Free Beta-catenin Research Articles

Role of Wnt signaling pathway in type 2 diabetes mellitus: An overview

The Wnt signaling pathway was discovered in 1982 by Nusse and Varmus during their breast cancer research. The key effect or of this pathway the bipartite transcription factor beta-catTCF is formed by free beta-catenin beta-cat and a TCF protein including TCF7L2. The Wnt signaling pathway regulates cell survival migration proliferation differentiation and organ development. Extensive recent investigations have shown that several key components of the Wnt signaling pathway are implicated in metabolic homeostasis and the development of type 2 diabetes mellitus. ThsWnt signaling is critical for glucagon like peptide-1 GLP-1 secretion by the intestinal endocrine L-cells. Thus alteration in this pathway could lead to reduced secretion of GLP-1 which in turn could have effect on insulin secretion. The GLP-1 in concert with insulin plays a critical role in blood glucose homeostasis. In this review we introduce background knowledge of Wnt signaling pathway and its role in type 2 diabetes mellitus.nbsp

The curry spice curcumin attenuates beta-amyloid-induced toxicity through beta-catenin and PI3K signaling in rat organotypic hippocampal slice culture

Objective: Accumulating evidence indicates that curcumin potently protects against beta-amyloid (Abeta) due to its oxygen free radicals scavenging and anti-inflammatory properties. However, cellular mechanisms that may underlie the neuroprotective effect of curcumin in Abeta-induced toxicity are not fully understood yet. The present study was undertaken to investigate the mechanisms involved in neuroprotective effects of curcumin, particularly involving Wnt/beta-catenin and PI3K pathways.Methods: Organotypic hippocampal slice cultures were treated with curcumin and exposed to Abeta1–42 for 48 hours. Synaptic dysfunction, cell death, ROS formation, neuroinflammation and beta-catenin, Akt, and GSK-3beta phosphorylation were measured to determine the effects of curcumin against Abeta toxicity.Results: Curcumin significantly attenuated Abeta-induced cell death, loss of synaptophysin, and ROS generation. Furthermore, curcumin was able to decrease IL-6 release and increase IL-10 release, and prevented glial activation. The phosphorylation of beta-catenin was avoided and the levels of free beta-catenin were increased by curcumin to promote cell survival upon treatment with Abeta. Curcumin, in the presence of Abeta, activated Akt which in turn phosphorylates GSK-3beta, and resulted in the inhibition of GSK-3beta. The presence of LY294002, an inhibitor of PI3K pathway, blocked the pro-survival effect of curcumin.Discussion: These results reinforce the neuroprotective effects of curcumin on Abeta toxicity and add some evidence that its mechanism may involve beta-catenin and PI3K signaling pathway in organotypic hippocampal slice culture.

Examination of effects of GSK3β phosphorylation, β-catenin phosphorylation, and β-catenin degradation on kinetics of Wnt signaling pathway using computational method

BackgroundRecent experiments have explored effects of activities of kinases other than the well-studied GSK3β, in wnt pathway signaling, particularly at the level of β-catenin. It has also been found that the kinase PKA attenuates β-catenin degradation. However, the effects of these kinases on the level and degradation of β-catenin and the resulting downstream transcription activity remain to be clarified. Furthermore, the effect of GSK3β phosphorylation on the β-catenin level has not been examined computationally. In the present study, the effects of phosphorylation of GSK3β and of phosphorylations and degradation of β-catenin on the kinetics of the wnt signaling pathway were examined computationally.MethodsThe well-known computational Lee-Heinrich kinetic model of the wnt pathway was modified to include these effects. The rate laws of reactions in the modified model were solved numerically to examine these effects on β-catenin level.ResultsThe computations showed that the β-catenin level is almost linearly proportional to the phosphorylation activity of GSK3β. The dependence of β-catenin level on the phosphorylation and degradation of free β-catenin and downstream TCF activity can be analyzed with an approximate, simple function of kinetic parameters for added reaction steps associated with effects examined, rationalizing the experimental results.ConclusionThe phosphorylations of β-catenin by kinases other than GSK3β involve free unphorphorylated β-catenin rather than GSK3β-phosphorylated β-catenin*. In order to account for the observed enhancement of TCF activity, the β-catenin dephosphorylation step is essential, and the kinetic parameters of β-catenin phosphorylation and degradation need to meet a condition described in the main text. These findings should be useful for future experiments.

