The leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l are identified as Tcf4/β-catenin co-activators and shown to be essential for Wnt-driven endogenous gene expression, intestinal development and homeostasis. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/β-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/β-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and β-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/β-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a β-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published β-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways.  The canonical Wnt pathway is a key regulatory pathway controlling intestinal cell proliferation, differentiation, and stem cell maintenance, and its deregulation leads to malignancies in the mammalian gut. A decade has passed since the discovery of the transcription factors TCF4-β-catenin as the downstream intestinal molecular effectors of Wnt, but few transcriptional activators essential and unique to the regulation of this transcription program have been found. In this study, using a proteomics approach, we identify the leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l as interactors with Tcf4/β-catenin in the mouse small intestinal epithelium. We demonstrate that Mllt10/Af10–Dot1l are recruited to Wnt target genes in intestinal epithelial cells and are essential to regulate expression of these targets. We also show a genetic link between the Wnt pathway and Mllt10/Af10-Dot1l in zebrafish and delineate their essential role in Wnt-driven endogenous gene expression. Finally, we demonstrate the physiological role of Mllt10/Af10-Dot1l in Wnt-driven intestinal development and homeostasis; depletion of Mllt10/Af10-Dot1l in zebrafish embryos mimics the Tcf4-depleted phenotype in which significant intestinal proliferation defects accompany a decrease in total number of intestinal cells. We conclude that the enzyme Dot1l may present an attractive candidate for drug targeting in colorectal cancer. The canonical Wnt signaling pathway has been shown to play a central role in cell proliferation, differentiation, and stem cell maintenance In colorectal cancer, activating mutations in Wnt pathway components, such as APC, AXIN1, or β-catenin Drosophila have led to the identification of Pygopus and Lgs/BCL9 as cofactors of β-catenin/TCF mediated transcription The β-catenin/TCF4 complex is thought to drive TCF4 target gene expression by recruitment of activating cofactors Despite the passage of a decade after the discovery of TCF4 and β-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. The identification and study of the complete repertoire of nuclear TCF4/β-catenin coactivator complex components dedicated to this transcriptional program is both required to unveil the key targets and mechanistic events responsible for initiation and progression of cancer and also critical to the definition of potential therapeutic targets in colorectal cancer.We applied a Mass Spectrometry approach to identify novel components of the endogenous Tcf4 complex in the mouse small intestinal epithelium. Working under the premise that intestinal crypt cells contain activating Tcf4/β-catenin complexes and that villus cells contain repressive Tcf4 complexes, we obtained separated mouse intestinal crypt and villus cells, using a mechanical fractionation method To confirm that Mllt10/Af10 interacts with Tcf4, we immunoprecipitated Tcf4 complexes from crypt lysates and probed for the association with endogenous Mllt10/Af10 by Western blotting using an antibody specific for Mllt10/Af10. The Tcf4 complex contained Mllt10/Af10 in the crypts of the small intestinal epithelium . ImmunopIn the mouse small intestine, Mllt10/Af10 was found to be expressed specifically in the crypt proliferative compartment and not the villus as shown by Western blotting on purified crypt and villus fractions  and by iAxin2  assays on mouse small intestinal epithelium . The purAxin2  and mous2 . In this cell line a point mutation in β-catenin results in its constitutive stabilization and activation of the Wnt pathway. This Wnt “on” system mimics the proliferative crypt compartment, in which the Wnt pathway is active. MLLT10/AF10 was immunoprecipitated from Ls174T CRCs and probed for interaction with TCF4, β-catenin, and DOT1L by Western blotting . Both TCAXIN2 and c-MYC in vivo by ChIP in the presence or absence of β-catenin  Wnt targets were downregulated upon BRG1 and p300 depletion.To examine the specificity of MLLT10/AF10 in Wnt transcriptional regulation, we performed microarray analysis in Ls174T colorectal cancer cells comparing the effect of MLLT10/AF10 depletion with two other known Wnt coactivators, BRG1 r panels . Our datlidation . All selAXIN2 and ZCCHC12Our data using the constitutive Wnt activated Ls174T CRC demonstrated that depletion of MLLT10/AF10-DOT1L reduced Wnt target gene expression. To demonstrate that activation of Wnt signaling in unstimulated cells results in a time-dependent association of MLLT10/AF10 and DOT1L on Wnt target gene promoters, we extended our studies to the physiologically relevant HEK293T system. In the latter, Wnt pathway components are intact but not mutationally activated. Wnt stimulation in these cells results in a time-dependent activation of a Wnt target gene program. ChIP analysis in HEK293T cells indicates that while TCF4 is enriched on the Wnt target genes AXIN2 and c-MYC  as a model system. Zebrafish has been shown to be an important and relevant model system for the study of development of the intestinal epithelium and tumorigenesis tcf4/tcf7l2 mutant zebrafish were shown to recapitulate the phenotype of the Tcf4 knockout mouse tcf4/tcf7l2 plays a similar critical role in zebrafish intestinal development as it does in mouse. Using the Mllt10/Af10 mouse protein sequence, we identified the full zebrafish Mllt10/Af10 protein sequence  demonstrated expression of tcf7l2 in embryos by 13 hpf, which remained strong at all subsequent time points examined  to deplete these genes , and dot1l (two independent sequences)  . In all t 56 hpf , indicathpf lef1. Similarmidbrain , highligtcf7l2, mllt10/af10, and dot1l depletion on Wnt activity in the intestine of TOPdGFP embryos by in situ hybridization for GFP  embryos display a variety of developmental defects i-fabp) tcf7l2 mutant zebrafish line, we showed recently that Tcf4 is a main effector of Wnt signaling during zebrafish intestine organogenesis and adult homeostasis tcf7l2 morpholino in mcr/mcrapc embryos rescued the expression of the intestinal differentiation marker i-fabp, as was observed previously using the tcf7l2 mutant zebrafish line i-fabp expression  incorporation analysis to mark cells progressing through the S-phase of the cell cycle at 124 hpf . Despite the inability to revert all of the mcr/mcrapc defects, MO depletion of Tcf4, Mllt10, and Dot1l in mcr/mcrapc fish rescues the block in intestinal differentiation as well as the mis-expression of the Wnt target genes cyp26a1 and axin2. Thus, Tcf4 plays a crucial role during intestinal development and acts as the main downstream effector of the Wnt pathway in this tissue Abrogation of Apc function during zebrafish development arrests affected cells in a proliferative state, likely due to inappropriate activation of Tcf target genes, leading to a blockage in differentiation of the intestinal epithelium tcf7l2 as the principle tcf/lef family member involved in expression of axin2 in the intestinal epithelium and GFP in the hindbrain in wild type and TOPdGFP line, respectively. We observe similar down-regulation of GFP expression in the TOPdGFP line as well as down-regulation of the target gene axin2 also upon depletion of mllt10 and dot1l. While our data suggest that the expression of axin2 in the intestinal epithelium of wild-type embryos and GFP in the hindbrain of TOPdGFP line is highly depended on tcf7l2, we cannot exclude possible redundant regulation of other developmentally important Wnt-targets by other tcf/lef members. The observed residual expression of GFP in the TOPdGFP Tcf4, Mllt10, and Dot1l morphant embryos may reflect the role of other tcf/lef members in controlling the expression of Tcf/Lef-derived genes in the hindbrain.Our data are consistent with tcf7l2 mutant zebrafish line does not show any strong morphological defects during early embryogenesis tcf7l2 mimics the absence of morphological defects during early embryogenesis. The overall lack of early macroscopic morphological defects upon mllt10 and dot1l depletion together with their requirement for intestinal homeostasis is consistent with their specific functional dependence on tcf7l2 and not other tcf/lef family members  supplemented with protease inhibitors . Cellular lysates were precleared with IgG-agarose beads (Sigma) for 6 h at 4°C. Immunoprecipitations of endogenous complexes from mouse crypt, villus, and Ls174T cells were carried out overnight at 4°C, with anti-TCF4 (Santa Cruz), anti β-catenin (BD transduction), anti-Mllt10/Af10 (Santa Cruz or abcam), or anti-DOT1L (Abgent) antibodies at concentrations of 2 µg/mL in combination with a 50% protein G-Agarose slurry (Sigma). Immunoprecipitated material was washed four times in PLB buffer. Bound proteins were subjected to SDS-PAGE and Western blot analysis or Mass Spectrometry Analysis. Western blot analysis was also conducted using antibodies against TCF-1 (upstate), EPHB2 (Abgent), and p300 and BRG1 (SantaCruz).www.matrixscience.com), with trypsin set as enzyme.For details of the mass spectrometry analysis, please see Mouse small intestine tissue was fixed in 10% formalin, paraffin embedded, and sectioned. Human colon samples were obtained immediately after resection. The tissue was fixed in 4.0% formaldehyde in PBS for 2 h at 4°C. Tissues were cryo protected in 30% Sucrose o/n at 4°C after which samples were frozen in Tissue Tec. Sections of 16 um were made with a Leica CryoStat. Sections were immunostained after permeabilization with 0.5% Triton X-100 in 1% BSA, 1% Glycine, 0.1% Lysine in PBS. Immunostaining was done in the permeabilization buffer with Mllt10/Af10 (Santa Cruz) and E-Cadherin (Transduction Laboratories) antisera o/n at 4°C. Secondary antibody (InVitrogen Alexa 488 and 568) staining was done for 1 h at 4°C. Fluorescent imaging was done with a Leica confocal laser scanning microscope.For histology, 124 hpf zebrafish embryos were fixed in 4% (w = v) paraformaldehyde in PBS overnight at 4°C with agitation, rinsed in PBS, dehydrated through a standard ethanol series to 100%, embedded in paraffin, and sectioned at 6 µm intervals for staining.GFP imaging in TOPdGFP zebrafish line was performed using a Leica SP5 confocal microscope and images were processed with Leica Application Suite.5 cells, and protein levels were examined by Western blot analysis 72–96 h after transfection.Pre-designed siRNA pools targeting transcripts of the human β-catenin (L-003482-00), MLLT10/AF10 (L-019827-00), DOT1L (L-014900-01), BRG1 (SMARCA4) (L-010431-00), p300 (L-003486-00), and non-target control siRNA pool (D-001810-10-20) were purchased from Dharmacon and used to knock down the corresponding genes in Ls174T CRC and HEK293T cells. siRNA was delivered into Ls174T and HEK293T cells using either the siLentFect Lipid Reagent (BioRad) or INTERFERin reagent (Polyplus Transfections). siRNA (15 nM) was used to transfect 2×10Ls174T CRC cells containing integrated TOPFLASH and FOPFLASH  AXIN2, c-MYC, and ZCCHC12 genes.ChIP was performed on Ls174T CRC and HEK293T cells or purified mouse crypt and villus fractions as previously described Ls174T and HEK293T cells were transfected with siRNA . HEK293T cells were then induced with Wnt3A or control medium to measure direct Wnt regulated activity. Total RNA was extracted from samples using RNeasy Mini Kit (Qiagen). 