Expression of WNT signalling pathway genes during chicken craniofacial development

A comprehensive expression analysis of WNT signalling pathway genes during several stages of chicken facial development was performed. Thirty genes were surveyed including: WNT1, 2B, 3A, 4, 5A, 5B, 6, 7A, 7B, 8B, 8C, 9A, 9B, 11, 11B, 16, CTNNB1, LEF1, FRZB1, DKK1, DKK2, FZD1‐8, FZD10. The strictly canonical WNTs (2B, 7A, 9B, and 16) in addition to WNT4 WNT6 (both canonical and non‐canonical) are epithelially expressed, whereas WNT5A, 5B, 11 are limited to the mesenchyme. WNT16 is limited to the invaginating nasal pit, respiratory epithelium, and lip fusion zone. Antagonists DKK1 and FRZB1 are expressed in the fusing primary palate but then are decreased at stage 28 when fusion is beginning. This suggests that canonical WNT signalling may be active during lip fusion. Mediators of canonical signalling, CTNNB1, LEF1, and the majority of the FZD genes are expressed ubiquitously. These data show that activation of the canonical WNT pathway is feasible in all regions of the face; however, the localization of ligands and antagonists confers specificity. Developmental Dynamics 238:1150–1165, 2009. © 2009 Wiley‐Liss, Inc.     

The role of WNT1 mutant variant (WNT1c.677C>T) in osteogenesis imperfecta

Osteogenesis imperfecta (OI), also known as “brittle bone disease,” is a rare inherited genetic disorder characterized by bone fragility and often associated with short stature. The mutation in WNT1 causes autosomal recessive OI (AR-OI) due to the key role of WNT/β-catenin signaling in bone formation. WNT1 mutations cause phenotypes in OI of varying degrees of clinical severity, ranging from moderate to progressively deforming forms. The nucleotide change c.677C > T is one of the recurrent variants in the WNT1 alleles in Chinese AR-OI patients. To explore the effects of mutation c.677C > T on WNT1 function, we evaluated the activation of WNT/β-catenin signaling, cell proliferation, osteoblast differentiation, and osteoclast differentiation in WNT1^c.677C>T, WNT1^c.884C>A, and wild type WNT1 transfected into MC3T3-E1 preosteoblasts. Plasmids containing wild type WNT1, WNT1^c.677C>T, and WNT1^c.884C>A cDNAs were constructed. Protein levels of phosphorylation at serine 9 of GSK-3β (p-GSK-3β), GSK-3β, nonphosphorylated β-catenin (non-p-β-catenin), and β-catenin were detected with western blot. Cell proliferation was determined using MTS. BMP-2 and RANKL mRNA and protein levels were detected by qPCR and western blot. Our results showed that WNT1^c.677C>T failed to activate WNT/β-catenin signaling and impaired the proliferation of preosteoblasts. Moreover, compared to wild type WNT1, WNT1^c.677C>T downregulated BMP-2 protein expression and was exhibited a diminished capacity to suppress the RANKL protein level. In conclusion, mutation c.677C > T hindered the ability of WNT1 to induce the WNT/β-catenin signaling pathway and it affected the WNT/β-catenin pathway which might potentially contribute to hampered bone homeostasis.     

WNT10B mutations associated with isolated dental anomalies

Isolated hypodontia is the most common human malformation. It is caused by heterozygous variants in various genes, with heterozygous WNT10A variants being the most common cause. WNT10A and WNT10B are paralogs that likely evolved from a common ancestral gene after its duplication. Recently, an association of WNT10B variants with oligodontia (severe tooth agenesis) has been reported. We performed mutational analysis in our cohort of 256 unrelated Thai families with various kinds of isolated dental anomalies. In 7 families afflicted with dental anomalies we detected 4 heterozygous missense variants in WNT10B. We performed whole exome sequencing in the patients who had WNT10B mutations and found no mutations in other known hypodontia‐associated genes, including WNT10A, MSX1, PAX9, EDA, AXIN2, EDAR, EDARADD, LPR6, TFAP2B, LPR6, NEMO, KRT17, and GREM2. Our findings indicate that the variants c.475G>C [p.(Ala159Pro)], found in 4 families, and c.1052G>A [p.(Arg351His)], found in 1 family, are most probably causative. They also show that WNT10B variants are associated not only with oligodontia and isolated tooth agenesis, but also with microdontia, short tooth roots, dental pulp stones, and taurodontism.     

