In addition, MPT0G009 markedly inhibited cytokine secretion and macrophage colony-stimulating factor/receptor activator of nuclear factor kappa B ligand-induced osteoclastogenesis by macrophages (50 ng/ml each). These MPT0G009 effects on cytokine secretion and osteoclast formation were reduced by the overexpression of HDAC 1 (class I HDAC) and 6 (class II HDAC) in cells, suggesting that these effects were due to the inhibition of its activity. In an in vivo rat model, oral administration of MPT0G009 (25 mg/kg) significantly inhibited paw swelling and bone destruction. Furthermore, compared with SAHA, MPT0G009 exhibited longer half-life  and higher oral bioavailability (13%) in rats. These results established the preclinical anti-arthritic efficacy and pharmacokinetic parameters of MPT0G009, which may provide a new therapeutic approach for treating inflammatory arthritis.The pathology of rheumatoid arthritis includes synoviocyte proliferation and inflammatory mediator expression, which may result from dysregulated epigenetic control by histone deacetylase (HDAC). Thus, HDAC inhibitors may be useful for treating inflammatory disease. This was a preclinical study of the HDAC inhibitor, MPT0G009. The IC However, some adverse effects of these drugs remain major concerns. Recently developed therapies that targeted cytokines have a major impact on the disease course of RA,3 however, its usage may be difficult because of increased risks of infection and nonresponse rates.5 Therefore, novel treatments that target critical intracellular molecules in synovial inflammation are required.Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease that results in destructive arthropathy. The complex interactions between the synovial and immune system cells result in synoviocyte proliferation, release of inflammatory cytokines/chemokines that recruit immune cells into the affected joints and activate infiltrated cells, and expression of degradative enzymes, resulting in progressive joint damage.7 Histone deacetylases (HDACs) are categorized into four categories: class I ; class IIa  and class IIb (HDAC6 and 10); class III (SIRT1–7); and class IV (HDAC11). These are involved in the post-translational modifications of core histone and nonhistone proteins. Recent proteomic analyses have shown that a substantial number of key signal transduction components and transcription factors that regulate immune responses and inflammation are HDAC substrates.9 Thus, HDAC inhibitors have been examined as promising anti-inflammatory agents.10 However, there are very few HDAC inhibitors that have been sufficiently developed to undergo clinical trials for RA treatment. One study showed that ITF2357 (givinostat) ameliorated joint inflammation and prevented cartilage and bone destruction in an animal model.11 However, a phase II safety and efficacy clinical trial of ITF2357 that evaluated patients with active systemic onset of juvenile idiopathic arthritis, but not those with RA, suggested that HDAC inhibitors still require considerable development for use as RA therapeutics.The imbalance in proinflammatory cytokine networks is central to disease development.N-hydroxyacrylamide) indolines derivative HDAC inhibitors. Among these, MPT0G009 (3--N-hydroxyacrylamide) demonstrated potent cytokine release inhibitory effects.12 In this study, we evaluated the anti-arthritic effects of MPT0G009 in vitro and in an in vivo model and determined the pharmacokinetics and its maximum tolerated dose (MTD). Our results showed that MPT0G009 was >10 times potent than the marketed HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on HDACs inhibition in the human RA fibroblast-like synoviocytes and rabbit synovial fibroblast cell line, HIG-82. MPT0G009 also had a longer half-life, higher systemic exposure and oral bioavailability than SAHA. Our results show that MPT0G009 is a potential candidate for clinically treating RA.We have synthesized 1-arylsulfonyl-5- or SAHA  for 30 min before and during incubation for 24 h with lipopolysaccharide  or IL-1β (10 ng/ml). These supernatants were then assayed for prostaglandin E2 (PGE2), NO and IL-6.To evaluate the anti-inflammatory effects of MPT0G009, supernatants from cultures of RAW264.7 cells  and RA f2 production by both cell types, NO production by RAW264.7 cells and IL-6 production by RA-FLS in a concentration-dependent manner; MPT0G009 was more effective than SAHA. As synoviocyte proliferation has a pivotal role in RA pathogenesis, we assessed the effects of MPT0G009 and SAHA at the above mentioned concentrations on the proliferation of HIG-82 synoviocytes  had a similar effect. In addition, treating RA-FLS cells, but not HIG-82 cells, with MPT0G009 for 12 or 24 h resulted in decreased levels of HDAC3 but not of the other isoforms, whereas SAHA had no effect  was added for 24 h, and nitrite and PGE2 levels were measured in culture supernatants. These results showed that overexpression of HDAC significantly reduced the inhibitory effects of MPT0G009 or SAHA on LPS-mediated NO or PGE2 release =13.0% (The pharmokinetic parameters of MPT0G009 in rats after single-dose intravenous (i.v.) and oral administration are summarized in F)=13.0% . Table 3in vitro and in an in vivo model. MPT0G009 was found to inhibit cytokine release by LPS-stimulated macrophages, inhibit synoviocyte proliferation, reduce osteoclast differentiation and ameliorate arthritis progression. In addition, we found that the overexpression of HDAC reduced the inhibitory effects of MPT0G009, showing that its effects were primarily due to a decrease in HDAC activity. Furthermore, we determined the pharmacokinetic parameters and MTD of MPT0G009.In this study, we evaluated the preclinical anti-arthritic effects of an orally available HDAC inhibitor, MPT0G009, both 16 Recent proteomics analyses have shown that a substantial number of key signal transduction components and transcription factors that regulate immune responses and inflammation are HDAC substrates.10 Consistent with these findings, HDAC inhibitors reduce LPS-induced or IL-1-induced expression levels of inflammatory mediators18 and inflammatory mediator-induced synovial inflammation and subsequent cartilage destruction.19 A majority of these are pan-HDAC inhibitors and target the following two primary classes of HDAC enzymes: class I and class II. Although which HDACs are involved in the processes associated with RA inflammation remains unclear, it has been suggested that several isoforms may have important roles in RA pathogenesis. High HDAC1 levels were observed in RA synovial tissues; these levels correlated with the higher expression of tumor necrosis factor.20 HDAC3 knockdown inhibited the expression of VCAM-1 in a human endothelial cell line, thereby reducing monocyte adhesion.21 HDAC3 knockdown also inhibited osteoclast differentiation by mouse bone marrow and RAW264.7 cells.22 An HDAC6 inhibitor promoted T-regulatory-dependent suppressive activity in an autoinflammatory model.23 HDAC8 was upregulated during the later stages of osteoclast development after RANKL treatment.24 Therefore, pan-HDAC inhibitors probably exert their therapeutic effects in multiple ways.9 In this study, MPT0G009 significantly inhibited the activity of all the four class I HDACs  and a class IIb HDAC (HDAC6). Thus, it was not unexpected that it suppressed RA development through multiple mechanisms by inhibiting RA-FLS proliferation, osteoclast differentiation and inflammatory mediator release.HDAC inhibitors were originally developed as anticancer agents that functioned through multiple mechanisms, such as inducing cancer cell apoptosis and cell cycle arrest.25 However, the question of why MPT0G009 induces proteasome-dependent downregulation of HDAC3 cannot be explained. We speculate that it may be because of the inhibition of HDAC6 activity, as HDAC6 regulates α-tubulin acetylation and facilitates transportation of degradable proteins from the aggresome to the lysosome.27 In addition, an HDAC6 inhibitor resulted in α-tubulin hyperacetylation, disruption of the interaction between HDAC6 and dynein, and an increase in the delivery of ubiquitinated proteins to the proteasome.28 Our results showed that in RA-FLS, MPT0G009 inhibited HDAC6 activity and also reduced the levels of HDAC3 by proteasomal-mediated degradation. This latter effect was inhibited by the 26S proteasome inhibitor MG-132. However, why MPT0G009 treatment only downregulated the expression of HDAC3 and not that of other HDAC isoforms will require further investigation.We also found that MPT0G009 treatment resulted in proteasome-dependent downregulation of HDAC3. Interestingly, SAHA treatment was found to decrease the levels of HDAC2 and HDAC4 in the cortex and brain stem in the R6/2 Huntington's mouse model.7 A recent study showed that a combination of class I and II HDAC inhibitors synergistically inhibited osteoclast activity, and this combination was more effective than a class I or class II inhibitor alone,24 suggesting that a pan-HDAC inhibitor may be more effective in suppressing the osteoclast activity. Our results showed that MPT0G009 significantly inhibited RANKL-induced osteoclast formation at a concentration as low as 5 nM, whereas SAHA had little effect even at a concentration as high as 50 nM. We suggest that this may be because MPT0G009 has a greater inhibitory effect than SAHA on both class I and II HDACs. In addition, downregulation of MPT0G009-induced HDAC3 may contribute to its inhibitory effect on osteoclast differentiation because HDAC3 downregulation inhibits osteoclast differentiation in mouse bone marrow and RAW264.7 cells.22 Another study showed that SAHA could suppress osteoclastogenesis in vitro but at a concentration (300 nM) that is higher than the maximum concentration used in our study (50 nM).29Bone erosion is associated with RA severity and poor functional outcomes.30 as shown in A previous study established the pharmacokinetic parameters for SAHA in rats,Taken together, the superior potency and pharmacokinetic properties of MPT0G009 compared with those of SAHA in this preclinical setting makes it a candidate for clinically treating RA.12 The non-conjugated primary antibodies used were against HDAC6 or β-actin , HDAC1, HDAC2, HDAC3 or histone H3 , or acetyl-histone H3 or p21/WAF1/Cip1 . Fluorescein isothiocyanate (FITC)-conjugated anti-CD51/61 antibodies was from BD Biosciences . The labeled secondary antibodies were horseradish peroxidase- or FITC-conjugated anti-mouse or anti-rabbit IgG antibodies . Fluorogenic HDAC1, 2, 3, 4, 6 or 8 assay kits were from BPS Bioscience Corp. , colorimetric HDAC activity kits from BioVision Inc. , PGE2 immunoassay kits from R&D Systems  and IL-6 ELISA kits from eBioscience Inc. . The pGL4.32[luc2P/NF-κB-RE/Hygro] and pGL4.30[luc2P/NFAT-RE/Hygro] plasmids were from Promega Corp.  and the HDAC1-Flag (plasmid 13820) and HDAC6-Flag (plasmid 13823) plasmids from Addgene Inc. . TurboFect transfection reagent was from Fermentas . Unless otherwise stated, all other chemicals were from Sigma-Aldrich .MPT0G009 and SAHA were synthesized by Dr. Jing-Ping Liou to >98% purity.2/95% air in, respectively, 90% Ham's F-12 or Dulbecco's modified Eagle medium, both containing 10% heat-inactivated fetal bovine serum (FBS)  and 1% penicillin/streptomycin . Human RA-FLSs from Cell Application Inc.  were grown in synoviocyte growth medium from the same supplier.The HIG-82 rabbit synovial fibroblast and RAW264.7 mouse macrophage cell lines were purchased from the Bioresource Collection and Research Center  and the cells cultured at 37 °C in 5% COFluorogenic HDAC assay kits were used to assess the ability of HDAC inhibitors to inhibit deacetylation of lysine residues on the substrate by recombinant HDAC1, 2, 3, 4, 6 or 8 according to the manufacturer's instructions.Colorimetric HDAC activity kits were used to assay HDAC activity in cell lysates according to the manufacturer's instructions.HIG-82 synoviocytes or RA-FLSs were incubated for 48 h with the indicated concentrations of MPT0G009 or SAHA, then were fixed with 10% trichloroacetic acid, stained for 30 min with sulforhodamine B , and washed repeatedly with 1% acetic acid, then protein-bound dye was dissolved in 10 mM Tris base solution and the optical density at 510 nm measured.Osteoclast formation was measured by quantifying cells stained for the osteoclast marker TRAP using an acid phosphatase kit . Briefly, the specimens were fixed for 30 s in fixative