The diagnosis of solid pseudopapillary neoplasms (SPNs) is challenging because some SPNs share many similar morphological and immunohistochemical features with other pancreatic neoplasms. In this study, we investigated potential diagnostic markers of SPN.Based on the SPN-specific upregulated genes from a previous DNA microarray and proteome study, we selected six immunohistochemical markers . We also evaluated the Ki-67 proliferative index to investigate its associations with prognosis. To validate these markers, we studied 91 SPNs as well as 51 pancreatic ductal carcinomas (PDC) and 48 neuroendocrine tumors (NET) as controls.P < 0.001). When beta-catenin, LEF1, and TFE3 staining were combined, the sensitivity and specificity were 100% and 91.9%, respectively. Four (4.4%) SPNs showed distant metastasis and these tumors were associated with a relatively high Ki-67 proliferative index .We found frequent and diffuse nuclear expressions of β-catenin (98.9%), AR (81.3%), LEF1 (93.4%), TFE3 (74.7%), FUS (84.6%), and cytoplasmic expression of WIF-1 (96.7%) in SPNs. In contrast, PDCs and NETs showed no expression. (We identified LEF1, TFE3, and AR as putative diagnostic markers of SPN, auxiliary to β-catenin. Incorporated into an immunohistochemical panel, these markers could be beneficial to distinguish SPN from PDC and NET. In addition, we suggest that the Ki-67 proliferative index can be a predictive marker of metastasis in SPNs. Among pancreatic tumors, solid pseudopapillary neoplasm (SPN) is very rare and comprises only 1–3% of primary pancreatic tumors. In spite of its clinical rarity, the clinical and pathologic findings are well recognized. SPN is composed of poorly cohesive monomorphic epithelial cells that form solid, pseudopapillary structures, and it occurs predominantly in young women [CTNNB1, which encodes β-catenin [SPNs are genetically characterized by somatic mutations in exon 3 of -catenin , 3. In a-catenin -7. Throu-catenin , 8.However, there are several unresolved issues in SPNs. First, putative diagnostic immunohistochemical markers are necessary. The pathologic diagnosis of SPNs is usually made using a combination of histologic and immunohistochemical markers, including beta-catenin, CD10, chromogranin, and E-cadherin , 10. DifIn this study, we selected several immunohistochemical markers from the SPN-specific up-regulated genes and validated their diagnostic usability in SPNs. Additionally, we investigated clinicopathologic parameters associated with the malignant behavior of SPNs.The basic clinicopathologic findings are listed in Table The rim of most SPNs is known to be infiltrative. However, since some SPNs show totally expanding borders, we examined whether clinicopathologic parameters differ depending on whether the margin of tumor invades surrounding parenchyma. When the border of SPNs was divided into at least focally infiltrative pattern and totally expanding pattern, male sex (p = 0.035), smaller tumor size p < 0.001), non-extensive hemorrhage and/or necrosis (p = 0.001), and frequent perineural invasion (p = 0.034) were found in the infiltrative SPNs , AR (81.3%), LEF1 (93.4%), TFE3 (74.7%), and FUS (84.6%), and cytoplasmic expression of WIF-1 (96.7%) were noted . However, there were no other significant correlations between the expression of other markers and clinicopathologic parameters.In our SPN cohort, distant metastasis was noted in 4 (4.4%) cases. The sites of metastasis were the liver  and specific (97%) marker for SPN. Additionally, we demonstrated that 3 putative markers  showed high rates of sensitivity and specificity. When beta-catenin, LEF1, and TFE3 were combined, the sensitivity and specificity were 100% and 91.9%, respectively. Based on these findings, we suggest that LEF1, TFE3, and AR are useful for the diagnosis of SPN, and the combination of these markers with beta-catenin is helpful to improve their sensitivity and specificity in the diagnosis of SPNs.CTNNB1 and act as a regulator of the Wnt/CTNNB1 signaling pathway [et al. [LEF1 is a member of the lymphoid enhancer-binding factor 1/T-cell factor (LEF1/TCF) complex . It inte pathway , 27. It  pathway , PDCs, a [et al.  have demTFE3 is a member of the microphthalmia (MiT) family of transcription factors, which is composed of MITF, TFE3, TFEB, and TFEC. MiT transcription factors form homodimers or heterodimers that bind target promoters and act as regulators of melanocyte development, cellular proliferation, survival, motility, and metabolism, which are deregulated during oncogenic processes , 30. TheAR is associated with cell cycle regulation and with the AR signaling pathway, which is activated in SPN , 7. MoreIn this study, we aimed to identify useful biomarkers for predicting the malignant behavior of SPN. There remains a lack of consensus of the criteria for “malignant” SPN. The concerning features, including perineural invasion, vascular invasion, or deep infiltrative growth, that are commonly found in other malignant neoplasms do not indicate malignant behavior in SPNs . ConsequIn summary, we found that LEF1, TFE3, and AR were putative diagnostic markers of SPN auxiliary to β-catenin. Incorporated into an immunohistochemical panel, they could be beneficial to distinguish SPN from PDC and NET. In addition, we suggest that the Ki-67 proliferative index could be a predictive marker of metastasis in SPN.Ninety-one formalin-fixed, paraffin-embedded (FFPE) specimens of SPN were obtained from the archives of the Department of Pathology, Yonsei University, Seoul, Korea and from the Liver Cancer Specimen Bank of the National Research Resource Bank Program of the Korean Science and Engineering Foundation of the Ministry of Science and Technology; the cases were encountered between 1999 and 2016. From consecutive cases of surgically resected pancreases, 51 PDCs were retrieved from 2009 to 2011 and 48 NETs were obtained from 2009 to 2015. Hematoxylin and eosin (H&E)-stained slides of all cases were reviewed. Histologic diagnosis of SPN was made based on microscopic features and one or more immunohistochemical markers including beta-catenin, CD10, E-cadherin, and chromogranin [Ninety-one cases of SPN, 51 of PDC, and 48 of NET were used for tissue microarray (TMA) construction using FFPE samples. A representative area of each case was selected under the microscope. Core tissues (3 mm in diameter) were taken from the individual FFPE blocks (donor blocks) and arranged in recipient paraffin blocks (tissue array blocks) using a trephine apparatus. Thirty-eight SPNs were selected and examined using conventional whole sections to rule out intra-tumoral heterogeneity.In our previous study, we found 2,026 up-regulated genes in SPNs compared with non-neoplastic pancreas. Among them, 1,119 genes were exclusively up-regulated in SPNs compared to PDCs and NETs. Beta-catenin, AR and WNT inhibitory factor 1 (WIF-1) have been identified by western blotting and immunohistochemical analysis . We chosImmunohistochemistry was conducted on 4-μm TMA tissue sections by a Ventana Bench Mark XT Autostainer . Details of the tested primary antibodies are shown in We also evaluated Ki-67 proliferative index using whole section slides to investigate its associations with prognosis. For calculating Ki-67 proliferative index , each tumor slide was manually scanned with a microscope at × 20 objective, and the area of greatest Ki67 positivity (hot spot) was selected for photography. Colored image of the hot spot was captured, and Ki67-negative and -positive tumor cells were marked in different colors. At least 500 tumor cells were counted. Pale staining nuclei were ignored during counting .Comparison of qualitative variables between groups was performed using the chi-square or Fisher’s exact test. The diagnostic sensitivity and the specificity for each of the markers individually, as well as in different combinations, were calculated. Using a logistic regression model, univariate analysis was conducted and odds ratios were estimated to assess predictive markers for distant metastasis. Two-sided P-values of less than 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS 22 software for Windows .


