It is now well documented that natural products have played an important role in anticancer therapy. Many studies focus on the ability of these natural compounds to modulate tumor-related signaling pathways and the relationship of these properties to an anticancer effect. According to the World Health Organization (WHO), colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death among men and women. Therefore, finding strategies to fight against CRC is an emergent health problem. CRC has a strong association with deregulation of Wnt/β-catenin signaling pathway. As some types of natural compounds are capable of modulating the Wnt/β-catenin signaling, one important question is whether they could counteract CRC. In this review, we discuss the role of flavonoids, a class of natural compounds, on Wnt/β-catenin regulation and its possible potential for therapeutic usage on colorectal cancer. The theory that natural molecules might inhibit cancer development started more than 100 years ago, when laboratories began to investigate the effect of small molecules extracted from plants and fungi on tumor development and growth ,3. CurreUncontrolled cell growth has been a feature of many types of tumors, in particular colorectal cancer which is, according to the WHO, the third most common cancer with 1,361,000 cases in 2012 . DespiteA great effort has been made to understand the molecular mechanism by which these natural compounds are acting to inhibit tumor growth ,6,10,15.Wnts are an evolutionary conserved family of secreted glycoproteins found as 19 distinct Wnt ligands in mammalians . The WntDuring canonical Wnt signaling, binding of Wnt ligands to Frizzled/low-density lipoprotein-related protein 5/6 (LRP5/6) receptor complexes causes stabilization of β-catenin. Activation of the Wnt pathway is controlled by regulation in disassembly of the destruction complex, which stabilizes β-catenin. The destruction complex, composed of APC (Adenomatous polyposis coli), Axin1, GSK-3β (Glycogen synthase kinase 3-β), and CK1 (casein kinase 1), phosphorylates serine residues in β-catenin leading to its ubiquitination by β-Trcp (F-box/WD repeat-containing protein 1A) and degradation by the proteasome. Stabilized β-catenin is then able to translocate to the nucleus and, through interactions with the T-cell factor (Tcf)/lymphoid enhancer factor 1 (LEF-1), modulates the expression of specific target genes that control developmental and cell cycle genes and oncogenes  20,21].,21.20,21Conversely, mutations in components of the Wnt/β-catenin are often associated with cancer, in particular in CRC ,25. The CRC is associated with polyps, not detected in early stages, leading to tumor development . Polyps in situ or intramucosal carcinoma [In the stage 0, cancer is in the earliest stage and it has not grown beyond the inner layer (mucosa) of the colon or rectum. This stage is also known as carcinoma arcinoma ,35. In sarcinoma ,35. WhenWnt, RAS2MAPK, PI3K, TGF-β, p53 and DNA mismatch-repair pathways [APC gene. Without APC, β-catenin accumulates to high levels, translocates into the nucleus, associates with TCF/LEF leading to its binding to DNA and consequent transcription of genes that are important for stem cell renewal and differentiation. However, when inappropriately expressed, APC can lead to cancer [A recent analysis published in 2012 by The Cancer Genome Atlas Network, has uncovered several critical genes and important pathways in the initiation and progression of CRC . These ipathways  They deso cancer . Anothero cancer . In addio cancer ,37. The in vitro and in vivo on APC mutant mice [Since Wnt signaling is critical for some carcinogenic development, many studies have been searching molecules capable of modulating this signaling pathway . Severalant mice . IWR-1 iant mice . G007-LKant mice . Interesant mice ,44.et al. showed ICG-001 as a selective inhibitor of Wnt/β-catenin signaling through specific binding of CREB-binding protein (CBP), disrupting its interaction with β-catenin leading to Wnt signaling inhibition [Emami KH hibition . While whibition . NSC6680hibition ,47,48,49Nuclear inhibitors of Wnt signaling are also very pursued, since upstream inhibition of Wnt signaling may have collateral effects on others signaling pathways. iCRT3, iCRT5, iCRT14, PKF118-310, PKF115-584 and CGP049090, BC21 are capable of inhibiting Wnt signaling by disrupting the interaction of β-catenin with its transcription factor TCF4 ,51. iCRTFlavonoids, polyphenolic compounds, constitute a very large group of natural products and one of the most characteristic classes of compounds in plants metabolism . More thin vitro [in vivo using Xenopus embryos as model system [Quercetin is one of the most studied and has been submitted to clinical trials. Quercetin has been pointed as a potential anti-cancer drug in colorectal cancer ,72 and iin vitro ,74. Morein vitro . This kiin vitro . Our grol system . Many adl system ,76,77,78wif-1 gene (Wnt inhibitory factor 1) expression through its demethylation. WIF-1 is a Wnt antagonist that directly binds to Wnt molecules, blocking signaling [−/+ mouse, a model for intestinal tumorigenesis, by reducing nuclear β-catenin levels, suggesting an inhibition of the Wnt mediator β-catenin translocation to the nucleus [SFRP1 gene in hepatoblastoma [Another vastly studied anticancer flavonoid is EGCG, which inhibited Wnt signaling in colorectal cancer cells ,79. The ignaling ,57,58,59 nucleus ,81. Receblastoma .O-β-d-glucopyranoside), a glycosylated derivative of quercetin, inhibits growth of different colorectal cancer cell lines (unpublished data). Genistein also suppressed β-catenin/TCF transcriptional activity in SW480 cells in a dose-dependent manner. This flavonoid affects upstream components of the β-catenin/TCF pathway by suppression of AKT phosphorylation, thus inhibiting GSK-3β dephosphorylation. Then, phosphorylated GSK-3β is able to phosphorylate β-catenin facilitating its ubiquitylation and degradation [APC gene. However, in HCT116 cells, which harbor wild-type APC but mutant β-catenin, silibinin has no effect, suggesting its selective effects on Wnt/β-catenin pathway [Scutellaria radix, a traditional Chinese medicine, has been shown to decrease expression of Wnt target genes Wnt3A, LRP6, Cyclin D1, c-Myc and to increase expression of Axin1 [et al. showed flavonone as an inhibitor of Wnt signaling by modulating TNKS activity [Ongoing studies of our group show that isoquercitrin (quercetin 3-radation . In 2010radation . They suradation . Interes pathway . Wogoninof Axin1 . Wogoninactivity . The autactivity , illustrDespite of the significant advances in diagnostic of CRC, current treatments consist mostly of surgery and conventional chemotherapy. These treatments confer limited benefit, which makes CRC the third leading cause of cancer death among men and women. CRC development and progression involve silencing of genes and alteration of regulatory mechanisms in one or more members of the Wnt/β-catenin signaling pathway. Thus, the identification of substances capable of modulating the Wnt/β-catenin signaling has been a major effort for many research groups. Notably several new compounds have recently been described as inhibitors of different components of the Wnt/β-catenin signaling pathway. Particular attention has been given to flavonoids since case-control, cell culture and animal studies have revealed a plethora of bona fide compounds with protective role against CRC malignancy. However a long way has still to be paved to achieve treatment success of CRC. One problem to overcome is the bioavailability of flavonoids during cellular metabolism. In this regard, chemical structure modification and the use of nanoparticles may be a good strategy to develop more effective flavonoids to target Wnt/β-catenin and fight CRC growth and progression.In summary, the recent advances in the understanding of the mechanisms of action of flavonoids targeting Wnt/β-catenin may shed light on future CRC therapies.


