Wnt Receptor Activation Research Articles

Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells.

The differential activation of Wnt pathways (canonical: Wnt/β-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca2+) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD–co-receptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6–LRP6 and FZD6–ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6–LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of β-catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6–ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/β-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes.

Genome-wide identification of long noncoding RNAs and their competing endogenous RNA networks involved in the odontogenic differentiation of human dental pulp stem cells

BackgroundLong noncoding RNAs (lncRNAs) play an important role in the multiple differentiations of mesenchymal stem cells (MSCs). However, few studies have focused on the regulatory mechanism of lncRNAs in the odontogenic differentiation of human dental pulp stem cells (hDPSCs).MethodshDPSCs were induced to differentiate into odontoblasts in vitro, and the expression profiles of lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in differentiated and undifferentiated cells were obtained by microarray. Bioinformatics analyses including Gene Ontology (GO) analysis, pathway analysis, and binding site prediction were performed for functional annotation of lncRNA. miRNA/odontogenesis-related gene networks and lncRNA-associated ceRNA networks were constructed. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the expression of selected genes. RNA fluorescence in situ hybridization (FISH), qRT-PCR, and western blot analysis were used to explore the location and function of lncRNA-G043225. Dual-luciferase reporter assay was performed to confirm the binding sites of miR-588 with G043225 and Fibrillin 1 (FBN1).ResultsWe identified 132 lncRNAs, 114 miRNAs, and 172 mRNAs were differentially expressed. GO analysis demonstrated that regulation of the neurogenic locus notch homolog (Notch), Wnt, and epidermal growth factor receptor (ERBB) signaling pathways and activation of mitogen-activated protein kinase (MAPK) activity were related to odontogenic differentiation. Pathway analysis indicated that the most significant pathway was the forkhead box O (FoxO) signaling pathway, which is related to odontogenic differentiation. Two odontogenesis-related gene-centered lncRNA-associated ceRNA networks were successfully constructed. The qRT-PCR validation results were consistent with the microarray analysis. G043225 mainly locating in cytoplasm was proved to promote the odontogenic differentiation of hDPSCs via miR-588 and FBN1.ConclusionThis is the first study revealing lncRNA-associated ceRNA network during odontogenic differentiation of hDPSCs using microarray, and it could provide clues to explore the mechanism of action at the RNA-RNA level as well as novel treatments for dentin regeneration based on stem cells.

A Transcription-uncoupled Negative Feedback Loop for the 1 WNT Pathway: WNT Activates the AAK1 Kinase to Promote Clathrin-mediated Endocytosis of LRP6

β-catenin-dependent WNT signal transduction governs development, tissue homeostasis and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome we report that the AP2 Associated Kinase 1 (AAK1), a known CME enhancer, strongly inhibits WNT signaling. Reciprocally, silencing of AAK1 expression or pharmacological inhibition of AAK1 activity using a new potent and selective AAK1 inhibitor activates WNT signaling. We show that AAK1 and LRP6 co-complex, and that AAK1 promotes clathrinmediated endocytosis of LRP6 to suppress the WNT pathway. Our data reveal a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, thus promoting clathrin-coated pit maturation. We propose that following WNT receptor activation and the ensuing transcriptional response, increased AAK1 function limits WNT signaling longevity.

WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop

Summaryβ-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.

Wnt lipidation: Roles in trafficking, modulation, and function.

The Wnt signaling pathway consists of various downstream target proteins that have substantial roles in mammalian cell proliferation, differentiation, and development. Its aberrant activity can lead to uncontrolled proliferation and tumorigenesis. The posttranslational connection of fatty acyl chains to Wnt proteins provides the unique capacity for regulation of Wnt activity. In spite of the past belief that Wnt molecules are subject to dual acylation, it has been shown that these proteins have only one acylation site and undergo monounsaturated fatty acylation. The Wnt monounsaturated fatty acyl chain is more than just a hydrophobic coating and appears to be critical for Wnt signaling, transport, and receptor activation. Here, we provide an overview of recent findings in Wnt monounsaturated fatty acylation and the mechanism by which this lipid moiety regulates Wnt activity from the site of production to its receptor interactions.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting β-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wntsignaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased β-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote β-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.

