Flt3 Internal Tandem Duplications Cooperate with Wnt Signaling in Leukemic Signal Transduction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 822-822
Author(s):  
Lara Tickenbrock ◽  
Joachim Schwable ◽  
Markus Wiedehage ◽  
Bjoern Steffen ◽  
Chunaram Choudhary ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Wnt Signaling ◽  
Fusion Proteins ◽  
Transcriptional Activity ◽  
Dominant Negative ◽  
Cell Surface Receptor ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Wnt Proteins ◽  
Tandem Duplications

Abstract Flt3 internal tandem duplications cooperate with Wnt signaling in leukemic signal transduction Lara Tickenbrock, Joachim Schwable, Markus Wiedehage, Bjoern Steffen, Chunaram Choudhary, Wolfgang E. Berdel, Carsten Muller-Tidow and Hubert Serve Department of Medicine, Hematology and Oncology, University of Muenster, D-48149 Muenster, Germany The type III receptor tyrosine kinase (RTK) Flt3 plays an important role in survival and proliferation of hematopoietic progenitor cells. Somatic mutations of Flt3 consisting of internal tandem duplications (ITD) occur in 25% of patients with acute myeloid leukemia (AML), and are associated with a poor prognosis. They result in Flt3-ligand (FL) independent kinase activation of Flt3. The Wnt signal transduction pathway has recently been implicated to be an important regulator of early hematopoietic stem cell fate decisions. Also, we previously showed that activation of Wnt-dependent Tcf transcriptional activity is induced by the leukemia-associated fusion proteins PML-RAR? and AML1-ETO. Here, we show that Flt3-ITD mutations enhance basal and Wnt3a-stimulated Tcf activity in myeloid cells. Microarray experiments analyzing Flt3-ITD target genes in 32D cells revealed up to 8-fold induction of Frizzled-4, a cell surface receptor for Wnt proteins, by Flt3-ITD mutations. This could be verified by real-time RT-PCR and Western Blot analyses. In functional analyses, we explored the synergism of Flt3-ITD and the activation of Wnt signaling. Flt3-ITD mutations induced the accumulation of ?-Catenin and TCF-dependent transcriptional activity. Also, the presence of Flt3-ITD highly sensitized cells for ?-Catenin-induction by the Wnt3a. Wnt3a incubation enhanced 32D cell proliferation in the presence of activated Flt3 receptors. More importantly, Flt3-ITD-mediated clonogenic growth highly depended on the activity of Tcf transcription factors, since transient transfection of Flt3-ITD cells with dominant negative TCF4 almost abolished 32D cell colony growth. Our results indicate that Flt3-ITD mutations mediate their leukemogenic effects in part through the activation of Wnt-dependent signaling pathways, possibly defining this signal system as a converging point of leukemogenic events elicited by Flt3-mutations and leukemia-associated fusion proteins.

scholarly journals Wnt-inhibitory factor 1 dysregulation of the bone marrow niche exhausts hematopoietic stem cells

Blood ◽  
2011 ◽  
Vol 118 (9) ◽  
pp. 2420-2429 ◽  
Author(s):  
Christoph Schaniel ◽  
Dario Sirabella ◽  
Jiajing Qiu ◽  
Xiaohong Niu ◽  
Ihor R. Lemischka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Cell Fate ◽  
Sonic Hedgehog ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cell Niche ◽  
Wnt Inhibitory Factor 1 ◽  
Stem Cell Pool

