Negative Regulator Of Wnt Signaling Research Articles

Methylation by protein arginine methyltransferase 1 increases stability of Axin, a negative regulator of Wnt signaling

Axin, a negative regulator of Wnt signaling, is a key scaffold protein for the β-catenin destruction complex. It has been previously shown that multiple post-translational modification enzymes regulate the level of Axin. Here, we provide evidence that protein arginine methyltransferase 1 (PRMT1) directly interacts with and methylates the 378th arginine residue of Axin both in vitro and in vivo. We found that the transient expression of PRMT1 led to an increased level of Axin and that knockdown of endogenous PRMT1 by short hairpin RNA reduced the level of Axin. These results suggest that methylation by PRMT1 enhanced the stability of Axin. Methylation of Axin by PRMT1 also seemingly enhanced the interaction between Axin and glycogen synthase kinase 3β, leading to decreased ubiquitination of Axin. Consistent with the role of PRMT1 in the regulation of Axin, knockdown of PRMT1 enhanced the level of cytoplasmic β-catenin as well as β-catenin-dependent transcription activity. In summary, we show that the methylation of Axin occurred in vivo and controlled the stability of Axin. Therefore, methylation of Axin by PRMT1 may serve as a finely tuned regulation mechanism for Wnt/β-catenin signaling.

The Protein Stability of Axin, a Negative Regulator of Wnt Signaling, Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys(505) of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.

P112. MEST/Peg1 inhibits Wnt signaling via regulating maturation of LRP6

Wnt/β-catenin signaling plays negative roles in adipogenesis. Previously it was shown that the expressions of SFRP5 and Naked1, which are negative regulators of Wnt signaling, along with MEST/Peg1 were increased in diet-induced obesity mice. MEST/Peg1 is an imprinted gene and plays important roles in embryo development while its biochemical role was not determined yet. We tested if MEST/Peg1 has any roles in the regulation of Wnt signaling and found that ectopic expression of MEST/Peg1 inhibited Wnt-mediated reporter activity via enhancing ubiquitination of β-catenin. Most interestingly, a Wnt co-receptor LRP6 co-localized with MEST/Peg1 in ER and the maturation and membrane localization of LRP6, which are necessary for the signal transduction of Wnt, were blocked by the ectopic expression of MEST/Peg1. MEST/Peg1 inhibited maturation of LRP6 via controlling glycosylation on LRP6. Knockdown of MEST/Peg1, which might enhance Wnt signaling, blocked adipogenic differentiation of 3T3-L1 cells. Overall, our data suggest that MEST/Peg1 is a novel regulator of Wnt/β-catenin signaling during adipogenic differentiation. Supported by the National Research Foundation of Korea (2009-0076118) and Brain Korea 21 program.

P132. Methylation by protein arginine methyltransferase1 augments stability of Axin

Axin, a negative regulator of Wnt signaling, is a key scaffold protein for the β-catenin destruction complex. It has been previously shown that multiple post-translational modification enzymes regulate the level of Axin. Here, we provide evidence that protein arginine methyltransferase1 (PRMT1) directly interacts with and methylates the 378th arginine residue of Axin both in vitro and in vivo. We found that the transient expression of PRMT1 led to an increased level of Axin and that knockdown of endogenous PRMT1 by shRNA reduced the level of Axin. These results suggest that methylation by PRMT1 enhanced the stability of Axin. Methylation of Axin by PRMT1 also seemingly enhanced the interaction between Axin and GSK3β, leading to decreased ubiquitination of Axin. Consistent with the role of PRMT1 in the regulation of Axin, knockdown of PRMT1 enhanced the level of cytoplasmic β-catenin as well as β-catenin-dependent transcription activity. In summary, we show that the methylation of Axin occurred in vivo and controlled the stability of Axin. Therefore, methylation of Axin by PRMT1 may serve as a finely-tuned regulation mechanism for Wnt/β-catenin signaling.

