The inhibitory effects of SP141 on β-catenin were mediated by the ubiquitin–proteasome system in an MDM2-independent manner. In conclusion, these results suggest that SP141 exerts its anticancer activity by dually inhibiting β-catenin and MDM2. We envision that β-carboline derivatives can be developed as promising dual inhibitors of β-catenin and MDM2 for the treatment of advanced pancreatic cancer.The β-catenin and MDM2 oncoproteins are overexpressed and constitutively activated in human pancreatic cancer and contribute to its initiation, progression, and metastasis. The Wnt/β-catenin signaling pathway strongly interacts with the MDM2-p53 signaling pathway, accelerating the tumorigenesis and its development. Therefore, therapies inhibiting both β-catenin and MDM2 are suggested to be ideal treatments for patients with advanced pancreatic cancer. We have recently identified a novel class of β-carboline compounds as the specific and potent MDM2 inhibitors, including a lead compound SP141. In the present study, we utilized SP141 as an exemplary β-carboline compound to characterize β-catenin as a molecular target of the β-carboline compounds and to demonstrate an important role of β-catenin in the anticancer activity of β-carboline. We found that the silencing of either β-catenin or MDM2 largely reduced the anticancer activity of SP141 while the double silencing of both genes almost completely blocked SP141’s activity. SP141 directly bound to β-catenin and inhibited its expression and activity in pancreatic cancer cells  The Wnt/β-catenin signaling pathway plays multiple regulatory roles in development and tissue homeostasis . DeregulThe Wnt/β-catenin pathway strongly interacts with the murine double minute 2 (MDM2)-p53 pathway during the initiation and progression of human cancer. It has been discovered that β-catenin overexpression stabilizes the p53 protein and increases its transcriptional activity by protecting it from MDM2-mediated proteasomal degradation . Howeverin vitro and in vivo  were synthesized and purified as described previously ,c. Antib3 cells/well) and transfected with β-catenin siRNA, MDM2 siRNA, or both as indicated, followed by exposure to SP141 at various concentrations for 72 h. The treated cells were then incubated with MTT solution for an additional 3 h. Finally, the formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm.Human pancreatic cancer cell lines Panc-1 and AsPC-1 were purchased from American Type Culture Collection and cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The MTT assay was used to evaluate the effects of SP141 on pancreatic cancer cell viability as described , 2016b. The molecular modeling of SP141-β-catenin binding was performed with the SYBYL-X 2.0 program package (Tripos), and the results were analyzed using Pymol 1.7 software. The crystal structure of β-catenin (PDB entry: 2Z6H) was used to generate the SP141-β-catenin binding complex. The SYBYL/Sketch module was applied to construct the structure of SP141, which was further optimized using Powell’s method and then assigned to SYBYL-X 2.0 software using the Gasteiger–Hückel method .The streptavidin–agarose pull-down assay was performed as described previously . In brieCells and tumor tissues were lysed in NP-40 lysis buffer containing protease inhibitors (Sigma). The lysates were evaluated for their protein concentrations using the Bradford reagent (Bio-Rad) and subjected to Western blotting as described previously . Immunopc-Myc forward, 5′-GCTGCTTAGACGCTGGATT-3′; c-Myc reverse, 5′-TGCTGCTGCTGGTAGAAGTT-3′; CCND1 (cyclin D1) forward, 5′-GCATCTACACCGACAACTCC-3′; CCND1 (cyclin D1) reverse, 5′-CGGATGATCTGTTTGTTCTCC-3′; GAPDH forward, 5′-GGAGTCCACTGGCGTCTTCAC-3′; GAPDH reverse, 5′-GAGGCATTGCTGATGATCTTGAGG-3′.The total RNA was extracted from the control and SP141-treated cells using the Trizol reagent (Invitrogen) and the cDNA was synthesized by SuperScript reverse transcription-PCR (Invitrogen) according to the manufacturer’s protocols. The primer sequences were as follows: The Panc-1 and AsPC-1 human pancreatic cancer orthotopic models were developed by following the animal study protocols that have been approved by the Institutional Animal Use and Care Committee of the Texas Tech University Health Sciences Center. The orthotopic tumor-bearing mice were treated with SP141  or vehicle controls and monitored for tumor growth, changes in body weight, and other physical appearance as described previously .4 cells/well) were treated with SP141 for 12 h, followed by immunofluorescence detection as described previously  cell lines were used in these studies because both cell lines harbor high expression levels of MDM2 and β-catenin, without wild-type p53. As examined by Western blotting and quantified using IMAGEJ software, the transfected cells exhibited a β-catenin and MDM2 knockdown (KD) efficiency of >60% . MDM2 KD did not affect the endogenous β-catenin expression and did not alter the inhibitory effects of SP141 on β-catenin protein expression in both Panc-1 and AsPC-1 cells . These results indicated that MDM2 was not involved in the inhibition of β-catenin by SP141. Similar results were obtained in β-catenin KD cells .To assess a role of β-catenin in SP141’s anticancer activity, we performed transient transfection experiments using β-catenin siRNA, MDM2 siRNA, or both. The pancreatic cancer Panc-1 (p53-mutant) and AsPC-1 . Consistent with our previous study . The double KD of β-catenin and MDM2 almost completely blocked the inhibitory effects of SP141 on pancreatic cancer cell viability .We further examined the effects of the single KD of either β-catenin or MDM2 on SP141’s anticancer activity. As shown in us study , MDM2 KDin vitro. As shown in Figure 2A, SP141 decreased the protein levels of β-catenin and its downstream target genes, c-Myc and cyclin D1 in both Panc-1 and AsPC-1 cells in a concentration-dependent manner. However, the compound did not change the expression levels of phosphorylated β-catenin proteins at Ser45 and at Ser33/37/Thr41. Similar observations were obtained with the decreased mRNA expression of both c-Myc and CCND1 (cyclin D1) in Panc1 and AsPC1 cells in response to SP141 treatment . It was further observed that SP141 largely reduced the β-catenin expression levels in nucleus . An immunofluorescence assay was performed to detect the expression and distribution of β-catenin in SP141-treated cells. The results  were similar to those observed by Western blot analysis. Of note, SP141 treatment did not affect the membrane-bound β-catenin protein.We further examined the effects of SP141 on the expression, subcellular distribution, and activity of β-catenin in pancreatic cancer cells Figure 3A) and Western blotting . Also, SP141 treatment did not affect the expression of phosphorylated β-catenin proteins at Ser45 and Ser33/37/Thr41 .We also examined the effects of SP141 on the β-catenin expression in the tumors from pancreatic cancer orthotopic models that were used in the previous study . In compFigure 4A, both the β-catenin and MDM2 proteins in the Panc-1 and AsPC-1 cell lysates were specifically precipitated by biotin-SP141, but not the biotin tag. We further observed that a preincubation of non-biotinylated SP141 significantly reduced the formation of SP141-β-catenin and SP141-MDM2 complexes in the cell lysates . These results indicated that SP141 could directly bind to β-catenin protein, with an affinity similar to the SP141-MDM2 binding. Similar results were obtained with recombinant His-β-catenin protein. As shown in Figure 4B, an increase in the formation of SP141-β-catenin complex was observed with the increase of the preincubated His-β-catenin protein. The SP141-β-catenin complex was further reduced by a preincubation of the non-biotinylated SP141, suggesting a specificity of the SP141-β-catenin binding.To further understand the molecular mechanisms of SP141’s inhibitory effects on β-catenin, we performed a pull-down assay using biotinylated SP141 (biotin-SP141)-bound beads and pancreatic cancer cell lysates. As shown in Figures 4C,D, SP141 could directly and specifically bind to the Armadillo repeat domain of β-catenin. The SP141-β-catenin binding mode indicated that the naphthyl group and methoxyl group of SP141 displayed hydrophobic interaction with LYS508 and HIS470 in the β-catenin, respectively . The nitrogen at the 9-position of SP141 might directly interact with ARG469 via a hydrogen bond . As shown in Figure 4E, SP141 directly bound to the full length β-catenin and the β-catenin construct harboring Armadillo repeat domain and C-terminal domain. However, the compound did not show any binding to the N-terminal and C-terminal domains of β-catenin. These results further validated a specific binding of SP141 to the Armadillo repeat domain.To further examine the binding mechanism of SP141 with β-catenin, we performed a molecular docking study. As shown in Figure 5A, SP141 treatment shortened the half-life of β-catenin and accelerated its protein degradation in Panc-1 and AsPC-1 cells. SP141-induced destabilization of β-catenin was significantly inhibited by a proteasome inhibitor MG-132, suggesting that SP141 could enhance β-catenin degradation via the ubiquitin–proteasome pathway . To test this hypothesis, we transfected the Panc-1 and AsPC-1 cells with an ubiquitin plasmid and treated the transfected cells with SP141. As shown in Figure 5C, a Western blot analysis of immunoprecipitated β-catenin exhibited a concentration-dependent enhancement of β-catenin ubiquitination by SP141 treatment. We further assessed a role of MDM2 in SP141’s inhibitory effects on β-catenin. As shown in Figure 5D, no significant change of SP141-induced β-catenin degradation was observed in both Panc-1 and AsPC-1 cells with or without MDM2 silencing. We, therefore, concluded that SP141 destabilized β-catenin via a proteasome-dependent and MDM2-independent pathway.We further examined whether the binding of SP141 to β-catenin could induce its destabilization. As shown in APC, TP53, etc.)    and the 2, etc.) . It has 2, etc.) . Therefoin vitro and in vivo. The binding of SP141 to β-catenin could cause its ubiquitination and proteasomal degradation. The SP141-induced β-catenin degradation occurred without affecting the expression level of phosphorylated β-catenin, which was distinct from previously reported small molecule inhibitors (null pancreatic cancer cells.The main aim of the present study was to investigate a role of β-catenin in the anticancer activity of a β-carboline-type MDM2 inhibitor SP141 as well as the molecular mechanisms that are responsible for the inhibition of β-catenin by SP141. In this study, we demonstrated that the inhibitory effects of SP141 on β-catenin played a crucial role in its anticancer activity, as evidenced by the β-catenin KD pancreatic cancer cell lines. Importantly, the double KD of β-catenin and MDM2 almost entirely blocked SP141’s anti-pancreatic cancer activity, indicating that the dual inhibition of β-catenin and MDM2 by this compound might exhibit a synergistic activity against tumor growth. It was further discovered that SP141 directly bound to the Armadillo repeat domain of β-catenin and inhibited its expression and activity in pancreatic cancer cells hibitors . FurtherTo date, there is no clinically approved small molecule β-catenin inhibitor. The majority of previously reported β-catenin inhibitors have been developed to target the binding of β-catenin to TCF, CBP or BCL-9, inhibiting the transcription of its downstream target genes . ICG-001Our data indicated that the β-carboline derivative SP141 specifically bound to β-catenin and induced its degradation without affecting the phosphorylated β-catenin expression. These results suggested that SP141 could function as an allosteric β-catenin inhibitor that induces its conformational changes and modulates the bindings of β-catenin with its regulators other than the destruction complex . It has In summary, this study demonstrates β-catenin as a molecular target of the carboline compound SP141. These results provide new insights into the strategic development of β-carboline derivatives as novel inhibitors of β-catenin. It is envisioned that SP141 can be a useful tool to explore the β-catenin and MDM2 dual-targeting approach for cancer therapy.WW and RZ organized, conceived, designed, and supervised the study. J-JQ and XL designed and conducted the experiments, and drafted the manuscript. HD and JKB synthesized test compounds and helped study design and interpretation of data. All authors read and approved the manuscript.JKB, RZ, and WW hold a US patent for the use of β-carboline derivative in treating cancer. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Furthermore, we demonstrate that Ac2-26 induces IKKβ accumulation in lysosomes and that lysosomal-associated membrane protein 2A (LAMP-2A), not LC-3, is enhanced in microglia exposed to Ac2-26. We hypothesize that Ac2-26 mediates IKKβ degradation in lysosomes through chaperone-mediated autophagy (CMA). Interestingly, ANXA1 in the cytoplasm does not interact with IKKβ but with HSPB1, and Ac2-26 promotes HSPB1 binding to IKKβ. Furthermore, both ANXA1 and HSPB1 can interact with Hsc70 and LAMP-2A, but IKKβ only associates with LAMP-2A. Downregulation of HSPB1 or LAMP-2A reverses the degradation of IKKβ induced by Ac2-26. Taken together, these findings define an essential role of exogenous Ac2-26 in microglia and demonstrate that Ac2-26 is associated with HSPB1 and promotes HSPB1 binding to IKKβ, which is degraded by CMA, thereby reducing TNF-α expression.Annexin A1 (ANXA1) is an endogenous protein with potent anti-inflammatory properties in the brain. Although ANXA1 has been predominantly studied for its binding to formyl peptide receptors (FPRs) on plasma membranes, little is known regarding whether this protein has an anti-inflammatory effect in the cytosol. Here, we investigated the mechanism by which the ANXA1 peptide Ac2-26 decreases high TNF-α production and IKKβ activity, which was caused by oxygen glucose deprivation/reperfusion (OGD/R)-induced neuronal conditioned medium (NCM) in microglia. We found that exogenous Ac2-26 crosses into the cytoplasm of microglia and inhibits both gene expression and protein secretion of TNF-α. Ac2-26 also causes a decrease in IKKβ protein but not IKKβ mRNA, and this effect is inverted by lysosome inhibitor NH Worldwide, stroke is a leading cause of death and disability with limited therapeutic options , an endogenous glucocorticoid mediator implicated in mediation of a broad range of anti-inflammatory actions -induced neuronal conditioned medium (NCM). We demonstrate that Ac2-26 provokes the association of IKKβ with the small molecular chaperone HSPB1, which leads to degradation of IKKβ by CMA. Inhibition of HSPB1 or LAMP-2A leads to excessive IKKβ activation and TNF-α secretion. Importantly, our data show that ANXA1, HSPB1 and IKKβ all bind to LAMP-2A and are degraded through CMA. Thus, for the first time, this study highlights the importance of the CMA pathway in microglia and provides a new mechanism by which exogenous Ac2-26 may be exploited as an anti-inflammatory therapy by inducing HSPB1 and promoting the degradation of IKKβ in microglia following stroke.Sprague-Dawley rats  were obtained from the Experiment Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Rats were housed in temperature-controlled rooms and maintained on a 12-h/12-h light/dark cycle. All animal experiments were approved by Tongji Medical College Experimental Animal Ethics Committee . All procedures complied with the guidelines of the National Institutes of Health for the Care and Use of Laboratory animals .d-lysine (Sigma)-coated plates in DMEM containing 10% FBS for attachment overnight.Primary neuronal cultures were obtained from embryos at embryonic d16–18 by a standard procedure as previously described  incubator at 37°C for 2 h. Control cells were incubated in high-glucose DMEM under normoxic conditions (95%O2/5%CO2) for the same time period. The cells were returned to normoxic conditions with regular medium to terminate OGD and begin reperfusion. NCM was collected 24 h later.Oxygen glucose deprivation (OGD) was induced in primary neuronal or microglial cultures. Briefly, culture medium was replaced by glucose-free DMEM (ThermoFisher Scientific), and cells were placed in an oxygen-deprived (94%N4CL and chloroquine were acquired from Sigma-Aldrich. Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK) and FITC-Ac2-26 were synthesized by Bioyeargene Biotechnology .The primary antibodies used were as follows: rabbit monoclonal antibody (mAb) against IKKβ (#8943) and polyclonal antibodies against LC3(#12741), IKKβ (#2678), ANXA1 (#3299) and p62 (#5114) were obtained from Cell Signaling Technology. Goat polyclonal antibodies against HSPB1 (sc-1048) and TNFα (sc-1350); mouse polyclonal antibodies against Hsc70 (sc-7298), β-actin (sc-47778) and GADPH (sc-365042); and rabbit or mouse immunoglobulin G(IgG) were from Santa Cruz Biotechnology. Rabbit mAb against LAMP-2A (ab125068) and mouse mAb LAMP-1 (ab25630) were from Abcam. Rabbit polyclonal antibodies against phospho-IκBα were purchased from Ruiying Biotechnology . Alexa Fluor probe-labeled secondary antibodies were from Jackson ImmunoResearch Labs. The nFPR antagonist, N-tert-butoxy-carbonyl-methionyl-leucyl-phenylalanine (BOC-1), was obtained from MP Biomedicals. NHLAMP-2A shRNA (shLAMP-2A)-expressing adenovirus -expressing adenovirus (target sequence: GGAGATCACCATTCCGGTTAC) and scrambled sequence adenovirus were supplied commercially by Vigene Biosciences . mRNA was knocked down by transfection of microglia cells with the indicated adenovirus. Infected cells were sorted by GFP expression. LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. , as recommended by the manufacturer. At 48 h after transfection with various plasmids, the cells were subjected to Western blotting analysis. For Ac2-26 treatment, the cells were treated with 10 μM Ac2-26 for 24 h before harvesting  for 2 h  FBS (ThermoFisher Scientific) and incubated at 37°C under humidified atmosphere of 5% (v/v) COg for 10 min at 4°C. The obtained supernatant (4 ml) was then centrifuged at 20,000× g for 20 min at 4°C. The supernatant was diluted in a solution containing 19% OptiPrep density gradient medium. The mixed medium was separated by density gradient centrifugation  with Beckman Coulter Optimal L-80 XP Ultracentrifue and SW 55Ti Swing out Rotor in a multistep OptiPrep gradient, and calcium chloride was added to a final concentration of 8 mM for low-speed (5000× g for 15 min) centrifugation. The top 3 ml was collected as the lysosomal fraction and stored at 4°C until use for Western blotting. Lysosomal integrity was assessed using Neutral Red dye (Sigma).Lysosomes were isolated from brain tissue using a Lysosome Isolation kit (Sigma) according to the kit protocol. Briefly, 4 g rat brain tissue was homogenized in 16 ml ice-cold extraction buffer containing 1× protein inhibitor cocktail at 8000 rpm for 5 s, followed by 9500 rpm for two additional 5-s periods. The homogenate was centrifuged at 1000× TM Software, Version 5.1 (Bio-Rad). All Western blot data are expressed as the ratio of the levels of the protein of interest and β-actin.Cells were lysed in RIPA buffer  supplemented with a protease inhibitor mixture. Protein samples (60 μg) were heated at 98°C for 10 min and separated on 10% or 12% SDS-PAGE gels. The proteins were then transferred to a polyvinylidene fluoride membrane . The membranes were incubated with 5% fat-free milk in TBST for 1 h and probed with the antibody (Ab) of interest overnight at 4°C, followed by HRP-conjugated secondary Ab and developed by chemiluminescence detection  with autoradiography (Bio-Rad). Band densitometry was analyzed by Image Labg) at 4°C for 15 min. Proteins were immunoprecipitated with indicated antibodies and shaken at 4°C overnight. Then, 40 μL precleared Protein A/G Plus-agarose beads  was incubated with immunocomplexes for 3 h at 4°C and centrifuged at 1000× g for 5 min. The supernatant was discarded, and the pellet was washed 5 times with PBS. Next, the precipitates were resuspended with 1× sample buffer and heated at 98°C for 5 min. The samples were centrifuged for 5 s, and the supernatants were collected for Western blotting. Immunoblotting analysis was performed as previously described.Cell lysates were centrifuged  according to the manufacturer’s protocols. Real-time PCR was performed using SYBR Green I PCR Master Mix in a StepOnePlus Real-Time PCR System (Applied Biosystems). The data were analyzed using StepOne System software with a cycle threshold (Ct) in the linear range of amplification and then processed by the 2The following primers were used:TNF-α: forward, 5′- CGTGTTCATCCGTTCTCTACC-3′;TNF-α: reverse, 5′- CGTGTGTTTCTGAGCATCGT-3′;IKKβ: forward, 5′- TTGTGGGGACCCTGCAATAC-3′;IKKβ: reverse, 5′- GCCGAAGCTCCAGTAGTCAA-3′;β-actin: forward, 5′- CACCCGCGAGTACAACCTTC-3′;β-actin: reverse, 5′- CCCATACCCACCATCACACC-3′.All data were reinterpreted as fold changes relative to untreated control samples.4 cells/well, and TNF-α levels were determined by a rat TNF-α ELISA kit (BioLegend) according to the kit protocol.Microglia were seeded in PDL-coated 24-well plates at 3 × 10Primary microglia were collected and washed with PBS 3 × 5 min, followed by fixation with 4% paraformaldehyde (PFA) at room temperature for 15 min and permeabilization with 0.1% Triton X-100 for 10 min. Cells were then blocked in 10% goat serum (ThermoFisher Scientific) in PBS for 1 h at 22°C, followed by staining with a mAb for LAMP-2A  and LAMP-1  at 4°C overnight. Cultures were subsequently washed three times in PBS and counterstained with Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG for 1 h at room temperature. The cells were subsequently washed three times in PBS and stained with DAPI (Abcam) for 5 min at room temperature. Finally, the cultures were analyzed with a confocal microscope . Images were digitally analyzed by Image-Pro Plus software to quantify the fluorescence intensity of cells.t-test. P < 0.05 was considered statistically significant. The statistical software used was Prism v.6 (GraphPad Software).All experiments were performed a minimum of three times (independently) and are expressed as the mean values ± SEM. Statistically significant differences between groups were identified by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests or unpaired Student’s 4CL but not the FPR receptor antagonist BOC-1  are a family of molecular chaperones that may exhibit immunomodulatory and anti-inflammatory functions (Shao et al., Regarding IKKβ degradation, we explored whether the high level of HSPB1 triggered by Ac2-26 may assist IKKβ as a molecular chaperone for autophagy. The above data showed that both IKKβ reduction and HSPB1 elevation were induced by Ac2-26 in microglia; these results suggested a possible interaction between HSPB1 and IKKβ. First, we examined the association in NCM-treated microglia with or without Ac2-26. Immunoprecipitation experiments demonstrated that HSPB1 was slightly associated with IKKβ under resting physiological conditions in microglia Figure , which iAn important question regarding autophagy related to IKKβ reduction is whether an autophagic pathway is responsible for Ac2-26-mediated inflammation. We monitored the shift of LC3-I to LC3-II, a canonical autophagosome marker. Primary microglia were cocultured with Ac2-26 for 24 h, and the results showed no changes in the conversion to LC3-II and accumulation of p62 Figure , which iProteins undergoing CMA-mediated lysosomal degradation often contain a loosely defined KFERQ motif recognized by Hsc70 and bind to the cytosolic tail of LAMP-2A (Kaushik et al., To further evaluate the degradation of these proteins by potential uptake in lysosomes, we assessed the accumulation of IKKβ, ANXA1 and HSPB1 in lysosomes from rat brains by immunoblotting. We pretreated brain tissue with chloroquine, a lysosomal inhibitor, prior to treatment with Ac2-26 as any lysosome-internalized substrate is rapidly degraded by the luminal protease. The integrity of the lysosomes was assessed by Neutral Red dye Figure . As showMicroglia are the major resident immune cells involved in the defense against brain damage. When microglia are excessively activated by damage-associated molecular patterns following stroke, they can produce many proinflammatory cytokines that can disrupt neural cells and influence neurogenesis. We have reported the importance of ANXA1 in driving microglial activation and migration to protect neurons from ischemia-like injury (Luo et al., Endogenous ANXA1 is associated with the treatment of unresolved inflammation (Vago et al., We observed that exogenous Ac2-26 was mainly located in the cytosol of microglia. The primary mechanism by which ANXA1 is known to mediate its effects is through FPRs. Exogenous ANXA1 functions through FPRs to initiate endogenous pro-resolving and anti-inflammatory pathways following ischemic stroke (Vital et al., The small molecular weight heat shock protein, HSPB1, is upregulated in both neurons and astrocytes of different animal species under stress and neurodegenerative conditions (Filipcik et al., CMA, one of the lysosomal proteolysis pathways, is characterized by its specificity for the selective degradation of substrate proteins. Basal levels of CMA activity are detectable in almost all mammalian cells, and contribute to the maintenance of cellular homeostasis as well as specialized functions depending on the cell type and degraded substrate. In contrast to the idea that CMA is upregulated under various conditions ranging from prolonged starvation to different cellular stresses (Cuervo et al., In conclusion, our data described a novel mechanism by which exogenous ANXA1 regulates TNF-α expression and provided the first evidence that CMA plays a fundamental role in driving Ac2-26-induced rapid loss of IKKβ protein in microglia. Mechanistically, ANXA1 attenuates IKKβ activity by inducing HSPB1, a small chaperone, to bind to both ANXA1 and IKKβ for degradation in lysosomes. This study presented the first observation that HSPB1 is involved in Ac2-26-induced CMA in microglia. Hence, these results highlighted the important role of CMA in controlling inflammation in microglia and provided a promising therapeutic target pathway applicable to exogenous Ac2-26 in stroke.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.LL designed the experimental project, carried out most of the experiments and prepared the manuscript. DA and JX participated in the primary microglia culture and performed the HSPB1 analysis in tissues and cell. BS and XL contributed to the interpretation of the results. JS provided oversight for the study including experimental design, data interpretation and manuscript preparation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LP declared a shared affiliation, though no other collaboration with the authors to the handling Editor.