Abstract The role of Wnt signaling in hematopoietic stem cell fate decisions remains controversial. We elected to dysregulate Wnt signaling from the perspective of the stem cell niche by expressing the pan Wnt inhibitor, Wnt inhibitory factor 1 (Wif1), specifically in osteoblasts. Here we report that osteoblastic Wif1 overexpression disrupts stem cell quiescence, leading to a loss of self-renewal potential. Primitive stem and progenitor populations were more proliferative and elevated in bone marrow and spleen, manifesting an impaired ability to maintain a self-renewing stem cell pool. Exhaustion of the stem cell pool was apparent only in the context of systemic stress by chemotherapy or transplantation of wild-type stem cells into irradiated Wif1 hosts. Paradoxically this is mediated, at least in part, by an autocrine induction of canonical Wnt signaling in stem cells on sequestration of Wnts in the environment. Additional signaling pathways are dysregulated in this model, primarily activated Sonic Hedgehog signaling in stem cells as a result of Wif1-induced osteoblastic expression of Sonic Hedgehog. We find that dysregulation of the stem cell niche by overexpression of an individual component impacts other unanticipated regulatory pathways in a combinatorial manner, ultimately disrupting niche mediated stem cell fate decisions.

Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3414-3414 ◽  
Author(s):  
Marijke W Maijenburg ◽  
Marion Kleijer ◽  
Kim Vermeul ◽  
Erik P.J. Mul ◽  
Floris P.J. van Alphen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

Mastermind critically regulates Notch-mediated lymphoid cell fate decisions

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1696-1702 ◽  
Author(s):  
Ivan Maillard ◽  
Andrew P. Weng ◽  
Andrea C. Carpenter ◽  
Carlos G. Rodriguez ◽  
Hong Sai ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Fate ◽  
Critical Role ◽  
Family Members ◽  
Notch Pathway ◽  
Dominant Negative ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions

Abstract During lymphoid development, Notch1 plays a critical role in the T-cell/B-cell lineage decision, while Notch2 is essential for marginal zone B-cell (MZB) development. Notch pathway activation induces translocation of intracellular Notch (ICN) to the nucleus, where it interacts with the transcription factor CSL (CBF1/RBP-Jk, Suppressor of Hairless, Lag-1). In vitro, ICN binds Mastermind-like proteins, which act as potent Notch coactivators. Three MAML family members (MAML1-3) have been identified in mammals, but their importance in vivo is unknown. To investigate the function of MAMLs in hematopoietic development, we introduced a dominant negative (DN) mutant of MAML1, capable of inhibiting Notch1-4, in murine hematopoietic stem cells. DNMAML1 resulted in early inhibition of T-cell development and the appearance of intrathymic B cells, phenotypes consistent with Notch1 inhibition. The T-cell differentiation block was as profound as that produced by enforced expression of the Notch modulator Deltex1. In DNMAML1-transduced spleen cells, a dramatic decrease in MZB cells was present, consistent with Notch2 inhibition. In contrast, Deltex1 did not decrease MZB cell numbers. These results suggest a critical role for MAMLs during Notch-mediated cell fate decisions in vivo and indicate that DNMAML1, but not Deltex1, can be used to interfere with the function of multiple Notch family members. (Blood. 2004;104:1696-1702)

scholarly journals Wnt-β-Catenin Signaling Promotes the Maturation of Mast Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Tomoko Yamaguchi ◽  
Misae Nishijima ◽  
Katsuhisa Tashiro ◽  
Kenji Kawabata
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Wnt Signaling ◽  
Molecular Mechanisms ◽  
Histidine Decarboxylase ◽  
Wnt Signaling Pathway ◽  
Hematopoietic Stem ◽  
Peripheral Tissues ◽  
Wnt Proteins

Mast cells play an important role in the pathogenesis of allergic diseases. Immature mast cells migrate into peripheral tissues from the bone marrow and undergo complete maturation. Interestingly, mast cells have characteristics similar to hematopoietic stem cells (HSCs), such as self-renewal and c-kit expression. In HSCs, Wnt signaling is involved in their maintenance and differentiation. On the other hand, the relation between Wnt signaling and mast cell differentiation is poorly understood. To study whether Wnt signals play a role in the maturation of mast cells, we studied the effect of Wnt proteins on mast cell maturation of bone marrow-derived mast cells (BMMCs). The expression levels of CD81 protein and histidine decarboxylase mRNA and activity of mast cell-specific protease were all elevated in BMMCs treated with Wnt5a. In addition, Wnt5a induced the expression of Axin2 and TCF mRNA in BMMCs. These results showed that Wnt5a could promote the maturation of mast cells via the canonical Wnt signaling pathway and provide important insights into the molecular mechanisms underlying the differentiation of mast cells.

