FZD7, which may activate all Wnt pathways, but favors non-canonical signaling, and NFATc3, a downstream transcription factor and readout of the non-canonical Wnt/Ca2+ pathway, were significantly increased in SCZ and BD (p < 3 × 10−4). Furthermore, patients had lower plasma levels of soluble dickkopf 1 and sclerostin (p < 0.01) compared with HC. Our findings suggest that SCZ and BD are characterized by abnormal Wnt gene expression and plasma protein levels, and we propose that drugs targeting the Wnt pathway may have a role in the treatment of severe mental disorders.The Wnt signaling pathway plays a crucial role in neurodevelopment and in regulating the function and structure of the adult nervous system. Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders with evidence of subtle neurodevelopmental, structural and functional neuronal abnormalities. We aimed to elucidate the role of aberrant regulation of the Wnt system in these disorders by evaluating plasma levels of secreted Wnt modulators in patients (SCZ = 551 and BD = 246) and healthy controls (HCs = 639) using enzyme immune-assay. We also investigated the expression of 141 Wnt-related genes in whole blood in a subsample  using microarray analysis. Both SCZ and BD had dysregulated mRNA expression of Wnt-related genes favoring attenuated canonical (beta-catenin-dependent) signaling, and there were also indices of enhanced non-canonical Wnt signaling. In particular,  Early brain morphological findings , behavioral and cognitive alterations, and nonspecific motor development abnormalities in SCZ were among the first observations that underpinned the neurodevelopmental hypothesis of SCZ6. In BD, subtle brain morphological changes, behavioral alterations prior to onset of illness, and attenuations in neurogenesis may indicate disrupted neurodevelopment7. Further, in the adult brain, structural alterations, aberrant brain connectivity, and biochemical changes suggest patterns of subtle structural and functional brain abnormalities in both illnesses9. In addition, some studies have also indicated signs of neurodegenerative processes in SCZ10, with weaker evidence in BD11.Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental illnesses that are leading causes of worldwide disability13. Secreted modulators such as the dickkopf (DKKs) and the secreted frizzled related proteins (sFRPs) regulate both the canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) Wnt signaling, modulating the effects of various Wnt ligands14. Initial studies investigating the Wnt signaling pathway in severe mental disorders almost two decades ago focused on the hippocampus, where immunoreactivity of the Wnt ligand Wnt-1 was observed15. Aberrant findings in SCZ included reduced β-catenin16, increased Wnt-1 expression15, and reduced glycogen synthase kinase 3 beta (GSK-3β), a multifaceted kinase in Wnt signaling17. Later, large genome-wide association studies identified several polymorphisms in Wnt signaling in SCZ and BD suggesting a strong genetic component18. In particular, secreted Wnt antagonists, such as DKK419, sFRP1, and FZD321, have been associated with brain volumes22 and increased susceptibility to SCZ. In addition, these Wnt antagonists are located in the chromosomal region 8p, which is a genetically implicated region in neuropsychiatric disorders18. Furthermore, downstream modulators of β-catenin-dependent signaling have also been linked to increased susceptibility to both SCZ24 and BD, as well as suicidal behavior25 and body composition26. The Wnt signaling pathway is also linked to BD through investigations identifying that lithium impairs GSK-3β expression27. Although alterations in the Wnt signaling pathway have been evident for the past couple of decades a thorough understanding is still lacking. In particular, whereas most studies on the potential involvement of Wnt signaling in severe mental disorders have focused the canonical pathway, few studies has investigated the role of the non-canonical pathways that may be equally relevant in the pathogenesis of psychiatric disorders14. Furthermore, no studies have investigated serum levels of central secreted Wnt antagonists such as DKKs or frizzled related proteins.During neurodevelopment and in the adult brain the Wnt signaling pathway plays a crucial role in neural stem cell proliferation, differentiation and migration, neuroplasticity, and neurogenesisIn the present study we aimed to further elucidate the aberrations in the Wnt signaling pathway by conducting a pathway analysis on leukocyte mRNA in a large sample of SCZ and BD patients, and healthy controls (HCs) to identify differentially expressed genes as well as comparing circulating levels of secreted Wnt agonists and antagonists. We also aimed to explore associations between the Wnt signaling pathway and the use of medication.28 is approved by the Regional Committee for Medical Research Ethics and the Norwegian Data Inspectorate. The biobank is approved by the Norwegian Directorate of Health. All participants provided written informed consent after receiving a complete description of the study.The TOP Study at the NORMENT Centre, Oslo University Hospital, and collaborating Norwegian hospitals28). Healthy volunteers without any history of severe psychiatric disorders (or in any of their first-degree relatives) or substance/alcohol abuse/dependency from the same catchment area were randomly selected from the National Population Registry (www.ssb.no). For the present analyses, patients and controls were not included if they had coexisting autoimmune or inflammatory disease, cancer, ongoing infections, used anti-inflammatory drugs, or had C-reactive protein levels above 20 mg/L. In total, 1625 participants included in the study had available plasma for protein assessment. Of these, 1436 participants  met criteria for inclusion. A subsample of patients and controls  had available blood samples and passed quality control for microarray analyses.The main inclusion criteria were having a Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) diagnosis of SCZ spectrum disorders or bipolar spectrum disorders, intelligence quotient > 70, and age between 18 and 65 years , and we also measured serum levels of medication from the same blood sample that was used for gene expression and protein level analyses.Diagnosis was obtained using the Structured Clinical Interview for DSM-IV Axis I Disorders36. We selected stabile plasma ligands that circulate at a detectable level: DKK1; DKK3; sclerostin (SOST), R-spondin-3 (RSPO3) and sFRP3. We measured their plasma levels in duplicate using commercially available antibodies  in a 384 format using a combination of a SELMA  pipetting robot and a BioTek  dispenser/washer. For details see supplementary text We have previously described the blood sampling methodBlood samples were collected using Tempus Blood RNA Tubes. Total RNA was extracted with ABI PRISM 6100 Nucleic Acid PrepStation and TEMPUS 12-port RNA Isolation Kit according to the manufacturer’s protocol. High-Capacity cDNA Reverse Transcription Kit was used for reverse transcription of 1 µg RNA.http://www.genome.jp/kegg/pathway.html). For each sample, 200 ng of total RNA was biotin-labeled and amplified using the Illumina TotalPrep-96 RNA Amplification Kit . Global analysis of gene expression was performed with Illumina HumanHT-12 v4 Bead Chip  consisting of more than 47 000 probes . For this purpose, 842 samples  passed labeling and scanning. Raw microarray scan files were exported using the Illumina GenomeStudio software and loaded into R for downstream analysis using specific packages provided by BioConductor37. Lumi was used to detect outliers38. R package (version 3.24.4.) was used to correct for technical batch effects, like RNA extraction batch, RNA extraction method, DNase treatment batch, cRNA labeling batch, and chip hybridization. Further quality control, quantile-normalization, and log2-transformation were done using Limma39.We selected 141 Wnt pathway-related genes using the Kyoto Encyclopedia of Genes and Genomes database  for skewed distributions to investigate differences between groups. Correlations were examined by using Spearman’s rank correlation. We controlled for age and sex in linear regression models.Statistical analyses were performed using the SPSS software package for Windows, version 24.0 for plasma analyses. Data normality was assessed using the Kolmogorov–Smirnov and Shapiro–Wilk tests. We investigated differences in demographic data between groups using the chi-square test for categorical variables, the Kruskal–Wallis test for continuous variables, and the Mann–Whitney Bmal1 as covariates. Bmal1 was included in the analyses to adjust for differences in time of blood sampling and circadian rhythm between patients and controls.To find associations between expression and the diagnostic group, a linear model was fitted in the R software environment using age, sex, and the expression level of https://www.whocc.no/atcdd). The defined daily dose is the assumed average maintenance dose per day for a drug used for its main indication in adults. We used serum concentration levels for lithium instead of defined daily dose. We selected significantly regulated Wnt antagonists in plasma  or major upstream or downstream differentially expressed genes . Associations were explored using analysis of covariance where we controlled for age, sex, and other medication groups, and we also investigated whether medication groups would influence these Wnt members  according to the guidelines from the World Health Organization Collaborating Center for Drug Statistics Methodology  and p < 0.01 for plasma ligand analyses (adjusting for 5 tests).We corrected for multiple testing according to the Bonferroni method and alpha was set at https://gtexportal.org/home/gene/FZD7] the GTEx Portal on 06/25/2017 and/or dbGaP accession number phs000424.vN.pN on 06/25/2017. NFATC3 protein expression in the brain was evaluated using The Human Protein Atlas (www.proteinatlas.org)40.In addition, we evaluated the tissue expression of FZD7 using the publically available Genotype-Tissue Expression (GTEx) dataset. The GTEx Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from [n = 1436) and the leukocyte (n = 842) cohorts. Patients with SCZ had higher symptom load and lower functioning than BD.The socio-demographic and clinical characteristics of the participants are presented in Table p < 0.01) and SOST (p < 0.01 for SCZ and BD), also significant in adjusted analyses . In the SCZ group, the levels of the Wnt antagonist sFRP3 were nominally decreased compared to HC (p = 0.04), with no significant differences in plasma ligand levels between SCZ and BD. As for the Wnt agonist RSPO3, we found nominally significant lower levels in SCZ, and no significant differences in BD compared to HC.The plasma levels of Wnt ligands in absolute values and group comparisons are summarized in Table p < 3 × 10−4) are described in Table −4 < p < 0.05) in Supplementary table p < 3 × 10−4), and between |0.02| and |0.13| in trend level findings (3 × 10−4 < p < 0.05).The expression of Wnt pathway genes in whole blood are summarized in Figs. FZD7 expression was significantly increased in SCZ compared to HC (p < 3 × 10−4) with similar regression coefficients, but trend level changes in BD (p < 0.001). No other frizzled receptors or Wnt ligands were significantly regulated in our patient population.In general, gene expression in patients compared to HCs was quite similar for SCZ and BD with more modest changes in BD. For example, the Wnt receptor FRAT2 decreased in SCZ, CSNK2A2 decreased in BD, and CSNK2B increased in both), degradation complex , and nuclear regulation of β-catenin . In addition, the transcriptional target JUN was decreased in SCZ and BD while CCND3 was increased in SCZ. The only significant differentially expressed genes between SCZ and BD were increased ICAT and decreased CAMK2G mRNA expression in SCZ  were increased in SCZ, and CAMK2G in BD. Second, isozymes belonging to the catalytic subunit of CaN and protein kinase C (PKC) were downregulated . Finally, NFAT3C was increased in both SCZ and BD.The only gene involved in the non-canonical planar cell polarity pathway that was differently expressed was decreased HC Table . For theFZD7 and NFAT3C, an important transcriptional factor of the non-canonical Wnt signaling pathway, may be major characteristics of SCZ and BD. To further elucidate these issues, we investigated tissue expression of these proteins in human brain based on available databases. Evaluation of tissue expression of FZD7 using the GTEx portal revealed the highest expression of FZD7 in the cerebellum compared to other tissues  had significantly lower levels of SOST  and increased FZD7 expression .We found a positive association between defined daily dose of antipsychotics and plasma levels of DKK1 , and NFAT is an important inducer of inflammatory responses in neutrophils54. Non-canonical Wnt ligands can trigger the Wnt/Ca2+ pathway leading to activation of calcium-sensitive enzymes, including CaN, Ca2+/calmodulin-regulated kinase II, and PKC, which may activate NFAT signaling55. We and others have demonstrated that non-canonical WNT5A stimulation enhanced NFAT activity57. Based on the complex expression pattern of CaN and PKC transcripts, but increased CAMK2 in SCZ and BD, it is tempting to hypothesize that CAMK2 could activate NFAT as has been demonstrated in lymphocytes59. Regardless of the mechanisms for upregulation, enhanced NFAT4 activity in the brain and/or immune cells could potentially induce neuro-inflammatory responses in SCZ and BD. Of relevance, both NFAT60 and CAMK261 may antagonize the canonical pathway.Our second major finding regarding gene expression was increased 53 we found evidence of attenuated canonical Wnt signaling in SCZ and BD. This is suggested by decreased mRNA levels of members belonging to the β-catenin destruction complex , essential for Wnt/β-catenin signaling63. Aberrant CK2 signaling in the cortex has been linked to altered neurotransmitter release in SCZ64. Decreased PSEN1 would favor enhanced canonical Wnt signaling, but may also have multiple functions outside of the γ-secretase complex. In addition, regulated levels of components involved in p-53-mediated degradation of β-catenin  could reflect altered turnover of β-catenin, but are difficult to interpret as multiple non-Wnt substrates are processed by this complex. We identified several differentially expressed genes involved in nuclear transport and regulation of β-catenin, such as increased CHD8, a nuclear protein that inhibits β-catenin signaling65, and decreased MAP3K7. MAP3K7 is also known as TAK1, and studies in TAK(−/−) mice reveal that low levels may adversely affect cerebellar development and neuro-developmentally regulated behavior66. Decreased expression of transcriptional co-activators of canonical Wnt target genes such as CtBP1 (both co-repressor and co-activator), CREBBP, a protein linked to SCZ susceptibility67, and the transforming growth factor transcription factor SMAD4 are also compatible with attenuated canonical signaling. These finding are supported by decreased expression of the Wnt target gene JUN, which may further reinforce Wnt signaling68 and increased CCND3, which in contrast to cyclin D1/D2, is increased in response to inhibition of canonical Wnt signaling70.In support of a previous study71 and the decreased circulating levels of DKK1, an antagonist of the canonical Wnt pathway, observed in SCZ and BD could reflect a downregulation of DKK1in an attempt to enhance canonical Wnt signaling. In an analogous fashion, we have shown WNT5A stimulates the release of sFRP3, which antagonizes WNT5A-induced NFAT and ERK activity72. Alternatively, DKK1 may reflect decreased activity in the Wnt canonical pathway as DKK1 is directly regulated by the β-catenin/TCF complex under physiological circumstances73. Low serum levels of DKK1 have been associated with increased risk of somatic diseases, and to predict increased mortality in older patients74. SOST is also a potent inhibitor of the Wnt canonical pathway75, and low circulating levels are frequently seen in relation to enhanced calcification76, which is relevant as calcification of the choroid plexus has been associated with regional brain atrophies and neurodegenerative phenotype in SCZ and BD77. Serum levels of SOST have not been investigated in severe mental disorders, but are inversely correlated with vitamin D levels78, and low vitamin D associates with symptoms in severe mental disorders79.Interpretation of signaling networks in cross-sectional studies is further complicated by that changes in expression may reflect compensatory feedback mechanisms in response to dysregulated signaling. Multiple families of secreted antagonists or modulators may regulate Wnt signaling80.We investigated whether our findings may be attributed to the use of medication. Due to the naturalistic nature of our study most patients were using a combination of psychotropic medication. We detected a significant dosage-dependent association between antipsychotics and DKK1, however we found no group effects of antipsychotics. SOST levels were lower in the group using antidepressants, which is compatible with an activation in canonical Wnt signalingOur study has some limitations that should be considered. First, we did not measure phosphorylation status or protein levels of intracellular components of the Wnt pathways, thus our conclusions rely on gene expression data and plasma protein levels. Second, although a post hoc power evaluation revealed an observed power above 0.90 for most transcripts and circulating proteins, observed power was low (below 0.60), for several low-abundant transcripts. Third, many patients were using a combination of psychotropic medications, which made it impossible to investigate effects of medication in monotherapy. We did, however, control for co-medication in the statistical analyses. Lastly, the cross-sectional design of the study hinders us from making strong claims about causality.NFATC3 and FZD7 mRNA expression is increased in peripheral blood of patients, while they have lower serum levels of DKK1 and SOST compared to HCs. Our findings could suggest that drugs blocking the non-canonical Wnt Ca pathway  could have a role in the treatment of severe mental disorders and warrants further investigation.In summary, we provide further evidence of altered Wnt signaling in SCZ and BD in a well-powered sample. In particular, we show that Revised supplemetary file


We established a titanium (Ti) particle-stressed murine MC3T3-E1 cell culture system and calvariae osteolysis model to test the hypothesis that Ti particle-induced osteogenic inhibition and bone destruction are mediated by the GSK-3β/β-catenin signal pathway. Our findings showed that Ti particles reduced osteogenic differentiation induced by osteogenesis-related gene expression, alkaline phosphatase activity and matrix mineralization, as well as pSer9-GSK-3β expression and β-catenin signal activity. Downregulation of GSK-3β activity attenuated Ti particle-induced osteogenic inhibition, whereas the β-catenin inhibitor reversed this protective effect. Moreover, the GSK-3β/β-catenin signal pathway mediated the upregulation of RANKL and downregulation of OPG in Ti particle-stressed MC3T3-E1 cells. In addition, our in vivo results showed that Ti particles induced bone loss via regulating GSK-3β and β-catenin signals. Based on these results, we concluded that the GSK-3β/β-catenin signal pathway mediates the adverse effects of Ti particles on osteoblast differentiation and bone destruction, and can be used as a potential therapeutic target for the treatment of peri-prosthetic osteolysis.Wear debris-induced osteogenic inhibition and bone destruction are critical in the initiation of peri-prosthetic osteolysis. However, the molecular mechanism underlying this phenomenon is poorly understood. In this study, we analyzed the involvement of the GSK-3 However, the molecular mechanisms underlying the particle-induced osteogenic inhibition are poorly understood.Peri-prosthetic osteolysis and subsequent aseptic loosening are the most common and potentially devastating long-term complications of total joint arthroplasty.