To explore the modulatory effects and mechanism of secretory clusterin (sCLU) on cancer stem cell (CSC) properties in hepatocellular carcinoma (HCC).The effects of sCLU repression or overexpression on chemoresistance, migration, invasion, and tumor growth were detected by MTT, wound healing, transwell assays, and xenograft assay, respectively. The tumor sphere assay was performed to evaluate the self-renewal ability of HCC cells. In addition, the molecular regulation between sCLU and AKT/GSK-3β/β-catenin axis in HCC cells were discovered by western blotting, quantitative real-time PCR (qRT-PCR), and immunofluorescence. The expression status of sCLU and β-catenin in HCC tissues were investigated by immunohistochemistry.Knockdown or overexpressing sCLU remarkably inhibited or promoted the chemoresistance against sorafenib/doxorubicin, metastasis, and tumor growth of HCC cells, respectively. HepG2 and HCCLM3-derived spheroids showed higher expression of sCLU than that in attached cells. Additionally, repressing sCLU impaired the self-renewal capacity of HCC cells and CSC-related chemoresistance while overexpression of sCLU enhanced these CSC properties. Knockdown or overexpression of sCLU inhibited or increased the expressions of β-catenin, cyclinD1, MMP-2 and MMP-9, and the phosphorylation of AKT or GSK3β signaling, respectively. However, LiCl or LY294002 abrogated the effects mediated by sCLU silencing or overexpression on chemoresistance, metastasis, and CSC phenotype. Furthermore, co-expression of sCLU and β-catenin in HCC tissues indicated poor prognosis of HCC patients.Taken together, the oncogenic sCLU might promote CSC phenotype via activating AKT/GSK3β/β-catenin axis, suggesting that sCLU was a potential molecular-target for HCC therapy. Hepatocellular carcinoma (HCC) accounts for 80–90% liver cancers, which ranks the sixth most common neoplasm and the third in terms of cancer-caused death . PatientIn past decades, accumulating evidence indicated that cancer stem cells (CSCs) within the tumor bulk were implicated in the recurrence, chemoresistance and metastasis of various cancer types, which is the major hurdles for tumor therapy . AcquisiRecent evidence indicated that secretory clusterin (sCLU) was correlated with CSC properties and chemoresistance in breast cancer cells , 11. sCLLiver specimens were collected from 72 patients with HCC underwent hepatotomy at the Affiliated Hospital of Nantong University between January 2007 and September 2010. Clinical information was recorded in detail, including each patient’ clinical factors and 5-year post surgery follow-up. The research included 55 men (76.39%) and 17 (23.61%) women with a mean age of 52.3 years (range 31–74 years). The mean diameter of tumors was 4.36 cm (range 0.8–14 cm), and 28 cases (38.89%) were larger than 5 cm. Alpha-fetoprotein (AFP) was elevated (≥ 50 ng/mL) in 36 (50%) patients. Of the patients, there were 22 cases (30.56%) with metastasis and 7 (9.72%) with portal vein invasion; 56 cases (77.78%) with well and moderate differentiation and 16 (22.22%) with poor differentiation; 43 cases (59.72%) at stages I-II and 29 (40.28%) at stages III-IV. HCC was diagnosed histologically in all patients. The study protocol was approved by the ethical committee of Affiliated Hospital of Nantong University. The informed consent was acquired from each participant according to World Medical Association Declaration of Helsinki .The tissue microarray (TMA) chip was performed by Outdo Biotech Company . 72 cancerous tissues and self-control para-cancerous tissues were involved in the study. After the conformation of tumor area by hematoxylin and eosin (H&E), the target tissues were arrayed into a recipient paraffin block to construct a TMA chip. Immunohistochemistry (IHC) was conducted using Autostainer Universal Staining System . TMAs were deparaffinized in xylene, dehydrated in gradient concentrations of ethanol, followed by incubating in 0.3% hydrogen peroxide solution. After that, TMAs were exposed to 10 mM citrate buffer for antigen retrieval, and then blocked in phosphate buffered solution (PBS) containing 10% goat serum at room temperature. Then TMAs were subsequently incubated overnight in solution containing mouse human anti-sCLU monoclonal antibody  or mouse anti-β-catenin monoclonal antibody  at 4 °C. Next, slides were incubated in a horseradish peroxidase-conjugated secondary antibody  at room temperature for 30 min. At last, TMA sections were visualized by 3,3′-diaminobenzidine , and counterstained by hematoxylin after dehydration and clearing with xylene. Control samples were treated with PBS instead of primary antibody.The IHC results were determined independently by two experienced pathologists under the microscopes , who were blind to each other’s reading. For sCLU, the sum of staining intensity and positive percentages of cells was defined as the final score. In detail, the positive percentages were scored as follows: 0 (0%), 1 for 1% to 33%, 2 for 34% to 66%, and 3 for 67% to 100%; and staining intensity was classified to four categories: 0 for negative, 1 for weak staining, 2 for moderate and 3 for strong staining. Score of 0 to 2 was defined as no or low expression and 3–6 as positive with high expression. The higher score was chosen as the final score in case of a difference between duplicate tissue cores. Meanwhile, it was different for β-catenin: intact membranous expression of β-catenin was defined as negative expression; otherwise, discontinuous membranous staining, cytoplasmic staining, or nuclear staining was regarded as positive expression .2 incubator with humidified atmosphere.HCC cell lines  were purchased from Type Culture Collection of the Chinese Academy of Sciences . Cells were incubated in Dulbecco’s modified eagle medium (DMEM) or Roswell Park Memorial Institute-1640  with solution of penicillin/streptomycin, and 10% fetal bovine serum (FBS) at a 5% COFor sCLU silencing, the pRNAT-U6.1/Neo vector was generated previously by Biomics Company  . The seq3/well) were seeded in 96-well plates. The cells were cultured overnight, followed by incubation with sorafenib or doxorubicin at indicated concentrations for 24 h. Then MTT solution was administrated to each well and incubated for 4 h. Optical density (OD) was determined by using a microplate reader at 490 nm.Cell viability was detected by the MTT assay as described previously . In brieHepG2 and HCCLM3 cells were plated in 6-well plates with 500 cells per well. The cells were cultured overnight, followed by incubation with sorafenib or doxorubicin and cultured for 12 days. Then the colonies were washed by PBS, followed by fixing in 4% paraformaldehyde, and staining in 0.5% gentian violet. Each experiment was independently conducted at least three times.5 cells/mL. Until confluence, a vertical wound was made by using a sterile pipette tip, followed by washing with medium to remove cell debris. Following the initial picture, the plates were incubated and snapped at indicated time points under a microscope . The width of the wound was measured using a scale bar to detect the relative migration of HCC cells.Cells were seeded in 6-well plates at the density of 2 × 105 cells/mL. After incubation for 24 h, cells were rinsed in PBS for 3 times, fixed in 4% paraformaldehyde and stained in 0.5% gentian violet. The invasive cells were visualized and counted in 3 random fields under a microscope .HepG2 and HCCLM3 cells were collected and resuspended in serum-free medium. Transwell chambers  were pre-treated with Matrigel . The lower chamber was added with 600 μL DMEM medium supplemented with 10% FBS, while the upper chambers were plated with HCC cells at the density of 1 × 10®Premix Ex TaqTMII  according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as a reference gene. The melting curves were observed for the non-specific amplification. The messenger RNA (mRNA) expression was determined by using the 2−ΔΔCt method and normalized to respective controls. The primer sequences were as follows. sCLU, Forward (F): 3′-ATCACTGTGACGGTCCCTGTA-5′ and Reverse (R): 3′-TCACTC CTCCCGGTGCTT-5′; CTNNB1, F: 3′-AAGACATCACTGAGCCTGCCAT-5′ and R: 3′-CGATTTGCGGGACAAAGGGCAA-5′; GAPDH, F: 3′-CAAGGTCATCCAT GACAACTTTG-5′ and R: 3′-GTCCACCACCCTGTTGCTGTAG-5′.Total RNA was extracted by using Trizol kit  according to the manufacturer’s instructions. RNA of each sample was subjected into reverse transcription into synthesized complementary DNA (cDNA) with RevertAidTM cDNA Synthesis Kit . Quantitative real-time PCR (qRT-PCR) was conducted by using SYBRTotal protein was extracted by using a radioimmunoprecipitation assay (RIPA) kit  with protease inhibitors. Protein samples were loaded in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring onto polyvinylidene difluoride (PVDF) membranes. Then the samples were blocked with 5% bovine serum albumin (BSA) for 3 h, and incubated in the primary antibody overnight at 4 °C. Following washing with Tris-buffered Saline with Tween (TBST) and incubating in the corresponding secondary antibody, the membranes were detected by the enhanced chemiluminescence (ECL) kit . Antibodies involved in the study were diluted as follows: GAPDH, β-catenin, GSK-3β, and Cyclin D1 ; sCLU ; AKT, p-AKT, p-β-catenin, p-GSK-3β, MMP-2, MMP-9, CD133, CD44, CD90, CD24, and EpCAM ; IgG horseradish peroxidase conjugate .HCCLM3 or HepG2 Cells transfected with shRNA-sCLU or OE-sCLU were plated in Ultra-Low Attachment 24-well plate  and cultured in DMEM/F12  supplemented with 2% B27 , N-2, 20 ng/mL bFGF/EGF . Following the incubation for 2 weeks, the tumor spheres were observed and counted under an inverted microscope .HCCLM3 or HepG2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by blocking in 4% BSA solution, and incubated with primary antibody against β-catenin (dilution ratios: 1: 500) at 4 °C overnight. Then cells were washed and incubated in solution of Alexa Fluor 488 goat anti-rabbit secondary antibody . After being washed for 3 times and stained in 4′,6-diamidino-2-phenylindole  solution for 10 min, the samples were visualized using a fluorescence microscope .6 HepG2 cells transfected with OE-sCLU or control plasmids were suspended in 100 μl PBS and injected subcutaneously into mice. The tumor volume was measured every 4 days and calculated according to the formula: Volume = 0.5 × Length × Width2. At the 35th day after injection, the mice were sacrificed. Each resected tumor was analyzed for sCLU and β-catenin protein levels by immunohistochemistry.The animal study was approved by the Animal Ethics Committee of Affiliated Hospital of Nantong University. Male BALB/c nude mice at the age of 6 weeks were obtained from the animal center of Nantong University. 