Soy isoflavone genistein upregulates epithelial adhesion molecule E-cadherin expression and attenuates -catenin signaling in mammary epithelial cells

Breast cancer risk is highly modifiable by diet; however, mechanisms underlying dietary protection against mammary tumorigenesis remain poorly understood. A proportion of breast carcinomas is associated with deregulation of beta-catenin stability and amplification of c-Myc expression. We recently showed that dietary exposure to the soy isoflavone genistein (Gen) inhibited Wnt transduction in rat mammary epithelial cells in vivo. Here, we explored the role of Gen on cell adhesion protein, E-cadherin, expression to downregulate beta-catenin proto-oncogene function. In mammary glands of female rats exposed to dietary Gen, E-cadherin and beta-catenin protein levels were increased, concurrent with higher beta-casein gene expression. In HC11 mouse mammary epithelial cells, Gen diminished basal and Wnt-1-induced cell proliferation and attenuated Wnt-1 targets c-Myc and Cyclin D1 expression. Whereas, Gen had no effect on E-cadherin transcript levels, the abundance of membrane E-cadherin protein and of E-cadherin-beta-catenin adhesion complex was increased by Gen, attendant with downregulation of Wnt-1-induced free beta-catenin accumulation in cytosol. Gen inhibition of Wnt-induced c-Myc expression was mimicked by an estrogen receptor (ER)-beta-specific but not ER-alpha-specific agonist and was attenuated with loss of ER-beta expression, concordant with decreased E-cadherin expression. E-cadherin small-interfering RNA targeting eliminated Gen inhibition of Wnt-stimulated c-Myc expression and promoted Gen induction of basal c-Myc transcript levels and subsequent proliferation. Our studies identify E-cadherin as a Gen cellular target and demonstrate that the dichotomy in mammary epithelial response to Gen may be a function of cellular E-cadherin expression.

Lithium inhibits proliferation of human esophageal cancer cell line Eca-109 by inducing a G2/M cell cycle arrest

To investigate the effect of lithium on proliferation of esophageal cancer (EC) cells and its preliminary mechanisms. Eca-109 cells were treated with lithium chloride, a highly selective inhibitor of glycogen synthase kinase 3beta (GSK-3beta), at different concentrations (2-30 mmol/L) and time points (0, 2, 4, 6 and 24 h). Cell proliferative ability was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and cell cycle distribution was examined by flow cytometry. Expressions of p-GSK-3beta, beta-catenin, cyclin B1, cdc2 and cyclin D1 protein were detected by Western blotting, and the subcellular localization of beta-catenin was determined by immunofluorescence. The mRNA level of cyclin B1 was detected by reverse transcription polymerase chain reaction (RT-PCR). Lithium could inhibit the proliferation of Eca-109 cells. Lithium at a concentration of 20 mmol/L lithium for 24 h produced obvious changes in the distribution of cell cycle, and increased the number of cells in G(2)/M phase (P<0.05 vs control group). Western blotting showed that lithium inhibited GSK-3beta by Ser-9 phosphorylation and stabilized free beta-catenin in the cytoplasm. Immunofluorescence further confirmed that free beta-catenin actively translocated to the nucleus. Moreover, lithium slightly elevated cyclin D1 protein expression, whereas lowered the cyclin B1 expression after 24 h lithium exposure and no obvious change was observed for cdc2 protein. Lithium can inhibit the proliferation of human esophageal cancer cell line Eca-109 by inducing a G(2)/M cell cycle arrest, which is mainly mediated through the inhibition of lithium-sensitive molecule, GSK-3beta, and reduction of cyclin B1 expression.

Expression of Wnt-3a in Myeloma Cells Inhibits the Osteolytic Phenotype In-Vivo.

