We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1  Wnt signaling is involved in many aspects of development and again in the homeostasis of certain stem cells in the adult, such as those found in hematopoietic, hair follicle and intestinal crypt stem cells nkd1 expression recapitulates many, if not all, of the known Wnt signaling events during development nkd2 expression does not appear to be under the control of Wnt signaling Whereas it has been demonstrated that Axin2 functions similarly to Axin1, by binding to β-catenin and inducing its degradation Drosophila has established that Nkd acts between Dvl and β-catenin, and its inhibitory activity is dependent on intact and active Wnt signaling Drosophila and in mammalian in vitro assays, Nkd was found to interact physically with the PDZ domain of Dvl Drosophila, the EF-Hand of Nkd alone is required for binding to Dvl nkd-/- mutants to adulthood Previous work in Further insight into Nkd1 function was recently gained using an integrated physical and functional screen to identify Wnt inhibitors in vivo, we employed the zebrafish model. At dome stage, which is approximately 4.3 hours post fertilization (hpf), the zebrafish blastula contains approximately 4000 to 8000 pluripotent cells that have yet to undergo differentiation or gastrulation in vivo model, specifically at the level of cyto-nuclear distribution of β-catenin.To probe further the functional significance of the Nkd1-β-catenin interaction GFP  showed enriched protein expression at the plasma membrane at dome stage (4.3 hpf)  alone or with Wnt8 were homogenized and the lysate was fractionated to isolate the plasma membrane and cytoplasmic fractions  to generate Nkd1G2A-GFP or Nkd1G2A-myc. This mutation abolishes the myristoylation activity of human Nkd2 GFP, Nkd1G2A-GFP was no longer plasma membrane-enriched and no longer formed the cytoplasmic puncta as assayed by immunohistochemistry  . In addi4.3 hpf)  [35], [3ractions . Consistfraction . Co-injehemistry . Instead Nkd1myc . The frahemistry .G2A proteins did not add up to the levels detected in the whole cell lysate when compared to wild-type Nkd1 by Western blot analysis. We generated a Nkd1G2P mutant form and observed that it was also unstable in our fractionation experiments (not shown), even though a proline at the N-terminal end of proteins is predicted to be stable G2A/P stability. In general, we found that Nkd1 was more stable than Nkd1G2A (TVR submitted) and so more Nkd1G2A RNA was injected to compensate for this difference.We also noticed that the levels of cytoplasmic plus membrane Nkd1G2A and assayed for mkp3 expression by whole mount in situ hybridization at sphere stage (3.8. hpf). At this stage, mkp3 is a direct target of the maternal Wnt/β-catenin pathway G2A had very little effect on mkp3 expression in contrast to Nkd1 overexpression, which led to a reduction in mkp3 expression (GFPnkd1 or G2A-GFPnkd1 RNA was assayed for endogenous nkd1 expression at dome stage (4.3 hpf) as readout of active Wnt signaling  was less effective at doing so.To ascertain if the myristoylation sequence of Nkd1 was required for antagonizing Wnt activity, we overexpressed Nkd1 and Nkd1pression . This waignaling . ConsistG2A could prevent the activation of target genes induced by excess Wnt signaling, we performed qRT-PCR on embryos co-injected with wnt8 and GFPnkd1 or G2A-GFPnkd1 RNAs. For comparison we also co-injected wnt8 and axin1 RNA, the latter encoding a potent inhibitor of Wnt signaling  with or without wnt8 RNA, isolated the cytoplasmic fraction and assayed for β-catenin by Western blot . GFP mock-injected embryos displayed nuclear β-catenin around the perimeter of the embryo as a result of endogenous ventro-lateral Wnt8 activity  into 1 of 4 blastomeres at the 4-cell stage. We reasoned that if Nkd1GFP somehow affects nuclear β-catenin, we would predict GFP positive cells to have reduced levels of nuclear β-catenin, compared to juxtaposed, GFP negative cells, which would have elevated levels of nuclear β-catenin due to the non-cell autonomous effects of Wnt8. Accordingly, we observed a decrease in nuclear β-catenin in Nkd1GFP positive cells, compared to juxtaposed and surrounding cells that were GFP negative  increases in cytoplasmic β-catenin in the presence of Nkd1, without Wnt stimulation. Further, we observed that Nkd1 physically interacts with β-catenin and that the strength of this interaction is dependent on an intact myristoylation sequence in Nkd1. This suggests that Nkd1 needs to associate with the plasma membrane before it can interact with β-catenin. Also, we have determined that β-catenin and Dvl overlap in Nkd1 binding domains, that they likely compete for binding to Nkd1, and that Wnt signaling may promote binding between Nkd1 and β-catenin. Finally, our observation that Nkd1 inhibits accumulation of nuclear β-catenin suggests that this effect of Nkd1 takes place in the cytoplasm.Recently, Axin1 and Axin2 were also found to interact with Nkd1 through the C-terminal poly-histidine tail of Nkd1 In Drosophila, Nkd was also tested for interactions with Axin and β-catenin by yeast-2-hybrid but no interaction was detected Myristoylation of Nkd1 is critical for its activity and to understand this further, we generated Nkd1 chimeras to target Nkd1 to the membrane in the absence of myristoylation to determine if these chimera's could recapitulate wild type Nkd1 activity. Tagging Nkd1 with alternate membrane localization motifs  could not recapitulate Nkd1 activity and in all cases could not localize, or only weakly localize, to the plasma membrane . This suggests that myristoylation is specifically effective at bringing Nkd1 to the membrane.Drosophila it was found that Nkd Cuticle binds to Importin alpha3 via its ARM repeats and that this region in Nkd is critical for its function Drosophila cells did not result in nuclear accumulation of Drosophila Nkd The exact mechanism of Nkd1 action is still unknown, but may involve the nuclear import machinery. In nkd-/- phenotypes in the developing Drosophila embryo.Our observations that Nkd1 has a more dramatic effect on ectopic Wnt signaling compared with endogenous Wnt signaling appears to reflect an evolutionarily conserved phenomenon. In fly, Nkd is only required during early segmentation of the Drosophila embryo and not during other aspects of development involving Wg signaling In conclusion, we have demonstrated that in zebrafish blastula cells, Nkd1 prevents nuclear accumulation of β-catenin and that this function of Nkd1 is dependent on its plasma membrane localization via myristoylation. Furthermore, we also found Nkd1 to interact with β-catenin and that Dvl2 and β-catenin may compete for binding to Nkd1. The functional significance of Nkd1 interacting with β-catenin, Dvl2 and Axin is currently under investigation.5′-ATGCCCCTCGTGCTGTTTT-3′; β-Actin Reverse: 5′-TCTGTCCCATGCCAACCAT-3′; Nkd1-5′UTR Forward 5′- AAGCCGCGCGCTCTTC-3′; Nkd1-5′UTR Reverse 5′- GAAACATCAGAAACAAAAAACAATCC-3′; Axin2 Forward: 5′-ATGCCCCTCGTGCTGTTTT-3′; Axin2 Reverse 5′-TCTGTCCCATGCCAACCAT-3′. Absolute quantification was determined using a standard curve generated by β-actin. Relative quantitative RT-PCR (qRT-PCR) was determined by normalizing to uninjected.Total RNA from embryos was prepared using Trizol (Gibco). cDNA was synthesized with Superscript II (Invitrogen). Quantitative RTPCR (qRT-PCR) was performed using IQ Syber green (BioRad). Three replicates were performed for each experiment. Sequences of primers were as follows. β-Actin Forward: G2A constructs were generated by site directed mutagenesis. Constructs were cloned into the ClaI site of pCS2+myc or pCS2+GFP. Mutagenesis primers are as follows Nkd1G2A For 5′- GCAGGATCCCATCGATGATGGCTAAACTTCATTCC-3′; Nkd1G2A Rev 5′- GGAATGAAGTTTAGCCATCATCGATGGGATCCTGC-3′; Orientation and sequence integrity were confirmed by sequencing. pCS2+Axin1 was kindly provided by Toshio Hirano Nkd1nkd1myc, GFP; nkd1N-GFP; ornkd1 1200 pg G2A-myc or nkd1G2A-GFP;nkd1 25 pg wnt8; 100 pg HAdsh2-; 400 pg axin1 in 1% phenol red were pressure injected into the yolk cell of one- to two-cell-stage embryos. For immunohistochemistry on nuclear β-catenin, a mixture of 25 pg wnt8 and 100 pg of GFPnkd1 or N-GFPnkd1 was pressure injected into one of four blastomeres.Capped mRNA was synthesized and purified using Ambion's mMessage mMachine kit following their protocol. mRNA was diluted in RNase free water, and its quantity and quality was analyzed by spectrophotometer and RNase free gel analysis. For all of the experiments, except where noted, 800 pg of 2; 1 mM EGTA; 0.02% Na Azide) containing Complete® Protease Inhibitor (Roche). Lysates were centrifuged at 1000g for 5 min at 4°C to pellet nuclei, large membrane fragments and whole cells. The resulting lysate was further fractionated into crude plasma membrane and cytoplasmic fractions by centrifugation at 100,000g for 1 hr at 4°C.Ten embryos were harvested at dome stage (4.3 hpf), mechanically dechorionated and deyolked in 0.3X Danieau. Deyolked animal caps were lysed by trituration in TKM buffer  1∶1000, polyclonal anti-pan-cadherin (Abcam) 1∶1000, monoclonal anti-actin (Sigma) 1∶2000; polyclonal anti-Dvl2 1∶5000; anti-HA (Vanderbilt Antibody Core) 1∶1000.Primary antibodies: mouse anti-myc 1∶500; Rat anti-HA 1∶200; Rabbit anti-zDvl2 1∶1000 or 1∶5000; mouse anti-β-catenin 1∶250). Secondary antibodies: Cy2 Donkey anti-Rat 1∶200; Cy3 Goat anti-mouse 1∶200; Cy3 Goat anti-Rabbit 1∶200; Cy2 Goat anti-Rabbit 1∶200; AlexaFluor 594 1∶200; syto59 1∶10,000.Whole mount immunhistochemistry: Nuclear β-catenin staining was carried out using the Vectastain Elite ABC kit (Vectastain Laboratories) along with Avidin D  and ImmPact DAB (Vectastain Laboratories).GFP positive and 6 juxtaposted GFP negative cells, and 10 cells for Nkd1N-GFP positive and juxtaposed GFP negative cells were analyzed. Grey scales for Nuclear Hoechst and nuclear β-catenin where calculated using Image J. They grey levels of signal intensity were measured across the cell for both the Hoechst staining and the β-catenin staining. For each cell, the average level of β-catenin in the nuclei was normalized to its Hoechst staining. The adjusted averages were then plotted and compared by students T-test.Immunohistochemistry: To quantify the changes in nuclear β-catenin, 6 cells Nkd1Immunoprecipitation (IP) and immunoblotting (IB) were carried out using an anti-flag antibody . LUMIER experiments were performed as previously described Figure S1Nkd1 inhibits nuclear β-catenin. (A) Whole embryo analysis of nuclear β-catenin staining as per GFP and Nkd1N-GFP co-injections with wnt8. Injections are labeled along the left side, staining is identified along the top.(TIF)Click here for additional data file.Figure S2Dvl2 and β-catenin bind to similar domains on Nkd1. HEK293T cells were transfected with luciferase-tagged β-catenin (left panel) or Dvl2 (right panel) and the indicated 3XFlag-Nkd1 constructs. Cell lysates were subject to anti-Flag immunoprecipitation and the presence of β-catenin or Dvl2 was assessed by luciferase assay. Luciferase activity of the immunoprecipitate was normalised to that of the total cell lysate (left panel) or plotted separately (right panel). Data are the average of at least 4 independent experiments expressed as % wild type +/− SEM (left panel) or as the mean of 2 samples +/− standard deviation of a representative experiment (right panel).(TIF)Click here for additional data file.


Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach  in vitro and in vivo models, we show that TCF/LEF proteins can be activated independently of β-catenin through cooperation with members of the ATF2 subfamily of transcription factors. This novel alternative mechanism of TCF/LEF activation is constitutively up-regulated in certain hematopoietic tumor cells, where it regulates the expression of TCF/LEF target genes and promotes cell growth.The Wnt signaling pathway plays a crucial role during embryonic development and in the maintenance of stem cell populations in various organs and tissues. Aberrant activation of this pathway through different mechanisms participates in the onset and progression of several types of human cancers. In the presence of Wnt ligands, stabilized β-catenin acts as a transcriptional activator to induce the expression of target genes through binding to the TCF/LEF family of transcription factors. Using  The Wnt/β-catenin signaling pathway plays an essential role during embryonic development and as a major regulator of stem/progenitor cell maintenance in a number of postnatal organs and tissues, including the gastrointestinal tract, the skin and the hematopoietic system Myc, cyclin D1, Axin 2 and Lef1The Wnt/β-catenin, or canonical, pathway is initiated by Wnt-mediated coupling of the seven transmembrane domain receptor Frizzled and the single-membrane-spanning low-density receptor-related protein 5/6 (LRP5/6), followed by phosphorylation of the LRP5/6 intracellular domain While its role in the control of gene expression is thought to depend mainly on the interaction with TCF/LEF proteins, β-catenin can act in some contexts through binding to other transcription factors, including the homeodomain protein Prop1 in vitro and in vivo approaches, we further demonstrate that TCF1 retains transcriptional activity in the absence of β-catenin. Finally, we establish that ATF2 family members physically and functionally interact with TCF1/LEF1 factors to promote target gene expression and hematopoietic tumor cell growth. Together, our results uncover a new mechanism that activates TCF1/LEF1 transcriptional activity independently of β-catenin and the Wnt canonical pathway.While the β-catenin pathway has been reported to be upregulated in cancer stem cells of different leukemias Using lentiviral wild-type (Top) and mutant (Fop) TCF/LEF reporters, we investigated the status of Wnt signaling in cells derived from several types of human cancer. As a result of this screen, we identified up-regulated TCF/LEF activity in different hematopoietic tumor cells, including Ramos, K562 and Jurkat . As showTo gain insights into the biological relevance of this β-catenin-independent TCF/LEF activity, we tested the effects of a GFP-fused DN-TCF4 in Ramos lymphoma cells using a lentiviral tetracycline (Tet-off) inducible system. As a negative control for these experiments, we utilized U937 lymphoma cells, which lacked detectable constitutive TCF/LEF activity . DoxycylWe extended our analysis to other hematopoietic tumor cell lines as well as primary tumors. Several mantle cell lymphoma lines, including Granta-519, HBL-2, JEKO-1 and JVM-2, exhibited TCF/LEF reporter levels as high or higher than Ramos  and alsoTCF1 and LEF1 were cloned in 1991 from hematopoietic cells Based on the crystal structure of the β-catenin-TCF3 complex, we mutated two residues in the TCF1 β-catenin binding domain, D21A and E29K, which should impair the ability of this transcription factor to bind to β-catenin Xenopus embryonic development, locally activated Wnt signaling induces the asymmetric expression of genes of the dorsal organizer, thus allowing the establishment of dorsal-ventral polarity in vivo model to assess the β-catenin-independent activity of TCF1. As previously reported tcf1, but not tcf4, mRNA induced ectopic expression of the Wnt direct target gene Xnr3 in animal cap explants , U937 (histiocytic lymphoma), K562 (chronic myelogenous leukemia) and Jurkat (acute T-cell leukemia) cells were maintained in RPMI-1640 (Lonza) supplemented with 10% fetal bovine serum . Human mantle cell lymphoma lines including Granta-519, HBL-2, JEKO-1 and JVM-2 were generously provided by Dr. Samir Parekh, Icahn School of Medicine at Mount Sinai, and were also cultured in this medium. AB5 , PA1 (ovarian teratocarcinoma), 293T (human embryonic kidney), NIH-3T3 (mouse fibroblasts) and Mel888 (melanoma) cells were maintained in DMEM (Lonza) supplemented with 10% FBS.2. Two days after transduction, the cells were selected in 2 µg/ml puromycin or 10 µg/ml blasticidin.Transient transfection was performed using Fugene 6 (Roche) according to the manufacturer's instructions or with polyethylenimine (Polysciences). For lentivirus production, 293T cells were co-transfected with the lentiviral vector, pCMV Δ8.91 and pMD VSV-G plasmids. The conditioned medium containing the viral particles was collected two, three and four days after transfection, supplemented with 8 µg/ml polybrene and added to a pellet of hematopoietic tumor cells, followed by centrifugation for 1 h at 500 g, 4°C and overnight incubation at 37°C, 5% COPrimary human lymphoma samples were obtained either as part of standard excisional biopsy or from peripheral blood samples from patients at the Icahn School of Medicine at Mount Sinai with informed consent reviewed and approved by the Institutional Review Board and in accordance with the Declaration of Helsinki. Specimens were processed to viable, sterile single-cell suspensions. Briefly, lymph node tissue was diced and forced through a metal sieve in a laminar flow hood into RPMI tissue culture medium. Peripheral blood mononuclear cells or disaggregated follicular lymphoma biopsy cells were pelleted by low-speed centrifugation, resuspended in media composed of 90% fetal calf serum and 10% DMSO (Sigma), frozen slowly in the vapor phase of liquid nitrogen in multiple cryotubes, and stored in liquid nitrogen. The frozen cells were thawed and maintained for 1–3 days in RPMI-1640 supplemented with 10% fetal bovine serum prior to RNA extraction for qPCR analysis or preparation of cell lysates for analysis of uncomplexed β-catenin (see below).GTACGAGCTGCTATGTTCC (β-catenin), CACCATTCCCCTGTTTGTG (γ-catenin), AGCCCTCAGGAAGTTGATTAAA (ATF2) and CGAAGAACTCACTTCTCAGAA (ATF7). All constructs were sequence verified.Lentiviral TCF/LEF luciferase and GFP reporters, β-catenin and Dvl shRNAs, inducible and constitutive DN-TCF4 vectors and the SuperTop reporter (pTA-Luc vector) were previously described The following antibodies were purchased from commercial sources: mouse anti-β-catenin, mouse anti-γ-catenin (BD Biosciences), mouse anti-tubulin, mouse anti-Flag, mouse anti-HA (Sigma), rabbit anti-ATF2, rabbit anti-HA (Santa Cruz), goat anti-Axin (R&D Systems), rabbit anti-ATF7 (Abcam), mouse anti-LEF1 (Millipore). Mouse anti-myc clone 9E10 was obtained from the Mount Sinai Hybridoma Core Facility.For immunoblot, cells were washed once with phosphate-buffered saline (PBS) and lysed on ice in lysis buffer containing 50 mM Hepes pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 20 mM NaF, 2 mM sodium orthovanadate, supplemented with the Complete Mini proteinase inhibitor cocktail tablets (Roche). Lysates were cleared by centrifugation at 20,000× g for 15 min at 4°C and protein concentrations were determined by using the Bio-Rad protein assay (Bio-Rad). Sodium dodecyl sulfate (SDS) loading buffer was added to equal amounts of lysate, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to Immobilon-P membranes (Millipore). Antibodies used in immunoblot analysis were revealed by chemiluminescence using ECL Western Blotting Substrate (Thermo Fisher Scientific) or using the Odyssey Infrared Imaging System (LI-COR Biotechnology). For immunoprecipitation, equal amounts of cell lysates were incubated either with anti-Flag M2 agarose beads (Sigma) for 3 hrs at 4°C or with mouse anti-myc or mouse anti-HA antibodies for 1 hr at 4°C, followed by 3 hr incubation with Protein G Sepharose 4 Fast Flow beads . For LEF1 immunoprecipitation, equal amounts of cell lysates were incubated with aLEF1 antibody for 2 hrs at 4°C, followed by overnight incubation with Protein G Sepharose 4 Fast Flow beads. Beads were washed three times with lysis buffer and resuspended in SDS loading buffer, followed by SDS-PAGE and immunoblot.Uncomplexed β-catenin was measured using glutathione S-transferase (GST)–E-cadherin or GST-TCF1 beads as described previously 3 cells per 60-mm dish in 0.5% sea plaque agarose (Cambrex) in RPMI supplemented with 10% FBS on a semisolid bottom layer of growth medium containing 1% agarose. Cells were fed once weekly with 0.3 mL of medium and stained after 17 days with iodonitrotetrazolium (Sigma). The cells containing Tet-off inducible DN-TCF4-GFP vector were grown overnight in the presence or the absence of 100 ng/ml doxycycline, followed by extensive washing in PBS. The cells were then counted and seeded in soft agarose dishes in the presence or the absence of 100 ng/ml doxycyline. The remaining cells were grown for one additional day with or without 100 ng/ml doxycyline, and FACS analysis was performed to assess the level of induction of DN-TCF4-GFP.Growth of hematopoietic tumor cells in soft agarose was determined by seeding 2×105′-ATCAGTGCCGTGGTTCGT and 5′-TTCGGAGAGTTCTGGGATTG), 18S (5′-GTAACCCGTTGAACCCAT and 5′-CCATCCAATCGGTAGTAG), Axin 2 (5′-ACTGCCCACACGATAAGGAG and 5′-CTGGCTATGTCTTTGGACCA), LEF1 (5′-CTTTATCCAGGCTGGTCTGC and 5′-TCGTTTTCCACCATGTTTCA).Total RNA was extracted using the Trizol Reagent (Invitrogen), incubated with DNase I (Invitrogen) and reverse transcribed in the presence of random primers using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol. Quantitative PCR was performed using FastStart SYBR Green Master (Roche) on a MJ Opticon (Bio-Rad). The primers used for qPCR were: TATA Binding Protein . RT-PCR was performed as described 5′-GGATCGTTATCACCTCTG and 5′-GTGTAGTCTGTAGCAGCA), chordin (5′-CAGTCAGATGGAGCAGGATC and 5′-AGTCCCATTGCCCGAGTTGC), EF1-α (5′-CAGATTGGTGCTGGATATGC and 5′-ACTGCCTTGATGACTCCTAG), Xnr3 (5′-GTGAATCCACTTGTGCAGTT and 5′-ACAGAGCCAATCTCATGTGC).RNA was synthesized in vitro in the presence