solution , then incubated for 1 h at 37 °C with a mixture of the provided naphthol AS-BI phosphate and tartrate solutions, followed by counterstaining with hematoxylin solution. Osteoclasts were counted as TRAP-positive multinuclear (>3 nuclei) cells using light microscopy. The morphological features of osteoclasts were also photographed.2HPO4 and 0.1 M citric acid buffer, pH 7.8), resuspended in 1 ml of propidium iodide staining buffer , incubated at 37 °C for 30 min in the dark, sorted by FACScan, and analyzed using CellQuest software (BD Biosciences).RAW264.7 cells and RA-FLSs were harvested and washed twice with FACS washing buffer (1% FBS in PBS), followed by incubation at 4 °C for 30 min with antibodies. After three washes with FACS washing buffer, the fluorescence of the cells was analyzed on a FACScan flow cytometer (BD Biosciences). To detect cell cycle progression, cells were incubated with or without the indicated agent for 24 h, washed twice with ice-cold PBS, collected by centrifugation and fixed in 70% (v/v) ethanol for 2 h at −20 °C. They were then incubated for 30 min at room temperature with 0.2 ml of DNA extraction buffer  and 1 μl of TurboFect transfection reagent were mixed for 20 min at room temperature, then were added to the cells and the mixtures incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2 in air. For the reporter gene assay, 100 μl of reporter lysis buffer (Promega Corp.) was added, then the cells were scraped off the dishes, centrifuged at 17 000 g for 30 s at 4 °C and the supernatants collected. Aliquots of cell lysates (20 μl) were placed in the wells of an opaque black 96-well microtiter plate, then 40 μl of luciferase substrate (Promega Corp.) was added and the luminescence immediately measured in a microplate luminometer .RAW264.7 cells or RA-FLSs were seeded 1 day before transfection. The plasmids pGL4.32[n=3 per group) i.v. (2 mg/kg dose) by a bolus injection to jugular vein or periorally (20 mg/kg dose). Blood sample (0.15 ml) from 0 h (before dosing) to 24 h was collected from each animal via the jugular vein cannula and stored in ice. Plasma was separated from the blood and centrifuged at 14 000 g for 30 min at 4 °C. All samples were analyzed for the test compound by LC-MS/MS . Data were acquired via multiple reactions monitoring. Plasma concentration data were analyzed with standard noncompartmental method.A 1–5 mg/ml dosing solution was preparing by dissolving appropriate amount compound in a mixture of PEG400/DMSO  for i.v. administration and 1% carboxylmethyl cellulose, 0.5% Tween 80 for oral dosing, respectively. Eight to 10-week-old male Sprague–Dawley rats were obtained from BioLASCO Corp. , and tested compound was separately administered to group of rats  in mineral oil at 3 mg/ml. CFA-induced arthritis was induced by intradermal injection of 100 μl of the CFA emulsion into the base of the right hind paw on day 0. MPT0G009 (25 mg/kg), SAHA (200 mg/kg) or vehicle was given daily by gavage from days 2 to 21. On days 0, 2, 6, 9, 13, 17 and 21, the animals were weighed and both hind paw volumes measured using a digital plethysmometer . On day 21, micro-CT of the paws was performed by the Core Facilities Center of the National Research Program for Biopharmaceuticals using an in vivo micro-CT scanner  at 18 μm resolution and 180° scanning with a rotation step of 0.8° per image, 300 ms integration time, 70 keV photon energy and 350 μA current. The reconstructed volumes from micro-CT scans were analyzed by CT Analysis Software . Quantification of volumetric BMD and bone volume (BV) was performed in a defined bone area ranging 12 mm from tarsals to the end of the calcaneus. The BMC was described by the product of BV and BMD. On day 21, the rats were killed and specimens from the phalanges and ankle joints stained with hematoxylin–eosin or safranin O for histopathological assessment by the National Laboratory Animal Center. Intensity of arthritis was scored as described previously from 0 to 4.19 All four legs were scored, so the highest possible arthritic score was 16.Five-week-old male Lewis rats were obtained from the National Laboratory Animal Center . Complete Freund's adjuvant (CFA) was prepared by suspending heat-killed Animal experiments were approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (IACUC number: 20110376 and 20110303).post hoc test was used to determine the pairs of groups showing statistically significant differences. A P-value of <0.05 was considered statistically significant.The data are expressed as the mean±S.E.M. and were analyzed using one-way ANOVA. When ANOVA showed significant differences between groups, Tukey's


The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. Additionally, Wnt/β-catenin signaling pathway is one of the most important intracellular pathways that controls cancer progression. To date most of the identified targets of this pathway are shown to harbor tumorigenic properties. We previously showed that Mannosyl glycoprotein acetylglucosaminyl-transferase (MGAT1) enzyme is among the Wnt/β-catenin signaling putative target genes in hepatocellular carcinoma cell lines (Huh7).MGAT1 protein levels were determined by Western Blotting from Huh7 cell lines in which Wnt/β-catenin pathway was activated by means of different approaches such as LiCl treatment and mutant β-catenin overexpression. Luciferase reporter assay was used to analyze the promoter activity of MGAT1. The mRNA levels of MGAT1 were determined by quantitative real-time PCR from Huh7 cells that were treated with either Wnt agonist or GSK-3β inhibitor. Wound healing and XTT cell proliferation assays were performed in order to determine the proliferation and migration capacities of MGAT1 overexpressing stable Huh7 cells. Finally, xenograft experiments were carried out to measure the tumor formation capacities in vivo.In this study we showed that the activation of Wnt/β-catenin pathway culminates in the upregulation of MGAT1 enzyme both at transcriptional and post-transcriptional levels. We also showed that overexpression of the β-catenin gene (CTNNB1) increased the promoter activity of MGAT1. We applied a set of complementary approaches to elucidate the functional importance of MGAT1 as a vital target of Wnt/β-catenin signaling in Huh7 cells. Our analyses related to cell proliferation and migration assays showed that in comparison to the control cells, MGAT1 expressing Huh7 cells have greater proliferative and invasive capabilities. Furthermore, the stable overexpression of MGAT1 gene in Huh7 cell lines lead to a significant increase in tumor growth rate in Severe Combined Immunodeficient (SCID) mice.Taken together, we showed for the first time that MGAT is a novel Wnt/β-catenin pathway target that has important implications for tumorigenesis. The Wnt Signaling pathway is conserved in various organisms from worms to mammals, and play important roles in development, differentiation, cellular proliferation, morphology, motility and fate . Wnt proThe canonical Wnt/β-catenin signaling pathway is the best understood Wnt signaling pathway. β-catenin, which is associated with the membrane bound E-cadherin, or is free in the cytoplasm, plays a major role in the transduction of the canonical Wnt/β-catenin signal , 6. CytoIn various types of cancers  Wnt/β-catenin associated molecules, such as proto-oncogene CTNNB1, and tumor suppressor genes such as APC or AXIN show alterations . Among tIn a quest for Wnt signaling targets we identified MGAT1 -glycoprotein beta-1,2-N-acetylglucosaminyl-transferase), which is a medial Golgi enzyme that catalyzes the first step in the conversion of oligomannose-type N-glycans into complex and hybrid N-glycans . The dedMGAT1 is essential for normal embryogenesis in the mouse. Previous studies showed that mice lacking a functional MGAT1 gene die at approximately 10 days after fertilization with multiple developmental abnormalities, indicating the importance of complex and hybrid type N-glycans in cell-cell interactions .Null mutations in Drosophila MGAT1 have been shown to produce defects in adult locomotory activity when compared to wild type flies. Moreover, the null mutant males are sterile and determined to have a severely reduced mean life span. Thus it is stated that, MGAT1-dependent N-Glycans are required for locomotory activity, life span and brain development in flies .In our previous study , we perf2. Passages 6–15 were used for the experiments. Cells were routinely passaged before reaching ~90% confluence. Transfections were done using the Turbofect transfection reagent (Thermo) according to DNA: reagent ratios suggested by the manufacturer.Cell lines were cultured in Dulbecco’s modified Eagle’s medium (Hyclone) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Hyclone) at 37 °C in 5% COHuh7 cells (Cat no. PTA-4583) were treated with Wnt agonist  and GSK-3β Inhibitor XII, TWS 119  in concentration-dependent manner. DMSO was used as negative control.For the generation of stable cell lines; Huh7 cells in 6-well plates were transfected separately with the following constructs: pIRES2-EGFP-MGAT1 and pIRES2-EGFP-GFP (control). Transfected cells were cultivated in the presence of G-418 geneticin sulfate solution (Hyclone). 500 μg/ml geneticin was used as working solution for selection of stable cell lines. The selection was carried on for 3–4 weeks, while the cells were passaged when confluency was reached. 250 μg/ml geneticin was used as working solution for the maintenance of stable cell lines.Huh7 cells stably expressing the corresponding genes were grown to full confluency on a 6-well cell culture plate. A wound is introduced by scratching the confluent monolayer cells with a pipette tip (time = 0). Plates were washed twice with 1X PBS in order to remove detached cells and incubated with complete growth medium. Cell migration into the scraped empty space was followed at 24 h, 48 h and 72 h time intervals using bright field microscopy. At the end of 72 h, the distances across the wound were quantified by using the ImageJ software.Stable Huh7 cells were cultivated in DMEM without any antibiotics for 48 h before in vivo studies. For tumor formation assay, the confluent cells in 10-cm culture plates were trypsinized and washed twice with PBS. The cell pellets were resuspended in 150 μl PBS and kept on ice until injection. The cells were injected subcutaneously and bilaterally (control on one side) into the abdominal region of 6–14-week-old female SCID mice. Tumors at the injection sites were collected at different days post-injection to determine tumor volume and weight. The isolated tumors were snap-frozen in liquid nitrogen and stored at −80 °C for further use.Total RNA isolation was performed with GeneJet RNA Purification kit (Thermo) following the manufacturer’s instruction. Adherent cells were washed with PBS, trypsinized and centrifuged at 1600 rpm for 5 min. Cell pellets were dissolved in 600 μl lysis buffer and vortexed for 10 s. Homogenization of the lysate was accomplished by passing through a RNase-free syringe several times. Total RNA concentration was measured by nanodrop and absence of DNA contamination is verified (OD 260/280 ratio > 2). Total RNA integrity was checked by loading the samples on 1% agarose gel and confirming the presence of 18S and 28S ribosomal RNAs. Purified RNA samples were stored at −80 °C until use.Luciferase assay was performed according to Dual-Glo Luciferase Assay System (Promega) protocol. For the assay, cells were co-transfected with 1 μg of a pGL3-luciferase reporter plasmid including promoter of interest, 1 μg β-catenin, TCF4 or dNTCF4 expression plasmid and 100 ng of pGL3-SV40-Renilla  per well of a 12-well plate. Transfection is done by using Turbofect transfection reagent (Thermo) according to the manufacturer’s instructions. About 48 h post-transfection, cells were collected by trypsin, and then washed with 1X PBS twice. Pellet was completely resuspended in 100 μl 1X PBS and placed into 96-well plate. Firefly and Renilla luciferase activities were measured respectively, using a fluorometer. Firefly luciferase readings were normalized to Renilla luciferase readings and graphs were plotted in Microsoft Excel.t = 0 h). For protein isolation from the treated cell lines, cell lysis buffer with freshly added protease inhibitors was added on to the cells (100 μl for per well of 6-well plate and 400 μl for 10 cm plate) and the cells were subjected to protein extraction procedure.Huh7 cells were seeded into 10 cm culture dishes or 6-well plates based on the experiments. Following day, lithium chloride (LiCl) or sodium chloride (NaCl) was added to the medium of the cells to a final concentration of 25 mM. Reverse treatment was employed where all the cells treated at different time points were harvested at the same time  according to the manufacturer’s protocol. The primers were chosen among the primers of MGH primer bank encompassing intron/exon junctions and blasted against the target genome. The reaction mixtures were distributed into the wells of 96-well qPCR plate and cDNAs were added last. The plate was centrifuged at 2000 rpm for 2 min in order to collect the components to the bottom of the wells. PCR reaction was started with initial denaturation at 95 °C for 10 min. Amplification cycle consists of three steps; a denaturation step at 95 °C for 15 s, an annealing step at 60 °C for 30 s and an elongation step at 72 °C for 30 s. It was repeated for 40 cycles and finished by a melting curve step. For each gene, expression levels were normalized to GAPDH as the control. Relative expression levels were calculated with the 2-ΔΔCt method, comparing the expression level of the gene of interest between the test sample and control sample.3 cells per well. The cells were incubated at 37 °C incubator for 3 days. At the day of the experiment, the XTT labeling reagent was mixed with an electron-coupling reagent in a 50:1 ratio. 