scholarly journals Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation

Blood ◽  
2009 ◽  
Vol 113 (3) ◽  
pp. 546-554 ◽  
Author(s):  
Tiago C. Luis ◽  
Floor Weerkamp ◽  
Brigitta A. E. Naber ◽  
Miranda R. M. Baert ◽  
Edwin F. E. de Haas ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Cell Fate ◽  
Fetal Liver ◽  
In Vitro Assays ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Thymocyte Differentiation ◽  
B Lymphoid

Abstract Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a−/− HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis.

Expansion of Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells Requires Rac-Mediated Interaction with Stromal Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1398-1398
Author(s):  
Jan Jacob Schuringa ◽  
Marjan Rozenveld-Geugien ◽  
Inge Baas ◽  
Djoke van Gosliga ◽  
Edo Vellenga
Keyword(s):  
Signal Transduction ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Dramatic Decrease ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract We have studied Rac signal transduction in human cord-blood (CB) and acute myeloid leukemia (AML) CD34+ cells and determined that Rac proteins are critically involved in the interaction between human stem/progenitor cells and stroma. Constitutive activation of Rac signaling was achieved by retroviral introduction of Rac1-V12 into CB-derived CD34+ cells, while inhibition of Rac signaling was established by retroviral introduction of dominant negative Rac1-N17 or by utilizing the Rac inhibitor NSC23766. Inhibition of Rac signaling resulted in a proliferative disadvantage of CB-CD34+ cells when cultured on MS5 stromal cells. Cells were severely disturbed in their migration towards and direct association with MS5 stroma when Rac signaling was inhibited. The Long Term Culture-Initiating Cells (LTC-ICs) migrated underneath the stromal MS5 layer within 24 hrs after plating, and similar results were obtained for about 50% of the Colony Forming Cells (CFCs). However, transient inhibition of Rac signaling for 24–72 hrs resulted in a shift of LTC-ICs and CFCs to the suspension phase as determined by colony assays and CAFC week 5 enumeration. When Rac signaling signal transduction was inhibited during the 5 week coculture period a dramatic decrease in LTC-IC frequency from 0.6% to 0.15% was observed. Many of these phenotypes were reversed in the presence of activated Rac1-V12, including improved migration towards and association with MS5 cells and elevated LTC-IC frequencies. Importantly, in CAFC assays using AML cells (n=8) that were enriched for leukemic stem cells on the basis of a CD34+/CD38low phenotype we observed a dramatic decrease in leukemic CAFC formation as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766. Taken together, our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.

Flt3 tandem duplication mutations cooperate with Wnt signaling in leukemic signal transduction

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3699-3706 ◽  
Author(s):  
Lara Tickenbrock ◽  
Joachim Schwäble ◽  
Markus Wiedehage ◽  
Björn Steffen ◽  
Bülent Sargin ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Wnt Signaling ◽  
Tandem Duplication ◽  
Strong Dependence ◽  
Wnt Signaling Pathway ◽  
Dominant Negative ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Protein Levels ◽  
Primary Mouse

