V12. The expression of CARF was up-regulated in both HCC mouse model  and human HCC clinical samples. Overexpression of CARF promoted the growth and migration of HCC cells, while knocking down the expression of CARF inhibited the growth and migration of HCC cells. In the mechanism study, CARF was found to interact with beta-catenin, impaired the interaction between beta-catenin and ICAT, and activated beta-catenin/TCF signaling. Moreover, knocking down the expression of CARF inhibited the tumorigenesis in the HCC mouse model. Taken together, this study revealed the oncogenic functions of CARF in the tumorigenesis of HCC by activating beta-catenin/TCF signaling, and suggested CARF might be a therapeutic target in the treatment of HCC.Overactivation of Ras signaling is very common in the hepatocellular carcinoma (HCC) due to its constitutive active mutation, which makes it a big challenge to target Ras signaling. Therefore, identifying effectors downstream of Ras signaling would benefit the development of novel therapeutic strategies. In this study, it was found that the expression of CARF (collaborate of ARF) was induced by oncogenic Ras Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and its incidence is increasing yearly . AlthougOver-activation Ras signaling due to its constitutive mutation on the 12th code is frequently observed in HCC clinical samples , suggestBesides oncogenic Ras signaling, dys-regulation of beta-catenin/TCF signaling could also be found in most of HCC clinical samples . Wnt ligCross-talk between oncogenic Ras signaling and beta-catenin/TCF signaling is very common in the tumorigenesis. Ras-activated Dsor1, the homolog of Raf, has been reported to promote the beta-catenin signaling in Drosophila development . ConsistCARF, collaborator of ARF , activates p53 functions by direct interacting and stabilizing of ARF as well as p53 proteins . In addiV12 (HA-tagged RasV12) in L02  and 7404 (HCC cells) cells dramatically up-regulated the expression of CARF . It was found that the mRNA level and protein level of CARF were increased in the HCC mouse model compared with their control littermates  . Therefore, we firstly examined whether the expression of CARF was regulated by the oncogenic Ras signaling. Over-expression of HA-RasF Figure . Next, w) Figure . These on Figure . Taken tWe next extended our study to the human HCC samples. The mRNA level of CARF in 30 HCC tissues and paired adjacent tissues was examined using Real-time PCR. As shown in Figure To study the biological functions of CARF in the progression of HCC, we firstly forced the expression of CARF in 7404 and Hep3B cells Figure . Up-reguIn the next study, we investigated the roles of endogenously expressed CARF. At least 50% CARF expression was knocked down by two independent siRNA sequences Figure . Down-reTo find out the molecular mechanism through which CARF promoted the growth and migration of HCC cells, we performed the screening using reporter assay targeting various signaling pathways. In the screening, CARF was found to activate Topflash reporter  while exerted few effects on the activation NF-kappB and YAP/TEAD signaling Figure . In the In the next study, we investigated how CARF regulated beta-catenin/TCF signaling. We first examined the interaction between CARF and beta-catenin. As shown in Figure f/f; Ras). About 50% CARF expression was knocked down using adeno-associated virus (AAV)  six weeks after adenovirus injection, which was demonstrated by the liver morphology and tumor number  Figure . The knor Figure . Moreover Figure . Taken tV12, suggesting that targeting CARF might be a promising strategy for blocking onogenic Ras signaling. Furthermore, previous reports have shown that CARF promoted carcinogenesis in cells losing wild type P53 function [The inactive mutation of P53 and constitutive activation of Ras signaling are very common in the carcinogensis of hepatocellular carcinoma (HCC) . Restorafunction . Due to Previous studies demonstrated that the function of CARF depended on the status of P53. CARF induced cell growth arrest in cells with wild type P53, whereas it induced carcinogenesis in cells with absence of P53. In this study, it has been observed that the expression of CARF was up-regulated . CombiniIn this study, we have shown that CARF, a Ras-induced gene, positively regulating beta-catenin/TCF signaling. These observations suggested that CARF might be a knot which mediated the cross-talk between Ras signaling and beta-catenin/TCF signaling. In fact, a few reports have shown the cross-talk between Ras signaling and beta-catenin/TCF signaling in HCC. For example, Sorafenib, the inhibitor of Ras signaling, inhibited Wnt/beta-catenin signaling and sensitized cells to the cisplatin treatment, suggesting the regulation of Wnt/beta-catenin signaling by Ras signaling . HoweverNumerous studies have demonstrated that beta-catenin/TCF signaling is activated in HCC. Genetic disruption of APC leads to the activation of beta-catenin/TCF signaling and tumorigenesis of HCC . HoweverIn summary, the present study has shown the oncogenic roles of CARF in HCC by positively regulating beta-catenin/TCF signaling and suggesting that CARF might be a target for HCC treatment. Moreover, based on the expression pattern of CARF in HCC samples, it is very important to evaluate whether CARF might be served as tumor marker using HCC tissue array.2.The normal hepatic cells L02 and HCC cells  were purchased from ATCC  and cultured in DMEM medium containing 10% FBS  and antibiotics (Sigma). Cells were incubated in the 37°C atmosphere containing 5% COf/f; Loxp-Stop-Loxp-RasG12D) were generated by crossing Alb-Cre mice with P53f/f; Loxp-Stop-Loxp-RasG12D mice. The expression of Alb-Cre led to the inactivation of P53, deleted the stop codon in the front of Ras locus and activated the expression of oncogenic Ras. Alb-Cre mice and P53f/f; Loxp-Stop-Loxp-RasG12D mice were bought from Jackson Lab . Littermates without the expression of Alb-Cre were used as control. All mouse experiments were performed under the approval of the Jiangsu University IACUC.HCC mouse models  and fused with the myc tag. The pcDNA3.1 plasmid and the CARF expression vector were transfected into HCC cells using lipofectamin 2000 (Invitroen). The transfected cells were selected with G418 (Gibco) for two weeks. The resist cells were pooled and examined the expression of myc tagged CARF.The complementary DNA (cDNA) was prepared using the kit purchased from Promega after the total RNA was extracted using Trizol (Invitrogen). The mRNA level of CARF in the HCC tissues and paired non-cancerous tissues was examined by quantitative real-time PCR using SYBR Green Realtime PCR Master Mix (TOYOBO) following the instructions of the manufacturer. Sequences of quantitative real-time PCR primers are listed as follows:18S Forward primer: 5′-TAAATCAGTTATGGTT CCTT -3′18S Reverse primer: 5′-CGACTACCATCGAAAG TTGA-3′CARF Forward primer: 5′-TCATCTCCTTTTCCAT GGCC-3′CARF Reverse primer: 5′-TCTTGGCAACCAGTTC ATCT-3′3 cells were seeded in 12-well plates. Medium was changed every two days. Two weeks later, the cells were stained with 0.5% crystal violet solution. After incubation for 5 min, the cells were washed twice with phosphate-buffered saline (PBS) and photographed. Cells were resolved with 1% SDS solution. The absorbance at 600 nm was measured using a microplate reader.1 × 105) was suspended in 50 μl medium containing 1% FBS and placed in the upper chamber. The lower chamber was loaded with 152 μl medium containing 10% FBS. 8 hours later, cells migrated to the lower surface of filters was detected with traditional hematoxylin and eosin (H&E) staining. The experiments were repeated for thrice. Four random visual fields were counted for each sample and the average was determined.Boyden chamber (Neuro probe) was used to evaluate the migratory ability of HCC cells. Cells , and digested with RNase. Then, the cell cycle distribution was analyzed with flow cytometry (BD Bioscience).Cells were lysed in lysis buffer . After being determined the protein concentration, the cellular protein was subjected to SDS-PAGE and western blot analysis with the following primary antibodies. HA (1:5000) and Flag HA (1:5000) tag antibodies were obtained from Sigma, CARF antibody was bought from Abcam HA (1:3000), GAPDH antibody was bought from Santa Cruz (1:3000), c-Myc, CyclinD1, Twist and beta-catenin (1:1000) antibodies were obtained from Cell Signaling Technology.9 PFU si CARF adeno-associated virus (AAV) and control virus were injected into the tail vein. The down-regulation of CARF was examined.The lentivirus and adeno-associated virus (AAV) for knocking down the expression of CARF were purchased from Genechem . For down-regulating the expression of CARF in HCC cells, si CARF lentivirus and control lentivirus were incubated with 7404 and Hep3B cells overnight, then cells were sorted by GFP. For down-regulating the expression of CARF in HCC mouse model, 2 × 102HPO4, 2 mmol/l NaH2PO4 and 150 mmol/l NaCl) for 5 min each, blocked for 1 h in 0.01 mol/l PBS supplemented with 0.3% Triton X-100 and 5% normal goat serum, followed by addition of anti-CARF  antibody at 4°C overnight. After brief washes in 0.01 mol/l PBS, sections were exposed for 2 h to 0.01 mol/l PBS containing horseradish peroxidase-conjugated rabbit anti-goat IgG (1:500), followed by development with 0.3% H2O2 and 0.03% 3,30-diaminobenzidine in 0.05 mol/l Tris-HCl (pH 7.6). Immunohistochemistry for each sample was performed at least three separate times, and all sections were counterstained with hematoxylin.5 μm-thick consecutive sections of HCC tumor tissues and paired normal tissues were cut and mounted on glass slides. After deparaffinizing, rehydrateing, antigen retrieval and blocking endogenous peroxidases, the sections were washed thrice with 0.01 mol/l PBS  benzenesulfonyl fluoride hydrochloride) and 10 mM iodoacetate. Cell lysate was incubated on ice for 15 min before cellular debris and nuclei were removed by centrifugation at 10000 g for 15 min. Cell lysate was incubated with the antibody overnight at 4°C. Protein A-Sepharose  beads in a 50:50 mixture in 50 mM Tris buffer (pH 7.0) were added to the cell lysate and further incubated for another 4 h at 4°C. The immunoprecipitates were washed four times with Tris-buffered saline and boiled for 5min in 40 μl Laemmli buffer containing 0.02% blue bromophenol and 2% bmercaptoethanol.Cells were washed with ice-cold PBS and harvested in Tris-buffered saline (pH 7.4), containing 50 mM Tris, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Nat-test (two-tailed) using Prism GraphPad software. Differences with P < 0.05 were considered statistically significant. Data were represented as mean ± SEM.Statistical analyses were performed by the Student