Coronary artery disease is treated by vein grafting and stent implantation. Late vein graft failure and restenosis of stented arteries reduce the success rates of these approaches and are caused by neointima formation. We have previously shown that Wnt proteins are up-regulated during intimal thickening, and have speculated that these lead to activation of downstream genes with β-catenin/T-cell factor (TCF)-responsive promoters. In the present study, we aimed to provide evidence that β-catenin/TCF signalling promotes neointima formation and assess whether targeting this pathway has potential for reducing neointima formation. We utilized a gene therapy approach selectively targeting cells in which the β-catenin/TCF pathway is activated by using a recombinant adenovirus Ad-TOPTK, which carries a herpes simplex virus thymidine kinase (HSV-TK) gene under the control of a β-catenin/TCF-response promoter. Cells with activated β-catenin will therefore be selectively killed. Ad-TOPTK and ganciclovir (GCV) treatment significantly suppressed the growth of the neointima in a murine model of left carotid artery ligation. In summary, we demonstrated that Wnt/β-catenin/TCF signalling promotes neointima formation, by showing that the selective death of cells with activated β-catenin suppressed neointima formation. This highlights the therapeutic potential for reducing late vein graft failure and in-stent restenosis by targeting β-catenin/TCF signalling. Our recent work has shown the involvement of the Wnt/β-catenin pathway in both migration ,2 and prWe aimed to show that β-catenin/TCF signalling is required for neointima formation and to examine whether using a suicide gene approach, similar to that previously utilized by Kwong et al.  in a mouIn summary, the present study will not only test directly whether β-catenin/TCF signalling is required for neointima formation but also be a proof of principle study to demonstrate whether a β-catenin /TCF signalling driven suicide gene approach retards neointima formation. If this is the case, future approaches could be developed to utilize this strategy to reduce vein graft failure and in-stent restenosis.Ad-TK and Ad-TOPTK viruses were generously provided by Mien-Chie Hung . These viruses have been described in detail previously . ProductSurplus de-identified segments of human saphenous vein were collected at the completion of bypass surgery from patients that gave written informed consent for the use of their tissues for research. The use of these vein segments for the present study was approved by NHS National Research Ethics Service (NRES number 04/Q2007/6: Frenchay Ethics Research Committee). It was not necessary for the Faculty of Health Sciences Ethics Board, University of Bristol to approve the present study as it was approved by NRES. However, the University of Bristol Research Governance approved, registered and sponsored the present study. Human saphenous vein smooth muscle cells (SMCs) were isolated from the saphenous vein segments using the explant technique  and main10 pfu Ad-β-gal, Ad-TK or Ad-TOPTK adenovirus. An osmotic mini-pump  was inserted subcutaneously that released GCV at 25 mg/kg per day or PBS as control.C57bl6/J mice were obtained and housed in the animal facilities at the University of Bristol. Housing, care and all procedures were performed in accordance with the guidelines and regulations of the University of Bristol and the United Kingdom Home Office. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The University of Bristol, Animal Welfare and Ethical Review Body (AWERB) approved the present study. The left common carotid artery was ligated near the carotid bifurcation, as described previously [The ligated carotid arteries were removed 3 and 21 days later and embedded in agar blocks vertically or horizontally respectively for sectioning. Agar blocks were processed and embedded in paraffin wax. Sections were cut at 3 μm thickness and stained using elastin Van Gieson (EVG) for visualization and quantification of the vessel and neointima by image analysis (Image Pro). The size of the intima was measured and then expressed as a percentage of the lumen area. Immunofluorescence staining for TK was performed using 4 μg/ml goat anti-HSV-1 TK (Santa Cruz Biotechnology: VN-20 SC-20837) to show expression of the virus following adenoviral infection and ligation. Briefly, sections were deparaffinized, rehydrated and subjected to antigen retrieval using citrate buffer and microwaving, sections were blocked with Image-FX and then incubated with the primary antibody overnight. The following day the bound primary antibody was detected with anti-goat Alexa Fluor-488 and then mounted with prolong gold with DAPI. Non-immune goat IgG was used as negative control.Carotid arteries were removed, rinsed in PBS, chopped finely and lysed in 5% (w/v) SDS lysis buffer with agitation for 15 min, before centrifugation at 13.000 g for 5 minutes and removal of supernatant for Western blotting using 50 ng/ml goat anti-HSV-1 TK (Santa Cruz Biotechnology: VN-20 SC-20837). Bound primary antibodies were detected using HRP-conjugated secondary antibodies and ECL reagent . Protein loading was observed using Bio-Rad stain-free technology.t test when two groups were analysed. A significant difference accepted when P<0.05.Results are expressed as mean ± S.E.M. Data were analysed by ANOVA and Student–Newman–Keuls post hoc test when more than three groups were analysed and Student's In order to test the hypothesis that VSMCs can be selectively killed by targeting β-catenin/TCF pathway with a responsive promoter, VSMCs were infected with Ad-TOPTK with or without Ad-β-catenin and cultured in the presence or absence of GCV. Cell number was measured by WST-1 assay, to assess cell viability. The efficacy of Ad-TOPTK in cell killing was compared with the adenovirus with TK under the control of the complete CMV promoter (Ad-TK). As shown in The effectiveness of infecting the carotid artery of mice with adenoviruses using pluronic gel was examined using the Ad-TK and Ad-TOPTK virus; Ad-β-gal was used as a negative control. The virus was applied to the adventitia immediately after ligation in pluronic gel. The animal was allowed to recover and 3 days later the carotid arteries were removed. Arteries were either fixed in formalin and subjected to immunofluorescence for TK or chopped finely and lysed in 5% SDS lysis buffer. To demonstrate activation of the β-catenin signalling pathway after carotid ligation, Ad-TOPTK was delivered to the adventitia of ligated carotid arteries using pluronic gel. Ad-β-gal was used as a negative control. 2 and 63±12 compared with 77±8% respectively).To test the effectiveness of Ad-TOPTK/GCV in suppressing neointima formation in animals, the adenovirus was delivered to the adventitia of ligated carotid arteries using pluronic gel. In addition, these animals received mini-osmotic pumps to continuously deliver GCV for 21 days and the size of the neointima was measured from paraffin wax embedded sections. As shown in in vivo. Although previous studies have implied the involvement of this pathway using reporter animals, detection of β-catenin activity and up-regulation of β-catenin target genes [The present study shows that selective death of cells with activated β-catenin can reduce neointima formation et genes ,4,11; diet genes . Furtherin vivo, using TOPgal transgenic mice that  β-catenin activity is increased in the media of left carotid arteries 3 days after carotid ligation injury and in the neointima formed 28 days after injury [When embarking on the present study, we considered the β-catenin/TCF signalling pathway as a very promising approach due to the expanding evidence that was suggestive of a role for β-catenin/TCF signalling in neointima formation. β-catenin is usually sequestered at the cell membrane by N-cadherin, where it is a component of the cadherin cell–cell contacts. During cell migration and proliferation, we have shown that the cell–cell contacts are lost, N-cadherin is shed from the cell extracellular membrane by proteases including matrix degrading metalloproteinases, and β-catenin is released into the cell ,13. Thisr injury .Fadd. Beneficial effects have also been observed by inhibition of proliferation of pancreatic and gastric cancer cells using the β-catenin promoter to drive the expression of suicide genes [The present study uses a targeted cell death approach, which has been successfully utilized by groups working in cancer biology ,14–16. Ide genes ,18.Although it is unlikely to be feasible to directly translate this approach to humans due to the requirement for continual GCV infusion, we consider that this proof of principle study was critical to provide the foundation to examine other suicide gene approaches for the use in restenosis and late vein graft failure. Accordingly, future approaches should avoid the requirement for GCV in order to be useful therapeutics. In addition, it is important that the suicide gene chosen for future work is not secreted, to ensure containment of the cell death to only cells with β-catenin/TCF signalling and not within non-activated cells in the close vicinity. Further work is needed to investigate the effect this approach has on endothelial cell function since this is an important mediator of neointima formation. Wnt/β-catenin signalling is known to affect endothelial cell migration and proliferation , so it iin vivo animal model (summarized in In conclusion, we have demonstrated that β-catenin/TCF signalling was activated within the neointima of ligated arteries. We also showed that the combination of Ad-TOPTK adenovirus and GCV treatment selectively killed β-catenin-activated cells, both in tissue culture and in an


Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. •Genome correction of SOD1 E100G mutation corrects ALS phenotypes in MNs•Activation of MAPK, AP1, WNT, cell-cycle, and p53 signaling in ALS MNs•Pharmacological screening uncovers ERK and JNK signaling as therapeutic targets•Susceptibility of MNs to degeneration may be due to heightened JUN activity in MNs In this article, Bhinge, Stanton, and colleagues use genome editing of patient-derived iPSCs to model ALS phenotypic defects in vitro. Transcriptomic analysis of disease MNs reveals activation of MAPK, AP1, WNT, cell-cycle, and p53 signaling in ALS MNs. Pharmacological screening uncovers activated ERK and JNK signaling as therapeutic targets in ALS. SOD1) gene have been linked to ALS  is a devastating neurodegenerative condition characterized by a progressive loss of upper and lower MNs . There id to ALS . Althougd to ALS . Spinal d to ALS . In thisSOD1 iPSC using the CRISPR-Cas9 system  into spinal motor neurons by adapting a recently published protocol capable of generating MNs with high efficiency . For allSince the over-arching phenotype observed in ALS patients is the specific loss of MNs, we asked whether ALS MNs would reveal disease-associated decline of survival in vitro. ALS is an adult-onset disease, therefore we monitored survival of MNs after they had attained maturity. Accordingly, we followed MN survival in low-density cultures from day 30 to day 44 A. ALS muXBP1 to total XBP1 transcript levels (sXBP1:XBP1) via qRT-PCR. We found an increased sXBP1:XBP1 ratio in SOD1 MNs compared with the control MNs that normalized upon genetic correction  to avoid transcriptional changes associated with cell death. Unsupervised hierarchical clustering of the RNA-seq data showed that the diseased MNs