neuronal positioning during cortical development. PMID:25732825

  18. Hairy Math: Add Wnt-3a to Multiply Bulge Cells

    PubMed Central

    Hossain, M. Zulfiquer; Garza, Luis A.

    2015-01-01

    Canonical Wnt signals are important for activation of epithelial skin stem cells, but the role of individual Wnt ligands remains uncertain. Ouji et al. demonstrate a key role for Wnt-3a in partial maintenance and long-term expansion of epithelial skin stem cells in vitro. They also report a method for expanding these cells in vitro without feeder cells. PMID:25964269

  19. CD151-α3β1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling.

    PubMed

    Baldwin, Lauren A; Hoff, John T; Lefringhouse, Jason; Zhang, Michael; Jia, Changhe; Liu, Zeyi; Erfani, Sonia; Jin, Hongyan; Xu, Mei; She, Qing-Bai; van Nagell, John R; Wang, Chi; Chen, Li; Plattner, Rina; Kaetzel, David M; Luo, Jia; Lu, Michael; West, Dava; Liu, Chunming; Ueland, Fred R; Drapkin, Ronny; Zhou, Binhua P; Yang, Xiuwei H

    2014-12-15

    Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling. PMID:25356755

  20. CD151-α3β1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling

    PubMed Central

    Zhang, Michael; Jia, Changhe; Liu, Zeyi; Erfani, Sonia; Jin, Hongyan; Xu, Mei; She, Qing-Bai; van Nagell, John R.; Wang, Chi; Chen, Li; Plattner, Rina; Kaetzel, David M.; Luo, Jia; Lu, Michael; West, Dava; Liu, Chunming; Ueland, Fred R.; Drapkin, Ronny; Zhou, Binhua P.; Yang, Xiuwei H.

    2014-01-01

    Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling. PMID:25356755

  1. Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2-JNK signalling in breast cancer.

    PubMed

    Puvirajesinghe, Tania M; Bertucci, François; Jain, Ashish; Scerbo, Pierluigi; Belotti, Edwige; Audebert, Stéphane; Sebbagh, Michael; Lopez, Marc; Brech, Andreas; Finetti, Pascal; Charafe-Jauffret, Emmanuelle; Chaffanet, Max; Castellano, Rémy; Restouin, Audrey; Marchetto, Sylvie; Collette, Yves; Gonçalvès, Anthony; Macara, Ian; Birnbaum, Daniel; Kodjabachian, Laurent; Johansen, Terje; Borg, Jean-Paul

    2016-01-01

    The non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway plays a crucial role in embryonic development. Recent work has linked defects of this pathway to breast cancer aggressiveness and proposed Wnt/PCP signalling as a therapeutic target. Here we show that the archetypal Wnt/PCP protein VANGL2 is overexpressed in basal breast cancers, associated with poor prognosis and implicated in tumour growth. We identify the scaffold p62/SQSTM1 protein as a novel VANGL2-binding partner and show its key role in an evolutionarily conserved VANGL2-p62/SQSTM1-JNK pathway. This proliferative signalling cascade is upregulated in breast cancer patients with shorter survival and can be inactivated in patient-derived xenograft cells by inhibition of the JNK pathway or by disruption of the VANGL2-p62/SQSTM1 interaction. VANGL2-JNK signalling is thus a potential target for breast cancer therapy. PMID:26754771

  2. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling.

    PubMed

    Liu, Jessica A J; Wu, Ming-Hoi; Yan, Carol H; Chau, Bolton K H; So, Henry; Ng, Alvis; Chan, Alan; Cheah, Kathryn S E; Briscoe, James; Cheung, Martin

    2013-02-19

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination. PMID:23382206

  3. Rescuing failed oral implants via Wnt activation

    PubMed Central

    Yin, Xing; Li, Jingtao; Chen, Tao; Mouraret, Sylvain; Dhamdhere, Girija; Brunski, John B.; Zou, Shujuan; Helms, Jill A.

    2016-01-01

    Aim Implant osseointegration is not always guaranteed and once fibrous encapsulation occurs clinicians have few options other than implant removal. Our goal was to test whether a WNT protein therapeutic could rescue such failed implants. Material and Methods Titanium implants were placed in over-sized murine oral osteotomies. A lack of primary stability was verified by mechanical testing. Interfacial strains were estimated by finite element modelling and histology coupled with histomorphometry confirmed the lack of peri-implant bone. After fibrous encapsulation was established peri-implant injections of a liposomal formulation of WNT3A protein (L-WNT3A) or liposomal PBS (L-PBS) were then initiated. Quantitative assays were employed to analyse the effects of L-WNT3A treatment. Results Implants in gap-type interfaces exhibited high interfacial strains and no primary stability. After verification of implant failure, L-WNT3A or L-PBS injections were initiated. L-WNT3A induced a rapid, significant increase in Wnt responsiveness in the peri-implant environment, cell proliferation and osteogenic protein expression. The amount of peri-implant bone and bone in contact with the implant were significantly higher in L-WNT3A cases. Conclusions These data demonstrate L-WNT3A can induce peri-implant bone formation even in cases where fibrous encapsulation predominates. PMID:26718012

  4. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  5. Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals.

    PubMed

    Shao, Jian-Su; Cheng, Su-Li; Pingsterhaus, Joyce M; Charlton-Kachigian, Nichole; Loewy, Arleen P; Towler, Dwight A

    2005-05-01

    In diabetic LDLR-/- mice, an ectopic BMP2-Msx2 gene regulatory program is upregulated in association with vascular calcification. We verified the procalcific actions of aortic Msx2 expression in vivo. CMV-Msx2 transgenic (CMV-Msx2Tg(+)) mice expressed 3-fold higher levels of aortic Msx2 than nontransgenic littermates. On high-fat diets, CMV-Msx2Tg(+) mice exhibited marked cardiovascular calcification involving aortic and coronary tunica media. This corresponded to regions of Msx2 immunoreactivity in adjacent adventitial myofibroblasts, suggesting a potential paracrine osteogenic signal. To better understand Msx2-regulated calcification, we studied actions in 10T1/2 cells. We found that conditioned media from Msx2-transduced 10T1/2 cells (Msx2-CM) is both pro-osteogenic and adipostatic; these features are characteristic of Wnt signaling. Msx2-CM stimulated Wnt-dependent TCF/LEF transcription, and Msx2-transduced cells exhibited increased nuclear beta-catenin localization with concomitant alkaline phosphatase induction. Msx2 upregulated Wnt3a and Wnt7a but downregulated expression of the canonical inhibitor Dkk1. Dkk1 treatment reversed osteogenic and adipostatic actions of Msx2. Teriparatide, a PTH1R agonist that inhibits murine vascular calcification, suppressed vascular BMP2-Msx2-Wnt signaling. Analyses of CMV-Msx2Tg(+) mice confirmed that Msx2 suppresses aortic Dkk1 and upregulates vascular Wnts; moreover, TOPGAL(+) (Wnt reporter); CMV-Msx2Tg(+) mice exhibited augmented aortic LacZ expression. Thus, Msx2-expressing cells elaborated an osteogenic milieu that promotes vascular calcification in part via paracrine Wnt signals. PMID:15841209

  6. Whole-exome sequencing reveals LRP5 mutations and canonical Wnt signaling associated with hepatic cystogenesis

    PubMed Central

    Cnossen, Wybrich R.; te Morsche, René H. M.; Hoischen, Alexander; Gilissen, Christian; Chrispijn, Melissa; Venselaar, Hanka; Mehdi, Soufi; Bergmann, Carsten; Veltman, Joris A.; Drenth, Joost P. H.

    2014-01-01

    Polycystic livers are seen in the rare inherited disorder isolated polycystic liver disease (PCLD) and are recognized as the most common extrarenal manifestation in autosomal dominant polycystic kidney disease. Hepatic cystogenesis is characterized by progressive proliferation of cholangiocytes, ultimately causing hepatomegaly. Genetically, polycystic liver disease is a heterogeneous disorder with incomplete penetrance and caused by mutations in PRKCSH, SEC63, PKD1, or PKD2. Genome-wide SNP typing and Sanger sequencing revealed no pathogenic variants in hitherto genes in an extended PCLD family. We performed whole-exome sequencing of DNA samples from two members. A heterozygous variant c.3562C > T located at a highly conserved amino acid position (p.R1188W) in the low density lipoprotein receptor-related protein 5 (LRP5) gene segregated with the disease (logarithm of odds score, 4.62) but was not observed in more than 1,000 unaffected individuals. Screening of LRP5 in a PCLD cohort identified three additional mutations in three unrelated families with polycystic livers (p.V454M, p.R1529S, and p.D1551N), again all undetected in controls. All variants were predicted to be damaging with profound structural effects on LRP5 protein domains. Liver cyst tissue and normal hepatic tissue samples from patients and controls showed abundant LRP5 expression by immunohistochemistry. Functional activity analyses indicated that mutant LRP5 led to reduced wingless signal activation. In conclusion, we demonstrate that germ-line LRP5 missense mutations are associated with hepatic cystogenesis. The findings presented in this study link the pathophysiology of PCLD to deregulation of the canonical wingless signaling pathway. PMID:24706814

  7. Whole-exome sequencing reveals LRP5 mutations and canonical Wnt signaling associated with hepatic cystogenesis.

    PubMed

    Cnossen, Wybrich R; te Morsche, René H M; Hoischen, Alexander; Gilissen, Christian; Chrispijn, Melissa; Venselaar, Hanka; Mehdi, Soufi; Bergmann, Carsten; Veltman, Joris A; Drenth, Joost P H

    2014-04-01

    Polycystic livers are seen in the rare inherited disorder isolated polycystic liver disease (PCLD) and are recognized as the most common extrarenal manifestation in autosomal dominant polycystic kidney disease. Hepatic cystogenesis is characterized by progressive proliferation of cholangiocytes, ultimately causing hepatomegaly. Genetically, polycystic liver disease is a heterogeneous disorder with incomplete penetrance and caused by mutations in PRKCSH, SEC63, PKD1, or PKD2. Genome-wide SNP typing and Sanger sequencing revealed no pathogenic variants in hitherto genes in an extended PCLD family. We performed whole-exome sequencing of DNA samples from two members. A heterozygous variant c.3562C > T located at a highly conserved amino acid position (p.R1188W) in the low density lipoprotein receptor-related protein 5 (LRP5) gene segregated with the disease (logarithm of odds score, 4.62) but was not observed in more than 1,000 unaffected individuals. Screening of LRP5 in a PCLD cohort identified three additional mutations in three unrelated families with polycystic livers (p.V454M, p.R1529S, and p.D1551N), again all undetected in controls. All variants were predicted to be damaging with profound structural effects on LRP5 protein domains. Liver cyst tissue and normal hepatic tissue samples from patients and controls showed abundant LRP5 expression by immunohistochemistry. Functional activity analyses indicated that mutant LRP5 led to reduced wingless signal activation. In conclusion, we demonstrate that germ-line LRP5 missense mutations are associated with hepatic cystogenesis. The findings presented in this study link the pathophysiology of PCLD to deregulation of the canonical wingless signaling pathway. PMID:24706814

  8. Dact2 Represses PITX2 Transcriptional Activation and Cell Proliferation through Wnt/beta-Catenin Signaling during Odontogenesis

    PubMed Central

    Li, Xiao; Florez, Sergio; Wang, Jianbo; Cao, Huojun; Amendt, Brad A.

    2013-01-01

    Dact proteins belong to the Dapper/Frodo protein family and function as cytoplasmic attenuators in Wnt and TGFβ signaling. Previous studies show that Dact1 is a potent Wnt signaling inhibitor by promoting degradation of β-catenin. We report a new mechanism for Dact2 function as an inhibitor of the canonical Wnt signaling pathway by interacting with PITX2. PITX2 is a downstream transcription factor in Wnt/β-catenin signaling, and PITX2 synergizes with Lef-1 to activate downstream genes. Immunohistochemistry verified the expression of Dact2 in the tooth epithelium, which correlated with Pitx2 epithelial expression. Dact2 loss of function and PITX2 gain of function studies reveal a feedback mechanism for controlling Dact2 expression. Pitx2 endogenously activates Dact2 expression and Dact2 feeds back to repress Pitx2 transcriptional activity. A Topflash reporter system was employed showing PITX2 activation of Wnt signaling, which is attenuated by Dact2. Transient transfections demonstrate the inhibitory effect of Dact2 on critical dental epithelial differentiation factors during tooth development. Dact2 significantly inhibits PITX2 activation of the Dlx2 and amelogenin promoters. Multiple lines of evidence conclude the inhibition is achieved by the physical interaction between Dact2 and Pitx2 proteins. The loss of function of Dact2 also reveals increased cell proliferation due to up-regulated Wnt downstream genes, cyclinD1 and cyclinD2. In summary, we have identified a novel role for Dact2 as an inhibitor of the canonical Wnt pathway in embryonic tooth development through its regulation of cell proliferation and differentiation. PMID:23349981

  9. Activated Wnt signaling induces myofibroblast differentiation of mesenchymal stem cells, contributing to pulmonary fibrosis.

    PubMed

    Sun, Zhaorui; Wang, Cong; Shi, Chaowen; Sun, Fangfang; Xu, Xiaomeng; Qian, Weiping; Nie, Shinan; Han, Xiaodong

    2014-05-01

    Acute lung injury may lead to fibrogenesis. However, no treatment is currently available. This study was conducted to determine the effects of bone marrow-derived mesenchymal stem cells (MSCs) in a model of HCl-induced acute lung injury in Sprague-Dawley (SD) rats. Stromal cell-derived factor (SDF)-1 and its receptor CXC chemokine receptor (CXCR)4 have been shown to participate in mobilizing MSCs. Adenovirus carrying the CXCR4 gene was used to transfect MSCs in order to increase the engraftment numbers of MSCs at injured sites. Histological examination data demonstrated that the engraftment of MSCs did not attenuate lung injury and pulmonary fibrosis. The results showed that engraftment of MSCs almost differentiated into myofibroblasts, but rarely differentiated into lung epithelial cells. Additionally, it was demonstrated that activated canonical Wnt/β-catenin signaling in injured lung tissue regulated the myofibroblast differentiation of MSCs in vivo. The in vitro study results demonstrated that activation of the Wnt/β-catenin signaling stimulated MSCs to express myofibroblast markers; however, this process was attenuated by Wnt antagonist DKK1. Therefore, the results demonstrated that the aberrant activation of Wnt signaling induces the myofibroblast differentiation of engrafted MSCs, thus contributing to pulmonary fibrosis following lung injury. PMID:24573542

  10. Wnt signaling in osteosarcoma.

    PubMed

    Lin, Carol H; Ji, Tao; Chen, Cheng-Fong; Hoang, Bang H

    2014-01-01

    Osteosarcoma (OS) is the most common primary bone malignancy diagnosed in children and adolescents with a high propensity for local invasion and distant metastasis. Despite current multidisciplinary treatments, there has not been a drastic change in overall prognosis within the last two decades. With current treatments, 60-70 % of patients with localized disease survive. Given a propensity of Wnt signaling to control multiple cellular processes, including proliferation, cell fate determination, and differentiation, it is a critical pathway in OS disease progression. At the same time, this pathway is extremely complex with vast arrays of cross-talk. Even though decades of research have linked the role of Wnt to tumorigenesis, there are still outstanding areas that remain poorly understood and even controversial. The canonical Wnt pathway functions to regulate the levels of the transcriptional co-activator β-catenin, which ultimately controls key developmental gene expressions. Given the central role of this mediator, inhibition of Wnt/β-catenin signaling has been investigated as a potential strategy for cancer control. In OS, several secreted protein families modulate the Wnt/β-catenin signaling, including secreted Frizzled-related proteins (sFRPs), Wnt inhibitory protein (WIF), Dickkopf proteins (DKK-1,2,3), sclerostin, and small molecules. This chapter focuses on our current understanding of Wnt/β-catenin signaling in OS, based on recent in vitro and in vivo data. Wnt activates noncanonical signaling pathways as well that are independent of β-catenin which will be discussed. In addition, stem cells and their association with Wnt/β-catenin are important factors to consider. Ultimately, the multiple canonical and noncanonical Wnt/β-catenin agonists and antagonists need to be further explored for potential targeted therapies. PMID:24924167

  11. How targets select activation or repression in response to Wnt.

    PubMed

    Murgan, Sabrina; Bertrand, Vincent

    2015-01-01

    In metazoans, the Wnt signaling pathway plays a key role in the regulation of binary decisions during development. During this process different sets of target genes are activated in cells where the Wnt pathway is active (classic target genes) versus cells where the pathway is inactive (opposite target genes). While the mechanism of transcriptional activation is well understood for classic target genes, how opposite target genes are activated in the absence of Wnt remains poorly characterized. Here we discuss how the key transcriptional mediator of the Wnt pathway, the TCF family member POP-1, regulates opposite target genes during C. elegans development. We examine recent findings suggesting that the direction of the transcriptional output (activation or repression) can be determined by the way TCF is recruited and physically interacts with its target gene. PMID:27123368

  12. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins.

    PubMed

    Rios-Esteves, Jessica; Resh, Marilyn D

    2013-09-26

    Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. PMID:24055053

  13. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins

    PubMed Central

    Rios-Esteves, Jessica; Resh, Marilyn D.

    2013-01-01

    Summary Wnt proteins contain an unusual lipid modification, palmitoleic acid. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate which is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a, and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. PMID:24055053

  14. Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity*

    PubMed Central

    Borgal, Lori; Rinschen, Markus M.; Dafinger, Claudia; Hoff, Sylvia; Reinert, Matthäus J.; Lamkemeyer, Tobias; Lienkamp, Soeren S.; Benzing, Thomas; Schermer, Bernhard

    2014-01-01

    Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner. PMID:25100726

  15. Kdm2a/b Lysine Demethylases Regulate Canonical Wnt Signaling by Modulating the Stability of Nuclear β-Catenin.

    PubMed

    Lu, Lei; Gao, Yan; Zhang, Zan; Cao, Qing; Zhang, Xuena; Zou, Jianghuan; Cao, Ying

    2015-06-22

    In the absence of Wnt activation, cytosolic β-catenin is degraded through GSK3/CK1-mediated phosphorylation at the N terminus. Here, we show that, upon Wnt activation, the stability of nuclear β-catenin is regulated via methylation/demethylation. The protein lysine demethylases Kdm2a and Kdm2b regulate the turnover of non-phosphorylated β-catenin specifically within the nucleus via direct interaction with the fourth and fifth armadillo repeats. The lysine residues within this region are required for the methylation of non-phosphorylated β-catenin, which is demethylated by Kdm2a/b and subsequently ubiquitylated. During Xenopus embryogenesis, kdm2a/b genes are transcribed during early embryogenesis and are required for the specification of the body axis. Kdm2a/b knockdown in Xenopus embryos leads to increases in non-phosphorylated and methylated β-catenin, concurrent with the upregulation of β-catenin target genes. This mechanism is required for controlling the output of the Wnt/β-catenin signaling pathway to maintain normal cellular functions. PMID:26004508

  16. Wnt Signaling in Neurogenesis during Aging and Physical Activity

    PubMed Central

    Chen, Michael; Do, Huong

    2012-01-01

    Over the past decade, much progress has been made regarding our understanding of neurogenesis in both young and old animals and where it occurs throughout the lifespan, although the growth of new neurons declines with increasing age. In addition, physical activity can reverse this age-dependent decline in neurogenesis. Highly correlated with this decline is the degree of inter and intracellular Wnt signaling, the molecular mechanisms of which have only recently started to be elucidated. So far, most of what we know about intracellular signaling during/following exercise centers around the CREB/CRE initiated transcriptional events. Relatively little is known, however, about how aging and physical activity affect the Wnt signaling pathway. Herein, we briefly review the salient features of neurogenesis in young and then in old adult animals. Then, we discuss Wnt signaling and review the very few in vitro and in vivo studies that have examined the Wnt signaling pathways in aging and physical activity. PMID:24961268

  17. Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

    SciTech Connect

    Carmon, Kendra S.; Loose, David S.

    2008-04-04

    Wnts are secreted glycoproteins that regulate important cellular processes including proliferation, differentiation, and cell fate. In the {beta}-catenin/canonical pathway, Wnt interacts with Fzd receptors to inhibit degradation of {beta}-catenin and promote its translocation into the nucleus where it regulates transcription of a number of genes. Dysregulation of this pathway has been attributed to a host of diseases including cancer. As a result, components of the {beta}-catenin/canonical pathway have been gaining recognition as promising targets for the discovery of novel therapeutic agents. Here, we show, using an ELISA-based protein-protein binding assay that purified Wnt7a binds to the extracellular cysteine-rich domain of Fzd5 in the nanomolar range. We have developed a novel split eGFP complementation assay to visually detect Wnt7a-Fzd5 interactions and subsequent pathway activation in cells. These biological tools could help lead to a better understanding of Wnt-Fzd interactions and the identification of new modulators of Wnt signaling.

  18. Wnt/β-catenin signaling: components, mechanisms, and diseases

    PubMed Central

    MacDonald, Bryan T.; Tamai, Keiko

    2010-01-01

    Signaling by the Wnt family of secreted glycolipoproteins via the transcription co-activator β-catenin controls embryonic development and adult homeostasis. Here we review recent progresses in this so-called canonical Wnt signaling pathway. We discuss Wnt ligands, agonists and antagonists and their interactions with Wnt receptors. We also dissect critical events that regulate β-catenin stability from Wnt receptors to the cytoplasmic β-catenin destruction complex, and nuclear machinery that mediates β-catenin-dependent transcription. Finally we highlight some key aspects of Wnt/β-catenin signaling in human diseases including congenital malformations, cancer and osteoporosis and potential therapeutic implications. PMID:19619488

  19. Biochemical Methods to Analyze Wnt Protein Secretion.

    PubMed

    Glaeser, Kathrin; Boutros, Michael; Gross, Julia Christina

    2016-01-01

    Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading. PMID:27590148

  20. Protective LRRK2 R1398H Variant Enhances GTPase and Wnt Signaling Activity

    PubMed Central

    Nixon-Abell, Jonathon; Berwick, Daniel C.; Grannó, Simone; Spain, Victoria A.; Blackstone, Craig; Harvey, Kirsten

    2016-01-01

    Mutations in LRRK2 are a common cause of familial and idiopathic Parkinson’s disease (PD). Recently, the LRRK2 GTPase domain R1398H variant was suggested in genetic studies to confer protection against PD but mechanistic data supporting this is lacking. Here, we present evidence that R1398H affects GTPase function, axon outgrowth, and Wnt signaling in a manner opposite to pathogenic LRRK2 mutations. LRRK2 R1398H GTPase domain dimerization and GTP hydrolysis were increased whereas GTP binding was reduced, leading to a decrease in active GTP-bound LRRK2. This protective variant also increased axon length of primary cortical neurones in comparison to wild-type LRRK2, whereas the R1441G LRRK2 pathogenic mutant decreased axon outgrowth. Importantly, R1398H enhanced the stimulatory effect of LRRK2 on canonical Wnt signaling whereas the G2385R risk variant, in accordance with all previously tested pathogenic LRRK2 mutants, had the opposite effect. Molecular modeling placed R1398H in close proximity to PD-causing mutations suggesting that this protective LRRK2 variant, like familial mutations, affects intramolecular RocCOR domain interactions. Thus, our data suggest that R1398H LRRK2 is a bona fide protective variant. The opposite effects of protective versus PD associated LRRK2 variants on GTPase function and canonical Wnt signaling activity also suggests that regulation of these two basic signaling mechanisms is important for neuronal function. We conclude that LRRK2 mediated Wnt signaling and GTPase function are fundamental in conferring disease susceptibility and have clear implications for therapeutic target identification. PMID:27013965

  1. Disruption of the non-canonical Wnt gene PRICKLE2 leads to autism-like behaviors with evidence for hippocampal synaptic dysfunction

    PubMed Central

    Sowers, L. P.; Loo, L.; Wu, Y.; Campbell, E.; Ulrich, J. D.; Wu, S.; Paemka, L.; Wassink, T.; Meyer, K.; Bing, X.; El-Shanti, H.; Usachev, Y. M.; Ueno, N.; Manak, R. J.; Shepherd, A. J.; Ferguson, P. J.; Darbro, B. W.; Richerson, G. B.; Mohapatra, D. P.; Wemmie, J. A.; Bassuk, A. G.

    2014-01-01

    Autism spectrum disorders (ASDs) have been suggested to arise from abnormalities in the canonical and non-canonical Wnt signaling pathways. However, a direct connection between a human variant in a Wnt pathway gene and ASD-relevant brain pathology has not been established. Prickle2 (Pk2) is a post-synaptic non-canonical Wnt signaling protein shown to interact with post synaptic density 95 (PSD-95). Here we show that mice with disruption in Prickle2 display behavioral abnormalities including altered social interaction, learning abnormalities, and behavioral inflexibility. Prickle2 disruption in mouse hippocampal neurons led to reductions in dendrite branching, synapse number, and post-synaptic density size. Consistent with these findings, Prickle2 null neurons show decreased frequency and size of spontaneous miniature synaptic currents. These behavioral and physiological abnormalities in Prickle2 disrupted mice are consistent with ASD-like phenotypes present in other mouse models of ASDs. In 384 individuals with autism, we identified two with distinct, heterozygous, rare, non-synonymous PRICKLE2 variants (p.E8Q and p.V153I) that were shared by their affected siblings and inherited paternally. Unlike wild-type PRICKLE2, the PRICKLE2 variants found in ASD patients exhibit deficits in morphological and electrophysiological assays. These data suggest that these PRICKLE2 variants cause a critical loss of PRICKLE2 function. The data presented here provide new insight into the biological roles of Prickle2, its behavioral importance, and suggest disruptions in non-canonical Wnt genes such as PRICKLE2 may contribute to synaptic abnormalities underlying ASDs. PMID:23711981

  2. Noncanonical Wnt5a enhances Wnt/β-catenin signaling during osteoblastogenesis.

    PubMed

    Okamoto, Masanori; Udagawa, Nobuyuki; Uehara, Shunsuke; Maeda, Kazuhiro; Yamashita, Teruhito; Nakamichi, Yuko; Kato, Hiroyuki; Saito, Naoto; Minami, Yasuhiro; Takahashi, Naoyuki; Kobayashi, Yasuhiro

    2014-01-01

    Wnt regulates bone formation through β-catenin-dependent canonical and -independent noncanonical signaling pathways. However, the cooperation that exists between the two signaling pathways during osteoblastogenesis remains to be elucidated. Here, we showed that the lack of Wnt5a in osteoblast-lineage cells impaired Wnt/β-catenin signaling due to the reduced expression of Lrp5 and Lrp6. Pretreatment of ST2 cells, a stromal cell line, with Wnt5a enhanced canonical Wnt ligand-induced Tcf/Lef transcription activity. Short hairpin RNA-mediated knockdown of Wnt5a, but not treatment with Dkk1, an antagonist of Wnt/β-catenin signaling, reduced the expression of Lrp5 and Lrp6 in osteoblast-lineage cells under osteogenic culture conditions. Osteoblast-lineage cells from Wnt5a-deficient mice exhibited reduced Wnt/β-catenin signaling, which impaired osteoblast differentiation and enhanced adipocyte differentiation. Adenovirus-mediated gene transfer of Lrp5 into Wnt5a-deficient osteoblast-lineage cells rescued their phenotypic features. Therefore, Wnt5a-induced noncanonical signaling cooperates with Wnt/β-catenin signaling to achieve proper bone formation. PMID:24670389

  3. Wnt3a suppresses Wnt/β-catenin signaling and cancer cell proliferation following serum deprivation.

    PubMed

    He, Qingqing; Yan, Hongwei; Wo, Da; Liu, Junjun; Liu, Peng; Zhang, Jiankang; Li, Limei; Zhou, Bin; Ge, Jin; Li, Huashun; Liu, Shangfeng; Zhu, Weidong

    2016-02-01

    Canonical Wnt/β-catenin signaling is often aberrantly activated in tumor cells and required for tumor growth. The internalization of Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) induced by Wnt ligands is commonly thought to be critical for Wnt/β-catenin signaling activation. However, in contrast to theses previous studies, we here show that persistent excessive stimulation with a canonical Wnt ligand Wnt3a could induce a long-term decreased expression level of membrane LRP6, which prevented nuclear β-catenin accumulation and tumor cell;proliferation. Importantly, Wnt3a was robustly upregulated following serum deprivation. The upregulated Wnt3a under serum deprivation was responsible for LRP6 internalization, decreased accumulation of nuclear β-catenin, and further inhibition of tumor cell proliferation. It has well been known that insufficient blood supply during tumor development occurs frequently, causing a worsening environment for tumor growth. Therefore, these results reveal a novel inhibitory role of Wnt3a on canonical Wnt/β-catenin signaling and cancer cell proliferation when there is an insufficient blood supply during tumor development, which might be a potential mechanism for tumor evasion within a worsening environment. PMID:26643293

  4. Wnt5a Signaling in Cancer.

    PubMed

    Asem, Marwa S; Buechler, Steven; Wates, Rebecca Burkhalter; Miller, Daniel L; Stack, M Sharon

    2016-01-01

    Wnt5a is involved in activating several non-canonical WNT signaling pathways, through binding to different members of the Frizzled- and Ror-family receptors. Wnt5a signaling is critical for regulating normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However, the aberrant activation or inhibition of Wnt5a signaling is emerging as an important event in cancer progression, exerting both oncogenic and tumor suppressive effects. Recent studies show the involvement of Wnt5a in regulating cancer cell invasion, metastasis, metabolism and inflammation. In this article, we review findings regarding the molecular mechanisms and roles of Wnt5a signaling in various cancer types, and highlight Wnt5a in ovarian cancer. PMID:27571105

  5. A novel function for Wnt signaling modulating neuronal firing activity and the temporal structure of spontaneous oscillation in the entorhinal-hippocampal circuit.

    PubMed

    Oliva, Carolina A; Inestrosa, Nibaldo C

    2015-07-01

    During early and late postnatal developments, the establishment of functional neuronal connectivity depends on molecules like Wnt that help the recently formed synapses to establish and consolidate their new cellular interactions. However, unlike other molecules, whether Wnt can modulate the firing properties of cells is unknown. Here, for the first time we explore the physiological effect of the canonical and non-canonical Wnt pathways on a circuit that is currently generating oscillatory activity, the entorhinal cortex-hippocampal circuit. Our results indicate that Wnt pathways have strong influence in the circuital and cellular properties depending on the Wnt protein isoforms, concentration, and type of neuronal circuit. Antibodies against canonical and non-canonical ligands, as well as WASP-1 and sFRP-2, demonstrate that constitutive release of Wnts contributes to the maintenance of the network and intrinsic properties of the circuit. Furthermore, we found that the excess of Wnt3a or the permanent intracellular activation of the pathway with BIO-6 accelerates the period of the oscillation by disrupting the oscillatory units (Up states) in short units, presumably by affecting the synaptic mechanisms that couples neurons into the oscillatory cycle, but without affecting the spike generation. Instead, low doses of Wnt5a increase the period of the oscillation in EC by incorporating new cells into the network activity, probably modifying firing activity in other places of the circuit. Moreover, we found that Wnt signaling operates under different principles in the hippocampus. Using pyrvinium pamoate, a Wnt/β-catenin dependent pathway inhibitor, we demonstrated that this pathway is essential to keep the firing activity in the circuit CA3, and in less degree of CA1 circuit. However, CA1 circuit possesses homeostatic mechanisms to up-regulate the firing activity when it has been suppressed in CA3, and to down-modulate the cellular excitability when exacerbated

  6. FZD7 drives in vitro aggressiveness in Stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway

    PubMed Central

    Asad, M; Wong, M K; Tan, T Z; Choolani, M; Low, J; Mori, S; Virshup, D; Thiery, J P; Huang, R Y-J

    2014-01-01

    Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups: epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that frizzled family receptor 7 (FZD7), a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 sub-population, with no effect on apoptosis. The cells also displayed a distinctive morphologic change by colony compaction to become more epithelial-like and polarised with smaller internuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and β-catenin suggested an increase in cadherin-based cell–cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell–cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA (Ras homolog family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1 (Rho family, small GTP-binding protein Rac1)) activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant upregulation in Rac1 activity. These changes in pMLC and RhoA, as well as the increased TopFlash reporter activities in si-FZD7 cells, suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), prickle homolog 4 (Drosophila) (PRICKLE4), dishevelled-associated activator of morphogenesis 1 (DAAM1

  7. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    SciTech Connect

    Kim, Inae; Hur, Jung; Jeong, Sunjoo

    2015-01-30

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  8. Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea.

    PubMed

    Liu, L; Chen, Y; Qi, J; Zhang, Y; He, Y; Ni, W; Li, W; Zhang, S; Sun, S; Taketo, M M; Wang, L; Chai, R; Li, H

    2016-01-01

    Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death. PMID:26962686

  9. Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea

    PubMed Central

    Liu, L; Chen, Y; Qi, J; Zhang, Y; He, Y; Ni, W; Li, W; Zhang, S; Sun, S; Taketo, M M; Wang, L; Chai, R; Li, H

    2016-01-01

    Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death. PMID:26962686

  10. Use of an Activated Beta-Catenin to Identify Wnt Pathway Target Genes in Caenorhabditis elegans, Including a Subset of Collagen Genes Expressed in Late Larval Development

    PubMed Central

    Jackson, Belinda M.; Abete-Luzi, Patricia; Krause, Michael W.; Eisenmann, David M.

    2014-01-01

    The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin−dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle. PMID:24569038

  11. Newly Constructed Network Models of Different WNT Signaling Cascades Applied to Breast Cancer Expression Data

    PubMed Central

    Kramer, Frank; Pukrop, Tobias; Beißbarth, Tim; Bleckmann, Annalen

    2015-01-01

    Introduction WNT signaling is a complex process comprising multiple pathways: the canonical β-catenin-dependent pathway and several alternative non-canonical pathways that act in a β-catenin-independent manner. Representing these intricate signaling mechanisms through bioinformatic approaches is challenging. Nevertheless, a simplified but reliable bioinformatic WNT pathway model is needed, which can be further utilized to decipher specific WNT activation states within e.g. high-throughput data. Results In order to build such a model, we collected, parsed, and curated available WNT signaling knowledge from different pathway databases. The data were assembled to construct computationally suitable models of different WNT signaling cascades in the form of directed signaling graphs. This resulted in four networks representing canonical WNT signaling, non-canonical WNT signaling, the inhibition of canonical WNT signaling and the regulation of WNT signaling pathways, respectively. Furthermore, these networks were integrated with microarray and RNA sequencing data to gain deeper insight into the underlying biology of gene expression differences between MCF-7 and MDA-MB-231 breast cancer cell lines, representing weakly and highly invasive breast carcinomas, respectively. Differential genes up-regulated in the MDA-MB-231 compared to the MCF-7 cell line were found to display enrichment in the gene set originating from the non-canonical network. Moreover, we identified and validated differentially regulated modules representing canonical and non-canonical WNT pathway components specific for the aggressive basal-like breast cancer subtype. Conclusions In conclusion, we demonstrated that these newly constructed WNT networks reliably reflect distinct WNT signaling processes. Using transcriptomic data, we shaped these networks into comprehensive modules of the genes implicated in the aggressive basal-like breast cancer subtype and demonstrated that non-canonical WNT signaling is

  12. Polycomb group genes Psc and Su(z)2 restrict follicle stem cell self-renewal and extrusion by controlling canonical and noncanonical Wnt signaling

    PubMed Central

    Li, Xinghua; Han, Yue; Xi, Rongwen

    2010-01-01

    Stem cells are critical for maintaining tissue homeostasis and are commonly governed by their niche microenvironment, although the intrinsic mechanisms controlling their multipotency are poorly understood. Polycomb group (PcG) genes are epigenetic silencers, and have emerged recently as important players in maintaining stem cell multipotency by preventing the initiation of differentiation programs. Here we describe an unexpected role of specific PcG genes in allowing adult stem cell differentiation and preventing stem cell-derived tumor development. We show that Posterior sex combs (Psc), which encodes a core Polycomb-repressive complex 1 (PRC1) component, functions redundantly with a similar gene, Suppressor of zeste two [Su(z)2], to restrict follicle stem cell (FSC) self-renewal in the Drosophila ovary. FSCs carrying deletion mutations of both genes extrude basally from the epithelium and continue to self-propagate at ectopic sites, leading to the development of FSC-like tumors. Furthermore, we show that the propagation of the mutant cells is driven by sustained activation of the canonical Wnt signaling pathway, which is essential for FSC self-renewal, whereas the epithelial extrusion is mediated through the planar cell polarity pathway. This study reveals a novel mechanism of epithelial extrusion, and indicates a novel role of polycomb function in allowing adult stem cell differentiation by antagonizing self-renewal programs. Given evolutionary conservation of PcG genes from Drosophila to mammals, they could have similar functions in mammalian stem cells and cancer. PMID:20439432

  13. Presenilin Deficiency or Lysosomal Inhibition Enhance Wnt Signaling Through Relocalization of GSK3 to the Late Endosomal Compartment

    PubMed Central

    Dobrowolski, Radek; Vick, Philipp; Ploper, Diego; Gumper, Iwona; Snitkin, Harriet; Sabatini, David D.; De Robertis, Edward M.