Cementum is a mineralized layer on the tooth’s root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of β-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/β-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or β-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/β-catenin complex in the plasminogen proximal promoter regions (−900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation. As the paradigm shifts from tissue replacement by prosthetics to tissue regeneration in orthopedic or dental research, engineered microenvironments and localized delivery systems of biological factors have rapidly been developed for preclinical or clinical wound healing ,2,3. ForFor physiological restorations in a functional periodontal complex, interfacial tissues are significantly required to structurally integrate individual tissues and to form hierarchical constructs, like the bone–periodontal ligament (PDL)–cementum–tooth surface . In partThe Wnt signaling pathway has been known to affect tooth morphogenesis ,28,29,30Previously, we investigated the differentiation of MC3T3-E1 pre-osteoblasts by natural biopolymeric materials like fibrin and collagen ,39. In pIt was investigated whether fibrin enhanced canonical Wnt signaling by comparing OCCM30 cells cultured on fibrin (OCCM30-fibrin) with cells cultured on TCD (OCCM30-TCD). Expression of Wnt3a, which can trigger the canonical Wnt signaling pathway, was checked in cells cultured for three days in a differentiation medium. The immunofluorescence microscope images showed that β-catenin was more intensely stained in the OCCM30-fibrin and that its nuclear location was increased A. As a pIn addition, quantitative real-time PCR was used to analyze the transcript levels of cementoblast/osteoblast differentiation markers in the OCCM30-fibrin and OCCM30-TCD. The results show that the OCCM30-fibrin had statistically higher levels of the molecular expression of BSP, OCN, and Runx2, which were identified for biomineralization or mineralized tissue formations A–C. RegaReal-time qPCRs and western blots showed that the expression of plasminogen was significantly higher in the OCCM30-fibrin than in the OCCM30-TCD A. PlasmiThe relationship between the fibrin-enhanced canonical Wnt signaling and the expression of plasminogen was determined. The activation of canonical Wnt signaling with LiCl treatment led to a further increase in the transcript and protein levels for plasminogen expression in the OCCM30-fibrin A,B. In tIn order to determine the correlation between the canonical Wnt signaling pathway and the plasminogen gene expression in the OCCM30-fibrin, plasminogen expression was analyzed after knockdown of Wnt3a and β-catenin with siRNAs. From the results, the knockdown of Wnt3a and β-catenin dramatically reduced the expression of plasminogen in the OCCM30-fibrin . Based oTo address whether the canonical Wnt signaling in the OCCM30-fibrin directly targets plasminogen expression, the mouse plasminogen gene proximal promoter was analyzed by TESS, and putative LEF1 binding sites were found between −900 and the putative transcription start site (+1) denoted as follows: L1, −291 to −286, L2, −282 to −277, and L3, −53 to −48 respectively A. Site-dFibrin is a natural biopolymer material from critical blood-clotting components and formed by polymerization upon the thrombin-mediated cleavage of fibrinogen. This biomedical material is generally used to induce tissue formation as injectable biodegradable scaffolds or to deliver cell-encapsulated particles to target wound defects ,45. In aWith the modification to slow fibrinolysis using aminocaproic acid ,48 in thCementogenic differentiation in OCCM30-fibrin can be simultaneously affected by fibrin degradation, which can lead to a loss of cell attachment and ultimately apoptosis. Therefore, natural fibrin matrices should be optimally modified to slow the rate of fibrin biodegradation, to enhance cementoblast differentiation, and to promote mineralization. We reported phenomenological results about the interactions of a fibrin biomaterial and OCCM30 cells for fibrinolysis and the promotion of cementogenesis in vitro and in vivo . This stl-ascorbic acid, lithium chloride (LiCl), plasminogen-free fibrinogen from bovine plasma, thrombin, 6-aminocaproic acid, and Cholroform:Isoamyl alcohol 24:1 was purchased from Sigma-Aldrich Co. . Calcium chloride was purchased from Georgia Chemical Inc. . The WEST-ZOL™ Western Blotting System was purchased from iNtRON Biotechnology . RNAiso Plus reagents, PrimeScripts™ RT reagent kit, and SYBR® Premix Ex Taq™ were purchased from Takara Bio Inc . VECTASHIELD® Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories, Inc. . The Dual-Glo® Luciferase assay kit and pRL Renilla Luciferase Control Reporter Vectors were purchased from Promega Co. . Genefectine™ reagent was purchased from Genetrone Biotech . siRNA against Wnt3a and β-catenin of siGENOME SMARTpool, siGENOME Non-Targeting siRNA Pools #1, and ON-TARGETplus siRNA reagents were purchased from Dharmacon Inc. . The Protein G Agarose/Salmon Sperm DNA kit was purchased from Millipore . cOmplete™, an ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail tablet was purchased from Roche Diagnostics GmbH. . The DNA Clean & Concentrator™ kit was purchased from Zymo Research . Phenol:Cholorform pH 6.7/8.0 was purchased from Amresco. Anti β-catenin polyclonal rabbit antibody was purchased from Cell Signaling Technology Inc. . Anti-Wnt3 rabbit polyclonal antibody, anti-plasminogen rabbit polyclonal antibody, β-actin horseradish peroxidase (HRP) conjugated mouse monoclonal IgG1 antibody, and HRP conjugated anti-rabbit goat polyclonal antibody were purchased from Santa Cruz Biotechnology Inc. . Alexa Fluor® 488 conjugated anti-rabbit IgG goat polyclonal IgG and rhodamine phalloidin were purchased from Molecular probes® .Glycerol-2-phosphate, Fibrinogen was dissolved in distilled water at a concentration of 20 mg/mL. The thrombin solution was prepared composed of 4 Unit/mL of thrombin, 5 mM of calcium chloride, and 500 μg/mL of 6-aminocaproic acid. Each of the fibrinogen and thrombin solutions was sterilized using 0.2 μm sterilizing filter. The sterilized solutions were mixed gently with a ratio of 5:1, and 2 mL of the mixture were immediately added to 60 mm TCD and incubated at 37 °C.v/v) fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin (P/S). For the experiments, the cells were seeded onto 60 mm TCD and a fibrin gel described above. After one day of seeding, the cultured media was changed to differentiation media . There were 5.25 × 105 cells per 60-mm tissue culture dish which was exactly same as our previously published study [2.Murine cementoblast cells (OCCM-30) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%  mRNA expression. The primer sequences are showed in Total RNA was isolated using RNA extraction reagent (RNAiso Plus) from cells cultures for three days in DM on TCD or Fibrin gel. The concentration of RNA was determined with the Nanodrop™ 2000 . The cDNA was synthesized from the total RNA with a reverse transcription kit (PrimeScripts™ RT reagent kit). Quantitative polymer chain reaction (qPCR) was performed on 7500 Real-Time PCR System  with the SYBRCell lysates were prepared with a lysis buffer consisting of 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 30 μL/mL aprotinin, 10 mg/mL phenylmethylsulfonyl fluoride, 100 mM, and sodium orthovanadate in phosphate buffered saline (PBS). The samples were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) acrylamide gels and then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dried milk in tris buffered saline with 0.1% tween 20 (TBS-T) for 1 h at room temperature, and incubated with each primary antibody overnight at 4 °C. This procedure was followed by incubation with an HRP-conjugated secondary antibody for 2 h. After treatment with an enhanced chemiluminescence reagent (WEST-ZOL™) treatment, the luminescence was detected with a Fujifilm LAS-2000 .® 488 conjugated-goat anti-rabbit secondary antibody for 1 h. To visualize the actin filaments, the samples were incubated with rhodamine phalloidin for 20 min. Cover slips were mounted in a mounting medium contained DAPI, to identify the nuclei. The signal was detected by immunofluorescence microscopy (OLYMPUS FV300). For all of the above procedures, after each incubation step, the cells were washed for 5 min × 3 times with PBS.To determine β-catenin translocation to the nucleus, OCCM-30 cells were seeded onto fibrin and TCD in growth medium. After overnight culturing, the medium was changed to a differentiation medium and cultured for three more days. A 20 mM LiCl treatment was done for the last 24 h as a control group. The cultured cells were fixed with 4% formaldehyde in PBS, for 15 min. For permeabilization, the fixed cells were incubated in PBS containing 0.1% Triton X-100 (PBS-T), for 10 min. Then, the cells were blocked with 5% BSA in PBS-T for 1 h. Samples were then incubated with a 1:200 dilution of anti-β-catenin rabbit polyclonal antibody for 2 h followed by a 1:100 dilution of Alexa Fluor5 cells per well. After one day, the medium was changed to the differentiation medium. On the second day of differentiation, the cells were transfected with TopFlash or FopFlash constructs with the Genefectin™ reagent. Renilla luciferase constructs were used as a control. After 6 h of transfection, the medium was changed to a fresh differentiation medium without antibiotics. After 36 h of transfection, 300 µL of Dual-Glo® Luciferase reagent were added to the wells containing 300 µL of medium and mixed for 10 min to lyse the cells completely. Then, 150 µL of each lysate were dispensed into a well on a 96-well assay plate and measured for firefly luminescence with the GloMax®-Multi Detection System from Promega Co. . The Renilla luminescence was recorded after addition of 75 µL of the Ducal-Glo® Stop & Glo® reagent following the manufacturer’s instructions. The TOP/FOP activity was calculated by the ratio of firefly: Renilla luminescence. The experiment was repeated two times independently.Fibrin gel was prepared for 24 well plates using 300 μL per well. OCCM-30 cells were seeded onto the fibrin and TCD (well without fibrin gel) at a density of 1 × 10The siRNAs against Wnt3a and β-catenin were used to knockdown expressions of Wnt3a and β-catenin. A scrambled siRNA (siGENOME Non-Targeting siRNA #1) was used as a control. After OCCM-30 cells were seeded on fibrin and in 60 mm TCD and incubated overnight, the growth medium was changed to the differentiation medium. One day later, each specific siRNA was transfected into the cells following the manufacturer’s instruction. After 48 h of transfection, the cells were harvested and the expression of proteins and DNA were determined.The plasminogen promoter regions D-900 (bp-900 to +54) and D-100 (bp-100 to +54) were cloned in to the pGL3 vector following standard protocols. The primers used for the cloning of the plasminogen promoter are described in ® Luciferase reagent as described above in the β-catenin/Tcf luciferase assay section.For the transient transfection studies, plasminogen promoter constructs of D-900, D-100, mutants  or an empty basic plasmid pGL-3 as a control were co-transfected with the pRL-TK vector using the Genefectine™ reagent into the OCCM-30 cells cultured on fibrin and TCD. Six hours after the transfection, the cultured medium was changed to the differentiation medium without any antibiotics and cultured for an additionally 36 h prior to harvesting. The luciferase activity was measured with the Dual-Glo2, 10 mM KCl, and 0.1% NP-40, pH 7.8) with protease inhibitor was added to the cells and