LRRK2 are a common cause of familial and idiopathic Parkinson’s disease (PD). Recently, the LRRK2 GTPase domain R1398H variant was suggested in genetic studies to confer protection against PD but mechanistic data supporting this is lacking. Here, we present evidence that R1398H affects GTPase function, axon outgrowth, and Wnt signaling in a manner opposite to pathogenic LRRK2 mutations. LRRK2 R1398H GTPase domain dimerization and GTP hydrolysis were increased whereas GTP binding was reduced, leading to a decrease in active GTP-bound LRRK2. This protective variant also increased axon length of primary cortical neurones in comparison to wild-type LRRK2, whereas the R1441G LRRK2 pathogenic mutant decreased axon outgrowth. Importantly, R1398H enhanced the stimulatory effect of LRRK2 on canonical Wnt signaling whereas the G2385R risk variant, in accordance with all previously tested pathogenic LRRK2 mutants, had the opposite effect. Molecular modeling placed R1398H in close proximity to PD-causing mutations suggesting that this protective LRRK2 variant, like familial mutations, affects intramolecular RocCOR domain interactions. Thus, our data suggest that R1398H LRRK2 is a bona fide protective variant. The opposite effects of protective versus PD associated LRRK2 variants on GTPase function and canonical Wnt signaling activity also suggests that regulation of these two basic signaling mechanisms is important for neuronal function. We conclude that LRRK2 mediated Wnt signaling and GTPase function are fundamental in conferring disease susceptibility and have clear implications for therapeutic target identification.Mutations in  LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the most common known cause of inherited Parkinson’s disease . The ‘catalytic core’ is contained within the Roc (Ras of complex proteins), COR  and kinase domains  and appears essential for LRRK2 function  class of molecular switches . Binding of GTP to the Roc domains results in protein dimerization allowing binding of effector proteins. GTP hydrolysis disrupts Roc dimerization, leading to the dissociation of effector proteins (Based on homology to switches . Under tproteins .Table 1) supporting the idea that shifting LRRK2 to the GTP-bound ‘on’-state promotes neurodegeneration.All known PD-causing mutations located within the LRRK2 RocCOR domain have been reported to increase GTP-binding, decrease GTPase activity, or both  provide strong experimental support for the status of LRRK2 R1398H as a genuine protective variant; (2) increase the weight of evidence that GTP-bound LRRK2 is pathogenic; and (3) provide further data indicating that decreased canonical Wnt signaling is a key pathomechanism underlying PD.Here, we report the behavior of the LRRK2 R1398H variant in five assays for which the effect of a pDS-BAIT  plasmids containing the LRRK2 Roc and RocCOR domains , pACT2 (Clontech) containing the LRRK2 RocCOR domain and pYTH16 containing the intracellular domain of LRP6 (amino acids 1416–1613) have been described previously . pCHMWS 2. Transient transfection was performed using FuGENE HD (Roche) according to the manufacturer’s instructions, using a 2.5 μL transfection reagent to 1 μg DNA ratio. In all cases, cells were harvested 24 h after transfection.HEK293 cells and SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin G and 100 μg/ml streptomycin at 37°C and 5% CO2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4.7H2O) containing 40 mM β-mercaptoethanol, followed by lysis in 0.1% (w/v) SDS (Sigma-Aldrich) and 0.1% (v/v) chloroform (Sigma-Aldrich). After the addition of chloropheno-red-β-D-galactopyranoside (Sigma-Aldrich), the color change was recorded at 540 nm and readings adjusted for turbidity of the yeast suspension at 620 nm. The background signal (bait plus empty pACT2 vector) was subtracted from each reading and values were normalized to the wild-type RocCOR response, which was set at 100%. All protein interactions were assayed in three to five independent experiments in triplicate.The L40 yeast strain (Invitrogen) was co-transformed with pDS Roc or RocCOR bait and pACT2 wild-type or mutant RocCOR prey constructs, and the Y190 yeast strain (Clontech) was co-transformed with the pYTH16 LRP6 intracellular domain bait and pACT2 wild-type or mutant RocCOR prey constructs. Transformations were spread on selective dropout media (Clontech) lacking leucine and tryptophan for transformation controls, or leucine, tryptophan and histidine, supplemented with 0.5 mM 3-aminotriazole (L40 strain) or 10 mM 3-aminotriazole  for nutritional selection. After incubation at 30°C for 3 days, prototrophic colonies were picked and used to inoculate minimal SD (Clontech) media lacking leucine and tryptophan. Samples were subsequently incubated shaking at 30°C overnight. Cell pellets were then resuspended in Z-buffer , and lysates were centrifuged for 10 min at 20,000 g and 4°C. Supernatants containing 100 μg protein each, as assessed by QuickStart Bradford assay (Bio-Rad), were incubated for 80 min at 4°C with 30 μl of GTP-Sepharose bead suspension (Sigma) that was pre-treated with 100 μg/ml BSA (Pierce) at 4°C for 1 h. Samples were washed three times with 500 μl lysis buffer G, before bound protein was eluted using 100 μM GTP in lysis buffer G. The resulting eluate was added to 1× NuPAGE sample buffer (Invitrogen) and heated for 6 min at 96°C. The eluates were analyzed by SDS-PAGE and immunoblotting. Initially, protein was loaded into 4–12% (w/v) BisTris pre-cast gels (Invitrogen), prior to transfer to polyvinylidine fluoride membranes (Millipore). Non-specific bands were blocked for 1 h at 37°C with 5% (w/v) skimmed milk in PBS plus 0.1% (v/v) Tween 20. Anti-Calnexin antibody (Abcam) was used at 1:4000, and anti-FLAG antibody (Sigma-Aldrich) was used at 1:3000 at 4°C overnight. For detection, an HRP-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology) was used at a final dilution of 1:2000, together with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).HEK293 cells were transfected with 3× FLAG-tagged T1348N, R1398H or wild-type LRRK2. The GTP binding assay was performed similarly as described by others . Brieflyg, 3 min), followed by two washes in GTPase lysis buffer and a brief rinse in GTPase buffer . Proteins were eluted from beads with 3× FLAG peptide (Sigma-Aldrich) in GTPase buffer according to manufacturer’s instructions. The protein concentration of eluates were calculated from a serial BSA dilution curve, with purity assessed by running a small volume of each sample on an SDS-PAGE gel and staining with GelCode Blue Stain Reagent (Pierce). GTPase assays were performed according to GTPase assays were carried out according to 2 (in cylinders) then decapitated using a guillotine. All animal studies were approved by the National Institute of Neurological Disorders and Stroke/National Institute on Deafness and Other Communication Disorders Animal Care and Use Committee (Protocol 1151-12). Neurones were transfected with wild-type and mutant myc-tagged LRRK2 using the Amaxa Rat Neuron Nucleofector Kit, program 0-03, according to the manufacturer’s protocol . Neurones were then plated at a density of approximately 2.6 × 104/cm2 on cover slips and maintained as described previously (in vitro (DIV) neurones were fixed for 10 min with 4% paraformaldehyde, and permeabilized for 15 min with 0.05% Triton-X (Sigma-Aldrich) prior to a 1 h block in 5% NGS (GIBCO). Slides were then immunostained with primary and Alexa Fluor secondary antibodies, mounted using ProLong gold (Life Technologies), and imaged using a Zeiss LSM710 laser-scanning confocal microscope. Primary antibodies against the following proteins were used: myc-epitope (Santa Cruz Biotechnology), MAP2 (Abcam) and Tau-1 (Abcam). Alexa Fluor 488 (rabbit), 555 (mouse) and 633 (goat) secondary antibodies (Thermo Scientific) were used against myc, tau and MAP-2 epitopes respectively.Primary cultures of rat cortical neurones were prepared from E18 embryos obtained from timed-pregnant Sprague-Dawley rats  narcotized with COeviously . At 7 daAxon outgrowth properties of neurones were quantified manually. Three to six coverslips from three independent experiments for each genotype were analyzed. Axonal outgrowth and branching of dissociated neurones were quantified manually and verified using the NeuronJ plugin for ImageJ. Transfected neurones were identified using the myc-epitope antibody, whilst the length of the longest Tau-1 stained process from each neurone was measured. At least 40 neurones were quantified for each genotype, myc vector control, myc-LRRK2 wild-type, myc-LRRK2 R1398H, myc-LRRK2 R1441G, and myc-LRRK2 R1398H/R1441G from at least three coverslips.Canonical Wnt activity was measured using the TOPflash reporter plasmid  in humanFigure 4D) were tested by two-way ANOVA with repeated measures, with the independent variables genotype, time, and time × genotype, followed by post hoc analysis by two-sided Dunnett’s testing. Axonal branching was assessed by Kruskal–Wallis  or Mann–Witney  tests. Axon length was analyzed by one-way ANOVA followed by Bonferroni post hoc analysis  or Student’s t-test . For axon length analysis, outliers  were first excluded. All other experiments were analyzed by one-way ANOVA for the effect of genotype followed by a two-sided Dunnett’s test with wild-type LRRK2 considered the control. Student’s t-test was performed using Excel, all other statistics were performed with SPSS software. All error bars represent the standard error of the mean.GTPase assays . The R1441G mutant was studied as a pathogenic control showing as expected a significant decrease in RocCOR dimerisation . Consistently, R1398H also strengthened interaction between the LRRK2 RocCOR tandem domain and an isolated wild-type LRRK2 Roc domain, whilst the R1441G, R1441H, and Y1699C pathogenic mutations weakened this interaction . R1441C was also studied in this experiment, displaying a non-significant trend toward decreased interaction . These effects were not due to changes in protein expression . Thus, in contrast to