Wnt signaling affects fundamental development pathways by regulating cell proliferation and differentiation. Aberrant activation of Wnt/β-catenin signaling promotes the development of several cancers and is an attractive target for chemopreventive and chemotherapeutic agents.In order to identify the novel antagonists for the Wnt/β-catenin pathway, we employed a cell-based Wnt reporter system (TOPflash) to screen a library of 960 known drugs. We identified spiperone, a psychotropic drug, as a novel Wnt inhibitor, which specifically blocks canonical Wnt signaling prior to the activation of β-catenin. The Wnt inhibitory function of spiperone is not associated with its dopamine-, serotonin- and sigma-receptor antagonist properties. Instead, spiperone increases intracellular calcium levels in a similar manner to thapsigargin, that also impedes Wnt signal transduction. Inhibition of protein kinase C had no effect on spiperone-mediated antagonism of Wnt signaling.Spiperone is a calcium regulator. It inhibits Wnt signaling by enhancing intracellular calcium levels. The Wnt signaling pathway plays important roles in the regulation of cell proliferation, differentiation, and apoptosis -4. In thThe β-catenin molecule is a key effector in the canonical Wnt pathway. However, not all Wnt proteins activate the β-catenin complex. Some Wnt family members, such as Wnt4, Wnt5a and Wnt11, are able to initiate β-catenin-independent Wnt signaling by binding to a Frizzled receptor and possibly the coreceptor, Knypek (Kny) or Ror1 or Ror2 . This leSpiperone is a butyrophenone antipsychotic agent with dopamine and serotonin (5-HT) receptor antagonist properties -11. It iTo identify antagonists of canonical Wnt signaling, we used a cell-based TOPflash reporter system to screen the Gen-plus drug library (Microsource) that contains 960 FDA-approved drugs. In this system, transfected Dvl (an upstream activator of the Wnt pathway) stimulated TCF/LEF response elements in the TOPflash reporter gene. Inhibitors of Wnt signaling were identified based on their ability to block the transcription of the reporter gene, but not a control gene. Small molecular compounds were screened at 10 μM and 50 μM. The initial screen identified spiperone as an antagonist of Wnt signaling. To confirm the Wnt inhibitory effect of spiperone, the TOPflash reporter was activated by Wnt1/LRP6 or Wnt3/LRP6, Dvl and β-catenin, respectively, in transient transfection assays. Treatment with 5 μM spiperone only weakly inhibited Dvl- or β-catenin-mediated signaling Figure. &1C, wherTo determine whether spiperone-mediated inhibition of Wnt signaling is associated with its antipsychotic effect, we examined other psychotropic drugs for their effects on Wnt signaling. These ligands include serotonin 5-HT1 receptor antagonists , 5-HT1 receptor agonist (8-OH-DPAT), 5-HT2 receptor antagonists (ketaserin and SB-204741), additional serotonin receptor antagonists (clozapine and SB-269970) and a sigma receptor ligand (DTG) -15. NAN-The TOPflash reporter was transfected into HEK293 cells with expression plasmids for Wnt1 and LRP6. The cells were treated with increasing concentrations of the various psychotropic drugs. Surprisingly, none of the drugs tested except spiperone had the ability to block Wnt1/LRP6-mediated signaling , calmodulin, and with other intracellular molecules, to activate downstream signaling pathways. To explore the role of PKC in spiperone-mediated inhibition of Wnt signaling, a PKC inhibitor GF109203X was tested. As indicated in Figure Previous studies have demonstrated that noncanonical Wnt family members repress canonical Wnt signaling by inducing a calcium influx ,7,20-22.A recent study showed that ionomycin is a negative regulator of β-catenin/TCF signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear β-catenin products and to the suppressed binding of TCF complexes to consensus DNA . However2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist [Spiperone is a psychotropic agent that acts as a potent dopamine Dtagonist -11, and tagonist . Spiperotagonist . Among tDuring the preparation of this manuscript, Liang et al.  reportedIn summary, our result clearly demonstrated that spiperone is a calcium regulator. It specifically blocks canonical Wnt signaling by elevating intracellular calcium levels. This drug may have chemopreventive or chemotherapeutic utility in malignancies associated with abnormal Wnt activation.Spiperone, ionomycin, thapsigargin (Tg), pindolol, UH-301, WAY100635, 8-OH-DPAT, SB-269970, NAN-190, SB-204741, Ketaserin, Clozapine, DTG  and GF109203X were purchased from Sigma-Aldrich . A Gen-plus collection of 960 known drugs was obtained from Microsource .The human embryonic kidney cell line HEK293  was transfected using the FuGene transfection reagent  according to the manufacturer's instruction.V12 have been described previously [The reporter plasmid TOPflash was a gift from H. Clevers . The NFAT-Luc and AP1-Luc reporters were purchased from BD Biosciences. The expression plasmids encoding Wnt1, Wnt3, LRP6, Dvl, β-catenin, NFATc and H-raseviously ,28.For screening of the drug library, HEK293 cells were grown for at least 24 h in 10 cm plates prior to transfection. At ~50% confluence, cells were transfected with 5 μg of TOPflash reporter, 1 μg expression vector for Dvl, 1 μg of control plasmid pCMXβgal and carrier DNA pcDNA3 plasmid for a total of 10 μg/plate. After transfection for 24 h, cells were harvested and dispersed in 96-well microtiter plates. Then the cells were treated with the different agents, generally at 10 μM and 50 μM for the initial screen. After overnight incubation, the cells were lysed in 1× potassium phosphate buffer, pH 7.8, containing 1% Triton X-100, and luciferase activities were assayed in the presence of substrate using a microtiter plate luminometer . The luciferase values were normalized for variations in transfection efficiency using the β-galactosidase internal control. Spiperone, and other compounds that were scored positive, had ≥30% inhibition of TOPflash activity when compared to the designated control cultures. In other experiments, transient transfections were performed in 12-well plates. HEK293 cells were transfected with 0.5 μg of reporter plasmid, 0.1 μg of control plasmid pCMXβgal, 0.1-0.2 μg expression plasmids, and carrier DNA pcDNA3 plasmid for a total of 1 μg/well. After 16 h, the cells were washed and treated with 5 μM spiperone or solvent (DMSO) for 24 h. Then luciferase values were determined. In the Results section, data are expressed as fold stimulation of luciferase activity compared to the basal level. All the transfection results represent means of a minimum of three independent transfections assayed in duplicate, ± the standard error of the mean (SEM).2+ concentration were measured with the calcium sensitive dye Fluo-4 . Briefly, HEK293 cells (2 × 106 cells/ml) were incubated with 2 μM Fluo-4/AM at 37°C for 30 min in Hanks balanced salt solution (HBSS) containing 5 mM KCl, 0.4 mM KH2PO4, 0.8 mM MgSO4, 137 mM NaCl, 0.3 mM Na2HPO4, 5.5 mM glucose, 1.26 mM CaCl2, 0.5 mM MgCl2. Cells were washed 2 times with HBSS and then suspended in 2 ml HBSS. Samples were analyzed for 30 sec to establish a base line and then stimulated with 2 μM ionomycin, 10 μM DTG, 1 μM thapsigargin and different concentrations of spiperone as indicated in figure Changes in intracellular CaThe authors declare that they have no competing interests.DL participated in the design of the study, carried out the experiments and drafted the manuscript. DAC participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.


