Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions  These iKT cells . The convia β-catenin-dependent or -independent signaling pathways. In the absence of Wnt ligation, casein kinase-1 and glycogen synthase kinase-3β phosphorylate β-catenin within the β-catenin destruction complex, which also contains the scaffold proteins adenomatous polyposis coli and axis inhibition (Axin). Phosphorylated β-catenin is targeted for proteasomal degradation .The complex phenotypical changes in genetically targeted mouse models have thus far made it difficult to separate effects on NKT cell development from functional effects on NKT cells upon antigen encounter. Furthermore, whether Wnt ligands are directly involved in the governance of NKT cell functions  of APCs , 23, ledWls knockout mice (Wlsfl/fl LysM-Cre+) and their littermate controls (Wlsfl/fl LysM-Cre−) were obtained by backcrossing Wlsfl/fl mice  and the University of Melbourne Animal Ethics Committee (06089).C57BL/6 mice were bred in house or obtained from the Animal Resources Centre . Tcf/Lef TOPFlash reporter mice, which express luciferase as a reporter of Tcf/Lef transcriptional activity , were br/fl mice  with C57l-histidine/0.5% Tween 20 in PBS). ICG001  and IWP2 , or an equivalent volume of DMSO (Sigma-Aldrich) as a vehicle control, were diluted in PBS and injected intraperitoneally 16 h prior to α-GalCer injection.Male and female mice aged 8–14 weeks were housed in microisolator cages (Technoplast) with corncob bedding, nesting material, and houses. Mice were injected intravenously, during the light cycle, with 2 µg α-GalCer , or an equivalent volume of vehicle control (5.6% sucrose/0.75% g. Leukocytes and red blood cells (RBCs) were harvested, and RBCs lysed in lysis buffer . Intracellular cytokine staining was performed on leukocytes using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Fc receptor binding was blocked with anti-mouse CD16/32  before addition of antibodies against mouse CD3 , NK1.1 , Fzd1 , Fzd7 , TCRβ , CD11b , Ly-6G , Ly-6C , IFN-γ , and IL-4 . Streptavidin-PE-Cy7 (BD Pharmingen) was used for detection of biotinylated antibodies. α-GalCer-loaded CD1d tetramer (PE) was generated in-house using a baculovirus expression system, similar to that previously described , and centrifuged at room temperature for 15 min at 760 × escribed . Samplesl-glutamine, 1 mM sodium pyruvate, 10 mM HEPES , and 20% L cell-conditioned medium. Fresh medium  was added every second day. After 6 days, adherent cells were harvested by washing with warm PBS, followed by incubation with cold 0.1 M EDTA in PBS for 10 min at 4°C. Cells were washed with cold PBS, centrifuged, and resuspended in TRIzol (Life Technologies).Bone marrow cells from femurs and tibias of male and female mice aged 8–14 weeks were cultured in DMEM (Life Technologies) containing 10% FBS , 2 mM Hprt) [2] and is depicted as relative gene expression. Gene-specific primers were designed using the Integrated DNA Technologies online primer design tool or were previously published . Quantitative real-time PCR was performed with SYBR Green PCR master mix (Life Technologies) using an ABI Prism 7900HT (PerkinElmer). Expression of genes of interest was normalized to the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (2SO4 (Sigma-Aldrich).Mouse IFN-γ (R&D Systems) and IL-12p40 (BD Biosciences) ELISA kits were used according to the manufacturer’s protocols to determine cytokine concentrations in mouse serum. Serum concentrations of IL-4 were determined using a sandwich ELISA. Maxisorp plates (Nunc) were coated with anti-mouse IL-4 capture antibody  in 50 mM sodium bicarbonate buffer (pH 9.4) overnight. Plates were washed with 0.05% Tween 20 (Sigma-Aldrich) in PBS, blocked with 4% bovine serum albumin  in PBS for 2 h, and then samples and standards , serially diluted two-fold from 2,000 pg/mL, were added for 1 h. Anti-mouse IL-4 detection antibody  diluted in 4% BSA in PBS was added for 1 h, followed by HRP-conjugated streptavidin  for 30 min. The plates were developed with TMB substrate (BD Biosciences), and the reaction stopped with 1 M H+ TCRβ+ NK1.1+ cells and further divided on the basis of CD4 expression. The purity of CD4− and CD4+ NKT cell subsets analyzed by flow cytometry post-sort was >96%. Sorted NKT cell subsets (four samples/group) were stimulated with Dynabeads Mouse CD3/CD28 T Cell Expander Beads (Invitrogen) at a 1:1 ratio of beads to cells for 2 h at 37°C. RNA was extracted from stimulated NKT cell subsets (Qiagen RNeasy and Qiashredder), RNA yield quantified spectrophotometrically on a Nanodrop ND-1000 and aliquots electrophoresed for determination of sample concentration and purity. Microarray hybridizations were performed using 100 ng RNA and the WT Expression kit (Life Technologies), WT Terminal Labeling and Controls Kit (Affymetrix), and Affymetrix Mouse Gene_1.0ST arrays . The probed arrays were washed and stained using the GeneChip Hybridization Wash and Stain Kit (Affymetrix), and scanned using the GeneChip Scanner 3000. Images (.dat files) were processed using GeneChip Command Console (Affymetrix) and CEL files imported into Partek Genomics Suite 6.6 (Partek SG) for further analysis as published previously . P values <0.05 were considered statistically significant.One- or two-way ANOVA with Dunnett’s or Bonferroni’s correction for multiple comparisons, or unpaired two-sided Student’s +NK1.1+ NKT cells in α-GalCer-challenged mice, we established an intracellular flow cytometry protocol for CD3 and NK1.1 to track NKT cells in vivo early after α-GalCer challenge . This allows for better separation of the CD3+NK1.1+ population from other cell populations, particularly after challenge. At 1.5 h after challenge, a greater proportion of CD3+NK1.1+ was detected using the intracellular staining protocol, compared to surface only staining. However, similar percentages of CD3+NK1.1+ cells were detected at 6 h using either staining protocol, indicating that these cells have either degraded CD3 and NK1.1, or are no longer present in liver tissue.α-Galactosylceramide rapidly induces IFN-γ and IL-4 expression by NKT cells , accompae Figure , CD3, NK+NK1.1+ cells after systemic administration of α-GalCer. A rapid increase of IFN-γ+CD3+NK1.1+ cells with 57.8 ± 4.6% cells expressing IFN-γ was observed at 1.5 h and 51.6 ± 4.6% at 3 h post challenge   in the liver in DMSO- and ICG001-treated mice, after both control and α-GalCer challenge  and recruited inflammatory (Ly6Chi) monocyte populations. In contrast to the early effects on IFN-γ responses, liver Il4 mRNA expression was reduced in ICG001-treated mice compared to DMSO controls at 4.5 h  and CD3−NK1.1+ (NK) cells in the liver .e Figure A. This ps Figure A. As thith IWP-2   Wnt/β-catenin signaling in the liver environment regulates IFN-γ responses; (ii) active suppression of hepatic Wnt/β-catenin signaling shortly after α-GalCer exposure biases cytokine responses toward IFN-γ production; (iii) β-catenin activity independent of APC-derived Wnt ligands contributes to IL-4 expression by NKT cells; (iv) Wnt ligands that act via β-catenin-independent signaling contribute to the perpetuation of IFN-γ responses within several hours post challenge; and (v) myeloid-derived Wnt ligands are, in part, responsible for these regulatory effects on NKT cells.Studies on contributions of β-catenin activity and Wnt ligands to NKT cell functions in vivo model of α-GalCer challenge. It is important to note that β-catenin activation is not exclusively driven by Wnt ligands and can, for example, be induced by microbial ligands and hepatic growth factor . Whereas this dual-functionality of IL-4+ NKT cells gradually declined (54.3 ± 10.9% at 6 h) neither Porcupine nor β-catenin inhibition affected the percentage of IFN-γ+ cells in the IL-4+ CD3+ NK1.1+ population (data not shown). Thus, functionally distinct NKT cell subsets may be selectively responsive to Wnt-mediated regulation of IFN-γ.Our findings and previous reports emphasize the plethora of Wnt ligands and components of the Wnt signaling machinery that are expressed in liver tissue and are dynamically regulated in response to pathological challenges , 35, 47.Wnt3a, Wnt5a, Wnt7a, Wnt7b, and Wnt10a) , or no significant changes in their expression (Wnt5a) during the first 6 h following a single α-GalCer injection. This indicates that different subsets of Wnt ligands in the liver microenvironment, in concert with other cytokines and growth factors, may shape NKT cell functions at different stages of activation when compared to anergy. While the specific functional contributions of individual Wnt ligands remain to be established, it is tempting to speculate that this may be reflective of the interaction of NKT cells with different cellular sources of Wnt proteins. There is indeed precedence for differential roles of Wnt proteins in the liver environment depending on the cellular source. Whereas macrophage-derived Wnt proteins promote liver regeneration after partial hepatectomy, Wnt proteins expressed by epithelial cells did not contribute to this process  , displayTaken together, the data presented in this study implicate Wnt proteins as regulators of NKT cell functionality in the liver environment Figure . SeveralAll procedures involving animals adhered to the guidelines of the National Health and Medical Research Council Australian Code for the Care and Use of Animals for Scientific Purposes, and were approved by the Animal Ethics Committee of The University of Queensland  and the University of Melbourne Animal Ethics Committee (06089).JK, MJ, LP, JM, TT-T, LT, TN, and DM performed experiments. JK, MJ, LP, DM, SB, AGB, DG, and AB designed experiments. JK and AB analyzed the data. RV, RS, and KK provided reagents and protocols. JK and AB wrote the manuscript, and all authors provided editorial input.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.