2 µg of RNA from each sample, together with universal human reference RNA (Strategene), was labeled by Quick Amp Labelling Kit, two color (Agilent) with Cy3 and Cy5, respectively. Samples were hybridized to 4×44K Whole Human Genome dual color Microarrays . Microarray signal and background information were retrieved using Feature Extraction program . For each pair of experiments, fluorescent signals in either channel with greater than 2-fold above the local background were considered as well-measured. Corresponding fold changes of Ls174T cells after siβ-catenin, si MLLT10/AF10, si BRG1, or si p300 suppression were calculated by normalizing with the siControl samples. In HEK293T cells, Wnt regulated genes after 9 h Wnt induction were obtained by normalizing the fold change between 9 h Wnt3A and Control medium induction in siControl samples (no Wnt), and the corresponding fold changes of si MLLT10/AF10, si DOT1L, si BRG1, and si p300 upon 9 h Wnt induction were obtained by normalizing with siControl samples after 9 h Wnt induction. The raw data were submitted to Gene Expression Omnibus, accession number GSE21367.Axin2 and c-Myc genes and human AXIN2, c-MYC, and ZCCHC12 genes were used in qPCR analysis. Primer sequences are provided in ChIP experiments were analyzed by qPCR in an iCycler iQ real-time PCR detection system (BioRad) using iQ Sybergreen Supermix (BioRad). ChIP values were normalized as a percentage of input and presented as the percentage of input immunoprecipitated. Primer pairs spanning the upstream regulatory regions, gene body, and downstream regulatory regions of mouse mcrapc and TOPdGFP lines were previously described mcrapc mutants were genotyped as described Zebrafish were maintained on a 14-h-light/10-h-dark cycle at 28.5°C. Fertilized embryos were collected after natural spawning, cultured, and staged by developmental time and morphological criteria. Drosophila melanogaster, Homo sapiens, Mus musculus, and trace cDNA sequences for Danio rerio mllt10/af10 were analyzed with ENSEMBL. Full exon-intron structures of mllt10/af10 danio rerio prediction were performed with Genome Scan. Phylogenetic tree of Mllt10/Af10 and domain alignment were performed with CLUSTAL V (DNAstar).n>50 embryos.Embryos at different developmental stages were homogenized in TRIzol reagent (Invitrogen) using a homogenizer. Total RNA was isolated according to the manufacturer's instructions. Single-stranded cDNA was synthesized from 1 µg total RNA using Superscript III (Invitrogen). PCR primers used  were desc-MYC, AXIN2, LGR5, EPHB3, and ASCL2 in Ls174T CRC are provided in RT-PCR primers used to detect mRNA expression of Wnt target genes human tcf7l2, mllt10, dot1l, tbp, tcf7, tcf7l1a, tcf7l1b, and lef1 are provided in RT-PCR primers used to detect mRNA expression of zebrafish genes tcf7l2, mllt10, and dot1l were designed in house and obtained from Gene Tools LLC . The blocking MO against p53 has been previously described , dot1l (two different sequences), dGFP, i-fabp, cyp26a1, or axin2 cDNA followed by in vitro transcription and purified using NucleoSpin RNA clean-up columns (Machery-Nagel). Pictures were obtained using a Leica microscope (TCS NT), digitized with a camera , and processed with IM500 Image Manager (Leica). WISH was preformed for i-fabp in mcr/mcrapc embryos after MO injection, expression scored, and each embryo genotyped. Experiments were in triplicate; in each replica, n>100 embryos were analyzed.Embryos were collected at 56, 72, and 80 hpf, fixed in 4% paraformaldehyde in PBS overnight at 4°C, rinsed in PBS, dehydrated through a standard methanol series to 100%, and stored at −20°C. Whole-mount in situ hybridizations (WISH) were carried out as described 122 hpf live embryos (previously MO injected at one-cell stage) were immersed in embryo medium with BrdU (Sigma) at 3 mg/mL for 30 min at 28.5°C. After several washes with pre-warmed embryo medium, embryos were left for 1 h at 28.5°C in embryo medium, euthanized, and fixed in 4% paraformaldehyde in PBS overnight at 4°C. Detection of BrdU was carried out by immunostaining with a mouse anti-BrdU antibody .Figure S1Peptides identified and coverage of (A) Mllt10 and (B) Dot1l in Tcf4 complex in mouse small intestinal crypt. Amino acid sequences of Mllt10 and Dot1l detected in the Tcf4 immunoprecipitate from crypt lysates are underlined.(0.03 MB PDF)Click here for additional data file.Figure S2Specific enrichment of Mllt10 and Dot1l, and H3K79 di-/tri-methylation at c-Myc locus in mouse crypts. (A) Schematic representation of the mouse amplicons scanned in ChIP experiments by qPCR. Purified crypt and villus fractions from mouse intestine were subjected to ChIP using antibodies directed against Tcf4 (B), β-catenin (C), Mllt10/Af10 (D), Dot1l (E), di-methyl H3K79 (F), and tri-methyl H3K79 (G). Chromatin was immunoprecipitated with the specified antibodies followed by qPCR using primer pairs spanning the c-Myc locus as indicated in (A). Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.(0.07 MB PDF)Click here for additional data file.Figure S3β-catenin recruits Mllt10/Af10 to Wnt targets. (A) Ls174T CRC lysates were immunoprecipitated with antibodies against TCF4 (left panel) and β-catenin (right panel) and analyzed by Western blotting with the indicated antibodies for binding to MLLT10/Af10 and DOT1L. (B–H) β-catenin-dependent H3K79 methylation and recruitment of MLLT10 and DOT1L to c-MYC gene in Ls174T CRC. (B) Schematic representation of human c-MYC locus and amplicons scanned in ChIP assays by qPCR. ChIP experiments in Ls174T CRC uninduced or induced with Dox using antibodies against (C) TCF4, (D) β-catenin, (E) MLLT10, (F) DOT1L, (G) H3K79 dimethyl, and (H) H3K79 trimethyl. Immunoprecipitated DNA was analyzed by qPCR using primer pairs specific for the c-MYC locus as indicated. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.(0.08 MB PDF)Click here for additional data file.Figure S4Mllt10/Af10 interacts directly with β-catenin. (A) Recombinant GST-fused TCF4 and β-catenin proteins were used in pull-down assays with in vitro translated S35 labeled MLLT10 to examine direct interaction. (B) Schematic representation of N-Terminal and C-Terminal MLLT10 deletion mutants and S35 labeled ΔC- and C-Terminal β-catenin deletion mutants used in GST pulldown assays (C). N-Terminal MLLT10 interacts directly with the β-catenin C-terminal domain.(0.03 MB PDF)Click here for additional data file.Figure S5(A) Significant overlap between differentially regulated genes in response to MLLT10 or DOT1L depletion in HEK293T cells after Wnt stimulation. Comparison of the corresponding expression pattern after siRNA suppression of MLLT10/AF10 or DOT1L in Wnt induced condition. Heatmap showing 1,116 transcripts after siRNA depletion of MLLT10 and 9 h Wnt stimulation in HEK293T cells with greater than 1.5-fold variation. Also shown is comparison of the corresponding expression pattern of genes induced after 9 h Wnt treatment and after siRNA suppression of DOT1L. Red, upregulated after MLLT10 suppression; green, downregulated after MLLT10 suppression; grey, missing data. (B) Venn diagram comparatively depicting genes suppressed upon MLLT10 and DOT1L depletion in HEK293T cells under Wnt-induced conditions.(0.03 MB PDF)Click here for additional data file.Figure S6mRNA expression of tcf7l2, mllt10, and dot1l during zebrafish embryonic development and their depletion by splice-inhibiting MO sequences. (A) Phylogenetic tree analysis places zebrafish (Danio rerio) Mllt10 close to mouse and human sequences. (B–C) Amino acid sequence alignment of the Leucine zipper and PHD finger domains show high conservation of Mllt10 between species. (D) RT-PCR analysis of tcf7l2, mllt10, dot1l, and tbp RNA expression levels in whole embryos at different stages of embryonic development. (E) Representation of the splice-blocking MO sequences (blue) generated to deplete mRNA levels for tcf7l2, mllt10, and dot1l.(0.03 MB PDF)Click here for additional data file.Figure S7Depletion of tcf7l2, mllt10/af10 and dot1l rescues mis-expression of axin2mcr/mcr zebrafish, placing these genes downstream of Apc as Wnt target gene activators (A–N). in apc Representative whole mount in situ hybridizations for axin2 in wild type and mcr/mcrapc mutant embryos at 80 hpf injected with  buffer alone,  MO against tcf7l2, (E–H) two independent mllt10/af10 MOs, (I–L) two independent dot1l MO, and  control MO. All MOs have been coinjected with a MO against p53. All images were captured using the same exposure and represent at least three independent experiments. In parentheses number of embryos showing described phenotype per number of total embryos analyzed.(0.06 MB PDF)Click here for additional data file.Figure S8Depletion of tcf7l2, mllt10, or dot1l does not affect the expression levels of other tcf/lef family members in zebrafish embryos. RT-PCR analysis of lef1, tcf7, tcf7l1a, tcf7l1b, and tbp RNA expression levels in whole embryos injected with MO against p53 alone, or MO against p53 coinjected with MOs against tcf7l2, mllt10, dot1l, or control MO, respectively.(0.02 MB PDF)Click here for additional data file.Table S1List of proteins with highest scores identified by MS in Tcf4 crypt complex.(0.03 MB XLS)Click here for additional data file.Table S2Ls174T Wnt regulated gene list—1,003 transcripts.(0.24 MB XLS)Click here for additional data file.Table S3HEK293T Wnt-induced gene list after 9 h Wnt induction—1,189 transcripts.(0.30 MB XLS)Click here for additional data file.Table S4Primer and Morpholino sequences.(0.03 MB XLS)Click here for additional data file.


