Dietmar Gradl

The Wnt/Wg signal transducer beta-catenin controls fibronectin expression.

ABSTRACT β-Catenin stabilizes the cadherin cell adhesion complex but, as a component of the Wnt/Wg signaling pathway, also controls gene expression by forming a heterodimer with a transcription factor of the LEF-TCF family. We demonstrate that the substrate adhesion molecule fibronectin is a direct target of Wnt/Wg signaling. Nuclear depletion of β-catenin following cadherin transfection in Xenopusfibroblasts resulted in downregulation of fibronectin expression which was restored by activating the Wnt/Wg signaling cascade via LiCl treatment or transfection of either Xwnt-8 or β-catenin. We isolated the Xenopus fibronectin gene (FN) promoter and found four putative LEF-TCF binding sites. By comparing the activities of different fibronectin gene reporter constructs in fibroblasts and cadherin transfectants, the LEF-TCF site at position −368 was identified as a Wnt/Wg response element. LEF-1-related proteins were found in nuclei of the fibroblasts but were absent in a kidney epithelial cell line. Consistent with the lack of these transcription factors, the FN promoter was silent in the epithelial cells but was activated upon transfection of LEF-1. Wild-typeXenopus Tcf-3 (XTcf-3) was unable to activateFN promoter reporter constructs, while a mutant lacking the groucho binding region behaved like LEF-1. In contrast to XTcf-3, LEF-1 does not interact with groucho proteins, which turn TCFs into activators or repressors (J. Roose, M. Molenaar, J. Hurenkamp, J. Peterson, H. Brantjes, P. Moerer, M. van de Wetering, O. Destreé, and H. Clevers, Nature 395:608–612, 1998). Together these data provide evidence that expressing LEF-1 enables fibroblasts, in contrast to epithelial cells, to respond to the Wnt/Wg signal via β-catenin in stimulating fibronectin gene transcription. Our findings further promote the idea that due to its dual function, β-catenin regulates the balance between cell-cell and cell-substrate adhesion.

A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice

Increased nuclear accumulation of β-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/β-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the β-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the β-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the β-catenin destruction complex, followed by increased degradation of β-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of β-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/β-catenin signaling through inhibiting the PARP domain of TNKS1/2.

Beta-arrestin is a necessary component of Wnt/beta-catenin signaling in vitro and in vivo.

The Wnt/β-catenin signaling pathway is crucial for proper embryonic development and tissue homeostasis. The phosphoprotein dishevelled (Dvl) is an integral part of Wnt signaling and has recently been shown to interact with the multifunctional scaffolding protein β-arrestin. Using Dvl deletion constructs, we found that β-arrestin binds a region N-terminal of the PDZ domain of Dvl, which contains casein kinase 1 (CK1) phosphorylation sites. Inhibition of Wnt signaling by CK1 inhibitors reduced the binding of β-arrestin to Dvl. Moreover, mouse embryonic fibroblasts lacking β-arrestins were able to phosphorylate LRP6 in response to Wnt-3a but decreased the activation of Dvl and blocked β-catenin signaling. In addition, we found that β-arrestin can bind axin and forms a trimeric complex with axin and Dvl. Furthermore, treatment of Xenopus laevis embryos with β-arrestin morpholinos reduced the activation of endogenous β-catenin, decreased the expression of the β-catenin target gene, Xnr3, and blocked axis duplication induced by X-Wnt-8, CK1ε, or DshΔDEP, but not by β-catenin. Thus, our results identify β-arrestin as a necessary component for Wnt/β-catenin signaling, linking Dvl and axin, and open a vast array of signaling avenues and possibilities for cross-talk with other β-arrestin-dependent signaling pathways.

Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

Canonical Wnt signaling is deregulated in several types of human cancer where it plays a central role in tumor cell growth and progression. Here we report the identification of 2 new small molecules that specifically inhibit canonical Wnt pathway at the level of the destruction complex. Specificity was verified in various cellular reporter systems, a Xenopus double-axis formation assay and a gene expression profile analysis. In human colorectal cancer (CRC) cells, the new compounds JW67 and JW74 rapidly reduced active β-catenin with a subsequent downregulation of Wnt target genes, including AXIN2, SP5, and NKD1. Notably, AXIN2 protein levels were strongly increased after compound exposure. Long-term treatment with JW74 inhibited the growth of tumor cells in both a mouse xenograft model of CRC and in Apc(Min) mice (multiple intestinal neoplasia, Min). Our findings rationalize further preclinical and clinical evaluation of these new compounds as novel modalities for cancer treatment.

Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1

Pontin (Tip49) and Reptin (Tip48) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. Here we show that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. We conclude that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. We propose a novel function of xPontin and xReptin as co-repressors in the c-Myc/Miz-1 pathway.

Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling.

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.