We investigated here whether β-catenin interacts with and regulates endothelial NOS (eNOS) and whether eNOS activation promotes β-catenin signalling.in situ proximity ligation assay. eNOS activity, assessed by cGMP production and eNOS phosphorylation (Ser1177), was enhanced in β-catenin−/− mouse pulmonary endothelial cells (MPECs) relative to wild-type MPECs. eNOS activation , or application of an NO donor (spermine NONOate) or cGMP-analogue (8-bromo-cGMP) caused nuclear translocation of β-catenin in HUVEC as shown by western blotting of nuclear extracts. Exposure to spermine NONOate, 8-bromo-cGMP, or sildenafil (a phosphodiesterase type 5 inhibitor) also increased the expression of β-catenin-dependent transcripts, IL-8, and cyclin D1. Stimulation of wild-type MPECs with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), spermine NONOate, 8-bromo-cGMP, or sildenafil increased tube length relative to controls in an angiogenesis assay. These responses were abrogated in β-catenin−/− MPECs, with the exception of that to bFGF which is NO-independent. In C57BL/6 mice, subcutaneous VEGF-supplemented Matrigel plugs containing β-catenin−/− MPECs exhibited reduced angiogenesis compared with plugs containing wild-type MPECs. Angiogenesis was not altered in bFGF-supplemented Matrigel.We identified β-catenin as a novel eNOS binding protein in human umbilical vein endothelial cells (HUVECs) by mass spectroscopy and western blot analyses of β-catenin and eNOS immunoprecipitates. This was confirmed by These data reveal bidirectional cross-talk and regulation between the NO-cGMP and β-catenin signalling pathways. As examples, activity of the transcription factor c-jun is inhibited by NO,17 and NF-κB activity is down-regulated due to NO-induced S-nitrosylation.18 A potential link between transcriptional regulation by NO and β-catenin has been revealed in conditionally immortalized murine colonic epithelial cells, where the NO donor NOR-1 was reported to induce β-catenin association with LEF in the nucleus.19Aside from its importance in the regulation of permeability, β-catenin in concert with T-cell factor/lymphocyte enhancing factor (TCF/LEF) transcription factors, also plays an important role in transcriptional regulation of various genes and is a key modulator in the Wnt signalling pathway.We hypothesized that endothelium-derived NO may induce β-catenin to translocate from the cytosol and/or plasmalemma of endothelial cells to the nucleus, thereby inducing changes in gene transcription. The aims of this study were three-fold: first to investigate whether β-catenin interacts directly with eNOS in endothelial cells; secondly to ascertain whether such interaction modulates eNOS activity; and thirdly to determine whether activation of NO-cGMP signalling—either through endogenous eNOS stimulation or through other means—gives rise to nuclear translocation of β-catenin with resultant gene transcriptional and functional effects.2.Supplementary material online.Detailed methods are available in the 2.120 Wild-type and β-catenin−/− mouse pulmonary endothelial cells (MPECs) were a generous gift from Professor Elisabetta Dejana, University of Milan, Italy.21Human umbilical vein endothelial cells (HUVECs) were isolated as previously described.2.2In situ proximity ligation assay (PLA) was carried out using rabbit anti-eNOS and mouse anti-β-catenin primary antibodies in combination with Duolink In Situ Red Detection Kit (Sigma). PLA was carried out according to the manufacturer's instructions.HUVECs cultured in 16-well chamber slides (30 000 cells/well) were fixed with 4% paraformaldehyde then permeabilized with 0.5% TX-100. 2.320 For preparation of nuclear lysates, hypotonic buffer followed by centrifugation was used to collect the cytosolic fraction (supernatant). Pellets (containing the nuclei) were lysed with high-salt buffer.Cell lysates were prepared as previously described.2.44Immunoprecipitation of eNOS or β-catenin from 400 µg of cell lysate with 1 µg of antibody was carried out as previously described using Protein G Sepharose beads. Electrophoresis and electroblotting were performed as described.2.5MPEC lysates were extracted in the presence of 3-isobutyl-1-methylxanthine  by addition of 0.1 mol/L HCl. cGMP concentration was assessed following acetylation using Cyclic GMP EIA Kit  according to the manufacturer's instructions. Resulting cGMP concentrations were normalized to protein content per sample.2.6in vitro, MPECs were seeded onto Matrigel; total tube length was assessed after 6 h. To investigate in vivo angiogenesis, Matrigel supplemented with either VEGF or basic fibroblast growth factor (bFGF) was mixed with MPECs loaded with Vybrant® DiO cell labelling solution (Life Technologies) and injected into C57BL/6 mice anaesthetized using isoflurane. Following sacrifice, plugs were excised, snap-frozen, and cryosectioned for immunostaining with an anti-CD31 antibody and imaged by confocal microscopy. Capillary formation was assessed by determining the percentage of CD31-positive Vybrant-labelled MPECs compared with the total number of CD31-positive cells within four randomly selected fields of view per plug. Animals were studied in accordance with Directive 2010/63/EU of the European Parliament.To evaluate angiogenesis (tube formation) 2.7RNA was isolated using QIAGEN RNeasy Kit according to the manufacturer's instructions. Transcript levels were assessed by quantitative real-time PCR using gene-specific primers.2.8post-hoc pairwise comparisons. Two-way ANOVA was carried out where inhibitors were used in conjunction with agonists. Where responses of wild-type and β-catenin−/− MPECs were examined, responses within each cell type were analysed separately by ANOVA as appropriate. In all cases, P < 0.05 (two-sided) was taken to indicate statistical significance.All data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism (Version 6.0) software. Unless indicated otherwise, all statistical comparisons were by one-way analysis of variance (ANOVA) with repeated measures and the Geisser-Greenhouse correction for sphericity. Fisher's LSD test was used for planned 3.3.1Figure 1A). Immunoprecipitation of eNOS resulted in the detection of both eNOS and β-catenin, and similarly immunoprecipitation of β-catenin resulted in the detection of both proteins. The association between eNOS and β-catenin was confirmed in situ in fixed cells by PLA . Amplification products were detected in HUVEC exposed to both eNOS and β-catenin antibodies, indicative of close proximity (<40 nm) of the two proteins; in contrast, controls where antibodies were omitted or used individually did not yield amplification products. Furthermore, peptide mass fingerprinting confirmed the presence of β-catenin in eNOS immunoprecipitates .To investigate whether β-catenin associates with eNOS, eNOS was immunoprecipitated from HUVEC lysates, and the resulting immunoprecipitates were subjected to western blotting for both eNOS and β-catenin . cGMP was higher in β-catenin−/− compared with wild-type MPECs . We next assessed eNOS phosphorylation status on Ser1177, a sensitive index of eNOS activity, by western blotting of cell lysates using an antibody that specifically recognizes phosphoSer-1177-modified eNOS; this was performed both on unstimulated and stimulated  wild-type and β-catenin−/− MPECs. Stimulation with either agent increased eNOS phosphorylation in wild-type MPECs, and this was abrogated by pre-treatment with Akt inhibitor IV . Moreover, we found that β-catenin−/− MPECs exhibited significantly higher levels of eNOS phosphorylation under non-stimulated conditions, which were only partially reduced by the Akt inhibitor, and that stimulation of these cells with adenosine or histamine did not produce any further rise in eNOS phosphorylation . These data suggest that β-catenin may function as an endogenous negative regulator of eNOS in endothelial cells.To determine whether β-catenin binding to eNOS may serve to regulate eNOS function, we first measured intracellular cGMP, a well-validated index of bioactive NO, in wild-type and β-catenin3.3Figure ).Having demonstrated that β-catenin associates with, and tonically inhibits, eNOS activity in unstimulated cells we sought to determine, using PLA, whether the interaction is maintained following pharmacological activation of eNOS. Amplification products were observed throughout the cell and localized close to cell borders in vehicle-treated HUVECs; however, following stimulation for 2.5 min with adenosine (100 µmol/L), salbutamol (1 µmol/L), histamine (100 µmol/L), or thrombin (1 IU/mL), few amplification products were observed, suggesting a loss of interaction between eNOS and β-catenin following eNOS activation , with no obvious change in plasmalemmal staining.We next determined whether eNOS stimulation or NO-cGMP pathway activation by other means induces β-catenin to translocate to the nucleus, an initial first step in its mediating effects on downstream gene transcription. HUVECs were treated with adenosine (100 µmol/L), salbutamol (1 µmol/L), histamine (100 µmol/L), or thrombin (1 IU/mL)—all at concentrations which we have previously found to cause maximal eNOS activation—or vehicle for 2.5 min, following which cells were fixed and immunostained for β-catenin, and imaged by confocal microscopy. In vehicle-treated cells, β-catenin was found mainly in the plasma membrane, in an even distribution, with minimal nuclear staining. In contrast, treatment with any of the eNOS agonists caused clear accumulation of β-catenin staining in the nuclear and/or peri-nuclear region . However, whereas the nuclear accumulation of β-catenin induced by adenosine and salbutamol (which do not elevate cytosolic Ca2+) was completely inhibited by the NOS inhibitor NG-nitro-L-arginine methyl ester , that induced by histamine and thrombin (which activate eNOS by elevating cytosolic Ca2+) was not . This suggests that both NO and an elevated cytosolic Ca2+ can independently induce translocation of β-catenin to the nucleus, the latter irrespective of eNOS activation. Consistent with this, translocation of β-catenin to the nucleus induced by the Ca2+ ionophore ionomycin (10 µmol/L) was similarly unaffected by the presence of L-NAME . Glycogen synthase kinase (GSK)-3β phosphorylates β-catenin on Ser37 and Thr41, promoting its ubiquitination and degradation.22 We therefore also examined the effect of LiCl (which inhibits GSK-3β23) on nuclear accumulation of β-catenin, as a positive control. As expected, LiCl (10 mmol/L) increased nuclear β-catenin in HUVEC which was unaffected by co-incubation with L-NAME .To confirm that eNOS activation gives rise to accumulation of β-catenin within the nucleus, HUVECs were treated as above and nuclear proteins were extracted. Levels of nuclear β-catenin were determined by western blotting of nuclear extracts, with normalization to the nuclear membrane protein lamin A/C. All eNOS agonists tested induced an increase of nuclear β-catenin , whereas treatment with adenosine and salbutamol did not . These results show that, although β-catenin and eNOS co-associate in the plasma membrane, β-catenin translocation to the nucleus following eNOS activation (at least by agonists that do not raise intracellular Ca2+) does not require translocation of eNOS.We also examined whether eNOS, following its activation, co-translocates to the nucleus with β-catenin. Treatment with the Ca2+ clearly activates a separate pathway (as shown earlier), we used salbutamol (which activates eNOS without elevating cytosolic Ca2+23) as the agonist. HUVECs were treated with salbutamol for 2.5 min, in the presence or absence of either L-lysine (1 mmol/L), which competes with L-arginine for transport into the cell and suppresses Ca2+-independent activation of eNOS,23 or 1H-oxadiazoloquinoxalin-1-one , a specific inhibitor of sGC. To