The Dkk3 gene encodes a vital intracellular regulator of cell proliferation.

Members of the Dickkopf (Dkk) family of Wnt antagonists interrupt Wnt-induced receptor assembly and participate in axial patterning and cell fate determination. One family member, DKK3, does not block Wnt receptor activation. Loss of Dkk3 expression in cancer is associated with hyperproliferation and dysregulated ß-catenin signaling, and ectopic expression of Dkk3 halts cancer growth. The molecular events mediating the DKK3-dependent arrest of ß-catenin-driven cell proliferation in cancer cells are unknown. Here we report the identification of a new intracellular gene product originating from the Dkk3 locus. This Dkk3b transcript originates from a second transcriptional start site located in intron 2 of the Dkk3 gene. It is essential for early mouse development and is a newly recognized regulator of ß-catenin signaling and cell proliferation. Dkk3b interrupts nuclear translocation ß-catenin by capturing cytoplasmic, unphosphorylated ß-catenin in an extra-nuclear complex with ß-TrCP. These data reveal a new regulator of one of the most studied signal transduction pathways in metazoans and provides a novel, completely untapped therapeutic target for silencing the aberrant ß-catenin signaling that drives hyperproliferation in many cancers.

Phosphorylated low-density lipoprotein receptor-related protein 6 is prevalent in hippocampal progenitor cells and circuits of aged human hippocampus

Wnt signaling has been implicated in Alzheimer’s disease (AD) pathogenesis, but no studies have described Wnt signaling in aging brain. Phosphorylation of the Wnt coreceptor, low-density lipoprotein receptor-related protein 6 (Lrp6), is a sensitive indicator of Wnt ligand-receptor interaction and canonical Wnt signaling. We report that in aged human temporal lobe, the phospho-Lrp6 (pLrp6) epitope localizes to neurons in the entorhinal cortex (EC), the dentate gyrus (DG), and the hippocampal formation, especially in the CA3 field. Activated Lrp6 is detected in neuronal soma and in neuronal processes, particularly in the mossy fiber terminals in the stratum lucidum of CA3. These three regions and their connectivity represent the afferent arm of the major hippocampal circuit. In the DG, cells positive for pLrp6 include Type 1 and Type 2 hippocampal progenitor cells. Overall, these data indicate regional Wnt receptor activation in the human hippocampus that is most prominent in the cells comprising the afferent arm of the major hippocampal circuit that is associated with learning and memory functions. These findings are consistent with data from rodent studies which suggest an important role for Wnts in adult neurogenesis in the human DG. We speculate that Wnt signaling may be an activity-dependent trophic influence in the hippocampus.

Targeting the Wingless Signaling Pathway with Natural Compounds as Chemopreventive or Chemotherapeutic Agents

The aberrant activation of the wingless (Wnt) signaling pathway is a key element involved in carcinogenesis as Wnt regulates a variety of cellular processes including proliferation, differentiation, survival, apoptosis and cell motility. Upon Wnt receptor activation, the canonical "Wnt/beta-catenin" as well as the non canonical "Wnt/planar cell polarity, Wnt/Ca²⁺" pathways are activated. This offers multiple possibilities to target the aberrant regulation of this signaling pathway in order to counteract cancer proliferation. During the last decade, natural compounds from both marine and terrestrial origins were tested for their potential to modulate the expression of specific genes related to the Wnt signaling cascade but also for their anti-carcinogenic properties. It appears that phenolic compounds (e.g., caffeic acid phenethyl ester, curcumin and derivatives, green, white and black tea, resveratrol, quercetin, isoflavone, fisetin, and isoflavone) as well as other small molecules were able to inhibit the Wnt signaling through the modulation of beta-catenin expression, transcriptional activity and of the subsequent expression of Wnt target genes. Altogether, these findings underline the fact that Wnt signaling could be considered as a promising target for innovative strategies for cancer treatment and prevention.

