Wingless-type MMTV Integration Site Family Research Articles

CLC– a novel susceptibility gene for allergic rhinitis?

Studies of the nasal lavage fluid proteome have previously identified proteins differently expressed in patients with symptomatic allergic rhinitis, e.g. S100A7, prolactin-inducible protein (PIP), wingless-type MMTV integration site family, member 2B (WNT2B), Charcot-Leyden crystal protein (CLC) and palate lung nasal epithelial clone (PLUNC). The aim of the present study was to investigate if genetic variation associated with allergic rhinitis can be found in these genes. Peripheral blood was collected from 251 patients with birch and/or grass pollen-induced allergic rhinitis and 386 nonatopic healthy controls. A total of 39 single nucleotide polymorphisms (SNPs) distributed over the genes PIP, WNT2B, CLC and PLUNC were selected from dbSNP, genotyped and investigated for associations with allergic rhinitis. Twelve additional SNPs were subsequently analysed for CLC. All 22 investigated SNPs in CLC were polymorphic. Ten SNPs yielded significant differences between cases and controls with respect to genotype frequencies. Homozygotes for the minor allele were more common in allergic individuals compared to healthy controls. The minor alleles of these SNPs were all located on the same haplotype. Furthermore, homozygotes for the minor allele of two of the promoter SNPs had higher average scores for birch in skin prick test. In contrast, for seven SNPs within the gene, heterozygotes and homozygotes for the major allele had higher average scores for grass. None of the other three genes showed association. Genetic variation in CLC was found to be associated with allergic rhinitis. The pattern of variation is compatible with a recessive inheritance model and the previously observed altered protein levels detected in patients with allergic rhinitis.

Thiazolidinediones increase the wingless-type MMTV integration site family (WNT) inhibitor Dickkopf-1 in adipocytes: a link with osteogenesis

Dickkopf-1 (DKK1) is a secreted inhibitor of canonical wingless-type MMTV integration site family (WNT) signalling; the key pathway for cell fate and development. Inhibition of WNT signalling by DKK1 in precursor cells promotes adipogenesis and inhibits osteogenesis. Previous studies have shown that treatment of type 2 diabetic patients with thiazolidinediones (TZDs) reduces bone density and increases risk of bone fractures, while body fat is increased. We examined the effect of TZDs on secretion and DKK1 levels in pre-adipocytes and mature adipose cells and also measured circulating DKK1 levels in 11 patients with type 2 diabetes before and after treatment with the TZD rosiglitazone for 90 days. TZDs added in vitro rapidly increased DKK1 protein levels and secretion in both fully differentiated adipose cells and pre-adipocytes undergoing differentiation. In parallel, beta-catenin levels, a marker of canonical WNT signalling, were reduced. Serum levels of DKK1 were also increased in several of the patients with type 2 diabetes after treatment with rosiglitazone for 90 days. These results provide a novel mechanism whereby peroxisome proliferator-activated receptor-gamma activation can terminate WNT signalling and promote adipogenesis. Furthermore, they provide an explanation for why TZD treatment can lead to reduced bone formation and increased risk of fractures, since inhibited WNT signalling in progenitor cells promotes adipogenesis while osteogenesis is reduced.

Association between autism and variants in the wingless-type MMTV integration site family member 2 ( WNT2) gene

Autism is a severe neurodevelopmental disorder with a complex genetic aetiology. The wingless-type MMTV integration site family member 2 (WNT2) gene has been considered as a candidate gene for autism. We conducted a case-control study and followed up with a transmission disequilibrium test (TDT) analysis to confirm replication of the significant results for the first time. We conducted a case-control study of nine single nucleotide polymorphisms (SNPs) within the WNT2 gene in 170 patients with autism and 214 normal controls in a Japanese population. We then conducted a TDT analysis in 98 autistic families (trios) to replicate the results of the case-control study. In the case-control study, three SNPs (rs3779547, rs4727847 and rs3729629), two major individual haplotypes (A-T-C and G-G-G, consisting of rs3779547, rs4727847, and rs3729629), and global probability values of the haplotype distributions in the same region (global p=0.0091) showed significant associations with autism. Furthermore, all of these significant associations were also observed in the TDT analysis. Our findings provide evidence for a significant association between WNT2 and autism. Considering the important role of the WNT2 gene in brain development, our results therefore indicate that the WNT2 gene is one of the strong candidate genes for autism.

Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice

TCF7L2 is a type 2 diabetes susceptibility gene and downstream effector of canonical wingless-type MMTV integration site family (WNT) signalling. However, it is unknown whether this pathway is active in adult pancreatic islets in vivo, and whether it is regulated in obesity. We analysed activation of endogenous WNT signalling in the endocrine pancreas from wild-type and obese mice (ob/ob) using a reporter transgene (Topgal). Regulation of WNT signalling was compared using gene chip experiments from isolated pancreatic islets. Activation of canonical WNT signalling in pancreatic islets and the mouse beta cell line MIN6 was measured using immunoblotting for cytosolic beta-catenin. Endogenous canonical WNT signalling was absent in the adult endocrine pancreas in both wild-type and obese mice. We identified WNT4 as an abundant WNT signalling molecule in adult pancreatic islets that is induced in two different insulin-resistant mouse models. Increased expression of WNT4 inhibited canonical WNT signalling in pancreatic islets and MIN6 cells. Canonical WNT signalling is not active in adult beta cells in vivo. WNT4 provides a potential mechanism for suppression of canonical WNT signalling in obese mice.

Wingless-type MMTV integration site family member 5A (WNT5A)

Wingless-type MMTV integration site family member 5A (WNT5A)

Chronic Pancreatitis and Pancreatic Cancer: Prediction and Mechanism

We investigated the SPINK 1 mutations in 156 sporadic pancreatic cancer (PCa), and 8 pancreatic cancer with chronic pancreatitis (CPPCa) patients, and in 527 healthy subjects. The results demonstrated that 3 of 8 patients with CPPCa (37.5%) had the SPINK 1 gene N34S mutation. In addition, 3 of 156 sporadic PCa patients (1.9%) and 1 of them (0.6%) had the N34S and IVS3+2T>C mutation, respectively. The combined frequency of 2.5% was significantly higher than that of healthy subjects (0.38%), suggesting that the SPINK 1 mutation is an important risk factor for the development of pancreatic cancer. To investigate the genetic difference between sporadic PCa and CPPCa, we investigated several factors involved in the pathogenesis of PCa in 6 CPPCa and 15 sporadic PCa patients. The factors examined were genes including K-ras, p53, smad 4, p-smad 1, CXCL 14, NF-kB subunit p65 and Wnt 5a. No significant difference was found in the comparative examination of these factors, suggesting that the molecular disorders appeared to occur similarly in CPPCa as well as sporadic PCa. To assess the role of fibrosis in pancreatic carcinogenesis, we investigated the effects of pancreatic stellate cells (PSCs), which are largely responsible for pancreatic fibrogenesis, on duct cells, in vitro and in vivo. Activated PSCs were found surrounding precancerous duct cells in the tissues of a dimethylbenzanthracene mouse model and those of human PCa. Consistently, human pancreatic epithelial duct cells cultured with PSC conditioned media showed increased cell proliferation and colony formation, suggesting that PSCs may promote pancreatic ductal tumorigenesis.