AbstractActivating Flt3 mutations occur in about 30% of patients with acute myeloid leukemia (AML), often as in-frame internal tandem duplication (ITD) at the juxtamembrane domain of the receptor. These mutations transform hematopoietic cell lines and primary mouse bone marrow. Here, we analyzed the interaction between oncogenic Flt3-ITD mutations and the Wingless-type (Wnt) signaling pathway in the myeloid progenitor cell line 32D. Microarray analyses revealed higher mRNA expression of Frizzled-4, a receptor for Wnt ligands in 32D/Flt3-ITD cells. Findings were verified by quantitative realtime reverse transcription–polymerase chain reaction (RT-PCR) and on the protein level. Compared with 32D/Flt3-WT (wild-type) cells, 32D/Flt3-ITD cells also showed greatly enhanced β-catenin protein levels, irrespective of their exposure to Wnt3a, a ligand inducing the canonical Wnt signal transduction pathway. In addition, 5 of 7 AML samples with Flt3-ITD mutations expressed high β-catenin protein levels, whereas patients with wild-type Flt3 did not. Also, Flt3-ITD induced enhanced T-cell factor (TCF)–dependent transcriptional activity and the induction of the Wnt target gene c-myc. In the presence of Flt3-WT or Flt3-ITD signaling, Wnt3a slightly increased 32D cell proliferation. However, transfection experiments with dominant-negative (dn) TCF4 revealed a strong dependence of Flt3-ITD–mediated clonogenic growth on TCF activity. Taken together, our results indicate that Flt3-ITD and Wnt-dependent signaling pathways synergize in myeloid transformation.

Transcription Factor Dosage: Maximal STAT5-Induced Proliferation and Stem Cell Self-Renewal at Intermediate STAT5 Activity Levels.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2242-2242
Author(s):  
Albertus T.J. Wierenga ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Stem Cell ◽  
Fusion Proteins ◽  
Activity Levels ◽  
Dependent Manner ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract The level of transcription factor activity critically regulates cell fate decisions such as hematopoietic stem cell self-renewal and differentiation. Here, we describe a model in which STAT5 activity is introduced into human hematopoietic stem/progenitor cells in a dose-dependent manner by overexpression of tamoxifen-inducible STAT5A-ER fusion proteins that can be activated upon treatment with 4-OHT. For this study, we tested STAT5A(wt), STAT5A(1*6) and STAT5A(S711F)-ER fusion proteins. Introduction of STAT5A activity in cord blood-derived CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased CAFC and LTC-IC frequencies that were predominantly elevated at intermediate STAT5A activity levels, but not at high STAT5A activity levels. Self-renewal of progenitors was addressed by serial replating of CFUs grown in methylcellulose, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. Extensive gene expression profiling in CB CD34+ cells over a range of STAT5A activation levels identified subsets of genes that were induced by low, intermediate and high STAT5A transcriptional activity. Thus, we could cluster gene expression levels of STAT5 target genes into a ‘differentiation signature’ versus a ‘proliferation/self-renewal signature’. In conclusion, our data suggest that intermediate rather than high STAT5 activity levels might be required to induce leukemic transformation.

The Short Lef-1 Isoform, Lacking The β-Catenin Binding Domain, Is Not Acting As a Dominant Negative Lef-1 Variant, But Is a Hematopoietic Active Protein With Unique DNA Binding Properties