segregated distinctly from the corrected ones, indicating that correction of the SOD1 mutant allele had induced significant changes in the transcriptome A. We ideGSEA identified several pathways as differentially regulated in ALS MNs. For SOD1, significantly upregulated pathways were associated with p53 activation, cell-cycle regulation, WNT signaling, AP1 activation, and the unfolded protein response (UPR) B. StrikiFor further investigation, we decided to focus on the pathways that were activated in ALS MNs. qRT-PCR confirmed differential expression of several genes identified from RNA-seq analysis and representative of pathways found to be activated by GSEA C. We hypTo investigate whether the activated pathways identified in ALS MNs were causal or merely associated with the disease phenotype, we assayed MN survival in vitro after pharmacological inhibition of the identified pathways. MNs differentiated from mutant SOD1 iPSCs were treated with small-molecule inhibitors targeting the p38, ERK, JNK, and CDK kinases, as well as p53 and WNT pathways, at the indicated concentrations starting at day 30 and continued until day 44 . UntreatWe performed immunofluorescence assays in ALS and isogenic control MNs to confirm activation of the AP1 and WNT pathways. For a given target protein , we measured the median fluorescent signal in the nuclei of diseased and isogenic control MNs and plotted the distribution of intensities C. ImmunoALS is characterized by selective loss of MNs, while other neurons remain viable even though they harbor the same germline mutations. Using our in vitro model, we asked if there was differential activation of signaling pathways in MNs and non-MNs that might confer disease-specific loss of MNs. We analyzed nuclear localization of JUN and β-catenin in MNs (ISL1+) and non-MNs (ISL1−). Strikingly, we found that the levels of nuclear JUN were almost five times higher in MNs compared with non-MNs D. This dFUS gene  mutation as well as iPSCs derived from a healthy individual (GM23280A) were obtained from the Coriell Institute for Medical Research. Cells were maintained as colonies on human ES qualified Matrigel (Corning) in mTeSR (STEMCELL Technologies). Plasmids for the hCas9 and gRNA were obtained from Addgene and were a kind gift from the Church lab . Optical stimulation was performed by the system setup as previously described (2. Transistor-transistor logic pulses were sent to control the beginning and end of laser stimulus precisely.For multi-electrode array (MEA) recordings, progenitors derived from GM23280A iPSCs were plated directly on an MEA and differentiated as described above. Extracellular electrophysiological recordings of D34 neurons were performed in neuronal growth medium using a 60-channel MEA platform  as previously described . MC_Rackescribed . A 500 m2+ indicator Fluo-4, incubated for 15 min, and fluorescence intensities were recorded for the same field.GM23280A iPSC-derived D14 MNs were plated on glass bottom imaging dishes and infected with lentiviral particles expressing GCaMP6 for 6 hr, washed, and the media replaced with neuronal media as described above. At D21 of differentiation, cortical neurons obtained from embryonic day 18 (E18) rat pups were plated onto the MN monolayer. Cortical tissue was dissected from rat pups as described previously . CorticaDay 10 MN progenitors were plated on poly-D-lysine/laminin-coated 96-well plates at a density of 5,000 cells per well and differentiated into neurons. Cells were immunostained at day 30  and imaghttp://software.broadinstitute.org/gsea/index).RNA was extracted from day 30 neuronal cultures as described above. Sequencing libraries were generated from 1 μg of RNA using the NEBnext Ultra kit (NEB) according to the manufacturer's instructions and sequenced on an Illumina HiSeq instrument. Raw reads were mapped to the human genome hg19 using bwa, and only reads mapping to unique locations retained. The DESeq2 R package  and custFor immunostaining, cells were fixed with 4% paraformaldehyde, permeabilized with ice-cold methanol for 5 min, and washed with PBS containing 10% serum for 1 hr at room temperature. Cells were incubated with primary antibodies  diluted Cell lysates were separated on 12% SDS-PAGE gels, and proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% milk in TBST  and probed with corresponding primary antibodies against specific proteins . HorseraRNA was extracted with TRIzol reagent (Invitrogen) and reverse transcribed using random hexamers and the high-capacity reverse transcription system from Applied Biosystems. PCR was performed using the SYBR GREEN PCR Master Mix from Applied Biosystems. The target gene mRNA expression was normalized to the expression of GAPDH, and relative mRNA fold changes were calculated by the ΔΔCt method. Primer sequences have been included in 2+ imaging and analyzed the data. A.M.J.V. analyzed and interpreted the MEA recordings and Ca2+ imaging data and contributed to the experimental design.Conception and Design, A.B., L.W.S.; Collection and Assembly of Data, A.B., S.C.N.; Data Analysis and Interpretation, A.B., S.C.N., L.W.S.; Manuscript writing, A.B., S.C.N., L.W.S. Financial Support, L.W.S. X.Z. performed the MEA recordings and Ca


Activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) has been implicated in diverse kidney diseases, yet its in vivo significance in diabetic nephropathy (DN) is largely unknown. In the present study, we demonstrated a podocyte-specific Rac1-deficient mouse strain and showed that specific inhibition of Rac1 was able to attenuate diabetic podocyte injury and proteinuria by the blockade of Rac1/PAK1/p38/β-catenin signaling cascade, which reinstated the integrity of podocyte slit diaphragms (SD), rectified the effacement of foot processes (FPs), and prevented the dedifferentiation of podocytes. In vitro, we showed Rac1/PAK1 physically bound to β-catenin and had a direct phosphorylation modification on its C-terminal Ser675, leading to less ubiquitylated β-catenin, namely more stabilized β-catenin, and its nuclear migration under high-glucose conditions; further, p38 activation might be responsible for β-catenin nuclear accumulation via potentiating myocyte-specific enhancer factor 2C (MEF2c) phosphorylation. These findings provided evidence for a potential renoprotective and therapeutic strategy of cell-specific Rac1 deficiency for DN and other proteinuric diseases. Podocytes are terminally differentiated epithelial cells of the glomerulus, essential for the maintenance of an intact glomerular filtration barrier (GFB), and damaged podocytes are a key contributor to the onset of proteinuria and the progression of DN4. To date, mechanisms that govern diabetic podocyte damage and kidney injury remain poorly understood.Diabetic nephropathy (DN) is one of the leading causes for chronic kidney diseases5. Abnormal Rac1 signaling is involved in ROS production and inflammatory responses and reportedly linked to a number of debilitating human diseases, including cancer, diabetes, and kidney disorders8. Kolavennu et al. demonstrated in vivo targeting RhoA signaling, another pivotal member of Rho GTPases, could ameliorate albuminuria in a rodent model of diabetes via downstream signaling ROCK9. And we previously showed in vitro that Rac1 interference was protective against high-glucose (HG)-induced podocyte damage10, hence we postulated that manipulating Rac1 expression might as well be of a curative potentiality in vivo. Notably, systemic KO of Rac1 would lead to embryonic lethality in mice due to germ-layer formation defects12. Thus in the present study, we described a podocyte-specific Rac1-deficient mouse strain and generated diabetes models in these mice, aiming to uncover a renoprotective and therapeutic role of cell-specific Rac1 deficiency in DN and related podocyte damage.Rac1, a member of Rho family small GTPases, is a multi-functional molecule implicated in various cellular processes involving cell adhesions, proliferation, and plasticity13. Hyperphosphorylated p38 is found in renal proximal tubular epithelial cells (PTECs) and contributes to their epithelial–mesenchymal transition (EMT)14. Aberrant p38 phosphorylation was also correlated with the modulation of podocyte cytoskeletal dynamics16. Additionally, p38 could be activated by PAK1, a major downstream target of Rac1, as reported in several cancer cell lines and tracheal smooth muscle cell18. However, interplays between Rac1/PAK1 and p38 in diabetic podocytes and how would these interactions contribute to podocyte damage and proteinuria is not fully clarified.Among many factors implicated in the pathogenesis of DN, p38 MAPK (p38), which belongs to MAPK family, is typically involved in diabetes as a critical mediator of inflammatory reactions and mitochondrial malfunction19. In our previous study, Rac1/PAK1 activation was sufficient to trigger enhanced β-catenin dephosphorylation in podocytes, which later provoked elevated Snail expression upon HG stimulation10. Intriguingly, p38 could also regulate β-catenin signaling by inactivation of GSK3β in mouse F9 cells20, whereas little information is available on crosstalk between p38 and β-catenin in podocytes. Recent studies indicated that C-terminus domain of β-catenin might constitute the minimum region necessary for β-catenin shuttling between the cytosol and nucleus21. Thus it would be of interest to further identify whether there are modifications at β-catenin C-terminus in podocytes under HG conditions. Collectively, we tested our hypothesis in the present study that podocyte-specific transgenic ablation of Rac1 might be renoprotective against podocyte damage and proteinuria via prohibiting a signaling cascade of Rac1/PAK1/p38; and that epic activation of this signaling might also contribute to β-catenin activation and nuclear translocation in damaged podocytes both in vivo and in vitro.It was reported that Rac1 activation controlled nuclear localization of β-catenin, a key intracellular signal transducer involved in kidney fibrosis, during canonical Wnt signaling, depending on phosphorylation at its Ser191 and Ser605P < 0.01) without altering synaptopodin expression (P > 0.05) between wild-type WT and TG mice . In primary podocytes, Rac1 expression was significantly reduced in TG mice compared with WT mice on both protein and mRNA levels . There were no distinguishable differences in hair growth or appearance between WT and TG mice .TG mice were characterized by immunofluorescence of the kidney cortex, and real-time PCR and western blot analysis of primary cultured podocytes. Immunofluorescence demonstrated a marked decrease in Rac1 staining in TG glomerular podocytes . In WT-STZ mice, TEM demonstrated a mild degree of foot process (FP) effusion at 6 weeks      .By generating diabetes models, we showed significant differences between WT diabetic mice and their nondiabetic controls in both weight and blood glucose (BG) at 4, 6, 8, and 12 weeks after the onset of diabetes  Fig. , and a m01) Fig. . Mutatio01) Fig. . At 12 w01) Fig. ; and the01) Fig. . MoreoveP < 0.01). At 12 weeks, there occurred moderately mesangial proliferation and matrix accumulation, capillaries stenosis and parietal endothelial proliferation, indicative of advanced renal injury (P < 0.01)   and ZO-1 (P < 0.01) were both rectified, with diminished desmin staining compared with WT-STZ mice at 6 (P < 0.01)   , without alterations of BG or systolic blood pressure (SBP) (Table P > 0.05). The weight loss was also rescued in TG diabetic mice compared with WT hyperglycemic littermates at 4, 6, and 8 weeks (Table P < 0.05). Moreover, WT-1 staining revealed that podocyte numbers were not altered among four groups at 12 weeks .At 6 weeks, PAS staining showed mesangial areas mildly expanded, with swelling endothelial cells and capillaries stenosis, in WT-STZ mice Fig.  