We established a cell line that constitutively expresses a dominant-negative LEF-1 and it was used in a gene expression microarray analysis. In this way, genes that respond to estradiol or Wnt3a, sensitive to LEF-1, could be identified and validated. Together, these data demonstrate the existence of a new signaling pathway controlled by estradiol in neurons. This pathway shares some elements of the insulin-like growth factor-1/Insulin and Wnt signaling pathways, however, our data strongly suggest that it is different from that of both these ligands. These findings may reveal a set of new physiological roles for estrogens, at least in the Central Nervous System (CNS).Estradiol may fulfill a plethora of functions in neurons, in which much of its activity is associated with its capacity to directly bind and dimerize estrogen receptors. This hormone-protein complex can either bind directly to estrogen response elements (ERE's) in gene promoters, or it may act as a cofactor at non-ERE sites interacting with other DNA-binding elements such as AP-1 or c-Jun. Many of the neuroprotective effects described for estrogen have been associated with this mode of action. However, recent evidence suggests that in addition to these “genomic effects”, estrogen may also act as a more general “trophic factor” triggering cytoplasmic signals and extending the potential activity of this hormone. We demonstrated that estrogen receptor alpha associates with β-catenin and glycogen synthase kinase 3 in the brain and in neurons, which has since been confirmed by others. Here, we show that the action of estradiol activates β-catenin transcription in neuroblastoma cells and in primary cortical neurons. This activation is time and concentration-dependent, and it may be abolished by the estrogen receptor antagonist ICI 182780. The transcriptional activation of β-catenin is dependent on lymphoid enhancer binding factor-1 (LEF-1) and a truncated-mutant of LEF-1 almost completely blocks estradiol TCF-mediated transcription. Transcription of a TCF-reporter in a transgenic mouse model is enhanced by estradiol in a similar fashion to that produced by Wnt3a. In addition, activation of a luciferase reporter driven by the  Estrogens fulfill a wide range of functions during development and differentiation in mammals of both sexes. In addition to these functions, they are also thought to play an important role in neuroprotection We recently demonstrated that ERα is linked to PI3K associated “cytoplasmic signaling” in the brain and in primary neurons, where estradiol can induce the immediate activation of Akt/PKB and the subsequent inhibition of glycogen synthase kinase 3 (GSK3). On the light of the important role proposed for GSK3 in neuronal survival We also identified novel complexes in which ERα, GSK3 and β-catenin were associated, demonstrating that the action of estradiol transiently stabilized β-catenin, which is disassociated from the complex with ERα In this study we have assessed whether the β-catenin, stabilized by estradiol in neurons, may exert a significant effect at the transcriptional level. We show that the stabilization of β-catenin by estradiol is correlated with the inhibition of GSK3 in neuronal-like cells (N2a-m), as well as in cortical neurons. Indeed, estradiol increased TCF/LEF-1 transcription in a dose-dependent manner. This increase in transcription was partially prevented by the addition of an estradiol antagonist and was mimicked by an alpha and beta specific-agonist. In neuronal cells, estradiol induced the formation of a TCF-DNA complex that was impaired by the presence of antibodies against LEF-1. Moreover, in cortical neurons from a TCF/LEF-1–βgal transgenic mouse, β-galactosidase activity was up-regulated by the action of estradiol. Finally, in a LEF-1 mutant cell line, we identified some genes that were differentially regulated by estradiol.Together, all these data demonstrate that in addition to its nuclear action mediated through the ER, estradiol triggers a signal that recruits β-catenin and LEF-1 and that may be responsible for more wide-ranging actions of this hormone, at least in neurons.N2a-m cells, a clone derived from NB2a (ATCC: CCL 131) and that has been maintained for several years in our laboratory, responded to estradiol in a similar manner as primary neurons, as demonstrated below.We recently demonstrated that estradiol inhibit GSK3 and stabilize β-catenin in the hippocampus and in hippocampal neurons. To further analyze this phenomenon, it was necessary to transfect neuronal cells. We first examined whether neuroblastoma cells behave like primary neurons concerning this estrogenic action. We found that The effect of estradiol on this cell line was examined and we initially determined whether N2a-m cells expressed both α and β estrogen receptors when maintained in serum–free medium (Supplementary ICI/estradiol ratio was maintained 100×)  .A complex of ER α and β-catenin was recently detected in the hippocampus of female ovariectomized rats Given that β-catenin may fulfill two distinct cellular functions, as an adhesion molecule or as co-transcription factor. Two different pools have been described associated with different functions luciferase activity  in combination with an EGFP reporter. When estradiol (100 nM) was added after cell transfection, luciferase activity increased 24±4 fold n = 4, , reachinactivity .To determine whether this transcriptional activation was associated with ER α or ER β, we used two specific agonists of either the α or β receptor isoforms. Both 4,4′,4″-(propyl-[(1)H]-pyrazole-1,3,5-triyl) trisphenol  and 2,3-bis (4-hydroxyphenyl) propionitrile (DPN (β-selective) agonists increased transcriptional activity in N2a-m cells, although the activity mediated by PPT was more prominent that that mediated by DPN, suggesting that ER α is more effectively coupled to this novel pathway . As a poTo determine whether estradiol can regulate the expression of a reporter controlled by TCF in primary neurons, we used two complementary approaches. First, we transfected TOPFlash or FOPFlash into primary neurons  and afteLucAs a complementary alternative we try to analyze whether an endogenous promoter may be regulated by estradiol in a similar fashion as Wnt3a. For that purpose we used a construct containing a 2.8 Kb region of the Engrailed-1 promoter that has three LEF-1 sites situated upstream of a luciferase gene pENP1versus estradiol) (versusluciferase expression (mock transfected cells responded to estradiol as previously shown.The mechanism by which Wnt modulates β-catenin-mediated transcription is sensitive to the phosphorylation of β-catenin as well as to β-catenin/TCF binding (reviewed in ) versus. In all ) versus. Hence, pression , whereasLuc, the luciferase expression estradiol-mediated was severely reduced by the expression of LEF1-mutant . We detected a DNA-protein complex that was more evident after estradiol or Wnt3a treatment , the first three of which are found in neuronal tissues Together, the data obtained so far suggested that estradiol might activate a β-Catenin/LEF-1 complex that is similar to that activated by Wnt in other systems. Thus, to shed further light on this new molecular mechanisms, we took advantage of the fact that the Δ56LEF-1 construct may act as a dominant-negative construct and we generated N2a-m cells stably transfected with Δ56LEF-1 . This cemock-transfected with the empty pcDNA3.1 vector  or Δ56LEF-1/N2a-m (group B). In both cases, cells were treated with estradiol or Wnt3a for 60 minutes and then total RNA was purified. Gene expression from the two stable cell types (A and B) was then analyzed using the Applied Biosystems Mouse Genome Survey Microarray (Applied Biosystems).Subsequently, stably transfected N2a-m cells were utilized to obtain the total RNA, either from A versus group B, for each treatment. From these we selected those with a log2R higher or lower than 1, being 102 genes upregulated and 121 genes downregulated by estradiol; and 137 genes upregulated and 166 genes downregulated by Wnt3a. Ref Seq and the Gene Name. In contrast, the genes modified only after estradiol treatment or only after Wnt3a treatment that presented no significant differences between A and B were discarded. Initially, for the confirmation of the putative deferential response of both cells (A vs B) we selected two genes presented in both arrays that were modified by Wnt as well as by estradiol (Plasminogen and LEF-1). A final validation was performed using specific antibodies against the selected proteins (at http://www.stanford.edu/~rnusse/pathways/targets.html).Genes were normalized with respect to control untreated cells, and divided in different groups. From these groups, we only consider those with differences when compared group proteins  and 8C. proteins . In addi2 R of some of these genes was low, and certainly, the protein expression showed no differences. Considering that the low level of detection may be related with the short exposure time (60 minutes), we extended the treatment to confirm the differential regulation of some genes mediated by Δ56LEF-1. Accordingly, we treated the cell lines with estradiol or Wnt3a over a period of 24 h to allow for the accumulation of the protein. Soluble fraction, or nuclear fractions, or conditioned medium was obtained from treated cells and we analyzed the genes identified as differentially expressed in the microarray (when antibodies were available) as well as some genes known to be regulated by Wnt, such as E-cadherin, β-catenin or cyclin D.As shown in Our data confirmed some of the variations in the genes described. Interestingly, MMP-2 secretion was most prominent in Wnt-treated cells whereas the proactive form appeared to be secreted prominently by Δ56LEF-1/N2a-m cells. Similarly, β-catenin displayed weaker protein expression in Δ56LEF-1/N2a-m cells, independently of the treatment.A), whereas the presence of Δ56LEF-1 generated an uncontrolled upregulation. This result revealed an important role for LEF-1 in estrogen-regulated E-cadherin expression.Finally, a second set of genes were found, those that respond to estradiol (and/or Wnt) in control cells and whose regulation was modified by the presence of Δ56LEF-1. Indeed, longer treatment enabled clear differences to be detected at the protein level in genes such as N-cadherin and Cyclin D2. Our data indicated that N-cadherin was upregulated by estradiol and Wnt, and that the expression of Δ56LEF-1 almost completely blocked these effects . While CEstrogens may act through different mechanisms, the best known of which involves their capacity to directly bind to and dimerize estrogen receptors (ERs). This hormone-protein complex can either bind directly to promoter estrogen response elements (EREs), or act as a cofactor at non-ERE sites through the interaction with other DNA-binding elements, such as AP-1, or c-Jun We had initially found that the inhibition of GSK3 by estradiol was correlated with the stabilization of β-catenin. Thus, our first aim here was to determine whether the pool of β-catenin regulated by estradiol acts as a co-transcriptional modulator using canonical TCF-mediated transcription or alternatively, whether the stabilization of β-catenin plays some other role. With this aim, we selected a neuronal cell line easily amenable to transfection with a TCF-reporter: the N2a-m cell line which expresses the α and β ER receptors and accumulates nuclear ER when treated with estradiol. Significantly, we detected a complex of β-catenin and GSK3 in these cells following exposure to either ERα or ERβ. When N2a-m or cortical neurons were treated with estradiol, we detected a similar biochemical response with an increase of GSK3 serine phosphorylation as well as the stabilization of β-catenin. This inhibition of GSK3 was time- and concentration-dependent, and these data are in essence similar to those described in the hippocampus of ovariectomiced rats after estradiol treatment, or in hippocampal neurons −9 M. This nuclear activation of luciferase was time- and concentration-dependent, it was maximal at 10–20×10−8 M, and it could be abolished by the ER antagonist ICI 182780. Similar, but slightly weaker transcriptional activation could be obtained with the ERα-selective agonist, PPT (5–10 nM), as well with the ERβ-selective agonist, DPN (5–10 nM). All these data strongly suggested that estradiol stabilizes β-catenin through ERα and ERβ, and that at least a portion of this protein pool can activate