proven PD-causing RocCOR mutations, R1398H enhanced intermolecular dimerization within the LRRK2 GTPase domain.To investigate functional effects of R1398H, we studied this variant in assays of LRRK2 RocCOR domain dimerization. These sensitive quantitative yeast-two hybrid (Q-YTH) assays have been used previously in our laboratory to show that the PD-causing R1441C, R1441G, R1441H, and Y1699C mutations significantly weaken RocCOR domain dimerization . IntriguC. tepidum Roco protein . Using the most probable rotamer conformation, the long basic side chain of arginine projected toward the COR domain of the same molecule. Indeed, R1398 was predicted to bond with the hydroxyl group of a conserved serine in the COR domain . This is an intriguing possibility, since the R1441C/G and Y1699C pathogenic mutations have also been predicted to modify intramolecular RocCOR interactions . Taken together, our molecular modeling suggests that the observed increase in intermolecular RocCOR dimerization caused by R1398H  likely occurs via an indirect mechanism, involving changes to intramolecular interactions between Roc and COR domains.To examine the molecular mechanism underlying altered RocCOR dimerization, the predicted location of R1398 was examined by molecular modeling of the closest available protein structure: the RocCOR tandem domain of  protein . Supportractions . In agreTable 1). In principle, a protective amino acid substitution located within this portion of LRRK2 would be expected to have the opposite effect. Thus, the effect of the R1398H variant on LRRK2 GTPase function was examined directly, using full-length human LRRK2 expressed in mammalian cells. Firstly, this variant was studied in GTP binding assays. As reported by others, wild-type LRRK2 bound strongly to immobilized GTP, whilst a well-characterized mutant, LRRK2 T1348N, had negligible GTP-binding capacity . Strikingly, the R1398H variant also displayed weakened GTP binding (∼47% of wild-type), although this value was more than threefold greater than for T1348N LRRK2, indicating that GTP binding is not abolished entirely.Numerous reports indicate that PD-causing mutations in the LRRK2 RocCOR tandem domain increase the ratio of GTP-bound (‘on’) LRRK2 to GDP-bound (‘off’) LRRK2, by weakening GTPase activity and/or facilitating GTP binding , and equimolar amounts were used in subsequent experiments . This protein produced a steady turnover of GTP, confirming that wild-type LRRK2 possesses intrinsic GTPase activity . Unsurprisingly, since T1348N LRRK2 is almost unable to bind GTP, this mutant possessed very little GTPase activity, with GTP hydrolysis undetectable until the 20 min time point . However, R1398H LRRK2 showed a marked increase in GTP hydrolysis relative to wild-type LRRK2 . In summary, the protective R1398H LRRK2 variant weakens GTP binding but increases steady-state GTP hydrolysis, both of which are consistent with a decrease in the proportion of GTP-bound LRRK2.We next examined the effect of the LRRK2 R1398H variant on GTP hydrolysis Figure 5A). By contrast, LRRK2 R1398H enhanced DVL1-driven Wnt activation almost twice as much as wild-type LRRK2 . This opposing effect of the protective LRRK2 variant to that described for pathogenic variants in four distinct LRRK2 domains is notable, and cannot be attributed to altered expression levels . These experiments suggest a strong correlation between PD risk conferred by LRRK2 variants and regulation of canonical Wnt signaling activity.We have previously reported that PD-causing mutations in the Roc, COR and kinase domains of LRRK2 weaken the activation of canonical Wnt signaling that is elicited by disheveled (DVL) proteins . ConsistTo further examine a possible causal mechanism that affects canonical Wnt signaling, we investigated the interaction between the LRRK2 R1398H variant and the canonical Wnt co-receptor LRP6 in Q-YTH experiments. Using the LRP6 intracellular domain as bait and the LRRK2 RocCOR tandem domain as prey, we observed a decrease in the interaction strength with the R1398H protective variant relative to wild-type LRRK2  or axon branching (p = 0.695) relative to neurones transfected with empty vector control . Furthermore, no difference in axon branching was observed between the different LRRK2 genotypes . However, the overexpression of LRRK2 mutants had a marked effect on axon length . In agreement with previous work, neurones overexpressing the pathogenic LRRK2 R1441G mutant showed a reduction in axon length relative to wild-type LRRK2 .Temporary differences in neurite outgrowth between LRRK2 knockout, mutant and wild-type neurones have been reported in various experimental systems . As LRRKpe LRRK2 . By contFigure 2A; Figure 2). Secondly, our molecular modeling suggests that this amino acid substitution will affect the interaction between the Roc and COR domains of the same molecule . Similarly, the R1441C/G/H and Y1699C mutations, and indirectly the N1437H mutation, have been suggested to affect intramolecular RocCOR interactions and consequently RocCOR dimerization as a proposed molecular pathomechanism (Table 1). Fourthly, the R1398H protective variant increases axon length in cortical neurones, whereas LRRK2 mutants cause shortening of axons in equivalent assays .As mentioned, PD-causing mutations outside the RocCOR tandem domain do not appear to operate via the same mechanism as those affecting GTPase function, i.e., they do not shift LRRK2 GTPase toward the GTP-bound ‘on’-state. This is curious, since logic would dictate that the effects of all PD-causing mutations in of LRRK2 , and a P2+-signaling (in vivo and associates with the Wnt co-receptor LRP6 at membranes. Importantly, expression of familial LRRK2 mutants results in decreased activation of Wnt/β-catenin signaling (Our data add to the growing body of evidence indicating that deregulation of canonical Wnt signaling is involved in LRRK2 PD . Previouignaling . LRRK2 aignaling . Strikinsubstantia nigra promotes dopaminergic neurone death (Our data also have implications beyond LRRK2 PD. Wnt signaling pathways have emerged as essential regulators of neuronal development and maintenance . Wnt ligne death . Wnt signe death . FurtherOur data support a model where pathogenic RocCOR mutants display altered intramolecular RocCOR interactions, leading to weakened RocCOR dimerization, increased GTP binding and decreased GTP hydrolysis. Together, these effects lead to a greater proportion of LRRK2 molecules existing in the GTP-bound ‘on’-state. The protective R1398H Roc domain mutation also affects RocCOR interactions but with the opposite result, leading to more LRRK2 in the ‘off’-state. As such, developing small molecules to decrease LRRK2 GTP-binding and/or stimulate LRRK2 GTPase activity seems a promising strategy for the development of PD modifying treatments and has already shown some encouraging results in LRRK2 GTP and kinase domain mutant PD models , 2015.JN-A, DB, CB, and KH designed the experiments; JN-A, DB, SG, and VS performed the experiments; JN-A, DB, SG, and KH analyzed the data; DB and KH wrote the paper. All authors were involved in revising the paper for important intellectual content, and gave final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO), a specific inhibitor of GSK-3 activity.Results: Overactivating the Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh), Epiprofin (Epfn), and Fibroblast growth factor8 (Fgf8), which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4), Fibroblast growth factor10 (Fgf10), Muscle segment homeobox 1 (Msx-1), Bone Morphogenetic protein 4 (Bmp4), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1) were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b) and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis.Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp, and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts. The mouse tooth is a good model for the study of regulatory pathways involved in cell differentiation, proliferation and organogenesis. Mouse tooth development begins around embryonic day 10.5  with a local thickening and oral epithelial invagination. Continuation of this invagination process results in the formation of epithelial tooth buds at E12.5–13.5. The mesenchymal cells condense around the buds, and after the bud stage, the tooth germ progresses to the cap (E14.5), early bell (E17.5) and late bell (E19.5) stages. From the bell stage onwards, the epithelial cells in contact with dentin differentiate into the enamel-producing ameloblasts , Sonic hedgehog (SHH), Wingless integration site (Wnt), and Transforming growth factor β (TGFβ) superfamilies are essential in tooth development  is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and ameloblast and odontoblast differentiation. However, its mechanism of action is still not fully understood. During odontogenesis, Epfn and of other downstream signaling effectors such as Bmp, Fgf, and Shh. Our model consists of transient application of BIO, a highly potent, selective, reversible, and competitive inhibitor of GSK-3 -6-bromoindirubin-3′-oxime  is a cell-permeable bis-indolo(indirubin) compound that acts as a highly potent, selective, reversible, and ATP-competitive inhibitor of GSK-3.in vitro experiments.1-Methyl-BIO  is a cell-permeable N-methylated analog of BIO that serves as a relevant inactive control of kinase activation. BIO and MetBIO were dissolved in dimethyl sulfoxide  and diluted in culture media at 20 μM for TM filters  and cultured in 1 ml Roswell Park Memorial Institute (RPMI) medium (Gibco) supplemented with 0.18 mg ascorbic acid, 2 mM L-glutamine (Gibco), 0.1 mg kanamycin (Thermo-Fisher Scientific)and 20% fetal bovine serum (Biochrom), at 37°C and 5% CO2. Explants were treated with BIO and MetBIO (control) at 20 μM for 24 or 48 h or for 4 or 6 days. E19.5 teeth were not maintained in culture for more than 4 days in vitro due to size-related limitations on nutrient and oxygen exchange. The culture medium was changed every 2 days until the end of the culture. Some explants were cultured with 10 μM 