2 × 10χ2 tests. Survival curves were performed by Kaplan–Meier method with a log-rank test. The analyses of univariate and multivariate Cox regression were conducted to evaluate potential prognostic factors. t test was evaluated between two groups of continuous variables; the comparisons among three groups were conducted by Student’s Newman-Keuls (SNK) test. P value (two-sided tests) less than 0.05 was considered to be statistically significant.The data in this study were presented as mean ± SD and analyzed by the SPSS 19.0 or Stata 12.0 software. Correlations of clinicopathologic variables with sCLU and β-catenin expressions were examined by t = 13.92, P = 0.0002, Fig. t = 6.988, P = 0.0022, Fig. 50: 11.51 µM vs. 4.062 µM) and doxorubicin (IC50: 1.525 µM vs. 0.72 µM) was obviously enhanced following sCLU repression . Moreover, sCLU expression was elevated in advanced clinical grades of HCC .Of the 72 HCC patients, 37(51.39%) were positive for simultaneous expressions of sCLU and β-catenin, 18(25.00%) were single-positive for sCLU, 3(4.17%) were single-positive for β-catenin, and 14(19.44%) were negative for either sCLU or β-catenin expression, respectively. Furthermore, the Spearman rank correlation test revealed a positive correlation between the expressions of sCLU and β-catenin in HCC tissues , multifocal nodules , and advanced TNM stage . No statistically significant correlation was found with AFP, portal vein invasion, differentiation, HBV infection, tumor size, or liver cirrhosis.Correlations of sCLU or/and β-catenin expressions with clinicopathologic features in HCC are presented in Table χ2= 4.433, P = 0.047). On the other hand, β-catenin-positive patients had a poorer OS, although the difference was not significant . Moreover, co-expression of sCLU and β-catenin revealed an obviously shorter overall survival . Furthermore, patients were split into 4 groups according to the expressions of the sCLU and β-catenin: sCLU (+)/β-catenin (+), sCLU (+)/β-catenin (−), sCLU (−)/β-catenin (+), and sCLU (−)/β-catenin (−); the Kaplan–Meier assay showed that patients with sCLU (+)/β-catenin (+) had the poorest OS among the groups .As shown in Fig. P = 0.035).The univariate and multivariate Cox Regression analyses are presented in Table Carcinogenesis of HCC is a complicated, multi-factorial, and multi-staged process, and oncogenes always contribute to tumor progression through varieties of cancer-related pathways . sCLU, aVarious pathways are implicated in the CSC features, of which hyperactivated Wnt/β-catenin pathway plays indispensable roles. Wnt/β-catenin transduction pathway is considered to regulate cell morphology and differentiation in normal condition , 27. HowBased on these results above, sCLU-mediated AKT/GSK3β/β-catenin activation contributed to the malignant behaviors and CSC phenotype of HCC cells. Then we further explored expression characteristics of sCLU and the key factor of Wnt pathway in HCC tissues. sCLU and β-catenin had a parallelly upregulated expression trend in tissues at advanced stages than in para-cancerous tissues or early stages, indicating that both of them were involved in HCC staging. However, despite that the prognostic value of the two proteins was previously reported, the current analysis found that neither of sCLU nor β-catenin alone was an independent prognostic marker. We speculated that the discrepancy might be attributed to the sample size and differences in evaluation of IHC. Interestingly, the subset of HCC patients with simultaneous expression had poorest prognosis compared with other patients, further indicating sCLU and β-catenin contributed to the progression of HCC. Given above, it was speculated that there might be a positive correlation between sCLU and AKT/GSK-3β/β-catenin pathway during HCC progression, which could be activated to cause CSC phenotype and other malignant behaviors Fig. .Fig. 7HyIn conclusion, our study demonstrated for the first time that sCLU activated AKT/GSK3β/β-catenin axis, followed by inducing chemoresistance, metastasis, CSC phenotype, and even poor prognosis of HCC patients. While more assays should be conducted to validate the exact mechanisms, the study sheds lights on the novel approach to block liver CSC phenotype and an attractive strategy for HCC therapy.Additional file 1: Fig. S1. sCLU regulated the expression of CSC markers of HCC cells. A, the expression of CSC markers in HCCLM3 cells transfected with NC or sh-sCLU were detected by western blotting. B, relative protein intensities in A were detected by Image J software. C, the expression of CSC markers in HepG2 cells transfected with NC or OE-sCLU were detected by western blotting. D, relative protein intensities in C were detected by Image J software. GAPDH was used as a loading control. **P < 0.01; *P < 0.05. Fig. S2. The expression features of sCLU and β-catenin in HCC tissues. A, sCLU staining scores in HCC and para-cancerous tissues. B, β-catenin staining scores in HCC and para-cancerous tissues. C, positive ratio of sCLU and β-catenin expression in HCC tissues at different TNM stages. *P < 0.05, **P < 0.01. TNM, tumor-node-metastasis.


Results demonstrated an elevated expression of key components of Wnt/β-catenin signaling and NOX4 in the lungs of silicon dioxide- (SiO2-) induced silicosis mice. Furthermore, the activated Wnt/β-catenin and NOX4 signaling are accompanied by an inhibition of cell proliferation, an increase of ROS production and cell apoptosis, and an upregulation of profibrogenic factors in BEAS-2B human lung epithelial cells exposed to SiO2. A mechanistic study further demonstrated that the Wnt3a-mediated activation of canonical Wnt signaling could augment the SiO2-induced NOX4 expression and reactive oxygen species (ROS) production but reduced glutathione (GSH), while Wnt inhibitor DKK1 exhibited an opposite effect to Wnt3a. Vice versa, an overexpression of NOX4 further activated SiO2-induced Wnt/β-catenin signaling and NFE2-related factor 2 (Nrf2) antioxidant response along with a reduction of GSH, whereas the shRNA-mediated knockdown of NOX4 showed an opposite effect to NOX4 overexpression. These results imply a positive feed forward loop between Wnt/β-catenin and NOX4 signaling that may promote epithelial-mesenchymal transition (EMT) of lung epithelial cells in response to an exposure of silica dust, which may thus provide an insight into the profibrogenic role of Wnt/β-catenin and NOX4 crosstalk in lung epithelial cell injury and pathogenesis of silicosis.Silicosis is a chronic fibrotic lung disease caused by the accumulation of silica dust in the distal lung. Canonical Wnt signaling and NADPH oxidase 4 (NOX4) have been demonstrated to play a crucial role in the pathogenesis of pulmonary fibrosis including silicosis. However, the underlying mechanisms of crosstalk between these two signalings are not fully understood. In the present study, we aimed to explore the interaction of Wnt/ Similarilicosis , 11–13. s models –16. ThesIn addition to the dysregulation of cellular signaling activities, the inhalation of silica dust also causes oxidative stress by the production of reactive oxygen species (ROS) that contributes to chronic airway inflammation and epithelial cell injury . Indeed,2O2 after a catalase activity [NOX4-deficient mice developed significantly less bleomycin-induced pulmonary fibrosis and alveolar epithelial cell death [The major ROS are generated by NOXs, membrane-bound enzyme complexes present in both phagocytes and nonphagocytic cells . There aactivity , 24. Moractivity , 26. Thell death , suggestll death . Mechanill death , 25.β-catenin signaling in pulmonary fibrotic procession, and involvements of silica-induced ROS production in pathogenesis of silica-related diseases, we hypothesized that the interaction between the NOX4 and Wnt/β-catenin signaling may contribute to the pathogenetic process of silica-related lung disease. However, the link between Wnt/β-catenin signaling and NOX4-mediated ROS in the development of silicosis has not been established. Here, we demonstrate that a positive feed forward loop of NOX4 and Wnt/β-catenin signaling promotes the fibrotic property in airway epithelial cells in response to silica dust exposure.In view of the widespread evidence for hyperactivated NOX4 and Wnt/2, mesoporous, 2 μm particle size, CAS number 7631-86-9) and silicon dioxide  were products of Sigma-Aldrich . The SiO2 particles were baked at 200°C for 2 h to inactivate endotoxin prior to being suspended in saline at a concentration of 100 mg/mL. The SiO2 saline stock was further dispersed for 15 min in a water bath sonicator followed by being triturated through a 25G needle before use as described in the previous report [Silica . For the generation of silicosis lungs in mice, the protocol, dose, and delivery method described in the previous study were employed with a slight modification [ad libitum at Ningxia Medical University . The male and female mice were randomly divided into two groups : (1) saline control group: mice were intratracheally instilled 50 μL of saline and (2) silica group: animals were intratracheally instilled 50 μL of 50 mg/mL silica in saline . The mice were euthanized at 2 weeks (14 days) after the exposure of silica dust for pathohistological and molecular analysis [The protocol and use of mice were approved by the Laboratory Animal Committee of College of Life Science at Ningxia University, in accordance with guidelines of the National Institutes of Health fication . Twelve analysis .2) at 37°C. When cells reached ~80% confluence, they were utilized for infection with adenoviral vectors and/or exposed to different concentrations of SiO2 dust for various time periods for analysis. Adenoviral vectors expressing mouse Wnt3a (AdWnt3a), Ad.shRNA-NOX4 and adenoviral backbone vector control (AdC) were kindly provided by Dr. John F. Engelhardt at the University of Iowa  [2 (2 μm in size) at a concentration of 100 μg/cm2. The cells were then harvested for analysis. For the treatment of ROS scavenger N-acetyl-L-cysteine (NAC), cells were refreshed with media containing 10 mmol/L of NAC for 2 h before SiO2 was added into the culture