MiR-29 Modulates Wnt Signaling in Human Osteoblasts through a Positive Feedback Loop

Differentiation of human mesenchymal stem cells into osteoblasts is controlled by extracellular cues. Canonical Wnt signaling is particularly important for maintenance of bone mass in humans. Post-transcriptional regulation of gene expression, mediated by microRNAs, plays an essential role in the control of osteoblast differentiation. Here, we find that miR-29a is necessary for human osteoblast differentiation, and miR-29a is increased during differentiation in the mesenchymal precursor cell line hFOB1.19 and in primary cultures of human osteoblasts. Furthermore, the promoter of the expressed sequence tag containing the human miR-29a gene is induced by canonical Wnt signaling. This effect is mediated, at least in part, by two T-cell factor/LEF-binding sites within the proximal promoter. Furthermore, we show that the negative regulators of Wnt signaling, Dikkopf-1 (Dkk1), Kremen2, and secreted frizzled related protein 2 (sFRP2), are direct targets of miR-29a. Endogenous protein levels for these Wnt antagonists are increased in cells transfected with synthetic miR-29a inhibitor. In contrast, transfection with miR-29a mimic decreases expression of these antagonists and potentiates Wnt signaling. Overall, we demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that can modulate osteoblast differentiation. Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down-regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a potentiates Wnt signaling, contributing to a gene expression program important for osteoblast differentiation. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during osteoblast differentiation, allowing for fine-tuning of intricate cellular processes.

Abstract 173: Frequent concomitant epigenetic silencing of the SFRPs and SOX1, Wnt/β-catenin regulators, in human hepatocellular carcinoma

Abstract Oncogenic activation of the Wnt/β-catenin signaling pathway is common in hepatocellular carcinoma (HCC). The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications for carcinogenesis. Recently, we have shown that SFRPs are frequently downregulated through promoter hypermethylation in HCC cell lines and clinical HCC tissues. Furthermore, we have demonstrated that restoration of SFRPs could attenuate Wnt signaling in HCC cells with β-catenin point mutation, decrease abnormal accumulation of free β-catenin in the nucleus, and then suppress cell growth. Sox1 encodes a transcription factor involved in the regulation of embryonic development and cell fate determination. Structurally related to TCF/LEFs, several members of the Sox family, including Sox17, Sox3, Sox7, and Sox9, have also been implicated in repressing β-catenin activity by a mechanism that is not well understood. In this study, we found that the Sox1 was severely down-regulated by quantitative reverse transcription polymerase chain reaction (QRT-PCR). Next, we employed bisulfite modification and methylation-specific PCR (MS-PCR) to analyze the feature of Sox1 promoter methylation in the HCC cell lines and found that the majority of the tested cell lines exhibited hypermethylation. Meanwhile, we also examined sixty primary HCC surgical samples and adjacent nonturmorous liver tissues from Taiwan Liver Cancer Network (TLCN). These results showed that aberrant promoter methylation of SOX1 were found in 32 of 60 HCCs (53.3%), in 13 of 50 cirrhotic livers (26%), and in one of 30 normal control tissues (3.3%). Concomitantly, SFRPs was epigenetically inactivated in HCC cell lines and most of all the tumors with methylated SOX1. A significant correlation between the methylation of both genes was found (p >0.05). Furthermore, overexpression of SOX-1 in HCC cells could suppress TCF-dependent transcriptional activity and suppress colony formation. Taken together, our data suggest that SOX1 is a negative modulator of Wnt/β-catenin signaling, and that SOX1 silencing due to promoter hypermethylation is an early event during tumorigenesis and may contribute to aberrant activation of Wnt signaling in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 173.

Negative regulation of Wnt signaling mediated by CK1‐phosphorylated DishevelledviaRor2

Dishevelled (Dvl) is a multifunctional effector of different Wnt cascades. Both canonical Wnt3a and noncanonical Wnt5a stimulate casein-kinase-1 (CK1) -mediated phosphorylation of Dvl, visualized as electrophoretic mobility shift [phosphorylated and shifted Dvl (ps-Dvl)]. However, the role of this phosphorylation remains obscure. Here we report the functional interaction of ps-Dvl with the receptor tyrosine kinase Ror2, which is an alternative Wnt receptor and is able to inhibit canonical Wnt signaling. We demonstrate interaction between Ror2 and ps-Dvl at the cell membrane after Wnt3a or Wnt5a stimulus dependent on CK1. Ps-Dvl interacts with the C-terminal proline-serine-threonine-rich domain of Ror2, which is required for efficient inhibition of canonical Wnt signaling. We further show that the Dvl C terminus, which seems to be exposed in ps-Dvl and efficiently binds Ror2, is an intrinsic negative regulator of the canonical Wnt pathway downstream of beta-catenin. The Dvl C terminus is necessary and sufficient to inhibit canonical Wnt/beta-catenin signaling, which is dependent on the presence of Ror2. Furthermore, both the Dvl C terminus and CK1epsilon can inhibit the Wnt5a/Ror2/ATF2 pathway in mammalian cells and Xenopus explant cultures. This suggests that phosphorylation of Dvl triggers negative feedback regulation for different branches of Wnt signaling in a Ror2-dependent manner.