WNT10A gene is the second molecular candidate in a cohort of young Italian subjects with ectodermal derivative impairment (EDI)

Ectodermal dysplasias are a group of genetic disorders defined by ectodermal derivative impairment (EDI). To test the impact of the Wnt/beta‐catenin pathway in the genetic screening of EDI, we performed a molecular gene study of WNT10A in 60 subjects from a population of 133 young Italian patients referred for the impairment of at least one major ectodermal‐derived structure and who had a previous negative molecular screen for ectodysplasin signaling pathway genes ED1, EDAR, and EDARADD. Fourteen WNT10A mutations were identified in 33 subjects (24.8%), 11 of which were novel variants. The phenotype was evaluated through a detailed clinical examination of the major and minor ectodermal‐derived structures. This study is the first to show that, after ED1, WNT10A is the second molecular candidate for EDI in a large Italian Caucasian population. The study confirmed that Phe228Ile is the most frequent WNT10A variant in Caucasian populations, and that WNT10A mutations are associated with large variability in EDI.     

Frizzled-10, up-regulated in primary colorectal cancer, is a positive regulator of the WNT - beta-catenin - TCF signaling pathway.

WNT signaling pathway is implicated in embryogenesis as well as in carcinogenesis. We have previously cloned and characterized Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, and FZD10, encoding seven-transmembrane-type WNT receptors. Here, expression of FZD10 mRNA in various types of human cancer and effects of FZD10 mRNA microinjection into Xenopus early embryos were investigated. Northern blot analyses revealed relatively high-level expression of 4.0-kb FZD10 mRNA in cervical cancer cell lines HeLa S3, SKG-I, SKG-IIIa, and in a glioblastoma cell line A-172. Matched tumor/normal expression array analysis revealed significant up-regulation of FZD10 mRNA in 2 cases of primary colon cancer. Function of FZD10 was next investigated by using Xenopus axis duplication assay, in which positive regulators of the WNT - beta-catenin - TCF signaling pathway induce axis duplication. Injection of wild-type FZD10 mRNA into the ventral marginal zone of 4-cell-stage Xenopus embryos induced partial axis duplication in 40% of embryos. Ventral injection of Thr579Ala FZD10 mRNA or Val581Leu FZD10 mRNA with mutations in the C-terminal Ser/Thr-X-Val motif also induced partial axis duplication in about 40% of embryos. Furthermore, ventral injection of FZD10 mRNA significantly augmented the potential of co-injected Xenopus wnt-8 (Xwnt-8) mRNA to induce complete axis duplication. These results suggest that up-regulation of FZD10 mRNA in several types of human cells might lead to carcinogenesis through activation of the beta-catenin - TCF signaling pathway synergistically with some class of WNTs.     

Regulation of WNT signaling molecules by retinoic acid during neuronal differentiation in NT2 cells: threshold model of WNT action (review)

NT2/NTera2 cells, derived from human embryonal tumor, differentiate into neuronal cells after treatment with all-trans-retinoic acid (ATRA). We have cloned and characterized 13 out of 19 human WNT genes, and also 9 out of 10 human Frizzled (FZD) genes encoding seven-transmembrane-type WNT receptors, which are potent targets for pharmacogenomics in the post-genomic era, especially in the field of regenerative medicine and clinical oncology. Because WNT signals are implicated in morphogenesis of neural tissues, regulation of 19 WNT genes and 10 FZD genes during the early phase of neuronal differentiation in NT2 cells is reviewed. Multiple WNTs and FZDs are expressed in NT2 cells. WNT2B/WNT13 gene encode 2 isoforms due to alternative splicing of alternative promoter type, and WNT2B isoform 2 (WNT2B2) rather than WNT2B isoform 1 (WNT2B1) is expressed in NT2 cells. WNT3A, WNT8A, WNT8B, WNT10B and WNT11 are down-regulated in NT2 cells after ATRA treatment, while WNT2, WNT7B and WNT14B are up-regulated. FZD4 and FZD10 are up-regulated in NT2 cells after ATRA treatment. Expression of multiple WNT signaling molecules are dramatically changed during the early phase of neuronal differentiation in NT2 cells. Each WNT activates the beta-catenin - TCF pathway, the JNK pathway or the Ca2+-releasing pathway in NT2 cells, and summed effects of multiple WNTs might determine the fate of NT2 cells (self-renewal or differentiation) through switching intracellular WNT signaling pathways. The author proposes the threshold model of WNT action.     