    2012-01-01

    SUMMARY Sustained canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity through its sequestration inside multivesicular endosomes (MVEs). Here we show that Wnt signaling is increased by the lysosomal inhibitor Chloroquine, which causes accumulation of MVEs. A similar MVE expansion and increased Wnt responsiveness was found in cells deficient in Presenilin, a protein associated with Alzheimer's disease. The Wnt-enhancing effects were entirely dependent on functional endosomal sorting complex required for transport (ESCRT), which are needed for formation of intraluminal vesicles in MVEs. We suggest that accumulation of late endosomal structures leads to enhanced canonical Wnt signaling through increased Wnt-receptor/GSK3 sequestration. The decrease in GSK3 cytosolic activity stabilized cytoplasmic GSK3 substrates such as β-Catenin, the microtubule associated protein Tau and other proteins. These results underscore the importance of the endosomal pathway in canonical Wnt signaling and reveal a new mechanism for regulation of Wnt signaling by Presenilin deficiency. PMID:23122960

  14. Effect of Pulsed Electromagnetic Field on Bone Formation and Lipid Metabolism of Glucocorticoid-Induced Osteoporosis Rats through Canonical Wnt Signaling Pathway.

    PubMed

    Jiang, Yuan; Gou, Hui; Wang, Sanrong; Zhu, Jiang; Tian, Si; Yu, Lehua

    2016-01-01

    Pulsed electromagnetic field (PEMF) has been suggested as a promising method alternative to drug-based therapies for treating osteoporosis (OP), but the role of PEMF in GIOP animal models still remains unknown. This study was performed to investigate the effect of PEMF on bone formation and lipid metabolism and further explored the several important components and targets of canonical Wnt signaling pathway in GIOP rats. After 12 weeks of intervention, bone mineral density (BMD) level of the whole body increased significantly, serum lipid levels decreased significantly, and trabeculae were thicker in GIOP rats of PEMF group. PEMF stimulation upregulated the mRNA and protein expression of Wnt10b, LRP5, β-catenin, OPG, and Runx2 and downregulated Axin2, PPAR-γ, C/EBPα, FABP4, and Dkk-1. The results of this study suggested that PEMF stimulation can prevent bone loss and improve lipid metabolism disorders in GIOP rats. Canonical Wnt signaling pathway plays an important role in bone formation and lipid metabolism during PEMF stimulation. PMID:26941827

  15. Effect of Pulsed Electromagnetic Field on Bone Formation and Lipid Metabolism of Glucocorticoid-Induced Osteoporosis Rats through Canonical Wnt Signaling Pathway

    PubMed Central

    Jiang, Yuan; Gou, Hui; Wang, Sanrong; Zhu, Jiang; Tian, Si; Yu, Lehua

    2016-01-01

    Pulsed electromagnetic field (PEMF) has been suggested as a promising method alternative to drug-based therapies for treating osteoporosis (OP), but the role of PEMF in GIOP animal models still remains unknown. This study was performed to investigate the effect of PEMF on bone formation and lipid metabolism and further explored the several important components and targets of canonical Wnt signaling pathway in GIOP rats. After 12 weeks of intervention, bone mineral density (BMD) level of the whole body increased significantly, serum lipid levels decreased significantly, and trabeculae were thicker in GIOP rats of PEMF group. PEMF stimulation upregulated the mRNA and protein expression of Wnt10b, LRP5, β-catenin, OPG, and Runx2 and downregulated Axin2, PPAR-γ, C/EBPα, FABP4, and Dkk-1. The results of this study suggested that PEMF stimulation can prevent bone loss and improve lipid metabolism disorders in GIOP rats. Canonical Wnt signaling pathway plays an important role in bone formation and lipid metabolism during PEMF stimulation. PMID:26941827

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

    PubMed

    Zhou, C-J; Borello, U; Rubenstein, J L R; Pleasure, S J

    2006-11-01

    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. PMID:16920270

  17. Thyroid hormone activates Wnt/β-catenin signaling involved in adult epithelial development during intestinal remodeling in Xenopus laevis.

    PubMed

    Hasebe, Takashi; Fujimoto, Kenta; Kajita, Mitsuko; Ishizuya-Oka, Atsuko

    2016-08-01

    During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including β-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear β-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling. PMID:27068920

  18. Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2–JNK signalling in breast cancer

    PubMed Central

    Puvirajesinghe, Tania M.; Bertucci, François; Jain, Ashish; Scerbo, Pierluigi; Belotti, Edwige; Audebert, Stéphane; Sebbagh, Michael; Lopez, Marc; Brech, Andreas; Finetti, Pascal; Charafe-Jauffret, Emmanuelle; Chaffanet, Max; Castellano, Rémy; Restouin, Audrey; Marchetto, Sylvie; Collette, Yves; Gonçalvès, Anthony; Macara, Ian; Birnbaum, Daniel; Kodjabachian, Laurent; Johansen, Terje; Borg, Jean-Paul

    2016-01-01

    The non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway plays a crucial role in embryonic development. Recent work has linked defects of this pathway to breast cancer aggressiveness and proposed Wnt/PCP signalling as a therapeutic target. Here we show that the archetypal Wnt/PCP protein VANGL2 is overexpressed in basal breast cancers, associated with poor prognosis and implicated in tumour growth. We identify the scaffold p62/SQSTM1 protein as a novel VANGL2-binding partner and show its key role in an evolutionarily conserved VANGL2–p62/SQSTM1–JNK pathway. This proliferative signalling cascade is upregulated in breast cancer patients with shorter survival and can be inactivated in patient-derived xenograft cells by inhibition of the JNK pathway or by disruption of the VANGL2–p62/SQSTM1 interaction. VANGL2–JNK signalling is thus a potential target for breast cancer therapy. PMID:26754771

  19. Wnt3 and Wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain

    PubMed Central

    2012-01-01

    Background A fundamental requirement for development of diverse brain regions is the function of local organizers at morphological boundaries. These organizers are restricted groups of cells that secrete signaling molecules, which in turn regulate the fate of the adjacent neural tissue. The thalamus is located in the caudal diencephalon and is the central relay station between the sense organs and higher brain areas. The mid-diencephalic organizer (MDO) orchestrates the development of the thalamus by releasing secreted signaling molecules such as Shh. Results Here we show that canonical Wnt signaling in the caudal forebrain is required for the formation of the Shh-secreting MD organizer in zebrafish. Wnt signaling induces the MDO in a narrow time window of 4 hours - between 10 and 14 hours post fertilization. Loss of Wnt3 and Wnt3a prevents induction of the MDO, a phenotype also observed upon blockage of canonical Wnt signaling per se. Pharmaceutical activation of the canonical Wnt pathways in Wnt3/Wnt3a compound morphant embryos is able to restore the lack of the MDO. After blockage of Wnt signaling or knock-down of Wnt3/Wnt3a we find an increase of apoptotic cells specifically within the organizer primordium. Consistently, blockage of apoptosis restores the thalamus organizer MDO in Wnt deficient embryos. Conclusion We have identified canonical Wnt signaling as a novel pathway, that is required for proper formation of the MDO and consequently for the development of the major relay station of the brain - the thalamus. We propose that Wnt ligands are necessary to maintain the primordial tissue of the organizer during somitogenesis by suppressing Tp53-mediated apoptosis. PMID:22475147

  20. Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

    PubMed Central

    2013-01-01

    Introduction Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. Methods Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. Results Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice

  1. Wnt Signaling Activates Shh Signaling in Early Postnatal Intervertebral Discs, and Re-Activates Shh Signaling in Old Discs in the Mouse

    PubMed Central

    Sinner, Debora; Wylie, Christopher C.; Dahia, Chitra Lekha

    2014-01-01

    Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease. PMID:24892825

  2. Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    PubMed Central

    2012-01-01

    Background Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the role of LRP5 is further investigated here. Methods LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures) but who lacked the clinical features of osteogenesis imperfecta (OI) or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA). In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR) to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1) and 5-hydroxytryptamine (5-Htr1b). Results Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R) were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q) significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q) reduced expression of serotonin receptor 5-Htr1b (p < 0.002). Conclusions Our results provide additional information on the role of LRP5 mutations and their effects on

  3. The Wnt pathway: emerging anticancer strategies.

    PubMed

    Gupta, Aman; Verma, Anukriti; Mishra, Ashutosh K; Wadhwa, Gulshan; Sharma, Sanjeev K; Jain, Chakresh K

    2013-05-01

    The canonical Wnt cascade has emerged as a critical regulator of cancer cells. Activation of the Wnt signaling pathway has also been associated with stem cell, thus raising the possibility of its role in embryogenesis and in the proliferation of malignant cancer cells. Wnt pathway has been reported to be involved in normal physiological processes in adult animals and integrally associated with cancer cell growth and maintenance, thus has been harnessed to devise strategies for anticancer therapy. The presence or absence of some members in this pathway, such as β-catenin, Axin or APC, has been found to involve in different types of tumors in human beings. Dysregulation of the canonical Wnt/β-catenin signaling pathway, mostly by inactivating mutations of the APC tumor suppressor, or oncogenic mutations of β-catenin, has been implicated in colorectal tumorigenesis. Further, elevated levels of β-catenin protein, a hallmark of activated canonical Wnt pathway, have been significantly observed in common forms of human malignancies, indicating that activation of the Wnt pathway may play an important role in tumor development and hence could be a crucial consideration for drug development. The paper discusses the potential therapeutic and diagnostic strategies directing on Wnt pathways on the basis of recent patents and their analysis. PMID:23432158

  4. Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

    SciTech Connect

    Santiago, Francisco; Oguma, Junya; Brown, Anthony M.C.; Laurence, Jeffrey

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. Black-Right-Pointing-Pointer Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. Black-Right-Pointing-Pointer Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. Black-Right-Pointing-Pointer Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/{beta}-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of {beta}-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, {beta}-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in

  5. Resveratrol Enhances Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells through Inhibiting Canonical WNT Signal Pathway and Enhancing Serum Response Factor-miR-1 Axis

    PubMed Central

    Liu, Hui; Zhang, Shaoli; Zhao, Lihua; Zhang, Yan; Li, Qiuping; Chai, Xiaoyan; Zhang, Yongchun

    2016-01-01

    Resveratrol (trans-3,5,4′-trihydroxystilbene) (RSV) is a natural polyphenol with protective effects over cardiac tissues and can affect cell survival and differentiation in cardiac stem cells transplantation. However, whether this agent can affect cardiomyocytes (CMs) differentiation of induced pluripotent stem cells (iPSCs) is not yet clear. This study explored whether RSV can affect CMs differentiation of human iPSCs. Under embryoid bodies (EBs) condition, the effect of RSV on the change of pluripotent markers, endoderm markers, mesoderm markers, and ectoderm markers was measured using qRT-PCR. Under CM differentiation culture, the effect of RSV on CM specific markers was also measured. The regulative role of RSV over canonical Wnt signal pathway and serum response factor- (SRF-) miR-1 axis and the functions of these two axes were further studied. Results showed that RSV had no effect on the self-renewal of human iPSCs but could promote mesoderm differentiation. Under CM differentiation culture, RSV could promote CM differentiation of human iPSCs through suppressing canonical Wnt signal pathway and enhancing SRF-miR-1 axis. PMID:26798354

  6. Histone Chaperone SSRP1 is Essential for Wnt Signaling Pathway Activity During Osteoblast Differentiation.

    PubMed

    Hossan, Tareq; Nagarajan, Sankari; Baumgart, Simon J; Xie, Wanhua; Magallanes, Roberto Tirado; Hernandez, Céline; Chiaroni, Pierre-Marie; Indenbirken, Daniela; Spitzner, Melanie; Thomas-Chollier, Morgane; Grade, Marian; Thieffry, Denis; Grundhoff, Adam; Wegwitz, Florian; Johnsen, Steven A

    2016-05-01

    Cellular differentiation is accompanied by dramatic changes in chromatin structure which direct the activation of lineage-specific transcriptional programs. Structure-specific recognition protein-1 (SSRP1) is a histone chaperone which is important for chromatin-associated processes such as transcription, DNA replication and repair. Since the function of SSRP1 during cell differentiation remains unclear, we investigated its potential role in controlling lineage determination. Depletion of SSRP1 in human mesenchymal stem cells elicited lineage-specific effects by increasing expression of adipocyte-specific genes and decreasing the expression of osteoblast-specific genes. Consistent with a role in controlling lineage specification, transcriptome-wide RNA-sequencing following SSRP1 depletion and the induction of osteoblast differentiation revealed a specific decrease in the expression of genes involved in biological processes related to osteoblast differentiation. Importantly, we observed a specific downregulation of target genes of the canonical Wnt signaling pathway, which was accompanied by decreased nuclear localization of active β-catenin. Together our data uncover a previously unknown role for SSRP1 in promoting the activation of the Wnt signaling pathway activity during cellular differentiation. Stem Cells 2016;34:1369-1376. PMID:27146025

  7. Wnt5a can both activate and repress Wnt/β-catenin signaling during mouse embryonic development

    PubMed Central

    van Amerongen, Renée; Fuerer, Christophe; Mizutani, Makiko; Nusse, Roel

    2012-01-01

    Embryonic development is controlled by a small set of signal transduction pathways, with vastly different phenotypic outcomes depending on the time and place of their recruitment. How the same molecular machinery can elicit such specific and distinct responses, remains one of the outstanding questions in developmental biology. Part of the answer may lie in the high inherent genetic complexity of these signaling cascades, as observed for the Wnt-pathway. The mammalian genome encodes multiple Wnt proteins and receptors, each of which show dynamic and tightly controlled expression patterns in the embryo. Yet how these components interact in the context of the whole organism remains unknown. Here we report the generation of a novel, inducible transgenic mouse model that allows spatiotemporal control over the expression of Wnt5a, a protein implicated in many developmental processes and multiple Wnt-signaling responses. We show that ectopic Wnt5a expression from E10.5 onwards results in a variety of developmental defects, including loss of hair follicles and reduced bone formation in the skull. Moreover, we find that Wnt5a can have dual signaling activities during mouse embryonic development. Specifically, Wnt5a is capable of both inducing and repressing β-catenin/TCF signaling in vivo, depending on the time and site of expression and the receptors expressed by receiving cells. These experiments show for the first time that a single mammalian Wnt protein can have multiple signaling activities in vivo, thereby furthering our understanding of how signaling specificity is achieved in a complex developmental context. PMID:22771246

  8. Transgenic flash mice for in vivo quantitative monitoring of canonical Wnt signaling to track hair follicle cycle dynamics.

    PubMed

    Hodgson, Samantha S; Neufeld, Zoltan; Villani, Rehan M; Roy, Edwige; Khosrotehrani, Kiarash

    2014-06-01

    Hair follicles (HFs) upon development enter a lifelong cycle of growth, regression, and resting. These phases have been extensively studied at the cellular and molecular levels for individual HFs. However, HFs group into domains with coordinated cycling strongly influenced by their environment. These macroscopic hair domains have been difficult to study and can be influenced by physiological or pathological conditions, such as pregnancy or skin wounds. To robustly address this issue, we generated a mouse model for quantitative monitoring of β-catenin activity reflecting HF cycle dynamics macroscopically by using live bioluminescence imaging. These mice allowed live tracking of HF cycles and development, and highlighted hair regenerative patterns known to occur through macro-environmental cues, including initiation events, propagating anagen and border stability, and allowed refinement of a mechanistic mathematical model that integrates epidermal cell population dynamics into an excitable reaction-diffusion model. HF cycling could be studied in situations of pregnancy, wound healing, hair plucking, as well as in response to cyclosporine or Wnt3a stimulation. In conclusion, we developed a model for analysis of HF cycling at the macroscopic level that will allow refined analysis of hair cycle kinetics as well as its propagation dynamics. PMID:24531689

  9. Effects of fulvestrant on biological activity and Wnt expression in rat GH3 cells☆

    PubMed Central

    Bai, Jiwei; Wang, Yan; Li, Chuzhong; Zhang, Yazhuo

    2012-01-01

    The present study investigated the influence of anti-estrogen treatment (fulvestrant) on pituitary adenoma cell line GH3 biological activity, the estrogen receptor α pathway, the WnT pathway, and mechanisms of decreased Wnt inhibitory factor-1 expression in GH3 cells. Results showed that fulvestrant suppressed GH3 cell proliferation and reduced hormone secretion in a dose-dependent manner. Estrogen receptor α and Wnt4 expression decreased, but Wnt inhibitory factor-1 expression increased in a dose-dependent manner following fulvestrant treatment, and β-catenin expression remained unchanged. Inhibitors of DNA methylation and histone modification upregulated Wnt inhibitory factor-1 expression. Results suggested that fulvestrant suppressed biological activity of GH3 cells via the estrogen receptor α and Wnt pathways. These results suggested that decreased Wnt inhibitory factor-1 expression in GH3 cells played a role in epigenetic mechanisms. Anti-estrogen therapies could provide novel treatments for growth hormone adenomas. PMID:25806070

  10. Molecular hydrogen suppresses activated Wnt/β-catenin signaling.

    PubMed

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

  11. Molecular hydrogen suppresses activated Wnt/β-catenin signaling

    PubMed Central

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

  12. TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

    PubMed Central

    He, Xiaoqing; Wang, Hai; Jin, Tao; Xu, Yongqing; Mei, Liangbin; Yang, Jun

    2016-01-01

    Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity. PMID:26930594

  13. Advanced bone formation in mice with a dominant-negative mutation in the thyroid hormone receptor β gene due to activation of Wnt/β-catenin protein signaling.

    PubMed

    O'Shea, Patrick J; Kim, Dong Wook; Logan, John G; Davis, Sean; Walker, Robert L; Meltzer, Paul S; Cheng, Sheue-yann; Williams, Graham R

    2012-05-18

    Thyroid hormone (T(3)) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance, but the signaling pathways mediating these effects are poorly understood. Thrb(PV/PV) mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T(3) receptor (TRβ(PV)) that cannot bind T(3) and interferes with the actions of wild-type TR. Thrb(PV/PV) mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and Thrb(PV/PV) mice. Activation of the canonical Wnt signaling in Thrb(PV/PV) mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during postnatal growth. By contrast, T(3) treatment inhibited Wnt signaling in osteoblastic cells, suggesting that T(3) inhibits the Wnt pathway by facilitating proteasomal degradation of β-catenin and preventing its accumulation in the nucleus. Activation of the Wnt pathway in Thrb(PV/PV) mice, however, results from a gain of function for TRβ(PV) that stabilizes β-catenin despite the presence of increased thyroid hormone levels. These studies demonstrate novel interactions between T(3) and Wnt signaling pathways in the regulation of skeletal development and bone formation. PMID:22442145

  14. Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib

    PubMed Central

    Lachenmayer, Anja; Alsinet, Clara; Savic, Radoslav; Cabellos, Laia; Toffanin, Sara; Hoshida, Yujin; Villanueva, Augusto; Minguez, Beatriz; Newell, Philippa; Tsai, Hung-Wen; Barretina, Jordi; Thung, Swan; Ward, Stephen C.; Bruix, Jordi; Mazzaferro, Vincenzo; Schwartz, Myron; Friedman, Scott L.; Llovet, Josep M.

    2012-01-01

    Purpose Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt-signaling. Underlying biological mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer. Experimental Design The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs) and CTNNB1-mutation data (91 HCCs). Effects of sorafenib on Wnt-signaling were evaluated in four liver cancer cell lines with active Wnt signaling and a tumor xenograft model. Results Evidence for Wnt activation was observed for 315 (49.1%) cases, and was further classified as CTNNB1-class [138 cases (21.5%)] or Wnt-TGFβ-class [177 cases (27.6%)]. CTNNB1-class was characterized by up-regulation of liver-specific Wnt-targets, nuclear β-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, while Wnt-TGFβ-class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear β-catenin. Sorafenib decreased Wnt-signaling and β-catenin protein in HepG2 (CTNNB1-class), SNU387 (Wnt-TGFβ-class), SNU398 (CTNNB1-mutation) and Huh7 (Lithium-chloride-pathway activation) cell lines. Additionally, sorafenib attenuated expression of liver-related Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1-class-specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals. Conclusions Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGFβ), accounting for half of all HCC patients. Sorafenib modulates β-catenin/Wnt-signaling in experimental models that harbor the CTNNB1-class-signature. PMID:22811581

  15. Early Stages of Zebrafish Eye Formation Require the Coordinated Activity of Wnt11, Fz5, and the Wnt/β-Catenin Pathway

    PubMed Central

    Cavodeassi, Florencia; Carreira-Barbosa, Filipa; Young, Rodrigo M.; Concha, Miguel L.; Allende, Miguel L.; Houart, Corinne; Tada, Masazumi; Wilson, Stephen W.

    2009-01-01

    Summary During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/β-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/β-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells. PMID:15996547

  16. Structural Insights into the Inhibition of Wnt Signaling by Cancer Antigen 5T4/Wnt-Activated Inhibitory Factor 1

    PubMed Central

    Zhao, Yuguang; Malinauskas, Tomas; Harlos, Karl; Jones, E. Yvonne

    2014-01-01

    Summary The tumor antigen 5T4/WAIF1 (Wnt-activated inhibitory factor 1; also known as Trophoblast glycoprotein TPBG) is a cell surface protein targeted in multiple cancer immunotherapy clinical trials. Recently, it has been shown that 5T4/WAIF1 inhibits Wnt/β-catenin signaling, a signaling system central to many developmental and pathological processes. Wnt/β-catenin signaling is controlled by multiple inhibitors and activators. Here, we report crystal structures for the extracellular domain of 5T4/WAIF1 at 1.8 Å resolution. They reveal a highly glycosylated, rigid core, comprising eight leucine-rich repeats (LRRs), which serves as a platform to present evolutionarily conserved surface residues in the N-terminal LRR1. Structural and cell-based analyses, coupled with previously reported in vivo data, suggest that Tyr325 plus the LRR1 surface centered on a second exposed aromatic residue, Phe97, are essential for inhibition of Wnt/β-catenin signaling. These results provide a structural basis for the development of 5T4/WAIF1-targeted therapies that preserve or block 5T4/WAIF1-mediated inhibition of Wnt/β-catenin signaling. PMID:24582434

  17. Cucurbitacin B inhibits the stemness and metastatic abilities of NSCLC via downregulation of canonical Wnt/β-catenin signaling axis

    PubMed Central

    Shukla, Samriddhi; Sinha, Sonam; Khan, Sajid; Kumar, Sudhir; Singh, Kavita; Mitra, Kalyan; Maurya, Rakesh; Meeran, Syed Musthapa

    2016-01-01

    Lack of effective anti-metastatic drugs creates a major hurdle for metastatic lung cancer therapy. For successful lung cancer treatment, there is a strong need of newer therapeutics with metastasis-inhibitory potential. In the present study, we determined the anti-metastatic and anti-angiogenic potential of a natural plant triterpenoid, Cucurbitacin B (CuB) against non-small cell lung cancer (NSCLC) both in vitro and in vivo. CuB demonstrated a strong anti-migratory and anti-invasive ability against metastatic NSCLC at nanomolar concentrations. CuB also showed significant tumor angiogenesis-inhibitory effects as evidenced by the inhibition of migratory, invasive and tube-forming capacities of human umbilical vein endothelial cells. CuB-mediated inhibition of angiogenesis was validated by the inhibition of pre-existing vasculature in chick embryo chorio-allantoic membrane and matrigel plugs. Similarly, CuB inhibited the migratory behavior of TGF-β1-induced experimental EMT model. The CuB-mediated inhibition of metastasis and angiogenesis was attributable to the downregulation of Wnt/β-catenin signaling axis, validated by siRNA-knockdown of Wnt3 and Wnt3a. The CuB-mediated downregulation of Wnt/β-catenin signaling was also validated using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis model in vivo. Collectively, our findings suggest that CuB inhibited the metastatic abilities of NSCLC through the inhibition of Wnt/β-catenin signaling axis. PMID:26905250

  18. RhoA Controls Wnt Upregulation on Microstructured Titanium Surfaces

    PubMed Central

    Mazzotta, Silvia; Piergianni, Maddalena; Piemontese, Marilina; Passeri, Giovanni

    2014-01-01

    Rough topography enhances the activation of Wnt canonical signaling in vitro, and this mediates its effects on cell differentiation. However, the molecular mechanisms underlying topography-dependent control of Wnt signaling are still poorly understood. As the small GTPase RhoA controls cytoskeletal reorganization and actomyosin-induced tensional forces, we hypothesized that RhoA could affect the activation of Wnt signaling in cells on micropatterned titanium surfaces. G-LISA assay revealed that RhoA activation was higher in C2C12 cells on rough (SLA) surfaces under basal conditions than on smooth (Polished) titanium. Transfection with dominant negative RhoA decreased Wnt activation by normalized TCF-Luc activity on SLA, whilst transfection with constitutively active RhoA increased TCF-Luc activation on Polished titanium. One mM Myosin II inhibitor Blebbistatin increased RhoA activation but decreased Wnt activation on SLA surfaces, indicating that tension-generating structures are required for canonical Wnt modulation on titanium surfaces. Actin inhibitor Cytochalasin markedly enhanced RhoA and TCF-Luc activation on both surfaces and increased the expression of differentiation markers in murine osteoblastic MC3T3 cells. Taken together, these data show that RhoA is upregulated in cells on rough surfaces and it affects the activation of Wnt canonical signaling through Myosin II modulation. PMID:24949442

  19. Concurrent Transient Activation of Wnt/{beta}-Catenin Pathway Prevents Radiation Damage to Salivary Glands

    SciTech Connect

    Hai Bo; Yang Zhenhua; Shangguan Lei; Zhao Yanqiu; Boyer, Arthur; Liu, Fei

    2012-05-01

    Purpose: Many head and neck cancer survivors treated with radiotherapy suffer from permanent impairment of their salivary gland function, for which few effective prevention or treatment options are available. This study explored the potential of transient activation of Wnt/{beta}-catenin signaling in preventing radiation damage to salivary glands in a preclinical model. Methods and Materials: Wnt reporter transgenic mice were exposed to 15 Gy single-dose radiation in the head and neck area to evaluate the effects of radiation on Wnt activity in salivary glands. Transient Wnt1 overexpression in basal epithelia was induced in inducible Wnt1 transgenic mice before together with, after, or without local radiation, and then saliva flow rate, histology, apoptosis, proliferation, stem cell activity, and mRNA expression were evaluated. Results: Radiation damage did not significantly affect activity of Wnt/{beta}-catenin pathway as physical damage did. Transient expression of Wnt1 in basal epithelia significantly activated the Wnt/{beta}-catenin pathway in submandibular glands of male mice but not in those of females. Concurrent transient activation of the Wnt pathway prevented chronic salivary gland dysfunction following radiation by suppressing apoptosis and preserving functional salivary stem/progenitor cells. In contrast, Wnt activation 3 days before or after irradiation did not show significant beneficial effects, mainly due to failure to inhibit acute apoptosis after radiation. Excessive Wnt activation before radiation failed to inhibit apoptosis, likely due to extensive induction of mitosis and up-regulation of proapoptosis gene PUMA while that after radiation might miss the critical treatment window. Conclusion: These results suggest that concurrent transient activation of the Wnt/{beta}-catenin pathway could prevent radiation-induced salivary gland dysfunction.

  20. Increased Activation of the Wnt/β-Catenin Pathway in Spontaneous Hepatocellular Carcinoma Observed in Farnesoid X Receptor Knockout Mice

    PubMed Central

    Wolfe, Andy; Thomas, Ann; Edwards, Genea; Jaseja, Reshma; Guo, Grace L.

    2011-01-01

    Farnesoid X receptor (FXR), the primary bile acid-sensing nuclear receptor, also is known for its anticancer properties. It is known that FXR deficiency in mice results in spontaneous hepatocellular carcinoma (HCC), but the mechanisms are not completely understood. We report that sustained activation of the Wnt/β-catenin pathway is associated with spontaneous HCC in FXR-knockout (KO) mice. HCC development was studied in FXR-KO mice at 3, 8, and 14 months of age. No tumors were observed at either 3 or 8 months, but the presence of HCC was observed in 100% of the FXR-KO mice at the age of 14 months. Further analysis revealed no change in β-catenin activation in the livers of 3-month-old FXR-KO mice, but a moderate increase was observed in 8-month-old FXR-KO mice. β-Catenin activation further increased significantly in 14-month-old tumor-bearing mice. Further analysis revealed that two independent mechanisms might be involved in β-catenin activation in the livers of FXR-KO mice. Activation of canonical Wnt signaling was evident as indicated by increased Wnt4 and dishevelled expression along with glycogen synthase kinase-3β inactivation. We also observed decreased expression of E-cadherin, a known regulator of β-catenin, in FXR-KO mice. The decrease in E-cadherin expression was accompanied by increased expression of its transcriptional repressor, Snail. Consistent with the increased HCC in FXR-KO mice, we observed a significant decrease in FXR expression and activity in human HCC samples. Taken together, these data indicate that a temporal increase in the activation of Wnt/β-catenin is observed during spontaneous HCC development in FXR-KO mice and is potentially critical for tumor development. PMID:21430080

  1. GSK-3 and Wnt Signaling in Neurogenesis and Bipolar Disorder

    PubMed Central

    Valvezan, Alexander J.; Klein, Peter S.

    2012-01-01

    The canonical Wnt signaling pathway is critical for development of the mammalian central nervous system and regulates diverse processes throughout adulthood, including adult neurogenesis. Glycogen synthase kinase-3 (GSK-3) antagonizes the canonical Wnt pathway and therefore also plays a central role in neural development and adult neurogenesis. Lithium, the first line of therapy for bipolar disorder, inhibits GSK-3, activates Wnt signaling and stimulates adult neurogenesis, which may be important for its therapeutic effects. GSK-3 also regulates other critical signaling pathways which may contribute to the therapeutic effects of lithium, including growth factor/neurotrophin signaling downstream of Akt. Here we will review the roles of GSK-3 in CNS development and adult neurogenesis, with a focus on the canonical Wnt pathway. We will also discuss the validation of GSK-3 as the relevant target of lithium and the mechanisms downstream of GSK-3 that influence mammalian behavior. PMID:22319467

  2. Wnt4 Enhances Murine Hematopoietic Progenitor Cell Expansion Through a Planar Cell Polarity-Like Pathway

    PubMed Central

    Heinonen, Krista M.; Vanegas, Juan Ruiz; Lew, Deborah; Krosl, Jana; Perreault, Claude

    2011-01-01

    Background While the role of canonical (β-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. Methodology/Principal Findings Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while β-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. Conclusions/Significance Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis through a pathway involving Wnt4, Fz6, Rac1 and Jnk kinases. PMID:21541287

  3. CTHRC1 Acts as a Prognostic Factor and Promotes Invasiveness of Gastrointestinal Stromal Tumors by Activating Wnt/PCP-Rho Signaling1

    PubMed Central

    Ma, Ming-Ze; Zhuang, Chun; Yang, Xiao-Mei; Zhang, Zi-Zhen; Ma, Hong; Zhang, Wen-Ming; You, Haiyan; Qin, Wenxin; Gu, Jianren; Yang, Shengli; Cao, Hui; Zhang, Zhi-Gang

    2014-01-01

    Gastrointestinal stromal tumors (GISTs) are the major gastrointestinal mesenchymal tumors with a variable malignancy ranging from a curable disorder to highly malignant sarcomas. Metastasis and recurrence are the main causes of death in GIST patients. To further explore the mechanism of metastasis and to more accurately estimate the recurrence risk of GISTs after surgery, the clinical significance and functional role of collagen triple helix repeat containing-1 (CTHRC1) in GIST were investigated. We found that CTHRC1 expression was gradually elevated as the risk grade of NIH classification increased, and was closely correlated with disease-free survival and overall survival in 412 GIST patients. In vitro experiments showed that recombinant CTHRC1 protein promoted the migration and invasion capacities of primary GIST cells. A luciferase reporter assay and pull down assay demonstrated that recombinant CTHRC1 protein activated noncanonical Wnt/PCP-Rho signaling but inhibited canonical Wnt signaling. The pro-motility effect of CTHRC1 on GIST cells was reversed by using a Wnt5a neutralizing antibody and inhibitors of Rac1 or ROCK. Taken together, these data indicate that CTHRC1 may serve as a new predictor of recurrence risk and prognosis in post-operative GIST patients and may play an important role in facilitating GIST progression. Furthermore, CTHRC1 promotes GIST cell migration and invasion by activating Wnt/PCP-Rho signaling, suggesting that the CTHRC1-Wnt/PCP-Rho axis may be a new therapeutic target for interventions against GIST invasion and metastasis. PMID:24726140

  4. Re-programming of C. elegans male epidermal precursor fates by Wnt, Hox, and LIN-12/Notch activities

    PubMed Central

    Yu, Hui; Seah, Adeline; Sternberg, Paul W.

    2012-01-01

    In C. elegans males, different subsets of ventral epidermal precursor (Pn.p) cells adopt distinct fates in a position-specific manner: three posterior cells, P(9–11).p, comprise the hook sensillum competence group (HCG) with three potential fates (1°, 2°, or 3°), while eight anterior cells, P(1–8).p, fuse with the hyp7 epidermal syncytium. Here we show that activation of the canonical BAR-1 β-catenin pathway of Wnt signaling alters the competence of P(3–8).p and specifies ectopic HCG-like fates. This fate transformation requires the Hox gene mab-5. In addition, misexpression of mab-5 in P(1–8).p is sufficient to establish HCG competence among these cells, as well as to generate ectopic HCG fates in combination with LIN-12 or EGF signaling. While increased Wnt signaling induces predominantly 1° HCG fates, increased LIN-12 or EGF signaling in combination with MAB-5 overexpression promotes 2° HCG fates in anterior Pn.p cells, suggesting distinctive functions of Wnt, LIN-12, and EGF signaling in specification of HCG fates. Lastly, wild-type mab-5 function is necessary for normal P(9–11).p fate specification, indicating that regulation of ectopic HCG fate formation revealed in anterior Pn.p cells reflect mechanisms of pattern formation during normal hook development. PMID:20478294

  5. Novel perspectives on non-canonical inflammasome activation

    PubMed Central

    Diamond, Catherine Emma; Khameneh, Hanif Javanmard; Brough, David; Mortellaro, Alessandra

    2015-01-01

    Inflammasomes are cytosolic multi-protein complexes that regulate the secretion of the proinflammatory cytokines, IL-1β and IL-18, and induce pyroptosis, an inflammatory form of cell death. The NLRP3 inflammasome is the most well-characterized member of this family and functions by sensing intracellular pathogen- and damage-associated molecular patterns and activating caspase-1, which processes the biologically inactive IL-1β and IL-18 precursors into active cytokines. Recent studies have identified an alternative mechanism of inflammasome activation, termed the non-canonical inflammasome, which is triggered by cytosolic sensing of lipopolysaccharide (LPS) derived from bacteria that have escaped phagolysosomes. This pathway is independent of Toll-like receptor 4 (TLR4), the well-known extracellular receptor for LPS, but instead depends on the inflammatory protease, caspase-11. Although our understanding of caspase-11 activation is still in its infancy, it appears to be an essential mediator of septic shock and attenuates intestinal inflammation. In this review, we bring together the latest data on the roles of caspase-11 and the mechanisms underlying caspase-11-mediated activation of the non-canonical inflammasome, and consider the implications of this pathway on TLR4-independent immune responses to LPS.

  6. Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity.