50 μl of XTT labeling mixture was added per well and the cells were incubated with this mixture for 2 h at 37 °C. The spectrophotometric absorbance of the samples at 475 nm were measured in a plate reader. A second measurement was performed at 650 nm as the reference wavelength.XTT Cell Proliferation Assay Kit II (Roche) was used according to manufacturer’s instruction. The stable Huh7 cells were seeded into 96-well cell culture plates at a concentration of 4 × 10The candidate transcriptional targets of the canonical Wnt/β-catenin pathway were determined previously in our laboratory by using genome wide microarray analyses and SAGE techniques. MGAT1 gene has been included among the candidate Wnt/β-catenin target genes based on the data obtained from SAGE and Microarray analyses. It is one of the genes that were shown to be upregulated in response to mutant β-catenin overexpression in the Huh7 cell line .The basal protein levels of MGAT1, along with a few other key proteins of cell proliferation were determined in various cell types, including carcinoma and non-carcinoma cell lines Fig. . We obseLithium chloride (LiCl) is a widely used GSK-3β inhibitor that results in the activation of Wnt/β-catenin signaling. It interferes with the function of GSK-3β degradation complex and prevents β-catenin phosphorylation and degradation . Huh7 ceUnlike other hepatocellular carcinoma cell lines, Huh7 cells do not show abnormal activity of Wnt/β-catenin pathway. In order to activate the pathway, Huh7 cells were transfected with the plasmids expressing either the wild-type β-catenin, S33Y-β-catenin or the combination of S33Y-β-catenin and TCF4. The protein levels of MGAT1 were determined by Western Blotting Fig. . HSF2 anIn order to determine MGAT1 mRNA levels in response to Wnt/β-catenin pathway activation, Huh7 cells were treated with either Wnt agonist or GSK3β inhibitor XII (TWS 119). TWS 119 is a synthetic chemical inhibitor of GSK3β and used commonly for the activation of Wnt/β-catenin signaling . QuantitThe results of q-RTPCR indicate that MGAT1 mRNA levels increased gradually with increasing concentrations of the Wnt agonist. As expected, AXIN2 mRNA was upregulated by Wnt agonist treatment Fig. . MGAT1 mTo get insights into the transcriptional regulation of MGAT1 gene under modulated Wnt/β-catenin pathway activation we performed luciferase reporter assay to monitor the promoter activity of this gene. For positive control of luciferase activity, TOPFLASH vector which contains three copies of wild-type TCF4 binding sites was used. On the other hand, FOPFLASH vector which contains mutant TCF4 binding sites, was used as negative control. The ratio of firefly luciferase to renilla luciferase was determined for each case, and plotted as relative luciferase activity.Overexpression of the degradation-resistant β-catenin mutant (S33Y-β-catenin) in Huh7 cells resulted in higher luciferase activity from MGAT1 promoter compared to the negative control vector FOPFLASH, or the promoterless luciferase vector pGL3-basic Fig. .Fig. 3LuFurthermore, S33Y-β-catenin was overexpressed in Huh7 cells in combination with either wild-type transcription factor TCF4 or its dominant negative form (dN-TCF4). Relative luciferase activity from MGAT1 promoter was determined together with the activity from the promoterless pGL3-basic vector as the negative control. Overexpression of S33Y-β-catenin together with TCF4 led to more than 8-fold increase in MGAT1 promoter activity; however, when dominant negative form of TCF4 (dN-TCF4) is overexpressed with S33Y-β-catenin, this activity was reduced nearly in half Fig. .In order to activate the pathway, three different ligands of the canonical Wnt/β-catenin signaling pathway  were overexpressed in Huh7 cell lines by transfecting the cells with each of the pLNCX-Wnt1, pLNCX-Wnt3a and pLNCX-Wnt5 plasmids. Relative luciferase activity from the MGAT1 promoter was determined in each case. Overexpression of Wnt1 led to the highest luciferase activity from MGAT1 promoter (approximately 15-fold increase compared to control), whereas overexpression of Wnt3a and Wnt5 also led to more than 10-fold increase in luciferase activity compared to the one obtained from mock transfection Fig. . As a reStable expression of MGAT1 and GFP (as negative control) genes in Huh7 cells was established as a first step, in order to analyze the effect of MGAT1 both in vitro and in vivo.Wound healing assay was performed in order to determine whether there existed any difference in the cell migration capacities of the stable Huh7 cells. MGAT1 and GFP overexpressing Huh7 cells were used for this assay. After introducing a scratch across the confluent and adherent layer of cells in 6-well plates, the cells were visualized under the microscope at different time points starting from 0 h up to 72 h. The widths of the scratches indicated by yellow lines were quantified by Image J software Fig. .Fig. 4WoDifferences between wound closure capacities of the stable cells were observed after 72 h. Distances across the wound were measured at each time point, and difference between the initial and final values were computed as percent wound closure for each stable cell line. Overall, MGAT1 overexpressing stable Huh7 cells demonstrated a higher percentage of wound closure  compared to the cells overexpressing GFP as the control  Fig. . These rIn order to determine whether there is any difference in the cellular proliferation properties of the stable cell lines, we perform XTT Assay. The stable Huh7 cell lines overexpressing MGAT1 and GFP (control) were grown at 96-well culture plates for three days under two different sets of serum starvation conditions (2% FBS and 5% FBS). At the end of three days, absorbance spectra for the cells treated with XTT reagent was recorded at 475 nm using an ELISA plate reader. Normalization was done by taking another set of measurements at the reference wavelength of 650 nm. The results indicate that the relative absorbance values are approximately 30% higher in MGAT1 overexpressing cells compared to the GFP overexpressing cells in both of the serum starvation conditions Fig. .In order to test the tumor forming abilities of the stable Huh7 cell lines in vivo, we performed Xenograft assays in SCID mice. The mice were injected subcutaneously and bilaterally from their flank regions. MGAT1 overexpressing Huh7 cells were injected on the left flank of the mice, while GFP overexpressing Huh7 cells were injected on the right flank as the control Fig..Fig. 5MGThe mice were checked for tumor formation regularly. These experiments clearly indicate visible differences in tumor sizes 3–4 weeks after the injection of MGAT1 overexpressing Huh7 cells into the mice. At this point, the mice were sacrificed and tumors were isolated Fig. . DiffereThe canonical Wnt/β-catenin signaling pathway is a highly conserved pathway and it is involved in various differentiation events during embryonic development, such as axis formation, cellular proliferation, differentiation and morphogenesis. β-catenin is considered to be the key molecule in this pathway. In addition to its functions in vertebrate early development, Wnt/β-catenin pathway has the potential to initiate tumor formation when aberrantly activated. Those characteristics of the Wnt/β-catenin signaling pathway makes the pathway itself and its targets important subjects in cancer research fields. In fact, the genes regulated by this pathway are potential drug and gene therapy targets. Therefore, we previously performed SAGE and microarray screens in our laboratory in order to identify novel transcriptional targets of the Wnt/β-catenin pathway. As the result of these analyses, a number of genes were determined to be either upregulated or downregulated significantly in an experimental setup mimicking the constitutively active Wnt/β-catenin pathway.MGAT1 was one of the genes that showed differential expression levels in response to β-catenin activation. Moreover, it was previously included among the candidate target genes of Wnt/β-catenin signaling in SAGE analyses . MGAT1 iTo address this more closely, we used various carcinoma cell lines (mostly hepatocellular carcinoma) and non-carcinoma cell lines to monitor the basal protein expression levels of MGAT1 alongside other important key cell proliferation proteins Fig. . MGAT1 pTo better complement these findings, a series of cell migration and proliferation assays were performed to assess the proliferative potentials of the stable cell lines in vitro. The results of wound healing assay suggest that MGAT1 overexpressing stable cells were more effective in closing the wound compared to GFP overexpressing cells, which gives us a hint about their higher migration and proliferation abilities. The stable cell lines were also analyzed by XTT cell proliferation assay to test for proliferation related to metabolic activities of the cells. At normal growth conditions (10% FBS) there appeared to be no significant difference between the absorbance values of the stable cell lines (data not shown). However, under serum starvation conditions (2% or 5% FBS), MGAT1 overexpressing cells displayed higher proliferation rate compared to GFP overexpressing cells.To further substantiate the impact of MGAT1 on tumorigenesis, and assess its role in vivo tumorigenesis, the stable cell lines were subsequently used for xenograft assays. The injections were made subcutaneously and bilaterally such that the left flank regions of mice were injected with MGAT1 overexpressing cells, whereas the right flank regions were injected with control cells (GFP overexpressing Huh7 cells). In vivo xenograft experiments demonstrated that MGAT1 overexpressing cells were able to generate larger tumors compared to GFP overexpressing cells.MGAT1 is one of the proteins functioning in the process of N-glycan synthesis, which play important roles in cell-cell and cell-matrix interactions essential for the development of multicellular organisms. Proteoglycans are the key regulators of cell-matrix dynamics and there are numerous studies showing that abnormal or erroneous expression of proteoglycans can lead to various types of carcinoma –22. WithTaken together, we identified for the first time that MGAT1 gene is a novel transcriptional target of Wnt/β-catenin pathway. Furthermore, we have shown MGAT1 to be upregulated in response to Wnt/β-catenin pathway activation. Since this pathway is one of the most important intracellular pathways for cancer progression, we tested this novel target gene in terms of its tumorigenic capacity. As a result, we were able to characterize MGAT1 gene as a potential oncogene, since the overexpression of this gene in hepatocellular carcinoma cell lines leads to the formation of larger tumors when transplanted into SCID mice. Future studies would need to be carried out, such as transcriptomic analysis of the tumors by RNA Sequencing technique, in order to determine which pathways and biological processes contribute to these mechanisms.


