Β-Catenin-Tcf/Lef Signaling Promotes Steady State and Emergency Granulopoiesis through G-CSF Receptor Upregulation

The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the Tcf/Lef transcription factors and subsequent transcriptional activation of Wnt-target genes. This pathway acts as an essential regulator of differentiation and cell fate decisions in various tissues. In the hematopoietic system, the function of the pathway has been investigated mainly by genetic manipulation of β-catenin. However, this approach does not allow to discriminate between Tcf/Lef dependent or independent β-catenin activity. In order to specifically identify the function of β-catenin-Tcf/Lef signaling in hematopoietic cells, we employed a transgenic mouse model expressing a dominant negative form of the human TCF4 transcription factor (dnTCF4). dnTCF4, a truncated protein lacking the β-catenin binding domain, abrogates activation of Wnt target genes, even when β-catenin is stabilized and translocated into the nucleus. In our model, expression of dnTCF4 is activated from the Rosa26 locus only in cells producing Cre recombinase (driven by Vav-iCre). Importantly, all components of Wnt signaling, including endogenous Tcf/Lef proteins and β-catenin, are intact in Cre-expressing cells. We observed that dnTCF4 transgenic mice have reduced numbers of granulocytes together with accumulation of short-term hematopoietic stem cells (ST-HSC) and common myeloid progenitors (CMPs) in bone marrow. Accordingly, dnTCF4-expressing bone marrow consistently showed a block of granulocytic differentiation and retention of an immature phenotype in colony forming assays. This differentiation arrest and accumulation of immature cells was also observed when wild type cells were cultured in semi-solid medium in the presence of a cell-penetrating peptide that disrupts β-catenin-Tcf/Lef interaction. Together, these results indicate that disruption of the β-catenin/Tcf-Lef interaction, either by genetic manipulation or a drug based approach, alters steady-state hematopoiesis. To identify a mechanism through which β-catenin-Tcf/Lef signaling affects granulopoiesis, wild type and dnTCF4 expressing ST-HSCs were subjected to RNA sequencing. Several genes related to myeloid development were differentially expressed in dnTCF4 expressing cells, including downregulation of Csf3r, the gene encoding for the G-CSF receptor. Publicly available datasets from ChIP-seq experiments on human cell lines confirmed TCF4 enrichment in the distal promoter of the CSF3R gene, suggesting that CSF3R is directly regulated by canonical Wnt signaling. Using flow cytometry we verified reduced levels of G-CSF receptor on the cell surface of dnTCF4 progenitor cells, and attenuation of downstream Stat3 phosphorylation after G-CSF treatment. Finally, when grown in the presence of G-CSF, dnTCF4-expressing bone marrow cells showed impaired differentiation abilities and reduced granulocytic counts compared to wild type bone marrow cells. These results encouraged us to investigate the role of the β-catenin-Tcf/Lef signaling pathway during emergency granulopoiesis by challenging mice with lipopolysaccharide (LPS). Remarkably, dnTCF4 mice showed defects upon LPS stimulation, and completely failed to maintain and expand myeloid progenitor populations, a critical step during emergency granulopoiesis. Altogether, we showed that β-catenin-Tcf/Lef signaling axis is crucial for proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Mechanistically, we demonstrated that the β-catenin-Tcf/Lef interaction controls expression of genes involved in myeloid differentiation, and directly enhances expression of the G-CSF receptor, a crucial molecule for proper development of granulocytes. Disclosures No relevant conflicts of interest to declare.

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β-Catenin Expression in Bone Marrow Stromal Cells Is Required for Normal Proliferation and Differentiation of Primitive Hematopoietic Cells.

Blood ◽

10.1182/blood.v110.11.608.608 ◽

2007 ◽

Vol 110 (11) ◽

pp. 608-608

Author(s):

