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Results for “Wnt Signaling Pathway”

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PubMed2005

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation.

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Retrograde signalling at the synapse: a role for Wnt proteins.

The formation of functional synapses requires a proper dialogue between incoming axons and their future synaptic targets. As axons approach their target, they are instructed to slow down and remodel to form proper presynaptic terminals. Although significant progress has been made in the identification of the mechanisms that control axon guidance, little is known about the mechanisms that regulate the conversion of actively growing axon into a presynaptic terminal. We found that Wnt secreted proteins are retrograde signals that regulate the terminal arborization of axons and synaptic differentiation. Wnts released from postsynaptic neurons induce extensive remodelling on incoming axons. This remodelling is manifested by a decrease in axon extension with a concomitant increase in growth-cone size. This morphological change is correlated with changes in the dynamics and organization of microtubules. Studies of a vertebrate synapse and the Drosophila neuromuscular junction suggest that a conserved Wnt signalling pathway modulates presynaptic microtubules as axons remodel during synapse formation. In this paper I discuss the role of the Wnt-Dvl (Dishevelled protein)-GSK-3beta (glycogen synthase kinase-3beta) signalling pathway in axon remodelling during synapse formation in the central nervous system.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Expression of Wnt genes and frizzled 1 and 2 receptors in normal breast epithelium and infiltrating breast carcinoma.

The Wnt genes encode a family of related, secreted proteins which initiate a signal cascade upon binding to cell surface receptor molecules. The signaling pathway has been shown to be critical for normal growth and development in model organisms and is implicated in the genesis of numerous human cancers. Wnt proteins regulate mammary development in the mouse but their precise role in normal breast development and malignant transformation in humans remains poorly defined. In this study, we have examined the expression of several Wnt ligands by in situ anti-sense RNA hybridization in normal and malignant human breast tissue, as well as in several estrogen-responsive and estrogen-independent human breast cancer cell lines. The specific Wnt genes tested included Wnt1, Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7b and Wnt10b. We have also studied the expression of frizzled receptors 1 and 2 by immunohistochemistry in these tissues. Our results indicate that several of the Wnt ligands, especially Wnt1 and Wnt6, are strongly expressed in both normal and malignant breast tissue and that Wnt7b is down-regulated in breast cancer, compared to normal breast epithelium. The expression of frizzled 1 and 2 receptors was found to be up-regulated in breast cancer. These studies provide additional support to the hypothesis that the Wnt signaling pathway is involved in human breast cancer.

Breast Neoplasms↗

PubMed2005

[SFRP, a family of new colorectal tumor suppressor candidate genes].

Aberrant Wnt pathway signaling is an early progression event in up to 90% of colorectal cancers (CRC). It occurs mainly through mutations of APC or beta-catenin. We have identified frequent promoter hypermethylation and gene silencing of the secreted frizzled-related protein (SFRP) genes in CRC. SFRPs possess a domain similar to one in the Wnt receptor Frizzled proteins and can inhibit Wnt receptor binding to downregulate pathway signaling in development. We next found that restoration of SFRP function in CRC cells attenuates Wnt signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in CRC progression and may, thus, provide constitutive Wnt signaling which is required to complement downstream mutations in the evolution of CRC.

Colorectal Neoplasms↗

PubMed2005

Adenomatous polyposis coli (Apc) tumor suppressor gene as a multifunctional gene.

The adenomatous polyposis coli (Apc) gene is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. The Apc gene product (APC), basically a cytoplasmic protein, blocks cell cycle progression and plays crucial roles in development. The APC binds to beta-catenin, axin and glycogen synthase kinase 3beta to form a large protein complex, in which beta-catenin is phosphorylated and broken down, resulting in negative regulation of the Wnt signaling pathway. Most of the mutated Apc genes in colorectal tumors lack beta-catenin-binding regions and fail to inhibit Wnt signaling, leading to overproliferation of tumor cells. The APC, having some nuclear localizing signals in its molecule, can also be localized in the nucleus. The nuclear APC exports excess beta-catenin to the cytoplasm. Through its C-terminus, APC binds to post-synaptic density discs large zonula occludens domain-containing proteins, such as discs large (DLG) and post-synaptic density (PSD)-95, and may play important roles in epithelial morphogenesis, brain development and neuronal functions. In addition, APC is involved in cell motility through its association with microtubules and APC-stimulated guanine nucleotide exchange factor. Colocalization of APC and DLG is dependent on microtubules. The Apc gene is highly expressed in the embryonic and postnatal developing brain. Recently, we found that APC is required for the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by facilitating the clustering of PSD-95 and these receptors at the postsynapse. In addition, APC is present in astrocytes, although its role in astrocytes is, as yet, unknown.

Adenomatous Polyposis Coli↗

PubMed1999

EGL-27 is similar to a metastasis-associated factor and controls cell polarity and cell migration in C. elegans.