Sox9, become differentially expressed. Many of these differentially expressed genes are known targets of the Polycomb Repressive Complex 2 (PRC2). Thus, we hypothesized that PRC2 is required for Wnt/β-catenin-mediated repression of chondrogenesis in the embryonic CM. We find that β-catenin can physically interact with PRC2 components in the CM in vivo. However, upon genetic deletion of Enhancer of Zeste homolog 2 (EZH2), the catalytic component of PRC2, chondrogenesis remains repressed and the bone and dermis cell fate is preserved in the CM. Furthermore, loss of β-catenin does not alter either the H3K27me3 enrichment levels genome-wide or on cartilage differentiation determinants, including Sox9. Our results indicate that EZH2 is not required to repress chondrogenesis in the CM downstream of Wnt/β-catenin signaling.A hallmark of craniofacial development is the differentiation of multiple cell lineages in close proximity to one another. The mouse skull bones and overlying dermis are derived from the cranial mesenchyme (CM). Cell fate selection of the embryonic cranial bone and dermis in the CM requires Wnt/β-catenin signaling, and loss of β-catenin leads to an ectopic chondrogenic cell fate switch. The mechanism by which Wnt/β-catenin activity suppresses the cartilage fate is unclear. Upon conditional deletion of β-catenin in the CM, several key determinants of the cartilage differentiation program, including  Embryonic craniofacial development involves the formation of complex structures from two progenitor stem cell pools: the cranial neural crest (CNC) and cephalic paraxial mesoderm (PM)  to chick limb bud micromass cultures  (Rosa26 Reporter (R26R) (-catenin null (β-catenin∆) (-catenin floxed (βfl-catenin) (Twist2Cre(Dermo1Cre) (Ezh2 floxed (flEzh2)  0.5. For each experiment, a minimum of three mutants with litter-matched controls were studied unless otherwise noted. Animals of both sexes were randomly assigned to all the studies. The Case Western Reserve University (CWRU) Institutional Animal Care and Use Committee approved all animal procedures in accordance with AVMA guidelines .The following strains were used in this study: r (R26R) , β-catenEn1Cre-positive, and also contained the overlying ectoderm, which is negative for En1Cre (CM+ectoderm). Each embryo yielded ∼500,000 CM cells for the controls and 250,000–500,000 CM cells for the mutants. Individual embryos were kept separate and considered as single biological replicates. The wild-type samples isolated for co-immunoprecipitation were dissociated by incubating the tissue in 0.25% Trypsin-EDTA (Thermo Fisher Scientific 25200056) at 37° for 5–7 min, and the CM was selectively enriched from the ectoderm using an Invitrogen FlowComp Flexi Kit (Invitrogen 11060D) and a PDGFRα antibody (5–10 µg/2.5 million cells) (R&D Systems AF1062) , Suz12 (Mm01304152_m1), Eed (Mm00469660_m1), Sox9 (Mm00448840_m1), Axin2 (Mm00443610_m1) Twist2 (Mm00492147_m1), Keratin14 (mm00516876_m1), and Pdgfrα (Mm00440701_m1). CT values obtained for specific genes were normalized to those of β-actin (Invitrogen 4352663).The CM+ectoderm was manually dissected from E13.5 embryos (described above). RNA was isolated as previously described  and disrupted with a 23-gauge needle and syringe. The precipitating antibody was added to the lysate and incubated overnight at 4°. Immunoprecipitation was performed by incubating 100 µl of Dynabeads Protein G beads (Invitrogen 10003D) with the lysate and antibody for 1 hr at 4°. Beads were then washed four times with 1 ml wash buffer . The sample was collected in NuPage LDS Sample Buffer (Thermo Fisher Scientific NP0007) supplemented with β-mercaptoethanol, and was heated at 90° for 5 min. Rabbit polyclonal antibodies against nonphospho-β-catenin, 3.43 µg ; EZH2, 10 µg (Diagenode C15410039); and IgG, 6 µg (Abcam ab46540) were used for immunoprecipitation. Protein species were separated by SDS-PAGE using Mini-PROTEAN TGC gels (Bio-Rad #456-1084). Western blots were performed with primary antibodies against β-catenin  or EZH2 . Clean-Blot IP Detection Reagent (HRP)  was used as secondary antibody.E13.5 CM was collected by manual dissection and the CM was enriched from the ectoderm (described above). At least 2 million cells from multiple embryos were incubated with 1 ml lysis buffer . Protein species were separated by SDS-PAGE using Mini-PROTEAN TGC gels (Bio-Rad #456-1084). Western blots were performed with polyclonal rabbit primary antibodies against H3K27me3 , EZH2 , and SUZ12 . Species-specific HRP-conjugated secondary antibodies were used at a 1:10,000 dilution. Immunoblots were probed with a rabbit anti-β-tubulin antibody  as a loading control. Signals were detected using an Amersham ECL Western Blotting Analysis System , and imaged using an Odyssey FC Imaging System (Li-Cor). Relative protein levels were quantitated using ImageJ/Fiji software . Total RNA was isolated from individual embryos as previously described  . Next, 14 µg chromatin was immunoprecipitated with 4 µg rabbit anti-H3K27me3 (Millipore #07-449). Sequencing was performed on an Illumina NextSequation 500 producing 75-nucleotide, single-end reads. Drosophila DNA was “spiked in.” The ratio of aligned Drosophila reads in the mutant vs. control samples  was used to normalize the number of reads in the mouse samples by downsampling the larger sample . Sequences were aligned and analyzed twice independently. The analysis was first performed using a custom pipeline consisting of Bowtie2 for genome alignment to the mouse mm9 genome and Macs 1.4 at default settings for peak calling  at 37° for 5–7 min, and then plated in DMEM with 10% fetal bovine serum. Fibroblasts were allowed to adhere to the plate for 1–2 hr, after which the media was removed and fresh media was added. Chemical inhibition was performed at no later than passage 3. Next, 10% Wnt3a-conditioned media and the chemical inhibitor, UNC1999 (Sigma SML0778) or GSK126 , were added simultaneously. The cells were incubated for the indicated amount of time. Following incubation, the cells were trypsinized and processed for protein or mRNA analysis.P-values for statistical tests in all figures are represented as * P < 0.05 and ** P < 0.01.Graphs and statistical analyses were generated using Prism 6 (GraphPad Software). Data are presented as mean ± SEM in all graphs unless otherwise stated. All pairwise sample comparisons were performed using a Mann–Whitney test. The Strains are publicly available at the Jackson Laboratory. Sequencing data are available at GEO with the accession number GSE96872.in vivo, we conditionally deleted β-catenin in the CM using Engrailed1Cre (En1Cre), manually dissected the CM along with the ectoderm (CM+ectoderm), and collected all the CNC- and PM-derived mesenchyme surrounding the brain . The analysis of the data revealed 521 genes that were differentially expressed by at least 1.4-fold in the two experimental groups (P < 0.05). Of the 521 differentially expressed genes, 322 were downregulated and 199 were upregulated in the mutants relative to the controls. Validating the approach, changes in expression of known Wnt/β-catenin targets were observed despite the presence of ectodermal cells, in which canonical Wnt signaling is known to be active  (File S1) (File S1) (File S1). Thus, the differential expression of PRC2 targets in the β-catenin mutant CM+ectoderm and dorsal mesenchyme reveals a potential functional link between the two pathways.In an effort to determine a functional link between β-catenin and PRC2 he brain  . This assay allowed us to unbiasedly and comprehensively map the genome-wide distribution of the H3K27me3 modification (Active Motif Technology) (GSE96872). In the CM+ectoderm of E13.5 controls, the transcriptional start sites of multiple cartilage markers, such as Sox9, Col2a1, Col9a2, and Col11a2 , we set out to test the hypothesis that β-catenin and PRC2 components physically interact at native protein levels in the mouse CM extracts. We manually dissected the CM, made a cell suspension, and used a PDGFRα antibody bound to magnetic beads to enrich for the CM population  in the purified sample, confirming enrichment for CM . We then conditionally deleted Ezh2 in the CM using Dermo1Cre, which is expressed in the CM by E10.0   . Consistix (OSX) . Based oin vitro . Sequencing of the CM+ectoderm revealed, by two independent analyses, 14,337 peaks in the control and 10,752 peaks in the mutant, thus 25% fewer peaks in the mutant. Surprisingly, genome-wide comparisons between individual mutant and control H3K27me3 peaks revealed modest differences in enrichment fold between the two samples . Representative enrichment for strong, medium, and weak peaks can be found on the HoxA cluster, Sept9, and Lmtk3, respectively . The genomic location of each class of peak using GREAT revealed that the large majority of strong and medium peaks were within 5 kb of the transcription start site (TSS), and the weak peaks had a more even distribution spanning out 500 kb from the TSS . Between controls and β-catenin mutants, the number of strong and medium peaks was comparable, with most of the variation found in the weak peaks . To further characterize each class of peak, we performed gene ontology analysis on the control H3K27me3 ChIP-seq data set. Gene ontology analysis revealed distinct functions for the strong peaks such as DNA-binding/transcription regulation and conserved homeobox sites, and the medium and weak peaks shared functions such as Wnt signaling and ion transport . Furthermore, comparisons between each class of peak found near a TSS (±5 kb) and the genes identified in our RNA-seq data set revealed that 70% of strong peaks, 53% of medium peaks, and 47% of weak peaks were associated with transcriptional repression (<1 FPKM) . These results indicate that the level of H3K27me3 enrichment may be predictive of its transcriptional repressive function. When we intersected genes bound by each class of H3K27me3 peak and differentially expressed genes, we found that each class of peak had similar enrichment between the down- and upregulated genes, indicating that H3K27me3 enrichment does not predict transcriptional repression by β-catenin .From our ChIP-seq data set, we observed variations of the H3K27me3 enrichment throughout the genome ranging from large peaks blanketing an entire gene body to smaller peaks located just on the promoter. To further investigate the connection between H3K27me3 peak enrichment strength and gene expression, we divided the H3K27me3 peaks into three categories based on the level of enrichment: strong (>20-fold enrichment), medium (10–20-fold enrichment), and weak (≤ fivefold enrichment) . More importantly, H3K27me3 enrichment did not change on Sox9, Col2a1, Col9a2, or Col11a2 (File S1). Furthermore, H3K27me3 enrichment was similar between β-catenin controls and mutants on the TSS of critical bone and dermal marker genes, such as Runx2, Twist1, Twist2, Axin2, and Lef1 . Mcm6 serves as a negative control and lacks H3K27me3 enrichment in either the control or mutant . It is worth noting that Cdkn2a, HoxA, and T loci had strong H3K27me3 enrichment peaks, while Sox9 and Col2a1 loci had medium enrichment peaks in both controls and β-catenin mutants (File S1). Our results showed that H3K27me3 enrichment is not depleted from the TSS of chondrocyte differentiation determinants in β-catenin mutants and remains enriched in actively transcribed genes.To determine if the loss of β-catenin resulted in depletion of H3K27me3 on chondrocyte differentiation determinants, we examined the enrichment of H3K27me3 on utant CM  . These results suggest that the role of PRC2 in embryonic development may be cell type- and developmental stage-specific. Future studies in vivo are required to tease out the timing and dynamics of developmental gene regulation by PRC2.The lack of cell fate changes in the supraorbital CM of in vivo RNA-seq and ChIP-seq studies revealed legitimate H3K27me3 peaks in genes that did not correlate with transcription levels. We found that both expressed and repressed genes in control CM+ectoderm can be enriched for H3K27me3, demonstrating that H3K27me3 is not sufficient to indicate repression. The H3K27me3 marks remain at Sox9 and other cartilage marker genes in β-catenin mutants, suggesting that these marks may be carried over and reflective of a past transcriptional off state.There are recent data from several groups refining the role of PRC2 and H3K27me3 enrichment. According to the histone code hypothesis, H3K27me3 is often found on transcriptionally repressed genes and is widely considered to be a sign of transcriptional repression . In earlSox9 promoter (Sox9 and Col2a1 mRNA levels in primary CM+ectoderm cells cultured in vitro (data not shown). Further studies in vivo will be required to investigate this model. The second model postulates that β-catenin activates yet-to-be-identified signaling pathways or transcription factors that would be directly involved in repression. For example, Twist1 is positively regulated by Wnt/β-catenin signaling, and conditional deletion of Twist1 partially phenocopies the ectopic chondrogenesis found in the En1Cre/+;R26R/+;βfl/-cateninΔ mutants , or other histone modification-related mechanisms, such as G9a-associated K9me3 repression. A study in chick limb bud micromass cultures showed that the addition of exogenous Wnt3a led to an increase in DNA methylation by DNMT3a on the promoter . HoweverOverall, our data suggested a model whereby the repression of the chondrogenic fate by Wnt/β-catenin signaling does not rely on EZH2 and H3K27me3, but implies other yet-to-be-identified transcriptional or post-transcriptional mechanisms.www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300311/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.


