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Results for “Wnt Proteins”

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At least 19 recordsLinked to original sources
PubMed1999

A new secreted protein that binds to Wnt proteins and inhibits their activities.

The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion.

The secretion and extracellular transport of Wnt protein are thought to be well-regulated processes. Wnt is known to be acylated with palmitic acid at a conserved cysteine residue (Cys77 in murine Wnt-3a), and this residue appears to be required for the control of extracellular transport. Here, we show that murine Wnt-3a is also acylated at a conserved serine residue (Ser209). Of note, we demonstrated that this residue is modified with a monounsaturated fatty acid, palmitoleic acid. Wnt-3a defective in acylation at Ser209 is not secreted from cells in culture or in Xenopus embryos, but it is retained in the endoplasmic reticulum (ER). Furthermore, Porcupine, a protein with structural similarities to membrane-bound O-acyltransferases, is required for Ser209-dependent acylation, as well as for Wnt-3a transport from the ER for secretion. These results strongly suggest that Wnt protein requires a particular lipid modification for proper intracellular transport during the secretory process.

Acylation↗
PubMed2003

Wnt proteins are lipid-modified and can act as stem cell growth factors.

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.

Amino Acid Sequence↗
PubMed2006

Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells.

Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu.

Amino Acid Sequence↗
PubMed1999

Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.

Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.

Animals↗
PubMed2004

Wnt proteins in mammary development and cancer.

Secreted proteins of the Wnt family play widespread roles in the regulation of embryonic development, and aberrant activation of the canonical Wnt/beta-catenin pathway is one of the most frequent signaling abnormalities known in human cancer. While the consequences of Wnt signaling in development are diverse at the cellular level, they are often concerned with cell fate determination. Recent data also indicate that Wnt proteins influence the self-renewal of stem cells in certain tissues. In the mammary gland, Wnt signals are strongly implicated in initial development of the mammary rudiments, and in the ductal branching and alveolar morphogenesis that occurs during pregnancy. Transgenic expression of Wnt1 or Wnt10b in the mouse mammary gland leads to lobuloalveolar hyperplasia with a major risk of progression to carcinoma. Recent evidence suggests that this phenotype is associated with expansion of a multipotent progenitor cell population. In human breast cancer, evidence of beta-catenin accumulation implies that the canonical Wnt signaling pathway is active in over 50% of carcinomas. However, specific mutations that might account for this activation of signaling have not yet been identified.

Breast↗
PubMed1995

Biochemical analysis of murine Wnt proteins reveals both shared and distinct properties.

The murine Wnt family of proteins consists of at least 12 members that possess significant amino acid homology. Current evidence suggests that these proteins are secreted cell-signaling molecules which are likely to have multiple roles during both embryonic development and oncogenesis. Although the biochemical properties of Wnt-1 have been thoroughly examined, less is known about the characteristics of other Wnt family members. We have compared the properties of six murine Wnt proteins (Wnt-1, Wnt-3a, Wnt-5a, Wnt-5b, Wnt-6, and Wnt-7b) transiently expressed in COS cells. All members enter the endoplasmic reticulum (ER) and are glycosylated. However, all six Wnt proteins are primarily retained in the ER in association with BiP, a resident ER protein that binds to improperly folded proteins and prevents their secretion and/or promotes proper folding. Although all Wnt family members examined are similarly processed, one notable difference was identified. Whereas addition of suramin to COS cell cultures significantly increases the levels of all six Wnts in the medium, the addition of heparin only influences the levels of Wnt-1, Wnt-6, and Wnt-7b.

Amino Acid Sequence↗
PubMed2004

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin.

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Chimeric Wnt proteins define the amino-terminus of Wnt-1 as a transformation-specific determinant.

Wnt-1 induces morphological transformation of C57MG mammary epithelial cells and accumulation of cytosolic beta-catenin whereas Wnt-5a has no effect. To identify regions within the 370 amino acid Wnt-1 protein required for these functions we tested eleven chimeric genes that contained variable amounts of Wnt-1 and Wnt-5a sequence. Transformation and beta-catenin regulation in C57MG cells is controlled by amino acids that lie within 186 residues of the amino terminus of Wnt-1. Small substitutions between residues 186 and 292 reduced Wnt-1 activity. Replacement of the carboxy terminal 79 amino acids of Wnt-1 by Wnt-5a did not affect function. These results were supported by transient expression assays in 293 cells wherein beta-catenin accumulated in the cytoplasm in response to ectopic Wnt-1 expression. In 293 cells, a larger region of the amino-terminus of Wnt-1 was found to be required for beta-catenin regulation. Nonfunctional chimeras that contained at least 99 amino terminal Wnt-1 residues inhibited Wnt-1 stimulation of 293 cells. One of these chimeras inhibited both Wnt-1 and Wnt-3 activity suggesting that Wnt-1 and Wnt-3 interact with a common signaling component.