MAP3K1 functionally interacts with Axin1 in the canonical Wnt signalling pathway

A central point of regulation in the Wnt/beta-catenin signalling pathway is the formation of the beta-catenin destruction complex. Axin1, an essential negative regulator of Wnt signalling, serves as a scaffold within this complex and is critical for rapid turnover of beta-catenin. To examine the mechanism by which Wnt signalling disables the destruction complex, we used an immunoprecipitation-coupled proteomics approach to identify novel endogenous binding partners of Axin1. We found mitogen-activated protein kinase kinase kinase 1 (MAP3K1) as an Axin1 interactor in Ls174T colorectal cancer (CRC) cells. Importantly, confirmation of this interaction in HEK293T cells indicated that the Axin1-MAP3K1 interaction is induced and modulated by Wnt stimulation. siRNA depletion of MAP3K1 specifically abrogated TCF/LEF-driven transcription and Wnt3A-driven endogenous gene expression in both HEK293T as well as DLD-1 CRC. Expression of ubiquitin ligase mutants of MAP3K1 abrogated TCF/LEF transcription, whereas kinase mutants had no effect in TCF-driven activity, highlighting the essential role of the MAP3K1 E3 ubiquitin ligase activity in regulation of the Wnt/beta-catenin pathway. These results suggest that MAP3K1, previously reported as an Axin1 inter-actor in c-Jun NH(2)-terminal kinase pathway, is also involved in the canonical Wnt signalling pathway and positively regulates expression of Wnt target genes.

Apical constriction and invagination downstream of the canonical Wnt signaling pathway require Rho1 and Myosin II

The tumor suppressor Adenomatous polyposis coli (APC) is a negative regulator of Wnt signaling and functions in cytoskeletal organization. Disruption of human APC in colonic epithelia initiates benign polyps that progress to carcinoma following additional mutations. The early events of polyposis are poorly understood, as is the role of canonical Wnt signaling in normal epithelial architecture and morphogenesis. To determine the consequences of complete loss of APC in a model epithelium, we generated APC2 APC1 double null clones in the Drosophila wing imaginal disc. APC loss leads to segregation, apical constriction, and invagination that result from transcriptional activation of canonical Wnt signaling. Further, we show that Wnt-dependent changes in cell fate can be decoupled from Wnt-dependent changes in cell shape. Wnt activation is reported to upregulate DE-cadherin in wing discs, and elevated DE-cadherin is thought to promote apical constriction. We find that apical constriction and invagination of APC null tissue are independent of DE-cadherin elevation, but are dependent on Myosin II activity. Further, we show that disruption of Rho1 suppresses apical constriction and invagination in APC null cells. Our data suggest a novel link between canonical Wnt signaling and epithelial structure that requires activation of the Rho1 pathway and Myosin II.

High Expression of Pitx-2 in the ICAT-deficient Metanephros Leads to Developmental Arrest

ICAT (Inhibitor of β-catenin and T cell factor) inhibits the interaction between β-catenin and TCF/LEF transcription factor and serves as a negative regulator of Wnt signaling. In a subset of ICAT knockout mice, significant delay in the ureteric bud branching and renal agenesis are observed. In order to examine the process of this developmental defect, molecular changes were analyzed in fetal ICAT–/– kidneys with a focus on Wnt-signaling associated factors. The protein level of active β-catenin was elevated in ICAT–/– kidneys. DNA microarray and immunohistochemical analyses revealed that the expression of a Wnt target gene Pitx-2 was enhanced in ICAT–/– kidneys. There was no genotypic difference in the expression level of another Wnt target gene, c-Ret. These results suggest that the enhancement of Pitx-2 expression induced by activated Wnt signaling leads to delays in ureteric bud branching and subsequent renal agenesis. In the ICAT–/– kidneys which developed to E18.5 without any apparent defect, renal glomeruli, convoluted tubules and collecting ducts were decreased in density and showed abnormal structure. ICAT may be required for various developmental stages during renal development.

