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1
Krauss, S., V. Korzh, A. Fjose, and T. Johansen. "Expression of four zebrafish wnt-related genes during embryogenesis." Development 116, no. 1 (September 1, 1992): 249–59. http://dx.doi.org/10.1242/dev.116.1.249.
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The wnt gene family codes for a group of cysteine-rich, secreted proteins, which are differentially expressed in the developing embryo and are possibly involved in cellular communication. Here, we describe the polymerase chain reaction based cloning and embryonic expression patterns of four zebrafish wnt-related sequences; wnt[a], wnt[b], wnt[c] and wnt[d]. One of these genes, wnt[a], is a potential homologue of murine Wnt-3, while the other three genes most likely represent new members of the vertebrate wnt gene family. In zebrafish embryos, transcripts of wnt[a] are confined to the dorsal diencephalon, the dorsal midbrain, the rhombic lips and the dorsal portions of the spinal cord. wnt[b] is expressed in the tail bud and at considerably lower levels in the mesoderm of the head. wnt[c] transcripts are present within the diencephalon and the posterior midbrain whereas wnt[d] shows a surprisingly similar expression pattern to zebrafish wnt-1. By analogy to wnt-1, it is likely that the members of the zebrafish wnt gene family play an important role in cell-to-cell signalling during pattern formation in the neural tube and the tail bud.
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Caspi, Elanite, and Rina Rosin-Arbesfeld. "A Novel Functional Screen in Human Cells Identifies MOCA as a Negative Regulator of Wnt Signaling." Molecular Biology of the Cell 19, no. 11 (November 2008): 4660–74. http://dx.doi.org/10.1091/mbc.e07-10-1046.
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Aberrant Wnt signal transduction is involved in many human diseases such as cancer and neurodegenerative disorders. The key effector protein of the canonical Wnt pathway is β-catenin, which functions with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate gene transcription that leads to expression of Wnt target genes. In this study we provide results obtained from a novel functional screen of a human brain cDNA library used to identify 63 genes that are putative negative Wnt regulators. These genes were divided into eight functional groups that include known canonical and noncanonical Wnt pathway components and genes that had not yet been assigned to the Wnt pathway. One of the groups, the presenilin-binding proteins, contains the modifier of cell adhesion (MOCA) gene. We show that MOCA is a novel inhibitor of Wnt/β-catenin signaling. MOCA forms a complex with β-catenin and inhibits transcription of known Wnt target genes. Epistasis experiments indicate that MOCA acts to reduce the levels of nuclear β-catenin, increase the levels of membrane-bound β-catenin, and enhances cell–cell adhesion. Therefore, our data indicate that MOCA is a novel Wnt negative regulator and demonstrate that this screening approach can be a rapid means for isolation of new Wnt regulators.
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Khan, Naveed I., Kenneth Francis Bradstock, and Linda J. Bendall. "The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells." Blood 108, no. 11 (November 16, 2006): 1850. http://dx.doi.org/10.1182/blood.v108.11.1850.1850.
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Abstract Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.
4
Gerlach, Jan P., Ingrid Jordens, Daniele V. F. Tauriello, Ineke van ‘t Land-Kuper, Jeroen M. Bugter, Ivar Noordstra, Johanneke van der Kooij, et al. "TMEM59 potentiates Wnt signaling by promoting signalosome formation." Proceedings of the National Academy of Sciences 115, no. 17 (April 9, 2018): E3996—E4005. http://dx.doi.org/10.1073/pnas.1721321115.
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Wnt/β-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt–FZD assemblies via intramembrane interactions. Subsequently, these Wnt–FZD–TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.
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Wöhrle, Simon, Britta Wallmen, and Andreas Hecht. "Differential Control of Wnt Target Genes Involves Epigenetic Mechanisms and Selective Promoter Occupancy by T-Cell Factors." Molecular and Cellular Biology 27, no. 23 (October 8, 2007): 8164–77. http://dx.doi.org/10.1128/mcb.00555-07.
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ABSTRACT Canonical Wnt signaling and its nuclear effectors, β-catenin and the family of T-cell factor (TCF) DNA-binding proteins, belong to the small number of regulatory systems which are repeatedly used for context-dependent control of distinct genetic programs. The apparent ability to elicit a large variety of transcriptional responses necessitates that β-catenin and TCFs distinguish precisely between genes to be activated and genes to remain silent in a specific context. How this is achieved is unclear. Here, we examined patterns of Wnt target gene activation and promoter occupancy by TCFs in different mouse cell culture models. Remarkably, within a given cell type only Wnt-responsive promoters are bound by specific subsets of TCFs, whereas nonresponsive Wnt target promoters remain unoccupied. Wnt-responsive, TCF-bound states correlate with DNA hypomethylation, histone H3 hyperacetylation, and H3K4 trimethylation. Inactive, nonresponsive promoter chromatin shows DNA hypermethylation, is devoid of active histone marks, and additionally can show repressive H3K27 trimethylation. Furthermore, chromatin structural states appear to be independent of Wnt pathway activity. Apparently, cell-type-specific regulation of Wnt target genes comprises multilayered control systems. These involve epigenetic modifications of promoter chromatin and differential promoter occupancy by functionally distinct TCF proteins, which together determine susceptibility to Wnt signaling.
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Eisenberg, Leonard M., and Carol A. Eisenberg. "Evaluating the Role of Wnt Signal Transduction in Promoting the Development of the Heart." Scientific World JOURNAL 7 (2007): 161–76. http://dx.doi.org/10.1100/tsw.2007.71.
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Wnts are a family of secreted signaling proteins that are encoded by 19 distinct genes in the vertebrate genome. These molecules initiate several signal transduction pathways: the canonical Wnt, Wnt/Ca2+, and Wnt/planar cell polarity pathways. Wnt proteins have major impact on embryonic development, tumor progression, and stem cell differentiation. Wnt signal transduction also influences the formation of the heart, yet many issues concerning the involvement of Wnt regulation in initiating cardiac development remain unresolved. In this review, we will examine the published record to discern (a) what has been shown by experimental studies on the participation of Wnt signaling in cardiogenesis, and (b) what are the important questions that need to be addressed to understand the importance and function of Wnt signal transduction in facilitating the development of the heart.
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Zhang, Shu, Tolga Çagatay, Manami Amanai, Mei Zhang, Janine Kline, Diego H. Castrillon, Raheela Ashfaq, Orhan K. Öz, and Keith A. Wharton. "Viable Mice with Compound Mutations in the Wnt/Dvl Pathway Antagonists nkd1 and nkd2." Molecular and Cellular Biology 27, no. 12 (April 16, 2007): 4454–64. http://dx.doi.org/10.1128/mcb.00133-07.
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ABSTRACT Gradients of Wnt/β-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/β-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd lacZ allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/β-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.
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Ploper, Diego, Vincent F. Taelman, Lidia Robert, Brian S. Perez, Björn Titz, Hsiao-Wang Chen, Thomas G. Graeber, Erika von Euw, Antoni Ribas, and Edward M. De Robertis. "MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells." Proceedings of the National Academy of Sciences 112, no. 5 (January 20, 2015): E420—E429. http://dx.doi.org/10.1073/pnas.1424576112.
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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.
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Ye, Mittag, Schmidt, Simm, Horstkorte, and Huber. "Wnt Glycation Inhibits Canonical Signaling." Cells 8, no. 11 (October 25, 2019): 1320. http://dx.doi.org/10.3390/cells8111320.
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Glycation occurs as a non-enzymatic reaction between amino and thiol groups of proteins, lipids, and nucleotides with reducing sugars or -dicarbonyl metabolites. The chemical reaction underlying is the Maillard reaction leading to the formation of a heterogeneous group of compounds named advanced glycation end products (AGEs). Deleterious effects have been observed to accompany glycation such as alterations of protein structure and function resulting in crosslinking and accumulation of insoluble protein aggregates. A substantial body of evidence associates glycation with aging. Wnt signaling plays a fundamental role in stem cell biology as well as in regeneration and repair mechanisms. Emerging evidence implicates that changes in Wnt/-catenin pathway activity contribute to the aging process. Here, we investigated the effect of glycation of Wnt3a on its signaling activity. Methods: Glycation was induced by treatment of Wnt3a-conditioned medium (CM) with glyoxal (GO). Effects on Wnt3a signaling activity were analyzed by Topflash/Fopflash reporter gene assay, co-immunoprecipitation, and quantitative RT-PCR. Results: Our data show that GO-treatment results in glycation of Wnt3a. Glycated Wnt3a suppresses -catenin transcriptional activity in reporter gene assays, reduced binding of -catenin to T-cell factor 4 (TCF-4) and extenuated transcription of Wnt/-catenin target genes. Conclusions: GO-induced glycation impairs Wnt3a signaling function.
10
Blache, Philippe, Marc van de Wetering, Isabelle Duluc, Claire Domon, Philippe Berta, Jean-Noël Freund, Hans Clevers, and Philippe Jay. "SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes." Journal of Cell Biology 166, no. 1 (July 5, 2004): 37–47. http://dx.doi.org/10.1083/jcb.200311021.