Analyses of the molecular profile of IGCNU and seminoma show similarities to the expression profile of fetal germ cells/gonocytes. In murine PGCs, expression and interaction of Blimp1 and Prmt5 results in arginine 3 dimethylation of histone H2A and H4. This imposes epigenetic modifications leading to transcriptional repression in mouse PGCs enabling them to escape the somatic differentiation program during migration, while expressing markers of pluripotency.Most testicular germ cell tumors arise from intratubular germ cell neoplasia unclassified  and other nonseminomatous tumors.These data reveal similarities in marker expression and histone modification between murine and human PGCs. Moreover, we speculate that the histone H2A and H4 arginine 3 dimethylation might be the mechanism by which IGCNU and seminoma maintain the undifferentiated state while loss of these histone modifications leads to somatic differentiation observed in nonseminomatous tumors. The tumis model .HoxB1 [Expression of pluripotency genes is detected in embryonic stem cells (ES) and the inner cell mass of the early embryo. Additionally murine and human ES cells need to be cultured in the presence of factors inhibiting differentiation, although there are species specific differences ,12. In PHoxB1 . Blimp1 HoxB1 .In the present study, we investigated the expression of BLIMP1/PRMT5 during human fetal germ cell development and in testicular germ cell tumors. Analyzing human fetal tissues, we found BLIMP1/PRMT5 colocalized in gonocytes at weeks 12 – 19 of pregnancy, supporting a role in human germ cell development. Furthermore BLIMP1/PRMT5 is expressed in IGCNU and seminoma, but downregulated in nonseminomatous GCTs. Since the nuclear localization of BLIMP1 correlated with the presence of the histone modifications H2AR3me2s and H4R3me2, our data help in explaining the undifferentiated/fetal state of IGCNU and seminoma.th week of pregnancy migrating gonocytes coexpressing PRMT5 and BLIMP1 were detected,  set at 1. Expression of BLIMP1 was significantly higher in IGCNU (p = 0.029) containing testicular parenchyma and seminoma Fig. , but notFinally, we asked whether BLIMP1/PRMT5 and modification of histone H2A and H4 could be detected in TCam-2, a cell line derived from a seminoma patient ,22. HereWe had shown, that nuclear BLIMP1 and methylated H2A and H4 are expressed in IGCNU and seminoma, yet these cells express either little or cytoplasmic PRMT5 Fig. . We specIn this study, we analyzed the expression of the putative inhibitor complex of germ cell differentiation BLIMP1 and PRMT5 on mRNA and protein level and the presence of the resulting repressive histone modifications H2A/H4R3me2s in human fetal and adult germ cells as well as TGCTs. We found BLIMP1 and PRMT5 localized in the nuclei of gonocytes, and the latter also in the cytoplasm, and could show the presence of the resulting dimethylation of H2A/H4 at arginine 3. In IGCNU a strong nuclear signal of BLIMP1 and of H2K3me2s/H4K3me2s was detected, whereas PRMT5 signal was cytoplasmatic in IGCNU and heterogeneous in seminomas.HoxB1, a hallmark of germ cell specification and fail to express Stella a marker of undifferentiated germ cells [Drosophila melanogaster homolog of PRMT5, Capsuleen/dart5, is essential for germ cell specification and maintenance [The expression in fetal gonocytes in humans described here is in concordance to the observations made in mouse  indicatirm cells . Also, rntenance ,26. Intentenance  and gradntenance -29. HencIt is believed that those persisting gonocytes give rise to IGCNU the common precursor lesion of all type II TGCTs . Indeed,Upon progression of IGCNU to nonseminomas signal intensity of BLIMP1 decreased and subcellular localization changed. As a consequence, H2A/H4 modification decreased and became heterogeneous in nonseminomas. Hence, the loss of the repressive histone modifications allows further uncontrolled differentiation observed in nonseminomas.Taken together we propose the following model for development of germ cell neoplasia. First, coexpression and nuclear localization of the BLIMP1/PRMT5 complex leads to histone H2A/H4 dimethylation which results in transcriptional silencing of genes responsible for somatic differentiation in PGCs. Upon differentiation to prespermatogonia, this complex is downregulated and the H2A/H4 marks are lost. Aberrant constitutive histone H2A/H4 arginine 3 dimethylation allows the cells to escape the regular differentiation program resulting in their persistence into adulthood. These cells eventually progress into IGCNU, displaying the H2A/H4R3me2s modification as well. Since the subcellular localization of PRMT5 excludes PRMT5-dependent histone H2A/H4 modification in IGCNU we propose that BLIMP1 might act in cooperation with PRMT7. This mechanism persists in seminoma where the H2A/H4R3me2s modifications can be observed which explains the undifferentiated nature of the tumor cells. Translocation of BLIMP1 into the cytoplasm leads to breakdown of histone H2A/H4 dimethylation and subsequently to the activation of the differentiation programs and therefore the conversion from IGCNU into a nonseminomatous germ cell tumors.Formalin fixed, paraffin embedded testicular tissues from 46 patients with GCTs  and 100 ng of total RNA according to manufacturers instructions. PCRs were carried out in triplicates with following Primers: BLIMP1 F: 5'-GGGTGCAGCCTTTATGAGTC-3'; BLIMP1 R: 5'-CCTTGTTCA TGCCCTGAGAT-3'; PRMT5 F: 5'TTGCCGGC TACTTTGAGACT-3'; PRMT5 R: 5'-AAGGCAGGA AAGCAGATTGA-3'; GAPDH-F: 5'-TGGTATCGTGGAA GGACTCATG AC-3; GAPDH R: 5'-ATGCC AGTGAGCTTCCCGTTCAGC-3'. (β-Act: 25 cycles BLIMP1 and PRMT5: 30 cycles). After agarose gel electrophoresis of the PCR-products band intensity was measured after RT-PCR with the image analysis software ImageJ 1.37 v  in triplicates and normalized to the according GAPDH band.Total RNA from at least three samples per tumor entity was extracted with TRIzol  according to manufacturer's instruction. cDNA-syntesis was performed using SuperScript III reverse transcriptase  and Oligo d(T)®  following manufacturers instructions. Immunopreciptation was performed with 1,5 μg anti-PRMT5 antibody  or anti-PRMT7 . Western Blot with anti-BLIMP1 antibody followed (provided by H. M. Jäck).Co-IP was performed with DYNABEADSFor protein analysis Mini-PROTEAN Electrophoresis Cell and Mini Trans-Blot system was used . Proteins were isolated using RIPA-buffer and prepared using standard protocol and finaly electrophoresed at 30 mA for 90 min. The gel was blotted onto a PVDF membrane in a BioRad blotting chamber overnight at 30 V at 4°C according to published protocols. After blocking in PBSTM  primary antibodies (anti-BLIMP1 1:400 (kind gift from H. Jäck), anti-PRMT5 1:200, Chemicon International, USA) were incubated in PBSTM for 3 h at RT. The secondary antibodies  were diluted 1:2000. Finally the membrane was incubated in 2 ml PierceSuper Signal West Pico chemiluminescent substrate  and the signal was detected using Kodak X-Ray film .DNA Array Dataset used to analyze BLIMP1/PRMT5 expression in Seminoma, embryonal carcinoma, TCam2 and JKT1 were generated as described .2O2 (diluted in distilled water). Sections were washed in Tris-buffered saline  and incubated with primary antibodies overnight at 4°C. Thereafter, a HRP-labeled polymer conjugated with a secondary antibody was applied (DAKO EnVision-AEC KIT). Pictures were taken using a Leica microscope fitted with a JVC digital camera . Figures were assembled using Adobe CS3 software package. Merge of pictures was performed using ImageJ .For immunohistochemistry on paraffin-embedded tissue, dewaxed, 4-μm thick tissue sections were microwave-pretreated in citrate-buffer. Primary antibodies to PRMT5 , PRMT7  BLIMP1  and H2AR3me2s/H4R3me2s  were used for detection. Immunohistochemistry was performed using the DAKO EnVision-AEC Kit and manufacturers protocol  as previously described [DE, KB, DN, AG performed experiments, AG, HMJ, AMM and LL contributed material, KS helped in analysis of the Immunohistochemistry. DE, KB and HS conceived the experiments. DE, KB, LL and HS wrote the manuscript. All authors have seen and approved the final version.


The Wnt/β-catenin signal transduction pathway regulates a broad range of developmental processes. Aberrant activation of the Wnt pathway leads to cancer and degenerative diseases. β-catenin is a key signaling molecule that is frequently used as a direct monitor of Wnt pathway activation. This paper describes a multi-parametric method for quantitative analysis of cellular β-catenin protein levels in a rapid and high-throughput manner. The assay offers temporally resolved detection of Wnt-stimulated accumulation of β-catenin, simultaneously detecting cell number, and it sheds light onto the kinetics of posttranslational stabilization of β-catenin. The Wnt/β-catenin canonical pathway plays key roles in development and in the progression of several types of human cancer 2 = 0.997) between the measured DNA fluorescence and the actual number of cells plated in each well  for half-maximal accumulation of intracellular β-catenin was calculated and shown to depend on the period of stimulation  screening and validating the mechanism of action of modulators of β-catenin or GSK3β, and (2) quantifying the basal and Wnt-induced expression levels of β-catenin across different cell types 2. Cell-Bind™ 384-well plates and monoclonal anti-β-catenin antibody [cat # 610154] were purchased from Becton-Dickinson . Anti-mouse IRDye800CW and DRAQ5 (far-red) were obtained from Rockland . All chemicals were purchased from Sigma . ICG-001, prepared according to literature precedence Mouse fibroblast L-cells were obtained from ATCC, maintained as per the manufacturer's recommendations in growth medium  in an incubator at 37°C and 5% CO2  and U2OS (osteosarcoma); 2. For treatment with various stimuli or inhibitors, 20 µl of 2× stock of chemicals dissolved in growth medium such that the DMSO concentration did not exceed 0.5% were added to the wells and then incubated for the desired amount of time. The cells were then fixed in 4% PFA by adding 20 µl of 12% PFA directly to the wells for 1 h at room temperature. The wells were washed three times with PBS (50 µl/well), permeabilized with PBS/0.1% Triton X-100 , and blocked in LI-COR buffer (50 µl/well) for 2 hours at room temperature . The wells were then incubated with mouse anti-β-catenin antibody  in LI-COR blocking buffer for 2 hours at room temperature (20 µl/well) and subsequently washed with PBS/0.1% Tween-20 . Infrared anti-mouse IRDye800CW secondary antibody (1: 200) and DRAQ5  in PBS/0.5% Tween-20 were then added (20 µl/well). The plates were incubated for 1 hour at room temperature, and the wells were washed with PBS/0.1% Tween-20 (three times) and incubated in PBS (50 µl/well). The plates were covered with black seals and imaged on an Odyssey infrared scanner using microplate2 settings with sensitivity of 5 in both the 700 and 800 nm wavelength channels. Data were acquired by using Odyssey software, exported and analyzed in Excel  or KaleidaGraph (Synergy Software). β-catenin values were background subtracted from wells treated only with secondary antibody, and then normalized to cell numbers by dividing by the total DNA fluorescence signal.On Day 1, L-cells (5000 cells/20 µl/well) were seeded in a clear bottomed, black walled 384-well plate and grown for 24 h at 37°C/5% COFigure S1Monitoring cellular β-catenin accumulation in response to the Wnt/β-catenin pathway inhibitor ICG-001. L-cells were treated with Wnt3a (50 ng/ml) in the absence or presence of 10 µM ICG-001 for the indicated times. Each point represents mean±SEM (n = 4).(0.27 MB TIF)Click here for additional data file.Figure S2Detection of cellular β-catenin in osteosarcoma U2OS cells, and SW480 and HCT15 colon cancer cell lines. (A) Titration of β-catenin mAb. U2OS cells were incubated in the absence of presence of Wnt3a (50 ng/ml) for 24 h. (B) Detection of β-catenin in SW480 and HCT15 cell lines. m denotes mouse species.(0.20 MB TIF)Click here for additional data file.


