Oligomeric assemblies of Amyloid-β (Aβ) are suggested to be central in the pathogenesis of Alzheimer’s disease, since levels of soluble Aβ much better correlate with the extent of cognitive dysfunctions than senile plaque counts do. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit maintenance of hippocampal long term potentiation. The tg-ArcSwe model, transgenic mice with the Arctic and Swedish Alzheimer mutations, expresses elevated levels of Aβ protofibrils in the brain, making tg-ArcSwe a highly suitable model to investigate the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg-ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg-ArcSwe mice, as compared to several transgenic models lacking the Arctic mutation. In aged tg-ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50 percent higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg-ArcSwe mice showed deficits in spatial learning and individual performance in Morris water maze correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age-dependent manner in tg-ArcSwe mice, although to a far less extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.
Alzheimer’s disease; amyloid-β protofibrils; Arctic mutation; transgenic mice; spatial learning
The Wnt/β-catenin signaling pathway controls cellular proliferation in the intestines. In response to Wnt, β-catenin transits into the nucleus and associates with members of the T-cell factor (TCF) family of transcription factors. β-Catenin/TCF complexes bind Wnt responsive DNA elements (WREs) to activate target gene expression. The c-MYC proto-oncogene (MYC) is a direct target of β-catenin/TCF complexes. We recently identified the MYC 3′ WRE, which maps 1.4-kb downstream from the MYC transcription stop site. To investigate the role of the Myc 3′ WRE in the intestines, we generated a mouse model with a germ line deletion of this element. The intestinal architecture was largely preserved in knockout mice; however, removal of the Myc 3′ WRE compromised the crypt microenvironment. In comparison to wild-type intestines, knockout intestines contained an increased number of proliferative cells and a reduced number of differentiated cells comprising both absorptive and secretory lineages. Using a model of colitis, we found that knockout colons repaired more rapidly during the recovery period of the protocol. These results indicate that regulation of MYC expression through the Myc 3′ WRE contributes to intestinal homeostasis. Furthermore, our study implicates MYC as an important regulator of intestinal regeneration following injury.
The Wnt pathway effector gene TCF7L2 has been linked to type II diabetes, making it important to study the role of Wnt signaling in diabetes pathogenesis. We examined the expression of multiple Wnt pathway components in pancreases from normal individuals and type II diabetic individuals. Multiple members of the Wnt signaling pathway, including TCF7L2, Wnt2b, β-catenin, pGSK3β, TCF3, cyclinD1, and c-myc, were undetectable or expressed at low levels in islets from nondiabetic individuals, but were also upregulated specifically in islets of type II diabetic patients. Culture of pancreatic tissue and islet isolation led to Wnt activation that was reversed by the Wnt antagonist sFRP, demonstrating that Wnt activation in that setting was due to soluble Wnt factors. These data support a model in which the Wnt pathway plays a dynamic role in the pathogenesis of type II diabetes and suggest manipulation of Wnt signaling as a new approach to β-cell-directed diabetes therapy.
Inappropriate activation of c-Myc (MYC) gene expression by the Wnt/ß-catenin signaling pathway is required for colorectal carcinogenesis. The elevated MYC levels in colon cancer cells are attributed in part to ß-catenin/TCF4 transcription complexes that are assembled at proximal Wnt/ß-catenin responsive enhancers (WREs). Recent studies suggest that additional WREs that control MYC expression reside far upstream of the MYC transcription start site. Here, I report the characterization of five novel WREs that localize to a region over 400 kb upstream from MYC. These WREs harbor nucleosomes with post-translational histone modifications that demarcate enhancer and gene promoter regions. Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops. The chromatin loops are not restricted to colon cancer cells, but are also found in kidney epithelial and lung fibroblast cell lines that lack de-regulated Wnt signaling and nuclear ß-catenin/TCF4 complexes. While each chromatin loop is detected in quiescent cells, the positioning of three of the five distal enhancers with the MYC promoter is induced by serum mitogens. These findings suggest that the architecture of the MYC promoter is comprised of distal elements that are juxtaposed through large chromatin loops and that ß-catenin/TCF4 complexes utilize this conformation to activate MYC expression in colon cancer cells.