Michael Nemeth ◽

Yingzi Yang ◽

David Bodine

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Wnt Signaling ◽

Stromal Cells ◽

Bone Marrow Cells ◽

Wild Type ◽

Canonical Wnt Signaling ◽

Proliferation And Differentiation ◽

Canonical Wnt ◽

Marrow Cells

Abstract Normal function of adult hematopoietic stem cells (HSC) is dependent upon the bone marrow microenvironment, which is comprised of multiple cell types, including fibroblasts, endothelial cells, osteoblasts and stromal progenitors. We hypothesized that canonical Wnt signaling, which is necessary for the development of mesenchymal tissue, regulates the cellular structure and function of the microenvironment. We tested this hypothesis using an in vitro model of bone marrow stroma that is deficient in β-catenin, the critical transactivator of canonical Wnt signals. β-catenin−/ − Dexter stroma cultures were established from whole bone marrow harvested from β-cateninlox/lox Mx-cre+/cre mice treated with 7–10 doses of 300 μg pIpC. PCR analysis of stromal cell DNA showed nearly 100% deletion of β-catenin. Confluent stroma cultures were irradiated and seeded with 4 x 104 lin cells/cm2. We have reported (Nemeth, et al, Blood, 108, 29a) that β-catenin−/ − stroma exhibit decreased ability to support CFU formation and generate osteoblasts. The reduction in CFU-C correlates with a 75% increase in the percentage of lin− progenitors cultured on β-catenin−/ − stroma undergoing apoptosis (23.6 ± 3.4%) compared to wild-type (13.6 ± 1.3% ; n = 3; p < .01). To determine the mechanism by which canonical Wnt signaling regulates microenvironment function, we used a cytokine antibody array to analyze protein levels of 30 different hematopoietic growth factors and adhesion molecules. We observed decreased amounts of the soluble factors bFGF (3.3 ± 0.6-fold) and SCF (2.3 ± 0.3-fold), and the adhesion factor VCAM-1 (2.7 ± 0.3-fold) (n = 3; p < .01) in β-catenin−/ − stroma. The decrease in VCAM-1 corresponded with decreased percentages of VCAM-1+ osteoblasts (26.8 ± 0.9% vs. 43.9 ± 5.7%) and endothelial cells (31.6 ± 5.7% vs. 76.5 ± 10.9%) in β-catenin−/ − stroma compared to wild-type (n = 4, p < .01). From these data, we hypothesized that β-catenin is necessary for maintaining the stromal cells that support HSCs in vivo. We tested this hypothesis by transplanting 2 x 106 wild-type whole bone marrow cells (CD45.1) into lethally-irradiated β-cateninlox/lox Mx-cre+/cre mice and littermate controls. 8 weeks later, transplanted mice were treated with pIpC, resulting in mice with a wild-type hematopoietic system and a β-catenin−/ − microenvironment. Two weeks after the final treatment, we observed a 2.7 ± 0.1-fold reduction in the percentage of long- and short-term HSCs (lin−, c-kitHI, Sca-1HI) in bone marrow from mice with a β-catenin−/ − microenvironment compared to wild-type (n = 4, p < .05). We performed a competitive repopulation assay, transplanting 1 x 106 whole bone marrow cells harvested from primary recipients with a wild-type or β-catenin−/ − microenvironment with 1 x 106 CD45.2 whole bone marrow cells into lethally-irradiated secondary recipient mice. After 16 weeks, there was no difference in mean repopulation by bone marrow cells from the β-catenin−/ − microenvironment (9.2 ± 2.8%) compared to wild-type (10.7 ± 0.6%), indicating that self-renewal was unaffected. However, we did observe a significant 4-fold increase in variability of repopulation by bone marrow cells from the β-catenin−/ − microenvironment (F-test = .01). Since smaller numbers of HSCs will yield greater variability in repopulation than larger numbers, this is consistent with the observation that the β-catenin−/ − microenvironment supports fewer HSCs. From these data, we propose a model in which canonical Wnt signaling in the microenvironment is necessary for hematopoietic proliferation and differentiation.

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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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Gene expression in peripheral blood in treatment-free major depression

Acta Neuropsychiatrica ◽

10.1017/neu.2020.5 ◽

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.

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

Biochemistry and Cell Biology ◽

10.1139/o11-048 ◽

2011 ◽

Vol 89 (6) ◽

pp. 515-521 ◽

Cited By ~ 16

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.

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Icariin Regulates the Bidirectional Differentiation of Bone Marrow Mesenchymal Stem Cells through Canonical Wnt Signaling Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/8085325 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 13

Author(s):

Jun-ming Huang ◽

Yuan Bao ◽

Wei Xiang ◽

Xing-zhi Jing ◽

Jia-chao Guo ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Western Blot ◽

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Canonical Wnt Signaling ◽

Marrow Mesenchymal Stem Cells ◽

Canonical Wnt

Fat infiltration within the bone marrow is easily observed in some postmenopausal women. Those fats are mainly derived from bone marrow mesenchymal stem cells (BMMSCs). The increment of adipocytes derived from BMMSCs leads to decreased osteoblasts derived from BMMSCs, so the bidirectional differentiation of BMMSCs significantly contributes to osteoporosis. Icariin is the main extractive of Herba Epimedii which is widely used in traditional Chinese medicine. In this experiment, we investigated the effect of icariin on the bidirectional differentiation of BMMSCs through quantitative real-time PCR, immunofluorescence, western blot, and tissue sections in vitro and in vivo. We found that icariin obviously promotes osteogenesis and inhibits adipogenesis through detecting staining and gene expression. Micro-CT analysis showed that icariin treatment alleviated the loss of cancellous bone of the distal femur in ovariectomized (OVX) mice. H&E staining analysis showed that icariin-treated OVX mice obtained higher bone mass and fewer bone marrow lipid droplets than OVX mice. Western blot and immunofluorescence showed that icariin regulates the bidirectional differentiation of BMMSCs via canonical Wnt signaling. This study demonstrates that icariin exerts its antiosteoporotic effect by regulating the bidirectional differentiation of BMMSCs through the canonical Wnt signaling pathway.