    PubMed

    Shono, Takumi; Ishikawa, Naoki; Toume, Kazufumi; Arai, Midori A; Ahmed, Firoj; Sadhu, Samir K; Ishibashi, Masami

    2015-07-15

    The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 μM, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear β-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing β-catenin levels. PMID:26026364

  7. Activation of Wnt3a signaling promotes myogenic differentiation of mesenchymal stem cells in mdx mice

    PubMed Central

    Shang, Yan-chang; Wang, Shu-hui; Xiong, Fu; Peng, Fu-ning; Liu, Zhen-shan; Geng, Jia; Zhang, Cheng

    2016-01-01

    Aim: Duchenne muscular dystrophy (DMD) is an X-linked genetic muscular disorder with no effective treatment at present. Mesenchymal stem cell (MSC) transplantation has been used to treat DMD, but the efficiency is low. Our previous studies show that activation of Wnt3a signaling promotes myogenic differentiation of MSCs in vitro. Here we report an effective MSC transplantation therapy in mdx mice by activation of Wnt3a signaling. Methods: MSCs were isolated from mouse bone marrow, and pretreated with Wnt3a-conditioned medium (Wnt3a-CM), then transplanted into mdx mice. The recipient mice were euthanized at 4, 8, 12, 16 weeks after the transplantation, and muscle pathological changes were examined. The expression of dystrophin in muscle was detected using immunofluorescence staining, RT-PCR and Western blotting. Results: Sixteen weeks later, transplantation of Wnt3a-pretreated MSCs in mdx mice improved the characteristics of dystrophic muscles evidenced by significant reductions in centrally nucleated myofibers, the variability range of cross-sectional area (CSA) and the connective tissue area of myofibers. Furthermore, transplantation of Wnt3a-pretreated MSCs in mdx mice gradually and markedly increased the expression of dystrophin in muscle, and improved the efficiency of myogenic differentiation. Conclusion: Transplantation of Wnt3a-pretreated MSCs in mdx mice results in long-term amelioration of the dystrophic phenotype and restores dystrophin expression in muscle. The results suggest that Wnt3a may be a promising candidate for the treatment of DMD. PMID:27133298

  8. The influence of Leucine-rich amelogenin peptide on MSC fate by inducing Wnt10b expression.

    PubMed

    Wen, Xin; Cawthorn, William P; MacDougald, Ormond A; Stupp, Samuel I; Snead, Malcolm L; Zhou, Yan

    2011-09-01

    Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein indispensable for enamel formation. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) induces osteogenesis in various cell types. Previously, we demonstrated that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists. There is a reciprocal relationship between osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). Wnt10b-mediated activation of canonical Wnt signaling has been shown to regulate mesenchymal stem cell fate. Using the bipotential bone marrow stromal cell line ST2, we have demonstrated that LRAP activates the canonical Wnt/β-catenin signaling pathway. A specific Wnt inhibitor sFRP-1 abolishes the effect of LRAP on the stimulation of osteogenesis and the inhibition of adipogenesis of ST2 cells. LRAP treatment elevates the Wnt10b expression level whereas Wnt10b knockdown by siRNA abrogates the effect of LRAP. We show here that LRAP promotes osteogenesis of mesenchymal stem cells at the expense of adipogenesis through upregulating Wnt10b expression to activate Wnt signaling. PMID:21663957

  9. Wnt/{beta}-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    SciTech Connect

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. Black-Right-Pointing-Pointer Wnt3a induces Id3 expression via canonical Wnt/{beta}-catenin pathway. Black-Right-Pointing-Pointer Wnt3a-induced Id3 expression does not depend on BMP signaling activation. Black-Right-Pointing-Pointer Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a {beta}-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/{beta}-catenin induced gene in myoblast cell fate determination.

  10. MiR-146a-5p suppresses activation and proliferation of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis through directly targeting Wnt1 and Wnt5a

    PubMed Central

    Du, Jinghua; Niu, Xuemin; Wang, Yang; Kong, Lingbo; Wang, Rongqi; Zhang, Yuguo; Zhao, Suxian; Nan, Yuemin

    2015-01-01

    Nonalcoholic fibrosing steatohepatitis is a uniform process throughout nonalcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) have been suggested to modulate cellular processes in liver diseases. However, the functional role of miRNAs in nonalcoholic fibrosing steatohepatitis is largely unclear. In this study, we systematically analyzed the hepatic miRNAs by microarray analysis in nonalcoholic fibrosing steatohepatitis in C57BL/6J mice induced by methionine-choline deficient (MCD) diet. We identified 19 up-regulated and 18 down-regulated miRNAs in liver with fibrosis. Among these dysregulated miRNAs, miR-146a-5p was the most significant down-regulated miRNA. Luciferase activity assay confirmed that Wnt1 and Wnt5a were both the target genes of miR-146a-5p. Hepatic miR-146a-5p was down-regulated in fibrosing steatohepatitis, but its target genes Wnt1 and Wnt5a and their consequent effectors α-SMA and Col-1 were significantly up-regulated. In addition, miR-146a-5p was downregulated, whilst Wnt1 and Wnt5a were up-regulated in the activated primary hepatic stellate cells (HSCs) compared to the quiescent primary HSCs. Overexpression of miR-146a-5p in HSCs inhibited HSC activation and proliferation, which concomitant with the decreased expressions of Wnt1, Wnt5a, α-SMA and Col-1. In conclusion, miR-146a-5p suppresses activation and proliferation of HSCs in the progress of nonalcoholic fibrosing steatohepatitis through targeting Wnt1 and Wnt5a and consequent effectors α-SMA and Col-1. PMID:26537990

  11. Wnt/β-Catenin and noncanonical Wnt signaling interact in tissue evagination in the simple eumetazoan Hydra

    PubMed Central

    Philipp, Isabelle; Aufschnaiter, Roland; Özbek, Suat; Pontasch, Stefanie; Jenewein, Marcell; Watanabe, Hiroshi; Rentzsch, Fabian; Holstein, Thomas W.; Hobmayer, Bert

    2009-01-01

    In and evaginations of 2D cell sheets are major shape generating processes in animal development. They result from directed movement and intercalation of polarized cells associated with cell shape changes. Work on several bilaterian model organisms has emphasized the role of noncanonical Wnt signaling in cell polarization and movement. However, the molecular processes responsible for generating tissue and body shape in ancestral, prebilaterian animals are unknown. We show that noncanonical Wnt signaling acts in mass tissue movements during bud and tentacle evagination and regeneration in the cnidarian polyp Hydra. The wnt5, wnt8, frizzled2 (fz2), and dishevelled-expressing cell clusters define the positions, where bud and tentacle evaginations are initiated; wnt8, fz2, and dishevelled remain up-regulated in those epithelial cells, undergoing cell shape changes during the entire evagination process. Downstream of wnt and dsh expression, JNK activity is required for the evagination process. Multiple ectopic wnt5, wnt8, fz2, and dishevelled-expressing centers and the subsequent evagination of ectopic tentacles are induced throughout the body column by activation of Wnt/β-Catenin signaling. Our results indicate that integration of axial patterning and tissue morphogenesis by the coordinated action of canonical and noncanonical Wnt pathways was crucial for the evolution of eumetazoan body plans. PMID:19237582

  12. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  13. Targeted Overexpression of TGF-α in the Corneal Epithelium of Adult Transgenic Mice Induces Changes in Anterior Segment Morphology and Activates Noncanonical Wnt Signaling

    PubMed Central

    Yuan, Yong; Yeh, Lung-Kun; Liu, Hongshan; Yamanaka, Osamu; Hardie, William D.; Kao, Winston W.-Y.; Liu, Chia-Yang

    2013-01-01

    Purpose. Transforming growth factor-alpha (TGF-α) transduces its signal through the epidermal growth factor receptor and is essential for corneal epithelial homeostasis. Previous studies have demonstrated that overexpression of TGF-α in the developing eye leads to anterior segment dysgenesis. However, the underlying mechanisms remain unclear. Here we examined the effects of TGF-α overexpression on adult ocular surface homeostasis. Methods. Binary Tet-On transgenic Krt12rtTA/tet-O-TGF-α mice were subjected to doxycycline (Dox) induction to overexpress TGF-α in the corneal epithelium. Intraocular pressure (IOP) was measured by noninvasive tonometry. The enucleated eyes of the experimental mice were subjected to histopathology, immunohistochemistry, and biochemistry examination. Results. Histologic and immunofluorescent examination showed that double-transgenic mice overexpressing TGF-α manifested peripheral anterior synechiae. Elevation of IOP, activation of glial cells, and loss of retinal ganglion cells were also observed. Quantitative real-time PCR revealed that the expressions of genes (RXRα, PITX2, and FOXC1) related to anterior segment dysgenesis were downregulated. Canonical Wnt signaling was suppressed, whereas noncanonical Wnt ligands (Wnt4 and Wnt5a) were upregulated. Increased myosin light chain phosphorylation suggested that noncanonical Wnt signaling is activated in affected eyes. Conclusions. Overexpression of TGF-α in the corneal epithelium induces changes in anterior segment morphology. Corneal endothelial abnormalities are associated with the activation of the noncanonical Wnt and RhoA/ROCK signaling axis, indicating a potential application of RhoA/ROCK inhibitors as a therapeutic strategy for certain types of secondary angle-closure glaucoma. PMID:23412089

  14. Wnt5a uses CD146 as a receptor to regulate cell motility and convergent extension

    NASA Astrophysics Data System (ADS)

    Ye, Zhongde; Zhang, Chunxia; Tu, Tao; Sun, Min; Liu, Dan; Lu, Di; Feng, Jing; Yang, Dongling; Liu, Feng; Yan, Xiyun

    2013-12-01

    Dysregulation of Wnt signalling leads to developmental defects and diseases. Non-canonical Wnt signalling via planar cell polarity proteins regulates cell migration and convergent extension; however, the underlying mechanisms are poorly understood. Here we report that Wnt5a uses CD146 as a receptor to regulate cell migration and zebrafish embryonic convergent extension. CD146 binds to Wnt5a with the high affinity required for Wnt5a-induced activation of Dishevelled (Dvl) and c-jun amino-terminal kinase (JNK). The interaction between CD146 and Dvl2 is enhanced on Wnt5a treatment. Mutation of the Dvl2-binding region impairs its ability to activate JNK, promote cell migration and facilitate the formation of cell protrusions. Knockdown of Dvls impairs CD146-induced cell migration. Interestingly, CD146 inhibits canonical Wnt signalling by promoting β-catenin degradation. Our results suggest a model in which CD146 acts as a functional Wnt5a receptor in regulating cell migration and convergent extension, turning off the canonical Wnt signalling branch.

  15. Noncanonical WNT-5B signaling induces inflammatory responses in human lung fibroblasts.

    PubMed

    van Dijk, Eline M; Menzen, Mark H; Spanjer, Anita I R; Middag, Laurens D C; Brandsma, Corry-Anke A; Gosens, Reinoud

    2016-06-01

    COPD is a progressive chronic lung disease characterized by pulmonary inflammation. Several recent studies indicate aberrant expression of WNT ligands and Frizzled receptors in the disease. For example, WNT-5A/B ligand expression was recently found to be increased in lung fibroblasts of COPD patients. However, possible effects of WNT-5A and WNT-5B on inflammation have not been investigated yet. In this study, we assessed the regulation of inflammatory cytokine release in response to WNT-5A/B signaling in human lung fibroblasts. Primary human fetal lung fibroblasts (MRC-5), and primary lung fibroblasts from COPD patients and non-COPD controls were treated with recombinant WNT-5A or WNT-5B to assess IL-6 and CXCL8 cytokine secretion and gene expression levels. Following WNT-5B, and to a lesser extent WNT-5A stimulation, fibroblasts showed increased IL-6 and CXCL8 cytokine secretion and mRNA expression. WNT-5B-mediated IL-6 and CXCL8 release was higher in fibroblasts from COPD patients than in non-COPD controls. In MRC-5 fibroblasts, WNT-5B-induced CXCL8 release was mediated primarily via the Frizzled-2 receptor and TAK1 signaling, whereas canonical β-catenin signaling was not involved. In further support of noncanonical signaling, we showed activation of JNK, p38, and p65 NF-κB by WNT-5B. Furthermore, inhibition of JNK and p38 prevented WNT-5B-induced IL-6 and CXCL8 secretion, whereas IKK inhibition prevented CXCL8 secretion only, indicating distinct pathways for WNT-5B-induced IL-6 and CXCL8 release. WNT-5B induces IL-6 and CXCL8 secretion in pulmonary fibroblasts. In summary, WNT-5B mediates this via Frizzled-2 and TAK1. As WNT-5 signaling is increased in COPD, this WNT-5-induced inflammatory response could represent a therapeutic target. PMID:27036869

  16. SOX9 drives WNT pathway activation in prostate cancer

    PubMed Central

    Ma, Fen; Ye, Huihui; He, Housheng Hansen; Gerrin, Sean J.; Chen, Sen; Tanenbaum, Benjamin A.; Sowalsky, Adam G.; He, Lingfeng; Wang, Hongyun; Balk, Steven P.; Yuan, Xin

    2016-01-01

    The transcription factor SOX9 is critical for prostate development, and dysregulation of SOX9 is implicated in prostate cancer (PCa). However, the SOX9-dependent genes and pathways involved in both normal and neoplastic prostate epithelium are largely unknown. Here, we performed SOX9 ChIP sequencing analysis and transcriptome profiling of PCa cells and determined that SOX9 positively regulates multiple WNT pathway genes, including those encoding WNT receptors (frizzled [FZD] and lipoprotein receptor-related protein [LRP] family members) and the downstream β-catenin effector TCF4. Analyses of PCa xenografts and clinical samples both revealed an association between the expression of SOX9 and WNT pathway components in PCa. Finally, treatment of SOX9-expressing PCa cells with a WNT synthesis inhibitor (LGK974) reduced WNT pathway signaling in vitro and tumor growth in murine xenograft models. Together, our data indicate that SOX9 expression drives PCa by reactivating the WNT/β−catenin signaling that mediates ductal morphogenesis in fetal prostate and define a subgroup of patients who would benefit from WNT-targeted therapy. PMID:27043282

  17. Novel effects of sphingosylphosphorylcholine on invasion of breast cancer: Involvement of matrix metalloproteinase-3 secretion leading to WNT activation.

    PubMed

    Kim, Hyun Ji; Kang, Gyeoung Jin; Kim, Eun Ji; Park, Mi Kyung; Byun, Hyun Jung; Nam, Seungyoon; Lee, Ho; Lee, Chang Hoon

    2016-09-01

    Sphingosylphosphorylcholine (SPC) participates in several cellular processes including metastasis. SPC induces keratin reorganization and regulates the viscoelasticity of metastatic cancer cells including PANC-1 cancer cells leading to enhanced migration and invasion. The role of SPC and the relevant mechanism in invasion of breast cell are as yet unknown. SPC dose-dependently induces invasion of breast cancer cells or breast immortalized cells. Reverse transcription polymerase chain reaction and Western blot analyses of MCF10A and ZR-75-1 cells indicated that SPC induces expression and secretion of matrix metalloproteinase-3 (MMP3). From online KMPLOT, relapse free survival is high in patients having low MMP3 expressed basal breast cancer (n=581, p=0.032). UK370106 (MMP3 inhibitor) or gene silencing of MMP3 markedly inhibited the SPC-induced invasion of MCF10A cells. An extracellular signal-regulated kinase (ERK) inhibitor, PD98059, significantly suppressed the secretion and the gelatinolytic activity of MMP3, and invasion in MCF10A cells. Over-expression of ERK1 and ERK2 promoted both the expression and secretion of MMP3. In contrast, gene silencing of ERK1 and ERK2 attenuated the secretion of MMP3 in MCF10A cells. The effects of SPC-induced MMP3 secretion on β-catenin and TCF/lymphoid enhancer factor (LEF) promoter activity were examined since MMP3 indirectly activates canonical Wnt signaling. SPC induced translocation of β-catenin to nucleus and increased TCF/LEF promoter activity. These events were suppressed by UK370106 or PD98059. Wnt inhibitor, FH535 inhibited SPC-induced MMP3 secretion and invasion. Taken together, these results suggest that SPC induces MMP3 expression and secretion via ERK leading to Wnt activation. PMID:27216977

  18. Role of the Wnt-Frizzled system in cardiac pathophysiology: a rapidly developing, poorly understood area with enormous potential

    PubMed Central

    Dawson, Kristin; Aflaki, Mona; Nattel, Stanley

    2013-01-01

    The Wnt-Frizzled (Fzd) G-protein-coupled receptor system, involving 19 distinct Wnt ligands and 10 Fzd receptors, plays key roles in the development and functioning of many organ systems. There is increasing evidence that Wnt-Fzd signalling is important in regulating cardiac function. Wnt-Fzd signalling primarily involves a canonical pathway, with dishevelled-1-dependent nuclear translocation of β-catenin that derepresses Wnt-sensitive gene transcription, but can also include non-canonical pathways via phospholipase-C/Ca2+ mobilization and dishevelled-protein activation of small GTPases. Wnt-Fzd effects vary with specific ligand/receptor interactions and associated downstream pathways. This paper reviews the biochemistry and physiology of the Wnt-Fzd complex, and presents current knowledge of Wnt signalling in cardiac remodelling processes such as hypertrophy and fibrosis, as well as disease states such as myocardial infarction (MI), heart failure and arrhythmias. Wnt signalling is activated during hypertrophy; inhibiting Wnt signalling by activating glycogen synthase kinase attenuates the hypertrophic response. Wnt signalling has complex and time-dependent actions post-MI, so that either beneficial or harmful effects might result from Wnt-directed interventions. Stem cell biology, a promising area for therapeutic intervention, is highly regulated by Wnt signalling. The Wnt system regulates fibroblast function, and is prominently altered in arrhythmogenic ventricular cardiomyopathy, a familial disease involving excess deposition of fibroadipose tissue. Wnt signalling controls connexin43 expression, thereby contributing to the regulation of cardiac electrical stability and arrhythmia generation. Although much has been learned about Wnt-Fzd signalling in hypertrophy and infarction, its role is poorly understood for a broad range of other heart disorders. Much more needs to be learned for its contributions to be fully appreciated, and to permit more effective

  19. A truncated Wnt7a retains full biological activity in skeletal muscle

    NASA Astrophysics Data System (ADS)

    von Maltzahn, Julia; Zinoviev, Radoslav; Chang, Natasha C.; Bentzinger, C. Florian; Rudnicki, Michael A.

    2013-11-01

    Wnt signaling has essential roles during embryonic development and tissue homoeostasis. Wnt proteins are post-translationally modified and the attachment of a palmitate moiety at two conserved residues is believed to be a prerequisite for the secretion and function of Wnt proteins. Here we demonstrate that a mammalian Wnt protein can be fully functional without palmitoylation. We generate a truncated Wnt7a variant, consisting of the C-terminal 137 amino acids lacking the conserved palmitoylation sites and show that it retains full biological activity in skeletal muscle. This includes binding to and signaling through its receptor Fzd7 to stimulate symmetric expansion of satellite stem cells by activating the planar-cell polarity pathway and inducing myofibre hypertrophy by signaling through the AKT/mTOR pathway. Furthermore, this truncated Wnt7a shows enhanced secretion and dispersion compared with the full-length protein. Together, these findings open important new avenues for the development of Wnt7a as a treatment for muscle-wasting diseases and have broad implications for the therapeutic use of Wnts as biologics.

  20. Wnt4 is required for ostia development in the Drosophila heart.

    PubMed

    Chen, Zhimin; Zhu, Jun-Yi; Fu, Yulong; Richman, Adam; Han, Zhe

    2016-05-15

    The Drosophila ostia are valve-like structures in the heart with functional similarity to vertebrate cardiac valves. The Wnt/β-catenin signaling pathway is critical for valve development in zebrafish and mouse, but the key ligand(s) for valve induction remains unclear. We observed high levels of Wnt4 gene expression in Drosophila ostia progenitor cells, immediately prior to morphological differentiation of these cells associated with ostia formation. This differentiation was blocked in Wnt4 mutants and in flies expressing canonical Wnt signaling pathway inhibitors but not inhibitors of the planar cell polarity pathway. High levels of Wnt4 dependent activation of a canonical Wnt signaling reporter was observed specifically in ostia progenitor cells. In vertebrate valve formation Wnt signaling is active in cells undergoing early endothelial-mesenchymal transition (EMT) and the Wnt9 homolog of Drosophila Wnt4 is expressed in valve progenitors. In demonstrating an essential role for Wnt4 in ostia development we have identified similarities between molecular and cellular events associated with early EMT during vertebrate valve development and the differentiation and partial delamination of ostia progenitor cells in the process of ostia formation. PMID:26994311

  1. Role of Sulf1A in Wnt1- and Wnt6-induced growth regulation and myoblast hyper-elongation.

    PubMed

    Hitchins, L; Fletcher, F; Allen, S; Dhoot, G K

    2013-01-01

    Sulf1A expression, which is a characteristic of embryonic muscle, is undetectable in mature muscle fibres and quiescent satellite cells, but is re-activated in vivo upon injury and in vitro following activation of satellite cells. Sulf1A is known to enhance canonical Wnt signalling, and its association with Wnt1-induced satellite cell proliferation in vitro in the present study further confirmed this. However, exogenous Wnt6 decreased satellite cell proliferation but promoted the adoption of a hyper-elongated cell morphology in myoblasts on isolated single fibres in culture. Such Wnt6-induced cellular hyper-elongation and inhibition of proliferation was found to be dependent upon Sulf1A, as treatment with Sulf1A neutralising antibodies abolished both these effects. This indicates that Sulf1A can regulate Wnt6 signalling and cellular differentiation in skeletal muscle. PMID:23772371

  2. The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity

    PubMed Central

    Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T.; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

    2015-01-01

    ABSTRACT Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. PMID:25616895

  3. C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells.

    PubMed

    Fujiwara, Miho; Kato, Shintaro; Niwa, Yuki; Suzuki, Takehiro; Tsuchiya, Miyu; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

    2016-08-01

    R-spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β-catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C-mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C-mannosylation sites at Trp(153) and Trp(156) ; however, it had been unclear whether these sites are C-mannosylated or not. We demonstrated that Rspo3 was C-mannosylated at both Trp(153) and Trp(156) by mass spectrometry. Using C-mannosylation-defective Rspo3 mutant-overexpressing cell lines, we found that C-mannosylation of Rspo3 promotes its secretion and activates Wnt/β-catenin signaling. PMID:27350215

  4. Wnt5a Supports Osteogenic Lineage Decisions in Embryonic Stem Cells.

    PubMed

    Keller, Kevin C; Ding, Huawen; Tieu, Rudy; Sparks, Nicole R L; Ehnes, Devon D; Zur Nieden, Nicole I

    2016-07-01

    The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: β-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of β-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine. PMID:26956615

  5. Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration.

    PubMed

    Harris, Katherine E; Beckendorf, Steven K

    2007-06-01

    Guided cell migration is necessary for the proper function and development of many tissues, one of which is the Drosophila embryonic salivary gland. Here we show that two distinct Wnt signaling pathways regulate salivary gland migration. Early in migration, the salivary gland responds to a WNT4-Frizzled signal for proper positioning within the embryo. Disruption of this signal, through mutations in Wnt4, frizzled or frizzled 2, results in misguided salivary glands that curve ventrally. Furthermore, disruption of downstream components of the canonical Wnt pathway, such as dishevelled or Tcf, also results in ventrally curved salivary glands. Analysis of a second Wnt signal, which acts through the atypical Wnt receptor Derailed, indicates a requirement for Wnt5 signaling late in salivary gland migration. WNT5 is expressed in the central nervous system and acts as a repulsive signal, needed to keep the migrating salivary gland on course. The receptor for WNT5, Derailed, is expressed in the actively migrating tip of the salivary glands. In embryos mutant for derailed or Wnt5, salivary gland migration is disrupted; the tip of the gland migrates abnormally toward the central nervous system. Our results suggest that both the Wnt4-frizzled pathway and a separate Wnt5-derailed pathway are needed for proper salivary gland migration. PMID:17507403

  6. Wnt/β-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance

    PubMed Central

    Mohammed, Maryam K.; Shao, Connie; Wang, Jing; Wei, Qiang; Wang, Xin; Collier, Zachary; Tang, Shengli; Liu, Hao; Zhang, Fugui; Huang, Jiayi; Guo, Dan; Lu, Minpeng; Liu, Feng; Liu, Jianxiang; Ma, Chao; Shi, Lewis L.; Athiviraham, Aravind; He, Tong-Chuan; Lee, Michael J.

    2016-01-01

    Wnt signaling transduces evolutionarily conserved pathways which play important roles in initiating and regulating a diverse range of cellular activities, including cell proliferation, calcium homeostasis, and cell polarity. The role of Wnt signaling in control of cell proliferation and stem cell self-renewal is primarily carried out through the canonical pathway, which is the best characterized among the multiple Wnt signaling branches. The past 10 years has seen a rapid expansion in our understanding of the complexity of this pathway, as many new components of Wnt signaling have been identified and linked to signaling regulation, stem cell functions, and adult tissue homeostasis. Additionally, a substantial body of evidence links Wnt signaling to tumorigenesis of many cancer types and implicates it in the development of cancer drug resistance. Thus, a better understanding of the mechanisms by which dysregulation of Wnt signaling precedes the development and progression of human cancer may hasten the development of pathway inhibitors to augment current therapy. This review summarizes and synthesizes our current knowledge of the canonical Wnt pathway in development and disease. We begin with an overview of the components of the canonical Wnt signaling pathway and delve into the role this pathway has been shown to play in stemness, tumorigenesis, and cancer drug resistance. Ultimately, we hope to present an organized collection of evidence implicating Wnt signaling in tumorigenesis and chemoresistance to facilitate the pursuit of Wnt pathway modulators that may improve outcomes of cancers in which Wnt signaling contributes to aggressive disease and/or treatment resistance. PMID:27077077

  7. Clipping the Wings of Glioblastoma: Modulation of WNT as a Novel Therapeutic Strategy.

    PubMed

    Suwala, Abigail K; Hanaford, Allison; Kahlert, Ulf D; Maciaczyk, Jaroslaw

    2016-05-01

    Glioblastoma (GBM) is the most malignant brain tumor and has a dismal prognosis. Aberrant WNT signaling is known to promote glioma cell growth and dissemination and resistance to conventional radio- and chemotherapy. Moreover, a population of cancer stem-like cells that promote glioma growth and recurrence are strongly dependent on WNT signaling. Here, we discuss the role and mechanisms of aberrant canonical and noncanonical WNT signaling in GBM. We present current clinical approaches aimed at modulating WNT activity and evaluate their clinical perspective as a novel treatment option for GBM. PMID:26979081

  8. Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis.

    PubMed

    Tian, Hua; Biehs, Brian; Chiu, Cecilia; Siebel, Christian W; Wu, Yan; Costa, Mike; de Sauvage, Frederic J; Klein, Ophir D

    2015-04-01

    Proper organ homeostasis requires tight control of adult stem cells and differentiation through the integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a derepression of the Wnt signaling pathway, leading to misexpression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology. PMID:25818302

  9. AP1- and NF-kappaB-binding sites conserved among mammalian WNT10B orthologs elucidate the TNFalpha-WNT10B signaling loop implicated in carcinogenesis and adipogenesis.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-04-01

    WNT signals are context-dependently transduced to canonical and non-canonical signaling cascades. We cloned and characterized wild-type human WNT10B, while another group cloned aberrant human WNT10B with Gly60Asp amino-acid substitution. Proto-oncogene WNT10B is expressed in gastric cancer, pancreatic cancer, breast cancer, esophageal cancer, and cervical cancer. Because WNT10B blocks adipocyte differentiation, coding SNP of WNT10B gene is associated with familial obesity. In 2001, we reported WNT10B upregulation by TNFalpha. Here, comparative integromics analyses on WNT10B orthologs were performed to elucidate the transcriptional mechanism of WNT10B. Chimpanzee WNT10B and cow Wnt10b genes were identified within NW_001223159.1 and AC150975.2 genome sequences, respectively, by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee WNT10B and cow Wnt10b showed 98.7% and 95.1% total-amino-acid identity with human WNT10B, respectively. N-terminal signal peptide, 24 Cys residues, two Asn-linked glycosylation sites, and Gly60 of human WNT10B were conserved among mammalian WNT10B orthologs. Transcription start site of human WNT10B gene was 106-bp upstream of NM_003394.2 RefSeq 5'-end. Number of GC di-nucleotide repeats just down-stream of WNT10B transcription start site varied among primates and human population. Comparative genomics analyses revealed that double AP1-binding sites in the 5'-flanking promoter region and NF-kappaB-binding site in intron 3 were conserved among human, chimpanzee, cow, mouse, and rat WNT10B orthologs. Because TNFalpha signaling through TNFR1 and TRADD/RIP/TRAF2 complex activates JUN kinase (JNK) and IkappaB kinase (IKK) signaling cascades, conserved AP1- and NF-kappaB-binding sites explain the mechanism of TNFalpha-induced WNT10B upregulation. TNFalpha-WNT10B signaling loop is the negative feedback mechanism of adipogenesis to prevent obesity and metabolic syndrome. On the other hand, TNFalpha-WNT10B signaling loop is

  10. Wnt Pathway Stabilizes MeCP2 Protein to Repress PPAR-γ in Activation of Hepatic Stellate Cells.

    PubMed

    Kweon, Soo-Mi; Chi, Feng; Higashiyama, Reiichi; Lai, Keane; Tsukamoto, Hidekazu

    2016-01-01

    PPAR-γ is essential for differentiation of hepatic stellate cells (HSC), and its loss due to epigenetic repression by methyl-CpG binding protein 2 (MeCP2) causes HSC myofibroblastic activation mediated in part via Wnt pathway, the key cellular event in liver fibrosis. Decreased miR-132 was previously proposed to promote MeCP2 protein translation for Ppar-γ repression in activated HSC (aHSC). The present study aimed to test this notion and to better understand the mechanisms of MeCP2 upregulation in aHSC. MeCP2 protein is increased on day 3 to 7 as HSC become activated in primary culture on plastic, but this is accompanied by increased but not reduced miR-132 or miR-212 which is also expected to target MeCP2 due to its similar sequence with miR-132. The levels of these mRNAs are decreased 40~50% in aHSCs isolated from experimental cholestatic liver fibrosis but increased 6-8 fold in aHSC from hepatotoxic liver fibrosis in rats. Suppression of either or both of miR132 and miR212 with specific anti-miRNA oligonucleotides (anti-oligo), does not affect MeCP2 protein levels in aHSCs. The Wnt antagonist FJ9 which inhibits HSC activation, increases miR-132/miR-212, reduces MeCP2 and its enrichment at 5' Ppar-γ promoter, and restores Ppar-γ expression but the anti-oligo do not prevent Ppar-γ upregulation. The pan-NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI) also reduces both MeCP2 and stabilized non-(S33/S37/Thr41)-phospho β-catenin and reverts aHSC to quiescent cells but do not affect miR-132/miR-212 levels. Wnt antagonism with FJ9 increases MeCP2 protein degradation in cultured HSC, and FJ9-mediated loss of MeCP2 is rescued by leupeptin but not by proteasome and lysozome inhibitors. In conclusion, canonical Wnt pathway increases MeCP2 protein due to protein stability which in turn represses Ppar-γ and activates HSC. PMID:27214381

  11. Wnt Pathway Stabilizes MeCP2 Protein to Repress PPAR-γ in Activation of Hepatic Stellate Cells

    PubMed Central

    Kweon, Soo-Mi; Chi, Feng; Higashiyama, Reiichi; Lai, Keane; Tsukamoto, Hidekazu

    2016-01-01

    PPAR-γ is essential for differentiation of hepatic stellate cells (HSC), and its loss due to epigenetic repression by methyl-CpG binding protein 2 (MeCP2) causes HSC myofibroblastic activation mediated in part via Wnt pathway, the key cellular event in liver fibrosis. Decreased miR-132 was previously proposed to promote MeCP2 protein translation for Ppar-γ repression in activated HSC (aHSC). The present study aimed to test this notion and to better understand the mechanisms of MeCP2 upregulation in aHSC. MeCP2 protein is increased on day 3 to 7 as HSC become activated in primary culture on plastic, but this is accompanied by increased but not reduced miR-132 or miR-212 which is also expected to target MeCP2 due to its similar sequence with miR-132. The levels of these mRNAs are decreased 40~50% in aHSCs isolated from experimental cholestatic liver fibrosis but increased 6–8 fold in aHSC from hepatotoxic liver fibrosis in rats. Suppression of either or both of miR132 and miR212 with specific anti-miRNA oligonucleotides (anti-oligo), does not affect MeCP2 protein levels in aHSCs. The Wnt antagonist FJ9 which inhibits HSC activation, increases miR-132/miR-212, reduces MeCP2 and its enrichment at 5’ Ppar-γ promoter, and restores Ppar-γ expression but the anti-oligo do not prevent Ppar-γ upregulation. The pan-NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI) also reduces both MeCP2 and stabilized non-(S33/S37/Thr41)-phospho β-catenin and reverts aHSC to quiescent cells but do not affect miR-132/miR-212 levels. Wnt antagonism with FJ9 increases MeCP2 protein degradation in cultured HSC, and FJ9-mediated loss of MeCP2 is rescued by leupeptin but not by proteasome and lysozome inhibitors. In conclusion, canonical Wnt pathway increases MeCP2 protein due to protein stability which in turn represses Ppar-γ and activates HSC. PMID:27214381

  12. Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans.