This paper examines the effect of surface treatment and filler shape factor on the fracture toughness and elastic modulus of epoxy-based nanocomposite. Two forms of nanofillers, polydopamine-coated montmorillonite clay (D-clay) and polydopamine-coated carbon nanofibres (D-CNF) were investigated. It was found that Young’s modulus increases with increasing D-clay and D-CNF loading. However, the fracture toughness decreases with increased D-clay loading but increases with increased D-CNF loading. Explanations have been provided with the aid of fractographic analysis using electron microscope observations of the crack-filler interactions. Fractographic analysis suggests that although polydopamine provides a strong adhesion between the fillers and the matrix, leading to enhanced elastic stiffness, the enhancement prohibits energy release via secondary cracking, resulting in a decrease in fracture toughness. In contrast, 1D fibre is effective in increasing the energy dissipation during fracture through crack deflection, fibre debonding, fibre break, and pull-out. Epoxy has been used extensively for engineering applications due to its combined advantages of high stiffness, strength, creep resistance, and chemical resistance. However, its fracture resistance is relatively low because of the rigid network of cross-linked polymer chains. Significant effort has been made in the past to improve various properties of epoxy, such as changing the backbone chemistries of the epoxy and curing agent , modifyiTowards the end of the 20th century, the concept of nanocomposites started to emerge, offering the industry with promising results and a unique level of property improvement. This method involves the incorporation of nano-sized organic and inorganic nanoparticles to the epoxy matrix. Researchers from Toyota  increaseGenerally, surface treatment of nanofillers is required for better interaction with hydrophobic polymer matrices. Mussel adhesive proteins (MAPs) have received a great amount of attention for their capability to adhere effectively to different types of surfaces. MAP contains a large quality of a particular amino acid known as dopamine (DOPA) that is responsible for the strong adhesion of mussels on different types of marine surfaces . PodsiadDespite these efforts, there have been very limited studies on the fundamental root causes that contribute to the cracking process, i.e., the micro-mechanisms of a fracture in the nanocomposite, especially the filler and filler-matrix interaction. In this study, we investigated how DOPA coating treatment affects the fracture toughness and elastic modulus of epoxy-based nanocomposites. Two types of nanofillers were used: montmorillonite (MMT) clay as a 2D filler, and carbon nanofibre (CNF) as a 1D filler. Composites with and without DOPA coating on the fillers were tested and analysed. The comparison between the 1D and 2D fillers enables us to understand how the form factor affects the fracture toughness and fracture process.−1 is due to in-plane stretching of Si–O, and the out-of-plane stretching will appear as a tiny shoulder at 1115 cm−1. Additionally, bending vibrations of the hydroxyl groups of water generally occur at 1641 cm−1 in MMT (stretching will be at ~3430 cm−1). The relatively broad band between 3200 and 3500 cm−1 that appeared in D-Clay compared to MMT is a combination of stretching vibrations of –NH and –OH. The band at 1618 cm−1 in D-Clay assigned to C=C further confirms the presence of dopamine.The DOPA-coated clay (D-clay) particles were examined using X-ray photoelectron spectroscopy (XPS) to verify the presence of DOPA on the MMT surface. The results from XPS revealed that the sodium atoms were no longer detected and the signals for oxygen and silicon were largely reduced as compared to the untreated MMT a. At theThermogravimetric analysis (TGA) was used to determine the amount of DOPA polymerized on the surface of the MMT. From the TGA curve shown in The D-spacing of the samples were measured using X-ray diffraction (XRD) and results are shown in After the epoxy resin was cured with the amine hardener, the peak at 2θ = 3.8° weakened significantly. This result shows that during curing, intercalation of D-clay has taken place. This extent of intercalation could lead to great improvement in both mechanical and physical properties of the polymer clay composite ,25,26,27On the other hand, TEM images taken from the cured DOPA-coated carbon nanofibre (D-CNF) epoxy nanocomposites show that the D-CNF are individually and randomly dispersed throughout the epoxy matrix . PDOPA cAs shown by the dynamic mechanical analysis in As shown in Similarly, as shown in The contrast between DOPA-coated fillers and virgin fillers clearly indicates the importance of interfacial adhesion between the matrix and the fillers. The D-clay and D-CNF, having a strong adhesion with the matrix, are able to enable effectively transfer stress from the epoxy matrix to the filler (which has a higher stiffness). As a result, the overall elastic constant increases because the filler is able to undertake some of the stress. Untreated filler, on the other hand, does not have the strong interface with the matrix. Therefore, the stress is largely undertaken by the matrix only. The Young’s modulus decreases because of loss of volumetric percentage of the load bearing phase of the epoxy matrix.IcK and IcG values of the D-clay epoxy nanocomposites are lower than that of the neat epoxy. The addition of 2 wt % clay loading in the D-clay nanocomposites exhibits a toughness of 0.58 MPa∙m1/2 and 88.74 J/m2 for IcK and IcG, respectively. This represents a drop of 11.8% for IcK and 36.9% for IcG as compared to neat epoxy. However, the addition of 2 wt % untreated V-clay exhibits a toughness of 0.834 MPa∙m1/2 and 290.1 J/m2 for IcK and IcG, respectively. This is an increase of 23.1% for IcK and 104% for IcG as compared to neat epoxy.The fracture toughness obtained by the single-edge notched 3-point bending (SEN-3PB) test is shown in IcK and IcG values of both D-CNF and V-CNF epoxy nanocomposites are higher than that of the neat epoxy. The addition of 2 wt % clay in the D-CNF nanocomposite exhibits a toughness of 0.878 MPa∙m1/2 and 202.5 J/m2 for IcK and IcG, respectively. This is an increase of 33.0% for IcK and 43.0% for IcG as compared to neat epoxy. The addition of 2 wt % V-CNF exhibits a toughness of 0.836 MPa∙m1/2 and 280.4 J/m2 for IcK and IcG, respectively. This represents an increase of 26.7% for IcK and 98.0% for IcG as compared to neat epoxy.With CNF, A comparison of neat epoxy, V-clay, and D-clay epoxy composites fracture surfaces are shown in TEM images were taken at sub-critical points of the crack initiation site of D-clay epoxy nanocomposites a. SeveraIn contrast, in V-clay epoxy composites as the degree of intercalation is poor c and witIn summary, the fracture toughness of the D-clay epoxy nanocomposites is lower than that of the pure epoxy and V-clay epoxy composites. Although the DOPA modification on the clay layers provides very strong adhesion between the clay layers and epoxy matrix, it forces the microcracks to initiate within the clay layers gallery. These weakly bonded clay layers provide an easy path for the fracture to break through. The unmodified V-clay on the other hand, makes it easy to induce secondary cracking. This results in the fracture path to go through a much tortuous path and, thus, increases the fracture toughness of V-clay epoxy composites. We wish to emphasize that the current findings are limited to 2D clay fillers that have the intrinsically weak bonding between its adjacent layers. Such findings are not applicable if a 2D monolayer is used.IcG) is higher for the V-CNF composite than the D-CNF composite. Crack-tip stress relief and possible formation of secondary cracking could be the main reason for such an enhancement as they are favoured by weak filler-matrix interface as explained in the V-clay case.D-CNF and V-CNF epoxy composites have quite similar morphologies on the fractured surface . The V-CThe sub-critical point of the crack initiation site of D-CNF nanocomposites is shown in The clay used for this experiment was pristine montmorillonite (MMT) clay from Nanocor Inc., , with a cationic exchange capacity of 145 mmol/100 g. MMT has a layered monoclinic crystal structure with a large space between the layers along the c-axis. The mean particle size of NaMMT was calculated to be ~200 μm using the sieve mesh method. The carbon nanofibre (CNF) used was Pyrograf-III  with a diameter in the range of 70–150 nm and a length of 50–200 μm, obtained from Applied Science Inc. . Tris(hydroxymethyl)aminomethane , dopamaine hydrochloride, 3,4-Di-hydroxyphenethylamine hydrochloride  and acetone (Tech grade) were obtained from Sigma-Aldrich (Singapore). Epoxy precursor DER 332 was supplied by Dow Chemicals (Singapore) and amine hardener Ethacure-100LC was supplied by Albemarle Corp . All chemicals were used as received.One gram of MMT clay was added into 100 mL of deionized water and mechanically stirred for 24 h. This was followed by resting it under ambient condition for another 24 h. After that, the settlement at the bottom of the beaker was removed. The remaining suspension was added into 250 mL 10 nM TRIS buffer solution and mechanically stirred for 15 min to achieve a homogenous clay dispersion in the new solution. Different amounts of DOPA, corresponding to 0.1 to 2.0 mg/mL solution concentration, was then added and continuously stirred for 2 h at ambient condition. The thermal stability was determined by the starting temperature of degradation using TGA. It was found that 1.5 mg/mL was able to provide the most stable DOPA coating and sufficient coating thickness, and the fillers were well dispersed. Therefore, this concentration was used for all the experiments unless otherwise stated. The suspension was then centrifuged at 7500 rpm for 20 min. This step of wash-centrifuge was repeated for four times to remove the unattached DOPA oligomers. A dark slurry-like solution was obtained after the washing. Finally, D-clay was produced by drying the solution in a vacuum oven for 24 h at 70 °C. In the same way, D-CNF slurry was prepared.