5-bromo-2′-deoxyuridine (SigmaAldrich) for 2 h. Samples were processed for histology, in situ hybridization or immunofluorescence. The experiments with mice have been approved by the ethic committee of the University of the Basque Country, UPV/EHU (346/2014/UNDA).Mouse first branchial arches at E11.5 and first lower molars at three stages of tooth development  were dissected by microsurgery with scalpels, in Hank's buffered saline solution (Gibco) under a stereomicroscope. Teeth were placed on hydrophilized polytetrafluoroethylene (PTFE) MillicellTooth cultures were fixed overnight at 4°C in 4% paraformaldehyde (SigmaAldrich). Subsequently, they were cryoprotected overnight at 4°C in 30% sucrose and then embedded in OCT (Tissue-Tek) and stored at −80°C until use. Frozen sections (15 μm) were obtained using a cryostat (Shandon) and post-fixed in 4% paraformaldehyde for 10 min. Some sections were stained with hematoxylin-eosin or permeabilized using 1% Triton X-100 (SigmaAldrich) in PBS (SigmaAldrich) and blocked with 10% fetal bovine serum for 1 h at room temperature. These samples were incubated overnight at 4°C with the following antibodies:mouse anti-bromodeoxyuridine-fluorescein monoclonal antibody 1:200 (Roche 11202693001), rabbit anti-cleaved Caspase3 1:200  anti-Nestin 1:100  and anti-Amelogenin 1:200 (ab59705 Abcam) antibodies. The secondary antibody used was Alexa Fluor 488-conjugated anti-rabbit antiserum (dilution 1:500). Cell nuclei were stained with DAPI  0.5 μg/ml. Immunofluorescences were performed by triplicate and images were obtained using an Olympus FluoView FV500 confocal microscope or a Zeiss Axioskop fluorescence microscope with Nikon DS-Qi1Mc and Nikon NIS-Elements software, respectively.Lef1, Bmp4, Msx1, Fgf9, Fgf10, Fgf4, Epfn, Dkk1, Wnt10b, and Shh were prepared using a DIG RNA labeling kit (Roche Applied Science) according to the manufacturer's instructions. For whole-mount in situ hybridization, E11.5 branchial arches and E14.5 and E17.5 first molars cultured with 20 μM BIO or MetBIO for 24 or 48 h were fixed in 4% paraformaldehyde/PBS overnight, dehydrated in methanol, and kept at −20°C until use. Whole-mount RNA in situ hybridization was carried out according to Nieto et al.  software. Images were edited using Photoshop CS5 software.Constitutive activation of β-catenin in the epithelial tooth buds or the deletion of β-catenin in the dental mesenchyme results in the formation of supernumerary teeth  activity is an important characteristic of mineralizing cells of mesenchymal origin. Thus, we wanted to assess whether the change in Nestin expression would be paralleled by that of ALP in BIO-treated molar teeth. In control molars, ALP activity was detected in the stellate reticulum, odontoblasts, and subodontoblastic cells, whereas preameloblasts were negative for ALP activity Figure . ALP actn Figure  and theiLef1, after culture with BIO, indicates the effectiveness of our treatment to overactivate the canonical Wnt pathway at the morphogenesis stages of odontogenesis ; later on, its expression is restricted to the dental mesenchyme  of this pathway  Figures .in vivo tooth development, Wnt10b is expressed in the presumptive regions of incisors and molars in the first branchial arches  and treated with BIO for 6 or 4 days (only for E19.5 molars). First, quantification of BrdU immunohistochemical labeling revealed a significant increase in cell proliferation in the dental mesenchyme of BIO-treated E17.5 teeth compared with the control. BIO-treated E14.5 molars increased cell proliferation but not significantly, and 19.5 molars showed cell proliferation similar to that observed under control conditions  and ameloblasts (E19.5). Thus, in the presence of BIO or Met-BIO (control) we cultured E17.5 and E19.5 first molars for 6 and 4 days, respectively. In control molars, dental mesenchymal cells differentiated into functional odontoblasts secreting dentin, and the inner dental epithelial cells became well-polarized cells in the ameloblastic lineage. Moreover, these molars exhibited developed tooth cusps with predentin/dentin Figures , mesenchThe increased expression of Lef1 after culture of E17.5 molars with BIO for 48 h indicated the effectiveness of our treatment to overactivate the canonical Wnt pathway during differentiation stages in odontogenesis Figures .Dkk1, Shh, Bmp4, Wnt10b, and Epfn. At E17.5, compared with the controls, expression of Dkk1 and Bmp4 was markedly increased in the dental mesenchyme of molars treated with BIO  did not generate extra teeth , during which treatment with BIO could cause an expansion of tooth inductive potential to other neighboring oral epithelial areas. BIO exposure at the morphogenesis and differentiation stages did not cause any increase in tooth number.In our Shh expression in the inner dental epithelium of cultured molars, whereas the expression of both Fgf4 and Fgf10 increased in the enamel knots and dental mesenchyme. Importantly, these changes correlated with some of the dysfunctions observed at later developmental stages, such as the impairment in ameloblast and odontoblast cell differentiation and increased dental size.Wnt/β-catenin signaling plays an important role in setting the size and morphology of the adult teeth  and during dental differentiation (E17.5), based on the responses to stimulation with BIO. These findings reinforce the view that precise control of the activity of the Wnt/β-catenin pathway is necessary for proper tooth development. Therefore, isolated cultured molars react to BIO treatment by switching off canonical Wnt activity in an attempt to counterbalance this pharmacologically induced hyperactivity.One gene that is consistently upregulated after β-catenin stabilization is Msx1 and Bmp4 in the dental mesenchyme during the transition from initiation to morphogenesis stage of odontogenesis  and the Basque Government (IT831-13). MA and PG received a fellowship from the University of the Basque Country.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite decades of effort to develop effective therapy and to identify promising new drugs, prostate cancer is lethal once it progresses to castration-resistant disease. Studies show mis-regulation of multiple pathways in castration-resistant prostate cancer (CRPC), reflecting the heterogeneity of the tumors and also hinting that targeting androgen receptor (AR) pathway alone might not be sufficient to treat CRPC. In this study, we present evidence that the Wnt/β-catenin pathway might be activated in prostate cancer cells after androgen-deprivation to promote androgen-independent growth, partly through enhanced interaction of β-catenin with TCF4. Androgen-independent prostate cancer cells were more prone to activate a Wnt-reporter, and inhibition of the Wnt/β-catenin pathway increased sensitivity of these cells to the second-generation antiandrogen, enzalutamide. Combined treatment of enzalutamide and Wnt/β-catenin inhibitor showed increased growth repression in both androgen-dependent and -independent prostate cancer cells, suggesting therapeutic potential for this approach. Prostate cancer is the most common form of cancer in males . Given tDue to the complex signaling networks in advanced disease, inhibition of one pathway might cause unpredictable responses , 7. In tRecent genome sequencing data has shown misregulation of the Wnt-pathway in prostate cancer with disease progression. The comparative analysis of two separate whole-exome sequencing sets of data, one from primary tumors  and the Our group previously published proof of concept studies showing that a small molecule inhibitor of the Wnt/β-catenin pathway, iCRT3 (shortened to C3) could decrease AR mRNA expression and transcription of downstream target genes by interfering with β-catenin/TCF interaction on the AR promoter. We also showed that C3 could interfere with AR and β-catenin protein interaction. The later protein interaction studies were performed in the presence of high levels of androgen to stabilize AR protein levels so that they were not decreased in the presence of β-catenin inhibitor .Work described in this manuscript shows that the activity of the Wnt/β-catenin pathway is low in prostate cancer cells likely due to the preference for β-catenin interaction with AR rather than TCF4 in these cells. We observe that suppression of AR activity by androgen-deprivation, antiandrogen treatment or AR knockdown promoted Wnt/β-catenin-target gene expression and this correlated with increased interaction between TCF4 and β-catenin. The enhanced activation of the Wnt/β-catenin pathway caused growth of androgen-dependent LNCaP cells in the absence of androgen or in the presence of antiandrogen. Activation of the Wnt/β-catenin pathway was also examined in an androgen-independent subline of LNCaP cells, LNCaP-abl (abl). Abl cells were generated by continuous passage in androgen depleted media and selected for their ability to proliferate in the androgen deprived condition . Abl celLNCaP  and LNCaP-abl (abl)  . Packaging cells (HEK293T/17)  were transiently transfected with 6 μg of eGFP Wnt reporter construct, 4 μg of packaging plasmid (psPAX2) and 2 μg of envelope expressing plasmid (pMD2.G) using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instructions. Media conditioned by the transfectants was collected after 24 and 48 h. LNCaP, abl and 22Rv1 cells were infected by incubation in conditioned media overnight and allowed to recover for a day. Infected cells were passaged and selected by mCherry expression using flow cytometry.Total RNA was isolated using the RNeasy kit (Qiagen), and then reverse transcribed at 55°C for 1 h using Superscript III reverse transcriptase and oligo- (dT) 20 primers (Invitrogen). Real-time PCR was performed using gene-specific primers and 2XSYBR green Taq-ready mix (Sigma). Data were analyzed by the DDCT method using RPL19 as a control gene and normalized to control samples, which were arbitrarily set to 1.3VO4, 10 mg/ml of leupeptin and 10 mg/ml of aprotinin. Protein lysates were subjected to SDS-PAGE and immunoblotted with