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

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.

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

Blood ◽  
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.

scholarly journals Deregulation of the histone H3K9 di-methylation landscape suppresses canonical Wnt signaling in embryonal rhabdomyosarcoma

2020 ◽  
Author(s):  
Ananya Pal ◽  
Jia Yu Leung ◽  
Gareth Chin Khye Ang ◽  
Vinay Kumar Rao ◽  
Luca Pignata ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Myogenic Differentiation ◽  
Wnt Signaling Pathway ◽  
Embryonal Rhabdomyosarcoma ◽  
Dependent Manner ◽  
Canonical Wnt Signaling ◽  
Therapeutic Modalities ◽  
Canonical Wnt

AbstractThe Wnt signaling pathway is down-regulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of myogenic differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way towards novel therapeutic modalities. In this study, we demonstrate that the H3K9 lysine methyltransferase G9a suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of G9a expression or activity reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in vivo. Mechanistically, G9a impacted Sp1 and p300 enrichment at the DKK1 promoter in a methylation-dependent manner. The reduced tumor growth upon G9a deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among thirteen drugs targeting chromatin modifiers, G9a inhibitors were highly effective in reducing ERMS cell viability. Together, our study demonstrates that ERMS cells are vulnerable to G9a inhibitors and suggest that targeting the G9a-DKK1-β-catenin node holds promise for differentiation therapy.

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

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.

Role of Androgen Dependent TFPI-Regulating Protein (ADTRP) in Vascular Development and Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 556-556 ◽  
Author(s):  
Maulin Mukeshchandra Patel ◽  
Robert Silasi-Mansat ◽  
Ravi Shankar Keshari ◽  
Christopher L. Sansam ◽  
David A. Jones ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Target Genes ◽  
Vascular Development ◽  
The Novel ◽  
Canonical Wnt Signaling ◽  
And Function ◽  
Canonical Wnt

Abstract We used in vitro and in vivo models to characterize the physiological role of the novel protein encoded by C6ORF105. This gene's expression is androgen-responsive, and the encoded protein is predicted to be palmitoylated and membrane multi-spanning. Previously we showed that C6ORF105 expression co-regulates with tissue factor pathway inhibitor (TFPI)in human endothelial cells (EC); hence we named this protein "androgen-dependent TFPI-regulating protein" (ADTRP). Using in vitro cell-based TOP-Flash reporter assay we identified ADTRP as a negative regulator of canonical Wnt signaling in human cells. Overexpressing ADTRP in HEK293T cells inhibited the activity of beta-catenin/TCF-dependent transcriptional reporter, while silencing ADTRP increased the expression of Wnt target genes LEF-1, AXIN-2, IL-8 and DKK-2 in EA.hy926 EC line and HUVEC. Addition of LiCl showed that the effect of ADTRP was upstream of GSK3, therefore we focused the investigations on the Wnt signalosome proteins. ADTRP expression in HEK293T cells led to decreased phosphorylation of Wnt co-receptor LRP6, suggesting that ADTRP can affect this critical membrane-located event of Wnt signaling. Furthermore, ADTRP expression in reporter cells transfected with a constitutively phosphorylated form of LRP6 (LRP6DN mutant) inhibited Wnt3a- induced signaling, which suggests that ADTRP can interfere with events downstream of LRP6 phosphorylation, such as Axin-2 binding. Altogether, these data indicate that the Wnt signaling inhibitory activity of ADTRP takes place at the plasma membrane level. Site directed mutagenesis of the predicted palmitoylation site Cys61 showed that Wnt inhibitory effects of ADTRP require palmitoyl-mediated anchoring, highlighting the importance of proper membrane location of ADTRP for Wnt pathway inhibition. In vivo morpholino-based knockdown of adtrp in zebrafish embryos produced aberrant angiogenesis, defective branching and ruptured vessels, hemorrhage spots, pericardial edema and slow heart-beat, all reminiscent of defects caused by activation of canonical Wnt signaling. Indeed, adtrp knock down increased Wnt mediated lef-1 and pax-2a as well as mmp2 and mmp9 mRNA expression. Co-injection of ADTRP mRNA partially recovered the adtrp morpholino- induced morphologic abnormalities. Also, knock down of adtrp in a Wnt reporter zebrafish showed increased expression of ectopic Wnt signaling. Furthermore, our recently established Adtrp-/- mice also display some typical Wnt-mediated vascular defects, including: (i) abnormal patterning, increased capillary tortuosity, abnormal branching and increased density of the capillary network; (ii) dilated vessels, especially venules and veins; (iii) increased leakeage of permeability tracers (Evans blue and fluorescent dextran) without evident changes in endothelial junctions; (iv) hemorrhage spots in the skin, meningeal layers, heart, bladder and kidneys; (v) intravascular and interstitial fibrin deposition in the lung, liver and kidney. ADTRP deficiency decreased plasma TFPI antigen by ~2-times. Furthermore, TFPI antigen and anticoagulant activity in lung extracts and isolated lung EC were similarly decreased, which confirms our previous in vitro data. We aslo noticed increased tail bleeding time (>500 sec vs. 200 sec in WT littermates) and blood volume loss, which likely was caused by increased dilation of the tail vein. Gene expression analysis of whole organs showed upregulation of Wnt target genes involved in vascular contractility (Nos3), and extracellular matrix remodeling (Mmp2). Similarly, skin fibroblasts and lung EC isolated from Adtrp-/- mice showed increased expression of Wnt target genes (Lef-1, Cyclin D, Dkk2, c-Myc), which indicates constitutive activation of canonical Wnt signaling. In conclusion, we used genetic animal models and cell culture systems to show for the first time that the novel protein ADTRP plays major roles in vascular development and function. Lack of, or low levels of ADTRP associate with activation of coagulation and vascular development defects, which may be due, at least in part, to intrinsic high levels of ectopic canonical Wnt signaling. Disclosures No relevant conflicts of interest to declare.