We show that β-catenin stabilization in mouse midbrain-rhombomere1 region leads to robust up-regulation of several Wnt signalling target genes, including Fgf8. Suggestive of direct transcriptional regulation of the Fgf8 gene, β-catenin stabilization resulted in Fgf8 up-regulation also in other tissues, specifically in the ventral limb ectoderm. Interestingly, stabilization of β-catenin rapidly caused down-regulation of the expression of Wnt1 itself, suggesting a negative feedback loop. The changes in signal molecule expression were concomitant with deregulation of anterior-posterior and dorso-ventral patterning. The transcriptional regulatory functions of β-catenin were confirmed by β-catenin loss-of-function experiments. Temporally controlled inactivation of β-catenin revealed a cell-autonomous role for β-catenin in the maintenance of cell-type specific gene expression in the progenitors of midbrain dopaminergic neurons. These results highlight the role of β-catenin in establishment of neuroectodermal signalling centers, promoting region-specific gene expression and regulation of cell fate determination. Wnts are a family of secreted lipoproteins, which play a crucial role during embryogenesis via the regulation of patterning, cell fate decision and cell polarity. Wnts mediate their intracellular effects by inducing stabilization and nuclear translocation of β-catenin. In the absence of Wnt ligands, cytoplasmic β-catenin is phosphorylated by glycogen synthase kinase 3β (GSK3β). Phosphorylated β-catenin is targeted for ubiquitination and proteasomal degradation. Binding of Wnt molecules to their cell surface receptors releases β-catenin from the destruction complex followed by accumulation and nuclear translocation of β-catenin. Nuclear β-catenin complexes with the TCF/LEF family of transcription factors to regulate Wnt target gene expression  patterning requires anterior inhibition of Wnt pathway in vivo and in vitroIn the midbrain, early Wnt activity is responsible for establishment of a local organizing centre, the isthmic organizer In this work we studied the role of β-catenin in neuronal development in the midbrain. For this, we applied spatially and temporally controlled stabilization and inactivation of β-catenin in mouse embryos. Our findings underscore β-catenin as important transcriptional co-factor regulating midbrain gene expression and patterning.Engrailed 1 (En1) allele carrying Cre-recombinase knock-in R26cre-ert-catenin loss-of-function allele -catenin loss-of-exon3 allele R26cre-ert mice, pregnant females were given intraperitoneal injection of tamoxifen (Sigma) (8 mg/40 g body weight). All animal work has been conducted according to relevant national and international guidelines. Approval has been obtained from the Finnish Committee of Experimental Animal Research.Generation and genotyping of an in situ analysis was performed by a modified protocol in situ hybridization on paraffin sections was performed as described previously 35S-labeled antisense probes. Probes used were: Drapc1Fgf8Otx2Aldh1a1Pax3 (IMAGE RZPDp981A09196D), Ngn2 (IMAGE 2922473), Lmx1a (IMAGE 317647), Phox2a (IMAGE 480100), Mash1 (IMAGE 6415061), Wnt1 (gift from Klaus Schughart), Shh (gift from Andrew McMahon), Gli1 (gift from David Rice), Axin2, Tcf1, Dkk1 (gift from Irma Thesleff).Whole-mount mRNA Immunfluorescent staining on paraffin sections was performed as described previously Whole-mount staining was visualized with a Leica MZFLIII microscope and photographed using an Olympus DP50-CU camera. Staining on paraffin sections were visualized with an Olympus AX70 microscope and photographed using an Olympus DP70 camera. Images were processed and assembled using Adobe Photoshop software. Confocal images were acquired using the Leica TCS SP5 confocal system and LAS-AF software. Confocal stacks and images were processed and deconvoluted using Imaris 6.1 (Bitplane) and AutoQuantX (AutoQuant) software.En1cre/+; β-cateninflox(ex3)/+ embryos excision of exon 3, containing the GSK3β phosphorylation sites, prevents targeting of β-catenin for proteosomal degradation and is expected to lead to β-catenin protein accumulation in the midbrain and rhombomere1 (r1) -catenin in R26cre-ert/+; β-cateninflox/flox embryos was induced by intraperitoneal injection of tamoxifen into pregnant dams β-cateninflox allele. In the following, we call the En1cre/+; β-cateninflox(ex3)/+ and R26cre-ert/+; β-cateninflox/flox embryos as β-cateninstab (stabilized) and β-cateninlof (loss of function), respectively.To study the role of β-catenin in midbrain neurogenesis we used conditional stabilization and inactivation of β-catenin. In the stab embryos, we crossed transgenic mice expressing LacZ gene under control of β-catenin/TCF responsive elements  with β-cateninflox(ex3) mice stab embryos, β-galactosidase staining was drastically increased in rostral rhombomere1 and caudal midbrain – correlating with the domain of Engrailed 1 expression  embryos, was strongly induced in dorsal midbrain and rhombomere1 in β-cateninstab embryos  allele and stabilization of the β-catenin protein in the β-cateninstab embryos . Consistent with the spreading of the Shh, expression of Gli1, a target and a mediator of Shh signalling, was enhanced in the dorsal midbrain of β-cateninstab embryos  in the midbrain and trochlear (IV) nucleus in the rhombomere 1. Phox2a expression was expanded to dorsal midbrain of β-cateninstab embryos  and at E10.5   is detected in few β-catenin protein-null cells (stab embryos completely abolishes TH expression (data not shown). This is likely due to general mispatterning of the ventral midbrain.We have shown previously that, consistent with incomplete inactivation of β-catenin mRNA in β-cateninat E10.5 . Importat E12.5) . Howevert E12.5)  in E12.5ll cells . HoweverWe have analyzed the role of β-catenin in development of the midbrain-rhombomere 1 region using spatially and temporally controlled stabilization and inactivation of the β-catenin locus in mice. We have shown that β-catenin regulates anterior-posterior and dorso-ventral patterning in the midbrain-rhombomere 1 region at least partly by activating expression of Fgf8 and Shh, two other important patterning signals. In addition, our results suggest direct cell-autonomous effects of β-catenin on the expression in the progenitor cells of dopaminergic neurons in the ventral midbrain.Drapc1, Axin2 and Tcf1. High level of Wnt/β-catenin signalling in β-cateninstab embryos also coincides with increase and caudal expansion of Fgf8 expression. In the wild-type embryos, Fgf8 is induced broadly in the rhombomere 1 but gets restricted to the most anterior rhombomere 1 next to the Wnt expressing posterior midbrain by E9.0. In the β-cateninstab embryos, Fgf8 is expressed in the entire rhombomere 1 still at E10.5, but is not induced in the midbrain despite robust activation of the β-catenin targets. This strongly suggests that the canonical Wnt signalling pathway, involving transcriptional regulation by β-catenin, maintains Fgf8 expression in the anterior rhombomere 1. Regulation of Fgf8 expression by β-catenin does not appear to be restricted to the rhombomere 1. β-catenin stabilization is sufficient to turn on Fgf8 expression in the ectoderm of ventral limb bud. Furthermore, analyses of the β-cateninlof mutants indicated that in addition to the rhombomere 1, also many other domains of Fgf8 expression are dependent on β-catenin activity. These observations together suggest a rather direct role for β-catenin in Fgf8 regulation.Accumulation of the degradation resistant β-catenin in the midbrain and rhombomere1 leads to drastic up-regulation of the expression of Wnt target genes, including Wnt1 in midbrain-hindbrain border . Our results are consistent with a recently published work showing that β-catenin signalling is required for dopaminergic neuron development in the ventral midbrain Ventral midbrain is the location where the dopaminergic neurons (DA) of substantia nigra and ventral tegmental area are generated. Lmx1a is an early and crucial activator of differentiation of ventral midbrain progenitors into DA neurons Loss of β-catenin in neuroepithelium has been shown to cause the defects in adherent junctions Taken together our results emphasize the role of β-catenin as a transcriptional co-regulator involved in anterior-posterior and dorso-ventral patterning in developing midbrain and rostral hindbrain. In part, this is achieved by interactions with other signalling pathways. We further identify β-catenin as a cell-autonomous regulator of cell fate specification in the ventral mouse midbrain.