transcription. This transcriptional activation depended on the phosphorylation status of β-catenin. Indeed, the highest levels of transcription were obtained with a mutated form of β-catenin (S33Y) that was virtually unaffected by estradiol. These data raise the question as to whether this TCF- β-catenin mediated transcription is similar to that obtained with Wnt proteins.Taking into account these initial results, the question arose as to whether β-catenin stabilized by estradiol might modulate transcription. It is believed that β-catenin has two different and complementary roles in cells; it may contribute to the cell-cell adhesion or it may act as a co-transcriptional regulator of the TCF family engrailed-1 promoter that contains well characterized TCF elements (pENP1-luc) , and also activate transcription in primary neurons derived from mice transgenic for a TCF-β-galactosidase reporter driving the expression of a luciferase reporter. It is important to remember that the estradiol-dependent transcription mediated by pENP1-luc is severely inhibited by the expression a LEF-1 truncated-mutant (Δ56LEF-1) We have also showed that estradiol may activate transcription from a portion of the It was recently demonstrated that estradiol regulates LEF-1 and Tcf3, and more importantly, that a complex containing ERα and Tcf3/LEF-1 may be immunoprecipitated from mouse uterus extracts versus proteolysis??) Further studies will be required to clarify whether the same ERα/β-catenin/Tcf-3 complex observed in the uterus is also present in neurons or in some specific brain regions. It will be interesting analyzing other possible components that might mediate the effect of the estrogen receptor on TCF/LEF-1 transcription, such as transducin beta-like protein 1 (TBL1) and its highly related family member TBLR1 Several publications have recently emerged in which new elements have been described that regulate the Wnt-β-catenin pathway. For example, c-Jun forms a complex with β-catenin and Dvl in the nucleus, and this association regulates the transcriptional activity of β-catenin To determine more specifically whether estradiol can regulate gene expression using the TCF/LEF-1-β-catenin system in a way similar to that of Wnt3a, we generated N2a-m cells stably transfected with Δ56LEF-1 cDNA. By analyzing the gene expression profile of these cell lines on an Aplied Biosystems Mouse Genome Survey Microarray, we detected 223 genes modified by estradiol; whereas Wnt3a modified 303 genes, sensitive to Δ56LEF-1 . Once 8–10 embryonic cortices were obtained, they were finely cited, washed twice in HBSS 1× buffer,and incubated in 0.25% trypsin (GIBCO) and 1 mg/ml DNAse (Roche) for 15 min at 37°C. Trypsin and DNAse were then eliminated by washing three times, with HBSS 1×, and the cut tissue was then triturated using a siliconized pipette. The cells were counted and plated in a poly-lysine coated  60 mm dish containing plating medium . After 3 hours plating, the medium was changed to phenol red-free Neurobasal medium supplemented with B-B27 GIBCO) and the neurons were maintained under these conditions for 2 days. Murine neuroblastoma N2a-m cells were grown at 37°C in 7% CO2, in DMEM supplemented with 10% fetal calf serum (GIBCO) and 2 mM glutamine.Mice were treated following the guidelines of Council of Europe Convention ETS123, recently revised as indicated in the Directive 86/609/EEC. In addition all protocols were approved by the institutional animal care and use committee. Cortical neurons were obtained from E18 mouse embryos after isolating the cortex in CaOne day before treatment, the medium was changed to serum and phenol red free-medium The compounds used for treatments were: 17-β-estradiol (stored in ethanol at a concentration of 1 mM), ICI 182780 , Wnt3a recombinant protein , and the estrogen receptor agonists, 4,4′,4″-(propyl-[(1)H]-pyrazole-1,3,5-triyl) trisphenol  and 2,3-bis (4-hydroxyphenyl) propionitrile (DPN (β-selective) agonists, both dissolved in ethanol. Different concentrations of these compounds were used as indicated for each experiment and the controls received the vehicle of each compound alone, ethanol in the case of estradiol, DMSO for ICI 182780 and PBS-BSA 0.1% as control for Wnt3a. The final concentration of ICI 182780 used was depending on the concentration of estradiol, in a ICI/estradiol ratio of 100 times.3VO4, 5 mM EDTA, 1% Triton and a protease inhibitor cocktail (Roche). The cells were left for 30 min on ice in this lysis buffer and then collected with a cell scraper. After adding loading buffer, the samples were boiled for 10 min and resolved by Tris/Glycine SDS-Polyacrylamide gel electrophoresis, and the proteins were then transferred to a nitrocellulose membrane (Amersham) in the presence of 20% methanol and 0.1% SDS. Non-specific signals were blocked by incubating the membrane in PBS-Tween-20 (PBT) and 5% milk for 2 h. The antibodies used to probe the membranes were raised against: Ct ERα 1∶800 , ERβ 1∶1000 , GSK3 α/β 1∶1000 , GSKPSer9, 21 1∶1000 , β-catenin 1∶800 (Transduction Labs), non-phospho β-catenin 1∶800 (Upstate), actin 1∶2000 (Sigma), LEF-1 HMG box 1∶800 (Sigma), cyclin D1 1∶1000 (Santa Cruz), cyclin D2 1∶1000 (MBL), N cadherin 1∶1000 (Sigma), E cadherin 1∶800 (Santa Cruz), MMP2 1∶200 (Santa Cruz), nucleolin 1∶1000 (Santa Cruz), beta-galactosidase (ICN Biomed-Cappel), β-III tubulin (Chemicon). These antibodies were then detected with horseradish peroxidase conjugated secondary antibodies (Amersham) used at a dilution of 1∶5000.After two washes with PBS, cell extracts were prepared in lysis buffer containing 200 mM Hepes pH 7.4, 100 mM NaCl, 100 mM NaF, 1 mM Na4Cl and then blocked and permeabilized for 30 min with 1% fetal calf serum and 0.1% Triton-X-100 in PBS. The cells were incubated with the primary antibodies for 2 h at RT and after 3 washes with blocking solution, the secondary Alexa fluor-488 or 594 antibodies were added . The primary antibodies used were raised against β-catenin  or ERα . To visualize the distribution of polymerized actin, fixed cells were stained with phalloidin conjugated to Alexa 488 or 594, as indicated in each experiment . Coverslips were mounted with Fluoromount G  and photographs were taken on a confocal microscope .To study the localization of proteins by immunocytochemistry, we fixed cells plated at low density with 4% paraformaldehyde for 20 min at RT. After 3 washes for 10 min in PBS, fixed-cells were incubated with 50 mM NHThe plasmids used for transient expression were: pTOPFlash (Upstate), containing three copies of the consensus sequence recognized by the TCF/LEF transcription factors (CCTTTGATC) and with the c-Fos promoter driving the expression of luciferase gene; pFOPFlash (Upstate) that contains three non-functional copies of the TCF/LEF binding site (CCTTTGGCC); and EGFP-N1 encodes an enhanced green-fluorescent protein used to quantify the efficiency of transfection when co-transfected with TOP/FOPFlash. N2a-m cells were transfected using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. For transfection, 600,000 cells were plated in a 60 mm-culture dish with growth medium and the day before transfection, the medium was changed to DMEM without FCS or antibiotics. Co-transfections were performed with 2 µl LipofectAMINE (Invitrogen) and 1 µg of total-DNA (0.75 µg TOP/FOP plus 0.25 µg EGFP-N1). After transfection, the medium was changed to DMEM without phenol red or FCS, supplemented with an antibiotic cocktail. Treatments were started 48 h after transfection to coincide with the maximum expression levels of the co-transfected proteins.Other plasmids used for transfection were: empty pcDNA3 as an internal control; Δ45 β-catenin, a deleted form of β-catenin lacking the N-terminal (kindly provided by Dr. R. Moon); and LEF-1Δ56 (kindly provided by Dr. R. Grosschedl) that expresses a form of LEF-1 that is unable to interact with β-catenin.6 cells, and both luciferase activity and the efficiency of nucleofection were analyzed after 2 days in culture.The TOPFlash plasmid was introduced into cortical neurons by nucleofection  according to the manufacturer instructions. A total of 3 µg TOPFlash plus 1 µg GFP was introduced into 4×104, 5 mM ATP, 1 mM DTT, 100 µg/ml BSA) mixed with 20 µl of the cell extract. The luciferine reaction substrate (Promega) was used at a concentration of 1 mM (dissolved in sterile water), and we determined the quantity of luciferase expressed in the cells by measuring the reaction at 560 nm with a luminometer . The values obtained correspond to the mean and standard deviation of three sets of independent experiments. To analyze the induction of β-catenin co-activator function, the values were normalized to controls (corresponding to the ratio transfected/non treated cells).After treatment of the TOPFlash transfected N2a-m cells, they were incubated in 60 mm plate with 1 ml of TEN buffer  for 15 min at RT to detach the cells from the dish. Once the cells had been collected and pelleted, they were resuspended in 100 µl of lysis-buffer  and incubated on ice for 30 min. The lysate was then centrifuged at 4°C, 5 min to eliminate aggregates and the supernatant was transferred to a fresh tube. A fraction of this supernatant was resolved by electrophoresis to check the cellular mass and the amount of protein was quantified. The rest of the cell extract was used to measure the luciferase activity in 200 µl of luciferase buffer . After incubation on ice for 30 min, the lysate was centrifuged  to eliminate possible aggregates. The supernatant  was incubated in the following buffer: 2% Triton X-100, 20 mM Tris pH 7.4, 2 mM EDTA pH 8, 2 mM EGTA pH 8, 0.4 mM sodium ortho-vanadate, 0.4 mM PMSF, 1% NP-40) in the presence of 5 µg of anti-ERα antibody in a total volume of 500 µl. The mixture was incubated for 1 h at 4°C and subsequently, 10 ul of Protein A-Agarose solution (Sigma) was added and incubated at 4°C for 30 min with agitation. The agarose beads were recovered by centrifuging at 16000×g for 4 min at 4°C, and the pellet was washed three times with buffer A and centrifuged (16000×g at 4°C). Finally, the pellet was resuspended in 30 µl of 2× electrophoresis sample buffer , boiled for 10 min and loaded onto an SDS-PAGE gel to be analyzed by western blotting.Inmunoprecipitation assays we carried out on a confluent culture dish of N2a-m cells that were collected in 0.5 ml of immunoprecipitation buffer A . The homogenate was centrifuged at 2500 rpm for 10 min at 4°C, the supernatant was discarded and the pellet was resuspended in buffer A without Triton, and centrifuged in the same conditions. The pellet obtained was resuspended in buffer B  and homogenized again. The extract was carefully loaded over buffer C  and spun at 12000 rpm for 60 min at 4°C. The final pellet of the nuclei was resuspended in 50 µl of buffer A, 150 µl of protein extraction buffer  was added and the mix was incubated on ice for 30 min. To finally obtain the soluble fraction of nuclear proteins, the extract was centrifuged at 35000 rpm for 30 min at 4°C and the protein concentration in the extracts was measure using the Lowry reaction.After the dissection of the cortex from 12–16 E18 embryos, the tissue was homogenized in buffer A . Once two confluent P100 mm dishes were collected in a fresh tube , cells were incubated for 15 min on ice and then 40 µl of 10% NP-40 was added and vortexed for 20 sec. The supernatant was centrifuged for 30 sec at 15000 rpm to obtain the cytoplasm fraction. Once dry-resuspended the pellet containing the nuclei, 50 µl of EMSA buffer B  was added at 4°C with vigorous shaking for 15 min. The samples were then centrifuged for 30 sec at 15000 rpm and the supernatant recovered as the nuclear extract. The EMSA assay was performed as described previously 5′-GTCGCCCTTTGATCTTACC-3′, reverse: 5′-GTCGGGTAAGATCAAAGGG-3′, synthetic oligonucleotides from Invitrogen). When antibodies were used in the EMSA assays, 2 µg of antibody against TCF-1-X, TCF-3-X and LEF-1-X (from Santa Cruz) and 1 µg of antibody against β-catenin (Transduction Labs) or 0.8 µg of antibody against ERα  was added 20 min after the binding reaction had terminated. Competition experiments were performed to recognize the specific bands corresponding to the TCF/LEF-1-DNA complex using TCF/LEF containing the mutated sequence  and unlabelled-wild type TCF/LEF sequence at different final concentrations (20 ng and 100 ng). In all cases, the binding reaction was carried out at a final concentration of 60–65 mM KCl and in the