New insights into the regulation of Axin function in canonical Wnt signaling pathway.
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ABSTRACT:
SUBMITTER: Song X
PROVIDER: S-EPMC3967064 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Wnts regulate important intracellular signaling events, and dysregulation of the Wnt pathway has been linked to human disease. Here, we uncover numerous Wnt canonical effectors in human platelets where Wnts, their receptors, and downstream signaling components have not been previously described. We demonstrate that the Wnt3a ligand inhibits platelet adhesion, activation, dense granule secretion, and aggregation. Wnt3a also altered platelet shape change and inhibited the activation of the small GTPase RhoA. In addition, we found the Wnt-beta-catenin signaling pathway to be functional in platelets. Finally, disruption of the Wnt Frizzled 6 receptor in the mouse resulted in a hyperactivatory platelet phenotype and a reduced sensitivity to Wnt3a. Taken together our studies reveal a novel functional role for Wnt signaling in regulating anucleate platelet function and may provide a tractable target for future antiplatelet therapy.
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Project description:The stability of β-catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3β phosphorylates β-catenin to be degraded by ubiquitination with β-TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected β-catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of β-catenin. These results suggest that WDR26 plays a negative role in β-catenin degradation in the Wnt signaling pathway.
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Project description:The canonical Wnt pathway regulates numerous fundamental processes throughout development and adult physiology and is often disrupted in diseases [1-4]. Signal in the pathway is transduced by β-catenin, which in complex with Tcf/Lef regulates transcription. Despite the many processes that the Wnt pathway governs, β-catenin acts primarily on a single cis element in the DNA, the Wnt-responsive element (WRE), at times potentiated by a nearby Helper site. In this study, working with Xenopus, mouse, and human systems, we identified a cis element, distinct from WRE, upon which β-catenin and Tcf act. The element is 11 bp long, hundreds of bases apart from the WRE, and exhibits a suppressive effect. In Xenopus patterning, loss of the 11-bp negative regulatory elements (11-bp NREs) broadened dorsal expression of siamois. In mouse embryonic stem cells, genomic deletion of the 11-bp NREs in the promoter elevated Brachyury expression. This reveals a previously unappreciated mechanism within the Wnt pathway, where gene response is not only driven by WREs but also tuned by 11-bp NREs. Using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), we found evidence for the NREs binding to β-catenin and Tcf-suggesting a dual action by β-catenin as a signal and a feedforward sensor. Analyzing β-catenin ChIP sequencing in human cells, we found the 11-bp NREs co-localizing with the WRE in 45%-71% of the peaks, suggesting a widespread role for the mechanism. This study presents an example of a more complex cis regulation by a signaling pathway, where a signal is processed through two distinct cis elements in a gene circuitry.
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Project description:Adenomatous polyposis coli (APC) and Axin interactions serve an important role in colorectal cancer (CRC) pathogenesis. The aim of the present study was to assess the combined effects of Axin and APC co‑expression in CRC cells, and to determine the underlying mechanisms involved. SW480 cells were divided into the following groups: Untransfected (SW480 group), transfected with pEGFP‑N3plus pCS2‑MT (SW480/vector‑vector), transfected with pEGFP‑N3‑APC5 (SW480/APC5), and transfected with pEGFP‑N3‑APC5 pluspCS2‑MT‑Axin (SW480/APC5‑Axin). APC5 and Axin mRNA levels were determined by reverse transcription‑polymerase chain reaction. MTT assays and flow cytometry analysis were performed to assess cell growth and cell cycle distribution, respectively. Quantitative PCR and western blot analyses were conducted to evaluate the mRNA and protein levels, respectively, of Wnt signaling effectors, including β‑catenin, c‑myc and survivin. Successful transfection of SW480 cells was determined with APC and APC‑Axin plasmids as indicated by the green fluorescence signals. Notably, SW480/APC5 cell growth was inhibited by 40.33%, and cells co‑expressing APC5 and Axin demonstrated 61.27% inhibition of cell growth compared with SW480 control cells. The results demonstrate that APC5 may induce G1/S arrest in SW480 cells, and Axin may enhance cell growth arrest induced by APC5. The mRNA and protein levels of β‑catenin, c‑myc and survivin were significantly reduced in SW480/APC‑Axin cells when compared with the SW480/APC group. In conclusion, co‑expression of APC5 and Axin genes significantly downregulated Wnt signaling in human SW480 CRC cells and inhibited cell growth, when compared with cells transfected with APC5 alone. These results may provide experimental evidence to support combined gene therapy in CRC.
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Project description:Axin, APC, and the kinase GSK3 beta are part of a destruction complex that regulates the stability of the Wnt pathway effector beta-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3 beta, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. We have shown previously that the EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/beta-catenin and POP-1/Tcf. Here, we have investigated how BAR-1 is regulated by the EGL-20 pathway. First, we have characterized a negative regulator of the EGL-20 pathway, pry-1. We show that pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. Our results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. We show that PRY-1 interacts with BAR-1, SGG-1, and APR-1 and that overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that it can functionally interact with vertebrate destruction complex components. Finally, we show that SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. We conclude that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/beta-catenin signaling in C. elegans.
| S-EPMC186271 | biostudies-literature
Project description:Wnt/?-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs) are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/?-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/?-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC) to upregulate Wnt/?-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/?-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding ?-catenin) through binding to its 3' untranslated region (3' UTR). Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/?-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/?-catenin signaling, which help to understand the complex regulation of Wnt/?-catenin signaling.