the following antibodies against: AR (441), β-catenin (H-102), TCF4 ; active β-catenin ; tubulin (Covance). Protein bands were visualized using ECL Western Blotting detection reagents . Image J (NIH) software was used to quantitate protein levels.For immunoblot analysis, cells were lysed in Triton buffer and supplemented with 1 mM PMSF, 1 mM NaIn immunoprecipitation experiments cells were lysed as described above. Primary antibodies listed above were added to at least 1.5 mg of total protein and incubated overnight at 4°C followed by the addition of Protein A/G agarose beads (Santa Cruz Biotechnology) for 2 h. Immune complexes were extensively washed with Triton buffer and solubilized using Laemmli sample buffer (BioRad). Normal mouse IgG (Santa Cruz Biotechnology) or normal rabbit sera (Sigma) were used as controls.For immunofluorescence staining, cells were fixed with 4% formaldehyde in PBS for 20 min, washed with PBS three times, permeabilized with 0.2% Triton-X in PBS for 20 min, blocked with 5% normal goat and 5% normal horse serum in PBS for 1 h, and incubated with antibody against active β-catenin (Millipore) diluted in blocking buffer, overnight at 4°C. The following day, cells were washed with PBS three times and then incubated with secondary antibodies conjugated with FITC (Vector Lab) at room temperature for 1 h. Subsequently, cells were washed in PBS three times and mounted with DAPI mounting solution (Vector Lab). To analyze eGFP and mCherry levels in cells stably integrated with 7xTcf-eGFP/SV40-mCherry constructs, cells were fixed, washed and permeabilized as described above and mounted with DAPI mounting solution.3 cells were plated in each well of black 96 well plate. Cells were plated in hormone-deprived media containing 5% CFBS and cultured for 2 to 3 days, and then treated with indicated reagents for each experiment. The reagents were added every two days with more media and an equal amount of vehicle added to the control group. At 2–3 day intervals, CyQUANT assay  was performed according to manufacturer’s instructions and analyzed on a SpectraMax M5 plate reader (Molecular Devices) running SoftMax Pro® software.For the CyQUANT cell proliferation assay, 3 to 4 X 10ChIP was performed as previously described . Protein5 cells were plated in each well of a 24 well plate in hormone-deprived media containing 5% CFBS and the mixture of HiPerFect reagent and siRNAs were added directly on top of the cells. Cells were cultured for 2 days and then treated with indicated reagents for each experiment. For CyQUANT proliferation assay, 3 to 4 X 103 cells were plated in each well of black 96 well plates in hormone-deprived media containing 5% CFBS and the mixture of HiPerFect reagent and APC siRNAs were added directly on top of the cells. Cells were cultured for 2 days and then treated with indicated reagents for each experiment. CyQUANT assay was performed at 2–3 day intervals as described above.For β-catenin and AR knockdown, siGENOME SMARTpool siRNAs against β-catenin or AR were used (Dharmacon). For APC knockdown, the pool of three Silencer® Select siRNAs against APC  were used. Cells were transfected with HiPerFect transfection reagent (Qiagen) according to manufacturer’s instructions. 1 X 10The cell lines stably depleted of β-catenin were generated with lentiviral pGIPZ shRNA against β-catenin  or a control shRNA . After infection, cells were plated at a very low density and selected for 10 days with 1 μg/ml puromycin (Sigma). Each resistant cell colony was collected and expanded in the selection media to screen for beta-catenin protein levels. Two cell lines showing moderate reduction of β-catenin were selected to test enzalutamide sensitivity (sh-β-cat-1 and -2) to minimize the robust growth inhibitory effect of β-catenin knockdown (25).To study the Wnt/β-catenin pathway activity in prostate cancer, we monitored endogenous Wnt/β-catenin activity on a Wnt-reporter upstream of eGFP (7xTcf-eGFP/SV40-mCherry). The construct also expresses mCherry under the constitutively active SV40 promoter indicating positively infected cells . LNCaP, Given that β-catenin interacts with AR in an androgen-dependent manner –28, we rAxin2, were analyzed. Compared to control cells, higher induction of Wnt-reporter and Axin2 mRNA levels were observed in LNCaP cells cultured in hormone-deprived media, in the presence of enzalutamide or treated with AR siRNA  or siRNAR siRNA . As an aAR siRNA , APC knoed cells , suggestlutamide  with theol cells . Depletibl cells  suggestiAltogether, these results suggest that in androgen-dependent LNCaP cells, inhibition of AR may allow the tumor cells to activate the Wnt/β-catenin pathway. Under conditions of hormone deprivation this may occur upon exposure to Wnt-signals likely present in the tumor microenvironment , 31, 32.Axin2) and AR-target (PSA) genes in each condition were analyzed  [Since one of the cellular responses of the Wnt-pathway is to promote proliferation , 38, we er cells . The GSKreatment , as welled cells . These rRT3 (C3) , diminisSince inhibition of AR activity resulted in enhanced Wnt/β-catenin-responsive transcription  and cellContrary to what we observed in LNCaP cells, treatment of abl cells with GSK-3 inhibitor showed no effect on proliferation in either hormone-deprived or enzalutamide treated culture conditions . In factExtensive effort has been spent to develop more effective next-generation AR therapies to treat CRPC, including enzalutamide  and abirThe crosstalk between AR and Wnt/β-catenin pathways in prostate cancer has been well studied , 48. In Here we showed that the Wnt/β-catenin pathway is repressed by AR in androgen-dependent LNCaP cells. We also showed that hormone-deprivation or antiandrogen treatment led to Wnt/β-catenin driven cellular proliferation . SimultaPrevious studies indicate that AR and TCF4 have overlapping interaction domains on β-catenin , 50 and Here, we activated endogenous β-catenin through inhibition of the β-catenin destruction complex via GSK-3 inhibition. We used GSK-3 inhibitor or siRNA against APC to show that the activation of Wnt/β-catenin responsive transcription was enhanced when AR activity was repressed . This waOne promising strategy to overcome acquired resistance is a rationally designed combination therapy against the resistant pathways based on pre-clinical studies . CombinaIn summary, this study showed that inhibition of AR activity caused increased activation of Wnt/β-catenin pathway and this increased activation promoted androgen-independent growth of prostate cancer cells. Simultaneous inhibition of AR and Wnt/β-catenin pathways showed increased growth repression of LNCaP and enzalutamide-resistant abl cells, indicating the therapeutic potential of targeting both pathways. The development of Wnt/β-catenin-inhibitors optimized for in vivo use will facilitate this approach.


The Wnt/beta-catenin and the Hedgehog (Hh) pathway interact in various cell types while eliciting opposing or synergistic cellular effects. Both pathways are known as exclusive drivers of two distinct molecular subtypes of medulloblastoma (MB).In sonic hedgehog (Shh)-driven MB, activation of Wnt signaling has been shown to suppress tumor growth by either beta-catenin-dependent or -independent inhibition of Shh signaling. However, mechanistic insight in how beta-catenin inhibits the Hh pathway is not known.+/-) in vitro. LiCl treatment of MB spheres down-regulated the Hh target Gli1, whereas the repressive Gli3 protein (Gli3R) was increased. Mechanistically, we show by co-immunoprecipitation and proximity ligation assay that stabilized beta-catenin physically interacts with Gli1, leading to Gli1 sequestration and inhibition of its transcriptional activity. Reduction of Hh signaling upon LiCl stimulation resulted in reduced proliferation, sphere self renewal, a G2/M arrest and induction of a senescent-like state, indicated by p21 upregulation and by increased staining of senescence-associated beta-galactosidase . Moreover, LiCl treatment of subcutaneously transplanted MB cells significantly reduced tumor initiation defined as “tumor take”. Although tumor progression was similar, LiCl-treated tumors showed decreased mitotic figures and phospho-histone H3 staining.Here we show that beta-catenin stabilization by the glycogen synthase kinase 3 inhibitor lithium chloride (LiCl) reduced growth of primary hedgehog-driven MB tumor spheres from patched heterozygous mice  contains supplementary material, which is available to authorized users. MB of (ptch1) ,5-7. The (ptch1) ,9.In the “canonical” Wnt pathway β-catenin acts as a transcription factor with members of the lymphoid enhancer factor (Lef)/T-cell factor family . On Wnt Interestingly, Wnt and Shh were shown to interact in various cell types and organs during development and in the adult, while eliciting opposing or synergistic cellular effects. Generally, it has previously been shown that Shh inhibits the Wnt pathway during tongue papilla development and in squamous cell carcinoma ,4,13,14.Recently, also in MB interaction of the Wnt and Hh pathway has been shown, describing either a β-catenin-dependent or -independent inhibition of Shh signaling by Wnt ,6,15.However, neither mechanistic insight into β-catenin-mediated inhibition of the Hh pathway, nor the therapeutic potential of Wnt/β-catenin-activating drugs has been examined specifically in MB.