of cap analog using the mMessage mMachine kit (Ambion). Microinjection, explant dissection, cell culture and whole-mount antibody staining were performed as described Studies on Xenopus laevis embryos were performed in accordance with the guidelines of the American Veterinary Medical Association, and under the auspices of the Queens College Institutional Animal Care and Use Committee (IACUC). All experiments were undertaken with the highest regard for scientific, ethical, and humane principles.Figure S1Endogenous TCF/LEF activation in hematopoietic cancer cells. (A) K562 and U937 cells were transduced with wild-type (Top) or mutant (Fop) TCF/LEF lentiviral reporter driving the expression of GFP, followed by FACS analysis. Breast MDAMB157 and lung H23 cells, which display constitutive autocrine Wnt activation, were used as positive controls. (B) β-catenin or γ-catenin knockdown in K562 cells did not affect TCF/LEF activity. K562 cells containing Top or Fop luciferase lentiviral reporter were transduced with β-catenin or γ-catenin shRNAs, and the TCF/LEF reporter activity was measured (lower panel). β-catenin and γ-catenin down-regulation was assessed by immunoblot (upper panel). (C) K562 cells were transduced with Top- or Fop-GFP lentiviral reporter, followed by transduction with luciferase (control) or DN-TCF4 lentiviruses. The GFP levels, corresponding to TCF/LEF activation, were measured by FACS. (D) The mRNA levels of Lef1 and Axin 2 in the cells depicted in C were quantified by real-time PCR, and the expression of DN-TCF4 was assessed by immunoblot. Bcl-xL was used as a loading control. (E) The indicated mantle cell lymphoma (MCL) cells were transduced with Top or Fop luciferase reporter and a renilla luciferase virus, and the TCF/LEF transcriptional activity was calculated by dividing the Top/renilla ratio by the Fop/renilla ratio. Ramos and U937 cells were used as positive and negative controls, respectively. (F) Lack of uncomplexed β-catenin in the MCL cells used in E, as measured by GST-TCF1 pull-down. Ovarian PA1 cells were used as positive control, while Ramos and U937 cells were used as negative controls. (G) Lack of uncomplexed β-catenin in primary hematopoietic tumors. The type of tumor corresponding to each sample is indicated in (PDF)Click here for additional data file.Figure S2TCF1 expression triggers TCF/LEF reporter activity in 293T cells. (A) 293T cells were co-transfected with the indicated wild-type (T) or mutant (F) TCF/LEF reporters, the renilla plasmid pRL-CMV and increasing amounts of TCF1 construct or empty pcDNA3HA vector (500 ng). The TCF/LEF activity is expressed as relative luciferase units normalized by the renilla luciferase reading. (B) 293T cells were co-transfected with SuperTop reporter, pBind renilla plasmid and HA-tagged TCF1 in the absence or the presence of ten-fold amount of HA-tagged DN-TCF4. Two days after transfection, the cells were lysed, followed by luciferase assay (lower panel) and immunoblot using anti-HA antibody to assess the expression levels of TCF1 and DN-TCF4 (upper panel). (C) Empty vector, TCF1, LEF1 or TCF4 constructs were co-transfected with SuperTop or SuperFop and renilla luciferase plasmids in 293T cells and the Top/Fop ratio was calculated.(PDF)Click here for additional data file.Figure S3Lack of physical and functional interaction of D21A;E29K mutant TCF1 (TCF1mt) with β-catenin or γ-catenin. (A) 293T cells were transfected as indicated with myc-tagged β-catenin and HA-tagged wild-type or mutant TCF1, followed by immunoprecipitation using anti-myc antibody and immunoblot with anti-myc or anti-HA antibodies. (B) Mel888 melanoma cells containing a β-catenin mutation resulting in constitutive Wnt activation were co-transfected with SuperTop reporter, the renilla pRL-CMV plasmid and equal amounts of empty vector, TCF1, d36TCF1 or TCF1mt. Two days after transfection, luciferase assay was performed and the TCF/LEF reporter activity is represented as renilla normalized relative luciferase units. (C) 293T cells were co-transfected with SuperTop reporter, the renilla pRL-CMV plasmid, γ-catenin and equal amounts of empty vector, TCF1 or TCF1mt, followed by luciferase assay two days after transfection.(PDF)Click here for additional data file.Figure S4Exon IVa is not required for TCF1 transcriptional activity. 293T cells were co-transfected with the SuperTop or SuperFop reporter, the renilla plasmid pRL-CMV and the indicated TCF1 or TCF4 constructs. Two days after transfection, luciferase assay was performed and TCF/LEF activity expressed as the Top/Fop ratio of the renilla normalized luciferase values.(PDF)Click here for additional data file.Figure S5aa 101–211 are involved in β-catenin-independent, but not β-dependent, TCF1 transcriptional activity. (A) 293T cells were co-transfected with the indicated SuperTop reporter, the renilla plasmid pRL-CMV and the indicated TCF1 construct followed two days later by luciferase assay. The TCF/LEF activity is expressed as relative luciferase units normalized by the renilla luciferase reading (lower panel). The expression of the different TCF1 constructs was assessed by immunoblot using the same cell lysates (upper panel). (B) 293T cells were co-transfected with SuperTop reporter, pRL-CMV and β-catenin (S33Y) with or without the indicated TCF1 constructs, followed two days later by luciferase assay.(PDF)Click here for additional data file.Figure S6ATF2 synergizes with TCF1mt independently of β-catenin. 293T cells were co-transfected with TCF1mt, ATF2 or Wnt3a in combination with shRNA vectors targeting either CHK1 (control), the three Dvl isoforms or β-catenin, in the presence of the SuperTop reporter and the renilla plasmid pRL-CMV. Three days after transfection, luciferase assay was performed and TCF/LEF activity expressed as the mean +/− SD of the renilla normalized luciferase values. The Wnt3a construct was used to assess the efficiency of shDvl and shβ-catenin in inhibiting Wnt canonical signaling.(PDF)Click here for additional data file.Figure S7Lack of synergy between TCF4 and ATF2 factors. (A) 293T cells were co-transfected with TCF4, ATF2, CREB5 or ATF7 as indicated, in the presence of the SuperTop reporter and the renilla plasmid pRL-CMV. Two days after transfection, luciferase assay was performed and TCF/LEF activity expressed as the mean +/− SD of the renilla normalized luciferase values (lower panel). The same lysate was used to assess the expression of TCF4 and ATF2 factors by immunoblot (upper panel). (B) 293T cells were co-transfected as indicated with Flag-tagged CREB5, HA-tagged TCF1 and HA-tagged TCF4, followed by immunoprecipitation using anti-Flag antibody and immunoblot with anti-HA or anti-Flag antibodies.(PDF)Click here for additional data file.Figure S8Interaction between endogenous LEF1 and ATF2/7 in hematopoietic tumor cells. 2 mg of Lysate from K562 (A) or Ramos (B) cells was immunoprecipitated using 10 ug of anti-LEF1 or control antibody, followed by immunoblot using the indicated antibodies. The band corresponding to immunoprecipitated LEF1 (∼55 kDa) was masked by the IgG heavy chain used for the immunoprecipitation.(PDF)Click here for additional data file.


Burkholderia mallei strain isolated from a diseased dromedary in Bahrain revealed close genetic proximity to strain Dubai 7, which caused an outbreak of glanders in horses in the United Arab Emirates in 2004.We confirm a natural infection of dromedaries with glanders. Multilocus variable number tandem repeat analysis of a  Burkholderia mallei –listed disease, is a contagious, life-threatening disease of equids caused by the gram-negative bacterium B. mallei infections are known to occur in various mammals . Camels are also susceptible to B. mallei, as experimental infection has demonstrated .Natural B. mallei infection has not yet been confirmed. Here we provide evidence for a B. mallei infection in 1 of the diseased dromedaries.An outbreak of glanders is ongoing in equids in Bahrain  and were positive for nitrate, glucose assimilation, arginine dehydrolase (after 4 days of incubation), N–acetyl glucosamine, and potassium gluconate. The API 20 NE-test identified the colonies as B. mallei because the same API ID number (1140504) occurred as in the previously isolated Dubai 7 strain  until it became positive. This fluid was then cultured on sheep blood agar at 37°C for 72 h. The isolate stained poorly gram-negative, was rod shaped, and tested oxidase positive. Suspected During necropsy, typical glanderous lesions in the lung, choanae, and nasal septae were observed. Golf ball–sized reddish-gray nodules resembling tubercles with a central gray necrotic zone were detected in the lungs. In the choanae and nasal septae, stellate scars, ulcers, and honeycomb necrotic patches covered with yellow pus , panel BB. mallei in lung and choanae specimens was examined by using standard culturing techniques as described by Wittig et al. , as previously described by Godoy et al. . Sequence analysis of the 16S rRNA  gene displayed the B. mallei strains , and the Bactrian camel (C. bactrianus) are susceptible to B. mallei (glanders) and B. pseudomallei (melioidosis) infection .B. mallei was isolated from venous blood, indicating septicemia. The pathogen was also directly isolated from the pus, which had accumulated in the choanae, but not from nasal and eye swabs and, unexpectedly not from the lung lesions. A possible explanation for the failure to isolate B. mallei from the nasal swabs was the heavy growth of various other contaminating bacteria because no selective culture medium exists for B. mallei. It could also be explained by storage of the samples at –20°C for >20 days, which probably destroyed the bacteria.The genetic relatedness of the strain isolated from the dromedary to the strain isolated in 2004 from horses in the United Arab Emirates suggests that this strain might be endemic to this region. It also appears to be genetically distinct from a recent outbreak in Pakistan, demonstrating the persistence of multiple strains on a larger geographic scale. Isolation of this pathogen from both camels and horses poses new challenges to the international trade of equids from and to countries where camels are raised.