−1 to 4000 cm−1 with a resolution of 4 cm−1. Crystal structure was determined using X-ray diffraction (XRD) on a PANalytical X’Pert PRO X-ray diffractometer  with Cu K radiation (λ = 0.154 nm). D-clay was pounded and grinded into powder form before the XRD measurement. Samples were scanned with a scanning rate of 2°/min from 2° to 35° in ambience. The average D-spacing was then calculated using the Bragg’s law based on the measured diffraction angle.X-ray photoelectron spectroscopy (XPS) measurement was carried out using a Kratos Analytical AXIS HIS spectrometer  with a monochromatized Al Ka X-ray source (1486.6 eV photons) to verify the polydopamine coating on the clay. Thermogravimetric analysis (TGA) was carried out using a TGA Q2950  under an atmosphere environment where the samples were heated from 25 °C to 140 °C and kept isothermally for 40 min before being further heated at a rate of 10 °C/min to 850 °C. Fourier transform infrared spectroscopy (FTIR) was carried out using a Perkin-Elmer 2000 spectrometer  scanning in the range of 400 cmw:w). The mixture was then stirred using a mechanical stirrer for 30 min to achieve a homogenous mixture of the epoxy and amine hardener. The mixture was then degassed at 50 °C and cured using a curing profile of 100 °C for 2 h, then 180 °C for 5 h.To prepare the D-clay and D-CNF epoxy nanocomposites, precursor DER 332 was introduced into the slurry and the solution was vigorously stirred for 2 h using a homogeniser, after which acetone was removed using a rotary evaporator and the remaining slurry mixture was dried in a vacuum oven at 85 °C for 48 h. The amine hardener Ethacure-100LC was then added to the dried epoxy at the epoxy/amine hardener ratio of 3.8:1  in the single cantilever mode. The samples were moulded into a rectangular size of 30 mm × 10 mm × 3 mm and scanned from 25 °C to 250 °C at a heating rate of 3 °C/min using a frequency of 1 Hz. Young’s modulus of the nanocomposites was determined on an Instron 5567 Universal Tester  at a cross-head speed of 1 mm/min according to ASTM D-5083-10 test standard. The length and thickness of the tested portion is 50 mm × 3 mm, and the total length is 80 mm. The strain was recorded using a strain gauge at a gauge length of 25 mm. IcK). The dimensions of the specimens are 60 mm × 12.7 mm × 4.0 mm, and the span is 50 mm. A sharp notch was introduced to the specimen by tapping a hammer on a razor blade inserted in to the sample. At least five valid results were obtained for each condition. The critical strain energy release rate (IcG) was calculated from the IcK, assuming a Poisson’s ratio of 0.35.Fracture toughness was measured using the SEN-3PN test to obtain the Mode-I critical stress intensity factor (A scanning electron microscope (SEM) was used to examine the fracture surface of the tested SEN-3PB specimens. The samples were coated with a thin layer of gold and observed using a JEOL 6700 SEM . The accelerating voltage was 5 kV. To study the microstructure at the crack tip damage zone, the area in front of the crack tip was located and carefully trimmed down manually with a sharp razor blade to the size suitable for ultra-microtoming. Subsequently, the face of the sample was trimmed by a freshly-made glass knife in the Leica Ultracut Microtome. Finally, the sample was carefully sectioned to 50–70 nm thick sections at a rate of 0.3 mm/s using a diamond knife in the same microtome machine at ambient temperature. The thin sections were then collected by a water-loop onto a carbon-coated copper grid. These sections were observed under a JOEL 2100 transmission electron microscope  at 200 kV in bright field mode.The effects of PDOPA-coated MMT clay, which has a weak bonding between adjacent layers, and carbon nanofibre as fillers on the mechanical properties of their epoxy nanocomposites are evaluated and compared with the unmodified counterparts. The storage modulus is enhanced at very low filler loadings. Young’s modulus of D-clay and D-CNF nanocomposites are enhanced while the unmodified counterparts display a reduction. The decrease in the Young’s modulus of the untreated filler composite proves the importance of filler-matrix adhesion in effective stress transfer when the elasticity constant is concerned. IcK and IcG values of the D-clay epoxy nanocomposites are lower than that of the neat epoxy and that of V-clay composites. Through fractographic analysis and crack tip clay-epoxy interaction analysis, we were able to conclude that the strong adhesion of the clay surface with matrix forces the clay layers to delaminate under crack tip stresses. Since the delamination toughness of the clay layers is much lower, the low energy consumption of clay delamination and the strong adhesion between clay and the matrix lead to a lower toughness of the composites. On the other hand, virgin clay composites possess increased fracture toughness mainly due to the formation of secondary cracking induced by the weak interface between the clay and the epoxy matrix.The effects of DOPA surface treatment and filler shape on the fracture properties of epoxy nanocomposites, however, prove to be rather more complex. The The fracture toughness increases with both modified and unmodified one-dimensional CNF nanocomposites. Through fracture surface and crack tip damage investigation using electron microscopy, it was found that the 1D filler possesses more versatile means to enhance the fracture resistance regardless of the strength of filler-matrix interface. For the brittle epoxy matrix, the current study suggests that both the form factor and the intrinsic properties of the nanofillers have strong effects on the fracture energy of the epoxy composites. The outcome of this study could serve as a guideline for engineering the mechanical properties of future nanocomposites using a brittle matrix.


Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2  and Mmp13 . Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of  Osteoarthritis (OA) is a progressively degenerative joint disorder and causes chronic disability in elderly people and is one of the major health problems worldwide . OA is cMmp13 gene in mouse primary chondrocytes [FRZB, a secreted antagonist for Wnts, are risk factors for hip and knee OA [Wnt/β-catenin signaling regulates crucial aspects of determination of cell fate during adult tissue homeostasis by regulating expressions of target genes. Most Wnt ligands activate the canonical Wnt/β-catenin signaling, whereas some may not , 6. In tdrocytes . High sedrocytes . In addi knee OA , 24. Ind knee OA , 25. On  knee OA . These s knee OA , 28. In  knee OA , 11, 19.Drug repositioning strategy, in which a drug already used for a specific disease is applied to another disease, has been gaining increasing attention from academia and industry , 30. TheSox9. We found that fluoxetine directly enhanced binding of β-catenin with Axin1 in vitro. Intraarticular injection of fluoxetine ameliorated cartilage damage in a rat OA model.The purpose of this study is to identify a clinically applicable drug, which inhibits Wnt/β-catenin signaling for OA. Using the drug repositioning strategy, we identified that fluoxetine, an antidepressant with a selective serotonin-reuptake inhibiting activity, suppressed activated Wnt/β-catenin signaling in chondrocytes. In chondrogenically differentiated ATDC5 cells, fluoxetine decreased activated Wnt/β-catenin signaling, suppressed degradation of proteoglycans, and increased expression of Wnt3a, according to the manufacturer’s protocol. Briefly, L Wnt-3A cells were cultured in 10 ml DMEM supplemented with 10% FBS in a 10-cm dish, and were grown to confluency. The cells were cultured two additional days, and the medium was harvested as the first batch. Ten ml of fresh culture medium were added to the cells, and harvested 2 days later as the second batch. The first and second batches were mixed in a ratio of 1:1 to make Wnt3A-CM. We screened 1,186 FDA-approved chemical compounds  for reduction of Wnt/β-catenin signaling activated by 25% Wnt3A-CM. We used TOPFlash firefly luciferase reporter plasmid, which has eight TCF/LEF-binding sites in pTA-Luc vector  to measure β-catenin-mediated transcriptional activation in HCS-2/8 cells treated with Wnt3A-CM [4 cells/well in a 96-well culture plate  in the presence of 10 μM each drug and 25% Wnt3A-CM, and were cultured for 24 additional hours. Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) and PowerScan MX . Firefly luciferase activity was normalized by Renilla luciferase activity, and relative luciferase units (RLU) were calculated.Human chondrosarcoma (HCS-2/8) cells, which were kindly provided by Dr. Masaharu Takigawa at Okayama University , were cuWnt3A-CM . HCS-2/82 culture flasks and incubated in DMEM containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37°C in an atmosphere of 5% CO2. At confluence, the cells were detached and seeded in a 12-well plate at a density of 2 × 105/well [Human studies were approved by the Ethical Review Committee of the Nagoya University Graduate School of Medicine. Human OAC cells were isolated from the dissected cartilage in patients with knee OA at the time of joint replacement surgery after appropriate written informed consent was obtained. Articular cartilage from the femoral condyles, tibial plateaus, and articular surface of the patella was cut into small pieces with a scalpel. Cartilage samples were minced and digested at 37°C with trypsin–EDTA solution for 30 min, incubated with 3 mg/ml type II collagenase  in Dulbecco’s Modified Eagle’s Medium  for 18 hours, filtered through a nylon mesh, and washed extensively. Next, the isolated chondrocytes were seeded in 75 cm105/well . OAC celAXIN2, and downregulated SOX9 and MMP13 in OAC cells  rats (8 weeks old) were anesthetized with an intraperitoneal injection of pentobarbital sodium (0.1 ml of 10 mg/ml). Under sterile conditions, the right knee of the rat was induced to OA by resection of the menisco-tibial ligament to destabilize medial meniscus (DMM surgery) . Skin anSections at the medial compartment  or the lateral compartment  in a rat model were stained with Safranin O and Fast-green. OA progressions were graded according to the modified Mankin histologic score on both tibial and femoral sides of articular cartilage , 36. The2O2 for 15 min to inactivate endogenous peroxidases. Sections were blocked with 5% goat serum in TBS-T for 1 hour at room temperature followed by overnight incubation with antibodies against β-catenin (dilution 1:100), TNF, or IL6  at 4°C. After washing with TBS-T, sections were incubated with Alexa 488-conjugated donkey anti-rabbit IgG  for 1 hour at room temperature. Finally, the specimens were mounted in VectaShield containing 2 μg/ml diamidino-2-phenylindole  and visualized using the IX71 (Olympus) microscope.We confirmed that, unlike a mouse rheumatoid arthritis (RA) model , immunos2 for each section. Signals less than 4 μm in diameter were discarded as non-specific signals, and signals more than 4 μm in diameter were taken as positive signals. DAPI-staining was used to localize the nucleus and to count the number of cells. When the intensity of β-catenin in the nucleus was similar to or more than that in the cytoplasm, the cell was counted as a nuclear