Malus pumila Miller cv. ‘Annurca’ and Malus domestica cv ‘Limoncella’; (ii) identify the mechanisms underpinning their activities and; (iii) evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract.Inhibitors of the Wingless-related Integration site (WNT)/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP). This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i) test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples,  Familial adenomatous polyposis (FAP) is an inherited gastrointestinal syndrome, characterized by the formation of a large number of adenomas throughout the small intestine and the large bowel. FAP has a birth incidence of about 1/8300, manifests equally in both sexes, and accounts for less than 1% of colorectal cancer (CRC) cases . While cAdenomatous Polyposis Coli (APC) gene on chromosome 5q21–q22. This locus contains a tumor suppressor gene encoding for the protein APC, that functions intracellularly as a scaffold in a large protein complex, known as “β-catenin destruction complex” -1H-indol-3-yl]-1H-pyrrole-2,5-dione, #137592-45-1, Tocris, Bristol, UK) was dissolved in DMSO.Chemicals and reagents used were either analytical-reagent or HPLC grade. The water was treated in a Milli-Q water purification system  before use. The standards used for the identification and quantification of phenolic acids and flavonoids were chlorogenic acid, (+)-catechin, (−)-epicatechin, phloretin, phloridzin, procyanidin B2, quercetin, rutin  apple fruits and Limoncella  were collected from Valle di Maddaloni , in October 2016, when fruits had just been harvested. Annurca fruits were reddened for about 30 days  (interspaced by the triplet 5′-GTA-3′) were positioned upstream to a minimal TATA box promoter [5′-tagagggtatataatggaagctcgaattccag-3′] and a KOZAC region [5′-cttggcattccggtactgttggtaaaaagcttggcattccggtactgttggtaaagccacc-3′]. The sequence was cloned in the vector pCDNA 3.1 (+) GFP between the restriction sites for NruI and HindIII. This replacement substitutes the cytomegalovirus (cmv) promoter of the original vector with the TCF/LEF responsive sequences. The correctness of the sequences were verified by DNA sequencing. The reporter construct (TCF-mut GFP) was used as a negative control. This was obtained by the mutagenesis of TCF-wt GFP and presented the 8 repeats of the TCF/LEF binding sequence mutated to [5′-AGGCCAAAGGGG-3′]. The reporter construct, cmv GFP, used in this manuscript as the transfection control, was obtained by replacing the TCF/LEF consensus region in TCF-wt GFP with a cmv promoter.3 per well) in 96-well black Optyplates . After 24 h, cells were co-transfected with both WNT reporter GFP construct and HA-FZD4-wt. Transfection mixtures were prepared as follows: for each well, 0.25 µg of PEI (pH 7.0) was supplemented with 0.08 µg of DNA (both diluted in 4 μL of DMEM). The mixture was incubated at room temperature (RT) for 30 min, to be then diluted in culture medium and added to the cells. Twenty-four hours after transfection, the medium was replaced and cells were incubated with WNT5A conditioned medium. Human glyoblastoma U87MG cells were used as a source of WNT5A conditioned medium [HEK293 cells were seeded  for 30 min. Formaldehyde was quenched by incubating the cells for 30 min in 0.1 M glycine in PBS 1x. Then, cells were permeabilized in 0.1% Triton/PBS, pH 7.4, for 10 min at 25 °C and then incubated with a rabbit polyclonal anti-β-catenin antibody  diluted 1:200 in PBS and a goat anti-rabbit IgG (H&L), DyLight 594 conjugate,  (diluted 1:500 in PBS), for 1 h and 45 min, respectively.t-test or two-way ANOVA, followed by the Tukey–Kramer multiple comparison test, to evaluate significant differences between a pair of means. For dose-response data, half maximal effective concentrations (EC50) were calculated using nonlinear regression analysis with Prism software 6.0 . EC50 are indicated as mean ± standard error mean (SEM). All data were analyzed using the two-tailed Student’s t-test and p < 0.05 indicated a statistically significant result.Unless otherwise stated, all of the experimental data are shown as mean ± standard deviation (SD) of at least three replications . StatistThe total (flesh and peel) polyphenolic content in AAE and LAE prepared for this study was determined by HPLC-DAD analysis and is reported in They contained a similar amount of chlorogenic acid, the most abundant hydroxycinnamic acid in apples, as well as of the flavan-3-ols, (+) catechin and (–) epicatechin. The quercetin-glycoside, rutin and the phloretin–glycoside, phloridzin, were more abundant in AAE than in LAE. The latter, on the contrary, contained higher amounts of procyanidin B2 and isoquercetin ,26. The The activity of the apple extracts on WNT/β-catenin signaling was assayed in a reconstituted recombinant system. We used, as a biological platform, human embryonic HEK293 cells, transiently expressing both the WNT receptor Frizzled 4 (FZD4)  and a WNThe FZD agonist, WNT5A, induced GFP expression in HEK293 cells, transiently transfected with TCF-wt GFP . On the 2+ pathway [WNT5A has been shown to activate both the WNT/β-catenin pathway as well as one of the “non-canonical” branches of WNT signaling, the WNT/Ca2+ pathway) did not affect GFP expression induced by WNT5A (Inhibitors of Nuclear Factor of Activated T-cells (NFAT) and Protein Kinase C (PKC)  and PEF(LAE))  were ass50 of 1.8 ± 0.1 μM and 3.1 ± 0.4 μM, respectively. Phloridzin and procyanidin B2 showed EC50 of 12.9 ± 0.5 μM and 1.4 ± 0.3 μM, respectively. Considering their low abundance in both the apple extracts, none of the abovementioned molecules would reach effective concentrations in AAE and LAE solutions diluted at their EC50 for WNT inhibition  ,35. OnceWe thus challenged AAE and LAE to compete with LiCl and EGF, two inducers of the WNT pathway, both acting downstream to APC. LiCl binds directly to GSK-3β and inhibits the β-catenin destruction complex. In contrast, EGF activates the EGFR pathway, that, via AKT, promotes β-catenin detachment from the Plasma Membrane and its nuclear translocation. In our biological system, both LiCl and EGF induced, in a dose-response manner, GFP expression in HEK293 transfected with TCF-wt GFP A–C. On tAAE and LAE failed to inhibit activation of the WNT pathway induced by 15 and 30 mM LiCl. However, they both reduced WNT pathway activity induced by 5 and 10 mM LiCl  . IndLAE presents a WNT inhibitory activity increase compared to LAE (EC.9 mg/L) A. A HPLC.9 mg/L) . HoweverTo achieve WNT inhibitory activity in the colon, apple polyphenolic must be bio-accessible, i.e., they should be extracted from the food matrix, resist to GI-digestion and reach the colon-rectal section of the intestine. However, GI digestion may affect substantially native apple polyphenolic patterns and concentrations as well as induce drastic structural changes of the food constituents . During Polyphenol consumption has been related to several health beneficial effects, such as reduced overall mortality , incidenWe here showed that apple extracts from both Annurca (AAE) and Limoncella (LAE) are endowed with inhibitory activity toward WNT/β-catenin signaling, an intracellular pathway involved in many forms of cancer. Moreover, we showed that both the extracts are potentially suitable for the treatment of FAP, a WNT related disease. In this syndrome, a mutation in the protein APC, leads to hyperactivation of the WNT/β-catenin signaling pathway and, in turn, to uncontrolled intestinal cell proliferation and a high risk for the formation of adenocarcinomas. We showed that both AAE and LAE are able to inhibit WNT signaling in cells carrying APC mutations. In both in vitro cultures of cells as well as in ex vivo biopsies of FAP patients, AAE and LAE blocked the proliferation and duplication rates of colon-rectal cancer cells.50 for WNT pathway inhibition in the micromolar range [AAE and LAE WNT inhibitory activity can be partially ascribed to their polyphenolic content. However, none of the major constituents of their polyphenolic fractions, tested as pure molecules, exerted strong WNT inhibitory activities, suggesting the importance of the whole pool of polyphenols for the activity of the extracts. WNT pathway inhibitory activity has been described for many individual polyphenols. Epigallocatechin gallate (EGCG) , isoquerar range ,52,53. Aar range , upon coar range ,56,57, nar range ,58,59,60Our preliminary results suggest that the WNT inhibitory activity of AAE and LAE can be ascribed either to the antioxidant activity of the entire polyphenolic mixture or to the effects that the latter may exert on its own components, protecting them from detrimental chemical modifications occurring in physiological environments.We here showed that food-grade large-scale manufacturing alters the flavonoid composition of LAE, drastically increasing the WNT inhibitory activity of the extract. This could be ascribed to the presence of quercetin, a potent β-catenin inhibitor, absent in the original extract, which probably originated from the conversion of quercetin-glycosides into their aglycone form. This result should encourage researchers to test the activity of the extract after industrial manufacturing. The latter drastically alters the composition of the extract, even if this event, as we have here shown, does not always have to be detrimental to the properties of the extract.We finally showed that both LAE and AAE lose activity upon in vitro simulated GI digestion as a result of a severe loss of polyphenols. This strongly points towards encapsulation in gastro-resistant tablets (for example enteric coatings) as an essential requirement to preserve apple extract WNT inhibitory activity in the colon-rectal section of the intestinal tract.