incubated for 20 min on ice. Under the cold condition, the nuclei were separated with the Dounce Homogenizer (ten strokes on ice) and spun down . The nuclei lysis buffer contained 1% SDS, 10 mM EDTA, and 50 mM Tris-Cl (pH 8.0), and protease inhibitors were added to the collected nuclei pallets. The lysate was sonicated for 4 s 12 times under an ice-cold condition, to break the DNA into fragments. Then, 1 μL (1%) of each sample was saved as the input fraction. The remaining samples were diluted 10-fold with the ChIP dilution buffer  containing a protease inhibitor. Immunoprecipitation was performed by adding protein G beads (1:10) and anti β-catenin rabbit polyclonal antibody (1:50) or protein G beads only as the negative control and incubated on a rotating shaker for 1 h at 4 °C. The bead-samples were washed three times with 0.1% SDS in dilution buffer, and one time with final washing buffer  containing a protease inhibitor and two times more with TE buffer . The cross-link was reversed by incubation in TE buffer containing 1% SDS at 65 °C for 5 h. Then, proteinase K was added  and incubated at 45 °C for 1 h. After that, the DNA fragments were extracted with Phenol/Chloroform/Isoamyl methods, and purified with DNA Clean & Concentrator™. The purified DNA was amplified by PCR using the primers which are listed in The ChIP assay was performed according to the instructions provided by the Protein G Agarose/Salmon Sperm DNA kit. After three days of differentiation, cells were fixed by adding 37% formaldehyde stock directly into the media in cultured dishes with a final concentration of 1%, incubated for 10 min at room temperature, and quenched with 0.125 M glycine. The cell were collected by scraping and washed with cold PBS. Swelling buffer  was used to statistically analyze data which were presented using means ± standard deviation (S.D.). All data were analyzed with the Student Fibrin positively affected OCCM30 differentiation through the canonical Wnt signaling pathway; however, canonical Wnt signaling also influenced the enzymatic degradation of fibrin. Wnt3a expression and the sequential canonical Wnt signaling were increased, and plasminogen expression was also upregulated. Therefore, fibrin has a critical limitation in supporting mineralized tissue neogenesis and possibly leads to attachment loss and apoptosis, simultaneously. Although our previous study used aminocaproic acid to temporally inhibit rapid fibrinolysis, these findings provide critical insights that spatiotemporal and precise controls of fibrinolysis are significantly required for cementoblast differentiation and tissue formation at optimal time periods.


POMC) gene expression in the hypothalamus. In this study, we examined the potential neural mechanisms by which these effects occur. Male Sprague Dawley rats were implanted with a cannula in the third ventricle of the brain through which an inhibitor of phosphatidylinositol-3 kinase (PI3K) (wortmannin) was administered, and EX-4 or vehicle was administered via intraperitoneal (IP) injection. The activity of PI3K/protein kinase B (AKT) and insulin receptor substrate-1 (IRS-1) in the hypothalamic arcuate was determined. We found that EX-4 treatment significantly decreased food intake and body weight. However, there were almost no changes in food intake and body weight when wortmannin injection (into the third ventricle) occurred prior to EX-4 IP injection. EX-4 not only increased the activity of PI3K/AKT, but it also increased IRS-1 activity. These results show that EX-4 likely suppresses food intake due to its ability to enhance insulin signaling.Exendin-4 (EX-4), a glucagon-like peptide-1 (GLP-1) receptor agonist, has been shown to reduce food intake and to increase proopiomelanocortin ( Human and nonhuman animal experiments have shown that GLP-1 affects the central nervous system (CNS) to alter food intake and body weight regulation. In human studies, the GLP-1 receptor (GLP-1R) ligand greatly enhances insulin release6 and reduces appetite and energy intake7, when it is peripherally administrated. In animal experiments, either peripheral or central administration of GLP-1R suppressed food intake at least in part via CNS GLP-1R activation10. In addition, centrally injected GLP-1R antagonist exendin-(9–39) can attenuate the intake suppression effects of the GLP-1R agonist12; centrally, viral knockdown of endogenous CNS GLP-1-producing preproglucagon (PPG) neurons causes hyperphagia and increased body weight13. Recently, a study by Rupprecht et al.14 showed that GLP-1R activation in the hindbrain suppressed food intake via a phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)-dependent pathway, which started to elucidate the specific CNS GLP-1R-expressing nuclei and the intracellular signaling mechanisms through which food intake suppression occurs. However, additional studies are needed to identify the role(s) of CNS GLP-1R in food intake and energy expenditure.The glucagon-like peptide-1 (GLP-1) system was recently established as a target for type 2 diabetes (T2D) and obesity treatment16. Orexigenic neuropeptides, such as neuropeptide Y (NPY) and agouti-related protein (AgRP), increase food intake and body weight, while α-melanocyte-stimulating hormone (α-MSH), a product of proopiomelanocortin (POMC), is an anorexigneic neuropeptide that reduces food intake. α-MSH is expressed in neurons of the hypothalamic arcuate nucleus (ARC), which are perfectly positioned to integrate short-term satiety and long-term adiposity signals for energy homeostasis17. PI3K/AKT signaling is thought to mediate leptin and insulin action in POMC and AgRP-expressing neurons18. It has been suggested that insulin helps to suppress the urge for food intake in the CNS via PI3K/AKT signaling19. Upstream to PI3K/AKT in the insulin pathway, the activities of the β-subunit of the insulin receptor and insulin receptor substrate-1 (IRS-1)21 are elevated in response to the ratio of phospho-insulin receptor/total insulin receptor and phospho-IRS-1/total IRS-1.Food intake and energy expenditure is mediated in part by neuropeptides expressed in neurons within nuclei of the mediobasal hypothalamusPOMC gene expression in the hypothalamus of rats8. However, whether insulin signaling is involved in the mechanism by which EX-4 reduces food intake remains unknown. CNS GLP-1R signaling is involved in energy balance22, while PI3K/AKT signaling is also required in the regulation of energy expenditures within the hypothalamus23. In addition, PI3K/AKT signaling is both the direct and indirect upstream target of POMC gene expression26. These data suggest that the intake suppressive effects of GLP-1R require PI3K/AKT signaling regulation.Our previous study suggested that GLP-1R agonist exendin-4 (EX-4) reduced food intake and body weight by altering in vivo. We found that the effects of ICV wortmannin on EX-4-induced reduced food intake occurred by increasing ARC PI3K signaling.To test our hypothesis, intracerebroventricular (ICV) administration of a PI3K inhibitor via the third cerebral ventricle and intraperitoneal (IP) injection of EX-4 was used to explore the mechanisms by which peripheral administration of EX-4 reduces food intake in the hypothalamic ARC P < 0.001] and Time . There was no significant Dose × Time effect . The results are summarized in Fig. To identify the highest dose of wortmannin that inhibits PI3K in ARC without increasing food intake, we first conducted a dose-response test. Two-way ANOVA, Dose × Time, revealed a main effect of Dose . Finally, ICV administration of 5 ng wortmannin significantly increased food intake at 0–1 h , 0–2 h , 0–4 h , and 0–20 h  after a 4-hour fasting period.Administration of vehicle or 0.5 ng wortmannin resulted in similar food intake at every time point measured: 0–1 h  = 18.6, P < 0.001], Time , wortmannin × EX-4  and EX-4 × Time  . This reduction in food intake can be reversed by central 1.5 ng wortmannin injection prior to peripheral EX-4 treatment (wor/EX-4). Compared with the veh/EX-4 combination, wor/EX-4 resulted in significantly greater food intake  revealed a significant effect of wortmannin × EX-4 . The wortmannin  or the EX-4 effect  was not significant. EX-4 treatment significantly reduced body weight , and such an effect was attenuated by wortmannin  . Food intake was measured at 0–1, 0–2, 0–4, and 0–20 h after dark cycle initiation. Three-way repeated measures ANOVA (wortmannin × EX-4 × Time) revealed significant effects of wortmannin  Fig. . The res05) Fig. .Figure 2P < 0.01], EX-4  and wortmannin × EX-4 . Third ICV wortmannin had no effect on AKT phosphorylation in ARC tissue at 3 h post-administration compared with central vehicle injection . Intraperitoneal injection of EX-4 robustly increased phosphorylation of AKT 1 h post-administration compared with vehicle peripheral injection . This Ex-4 caused increase in AKT phosphorylation was inhibited by replacing the central vehicle with wortmannin . Together, these results indicate that the activity of PI3K/AKT was increased after peripheral injection of EX-4, and this change was reversed by a PI3K inhibitor  revealed significant effects of wortmannin [F = 8.62, P < 0.05). These findings indicated that peripheral EX-4 injection might enhance ARC insulin signaling via insulin receptor signaling to PI3K/AKT . Furthermore, we focused on different region of the brain (ARC vs. hindbrain), and PI3K inhibitor was applied via 3rd ICV in the current study, but via 4th ICV in their study.In the current study, we aimed to determine whether the PI3K/AKT pathway also mediates the actions of EX-4 in the hypothalamus. We found that EX-4, at a dose of 3.2 μg/kg, significantly increased the phosphorylation of AKT in the ARC one hour after EX-4 administration, and at the same time food intake was notably decreased. If the phosphorylation of AKT is mediated by the PI3K signaling pathway, the increased phosphorylation of AKT induced by EX-4 should be reduced by pharmacological inhibition of the PI3K signaling pathway. Consistent with this hypothesis, injection of the PI3K inhibitor, wortmannin, into the third ventricle of the brain notably reduced the phosphorylation of AKT induced by EX-4, and the ability of EX-4 to decrease food intake was blocked. These data suggested that PI3K signaling mediates the downstream actions of EX-4 in ARC neurons. The study by Rupprecht 21. The activity of IRS-1 is increased after insulin binds with its receptor, which subsequently increases the activity of PI3K/AKT21. It has also been reported that GLP-1 increased IRβ and IRS-1 in adipocytes37. To determine whether insulin signaling is involved in the activation of PI3K/AKT by EX-4, we assessed the activity of IRS-1 after peripheral EX-4 injection. Our results showed that phosphorylation of IRS-1 was markedly increased in ARC neurons one hour after EX-4 administration. These results provide evidence that EX-4 has a positive effect on IRS-1/PI3K/AKT signaling pathway in the ARC.IRS-1 is an important component of insulin signaling, which is located upstream of PI3K/AKTet al. previously described the production and secretion of insulin by fetal neurons in culture and demonstrated that neuronal synthesized insulin promoted neurofilamant distribution and