Vascularization of the vertebrate brain takes place during embryonic development from a preformed perineural vascular plexus. As a consequence of the intimate contact with neuroectodermal cells the vessels, which are entering the brain exclusively via sprouting angiogenesis, acquire and maintain unique barrier properties known as the blood-brain barrier (BBB). The endothelial BBB depends upon the close association of endothelial cells with pericytes, astrocytes, neurons and microglia, which are summarized in the term neuro-vascular unit. Although it is known since decades that the CNS tissue provides the cues for BBB induction and differentiation in endothelial cells, the molecular mechanism remained obscure.Sonic hedgehog are discussed. Finally, we considered the potential role of the Wnt pathway in vascular brain pathologies in which BBB function is hampered, as for example in glioma, stroke and Alzheimer's disease.Only recently, the canonical Wnt/β-catenin pathway and the Wnt7a/7b growth factors have been implicated in brain angiogenesis on the one hand and in BBB induction on the other. This breakthrough in understanding the differentiation of the brain vasculature prompted us to review these findings embedded in the emerging concepts of Wnt signaling in the vasculature. In particular, interactions with other pathways that are crucial for vascular development such as VEGF, Notch, angiopoietins and  In vertebrate ontogenesis, blood vessels arise through differentiation of mesodermal hemangioblasts into endothelial cells (ECs), which assemble into so-called blood islands of the extraembryonic yolk sac and into the aortic primordia of the embryo proper . The firThe central nervous system (CNS) however, is exclusively vascularized by angiogenesis starting at embryonic day 9 in rodents from the perineural vascular plexus (PVP), covering the entire surface of the neural tube . In the ctn have been described to be crucial for the functional state of AJs including those of brain ECs (for review see [2) that is the hallmark of the BBB [The mature BBB consists of a complex cellular system in which capillaries are regularly covered by a high number of pericytes embedded in a common basal membrane and by astrocytic endfeet. The circumference of brain capillaries is lined by a single endothelial cell connected with itself and neighboring endothelial cells by complex tight junctions (TJs), intermingled with non-occluding adherens junctions (AJs) . AJs areview see ) , members of the ATP binding cassette (ABC) transporter family (ABCB1/P-glycoprotein/MDR1 and ABCG2), and enzymes which together establish the complex phenotype of the BBB (for review see ).In contrast to the genes, which become upregulated in brain endothelia during BBB maturation, the panendothelial cell antigen MECA-32  becomes specifically down regulated . As a co✝ hypothesized that soluble factors produced by the brain and recognized by specific receptors on ECs of the PVP are responsible for brain angiogenesis. Although the initial candidates aFGF/bFGF were potent inducers of EC proliferation in vitro and of angiogenesis in vivo [Driven by the question what makes endothelial cells grow into the neuroectoderm, in the 1980s Werner Risau in vivo -13, thei in vivo -16. When in vivo ). Althou in vivo . FurtherOne major drawback for understanding brain angiogenesis is that these mechanisms also apply outside the CNS. Therefore, it seems unlikely that they are specifically involved in BBB differentiation creating highly specialized ECs.The motivation for this review is drawn from recent reports indicating that in particular the Wnt ligands Wnt 7a/b, signaling via the Wnt/β-catenin pathway, could act as CNS-specific morphogens regulating particularly steps of angiogenesis and vascular differentiation -22.armadillo in Drosophila sp.) mediated pathway, as opposed to two "non-canonical" or β-catenin-independent pathways (see below). Canonical Wnt signaling involves binding of Wnt growth factors to the cystein-rich domain (CRD) of Fzd, which forms a receptor complex with the co-receptor LDL-receptor related protein 5/6 . In the absence of Wnt ligands the degradation complex, formed by Axin, glycogen syntase kinase 3β , casein kinase 1α (CK1α) and adenomatous poliposis coli (APC), accomplishes the N-terminal phosphorylation of β-catenin at Ser45 (by CK1-α) and subsequently, at Thr41, Ser37, and Ser33 (by GSK-3β) [Slimb in Drosophila sp.) and degraded in the proteasome [Wnt signaling regulates various biological processes on a cellular level such as proliferation, apoptosis, polarity and differentiation as well as pluripotency of stem cells. These functions have consequences on a tissue or organ level, influencing axis determination, segmentation as well as branching of tubular structures . Wnts ar GSK-3β) . Phosphooteasome . Wnt ligoteasome ,29.2+ release and activation of the protein kinase C (PKC) or Ca2+/calmodulin-dependent protein kinase (CamkII), both summarized as the Ca2+-pathway  family , and strabismus/Van Gogh .).2+ releMoreover, secreted antagonists, including Dickkopf homologs (Dkk1-4), secreted frizzled-related protein families , and atypical Wnt receptors, such as the receptor-like tyrosine kinase (Ryk) and receptor tyrosine kinase-like orphan receptors (Ror), can regulate the Wnt signaling output .Recently, it could be demonstrated that the combined inactivation of Wnt7a and Wnt7b leads to severely defective angiogenesis in the central nervous system (CNS) ,22. Wnt7It is obvious that, when brain angiogenesis starts, endothelial cells get in contact with various neuroectodermal cells like neuroblasts and various glial cells and/or their precursors . That enAlthough numerous aspects of BBB development in brain capillary ECs have been monitored and described, the crucial question concerning the mechanism behind the induction of this differentiation process remained to be elucidated.In this regard, it was of major interest whether Wnts, in addition to their function in brain angiogenesis, might participate also in the differentiation of the BBB phenotype in brain ECs. Indeed, Wnt7a and Wnt7b were also shown to induce expression of the glucose transporter 1 (Glut1), an early marker of endothelial BBB specialization , which is structurally and functionally similar to the BBB. In the retina, Wnt signaling has been reported to regulate specification of stalk cells in the expanding vascular network . Herein feration .corpora lutea angiogenesis leading to infertility [Fzd4 KO mice display retarded retinal and ear vessel growth and inappropriate vessel pattering, delayed hyaloid vessel regression, dead-ended aneurysm-like capillaries, vascular leakage and abnormal ertility ,44. Furtertility -47.Also inactivation of the Lrp5 gene in mice leads to delayed hyaloid vessel regression and defective migration of retinal blood vessels ,49. AppaAnother interesting question is how barrier properties are induced in retinal blood vessels, which form the BRB. Recently, Norrin has been implicated in this function . Junge aβ inhibition in immortalized human ECs [β-catenin signaling regulates induction and maintenance of BBB genes also in human ECs.Although by now the involvement of Wnt/β-catenin signaling in brain angiogenesis and endothelial barrier induction has been established, it remains to be clarified to which extend the plethora of BBB associated genes are directly or indirectly dependent on the Wnt pathway. In this regard Lim and co-workers described the up-regulation of P-glycoprotein  upon GSK3uman ECs , supportSonic hedgehog (Shh) will be brought to light by future work.It is known that Wnt signaling does not regulate cell and tissue differentiation on its own, but interacts with other major pathways, which play a crucial role herein. Although by now we are still scratching the surface of the Wnt pathway in endothelial biology, it is more than likely that extensive crosstalk to pathways such as receptor tyrosine kinases , Notch, TGFβ/BMP and in vitro.Below the currently known as well as some hypothetical interactions are discussed. The most prominent finding for vascular biology was that the Wnt pathway regulates VEGF in colon carcinoma . This haConversely, PI3K/Akt signaling, which is activated by receptor tyrosine kinases (RTKs), may positively regulate β-catenin transcriptional activity independent of Wnt-Fzd interaction .The process of vascular sprouting is regulated by numerous signals and recent studies identified the Dll4/Notch1 signaling axis as the key endothelial mechanism controlling the appropriate numbers of sprouting tip and following stalk cells (for review see ). IntereSonic hedgehog (Shh) seems to upregulate angiogenic factors [Wnts, and in particular Wnt7a/b have recently been implicated in vascularization of the developing neuroectoderm ,22. Simi factors  and to b factors . The ava factors ,51. Whet factors . WhetherThere is ample evidence that the NF-κB pathway and the Wnt/β-catenin pathway interact to differentially regulate inflammation, tumor formation, metastasis and resistance to chemotherapy in epithelial cells . Apparensrc-suppressed C-kinase substrate , which was upregulated upon increasing oxygen tension [in vitro, thereby leading to increased junction localization of ZO-1 and claudin-1 in ECs. Furthermore, SSeCKS can be induced in astrocytes by PDGF-B, which in turn is produced by endothelial cells and plays also an important role in pericyte recruitment to blood vessels [Recent studies link Wnt/β-catenin to hypoxia signaling by the expression of von Hippel Lindau (VHL) via the β-catenin-TCF-4 complex . VHL is  tension . The ove vessels .It is tempting to speculate that a molecular link between Wnt and hypoxia signaling may also be relevant for the endothelial cell response in physiological and pathological sprouting angiogenesis.Since Wnt signaling was identified to play a major role in vascular development and differentiation, it is of particular interest to understand its involvement in pathological angiogenesis in which hypoxia is known to be one of the major driving forces.Tissue hypoxia takes also place in tumors and is described as a prerequisite for the angiogenic switch of various cancers, leading to the massive expression of VEGF-A by hypoxic tumor cells . The mosNevertheless, glioblastoma tumor vessels were demonstrated to regularly show nuclear β-catenin in human samples and in an N-ethyl-N-nitrosourea-induced rat glioma model ,77. The in vitro through PI3K/GSK-3β-dependent and -independent pathways, respectively [After stroke, independent of its etiology from ischemic or hemorrhagic insult, BBB integrity is focally abolished and during glia scar formation, inflammation and neo-angiogenesis occurs . Lithiumectively . This miectively . VEGF inPSEN1) gene is mutated, has been correlated with reduced levels of active β-catenin [Aβ), the major product of the presenilin/γ-secretase complex, was demonstrated to bind to the CRD of Fzd, thereby inhibiting Wnt mediated signaling [Aβ by EC-specific transporters , in which the presenilin-1 . HereinImportant clinical insight into the involvement of Wnt signaling in vascular pathogenesis came from studies of the human disorders Norrie disease (ND), Coat's disease and familial exudative vitreoretinopathy (FEVR) as mentioned above . FEVR haNorrie disease is caused by a X-linked recessive mutation of Norrie disease pseudoglioma gene (NDP), coding for Norrin/Norrie disease protein (Ndp) ,86. The In contrast to NDP mutation, Fzd4 mutations are autosomal dominant and those of Lrp5 are autosomal recessive, but phenotypes are largely overlapping.With the identification of TSPAN12 as a modulator of Norrin signaling that genetically and functionally interacts with Fzd4, another gene could potentially be mutated in the FEVR family of diseases, although no mutation has been discovered in humans so far .in vitro and/or in vivo makes the interpretation of signal specificity even more complicated.The underlying mechanisms for CNS vascularization in general and for endothelial barrier differentiation in particular have been obscure for about one century, since Paul Ehrlich and Edwin Goldmann established the concept of the BBB. The recent identification of the Wnt/β-catenin pathway as a driving force for brain angiogenesis and BBB differentiation has shed first light onto this burning question. Although Wnt7a/7b were confirmed to be the major ligands activating β-catenin signaling in brain and spinal cord ECs, it remains elusive if exactly the same molecular pathway operates in the entire CNS to induce the BBB phenotype in ECs. This hypothesis is challenged in the retina, in which Norrin via Fzd4-Lrp5-TSPAN12 appears to be the predominant activator of β-catenin signaling in ECs. The finding that ECs can express the majority of Wnt pathway related genes Sonic hedgehog to regulate BBB formation. The challenges for the further understanding of brain angiogenesis and endothelial barrier differentiation are the identification of cell and tissue specificity of the Wnt pathway for CNS vascular physiology, as well as the definition of Wnt signaling as a bona fide target to modulate barrier properties under pathological conditions.Furthermore, Wnt signals in the brain vasculature will integrate with other pathways such as Notch, FGF, TGFβ and The authors declare that they have no competing interests.SL was involved in preparation of the manuscript. All authors read and approved the final manuscript.