α and GSK-3β, which share 97% amino-acid sequence similarity within their kinase catalytic domains.11 These proteins regulate multiple biological processes, including bone formation and remodeling during growth and development. Of the two isoforms, GSK-3β is more important for bone remodeling, as demonstrated by increased bone formation in GSK-3β+/– mice.12 GSK-3β is a member of the β-catenin destruction system (APC and axin), which initiates the degradation of β-catenin in the absence of Wnt signals. Furthermore, inactivation of GSK-3β decreases the phosphorylation of β-catenin and prevents the degradation of β-catenin by proteasome. Stabilized β-catenin accumulates and translocates to the nucleus, where it binds the TCF/LEF family of transcription factors that subsequently activate specific genes.14 It is known that β-catenin is critical for Wnt/β-catenin pathway activation, which is essential for proper bone development, and inhibition of this signal pathway downregulates bone formation.14 Clinical results using pharmacological antagonists of GSK-3β showed that the Wnt/β-catenin signal increased bone stock via the β-catenin signal pathway.15 Our previous studies have demonstrated that during wear debris-induced osteolysis, GSK-3β is constitutively expressed in the form of phosphor-Ser-9-GSK-3β at a low level, but not GSK-3β.16 However, to our knowledge, studies have not reported the role of GSK-3β/β-catenin signals in osteoblast differentiation and bone destruction during aseptic loosening initiated by wear debris.Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase, is a critical regulator of glycogen metabolism. GSK-3 has two isoforms, GSK-3β/β-catenin signal pathway. We established a murine clavarial model of titanium (Ti) particle-induced bone destruction, along with a murine MC3T3-E1 osteoblast cell culture system to verify this hypothesis.Therefore, in this study, we hypothesized that wear particles may reduce osteoblast function and promote bone destruction via the GSK-3in vitro study. RT-PCR results showed that Ti particles (0.1 mg/ml) significantly reduced the gene expression of runx2, sp7 and bgalp compared with that in control group , and then stimulated with Ti particles for different durations. As shown in β expression, promoted β-catenin nuclear translocation and further upregulated β-catenin signal activity even in the presence of Ti particles. After co-culture with LiCl for 3 days, ALP activity was 32.4±2.4 nmol pNPP/min/μg protein, which was significantly higher than in cells treated with Ti particles alone (24.7±1.8 nmol pNPP/min/μg protein). LiCl treatment markedly raised the mRNA levels of osteoblastogenesis-related genes, including runx2, sp7 and bgalp, compared with the group treated with Ti particles alone. Moreover, LiCl stimulated cell mineralization by approximately 122.7% when compared with the Ti particle-treated group  for 30 min, and then stimulated with LiCl and Ti particles for 24 h. Western blots demonstrated that LiCl significantly increased β-catenin accumulation in the nucleus. However, the positive effects of LiCl on β-catenin accumulation, as well as ctnnb1 and axis-2 gene copies were significantly impaired by the addition of exogenous ICG-001  was reduced, whereas the secretion of OPG (2.45±0.32 ng/ml) was increased. Consequently, the mean RANKL/OPG ratio decreased from (25.75±3.21)/103 (Ti group) to (4.76±0.89)/103 (Ti+LiCl group). In addition, pretreatment with ICG-001 reversed the effects of the GSK-3β inhibitor.As shown in β/β-catenin signal pathway in vivo, the murine calvarial model was used to mimic the molecular pathogenesis of peri-prosthetic osteolysis. Daily injection of the GSK-3β inhibitor evidently increased bone mineral density (BMD) within a region of interest (ROI) of calvaria exposed to Ti particles . Micro-CT also revealed a marked increase in bone volume (BV) and BV against tissue volume (BV/TV) ratio in mice stimulated with the GSK-3β inhibitor. In ICG-001-treated mice, BMD, BV and BV/TV were markedly decreased compared with mice stimulated with GSK-3β inhibitor (β inhibitor decreased the area of eroded bone surface (EBS) and increased bone thickness (BT) within an ROI in the calvaria of mice in the Ti particle-stimulated group. Tartrate-resistant acid phosphatase (TRAP) staining revealed the presence of lots of TRAP-positive cells within ROI in Ti particle-treated group. When LiCl was administered, TRAP-positive cell numbers decreased by 58.7%, and osteoclast surface per bone surface (OCS/BS) decreased by 64.0%, compared with those in Ti particle-treated mice. However, local treatment with the β-catenin selective inhibitor reversed the protective effects of LiCl on bone destruction  Guide for the Care and Use of Laboratory Animals and the Animal Care Committee of the First Affiliated Hospital of Soochow University. All experimental protocols in this study were approved by the ethics committee of the First Affiliated Hospital of Soochow University.30 Briefly, these particles were baked at 180 °C for 12 h, and then immersed in ethanol (75%) for 2 days. Endotoxin levels in the particles were determined using a commercial detection kit , and particles with <0.02 EU/ml endotoxin were used.Ti particles were obtained from Alfa Aesar  and were prepared as previously described.α-MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM l-glutamine. For differentiation experiments, cells were cultured in the above α-MEM and 50 μg/ml ascorbic acid , 10 mM β-glycerophosphate (Sigma), Ti particles, 10 mM lithium chloride  or 10 μm ICG-001 . The control cultures did not contain Ti particles.The murine MC3T3-E1 osteoblast cell line was obtained from ATCC  and maintained in alpha-minimum essential medium (4/ml) were seeded on 96-well plates and allowed to attach for 12 h in α-MEM containing 10% FBS. Cells were then cultured with 0.1 mg/ml Ti particles for 24, 48 or 72 h. At each time point, cells were incubated with fresh serum-free medium containing the CCK-8  reagent for 2 h at 37 °C. The culture medium was replaced with equal volumes of DMSO to dissolve formazan crystals, and then shook at room temperature for 10 min. Absorbance at 450 nm was obtained by using a microplate reader. Each reaction had three replicates and all experiments were repeated at least three times.MC3T3-E1 cells (10μg) was used to synthesize first-strand cDNA with an RT-PCR kit (Invitrogen). Glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used as an internal control. The mRNA levels of each gene from each experimental sample were normalized by comparison with a corresponding internal control using previously described methods.31 The primers used in this study are shown in Total RNA was extracted from MC3T3-E1 cells using TRIzol Reagent . RNA integrity was determined by separating the RNA on an agarose, and quality was assessed by having the A260/A280 ratio cutoff higher than 1.7. Total RNA . Briefly, the cell layers were washed in ice-cold PBS to remove residual particles and detached cells. Then, the cells were lysed in a solution containing 10 mM Tris and 0.1% TritonX-100 (pH 7.5). After centrifugation, the supernatants were collected and incubated in an alkaline buffer (pH 10.5) containing 5 mM pNPP (substrate) and 2 mM MgCl2O before the addition of 40 mM ARS solution  for 30 min at room temperature with gentle agitation. The stained cells were observed through a microscope. To quantify mineralization, the stained layers were washed five times for 5 min each with excess dH2O, and three times with PBS for 5 min each at room temperature. Mineral bound ARS was solubilized by the addition of 10% cetylpyridinium chloride . Dye absorbance was measured at 570 nm. Six wells were analyzed per experiment. Experiments were repeated at least three times.ARS was used to quantify the mineralization of MC3T3-E1 cultures in response to Ti particles and/or LiCl. Briefly, MC3T3-E1 cells were grown to confluence in culture media as previously described, and then grown for an additional 21 days in mineralization media in the presence or absence of Ti particles, and/or LiCl, at the indicated concentrations. Following treatment, mineralized osteoblasts were washed three times with PBS, and fixed in 4% paraformaldehyde for 1 h. The fixed layers were washed three times with excess dHμg of nuclear or total cell protein was used for western blotting. Samples were separated on SDS-PAGE, and proteins were then transferred to PVDF membranes. Immunoblotting was performed as described previously using specific antibodies raised against β-catenin , GSK-3β  and pSer9-GSK-3β . Protein quantification was performed using the Odyssey IR software, version 1.2 . Relative protein levels were calculated as the ratio of treated versus control, after normalizing against the housekeeping protein. Results are representative of three independent experiments.Protein quantification was performed using the BCA protein assay reagent, and 20 β-catenin/TCF target genes, MC3T3-E1 cells were transfected transiently with 2 μg TopFlash constructs by electroporation using Nucleofector technology. These constructs contained a firefly luciferase reporter under the control of a wild-type TCF/LEF-binding site. Cells were co-transfected with 0.2 μg of β-galactosidase reporter vector as an internal standard for transfection. Twenty-four hours post-transfection, cells were stimulated with the indicated concentrations of Ti particles, LiCl and/or Wnt3a. Following 12 h of stimulation in α-MEM with 5% FBS, luciferase and β-galactosidase reporter activities were assayed using Bright Glo and β-Glo assay kits, respectively . Topflash results were normalized to the activity of β-galactosidase.To assay for the activation of MC3T3-E1 cells with or without LiCl or ICG-001 pretreatment were incubated with 0.1 mg/ml Ti particles at 37 °C for 24 h. The culture medium was harvested for ELISA analysis. Mouse RANKL and OPG ELISA kits  were used to determine the levels of the two cytokines according to the manufacture’s instruction.