presence of poly dIdC (1 mg/ml) and CHAPS (20%). The results of the binding reaction were analyzed in 4.5% acrylamide/bisacrylamide gels run at constant 130 V and dried .To obtain nuclear extracts of N2a-m or cortical neurons, cells were grown in serum free-DMEM, without phenol red, to 80% confluence. After washing twice with PBS, 400 µl of EMSA buffer A was added to each P100 dish . PCR was performed under optimal conditions for each primer pair. The oligonucleotide sequences and PCR conditions used were as follows: actin: 94°C-15 s, 57°C-10 s, 72°C-20 s , cyclin D1: 94°C-15 s, 55°C-10 s, 72°C-20 s , β-galactosidase: 94°C-15 s, 64°C-5 s, 72°C-15 s .Total RNA was isolated from N2a-m cells from a confluent P100 mm dish or from 3 P60 mm dishes in the case of cortical neurons. An additional set of RNA was obtained from N2a-m stable-transfected with pcDNA3-LEF-1 56 or with empty-pcDNA3, either untreated or exposed to estradiol or Wnt3a. RNA was obtained using TRIzol (GIBCO) and it was finally resuspended in sterile-DEPC treated water. The RNA concentration was determined by spectrophotometry at 260 nm, and its integrity was checked using a Bioanalyzer Chip (Agilent). First strand cDNA synthesis was performed on 2 µg of RNA and using the reverse primer as the priming site, hybridized at 37–40°C during 5 min before the remaining reaction components were added: MMLV Reverse-Transcriptase, 2.5 mM DTT, and RT-Buffer (GIBCO) with RNAsin and 10 mM dNTPs. Elongation was performed at 37°C  and the reaction was finally heated to 94°C (5 min) to inactivate the RT enzyme. RT-PCR amplification was performed to analyze cyclin D1, c-myc, actin, β-galactosidase, actin and GADPH expression using the Master SYBR Green I mix (Roche) with 2 mM MgCl5′-ATCCTCTGCATGGTCAGGTC-3′, reverse: 5′-CGTGGCCTGATTCATTCC-3′, 315 bp), endogenous gene . The mouse colony was maintained in heterozygosis and it was necessary to check each embryo when used for primary neuronal cultures. Accordingly, a portion of the cortex was placed in a mixture containing “Dye Solution” (5 mM K4[Fe(CN)6].3H20, 5 mM K3[Fe(CN)6], 2 mM MgCl2) and X-Gal (1 mg/ml).The transgenic mice containing the lacZ gene under the control of three consensus TCF/LEF-binding motifs upstream of the c-fos promoter have been described previously Gene expression profiles were generated using Applied Biosystems Mouse Genome Survey Microarray. Total RNA pools were cleaned using the RNeasy kit from Qiagen . Digoxigenin-UTP labeled cDNA probes were generated and linearly amplified from 1 µg of each RNA pool using Applied Biosystems Chemiluminescent RT-IVT labeling Kit. Microarray hybridization, chemiluminescence detection, image acquisition and analysis were done according to Applied Biosystems protocols and the 1700 Chemiluminescent Microarray Analyzer.log transformed, loess-normalized (http://www.bioconductor.org) and further filtered by standard Expression Array System Signal to Noise threshold (S/N greater than 3 in at least one sample). The expression profiles of the two control populations (N2a-m stable-cell lines with or without ΔLEF-1) were compared with the corresponding population, treated with either estradiol or Wnt3a.Applied Biosystems Expression System software was used to extract Assay Signal, and Assay Signal to Noise ratio values from the microarray images. Bad spots flagged by the software were removed from the analysis. The Assay Signal of the represented genes was Figure S1N2a-m cells express both the ERα and ER{lower caes beta} isoforms of the estrogen receptor and they respond to estradiol. Immunocytochemistry with anti-ERα and ERβ identified a different cytoplasmic location for the two isoforms in both cell lines. While ERα presented was diffusely distributed throughout the cytosol and nucleus, and it accumulated in the nucleus after the addition of estradiol (A–B). ERβ distribution was more heterogeneous and it could be found in aggregates, diffusely distributed in the cytosol or in the nucleus(C–D). The addition of estradiol (B and D) increases the immunoreactivity against two receptors in the nucleus. Images were analyzed by confocal microscopy and western blotting confirmed the expression of both isoforms in total cell extracts from control cells.(9.68 MB EPS)Click here for additional data file.Figure S2Distribution of β-catenin after exposure of N2a-m cells to estradiol does not reveal the translocation of β-catenin to the nucleus. Cell fractions corresponding to the soluble, membrane and nuclear fractions were analyzed in control and estradiol treated cells. Neither 30 nor 60 min exposure to estradiol provoked the nuclear accumulation of β-catenin, statistically significant. In contrast, recruitment of β-catenin to the membrane fraction was more evident. We also checked the movement of ERα as a control of the assay, which clearly accumulated in the nuclear fraction see .(2.36 MB EPS)Click here for additional data file.Figure S3N2a-m cells are responsive to Wnt3a protein. (A)- N2a-m cells respond to Wnt3a (20 ng/ml) with the accumulation of β-catenin and with a maximal effect observed after 90–120 minutes. (B)- The transcriptional activity of β-catenin was analyzed with the TCF-luc reporter. Luciferase activity was calculated at different concentrations of Wnt3a protein, as indicated above, and Wnt3a produced a significant increase in β-catenin/TCF dependent transcription. Asterisk represents P value from Student's t-test: * (P≤0.05), when compared the two Wnt concentrations; and ** (P≤0.01) when compared with control.(1.61 MB EPS)Click here for additional data file.Figure S4Neither ERα nor TCF-3 antibodies modify the migration of nuclear protein extracts from N2a-m cells exposed to estradiol or Wnt3a. Nuclear protein extracts were obtained from control or estradiol/Wnt3a treated cells as indicated in each lane. The incubation with specific antibodies against ERα or TCF3 did not produce a higher molecular weight band that migrated more slowly than that previously identified as a TCF-DNA complex, nor did they prevent the formation of the complex.(2.50 MB EPS)Click here for additional data file.


However, the molecular defects that contribute to the activation of this pathway have not been elucidated. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) is a candidate for the regulation of cytoplasmic β-catenin. In this study, we developed in situ hybridisation probes to evaluate the presence of FRAT1 and used an anti-β-catenin antibody to evaluate by immunohistochemistry the expression levels and subcellular localisation of β-catenin in ovarian cancer tissue microarrays. Expression of FRAT1 was found in some human normal tissues and 47% of ovarian adenocarcinomas. A total of 46% of ovarian serous adenocarcinomas were positive for FRAT1 expression. Accumulation of β-catenin in the nucleus and/or cytoplasm was observed in 55% ovarian adenocarcinomas and in 59% of serous adenocarcinomas. A significant association was observed in ovarian serous adenocarcinomas between FRAT1 and β-catenin expression (P<0.01). These findings support that Wnt/β-catenin signalling may be aberrantly activated through FRAT1 overexpression in ovarian serous adenocarcinomas. The mechanism behind the overexpression of FRAT1 in ovarian serous adenocarcinomas and its significance is yet to be investigated.The mechanisms involved in the pathogenesis of ovarian cancer are poorly understood, but evidence suggests that aberrant activation of Wnt/ Ovarian cancer is the sixth most common cancer in women worldwide and a highly aggressive gynaecological malignancy producing almost 125 000 deaths yearly . Furtherβ-catenin has been observed in ovarian cancer  family and activates the transcription of Wnt/β-catenin target genes, which are involved in cellular differentiation and proliferation  or Axin occur rarely in ovarian cancer (β-catenin levels in other types of ovarian tumours. Previous studies have shown that frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) is strikingly overexpressed in some human cancer cells  activity towards β-catenin, at least in part, by preventing Axin binding to GSK-3  were cut from the array blocks using tape sectioning materials from Instrumedics .TARP5-T-BO-1 tumour tissue arrays were constructed by the NCI TARP Lab using anonymous donor blocks obtained from the Cooperative Human Tissue Network. Detailed information about TARP5-T-BO-1 tumour tissue arrays is available at eviously , using aFRAT1 cDNA obtained from HEK293 cells into pcDNA3 vector . Correct cloning of FRAT1 cDNA fragments was confirmed by sequencing. The resulting plasmid was designated FRAT1/pcDNA3. Probes for in situ hybridisation were generated by in vitro transcription using SP6 polymerase  as described below.The FRAT1 expression vector was constructed by cloning the open reading frame of the human in situ hybridisation, digoxigenine (DIG)-labelled FRAT1 probes were prepared using a DIG-RNA-labelling kit (Roche), according to the manufacturer's instructions. In brief, FRAT1/pcDNA3 plasmids were linearised with HindIII and DIG-labelled probes were generated by in vitro transcription using SP6 polymerase (Roche). The amount of transcripts was monitored by 2% agarose gel electrophoresis and determined by spectrophotometry. Labelling efficiency was controlled by dot blot analysis of serial probe dilutions.For In situ hybridisation was performed according to the method described previously (−1 HCl for 10 min at room temperature and then permeabilised using proteinase K (100 mg l−1) at 37°C for 15 min. Digestion was stopped by washing the samples in phosphate-buffered saline (PBS) (pH 7.4). The samples were then dehydrated in serial dilutions of ethanol. DIG-labelled FRAT1 probes were diluted in hybridisation buffer . After probes were applied, the samples were covered with sterile coverslips. Hybridisation was performed overnight at 42°C in a sealed humidified chamber containing 50% formamide. Nonspecific binding or unbound probes were removed by the following posthybridisation washes: 2 × SSC at room temperature (2 × 10 min), 1 × SSC at room temperature (2 × 10 min), and finally, the sections were washed in TBS containing 0.1% Tween-20 (TTBS). Hybridisation signals were detected using an alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche). After washing in TTBS, the slides were incubated with nitro blue tetrazolium/5-bromo-4-chloroindol-3-yl phosphate (NBT/BCIP) (Roche) for 10 min. The FRAT1-positive cells were purple–blue stained. The negative controls were incubated with hybridising solution without FRAT1 probes.M, pH=6.0) in a microwave oven. To reduce background nonspecific staining, slides were incubated with normal nonimmune serum for 30 min. The excess serum was removed and slides were incubated for 60 min with anti-β-catenin . Each incubation step was carried out at room temperature and was followed by three sequential washes (5 min each) in PBS. Slides were then incubated with biotin-conjugated second antibody for 30 min, followed by incubation with streptavidin–peroxidase for 30 min. Peroxidase activity was identified by applying 3,3′-diaminobenzidine tetrachloride (DAB). Sections were rinsed in water and counterstained with haematoxylin. Finally, slides were dehydrated in alcohol, cleared in xylene. Slides were also stained in the absence of primary antibody to evaluate nonspecific secondary antibody reactions.Immunohistochemistry was performed using an UltraSensitive™ Kit (Maixin-Bio) according to the manufacturer's protocol as described previously  and high (scores 1–3) expression of FRAT1. Meanwhile, β-catenin protein expression profiles in cancer tissues were further grouped into cytoplasm/nucleus accumulation or no accumulation.All FRAT1 expression levels and β-catenin expression profiles on a per case basis was further analysed by Spearman's ρ correlation coefficient test. Values of P<0.05 were considered statistically significant.SPSS for Windows (SPSS Inc.) was used for statistical analysis. Correlation between the in situ hybridisation with the probes directed at FRAT1 was located in the cytoplasm of the cells  ovarian adenocarcinomas investigated. In total, 46% (19 out of 41) of all ovarian serous adenocarcinomas were positive for FRAT1 expression, followed by four out of 11 clear cell, four out of five endometrioid and one out of three mucinous adenocarcinomas (β-catenin expression with variable extension and intensity. Accumulation of β-catenin in the nucleus and/or cytoplasm was observed in 33 of 60 (55%) ovarian