The incidence and mortality of intrahepatic cholangiocarcinoma (ICC) are increasing worldwide in recent decades. Osteopontin (OPN) plays an important role in cancer metastasis, but its functional mechanism in ICC is not clear yet. In this study, we found that OPN level was elevated both in plasma and tumor tissues of ICC patients, which was closely related to a shorter overall survival (OS) and high probability of tumor relapse after curative resection. The gain- and loss-of-function studies determined that OPN could promote ICC growth and metastasis. OPN selectively interacted with β-Catenin and knockdown of β-Catenin abrogated the effects induced by OPN. OPN recruited MAPK1 and activated MEK–MAPK1 pathway to mediate the S675 phosphorylation of β-Catenin and nucleus accumulation, which induced the activation of Wnt signaling. Moreover, a significant correlation between OPN and β-Catenin was found in ICC tissues. OPN, β-Catenin, and their combination were independent prognostic indicator for ICC patients. In conclusion, OPN promotes ICC progression through recruiting MAPK1 and activating the Wnt/β-Catenin pathway and can serve as a novel prognostic marker and therapeutic target for ICC. In recent decades, the incidence and mortality rate of ICC have been increasing in the world3. ICC is characterized as a devastating disease with very dismal prognosis4. Currently, surgical resection and liver transplantation are the main treatment options; however, the 5-year survival rate is still very low due to the high probability of metastasis and relapse6. The pathogenesis of ICC is very complicated. Several signaling pathway deregulations that are related to the inflammatory, stress response, tumor growth, and metastasis have been reported to be involved, such as JAK–IL-6-STAT3, EGFR and HGF/MET, RAS/MAPK, NOTCH and WNT signaling7. Unfortunately, to date no molecular targeted therapy is effective for ICC6. Therefore, it is urgent to define the molecular mechanism involved in the progression of ICC.Intrahepatic cholangiocarcinoma (ICC), arising from the intrahepatic bile ducts, is the second most common primary liver cancer11. In our previous studies, we have confirmed that OPN plays important roles in promoting metastasis of hepatocellular carcinoma (HCC)15. Recent evidence from other studies showed that OPN was highly expressed in the tumor and stroma of ICC tissues17. Moreover, its expression in the stroma was closely associated with the overall survival (OS) of ICC patients17. However, little is understood about the function role and mechanism of OPN in ICC growth and metastasis.Osteopontin (OPN) is a chemokine-like phosphorylated glycoprotein, remaining intracellular or secreted, which is frequently upregulated in numerous human cancers and plays a pivotal role in proliferation, stemness, inflammatory response, extracellular matrix (ECM) degradation, angiogenesis, invasion, and metastasisC-MYC, CYCLIN-D1, and PROX1. These results provide a better understanding of the underlying mechanism by which OPN involved in the progression and metastasis of ICC.In the present study, we found that OPN was over-expressed in ICC tissues, which was closely associated with worse clinical outcomes and played a crucial role in tumor growth and metastasis. OPN directly interacted with β-Catenin and mediated its S675 phosphorylation and nucleus translocation by recruiting and activating MAPK1, which could upregulate Wnt target genes 19. OPN levels of five established ICC cell lines were much higher than that of HiBEpiC cells, and the OPN level was significantly associated with the metastatic abilities of ICC cells  Fig. . And qualls Fig. . These sTo further explore the functional roles of OPN in ICC, OPN was knocked down in HuCCT1 and CCLP1 cells, which showed higher invasive capabilities and OPN expression. Three OPN-specific shRNAs were designed to silence the endogenous OPN expression in ICC cells and shOPN-3 which induced a 90% reduction of OPN expression was used for further studies  (SP600125), PI3 kinase-AKT (LY294002), and Rac1 (EHop-016)  Fig. . Western16) Fig. . These f21, we assumed that OPN could recruit and activate MAPK1 to regulate Wnt signaling. Co-IP demonstrated that both exogenous and endogenous OPN interacted with MAPK1 in CCLP1 and HEK293T cells  has been reported to play a crucial role in regulating the Wnt/β-Catenin signaling pathwaylls Fig. . Confocalls Fig. . Moreovells Fig.  left, OPlls Fig. .Fig. 7OPNext, we detected whether OPN induced the Ser675 phosphorylation of β-Catenin through recruiting and activating MAPK1. Over-expressing OPN in RBE cells could remarkably induce β-Catenin Ser675 phosphorylation, which could be blocked by U0126 Fig. , right. Down-regulation of β-Catenin blocked the TOP/FOP reporter activity mediated by OPN overexpression. However, there is no significant effect on Wnt/β-Catenin transcriptional activity in RBE cells when re-expression of β-Catenin shRES following with U0126 treatment or re-expression of β-Catenin S675A, which mimicked unphosphorylation of β-Catenin at S675 Fig. . OPN up-As expected, re-introduction of β-Catenin could restore in vitro colony formation  containing 180 patients from an independent cohort. Patients with high OPN levels exhibited higher preoperative GGT level, advanced pTNM stage, and more regional lymph node metastasis , GS3Kβ (glycogen synthase kinase-3b), axin, and casein kinase 1a for proteasome degradation after phosphorylation. Recently, different studies have proved that the phosphorylation of specific sites could promote the stability and nucleus translocation of β-Catenin. The JNK2-mediated Ser191 and Ser605 phosphorylation13. Here, we revealed that OPN could recruit and activate MAPK1 to induce β-Catenin Ser675 phosphorylation and promote ICC progression. Inhibition of the MEK/MAPK1 pathway by U0126, an inhibitor for MEK/MAPK1 signaling, or overexpression of β-Catenin S675A  could block the activation of Wnt signaling and promotion of growth and metastasis of ICC cells induced by OPN.Our previous study found that OPN promoted HCC growth and metastasisIn conclusion, our study revealed OPN as a novel promoter of ICC malignant progression. Moreover, OPN selectively interacted with β-Catenin and mediated its S675 phosphorylation through recruiting and activating MAPK1 resulting in activation of the Wnt signaling  by using FuGene HD reagent . In order to normalize transfection efficiency, cells were cotransfected with 0.2 mg Renilla reniformis luciferase driven under the TK promoter , which was usually used as an internal control reporter. Luciferase assay was performed by using the Dual Luciferase Assay System kit 36 h after transfection according to the manufacturer’s protocols (Promega). Relative luciferase activity was reported as fold induction after normalization for transfection efficiency.37. Details about in vitro and in vivo assays can be found in the The in vivo mice model was established as described previouslyt-test or Mann–Whitney test, when appropriate. The impact of prognostic factors on OS and recurrence of ICC patients was assessed using Cox regression and Kaplan–Meier analysis. ROC analysis was used to find the predictive value of each indicator. Two-tailed P < 0.05 was regarded as of statistical significance marked with *, similarly P < 0.01 with ** and P < 0.001 with ***.Statistical analysis was performed using SPSS version 13.0 and Graphpad 6.0. Qualitative data are presented as number (percent), and quantitative values were shown as mean ± SD. The association between qualitative variables was analyzed using the Chi-square test or Fisher’s exact test, while quantitative values using Student's Detailed methods for immunohistochemistry, ELISAs, RNA isolation and real-time qPCR, cell culture and plasmids, immunoprecipitation, silver staining and mass spectrometry, nuclear and cytosol fractionation assay, immunofluorescence, cell proliferation assay, colony formation assay, wound-healing assay, migration and invasion assay, in vivo tumor growth, and metastasis assays are described in the Supplementary materialSupplementary Figure1Supplementary Figure2Supplementary Figure3Supplementary Figure4Supplementary Figure5Supplementary Figure6Supplementary Figure7Supplementary Figure8Supplementary Figure 9


As the number of patients with cardioembolic ischemic stroke is predicted to be double by 2030, increased burden of warfarin-associated hemorrhagic transformation (HT) after cerebral ischemia is an expected consequence. However, thus far, no effective treatment strategy is available for HT prevention in routine clinical practice. While the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) is known to protect against oxidative stress and neuronal cell death caused by ischemic brain damage, its effect on preventing warfarin-associated HT after cerebral ischemia is yet unknown. Therefore, we hypothesized that Ex-4 would stabilize the blood-brain barrier (BBB) and suppress neuroinflammation through PI3K-Akt-induced inhibition of glycogen synthase kinase-3β (GSK-3β) after warfarin-associated HT post-cerebral ischemia.We used male C57BL/6 mice for all experiments. A 5-mg warfarin sodium tablet was dissolved in animals’ drinking water (effective warfarin uptake 0.04 mg (2 mg/kg) per mouse). The mice were fed for 0, 6, 12, and 24 h with ad libitum access to the treated water. To study the effects of Ex-4, temporary middle cerebral artery occlusion (MCAO) was performed. Then, either Ex-4 (10 mg/kg) or saline was injected through the tail vein, and in the Ex-4 + wortmannin group, PI3K inhibitor wortmannin was intravenously injected, after reperfusion. The infarct volume, neurological deficits, and integrity of the BBB were assessed 72 h post MCAO. One- or two-way ANOVA was used to test the difference between means followed by Newman–Keuls post hoc testing for pair-wise comparison.We observed that Ex-4 ameliorated warfarin-associated HT and preserved the integrity of the BBB after cerebral ischemia through the PI3K/Akt/GSK-3β pathway. Furthermore, Ex-4 suppressed oxidative DNA damage and lipid peroxidation, attenuated pro-inflammatory cytokine expression levels, and suppressed microglial activation and neutrophil infiltration in warfarin-associated HT post-cerebral ischemia. However, these effects were totally abolished in the mice treated with Ex-4 + the PI3K inhibitor—wortmannin. The PI3K/Akt-GSK-3β signaling pathway appeared to contribute to the protection afforded by Ex-4 in the warfarin-associated HT model.GLP-1 administration could reduce warfarin-associated HT in mice. This beneficial effect of GLP-1 is associated with attenuating neuroinflammation and BBB disruption by inactivating GSK-3β through the PI3K/Akt pathway.The online version of this article (doi:10.1186/s12974-016-0661-0) contains supplementary material, which is available to authorized users. Globally, ischemic stroke is one of the leading causes of death and long-term disability . The numEarly HT can occur as a complication of cardioembolic ischemic stroke . AdditioThe glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) is a long-acting analog of the endogenous insulinotropic peptide GLP-1. Both GLP-1 and Ex-4 have multiple physiologic functions, such as the induction of glucose-dependent insulin release, inhibition of glucagon secretion, stimulation of B cell replication, and antiapoptotic action . Owing tPrevious studies have shown that after a hemorrhagic stroke, cytotoxic events activate the ubiquitously expressed glycogen synthase kinase-3β (GSK-3β), which increases the expression of β-catenin , 17 and All experiments were conducted using male C57BL/6 mice (body weight 18–25 g) at a constant temperature and with a consistent light cycle (from 07:00 to 18:00) under normal diet. This study was carried out in accordance with the Guide for the National Science Council of the Republic of China. All animals were treated according to protocols approved by the Institutional Animal Care and Use Committee of Fudan University.