media. The cells were then continuously cultured in the presence of NAC for an additional 24 h or 48 h prior to being harvested for analysis.A human bronchial epithelial cell line, BEAS-2B (ATCC CRL-9609), was purchased from American Type Culture Collection (ATCC) . Cells were cultured in DME/F12 (50%/50% volume) basal medium (HyClone) supplemented with 10% Fetal Bovine Serum (FBS)  in a humidified atmosphere of 95% air-5% carbon dioxide  . Adenovi3/well) were seeded in a 96-well plate and grown overnight prior to exposure to silica dust (2 μm particle size) at a density of 0, 50, 100, 150, or 200 μg/cm2 for 24 or 48 h. Subsequently, 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for an additional 2 h. The absorbance of wavelength at 450 nm was read on a microplate reader . The relative cell viability was expressed as the percentage of (OD2−cellsSiO − OD2−mediumSiO)/(ODcells − ODmedium) × 100, where the OD2−cellsSiO, OD2−mediumSiO, ODcells, and ODmedium represented the values of OD450 nm of wells of SiO2-treated cells, medium containing SiO2, untreated control cell culture, and blank medium alone, respectively. All experiments were performed with biological triplicates, and data were representative of at least three independent experiments.The cell viability was accessed using the Cell Counting Kit- (CCK-) 8 as per the manufacturer's instruction . Briefly, the cells . The nuclear protein was extracted with a NE-PER™ Nuclear Extraction kit . The total protein of the mouse lung was isolated by homogenizing the tissue in Enhanced RIPA Lysis Buffer , followed by centrifugation. The cell lysate or homogenized lung tissue was then centrifuged at 12,000 × rpm for 20 min at 4°C; the supernatants were collected as total proteins. The concentration of protein was detected using the BCA Protein Assay Kit . The proteins (30 μg) were resolved by 8%-10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel (SDS-PAGE) and transferred to a PVDF membrane . The membrane was then blocked in 5% nonfat milk in TBS for 1 hour at room temperature (RT). The protein of interest was probed with its specific antibodies, and the blots were then developed using the enhanced chemiluminescence (ECL) reagent  as described elsewhere. The levels of protein expression were semiquantified by optical densitometry using ImageJ Software version 2.0.0 (http://rsb.info.nih.gov/ij/). The ratio between the net intensity of each sample divided by the GAPDH internal control was calculated as a densitometric arbitrary unit (A.U.), which served as an index of the relative expression of the protein of interest. The use and information of primary antibodies employed in the present study are listed in Suppl. Tables The total protein of cells was extracted from cells treated with 100 μm frozen tissue sections. For IF staining cells on coverslips, cells were seeded in the Glass Bottom Cell Culture Dish (diameter of 15 mm) at a density of 5 × 104 cells/dish and cultured overnight prior to exposure to silica dust (100 μg/cm2) for an additional 24 h or 48 h. Then, the cells were fixed with 4% PFA for 15 min. For IF staining, the 4% PFA-fixed slides were permeabilized by 0.2% TritonX-100/PBS for 20 min at RT, followed by being blocked with 5% normal donkey serum in PBS for 1 h at RT. The appropriately diluted primary antibody to the protein of interest was then applied to the section and incubated at 4°C overnight. The binding of primary antibody was detected by Alexa Flour fluorescence (488 or 565)-conjugated secondary antibodies. The EdU corporation assay was performed using the Click-iT™ EdU Cell Proliferation Kit as per the manufacturer's instruction . The slides were mounted with VectShield with DAPI medium  for visualizing and imaging using a Leica TCS SP2 A0BS Confocal System and processed on Leica Confocal Software v.2.6.1 .In order to histologically examine the pathology of the lungs of mice exposed to silica, the lung tissue of mice was fixed in 10% neutral formalin and processed paraffin embedding and section for pathohistological analysis by hematoxylin and eosin (HE) staining. Meanwhile, a part of the lung tissue was fixed in 4% paraformaldehyde (PFA) in PBS for 2 days prior to being embedded in an Optimal Cutting Temperature (OCT) compound for immunofluorescence (IF) staining on 10 5 cells/well) cultured in a 6-well plate were infected with adenoviral vector for 24 h and/or treated with or without silica for an additional 48 h before the intercellular ROS was assessed by a flow cytometry assay. Briefly, the cells were dissociated from the plate and washed with 1x PBS, followed by being incubated with the CellROX® Orange Reagent  at a final concentration of 5 μM that was prediluted in phenol red-free DMEM in the dark at 37°C for 30 min. After the incubation, the CellROX® Orange Reagent solution was removed, and the removed medium and the cells were washed with PBS three times prior to being resuspended in PBS for flow cytometry analysis on a BD FACSCanto II. At least 10,000 events were analyzed for each condition. For ROS staining, cells were incubated with a 5 mmol/L CellROX® Orange Reagent in the dark at RT for 30 min, before they were imaged under fluorescence microscopy. All experiments were performed with biological triplicates, and data are representative of at least three independent experiments.Cells . The reduced GSH were normalized by protein concentrations. All experiments were performed with biological triplicates, and data are representative of at least three independent experiments.Cells were rinsed with PBS and lysed with 1% TritonX-100 in PBS for 30 min at 4°C. The lysis was collected and centrifuged at 3500 rpm for 10 min. The supernatant was harvested for analysis. A total of 100 β-catenin activity, BEAS-2B cells cultured in 12-well plates were cotransfected with TCF Reporter Plasmid (TopFlash) and pCMV-renilla luciferase plasmid  at a ratio of 50 : 1 using X-tremeGENE HP . The cells were exposed to silica dust (2.0 nm size) at 24 h post the transfection and continued to culture for an additional 24 h or 48 h before they were harvested for analysis. The cells were lysed in 1x Passive Reporter Lysis Buffer (Promega). Protein concentrations were determined using the Bradford method, and all lysates were normalized to the same protein concentration using the lysis buffer. Two microliters of normalized cell lysate was used for measurement of the relative luciferase activity units (RLU), for both firefly and renilla luciferase, using the dual luciferase assay kit (Promega). Transfection efficiencies were normalized by dividing the relative firefly luciferase units by the relative renilla luciferase units. Following normalization, values were represented as RLU. All experiments were performed with biological triplicates, and data are representative of at least three independent experiments.The Wnt signaling TCF Reporter Plasmid (TopFlash) was a product of Millipore , and the pCMV-renilla luciferase plasmid was purchased from Promega . In order to access the Wnt/p < 0.05.All of the experiments were performed for at least three biological repeats. Data are presented as the mean ± standard error of the mean (SEM). All analyses were assessed using GraphPad Prism version 5 software . Statistical significance was defined as β-catenin signaling cascade and NOX4 protein and the fibrotic and fibrogenic factors in the lungs of SiO2-induced silicosis mice were ascertained by immunoblotting (IB) and immunofluorescent staining (IF) assays. The mice that intratracheally received silica dust exhibited abundant silicosis nodules in the parenchyma of lungs as evaluated by HE histological staining , and Axin2 but decreased Wnt inhibitor DKK1 protein  and vimentin, in lungs exposed to silica  assay  assay . These rβ-catenin signaling activity and NOX4 expression of BEAS-2B lung epithelial cells in response to silica dust was examined. The cell viability assay suggested a 50% decrease of cell viability in lung epithelial BEAS-2B cells exposed to 200 μg/cm2 of SiO2 and revealed a dose-dependent inhibition of cell proliferation in cells exposed to SiO2 for 24 h and 48 h in a range of 0-200 μg/cm2 (2 (100 μg/cm2) led to a significantly enhanced activation of Wnt/β-catenin signaling in BEAS-2B cells compared to the saline control (p < 0.01), as assessed by IF staining of Wnt3a ligand in the cytoplasm and ABC in nuclei , which was corroborated by the IF staining  or myofibrogenesis of lung epithelial cells is a hallmark of pulmonary fibrosis, a feature of silicosis , the alt0 μg/cm2 . As expen nuclei , the Wntignaling , and immignaling . The elestaining  and IB astaining .2 in the induction of ROS production and EMT of epithelial cells, we examined the expression of NOX proteins and EMT-related molecules in cells exposed to silica for both 24 h and 48 h, although a significant reduction of cell viability was induced by the exposure of SiO2 at 100 μg/cm2 for 48 h. As expected, a dose-dependent increase of ROS production was observed in BEAS-2B cells treated with SiO2 for 48 h, but not 24 h post the exposure of silica dust relative to the saline  (10 mmol/L) exhibited a capacity to diminish SiOAC alone  . Notabl2B cells . Of note Figures . The pre Figures , suggestβ-catenin signaling and NOX4 in lung epithelial cells, the signaling activity was altered in BEAS-2B cells by the infection of adenoviral vector expressing mouse Wnt3a (AdWnt3a) or DKK1 (AdDKK1), and the change of ROS production in response to SiO2 exposure was measured. The IB assay showed a robust expression of Wnt3a and DKK1 proteins in cells infected with AdWnt3a and AdDKK1, along with an increase and decrease of ABC protein in comparison to cells infected by control AdC vector, respectively  or short hairpin RNA (shRNA) to NOX4 gene (AdshRNA). As expected, an increased abundance of NOX4 protein was found in cells infected with AdNOX4, and a decreased NOX4 protein was detected in cells infected with AdshRNA, suggesting that these adenoviral vectors were capable of overexpressing and knocking down NOX4 in BEAS-2B cells  in BEAS-2B cells, but the expression of Nfr2-regulated gene heme oxygenase-1 (HO-1) has not altered the ectopic expression of NOX4  antioxidant response was involved in the crosstalk between the Wnt/β-catenin and NOX4 signaling in BEAS-2B cells in response to SiO2 challenge.In the present study, the interaction between the Wnt/β-catenin-mediated canonical signaling and