adenocarcinomas. In serous adenocarcinomas, the percentage of cases with β-catenin accumulation was approximately 59%. Nuclear and/or cytoplasmic expression of β-catenin was also observed in four out of 11 clear cell, five out of five endometrioid and zero out of three mucinous adenocarcinomas . These results suggest that FRAT1 may modulate the Wnt/β-catenin pathway in ovarian serous adenocarcinomas.To examine whether of FRAT1 ; Table 2β-catenin in ovarian cancer are a little confusing. However, it is consistent that nuclear β-catenin is almost exclusively present in endometriod type of ovarian cancer  inactivation by methylation -infected Eì-Pim1 or H2-K-Myc transgenic mice (Xenopus homologue GSK-3-binding protein (GBP) was identified as a GSK-3-binding protein  (β-catenin pathway. Since the identification of human FRAT1, an additional homologue has been discovered in the human genome. FRAT2, just like FRAT1, functions as positive regulator of the Wnt pathway. FRAT1 and FRAT2 are more homologous in the N-terminal region, but are completely divergent in the C-terminal region (FRAT1 span the C-terminal coding region of this gene, minimising the potential for crossreactivity between human FRAT1 and FRAT2. Recently, results from ne Frat1 . Frat1 wyclin D1  and humain situ hybridisation probes to demonstrate the presence of FRAT1 in paraffin-embedded human normal tissues and ovarian cancer tissues. To our knowledge, no other research group has been able to produce in situ hybridisation probes against FRAT1 to date. In situ hybridisation seems to be a more selective and more specific technique than immunohistochemistry, but requires greater care in preparing nuclease-free solutions. Tissue microarrays were also used for the rapid and efficient analysis of large numbers of paraffin-embedded tissues (in situ hybridisation to detect the expression of FRAT1 and by using immunohistochemistry to detect the expression of β-catenin on tissue microarrays of paraffin-embedded ovarian adenocarcinomas, we report here that cytoplasmic/nuclear accumulation of β-catenin is closely associated with FRAT1 overexpression in ovarian serous adenocarcinomas. Although it is uncertain whether FRAT1 expression is the cause or effect of cytoplasmicβ-catenin in certain circumstances, until now, almost all data shown in the literatures indicate that FRAT1 is a positive regulator of the Wnt/β-catenin pathway. No reports now suggest that Wnt/β-catenin pathway can regulate the expression of FRAT1. Thus, a strong correlation between expression of FRAT1 and the subcellular localisation of β-catenin indicates that FRAT1 may modulate the Wnt/β-catenin pathway in ovarian serous adenocarcinomas. A study with larger cohort seems mandatory to clarify whether the same role of FRAT1 exists in other histological subtypes of ovarian cancer, such as clear cell, endometrioid and mucinous adenocarcinomas. In addition, we report that FRAT1 is detected in some normal human tissues, including endometrium, testis, pancreas and prostate. Similar results have been obtained by other researchers (FRAT1 expression levels in the ovary were relatively low but detectable. Frat is expressed in rapidly dividing cells (FRAT1 may also be observed, but it needs further investigation.In this study, we used  tissues . By usinFRAT1 mRNA and β-catenin protein expression were studied in 60 ovarian adenocarcinomas, including 41 serous, 11 clear cell, five endometrioid and three mucinous adenocarcinomas. We report the expression pattern of FRAT1 in some normal human tissues and that overexpression of FRAT1 in ovarian serous adenocarcinomas is significantly associated with cytoplasmic/nuclear accumulation of β-catenin. Wnt/β-catenin signalling may be aberrantly activated through FRAT1 overexpression in ovarian serous adenocarcinomas. The mechanism behind the overexpression of FRAT1 in ovarian serous adenocarcinomas and its significance is yet to be investigated.In conclusion,


Exenatide is a unique agent which can effectively control blood glucose levels in type 2 diabetes mellitus without producing dangerous adverse effects. In addition, it can lower body weight which is very essential for the treatment of obese type 2 diabetes mellitus patients. Since it can delay the destruction of islet beta-cells, type 2 diabetes mellitus patients are not rapidly converted to type 1 diabetes mellitus and ultimately appearance of complications of the disease is halted or delayed. Its long-acting-release formula, which would be used once per week, simultaneously retaining all the properties of twice-daily subcutaneous administration, is undergoing clinical trial. This drug is considered as an adjunct to metformin/sulfonylureas/insulin. Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder, where, the currently used antidiabetic drugs could not retard the progression of the disease. Therefore, agents, effective in this respect, are needed which can delay such progression. Otherwise, diabetes-associated complications will appear within a short period and there is the possibility of such patients being converted to type 1 diabetes mellitus (T1DM) individuals. Moreover, in spite of multidrug therapy, even with insulin in various combinations and dosage regimens, it has not been possible to achieve proper glycemic control in a significant number of T2DM patients. As the pathogenesis and pathophysiology of the disease is multifactorial, oral antidiabetic agents with different modes of actions have been developed and are in use with various degrees of success. Recently, the role of incretins, particularly that of glucagon-like peptide-1 (GLP-1), in metabolic homeostasis in general and that of glucose in particular, has been firmly established. But, pharmacokinetic studies of endogenous GLP-1 have shown the incretin to have a plasma half-life of only few minutes which makes it unsuitable for routine therapy in T2DM. Hence, attempts were made to find out GLP-1 like substances in other animal species and/or synthesize compounds with GLP-1 like (GLP-1 agonists) action having longer plasma half-life[c], and need for pharmacotherapy. Unfortunately, most of the currently available antidiabetic drugs, including insulin, cause weight gain. Therefore, pharmacotherapy for obesity, as part of an integrated management plan, is beneficial for maintaining wt loss, optimising glycemic control and probably delaying progression of the disease[T2DM, being a disease of overnutrition, its onset and progression are associated intimately with obesity in which there is excess fat accumulation in the abdomen, muscles and liver. Hence, modest weight (wt) loss (approximately 7%) by restricted diet and exercise can check or delay the onset of this disease. It has been observed that in such patients, weight loss decreases fasting and postprandial (pp) plasma glucose levels, glycosylated hemoglobin [HbA1Heloderma suspectum). This lizard takes food four times a year and during feeding, EX-4, secreted in the saliva, is thought to help its pancreas to switch on[Exendin-4 (EX-4), an incretin-mimetic peptide hormone (containing 39 aminoacid residues) having GLP-1 like action3 and concswitch on. Exenatiswitch on7 and actswitch on.EX is the first incretin-mimetic compound that has been approved by USA Food and Drug Administration (FDA) in April, 2005, as an adjunctive therapeutic agent to improve glycemic control in T2DM patients who have suboptimal glycemic control with either sulfonylurea or metformin monotherapy–58 or wi5EX stimulates mammalian receptors for truncated GLP-1 (tGLP-1) with a relatively prolonged action and much44555et al[Couto et al have recThe N-terminal region of GLP-1 and EX-4 are almost identical (they share 53 per cent amino acid sequence similarity), a significant difference being in the second amino acid residue alanine in GLP-1 and glycine in EX-4. Another important difference is that EX-4 has an extra nine amino acid residues at its C-terminus as shownet al[Some researchers have shown that both of them (GLP-1 and EX) bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency–15. ContAs far as glucose-lowering activity of EX  is concerned, it is up to 3000-fold more than exogenous GLP-1. While iEX has several mechanisms of action, the ultimate outcome being better glycemic control. Some of the actions are acute (immediate), glucose-dependent insulin secretion, suppression of pp high glucagon concentrations, delayed gastric emptying, inhibition of food intake, and modulation of glucose trafficking in peripheral tissues, while others appear late: weight loss, improved beta-cell mass and function916.9EX increases pancreatic response to food leading to enhanced secretion of higher and more appropriate amount of insulin that assists in lowering the pp rise in blood sugar. It cont45517EX also checks pancreatic release of glucagon in response to food  in T2DM patients, some workers attempted to see the response of it on beta-cell in animals with T1DM. As T1DM is an autoimmune disease, it is associated with near complete beta-cell destruction. At present some evidences suggest that beta-cell regeneration is possible, but ongoing autoimmune damage checks restoration of beta-cell mass. A novel approach to reverse T1DM may be possible by simultaneously blocking autoimmune cytokine damage and supplying a growth-promoting stimulus for beta-cells. For that reason, in one study, researchers combined lisofylline to suppress autoimmunity and EX-4 to increase beta-cell proliferation for treating autoimmune-mediated diabetes in non-obese diabetic (NOD) mouse model. They observed that this therapy effectively checked new-onset diabetes within a week of combined treatment, and even mediated euglycemia up to 145 d after drugs withdrawal. The resultant effect of this therapy was associated with improved beta-cell metabolism and insulin secretion, while decreasing beta-cell apoptosis. It is possible that such unique therapy could become a new strategy to control T1DM in humans.max) of 211 pg/ml is achieved in 2.1 h  along with a mean area under the curve (AUC) of 1036 pgh/ml[281/2) of EX is 26 min in humans, it is 1-2 min for biologically active GLP-1. As has been mentioned earlier, this difference is because of penultimate NH2-terminal glycine (in EX) instead of alanine (as in GLP-1)[Since EX is a peptide, it needs to be administered parenterally. It is a836 pgh/ml. After a36 pgh/ml28. The k36 pgh/ml2829. Its pgh/ml28. Its dos pgh/ml28 as found pgh/ml288. While Adverse drug reactions of EX are limited and mild to moderate in nature1930. The195288323232According to one study report, even doses up to 10 times the recommended dose were accidentally administered in three of the subjects, resulted in severe hypoglycemia requiring parenteral glucose administration. But the recovery in all the cases was uneventful.The use of metformin and EX combination does not increase the incidence of nausea and the 3321Approximately 45 per cent of T2DM patients receiving EX were positive for antiexenatide antibodies, with the majority of them having low titres range (<1/125). It has As EX delays gastric emptying, caution should be taken when the drug is co-administered with certain drugs like digoxin, lovastatin, lisinopril, acetaminophen, antiinfectives and oral contraceptives28. It haEX is not indicated in T1DM or diabetic ketoacidosis as it is not an insulin substitute. It has not been recommended for diabetics with end stage renal disease (creatinine clearance <30 ml/min) and severe gastrointestinal disorder (like gastroparesis)3.EX is administered in prefilled pen. The pen5EX has undergone extensive clinical trials, being used alone or in combination with metformin/sulfonylurea, by several groups of researchers over different time periods with various doses. Such trial results were found to be highly encouraging for therapeutic use of the compound in T2DM either alone or as an adjunct to metformin/sulfonylurea with tolerable adverse effects.et al[c (P<0.009) after one month of treatment. In another clinical trial lasting 30 w, EX therapy was associated with moderate decrease in mean HbA(1)c level of approximately 0.8 per cent and an average weight loss of about 2 kg compared with baseline[c levels (0.3 to 0.7% more than placebo), fasting plasma glucose and body weight (1.4 to 2.3 kg).Administering EX once- or twice-daily bolus s.c. injections, Egan et al conducte baseline. Similar baseline reported baseline describeCvetkovic and Polsker have menet al[Fineman et al performeet al. Incidenet al.c of about 8.5 per cent and mean body weight of approximately 99 kg, the use of EX 5 μg and 10 μg s.c. twice-daily with either metformin/sulfonylurea or metformin and sulfonylurea, indicated a decrease in HbA(1)c by 0.6 and 0.9 per cent and body wt by 3.1 and 4.2 kg, respectively as compared to placebo. Long-term extension data in 265 patients showed that the reduction of HbA(1)c was maintained even at 82 w (-1.1% from baseline) and the wt gradually continued to decrease over time (-4.5 kg from baseline). In addition, there were useful effects on lipid profile with small but important reductions in LDL and triglycerides and a rise in HDL cholesterol[c, fasting and pp plasma glucose compared with baseline when added to metformin/sulfonylureas or a combination of both, with a mean weight loss of about 2 kg. Especially during initiation of therapy with sulfonylureas (not with metformin), hypoglycemia was encountered[c and body weight which was maintained up to varying periods (from 30 to 82 w). Weight loss was more with EX and metformin than EX and sulphonylureas[38c[333833In three 30-w double blind, placebo controlled studies, in more than 1400 patients with a mean HbA(1)countered. In a pronylureas. Adjunctonylureas. In someonylureas and fastonylureas3843, HbAureas[38c333843 an38c333833.et al[c and a progressive decrease in weight over 82 w with T2DM failing to achieve glycemic control with metformin alone. In one placebo controlled blind study, Fineman et al.