β-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular β-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in β-catenin: mutations of β-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of β-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by β-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of β-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the β-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of β-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.
The wnt pathway regulates the steady state level of β-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit β-catenin turnover via its competition with axin and adenomatous polyposis for β-catenin binding. A mutant of β-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of β-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between β-catenin and Tcf3 stimulate β-catenin degradation, indicating this interaction normally plays an important role in regulating β-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1ε, and phosphorylation of Tcf3 by CKIε stimulates its binding to β-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1ε to inhibit β-catenin degradation, whereas CKI-7, an inhibitor of CK1ε, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1ε stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1ε can modulate wnt signaling in vivo by regulating both the β-catenin-Tcf3 and the GBP-dsh interfaces.
β-catenin; Tcf; wnt; casein kinase; Xenopus
Wnt3a stimulates cellular trafficking of key signaling elements (e.g., Axin, Dishevelled-2, β-catenin, and glycogen synthase kinase-3β) and primitive endoderm formation in mouse F9 embryonic teratocarcinoma cells.
The role of phosphoprotein phosphatase-2A in signaling of the Wnt/β-catenin/Lef-Tcf-sensitive gene activation pathway was investigated. Wnt3a action attenuates phosphoprotein phosphatase-2A activity and stimulates the Lef/Tcf-sensitive gene transcription. Inhibiting phosphoprotein phosphatase-2A by okadaic acid, by treatment with siRNA (targeting the C-subunit of the enzyme), or by expression of SV40 small t antigen mimics Wnt3a action, increasing the cellular abundance of Axin and phospho-glycogen synthase kinase-3β as well as the trafficking of signaling elements in the Wnt/β-catenin pathway. Although mimicking effects of Wnt3a on the cellular abundance and trafficking of key signaling elements in the Wnt canonical pathway, suppression of phosphatase-2A alone did not provoke activation of the Lef/Tcf-sensitive transcriptional response, but did potentiate its activation by Wnt3a. Phosphoprotein phosphatase-2A and the scaffold phosphoprotein Dishevelled-2 display similarities in cellular trafficking in response to either Wnt3a or suppression of the phosphatase. A docking site for phosphoprotein phosphatase-2A in the DEP domain of Dishevelled-2 was identified.
In current study, we showed new roles of phosphoprotein phosphatase-2A in Wnt/β-catenin signaling pathway: effect on protein expression, effect on protein trafficking, retention of molecules in subcellular compartments, and regulation of enzymatic activity of several key players. Docking of phosphoprotein phosphatase-2A by Dishevelled-2 suppresses phosphatase activity and explains in part the central role of this phosphatase in the counterregulation of the Wnt/β-catenin signaling pathway.
Self-renewal of rodent embryonic stem (ES) cells is enhanced by partial inhibition of glycogen synthase kinase-3 (Gsk3)1
2. This effect has variously been attributed to stimulation of Wnt signalling via β-catenin1, stabilisation of cMyc3, and global de-inhibition of anabolic processes4. Here we demonstrate that β-catenin is not necessary for ES cell identity or expansion, but its absence eliminates the self-renewal response to Gsk3 inhibition. Responsiveness is fully restored by truncated β-catenin lacking the C-terminal transactivation domain5. However, requirement for Gsk3 inhibition is dictated by expression of Tcf3 and mediated by direct interaction with β-catenin. Tcf3 localises to many pluripotency genes6 in ES cells. Our findings confirm that Tcf3 acts as a transcriptional repressor and reveal that β-catenin directly abrogates Tcf3 function. We conclude that Gsk3 inhibition stabilises the ES cell state primarily by reducing repressive influence on the core pluripotency network.
