β-Catenin-Independent Activation of TCF1/LEF1 in Human Hematopoietic Tumor Cells through Interaction with ATF2 Transcription Factors

The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling

Canonical Wnts function as potent regulators of osteogenesis by human mesenchymal stem cells

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study, we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis, which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation, which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling, the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model, high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However, hMSCs with exogenous expression of Wnt1 but not stabilized β-catenin markedly stimulate bone formation by naive hMSCs, arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo

β-Catenin-Independent Activation of TCF1/LEF1 in Human Hematopoietic Tumor Cells through Interaction with ATF2 Transcription Factors

International audienceThe role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of b-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to b-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of b-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of b-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to b-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the b-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of b-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling

Mapping of the TCF1 domains involved in β-catenin-independent transcriptional activity.

<p>(A) Diagram of the different constructs used for the mapping. (B–D) 293T cells were co-transfected with the SuperTop reporter, the renilla plasmid and the indicated constructs, followed by reporter assay two days after transfection. (B) TCF1 N-terminal domain is required, but not sufficient, for β-catenin-independent full transcriptional activity. (C) Deletion of either aa 101–211 or 212–299 inhibits the β-catenin-independent transcriptional activity of TCF1. (D) Transcriptional activity of two different TCF1 domains fused to the Gal4 DNA binding domain. 293T cells were co-transfected with a Gal4 responsive reporter and the indicated TCF1-Gal4 fusion constructs, followed by reporter assay. (E) Expression of TCF1 aa 37–122, but not aa 100–211, inhibits TCF1-induced reporter activity. 293T cells were co-transfected with the Supertop reporter and the indicated TCF1-Gal4 fusion constructs, in the presence or the absence of TCF1, followed by reporter assay two days after transfection.</p

ATF2 and ATF7 knockdown in Ramos cells decreases TCF/LEF activity, Axin 2 expression and cell growth.

<p>(A–C) Ramos cells containing the TCF/LEF firefly and renilla luciferase lentiviral reporters were transduced with shATF2 and shATF7 or control lentiviral vectors. ATF2 and ATF7 down-regulation assessed by immunoblot analysis. (A) shATF2/7 inhibits constitutive TCF/LEF reporter activity in Ramos cells. (B) Knockdown of ATF2 and ATF7 reduces the expression of the Wnt target gene Axin 2 in Ramos cells as measured by quantitative real-time PCR. The results normalized to the control represent the mean values ± SEM of three independent experiments. (*) P<0.05; (**) P<0.001 compared with control (two-way Anova test) (C) shATF2/7 inhibits Ramos cell colony formation. (D) Model for β-catenin-dependent and β-catenin-independent canonical Wnt signaling. See the text for details.</p

β-catenin-independent transcriptional activity of TCF1/LEF1 factors.

<p>(A) Empty pcDNA3HA vector or increasing amounts of HA-tagged TCF1, LEF1 or TCF4 constructs were co-transfected with SuperTop or SuperFop and renilla luciferase plasmids in 293T cells and the Top/Fop ratio was calculated. (B–C) Effects of a decoy construct containing TCF1 β-catenin binding domain (BCBD) on Wnt3a- and TCF1-induced TCF/LEF reporter activity. 293T cells were co-transfected with SuperTop and renilla reporter plasmids, together with the Wnt3a, TCF1 and BCBD vectors as indicated. (D–E) Mutant TCF1 (mtTCF1; D21A, E29K) is unable to bind to β-catenin. (D) 293T cells were transfected with the indicated HA-tagged TCF1 or TCF4 constructs and the myc-tagged β-catenin, followed by immunoprecipitation using anti-HA antibody and immunoblot with anti-myc or anti-HA antibodies. (E) 3T3 cells were co-transfected with β-catenin, the indicated amounts of TCF1, d36TCF1 or mtTCF1, together with SuperTop and renilla luciferase plasmids. (F) Effects of TCF1 and mtTCF1 expression on TCF/LEF reporter activity. 293T cells were co-transfected with SuperTop and renilla luciferase plasmids and different amounts of TCF1 or mtTCF1, followed by reporter assay.</p