μl PBS or ICG-001 (10 μg) at the surgery site before particle implantation, and then daily. After 2 weeks, mice were killed, and calvariae were collected for further analysis.For this study, we established a murine calvariae osteolysis model was established. Briefly, 40 C57BL/6 female mice (8-weeks-old) were divided into four groups: (a) control; (b) Ti; (c) LiCl; and (d) LiCl and ICG-001. The mice were first anesthetized, and then Ti particles (20 mg) were placed on the surface of calvariae of mice in groups (b), (c) and (d) groups. After surgery, mice in groups (c) and (d) were gavage-fed 200 mg/kg LiCl every day. In addition, the LiCl-treated mice were injected with 10 n=5 per group) were scanned and reconstructed into a 3D structure with micro-CT  at a normal resolution of 18 μm. The X-ray source was set at 80 kV and 100 μA, and exposure time was 100 ms. X-ray projections were obtained at 0.9 intervals with a scanning angular rotation of 360°. A cylindrical ROI (3 × 3 × 1 mm) was identified to reduce the bias of quantitative analysis of the calvaria.34 Parameters including BMD (mg/mm2), BV (mm3) and BV/TV (%) were calculated using the Skyscan CT-analyzer software.The calvariae (n=5 per group) were embedded in paraffin, and five sections were prepared for H&E and TRAP staining. EBS and BT were quantified from five consecutive sections per animal using Image Pro-Plus 6.0. 35 Then, TRAP-positive cells were identified, and OCS/BS (%) was calculated according to the methods established by Sawyer et al.36After decalcification in 10% EDTA for 21 days, calvariae (P-value <0.05.Data were obtained from three or more independent experiments. The significance of difference between groups was assessed by one-way ANOVA with post-hoc Tukey for multiple comparisons. Statistical significance was set as


Neurological disorders have been linked to abnormal excitatory neurotransmission. Perturbations in glutamate cycling can have profound impacts on normal activity, lead to excitotoxicity and neuroinflammation, and induce and/or exacerbate impairments in these diseases. Astrocytes play a key role in excitatory signaling as they both clear glutamate from the synaptic cleft and house enzymes responsible for glutamate conversion to glutamine. However, mechanisms responsible for the regulation of glutamate cycling, including the main astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2 or GLT-1 in rodents) and glutamine synthetase (GS) which catalyzes the ATP-dependent reaction of glutamate and ammonia into glutamine, remain largely undefined.Gain and loss of function for β-catenin in human progenitor-derived astrocyte (PDAs) was used to assess EAAT2 and GS levels by PCR, western blot, luciferase reporter assays, and chromatin immunoprecipitation (ChIP). Further, morpholinos were stereotaxically injected into C57BL/6 mice and western blots measured the protein levels of β-catenin, GLT-1, and GS.β-Catenin, a transcriptional co-activator and the central mediator of Wnt/β-catenin signaling pathway, positively regulates EAAT2 and GS at the transcriptional level in PDAs by partnering with T cell factor 1 (TCF-1) and TCF-3, respectively. This pathway is conserved in vivo as the knockdown of β-catenin in the prefrontal cortex results in reduced GLT-1 and GS expression.These studies confirm that β-catenin regulates key proteins responsible for excitatory glutamate neurotransmission in vitro and in vivo and reveal the therapeutic potential of β-catenin modulation in treating diseases with abnormal glutamatergic neurotransmission and excitotoxicity. The glutamate/glutamine cycle is an integral component of brain homeostasis. Neurons release glutamate, which is the most abundant neurotransmitter in the brain involved in learning, memory, and cognition. In excess, glutamate is neurotoxic and is linked to a number of neurodegenerative diseases such as Alzheimer’s disease , ParkinsAstrocytes have a dominant role in the glutamate/glutamine cycle. They clear >90 % of excess glutamate , 6 throuUsing microarray analysis, we previously showed that β-catenin knockdown in human progenitor-derived astrocyte (PDAs) leads to modulation of genes relevant in regulating aspects of glutamate neurotransmission . This fiWe evaluated here the role of β-catenin in regulating the expression of EAAT2 and GS in human astrocytes and mouse medial prefrontal cortex (mPFC). We show that β-catenin positively regulates EAAT2 and GS expression but through association with different TCF/LEF members. In regulating EAAT2 and GS expression, β-catenin interacts with TCF-1 and TCF-3, respectively. These findings demonstrate that the β-catenin pathway is a critical regulator of EAAT2 and GS expression, which can be harnessed to remove excess glutamate in neurodegenerative diseases.Progenitor-derived astrocyte (PDAs) were generated from neural progenitor cells  as previously described . BrieflyThe TOPflash plasmid (Millipore) detecting β-catenin signaling contains multiple TCF/LEF binding sites linked to a firefly luciferase reporter. An approximate 2.5-kb human EAAT2 promoter linked to firefly luciferase plasmid (EAAT2-prom) was a kind gift from Dr. Paul B. Fisher . A pcDNA plasmid harboring the constitutively active β-catenin gene termed as pABC  or control pcDNA (10792) was obtained from Addgene. Plasmids were transfected using Lipofectamine 2000 as per manufacturer’s instruction (Invitrogen). ON-TARGET plus SMART pool siRNAs specific for TCF-1 (L-019735-00), TCF-3 (L-014703-00), TCF-4 (L-003816-00), LEF1 (L-015396-00), β-catenin (L-003482-00), and scrambled (D-001810-10) all from Thermo Fisher Dharmacon were transfected using Lipofectamine siRNAmax (Invitrogen Life Technologies) according to the reagent protocol. Cells were approximately 70–80 % confluent at the time of transfection.Twenty-four hours post transfection, the culture medium was removed, cells were gently washed with PBS once, and 100 μl of passive lysis buffer (PLB) was added and incubated at 37 °C for 5 min. Cells were lysed by pipetting up and down several times and spun at 5000 rpm for 4 min to remove debris, and 10–20 μl was used to assay for luciferase activity using luciferase assay system (Promega) in a single injector luminometer. Total protein concentration was measured using bicinchoninic acid assay (BCA) protein assay kit (Thermo Fisher), and relative light units were normalized to micrograms of protein. Graphs were plotted from data obtained as a mean of three independent experiments with standard deviation as error bars.In vitro cell samples were lysed by incubating with 50 μl of PLB for 5 min at 37 °C and pipetting up and down several times. In vivo brain tissue samples were lysed by incubating with 100 μl Radioimmunoprecipitation Assay (RIPA) buffer and homogenized. Both lysates were cleared for cell debris by spinning at 5000 rpm for 5 min. Total protein content of the cleared lysate was estimated by BCA, and 5–20 μg of total protein was separated on a 10 % SDS PAGE, transferred to a nitrocellulose membrane, blocked with superblock (Thermo Fisher) containing 0.1 % Tween 20 (T20) for 1 h, and incubated with appropriate primary antibodies overnight at 4 °C in superblock-0.1 % T20. Membranes were washed extensively with Tris buffer saline containing 0.1 % T20 (TBST) and incubated with appropriate secondary antibody in Superblock-0.1 % T20 for 45 min at room temperature (RT). Membranes were again washed extensively in TBST and developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) according to instructions. Relative quantification of proteins were carried out using ImageJ software and normalized to GAPDH . Primary antibodies used were as follows: anti-rabbit β-catenin antibody , anti-mouse EAAT2 antibody , anti-mouse GS antibody , and anti-rabbit GAPDH antibody . Secondary antibodies were enhanced chemiluminescence horseradish peroxidase (ECL HRP)-linked rabbit IgG whole antibody (7074) or ECL HRP-linked mouse IgG whole antibody (7076) from Cell Signaling.CT method with GAPDH as the endogenous control.RNA was isolated using RNeasy mini kit (Qiagen). Digested with DNaseI (Sigma-Aldrich) for 15 min at RT to remove DNA contamination, and subsequently, DNaseI was inactivated by heating at 70 °C for 10 min. cDNA was synthesized using Qscript supermix (Quanta Biosciences). Real-time PCR was performed using SSO fast SYBR green supermix (Bio-Rad) in a 7500 real-time PCR system (Applied Biosystems) using 7500 software v2.0.1. Melting curve analysis was performed to ensure the amplification of a single product. Primers used were the following: TCF-1-F-5′-AGGCCAAGAAGCCAACCATCAAGA and TCF-1-R-5′-ACTCTGCAATGACCTTGGCTCTCA; TCF-3-F-5′-TGCAGTGAGCGTGAAATCACCAGT and TCF-3-R-5′-AATGGCTGCACTTTCCTTCAGGGT; TCF-4-F-5′-TCGGCAGAGAGGGATTTAGCTGATGT and TCF-4-R-5′-CTTTCCCGGGATTTGTCTCGGAAACT; LEF1-F-5′-AAGCATCCAGATGGAGGCCTCTACAA and LEF1-R-5′-TGATGTTCTCGGGATGGGTGGAGAAA; β-catenin-F-5′-TCTTGCCCTTTGTCCCGCAAATCA and β-catenin-R-5′-TCCACAAATTGCTGCGTCCCA; EAAT2-F-5′-CCAAGCTTGGATCACTGCCCTGG and EAAT2-R-5′-CCAGCCCCAAAAGAGTCACCCACAA; GS-F-5′-TTGAGAAACTAAGCAAGCGGCACC and GS-R-5′-ATCCAGTTAGACGTCGGGCATTGT; and GAPDH-F-5′-CTTCAACGACCACTTTGT and GAPDH-R-5′-TGGTCCAGGGGTCTTACT. Fold change in messenger RNA (mRNA) expression was calculated by relative quantification using the comparative 6 cells and 5 μg of antibody were used. Samples were analyzed by real-time PCR as explained earlier. Primers used to amplify EAAT2 promoter regions were divided into three regions: proximal (F-AAGACACACACTTACCCTTGACGG and R-CGTCTTAGGGCATTTGACTTTGGG), middle (F-TGTGGCCCTCCAAGTGAGTTCTTT and R-TGACGAGACCTGTGCAGCTTTGAT), and distal (F-GGGTGATGTCAGCTCTCGACGAA and R-AGGGAGGGATTGCAAGGTTTAGC). Primers used to amplify the putative GS promoter region were GS-F-TCTCTTGATGGTGCTGCTGTCACT and GS-R-AGAACGGAATGGTAAGGCGTGAGT. Data was normalized to IgG and represented as fold change with respect to IgG.PDAs were grown for 24–48 h and ChIPed for the presence of TCF-1 and β-catenin on the endogenous promoter region of EAAT2 or GS using a Millipore kit with antibodies for TCF-1, TCF-3 , and β-catenin . A suitable mouse IgG1 isotype control (5415) or rabbit IgG control (3900) from Cell Signaling was included in the experiments. Similarly, ChIP was performed on PDA cells transfected