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TCF and SOX proteins belong to the high mobility group box transcription factor family. Whereas TCFs, the transcriptional effectors of the Wnt pathway, have been widely implicated in the development, homeostasis and disease of the intestine epithelium, little is known about the function of the SOX proteins in this tissue. Here, we identified SOX9 in a SOX expression screening in the mouse fetal intestine. We report that the SOX9 protein is expressed in the intestinal epithelium in a pattern characteristic of Wnt targets. We provide in vitro and in vivo evidence that a bipartite β-catenin/TCF4 transcription factor, the effector of the Wnt signaling pathway, is required for SOX9 expression in epithelial cells. Finally, in colon epithelium-derived cells, SOX9 transcriptionally represses the CDX2 and MUC2 genes, normally expressed in the mature villus cells of the intestinal epithelium, and may therefore contribute to the Wnt-dependent maintenance of a progenitor cell phenotype.
11
Salinas, P. C., C. Fletcher, N. G. Copeland, N. A. Jenkins, and R. Nusse. "Maintenance of Wnt-3 expression in Purkinje cells of the mouse cerebellum depends on interactions with granule cells." Development 120, no. 5 (May 1, 1994): 1277–86. http://dx.doi.org/10.1242/dev.120.5.1277.
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Wnt genes encode secreted proteins implicated in cell fate changes during development. To define specific cell populations in which Wnt genes act, we have examined Wnt expression in the cerebellum. This part of the brain has a relatively simple structure and contains well-characterized cell populations. We found that Wnt-3 is expressed during development of the cerebellum and that expression is restricted to the Purkinje cell layer in the adult. Wnt-3 expression in Purkinje cells increases postnatally as granule cells start to make contacts with Purkinje cells. To investigate whether interactions with granule cells influence Wnt-3 expression in Purkinje cells, we examined gene expression in several mouse mutants, using the expression of En-2 to follow the fate of granule cells. In the weaver mutant, in which granule cells fail to migrate and subsequently die in the external granular layer, Wnt-3 expression was normal at postnatal day 15 (P15). At that time, some granule cells are still present in the external granular layer. At P28, however, when granule cells could no longer be detected, Wnt-3 expression was almost absent. In the meander tail mutant, in which the anterior cerebellar lobes lack granule cells, Wnt-3 expression was only detected in the normal posterior lobes. Since En genes are implicated in cell-cell interactions mediated by Wnt genes, we examined En-2/En-2 mutant mice, finding normal Wnt-3 expression, indicating that the effect of granule cells on the maintenance of Wnt-3 is not mediated by En-2. Our results show that Wnt-3 expression in Purkinje cells is modulated by their presynaptic granule cells at the time of neuronal maturation.
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Hinck, L., WJ Nelson, and J. Papkoff. "Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-catenin binding to the cell adhesion protein cadherin." Journal of Cell Biology 124, no. 5 (March 1, 1994): 729–41. http://dx.doi.org/10.1083/jcb.124.5.729.
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Wnt-1 homologs have been identified in invertebrates and vertebrates and play important roles in cellular differentiation and organization. In Drosophila, the products of the segment polarity genes wingless (the Wnt-1 homolog) and armadillo participate in a signal transduction pathway important for cellular boundary formation in embryonic development, but functional interactions between the proteins are unknown. We have examined Wnt-1 function in mammalian cells in which armadillo (beta-catenin and plakoglobin) is known to bind to and regulate cadherin cell adhesion proteins. We show that Wnt-1 expression results in the accumulation of beta-catenin and plakoglobin. In addition, binding of beta-catenin to the cell adhesion protein, cadherin, is stabilized, resulting in a concomitant increase in the strength of calcium-dependent cell-cell adhesion. Thus, a consequence of the functional interaction between Wnt-1 and armadillo family members is the strengthening of cell-cell adhesion, which may lead to the specification of cellular boundaries.
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Levay-Young, B. K., and M. Navre. "Growth and developmental regulation of wnt-2 (irp) gene in mesenchymal cells of fetal lung." American Journal of Physiology-Lung Cellular and Molecular Physiology 262, no. 6 (June 1, 1992): L672—L683. http://dx.doi.org/10.1152/ajplung.1992.262.6.l672.
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The wnt gene family encodes a group of proteins implicated as intercellular signaling molecules in vertebrate development. Because many wnt genes are also expressed in the lung, we have examined whether the wnt family member wnt-2 (irp) plays a role in lung development. We have cloned rat wnt-2 and found that this cDNA detects multiple mRNAs expressed at high levels in fetal rat lung. Much lower levels were found in adult rat lung and other tissues, including, surprisingly, the mammary gland. The wnt-2 mRNA was also detected in human fetal lung fibroblast cell lines, where the mRNA levels were dramatically regulated by growth state as well as growth factor stimulation. In situ hybridization showed that, in fetal rat lung, wnt-2 mRNA expression is restricted to the mesenchyme; levels in the developing epithelium were indistinguishable from background. Based on the known properties of other wnt proteins, our data lead us to propose that wnt-2 may play a role in lung development by mediating intercellular interaction(s) between mesenchyme and epithelium.
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Kamdem, Nestor, Yvette Roske, Dmytro Kovalskyy, Maxim O. Platonov, Oleksii Balinskyi, Annika Kreuchwig, Jörn Saupe, et al. "Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling." Magnetic Resonance 2, no. 1 (June 2, 2021): 355–74. http://dx.doi.org/10.5194/mr-2-355-2021.
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Abstract. Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ–Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure–activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50∼50 µM) and Western blotting of β-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (KD=2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.
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Tran, Bang Manh, Dustin James Flanagan, Gregor Ebert, Nadia Warner, Hoanh Tran, Theodora Fifis, Georgios Kastrappis, et al. "The Hepatitis B Virus Pre-Core Protein p22 Activates Wnt Signaling." Cancers 12, no. 6 (May 31, 2020): 1435. http://dx.doi.org/10.3390/cancers12061435.
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An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/β-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/β-catenin transcription. The effect of p22 on TCF/β-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/β-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or β-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/β-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely APC and CTNNB1. Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer.
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Brantjes, H., N. Barker, J. van Es, and H. Clevers. "TCF: Lady Justice Casting the Final Verdict on the Outcome of Wnt Signalling." Biological Chemistry 383, no. 2 (February 15, 2002): 255–61. http://dx.doi.org/10.1515/bc.2002.027.
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Abstract The Wnt signalling cascade plays an important role during embryonic patterning and cell fate determination and is highly conserved throughout evolution. Factors of the TCF/LEF HMG domain family (Tcfs) are the downstream effectors of this signal transduction pathway. Upon Wnt signalling, a cascade is initiated that results in the translocation of βcatenin to the nucleus, where it interacts with Tcf to generate a transcriptionally active complex. This bipartite transcription factor is targeted to the upstream regulatory regions of Tcf target genes. In the absence of Wnt signals, βcatenin is degraded in the cytoplasm via the ubiquitinproteasome pathway. Several proteins are instrumental in achieving this tight regulation of βcatenin levels in the cell, including adenomatous polyposis coli (APC), GSK3 β, and Axin/Conductin. Deregulation of the Wnt signalling pathway is implicated in several forms of cancer, such as colon carcinoma and melanoma. This deregulation is achieved via mutation of APC, βcatenin or Axin, resulting in elevated βcatenin levels and the presence of constitutively active Tcfβcatenin complexes in the nucleus. The accompanying inappropriate activation of target genes is considered to be a critical, early event in this carcinogenesis. In addition to regulating βcatenin levels, normal healthy cells have evolved a second level of regulation, by manipulating the activity of the Tcf proteins themselves. In the absence of Wnt signalling, Tcf complexes with several transcriptional repressor proteins ensuring active repression of Tcf target genes. In this review the dual role of Tcf proteins in the Wnt signalling cascade will be discussed.
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Dorfman, Tatiana, Yulia Pollak, Rima Sohotnik, Arnold G. Coran, Jacob Bejar, and Igor Sukhotnik. "Enhanced intestinal epithelial cell proliferation in diabetic rats correlates with β-catenin accumulation." Journal of Endocrinology 226, no. 3 (June 25, 2015): 135–43. http://dx.doi.org/10.1530/joe-14-0725.
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The Wnt/β-catenin signaling cascade is implicated in the control of stem cell activity, cell proliferation, and cell survival of the gastrointestinal epithelium. Recent evidence indicates that the Wnt/β-catenin pathway is activated under diabetic conditions. The purpose of this study was to evaluate the role of Wnt/β-catenin signaling during diabetes-induced enteropathy in a rat model. Male rats were divided into three groups: control rats received injections of vehicle; diabetic rats received injections of one dose of streptozotocin (STZ); and diabetic–insulin rats received injections of STZ and were treated with insulin given subcutaneously at a dose of 1 U/kg twice daily. Rats were killed on day 7. Wnt/β-catenin-related genes and expression of proteins was determined using real-time PCR, western blotting, and immunohistochemistry. Among 13 genes identified by real-time PCR, seven genes were upregulated in diabetic rats compared with control animals including the target genes c-Myc and Tcf4. Diabetic rats also showed a significant increase in β-catenin protein compared with control animals. Treatment of diabetic rats attenuated the stimulating effect of diabetes on intestinal cell proliferation and Wnt/β-catenin signaling. In conclusion, enhanced intestinal epithelial cell proliferation in diabetic rats correlates with β-catenin accumulation.