directly determine the effects of cGMP and NO, we also stimulated the cells with the cell-permeable cGMP analogue, 8-bromo-cGMP (10 µmol/L), or the NO donor, spermine NONOate (10 µmol/L), for 2.5 min. Consistent with the hypothesis that NO and cGMP mediate translocation of β-catenin, the salbutamol-induced elevation of nuclear β-catenin was abolished by both L-lysine and ODQ, and both 8-bromo-cGMP and spermine NONOate induced elevation of nuclear β-catenin .On this basis, we hypothesized that eNOS-mediated translocation of β-catenin to the nucleus was caused by NO production with consequent activation of soluble guanylyl cyclase (sGC) and cGMP generation. As elevation of cytosolic Ca3.524 All four of the eNOS agonists tested induced an increase in active β-catenin in whole cell lysates, with no change in total β-catenin, as did LiCl which was used as a positive control .Nuclear translocation of β-catenin is typically associated with its activation, requiring dephosphorylation of Ser37 and Thr41. We examined activation of β-catenin using the clone 8E7 antibody , which recognizes active β-catenin dephosphorylated on Ser37 and Thr41.3.625 HUVECs were treated with adenosine (a Ca2+-independent activator of eNOS23) or spermine NONOate for 2.5 min, in the presence or absence of either ODQ or the PKG inhibitor Rp-8-bromo-guanosine 3′5′-cyclic monophosphorothioate . Phosphorylation of GSK-3β was assessed using an antibody that recognizes GSK-3β phosphorylated on Ser9 . Adenosine and spermine NONOate both increased the ratio of phospho-GSK-3β/total GSK-3β, and these increases were abolished by either ODQ or Rp. In contrast, although LiCl increased the phospho-GSK-3β/total GSK-3β ratio, this was unaffected by either ODQ or Rp .Having established a role for sGC activation and cGMP generation in inducing translocation of β-catenin to the nucleus, we reasoned that cGMP-dependent protein kinase (PKG) may be important in this process by stabilizing β-catenin through phosphorylation (and hence inhibition) of GSK-3β.3.711 and interleukin 8 (IL-8),27 which are β-catenin/TCF target genes involved in the regulation of angiogenesis, 12 h after stimulation with spermine NONOate or the phosphodiesterase inhibitor sildenafil (100 nmol/L). Both agents increased expression of IL-8  and cyclin D1  relative to vehicle, as did LiCl (included as a positive control).To ascertain whether nuclear translocation of β-catenin induced by the NO-sGC-cGMP pathway gives rise to transcriptional activation, we assessed the expression of cyclin D13.830 and also regulating VEGF expression.32 We hypothesized that, since NO and cGMP increase transcriptional activity of β-catenin, loss of β-catenin may impair the NO-mediated angiogenic response. We assessed the expression of VEGF in wild-type and β-catenin−/− MPECs following stimulation for 12 h with spermine NONOate, 8-bromo-cGMP or sildenafil, by western blotting of cell lysates. All three agents increased expression of VEGF compared with vehicle in wild-type, but not β-catenin−/−, MPECs . A scratch-wound assay was performed to ascertain the migratory capacity of wild-type and β-catenin−/− MPECs in response to stimulation (as discussed earlier), as well as to VEGF. Wound closure was increased by all agents in wild-type MPECs relative to vehicle, but not in β-catenin−/− MPECs .NO-cGMP signalling plays a pivotal role in VEGF-induced angiogenesis, acting as a downstream effector of VEGF,in vitro by seeding MPECs onto Matrigel following stimulation with VEGF, spermine NONOate, 8-bromo-cGMP, or sildenafil. Tube length was increased by all agents in wild-type, but not β-catenin−/−, MPECs relative to vehicle . On the other hand both wild-type and β-catenin−/− MPECs exhibited increased tube length following stimulation with bFGF , an agent which stimulates angiogenesis independently of NO-cGMP signalling.30Angiogenesis (tube formation) was assessed in vivo, a Matrigel plug assay was performed to assess capillary formation in plugs containing fluorescent-labelled wild-type or β-catenin−/− MPECs in the presence of either VEGF or bFGF. In plugs supplemented with bFGF, capillary formation  was similar between wild-type and β-catenin−/− MPECs; however, in VEGF-supplemented plugs, β-catenin−/− MPECs exhibited less Vybrant-positive CD31-positive cells and lower numbers of capillary-like structures or vascular networks compared with wild-type MPECs . In parallel experiments, Matrigel was injected containing VEGF and either scrambled or β-catenin siRNA. The efficacy of β-catenin siRNA to reduce β-catenin protein levels was initially confirmed by western blotting of lysates following transfection of wild-type MPECs. Plugs containing β-catenin siRNA exhibited fewer CD31-positive cells per plug  compared with those containing non-targeting sequences . These data suggest that angiogenesis, driven by NO signalling, is attenuated following loss or knockdown of β-catenin.To determine whether loss of β-catenin affects NO-mediated angiogenesis 4.We here provide the first evidence that eNOS associates with β-catenin in endothelial cells, and that this association serves as a negative regulator of eNOS activity such that, when β-catenin is absent, basal eNOS activity/Ser1177 phosphorylation is increased and is unable to increase further in response to standard agonists (adenosine and histamine). We also show for the first time that NO, derived either from eNOS or exogenously, induces β-catenin translocation to the nucleus with resultant effects on gene transcription and endothelial function, in particular with regard to angiogenesis.In situ proximity ligation assay revealed a pattern of co-localization of eNOS and β-catenin predominantly close to cell borders, which may be consistent with localization to caveolae. Both proteins are known to associate with caveolin-135 and this newly identified interaction between eNOS and β-catenin may indicate a novel signalling complex. Caveolin-1 is a negative regulator of eNOS,36 and it is possible that the inhibitory action of β-catenin on eNOS function may be mediated through co-interaction with caveolin-1. This possibility deserves further investigation in future work. Analysis of proximity ligation assays also revealed that pharmacological activation of eNOS disrupts the interaction with inhibitory β-catenin, thus permitting NO production. This observation supports the concept that eNOS is subject to allosteric regulation by β-catenin, and also warrants further study in the future.6 Conversely, it has previously been shown in colon carcinoma cells, where β-catenin is constitutively active, that application of NO-acetylsalicylic acid reduces β-catenin transcriptional activity due to an inhibitory action of NO on the interaction between β-catenin and TCF-4.37 This suggests that in neoplastic cells, the communication between NO and β-catenin differs in comparison to contact-inhibited quiescent endothelial cells, where β-catenin expression and localization is highly regulated.Our demonstration that NO-cGMP signalling induces nuclear translocation of β-catenin as well as downstream gene transcriptional effects supports previous work revealing induction of β-catenin signalling following activation of iNOS.S-nitrosylate β-catenin.8 Although we found that application of an exogenous NO donor mimicked the effect of eNOS activation on β-catenin nuclear translocation, which might be consistent with such a mechanism, we also found that inhibition of sGC (with ODQ) prevented this translocation in response to salbutamol; moreover, elevation of cGMP (with 8-bromo-cGMP or sildenafil) promoted nuclear translocation of β-catenin. These data suggest that the effect of eNOS activation or exogenous NO on β-catenin translocation is mediated through cGMP elevation rather than through direct NO-mediated S-nitrosylation of β-catenin, or indeed translocation of β-catenin released from active eNOS. cGMP exerts its intracellular signalling through activation of PKG. PKG has previously been reported to phosphorylate GSK-3β inhibiting its activity.25 We here show that activation of eNOS or exogenous application of spermine NONOate, increases phosphorylation of GSK-3β and that this is attenuated following inhibition of sGC or PKG. Although the PKG inhibitor may have non-specific effects at higher concentrations, the concentration used in this study is highly selective for PKG.38 Our results are therefore consistent with NO, derived either from eNOS or from an exogenously applied NO donor, leading to sGC/cGMP/PKG activation with resultant GSK-3β inhibition and thereby activation and stabilization of β-catenin through a decrease in its phosphorylation .NO has previously been shown to directly 2+-elevating eNOS agonists, such as histamine and thrombin, increase vascular permeability through disruption of the VE-cadherin/β-catenin complex.40 A recent study reported that exposure of microvascular endothelial cells to histamine resulted in increased levels of β-catenin in the nucleus.41 Similarly, stimulation with thrombin has also been found to promote nuclear translocation of β-catenin with consequent changes in gene expression.42 Consistent with these findings, our study has shown that histamine and thrombin induce β-catenin nuclear translocation, apparently through increasing intracellular Ca2+. However, this occurs independently of any effects of histamine and thrombin on eNOS activity or NO generation, since L-NAME co-incubation did not alter the effect of histamine or thrombin  on β-catenin nuclear translocation. It is possible that the nuclear translocation of β-catenin seen with these agents occurs secondary to nuclear translocation of associated eNOS. However, translocation is more likely to occur from cytosolic β-catenin (due to an increase in stabilization as a consequence of inhibition of the destruction complex) rather than plasmalemmal (and hence eNOS-associated) β-catenin, since we found no obvious changes in plasmalemmal distribution of β-catenin following 2.5 min treatment, although relocation from this compartment cannot be ruled out.Many Ca45 This has been described as occurring through cGMP-mediated activation of transcription factors  and through S-nitrosylation of transcription factors , as reviewed recently.46 Our data define a novel hitherto unrecognized mechanism by which NO regulates gene expression, namely through cGMP-dependent β-catenin nuclear translocation.On the other hand, NO-mediated translocation of β-catenin to the nucleus does appear to result in transcriptional activation. Our data demonstrate that activation of NO-cGMP signalling using either an NO donor (spermine NONOate) or sildenafil, which inhibits cGMP phosphodiesterases and thereby elevates cGMP, results in the expression of β-catenin/TCF target genes, namely IL-8 and cyclin D1, which are important in regulating angiogenesis and proliferation. NO is well documented to modulate gene regulation at the transcriptional level in endothelial cells.30 and it has been shown previously that angiogenesis and wound healing are impaired in eNOS-deficient mice.47 We provide evidence here that β-catenin is also required for NO-driven angiogenesis. Endothelial cells lacking the β-catenin gene exhibited reduced expression of VEGF in response to exogenously applied NO or elevation of cGMP. These cells also exhibited attenuated angiogenic and migratory responses to exogenous NO or to increased cGMP, as well as to VEGF . On the other hand, angiogenic responses to the NO-independent pro-angiogenic stimulus bFGF30 were intact in these cells. In vivo capillary formation by β-catenin−/− MPECs was also attenuated in VEGF-supplemented Matrigel plugs when compared with capillary formation by wild-type MPECs, while there was no difference between these two cell types in Matrigel supplemented with bFGF. These data support the observation that β-catenin is required to mediate the pro-angiogenic effects of NO-cGMP signalling.NO-cGMP signalling plays a key role in VEGF-induced angiogenesis,In conclusion, we have found in endothelial cells that β-catenin associates with eNOS under basal conditions, and that eNOS activation leads to β-catenin translocation to the nucleus through a cGMP-dependent mechanism with resultant effects on gene transcription and downstream functional responses, specifically as regards angiogenesis. We have also shown that β-catenin acts as an endogenous negative regulator of eNOS. Our findings provide novel evidence of bidirectional cross-talk and regulation between the NO-cGMP and the β-catenin signalling systems.Cardiovascular Research online.Supplementary Material is available at the British Heart Foundation  and Wellcome Trust (WT087776MA).This work was supported by grants from the China Scholarship Council (to N.C.),