+/- MB tumor spheres as well as tumor take in mice. We provide mechanistic evidence that this effect is dependent on the physical interaction of Gli1 and β-catenin, leading to Gli1 degradation, G2/M cell cycle arrest and cellular senescence.Here we show that the FDA-approved drug LiCl, which results in β-catenin stabilization via GSK-3 inhibition, suppressed formation of Ptch+/- mice [in vitro. Inhibition of GSK-3 by LiCl, evidenced by GSK-3 phosphorylation .As shown in the developing spinal cord, activated Wnt/β-catenin signaling increases the expression of repressive Gli3 (Gli3R), which in turn inhibits the Hh pathway ,11. We cAlthough the up-regulation of Gli3R and the concomitant down-regulation of Ptch1, Hhip and Gli1 either on mRNA or protein level fit to the interpretation of Hh pathway inhibition by LiCl-mediated β-catenin stabilization, the mechanism of Gli1 down-regulation remained elusive. To evaluate if proteasomal degradation diminishes Gli1 we investigated the effect on Gli1 protein by the proteasome inhibitor MG-132. LiCl-mediated decrease of Gli1 was partly reversed by proteasomal inhibition -positive cells . However this aspect requires further investigation.Along this line, here we show that in primary Ptchs Figure e and uprs Figure f. These +/- MB cultures after 8 h LiCl stimulation and blotted for Gli1, which was significantly increased upon β-catenin stabilization by LiCl, although low amounts of co-precipitation was also detectable in the NaCl controls  with antibodies against β-catenin and Gli1. Indeed, this approach corroborated the β-catenin-Gli1 interaction observed in co-precipitation experiments . MEFs were transfected with the chimeric construct LEFΔN-βCTA, conferring dominant β-catenin-LEF transcription, and subsequently stimulated with control or Shh-conditioned medium (ShhCM) [-/- MEFs  . As a re (ShhCM) . Interess Figure d, suggesFurthermore, we investigated the role of β-catenin for hedgehog-mediated MB sphere growth by knocking down β-catenin by a siRNA approach. Interestingly, knock-down of β-catenin abrogated the growth inhibitory function of LiCl treatment  or NaCl  as control [Finally, in order to understand if β-catenin activation might be beneficial for MB patients, we transplanted primary mouse Ptch control ,22.LiCl treatment resulted in a significant delay in initial tumor incidence that we defined as “tumor take”, ranging between day 22 and 26 Figure b. Nevertin vitro findings -positive cells Figures c, d. ThiIt is interesting however, that LiCl tumors showed decreased necrotic areas (data not shown), although tumor size did not significantly differ from controls. In this regard it is worth mentioning that we recently reported on a normalizing effect of β-catenin activation in endothelial cells of glioma vasculature . This wo+/- tumors did not differ with respect to extracellular matrix and GFAP staining (Additional file The overall tumor histopathology of the subcutaneous PtchThe systemic treatment with LiCl and the resulting stabilization of β-catenin will always target more cell types then the tumor cell itself. Consequently, we have to estimate the benefit of the treatment by the net outcome of tumor burden. It should be noted that the clinical impact of these findings might relate to some types of Shh-driven MB that occur in infants and young adults, which are difficult to treat with established regimes or by Smo inhibitors that are currently in clinical trials . It seemIn summary our data show for the first time that activation of Wnt/β-catenin signaling by LiCl treatment inhibits proliferation of Shh-driven mouse MB via the interaction of β-catenin and Gli1.This supports the concept that Gli1 interacts with β-catenin by default, and that an increase of stabilized β-catenin is able to sequester an increased amount of Gli1 and vice versa. This scheme of a balanced interaction implies that β-catenin stabilization could be titrated to a level at which virtually all Gli1 becomes sequestered, without affecting canonical Wnt pathway activation. These findings may open novel possibilities for therapeutic interventions for MB patients.


Background: γ-Catenin is a tumor suppressor in lung cancer.Results: γ-Catenin is a novel regulator of HAI-1 and cell migration.Conclusion: γ-Catenin and HAI-1 regulate cell migration and sensitize NSCLC cells to c-MET inhibitor.Significance: Targeting γ-catenin-mediated HAI-1 expression might be a new strategy to treat lung cancer. γ-catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors. Lung cancer is the leading cause of cancer mortality in the United States , 2. The One signaling pathway that has been a focus of active research in lung cancer is the c-MET signaling pathway –6. The c–3–10–Plakoglobin (γ-catenin) is a member of the armadillo repeats containing proteins  that exhIn the current study we have identified a novel role for γ-catenin in the regulation of cell migration, which is an important step for tumor progression and metastasis. Interestingly, re-expression of γ-catenin in NSCLC cell lines resulted in reduced cell migration as determined by both scratch and trans-well cell migration assays. Additionally, we demonstrate that the γ-catenin-induced anti-migratory effects were mediated via the expression of HAI-1 in a p53-dependent manner. Taken together, γ-catenin is shown to be a novel regulator of HAI-1 that is a critical regulator of HGF/c-MET signaling. Therefore targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors.2 incubator at 37 °C. Cell lines were cultured bi-weekly and stocks of cell lines were passaged no more than ten times for use in experiments. H157 and A549 cells stably expressing pLNCX and pLNCX-γ-catenin plasmids were developed as described earlier  cells and the NSCLC cell lines  were obtained from the tissue culture core of the University of Colorado, Anschutz Medical Campus. All cell lines were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO earlier .m siRNAs by using Lipofectamine 2000 reagent according to the manufacturer's protocol. p53 shRNA construct in TRC1 pLKO-puro backbone was obtained from Dr. Chris Porter  and was used to transfect NSCLC cells with Lipofectamine reagent as per the manufacturer's recommendation.Double-stranded RNAs (siRNAs) targeting human γ-catenin (CCCUCGUGCAGAUCAUGCGUAACUA) were procured from Invitrogen. Control siRNA (sc-37007), p53 siRNA (sc-29435), and HAI-1 siRNA (sc-39554) were purchased from Santa Cruz Biotechnology. NSCLC cells were treated with 50 nThe reporter plasmid (p53-RE-luciferase reporter), expression plasmids , or siRNA  and CMV-β-galactosidase control plasmids were transiently transfected into cells using Lipofectamine 2000 Reagent  as per manufacturer's recommendations. p53-luciferase reporter, with luciferase under the control of 14 repeats of p53-binding sequence (TGCCTGGACTTGCCTGG) was obtained from Stratagene. After 24 h, the lysates were assayed for luciferase activities. The luciferase values were normalized to β-galactosidase values and are represented in the graphs.m β-glycerophosphate, pH 7.2, 0.1 mm sodium vanadate, 2 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 2 μg/ml leupeptin, and 4 μg/ml aprotinin) and the Western blot analysis was carried out as previously described  and 3% nonfat dry milk and then incubated with the same blocking solution containing the indicated antibodies at 0.5 μg/ml for 16 h. Filters were extensively washed in TTBS, and bound antibodies were visualized with horseradish peroxidase (HRP)-coupled secondary antibodies.Cell extracts were prepared in a lysis buffer  for 1 h at room temperature. After de-staining in water and drying, colonies were quantified using Bio-Rad Chemidoc Imaging System and Quantity One Software. Cloning efficiency represents the mean number of colonies formed per well.Clonogenic assays were performed in triplicates by seeding 1000 cells per well in a 12-well culture plate followed by incubation at 37 °C in a 5% CO2 incubator. Cell proliferation was measured at 24, 48, and 72 h by adding 20 μl of MTS reagent  to each well, followed by incubation at 37 °C. After 1 h, the absorbance of the formazan product was measured at 490 nm using a plate reader. Normalized absorbance values (sample readings-readings of medium only blank) were represented in the graphs.MTS cell growth assays were performed in triplicates by seeding 500 cells per well in a 96-well culture plate, followed by incubation at 37 °C in a 5% COm), Tivantinib (0.5 μm), or 5-Aza + Tivantinib. After 48 h of treatment, cells were trypsinized from the wells with 100 μl of trypsin, diluted with 400 μl of growth medium, and counted using a hemocytometer.For the measurement of cell growth rates, H157 cells  were seeded in a 24-well culture plate. After 24 h, the cells were treated with either vehicle control (DMSO), 5-Aza (3 μm). On subsequent days, cells were trypsinized from the wells with 100 μl of trypsin, diluted with 400 μl of growth medium, and counted using a hemocytometer.Similarly, H157 cells stably expressing either empty vector or γ-catenin were seeded in a 24-well plate . After 24 h, the cells were treated with either vehicle control (DMSO), or Tivantinib  containing 8 μm permeable pores and allowed to migrate toward 10% FBS containing medium. Later, the cells in the trans-well inserts were removed and the inserts were washed in PBS for three times. The migrated cells on the bottom of the insert were fixed with 2% glutaraldehyde solution followed by crystal violet (1%) staining. After washing the inserts three times with PBS, the inserts were allowed to air dry and pictures were taken using an inverted microscope. Ten independent fields were counted for each trans-well and the average number of cells/field were represented in the graphs.For measuring apoptosis, FITC Annexin V apoptosis detection kit I was used . Briefly, H157 cells stably expressing γ-catenin were trypsinized, and washed three times with PBS. Later, cells were stained with Annexin V according to manufacturer's protocol. Flow cytometry was performed using a Beckman Coulter Gallios  flow cytometer at the UIC flow cytometry core facility.supplementary table containing the Affy probeset ID and additional annotation for each commonly correlated transcript along with their associated r- and p values for each dataset.Lung cancer cell line gene expression datasets from the Cancer Cell Line Encyclopedia (CCLE)  and UT Southwestern Medical Center  were downloaded from Gene Expression Omnibus (GEO) and used to find genes correlated to γ-catenin expression. There were 42 cell lines shared between the lung cancer cell line datasets (∼60% of the UT study) and both use the same platform (Affymetrix) to assess global gene expression. An absolute r-value of greater than 0.5 (Pearson) was used to define significant correlation to γ-catenin independently in each dataset. There were 324 and 252 transcripts correlated to γ-catenin  in the CCLE and UT datasets, respectively, of which 152 were common to both the datasets. We have uploaded an excel spreadsheet as a www.ingenuity.com). IPA takes a select list of genes and creates limited, interconnected networks (35 genes maximum) based on the evidence of direct or indirect biological relationships contained in the Ingenuity Knowledge Base. The IPA network algorithm seeks to maximize the interconnectivity within a group of selected genes and scores networks based on a right tailed Fisher's exact test that calculates the probability that the given relationships can be explained by a random model. The networks do not include all possible relationships for each member, because of size constraints placed on the network, and specific genes may appear in multiple networks.The 152 transcripts correlated to γ-catenin were later analyzed for over-representation of biological functions, pathways and connections using the bioinformatics software Ingenuity Pathways Analysis  is denoted with an “asterisk” symbol, while a decrease in statistical significance  is denoted with a “hash” symbol.Data were compiled from at least three independent, replicate experiments, each performed on separate cultures and on separate occasions. The responses are displayed as “fold-changes.” Comparisons of data among experimental groups were performed using Student's A) and observed a striking decrease in γ-catenin expression in 82% of NSCLC cell lines tested when compared with a non-transformed bronchial epithelial cell line i.e. Beas2B . However, the role of γ-catenin in NSCLC cell migration has not been completely understood. We chose H157 and H1299 cell lines for our studies since: 1) they express relatively low levels of γ-catenin, and 2) they were amenable to transfections for overexpression and gene knockdown studies. To evaluate the role of γ-catenin in NSCLC cell migration, γ-catenin was expressed in H157 and H1299 cell lines (B and C). Following transient transfections, cell migration was assessed via a wound-healing assay (scratch assay) by creating 3 mm wounds on confluent cultures (D and E). The abilities of the cells to fill the wound under different treatment conditions were later determined in the presence of a mitosis/cell division inhibitor (mitomycin C). In comparison to control vector transfected cells, H157 cells transfected with γ-catenin displayed reduced cell migration (B and D). On the contrary, expression of γ-catenin in H1299 cells failed to reduce cell migration, (C and E). To clarify the role of γ-catenin in cell migration, we also performed trans-well migration assays using 8-μm pore size cell culture inserts. Consistent with the scratch assays, expression of γ-catenin in H157 cells, but not in H1299 cells, reduced cell migration (F and G). It was interesting to note that H157 and H1299 differ in their p53 mutation status. While, H157 expressed p53 tumor suppressor (harbors a heterozygous non-sense mutation at codon 298), H1299, on the contrary, was p53 null . Furthermore, the p53-dependent effects of γ-catenin were not restricted to cell migration. γ-catenin also induced strong anti-proliferative effects in H157 cells, but not in H1299 cells, as determined by clonogenic cell proliferation and survival assays (H and I). Taken together, these findings suggest that the anti-proliferative and anti-migratory effects of γ-catenin in NSCLC are mediated by p53.Plakoglobin (γ-catenin) expression was shown to be either lost or down-regulated in a subset of human lung cancers . We furtFig. 1A), and anti-migratory (B) effects in p53 siRNA or p53 shRNA treated H157 cells, suggesting that the effects of γ-catenin were indeed p53-dependent.To further explore the p53-dependent effects of γ-catenin on cell proliferation and cell migration, we utilized short hairpin RNA (shRNA) or short interference RNAs (siRNAs) against p53. Interestingly, expression of γ-catenin failed to induce anti-proliferative A, and anigratory B effectsA). For knockdown of γ-catenin, siRNAs specifically targeting γ-catenin were designed, tested for their specificity and then employed to selectively suppress γ-catenin. The siRNAs specifically suppressed γ-catenin achieving more than 90% reduction in the expression of γ-catenin (A). Interestingly, the suppression of γ-catenin provoked a dramatic loss in the expression of p53 (A).We explored next if γ-catenin could regulate p53 expression. To test our hypothesis, we utilized two different strategies: 1) The knockdown of γ-catenin in a non-transformed bronchial epithelial cell line (Beas2B) and 2) The expression of γ-catenin in H157 cells, which have reduced γ-catenin expression A. For kn-catenin A. Interen of p53 A.B). Regulation of gene expression is mediated by several mechanisms, including transcriptional regulation and/or post-translational regulation. Interestingly, expression of γ-catenin in H157 cells also induced robust p53 mRNA expression, suggesting that γ-catenin might be regulating p53 expression at the transcriptional level (C). We also determined whether γ-catenin-mediated p53 expression was due to altered protein stability (D). For these studies Beas2B cells were first transfected with either control siRNA or γ-catenin siRNA, followed by the treatment with cycloheximide, which blocks de novo protein synthesis. The p53 half-lives were later determined via immunoblotting protein lysates with p53 antibodies (D). Interestingly, siRNA mediated knockdown of γ-catenin resulted in the rapid decay of p53 protein in comparison to control siRNA treated cells (D). These experiments indicated that γ-catenin also plays an important role in p53 protein stabilization.If the hypothesis that γ-catenin mediates p53 expression was correct, expression of a γ-catenin in H157 cells would be expected to induce p53 expression. Transient expression of γ-catenin in H157 cells indeed resulted in an increased expression of p53 B. Regulaal level C. We alstability D. For thtibodies D. Intereed cells D. These E). For these experiments, we made use of a luciferase reporter vector with luciferase under the control of 14 repeats of p53-binding sequence . Consistent with the stabilization of p53 (B-D), expression of γ-catenin in H157 induced a 30-fold increase in p53 activity in comparison to control vector transfected cells (E). While expression of γ-catenin in a p53 null cell line, H1299, had no effect on p53 activity (E). Taken together, these data suggest that γ-catenin is a novel regulator of p53 tumor suppressor protein via transcriptional and post-translational mechanisms.We next asked if γ-catenin expression could also induce p53 activation E. For thn of p53 , B-D, exed cells E. While activity E. Taken Supplemental Table S1). When we use IPA to connect these genes based on previously known biological associations, a very robust signaling network is produced , with the tumor suppressors HAI-1/SPINT-1 and Stratifin (SFN) central to these connections (A).To investigate the mechanism by which γ-catenin exerts its anti-proliferative and anti-migratory effects, we performed comprehensive pathway analysis and built a gene interaction network based on publicly available gene expression datasets containing an extensive number of lung tumor cell lines. We identified 152 common transcripts, out of 324 and 252 transcripts independently correlated to γ-catenin  in two different datasets. There was complete concordance in the direction of correlation between the two datasets, with the tumor suppressor protein, Serine Protease Inhibitor, Kunitz Type 1 (HAI-1 or SPINT1), being the top correlated gene in both datasets (nections A.B). Expression of γ-catenin in H1299 cells, on the contrary, failed to induce HAI-1 mRNA expression (C). Although γ-catenin failed to induce HAI-1 mRNA expression in H1299 cells, expression of wild-type p53 cDNAs in H1299 stimulated robust HAI-1 mRNA expression (C), suggesting that γ-catenin-induced HAI-1 expression is p53-dependent. Consistent with the effects of γ-catenin expression on HAI-1 mRNA expression, γ-catenin expression also induced HAI-1 protein expression in a p53-dependent fashion (D and E). In support of our hypothesis, γ-catenin/p53 regulation of HAI-1 expression was also observed in another NSCLC cell line i.e. A549 (F). Moreover, γ-catenin-induced HAI-1 expression in H157 cells was also attenuated in the presence of p53 siRNAs (G). Of note, in silico analysis of HAI-1 promoter for potential transcription factor binding sites also revealed p53 binding sites. Taken together, these data suggest that γ-catenin is a novel regulator of HAI-1 through a p53-dependent mechanism.Having identified this association, we evaluated if γ-catenin could regulate the expression of HAI-1. Interestingly, the expression of γ-catenin in H157 cells induced strong expression of HAI-1 transcripts, as revealed by quantitative PCR qPCR, B. Exprespression C. Althoupression C, sugges fashion , D and E.e. A549 F. Moreov3 siRNAs G. Of notH), suggesting that the effects of γ-catenin were indeed mediated via the induction of HAI-1.To test if the anti-proliferative effects of γ-catenin were mediated via the induction of HAI-1, we have co-transfected H157 cells with γ-catenin and HAI-1 specific siRNAs. Interestingly, γ-catenin-induced anti-proliferative effects were abolished in the presence of HAI-1 siRNAs H, suggesA and B). The expression of HAI-1 resulted in reduced cell proliferation rates in H157 cells as determined by an MTS (C) and clonogenic cell proliferation assays (E). Interestingly, expression of HAI-1 in H1299 cells also affected cell proliferation as determined by an MTS (D) and clonogenic assays (F), further supporting our earlier assertion that HAI-1 is operating downstream of p53. Furthermore, HAI-1 expression also resulted in reduced cell migration as determined by a scratch assay in both H157 and H1299 cells (G and H). Interestingly, expression of WT p53 in H1299 was also