Wnt signaling is a highly conserved signal transduction pathway involved in embryonic development. Inappropriate canonical Wnt signaling resulting in beta-catenin stabilization, is associated with several types of human cancers. Multiple myeloma plasma cells express Wnt receptors, Wnt ligands and soluble Wnt inhibitors. Wnt signaling is central to osteoblast and osteoclasts development and secretion of Wnt signaling inhibitors by myeloma cells is thought to contribute to the osteolytic phenotype seen in this disease and prostate cancer. While it is now clear that MM cells can signal through both canonical and non-canonical mechanisms, there are conflicting data as to the direct role of Wnt signaling in myeloma cell biology. Others have shown that Wnts cause proliferation of myeloma cells; while we have shown that canonical Wnts cause morphological changes and migration, but not cell proliferation. To further elucidate the role of canonical Wnt signaling in myeloma and myeloma bone disease we used limiting dilutions in the presence of G418 to create two independent stable clones of the myeloma cell line NCI-H929 expressing Wnt-3A (H929/W3A), which is not expressed in myeloma, and an empty vector (H929/EV). Because Wnt antibodies are not available we cloned Wnt-3A as a fusion protein with hemagglutinin (HA). Western blots against HA revealed a positive band of the expected size only in the H929/W3A clones. GST-E-cadherin binding assay and Western blot analysis revealed elevated levels of total and free beta-catenin in H929/W3A relative to H929/EV, however, there this was not associated with increased growth or proliferation by MTT assay. To determine the in-vivo growth characteristics and effects on bone resorption of Wnt-3A producing cells, we transplanted the lines into a human bone implanted the flank of SCID mice. Tumor growth rate as determined by increased production of human immunoglobulin in mice serum was significantly slower in the Wnt-3A transfected cells relative to controls (P &lt; .05). Loss of bone mineral density (BMD) of the implanted bones engrafted with H929/W3A cells was lower than in bones engrafted with H929/EV cells (P &lt; .05). Reduced tumor burden and BMD loss was also visualized on x-ray radiographs. Taken together these data indicate that all factors promoting bone resorption produced by or elicited by the myeloma cell line H929 are subordinate to canonical Wnt signaling and that prevention of bone destruction may help control myeloma progression.

Matrilysin (MMP-7) degrades VE-cadherin and accelerates accumulation of beta-catenin in the nucleus of human umbilical vein endothelial cells

Matrilysin, MMP-7, is an important target for anti-metastasis therapy of colorectal cancer because it is a strong proteolytic factor secreted from the cancer cell itself and it induces tumor angiogenesis. In a previous report, we showed that matrilysin accelerated human umbilical vein endothelial cell (HUVEC) proliferation in low serum conditioned medium. In the present study, we show that matrilysin stimulation decreased VE-cadherin expression, induced accumulation of beta-catenin in the nucleus of the HUVEC, and up-regulated matrilysin mRNA expression. These results compel a hypothesis that matrilysin cleaves VE-cadherin and releases beta-catenin from the VE-cadherin/catenin complex; the free beta-catenin can activate T-cell factor (Tcf) DNA binding protein, which accelerates cell proliferation and matrilysin expression.

Mutations of the APC, beta-catenin, and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma

The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC, beta-catenin and Axin are mutated in some primary human cancers. Mutations in these genes can impair the down regulation of beta-catenin, which results in the stabilization of beta-catenin, accumulation of free beta-catenin and subsequent activation of the Wnt pathway. To clarify the genetic alterations of components of the Wnt pathway in oral squamous cell carcinoma (SCC), we examined mutations in the APC, beta-catenin and Axin genes and subcellular localization of beta-catenin. 20 oral SCC tissues and four cell lines derived from oral SCC were used. Mutational analysis was performed by a single-strand conformation polymorphism (SSCP) method and direct sequencing analysis. The samples were also examined by immunohistochemical staining and immunoblot analysis. In 3 of 4 cell lines, mutations were observed in the APC and Axin1 genes without amino acid substitutions. In a clinical sample, a mutation in the Axin1 gene was detected; a T insertion at codon 250 resulted in the formation of a stop codon at codon 259. In addition, cytoplasmic accumulation of beta-catenin was observed in 3 (75%) of 4 cell lines and 18 (90%) of 20 cancer tissue samples. The Axin1 gene may be one of the mutational target in oral SCC. In addition, the cytoplasmic accumulation of beta-catenin is a common characteristic of oral SCC, but is not closely associated with mutational alterations in the APC, beta-catenin and Axin1 genes.

Role of the intracellular domains of LRP5 and LRP6 in activating the Wnt canonical pathway