To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers , were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10−9 to 10−8 M) significantly up-regulated the expressions of osteoblastic genes. SP (10−8 M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10−8 M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10 They coStability and localization of β-catenin is crucial for signal transduction in the Wnt/β-catenin pathway ,14. Canoin vitro model with osteogenic properties and fast growth. Our findings revealed that SP enhanced the differentiation of MC3T3-E1 cells and activated the Wnt/β-catenin signaling pathway.Recent reports showed that calcitonin gene-related peptide, another neurotransmitter released by peripheral nerve system, promoted the differentiation of MC3T3-E1 osteoblast-like cells . However2.2.1.2.1.1.To show the osteoblastic features of the MC3T3-E1 line, cells were used for alkaline phosphatase (ALP) and Alizarin red staining before exposure to the differentiation medium. ALP activity and presence of Ca deposits in extracellular matrix indicate that the MC3T3-E1 cells possess osteogenic properties .2.1.2.collagen type I, alkaline phosphatase, osteocalcin, and Runx2) using quantitative polymerase chain reaction (qPCR). Collagen type I and alkaline phosphatase were selected as early markers of osteoblastic differentiation. Osteocalcin was selected as a late marker of that process. Furthermore, the expression of Runx2, a transcription factor crucial in osteoblast differentiation, was examined. Three different concentrations of SP  were tested in MC3T3-E1 cells at days 4, 7, and 14 in the osteoinductive medium. SP (10−8 M) significantly increased the level of alkaline phosphatase and collagen type I mRNA at day 4, significantly increased the levels of osteocalcin and Runx2 mRNA at days 4 and 7. SP at concentration of 10−9 M, significantly increased the levels of alkaline phosphatase and Runx2 mRNA at days 4 and 14, significantly increased the level of osteocalcin mRNA at days 4, 7, and 14 and collagen type I mRNA at day 4. Treatment with 10−10 M SP resulted only in elevated collagen type I and osteocalcin mRNA level at day 4 compared with the control group (group 4) (The impact of SP at different concentrations on the differentiation of MC3T3-E1 cells into osteoblasts was assessed by testing the osteoblastic gene expressions (group 4) .2.1.3.−8 M) significantly increased the expression of alkaline phosphatase and collagen type I mRNA at day 4 compared with the control group (group D) and also significantly increased the levels of osteocalcin and Runx2 mRNA on days 4 and 7  was evaluated by qPCR. The addition of SP (10−8 M) to the osteoinductive medium significantly increased the expression of c-myc and Lef1 on days 4, 7, and 14 compared with the control group (group D). Moreover, it significantly increased the expression of cyclin D1 on days 4 and 7 but decreased the expression of Tcf7 on days 4 and 7. These effects of SP were significantly inhibited by the pretreatment with NK1 antagonist and DKK1. SP also significantly increased the expression of β-catenin mRNA on day 4 but, instead of down-regulating the expression of β-catenin mRNA, the pretreatment with NK1 antagonist and DKK1 increased the expression of β-catenin mRNA compared with the SP group (group A)  .2.1.5.−8 M) to the osteoinductive medium increased the level of c-myc and β-catenin protein on days 4, 7, and 14 compared with the control group (group D). Pretreatment with NK1 antagonist and DKK1 inhibited the SP induced c-myc and β-catenin protein level increase on days 4, 7, and 14  to the osteoinductive medium resulted in transfer of β-catenin (green) into the nucleus (blue). Pretreatment with NK1 antagonist and DKK1 prevented the SP induced transfer of β-catenin into the nucleus . Addition of SP  led to an increase in osteoblastic gene expression including collagen type I, alkaline phosphatase, Runx2, and osteocalcin on day 4 and to a less extent on day 7. The role of SP was in turn inhibited by the NK1 receptor antagonist and DKK1, the antagonist of Wnt/β-catenin signaling pathway. SP treatment significantly increased the expressions of genes and levels of proteins associated with activation of the Wnt/β-catenin signaling pathway, but down-regulated the genes associated with inhibition of the Wnt/β-catenin signaling pathway on days 4, 7, and 14. The NK1 receptor antagonist and DKK1 inhibited these effects of SP. These findings indicate that SP may stimulate the differentiation of MC3T3-E1 cells via the Wnt/β-catenin signaling, whereas the NK1 antagonist and DKK1 may weaken the role of SP inducing the differentiation of MC3T3-E1 cells. Our data suggests a potential mechanism of nerves involvement in bone repair.In this study we explored the osteogenic effects of SP treatment on MC3T3-E1 osteoblast-like cells. We found that SP significantly promoted the differentiation of MC3T3-E1 cells. The osteoblastic differentiation Previous studies have established the role of β-catenin as a key element of Wnt/β-catenin signaling pathway in control of developmental gene expression programs. Upon activation of the destruction complex, phosphorylation of β-catenin by GSK-3 triggers subsequent ubiquitination and degradation of β-catenin by the proteasome. This mechanism allows maintaining low level of β-catenin in the cytoplasm ,19. When−8 M) for 4, 7, and 14 days led to increased expression of Lef1 and decreased expression of Tcf7. Therefore, we speculate that the cooperation of Lef1 and Tcf7 may be required in the process of activation of Wnt/β-catenin signaling induced by SP.The role of Tcf7/Lef1 in the Wnt/β-catenin signaling is still controversial. Tcf7 may act as both a repressor and an activator while Lef1 might be usually an activator but sometimes a repressor –31. In tAs Wnt/β-catenin signaling plays an important role in proliferation and differentiation in numerous developmental stages as well as adult tissue homeostasis, Wnt target genes have to be context specific ,34. In t3.3.1.2 atmosphere at 37 °C in α-MEM  containing 10% heat-inactivated fetal bovine serum (FBS). The medium was supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. After reaching 80% confluence, cells were passaged with 0.02% trypsin  and transferred to new culture flasks in a ratio of 1:3. To induce osteogenic differentiation culture medium was changed to differentiation medium (α-MEM containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid). The cell culture was performed in two steps. To find optimal concentration for promotion of cellular differentiation, the cells were divided into 4 groups according to different concentrations of SP : Group 1, incubated with SP (10−8 M); Group 2, incubated with SP (10−9 M); Group 3 incubated with SP (10−10 M); and group 4, the control group, treated with the same volume of PBS. To determine the effects of SP, NK1 antagonist, and DKK1 on the differentiation of MC3T3-E1 cells, cells were divided into 4 groups: Group A, incubated with SP (10−8 M); Group B, incubated with a mixture of SP and NK1 antagonist  [Murine osteoblastic MC3T3-E1 cells  were cultured in 5% COP-96345) ; Group CP-96345) ; and Gro3.2.Medium from the cultures before the shifting to differentiation medium was discarded and the cells were fixed in 4% paraformaldehyde for 30 min. After washing three times with ice-cold PBS, cells were stained for 5 min with alizarin red (Sigma) and analyzed under a light microscope.N,N-dimethyl formamide for 30 min or until a yellow color appeared. Cells were washed with PBS and photographed using a phase-contrast microscope. The alkaline phosphatase activity was assessed as red stains indicating the products of enzyme activity [The alkaline phosphatase (ALP) activity staining was performed with the cells before exposure to the differentiation medium. Cells were rinsed once with PBS, fixed in 2% formaldehyde and subsequently stained at 37 °C in solution containing naphthol AS-MX phosphate disodium salt, fast red salt (Sigma) and activity .3.3.GAPDH) mRNA. Data represent mean expression from 3 experiments.Total RNA of the MC3T3-E1 cells from the eight groups (groups 1–4 and groups A–D) at the 3 time points  was extracted using a RNeasy Mini Kit  according to the manufacturers protocol. After subsequent DNase digestion, 1 μg of RNA was used to synthetize 20 μL of cDNA using an iScript cDNA Synthesis Kit . Quantitative real time PCR reactions were performed in the ABI prism 7900 real time PCR system using the SYBR Green PCR master mix . Expression in each sample was evaluated in three technical replicates. Sequences of the oligonucleotides used in this study are listed in 3.4.The cells from groups A–D at indicated time points  were used for protein extraction. Cells were treated with the lysis buffer  and protein extracts were dissolved in sample buffer containing 50 mM Tris-HCl, 2% SDS, 10% glycerol, 100 mM dithiothreitol (pH = 6.80). Proteins were separated by SDS-PAGE in 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were decorated with anti-β-catenin antibodies and anti-c-myc antibodies . Bands were captured and documented through a CCD system . Blots were stripped and re-probed with anti-tubulin antibodies to demonstrate equal loading and to allow normalization of the protein content. Densitometry of the bands was performed using Molecular Imaging Software Version 4.0 (Kodak).3.5.co-butylene terephthalate (PBST) for 30 min. After washing three times in PBS, cells were incubated overnight with rabbit anti-mouse anti-β-catenin antibodies (Santa Cruz Biotechnology) diluted 1:100 in PBST. Cells were washed 3 times with PBS and incubated for 1 h with fluorescein isothiocyanate (FITC)-linked rabbit anti-mouse IgG antibody diluted 1:100 (USCN) and again washed three times in PBS. 4′-6-Diamidino-2-phenylindole (DAPI) was added to mark the nuclei. Slides were examined in Fluoview 300  fluorescent microscope and images were acquired [The cells from groups A–D at day 4 were fixed , permeabilized  and blocked in 1% bovine serum albumin (BSA) in poly butylene succinate-acquired .3.6.post hoc test was used to evaluate the statistical significance of the differences. Significance was achieved when p < 0.05. Presented error bars indicate standard error of the mean (SEM). * is indicative of p < 0.05, **p < 0.01 and ***p < 0.001.Statistical analyses were performed in SPSS . One-way ANOVA test followed by Dunnett (2-tailed t) 4.In conclusion, this study demonstrates that SP may stimulate the osteogenic differentiation of MC3T3-E1 cells and the canonical Wnt signaling may contribute to this process. The role of SP and Wnt/β-catenin signaling in osteogenesis should be further and depth studied by detecting more genes and proteins, such as GSK-3β, Axin, and APC, to elucidate the role of SP and Wnt/β-catenin signaling in bone development and remodeling to improve our understanding of the role of nerves in bone repair.