The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway

AbstractCOVER PHOTOGRAPH: Signaling by the Wingless morphogen (a member of the Wnt family of secreted glycoproteins) is required for the proper development of appendages in Drosophila. Overexpression of Axin, a negative regulator of Wnt signaling, leads to severe defects in Drosophila wing growth and patterning (the affected wing is shown in a green pseudocolor, compared to a wild‐type wing in pink). The inhibitory activity of Axin can be suppressed by co‐expression of the GTP‐binding protein Gαo, leading to a partial rescue in wing development (yellow). Gαo physically binds the RGS domain of Axin and recruits Axin it to the plasma membrane, antagonizing the Axin‐based destruction complex. From Egger‐Adam and Katanaev, Developmental Dynamics 239:168–183, 2010. © 2009 Wiley‐Liss, Inc.

CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.

A critical role for sFRP proteins in maintaining caudal neural tube closure in mice via inhibition of BMP signaling

Both the BMP and Wnt pathways have been implicated in directing aspects of dorsal neural tube closure and cell fate specification. However, the mechanisms that control the diverse responses to these signals are poorly understood. In this study, we provide genetic and functional evidence that the secreted sFRP1 and sFRP2 proteins, which have been primarily implicated as negative regulators of Wnt signaling, can also antagonize BMP signaling in the caudal neural tube and that this function is critical to maintain proper neural tube closure and dorsal cell fate segregation. Our studies thus reveal a novel role for specific sFRP proteins in balancing the response of cells to two critical extracellular signaling pathways.

Vascular Progenitor Cells in Diabetes Mellitus

See related article, pages 1095–1102 Over the centuries, diabetes mellitus–associated ischemic ulcers have driven physicians to continue to improve their skills in healing the ulcers to prevent devastating complications.1 Unfortunately, extremity amputation still remains the outcome in many cases. The recent development of vascular progenitor cell (PC) transplantation aimed at enhancing angiogenesis and wound healing may, however, provide new hope in the treatment of this old ailment.2,3 In this issue of Circulation Research , Barcelos et al show in a murine diabetes mellitus model that topical transplantation of human fetal CD133+ PCs significantly accelerates the healing of skin wounds on ischemic hind limbs.4 Their data suggest that the reparative angiogenesis is mediated by the wingless (Wnt) signaling pathway. The prominin-1 (PROM1)/CD133 gene encodes a pentaspan transmembrane glycoprotein expressed in a variety of stem cells, including hematopoietic stem cells, endothelial PCs (EPCs), and neuronal/glial stem cells.5,6 By suppressing further differentiation, PROM1 helps these cells maintain their stem cell properties. Mutations in PROM1 may result in retinitis pigmentosa,7 and its overexpression is also associated with cancer formation.8 In view of this cancer potential, overstimulation of CD133+ cells may also lead to unwanted consequences. Additionally, that human fetal CD133+ cells are not easily accessible …

P002 Epidemiological characteristics of MDS in Poland in 2008

The CXXC domain protein 4 (CXXC4) functions as a negative regulator of Wnt signaling and also regulates expression of the ten-eleven translocation 2 (TET2) protein for DNA methylation. This study detected levels of CXXC4 and TET2 mRNA to determine their association with survival of patients with myelodysplastic syndrome (MDS).Levels of TET2 and CXXC4 mRNA were analyzed in bone marrow samples from 154 MDS patients and 50 control subjects using qRT-PCR and subsequently associated these levels with clinicopathological characteristics and survival of MDS patients.Levels of TET2 and CXXC4 mRNA were significantly lower in MDS patients than that in controls (P = 0.009 and P < 0.001, respectively). Patients with advanced WHO subtypes (e.g., RAEB-1 and RAEB-2) exhibited a higher level of CXXC4 mRNA (P = 0.020) compared to those with early stage subtypes (i.e., RA, RARS, RCMD, RCMD-RS, 5q-syndrome, and MDS-U). Moreover, levels of CXXC4 mRNA were associated with marrow blast levels (P = 0.014) and neutrophil counts (P = 0.039). Levels of CXXC4 mRNA and hemoglobin and IPSS cytopenias were associated with the overall survival (P = 0.025) but not with the leukemia-free survival of MDS patients. The multivariate analysis demonstrated that the age of patients and levels of hemoglobin and marrow blast were independent risk factors for survival of MDS patients.This study demonstrated that the age of patients and levels of CXXC4 mRNA, hemoglobin, and marrow blast associated with survival of MDS patients.