β-catenin-independent noncanonical signaling were altered in human airway epithelial cells upon silica stimulation; the Wnt inhibitor SFRP1 and noncanonical ligand Wnt5a were downregulated, while another Wnt inhibitor DKK1 was upregulated [β-catenin signaling was reactivated in silicosis lungs [β-catenin signaling by shRNA to β-catenin [β-catenin signaling was also reported in silicosis rats that were administrated with rat bone marrow mesenchymal stromal cells (BMSCs) [β-catenin signaling, along with the increased abundance of NOX4 and profibrogenic proteins but reduced DKK1, was also observed in a SiO2-induced silicosis mouse lung, and BEAS-2B human lung epithelial cells exposed to SiO2.Wnt signaling is pivotal to lung development and homeostatic maintenance of the mature lung, by mediating stem cell self-renewal, turnover, and injury/repair of epithelia , 35. Howegulated , despiteis lungs , 15. The-catenin , 15 disp-catenin . Such at (BMSCs) . In lineNOX4 exhibited a significantly less severe fibrotic phenotype in the lungs of a bleomycin-induced pulmonary fibrosis mouse model [2 stimulations. Of note, the shRNA-mediated knockdown of NOX4 led to a significant decrease of SiO2-induced profibrogenic molecules  in lung epithelial cells.Repeated oxidative stress is one of the detrimental factors of lung injury. The NOX-mediated overproduction of ROS due to excessive stimulation of proinflammatory cytokines or environmental insults such as silica dusts causes a major part of oxidative stress in the lungs. Among different isoforms of NOXs, the pathophysiological roles of NOX4 isoform have a great implication in lung epithelial cell death, (myo)fibroblast differentiation, and collagen deposition , 25, 41.se model . In thisβ-catenin and ROS in the regulation of cell proliferation and differentiation [β-catenin was able to modulate redox regulatory protein p66(Shc), which in turn regulate a NOX4-mediated ROS and ultimately lead to vascular endothelial dysfunction [β-catenin in the endothelium resulted in an increased vascular ROS production and endothelial dysfunction [β-catenin signaling in mouse extraembryonic endoderm patterning; a sustained exposure of H2O2 (ROS) enhanced Wnt/β-catenin activity in retinoic acid-treated F9 teratocarcinoma cells and promoted cell differentiation into primitive endoderm, while exposure of these cells to antioxidant NAC impeded cell differentiation [β-catenin signaling was also observed in podocyte dysfunction, and a podocyte-specific knockout of β-catenin protected podocytes from injury and albuminuria induced by advanced oxidation protein products in mice. Mechanistically, the Wnt/β-catenin exerted its function by inducing the receptor of advanced glycation end products- (RAGE-) mediated NOX expression, ROS production, and activation of nuclear factor-kappaB (NFκB) [β-catenin was also reported in intestinal and colon epithelial cells, in which the NOX1 regulated Wnt signaling in a redox-dependent manner [β-catenin and NOX4 was also found in BEAS-2B lung epithelial cells in response to silica dust exposure. The Wnt3a-activated Wnt/β-catenin signaling augmented NOX4 expression and ROS production and reduced GSH production, accompanied by an increased expression of profibrogenic proteins; conversely, the DKK1-mediated inhibition of Wnt/β-catenin exerted an opposite effect of Wnt3a on lung epithelial cells exposed to SiO2. Vice versa, an overexpression of NOX4 enhanced Wnt/β-catenin signaling activity, along with an augmentation of profibrogenic proteins in cells. NOX4 also reduced GSH production and increased the expression of Nrf2, a key intracellular antifibrotic factor that maintains the homeostasis of ECM [An increasing number of evidences have demonstrated a crosstalk of Wnt/ntiation –46. For function . In thisfunction . In contntiation . SimilarB (NFκB) . Such a s of ECM , while ts of ECM . It is wβ-catenin-mediated decline of reduced glutathione (GSH) was also determined in lung epithelial cells, which was in line with findings in lens epithelial cells [β-catenin and NOX4 promotes the EMT and fibrogenesis in lung epithelial cells in response to the exposure of silica dust.In addition, reduced glutathione (GSH) is a ubiquitous tripeptide thiol, which has been recognized as a protective antioxidant against oxidative stresses . A defical cells , zebrafial cells , and mural cells . Taken t2 particles. The in vivo results demonstrated an enhanced activity of Wnt/β-catenin and NOX4 signaling in SiO2-induced mouse silicosis lungs. The in vitro study revealed the inhibition of cell proliferation, along with enhanced Wnt/β-catenin signaling activity, increased ROS production, expression of NOX4, and profibrogenic proteins in BEAS-2B cells exposed to SiO2. A molecular study further demonstrated the crosstalk between canonical Wnt/β-catenin signaling and NOX4 in lung epithelial cells exposed to silica dust; an activation of Wnt/β-catenin signaling increased NOX4 and ROS production but reduced GSH; vice versa, an increased NOX4 enhanced the canonical Wnt signaling. Mechanistically, the Nrf2-mediated antioxidant activity may be involved in the interaction between the Wnt/β-catenin and NOX4 signaling and play roles in the EMT and fibrogenic processes of lung epithelial cells in response to a silica exposure (β-catenin and NOX4 signaling promotes EMT property in BEAS-2B cells exposed to silica stimulation, which thus emphasize the profibrogenic role of the crosstalk of Wnt and NOX-mediated ROS in lung epithelial cell injury and development of silicosis, which warrants further investigations.Collectively, in the present study, the crosstalk between canonical Wnt signaling and NOX4 was investigated in BEAS-2B human lung epithelial cells in response to SiOexposure . These f


In time-based visual selection, task-irrelevant, old stimuli can be inhibited in order to allow the selective processing of new stimuli that appear at a later point in time (the preview benefit; Watson & Humphreys,  Their ages ranged from 18–25 years . All participants reported normal or corrected to normal visual acuity in this and the remaining experiments.Stimuli were presented on a 22” LCD panel at a resolution of 1680 × 1050 pixels. A custom written computer program generated the stimuli and recorded participants’ responses. The target was a vertical rectangle defined by Kanizsa-type illusory contours and the distractors were horizontal Kanizsa-type rectangles displayed against the white background of the computer monitor. Four black pacman shapes formed the Kanizsa-type rectangles that measured 25.2 × 37.8 mm . Each pacman had a diameter of 16.8 mm (1.69°)There were three main conditions: a half-element baseline (HEB), a full-element baseline (FEB) and a preview condition. A trial in the FEB condition consisted of a blank screen for 500 ms, followed by a fixation cross for 750 ms, after which a search display of 4, 8, or 16 items was presented. Search displays remained visible until the participant indicated the location of the target by pressing the Z key if the target was on the left side of the display, or the M key if it was on the right side of the display, on a standard computer keyboard  catch trials on which there was no target (the target was replaced by a distractor). Participants responded to these trials by pressing the space bar on the keyboard. The purpose of the catch trials was to ensure that participants did not search only half of the display, by concluding that the target was on the opposite side if not present on the display side they searched  in the preview condition was compared with that in the two baseline conditions. In the FEB and preview conditions, slopes were calculated using the actual display size. In the HEB condition, slopes were calculated using twice the true number of items. The search rate in the HEB then represents the time that would be needed to search through only the new items in the preview condition. Therefore, if search in the preview condition corresponds to that of the HEB, the old items have been fully excluded from search. However, if search rates in the preview condition match those of the FEB, the old items have not been ignored and were included in the search.F = 42.92, MSE = 41023.37, p < .001, display size, F = 226.86, MSE = 47031.34, p < .001, and a significant Condition × Display Size interaction, F = 10.48, MSE = 9494.61, p < .001. As shown in Fig. Trials with RTs less than 200 ms or greater than 10 s were removed as outliers (0.01% of the data) and catch trials were also removed. Search slopes are presented in Fig. F2,4 = 42.92Following previous work, we compared search efficiency in the preview condition with that in each of the two baselines in order to determine if a preview benefit had occurred. In addition, given the apparent difference between efficiency at large and small display sizes, we also assessed search efficiency at the smaller (4–8) and larger (8–16) display sizes individually  = 9.59, MSE = 23785.68, p < .01, and increased with display size, F = 200.98, MSE = 33157.56, p < .001. The Condition × Display Size interaction was also significant, F =10.92, MSE = 8203.22, p < .005, indicating that search was less efficient overall in the preview condition than in the HEB. Considering only small display sizes (4–8 items), RTs were shorter for a display size of four than eight, F = 204.63, MSE = 3556.32, p < .001, however, neither the main effect of condition, F = 3.82, MSE = 10136.62, p = .067, nor the Condition × Display size interaction proved significant, F < 1. Considering only large display sizes (8–16 items), RTs were longer for a display size of 16 than eight items, F =186.77, MSE = 16052.18, p < .001. Overall RTs were longer in the preview condition than in the HEB, F = 9.47, MSE = 25057.28, p < .01, and RTs increased more from eight to 16 items in the preview condition than in the HEB, F = 14.20, MSE = 5883.53, p < .005.Overall RTs were longer in the preview condition than in the HEB, F = 38.29, MSE = 46404.27, p < .001, increased with display size, F = 179.25, MSE = 44849.85, p < .001, and the Condition × Display size interaction was also significant F = 5.09, MSE = 8742.74, p < .05. Considering small display sizes (4–8 items), RTs were faster overall in the preview condition than in the FEB, F = 47.87, MSE = 23011.23, p < .001, and were faster for a display size of four than of eight items F = 106.04, MSE = 12367.99, p < .001. There