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Wang, Lili, Nathalie Pochet, Kristian Cibulskis, Wandi Zhang, Mike Lawrence, Quinlan L. Sievers, Erica Shefler, et al. "Whole Genome Sequencing Identifies Functional Mutations In the Wnt Pathway In CLL." Blood 116, no. 21 (November 19, 2010): 693. http://dx.doi.org/10.1182/blood.v116.21.693.693.
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Abstract Abstract 693 The molecular pathogenesis of chronic lymphocytic leukemia (CLL) has not been well-characterized. Next generation sequencing technologies provide an unprecedented opportunity to screen entire genomes for genetic changes associated with tumorigenesis. To examine the mutational spectrum in CLL, we completed Illumina sequencing of 2 genomes and 4 exomes from patients with advanced CLL in parallel with germline DNA from autologous skin fibroblasts (average coverage 66x/59x for CLL/normal). While the somatic mutation rates varied widely between individuals, ranging from 1–10 per megabase, we discovered that 2 of 6 tumors harbored novel nonsynonymous mutations in Wnt pathway members (DKK2 [R197H] from Patient 1 and BCL9 [G458S] from Patient 3), suggesting that perturbation of this pathway may be critical in CLL. By targeted quantitative RNA sequencing, we detected both mutated DKK2 and BCL9 as heterozygous mutations in tumor RNA from Patients 1 and 3, respectively. We confirmed that the Wnt pathway is in fact highly dysregulated in CLL based on gene expression analysis of 141 Wnt pathway members, in a dataset comprised of 155 CLL and 20 normal CD19+ B cell samples. In CLL cells, Wnt family members (Wnt1, Wnt2, Wnt2B, Wnt3, Wnt4, Wnt5B, Wnt9A, Wnt10A, and Wnt16) and Wnt receptors (FZD3, FZD8, FZD9, LRP6) were overexpressed. Additionally, we observed downregulation of Wnt pathway repressors (TLE1, SOX4 and NKD1) and upregulation of activators (SMAD2, DVL2, DVL3 and PYGO1). Expression of the downstream transcription factor LEF1 was strikingly elevated in CLL, and we measured consistent 3–4 fold elevation of LEF1 protein in CLL-B cells (n=10) compared to normal B cells (n=5) by intracellular staining (p= 0.002). Patient CLL cells demonstrated 5-fold greater beta-catenin mediated activation of the Wnt pathway in CLL compared to normal B cells (p=0.01), as measured by direct introduction of the LEF1/TCF1 luciferase reporter genes into primary cells. Silencing of LEF1 in CLL cells (n=5) led to increased cell death in vitro by 70–90%. Collectively, these results demonstrate that Wnt pathway activation and dysregulation contributes to CLL survival. To examine the possible effects of mutated DKK2 and BCL9 on Wnt pathway activity, we introduced plasmids encoding wild-type or mutated genes, together with those encoding Wnt1 protein and Wnt reporter genes, into 293T cells. DKK2 is a secreted factor that normally represses Wnt activation by competing with Wnt proteins for binding to its surface receptors. We observed that DKK2 [R197H] is mutated in its second cysteine-rich domain, which is necessary for its interaction with the Wnt receptor. BCL9 interacts with the LEF1/TCF transcriptional factor complex and has been described to have both repressive and activating effects. In 3 of 3 experiments, we observed that the mutated forms of both genes lost their repressive activity. The loss of repression generated for both mutated genes was most marked in the presence of low concentrations of Wnt protein. Moreover, both mutated genes act as dominant alleles, especially in the presence of low concentrations of Wnt protein. Since DKK2 is a secreted factor, we examined the effects of plasma of Patient 1 on Wnt pathway activity. In both normal donors (n=4) and CLL patients (n=4), DKK2 protein could be consistently detected at equivalent amounts in blood plasma following immunoprecipitation and immunoblotting with DKK2-specific antibodies Co-culture of 293T cells expressing the Wnt pathway luciferase reporters with Patient 1 plasma (2-10%) resulted in dramatic elevation of Wnt activation, which was abolished by prior depletion of DKK2. We observed similar Wnt activation when testing plasma from 22 of 93 CLL patients, but not from 5 normal volunteers. Further sequencing of 100 Wnt pathway members from 60 additional CLL patients is in progress to define the extent to which somatic mutation is a common mechanism by which this pathway is altered. Our studies suggest that Wnt pathway inhibitors represent a promising therapeutic approach for CLL. Disclosures: No relevant conflicts of interest to declare.
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Graba, Y., K. Gieseler, D. Aragnol, P. Laurenti, M. C. Mariol, H. Berenger, T. Sagnier, and J. Pradel. "DWnt-4, a novel Drosophila Wnt gene acts downstream of homeotic complex genes in the visceral mesoderm." Development 121, no. 1 (January 1, 1995): 209–18. http://dx.doi.org/10.1242/dev.121.1.209.
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Wnt genes encode putative cell signalling proteins which play crucial roles during development. From a library of DNA fragments associated, in vivo, with Ultrabithorax proteins, we isolated a novel Drosophila Wnt gene, DWnt-4. Neither a paralog nor an ortholog of the gene exist in the current repertoire of full-length Wnt sequences. DWnt-4 maps close (30 kb) to wingless, suggesting that the two Wnt genes derive from a duplication that occurred early in evolution, since they are significantly diverged in sequence and structure. Developmental expression of DWnt-4 partially overlaps that of wingless. The gene is transcribed following a segment polarity-like pattern in the posterior-most cells of each parasegment of the ectoderm, and at two locations that correspond to parasegments 4 and 8 of the visceral mesoderm. The control of DWnt-4 expression in the visceral mesoderm involves a network of regulatory molecules that includes Ultrabithorax and other proteins from the homeotic complex (HOM-C), as well as the TGF-beta decapentaplegic gene product.
20
Atalar, Fatmahan, Ozden Hatirnaz, Yucel Erbilgin, Ugur Ozbek, and Muge Sayitoglu. "Upregulation of FZ5 Results in Abberant Expression of Beta-Catenin and LEF/TCF Complex in Acute Myeloid Leukemia." Blood 108, no. 11 (November 16, 2006): 4316. http://dx.doi.org/10.1182/blood.v108.11.4316.4316.
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Abstract Wnt proteins form a family of secreted signalling proteins that play a key role in various developmental events such as cell differentiation, cell migration, cell polarity and cell proliferation. Activation of the Wnt/beta-catenin through canonical and non-canonical pathways has recently been shown to be crucial to the self-renewal of hematopoietic progenitors and stem cells. WNT proteins initiate the canonical (beta-catenin-regulated) signaling cascade by binding to seven-transmembrane spanning receptors of the Frizzled (FZ) family. WNT stimulation leads to beta-catenin accumulation, nuclear translocation and interaction with T-cell factor/lymphoid enhancer factor (TCF/LEF) to regulate genes important for embryonic development and proliferation. Also beta-catenin is thought to have a role in the self renewal potential of leukemic cells in AML. To determine the role of the WNT pathways in the development of acute myeloid leukaemia (AML), we studied seven WNT pathway genes (WNT10b, WNT5a, FZ5, Beta-catenin, APC, TCF1 and LEF1) in 34 AML patients by quantitative real-time PCR. Results were compared by peripheral blood samples from healthy donors by using Ct values. FZ5 gene expression showed 10 fold and LEF1gene expression showed 3 fold increase when compared to controls (p>0.01 and p=0.02 respectively). Beta-catenin was 5 fold higher in patients than normal peripheral blood samples. The WNT10b expression was slightly lower (1.5 fold) comparing to other genes. TCF1 expression level also seemed to be higher in AML patients (OR=4.2 95%CI=0.04–2.82, p=0.06). These results may represent that canonical WNT signaling takes place in AML development and provide the evidence for the involvement of FZ5 upregulation in the pathogenesis of AML through activation of the TCF/LEF mediated signaling.
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Ellwanger, Kristina, Hiroaki Saito, Philippe Clément-Lacroix, Nicole Maltry, Joachim Niedermeyer, Woon Kyu Lee, Roland Baron, Georges Rawadi, Heiner Westphal, and Christof Niehrs. "Targeted Disruption of the Wnt Regulator Kremen Induces Limb Defects and High Bone Density." Molecular and Cellular Biology 28, no. 15 (May 27, 2008): 4875–82. http://dx.doi.org/10.1128/mcb.00222-08.
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ABSTRACT Kremen1 and Kremen2 (Krm1 and Krm2) are transmembrane coreceptors for Dickkopf1 (Dkk1), an antagonist of Wnt/β-catenin signaling. The physiological relevance of Kremen proteins in mammals as Wnt modulators is unresolved. We generated and characterized Krm mutant mice and found that double mutants show enhanced Wnt signaling accompanied by ectopic postaxial forelimb digits and expanded apical ectodermal ridges. Triple mutant Krm1 −/ − Krm2 −/ − Dkk1 +/ − mice show enhanced growth of ectopic digits, indicating that Dkk1 and Krm genes genetically interact during limb development. Wnt/β-catenin signaling also plays a critical role in bone formation. Single Krm mutants show normal bone formation and bone mass, while double mutants show increased bone volume and bone formation parameters. Our study provides the first genetic evidence for a functional interaction of Kremen proteins with Dkk1 as negative regulators of Wnt/β-catenin signaling and reveals that Kremen proteins are not universally required for Dkk1 function.