Mutations in the C. elegans gene egl-27 cause defects in cell polarity and cell migration: the polarity of the asymmetric T cell division is disrupted and the descendants of the migratory QL neuroblast migrate incorrectly because they fail to express the Hox gene mab-5. Both of these processes are known to be controlled by Wnt pathways. Mosaic analysis indicates that egl-27 function is required in the T cell for proper cell polarity. We cloned egl-27 and discovered that a domain of the predicted EGL-27 protein has similarity to Mta1, a mammalian factor overexpressed in metastatic cells. Overlaps in the phenotypes of egl-27 and Wnt pathway mutants suggest that the EGL-27 protein interacts with Wnt signaling pathways in C. elegans.

Amino Acid Sequence↗

PubMed1997

WNTs modulate cell fate and behavior during vertebrate development.

Wnt genes encode a family of secreted glycoproteins that modulate cell fate and behavior in embryos through activation of receptor-mediated signaling pathways. Wnt sequences, patterns of expression and activities are highly conserved in evolution, so it has been possible to gain insights into the functions, and mechanisms of action, of the Wnt genes through a synthesis of genetic and cell biological approaches in different organisms. These studies suggest that there are functionally distinct WNT proteins as assayed by the ability to transform cells and by differences in embryonic responses to ectopic WNT signals. Moreover, gain-of-function and loss-of-function studies both support the involvement of Wnt proteins in modulating cell fate and cell behavior during vertebrate development, often through combinatorial interactions with other signaling pathways to regulate gene expression.

Animals↗

PubMed2004

Gene expression after crush injury of human saphenous vein: using microarrays to define the transcriptional profile.

BACKGROUND: Vein graft stenosis is believed to be the pathophysiologic response of vascular tissue to injury and is the major cause of vein graft failure. Therapeutic interventions might improve with knowledge of the physiologic pathways involved in the hyperplastic response to vascular injury. In this study, our purpose was to identify induced, early pathways that might be important in the human response to vascular injury. STUDY DESIGN: Human saphenous vein from 7 patients was organ cultured or crush injured and cultured for 48 or 72 hours after harvest. Gene expression was determined for syngeneic veins at harvest and at the experimental time points and compared to determine which genes were induced or repressed. Expressed genes (the transcriptional profile) were then assigned to functional physiologic classes. RESULTS: At 72 hours, in both organ-cultured and crush-injured vein, the gene for the Wnt ligand protein (WNT5A) was induced. At 48 hours in the organ-cultured vein only, the gene for the Frizzled protein (FZD2), a subunit of the Wnt receptor complex, was repressed. At 72 hours in injured vein only, the gene for the product of Wnt signaling (WISP1) was induced; the gene for the Wnt-binding, soluble Frizzled-related protein (FRZB) was repressed; and the gene for Dickkopf (DKK1) protein, which binds to the low density lipoprotein receptor-related protein subunit of the Wnt receptor complex, was induced. CONCLUSIONS: Early induction of WNT5A, coupled with the coordinated induction and repression of genes that modulate the Wnt signaling pathway, led to the early, selective induction of WISP1 and no other Wnt-inducible genes. This early, selective expression of a limited gene set might characterize the human vascular response to injury, and could enable development of therapies to treat the clinical sequelae of this response.

CCN Intercellular Signaling Proteins↗

PubMed2005

Nrarp functions to modulate neural-crest-cell differentiation by regulating LEF1 protein stability.

Nrarp (Notch-regulated ankyrin repeat protein) is a small protein that has two ankyrin repeats. Although Nrarp is known to be an inhibitory component of the Notch signalling pathway that operates in different developmental processes, the in vivo roles of Nrarp have not been fully characterized. Here, we show that Nrarp is a positive regulator in the Wnt signalling pathway. In zebrafish, knockdown of Nrarp-a expression by an antisense morpholino oligonucleotide (MO) results in altered Wnt-signalling-dependent neural-crest-cell development. Nrarp stabilizes LEF1 protein, a pivotal transcription factor in the Wnt signalling cascade, by blocking LEF1 ubiquitination. In accordance with this, the knockdown phenotype of lef1 is similar to that of nrarp-a, at least in part, in its effect on the development of multiple tissues in zebrafish. Furthermore, activation of LEF1 does not affect Notch activity or vice versa. These findings reveal that Nrarp independently regulates canonical Wnt and Notch signalling by modulating LEF1 and Notch protein turnover, respectively.

Animals↗

PubMed2000

The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of BMP2/4 and Wnt pathways.

The neuroectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be regionalized into forebrain, midbrain, hindbrain and spinal cord by a two-step process. In the activation step, the Spemann gastrula organizer induces neuroectoderm with anterior character, followed by posteriorization by a transforming signal. Recently, simultaneous inhibition of BMP and Wnt signaling was shown to induce head formation in frog embryos. However, how the inhibition of BMP and Wnt signaling pathways specify a properly patterned head, and how they are regulated in vivo, is not understood. Here we demonstrate that the loss of anterior neural fates observed in zebrafish bozozok (boz) mutants occurs during gastrulation due to a reduction and subsequent posteriorization of neuroectoderm. The neural induction defect was correlated with decreased chordino expression and consequent increases in bmp2b/4 expression, and was suppressed by overexpression of BMP antagonists. Whereas expression of anterior neural markers was restored by ectopic BMP inhibition in early boz gastrulae, it was not maintained during later gastrulation. The posteriorization of neuroectoderm in boz was correlated with ectopic dorsal wnt8 expression. Overexpression of a Wnt antagonist rescued formation of the organizer and anterior neural fates in boz mutants. We propose that boz specifies formation of anterior neuroectoderm by regulating BMP and Wnt pathways in a fashion consistent with Nieuwkoop's two-step neural patterning model. boz promotes neural induction by positively regulating organizer-derived chordino and limiting the antineuralizing activity of BMP2b/4 morphogens. In addition, by negative regulation of Wnt signaling, boz promotes organizer formation and limits posteriorization of neuroectoderm in the late gastrula.

Animals↗

PubMed2002

The JNK signal transduction pathway.

The c-Jun NH(2)-terminal kinase (JNK) is a member of an evolutionarily conserved sub-family of mitogen-activated protein (MAP) kinases. Recent studies have led to progress towards understanding the physiological function of the JNK signaling pathway, including the analysis of the phenotype of knockout mice. An important role for JNK in the non-canonical Wnt-signaling pathway has been established. Insight into the role of scaffold proteins that may assemble functional JNK modules has been achieved. In addition, a small molecule pharmacological inhibitor of JNK has been described and it is likely that this drug will facilitate future studies of JNK function.

Amino Acid Sequence↗

PubMed2003

A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

BACKGROUND: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers. RESULTS: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice. CONCLUSION: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

Animals↗

PubMed2000

Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein.

Wnt signaling has an important role in both embryonic development and tumorigenesis. beta-Catenin, a key component of the Wnt signaling pathway, interacts with the TCF/LEF family of transcription factors and activates transcription of Wnt target genes. Here, we identify a novel beta-catenin-interacting protein, ICAT, that was found to inhibit the interaction of beta-catenin with TCF-4 and represses beta-catenin-TCF-4-mediated transactivation. Furthermore, ICAT inhibited Xenopus axis formation by interfering with Wnt signaling. These results suggest that ICAT negatively regulates Wnt signaling via inhibition of the interaction between beta-catenin and TCF and is integral in development and cell proliferation.

Adaptor Proteins, Signal Transducing↗

PubMed2007

WIF1, an inhibitor of the Wnt pathway, is rearranged in salivary gland tumors.

Chromosome rearrangements involving 12q13-15 are frequent among several tumors, including pleomorphic adenomas. The common molecular target for these aberrations is the HMGA2 gene, but various fusion partners of HMGA2 have been reported in tumors. Here we report the identification of the WNT inhibitory factor 1 (WIF1) gene as a novel HMGA2 fusion partner in a salivary gland pleomorphic adenoma. In normal salivary gland tissue WIF1 is expressed at a high level and HMGA2 is not expressed. However, in the pleomorphic adenoma expressing the HMGA2/WIF1 fusion transcript, we observed re-expression of HMGA2 wild-type transcripts and very low levels of WIF1 expression. These data suggest a possible synergistic effect between upregulation of HMGA2 and downregulation of WIF1. We screened 13 additional benign and malignant salivary gland tumors and detected WIF1 rearrangement in one out of two carcinomas ex-pleomorphic adenoma analyzed. In this malignant tumor, the rearrangement of one WIF1 allele coexists with loss of the other allele, a classic signature of a tumor suppressor gene. WIF1 is an antagonist of the Wnt signaling pathway, which plays a critical role in human cancer. In transgenic mouse models, Wnt activation leads to a high frequency of benign and malignant salivary gland tumors. To our knowledge, this is the first report suggesting that WIF1 is a recurrent target in human salivary gland oncogenesis and that downregulation of WIF1 plays a role in the development and/or progression of pleomorphic adenomas.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Molecular cloning and characterization of human WNT5B on chromosome 12p13.3 region.