Conditional gene manipulation in mice becomes a routine for genetic studies of mammalian gene functions. Additional site-specific recombinases such as FLP or φ31 provide one more level of gene manipulation flexibility. The recombination activity of the currently available FLP deleter mice remains low. We generated a new FLP deleter mouse line with the mouse codon-optimized FLPo gene in C57BJ/6 background, which showed superior recombination efficacy in comparison to FLPe deleter mice. 100% complete removal of FRT-flanked Neo cassette was observed in all F1 progeny mice carrying both FLPo and Neo cassette, which can be transmitted to F2 generation independent of FLPo activity. Our new FLPo transgenic mice (on pure C57BJ/6 background) will largely facilitate the gene targeting process and is valuable for conditional gene manipulation. Gene targeting via homologous recombination remains a powerful tool for functional analyses of mammalian genes. The advent of multiple site-specific recombinases  allows delicate gene manipulation in a tissue- or time-specific manner. These conditional knockout (CKO) mice are not only desired when embryonic lethality caused by deletion of essential genes, but also are better models for human diseases. The Cre-LoxP system alone could be used for conditional knockout. However, removal of the selection marker from the genome by partial Cre activity in vivo is complicated, time-consuming and may be gene-dependent EFα promoter, and achieved up to 71% recombination efficiency in the C57BL/6N genetic background Initial efforts were made to enhance the thermo-stability of FLP enzyme by random mutagenesis through molecular evolution. As a result, a new version of FLP, FLPe exhibited 4–10 fold higher activity than FLP at 37°C Recently, Raymond et al synthesized de novo a mouse codon-optimized FLP version, FLPo, which was shown to exhibit significantly enhanced recombinase activity comparable to Cre in ROSA26-based ES reporter cells pPGKFLPobpA expresses de novo synthesized mouse codon-optimized FLP (FLPo) under the PGK promoter , and is PGK-FLPo transgenic founder mice  were crossed with mice that carry the FRT-flanked PGK-Neo cassette (GHRNeo/+) in intron 5 of the growth hormone receptor GHR gene ; PGK-FLPo/+; GHRNeo/+ and GHRNeo/PGK-FLPo. FLPo-mediated recombination can only be evaluated in mice carrying both the FLPo transgene and the Neo cassette. In these mice, if FLPo exerts its enzymatic activity in all cells and results in a complete removal of the FRT-flanked Neo cassette through recombination-based FRT site excision, the genotype of these double transgenic mice should become GHRLox/PGK-FLPo. However, if FLPo exhibits partial enzymatic activity, it will result in a mosaic of cells retaining the Neo cassette or lacking Neo cassette. In this case, a chimera of GHRNeo/GHRLox/PGK-FLPo will be detected. The ratio between GHRLox and GHRNeo can be used for estimation of the efficiency of FLPo-mediated recombination in vivo.To determine the efficiency of FLPo-mediated recombination, 4 heterozygous GHR gene . As a coAs shown in The FLP activity in the tail tips may not represent recombination in the germline. Since only removal of Neo cassette occurring in the germline can be transmitted permanently to the next generation, we first sought to examine whether removal of the Neo cassette also took place in other tissues in F1 generation. Of note, a FRT-flanked GFP reporter line for FLP mediated recombination would be optimal to address this question. However, such a reporter line is not available commercially, therefore, we performed PCR in various tissues. As shown in The recombination efficiency for each line is summarized in We report here the generation of highly efficient FLPo deleter mice in C57BL/6 background. It exhibited 100% complete removal of FRT-flanked Neo cassette in the F1 progeny mice carrying both FLPo transgene and the Neo cassette in different genetic loci. Importantly, the complete removal of Neo cassette was maintained in F2 generation independently of FLPo activity. In the subsequent six generations of crosses with the wild type mice, 100% germline transmission of Lox allele (after Neo cassette removed by FLPo) was observed in each generation in the absence of FLPo transgene, indicating the superior recombination efficacy of our newly developed FLPo deleter mice.The predominant use of conditional knockout in mouse genetic studies demands additional effective site-specific recombinase other than Cre for more delicate and efficient gene manipulation. Although FLP was the first site-specific recombinase used in genetic engineering of higher eukaryotes, successful site-specific recombination in mammalian cells and mice was first achieved with and mostly involved Cre, largely due to its highly efficient recombination activity in mammals. Despite initial reports that the efficacy of Flp recombination could be as high as 90% in mouse ES cells (AK-7) The improved version of FLP, FLPe exhibits 4–10 fold enhanced thermal stability at 37°C In contrast, the mouse codon-optimized FLPo gene has been shown to be highly active in mouse ES cells, resulting in comparable recombination efficiency as Cre www.jaxsonlab.In summary, our FLPo deleter mouse line is valuable for gene manipulation and will facilitate the general process of generation of conditional knockout mice. This line will be donated to The Jackson Laboratory. For information on obtaining the animals, please refer to the URL: hrttp://All mouse models used in the study were bred on a C57B6/J background. Animal care and maintenance were provided through the Mount Sinai School of Medicine AAALAC Accredited Animal Facility. All procedures were approved by the Animal Care and Use Committee of the Mount Sinai School of Medicine.pPGKFLPobpA plasmid was constructed by Raymond et al (2008) and purchased from Addgene (Addgene plasmid 13793) The 2.2-kb Sal I/Not I fragment from the pPGKFLPobpA plasmid was purified by QIAquick Gel Extraction Kit . The linearized DNA solution (2 ug/ml) was microinjected into pronuclei of fertilized one-cell embryos of C57BL/6N mice. Injected embryos were transplanted into the pseudopregnant mice. Breeding and maintenance of mice were performed under institutional guidelines. Mice were fed ad libitum with standard laboratory chow and water under a 12-h light/dark cycle. FLPo transgenic mice were genotyped by PCR with a primer set: PGK-P and Flpo-A or Flpo 5 and Flpo3. (Information of primer sequences can be obtained upon request).PGK-FLPo transgenic mice FLPo10, FLPo17, FLPo15 and FLPo6 were crossed with mice that carry the FRT-flanked PGK-Neo cassette. The F1 pups from this mating were genotyped for the PGK-FLPo transgene and the FRT-Neo cassette. F1 progeny mice carrying both the FLPo transgene and the Lox allele were backcrossed to wild-type mice to remove the FLPo transgene. Genomic DNA of F1 and F2 progeny was subjected to PCR analysis. S6/D452 primer set was used for distinguish wild type from Neo and Lox alleles . Primer set S6/R2 was used for distinguish wild type and Lox allele . Genomic DNA isolation from tissues and tail is following the standard protocol.The heterozygous Total RNA from tissues was extracted using TRIzol reagent according to the manufacturer's instructions . RNA integrity was verified using Bioanalyzer . One ug of RNA was reverse-transcribed to cDNA using oligo(dT) primers with RT-PCR kit according to the manufacturer's instructions . RT-PCR was performed and b-actin was used as control.