able to restore the ability of γ-catenin to inhibit cell migration (I). Furthermore, WT p53-induced effects on H1299 cell migration was also HAI-1-dependent (J). Taken together, these data suggest novel anti-proliferative and anti-migratory roles for HAI-1 in NSCLC.HAI-1 was shown to inhibit cell proliferation, migration, and cellular invasion in uterine leiomyosarcoma cells and intestinal tumorigenesis , 26. Howy an MTS C and clon assays E. Interey an MTS D and cloc assays F, furthe99 cells , G and Higration I. Furtheependent J. Taken A). Stable expression of γ-catenin in H157 also induced p53 expression  and p53 activation . Furthermore, stable expression of γ-catenin did not effect cell viability since there was no difference in Annexin V/propidium iodide staining between control cells and H157 cells stably expressing γ-catenin . As shown earlier (H), stable expression of γ-catenin resulted in reduced cell proliferation in H157 cells . Interestingly, treatment of H157 cells stably expressing γ-catenin with the c-MET inhibitor, Tivantinib, further reduced γ-catenin-mediated growth inhibition .HAI-1 is known to bind HGFA and repress HGF, the ligand for c-MET receptor signaling –14. In t–Fig. 6 earlier H, stableE) and reduced cell proliferation (F), which concur with our earlier findings (F). We next explored if the treatment of H157 cells with 5-Aza would further sensitize H157 cells to Tivantinib-mediated growth inhibition (G). For these experiments, we treated H157 cells with the vehicle (DMSO), 5-Aza, Tivantinib, or with a combination of 5-Aza and Tivantinib (G). Notably, treatment of H157 cells with 5-Aza and Tivantinib showed a significant reduction in growth when compared with H157 cells treated with either 5-Aza or Tivantinib alone. In total, these data suggest that the induction of γ-catenin expression, via 5-Aza treatment, might represent a novel strategy to treat NSCLC in combination with c-MET inhibitors.Interestingly, treatment of H157 cells with 5-Aza-2′-deoxycytidine , a DNA methyl transferase inhibitor, induced γ-catenin expression E and redferation F, which findings . Furtherfindings F. We nexhibition G. For thvantinib G. NotablPlakoglobin (γ-catenin) has a well-defined role as a structural protein , where iSince the tumor suppressive activities of γ-catenin were apparent , we explG).The role of γ-catenin in signal transduction is not fully understood. It has been previously shown that γ-catenin expression in NSCLC cells could reduce the activity of the Lymphoid enhancer factor (LEF)/T-cell factor (TCF)-dependent gene transcription , 19, 22.Fig. 6Recent work has also shown that γ-catenin could interact with p53 and induce the gene expression of 14-3-3 (SFN), a tumor suppressor protein in human tongue squamous cell carcinoma SCC9 cells . In thisThe current study also identified HAI-1 as a novel downstream effector of γ-catenin , most imA). 2) γ-catenin-mediated HAI-1 regulation also offers a promising novel strategy to inhibit the c-MET signaling pathway (B). In addition, the expression levels of γ-catenin and HAI-1 may also serve as novel biomarkers for predicting responses to 5-Aza and/or c-MET inhibitors. Although high doses of 5-Aza showed promise in patients with metastatic lung cancer, low dose 5-Aza was not very effective  given the regulation of γ-catenin expression via promoter methylation, restoration of γ-catenin expression through the treatment of 5-Aza might be a potential therapeutic option in the treatment of NSCLC. Gauging the expression levels of γ-catenin and p53 mutation status, it is possible to define the sensitivity of NSCLC to 5-Aza treatment A. 2) γ-c pathway B. In addffective , 37. How


IkkβΔCS mice in which the Ikkβ gene was specifically deleted in the corneal stromal keratocytes. The IkkβΔCS corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the IkkβF/F corneas that restored transparency in 2 weeks after injury, over 50% of the IkkβΔCS corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence β-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the IkkβΔCS corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKβ in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing.IkB kinase β (IKKβ) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKβ in the cornea using  IκB kinase β (IKKβ) is a key catalytic subunit of the IKK complex . It playIkkβ gene is deleted in specific cell types, it has become evident that IKKβ has diverse roles in the regulation of homeostasis, stress responses, survival and apoptosis [With the advent of genetic mutant mice in which the poptosis . Studiespoptosis ,6, to inpoptosis , to reprpoptosis , and to poptosis . The phypoptosis . Similarpoptosis .The cornea consists of five distinct layers: a stratified non-keratinized epithelial cell layer, the Bowman’s membrane, a highly organized collagenous stroma layer interspersed with keratocytes, the Descemet’s membrane and a single endothelial cell layer . PreviouHere we have applied the similar approach to characterize the roles of IKKβ in keratocytes, the residential cells of the corneal stroma. We show that the keratocyte IKKβ is also dispensable for corneal development, but is required for wound healing. In response to mild alkaline burn injury , IKKβ-deIkkβF/F mice were a gift from Dr. Michael Karin at the University of California at San Diego and the Kera-Cre mice were described before [The d before , The micThe following antibodies were used in the study: anti-p-SMAD 2 , anti-SMAD 3  and anti-p-Jun  were from Cell Signaling, anti-α-SMA from Abcam, anti-β-actin from Sigma-Aldrich, anti-CD45 and anti-CD11b from Invitrogen, anti-p-JNK  from Promega, and anti-HO-1 from StressGen BiotechnologiesAlkali burn corneal injuries were done following protocols described before with minor modifications . BrieflyCorneal opacity was evaluated by stereoscopic microscopy and slit lamp. The opacity was scored as: 0, completely clear cornea, +1, slight opacity, +2, mild opacity with iris and lens visible, +3, severe opacity with iris and lens invisible, but opacity was limited to the cauterized area, +4, extensive opacity throughout the entire cornea. Cryosections of the eye tissues were subjected to H&E staining following standard procedures. Immunohistochemical analysis was done as described previously . TUNEL wt-test or ANOVA. * p< 0.05, ** p< 0.01 and *** p< 0.001 were considered statistically significant.The data were analyzed by either two-tailed student Kera-Cre transgenic mice carry Cre recombinase gene controlled by the Keratocan promoter [IkkβF/F and Kera-Cre mice and identified the IkkβF/F and Kera-Cre genes in the offspring by genotyping of tail genomic DNA , respectively. Hence, IKKβ expression in keratocytes is dispensable for corneal development and maintenance.To assess if the keratocyte IKKβ might be required for corneal development, we used stereoscopic examination of the eyes of kβF mice . IkkβF astaining . NeitherIkkβF and IkkβΔCS corneas and examined healing stereoscopically. Both IkkβF and IkkβΔCS eyes displayed haze at 1 day post-injury and gradual resolution of the haze 4–7 days after injury  had recurrent haze and became cloudy. The opacity score was high at day 0–1 and reduced at 4–14 days after injury in all of the eyes examined. It remained low in IkkβF, but became significantly higher in IkkβΔCS corneas at 4 weeks after injury , the cloudy IkkβΔCS corneas were swollen with increased stroma thickness  had a few detectable inflammatory cells; however, the opaque IkkβΔCS corneas (eye 1) were filled with macrophages and neutrophils (IkkβΔCS corneas were transparent (eye 2) at 28 days, they were similar to the IkkβF eyes with little, if any, detectable inflammatory cells. The number of inflammatory cells in IkkβΔCS corneas was more abundant than that in IkkβF corneas , a marker of myofibroblasts. There was indeed abundant α-SMA expression in the opaque IkkβΔCS, but not in the IkkβF and transparent IkkβΔCS corneas at 28 days after injury . Studies in fibroblasts have shown that loss of IKKβ leads to oxidative stress, which induces c-Jun binding and activation of the Tgfβ promoter and gene expression; TGFβ in turn potentiates myofibroblast transformation and senescence . It is possible that oxidative stress also serves as a molecular link between IKKβ and TGFβ signaling in the IkkβΔCS corneas.The corneal keratocytes are quiescent in the absence of external insults, but enter cell cycle and become active under pathologic conditions ,36. In rntiation . InteresCorneal wound healing involves an early inflammatory phase followed by a late remodeling phase. In the early phase, tissue damage triggers neutrophil infiltration and macrophage invasion. These inflammatory cells produce cytokines, chemokines and molecules with microbicidal activity important for protecting the cornea from infection and environmental insults. Previous studies by Saika, et. al. have shown that activation of the IKK-NF-κB pathways in the neutrophil and macrophage makes the major contribution to the inflammatory responses in corneal wound healing . ConsistIkkβΔCS corneas.The mechanisms through which IKKβ regulates redox homeostasis and TGFβ signaling have been investigated in fibroblasts. In essence, IKKβ is required for optimal expression of redox scavengers, and IKKβ-null cells have decreased capacity to counteract oxidative stress elicited by environmental insults. When oxidative stress increases to a threshold level, it activates the JNK-c-Jun pathway, which induces TGFβ expression and activity; TGFβ in turn can act through NADH oxidase to further potentiate oxidative stress. Due to activation of the autocrine amplification of the ROS-TGFβ-NOX loop, IKKβ ablation in fibroblasts leads to a progressive increase of oxidative stress and TGFβ signaling, and a gradual myofibroblast transformation and premature senescence . It is possible that IKKβ also regulates redox homeostasis in keratocytes, where the activation of the ROS-TGFβ-NOX loop leads to the more severe wound healing defects observed in the S1 File(PDF)Click here for additional data file.