Identification of Two Regulatory Elements within the High Mobility Group Box Transcription Factor XTCF-4

Some members of the Wnt family of extracellular glycoproteins regulate target gene expression by inducing stabilization and nuclear accumulation of β-catenin, which functions as a transcriptional activator after binding to transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family. Three different members of this family have been identified in Xenopus laevis thus far that differ in their ability to influence mesodermal differentiation and to activate expression of the Wnt target gene fibronectin. Here we report on the isolation and characterization of additional variants of XTCF-4. We show that the differential ability of these proteins and other members of the TCF family to activate target genes is neither due to preferential interaction with transcriptional cofactors of the groucho family or SMAD4 nor to different DNA binding affinities. Expression of these proteins in an epithelial cell line reveals differences in their ability to form a ternary complex with DNA and β-catenin. Interestingly, formation of this ternary complex was not sufficient to activate target gene expression as previously thought. Our experiments identify two amino acid sequence motifs, LVPQ and SFLSS, in the central domain of XTCF-4 that regulate the formation of the DNA-TCF-β-catenin complex or activation of target genes, respectively. Biochemical studies reveal that the phosphorylation state of these XTCF-4 variants correlates with their ability to form a ternary complex with β-catenin and DNA but not to activate target gene expression. The described variants of XTFC-4 with their different properties in complex formation provide strong evidence that in addition to the regulation of β-catenin stability the isoforms of TCF/LEF transcription factors and their posttranslational modifications define the cellular response of a Wnt/wingless signal.

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from raster-scanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10-100 times higher fluorophore concentrations.

Keeping a close eye on Wnt-1/wg signaling in Xenopus.

Nearly two decades have past since description of the ®rst member of the Wnt family by Nusse and Varmus in 1982. Wnt-1 (at that time int-1) was shown to encode a secreted glycoprotein, and misexpression of Wnt-1 was found to promote mammary tumors in mice (Nusse and Varmus, 1982). Tremendous efforts have been undertaken to decipher the intracellular signaling events triggered by this family of extracellular glycoproteins, and now many of the principal mechanisms have been elucidated. Results from different experimental systems have contributed to our present understanding of the Wnt signaling cascade, but two organisms, Drosophila melanogaster and Xenopus laevis have played a critical role in this race. The observation that Wnt-1 is the mammalian homolog of the Drosophila segment polarity gene wingless (wg) (Rijsewijk et al., 1987) was the basis for the hypothesis that both, Wnt-1 and Wg, trigger the same signaling events. Xenopus laevis came into play as a result of the observation by McMahon and Moon (1989) that overexpressing Wnt-1 in ventral blastomeres of early Xenopus embryos elicits a duplication of the embryonic axis. This axis induction assay also showed the central role of b -catenin in Wnt-1/wg signaling (Funayama et al., 1995; Guger and Gumbiner, 1995). Even homologs of pathway genes in other species were found to be suf®cient to trigger axis duplication, e.g. Drosophila armadillo or dishevelled. In the last few years, this cross species axis duplication assay has been widely accepted to allocate newly identi®ed proteins to the Wnt-1/wg signaling cascade (for example see Fig. 2). The family of Wnt proteins is divided into two functional classes based on various activity assays. Only some members of the Wnt family, called Wnt-1/wg class, are able to induce the formation of a secondary axis when injected ventrally into a 4-cell stage Xenopus embryo (review by Moon and Kimelman, 1998). The same subset of Wnt members has been shown to transform C57mg cells (Wong et al., 1994). Both of these effects are thought to be mediated by the Wnt-1/wg signaling cascade which will be described later in this review. The Wnt-5A class of Wnts failed in both assays; i.e. Xwnt-5A, Xwnt-4 and Xwnt-11 do not possess axis inducing capacity, and Wnt-4 and Wnt5A do not transform C57mg cells. In addition, members of this class (Xwnt-4, -5A, -11) are able to antagonize the axis inducing effect of the Wnt-1/wg class in the Xenopus embryo, and Wnt-5A is able to reverse the transforming properties of Wnt-1 in C57 mg cells (Olson and Gibo, 1998). For these reasons, they are discussed as tumor suppressors. In Xenopus assays, the overexpression of the three Wnt-5A class members resulted in disruption of morphogenetic movements. In zebra®sh embryos, Xwnt5A has been shown to signal via intracellular release of calcium ions (Slusarski et al., 1997a,b). The effects of Xwnt-4, Xwnt-5A and Xwnt-11 on cell migration can be Mechanisms of Development 86 (1999) 3±15

Near-Field Optical Study of Protein Transport Kinetics at a Single Nuclear Pore

The kinetics of proteins passing through individual nuclear pore complexes (NPCs) of the nuclear envelope (NE) was studied using near-field scanning optical microscopy (NSOM) in combination with fluorescence correlation spectroscopy (FCS). The NSOM probe was placed over a single pore in an unsupported native NE to observe fluorescence-labeled NTF2 moving in the transport channel. A correlation analysis of the arising fluorescence fluctuations enabled us to characterize the translocation as driven by Brownian motion and to determine the related kinetic constants. Though trapped in the pore, NTF2 turned out to be highly mobile within a large axial extension. Our findings support the idea that molecules in transit interact with NPC proteins containing phenylalanine-glycine-repeat domains at the periphery of the channel. NSOM-FCS may help to understand the facilitated translocation in more detail and offers a new way to study single molecule mobility on a nanoscale.