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Anthony, Christin C., David J. Robbins, Yashi Ahmed, and Ethan Lee. "Nuclear Regulation of Wnt/β-Catenin Signaling: It’s a Complex Situation." Genes 11, no. 8 (August 4, 2020): 886. http://dx.doi.org/10.3390/genes11080886.
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Wnt signaling is an evolutionarily conserved metazoan cell communication pathway required for proper animal development. Of the myriad of signaling events that have been ascribed to cellular activation by Wnt ligands, the canonical Wnt/β-catenin pathway has been the most studied and best understood. Misregulation of Wnt/β-catenin signaling has been implicated in developmental defects in the embryo and major diseases in the adult. Despite the latter, no drugs that inhibit the Wnt/β-catenin pathway have been approved by the FDA. In this review, we explore the least understood step in the Wnt/β-catenin pathway—nuclear regulation of Wnt target gene transcription. We initially describe our current understanding of the importation of β-catenin into the nucleus. We then focus on the mechanism of action of the major nuclear proteins implicated in driving gene transcription. Finally, we explore the concept of a nuclear Wnt enhanceosome and propose a modified model that describes the necessary components for the transcription of Wnt target genes.
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Xiao, Hualiang, Ying Zeng, Qiushi Wang, Shirong Wei, and Xiangfeng Zhu. "A Novel Positive Feedback Loop Between NTSR1 and Wnt/β-Catenin Contributes to Tumor Growth of Glioblastoma." Cellular Physiology and Biochemistry 43, no. 5 (2017): 2133–42. http://dx.doi.org/10.1159/000484232.
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Background/Aims: Neurotensin (NTS), an intestinal hormone, is profoundly implicated in cancer progression through binding its primary receptor NTSR1. The conserved Wnt/β-Catenin pathway regulates cell proliferation and differentiation via activation of the β-catenin/T-cell factor (TCF) complex and subsequent modulation of a set of target genes. In this study, we aimed to uncover the potential connection between NTS/NTSR1 signaling and Wnt/β-Catenin pathway. Methods: Genetic silencing, pharmacological inhibition and gain-of-function studies as well as bioinformatic analysis were performed to uncover the link between NTS/ NTSR1 signaling and Wnt/β-Catenin pathway. Two inhibitors were used in vivo to evaluate the efficiency of targeting NTS/NTSR1 signaling or Wnt/β-Catenin pathway. Results: We found that NTS/NTSR1 induced the activation of mitogen-activated protein kinase (MAPK) and the NF-κB pathway, which further promoted the expression of Wnt proteins, including Wnt1, Wnt3a and Wnt5a. Meanwhile, the mRNA and protein expression levels of NTSR1 were increased by the Wnt pathway activator Wnt3a and decreased by the Wnt inhibitor iCRT3 in glioblastoma cells. Furthermore, pharmacological inhibition of NTS/NTSR1 or Wnt/β-Catenin signaling suppressed tumor growth in vitro and in vivo. Conclusion: These results reveal a positive feedback loop between NTS/NTSR1 and Wnt/β-Catenin signaling in glioblastoma cells that might be important for tumor development and provide potential therapeutic targets for glioblastoma.
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Nwaokorie, Annabelle, and Dirk Fey. "Personalised Medicine for Colorectal Cancer Using Mechanism-Based Machine Learning Models." International Journal of Molecular Sciences 22, no. 18 (September 15, 2021): 9970. http://dx.doi.org/10.3390/ijms22189970.
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Gaining insight into the mechanisms of signal transduction networks (STNs) by using critical features from patient-specific mathematical models can improve patient stratification and help to identify potential drug targets. To achieve this, these models should focus on the critical STNs for each cancer, include prognostic genes and proteins, and correctly predict patient-specific differences in STN activity. Focussing on colorectal cancer and the WNT STN, we used mechanism-based machine learning models to identify genes and proteins with significant associations to event-free patient survival and predictive power for explaining patient-specific differences of STN activity. First, we identified the WNT pathway as the most significant pathway associated with event-free survival. Second, we built linear-regression models that incorporated both genes and proteins from established mechanistic models in the literature and novel genes with significant associations to event-free patient survival. Data from The Cancer Genome Atlas and Clinical Proteomic Tumour Analysis Consortium were used, and patient-specific STN activity scores were computed using PROGENy. Three linear regression models were built, based on; (1) the gene-set of a state-of-the-art mechanistic model in the literature, (2) novel genes identified, and (3) novel proteins identified. The novel genes and proteins were genes and proteins of the extant WNT pathway whose expression was significantly associated with event-free survival. The results show that the predictive power of a model that incorporated novel event-free associated genes is better compared to a model focussing on the genes of a current state-of-the-art mechanistic model. Several significant genes that should be integrated into future mechanistic models of the WNT pathway are DVL3, FZD5, RAC1, ROCK2, GSK3B, CTB2, CBT1, and PRKCA. Thus, the study demonstrates that using mechanistic information in combination with machine learning can identify novel features (genes and proteins) that are important for explaining the STN heterogeneity between patients and their association to clinical outcomes.
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Hartmann, C., and C. J. Tabin. "Dual roles of Wnt signaling during chondrogenesis in the chicken limb." Development 127, no. 14 (July 15, 2000): 3141–59. http://dx.doi.org/10.1242/dev.127.14.3141.
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Long bones of the appendicular skeleton are formed from a cartilage template in a process known as endochondral bone development. Chondrocytes within this template undergo a progressive program of differentiation from proliferating to postmitotic prehypertrophic to hypertrophic chondrocytes, while mesenchymal cells immediately surrounding the early cartilage template form the perichondrium. Recently, members of the Wnt family of secreted signaling molecules have been implicated in regulating chondrocyte differentiation. We find that Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb: Wnt-5a is expressed in the perichondrium, Wnt-5b is expressed in a subpopulation of prehypertrophic chondrocytes and in the outermost cell layer of the perichondrium, and Wnt-4 is expressed in cells of the joint region. Misexpression experiments demonstrate that two of these Wnt molecules, Wnt-5a and Wnt-4, have opposing effects on the differentiation of chondrocytes and that these effects are mediated through divergent signaling pathways. Specifically, Wnt-5a misexpression delays the maturation of chondrocytes and the onset of bone collar formation, while Wnt-4 misexpression accelerates these two processes. Misexpression of a stabilized form of beta-catenin also results in accelerated chondrogenesis, suggesting that a beta-catenin/TCF-LEF complex is involved in mediating the positive regulatory effect of Wnt-4. A number of the genes involved in Wnt signal tranduction, including two members of the Frizzled gene family, which are believed to encode Wnt-receptors, show very dynamic and distinct expression patterns in cartilaginous elements of developing chicken limbs. Misexpression of putative dominant-negative forms of the two Frizzled proteins results in severe shortening of the infected cartilage elements due to a delay in chondrocyte maturation, indicating that an endogenous Wnt signal does indeed function to promote chondrogenic differentiation.
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Berwick, Daniel C., and Kirsten Harvey. "The importance of Wnt signalling for neurodegeneration in Parkinson's disease." Biochemical Society Transactions 40, no. 5 (September 19, 2012): 1123–28. http://dx.doi.org/10.1042/bst20120122.
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PD (Parkinson's disease) is a devastating progressive motor disorder with no available cure. Over the last two decades, an increasing number of genetic defects have been found that cause familial and idiopathic forms of PD. In parallel, the importance of Wnt signalling pathways for the healthy functioning of the adult brain and the dysregulation of these pathways in neurodegenerative disease has become apparent. Cell biological functions disrupted in PD are partially controlled by Wnt signalling pathways and proteins encoded by PARK genes have been shown to modify Wnt signalling. This suggests the prospect of targeting Wnt signalling pathways to modify PD progression.
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Działo, Edyta, Michał Rudnik, Roman Koning, Marcin Czepiel, Karolina Tkacz, Monika Baj-Krzyworzeka, Oliver Distler, Maciej Siedlar, Gabriela Kania, and Przemysław Błyszczuk. "WNT3a and WNT5a Transported by Exosomes Activate WNT Signaling Pathways in Human Cardiac Fibroblasts." International Journal of Molecular Sciences 20, no. 6 (March 21, 2019): 1436. http://dx.doi.org/10.3390/ijms20061436.
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WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20–150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3β (GSK3β), induced nuclear translocation of β-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/β-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate β-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.
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Theil, Thomas, Songül Aydin, Silke Koch, Lars Grotewold, and Ulrich Rüther. "Wnt and Bmp signalling cooperatively regulate graded Emx2 expression in the dorsal telencephalon." Development 129, no. 13 (July 1, 2002): 3045–54. http://dx.doi.org/10.1242/dev.129.13.3045.
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Pattern formation of the dorsal telencephalon is governed by a regionalisation process that leads to the formation of distinct domains, including the future hippocampus and neocortex. Recent studies have implicated signalling proteins of the Wnt and Bmp gene families as well as several transcription factors, including Gli3 and the Emx homeobox genes, in the molecular control of this process. The regulatory relationships between these genes, however, remain largely unknown. We have used transgenic analysis to investigate the upstream mechanisms for regulation of Emx2 in the dorsal telencephalon. We have identified an enhancer from the mouse Emx2 gene that drives specific expression of a lacZ reporter gene in the dorsal telencephalon. This element contains binding sites for Tcf and Smad proteins, transcriptional mediators of the Wnt and Bmp signalling pathway, respectively. Mutations of these binding sites abolish telencephalic enhancer activity, while ectopic expression of these signalling pathways leads to ectopic activation of the enhancer. These results establish Emx2 as a direct transcriptional target of Wnt and Bmp signalling and provide insights into a genetic hierarchy involving Gli3, Emx2 and Bmp and Wnt genes in the control of dorsal telencephalic development.