Wnt proteins comprise a large class of signaling molecules that regulate a variety of developmental processes, including synapse formation. Previous studies have shown Wnts to be involved in both the induction and prevention of synapses in a number of different organisms. However, it is not clear whether the influence of Wnts on synapses is a result of Wnts' behavior in different organisms or differences in the activity of different Wnt ligands.in situ hybridization to show that several Wnt ligands  and their receptors, Frizzled, are expressed in the developing hippocampus during the period of synapse formation in rodents. We used recombinant Wnt protein or Wnt conditioned media to explore the effects of Wnts on synapses in hippocampal cultures. We found that Wnt7a and Wnt7b activate canonical signaling, whereas Wnt5a activates a noncanonical pathway. The activation of the canonical pathway, either through pathway manipulations or through Wnt stimulation, increases presynaptic inputs. In contrast, exposure to Wnt5a, which activates a noncanonical signaling pathway, decreases the number of presynaptic terminals.We used Our observations suggest that the pro- and antisynaptogenic effects of Wnt proteins are associated with the activation of the canonical and noncanonical Wnt signaling pathways. Wnts comprise a large family of 19 different secreted proteins that have been described in numerous developmental processes. Wnts can signal through several different types of receptors, but the most widely recognized Wnt receptors are Frizzled proteins (Fzd). Fzds comprise a family of 10 different G-coupled protein receptors . SeveralDrosophila, Wnts act both pre- and postsynaptically to positively regulate presynaptic bouton number, presynaptic active zone assembly, and postsynaptic glutamate receptor distribution [Caenorhabditis elegans, Wnts are antisynaptogenic molecules and prevent synapse formation near the tail of the animal [Several observations suggest that Wnt proteins are involved in synapse formation. First, many Wnt signaling components are present at the synapse ,6. Seconribution . Converse animal . It is nIn this study we explore the function of Wnt proteins in synapse formation by examining the effects of several developmentally expressed Wnts on hippocampal cultures. We show here that several Wnts and Fzds are expressed in the developing hippocampus and that Wnts have differential synaptogenic activity according to the ability of Wnts to stabilize β-catenin.in situ hybridization screen to identify Wnts that are expressed in the hippocampus during synaptogenesis. In rodents, most synapses in the brain are formed within the first two weeks after birth [Wnt5a, Wnt7a, and Wnt7b, and low levels of Wnt3, were expressed . We found that Fzd3 was heavily expressed in cell bodies, dendrites and axons  transfected neurons were immunostained for GFP and Fzd3 at 14 days s Figure . To expls Figure , arrows,in vitro, the number of presynaptic inputs increased dramatically  . Dkk-1 pNext, we examined the consequences of manipulating downstream canonical signaling components Figure . To mimiOf the Wnts expressed in the developing hippocampus, only one, Wnt5a, has been strongly implicated as a noncanonical Wnt pathway activator. However, Wnt5a has also recently been shown to signal through β-catenin . TherefoTo determine if Wnt5a affects hippocampal synapse formation, we exposed cultures to Wnt5a at 8 DIV for 36 hours and observed the effect on the number of VGLUT1 puncta. In contrast to canonical Wnts, exposure to Wnt5a led to a significant decrease in the number of presynaptic terminals Figure , implyinFinally, we wanted to examine the net effect of Wnt signaling on synapse formation given the differential effect of Wnts on VGLUT1 puncta number. To broadly inhibit Wnt signaling, we applied secreted frizzled related protein 2 (sFRP2) to hippocampal cultures. sFRP2 is an endogenous secreted protein that has a binding domain similar to Fzd . We founin situ hybridization, we found that of the 19 Wnt ligands, only a few are expressed in the hippocampus, including Wnt3, Wnt5a, Wnt7a, and Wnt7b. Previous studies investigating the developmental regulation of Wnt expression have shown similar results. Shimogori et al. [et al. [et al. [in situ hybridization may not be sensitive enough to detect low signal levels from background.Our results indicate that several Wnts are expressed in the hippocampus during the peak of synapse formation in development. Using i et al.  examined [et al.  also rep [et al.  reportedWe also found that three of the six Fzd receptors we tested are expressed during hippocampal development. These Fzds were Fzd3, Fzd5, and Fzd8. At present, we do not know which Fzd interacts with each Wnt, although it is likely that the Wnts present in the hippocampus bind to multiple Fzd receptors . Indeed,We find that the Wnts expressed in the developing hippocampus have differential effects on stabilization of β-catenin. Based on our evidence of increased β-catenin levels when Wnt7a and Wnt7b is applied, and that of previous work ,32, it iWe found that activation of the canonical Wnt pathway promotes synapse formation based on the evidence that Wnts that activate canonical Wnt signaling increase the number of presynaptic terminals per area of dendrite. The canonical pathway regulates presynaptic inputs through a series of events that begins with Wnts binding to the Fzd-LRP5/6 coreceptor, as indicated by the reduction of presynaptic inputs when this complex is inhibited by Dkk-1. The activation of the coreceptors leads to stabilization of β-catenin, indicated by the increase in presynaptic inputs when β-catenin levels are increased either by inhibition of GSK3β by LiCl or transfection of a constitutively active form of β-catenin. Additionally, when Wnt signals are blocked by transfection of axin, which is part of the complex that degrades β-catenin, we see a reduction of inputs.There are several mechanisms by which increased β-catenin levels might promote synapse formation. First, Wnts that activate the canonical signaling pathway might act solely to stabilize β-catenin, which can in turn stabilize N-cadherin, and therefore act as part of a cell adhesion mechanism ,39. SecoAn interesting observation is that Wnt3a activates β-catenin much more strongly than Wnt7a or Wnt7b and yet, the degree to which Wnt3a increases synapses is approximately the same as Wnt7a or Wnt7b. It therefore appears that the level of nuclear Wnt signaling is not the sole determinant of synaptogenic activity. It is likely that Wnt7a and Wnt7b activate other signaling pathways, such as the Wnt-GSK3-microtubule or Wnt-mTOR  pathways. These pathways might cooperate with the canonical signaling pathway to regulate synapse number.In addition to effects of the canonical Wnt pathway, we also wanted to explore the effect of a noncanonical Wnt pathway on the development of synapses. We found that in hippocampal cultures, Wnt5a decreased β-catenin levels and reduced the number of presynaptic puncta. We do not yet know the pathway by which Wnt5a exerts its effect, but it is likely to be a noncanonical pathway that antagonizes the canonical pathway . By antaet al. [et al. [et al. [et al. [et al. [Our observation that Wnt5a decreases the number of synaptic inputs differs with a study by Cerpa et al.  in which [et al. . The tim [et al.  applied  [et al.  used neu [et al.  were stuet al. [One additional difference between our study and the study by Cerpa et al.  is the tOur results show that inhibition of endogenous Wnts by sFRP2 leads to an increase in presynaptic inputs, suggesting that Wnt5a is the dominant Wnt during synapse formation in hippocampal cultures. This could be due to higher Wnt5a expression than Wnt7a and Wnt7b combined or a higher affinity of Wnt5a for Fzd receptors, either of which would lead to more noncanonical pathway activation than β-catenin activation. While the mechanism of competition between canonical and noncanonical pathway activators is not yet known, there have been two studies that would suggest that Wnt5a is dominant over canonical Wnts. Both studies applied Wnt5a along with Wnt3a. One study found that Wnt5a lowers β-catenin levels, even when Wnt3a is added along with Wnt5a . AnotherC. elegans by a mechanism that does not involve the canonical pathway [Drosophila embryos also has an antisynaptogenic activity [Previous studies have reported divergent effects of Wnts in synapse formation. Wnts have been shown to be antisynaptogenic in  pathway . Wnt4 inactivity . Althougactivity . Conversactivity . Based oThe most striking observation from our experiments is that canonical Wnt pathway activation has the opposite effect on presynaptic inputs as noncanonical Wnt pathway activation. Previous work has shown Wnts to be both pro- and anti-synaptogenic, although these studies were done in several different systems with different Wnt proteins. We show here, in the same system, that Wnts exert a bidirectional influence on synapse formation.All experiments have been carried out under the University of California, San Diego's Animal Care and Use Committee guidelines.Hippocampi were dissected from P0 Long Evans rats, dissociated, and plated onto 8-well LabTek chamber slides  coated with poly-d-lysine  and laminin  at a density of 80,000 cells/well. Cultures were grown for 8 or 12 days at 37°C with 5% carbon dioxide atmosphere. Cells were then treated with for 36 hours with lithium chloride (4 mM), sFRP2 (200 ng/mL), recombinant Dkk-1 (200 ng/mL), recombinant Wnt3a (150 ng/mL), recombinant Wnt5a (200 ng/mL), recombinant Wnt7a (200 ng/mL) or Wnt7b conditioned media. All proteins were obtained from R&D Systems  and reconstituted in 0.1% bovine serum albumin in 1× phosphate-buffered saline (PBS). Cells treated with Wnt proteins and their control conditions were changed into 5% fetal bovine serum (FBS) media upon addition of the protein or control buffer. To control for nonspecific binding of sFRP2, heat inactivated protein was added to the control condition.Wnt7b conditioned media was prepared by plating HEK293T cells in 60 mm dishes at a density of 2 million cells/dish in 10% FBS media. HEK293T cells were then transfected 24 hours after plating with either Wnt7b or GFP using Fugene . The Fugene transfectant was washed off 24 hours later and cells were changed into a 2% FBS media. HEK293T cells were incubated for 72 hours. Wnt7b or control conditioned medium was then supplemented with B27 and Glutamax , and glucose (to 120 mM) and then added directly to hippocampal cultures.The constitutively active β-catenin and full length axin constructs were generously donated by Dr Tannishtha Reya. PCR amplification was used to purify the inserts with the following