with EAAT2-prom for 24 h using anti-TCF-1 antibody. Per immunoprecipitation, approximately 2 × 10Cells either transfected with plasmids for 24 h or siRNAs for 72 h were spiked with 2 mM L-glutamic acid (glutamate) and incubated for 30 min, and the final concentration of glutamate in the supernatant was measured by glutamate assay kit (Biovision) as per instructions. A standard curve was generated by diluting the provided glutamate standard in complete DMEM media.2 inhalation followed by rapid decapitation 4 days following the second surgery, and brain punches were isolated from the region targeted with the injections. Punches were homogenized in RIPA buffer, and western blots were run as described above.Animal studies were conducted with approval from the Rush University Medical Center Institutional Animal Care and Use Committee (IACUC). Male 10C57bl/6J mice aged 4–6 weeks were purchased from Jackson Laboratories. Approximately 1 week after arrival, animals were anesthetized with ketamine/xylazine  and placed in a stereotaxic apparatus. Bilateral injections of vivo-morpholinos  were targeted to the mPFC with coordinates relative to Bregma +2.0 A/P, +0.3 M/L, −1.0 D/V. The antisense (AS) sequences for mouse β-catenin was designed from the larger of the two mRNA variants, one near the 5′ region (AS) 5′-GCCGCACAAGGAGCGATTTATAAGC-3′ and one spanning the translational start site (AS2) 5′-CTTGAGTAGCCATTGTCCACGCAGC-3′. A control morpholino sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′ or PBS was injected as a control. All morpholinos were at a concentration of 500 nM per 1.0 μl. Mice received 1 μl/hemisphere microinjections on day 1 and day 3. Mice were sacrificed by COWe performed our studies in human progenitor-derived astrocyte (PDAs). PDAs exhibit prototypical characteristics of astrocytes including expression of GFAP, EAAT2, and glutamate synthetase and are capable of glutamate uptake in a standard glutamate uptake assay . To asseTo determine if the ability of β-catenin to induce EAAT2 is mediated at the promoter level, an approximate 2.5-kb human promoter for EAAT2 linked to luciferase was transfected into PDAs. This EAAT2 promoter is well characterized . PDAs weIn the canonical Wnt pathway, β-catenin regulates its target genes by partnering with one of its downstream transcription factors such as TCF-1, TCF-3, TCF-4, or LEF1. To determine the partner of β-catenin in regulating EAAT2 gene expression, we performed bioinformatic studies to identify putative TCF/LEF sites on the EAAT2 promoter. Analysis of the 2.5-kb promoter revealed the presence of at least eight TCF/LEF binding consensus sites (CAAAGA) clustered into three regions, which we designated as proximal, middle, and distal regions with respect to the transcription initiation site Fig. . PreviouIn order to assess the role of the β-catenin pathway in regulating the expression of GS, we KD β-catenin in PDAs for 72 h and evaluated the expression of GS at the mRNA and protein levels. We show that β-catenin KD significantly inhibited the expression of GS mRNA (~80 %) and protein Fig. , b whereβ-Catenin regulation of EAAT2 and GS is not limited to PDAs. Using an additional primary human astrocyte, we show that β-catenin KD by siRNA in NHAs resulted in a significant reduction in EAAT2 mRNA by 60 % and in GS mRNA by 50 % Fig. . Given tWe next sought to determine whether β-catenin regulates GLT-1 (EAAT2) and GS expression in vivo. Vivo-morpholinos  were designed to block translation initiation of β-catenin in the cytosol Fig. . Mice 1. Among tThe β-catenin signaling pathway is vital to various functions in the CNS ranging from memory consolidation in astrocytes, neurogenesis, and neurotransmitter release to induction of long-term potentiation and depolarization resulting in increased synaptic strengths , 23. DisThrough gain and loss of function studies as well as ChIP assays, we demonstrate the molecular mechanism by which β-catenin regulates EAAT2 and GS expression in astrocytes. We show that β-catenin partners with TCF-1 to induce EAAT2 expression and partners with TCF-3 to induce GS gene expression. Although we show that TCF-1 binds to EAAT2 promoter and regulates its expression, other members of TCF/LEF could also be binding to this promoter. In regulating GS gene expression, β-catenin may partner with additional transcriptional factors in addition to TCF-3. This is partly because, although we observed a statistically significant decrease in GS mRNA expression in TCF-3 KD astrocytes, this KD was only 35–40 % less than the control suggesting that other transcription factors may also be involved.Our in vivo studies also confirmed that β-catenin positively regulates EAAT2/GLT-1 and GS expression. We used vivo-morpholinos to KD β-catenin in the prefrontal cortex of mice. Vivo-morpholinos are well established to effectively KD proteins of interest in many target organs in small rodents –26. HoweThe finding that β-catenin positively regulates EAAT2 and GS has a broader applicability in understanding mechanisms of neurodegenerative diseases which can be related to chronic neuroinflammation. The role of astrocytic β-catenin in neuroinflammation has not been well characterized. Total β-catenin has been shown to be dysregulated in a number of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and neuroAIDS, in neuropsychiatric disorders such as schizophrenia as well as a decline in Wnt/β-catenin signaling due to aging processes , 27–30. Past strategies to eliminate excess glutamate signaling via antagonizing NMDAR receptor (memantine) have had many side effects  with litIn summary, we demonstrate that β-catenin signaling in primary human astrocytes regulates key proteins of glutamate/glutamine cycling. β-Catenin partners with transcription factor TCF-1 to positively regulate EAAT2 protein expression. Further, β-catenin KD decreases GS in part through partnering with TCF-3 although there are likely other transcriptional binding partners as TCF-3 KD resulted in a partial decrease in GS expression. Using vivo-morpholinos to KD β-catenin expression in the prefrontal cortex of mice, we show that KD of β-catenin results in KD of GLT-1/GS. By showing here that β-catenin regulates glutamate/glutamine cycle, our findings can now be exploited as a novel and viable target to induce EAAT2/GS on astrocytes and reduce excess glutamate potentially diminishing neuroinflammation/neurodegeneration.


KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutation. The lineage transcription factor ETV1 is expressed similarly in GISTs regardless of malignant potential. Although the related transcription factor ETV4 has been associated with metastasis and tumor progression in other cancers, its role in GIST is unknown. In this study, we found that ETV4 levels were high in a subset of human GISTs and correlated with high mitotic rate. Through Gene Set Enrichment Analysis in selected human GISTs, we identified a relationship between ETV4 levels and β-catenin signaling, especially in advanced GISTs. GIST specimens with high ETV4 levels overexpressed cell cycle regulating genes and had aberrant activation of the canonical Wnt pathway. In human GIST cell lines, ETV4 RNA interference suppressed cell cycle genes and Wnt/β-catenin signaling. ETV4 knockdown also reduced tumor cell proliferation, invasion, and tumor growth in vivo. Conversely, ETV4 overexpression increased cyclin D1 expression and Wnt/β-catenin signaling. Moreover, we determined that ETV4 knockdown destabilized nuclear β-catenin and increased its degradation via COP1, an E3 ligase involved in both ETV4 and β-catenin turnover. Aberrant accumulation of ETV4 and nuclear β-catenin was found in patient derived xenografts created from metastatic GISTs that became resistant to tyrosine kinase inhibitors. Collectively, our findings highlight the significance of ETV4 expression in GIST and identify ETV4 as a biomarker in human GISTs.Gastrointestinal stromal tumor (GIST) is the most common sarcoma, often resulting from a  KIT or PDGFRA [KIT mutation [p16 (CDKN2A) gene deletion and inactivation of myc-associated protein (MAX) are found to be common genetic aberrations in GIST progression [Gastrointestinal stromal tumor (GIST) is the most common subtype of human sarcoma and typically occurs in the stomach or small intestine . The majr PDGFRA , 3. The r PDGFRA . Unfortumutation . Althougmutation , 7, the gression , 9, a beETV1, ETV4, ETV5, and ERG are members of the erythroblast transformation specific (ETS) transcription factor family and are well established as oncogenes in certain cancers , 11, whiThe role of ETV4 in GIST has not been defined. In this study, we discovered that ETV4 was overexpressed in a subset of human GISTs and modulated cell cycle regulation and Wnt/β-catenin signaling, which we previously demonstrated to be important in GIST malignancy . KnockdoP < 0.05). Conversely, expression of the GIST lineage survival factor ETV1 did not correlate with mitotic rate or whether a tumor was metastatic  cells  with either ETV4-specific siRNA or non-target control siRNA. Effective knockdown of ETV4 with different constructs was confirmed by real-time PCR Figure . NotablyA Figure . Using KA Figure , consistA Figure . We alsoA Figure .To extend our observations, we examined cell cycle and Wnt signaling components in GIST T1 cells after 96h of transfection using control siRNA or two ETV4 siRNA constructs. We found that ETV4 knockdown reduced the phosphorylation of AKT and GSK3β serine 9 in GIST T1 cells Figure , which fTo test whether overexpression of ETV4 affects the cell cycle and Wnt/β-catenin signaling, we transfected GIST T1 cells with a control or an ETV4-overexpression plasmid. ETV4 overexpression was confirmed in the nuclear extract Figure , left, aETS family members have been shown to regulate Wnt/β-catenin signaling by directly binding to the promoter of the co-transcription factor LEF1 or by controlling β-catenin stability , 27. HowTo further assess whether nuclear β-catenin stability is affected by ETV4, we examined nuclear β-catenin turnover in control or ETV4 siRNA-transfected GIST T1 cells in a cycloheximide (CHX) chase