LDL-receptor related proteins 5 and 6 (LRP5/6) are co-receptors of Frizzled receptors that mediate Wnt-induced activation of the transcription factor family TCF/LEF-1. Even though LRP5 and LRP6 are highly homologous, LRP6, but not LRP5, is expressed primarily in the nervous system and deletion of the LRP6 gene results in significant brain abnormalities, while deletion of LRP5 results in primarily decreased bone density. Additionally, the exact function of LRP5 and LRP6 have not been clearly defined, although it is clear that they both play key roles in the Wnt canonical pathway. In this study the role of the intracellular domains of LRP5/6 in mediating Wnt signaling was examined. In the absence of exogenous Wnt 3a, full-length (FL) LRP6, but not LRP5, increased TCF/LEF-1 transcriptional activity, however both significantly potentiated Wnt 3a-induced TCF/LEF-1 activation. In contrast to the findings with the FL constructs, the intracellular domains (membrane-anchored and cytosolic) of both LRP5 and LRP6 significantly increased TCF/LEF-1 activation in the absence of Wnt 3a, and potentiated the Wnt 3a-induced decrease in beta-catenin phosphorylation, increase in free beta-catenin levels and the increase in TCF/LEF-1 activity. These findings demonstrate that: (1) LRP5 and LRP6 differentially modulate TCF/LEF-1 activation in the absence of Wnt 3a and (2) the intracellular C-terminal domains of LRP5/6 potentiate Wnt 3a-induced TCF/LEF-1 activation whether or not they are membrane-anchored. These findings provide significant new insights into the roles of LRP5/6 in modulating canonical Wnt signaling.

Cytoplasmic expression of E‐cadherin and β‐Catenin correlated with LOH and hypermethylation of the APC gene in oral squamous cell carcinomas

Inactivation of the adenomatous polyposis coli (APC) gene results in accumulation and translocation of beta-Catenin, which are important for malignant development. The aim of the present study is to investigate the possible role of APC/beta-Catenin pathway in oral squamous cell carcinomas. The DNA from 34 patients was examined for loss of heterozygosity (LOH) at two markers surrounding the APC, and for hypermethylation of the APC promoter by using methylation-specific polymerase chain reaction (MS-PCR). Fifteen of 34 samples were stained immunohistochemically to show the expression of E-cadherin and beta-Catenin. We found that cytoplasmic rather than membrane staining of E-cadherin and beta-Catenin was a prominent aberrant tumour-related alteration, and that this expression was mainly present in moderately and poorly differentiated tumours. LOH and hypermethylation of the APC promoter was found in four of 31 and five of 34 carcinoma samples, respectively. Four of five cases presenting LOH/hypermethylation showed cytoplasmic expression of E-cadherin and beta-Catenin by immunohistochemical (IHC) staining. The present results indicate that LOH at the APC locus or hypermethylation of the APC promoter 1a may lead to free beta-Catenin accumulation in cytoplasm of oral carcinoma cells and thereby to oral malignant progression.

Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer.

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

Requirement for a nuclear function of beta-catenin in Wnt signaling.

Wnt signaling stabilizes beta-catenin, which in turn influences the transcription of Wnt-responsive genes in conjunction with T-cell factor (TCF) transcription factors. At present, there are two models for the actions of beta-catenin. The conventional nuclear model suggests that beta-catenin acts in the nucleus to form a heterodimeric transcriptional factor complex with TCF, with TCF providing DNA-specific binding and the C and N termini of beta-catenin stimulating transcription. The alternative cytoplasmic model postulates that beta-catenin exports TCF from the nucleus to relieve its repressive activity or activates it in the cytoplasm. We have generated modified forms of beta-catenin and used RNA interference against endogenous beta-catenin to distinguish between these models in cultured mammalian and Drosophila cells. We show that the VP16 transcriptional activation domain can replace the C terminus of beta-catenin without loss of function and that the function of beta-catenin is compromised by fusion to a transcriptional repressor domain from histone deacetylase, favoring the direct effects of beta-catenin in the nucleus. Furthermore, membrane-tethered beta-catenin requires interaction with the adenomatous polyposis coli protein but not with TCF for its function, whereas untethered beta-catenin requires binding to TCF for its signaling activity. Importantly, by using RNA interference, we show that the signaling activity of membrane-tethered beta-catenin, but not free beta-catenin, requires the presence of endogenous beta-catenin, which is able to accumulate in the nucleus when stabilized by the binding of the beta-catenin degradation machinery to the membrane-tethered form. All of these data support a nuclear model for the normal function of beta-catenin.

Beta-Catenin is expressed aberrantly in tumors expressing shadow cells. Pilomatricoma, craniopharyngioma, and calcifying odontogenic cyst.