β-catenin-positive cell. All quantification was performed by a blinded observer.We evaluated cytoplasmic and nuclear accumulation of β-catenin in articular chondrocytes using the MetaMorph software (Molecular Device). We analyzed the entire articular chondrocytes except for the areas where articular cartilage was severely damaged and no or only a few chondrocytes were observed. We quantified ~200–300 μmt-test. The comparison among multiple groups was carried out using one-way analysis of variance (ANOVA) and followed by Tukey-Kramer test. All statistical analyses were performed with SPSS Statistics 23 (IBM) and SAS version 9.4 .The continuous variables are expressed as mean ± standard deviation (SD). The comparison between two conditions was carried out using Student’s Details of other materials and methods are available in Supplementary Information, which includes the following methods: Alcian blue staining, total RNA extraction and real-time RT-PCR analysis, Western blotting, co-immunoprecipitation assay for Axin1, immunofluorescence staining of OAC cells, and MTS assay.n = 5) reduction of relative TOPFlash reporter activity. In addition, fluoxetine suppressed the TOPFlash reporter activity in a dose-dependent manner (n = 5), respectively. Thus, the inhibitory effect on TOPFlash by fluoxetine is unlikely due to its inhibitory effect on serotonin reuptake. As fluoxetine is the first anti-depressant in the class of serotonin-reuptake inhibitor and has been long used in clinical practice [To identify a clinically applicable drug for OA, chondrogenic HCS-2/8 human chondrosarcoma cells  were trat manner . We alsopractice , we pursIn articular cartilage, chondrocytes are embedded in an abundant extracellular matrix composed of proteoglycans and collagens. Mouse ATDC5 cells recapitulate chondrogenic differentiation, and produce more proteoglycans than HCS-2/8 cells . We thusAxin2 mRNA, a specific marker for Wnt/β-catenin signaling, (ii) decreased Sox9 mRNA, a master gene for chondrocyte differentiation, and (iii) upregulated Mmp13 mRNA, coding for a proteinase for collagen type II. Similarly, LiCl upregulated Matn1 mRNA, encoding one of extracellular cellular matrix proteins upregulated by Sox9 [We next confirmed in chondrogenically differentiated ATDC5 cells that 20% Wnt3A-CM , recombi by Sox9 , in chonSox9 induced by 20% Wnt3A-CM  in SD rats, and histologically evaluated progression of OA. To examine the effects of fluoxetine on articular chondrocytes of 50 μM , 100 μM of 50 μM , and 200of 50 μM  fluoxetiof 50 μM  Only 50 of 50 μM . The difn = 6 rats) in the PBS group and 16.3 ± 5.7 in the fluoxetine group . In a previous report, intraperitoneal injection of 25 mg/kg/day (0.625 mg/25 g mouse/day) fluoxetine, which was 19–75 times higher than human doses, exhibited potent anti-inflammatory activity in a mouse model of rheumatoid arthritis . Assuminne group . In a sene group  Immunoflne group  and its ne group  showed tIn contrast to rheumatoid arthritis (RA), no disease-modifying drugs are currently available for OA . AnalgesFRZB in cultured cells. Verapamil thus suppresses Wnt/β-catenin signaling extracellularly, and should have no direct intracellular effect. In addition to activation of β-catenin, Wnt ligands also activate non-canonical PCP and Ca2+ pathways [FRZB [We previously reported that verapamil, a calcium channel blocker, protected against cartilage degradation by inducing expression of FRZB in a rat OA model . FRZB ispathways , the rolys [FRZB . InsteadMmp13  OAC cells are treated with conditioned medium containing Wnt3A  for 48 hours. Expression levels of AXIN2(A), SOX9(B), and MMP13(C) mRNA are normalized to that without treatment. The mean and SD in three wells in a single experiment are indicated. *p < 0.05 and ***p < 0.005 by Student t-test.(TIF)Click here for additional data file.S2 Fig Three rats in each group had DMM and sham surgeries in the right and left knees, respectively, as described in (C) Human OAC cells are treated with or without fluoxetine (10 μM) in the presence of 10 mM LiCl for 48 hours. Expression levels of each mRNA are normalized to that without treatment. The mean and SD (n = 2 patients) are indicated.(TIF)Click here for additional data file.S3 Fig ATDC5 cells are treated with indicated concentrations of fluoxetine and LiCl for 48 hours. Cell growth is quantified using the MTS assay. Data are presented as the mean and SD of 490-nm absorbance normalized by that without fluoxetine (n = 6 wells in an experiment). Fluoxetine shows slight toxicity at 25 and 50 μM. (A)P-value by one-way ANOVA of each condition is <0.0001. (B)P-value by Student’s t-test is 0.42.  Differentiated ATDC5 cells are treated with mouse Wnt3A proteins  (C), 10 mM NaCl (D), or 10 mM LiCl (D) for 48 hours. Expression levels of each mRNA are normalized to that without treatment. The mean and SD in two wells each in two independent experiments (n = 4) are indicated. (C)P-values by one-way ANOVA of Axin2, Sox9, and Mmp13 expressions are 0.011, 0.010, and 0.024, respectively. (D)P-values by one-way ANOVA of Axin2, Sox9 and Mmp13 expressions are 0.036, 0.028, and 0.018, respectively. *p < 0.05, **p < 0.01, and ***p < 0.005 by posthoc Tukey-Kramer test.(TIF)Click here for additional data file.S4 Fig(A) or 10 μM Akt inhibitor (B-D) with or without 10 μM fluoxetine for 48 hours. Expression levels of each mRNA are normalized to that without treatment. The mean value of three wells in a single experiment is calculated first. Then, the means of three wells in three independent experiments are used to calculate the mean and SD (n = 3). P-values by one-way ANOVA are 0.016 (A), 0.32 (B), 0.089 (C), 0.066 (D), respectively. *p < 0.05 by posthoc Tukey-Kramer test.ATDC5 cells are treated with ITS to induce chondrogenic differentiation. Differentiated ATDC5 cells are treated with 10 mM LiCl (TIF)Click here for additional data file.S5 Fig(A) Representative staining of knee joints with Safranin O and Fast-green. Scale bar = 400 μm. (B) OA progressions are evaluated by modified Mankin score at 8 weeks after the surgery. The mean and SD (n = 3 rats for each) are indicated. P-values by one-way ANOVA of each condition (DMM surgery and Sham surgery) are 0.035 and 0.42, respectively. *p < 0.05 by posthoc Tukey-Kramer test. The data suggest 200 μM fluoxetine rather worsens OA pathology.Three rats in each group have DMM and sham surgeries in the right and left knees, respectively. (TIF)Click here for additional data file.S6 Fig(B) Larger areas of stained sections of knee joints shown in Sections crossing the narrowest interarticular space at the lateral compartment comprised of the lateral femoral condyle and the lateral tibial plateau are evaluated by modified Mankin score in three sham-operated knees and four DMM-operated knees. The modified Mankin scores at the lateral compartments are lower than those at the medial compartments indicated in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. However, prior phosphorylation of β-catenin at Tyr654 was essential for further phosphorylation of β-catenin by Jak3. Interaction studies indicated that phosphorylated Jak3 bound to phosphorylated β-catenin with a dissociation constant of 0.28 μm, and although both the kinase and FERM  domains of Jak3 interacted with β-catenin, the NTD domain of β-catenin facilitated its interactions with Jak3. Physiologically, Jak3-mediated phosphorylation of β-catenin suppressed EGF-mediated epithelial–mesenchymal transition and facilitated epithelial barrier functions by AJ localization of phosphorylated β-catenin through its interactions with α-catenin. Moreover, loss of Jak3-mediated phosphorylation sites in β-catenin abrogated its AJ localization and compromised epithelial barrier functions. Thus, we not only characterize Jak3 interaction with β-catenin but also demonstrate the mechanism of molecular interplay between AJ dynamics and EMT by Jak3-mediated NTD phosphorylation of β-catenin.Compromise in adherens junctions (AJs) is associated with several chronic inflammatory diseases. We reported previously that Janus kinase 3, a non-receptor tyrosine kinase, plays a crucial role in AJ formation through its interaction with β-catenin. In this report, we characterize the structural determinants responsible for Jak3 interactions with β-catenin and determine the functional implications of previously unknown tyrosine residues on β-catenin phosphorylated by Jak3. We demonstrate that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of β-catenin, where Jak3 directly phosphorylated three tyrosine residues,  An adherens junctions (AJ)4–β-Catenin, originally identified as AJ-associated protein, is a member of the catenin family of proteins that links the cytoplasmic domain of cadherins to actin cytoskeleton–bound α-catenin . Structu4.1, ezrin, radixin, and moesin) domain, found in molecules such as Band 4.1, ezrin, radixin, and moesin . We also expressed and purified the non-phosphorylated form of GST-tagged Jak3-WT using BL-21 expression systems . Because recombinant Jak3-WT autophosphorylates in a time-dependent manner, with a t½  of autophosphorylation of 135 s . Because β-catenin was a direct substrate for Jak3, we determined the kinetics of β-catenin binding to Jak3. Pairwise binding studies showed that Jak3-WT interacted with β-catenin in a dose-dependent manner with a dK of 0.28 μm .Previously we reported that Jak3 facilitated intestinal mucosal barrier functions through its interactions with the adherens junction protein β-catenin , 18, 22.Fig 1of 135 s , we detereported  Jak3 inh of 33 s  and 3.2  of 33 s , respectA shows a schematic of β-catenin and its mutants. The wild type and these mutant proteins of β-catenin were expressed and purified using the BL21 expression system  and were used as substrates for an in vitro kinase assay using autophosphorylated Jak3 as an enzyme. Fig. 2C shows that Jak3 trans-phosphorylated β-catenin; however, deletion of the CTD of β-catenin resulted in a 1.5-fold increase in tyrosine phosphorylation. Moreover, deletion of both the ARM domain and CTD did not change phosphorylation by Jak3, indicating that Jak3-mediated phosphorylation of β-catenin was due to tyrosine residues present in the NTD of β-catenin. Because the NTD of β-catenin has four tyrosine residues, and we reported previously that, in human intestinal epithelial cells, Jak3 does not associate with Tyr142-phosphorylated β-catenin . To further confirm Tyr30, Tyr64, and Tyr86 as Jak3-mediated tyrosine phosphorylation sites in β-catenin, triple mutation studies using either the NTD alone  or full-length β-catenin  showed that mutation of all three resulted in complete loss of Jak3-mediated β-catenin phosphorylation .Because β-catenin not only interacted with but