Orphan receptor tyrosine kinase ROR2 as a potential therapeutic target for osteosarcoma

Osteosarcoma is the most prevalent bone malignant tumor in children and adolescents, and displays heterogeneous histology and high propensity for distant metastasis. Although adjuvant chemotherapy remarkably improved treatment outcome over the past few decades, prognosis for osteosarcoma patients with pulmonary metastasis is still unsatisfactory. To identify novel therapeutic targets for osteosarcoma, we investigated the gene expression profile of osteosarcomas by cDNA microarray analysis and found transactivation of receptor tyrosine kinase-like orphan receptor 2 (ROR2) expression in the majority of osteosarcoma samples. Treatment of osteosarcoma cell lines with siRNA against ROR2 significantly inhibited cell proliferation and migration. We also identified wingless-type MMTV integration site family, member 5B (WNT5B) as a putative ROR2 ligand and that the physiological interaction of WNT5B and ROR2 could enhance cell migration, indicating the possible roles of ROR2 and WNT5B in the metastatic property of osteosarcoma cells. Taken together, our findings revealed that the WNT5B/ROR2 signaling pathway is a promising therapeutic target for osteosarcoma.

Stromal cell-derived factor-1 promotes survival of pancreatic beta cells by the stabilisation of beta-catenin and activation of transcription factor 7-like 2 (TCF7L2)

Stromal cell-derived factor-1 (SDF-1) is a chemokine produced in stromal tissues in multiple organs. Earlier we reported on levels of SDF-1 and SDF-1 receptor (CXCR4) in the insulin-producing beta cells of the mouse pancreas and determined that the SDF-1/CXCR4 axis is important for beta cell survival through activation of the prosurvival kinase, protein kinase B (AKT). Since AKT is known to modulate the wingless-type MMTV integration site family (WNT) signalling cascade, we examined the effects of SDF-1/CXCR4 on WNT signalling in beta cells and whether this signalling is important for cell survival. Activation of downstream WNT signalling (beta-catenin and transcription factor 7-like 2, [TCF7L2]) in response to SDF-1 was examined in the islets of WNT signalling reporter (Tcf-optimal promoter beta-galactosidase) mice and in INS-1 and MIN6 beta cells. Cytoprotection of beta cells by SDF-1 in response to the induction of apoptosis was assessed by caspase 3 and TUNEL assays. SDF-1 induced WNT signalling in beta cells of isolated islets and in INS-1 cells via CXCR4-mediated activation of Galphai/o-coupled signalling and the phosphatidylinositol 3-kinase/AKT signalling cascade resulting in the inhibition of glycogen synthase kinase 3-beta. The key WNT signalling regulators, beta-catenin and AKT, were activated by SDF-1 at the transcriptional and post-translational levels. Specific inhibition of beta-catenin in the WNT signalling cascade reversed the anti-apoptotic effects of SDF-1. SDF-1 promotes pancreatic beta cell survival via activation of AKT and downstream WNT signalling via the stabilisation and activation of beta-catenin/TCF7L2 transcriptional activators. These findings suggest a mechanism for SDF-1 based glucose-lowering therapies by enhancing beta cell mass through increasing cell survival.

Differentiation of a Highly Tumorigenic Basal Cell Compartment in Urothelial Carcinoma

Highly tumorigenic cancer cell (HTC) populations have been identified for a variety of solid tumors and assigned stem cell properties. Strategies for identifying HTCs in solid tumors have been primarily empirical rather than rational, particularly in epithelial tumors, which are responsible for 80% of cancer deaths. We report evidence for a spatially restricted bladder epithelial (urothelial) differentiation program in primary urothelial cancers (UCs) and in UC xenografts. We identified a highly tumorigenic UC cell compartment that resembles benign urothelial stem cells (basal cells), co-expresses the 67-kDa laminin receptor and the basal cell-specific cytokeratin CK17, and lacks the carcinoembryonic antigen family member CEACAM6 (CD66c). This multipotent compartment resides at the tumor-stroma interface, is easily identified on histologic sections, and possesses most, if not all, of the engraftable tumor-forming ability in the parental xenograft. We analyzed differential expression of genes and pathways in basal-like cells versus more differentiated cells. Among these, we found significant enrichment of pathways comprising "hallmarks" of cancer, and pharmacologically targetable signaling pathways, including Janus kinase-signal transducer and activator of transcription, Notch, focal adhesion, mammalian target of rapamycin, epidermal growth factor receptor (erythroblastic leukemia viral oncogene homolog [ErbB]), and wingless-type MMTV integration site family (Wnt). The basal/HTC gene expression signature was essentially invisible within the context of nontumorigenic cell gene expression and overlapped significantly with genes driving progression and death in primary human UC. The spatially restricted epithelial differentiation program described here represents a conceptual advance in understanding cellular heterogeneity of carcinomas and identifies basal-like HTCs as attractive targets for cancer therapy.

