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Concept: Wnt signaling pathway

169

Members of the Frizzled family of sevenpass transmembrane receptors signal via the canonical Wnt pathway and also via noncanonical pathways of which the best characterized is the planar polarity pathway. Activation of both canonical and planar polarity signaling requires interaction between Frizzled receptors and cytoplasmic proteins of the Dishevelled family; however, there has been some dispute regarding whether the Frizzled-Dishevelled interactions are the same in both cases. Studies looking at mutated forms of Dishevelled suggested that stable recruitment of Dishevelled to membranes by Frizzled was required only for planar polarity activity, implying that qualitatively different Frizzled-Dishevelled interactions underlie canonical signaling. Conversely, studies looking at the sequence requirements of Frizzled receptors in the fruit fly Drosophila melanogaster for canonical and planar polarity signaling have concluded that there is most likely a common mechanism of action. To understand better Frizzled receptor function, we have carried out a large-scale mutagenesis in Drosophila to isolate novel mutations in frizzled that affect planar polarity activity and have identified a group of missense mutations in cytosolic-facing regions of the Frizzled receptor that block Dishevelled recruitment. Interestingly, although some of these affect both planar polarity and canonical activity, as previously reported for similar lesions, we find a subset that affect only planar polarity activity. These results support the view that qualitatively different Frizzled-Dishevelled interactions underlie planar polarity and canonical Wnt signaling.

Concepts: Protein, Mutation, Point mutation, Missense mutation, Drosophila melanogaster, Drosophila, Wnt signaling pathway, Frizzled

156

Abstract β-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of β-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of β-catenin’s role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of β-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous β-catenin complexes. In addition, we engineered intracellularly functional anti-β-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous β-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated β-catenin in response to compound treatment in real time using High Content Imaging. The anti-β-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular β-catenin in biochemical and cell biological assays.

Concepts: Antibody, DNA, Protein, Protein structure, Organism, Cell biology, Wnt signaling pathway, Single domain antibody

33

Although intestinal homeostasis is maintained by intestinal stem cells (ISCs), regeneration is impaired upon aging. Here, we first uncover changes in intestinal architecture, cell number, and cell composition upon aging. Second, we identify a decline in the regenerative capacity of ISCs upon aging because of a decline in canonical Wnt signaling in ISCs. Changes in expression of Wnts are found in stem cells themselves and in their niche, including Paneth cells and mesenchyme. Third, reactivating canonical Wnt signaling enhances the function of both murine and human ISCs and, thus, ameliorates aging-associated phenotypes of ISCs in an organoid assay. Our data demonstrate a role for impaired Wnt signaling in physiological aging of ISCs and further identify potential therapeutic avenues to improve ISC regenerative potential upon aging.

Concepts: Gene, Cell, Human, Bacteria, Organism, Cell division, Stem cell, Wnt signaling pathway

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We have recently unravelled a novel function for CD82 in E-cadherin-mediated cellular adhesion. CD82 inhibits β-catenin tyrosine phosphorylation and stabilizes E-cadherin-β-catenin complexes at the cell membrane. This function inhibits cancer cell dissociation from the primary cancer nest and limits metastasis. In this study, we focused on the effect of CD82 on the Wnt/β-catenin (canonical) pathway, which controls the cellular distribution of β-catenin. CD82 had no effect on the expression of Wnt proteins but led to significant downregulation of Frizzled (Fzd) 2, 3, 5, 7 and 9, suggesting downregulation of the Wnt/β-catenin pathway. CD82 also inhibited phosphorylation of β-catenin at Ser45, Ser33, Ser37 and Thr41 by downregulation of glycogen synthase kinase-3β (GSK-3β) and kinase casein kinase 1α (CK1α). Downregulation of GSK-3β and CK1α also led to accumulation of β-catenin in the cytoplasm or at the cell membrane. CD82 translocated β-catenin to the cell membrane, suggesting that CD82 strengthens the interaction between E-cadherin and β-catenin. We concluded that CD82 attenuates Wnt signalling by controlling β-catenin cellular distribution at multiple levels: i) inhibition of β-catenin nuclear translocation by downregulation of Fzd receptor proteins; ii) accumulation of β-catenin at the cell membrane by downregulation of GSK-3β and CK1α; and iii) stabilization of the E-cadherin-β-catenin complex.

Concepts: Protein, Cell, Cancer, Signal transduction, Enzyme, Lung cancer, Cell biology, Wnt signaling pathway

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Tooth agenesis is one of the most common dental anomalies, with a complex and not yet fully elucidated aetiology. Given the crucial role of the WNT signalling pathway during tooth development, the purpose of this study was to determine whether nucleotide variants of genes encoding components of this signalling pathway might be associated with hypodontia and oligodontia in the Polish population. A set of 34 SNPs in 13 WNT and WNT-related genes were analyzed in a group of 157 patients with tooth agenesis and a properly matched control group (n=430). In addition, direct sequencing was performed to detect mutations in the MSX1, PAX9 and WNT10A genes. Both single-marker and haplotype analyses showed highly significant association between SNPs in the WNT10A gene and the risk for tooth agenesis. Moreover, nine pathogenic mutations within the coding region of the WNT10A gene were identified in 26 out of 42 (62%) tested patients. One novel heterozygous mutation was identified in the PAX9 gene. Borderline association with the risk of non-syndromic tooth agenesis was also observed for the APC, CTNNB1, DVL2 and WNT11 polymorphisms. In conclusion, nucleotide variants of genes encoding important components of the WNT signalling pathway might influence the risk of tooth agenesis.

