Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling

AbstractThe ability of the extracellular matrix (ECM) to instruct progenitor cell differentiation has generated excitement for the development of materials-based regenerative solutions. We previously described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material capable of inducing in vivo skull regeneration approaching 60% of the biomechanical properties of native calvarium without exogenous growth factors or ex vivo progenitor cell-priming, suggesting promise as a first-generation material for skull regeneration. Here, we evaluated the contribution of titrating stiffness to osteogenicity by comparing non-crosslinked (NX-MC) and crosslinked (MC) forms of MC-GAG. While both materials were osteogenic, MC demonstrated an increased expression of osteogenic markers and mineralization compared to NX-MC. Both materials were capable of autogenously activating the canonical bone morphogenetic protein receptor (BMPR) signaling pathway with phosphorylation of Smad1/5 (small mothers against decapentaplegic-1/5). However, unlike NX-MC, hMSCs cultured on MC demonstrated significant elevations in the major mechanotransduction mediators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) expression coincident with β-catenin activation in the canonical Wnt signaling pathway. Inhibition of YAP/TAZ activation reduced osteogenic marker expression, mineralization, and β-catenin activation in MC with a much lesser of an effect on NX-MC. YAP/TAZ inhibition also resulted in a reciprocal increase in Smad1/5 phosphorylation as well as BMP2 expression. Our results indicate that increasing MC-GAG stiffness induces osteogenic differentiation via the mechanotransduction mediators YAP/TAZ and the canonical Wnt signaling pathway, whereas the canonical BMPR signaling pathway is activated in a manner independent of mechanical cues.

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AML Survival and In Vivo Progression Is Dependent On β-Catenin Signaling.

Blood ◽

10.1182/blood.v114.22.2398.2398 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2398-2398

Author(s):

Elena K Siapati ◽

Magda Papadaki ◽

Zoi Kozaou ◽

Erasmia Rouka ◽

Evridiki Michali ◽

...

Keyword(s):

Bone Marrow ◽

Wnt Signaling ◽

Signaling Pathway ◽

Cell Lines ◽

Wnt Signaling Pathway ◽

Canonical Wnt Signaling ◽

Down Regulation ◽

Canonical Wnt

Abstract Abstract 2398 Poster Board II-375 B-catenin is the central effector molecule of the canonical wnt signaling pathway which governs cell fate and differentiation during embryogenesis as well as self-renewal of hematopoietic stem cells. Deregulation of the pathway has been observed in various malignancies including myeloid leukemias where over-expression of β-catenin is an independent adverse prognostic factor. In the present study we examined the functional outcome of stable β-catenin down-regulation through lentivirus-mediated expression of short hairpin RNA (shRNA). Reduction of the β-catenin levels in AML cell lines and patient samples diminished their in vitro proliferation ability without significantly affecting cell viability. In order to study the role of β-catenin in vivo, we transplanted leukemic cell lines with control or reduced levels of β-catenin in NOD/SCID animals and analyzed the engraftment levels in the bone marrow. We observed that while the immediate homing of the cells was not affected by the β-catenin levels, the bone marrow engraftment was directly dependent on its levels. Subsequent examination of bone marrow sections revealed that the reduced engraftment was partly due to the inability of the cells with lower β-catenin levels to dock to the endosteal niches, a finding that was confirmed in competitive repopulation assays with untransduced cells. When we examined the expression levels of adhesion molecules and integrins in engrafted cells in vivo, we observed a significant down-regulation of CD44 expression, a molecule that participates in the interaction of HSCs with the niche. Gene expression analysis of the components of the wnt signaling pathway showed that the pathway is subject to tight transcriptional regulation with minor expression deviations. We did, however, observe an up-regulation in components that participate in the non-canonical wnt signaling pathways such as the WNT5B ligand. Ongoing experiments in normal cord blood CD34+ cells will determine the in vivo role of β-catenin signaling in normal hematopoietic progenitors. In conclusion, our study showed that β-catenin comprises an integral part in the development and progression of AML in vivo, indicating that manipulation of the wnt pathway may hold a therapeutic potential in the management of AML. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of canonical WNT signaling pathway by β-catenin/CBP inhibitor ICG001 ameliorates liver fibrosis in vivo through suppression of stromal CXCL12