Gene expression in peripheral blood in treatment-free major depression

2020 ◽  
Vol 32 (3) ◽  
pp. 135-144
Author(s):  
Alfredo B. Cuellar-Barboza ◽  
Jorge A. Sánchez-Ruiz ◽  
Iram P. Rodriguez-Sanchez ◽  
Sarai González ◽  
Geovana Calvo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Target Genes ◽  
Wnt Signaling Pathway ◽  
Mexican Descent ◽  
Population Diversity ◽  
Canonical Wnt Signaling ◽  
Lineal Regression ◽  
Canonical Wnt

AbstractBackground:Peripheral gene expression of several molecular pathways has been studied in major depressive disorder (MDD) with promising results. We sought to investigate some of these genes in a treatment-free Latino sample of Mexican descent.Material and Methods:The sample consisted of 50 MDD treatment-free cases and 50 sex and age-matched controls. Gene expression of candidate genes of neuroplasticity (BDNF, p11, and VGF), inflammation (IL1A, IL1B, IL4, IL6, IL7, IL8, IL10, MIF, and TNFA), the canonical Wnt signaling pathway (TCF7L2, APC, and GSK3B), and mTOR, was compared in cases and controls. RNA was obtained from blood samples. We used bivariate analyses to compare subjects versus control mean mRNA quantification of target genes and lineal regression modelling to test for effects of age and body mass index on gene expression.Results:Most subjects were female (66%) with a mean age of 26.7 (SD 7.9) years. Only GSK3B was differentially expressed between cases and controls at a statistically significant level (p = 0.048). TCF7L-2 showed the highest number of correlations with MDD-related traits, yet these were modest in size.Discussion:GSK3B encodes a moderator of the canonical Wnt signaling pathway. It has a role in neuroplasticity, neuroprotection, depression, and other psychiatric phenotypes. We found that adding population diversity has the potential to elicit distinct peripheral gene expression markers in MDD and MDD-related traits. However, our results should only be considered as hypothesis-generating research that merits further replication in larger cohorts of similar ancestry.

Wnt-signaling mediates the anti-adipogenic action of lysophosphatidic acid through cross talking with the Rho/Rho associated kinase (ROCK) pathway

2011 ◽  
Vol 89 (6) ◽  
pp. 515-521 ◽  
Author(s):  
L. Li ◽  
L. Tam ◽  
L. Liu ◽  
T. Jin ◽  
D.S. Ng
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Lysophosphatidic Acid ◽  
Target Genes ◽  
Biological Activities ◽  
Wnt Signaling Pathway ◽  
Canonical Wnt Signaling ◽  
Diverse Range ◽  
Canonical Wnt ◽  
Pparγ Expression

Lysophosphatidic acid (LPA) is a bioactive phospholipid with a diverse range of biological activities including the modulation of adipogenesis. Treatment of 3T3-L1 cells and 3T3F44A cells with LPA inhibits adipogenesis and reduces expression of PPARγ through activation of RhoGTPase and its downstream Rho associated kinase (ROCK). The mechanism of suppression of PPARγ expression by Rho/ROCK is poorly understood. By treating the differentiating 3T3-L1 cells with various combinations of LPA and ROCK inhibitors, Y-27632 and fasudil, we observed that LPA treatment resulted in attenuation of adipogenesis and a significant reduction in PPARγ mRNA as early as 3 d post-induction. LPA treatment also resulted in significant but delayed upregulation of components of the canonical Wnt signaling, namely Wnt10b mRNA, β-catenin protein, and mRNA expression of β-catenin target genes, detectable at day 7, but not day 3. Treatment of the 3T3-L1 cells with ROCK inhibitors Y-27632 and fasudil revealed a tonic activation of β-catenin/target genes by ROCK. This study identified the existence of a novel cross talk between the Rho/ROCK pathway and the Wnt-signaling pathway. The LPA/Rho/ROCK pathway inhibits expression of PPARγ and adipogenesis in part through a delayed activation of the canonical Wnt-signaling pathway based on increased Wnt10b expression and β-catenin induction.