Concepts: DNA, Gene, Genetics, Mutation, Evolution, Tooth development, Wnt signaling pathway, Hypodontia

27

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a poor response to treatment and prognosis. Constitutive activation of different signaling pathways in subsets of MCLs, through genetic and/or nongenetic alterations, endows tumor cells with enhanced proliferation and reduced apoptosis. The canonical Wnt pathway (β-catenin/TCF-LEF), implicated in the pathogenesis of numerous cancers, is constitutively active in half of MCLs. Here, we show that ZEB1, a transcription factor better known for promoting metastasis in carcinomas, is expressed in primary MCLs with active Wnt signaling. ZEB1 expression in MCL cells depends on Wnt, being downregulated by β-catenin knockdown or blocking of Wnt signaling by salinomycin. Knockdown of ZEB1 reduces in vitro cell viability and proliferation in MCL cells, and, importantly, tumor growth in mouse xenograft models. ZEB1 activates proliferation-associated (HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1) and anti-apoptotic (MCL1, BCL2, and BIRC5) genes and inhibits pro-apoptotic ones (TP53, BBC3, PMAIP1, and BAX). We show that ZEB1 expression in MCL cells determines differential resistance to chemotherapy drugs and regulates transporters involved in drug influx/efflux. Downregulation of ZEB1 by salinomycin increases the sensitivity of MCL cells to the cytotoxic effect of doxorubicin, cytarabine and gemcitabine. Lastly, salinomycin and doxorubicin display a synergistic effect in established and primary MCL cells. These results identify ZEB1 in MCL where it promotes cell proliferation, enhanced tumor growth and a differential response to chemotherapy drugs. ZEB1 could thus potentially become a predictive biomarker and therapeutic target in this lymphoma.Cell Death and Differentiation advance online publication, 6 September 2013; doi:10.1038/cdd.2013.123.

Concepts: DNA, Gene, Gene expression, Cancer, Oncology, Bcl-2, Mantle cell lymphoma, Wnt signaling pathway

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Introduction: Androgenetic alopecia (AGA) is the most common form of hair loss, however current treatment options are limited and moderately effective. In the past few years, there has been an increased interest in deciphering the molecular mechanisms responsible for this disorder, which has opened the possibility of novel treatments that promise to not only stimulate hair growth, but also to induce formation of new hair follicles. Areas covered: The future holds more effective topical treatments with less systemic side effects (such as topical 5-alfa-reductase inhibitors), prostaglandin analogs and antagonists, medications which act through the Wnt signaling pathway, stem cells for hair regeneration, platelet-rich plasma (PRP) and more effective ways of transplanting hair. A comprehensive search was made using PubMed, GoogleScholar and Clinicaltrial.gov using different combination of key words, which included AGA treatment, new treatments for AGA, Wnt pathway, prostaglandins, PRP and stem cells for hair regrowth. Expert opinion: In the near future, treatments with topical 5-alfa-reductase inhibitors and prostaglandin agonists or antagonists are expected. More evidence is needed to verify the efficacy of PRP. Although hair follicle bioengineering and multiplication is a fascinating and promising field, it is still a long way from being available to clinicians.

Concepts: Protein, Pharmacology, Chemotherapy, Hair, Wnt signaling pathway, Alopecia, Baldness, Androgenic alopecia

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With continuous accumulation of research on the molecular pathogenesis of autism, more attention is being focused on the canonical Wnt signaling pathway. The canonical Wnt signaling pathway has been implicated in a variety of functions in the cell, including proliferation, differentiation, apoptosis and neuronal migration during embryonic development. Given its wide range of roles, dysregulation of the canonical Wnt signaling pathway could have any number of deleterious effects on neural development and thereby contribute to the pathogenesis of neurodevelopmental disorders, such as autism. Here, we review human and animal studies that implicate the canonical Wnt signal transduction pathway in the pathogenesis of autism. We also describe the crosstalk between the canonical Wnt pathway with the Notch signaling pathway in several types of autism spectrum disorders, including Asperger syndrome and Fragile X. Further research on the crosstalk between the canonical Wnt signaling pathway and other signaling cascades in autism may be an efficient avenue to understand the etiology of autism and ultimately lead to alternative medications for autism-like phenotypes.

Concepts: Immune system, Signal transduction, Autism, Pervasive developmental disorder, Asperger syndrome, Wnt signaling pathway, Neural development, Neurodevelopmental disorder

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

Concepts: Gene, Cell, Species, Developmental biology, Regulation, Cilium, Wnt signaling pathway, Ciliopathy

23

Wnt signaling is a fundamental pathway that drives embryonic development and is essential for stem cell maintenance and tissue homeostasis. Dysregulation of Wnt signaling is linked to various diseases and a constitutively active Wnt pathway drives tumorigenesis. Thus, disruption of the Wnt response is deemed a promising strategy for cancer drug discovery. However, only few clinical drug candidates that target Wnt signaling are available so far and new small molecules modulators of Wnt-related processes are in high demand. Here we describe the synthesis of a withanolide-inspired compound collection generated using a pregnenolone-derived -lactone as the key intermediate which was transformed into a δ-lactone appended to the D-ring of the steroidal scaffold. This natural product-inspired compound library contained potent inhibitors of Wnt signaling that act upstream of the destruction complex to stabilize Axin in a tankyrase-independent manner.

Concepts: Pharmacology, Cancer, Chemistry, Chemical substance, The Key, Drug discovery, Natural product, Wnt signaling pathway