The expression of Wnts, β-catenin, secreted frizzled-related proteins (sFRPs) and Dkks was analyzed by quantitative RT-PCR and western blotting. The canonical Wnt signaling pathway was examined by immunofluorescence and western blot detection of nuclear β-catenin expression. The effect of recombinant mouse Dkk-1 (rmDkk-1) on cell proliferation and apoptosis was assessed by flow cytometry, immunofluorescence, immunocytochemistry and Cell Counting Kit-8 (CCK-8) analysis. The expression of β-catenin, collagen1α1, TGFβRII, PDGFRβ and α-SMA in PSCs treated with different concentrations of rmDkk-1 or siRNA against β-catenin was determined by quantitative RT-PCR and western blotting. Wnt2 was the only Wnt whose expression was significantly upregulated in response to PSC activation, and Wnt2 and β-catenin protein levels were significantly increased in the pancreas of CP mice, whereas Dkk-1 expression was evidently decreased. Nuclear β-catenin levels were markedly increased in activated PSCs, and rmDkk-1 suppressed the nuclear translocation of β-catenin and the proliferation and extracellular matrix production of PSCs through the downregulation of PDGFRβ and TGFβRII. Upregulation of Dkk-1 expression increased apoptosis in cultured PSCs. These results indicate that Wnt signaling may mediate the profibrotic effect of PSC activation, and Wnt2/Dkk-1 could be potential therapeutic targets for CP.The role of persistent activation of pancreatic stellate cells (PSCs) in the fibrosis associated with chronic pancreatitis (CP) is increasingly being recognized. Recent studies have shown that Wnt signaling is involved in the development of fibrosis in multiple organs, however, the role of specific Wnts in pancreatic fibrosis remains unknown. We investigated the role of Wnt signaling during PSC activation in CP and the effect of β-catenin inhibition and Dickkopf-related protein 1 (Dkk1) restoration on the phenotype of PSCs. CP was induced in mice by repetitive caerulein injection and mouse PSCs were isolated and activated  Chronic pancreatitis (CP) is characterized by progressive pancreatic fibrosis. The persistent activation of pancreatic stellate cells (PSCs) plays a pivotal role in pancreatic fibrogenesis, while the redifferentiation and outcome of activated PSCs determines the pathological outcome after pancreatic injury Although several inflammatory mediators released during the development of pancreatitis have the potential to regulate PSCs, accumulating evidence supports major roles for platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1 and angiotensin II as key modulators of the persistently activated and profibrotic phenotype of these cells in vivo and in vitro. Our findings showed that the canonical Wnt signaling is involved in the activation of PSCs in CP. Furthermore, the Wnt signaling antagonist Dkk1 suppressed the proliferative and profibrotic phenotype of PSCs by downregulating the expression of collagen1α1, TGFβRII and PDGFRβ through the inhibition of Wnt/β-catenin signaling.The Wnt signaling pathway is highly conserved in multicellular organisms and regulates cell fate in development and disease All the animal related procedures were approved by the Animal Care and Use Committee of The Tenth People's Hospital of Shanghai(permit number: 2011-RES1). This study was also approved by Science and Technology Commission of Shanghai Municipality (ID: SYXK 2007-0006). The mice were kept at 18°C–26°C on a 12 h light and dark cycle with free access to water and standard mice chow. They were allowed to acclimatize for a minimum of 1 week. The environment was maintained at a relative humidity of 30%–70%.Male Balb/c mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . Trizol reagent was from Invitrogen . The RT reagent kit and Premix Ex Taq were from TAKARA . Caerulein, Hoechst 33342, propidium iodide (PI), Cell Counting Kit-8 (CCK-8), 4′,6-diamidino-2-phenylindole (DAPI) and the antibody against bromodeoxyuridine (BrdU) were purchased from Sigma Chemical . The antibody against β-catenin was from Cell Signaling . The antibody against Dickkopf-1(Dkk-1) was from Abcam . Antibodies against desmin, α-smooth muscle actin (α-SMA), Ki-67 and Lamin A were from Santa Cruz Biotechnology . The antibodies against Wnt2 and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were from Epitomics , and HRP-conjugated secondary antibodies were from Jackson ImmunoResearch . Recombinant mouse Dkk-1 was from R&D Systems . Unless otherwise stated, all other chemicals were purchased from Sigma and cell culture reagents were from Gibco-BRL.All animal experiments were approved by the Animal Care and Use Committee of Tongji University. Chronic pancreatitis was induced in mice by repetitive acute pancreatitis as previously described 2. On the second day and every day thereafter, the culture medium was changed and treated with or without rmDkk-1 at different doses as described in the relevant figures.Primary PSCs were isolated from normal male Balb/c mice by digestion of pancreatic tissue and Nycodenz density gradient centrifugation The phenotype of PSCs was evaluated by immunofluorescence using antibodies specific for mouse desmin and α-SMA. PSCs were probed against a primary monoclonal anti-Ki-67 antibody to examine Ki-67 reactivity in addition to anti-β-catenin and anti-Wnt2 antibodies. For desmin, the staining sequence was goat-anti-mouse desmin (1∶50) and rhodamine-labeled anti-goat (1∶800). The staining sequence for α-SMA was mouse-anti-mouse α-SMA (1∶100) and rhodamine-labeled anti-mouse (1∶800). The staining sequence for Ki-67 was goat-anti-mouse Ki-67 (1∶50), and rhodamine-labeled anti-goat (1∶800). Rabbit-anti-mouse β-catenin (1∶100) and rabbit-anti-mouse Wnt2 (1∶100) were also used for immunofluorescence. Nuclear counterstaining was performed using DAPI, and staining was observed by confocal microscopy .−ΔΔCT) method PSC mRNA levels were detected by qRT-PCR for desmin, α-SMA, β-catenin, PDGFRβ, TGFβRII, Collagen1α1, Wnts, Dickkopf (Dkk) and secreted frizzled-related protein (sFRP) family. Briefly, total cellular RNA was extracted from PSCs using the Trizol reagent  and then quantified. RT reactions were performed with total RNA (2 µg) according to the ExScript RT reagent kit. Real-time PCR was performed in triplicate for each gene of interest under each triplicate experimental condition using the ABI Prism 7900HT Sequence Detection System . GAPDH was used as a separate endogenous control to which each gene of interest was normalized. Fold changes and subsequent percentage gene expression levels relative to the designated control groups were calculated using the comparative CT , anti-α-SMA antibody (1∶200), anti-β-catenin antibody (1∶1000), anti-Wnt2 antibody (1∶1000) and anti-Dkk-1 antibody (1∶200) overnight at 4°C. The next day, membranes were washed and incubated with HRP-conjugated mouse-anti-goat IgG (1∶2000), HRP-conjugated goat anti-mouse IgG (1∶2000), and HRP-conjugated goat anti-rabbit IgG (1∶2000) for 1 h at room temperature. After washing, the membranes were developed using the ECL-detection system , quickly dried, and exposed to ECL film.6 cells in 25 cm2 coated plastic bottles and cultured as previously described. On day 5, cells were collected, washed twice in cold PBS, mixed in 100 µl of 1× binding buffer, and incubated at room temperature for 15 min with an annexin-V/PI (BD Biosciences) double staining solution. Stained cells were analyzed by flow cytometry, and the percentage of apoptotic cells was calculated using ModFitLT software (Verity Software House).Freshly isolated mouse PSCs were seeded at a density of 2×10Apoptosis in PSCs was determined using Hoechst 33342 staining. Briefly, cells were stained with 8 mg/ml Hoechst 33342 and examined directly by fluorescence microscopy. Cells with nuclei containing condensed and/or fragmented chromatin were considered to be apoptotic 5 cells per well in 24-well plates in DMEM/F12 supplemented with 10% FBS and 1% penicillin–streptomycin. After 24 h, the cells were treated as indicated and incubated for 3 days. Proliferation was determined by the Cell Counting Kit-8 according to the manufacturer's instructions.Freshly isolated mouse PSCs were seeded at a density of 2×10For BrdU incorporation assay, 30 mM BrdU  was added to the culture medium for incorporation into the DNA of replicating cells. After 2 h of incubation, cells were fixed in 4% paraformaldehyde. Then cells were immunostained for BrdU according to the manufacturer's instructions. Finally, the proliferative cells were counted in 10 different fields at ×200 magnification in three independent experiments by two persons.RNA interference was performed to silence β-catenin expression. PSCs at 1 day were incubated in Opti-MEM Reduced Serum Medium, transferred to a six-well culture plate until reaching 60%–80% confluence, and transfected with 80 nM siRNA negative control or β-catenin specific FAM-siRNA for 6 h using Lipofectamine 2000 . Cells were then transferred to DMEM/F12 medium containing 10% FBS without antibiotics for 72 h. Pre-designed siRNAs targeting the coding region of the β-catenin gene   and silencer select negative control siRNA (GenePharma) were used in this study. The sequences are as follows: #9371-5′>3′GCCUCUGAUAAAGGCAACUTT, #9558-5′>3′ GCCUUAGUAAACAUAAUGATT, #9370-5′>3′GCACCAUGCAGAAUACAAATT, negative control-5′>3′ UUC UCC GAA CGU GUC ACG UTT. Specific silencing of the targeted gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. Cells were harvested and used for experiments as described below.All results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test for comparison of two groups, and ANOVA for multiple comparisons. In both cases, differences of P<0.05 were considered statistically significant.6 PSCs were harvested from four mice. Freshly isolated PSCs retained a quiescent phenotype for 1–2 days. After 48 h, they began to be activated and became fully activated by day 7 Approximately 1×10Assessment of Wnt gene expression in day 1 (quiescent) and day 7 (fully activated) PSCs by qRT-PCR showed that Wnt2 was notably increased in day 7 PSCs compared with day 1 PSCs . WesternHistopathological signs of CP such as abnormal architecture, glandular atrophy, pseudotubular complexes, fibrosis, and inflammatory cell infiltrates were observed in caerulein treated mice at the time of sacrifice (week 4) . To detein vitro, resulting in a reduction of cell number.Day 1 PSCs were incubated with or without rmDkk-1 at different doses  as previously described. After day 5 PSCs were harvested and cell proliferation was determined by CCK-8 analysis, which showed that high doses of rmDkk-1 (200 ng/ml) inhibited the proliferation of PSCs .FurthermDay 5 PSCs were harvested as previously described for quantitative RT-PCR and western blot analysis. Treatment with rmDkk-1 decreased β-catenin mRNA and protein levels in a concentration dependent manner, supporting our findings that the Wnt/β-catenin pathway was partly antagonized by rmDkk-1 . α-SMA,aTo determine whether rmDkk-1 inhibits the nuclear translocation of β-catenin, day 5 PSCs were harvested for immunofluorescence and western blot analysis. Administration of rmDkk-1 (200 ng/ml) for 3 days resulted in a marked decrease in nuclear β-catenin levels , which fin vitro.SiRNA mediated knockdown of β-catenin in day 1 PSCs and assessment of protein levels on day 5 showed a 90% inhibition of β-catenin expression . Knockdoin vitro.The Wnt signaling pathway plays an important role in a range of developmental and physiological processes including cell fate specification, tissue morphogenesis and homeostasis in vitro. The upregulated genes included Wnt1, 2, 3a and 10b and β–catenin, whereas Dkk-1, 2, 3 and 4 were significantly downregulated. Our screen for Wnts upregulated following the activation of PSCs showed that Wnt2 was the only Wnt that was significantly upregulated after the activation of PSCs. Furthermore, we also found that the levels of Wnt2 and β-catenin proteins were significantly increased in the pancreas of caerulein-induced CP mice. Duan et al reported that a pro-fibrotic Wnt1/β-catenin injury response is critically required for preserving cardiac function after acute ischemic cardiac injury Our results showed, for the first time, that Wnt/β-catenin signaling is activated during the activation of PSCs in vitro and in a mouse model of CP. Our results suggested that the Wnt/β-catenin pathway was activated during the activation of PSCs. The activation of Wnt signaling in PSCs and in an animal model of pancreatic fibrosis has not been described previously, and it suggests an important role for Wnt2/β-catenin signaling in the development of pancreatic fibrosis.Activation of the canonical Wnt/β-catenin pathway results in the accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus to act as a transcriptional coactivator of transcription factors belonging to the T cell factor/lymphocyte enhancer factor (TCF/LEF) family Wnt signals are negatively regulated by secreted antagonists that bind directly to the ligand, such as the secreted frizzled-related protein (sFRP) family, or that prevent LRP coreceptor association with Fz. Dickkopf (Dkk), the best-characterized of the latter group of antagonists, binds to LRP6 and inhibits the formation of the Wnt-induced Fz-LRP5/6 complex that is essential to the canonical Wnt/β-catenin pathway in vitro. Dkk-1 is a member of the Dickkopf family of secreted proteins, which play a significant role as negative regulators of the canonical Wnt/β-catenin pathway. Dkk-1 binds to low-density lipoprotein receptor-related protein (LRP) 5/6 with high affinity. In addition, Dkk-1 binds to another class of receptors, Kremen1 and 2 (Krm1/2). Dkk-1 can form a ternary complex with Krm2 and LRP6 and induce rapid endocytosis and removal of LRP6 from the plasma membrane Although Dkk-1 expression in CP tissues is difficult to detect by western blotting (data not shown), we showed that Dkk-1 levels were decreased during the activation of PSCs in vitro. Knockdown of β-catenin and restoration of Dkk-1 expression downregulated the expression of α-SMA, collagen1α1, TGFβRII and PDGFRβ, inhibited proliferation and induced apoptosis. These results provide further evidence of the important role of the Wnt/β-catenin pathway in PSC activation and shed light on the molecular mechanisms of PSC activation.In the present study, we hypothesized that an imbalance of the Wnt/Dkk negative feedback loop may promote the persistent activation of PSCs. We therefore corrected the imbalance of Wnt/Dkk negative feedback by restoring the level of Dkk-1 using rmDkk-1, which resulted in the partial inhibition of the Wnt signaling pathway. In addition, rmDkk-1 inhibited PSC activation and collagen synthesis, and high levels of rmDkk-1 expression inhibited proliferation and induced apoptosis in PSCs. Inhibition of Wnt signaling inhibited PSC activation in vivo. β-catenin may be involved in pancreatic repair in CP, which is particularly important because global Dkk-1 expression may inhibit pancreatic regeneration requiring canonical Wnt signaling In the present study, treatment of PSCs with the Wnt antagonist Dkk-1 inhibited their activation and attenuated canonical Wnt/β-catenin signaling. The β-catenin-dependent pathway, which is triggered by the interaction of Wnt with Frizzled and LRP5 or LRP6, is prominently involved in the regulation of cell differentiation and proliferation in vivo and in vitro. Our data suggested that an imbalance in Wnt/Dkk negative feedback signaling promotes the persistent activation of PSCs in CP, although the exact underlying mechanism remains unclear. We showed that Dkk inhibited the proliferation and profibrotic phenotype of PSCs by downregulating the expression of collagen1α1, TGFβRII and PDGFRβ via a block of the Wnt/β-catenin pathway. These results indicate that targeting Wnt2/β-catenin/Dkk1 may be a promising therapeutic strategy for the treatment of CP.In conclusion, we showed that the Wnt2/β-catenin pathway was activated during the activation of PSCs


Aberrant activation of β-catenin/Tcf-4 signaling has been implicated in human carcinogenesis, including colorectal cancer. In this study, we compared the effects of Tcf-4 knockdown with β-catenin knockdown on cell proliferation, apoptosis, and chemosensitivity in SW480 and HCT116 colon cancer cells using adenoviral vector-mediated short hairpin RNA (shRNA). Our results show that, compared to β-catenin knockdown, Tcf-4 knockdown more effectively inhibited colony formation, induced apoptosis, and increased 5-FU and oxaliplatin-mediated cytotoxicity in colon cancer cells. We further investigated the mechanisms involved in the different efficacies observed with β-catenin and Tcf-4 knockdown in colon cancer cells. FOXO4 is a member of the subfamily of mammalian FOXO forkhead transcription factors and plays a major role in controlling cellular proliferation, apoptosis, and DNA repair. Our data showed that the protein level of FOXO4 did not change after treatment with both β-catenin and Tcf-4 shRNA. However, β-catenin shRNA was found to increase the accumulation of phosphorylated FOXO4 S193 and decrease the expression of FOXO target genes p27Kip1 and MnSOD, whereas Tcf-4 shRNA showed the opposite effect. Therefore, compared to β-catenin knockdown, Tcf-4 knockdown shows better efficacy for inhibiting proliferation and inducing apoptosis of colorectal cancer cells, which may be related to increased FOXO4 transcriptional activity. These results suggest that Tcf-4 is an attractive potential therapeutic target for colorectal cancer therapy. The canonical Wnt signaling pathway plays a central role in numerous cellular processes, from embryonic development to adult tissue homeostasis β-catenin gene (CTNNB1) is mutated in many cancer cells, including CRC, resulting in the activated transcriptional activity of β-catenin/Tcf signaling et al. reported that knockdown of β-catenin by small interfering RNAs (siRNAs) or knockdown of TCF-4 by dominant negative TCF-4 (dnTCF) efficiently inhibited the activity of the TCF-reporter TOPFlash and induced cell cycle arrest and growth arrest in Ls174T colon cancer cells et al. reported that knockdown of TCF-4 by siRNAs increased cell growth in DLD-1 colon cancer cells Aberrant Wnt/β-catenin signaling has been reported to contribute to various human diseases, including colorectal cancer (CRC) FOXO4 is one member of the subfamily of mammalian FOXO forkhead transcription factors APC, wild-type CTNNB1) and HCT116  colon cancer cell lines using short hairpin RNA (shRNA). We show that compared to β-catenin knockdown, Tcf-4 knockdown significantly inhibits cell proliferation, induces cell apoptosis, and enhances the chemosensitivity of colon cancer cells through the upregulation of FOXO4 transcriptional activity.In this study, we hypothesized that the downstream transcription factor Tcf-4 is a more promising therapeutic target than β-catenin for treating CRC. Our aim was to compare the effects of Tcf-4 knockdown and β-catenin knockdown on cell proliferation, apoptosis, and chemosensitivity in the SW480  and maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 100 U/ml of penicillin and streptomycin under standard culture conditions. Oxaliplatin, 5-fluorouracil (5-FU), and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich . The plasmid pDC316-EGFP-U6 was provided by Vector Gene Technology Company Limited  and the plasmid pBHGloxΔE1, 3Cre was provided by Microbix Biosystems, Inc. .Tcf-4 GenBank accession no. NM_030756), one specific pair of oligonucleotides with a short hairpin and its negative control sequence were designed and synthesized, and then inserted into a small shuttle plasmid pDC316-EGFP-U6 at the BamH I and Hind III restriction enzyme sites. Oligonucleotides were designed with the following primers NM_001098209, 5'-AGCTTTTCCAAAAAGTGGGTGGTATAGAGGCTCTCTCTTGAAGAGCCTCTATACCACCCACGG-3′;′-GATCCCGTGGGTGGTATAGAGGCTCTTCAAGAGAGAGCCTCTATACCACCCACTTTTTGGAAA-3′, reverse β-catenin shRNA primer: 5'-GATCCCCGGAGCGACAGCTTCATATGTTCAAGAGACATATGAAGCTGTCGCTCCTTTTTGGAAA-3′, reverse Tcf-4 shRNA primer:5'-AGCTTTTCCAAAAAGGAGCGACAGCTTCATATGTCTCTTGAACATATGAAGCTGTCGCTCCGGG-3;5′-AGCTTTTCCAAAAACAGTAACTGAATAGCTACCTCTCTTGAAGGTAGCTATTCAGTTACTGGGG-3′. The control shRNA did not have homology to any relevant human genes. All constructs were verified by DNA sequencing. The resulting shuttle plasmids were co-transfected with the adenovirus rescue plasmid pBHGloxΔE1, 3Cre into 293 cells to acquire recombinant adenovirus. Recombinant adenovirus efficiency was detected by examining the cytopathic effect and EGFP expression in the cells. The adenovirus titers were measured after amplification and purification using TCID50 assays.5′-GATCCCCCAGTAACTGAATAGCTACCTTCAAGAGAGGTAGCTATTCAGTTACTGTTTTTGGAAA-3′, reverse direction. SW480 cells were infected with recombinant adenoviruses for 90 min, and after 24 h, they were seeded at 300 cells/well in 6-well plates and allowed to attach for 24 h. After incubation, the medium was changed and the plates were incubated for an additional 10 days under the same culture conditions. Colonies were fixed and stained with 0.1% crystal violet in 100% ethanol and then counted. Clones of at least 50 cells were counted as one colony.SW480 cells were infected with the recombinant adenoviruses for 90 min, and after 24 h, they were plated in 96-well plates at 4000 cells/well. Following 24 h culture, cells were treated with the indicated concentrations of 5-FU or oxaliplatin for 72 h. Then, 20 µL MTT (5 g/L) was added to each well and incubated for an additional 4 h. The culture media were then discarded, 0.15 mL dimethyl sulfoxide (DMSO) was added, and the plates were incubated for an additional 10 min with vibration. The absorbance was measured at 490 nm using a microplate reader .6 cells/well. Twenty-four hours after plating, cells at 70% confluency were infected with adenoviruses for 90 min and then washed to remove the adenoviruses. Following an additional 24 h culture, the apoptotic index was assessed by flow cytometry using Annexin-V-FITC kit as previously described SW480 cells were plated in six well plates at a density of 0.5×10Cells were harvested and total proteins were extracted with RIPA buffer containing protease inhibitors. Western blot was carried out as previously described For measurements of β-catenin/Tcf-4 transcriptional activity, a pair of luciferase reporter plasmids  were used. The pRL-TK luciferase reporter gene plasmid (Promega) was co-transfected to normalize for transfection efficiency. Transient transfection was performed using Fugene 6  according to the manufacturer’s instructions. The cells were infected with the recombinant adenoviruses for 90 min, and after 24 h, were subsequently co-transfected with TOPflash luciferase reporter (or mutant control FOP flash vector) and pRL-TK. After an additional 24 h incubation, cells were harvested for luciferase activity measurement using the dual-luciferase reporter assay system (Promega). Three replicate experiments were performed.P<0.05 was considered statistically significant.Data were expressed as the mean ± standard deviation (s.d.). The statistical significance of differences was determined by one way analysis of the variance (ANOVA) using SPSS v12.0 software . A value of To knockdown the expression of Tcf-4 or β-catenin, adenoviral vectors were generated to express a shRNA targeting Tcf-4 or β-catenin. Western blot analysis revealed a dose-dependent decrease in Tcf-4 or β-catenin protein expression at 48 h post-infection with the respective adenoviral vectors in SW480 and HCT116 cells, while no change in the protein expression was observed after infection with an adenovirus containing scrambled shRNA .SW480 and HCT116 cell lines have constitutively active β-catenin/Tcf-4 transcriptional activity. Therefore, cells were transiently transfected with a TCF reporter plasmid, TOPflash, which consists of three TCF binding sites upstream of a minimal tk promoter and the luciferase open reading frame, or control plasmid, FOPflash, which is identical to TOPflash except that it contains mutant inactive TCF binding sites. Cyclin D1 and c-Myc are known β-catenin/Tcf-4 target genes, and therefore we also investigated the protein expression of cyclin D1 and c-Myc by western blot. To analyze the protein level and activation of FOXO4 protein, SW480 and HCT116 cells were grown for 48 h after infection with adenoviruses, and the protein level was assessed by western blot. The protein level of FOXO4 was not significantly changed after treatment with β-catenin shRNA or Tcf-4 shRNA . HoweverWe next investigated the effect of Tcf-4 knockdown on cell proliferation and apoptosis in SW480 and HCT116 cells. Cell proliferation was determined using a colony formation assay. As shown in SW480 and HCT116 cells were pretreated with the recombinant adenoviruses carrying the respective shRNA and were then treated with 5-FU or oxaliplatin at various concentrations for 72 h. Cell viability was determined using an MTT assay. As shown in APC (up to 80%) and less often of β-catenin (around 10%) or AXIN2 APC/β-catenin genes, which results in aberrant activation of the β-catenin/Tcf-4 pathway, are common in CRC, suggesting that targeted inhibition of this pathway could be a potential therapeutic approach to control CRC. We and others have previously reported that natural compounds and non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the Wnt/β-catenin signaling pathway Colorectal cancer (CRC) is one of the most common adult malignant tumors occurring worldwide in terms of both morbidity and mortality. Despite improvements in medical therapy, the outcomes of treatment for locally advanced and metastatic disease remains disappointing, with 5-year survival rates lower than 10% in patients with metastasis. Aberrant WNT pathway signaling is an early progression event in 90% of CRC. It occurs through mutations mainly of In the present study, we specifically focused on comparing the efficacies of β-catenin knockdown and Tcf-4 knockdown in colon cancer cells, and investigated the possible molecular mechanisms responsible for these effects. We have shown that Tcf-4 knockdown induces a significantly stronger inhibition of cell proliferation, induction of apoptosis, and enhancement of chemosensitivity in colon cancer cells compared to β-catenin knockdown.APC or β-catenin itself. At least 60% of sporadic CRC cases contain one APC mutation, and almost half of them show abnormalities in both APC alleles in vitro.Activated β-catenin/Tcf-4 signaling by accumulation of β-catenin in the nucleus has been implicated in human carcinogenesis, including CRC. This accumulation may result from the mutation of either the tumor suppressor gene Coordination between cell proliferation and apoptosis plays a very important role in the carcinogenesis and development of colon cancer. The β-catenin/Tcf-4 pathway is an important signaling pathway that tips the balance of cell proliferation and apoptosis in cancer cells. In our study, β-catenin knockdown inhibited cell proliferation and induced cell apoptosis in both SW480 and HCT116 cells. Compared to β-catenin knockdown, Tcf-4 knockdown induced a significantly greater inhibition of cell proliferation and induction of apoptosis. Furthermore, we found that Tcf-4 knockdown significantly enhanced the chemosensitivity of both SW480 and HCT116 cells to 5-FU and oxaliplatin.We further investigated the mechanisms involved in the differential efficacies of β-catenin knockdown and Tcf-4 knockdown in SW480 and HCT116 cells. Our data showed that the protein level of FOXO4 did not change after treatment with both β-catenin and Tcf-4 shRNA. However, β-catenin shRNA was found to increase the accumulation of phosphorylated FOXO4 S193 and decrease the expression of FOXO target genes p27Kip1 and MnSOD, which is consistent with an earlier report showing that β-catenin-specific siRNA inhibited TCF reporter activity and FOXO-dependent signaling in LS174 colon cancer cells FOXO factors play major roles in controlling cell proliferation, apoptosis, and oxidative stress Therefore, we hypothesize that the upregulation of FOXO4 activity is responsible for the greater efficacy of Tcf-4 shRNA effects in colon cancer cells compared to β-catenin shRNA. On the one hand, Tcf-4 and FOXO4 may be competing for β-catenin In conclusion, we have shown that targeted inhibition of β-catenin/Tcf-4 signaling inhibits cell proliferation, induces apoptosis, and enhances chemosensitivity of colon cancer cells. In addition, compared to β-catenin knockdown, Tcf-4 knockdown showed greater efficacy for inhibiting CRC cell growth and inducing apoptosis, which may be related to an increase in FOXO4 transcriptional activity. These results suggest that Tcf-4 may be an attractive therapeutic target for CRC therapy. Future studies are needed to confirm these findings and ascertain the utility of targeting Tcf-4.


Signals that disrupt β-catenin association to cadherins may influence the translocation of β-catenin to the nucleus to regulate transcription. Post-translational modification of proteins is a signalling event that may lead to changes in structural conformation, association or function of the target proteins. NO and its derivatives induce nitration of proteins during inflammation. It has been described that animals treated with NO donors showed increased permeability due to modulation of VE-cadherin/catenin complex. We, therefore, aim to evaluate the effect of iNOS activation on the expression, nuclear localisation and function of β-catenin in endothelial cells.Expression, nuclear localisation, post-translational modifications and function of β-catenin was analysed by cell fractionation, immunoprecipitation, immunoblots, QRT-PCR and permeability assays in murine endothelial cells (H5V). Influence of macrophage activation on expression of VE-cadherin/p120-catenin/β-catenin complex in co-cultured H5V cells was also assessed. Activation of macrophages to produce NO provoked a decrease in VE-cadherin/p120-catenin/β-catenin expression in H5V cells. Phosphorylation of β-catenin, p120-catenin and VE-cadherin, and reduction in the barrier properties of the cell monolayer was associated with iNOS induction. Moreover, high NO levels provoked nitration of β-catenin, and induced its translocation to the nucleus. In the nucleus of NOS activated cells, nitration levels of β-catenin influenced its association with TCF4 and p65 proteins. High levels of NO altered β-catenin mediated gene expression of NFκB and Wnt target genes without affecting cell viability.NOS activity modulates β-catenin post-translational modifications, function and its association with different partners to promote endothelial cell survival. Therapeutic manipulation of iNOS levels may remove a critical cytoprotective mechanism of importance in tumour angiogenesis. Nitric oxide (NO), a free radical that mediates cytotoxic effects against host tissues and cells, plays a vital role in the regulation of inflammation. Detrimental effects of NO that are observed in the advanced stages of the inflammatory process include tissue injury and exacerbation of inflammation through activation of inducible nitric oxide synthases (iNOS) β-catenin is a ubiquitously expressed protein that plays at least two important functions in the cell. First, as a protein located at cell-cell adherent junctions (AJ) associated with cadherins  stabilizing their association with the cytoskeleton In the cytoplasm, β-catenin associates with a destruction complex composed of tumour suppressor protein adenomatous polyposis coli (APC), axin proteins, and serine-threonine glycogen synthase kinase-3β (GSK-3β) in vitro and in vivoGiven that the inflammatory process is characterized by high levels of NO, a leaky endothelium and expression of NFκB pro-inflammatory target genes, we hypothesized that NO may influence β-catenin function during chronic and acute inflammatory processes. We have previously reported that NO released by NO donors modulates the expression of VE-cadherin/catenin complex and increases endothelial cell permeability both Activated macrophages are a significant source of iNOS-derived NO at the site of inflammation Induction of iNOS expression and activity in macrophages, stimulated with the IFNγ/LPS combination, was confirmed respectively by immublots and the increase of nitrite levels in the media . In paraBased on the observations described we decided to assess the effect of iNOS induction in our endothelial model. iNOS induction has been observed in endothelial cells from different vascular origins NO-dependent regulation of the VE-cadherin/p120-catenin/β-catenin complex was observed in both TX-soluble and insoluble fractions, as well as in total cell lysates . Using qThe L-arginine analogue LNMMA was used to cause irreversible inactivation of the NOS enzyme We then tested if iNOS induction provoked any alterations on the function of VE-cadherin/p120-catenin/β-catenin complex regulating barrier properties of the endothelial monolayer. Indeed, iNOS induction results in a time dependent increase in the permeability of the H5V monolayers to large biomolecules (Ig-HRP), with highest permeability values observed after 24 h iNOS activation . This efWe also studied the effect of NO on human VE-cadherin/p120-catenin/β-catenin complex expression and endothelial barrier properties in confluent HUVEC monolayers. Analysis of HUVEC TX-fractions, as well as total cell lysates, was done as described for H5V cells. VE-cadherin levels were markedly reduced in both TX fractions and total cell lysates. p120-catenin levels mirrored VE-cadherin levels in all samples . β-catenThese observations suggest that IFNγ/LPS effect on the levels of β-catenin, p120-catenin and VE-cadherin are attributable to the presence of an active iNOS and the production of high levels of NO  and S2. Post-translational modifications of AJ proteins are associated with impaired barrier function. For instance, inflammatory mediators such as TGF-β, VEGF, histamine and peroxynitrite increase vascular permeability via AJ phosphorylation or nitration We also determined whether IFNγ/LPS treatment promotes nitration of AJ proteins in H5V cells. Protein tyrosine phosphorylation and nitration might be mutually exclusive. Therefore, we decided to establish protein nitration in cells stimulated for longer periods (12 h–24 h). Tyrosine nitrated proteins were immunoprecipitated from cell lysates with an antibody that specifically recognises nitrotyrosine. The immuprecipitate was then immunoblotted for VE-cadherin, p120-catenin and β-catenin. We failed to detected nitration of either VE-cadherin or p120-catenin in IFNγ/LPS treated cells, or in cells exposed to NO releasing compound SNAP . Howeverβ-catenin phosphorylation reduces its affinity for the cadherin cytoplasmic tail and targets the protein either for degradation by the ubiquitin/proteasome pathway (serine/threonine phosphorylation) or for translocation into the nucleus (tyrosine phosphorylation) Post-translational modification of β-catenin influences its cellular location and interaction with different partners such as TCF4 and NFκB proteins Nitration seems to influence the abundance of transcription factors associated with endothelial β-catenin . In unstThe observed effects seem to be mediated by NO, since both NAP and LNMMA restored the levels of TCF4 protein associated with β-catenin to those observed in unstimulated samples . NonetheGiven that nitration influences association of β-catenin with NFκB and Wnt pathway transcription factors, we checked whether iNOS activation influences the transcription levels of Wnt and NFκB pathway target genes in endothelial cells.We selected the antiapoptotic molecule A20 and iNOS, both known targets of the NFκB pathway, to verify NFκB downstream target gene expression in stimulated H5V cells In the case of iNOS transcript, its level at 12 h resembles the abundance of β-catenin/p65 complexes; whereas a reduction of its transcription is observed at longer stimulations times. It has been widely described that high levels of NO inhibit NFκB transcriptional activity on the iNOS promoter, a feedback mechanism that protects the cell from sustained iNOS activation Since both murine  and human  VE-cadherin promoter region contains several binding sites for TCF4 proteins; we investigated the expression of VE-cadherin as a target for the Wnt pathway Finally, the levels of the antiapoptotic protein Survivin were also measured, as it is a target for both the NFκB and Wnt pathways Given that expression of genes involved in cell survival and the antiapoptotic response were upregulated in conditions were nitration was favoured, we tested whether iNOS activation influences viability of the cells. We observed that although IFNγ/LPS stimulations slightly reduced the percentage of viable cells, these reductions were not statistically significant. Viability of the cells was not altered by IFNγ/LPS/LNMMA, NAP or SNAP treatments. In conclusion, conditions that favour release of high levels of NO and β-catenin nitration did not affect endothelial cell viability. Overall these results suggest that, in endothelial cells, β-catenin acts as a sensor of nitrating stress favouring the transcription of cytoprotective genes to limit endothelial cell damage.NO, either derived from endothelium or inflammatory cells, is an important signalling mediator in many endothelial cell (EC) processes β-catenin interacts with different transcription factors depending on the functional condition of the cells  determining the downstream cellular responses Endothelial cell survival is also required during vascular remodelling and angiogenesis Integrity of the endothelium and control of its barrier properties is critical for modulating the inflammatory response In several systems, the tyrosine phosphorylation of β-catenin reduces its affinity for cadherin’s cytoplasmic tail Recruitment of p120-catenin to AJs promotes cadherin clustering and strengthens adhesion Tyrosine phosphorylation of AJs in response to different stimuli is mediated by several tyrosine kinases including Src, PYK2, PAK and RAC kinases Nitration of proteins has been described in inflammatory pathologies characterized by high levels of NO Loss of VE-cadherin at cell-cell contacts, vascular injury and increase in nitrotyrosine formation are among the detrimental effects caused by oxidative stress Exposure of cells to NO increases the production of superoxide anion and consequently of the pool of nitrogen dioxide radical, involve in tyrosine nitration of proteins β-catenin/TCF signalling promotes vascular remodelling after injury and survival of ECs Human and murine endothelial cells have a functional Wnt signalling pathway that could play a role in growth and differentiation of normal endothelium as well as pathological angiogenesis In conclusion, we demonstrated that iNOS-derived NO regulates endothelial β-catenin functions. Our data suggest that a NO signalling pathway exist in ECs that recognizes the nitrating stress. β-catenin acts as sensor protein transmitting NO signal by switching its role from adherent protein to nuclear transcription factor, regulating gene expression. This mechanism might support EC survival during inflammation limiting cell injury associated with high levels of NO. Conversely in cancer cells, NO regulation of β-catenin may provide a mechanism of cytoprotection to maintain a pool of ECs necessary to promote tumour growth. This study may help to increase the understanding of the pluripotential roles of NO in vascular physiology and pathophysiology.2 atmosphere. Human umbilical vein endothelial cells (HUVEC) , were cultured as described before 2 atmosphere.H5V, a murine microvascular endothelial cell line Escherichia coli lipopolisacaride , L-N-monomethyl arginine citrate (LNMMA), S-nitroso-N-acetylpenicillamine (SNAP) and N-acetylpenicillamine (NAP) were from Sigma (UK).Murine recombinant interferon gamma (IFNγ), Mouse monoclonal antibody (mAb) to β-catenin (clone 14), mouse mAb to NOS II (clone 6) and mouse mAb to phosphotyrosine residues (clone PY69) were from Transduction Laboratories (USA). Rabbit polyclonal anti-p120 catenin (H-90) was from Santa Cruz Biotechnology . Rat mAb to murine VE-cadherin (clone BV13) and mouse mAb to PECAM-1 (clone 7.46) were from Abcam (UK). Mouse anti-TCF4 (clone 6H5-3) and rabbit anti-p65 antibodies were from Millipore (UK). IgG-HRP peroxidase conjugates were from Amersham (UK). Rabbit polyclonal anti-nitrotyrosine antibody was from Cayman Chemical  and rabbit polyclonal Histone (H3) antibody and rabbit mAb to α-tubulin (11H10) were from Cell Signaling Technology (UK).The nitrite content in cell culture supernates was determined using the Griess reagent containing 0.5% sulfanilamide and 0.05% N-(1-naphthyl) ethylenediamine hydrochloride in 45% acetic acid. Nitrite reacts with Griess colour reagent to give a red-violet diazo-dye which is measured spectrophotometrically at 543 nm and nitrite concentration is calculated from a sodium nitrite standard curve. Briefly, 500 µl of cell culture media were taken and added to a plastic cuvette containing 500 µl of Griess reagent and the absorbance was measured at 543 nm 3 cells/well) were seeded in triplicate into 96-well plates. On the following day, cells were incubated with IFNγ/LPS, IFNγ/LPS/LNMMA, SNAP or NAP. At various time points  viable cells were estimated by 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 20 µl of MTT  solution was added to each well  4 hours before the end of the experiment. The medium was then aspirated and the formazan crystals were solubilized with dimethylsulfoxide. Absorbance at 630 nm was subtracted (substrate background) from absorbance at 570 nm in each well. Values of viability of treated cells were expressed as a percentage of that from corresponding control cells (assumed to be 100%).For viability assays, H5V cells (15×102+- and Mg2+-containing PBS. Cells were extracted for 20 min on ice in lysis buffer containing 40 mM Tris (pH 7.6), 500 mM NaCl, 2 mM CaCl2, 1% Nonidet P-40, 1% Triton X-100, 2 mM Na3VO4, 1 mM PMSF, 20 U/ml aprotinin and 15 µg/ml leupeptin. Cell extracts were then centrifuged at 14000 g for 5 min (4°C) and the supernatant was defined as the TX soluble fraction while the pellets (TX insoluble fraction) were further solubilised in lysis buffer supplemented with 0.02% SDS. For total cell lysates, monolayers were extracted in lysis buffer containing 0.5% SDS. For experiments in which tyrosine phosphorylation was studied, cells were then washed twice in Ca2+- and Mg2+-containing PBS and disrupted on ice for 20 min with lysis buffer. Cell extracts were then centrifuged at 14000 g for 5 min (4°C) and used in immunoprecipitation assays.Triton X-100 (TX) soluble and insoluble fractions, as well as total cell extracts, were obtained from long confluent endothelial cells as described previously Nuclear proteins were isolated using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer protocol (Pierce Biotech).Immunoprecipitation was performed as described previously 2. Separated proteins were electroblotted onto a PVF membrane. Membranes were incubated overnight with blocking buffer (5% BSA dilute in 1 mM Ca+2- and Mg+2-containing 0.1% Tween-20-TBS (CaMgTTBS) and subsequently incubated for 1 hour at room temperature with the appropriate primary antibody diluted in blocking buffer. Blots were then incubated for 1 h at room temperature with the correspondent IgG horseradish peroxidase secondary antibody diluted in blocking buffer. Between incubation steps, membranes were washed several times with CaMgTTBS buffer. Blots were analysed for α-tubulin or H3  to normalize the protein load in each well. Immunoreactive bands were visualized by ECL using a Chemidoc System Bio-Rad Imager and quantified by Quantity One Imaging software  as a function of volume data (intensity/mm2). The Volume Rectangle Tool was used to measure the total signal intensity inside a boundary drawn around the bands without overlapping adjacent bands. Background was subtracted from each band volume by using local background subtraction. Intensities of bands acquired from each protein extract were normalised against corresponding values for bands of the house-keeping protein. Results were expressed as Band Density normalised to a house-keeping protein and are expressed as Intensity per square millimetres (INT*mm2).For immunoblotting 10 µg of protein samples diluted in Laemmli buffer  were electrophoresed on a 7.5% SDS-polyacrylamide gel. The gel was then incubated for 30 min (2×15 min) in transfer buffer containing 1 mM CaCl4 cell/cm2 at seeding) were cultured for 6 days in Transwell units  To analyse the effect of iNOS activation on cellular permeability, H5V cells (3×10).Dnase-I treated RNA was reverse transcribed into cDNA before assessing VE-cadherin, antiapoptotic molecule A20, survivin and iNOS expression using specific primer pairs . RT-PCR amplification was performed in triplicate in 96-well plates in a BioRad IQ iCycler. Serial dilutions of cDNA were used to plot a calibration curve, and gene expression quantified by plotting threshold cycle values. Expression levels were normalized to values obtained for the housekeeping gene . For normally distributed data, a t-test was used to determine the significance of differences between groups. All data analysis was performed using SPSS version 10.0 (SPSS Chicago IL). Differences were considered significant at the P≤0.05 levels.Figure S1Densitometric analysis of immunoblots shown in . Protein bands were visualized using a ChemiDoc System Bio-Rad Imager (Bio-Rad) and quantified by Quantity One® Imaging software (Bio-Rad) as described in 2). Statistical analysis was done using a t-test. The significance level was set at P<0.05 . A. Quantification of iNOS induction in H5V cells stimulated with IFNγ/LPS Click here for additional data file.Figure S2NO regulates the expression and function of VE-cadherin/p120catenin/β-catenin complex in H5V cells. Panel A. Expression of VE-cadherin, p120-catenin (p120-ctn) and β-catenin is reduced in HUVEC cells incubated with IFNγ/LPS. NOS inhibitor LNMMA attenuates NO effect on VE-cadherin/catenin complex. HUVEC TX fractions and total cell lysates were analysed by western blot for VE-cadherin, p120-catenin (p120-cnt) and β-catenin levels using specific antibodies. α-tubulin levels were used as a loading control. Nitrite production was measured using the Griess method and nitrite concentrations expressed in µM. B–D. Graphs represent the densitometry analysis of Panel A western blots. α-tubulin was used as a loading control. Protein bands were visualized using a ChemiDoc System Bio-Rad Imager (Bio-Rad) and quantified by Quantity One® Imaging software (Bio-Rad) as described in 2). Statistical analysis was done using a t-test. The significance level was set at P<0.05 . E. NO stimulates paracellular permeability to IgG-HRP in HUVEC cells. HUVEC cells were grown to confluence in Transwell units and stimulated to produce NO. Monolayer permeability to a tracer  was measured as described in (TIF)Click here for additional data file.


The IC50 values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC Wnt/β-catenin signaling plays an important role in embryonic development and can lead to tumor formation when aberrantly activated. A hallmark of the Wnt/β-catenin signaling activation is the stabilization of cytosolic β-catenin, which enters the nucleus to activate Wnt target genes by binding transcription factors of the T-cell factor/lymphoid enhancing factor (TCF/LEF) family Experiments performed in drosophila Niclosamide (trade name Niclocide) is a teniacide in the antihelmintic family that is especially effective against cestodes, which infects humans. Niclosamide has been FDA approved for such indications and has been used in humans for nearly 50 years Uncomplexed cytosolic β-catenin (free β-catenin) is the active form of β-catenin that is translocated to the cell nucleus to activate transcription factors of the TCF/LEF family, leading to the transcription of Wnt target genes. To determine if niclosamide can inhibit Wnt/β-catenin signaling, HEK293 cells were treated for with Wnt3A conditioned medium (CM) in the presence of niclosamide or vehicle (DMSO). The levels of cytosolic free β-catenin and total cellular β-catenin were then examined by Western blotting. As shown in LRP6 is an essential Wnt co-receptor for the Wnt/β-catenin signaling pathway, and LRP6 phosphorylation is critical for Wnt/β-catenin signaling activation induced by Wnt proteins To confirm the inhibitory effect of niclosamide on LRP6 expression and phosphorylation, we performed a Wnt/β-catenin signaling reporter assay to test whether niclosamide is able to inhibit Wnt/β-catenin signal activation by LRP6 and Wnt3A. HEK293 cells were transiently transfected with LRP6 along with Wnt/β-catenin signaling reporter TOPFlash, and treated with Wnt3A CM and niclosamide. As shown in To determine if niclosamide can block Wnt/β-catenin signaling in cancer cells, we examined the level of cytosolic free β-catenin after niclosamide treatment. We found that cytosolic free β-catenin levels in human prostate PC-3 and breast MDA-MB-231 cancer cells were significantly reduced after niclosamide treatment . FurtherIt is well recognized that axin2 is a specific transcriptional target of the Wnt/β-catenin signaling pathway, and that the expression level of axin2 is the signature of the activation of Wnt/β-catenin signaling To determine if the inhibitory effect of niclosamide on Wnt/β-catenin signaling is related to LRP6 expression, we examined LRP6 expression and phosphorylation after niclosamide treatment. As shown in To define the molecular mechanism underlying the effect of niclosamide on LRP6 protein level, we studied LRP6 turnover. PC-3 cells were treated with niclosamide at 1.2 µM for 0, 3, 6, 10 and 24 h in the presence of cycloheximide, a protein synthesis inhibitor. As shown in It has been reported that niclosamide downregulates components of the Wnt pathway, specifically cytosolic Dvl2 expression, resulting in diminished downstream Wnt/β-catenin signaling in colorectal cancers The Wnt/β-catenin pathway is important for cancer cell apoptosis 50 values less than 1 µM for all four tested cell lines. The IC50 values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells.Given that niclosamide can block Wnt/β-catenin signaling in cancer cells and can induce apoptosis, we next examined the effect of niclosamide on cancer cell proliferation. As shown in APC and CTNNB1, are significant contributing factors for colorectal cancers, they are typically not the predominate mechanism associated with many other types of cancer such as breast and prostate cancers. Instead, it appears that dysregulation of Wnt/β-catenin signaling on the cell surface leads to aberrant activation of this pathway in these types of cancer Compelling evidence indicates that there is an abnormal up-regulation of the Wnt/β-catenin pathway in tumorigenesis of many types of cancer. While genetic mutations of certain components of the Wnt/β-catenin pathway, such as +/- mice in vivoin vivo50 values less than 1 µM. Our results support the concept that LRP6 is a promising therapeutic target for breast cancer including TNBC.Wnt/β-catenin signaling plays an important role in mammary gland development and carcinogenesis in vitro and tumor growth in vivo50 values less than 1 µM, which are comparable to those shown to suppress LRP6 expression and the activities of Wnt/β-catenin signaling in prostate cancer cells. Together, these findings suggest that LRP6 is a potential therapeutic target for prostate cancer.Accumulated evidence has demonstrated a significant role for Wnt/β-catenin signaling in the development and progression of human prostate cancer APC and CTNNB1 are two major factors for Wnt/β-catenin signaling activation in colorectal cancers, however recent studies indicate that the levels of Wnt/β-catenin signaling in colorectal cancer cells could be modulated on the cell surface et al. demonstrated that Wnt receptor frizzled-7 activates the Wnt/β-catenin pathway in colorectal cancer cells despite the presence of APC or CTNNB1 mutation and that frizzled-7-siRNA may be used as a therapeutic reagent for colorectal cancer et al. reported that siRNA silencing of Wnt-2, which is well known for its overexpression in colorectal cancer, inhibited Wnt/β-catenin signaling and induced apoptosis in human colorectal cancer cells containing downstream mutations et al. reported that loss of WIF-1 expression was associated with promoter hypermethylation in colorectal cancer, and that restoration of WIF-1 function, Wnt-1 siRNA, or a monoclonal anti-Wnt-1 antibody induces significant apoptosis in colorectal cancer cells which contain downstream mutations and expressing Wnt-1 Mutations in in vitro and displays no obvious side effects in niclosamide-treated mice in vitro. Therefore, as a FDA approved antihelminthic drug, niclosamide will be an interesting comoound to be optimized to increase its bioavailability and to be tested for its in vivo cancer preventive and therapeutic roles in the future.The development of FDA approved drugs that are used for other indications is an attractive strategy for developing new drugs for cancer chemoprevention or therapy given their proven safety record. Disruption of Wnt/β-catenin signaling represents a great opportunity to determine if FDA approved drugs might have potential anticancer efficacy Niclosamide was purchased from Sigma. Polyclonal anti-phospho-LRP6 (Ser1490) and anti-Dvl2 and monoclonal anti-Axin2 were purchased from Cell Signaling Technology. Monoclonal anti-LRP6 was from Santa Cruz Biotechnology. Polyclonal rabbit anti-Cyclin D1 was from Chemicon International. Monoclonal anti-β-catenin was from BD Biosciences. Monoclonal anti-actin was from Sigma. Peroxidase labeled anti-mouse antibody and ECL system were purchased from Amersham Life Science. Plasmid pCS-Myc-hLRP6 containing the full-length human LRP6 cDNA was from Dr. Christof Niehrs . Plasmid Glutathione S-transferase (GST)-E-cadherin was kindly provided by Dr. Gail Johnson . The TOPFlash luciferase construct was from Upstate Biotechnology. The β-galactosidase-expressing vector, the luciferase assay system and the β-galactosidase assay system were purchased from Promega. Tissue culture media, fetal bovine serum (FBS), and plastic-ware were obtained from Life Technologies, Inc. Proteinase inhibitor cocktail Complete™ was obtained from Boehringer Mannheim. All cell lines were obtained from ATCC and were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM of L-glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin. Cells were grown in antibiotic-free medium for cell proliferation assays. Cell Titer Glo assay systems were from Promega. Cell death detection ELISA kit was purchased from Roche Diagnostics Corporation.Cells in 6-well plates were lysed in 0.5 ml of lysis buffer  at 4°C for 10 min. Equal quantities of protein were subjected to SDS-PAGE under reducing conditions. Following transfer to immobilon-P transfer membrane, successive incubations with a primary antibody, and a horseradish peroxidase-conjugated secondary antibody were carried out for 60–120 min at room temperature. The immunoreactive proteins were then detected using the ECL system. Films showing immunoreactive bands were scanned by Hp Scanjet 5590.The GST-E-cadherin binding assay was carried out exactly as previously described Total RNA was isolated from cell cultures using RNA-Bee (Tel-Test), and first-strand cDNA synthesis was performed using ProSTARTM Ultro HF RT-PCR Kit (Strategene) primed with oligo(dT) primer in a 20 µl reaction mixture containing 1 µg total RNA. For analysis of LRP6 mRNA levels, real-time RT-PCR was performed with the specific LRP6 and GAPDH primers purchased from SABioscience/Qiagene. The LRP6 mRNA level was normalized to the GAPDH mRNA level.Cytosolic Dvl2 expression was examined with the method as described for elsewhere Cancer cells were plated into 24-well plates. After overnight culture, the cells were transiently transfected with 0.1 µg of the TOPFlash luciferase construct (Upstate Biotechnology) and 0.1 µg of β-galactosidase-expressing vector . After 24 h incubation, cells were treated with niclosamide. Cells were then lysed 24 h later and both luciferase and β-galactosidase activities were determined. The luciferase activity was normalized to the β-galactosidase activity.Apoptosis was assessed with cell death detection ELISA kit purchased from Roche Diagnostics Corporation, Indianapolis. This assay detects oligonucleosomes released after gentle lysis of the cell. Cells were cultured in T25 flasks for the desired duration. The spent medium containing floating cells was saved and kept on ice. The adherent cells were collected by gentle trypsinization and were combined with the floaters for pelleting by centrifugation. After gentle lysis of the cells with the buffer provided with the detection kit, the cell lysate was used for the ELISA test. The results were normalized by the protein content obtained from parallel flasks with the cells being lysed using the buffer as described above for Western blotting.Cells were seeded into 96-well tissue culture treated microtiter plates at a density of 5000 cells/well in a total volume of 50 µl. After overnight incubation, the cells were treated with niclosamide for 72 h by adding 50 µl of 2X stock solutions to appropriate wells already containing 50 µl of cells and medium to expose cells to the final concentrations of niclosamide required. Cell viability was measured by the Cell Titer Glo Assay (Promega).Statistical analyses were performed using Student's unpaired t-test. Data were presented as mean ± SD. Differences at P<0.05 were considered statistically significant.