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2415-2415
Author(s):  
Katrin Elisabeth Edmaier ◽  
Kira Stahnke ◽  
Naidu M Vegi ◽  
Medhanie A Mulaw ◽  
Christian Buske
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Wnt Signaling Pathway ◽  
Normal Karyotype ◽  
Binding Domain ◽  
Dominant Negative ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Abstract Studies in the hematopoietic system of human and mice have demonstrated that the Wnt signaling pathway is essential for maintenance, activation and proliferation of normal hematopoietic stem cells. Lef-1 is a member of the Lef-1/T-cell-factor (Tcf) family of transcription factors regulated by the canonical Wnt signaling pathway and generally directly interacts with β-catenin in transcriptional complexes to induce expression of target genes, including the cell cycle regulators cyclin D1 and c-myc. We have previously shown that aberrant expression of Lef-1 perturbs normal hematopoietic stem cell (HSC) and progenitor function and induces acute myeloid leukemia in the murine bone marrow transplantation model and that it is a novel prognostic independent factor in patients with normal karyotype AML (Petropoulos et al. JEM, 2008; Metzeler et al., Blood 2012). Furthermore, we could recently demonstrate that shRNA mediated depletion of Lef-1 drastically compromises the function of long-term repopulating stem cells (Edmaier et al., Leukemia, in press), indicating a vital function of Lef-1 at the level of the most primitive stem cell compartment. So far, all these Lef-1 functions are ascribed to the full-length transcript of Lef-1, which acts as transcriptional mediator of Wnt signaling via its β-catenin binding domain, whereas the shorter Lef-1 isoform, deriving from an intronic promoter is thought to function as a dominant negative variant. To dissect the role of the ‘dominant negative’ Lef-1 isoform we first retrovirally engineered primary murine bone marrow cells to express the N-terminal deleted Lef-1 (Lef-1d56), lacking the β-catenin binding domain in comparison to the full-length Lef-1 (Lef-1WT) and the empty retroviral EGFP control. Loss of binding to β-catenin was validated by Co-IP for Lef-1d56. Of note, Lef-1d56 induced a 2,05fold and significant increase at the level of clonogenic cells in vitro compared to the control (n=3; p<0,05) and did not differ substantially from the activity of Lef-1 WT. β-catenin binding did not impact Lef-1 activity at the level of the short-term repopulating stem cells as documented in the CFU-S assay with almost comparable CFU-S frequencies between the wild-type and the mutant Lef-1 (d56) (73 CFU-S/1x105 and 64 CFU-S/1x105 , respectively, compared to 38 CFU-S/1x105 for the EGFP control; p<0.05). In clear contrast, loss of β-catenin interaction reduced the CRU frequency dramatically (1:597.197(Lef-1 WT) and 1:1.675.238 (Lef-1 d56), respectively and 1: 1.233.152 for the EGFP control; p=0,066), indicating the necessity of Lef-1 to collaborate with β-catenin at the level of the long-term repopulating stem cell. Retroviral expression of Lef-1 d56 in murine bone marrow stem and progenitor cells for 48h induced a distinct gene expression profile and deregulated more genes than Lef-1WT compared to the EGFP control. Differentially expressed genes between Lef-1d56 and Lef-1WT comprised known hematopoietic factors such as like Gata2, Ets1 and genes associated to the G-protein coupled receptor protein signaling. ChIP-Seq in hematopoietic murine cells revealed unique binding sites for Lef-1d56 compared to Lef-1WT with binding of the Lef-1d56 to promoters of Wnt5a and CD81. Taken together, our data clearly indicate that loss of the β-catenin binding site does not convert Lef-1 into an overall dominant negative variant, but creates a ‘neomorphic’ isoform with distinct biological and DNA binding properties. Analyses are ongoing which quantify the expression of LEF-1WT and the β-catenin domain lacking LEF-1 isoform in over 100 patients with normal karyotype AML in comparison to normal human stem and progenitor cells to test potential differences between normal and leukemic cells as well as associations of the two LEF-1 transcripts with the mutational status, gene expression and treatment outcome in this AML patient group. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Wnt Proteins Induce Dishevelled Phosphorylation via an LRP5/6- Independent Mechanism, Irrespective of Their Ability To Stabilize β-Catenin

2004 ◽  
Vol 24 (11) ◽  
pp. 4757-4768 ◽  
Author(s):  
José M. González-Sancho ◽  
Keith R. Brennan ◽  
Leslie A. Castelo-Soccio ◽  
Anthony M. C. Brown
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Protein Function ◽  
Wnt Signaling Pathway ◽  
Dominant Negative ◽  
Biochemical Assay ◽  
Wnt Proteins ◽  
Signaling Mechanisms ◽  
Independent Mechanism ◽  
Canonical Wnt

ABSTRACT Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of β-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve β-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/β-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with β-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/β-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/β-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to β-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/β-catenin pathway.

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