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Kasprzak, Aldona. "Angiogenesis-Related Functions of Wnt Signaling in Colorectal Carcinogenesis." Cancers 12, no. 12 (December 2, 2020): 3601. http://dx.doi.org/10.3390/cancers12123601.
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Aberrant activation of the Wnt/Fzd/β-catenin signaling pathway is one of the major molecular mechanisms of colorectal cancer (CRC) development and progression. On the other hand, one of the most common clinical CRC characteristics include high levels of angiogenesis, which is a key event in cancer cell dissemination and distant metastasis. The canonical Wnt/β-catenin downstream signaling regulates the most important pro-angiogenic molecules including vascular endothelial growth factor (VEGF) family members, matrix metalloproteinases (MMPs), and chemokines. Furthermore, mutations of the β-catenin gene associated with nuclear localization of the protein have been mainly detected in microsatellite unstable CRC. Elevated nuclear β-catenin increases the expression of many genes involved in tumor angiogenesis. Factors regulating angiogenesis with the participation of Wnt/β-catenin signaling include different groups of biologically active molecules including Wnt pathway components (e.g., Wnt2, DKK, BCL9 proteins), and non-Wnt pathway factors (e.g., chemoattractant cytokines, enzymatic proteins, and bioactive compounds of plants). Several lines of evidence argue for the use of angiogenesis inhibition in the treatment of CRC. In the context of this paper, components of the Wnt pathway are among the most promising targets for CRC therapy. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.
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Isani, Mubina A., Kristin Gee, Kathy Schall, Christopher R. Schlieve, Alexa Fode, Kathryn L. Fowler, and Tracy C. Grikscheit. "Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish." American Journal of Physiology-Gastrointestinal and Liver Physiology 316, no. 6 (June 1, 2019): G679—G691. http://dx.doi.org/10.1152/ajpgi.00343.2018.
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Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/β-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/β-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/β-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.
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Deogharkar, Akash, Satishkumar Vishram Singh, Harish Shrikrishna Bharambe, Raikamal Paul, Aliasgar Moiyadi, Atul Goel, Prakash Shetty, et al. "Downregulation of ARID1B, a tumor suppressor in the WNT subgroup medulloblastoma, activates multiple oncogenic signaling pathways." Human Molecular Genetics 30, no. 18 (May 5, 2021): 1721–33. http://dx.doi.org/10.1093/hmg/ddab134.
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Abstract Medulloblastoma, a common pediatric malignant brain tumor, consists of four distinct molecular subgroups WNT, SHH, Group 3 and Group 4. Exome sequencing of 11 WNT subgroup medulloblastomas from an Indian cohort identified mutations in several chromatin modifier genes, including genes of the mammalian SWI/SNF complex. The genome of WNT subgroup tumors is known to be stable except for monosomy 6. Two tumors, having monosomy 6, carried a loss of function mutation in the ARID1B gene located on chromosome 6. ARID1B expression is also lower in the WNT subgroup tumors compared to other subgroups and normal cerebellar tissues that could result in haploinsufficiency. The short hairpin RNA-mediated knockdown of ARID1B expression resulted in a significant increase in the malignant potential of medulloblastoma cells. Transcriptome sequencing identified upregulation of several genes encoding cell adhesion proteins, matrix metalloproteases indicating the epithelial–mesenchymal transition. The ARID1B knockdown also upregulated ERK1/ERK2 and PI3K/AKT signaling with a decrease in the expression of several negative regulators of these pathways. The expression of negative regulators of the WNT signaling like TLE1, MDFI, GPX3, ALX4, DLC1, MEST decreased upon ARID1B knockdown resulting in the activation of the canonical WNT signaling pathway. Synthetic lethality has been reported between SWI/SNF complex mutations and EZH2 inhibition, suggesting EZH2 inhibition as a possible therapeutic modality for WNT subgroup medulloblastomas. Thus, the identification of ARID1B as a tumor suppressor and its downregulation resulting in the activation of multiple signaling pathways opens up opportunities for novel therapeutic modalities for the treatment of WNT subgroup medulloblastoma.
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Heijink, Irene H., Harold G. de Bruin, Robin Dennebos, Marnix R. Jonker, Jacobien A. Noordhoek, Corry-Anke Brandsma, Maarten van den Berge, and Dirkje S. Postma. "Cigarette smoke-induced epithelial expression of WNT-5B: implications for COPD." European Respiratory Journal 48, no. 2 (April 28, 2016): 504–15. http://dx.doi.org/10.1183/13993003.01541-2015.
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Wingless/integrase-1 (WNT) signalling is associated with lung inflammation and repair, but its role in chronic obstructive pulmonary disease (COPD) pathogenesis is unclear. We investigated whether cigarette smoke-induced dysregulation of WNT-5B contributes to airway remodelling in COPD.We analysed WNT-5B protein expression in the lung tissue of COPD patients and (non)smoking controls, and investigated the effects of cigarette smoke exposure on WNT-5B expression in COPD and control-derived primary bronchial epithelial cells (PBECs). Additionally, we studied downstream effects of WNT-5B on remodelling related genes fibronectin, matrix metalloproteinase (MMP)-2, MMP-9 and SnaiI in BEAS-2B and air–liquid interface (ALI)-cultured PBECs.We observed that airway epithelial WNT-5B expression is significantly higher in lung tissue from COPD patients than controls. Cigarette smoke extract significantly increased mRNA expression of WNT-5B in COPD, but not control-derived PBECs. Exogenously added WNT-5B augmented the expression of remodelling related genes in BEAS-2B cells, which was mediated by transforming growth factor (TGF)-β/Smad3 signalling. In addition, WNT-5B upregulated the expression of these genes in ALI-cultured PBECs, particularly PBECs from COPD patients.Together, our results provide evidence that exaggerated WNT-5B expression upon cigarette smoke exposure in the bronchial epithelium of COPD patients leads to TGF-β/Smad3-dependent expression of genes related to airway remodelling.
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Cheng, Chi Keung, Libby Li, Suk Hang Cheng, Kitty Ng, Natalie P. H. Chan, Rosalina K. L. Ip, Raymond S. M. Wong, Matthew M. K. Shing, Chi Kong Li, and Margaret H. L. Ng. "Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia." Blood 118, no. 25 (December 15, 2011): 6638–48. http://dx.doi.org/10.1182/blood-2011-05-354712.
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Abstract Secreted-frizzled related proteins (SFRPs) are modulators of the Wnt signaling pathway that is closely involved in normal and malignant hematopoiesis. Epigenetic deregulation of Wnt modulators leading to aberrant signaling has been reported in adult patients with acute myeloid leukemia (AML), but its occurrence in childhood patients with AML and the role of individual modulators are unclear. In this study, we examined SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in 83 patients with AML (59 children and 24 adults) and found preferential SFRP1 methylation and mRNA down-regulation in the prognostically favorable subgroup of AML with t(8;21) translocation. Among the 4 genes, SFRP1 methylation independently predicted prolonged event-free and relapse-free survivals in childhood patients with nonacute promyelocytic leukemia with nonadverse cytogenetics. Mechanistically, we further demonstrated that RUNX1-ETO, the t(8;21) fusion product, specifically bound the SFRP1 promoter and repressed its transcription via a consensus RUNX binding site. In t(8;21)–leukemia cells, SFRP1 selectively inhibited canonical Wnt signaling and cellular proliferation that were associated with concomitant down-regulation of Wnt/β-catenin target genes, including CCND1 and MYC. Taken together, we identified SFRP1 as a transcriptional repression target of the t(8;21) fusion protein and demonstrated a novel mechanism of Wnt activation in a specific subtype of AML.
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Müller-Tidow, Carsten, Björn Steffen, Thomas Cauvet, Lara Tickenbrock, Ping Ji, Sven Diederichs, Bülent Sargin, et al. "Translocation Products in Acute Myeloid Leukemia Activate the Wnt Signaling Pathway in Hematopoietic Cells." Molecular and Cellular Biology 24, no. 7 (April 1, 2004): 2890–904. http://dx.doi.org/10.1128/mcb.24.7.2890-2904.2004.
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ABSTRACT The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARα), and PLZF-RARα encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARα, or PLZF-RARα. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (γ-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. β-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.
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Park, Hong-Beom, Ju-Won Kim, and Kwang-Hyun Baek. "Regulation of Wnt Signaling through Ubiquitination and Deubiquitination in Cancers." International Journal of Molecular Sciences 21, no. 11 (May 30, 2020): 3904. http://dx.doi.org/10.3390/ijms21113904.
