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Biochem Biophys Res Commun
2017 May 27;4872:433-437. doi: 10.1016/j.bbrc.2017.04.080.
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NRAGE induces β-catenin/Arm O-GlcNAcylation and negatively regulates Wnt signaling.
Chen Y
,
Jin L
,
Xue B
,
Jin D
,
Sun F
,
Wen C
.
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The Wnt pathway is crucial for animal development, as well as tumor formation. Understanding the regulation of Wnt signaling will help to elucidate the mechanism of the cell cycle, cell differentiation and tumorigenesis. It is generally accepted that in response to Wnt signals, β-catenin accumulates in the cytoplasm and is imported into the nucleus where it recruits LEF/TCF transcription factors to activate the expression of target genes. In this study, we report that human NRAGE, a neurotrophin receptor p75 (p75NTR) binding protein, markedly suppresses the expression of genes activated by the Wnt pathway. Consistent with this finding, loss of function of NRAGE by RNA interference (RNAi) activates the Wnt pathway. Moreover, NRAGE suppresses the induction of axis duplication by microinjected β-catenin in Xenopus embryos. To our surprise, NRAGE induces nuclear localization of β-catenin and increases its DNA binding ability. Further studies reveal that NRAGE leads to the modification of β-catenin/Arm with O-linked beta-N-acetylglucosamine (O-GlcNAc), and failure of the association between β-catenin/Arm and pygopus(pygo) protein, which is required for transcriptional activation of Wnt target genes. Therefore, our findings suggest a novel mechanism for regulating Wnt signaling.
Fig. 1. NRAGE suppresses the expression of Wnt pathway target genes. a, b, U2-OS cells in 6-well plates were co-transfected with a mixture of plasmids including 0.5 μg β-catenin, 0.2 μg TOPFlash or FOPFlash -luciferase (a) or cyclin D1-luciferase (b) reporter construct and 0.1 μg pCMV-β-gal as an internal standard. 24 h later, 10 MOI Ad-mycNRG or Adv were added to the medium for another 24 h. RLU indicates relative luciferase units. c, HEK293 cells were co-transfected with a mixture of plasmids including 0.5 μg β-catenin, 0.2 μg TOPFlash-luciferase reporter, 0.1 μg pCMV-β-gal and indicated amount of pSupper-EGFP1/NRG (RNAi) for 48 h d, Cell lysates from U2-OS cells infected with different doses of Ad-mycNRG and control Adv for 36 h were subjected to western blotting for detection of cyclin D1, Myc-NRAGE (Myc-NRG) and beta-actin.
Fig. 2. NRAGE induces nuclear translocation of β-catenin and it's binding to target promoters. a, Indirect immunofluorescence analysis of β-catenin (red) was performed in U2-OS cells. Nucleus (blue) was stained with DAPI. b, c, Gel retardation assays were performed with 5 μg nuclear extracts from the 293 cells. Nuclear extracts were from 293 cells infected with 30 MOI of Ad-mycNRG. Non-radiolabeled probe was added in 10 and 50 time the radiolabeled probe. Mt indicates mutant probe (b). Nuclear extracts were from 293 cells with indicated treatments (c). d, ChIP analysis of the DNA binding ability of β-catenin in response to NRAGE expression. Soluble chromatin was prepared from U2-OS cells, followed by immunoprecipitation (IP) with anti-β-catenin IgG or normal IgG. The DNA extracted from the respective immunoprecipitates was amplified using the primers that cover the promoter regions of cyclin D1 and MMP7 genes. Input represents PCR product amplified from 1% of soluble chromatin DNA. Chromatin solution incubated with normal antibody was a mixture of the three lysates with different treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. NRAGE disrupts the transcriptional complex of β-catenin/Arm and Pygo proteins. 293A cells were transfected with plasmids expressing Myc-tagged Pygo, HA-tagged Arm and pEGFPc3-NRG or pSuper-NRG (RNAi) as control (CTL). Whole-cell lysates were prepared 40 h after transfection. Lysates were immunoprecipitated by an anti-HA antibody (a) or an anti-Myc antibody (b). Immunoprecipitated materials and a fraction of each lysate were resolved by SDS-PAGE and analyzed by western blotting with antibodies indicated. IP, immunoprecipitation; IB, immunoblot. c, U2-OS cells were co-transfected with a mixture of plasmids including 0.5 μg β-catenin, 0.5 μg human pygopus2, 0.2 μg TOPFlash or FOPFlash -luciferase reporter constructs and 0.1 μg pCMV-β-gal as an internal standard. 24 h later, 10 MOI Ad-mycNRG or Adv were added to the medium for another 24 h.
Fig. 4. NRAGE induces O-GlcNAcylation of β-catenin/Arm. a, Whole-cell lysates from U2-OS cells infected with 10, 30, 50 MOI of Ad-mycNRG and 30 MOI of Adv for 24 h were immuoprecipitated by a rabbit polyclonal anti-β-catenin antibody and immunoblotted by an O-GlcNAc antibody. The loading amount of β-catenin was detected with a mouse anti-β-catenin antibody. b, Two hundred micrograms of total proteins were precipitated with WGA-agarose and subjected to immunoblotting for β-catenin. Input represents 10% of total lysate used for WGA-agarose precipitation. c, HA-tagged Arm was cotransfected with pEGFPc3-NRG, pSuper-NRG (RNAi) and pEGFPc3 vector, respectively. HA-tagged Arm was immuoprecipitated and immunoblotted by HA and O-GlcNAc antibodies. d, Cell lysates from U2-OS cells treated with DMSO or GlcNAc(to a final concentration of 10 μM) were immuoprecipitated by anti-β-catenin antibodies and immunoblotted by anti-O-GlcNAc, anti-p(S675)- β-catenin, anti-p(T41) -β-catenin and anti-β-catenin antibodies, respectively.
Figure S1: NRAGE prevents the second axis formation induced by beta-catenin in Xenopus embryos. a) Formation of the second axis in embryos injected with 40 pg of beta- catenin mRNA. GFP mRNA was co-injected in each embryo as tracer. b) The second axis induced by beta-catenin were rescued when 700 pg of NRG mRNA were co-injected with 40 pg of beta-catenin mRNA. GFP fluorescence indicates the injected site. c) Percentage of secondary axis formation in beta-catenin and beta-catenin+NRG injected embryos. Control (c) indicated beta-catenin injection; NRG indicates beta-catenin+NRG co-injection.
Figure S2: RNAi of NRAGE effectively eliminates the expression of GFP-NRAGE in HEK293A. pEGFP-hNRAGE (7 micrograms) was co-transfected with siRNA plasmid (5 micrograms) or pSupper-GFP vector (5 micrograms). The expression of GFP-hNRAGE was tested by GFP antibody. The low bands of GFP come from pSupper-GFP. NRG indicates the GFP-NRAGE fusion protein.
Figure S3: RT-PCR were performed using total RNA from U2-OS cells infected with Ad-mycNRG and control Adv for 40 hours.
Figure S4: Proposed model for the down regulation of Wnt signaling by NRAGE. NRAGE activity leads to the O-GlcNAcylation of beta-catenin/ Arm through an unknown mechanism and induces nuclear localization of beta-catenin/Arm. This O-GlcNAc modified protein binds to target promoter but fails to recruit co-activator Pygo, and then shuts down the expressions of Wnt target genes.