assay. ETV4 silencing significantly decreased the half-life of β-catenin Figure . In our + cells isolated from 4 metastatic, imatinib-resistant tumors and 8 primary, untreated tumors [CDC20 findings in ETV4-low and ETV4-high human GISTs , as we have demonstrated before as a negative regulator of the Wnt pathway in GIST [To determine the relevance of our ETV4 findings in GIST cell lines, 36 selected human GISTs  vs. ) were analyzed by high throughput RNA-sequencing. Gene Set Enrichment Analysis (GSEA) was performed to probe the pathway differences between these two groups. Similar to our findings in GIST T1 cells, the results showed upregulation of cell cycle genes and activation of the canonical Wnt pathway in metastatic, imatinib-resistant GIST with a high mitotic rate Figure  and 6B. d tumors . We valis Figure , left. D in GIST , was als in GIST . These rP < 0.001) between ETV4 and β-catenin expression , moderate (1+), or high (2+), and varied among specimens Figure . There wn Figure . Previoun Figure . Consistn Figure . Thus, hAlthough the ETS transcription factors ETV1 and ETV4 exhibit highly conserved homology within their DNA-binding domains, they seem to have distinct roles in GIST. ETV1 appears to be a lineage survival factor in GIST as it is involved in tumor initiation and proliferation , 14. Accin vitro, as well as tumor growth in vivo. ETV4 is known to activate the transcription of multiple tumor-associated genes, including MMP genes, Cox2, Cyclin D3, and Snail, suggesting a role in tumor progression and metastasis [MYC and other proliferation genes in prostate cancer cell lines [We found that ETV4 is an important oncogenic contributor to GIST since knockdown of ETV4 expression by RNA interference in both human and murine GIST cell lines suppressed tumor cell proliferation and invasion tastasis . Transcrll lines . ETV4 inll lines , 36. Accll lines . FurtherOur GSEA data linked ETV4 expression with Wnt pathway deregulation in metastatic, imatinib-resistant human GIST. It is important to note that since most metastatic, imatinib-resistant patients were also recurrent GISTs in our cohort, we could not analyze these groups separately although recent literature suggests there may be significant differences between these two groups . NeverthAnother potential mechanism to explain the relationship of ETV4 and β-catenin is that ETV4 is associated with the expression of pathway components and regulators of Wnt/β-catenin signaling. ETV4-high human GISTs had increased pathway activation compared to ETV4-low tumors, as evidenced by dephosphorylated active β-catenin, the transcription factor TCF4, and the co-receptor LRP6. Moreover, the Wnt pathway antagonist DKK4 was substantially lower in ETV4-high human GISTs. Our previous work showed that silencing of DKK4 in GISTs contributed to aberrant Wnt pathway activation .KitV558Δ/+ tumors [in vitro knockdown of ETV4 significantly decreased activated AKT signaling with a concomitant decrease of phospho-GSK3β in GIST T1, GIST882, and murine S2 GIST cell lines. In contrast, overexpression of ETV4 in GIST cell lines increased phospho-AKT activation and active β-catenin. A specific dual PI3K/AKT inhibitor, NVP-BEZ235, also inhibited GSK3β phosphorylation and β-catenin accumulation in the presence or absence of Wnt pathway activators in GIST cells  and KIT- populations using human CD117 microbeads (Miltenyi Biotec). The purity of KIT+ cells was greater than 90% by flow cytometry.Under an IRB approved protocol, we obtained 55 GIST specimens from consenting patients undergoing tumor resection at Memorial Sloan Kettering Cancer Center. Mitotic rate (number of mitoses per 50 high power fields) and mutation status for each specimen were obtained from pathology reports. Tumors were categorized into 2 groups based on the type of tumor (primary or metastatic) and the radiologic tumor response to tyrosine kinase inhibitors at the time of surgery ). Single cell suspensions were generated from fresh tumor tissues by a modified collagenase method as before , and sepFormalin-fixed and paraffin-embedded human GISTs were sectioned at 5 μm thickness. Antigen retrieval was achieved with citrate buffer. Immunohistochemistry was performed using anti-human ETV4 IgG  and anti-human β-catenin IgG  or the corresponding isotype control IgG (DAKO).KIT exon 13 mutant) as well as human GIST T1 and murine S2 GIST cells (both KIT exon 11 mutant) have been described previously [The human GIST882 cell line . Wells contained 15% FCS growth medium to serve as a chemoattractant. After 24h, the non-invading cells were gently removed from the upper chamber by a cotton swab. Cells that invaded into the lower chamber were fixed and stained with Diff-Quick (Siemens). The number of invading cells in the 2 groups was compared according to the manufacturer’s instruction. To determine the effect of ETV4 knockdown on tumor growth in vivo, 1x105 stably transfected GIST882 cells or murine S2 cells were injected subcutaneously (s.q.) into the right flank of NOD.Cg-PrkdcscidIL12rgtm1Wjl/SzJ (NOD scid gamma (NSG), Jackson Laboratory) mice. All mouse studies were approved by the Institutional Animal Care and Use Committee.Cell viability  of the stable GIST882 or murine S2 cells with control shRNA or ETV4 shRNA in 96-well plates was determined at 48 and 72h according to manufacturer’s instructions by optical density at 450 nm. Cell invasion of 1x10Total RNA was extracted from human GIST tissues or transfected cells using the RNAeasy Plus Mini Kit (Qiagen). Real-time PCR was performed as before . The folWestern blot of whole protein lysates or nuclear proteins from frozen tumor tissues or cells was performed as previously described . Antibodt = 0h negative siRNA control.GIST T1 cells were transiently transfected with control siRNA or ETV4 SMARTpool siRNA for 48h. Cells were collected after the addition of 200 μg/ml CHX at the indicated time points with or without 10 μM MG132. Cell lysates from nuclear extracts were assessed for signaling. Quantification was achieved by Image J software. The final β-catenin turnover rate at each time point is the percentage of β-catenin/lamin B1, then expressed relative to the GIST T1 cells were transiently transfected with neg siRNA and ETV4 siRNA for 48 hours. Cells were harvested, washed in PBS and fixed with ice-cold 70% ethanol at 4°C for at least 30 min. After washing with PBS, cells were incubated with propidium iodide (PI) (0.05 mg/ml) and ribonuclease (Sigma) at 37°C for 30 min. Flow cytometry was performed for cell cycle distribution.http://software.broadinstitute.org/gsea) [RNA was isolated from 36 selected human GIST specimens using the RNAeasy Plus Mini Kit (Qiagen). High throughput RNA sequencing was performed by the Integrated Genomics Core laboratory of Sloan Kettering Institute using the Illumina platform. GSEA was performed on the RNA sequencing data using gene sets from the MSigDB (Molecular Signatures Database) using the GSEA software package (rg/gsea) . The gen6 cells were injected into the flanks of NSG mice. Mutation status of each tumor was determined. GIST T1 xenografts were used as a comparison with these PDXs, since we have not been able to generate a PDX from a primary, untreated human GIST.PDXs were established from 4 metastatic, imatinib-resistant human GISTs. Bulk tumor cells were freshly isolated from GIST tumors using collagenase as above and 1x10t tests unless otherwise indicated using Prism 6.0 software (Graph Pad Software). P < 0.05 was considered significant.Data are expressed as mean ± SEM or median. Differences were detected by unpaired 2-tailed Student’s


The Wnt/β-catenin pathway is an evolutionarily conserved signaling pathway critical for several biological processes. An aberrant Wnt/β-catenin signaling is linked to several human diseases. Emerging studies have highlighted the regulatory role of the Wnt/β-catenin signaling pathway in normal physiological processes of parenchymal and hematopoietic cells. Recent studies have shown that the activation of Wnt/β-catenin pathway in dendritic cells (DCs) play a critical role in mucosal tolerance and suppression of chronic autoimmune pathologies. Alternatively, tumors activate Wnt/β-catenin pathway in DCs to induce immune tolerance and thereby evade antitumor immunity through suppression of effector T cell responses and promotion of regulatory T cell responses. Here, we review our work and current understanding of how Wnt/β-catenin signaling in DCs shapes the immune response in cancer and autoimmunity and discuss how Wnt/β-catenin pathway can be targeted for successful therapeutic interventions in various human diseases. The immune system functions constantly to defend from pathogens while preserving tolerance to self-antigens and commensal microbes , 2. ActiDendritic cells are crucial in the induction of immunity and tolerance. Every tissue in humans is armed with DCs to sense and induce inflammatory immune responses against pathogens or to preserve a tolerogenic environment under steady state conditions. DCs equipped with pattern-recognition receptors (PRRs) play a central role in detection of pathogen-associated molecular patterns (PAMPs) and induction of robust host immunity through a controlled activation of different arms of the innate and adaptive immune responses , 6. DCs + T helper 1 (Th1) and CD8+ cytotoxic T cell responses, whereas extracellular pathogens activate DCs to induce IL-4-producing CD4+ T helper 2 (Th2) cell responses (+CD4+ T helper 17 (Th17) cell responses against fungi and extracellular bacteria through increased production of IL-6, TGF-β, and IL-23  such as ulcerative colitis and Crohn’s disease . In IBD,+ T cells to myelin-specific effector Th1 and Th17 cells (+ T cells in causing EAE and MS pathogenesis (Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory disease that leads to multifocal demyelination in the white matter of the human CNS with debilitating motor and sensory dysfunction , 45. Pre17 cells , 46. Furogenesis , 48. Altogenesis , 4, 6. H+ T cell responses and diminished production of anti-inflammatory cytokines such as IL-10, TGF-β, and IL-27 , a negative regulator of the Wnt/β-catenin pathway, is highly upregulated in the serum during lupus progression . Similar2+ non-canonical pathway has been correlated with the pathophysiology of psoriasis  and co-inhibitory (PD-L1 and PD-L2) molecules . Our datt tumors , 75. Thot tumors , it remand IL-10 .Since Wnt-mediated activation of β-catenin in the TME promotes tumor progression through multiple mechanisms, pharmacological suppression of the β-catenin pathway represents a promising target for effective antitumor immunotherapy. Accordingly, treatment of mice with inhibitors that blocks the β-catenin/TCF pathway results in delayed tumor growth through suppression of regulatory T cell responses and increased antitumor CD4 and CD8 T cell responses –73, 75. 