Actin-related protein 2/3 complex, subunit 2 (ARPC2); fibronectin 1 (FN1); osteopontin (OPN; SPP1); regulator of G-protein signaling 1 (RGS1); wingless-type MMTV integration site family member 2 (WNT2)

Actin-related protein 2/3 complex, subunit 2 (ARPC2); fibronectin 1 (FN1); osteopontin (OPN; SPP1); regulator of G-protein signaling 1 (RGS1); wingless-type MMTV integration site family member 2 (WNT2)

Intramucosal Distribution of WNT Signaling Components in Human Esophagus

The molecular mechanisms governing the biology and pathobiology of esophageal squamous mucosa in health and disease are not completely understood. Earlier genome-wide expression study of normal-looking esophageal squamous mucosa has shown differential expression of the Wingless-type MMTV integration site family (Wnt) modulators Dickkopf (Dkk) homologs among healthy individuals and patients with reflux esophagitis and Barrett metaplasia suggesting that the Wnt pathway may be involved in esophageal mucosal biology. Seven full-thickness human donor esophagi were cryosectioned for immunohistochemical analysis, and lamina propria (LP), basal (BC), intermediate (IC), and superficial (SC) cells were also dissected by laser-capture microdissection for real-time polymerase chain reaction. Wnt1, 2b, and 3a were expressed primarily in BC, Wnt3, and 5b in LP, and Wnt5a in IC. Frizzled 1, low-density lipoprotein receptor-related protein 6, secreted frizzled-related protein 1, T-cell-specific transcription factor 3, and dishevelled 3 were expressed highest in LP decreasing precipitously medially toward SC. Dkk1 predominantly expressed in SC was more than 100-folds greater than other layers (P<0.001). Dkk4 was expressed primarily in SC but Dkk3 was opposite with greatest expression in LP. Immunohistochemical analysis showed Wnt1 and 3a in BC, Wnt5a in IC and SC, Dkk1 predominantly in SC, Dkk4 in SC and IC, and Dkk3 and SFRP1 in LP and BC

β-catenin (CTNNB1); wingless-type MMTV integration site family, member 3A (WNT3A)

β-catenin (CTNNB1); wingless-type MMTV integration site family, member 3A (WNT3A)

Quantitative Analysis of mRNA Transcripts of Hox, SHH, PTCH, Wnt, and Fzd Genes in Canine Hematopoietic Progenitor Cells and Various in vitro Colonies Differentiated from the Cells

Homeobox (Hox), Sonic hedgehog (SHH), and Wingless-type MMTV integration site family (Wnt) are known to modulate the self-renewal and expansion of hematopoietic progenitor/stem cells in humans and mice. Frizzled (Fzd) and Patched1 (PTCH1) represent the receptors of Wnt and SHH, respectively. In this study, the amounts of mRNA transcripts of the genes associated with the self-renewal of hematopoietic stem cells, HoxB3, HoxB4, HoxA10, Wnt5a, Wnt2b, Fzd1, Fzd6, SHH, and PTCH1, were measured in canine unfractionated bone marrow cells, CD34-enriched cells, and various colony-forming units in culture (CFU-C). Partial cDNA sequences of these 9 canine genes were determined in this study. Quantitative real-time polymerase chain reaction was employed to indicate their relative amounts of mRNA transcripts. Amounts of mRNA transcripts of HoxB3, HoxA10, PTCH1, and Wnt5a genes in canine CD34-enriched cell fraction were significantly larger than those in the CD34-depleted cell fraction. Amounts of mRNA transcripts of HoxB3, HoxA10, PTCH1, Wnt5a, and Wnt2b genes in various CFU-C cells were significantly smaller than those in the seeded CD34-enriched cell fraction. These results suggested important roles of the products of these genes in self-renewal, expansion, and survival of hematopoietic progenitor cells in dogs as shown in humans and rodents.