The urchin-like γ-MnS microstructures exhibit excellent cycling stability . And the discharge voltage is about 0.75 V, making it a good candidate for the application as the anode material in LIBs. SEM, TEM, and XRD were employed to inspect the changes of the active materials during the electrochemical process, which clearly indicate that the structural pulverization and reformation of the γ-MnS microstructures play important roles for the maintenance of the electrochemical performance during the charge/discharge process.MnS has been attracting more and more attentions in the fields of lithium ion batteries (LIBs) because of its high energy density and low voltage potential. In this paper, we present a simple method for the preparation of urchin-like  Thougs fields . Thus, m−1) and low voltage potential (average discharge voltage at ~0.65 V and charge voltage at ~1.25 V) [−1 at a current density of C/5 after 100 cycles [−1, the as-synthesized coral-like α-MnS can still deliver a reversible capacity of 699 mA h g−1 after 400 cycles [−1 at a current density of 500 mA g−1 after 100 cycles [Recently, transitional metal sulfides such as cobalt sulfides –8, nicke~1.25 V) , 20, 21.0 cycles . Zhang’s0 cycles . Kang’s 0 cycles . Despiteγ-MnS via a simple solvothermal method using l-cysteine and MnCl2 · 4H2O as the raw materials. Thanks to the unique 3D structure, the as-synthesized urchin-like γ-MnS exhibits excellent cycling stability. The discharge capacity can still reach 823.4 mA h g−1 after discharging for 1000 cycles at the current density of 500 mA g−1. Besides the cycling stability, the as-prepared urchin-like γ-MnS structures also exhibit satisfying rate performance. SEM, TEM, and XRD were employed to inspect the changes of the active materials during the electrochemical process, which clearly indicate that the structural pulverization and reformation of the γ-MnS structures play importance roles for the maintenance of the electrochemical performance during the charge/discharge process.It is well known that the electrochemical performance of the electrode material is highly dependent on its morphology and crystalline texture , 6. Thusγ-MnS nanostructures were synthesized via a facile solvothermal method. In a typical experiment, 1 mmol MnCl2 · 4H2O was firstly dissolved in the mixed solvent composing of 10 mL double-distilled water and 20 mL diethylene glycol (DEG). The mixed solution was then heated to 70 °C under constant stirring. In the next step, 2 mmol l-cysteine was added to the above solution. After heating at 70 °C for 2 h, the white turbid liquid was transferred into a 50-mL Teflon-lined stainless steel autoclave and heated to 180 °C for 18 h. After cooling down to room temperature, the ochre product was collected by centrifugation and washed three times with water and ethanol. The as-obtained product was finally dried in vacuum at 60 °C for 12 h.The urchin-like Phase purities of the as-prepared samples were characterized using X-ray powder diffraction . The sizes and morphologies of the samples were investigated using the field emission scanning electron microscopy  and transmission electron microscope . The high-resolution transmission electron microscopy (HRTEM) images were taken using a transmission electron microscopy  with an accelerating voltage of 200 kV. The surface information as well as chemical composition of the samples were examined using the X-ray photoelectron spectrum . The specific surface area and pore size distribution were determined by the Brunauer-Emmett-Teller (BET) nitrogen adsorption and desorption apparatus (Quantachrome autosorb IQ-C).γ-MnS, carbon black (Super-P) and carboxyl methyl cellulose binders (CMC) with a weight ratio of 5:3:2. The slurry was coated on copper foil and then dried in vacuum at 100 °C for 12 h. And the active material on each copper foil was weighted to be ~1.46 mg cm−2. The CR2032 coin cells were assembled in an argon-filled glove box ) with moisture and oxygen concentrations below 0.1 ppm. The electrolyte was a solution of 1 M LiPF6 in a mixture of ethylene carbonate (EC), ethyl methyl carbonate (EMC), and diethyl carbonate (DEC) at volume ratio of 4:2:4. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were measured by electrochemical workstation (CHI760E). The galvanostatic charged and discharged characteristics were tested in a voltage range from 0.01 to 3.0 V with LAND CT2001A battery tester.The working electrodes were prepared by mixing γ-MnS. The lattice constants of the as-synthesized sample are calculated to be a = b = 3.9792 Å, c = 6.4469 Å, α = β = 90.0°, and γ = 120.0°, which is consistent with the literature value (JCPDS card no. 40-1289). No other peak is detected, indicating high purity of the as-prepared sample.The phase purity of the as-prepared product was investigated using the power X-ray diffraction (XRD) and energy-dispersive spectrometer (EDS). Figure γ-MnS are shown as Fig. γ-MnS clearly indicates the existence of elements Mn and S , and the corresponding result is shown in Fig. d S Fig. . The Mn d S Fig.  consistsectively . Similarely [3/2 . The XPSγ-MnS nanostructures were further investigated using FESEM, TEM, and HRTEM. Figure γ-MnS microstructures, which clearly indicate that the as-obtained sample is mainly composed of urchin-like structures. The high-magnification SEM image of the as-prepared sample further indicates that the as-obtained urchin-like structure has a diameter of ca. 5 μm with acicular crystallites radiating from the center with a uniform size distribution , l-cysteine molecule is commonly employed for the preparation of metal sulfides with different morphologies [l-cysteine during the synthetic process, such as sulfur sources, polydentate ligands, self-assembly reagent, or even shape-controlling agent [2+ and l-cysteine were mixed in the solvent, a complex between Mn2+ and l-cysteine will form, leading to the formation of nanoplates at the initial stage. When the solvothermal reaction began, the initial formed Mn complex will decompose, leading to the formation γ-MnS microparticles. The whole chemical process can be described asBased on the previous reports and the time-dependent experiments, a two-step formation mechanism for the urchin-like d Scheme . Owing thologies , 24. Theng agent –32. Whenl-cysteine is excessive. The free thiol groups of the excessive cysteine molecule will bind to the surfaces of the initial formed γ-MnS nanoparticles. Meanwhile, the hydrogen bonds and S-S bonds will form between the cysteine molecules. Driven by the interactions of hydrogen bonds among tiny particles, H2O and DEG, the as-formed γ-MnS tiny nanoparticles will cross-link together, leading to the formation of irregular microparticles when the solvothermal reaction has been conducted for 1 h [γ-MnS microspheres via the Ostwald ripening process [2+, the growth rate of γ-MnS can be effectively controlled. The growth rate in all the direction is nearly the same under the influence of l-cysteine, leading to the formation of γ-MnS microspheres in this step. As the reaction went on, the influence of l-cysteine gradually weakens because of the thermal decomposition process during the solvothermal process. Driven by the intrinsic anisotropic growth habit of γ-MnS, the 1D γ-MnS nanorods will gradually form on the surfaces of the γ-MnS microspheres. As the solvothermal reaction went on, the as-formed nanorods grow longer and longer. When the reaction has been conducted for 18 h, the urchin-like microstructures finally form.In our synthesis,  for 1 h . As the  process , 33–36. l-cysteine during the synthetic process, comparative experiments employing different amount of l-cysteine were carried out. According to the XRD patterns of the samples when different amount of l-cysteine was employed, the final products under all conditions can be designated to be the hexagonal phased γ-MnS (JCPDS card no. 40-1289). Figure γ-MnS microstructure when 1 mmol l-cysteine was employed, which clearly indicate that the sample is composed of irregular microspheres. Careful observation further indicates that there are some small nanorods on the surfaces of these microspheres. When the amount of l-cysteine increased to 3 mmol, the products are mainly composed of some urchin-like structures as well as some irregular microparticles. As we have mentioned above, l-cysteine not only acts as the sulfur source but also acts as a morphological-controlling agent during the synthetic process. The formation of the complex between Mn and l-cysteine exerts great influence on the formation rate of MnS, which is of vital importance for the morphological control of the final product. When the concentration of l-cysteine is low, the reaction speed is high, which will result in the formation of the irregular microspheres. When the concentration of l-cysteine is high, the reaction rate becomes low, which would be beneficial for the morphological control of the final product. However, the low reaction rate will also inevitably lead to the incomplete transformation from the irregular microparticles to the urchin-like structures. And this can be used to explain the phenomenon why irregular microparticles as well as urchin-like structures co-exist in the obtained sample when 3 mmol l-cysteine was employed.To further investigate the effect of γ-MnS exhibits excellent electrochemical performance. Figure −1. Two peaks can be observed during the first cycles. The reduction peak centering at 0.49 V can be assigned to the reduction of Mn2+ to metallic Mn, while the oxidation peak centering at 1.3 V corresponds to the insertion of Li+ into the γ-MnS lattice to form a homogeneous phase of Li2MnS [As it is shown in Fig. f Li2MnS , 37, 38.f Li2MnS –41. The γ-MnS of different cycles at the current of 500 mA g−1 between 0.01 and 3.0 V. According to the discharge/charge profiles, the initial discharge capacity is 825.5 mA h g−1 and the corresponding columbic efficiency is about 69.06 %. The loss in initial columbic efficiency can be attributed to be the formation of the SEI layer and decomposition of the electrolyte, which have been widely reported for many anode materials [γ-MnS electrode are ~0.65 and ~1.25 V, respectively, which are in well agreement with the CV results. The discharging voltage plateau in the second cycle is much higher than the corresponding value in the first cycle, indicating the irreversible reactions of γ-MnS and formation of unstable SEI layer [Figure aterials –45. The EI layer , 46, 47.