was also a significant Condition × Display Size interaction, F = 11.95, MSE = 7067.09, p < .005, indicating more efficient search in the preview condition. At the large display sizes (8–16 items), overall RTs were shorter in the preview condition than in the FEB, F = 31.16, MSE = 50391.45, p < .001, and increased between eight and 16 items, F = 185.20, MSE = 20186.19, p < .001. However, the Condition × Display Size interaction did not approach significance, F = 1.04, MSE = 7304.89, p = .323.Overall RTs were shorter in the preview condition than in the FEB, F = 12.28, MSE = 5.43, p < .001, the number of errors increased with display size, F = 24.74, MSE = 11.39, p < .001, and there was a significant Condition × Display Size interaction, F = 8.09, MSE = 7.42, p < .001.Overall error rates were low (2.75%) and as shown in Table F = 2.51, MSE = 1.87, p = .096, or display size, F = 3.83, MSE = 5.45, p = .067, nor the Condition × Display Size interaction, F < 1, reached significance. At large display sizes (8–16 items), there was a significant main effect of condition, F = 12.22, MSE = 8.09, p < .001, display size, F = 28.77, MSE = 11.02, p < .001, and a significant Condition × Display Size interaction, F = 8.71 , MSE = 10.13, p < .005; there was a greater number of errors in the preview condition at the largest display size followed by the FEB and HEB conditions. Given that there was no reliable difference in (RT-based) search efficiency between FEB and preview at large set sizes, the conclusions based on the RT data were not compromised by a speed–accuracy trade-off. Indeed, the error rates further suggest the lack of a preview benefit at the larger display sizes. More errors in the preview at large display sizes in comparison to FEB may also be indicative of greater resource use in the preview condition, as a consequence of trying to suppress the previewed items as well as performing perceptual grouping.Given that most errors were found in the preview condition but that different search patterns were found at small and large display sizes, we conducted an analysis for each display size separately to identify any speed/accuracy trade-offs. For small display sizes of four and eight items, neither the main effects of condition, The overall error rate on catch trials was low (2.62%), confirming that participants were searching over the whole display. Given the small number of catch trials, these data were not analyzed further.The search slopes in the FEB condition numerically replicate those of Li and colleagues , providiThese findings lend support to high-level accounts for both time-based visual selection . Instead, such overall differences could be the result of changes in alertness, the presence of a warning signal or arousal effects  from the University of Warwick, aged 18–49 years  who participated for course credit or payment.The stimuli, apparatus and procedure were similar to those of Experiment F = 23.02, MSE = 47916.74, p <.001. Follow-up t- tests showed that overall RTs in the preview condition were significantly longer than in the HEB, t(17) = 2.79, p < .05, but significantly shorter than in the FEB, t(17) = 4.56, p < .001, and overall FEB RTs were longer than overall HEB RTs, t(17) = 6.37, p < .001. There was also a main effect of display size, F = 415.51, MSE = 14437.65, p < .001. However, of most interest, the Condition × Display Size interaction did not approach significance, F = 1.48, MSE = 5521.38, p = .218, suggesting no reliable difference in search rates between the three conditions.Outlier RTs less than 200ms or greater than 10s were removed from the analysis (0.01% of the data) as well as catch trials. Search slopes are presented in Fig. Errors. Errors were low overall (1.93%) and are presented in Table F = 18.74, MSE = 11.33, p < .001. There was no main effect of condition, F = 2.38, MSE = 7.88, p = .107, and no Condition × Display size interaction, F = 1.22, MSE = 5.41, p = .313. Error rates on catch trials were low (3.70%) and were not analyzed further. overall .93% and Experiment However, the results were unexpected—measured in terms of search slopes, there was no reliable difference in search efficiency across all three conditions. That is, search was as efficient in the FEB condition as it was in the HEB condition. This suggests that the set of stimuli formed from illusory contours could be selectively searched and that the simultaneous presence of non-illusory-perceptual groups of pacmen stimuli were not considered in the search process. In other words, even in standard space-based search conditions, the Kanizsa-type illusory stimuli could be segmented from the other items and prioritized. This provides possibly the first demonstration in preview search conditions where separating stimuli in time and space produces no benefit in search rates over the FEB condition, because search in the FEB is already as efficient as search in the HEB condition. One might note however, that although search in the FEB was just as efficient as in the HEB, RTs were nonetheless longer in the FEB condition. This might reflect an overall attentional cost of illusory contour formation that occurs before the onset of search. Similar to the large preview display sizes in Experiment 1, overall search times in Experiment 2 were shorter in the preview condition than in the FEB condition. However, this is not indicative of a preview benefit as it does not show differences in search efficiency between the two conditions but rather is suggestive of a faster onset of search. A faster search onset in preview conditions is likely due to changes in alertness, the presence of a warning signal or arousal effects . As with our predictions for Experiment 2, one possibility is that groups of non-illusory contour forming pacmen will consume fewer resources and this will allow a preview benefit to occur even at the large display sizes. The other possibility which has not been explored in previous studies with Kanizsa-type illusory stimuli, is that inefficient search patterns for Kanizsa-type illusory contours , aged 18 to 20 years  completed the experiment for course credit or payment.The stimulus displays, apparatus, and procedure were similar to those of Experiment 1 and 2, except that all pacmen forming the distractor stimuli were randomly oriented so that they did not form an illusory percept. The target was defined by a stimulus in which all the pacmen were oriented rightward see Fig. .Fig. 7ScA total of 0.14% outlier RTs that were less than 200 ms or greater than 10 s, as well as catch trials were excluded from the analysis. Search slope statistics are presented in Fig. F = 26.87, MSE = 85173.76, p < .001, display size, F = 261.18, MSE = 44179.47, p < .001, and a significant Condition × Display Size interaction, F = 6.21, MSE =17344.91, p < .001, indicating a difference in search rates across the conditions. Following Experiment 1, search efficiency in the preview condition was compared with both baselines and over a range of display sizes.A 3 × 3  within-subjects ANOVA revealed significant main effects of condition, F = 4.12, MSE = 62550.55, p = .058, a significant effect of display size, F = 283.05, MSE =16237.69, p < .001, and a significant Condition × Display size interaction indicating that overall, search in the preview condition was less efficient than in the HEB, F = 5.05, MSE =15406.95, p < .05. Considering only the small display sizes (4–8 items), RTs increased with display size, F = 137.39, MSE = 8247.92, p < .001. However, neither the main effect of condition, F = 2.49, MSE = 21928.41, p = .133, nor the Condition × Display Size interaction, F < 1, were significant. At the larger display sizes there was a trend for an effect of condition, F = 4.30, MSE = 59898.34, p = .054, and RTs increased with display size, F = 220.19, MSE = 16850.81, p < .001. There was also a significant Condition × Display Size interaction, F = 5.63, MSE = 12869.78, p < .05, indicating that, at large display sizes, search was less efficient in the preview condition than in the HEB.There was a trend towards significance regarding the main effect of condition, F = 23.53, MSE = 101612.09, p < .001, and increased with display size, F = 241.79, MSE = 26369.39, p < .001. The Condition × Display Size interaction was not significant, F = 2.51, MSE = 15845.67, p =.097. However, given past results and the findings from Experiment 1, we would expect search in the preview condition to be more efficient than in the FEB  and so there is some justification for treating this test as directional, in which case it would be significant at the .05 level.Overall RTs were shorter in the preview condition, F = 24.54, MSE = 54765.33, p < .001, and increased with display size, F = 69.09, MSE = 23975.51, p < .001. The Condition × Display Size interaction was also significant, F = 5.86, MSE = 8096.91, p < .05, indicating more efficient search in the preview condition than in the FEB. Considering the larger display sizes (eight and 16 items), preview search produced shorter overall RTs, F = 20.45, MSE = 99003.31, p < .001, and RTs increased with display size, F = 334.55, MSE = 15013.83, p < .001. However, the Condition × Display Size interaction did not approach significance, F < 1, providing no evidence for the presence of a preview benefit at the larger display sizes.At the smaller display sizes, overall RTs were shorter in the preview condition, F = 4.86, MSE = 20.56, p < .05, and error rate increased with display size, F = 42.93, MSE = 17.75, p < .001. Errors increased the most with display size in the preview condition, followed by the FEB, and then the HEB, F = 4.86, MSE = 9.64, p < .005. As in Experiment 1, we conducted additional analyses treating small and large display sizes separately. At small display sizes, errors increased with display size, F = 4.72, MSE = 6.99, p < .05. However, neither the main effect of condition, F< 1, nor the Condition × Display Size interaction, F = 1.39, MSE = 5.39, p = .26 proved significant. At large display sizes, more errors were made in the FEB and preview condition than in the HEB, F = 6.31, MSE = 22.75, p < .01, and error rates increased at the largest display size, F = 43.91, MSE = 21.26, p < .001. There was also a significant Condition × Display Size interaction, F = 3.95, MSE = 12.58, p < .05, indicating that errors increased most in the preview condition at large display sizes. The overall error rate on catch