axonal growth38. At the same time, exogenous insulin promoted neural differentiation and growth beyond central insulin39. The detailed molecular mechanisms by which EX-4 promotes insulin signaling in the ARC are still unclear. A possible explanation for the positive effect of EX-4 on IRS-1/PI3K/AKT signaling is that EX-4 promotes insulin secretion from neuronal cells or enhances insulin translocation through the blood-brain-barrier, which leads to higher brain insulin levels, and stronger activation of insulin signaling. However, there is also the possibility that EX-4 increases the activity of IRS-1 directly without the involvement of insulin. Further research is required to elucidate the full effects of EX-4 on IRS-1/PI3K/AKT signaling.Schechter In summary, this study demonstrates a critical role of the PI3K/AKT pathway in regulating food intake in the ARC of rats and suggests that the effect of EX-4 in suppressing food intake is most likely due to its positive effect on IRS-1/PI3K/AKT signaling in the ARC.Forty-eight adult male Sprague-Dawley rats  weighing 250–275 grams were individually housed under standard conditions . First, 16 rats were used for identifying the dose of ICV wortmannin that produced minimal effects on food intake. The remaining 32 rats were used for feeding tests. Rats were presented ad libitum access to standard rat chow  and tap water except where noted. All animal procedures were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University and conformed to the guidelines of the National Institutes of Health.40. Wortmannin  was dissolved in 5% DMSO in artificial cerebrospinal fluid (aCSF)  for ICV injections.EX-4  was dissolved in sterile water for injections, and a dose of 3.2 μg/kg was chosen based on our previous studyAt least one week after arrival, rats were anesthetized using a mixture of ketamine (90 mg/kg) and xylazine (2.7 mg/kg) and placed in a stereotaxic device (Kopf Instruments). A 23-gauge sterile guide cannula (Plastics One) was then implanted into the third ventricle . Cannulae were attached to the skull by dental acrylic and jeweler’s screws. After 7 days of recovery, a third ventricle cannula was placed and verified by 30-minute water intake in response to administration of 10 ng (in 2 µl) angiotensin II. Only data from rats that drank at least 5 ml more water in response to angiotensin II than to vehicle (2 µl aCSF) were included in the analyses.29. Based on the results of this test, we selected a dose of 1.5 ng wortmannin to test its effect on EX-4-associated food intake reduction.The purpose of this experiment was to determine whether ICV administration of wortmannin could block or attenuate the food intake suppressive effect of IP EX-4 injection. Sixteen rats were randomly assigned to 4 groups, and named as group “veh”, “0.5 ng”, “1.5 ng”, “5 ng”, respectively. After 7 days of recovery following cannulation surgery, food was taken away 4 hours before dark cycle onset. Two hours before dark cycle onset, three doses of wortmannin, 0.5, 1.5, 5 ng (in 2 µl/injection) or vehicle, were delivered to the third ventricle with a micropump  over a 2-minute period. Food was returned at dark cycle onset, and food intake was measured at 1, 2, 4, and 20 hours post-food presentation. Three experimental doses of wortmannin were chosen based on previous reportsSimilar procedures were used to examine the effects of 1.5 ng wortmannin on EX-4-induced food intake reduction. Thirty-two rats were randomly assigned to 4 groups, with 8 rats in each group, and named as group “veh/veh”, “veh/EX-4”, “wor/veh”, and “wor/EX-4”. On the day of drug administration, food was taken away 4 hours before dark cycle onset. Two hours prior to dark cycle onset, rats received ICV injections of the PI3K inhibitor wortmannin  or vehicle (2 μl 5% DMSO in aCSF). Five minutes prior to dark cycle onset, rats were injected with EX-4 intraperitoneally at a dose of 3.2 μg/kg or vehicle (water at a volume of 1 ml/kg). When the dark cycle began, rats were provided with standard chow, and measurements of food intake were taken 1, 2, 4, and 20 hours post-food presentation.Seven days after the feeding test, PI3K signaling was examined after wortmannin and EX-4 administration. Food intake in response to wortmannin and EX-4 were measured using the previously described procedures. One hour after dark cycle onset, rats were sacrificed with rapid decapitation, and the brains were collected and flash frozen on dry ice and stored in −80 °C freezer until processing. Coronal hypothalamic brain sections (500 µm thick) were cut with a cryostat. Hypothalamic nuclei punches were then taken using a blunt 19-G needle. Punches for ARC were taken at −3.20mm relative to the bregma, and the tissue was immediately homogenized on ice in 50 µl tissue lysis buffer  supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). After 1 hour, the homogenates were centrifuged , and supernatants were retained for protein determination. A protein assay kit  was used to determine the protein content. Whole tissue (13.7–26.1 μg) extracts were separated on 10% Bis-Tris SDS-PAGE gels , followed by electrophoretic transfer to polyvinylidene fluoride membranes. Membranes were incubated with primary antibodies overnight at 4 °C and with secondary antibodies, and then they were conjugated to alkaline phosphatase at room temperature for 1 hour. Imaging was performed using a Typhoon FLA9500 imager , and densitometry was analyzed using Image J software. Primary antibodies for AKT, the phosphorylated site of Ser473 on AKT, IR, the phosphorylated site of Tyr1345 on IR, IRS-1, the phosphorylated site of Ser307 on IRS-1, and the secondary antibodies were purchased from Cell Signaling Technology . The primary antibody against β-actin was purchased from Santa Cruz .t test where only two conditions were compared. Data from Western blots were analyzed by two-way ANOVA. All statistical analyses were conducted using Statistica . Differences were considered significant at P ≤ 0.05.All data are presented as mean ± SEM. All behavioral parameters were analyzed by two-way or three-way repeated measures ANOVA followed by a post-hoc Fisher LSD test as appropriate or Student


Apc2 loss to meet this aim. Apc2 is a homolog of Apc which can also form a destruction complex capable of binding β‐catenin, albeit less efficiently than Apc. We show that systemic loss of Apc2 results in an increase in the number of cells displaying nuclear β‐catenin at the base of the intestinal crypt. This subsequently impacts the expression levels of several ISC markers and the fitness of ISCs as assessed by organoid formation efficiency. This work provides the first evidence that the function and fitness of ISCs can be altered by even minor misregulation of the Wnt‐signaling pathway. Our data highlights the importance of correct maintenance of this crucial signaling pathway in the maintenance and function of the ISC population. Stem Cells2018;36:114–122The importance of the Wnt‐signaling pathway on the regulation and maintenance of the intestinal stem cell (ISC) population is well recognized. However, our current knowledge base is founded on models using systems of gross deregulation of the Wnt‐signaling pathway. Given the importance of this signaling pathway on intestinal homeostasis, there is a need to explore the role of more subtle alterations in Wnt‐signaling levels within this tissue. Herein, we have used a model of  It was shown that the ISC population is highly sensitive to changes in Wnt‐signaling levels even when such changes are below the threshold that affects the maintenance of intestinal homeostasis.To address the importance of subtle Wnt‐signaling deregulation on intestinal stem cell (ISC) function, this population of cells was analyzed within an Apc2The rapid turn‐over of cells within the intestinal epithelium is sustained by the presence of intestinal stem cells (ISCs) at the base of the intestinal crypt. The ISC population is tightly regulated in order to maintain intestinal homeostasis, and much of this regulation is controlled by the canonical Wnt‐signaling pathway. The canonical Wnt‐signaling pathway functions through β‐catenin, which builds up within the nucleus when a Wnt ligand is bound. Upon entering the nucleus, β‐catenin acts as a transcriptional co‐activator for Transcription Factor 4 (Tcf4), resulting in expression of a range of genes. In the absence of a Wnt ligand, a β‐catenin destruction complex consisting of GSK3β, Axin and adenomatous polyposis coli (APC), targets β‐catenin for phosphorylation, thereby depleting intracellular levels and preventing nuclear accumulation The role of Wnt signaling in the maintenance of the ISC compartment has been well described. Previous studies have shown that deficiency of Tcf4  results in a lack of ISCs during development Apc, Apc2, was first identified in drosophila and the structural similarities between the two proteins have led to the hypothesis that there may be some functional redundancy in the system A homolog of Of the structural similarities between APC2 and APC, the presence of several 20‐amino acid repeat motifs is the most striking. It is these regions within Apc and Apc2 which are capable of binding β‐catenin within the destruction complex and thereby regulate Wnt signaling APC are common in this disease, being seen in more than 80% of all cases APC2 are rarer , or the Taqman Universal mastermix system (Applied Biosystems) for genes with lower levels of expression. All samples were run in triplicate on the StepOne Plus PCR machine and the threshold cycle values of each gene were normalized to expression of β‐actin. Primer details can be found in the Supporting Information Methods.After dissection of the 10 cm of small intestine required for epithelial cell extraction, the remaining small intestine and colon was flushed with cold water, opened longitudinally and rolled from the proximal to the distal end. The resulting “swiss roll” was secured with a pin and placed in 10% formalin for 24 hours at 4°C, and processed into paraffin blocks. Five micrometer sections were cut and rehydrated into water. The sections were then either stained with haematoxylin and eosin for counting to enable histological analysis of apoptotic and mitotic bodies, or immunohistochemistry (IHC) was performed for β‐catenin expression. IHC was performed using Mouse Envision+ kit according to manufacturer's instructions with mouse mAB anti‐β‐catenin antibody 1:200 .Apoptotic bodies were identified by their distinctive morphology of detachment from neighbors combined with highly condensed chromatin using H&E sections, as described by Potten et al. In situ hyrbidization for Olfm4 was performed on formalin‐fixed paraffin‐embedded tissue prepared as for IHC. Protocol used was as outlined by Parry et al. Intestinal organoid culture was performed from small intestinal crypt preparations as previously described At day 10, organoids were triturated to single crypts and re‐seeded as before. Self‐renewal was measured as the number of organoids which had grown after 10 days post passage as a percentage of the number which were seeded.Prestoblue (Invitrogen) was incubated 1:10 in the organoid media (and negative controls) for 2 hours at 37°C, then removed and placed into a 96 well flat‐bottomed plate for reading levels of fluorescence.