®) containing sodium citrate as the anticoagulant. Measurements of INR values and prothrombin time were performed in the Department of Central Laboratory, Jingan District Centre Hospital, Shanghai, China.A 5-mg warfarin sodium tablet  was dissolved in 375 mL of water. The C57 BL/6 mice were fed for 0, 6, 12, and 24 hours with ad libitum access to the treated water. Assuming a mouse body weight of 20 g and a water consumption rate of 15 mL/100 g per 24 h, this dosage corresponds to a warfarin uptake of 0.04 mg (2 mg/kg) per mouse over a 24-h period. Similar doses of warfarin have been previously used . After 2Mice were anesthetized with ketamine/xylazine , and their body temperature was maintained at 37 °C by a heating pad and feedback control system . A laser Doppler probe was fixed on the skull 5 mm lateral and 2 mm posterior to the bregma. A coated filament was placed on the right middle cerebral artery (MCA) with concurrent recording of laser Doppler cerebral blood flow to ensure that the cerebral blood flow decreased to below 25 % of the baseline. After 45 min, the filament was removed ; incubated for 18 h at 55 °C; and centrifuged. The supernatants were subjected to spectrophotometry at 620 nm.All the mice were killed 72 h after temporary middle cerebral artery occlusion (MCAO), and brain tissues were incubated in 2,3,5-triphenyltetrazolium chloride (TTC) for 1 h. The infarct area in each slice was analyzed by a computerized image analysis system, and the infarct volume was calculated by multiplying the distance between sections . Neurolog for 30 min. The hemoglobin-containing supernatant was collected, 80 μL of Drabkin reagent (Sigma) was added to 20-μL supernatant aliquots, and the sample was kept standing for 15 min at room temperature. The optical density in each group was measured at 540 nm, and hemorrhage volume was expressed in equivalent units by comparison with a reference curve generated using homologous blood.The hemoglobin content in brain tissue was quantified by spectrophotometric assay. The hemispheric brain tissue was homogenized with phosphate-buffered saline (PBS) and centrifuged at 13,000×Striatal brain tissues from the MCA were lysed with radioimmunoprecipitation assay buffer (RIPA) containing protease inhibitors . Proteins were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane. The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-p-GSK-3β ; anti-GSK-3β ; anti-β-actin ; anti-p-β-catenin ; anti-β-catenin , anti-claudin-3 ; anti-claudin-5 ; anti-p-Akt ; anti-Akt ; anti-ICAM-1 ; anti-VCAM-1 ; anti-IKK-β ; anti-NF-kB ; anti-HHE ; anti-Iba1 ; and anti-myeloperoxidase (MPO) . Secondary antibodies conjugated with horseradish peroxidase were used, and immunoreactivity was visualized by chemiluminescence . Bands of interest were analyzed and quantified using Scion Image.The small interfering RNA (siRNA)-mediated GSK-3β gene knockdown was performed as previously described . Brieflyg for 30 min at 4 °C and then stored at −70 °C until the assay was performed. The supernatant was assayed for tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) using enzyme-linked immunosorbent assays  as described previously [Striatal brain tissues from the MCA were homogenized and collected by centrifugation at 15,000×eviously .Concentration of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in brain DNA was measured by Piao et al.’s method , with slSeventy-two hours after MCAO, the mice were anesthetized and first perfused with saline followed by fixation with buffered paraformaldehyde (4 %). The brains were removed and post-fixed in 4 % paraformaldehyde; the paraformaldehyde was then removed and replaced with 30 % sucrose solution overnight. Then 15-μm coronal sections were obtained on a cryostat. The slices were blocked with PBS containing 5 % bovine serum albumin (BSA), 10 % goat serum, and 0.3 % Triton-X 100. Next, the slices were incubated with the primary antibodies anti-Iba1  and anti-TNF-α  overnight at 4 °C. Then Alexa Fluor 488 or 595 labeled secondary antibody  for 2 h at room temperature. The tissue sections were washed twice in PBS and then immersed in DAPI (Molecular Probes) solution (1:1000 dilution) for 10 min. The sections were finally rinsed in distilled water and fixed with a coverslip with anti-fade mounting medium.First, microglia activation were counted and morphologically characterized based on the following criteria. Cells with an oval cell body containing a small volume of cytoplasm and long, thin, delicate, and radially branched processes were classified as ramified microglia . Activat®. A P value <0.05 was considered statistically significant.All values are expressed as mean ± standard deviation (SD). Differences between means were analyzed using either one-way or two-way ANOVA followed by Newman–Keuls post hoc testing for pair-wise comparison using SigmaStat v 3.5n = 6) and reached the therapeutic span used in humans. These results were consistent with those previously reported [To examine the influence of warfarin on animal PT-INR values, the mice were killed at the indicated time points and the PT-INR values were measured. After warfarin administration, the PT-INR values increased in a time-dependent manner Fig. . After 2reported . In viewMCAO induced a sharp drop of rCBF, leading to extensive infarction in the cerebral cortical and subcortical areas over a series of brain sections in the mice Fig. . CompareFunctional barrier properties were evaluated using Evans blue assays, 72 h after surgery. Significantly more extravasated dye was measured in the ischemic hemispheres of mice subjected to warfarin treatment compared with the control group. Ex-4 preserved BBB integrity in the model of warfarin-associated HT after MCAO, which was associated with significantly reduced dye extravasation in Ex-4-treated animals Fig. .Claudin-3 and claudin-5 are transmembrane proteins essential for maintaining the diffusion barrier provided by tight junctions , 38. PreIt has been reported that activated Akt (p-Akt) can inactivate GSK-3β and reduce the amount of GSK-3β available for phosphorylation (through the tyrosine-216 form) , 41. TheTo examine if warfarin and Ex-4 could phosphorylate Akt after cerebral ischemia, the phosphorylation of Akt was examined 72 h after MCAO. After normalizing the values of the active p-Akt with the amount of total Akt (Akt) in each sample, we observed an increase in the Ex-4-treated mice compared to warfarin treatment alone. We indirectly studied the activation of Akt by measuring the phosphorylation of its downstream target GSK-3β in the same brain areas. As compared with the warfarin-treated MCAO mice, Ex-4 treatment significantly suppressed the phosphorylation of GSK-3β. These phosphorylation changes of Akt and GSK-3β were totally abolished when the mice were treated with Ex-4 in combination with the PI3K inhibitor—wortmannin  and interstitial cell adhesion molecule-1 (ICAM-1) , 45. WarNext, we investigated whether Ex-4 can control oxidative stress in warfarin-associated HT using lipid peroxidation indicator (HHE) and DNA oxidative injure indicator (8-OHdG). 8-OHdG is a major form of oxidative DNA damage product, and 4-hydroxyhexenal (HHE) is one of the major lipid peroxidation products that are formed by n-3 polyunsaturated fatty acids in cells exposed to oxidative stress . The expWe additionally examined the role of Ex-4 in modulating neuroinflammation by measuring expression levels of several cytokines such as IKK-β, NF-kB, TNF-α, and IL-1β. The expression levels of IKK-β and NF-kB were significantly increased after warfarin-associated HT compared to MCAO alone, while Ex-4 treatment reduced the effect and wortmannin blocked the reduction induced by Ex-4 Fig. .Fig. 8ExThe expression levels of TNF-α and IL-1β were evaluated by ELISA. Both these cytokines were upregulated in warfarin-associated HT mice, and Ex-4 blocked the increase concordantly with a similar pattern for IKK-β and NF-kB. The modulating effect of Ex-4 on the cytokines’ expression levels were reversed by co-treatment with wortmannin Fig. .Consistent with the changes in cytokine levels, immunofluorescence analysis also showed that warfarin-associated HT robustly enhanced immunofluorescence intensity of Iba1 staining (a marker of microglia/macrophages) in the MCA area compared to the MCAO group Fig. . The quaIn addition to brain resident microglia, hematogenous leukocytes have been shown to play a pivotal role in post-stroke neuroinflammation. Among white blood cells, neutrophils have attracted much interest recently and have been intensively studied. The level of myeloperoxidase (MPO) was significantly increased in the warfarin-associated HT group compared to MCAO. Ex-4 treatment reversed the MPO level. The inhibition of Akt by wortmannin restored the MPO level back to that of the warfarin-associated HT group Fig. .Atrial fibrillation is a severe independent risk factor of stroke, its attributable risk increasing with age up to more than 20 % . INR-driGLP-1 and long-acting Ex-4 induce numerous biological actions through the G protein-coupled GLP-1 receptor (GLP-1R). GLP-1R is reportedly expressed in a wide range of tissues, including the brain. Moreover, GLP-1R stimulation has shown neuroprotective actions in previous findings, thereby establishing that GLP-1R stimulation protects hippocampal neurons from amyloid-β peptide and glutamate-induced toxicity , 51. As PI3K/Akt plays a crucial role in the cell death/survival pathway through several different downstream targets including GSK-3β . A tempoIn this study, Akt phosphorylation at Ser473 and GSK-3β dephosphorylation at tyr216 were increased in warfarin-associated HT after cerebral ischemia. Administration of Ex-4 substantially decreased HT and maintained the stability of BBB. The reduced dye extravasation and brain hemoglobin level were similar to that achieved by inhibition of GSK-3β. Evidence supporting enhanced BBB stabilization by Ex-4 including decreased adherens (VCAM-1 and ICAM-1) and increased tight junction (claudin-3 and claudin-5) proteins could be totally abolished by wortmannin, a specific PI3K inhibitor. These results suggest that warfarin-associated HT reduced the expression of tight junction proteins. This effect was prevented by treatment with Ex-4 through the PI3K/Akt-GSK-3β pathway. Furthermore, Ex-4 reduced the warfarin-induced hemorrhage volume via a protective effect on vascular endothelial cells.Inflammation has been recognized as a key contributor to the pathophysiology of cerebral ischemia . InflammOur study results showed that administration of GLP-1 could reduce warfarin-associated HT in mice. This beneficial effect of GLP-1 was associated with attenuating neuroinflammation and BBB disruption by inactivating GSK-3β through the PI3K/Akt pathway. These findings have important clinical implications and would be particularly beneficial in those receiving anticoagulant therapy. Future clinical trials should focus on confirming the efficacy and safety of this therapy.