P < 0.001) Fig. . These m01) Fig. . Rac1 de1) Figs.  and 12 w1) Figs.  and 2c. 01) Fig.  as well 01) Fig.  was both01) Fig. . At 12 w01) Fig. . In line Fig. . Teks Fig.  (P > 0.0P < 0.01), which were both abrogated in Rac1-deficient diabetic podocytes ; p38 was also heavily phosphorylated in WT diabetic podocytes  but suppressed in TG-STZ counterparts at 6 weeks .In primary podocytes from WT-STZ mice, Rac1 and PAK1 activities were significantly higher compared with normoglycemic controls at 6 weeks Fig.  , despite a relatively higher apoptotic rate compared with cells treated with LG , whereas injury hallmarks α-SMA and FSP-1 were boosted on both protein and mRNA levels . Immunofluorescence illustrated decreased staining of p-cadherin and ZO-1 . Simultaneously, there was also a remarkable elevation in p38 activation stemming from HG stimulation .In vitro, Rac1 and PAK1 were activated in cultured podocytes after HG stimulation for 48 h Fig.  . Consistent with in vivo data, Rac1 shRNA transfection prevented activation of Rac1, PAK1, and p38, compared with cells without transfection ; and ameliorated podocyte cell markers, evidenced by enhanced expression of p-cadherin and ZO-1 . Blunted staining of α-SMA and FSP-1 as well as restored podocyte hallmarks were further confirmed by Immunofluorescence , whereas hardly affected Rac1 activity ; SB203580 showed limited impacts on Rac1 or PAK1 activation , albeit its inhibitory role in p38 phosphorylation driven by HG . Either IPA-3 or SB203580 were able to preserve expression of p-cadherin and ZO-1, and abrogate expression of dedifferentiation hallmarks desmin, α-SMA and FSP-1 . Rac1 shRNA transfection or SB203580 pretreatment reinstated forced F-actin re-assembly, as F-actin bundles at the sub-membrane region were decreased, compared with HG podocytes .Potential interactions between these proteins were determined. Rac1 knockdown efficiency was first confirmed Figure  , and reduced in Rac1-deficient hyperglycemic littermates (P < 0.01)  Fig. , suggest01) Fig. . By CO-I01) Fig. , thus we01) Fig.  (P < 0.001) Fig. ; in cont01) Fig. . Moreove01) Fig. .Fig. 6In22, after HG treatment (P < 0.01)   Fig. , which w05) Fig. , when β-05) Fig. . Next, w05) Fig. .5. In the present study, we described a podocyte-specific Rac1-deficient transgenic mouse strain, which exhibited neither embryonic lethality nor spontaneous proteinuria or diabetes. By generating diabetes models in these TG diabetic mice as well as their littermate controls, we were able to show that podocyte-specific Rac1 depletion might be of therapeutic importance to the pathogenesis of DN.Dysfunction of glomerular podocytes has been implicated as a critical driving force for DN23. SD disruption has been recognized as a key initial theme shared in various kidney diseases arising at the podocyte level24. SD generates synchronous signaling for the precise coordination of the neighboring actin-based FPs; damage to the SD was associated with uncoupled signals27, thus leading to a disrupted cytoskeletal structure, i.e. effaced FPs as was demonstrated here. In vitro, HG induced F-actin rearrangement and cell hypermotility, the latter of which, is recognized as the in vitro manifestation of dynamic reorganization of the FP structure27; and these were rectified by Rac1 knockdown. These in vivo and in vitro observations demonstrated Rac1 depletion benefited diabetic podocytes through a major and early attenuation on plasticity of podocyte cytoskeleton via preserving SDs and FPs both structurally and functionally in the challenge of diabetes.We showed that consistent hyperglycemia caused striking structural mutations of podocyte-specific proteins p-cadherin and ZO-1, which are also key components of the SD, and a simultaneous retraction of FPs in podocytes at as early as 6 weeks; in diabetic mice where Rac1 was depleted, these alterations were significantly restored. Podocytes elaborate FPs surrounding glomeruli with SDs bridging interdigitations and comprising the final layer of the GFB28. Again, we confirmed PAK1 being a downstream target for Rac1 activation in vitro and in vivo: PAK1 activation was found in podocytes of WT-STZ mice as soon as SD proteins were jeopardized, but was dampened in Rac1 TG-STZ mice. Reportedly, Rac1 contributed to LPS-mediated p38 activation in GDIα knockdown podocytes;29 and PAK1 could activate MAPK cascades in oncogenic transformation of a variety of cancer cells17. Likewise, we demonstrated p38 being a downstream signaling of PAK1 rather than these two proteins serving as two parallel downstream substrates in injured podocytes upon Rac1 activation, namely a signaling cascade of Rac1/PAK1/p38 in cultured podocytes. Presumably this cascade might as well exist in diabetic podocytes and play a pivotal role based on our in vivo data that simultaneous p38 activation was detected when PAK1 was also activated, and that Rac1 ablation led to reduced activation of both PAK1 and p38 in diabetic glomeruli, further showing reversal of PAK1/p38 activation was an indispensable step to reach the therapeutic effect.Others and we demonstrated previously that PAK1, a key modulator in regulation of actin polymerization and cell motility in mammalian fibroblasts, served as a major downstream effector of Rac1 in several cell lines including podocytes in vitro30. Excess cytoplasmic β-catenin is often targeted by a N-terminal phosphorylation/ubiquitylation-mediated degradation system35. N-terminus dephosphorylation of β-catenin is sufficient to induce stabilized β-catenin migrating into the nucleus28. Recent studies indicated β-catenin C-terminal tail constitutes the minimum region necessary for both import and export of cell nucleus in a receptor-free and energy-independent pattern21. Consistent with these studies, we demonstrated C-terminal β-catenin was significantly phosphorylated under HG conditions, when β-catenin nuclear accumulation was marked. We further showed that Ser675 at C-terminal β-catenin was a direct phosphorylation target of Rac1 signaling: both WT- and CA-Rac1 (mimicking the pathogenic Rac1 activation upon stimuli) phosphorylated β-catenin at Ser675; this site modification was linked to obliterated ubitquitylation level of β-catenin, which meant more stabilized cytoplasmic β-catenin that could possibly enter the nucleus later on. MEF2c is a key transcription factor involved in controlling gene expression in several cell types including myocytes and neurons22. Its expression was elevated in cardiac tissues under experimental diabetic conditions and cardiomyocytes in exposure to high glucose36; and in a rodent model of diabetes, increased MEF2c expression was found in the retina and kidney37. Likewise, we showed the HG stimuli potentiated MEF2c activation in cultured podocytes. Also we showed that rather than directly bound to β-catenin, p38 interacted with β-catenin in a MEF2c phosphorylation-dependent manner; in other words, MEF2c could only bind to and phosphorylate β-catenin in the premise that p38 was activated. These aforementioned results led us to a potential mechanism that upon the stimuli of hyperglycemia, Rac1/PAK1 signaling was activated in podocyte; β-catenin was then modified, either dephosphorylated at N-terminus or phosphorylated at C-terminus, leading to elevated level of active β-catenin; the downstream p38 further ensured β-catenin transcriptional activities via activating MEF2c; and active β-catenin contributed to the dysregulation of a series of target genes, and imbalanced expression of SD proteins such as p-cadherin, as reported here.Intriguingly, we found β-catenin expression and nuclear accumulation was under the modulation of Rac1/PAK1/p38 cascade in vitro and in vivo. β-catenin is a key component of Wnt signaling pathway and active β-catenin is found in diverse kidney diseases including focal segmental glomerulosclerosis (FSGS) and DN38. EMT is a phenotypic conversion in epithelial cells, characterized by the loss of epithelial cell markers and the acquisition of fibroblastic hallmarks37. We presented in vivo and in vitro the reduction in podocyte epithelial markers (p-cadherin and ZO-1), acquisition of fibroblastic or mesenchymal hallmarks (desmin and FSP-1), and upregulation of β-catenin expression and activation, together with a disordered cytoskeleton , which were, to some extent, prevented by the blockade of Rac1/PAK1/p38 signaling. Mature podocytes, unlike typical epithelial cells, express low levels of an intermediate filament vimentin, and retain a more “motile” cytoskeletal structure applicably assembled and disassembled to meet dynamic requirements of FPs41. However, we did not show other significant fibroblastic markers such as α-SMA expressed in glomerular podocytes during the course of experimental diabetes in WT or TG mice (not shown). We hypothesized that in vivo diabetic podocytes might undergo a partial EMT or dedifferentiation process; this distinction might be attributed to far more complex circumstances in vivo instead of its direct exposure to the stimuli in vitro. Notably, there was no difference in podocyte numbers between diabetic or nondiabetic mice at this stage of diabetic nephropathy, implying that podocyte were undergoing a relatively early and adaptive mutation rather than apoptosis under such diabetic conditions, which was partially reversible, as indicated in the present study, by Rac1 signaling deficiency. The curative effect was further confirmed by the finding that the onset of proteinuria, which succeeded the podocyte damage, was as well reduced by the rectification of podocyte impairment by the blockade of Rac1 signaling. In addition, we showed Rac1 depletion had a mild attenuation on mesangial areas, implying a pleiotropic effect of podocyte-specific Rac1 deficiency beyond its self-protecting mechanisms. This might be presumably attributed to reduction of mmp-9 in both podocytes and mesangial cells, which played a key role in the extracellular matrix (ECM) turnover42.Nuclear β-catenin is responsible for the modulation of a series of EMT genes such as Twist, Snail and Slug43. Since aldosterone-mineralocorticoid-receptor system has long been recognized as a pivotal player in the salt homeostasis and regulation of blood pressure44, it would be of interest to further identify whether there are any compensatory effects after Rac1 depletion. Taken together, we demonstrated podocyte-specific Rac1 depletion attenuated diabetic podocyte injury through the blockade of Rac1/PAK1/p38/β-catenin cascade, which favored DN in following aspects: (a) preserving the structure and function of SDs; (b) rectifying the sophisticated cytoskeleton and FP effacement; (c) reversing podocyte dedifferentiation; (d) reducing early mesangial injury; (e) and reducing proteinuria, without SBP or BG interference nce Fig. . These fThe nephrin promoter-Rac1 shRNA was linearized and microinjected into C57BL/6 mouse embryos that were transferred to the oviduct of pseudopregnant recipient mice. By DNA genotyping, 3 of 19 pups were identified positive  and were crossed with C57BL/6 mice to generate mice used in the present study. Forward primer: TTCGGTCGACTAGGGATAACAGG; reverse primer: TAATCCAGAGGTTGATTATCGGAA. PCR