Extensive renal ablation is an experimental model widely used to gain insight into the mechanisms responsible for the development of CKD, but it was not evaluated for Wnt/β-catenin pathway. This study aimed to elucidate if the rat 5/6 renal mass reduction model (RMR) is a good model for the Wnt/β-catenin activation and possible next modulation. RMR model was evaluated at 12 and 18 weeks after the surgery, when CKD is close to end-stage kidney disease demonstrated by molecular and histological studies. Wnt pathway components were analyzed at mRNA and protein level. Our results demonstrate that Wnt pathway is active by increase of β-catenin at mRNA level and nuclear translocation in tubular epithelium as well as some target genes. These results validate the RMR model for future modulation of Wnt pathway, starting at shorter time after the surgery.Progression of chronic kidney disease (CKD) is characterized by deposition of extracellular matrix. This is an irreversible process that leads to tubulointerstitial fibrosis and finally loss of kidney function. Wnt/ Progression of chronic kidney disease (CKD) is characterized by deposition of extracellular matrix (ECM). This is an irreversible process that leads to tubulointerstitial fibrosis and finally loss of kidney function. It is widely accepted that epithelial-to-mesenchymal transition (EMT) is the main factor responsible for organ fibrosis. EMT is defined as phenotypic change of fully differentiated epithelial cells to matrix-producing fibroblasts .γ, tumor necrosis factor-α, interleukin-1-β, and transforming growth factor-β-1 (TGF-β) [Several studies have suggested that specific signal pathways may affect the development of fibrosis in the kidney and contribute to epithelial cell injury and fibroplasia. Some of the signaling mediators involved include proinflammatory and profibrotic cytokines produced by infiltrating and resident cells, such as interferon- (TGF-β) .β signaling pathway plays a pivotal role in embryonic development and organogenesis and is a key fibrogenic cytokine in several fibrotic diseases of the kidney, lung, and other organs. Campistol et al. have previously demonstrated that TGF-β serum levels were significantly higher in kidney transplant patients with interstitial fibrosis and tubular atrophy than in those with normal renal function [The TGF-function , 4.β has been shown to be critical for extracellular matrix turnover and for cellular plasticity, such as epithelial-to-mesenchymal transition. This transition represents the phenotypic, reversible switching of epithelial to fibroblast-like cells and may play a major role in the pathogenesis of CKD, increasing the number of activated fibroblasts derived from epithelial cells [β in fibrogenic processes and renal fibrosis has been widely described, as has been the role of crosstalk between TGF-β and Wnt/β-catenin pathways in cell fate determination during embryonic development and in the adult [β pathway, the SMAD proteins, can also be regulated by axin, a regulator of Wnt signaling. However, there is little evidence of interdependence or retroregulation between the components of the two pathways in transcriptional regulation of target genes in mammals, particularly in the context of epithelial-to-mesenchymal transition and organ fibrosis [TGF-al cells –7. The rhe adult . Crosstafibrosis .β-catenin pathway was reported to be aberrantly activated in the progressive damage associated with chronic organ failure [β-catenin. In the absence of Frizzled (FZD) receptor binding of Wnt ligands, β-catenin is constitutively phosphorylated by glycogen sintase kinase-3β (GSK-3β) and subsequently degrades by the complex composed of the adenomatous polyposis coli protein (APC) and axin. In the presence of Wnt ligand binding to a FZD receptor and a low density receptor (LRP) coreceptor leads to the phosphorylation of disheveled protein in the cytoplasm (DVL). The activated DVL binds to axin in order to antagonize the action of GSK-3β; subsequently β-catenin phosphorylation and ubiquitination are inhibited. This causes a citoplasmatic accumulation of β-catenin in the cytoplasm and translocation to the nucleus where it regulates target gene expression through interaction with members of the TCF/LEF family of HGM-domain transcription factors [Over the last years, the Wnt/ failure , 10. Wnt factors , 10.β-catenin signaling has been shown to be involved in epithelial cell injury and hyperplasia, as well as in impaired epithelial-mesenchymal crosstalk in fibrosis. Modulation of Wnt signaling led to an attenuation of lung fibrosis in vivo [Increased Wnt/ in vivo , while a in vivo  and anim in vivo , unilate in vivo , lupus n in vivo , or diab in vivo .Several CKD animal models have been used to study the mechanisms responsible for the disease progression. Extensive renal ablation is an experimental model widely used to gain insight into the mechanisms responsible for the development of CKD since it causes a combination of glomerular hemodynamic alterations and nonhemodynamic events that result in progressive glomerulosclerosis, tubulointerstitial damage, proteinuria, and renal functional impairment in the remnant kidney. There is early glomerulosclerosis by week 4, with segmental sclerosis with tubulointerstitial fibrosis by week 8 , 16.β-catenin activation and possible next modulation. To do this, we used 5/6 renal mass reduction model animals and we sacrificed animals at 12 and 18 weeks after the surgery, when CKD is well established, and then we characterized the fibrosis establishment and the Wnt activation.This study aimed to elucidate if the 5/6 renal mass reduction model is a good model for the Wnt/β-catenin (number 9562), phospho-S9- and total GSK-3β (numbers 9336 and 9315), phospho- and total LRP6 (numbers 2568 and 2560), and phospho-SMAD3 (C25A9) ; Wnt1 (600-401-A37) ; α-SMA (A2547); Ca2+ATPase (A7952); cyclin D1 (C7464); GADPH (G9545); β-actin (A3854); Pan CK (F3418); Desmin (D1033) and SIS3 (S0447) ; FBPase (sc-166097) and factor VIII (sc-27647) ; Alexa Fluor 568 phalloidin (A-12380) (Invitrogen); vimentin (AB1620) and TGF-β (MAB1032) .In this study, the following antibodies were used: total Male Wistar rats  weighing approximately 225 g were used. The rats were kept at a constant temperature and humidity and at a 12 h light/dark cycle. The animals had free access to standard rat chow  and water.This study was approved by and conducted according to the guidelines of the Local Animal Ethics Committee .n = 33) were randomly assigned to two groups according to the surgical intervention. One group was assigned to undergo renal mass reduction (nephrectomized group (Nx)) and the other to have a sham operation (sham group (S)). Surgical procedures were performed under general anesthesia with isoflurane . After an abdominal incision, the left kidney was exposed and separated from the adrenal gland. The lower and upper poles of the left kidney were frozen by application of a cylinder of dry ice of standard size for 2 minutes on each pole. After one week, the right kidney was removed. The sham group rats underwent the same abdominal incision and manipulation of the left and the right kidneys without tissue destruction as described by Rovira et al. [Animals . Relative changes in transcript levels compared with controls are expressed in DDCt values (DDCt = DCttreated − DCtcontrol). All DDCt values correspond approximately to the binary logarithm of the fold change, as mentioned in Total RNA was extracted by using a Perfect Pure RNA fibrous Tissue Kit  from renal tissue (human and rat) and by using RNeasy Mini Kit  extraction kits according to the manufacturer's protocol. cDNAs were generated by reverse transcription using MuLV reverse transcriptase . Quantitative (q)RT-PCR was performed using Light Cycler 480 SyBr Green I master (Roche), as previously described. r_RPL19, ubiquitously and equally expressed gene free of pseudogenes, was used as a reference gene in all qRT-PCR reactions. PCR was performed using the primers at a final concentration of 200 nM. The relative transcript abundance of a gene is expressed in DCt values , and the whole proteins were extracted by centrifugation  for 10 min at 4°C, as recommended by the supplier. Proteins were quantified using the Micro BCA Protein Assay Kit (PIERCE).μg of protein were separated by electrophoresis on SDS polyacrylamide gels. The separated proteins were transferred to PVDF membranes (BioRad), blocked with 5% skimmed milk or BSA depending on customer indication, and incubated with the indicated antibodies. The proteins were then visualized by enhanced chemiluminescence detection  with Image Quant LASS 4000 . Densitometry quantification was done using Image J.Samples containing 30 μm) were cut and mounted on slides. Light microscopic evaluation was performed on sections stained by Masson trichrome to evaluate fibrotic changes in representative renal allografts [Human biopsies and rat kidneys were placed in 4% (w/v) paraformaldehyde after explantation and were processed for paraffin embedding. Sections 3  μm were μm sections were subjected to antigen retrieval and quenching of endogenous peroxidase activity using 3% (v/v) H2O2 for 20 minutes. Immune complexes were visualized using suitable peroxidase-coupled secondary antibodies, according to the manufacturer's protocol . FBPase staining was made to discriminate the proximal tubule, and double staining with β-catenin was performed. Additionally, to exclude false-positive results for β-catenin nuclear translocation, we used histological slides of β-catenin from the next section to perform single staining with Vulcan fast red. The whole area of the biopsy samples was examined to detect cells with nuclear localization of β-catenin.The 3 Protein extracts from rat kidneys were used. Total collagen content was determined using the Sircol Collagen Assay kit  following the enhanced protocol. Samples and collagen standards were then read at 540 nm. Collagen concentrations were calculated using a standard curve generated by using acid-soluble type 1 collagen.All animals that undergo renal mass reduction (RMR) showed clear loss of renal function that it is increasing across the time. Diuresis, proteinuria, BUN, and creatinine were highly increased in nephrectomized animals as well as urinary creatinine and creatinine clearance was markedly reduced after renal mass reduction compared with sham animals .