+/+Apc2 and −/−Apc2 mice carrying the Lgr5Cre‐GFPIntestinal epithelia of both t test was performed for all normally distributed data, while a nonparametric Mann‐Whitney test was used for all other data sets.All data analysis was carried out using SPSS statistical software (IBM). A two‐tailed Student's −/−Apc2 and +/+Apc2 litermates. This analysis uncovered a significant increase in the number of cells possessing nuclear β‐catenin in an Apc2 deficient environment , and Wif1 are also significantly upregulated in response to Apc2 deficiency, indicating that the normal regulatory feedback loops are stimulated in response to the subtly elevated levels of Wnt signaling . However, detailed analysis of the number of cells and location of apoptotic cells revealed subtle changes in intestinal homeostasis. Other than cell shedding which occurs at the tip of the villus, most apoptosis within the small intestine occurs at the crypt base −/−Apc2 mice .A detailed analysis of the intestines from the Apc has a known role in the maintenance of genomic stability and loss of Apc can drive cell death through a number of mechanisms, not solely because of its role in regulating Wnt signaling. Due to the potential functional redundancy between the Apc proteins, DNA damage markers were assessed as a proxy for genomic instability within the small intestine. Apc2 deficiency had no apparent effect on levels of genomic instability, indicating that the increased cell death observed at the base of the crypt is not due to a potential role of Apc2 in regulating DNA damage .Lgr5, Ascl2, Olfm4, and Msi1 expression representing the CBC population and Bmi1 expression representing the +4 population. Interestingly, only Lgr5 and Ascl2 were significantly upregulated in response to loss of Apc2  or size of the organoids, indicating that the organoids which did grow were normal. IHC on formalin fixed, paraffin embedded organoids, and immunofluorescence on wholemount organoids revealed that lls Fig. A, 5B. Thflox/floxApc mice are not Apc2 deficiency, intestinal crypts from both wildtype and −/−Apc2 mice were cultured in 6.5 μg/ml, 3.25 μg/ml, and 0 μg/ml R‐spondin. Wildtype crypts die in culture in the absence of R‐spondin by day 2–3, but interestingly −/−Apc2 organoids survived until day 3 and only began to die by day 4 , the ISCs which have overcome the apoptotic effect of increased Wnt signaling are less dependent of R‐spondin, indicating that they are more highly Wnt‐activated than wildtype.Wildtype organoids cultures are only capable of growth in the presence of R‐spondin within the culture medium for cell survival and organoid growth, whereas organoids grown from Wnt‐activated stem cells such as those derived from It is widely accepted that Wnt signaling levels are tightly regulated within the intestine in order to maintain homeostasis, with changes in Wnt signaling having potentially disastrous effects on the structural integrity of the tissue. Although the relationship between Wnt signaling and the maintenance of the ISC population has been investigated when Wnt‐signaling levels are grossly altered, the effect of subtly altered Wnt signaling on the ISC compartment has so far remained unexplored.Apc2 resulted in an increased expression of the ISC markers Lgr5 and Ascl2 but no change in the percentage of +Lgr5 cells. Functionality of the ISCs was explored through the use of organoid culture techniques, and showed that loss of Apc2 leaves intestinal crypts less efficient at forming organoids in culture, a capability which is associated with functionality of the ISC compartment. Of the −/−Apc2 organoids which did grow however there was no difference in self‐renewal efficiency compared to wildtype. These observations lead to the hypothesis that within an −/−Apc2 murine intestine there is a heterogeneous population of ISCs, a proportion of which can behave normally and form organoids which grow in the same manner to wildtype organoids, and others which are more highly Wnt activated are more likely to die, (as observed by an increase in apoptosis within the first four cell positions within the crypt) and are less fit as ISCs .A constitutive Apc2 knockout proved to be a suitable model to represent subtly altered levels of Wnt signaling within the intestine. In order to analyze the effect of these altered Wnt levels on the ISC compartment, a wide range of techniques were used, which showed that loss of Msi1. Recently, it has been proposed that Msi protein levels, although not a good indication of the CBC population, play an important role in the activation of the quiescent ISC population into the cell cycle in response to injury Apc2 loss, and the associated reduced Msi1 expression, may well impact the ability of intestinal crypts to activate their quiescent stem cell population in response to trauma. This could explain why intestinal homeostasis was largely unaffected by Apc2 deficiency, but had an impact on ISC fitness.Given that the levels of ISC markers did not correlate with intestinal organoid formation efficiency, it must be assumed that either levels of expression of these markers do not directly impact ISC fitness, or that organoid formation is a poor readout of ISC activity levels. The one marker which did show a correlation was expression of This hypothesis is supported by the idea that levels of Wnt signaling within the intestinal epithelium must be tightly regulated M.A.Y.: designed the research, managed the mouse intercrosses, performed organoid culture and associated imaging, performed IHC, performed qRT‐PCR, analyzed data, manuscript drafting; C.S.D.: designed the research, performed IHC; K.R.R.: designed the research; A.R.C.: designed the research; E.T.: managed the mouse intercrosses; R.J. performed qRT‐PCR, performed western blotting; All authors: critically reviewed the manuscript, read and approved the final manuscript. The now late Professor Alan Clarke was aware of the results of the study and had approved of the manuscript plan.The authors indicated no potential conflicts of interest.This article was published online on 13 October 2017. Minor edits have been made that do not affect data. This notice is included in the online and print versions to indicate that both have been corrected 29 December 2017.Supplementary FiguresClick here for additional data file.


The crystal phagocytic rate of bone marrow-derived M2Mφs was higher than that of bone marrow-derived Mφs and M1Mφs and increased on co-culture with renal tubular cells (RTCs). However, the amount of crystal attachment on RTCs reduced on co-culture with M2Mφs. In six hyperoxaluric C57BL/6J mice, M1Mφ transfusion and induction by LPS and IFN-γ facilitated renal crystal formation, whereas M2Mφ transfusion and induction by IL-4 and IL-13 suppressed renal crystal formation compared with the control. These M2Mφ treatments reduced the expression of crystal-related genes, such as osteopontin and CD44, whereas M1Mφ treatment increased the expression of pro-inflammatory and adhesion-related genes such as IL-6, inducible NOS, TNF-α, C3, and VCAM-1. The expression of M2Mφ-related genes was lower whereas that of M1Mφ-related genes was higher in papillary tissue of CaOx stone formers. Overall, our results suggest that renal crystal development is facilitated by M1Mφs, but suppressed by M2Mφs.In our previous report, M2-macrophage (Mφs) deficient mice showed increased renal calcium oxalate (CaOx) crystal formation; however, the role of Mφs-related-cytokines and chemokines that affect kidney stone formation remains unknown. Here, we investigated the role of M1/M2s in crystal development by using  Mφs have two major poles, one classically activated (M1) and another alternatively activated (M2), which are probably associated with crystal formation or disappearance and need to be considered as two opposite Mφ types11Kidney stone disease has markedly increased both in adult and pediatric populations12in vivo models of nephropathy and ischemia/reperfusion acute kidney injury16171820The population of renal mononuclear phagocytes, including Mφs, has diverse reactions in kidney disease1314ex vivo induction of bone-derived Mφs (BMMs) with both in vitro and in vivo approaches. Ex vivo M2Mφs showed significant ability of COM crystal phagocytosis and anti-adherence on RTCs. The adoptive transfusion and selective induction by M2Mφs attenuated renal crystal formation, whereas those by M1Mφs facilitated renal crystal formation. We further discovered the gene expression profile of human renal papillae related to Mφs, and showed that CaOx stone formers had lower expression of M2Mφ-related genes than the controls.In this study, we investigated the role of M1Mφs and M2Mφs in renal CaOx crystal development using The amount of adherent fluorescent-labeled COM crystals  was consSpp1, encoding osteopontin [OPN]) in the RTC+M2Mφ group was lower than that in the RTC group, whereas the expression of Cd44 and chemokine (CC motif) ligand 2  was higher in the RTC+M1Mφ group than in the other groups. The expression of tumor necrosis factor  in the RTC+M1Mφ group was the highest, whereas that in the RTC+M2φ group was the lowest among the four groups (C3) and vascular cell adhesion molecule 1 (Vcam1) in the RTC+M1Mφ group was significantly higher than that in the other groups; however, no significant differences were identified in the expression of fibronectin 1 (Fn1) among groups , Tnf, and interleukin 10 (IL10) was considerably higher in the M1Mφ group than in the BMM and M2Mφ groups, and increased by COM exposure, whereas it decreased on co-culture with RTCs (The expression of φ groups . The expith RTCs . Enzyme-ith RTCs .Arg1), chitinase 3-like 3 , and peroxisome proliferator activated receptor gamma (Pparγ) was considerably higher in the M2Mφ group than in the BMM and M1Mφ groups. The expression of Arg1 and Chi3l3 in the M2Mφ group increased on co-culture with RTCs. The expression of Pparg in the M1Mφ group was the lowest among the three Mφ groups -treated mice under the polarized light optical microscope with Pizzolato staining .p < 0.03). The amount of renal crystals in the GOX+M2 (I by daily i. p. administration of IL-4 and IL-13) group was lower than that in the GOX group  and GOX+M1  groups than in the other groups (OX group . The amoOX group . The ratOX group .Serum creatinine and phosphorus levels in the GOX+M1 (I) group were significantly higher than those in the control, GOX, and GOX (I) groups (p < 0.05). Serum sodium levels in the GOX+M1 (I) and M2 (I) groups were higher than those in the control group.Urinary volume in the GOX and GOX+M2 (I) groups was higher than that in the control groups. All GOX-treated groups had considerably higher levels of urinary oxalate compared to the control group. There were no significant differences in the other serum and urinary variables among the 6 experiment groups .OPN, CD44, and MCP-1 were detected in the renal tubular cells around the crystals, whereas FN and Vcam1 in the interstitial spaces of the renal tubular cells. The staining intensity of OPN, CD44, MCP-1, and Vcam1 was stronger in the GOX+M1 (T) and GOX+M1 (I) groups, whereas that of FN was stronger in the GOX+M2 (T) group than in other groups .Spp1, Cd44, Ccl2, and C3 in the GOX group. In the M1Mφ-treated groups, the expression of Spp1, Cd44, Ccl2, C3, and Vcam1 was considerably higher than that in the M2Mφ-treated groups. The expression of Fn1 was lower in the GOX+M1 (I) group, but higher in the GOX+M2 (I) group compared with the control and GOX groups (Quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed a marked increase in the expression of X groups .