Tcf transcription factors interact with β-catenin and Armadillo to mediate Wnt/Wingless signaling. We now report the characterization of genes encoding two murine members of the Tcf family, mTcf-3 and mTcf-4. mTcf-3 mRNA is ubiquitously present in embryonic day 6.5 (E6.5) mouse embryos but gradually disappears over the next 3 to 4 days. mTcf-4 expression occurs first at E10.5 and is restricted to di- and mesencephalon and the intestinal epithelium during embryogenesis. The mTcf-3 and mTcf-4 proteins bind a canonical Tcf DNA motif and can complex with the transcriptional coactivator β-catenin. Overexpression of Wnt-1 in a mammary epithelial cell line leads to the formation of a nuclear complex between β-catenin and Tcf proteins and to Tcf reporter gene transcription. These data demonstrate a direct link between Wnt stimulation and β-catenin/Tcf transcriptional activation and imply a role for mTcf-3 and -4 in early Wnt-driven developmental decisions in the mouse embryo.
Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC) development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif.
We investigated the TCF-4J and K isoform pair characterized by the presence (K) or absence (J) of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells) and K (K cells) to grow as solid tumors in nude mice was explored.
TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells) revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells). The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL) protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors.
Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions.
Wnt signaling is a fundamental pathway in embryogenesis which is evolutionary conserved from metazoans to humans. Much of our understanding of Wnt signaling events emerged from key developmental studies in drosophila, zebra fish, xenopus, and mice. Considerable data now exists on the role of Wnt signaling beyond these developmental processes and in particular its role in health and disease. The focus of this special issue is on Wnt/β-catenin and its diverse physiological cell signaling pathways in neurodegenerative and neuropsychiatric disorders. This special issue is composed of six reviews and two original articles selected to highlight recent advances in the role of Wnt signaling in CNS embryonic development, in adult brain function, in neurodegenerative conditions such as Alzheimer’s disease, schizophrenia, NeuroAIDS, and in gliomas. The finding that β-catenin can translocate to the nucleus where it binds to TCF/LEF transcription factors to regulate target gene expression was a seminal observation that linked β-catenin/LEF to T cell development and differentiation. We also provide a nostalgic look on recent advances in role of Wnts in T cell development and maturation. These reviews highlight the extensive body of work in these thematic areas as well as identify knowledge gaps, where appropriate. Understanding Wnt function under healthy and diseased conditions may provide a therapeutic resource, albeit it a challenging one, in diseases where dysfunctional and/or diminished Wnt signaling is a prominent player in the disease process.
Wnt signaling; β-catenin; Neurodegenerative diseases; T cell differentation; NeuroAIDS
TCF7L2 transcription factor is a downstream effector of the canonical Wnt/β-catenin signaling, which controls cell fate and homeostasis. However, the complexity of TCF7L2 expression with numerous mRNA isoforms coding for proteins with distinct N- and C-termini allows variability in TCF7L2 functions and regulations. Here, we show that although TCF7L2 mRNA isoforms distinguish fetal, immortalized and adult differentiated endothelial cells (EC), they cannot explain the lack of significant β-catenin/TCF7 activities in ECs. Lithium, a Wnt-signaling activator, increases TCF7L2 mRNA levels and induces an RNA isoform switch favoring the expression of TCF7L2-short forms lacking the C-termini domains. Although the latter occurs in different cell types, its extent depends on the overall increase of TCF7L2 transcription, which correlates with cell-responsiveness to Wnt/β-catenin signaling. While GSK3β down-regulation increases TCF7L2 expression, there is no concomitant change in TCF7L2 mRNA isoforms, which demonstrate the dual effects of lithium on TCF7L2 expression via a GSK3β-dependent up-regulation and a GSK3β-independent modulation of RNA-splicing. TCF7L2E-long forms display a repressor activity on TCF7L2-promoter reporters and lithium induces a decrease of the endogenous TCF7L2 forms bound to native TCF7L2-promoter chromatin at two novel distal TCF7-binding sites. Altogether our data reveal a lithium-induced RNA switch favoring the expression of TCF7L2-short forms, which results in a transcriptional de-repression of lithium-target genes negatively regulated by TCF7L2-long forms, like TCF7L2, and thus to an amplification of Wnt-signaling in responsive cells.