was also phosphorylated by Jak3, we determined the structural determinants of β-catenin phosphorylated by Jak3. Fig. 2-catenin , using aeviously  and p52SD shows that the non-phosphorylated form of β-catenin interacted with non-phosphorylated Jak3; however, tyrosine phosphorylation of β-catenin (P–β-catenin) increased the interactions between Jak3 and P–β-catenin by 1.6-fold. Surprisingly, non-phosphorylated forms of β-catenin minimally interacted with the phosphorylated forms of Jak3, indicating that prior phosphorylation at the tyrosine residues of β-catenin was necessary for enhanced interaction and further phosphorylation by P-Jak3. Because we reported previously that, in human IECs, Jak3-associated β-catenin had phosphorylation at Tyr654 of the CTD  form of non-phosphorylated, full-length β-catenin. Moreover, phosphomimetic (Y654E) full-length β-catenin also overcame the inhibitory effects of CTD toward Jak3-mediated phosphorylation, which was equivalent to the maximum phosphorylation level of NTD by P-Jak3 . To further confirm the role of Tyr654, our data show that tyrosine-phosphorylated forms of the Y654F mutant of β-catenin failed to interact with P-Jak3 . Fig. 2E shows the Western blot analysis of expression and purification of phosphomimetic (Y654E) and phosphomutant (Y654F) full-length β-catenin.Because the CTD of β-catenin partially inhibited Jak3-mediated phosphorylation of β-catenin, we determined the effects of tyrosine phosphorylation on the interactions between Jak3 and β-catenin. Fig. 2 the CTD , and FigF shows that Jak3 interacted with phosphorylated β-catenin; however, deletion of the kinase domain of Jak3 resulted in more than a 4-fold decrease in their interactions. Moreover, further deletion of both the kinase and pseudokinase domains did not affect the level of interaction; however, deletion of the kinase, pseudokinase, and SH2 domains altogether resulted in a 2-fold increase in their interactions. These results indicate that both the kinase and FERM domains of Jak3 were responsible for Jak3 interactions with β-catenin, where the SH2 domain of Jak3 partially inhibited the interactions between the FERM domain of Jak3 and β-catenin. Next we determined whether truncation of β-catenin had an impact on the interactions between β-catenin and P-Jak3. Fig. 2G shows that β-catenin interacted with Jak3; however, deletion of the CTD domain increased their interactions, which remained unaffected upon further deletion of ARM domains. This indicated that the NTD domain of β-catenin was necessary and sufficient for its interaction with Jak3.Because Jak3 not only interacted with β-catenin but also phosphorylated it, we determined the structural determinants of Jak3 responsible for its interactions with β-catenin. Fig. 2A). To determine whether Jak3 had an effect on the phosphorylation of specific tyrosine residues on β-catenin, these stably transfected cells were treated with IL-2, and cell lysates were immunoprecipitated with HA and immunoblotted either with phosphotyrosine or β-catenin antibody. We reported previously that treatment with IL-2 led to activation (–B) and the NTD of β-catenin (C) but not of NTD with a triple tyrosine mutation  (C). Moreover, treatment of IL-2 stimulated IECs with a Jak3 inhibitor led to decrease in β-catenin–WT phosphorylation (B) and complete loss of β-catenin–NTD phosphorylation, confirming (C) that Jak3 phosphorylates the NTD domain of β-catenin, where Tyr30, Tyr64, and Tyr86 are the tyrosine residues phosphorylated by Jak3 in a human IEC. Moreover, cells with mutation of Tyr30, Tyr64, and Tyr86 to Phe in full-length β-catenin did not show a change in phosphorylation either by IL-2 stimulation or by Jak3 inhibition in IL-2–stimulated cells (D), further confirming Tyr30, Tyr64, and Tyr86 as the Jak3-mediated phosphorylation sites in full-length β-catenin.Because P–β-catenin not only interacted with P-Jak3, but Jak3 also phosphorylated three tyrosine residues in the NTD of β-catenin, we determined the physiological significance of these in a human intestinal epithelial cell (IEC) model. Human IECs, HT-29 Cl-19A, were stably transfected either with pCDNA–HA–β-catenin–WT, pCDNA–HA–β-catenin–Y30F,Y64F,Y86F, pCDNA-HA-NTD, or pCDNA-HA-NTD-Y30F,Y64F,Y86F, and clones expressing comparable amounts of recombinant proteins were selected A. To dettivation –20 of Jaivation –, includiivation –. We showivation –B and the-catenin C but not4F,Y86F) C. Moreovrylation B and comnfirming C that Jaed cells D, furtheA and E, respectively). F and G, shows that expression of HA–β-catenin increased the trans-epithelial electrical resistance (TEER) in both IEC models, whereas expression of the Jak3-mediated phosphorylation  mutant of HA–β-catenin led to a reversal of these effects, indicating that the use of transformed IEC did not influence the physiological significance of Jak3-mediated phosphorylation of β-catenin. To further confirm that these physiological effects were due to loss of Jak3-mediated tyrosine phosphorylation sites in β-catenin and associated impairment of AJ formation, using confocal microscopy, we determined the AJ localization HA–β-catenin and its triple mutation counterparts. Fig. 3H shows that HA–β-catenin mostly localized to the AJ in IECs, as indicated by strong β-catenin staining at the cellular junctions. However, mutation of Jak3-mediated tyrosine phosphorylation sites  in β-catenin led to a substantial decrease in β-catenin localization to the AJ, as indicated by punctate β-catenin staining at the cellular junctions. Because prior phosphorylation at Tyr654 of β-catenin was essential for its interactions and subsequent phosphorylation by Jak3 (C) that led to AJ localization of phosphorylated-β-catenin, to demonstrate the in vivo significance of these findings, we determined whether absence of Jak3 affected the AJ localization of Tyr654-phosphorylated β-catenin in the mouse colon. Fig. 3I, top panels, shows that Jak3-expressing WT mice had Tyr654-phosphorylated-β-catenin mostly localized to the cellular junctions; however, lack of Jak3  resulted in loss of junctional localization of Tyr654-phosphorylated β-catenin. Moreover, Jak3 KO mice showed diffused and substantially reduced pTyr654–β-catenin expression in the colonic epithelium. This further corroborates our earlier reports that lack of Jak3 leads to compromised colonic barrier functions in mice  and normal FHC-CRL-1813) human IEC models transfected with either HA–β-catenin or HA–β-catenin–Y30F,Y64F,Y86F  of Tyr654-phosphorylated β-catenin with Jak3 was dependent on EGF-induced activation of Src, followed by Src-mediated phosphorylation of Tyr654 of β-catenin. For these experiments, IP with IgG represented as negative control, IP followed by IB with pTyr654–β-catenin antibody represented a positive control, and IB for total β-catenin was used as an input control. Fig. 4C, bottom panel, shows that, EGF stimulation of HA-tagged β-catenin led to a similar level of Tyr654 phosphorylation of β-catenin; however, co-stimulation with IL-2 led to disruption of interactions between HA-tagged β-catenin and Hif1α, which were reversed by mutation of Jak3-mediated phosphorylation sites  in β-catenin . Furthermore, EGF also promoted disruption of HA-tagged β-catenin with the AJ protein α-catenin, which was again reversed by mutation of Jak3-mediated phosphorylation sites  in β-catenin (center panel). To determine that this was not due to transformation-related factors of epithelial cells, Fig. 4D shows that activation of CRL-1813cells, a normal epithelial cell line, or HT-29cl-19a cells , with EGF led to expression of the EMT marker N-cadherin, which was associated not only with co-immunoprecipitation of Tyr654-phosphorylated β-catenin with Hif1α but also dissociation of Tyr654-phosphorylated β-catenin from the AJ protein α-catenin, indicating a disruption of the AJ. However, simultaneous activation by the Src activator EGF and the Jak3 activator IL-2 led to a reversal of these effects, as indicated by increased association of Tyr654-phosphorylated β-catenin with α-catenin, reflecting AJ localization of β-catenin, which was associated with decreased expression of the EMT marker N-cadherin. To further confirm that these effects were due to Jak3 activation, the experiments were performed in the presence of a Jak3 inhibitor (CP690550). D and E, shows that inhibition of Jak3 in EGF and IL-2–stimulated cells not only promoted EMT, as reflected by the expression of N-cadherin, but also prevented association of Tyr654-phosphorylated β-catenin with the AJ protein α-catenin, indicating AJ destabilization. This indicated that, irrespective of IEC types, Jak3 activation was required for the prevention of Src-mediated EMT in these epithelial cells. Because α-catenin interaction with β-catenin was necessary for AJ localization of α-catenin, and EGF-mediated tyrosine phosphorylation of β-catenin at Tyr654 disrupts these interactions, using confocal microscopy, we determined the effects Jak3 activation on α-catenin localization to the AJ. Fig. 4F shows that, in control unstimulated cells, α-catenin was mostly localized to cellular junctions. Moreover, stimulation by EGF led to loss of AJ localization of α-catenin, indicating Tyr654 phosphorylation of β-catenin. However, these effects were reversed by co-stimulation with EGF and IL-2, as indicated by increased junctional localization of α-catenin. To confirm that these reversals are due to Jak3-mediated β-catenin phosphorylation, the experiments were carried out in the presence of the Jak3 inhibitor tofacitinib. As shown in Fig. 4F, fourth panel, inhibition of Jak3 led to significant loss of AJ localization of α-catenin, as indicated by punctate α-catenin staining at the cellular junctions. To further confirm that Jak3-mediated changes in α-catenin localization were due to its interactions with β-catenin, using confocal microscopy, we showed that EGF activation of IECs led to loss in junctional localization of α-catenin , which was significantly reversed by co-stimulation of these cells with IL-2 , as indicated by strong yellow staining in the merged panel. To confirm that these effects were due to Jak3 activation, these experiments were performed in the presence of Jak3 inhibitor. Fig. 4G, fourth row, shows that inhibition of Jak3 significantly decreased α-catenin co-localization with Tyr654-phosphorylated β-catenin.Src kinase phosphorylates β-catenin at Tyrth Hif1α . Because654-phosphorylated β-catenin–mediated reversal of EMT, using flow cytometry, we determined the effects of EGF, EGF+IL-2, and EGF+IL-2+tofacitinib on the relative expression of EMT markers. The representative flow data in Fig. 5A and the corresponding statistical analysis from three independent experiments in