WNT signaling pathway plays a key role in carcinogenesis and embryogenesis. We have cloned and characterized the human WNT5B. Overlapping WNT5B cDNAs, containing 1080-bp ORF, were isolated. WNT5B encoded a 359-amino-acid polypeptide with the N-terminal signal peptide, four N-linked glycosylation sites, and consensus amino-acid residues conserved among the WNT family. WNT5B showed 80.5% total-amino-acid identity with WNT5A. Comparison between nucleotide sequence of WNT5B cDNA and human genome draft sequences revealed that the WNT5B gene, consisting of 4 exons, was located on human chromosome 12p13.3 region. Northern blot analyses with W5B2 probe detected the 2.8- and 2.4-kb WNT5B mRNAs. WNT5B was moderately expressed in adult prostate and fetal brain, and weakly expressed in fetal lung, kidney, adult liver, ovary, and small intestine. Among human cancer cell lines, WNT5B was expressed in gastric cancer cell lines MKN7, MKN45, KATO-III, and a teratocarcinoma cell line NT2. WNT5B might be implicated in human carcinogenesis through activation of the WNT-beta-catenin-TCF signaling pathway, just like Wnt5a.

Amino Acid Sequence↗

PubMed2005

CK2 as a positive regulator of Wnt signalling and tumourigenesis.

CK2 is upregulated in rapidly dividing cells including most human tumours. Transgenic overexpression of CK2 in lymphoid or mammary lineages predisposes to transformation. Multiple signalling and oncogene pathways could be regulated by CK2 in this process. Our studies suggest that phosphorylation of critical oncogenes by CK2, as well as by other serine-threonine kinases, regulates their stability via susceptibility to the proteasomal degradation system. Beta-catenin is a transcriptional co-factor in the Wnt signalling pathway that is regulated in this fashion. Inactivating mutations in the adenomatosis polyposis coli (APC) gene, which encodes a carrier protein for beta-catenin, or stabilizing mutations in beta-catenin itself, frequently occur in human tumours. CK2 and the monomeric serine-threonine kinase GSK3 have opposing actions on beta-catenin: GSK-3 phosphorylation of the N-terminus of beta-catenin promotes degradation; while phosphorylation by CK2 in the armadillo repeat protein interaction domain protects it. Beta-catenin is overexpressed in mammary tumours occurring in mice transgenic for CK2 or a dominant negative form of GSK3, and also in mammary tumours arising following treatment with the environmental carcinogen DMBA. Experiments are underway to determine whether expression of both CK2 and kinase inactive GSK3 further accelerates tumorigenesis. Inhibitors of GSK3 under development for treatment of diabetes could promote tumours, while CK2 inhibitors should be useful agents for treatment of cancer.

Animals↗

PubMed2002

Activation of an adipogenic program in adult myoblasts with age.

Myoblasts isolated from mouse hindlimb skeletal muscle demonstrated increased adipogenic potential as a function of age. Whereas myoblasts from 8-month-old adult mice did not significantly accumulate terminal markers of adipogenesis regardless of culture conditions, myoblasts from 23-month-old mice accumulated fat and expressed genes characteristic of differentiated adipocytes, such as the fatty acid binding protein aP2. This change in differentiation potential was associated with a change in the abundance of the mRNA encoding the transcription factor C/EBPalpha, and in the relative abundance of PPARgamma2 to PPARgamma1 mRNAs. Furthermore, PPARgamma activity appeared to be regulated at the level of phosphorylation, being more highly phosphorylated in myoblasts isolated from younger animals. Although adipogenic gene expression in myoblasts from aged animals was activated, presumably in response to PPARgamma and C/EBPalpha, unexpectedly, myogenic gene expression was not effectively repressed. The Wnt signaling pathway may also alter differentiation potential in muscle with age. Wnt-10b mRNA was more abundantly expressed in muscle tissue and cultured myoblasts from adult compared with aged mice, resulting in stabilization of cytosolic beta-catenin, that may potentially contribute to inhibition of adipogenic gene expression in adult myoblasts. The changes reported here, together with those reported in bone marrow stroma with age, suggest that a default program may be activated in mesenchymal cells with increasing age resulting in a more adipogenic-like phenotype. Whether this change in differentiation potential contributes to the increased adiposity in muscle with age remains to be determined.

Adipocytes↗

PubMed2004

Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine.

Wnt signaling pathways regulate many developmental responses; however, little is known about how Wnt ligands function on a biochemical level. Recent studies have shown that Wnt-3a is palmitoylated before secretion. Here we report that Drosophila Wnt-1 (Wingless) also undergoes a lipid modification. Lipidation occurs in the endoplasmic reticulum and is dependent on Porcupine, a putative O-acyltransferase. After modification, DWnt-1 partitions as a membrane-anchored protein and is sorted into lipid raft detergent-insoluble microdomains. Lipidation, raft targeting, and secretion can be blocked by the addition of 2-bromopalmitate, a competitive inhibitor of O-acyltransferase activity. Based on these results we propose a model whereby lipidation targets Wnt-1 to secretory vesicles that deliver the ligand to specialized microdomains at the cell surface where it can be packaged for secretion.

Animals↗