α-smooth muscle actin (α-SMA), fibroblast-specific protein-1 (FSP1), collagen 1a1 (col1a1), and vimentin (Vim) were analyzed. In addition, mRNA expression of the profibrotic mediators transforming growth factor-β (TGF-β) and plasminogen activator inhibitor-1 (Pai1) as well as the antifibrotic bone morphogenic proteins 4 and 7 (BMP4 and BMP7) were analyzed.Left kidney from sham (S) and 5/6 nephrectomized (Nx) rats was explanted at 12 or 18 weeks after surgery and underwent qRT-PCR analysis to assess renal fibrosis. General markers for fibroblast activation including α-SMA and Fn1 did not show expression regulation. The antifibrotic gene analysis did not show significative changes of either TGF-β or Pai1, while the antifibrotic proteins BMP4 and BMP7 were downregulated , while the Wnt1 protein level detected by Western blotting was significantly increased . The goal of this study was to characterize and validate the utility of the remnant kidney model with 5/6 nephrectomy (5/6 Nx) for the study of the Wnt pathway. Histological and molecular examination of the model demonstrated the damage and the Wnt pathway activation at 12 and 18 weeks after the surgery where we can mimic CKD. This confirms to us that 5/6 Nx is an easy, reliable, and good tool where Wnt pathway is activated.β in human and experimental models for renal fibrosis has been well described [Different models of renal mass reduction have been used to study the mechanisms of the progression of CKD in humans. In 5/6 Nx, the kidney's adaptive response to this surgical reduction in nephron number appears to be close enough to the pathophysiologic characteristics of human progressive nephropathies. 5/6 Nx is a well-described low nephron number model of chronic progressive renal disease with renal function impairment, proteinuria, glomerular sclerosis, and interstitial fibrosis , 16. Ourescribed , 17, 18.β-catenin accumulation, nuclear translocation, and interaction with TCF/LEF complex resulting in target gene regulation. We found upregulation at mRNA level of β-catenin and LEF1. The Wnt/β-catenin canonical pathway plays an essential role during development, driving to branching nephrogenesis in fetal kidney. In the adult, Wnt signaling plays role in the control of tissue homeostasis. Wnt pathway promotes cell proliferation, tissue expansion, cell fate determination, and terminal differentiation. mRNA analysis of Wnt ligands showed no significant upregulation of the canonical Wnt ligands Wnt1 and Wnt3a and downregulation of Wnt10b. Königshoff et al. described upregulation of Wnt1 on day 7 after obstructive injury in mice followed by a decline in mRNA levels [It is described that stimulation of canonical Wnt pathway led to β-catenin was analyzed in the rat renal tissue, β-catenin was expressed in proximal and distal tubules, but the proximal tubules showed basal localization and rarely citoplasmatic distribution, while the distal tubules showed strong cytoplasmic signal. Some nuclei of both tubules showed nuclear translocation but less frequently than in human samples. To confirm Wnt signaling activation in fibrotic rat kidneys, the key components of canonical Wnt signaling were analyzed at the protein level. Our data confirm Wnt activation, as demonstrated by an increase in the total content of β-catenin, LRP6, and GSK-3β phosphorylation, the target gene cyclin D1, and the Wnt1 ligand, indicating that Wnt1 plays a role in IFTA progression in the kidney, as previously described in heart and lung animal models. Duan et al. reported that Wnt1 was upregulated eightfold in the heart within 48 h after acute ischemic cardiac injury. These authors describe Wnt1 fibroblast proliferation and profibrotic gene induction [When the expression distribution of nduction . As in tβ-catenin pathway but not as strong as we expected. The 5/6 Nx model with the sacrifice at 12 and 18 weeks is a really advanced chronic damage. von Toerne et al. described CAD model and they analyzed the Wnt pathway activation across CAD development but until 8 weeks after the transplantation, and they observed upregulation across the time [In our model we observed an activation of the Wnt/the time . He et aTo our knowledge there are no reports about the Wnt regulation in a long term CKD. Our results demonstrate that pathway is active, but this is not a dramatic upregulation. That could be attributed to the functional tissue loss, because of the tubular and glomerular atrophy, indicating that time points analyzed could be too late for pathway modulation. Shorter time points will be necessary to set Wnt activation, in order to modulate its activation and couls study their effect on CKD activation.


Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell–cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue. Cell-cell adhesion of endothelial tissue, mediated by the adhesion molecule VE-cadherin, is tightly regulated. Here the authors show that the F-BAR domain protein pacsin2 is recruited to the trailing end of mechanically unbalanced Focal Adherens Junctions, where it inhibits internalization of VE-cadherin and protects cell-cell adhesion. The endothelium is a cell monolayer that covers the luminal side of blood vessels and maintains vascular barrier function. Regulation of endothelial monolayer integrity in homoeostasis, as well as its remodelling during angiogenesis, and transendothelial trafficking of immune cells, occurs at the VE-cadherin adhesion complex, the central component of endothelial cell–cell junctions1345To keep barrier integrity in vascular homoeostasis, perturbations at endothelial cell–cell junctions, induced by the highly dynamic mechanical changes that blood vessels experience910121314The VE-cadherin complex, which comprises the core catenins that connect the transmembrane cadherins to the actin cytoskeleton171920212214152728The F-BAR protein pacsin2  is known to regulate the actin cytoskeleton313230FAJs are not only phenotypically distinct from stable linear adherens junctions, but are also distinguished by the presence of vinculin, which interacts with the VE-cadherin–α-catenin complex upon force-induced tension23To understand how pacsin2 is recruited to cell–cell junctions, we first investigated the formation of FAJs by increasing cytoskeletal-derived tension on junctions, following thrombin treatments as described previously3738We next addressed the mechanism that underlies the asymmetric presence of pacsin2 at junctions. A curve of the double membrane at AJs is intracellularly perceived both as convex and concave shapes, depending from which cellular side of the junction this is sensed. To investigate if the recruitment of pacsin2, known to interact with convex membranes33Pacsin2, like many other F-BAR proteins, contains an F-BAR domain that is crucial for its binding to phospholipids in curved membranes and an SH3 domain, which interacts with effector proteins33To assess pacsin2 recruitment in relation to FAJ remodelling, we performed live imaging of HUVECs with lentiviral expression of pacsin2-GFP and α-catenin-mCherry (as marker for adherens junctions). Imaging of these transduced cells indicates that when pulling activity between cells is unbalanced, FAJs themselves displace in the direction of the cell that pulls away. This provides FAJs with a front  and a trailing end that follows the movement of the FAJ. Pacsin2 is consistently recruited to FAJ trailing ends  and 5. WTaken together, these data show that asymmetric remodelling of FAJs, mediated by local actomyosin changes, induces pacsin2 recruitment to the trailing ends of FAJs in an F-BAR domain-dependent manner.R) of these proteins with VE-cadherin at FAJs: α-catenin (R=0.72), β-catenin (R=0.77) and p120-catenin . By contrast, pacsin2 molecules show a significantly lower correlation with VE-cadherin (R=0.16) and the super-resolution imaging reveals that pacsin2 is decorating the invaginating ends of FAJs (Because (i) recruitment of pacsin2 to FAJs occurs via a different mechanism than recruitment of vinculin, and (ii) pacsin2 recruitment depends on a functional F-BAR domain, which is known to interact with the plasma membrane, we next used stimulated emission depletion (STED) super-resolution microscopy to further dissect the molecular organization of FAJs. First, we analysed the colocalization of endogenous VE-cadherin with the core catenin proteins in STED images and, as expected, found a high Pearson's correlation coefficient  of the internalized VE-cadherin-positive vesicles are endocytosed, whereas 9% (±1.4 s.e.m.) of the internalized vesicles were transferred from adjacent VE-cadherin expressing cells . Two of the five tested shRNA constructs induced robust pacsin2 knockdown in HUVECs . We deteTo investigate whether pacsin2 has a role in the polarized trafficking of α-catenin at FAJs, we next studied the mobility of α-catenin-mCherry at the trailing end of FAJs by FRAP in shControl and shPacsin2 transduced HUVECs. We find that the average mobile fraction of α-catenin-mCherry at the trailing end of FAJs is significantly higher in shPacsin2 HUVECs (51%±3.5 s.e.m.) than in shControl HUVECs  across endothelial cells transduced with pacsin2 or control shRNA. These experiments show that shPacsin2 cells have a significantly reduced capacity to form endothelial barriers, which is apparent both during cell–cell junction formation between recently plated cells and within established monolayers  was shown to regulate membrane dynamics during cell–cell junction formation, and its function is needed for efficient cadherin-based cell–cell adhesionDrosophila; the F-BAR proteins CIP4 and nostrin also appear to function as cadherin internalization suppressorsBecause pacsin2 is ubiquitously expressed30One of the key events implicated in inflammation, as well as in mechanical-induced remodelling of cell–cell junctions, is the trafficking of the VE-cadherin complex. Internalization of VE-cadherin occurs in response to permeability factors , actomyosin contraction, changes in (hemodynamic) forces or transendothelial migration of immune cells435354L-glutamine (Lonza) supplemented with 10% fetal calf serum and antibiotics. Bone marrow-derived human mesenchymal stromal cells, a kind gift from Dr Carlijn Voermans, were isolated and cultured as described previouslyPooled HUVECs (cultured up to passage six) from different donors (purchased from Lonza), immortalized HMEC-1 (described in ref. TransIT-LT1 (Mirus) according to the manufacturers' standard protocol. Pacsin2 expression was knocked down using shRNA expression plasmids from The RNAi Consortium (TRC) libraryFor lentiviral transductions, human pacsin2 fused at its C terminus to a GFP tag was amplified by PCR from a pcDNA6.2/C-EmGFP-DEST-pacsin2 