γ-MnS at current density of 500 mA g−1. An obvious decrease in the discharge capacity can be clearly observed in the initial 60 cycles. The discharge capacity of the as-prepared γ-MnS fades significantly in the following cycles, decreasing from 587.8 mA h g−1 at second cycle to 379.6 mA h g−1 at 60th cycle. This phenomenon could be attributed to the lithiation-induced mechanical degradation and the formation of the unstable SEI layer [−1 at the 60th cycle to 823.4 mA h g−1 in the 1000th cycle. It is interesting to note that the discharge capacity after 1000 cycles is higher than the theoretical value of γ-MnS, which may result from the so-called “pseudo-capacitance behavior.” This phenomenon is attributed to the reactivated process and stable SEI layer optimization [Figure EI layer , 48. To mization , 50. To mization . What ismization .Fig. 7SEγ-MnS in LIB applications. Herein, the as-prepared γ-MnS was tested under different charge current densities from 100 to 2000 mA g−1. When the current density is 100 mA g−1, the reversible capacity keeps at ~620 mA h g−1 after 10 cycles. When the current density increased to 500, 1000, and 2000 mA g−1, the corresponding reversible capacities are 450, 370, and 300 mA h g−1, respectively. Obviously, the γ-MnS anode material shows excellent performance at a different current density. Even being cycled at 2000 mA g−1, the capacity can still reach 300 mA h g−1. When the current density returns to 200 mA g−1, the capacity of γ-MnS can reach to 490 mA h g−1, which is much higher than that of graphite in the same condition [γ-MnS in this work shows good electrochemical performance.Figure ondition , 52. Theondition , 18, 19 γ-MnS, electrochemical impedance spectroscopy (EIS) was measured at frequencies from 0.01 Hz to 100 kHz and the result is shown as Fig. γ-MnS without charge/discharge process is larger than the corresponding value after 200 cycles, which clearly indicates the formation of the lithium channels and the degradation of the anode material, which is well in accordance with SEM and TEM images at 200th cycle as shown in Fig. To further understand the electrochemical properties of the urchin-like γ-MnS structures were successfully synthesized by a facile solvothermal method using l-cysteine and MnCl2 · 4H2O as the raw materials. A two-step growth mechanism has been proposed for the formation of the urchin-like γ-MnS structures. The product exhibits excellent cycling stability (823.4 mA h g−1 at the current density of 500 mA g−1 after 1000 cycles) and the discharge voltage is ~0.75 V. SEM, TEM, and XRD were employed to investigate the transformation of the active materials during the charge/discharge process, which clearly indicate that the structural degradation and reformation of the active material play the key roles for the maintenance of the cycling stability. Considering the simple preparation process, excellent cycling stability, and low voltage potential of the as-prepared sample, the product is considered to be a potential candidate anode material in LIBs.In summary, urchin-like


However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.Molecular hydrogen (H In the absence of Wnt ligands, β-catenin is steadily phosphorylated by casein kinase 1 (CK1) at Ser45 and glycogen synthase kinase 3 (GSK3) at Ser33/Ser37/Thr41 at its N-terminus in a degradation complex assembled by Axin1 and adenomatous polyposis coli (APC), and is subsequently degraded through the β-transducin repeat-containing protein (β-TrCP)-mediated ubiquitin/proteasome pathway2 is beneficial for musculoskeletal diseases including inflammatory and mitochondrial myopathies142 on OA to the best of our knowledge.Aberrant activation of Wnt/β-catenin signaling is associated with a number of diseases including cancers and degenerative diseases892 inhibited Wnt/β-catenin signaling activated by Wnt3a, LiCl, or BIO in L and HeLa cells. H2 promoted phosphorylation, ubiquitination, and subsequent degradation of β-catenin without directly affecting mRNA level of β-catenin. The effect of H2 required CK1/GSK3-phosphorylation sites on β-catenin, the CK1/GSK3 activities, as well as APC and Axin1 activities. We confirmed the suppressive effect of H2 on Wnt/β-catenin signaling in chondrocytes and observed a protective effect of H2 on OA progression. We report that H2 is an inhibitor for activated Wnt/β-catenin signaling, which provides additional evidence that H2 is a gaseous signal modulator.In this study, we observed that H2 affects Wnt/β-catenin signaling, we first conducted Topflash luciferase reporter assay in L cells with 10% H2 or 10% nitrogen (N2) gas. Topflash luciferase reporter plasmid carries 8 copies of TCF-binding sites in the promoter region and the firefly luciferase cDNA to quantify activation of Wnt/β-catenin signaling. Addition of Wnt3a or a GSK3 inhibitor, LiCl or BIO, to the culture medium for 24 h increased Topflash reporter activity, which, however, was attenuated by H2  kinase signaling in cell lines and rodent disease models342 suppressed Wnt/β-catenin signaling, we next asked whether H2 suppresses Wnt/β-catenin signaling through inhibition of JNK signaling. We pretreated L cells with 40 μM SP600125, which efficiently inhibited JNK-mediated phosphorylation of c-Jun  in L cells  to block protein biosynthesis of β-catenin and conducted the CHX chase assay to determine whether H2 accelerates the degradation rate of β-catenin. Because endogenous β-catenin is hard to be detected in L cells, we expressed myc-β-catenin in L cells and found that H2 accelerated degradation of myc-β-catenin  but not by high BIO concentrations (4 μM and 6 μM)  (http://www.genenames.org/), CTNNB1 and Ctnnb1 are used in this communication to indicate the genes for β-catenin in human and mouse, respectively. HCT-116 cells are heterozygous for a deletion mutation at Ser45, the CK1 regulatory site, in the β-catenin gene2 on Wnt3a- or BIO-induced β-catenin accumulation in HCT-116 cells  . These r16 cells  was less16 cells  and HeLa16 cells . The red-catenin . In conttenin (2 . To furtin (2 (2 . These r2 regulates phosphorylation of glycogen synthase (GS), which is another substrate of GSK3. We found that H2 did not affect GSK3-mediated phosphorylation of GS in L cells , promotes β-catenin hyperphosphorylation on serine-threonine residuesed cells , indicat2-medicated β-catenin degradation. First, in HT-29 human colon cancer cells carrying truncated APC2 failed to decrease β-catenin levels  cells. We first confirmed that H2 downregulated Topflash reporter activity in HCS-2/8 cells stimulated by Wnt3a or BIO  or unfavorable directions from clone to clone. To summarize, H2 suppresses Wnt/β-catenin signaling in human OAC cells, but the effect of H2 on ECM production cannot be evaluated due to variable responses of human OAC cells to Wtn3a and BIO. We also conducted Alcian blue staining in differentiated ATDC5 mouse chondrogenic cells, and found that both Wnt3a- and BIO-induced loss of proteoglycans were marginally reversed by H2 -induced OA cartilage in rats, where Wnt/β-catenin signaling is abnormally activated2 water (7 ppm) ad libitum from days 0 to 56 after DMM surgery. No difference in body weights was noted between the control and H2 groups 2. However, H2 is a stable gas and the reaction rate constant of H2 and •OH is in the order of 107 M−1·s−1, which is much lower than the reaction rate constants of •OH with other molecules (109 to 1010 M−1·s−1)2 concentration comes to the baseline level in 30 min after taking 200 ml saturated H2 water in healthy individuals2 is low and the dwell time of H2 in our body is short, H2 is unlikely to efficiently remove •OH. Thus, yet unidentified mechanisms should underlie the therapeutic effect of H2. A previous report showed that oral intake of H2 water increases gastric secretion of ghrelin in mice2 gas ameliorates a rat model of Parkinson’s disease2 alters signaling activities in mast cells and macrophages without directly scavenging reactive oxygen/nitrogen species3Although more than 321 studies have demonstrated beneficial effects of H2 inhibited endogenous β-catenin accumulation induced by Wnt3a and GSK3 inhibitors (LiCl and BIO), as well as exogenous β-catenin accumulation induced by a transgene, but had no effect on the basal endogenous β-catenin level. The effect of H2 was thus likely to be observed when Wnt/β-catenin signaling was abnormally activated. Then, we found that H2 increased β-catenin phosphorylation without attenuating the activity of PP2A, and accelerated β-catenin degradation without decreasing its mRNA level. Given that complete GSK3 inhibition and mutations at the CK1- or GSK3-phosphorylation sites of β-catenin nullified the H2 effect, the dual CK1 and GSK3 activities were required for H2-medicated β-catenin degradation. As H2 had no effect on GSK3-mediated phosphorylation of GS, which is another substrate of GSK3, H2 was likely to enhance GSK3 activity only in the degradation complex with APC and Axin1, but had no direct effect on GSK3 itself. Enhancement of CK1-mediated β-catenin phosphorylation by H2 is also in accordance with the assumption that the H2 works on the degradation complex, not on CK1 or GSK3. Additionally, as both CK1 and GSK3 are constitutively active in the resting state and as no allosteric activators are known, H2 is unlikely to be able to allosterically upregulate activities of CK1 and GSK3. To further confirm that the effect of H2 is on phosphorylation and not ubiquitination of β-catenin, we examined the effect of H2 on HT-29 human colon cancer cells harboring truncated APC. The truncated APC retains all three 15-aa repeats and three of seven 20-aa repeats, and lacks all three Axin1-binding sites and four of seven 20-aa repeats. A previous study shows that the three 20-aa repeats that are retained in the truncated APC are sufficient for β-catenin ubiquitination, and the interaction between the truncated APC and E3-ligase β-TrCP is not affected in HT-29 cells212 in HT-29 cells thus indicates that H2 enhances phosphorylation but not ubiquitination of β-catenin. In addition, our observation that knock-down of either APC or Axin1 minimizes the suppressive effect of H2 on β-catenin accumulation indicates that both APC and Axin1 are required for the H2 effect. We next examined the protein-protein interactions within the degradation complex using co-immunoprecipitation assay, and found that H2 had no effect on the interactions among Axin1, APC, β-catenin, and GSK3, but still could enhance CK1/GSK3-mediated phosphorylation of β-catenin. Water structure plays an active role in protein folding, enzyme catalysis, and cell signaling2O-H2 interaction accompanied by charge transfer is stronger