2+ pathway has been associated with the pathophysiology of psoriasis (The Wnt/β-catenin pathway in DCs is a key player in the pathogenesis of a wide amalgam of inflammatory and infectious diseases . Wnt/β-csoriasis , 62. Pha+ T cells and different subsets of CD4+ T cells are warranted for better therapeutic targeting of this pathway in autoimmunity and cancer. In this regard, recent advances in targeted delivery of small molecules and biologics represent a novel therapeutic approach to deliver Wnt/β-catenin signaling modulator specifically to DCs for clinical management of autoimmunity and cancer. Additionally, combining DCs-specific Wnt/β-catenin pathway inhibition with recent successful therapies targeting immune checkpoints represents a promising approach to promote antitumor immune responses.Optimal functioning of Wnt/β-catenin pathway in different type of non-immune and immune cells including DCs is vital for a range of cellular functions and homeostasis. Consequently, targeting Wnt/β-catenin pathway specifically in DCs and particularly in those DCs, which play a central role in the induction of autoimmune pathology or tumor progression is critical for successful therapeutic intervention to minimize off-target side effects. Therefore, it is crucial to understand the biology of different subsets of DCs and how Wnt/β-catenin pathway in these DCs subsets contributes to normal health and immunopathology. Moreover, further studies on understanding the biology Wnt/β-catenin signaling in other immune cells such as B cells, CD8The normal immune system functions by instigating an effective immune response against infectious agents while promoting tolerance toward commensal microbiota and self-antigens. The Wnt/β-catenin pathway participates in this balance through diverse mechanisms including the generation of tolerogenic DCs to regulate inflammatory responses. Inadequacies in the signaling of the Wnt/β-catenin pathway may result in allergic and autoimmune diseases through chronic and uncontrolled activation of the immune system or cancer due to inadequate immune surveillance resulting from excessive immune tolerance. Thus, Wnt/β-catenin signaling serves as a molecular switch between opposing immune functions, and targeting different elements of this pathway may provide therapeutic benefits in the remediation of autoimmune diseases, cancer, and infectious diseases. While it is clear that Wnt-signaling programs DCs to induce robust regulatory T cell responses and cytokines to contain inflammation, several important questions remain unanswered. It is unknown, for example, how the canonical and non-canonical pathways act in tandem to regulate immunity and tolerance, and how much crosstalk is involved with other pathways involved in the regulation of immune responses. Dissecting many of these issues around Wnt/β-catenin signaling in immune cells potentially helps in developing novel therapeutic strategies against various diseases stemming from an imbalance in the immune system.AS and RT have performed bibliographic researches and drafted the manuscript. DS has performed bibliographic researches. SM has performed bibliographic researches and drafted the 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.


Lithium salt is a classic glycogen synthase kinase 3 (GSK3) inhibitor. Beryllium is a structurally related inhibitor that is more potent but relatively uncharacterized. This study examined the effects of these inhibitors on the phosphorylation of endogenous GSK3 substrates. In NIH-3T3 cells, both salts caused a decrease in phosphorylated glycogen synthase, as expected. GSK3 inhibitors produce enhanced phosphorylation of Ser9 of GSK3β via a positive feedback mechanism, and both salts elicited this enhancement. Another GSK3 substrate is β-catenin, which has a central role in Wnt signaling. In A172 human glioblastoma cells, lithium treatment caused a surprising increase in phospho-Ser33/Ser37-β-catenin, which was quantified using an antibody-coupled capillary electrophoresis method. The β-catenin hyperphosphorylation was unaffected by p53 RNAi knockdown, indicating that p53 is not involved in the mechanism of this response. Lithium caused a decrease in the abundance of axin, a component of the β-catenin destruction complex that has a role in coordinating β-catenin ubiquitination and protein turnover. The axin and phospho-β-catenin results were reproduced in U251 and U87MG glioblastoma cell lines. These observations run contrary to the conventional view of the canonical Wnt signaling pathway, in which a GSK3 inhibitor would be expected to decrease, not increase, phospho-β-catenin levels.This article has an associated First Person interview with the first author of the paper. Summary: GSK3 inhibitors have potential use against Alzheimer's disease and other conditions. In this study, a classic inhibitor produced unexpected molecular effects on key components of the Wnt signaling pathway. Glycogen synthase kinase 3 (GSK3) has multiple functions in cell growth and differentiation . It has GSK3 is constitutively active, and hormones and other molecules that mediate signaling through GSK3-containing pathways typically trigger a reduction in GSK3 kinase activity. The insulin signal transduction pathway provides a clear example of this. GSK3 phosphorylates glycogen synthase (GS) at a cluster of residues  to maintain GS in a dormant state . InsulinIn the canonical Wnt signaling pathway, GSK3 partners with APC, axin, and other proteins to form a β-catenin ‘destruction complex’ . With thModulators of GSK3 activity have potential as therapeutic agents. GSK3 inhibitors could be useful in the treatment of Type 2 diabetes. GSK3 inhibitors are also being considered for their potential to ameliorate the progression of Alzheimer's disease. Neurofibrillary tangles and amyloid plaques are histological hallmarks of this disease. The neurofibrillary tangles are composed of hyperphosphorylated tau protein, and GSK3 is thought to be the principal kinase responsible for tau phosphorylation. Therefore, GSK3 inhibitors could suppress tau phosphorylation in brain tissue and thereby decrease the prevalence of neurofibrillary tangles. With respect to Wnt signaling, the effects of GSK3 inhibitors could be problematic. Turnover of β-catenin protein requires the phosphorylation of β-catenin by GSK3. Inhibition of GSK3 should lead to hypophosphorylation of β-catenin, increased abundance, and nuclear accumulation, which would promote the transcription of growth promoting gene products. In the context of the conventional view of Wnt signaling, one would have to consider the possibility that GSK3 inhibitors could be carcinogenic.Lithium salt is a classic GSK3 inhibitor . Synthet2+ ion as a cofactor. Lithium and beryllium (atomic numbers 3 and 4) have an ionic radius smaller than that of the magnesium ion (atomic number 12). Like Li+, Be2+ inhibits GSK3 kinase activity, but beryllium is about 1000-fold more potent . Be2+ at 30 and 100 µM produced a similar decrease, whereas 100 µM Ca2+ had no effect. Li+ and Be2+ did not change the levels of total GS, total GSK3α, or total GSK3β.This study aimed to investigate the effects of lithium and beryllium salts on the phosphorylation of endogenous GSK3 substrates. In order to assess more normal physiological conditions, untransfected cells were used instead of cells overexpressing recombinant substrates. In NIH-3T3 mouse embryo fibroblast cells, 20 mM Li+ and Be2+ in NIH-3T3 cells . However, previous studies using A172 cells showed that, in that cell type, increased Ser9-GSK3β phosphorylation can be elicited by 20 mM Li+ but not by 30 or 100 µM Be2+ , in which the normalization was based on the total protein concentration of the cell lysates, and by flow cytometry analysis , in which the normalization was on a per cell basis. In the flow cytometry assay, the mean fluorescence increased by 113%, 24%, and 143% for 20 mM Li+, 30 µM Be2+, and 100 µM Be2+, respectively, compared to untreated control cells. Thus, the consequences of Be2+ treatment with respect to this phosphorylation event appear to be cell type-dependent.GSK3 inhibitors elicit an increase in the phosphorylation of GSK3β at the Ser9 position. This is because GSK3 inhibitory effects are amplified via a positive feedback loop involving a GSK3-dependent phosphatase activity . Phospho µM Be2+ . Conside+ and Be2+ act as GSK3 inhibitors when added to cell culture media. We next sought to examine the effects of these salts on β-catenin. In western blots, β-catenin bands were faint with NIH-3T3 cells. Therefore, A172 human glioblastoma cells were selected for study. These cells express the protein well, and the responsiveness of this cell type to the cytostatic effects of Li+ and Be2+ has been characterized . Next, the capillary electrophoresis method was validated as a means to quantitate phosphorylation levels in differentially treated cells . At 50 mM, the highest concentration tested, cytotoxicity was evident. Lithium caused increased phospho-Ser9-GSK3β . However, the electropherogram tracing is a more accurate representation of the raw data. The data are shown in this format for the most important subset of the lithium dose-response results, pertaining to phosphorylation of β-catenin (Table S1). Depictions of normalized phospho-Ser33/Ser37-β-catenin and normalized phospho-Ser9-GSK3β [i.e. (phosphoprotein)/ ratios] are also