products (548 bp) were analyzed by agarose gel electrophoresis. The littermates lacking the transgene were control wild type (WT) mice.All animal studies were carried out with the review and approval of the animal care and use committee of Shandong University. At 8 weeks of age, 160 male WT and TG mice randomly allocated into two groups, either intraperitoneally injected with STZ  dissolved in citrate buffer (50 mg/kg for 5 consecutive days) (pH 4.5) or citrate buffer alone. Hyperglycemic state was monitored and maintained on a standard rodent diet with water ad libitum. SBP was examined by tail-cuff manometry . Urine was collected for determination of urinary protein when mice were weighed and placed in metabolic cages for 24 h. At the end of 4, 6, 8, and 12 weeks, mice were sacrificed and kidneys were harvested for following experiments.Small pieces of renal cortex were fixed in glutaraldehyde and osmic acid, dehydrated with an ethanol gradient, soaked in ethoxyline resin overnight, and mounted at 60 °C for 48 h. Ultrathin sections were cut and viewed under H-7500 TEM .45. Isolated glomeruli were maintained on type I collagen in RPMI 1640  . After 7 days, glomeruli were removed and cells were trypsinized and passed through a 40-μm-pore size sieve to remove mesangial and endothelial cells46. Podocytes identity was then done by synaptopodin and WT-1 immunofluorescence. Conditionally immortalized mouse podocytes were kindly provided by Professor Peter Mundel  via Professor Jie Ding (Peking University). Podocytes were cultured as described previously46.Isolation of glomeruli was done by three-step sieving  of kidney cortices6 cells/well) with indicated plasmids using Lipofectamine3000 reagent . After Rac1 shRNA incubation, podocytes were either treated with low glucose (LG) (5.6 mM) or HG (30 mM) for an additional 48 h. Cells transfected with scrambled shRNA and incubated with HG for 48 h were controls.Rac1 shRNA 5′−GACGTGTTCTTAATTTGCTT-3′, a scrambled shRNA 5′−GCGCGCTTTGTAGGATTCG-3′, Flag-β-catenin vectors, GFP-WT-Rac1, GFP-constitutive active (CA)-Rac1 (CA) (V12), GFP-dominant negative (DN)-Rac1 (N17), and an empty vector, were from Cyagen Biosciences . Transfection was performed . Briefly, cells were harvested and then lysed with assay/lysis buffer containing protease inhibitors on ice. The cell lysates were then centrifuged and protein content was estimated using the Bio-Rad protein assay. To analyze active Rac1 levels, each sample was adjust the volume of to 1 ml with 1× assay/lysis Buffer Lysate and incubated with 1 μl anti-active Rac1 monoclonal antibody, bound with protein A/G agarose beads for 1 h at 4 °C. The GTP-bound forms of Rac1 in the pull-down samples were precipitated, run in a 10% SDS-PAGE gel and subjected to western blot.Co-IP was performed using Universal Magnetic Co-IP Kit according to the manufacturer’s instructions .Recombinant GST-p38 and GST-myocyte-specific enhancer factor 2C (MEF2c)  were incubated in kinase buffer for 3 h at 30 °C with or without ATP addition , followed by 6× His-β-catenin , glutathione-agarose beads , and NP-40 lysis buffer . Beads were heated, run in SDS-PAGE gel, and subjected to western blot.10. Primary antibodies used were as follows: p-cadherin , FSP-1, α-SMA, β-catenin, mmp-9, Rac1 ; phospho-β-catenin (Ser552), phospho-β-catenin (Ser675), phospho-p38 (Thr180/Tyr182), p38, PAK1 and phospho-PAK1 (Thr423) , β-actin , ZO-1  and desmin (Immunoway). Bands were detected using ECL system  and Bio-Rad electrophoresis image analyzer .The protocol was described previously10. Primers were from Sangon Biotech . Sequences were designed as follows  was used for F-actin staining. All analyses were performed in a blinded fashion. Images were captured on an inverted phase/fluorescence microscope .4/ml), which were allowed to attach at 37 °C for 48 h. Non-migratory cells were removed; migrated cells were stained with haematoxylin. Cell number in the center of a membrane (one field) was counted using phase contrast microscopy (Leica Microsystems GmbH). Data presented denote the mean ± s.d. of three independent experiments.Transwell cell-culture inserts (8 μm)  were placed in RPMI1640 (10% FBS) in lower compartment. Podocytes were seeded in upper chambers (1 × 10t-test, and one-way ANOVA and LSD-t test; and P < 0.05 were considered to be statistically significant.Experiments were performed at least three times. Values were reported as mean ± s.d. Data were analyzed using SPSS 19.0 software. Statistical significance was assessed using Student’s Supplementary figureFigure S


P=0.024), while higher MAML3 expression (n=76) was associated with shorter recurrence-free survival (P=0.022). Immunohistochemical and immunoprecipitation analyses revealed that SOX17 and MAML3 co-localized in EC cell nuclei, and the MAML3 C-terminal region was necessary for SOX17 binding. SOX17 regulated MAML3 transcription via binding to the MAML3 promoter, decreasing Wnt pathway protein expression and suppressing EC cell growth and colony formation in vitro. In nude mice, SOX17 over-expression inhibited tumor growth, and co-inhibition or co-overexpression of SOX17 and MAML3 rescued this response. Our results suggest that decreasing SOX17 levels may promote EC development and progression, and that by downregulating MAML3 expression and Wnt signaling, SOX17 acts as a tumor suppressor that may improve outcome in patients with EC.β-catenin is a key regulatory factor for the Wnt signaling pathway. SOX17 is an important β-catenin inhibitor, while MAML3 is a co-activator of β-catenin-mediated transcription. Out of 120 endometrial cancer (EC) patients, we found that those with tumors expressing higher SOX17 (n=68) had longer recurrence-free survival ( Endometrial cancer (EC) is the most common gynecologic malignancy, with an estimated 47,130 new cases and 8,010 deaths occurring in 2014 .Maml3 [SOX17 (SRY-box containing gene 17) is a member of the SRY-related High Mobility Group (HMG) box transcription factor superfamily and regulates a variety of developmental processes and diseases . SOX17 eMaml3 , the rolMAML3 is a critical transcriptional co-activator in the Notch signaling pathway, and is encoded by a family of Mastermind like (MAML) genes . MAML3 iin vivo and in vitro via MAML3 and SOX17 overexpression and downregulation.Based on these observations, we hypothesized that SOX17 downregulates MAML3, modulating nuclear β-catenin and antagonizing Wnt signaling in EC. We assessed the functional significance of SOX17 in EC P=0.001). These observations suggested that decreased SOX17 expression and subsequent high MAML3 expression, along with associated Wnt signaling abnormalities, could promote EC development and progression.Immunohistochemical (IHC) analyses of human normal endometrium (n=30), atypical hyperplasia (n=30) and EC tumor specimens (n=60) showed that SOX17 expression was very low or absent in late EC stages, with high expression of Wnt pathway proteins. MAML3 expression was high in well-, moderately- and poorly- differentiated EC tissue samples Figure . SOX17 aP=0.001)  Figure .P=0.024)   had significantly longer recurrence-free survival  Figure , while h) Figure .Together, these results suggested a functional interaction between SOX17 and MAML3 during EC progression.SOX17 and MAML3 nuclear co-localization was confirmed using a laser scanning confocal microscope Figure , suggestWe then developed plasmids encoding either the N- or C-termini of SOX17 or MAML3, with HA or c-Myc tags Figure . HEK293 Western blot and real-time PCR results showed that decreased SOX17 expression increased MAML3 mRNA and protein levels in AN3CA Figure , Ishikawhttp://snpper.chip.org/mapper/mapper-main) identified a putative SOX17 binding site located −803 to −809 bp from the transcription start site in the MAML3 promoter region , suggesting that SOX17 could influence Wnt/β-catenin signaling.We evaluated SOX17, MAML3, β-catenin, CyclinD1 and c-Myc expression by IHC analysis in paraffin-embedded human ECsamples Figure . SOX17 eThe β-catenin promoter luciferase reporter, TOPflash, was co-transfected into HEK293 cells with SOX17cDNA or SOX17 shRNA. TOPflash activity was decreased by co-transfection of SOX17 cDNA and increased by SOX17 shRNA. We then introduced point mutations in TOPflash  using site-directed mutagenesis by base substitution. The mutants were no longer responsive to SOX17 Figure  & 4B. FOFurther validating this observed SOX17 inhibitory effect, MAML3 expression was down-regulated by SOX17 as measured by western blot. c-Myc and CyclinD1 were down-regulated by SOX17 indirectly Figure . In contWe transfected SOX17 cDNA or SOX17 shRNA into HEC-1B and Ishikawa cells Figure . MTT anaNext, because SOX17 overexpression reduced MAML3 levels, we then transfected MAML3 cDNA into SOX17 over-expressing HEC-1B and Ishikawa cells to rescue MAML3 expression Figure . MTT anaFinally, HEC-1B and Ishikawa cells co-transfected with MAML3 shRNA and SOX17 cDNA grew slower the controls and formed smaller colonies Figure .6 cells/ml and 100 μl was injected into the flanks of nude mice (n=5). We measured tumor sizes starting 5 days post-injection using the formula (2W × L) every 5 days for 32 days. Mice were then euthanized and tumors were excised. Tumor weights Stably transfected cells were subcutaneously transplanted into BALB/c nude mice. Cells were suspended at 5×10s Figure  and sizes Figure  from SOXs Figure . When SOs Figure  (P>0.05)In this study, we evaluated SOX17 expression in EC patient tumor samples and cell lines. High SOX17 levels were observed in benign endometrial tumor tissues, with low expression in malignant EC tissues. As a canonical Wnt antagonist, SOX17 is epigenetically inactivated by promoter methylation in many cancers , 12, 14,in vivo studies demonstrated significantly reduced growth in tumors overexpressing SOX17 compared with controls.SOX17 inhibits β-catenin/TCF-dependent transcription  and inhiTight control of nuclear β-catenin levels is key for regulation of Wnt/β-catenin signaling, and Wnt/β-catenin pathway dysregulation plays an essential role in type I EC in particular . One conc-Myc is reportedly an important effector of β-catenin, while Cyclin D1 is a critical β-catenin target . Our obsMAML3 promoter as the mechanism for inhibiting β-catenin/TCF activity and Wnt signaling. First, SOX17 decreased MAML3 mRNA and protein levels in EC cell lines , atypical hyperplasia (n=30) and EC tumors (n=60). The average age of patients with EC was 51.8±2.8 years . Tumor stages (I–III) and histological grades (G1–G3) were established according to the criteria of the International Federation of Gynecology and Obstetrics (FIGO) surgical staging system (2009) . None of6 cells per well were seeded and then harvested at 24 h for real-time PCR analysis, or at 72 h for western blot analysis. SOX17-overexpressing and knockdown cell lines were established as previously described [5) was seeded and analyzed at 24 h post transfection. All experiments were repeated three times unless stated otherwise.The human EC cell lines Ishikawa, HEC-1B and AN3CA, were obtained from and maintained as recommended by the China Center for Type Culture Collection (CCTCC). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum  at 37°C with 5% CO2. Transfections were performed with Lipofctamine 2000 transfection reagent  in 6-well plates following the manufacturers' protocols. For transfection efficiency analysis, prior to transfection, 1×10escribed . For immIHC staining was performed as previously described . AntibodTotal RNA extraction and reverse transcription (RT) reactions were performed as described previously . Real-tiImmunoprecipitation was performed as previously described . ProteinWestern blot analysis to assess protein expression was performed as previously described . ProteinChIP assay was performed using a Chromatin Immunoprecipitation (ChIP) Assay Kit  as described previously . DNA wasThe SOX17cDNA plasmid was purchased from Origene  and the MAML3cDNA plasmid was purchased from Genecopoeia . RNAi was performed as previously described . siRNA t2) were transfected and incubated for 14 days, fixed with 100% methanol and stained with hematoxylin. Colonies (>50 cells) were counted manually and plotted as described [Cells were plated for 24 h and transfected with SOX17 plasmid or MAML3 plasmid. The number of viable cells was determined at 24, 48, 72 and 96 h using the 3--2,5-diphenyltetrazolium bromide (MTT) assay as described . For theescribed .5 cells/well in a 24-well cell plate one day prior to transfection with Superfect according to the manufacturer's protocol . Luciferase activity was normalized for transfection efficiency using the mutant promoter reporter plasmid, FOPflash , as an internal control [Cells were seeded at 1×10 control .6 HEC-1B cells stably overexpression SOX17 or with SOX17 knocked down were implanted subcutaneously into 4–6-week-old BALB/c nude mice purchased from the Shanghai Laboratory Animal Center (SLAC) . Tumor growth was measured using a digital caliper every 5 days for 32 days. Tumor weight was measured when mice were sacrificed on day 32.5×10t-test. Associations between SOX17 expression and clinicopathological parameters were examined by the Chi-square test. P<0.05 was considered statistically significant.All experiments were repeated in triplicate. Data were expressed as the mean±SD. Statistical significance between two groups was determined by Student's


The roles in photosystem I (PSI) assembly of the nucleus-encoded thylakoid protein Y3IP1 who interacts with the plastid-encoded Ycf3 protein that has been well-characterized in plants. However, its function and potential mechanisms in other aspects remain poorly understood.MdY3IP1 gene, which encodes a protein highly homologous to the Arabidopsis Y3IP1 (AtY3IP1). Ectopic expression of MdY3IP1 triggered early-flowering and enhanced salt tolerance in Arabidopsis plants. MdY3IP1 controlled floral transition by accelerating sugar metabolism process in plant cells, thereby influencing the expression of flowering-associated genes. The increase in salt stress tolerance in MdY3IP1-expressing plants correlated with reduced reactive oxygen species (ROS) accumulation, and an increase in lateral root development by regulating both auxin biosynthesis and transport, as followed by enhancement of salt tolerance in Arabidopsis. Overall, these findings provide new evidences for additional functions of Y3IP1-like proteins and their underlying mechanisms of which Y3IP1 confers early-flowering and salt tolerance phenotypes in plants.We identified the apple MdY3IP1 gene. Further molecular and genetic approaches will accelerate our knowledge of MdY3IP1 functions in PSI complex formation and plants stress resistance, and inform strategies for creating transgenic crop varieties with early maturity and high-resistant to adverse environmental conditions.These observations suggest that plant growth and stress resistance can be affected by the regulation of the The online version of this article (10.1186/s12870-018-1232-6) contains supplementary material, which is available to authorized users. Growth and development of higher plants largely depends on the photosynthetic activity derived from cooperating photosystem I (PSI) and photosystem II (PSII) complexes –3. PSI iPSI has a molecular mass of approximately 600 kDa, and is composed of two subcomplexes: the PSI core complex and the light-harversting complex I (LHCI). It has at least twelve core subunits in higher plants, including three peripheral  and nine membrane-intrinsic subunits  –14. AlthArabidopsis and tobacco -IAA was added. IAA content measurement was performed using GC-QQQ .2-week-old seedlings of the control and t-test. A difference at P ≤ 0.01 was considered significant (*), and P ≤ 0.001 was considered extremely significant (**).Samples were analyzed in triplicates, and the data expressed as the mean ± standard error (SE) unless noted otherwise. Statistical significance was determined using Student’s Additional file 1: Figure S1.Analysis of the deduced amino acid of MdY3IP1. (DOC 1372 kb)Additional file 2: Figure S2.MdY3IP1-expressing transgenic Arabidopsis plants under short-days condition. (DOC 702 kb)Early flowering phenotype in the Additional file 3: Figure S3.MdY3IP1 under 4 °C, 2% PEG and 100 μM ABA. (ZIP 292 kb)Expression of Additional file 4: Figure S4.MdY3IP1 transgenic Arabidopsis plants. (ZIP 476 kb)Effect of the auxin transport inhibitor NPA on primary root length in control and Additional file 5: Figure S5.The deduced functional domains of MdY3IP1 protein. (DOC 44 kb)Additional file 6: Figure S6.AtSOS1, AtSOS2 and AtSOS3 in the control and MdY3IP1 transgenic Arabidopsis plants by qPCR assay. (ZIP 275 kb)Expression levels of Additional file 7: Table S1.The primers used for RT-PCR and qRT-PCR in this study. (PDF 18 kb)


These results suggest that DMP-PYT may have beneficial effects for preventing and for treating osteoporosis.Osteoblast differentiation is regulated through the successive activation of signaling molecules by a complex interplay of extracellular signals such as bone morphogenetic protein (BMP) and Wnt ligands. Numerous studies have identified natural as well as synthetic compounds with osteogenic activity through the regulation of either BMP/SMADs or the Wnt/β-catenin pathway. Here, we attempted to isolate small molecules that concurrently activated both SMADs and β-catenin, which led to the discovery of a novel potent osteogenic compound, DMP-PYT. Upon BMP2 stimulation, DMP-PYT substantially increased osteoblast differentiation featured by enhanced expression of osteoblast-specific genes and accelerated calcification through activation of BMPs expression. DMP-PYT promoted BMP2-induced SMAD1/5/8 phosphorylation and β-catenin expression, the latter in a BMP2-independent manner. DMP-PYT alone enhanced nuclear localization of β-catenin to promote the DNA-binding and transcriptional activity of T-cell factor, thereby resulting in increased osteoblast differentiation in the absence of BMP2. Most importantly, DMP-PYT advanced skeletal development and bone calcification in zebrafish larvae. Conclusively, DMP-PYT strongly stimulated osteoblast differentiation and bone formation  Osteoblasts express receptor activator of NF-κB ligand (RANKL), which binds to its receptor, RANK, on the extracellular surface of osteoclasts4. RANKL–RANK interaction stimulates osteoclast differentiation and bone resorption activity, whereas osteoprotegerin, produced by osteoblastic stromal cells, interrupts this interaction through competitive binding to RANKL, thus inhibiting osteoclast differentiation5. Additionally, osteoclasts regulate osteoblast differentiation and bone-forming activity by producing cytokines, such as transforming growth factor-beta (TGFβ)6. Although it is argued that TGFβ is critically required for osteoclast differentiation, TGFβ and bone morphogenetic proteins (BMPs) that belong to the TGFβ superfamily stimulate osteoblast differentiation through activation of the TGFβ receptor or BMP receptor signaling pathways7. Imbalance between bone formation and resorption causes various bone disorders, including osteoporosis and osteopetrosis.Bone tissue is composed of bone-forming osteoblasts and bone-resorbing osteoclasts. Bone mass is homeostatically regulated by the interplay of osteoblasts and osteoclasts8. In addition, BMP signaling activates β-catenin, a Wnt signal transducer, to induce osteoblast differentiation9. Upon BMP stimulation, β-catenin accumulates and locates to the nucleus. Nuclear β-catenin interacts with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins to promote TCF/LEF-mediated gene transcription10. BMP and Wnt cooperatively activate SMAD- and β-catenin-mediated osteoblast gene expression and accelerate osteoblast differentiation, implicating the importance of the BMP and Wnt signaling pathways in bone formation11.Osteoblasts are differentiated from bone marrow mesenchymal stem cells upon stimulation with extracellular signals that activate intracellular signaling molecules. In particular, extracellular BMPs bind to their receptors and activate receptor kinases, resulting in the phosphorylation of specific SMAD proteins. Activated SMADs translocate into the nucleus to increase the transcription of genes encoding osteoblast-specific factors, such as runt-related transcription factor 2 (RUNX2), osteocalcin, matrix extracellular phosphoglycoprotein, and alkaline phosphatase15, and several compounds were newly characterized to down- or up-regulate Wnt/β-catenin signaling17. However, these compounds mainly control the ligand-receptor binding complex or receptor-associated membrane proteins as agonists or antagonists. It would be useful to isolate cell-permeable small compounds that can directly modulate BMP- and Wnt-mediated signaling molecules. To this end, we screened a drugable chemical library and investigated the bioactive compounds that activated both BMP/SMADs and β-catenin. We identified a novel compound, DMP-PYT, which strongly promoted bone formation in vitro as well as in vivo through phosphorylation of BMP/SMADs and nuclear accumulation of β-catenin.Various studies have attempted to isolate small molecules that activate the BMP and Wnt signaling pathways. Dorsomorphin derivatives and flavonoids were identified as BMP inhibitors or activators26, 5-(3-(4-(dimethylamino)phenyl)allylidene)-1-pyrimidine-2,4,6 -trione (MP-PYT), more potently increased BMP2-induced β-catenin expression than the others  zebrafish, which selectively expresses red fluorescence in the liver during larval development19. Administration of DMP-PYT did not affect hepatic red fluorescence in the zebrafish larvae , chemical derivatives (H1 through H19) were purchased from InterBioScreen . The structure and stereochemistry of all compounds including DMP-PYT (InterBioScreen) were confirmed by various physicochemical analytical methods and provided by KRICT and InterBioScreen with 91– 98% purity and endotoxin-free. Recombinant human BMP2, ascorbic acid, β-glycerophosphate, and ALP staining kit were purchased from R&D  and Fluka , and Sigma-Aldrich Corp. .5 cells/ml) were grown to confluence for 48 h and replaced with DMEM containing BMP-2  for inducing osteoblast differentiation. Cells were replaced with fresh DMEM containing BMP2 at day 3 and cultured for 6 days. MC3T3-E1 cells (105 cells/ml) were cultured and grown to confluence in αMEM supplemented with 10% FBS, glutamine (2 mM), and penicillin and streptomycin (100 U/ml). Cells were then replaced with complete αMEM containing ascorbic acid (50 µg/ml) and β-glycerophosphate (10 mM) for osteoblast differentiation. Cells were refreshed every 3 days and induced to differentiate into osteoblasts for 10 days.Mouse cell lines C2C12  and MC3T3-E1 subclone 4  were maintained in DMEM containing 10% heat inactivated FBS . C2C12 cells . Cells were then rinsed with deionized water and subjected to a microscopic observation. For alizarin red S staining, cells were washed with distilled water, fixed