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1.  Amyloid-β protofibril levels correlate with spatial learning in Arctic Alzheimer’s disease transgenic mice 
The FEBS journal  2009;276(4):995-1006.
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.
PMCID: PMC2752010  PMID: 19215301
Alzheimer’s disease; amyloid-β protofibrils; Arctic mutation; transgenic mice; spatial learning
2.  The Myc 3′ Wnt-Responsive Element Regulates Homeostasis and Regeneration in the Mouse Intestinal Tract 
Molecular and Cellular Biology  2012;32(19):3891-3902.
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.
PMCID: PMC3457533  PMID: 22826434
3.  Deregulated WNT signaling in childhood T-cell acute lymphoblastic leukemia 
Blood Cancer Journal  2014;4(3):e192-.
WNT signaling has been implicated in the regulation of hematopoietic stem cells and plays an important role during T-cell development in thymus. Here we investigated WNT pathway activation in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients. To evaluate the potential role of WNT signaling in T-cell leukomogenesis, we performed expression analysis of key components of WNT pathway. More than 85% of the childhood T-ALL patients showed upregulated β-catenin expression at the protein level compared with normal human thymocytes. The impact of this upregulation was reflected in high expression of known target genes (AXIN2, c-MYC, TCF1 and LEF). Especially AXIN2, the universal target gene of WNT pathway, was upregulated at both mRNA and protein levels in ∼40% of the patients. When β-CATENIN gene was silenced by small interfering RNA, the cancer cells showed higher rates of apoptosis. These results demonstrate that abnormal WNT signaling activation occurs in a significant fraction of human T-ALL cases independent of known T-ALL risk factors. We conclude that deregulated WNT signaling is a novel oncogenic event in childhood T-ALL.
PMCID: PMC3972698  PMID: 24632884
4.  Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the β-Catenin Pathway 
Molecular and Cellular Biology  2001;21(17):5857-5868.
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.
PMCID: PMC87305  PMID: 11486025
5.  Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling 
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.
PMCID: PMC2211464  PMID: 17961225
6.  Islet Specific Wnt Activation in Human Type II Diabetes 
Experimental Diabetes Research  2009;2008:728763.
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.
PMCID: PMC2628766  PMID: 19165345
7.  Multiple Wnt/ß-Catenin Responsive Enhancers Align with the MYC Promoter through Long-Range Chromatin Loops 
PLoS ONE  2011;6(4):e18966.
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.
PMCID: PMC3080403  PMID: 21533051
8.  The balance of TCF7L2 variants with differential activities in Wnt-signaling is regulated by lithium in a GSK3β-independent manner 
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.
PMCID: PMC2926262  PMID: 20654575
β-catenin; alternative-splicing; endothelial cells; immortalization
9.  Physiological regulation of β-catenin stability by Tcf3 and CK1ε 
The Journal of Cell Biology  2001;154(5):983-994.
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.
PMCID: PMC2196183  PMID: 11524435
β-catenin; Tcf; wnt; casein kinase; Xenopus
10.  Efficient Wnt mediated intestinal hyperproliferation requires the cyclin D2-CDK4/6 complex 
Cell Division  2011;6:3.
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.
PMCID: PMC3040687  PMID: 21288356
11.  Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling 
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.
PMCID: PMC1976611  PMID: 17634137
12.  Control of TCF-4 Expression by VDR and Vitamin D in the Mouse Mammary Gland and Colorectal Cancer Cell Lines 
PLoS ONE  2009;4(11):e7872.
The vitamin D receptor (VDR) pathway is important in the prevention and potentially in the treatment of many cancers. One important mechanism of VDR action is related to its interaction with the Wnt/β-catenin pathway. Agonist-bound VDR inhibits the oncogenic Wnt/β-catenin/TCF pathway by interacting directly with β-catenin and in some cells by increasing cadherin expression which, in turn, recruits β-catenin to the membrane. Here we identify TCF-4, a transcriptional regulator and β-catenin binding partner as an indirect target of the VDR pathway.