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Project description:The Wnt signaling pathway is an evolutionary conserved system, having pivotal roles during animal development. When over-activated, this signaling pathway is involved in cancer initiation and progression. The canonical Wnt pathway regulates the stability of β-catenin primarily by a destruction complex containing a number of different proteins, including Glycogen synthase kinase 3β (GSK-3β) and Axin, that promote proteasomal degradation of β-catenin. As this signaling cascade is modified by various proteins, novel screens aimed at identifying new Wnt signaling regulators were conducted in our laboratory. One of the different genes that were identified as Wnt signaling activators was Aldolase C (ALDOC). Here we report that ALDOC, Aldolase A (ALDOA) and Aldolase B (ALDOB) activate Wnt signaling in a GSK-3β-dependent mechanism, by disrupting the GSK-3β-Axin interaction and targeting Axin to the dishevelled (Dvl)-induced signalosomes that positively regulate the Wnt pathway thus placing the Aldolase proteins as novel Wnt signaling regulators.
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Project description:Embryonic stem cells (ESCs) have both the ability to self-renew and to differentiate into various cell lineages. Retinoic acid (RA), a metabolite of Vitamin A, has a critical function in initiating lineage differentiation of ESCs through binding to the retinoic acid receptors. Additionally, the Wnt signaling pathway plays a role in pluripotency and differentiation, depending on the activation status of the canonical and noncanonical pathways. The activation of the canonical Wnt signaling pathway, which requires the nuclear accumulation of ?-catenin and its interaction with Tcf1/Lef at Wnt response elements, is involved in ESC stemness maintenance. The noncanonical Wnt signaling pathway, through actions of Tcf3, can antagonize the canonical pathway. We show that RA activates the noncanonical Wnt signaling pathway, while concomitantly inhibiting the canonical pathway. RA increases the expression of ligands and receptors of the noncanonical Wnt pathway (Wnt 5a, 7a, Fzd2 and Fzd6), downstream signaling, and Tcf3 expression. RA reduces the phosphorylated ?-catenin levels by fourfold, although total ?-catenin levels do not change. We show that RA signaling increases the dissociation of Tcf1 and the association of Tcf3 at promoters of genes that regulate stemness (e.g., NR5A2, Lrh-1) or differentiation (e.g. Cyr61, Zic5). Knockdown of Tcf3 increases Lrh-1 transcript levels in mESCs and prevents the RA-associated, fourfold increase in Zic5, indicating that RA requires Tcf3 to effect changes in Zic5 levels. We demonstrate a novel role for RA in altering the activation of these two Wnt signaling pathways and show that Tcf3 mediates some actions of RA during differentiation.
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Project description:Foxp3 is crucial for both the development and function of regulatory T cells (Treg),however the post-translational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that TCF1 and Foxp3 interact in the nucleus of Treg, and that active Wnt-signaling disrupts Foxp3 transcriptional activity. Utilizing a global ChIP-seq comparison we demonstrate considerable overlap between Foxp3 and Wnt target genes in Treg. Activation of Wnt signaling significantly reduces Treg-mediated suppression both in vitro and in vivo, while disruption of Wnt signaling in Treg enhances their suppressive capacity. Activation of effector T cells increases Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced by activated mononuclear cells under inflammatory conditions represses Treg function allowing a productive immune response, but if uncontrolled could lead to the development of autoimmunity. Beta catenin ChIP-seq in Treg
2013-07-26 | E-GEOD-49199 | biostudies-arrayexpress
Project description:Glucocorticoid-induced glaucoma is a secondary open-angle glaucoma. About 40% of the general population may develop elevated intraocular pressure on prolonged glucocorticoid treatment secondary to damages in the trabecular meshwork (TM), a tissue that regulates intraocular pressure. Therefore, identifying the key molecules responsible for glucocorticoid-induced ocular hypertension is crucial. In this study, Dickkopf-related protein 1 (Dkk1), a canonical Wnt signaling inhibitor, was found to be elevated in the aqueous humor and TM of glaucoma patients. At the signaling level, Dkk1 enhanced glucocorticoid receptor (GR) signaling, whereas Dkk1 knockdown or Wnt signaling activators decreased GR signaling in human TM cells as indicated by luciferase assays. Similarly, activation of the GR signaling inhibited Wnt signaling. At the protein level, glucocorticoid-induced extracellular matrix was inhibited by Wnt activation using Wnt activators or Dkk1 knockdown in primary human TM cells. In contrast, inhibition of canonical Wnt signaling by β-catenin knockdown increased glucocorticoid-induced extracellular matrix proteins. At the physiological level, adenovirus-mediated Wnt3a expression decreased glucocorticoid-induced ocular hypertension in mouse eyes. In summary, Wnt and GR signaling inhibit each other in the TM, and canonical Wnt signaling activators may prevent the adverse effect of glucocorticoids in the eye.
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