in ice-cold 70% ethanol for 1 h, and then stained with 2% Alizarin red S stain solution (adjusted to pH 4.2 with 10% ammonium hydroxide) (Sigma-Aldrich) for 30 min. Stained cells were washed and observed using a light microscope. At least 10 cell images were captured under a microscope  fitted with a DP70 camera system (Olympus) and used for a quantitative analysis with the Image J software. To determine ALP activity, cells were washed and lysed with Triton X-100 lysis buffer . Cell supernatants were incubated with an ALP substrate. ALP activity assay was performed in triplicate using the LabAssay ALP Kit . Relative ALP activity was calculated by comparing with standard solution and expressed as units per mg of protein according to the manufacturer’s instructions.Total cell lysates were obtained by lysis in RIPA lysis buffer with protease inhibitors . Nuclear and cytosolic proteins were fractionated using NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific) and protein concentration was determined by bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Proteins (20 µg) were resolved by SDS-PAGE and transferred to an Immobilon-P membrane . Protein blots were incubated with antibodies against pSMAD1/5/8 , β-catenin (Millipore), RUNX2 , SMAD1/5/8, BMP4, BMP7, Wnt3a, Lamin B, and β-actin . Specific protein bands were detected by chemiluminescence imaging with LAS-3000 .Total RNA was isolated using a TRIzol reagent  and reversely transcribed using a cDNA synthesis kit . The cDNA was then used as template for real-time PCR using SYBR Green pre-mix buffer  and a specific primer set. Primers were as follows: 5′-gccgggaatgatgagaacta-3′, 5′-ggaccgtccactgtcacttt-3′ for RUNX2; 5′-gggacccgctgtcttctagt-3′, 5′-tcaactcaaattcgctgagga-3′ for BMP2; 5′-cctggtaaccgaatgctgat-3′, 5′-agccggtaaagatccctcat-3′ for BMP4; 5′-caacgccctgtccaatgac-3′, 5′-actcttgcggttcaaggagtg-3′ for BMP6; 5′-cgataccaccatcgggagttc-3′, 5′-aaggtctcgttgtcaaatcgc-3′ for BMP7; 5′-tgctgtattgctgacctggg-3′, 5′-gttccagcggttagacacga-3′ for BMPR1α; 5′-atgcctgttgtcacctctgg-3′, 5′-acttcccgagctctgagact-3′ for BMPR1β; 5′-agcacagaggcccaattctc-3′, 5′-ttagacactgtggtcgtggc-3′ for BMPRII; 5′-atgggcgtctccacagtaac-3′; 5′-tcacccgagtggtagtcaca-3′ for ALP; and 5′- aactttggcattgtggaagg-3′, 5′-acacattgggggtaggaaca-3′ for GAPDH. Real-time PCR was performed using an ABI-Prism 7700 sequence detector (Applied Biosystems) and the relative expression level was calculated after normalization to the level of GAPDH.C2C12 cells were transfected with TOP-FLASH, a reporter gene containing the β-catenin/TCF binding element linked to the luciferase gene by liposome-mediated transfection (Invitrogen) and then treated with DMP-PYT compound for an additional 24 h. A pCMVβ vector was used as an internal control to compensate for the transfection efficiency. Cell extracts were harvested in a reporter lysis buffer. Reporter activity was assayed with a luciferase assay kit  or a β-galactosidase assay kit (Thermo Fisher Scientific). Relative luciferase activity was determined after normalization to the β-galactosidase activity and expressed as a fold induction.MC3T3-E1 cells were treated with DMP-PYT and fixed in formalin. Cells were then permeabilized with 0.1% Triton X-100 in PBS and subsequently incubated with antibodies against active β-catenin (Millipore). Cells were incubated with secondary antibody conjugated with Alexa Fluor 488 . Nuclei were counterstained using Hoechst 33342 . Cells that express β-catenin in the nucleus were counted from 10 different images of three independent experiments. Results are mean ± SE and expressed as percentage compared to the control group. Nuclear localization of β-catenin was determined in Wnt3a-treated cells as a positive control.ChIP assays were performed using MAGnify™ ChIP according to the manufacturer’s instructions (Thermo Fisher Scientific). In brief, C2C12 cells were incubated with DMP-PYT for 24 h and fixed in formaldehyde for 10 min. Cell suspensions were sonicated using High power sonication under the optimized condition . Cell lysates were incubated with anti-TCF4 antibody and chromatin-immune complexes were captured to magnetic beads and eluted in PBS after washing. Quantitative real-time PCR was performed using primer sets detecting TCF4 binding elements in distal promoter of BMP2 gene. The following primers were used for ChIP-PCR: 5′-gacctctacagctctagaaacag-3′ and 5′-cattcaggagccttcatagtacc-3′.WT and Tg (L-fabp:dsRed) zebrafish were maintained at 28 °C with a photoperiod of 14:10 h (light:dark), in 10 L glass tanks with freshwater and aeration. Spawning of zebrafish was carried out by pairing one male with two females and fish eggs were incubated in egg water (Sigma Aldrich). Adult zebrafish experiments were conducted in accordance with the specific guidelines approved by the Institutional Animal Care and Use Committee of Chungnam National University (CNU-00866). At 72 h post fertilization, the zebrafish larvae were exposed to DMP-PYT for 4 days in KRICT. At day 7 post fertilization, Tg (L-fabp:dsRed) zebrafish were then euthanized and mounted on glass slides, followed by microscopic observation using a stereomicroscope . L-FABP-RFP was captured and quantitatively determined from 10 larvae images by calculating the fluorescent area of the liver (Leica Application Suite software). WT zebrafish larvae were incubated with DMP-PYT for an additional 4 days at 72 h post fertilization and immersed by deionized water containing 0.2% calcein (Sigma Aldrich) for 20 min for determining bone formation. Zebrafish larvae were then mounted on glass slides and observed under a fluorescent microscope. Calcification of axial skeletal was determined by measuring fluorescence intensity from 10 larvae images in each group using the Image J software and expressed as the percentage of the control.All experiments were performed at least three times and data are expressed as mean ± SE. A statistical significance was determined by the one-way analysis of variance. P value under 0.05 (P < 0.05) was considered statistically significant.in vitro and in vivo, suggesting its beneficial effect in treating bone defects such as osteoporosis.In conclusion, we screened chemical compounds for osteogenic activity as evidenced by increases in SMAD phosphorylation and β-catenin expression. A novel pyrimidine trione derivative, DMP-PYT, was characterized to enhance BMP-induced osteoblast differentiation through an induction of BMP-dependent phosphorylation of SMADs and BMP-independent activation of β-catenin. DMP-PYT increased the nuclear expression of β-catenin and DNA-binding activity of TCF/LEF, thereby upregulating osteoblast-specific markers. DMP-PYT significantly promoted calcium deposition and bone formation Supplementary Info


These results suggest that DISC1 promotes NSCLC growth, likely through GSK3β/β-catenin signaling, and that DISC1 may function as an oncogene and novel anti-NSCLC therapeutic target.Neuropsychiatric disorder-associated disrupted-in-schizophrenia-1 (DISC1) activates Wnt/β-catenin signaling by inhibiting glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and may promote neural progenitor cell and pancreatic β-cell proliferation. The present study found that DISC1 promotes non-small cell lung cancer (NSCLC) cell growth. Western blotting and immunohistochemistry analyses showed that DISC1 was highly expressed in NSCLC cell lines and patient tissues. DISC1 expression was negatively associated with phosphorylated (p-) GSK3β, but positively correlated with a more invasive tumor phenotype and predicted poor NSCLC patient prognosis. siRNA-mediated DISC1 silencing increased p-GSK3β expression and decreased expression of β-catenin and Cyclin D1, while DISC1 upregulation produced the opposite results. DISC1 knockdown also reduced NSCLC cell proliferation rates  Lung cancer is the most frequently occurring cancer type, and the leading cause of cancer death globally , 2. MostDisrupted-in-schizophrenia-1 (DISC1) is a risk gene associated with major mental illnesses, including schizophrenia and depression . A balanThe canonical Wnt/β-catenin pathway modulates cell proliferation, which is negatively regulated by the β-catenin destruction complex. GSK3β promotes β-catenin phosphorylation, resulting in β-catenin ubiquitylation-mediated degradation in the proteasome –18. HoweThe present study assessed DISC1 expression in human NSCLC cell lines and tumor tissues, and evaluated relationships between DISC1 expression, clinicopathological features, and patient prognosis. We also investigated the role of DISC1 in cancer cell proliferation. We demonstrated that high DISC1 expression promotes NSCLC growth, possibly through GSK3β/β-catenin signaling.Western blotting results showed that DISC1 and PCNA (a cancer cell proliferation marker) levels were higher in NSCLC tissues than in paired adjacent non-tumor tissues Figure , and exhDISC1 and the Wnt pathway molecules, p-GSK3β (Phosphorylation at Tyr216), β-catenin, Cyclin D1, and Ki-67, were measured in 140 NSCLC patient tissue samples via immunohistochemical (IHC) analysis. DISC1 and β-catenin were expressed in the cytoplasm and nucleus, p-GSK3β was prominently expressed in cytoplasm, and Ki-67 was expressed in the nucleus. DISC1, β-catenin, Cyclin D1, and Ki-67 levels increased from well- to poorly-differentiated NSCLC tissues , Cyclin D1 , or Ki-67  expression, and a negative correlation between DISC1 and p-GSK3β expression   or high (≥50%) level of expression [P=0.049), tumor size (P=0.013), pathology grade (P<0.001), grade (P=0.020), lymph node metastasis (P=0.032), and Ki-67 (P<0.001), p-GSK3β (P<0.001), β-catenin (P<0.001), and Cyclin D1 expression (P<0.001) (Table P=0.800), age (P=0.375), clinical stage (P=0.123), or distant metastasis status (P=0.143).Spearman’s rank correlation identified positive associations between DISC1 and β-catenin , tumor size (P=0.040), pathology grade (P<0.01) were correlated with patient survival status (Table P<0.01) and Ki-67 (P=0.030) levels were independent prognostic factors for patient overall survival  of the 140 NSCLC patients analyzed survived from the high DISC1 expression group, while 66% (44/67) survived from the low expression group  and a negative control siRNA (NC-siRNA) were used to assess the impacts of DISC1 on cell proliferation. DISC1 levels decreased in DISC1-silenced A549 cells, as shown by western blotting Figure . DISC1-sDuring brain development, DISC1 amino acids 195–238 regulate GSK3β/β-catenin/Wnt signaling by inhibiting GSK3β phosphorylation, which in turn reduces β-catenin phosphorylation and stabilizes β-catenin . siRNA-mUncontrolled cell proliferation is a major factor in lung cancer development and progression . A more DISC1 was first associated with a variety of brain disorders, including schizophrenia, mood disorders, and autism –12. DISCNeurological studies reported that DISC1 directly interacts with GSK3β, and suggested that GSK3β promotes β-catenin phosphorylation and ubiquitylation-mediated β-catenin degradation in the proteasome –19. HoweEight paired NSCLC and adjacent non-tumor tissues were collected immediately after surgical removal and stored at -80°C for western blot analysis. Additionally, 140 NSCLC specimens, along with patient demographic information and clinical data  were obtained from the Institute of Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium  containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2 .Western blotting, and IHC analysis and evaluation were performed as described