Loss of key components that form cell-cell adherens junctions, such as α-catenin, triggers severe epidermal hyperproliferation. However, the underlying molecular mechanisms remain largely unknown. We report here that neuroblastoma breakpoint family (NBPF) genes are upregulated and that NBPF7 specifically promotes cellular proliferation of α-catenin-silenced HaCaT cells through functional linkage with the NF-κB pathway. Genome-wide profiling of HaCaT cells shows that NBPF genes are upregulated following α-catenin knockdown. Data from western blot analyses are consistent with the activation of the NF-κB pathway as well as increased expression of NBPF7 by α-catenin knockdown. Co-immunoprecipitation assays indicate that NBPF7 could be detected in endogenous activated NF-κB immunoprecipitates. Immunoflurence analyses demonstrate that NBPF7 co-localizes with activated NF-κB in the nucleus after α-catenin silencing. Moreover, inhibition of NBPF7 decreases the proliferation of HaCaT cells and abolishes the enhanced proliferation associated with α-catenin knockdown in HaCaT cells. These results indicate that NBPF7 plays a key role in the α-catenin signaling pathway that regulates cell proliferation of keratinocytes. Our findings suggest that the classical NF-κB pathway plays a critical role in cellular proliferation and that NBPF7 is a functional mediator for α-catenin in the regulation of keratinocyte growth. Duriin vitro , 11. A pin vitro . The undThe nuclear factor κB (NF-κB) is a nuclear transcription factor that regulates the expression of a large number of genes critical for cell proliferation, apoptosis, survival, viral replication and inflammation. Indeed, dysregulation of NF-κB has been found in several types of cancers and autoimmune diseases , 13. MorIn the present study, we genetically silenced α-catenin in a HaCaT cell model and performed genome-wide analysis of the gene expression profile of these cells. We found that NBPF genes were indeed upregulated. We further investigated the functional linkage between NBPF7 and the NF-κB pathway in the proliferation of α-catenin-knockdown HaCaT cells.A recent study demonstrated that α-catenin modulates gene transcription by controlling actin dynamics in the nucleus . To idenin vitro and found that stable knockdown of α-catenin resulted in enhanced cell growth compared with control cells  assays with a NBPF antibody that can recognize most NBPF members . We found that NBPF7 could be detected by the NBPF antibody in endogenous p65 immunoprecipitates from the α-catenin-knockdown HaCaT cell line Figure . This waGiven the upregulation of NBPF7, we tested the possibility that it may promote cell proliferation. To that end, we used shRNAs to stably silence NBPF7 in wild type HaCaT cells and examined the ensuing rate of cell growth. We found that the mRNA expression levels of NBPF7 were significantly decreased by NBPF7 shRNAs in HaCaT cells Figure . SimilarOur results thus far suggest that inhibition of NBPF7 might abolish the enhanced cell proliferation following loss of function of α-catenin in HaCaT cells. To test this possibility, we used the same shNBPF7 lentiviruses to infect α-catenin-knockdown HaCaT cells (α-catkd1). The mRNA Figure  and protIn this study, we presented the gene expression profile of HaCaT cells following deletion of α-catenin and found that NBPF genes were upregulated. We further showed that the NF-κB pathway was activated and NF-κB interacted with NBPF7 in α-catenin-knockdown HaCaT cells. Importantly, we found that inhibition of NBPF7 decreased the proliferation of HaCaT cells and abolished the enhanced proliferative capacity of α-catenin-knockdown HaCaT cells. These results indicated that NBPF7 plays key roles in the α-catenin signaling pathway that regulates cell proliferation of keratinocytes.Although previous studies reported enhanced cell proliferation rates in α-catenin-deleted HaCaT cells , 19, theOur further validation analyses showed that silencing α-catenin led to activation of the NF-κB pathway, accompanied with upregulation of NBPF7. The pattern of α-catenin-mediated destabilization of IκBα was also observed in breast cancer cell lines . In contOur results also suggest that NBPF plays key roles in the regulation of cell proliferation by the α-catenin/NF-κB pathways. In agreement with our findings, it was shown that NBPF genes were densely covered by many high-confidence ChIP-Seq peaks of NF-κB . The NBPIn summary, we found that NBPF genes were upregulated in α-catenin-knockdown HaCaT cells and NBPF7 through functional linkage with the NF-κB pathway promoted the cell proliferation of α-catenin-knockdown HaCaT cells. These results indicated that NBPF7 plays key roles in the α-catenin signaling pathway in the regulation of cell proliferation of keratinocytes.The shRNA for α-catenin and scramble control RNA oligonucleotides were chemically synthesized and purified with high-performance liquid chromatography by GenePharma . The sequences of the RNA oligonucleotides were as follows: shα-catenin-196 5’-CAGGUUACAACCCUUGUAATT-3’; shα-catenin-1254 5’-GGACCACGUUUCAGAUUCUTT-3’; shα-catenin-2555 5’-CAGCCAAGAACUUGAUGAATT-3’; Scramble: 5’-CAGUACUUUUGUGUAGUACAA-3’. RNA oligonucleotides were transiently transfected using Lipofectamine 2000 . HaCaT cell culture was performed as previously described .α-catenin knockdown in HaCaT cells was achieved by shRNA infection  as previously described . The shRTotal RNA was extracted from HaCaT cells 3 days post-transduction of α-catenin RNA oligonucleotides using TRIzol reagent according to the manufacturer’s instructions . cDNA was synthesized using GoScript™ Reverse Transcription System according to the manufacturer’s instructions . Labeling of cDNA and hybridization to Affymetrix PrimeView human gene expression array was performed using GeneChip® Hybridization, Wash and Stain Kit  in Hybridization Oven 645  and Fluidics Station 450  according to the manufacturer’s instructions. Slides were scanned by GeneChip® Scanner 3000  and the data were analyzed with Command Console Software 3.1 (Affymetrix) using the default settings. Raw data were normalized by RMA algorithm with Gene Spring Software 11.0 .The following antibodies were used for western blot analysis: anti-NBPF15 rabbit polyclonal , anti-NBPF7 goat monoclonal , anti-α-catenin mouse monoclonal , anti-NF-κB (p65) mouse monoclonal , anti-IκBα mouse monoclonal  and anti-β-actin mouse monoclonal . Signals were developed with the enhanced chemiluminescence detection system .Cells were fixed in 4% paraformaldehyde for 15 min followed by blocking in PBS with 2.5% normal goat serum, 0.3% triton X100, and 2% bovine serum albumin for 30 min. Sections were incubated in primary antibodies against NBPF7 (1:100) and NF-κB (p65) (1:200) for 1 hour at room temperature. The nucleus was counterstained by DAPI and imaged using a Nikon eclipse E600 microscope with DP Manager Version 1.2.1.107 software.Total RNA was extracted from cultured cells using TRIzol reagent  according to manufacturer’s instructions. 1 mg of RNA was employed to synthesize cDNA using the PrimeScript RT reagent kit perfect real time  or the miScript II RT Kit . Samples were run in triplicate and normalized to GAPDH. Primer sequences are as follows: α-catenin forward primer: 5’- GGGGATAAAATTGCGAAGGAGA-3’; reverse primer: 5’- GTTGCCTCGCTTCACAGAAGA-3’; NBPF7 forward primer: 5’- CTGTGGATTTGTGGGCTGAAG-3’; reverse primer: 5’- GGAGTGTTCGCTGGCTACAT-3’; GAPDH forward primer: 5’- ATTTGGTCGTATTGGGCG-3’; reverse primer: 5’- CTCGCTCCTGGAAGATGG-3’;Co-immunoprecipitation studies were performed by lysing cells in RIPA buffer followed by pre-clearing with protein A/G agarose beads. Primary antibodies of interest (NF-κB p65 at 1:100) were added, and protein/antibody complexes were incubated overnight at 4°C, and captured on protein A/G beads. Samples were then eluted in laemmli buffer and analyzed by Western blot.Cells were plated at concentration of 2000 cell/well in 96-well plates in triplicate. The cells were monitored for cell proliferation with the Cellomics arrayscan VT1  according to the manufacturer’s directions for 5 days. Cellomics scan was performed at each time point in 5 fields. Captured images were analyzed for cell counts.All data are expressed as the mean ± SEM of at least three separate experiments. The statistical significance between two experimental groups was indicated in the figures by asterisks, and comparisons were made using the Student’s t test. Calculated P-values less than or equal to 0.05 were considered to be of statistical significance. Data were analyzed with the PRISM software version 4 .