scholarly journals Evolutionary Turnover in Wnt Gene Expression but Conservation of Wnt Signaling during Ovary Determination in a TSD Reptile

2021 ◽  
pp. 1-22
Author(s):  
Turk Rhen ◽  
Zachary Even ◽  
Alaina Brenner ◽  
Alexandra Lodewyk ◽  
Debojyoti Das ◽  
...  
Keyword(s):  
Sex Determination ◽  
Wnt Signaling ◽  
Planar Cell Polarity ◽  
Target Genes ◽  
Calcium Release ◽  
Canonical Wnt Signaling ◽  
Snapping Turtle ◽  
Canonical Wnt

Temperature-dependent sex determination (TSD) is a well-known characteristic of many reptilian species. However, the molecular processes linking ambient temperature to determination of gonad fate remain hazy. Here, we test the hypothesis that Wnt expression and signaling differ between female- and male-producing temperatures in the snapping turtle <i>Chelydra serpentina</i>. Canonical Wnt signaling involves secretion of glycoproteins called WNTs, which bind to and activate membrane bound receptors that trigger β-catenin stabilization and translocation to the nucleus where β-catenin interacts with TCF/LEF transcription factors to regulate expression of Wnt targets. Non-canonical Wnt signaling occurs via 2 pathways that are independent of β-catenin: one involves intracellular calcium release (the Wnt/Ca<sup>2+</sup> pathway), while the other involves activation of RAC1, JNK, and RHOA (the Wnt/planar cell polarity pathway). We screened 20 Wnt genes for differential expression between female- and male-producing temperatures during sex determination in the snapping turtle. Exposure of embryos to the female-producing temperature decreased expression of 7 Wnt genes but increased expression of 2 Wnt genes and <i>Rspo1</i> relative to embryos at the male-producing temperature. Temperature also regulated expression of putative Wnt target genes in vivo and a canonical Wnt reporter (6x TCF/LEF sites drive H2B-GFP expression) in embryonic gonadal cells in vitro. Results indicate that Wnt signaling was higher at the female- than at the male-producing temperature. Evolutionary analyses of all 20 Wnt genes revealed that thermosensitive Wnts, as opposed to insensitive Wnts, were less likely to show evidence of positive selection and experienced stronger purifying selection within TSD species.

scholarly journals Nuclear Dvl, c-Jun, β-catenin, and TCF form a complex leading to stabiLization of β-catenin–TCF interaction

2008 ◽  
Vol 180 (6) ◽  
pp. 1087-1100 ◽  
Author(s):  
Xiao-qing Gan ◽  
Ji-yong Wang ◽  
Ying Xi ◽  
Zhi-li Wu ◽  
Yi-ping Li ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Gene Transcription ◽  
Mammalian Cells ◽  
Target Genes ◽  
Wnt Signaling Pathway ◽  
Gene Promoters ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt ◽  
Regulate Gene

In canonical Wnt signaling, Dishevelled (Dvl) is a critical cytoplasmic regulator that releases β-catenin from degradation. Here, we find that Dvl and c-Jun form a complex with β-catenin–T-cell factor 4 (TCF-4) on the promoter of Wnt target genes and regulate gene transcription. The complex forms via two interactions of nuclear Dvl with c-Jun and β-catenin, respectively, both of which bind to TCF. Disrupting the interaction of Dvl with either c-Jun or β-catenin suppresses canonical Wnt signaling–stimulated transcription, and the reduction of Dvl diminished β-catenin–TCF-4 association on Wnt target gene promoters in vivo. Expression of a TCF-Dvl fusion protein largely rescued the c-Jun knockdown Wnt signaling deficiency in mammalian cells and zebrafish. Thus, we confirm that c-Jun functions in canonical Wnt signaling and show that c-Jun functions as a scaffold in the β-catenin–TCFs transcription complex bridging Dvl to TCF. Our results reveal a mechanism by which nuclear Dvl cooperates with c-Jun to regulate gene transcription stimulated by the canonical Wnt signaling pathway.

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