than predicted2 can easily diffuse into every cellular compartment, solubilized H2 interfacing with other biomolecules possibly alters their hydration structures and subsequently their activities. In Wnt/β-catenin signaling, H2 may dynamically enhance protein-protein interactions during the process of β-catenin phosphorylation, which, however, could not be detected in our co-immunoprecipitation assays because of a rapid decrease of H2 concentration in a test tube2 may modulate interaction of one of molecules constituting the degradation complex with a yet unidentified molecule, which leads to enhancement of β-catenin phosphorylation.In this study, we first showed that H2 prevents degranulation of mast cells not by a radical scavenging effect, but by downregulating NADPH oxidase activity via attenuating the phosphorylation of the FcεRI-associated Lyn and its downstream signal transduction molecules  without affecting other signaling molecules 2 reduces lipopolysaccharide/interferon γ (LPS/IFNγ)-induced nitric oxide (NO) release by suppressing the activity of inducible nitric oxide synthase (iNOS) not by affecting NADPH oxidase activity but by suppressing phosphorylation of ASK1 and its downstream signaling molecules 2 suppresses Wnt/β-catenin signaling without scavenging hydroxyl radicals or peroxynitrite. Inhibition of JNK, but not of ERK or p38 MAP kinase, suppresses Wnt3a-induced β-catenin accumulation2 was unlikely to be dependent on JNK because inhibition of JNK did not attenuate the effect of H2. Akt phosphorylates GSK3 on Ser9 to inactivate it and inhibition of Akt activates GSK32 had no effect on GSK3-mediated GS phosphorylation, it is unlikely that H2 activates GSK3 via suppression of Akt. Accordingly, H2 is able to specifically modulate signaling pathways in cell- and disease-specific manners.We have previously shown that H2 could not decrease abnormally elevated β-catenin in HCT-116, HepG2, and HT-29 cancer cells. Thus, H2 is unlikely to suppress proliferation of these cells. Previous studies have shown that H2 inhibited cell proliferation of human tongue carcinoma cells HSC-42 and 5-fluorouracil induced apoptosis of colon 26 cells2 suppressed the expression of vascular endothelial growth factor (VEGF) in human lung adenocarcinoma cells A5492 on Wnt/β-catenin signaling has not been dissected in these cells, H2 might have achieved the tumor-suppressing effects by suppressing Wnt/β-catenin signaling. Alternatively, H2 might have suppressed cell proliferation by modulating other signaling pathway(s) and/or molecules.In this study, we showed that H2 suppresses Wnt/β-catenin signaling in human OAC cells and also rescues Wnt3a- or BIO-induced loss of proteoglycan in differentiated ATDC5 chondrogenic cells. Additionally, H2 tended to protect against cartilage degradation in the DMM-induced OA model in rats, although statistical significance was not observed. The insignificant effect of H2 on the OA model may be partly accounted for by a limited effect of H2 on Wnt/β-catenin signaling in cartilage chondrocytes. Alternatively, the DMM-induced OA progression is accelerated by other signaling pathways that are insensitive to H2. In contrast to the deleterious effect of aberrantly activated Wnt/β-catenin signaling in OA, excessive inhibition of Wnt/β-catenin signaling also worsens OA2 did not change the levels of endogenous β-catenin and Sox9 on the sham-operated side, H2 is expected to have no effect on cartilage development and homeostasis. Our results suggest that H2 may be able to attenuate OA progression in humans. However, other signaling pathways and/or molecules, including transforming growth factor β (TGF-β) signaling and inflammatory responses, are also involved in OA development and progression2 modulated other signaling pathways or molecules to ameliorate OA.As stated in introduction, abnormal activation of Wnt/β-catenin signaling deteriorates OA and thus Wnt/β-catenin signaling can be a therapeutic target for OA. In this study, we first show that H2 suppresses abnormally activated Wnt/β-catenin signaling, which plays pivotal roles in diverse pathophysiologic processes, by enhancing β-catenin phosphorylation in the degradation complex  was mixed with CO2-added air  to make 5% or 10% H2 gas, which was delivered into the box via an afferent tube and out of the box via an efferent tube connected to a draft chamber. In the control group, 3 ml/min or 6 ml/min of N2 gas was used instead of H2 gas to control for O2 concentrations. We measured the hydrogen concentration in the medium by equilibrating 1 ml medium with 100 ml of 100% N2 gas and by injecting 1 ml equilibrated gas into a gas chromatography connected to a semiconductor gas detector . As shown in 2 concentrations were detectable in culture medium in 2 min after administration of H2 gas. The H2 concentration in the culture medium stayed stable after 20 min of H2 administration. The amount of H2 dissolved in the culture medium doubled by increasing H2 concentration from 5% to 10%. The pH of the culture medium after culturing L cells for 24 h with 10% N2 or 10% H2 gas administration was 7.71 ± 0.03 and 7.67 ± 0.02 , respectively, with no statistical difference by Student’s t-test.Cells were cultured in a culture dish in a 560-ml closed plastic box that was covered with aluminum and humidified with water at the base of the box. The box was put in a convection incubator . We adjusted the temperature of the incubator to make the temperature inside the box 37 °C using an electronic thermometer. In the HRenilla luciferase plasmid . L cells were transfected using Lipofectamine 2000 (Invitrogen), whereas HeLa and HCS-2/8 cells were transfected using FuGENE 6 (Roche). Twenty-four hours later, cells were treated with either 50% control CM, 50% Wnt3a CM, 30 mM LiCl, or 2–4 μM BIO in 10% H2 or 10% N2 gas for 24 h. Luciferase activity was measured in triplicate by the Dual Luciferase Reporter Assay System (Promega).L, L Wnt3a, HeLa, HCT-116, and HT-29 cells were obtained from ATCC. HepG2 and ATDC5 cells were from RIKEN BioResource Center. HCS-2/8 cells were kindly provided by Dr. Masaharu Takigawa at Okayama University. Studies using human OAC cells were approved by the Ethical Review Committee of Nagoya University Graduate School of Medicine, and were performed in accordance with the relevant guidelines by MHLW, Japan. After a written informed consent was given, OAC cells were obtained from patients who underwent total joint replacement for severe knee OA. L, L Wnt3a, HeLa, HCT-116, HT-29, HepG2, HCS-2/6, and human OAC cells were cultured in the Dulbecco’s Modified Eagle’s medium  supplemented with 10% fetal bovine serum . ATDC5 cells were cultured in DMEM/F12  supplemented with 5% FBS. L and L Wnt3a cells were cultured for 4 d to make control conditioned medium (CM) and Wnt3a CM, respectively. Lithium chloride (LiCl), cycloheximide (CHX), N-benzyoloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), dimethyl sulfoxide (DMSO), and SP600125 were purchased from Wako. Okadaic acid was bought from Research Biochemicals International (RBI), and 6-bromoindirubin-3′-oxime (BIO) was from Sigma-Aldrich. To quantify the canonical Wnt/β-catenin signaling activity, cells were transfected with the Topflash luciferase reporter plasmid  and the 2 or 10% N2 gas for 24 h. To knock-down Axin1, we performed siRNA transfection in two consecutive days as described previously2 or 10% N2 gas for 24 h.A plasmid carrying myc-β-catenin (XE28 XBC 40) was kindly gifted from Dr. Takamasa Yamamoto at National Institute for Basic Biology. Plasmid carrying HA-WT-β -catenin, HA-ΔN-β-catenin, and HA-S37A-β-catenin were kindly provided by Dr. Eisuke Nishida at Kyoto University. L cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen). Sequences of siRNAs against APC and Axin1 were adopted from previous studies422, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 μg/μl aprotinin, 1 μg/μl leupeptin, 1 μg/μl pepstatin A, 1 mM PMSF, 1 mM sodium orthovanadate, and the Phosphatase Inhibitor Cocktail . The lysates were incubated on ice for 15 min, sonicated for 10 sec, and then centrifuged at 20,600 × g at 4 °C for 15 min. Preparation of nuclear fraction is described in details in the Cells were washed with PBS twice and were harvested with PLC buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 10% glycerol, 1% TritonX-100, 1.5 mM MgClProteins were immunoprecipitated from L cell lysates as shown in the Total RNAs from cells were extracted by RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and then reverse-transcribed into complementary DNA (cDNA) using an oligo-dT primer (Thermo Fisher Scientific) and ReverTraAce (Toyobo). Real-time quantitative PCR (qPCR) was performed with the LightCycler 480 (Roche Applied Science) using the SYBR Premix ExTaq (Takara Bio). The expression level of a specific gene was normalized by the levels of β2-microglobulin (β2-MG). PCR primers are shown in Details are shown in the Alcian blue staining in differentiated ATDC5 cells was performed as described previously with slight modificationsCell proliferation of ATDC5 cells were estimated by a BrdU cell proliferation ELISA kit (Roche). Details are shown in the 2 water group with unlimited access to degassed water and H2 water after surgery, respectively. H2 water was freshly prepared every evening using Hydrogen Water 7.0 , which was kindly provided by MiZ Co. Ltd. The concentration of dissolved H2 was 5 to 7 ppm, whereas the concentration of saturated H2 under the standard ambient temperature and pressure (SATP) is 1.6 ppm. The H2 concentrations in the glass vessel for rodents exponentially decreased with a half-life of 1.09 h2 concentration in the water that the rodents drank was predicted to be 1.66 ppmAll animal studies were approved by the Animal Care and Use Committee of the Nagoya University Graduate School of Medicine, and were performed in accordance with the relevant guidelines by MEXT, Japan. Details of experiments with fed and starved mice are shown in the in cellulo studies, values are normalized to those of cells treated with 50% control CM and 10% N2 gas, unless otherwise indicated. Statistical significance was estimated either by Student’s t-test or two-way repeated measures ANOVA test. Bonferroni correction was applied to Student’s t-test for multiple comparisons. P-values less than 0.05 were considered significant.All values were presented as the mean and SEM. For How to cite this article: Lin, Y. et al. Molecular hydrogen suppresses activated Wnt/β-catenin signaling. Sci. Rep.6, 31986; doi: 10.1038/srep31986 (2016).