    PubMed

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2015-10-27

    Wnt proteins regulate axonal outgrowth along the anterior-posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, we found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled receptor, instead of controlling neuronal polarity as previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1) and Prickle (PRKL-1). Our results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology. PMID:26460008

  13. Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans

    PubMed Central

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2015-01-01

    Wnt proteins regulate axonal outgrowth along the anterior–posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, we found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled receptor, instead of controlling neuronal polarity as previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1) and Prickle (PRKL-1). Our results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology. PMID:26460008

  14. Wnt signaling during tooth replacement in zebrafish (Danio rerio): pitfalls and perspectives.

    PubMed

    Huysseune, Ann; Soenens, Mieke; Elderweirdt, Fien

    2014-01-01

    The canonical (β-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble Wnt inhibitor dickkopf1 (dkk1) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of Wnt signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axin1, mimic overexpression of Wnt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LiCl had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical Wnt pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of Wnt signaling in tooth development in polyphyodonts. PMID

  15. Wnt signaling during tooth replacement in zebrafish (Danio rerio): pitfalls and perspectives

    PubMed Central

    Huysseune, Ann; Soenens, Mieke; Elderweirdt, Fien

    2014-01-01

    The canonical (β-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble Wnt inhibitor dickkopf1 (dkk1) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of Wnt signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axin1, mimic overexpression of Wnt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LiCl had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical Wnt pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of Wnt signaling in tooth development in polyphyodonts. PMID

  16. The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD.

    PubMed

    Cosín-Roger, J; Ortiz-Masiá, D; Calatayud, S; Hernández, C; Esplugues, J V; Barrachina, M D

    2016-07-01

    The complete repair of the mucosa constitutes a key goal in inflammatory bowel disease (IBD) treatment. The Wnt signaling pathway mediates mucosal repair and M2 macrophages that coordinate efficient healing have been related to Wnt ligand expression. Signal transducer and activator of transcription 6 (STAT6) mediates M2 polarization in vitro and we hypothesize that a STAT6-dependent macrophage phenotype mediates mucosal repair in acute murine colitis by activating the Wnt signaling pathway. Our results reveal an impaired mucosal expression of M2 macrophage-associated genes and delayed wound healing in STAT6(-/-) mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These mice also exhibited decreased mucosal expression of Wnt2b, Wnt7b, and Wnt10a, diminished protein levels of nuclear β-catenin that is mainly located in crypts adjacent to damage, and reduced mRNA expression of two Wnt/β-catenin target molecules Lgr5 and c-Myc when compared with wild-type (WT) mice. Murine peritoneal macrophages treated with interleukin-4 (IL-4) and polarized toward an M2a phenotype overexpressed Wnt2b, Wnt7b, and Wnt10a in a STAT6-dependent manner. Administration of a Wnt agonist as well as transfer of properly polarized M2a macrophages to STAT6(-/-) mice activated the Wnt signaling pathway in the damaged mucosa and accelerated wound healing. Our results demonstrate that a STAT6-dependent macrophage phenotype promotes mucosal repair in TNBS-treated mice through activation of the Wnt signaling pathway. PMID:26601901

  17. Wnt Signaling in Bone

    PubMed Central

    Kubota, Takuo; Michigami, Toshimi; Ozono, Keiichi

    2010-01-01

    Wnt signaling is involved not only in embryonic development but also in maintenance of homeostasis in postnatal tissues. Multiple lines of evidence have increased understanding of the roles of Wnt signaling in bone since mutations in the LRP5 gene were identified in human bone diseases. Canonical Wnt signaling promotes mesenchymal progenitor cells to differentiate into osteoblasts. The canonical Wnt/β-catenin pathway possibly through Lrp6, a co-receptor for Wnts as well as Lrp5, in osteoblasts regulates bone resorption by increasing the OPG/RANKL ratio. However, endogenous inhibitors of Wnt signaling including sclerostin block bone formation. Regulation of sclerostin appears to be one of the mechanisms of PTH anabolic actions on bone. Since sclerostin is almost exclusively expressed in osteocytes, inhibition of sclerostin is the most promising design. Surprisingly, Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum, but not by directly promoting bone formation. Pharmacological intervention may be considered in many components of the canonical Wnt signaling pathway, although adverse effects and tumorigenicity to other tissues are important. More studies will be needed to fully understand how the Wnt signaling pathway actually influences bone metabolism and to assure the safety of new interventions. PMID:23926379

  18. Activation of Wnt/β-catenin signaling by hydrogen peroxide transcriptionally inhibits NaV1.5 expression.

    PubMed

    Wang, Ning; Huo, Rong; Cai, Benzhi; Lu, Yan; Ye, Bo; Li, Xiang; Li, Faqian; Xu, Haodong

    2016-07-01

    Oxidants and canonical Wnt/β-catenin signaling have been shown to decrease cardiac Na(+) channel activity by suppressing NaV1.5 expression. Our aims are to determine if hydrogen peroxide (H2O2), one oxidant of reactive oxygen species (ROS), activates Wnt/β-catenin signaling and promotes β-catenin nuclear activity, leading to suppression of NaV1.5 expression and if this suppression requires the interaction of β-catenin with its nuclear partner, TCF4 (also called TCF7L2) to decrease SCN5a promoter activity. The results demonstrated that H2O2 increased β-catenin, but not TCF4 nuclear localization determined by immunofluorescence without affecting total β-catenin protein level. Furthermore, H2O2 exerted a dose- and time-dependent suppressive effect on NaV1.5 expression. RT-PCR and/or Western blot analyses revealed that overexpressing active form of β-catenin or stabilizing β-catenin by GSK-3β inhibitors, LiCl and Bio, suppressed NaV1.5 expression in HL-1 cells. In contrast, destabilization of β-catenin by a constitutively active GSK-3β mutant (S9A) upregulated NaV1.5 expression. Whole-cell recording showed that LiCl significantly inhibited Na(+) channel activity in these cells. Using immunoprecipitation (IP), we showed that β-catenin interacted with TCF4 indicating that β-catenin as a co-transfactor, regulates NaV1.5 expression through TCF4. Analyses of the SCN5a promoter sequences among different species by using VISTA tools indicated that SCN5a promoter harbors TCF4 binding sites. Chromatin IP assays demonstrated that both β-catenin and TCF4 were recruited in the SCN5a promoter, and regulated its activity. Luciferase promoter assays exhibited that β-catenin inhibited the SCN5a promoter activity at a dose-dependent manner and this inhibition required TCF4. Small interfering (Si) RNA targeting β-catenin significantly increased SCN5a promoter activity, leading to enhanced NaV1.5 expression. As expected, β-catenin SiRNA prevents H2O2 suppressive effects

  19. WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in Xenopus gastrulation.

    PubMed

    Tanegashima, Kosuke; Zhao, Hui; Dawid, Igor B

    2008-02-20

    The Wnt-PCP (planar cell polarity, PCP) pathway regulates cell polarity and convergent extension movements during axis formation in vertebrates by activation of Rho and Rac, leading to the re-organization of the actin cytoskeleton. Rho and Rac activation require guanine nucleotide-exchange factors (GEFs), but the identity of the GEF involved in Wnt-PCP-mediated convergent extension is unknown. Here we report the identification of the weak-similarity GEF (WGEF) gene by a microarray-based screen for notochord enriched genes, and show that WGEF is involved in Wnt-regulated convergent extension. Overexpression of WGEF activated RhoA and rescued the suppression of convergent extension by dominant-negative Wnt-11, whereas depletion of WGEF led to suppression of convergent extension that could be rescued by RhoA or Rho-associated kinase activation. WGEF protein preferentially localized at the plasma membrane, and Frizzled-7 induced colocalization of Dishevelled and WGEF. WGEF protein can bind to Dishevelled and Daam-1, and deletion of the Dishevelled-binding domain generates a hyperactive from of WGEF. These results indicate that WGEF is a component of the Wnt-PCP pathway that connects Dishevelled to Rho activation. PMID:18256687

  20. EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells

    PubMed Central

    Zhang, Yujie; Du, Jun; Zheng, Jianchao; Liu, Jiaojing; Xu, Rui; Shen, Tian; Zhu, Yichao; Chang, Jun; Wang, Hong; Zhang, Zhihong; Meng, Fanqing; Wang, Yan; Chen, Yongchang; Xu, Yong; Gu, Luo

    2015-01-01

    Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells. PMID:25779663

  1. Dual functions for WNT5A during cartilage development and in disease.

    PubMed

    Hosseini-Farahabadi, Sara; Geetha-Loganathan, Poongodi; Fu, Katherine; Nimmagadda, Suresh; Yang, Hoe Joong; Richman, Joy M

    2013-06-24

    Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis is unknown. We mapped expression of WNT5A in the developing chicken skull and found that the highest expression was in early Meckel's cartilage but by stage 35 expression was decreased to background. We focused on chondrogenesis by targeting a retrovirus expressing WNT5A to the mandibular prominence prior to cell differentiation. Unexpectedly, there were no phenotypes in the first 6days following injection; however later the mandibular bones and Meckel's cartilage were reduced or missing on the treated side. To examine the effects on cartilage differentiation we treated micromass cultures from mandibular mesenchyme with Wnt5a-conditioned media (CM). Similar to in vivo viral data, cartilage differentiates normally, but, after 6days of culture, nearly all Alcian blue staining is lost. Collagen II and aggrecan were also decreased in treated cultures. The matrix loss was correlated with upregulation of metalloproteinases, MMP1, MMP13, and ADAMTS5 (codes for Aggrecanase). Moreover, Marimastat, an MMP and Aggrecanase inhibitor rescued cartilage matrix in Wnt5a-CM treated cultures. The pathways mediating these cartilage and RNA changes were investigated using luciferase assays. Wnt5a-CM was a potent inhibitor of the canonical pathway and strongly activated JNK/PCP signaling. To determine whether the matrix loss is mediated by repression of canonical signaling or activation of the JNK pathway we treated mandibular cultures with either DKK1, an antagonist of the canonical pathway, or a small molecule that antagonizes JNK signaling (TCS JNK 6o). DKK1 slightly increased cartilage formation and therefore suggested that the endogenous canonical signaling represses chondrogenesis. To test this further we added an excess of Wnt3a-CM and found that far fewer cartilage nodules differentiated. Since DKK1 did not mimic the effects of Wnt5a we excluded the

  2. METABOLISM Wnt Signaling Regulates Hepatic Metabolism

    PubMed Central

    Liu, Hongjun; Fergusson, Maria M.; Wu, J. Julie; Rovira, Ilsa I.; Liu, Jie; Gavrilova, Oksana; Lu, Teng; Bao, Jianjun; Han, Donghe; Sack, Michael N.; Finkel, Toren

    2011-01-01

    The contribution of the Wnt pathway has been extensively characterized in embryogenesis, differentiation, and stem cell biology but not in mammalian metabolism. Here, using in vivo gain- and loss-of-function models, we demonstrate an important role for Wnt signaling in hepatic metabolism. In particular, β-Catenin, the downstream mediator of canonical Wnt signaling, altered serum glucose concentrations and regulated hepatic glucose production. β-catenin also modulated hepatic insulin signaling. Furthermore, β-catenin interacted with the transcription factor FoxO1 in livers from mice under starved conditions. The interaction of FoxO1 with β-catenin regulated the transcriptional activation of the genes encoding glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), the two rate-limiting enzymes in hepatic gluconeogenesis. Moreover, starvation induced the hepatic expression of mRNAs encoding different Wnt isoforms. In addition, nutrient deprivation appeared to favor the association of β-catenin with FoxO family members, rather than with members of the T cell factor of transcriptional activators. Notably, in a model of diet-induced obesity, hepatic deletion of β-catenin improved overall metabolic homeostasis. These observations implicate Wnt signaling in the modulation of hepatic metabolism and raise the possibility that Wnt signaling may play a similar role in the metabolic regulation of other tissues. PMID:21285411

  3. GATA2 regulates Wnt signaling to promote primitive red blood cell fate.

    PubMed

    Mimoto, Mizuho S; Kwon, Sunjong; Green, Yangsook Song; Goldman, Devorah; Christian, Jan L

    2015-11-01

    Primitive erythropoiesis is regulated in a non cell-autonomous fashion across evolution from frogs to mammals. In Xenopus laevis, signals from the overlying ectoderm are required to induce the mesoderm to adopt an erythroid fate. Previous studies in our lab identified the transcription factor GATA2 as a key regulator of this ectodermal signal. To identify GATA2 target genes in the ectoderm required for red blood cell formation in the mesoderm, we used microarray analysis to compare gene expression in ectoderm from GATA2 depleted and wild type embryos. Our analysis identified components of the non-canonical and canonical Wnt pathways as being reciprocally up- and down-regulated downstream of GATA2 in both mesoderm and ectoderm. We show that up-regulation of canonical Wnt signaling during gastrulation blocks commitment to a hematopoietic fate while down-regulation of non-canonical Wnt signaling impairs erythroid differentiation. Our results are consistent with a model in which GATA2 contributes to inhibition of canonical Wnt signaling, thereby permitting progenitors to exit the cell cycle and commit to a hematopoietic fate. Subsequently, activation of non-canonical Wnt signaling plays a later role in enabling these progenitors to differentiate as mature red blood cells. PMID:26365900

  4. Activation of the wnt/β-Catenin Signaling Pathway in Polymyositis, Dermatomyositis and Duchenne Muscular Dystrophy

    PubMed Central

    Liu, Fuchen; Liang, Zonglai; Xu, Jingwen; Li, Wei; Zhao, Dandan; Zhao, Yuying

    2016-01-01

    Background and Purpose The wnt/β-catenin signaling pathway plays a critical role in embryonic development and adult-tissue homeostasis. Recent investigations implicate the importance of wnt/β-catenin signaling in normal wound healing and its sustained activation being associated with fibrogenesis. We investigated the immunolocalization and activation of wnt/β-catenin in polymyositis (PM), dermatomyositis (DM), and Duchenne muscular dystrophy (DMD). Methods Immunofluorescence staining and Western blot analysis of β-catenin were performed in muscle specimens from 6 PM, 8 DM, and 6 DMD subjects. The β-catenin/Tcf4 DNA-binding activity in muscle was studied using an electrophoretic mobility shift assay (EMSA), and serum wnt/β-catenin/Tcf transcriptional activity was measured using a luciferase reporter gene assay. Results Immunoreactivity for β-catenin was found in the cytoplasm and nuclei of muscle fibers in PM, DM, and DMD. The protein level of β-catenin was elevated, and EMSA analysis confirmed the activation of wnt/β-catenin signaling. The transcriptional activities of β-catenin/Tcf in the circulation were increased in patients with PM, DM, and DMD, especially in those with interstitial lung disease, and these transcriptional activities decreased when PM or DM patients exhibited obvious clinical improvements. Conclusions Our findings indicate that wnt/β-catenin signaling is activated in PM, DM, and DMD. Its activation in muscle tissue and the circulation may play a role in modulating muscle regeneration and be at least partly involved in the process of muscle and pulmonary fibrosis. PMID:27165423

  5. Proinflammatory Cytokines Regulate Cementogenic Differentiation of Periodontal Ligament Cells by Wnt/Ca(2+) Signaling Pathway.

    PubMed

    Han, Pingping; Lloyd, Tain; Chen, Zetao; Xiao, Yin

    2016-05-01

    Periodontal inflammation can inhibit cell differentiation of periodontal ligament cells (PDLCs), resulting in decreased bone/cementum regeneration ability. The Wnt signaling pathway, including canonical Wnt/β-catenin signaling and noncanonical Wnt/Ca(2+) signaling, plays essential roles in cell proliferation and differentiation during tooth development. However, little is still known whether noncanonical Wnt/Ca(2+) signaling cascade could regulate cementogenic/osteogenic differentiation capability of PDLCs within an inflammatory environment. Therefore, in this study, human PDLCs (hPDLCs) and their cementogenic differentiation potential were investigated in the presence of cytokines. The data demonstrated that both cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) inhibited cell proliferation, relative alkaline phosphatase activity, bone/cementum-related gene/protein expression, and canonical Wnt pathway-related gene/protein expression in hPDLCs. Interestingly, both cytokines upregulated the noncanonical Wnt/Ca(2+) signaling-related gene and protein expression in hPDLCs. When the Wnt/Ca(2+) pathway was blocked by Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN93, even in the presence of IL-6 and TNF-α, cementogenesis could be stimulated in hPDLCs. Our data indicate that the Wnt/Ca(2+) pathway plays an inhibitory role on PDLC cementogenic differentiation in inflammatory microenvironments. Therefore, targeting the Wnt/Ca(2+) pathway may provide a novel therapeutic approach to improve periodontal regeneration for periodontal diseases. PMID:27074616

  6. Wnt induction of chondrocyte hypertrophy through the Runx2 transcription factor.

    PubMed

    Dong, Yu-Feng; Soung, Do Y; Schwarz, Edward M; O'Keefe, Regis J; Drissi, Hicham

    2006-07-01

    We investigated the molecular mechanisms underlying canonical Wnt-mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over-expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP-2)-induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF-beta-induced expression of Sox9 and col2a1. Over-expression of beta-catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and beta-catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical beta-catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of beta-catenin-TCF/Lef on Runx2 promoter. Co-transfection of lymphoid enhancer factor (Lef1) and beta-catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt-mediated induction of Runx2. Mutation of the TCF/Lef binding site in the -128 fragment of the Runx2 promoter resulted in loss of its responsiveness to beta-catenin. Additionally, gel-shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site-directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/beta-catenin signaling is regulated by TGF-beta and BMP-2 in chick upper sternal chondrocytes, and mediates

  7. Loss of Wnt8b has no overt effect on hippocampus development but leads to altered Wnt gene expression levels in dorsomedial telencephalon

    PubMed Central

    McLaughlin, David; Nichols, Jennifer; Price, David J.; Theil, Thomas; Mason, John O.

    2015-01-01

    Wnt signalling proteins regulate many aspects of animal development. We have investigated the function of mouse Wnt8b during forebrain development. Wnt8b is expressed in a highly restricted pattern including the prospective hippocampus and hypothalamus. Mutant mice lacking Wnt8b are viable and healthy. The size and morphology of the hippocampus appeared normal in mutant embryos and adults and we found no evidence of hypothalamic defects in mutants. Wnt8b is also expressed in the neurogenic region of the adult dentate gyrus, however cell proliferation was unchanged in Wnt8b−/− mutants. Mutant embryos did, however, display altered levels of expression of other Wnt genes normally expressed in forebrain. The spatial expression patterns of other Wnt genes and the overall level of canonical Wnt activity were indistinguishable from wild types. Thus, loss of Wnt8b does not give rise to an overt morphological phenotype, but does affect expression levels of other Wnts in developing forebrain. PMID:19890917

  8. Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness

    PubMed Central

    Avgustinova, Alexandra; Iravani, Marjan; Robertson, David; Fearns, Antony; Gao, Qiong; Klingbeil, Pamela; Hanby, Andrew M.; Speirs, Valerie; Sahai, Erik; Calvo, Fernando; Isacke, Clare M.

    2016-01-01

    Stromal fibroblast recruitment to tumours and activation to a cancer-associated fibroblast (CAF) phenotype has been implicated in promoting primary tumour growth and progression to metastatic disease. However, the mechanisms underlying the tumour:fibroblast crosstalk that drive the intertumoural stromal heterogeneity remain poorly understood. Using in vivo models we identify Wnt7a as a key factor secreted exclusively by aggressive breast tumour cells, which induces CAF conversion. Functionally, this results in extracellular matrix remodelling to create a permissive environment for tumour cell invasion and promotion of distant metastasis. Mechanistically, Wnt7a-mediated fibroblast activation is not dependent on classical Wnt signalling. Instead, we demonstrate that Wnt7a potentiates TGFβ receptor signalling both in 3D in vitro and in vivo models, thus highlighting the interaction between two of the key signalling pathways in development and disease. Importantly, in clinical breast cancer cohorts, tumour cell Wnt7a expression correlates with a desmoplastic, poor-prognosis stroma and poor patient outcome. PMID:26777421

  9. Crosstalk between TGFβ and Wnt signaling pathways in the human trabecular meshwork.

    PubMed

    Webber, Hannah C; Bermudez, Jaclyn Y; Sethi, Anirudh; Clark, Abbot F; Mao, Weiming

    2016-07-01

    Primary Open Angle Glaucoma (POAG) is an irreversible, vision-threatening disease that affects millions worldwide. The principal risk factor of POAG is increased intraocular pressure (IOP) due to pathological changes in the trabecular meshwork (TM). The TGFβ signaling pathway activator TGFβ2 and the Wnt signaling pathway inhibitor secreted frizzled-related protein 1 (sFRP1) are elevated in the POAG TM. In this study, we determined whether there is a crosstalk between the TGFβ/Smad pathway and the canonical Wnt pathway using luciferase reporter assays. Lentiviral luciferase reporter vectors for studying the TGFβ/Smad pathway or the canonical Wnt pathway were transduced into primary human non-glaucomatous TM (NTM) cells. Cells were treated with or without a combination of 5 μg/ml TGFβ2 and/or 100 ng/ml Wnt3a recombinant proteins, and luciferase levels were measured using a plate reader. We found that TGFβ2 inhibited Wnt3a-induced canonical Wnt pathway activation, while Wnt3a inhibited TGFβ2-induced TGFβ/Smad pathway activation (n = 6, p < 0.05) in 3 NTM cell strains. We also found that knocking down of Smad4 or β-catenin using siRNA in HTM5 cells transfected with similar luciferase reporter plasmids abolished the inhibitory effect of TGFβ2 and/or Wnt3a on the other pathway (n = 6). Our results suggest the existence of a cross-inhibition between the TGFβ/Smad and canonical Wnt pathways in the TM, and this cross-inhibition may be mediated by Smad4 and β-catenin. PMID:27091054

  10. Wnt/β-catenin signaling in bone marrow niche.

    PubMed

    Ahmadzadeh, Ahmad; Norozi, Fatemeh; Shahrabi, Saeid; Shahjahani, Mohammad; Saki, Najmaldin

    2016-02-01

    The bone marrow (BM) niche is a specific physiological environment for hematopoietic and non-hematopoietic stem cells (HSCs). Several signaling pathways (including Wnt/β-catenin) regulate various aspects of stem cell growth, function and death in the BM niche. In addition, the canonical Wnt pathway is crucial for directing self-renewal and differentiation as important mechanisms in many types of stem cells. We review the role of the Wnt/β-catenin pathway in the BM niche and its importance in stem cells. Relevant literature was identified by a PubMed search (1997-2014) of English-language literature by using the following keywords: BM niche, Wnt/β-catenin signaling, osteoblast, osteoclast and bone disease. The Wnt/β-catenin pathway regulates the stability of the β-catenin proto-oncogene. The stabilized β-catenin then translocates to the nucleus, forming a β-catenin-TCF/LEF complex regulating the transcription of specific target genes. Stem cells require β-catenin to mediate their response to Wnt signaling for maintenance and transition from the pluripotent state during embryogenesis. In adult stem cells, Wnt signaling functions at various hierarchical levels to contribute to the specification of the diverse tissues. Aberrant Wnt/β-catenin signaling and its downstream transcriptional regulators are observed in several malignant stem cells and human cancers. Because Wnt signaling can maintain stem cells and cancer cells, the ability to modulate the Wnt pathway either positively or negatively may be of therapeutic relevance. The controlled activation of Wnt signaling might allow us to enhance stem and progenitor cell activity when regeneration is needed. PMID:26475718

  11. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

    PubMed Central

    Gu, Bai-Wei; Apicella, Marisa; Mills, Jason; Fan, Jian-Meng; Reeves, Dara A.; French, Deborah; Podsakoff, Gregory M.; Bessler, Monica; Mason, Philip J.

    2015-01-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed “corrected” lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human

  12. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

    PubMed

    Gu, Bai-Wei; Apicella, Marisa; Mills, Jason; Fan, Jian-Meng; Reeves, Dara A; French, Deborah; Podsakoff, Gregory M; Bessler, Monica; Mason, Philip J

    2015-01-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human

  13. Activation and Inhibition of The Wnt3A Signaling Pathway in Buffalo (Bubalus bubalis) Embryonic Stem Cells: Effects of WNT3A, Bio and Dkk1

    PubMed Central

    Zandi, Mohammad; Shah, Syed Mohamad; Muzaffar, Musharifa; Kumar Singh, Manoj; Palta, Prabhat; Kumar Singla, Suresh; Sham Manik, Radhey; Chauhan, Manmohan Singh

    2015-01-01

    Background This research studies the effects of activation and inhibition of Wnt3A signaling pathway in buffalo (Bubalus bubalis) embryonic stem (ES) cell-like cells. Materials and Methods To carry on this experimental study, the effects of activation and inhibition of Wnt3A signaling in buffalo ES cell-like cells were examined using Bio (0.5 mM) combined with WNT3A (200 ng/ml), as an activator, and Dickkopf-1 (Dkk1, 250 ng/ml), as an inhibitor, of the pathway. ES cells were cultured up to three weeks in ES cell medium without fibroblast growth factor-2 (FGF-2) and leukemia inhibitory factor (LIF), but in the presence of Bio, WNT3A, Bio+WNT3A and Dkk1. The effects of these supplements were measured on the mean area of ES cell colonies and on the expression levels of a number of important genes related to pluripotency (Oct4, Nanog, Sox2 and c-Myc) and the Wnt pathway (β-catenin). ES cell colonies cultured in ES cell medium that contained optimized quantities of LIF and FGF-2 were used as the control. Data were collected for week-1 and week-3 treated cultures. In addition, WNT3A-transfected ES cells were compared with the respective mock-transfected colonies, either alone or in combination with Dkk1 for expression of β-catenin and the pluripotency-related genes. Data were analyzed by ANOVA, and statistical significance was accepted at P<0.05. Results Among various examined concentrations of Bio (0.5-5 mM), the optimum effect was observed at the 0.5 mM dose as indicated by colony area and expressions of pluripotency-related genes at both weeks-1 and -3 culture periods. At this concentration,the expressions of Nanog, Oct3/4, Sox2, c-Myc and β-catenin genes were nonsignificantly higher compared to the controls. Expressions of these genes were highest in the Bio+WNT3A treated group, followed by the WNT3A and Bio-supplemented groups, and lowest in the Dkk1-treated group. The WNT-transfected colonies showed higher expressions compared to both mock and Dkk1-treated mock

  14. Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

    PubMed Central

    Kumar, Pankaj; Magala, Pearl; Geiger-Schuller, Kathryn R.; Majumdar, Ananya; Tolman, Joel R.; Wolberger, Cynthia

    2015-01-01

    Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity. PMID:26286193

  15. Wnt6 regulates epithelial cell differentiation and is dysregulated in renal fibrosis.

    PubMed

    Beaton, Hayley; Andrews, Darrell; Parsons, Martin; Murphy, Mary; Gaffney, Andrew; Kavanagh, David; McKay, Gareth J; Maxwell, Alexander P; Taylor, Cormac T; Cummins, Eoin P; Godson, Catherine; Higgins, Debra F; Murphy, Paula; Crean, John

    2016-07-01

    Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3β (Ser9), nuclear accumulation of β-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-β (TGF-β); Wnt6 reversed TGF-β-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-β-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/β(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis. PMID:27122540

  16. Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin.

    PubMed

    Mihara, Emiko; Hirai, Hidenori; Yamamoto, Hideki; Tamura-Kawakami, Keiko; Matano, Mami; Kikuchi, Akira; Sato, Toshiro; Takagi, Junichi

    2016-01-01

    Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years. PMID:26902720

  17. Lithium stimulates human bone marrow derived mesenchymal stem cell proliferation through GSK-3β-dependent β-catenin/Wnt pathway activation.

    PubMed

    Zhu, Zhenzhong; Yin, Junhui; Guan, Junjie; Hu, Bin; Niu, Xin; Jin, Dongxu; Wang, Yang; Zhang, Changqing

    2014-12-01

    Mesenchymal stem cells (MSCs) are multipotent cells that have been widely used in cell based transplantation therapy. The use of MSCs requires in vitro expansion in order to fulfill their regenerative capacity. Therefore the proliferative ability of MSCs is one of the key factors which determine MSC therapeutic efficacy. In the present study, we showed for the first time that lithium, a well-known antidepressant, reversibly promoted the proliferation of human bone marrow derived MSCs in vitro. MSCs treated with 5 mm lithium proliferated more rapidly than untreated cells without undergoing apoptosis. Lithium increased the proportion of cells in S phase as well as cyclin D1 expression. Mechanistic studies revealed that these effects were dependent upon the activation of the glycogen synthase kinase 3β (GSK-3β) mediated canonical Wnt pathway. Lithium induced Ser9 phosphorylation, which results in the inhibition of GSK-3β activity, β-catenin accumulation and Wnt pathway activation. Utilizing a specific GSK-3β inhibitor SB216763 or siRNA-mediated inhibition of GSK-3β produced effects similar to those induced by lithium. In contrast, either quercetin, an inhibitor of the β-catenin/TCF pathway, or siRNA-mediated knockdown of β-catenin abolished the proliferative effect of lithium, suggesting that lithium stimulates MSC proliferation via the GSK-3β-dependent β-catenin/Wnt pathway. Collectively, these studies elucidate a novel role of lithium, which may not only provide a simple and effective way to strengthen MSC transplantation therapy efficacy but also shed light on lithium's clinical application for the treatment of certain disorders resulting from β-catenin/Wnt pathway suppression. PMID:25265417

  18. Molecular cloning, characterization and expression analysis of Wnt4, Wnt5, Wnt6, Wnt7, Wnt10 and Wnt16 from Litopenaeus vannamei.

    PubMed

    Zhang, Shuang; Li, Chao-Zheng; Yang, Qi-Hui; Dong, Xiao-Hui; Chi, Shu-Yan; Liu, Hong-Yu; Shi, Li-Li; Tan, Bei-Ping

    2016-07-01

    The Wnt (Wg-type MMTV integration site) signaling represents as the negative regulator of virus-induced innate immune responses. Wnt genes act as ligands to activate the Wnt signaling. To know more about the information of Wnt genes in invertebrates, Litopenaeus vannamei Wnt genes (LvWnts) were identified and characterized. In this study, Six Wnt genes (LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16) were obtained in L. vannamei. The complete cDNAs open reading frames (ORF) of LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16 were 1077 bp, 1107 bp, 1350 bp, 1047 bp, 1509 bp and 1158 bp (GenBank accession no. KU169896, KU169897, KU169898, KU169899, KU169900 and KU169901), encoding 358, 368, 449, 348, 502 and 385 amino acid (aa) residues respectively. All the six members of LvWnts contain a Wnt1 domain, which is considered as an important feature of Wnt gene family. ClustalW analysis with amino acid sequences revealed that the proportion of identity with other species was more than 48% for all the LvWnts except LvWnt10 (36-41%). The phylogenetic relationship analysis illustrated that different subtype of Wnts formed their own separate branches and were placed in branch of invertebrates respectively with strong bootstrap support. The constitutive expressions of LvWnts were confirmed by RT-PCR in all the examined five developmental stages and eleven tissues of L. vannamei with different express patterns. LvWnt4, LvWnt5 and LvWnt10 were expressed highest in nerve while LvWnt6, LvWnt7 and LvWnt16 were expressed highest in intestine, stomach and gill, respectively. In addition, all the LvWnts were regulated by white spot syndrome virus (WSSV) challenges at different levels in hepatopancreas, gill and hemocytes, suggesting that Wnt genes may play a role in the defense against pathogenic virus infection in innate immune of L. vannamei. PMID:27153750

  19. Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB signalling

    PubMed Central

    Saldanha-Araujo, Felipe; Haddad, Rodrigo; de Farias, Kelen C R Malmegrim; Souza, Alessandra de Paula Alves; Palma, Patrícia V; Araujo, Amélia G; Orellana, Maristela D; Voltarelli, Julio C; Covas, Dimas T; Zago, Marco A; Panepucci, Rodrigo A

    2012-01-01

    Abstract Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3+ T cells were activated and cultured in the presence or absence of MSCs. CD4+ cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69+ cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69+ cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling. PMID:21777379

  20. Modulation of WNT/β-catenin pathway in melanoma by biologically active components derived from plants.

    PubMed

    Gajos-Michniewicz, Anna; Czyz, Malgorzata

    2016-03-01

    Metastatic melanoma is an aggressive cancer, often resistant to treatment. Therefore, it is essential to determine the molecular mechanisms leading to melanoma or underlying resistance to therapy, and the response to targeted inhibition of the RAS/BRAF/MEK/ERK pathway was a good lesson in this respect. Aberrant WNT/β-catenin pathway is observed in melanoma, and the modulators of this signaling cascade have been under investigation in the context of therapy as well as chemoprevention. Several natural compounds were recognized as being capable of targeting elements of the WNT/β-catenin pathway in various cancers, however, only a few of them can modulate this pathway in melanoma. This review examines recent research on the role of the WNT/β-catenin pathway in tumor development and maintenance, as well as summarizes the current knowledge concerning the modulation of this pathway in melanoma by active compounds of natural origin. PMID:26851176

  1. Wnt Signaling in Cartilage Development and Diseases: Lessons from Animal Studies

    PubMed Central

    Usami, Yu; Gunawardena, Aruni T.; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi

    2016-01-01

    Cartilage not only plays essential roles in skeletal development and growth during pre-and post-natal stages but also serves to provide smooth movement of skeletons throughout life. Thus dysfunction of cartilage causes a variety of skeletal disorders. Results from animal studies reveal that β-catenin-dependent canonical and independent non-canonical Wnt signaling pathways have multiple roles in regulation of cartilage development, growth and maintenance. β-catenin-dependent signaling is required for progression of endochondral ossification and growth of axial and appendicular skeletons while excessive activation of this signaling can cause severe inhibition of initial cartilage formation and growth plate organization and function in mice. In contrast, non-canonical Wnt signaling is important in columnar organization of growth plate chondrocytes. Manipulation of Wnt signaling causes or ameliorates articular cartilage degeneration in rodent osteoarthritis models. Human genetic studies indicate that Wnt/β-catenin signaling is a risk factor for osteoarthritis. Accumulative findings from analysis of expression of Wnt signaling molecules and in vivo and in vitro functional experiments suggest that Wnt signaling is a therapeutic target for osteoarthritis. The target tissues of Wnt signaling may be not only articular cartilage but also synovium and subchondral bone. PMID:26641070

  2. WNT/PCP signaling pathway and human cancer (review).

    PubMed

    Katoh, Masaru

    2005-12-01

    WNT/planar cell polarity (PCP) signaling pathway controls tissue polarity and cell movement through the activation of RHOA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades. PCP is induced in Drosophila by the asymmetrical localization of Frizzled-Dishevelled-Diego-Starry night (Flamingo) complex and Van Gogh (Strabismus)-Prickle complex. Here, WNT/PCP signaling pathway implicated in human carcinogenesis is reviewed. Human WNT5A, WNT5B, and WNT11 are representative non-canonical WNTs transducing PCP signals through FZD3 or FZD6 receptors, and ROR1, ROR2 or PTK7 co-receptors. Human VANGL1, VANGL2 (Van Gogh homologs), CELSR1, CELSR2, CELSR3 (Starry night homologs), DVL1, DVL2, DVL3 (Dishevelled homologs), PRICKLE1, PRICKLE2 (Prickle homologs), and ANKRD6 (Diego homolog) are core PCP signaling molecules. MAGI3 assembles FZD, VANGL, PTEN, and adhesion molecules. Dishevelled-dependent WNT/PCP signals are transduced to the RHOA signaling cascade through Formin homology proteins DAAM1 and DAAM2, and to the JNK signaling cascade through MAPKKKs and MAPKK4/7. Dishevelled-independent WNT/ PCP signals are transduced to the NLK signaling cascade through MAP3K7 (TAK1). ANKRD6, NKD1 and NKD2 induce class switch from the WNT/GSK3beta signaling pathway to the WNT/PCP signaling pathway. WNT5A is up-regulated in various types of human cancer, such as gastric cancer, lung cancer, and melanoma. FZD3/FZD6 receptor and ROR2 co-receptor transduce WNT5A signal in gastric cancer. Aberrant activation of WNT/PCP signaling pathway in human cancer leads to more malignant phenotypes, such as abnormal tissue polarity, invasion, and metastasis. cDNA-PCR, microarray or ELISA reflecting aberrant activation of WNT/PCP signaling pathway could be developed as novel cancer prognostics. Single nucleotide polymorphism (SNP) and copy number polymorphism (CNP) of WNT/PCP signaling molecules mentioned above are suitable for use in screening of cancer predisposition, especially

  3. The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis.

    PubMed

    Borello, Ugo; Berarducci, Barbara; Murphy, Paula; Bajard, Lola; Buffa, Viviana; Piccolo, Stefano; Buckingham, Margaret; Cossu, Giulio

    2006-09-01

    Canonical Wnt/beta-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical beta-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/beta-catenin pathway in somite progenitor cells, whereas expression of activated beta-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5' to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/beta-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/beta-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells. PMID:16936075

  4. Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/βcatenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek

    PubMed Central

    Caneparo, Luca; Huang, Ya-Lin; Staudt, Nicole; Tada, Masasumi; Ahrendt, Reiner; Kazanskaya, Olga; Niehrs, Christof; Houart, Corinne

    2007-01-01

    Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/βcatenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a “head inducer” during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/βcatenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a βcatenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the βcatenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/βcatenin and Wnt/PCP pathways. PMID:17322405

  5. Wnt1/βcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair

    PubMed Central

    Duan, Jinzhu; Gherghe, Costin; Liu, Dianxin; Hamlett, Eric; Srikantha, Luxman; Rodgers, Laurel; Regan, Jenna N; Rojas, Mauricio; Willis, Monte; Leask, Andrew; Majesky, Mark; Deb, Arjun

    2012-01-01

    Wnts are required for cardiogenesis but the role of specific Wnts in cardiac repair remains unknown. In this report, we show that a dynamic Wnt1/βcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. Acute ischaemic cardiac injury upregulates Wnt1 that is initially expressed in the epicardium and subsequently by cardiac fibroblasts in the region of injury. Following cardiac injury, the epicardium is activated organ-wide in a Wnt-dependent manner, expands, undergoes epithelial–mesenchymal transition (EMT) to generate cardiac fibroblasts, which localize in the subepicardial space. The injured regions in the heart are Wnt responsive as well and Wnt1 induces cardiac fibroblasts to proliferate and express pro-fibrotic genes. Disruption of downstream Wnt signalling in epicardial cells decreases epicardial expansion, EMT and leads to impaired cardiac function and ventricular dilatation after cardiac injury. Furthermore, disruption of Wnt/βcatenin signalling in cardiac fibroblasts impairs wound healing and decreases cardiac performance as well. These findings reveal that a pro-fibrotic Wnt1/βcatenin injury response is critically required for preserving cardiac function after acute ischaemic cardiac injury. PMID:22085926

  6. Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis.

    PubMed

    Tian, Yuan; Mok, Myth T S; Yang, Pengyuan; Cheng, Alfred S L

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients. PMID:27556491

  7. Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis

    PubMed Central

    Tian, Yuan; Mok, Myth T.S.; Yang, Pengyuan; Cheng, Alfred S.L.