vector (provided by Dr Ronen Zaidel-Bar) and cloned into a self-inactivating lentiviral pLV-CMV-ires-puro vector between the SnaBI and NheI restriction sites to generate pLV-CMV-pacsin2-GFP, and the pLV-CMV-pacsin2-Δ306 variant that expresses only its F-BAR domain: amino acids 1–306. The pLV-CMV-pacsin2-GFP construct was modified by replacing GFP with mCherry DNA sequence to generate pLV-CMV-pacsin2-mCherry. An arginine (CGC) to aspartic acid (GAC) mutation at amino acid position 50 in the pacsin2 F-BAR domain was generated by site-directed mutagenesis to generate pLV-pacsin2-[R50D]-GFP. A membrane localization mutant was generated by mutating methionine at position 124 and 125 (ATG ATG) to glutamic acid (GAG GAG). An SH3 mutant was generated by mutating proline at position 478 (CCG) to leucine (CTG) in pacsin2. Mouse α-catenin-mCherry and human Rab4a-GFP were cloned into the pLV-CMV-ires-puro vector using NdeI and NheI restriction sites. The lentiviral expression constructs pLV-α-catenin-Dendra2, pLV-VE-cadherin-GFP and pLV-p120-catenin-mCherry have been previously describedMouse monoclonal anti-actin  anti-vinculin antibody hVIN-1 ), rabbit anti-α-catenin serum  and anti-β-catenin serum  were obtained from Sigma-Aldrich. Mouse monoclonal anti-p120-catenin antibody , anti-β-catenin , anti-N-cadherin  and anti-Rho GDI  were from BD Bioscience. Rabbit polyclonal anti-pacsin2 antibody was from Abcam , and anti-pacsin1 and anti-pacsin3 rabbit sera were a kind gift from Dr Markus Ploman. Rabbit anti-nostrin was a generous gift of Dr Stefanie Oess. Monoclonal antibodies against serine 19 phosphorylated myosin light chain 2  and Rab5  were from Cell Signaling. To detect VE-cadherin, we used the following mouse monoclonal anti-VE-cadherin antibodies: clone 75 , clone F-8  and clone 55-7H1 directly coupled to Alexa-Fluor 647 ; rabbit polyclonal antibody  and goat polyclonal antibody . We used Acti-stain 555  or Promofluor 415-coupled phalloidin  to visualize F-actin. Secondary antibodies coupled to Alexa-Fluor 488, 568, 594 and 647 were obtained from Molecular Probes (Life Technologies) and all diluted 1/500. Bovine plasma-derived fibronectin was purchased from Sigma-Aldrich.−1 fibronectin and subsequently fixed in 4% paraformaldehyde in PBS  for 10 min, permeabilized with 0.5% Triton X100 in PBS for 5 min and blocked for 30 min with 2% bovine serum albumin (BSA) in PBS. For the scratch assays HUVECs were cultured overnight. A scratch was made across the cell layer using a sterile pipette tip, and cells were allowed to recover and migrate for 4 h before fixation and IF staining. Primary and secondary antibody stainings were performed in 0.5% BSA in PBS for 1 h, and coverslips were mounted in Mowiol4-88/DABCO solution. For the IF stainings in ++ at 4 °C for no longer than 24 h after surgery until further preparation as described previouslyFor standard IF staining, cells were cultured on coverslips coated with 5 μg ml−1 fibronectin and imaged within microscope incubators at 37 °C and 5% CO2. Widefield imaging (2) was photobleached by 200 iterations using the DPSS laser at 561 nm at maximal power (15 mW). Images were enhanced for display with an unsharp mask filter and adjusted for brightness/contrast in ImageJ.For live microscopy, cells were plated on Lab-Tek-chambered 1.0 borosilicated coverglass slides coated with 5 μg ml imaging  and 12 w imaging , a LEICA imaging  were perSTED microscopy was performed on a Leica TCS SP8 STED 3X microscope. HUVEC monolayers were stained using the following antibodies: rabbit polyclonal anti-pacsin2 (Abcam), mouse monoclonal anti-vinculin hVIN-1 (Sigma), mouse monoclonal anti-p120-catenin clone 98/pp120 (BD Bioscience), rabbit anti-α-catenin and anti-β-catenin serum (Sigma) combined with anti-VE-cadherin mouse monoclonal F-8 (Santa Cruz) or rabbit polyclonal (Cayman) antibodies. Secondary antibodies used were STAR 580 and STAR 635P (Abberior). Samples were irradiated with a pulsed white light laser at wavelengths 587 and 633 nm. A pulsed STED laser line at a wavelength of 775 nm was used for the depletion of the 587- and 633-nm fluorophore, reaching a lateral resolution of ∼45 nm to distinguish individual fluorescent signals in these IF stainings. The signal was detected using a gated hybrid detector in counting mode. STED images were acquired using a dedicated 100 × 1.4 NA oil objective. Finally, deconvolution was performed with Huygens Professional (Scientific Volume Imaging).Cell lysates were prepared in reduced sample buffer and analysed by standard western blotting and enhanced chemiluminescence detection. Uncropped scans of blots are presented in For flow cytometry analysis in L-cysteine (Sigma) for 15 min at 37 °C and subsequently coated with 10 μg ml−1 fibronectin (Sigma) in 0.9% NaCl for 1 h at 37 °C. Cells were seeded and electrical resistance was continuously measured during monolayer formation at 30 kHz using ECIS model 9600 (Applied BioPhysics) and 37 °C at 5% CO2 incubator conditions. Resistane values were corrected for background signal that was measured in ECIS wells without cells . In Endothelial barrier function was determined by measuring the electrical resistance of monolayers by ECIS. Electrode arrays  were treated with 10 mM 2 at the pacsin2-positive versus pacsin2-negative side of FAJs. The values are corrected for background and normalized to maximal intensities in the overall image. To determine the speed of FAJ movement .Supplementary Figures 1-3Pacsin2 localization at Focal Adherens Junctions. Confocal z-stack of single channel and merged IF high magnification images of FAJs in HUVECs stained for pacsin2 (green) and VE-cadherin (red). Z-stack is projected in basal-to-apical direction and each stack represents 0.227 μm in z-direction. Scale bar indicates 5 μm in xy direction. Images were acquired by confocal laser scanning microscopy (Zeiss LSM 510) using a 63x/1.4 NA oil objective.Pacsin2 dynamics during FAJ formation induced by thrombin. Time lapse recording of HUVECs expressing pacsin2-GFP (green) and α-catenin-mCherry (red). The formation of FAJs was induced by addition of thrombin at the first minute of the time lapse. Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 40 minutes.Pacsin2 dynamics during the release of cytoskeletal tension on FAJs by Y-27632. Time lapse recording of HUVECs expressing pacsin2-GFP (green) and α-catenin-mCherry (red). Tension at FAJs was reduced by addition of Rho-kinase inhibitor Y-27632 at the 90 seconds time frame of the movie. Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 20 minutes.Pacsin2 at FAJs is derived from one cell. Time lapse recording of mosaic monolayer of HUVECs expressing pacsin2-GFP (green) or pacsin2-mCherry (red). The movie shows that the FAJs contain pacsin2-GFP from the left cell, but not pacsin2-mCherry from the right cell that takes part in the FAJs. Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 18 minutes.Pacsin2 locates at the trailing ends of α-catenin at FAJs. Time lapse recording of HUVECs expressing pacsin2-GFP (green) and α-catenin-mCherry (red). The bottom cell pulls on the FAJs causing directional downwards movement of the FAJs, which is accompanied by the recruitment of pacsin2 to the trailing ends of the junctions (upper part of the junctions). Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 1 hour and 4 minutes.Pacsin2 locates at the trailing ends of VE-cadherin at FAJs. Time lapse recording of HUVECs expressing VE-cadherin-GFP (green) and pacsin2-mCherry (red). Pulling forces from the right cell cause movement of the FAJs. Pacsin2 recruitment occurs at trailing ends of the junctions (left side). Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 28 minutes.Pacsin2 locates at the trailing ends of p120-catenin at FAJs. Time lapse recording of HUVECs expressing pacsin2-GFP (green) and p120-catenin-mCherry (red). Pulling forces from the right cell cause movement of the FAJs. Pacsin2 recruitment occurs at trailing ends of the junctions (left side). Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 17 minutes.F-BAR point-mutation in pacsin2 prevents recruitment to moving FAJs. Time lapse recording of HUVECs expressing pacsin2[R50D]-GFP (green) and α-catenin-mCherry (red). The R50D point-mutation in pacsin2 prevents binding to curved membranes. The movie shows enlargements of two regions of interest (moving FAJs) from the overview image. Even 4 though clear remodeling and movement of FAJs occurs, no pacsin2[R50D] is recruited to the junctions. Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 34 minutes.Polarized dynamics of α-catenin within remodeling FAJs. Time lapse recording of HUVECs expressing α-catenin-Dendra2 shown before, and after photoswitching the front of an FAJ using a 405 confocal laser. After photoswitching a part of the FAJ, newly recruited α-catenin-Dendra2 protein (green) incorporate at the front of the FAJ, whereas α-cateninDendra2 photoswitched to red is relocated to the trailing end of the FAJ. Images were acquired by time-lapse confocal microscopy (LEICA TCS SP8) using a 63x 1.4 NA oil objective. Frames were taken every 1 minute for 12 minutes.Mobility of α-catenin differs between the front and the trailing ends of FAJs. Time lapse recording of mCherry signal in HUVECs expressing pacsin2-GFP and α-cateninmCherry. Movie shows α-catenin-mCherry signal before and after photobleaching the front of an pacsin2-positive FAJ (left movie) or the trailing end of a pacsin2-positive FAJ (right movie). In contrast to the front of FAJs, recovery of α-catenin fluorescence at the trailing end of FAJs is strongly reduced. Images were acquired by confocal laser scanning microscopy (Zeiss LSM 510) using a 63x/1.4 NA oil objective. Frames were taken every 1.57 sec for 224 sec.Pacsin2 dynamics during internalization from FAJ trailing ends. Time lapse recording of HUVECs expressing pacsin2-GFP (green) and α-catenin-mCherry (red). Movie shows the promotion of internalization from FAJs (open arrows) once recruited pacsin2 (filled arrows) dissociates from the junction trailing end. Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 32 minutes.VE-cadherin internalized from FAJs and targeted to Rab4 endosome. Time lapse recording of HUVECs expressing Rab4-GFP (green) and VE-cadherin-RFP (red). Movie shows VE-cadherin-RFP internalizing from a FAJ (followed by arrow) and being transported to Rab4-GFP-positive endosomes. Images were acquired by time-lapse widefield microscopy (Zeiss Observer Z1) using a 63x/1.4 NA oil objective. Frames were taken every 30 sec for 32 minutes.