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The Wnt signaling pathway plays important roles in embryonic development, homeostatic processes, cell differentiation, cell polarity, cell proliferation, and cell migration via the β-catenin binding of Wnt target genes. Dysregulation of Wnt signaling is associated with various diseases such as cancer, aging, Alzheimer’s disease, metabolic disease, and pigmentation disorders. Numerous studies entailing the Wnt signaling pathway have been conducted for various cancers. Diverse signaling factors mediate the up- or down-regulation of Wnt signaling through post-translational modifications (PTMs), and aberrant regulation is associated with several different malignancies in humans. Of the numerous PTMs involved, most Wnt signaling factors are regulated by ubiquitination and deubiquitination. Ubiquitination by E3 ligase attaches ubiquitins to target proteins and usually induces proteasomal degradation of Wnt signaling factors such as β-catenin, Axin, GSK3, and Dvl. Conversely, deubiquitination induced by the deubiquitinating enzymes (DUBs) detaches the ubiquitins and modulates the stability of signaling factors. In this review, we discuss the effects of ubiquitination and deubiquitination on the Wnt signaling pathway, and the inhibitors of DUBs that can be applied for cancer therapeutic strategies.
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Ziemer, Lisa Taneyhill, Diane Pennica, and Arnold J. Levine. "Identification of a Mouse Homolog of the Human BTEB2Transcription Factor as a β-Catenin-Independent Wnt-1-Responsive Gene." Molecular and Cellular Biology 21, no. 2 (January 15, 2001): 562–74. http://dx.doi.org/10.1128/mcb.21.2.562-574.2001.
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ABSTRACT The Wnt/Wg signaling pathway functions during development to regulate cell fate determination and patterning in various organisms. Two pathways are reported to lie downstream of Wnt signaling in vertebrates. The canonical pathway relies on the activation of target genes through the β-catenin–Lef/TCF complex, while the noncanonical pathway employs the activation of protein kinase C (PKC) and increases in intracellular calcium to induce target gene expression. cDNA subtractive hybridization between a cell line that overexpresses Wnt-1 (C57MG/Wnt-1) and the parental cell line (C57MG) was performed to identify downstream target genes of Wnt-1 signaling. Among the putative Wnt-1 target genes, we have identified a mouse homolog of the gene encoding human transcription factor basic transcription element binding protein 2 (mBTEB2). ThemBTEB2 transcript is found at high levels in mammary tissue taken from a transgenic mouse overexpressing Wnt-1 (both tissue prior to active proliferation and tumor tissue) but is barely detectable in wild-type mouse mammary glands. The regulation of mBTEB2 by Wnt-1 signaling in tissue culture occurs through a β-catenin–Lef/TCF-independent mechanism, as it is instead partially regulated by PKC. The Wnt-1-induced, PKC-dependent activation of mouse BTEB2 in C57MG cells, as well as the ability of Wnt-1 to stabilize β-catenin in these cells, is consistent with the hypothesis that both the noncanonical and canonical Wnt pathways are activated concomitantly in the same cell. These results suggest that mBTEB2 is a biologically relevant target of Wnt-1 signaling that is activated through a β-catenin-independent, PKC-sensitive pathway in response to Wnt-1.
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Patel, Maulin Mukeshchandra, Robert Silasi-Mansat, Ravi Shankar Keshari, Christopher L. Sansam, David A. Jones, Cristina Lupu, and Florea Lupu. "Role of Androgen Dependent TFPI-Regulating Protein (ADTRP) in Vascular Development and Function." Blood 128, no. 22 (December 2, 2016): 556. http://dx.doi.org/10.1182/blood.v128.22.556.556.
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Abstract We used in vitro and in vivo models to characterize the physiological role of the novel protein encoded by C6ORF105. This gene's expression is androgen-responsive, and the encoded protein is predicted to be palmitoylated and membrane multi-spanning. Previously we showed that C6ORF105 expression co-regulates with tissue factor pathway inhibitor (TFPI)in human endothelial cells (EC); hence we named this protein "androgen-dependent TFPI-regulating protein" (ADTRP). Using in vitro cell-based TOP-Flash reporter assay we identified ADTRP as a negative regulator of canonical Wnt signaling in human cells. Overexpressing ADTRP in HEK293T cells inhibited the activity of beta-catenin/TCF-dependent transcriptional reporter, while silencing ADTRP increased the expression of Wnt target genes LEF-1, AXIN-2, IL-8 and DKK-2 in EA.hy926 EC line and HUVEC. Addition of LiCl showed that the effect of ADTRP was upstream of GSK3, therefore we focused the investigations on the Wnt signalosome proteins. ADTRP expression in HEK293T cells led to decreased phosphorylation of Wnt co-receptor LRP6, suggesting that ADTRP can affect this critical membrane-located event of Wnt signaling. Furthermore, ADTRP expression in reporter cells transfected with a constitutively phosphorylated form of LRP6 (LRP6DN mutant) inhibited Wnt3a- induced signaling, which suggests that ADTRP can interfere with events downstream of LRP6 phosphorylation, such as Axin-2 binding. Altogether, these data indicate that the Wnt signaling inhibitory activity of ADTRP takes place at the plasma membrane level. Site directed mutagenesis of the predicted palmitoylation site Cys61 showed that Wnt inhibitory effects of ADTRP require palmitoyl-mediated anchoring, highlighting the importance of proper membrane location of ADTRP for Wnt pathway inhibition. In vivo morpholino-based knockdown of adtrp in zebrafish embryos produced aberrant angiogenesis, defective branching and ruptured vessels, hemorrhage spots, pericardial edema and slow heart-beat, all reminiscent of defects caused by activation of canonical Wnt signaling. Indeed, adtrp knock down increased Wnt mediated lef-1 and pax-2a as well as mmp2 and mmp9 mRNA expression. Co-injection of ADTRP mRNA partially recovered the adtrp morpholino- induced morphologic abnormalities. Also, knock down of adtrp in a Wnt reporter zebrafish showed increased expression of ectopic Wnt signaling. Furthermore, our recently established Adtrp-/- mice also display some typical Wnt-mediated vascular defects, including: (i) abnormal patterning, increased capillary tortuosity, abnormal branching and increased density of the capillary network; (ii) dilated vessels, especially venules and veins; (iii) increased leakeage of permeability tracers (Evans blue and fluorescent dextran) without evident changes in endothelial junctions; (iv) hemorrhage spots in the skin, meningeal layers, heart, bladder and kidneys; (v) intravascular and interstitial fibrin deposition in the lung, liver and kidney. ADTRP deficiency decreased plasma TFPI antigen by ~2-times. Furthermore, TFPI antigen and anticoagulant activity in lung extracts and isolated lung EC were similarly decreased, which confirms our previous in vitro data. We aslo noticed increased tail bleeding time (>500 sec vs. 200 sec in WT littermates) and blood volume loss, which likely was caused by increased dilation of the tail vein. Gene expression analysis of whole organs showed upregulation of Wnt target genes involved in vascular contractility (Nos3), and extracellular matrix remodeling (Mmp2). Similarly, skin fibroblasts and lung EC isolated from Adtrp-/- mice showed increased expression of Wnt target genes (Lef-1, Cyclin D, Dkk2, c-Myc), which indicates constitutive activation of canonical Wnt signaling. In conclusion, we used genetic animal models and cell culture systems to show for the first time that the novel protein ADTRP plays major roles in vascular development and function. Lack of, or low levels of ADTRP associate with activation of coagulation and vascular development defects, which may be due, at least in part, to intrinsic high levels of ectopic canonical Wnt signaling. Disclosures No relevant conflicts of interest to declare.
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Jiang, Tongmeng, Bo Zhou, Li Huang, Huayu Wu, Jiancheng Huang, Tihong Liang, Hui Liu, Li Zheng, and Jinmin Zhao. "Andrographolide Exerts Pro-Osteogenic Effect by Activation of Wnt/β-Catenin Signaling Pathway in Vitro." Cellular Physiology and Biochemistry 36, no. 6 (2015): 2327–39. http://dx.doi.org/10.1159/000430196.
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Background/Aims: Osteoporosis is a metabolic bone disorders that tortures about millions of people worldwide. Recent studies showed that Andrographolide (AP) is a promising natural compound for the treatment of osteoclast-related bone diseases. However, its potential in treatment of osteoporosis has not been fully explored. Methods: In this study, the effect of AP on osteoblasts metabolism was investigated via the detection of cell proliferation, cell viability, ALP activity, the expression of osteogenic specific genes including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), osteocalcin (OCN), Bone morphogenic protein-2 (BMP2) and Alkaline phosphatase(ALP) for 3, 5 and 7 days respectively. Further exploration of the association of AP with WNT/β-catenin signaling pathway was performed by examination of the expression of WNT related genes and proteins. Results: Results showed that AP of 4.46 and 8.92 µM, especially 8.92 µM was beneficial to osteogenic differentiation by upregulating ALP activity and expression of osteogenic related genes (P<0.05). Pathway analyses identify canonical WNT/β-catenin pathway as an important mediator in AP-induced osteogenesis. Conclusion: This study indicates that AP exerts its pro-osteogenic potential via activation of the WNT/β-catenin in osteoblasts and thus may represent a candidate of therapeutic agent for osteoporosis.
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Muñoz-Bello, J., Leslie Olmedo-Nieva, Leonardo Castro-Muñoz, Joaquín Manzo-Merino, Adriana Contreras-Paredes, Claudia González-Espinosa, Alejandro López-Saavedra, and Marcela Lizano. "HPV-18 E6 Oncoprotein and Its Spliced Isoform E6*I Regulate the Wnt/β-Catenin Cell Signaling Pathway through the TCF-4 Transcriptional Factor." International Journal of Molecular Sciences 19, no. 10 (October 13, 2018): 3153. http://dx.doi.org/10.3390/ijms19103153.