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients. PMID:27556491

  8. Euodia sutchuenensis Dode extract stimulates osteoblast differentiation via Wnt/β-catenin pathway activation

    PubMed Central

    Hwang, Jeong-Ha; Cha, Pu-Hyeon; Han, Gyoonhee; Bach, Tran The; Min, Do Sik; Choi, Kang-Yell

    2015-01-01

    The Wnt/β-catenin pathway has a role in osteoblast differentiation and bone formation. We screened 100 plant extracts and identified an extract from Euodia sutchuenensis Dode (ESD) leaf and young branch as an effective activator of the Wnt/β-catenin pathway. ESD extract increased β-catenin levels and β-catenin nuclear accumulation in murine primary osteoblasts. The ESD extract also increased mRNA levels of osteoblast markers, including RUNX2, BMP2 and COL1A1, and enhanced alkaline phosphatase (ALP) activity in murine primary osteoblasts. Both ESD extract-induced β-catenin increment and ALP activation were abolished by β-catenin knockdown, confirming that the Wnt/β-catenin pathway functions in osteoblast differentiation. ESD extract enhanced terminal osteoblast differentiation as shown by staining with Alizarin Red S and significantly increased murine calvarial bone thickness. This study shows that ESD extract stimulates osteoblast differentiation via the Wnt/β-catenin pathway and enhances murine calvarial bone formation ex vivo. PMID:25792220

  9. The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway*

    PubMed Central

    Martinez, Sébastien; Scerbo, Pierluigi; Giordano, Marilyn; Daulat, Avais M.; Lhoumeau, Anne-Catherine; Thomé, Virginie; Kodjabachian, Laurent; Borg, Jean-Paul

    2015-01-01

    The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements. PMID:26499793

  10. A Role for the Non-Canonical Wnt-β-Catenin and TGF-β Signaling Pathways in the Induction of Tolerance during the Establishment of a Salmonella enterica Serovar Enteritidis Persistent Cecal Infection in Chickens

    PubMed Central

    Kogut, Michael H.; Arsenault, Ryan J.

    2015-01-01

    Non-typhoidal Salmonella enterica induce an early pro-inflammatory response in chickens. However, the response is short-lived, asymptomatic of disease, resulting in a persistent colonization of the ceca, and fecal shedding of bacteria. The underlying mechanisms that control this persistent infection of chickens by Salmonella are unknown. Recently, we found an expansion of the Treg population and subsequent increased in vitro immunosuppressive functions of the CD4+CD25+ cells isolated from the ceca of the Salmonella-infected chickens by day 4 post-infection that increased steadily throughout the course of the 14 days of infection, whereas the number of CD4+CD25+ cells in the non-infected controls remained steady throughout the study. CD4+CD25+ cells from cecal tonsils of S. enteritidis-infected birds had greater expression of IL-10 mRNA content than the CD4+CD25+ cells from the non-infected controls at all the time points studied. These results suggest the development of a tolerogenic immune response in the cecum of Salmonella-infected chickens may contribute to the persistance of Salmonella cecal colonization. Using a chicken-specific kinome peptide immune array, we have analyzed the signaling pathways altered during the establishment of this tolerogenic state. This analysis has revealed a role for the non-canonical Wnt signaling pathway in the cecum at 4 days post-infection. Infection induced the significant (p < 0.01) phosphorylation of the G-protein-coupled transmembrane protein, Frizzled 1 (FZD1), resulting in an influx of intracellular Ca2+ and the phosphorylation of the Ca2+-dependent effector molecules calcium/calmodulin-dependent kinase II (CamKII), β-catenin, protein kinase C, and the activation of the transcription factor, NFAT. Nuclear translocation of NFAT resulted in a significant increase in the expression of the anti-inflammatory cytokines IL-10 and TGF-β. Increased expression of TGF-β4 mRNA activates the TGF-β signaling pathway that

  11. Functional interactions between the LRP6 WNT co-receptor and folate supplementation.

    PubMed

    Gray, Jason D; Nakouzi, Ghunwa; Slowinska-Castaldo, Bozena; Dazard, Jean-Eudes; Rao, J Sunil; Nadeau, Joseph H; Ross, M Elizabeth

    2010-12-01

    Crooked tail (Cd) mice bear a gain-of-function mutation in Lrp6, a co-receptor for canonical WNT signaling, and are a model of neural tube defects (NTDs), preventable with dietary folic acid (FA) supplementation. Whether the FA response reflects a direct influence of FA on LRP6 function was tested with prenatal supplementation in LRP6-deficient embryos. The enriched FA (10 ppm) diet reduced the occurrence of birth defects among all litters compared with the control (2 ppm FA) diet, but did so by increasing early lethality of Lrp6(-/-) embryos while actually increasing NTDs among nulls alive at embryonic days 10-13 (E10-13). Proliferation in cranial neural folds was reduced in homozygous Lrp6(-/-) mutants versus wild-type embryos at E10, and FA supplementation increased proliferation in wild-type but not mutant neuroepithelia. Canonical WNT activity was reduced in LRP6-deficient midbrain-hindbrain at E9.5, demonstrated in vivo by a TCF/LEF-reporter transgene. FA levels in media modulated the canonical WNT response in NIH3T3 cells, suggesting that although FA was required for optimal WNT signaling, even modest FA elevations attenuated LRP5/6-dependent canonical WNT responses. Gene expression analysis in embryos and adults showed striking interactions between targeted Lrp6 deficiency and FA supplementation, especially for mitochondrial function, folate and methionine metabolism, WNT signaling and cytoskeletal regulation that together implicate relevant signaling and metabolic pathways supporting cell proliferation, morphology and differentiation. We propose that FA supplementation rescues Lrp6(Cd/Cd) fetuses by normalizing hyperactive WNT activity, whereas in LRP6-deficient embryos, added FA further attenuates reduced WNT activity, thereby compromising development. PMID:20843827

  12. Wnt1 Participates in Inflammation Induced by Lipopolysaccharide Through Upregulating Scavenger Receptor A and NF-kB.

    PubMed

    Zhao, Wenting; Sun, Zewei; Wang, Shuai; Li, Zhenwei; Zheng, Liangrong

    2015-08-01

    The study investigated the role of wnt1 in the inflammatory response initiated by lipolysaccharide (LPS), and analyzed the association between wnt1, NF-KB, and inflammatory factors. THP-1 cells were activated with phorbol-12-myristate-13-acetate (PMA) and treated with LPS to induce inflammation. THP-1 cells were transfected with wnt1siRNA and overexpression plasmid to explore the relationship among wnt1, SRA, and NF-KB. Inhibitor of β-catenin and siRNA of FZD1were used to investigate the signaling events involved in SRA activation induced by wnt1. Levels of NF-kB protein and inflammatory cytokines were assessed followingwnt1 siRNA and LPS treatment. PMA activation and LPS treatment of THP-1 cells increased wnt1 protein levels. Wnt1 promoted SRA expression through activation of canonical wnt pathway. Wnt1 increased NF-kB protein levels and enhanced the secretion of IL-6, TNF-α, and iNOS through binding to SRA. These findings suggest that wnt1 increased SRA and NF-kB protein levels and participated in the inflammatory response. PMID:25749569

  13. Chronic hypoxia induces the activation of the Wnt/β-catenin signaling pathway and stimulates hippocampal neurogenesis in wild-type and APPswe-PS1ΔE9 transgenic mice in vivo

    PubMed Central

    Varela-Nallar, Lorena; Rojas-Abalos, Macarena; Abbott, Ana C.; Moya, Esteban A.; Iturriaga, Rodrigo; Inestrosa, Nibaldo C.

    2014-01-01

    Hypoxia modulates proliferation and differentiation of cultured embryonic and adult stem cells, an effect that includes β-catenin, a key component of the canonical Wnt signaling pathway. Here we studied the effect of mild hypoxia on the activity of the Wnt/β-catenin signaling pathway in the hippocampus of adult mice in vivo. The hypoxia-inducible transcription factor-1α (HIF-1α) was analyzed as a molecular control of the physiological hypoxic response. Exposure to chronic hypoxia (10% oxygen for 6–72 h) stimulated the activation of the Wnt/β-catenin signaling pathway. Because the Wnt/β-catenin pathway is a positive modulator of adult neurogenesis, we evaluated whether chronic hypoxia was able to stimulate neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus. Results indicate that hypoxia increased cell proliferation and neurogenesis in adult wild-type mice as determined by Ki67 staining, Bromodeoxyuridine (BrdU) incorporation and double labeling with doublecortin (DCX). Chronic hypoxia also induced neurogenesis in a double transgenic APPswe-PS1ΔE9 mouse model of Alzheimer’s disease (AD), which shows decreased levels of neurogenesis in the SGZ. Our results show for the first time that exposure to hypoxia in vivo can induce the activation of the Wnt/β-catenin signaling cascade in the hippocampus, suggesting that mild hypoxia may have a therapeutic value in neurodegenerative disorders associated with altered Wnt signaling in the brain and also in pathological conditions in which hippocampal neurogenesis is impaired. PMID:24574965

  14. Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.

    PubMed

    Wang, Ronghua; Sun, Qian; Wang, Peng; Liu, Man; Xiong, Si; Luo, Jing; Huang, Hai; Du, Qiang; Geller, David A; Cheng, Bin

    2016-02-01

    Human hepatocellular carcinoma (HCC) is driven and maintained by liver cancer stem cells (LCSCs) that display stem cell properties. These LCSCs are promoted by the intersecting of Notch and Wnt/β-Catenin signaling pathways. In this study, we demonstrate that LCSCs with markers CD90, CD24, CD13, and CD133 possess stem properties of self-renewal and tumorigenicity in NOD/SCID mice. The increased expression of these markers was correlated with advanced disease stage, larger tumors, and worse overall survival in 61 HCC cases. We also found that both Notch and Wnt/β-catenin signaling pathways played important roles in increasing the stem-ness characteristics of LCSCs. Our data suggested that Notch1 was downstream of Wnt/β-catenin. The active form of Notch1 intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notch1 negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notch1 with lentivirus N1ShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notch1 in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs. The central role of Notch and the Wnt/β-catenin signaling pathway in LCSCs may provide an attractive therapeutic strategy against HCC. PMID:26735577

  15. Involvement of Wnt, Eda and Shh at defined stages of sweat gland development.

    PubMed

    Cui, Chang-Yi; Yin, Mingzhu; Sima, Jian; Childress, Victoria; Michel, Marc; Piao, Yulan; Schlessinger, David

    2014-10-01

    To maintain body temperature, sweat glands develop from embryonic ectoderm by a poorly defined mechanism. We demonstrate a temporal cascade of regulation during mouse sweat gland formation. Sweat gland induction failed completely when canonical Wnt signaling was blocked in skin epithelium, and was accompanied by sharp downregulation of downstream Wnt, Eda and Shh pathway genes. The Wnt antagonist Dkk4 appeared to inhibit this induction: Dkk4 was sharply downregulated in β-catenin-ablated mice, indicating that it is induced by Wnt/β-catenin; however, its overexpression repressed Wnt target genes and significantly reduced gland numbers. Eda signaling succeeded Wnt. Wnt signaling was still active and nascent sweat gland pre-germs were still seen in Eda-null mice, but the pre-germs failed to develop further and the downstream Shh pathway was not activated. When Wnt and Eda were intact but Shh was ablated, germ induction and subsequent duct formation occurred normally, but the final stage of secretory coil formation failed. Thus, sweat gland development shows a relay of regulatory steps initiated by Wnt/β-catenin - itself modulated by Dkk4 - with subsequent participation of Eda and Shh pathways. PMID:25249463

  16. Involvement of Wnt, Eda and Shh at defined stages of sweat gland development

    PubMed Central

    Cui, Chang-Yi; Yin, Mingzhu; Sima, Jian; Childress, Victoria; Michel, Marc; Piao, Yulan; Schlessinger, David

    2014-01-01

    To maintain body temperature, sweat glands develop from embryonic ectoderm by a poorly defined mechanism. We demonstrate a temporal cascade of regulation during mouse sweat gland formation. Sweat gland induction failed completely when canonical Wnt signaling was blocked in skin epithelium, and was accompanied by sharp downregulation of downstream Wnt, Eda and Shh pathway genes. The Wnt antagonist Dkk4 appeared to inhibit this induction: Dkk4 was sharply downregulated in β-catenin-ablated mice, indicating that it is induced by Wnt/β-catenin; however, its overexpression repressed Wnt target genes and significantly reduced gland numbers. Eda signaling succeeded Wnt. Wnt signaling was still active and nascent sweat gland pre-germs were still seen in Eda-null mice, but the pre-germs failed to develop further and the downstream Shh pathway was not activated. When Wnt and Eda were intact but Shh was ablated, germ induction and subsequent duct formation occurred normally, but the final stage of secretory coil formation failed. Thus, sweat gland development shows a relay of regulatory steps initiated by Wnt/β-catenin – itself modulated by Dkk4 – with subsequent participation of Eda and Shh pathways. PMID:25249463

  17. Wnt signaling activation in adipose progenitors promotes insulin-independent muscle glucose uptake

    PubMed Central

    Zeve, Daniel; Seo, Jin; Suh, Jae Myoung; Stenesen, Drew; Tang, Wei; Berglund, Eric D.; Wan, Yihong; Williams, Linda J.; Lim, Ajin; Martinez, Myrna J.; McKay, Renée M.; Millay, Douglas P.; Olson, Eric N.; Graff, Jonathan M.

    2012-01-01

    SUMMARY Adipose tissues provide circulating nutrients and hormones. We present in vivo mouse studies highlighting roles for Wnt signals in both aspects of metabolism. β-catenin activation in PPARγ–expressing fat progenitors (PBCA) decreased fat mass and induced fibrotic replacement of subcutaneous fat specifically. In spite of lipodystrophy, PBCA mice did not develop the expected diabetes and hepatosteatosis, but rather exhibited improved glucose metabolism and normal insulin sensitivity. Glucose uptake was increased in muscle independently of insulin, associated with cell surface translocation of glucose transporters and AMPK activation. Ex vivo assays showed these effects were likely secondary to blood-borne signals since PBCA sera or conditioned media from PBCA fat progenitors enhanced glucose uptake and activated AMPK in muscle cultures. Thus, adipose progenitor Wnt activation dissociates lipodystrophy from dysfunctional metabolism and highlights a fat-muscle endocrine axis, which may represent a potential therapy to lower blood glucose and improve metabolism. PMID:22482731

  18. Mapping the dynamic expression of Wnt11 and the lineage contribution of Wnt11-expressing cells during early mouse development.

    PubMed

    Sinha, Tanvi; Lin, Lizhu; Li, Ding; Davis, Jennifer; Evans, Sylvia; Wynshaw-Boris, Anthony; Wang, Jianbo

    2015-02-15

    Planar cell polarity (PCP) signaling is an evolutionarily conserved mechanism that coordinates polarized cell behavior to regulate tissue morphogenesis during vertebrate gastrulation, neurulation and organogenesis. In Xenopus and zebrafish, PCP signaling is activated by non-canonical Wnts such as Wnt11, and detailed understanding of Wnt11 expression has provided important clues on when, where and how PCP may be activated to regulate tissue morphogenesis. To explore the role of Wnt11 in mammalian development, we established a Wnt11 expression and lineage map with high spatial and temporal resolution by creating and analyzing a tamoxifen-inducible Wnt11-CreER BAC (bacterial artificial chromosome) transgenic mouse line. Our short- and long-term lineage tracing experiments indicated that Wnt11-CreER could faithfully recapitulate endogenous Wnt11 expression, and revealed for the first time that cells transiently expressing Wnt11 at early gastrulation were fated to become specifically the progenitors of the entire endoderm. During mid-gastrulation, Wnt11-CreER expressing cells also contribute extensively to the endothelium in both embryonic and extraembryonic compartments, and the endocardium in all chambers of the developing heart. In contrast, Wnt11-CreER expression in the myocardium starts from late-gastrulation, and occurs in three transient, sequential waves: first in the precursors of the left ventricular (LV) myocardium from E7.0 to 8.0; subsequently in the right ventricular (RV) myocardium from E8.0 to 9.0; and finally in the superior wall of the outflow tract (OFT) myocardium from E8.5 to 10.5. These results provide formal genetic proof that the majority of the endocardium and myocardium diverge by mid-gastrulation in the mouse, and suggest a tight spatial and temporal control of Wnt11 expression in the myocardial lineage to coordinate with myocardial differentiation in the first and second heart field progenitors to form the LV, RV and OFT. The insights gained

  19. Paracrine activation of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth

    PubMed Central

    Ono, Masanori; Yin, Ping; Navarro, Antonia; Moravek, Molly B.; Coon, John S.; Druschitz, Stacy A.; Serna, Vanida Ann; Qiang, Wenan; Brooks, David C.; Malpani, Saurabh S.; Ma, Jiajia; Ercan, Cihangir Mutlu; Mittal, Navdha; Monsivais, Diana; Dyson, Matthew T.; Yemelyanov, Alex; Maruyama, Tetsuo; Chakravarti, Debabrata; Kim, J. Julie; Kurita, Takeshi; Gottardi, Cara J.; Bulun, Serdar E.

    2013-01-01

    Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15–30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas. PMID:24082114

  20. Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules.

    PubMed

    Nakamura, Yukio; de Paiva Alves, Eduardo; Veenstra, Gert Jan C; Hoppler, Stefan

    2016-06-01

    Key signalling pathways, such as canonical Wnt/β-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear β-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of β-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of β-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates β-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence β-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/β-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of β-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/β-catenin target genes subsequent to β-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated β-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts. PMID:27068107

  1. Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

    PubMed Central

    Nakamura, Yukio; de Paiva Alves, Eduardo; Veenstra, Gert Jan C.; Hoppler, Stefan

    2016-01-01

    Key signalling pathways, such as canonical Wnt/β-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear β-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of β-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of β-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates β-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence β-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/β-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of β-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/β-catenin target genes subsequent to β-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated β-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts. PMID:27068107

  2. Non-canonical NFκB activation promotes chemokine expression in podocytes

    PubMed Central

    Valiño-Rivas, Lara; Gonzalez-Lafuente, Laura; Sanz, Ana B.; Ruiz-Ortega, Marta; Ortiz, Alberto; Sanchez-Niño, Maria D.

    2016-01-01

    TNF-like weak inducer of apoptosis (TWEAK) receptor Fn14 is expressed by podocytes and Fn14 deficiency protects from experimental proteinuric kidney disease. However, the downstream effectors of TWEAK/Fn14 in podocytes are poorly characterized. We have explored TWEAK activation of non-canonical NFκB signaling in cultured podocytes. In cultured podocytes, TWEAK increased the expression of the chemokines CCL21, CCL19 and RANTES in a time-dependent manner. The inhibitor of canonical NFκB activation parthenolide inhibited the CCL19 and the early RANTES responses, but not the CCL21 or late RANTES responses. In this regard, TWEAK induced non-canonical NFκB activation in podocytes, characterized by NFκB2/p100 processing to NFκB2/p52 and nuclear migration of RelB/p52. Silencing by a specific siRNA of NIK, the upstream kinase of the non-canonical NFκB pathway, prevented CCL21 upregulation but did not modulate CCL19 or RANTES expression in response to TWEAK, thus establishing CCL21 as a non-canonical NFκB target in podocytes. Increased kidney Fn14 and CCL21 expression was also observed in rat proteinuric kidney disease induced by puromycin, and was localized to podocytes. In conclusion, TWEAK activates the non-canonical NFκB pathway in podocytes, leading to upregulation of CCL21 expression. The non-canonical NFκB pathway should be explored as a potential therapeutic target in proteinuric kidney disease. PMID:27353019

  3. Characterization of Wnt/β-catenin signaling in rhabdomyosarcoma.

    PubMed

    Annavarapu, Srinivas R; Cialfi, Samantha; Dominici, Carlo; Kokai, George K; Uccini, Stefania; Ceccarelli, Simona; McDowell, Heather P; Helliwell, Timothy R

    2013-10-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. β-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/β-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed β-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of β-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of β-catenin, stabilization of the active cytosolic form and nuclear translocation of β-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/β-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target. PMID:23999248

  4. Wnt-11 signaling leads to down-regulation of the Wnt/{beta}-catenin, JNK/AP-1 and NF-{kappa}B pathways and promotes viability in the CHO-K1 cells

    SciTech Connect

    Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka Vainio, Seppo

    2008-08-01

    The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical {beta}-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical {beta}-catenin mediated Wnt signaling but also JNK/AP-1 and NF-{kappa}B signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-{kappa}B pathway. Consistent with the central role of Akt, JNK and NF-{kappa}B in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.

  5. p38 MAP kinase is required for Wnt3a-mediated osterix expression independently of Wnt-LRP5/6-GSK3β signaling axis in dental follicle cells.

    PubMed

    Sakisaka, Yukihiko; Kanaya, Sousuke; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

    2016-09-16

    Wnt3a is a secreted glycoprotein that activates the glycogen synthase kinase-3β (GSK3β)/β-catenin signaling pathway through low-density-lipoprotein receptor-related protein (LRP)5/6 co-receptors. Wnt3a has been implicated in periodontal development and homeostasis, as well as in cementum formation. Recently, we have reported that Wnt3a increases alkaline phosphatase expression through the induction of osterix (Osx) expression in dental follicle cells, a precursor of cementoblasts. However, the molecular mechanism by which Wnt3a induces Osx expression is still unknown. In this study, we show that Wnt3a-induced Osx expression was inhibited in the presence of p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203580 and SB202190) at gene and protein levels, as assessed by real-time PCR and immunocytohistochemistry, respectively. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist binding to LRP5/6 co-receptors, did not influence Wnt3a-mediated p38 MAPK phosphorylation, suggesting that Wnt3a activates p38 MAPK through LRP5/6-independent signaling. On the other hand, pretreatment with p38 MAPK inhibitors had no effects on the phosphorylated status of GSK3β and β-catenin as well as β-catenin nuclear translocation, but inhibited Wnt3a-mediated β-catenin transcriptional activity. These findings suggest that p38 MAPK modulates canonical Wnt signaling at the β-catenin transcriptional level without any crosstalk with the Wnt3a-mediated LRP5/6-GSK3β signaling axis and subsequent β-catenin nuclear translocation. These findings expand our knowledge of the mechanisms controlling periodontal development and regeneration. PMID:27450807

  6. Statins activate the canonical hedgehog-signaling and aggravate non-cirrhotic portal hypertension, but inhibit the non-canonical hedgehog signaling and cirrhotic portal hypertension

    PubMed Central

    Uschner, Frank E.; Ranabhat, Ganesh; Choi, Steve S.; Granzow, Michaela; Klein, Sabine; Schierwagen, Robert; Raskopf, Esther; Gautsch, Sebastian; van der Ven, Peter F. M.; Fürst, Dieter O.; Strassburg, Christian P.; Sauerbruch, Tilman; Mae Diehl, Anna; Trebicka, Jonel

    2015-01-01

    Liver cirrhosis but also portal vein obstruction cause portal hypertension (PHT) and angiogenesis. This study investigated the differences of angiogenesis in cirrhotic and non-cirrhotic PHT with special emphasis on the canonical (Shh/Gli) and non-canonical (Shh/RhoA) hedgehog pathway. Cirrhotic (bile duct ligation/BDL; CCl4 intoxication) and non-cirrhotic (partial portal vein ligation/PPVL) rats received either atorvastatin (15 mg/kg; 7d) or control chow before sacrifice. Invasive hemodynamic measurement and Matrigel implantation assessed angiogenesis in vivo. Angiogenesis in vitro was analysed using migration and tube formation assay. In liver and vessel samples from animals and humans, transcript expression was analyzed using RT-PCR and protein expression using Western blot. Atorvastatin decreased portal pressure, shunt flow and angiogenesis in cirrhosis, whereas atorvastatin increased these parameters in PPVL rats. Non-canonical Hh was upregulated in experimental and human liver cirrhosis and was blunted by atorvastatin. Moreover, atorvastatin blocked the non-canonical Hh-pathway RhoA dependently in activated hepatic steallate cells (HSCs). Interestingly, hepatic and extrahepatic Hh-pathway was enhanced in PPVL rats, which resulted in increased angiogenesis. In summary, statins caused contrary effects in cirrhotic and non-cirrhotic portal hypertension. Atorvastatin inhibited the non-canonical Hh-pathway and angiogenesis in cirrhosis. In portal vein obstruction, statins enhanced the canonical Hh-pathway and aggravated PHT and angiogenesis. PMID:26412302

  7. Aberrant activation of Wnt/β-catenin signaling pathway contributes to the sequential progression of DMBA-induced HBP carcinomas.

    PubMed

    Vidya Priyadarsini, Ramamurthi; Senthil Murugan, Ramalingam; Nagini, Siddavaram

    2012-01-01

    Wnt signaling pathway mediated via interactions between β-catenin and members of the TCF/LEF-1 family of transcription factors plays a central role in the regulation of epithelial cell proliferation, apoptosis, differentiation, adhesion, epithelial-mesenchymal transition, and invasion. Aberrant activation of the Wnt/β-catenin signaling pathway with overexpression of Wnt and Fz, mutations of APC, β-catenin, and axin 1, and cytoplasmic accumulation of β-catenin have been frequently reported in a broad spectrum of human malignancies including oral squamous cell carcinomas (OSCCs). However, changes in the components of the Wnt signaling pathway have not been documented during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis, a paradigm for oral oncogenesis and chemointervention. In this study, we evaluated the role of β-catenin accumulation and Wnt ligands, Wnt signaling members (Fz, Dvl, APC, GSK-3β, axin, and WIF) and the downstream targets of Wnt (cyclin D1, MMP-2, and MMP-9) during the sequential progression of DMBA-induced HBP carcinomas by semi-quantitative RT-PCR and western blot analyses. Our data reveal a correlation between β-catenin accumulation and activation of Wnt signaling, and its downstream effector molecules during the sequential development of HBP carcinomas from hyperplasia to invasive carcinoma through dysplasia. Our data also support a pivotal role for β-catenin in the malignant transition of the HBP. Aberrant Wnt signaling may be a hallmark of progression to malignancy during DMBA-induced HBP carcinogenesis and could be a potential preventive and therapeutic target for suppression of OSCC. PMID:21924667

  8. Continuous tooth generation in mouse is induced by activated epithelial Wnt/β-catenin signaling

    PubMed Central

    Järvinen, Elina; Salazar-Ciudad, Isaac; Birchmeier, Walter; Taketo, Makoto M.; Jernvall, Jukka; Thesleff, Irma

    2006-01-01

    The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized β-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of β-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes. PMID:17121988

  9. Continuous tooth generation in mouse is induced by activated epithelial Wnt/beta-catenin signaling.

    PubMed

    Järvinen, Elina; Salazar-Ciudad, Isaac; Birchmeier, Walter; Taketo, Makoto M; Jernvall, Jukka; Thesleff, Irma

    2006-12-01

    The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized beta-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of beta-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes. PMID:17121988

  10. Ras-activated Dsor1 promotes Wnt signaling in Drosophila development.

    PubMed

    Hall, Eric T; Verheyen, Esther M

    2015-12-15

    Wnt/Wingless (Wg) and Ras-MAPK signaling both play fundamental roles in growth and cell fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras-MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. We find novel evidence that the Drosophila homolog of the dual specificity kinases MEK1/2 (also known as MAP2K1/2), Downstream of Raf1 (Dsor1), is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wg target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila β-catenin). We identify a close physical interaction between Dsor1 and Arm, and find that catalytically inactive Dsor1 causes a reduction in active Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. We find that Ras-Dsor1 activity is independent of upstream activation by EGFR, and instead it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Taken together, our results suggest that there is a new crosstalk pathway between insulin and Wg signaling that is mediated by Dsor1. PMID:26542023

  11. Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: implications for autism spectrum disorders

    PubMed Central

    2014-01-01

    Prostaglandin E2 (PGE2) is a natural lipid-derived molecule that is involved in important physiological functions. Abnormal PGE2 signalling has been associated with pathologies of the nervous system. Previous studies provide evidence for the interaction of PGE2 and canonical Wnt signalling pathways in non-neuronal cells. Since the Wnt pathway is crucial in the development and organization of the brain, the main goal of this study is to determine whether collaboration between these pathways exists in neuronal cell types. We report that PGE2 interacts with canonical Wnt signalling through PKA and PI-3K in neuroectodermal (NE-4C) stem cells. We used time-lapse microscopy to determine that PGE2 increases the final distance from origin, path length travelled, and the average speed of migration in Wnt-activated cells. Furthermore, PGE2 alters distinct cellular phenotypes that are characteristic of Wnt-induced NE-4C cells, which corresponds to the modified splitting behaviour of the cells. We also found that in Wnt-induced cells the level of β-catenin protein was increased and the expression levels of Wnt-target genes (Ctnnb1, Ptgs2, Ccnd1, Mmp9) was significantly upregulated in response to PGE2 treatment. This confirms that PGE2 activated the canonical Wnt signalling pathway. Furthermore, the upregulated genes have been previously associated with ASD. Our findings show, for the first time, evidence for cross-talk between PGE2 and Wnt signalling in neuronal cells, where PKA and PI-3K might act as mediators between the two pathways. Given the importance of PGE2 and Wnt signalling in prenatal development of the nervous system, our study provides insight into how interaction between these two pathways may influence neurodevelopment. PMID:24656144

  12. Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin.

    PubMed

    Chinison, Jessica; Aguilar, Jose S; Avalos, Alan; Huang, Ying; Wang, Zhijun; Cameron, D Joshua; Hao, Jijun

    2016-01-01

    Abnormal activation of canonical Wnt/β-catenin signaling is implicated in many diseases including cancer. As a result, therapeutic agents that disrupt this signaling pathway have been highly sought after. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and it has a broad spectrum of biological functions. Here we show that triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin. In addition, triptonide treatment robustly rescued the zebrafish "eyeless" phenotype induced by GSK-3β antagonist 6-bromoindirubin-30-oxime (BIO) for Wnt signaling activation during embryonic gastrulation. Finally, triptonide effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide. PMID:27596363

  13. Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin

    PubMed Central

    Chinison, Jessica; Aguilar, Jose S.; Avalos, Alan; Huang, Ying; Wang, Zhijun; Cameron, D. Joshua; Hao, Jijun

    2016-01-01

    Abnormal activation of canonical Wnt/β-catenin signaling is implicated in many diseases including cancer. As a result, therapeutic agents that disrupt this signaling pathway have been highly sought after. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and it has a broad spectrum of biological functions. Here we show that triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin. In addition, triptonide treatment robustly rescued the zebrafish “eyeless” phenotype induced by GSK-3β antagonist 6-bromoindirubin-30-oxime (BIO) for Wnt signaling activation during embryonic gastrulation. Finally, triptonide effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide. PMID:27596363

  14. The Wnt pathway limits BMP signaling outside of the germline stem cell niche in Drosophila ovaries.

    PubMed

    Mottier-Pavie, Violaine I; Palacios, Victor; Eliazer, Susan; Scoggin, Shane; Buszczak, Michael

    2016-09-01

    The mechanisms that modulate and limit the signaling output of adult stem cell niches remain poorly understood. To gain further insights into how these microenvironments are regulated in vivo, we performed a candidate gene screen designed to identify factors that restrict BMP signal production to the cap cells that comprise the germline stem cell (GSC) niche of Drosophila ovaries. Through these efforts, we found that disruption of Wnt4 and components of the canonical Wnt pathway results in a complex germ cell phenotype marked by an expansion of GSC-like cells, pre-cystoblasts and cystoblasts in young females. This phenotype correlates with an increase of decapentaplegic (dpp) mRNA levels within escort cells and varying levels of BMP responsiveness in the germline. Further genetic experiments show that Wnt4, which exhibits graded expression in somatic cells of germaria, activates the Wnt pathway in posteriorly positioned escort cells. The activation of the Wnt pathway appears to be limited by the BMP pathway itself, as loss of Mad in escort cells results in the expansion of Wnt pathway activation. Wnt pathway activity changes within germaria during the course of aging, coincident with changes in dpp production. These data suggest that mutual antagonism between the BMP and Wnt pathways in somatic cells helps to regulate germ cell differentiation. PMID:27364467

  15. Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities.

    PubMed

    Zhang, Tianyi; Liao, Yang; Hsu, Fu-Ning; Zhang, Robin; Searle, Jennifer S; Pei, Xun; Li, Xuan; Ryoo, Hyung Don; Ji, Jun-Yuan; Du, Wei

    2014-05-01

    Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor. PMID:24809668

  16. Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt.

    PubMed

    Shen, M; Wang, L; Wang, B; Wang, T; Yang, G; Shen, L; Wang, T; Guo, X; Liu, Y; Xia, Y; Jia, L; Wang, X

    2014-01-01

    Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl(-) currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel by 4,4'-diisothiocya-natostilbene-2,2'-disulfonic acid (DIDS), a non-selective Cl(-) channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl(-) channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl(-) channel blockers against ER stress-associated cardiac anomalies. PMID:25412307

  17. Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt

    PubMed Central

    Shen, M; Wang, L; Wang, B; Wang, T; Yang, G; Shen, L; Wang, T; Guo, X; Liu, Y; Xia, Y; Jia, L; Wang, X

    2014-01-01

    Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl− currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl− channel by 4,4'-diisothiocya-natostilbene-2,2'-disulfonic acid (DIDS), a non-selective Cl− channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl− channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl− channel blockers against ER stress-associated cardiac anomalies. PMID:25412307

  18. Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation

    PubMed Central

    Kandyba, Eve; Leung, Yvonne; Chen, Yi-Bu; Widelitz, Randall; Chuong, Cheng-Ming; Kobielak, Krzysztof

    2013-01-01

    Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell–dermal papilla-based epithelial–mesenchymal interaction layer and the hair follicle–intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation. PMID:23292934

  19. Matrix Rigidity Activates Wnt Signaling through Down-regulation of Dickkopf-1 Protein*

    PubMed Central

    Barbolina, Maria V.; Liu, Yiuying; Gurler, Hilal; Kim, Mijung; Kajdacsy-Balla, Andre A.; Rooper, Lisa; Shepard, Jaclyn; Weiss, Michael; Shea, Lonnie D.; Penzes, Peter; Ravosa, Matthew J.; Stack, M. Sharon

    2013-01-01

    Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling. PMID:23152495

  20. Impacts of non-canonical El Niño patterns on Atlantic hurricane activity

    NASA Astrophysics Data System (ADS)

    Larson, Sarah; Lee, Sang-Ki; Wang, Chunzai; Chung, Eui-Seok; Enfield, David

    2012-07-01

    The impact of non-canonical El Niño patterns, typically characterized by warmer than normal sea surface temperatures (SSTs) in the central tropical Pacific, on Atlantic tropical cyclone (TC) is explored by using composites of key Atlantic TC indices and tropospheric vertical wind shear over the Atlantic main development region (MDR). The highlight of our major findings is that, while the canonical El Niño pattern has a strong suppressing influence on Atlantic TC activity, non-canonical El Niño patterns considered in this study, namely central Pacific warming, El Niño Modoki, positive phase Trans-Niño, and positive phase Pacific meridional mode, all have insubstantial impact on Atlantic TC activity. This result becomes more conclusive when the impact of MDR SST is removed from the Atlantic TC indices and MDR wind shear by using the method of linear regression. Further analysis suggests that the tropical Pacific SST anomalies associated with the non-canonical El Niño patterns are not strong enough to cause a substantial warming of the tropical troposphere in the Atlantic region, which is the key factor that increases the wind shear and atmospheric static stability over the MDR. During the recent decades, the non-canonical El Niños have been more frequent while the canonical El Niño has been less frequent. If such a trend continues in the future, it is expected that the suppressing effect of El Niño on Atlantic TC activity will diminish and thus the MDR SST will play a more important role in controlling Atlantic TC activity in the coming decades.

  1. Impacts of non-canonical El Niño patterns on Atlantic hurricane activity

    NASA Astrophysics Data System (ADS)

    Larson, S.; Lee, S.; Wang, C.; Chung, E.; Enfield, D. B.

    2012-12-01

    The impact of non-canonical El Niño patterns, typically characterized by warmer than normal sea surface tempera- tures (SSTs) in the central tropical Pacific, on Atlantic tropical cyclone (TC) is explored by using composites of key Atlantic TC indices and tropospheric vertical wind shear over the Atlantic main development region (MDR). The highlight of our major findings is that, while the canonical El Niño pattern has a strong suppressing influence on Atlantic TC activity, non-canonical El Niño patterns con- sidered in this study, namely central Pacific warming, El Niño Modoki, positive phase Trans-Niño, and positive phase Pacific meridional mode, all have insubstantial impact on Atlantic TC activity. This result becomes more conclu- sive when the impact of MDR SST is removed from the Atlantic TC indices and MDR wind shear by using the method of linear regression. Further analysis suggests that the tropical Pacific SST anomalies associated with the non- canonical El Niño patterns are not strong enough to cause a substantial warming of the tropical troposphere in the Atlantic region, which is the key factor that increases the wind shear and atmospheric static stability over the MDR. During the recent decades, the non-canonical El Niños have been more frequent while the canonical El Niño has been less frequent. If such a trend continues in the future, it is expected that the suppressing effect of El Niño on Atlantic TC activity will diminish and thus the MDR SST will play a more important role in controlling Atlantic TC activity in the coming decades.

