An ATF2-based luciferase reporter to monitor non-canonical Wnt signaling in xenopus embryos

2010 ◽  
Vol 240 (1) ◽  
pp. 188-194 ◽  
Author(s):  
Bisei Ohkawara ◽  
Christof Niehrs
Keyword(s):  
Wnt Signaling ◽  
Luciferase Reporter ◽  
Canonical Wnt Signaling ◽  
Xenopus Embryos ◽  
Canonical Wnt

scholarly journals Maternal Wnt11 Activates the Canonical Wnt Signaling Pathway Required for Axis Formation in Xenopus Embryos

Cell ◽  
2005 ◽  
Vol 120 (6) ◽  
pp. 857-871 ◽  
Author(s):  
Qinghua Tao ◽  
Chika Yokota ◽  
Helbert Puck ◽  
Matt Kofron ◽  
Bilge Birsoy ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Axis Formation ◽  
Canonical Wnt Signaling ◽  
Xenopus Embryos ◽  
Canonical Wnt

Bortezomib Induces Activation of b-Catenin/TCF Signaling Pathway in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1851-1851
Author(s):  
Ya-Wei Qiang ◽  
Bo Hu ◽  
Yu Chen ◽  
Wei Qiang ◽  
Christoph Heuck ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt ◽  
Dose Dependent

Abstract Abstract 1851 Background: The proteasome inhibitor Bortezomib (Bz) shows significant activity in Multiple Myeloma (MM) by acting on MM cell directly as well as by augmenting bone formation in vitro and in vivo. Its effect on the bone could be traced to promoting differentiation of mesenchymal stem cells into osteoblast cells by regulating BMP2 and canonical Wnt signaling. However, the molecular mechanism mediating the direct anti-MM activity of Bz remains to be fully understood. Initially the rationale for the use of Bz in MM was inhibition of NF-kB signaling, yet subsequent studies showed that Bz actually induces activation of this pathway. In this study, we examined whether Bz regulates the activity of canonical Wnt signaling pathway in MM and whether the growth-inhibition effect of Bz was associated with activation of this pathway by using multiple MM cell lines including EJM, H929, INA6, KMS28BM, JJN3, L363, OPM1, OPM2, RPMI8226, UTMC, XG2 and XG6 as well as primary plasma cells (PC) from six patients with newly diagnosed MM. Methods/Results: Immunoblotting demonstrated that Bz induces stabilization of b-catenin protein in three MM cell lines (H929, OPM2 and UTMC) in a time- and dose-dependent manner. These changes were not seen when the same cell lysate were immunoblotted for other catenin family members, a-catenin and g-catenin. Increased levels of b-catenin protein response to Bz treatment were observed in other 9 MM cell lines (EJM, INA6, KMS28BM, JJN3, L363, OPM1, RPMI8226, XG2 and XG6) and in the 6 CD138+ sorted bone marrow PC from patients with MM. To determine if Bz regulation of b-catenin level is a specific effect of the inhibition of 26S proteasome subunit we treated the same MM cell lines with another proteasome inhibitor, MG132. Similar results were observed in response to MG132 for all four MM cell lines, suggesting the effect of Bz on b-catenin protein is 26S proteosome inhibitor specific. Increases in b-catenin protein levels in MM cells were not due to increased Ctnnb1/CTNNB (b-catenin) gene transcription as b-catenin mRNA did not change in these cells treated with Bz. These results indicate that proteasome inhibition increases b-catenin is independent of transcriptional upregulation. To determine whether Bz induces the nuclear localization and transcriptional activity of b-catenin, cells were incubated with Bz for 6 hours and then fractionated to separate the nuclear and cytoplasmic fractions. Treatment with Bz resulted an increase in nuclear b-catenin as well as b-catenin in cytoplasm in four cell lines including H929, INA6, OPM1 and MM144. Increase in cytoplasmic and nuclear b-catenin was further confirmed by immunofluorescence with antibodies specific for active form of b-catenin. To determine whether Bz affects b-catenin-mediated transcriptional activity, we used a TCF/LEF luciferase reporter construct cloned in lentiviral vector. OPM2 cells were infected with lentiviral particle containing the TCF reporter or containing empty vector and were then treated with serial concentrations of Bz. The luciferase activity exhibited a dose-dependent response to Bz analogous to the stabilization of b-catenin. Similar results were observed in 7 out of 8 MM cell lines compared with untreated control. Stimulation of TCF transcriptional activity by Bz was independent of modifiers of extracellular Wnt ligands, such as Frizzled receptors, LRP5/6 co-receptors and sFRPs or the activation of intracellular GSK3b. Conclusion: These results indicate that Bz augments activation of canonical Wnt signaling by preventing b-catenin protein from proteosome-mediated degradation in MM cells. Concentrations of Bz for stimulating TCF transcriptional activity are comparable to those being used to induce inhibition of MM proliferation. Experiments modulating cytoplasmic as well as the nuclear players and interactions of the Wnt-pathway are ongoing to determine if Bz mediated activation of b-catenin signaling is responsible for its direct anti-MM effect. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

scholarly journals Syndecan-4 in non-canonical Wnt signaling and gastrulation movements in Xenopus embryos

2007 ◽  
Vol 306 (1) ◽  
pp. 397-398
Author(s):  
Rosana Muñoz ◽  
Loreto Carvallo ◽  
Alejandro Burga ◽  
Juan Larraín
Keyword(s):  
Wnt Signaling ◽  
Canonical Wnt Signaling ◽  
Xenopus Embryos ◽  
Canonical Wnt

scholarly journals Identification of Compound Heterozygous Variants in LRP4 Demonstrates That a Pathogenic Variant outside the Third β-Propeller Domain Can Cause Sclerosteosis

Genes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 80
Author(s):  
Yentl Huybrechts ◽  
Eveline Boudin ◽  
Gretl Hendrickx ◽  
Ellen Steenackers ◽  
Neveen Hamdy ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Pathogenic Variant ◽  
Luciferase Reporter ◽  
Compound Heterozygous ◽  
Canonical Wnt Signaling ◽  
High Bone Mass ◽  
The Third ◽  
Compound Heterozygous Variants ◽  
Canonical Wnt ◽  
Signaling Activity

Sclerosteosis is a high bone mass disorder, caused by pathogenic variants in the genes encoding sclerostin or LRP4. Both proteins form a complex that strongly inhibits canonical WNT signaling activity, a pathway of major importance in bone formation. So far, all reported disease-causing variants are located in the third β-propeller domain of LRP4, which is essential for the interaction with sclerostin. Here, we report the identification of two compound heterozygous variants, a known p.Arg1170Gln and a novel p.Arg632His variant, in a patient with a sclerosteosis phenotype. Interestingly, the novel variant is located in the first β-propeller domain, which is known to be indispensable for the interaction with agrin. However, using luciferase reporter assays, we demonstrated that both the p.Arg1170Gln and the p.Arg632His variant in LRP4 reduced the inhibitory capacity of sclerostin on canonical WNT signaling activity. In conclusion, this study is the first to demonstrate that a pathogenic variant in the first β-propeller domain of LRP4 can contribute to the development of sclerosteosis, which broadens the mutational spectrum of the disorder.

Sign in / Sign up

Export Citation Format

Share Document