provided .The spatial distribution of chemiluminescence signal in each capillary can be depicted as a ‘virtual’ western blot. This is a convenient way to display a large set of results concisely . Instead, BeSO4 appeared to diminish phospho-Ser33/Ser37-β-catenin somewhat, especially at 100 µM, but a clear dose-dependent relationship was not evident. The phosphatase inhibitor calyculin A, which was used as a positive control, caused an increase in phospho-Ser33/Ser37-β-catenin, as expected . BeSO4 had no appreciable effect on phosphorylation of GSK3β at Ser9 in A172 , confirming previous observations for this cell type . Examination of GAPDH and total protein by capillary electrophoresis confirmed that the total protein levels in each sample were well-matched . BeSO4 at 10-1000 µM did produce an increase in p21 protein expression , which is a known response to Be2+ in this cell type .In contrast to LiCl, BeSOell type . Howeverell type , confirmIn the conventional model of Wnt signaling, diminished GSK3 kinase activity causes β-catenin hypophosphorylation, enabling the protein to escape phosphorylation-directed degradation, which leads to the entry of β-catenin into the nucleus. Considering that lithium causes β-catenin hyperphosphorylation in A172 cells, we wondered whether β-catenin would enter the nucleus after lithium treatment in this cell type. To address this question, confocal microscopy was used to examine the subcellular distribution of β-catenin . A172 ceTable S1).Although phosphorylation of β-catenin provides a signal for degradation in the conventional model of Wnt signaling, the phosphorylated form accumulated without degradation in lithium-treated A172 cells. One possible explanation for this observation could be that ubiquitination is somehow decoupled from phosphorylation in lithium-treated A172 cells. The scaffolding protein axin and the E3-ubiquitin ligase β-TrCP are each constituents of the β-catenin destruction complex . If lithIn order to gauge whether the unexpected effects of lithium treatment were specific to A172 cells or more broadly applicable, we chose four additional cell lines for analysis. Because A172 cells are from glioblastoma, we looked at two more human glioblastoma lines, U87MG and U251, to see whether glioma cells have a general tendency to respond to lithium in this manner. U87MG cells express wild-type p53 protein, whereas U251 cells express mutated p53. Both cell lines express β-catenin at moderate levels . To reprt-test P-values of 0.02, 0.03, and 0.04, respectively, but the ANOVA was just short of significance with P=0.056 (Table S1). The general trends in U87MG were fairly similar, but the magnitude of the effects was lower. Like A172, both U251 and U87MG displayed dose-dependent decreases in axin levels, down to 70% of control in the case of U87MG. The complete set of raw data and statistical analysis for the U251, U87MG, RKO, and HT-1080 experiments is provided (Table S1). Depictions of normalized phospho-Ser33/Ser37-β-catenin [i.e. (phospho-β-catenin)/ ratios] are also provided .The two additional glioma cell lines responded to lithium in a manner that was generally similar to A172 cells, although the effects in A172 were more pronounced. Lithium caused a large increase in phospho-β-catenin in U251 and U87MG cells, and a proportionally much smaller increase in total β-catenin . For U25Figs S8 and S13, Table S1). With respect to all of the antigens, the responses of HT-1080 cells were intermediate between the contrasting responses of RKO cells and glioma cells.The RKO cell type provided an interesting contrast to the glioma cell lines. Lithium produced a massive increase in total β-catenin, raising it to 353% of control in the case of 30 mM LiCl, representing a 253% increase . This is2+ at 100 µM decreased phospho-β-catenin as seen in western blots , but the magnitude of this effect relative to experimental variation was modest. In contrast, Li+ increased phospho-β-catenin . At 50 mM, a concentration that exhibits some cytotoxicity, phosphorylation began to decline yet still exceeded the levels seen in untreated cells.Lithium is a classic GSK3 inhibitor, and beryllium is a structurally related inhibitor. One of the most important GSK3 substrates is β-catenin. In A172 cells, Bern blots D and in -catenin . This wa-catenin ; Fig. S5Lithium-induced hyperphosphorylation of β-catenin was observed in two other glioma cell lines, showing that the response is not restricted to A172 cells, and suggesting that it may be common in this cell type. The phenomenon was also observed in the non-glial cell types RKO and HT-1080, but in these cell lines the change in phospho-β-catenin was more or less proportional to the change in total β-catenin. Even a proportional increase does not seem to comport with the standard view of canonical Wnt signaling. If the total pool of β-catenin increases because the subset of β-catenin that is phosphorylated is degraded, it is not clear why phospho-β-catenin should increase in proportion to total β-catenin, especially under conditions in which the kinase responsible for the phosphorylation is pharmacologically inhibited.A172 cells possess wild-type p53. Lithium treatment activates p53 in A172 cells. There are known interactions between p53 signaling and the Wnt/GSK3/β-catenin signal transduction pathway, so experiments were designed to test whether p53 has a role in the unexpected hyperphosphorylation of β-catenin in response to lithium. However, this possibility was discounted after comparing normal A172 cells and a p53 knockdown A172 cell line. In A172 cells, the presence or absence of p53 function did not fundamentally alter the effects of lithium on β-catenin hyperphosphorylation. This conclusion was further reinforced by comparing U87MG (wild-type p53) and U251 (mutated p53), which gave similar results.In vitro phosphatase assays show that lithium does not inhibit PP2A directly . The generation of stable p53 knockdown A172 cell lines using lentivirus has been previously described . These c4•4H2O  and LiCl (Sigma) were prepared in ultrapure water and sterile filtered. MG-132 and SB216763 were from Santa Cruz Biotechnology  and calyculin A was from Cell Signaling Technology . Hoechst 33342 dye was from Sigma.Stocks of BeSOp-nitrophenyl phosphate). Lysate was clarified by centrifugation. A portion of the supernatant was mixed with Laemmli SDS sample buffer and boiled immediately, and a portion was used in a BCA assay (Pierce #23227) to measure total protein concentration. Normalized protein samples were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore #IPFL20200 or Bio-Rad #162-0255), and blocked with 10% milk or, when probing with phospho-antibodies, StartingBlock™T20-TBS (Thermo Scientific Pierce #37543). Primary antibody-labeled blots were incubated with their respective HRP-conjugated secondary antibodies and developed with ECL-Plus  or Clarity Western ECL substrate (Bio-Rad #170-5061). The following primary antibodies were used for western blots: β-catenin mouse monoclonal , phospho-Ser33-β-catenin rabbit polyclonal , p53 mouse monoclonal , actin goat polyclonal (#sc-1615 Santa Cruz Biotechnology).Cells were grown in 100 mm culture dishes and harvested by trypsinization. Cells were washed twice with phosphate-buffered saline and total cell lysates were prepared using M-PER lysis reagent (Pierce #78501) supplemented with protease  and phosphatase inhibitors , however the instrument draws only a portion of this for analysis. Lysates were mixed with SDS-containing Sample Buffer to give a 1× final concentration, fluorescent internal control size markers  were included , and the sample mixture was heated to 95°C for 5 min. Samples were run on 12-230 kD size assay capillaries in a Wes capillary electrophoresis system (ProteinSimple). For each series, one capillary containing a biotinylated size ladder  was always included in the run. Primary antibody concentrations (described below) were optimized in pilot experiments. HRP-conjugated mouse and rabbit secondary antibodies, and chemiluminescence detection reagents, were from ProteinSimple. Capillary electrophoresis data analysis, including peak area determinations, was done using Compass software (ProteinSimple). Total protein labeling reagent was from ProteinSimple. The following primary antibodies were used for capillary electrophoresis, at the indicated dilutions: 1:500 GAPDH rabbit polyclonal , 1:100 GSK3β rabbit polyclonal , 1:50 phospho-Ser9-GSK3β rabbit polyclonal , 1:250 β-catenin rabbit monoclonal , 1:50 phospho-Ser33/Ser37-β-catenin rabbit polyclonal , 1:50 phospho-Ser33/Ser37/Thr41-β-catenin rabbit polyclonal , 1:10 p21 mouse monoclonal , 1:100 axin1 rabbit monoclonal .Cells were treated as indicated and harvested by trypsinization, then washed with PBS. In the ‘Development of a capillary electrophoresis method’ experiment, cells were lysed in 0.1× SDS-containing Sample Buffer  supplemented with protease and phosphatase inhibitors, and heated at 95°C for 3 min. For all other experiments, the cells were lysed in M-PER lysis reagent supplemented with protease and phosphatase inhibitors. The protein concentration of lysates was measured using the BCA assay, and the same amount of protein was loaded into each capillary for a particular antigen analysis series. The total protein amount per 5 µl is listed for each antigen , the log[total protein] was subtracted from the log[phosphoprotein] to obtain a log[difference]. This data transformation is preferable because, unlike ratios, arithmetic combinations of logs exhibit a Gaussian distribution . Cells were double-labeled overnight at 4°C with mouse monoclonal anti-β-catenin antibody  and goat polyclonal anti-lamin B antibody . Secondary antibodies were Alexa Fluor 647-conjugated anti-mouse IgG , and FITC-conjugated anti-goat IgG . The DNA dye Hoechst 33342 was used at 1 µg/ml to stain chromatin. Images were collected using a Nikon A1R confocal laser scanning microscopy system.A172 cells were grown in MatTek glass-bottom dishes (with No. 1.5 coverslip), treated as indicated, washed with phosphate-buffered saline (PBS) supplemented with 1 mM CaCl