APC controls Wnt-induced β-catenin destruction complex recruitment in human colonocytes
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Abstract Wnt/β-catenin signaling is essential for intestinal homeostasis and is aberrantly activated in most colorectal cancers (CRC) through mutation of the tumor suppressor Adenomatous Polyposis Coli ( APC ). APC is an essential component of a cytoplasmic protein complex that targets β-catenin for destruction. Following Wnt ligand presentation, this complex is inhibited. However, a role for APC in this inhibition has not been shown. Here, we utilized Wnt3a-beads to locally activate Wnt co-receptors. In response, the endogenous β-catenin destruction complex reoriented toward the local Wnt cue in CRC cells with full-length APC, but not if APC was truncated or depleted. Non-transformed human colon epithelial cells displayed similar Wnt-induced destruction complex localization which appeared to be dependent on APC and less so on Axin. Our results expand the current model of Wnt/β-catenin signaling such that in response to Wnt, the β-catenin destruction complex: (1) maintains composition and binding to β-catenin, (2) moves toward the plasma membrane, and (3) requires full-length APC for this relocalization.Keywords:
Adenomatous polyposis coli
WNT3A
Beta-catenin
LRP6
β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.
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Background: The Wnt/Wingless signalling pathway plays an important role in both embryonic development and tumorigenesis. β‐Catenin and Axin are positive and negative effectors of the Wnt signalling pathway, respectively. Results: We found that Axin interacts with β‐catenin and glycogen synthase kinase‐3β (GSK‐3β). Furthermore, the regulation of the G‐protein signalling (RGS) domain of Axin is associated with the colorectal tumour suppressor adenomatous polyposis coli (APC). Overexpression of Axin in the human colorectal cancer cell line SW480 induced a drastic reduction in the level of β‐catenin. Interaction with β‐catenin and GSK‐3β was required for the Axin‐mediated β‐catenin reduction. Conclusion: Axin interacts with β‐catenin, GSK‐3β and APC, and negatively regulates the Wnt signalling pathway, presumably by regulating the level of β‐catenin.
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Abstract Nuclear β‐catenin affects the developmental process and progression of tumors. However, the precise mechanism for the nuclear export of β‐catenin is not completely understood. We found that β‐catenin can bind directly to CRM1 through its central armadillo (ARM) repeats region, independently of the adenomatous polyposis coli (APC) protein. CRM1 overexpression transports nuclear β‐catenin into the cytoplasm and decreases LEF‐1/β‐catenin‐dependent transcriptional activity, which is also affected by the co‐overexpression of E‐cadherin. CRM1 competed with E‐cadherin and LEF‐1 for binding to β‐catenin. β‐catenin could interact directly with APC through its essential sequences between amino acids 342 and 350. The site‐directed β‐catenin mutant (NES2 − ), which could interact with CRM1, but not with APC, still retained its ability to export from the nucleus and its transactivational activity. This suggests that CRM1 can function as an efficient nuclear exporter for β‐catenin independently of APC. These results strongly suggest that the CRM1‐mediated pathway is involved in the efficient transport of nuclear β‐catenin in the nucleus of cells.
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Rationale: The WNT signaling pathways are important in lung repair and tissue remodeling. Canonical WNT signaling is defined by activation β-catenin signaling, whereas non-canonical WNT signaling does not. β-Catenin signaling is reduced in alveolar epithelial cells in emphysema, a pathological feature of COPD. The mechanisms involved in WNT/β-catenin down regulation are unknown. We investigated the cross-talk between canonical and non-canonical WNT signaling in lung epithelial cells. Methods: Human and murine lung epithelial cells were treated with the WNT ligands WNT3A and WNT5A. β-Catenin signaling was activated by GSK3 inhibition. WNT signaling was monitored by β-catenin sensitive luciferase assay (TOP/FOP-flash) and immunoblotting. Results: Stimulation of lung epithelial cells with WNT3A led to LRP6 phosphorylation, β-catenin stabilization and activation of TOP/FOP-flash reporter, confirming canonical WNT signaling. WNT5A did not activate β-catenin signaling, but induced DVL phosphorylation indicating non-canonical WNT signaling. Co-stimulation with WNT3A and various doses of WNT5A resulted in a dose-dependent decrease in TOP/FOP-flash reporter activation, due to attenuated LRP6 phosphorylation and decreased β-catenin stabilization. GSK3 inhibition activated TOP-flash luciferase activity, which was antagonized WNT5A. Basal signaling by WNT5A was mediated by PKC, but not JNK or TAK1. Importantly, WNT5A was up regulated in the lungs of mice after cigarette smoke exposure. Conclusion: Canonical WNT signaling activated by either WNT3A or by GSK3 inhibition is attenuated by the non-canonical WNT ligand WNT5A in lung epithelial cells. HB is supported by an ERS long-term fellowship (LTRF 79-2012).