[c to be less than 7 per cent in 15 per cent of patients as compared to only 4 per cent in the placebo group (P<0.006).According to Ratner et al, EX was an et al. took 109c. While EX therapy was associated with notable reductions in body weight and pp glucose excursions, insulin glargine caused significantly greater decrease in fasting serum glucose[In another study, treatment with EX or insulin glargine for 16 w was accompanied with similar significant improvements in HbA(1)m glucose. Based om glucose.After successful clinical trials, EX has been approved to be used in T2DM patients with either metformin or sulfonylurea or thiazolidinedione monotherapy or with metformin and sulfonylurea combination therapy. Some physicians also administered EX alone or in combination with insulin. During such use, the merits and demerits of the drug has been assessed while administered alone or as an adjunct with others, as mentioned earlier.c[et al[et al[In addition to positive therapeutic effects on fasting and pp glucose levels, EX treatment has been found to be associated with significant, dose-dependent decrease in HbA(1)c from basc19. It hac[et al also men al[et al reported al[et al. Since E al[et al. It is d al[et al.c. On the other hand, EX produced decrease in body weight while insulin analogue caused weight gain. So, EX is a treatment option in insulin-naïve patients with T2DM and who are overweight and suboptimally controlled by metformin and sulfonylurea[EX offers a unique treatment option for T2DM patients who are refractory to metformin or sulfonylurea or both32. Consifonylurea.c, systolic blood pressure, triglycerides and high-sensitivity C-reactive protein (CRP)[c concentrations fell just above 7 per cent in both groups (1.1% drop from baseline). But the body weight in patients using insulin glargine increased by a mean of 1.8 kg during the study, while that of subjects using EX declined by an average of 2.3 kg. Such investigation results suggest that insulin glargine and EX produce similar benefits as far as HbA(1)c levels are concerned, but EX has added advantage of weight loss. During this study it was also observed that nausea occurred in about 9 per cent of patients using insulin glargine, while it was found in 57 per cent of patients receiving EX. The incidence of vomiting was about 4 per cent of T2DM in insulin glargine group and 17 per cent in patients using EX (P<0.001). Comparable hypoglycemic rate was seen in both the groups, 6.3 events per patient-year in insulin glargine group and 7.3 cases per patient-year in the group using EX. However, the incidence of nocturnal hypoglycemia was slightly more in the patients taking insulin glargine[Another useful finding is that EX effectively treats obese subjects with T2DM on insulin, leading to weight loss and decrease in levels of HbA(1)ein (CRP). Chronicein (CRP). Adminisein (CRP). On the ein (CRP). Virji[5ein (CRP) has alsoein (CRP). Compari glargine.c decrease. Since EX therapy provided better glycemic control, it is considered to be a potential alternative for the treatment of T2DM. Moreover, its therapeutic advantage is wt reduction[c[Similar to biphasic insulin aspart, EX treatment also resulted in HbA(1)reduction. EX can reduction. Althougduction[c.c, the extra clinical effects of improved lipid levels, systolic blood pressure, and lowered body mass index all positively contributed to the cost-effectiveness of EX[In USA, one long-term cost-effectiveness of Ex was estimated in T2DM patients, where it was found to improve cost-effectiveness. In addition to sustained reduction in HbA(1)ess of EX.The initial recommended dose of EX (in combination with metformin or sulfonylureas) is 5 μg (s.c.) twice-a-day, administered within 60 min before the morning and evening meals52831. Th5283When EX is used with metformin in T2DM patients, metformin dose may not be adjusted. On the other hand, when it is administered with sulfonylurea, dose reduction of sulfonylurea should be considered because such combination is liable to cause more hypoglycemia. EX decrOne of the important drawbacks of EX therapy is its twice-daily parenteral administration. To overcome this disadvantage a long-acting injectable preparation of the compound has been formulated which can be used once-a-week62, even Phase 3 clinical trials have been conducted to find out the plasma concentration and effects of EX LAR preparations administered at different time intervals. In these dosage forms, EX is being placed inside a biodegradable polymeric microsphere, which produces a sustained-release formulation that can be injected once-a-week, once-every-other week, and once-a-month. Such preparations have been found to maintain adequate plasma concentrations of the active drug for weeks to months after a single dose. Following injection of EX LAR, a small amount of active drug diffuses out of this polymeric microsphere due to hydration of the polymer, and there is continued stable release of active drug due to progressive breakdown of the microsphere over time.c and body weight (loss of weight)[In a 16 w study, 2 doses of EX LAR administered once-weekly were well tolerated and simultaneously achieved dose-dependent improvements in HbA(1)f weight). In T2DMf weight).et al[c levels were decreased with 2 mg dose.Kim et al used twoc as effectively as EX LAR along with the wt loss profile, this may improve further with longer-term use. Considering patient's compliance, once-weekly formulation of the drug (EX LAR) may replace EX BID for the treatment of T2DM patients. Whether these useful effects of EX are maintained in the longer-term use and it has effects on pancreatic beta-cell regeneration in humans remain to be studied. So, longer-term large scale studies are needed to gain further insight into treatment with EX LAR.EX LAR once-weekly therapy is promising in T2DM, because the preparation retains all the benefits of EX BID intact without being administered frequently. No otheet al[Another option to bypass twice-daily use of EX is its intranasal administration. It requires an aqueous mixture of EX and a delivery enhancer selected from the group consisting of a solubilizer, a chelator and a surfactant. As a suet al have repEX is a synthetic GLP-1 receptor agonist. When administered subcutaneously, it was found to increase glucose-dependent insulin secretion, suppression of pp high glucagon concentration, delayed gastric emptying, inhibition of food intake, modulation of glucose trafficking in peripheral tissues and wt loss with mild to moderate hypoglycemia and gastrointestinal adverse effects.c[343234341et al[From the clinical trial results provided by different research groups and therapeutic responses observed by different physicians, EX appears to be a useful agent in the management of T2DM. Clinical trials on T2DM patients have been conducted both for short about 30 d)40 as wel0 d40 as 43234394142c–38, imprc3438 and c343238, alongc343234, nausec343234334 in coc343234340/metfor343234341, in addi34323434145/sulfon4341et al comparedc, fasting as well as pp blood glucose and body wt along with less hypoglycemia, nausea and vomiting [c, plasma glucose (both fasting and pp) and wt[39c, body weight, systolic blood pressure, triglycerides and high-sensitivity CRP[c and providing significant glycemic control like insulin, EX, in contrast to insulin, has been found to reduce wt[3et al[c in addition to improved lipid profile, decreased systolic blood pressure and body mass index; all of which positively contributed to the cost-effectiveness of the drug.After the FDA approval for marketing, EX was used for treatment of T2DM by many physicians either alone or in combination with metformin/sulfonylureas/thiazolidinediones/insulin, but its utility as a first line drug is yet to be defined. When ussymptoms). Moreovesymptoms). When EXp) and wt39. Moreo and wt39. Hence, ivity CRP along wiivity CRP. Though educe wt355–58, imeduce wt3. In addieduce wt3. Minshalwt[3et al found EXFrom the above observations it can be concluded that EX is an ideal adjunct to metformin/sulfonylureas/insulin in T2DM patients where it can potentiate the antidiabetic action without increasing hypoglycemia with the additional benefit of weight loss, thereby decreasing insulin resistance which is a major problem in obese T2DM patients. During such adjunctive therapy, though the patient requires additional daily s.c. injections and suffers from gastrointestinal adverse effects (mild nausea and vomiting)69, assoc3669One unique and exceptional property of EX has been demonstrated by several study groups, where the drug has been found to halt the progression of degeneration (apoptos


These mutations are associated with chromosomal stability, low histological grade, low tumor invasion and better patient survival. We hypothesized that canonical Wnt signaling is selectively activated in well-differentiated, but repressed in poorly differentiated HCCs. To this aim, we characterized differentiation status of HCC cell lines and compared their expression status of Wnt pathway genes, and explored their activity of canonical Wnt signaling.We classified human HCC cell lines into "well-differentiated" and "poorly differentiated" subtypes, based on the expression of hepatocyte lineage, epithelial and mesenchymal markers. Poorly differentiated cell lines lost epithelial and hepatocyte lineage markers, and overexpressed mesenchymal markers. Also, they were highly motile and invasive. We compared the expression of 45 Wnt pathway genes between two subtypes. TCF1 and TCF4 factors, and LRP5 and LRP6 co-receptors were ubiquitously expressed. Likewise, six Frizzled receptors, and canonical Wnt3 ligand were expressed in both subtypes. In contrast, canonical ligand Wnt8b and noncanonical ligands Wnt4, Wnt5a, Wnt5b and Wnt7b were expressed selectively in well- and poorly differentiated cell lines, respectively. Canonical Wnt signaling activity, as tested by a TCF reporter assay was detected in 80% of well-differentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant β-catenin was weak in poorly differentiated SNU449 cell line, suggesting a repressive mechanism. We tested Wnt5a as a candidate antagonist. It strongly inhibited canonical Wnt signaling that is activated by mutant β-catenin in HCC cell lines.Differential expression of Wnt ligands in HCC cells is associated with selective activation of canonical Wnt signaling in well-differentiated, and its repression in poorly differentiated cell lines. One potential mechanism of repression involved Wnt5a, acting as an antagonist of canonical Wnt signaling. Our observations support the hypothesis that Wnt pathway is selectively activated or repressed depending on differentiation status of HCC cells. We propose that canonical and noncanonical Wnt pathways have complementary roles in HCC, where the canonical signaling contributes to tumor initiation, and noncanonical signaling to tumor progression. Hepatocellular carcinoma (HCC) is an epithelial cancer that originates from hepatocytes or