  2. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation

    PubMed Central

    Xavier, Guilherme M.; Patist, Amanda L.; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T.; Pedro Martinez-Barbera, Juan; Thavaraj, Selvam; Cobourne, Martyn T.; Andoniadou, Cynthia L.

    2015-01-01

    In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma. PMID:26411543

  3. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

    PubMed

    Xavier, Guilherme M; Patist, Amanda L; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T; Martinez-Barbera, Juan Pedro; Thavaraj, Selvam; Cobourne, Martyn T; Andoniadou, Cynthia L

    2015-01-01

    In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma. PMID:26411543

  4. Genomic insights into WNT/β-catenin signaling

    PubMed Central

    Rosenbluh, Joseph; Wang, Xiaoxing; Hahn, William C.

    2014-01-01

    The canonical WNT pathway regulates the stability of the proto-oncogene β-catenin and is aberrantly activated in many cancer types. Studies in a wide range of experimental models confirm that β-catenin activity is required for tumor initiation in cancers where this pathway is deregulated. However, to date this pathway has proven to be challenging to target therapeutically. Moreover, several lines of evidence suggest that other components and regulators of β-catenin exist. Here we will describe recent structural and functional studies describing genomic alterations and new regulators of β-catenin that lead to aberrant activation of the WNT/β-catenin pathway. These findings provide new insights into the biology of WNT/β-catenin signaling and suggest potential therapeutic opportunities. PMID:24365576

  5. Monitoring Wnt/β-Catenin Signaling in Skin.

    PubMed

    Ku, Amy T; Miao, Qi; Nguyen, Hoang

    2016-01-01

    Wnt signaling through β-catenin plays a crucial role in skin development and homeostasis. Disruption or hyperactivation of this pathway results in skin defects and diseases (Lim and Nusse, Cold Spring Harb Perspect Biol 5(2), 2013). Monitoring Wnt signaling in skin under normal and abnormal conditions is therefore critical to understand the role of this pathway in development and homeostasis.In this chapter, we provide methods to detect Wnt/β-catenin (canonical) signaling in the skin. We present a comprehensive list of Wnt reporter mice and detail the processing of skin tissue to detect reporter genes. From this list, we focus on the three most recent lines that, according to reports, are the most sensitive in skin. Additionally, we describe a protocol to detect nuclear β-catenin, a hallmark of active Wnt signaling, although this technique should be used with caution due to its limited sensitivity. The techniques outlined below will be useful for detecting active Wnt signaling in skin. PMID:27590159

  6. Fezf2 promotes neuronal differentiation through localised activation of Wnt/β-catenin signalling during forebrain development

    PubMed Central

    Zhang, Siwei; Li, Jingjing; Lea, Robert; Vleminckx, Kris; Amaya, Enrique

    2014-01-01

    Brain regionalisation, neuronal subtype diversification and circuit connectivity are crucial events in the establishment of higher cognitive functions. Here we report the requirement for the transcriptional repressor Fezf2 for proper differentiation of neural progenitor cells during the development of the Xenopus forebrain. Depletion of Fezf2 induces apoptosis in postmitotic neural progenitors, with concomitant reduction in forebrain size and neuronal differentiation. Mechanistically, we found that Fezf2 stimulates neuronal differentiation by promoting Wnt/β-catenin signalling in the developing forebrain. In addition, we show that Fezf2 promotes activation of Wnt/β-catenin signalling by repressing the expression of two negative regulators of Wnt signalling, namely lhx2 and lhx9. Our findings suggest that Fezf2 plays an essential role in controlling when and where neuronal differentiation occurs within the developing forebrain and that it does so by promoting local Wnt/β-catenin signalling via a double-repressor model. PMID:25468942

  7. Conserved POU/OCT- and GATA-binding sites in 5'-flanking promoter region of mammalian WNT8B orthologs.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-05-01

    WNT family members are secreted-type glycoproteins regulating cell fate, planar cell polarity, cell adhesion, and cell movement. WNT signals are context-dependently transduced to the canonical pathway for the transcriptional up-regulation of MYC, CCND1, FGF20, JAG1, WISP1 and DKK1 genes, and also to the non-canonical pathway for the activation of RHOA, JNK, PKC, NFAT and NLK signaling cascades. We cloned and characterized the wild-type human WNT8B, while another group the aberrant human WNT8B with Gly230Ala and Arg284Leu amino-acid substitutions. Although WNT8B is undetectable in normal adult tissues by using Northern blot analyses, WNT8B is expressed in gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, and embryonal tumors. Here, comparative integromics on WNT8B orthologs were investigated by using bioinformatics (Techint) and human intelligence (Humint). Cow Wnt8b gene was identified within NW_001494361.1 genome sequence. Predicted sequence XM_582222.3 was an artificial cow Wnt8b with aberrant prediction for the first exon. Cow Wnt8b complete coding sequence was found to encode a 350-amino-acid protein, which showed 96.9% total-amino-acid identity with human WNT8B. Comparative proteomics revealed that N-terminal signal peptide, 22 Cys residues, two Asn-linked glycosylation sites, Gly230, and Arg284 of human WNT8B were conserved among mammalian WNT8B orthologs. Comparative genomics revealed that POU/OCT- and GATA-binding sites in the 5'-flanking promoter region were conserved among human, chimpanzee, cow, mouse, and rat WNT8B orthologs. In silico expression analyses revealed that human WNT8B was expressed in embryoid body derived from embryonic stem (ES) cells, hepatocyte progenitors derived from ES cells, fetal brain, diffuse-type gastric cancer, colorectal cancer, prostate cancer, and ovarian fibrotheoma. Based on the expression profiles of POU and GATA family transcription factors, it was revealed that WNT8B expression in hepatocyte

  8. Characterization and development of novel small-molecules inhibiting GSK3 and activating Wnt signaling†

    PubMed Central

    Zou, Haixia; Semenov, Mikhail V.; Moshinsky, Deborah; He, Xi; Huang, Haigen; Li, Song; Quan, Junmin; Yang, Zhen

    2010-01-01

    Glycogen synthase kinase 3 (GSK3) is an essential component of the Wnt signaling pathway and plays important roles in regulating cell proliferation, differentiation, and apoptosis. As GSK3 is abnormally upregulated in several diseases including type II diabetes, Alzheimer’s disease and cancer, it has been regarded as a potential drug target. During zebrafish development, inhibition of GSK3 leads to ectopic activation of the Wnt pathway, resulting in a headless embryo. Using this phenotype as an assay we screened a chemical library of 4000 compounds and identified one novel compound, 3F8, which specifically inhibits eye and forebrain formation in zebrafish embryos, resembling a typical Wnt overexpression phenotype. Cell reporter assays, chemical informatics analysis and in vitro kinase experiments revealed that 3F8 is a selective GSK3 inhibitor, which is more potent than SB216763, a commonly used GSK3 inhibitor. Based on the structure of 3F8, a new generation of compounds inhibiting GSK3 was synthesized and validated by biological assays. Together, 3F8 and its derivatives could be useful as new reagents and potential therapeutic candidates for GSK3 related diseases. PMID:19823752

  9. Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-catenin-dependent mechanism.

    PubMed

    Cawthorn, William P; Bree, Adam J; Yao, Yao; Du, Baowen; Hemati, Nahid; Martinez-Santibañez, Gabriel; MacDougald, Ormond A

    2012-02-01

    Wnt10b is an established regulator of mesenchymal stem cell (MSC) fate that inhibits adipogenesis and stimulates osteoblastogenesis, thereby impacting bone mass in vivo. However, downstream mechanisms through which Wnt10b exerts these effects are poorly understood. Moreover, whether other endogenous Wnt ligands also modulate MSC fate remains to be fully addressed. In this study, we identify Wnt6 and Wnt10a as additional Wnt family members that, like Wnt10b, are downregulated during development of white adipocytes in vivo and in vitro, suggesting that Wnt6 and/or Wnt10a may also inhibit adipogenesis. To assess the relative activities of Wnt6, Wnt10a and Wnt10b to regulate mesenchymal cell fate, we used gain- and loss-of function approaches in bipotential ST2 cells and in 3T3-L1 preadipocytes. Enforced expression of Wnt10a stabilizes β-catenin, suppresses adipogenesis and stimulates osteoblastogenesis to a similar extent as Wnt10b, whereas stable expression of Wnt6 has a weaker effect on these processes than Wnt10a or Wnt10b. In contrast, knockdown of endogenous Wnt6 is associated with greater preadipocyte differentiation and impaired osteoblastogenesis than knockdown of Wnt10a or Wnt10b, suggesting that, among these Wnt ligands, Wnt6 is the most potent endogenous regulator of MSC fate. Finally, we show that knockdown of β-catenin completely prevents the inhibition of adipogenesis and stimulation of osteoblast differentiation by Wnt6, Wnt10a or Wnt10b. Potential mechanisms whereby Wnts regulate fate of MSCs downstream of β-catenin are also investigated. In conclusion, this study identifies Wnt10a and Wnt6 as additional regulators of MSC fate and demonstrates that mechanisms downstream of β-catenin are required for Wnt6, Wnt10a and Wnt10b to influence differentiation of mesenchymal precursors. PMID:21872687

  10. Monitoring Wnt Signaling in Zebrafish Using Fluorescent Biosensors.

    PubMed

    Facchinello, Nicola; Schiavone, Marco; Vettori, Andrea; Argenton, Francesco; Tiso, Natascia

    2016-01-01

    In this chapter, we are presenting methods to monitor and quantify in vivo canonical Wnt signaling activities at single-cell resolution in zebrafish. Our technology is based on artificial enhancers, obtained by polymerization of TCF binding elements, cloned upstream to ubiquitous or tissue-specific promoters. The different promoter/enhancer combinations are used to drive fluorescent protein reporter constructs integrated in the zebrafish germline by microinjection of fertilized zebrafish eggs. Fish with a single integration site are selected by Mendelian analysis of fluorescent carriers, and heterozygous offspring are used to monitor and quantify canonical Wnt activities. Open source public domain software such as ImageJ/Fiji is used to calculate the integrated densities in the region of interest and compare the effect of experimental conditions on control and treated animals. PMID:27590154

  11. The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

    PubMed Central

    Kopan, Raphael; Ilagan, Ma. Xenia G.

    2009-01-01

    Notch signaling regulates many aspects of metazoan development and tissue renewal. Accordingly, misregulation or loss of Notch signaling underlies multiple human disorders, from developmental syndromes to adult onset diseases and cancer. Notch receptor activation is irreversible as it involves proteolysis-mediated release of the Notch intracellular domain, translocation to the nucleus, and association with a DNA-bound protein. Even though each Notch molecule signals only once without amplification by secondary messenger cascades, Notch signaling is remarkably robust in most tissues. In this review, we highlight the recent studies that reveal new molecular details involved in regulating ligand-mediated activation, receptor proteolysis and target selection. PMID:19379690

  12. How the Wnt signaling pathway protects from neurodegeneration: the mitochondrial scenario.

    PubMed

    Arrázola, Macarena S; Silva-Alvarez, Carmen; Inestrosa, Nibaldo C

    2015-01-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as "mitochondrial dynamics" is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration. PMID:25999816

  13. How the Wnt signaling pathway protects from neurodegeneration: the mitochondrial scenario

    PubMed Central

    Arrázola, Macarena S.; Silva-Alvarez, Carmen; Inestrosa, Nibaldo C.

    2015-01-01

    Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as “mitochondrial dynamics” is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration. PMID:25999816

  14. Two mutations in human BICC1 resulting in Wnt pathway hyperactivity associated with cystic renal dysplasia.

    PubMed

    Kraus, Marine R-C; Clauin, Séverine; Pfister, Yvan; Di Maïo, Massimo; Ulinski, Tim; Constam, Daniel; Bellanné-Chantelot, Christine; Grapin-Botton, Anne

    2012-01-01

    Bicaudal C homologue 1 (Bicc1) knockout in mice causes polycystic kidney disease and pancreas development defects, including a reduction in insulin-producing β-cells and ensuing diabetes. We therefore screened 137 patients with renal abnormalities or association of early-onset diabetes and renal disease for genetic alterations in BICC1. We identified two heterozygous mutations, one nonsense in the first K Homology (KH) domain and one missense in the sterile alpha motif (SAM) domain. In mice, Bicc1 blocks canonical Wnt signaling, mostly via its SAM domain. We show that the human BICC1, similar to its mouse counterpart, blocks canonical Wnt signaling. The nonsense mutation identified results in a complete loss of Wnt inhibitory activity. The point mutation in the SAM domain has a similar effect to a complete SAM domain deletion, resulting in a 22% loss of activity. PMID:21922595

  15. Identification and characterization of a small-molecule inhibitor of Wnt signaling in glioblastoma cells.

    PubMed

    De Robertis, Alessandra; Valensin, Silvia; Rossi, Marco; Tunici, Patrizia; Verani, Margherita; De Rosa, Antonella; Giordano, Cinzia; Varrone, Maurizio; Nencini, Arianna; Pratelli, Carmela; Benicchi, Tiziana; Bakker, Annette; Hill, Jeffrey; Sangthongpitag, Kanda; Pendharkar, Vishal; Liu, Boping; Ng, Fui Mee; Then, Siew Wen; Jing Tai, Shi; Cheong, Seong-Moon; He, Xi; Caricasole, Andrea; Salerno, Massimiliano

    2013-07-01

    Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased β-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics. PMID:23619303

  16. Identification and characterization of a small molecule inhibitor of WNT signaling in glioblastoma cells

    PubMed Central

    De Robertis, Alessandra; Valensin, Silvia; Rossi, Marco; Tunici, Patrizia; Verani, Margherita; De Rosa, Antonella; Giordano, Cinzia; Varrone, Maurizio; Nencini, Arianna; Pratelli, Carmela; Benicchi, Tiziana; Bakker, Annette; Hill, Jeffrey; Sangthongpitag, Kanda; Pendharkar, Vishal; Boping, Liu; Mee, Ng Fui; Wen, Then Siew; Jing, Tai Shi; Cheong, Seong-Moon; He, Xi; Caricasole, Andrea; Salerno, Massimiliano

    2013-01-01

    Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than one year. Although canonical WNT pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-CATENIN or AXIN proteins), an increasing number of studies suggest that elevated WNT signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of WNT signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to WNT inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical WNT signaling and associated proliferative responses in GBM cells. Here we show that the small molecule SEN461 inhibits the canonical WNT signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced AXIN stabilization, increased β-CATENIN phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized WNT signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small molecule-mediated inhibition of WNT signaling may be a potential approach for GBM therapeutics. PMID:23619303

  17. Activation of B cells by non-canonical helper signals

    PubMed Central

    Cerutti, Andrea; Cols, Montserrat; Puga, Irene

    2012-01-01

    Cognate interaction between T and B lymphocytes of the adaptive immune system is essential for the production of high-affinity antibodies against microbes, and for the establishment of long-term immunological memory. Growing evidence shows that—in addition to presenting antigens to T and B cells—macrophages, dendritic cells and other cells of the innate immune system provide activating signals to B cells, as well as survival signals to antibody-secreting plasma cells. Here, we discuss how these innate immune cells contribute to the induction of highly diversified and temporally sustained antibody responses, both systemically and at mucosal sites of antigen entry. PMID:22868664

  18. CAFET Algorithm Reveals Wnt/PCP Signature in Lung Squamous Cell Carcinoma

    PubMed Central

    Hu, Yue; Galkin, Anna V.; Wu, Chunlei; Reddy, Venkateshwar; Su, Andrew I.

    2011-01-01

    We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC) samples and developed a new algorithm called Coverage Analysis with Fisher’s Exact Test (CAFET) to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC) and adenocarcinoma (AC) subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP) pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis. PMID:22016777

  19. Wnt/Catenin Signaling in Adult Stem Cell Physiology and Disease

    PubMed Central

    Ring, Alexander; Kim, Yong-Mi

    2014-01-01

    Wnt signaling plays an important role in development and disease. In this review we focus on the role of the canonical Wnt signaling pathway in somatic stem cell biology and its critical role in tissue homeostasis. We present current knowledge how Wnt/β-catenin signaling affects tissue stem cell behavior in various organ systems, including the gut, mammary gland, the hematopoietic and nervous system. We discuss evidence that canonical Wnt signaling can both maintain potency and an undifferentiated state as well as cause differentiation in somatic stem cells, depending on the cellular and environmental context. Based on studies by our lab and others, we will attempt to explain the dichotomous behavior of this signaling pathway in determining cell fate decisions and put special emphasis on the interaction of β-catenin with two highly homologous co-activator proteins, CBP and p300, to shed light on the their differential role in the outcome of Wnt/β-catenin signaling. Furthermore, we review current knowledge regarding the aberrant regulation of Wnt/β-catenin signaling in cancer biology, particularly its pivotal role in the context of cancer stem cells. Finally, we discuss data demonstrating that small molecule modulators of the β-catenin/co-activator interaction can be used to shift the balance between undifferentiated proliferation and differentiation, which potentially presents a promising therapeutic approach to stem cell based disease mechanisms. PMID:24825509

  20. Wnt and the Wnt signaling pathway in bone development and disease

    PubMed Central

    Wang, Yiping; Li, Yi-Ping; Paulson, Christie; Shao, Jian-Zhong; Zhang, Xiaoling; Wu, Mengrui; Chen, Wei

    2014-01-01

    Wnt signaling affects both bone modeling, which occurs during development, and bone remodeling, which is a lifelong process involving tissue renewal. Wnt signals are especially known to affect the differentiation of osteoblasts. In this review, we summarize recent advances in understanding the mechanisms of Wnt signaling, which is divided into two major branches: the canonical pathway and the noncanonical pathway. The canonical pathway is also called the Wnt/β-catenin pathway. There are two major noncanonical pathways: the Wnt-planar cell polarity pathway (Wnt-PCP pathway) and the Wnt-calcium pathway (Wnt-Ca2+ pathway). This review also discusses how Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists affect both the bone modeling and bone remodeling processes. We also review the role of Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists in bone as demonstrated in mouse models. Disrupted Wnt signaling is linked to several bone diseases, including osteoporosis, van Buchem disease, and sclerosteosis. Studying the mechanism of Wnt signaling and its interactions with other signaling pathways in bone will provide potential therapeutic targets to treat these bone diseases. PMID:24389191

  1. Overexpression of Wnt-1 in thyrocytes enhances cellular growth but suppresses transcription of the thyroperoxidase gene via different signaling mechanisms.

    PubMed

    Kim, Won Bae; Lewis, Christopher J; McCall, Kelly D; Malgor, Ramiro; Kohn, Aimee D; Moon, Randall T; Kohn, Leonard D

    2007-04-01

    Wnt binding to cell surface receptors can activate a 'canonical' pathway that increases cellular beta-catenin or a 'noncanonical' Ca(++) pathway which can increase protein kinase C (PKC) activity. Although components of both Wnt/beta-catenin-signaling pathways exist in thyrocytes, their biological role is largely unknown. In evaluating the biological role of Wnt signaling in differentiated FRTL-5 thyroid cells, we showed that TSH increased canonical Wnt-1 but, surprisingly, decreased the active form of beta-catenin. Transient overexpression of Wnt-1 or beta-catenin in FRTL-5 cells increased active beta-catenin (ABC), decreased thyroperoxidase (TPO) mRNA, and suppressed TPO-promoter activity. The target of beta-catenin suppressive action was a consensus T cell factor/lymphoid enhancing factor (TCF/LEF)-binding site 5'-A/T A/T CAAAG-3', -137 to -129 bp on the rat TPO promoter. beta-Catenin overexpression significantly increased complex formation between beta-catenin/TCF-1 and an oligonucleotide containing the TCF/LEF sequence, suggesting that the beta-catenin/TCF-1 complex acts as a transcriptional repressor of the TPO gene. Stable over-expression of Wnt-1 in FRTL-5 cells significantly increased the growth rate without increasing beta-catenin levels. Increased growth was blunted by a PKC inhibitor, staurosporin. Wnt-1 overexpression increased serine phosphorylation, without affecting tyrosine phosphorylation, of signal transducers and activators of transcription 3 (STAT3) protein. In addition, these final results suggest that TSH-induced increase in Wnt-1 levels in thyrocytes contributes to enhanced cellular growth via a PKC pathway that increases STAT3 serine phosphorylation and activation, whereas TSH-induced decrease in activation of beta-catenin simultaneously relieves transcriptional suppression of TPO. We hypothesize that Wnt signaling contributes to the ability of TSH to simultaneously increase cell growth and functional, thyroid-specific, gene expression

  2. Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons*

    PubMed Central

    Godoy, Juan A.; Arrázola, Macarena S.; Ordenes, Daniela; Silva-Alvarez, Carmen; Braidy, Nady; Inestrosa, Nibaldo C.

    2014-01-01

    The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca2+), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca2+ signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders. PMID:25336659

  3. Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers.

    PubMed

    Chang, Chung-Hsing; Jiang, Ting-Xin; Lin, Chih-Min; Burrus, Laura W; Chuong, Cheng-Ming; Widelitz, Randall

    2004-02-01

    Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds. PMID:15037317

  4. WNT-3A Regulates an Axin1/NRF2 Complex That Regulates Antioxidant Metabolism in Hepatocytes

    PubMed Central

    Rada, Patricia; Rojo, Ana I.; Offergeld, Anika; Feng, Gui Jie; Velasco-Martín, Juan P.; González-Sancho, José Manuel; Valverde, Ángela M.; Dale, Trevor; Regadera, Javier

    2015-01-01

    Abstract Aims: Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. Results: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. Innovation: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. Conclusion: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism. Antioxid. Redox Signal. 22, 555–571. PMID:25336178

  5. Wnt signaling and the activation of myogenesis in mammals.

    PubMed

    Cossu, G; Borello, U

    1999-12-15

    In the amniote embryos, specification of skeletal myoblasts occurs in the paraxial mesoderm in response to a number of signaling molecules produced by neighboring tissues such as neural tube, notochord and dorsal ectoderm. Candidate molecules for this complex signaling activity include Sonic hedgehog, Wnts and Noggin as positive activators and BMP4 as a possible inhibitor. Recently, the receptors and the post-receptor pathways for Sonic hedgehog and Wnts have been characterized, and this has opened up the possibility of linking these signaling events to the activation of myogenic regulatory factor genes such as Myf5 and MyoD and functionally related genes such as Pax3. Here we focus on the role of Wnts, their putative receptors Frizzled and the soluble antagonist Frzb1 in regulating mammalian myogenesis. Although it is becoming evident that the signaling downstream of Frizzled receptors is much more complex than anticipated, it is conceivable that it may lead to transcriptional activation of Myf5 and MyoD and to initiation of myogenesis. However, the fact that both Wnts and Sonic hedgehog have a strong effect on cell proliferation and survival suggests that they may contribute to the overall process of myogenesis by a combination of these different biological activities. PMID:10601008

  6. Wnt signaling and the activation of myogenesis in mammals.

    PubMed Central

    Cossu, G; Borello, U

    1999-01-01

    In the amniote embryos, specification of skeletal myoblasts occurs in the paraxial mesoderm in response to a number of signaling molecules produced by neighboring tissues such as neural tube, notochord and dorsal ectoderm. Candidate molecules for this complex signaling activity include Sonic hedgehog, Wnts and Noggin as positive activators and BMP4 as a possible inhibitor. Recently, the receptors and the post-receptor pathways for Sonic hedgehog and Wnts have been characterized, and this has opened up the possibility of linking these signaling events to the activation of myogenic regulatory factor genes such as Myf5 and MyoD and functionally related genes such as Pax3. Here we focus on the role of Wnts, their putative receptors Frizzled and the soluble antagonist Frzb1 in regulating mammalian myogenesis. Although it is becoming evident that the signaling downstream of Frizzled receptors is much more complex than anticipated, it is conceivable that it may lead to transcriptional activation of Myf5 and MyoD and to initiation of myogenesis. However, the fact that both Wnts and Sonic hedgehog have a strong effect on cell proliferation and survival suggests that they may contribute to the overall process of myogenesis by a combination of these different biological activities. PMID:10601008

  7. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    SciTech Connect

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  8. Wnt Signaling in Cell Motility and Invasion: Drawing Parallels between Development and Cancer.

    PubMed

    Sedgwick, Alanna E; D'Souza-Schorey, Crislyn

    2016-01-01

    The importance of canonical and non-canonical Wnt signal transduction cascades in embryonic development and tissue homeostasis is well recognized. The aberrant activation of these pathways in the adult leads to abnormal cellular behaviors, and tumor progression is frequently a consequence. Here we discuss recent findings and analogies between Wnt signaling in developmental processes and tumor progression, with a particular focus on cell motility and matrix invasion and highlight the roles of the ARF (ADP-Ribosylation Factor) and Rho-family small GTP-binding proteins. Wnt-regulated signal transduction from cell surface receptors, signaling endosomes and/or extracellular vesicles has the potential to profoundly influence cell movement, matrix degradation and paracrine signaling in both development and disease. PMID:27589803

  9. MiR-328 promotes glioma cell invasion via SFRP1-dependent Wnt-signaling activation.

    PubMed

    Delic, Sabit; Lottmann, Nadine; Stelzl, Anja; Liesenberg, Franziska; Wolter, Marietta; Götze, Silke; Zapatka, Marc; Shiio, Yuzuru; Sabel, Michael C; Felsberg, Jörg; Reifenberger, Guido; Riemenschneider, Markus J

    2014-01-01

    Background Diffusely infiltrative growth of human astrocytic gliomas is one of the major obstacles to successful tumor therapy. Thorough insights into the molecules and pathways signaling glioma cell invasion thus appear of major relevance for the development of targeted and individualized therapies. By miRNA expression profiling of microdissected human tumor biopsy specimens we identified miR-328 as one of the main miRNAs upregulated in invading glioma cells in vivo and further investigated its role in glioma pathogenesis. Methods We employed miRNA mimics and inhibitors to functionally characterize miR-328, 3' untranslated region luciferase assays, and T-cell factor/lymphoid enhancer factor reporter assays to pinpoint miR-328 targets and signaling pathways, and analyzed miR-328 expression in a large panel of gliomas. Results First, we corroborated the invasion-promoting role of miR-328 in A172 and TP365MG glioma cells. Secreted Frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signaling, was then pinpointed as a direct miR-328 target. SFRP1 expression is of prognostic relevance in gliomas with reduced expression, being associated with significantly lower overall patient survival in both the Repository of Molecular Brain Neoplasia Data (REMBRANDT) and The Cancer Genome Atlas. Of note, miR-328 regulated both SFRP1 protein expression levels and Wnt signaling pathway activity. Finally, in human glioma tissues miR-328 appeared to account for the downregulation of SFRP1 preferentially in lower-grade astrocytic gliomas and was inversely related to SFRP1 promoter hypermethylation. Conclusion Taken together, we report on a novel molecular miR-328-dependent mechanism that via SFRP1 inhibition and Wnt activation contributes to the infiltrative glioma phenotype at already early stages of glioma progression, with unfavorable prognostic implications for the final outcome of the disease. PMID:24305703

  10. Identification of Wnt/β-catenin signaling pathway in dermal papilla cells of human scalp hair follicles: TCF4 regulates the proliferation and secretory activity of dermal papilla cell.

    PubMed

    Xiong, Ya; Liu, Yi; Song, Zhiqiang; Hao, Fei; Yang, Xichuan

    2014-01-01

    It is clear that the dermal papilla cell (DPC), which is located at the bottom of the hair follicle, is a special mesenchymal component, and it plays a leading role in regulating hair follicle development and periodic regeneration. Recent studies showed that the Wnt signaling pathway through β-catenin (canonical Wnt signaling pathway) is an essential component in maintaining the hair-inducing activity of the dermal papilla and growth of hair papilla cells. However, the intrinsic pathways and regulating mechanism are largely unknown. In the previous work, we constructed a cDNA subtractive library of DPC and first found that the TCF4 gene, as a key factor of Wnt signaling pathway, was expressed as the upregulated gene of the hair follicle in low-passage DPC. This study was to explore the role of TCF4 in regulating the proliferation and secretory activity of DPC. We constructed a pcDNA3.0-TCF4 expression vector and transfected it into DPC to achieve stable expression by bangosome 2000. Furthermore, we used the method of chemosynthesis to synthesize three pairs of TCF4 siRNA and transfected them into DPC. Meanwhile, we compared the transfection group and non-transfection group. We first proposed that there was expression difference in TCF4 in DPC under different biological condition. This study may have a high impact on the molecular mechanism of follicular lesions and provide a new vision for the treatment of clinic diseases. PMID:24354472

  11. TWEAK favors phosphate-induced calcification of vascular smooth muscle cells through canonical and non-canonical activation of NFκB

    PubMed Central

    Hénaut, L; Sanz, A B; Martin-Sanchez, D; Carrasco, S; Villa-Bellosta, R; Aldamiz-Echevarria, G; Massy, Z A; Sanchez-Nino, M D; Ortiz, A

    2016-01-01

    Vascular calcification (VC) is associated with increased cardiovascular mortality in aging, chronic kidney disease (CKD), type 2 diabetes mellitus (T2DM) and atherosclerosis. TNF-like weak inducer of apoptosis (TWEAK) recently emerged as a new biomarker for the diagnosis and prognosis of cardiovascular diseases. TWEAK binding to its functional receptor Fn14 was reported to promote several steps of atherosclerotic plaque progression. However, no information is currently available on the role of TWEAK/Fn14 on the development of medial calcification, which is highly prevalent in aging, CKD and T2DM. This study explored the involvement of TWEAK in human vascular smooth muscle cells (h-VSMCs) calcification in vitro. We report that TWEAK binding to Fn14 promotes inorganic phosphate-induced h-VSMCs calcification, favors h-VSMCs osteogenic transition, decreasing acta2 and myh11 and increasing bmp2 mRNA and tissue non-specific alkaline phosphatase (TNAP), and increases MMP9 activity. Blockade of the canonical NFκB pathway reduced by 80% TWEAK pro-calcific properties and decreased osteogenic transition, TNAP and MMP9 activity. Blockade of non-canonical NFκB signaling by a siRNA targeting RelB reduced by 20% TWEAK pro-calcific effects and decreased TWEAK-induced loss of h-VSMCs contractile phenotype and MMP9 activity, without modulating bmp2 mRNA or TNAP activity. Inhibition of ERK1/2 activation by a MAPK kinase inhibitor did not influence TWEAK pro-calcific properties. Our results suggest that TWEAK/Fn14 directly favors inorganic phosphate-induced h-VSMCs calcification by activation of both canonical and non-canonical NFκB pathways. Given the availability of neutralizing anti-TWEAK strategies, our study sheds light on the TWEAK/Fn14 axis as a novel therapeutic target in the prevention of VC. PMID:27441657

  12. TWEAK favors phosphate-induced calcification of vascular smooth muscle cells through canonical and non-canonical activation of NFκB.

    PubMed

    Hénaut, L; Sanz, A B; Martin-Sanchez, D; Carrasco, S; Villa-Bellosta, R; Aldamiz-Echevarria, G; Massy, Z A; Sanchez-Nino, M D; Ortiz, A

    2016-01-01

    Vascular calcification (VC) is associated with increased cardiovascular mortality in aging, chronic kidney disease (CKD), type 2 diabetes mellitus (T2DM) and atherosclerosis. TNF-like weak inducer of apoptosis (TWEAK) recently emerged as a new biomarker for the diagnosis and prognosis of cardiovascular diseases. TWEAK binding to its functional receptor Fn14 was reported to promote several steps of atherosclerotic plaque progression. However, no information is currently available on the role of TWEAK/Fn14 on the development of medial calcification, which is highly prevalent in aging, CKD and T2DM. This study explored the involvement of TWEAK in human vascular smooth muscle cells (h-VSMCs) calcification in vitro. We report that TWEAK binding to Fn14 promotes inorganic phosphate-induced h-VSMCs calcification, favors h-VSMCs osteogenic transition, decreasing acta2 and myh11 and increasing bmp2 mRNA and tissue non-specific alkaline phosphatase (TNAP), and increases MMP9 activity. Blockade of the canonical NFκB pathway reduced by 80% TWEAK pro-calcific properties and decreased osteogenic transition, TNAP and MMP9 activity. Blockade of non-canonical NFκB signaling by a siRNA targeting RelB reduced by 20% TWEAK pro-calcific effects and decreased TWEAK-induced loss of h-VSMCs contractile phenotype and MMP9 activity, without modulating bmp2 mRNA or TNAP activity. Inhibition of ERK1/2 activation by a MAPK kinase inhibitor did not influence TWEAK pro-calcific properties. Our results suggest that TWEAK/Fn14 directly favors inorganic phosphate-induced h-VSMCs calcification by activation of both canonical and non-canonical NFκB pathways. Given the availability of neutralizing anti-TWEAK strategies, our study sheds light on the TWEAK/Fn14 axis as a novel therapeutic target in the prevention of VC. PMID:27441657

  13. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

    PubMed

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  14. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

    PubMed Central

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  15. PLD1 regulates Xenopus convergent extension movements by mediating Frizzled7 endocytosis for Wnt/PCP signal activation.

    PubMed

    Lee, Hyeyoon; Lee, Seung Joon; Kim, Gun-Hwa; Yeo, Inchul; Han, Jin-Kwan

    2016-03-01

    Phospholipase D (PLD) is involved in the regulation of receptor-associated signaling, cell movement, cell adhesion and endocytosis. However, its physiological role in vertebrate development remains poorly understood. In this study, we show that PLD1 is required for the convergent extension (CE) movements during Xenopus gastrulation by activating Wnt/PCP signaling. Xenopus PLD1 protein is specifically enriched in the dorsal region of Xenopus gastrula embryo and loss or gain-of-function of PLD1 induce defects in gastrulation and CE movements. These defective phenotypes are due to impaired regulation of Wnt/PCP signaling pathway. Biochemical and imaging analysis using Xenopus tissues reveal that PLD1 is required for Fz7 receptor endocytosis upon Wnt11 stimulation. Moreover, we show that Fz7 endocytosis depends on dynamin and regulation of GAP activity of dynamin by PLD1 via its PX domain is crucial for this process. Taken together, our results suggest that PLD1 acts as a new positive mediator of Wnt/PCP signaling by promoting Wnt11-induced Fz7 endocytosis for precise regulation of Xenopus CE movements. PMID:26806705

  16. The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression.

    PubMed

    Ma, Yuanqing; Liu, Xiuli; Liu, Zhaoting; Wei, Shi; Shang, Hanqiao; Xue, Yu; Cao, Yu; Meng, Anming; Wang, Qiang

    2015-06-01

    During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-β signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-β signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-β/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization. PMID:26041917

  17. The HMG-box transcription factor SoxNeuro acts with Tcf to control Wg/Wnt signaling activity.

    PubMed

    Chao, Anna T; Jones, Whitney M; Bejsovec, Amy

    2007-03-01

    Wnt signaling specifies cell fates in many tissues during vertebrate and invertebrate embryogenesis. To understand better how Wnt signaling is regulated during development, we have performed genetic screens to isolate mutations that suppress or enhance mutations in the fly Wnt homolog, wingless (wg). We find that loss-of-function mutations in the neural determinant SoxNeuro (also known as Sox-neuro, SoxN) partially suppress wg mutant pattern defects. SoxN encodes a HMG-box-containing protein related to the vertebrate Sox1, Sox2 and Sox3 proteins, which have been implicated in patterning events in the early mouse embryo. In Drosophila, SoxN has previously been shown to specify neural progenitors in the embryonic central nervous system. Here, we show that SoxN negatively regulates Wg pathway activity in the embryonic epidermis. Loss of SoxN function hyperactivates the Wg pathway, whereas its overexpression represses pathway activity. Epistasis analysis with other components of the Wg pathway places SoxN at the level of the transcription factor Pan (also known as Lef, Tcf) in regulating target gene expression. In human cell culture assays, SoxN represses Tcf-responsive reporter expression, indicating that the fly gene product can interact with mammalian Wnt pathway components. In both flies and in human cells, SoxN repression is potentiated by adding ectopic Tcf, suggesting that SoxN interacts with the repressor form of Tcf to influence Wg/Wnt target gene transcription. PMID:17267442

  18. Wnt/β-catenin-signaling and the formation of Müller glia-derived progenitors in the chick retina.

    PubMed

    Gallina, Donika; Palazzo, Isabella; Steffenson, Lillia; Todd, Levi; Fischer, Andy J

    2016-09-01

    Müller glia can be stimulated to de-differentiate, proliferate, and form Müller glia-derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de-differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt-pathway. The purpose of this study was to investigate how Wnt-signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA-damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt-signaling, including dkk1, dkk3, axin2, c-myc, tcf-1, and cd44. We find accumulations of nuclear β-catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA-treatment. Inhibition of Wnt-signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt-signaling, with GSK3β-inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt-signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK-signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β-catenin. In FGF2-treated retinas, inhibition of Wnt-signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt-signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt-signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt-signaling is required for the formation of proliferating MGPCs. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 983-1002, 2016. PMID:26663639

  19. Wnt/Axin1/β-Catenin Signaling Regulates Asymmetric Nodal Activation, Elaboration, and Concordance of CNS Asymmetries

    PubMed Central

    Carl, Matthias; Bianco, Isaac H.; Bajoghli, Baubak; Aghaallaei, Narges; Czerny, Thomas; Wilson, Stephen W.