Wingless-type MMTV integration site family, member 7A (WNT7A); WNT7B

Wingless-type MMTV integration site family, member 7A (WNT7A); WNT7B

Enhanced Tau Phosphorylation in the Hippocampus of Mice Treated with 3,4-Methylenedioxymethamphetamine (“Ecstasy”)

3,4-Methylenedioxymethamphetamine (MDMA) ("Ecstasy") produces neurotoxic effects, which result into an impairment of learning and memory and other neurological dysfunctions. We examined whether MDMA induces increases in tau protein phosphorylation, which are typically associated with Alzheimer's disease and other chronic neurodegenerative disorders. We injected mice with MDMA at cumulative doses of 10-50 mg/kg intraperitoneally, which are approximately equivalent to doses generally consumed by humans. MDMA enhanced the formation of reactive oxygen species and induced reactive gliosis in the hippocampus, without histological evidence of neuronal loss. An acute or 6 d treatment with MDMA increased tau protein phosphorylation in the hippocampus, revealed by both anti-phospho(Ser(404))-tau and paired helical filament-1 antibodies. This increase was restricted to the CA2/CA3 subfields and lasted 1 and 7 d after acute and repeated MDMA treatment, respectively. Tau protein was phosphorylated as a result of two nonredundant mechanisms: (1) inhibition of the canonical Wnt (wingless-type MMTV integration site family) pathway, with ensuing activation of glycogen synthase kinase-3beta; and (2) activation of type-5 cyclin-dependent kinase (Cdk5). MDMA induced the expression of the Wnt antagonist, Dickkopf-1, and the expression of the Cdk5-activating protein, p25. In addition, the increase in tau phosphorylation was attenuated by strategies that rescued the Wnt pathway or inhibited Cdk5. Finally, an impairment in hippocampus-dependent spatial learning was induced by doses of MDMA that increased tau phosphorylation, although the impairment outlasted this biochemical event. We conclude that tau hyperphosphorylation in the hippocampus may contribute to the impairment of learning and memory associated with MDMA abuse.

Wnt4 inhibits β‐catenin/TCF signalling by redirecting β‐catenin to the cell membrane

During embryonic development, beta-catenin is central both to the transcriptional activation of Wnt [wingless-type MMTV (murine-mammary-tumour virus) integration site family] target genes and as a mediator of cell-cell adhesion. Signals that regulate its levels and subcellular localization are critical. One mechanism of Wnt signalling results in stabilization of beta-catenin protein, which leads to its translocation into the nucleus, where it interacts with TCF (T-cell factor, HMG box) and activates transcription of target genes. Less well understood are mechanisms of Wnt signalling that do not involve beta-catenin stabilization and result in inhibition of beta-catenin-mediated transcription. Here, we show that a member of the Wnt protein family, Wnt4 (Wnt, member 4), regulates the subcellular localization of beta-catenin, redirecting it to the cell membrane. Unique among Wnts, this action does not affect the stability of beta-catenin but does prohibit its involvement in TCF gene transactivation. This novel mechanism suggests that Wnt4 acts as a switch between the two modes of beta-catenin function, transcriptional activation and cell-cell adhesion.