Identification of WNT/{beta}-CATENIN signaling pathway components in human cumulus cells

Signaling via the conserved WNT/β-CATENIN pathway controlsdiverse developmental processes. To explore its potential rolein the ovary, we investigated the expression of WNTs, frizzled(FZD) receptors and other pathway components in human cumuluscells obtained from oocytes collected for in vitro fertilization.Proteins were detected in cultured cells using immunofluorescencemicroscopy. Protein–protein interactions were analyzedby means of immunoprecipitation. WNT2, FZD2, FZD3 and FZD9 wereidentified but WNT1, WNT4 and FZD4 were not detected. WNT2 isco-expressed with FZD2, FZD3 and FZD9. Co-immunoprecipitationusing WNT2 antibody demonstrated that WNT2 interacts with bothFZD3 and FZD9, but only FZD9 antibody precipitated WNT2. Wealso identified DVL (disheveled), AXIN, GSK-3β (glycogensynthase kinase-3β) and β-CATENIN. β-CATENINis concentrated in the plasma membranes. DVL co-localizes withFZD9 and AXIN in the membranes, but GSK-3β has little co-localizationwith AXIN and β-CATENIN. Interestingly, β-CATENINis highly co-localized with FZD9 and AXIN. CDH1 (E-cadherin)was also detected in the plasma membranes and cytoplasm, co-localizedwith β-CATENIN, and CDH1 antibody precipitated β-CATENIN.The results suggest that WNT2 could act through its receptorFZD9 to regulate the β-CATENIN pathway in cumulus cells,recruiting β-CATENIN into plasma membranes and promotingthe formation of adherens junctions involving CDH1.     

Dental and extra‐oral clinical features in 41 patients with WNT10A gene mutations: A multicentric genotype–phenotype study

WNT10A gene encodes a canonical wingless pathway signaling molecule involved in cell fate specification as well as morphogenetic patterning of the developing ectoderm, nervous system, skeleton, and tooth. In patients, WNT10A mutations are responsible for ectodermal‐derived pathologies including isolated hypo‐oligodontia, tricho‐odonto‐onycho‐dermal dysplasia and Schöpf‐Schulz‐Passarge syndrome (SSPS). Here we describe the dental, ectodermal, and extra‐ectodermal phenotypic features of a cohort of 41 patients from 32 unrelated families. Correlations with WNT10A molecular status (heterozygous carrier, compound heterozygous, homozygous) and patient's phenotypes were performed. Mild to severe oligodontia was observed in all patients bearing biallelic WNT10A mutations. However, patients with compound heterozygous mutations presented no significant difference in phenotypes compared with homozygous individuals. Anomalies in tooth morphology were frequently observed with heterozygous patients displaying hypodontia. No signs of SSPS, especially eyelids cysts, were detected in our cohort. Interestingly, extra‐ectodermal signs consisted of skeletal, neurological and vascular anomalies, the latter suggesting a wider phenotypic spectrum associated with WNT10A mutations. Indeed, the Wnt pathway plays a crucial role in skeletal development, lipid metabolism, and neurogenesis, potentially explaining patient's clinical manifestations.     