+CD11b+ cells, significantly increased in all the GOX-treated groups (p < 0.01); however, no significant differences were identified among them , and GOX+M1 (I) groups. The number of M1-like Mφs was also higher in the GOX, GOX+M1 (T), and GOX+M1 (I) groups than in the control group. However, the number of M2-like Mφs was higher, whereas that of M1-like Mφs was lower in the GOX+M2 (T) and GOX+M2 (I) groups compared with the GOX-treated and M1-treated groups  group than in the control and GOX+M2 (T) groups. The expression of Tnf, Arg1, Chi3l3, and Il10 was considerably higher in the GOX+M1 (I) group than in the control, GOX, and GOX+M2 (I) groups. The expression of interleukin 4 (IL4) and Pparg was lower in the GOX+M1(I) group than in the control and GOX groups. The expression of IL4 and resistin like alpha (Retnla found in inflammatory zone [FIZZ]1) was significantly higher in the GOX+M2 (I) group than in the GOX+M1 (I) group (The expression of I) group .The gene expression profiles of renal papillary tissues from CaOx stone formers were characterized, and no significant differences were identified in physical, serum, urinary backgrounds between CaOx stone formers and control patients .SPP1 was higher, whereas that of FN1 was lower in the normal and plaque groups compared with the control group , was higher in the normal and plaque groups compared with that in the control group , and CD163 was lower, whereas that of IL4 and RETNLB was higher in the normal and plaque groups compared with that in the control group  and interferon (IFN)-γ might cause renal dysfunction via acute renal injury; however, no changes in urinary variables were identified, revealing that there is no association between renal dysfunction and increased crystal deposition.Il4, Chi3l3, and Retnla, and decreased that of Nos2 and Tnf; whereas M2Mφ-transfusion did not affect any of these genes. It is assumed that crystal formation was differentially suppressed by M2Mφ transfusion or induction; the induction of M2Mφs by interleukins affected parenchymal cells, increased anti-inflammatory molecules, and reduced pro-inflammatory molecules, whereas the transfusion of M2Mφs improved crystal phagocytic and anti-adherent ability. Although a previous study reported that bone marrow-derived M2Mφs were difficult to maintain in their state compared with spleen-derived M2MφsPan-Mφs, which appeared in mice kidneys, increased with GOX treatment. Mφs in the GOX and M1Mφ-treated groups were mostly M1Mφs, indicating that they were responsible for the development and formation of crystal deposits in the kidney. However, when the number of M2Mφs reached that of M1Mφs, the renal crystal formation was suppressed. M1Mφ treatment increased the expression of pro-inflammatory molecules with crystal formation. M2Mφ induction increased the expression of anti-inflammatory genes, such as SPP1, and two anti-inflammatory-related genes was higher, whereas that of four anti-inflammatory-related genes, including FN1, was lower both in the normal and plaque groups compared with the control group. The upregulation of M1Mφ-related genes and downregulation of some M2Mφ-related genes contributed to the development of human nephrolithiasis and RPs. Therefore, if the M1Mφ-dominant environment in human renal papilla was consistent with the in vitro and in vivo results of the present study, differences in the role of Mφs might be responsible for the development of intraluminal crystals and RP-related nephrolithiasis.RPs provide a base for the development of idiopathic CaOx kidney stones, which begins in the basement membranes of thin Henle’s loops with calcium deposits4446in vitro and in vivo models do not accurately mimic the environment of kidney stones in patients who mostly have idiopathic CaOx stones related to RPs. Since both models have acute injuries and thus, excessive inflammation, M1/M2Mφs may easily affect crystal formation under specific circumstances. (2) Some parts of the role of Mφs are still unclear and need to be investigated in further studies. For instance, optimized capture of Mφ phagocytosis based on specific molecule activation both in vitro and in vivo would be useful for further understanding of their exact role with regard to crystals. (3) The M1/M2 Mφ treatment for patients with kidney stones needs further research because of the short-term effectiveness of bone marrow-derived M2Mφs and limited information on M1/M2 induction. (4) A relative higher number of human papillary samples is necessary for investigating CaOx stone formers and obtain accurate results.This study has some limitations: (1) The experimental ex vivo induction of BMMs with both in vitro and in vivo approaches. Our results showed that M2Mφs suppressed renal CaOx stone development by crystal phagocytosis, inhibited crystal attachment to renal tubular epithelial cells, and reduced the expression of pro-inflammatory genes. These findings supported the therapeutic possibility of targeted Mφ-phenotype shifting from M1 to M2.Here, we investigated the differential roles of M1/M2Mφs in the development of renal CaOx using L-conditioned medium were generated as described previously5 cells cm−2. For priming experiments, BMMs were stimulated for 20 h with 100 ng ml−1 of LPS  and 20 ng ml−1 of GM-CSF  for M1Mφs or 50 ng ml−1 of IL-4 (R&D Systems) and 10 ng ml−1 of M-CSF  for M2Mφs49BMMs grown in lymphocytes and 10% 5 cells cm−2) were grown in six-well culture plates . The cells were maintained in a humidified incubator at 37 °C with 5% CO2 for 24 h and then treated with 25 μg cm−2 of fluorescence-labeled COM crystals. Co-cultures (0.2 × 105 cells cm−2) were grown in the upper inserts of the transwell. Cultured RTCs were observed under a DMI4000 B phase contrast microscope  at 6 h after COM treatment.Fluorescence-labeled COM crystals were prepared as described previously5 cells cm−2) were mono-cultured or co-cultured with RTCs (0.2 × 105 cells cm−2) using transwell inserts as described above. Each group of cultured cells was harvested after 6 h of COM treatment and then used for flow cytometry and qRT-PCR.We divided each type of cell into nine groups. BMMs, M1Mφs, and M2Mφs without COM crystal exposure were used as control groups. The other six groups were incubated with COM crystals; three types of Mφs . The mice were fed standard chow  and had free access to water. Equal numbers of 8-week-old male mice were assigned to six groups (n = 6): control, GOX (treated with glyoxylate), GOX+M1(T) (treated by glyoxylate and M1Mφ transfusion), GOX+M2(T) (treated by glyoxylate and M2Mφ transfusion), GOX+M1(I) (treated by glyoxylate and M1Mφ induction), and GOX+M2(I) (treated by glyoxylate and M2Mφ induction). Each glyoxylate treatment was performed as a 6-d intra-abdominal injection of 80 mg kgg for 15 min, and 100 μl sediment samples were observed at 400 × magnification using the AX80 light optical microphotograph  to detect and quantify the number of CaOx crystals.Blood, 24-h urine samples, and kidney tissues were obtained from six mice from each group at d 6. Blood and urinary biochemistry was examined by Mitsubishi Chemical Medicine . Urinary pH and volumes were measured manually. Urinary oxalate concentrations were analyzed using FOM-110A 6 viable Mφs were separated with the medium. Mice were anesthetized by sevoflurane and transfused with either M1Mφs or M2Mφs via a single tail-vein injection using a 27 gauge needle at 1 d prior to glyoxylate administration as described previouslyAfter the culture and differentiation of M1Mφs and M2Mφs from BMMs, 1.0 × 10−1 LPS (Sigma-Aldrich) and 1.0 μg body−1 IFN-γ , 5.0 μg body−1 IL-4 (BioLegend), and 5.0 μg body−1 IL-13 (BioLegend) were performed concurrently with glyoxylate treatment in order to induce the migration of M1Mφs and M2Mφs1254555657Daily intra-abdominal injections of 50 μg body+ and/or CD11c+ cells as single-cell suspensions using mouse CD11b- or CD11c-microbeads and auto-MACS (Miltenyi Biotec), following the manufacturer’s protocol. Next, CD11b and/or CD11c cells from mouse kidneys and BMM-derived M1/M2Mφs were stained with Ly-6C CD45, CD11b, CD11c, CD163, CD206, and F4/80 of their antibodies  using the 7500 FAST Real-time PCR System (Applied Biosystems). After denaturing at 95 °C for 10 min, PCR cycling was performed with each cycle consisting of 95 °C for 15 s followed by 60 °C for 1 min. The PCR reaction was repeated 40 times. cDNA amplification was compared with that of control samples, and the expression ratios were determined using a standard curve prepared from a 35-dilution series of control samples and corrected for the amount of total RNAELISAs were performed to measure the levels of soluble OPN , MCP-1 , IL-6 , and TNFα  produced in the supernatant of each culture dish according to the manufacturer’s instructions.The genome-wide analysis of human papillary tissue was approved by the Nagoya City University Ethics Board (No. 929), and these protocols were conducted in accordance with the Declaration of Helsinki. All participants provided informed consent prior to the surgery. We obtained renal papillary tissue biopsies of six idiopathic CaOx stones from participants who underwent retrograde intrarenal surgery at our institutions in November 2013–April 2015. Patients with active urinary tract infection, metabolic and autoimmune disease, carcinoma, and severe hydronephrosis  were excluded. In each patient, samples were individually collected from the renal papillary tissue with RP (Plaque group) and the normal papillary tissue without RP . The control group consisted of normal renal papillary tissues collected from six patients who underwent ureteroscopy or nephrectomy either for hemorrhage screening or for adrenal tumor adhesion without urolithiasis.® using the RNeasy Micro Kit (Qiagen). cDNAs amplified using the Ovation Pico System  were subjected to transcriptome analysis using Agilent SurePrint G3 microarrays. Microarray data were analyzed using GeneSpring 13.1 . Changes in gene expression greater than 2-fold between groups were considered significant (p < 0.01). All microarray data were deposited in the Gene Expression Omnibus (Acc. No: GSE 73680).Total RNA was extracted from the human renal papillary tissues in RNAlaterU test for identifying differences between two groups. Categorical data were compared using Fisher’s exact test. Differences were considered statistically significant at p < 0.05. All statistical analyses were performed using SAS 9.1 .All data are expressed as mean ± standard deviation. Two-way analysis of variance was performed for identifying differences among three or more groups or the Mann-Whitney How to cite this article: Taguchi, K. et al. M1/M2-macrophage phenotypes regulate renal calcium oxalate crystal development. Sci. Rep.6, 35167; doi: 10.1038/srep35167 (2016).