Pegylated interferon‐alpha targets beta‐catenin by inducing its nuclear export

Treatment with Peg‐IFN, a first line therapy for chronic HCV infection, reduces the development of hepatocellular carcinoma (HCC). Furthermore, activation of the Wnt pathway is observed in a significant subset of HCC. We explored the possibility that Peg‐IFN may act through inhibition of Wnt signaling. To examine Peg‐IFN's effects in vivo, mice overexpressing β‐catenin were exposed to DEN and treated with Peg‐IFN (70,000 U) for six weeks. Treatment with Peg‐IFN decreased nuclear β‐catenin compared to saline treated and decreased Ki‐67 positive hepatocytes. No change in the transcription of β‐catenin was evident suggesting the effect to be through a post‐translational mechanism. We began in vitro studies to verify this finding and explore the mechanism of reduced Wnt signaling. Treatment of human hepatoma cells (HepG2 &amp; Hep3B) with Peg‐IFN led to a decrease in the active form of β‐catenin. Likewise, β‐catenin dependent transcriptional activity was decreased by up to 50% as measured by TOPflash luciferase reporter assay. Real‐time PCR showed an increase in two negative regulators of Wnt signaling, Dkk1 and RanBP3. Overexpression of RanBP3 in HepG2 cells significantly decreased β‐catenin transcriptional activity. Thus Peg‐IFN effectively downregulates Wnt/β‐catenin signaling at least partly through increased expression of RanBP3, which is an attractive means of therapeutically inhibiting mutated‐β ‐catenin.

Characterization of the Structural Features and Interactions of Sclerostin

The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.

Axin localizes to mitotic spindles and centrosomes in mitotic cells

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3β) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3β in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.

The structural and functional determinants of the Axin and Dishevelled DIX domains

BackgroundThe dishevelled and axin genes encode multi-domain proteins that play key roles in WNT signalling. Dishevelled prevents β-catenin degradation by interfering with the interaction of β-catenin with the degradation-mediating Axin-APC-GSK3β complex. This interference leads to an accumulation of cytoplasmic β-catenin, which enters the nucleus and interacts with transcription factors that induce expression of Wnt-target genes. Axin, as a component of the degradation-mediating complex, is a potent negative regulator of Wnt signalling, whereas Dishevelled is a potent activator. Both Dishevelled and Axin possess a DIX (Dishevelled/Axin) domain, which mediates protein-protein interactions, specifically homodimerization.ResultsAn evolutionary trace analysis of DIX domains identified conserved residues which, when mapped onto the crystal structure of the Axin DIX domain and a comparative model of the Dishevelled DIX domain, allow their categorization as residues of either structural or functional importance. We identify residues that are structural and functional determinants of the DIX domain fold, as well as those that are specific to homodimerization of Axin and Dishevelled.ConclusionThis report provides the first explanation of the mutant phenotypes caused by non-synonymous substitutions in the Dishevelled and Axin DIX domain by correlating their presumed functional significance with molecular structure.

Dickkopf-4 is frequently down-regulated and inhibits growth of colorectal cancer cells

Like Dickkopf-1 (DKK1), DKK4 is a target of β-catenin/Tcf-4 in colorectal cancer. However, as a negative regulator of Wnt signalling its function in colorectal cancer cells is not well understood. We report that DKK4 is frequently down-regulated in colorectal cancer cell lines with deregulated β-catenin/Tcf-4 and in primary colorectal cancers. Exposure of cancer cells to DKK4 strongly inhibits basal β-catenin/Tcf-4 signalling activity, cancer cell growth and cell cycle progression. Therefore, loss of this negative feed-back loop provides Wnt factor expressing cancer cells with a growth advantage. Our data demonstrate that DKK4 is an important negative regulator of colon cancer cell growth.