Journal of Hepatology ◽

10.1016/s0168-8278(17)31773-7 ◽

2017 ◽

Vol 66 (1) ◽

pp. S654

Author(s):

B.Ö. Akcora ◽

G. Storm ◽

J. Prakash ◽

R. Bansal

Keyword(s):

Liver Fibrosis ◽

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Canonical Wnt Signaling ◽

Canonical Wnt

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Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

10.1101/2020.05.03.072579 ◽

2020 ◽

Author(s):

Gabriele Colozza ◽

Yasaman Jami-Alahmadi ◽

Alyssa Dsouza ◽

Nydia Tejeda-Muñoz ◽

Lauren V. Albrecht ◽

...

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Secretory Pathway ◽

Wnt Signaling Pathway ◽

Canonical Wnt Signaling ◽

Labeling Approach ◽

Canonical Wnt ◽

Fused Gene ◽

Multivesicular Endosomes

AbstractThe canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.SignificanceWnt/β-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylated-enriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes. Xenopus embryo assays revealed that TFG is required in vivo for canonical Wnt signaling.

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Inhibition of canonical WNT signaling pathway by β-catenin/CBP inhibitor ICG-001 ameliorates liver fibrosis in vivo through suppression of stromal CXCL12

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease ◽

10.1016/j.bbadis.2017.12.001 ◽

2018 ◽

Vol 1864 (3) ◽

pp. 804-818 ◽

Cited By ~ 28

Author(s):

Büsra Öztürk Akcora ◽

Gert Storm ◽

Ruchi Bansal

Keyword(s):

Liver Fibrosis ◽

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Canonical Wnt Signaling ◽

Canonical Wnt

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Abstract 361: Activation of Wnt Pathway in Macrophages During Atherosclerosis Regression

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.361 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Prashanthi Menon ◽

Yulia Vengrenyuk ◽

Yoscar Ogando ◽

Stephen Ramsey ◽

Elizabeth Gold ◽

...

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Mouse Models ◽

Target Genes ◽

Wnt Signaling Pathway ◽

Macrophage Migration ◽

Canonical Wnt Signaling ◽

Canonical Wnt

Introduction and Objective: Transcriptome analysis of plaque macrophages in two different mouse models of atherosclerosis regression revealed an over representation of consensus binding site sequences for the T-cell factor (TCF)/Lymphoid enhancer binding factor (LEF) family of transcription factors, suggesting canonical Wnt signaling pathway activation during regression in vivo. The canonical Wnt/β-catenin signaling pathway is important for cardiac development and regulates processes such as migration, invasion and tissue repair. However, its function in plaque macrophages is unclear. The objective of the study was to understand the role of canonical Wnt signaling in macrophages during regression using in vivo and in vitro approaches. Methods and Results: Immunohistochemistry of atherosclerotic arterial sections in mouse models of atherosclerosis regression (Reversa and aortic arch transplant) showed a significant increase in β-catenin expression in regressing vs. progressing macrophages. Elevated transcript levels of canonical Wnt downstream targets Ctnnb1, Lrp1 and Gja1 were detected in regressing plaque macrophages isolated by laser capture microdissection (LCM). Canonical Wnt signaling was further investigated in Wnt3a-stimulated primary bone marrow-derived macrophages (BMDM) in vitro, revealing upregulation of pathway target genes Ctnnb1 and Axin2. Furthermore, immunofluorescence analysis of BMDM stimulated with Wnt3a showed increased nuclear expression of β-catenin. Macrophage cell migration evaluated by scratch wound assay revealed a significant increase in migration in Wnt3a-treated vs. untreated BMDM. Conclusions: Our findings demonstrate that canonical Wnt signaling is activated in regressing plaque macrophages and regulates macrophage migration in vitro. Future studies are aimed at understanding the mechanism by which Wnt modulates macrophage migration.

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