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Canonical and Non-Canonical Wnt Signaling Regulate the Balance between HSC Self-Renewal and Differentiation.

Blood ◽

10.1182/blood.v108.11.862.862 ◽

2006 ◽

Vol 108 (11) ◽

pp. 862-862

Author(s):

Michael Nemeth ◽

Yingzi Yang ◽

David Bodine

Keyword(s):

Bone Marrow ◽

Wnt Signaling ◽

Bone Marrow Cells ◽

Retroviral Vectors ◽

Canonical Wnt Signaling ◽

Self Renewal ◽

Hematopoietic Stem ◽

Serum Free ◽

Canonical Wnt ◽

Marrow Cells

Abstract We and others have implicated activation of canonical Wnt signaling by Wnt3a in promoting hematopoietic stem cell (HSC) self-renewal. Since Wnt5a can inhibit canonical Wnt signaling (Topol, et al. 2003), we hypothesized that Wnt3a and Wnt5a act as antagonists on HSC function. To examine the effect of Wnt3a and Wnt5a on canonical Wnt signaling in HSCs, we isolated HSCs (lin−, c-kitHI, Sca-1HI, IL-7Rα−) that contain a lacZ reporter gene that is induced by canonical signaling and cultured them in serum-free media in the presence of 30 ng/ml SCF and Flt3L and recombinant Wnt3a (3a; 100 ng/ml) with and without recombinant Wnt5a (5a; 500 ng/ml). We observed a significant 50% reduction (p < .05) in lacZ mRNA in HSCs cultured with 3a + 5a compared to 3a alone, indicating that Wnt5a inhibits canonical signaling in HSCs. Treatment with 3a, 5a, and 3a + 5a caused the percentage of actively cycling HSCs to decrease 1.4 to 1.6-fold compared to control (SCF + Flt3L) (p < .02). To examine the role of Wnt signaling in HSC self-renewal, wild-type CD45.1+ HSCs were cultured for 6 days before being transplanted into lethally irradiated recipients (1:100 ratio HSCs to CD45.2+ whole bone marrow cells; total number of CD45.1+ HSCs transplanted ranged from 3 to 5 × 103). There was no difference in long-term repopulation between control HSCs (6.7 ± 5.5% CD45.1+ bone marrow cells, n = 14) and HSCs cultured with 3a (4.1 ± 3.7%, n = 8, p = .24). However, HSCs cultured with 5a (24.1 ± 21.2%, n = 10) or 5a + 3a (38.5 ± 36.4%, n = 11) showed significant increases in repopulation compared to control (p < .01; Mann-Whitney test). To determine if additional survival signals were necessary for 3a to induce HSC self-renewal, we transplanted cultured HSCs isolated from transgenic mice that overexpressed the anti-apoptotic gene Bcl2. We observed that control HSCs engrafted 7/7 mice (average repopulation: 17.8 ± 6.8% CD45.1+ bone marrow) whereas when 3a was added, only 1/7 mice showed engraftment (1.2%). Together, these data suggest that induction of canonical signaling by Wnt3a results in decreased HSC expansion and self-renewal and that Wnt5a positively regulates HSC self-renewal by non-canonical Wnt signaling pathways independent of its ability to inhibit canonical signaling. We examined the effects of Wnt3a and Wnt5a on Hoxb4, Notch1, and c-myc expression in HSCs. We observed that adding 3a to serum-free cultures caused no change in Hoxb4 mRNA, a 2-fold reduction in Notch1 mRNA (p < .01) and a 2.4-fold increase in the canonical Wnt pathway target gene c-myc (p < .05; n = 3) compared to control. No changes in Hoxb4, Notch1, or c-myc mRNA were observed in HSCs cultured with 5a or 5a + 3a. Since overexpression of c-myc has been linked to loss of HSC self-renewal, we hypothesized that overexpression of Wnt3a would inhibit HSC self-renewal and repopulation. We transduced CD45.1+ bone marrow with Wnt3a-IRES-GFP or IRES-GFP retroviral vectors and sorted GFP+ cells were transplanted with equal numbers of CD45.2+ mock-transduced cells. Four months post transplantation, 3.4 ± 1.8% of bone marrow cells in IRES-GFP recipients were GFP+ (n = 8). In contrast, no GFP+ cells were detected in Wnt3a-GFP recipients (n = 8). Wnt3a-GFP retroviral DNA was detected by PCR, suggesting that vector silencing was required for transduced cell survival. We propose that imbalanced canonical Wnt signaling in HSCs deregulates hematopoiesis by inducing pro-differentiation genes such as c-myc and that additional signals, e.g. Wnt5a, are required to maintain the balance between HSC differentiation and self-renewal.

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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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