Distinct impacts of bi‐allelic WNT10A mutations on the permanent and primary dentitions in odonto‐onycho‐dermal dysplasia

Odonto‐onycho‐dermal dysplasia (OODD) is a rare autosomal recessive syndrome characterized by multiple ectodermal abnormalities. Mutations of the wingless‐type MMTV integration site family member 10A (WNT10A) gene have been associated with OODD. To date, only 11 OODD‐associated WNT10A mutations have been reported. In this report, we Characterized the clinical manifestations with focusing on dental phenotypes in four unrelated OODD patients. By Sanger sequencing, we identified five novel mutations in the WNT10A gene, including two homozygous nonsense mutations c.1176C>A (p.Cys392*) and c.742C>T (p.Arg248*), one homozygous frame‐shift mutation c.898‐899delAT (p.Ile300Profs*126), and a compound heterozygous mutation c.826T>A (p.Cys276Ser) and c.949delG (p.Ala317Hisfs*121). Our findings confirmed that bi‐allelic mutations of WNT10A were responsible for OODD and greatly expanded the mutation spectrum of OODD. For the first time, we demonstrated that bi‐allelic WNT10A mutations could lead to anodontia of permanent teeth, which enhanced the phenotypic spectrum of WNT10A mutations. Interestingly, we found that bi‐allelic mutations in the WNT10A gene preferentially affect the permanent dentition rather the primary dentition, suggesting that the molecular mechanisms regulated by WNT10A in the development of permanent teeth and deciduous teeth might be different.     

Ptosis as a unique hallmark for autosomal recessive WNT1‐associated osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder, mainly characterized by bone fragility and low bone mass. Defects in the type I procollagen‐encoding genes account for the majority of OI, but increasingly more rare autosomal recessive (AR) forms are being identified, which are caused by defects in genes involved in collagen metabolism, bone mineralization, or osteoblast differentiation. Bi‐allelic mutations in WNT1 have been associated with a rare form of AR OI, characterized by severe osteoporosis, vertebral compression, scoliosis, fractures, short stature, and variable neurological problems. Heterozygous WNT1 mutations have been linked to autosomal dominant early‐onset osteoporosis. In this study, we describe the clinical and molecular findings in 10 new patients with AR WNT1‐related OI. Thorough revision of the clinical symptoms of these 10 novel patients and previously published AR WNT1 OI cases highlight ptosis as a unique hallmark in the diagnosis of this OI subtype.     

Expression and regulation of WNT5A and WNT5B in human cancer: up-regulation of WNT5A by TNFalpha in MKN45 cells and up-regulation of WNT5B by beta-estradiol in MCF-7 cells

WNT signaling pathway plays key roles in carcinogenesis and embryogenesis, and WNT signaling molecules are potent targets for diagnosis, prevention and treatment of cancer as well as for regenerative medicine or tissue engineering. We have so far cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14 and WNT14B/WNT15 using bioinformatics and cDNA-PCR. We have also reported frequent up-regulation of WNT2 and WNT5A in primary gastric cancer, which is probably due to cancer-stromal interaction. Here, expression and regulation of WNT5A and WNT5B in human cancer were investigated. WNT5A was relatively highly expressed in TE6 and TE10 among 12 esophageal cancer cell lines, and WNT5B was expressed in the majority of esophageal cancer cell lines. Among 7 pancreatic cancer cell lines, WNT5A was up-regulated in Hs700T, and WNT5B in PANC-1. WNT5A, but not WNT5B, was up-regulated by TNFalpha in MKN45 cells derived from gastric cancer. WNT5B, but not WNT5A, was up-regulated by beta-estradiol in MCF-7 cells derived from breast cancer. WNT5A and WNT5B were expressed together in 5 embryonal tumor cell lines, and were slightly down-regulated by all-trans retinoic acid in NT2 cells. Up-regulation of WNT5A and WNT5B in several types of human cancer expressing FZD5 might lead to more malignant phenotype through activation of the beta-catenin - TCF pathway.     

SETDB1 accelerates tumourigenesis by regulating the WNT signalling pathway

We investigated the oncogenic role of SETDB1, focusing on non‐small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT–β‐catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Rare variations in WNT3A and DKK1 may predispose carriers to primary osteoporosis