SGO1 transcription and SGO1 expression tended to be higher in MYCN- or MYC-overexpressing cancers. Together, these findings indicate that SGO1 plays a role in the DNA damage response in interphase. Therefore, we propose that SGO1 represents a potential molecular target for treatment of MYCN-amplified neuroblastoma.Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of SGO1 impaired proliferation and induced DNA damage followed by a senescence-like phenotype only in MYCN-overexpressing neuroblastoma cells. In these cells, SGO1 knockdown induced DNA damage, even during interphase, and this effect was independent of cohesin. Furthermore, MYCN-promoted  SGO1 transcription have been reported. SGO1 also plays important roles in various cancers5678Shugoshin 1 (SGO1) is required to ensure the accuracy of chromosome segregation during mitosis and meiosisThe cohesin complex, which contains Structural maintenance of chromosome 1A (SMC1A), SMC3, RAD21, and Stromal antigen 2 (STAG2), forms a ring-like structure that holds sister chromatids togetherMYCN is a MYC family protein and neural tissue-specific transcription factor that contains a β-helix-loop-helix domain17181920MYCN is amplified in ~25% of cases of neuroblastoma, the most common extracranial solid tumor seen during childhood, and MYCN amplification correlates with poor prognosis. Because MYC or MYCN is required for fundamental cellular processes, MYC or MYCN inhibitors may cause undesirable side effects. Identifying the gene(s) which shows synthetic (dosage) lethal interactions2526272829303132333435Although MYCN cannot transform cells on its own22We previously reported that the condensin subunit SMC2 is a target of MYCN, and that SMC2 downregulation causes a synergistic phenotype in conjunction with MYCN amplification or overexpressionSGO1 is a transcriptional target of MYCN, and that SGO1 expression correlates with MYCN or MYC expression in various cancers. These results suggest that SGO1 represents a potential molecular target for therapeutics against MYCN- or MYC-overexpressing cancers.Here, we demonstrate that MYCN overexpression/amplification and SGO1 knockdown synergistically inhibit cell proliferation. The growth defect in SGO1-knockdown/MYCN-overexpressing/amplified cells is the result of persistent DNA damage, which leads to a senescence-like phenotype. In MYCN-overexpressing neuroblastoma cells, SGO1 knockdown induced DNA damage even in interphase, and this phenotype was independent of cohesin. In addition, we found that MYCN-Tg miceSgo1  using the R2 bioinformatics platform (http://r2.amc.nl). Consistent with the expression pattern in MYCN-Tg mice, SGO1 expression was elevated in human MYCN-amplified tumors  than in a neuroblastoma cell line harboring a single copy of MYCN (SK-N-AS)  to identify genes induced during progression of neuroblastoma in miceSgo1 . To conf-Tg mice . As exped tumors . LikewisSK-N-AS) . Based oSK-N-AS) . ExpressSGO1 mRNA levels, we measured changes in SGO1 mRNA levels using SH-EP cells harboring a single copy of MYCN. MYCN overexpression induced SGO1 upregulation at both the mRNA and protein levels  assays. ChIP analysis revealed that MYCN associated most strongly with E-box1 (P = 0.035), and tended to associate with E-box2 (P = 0.056). It also associated with E-box3, but much less strongly . SGO1 knockdown severely inhibited the proliferation of MYCN-overexpressing SH-EP cells, but had a much smaller effect in control SH-EP cells . Next, wWe next performed flow cytometry analysis to further investigate the mechanisms underlying inhibition of cell proliferation in SGO1-knockdown cells. A larger fraction of the cell population accumulated in G2/M phase in SGO1-knockdown cells than in control cells transfected with a non-targeting shRNA . The effMYCN-Tg mice (GSE43419). With the exception of Stag2, expression of all cohesin subunits increased during neuroblastoma progression  (P = 0.36), possibly because the viability of these cells was already quite low.Poly ADP-ribose polymerase (PARP) inhibitors show synergistic responses to cohesin knockdown= 0.007) . We deteMYCN-amplified cell lines IMR32 and NB39 Taken together, these data demonstrate that SGO1 knockdown induced DNA damage even in interphase, and that most of the damage was repaired primarily by NHEJ. In MYCN-overexpressing cells, however, persistent SGO1 knockdown-induced damage caused growth arrest. Although the underlying mechanism remains unknown, one possibility is that SGO1 knockdown induces greater DNA damage, which exceeds the capacity for repair.MYCN-amplified IMR32 cells. However, TUNEL-positive cells were barely detectable  assay to detect apoptosis in tectable . Therefotectable , the protectable . In additectable . These rHere, we showed that simultaneous downregulation of SGO1 and upregulation of MYCN induces higher levels of DNA damage, which in turn halts cell proliferation and causes cells to express a senescence-like phenotype . MYCN inThe phenotype of SGO1 knockdown varied depending on cell type, potentially due to differences in the expression level of MYC or MYCN. In this regard, SGO1 knockdown alone induces cellular senescence in human fibroblasts5SGO1 knockdown induced DNA damage in MYCN-overexpressing neuroblastoma cells , resultiIn SGO1-knockdown MYCN-overexpressing cells, some DNA damage occurred in interphase . SeveralMYC itself is also a target of YAP/TAZMYCN-amplified neuroblastoma and tumors bearing YAP/TAZ hyperactivation.Finally, we showed that, in addition to being a target of YAP/TAZ/TEAD (the Hippo pathway) and AP-1, SGO1 is also a target of MYCN . Like maMYCN Tg micehttp://www.ncbi.nlm.nih.gov/geo) under accession number GSE43419.Previously published datand generation packaging system based on psPAX2 and pMD2.G vectors.Cell lines used in this study are listed in 354038Commercially available non-targeting (Sigma) and SGO1- or cohesin subunit-specific shRNA vectors (Thermo Scientific Open Biosystems) were used. The shRNA sequences are listed in T method.Total RNA was isolated from cells using the RNeasy Plus Mini kit (Qiagen) or the ISOGEN II reagent (Nippongene). cDNA was prepared using ReverTra Ace qPCR RT kit with gDNA Remover (TOYOBO). Quantitative PCR analyses were performed using the KAPA SYBR Fast qPCR kit (KAPA Biosystems) on an Applied Biosystems 7500 Real-Time PCR System. Relative gene expression levels were determined using the ΔΔCWhole-cell lysates were prepared using Cell Lytic M solution  containing a protease inhibitor cocktail  and subjected to SDS-PAGE. Immunoblotting was performed as previously described® 488 conjugate ; anti-mouse IgG, HRP-linked ; and anti-rabbit IgG, HRP-linked  antibodies.The following antibodies were used: anti-SGO1 mouse monoclonal ; anti-SGO1 rabbit polyclonal ; anti-MYCN mouse monoclonals OP13 , NB200-109 , and sc-53993 (Santa Cruz Biotechnology); anti-MYCN rabbit polyclonal ; anti-phospho-histone H2A.X (Ser139) mouse monoclonal ; anti-SMC3 rabbit polyclonal ; anti-β-ACTIN (8H10D10) mouse monoclonal ; normal mouse IgG ; goat anti-mouse IgG (H+L) secondary antibody, Alexa FluorSGO1 gene. Cells immunoprecipitated with control IgGs were used as a negative control.Cultured sub-confluent SH-EP cells expressing Venus or MYCN were treated with 1% (v/v) formaldehyde for 10 min at room temperature. Cross-linking was stopped by addition of glycine to a final concentration of 125 mM. The cells were washed with cold PBS and then harvested. The cells were pelleted, frozen at −70 °C, and then lysed by mechanical disruption. The Halo-ChIP system (Promega) was then used according to the manufacturer’s instructions, with a slight modification: an anti-MYCN antibody and Dynabeads-protein G  were used. DNA was purified twice using phenol-CIAA and then PCR-amplified using primers spanning the MYCN-binding site upstream of the MYCN-overexpressing or control Venus-expressing SH-EP cells were infected with non-target or shSGO1 lentivirus. 4, 5, 6, 7, or 8 days later, the cells were counted using Cell Counting Kit-8, according to the manufacturer’s protocol (Dojindo Laboratories).DNA content was determined on a FACSCalibur instrument (BD Bioscience), and data were analyzed using Cell Quest software.Cells were grown on glass coverslips in 4 well plates and then fixed with 4% paraformaldehyde for 1 hr at room temperature. Fixed cells were permeabilized with PBS containing 1% NP-40 for 10 min at room temperature and incubated with an anti-γ-H2AX antibody (1:1000 dilution) for 1 hr at room temperature, followed by an Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes) (1:1000 dilution) for 30 min at room temperature. After incubation with 4′6-diamidino-2-phenylindole (0.1 μg/ml) for 5 min, cells were mounted in FluorSave Reagent (Millipore). All images were obtained using BZ-9000 (Keyence) or LSM710 (Carl-Zeiss) and subsequently processed using MetaMorph or Zen software and Adobe Photoshop.The NHEJ assay was performed as describedThe HR assay was performed as previously describedSGO1 and induce MYCN overexpression. After 24 hrs, nocodazole (0.9 μg/ml) was added to arrest cells at G2/M. After another 24 hrs, cells were counterstained with DAPI. Cells were then examined under an LSM710 microscope (Carl-Zeiss), and images were analyzed using Image Zen software (Zeiss).Cells were infected with lentivirus to introduce shThe TUNEL assay was performed using the APO-DIRECT kit (BD Pharmingen), and the cells were analyzed on a FACSCanto flow cytometer (BD Pharmingen) using the Diva software.SA-β-GAL assays were performed using the Senescence Detection kit .f test. Statistical significance was evaluated with a 2-tailed, unpaired t test and significance set at P < 0.05.Results are expressed as the mean ± SE. Homoscedasticities were checked using the How to cite this article: Murakami-Tonami, Y. et al. SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells. Sci. Rep.6, 31615; doi: 10.1038/srep31615 (2016).