trials was low (4.78%) and these data were not analyzed further.Error rates were low overall (4.23%) and showed a similar pattern to the RT data . Thus, here the perception of illusory contours had little overall impact even in standard visual search task conditions.In Experiment 4, we consider how local transient changes to grouped elements might influence the preview benefit obtained at small display sizes. This is theoretically interesting because it allows us to clarify whether inhibition is applied to the individual pacmen or holistically to the grouped representations.In Experiment 4, we examined the effect of making changes to the individual elements that form a perceptually grouped stimulus. In the preview condition, the placeholders were initially randomly rotated and did not elicit illusory contours. They were then subsequently rotated to form illusory contour rectangles when the second set of items was added. Thus, the final search display in the preview condition was comprised of illusory stimuli similar to those of Experiment 1. Previous research has shown that changes to the identity of previewed items can disrupt the preview benefit, whereas changes to surface properties, such as stimulus color or luminance, do not , aged 18–25 years  completed the experiment for course credit or payment. All had normal or corrected to normal vision.The stimuli, apparatus and procedure were similar to those of Experiment 1 except that in the preview condition, the previewed pacmen were initially orientated randomly. After 1s, the pacmen rotated so as to form Kanizsa figures, simultaneously with the onset of the second set of search items. The second set of items in the preview condition, as well as in the HEB and FEB, remained the same as in Experiment 1. That is, they consisted of horizontal Kanizsa-type rectangle distractors and a vertical Kanizsa-type rectangle target see Fig. .Fig. 10SRTs less than 200 ms or greater than 10s were removed from the analysis as outliers (0.08% of the data), as well as catch trials. Search slopes are shown in Fig. F = 51.75, MSE = 44306.64, p < .001, and that RTs increased with display size, F = 208.88, MSE = 68172.95, p < .001. Furthermore, RTs increased more with display size in the FEB and preview conditions than in the HEB condition, F=7.92, MSE=12437.43, p < .001. As shown in Fig. A two-way within-subjects ANOVA indicated that RTs were fastest overall in the HEB condition F = 69.97, MSE = 46985.70, p < .001, and increased with display size, F = 194.72, MSE = 38873.93, p < .001. In addition, RTs increased more with display size in the Preview condition than in the HEB, F = 10.16, MSE =13399.02, p < .001.RTs were longer overall in the preview condition than in the HEB, F = 165.72, MSE = 65683.86, p < .001. However, neither the main effect of condition, nor the Condition × Display Size interaction were significant, both Fs < 1.RTs increased with display size, F = 6.19, MSE = 11.65, p < .01, and the error rate increased with display size F = 29.87, MSE = 12.40, p < .001. Errors also increased more with display size in the FEB and preview conditions than in the HEB, F = 5.09, MSE = 6.98, p < .001. Catch trial errors were low (4.93%) and were not analyzed further.Error rates were low overall 3.16%) and are shown in Table 6% and arIn Experiment 4, search efficiency in the preview condition did not differ from that in the FEB at any display size, indicating that inhibition at the small display sizes was destroyed when the pacmen rotated. This is consistent with the idea that inhibition was applied to the perceptually grouped stimuli and that when their local elements rotated the inhibition was abolished because a perceptually new object identity emerged , illustrating a search context in which time-based selection had no opportunity to improve search efficiency. One interpretation for this finding is that Kanizsa-type illusory contours may produce sufficiently salient signals so as to allow those stimuli to be segmented in parallel when presented amongst non-illusory distractors  and new stimuli differing in their color. For example, in Watson and Humphreys , the preAlthough lab-based examples of visual illusions can be viewed as a by-product of our visual system, in natural environments this ability serves an adaptively vital function. Using luminance cues to detect object boundaries is crucial for object recognition in low-luminance environments, such as at night, in shadow, or to detect camouflaged or occluded objects. Here we show for the first time that perceptual grouping, regardless of illusory contours, can be a limiting factor in time-based visual attention. The results of the present study suggest that when such perceptual groups occur, attentional prioritization of new items is likely to be efficient only when there is a relatively small number of grouped items to be ignored. When larger numbers of distractors are present, the preview benefit is abolished. Such environments with complex stimuli that require perceptual grouping may thus impair the effectiveness of attentional mechanisms such as time-based visual selection and be more susceptible to distractor interference.


The Wnt/β-catenin signaling pathway exerts integral roles in embryogenesis and adult homeostasis. Aberrant activation of the pathway is implicated in growth-associated diseases and cancers, especially as a key driver in the initiation and progression of colorectal cancer (CRC). Loss or inactivation of Adenomatous polyposis coli (APC) results in constitutive activation of Wnt/β-catenin signaling, which is considered as an initiating event in the development of CRC. Increased Wnt/β-catenin signaling is observed in virtually all CRC patients, underscoring the importance of this pathway for therapeutic intervention. Prior studies have deciphered the regulatory networks required for the cytoplasmic stabilisation or degradation of the Wnt pathway effector, β-catenin. However, the mechanism whereby nuclear β-catenin drives or inhibits expression of Wnt target genes is more diverse and less well characterised. Here, we describe a brief synopsis of the core canonical Wnt pathway components, set the spotlight on nuclear mediators and highlight the emerging role of chromatin regulators as modulators of β-catenin-dependent transcription activity and oncogenic output. Wnt signaling is a growth control pathway that can regulate many biological processes ranging from development and evolution to adult homeostasis. Wnt signaling includes two branches, canonical (β-catenin-dependent activity) and non-canonical (β-catenin-independent activity) Wnt pathways. β-catenin is composed of a central region, including twelve imperfect Armadillo repeats, which are flanked by different domains in N- and C-terminus, respectively . It actsMoreover, many regulators have been identified which control β-catenin’s subcellular localisation, nuclear abundance or transcriptional activity . NegativMYC transcripts [Based on its fundamental functions in maintaining homeostasis, it is not surprising that inappropriate activation of the Wnt pathway contributes to many human diseases, including cancers . Loss-ofnscripts . Intrigunscripts . This de2+). Some components, such as FZD and DVL, are shared by both the canonical and non-canonical Wnt pathways. It has been identified that canonical WNTs can be antagonised by non-canonical WNTs [PLOD2 and HADH, which are necessary for the proliferation and viability of CRC cells [The non-canonical Wnt pathway (β-catenin-independent activity) contains two well-recognised cascades, the planar cell polarity pathway (Wnt/PCP) and the Wnt calcium signalling pathway  and TBL1-related protein (TBLR1) have been identified as a cofactor exchange complex for transcriptional activation . Wnt-indTRIB3 expression. TRIB3 brings β-catenin in close proximity to TCF4 to transactivate Wnt targets [TCF7L2 (encoding TCF4). It is assumed that the promoters of Wnt-regulated genes could be occupied by these two complexes to modulate their transcriptional outputs [Some regulators are capable of promoting the assembly of the β-catenin-TCF/LEF complex to enhance the transcriptional activity of the Wnt pathway. For example, a cell cycle regulator, Forkhead box protein M1 (FOXM1), stabilised upon Wnt activation, antagonises β-catenin-interacting protein 1 (ICAT), an inhibitor for β-catenin-TCF/LEF complex. In this manner, FOXM1 enables the localisation of β-catenin to promoter elements and facilitates the formation of the transcriptional complex . Similar targets . Special outputs   to TCF/LEF or binding of β-catenin-TCF/LEF complex to DNA leads to repressed expression of some genes, such as Cdh1 (encoding E-cadherin) [Sr (encoding Stripe) [In adherin)  and Sr ( Stripe) . This ef Stripe) . Additio Stripe) ,78. Wher Stripe) . Dapper  Stripe) . Chibby, Stripe) ,82. C-te Stripe) ,84 or re Stripe) ,86. Howe Stripe) ,88. The  Stripe) . In Wnt  Stripe) . Osterix Stripe)  . Consistent with this, Claudin-7 overexpression was found to promote loss of tumour cell polarity and tumourigenesis in a SOX9 dependent manner [SOX9 (sex-determining region Y (SRY)-box 9 protein), an intestinal crypt transcription factor, exerts crucial roles in regulating Paneth cell differentiation in the intestinal epithelium ,109. SOXhenotype . Howevert manner . Nevertht manner , and onct manner . Subsequt manner , which mBMP4 to inhibit its expression, ultimately restricting BMP signalling to differentiate tumour cells in CRC [LGR5 in CRC cells [Additionally, it has been demonstrated that some transcription factors can antagonise the binding of the β-catenin-TCF/LEF complex at relevant genomic loci to differentially regulate gene expression. GATA6, belonging to the GATA family of zinc-finger transcription factors, is one such example. GATA6 competes with the β-catenin-TCF4 complex to bind to the enhancer elements of s in CRC . FurtherRC cells . These oThe Mediator complex acts as a crucial component in transcription regulation, in light of its ability to transduce signals from pathway-specific transcription factors (TFs) to regulate RNA polymerase II (RNAPII) activity ,118. DepCDK8 and RB1 loci on 13q in colorectal cancer cells provide a clear explanation of how they select optimal conditions to antagonise E2F1 suppressions [MYC, provides a basis for understanding how Mediator kinase may enhance β-catenin transcriptional output [CDK8/19 has been implicated