β-catenin; alternative-splicing; endothelial cells; immortalization
A morphogenesis checkpoint in budding yeast delays nuclear division (and subsequent cell cycle progression) in cells that have failed to make a bud. We show that the ability of this checkpoint to delay nuclear division requires the SWE1 gene, encoding a protein kinase that inhibits the master cell cycle regulatory kinase Cdc28. The timing of nuclear division in cells that cannot make a bud is exquisitely sensitive to the dosage of SWE1 and MIH1 genes, which control phosphorylation of Cdc28 at tyrosine 19. In contrast, the timing of nuclear division in budded cells does not rely on Cdc28 phosphorylation, suggesting that the morphogenesis checkpoint somehow turns on this regulatory pathway. We show that SWE1 mRNA levels fluctuate during the cell cycle and are elevated in cells that cannot make a bud. However, regulation of SWE1 mRNA levels by the checkpoint is indirect, acting through a feedback loop requiring Swe1 activity. Further, the checkpoint is capable of delaying nuclear division even when SWE1 transcription is deregulated. We propose that the checkpoint delays nuclear division through post-translational regulation of Swe1 and that transcriptional feedback loops enhance the efficacy of the checkpoint.
High mobility group (HMG) transcription factors of the T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) family are a class of intrinsic regulators that are dynamically expressed in the embryonic mouse retina. Activation of TCF/LEFs is a hallmark of the Wnt/β-catenin pathway, however, the requirement for Wnt/β-catenin and noncanonical Wnt signaling during mammalian retinal development remains unclear. Our goal was to characterize more fully a TCF/LEF-responsive retinal progenitor population in the mouse embryo, and to correlate this with Wnt/β-catenin signaling.
TCF/LEF activation was analyzed in the TOPgal reporter mouse (TOP: TCF optimal promoter) at embryonic ages and compared to Axin2 mRNA expression, an endogenous readout of Wnt/β-catenin signaling. Reporter expression was also examined in embryos with a retina-specific deletion of the/β-catenin gene (Ctnnb1), using Six3-Cre transgenic mice. Finally, the extent to which TOPgal cells coexpress cell cycle proteins, basic helix-loop–helix (bHLH) transcription factors and other retinal cell type markers was tested by double-immunohistochemistry.
TOPgal reporter activation occurs transiently in a subpopulation of embryonic retinal progenitor cells. Axin2 is not expressed in the central retina, and TOPgal reporter expression persists in the absence of β-catenin. Although a proportion of TOPgal-labeled cells are proliferative, most coexpress the cyclin-dependent kinase inhibitor p27/Kip1.
TOPgal cells give rise to the four earliest cell types: ganglion cells, amacrines, horizontals and photoreceptors. TCF/LEF activation in the central retina does not correlate with Wnt/β-catenin signaling, pointing to an alternate role for this transcription factor family during retinal development.
A single nucleotide polymorphism (SNP) rs6983267, located within the 8q24 region, is strongly associated with risk of colorectal and prostate cancer. It has been suggested that the mechanism of this association is related to differential interaction of TCF7L2 protein (previously known as TCF-4) with alleles of rs6983267, influencing the expression of a well-known oncogene, MYC, located 335 Kb telomeric. Here, we tested the correlation between mRNA expression of MYC and several alternatively spliced forms of TCF7L2 in 117 non-cancer colon samples. We observed a strong correlation (r = 0.60, p < 10-6) between expression of MYC and a unique splicing form of TCF7L2. The level of MYC expression in these samples was associated with expression of some TCF7L2 splicing forms but not with genotypes of rs6983267, or interaction of rs6983267 with TCF7L2 expression. These findings suggest that some splicing forms of TCF7L2 may be functionally important for regulation of MYC expression in colon tissue but this regulation is not directly dependent on rs6983267.
The Wnt signaling pathway is deregulated in over 90% of human colorectal cancers. β-Catenin, the central signal transducer of the Wnt pathway, can directly modulate gene expression by interacting with transcription factors of the TCF/LEF family. In the present study we investigate the role of Wnt signaling in the homeostasis of intestinal epithelium by using tissue-specific, inducible β-catenin gene ablation in adult mice. Block of Wnt/β-catenin signaling resulted in rapid loss of transient-amplifying cells and crypt structures. Importantly, intestinal stem cells were induced to terminally differentiate upon deletion of β-catenin, resulting in a complete block of intestinal homeostasis and fatal loss of intestinal function. Transcriptional profiling of mutant crypt mRNA isolated by laser capture microdissection confirmed those observations and allowed us to identify genes potentially responsible for the functional preservation of intestinal stem cells. Our data demonstrate an essential requirement of Wnt/β-catenin signaling for the maintenance of the intestinal epithelium in the adult organism. This challenges attempts to target aberrant Wnt signaling as a new therapeutic strategy to treat colorectal cancer.
The transcription factor 7-like 2 (TCF7L2) is a critical component of the Wnt/β-catenin pathway. Aberrant TCF7L2 expression modifies Wnt signaling and mediates oncogenic effects through the upregulation of c-MYC and cyclin D. Genetic alterations in TCF7L2 may therefore affect cancer risk. Recently, TCF7L2 variants, including the microsatellite marker DG10S478 and the nearly perfectly linked SNP rs12233372, were identified to associate with type 2 diabetes.
We investigated the effect of the TCF7L2 rs12255372 variant on familial breast cancer (BC) risk by means of TaqMan allelic discrimination, analyzing BRCA1/2 mutation-negative index patients of 592 German BC families and 735 control individuals.
The T allele of rs12255372 showed an association with borderline significance (OR = 1.19, 95% C.I. = 1.01-1.42, P = 0.04), and the Cochran-Armitage test for trend revealed an allele dose-dependent association of rs12255372 with BC risk (Ptrend = 0.04).
Our results suggest a possible influence of TCF7L2 rs12255372 on the risk of familial BC.
Inactivation of the gene encoding the adenomatous polyposis coli (APC) tumour suppressor protein is recognized as the key early event in the development of colorectal cancers (CRC). Apc loss leads to nuclear localization of beta-catenin and constitutive activity of the beta-catenin-Tcf4 transcription complex. This complex drives the expression of genes involved in cell cycle progression such as c-Myc and cyclin D2. Acute loss of Apc in the small intestine leads to hyperproliferation within the intestinal crypt, increased levels of apoptosis, and perturbed differentiation and migration. It has been demonstrated that c-Myc is a critical mediator of the phenotypic abnormalities that follow Apc loss in the intestine. As it may be difficult to pharmacologically inhibit transcription factors such as c-Myc, investigating more druggable targets of the Wnt-c-Myc pathway within the intestine may reveal potential therapeutic targets for CRC. Recent work in our laboratory has shown that the cyclin D2-cyclin-dependent kinase 4/6 (CDK4/6) complex promotes hyperproliferation in Apc deficient intestinal tissue and ApcMin/+ adenomas. We showed that the hyperproliferative phenotype associated with Apc loss in vivo was partially dependent on the expression of cyclin D2. Most importantly, tumour growth and development in ApcMin/+ mice was strongly perturbed in mice lacking cyclin D2. Furthermore, pharmacological inhibition of CDK4/6 suppressed the proliferation of adenomatous cells. This commentary discusses the significance of this work in providing evidence for the importance of the cyclin D2-CDK4/6 complex in colorectal adenoma formation. It also argues that inhibition of this complex may be an effective chemopreventative strategy in CRC.