Childhood-onset primary osteoporosis is manifested as reduced bone mineral density, peripheral fractures and/or vertebral compression fractures. Until now, only mutations in LRP5 have been shown to cause the disorder. Candidate gene analyses were performed on 15 patients with primary osteoporosis and 80 healthy controls using CSGE and sequencing. The genes studied included DKK1, DKK2, WNT3A, WNT10B, AXIN1, SOST, TPH1 and 5-HTR1B. Two rare variants in WNT3A (c.152A > G, p.K51R) and DKK1 (c.359G > T, p.R120L) were identified in two patients and their affected family members, but not in control subjects, suggesting a significance for the skeletal phenotype. The in vitro studies of variants showed reduced signaling activity in p.K51R-Wnt3a, while no differences were observed between the WT and variant forms of DKK1. This study addresses the role of other components of the canonical Wnt signaling pathway besides LRP5 in primary osteoporosis, and putatively associates WNT3A and DKK1 variants with the disorder. Future functional studies are needed to elucidate the functional effects of the variants.     

WNT10A missense mutation associated with a complete Odonto-Onycho-Dermal Dysplasia syndrome

Wnt signalling is one of a few pathways that are crucial for controlling genetic programs during embryonic development as well as in adult tissues. WNT10A is expressed in the skin and epidermis and it has shown to be critical for the development of ectodermal appendages. A nonsense mutation in WNT10A was recently identified in odonto-onycho-dermal dysplasia (OODD; MIM 257980), a rare syndrome characterised by severe hypodontia, nail dystrophy, smooth tongue, dry skin, keratoderma and hyperhydrosis of palms and soles. We identified a large consanguineous Pakistani pedigree comprising six individuals affected by a complete OODD syndrome. Autozygosity mapping using SNP array analysis showed that the affected individuals are homozygous for the WNT10A gene region. Subsequent mutation screening showed a homozygous c.392C>T transition in exon 3 of WNT10A, which predicts a p.A131V substitution in a conserved α-helix domain. We report here on the first inherited missense mutation in WNT10A with associated ectodermal features.     

Disrupted WNT Signaling in Mouse Embryonic Stem Cells in the Absence of Calreticulin

The role of endoplasmic reticulum (ER) homeostasis and protein quality control in the regulation of WNT signaling is not understood. Here we provide evidence for a role of calreticulin in the regulation of WNT signaling. We show that a deficiency in calreticulin disrupted WNT signaling, and prevented cell cycle progression via the miR-302 microRNA family. These effects were dependent on the Ca²⁺ buffering capacity of calreticulin, as the protein is important in regulating ER Ca²⁺ release and activation of Ca²⁺-dependent kinase and phosphatase cascades (including c-Src, Akt, and PTP1B). We also show that calreticulin plays a role in the secretion and ER retention of WNT3a, thereby affecting downstream WNT signaling. In calreticulin-deficient ES cells, the WNT and miR-302 dependent maintenance of the naïve ES cell state and the transition to primed pluripotency transition were lost, preventing cells from undergoing accurate differentiation. Together, these findings demonstrate unexpected roles of calreticulin and ER Ca²⁺ homeostasis/signaling in the canonical WNT signaling pathway.     

Mutations in WNT10A are frequently involved in oligodontia associated with minor signs of ectodermal dysplasia

Ectodermal dysplasias (ED) are a clinically and genetically heterogeneous group of hereditary disorders that have in common abnormal development of ectodermal derivatives. Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of eccrine sweat glands, hair, and teeth. The X‐linked form of the disease, caused by mutations in the EDA gene, represents the majority of patients with the hypohidrotic form. Autosomal dominant and autosomal recessive forms are occasionally seen, and result from mutations in at least three genes (WNT10A, EDAR, or more rarely EDARADD). We have screened for mutations in EDAR (commonly involved in the hypohidrotic form) and WNT10A (involved in a wide spectrum of ED and in isolated hypodontia) in a cohort of 36 patients referred for EDA molecular screening, which failed to identify any mutation. We identified eight EDAR mutations in five patients (two with homozygous mutations, one with compound heterozygous mutations, and two with heterozygous mutation), four of which were novel variants. We identified 28 WNT10A mutations in 16 patients (5 with homozygous mutations, 7 with compound heterozygous mutations, and 4 with heterozygous mutations), seven of which were novel variants. Our study allows a more precise definition of the phenotypic spectrum associated with EDAR and WNT10A mutations and underlines the importance of the implication of WNT10A among patients with ED. © 2013 Wiley Periodicals, Inc.