We studied the beta-catenin immunohistochemical profile in tumors expressing shadow cells: pilomatricoma, 10 cases; calcifying odontogenic cyst, 6 cases; and craniopharyngioma, 9 cases. There was strong membranous, cytoplasmic, and nuclear staining of the immature basaloid cells in all of these tumors. Shadow cells were negative in all tumors. It has been documented that rising levels of free beta-catenin drive the formation of complexes with T-cell factor/lymphoid enhancer factor (TCF-Lef) and up-regulate the wingless-Wnt cell-cell signals. The end result is an abnormality of beta-catenin degradation and, thus, a buildup of free beta-catenin in the cytoplasm and/or nucleus, resulting in the stimulation of cellular proliferation and/or inhibition of cell death. beta-Catenin seems to have an important role in the oncogenesis of these tumors. The similar pattern of keratinization in these tumors and the similar pattern of beta-catenin immunoreactivity in the cytoplasm and the nucleus are important findings. It seems that the activation of a common cellular pathway, namely Wnt-beta-catenin-TCF-Lef, has a role in the pathogenesis of these tumors. The latter could be related to their shared method of keratinization or shared dysfunction of the cellular adhesion complex leading to tumorigenesis.

Adenomatous Polyposis Coli (APC)-independent Regulation of β-Catenin Degradation via a Retinoid X Receptor-mediated Pathway

Beta-catenin is a component of stable cell adherent complexes whereas its free form functions as a transcription factor that regulate genes involved in oncogenesis and metastasis. Free beta-catenin is eliminated by two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways regulated by glycogen synthase kinase 3beta (GSK3 beta) or p53-inducible Siah-1. Dysregulation of beta-catenin turnover consequent to mutations in critical genes of the APC-dependent pathways is implicated in cancers such as colorectal cancer. We have identified a novel retinoid X receptor (RXR)-mediated APC-independent pathway in the regulation of beta-catenin. In this proteasomal pathway, RXR agonists induce degradation of beta-catenin and RXR alpha and repress beta-catenin-mediated transcription. In vivo, beta-catenin interacts with RXR alpha in the absence of ligand, but RXR agonists enhanced the interaction. RXR agonist action was not impaired by GSK3 beta inhibitors or deletion of the GSK3 beta-targeted sequence from beta-catenin. In APC- and p53-mutated colorectal cancer cells, RXR agonists still inactivated endogenous beta-catenin via RXR alpha. Interestingly, deletion of the RXR alpha A/B region abolished ligand-induced beta-catenin degradation but not RXR alpha-mediated transactivation. RXR alpha-mediated inactivation of oncogenic beta-catenin paralleled a reduction in cell proliferation. These results suggest a potential role for RXR and its agonists in the regulation of beta-catenin turnover and related biological events.

Transcriptional Activation of the Proglucagon Gene by Lithium and β-Catenin in Intestinal Endocrine L Cells

The proglucagon gene encodes several peptide hormones that regulate blood glucose homeostasis, growth of the small intestine, and satiety. Among them, glucagon-like peptide 1 (GLP-1) lowers blood glucose levels in patients with diabetes and inhibits eating and drinking in fasted rats. Although proglucagon transcription and GLP-1 synthesis were shown to be activated by forskolin and other protein kinase A (PKA) activators, deleting or mutating the cAMP-response element (CRE) only moderately attenuates the proglucagon gene promoter in response to PKA activation. Therefore, PKA may activate proglucagon transcription via a mechanism independent of the CRE motif. Recently, PKA was shown to phosphorylate and inactivate GSK-3beta, a key mediator in the Wnt signaling pathway. We show here that lithium, an inhibitor of GSK-3beta, activates proglucagon gene transcription and stimulates GLP-1 synthesis in an intestinal endocrine L cell line, GLUTag. The activation was also observed in primary fetal rat intestinal cell (FRIC) cultures, but not in a pancreatic A cell line. Co-transfection of beta-catenin, a downstream effector of GSK-3beta activities, activated the proglucagon gene promoter without a CRE. Furthermore, forskolin and 8-Br-cAMP phosphorylated GSK-3beta at serine 9 in intestinal proglucagon-producing cells, and both lithium and forskolin induced the accumulation of free beta-catenin in these cell lines. These observations indicate that the proglucagon gene is among the targets of the Wnt signaling pathway.

Expression analysis of pediatric solid tumor cell lines using oligonucleotide microarrays

We identified patterns of differentially-expressed genes in cell lines derived from several pediatric solid tumors. Affymetrix Human Cancer G110 Arrays, carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell lines originating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of soft parts. Hierarchical clustering clearly differentiated these 3 entities and revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific up-regulation in these cell lines, respectively. Whereas ET lines demonstrated increased expression of microtubule-associated protein tau (MAPT), protein phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples exhibited high expression of wingless-type mouse mammary tumor virus integration site family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and adenomatous polyposis coli (APC) which are involved in regulating free beta-catenin levels. These genes likely maintain tumor-specific characteristics and participate in key downstream regulatory mechanisms. We also correlated the expression levels of up-regulated genes in ETs with their chromosomal localization and compared these data to the comparative genomic hybridization profiles of the cell lines. We demonstrate that gains of genetic material contribute essentially to differential gene expression.