In both colon cancer and type 2 diabetes, metabolic changes induced by upregulation of the Wnt/beta-catenin signaling and downregulation of peroxisome proliferator-activated receptor gamma (PPAR gamma) may help account for the frequent association of these two diseases. In both diseases, PPAR gamma is downregulated while the canonical Wnt/beta-catenin pathway is upregulated. In colon cancer, upregulation of the canonical Wnt system induces activation of pyruvate dehydrogenase kinase and deactivation of the pyruvate dehydrogenase complex. As a result, a large part of cytosolic pyruvate is converted into lactate through activation of lactate dehydrogenase. Lactate is extruded out of the cell by means of activation of monocarboxylate lactate transporter-1. This phenomenon is called Warburg effect. PPAR gamma agonists induce beta-catenin inhibition, while inhibition of the canonical Wnt/beta-catenin pathway activates PPAR gamma. In numerous mammalian living cells, PPAR gamma and the canonical Wnt/beta-catenin pathway behave in an opposite manner –5. Beta-The association between type 2 diabetes and cancer, including pancreatic and endometrial carcinoma, breast cancer, and colorectal and bladder cancers, has been known for many years. Epidemiological studies have reported a link between type 2 diabetes, obesity, and cancer, especially colon cancer –19. TypeThe underlying molecular basis for the link between type 2 diabetes and colon cancer is not fully understood. Hyperinsulinemia provides a link between diabetes, obesity, and cancer. Hyperinsulinemia and/or insulin resistance represent major factors in cancer pathogenesis . The hyp4.1.The Wnt/beta-catenin signaling plays an important role in cell fate, epithelial-mesenchymal transition (EMT) signaling, and embryonic development. Its dysfunction is involved in several pathologies such as carcinogenesis –34. The Importantly, glucose itself can directly impact the canonical Wnt pathway . In cancThe role of the Wnt pathway in driving cell proliferation during oncogenesis and especially colon cancer is well-known . On the In colon cancer, it has recently been shown that activation of the canonical Wnt/beta-catenin pathway partly decreases the oxidative metabolism in the TCA cycle and promotes cell proliferation . Both PDThus, in colon cancer, activation of canonical Wnt signaling directly acts on aerobic glycolysis and increases vessel development via the Wnt target gene PDK1 . Part ofMetabolic disorders combined with abnormal PDK activity are often associated with numerous diseases, such as type 2 diabetes, obesity, metabolic disorders, cardiomyopathies, neuropathies, and several types of cancer. PDKs play a key role in metabolic flexibility . They ar6.1.PPAR alpha, beta/delta, and gamma are ligand-activated transcriptional factors which belong to the nuclear hormone receptor superfamily. PPARs heterodimerize with the retinoid X receptor (RXR). PPAR gamma is expressed in various cell types, such as adipose tissues, muscles, brain, and immune cells. PPAR gamma is involved in the expression of many genes and contributes to glucose homeostasis, insulin sensitivity, lipid metabolism, immune responses, inflammation, and cell fate –75. The The functional crosstalk between PPAR gamma and the canonical Wnt/beta-catenin signaling involves the TCF/LEF binding domain of beta-catenin and a catenin binding domain (CBD) within PPAR gamma. In cells that express an APC-containing destruction complex, activation of PPAR gamma induces CPD . TZDs inInhibition of Wnt/beta-catenin signaling and upregulation of PPAR gamma have been reported in ARVC , 13. Gam APC gene mutations and this enables development of colon cancer [ APC- and p53-mutated colorectal cancer cells, RXR agonists inactivate beta-catenin via RXR alpha. RXR alpha-mediated inactivation of oncogenic beta-catenin occurs in parallel with a reduction in cell proliferation [ Min mice, used as a model for human familial adenomatous polyposis, TZDs increase the number of colon tumors [Beta-catenin-TCF/LEF signaling is activated in colon cancer . Nuclearn cancer . In colon cancer . Beta-caferation . Mutatioferation . In normferation . An earlferation . In tranferation . Activatferation . Mutatioferation . Conversn tumors , 93. Numn tumors . PPAR gamma gene is associated with severe hyperglycemia in patients. This provides a genetic link between PPAR gamma and type 2 diabetes [TZDs are potent insulin-sensitizers and certain TZDs represent a therapeutic target for the treatment of type 2 diabetes. However, the involvement of PPAR gamma in numerous pathways generates negative side-effects after PPAR gamma activation by TZDs in tissues or cells not concerned by the disease . PPAR gadiabetes . Humans diabetes . Expressdiabetes . PPAR gadiabetes .The Wnt/beta-catenin signaling pathway is involved in diabetes mellitus . Wnt sigPPAR gamma is downregulated while the canonical Wnt/beta-catenin pathway is upregulated in both type 2 diabetes and colon cancer. Wnt activates some crucial metabolic key enzymes, such as PDKs in the two pathologies. In colon cancer, this leads to aerobic glycolysis or the Warburg effect. Decreased PDH activity by upregulated PDK modifies metabolic flexibility that is, the capacity of the cell to adjust glucose and fatty acid oxidation. Competition between glucose and fatty acids for oxidation occurs at the level of the PDH complex, whose activity is decreased by PDKs. In colon cancer, partial deviation of pyruvate toward lactate contributes to protein synthesis, which are required for cell growth and proliferation. These major metabolic alterations induced by upregulated Wnt/beta-catenin signaling and downregulated PPAR gamma may partly account for the frequently encountered association between type 2 diabetes and colon cancer.


We found that Axin2 is rapidly upregulated in response to tooth damage and that these Axin2-expressing cells differentiate into new odontoblast-like cells that secrete reparative dentine. In addition, the Axin2-expressing cells produce a source of Wnt that acts in an autocrine manner to modulate reparative dentinogenesis.In non-growing teeth, such as mouse and human molars, primary odontoblasts are long-lived post-mitotic cells that secrete dentine throughout the life of the tooth. New odontoblast-like cells are only produced in response to a damage or trauma. Little is known about the molecular events that initiate mesenchymal stem cells to proliferate and differentiate into odontoblast-like cells in response to dentine damage. The reparative and regenerative capacity of multiple mammalian tissues depends on the activation of Wnt/β-catenin signaling pathway. In this study, we investigated the molecular role of Wnt/β-catenin signaling pathway in reparative dentinogenesis using an  In non-growing teeth, such as molars, primary odontoblasts can be lost if the dentine is breeched and the pulp is exposed. A second generation of odontoblast-like cells that originate from mesenchymal stem cells (MSCs) in the pulp can replace lost primary odontoblasts. The newly differentiated odontoblast-like cells secrete a form of tertiary dentine called  reparative dentine,  to seal the site of exposure and maintain pulp vitality. Continuously growing teeth, such as rodent incisors produce new dentine throughout life as an adaptation to the self-sharpening at their tips4. The continuous turnover of odontoblasts and pulp cells in the incisor is supported by mesenchyme stem cells (MSCs) that reside in a neurovascular niche at their proximal ends7. Unlike incisors, little is known about the molecular events that initiate MSCs to proliferate and differentiate into odontoblast-like cells in response to dentine damage in molars. The canonical Wnt/β-catenin signaling pathway is important for stem cell renewal, proliferation and differentiation9. Activation of Wnt/β-catenin signaling causes β-catenin to accumulate in the cytoplasm and translocate to the nucleus where it activates downstream target genes. During tooth development, Wnt/β-catenin signaling is required at various stages of tooth morphogenesis10. Wnt responsive genes, such as Axin2 are expressed in differentiating odontoblasts, implicating Wnt/β-catenin signaling in odontoblast development and maturation13. Inactivation of β-catenin expression during tooth development leads to the disruption of odontoblast differentiation and root formation whereas, the overexpression of β-catenin results in excessive dentine production from mature odontoblasts15. Limited information is available concerning the importance of Wnt/β-catenin signaling in the generation of new odontoblast-like cells in postnatal teeth. The reparative and regenerative capacity of multiple mammalian tissues depends on the activation of Wnt/β-catenin signaling pathway and both epithelial and mesenchymal stem cells depend on this pathway being active to drive tissue renewal and repair16. Several studies have shown that tissue specific stem cells involved in tissue regeneration and repair can be identified by genetically-labelled Wnt responsive genes21. It has previously been shown that elevated Wnt signaling  enhances reparative dentinogenesis in Axin2LacZ/LacZ mice22. We have shown that delivery of small molecule inhibitors of GSK3 activity  directly to exposed pulps promotes the production of reparative dentine in vivo23. In this study, we take advantage of genetically-modified mice to investigate the molecular role of Wnt/β-catenin signaling in the reparative dentinogenesis process. Our study has revealed that Axin2-expressing cells differentiate into new odontoblast-like cells that secrete reparative dentine. In addition, Axin2-expressing cells produce a source of Wnt ligands that acts in an autocrine manner to modulate reparative dentinogenesis.The complex structural composition of adult mammalian teeth provides a hard-outer barrier that protects the inner dental pulp from being exposed to the external environment. This barrier consists of two mineralised tissues, an outer layer of enamel and an inner thicker layer of dentine. If this mineralised barrier is damaged due to a trauma or dental caries, the dental pulp has the ability to produce a form of tertiary dentine called reactionary dentine by stimulation of specialised cells known as primary odontoblasts located at the periphery of the pulp chamber that are responsible for dentine secretionLacZ/LacZ, Axin2CreERT2, Rosa26-mTmG flox/+ (fl/+) and pCagCreERT2 Wntless(Wls)flox-flox (fl/fl) were obtained from the Jackson Laboratory. TCF/Lef:H2B-GFP reporter mice were a kind gift from Anna-Katerina Hadjantonakis24. To induce genetic recombination, adult mice (6 weeks) were injected intraperitoneally with tamoxifen dissolved in corn oil/10% (vol/vol) ethanol, corresponding to specific doses per gram body weight (0.1 mg of tamoxifen