Hyperactivation of Wnt/β-catenin signaling is one of the major causes of human colorectal cancer (CRC). A hallmark of Wnt signaling is the nuclear accumulation of β-catenin. Although β-catenin nuclear import and export have been widely investigated, the underlying mechanism of β-catenin's nuclear retention remains largely unknown. Here, we report that Twa1/Gid8 is a key nuclear retention factor for β-catenin during Wnt signaling and colorectal carcinogenesis. In the absence of Wnt, Twa1 exists together with β-catenin in the Axin complex and undergoes ubiquitination and degradation. Upon Wnt signaling, Twa1 translocates into the nucleus, where it binds and retains β-catenin. Depletion of Twa1 attenuates Wnt-stimulated gene expression, dorsal development of zebrafish embryos and xenograft tumor growth of CRC cells. Moreover, nuclear Twa1 is significantly upregulated in human CRC tissues, correlating with the nuclear accumulation of β-catenin and poor prognosis. Thus, our results identify Twa1 as a previously undescribed regulator of the Wnt pathway for promoting colorectal tumorigenesis by facilitating β-catenin nuclear retention. Dysfunction of the Wnt cascade is implicated in a variety of human diseases, most prominently in colorectal cancer (CRC). Mutation in the adenomatous polyposis coli (APC) gene, a key component of this pathway, is thought to be an initiating event in human colorectal carcinogenesis. In mouse models, inactivation of the APC gene causes intestinal hyperplasia and adenocarcinoma3.Canonical Wnt signaling plays pivotal roles in many biological processes, such as cell proliferation, cell fate determination and stem cell maintenance6. In the absence of Wnt, cytoplasmic β-catenin is constitutively degraded by the Axin complex, which is composed of APC, Axin, casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). CK1 and GSK3 sequentially phosphorylate β-catenin, resulting in its ubiquitination and proteasomal degradation. Upon Wnt stimulation, the Wnt ligand triggers the inactivation of the Axin complex to stabilize β-catenin, which subsequently translocates into the nucleus to form a transcriptional complex with T-cell factor (TCF) to activate Wnt target gene expression. Previous studies have reported that some proteins, including forkhead box protein M1 (FoxM1), mucin-1, insulin receptor substrate-1 (IRS-1) and B-cell lymphoma 9 (BCL9), facilitate β-catenin import into the nucleus, whereas other proteins, such as APC, Axin, Ran binding protein 3 (RanBP3) and Chibby, actively promote β-catenin export out of the nucleus16. These findings indicate that the nuclear β-catenin level is fine-tuned at multiple layers, such as protein stability and nuclear import and export. However, little is known about whether and how nuclear retention might provide another layer of regulation to control β-catenin nuclear accumulation.A hallmark of canonical Wnt signaling is the stabilization and nuclear translocation of β-catenin18. Using a comprehensive approach, we find that Twa1 is not only significantly upregulated in human CRC tissues, but also that this correlates with a poor prognosis for CRC patients. In response to Wnt signaling, Twa1 directly binds to β-catenin and promotes its nuclear retention. We exemplify the functions of Twa1 by showing its requirement for the dorsal development of zebrafish embryos and for the growth of CRC cells in a xenograft model. Although Twa1 associates with β-catenin in the nucleus, it does not interact with either TCF4 or Wnt-responsive elements (WREs), suggesting a chromatin-independent retention mechanism that is indispensible for the Wnt transcriptional program.Here we show that Twa1 (two hybrid-associated protein no.1 with RanBPM), also known as Gid8 (glucose-induced degradation protein 8 homolog), is a key nuclear retention factor for β-catenin during Wnt signaling and colorectal tumorigenesis. Twa1/Gid8 (Twa1) was initially described in the hunt for the proteins that can interact with Ran binding protein M (RanBPM), but its biological function remains largely unclear19. The results showed that the expression of 2 549 genes in CRC tissues was significantly changed when compared to nontumor tissues  < 0.01) (fork-head box Q1 (FoxQ1), c-Myc and leucine-rich repeat containing G protein-coupled receptor5 (LGR5), in our analysis . To confirm the upregulation of Twa1 in CRC tissues, we analyzed the RNA-seq data from the TCGA database and found that Twa1 mRNA expression levels were also significantly higher in CRC tissues than their pair-matched nontumor samples  < 0.01) . As expeanalysis 20,21,22. samples . Moreove samples . Taken t28. Further analyses showed that knockdown of Twa1 by two shRNAs targeting different regions of Twa1 mRNA significantly inhibited lymphoid enhancer-binding factor-luciferase (LEF-Luc) activity and reduced the mRNA levels of Wnt target genes, Axin2 and Cyclin D1  HEK-293 cell lines using the CRISPR/Cas9  system. Two Twa1 KO cell lines were obtained and the deletion was verified by genomic DNA PCR, Sanger sequencing and western blotting  domain of Twa1 was required for its interaction with β-catenin  and 3B17ytoplasm . Upon Wnytoplasm  and 3C. ytoplasm . Furtherytoplasm , but alsytoplasm  and 3F.To confirm if Twa1 promotes β-catenin nuclear accumulation through its CRA domain, we forced wild-type or mutant Twa1 to accumulate in the nucleus by fusing a nuclear localization signal (NLS) sequence at their C-terminus. Ectopic expression of nuclear-localized Twa1 (Twa1-NLS) and Twa1 mutants containing the CRA domain not only obviously induced nuclear translocation of exogenous or endogenous β-catenin, but also enhanced Wnt reporter activity and target gene expression even without Wnt3a stimulation . In contin vitro transport assays in digitonin-permeabilized cells revealed that addition of purified Twa1-NLS protein did not affect β-catenin nuclear import but significantly enhanced β-catenin nuclear retention, which was abolished by deletion of the CRA domain  experiments by bleaching GFP-β-catenin in HEK-293 cells expressing RFP-Twa1-NLS (RFP-fused Twa1-NLS) or RFP-Twa1-ΔCRA-NLS . After pA domain . MoreoveA domain 29. Toget30. Given that Twa1 binds and retains β-catenin in the nucleus, we assumed that Twa1 might also directly participate in β-catenin/TCF4-mediated transcription. Chromatin immunoprecipitation (ChIP) analysis showed that only TCF4 was constitutively bound to the WREs of Axin2 and Cyclin D1 in the absence of Wnt signals  or Twa1b-MO to knock down the expression of these two proteins. We found that both Twa1a and Twa1b morphants resembled β-catenin 2 morphants and exhibited loss or reduction of dorsal structures, characteristics of embryonic ventralization 40, and found that depletion of Twa1a markedly decreased boz expression , which harbor constitutively high levels of nuclear β-catenin due to APC mutations43, significantly reduced nuclear β-catenin levels, Wnt reporter activity, and target gene expression   (P < 0.0001)  . Further 0.0001) . More im 0.0001) . Taken tin vivo. Thus, Twa1 is a previously uncharacterized component of the Wnt signaling pathway that promotes β-catenin nuclear retention.In this study, we provide evidence that Twa1 is a critical β-catenin nuclear retention factor during Wnt signaling. In the absence of Wnt signals, both Twa1 and β-catenin are degraded via ubiquitination by the Axin complex. In response to Wnt stimulation, both cytoplasmic Twa1 and β-catenin are stabilized and translocated into the nucleus, where Twa1 retains β-catenin to ensure a sufficient amount of β-catenin for TCF4 binding, but does not directly participate in β-catenin/TCF4-mediated transcription . We exem2. β-catenin does not contain NLS or nuclear export signal (NES) sequences2 and earlier studies have shown that β-catenin enters into the nucleus by directly interacting with the nuclear pore complex, independently of the Ran GTPase or importin-mediated mechanism48. Recent reports demonstrate that some NLS-containing proteins, including mucin-1, IRS-1, BCL9 and FoxM1, are able to directly bind to β-catenin and transport β-catenin into the nucleus via the classical import pathway10. On the other hand, some NES-containing proteins, such as APC, Axin and Chibby, have been shown to associate with β-catenin and promote the export of nuclear β-catenin16.The molecular regulation of β-catenin nuclear accumulation during Wnt signaling remains enigmatic. Several steps have been implicated to be necessary for the nuclear accumulation of β-catenin: stabilization of β-catenin in the cytoplasm, its transport into the nucleus and subsequently its retention in the nucleus2. Overexpression of TCF4 shifts ectopic cytoplasmic β-catenin to the nucleus and slows its rate of nucleocytoplasmic shuttling50. Downregulation of LEF1 selectively reduces the chromatin-retained pool of nuclear β-catenin in NIH-3T3 cells51. Although nuclear β-catenin is captured by the chromatin-bound TCF family members on chromatin, recent kinetic ChIP analyses have shown that unlike TCF4 which maintains a steady state on WREs, β-catenin dynamically cycles on and off WREs during Wnt activation53. It is unclear how nuclear β-catenin that is dissociated from chromatin is retained in the nucleus to maintain sufficient levels for TCF4 binding.It is well-established that nuclear β-catenin directly binds to TCF family members such as TCF4 and LEF1 to activate Wnt target gene expressionHere our data clearly show that Twa1 suppresses β-catenin nuclear export, but has no significant effect on its nuclear import . Althoug18. RanBPM functions as a scaffolding protein in several signaling pathways and mediates multiple cellular processes55. However, unlike most members of the Ran-binding protein family, RanBPM does not contain the consensus domain for Ran binding in vivo56, consistent with its lack of involvement in nuclear-cytoplasmic shuttling57. Nevertheless, it will still be of interest to examine whether RanBPM participates in Twa1-mediated nuclear retention of β-catenin.Previous studies have reported Twa1 to be a partner of RanBPM in a yeast two-hybrid assay58. A high level of nuclear β-catenin is frequently found in CRC tissues and is correlated with poor prognosis of CRC patients60. Our results indicate that Twa1 is an essential β-catenin nuclear retention factor that contributes to colorectal carcinogenesis. Twa1 deficiency not only decreases β-catenin nuclear levels and Wnt target gene expression in CRC cells, but also inhibits the proliferation and tumorigenicity of CRC cells in nude mice  available in the Oncomine database. The cohort of human CRC tissues profiled on Affymetrix U133 Plus 2.0 arrays were normalized with the Robust Multi-array Average algorithm. The limma R package (v 3.2.3) was used to detect differentially expressed genes. The Linear models and Empirical Bayes methods were applied for analyzing these data, and the resulting P-values were adjusted using the Benjamini and Hochberg FDR algorithm61. Differentially expressed genes were thresholded at fold change > 2, P < 0.01 and FDR < 0.01.To identify differentially expressed genes in CRC, we analyzed the Hong CRC microarray data set . RNA-seq by Expectation-Maximization (RSEM) expression values were log2 transformed and used for statistical analysis with Student's t-test62.To confirm Twa1's upregulation in CRC tissues, we analyzed the CRC RNA-seq data set  obtained from the TCGA databases  27-59, the Twa1-ΔCTLH mutant deleted for amino-acids 65-122 and the Twa1-ΔCRA mutant deleted for amio-acids 118-215 were generated by PCR and subcloned into the pCS2+ vector containing an N-terminal Flag tag or the pCMV/myc/nuc vector containing a C-terminal NLS (Invitrogen). Myc-Ub, Myc-Ub-K48, Myc-Ub-K63, pCMV-Flag-YAP, pCMV-Flag-TAZ, pEF-Flag, pEP330X, pCS2+, LEF-Luc reporter, GLI-Luc reporter, NF-κB-Luc reporter, TEAD-Luc reporter, GFP-β-catenin and HA-GSK3β vectors were provided by Drs Yong Cang, Zongping Xia, Jinrong Peng, Ximei Wu (Zhejiang University), W James Nelson (Stanford University) or Jim R Woodgett (Toronto University). The luciferase reporters of various signaling cascades were constructed based on pGL3-basic vectors (Promega) as described previously63. Oligos corresponding to the following sequences were synthesized and cloned into pGV3-U6 , separately: 5′-GGAGAAGTTTCGAATGGAA-3′ and 5′-CAGCGGAGAAGTTTCGAAT-3′ for Twa1 RNAi (sh-Twa1 and sh-Twa1-2), 5′-GTACATTCTTGATAACAAT-3′ for Axin RNAi (sh-Axin), 5′-GGTGGTGGTTAATAAGGCT-3′ for β-catenin RNAi (sh-β-catenin) and 5′-GACGTTGCGAGAAGTTGGA-3′ for APC RNAi (sh-APC). Twa1 RNAi-resistant mutants were generated with a QuickChange Site-Directed Mutagenesis kit (Stratagene). All of these constructs were confirmed by DNA sequencing.Full-length human 29. Shh-conditioned medium (Shh-CM) was provided by Dr Ximei Wu (Zhejiang University). Wnt3a-expressing and L cells were purchased from American Type Culture Collection. The other cells were obtained from the Cell Bank of the Chinese Academy of Science .HEK-293, HEK-293T, HeLa, Wnt3a-expressing and control L cells were cultured in DMEM (Invitrogen) containing 10% fetal bovine serum . DLD1 and SW480 cells were grown in RPMI1640 (Invitrogen) containing 10% FBS. Wnt3a-conditioned medium (Wnt3a-CM) and control medium (Ctr-CM) were prepared as previously describedTransient transfection in HEK-293, DLD1 and SW480 cells were carried out using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Lentiviruses were prepared in HEK-293T cells by transfection with the indicated shRNA vectors and viral packaging constructs. The viral medium was collected, filtered and mixed with 4 μg/ml polybrene (Sigma) before addition to the target cells.Twa1 gene was inactivated in HEK-293 cells using the CRISPR/Cas9 system. Briefly, a pair of targeting sequences (5′-GAGAGCAGACATGAACCGCC-3′) was synthesized and introduced into a modified one-vector system pEP330X. HEK-293 cells transfected with this plasmid were treated with 1 μg/ml puromycin for 24 h and reseeded to 96-well plates to allow single-colony formation. After 12 days, genomic DNA was extracted from individual colonies. The candidate KO clones were verified by sequencing of the PCR fragments amplified by the primers (5′-ATTCTCCGGCTCACAGCTC-3′ and 5′-GCTACAGCACTCCTTATGTGTT-3′) and western blotting with anti-Twa1 antibody. The APC KO HEK-293 cells were prepared as previously described64. The candidate KO clones were verified by sequencing of the PCR fragments amplified by the primers (5′-AAACTCATTTGGCCCACAGG-3′ and 5′-TGCTTTGAAACATGCACTACGA-3′).The 4 cells per well and then transfected with LEF-Luc (200 ng) and Tk-renilla  vectors for 24 h. After treatment with Wnt3a-CM or 40 mM LiCl (Sigma) for 6 h, the cells were washed twice with phosphate buffered saline (PBS) and lysed in 200 μl passive lysis buffer (Promega). The luciferase activities were measured using dual luciferase reporter system (Promega). Firefly luciferase activities were normalized to Renilla luciferase activities.HEK-293 cells infected with lentiviruses containing sh-Twa1 or sh-ctr were seeded in 12-well plates at 2.5 × 1065. Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min, and blocked with 10% FBS for 30 min. The coverslips were then incubated with the indicated antibodies for 2 h, followed by Cy3-conjugated anti-mouse Ig or FITC-conjugated anti-rabbit Ig secondary antibody (Jackson Immuno Research) for 50 min. DNA was stained with 4′, 6-diamidino-2-phenylindole . The mounted coverslips were analyzed by Zeiss LSM510 microscopy.Immunoflourescence analysis was performed as described previouslyFRAP assays were performed in live cell chambers (Corning) at 37 °C using a Zeiss LSM510 microscopy. HEK-293, SW480 or DLD1 cells expressing GFP-catenin were bleached by the 488 nm laser line of the 20 mW argon laser at 100% power. About 90% of either nuclear or cytoplasmic GFP signal was bleached. Images were then taken with 35 frames at 25-s intervals. Average intensities in regions of interest were measured using MetaMorph software (Molecular Devices). The intensity in the pre-bleach image was set to 100%, and the first post-bleach image was set as time point 0. The recovery curves shown are averages of at least six cells (at least three independent experiments).66. The transport assay was performed in testing solution  containing an energy-regenerating system  and transport buffer . For β-catenin import to the nucleus, the cells were incubated in the testing solution with the indicated proteins  for 20 min and then fixed with 4% formaldehyde. For β-catenin retention in the nucleus, cells were treated with testing solution and the indicated proteins for 20 min, washed, rinsed with the transport buffer for another 20 min, and then fixed. The GFP intensity of each nucleus was measured by fluorescence microscopy (Olympus IX71). The mean intensity of 40 nuclei was plotted. The intensity of GFP signal in the nuclei incubated with His-GFP-β-catenin and GST-NLS in the import assay was set as 100%.Digitonin-permeabilized HeLa cells were prepared as described previously3 per well) were seeded in 96-well culture plates for 24 h and incubated with MTT  for 2 h. Dimethyl sulfoxide (100 μl) was then added into each well and resuspended until all crystals had been dissolved. The absorbance of the samples was measured with a spectrophotometer at 570 nm67.For MTT -2,5-diphenyl-2-H-tetrazolium bromide) assays, DLD1 or SW480 cells .For colony formation assays, DLD1 or SW480 cells were seeded at a density of 1 × 10Twa1, Axin2, Cyclin D1, GAPDH and U6 are listed: Twa1, 5′-CTGGAAACACTTGATGAACG-3′ and 5′-ATCTCTGTGAGGCACTCTCG-3′ Axin2, 5′-CTGGCTCCAGAAGATCACAAAG-3′ and 5′-ATCTCCTCAAACACCGCTCCA-3′ Cyclin D1, 5′-AGCTCCTGTGCTGCGAAGTGGAA-3′ and 5′-AGTGTTCAATGAAATCGTGCGGG-3′ APC, 5′-GTCCAAGGGTAGCCAAGGATG-3′ and 5′-CATCCTTGGCTACCCTTGGAC-3′ GAPDH, 5′-GCACCACCAACTGCTTA-3′ and 5′-AGTAGAGGCAGGGATGAT-3′ U6, 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′.Single-stranded cDNA was synthesized from total RNA using AMV reverse transcriptase (Takara). qRT-PCR was performed using SYBR Green PCR Master Mix (Takara) and 7500 Real-Time PCR System (Applied Biosystems). The primers for qRT-PCR of Anti-β-catenin (BD Bioscience), Twa1, lamin B (Proteintech), GSK3β, TCF4, Axin1 , Myc, GAPDH, GST, His, HA, Flag (Santa Cruz), actin and α-tubulin (Sigma) antibodies were acquired commercially.Lysates from cells or tissues were prepared in a radioimmunoprecipitation assay (RIPA) buffer  containing 50 mM NaF, 1 mM sodium vanadate, 1 μg/ml leupeptin, 0.1 μg/ml aprotinin and a cocktail of protease inhibitors (Roche), and then subjected to western analyses with the indicated antibodies and IRDye 700CW- or 800CW-conjugated secondary antibodies. The IRDye 700CW or 800CW activities were detected by the LI-COR Odyssey system (LI-COR Biosciences). Cytosolic and nuclear fractions were prepared using a Nuclear Extract Kit (Active Motif) according to the manufacturer's instructions. GAPDH, lamin B, α-tubulin and actin were used as loading controls for western analysis.69. In brief, GST, GST-Twa1, GST-Twa1-ΔLisH, GST-Twa1-ΔCTLH, GST-Twa1-ΔCRA or His-β-catenin expressed in Escherichia coli BL21 were purified and incubated in Tris buffered saline Nonidet P-40 buffer  supplemented with a cocktail of protease inhibitors at 4 °C for 4 h, and then added by glutathione-agarose beads (GST pull-down) or Ni-NTA-agarose beads (His pull-down) for another 2 h. The bound proteins were resolved by SDS-PAGE and subjected to western blotting.Pull-down assays were performed as describedCell lysates were incubated with the indicated antibodies at 4 °C overnight and added with 50 μl Protein A/G Sepharose beads (Santa Cruz) for another 4 h. The immunoprecipitates were washed and then processed for western blotting.For the sequential IP assay, lysates from HEK-293 cells transfected with HA-GSK3β, Myc-Twa1 and Flag-Axin were incubated with anti-Flag antibody bound to protein A/G-agarose beads at 4 °C overnight. The immunoprecipitates were washed and then eluted with Flag peptide for 2 h at 4 °C. The Flag eluates were subsequently incubated with anti-Myc antibody or control IgG at 4 °C overnight and added with protein A/G-agarose beads for another 4 h. The immunoprecipitates were washed and then processed for western blotting.For the ubiquitination assays, the cells were treated with 25 μM MG132 (Sigma) for 2 h before collection and lysed by RIPA buffer with 10 mM N-ethylmaleimide (Sigma) and a cocktail of protease inhibitors. The prepared lysates were then immunoprecipitated with anti-Flag antibody at 4 °C overnight and subjected to western analysis.70. Briefly, HEK-293 cells were harvested in Hank's buffer on ice, pelleted and lysed for 20 min in extraction buffer  containing 3 mM sodium ortho-vanadate, 2 mM phenylmethylsulfonyl fluoride, 100 nM okadaic acid and a cocktail of protease inhibitors. The supernatant was layered on top of a 15%-40% sucrose gradient with a buffer containing 30 mM Tris (pH 7.3), 0.02% Triton X-100, 140 mM sodium chloride, 25 mM sodium fluoride, 3 mM sodium ortho-vanadate and a cocktail of protease inhibitors. Ultracentrifugation was performed in a Beckman SW55 rotor at 100 000× g for 4 h at 4 °C. After centrifugation, fractions were collected from the bottom of the tube and subjected to western analysis.Sucrose gradient sedimentation was carried out as previously described6 cells were prepared with the ChIP assay kit  according to the manufacturer's instructions. The resulting precipitated DNA was analyzed by PCR as described previously71.For ChIP assays, 3 × 1040. Synthetic RNAs were transcribed in vitro using pCS2-Twa1a, Twa1b or Twa1a-ΔCRA vectors with MessageMachine (Ambion). All morpholinos (MOs) were purchased from Gene Tools. The following targeting sequences were used: Twa1a: 5′-CTGGCTTTTCAGCATAACTCATCAT-3′ Twa1b: 5′-CAGGCTTTTCTGAATAGCTCATCACA-3′ β-catenin 2: 5′-AGCCATCGTTGCGTCAATCCTTTAG-3′ standard control: 5′-CCTCTTACCTCAGTTACAATTTATA-3′. The doses of each MO and mRNA for injection were 6 ng and 15 pg, respectively.Wild-type zebrafish (strain AB) were maintained at 28.5 °C using standard protocols. Procedures for animal staging and injection were performed as describedin situ hybridization, the sequences of probes  were amplified from zebrafish cDNAs and cloned into pCS2+ vectors40. All constructs were confirmed by DNA sequencing. The probes were labeled using the Digoxigenin labeling kit (Roche). Whole-mount in situ hybridization was carried out as described previously40.For 6 cells per mouse). Tumor diameters were serially measured with calipers every 5 days, and tumor volumes were calculated using the formula V = (2L×W)/2, where V = volume (in mm3), L= length (in mm), W = width (in mm). The mice were killed and the tumors were isolated at 28 days after inoculation for further analysis.All mouse experiments were conducted in accordance with the Guide for the Care and Use of Animals for research purposes, and were approved by the Committee of Animal Ethics, Zhejiang University. Four-week-old female nude mice were bred in a specific pathogen-free environment in the Animal Facility, Zhejiang University. DLD1 cells were infected by lentiviral particles for 3 days, and then treated with 2 μg/ml puromycin for 1 day. The puromycin-resistant cells were trypsinized, washed and resuspended in PBS/Matrigel (1:1) and subcutaneously injected into the left flank of nude mice . All participants provided written informed consent for this study and the Ethics Committee of Zhejiang University School of Medicine approved the protocol. The clinical data were also obtained from medical records of the above hospitals. The clinicopathological characteristics of included patients were independently evaluated by at least two professional pathologists and summarized in t-tests were used for comparisons between two groups. A linear regression test was used to determine significance of the correlation between nuclear Twa1 and β-catenin levels. A log-rank test was used to determine significance for Kaplan-Meier analysis. χ2-tests were performed to determine significance of the relationship between expression of nuclear Twa1 and clinicopathologic features in CRC patients. A P-value < 0.05 was considered statistically significant.All experiments were repeated at least three times and representative images are shown. The two-tailed Student's TZ, YL and SX designed the experiments. YL, SX, WZ, CZ, CG, QS, YC and ZX performed the experiments. TZ and YL supervised direction of project, conducted experiments, interpretation of data and wrote the manuscript. All authors especially YK discussed the results and commented on the manuscript.The authors declare no competing financial interests.