Glycoprotein 90K  is a tumor-associated protein, and high 90K levels are associated with poor prognosis in some cancers. To clarify the role of 90K as an indicator for poor prognosis and metastasis in epithelial cancers, the present study investigated the effect of 90K on an adherens junctional protein, E-cadherin, which is frequently absent or downregulated in human epithelial cancers. Treatment of certain cancer cells with 90K significantly reduced E-cadherin levels in a cell-population-dependent manner, and these cells showed decreases in cell adhesion and increases in invasive cell motility. Mechanistically, 90K-induced E-cadherin downregulation occurred via ubiquitination-mediated proteasomal degradation. 90K interacted with the E-cadherin–p120-catenin complex and induced its dissociation, altering the phosphorylation status of p120-catenin, whereas it did not associate with β-catenin. In subconfluent cells, 90K decreased membrane-localized p120-catenin and the membrane fraction of the p120-catenin. Particularly, 90K-induced E-cadherin downregulation was diminished in p120-catenin knocked-down cells. Taken together, 90K upregulation promotes the dissociation of the E-cadherin–p120-catenin complex, leading to E-cadherin proteasomal degradation, and thereby destabilizing adherens junctions in less confluent tumor cells. Our results provide a potential mechanism to explain the poor prognosis of cancer patients with high serum 90K levels. The protein known as lectin galactoside-binding soluble 3-binding protein (LGALS3BP), Mac-2-binding protein (Mac-2BP), or 90K is a secretory glycoprotein, and its serum levels are observed to be elevated in several cancers and viral infections. As 90K plays a role in immune defense and immune regulation, its levels are therefore increased in patients with human immunodeficiency virus , hepatitRegarding the mechanisms underlying the effect of 90K on tumor progression, it was reported that 90K activates early immune responses to tumors and suppresses Wnt signaling via CD9/CD82-mediated ISGylation-dependent ubiquitination of β-catenin in CRC cells ,20. Howe2+ dependent cell–cell adhesion. It is associated with the actin cytoskeleton via cytoplasmic proteins, including α-, β-, and p120-catenin, forming the cadherin/catenin complex that is crucial to adherens junctions [E-cadherin is a type I transmembrane protein found at adherens junctions that mediates Caunctions . E-cadheunctions . Loss ofunctions ,26.In the present study, we aimed to elucidate the mechanism underlying the association of 90K with poor prognosis and metastasis of cancer. For this purpose, we examined the effect of 90K on the levels of E-cadherin, which forms the cadherin/catenin complex that is crucial to the function of adherens junctions, in various cancer cells. Our results showed that 90K destabilizes E-cadherin and affects cell adhesion and invasion in subconfluent cancer cells via dissociation of the E-cadherin–p120-catenin complex, suggesting that 90K drives less confluent tumor cells into the early steps of cancer metastasis. Namely, our results provide a mechanistic explanation of distant recurrence in cancer patients exhibiting high serum 90K levels: 90K promotes the release of cancer cells from tumor tissues through the destabilization of the adherens junction.As the upregulation of 90K in several cancers including colon, prostate, lung, and breast suggests an association between 90K and tumorigenesis ,9,11,12,l-lysine, and matrigel matrix  in ctrl/CM and 28.75  in 90K/CM-treated cells, indicating an approximately 18% reduction in membrane p120-catenin levels in 90K/CM-treated cells  or p120-catenin expression plasmid. Since 90K treatment primarily decreased the membrane/cytoplasm p120-catenin 1A ratio c,d, a p1In the present study, we investigated the effect of 90K on E-cadherin levels. Our main findings were as follows: (1) 90K negatively regulates E-cadherin levels in a cell-population-dependent manner; (2) 90K affects the adhesive and motile properties of subconfluent cancer cells; (3) the 90K modulation of E-cadherin levels is mediated by ubiquitination-dependent proteasomal degradation; (4) 90K interacts with adherens junction proteins, which affects the phosphorylation status of p120-catenin and the association between p120-catenin and E-cadherin; and (5) 90K affects the cellular localization of p120-catenin. However, it should be noted that other factors in the conditioned medium may be responsible to some degree for some of the described effects.The results of the present study indicate the identification of a new mechanism of destabilizing E-cadherin, especially in low-confluence cancer cells. 90K interacts with p120-catenin at the membrane, modulating p120-catenin phosphorylation and the interaction between p120-catenin and E-cadherin, resulting in changes in the subcellular localization of p120-catenin. Furthermore, 90K interacts with p120-catenin and promotes the dissociation of p120-catenin from E-cadherin. It was reported that the complex between E-cadherin and p120-catenin is critical for the formation and maintenance of the adherens junctions, and disruption of this complex is a hallmark feature of epithelially derived cancers . p120-CaE-cadherin stability and its interaction with p120-catenin are modulated by p120-catenin phosphorylation mediated by various plasma membrane kinases . In the The downregulation of E-cadherin by 90K was cell-density-dependent, namely, 90K regulated E-cadherin levels in low-confluence cells, whereas its effect was suppressed in confluent cells. Since the formation of stable junction complexes occurs at cell–cell contacts in confluent cells, these results suggest that the regulation of E-cadherin by 90K is junction-complex-status-dependent. Cancer cells show diminished cell–cell adhesion compared with normal epithelial cells , and E-cIn summary, we here show that E-cadherin associates with 90K and that destabilization of the E-cadherin–p120-catenin interaction by 90K alters p120-catenin subcellular localization and downregulates E-cadherin, resulting in decreased cell–cell adhesion. These effects were seen in low-confluence cells, which will promote escaping cancer cells from primary tumor tissues or blood vessels. Our present data suggest that high 90K levels play an oncogenic role by stimulating metastasis through the induction of E-cadherin degradation, especially in tumor tissues containing weak adherens junctions. Further study is required to completely reveal the roles of 90K in the progression and/or suppression of tumors in various microenvironments.The generation of the 90K-myc and ISG15-V5 constructs was described previously . HA-Ub w2 atmosphere. All cell lines used in the study were authenticated by the ATCC and BioMedicure using short tandem repeat (STR) PCR analysis. For the nonspecific scrambled siRNA (si-scr), we used the All Stars Negative Control siRNA . The siRNA duplexes were prepared and transfected according to a protocol provided with LipofectamineTM RNAiMAX . The short hairpin RNA (shRNA) for p120-catenin was kindly provided by M. Ikura .HEK293T (human embryonic kidney), CWR22Rv-1 and PC3 (prostate cancer), Caco2, DLD1, and SW620 , A549, H358, and H1650 (lung cancer), and MCF7 and BT474 (breast cancer) cell lines were purchased from the American Type Culture Collection  and the CSC221  cell line was purchased from BioMedicure . CWR22Rv-1, PC3, BT474, A549, H358, and H1650 cells were maintained in RPMI1640, and HEK293T, Caco2, DLD1, SW620, MCF7, and CSC221 cells were maintained in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% COFor RT-PCR, the primer set for E-cadherin was as follows: 5′-CGAGAGCTACACGTTCACGG-3′/5′-GGGTGTCGAGGGAAAAATAGG-3′, and that for GAPDH was as follows: 5′-ATCACCATCTTCCAGGAGCGA-3′/5′-AGTTGTCATGGATGACCTTGGC-3′.The 90K conditioned media (90K/CM) were prepared as previously described . BrieflyImmunoprecipitation and immunoblotting were performed as previously described . ImmunopImmunostaining and image acquisition were performed as previously described . BrieflyCytoplasmic and membrane proteins were extracted using a Subcellular Protein Fractionation Kit . To minimize contamination between fractions, pellets were washed in buffers from the previous step. The % membrane/cytoplasm ratios of p120-catenin 1A and 3A were determined by quantifying each band of membranous or cytoplasmic p120-catenin using the Multi Gauge 3.0 software program . All results were determined using at least three independent experiments.5/well) and Caco2 cells (1 × 105/well) were added to each well in triplicate and incubated for 24 h at 37 °C to prepare the cell-coated plate. While incubating the cell-coated plate, subconfluently plated MCF-7 and Caco2 cells were treated with ctrl/CM or 90K/CM for 16 h. After trypsinization, the cells were labeled with calcein green  in PBS for 10 min at 37 °C and then washed three times with PBS. A quantity of 3 × 105 of stained cells were then added to each well in triplicate and incubated for 1 h at 37 °C to allow the cells to adhere to the cell monolayer. After washing, cells remaining attached to the cell monolayer were visualized and counted under a fluorescence microscope.Cell–cell adhesion assays were performed in 48-well plates. MCF-7 cells (2 × 103 cells in 60 µL medium with 1% FBS were seeded on the Matrigel G (50 µg/mL) coated membrane with 8 µm pores. Fibronectin (20 µg/mL) was added to the lower compartment of the chemotaxis chamber. Images of each insert were taken every 30 min for a period of 40 h. The chemotactic migration from the top to the bottom side of the membrane was quantified as migrated cells on the bottom of the membrane in relation to the total cell number added. The calculation was performed with the IncuCyte ZOOM microscope software 2015A.For live-cell analysis of cell invasion, the IncuCyte ZOOM Chemotaxis Module and the IncuCyte ClearView 96-well cell migration plate were used. For each insert, 1 × 10t test. A p-value < 0.05 was considered significant unless indicated otherwise.All experiments were assayed in multiplicate. Data were expressed as mean ± standard error of the mean. All statistical analyses were performed using the SPSS version 17. Treatment effects were determined using one-way ANOVA post-hoc analysis or Student’s