    2007-01-01

    Summary Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/β-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries. PMID:17678853

  20. Norcantharidin inhibits Wnt signal pathway via promoter demethylation of WIF-1 in human non-small cell lung cancer.

    PubMed

    Xie, Junran; Zhang, Yaping; Hu, Xuming; Lv, Ran; Xiao, Dongju; Jiang, Li; Bao, Qi

    2015-05-01

    Wingless-type (Wnt) family of secreted glycoproteins is a group of signal molecules implicated in oncogenesis. Abnormal activation of Wnt signal pathway is associated with a variety of human cancers, including non-small cell lung cancer (NSCLC). Wnt antagonists, such as the secreted frizzled-related protein (SFRP) family, Wnt inhibitory factor-1 (WIF-1) and cerberus, inhibit Wnt signal pathway by directly binding to Wnt molecules. Norcantharidin (NCTD) is known to possess anticancer activity but less nephrotoxicity than cantharidin. In this study, we found that NCTD inhibited cell proliferation, induced apoptosis, arrested cell cycle and suppressed cell invasion/migration in vitro. Additionally, Wnt signal pathway transcription was also suppressed. NCTD treatment blocked cytoplasmic translocation of beta-catenin into the nucleus. Alterations of apoptosis-related proteins, such as Bax, cleaved caspase-3 (pro-apoptotic) and Bcl-2 (anti-apoptotic), had been detected. Furthermore, the expression levels of WIF-1 and SFRP1 were significantly increased in NCTD-treated groups compared with negative control (NC) groups. Abnormal methylation was observed in NC groups, while NCTD treatment promoted WIF-1 demethylation. The present study revealed that NCTD activated WIF-1 via promoter demethylation, inhibiting the canonical Wnt signal pathway in NSCLC, which may present a new therapeutic target in vivo. PMID:25814287

  1. Wnt7b is an important intrinsic regulator of hair follicle stem cell homeostasis and hair follicle cycling

    PubMed Central

    Kandyba, Eve; Kobielak, Krzysztof

    2014-01-01

    The hair follicle (HF) is an exceptional mini-organ to study the mechanisms which regulate HF morphogenesis, cycling, hair follicle stem cell (hfSCs) homeostasis and progeny differentiation. During morphogenesis, Wnt signaling is well characterized in the initiation of HF patterning but less is known about which particular Wnt ligands are required and whether individual Wnt ligands act in an indispensable or redundant manner during postnatal hfSCs anagen onset and HF cycle progression. Previously, we described the function of the Bone morphogenetic protein (BMP) signaling target gene WNT7a in intrinsic regulation of hfSCs homeostasis in vivo. Here, we investigated the role of Wnt7b, which was also intrinsically up-regulated in hfSCs during physiological and precocious anagen after BMP inhibition in vivo. We demonstrated Wnt7b to be a direct target of canonical BMP signaling in hfSCs and using Wnt7b conditional gene targeting during HF morphogenesis revealed disrupted HF cycling including a shorter anagen, premature catagen onset with overall shorter hair production and diminished HF differentiation marker expression. Additionally, we observed that postnatal ablation of Wnt7b resulted in delayed HF activation, affecting both the HG and bulge hfSCs but still maintaining a two-step sequence of HF stimulation. Interestingly, Wnt7b cKO hfSCs participated in re-formation of the new HF bulge, but with slower self-renewal. These findings demonstrate the importance of intrinsic Wnt7b expression in hfSCs regulation and normal HF cycling and surprisingly reveal a non-redundant role for Wnt7b in the control of HF anagen length and catagen entry which was not compensated by other Wnt ligands. PMID:24222445

  2. Wnt7b is an important intrinsic regulator of hair follicle stem cell homeostasis and hair follicle cycling.

    PubMed

    Kandyba, Eve; Kobielak, Krzysztof

    2014-04-01

    The hair follicle (HF) is an exceptional mini-organ to study the mechanisms which regulate HF morphogenesis, cycling, hair follicle stem cell (hfSCs) homeostasis, and progeny differentiation. During morphogenesis, Wnt signaling is well-characterized in the initiation of HF patterning but less is known about which particular Wnt ligands are required and whether individual Wnt ligands act in an indispensable or redundant manner during postnatal hfSCs anagen onset and HF cycle progression. Previously, we described the function of the bone morphogenetic protein (BMP) signaling target gene WNT7a in intrinsic regulation of hfSCs homeostasis in vivo. Here, we investigated the role of Wnt7b, which was also intrinsically upregulated in hfSCs during physiological and precocious anagen after BMP inhibition in vivo. We demonstrated Wnt7b to be a direct target of canonical BMP signaling in hfSCs and using Wnt7b conditional gene targeting during HF morphogenesis revealed disrupted HF cycling including a shorter anagen, premature catagen onset with overall shorter hair production, and diminished HF differentiation marker expression. Additionally, we observed that postnatal ablation of Wnt7b resulted in delayed HF activation, affecting both the hair germ and bulge hfSCs but still maintaining a two-step sequence of HF stimulation. Interestingly, Wnt7b cKO hfSCs participated in reformation of the new HF bulge, but with slower self-renewal. These findings demonstrate the importance of intrinsic Wnt7b expression in hfSCs regulation and normal HF cycling and surprisingly reveal a nonredundant role for Wnt7b in the control of HF anagen length and catagen entry which was not compensated by other Wnt ligands. PMID:24222445

  3. SNF2H promotes hepatocellular carcinoma proliferation by activating the Wnt/β-catenin signaling pathway

    PubMed Central

    Wang, Yanan; Qin, Juanxiu; Liu, Qian; Hong, Xufen; Li, Tianming; Zhu, Yuanjun; He, Lei; Zheng, Bing; Li, Min

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has an extremely poor prognosis. Surgical resection is always inapplicable to HCC patients diagnosed at an advanced tumor stage. The mechanisms underlying HCC cell proliferation remain obscure. In the present study, SWItch/sucrose nonfermentable catalytic subunit SNF2 (SNF2H) expression was tested in HCC tissues and Wnt/β-catenin pathway activation upon overexpression of SNF2H or knockdown of SNF2H expression was investigated in cultured HCC cells. It was demonstrated that SNF2H is a vital factor for HCC growth. The SNF2H expression level is increased in HCC tissues compared with paratumoral liver tissues. SNF2H promotes HCC cell proliferation and colony formation ability in vitro. SNF2H may increase the protein level of β-catenin and enhance its nuclear accumulation in HCC cells, thereby leading to the activation of the Wnt/β-catenin signaling pathway. In conclusion, the present results indicate that SNF2H plays a vital role in HCC cell growth, suggesting that SNF2H may be a promising therapeutic target for HCC treatment. PMID:27446433

  4. Fibulin-4 is a novel Wnt/β-Catenin pathway activator in human osteosarcoma.

    PubMed

    Li, Renzeng; Wang, Limin

    2016-06-10

    Fibulin-4, an extracellular glycoprotein implicated in connective tissue development and elastic fiber formation, draws increasing focuses in cancer research. However, little is known about the underlying oncogenic roles of Fibulin-4 in human osteosarcoma (OS). In this study, by immunohistochemical analysis, upregulated expression of Fibulin-4 was found in the OS clinical specimens and cell lines compared to their normal counterparts. Fibulin-4 was positively correlated with the T stage of OS patients, and the proliferation index Ki67. Based on informatics analysis and functional verification, microRNA-137 was identified as a potential upstream regulator of Fibulin-4. Knockdown of Fibulin-4 or introduction of microRNA-137 inhibited cell proliferation and promoted cell apoptosis, and adverse effects were observed by overexpression of Fibulin-4. Furthermore, the tumor-suppressive functions of microRNA-137 were markedly abolished by restoration of Fibulin-4 expression in OS cells. Mechanistically, Fibulin-4 activated Wnt/β-Catenin pathway and promoted the expression of its downstream targets, including CCND2, c-Myc and VEGF. Taken together, Fibulin-4 plays critical neoplastic roles in tumor growth of human OS by activating Wnt/β-Catenin signaling and may represent a potential therapeutic target. PMID:27157136

  5. WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum

    PubMed Central

    Pei, Yanxin; Brun, Sonja N.; Markant, Shirley L.; Lento, William; Gibson, Paul; Taketo, Makoto M.; Giovannini, Marco; Gilbertson, Richard J.; Wechsler-Reya, Robert J.

    2012-01-01

    The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition, WNT pathway mutations are associated with medulloblastoma, the most common malignant brain tumor in children. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover, mice that express activated β-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region, whereas expression of the same protein in GNPs impairs proliferation. Although β-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation. PMID:22461560

  6. Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures

    PubMed Central

    Movérare-Skrtic, Sofia; Henning, Petra; Liu, Xianwen; Nagano, Kenichi; Saito, Hiroaki; Börjesson, Anna E; Sjögren, Klara; Windahl, Sara H; Farman, Helen; Kindlund, Bert; Engdahl, Cecilia; Koskela, Antti; Zhang, Fu-Ping; Eriksson, Emma E; Zaman, Farasat; Hammarstedt, Ann; Isaksson, Hanna; Bally, Marta; Kassem, Ali; Lindholm, Catharina; Sandberg, Olof; Aspenberg, Per; Sävendahl, Lars; Feng, Jian Q; Tuckermann, Jan; Tuukkanen, Juha; Poutanen, Matti; Baron, Roland; Lerner, Ulf H; Gori, Francesca; Ohlsson, Claes

    2015-01-01

    The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need. PMID:25306233

  7. Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures.

    PubMed

    Movérare-Skrtic, Sofia; Henning, Petra; Liu, Xianwen; Nagano, Kenichi; Saito, Hiroaki; Börjesson, Anna E; Sjögren, Klara; Windahl, Sara H; Farman, Helen; Kindlund, Bert; Engdahl, Cecilia; Koskela, Antti; Zhang, Fu-Ping; Eriksson, Emma E; Zaman, Farasat; Hammarstedt, Ann; Isaksson, Hanna; Bally, Marta; Kassem, Ali; Lindholm, Catharina; Sandberg, Olof; Aspenberg, Per; Sävendahl, Lars; Feng, Jian Q; Tuckermann, Jan; Tuukkanen, Juha; Poutanen, Matti; Baron, Roland; Lerner, Ulf H; Gori, Francesca; Ohlsson, Claes

    2014-11-01

    The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need. PMID:25306233

  8. Runx2 Controls Bone Resorption through the Down-Regulation of the Wnt Pathway in Osteoblasts.

    PubMed

    Haxaire, Coline; Haÿ, Eric; Geoffroy, Valérie

    2016-06-01

    The transcription factor Runx2 and the Wnt/β-catenin pathway are major regulators of bone formation. Our aim was to assess the interactions between the Wnt/β-catenin pathway and Runx2 that contribute to bone resorption. Our results indicate that the activity of the canonical Wnt/β-catenin pathway depends on Runx2. Runx2 overexpression inhibited β-catenin levels and activity in vitro and in vivo. Inhibition of Gsk3b using lithium chloride in Runx2-overexpressing osteoporotic female mice rescued the Wnt/β-catenin signaling in vivo and completely restored trabecular bone volume by increasing bone formation and decreasing bone resorption. The activation of Wnt/β-catenin signaling by lithium chloride treatment reduced the number and activity of bone marrow-derived osteoclast-like cells in vitro, suggesting that the restoration of trabecular bone in vivo was due to decreased bone resorption, consistent with the reduced receptor activator of NF-κB ligand/osteoprotegerin ratio in Runx2-overexpressing osteoblasts. Lithium chloride also increased osteoblast differentiation and activity in vitro in agreement with the increase in mineral apposition rate and osteocalcin expression detected in vivo. Our results indicate that the activity of the canonical Wnt/β-catenin pathway in osteoblast is modulated by Runx2. To conclude, our in vivo and in vitro results highlight the role of Runx2 as a negative regulator of Wnt/β-catenin pathway activity in osteoblasts and indicate that the abnormal Wnt/β-catenin activity seen in Runx2 transgenic mice affects both osteoblast and osteoclast differentiation and activity. PMID:27083516

  9. Regeneration-associated WNT Signaling Is Activated in Long-term Reconstituting AC133bright Acute Myeloid Leukemia Cells12

    PubMed Central

    Beghini, Alessandro; Corlazzoli, Francesca; Del Giacco, Luca; Re, Matteo; Lazzaroni, Francesca; Brioschi, Matteo; Valentini, Giorgio; Ferrazzi, Fulvia; Ghilardi, Anna; Righi, Marco; Turrini, Mauro; Mignardi, Marco; Cesana, Clara; Bronte, Vincenzo; Nilsson, Mats; Morra, Enrica; Cairoli, Roberto

    2012-01-01

    Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/β-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133+ cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133bright AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133+ AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated β-catenin in vivo. We tested the LSC functional activity in AC133+ cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2-/-γc-/- mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133bright LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration. PMID:23308055

  10. Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells

    PubMed Central

    Akhade, Vijay Suresh; Dighe, Shrinivas Nivrutti; Kataruka, Shubhangini; Rao, Manchanahalli R. Satyanarayana

    2016-01-01

    Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis. Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes. PMID:26446991

  11. Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells.

    PubMed

    Akhade, Vijay Suresh; Dighe, Shrinivas Nivrutti; Kataruka, Shubhangini; Rao, Manchanahalli R Satyanarayana

    2016-01-01

    Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis. Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes. PMID:26446991

  12. TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways.

    PubMed

    Vivar, Raúl; Humeres, Claudio; Ayala, Pedro; Olmedo, Ivonne; Catalán, Mabel; García, Lorena; Lavandero, Sergio; Díaz-Araya, Guillermo

    2013-06-01

    Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia-reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways. PMID:23416528

  13. TGF-β-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8.

    PubMed

    Spanjer, Anita I R; Baarsma, Hoeke A; Oostenbrink, Lisette M; Jansen, Sepp R; Kuipers, Christine C; Lindner, Michael; Postma, Dirkje S; Meurs, Herman; Heijink, Irene H; Gosens, Reinoud; Königshoff, Melanie

    2016-05-01

    TGF-β is important in lung injury and remodeling processes. TGF-β and Wingless/integrase-1 (WNT) signaling are interconnected; however, the WNT ligand-receptor complexes involved are unknown. Thus, we aimed to identify Frizzled (FZD) receptors that mediate TGF-β-induced profibrotic signaling. MRC-5 and primary human lung fibroblasts were stimulated with TGF-β1, WNT-5A, or WNT-5B in the presence and absence of specific pathway inhibitors. Specific small interfering RNA was used to knock down FZD8. In vivo studies using bleomycin-induced lung fibrosis were performed in wild-type and FZD8-deficient mice. TGF-β1 induced FZD8 specifically via Smad3-dependent signaling in MRC-5 and primary human lung fibroblasts. It is noteworthy that FZD8 knockdown reduced TGF-β1-induced collagen Iα1, fibronectin, versican, α-smooth muscle (sm)-actin, and connective tissue growth factor. Moreover, bleomycin-induced lung fibrosis was attenuated in FZD8-deficient mice in vivo Although inhibition of canonical WNT signaling did not affect TGF-β1-induced gene expression in vitro, noncanonical WNT-5B mimicked TGF-β1-induced fibroblast activation. FZD8 knockdown reduced both WNT-5B-induced gene expression of fibronectin and α-sm-actin, as well as WNT-5B-induced changes in cellular impedance. Collectively, our findings demonstrate a role for FZD8 in TGF-β-induced profibrotic signaling and imply that WNT-5B may be the ligand for FZD8 in these responses.-Spanjer, A. I. R., Baarsma, H. A., Oostenbrink, L. M., Jansen, S. R., Kuipers, C. C., Lindner, M., Postma, D. S., Meurs, H., Heijink, I. H., Gosens, R., Königshoff, M. TGF-β-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8. PMID:26849959

  14. XEDAR activates the non-canonical NF-κB pathway

    SciTech Connect

    Verhelst, Kelly; Gardam, Sandra; Borghi, Alice; Kreike, Marja; Carpentier, Isabelle; Beyaert, Rudi

    2015-09-18

    Members of the tumor necrosis factor receptor (TNFR) superfamily are involved in a number of physiological and pathological responses by activating a wide variety of intracellular signaling pathways. The X-linked ectodermal dysplasia receptor (XEDAR; also known as EDA2R or TNFRSF27) is a member of the TNFR superfamily that is highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2), a member of the TNF family that is encoded by the anhidrotic ectodermal dysplasia (EDA) gene. Although XEDAR was first described in the year 2000, its function and molecular mechanism of action is still largely unclear. XEDAR has been reported to activate canonical nuclear factor κB (NF-κB) signaling and mitogen-activated protein (MAP) kinases. Here we report that XEDAR is also able to trigger the non-canonical NF-κB pathway, characterized by the processing of p100 (NF-κB2) into p52, followed by nuclear translocation of p52 and RelB. We provide evidence that XEDAR-induced p100 processing relies on the binding of XEDAR to TRAF3 and TRAF6, and requires the kinase activity of NIK and IKKα. We also show that XEDAR stimulation results in NIK accumulation and that p100 processing is negatively regulated by TRAF3, cIAP1 and A20. - Highlights: • XEDAR activates the non-canonical NF-κB pathway. • XEDAR-induced processing of p100 depends on XEDAR interaction with TRAF3 and TRAF6. • XEDAR-induced processing of p100 depends on NIK and IKKα activity. • Overexpression of XEDAR leads to NIK accumulation. • XEDAR-induced processing of p100 is negatively regulated by TRAF3 cIAP1 and A20.

  15. Regulation of tumorigenic Wnt signaling by cyclooxygenase-2, 5-lipoxygenase and their pharmacological inhibitors: A basis for novel drugs targeting cancer cells?

    PubMed

    Roos, Jessica; Grösch, Sabine; Werz, Oliver; Schröder, Peter; Ziegler, Slava; Fulda, Simone; Paulus, Patrick; Urbschat, Anja; Kühn, Benjamin; Maucher, Isabelle; Fettel, Jasmin; Vorup-Jensen, Thomas; Piesche, Matthias; Matrone, Carmela; Steinhilber, Dieter; Parnham, Michael J; Maier, Thorsten J

    2016-01-01

    Canonical Wnt signaling is a highly conserved pathway with a prominent role in embryogenic development, adult tissue homeostasis, cell polarization, stem cell biology, cell differentiation, and proliferation. Furthermore, canonical Wnt signaling is of pivotal importance in the pathogenesis of a number of cancer types and crucially affects tumor initiation, cancer cell proliferation, cancer cell apoptosis, and metastasis. Reports over the last decade have provided strong evidence for a pathophysiological role of Wnt signaling in non-malignant classical inflammatory and neurodegenerative diseases. Although, several agents suppressing the Wnt pathway at different levels have been identified, the development of clinically relevant Wnt-inhibiting agents remains challenging due to selectivity and toxicity issues. Several studies have shown that long-term administration of non-steroidal anti-inflammatory drugs protects against colon cancer and potentially other tumor types by interfering both with the COX and the Wnt pathway. Our own studies have shown that non-steroidal anti-inflammatory drugs suppress Wnt signaling by targeting the pro-inflammatory enzyme 5-lipoxygenase which is the key enzyme pathophysiologically involved in the synthesis of leukotrienes. Furthermore, we found a direct link between the 5-lipoxygenase and Wnt signaling pathways, which is essential for the maintenance of leukemic stem cells. Accordingly, genetic and pharmacological inhibition of 5-lipoxygenase led to an impairment of Wnt-dependent acute and chronic myeloid leukemic stem cells. We believe that 5-lipoxygenase inhibitors might represent a novel type of Wnt inhibitor activating a potentially naturally occurring novel mechanism of suppression of Wnt signaling that is non-toxic, at least in mice, and is potentially well tolerated in patients. PMID:26549540

  16. MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway.

    PubMed

    Ahmed, Mohammed I; Alam, Majid; Emelianov, Vladimir U; Poterlowicz, Krzysztof; Patel, Ankit; Sharov, Andrey A; Mardaryev, Andrei N; Botchkareva, Natalia V

    2014-11-24

    Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting β-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators β-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify β-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging. PMID:25422376

  17. NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis.

    PubMed

    Chung, H; Vilaysane, A; Lau, A; Stahl, M; Morampudi, V; Bondzi-Simpson, A; Platnich, J M; Bracey, N A; French, M-C; Beck, P L; Chun, J; Vallance, B A; Muruve, D A

    2016-08-01

    Nod-like receptor, pyrin containing 3 (NLRP3) is characterized primarily as a canonical caspase-1 activating inflammasome in macrophages. NLRP3 is also expressed in the epithelium of the kidney and gut; however, its function remains largely undefined. Primary mouse tubular epithelial cells (TEC) lacking Nlrp3 displayed reduced apoptosis downstream of the tumor necrosis factor (TNF) receptor and CD95. TECs were identified as type II apoptotic cells that activated caspase-8, tBid and mitochondrial apoptosis via caspase-9, responses that were reduced in Nlrp3-/- cells. The activation of caspase-8 during extrinsic apoptosis induced by TNFα/cycloheximide (TNFα/CHX) was dependent on adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and completely independent of caspase-1 or caspase-11. TECs and primary human proximal tubular epithelial cells (HPTC) did not activate a canonical inflammasome, caspase-1, or IL-1β secretion in response to TNFα/CHX or NLRP3-dependent triggers, such as ATP or nigericin. In cell fractionation studies and by confocal microscopy, NLRP3 colocalized with ASC and caspase-8 in speck-like complexes at the mitochondria during apoptosis. The formation of NLRP3/ASC/caspase-8 specks in response to TNFα/CHX was downstream of TNFR signaling and dependent on potassium efflux. Epithelial ASC specks were present in enteroids undergoing apoptosis and in the injured tubules of wild-type but not Nlrp3-/- or ASC-/- mice following ureteric unilateral obstruction in vivo. These data show that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells. PMID:26891693

  18. Wnt9A Induction Linked to Suppression of Human Colorectal Cancer Cell Proliferation

    PubMed Central

    Ali, Irshad; Medegan, Bani; Braun, Donald P.

    2016-01-01

    Most studies of Wnt signaling in malignant tissues have focused on the canonical Wnt pathway (CWP) due to its role in stimulating cellular proliferation. The role of the non-canonical Wnt pathway (NCWP) in tissues with dysregulated Wnt signaling is not fully understood. Understanding NCWP’s role is important since these opposing pathways act in concert to maintain homeostasis in healthy tissues. Our preliminary studies demonstrated that LiCl inhibited proliferation of primary cells derived from colorectal cancer (CRC). Since LiCl stimulates cell proliferation in normal tissues and NCWP suppresses it, the present study was designed to investigate the impact of NCWP components in LiCl-mediated effects. LiCl-mediated inhibition of CRC cell proliferation (p < 0.001) and increased apoptosis (p < 0.01) coincided with 23-fold increase (p < 0.025) in the expression of the NCWP ligand, Wnt9A. LiCl also suppressed β-catenin mRNA (p < 0.03), total β-catenin protein (p < 0.025) and the active form of β-catenin. LiCl-mediated inhibition of CRC cell proliferation was partially reversed by IWP-2, and Wnt9A antibody. Recombinant Wnt9A protein emulated LiCl effects by suppressing β-catenin protein (p < 0.001), inhibiting proliferation (p < 0.001) and increasing apoptosis (p < 0.03). This is the first study to demonstrate induction of a NCWP ligand, Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC cell proliferation. PMID:27049382

  19. Kirenol inhibits adipogenesis through activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes

    SciTech Connect

    Kim, Mi-Bo; Song, Youngwoo; Kim, Changhee; Hwang, Jae-Kwan

    2014-03-07

    Highlights: • Kirenol inhibits the adipogenic transcription factors and lipogenic enzymes. • Kirenol stimulates the Wnt/β-catenin signaling pathway components. • Kirenol inhibits adipogenesis through activation of the Wnt/β-catenin signaling pathway. - Abstract: Kirenol, a natural diterpenoid compound, has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, and anti-arthritic activities; however, its anti-adipogenic effect remains to be studied. The present study evaluated the effect of kirenol on anti-adipogenesis through the activation of the Wnt/β-catenin signaling pathway. Kirenol prevented intracellular lipid accumulation by down-regulating key adipogenesis transcription factors [peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and sterol regulatory element binding protein-1c (SREBP-1c)] and lipid biosynthesis-related enzymes [fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC)], as well as adipocytokines (adiponectin and leptin). Kirenol effectively activated the Wnt/β-catenin signaling pathway, in which kirenol up-regulated the expression of low density lipoprotein receptor related protein 6 (LRP6), disheveled 2 (DVL2), β-catenin, and cyclin D1 (CCND1), while it inactivated glycogen synthase kinase 3β (GSK3β) by increasing its phosphorylation. Kirenol down-regulated the expression levels of PPARγ and C/EBPα, which were up-regulated by siRNA knockdown of β-catenin. Overall, kirenol is capable of inhibiting the differentiation and lipogenesis of 3T3-L1 adipocytes through the activation of the Wnt/β-catenin signaling pathway, suggesting its potential as natural anti-obesity agent.

  20. Sp5 and Sp8 recruit β-catenin and Tcf1-Lef1 to select enhancers to activate Wnt target gene transcription.

    PubMed

    Kennedy, Mark W; Chalamalasetty, Ravindra B; Thomas, Sara; Garriock, Robert J; Jailwala, Parthav; Yamaguchi, Terry P

    2016-03-29

    The ancient, highly conserved, Wnt signaling pathway regulates cell fate in all metazoans. We have previously shown that combined null mutations of the specificity protein (Sp) 1/Klf-like zinc-finger transcription factorsSp5andSp8(i.e.,Sp5/8) result in an embryonic phenotype identical to that observed when core components of the Wnt/β-catenin pathway are mutated; however, their role in Wnt signal transduction is unknown. Here, we show in mouse embryos and differentiating embryonic stem cells that Sp5/8 are gene-specific transcriptional coactivators in the Wnt/β-catenin pathway. Sp5/8 bind directly to GC boxes in Wnt target gene enhancers and to adjacent, or distally positioned, chromatin-bound T-cell factor (Tcf) 1/lymphoid enhancer factor (Lef) 1 to facilitate recruitment of β-catenin to target gene enhancers. BecauseSp5is itself directly activated by Wnt signals, we propose that Sp5 is a Wnt/β-catenin pathway-specific transcripton factor that functions in a feed-forward loop to robustly activate select Wnt target genes. PMID:26969725

  1. Oestrogen and parathyroid hormone alleviate lumbar intervertebral disc degeneration in ovariectomized rats and enhance Wnt/β-catenin pathway activity

    PubMed Central

    Jia, Haobo; Ma, Jianxiong; Lv, Jianwei; Ma, Xinlong; Xu, Weiguo; Yang, Yang; Tian, Aixian; Wang, Ying; Sun, Lei; Xu, Liyan; Fu, Lin; Zhao, Jie

    2016-01-01

    To investigate the mitigation effect and mechanism of oestrogen and PTH on disc degeneration in rats after ovariectomy, as well as on Wnt/β-catenin pathway activity, thirty 3-month-old rats were ovariectomized and divided into three groups. Ten additional rats were used as controls. Eight weeks later, the rats were administered oestrogen or PTH for 12 weeks, and then discs were collected for tests. Results showed that nucleus pulposus cells in the Sham group were mostly notochord cells, while in the OVX group, cells gradually developed into chondrocyte-like cells. Oestrogen or PTH could partly recover the notochord cell number. After ovariectomy, the endplate roughened and endplate porosity decreased. After oestrogen or PTH treatment, the smoothness and porosity of endplate recovered. Compared with the Sham group, Aggrecan, Col2a and Wnt/β-catenin pathway expression in OVX group decreased, and either oestrogen or PTH treatment improved their expression. The biomechanical properties of intervertebral disc significantly changed after ovariectomy, and oestrogen or PTH treatment partly recovered them. Disc degeneration occurred with low oestrogen, and the underlying mechanisms involve nutrition supply disorders, cell type changes and decreased Wnt/β-catenin pathway activity. Oestrogen and PTH can retard disc degeneration in OVX rats and enhance Wnt/β-catenin pathway activity in nucleus pulposus. PMID:27279629

  2. Oestrogen and parathyroid hormone alleviate lumbar intervertebral disc degeneration in ovariectomized rats and enhance Wnt/β-catenin pathway activity.

    PubMed

    Jia, Haobo; Ma, Jianxiong; Lv, Jianwei; Ma, Xinlong; Xu, Weiguo; Yang, Yang; Tian, Aixian; Wang, Ying; Sun, Lei; Xu, Liyan; Fu, Lin; Zhao, Jie

    2016-01-01

    To investigate the mitigation effect and mechanism of oestrogen and PTH on disc degeneration in rats after ovariectomy, as well as on Wnt/β-catenin pathway activity, thirty 3-month-old rats were ovariectomized and divided into three groups. Ten additional rats were used as controls. Eight weeks later, the rats were administered oestrogen or PTH for 12 weeks, and then discs were collected for tests. Results showed that nucleus pulposus cells in the Sham group were mostly notochord cells, while in the OVX group, cells gradually developed into chondrocyte-like cells. Oestrogen or PTH could partly recover the notochord cell number. After ovariectomy, the endplate roughened and endplate porosity decreased. After oestrogen or PTH treatment, the smoothness and porosity of endplate recovered. Compared with the Sham group, Aggrecan, Col2a and Wnt/β-catenin pathway expression in OVX group decreased, and either oestrogen or PTH treatment improved their expression. The biomechanical properties of intervertebral disc significantly changed after ovariectomy, and oestrogen or PTH treatment partly recovered them. Disc degeneration occurred with low oestrogen, and the underlying mechanisms involve nutrition supply disorders, cell type changes and decreased Wnt/β-catenin pathway activity. Oestrogen and PTH can retard disc degeneration in OVX rats and enhance Wnt/β-catenin pathway activity in nucleus pulposus. PMID:27279629

  3. Human placental trophoblast invasion and differentiation: a particular focus on Wnt signaling

    PubMed Central

    Knöfler, Martin; Pollheimer, Jürgen

    2013-01-01

    Wingless ligands, a family of secreted proteins, are critically involved in organ development and tissue homeostasis by ensuring balanced rates of stem cell proliferation, cell death and differentiation. Wnt signaling components also play crucial roles in murine placental development controlling trophoblast lineage determination, chorioallantoic fusion and placental branching morphogenesis. However, the role of the pathway in human placentation, trophoblast development and differentiation is only partly understood. Here, we summarize our present knowledge about Wnt signaling in the human placenta and discuss its potential role in physiological and aberrant trophoblast invasion, gestational diseases and choriocarcinoma formation. Differentiation of proliferative first trimester cytotrophoblasts into invasive extravillous trophoblasts is associated with nuclear recruitment of β -catenin and induction of Wnt-dependent T-cell factor 4 suggesting that canonical Wnt signaling could be important for the formation and function of extravillous trophoblasts. Indeed, activation of the pathway was shown to promote trophoblast invasion in different in vitro trophoblast model systems as well as trophoblast cell fusion. Methylation-mediated silencing of inhibitors of Wnt signaling provided evidence for epigenetic activation of the pathway in placental tissues and choriocarcinoma cells. Similarly, abundant nuclear expression of β -catenin in invasive trophoblasts of complete hydatidiform moles suggested a role for hyper-activated Wnt signaling. In contrast, upregulation of Wnt inhibitors was noticed in placentae of women with preeclampsia, a disease characterized by shallow trophoblast invasion and incomplete spiral artery remodeling. Moreover, changes in Wnt signaling have been observed upon cytomegalovirus infection and in recurrent abortions. In summary, the current literature suggests a critical role of Wnt signaling in physiological and abnormal trophoblast function. PMID

  4. Wnt/β-Catenin Signaling Activation beyond Robust Nuclear β-Catenin Accumulation in Nondysplastic Barrett’s Esophagus: Regulation via Dickkopf-112

    PubMed Central

    Lyros, Orestis; Rafiee, Parvaneh; Nie, Linghui; Medda, Rituparna; Jovanovic, Nebojsa; Otterson, Mary F.; Behmaram, Behnaz; Gockel, Ιnes; Mackinnon, Alexander; Shaker, Reza

    2015-01-01

    INTRODUCTION: Wnt/β-catenin signaling activation has been reported only during the late steps of Barrett’s esophagus (BE) neoplastic progression, but not in BE metaplasia, based on the absence of nuclear β-catenin. However, β-catenin transcriptional activity has been recorded in absence of robust nuclear accumulation. Thus, we aimed to investigate the Wnt/β-catenin signaling in nondysplastic BE. METHODS: Esophageal tissues from healthy and BE patients without dysplasia were analyzed for Wnt target gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Esophageal squamous (EPC1-& EPC2-hTERT), BE metaplastic (CP-A), and adenocarcinoma (OE33) cell lines were characterized for Wnt activation by qRT-PCR, Western blot, and luciferase assay. Wnt activity regulation was examined by using recombinant Wnt3a and Dickkopf-1 (Dkk1) as well as Dkk1 short interfering RNA. RESULTS: Wnt target genes (AXIN2, c-MYC, Cyclin D1, Dkk1) and Wnt3a were significantly upregulated in nondysplastic BE compared with squamous mucosa. Elevated levels of dephosphorylated β-catenin were detected in nondysplastic BE. Nuclear active β-catenin and TOPflash activity were increased in CP-A and OE33 cells compared with squamous cells. Wnt3a-mediated β-catenin signaling activation was abolished by Dkk1 in CP-A cells. TOPFlash activity was elevated following Dkk1 silencing in CP-A but not in OE33 cells. Dysplastic and esophageal adenocarcinoma tissues demonstrated further Dkk1 and AXIN2 overexpression. CONCLUSIONS: Despite the absence of robust nuclear accumulation, β-catenin is transcriptionally active in nondysplastic BE. Dkk1 overexpression regulates β-catenin signaling in BE metaplastic but not in adenocarcinoma cells, suggesting that early perturbation of Dkk1-mediated signaling suppression may contribute to BE malignant transformation. PMID:26297437

  5. Induction and transport of Wnt 5a during macrophage-induced malignant invasion is mediated by two types of extracellular vesicles

    PubMed Central

    Gross, Julia Christina; Pukrop, Tobias; Wenzel, Dirk; Binder, Claudia

    2013-01-01

    Recently, we have shown that macrophage (MΦ)-induced invasion of breast cancer cells requires upregulation of Wnt 5a in MΦ leading to activation of β-Catenin-independent Wnt signaling in the tumor cells. However, it remained unclear, how malignant cells induce Wnt 5a in MΦ and how it is transferred back to the cancer cells. Here we identify two types of extracellular particles as essential for this intercellular interaction in both directions. Plasma membrane-derived microvesicles (MV) as well as exosomes from breast cancer cells, although biologically distinct populations, both induce Wnt 5a in MΦ. In contrast, the particle-free supernatant and vesicles from benign cells, such as platelets, have no such effect. Induction is antagonized by the Wnt inhibitor Dickkopf-1. Subsequently, Wnt 5a is shuttled via responding MΦ-MV and exosomes to the tumor cells enhancing their invasion. Wnt 5a export on both vesicle fractions depends at least partially on the cargo protein Evenness interrupted (Evi). Its knockdown leads to Wnt 5a depletion of both particle populations and reduced vesicle-mediated invasion. In conclusion, MV and exosomes are critical for MΦ-induced invasion of cancer cells since they are responsible for upregulation of MΦ-Wnt 5a as well as for its delivery to the recipient cells via a reciprocal loop. Although of different biogenesis, both populations share common features regarding function and Evi-dependent secretion of non-canonical Wnts. PMID:24185202

  6. Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche

    PubMed Central

    Luo, Lichao; Wang, Huashan; Fan, Chao; Liu, Sen

    2015-01-01

    Stem cell self-renewal versus differentiation is regulated by the niche, which provides localized molecules that favor self-renewal. In the Drosophila melanogaster female germline stem cell (GSC) niche, Decapentaplegic (Dpp), a fly transforming growth factor β molecule and well-established long-range morphogen, acts over one cell diameter to maintain the GSCs. Here, we show that Thickveins (Tkv; a type I receptor of Dpp) is highly expressed in stromal cells next to Dpp-producing cells and functions to remove excess Dpp outside the niche, thereby spatially restricting its activity. Interestingly, Tkv expression in these stromal cells is regulated by multiple Wnt ligands that are produced by the niche. Our data demonstrate a self-restraining mechanism by which the Drosophila ovarian GSC niche acts to define its own boundary. PMID:26008746

  7. Loss of WIF-1 and Wnt5a expression is related to aggressiveness of sporadic breast cancer in Tunisian patients.

    PubMed

    Trifa, Fatma; Karray-Chouayekh, Sondes; Jmal, Emna; Jmaa, Zeineb Ben; Khabir, Abdelmajid; Sellami-Boudawara, Tahia; Frikha, Mounir; Daoud, Jamel; Mokdad-Gargouri, Raja

    2013-06-01

    Activation of the Wnt/β-catenin signaling pathway is common in various human cancers. The aim of this study was to investigate the expression of 2 members of the Wnt family (WIF-1 and Wnt5a) in sporadic and hereditary breast cancer tissues. WIF-1, is a secreted antagonist that binds Wnt ligands, and therefore inhibits the canonical Wnt/β-catenin pathway. Wnt5a is one of the members of the noncanonical Wnt family that mainly acts through calcium signaling pathway. The expression of WIF-1 was analyzed by methylation-specific PCR and RT-PCR, and the level of Wnt5a ligand was quantified by RT-QPCR in breast cancer tissues. Methylation of WIF-1 was detected in 71.3 % and 81.8 % of sporadic and hereditary cases, respectively. Aberrant methylation of WIF-1 was associated with advanced TNM stage and triple negative cases in sporadic breast carcinoma (p=0.001 and p=0.037, respectively). In hereditary cases, methylation of WIF-1 correlated with age at diagnosis (p=0.027) and p53 status (p=0.035). Regarding patients' survival, WIF-1 methylated promoter conferred a reduced overall survival rate, and particularly in a group of patients with advanced TNM stage (p log rank=0.006). Furthermore, aberrant CpG methylation of the WIF-1 promoter was significantly associated with transcriptional silencing of this tumor suppressor gene in sporadic breast cancer tissues (p=0.036). On the other hand, in sporadic tumor tissues, the level of Wnt5a mRNA was significantly lower compared to normal tissues (p=0.031) and lower still in those showing more aggressive behavior, suggesting that Wnt5a, a ligand involved in the noncanonical Wnt/β-catenin pathway, could act as a tumor suppressor gene in breast cancer. PMID:23417837

  8. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    SciTech Connect

    Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. Black-Right-Pointing-Pointer Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. Black-Right-Pointing-Pointer Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. Black-Right-Pointing-Pointer Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through {beta}-catenin-dependent canonical and {beta}-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent

  9. Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active β-catenin

    PubMed Central

    Lavergne, Elise; Hendaoui, Ismaïl; Coulouarn, Cédric; Ribault, Catherine; Leseur, Julie; Eliat, Pierre-Antoine; Mebarki, Sihem; Corlu, Anne; Clément, Bruno; Musso, Orlando

    2011-01-01

    Constitutive activation of Wnt/β-catenin signaling in cancer results from mutations in pathway components, which frequently coexist with autocrine Wnt signaling or epigenetic silencing of extracellular Wnt antagonists. Among the extracellular Wnt inhibitors, the secreted frizzled-related proteins (SFRPs) are decoy receptors that contain soluble Wnt-binding frizzled domains. In addition to SFRPs, other endogenous molecules harboring frizzled motifs bind to and inhibit Wnt signaling. One of such molecules is V3Nter, a soluble SFRP-like frizzled polypeptide that binds to Wnt3a and inhibits Wnt signaling and expression of the β-catenin target genes cyclin D1 and c-myc. V3Nter is derived from the cell surface extracellular matrix component collagen XVIII. Here, we used HCT116 human colon cancer cells carrying the ΔS45 activating mutation in one of the alleles of β-catenin to show that V3Nter and SFRP-1 decrease baseline and Wnt3a-induced β-catenin stabilization. Consequently, V3Nter reduces the growth of human colorectal cancer xenografts by specifically controlling cell proliferation and cell cycle progression, without affecting angiogenesis or apoptosis, as shown by decreased [3H] thymidine (in vitro) or BrdU (in vivo) incorporation, clonogenesis assays, cell cycle analysis and magnetic resonance imaging in living mice. Additionally, V3Nter switches off the β-catenin target gene expression signature in vivo. Moreover, experiments with β-catenin allele-targeted cells showed that the ΔS45 β-catenin allele hampers, but does not abrogate inhibition of Wnt signaling by SFRP-1 or by the SFRP-like frizzled domain. Finally, neither SFRP-1 nor V3Nter affect β-catenin signaling in SW480 cells carrying non functional APC. Thus, SFRP-1 and the SFRP-like molecule V3Nter can inhibit tumor growth of β-catenin-activated tumor cells in vivo. PMID:20856206

  10. Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active β-catenin.