Methodology/Principal Findings
In this work, we show that TCF-4 (gene name TCF7L2) is decreased in the mammary gland of the VDR knockout mouse as compared to the wild-type mouse. Furthermore, we show 1,25(OH)2D3 increases TCF-4 at the RNA and protein levels in several human colorectal cancer cell lines, the effect of which is completely dependent on the VDR. In silico analysis of the human and mouse TCF7L2 promoters identified several putative VDR binding elements. Although TCF7L2 promoter reporters responded to exogenous VDR, and 1,25(OH)2D3, mutation analysis and chromatin immunoprecipitation assays, showed that the increase in TCF7L2 did not require recruitment of the VDR to the identified elements and indicates that the regulation by VDR is indirect. This is further confirmed by the requirement of de novo protein synthesis for this up-regulation.
Although it is generally assumed that binding of β-catenin to members of the TCF/LEF family is cancer-promoting, recent studies have indicated that TCF-4 functions instead as a transcriptional repressor that restricts breast and colorectal cancer cell growth. Consequently, we conclude that the 1,25(OH)2D3/VDR-mediated increase in TCF-4 may have a protective role in colon cancer as well as diabetes and Crohn's disease.
PMCID: PMC2774944  PMID: 19924301
13.  Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation 
Nature cell biology  2011;13(7):838-845.
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.
PMCID: PMC3160487  PMID: 21685889
14.  Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling 
PLoS ONE  2012;7(6):e39981.
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.
PMCID: PMC3386968  PMID: 22768190
15.  Evaluation of a Variant in the Transcription Factor 7-like 2 (TCF7L2) Gene and Prostate Cancer Risk, Progression, and Mortality in a Population-Based Study 
The Prostate  2008;68(7):740-747.
Transcription factor 7-like 2 (TCF7L2) is a high mobility group -box containing protein that is a critical member of the Wnt/β-catenin signaling pathway. In addition to its recently recognized role in diabetes, aberrant TCF7L2 expression has been implicated in cancer through regulation of cell proliferation and apoptosis by c-MYC and cyclin D. It has been hypothesized that germline variants within the TCF7L2 gene previously associated with diabetes may affect cancer risk through the Wnt/β-catenin signaling pathway. Specifically, the same risk allele of single nucleotide polymorphism (SNP) rs12255372 that is associated with diabetes (T allele) has recently been associated with an increased risk of breast cancer.
Here, we investigated associations between rs12255372 and prostate cancer risk among 597 cases and 548 controls from a population-based study. We also evaluated prostate cancer progression/recurrence and prostate-specific mortality among these cases under long-term surveillance. The risk allele was associated with cancer progression/recurrence after primary treatment [hazard ratio (HR) =1.48, 95% confidence interval (CI) =1.01-2.19), but this association was attenuated when the model was further adjusted for Gleason score and tumor stage. There were no associations between this SNP and the risk of developing disease or prostate cancer-specific mortality. Our findings suggest that this variant in the TCF7L2 gene may enhance tumor progression or metastasis, but it does not contribute significantly to tumor initiation. These findings are of clinical importance for identification of patients that may develop more aggressive/recurrent disease after primary treatment.
PMCID: PMC2765224  PMID: 18302196
16.  Two Members of the Tcf Family Implicated in Wnt/β-Catenin Signaling during Embryogenesis in the Mouse 
Molecular and Cellular Biology  1998;18(3):1248-1256.
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.
PMCID: PMC108837  PMID: 9488439
17.  The Wnt/β-catenin/TCF-4 pathway upregulates HMGA1 expression in colon cancer 
Cell biochemistry and function  2012;31(3):228-236.
High Mobility Group A1 (HMGA1) encodes proteins that act as mediators in viral integration, modification of chromatin structure, neoplastic transformation, and metastatic progression. Because HMGA1 is overexpressed in most cancers and has transcriptional relationships with several Wnt-responsive genes, we explored the involvement of HMGA1 in Wnt/β-catenin/TCF-4 signaling. In adenomatous polyposis coli (APCMin/+) mice, we observed significant upregulation of HMGA1 mRNA and protein in intestinal tumors when compared to normal intestinal mucosa. Conversely, restoration of Wnt signaling by zinc-induction of wt-APC resulted in HMGA1 downregulation in HT-29 cells. Because APC mutations are associated with mobilization of the β-catenin/TCF-4 transcriptional complex and subsequent activation of downstream oncogenic targets, we analyzed the 5′-flanking sequence of HMGA1 putative TCF-4 binding elements (TBEs). We identified two functional that specifically bind the β-catenin/TCF-4 complex in vitro and in vivo identifying HMGA1 as an immediate target of the β-catenin/TCF-4 signaling pathway in colon cancer. Collectively, these findings strongly implicate Wnt/β-catenin/TCF-4 signaling in regulating HMGA1 to further expand the extensive regulatory network affected by Wnt/β-catenin/TCF-4 signaling.