Fig. 5B show that there was approximately a 10-fold increase in cells expressing the EMT marker N-cadherin upon EGF-mediated  Src activation . Moreover, IL-2 alone did not influence N-cadherin expression, which was similar to that of the control. However, co-stimulation of IECs by EGF+IL-2 led to a more than 3-fold decrease in cells expressing EMT markers , which was correlated with increased AJ localization of β-catenin . To prove the specific role of Jak3-mediated β-catenin-phosphorylation, specific mutations of Jak3-mediated tyrosine phosphorylation residues to phenylalanine  in β-catenin resulted in abrogation of EMT prevention by IL-2 in the presence of EGF, as denoted by loss of responsiveness to IL-2 toward EGF-mediated β-catenin interactions with Hif1-α , loss of β-catenin interactions with α-catenin , and expression of the EMT marker N-cadherin . Taken together, these results indicate that Jak3 phosphorylates the Tyr30, Tyr64, and Tyr86 residues in Tyr654-phosphorylated β-catenin, which not only prevents β-catenin association with Hif1α and expression of the EMT marker N-cadherin but also facilitates Tyr30-, Tyr64-, and Tyr86-phosphorylated β-catenin association with α-catenin, thereby reinforcing AJ formation.To quantitate the extent of the effects of Jak3 on Tyr-catenin . To confD, left panels, shows that, compared with healthy epithelial tissues, there was a significant decrease in expression and localization of Jak3 that was associated with increased expression of the EMT marker N-cadherin in colonic polyps. These results show that a decrease in Jak3 expression and altered localization were associated with increased EMT in human colonic polyps.In humans, colonic polyps represent a transition state in the progression of normal epithelial cells to cancerous cells, where EMT plays an essential role in this progression, and sustained expression of the EMT marker N-cadherin is frequently reported , 25. BecFig. 5versus phosphomimetic mutants  of β-catenin. The simulations on β-catenin–WT and the β-catenin–Y654E mutant show that Y654E induced a significantly changed orientation of the β-catenin NTD (A), as indicated by stearic separation of the Gly85 and Ala80 residues in the NTD of the two superimposed structures of β-catenin–WT (gold) and β-catenin–Y654E (blue). Because it was reported that native β-catenin mostly localized to AJ through β-catenin–NTD  and β-catenin–Y30E,Y64E,Y86E , followed by stearic superimposition in the structures, show a close resemblance in the NTD, as indicated by the relative positions of Gly85 and Val166 in the superimposed proteins (B). This indicates that Y654E-mediated large shifts in NTD orientations were reversed by simultaneous mutations in Y30E,Y64E,Y86E, which was close to that of native β-catenin. Thus, the Y30E,Y64E,Y86E mutation that mimicked the phosphorylation state of these residues preserved the geometric flexibility of the NTD required for AJ localization of β-catenin.Because our results showed that Jak3 phosphorylation of β-catenin not only stabilized AJ but also suppressed EMT, using a phosphomimetic approach for Jak3-mediated β-catenin phosphorylation sites , we performed molecular simulations to compare the structural shifts in wild-type (Tyr) enin NTD A, as indenin–NTD , 27 inte-catenin , we dete-catenin B, gold a64E,Y86E B, blue, proteins B. This iAJs play a major role in maintaining the integrity of epithelial tissue architecture , 2. The dK of 0.28 μm but was also a direct substrate for Jak3 , in, inKd ofrylation  inhibitet½ of trans-phosphorylation of 34 s. Previously, we reported that Jak3 phosphorylates the cytoskeletal protein villin  was purchased from the ATCC. IL-2, CP-690550, and EGF were from Sigma); J67242 was from Alfaaesar, and the Pierce BCA protein assay kit was from Thermo Scientific. The following antibodies were used: pTyr20 ; Jak3 (Invitrogen); β-catenin, p52ShcA, villin, N-cadherin, β-actin, and Shc (Santa Cruz Biotechnology); p654–β-catenin (Abcam); Hif1α ; α-catenin and FITC (Sigma); His (Genescript); secondary fluorophore conjugate of anti-mouse FITC (Sigma); and anti-rabbit CY3 (Amersham Biosciences).The deidentified and discarded human colonic biopsy samples used in this study were obtained from CHRISTUS Spohn Health System laboratory. HT-29 Cl-19A, a permanently differentiated homogenous clone derived from human colonic epithelial cells (HT-29) was a kind gift from Dr. A. P. Naren (Cystic Fibrosis Research Center (CFRC), Cincinnati, OH) . FHC (CRTM) were done according to the specification sheet provided by the ATCC. However, treatment and transfection in FHC  were done according to the protocol of the supplier. The methods for HT-29 cell maintenance and treatment have been reported before  at a seeding density of 1 × 105 cells/insert and differentiated. Likewise, HT-29 CL19a cells stably transfected with pCDNA-HA β-catenin-WT and pCDNA-β-catenin-HA-Y30F,Y64F,Y86F were grown to confluence. TEER was measured using the Millicell electrical resistance system (Millipore) as reported previously (2) were subtracted from the observed resistance values. Cells without transfection were taken as the control.FHC . For IHC, the aforementioned sections from frozen tissue blocks were fixed with precooled acetone at 20 °C. Endogenous peroxidase activity was inactivated by incubating the tissue with 0.3% H2O2. Nonspecific binding was blocked by incubation with 10% fetal bovine serum prior to incubation with primary antibodies. The sections were then incubated with biotinylated secondary antibody followed by staining with the Vectastain ABC kit (Vector Laboratories). The reaction was visualized using 3,3-diaminobenzidine substrate. All experiments were performed at least in triplicate, and representative images are shown.Protein expression of N-cadherin, Hif1α, β-catenin, shc villin, and β-actin was examined by Western blotting. Cells with or without treatment were washed three times with 1× PBS and lysed with radioimmune precipitation assay lysis buffer that contained a mixture of protease inhibitors. Following quantification of total protein with the BCA® protein assay kit, 80 μg of the whole-cell lysate was denatured in Laemmli buffer at 95 °C for 10 min, resolved on 10–12% SDS-PAGE, and electrotransferred onto nitrocellulose membranes. The membranes were blocked in TBS that contained 0.05% Tween 20 and 1% BSA for 1 h at room temperature. Blots were then incubated with primary antibody followed by secondary antibody for 90 min at room temperature. Membrane-bound secondary antibodies were visualized with a chemiluminescence HRP substrate (Roche Applied Sciences) using a Chemilluminager. The methods for IP and IB using HT-29 CL-19A cell lysates under indicated experimental conditions were reported previously . For IFM654, and CP-690550 inhibits Jak3 activity. The treated cells were detached by using 2 mm EDTA in PBS, washed, and resuspended in 1:100 dilutions of primary antibodies for N-cadherin, Jak 3, p654 β-catenin, β-catenin, and Hif1α in a solution containing PBS with 1% BSA and 0.01% sodium azide for all experimental conditions. A secondary fluorophore conjugate of anti-mouse FITC for N-cadherin, p654 β-catenin, or anti-rabbit CY3 for Jak 3, β-catenin, and Hif 1α was used subsequent to incubation with primary antibody. The experimental samples were incubated at room temperature for 30 min and washed in PBS with 1% BSA and 0.01% sodium azide after incubation with primary and secondary antibodies. Flow cytometric data were acquired in FL 1  and FL 2  channels using a BD Accuri C6 flow cytometer (BD Biosciences) and analyzed using BD Accuri C6 software with appropriate unstained and gating controls. The percentages of cells determined to be positive on the FL1 or FL 2 channels were plotted in the form of a bar graph with Origin 8.6 (Originlab) software and statistically compared for all treatment groups.HT-29CL19A cells with or without treatment were trypsinized and seeded in 24-well cell culture–grade plates . To assess the role of Jak 3 in suppressing EMT induced by EGF, the cells were either treated with IL-2 alone (50 units/ml) or in combination with EGF  and CP-6654) or Jak3-mediated  phosphomimetic counterparts were modeled using the crystal structure of full-length β-catenin protein reported previously (2Z6G) as the template. The intensive modeling mode of the Phyre2 homology modeling software combines multiple template modeling and simplifies ab initio protein folding simulations to create the full-length model of the protein. To simulate the impact of phosphorylation of tyrosine residues  of β-catenin, these amino acids were replaced with the phosphomimetic residue Glu. The conformational changes induced in the structure of β-catenin were analyzed by superimposition and energy minimization using CHARMM Forcefield and visualized in Accelerys Discovery Studios (Biovia).Full-length β-catenin protein and its Src-mediated  and were analyzed using Microcal OriginA and B, and supplemental Figs S1–S3. All authors reviewed the results and approved the final version of the manuscript.N. K. and J. M. conceived and coordinated the study and wrote the paper. J. M. designed, performed, and analyzed the experiments shown in Figs. 1