per gram of body weight). Axin2CreERT2 mice received one dose of tamoxifen or corn oil (WT) on the day of tooth damage and another two doses over the next two consecutive days. Wlsfl/fl and Axin2CreERT2, Rosa26-mTmG fl/+; Wlsfl/fl mice received one dose of tamoxifen or corn oil (WT) over three consecutive days and tooth damage was performed 5 days post last tamoxifen or corn oil injection.All animals used in this study were handled in accordance with UK Home Office Regulations project license 70/7866 and personal license I6517C8EF. Experimental procedures were approved by the King’s College Ethical Review Process. Axin21/4) coupled to a high speed hand piece (Kavo Super Torque LUX 2 640B). Drilling was stopped when the pulp was visible under the dentine roof and a 27 G3/4 needle was used to expose the pulp chamber. Mineral Trioxide Aggregate  was applied to the exposed pulp and the cavity was sealed with glass ionomer Ketac™ Cem radiopaque (3 M ESPE). Post-op, the mice were given Vetergesic (Buprenorphine – Ceva) at the rate of 0.3 mg/kg intraperitonially as analgesic. The animals were sacrificed after at various time points post-damage.Mice were anaesthetised with a solution made with Hypnorm (Fentanyl/fluanisone - VetaPharma Ltd.), sterile water and Hypnovel (Midazolam - Roche) in the ratio 1:2:1 at the rate of 10 ml/kg intraperitonially. The oral cavity was opened with a mouth retractor to expose the molars. The superior first molars were cleaned using a sterile cotton plug soaked in phosphate buffered saline (PBS). A cavity was drilled in the centre of the superior first molar using a carbide bur  at −80 °C. For each condition, dental pulp tissue was pooled from at least 10 teeth and repeated in triplicate.−ΔΔCT method.Total RNA was extracted from the dental pulp using TRIzol (Invitrogen) as recommended by the manufacturer’s instructions. The RNA was reverse transcribed using random primers  according to the manufacturer’s instructions. Gene expression was then assayed by real-time qPCR using Kappa Syber Fast (Kappa Biosystems) on a Rotor-Gene Q cycler (Qiagen) system. Beta-actin primers  were used for the housekeeping gene, Axin2 primers  were used as the read-out of Wnt activity and for Gpr177 (Wls) expression, Forward – TCTAATGGTGACCTGGGTGTC and Reverse – TTCCAGCTCAGTGCCATACC primers were used. Reactions were performed in triplicate and relative changes to housekeeping gene were calculated by the 2TM 380 slides (Electron Microscopy Sciences). For Masson’s Trichrome staining, sections of adult teeth were deparaffinised in Neo-Clear and rehydrated through a series of graded ethanol. Sections were stained with Weigert’s Haematoxylin for 10 minutes, followed by 1% Biebrich-Scarlet-Acid Fuchsin solution for 15 minutes, differentiated in a mix of 5% phosphomolybdic acid and 5% phosphotungstic acid for 15 minutes and stained with 2.5% Aniline Blue solution for 10 minutes. Following staining, sections were differentiated in 1% acetic acid, dehydrated through 90% and 100% ethanol, cleared in Neo-Clear and permanently mounted in Neo-Mount.Teeth were fixed in 4% paraformaldehyde (PFA) for 24-hours at 4 °C, washed with PBS and decalcified in 19% EDTA, pH 8 for 4 weeks. Decalcified teeth were dehydrated through a graded series of ethanol, cleared in xylene and infused with wax at 60 °C in a Leica ASP300 tissue processor. Samples were embedded in wax and 8 μm sections were cut using a microtome . Sections were mounted on TrubondSections of adult teeth were deparaffinised in xylene and rehydrated in graded ethanol. To reduce endogenous peroxidase activity, sections were quenched with 3.5% hydrogen peroxide in PBS for 5 minutes and blocked with 10% goat serum in PBS with 0.1% Tween20 (PBST). No antigen retrieval was performed. The Primary antibodies were applied overnight at 4 °C. Proliferating cell nuclear antigen  and green fluorescent protein  antibodies were used at 1:200 and 1:500, respectively in PBST containing 5% goat serum. After washing the sections with PBST they were incubated with appropriate biotinylated secondary antibody, then horseradish peroxidase (HRP)-conjugated streptavidin-biotin antibody and washed with PBST. Immunoreactivity was visualised with ImmPACT DAB HRP Substrate (Vector Laboratories) or MenaPath green chromogen kit (Bio SB). Sections were counterstained with hematoxylin, dehydrated in graded ethanol, cleared in xylene and mounted in DPX. Sections were viewed in light-field using a Zeiss microscope (Axioskope 2 plus) and captured with an AxioCam HRC (Zeiss) using Axiovision software. For immunofluorescent co-staining, PCNA and GFP antibodies were used at 1:200 and 1:500, respectively in PBST containing 5% goat serum. After washing the sections with PBST they were incubated with Alexa Fluor® 647 to detect GFP and Alexa Fluor® 568 to detect PCNA and counterstained with Hoechst (40 μg/ml) in PBS before mounting with Citifluor. Immunofluorescence was visualised with a Leica TCS SP5 laser confocal microscope.In situ hybridisation for dentine sialo-phosphoprotein (Dspp) was performed on paraffin sections following standard procedures under RNase-free conditions25. Dspp was detected using TSA biotin system (PerkinElmer) in combination with the TSA Plus Cyanine 3.5 detection kit (PerkinElmer). After in situ hybridisation, slides were blocked with 10% goat serum in PBST, incubated with anti-GFP antibody  in PBST with 5% goat serum overnight at 4 °C. Sections were washed with PBST, incubated with Alexa Fluor® 647 secondary antibody and counterstained with Hoechst (40 μg/ml) in PBS before mounting with Citifluor. Immunofluorescence was visualised with a Leica TCS SP5 laser confocal microscope.t-test using GraphPad prism software was used to determine significance, a P-value < 0.05 was considered statistically significant. At least four independent experiments were performed for statistical analysis of dental pulp tissues described in the figure legends. Statistical data was presented as mean ± s.e.m.A two tailed unpaired Student’s The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.in situ hybridisation detection of Dspp gene was performed to identify odontoblasts27 and immunohistochemical staining PCNA was preformed to detect proliferating cells.To determine the timescale of reparative dentinogenesis in our model, damaged teeth were stained with Masson’s Trichrome to visualise dentine production, In situ hybridisation detection of Dspp showed primary odontoblasts located at the periphery of the dental pulp expressed Dspp, however no Dspp expression was detected at the site of exposure at 1 day post-damage, due to loss of local primary odontoblasts   mice do not express the Wls gene that encodes a sorting receptor required for Wnt secretion, thus cells cannot release Wnts to activate Wnt/β-catenin signaling. Real-time qPCR confirmed that the expression of the Wls gene was drastically reduced in the dental pulp tissue of Wlsfl/fl mice 5 days post-tamoxifen compared to WT mice  was created occlusally in the centre of the superior first molar crown to expose the dental pulp tissue with subsequent capping with mineral trioxide aggregate (MTA) and glass ionomer restoration. We used MTA as it is a biocompatible material that releases calcium ions that are thought to stimulate reparative dentine formation30. In endodontic procedures MTA is commonly used in vital pulp therapy to treat exposed dental pulp31. In our model, we observed robust proliferation and differentiation of a population of cells into new odontoblast-like cells that secrete reparative dentine at the site of damage.To study the molecular mechanisms of reparative dentinogenesis, we established a controlled molar-damage model LacZ and TCF/Lef:H2B-GFP reporter mice and real-time qPCR analysis of Axin2 expression, revealed that Wnt/β-catenin signaling was rapidly upregulated in response to damage. The upregulation of Wnt/β-catenin signaling corresponded with the peak of cell proliferation in the reparative dentinogenesis process, suggesting that Wnt/β-catenin signaling is mediating a proliferative expansion following damage. Furthermore, proliferating Wnt responsive cells could be detected at the site of damage. Our genetic tracing results indicated that Axin2-expressing cells undergo proliferative expansion following damage and some Axin2-expressing cells differentiate into odontoblast-like cells. At the end of the tracing period, the Axin2-expressing cells were in close association with the dentine bridge and presented long processes that extend into the dentine, a characteristic morphological feature of an odontoblast.To investigate the role of Wnt/β-catenin signaling in reparative dentinogenesis we took advantage of genetic mice models. Axin2LacZ/LacZ mice22.We confirmed that post-mitotic primary odontoblasts express Axin2 and that elevation of the level of Wnt/β-catenin signaling enhances the production of reparative dentine in Axin220. Furthermore, we showed that preventing Axin-2 expressing cells from secreting Wnts decreased the expansion of proliferative cells post-damage. This suggests that autocrine Wnt/β-catenin signaling stimulates cell proliferation in response to damage and provides an explanation for why reparative dentinogenesis is impaired in Axin2CreERT2; Wlsflox-flox mice.Interestingly, constitutive activation of the Wnt/β-catenin signaling did not promote primary odontoblasts to secrete reactionary dentine, but did enhance reparative dentinogenesis. Blocking Wnt/β-catenin signaling prevented the formation of reparative dentine in response to damage. Specific blocking of the ability of Axin2-expressing cells to release Wnts severely compromised the formation of reparative dentine. This finding indicates that Axin2-expressing cells are acting as their own source of Wnt ligands to drive reparative dentinogenesis via autocrine Wnt/β-catenin signaling. Autocrine Wnt/β-catenin signaling has been shown to be the mechanism by which some regenerating tissues renew. Axin2-expressing cells are responsible for skin and hair follicle renewal and these cells co-express Wnt ligands33. Alternately, Wnt/β-catenin signaling may be increasing the number of odontoblast-like cells, whereas primary odontoblast are unaffected as they are post-mitotic. Further studies to elucidate the dual roles of Wnt/β-catenin signaling in reactionary and reparative dentinogenesis could identify therapeutic strategies that actively promote stem cell driven tooth repair.Our study shows that Wnt/β-catenin signaling is important for the lifespan of primary odontoblasts as well as the generation of new odontoblast-like cells in response to tooth damage. We identify that Axin2 is expressed in odontoblast-like cells and that Axin2-expressing cells may be the source of their own proliferative signals in reparative dentinogenesis Fig. . The rolSupp data