Disruption of the Non-Canonical WNT Pathway in Lung Squamous Cell Carcinoma

Disruptions of beta-catenin and the canonical Wnt pathway are well documented in cancer. However, little is known of the non-canonical branch of the Wnt pathway. In this study, we investigate the transcript level patterns of genes in the Wnt pathway in squamous cell lung cancer using reverse-transcriptase (RT)-PCR. It was found that over half of the samples examined exhibited dysregulated gene expression of multiple components of the non-canonical branch of the WNT pathway. In the cases where beta catenin (CTNNB1) was not over-expressed, we identified strong relationships of expression between wingless-type MMTV integration site family member 5A (WNT5A)/ frizzled homolog 2 (FZD2), frizzled homolog 3 (FZD3) / dishevelled 2 (DVL2), and low density lipoprotein receptor-related protein 5 (LRP5)/ secreted frizzled-related protein 4 (SFRP4). This is one of the first studies to demonstrate expression of genes in the non-canonical pathway in normal lung tissue and its disruption in lung squamous cell carcinoma. These findings suggest that the non-canonical pathway may have a more prominent role in lung cancer than previously reported.

The Role ofFoxg1and Dorsal Midline Signaling in the Generation of Cajal-Retzius Subtypes

Cajal-Retzius (CR) cells, the earliest-born neurons in the neocortex, arise from discrete sources within the telencephalon, including the dorsal midline and the pallial-subpallial boundary (PSB). In particular, the cortical hem, a region of high bone morphogenetic proteins (BMPs) and Wnt (wingless-type MMTV integration site family) expression but lacking in Foxg1 (forkhead box G1) is a major source of CR neurons. Whether CR cells from distinct origins arise from disparate developmental processes or share a common mechanism is unclear. To elucidate the molecular basis of CR cell development, we assessed the role of both Foxg1 and dorsal midline signaling in the production of cortical hem- and PSB-derived CR cells. We demonstrate that the loss of Foxg1 results in the overproduction of both of these CR populations. However, removal of Foxg1 at embryonic day 13, although expanding the number of CR cells with a PSB phenotype, does not result in an expansion of BMPs or Wnts in the dorsomedial signaling center. Conversely, loss of the dorsal midline ligands as observed in Gli3 (glioma-associated oncogene homolog 3) mutants results in the loss of the cortical hem-derived CR character but does not affect the specification of PSB-derived CR cells. Hence, our findings demonstrate that, although the specification of cortical hem-derived CR cells is dependent on signaling from the dorsal midline, Foxg1 functions to repress the generation of both cortical hem- and PSB-derived CR cells.

Cullin4B/E3-ubiquitin ligase negatively regulates β-catenin

Beta-catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, beta-catenin is targeted to ubiquitin-proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in beta-catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased beta-catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and beta-catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of beta-catenin levels.

Gene amplifications at chromosome 7 of the human gastric cancer genome

Genetic aberrations at chromosome 7 are known to be related with diverse human diseases, including cancer and autism. In a number of cancer research areas involving gastric cancer, several comparative genomic hybridization studies employing metaphase chromosome or BAC clone microarrays have repeatedly identified human chromosome 7 as containing 'regions of changes' related with cancer progression. cDNA microarray-based comparative genomic hybridization can be used to directly identify individual target genes undergoing copy number variations. Copy number change analysis for 17,000 genes on a microarray format was performed with tumor and normal gastric tissues from 30 patients. A group of 90 genes undergoing copy number increases (gene amplification) at the p11 approximately p22 or q21 approximately q36 region of chromosome 7 is reported. The list of genes includes wingless-type MMTV integration site family member 2 (WNT2), a proto-oncogene and acyloxyacyl hydrolase (AOAH) that was amplified in >80% of the tested cases. The amplified genes are those functioning in the biological processes such as signal transduction pathways, cell proliferation, metabolism, transport, inflammatory response and protein folding or proteolysis. Also found in the list are genes that are targets for drug development, such as maltase-glucoamylase (MGAM), cyclin-dependent kinase 5 (CDK5), neuropeptide Y (NPY) and dopa decarboxylase (DDC). The current dataset can be used as one of the resources in understanding genetic aberrations of chromosome 7 in human gastric cancer.