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.

Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors

Wnt ligands signal through β-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through β-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.

Mutations in the Human naked cuticle Homolog NKD1 Found in Colorectal Cancer Alter Wnt/Dvl/β-Catenin Signaling

BackgroundMutation of Wnt signal antagonists Apc or Axin activates β-catenin signaling in many cancers including the majority of human colorectal adenocarcinomas. The phenotype of apc or axin mutation in the fruit fly Drosophila melanogaster is strikingly similar to that caused by mutation in the segment-polarity gene, naked cuticle (nkd). Nkd inhibits Wnt signaling by binding to the Dishevelled (Dsh/Dvl) family of scaffold proteins that link Wnt receptor activation to β-catenin accumulation and TCF-dependent transcription, but human NKD genes have yet to be directly implicated in cancer.Methodology/Principal FindingsWe identify for the first time mutations in NKD1 - one of two human nkd homologs - in a subset of DNA mismatch repair-deficient colorectal tumors that are not known to harbor mutations in other Wnt-pathway genes. The mutant Nkd1 proteins are defective at inhibiting Wnt signaling; in addition, the mutant Nkd1 proteins stabilize β-catenin and promote cell proliferation, in part due to a reduced ability of each mutant Nkd1 protein to bind and destabilize Dvl proteins.Conclusions/SignificanceOur data raise the hypothesis that specific NKD1 mutations promote Wnt-dependent tumorigenesis in a subset of DNA mismatch-repair-deficient colorectal adenocarcinomas and possibly other Wnt-signal driven human cancers.

β-Catenin Is a Mediator of the Response of Fibroblasts to Irradiation

Radiation causes soft tissue complications that include fibrosis and deficient wound healing. beta-Catenin, a key component in the canonical Wnt-signaling pathway, is activated in fibrotic processes and wound repair and, as such, could play a role in mediating cellular responses to irradiation. beta-Catenin can form a transcriptionally active complex with members of the Tcf family. A reporter mouse model, in addition to human cell cultures, was used to demonstrate that ionizing radiation activates beta-catenin-mediated, Tcf-dependent transcription both in vitro and in vivo. Furthermore, radiation activates beta-catenin via a Wnt-mediated mechanism, as in the presence of dickkopf-1, an inhibitor of Wnt receptor activation, beta-catenin levels did not increase after irradiation. Fibroblast cell cultures were derived from mice expressing either null or stabilized beta-catenin alleles. Cells expressing stabilized beta-catenin alleles had a higher proliferation rate and formed more colony-forming units than wild-type or null cells after irradiation. Wound healing was studied in these same mice after irradiation. There was a positive correlation between the tensile strength of the wound, the expression levels of type 1 collagen in the skin, and beta-catenin levels. Mice treated with lithium showed increased beta-catenin levels and increased wound strength. beta-Catenin mediates the effects of ionizing radiation in fibroblasts, and its modulation has the potential to decrease the severity of radiation-induced soft tissue complications.

R-spondins Signal Like Wnts, Sort Of

Wnt proteins are lipid-modified secreted molecules that initiate signaling by interacting with a receptor complex that includes the serpentine receptor Frizzled and low-density lipoprotein receptor—related protein 5 (LRP5) or LRP6. Wnt receptor activation leads to stabilization of β-catenin, which accumulates in the nucleus and activates transcription of Wnt target genes. Roof plate-specific spondins (R-spondins) are proteins unrelated in sequence to Wnts that, like Wnts, are implicated in regulation of development and that also activate signaling through β-catenin. Conflicting reports have been published as to whether R-spondins use the same receptors as Wnts and whether they bind to Frizzled or LRP6, both, or neither. Wei et al . present data showing that human R-spondin1 (hRspo1) binds to cultured human embryonic kidney (HEK293T) cells transfected with LRP6. Solid-phase binding assays showed that hRspo1 bound to the extracellular domain of LRP6 with high affinity (K d , 1.2 nM) but showed little if any binding to Frizzled. The hRspo1 protein also induced phosphorylation of LRP6 (as does Wnt). hRspo1 had synergistic effects with Wnt3A on phosphorylation of LRP6 in HEK293T cells and on phosphorylation of endogenous LRP6 in mouse embryo fibroblasts. R-spondins thus appear to work differently from Wnts in some ways (Wnts bind with high affinity to Frizzled, with weak or no binding to LRP6), but both ligands activate similar downstream signaling events. Furthermore, expression of R-spondins is stimulated by Wnts, leading the authors to propose that R-spondins may act in a positive feedback loop that reinforces Wnt signaling during development. Q. Wei, C. Yokota, M. V. Semenov, B. Doble, J. Woodgett, X. He, R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and β-catenin signaling. J. Biol. Chem. 282 , 15903-15911 (2007). [Abstract] [Full Text]