Amino Acid Sequence↗
PubMed2002

A transcriptional response to Wnt protein in human embryonic carcinoma cells.

BACKGROUND: Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway. RESULTS: We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP. CONCLUSIONS: Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

Animals↗
PubMed2004

Protein kinase A signalling via CREB controls myogenesis induced by Wnt proteins.

Select members of the Wnt family of secreted glycoproteins have been implicated in inducing the myogenic determinant genes Pax3, MyoD and Myf5 during mammalian embryogenesis, but the mechanism of induction has not been defined. We describe an unexpected role for protein kinase A (PKA) signalling via CREB in this induction. Using a combination of in vitro explant assays, mutant analysis and gene delivery into mouse embryos cultured ex vivo, we demonstrate that adenylyl cyclase signalling via PKA and its target transcription factor CREB are required for Wnt-directed myogenic gene expression. Wnt proteins can also stimulate CREB-mediated transcription, providing evidence for a Wnt signalling pathway involving PKA and CREB. Our findings raise the possibility that PKA/CREB signalling may also contribute to other Wnt-regulated processes in embryonic patterning, stem cell renewal and cancer.

Adenylyl Cyclases↗
PubMed1997

A role for WNT proteins in induction of dermomyotome.

Dorsoventral patterning of somites into sclerotome and dermomyotome involves antagonistic actions of ventralizing and dorsalizing signals originating from tissues surrounding the somites. The notochord and the floor plate of the neural tube provide a ventralizing signal(s) directing sclerotome development, whereas the surface ectoderm and dorsal neural tube provide a dorsalizing signal(s) directing dermomyotome development. Evidence has been provided that Sonic Hedgehog mediates the ventralizing effects of notochord and floor plate, but the dorsalizing signal(s) that patterns the dermomyotome has not been identified. The documented expression of Wnt1 and Wnt3a in the dorsal neural tube and of Wnt4 and Wnt6 in the surface ectoderm at the time of dermomyotome specification prompted us to investigate the involvement of WNT proteins in patterning the dermomyotome. Here we show that tissue culture cells expressing these WNT family members can maintain and induce dermomyotome marker expression in presomitic mesoderm explants, supporting the hypothesis that WNT proteins mediate the dorsalizing effects of the surface ectoderm and dorsal neural tube on somites.

Animals↗
PubMed2004

Wnt proteins promote neuronal differentiation in neural stem cell culture.

Wnt signaling is implicated in the control of cell growth and differentiation during CNS development from studies of mouse and chick models, but its action at the cellular level has been poorly understand. In this study, we examine the in vitro function of Wnt signaling in embryonic neural stem cells, dissociated from neurospheres derived from E11.5 mouse telencephalon. Conditioned media containing active Wnt-3a proteins are added to the neural stem cells and its effect on regeneration of neurospheres and differentiation into neuronal and glial cells was examined. Wnt-3a proteins inhibit regeneration of neurospheres, but promote differentiation into MAP2-positive neuronal cells. Wnt-3a proteins also increase the number of GFAP-positive astrocytes but suppress the number of oligodendroglial lineage cells expressing PDGFR or O4. These results indicate that Wnt-3a signaling can inhibit the maintenance of neural stem cells, but rather promote the differentiation of neural stem cells into several cell lineages.

Animals↗
PubMed2000

The informational content of gradients of Wnt proteins.

This perspective tackles the issues facing developmental biologists and cell biologists regarding how the molecular mechanisms for specifying cell fate are defined. This perspective focuses on members of the Wnt family. The author proposes that Wnt proteins may act as stabilizing signals for earlier inductive events in certain systems, for example, in Caenorhabditis elegans during the migration of two neurons and in Drosophila melanogaster during the patterning of the wing.

Animals↗
PubMed2006

Multiplicity of the interactions of Wnt proteins and their receptors.