their progenitors. It is the fifth most frequent neoplasm worldwide , and its incidence is steadily increasing in the West . HepatocCTNNB1 gene that encodes β-catenin, a key component of the Wnt/β-catenin  signaling pathway.Molecular changes involved in HCC dedifferentiation and invasiveness are known only partially. Epithelial markers such as hepatocyte nuclear factors and E-cadherin were reported to be down-regulated in HCC ,5 and thGLUL gene) as a sensitive and specific marker, Audard et al. . This data supports the hypothesis that well-differentiated HCC cells display an autocrine/paracrine canonical Wnt signaling, probably because they co-express Wnt3 and several canonical Frizzled receptors. However, the great majority of poorly differentiated cell lines failed to generate canonical Wnt signaling activity, although they similarly co-expressed Wnt3 and canonical Frizzled receptors.Taken together, we collected TCF activity data from 12 HCC cell lines. Independent of β-catenin or AXIN1 status, TCF activity was detected in four out of five (80%) well-differentiated cell lines, whereas only one out of seven (14%) poorly differentiated cell lines had constitutive TCF activity [The lack of canonical Wnt activity in poorly differentiated cells could be due to either lack of sufficient Wnt ligand activity. Alternatively, canonical Wnt signaling could be repressed in these cell lines. A number of proteins downstream to β-catenin such as Axin2, HTLE family, hAES, Chibby, CTBP and ICAT are known to display inhibitory activity on canonical Wnt signaling . We compNext, we compared TCF activity in Huh7, SNU449 and SNU182 cell lines following transient expression of a mutant (S33Y)-β-catenin Figure . TransfeIn order to confirm the data on weakened response in poorly differentiated cell lines, we generated a clone from SNU449 cells (SNU449-cl8) with Tet repressor controlled expression of N-terminally truncated β-catenin (aa 98-781). N-terminally truncated β-catenin forms are frequently detected in cancer cells including HCC cells. They lack Ser/Thr phosphorylation sites  that are critically involved in its ubiquitin-mediated degradation, and they accumulate in the cell nucleus leading to oncogenic activation canonical Wnt signaling . As showPresently, the mechanism of repression of canonical Wnt signaling in poorly differentiated HCC cells is unknown, but it is associated with a lack of nuclear accumulation of β-catenin. Among noncanonical Wnt ligands, Wnt5a is best known for its antagonistic effect on canonical Wnt signaling . TherefoSince the initial description of β-catenin mutations in HCCs in 1998 , Wnt sigMutational activation of canonical Wnt signaling is not a frequent event in HCC, in contrast to hepatoblastoma displaying very high rates . MutatioWe first classified 11 HCC cell lines into "well-differentiated" and "poorly differentiated" subtypes using hepatocyte lineage, epithelial and mesenchymal cell markers, and in vitro migration assays. Well-differentiated HCC cell lines shared many features with hepatocytes such as expression of HNF-1α, HNF-4α, and E-cadherin, and epithelial morphology. Poorly differentiated cell lines were usually deficient in the expression of hepatocyte lineage and epithelial markers, but they expressed different mesenchymal markers strongly. These two types of HCC cell lines were also distinguished from each other by their in vitro behaviors. Poorly differentiated cell lines were usually more motile and more invasive than well-differentiated cell lines  lacked detectable signaling activity. Thus, most probably, the canonical Wnt signaling was not only inactive, but also repressed in poorly differentiated HCC cell lines. In confirmation of this expectation, transient or Tet-regulated expression of mutant β-catenin failed to generate significant canonical Wnt signaling activity in two different poorly differentiated cell lines. Furthermore, we linked this weak activity to poor nuclear accumulation of β-catenin protein in SNU449.cl8 cell line. Thus, unlike well-differentiated cell lines, poorly differentiated HCC cells displayed strong resistance to canonical Wnt signal activation.The mechanisms of resistance to canonical Wnt signal activation in poorly differentiated HCC cells are presently unknown. We provide here one potential mechanism. Wnt5a has been previously implicated in canonical Wnt signaling as an antagonist and regulator of β-catenin levels in other cell types ,49. UsinThe role of Wnt5a in cancer is complex. It may play tumor-promoting or tumor-suppressing functions depending on cellular context. Wnt5a has been described as a tumor promoter in melanoma, gastric, pancreas, prostate cancer, but as a tumor suppressor in HCC, neuroblastoma, leukemia, colon, and thyroid cancers . The inaThe expression status of Wnt5a in HCC is not well known. To our knowledge, only one report addressed this issue . CompareTaken together, our studies demonstrate that canonical Wnt activity is active in well-differentiated, but repressed in poorly differentiated HCC cell lines. This correlates with in vivo tumor studies indicating that β-catenin mutations are prevelant in well-differentiated, but not in poorly differentiated tumors. In addition, we showed that poorly differentiated cell lines express noncanonical Wnt ligands such as Wnt5a acting as an antagonist of canonical Wnt signaling. Thus, it appears that HCC cells may activate or repress their canonical Wnt signaling, using autocrine/paracrine systems based on selective use of canonical and noncanonical Wnt ligands.We hypothesize that the active canonical Wnt signaling observed in well-differentiated HCC cells contributes to tumor initiation, but not necessarily to tumor progression. Instead, noncanonical Wnt signaling may be used by poorly differentiated HCC tumors to promote cell motility and invasion. Selective use of canonical and noncanonical Wnt signaling at different stages may be a key mechanism involved in hepatocellular carcinogenesis. Recent studies showed that canonical Wnt signaling contributes to the self-renewal and expansion of HCC-initiating cells with stem/progenitor cell features ,70. HoweOur observations support the hypothesis that Wnt pathway is selectively activated or repressed depending on differentiation status of HCC cells. We propose that canonical and noncanonical Wnt pathways have complementary roles in HCC, where the canonical signaling contributes to tumor initiation, and noncanonical signaling to tumor progression.Hepatocellular carcinoma cell lines Huh7, Hep40, Hep3B, Hep3B-TR, FOCUS, Mahlavu, SNU182, SNU 387, SNU 398, SNU423, SNU 449, SNU 475, PLC/PRF/5, SK-Hep-1, hepatoblastoma cell line HepG2 and colorectal cancer cell line SW480 were cultivated as described previously .Total RNAs were extracted from cultured cells using NucleoSpin RNA II Kit  according to the manufacturer's protocol. The cDNAs were prepared from total RNA (2 μg) using RevertAid First Strand cDNA Synthesis Kit . A negative control without reverse transcriptase (1 μl ddH2O instead) was also prepared for each sample. All PCR reactions were carried out using 1 μl cDNA from the reverse transcription mix, for 35 cycles except GAPDH, which was amplified for 24 cycles. Negative controls without reverse transcriptase were included for each set of primers (primer sequence information is available upon request). PCR products were analyzed on a 2% (w/v) agarose gel.Cells were cultured in six-well culture plates in RPMI 1640 or DMEM with 10% FBS. A single linear wound was made with a p200 pipette tip in confluent monolayer cells. The distances between wound edges were measured at fixed points in each dish according to standardized template. Debris were removed by washing the cells twice with PBS and then cells were incubated in RPMI 1640 or DMEM with 2% FBS. After 24 hours migration, cells were fixed with methanol and stained with 0.2% crystal violet. Cell migration into the wound was visualized using phase contrast microscopy (x20 magnification). The number of cells migrating beyond the wound edge was quantified microscopically in the randomly selected fields for each triplicate well.Cells were grown on coverslips, fixed in 4% formaldehyde, permeated with 0.5% saponin/0.1% Triton X-100, and stained with mouse monoclonal anti-human vimentin antibody (Dako) using Envision kit (Dako), developed with diaminobenzidine, and counterstained with hematoxylin.® 594 F(ab')2 fragment of rabbit anti-mouse IgG (H+L) (Invitrogen) (dilution 1:750). Cells were counterstained with DAPI (dilution 1:750), slides were mounted using ProLong® Gold antifade reagent (Invitrogen) and examined under Zeiss LSM 510 Meta laser scanning confocal microscope  using 488 nm and 543 nm laser excitation lines, and photographed.Cells were grown on slides in 6 well plates and were fixed in 3.5% paraformaldehyde (PFA) for 15 minutes, and permealized using 0,25% Triton-X-100 for 10 min. Non specific protein binding was blocked by 30 minutes of incubation with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature. Cells were then incubated 2 hours with monoclonal anti-β-catenin antibody M5.2 (1:200 dilution) in 1% BSA in PBS at room temperature in a moist chamber. Immunofluorescence staining was obtained by incubating for 1 hour with Alexa FluorThe pShuttle-IRES-Wnt5a expression plasmid was constructed by subcloning of an EcoRI-cut Wnt5a cDNA fragment from plasmid pGEMTz-Wnt5a vector (a gift from R. Kemler) into BglII site of the pShuttle-IRES-hrGFP-1 vector . pCI-Neo-mutant β-catenin (S33Y) expression plasmid, and pGL3-OT and pGL3-OF reporter plasmids were kindly provided by B. Vogelstein. Other plasmids were pCI-Neo (Promega) and pEGFP-N2 . The pAUCT-ΔN-β-catenin plasmid expressing N-terminally truncated β-catenin (aa 98-781) under the control of Tet repressor was constructed using pAUCT-CCW vector . A cDNA fragment of XhoI-NotI digestion from pCI-Neo-mutant β-catenin (S33Y) plasmid was inserted into XhoI-NotI site of pAUCT-CCW vector.® Microplate Luminometer  and data was normalized according to transfection efficiency obtained with each transfection, as described previously [Endogenous TCF/LEF-dependent transcriptional activity was tested by using pGL3-OT and pGL3-OF reporter plasmids, as described previously , except eviously . HepG2 cSNU449 cell line clone ectopically expressing N-terminally truncated (aa 98-781) β-catenin, under the control of Tet repressor was generated by stable transfection with pAUCT-ΔN-β-catenin plasmid. Briefly, SNU449 cells were plated onto 6-well plate and transfected with 2 μg of plasmid DNA using Lipofectamin 2000 reagent (Invitrogen). 24 hours post transfection, cells were transferred to 90 mm dishes and subjected to G418 (0.6 μg/ml) selection in the presence of tetracycline (1 μg/ml) until resistant cell colonies became visible. Several clones were tested by Western blot for the expression of N-terminally truncated (aa 98-781) β-catenin after withdrawing tetracycline from the culture medium to induce the expression of the transgene. Only one clone (SNU449-cl8) displayed Tet-dependent expression of mutant β-catenin, and it was used for further studies.Detergent-soluble cell lysates were prepared at 48 h post-transfection and used for western blot analysis, as described previously . Antibod.The statistical significance of the active TCF reporter activity between well-differentiated and poorly differentiated HCC cell lines was tested by Fisher Exact Probability Test using an on-line tool AFP: α-fetoprotein; EMT: epithelial to mesenchymal transition; FZD: Frizzled; HCC: hepatocellular carcinoma; HBV: hepatitis B virus; HNF: hepatocyte nuclear factor; PD: poorly differentiated; RT-PCR: reverse transcriptase-polymerase chain reaction; WD: well differentiatedThe authors declare that they have no competing interests.HY, KB, SS and EE carried our RT-PCR analyses. HY carried out confocal microscopy and western blot analyses. SS and MY carried out the immunocytochemistry. HY, HB, NO and NT carried out TCF reporter activity assays. KB constructed SNU440.cl8 cell line. EC, AT and NA carried out wound-healing and cell migration assays. KCA contributed to the design of the study. M.O. conceived of, designed and coordinated the study. MO., HY and KB drafted the manuscript. All authors read and approved the final manuscript.Nuri Ozturk - present address: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599. USA.Nilgun Tasdemir - present address: Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA.