The pathogenesis of pancreatic ductal adenocarcinoma (PDAC) remains poorly understood. S100 calcium-binding protein A6 (S100A6) has been associated with PDAC; however, the effect of S100A6 on PDAC migration and invasion has not yet been explored. In this study, Panc-1 cells were transfected with a plasmid to induce overexpression of S100A6, and β-catenin was knocked down using a specific short hairpin RNA (shRNA). The wound-healing and Transwell assays demonstrated that S100A6 promoted PDAC cell migration and invasion. Furthermore, β-catenin shRNA inhibited the migration and invasion of PDAC cells. We confirmed that S100A6 induces PDAC cell migration and invasion via activation of β-catenin in vitro. Assessment of mRNA and protein levels revealed that S100A6 induces increased expression of β-catenin, N-cadherin and vimentin, and decreased expression of E-cadherin in PDAC cells. β-catenin shRNA also altered the expression of epithelial-mesenchymal transition (EMT)-related markers in PDAC cells. Specifically, expression of E-cadherin was increased, whereas expression of N-cadherin and vimentin was decreased. Finally, we demonstrated that S100A6 alters the expression of EMT-related markers via β-catenin activation. In conclusion, S100A6 induces EMT and promotes cell migration and invasion in a β-catenin-dependent manner. S100A6 may therefore represent a novel potential therapeutic target for the treatment of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is a serious global health problem. It is the fourth most common cause of cancer-related death in the United States, with a 5-year overall relative survival rate of 6%. In ChinS100A6 belongs to the S100 family, expression of which is connected to tumorigenesis and metastasis. LogsdonWnt/β-catenin signaling influences cell fate, proliferation, polarity, and cell death during embryonic development, as well as tissue homeostasis in adults. AberranThere is a wealth of research concerning the attributes of circulating tumor cells that relate to the epithelial-mesenchymal transition (EMT),15. EMT In this report, we investigated the function and associated mechanism of S100A6 in relation to EMT induction, by assessing its influence on Panc-1 cell migration and invasion.2.The human pancreatic cancer cell line, Panc-1, was purchased from the American Type Culture Collection (USA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)  containing 10% fetal bovine serum , 1% penicillin and streptomycin  at 37°C with 5% COThe complementary DNAs for human S100A6 and the negative control DNA were inserted into a GV146 plasmid vector purchased from Genechem . A shRNA sequence targeting β-catenin (5′-ATCACTGAGCCTGCCATCTGTGCTCTTCG-3′) and a negative control sequence were synthesized and ligated into the pRFP-C-RS vector . The plasmids were amplified according to the manufacturer’s instructions. Sequence analysis after cloning revealed 100% homology to published sequences.The S100A6 overexpression plasmid and the control plasmid were transfected into Panc-1 cells using opti-MEM reduced-serum medium  and Lipofectamine 2000 , according to the manufacturer’s instructions.2.β-catenin shRNA and control shRNA were transfected into Panc-1 cells as stated above. After transient transfection, Panc-1 cells were selected using 1.0 μg/mL puromycin, and maintained in growth medium supplemented with 1.0 μg/mL puromycin. Clonal populations of cells were isolated by transferring single clumps of cells into individual wells of a six-well plate. Cells were then maintained at 37°C with 5% COThen S100A6 overexpression and control plasmids were transfected into β-catenin knockdown stable cell lines, as stated above.Cells were seeded into six-well plates and transfected with the S100A6 overexpression or control plasmid, and β-catenin shRNA or control shRNA. After 36 h, complete medium was replaced with FBS-free DMEM, and after a further 6 h, the cell monolayers were wounded with a 200-μL sterile plastic tip. The cultures were thereafter maintained in FBS-free DMEM, and wound areas were observed and photographed using an inverted microscope at 0, 24 and 48 h after wounding. The wound width was measured using Image J software. Wound healing rate was assessed as follows:5/mL in FBS-free DMEM, was seeded into the upper chambers. The lower chambers were filled with DMEM containing 10% FBS as a stimulatory factor. The chambers were incubated in a humidified tissue culture incubator at 37°C, 5% CO2 atmosphere, for 8 h. Cells were then fixed with 95% ethanol for 20 min, stained by crystal violet for 30 min, and counted using a microscope with a 40× objective.Transwell chambers  with an 8-μm pore were used to assess migration in 24-well plates. After transfection, a 200-μL volume of cells, at a density of 1 × 10To assess cell invasion, BioCoat Matrigel (BD Biosciences)  was applied to the upper insert chambers 5 h before following the procedure for the migration assay described above.−ΔΔCt[Total RNA from Panc-1 cells was isolated using TRIzol reagent . Total RNA was used as a template to synthesize cDNA, according to the manufacturer’s instructions . Real-time PCR was performed using a 7500 Real-Time PCR System , with a reaction mix consisting of 5-μL 2× SYBR Green Master Mix, 1-μL template, 0.4-μL forward primer, 0.4-μL reverse primer and 3.2-μL RNA-free water. The cycling conditions were 95°C for 10 min, followed by 40 cycles of 95˚C for 15 s and 60˚C for 1 min. Gene expression levels were normalized relative to that of GAPDH. All reactions were performed in triplicate. Relative mRNA levels were presented as 2−ΔΔCt. The priAfter transfection, cells were harvested using RIPA lysis buffer  supplemented with PMSF protease inhibitor . The protein concentrations of the samples were determined using a protein quantitation kit . Samples (40–100 μg) were then subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF). Membranes were blocked for 2 h in 5% nonfat dried milk. Membranes were incubated with specific antibodies at 4°C overnight. The following primary antibodies were used in this study: anti-β-catenin , anti-E cadherin , anti-N cadherin , anti-vimentin  and anti-β-actin . The membranes were then incubated with the corresponding fluorescent secondary antibodies (Odyssey). Western blots were quantified using Odyssey Infrared Imaging. Protein expression levels were normalized relative to that of β-actin.t-test was used for statistical analysis. All data were analyzed using the SPSS 17.0 statistical software package and figures were generated using the GraphPad Prism 5 software. For all tests, p < 0.05 was considered to indicate statistical significance.All experiments were carried out in triplicate and repeated independently at least three times. Data are presented as means ± SD (standard deviation). Student’s To assess whether S100A6 promotes PDAC cell migration and invasion, Panc-1 cells were transfected with a S100A6 overexpression plasmid or a control plasmid. Real-time RT-PCR was used to verify that the S100A6 overexpression plasmid increased the mRNA levels of S100A6 in Panc-1 cells. We detected a significant increase in the level of S100A6 mRNA in S100A6-overexpressing cells, compared to control cells (p < 0.001) .. Next, When investigating the possible downstream mediators of S100A6 responsible for promotion of PDAC cell migration and invasion, we noted that overexpression of S100A6 resulted in a significant increase in β-catenin mRNA levels in Panc-1 cells (p = 0.006) . Given tTo explore the effect of β-catenin on migration and invasion of PDAC cells, we used β-catenin shRNA to generate a stable β-catenin knockdown Panc-1 cell line. Panc-1 cells transfected with non-target shRNA were used as a control. Knockdown of β-catenin was confirmed by real-time PCR and Western blotting  .. A wounTo evaluate whether β-catenin is involved in the EMT, we assessed the expression of EMT-related markers at the mRNA and protein levels in β-catenin-knockdown Panc-1 cells. Knockdown of β-catenin resulted in increased levels of E-cadherin mRNA, and decreased levels of N-cadherin and vimentin mRNA  . WesternTo explore whether S100A6 promotes PDAC cell migration and invasion via regulation of β-catenin, we transfected stable β-catenin-knockdown Panc-1 cells with a S100A6 overexpression plasmid. We assessed β-catenin expression using real-time RT-PCR and Western blot analyses. There were no significant differences in the mRNA and protein levels between the two groups  .. These To investigate the potential mechanistic relationship between S100A6 and β-catenin in relation to EMT in pancreatic cancer, we transfected stable β-catenin-knockdown Panc-1 cells with a S100A6 overexpression plasmid or a control plasmid, and evaluated the expression of EMT-related markers. Real-time RT-PCR demonstrated no significant differences in the mRNA levels of E-cadherin, N-cadherin and vimentin between the two groups  . We confIn this study, we demonstrated that S100A6 overexpression promotes, and β-catenin shRNA inhibits, PDAC cell migration and invasion. We established that S100A6 promotes PDAC cell migration and invasion via β-catenin activation in vitro. We also confirmed that S100A6 expression correlates with that of β-catenin and that the expression of EMT-related markers in PDAC cells is modified by both S100A6 overexpression and β-catenin knockdown.S100A6 is reported to be upregulated in a number of cancers, including lung, colorectal, skin, gastric, pancreatic ductal adenocarcinoma and other epithelial cancers. It is rA wide range of experimental models are suggestive of an important role for β-catenin in pancreatic cancer metastasis. NowotnyIn numerous models, including mammary epithelial and carcinoma cell lines, the Wnt/β-catenin pathway is thought to induce EMT. β-catenin is usually bound to E-cadherin, which is important for cell-cell connections and the cadherin cytoskeleton. In addition, β-catenin transportation into the nucleus can promote the expression of EMT related genes. Some stA recent study reported that S100A4, a member of the S100 family, could be a key factor in promoting the EMT process in PDAC. In thisS100A6 can induce EMT and promote cell migration and invasion in a β-catenin-dependent manner. S100A6 may therefore represent a novel potential therapeutic target for PDAC.S1 Fig***p < 0.001.(TIF)Click here for additional data file.S2 Fig(A) Real-time RT-PCR revealed a significant decrease in the β-catenin mRNA level in β-catenin shRNA Panc-1 cells. (B) Western blotting revealed a significant decrease in β-catenin protein in the β-catenin shRNA group, relative to the control. (C) The level of β-catenin protein was significantly different between the two groups. **p < 0.01; ***p < 0.001.(TIF)Click here for additional data file.S3 Fig(A) There were no significant differences in β-catenin mRNA levels between stable β-catenin-knockdown cells overexpressing S100A6 and stable β-catenin knockdown cells expressing the control plasmid. (B) Western blotting revealed similar changes in β-catenin between the two groups. (C) There were no significant differences between the β-catenin protein levels between the two groups.http://www.textcheck.com/certificate/GZTsUkThe English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:(TIF)Click here for additional data file.


These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3β and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3β and Nur77 to allow β-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased β-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3β-9ser , Nur77 , and β-catenin  expressions. In conclusion, we identified GSK-3β and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of β-catenin and subsequent downstream signaling mediated by β-catenin in HCC cells, and provides potential targets for future therapeutic interventions.The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray Hepatocellular carcinoma (HCC) is the seventh most lethal malignancy worldwide, and the second leading cause of cancer related death  (2012 Globocan (WHO IARC)). It is typically fatal with a mortality-to-incidence ratio of 0.95 due to difficulties in early detection, limited therapeutic options, and high recurrence rates. Lack of reliable HCC biomarkers leads to late diagnosis, especially in high risk populations (chronic carriers of hepatitis B virus or hepatitis C virus). Meanwhile, there is no curative therapy for the majority of HCC patients who are diagnosed at advanced stages.Our previous gene expression data identified N-Myc downstream regulated gene 1 (NDRG1) as a clinically relevant biomarker of HCC. NDRG1 is a stress response protein whose expression is regulated by a variety of cellular stresses, especially hypoxia . SignifiWe aim to further elucidate the biological functions mediated by NDRG1 in HCC cells, so as to improve our understanding of hepatocarcinogenesis and tumor progression, which in turn may allow rational design of novel therapeutic approaches targeting NDRG1. To this end, we attempt to identify novel interaction partners of NDRG1 through protein array screening and to further study the molecular events regulated by these interactions in HCC cells.p values less than 0.05. Among these candidates, we selected GSK-3β and Nur77, two functionally important proteins on the top of list, for further investigations. To validate these potential hits, we first confirmed the interactions of NDRG1 with GSK-3β and Nur77 in a panel of HCC cell lines using Co-IP. The interactions of NDRG1 with GSK-3β, and of NDRG1 with Nur77 were only detected in Huh7 and HepG2 cells, but not in Hep3B cells which lack NDRG1 expression under normoxia , and Cyclin D1 (a key downstream target of β-catenin) Figure . NDRG1 sTo determine whether the hypoxia-induced nuclear accumulation of β-catenin is regulated by NDRG1, we transfected HepG2 and Hep3B cells with NDRG1-specific siRNA and cultured these cells under hypoxia. Subcellular fractionation followed by Western blotting showed that NDRG1 suppression decreased nuclear accumulation of β-catenin in both HepG2 and Hep3B cells under hypoxia, with accompanying decreases in GSK-3β 9ser and Cyclin D1 levels, and increased cytoplasmic accumulation of Nur77 Figure . ConsistSince both GSK-3β and Nur77 facilitate β-catenin degradation, and we observed that NDRG1 enhance β-catenin accumulation, we further hypothesized that NDRG1 interaction with GSK-3β and Nur77 may impede β-catenin degradation  in HCC cells. To test this hypothesis, we treated Hep3B cells  with 10 μM of the GSK-3β agonist LY294002, or the Nur77 agonist Cns-A for 48 hours, and then analyzed β-catenin expression and localization in HCC cells. LY294002 enhanced GSK-3β activity as observed by decreased levels of GSK-3β 9ser (the inactive form of GSK-3β), whereas Cns-A up-regulated Nur77 expression and also its nuclear expression in all three HCC cell lines. Even though both these events would be expected to promote β-catenin degradation, we observed reduced β-catenin only in Hep3B cells which have undetectable NDRG1 expression under normoxia Figure  and 4B. To further confirm this observation, we transfected HepG2 and Huh7 cells (with high endogenous NDRG1 levels) with NDRG1-specific siRNA, and examined the effects of NDRG1 protein suppression on GSK-3β or Nur77 agonist treatment under normoxia. As expected, NDRG1 suppression reduced β-catenin levels and its nuclear accumulation Figure  and 4D, To further understand the protective effect of NDRG1 against β-catenin degradation through its interactions with GSK-3β and Nur77, we examined the possibility that NDRG1 might disrupt GSK-3β-β-catenin and Nur77-β-catenin interactions through competitive binding. We treated Hep3B cells with either GSK-3β agonist LY294002, or Nur77 agonist Cns-A, and then used β-catenin antibody to pull down the complexes, which were detected using either GSK-3β or Nur77 respectively. We observed that each agonist alone enhanced the binding of GSK-3β to β-catenin Figure  and of Nin vivo, we inoculated Hep3B and HepG2 cells carrying inducible control or NDRG1 shRNA expression cassettes subcutaneously into nude mice. After 4 weeks, xenografts derived from NDRG1 shRNA cells were significantly smaller than those from control shRNA cells , Nur77 , and β-catenin  , and Hu-catenin . The tru-catenin , causing-catenin . Consistet al reported that Nur77 induced GSK-3β-independent degradation of β-catenin through direct interaction with β-catenin in the cytoplasm ytoplasm  and its ytoplasm , suppresr export . We repoin vitro, and to partially disrupt the activation of Wnt/β-catenin pathway in HCC xenografts [The expression of NDRG1 in HCC is induced by hypoxia, through the HIF-1α transcription factor. We previously demonstrated that NDRG1 expression is associated with vascular invasion in HCC patients, and that it regulates cell invasion, which are molecular events mediated by hypoxia , 27. We nografts . It was nografts . SorafenIn conclusion, we present new data that NDRG1 regulates the Wnt/β-catenin signaling pathway in HCC cells, through its direct interactions with GSK-3β and Nur77 to prevent β-catenin degradation. The disruption of protein-protein interactions, which are signaling hubs that link and transmit oncogenic signals across various molecular networks, offer a potentially effective means of halting oncogenic signaling (e. g. inhibition of p53 and MDM2 interaction by Nutlins) , 32. OurLuc(+) and Hep3B-Luc(+) were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum , and 1% penicillin-streptomycin. The cells were incubated in a humidified atmosphere containing 5% CO2 at 37°C. Where appropriate, cells were treated with the chemicals LY294002 (LY)  [The HCC cell lines HepG2, Hep3B, Huh7, PLC/PRF/5, HepG2-ers, MA) ; or Cns-ers, MA)  at 10 μMFull length NDRG1 protein was amplified from pcDNA 3.0 NDRG1 vector using the following primers: upstream (5′- TTT GGA TCC GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG ATG TCT CGG GAG ATG CAG GAT G -3′), and downstream (5′- TTT CTC GAG CTA GCA GGA GAC CTC CAT GGA CTT G -3′). The PCR reaction was performed in a GeneAmp PCR system 2700  under the following conditions: heat activation of the polymerase for 5 min at 95°C, followed by 30 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 2 min; with a final extension at 72°C for 10 min. The PCR-amplified product of the NDRG1 coding region is constructed into the BamHI and XhoI sites of vector pet28A . Recombinant V5-NDRG1 peptide was generated in E. coli  with IPTG induction, and native NDRG1 protein was purified using Ni-NTA Superflow Agarose  following the manufacturer's protocol. The concentration of V5-NDRG1 protein was measured by the BCA Protein Assay . Purified V5-NDRG1 protein was confirmed using Coomassie blue staining and Western blotting using V5-HRP secondary antibody .® protein microarray v5.0  according to the manufacturer's protocol. Briefly, ProtoArray slides were pre-blocked with blocking buffer, and then incubated for 90 min at 4°C with 1 μg V5-NDRG1 diluted in PBS buffer, with gentle shaking. Slides were then washed four times with 5 ml PBS. Alexa Fluor® 647 Anti-V5 Antibody  was then added and incubated for 90 min at 4°C with gentle shaking. Slides were then washed again four times with PBS, and dried. Dried slides were scanned using GenePix 4000B , and images analyzed using ProtoArray Prospector .The purified V5-NDRG1 protein was used to probe the Human ProtoArrayvia SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The membrane was incubated with the desired primary antibody, followed by incubation with appropriate HRP-conjugated secondary antibodies. The protein expression was detected by SuperSignal West Pico Chemiluminescent Substrate  according to the manufacturer's protocol. Primary antibodies used are: NDRG1, Nur77 ; p-Rb, GSK-3β, GSK-3β-9ser, Histone 3 ; β-catenin, p16, GAPDH . Secondary antibodies used are: goat anti-rabbit and goat anti-mouse .For Western blotting, cells were lysed with T-PER Tissue Protein Extraction Reagent . Protein lysates suspended in loading buffer were resolved Total protein (500 μg) in cell lysates were incubated with 2 μg anti-NDRG1 or anti-β-catenin antibody at 4°C for 2 hours; 20 μl A/G PLUS-Agarose beads  were then added into each sample and incubated at 4°C overnight. After incubation, the beads were separated from the lysis buffer and washed three times in cold PBS. Immunocomplexes were resuspended in 2x loading buffer, heat-denatured, and centrifuged; supernatants were collected and resolved by SDS-PAGE on a 10% polyacrylamide gel, and immunoblotting was performed with antibodies as indicated.Cells were seeded on the chamber slide and fixed with ice cold 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with 1% BSA for one hour. Cells were stained for indicated primary antibodies and secondary antibody, and then visualized using an Eclipse E600 image analysis system .Cells were plated at the desired density 24 hours prior to placement in a hypoxia chamber maintained at 0.5% oxygen for desired time periods depending on the experiment. Alternatively, hypoxia was induced chemically by incubating the plated cells in GasPak pouches for anaerobic culture  according to the manufacturer's instructions.Target specific siRNA (20 nM) against NDRG1  was transfected into cells in 6-well plates using Lipofectamine 2000  according to the manufacturer's instructions. The siRNA sequences are: NDRG1 siRNA: 5- GCU GAU CCA GUU UCC GGA Att-3.Clinical samples were collected from HCC patients who underwent hepatectomy at Stanford Hospital . This study was approved by the Institutional Review Board at Stanford University, and all patients signed informed consent forms prior to tissue collection. Immunohistochemical staining was performed following standard protocol as previously described . SectionLuc(+) and Hep3B-Luc(+) cells (1 × 106 each) stably transfected with control-shRNA or NDRG1-shRNA were injected into 4 weeks old nude mice  to induce subcutaneous tumor formation. Cells with control-shRNA were injected into the left shoulders and cells with NDRG1-shRNA were injected into the right shoulders (n = 8 for each group). Doxycycline (200 μg/ml)  was added daily into the drinking water of nude mice. Whole body luminescence intensity and tumor size were measured weekly. After 4 weeks, the mice were sacrificed and tumor tissues harvested for further investigations.HepG2-t-test and One-way ANOVA were used to calculate statistical significance. The correlation coefficient was calculated to measure the linear association among two sets of variables. Statistical significance is indicated by p < 0.05.Statistical analyses were performed using PRISM version 5.0 software . The Student's


By operating as both a subunit of the cadherin complex and a key component of Wnt signalling, β-catenin acts as the lynchpin between cell–cell contact and transcriptional regulation of proliferation, coordinating epithelial tissue homeostasis and regeneration. The integration of multiple growth-regulatory inputs with β-catenin signalling has been observed in cancer-derived cells, yet the existence of pathway crosstalk in normal cells is unknown. Using a highly regenerative normal human epithelial culture system that displays contact inhibition, we demonstrate that the receptor tyrosine kinase (RTK)-driven MAPK and Wnt–β-catenin signalling axes form a bidirectional positive-feedback loop to drive cellular proliferation. We show that β-catenin both drives and is regulated by proliferative signalling cues, and its downregulation coincides with the switch from proliferation to contact-inhibited quiescence. We reveal a novel contextual interrelationship whereby positive and negative feedback between three major signalling pathways – EGFR–ERK, PI3K–AKT and Wnt–β-catenin – enable autocrine-regulated tissue homeostasis as an emergent property of physical interactions between cells. Our work has direct implications for normal epithelial tissue homeostasis and provides insight as to how dysregulation of these pathways could drive excessive and sustained cellular growth in disease. Downstream of Wnt signalling, β-catenin that has translocated to the nucleus functions indirectly as a transcription factor by modulating the transcriptional activity of transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family to promote cell proliferation . β-catenIn the absence of Wnt ligand, β-catenin is phosphorylated by glycogen synthase kinase 3β (GSK3β), a component of the protein ‘destruction’ complex. This complex also contains the scaffold protein axin and the adenomatous polyposis coli (APC) protein. The destruction complex is responsible for the ubiquitylation and subsequent proteasomal degradation of β-catenin. Upon the binding of Wnt ligand to Frizzled (Fzd) receptors, an intracellular signalling cascade triggers the release of β-catenin from the destruction complex for subsequent trafficking to the nucleus or cell membrane .A further layer of complexity relates to the interaction of β-catenin with receptor tyrosine kinase (RTK) and the mitogen-activated protein kinase (MAPK) signalling pathways . Wnt ligin vitro. In low-Ca2+ (0.09 mM) serum-free medium, NHU cells adopt a highly proliferative phenotype driven by autocrine signalling, with amphiregulin recognised as the key EGFR-activating ligand (2+ (2 mM), wherein proliferation is promoted in juxtaposed cells that form stable intercellular junctions. This proliferation pathway is mediated by phospho-activation of AKT and is attenuated by PI3K antagonists or by functional inactivation of E-cadherin – either of which results in derepression of the EGFR–ERK pathway and activation of β-catenin–TCF signalling  cell culture system, in which tissue-isolated cells are maintained as multiple finite  cell lines g ligand . NHU celg ligand , althougg ligand . A secongnalling . Our aimsupplementary material Fig. S1.Gene arrays representing proliferating (24 hour) NHU cell cultures were analysed for the expression of Wnt pathway-related transcripts, and this analysis revealed that all the components necessary for a functional Wnt cascade were expressed. Seven Frizzled receptors  that have been implicated in transducing the canonical Wnt signal were present, as were the transcripts of four Wnt ligands . Transcripts for several downstream targets of Wnt signalling were also expressed by proliferative cultures, including Axin2 , survivin (BIRC5), Twist, SKP2 and cyclin D1. Archetypal extracellular Wnt antagonists, including soluble Fzd-related protein (sFrp) and Wif1, were not transcribed by proliferative cultures. A summary of the results from the expression arrays (and a comparison with results obtained from contact-inhibited quiescent cultures), along with an independent verification of the key findings by RT-PCR, is shown in In low-density culture, NHU cells homogeneously expressed nuclear-restricted active β-catenin, as shown using an antibody that recognised the Ser37 and Thr41 dephosphorylated form of β-catenin. This contrasted with growing cultures of the well-characterised Wnt-responsive SaOS-2 osteosarcoma cell line , which eThe highly specific GSK3β antagonist SB415286  was usedP<0.001) than that of a vehicle-control-treated sample (P>0.05) in TOPFLASH luciferase activity observed in NHU cells after SB415286 treatment . Western blotting results from parallel cultures corroborated the immunofluorescence microscopy findings, with active β-catenin expression peaking at 48 hours of culture  after treatment with the EGFR inhibitor PD153035 for 24, 48 and 72 hours in culture , implyinThe observation that an EGFR–ERK-driven β-catenin–TCF pathway dominated in proliferative NHU cells , combineThe blockade of EGFR in NHU cells by PD153035 reduced basal TCF promoter activity, as was reflected by low levels of luciferase reporter activity in the TOPFLASH assays. This allowed for the first time the detection of significant induction of TCF promoter activity and, upon Wnt activation with SB415286, there was an approximately threefold increase in TOPFLASH (but not FOPFLASH) reporter activity , signifyThe functional blockade of EGFR severely diminished NHU cell proliferation, but this could in part be overcome by pharmacological activation of Wnt signalling . Wnt stiTo investigate the role of β-catenin itself, we used a retroviral shRNA approach to generate NHU sub-lines carrying a stable β-catenin knockdown (NHU-β-cat-KD cells). Immunoblotting confirmed a substantial decrease in the expression of both active and total β-catenin in NHU-β-cat-KD cells in comparison with NHU-Con control-shRNA-expressing isogenic counterparts . ImmunofNHU-β-cat-KD cell cultures showed a significant reduction in growth compared with NHU-Con cells over a 6-day time-course , which w4 cells/cm2  results in the formation of stable E-cadherin-mediated cell–cell contacts . Notably, not only were strong cell contacts per se unable to sequester active β-catenin to the membrane, but they also did not alter the functional involvement of β-catenin in NHU cell proliferation as shown by Ca2+-switch experiments in control and NHU-β-cat-KD cells . Wnt3a has been previously shown to interact with several Fzd receptors, including Fzd2 and Fzd6, both expressed as transcripts by proliferating NHU cell cultures. Wnt5a has been shown to act as both an inhibitor and an activator of the canonical Wnt pathway, depending on receptor availability; if Fzd4 is present, Wnt5a can activate canonical signalling; however, if the tyrosine kinase ROR2 is present, Wnt5a inhibits the canonical Wnt cascade . AffymetWnt3a and Wnt5a ligands were obtained from engineered L cell derivatives that actively secrete high levels of soluble ligand into the culture medium . We firs2+ concentration of the DMEM (1.8 mM). In EGFR-blocked NHU cultures, incubation with either L-Wnt3a- or L-Wnt5a-conditioned medium resulted in strong nuclear active β-catenin localisation that was as intense as that observed in the presence of the GSK3β antagonist . Supplementation of L-cell derivative cultures with 80 µM palmitic acid restored the functionality of secreted Wnt ligand, as evidenced both by the clear induction of nuclear β-catenin expression in SaOS-2 cells  were unlikely to produce bioactive Wnt ligand. NHU cell cultures had been shown above to express Wnt3, Wnt5a, Wnt6 and Wnt7a transcripts, and to determine whether NHU cells had the potential to secrete functional Wnt ligand that could be used to drive canonical Wnt signalling in an autocrine/paracrine fashion, we prepared conditioned medium from NHU cells cultured in medium supplemented with either serum (NHU+FBS CM) or palmitic acid (NHU+PA CM). We initially tested these conditioned media for biological activity in SaOS-2 cells, where treatment with either NHU-derived conditioned medium resulted in nuclear translocation of active β-catenin, which was nearly as intense as that observed in cells treated with LiCl . By contThe effect of NHU+PA CM was tested on parallel homologous NHU cultures that were pre-treated with PD153035 inhibitor to block EGFR signalling; this was compared with vehicle and SB415286-treated control cultures. Treatment of EGFR-blocked NHU cells with NHU+PA CM did not result in population-wide nuclear active β-catenin , or any The capacity of Wnt and β-catenin to crosstalk with and modulate the function of the MAPK pathways and, conversely, the capacity of RTKs to trigger β-catenin signalling are characteristics that are generally associated with cancer-derived cell lines where, by definition, there is dysregulation of normal homeostatic growth regulatory pathways. Using an epithelial cell culture system that retains normal features by being both of finite lifespan and competent to undergo differentiation, we have shown here that bidirectional interactions between Wnt–β-catenin and RTK–MAPK pathways operate contextually within a single epithelial cell type to regulate proliferation.In conditions that inhibit the formation of stable adherens junctions, NHU cell cultures showed intense nuclear β-catenin staining associated with rapid population growth. We found a correlation between active β-catenin expression, ERK activity (phospho-42/44) and inactive (Ser9 phosphorylated) GSK3β, whereas blockade of EGFR or MAPKs reduced the expression and/or nuclear translocation of active β-catenin, ablated phospho-GSK3β and impaired TCF-mediated transcription. This crosstalk from MAPKs to β-catenin appears to be mediated through the phosphorylation of GSK3β and potentially by the ERK-mediated activation of the ribosomal proteins p70 S6  and p90 RSK , as described in a variety of tumour cells .supplementary material Fig. S1). Wif1 is frequently hypermethylated in urothelial carcinoma  signalling unmasked the potential for Wnt-pathway-mediated β-catenin activation, which, by triggering the activation of ERK, alleviated the ‘brake’ on proliferation exerted by EGFR blockade.The knockdown of β-catenin (in NHU-β-cat-KD cells) caused significant reduction in NHU cell proliferation, resulting in a reduced proportion of NHU-β-cat-KD cells in cell cycle (based on Ki67 expression). β-catenin knockdown caused a significant reduction in phospho-ERK by an unknown mechanism, although it has been speculated that Raf1 is involved in activating ERK downstream of β-catenin . As a reUnder adherens-junction-promoting conditions, active β-catenin is sequestered at intercellular junctions, resulting in the downregulation of β-catenin-regulated gene expression and increased cell–cell adhesion. Such sequestration of β-catenin is antagonised by a number of growth factor signalling mechanisms that either disrupt the adherens junction or downregulate E-cadherin expression and lead to a reduction in cell–cell contact coupled with an increase in TCF-mediated gene transcription. In this manner, β-catenin acts as a key coordinator of growth-factor-induced proliferation and cell–cell adhesion . When bo2+ growth medium, such as KSFMc, Ca2+-dependent cell–cell engagement is weak and β-catenin expression at the cell membrane is low. However, stimulating adherens junction engagement by increasing the concentration of extracellular Ca2+ did not reduce the levels of nuclear β-catenin or the activity of TCF. These results suggest that, once released from the destruction complex, β-catenin preferentially translocates to the nucleus. How this is regulated remains unclear, but evidence suggests that activated EGFR can phosphorylate β-catenin at Tyr654, negating its ability to interact with E-cadherin at the adherens junction  serum when harvesting conditioned medium, as serum contains albumin-bound palmitic acid that is required for Wnt post-translational palmitoylation . HoweverOur studies also provided some intriguing observations when we examined the ability of NHU cells to carry out autocrine/paracrine Wnt signalling by endogenously produced Wnt ligands. We demonstrated that EGFR-blocked NHU cells that had been incubated with conditioned medium from isogenic palmitic-acid-treated NHU cell cultures showed limited overall Wnt pathway activation, which was restricted to a small proportion of NHU cells that displayed high levels of active nuclear β-catenin after treatment. At present, the basis for this heterogeneity and its implications are unclear; however, the data imply that there might be a small subset of cells capable of driving self-renewal in response to autocrine/paracrine Wnt signalling. It is tempting to speculate that this might represent a subpopulation of cells with the ability to initiate their own programme of self-renewal, such as a stem cell population.in situ observations have been used to infer a basal progenitor , a ligand that is important during embryonic development. The relevance of these studies to human urothelium remains unclear, as there appear to be fundamental differences in the regulation of urothelial regeneration between human and rodent urothelium in vivo  would attenuate β-catenin signalling, thus switching off the signalling feedback loop and, subsequently, cell proliferation. Moreover, this efficient mechanism would represent a molecular target in carcinogenesis, as its dysregulation (constitutive activation) would provide a strong growth advantage during malignant transformation .Pharmacological inhibitors PD153035, U0126 and LY294002 were purchased from VWR (Merck). GSK3β inhibitors SB415286 and LiCl were from Sigma Aldrich. The antibodies used were against; active β-catenin dephosphorylated on Ser37 and Thr41 , total β-catenin , β-actin , E-cadherin , total ERK , phospho-42/44 MAPK , AKT , phospho-473 AKT  and phospho-9 GSK3β . The secondary antibodies were from Invitrogen. The secondary antibodies for immunofluorescence microscopy were Alexa-Fluor-488-conjugated goat anti-mouse-IgG and Alexa-Fluor-594-conjugated goat anti-rabbit-IgG, and those used for western blotting were Alexa-Fluor-680-conjugated goat anti-mouse-IgG, Alexa-Fluor-800-conjugated goat anti-rabbit-IgG and Alexa-Fluor-680-conjugated donkey anti-goat-IgG.Surgical specimens of normal ureteric urothelium were obtained with NHS Research Ethics Committee approval and with informed consent from patients with no histological evidence of urothelial dysplasia or malignancy. The preparation and maintenance of finite NHU cell lines was as detailed previously , and NHUThe β-catenin-knockdown shRNA-expressing sub-line (NHU-β-cat-KD) and its isogenic control-shRNA-expressing sub-line (NHU-Con) have been described previously . SaOS-2 Conditioned medium was collected from L-cell derivatives as described previously  and filtExpression array data analysed in the present study were selected from an experimental series analysed using Affymetrix™ GeneChip Human Genome U133 Plus 2.0 (HG-U133 Plus 2.0) arrays, as described previously , and thesupplementary material Table S1. RNA was isolated, cDNA synthesised and PCR performed using protocols and reaction profiles described elsewhere  or by semi-quantitative reverse transcription (RT)-PCR. Forward and reverse target gene primer sequences are provided in the lsewhere . GenomicImmunoblotting was performed as described elsewhere  and invo3 to 2×104 cells/well (depending on the experiment) on Teflon-coated 12-well glass slides. Cells were fixed either in a 1∶1 (v/v) mixture of methanol∶acetone or in 10% (v/v) formalin in PBS. Formalin-fixed slides were permeabilised in 0.1% (w/v) Triton X-100. Nuclei were visualised with 0.1 mg/ml Hoechst 33258, and proteins of interest were detected with the specific primary antibody followed by appropriate secondary conjugated to Alexa Fluor 488 or Alexa Fluor 594. Slides were examined under epi-fluorescence illumination on an Olympus BX60 microscope, and nuclear and cytoplasmic labelling were quantified using Photoshop (Adobe) as described previously  and were incubated for 24 hours at 37°C (day −1). Cells were treated with pharmacological inhibitors (single inhibitors or combinations thereof) in triplicate wells (day 0) and plates were assayed by MTT on days 0, 1, 3 and 6. A solvent balanced control was included at each time-point. Medium and inhibitors were replenished on day 3.Cell proliferation was indirectly determined by measurement of cell culture biomass using the MTT assay. Cells were seeded onto 96-well plates at a seeding density of 2×104 cells/well. The following day, 0.5 µg of TOPFLASH or FOPFLASH vector  was transfected into NHU cells using Fugene® HD reagent (Promega). Transfected cells were incubated for 16 hours, the medium was replaced with normal growth medium and cells were treated with Wnt ligand or GSK3β inhibitor for 24–72 hours. Lysates were prepared and Dual Luciferase Assays were performed as recommended by the manufacturer (Promega).NHU and SaOS-2 cells were seeded onto 24-well plates at a density of 4×10t-test – significance was assumed when P≤0.05.Statistics were performed using GraphPad InStat v3.05 (GraphPad). Parametric statistics [mean and s.d. (n−1)] were used for descriptive purposes, and tests of significance were by means of a two-tailed Student's


Drosophila. Here, we report the identification of Drosophila activated cdc42 kinase as a growth promoter and a novel Hippo signaling pathway regulator. We find that activated cdc42 kinase promotes tissue growth through modulating Yorkie activity. Furthermore, we demonstrate that activated cdc42 kinase interacts with Expanded and induces tyrosine phosphorylation of Expanded on multiple sites. We propose a model that activated cdc42 kinase negatively regulates Expanded by changing its phosphorylation status to promote tissue growth. Moreover, we show that ack genetically interacts with merlin and expanded. Thus, we identify Drosophila activated cdc42 kinase as a Hippo pathway regulator.Non-receptor tyrosine kinase activated cdc42 kinase was reported to participate in several types of cancers in mammals. It is also believed to have an anti-apoptotic function in  Activated cdc42 kinase (Ack) belongs to a conserved family of non-receptor tyrosine kinase [Drosophila, Cdc42 signaling that leads to dorsal closure of the epidermis during embryogenesis has been shown to regulate Ack protein level [Drosophila Ack during tumorigenesis remains elusive. Not until recently, a study reported that Ack has an anti-apoptotic function, and it genetically interacts with Yorkie (Yki) to regulate cell proliferation and apoptosis [e kinase . Severale kinase . During e kinase . Despitee kinase . In the in level . Moreovein level . Notwithpoptosis .The Hippo pathway controls organ size by orchestrating proliferation and apoptosis in diverse cells and tissues . Most, iDrosophila Ack as a growth promoter and a novel Hippo signaling pathway regulator. We show that Ack promotes tissue growth through enhancing Yki activity. Furthermore, we demonstrate that Ack interacts with Ex and negatively regulates Ex activity in a kinase-dependent manner by changing its phosphorylation status on multiple tyrosine residues. In addition, we determine that ack genetically interacts with merlin and ex. Taken together, our findings support that Drosophila Ack is a Hippo pathway regulator and promotes tissue growth via suppressing Ex-mediated Yki regulation.In this study, we report the identification of To identify novel regulators of the Hippo pathway, we performed mass spectrometry (MS) analysis using GST fused Yki tandem WW domains (refer to as GST-WW) as bait to identify its binding partners. As expected, some known Yki binding partners such as Wts, WWBP, Mop and Ack were found in the result . An inte3×Sd2-Luc), which was promoted by Ack expression ; and the expression level of Yki target genes was evaluated. ex is one of the Yki target genes and encodes an upstream Hippo pathway component that suppresses Yki’s function [Ex-LacZ (Ex-Z). Another well-known Yki target gene is diap1, whose transcriptional level can be monitored using a reporter gene diap1-GFP4.3 [hh-Gal4 enhanced Ex-Z signal and induced a significant upregulation of diap1-GFP signal in the P compartment  compartment of wing discs using function . Its tra1-GFP4.3 . Our respartment . Furtherpoptosis , 44. As  hh-Gal4 , indicatpression . In contpression . Togethein vitro and in vivo.Notably, cytoplasmic-nuclear translocation of endogenous Yki was affected when Ack (but not Ack-KD) was overexpressed . Yki wasin vivo  antibody was used to detect Ex phosphorylation status when Ack was coexpressed. We confirmed the specificity of the antibody by checking the autophosphorylation status of Ack (unpublished observations). As shown in To identify Ex tyrosine phosphorylation sites induced by Ack, we overexpressed Myc-tagged Ex with or without Ack in S2 cells. Ex-Myc protein was then immunoprecipitated and prepared for MS analysis. Five potential tyrosine phosphorylation sites of Ex were identified, including Y227, Y423, Y679, Y766 and Y1103 . To veriin vivo, we generated Ex and Ex-5YA transgenic flies using the phiC31 integration system to ensure that all of the transgenes were expressed from the same chromosomal location (attp at 25C6) so that position effects on gene expression were eliminated [MS1096-Gal4 on wing discs showed stronger activity compared with the wild-type Ex in regards to the reduction in wing size (To verify these findings iminated . Expressing size . Whereasing size . We beliattp flies containing a combination of Y679A and Y766A mutations (Ex-2YA). We found that Ex-2YA exhibited more severe growth defects than any of single mutants and in a level similar to Ex-5YA  exhibited stronger growth inhibition effect than those carrying wild-type Ex. We reasoned that, by abolishing Ack-induced Ex phosphorylation, Ex’s function on growth inhibition is enhanced. However, all of the Ex mutants bearing single mutation were partially suppressed by Ack overexpression in varying degrees . Notablyo Ex-5YA . Consisto Ex-5YA . These rutations . Of noteutations , suggestack and ex in vivo, we used ack mutant flies generated by excision of a P-element KG00869 [ack mutant flies are viable without obvious growth defect. In addition, expression of Hippo pathway target genes did not change in ack null imaginal discs. Such a striking discrepancy implies that there may be potential problems for the RNAi lines. The viable property of ack mutant flies made it a suitable tool for examining epistatic relationships with other components in double mutant analysis. In our analysis, ex mutant mosaic induced massive overgrowth not only in eyes but also at the base of antenna . Taken together with the adult phenotype, we concluded that Ack modulates Yki activity in eye discs. The anti-apoptotic function of Ack may be partially explained by increased Yki activity since increased Diap1 protein level may inhibit cell death [Schoenherr et al.  reportedll death , 55. TheDrosophila to human [Our results imply that Yki might not be a substrate for Ack tyrosine phosphorylation , lane 5.to human . So far,to human , while iIt was previously reported that ACK1 transduces extracellular signals to cytosolic and nuclear effectors to promote cell survival and growth, and several somatic ACK1 mutations discovered in tumors could increase the ability of tumor cells to proliferate and migrate . WhetherpUAST-V5 or pUAST-Flag vector. Ack and Ex mutants that contain point mutation were generated by PCR-based site-directed mutagenesis.The full-length Ack DNA fragment (3 222 bp) was amplified from a BDGP DGC clone (clone ID: GH10777) and was inserted into ack null allele (Ack10b) was a kind gift from Nicholas Harden, Simon Fraser University. kibra null allele (kibra4) was a kind gift from Hugo Stocker, Institute of Molecular Systems Biology. fj-lacZ was a kind gift from Kenneth D. Irvine, The State University of New Jersey. Other RNAi or null alleles used in this study were as following: UAS-AckRNAi (NIG 14992R1 and VDRC 39857), UAS-YkiRNAi (VDRC 40497), exe1 [mer4 [MS1096-Gal4, GMR-Gal4, hh-Gal4, hpoBF33, Ex-lacZ, diap1-GFP4.3 and bantam sensor mic32-GFP were described previously [Ex transgenic flies were generated by site-specific integration into the fly genome at 25C6 attp locus. 7), exe1  and mer4e1 [mer4 . MS1096-NAi VDRC 497, exe1eviously . Flies aDrosophila S2 cells were cultured for 3 days (10 dishes) and then collected and lysed in immunoprecipitation (IP) buffer  with protease inhibitor cocktail . Cell lysates were pre-cleared using Glutathione sepharose  and then incubated with 200 μg immobilized GST-WW protein, which was expressed in BL21 E. coli and was purified with Glutathione Sepharose. GST protein was used as a negative control. Proteins in pull-down samples were separated by SDS-PAGE, and were stained using Colloidal Blue staining . Compared with control sample, specific bands were selected for liquid chromatography coupled with tandem MS analysis in Protein Centre, SIBCB.For pull-down experiment, Drosophila Schneider’s Medium (Invitrogen) with 10% fetal bovine serum, 100 U ml−1 of penicillin and 100 mg ml−1 of Streptomycin. Plasmid transfection was carried out using LipofectAMINE (Invitrogen) according to the manufacturer’s instructions. The constructs transfected in S2 cells were all pUAST expression vectors unless otherwise indicated. A construct of ubiquitin-Gal4 was cotransfected with pUAST expression vectors for all transfection experiments.S2 cells were cultured in m Tris-HCl, pH 8.0, 100 mm NaCl, 1% NP-40, 10% Glycerol, 1.5 mm EDTA, 10 mm NaF, 1 mm Na3VO4) with protease inhibitor cocktail (Sigma). Cell lysates were then divided into two parts, and 2 μg antibody was used to do co-IP in each part. Proteins in IP samples were then washed with IP buffer and subjected for western blot analysis.For co-IP experiment, S2 cells expressing the indicated constructs were collected and lysed in IP buffer . Cells were collected 48 h after transfection and were lysed in IP buffer. Ex-Myc protein was immunoprecipitated by 50 μg immobilized mouse anti-Myc antibody (Sigma) and subjected to MS analysis at the Protein Center, SIBCB. The candidate sites were identified by the increased phosphorylation abundance in the Ex-Myc and V5-Ack transfected cells versus Ex-Myc transfected cells.t-test. The results were considered as statistically significant if P<0.05.All of the data in this study were expressed as the mean±s.d. and were analyzed using the Student’s Fluorescent microscopy was performed on a Leica LAS SP5 confocal microscope; confocal images were obtained using the Leica AF Lite system . Images were processed in Photoshop CS.


After activation by Wnt/β-Catenin ligands, a multi-protein complex assembles at the clustering membrane-bound receptors and intracellular signal transducers into the so-called Lrp6-signalosome. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt–Fz–Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The μ2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2μ2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2μ2-mediated endocytosis is important to maintain Wnt/β-catenin signaling in vertebrates. Wnt/β-Catenin signaling is an essential, evolutionarily conserved pathway in embryonic development, stem cell biology and human disease, including cancer . In the Xenopus gastrulation . This Clathrin adaptor is a heterotetramer with the four subunits α1, β1, μ2 and σ1 . It linkrulation . Since Drulation .in vivo. During endocytosis, Ap2μ2 protects Dvl2 from degradation to maintain signaling. Thus Ap2μ2 mediated endocytosis of the Lrp6-signalosome is an important step for Wnt/β-Catenin signaling maintenance in vertebrates.We find that endocytosis has an essential function during the process of Wnt/β-Catenin signal transduction in vertebrates. Overexpression of the Ap2μ2 subunit enhances the expression of direct Wnt/β-Catenin target genes and knockdown of the subunit decreases their expression. By using live confocal imaging analysis of intact zebrafish embryos, we show that Wnt8-induced Lrp6-signalosome formation requires the interaction of Ap2μ2 and Dvl2 at the plasma membrane. Wnt8, subsequently, becomes internalized in a non-cell autonomous fashion together with the receptors and the intracellular signal transducers. Along the endocytic route, however, their paths are quickly separated. We detect colocalization of the ligand-receptor complex by using endocytic Rab protein markers, such as Rab5 and Rab7, whereas the transducer complex, composed of Dvl2, Axin1, GSK3β and β-Catenin, forms independent cytoplasmic aggregates. Hence, we conclude that the transducer complex is dissociated from the vesicular membrane early after internalization. In summary, we show that interaction between Dvl2 and Ap2μ2 is required and sufficient for the formation and endocytosis of the signalosome including the dissociation of the transducer complex from the ligand-receptor complex supplementary material Figs S1, S2) In Ap2μ2a/μ2b double morphant embryos, we found that expression of bona-fide Wnt/β-Catenin target genes such as axin2 and lef1 was reduced . We confirmed Wnt signaling activity of Ap2μ2 by real-time quantitative PCR (RT-qPCR) for induction of axin2 and lef1 expression  for the analysis of canonical Wnt signaling because it is already expressed at 30% epiboly and, therefore, the first-acting Wnt/β-Catenin ligand during zebrafish development . We foun embryos . Hence, So far, the Lrp6-signalosome has been proposed to be necessary for Lrp6 phosphorylation and signaling in cell culture . Howeversupplementary material Fig. S3A-C) and its kinase CK1γ , as well as members of the transducer complex such as GSK3β , β-Catenin , Axin1  and Dvl2 , suggesting the formation of an active signalosome at the cytoplasmic membrane in zebrafish .We found that transiently expressed bio-active fluorescence-tagged zebrafish Wnt8 formed stable clusters at the cytoplasmic membrane of gastrula-stage embryos , therebysupplementary material Movie 1), which is comparable to the Wnt induced signalosomes described earlier . However, we found that only fragments of the signalosome were internalized, whereas the main part remained attached to the membrane. This suggests a flow-type process in which parts of the signalosome are endocytosed continuously and, in parallel, the signalosome becomes recharged by recruitment of new intracellular Dvl2. Noticeable, in all images analyzed, we barely detected intracellular colocalization of Dvl2 aggregates with Wnt8 or Lrp6 after the fast endocytosis of both proteins . Similarly, the co-receptor Lrp6 was enriched in the same cytoplasmic structures , suggesting the paracrine induction of signalosomes by Wnt8. Quantification of internalization events of Wnt8 together with Dvl2 revealed one endocytic event per receiving cell every 33 minutes . After treatment with bafilomycin A1, we observed overlapping intracellular accumulation of Wnt8 and Lrp6 compared to control embryos , whereas Dvl2 punctae rather appeared juxtaposed to Wnt8 aggregates  and strong overlap with Rab7  and another in the tyrosine-based YxxΦ sorting motive (AHEA) at the C-terminus, because both domains were described to be essential binding sites for Ap2μ2 in cell culture . We founxpressed . This suxpressed . Notablyxpressed . Next wexpressed , similarin vivo. The scaffold protein Axin1 was described to bind directly to the DIX domain of Dvl2 . We were also interested whether Ap2μ2 is able to compensate for the inhibited endocytosis of Axin1 after co-expression with the Dvl2K340M/AHEA double-mutant . However, Dvl2 recruited GSK3β into punctae upon overexpression . Expression of Dvl2 did not lead to obvious colocalization with β-Catenin  were maintained at 28°C on a 14 h light/10 h dark cycle  and the Karlsruhe Institute of Technology (KIT). Breeding zebrafish (rk cycle . To previn-vitro transcribed with the mMessage Machine Kit (Ambion).Embryos were harvested and enzymatically dechorionated with Pronase (Sigma-Aldrich) at one-cell stage before injection. All indicated constructs were injected at one-cell stage or, for clone formation, into one of 8–16 cells as capped mRNA, zAp2μ2a/b morpholino oligomers  spanning the ATG were obtained from GeneTools LTD.All expression constructs were subcloned in pCS2+ . zDvl2 ; # 17086Ap2μ2a was amplified from 24 hpf zebrafish embryo cDNA library with primer A (5′-CCATGGAATGTGAGGTTTGA-3′) and B (5′-GAGTATGACCGATGGGGAAA-3′) using Pfu polymerase (Promega) and the resulting product was introduced into pCS2+ for Ap2μ2a or into pGlyserteC-TFP [kind gift from Uwe Strähle ] for Ap2HindIII and KpnI, respectively; restriction sites are shown as lowercase letters) by amplification with primer A (5′-GGCaagcttGCACCATGGCGGAGACCAAGATAATTTATC-3′) and B (5′-CGGggtaccTTACATCACATCCACAAAAAACTCACTGg-3) and the resulting product introduced into pmCherry C-1 (Clontech). Mutagenesis to introduce the K340M mutation was performed in two steps. First a left flanking fragment was generated using primer A and primer C (5′-CATTGGGAATGGTGATCATCAGCCACATTCGATCT-3′). The right flanking fragment was generated with primer B and D (5′-AGATCGAATGTGGCTGATGATCACCATTCCCAATG-3′). The resulting products were gel-purified and added at equal molar concentration to a second reaction containing primers A and B. The product of this fusion PCR was purified, introduced into pZero, and confirmed by sequencing to yield pZero-zDVL K340M. The Y55A and L554A mutations were introduced simultaneously in a similar two-step manner, using primers E (5′-GTAGCTGTAGTTAGAGGCCTCATGGGCTGGCGGAGGCTGGGTG-3′) and F (5′-CACCCAGCCTCCGCCAGCCCATGAGGCCTCTAACTACAGCTAC-3′) to produce pZero-zDVL AHEA.For zDvl2-Cherry and zDvl2 mutants, all PCR reactions were performed per manufacturer directions using Phusion High Fidelity polymerase (New England Biolabs). Restriction sites were introduced to the N- and C-termini of zDvl2 (plasmid from Thermo Scientific)  in 1% DMSO for 1 hour before scanning or with 30 µM Vacuolin-1 in 0.3% DMSO for 1.5 hours before scanning .in situ hybridization was performed as described  and RNA was prepared using Direct-zol RNA Mini Prep Kit from Zymo Research. cDNA was prepared using MMLV reverse transcriptase from Promega and analysed in a real-time PCR system from LifeTechnologies (ABI StepOnePlus). Primers with the following sequences were used: β-Actin ; Axin2  and Lef1 .For luciferase reporter assay embryos were injected with 1.2 ng SuperTopFlash as in  and 1.2 in situ hybridization and phenotype analysis, embryos were imbedded in 70% Glycerol/PBS (v/v). Images were taken on an Olympus SZX16 microscope equipped with a DP71 digital camera by using Cell A imaging software. For confocal analysis, live embryos were embedded in 0.7% low melting Agarose (Sigma-Aldrich) dissolved in 1×Ringer's solution. Images were obtained by using a Leica TCS SP5 X confocal laser-scanning microscope with 40×or 63×dip-in objectives. Image processing was performed by using an Imaris 6.3.1 software (Bitplane AG).For


To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively  or negatively  control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/ Embryonic stem (ES) cells are pluripotent and self-renewing cells derived from the inner cell mass of preimplantation blastocysts. These cells can differentiate into the following three germ layers: the ectoderm, mesoderm, and endoderm. Due to these characteristics, ES cells are considered useful tools in both research and regenerative medicine. Therefore, it is important to understand how stem cells maintain their self-renewal capacity and differentiate into each specific lineage –3. Howev in vitro. Generally, for differentiation into neuronal cells, ES cells are aggregated to form embryoid bodies (EBs) in suspension culture for 4 days and treated with retinoic acid (RA) [ES cells can be differentiated into neuronal cell lineagecid (RA) , 6. Usincid (RA) , 6.β-catenin signaling is conserved in worms, flies, fish, frogs, mice, and humans [β-catenin forms a “destruction complex” along with the scaffold protein Axin, the tumor suppressor gene product APC, and GSK-3β that is phosphorylated by casein kinases (CK1) and glycogen synthase kinase 3β (GSK-3β). Phosphorylation of β-catenin triggers its ubiquitination by β-TrCP, followed by proteasomal degradation [β-catenin degradation, leading to the accumulation of β-catenin in the cytoplasm. Stabilized β-catenin then enters the nucleus where it interacts with TCF/LEF [ C-Myc, Cyclin D1, Brachury, and Twin.Wnts are secreted signaling proteins that regulate a variety of developmental processes, including cell growth, motility, and differentiation . Wnt/β-cd humans . In the radation , 10. How TCF/LEF –13 and aβ-catenin signaling has been shown to play important roles in ES cell self-renewal and differentiation [β-catenin signaling prevents differentiation of human and mouse ES cells. For example, activation of Wnt signaling by GSK3β inhibitor maintains self-renewal of human and mouse ES cells, and Wnt/β-catenin signaling upregulates Stat3 activity and prevents differentiation of mouse ES cells [β-catenin signaling by Wnt3a recombinants induces cell differentiation as well as proliferation [β-catenin signaling is also involved in regulating the neuronal differentiation of ES cells [β-catenin signaling and its components in the self-renewal and neuronal differentiation of ES cells remain controversial.Wnt/ntiation –17. ActiES cells , 19. FurES cells . Howeverferation . Wnt/β-cES cells . On the ES cells . Therefoβ-catenin signaling components during neuronal differentiation. The data using reporter ES cell lines representing endogenous Wnt/β-catenin signaling activity showed that Wnt/β-catenin signaling increased during neuronal differentiation. Interestingly, Wnt/β-catenin signaling increased until formation of neuronal precursor cells; however, it was reduced during later periods of neuronal differentiation. Differential regulation of neuronal differentiation by the treatment of activator or inhibitor of Wnt/β-catenin signaling suggested that Wnt signaling plays dual roles during neuronal differentiation.In this study, we examined the endogenous level of Wnt/ Sox1 gene, a reporter for differentiation into neural precursor cells) were cultured in mouse ES (mES) cell medium (DMEM (Gibco) supplemented with 15% FBS, 2 mM GlutaMAX (Gibco), MEM nonessential amino acids, β-mercaptoethanol (Gibco), tylosin, and 1% Pen/Strep (Gibco)) supplemented with LIF  on 0.2% gelatin-coated dishes. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.E14 and 46C ES cells (ES cell line in which EGFP is substituted into the ORF of theβ-mercaptoethanol (Gibco), and 1% Pen/Strep (Gibco)) and then plated on a 0.2% gelatin-coated tissue culture dish  containing DMSO (SIGMA), 0.75 μM MeBIO , 0.75 μM BIO  [μM IWR-1-endo  [To induce neuronal differentiation, undifferentiated 46C ES cells were trypsinized and neutralized with serum-containing medium. ES cells were dissociated in N2B27 medium (DMEM/F12 (Gibco), Neurobasal medium (Gibco), N2 supplement (Invitrogen), B27 supplement (Invitrogen), 1 mM GlutaMAX (Gibco), 0.1 M biochem) , and 0.7 Center) . N2B27 mTo analyze 46C ES cell-derived Sox1-GFP-positive neural precursors, 46C cells were differentiated for 6 days in N2B27 medium with MeBIO or BIO and DMSO or IWR. The cells were then trypsinized and neutralized with serum containing medium. After centrifugation, cells were washed two times with PBS and resuspended in PBS. Resuspended cells were analyzed using a FACScan. For data analysis, FACSDiva software was used.μg/mL of G418 (Gibco).Oct4 promoter-EGFP ES cells were stably transfected with TOP-mCherry and Axin2-promoter-mCherry using Amaxa nucleofector according to the manufacturer's instructions. Stably transfected clones were selected in media containing 800 μM BIO . Luciferase activity was measured by Dual-Luciferase reporter assay (Promega).To measure Wnt signaling activity, pTOP-Flash plasmid and pRL-TK plasmid were transfected into mES cells. Transfected ES cells were cultured in the presence or absence of LIF for 48 h. To measure Sox1 promoter activity under nondifferentiation and neuronal differentiation conditions, pGL3-mSox1 promoter plasmid and pRL-TK plasmid were transfected into 46C cells, which were cultured in mES medium or N2B27 medium with or without 0.75 μg) in a total reaction volume of 20 μL using ImProm-II Reverse Transcriptase (Promega) with random primers. cDNA was amplified under the following conditions: 94°C for 2 min, followed by 20, 25, or 30 cycles of 94°C for 45 s, 58°C for 45 s, and 72°C for 50 s.Examination of gene expression during neuronal differentiation was carried out by RT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was synthesized from total RNA , cells were incubated with Alexa Fluor 488 (Invitrogen) and DAPI (KPL Inc) for 1 h in the dark to stain the nucleus, followed by washing again five times with PBST. Wells were mounted in a drop of Mount Medium (KPL Inc). Signal was visualized by fluorescence using a microscope .Neuronally differentiated 46C ES cells for 15 days were fixed with 4% paraformaldehyde at room temperature for 20 min and then permeabilized for 20 min with permeabilization solution. Cells were incubated with anti-MapII (Chemicon) primary antibody for 1 h. After washing 10 times with PBST  or antiβ-actin (Santa Cruz Biotechnology) antibody. Immunocomplexes were visualized using an enhanced Chemiluminescence kit (Elpis Biotech).Mouse ES cells were seeded in 6-well plates and differentiated in ES medium or N2B27 medium without LIF. mES cells were washed with phosphate-buffered saline (PBS) and then lysed in RIPA buffer  on ice for 30 min. Lysates were cleared by centrifugation at 12,000 rpm for 10 min, after which supernatants were collected. Bradford (Bio-Rad) assay was used to determine the protein concentration. Equal amounts of protein were boiled and separated by SDS-polyacrylamide electrophoresis gels and transferred to a PVDF membrane . Blots were then incubated with antiactive-2) were swollen on ice for 10 min and then passed through a 26.5 gauge needle six times. After centrifugation at top speed for 5 min at 4°C, pellets were incubated with nuclei lysis buffer  for 10 min on ice with occasional vortexing. Chromatin was sheared to an average length size of 0.2~1 kb by sonication on ice. Supernatant was collected by centrifugation , and the supernatant concentration was determined by a spectrophotometer. The appropriate volume of chromatin was diluted 1 : 10 in ChIP dilution buffer , after which preclearing was performed at 4°C for 2 h with 10 μL of protein A/G plus-agarose beads (Santa Cruz Biotechnology). For immunoprecipitation, goat, rabbit-IgG (Bethyl), antiβ-catenin (BD biosciences), anti-TCF3 (Santa Cruz), and anti-TCF1  antibodies were administered at 4°C overnight. Immunoprecipitated chromatins were eluted, after which reverse cross-linking was carried out by addition of 0.3 M NaCl at 65°C overnight. Following phenol-chloroform extraction and ethanol precipitation, DNA was dissolved in 50 μL of TE buffer . Sox1-1 primers (Forward: 5′-AGTTCAGCCCTGAGTGAC-3′ and Reverse: 5′-TGGGTGCCTAGCGGAGAG-3′), Sox1-2 primers (Forward: 5′-TGGTCTGATCCCAAGTAG-3′ and Reverse: 5′-TTTCTGAAGCGATTCTCC-3′), and Axin primers (Forward: 5′-TAACGCGGGAGCTGAGTGTG-3′ and Reverse: 5′-AAATCCATCGCGAACGGCTG-3′) were used for PCR.Cells were cross-linked with 1% formaldehyde (Sigma) at room temperature for 10 min and then incubated with 0.125 M glycine for 5 min with gentle shaking. Cells were then washed twice with PBS before harvesting. Cells resuspended with hypotonic buffer  with the monolayer neural differentiation method. Upon being cultured in N2B27 medium, 46C ES cells started to express EGFP from day 4, and outgrowth of neurites was detected from day 8 after replating  and increased expression of an ectoderm marker (Sox1) during neuronal differentiation. Expression levels of many Wnts, Wnt receptors, Wnt coreceptors, and Wnts components also changed during differentiation  cells -driven reporter construct that contain responsive TCF binding elements [β inhibitor, enhanced expression of mCherry, indicating that this cell line reflects Wnt/β-catenin signaling. After stable cells were cultured in N2B27 medium for neural differentiation, expression of mCherry was significantly increased in neural precursor cells, whereas it was decreased in fully differentiated neurons  for indicated duration as described in β  [β-catenin signaling on days 4 to 6 is essential for the proper formation of neural precursor cells.As GSK3bilizer) , 29. Sim Sox1 mRNA expression during neural differentiation  analysis using antibodies specific for β-catenin, TCF1, and TCF3. Notably, β-catenin and TCF1 did not access to Sox1-1 and Sox1-2 region under nondifferentiation and early differentiation conditions, whereas they associated with all three potential binding sites from day 4 during monolayer differentiation. In addition, we found weak binding of β-catenin and TCF1 to Sox1-3 region in self-renewal stem cells. This weak β-catenin-TCF1 binding on Sox1-3 at day 0 may have a rheostatic role in transcriptionally controlling expression of Sox1 gene during differentiation. Sox1-3 region is a distal control element located around 3 kb from the transcription initiation site. Therefore, we expect that weak interaction of β-catenin-TCF1 keeps a ready state to easily activate genes. On the other hand, TCF3 occupancy, a known repressor protein for Wnt signaling, was not changed during differentiation (β-catenin/TCF1 complex activates Sox1 promoter through direct binding to the conserved TCF binding sites during the late stage of neural precursor differentiation.We found that BIO enhancesntiation . Therefoox1 gene . In ordentiation . These dβ-catenin signaling activity in undifferentiated ES cells seems to be low compared to differentiated stage (β-catenin signaling by the treatment of BIO or Wnt3a enhances stemness of ES cells [β-catenin signaling is necessary (β-catenin signaling than endogenous level in undifferentiated state is sufficient to enhance stemness, but other unknown changes during differentiation along with high level of Wnt/β-catenin signaling may be required for proper neuronal differentiation of ES cells.Here we showed that the expression of various Wnt signaling components is dynamically changed during neuronal differentiation , but theed stage . AlthougES cells , 19, 30,ecessary  but not β-catenin-TCF complex could regulate the expression of Sox1 in the middle of neuronal differentiation periods (β-catenin signaling induced Sox1 until generation of neural precursors and then increased level of Sox1 suppresses Wnt/β-catenin signaling during late neuronal differentiation period.Tsao et al.  showed t periods . Althougβ-catenin signaling until late neuronal differentiation stage blocked neuronal differentiation, which might be due to enhancing self-renewal of ES cells and inhibitory effect of Wnt/β-catenin signaling on late neuronal differentiation period, respectively, as shown before [β-catenin signaling activator along with small molecules regulating other signaling pathways will further enhance neuronal differentiation.We identified that treatment of BIO at specific time points during differentiation can enhance neuronal differentiation . Treatmen before , 19, 23.


Dishevelled (DVL) proteins, three of which have been identified in humans, are highly conserved components of canonical and noncanonical Wnt signaling pathways. These multifunctional proteins, originally discovered in the fruit fly, through their different domains mediate complex signal transduction: DIX  and PDZ  domains serve for canonical beta-catenin signaling, while PDZ and DEP  domains serve for non-canonical signaling. In canonical or beta-catenin signaling, DVL forms large molecular supercomplexes at the plasma membrane consisting of Wnt-Fz-LRP5/6-DVL-AXIN. This promotes the disassembly of the beta-catenin destruction machinery, beta-catenin accumulation, and consequent activation of Wnt signaling. Therefore, DVLs are considered to be key regulators that rescue cytoplasmic beta-catenin from degradation. The potential medical importance of DVLs is in both human degenerative disease and cancer. The overexpression of DVL has been shown to potentiate the activation of Wnt signaling and it is now apparent that up-regulation of DVLs is involved in several types of cancer. The members of the dishevelled (dsh/DVL) protein family are considered to be critical components of Wnt signaling, which are transducing signal into three different cellular routes , and Wnt-Car routes -5. The mr routes ,7, thereAmong three Wnt signaling cascades, the canonical Wnt signaling pathway is one of the basic mechanisms of the cell signaling, critical for embryonic development and adult tissue homeostasis, and is widely conserved in the animal kingdom. It is activated by binding of different Wnt ligands (19 were identified in humans) to specific receptors . In the The pathway is inactive when the levels of beta-catenin are kept low. This is achieved by beta-catenin’s degradation in a multiprotein destructive complex consisting of AXIN, APC (adenomatous polyposis coli), CK1 (casein kinase 1), and GSK3β (glycogen synthase kinase 3 beta). This results in beta-catenin phosphorylation, ubiquitination, and finally its degradation in the proteasome . The comDVL is considered to be a key regulator that rescues cytoplasmic beta-catenin from degradation. Binding of Wnt signaling molecule to membrane receptors activates DVL ,4. The rDrosophila melanogaster. In Drosophila, a single dsh gene is expressed and it is required for proper development. In contrast, three dsh homologue genes  have been identified in humans showing a high degree of similarity. DVL1 gene is located at 1p36 locus, and its protein is 695 amino acids long; DVL2 gene is located at 17p13.1, and its protein is 736 amino acids long; and DVL3 gene is located at 3q27, and its protein is 716 amino acids long (Dishevelled (dsh) is a multifunctional phosphoprotein originally discovered in the fruit fly ids long . The expids long . DVL2 isids long . Two isosrc homology 3) binding domain, which is situated downstream of PDZ (All dsh/DVL proteins (ranging from nematodes to humans) possess three conserved domains: an aminoterminal DIX , a central PDZ , and a carboxyl-terminal DEP  domain . In addim of PDZ . The prom of PDZ ,5. Dillmm of PDZ .The N-terminal DIX domain extends in humans for 85 amino acids for DVL1, 83 for DVL2, and 82 for DVL3, and is also present in the AXIN protein, which seems to be a scaffolding factor for Wnt signaling ,16. DIX The central PDZ domain is 73 amino acids long in all three human homologs and it provides a docking site for protein kinases, phosphatases, and adaptor proteins. The proteins that bind to the PDZ domain are best known for their roles in submembranous receptor assembly, where they integrate signaling molecules into larger complexes with subsequent signal transduction . The dirDEP domain located between PDZ domain and C-terminal region of DVL protein consists of 75 amino acids in all three human homologs. The same polypeptide motif can also be found in signaling factors such as the regulator of G-protein signaling (RGS) protein family, which harbors conserved, catalytic RGS domains as well as DEP domains in their N termini . It has vice versa  is also involved in the regulation of DVL activity. It can have a positive or negative influence, depending on which regulatory subunit realizes the binding to DVL ,7,8. EveDrosophila and Caenorhabditis elegans; biochemistry in cell culture; and ectopic gene expression in Xenopus embryos. Wnt signaling is essential for mammalian embryogenesis . The occurrence of EMT during tumor progression resembles the developmental scenario and sheds light on important mechanisms governing metastasis, where noninvasive tumor cells acquire motility and ultimately disseminate to places distant from the primary site. Wnt signaling pathway has a particularly tight link with EMT. Moreover, the stabilization and nuclear accumulation of beta-catenin can induce EMT ,60 by acDsh allele in Drosophila mutants, dishevelled genes and proteins have been assigned the central role in the mediation of Wnt signaling. The Wnt signal utilizes DIX and PDZ or PDZ and DEP domains of DVL proteins to channel signal into canonical or non-canonical downstream pathways.Ever since the first discovery of the Recent data suggest that in the canonical pathway DVL is responsible for the disassembly of the beta-catenin destruction complex and recruitment of AXIN to the membrane, where DVL forms large molecular supercomplexes. DVL is considered to be a key regulator that rescues cytoplasmic beta-catenin from degradation.APC, axin, and beta-catenin, which encode components of the beta-catenin destruction complex are very common in a variety of tumors.DVL activity is dynamically regulated by phosphorylation, ubiquitination, and degradation and it seems to be dependable on the cellular context as well. Constitutive activation of the Wnt pathway can lead to cancer. The mutations attributed to Therefore, molecular components of Wnt pathway (like beta-catenin for instance) are relevant biomarkers helpful in better diagnosis and treatment. DVLs are overexpressed in various tumor types, including lung cancer, prostate cancer, breast cancer, cervical squamous cell carcinoma, and gliomas. However, the importance of individual DVLs in tumor prognosis still needs elucidation. The approaches to decrease DVL expression, as well as agents blocking selected specific DVL interactions, may be of particular interest as potential targets for therapeutic interventions.


P/P > 38.41% could determine anti-islanding condition within 0.04 s; ΔP/P < −24.39% could determine anti-islanding condition within 0.04 s; −24.39% ≤ ΔP/P ≤ 38.41% could determine anti-islanding condition within 0.08 s. This method perturbed the system, only in the case of −24.39% ≤ ΔP/P ≤ 38.41% at which the control system of inverter injected a signal of undervoltage shift as necessary to check if the occurrence condition was an islanding condition or not.The mainly used local islanding detection methods may be classified as active and passive methods. Passive methods do not perturb the system but they have larger nondetection zones, whereas active methods have smaller nondetection zones but they perturb the system. In this paper, a new hybrid method is proposed to solve this problem. An over/undervoltage (passive method) has been used to initiate an undervoltage shift (active method), which changes the undervoltage shift of inverter, when the passive method cannot have a clear discrimination between islanding and other events in the system. Simulation results on MATLAB/SIMULINK show that over/undervoltage and undervoltage shifts of hybrid islanding detection method are very effective because they can determine anti-islanding condition very fast. Δ FeatureIslanding is a condition in which a portion of the utility system that contains both load and generation is isolated from the remainder of the utility system. Phenomena of islanding condition may occur for several reasons such a result of a fault that is detected by the utility but is not detected by the DG, a result of an accidental opening of the normal utility supply by an equipment failure, a result of human error or malicious mischief, an act of nature, and so forth. Cause of islanding condition should be avoided because the utility cannot control voltage and frequency in the island at which there is the possibility of damage to equipment because of voltage or frequency excursions outside of the acceptable ranges on which the utility has no control and so forth , 4.Islanding detection techniques can be divided into local and remote techniques. The local techniques can further be divided into passive, active, and hybrid techniques. Remote islanding detection techniques are follows these detection techniques are based on some kind of communication between the grid and the DG. They are more reliable than the local techniques, but they are more expensive to implement. Local islanding detection techniques are based on the measurement of some parameters  on the distributed generator side. They are classified as passive, based exclusively on the monitoring of these parameters, and active techniques, which intentionally introduce disturbances at the output of the inverter and observe whether the parameters outlined above are affected. Hybrid methods employ both the active and passive detection techniques .Local islanding detection techniques are interested in research and develop because they are suitable for small DGs , as remP/P after islanding condition occurs; the relationship between load and NDZ of OUV of inverter-based DG shows the change of load effect to enter or leave the NDZ; the relationship between voltage and NDZ of OUV of inverter-based DG shows phenomenon of overvoltage or undervoltage in the inverter; proposed methodology shows over/undervoltage and undervoltage shift islanding detection algorithm, demonstrating the methodology on MATLAB/SIMULINK which proves the new methodology by simulation and conclusion. The new hybrid method was used, an over/undervoltage has been used to initiate an undervoltage shift, when the over/undervoltage method cannot have a clear discrimination between islanding and other events in the system. Simulation results show that the new hybrid method is very effective because it can determine anti-islanding condition very fast.Presentation of a new hybrid islanding detection technique includes nondetection zone of over/undervoltage of inverter-based DG showing finding nondetection zone and showing the relationship between over/undervoltage and ΔIn this paper, over/undervoltage (OUV) method was used to initiate the islanding detection because it is highly effective in group of passive islanding detection methods. However, the OUV method has a weakness because it has a wide nondetection zone. This section analyzed the NDZ of the OUV technique. “Nondetection zone” (NDZ) can be defined as the range in terms of the difference between the power supplied by the DG inverter and that consumed by the load, in which an islanding detection scheme under test fails to detect this condition .The over/undervoltage technique allows detection of the islanding phenomenon through the measure of voltage at the point of common coupling (PCC) and subsequent comparison with the limits set proper operation. If the measured values are outside the established range, the inverter is stopped or disconnected. The OUV technique received the nondetection zone by analysis. To facilitate