primer pairs: axin forward, CGC CCG CGC CCA TGC AGA GTC CCA AAA TGA ATG TCC; axin reverse, CGC CCC GGG TCA GTC CAC CTT TTC CAC CTT GCC; constitutively active β-catenin, forward, CGC GGC CGC CCA TGG CTA CTC AAG CTC ACC TGA TG; constitutively active β-catenin, reverse, CGC CCC GGG TTA CAG GTC AGT ATC AAA CCA GGC CAG. Inserts were then ligated into an IRES-GFP construct.Constructs were transfected into hippocampal cultures at 7 DIV by calcium phosphate transfection. Briefly, low glucose DMEM  was added to cells just before preparation of transfectant. DNA was mixed with 250 mM calcium chloride and then added to an equal volume of 2× HEBS. Twenty five microliters of transfectant solution were added to each well. Cells were incubated at 37°C for approximately 20 minutes and then washed with low glucose DMEM. DMEM was replaced with fresh media after the last wash. Cells were incubated for an additional seven days before fixation.Hippocampal cultures were briefly washed with 1× PBS and then fixed in 4% paraformaldehyde/4% sucrose solution for 20 minutes. Cells were permeated with a blocking solution of 3% bovine serum albumin and 0.1% Triton-X for 30 minutes in 1× PBS. Cells were immunolabeled with anti-'active' β-catenin , anti-VGLUT1 , anti-Fzd3 , anti-pan MAGUK , and/or anti-MAP2  for 2 hours at room temperature or overnight at 4°C. Cultures were washed four times with blocking solution and then incubated with secondary antibodies  for 1 hour at room temperature. Cells were coverslipped using Fluormount-G .Z-stacks of random views of VGLUT1, MAP2, and MAGUKs or GluR2/3 immunofluorescence were captured with a Leica confocal microscope with conditions blinded to the experimenter. At least 10 images were taken per experiment and all experiments were repeated three or more times. Flattened Z stack images were first median filtered with radius of one pixel to reduce background noise and then thresholded to obtain discrete VGLUT1 puncta. The same threshold values were used throughout an entire set of images. The dendrite area was measured and the number of puncta that colocalized with the dendrite was quantified.For β-catenin experiments, random images were taken of cell bodies stained with MAP2 and 'active' β-catenin. At least six fields of view were taken of each experiment and experiments were repeated at least two times. A region of interest was identified within the nucleus and soma for each cell and the average intensity was measured for each region of interest.t-tests were used to determine significance between control and the experimental group. For results with more than two conditions, a single factor ANOVA was first performed and following statistical significance, a Tukey test was performed. Images prepared for figures have been enhanced in Photoshop 6.0 using a median filter to decrease background noise.All data analysis was performed using ImageJ. Student's Proteins from total cell extracts were separated on an 8% Bis-tris polyacrylamide gel. Blotting procedures were performed according to standard protocols and immunodection of 'active' β-catenin (1:5000) and β-tubulin  was carried out.P7 and P14 mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed from the skull and post-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 hours at 4°C. The tissue was then washed with 250 mM EDTA in 0.1 M phosphate buffer for 24 hours and then incubated in 25% sucrose for up to 72 hours at 4°C. Brains were embedded in TissueTek , frozen, and then stored at -80°C. Thirty micron horizontal sections were then cut using a cryostat.In situ hybridization was performed on sections with a probe concentration of 600 ng/mL in hybridization solution at 58°C for 36 hours. Hybridization was followed by sequential washes with 5× (5 minutes), 2× (1 minute), 50% formamide in 0.2× (30 minutes), and 0.2× (5 minutes) SSC at 58°C. Sections were then blocked for 1 hour in 1% Blocking reagent (Roche) followed by a three hour incubation at room temperature in anti-DIG-AP . Signals were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) for at least 2 hours and no more than 12 hours at room temperature. The hybridization signal was compared with sections using a sense probe under identical conditions. All probes were designed and made previously by the Yimin Zou lab [ Zou lab .in vitro; Dkk: Dickkopf; DMEM: Dulbecco's modified Eagle's medium; Dvl: Disheveled; FBS: fetal bovine serum; Fzd: Frizzled; GFP: green fluorescent protein; GSK: glycogen synthase kinase; LRP: low-density lipoprotein receptor-related protein; MAGUK: Membrane associated guanylate kinase; MAP: Microtubule associated protein; P: postnatal day; PBS: phosphate-buffered saline; sFRP: secreted frizzled related protein; VGLUT: Vesicular glutamate transporter.DG: dentate gyrus; DIV: days The authors declare that they have no competing interests.EKD performed all experiments. AG participated in the design of the experiments. YZ gave advice and reagents. All authors participated in the preparation of the manuscript.Wnt, Fzd3, and sFRP2 expression in the cortex. A nissl stained section of the cortex is included as reference. All sections were taken from P14 animals.Click here for fileWnt, sFRP2, and Fzd3 expression in the thalamus at P14. A nissl stained section is included for reference.Click here for fileLithium chloride increases 'active' β-catenin.  Images of control (A) and LiCl (B) treated neurons stained for Microtubule associated protein 2 (MAP2) and 'active' β-catenin. Scale bar = 6 μm.  Bar histograms showing normalized intensity of 'active' β-catenin immunofluorescence increases (± standard error of the mean) in the nucleus (C) and soma (D) with lithium chloride activation. n = 131 cells, *p < 0.001, Students t-test.Click here for fileWnt7a increases and Wnt5a decreases 'active' β-catenin in hippocampal cultures. (A) Representative images of Wnt7a versus control β-catenin levels (top). B-tubulin was used as a loading control (bottom). (B) Representative images of β-catenin levels in control and Wnt5a treated conditions (top). B-tubulin was used as a loading control (bottom). (C) Bar histograms showing the relative stabilization of β-catenin in control, and Wnt7a or Wnt5a treated neurons. Error bars represent standard error of the mean. Wnt7a and Wnt5a treated conditions are normalized to control. *p < 0.01, Student's t-test.Click here for fileDkk-1 decreases 'active' β-catenin.  Representative images of control (A) and Dkk-1 (B) treated neurons stained for Microtubule associated protein 2  and 'active' β-catenin.  Bar graphs showing normalized intensity of 'active' β-catenin immunofluorescence decreases in the nucleus (C) and soma (D). Error bars represent standard error of the mean. n = 348 cells, *p < 0.001, Student's t-test. Dkk-1 treatment condition was normalized to control.Click here for file


Many cancerous cells accumulate β-catenin in the nucleus. We examined the role of epidermal growth factor receptor (EGFR) signaling in the accumulation of β-catenin in the nuclei of oral cancer cells.We used two strains of cultured oral cancer cells, one with reduced EGFR expression (OECM1 cells) and one with elevated EGFR expression (SAS cells), and measured downstream effects, such as phosphorylation of β-catenin and GSK-3β, association of β-catenin with E-cadherin, and target gene regulation. We also studied the expression of EGFR, β-catenin, and cyclin D1 in 112 samples of oral cancer by immunostaining. Activation of EGFR signaling increased the amount of β-catenin in the nucleus and decreased the amount in the membranes. EGF treatment increased phosphorylation of β-catenin (tyrosine) and GSK-3β(Ser-(9), resulting in a loss of β-catenin association with E-cadherin. TOP-FLASH and FOP-FLASH reporter assays demonstrated that the EGFR signal regulates β-catenin transcriptional activity and mediates cyclin D1 expression. Chromatin immunoprecipitation experiments indicated that the EGFR signal affects chromatin architecture at the regulatory element of cyclin D1, and that the CBP, HDAC1, and Suv39h1 histone/chromatin remodeling complex is involved in this process. Immunostaining showed a significant association between EGFR expression and aberrant accumulation of β-catenin in oral cancer.EGFR signaling regulates β-catenin localization and stability, target gene expression, and tumor progression in oral cancer. Moreover, our data suggest that aberrant accumulation of β-catenin under EGFR activation is a malignancy marker of oral cancer. The Wnt/β-catenin pathway plays important roles in morphogenesis, normal physiological functions, and tumor formation. At the molecular level, β-catenin is involved in two apparently independent processes, cell-cell adhesion and signal transduction . In the Certain mutations of APC or β-catenin increase β-catenin signaling, leading to overexpression of oncogenes and promotion of neoplastic growth -15. HoweRecent reports have suggested that receptor tyrosine kinases (RTKs) can regulate β-catenin function ,21. EpidIn the present paper, we describe the effect of EGFR signaling on the nuclear accumulation of β-catenin in oral cancer. This extends our previous research into the mechanisms that underlie aberrant accumulation of β-catenin.2 at 37°C and the medium was changed three times per week. Cell lines were grown until they were 89-90% confluent. All cultures were negative for mycoplasma infection.All cell lines were maintained in DMEM or RPMI1640 media that were supplemented with 10% bovine serum and 1% gentamycin. Cells were maintained in a humidified atmosphere containing 5% CORecombinant human EGF was obtained from R&D Systems , EGFR inhibitor (AG1478) from A.G. Scientific , lithium chloride from Acros Organics Co. , Erbitux from Merck , mouse anti-E-cadherin and mouse anti-β-catenin from BD Transduction Lab , phospho-GSK-3β (Ser-9), phorpho-β-catenin (Ser33/37/Thr41), EGFR antibody, and phosphor-tyrosine antibody from Cell Signaling Technologies , anti-HDAC1, anti-cyclin D1, goat anti-rabbit IgG-HRP, donkey anti-goat IgG-HRP, and protein A/G Plus-Agarose immunoprecipitation reagent from Santa Cruz Biotechnology , anti-Suv39h1 (05-615), anti-acetyl histone H4 (06-88-66), anti-trimethyl-histone H3K9 (07-442), and anti-trimethyl-histone H3K4 (07-473) from Upstate Chemicon , rabbit anti-mouse IgG conjugated to HRP antibody from Novus Biologicals , anti-human EGFR and anti-CBP, anti-Lamin B1, and anti-alpha-tubulin from Abcam .All specimens were obtained from the archives of Tri-Service General Hospital  and included 112 samples of oral squamous cell carcinoma (HNSCC). The study design was approved by the Internal Review Board of Tri-Service General Hospital (TSGHIRB 095-05-116). More detailed information about the specimens was provided previously . A serie2, 0.5% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 2 μg/mL aprotinin). After incubation on ice for 10 min, the cells were homogenized by 20 strokes in a tightly fitting Dounce homogenizer. The homogenate was centrifuged at 1,500 × g for 5 min to sediment the nuclei. The supernatant was then centrifuged at 16,000 × g for 20 min, with the resulting supernatant considered the non-nuclear fraction. The nuclear pellet was washed three times with lysis buffer to remove contamination from cytoplasmic membranes. To extract nuclear proteins, isolated nuclei were resuspended in NETN buffer , and the mixture was sonicated briefly to facilitate nuclear lysis. Nuclear lysates were collected after centrifugation . Samples of each lysate were subjected to electrophoresis on an 8% SDS-polyacrylamide gel. Then, proteins were transferred to nitrocellulose membranes, immunoblotted with antibodies, and detected by electrochemiluminescence.Cellular fractionation was performed as described previously . Briefly® X-100 plus 1:100 protease inhibitor cocktail, P8340 from Sigma, and 1:100 phosphatase inhibitor cocktails, P5726 from Sigma). Before immunoprecipitation (IP), all samples were centrifuged at 12,000 × g for 30 minutes to remove insoluble cellular debris. For IP studies, lysates were pre-cleared for 1 h by use of protein A/G PLUS-agarose , incubated with antibodies at 4°C, and then treated with protein A/G PLUS-agarose for an additional 1 h. Immunoprecipitates were then washed 4 times with 1 mL TBS. After heating at 95°C for 10 minutes, proteins were resolved on SDS-PAGE, transferred to PVDF membranes for Western blot analysis, and immunoblotted with antibodies.Cell monolayers were rinsed with 1× Tris-based saline (TBS) and then scraped into 1 mL of TBS. After a brief centrifugation, cells were solubilized in 1 mL of cell lysis buffer  or β-catenin-LEF/TCF-insensitive (FOP) reporter vector using Lipofectamine 2000  according to the manufacturer's instructions. On the following day, cells were washed with serum-free medium and treated with EGF  or AG1478 . Reporter assays were performed using the luciferase reporter system .http://rsb.info.nih.gov/ij/. Signal intensities of the PCR data obtained from ChIP assays or from whole-cell lysates (Input DNA) were quantified from TIFF images by use of ImageJ, and then compared to the signal obtained for input control. Each ChIP experiment was repeated at least three times, and representative results are shown. Means and standard deviations (SDs) were calculated from the signal intensities.The chromatin immunoprecipitation assay (ChIP) was performed using a kit from Upstate  according to the manufacturer's instructions. Briefly, following treatment, cells were washed with PBS, cross-linked with 1% formaldehyde for 10 min, rinsed with ice-cold PBS, collected into PBS containing protease inhibitors, and then resuspended in lysis buffer . Cells were sonicated to produce 200-1000 bp of DNA fragments, followed by centrifugation to remove insoluble material. Samples were precleared for 1 h at 4°C with 60 μL of a 50% slurry of protein G agarose and salmon sperm. DNA immunoprecipitation was performed with indicated antibodies. Then, cross-links were reversed, and the bound DNA was purified by phenol:chloroform extraction. RT-PCR was performed using primers specific for human cyclin D1 promoter (5'-CCGACTGGTCAAGGTAGGAA-3' and 5'-CCAAGGGGGTAACCCTAAAA-3'). PCR reactions were run with PCR Master Mix (Promega), which consisted of 30 cycles of: 94°C × 30 s, 55°C × 30 s, and 72°C × 1 min, followed by 5 min at 72°C. PCR products were analyzed by 1.5% agarose gel electrophoresis, visualized with ethidium bromide, and then photographed. Images were saved as TIFF files and then analyzed with ImageJ Specimens that were embedded in paraffin blocks were cut into 5-μm sections. These were routinely stained with H&E for histological diagnosis, and additional sequential sections were selected for immunohistochemical studies. Immunodetection was performed with a standard DAKO EnVision stain system . Sections were dewaxed and subjected to antigen heat retrieval. Endogenous peroxidase activity and nonspecific binding were blocked by incubation with 3% hydrogen peroxide and nonimmune serum, respectively. Slides were then incubated sequentially with primary antibodies (16 h at 4°C) and DAKO labeled polymer secondary antibody . Diaminobenzidine hydrochloride (DAB) was used to visualize peroxidase activity. Then, sections were counterstained with hematoxylin and a cover slip was added prior to visualization.Using a semi-quantitative scale described previously , the sta2 test was used to assess the relationship of the results of the immunohistochemical determination of EGFR, β-catenin, and cyclin D1 expression and the clinical features of patients. P values less than 0.05 were considered statistically significant.A χIn many cancers, activation of the Wnt/β-catenin pathway is associated with mutations of APC and β-catenin, with exon 15 of APC and exon 3 of β-catenin the most common mutation sites ,31. Our To investigate the effect of the EGFR signal on β-catenin redistribution, we used two lines of oral cancer cells, SAS and OECM1. SAS cells have elevated levels of EGFR, and were used for loss-of-function assays; OECM1 cells have reduced levels of EGFR and were used for gain-of-function assay. When OECM1 cells were treated with EGF, which activates EGFR, the amount of nuclear β-catenin increased over time Figure . After oTo determine whether the phosphorylation of β-catenin or GSK-3β is associated with the nuclear translocation of β-catenin, we treated cells with AG1478 or EGF and examined the phosphorylation status of β-catenin and GSK-3β. Treatment of SAS cells with AG1478 markedly suppressed the phosphorylation of GSK-3β (Ser-9) and β-catenin (tyrosine) Figure ; treatmeGSK-3β phosphorylates cytosolic β-catenin at Ser-33, Ser-37, and Thr-41 prior to β-catenin degradation, but phosphorylation of GSK-3β at Ser-9 inhibits its kinase activity. Thus we measured the phosphorylation of β-catenin at these three residues by use of phosphorylation-specific antibodies. As shown in Figure Activation of the EGFR signal leads to nuclear translocation of β-catenin. Thus, we determined whether β-catenin-mediated promotion of transcription in cancer cells also depended on EGFR activity. Nuclear-localized β-catenin interacts with transcription factors of the TCF family, leading to increased expression of genes such as cyclin D1. Therefore, we tested RNA and protein levels of cyclin D1 using RT-PCR and Western blotting. EGF markedly stimulated cyclin D1 expression in OECM1 cells, and LiCl (a GSK-3β inhibitor) had a similar effect Figure . In contNext, we examined the effect of the EGFR signal on TCF transcriptional activity by transfected with the TCF luciferase reporter (TOP-FLASH) or a control vector (FOP-FLASH) in cells. Activated EGFR signal increased TCF transcriptional activity in OECM1 cells Figure , and inhTranscriptional activation is preceded by the formation of an activation complex with ATP-dependent chromatin remodeling enzymes and histone acetyltransferase in the promoter regions. Thus, we performed ChIP assays with primers that encompassed the cyclin D1 promoter region to test for site-specific histone modification and chromatin remodeling in the mediation of EGFR-regulated cyclin D1 expression. In OECM1 cells treated with EGF or LiCl, there was significantly increased association of the cyclin D1 promoter with CBP  and a decrease in HDAC1 (a histone deacetylase) and Suv39h1 (a histone methyltransferase) Figure . In addiTo investigate the clinical significance of our results with cultured cells, we performed immunohistochemical analysis of 112 samples of oral cancer and adjacent normal epithelium. In normal epithelial cells, there was weak expression of EGFR in basal and parabasal layers, homogeneous membranous staining of β-catenin, and rare or undetectable presence of cyclin D1 Figures . In tumoWe observed reduced EGFR immunostaining in 66 samples (59%), and elevated expression of EGFR in 46 samples (41%). A blinded observer scored 86 samples (77%) as membranous β-catenin staining and 26 cases (23%) as cytoplasmic/nuclear β-catenin staining. The observer scored 59 samples (53%) as having low cyclin D1 staining and 53 cases (47%) with high cyclin D1 staining.We examined the association of these results with various clinicopathologic features of the patients Table . We founDysregulation of the Wnt/β-catenin signaling pathway has been linked to various human cancers, and this dysregulation is often associated with mutations in the β-catenin destruction complex components or in β-catenin itself ,32. HoweNumerous cell signals can impact β-catenin function. It was recently demonstrated that numerous oncogenic tyrosine kinases promote accumulation of β-catenin in the nuclei of different types of cancer -36. EGFRThe identification of many nuclear partners of β-catenin indicates that this protein functions as a transcription regulator by covalent modification of chromatin ,41. ManyThe results of our experiments with cancer tissues corroborated the results of our experiments with cultured cells. In cancer tissues, EGFR expression correlated with the presence of nuclear β-catenin, and nuclear β-catenin correlated with the tumor malignancy index. This implicates the EGFR signal in mediating entry of β-catenin into the nucleus and progression of oral cancer. Our results are consistent with other studies which reported that nuclear β-catenin was present in 19-23% of oral cancer cells and associated with proliferation, invasiveness, and poor outcome of oral cancer ,48. In cIn summary Figure , our stuThe abbreviations used are: APC: Adenomatosis polyposis coli; ATP: Adenosine-5'-triphosphate; CBP: CREB binding protein; ChIP: Chromatin immunoprecipitation; DMEM: Eagle's minimal essential medium; EGF: Epidermal growth factor; EGFR: Epidermal growth factor receptor; GSK-3β: Glycogen synthase kinase 3 beta;HDAC1: Histone deacetylase 1; H3K4: Histone 3 lysines 4; H3K9: Histone 3 lysines 9; IHC: Immunohistochemistry; LEF-1: Lymphoid enhancer factor-1; LiCl: Lithium chloride; RTKs: Receptor tyrosine kinases; RT-PCR: Reverse transcription polymerase chain reaction; Ser: Serine; Suv39h1: Suppressor of variegation 3-9 homolog 1; TCF: T-cell factor; Thr: Threonine; Tyr: Tyrosine.The authors declare that they have no competing interests.CHL performed the experiments and prepared the draft version of the manuscript. HWH and PHH participated part of the experiments and data analysis. YSS designed the experiments, supervised the project, and prepared the manuscript. All authors have read and approved the final version of the manuscript.