PMCID: PMC3616152  PMID: 22961697
Wnt/β-catenin/TCF-4 Pathway; High Mobility Group A1 (HMGA1); colon cancer; oncogene; TCF-4 target gene; transcriptional activation
18.  No effect of cancer-associated SNP rs6983267 in the 8q24 region on co-expression of MYC and TCF7L2 in normal colon tissue 
Molecular Cancer  2009;8:96.
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.
PMCID: PMC2777153  PMID: 19895682
19.  Functional Analyses of Vertebrate TCF Proteins in C. elegans Embryos 
Developmental biology  2011;355(1):115-123.
In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognize specific promoter sequences upstream of endoderm genes and interact with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.
PMCID: PMC3104061  PMID: 21539828
C. elegans; Wnt signaling; POP-1; vertebrate TCF; A-P asymmetry
20.  Wnt/β-catenin and its Diverse Physiological Cell Signaling Pathways in Neurodegenerative and Neuropsychiatric Disorders 
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.
PMCID: PMC3690588  PMID: 23114888
Wnt signaling; β-catenin; Neurodegenerative diseases; T cell differentation; NeuroAIDS
21.  Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast. 
Molecular Biology of the Cell  1996;7(11):1657-1666.
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.
PMCID: PMC276016  PMID: 8930890
22.  Characterization of a transient TCF/LEF-responsive progenitor population in the embryonic mouse retina 
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.
PMCID: PMC2615067  PMID: 18599572
23.  Wnt/β-Catenin Is Essential for Intestinal Homeostasis and Maintenance of Intestinal Stem Cells▿ †  
Molecular and Cellular Biology  2007;27(21):7551-7559.
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.
PMCID: PMC2169070  PMID: 17785439
24.  Transcription factor 7-like 2 (TCF7L2) variant is associated with familial breast cancer risk: a case-control study 
BMC Cancer  2006;6:268.
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.
PMCID: PMC1665524  PMID: 17109766
25.  Abundance, complexation, and trafficking of Wnt/β-catenin signaling elements in response to Wnt3a 
Wnt3a regulates a canonical signaling pathway in early development that controls the nuclear accumulation of β-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes.
Using totipotent mouse F9 teratocarcinoma cells expressing Frizzled-1 and biochemical analyses, we detail the influence of Wnt3a stimulation on the expression, complexation, and subcellular trafficking of key signaling elements of the canonical pathway, i.e., Dishevelled-2, Axin, glycogen synthase kinase-3β, and β-catenin. Cellular content of β-catenin and Axin, and phospho-glycogen synthase kinase-3β, but not Dishevelled-2, increases in response to Wnt3a. Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions. Dishevelled-2, in contrast, is found predominately in the cytosol, whereas β-catenin is localized to the plasma membrane-enriched fraction. Wnt3a stimulates trafficking of Dishevelled-2, Axin, and glycogen synthase kinase-3β initially to the plasma membrane, later to the nucleus. Bioluminescence resonance energy transfer measurements reveal that complexes of Axin with Dishevelled-2, with glycogen synthase kinase-3β, and with β-catenin are demonstrable and they remain relatively stable in response to Wnt3a stimulation, although trafficking has occurred. Mammalian Dishevelled-1 and Dishevelled-2 display similar patterns of trafficking in response to Wnt3a, whereas that of Dishevelled-3 differs from the other two.
This study provides a detailed biochemical analysis of signaling elements key to Wnt3a regulation of the canonical pathway. We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules. In contrast, we observe little effect of Wnt3a stimulation on the level of protein-protein interactions among these constituents of Axin-based complexes themselves.
PMCID: PMC2211465  PMID: 17961224

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