The Wnt signaling pathway plays a major role in development, and upon deregulation it is implicated in neoplasia. The hallmark of the canonical Wnt signal is the protection of β-catenin from ubiquitination and proteasomal degradation induced by glycogen synthase kinase (GSK)-3β inhibition. The stabilized β-catenin translocates to the nucleus where it binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, activating the expression of Wnt target genes. In the absence of Wnt signal, TCF/LEF bind to Groucho (Gro)/TLE corepressors and repress Wnt target genes. Gro/TLE bind also to Engrailed (En) transcription factors mediating En-repressive activity on En target genes. Here, we present data suggesting that En-1 serves also as a negative regulator of β-catenin transcriptional activity; however, its repressive effect is independent of Gro/TLE. Our data suggest that En-1 acts by destabilizing β-catenin via a proteasomal degradation pathway that is GSK-3β–independent. Moreover, because En-1-mediated β-catenin degradation is also Siah independent, our data imply that En-1 exerts its repressive effect by a novel mechanism negatively controlling the level of β-catenin.
β-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3β (GSK-3β)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3β region of β-catenin stabilize the protein and activate β-catenin/TCF/LEF target genes. This study examined three oncogenic β-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3β. Compared with wild-type β-catenin (WT), the β-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of β-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the β-catenin mutants. Specifically, antiphospho-β-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated β-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant β-catenins, as well as on WT β-catenin. The relative order of ubiquitination was WT > D32G > D32N > D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of β-catenin.
β-catenin; TCF/LEF; APC; Wnt signaling; colorectal cancer; CTNNB1; human β-catenin gene; Ctnnb1; rat β-catenin gene
Clusterin (CLU) is an enigmatic molecule associated with various physiological processes and disease states. Different modes of cellular stress lead to increased CLU levels, and additionally numerous growth factors and cytokines affect the expression of the CLU gene. APC and c-MYC, both intimately linked to the Wnt signaling pathway have previously been shown to influence CLU levels, and we therefore investigated if changes in Wnt signaling activity in vitro could regulate the expression of one, or more, of several CLU mRNA and protein variants.
Over-expression of the cytoplasmic domain of E-cadherin tagged with GFP was used to abrogate Wnt signaling activity in LS174T and HCT116 colon carcinoma cells. This fusion construct sequestered signaling competent β-catenin whereby Wnt signaling was abrogated, and consequently cytoplasmic CLU protein levels increased as demonstrated by immunofluorescence. To determine which branch of the Wnt pathway was mediating the CLU response, we over-expressed dominant negative (dn) TCF1 and TCF4 transcription factors in stably transfected LS174T cells. We observed both intra- and extracellular levels of CLU protein to be induced by dnTCF1 but not dnTCF4. Subsequent analysis of the expression levels of three CLU mRNA variants by real time RT-PCR revealed only one CLU mRNA variant to be responsive to dnTCF1 over-expression. 5'-end RACE indicated that this CLU mRNA variant was shorter at the 5'-end than previously reported, and accordingly the translated protein was predicted to be shorter at the N-terminus and destined to the secretory pathway which fit our observations. Examination of the immediate expression kinetics of CLU after dnTCF1 over-expression using real time RT-PCR indicated that CLU might be a secondary Wnt target.
In conclusion, we have demonstrated that the Wnt signaling pathway specifically regulates one out of three CLU mRNA variants via TCF1. This CLU transcript is shorter at the 5' end than reported by the RefSeq database, and produces the intracellular 60 kDa CLU protein isoform which is secreted as a ~80 kDa protein after post-translational processing.