In this paper, we investigate if bNCSCs may improve survival of MNs harboring a mutant form of human SOD1 (SOD1G93A) in vitro under normal conditions and oxidative stress and in vivo after implantation to the spinal cord. We found that survival of SOD1G93A MNs in vitro was increased in the presence of bNCSCs under normal conditions as well as under oxidative stress. In addition, when SOD1G93A MN precursors were implanted to the spinal cord of adult mice, their survival was increased when they were co-implanted with bNCSCs. These findings show that bNCSCs support survival of SOD1G93A MNs in normal conditions and under oxidative stress in vitro and improve their survival in vivo, suggesting that bNCSCs have a potential for the development of novel stem cell-based therapeutic approaches in ALS models.ALS is a devastating disease resulting in degeneration of motor neurons (MNs) in the brain and spinal cord. The survival of MNs strongly depends on surrounding glial cells and neurotrophic support from muscles. We previously demonstrated that boundary cap neural crest stem cells (bNCSCs) can give rise to neurons and glial cells The online version of this article (doi:10.1007/s13311-016-0505-8) contains supplementary material, which is available to authorized users. G93A is the most widely studied model for ALS pathogenesis [Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disorder affecting primarily upper and lower motor neurons (MNs), and usually leading to the death of the patient, most commonly due to respiratory failure. The glutamate signaling antagonist Riluzole has a measurable positive effect on disease progression and is relatively free of side effects. Riluzole acts by attenuating excitotoxic impact on endangered MNs, and is currently the only available approved ALS treatment, but offers not more than a few months extended life expectancy . Riluzolin vitro [in vivo after transplantation into the spinal cord of rats [in vitro [Mutant SOD1 affects not only intrinsic properties of MNs, but also results in a neurotoxic astroglial phenotype, inducing degeneration of wild type MNs in vitro –7 and in of rats . Furtherin vitro , 10. Conin vitro . Wild tyin vitro . Implantin vitro , 14.in vitro and in vivo [in vitro and after transplantation into the immature mouse brain [in vitro (unpublished observation). Interestingly, another type of NCSCs, the hair follicle stem cells, did not have corresponding positive effects on co-cultured cells [Boundary cap neural crest stem cells (bNCSCs) is a transient neural crest-derived group of cells that are located at the dorsal root entry zone (DREZ) . These c in vivo , as wellse brain . We havese brain , 18 and se brain , as welled cells .G93AMNs, generated from SOD1G93A mouse embryonic stem cells (mESCs). These cell lines express green fluorescent protein (GFP) under the control of the promoter for the MN specific transcription factor HB9 (HB9::GFP cells), allowing their identification after directed differentiation to MN progenitors [G93A mESC line was previously analyzed in vitro under normal conditions [G93A MN survival. Generation of MNs from mESCs results also in abundant generation of astrocytes. These cells express glutamate aspartate transporter (GLAST) and can be identified by anti-GLAST antibodies [G93A astrocytes on SOD1G93A MNs, we used magnetic activated cell sorting (MACS) to eliminate GLAST-positive cells from SOD1G93A mESC cultures. To compare the effect of bNCSCs on SOD1G93A MN survival with mESC derived astrocytes, we used astrocytes differentiated from a non-SOD1 mutated glial fibrillary acidic protein (GFAP)::CD14 mESC line [These findings prompted us to test if bNCSCs have a beneficial effect on co-cultured and co-implanted SOD1genitors . Furthernditions . Here, wtibodies . To exclESC line .G93A MNs.Our findings show that bNCSCs exert a significant survival promoting effect on co-cultured and co-implanted SOD1WT) or mutant SOD1 (SOD1G93A) were a kind gift from Dr. Kevin Eggan (Harvard Stem Cell Institute). The risk of tumor formation for implanted cells from low passages is minimal. These cell lines carry green fluorescent protein (GFP) under the control of the promoter for the MN specific transcription factor HB9 (HB9::GFP cells) [WT and SOD1G93A mESC lines to derive GFP+ MNs.Mouse embryonic stem cell (mESC) lines harboring human wild type SOD1 (SOD1P cells) . We usedFor MN differentiation, a previously published protocol with small modifications was used . Cells win vitro MN differentiation, EBs were enzymatically dissociated with TrypLE™ Express (Gibco) and seeded on pre-coated coverslips with 0.01% poly-l-ornithine (Sigma) followed by 10 μg/mL laminin (Sigma). Cells were seeded at a density of 5 × 104 cells/coverslip in 24 well plates with ADFNB cell medium supplemented with 10 ng/mL of CNTF (Miltenyi Biotec) and GDNF (Miltenyi Biotec). 50% of the medium was replaced with fresh medium every other day until the cultures were fixed in 4% paraformaldehyde in phosphate buffered saline  at the indicated time points.For G93A MNs.bNCSCs were generated from transgenic mice harboring red fluorescent protein (RFP) under the universal actin promoter  accordinGFAP::CD14 mESCs , using MACS as has been previously described [Astrocytes were generated from escribed  with minGFAP::CD14 EBs were subjected to MACS to obtain astrocyte cultures. SOD1G93A EBs were subjected to MACS using anti-GLAST to deplete SOD1G93A MN cultures from SOD1G93A astrocytes.Anti-GLAST (ACSA-1) antibodies were used for MACS according to the manufacturers protocol (Miltenyi Biotec). bNCSCs and 6 cells were labeled with anti-GLAST antibodies. Thereafter, anti-mouse IgG magnetic microbeads (Miltenyi Biotec) were applied to the cells for 15 minutes at 4 °C. Cells were re-suspended in MACS buffer (Miltenyi Biotec) and the cell suspension was loaded onto a MACS separation column (Miltenyi Biotec) and placed in the magnetic field of a MACS separator.Culture suspensions containing around 3 × 10G93A cell line, the GLAST-positive fraction was removed to minimize the influence of SOD1G93A astrocytes on SOD1G93A MNs. For bNCSCs and GFAP::CD14 EBs, the GLAST positive fractions were used for the experiments.The column was rinsed three times with 0.5 mL of MACS buffer. In the case of the SOD14 of SOD1G93A cells were seeded on 0.01% poly-l-ornithine and 10 μg/mL laminin coated coverslips. In case of co-cultures, around 2 x 104 bNCSCs or GFAP::CD14 mESC-derived astrocytes were seeded first followed by SOD1G93A MNs.For differentiation assays, 5 × 102O2) in SOD1WT MNs and SOD1G93A MNs plated alone or together with bNCSCs. In addition, bNCSCs were treated alone with H2O2 at similar concentrations to test their survival capacity under these conditions. On day 4 of differentiation assay, H2O2  was added for 3 hours to the cultures. Thereafter, the coverslips were fixed and analyzed for MN or bNCSC survival . For each condition at least three experiments were performed.Oxidative stress was induced by hydrogen peroxide (Hhttp://www.taconic.com). The serial sections were from the spinal cords of six animals − three of which received bNCSCs and SOD1G93A MN precursors (treatment group) and three only SOD1G93A MN precursors (control group) − were analyzed under confocal microscope for MN survival and glial response. All animal experiments were approved by the Local Ethical Committee for Animal Experimentation, Uppsala, as required by Swedish Legislation and in accordance with European Union Directives.Transplantations were performed in Crl:NU(NCr)-Foxn1nu  NMRI adult male mice (n = 12),  attached to a stereotactic frame and connected to an infusion pump (KD Scientific Legato 130). For the control group an equal amount of cells containing only SOD1G93A MN precursors were injected. The needle was kept in place for 2 minutes before removal. The wound was closed in layers using ethilon nylon suture 6.0 and 4.0, respectively. The animals were given 50 μl buprenorphine (Temgesic©) subcutaneously every 12 hours for 3 days after the operation.The animals were anesthetized by spontaneous inhalation of Isoflurane. After onset of anesthesia the mouse was placed on its stomach on a heating pad (36 °C) and the skin cleaned with ethanol (70%). Following a procedure previously established in rats , an inciSeven days after surgery the animals were re-anesthetized with an intraperitoneal injection of a mixture of ketamine, xylazine and acepromazine , and per3 in PBS) for 60 minutes, and then incubated with primary antibodies overnight at 4 °C, followed by the appropriate secondary antibodies for 1 hour at RT  for 5 minutes, washed and left in blocking solution  of a Nikon Eclipse E800 epifluorescence microscope equipped with a Nikon DXM1200F CCD camera. For cultured cells, GFP and RFP positive cells were counted manually for each condition. GFP positive cells were counted in every 5th cryosection along the length of the transplant area . Sections were analysed using a Zeiss LSM700 or a Zeiss LSM780 confocal laser scanning microscope. Images were captured using a LD LCI Plan-Apochromat 20x (NA 0.8) or a LD C-Apochromat 40x W Corr (NA 1.1) objective.Datasets for oxidative stress and survival assays were analyzed using two-way ANOVA followed by the Bonferroni multiple comparisons tests. MN survival was analyzed using a two-tailed Students t-test. All statistical analyses were performed in GraphPad Prism 5.04. The confidence interval was stated at the 95% confidence level, placing statistical significance at p < 0.05.WT and SOD1G93A mESC lines formed EBs with abundant expression of HB9::GFP and gave rise to GFP+ MN precursors in culture , and continued to express RFP in differentiation assay in co-culture and co-implants   compared to SOD1WT MNs, indicating that SOD1G93A MNs are more susceptible than wildtype MNs to oxidative stress , brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) [G93A MNs to the spinal cord parenchyma and in some cases towards the central canal  , all of r (CNTF) . bNCSC-dr (CNTF) , which hr (CNTF) , 39, mayG93A MNs. Previous studies have shown that ALS is associated with disruption of the blood-CNS-barrier (B-CNS-B) [G93A resistant astrocytes may contribute to the formation of new blood vessels with an intact B-CNS-B in the diseased spinal cord. Implantation of bNCSCs to animal models of ALS may clarify the potential of these cells to recover B-CNS-B.The ability of bNCSCs to promote vascularization of co-implanted tissue  may alsoB-CNS-B) –46, a prG93A MNs in vitro and in vivo after co-implantation to the spinal cord. The beneficial effects by bNCSCs might be due to their resistance to oxidative stress and their neurotrophic and angiogenic support. These properties in combination with their resistance to SOD1G93A aggregates make them interesting candidates for further investigations on novel therapeutic approaches to achieve long-term neuroprotection in animal models of ALS.In conclusion, we show that bNCSCs promote survival of SOD1Supplementary Fig. 1WT and SOD1G93A EBs abundantly expressed HB9::GFP in MN precursors on day 7  and generated GFP+ MNs in vitro, expressing Beta-3-tubulin . Scale bar: 50 μm (d), 20 μm (h) (GIF 137 kb)SOD1High Resolution Image (TIF 3969 kb)Supplementary Fig. 2(a) and the bNCSC monolayer during differentiation stage (b). Immunostaining of bNCSC neurospheres shows expression of the glial specific marker GLAST (c) and the neural crest stem cell specific markers SOX2 (d). Scale bars: a,b 20 μm; c,d 50 μm. (GIF 164 kb)Overview of RFP-expressing bNCSC neurospheres in pre-differentiation High Resolution Image (TIF 6408 kb)Supplementary Fig. 32O2) on the survival of bNCSCs in vitro. Cultures were exposed to different concentrations of H2O2 for 3 hours. The survival of bNCSCs is not affected by different concentrations of H2O2 (GIF 5 kb)Effect of hydrogen peroxide (HHigh Resolution Image (TIF 18434 kb)Supplementary Fig. 4G93A MNs one week after implantation to the ventral horn. SOD1G93A MNs (green) are located in the grey matter close to the central canal and together with bNCSCs (red) in the white matter of the ventral funiculus. Scale bar: 50 μm (GIF 127 kb)bNCSCs and SOD1High Resolution Image (TIF 3407 kb)ESM 1(PDF 1224 kb)Below is the link to the electronic supplementary material.