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The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation and its aberrant activation in cervical cancer has been described. Persistent infection with high risk human papillomavirus (HR-HPV) is the most important factor for the development of this neoplasia, since E6 and E7 viral oncoproteins alter cellular processes, promoting cervical cancer development. A role of HPV-16 E6 in Wnt/β-catenin signaling has been proposed, although the participation of HPV-18 E6 has not been previously studied. The aim of this work was to investigate the participation of HPV-18 E6 and E6*I, in the regulation of the Wnt/β-catenin signaling pathway. Here, we show that E6 proteins up-regulate TCF-4 transcriptional activity and promote overexpression of Wnt target genes. In addition, it was demonstrated that E6 and E6*I bind to the TCF-4 (T cell factor 4) and β-catenin, impacting TCF-4 stabilization. We found that both E6 and E6*I proteins interact with the promoter of Sp5, in vitro and in vivo. Moreover, although differences in TCF-4 transcriptional activation were found among E6 intratype variants, no changes were observed in the levels of regulated genes. Furthermore, our data support that E6 proteins cooperate with β-catenin to promote cell proliferation.
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Halene, Stephanie, Ee-chun Cheng, Vincent Schulz, David Tuck, and Diane Krause. "OTT-MKL1 and MKL1 Inhibit Wnt Signaling." Blood 112, no. 11 (November 16, 2008): 2250. http://dx.doi.org/10.1182/blood.v112.11.2250.2250.
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Abstract The OTT-MKL1 fusion gene product is generated as a result of t(1;22) in a subset of acute megakaryoblastic leukemia predominantly encountered in young children. Due to myelofibrosis and the age at presentation, patient samples are scarce. We generated Human Erythroid Leukemia (HEL) cell derived cell lines with tet-inducible OTT, MKL1 and OTT-MKL1 to further elucidate the function of the respective proteins. HEL cells can be induced to differentiate down the megakaryocyte lineage by TPA. Induction with doxycycline resulted in transcription and translation of the respective genes within hours. While overexpression of MKL1 led to enhancement of megakaryocytic differentiation, both OTT and OTT-MKL1 overexpression led to cell death over the course of several days by apoptosis as evident by staining for Annexin V and morphology. The apoptotic cell death was greatly enhanced by concomitant induction of differentiation by TPA. We performed microarray analysis comparing uninduced and 8-hour tet-induced samples in the presence or absence of TPA. While overexpression of OTT had only a minimal effect on the transcriptome of the HEL cells, both MKL1 and OTT-MKL1 significantly affected the gene expression of many genes. Using a false discovery rate cut-off of p < 0.05, and assessing only those genes whose expression changed by greater than 2-fold, OTT-MKL1 and MKL1 induced the upregulation of 157 and 168 genes, respectively, and the downregulation of 56 and 62 genes, respectively. Only 20 genes were upregulated by both OTT-MKL1 and MKL1, and 12 genes were downregulated greater than 2-fold by both OTT-MKL1 and MKL1. GeneGo analysis comparing OTT-MKL1 over-expressing versus non-expressing cells revealed over-representation of the Wnt pathway. Among the differentially expressed genes implicated in the Wnt pathway were Frat1 and Frat2, which have been shown to inhibit GSK3β and lead to β-catenin nuclear accumulation, and thus stimulation of the Wnt pathway. At the same time, inhibitory NLK was upregulated and several down-stream targets of the Wnt pathway were downregulated. Spenito, the homolog of OTT in Drosophila, is known to have promoter-specific activating and inhibiting effects on Wnt target genes. We thus performed reporter assays to study the effects of OTT, MKL1 and OTT-MKL1 on the Wnt pathway. Using the TOP-/FOP-FLASH reporter system in 293T cells, there was a dose-dependent inhibition of β-catenin-mediated activation of the Tcf/Lef binding site promoter by MKL1 and OTT-MKL1. Full length OTT showed a minimal stimulatory effect only at low doses., while N-terminal OTT, lacking the SPOC domain and a dominant negative form of MKL1 lacking the transactivation domain each enhanced β-catenin induced Tcf/Lef mediated transcriptional activation. Studies to define the domains of OTT and MKL1 and the underlying mechanisms in hematopoietic cells are underway. These results suggest that MKL1 and OTT-MKL1 inhibit canonical Wnt signaling by inhibiting β-catenin induced transcription.
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Li, Jianyi, Guangzhen Wu, Yingkun Xu, Jiatong Li, Ningke Ruan, Yougen Chen, Qi Zhang, and Qinghua Xia. "Porcupine Inhibitor LGK974 Downregulates the Wnt Signaling Pathway and Inhibits Clear Cell Renal Cell Carcinoma." BioMed Research International 2020 (February 13, 2020): 1–16. http://dx.doi.org/10.1155/2020/2527643.
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Targeted therapy for kidney cancer has achieved significant clinical results. However, because most patients who use targeted therapy will develop drug resistance, we still need to constantly explore new therapeutic targets. Although porcupine (PORCN) as a palmitoyltransferase plays a crucial role in the activation and secretion of Wnt proteins and affects the activity of the Wnt signaling pathway, little is known about the role of PORCN in clear cell renal cell carcinoma (ccRCC). We found that PORCN is highly expressed in renal cancer cell lines and patients with renal cell carcinoma with high expression of PORCN have a poor prognosis. Pathway analysis of PORCN and its related proteins showed that PORCN played a role through the Wnt signaling pathway, and there was a strong coexpression relationship between PORCN and Wnt proteins. Therefore, PORCN may be a potential and effective target for ccRCC. In the present study, we found that LGK974 could inhibit proliferation and colony formation and induce apoptosis in ccRCC cells. We also found that LGK974 could inhibit the migration and invasion of renal cell carcinoma and reduce the expression of mesenchymal markers. After treatment with LGK974, the expression level of β-catenin, a key protein in the classical Wnt pathway, was significantly decreased, and the expression levels of the target genes cyclin D1, c-Myc, MMP9, and MMP2 in the Wnt signaling pathway were also significantly decreased, which represented a significant decrease in the activity of the Wnt signaling pathway. At the same time, the cycle of renal cancer cells was significantly blocked. In conclusion, our results indicate that LGK974 could significantly inhibit the progression of renal cancer cells in a safe concentration range, so PORCN may be a safe and effective target for patients with renal cancer.
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Sumanas, S., P. Strege, J. Heasman, and S. C. Ekker. "The putative wnt receptor Xenopus frizzled-7 functions upstream of beta-catenin in vertebrate dorsoventral mesoderm patterning." Development 127, no. 9 (May 1, 2000): 1981–90. http://dx.doi.org/10.1242/dev.127.9.1981.
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We have isolated one member of the frizzled family of wnt receptors from Xenopus (Xfz7) to study the role of cell-cell communication in the establishment of the vertebrate axis. We demonstrate that this maternally encoded protein specifically synergizes with wnt proteins in ectopic axis induction. Embryos derived from oocytes depleted of maternal Xfz7 RNA by antisense oligonucleotide injection are deficient in dorsoanterior structures. Xfz7-depleted embryos are deficient in dorsal but not ventral mesoderm due to the reduced expression of the wnt target genes siamois, Xnr3 and goosecoid. These signaling defects can be restored by the addition of beta-catenin but not Xwnt8b. Xfz7 thus functions upstream of the known GSK-3/axin/beta-catenin intracellular signaling complex in vertebrate dorsoventral mesoderm specification.
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Wang, Jiangbo, Xiu-rong Ren, Hailan Piao, Shengli Zhao, Takuya Osada, Richard T. Premont, Robert A. Mook, Michael A. Morse, Herbert Kim Lyerly, and Wei Chen. "Niclosamide-induced Wnt signaling inhibition in colorectal cancer is mediated by autophagy." Biochemical Journal 476, no. 3 (February 8, 2019): 535–46. http://dx.doi.org/10.1042/bcj20180385.
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Abstract The Wnt signaling pathway, known for regulating genes critical to normal embryonic development and tissue homeostasis, is dysregulated in many types of cancer. Previously, we identified that the anthelmintic drug niclosamide inhibited Wnt signaling by promoting internalization of Wnt receptor Frizzled 1 and degradation of Wnt signaling pathway proteins, Dishevelled 2 and β-catenin, contributing to suppression of colorectal cancer growth in vitro and in vivo. Here, we provide evidence that niclosamide-mediated inhibition of Wnt signaling is mediated through autophagosomes induced by niclosamide. Specifically, niclosamide promotes the co-localization of Frizzled 1 or β-catenin with LC3, an autophagosome marker. Niclosamide inhibition of Wnt signaling is attenuated in autophagosome-deficient ATG5−/− MEF cells or cells expressing shRNA targeting Beclin1, a critical constituent of autophagosome. Treatment with the autophagosome inhibitor 3MA blocks niclosamide-mediated Frizzled 1 degradation. The sensitivity of colorectal cancer cells to growth inhibition by niclosamide is correlated with autophagosome formation induced by niclosamide. Niclosamide inhibits mTORC1 and ULK1 activities and induces LC3B expression in niclosamide-sensitive cell lines, but not in the niclosamide-resistant cell lines tested. Interestingly, niclosamide is a less effective inhibitor of Wnt-responsive genes (β-catenin, c-Myc, and Survivin) in the niclosamide-resistant cells than in the niclosamide-sensitive cells, suggesting that deficient autophagy induction by niclosamide compromises the effect of niclosamide on Wnt signaling. Our findings provide a mechanistic understanding of the role of autophagosomes in the inhibition of Wnt signaling by niclosamide and may provide biomarkers to assist selection of patients whose tumors are likely to respond to niclosamide.