Wnts are secreted proteins that are essential for a wide array of developmental and physiological processes. They signal across the plasma membranes by interacting with serpentine receptors of the Frizzled (Fz) family and members of the low-density-lipoprotein receptor-related protein (LRP) family. Recent advances in the Wnt signaling field have revealed that Wnt-unrelated proteins activate or suppress Wnt signaling by binding to Fzs or LRP5/6 and that atypical receptor tyrosine kinases mediate Wnt signaling independently of Fz and/or function as a Fz co-receptor. This review highlights recent progress in our understanding of the multiplicity of Wnts and their receptors. We discuss how the interaction between the ligands and receptors activate distinct intracellular signaling pathways. We also discuss how intracellular trafficking of Wnt signaling components can regulate the sensitivity of cells to Wnts.

Animals↗
PubMed2005

Sustained Wnt protein expression in chondral constructs from mesenchymal stem cells.

Wnt genes encode a number of secreted glycoproteins which are closely associated with the cell surface and the extracellular matrix. Recently, members of Wnt family have been implicated in regulating chondrocyte differentiation, but their roles in the chondrogenic process are not fully understood. To contribute to an understanding of the roles of Wnts during chondrogenesis, we have analysed the spatiotemporal expression patterns of Wnt using in vitro models for chondrogenesis of human bone marrow-derived mesenchymal stem cells (hMSCs). In chondrogenic aggregate culture system, RT-PCR analysis revealed expression of Wnt5a and Wnt4 during late chondrogenesis (days 10 and 15). Immunohistochemical analysis showed widespread distribution of Wnt5a and Wnt4 throughout the aggregates at this late phase of culture (days 14 and 21). In addition, in this aggregate culture system, immunohistochemical staining of Wnt4 and Wnt5a showed similar spatiotemporal expression patterns to that of type II collagen or type X collagen. To confirm the results obtained by immunostaining, the specificity of the anti-Wnt4 or anti-Wnt5a antibody was assessed by Western blot analysis. Of Wnt4 and Wnt5a, only Wnt5a was immunodetectable by Western blot analysis. Western blot analysis showed that Wnt5a was expressed as two different molecular weight forms of 40 and 44 kDa. Treatment with PNGase F, which removes N-linked oligosaccharides, revealed that the mass difference between these two forms could be accounted for by the N-glycosylation status of the protein. When hMSCs were seeded on a porous gelatin sponge, immunolocalization studies showed that type II collagen and type X collagen were detected particularly at the periphery at day 7 of culture. In contrast, Wnt4 and Wnt5a showed even distribution throughout the hMSC/gelatin sponge constructs. Their different spatial expression patterns suggest that Wnt4 and Wnt5a proteins are not functionally linked to type II collagen and type X collagen synthesis in in vitro chondrogenic models of hMSCs.

Blotting, Western↗
PubMed2000

Roles of Wnt proteins in neural development and maintenance.

Many constituents of Wnt signaling pathways are expressed in the developing and mature nervous systems. Recent work has shown that Wnt signaling controls initial formation of the neural plate and many subsequent patterning decisions in the embryonic nervous system, including formation of the neural crest. Wnt signaling continues to be important at later stages of development. Wnts have been shown to regulate the anatomy of the neuronal cytoskeleton and the differentiation of synapses in the cerebellum. Wnt signaling has been demonstrated to regulate apoptosis and may participate in degenerative processes leading to cell death in the aging brain.

Aging↗
PubMed1995

The Drosophila Wnt protein DWnt-3 is a secreted glycoprotein localized on the axon tracts of the embryonic CNS.

The Wnt gene family encodes highly conserved cysteine-rich proteins which appear to act as secreted developmental signals. Both the mouse Wnt-1 gene and the Drosophila wingless (wg) gene play important roles in central nervous system (CNS) development. wg is also required earlier, in the development of the embryonic metameric body pattern. We have begun to characterize the developmental expression and role of another member of the Drosophila Wnt gene family, DWnt-3. Using antisera raised to the DWnt-3 protein, we show that the protein is secreted in vivo. The early protein expression domains include the limb and appendage primordia. Late expression domains comprise the ventral cord and supraesophageal ganglia of the CNS. Notably, DWnt-3 protein accumulates on the commissural and longitudinal axon tracts of the CNS. Ectopic expression of DWnt-3 in transgenic embryos bearing a HS-DWnt-3 construct leads to specific disruption of the commissural axon tracts of the CNS. We also show that DWnt-3 does not functionally replace wg in an in vivo assay. Experiments with a tissue culture cell line transfected with a construct encoding the DWnt-3 gene show that DWnt-3 protein is efficiently synthesized, glycosylated, proteolytically processed, and transported to the extracellular matrix and medium. DWnt-3, therefore, encodes a secreted protein, which is likely to play a role in development of the Drosophila CNS.

Animals↗