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Wnt/β-catenin signaling activity is maintained in homeostasis by an expanding list of molecular determinants. However, the molecular components and the regulatory mechanisms involved in its fine-tuning remain to be determined. Here, we identified C9orf140, a tumor-specific protein, as a novel Axin1-interacting protein by tandem-affinity purification and mass spectrometry. We further showed that C9orf140 is a negative regulator of Wnt/β-catenin signaling in cultured cells as well as in zebrafish embryos. It functions upstream of β-catenin, outcompetes PP2A for binding to Axin1, influences the balance between phosphorylation and de-phosphorylation of β-catenin, and ultimately compromises Wnt3A-induced β-catenin accumulation. Interestingly, Wnt-induced C9orf140 expression via β-catenin. We propose that C9orf140 mediates a negative feedback loop of Wnt/β-catenin signaling by interacting with Axin1. Our results advance the current understanding of the exquisite control of Wnt/β-catenin signaling cascade, and provide evidence of the new role of C9orf140.
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p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. We describe here that p120-catenin is required for Wnt pathway signaling. p120-catenin binds and is phosphorylated by CK1ε in response to Wnt3a. p120-catenin also associates to the Wnt co-receptor LRP5/6, an interaction mediated by E-cadherin, showing an unexpected physical link between adherens junctions and a Wnt receptor. Depletion of p120-catenin abolishes CK1ε binding to LRP5/6 and prevents CK1ε activation upon Wnt3a stimulation. Elimination of p120-catenin also inhibits early responses to Wnt, such as LRP5/6 and Dvl-2 phosphorylation and axin recruitment to the signalosome, as well as later effects, such as β-catenin stabilization. Moreover, since CK1ε is also required for E-cadherin phosphorylation, a modification that decreases the affinity for β-catenin, p120-catenin depletion prevents the increase in β-catenin transcriptional activity even in the absence of β-catenin degradation. Therefore, these results demonstrate a novel and crucial function of p120-catenin in Wnt signaling and unveil additional points of regulation by this factor of β-catenin transcriptional activity different of β-catenin stability.
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Wnt-beta-catenin signaling controls critical events in metazoan development, and its dysregulation leads to cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane co-receptors Frizzled (Fz) and low-density lipoprotein (LDL) receptor-related protein (LRP) 5 or LRP6 inhibits the degradation of the transcriptional coactivator beta-catenin, which translocates to the nucleus to regulate gene expression. The secreted protein Dickkopf1 (Dkk1) inhibits Wnt signaling by binding to LRP5 and LRP6 and blocking their interaction with Wnt and Fz. Kremen 1 and 2 (Krm1 and 2, collectively termed Krms) are single-pass transmembrane Dkk1 receptors that synergize with Dkk1 to inhibit Wnt signaling by promoting the endocytosis of LRP5 and LRP6. A study now suggests that Krms, in the absence of Dkk1, potentiate Wnt signaling by maintaining LRP5 and LRP6 at the plasma membrane. It is proposed that the absence or presence of Dkk1 determines whether Krms will activate or inhibit Wnt-beta-catenin signaling, respectively. Here, we speculate that the proposed context-dependent positive and negative roles for Krms could promote biphasic Wnt signaling in response to a shallow gradient of Dkk1, resulting in the generation of precise and robust borders between cells during development. Identification of a context-dependent role for Krms in Wnt-beta-catenin signaling offers insight into the mechanism of Wnt signaling and has important developmental implications.
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