Identification of a Wnt/beta-catenin signaling pathway in human thyroid cells.

beta-Catenin is a structural component of the adherens junctions. Outside the adherens junctions a complex consisting of glycogen synthase kinase 3beta, the tumor suppressor adenomatous polyposis coli, and axin constantly targets beta-Catenin for degradation to keep levels of free beta-Catenin low. Free beta-Catenin is able to bind to transcription factors of the T cell factor/lymphoid-enhancing factor family and to stimulate transcription of target genes. This signaling function of beta-Catenin is activated by extracellular Wnt factors that bind to Frizzled receptors and induce inhibition of beta-Catenin degradation. By RT-PCR and subcloning, we observed the expression of five Wnt factors, three members of the Frizzled receptor family, and all known Disheveled isoforms in thyroid cells. Immunoprecipitation studies demonstrated the formation of the complex targeting beta-Catenin for degradation. Introduction of a degradation resistant beta-Catenin into the thyroid carcinoma cell line WRO induced appearance of monomeric beta-Catenin as shown by size fractionation and nuclear beta-Catenin immunostaining. Reporter gene assays demonstrated a stimulation of T cell factor/lymphoid-enhancing factor-mediated transcription in these cells. In ARO cells, a thyroid carcinoma cell line carrying a mutated adenomatous polyposis coli gene, monomeric beta-Catenin and nuclear immunostaining were observed. In summary, our data indicate that elements of the Wnt signaling pathway are expressed in thyroid cells and that this pathway is functionally active.

Id2 Is a Target of the β-Catenin/T Cell Factor Pathway in Colon Carcinoma

Activation of beta-catenin/T cell factor (TCF) transcription as a result of mutations in the adenomatous polyposis coli (APC) and/or beta-catenin genes occurs in the majority of colon tumors. An increasing number of genes, including c-myc and cyclin D1, have been implicated as targets of this pathway. We now report that the dominant negative helix-loop-helix regulator Id2 is also a target of the beta-catenin/TCF transcription pathway in colon adenocarcinoma. Investigation of the mechanism for the overexpression of Id2 in colon carcinoma cells demonstrated that the Id2 promoter is activated, and the Id2 protein is up-regulated by beta-catenin. Conversely, reducing free beta-catenin blocked this induction of promoter activity. We have also used an electrophoretic mobility shift assay and supershift to identify a motif in the Id2 promoter that binds to TCF4 protein. Site-directed mutagenesis of this motif abolished promoter reporter activity. Both transfection of Id2 into SW480 cells and induction of Id2 in HT29 colon cells was found to increase anchorage-independent survival of these cells. Growing evidence associates disruption to Id2 expression with tumorigenesis, and our findings suggest that this dysregulation of Id2 expression is due to the activation of the beta-catenin/TCF pathway.

Differential Molecular Assemblies Underlie the Dual Function of Axin in Modulating the Wnt and JNK Pathways

Axin is a multidomain scaffold protein that exerts a dual function in the Wnt signaling and MEKK1/JNK pathways. This raises a critical question as to whether Axin-based differential molecular assemblies exist and how these may act to coordinate the two separate pathways. Here we show that both wild-type glycogen synthase kinase-3 beta (GSK-3 beta) and kinase-dead GSK-3 beta-Y216F (capable of binding to Axin), but not GSK-3 beta-K85M (incapable of binding to Axin in mammalian cells), prevented MEKK1 binding to the Axin complex, thereby inhibiting JNK activation. We further show that casein kinase I epsilon also inhibited Axin-mediated JNK activation by competing against MEKK1 binding. In contrast, beta-catenin and adenomatous polyposis coli binding did not affect MEKK1 binding to the same Axin complex. This suggests that even when Axin is "switched" to activate the JNK pathway, it is still capable of sequestering free beta-catenin, which is a critical aspect for cellular homeostasis. Our results clearly demonstrate that differential molecular assemblies underlie the duality of Axin functions in the negative regulation of Wnt signaling and activation of the JNK MAPK pathway.

Akt Participation in the Wnt Signaling Pathway through Dishevelled

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.