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Kierulf-Vieira, Kirsten Strømme, Cecilie Jonsgar Sandberg, Jo Waaler, Kaja Lund, Erlend Skaga, Birthe Mikkelsen Saberniak, Ioannis Panagopoulos, et al. "A Small-Molecule Tankyrase Inhibitor Reduces Glioma Stem Cell Proliferation and Sphere Formation." Cancers 12, no. 6 (June 19, 2020): 1630. http://dx.doi.org/10.3390/cancers12061630.
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Evidence suggests that the growth and therapeutic resistance of glioblastoma (GBM) may be enabled by a population of glioma stem cells (GSCs) that are regulated by typical stem cell pathways, including the WNT/β-catenin signaling pathway. We wanted to explore the effect of treating GSCs with a small-molecule inhibitor of tankyrase, G007-LK, which has been shown to be a potent modulator of the WNT/β-catenin and Hippo pathways in colon cancer. Four primary GSC cultures and two primary adult neural stem cell cultures were treated with G007-LK and subsequently evaluated through the measurement of growth characteristics, as well as the expression of WNT/β-catenin and Hippo signaling pathway-related proteins and genes. Treatment with G007-LK decreased in vitro proliferation and sphere formation in all four primary GSC cultures in a dose-dependent manner. G007-LK treatment altered the expression of key downstream WNT/β-catenin and Hippo signaling pathway-related proteins and genes. Finally, cotreatment with the established GBM chemotherapeutic compound temozolomide (TMZ) led to an additive reduction in sphere formation, suggesting that WNT/β-catenin signaling may contribute to TMZ resistance. These observations suggest that tankyrase inhibition may serve as a supplement to current GBM therapy, although more work is needed to determine the exact downstream mechanisms involved.
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Dharmarajan, A., N. Zeps, and S. McLaren. "003.Expression of secreted frizzled related protein-4 (sFRP-4) and associated Wnt signalling in cancer and apoptosis." Reproduction, Fertility and Development 17, no. 9 (2005): 63. http://dx.doi.org/10.1071/srb05abs003.
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We examined the interplay between Wnt and secreted frizzled related protein-4 (sFRP4) in estradiol induced cell growth in breast cancer cells (MCF-7), and also determined the in vivo distribution of sFRP-4 in human breast cancer. MCF-7 cells were treated with estradiol, sFRP-4 conditioned media and a combination of the two. Real-time RT-PCR and Western blot analysis were used to determine the expression of the sFRP-4 and its associated Wnt signalling molecules following treatment. Immunohistochemistry was performed to examine sFRP-4 expression patterns in human breast cancers. Estradiol treatment up-regulated the expression of the Wnt signalling genes Wnt-10b, beta-catenin and fz-4 (P < 0.001 for all genes). This up-regulation was not associated with an increase in the Wnt signalling pathway as measured by the levels of active beta-catenin. sFRP-4 conditioned media reduced MCF-7 cell proliferation, down-regulated the Wnt signalling genes beta-catenin and fz-4 as well as down-regulating wnt signalling activity. sFRP-4 was able to reduce the proliferation of estradiol stimulated MCF-7 cells. Cytoplasmic sFRP-4 protein was expressed in all breast tumours examined, with intense staining evident in the lobular carcinoma in situ and the ductal carcinoma. These data demonstrate that sFRP-4 is a potent inhibitor of the Wnt signalling pathway in MCF-7 cells, acting not only to down-regulate the activity of the wnt signalling pathway, but also down-regulate the transcription of Wnt signalling genes. The results of these in vitro and immunohistochemical experiments warrant further investigation as to whether sFRP-4 expression can be indicative of prognosis in human breast cancer. In addition to breast cancer, we have also examined the role of sFRP-4 in other cancers such as ovarian and prostate.
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Korinek, Vladimir, Nick Barker, Karl Willert, Miranda Molenaar, Jeroen Roose, Gerry Wagenaar, Marry Markman, Wout Lamers, Olivier Destree, and Hans Clevers. "Two Members of the Tcf Family Implicated in Wnt/β-Catenin Signaling during Embryogenesis in the Mouse." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1248–56. http://dx.doi.org/10.1128/mcb.18.3.1248.
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ABSTRACT Tcf transcription factors interact with β-catenin and Armadillo to mediate Wnt/Wingless signaling. We now report the characterization of genes encoding two murine members of the Tcf family,mTcf-3 and mTcf-4. mTcf-3 mRNA is ubiquitously present in embryonic day 6.5 (E6.5) mouse embryos but gradually disappears over the next 3 to 4 days. mTcf-4 expression occurs first at E10.5 and is restricted to di- and mesencephalon and the intestinal epithelium during embryogenesis. The mTcf-3 and mTcf-4 proteins bind a canonical Tcf DNA motif and can complex with the transcriptional coactivator β-catenin. Overexpression of Wnt-1 in a mammary epithelial cell line leads to the formation of a nuclear complex between β-catenin and Tcf proteins and to Tcf reporter gene transcription. These data demonstrate a direct link between Wnt stimulation and β-catenin/Tcf transcriptional activation and imply a role formTcf-3 and -4 in early Wnt-driven developmental decisions in the mouse embryo.
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Luo, Tian, Paige S. Dunphy, Taslima T. Lina, and Jere W. McBride. "Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival." Infection and Immunity 84, no. 3 (December 28, 2015): 686–700. http://dx.doi.org/10.1128/iai.01289-15.
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Ehrlichia chaffeensisinvades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined thatE. chaffeensistandem 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 byE. chaffeensisTRP effectors stimulates phagocytosis and promotes intracellular survival. AfterE. chaffeensisinfection, 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 thatE. chaffeensisexploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival.
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Chung, Jihyun, Vrajesh Karkhanis, Said Sif, and Robert A. Baiocchi. "Protein Arginine Methyltransferase 5 Supports MYC, Survivin and Cyclin D1 Activity in Aggressive Lymphomas By Regulating the WNT/β-Catenin Pathway." Blood 124, no. 21 (December 6, 2014): 58. http://dx.doi.org/10.1182/blood.v124.21.58.58.
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Abstract Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis. Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells. Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. Disclosures No relevant conflicts of interest to declare.
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Dominguez-Brauer, Carmen, Rahima Khatun, Andrew J. Elia, Kelsie L. Thu, Parameswaran Ramachandran, Shakiba P. Baniasadi, Zhenyue Hao, et al. "E3 ubiquitin ligase Mule targets β-catenin under conditions of hyperactive Wnt signaling." Proceedings of the National Academy of Sciences 114, no. 7 (January 30, 2017): E1148—E1157. http://dx.doi.org/10.1073/pnas.1621355114.
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Wnt signaling, named after the secreted proteins that bind to cell surface receptors to activate the pathway, plays critical roles both in embryonic development and the maintenance of homeostasis in many adult tissues. Two particularly important cellular programs orchestrated by Wnt signaling are proliferation and stem cell self-renewal. Constitutive activation of the Wnt pathway resulting from mutation or improper modulation of pathway components contributes to cancer development in various tissues. Colon cancers frequently bear inactivating mutations of the adenomatous polyposis coli (APC) gene, whose product is an important component of the destruction complex that regulates β-catenin levels. Stabilization and nuclear localization of β-catenin result in the expression of a panel of Wnt target genes. We previously showed that Mule/Huwe1/Arf-BP1 (Mule) controls murine intestinal stem and progenitor cell proliferation by modulating the Wnt pathway via c-Myc. Here we extend our investigation of Mule’s influence on oncogenesis by showing that Mule interacts directly with β-catenin and targets it for degradation under conditions of hyperactive Wnt signaling. Our findings suggest that Mule uses various mechanisms to fine-tune the Wnt pathway and provides multiple safeguards against tumorigenesis.
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Ding, Yu, Ying Xi, Ting Chen, Ji-yong Wang, Dong-lei Tao, Zhi-Li Wu, Yi-ping Li, Chen Li, Rong Zeng, and Lin Li. "Caprin-2 enhances canonical Wnt signaling through regulating LRP5/6 phosphorylation." Journal of Cell Biology 182, no. 5 (September 1, 2008): 865–72. http://dx.doi.org/10.1083/jcb.200803147.
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The low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) are coreceptors for Frizzled and transmit signals from the plasma membrane to the cytosol. However, the mechanism for LRP5/6 signal transmission remains undefined. Here, we identify cytoplasmic activation/proliferation-associated protein 2 (Caprin-2) as a LRP5/6-binding protein. Our data show that Caprin-2 stabilizes cytosolic β-catenin and enhances lymphoid enhancer-binding factor 1/T cell factor–dependent reporter gene activity as well as the expression of Wnt target genes in mammalian cells. Morpholino-mediated knockdown of Caprin-2 in zebrafish embryos inhibits Wnt/β-catenin signaling and results in a dorsalized phenotype. Moreover, Caprin-2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3, and thus enhances the interaction between Axin and LRP5/6. Therefore, Caprin-2 promotes activation of the canonical Wnt signaling pathway by regulating LRP5/6 phosphorylation.