Endostar, a novel modified recombinant human endostatin, is now widely studied for the treatment of diseases that are characterized or caused by pathological angiogenesis. However, its molecular mechanism remains unclear. In this study, we investigated the effects of Endostar on the Wnt/β-catenin signaling pathway, which has emerged as an important aspect of angiogenesis. We showed that Endostar significantly inhibited the proliferation, migration, invasion, and capillary-like tube formation of human umbilical vascular endothelial cells in a dose-dependent manner. Using a luciferase reporter assay, we also demonstrated that Endostar suppressed β-catenin-dependent T cell factor transcriptional activity in increasing doses. Moreover, we found that Endostar treatment also restricted the stabilized mutant β-catenin-mediated increase in transcriptional activity, suggesting that Endostar exerts its inhibitory influence on Wnt/β-catenin signaling by targeting β-catenin or its downstream molecules. Western blot and immunofluorescence results revealed that Endostar significantly decreased nuclear and total β-catenin levels. Finally, we discovered that Endostar down-regulated cyclin D1 and VEGF, two proteins that are known as the downstream targets of Wnt/β-catenin signaling and that also play important roles in angiogenesis. Our findings suggested that Endostar inhibits angiogenesis and that the downregulation of the Wnt/β-catenin signaling pathway may be involved in the inhibition of angiogenesis by Endostar. These results support the use of Endostar in further clinical applications. Angiogenesis, or the formation of new capillaries from preexisting blood vessels, is involved in the pathogenesis of many diseases such as cancer, atherosclerosis, and diabetic retinopathy Endostatin, a 20 kD C-terminal fragment of collagen XVIII, is one of the most effective anti-angiogenesis agents available. It has already been used to treat atherosclerosis and was also pushed into anti-cancer clinical trials Despite extensive studies of Endostar’s effects on these diseases, its molecular mechanism still remains ambiguous. Previous research showed that Endostar restricts angiogenesis by blocking VEGF-induced tyrosine phosphorylation of VEGFR-2 and inducing apoptosis through the activation of caspase-3 and decrease of Bcl-2 in human umbilical vein endothelial cells (HUVECs) Recent studies emphasize the important role of the Wnt/β-catenin signaling pathway in physiological and pathological angiogenesis In this study, we assessed the anti-angiogenic efficacy of Endostar in vitro and investigated its mechanism of action on the Wnt/β-catenin pathway in HUVECs.Endostar, expressed and purified in E.coli, was purchased from Simcere Pharmaceutical Research Co., Ltd. . Primary antibodies for β-catenin, cyclin D1, and β-actin were obtained from Cell Signaling Technology . Primary antibodies for VEGF and Histone H3 were obtained from Novus Biologicals  and Epitomics . pGL3-OT or pGL3-OF luciferase reporter gene plasmid and pcDNA3.1+ empty plasmid were kindly provided by Dr. Dai SD . The pcDNA3-S33Y-β-catenin plasmid and pRL-SV40 plasmid were obtained from Addgene (Plasmid 19286) and Promega , respectively.HUVECs were purchased from Sciencell  and were cultured in endothelial cell medium  which contained 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement (ECGS), 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C under a humidified 95%: 5% (v/v) mixture of air and CO2. The HUVECs were collected for use after 3–5 passages.4 cells/well for 12 hours, then incubated with different concentrations of Endostar  for 24 hours. HUVECs without Endostar treatment was used as a control. Cell viability was determined by the MTT method and calculated as % of control.As described previously, an MTT  assay was used to determine cell viability We used a wound healing assay to determine the chemotactic motility of HUVECs. The HUVECs were grown into full confluence in 6-well plates and starved with ECM containing 0.5% FBS for 6 hours to deactivate cell proliferation. After that, we wounded the HUVECs with pipette tips and washed them with PBS. We then incubated the cells in ECM with 5% FBS, 1% ECGS and different concentrations of Endostar  for 12 hours. We took images of the HUVECs and manually counted the ones that had migrated into the cell’s free space.4) in 100 µl ECM (0.5% FBS) and different concentrations of Endostar  for 12 hours. Noninvasive cells on the upper surface of the filter were then removed with cotton swabs. The Transwell inserts were fixed with cold 4% paraformaldehyde, stained with 0.1% crystal violet for 15 minutes, and washed with PBS for three times. Afterward, the filter was mounted onto glass slides and the invasive cells on the lower side of the filter were counted under 100× magnification from three random fields.We performed an invasion assay using the Transwell  with 6.5-mm diameter polycarbonate filters (8 µm pore size). Briefly, the Transwell was coated with 25 µg growth factor reduced Matrigel  and incubated for 2 hours at 37°C for gelling. The bottom chambers were filled with 500 µl ECM with 5% FBS, 1% ECGS to act as a chemoattractant 5/ml) were suspended in ECM  with different concentrations of Endostar , and 300 µl of the cell suspension was added to each well. After 16 hours, we photographed the tubular structure of endothelial cells with an inverted microscope  at 100× magnification. We then counted branch points in three random view-fields per well.We used a protocol from BD Biosciences to assess tube formation. Briefly, we coated pre-chilled 24-well plates with 289 µl growth factor reduced Matrigel and incubated them at 37°C for 45 minutes. Inactivated HUVECs . For our cotransfection experiments, we transfected cells with 200 ng of pcDNA3-S33Y-β-catenin plasmid or a control empty plasmid plus the reporter plasmid. After the cells were incubated for 12 hours, we added different concentrations of Endostar (50 and 100 µg/ml) into each well. After 24 hours, we used the Dual Luciferase Reporter Assay System (Promega USA) and a luminometer  to measure the luciferase activities. TCF/LEF-dependent transcriptional activity was normalized to Renilla luciferase activity from the control plasmid pRL-SV40.The TOPFlash/FOPFlash luciferase assay is widely used to evaluate β-catenin/TCF dependent signaling events that regulated by TCF/LEF family. TOPflash (pGL3-OT) construct driven by thymidine kinase minimal promoter contains three wild-type TCF binding sites upstream of a luciferase reporter gene. FOPflash (pGL3-OF) contains three mutated TCF binding sites and is a specificity control for TOPflash activity We further analyzed the effects of Endostar on Wnt/β-catenin signaling pathway. HUVECs were incubated in ECM  with different concentrations of Endostar  for 24 hours. The whole-cell extracts and the nuclear extracts were each prepared using RIPA buffer  and NE-PER nuclear and cytoplasmic extraction reagents  respectively. Proteins were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were incubated with primary antibodies anti-β-catenin, anti-VEGF, anti-cyclin D1, anti-β-actin and anti-Histone H3 followed by the addition of secondary antibody (Goat anti-rabbit or mouse IgG (H+L) antibody, KPL, USA). Protein bands were detected with an enhanced chemiluminescent substrate , scanned, and quantitated by a Bio-imaging analyzer (Bio-Rad).We analyzed the effects of Endostar on the cellular localization of β-catenin. HUVECs were grown overnight in confocal laser dishes and then treated with or without 20 mmol/l LiCl , a dose known to activate β-catenin All experiments were performed in duplicate and repeated at least three times. Quantitative data were presented as mean ± standard deviation (SD). Data comparison was based on analysis of variance (ANOVA), followed by a Bonferroni test to establish differences among groups (SPSS 17.0). Significant differences were accepted when p<0.05.An MTT assay showed that the proliferation of HUVECs in Endostar-treated groups  was suppressed by 25.63±2.85%, 45.92±2.50%, and 59.47±1.86%, respectively, after 24 hours . TherefoWe performed a wound healing assay to measure the effects of Endostar on HUVEC migration. As shown in Transwell assays were used to further assess Endostar’s effects on the chemotactic motility of HUVECs. Only a few invasive cells were observed in the 150 µg/mL Endostar-treated groups . TherefoEndothelial cell tube formation is an essential step in angiogenesis Angiogenesis is a very complex process that is mediated by many signaling routes, including the Wnt/β-catenin signaling pathway We then investigated the effects of Endostar on the expression of nuclear and total β-catenin and cyclin D1 (a downstream target gene) using a western blotting assay. The results in Pathological angiogenesis plays an important role in many diseases such as cancer, atherosclerosis, and diabetic retinopathy, and is a strategic target for future clinical therapy. Though Endostar, a novel recombinant human endostatin, is now widely studied for the treatment of these diseases, its molecular mechanism remains ambiguous. In the present report, we have shown that Endostar inhibits angiogenesis and that the downregulation of the Wnt/β-catenin signaling pathway may be involved in the inhibition of angiogenesis by Endostar.In this study, we first assessed the anti-angiogenic effects of Endostar in vitro. Our results showed that Endostar significantly inhibited multiple steps of angiogenesis, including endothelial cell proliferation, migration, invasion, and capillary structure formation in a dose-dependent manner, which is consistent with findings from previous studies Increasing evidence indicates that the Wnt/β-catenin signaling pathway plays an important role both in physiological and pathological angiogenesis Research that highlighted the emerging importance of the Wnt/β-catenin pathway in angiogenesis led us to assess the effects of Endostar on this pathway in HUVECs. In this study, we found that Endostar significantly inhibited TCF/LEF-dependent transcriptional activity in HUVECs in a dose-dependent manner. The phosphorylation of ser-33 is necessary for β-catenin degradation, and S33Y-β-catenin, a constitutively stabilized mutant β-catenin, is insensitive to the upstream antagonists of Wnt signaling VEGF is one of the most important regulators of angiogenesis In summary, our study shows that Endostar inhibits angiogenesis and that the downregulation of the Wnt/β-catenin signaling pathway may be involved in the inhibition of angiogenesis by Endostar. These results support the use of Endostar in further clinical applications.