this analysis, 2 (breaker/recloser). A local load is also connected at the PCC.It is usually assumed that the local load can be modeled as a parallel RLC circuit because, for most islanding detection methods (IDM), some types of RLC loads cause the most difficulty in detection. The equivalent circuit of the grid connected to the DG power generation system is shown in P + jQ flows from the DG system to node “PCC” and Pload + jQload flows from node “PCC” to the local load. The power flows from the utility grid to node “PCC” are ΔP + jΔQ.When the utility grid is connected (breaker is closed), the active and reactive power These power equations are shown in f0, and quality factor Qf are defined in Vmax = 115%, Vmin = 85%   isVVmax2−1Therefore, the NDZ of OUV is shown in P/P, at which ΔP/P can enter into NDZ of OUV shown in P/P; it is possible to use a simulator program with scale applications in MW instead of changing load with scale in kW or less. Therefore, this section used Powerworld Simulator Program for the proposed relationship between load and NDZ of OUV.Analysis in topic 2 showed that the OUV technique had a wide nondetection zone. When NDZ was wide, failure of islanding detection increased as well. This section shows that variation of load always occurs in real systems. The variation of load can affect ΔThe data used in the analysis of the IEEE 14-bus system was based on system data of the Provincial Electricity Authority (PEA) of Thailand. Some of the data are modified and converted to the format, and the per-unit for the data was used in Powerworld Simulator Program Version 16. The data entered into the program is a circuit of The 13-bus system has added DG type PV size 9 MW at bus 9. For easy analysis, off power flow of a transmission line connected bus 10 and bus 9 as shown in P when uneven ΔP directly affects uneven ΔP/P. Consider P/P, the ΔP/P enter nondetection zone at some time. If ΔP/P enters the nondetection zone when islanding condition occurs, voltage at PCC (VPCC) will not be less than undervoltage and will not be more than overvoltage. This phenomena are showed in the next section [From  section .P/P into NDZ of OUV. This section presents voltage at PCC (VPCC) after islanding condition occurs while ΔP/P enters or leaves the NDZ. Experiments use a real inverter.The experiment was the same as the anti-islanding testing diagram defined in UL 1741-1999, IEEE Std. 929-2000 and IEEE Std. 1547-2003 . There iThe experiment shows the relationship between voltage and NDZ of OUV by use of inverter as shown in Figures XL = 31.48 Ohm and XC = 31.48 Ohm. The load is resonant condition because XL = XC and this resonant condition makes Qload = 0. In the experiment, we have set the following: active power of DG is P = 1 kW, power factor of DG is PF = 1, power factor of load is PFload = 1, and active power of load changed from 600 W to 1,200 W (100 W per step). As for the worst case of islanding detection in the experiment, they should happen when active power of load is 1,000 W because active power of load is as valuable as active power of DG.In VPCC (voltage at the point of common coupling), before and after islanding condition happened, which can be seen in column 8 is off-grid voltage; VPCC decreases when ΔP increases. This result accords with P/P increases from negative to positive; it affects off-grid voltage at VPCC and decreases from overvoltage to undervoltage when is in comparison with on-grid voltage at VPCC (VGrid is the same as on-grid voltage at VPCC). The results show that a nondetection zone (NDZ) of OUV of inverter-based DG comes true [VPCC is less than on-grid voltage at VPCC multiply 0.85. As is wished, IEC 62116, normal voltage range is 85% ≤ V ≤ 115%, if V (V is the same as on-grid voltage at VPCC) was more than 115% or less than 85% that shows that islanding occurs and the control system of inverter will cease to energize the load. If the inverter set over/undervoltage of relays at the constant normal voltage of utility is 220 V, overvoltage of relay = 253 V and undervoltage of relay = 187 V. In VPCC was not more than 253 V or less than 187 V. The experimental results in VPCC happened before ΔP/P into positive which can be seen at row 4 in Pload = 900 W, P = 1,000 W, and ΔP/P = −10. VPCC before islanding condition was 229.8 V and off-grid voltage at VPCC was 191.0 V. Second, normal worst case range is Pload = P at which on-grid voltage at VPCC should be similar to off-grid voltage at VPCC but the experimental results show that VPCC between on-grid voltage at VPCC and off-grid voltage at VPCC was not similar. VPCC before and after islanding condition was most similar at Pload = 800 W, P = 1,000 W, and ΔP/P = −20 at row 3. On-grid voltage at VPCC was 231.6 V, and off-grid voltage at VPCC was 240.5 V.Nevertheless, there are some observations. Firstly, undervoltage of off-grid voltage at However, these experiments are tested as a parallel RLC circuit because for most islanding detection method some type of RLC load causes the most difficulty in detection. Therefore, investigation of the relationship between voltage and NDZ of OUV of local islanding detection technique by use of inverter-based DG , 13 is; then dV/dt of VPCC is calculated.The beginning of the algorithm detected islanding condition for anti-islanding condition. First, voltage is measured every period at PCC Next, dV/dt | >0, the algorithm compares VPCC after islanding condition occurs and over/undervoltage; mean VPCC (VPCC after islanding condition occurs) is more than 115%∗VPCC (VPCC before islanding condition occurs) = 253 V or less than 85%∗VPCC = 187 V. If it has more than 253 V or less than 187 V, the islanding condition occurs at which the algorithm will cease to energize to the load. If it is not more than 253 V or less than 187 V, the algorithm does the next process.When |This process will control the inverter to energize constant voltage at 84% of normal voltage, or 184.8 V (84%∗220 V) to power system. Variation of constant voltage at 84% is a new method in group of active islanding detection techniques. Normal voltage outputs of inverters can be changed, which changes the DC voltage to input of inverter or the gain of the inverter. This paper changes the DC voltage to input of inverter for the AC voltage output of the inverter less than or equal to 84%, So 184.8 V three cycles are injected into the system as shown in VPCC. The VPCC will same constant voltage (184.8 V) for three cycles. If VPCC has less than 187 V, the algorithm will cease to energize the load. If the occurrence condition is not an islanding condition, VPCC will remain the same, but P (active power of DG) will decrease inversely with ΔP (active power of grid) increasing that can prove the following.Afterwards, the inverter injected a constant voltage of 184.8 V for three cycles into the system. If this occurrence condition is an islanding condition, the voltage is measured at In normal conditions, the active power of load affects the active power of DG and the active power of grid in the point of common coupling. It is possible to calculate the active power variation versus the voltage variation injected in the load , 14, 15.PDG + PGrid and from (Deriving and from  (8) ∂(PDThe active power variation is expressed byR and Pload are constant. When the control system of the inverter injected constant voltage at 184.8 V (84%) for three cycles into the system after synchronizing the DG to the grid at which the occurrence condition is not islanding condition VPCC (ΔV) remains the same but ΔPDG decrease inversely with ΔPGrid increases. The changes between active power of DG and active power of grid are determined to be sufficient for the load. This paper shows the changing of active power in the next section.From  it can bThis section proves the over/undervoltage and undervoltage shift islanding detection algorithm for anti-islanding condition that this algorithm may need to develop in the future. This algorithm uses MATLAB/SIMULINK for the presentation. The model used in the demonstration, as in P/P and voltage after islanding condition occurs. The presentations include the case of ΔP/P > 38.41%, case of ΔP/P < −24.39%, and case of −24.39% ≤ ΔP/P ≤ 38.41%. In addition, this paper shows the case of injecting a signal to change the undervoltage but the occurrence condition is not an islanding condition.In the simulation to propose over/undervoltage and undervoltage shift block of hybrid islanding detection method, every case was a presentation with reference in P < Pload by enough to make  ΔP/P > 38.41%. When islanding condition occurs, VPCC will be less than 187 V (undervoltage of VPCC). The proposed method was proved by simulation on MATLAB/SIMULINK, and the model was set for islanding condition at 0.2 s. The results of simulation are show in This case will happen when VPCC decreased steadily. The control system of the inverter ordered DG off at the point of VPCC  (rms) < 187 V. In P/P > 38.41% were within 0.04 s.From P > Pload by enough to make  ΔP/P < −24.39%. When an islanding condition occurs, VPCC will be more than 253 V (overvoltage of VPCC). The model set the islanding condition at 0.2 s, which the simulation result show in This case will happen when VPCC increased steadily. The control system of the inverter ordered DG off at the point of VPCC  (rms) > 253 V. In P/P < −24.39% were within 0.04 s.From P ≈ Pload by enough to make −24.39% ≤ ΔP/P ≤ 38.41%. When an islanding condition occurs, VPCC will not be less than 187 V and not more than 253 V. The model set the islanding condition at 0.2 s, which the results of the simulation show in This case will happen when VPCC cannot be less than 187 V and not more than 253 V. Then the control system checked occurrence condition by injecting constant voltage (184.8 V (84%) for three cycles) into the load.From VPCC was the same constant voltage 184.8 V for three cycles. Then the control system of the inverter ordered DG off at the point of VPCC  (rms) < 187 V. In P/P ≤ 38.41% were within 0.08 s. However, if the occurrence conditions were not an islanding condition, the next case was used.If this occurrence condition was an islanding condition, the constant voltage of 184.8 V for three cycles was injected into the load. When the voltage was measured at the PCC, This case shows the change when the inverter injected constant voltage (184.8 V (84%) for three cycles) into the load, but the occurrence condition is not an islanding condition. In simulation, set signal of undervoltage shift was injected between 0.4 and 0.46 s.VPCC did not change, but at the moment the injected signal of undervoltage shift affected reduction of PDG and increment of PGrid, which is consistent with (From ent with . (10)ΔPDP/P > 38.41% could determine anti-islanding condition within 0.04 s; ΔP/P < −24.39% could determine anti-islanding condition within 0.04 s; −24.39% ≤ ΔP/P ≤ 38.41% could determine anti-islanding condition within 0.08 s. This method perturbed the system, only in the case of −24.39% ≤ ΔP/P ≤ 38.41% at which the control system of inverter injected a signal of undervoltage shift as necessary to check if the occurrence condition was an islanding condition or not.Simulation results on MATLAB/SIMULINK show that over/undervoltage and undervoltage shift of hybrid islanding detection method is very effective because it can determine anti-islanding condition very fast. Δ