    PubMed

    Lavergne, E; Hendaoui, I; Coulouarn, C; Ribault, C; Leseur, J; Eliat, P-A; Mebarki, S; Corlu, A; Clément, B; Musso, O

    2011-01-27

    Constitutive activation of Wnt/β-catenin signaling in cancer results from mutations in pathway components, which frequently coexist with autocrine Wnt signaling or epigenetic silencing of extracellular Wnt antagonists. Among the extracellular Wnt inhibitors, the secreted frizzled-related proteins (SFRPs) are decoy receptors that contain soluble Wnt-binding frizzled domains. In addition to SFRPs, other endogenous molecules harboring frizzled motifs bind to and inhibit Wnt signaling. One of such molecules is V3Nter, a soluble SFRP-like frizzled polypeptide that binds to Wnt3a and inhibits Wnt signaling and expression of the β-catenin target genes cyclin D1 and c-myc. V3Nter is derived from the cell surface extracellular matrix component collagen XVIII. Here, we used HCT116 human colon cancer cells carrying the ΔS45 activating mutation in one of the alleles of β-catenin to show that V3Nter and SFRP-1 decrease baseline and Wnt3a-induced β-catenin stabilization. Consequently, V3Nter reduces the growth of human colorectal cancer xenografts by specifically controlling cell proliferation and cell cycle progression, without affecting angiogenesis or apoptosis, as shown by decreased [(3)H]-thymidine (in vitro) or BrdU (in vivo) incorporation, clonogenesis assays, cell cycle analysis and magnetic resonance imaging in living mice. Additionally, V3Nter switches off the β-catenin target gene expression signature in vivo. Moreover, experiments with β-catenin allele-targeted cells showed that the ΔS45 β-catenin allele hampers, but does not abrogate, inhibition of Wnt signaling by SFRP-1 or by the SFRP-like frizzled domain. Finally, neither SFRP-1 nor V3Nter affect β-catenin signaling in SW480 cells carrying nonfunctional Adenomatous polyposis coli. Thus, SFRP-1 and the SFRP-like molecule V3Nter can inhibit tumor growth of β-catenin-activated tumor cells in vivo. PMID:20856206

  11. High glucose and hyperglycemic sera from type 2 diabetic patients impair DC differentiation by inducing ROS and activating Wnt/β-catenin and p38 MAPK.

    PubMed

    Gilardini Montani, Maria Saveria; Granato, Marisa; Cuomo, Laura; Valia, Sandro; Di Renzo, Livia; D'Orazi, Gabriella; Faggioni, Alberto; Cirone, Mara

    2016-04-01

    Type 2 is the type of diabetes with higher prevalence in contemporary time, representing about 90% of the global cases of diabetes. In the course of diabetes, several complications can occur, mostly due to hyperglycemia and increased reactive oxygen species (ROS) production. One of them is represented by an increased susceptibility to microbial infections and by a reduced capacity to clear them. Therefore, knowing the impact of hyperglycemia on immune system functionality is of utmost importance for the management of the disease. In this study, we show that medium containing high glucose reduced the in-vitro differentiation of monocytes into functional DCs and their activation mediated by PAMPs or DAMPs. Most importantly, the same effects were mediated by the hyperglycemic sera derived by type 2 diabetic patients, mimicking a more physiologic condition. DC dysfunction caused by hyperglycemia may be involved in the inefficient control of infections observed in diabetic patients, given the pivotal role of these cells in both the innate and adaptive immune response. Searching for the molecular mechanisms underlying DC dysfunction, we found that canonical Wnt/β-catenin and p38 MAPK pathways were activated in the DCs differentiated either in the presence of high glucose or of hyper-glycemic sera. Interestingly, the activation of these pathways and the DC immune dysfunction were partially counteracted by the anti-oxidant quercetin, a flavonoid already known to exert several beneficial effects in diabetes. PMID:26769359

  12. WNT10B/β-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer

    PubMed Central

    Wend, Peter; Runke, Stephanie; Wend, Korinna; Anchondo, Brenda; Yesayan, Maria; Jardon, Meghan; Hardie, Natalie; Loddenkemper, Christoph; Ulasov, Ilya; Lesniak, Maciej S; Wolsky, Rebecca; Bentolila, Laurent A; Grant, Stephen G; Elashoff, David; Lehr, Stephan; Latimer, Jean J; Bose, Shikha; Sattar, Husain; Krum, Susan A; Miranda-Carboni, Gustavo A

    2013-01-01

    Wnt/β-catenin signalling has been suggested to be active in basal-like breast cancer. However, in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of β-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active β-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical β-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/β-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients. PMID:23307470

  13. Insulin Receptor-Overexpressing β-Cells Ameliorate Hyperglycemia in Diabetic Rats through Wnt Signaling Activation

    PubMed Central

    Lee, Moon-Kyu

    2013-01-01

    To investigate the therapeutic efficacy and mechanism of β-cells with insulin receptor (IR) overexpression on diabetes mellitus (DM), rat insulinoma (INS-1) cells were engineered to stably express human insulin receptor (INS-IR cells), and subsequently transplanted into streptozotocin- induced diabetic rats. Compared with INS-1 cells, INS-IR cells showed improved β-cell function, including the increase in glucose utilization, calcium mobilization, and insulin secretion, and exhibited a higher rate of cell proliferation, and maintained lower levels of blood glucose in diabetic rats. These results were attributed to the increase of β-catenin/PPARγ complex bindings to peroxisome proliferator response elements in rat glucokinase (GK) promoter and the prolongation of S-phase of cell cycle by cyclin D1. These events resulted from more rapid and higher phosphorylation levels of insulin-signaling intermediates, including insulin receptor substrate (IRS)-1/IRS-2/phosphotylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog (AKT) 1, and the consequent enhancement of β-catenin nuclear translocation and Wnt responsive genes including GK and cyclin D1. Indeed, the higher functionality and proliferation shown in INS-IR cells were offset by β-catenin, cyclin D1, GK, AKT1, and IRS-2 gene depletion. In addition, the promotion of cell proliferation and insulin secretion by Wnt signaling activation was shown by 100 nM insulin treatment, and to a similar degree, was shown in INS-IR cells. In this regard, this study suggests that transferring INS-IR cells into diabetic animals is an effective and feasible DM treatment. Accordingly, the method might be a promising alternative strategy for treatment of DM given the adverse effects of insulin among patients, including the increased risk of modest weight gain and hypoglycemia. Additionally, this study demonstrates that the novel mechanism of cross-talk between insulin and Wnt signaling plays a primary role in the higher

  14. Bortezomib induces canonical nuclear factor-kappaB activation in multiple myeloma cells.

    PubMed

    Hideshima, Teru; Ikeda, Hiroshi; Chauhan, Dharminder; Okawa, Yutaka; Raje, Noopur; Podar, Klaus; Mitsiades, Constantine; Munshi, Nikhil C; Richardson, Paul G; Carrasco, Ruben D; Anderson, Kenneth C

    2009-07-30

    Bortezomib is a proteasome inhibitor with remarkable preclinical and clinical antitumor activity in multiple myeloma (MM) patients. The initial rationale for its use in MM was inhibition of nuclear factor (NF)-kappaB activity by blocking proteasomal degradation of inhibitor of kappaBalpha (IkappaBalpha). Bortezomib inhibits inducible NF-kappaB activity; however, its impact on constitutive NF-kappaB activity in MM cells has not yet been defined. In this study, we demonstrate that bortezomib significantly down-regulated IkappaBalpha expression and triggered NF-kappaB activation in MM cell lines and primary tumor cells from MM patients. Importantly, no inhibition of p65 (RelA) nuclear translocation was recognized after bortezomib treatment in a murine xenograft model bearing human MM cells. Bortezomib-induced NF-kappaB activation was mediated via the canonical pathway. Moreover, other classes of proteasome inhibitors also induced IkappaBalpha down-regulation associated with NF-kappaB activation. Molecular mechanisms whereby bortezomib induced IkappaBalpha down-regulation were further examined. Bortezomib triggered phosphorylation of IkappaB kinase (IKKbeta) and its upstream receptor-interacting protein 2, whereas IKKbeta inhibitor MLN120B blocked bortezomib-induced IkappaBalpha down-regulation and NF-kappaB activation, indicating receptor-interacting protein 2/IKKbeta signaling plays crucial role in bortezomib-induced NF-kappaB activation. Moreover, IKKbeta inhibitors enhanced bortezomib-induced cytotoxicity. Our studies therefore suggest that bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-kappaB activity in MM cells. PMID:19436050

  15. BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

    PubMed Central

    de Jesus Perez, Vinicio A.; Ali, Ziad; Alastalo, Tero-Pekka; Ikeno, Fumiaki; Sawada, Hirofumi; Lai, Ying-Ju; Kleisli, Thomas; Spiekerkoetter, Edda; Qu, Xiumei; Rubinos, Laura H.; Ashley, Euan; Amieva, Manuel; Dedhar, Shoukat

    2011-01-01

    We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)–β-catenin (βC) and Wnt–planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt–dependent manner, induces βC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt–PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1–mediated motility. Transfection of a Dvl mutant that binds βC without activating RhoA-Rac1 not only prevents BMP-2–mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent βC activity. Interfering with the Dvl-dependent Wnt–PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2–mediated tandem activation of Wnt–βC and Wnt–PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations. PMID:21220513

  16. Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

    PubMed Central

    He, Fenglei; Xiong, Wei; Wang, Ying; Li, Lu; Liu, Chao; Yamagami, Takashi; Taketo, Makoto M.; Zhou, Chengji; Chen, YiPing

    2010-01-01

    The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE. PMID:21185284

  17. Macrophage Activation in Pediatric Nonalcoholic Fatty Liver Disease (NAFLD) Correlates with Hepatic Progenitor Cell Response via Wnt3a Pathway

    PubMed Central

    Renzi, Anastasia; De Stefanis, Cristiano; Stronati, Laura; Franchitto, Antonio; Alisi, Anna; Onori, Paolo; De Vito, Rita; Alpini, Gianfranco; Gaudio, Eugenio

    2016-01-01

    Non-alcoholic fatty liver disease is one of the most important causes of liver-related morbidity in children. In non-alcoholic fatty liver disease, the activation of liver resident macrophage pool is a central event in the progression of liver injury. The aims of the present study were to evaluate the polarization of liver macrophages and the possible role of Wnt3a production by macrophages in hepatic progenitor cell response in the progression of pediatric non-alcoholic fatty liver disease. 32 children with biopsy-proven non-alcoholic fatty liver disease were included. 20 out of 32 patients were treated with docosahexaenoic acid for 18 months and biopsies at the baseline and after 18 months were included. Hepatic progenitor cell activation, macrophage subsets and Wnt/β-catenin pathway were evaluated by immunohistochemistry and immunofluorescence. Our results indicated that in pediatric non-alcoholic fatty liver disease, pro-inflammatory macrophages were the predominant subset. Macrophage polarization was correlated with Non-alcoholic fatty liver disease Activity Score, ductular reaction, and portal fibrosis; docosahexaenoic acid treatment determined a macrophage polarization towards an anti-inflammatory phenotype in correlation with the reduction of serum inflammatory cytokines, with increased macrophage apoptosis, and with the up-regulation of macrophage Wnt3a expression; macrophage Wnt3a expression was correlated with β-catenin phosphorylation in hepatic progenitor cells and signs of commitment towards hepatocyte fate. In conclusion, macrophage polarization seems to have a key role in the progression of pediatric non-alcoholic fatty liver disease; the modulation of macrophage polarization could drive hepatic progenitor cell response by Wnt3a production. PMID:27310371

  18. Organized emergence of multiple-generations of teeth in snakes is dysregulated by activation of Wnt/beta-catenin signalling.

    PubMed

    Gaete, Marcia; Tucker, Abigail S

    2013-01-01

    In contrast to mammals, most reptiles constantly regenerate their teeth. In the snake, the epithelial dental lamina ends in a successional lamina, which proliferates and elongates forming multiple tooth generations, all linked by a permanent dental lamina. To investigate the mechanisms used to control the initiation of new tooth germs in an ordered sequential pattern we utilized the polyphodont (multiple-generation) corn snake (Pantherophis guttatus). We observed that the dental lamina expressed the transcription factor Sox2, a multipotent stem cell marker, whereas the successional lamina cells expressed the transcription factor Lef1, a Wnt/β-catenin pathway target gene. Activation of the Wnt/β-catenin pathway in culture increased the number of developing tooth germs, in comparison to control untreated cultures. These additional tooth germs budded off from ectopic positions along the dental lamina, rather than in an ordered sequence from the successional lamina. Wnt/β-catenin activation enhanced cell proliferation, particularly in normally non-odontogenic regions of the dental lamina, which widely expressed Lef1, restricting the Sox2 domain. This suggests an expansion of the successional lamina at the expense of the dental lamina. Activation of the Wnt/β-catenin pathway in cultured snake dental organs, therefore, led to changes in proliferation and to the molecular pattern of the dental lamina, resulting in loss of the organised emergence of tooth germs. These results suggest that epithelial compartments are critical for the arrangement of organs that develop in sequence, and highlight the role of Wnt/β-catenin signalling in such processes. PMID:24019968

  19. Organized Emergence of Multiple-Generations of Teeth in Snakes Is Dysregulated by Activation of Wnt/Beta-Catenin Signalling

    PubMed Central

    Gaete, Marcia; Tucker, Abigail S.

    2013-01-01

    In contrast to mammals, most reptiles constantly regenerate their teeth. In the snake, the epithelial dental lamina ends in a successional lamina, which proliferates and elongates forming multiple tooth generations, all linked by a permanent dental lamina. To investigate the mechanisms used to control the initiation of new tooth germs in an ordered sequential pattern we utilized the polyphodont (multiple-generation) corn snake (Pantherophis guttatus). We observed that the dental lamina expressed the transcription factor Sox2, a multipotent stem cell marker, whereas the successional lamina cells expressed the transcription factor Lef1, a Wnt/β-catenin pathway target gene. Activation of the Wnt/β-catenin pathway in culture increased the number of developing tooth germs, in comparison to control untreated cultures. These additional tooth germs budded off from ectopic positions along the dental lamina, rather than in an ordered sequence from the successional lamina. Wnt/β-catenin activation enhanced cell proliferation, particularly in normally non-odontogenic regions of the dental lamina, which widely expressed Lef1, restricting the Sox2 domain. This suggests an expansion of the successional lamina at the expense of the dental lamina. Activation of the Wnt/β-catenin pathway in cultured snake dental organs, therefore, led to changes in proliferation and to the molecular pattern of the dental lamina, resulting in loss of the organised emergence of tooth germs. These results suggest that epithelial compartments are critical for the arrangement of organs that develop in sequence, and highlight the role of Wnt/β-catenin signalling in such processes. PMID:24019968

  20. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    DOE PAGESBeta

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active sitemore » metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.« less

  1. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    NASA Astrophysics Data System (ADS)

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-01

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5‧-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  2. Interactions between SOX factors and Wnt/beta-catenin signaling in development and disease.

    PubMed

    Kormish, Jay D; Sinner, Débora; Zorn, Aaron M

    2010-01-01

    The SOX family of transcription factors have emerged as modulators of canonical Wnt/beta-catenin signaling in diverse development and disease contexts. There are over 20 SOX proteins encoded in the vertebrate genome and recent evidence suggests that many of these can physically interact with beta-catenin and modulate the transcription of Wnt-target genes. The precise mechanisms by which SOX proteins regulate beta-catenin/TCF activity are still being resolved and there is evidence to support a number of models including: protein-protein interactions, the binding of SOX factors to Wnt-target gene promoters, the recruitment of co-repressors or co-activators, modulation of protein stability, and nuclear translocation. In some contexts, Wnt signaling also regulates SOX expression resulting in feedback regulatory loops that fine-tune cellular responses to beta-catenin/TCF activity. In this review, we summarize the examples of Sox-Wnt interactions and examine the underlying mechanisms of this potentially widespread and underappreciated mode of Wnt-regulation. PMID:19655378

  3. Wnt-signalling pathways and microRNAs network in carcinogenesis: experimental and bioinformatics approaches.

    PubMed

    Onyido, Emenike K; Sweeney, Eloise; Nateri, Abdolrahman Shams

    2016-01-01

    Over the past few years, microRNAs (miRNAs) have not only emerged as integral regulators of gene expression at the post-transcriptional level but also respond to signalling molecules to affect cell function(s). miRNAs crosstalk with a variety of the key cellular signalling networks such as Wnt, transforming growth factor-β and Notch, control stem cell activity in maintaining tissue homeostasis, while if dysregulated contributes to the initiation and progression of cancer. Herein, we overview the molecular mechanism(s) underlying the crosstalk between Wnt-signalling components (canonical and non-canonical) and miRNAs, as well as changes in the miRNA/Wnt-signalling components observed in the different forms of cancer. Furthermore, the fundamental understanding of miRNA-mediated regulation of Wnt-signalling pathway and vice versa has been significantly improved by high-throughput genomics and bioinformatics technologies. Whilst, these approaches have identified a number of specific miRNA(s) that function as oncogenes or tumour suppressors, additional analyses will be necessary to fully unravel the links among conserved cellular signalling pathways and miRNAs and their potential associated components in cancer, thereby creating therapeutic avenues against tumours. Hence, we also discuss the current challenges associated with Wnt-signalling/miRNAs complex and the analysis using the biomedical experimental and bioinformatics approaches. PMID:27590724

  4. Take the Wnt out of the inflammatory sails: modulatory effects of Wnt in airway diseases.

    PubMed

    Reuter, Sebastian; Beckert, Hendrik; Taube, Christian

    2016-02-01

    Bronchial asthma and chronic obstructive pulmonary disease (COPD) are chronic diseases that are associated with inflammation and structural changes in the airways and lungs. Recent findings have implicated Wnt pathways in critically regulating inflammatory responses, especially in asthma. Furthermore, canonical and noncanonical Wnt pathways are involved in structural changes such as airway remodeling, goblet cell metaplasia, and airway smooth muscle (ASM) proliferation. In COPD, Wnt pathways are not only associated with structural changes in the airways but also involved in the development of emphysema. The present review summarizes the role and function of the canonical and noncanonical Wnt pathway with regard to airway inflammation and structural changes in asthma and COPD. Further identification of the role and function of different Wnt molecules and pathways could help to develop novel therapeutic options for these diseases. PMID:26595171

  5. Dlx-2 is implicated in TGF-β- and Wnt-induced epithelial-mesenchymal, glycolytic switch, and mitochondrial repression by Snail activation.

    PubMed

    Lee, Su Yeon; Jeon, Hyun Min; Ju, Min Kyung; Jeong, Eui Kyong; Kim, Cho Hee; Yoo, Mi-Ae; Park, Hye Gyeong; Han, Song Iy; Kang, Ho Sung

    2015-04-01

    Epithelial-mesenchymal transition (EMT) and oncogenic metabolism (including glycolytic switch) are important for tumor development and progression. Here, we show that Dlx-2, one of distal-less (Dlx) homeobox genes, induces EMT and glycolytic switch by activation of Snail. In addition, it was induced by TGF-β and Wnt and regulates TGF-β- and Wnt-induced EMT and glycolytic switch by activating Snail. We also found that TGF-β/Wnt suppressed cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, in a Dlx-2/Snail-dependent manner. TGF-β/Wnt appeared to downregulate the expression of various COX subunits including COXVIc, COXVIIa and COXVIIc; among these COX subunits, COXVIc was a common target of TGF-β, Wnt, Dlx-2 and Snail, indicating that COXVIc downregulation plays an important role(s) in TGF-β/Wnt-induced COX inhibition. Taken together, our results showed that Dlx-2 is involved in TGF-β- and Wnt-induced EMT, glycolytic switch, and mitochondrial repression by Snail activation. PMID:25651912

  6. Promiscuous Mutations Activate the Non-Canonical NF-kB Pathway in Multiple Myeloma

    PubMed Central

    Keats, Jonathan J.; Fonseca, Rafael; Chesi, Marta; Schop, Roelandt; Baker, Angela; Chng, Wee-Joo; Van Wier, Scott; Tiedemann, Rodger; Shi, Chang-Xin; Sebag, Michael; Braggio, Esteban; Henry, Travis; Zhu, Yuan-Xiao; Fogle, Homer; Price-Troska, Tammy; Ahmann, Gregory; Mancini, Catherine; Brents, Leslie A.; Kumar, Shaji; Greipp, Philip; Dispenzieri, Angela; Bryant, Barb; Mulligan, George; Bruhn, Laurakay; Barrett, Michael; Valdez, Riccardo; Trent, Jeff; Stewart, A. Keith; Carpten, John; Bergsagel, P. Leif

    2007-01-01

    Summary Activation of NF-kB has been noted in many tumor types, however only rarely has this been linked to an underlying genetic mutation. An integrated analysis of high-density oligonucleotide array CGH and gene expression profiling data from 155 multiple myeloma samples identified a promiscuous array of abnormalities contributing to the dysregulation of NF-kB in approximately 20% of patients. We report mutations in ten genes causing the inactivation of TRAF2, TRAF3, CYLD, cIAP1/cIAP2, and activation of NFKB1, NFKB2, CD40, LTBR, TACI, and NIK that result primarily in constitutive activation of the non-canonical NF-kB pathway, with the single most common abnormality being inactivation of TRAF3. These results highlight the critical importance of the NF-kB pathway in the pathogenesis of multiple myeloma. PMID:17692805

  7. The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity.

    PubMed

    Surmann-Schmitt, Cordula; Sasaki, Takako; Hattori, Takako; Eitzinger, Nicole; Schett, Georg; von der Mark, Klaus; Stock, Michael

    2012-05-01

    Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment. PMID:21928342

  8. MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells

    PubMed Central

    Ploper, Diego; Taelman, Vincent F.; Robert, Lidia; Perez, Brian S.; Titz, Björn; Chen, Hsiao-Wang; Graeber, Thomas G.; von Euw, Erika; Ribas, Antoni; De Robertis, Edward M.

    2015-01-01

    Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer. PMID:25605940

  9. MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells.

    PubMed

    Ploper, Diego; Taelman, Vincent F; Robert, Lidia; Perez, Brian S; Titz, Björn; Chen, Hsiao-Wang; Graeber, Thomas G; von Euw, Erika; Ribas, Antoni; De Robertis, Edward M

    2015-02-01

    Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer. PMID:25605940

  10. Functional analysis of centipede development supports roles for Wnt genes in posterior development and segment generation.

    PubMed

    Hayden, Luke; Schlosser, Gerhard; Arthur, Wallace

    2015-01-01

    The genes of the Wnt family play important and highly conserved roles in posterior growth and development in a wide range of animal taxa. Wnt genes also operate in arthropod segmentation, and there has been much recent debate regarding the relationship between arthropod and vertebrate segmentation mechanisms. Due to its phylogenetic position, body form, and possession of many (11) Wnt genes, the centipede Strigamia maritima is a useful system with which to examine these issues. This study takes a functional approach based on treatment with lithium chloride, which causes ubiquitous activation of canonical Wnt signalling. This is the first functional developmental study performed in any of the 15,000 species of the arthropod subphylum Myriapoda. The expression of all 11 Wnt genes in Strigamia was analyzed in relation to posterior development. Three of these genes, Wnt11, Wnt5, and WntA, were strongly expressed in the posterior region and, thus, may play important roles in posterior developmental processes. In support of this hypothesis, LiCl treatment of S. maritima embryos was observed to produce posterior developmental defects and perturbations in AbdB and Delta expression. The effects of LiCl differ depending on the developmental stage treated, with more severe effects elicited by treatment during germband formation than by treatment at later stages. These results support a role for Wnt signalling in conferring posterior identity in Strigamia. In addition, data from this study are consistent with the hypothesis of segmentation based on a "clock and wavefront" mechanism operating in this species. PMID:25627713

  11. Triple SILAC to determine stimulus specific interactions in the Wnt pathway.

    PubMed

    Hilger, Maximiliane; Mann, Matthias

    2012-02-01

    Many important regulatory functions are performed by dynamic multiprotein complexes that adapt their composition and activity in response to different stimuli. Here we employ quantitative affinity purification coupled with mass spectrometry to efficiently separate background from specific interactors but add an additional quantitative dimension to explicitly characterize stimulus-dependent interactions. This is accomplished by SILAC in a triple-labeling format, in which pull-downs with bait, with bait and stimulus, and without bait are quantified against each other. As baits, we use full-length proteins fused to the green fluorescent protein and expressed under endogenous control. We applied this technology to Wnt signaling, which is important in development, tissue homeostasis, and cancer, and investigated interactions of the key components APC, Axin-1, DVL2, and CtBP2 with differential pathway activation. Our screens identify many known Wnt signaling complex components and link novel candidates to Wnt signaling, including FAM83B and Girdin, which we found as interactors to multiple Wnt pathway players. Girdin binds to DVL2 independent of stimulation with the ligand Wnt3a but to Axin-1 and APC in a stimulus-dependent manner. The core destruction complex itself, which regulates beta-catenin stability as the key step in canonical Wnt signaling, remained essentially unchanged. PMID:22011079

  12. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways

    PubMed Central

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P.; Monczor, Federico

    2015-01-01

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein βγ subunits and a parallel inhibitory effect mediated by Gαq-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the Gαq-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration. PMID:26635083

  13. Magnesium inhibits Wnt/β-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

    PubMed

    Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M; Madueño, Juan A; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R

    2014-01-01

    Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro

  14. Wnt Lipidation and Modifiers in Intestinal Carcinogenesis and Cancer

    PubMed Central

    Kaemmerer, Elke; Gassler, Nikolaus

    2016-01-01

    The wingless (Wnt) signaling is suggested as a fundamental hierarchical pathway in regulation of proliferation and differentiation of cells. The Wnt ligands are small proteins of about 40 kDa essentially for regulation and initiation of the Wnt activity. They are secreted proteins requiring acylation for activity in the Wnt signaling cascade and for functional interactivity with transmembrane proteins. Dual lipidation is important for posttranslational activation of the overwhelming number of Wnt proteins and is probably involved in their spatial distribution. The intestinal mucosa, where Wnt signaling is essential in configuration and maintenance, is an established model to study Wnt proteins and their role in carcinogenesis and cancer. The intestinal crypt-villus/crypt-plateau axis, a cellular system with self-renewal, proliferation, and differentiation, is tightly coordinated by a Wnt gradient. In the review, some attention is given to Wnt3, Wnt3A, and Wnt2B as important members of the Wnt family to address the role of lipidation and modifiers of Wnt proteins in intestinal carcinogenesis. Wnt3 is an important player in establishing the Wnt gradient in intestinal crypts and is mainly produced by Paneth cells. Wnt2B is characterized as a mitochondrial protein and shuttles between mitochondria and the nucleus. Porcupine and ACSL5, a long-chain fatty acid activating enzyme, are introduced as modifiers of Wnts and as interesting strategy to targeting Wnt-driven carcinogenesis. PMID:27438855

  15. Wnt Lipidation and Modifiers in Intestinal Carcinogenesis and Cancer.

    PubMed

    Kaemmerer, Elke; Gassler, Nikolaus

    2016-01-01

    The wingless (Wnt) signaling is suggested as a fundamental hierarchical pathway in regulation of proliferation and differentiation of cells. The Wnt ligands are small proteins of about 40 kDa essentially for regulation and initiation of the Wnt activity. They are secreted proteins requiring acylation for activity in the Wnt signaling cascade and for functional interactivity with transmembrane proteins. Dual lipidation is important for posttranslational activation of the overwhelming number of Wnt proteins and is probably involved in their spatial distribution. The intestinal mucosa, where Wnt signaling is essential in configuration and maintenance, is an established model to study Wnt proteins and their role in carcinogenesis and cancer. The intestinal crypt-villus/crypt-plateau axis, a cellular system with self-renewal, proliferation, and differentiation, is tightly coordinated by a Wnt gradient. In the review, some attention is given to Wnt3, Wnt3A, and Wnt2B as important members of the Wnt family to address the role of lipidation and modifiers of Wnt proteins in intestinal carcinogenesis. Wnt3 is an important player in establishing the Wnt gradient in intestinal crypts and is mainly produced by Paneth cells. Wnt2B is characterized as a mitochondrial protein and shuttles between mitochondria and the nucleus. Porcupine and ACSL5, a long-chain fatty acid activating enzyme, are introduced as modifiers of Wnts and as interesting strategy to targeting Wnt-driven carcinogenesis. PMID:27438855

  16. Enrichment of anammox from activated sludge and its application in the CANON process.

    PubMed

    Third, K A; Paxman, J; Schmid, M; Strous, M; Jetten, M S M; Cord-Ruwisch, R

    2005-02-01

    A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH (4) (+) (g biomass)(-1) h(-1) (0.58 kg total-N m(-3) day(-1)). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus 'Brocadia anammoxidans' strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m(-3) day(-1)) for an extended period of time. PMID:15735941

  17. Activation of Wnt/β-Catenin Signaling Increases Apoptosis in Melanoma Cells Treated with Trail

    PubMed Central

    Zimmerman, Zachary F.; Kulikauskas, Rima M.; Bomsztyk, Karol; Moon, Randall T.; Chien, Andy J.

    2013-01-01

    While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation. PMID:23869245

  18. A novel Wnt5a-Frizzled4 signaling pathway mediates activity-independent dendrite morphogenesis via the distal PDZ motif of Frizzled 4.

    PubMed

    Bian, Wen-Jie; Miao, Wan-Ying; He, Shun-Ji; Wan, Zong-Fang; Luo, Zhen-Ge; Yu, Xiang

    2015-08-01

    The morphology of the dendritic tree is critical to neuronal function and neural circuit wiring. Several Wnt family members have been demonstrated to play important roles in dendrite development. However, the Wnt receptors responsible for mediating this process remain largely elusive. Using primary hippocampal neuronal cultures as a model system, we report that Frizzled4 (Fzd4), a member of the Fzd family of Wnt receptors, specifically signals downstream of Wnt5a to promote dendrite branching and growth. Interestingly, the less conserved distal PDZ binding motif of Fzd4, and not its conserved proximal Dvl-interacting PDZ motif, is required for mediating this effect. We further showed that Dvl signaled parallel to and independent of Fzd4 in promoting dendrite growth. Unlike most previously described pathways, Wnt5a/Fzd4 signaling promoted dendrite development in an activity-independent and autocrine fashion. Together, these results provide the first identification of a Wnt receptor for regulating dendrite development in the mammalian system, and demonstrate a novel function of the distal PDZ motif of Fzd4 in dendrite morphogenesis, thereby expanding our knowledge of the complex roles of Wnt signaling in neural development. PMID:25424568

  19. EGF Inhibits Wnt/β-Catenin-Induced Osteoblast Differentiation by Promoting β-Catenin Degradation.

    PubMed

    Boonanantanasarn, Kanitsak; Lee, Hye-Lim; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa; Kim, Gwan-Shik

    2015-12-01

    Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/β-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/β-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3β inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/β-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect β-catenin mRNA expression levels, but reduced β-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted β-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with β-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/β-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/β-catenin signaling by enhancing proteasomal degradation of β-catenin. PMID:26015066