Metastasis from lung adenocarcinoma can occur swiftly to multiple organs within months of diagnosis. The mechanisms that confer this rapid metastatic capacity to lung tumors are unknown. Activation of the canonical WNT/TCF pathway is identified here as a determinant of metastasis to brain and bone during lung adenocarcinoma progression. Gene expression signatures denoting WNT/TCF activation are associated with relapse to multiple organs in primary lung adenocarcinoma. Metastatic subpopulations isolated from independent lymph node-derived lung adenocarcinoma cell lines harbor a hyperactive WNT/TCF pathway. Reduction of TCF activity in these cells attenuates their ability to form brain and bone metastases in mice, independently of effects on tumor growth in the lungs. The WNT/TCF target genes HOXB9 and LEF1 are identified as mediators of chemotactic invasion and colony outgrowth. Thus a distinct WNT/TCF signaling program through LEF1 and HOXB9 enhances the competence of lung adenocarcinoma cells to colonize the bones and the brain.
β-Catenin, a component of the Wnt signaling pathway, is a coactivator of human androgen receptor (hAR) transcriptional activity. Here, we show that Wnt signaling also influences androgen-mediated signaling through its ability to regulate hAR mRNA and protein in prostate cancer (PCa) cells. Three functional LEF-1/TCF binding sites lie within the promoter of the hAR gene as shown by CHIP assays that captured β-catenin-bound chromatin from Wnt-activated LNCaP cells. Chimeric reporter vectors that use the hAR gene promoter to drive luciferase expression confirmed that these LEF-1/TCF binding elements are able to confer robust upregulation of luciferase expression when stimulated by Wnt-1 or by transfection with β-catenin and that dominant-negative TCF or mutations within the dominant TCF-binding element abrogated the response. Semi-quantitative and real time RT-PCR assays confirmed that Wnt activation upregulates hAR mRNA in PCa cells. In contrast, hAR protein expression was strongly suppressed by Wnt activation. The reduction of hAR protein is consistent with evidence that Wnt signaling increased phosphorylation of Akt and its downstream target, MDM2 that promotes degradation of hAR protein through a proteasomal pathway. These data indicate that the hAR gene is a direct target of LEF-1/TCF transcriptional regulation in PCa cells but also show that the expression of the hAR protein is suppressed by a degradation pathway regulated by cross-talk of Wnt to Akt that is likely mediated by Wnt-directed degradation of the B regulatory subunit of protein phosphatase, PP2A.
androgen receptor; Wnt signaling; β-catenin; PCDH-PC; Akt; MDM2
As nuclear mediators of wnt/β-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/β-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants.
We identified cold-inducible RNA binding protein (CIRP) as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3.
Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.
Deregulated expression of myc proto-oncogenes is implicated in several human neoplasias. We analysed the expression of c-myc, N-myc, L-myc, max and RB1 mRNAs in a panel of human gliomas and glioma cell lines and compared the findings with normal neural cells. The max and RB1 genes were included in the study because their protein products can interact with the Myc proteins, being thus putative modulators of Myc activity. Several gliomas contained c/L-myc mRNAs at levels higher than those in fetal brain, L-myc predominantly in grade II/III and c-myc in grade III gliomas. High-level N-myc expression was detected. In one small-cell glioblastoma and lower levels in five other gliomas. In contrast, glioma cell lines totally lacked N/L-myc expression. The in situ hybridisations revealed mutually exclusive topographic distribution of myc and glial fibrillary acidic protein (GFAP) mRNAs, and a lack of correlation between myc expression and proliferative activity, max and RB1 mRNAs were detected in most tumours and cell lines. The glioma cells displayed interesting alternative splicing patterns of max mRNAs encoding Max proteins which either suppress (Max) or augment (delta Max) the transforming activity of Myc. We conclude that (1) glioma cells in vivo may coexpress several myc genes, thus resembling fetal neural cells; but (2) cultured glioma cells expression only c-myc; (3) myc, max and RB1 are regulated independently in glioma cells; and (4) alternative processing of max mRNA in some glioma cells results in delta Max encoding mRNAs not seen in normal fetal brain.