Dual Phosphorylation of LRP6

Wnt stimulates the stabilization of β-catenin through interactions with a receptor complex composed of the seven-transmembrane Frizzled and the single-transmembrane low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6). In the absence of Wnt, dual phosphorylation of β-catenin by two kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), leads to the destruction of β-catenin. Two groups (Davidson et al. and Zeng et al. ) report the identification of the kinases responsible for the dual phosphorylation of LRP5/6 in the presence of Wnt, which leads to sequestration of axin away from β-catenin, thereby allowing β-catenin to accumulate, enter the nucleus, and regulate transcription. In an expression screen, Davidson et al. identified CK1γ, which is a family of palmitoylated and membrane-associated isoforms of CK1, as a kinase that decreased the mobility of LRP6, indicative of phosphorylation. The interaction of LRP6 and CK1γ and the phosphorylation of LRP6 by CK1γ were confirmed. A positive role for CK1γ-mediated LRP6 phosphorylation in Wnt signaling was provided by gain-of-function and loss-of-function assays in cultured cells (Wnt reporter assays) and in Xenopus embryos. The cytoplasmic domain of LRP5/6 contains five PPPSP potential phosphorylation motifs; however, Wnt signaling can be observed in receptors with only one PPPSP site, which is flanked by CK1 sites. Point mutagenesis of a receptor with a CK site 1, a PPPSP, and a CK site 2 (1/S/2) cytoplasmic domain showed that in coimmunoprecipitation assays, all three phosphorylation sites synergized to promote axin binding, and in reporter assays, all three sites were required for maximal activity. Zeng et al. demonstrated that the PPPSP sites of the LRP6 were phosphorylated by GSK3 and that a second phosphorylation site that resembled a CK1 phosphorylation consensus motif followed each PPPSP site. Mutation of each of the CK1 sites inhibited activation of a Wnt reporter. In vitro binding of axin to LRP6 was maximal in the presence of GSK3 and CK1. GSK3 is generally associated with inhibition of Wnt signaling (β-catenin phosphorylation), and phosphorylation of LRP6 has a stimulatory effect on Wnt signaling. Thus, spatial localization may allow GSK3 to exert these differential effects. Zeng et al. found that a membrane-localized GSK3 activated a Wnt reporter gene and in Xenopus embryos promoted axis duplication, consistent with a positive effect on Wnt signaling. How Wnt signaling controls the dual phosphorylation of the LRP5/6 to promote axin binding and β-catenin stabilization remains to be clearly established (see Nusse for commentary). G. Davidson, W. Wu, J. Shen, J. Bilic, U. Fenger, P. Stannek, A. Glinka, C. Niehrs, Casein kinase 1 γ couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438 , 867-872 (2005). [PubMed] X. Zeng, K. Tamai, B. Doble, S. Li, H. Huang, R. Habas, H. Okamura, J. Woodgett, X. He, A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438 , 873-877 (2005). [PubMed] R. Nusse, Relays at the membrane. Nature 438 , 747-749 (2005). [PubMed]

Casein kinase 1 γ couples Wnt receptor activation to cytoplasmic signal transduction

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.