in several cancers, especially those induced by dysregulated gene expression rather than mutations . It has ressions ,130. E2Fressions . The phyl output . Togethel output . In concChromatin remodeling is an essential step for transcription regulation. The nucleosome, the basic subunit of chromatin, is composed of histones H2A, H2B, H3, H4 and 146 base pairs (bp) of DNA. These histone tails undergo diverse post-translational modifications, such as acetylation, methylation, phosphorylation, ubiquitination, sumoylation and ADP ribosylation. Such modifications can increase or decrease the accessibility of DNA for transcriptional regulators to coordinate gene expression ,135,136.Drosophila, dCBP can acetylate dTCF to decrease its affinity with Armadillo (β-catenin) to inhibit Wingless signalling [KAI1, a metastasis suppressor gene, is triggered by the P50-Pontin-TIP60 complex but is repressed by the P50-β-catenin-Reptin complex [HATs can transfer acetyl groups to lysine tails on histones to improve/inhibit gene expression . CBP andgnalling . Conversgnalling . In vertgnalling ,149,150 gnalling ,152,153.gnalling  and p300gnalling , respectgnalling . In addignalling . Lastly,gnalling . TIP49a gnalling ,160. For complex . In geneHDACs can remove acetyl groups from lysine tails on histones to counteract the activity of HATs. This causes chromatin to be compacted, thus inactivating the transcription of target genes. HDAC1 can combine with LEF1 to suppress Wnt/β-catenin target gene expression, whereas β-catenin can displace HDAC1, followed by interaction with LEF1 to render the new complex ready for activation . On the Histone methylation includes histone lysine methylation and histone arginine methylation. Histone lysine methylation can happen on lysine residues, such as Lys4, Lys9, Lys27, Lys36 and Lys79, of histone H3 and Lys20 of histone H4 via mono-, di-, or trimethylated manners . In addiKMT2A), the member of a mammalian trithorax-group (trx-G) gene, plays an essential role in maintaining the expression of the Hox gene during development [KMT2A gene, leading to the formation of new chimeric MLL1 fusion proteins, which lose the H3K4 methyltransferase domain [Lysine methyltransferases family 2 (KMT2) can methylate Lys 4 of histone H3 (H3K4), which is related to gene activation in eukaryotes . The famelopment . Nascente domain ,171,172.e domain . MLL1 ise domain . Subseque domain ,175. In e domain . Human Se domain .SFRP1 promoter, a Wnt inhibitory gene [KMT5A) is a member of the SET domain-containing methyltransferase family and is specifically responsible for H4K20 monomethylation to control gene expression [EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), targets H3K27 methylation to inactivate gene transcription . In hepaory gene ,180. Addpression . In Wnt/pression . Furtherpression . SET dompression .Unlike other lysine HMTases, DOT1L functions as H3K79 methyltransferase despite lacking a SET domain . In leukAXIN2 and WIF1, two antagonists of the pathway, and indirectly activating AKT/GSK3β signalling [MTDH), an oncoprotein, which regulates the Wnt pathway to promote HCC metastasis. MTDH overexpression causes the transport of PRMT5 from the nucleus to the cytoplasm, followed by that of β-catenin from the cytoplasm to the nucleus, which upregulates Wnt signalling [Additionally, histone arginine residues can also be methylated by protein arginine methyltransferases (PRMTs). PRMTs have nine isoforms in mammalian genomes and catalyze three different types of arginine methylation, including monomethylation as well as asymmetric and symmetric dimethylation. PRMT5 is responsible for symmetric demethylation, which modifies histones H3 and H4 to inhibit gene transcription. In lung and blood cancers, PRMT5 is overexpressed and works as a transcriptional repressor to inhibit tumour suppressor expression ,190. In gnalling . Howevergnalling . Moreovegnalling .Overall, HMTs play critical roles in promoting Wnt signalling by promoting the expression of agonists or inhibiting the expression of antagonists of the pathway. Moreover, dysregulation of HMT activity in the context of the Wnt pathway has been linked to many tumour types, but the prevalence of different enzymes is identified in specific organs, suggesting their tissue-specific tumour-promoting functions.DKK1, an antagonist of the Wnt pathway [Protein lysine demethylases (KDMs) can modify chromatin by demethylation of histone or non-histone protein lysine residues. Recently, some KDMs have been implicated in human CRC mainly through epigenetic modifications on H3K9, which augments Wnt/β-catenin signalling. H3K9 is involved in gene silencing, and KDM3 can directly remove suppressive H3K9me2 marks to activate Wnt downstream gene expression epigenetically. KDM3 can also drive MLL1-dependent H3K4 methylation to promote the binding of BCL9 and Pygo2 to chromatin, thus contributing to Wnt/β-catenin related transcription activation . Additio pathway . These dSWI/SNF is a conserved subfamily of ATP-dependent chromatin-remodeling complexes from yeast to human . They caThe BAF chromatin-remodeling complex, an analogue of the SWI/SWF complex, has also recently been reported to regulate Wnt signalling. For example, AT-rich interactive domain-containing protein 1 (ARID1), the largest subunit of the complex, possesses two isoforms—ARID1A and ARID1B. The deficiency of ARID1A can drive CRC without APC mutations owing to the dysregulation of enhancer activity . SubsequThe clinical implications of targeting the Wnt signalling pathway has been recognised for decades. Nevertheless, this has not been translated into the clinic for reasons detailed below. Dysregulated Wnt pathway can be targeted at three different levels: the Wnt-receptor complex, the cytoplasmic destruction complex and the nuclear β-catenin-TCF/LEF complex. Regulators involved in downstream transcriptional levels emerge as the most attractive targets for drug development. The implications of APC loss in the vast majority of CRC make inhibitors targeting Wnt signalling components upstream of the destruction complex ineffective. One exception is CRCs harbouring RSPO2/3 fusions . While aDisrupting protein–protein interactions in transcriptional complexes opens a new therapeutic window for anti-cancer drug development, although the transient nature of some interactions hinders effective drug development. Currently, most of the inhibitors targeting transcriptional level in Wnt pathway attempt to disrupt β-catenin-TCF4 interactions ,217,218.Epigenetic modifications are reversible, thus attracting extensive attention to targeting abnormal epigenetic events so as to revert the cancer phenotype to a normal state. Despite promising therapeutic potential in hematopoietic malignancies, it is not clear why epigenetic inhibitors exert less efficacy in solid tumours. Currently, drugs targeting epigenetic regulators directly in CRC are missing. Resistant mechanisms need to be explored to provide a better opportunity for the development of epigenetic drugs in solid cancers.Canonical Wnt signalling regulates multiple events involved in embryogenesis and adult homeostasis. In the adult intestine, the Wnt pathway is activated at the bottom of the crypt to maintain intestinal stem cell compartments. However, dysregulation of the Wnt pathway plays a primary role in driving colon cancer cell growth, invasion and survival. While the loss of APC and its consequence in leading to β-catenin accumulation in CRC is well described, mounting data highlight the importance of regulatory networks within the nucleus essential to mediate β-catenin activity. In this review, we have catalogued the main nuclear effectors of β-catenin activity in both tumourigenesis and normal homeostatic control. In addition to their diversity and complexity, as we have detailed, how some nuclear regulators can switch roles from positive to negative effectors based on their conformation , multimolecular assembly , subcellular localisation , enzyme activity  or specific tissue distribution . Furthermore, as we have shown, regulatory event at the level of chromatin configuration and structure expands the ability to control Wnt signalling at a functional and contextual level. Indeed, as illustrated in a number of examples in this review, the ability of regulators to act as activators or repressors may be distinctly contextual on whether the Wnt signal is maintaining gut homeostasis or driving tumourigenic outputs.Despite intense efforts, clinically approved drugs targeting this pathway are lacking, with a dearth of inhibitors in the clinical trial phases. One explanation centers on the difficulty of untangling Wnt signalling in normal tissue homeostasis from its role in cancer growth. This is underscored by the fact that Wnt pathway inhibitors have generally been found to exhibit toxic effects associated with their on-target activity on normal Wnt pathway signalling. The emerging theme of contextual effect in relation to the ability of nuclear regulators to modulate β-catenin activity offers hope in this regard. As we have illustrated, a number of nuclear β-catenin effectors seem to act in a cancer- or disease-specific manner, raising hope that their targeting may offer a more effective therapeutic window. Conversely, the plasticity of some nuclear β-catenin effectors with respect to their temporal, spatial or compositional features highlights challenges in effectively targeting components which, for example, may actively switch between oncoprotein and tumour suppressor roles. Clearly, additional studies are necessary to understand better the mechanistic underpinnings that may explain this observed plasticity. Indeed, the general concept of proteins changing shape and function to coordinate signalling pathways in response to specific stimuli is well described. As in other systems, a deeper and more comprehensive characterisation of the protein networks which regulate β-catenin transcription will eventually drive the identification of attractive therapeutic targets. In sum, despite the passing of three decades since the initial identification of the Wnt pathway, we have only touched the tip of the iceberg in understanding the intricacies of this signalling pathway. The rewards to be gained from further exploration and characterisation of nuclear β-catenin regulators are immeasurable for the millions of patients affected by Wnt pathway-related diseases every year.