Inflammatory and immune responses are generated locally by the selective invasion and accumulation of the immune cells into the lesion site. The infiltration process of the immune cells into the tissue from the blood through the vascular endothelial cells is closely regulated by a number of chemotactic factors and cell adhesion molecules.Fractalkine (FKN)/CX3CL1 is a membrane-bound chemokine possessing a chemokine/mucin hybrid structure and a transmembrane domain and has a dual function as an adhesion molecule and a chemoattractant. FKN is mainly expressed on activated endothelial cells, activated fibroblasts, and osteoblasts. Its receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, monocytes/macrophages, and osteoclasts. To date, a lot of key functional aspects of the FKN-CX3CR1 axis has been identified: (1) the rapid capture and firm adhesion of immune cells to vascular endothelial cells, (2) chemotaxis, (3) the enhancement of the transmigration to other chemokines, (4) the crawling behavior of the monocytes that patrol on vascular endothelial cells, (5) the retention of monocytes as the accessory cells of the inflamed endothelium to recruit inflammatory cells, and (6) the survival of the macrophage.In this review, we will focus on the pathological role of FKN in rheumatoid arthritis (RA) and the physiological role of FKN on osteoclast differentiation. Furthermore, we will discuss the therapeutic potential of anti-FKN mAb for RA patients and its distinct mode of action from other cytokine inhibitors. Rheumatoid arthritis (RA) is a long-lasting autoimmune disorder that primarily affects joints characterized by synovial hyperplasia and bone erosion associated with neovascularization, infiltration of pro-inflammatory cells, and increased cytokine production. Chemokines and their receptors control immune cell trafficking and are crucial for the inflammatory process. Fractalkine (FKN) is a unique membrane-bound chemokine possessing multiple biological functions. The FKN-CX3CR1 axis participates in the patrol for tissue damages and the quick mobilization and accumulation of immune cells to the sites of danger. The FKN-CX3CR1 axis is also involved in the pathogenesis in both bone-resorbing and inflammatory diseases. Taken together, FKN-CX3CR1 is expected to be a novel therapeutic target for RA by simultaneous direct inhibition of inflammation and bone resorption.Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the synovial hyperplasia, joint destruction, and massive infiltration of lymphocytes and macrophages into the synovium. Fibroblast-like synoviocytes (FLSs) also play a major role in the pathogenesis of RA by producing a variety of cytokines, chemokines, and matrix-degrading enzymes that mediate the interaction with neighboring inflammatory and endothelial cells. Chronic inflammatory environments are responsible for the progressive inflammation in the joints and the destruction of the articular cartilage and bone .Chemokines are a family of small (8–10 kDa) proteins that play an important role in the recruitment and activation of immune cells. They are subdivided into four subfamilies, C, CC, CXC, and CX3C chemokines, based on the number and spacing of the amino-terminal, conserved cysteine residues. The biological effects of chemokines are mediated by their binding to the cognate receptors, seven-transmembrane G protein-coupled receptors (GPCRs). Over 50 chemokines and 19 receptors have been identified, for which complex ligand-receptor relationships are revealed with high redundancy .Chemokines are originally identified as potent attractants for leukocytes such as neutrophils and monocytes and therefore are generally considered as mediators of acute inflammation (inflammatory chemokines). In addition, several chemokines have been found to be constitutively expressed in lymphoid and other tissues with individually characteristic patterns. Lymphocytes also express chemokine receptors with a cell-specific manner. Accumulating evidence indicates that chemokines are important not only in inflammation but also in the development, homeostasis, and functions of the immune system that should be shaped in a proper balance (immune or homeostatic chemokines).In this review, we will discuss the roles of FKN, the only member of the CX3C chemokine family, on inflammatory/immune diseases and its potential as a new therapeutic target for RA.FKN is a membrane-bound chemokine possessing a chemokine/mucin hybrid structure followed by a transmembrane domain . This inlow CX3CR1high monocytes scan capillaries and scavenge micrometric particles from their luminal side in a steady state in the kidney cortex. Importantly, a local TLR7-dependent danger signal increases the retention time of Ly6Clow CX3CR1high monocytes on the endothelium. These tethered monocytes are then activated and function as “accessory cells” of the endothelium by orchestrating the focal necrosis of endothelial cells by recruiting neutrophils and the in situ phagocytosis of cellular debris by monocytes [FKN is expressed on vascular endothelial cells and is strongly enhanced by the stimulation with pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interferon-γ (IFN-γ). CX3CR1 is expressed on monocytes/macrophages and perforin+/granzyme B+ cytotoxic lymphocytes including NK cells and terminally differentiated cytotoxic T cells . Solubleonocytes . The FKNonocytes . Lionakionocytes .These results indicate that the FKN-CX3CR1 axis participates in the patrol for tissue damages in normal conditions and the quick mobilization and accumulation of effector cells to the sites of danger  cells and endothelial cells in the RA synovium and contributes to the accumulation of T cells and macrophages, which express CX3CR1. The interaction between FKN and CX3CR1 is involved in the adhesion of the inflammatory cells to endothelial cells, their migration into the synovium, and cytokine production . FurtherMacrophages are the primary source of pro-inflammatory cytokines. A high percentage of macrophages within the rheumatoid synovium express the CX3CR1 receptor. Circulating CD16+ monocytes express higher levels of CX3CR1 than CD16− monocytes in both RA patients and healthy subjects. High levels of CX3CR1 expression are also seen in CD16+ monocytes localized to the lining layer in RA synovial tissue, and soluble FKN efficiently induced the chemotaxis of these cell populations , 13. YanFLSs, which are resident cells in the sublining region and markedly expanded in the RA synovium, have been linked with a number of deleterious effects in RA. FLSs produce pro-inflammatory cytokines, exhibit a capacity for antigen presentation, and induce T cell expansion . FKN is In the collagen-induced arthritis (CIA) model in mice, the prophylactic treatment of anti-FKN monoclonal antibody (mAb) significantly improves the clinical arthritis score and reduces the infiltration of inflammatory cells, and bone erosion in the synovium, suggesting that anti-FKN mAb ameliorates arthritis by inhibiting the infiltration of inflammatory cells into the synovium . In addiOsteoclast precursors selectively express CX3CR1, whereas FKN is expressed on osteoblasts. FKN on the osteoblasts is involved in the osteoblast-induced osteoclast differentiation . SolubleIn the analysis of the femoral bone tissues of heterozygous CX3CR1−EGFP knock-in mice, CX3CR1−EGFP+ cells are shown to differentiate into tartrate-resistant acid phosphatase (TRAP)+ mature osteoclasts. CX3CR1−EGFP+ but not CX3CR1−EGFP− cells sorted from bone marrow cells efficiently differentiate into TRAP+ mature osteoclast-like cells in vitro in the presence of RANKL, indicating that CX3CR1+ cells in the bone marrow are osteoclast precursors , 19.The role of the FKN-CX3CR1 axis in osteoclast recruitment and osteoclastogenesis is evaluated by an irradiated murine model. FKN is dramatically up-regulated in the skeletal vascular endothelium after ionizing radiation (IR). The induced FKN promotes the recruitment of circulating CX3CR1+ osteoclast precursors toward the bone remodeling surface in the irradiated bones and enhances subsequent bone resorption. In vivo experiments also show that the blockade of the FKN-CX3CR1 axis ameliorates osteoclastogenesis and prevented bone loss after IR .Collectively, FKN plays an important role in osteoclast recruitment and differentiation, possibly through its dual functions as a chemotactic factor and an adhesion molecule for CX3CR1+ osteoclast precursors. The FKN-CX3CR1 axis may be a novel target for the therapeutic intervention of bone-resorbing diseases such as RA and osteoporosis.KANAb001 (E6011) is the first humanized anti-FKN mAb, generated by KAN Research Institute, Inc. Currently, phase 1/2 clinical studies of E6011 are ongoing in both RA and Crohn’s disease in Japan by Eisai Co., Ltd. Recently, we have reported that E6011 is safe and well tolerated and has a promising efficacy in active RA patients with MTX or TNF inhibitor-inadequate response (MTX-IR or TNFi-IR) at the American College of Rheumatology 2015. While further clinical studies are required, the results obtained to date indicate that a novel biological DMARD targeting the FKN-CX3CR1 axis will be clinically beneficial for active RA patients.Most of current marketed mAbs inhibit specific cytokines and cytokine receptors, and JAK inhibitors block the effect of multiple cytokines by targeting cytokine signaling. On the other hand, E6011 targets the cell trafficking of immune cells, which produce multiple pro-inflammatory cytokines in the local inflamed sites. Previous studies have demonstrated that anti-FKN mAb inhibit the migration of both CX3CR1+ macrophages producing pro-inflammatory cytokines  and CX3CR1+ cytotoxic effector T cells containing cytotoxic molecules (granzyme B and perforin). These results indicate that anti-FKN mAb has a potential to block the most upstream step of the inflammation cascade in the local inflamed region. In addition, anti-FKN mAb suppresses the bone resorption by inhibiting osteoclast differentiation. Taken together, E6011 is expected to have strong preventive effects on joint destruction with a unique dual mode of action based on the inhibition of the immune cell trafficking and the direct suppression of osteoclastogenesis in local environments Fig. .Fig. 2E6FKN is a unique chemokine possessing a dual function as a chemoattractant and an adhesion molecule for CX3CR1-expressing monocytes, cytotoxic effector lymphocytes, and osteoclast precursors. Increasing evidence indicates that FKN is involved in the pathological roles of inflammatory disease such as RA. Now, clinical trials of E6011, the first humanized anti-FKN mAb, are ongoing in Japan. It is expected that E6011 will open up the possibility of a new therapeutic strategy for the treatment of RA with a novel mode of action distinct from other cytokine/cytokine receptor inhibitors  and modulator of T cell co-stimulation (abatacept).