RhoA loss- or gain-of-function in mouse embryonic limb bud ectoderms interacts genetically with Dkk1 gain-of-function to rescue the severe limb truncation phenotypes or to phenocopy the deletion of β-catenin, respectively. Likewise, RhoA loss-of-function in pre-osteoblasts robustly increases bone formation while gain-of-function decreases it. Importantly, high RhoA/Rock activity closely correlates with Jak and Gsk3β activities but inversely correlates with β-catenin signaling activity in bone marrow mesenchymal stromal cells from elderly male humans and mice, whereas systemic inhibition of Rock therefore activates the β-catenin signaling to antagonize aging-associated bone loss. Taken together, these results identify RhoA/Rock-dependent Gsk3β activation and subsequent β-catenin destabilization as a hitherto uncharacterized mechanism controlling limb outgrowth and bone homeostasis.The Wnt/β-catenin signaling pathway appears to be particularly important for bone homeostasis, whereas nuclear accumulation of β-catenin requires the activation of Rac1, a member of the Rho small GTPase family. The aim of the present study was to investigate the role of RhoA/Rho kinase (Rock)-mediated Wnt/β-catenin signaling in the regulation of aging-associated bone loss. We find that Lrp5/6-dependent and Lrp5/6-independent RhoA/Rock activation by Wnt3a activates Jak1/2 to directly phosphorylate Gsk3β at Tyr216, resulting in Gsk3β activation and subsequent β-catenin destabilization. In line with these molecular events, The online version contains supplementary material available at 10.1186/s13619-020-00071-3. Lef1), cyclin D1 (C.D1), Dickkopf-1 (Dkk1), and Axin2  pathway and the Wnt/CaDespite this discovery of a role for Rho small GTPases in the β-catenin-dependent Wnt signaling pathway, the importance and biological relevance of RhoA in the regulation of Wnt/β-catenin signaling remain obscure. Here, we report that the regulation of aging-associated bone loss involves the destabilization of β-catenin by RhoA/Rho kinase (Rock) through activation of Janus kinase (Jak) and its direct phosphorylation of the Tyr216 residue of glycogen synthase kinase-3β (Gsk3β).Antibodies against Jak1, Jak2, Gsk3β, β-catenin, p-Ser9-Gsk3β, p-Ser552-β-catenin, p-Ser33/37/Thr41-β-catenin (p-Ser33-β-catenin), and non-p-Ser45-β-catenin (active β-catenin) were from Cell Signaling . Antibodies against p-Tyr1022-Jak1 and p-Tyr1007-Jak2 were purchased from Bioworld Technology . Antibodies against Rock1, Rock2, Gsk3β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and Lamin B were obtained from Santa Cruz Biotechnology . Antibody against alkaline phosphatase (Alp), bone sialoprotein (Bsp), Lef1, Nestin, p-Ser1366-Rock2, and p-Tyr216-Gsk3β and recombinant human active Jak2 and Gsk3β were from Abcam . The IRDye 680, 700, and 800 s antibodies were from LI-COR Bioscience . Alexa555- and Alexa488-conjugated secondary antibodies were from Molecular Probes . Recombinant mouse active Wnt3a (rWnt3a), Wnt5a (rWnt5a), and Dkk1 (rDkk1) were from StemRD . Y27632 and Fasudil were from Selleckchem  and Pyridone 6 was from Medchem Express .Msx2-Cre, Col1-Cre (2.3 kb), CAT-dnRhoA+/−, R26-Dkk1+/−, and β-cateninf/f mouse strains were as reported  as described previously  knock-in mouse strain was generated by Cyagen Biosciences Inc.  as described previously  who suffered from femoral neck fracture and underwent the total hip replacement surgery and three young men  who suffered from traumatic fracture and underwent the internal fixation surgery in the Department of Orthopeadic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The young men were hurt in accidents and had no osteoporosis or other diagnosed diseases. The elderly men were satisfied with the diagnosis of osteoporosis according to the commonly accepted WHO definitions and criteria, and had no other diagnosed diseases. Bone marrows were used for preparation of smears or isolation of BMMSCs by the Human Marrow Lymphocyte Medium Kit . For quantification of bone marrow smear staining, phospho-ROCK22. BMMSCs were used for experiments after 4 ~ 7 passages.Mouse BMMSCs were isolated by a MagCellect Mouse Mesenchymal Stem Cell Isolation Kit  as described previously  were isolated from neonatal mice at postnatal day (P) 2 ~ 5, and expanded and maintained as described previously  as described previously . We determined significance by Student’s To determine the role of RhoA/Rock in Wnt/β-catenin signaling, we used an established binding assay to examine the levels of GTP-bound (active) form in primary murine calvarial osteoblasts (PMCOBs). Stimulation with recombinant mouse Wnt3a resulted in time-dependent and dose-dependent increases in the GTP-RhoA levels in PMCOBs Fig. a, b. Actcollagen1α1 (Col1) promoter. This generated Col1-Cre;dnRhoA+/− or Col1-Cre;caRhoA+/− neonates and their control littermates. We then isolated PMCOBs from these mouse strains (P2 ~ P5) and conducted western blot analyses to identify the cytosolic versus nuclear fractions of β-catenin. Activation of RhoA in the PMCOBs from the Col1-Cre;caRhoA+/− neonates and its inactivation in PMCOBs from Col1-Cre;dnRhoA+/− neonates were validated by determining the p-Rock2 levels  or the constitutively active form of RhoA  with the mice expressing Cre recombinase under the control of the 2.3-kb mouse 9N or the3a Fig. S. Thus, RRhoA loss-of function in the regulation of Wnt/β-catenin signaling. Fasudil treatment of PMCOBs slightly induced β-catenin expression in whole-cell lysates and in the cytosolic or nuclear fractions under basal conditions or robustly induced this expression in response to Wnt3a , an osteoblast marker, and the formation of mineralized nodules in either the presence or absence of Wnt3a, while inactivation enhanced these responses  of the mouse embryonic limb bud, where Wnt signaling is critical for limb outgrowth via β-catenin  exhibited normal forelimbs and varying lengths of hindlimbs but entirely lacked autopods  exhibited the same limb phenotypes as the Msx2-Cre;dnRhoA+/−;Dkk1+/+ embryos, including the varying hindlimb length and lack of autopods  did not show any obvious limb phenotype changes; however, the double-heterozygous embryos  (6/6) all failed to develop hindlimbs and nearly all the forelimbs (10/12 from 6 embryos) lacked structures distal to the scapula; these phenotypes are identical to those of the Msx2-Cre;Dkk1+/+ or Msx2-Cre;β-cateninn/f embryos  at E16.5 still lacked all hindlimb structures and exhibited truncations at various levels in the forelimbs; these defects were identical to those in the Msx2-Cre;β-cateninn/f embryos  in the AER and bone morphogenetic protein 4 (Bmp4) in the distal ventral ectoderm; both of these are target genes of Wnt/β-catenin signaling. As expected, the Msx2-Cre;Dkk1+/+ embryos expressed only residual Bmp4 and no Fgf8 in the forelimb of E10.5 (34–35 somites) embryos  and Gsk3β have been suggested to function as effectors of RhoA/Rock signaling and both are involved in the phosphorylation of β-catenin  and p-Ser9-Gsk3β and p-Tyr216-Gsk3β . Wnt3a increased p-Ser9-Gsk3β or decreased p-Ser33-β-catenin in a time-dependent manner within 60 min of stimulation, but time-dependently induced an increase in p-Tyr216-Gsk3β Fig. . ImportaLef1-luciferase expression by approximately 60% over the vector, whereas the Y216F variant of Gsk3β caused 100% and 85% increases, respectively, over the WT levels. In the presence of Wnt3a, the WT completely abolished this induction of β-catenin levels and Lef1-luciferase expression, but the Y216F variant only minimally affected the induction of β-catenin levels and Lef1-luciferase expression  and evaluating the capacity to mediate Wnt/β-catenin signaling. In the absence of Wnt3a, the wildtype Gsk3β (WT) decreased the β-catenin levels by approximately 50% and Rock1/2 is a serine/threonine kinase; however, it is unlikely to be directly responsible for phosphorylation of Gsk3β at Tyr216. We explored the underlying mechanism by which RhoA/Rock induces Gsk3β phosphorylation at Tyr216 by investigating the potential relevance of the Jak tyrosine kinase that physically interacts with and is activated by Rock , p-Rock2, p-Jak2, p-Tyr216-Gsk3β, and active β-catenin in longitudinal tibia sections of ns Fig. SA, B. Theias Fig. a-d.Fig.We investigated the physiological relevance of p-Jak and p-Tyr216-Gsk3β activation by Wnt3a using osteoblastic differentiation assays. C3H10T1/2 cells were transfected with either wild type or Gsk3β-Y216F or treated with 50 nM P6 and then examined for their ability to undergo osteoblastic differentiation in response to Wnt3a. Wildtype Gsk3β transfection reduced the Wnt3a-induced Alp activity and mineralized nodule formation by 63% and 89%, respectively, while transfection with the Y216F mutant Gsk3β decreased the Wnt3a-induced Alp activity and mineralized nodule formation by only 23% and 20%, respectively Fig. e-g. ConsdnRhoA or caRhoA caused severe limb phenotypes in the genetic background of Msx2-Cre;Dkk1+/+ or Msx2-Cre;Dkk1+/−, respectively. However, overexpression of dnRhoA or caRhoA alone resulted in no apparent limb phenotype changes in the otherwise wild type background. This observation led us to speculate that inactivation or activation of RhoA in the wild type background underscores both the robustness of the Wnt/β-catenin signaling system and the sensitivity of the AER to a threshold level of Wnt/β-catenin signaling.Overexpression of Col1-Cre;dnRhoA+/− or Col1-Cre;caRhoA+/− mice and their control littermates. Microcomputed tomography (μCT) analyses and three-dimensional reconstruction of the proximal tibias demonstrated that inactivation of RhoA in pre-osteoblasts increased the bone volume/trabecular volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) by 75%, 33%, and 56%, respectively, and decreased the trabecular separation (Tb.Sp) by 35%. By contrast, activation of RhoA in pre-osteoblasts decreased the BV/TV, Tb. Th, and Tb.N by 47%, 22%, and 35%, respectively, and increased the Tb.Sp by 65% , and Runx2, in femurs revealed that inactivation of RhoA robustly increased, while activation robustly decreased, these mRNA levels  and osteocalcin (OCN), whereas no effect was observed on the levels of bone resorption markers, including carboxyterminal collagen cross-links (CTX) and TRAP Fig. SF-I. Taken = 3, 28-, 32-, 33-year-old) or elderly  male patients and the tibial sections from 5- or 10-month-old male mice, with normal bone volumes or aging-associated bone loss, respectively, to determine the potential relevance between RhoA/Rock and Wnt/β-catenin signaling. We then performed the immunostaining of p-Rock2, p-Jak1, p-Tyr216-Gsk3β, p-Ser33-β-catenin , and non-p-Ser45-β-catenin , and quantified the mRNA levels of Lef1, Cyclin D1, and Axin2, the targets of Wnt/β-catenin signaling, and of Runx2, a osteoblastic gene marker.Low activity of Wnt/β-catenin signaling in BMMSCs contributes to the pathogenesis of type II osteoporosis or age-dependent bone loss  BMMSCs were scattered in the smears, and fewer NESTIN+ BMMSCs were obtained from the elderly men than from the young men. The immunosignals derived from p-ROCK2-, p-JAK1, p-Tyr216-GSK3β, p-β-CATENIN-, and active β-CATENIN were robustly detected, and the apparently overlapping signals between NESTIN and other molecules were readily observed in the cytoplasm of the BMMSCs  and of FZD1 ~ 10. BMMSCs from 8-month-old mice displayed almost the same mRNA levels of Wnt3a, Wnt5a, and Wnt10 as were found in BMMSCs from 2-month-old mice  as a hitherto uncharacterized signaling cascade that acts primarily to destabilize β-catenin. Thus, this study highlights a precise regulation of Wnt signaling and provides an additional mechanism for regulation of limb development, while also identifying a potential new therapeutic target for aging-associated bone loss.Additional file 1.Additional file 2.