previously , 25–26. siRNAs targeting DISC1 and negative control siRNA (NC-siRNA) were purchased from GenePharma Co., Ltd (China) Table , and a DA serum starvation and release process was used for cell cycle synchronization. Briefly, A549 cells were cultured in RPMI 1640 medium without FBS for 72 h, and were then replaced by 10% FBS-supplemented medium. Cells were then fixed in 70% ethanol for 1 h at 4°C and incubated with 1 mg/mL RNaseA for 30 min at 37°C. Cells were stained with propidium iodide   in PBS and 0.5% Triton X-100, and analyzed using a BD FACScan flow cytometer  and the CellQuest acquisition and analysis program.4 cells/well, cultured overnight, and then transfected with DISC1-siRNA2 and negative control siRNA. CCK-8 reagents  were added to each well at different time points, and wells were incubated for an additional 2 h at 37°C in the dark. Absorbency was measured at 450 nm (reference 650 nm) with a microplate reader . Experiments were repeated at least three times.Cell counting kit-8 (CCK8) assay was performed to evaluate cell proliferation. A549 cells were seeded into 96-well cell culture cluster plates  at 2×102 for 10 d. Surviving colonies (>50 cells/colony) were stained with 0.5% crystal violet and counted.Transfected A549 cells were seeded into 6-well cell culture cluster plates (Corning) at 200 cells/well, and incubated at 37°C with 5% CO2) test. Kaplan-Meier curves were constructed and Log-rank test was performed to analyze survival data. Multivariate analysis was performed using Cox’s proportional hazards model. The risk ratio and its 95% confidence interval were recorded for each marker. P<0.05 was considered statistically significant. All values were expressed as means ± SD. Each experiment was repeated at least three times.SPSS 19.0 software was used for statistical analysis. DISC1 expression and clinicopathological features were analyzed using the Chi-square (χ


Intraperitoneal administration of iCRT3 to C57BL/6 mice, subjected to cecal ligation and puncture-induced sepsis, decreases the plasma levels of proinflammatory cytokines and organ injury markers in a dose-dependent manner. The histological integrity of the lungs is improved with iCRT3 treatment, along with reduced lung collagen deposition and apoptosis. In addition, iCRT3 treatment also decreases the expression of the cytokines, neutrophil chemoattractants, as well as the MPO activity in the lungs of septic mice. Based on these findings we conclude that targeting the Wnt/β-Catenin pathway may provide a potential therapeutic approach for treatment of sepsis.The Wnt/β-catenin pathway has been involved in regulating inflammation in various infectious and inflammatory diseases. Sepsis is a life-threatening condition caused by dysregulated inflammatory response to infection with no effective therapy available. Recently elevated Wnt/β-catenin signaling has been detected in sepsis. However, its contribution to sepsis-associated inflammatory response remains to be explored. In this study, we show that inhibition of Wnt/β-catenin signaling reduces inflammation and mitigates sepsis-induced organ injury. Using  Sepsis is associated with high morbidity and mortality and is arguably one of the leading causes of preventable death in the world today5. In spite of the remarkable efforts and money spent with the objective of developing treatments, there is still no effective drug available, other than antibiotics, vasopressors and supportive care7. The pathophysiology of sepsis is still poorly understood, even after extensive basic research and clinical studies. Early phase of sepsis is characterized by a complex and dynamic systemic host response manifested by an array of pro-inflammatory mediators accompanied by numerous counter-regulatory mechanisms causing large scale organ damage and death9. The dysregulated inflammatory response in sepsis is a result of many diverse and cross-regulated pathways which gets altered10. Certainly, understanding the diverse signaling pathways activated and cross regulated during sepsis will impact the search for new therapeutic targets and agents with the compelling need to treat sepsis.Sepsis is now defined as a clinical syndrome due to dysregulated systemic immune response to infection that causes multiple organ failure and could be life-threatening12. Interestingly, Wnt signaling, classically regarded as the pathway involved in the control of cell proliferation, migration, and differentiation in embryonic development, has recently been involved in immunoregulatory mechanisms in various infectious and inflammatory diseases14.The canonical Wnt pathway is well characterized and involves β-catenin and members of T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family of transcription factors (TCF/LEF). Activation of the Wnt/β-catenin pathway via Wnt ligands prevents the proteasomal degradation of β-catenin by inhibiting the glycogen synthase kinase 3 β (GSK3β) phosphorylation activity, resulting in accumulation and translocation of β-catenin to the nucleus where it drives the expression of TCF/LEF-dependent genesvia a mechanism dependent on PI3K/AKT and Erk pathways, indicating towards its contribution to inflammation16. Like TLR4, other TLRs such as TLR2, TLR3, TLR5, and TLR9 are also known to activate or regulate β-catenin signaling in a pathogen or cell type-specific manner14. However, only limited evidence is available for a role of canonical Wnt/β-catenin signaling in sepsis. Activation of Wnt signaling has been reported in the lungs during the early stage of sepsis18. Interestingly, Wnt/β-catenin signaling is involved in the progression of inflammatory lung diseases including asthma20, emphysema21, and pulmonary fibrosis23. A recent study showed that Wnt inhibitors suppress LPS-induced inflammatory responses, hinting that a therapeutic strategy designed to attenuate Wnt/β-catenin signaling has the potential to prevent sepsis-induced inflammation24.The canonical Wnt signaling was shown to be activated by TLR4-ligand lipopolysaccharide (LPS) in monocytes and macrophages, 25. The effect of iCRT3 on Wnt/β-catenin signaling and proinflammatory activity in LPS-stimulated macrophages was first demonstrated by assessing the inhibition of TCF reporter activity, cytokine production and IκB degradation. We then examined the effect of treatment with iCRT3 on inflammation and organ injury in mice with sepsis induced by cecal ligation and puncture (CLP), a physiologically relevant model. This approach not only further elaborated the role of β-catenin-TCF mediated Wnt signaling in regulating sepsis-induced inflammation and organ injury but also evaluated the potential of using Wnt/β-catenin signaling-inhibitors as a therapeutic strategy for treating sepsis-induced lung damage.In the present study, we used iCRT3 , a small cell-permeable oxazole compound, which acts as a selective inhibitor of the Wnt pathway by blocking the interaction of β-catenin-TCF via direct binding to β-cateninRenilla luciferase reporter RL-TK. The tranfected cells were pre-treated with iCRT3 followed by LPS stimulation. Dual-luciferase assay demonstrated no detectable Wnt/β-catenin signaling activity as baseline in the naïve RAW264.7 cells. Wnt/β-catenin signaling reported by TOP-TK was increased by 3.2-fold in the cells treated with LPS, whereas FOP-TK showed no activation as expected  and keratinocyte-derived chemokine (KC), in the lungs. Similar to cytokines, mRNA levels of both chemokines MIP-2 and KC in the lungs were highly increased after CLP, but strikingly inhibited with iCRT3 treatment by 96.9% and 93.3%, respectively  are also attenuated in septic mice after iCRT3 treatment in a dose-dependent manner. Our results further validate the link between Wnt/β-catenin signaling and hyperinflammation/multiple organ injury in sepsis, with administration of iCRT3 effectively attenuating both.Based on 44. In accordance with these findings, increased collagen deposition in the lungs of septic mice was also evident in our study. Interestingly, effectiveness of iCRT3 in blocking Wnt/β-catenin pathway and attenuating collagen deposition in the bleomycin-induced fibrotic lung injury model has also been demonstrated previously23. These studies further support the approach of targeting Wnt/β-catenin signaling using small molecule inhibitors to control lung injury in sepsis. In addition to lung injury, acute kidney injury is also often observed in the septic patients45. Whether iCRT3 treatment can also attenuate kidney injury induced by sepsis needs further investigation.We examined the lung injury in septic mice, in more detail. CLP induced severe damage in the lung tissues as demonstrated by histologic analysis. After iCRT3 treatment, the structural integrity of the lung was better protected, with statistically significant improvement in lung injury score as judged by histologic examination. Early collagen deposition in rat lungs in CLP and LPS-induced sepsis model has been observed recently and associated with Wnt/β-catenin signaling activation46. However, when Wnt/β-catenin signaling was inhibited in bleomycin-induced lung fibrosis in mice using small molecule inhibitors XAV939 (works via β-catenin degradation) or ICG-001(works via binding to cyclic AMP response element-binding protein), the protective effect was attributed to increased epithelial differentiation of mesenchymal stem cells48. In our CLP model iCRT3 treatment not only attenuated lung collagen deposition but also decreased the expression of proinflammatory cytokines IL-6, TNF-α, and IL-1β and chemokines MIP-2 and KC. MIP-2 and KC are reported to be sufficient to induce neutrophil recruitment to the lung49. Indeed, iCRT3 treatment also reduced excessive infiltration of neutrophils observed as MPO activity in the lungs of septic mice.Of note, the protective effect of blocking Wnt/β-catenin pathway in pulmonary fibrosis via small interfering RNA (siRNA) for β-catenin, was attributed to the inhibition of collagen synthesis, by reducing TGF-β and matrix metaloproteinase-2 expression, with no significant effect on the inflammatory response51. By using TUNEL assay, we demonstrated that CLP induced significant apoptosis in the lungs of mice and iCRT3 treatment effectively decreased the number of apoptotic cells in the lungs of septic mice. Wnt/β-catenin signaling is known to regulate apoptosis through a variety of mechanisms, including TRAIL, caspases, STAT3, AKT, PTEN, PI3K, and MAPK pathways, enhancing or restraining it, depending upon cellular or environmental context52. Wnt/β-catenin-induced cell apoptosis has been reported in melanoma54, and hepatocellular carcinoma55. In addition, activated Wnt/β-catenin signaling was shown to promote macrophage apoptosis in response to mycobacterial infection via caspase-3 dependent pathway56. Apoptosis of macrophages has been implicated in the pathogenesis of sepsis57. This supports our study as blocking Wnt/β-catenin signaling resulted in reduced apoptosis.Apoptosis of immune and epithelial cells is an important mechanism contributing to acute lung injury and associated inflammationIn order to test the effectiveness of iCRT3 at the hyper-inflammatory stage in sepsis, we administered iCRT3 at 5 h after CLP when the levels of most proinflammatory cytokines are markedly elevated in mice. This study is the proof-of-concept to show the potential of using iCRT3 against sepsis. As for the treatment window, dose, and frequency of iCRT3 administration as well as its toxicity and pharmacokinetics, all these require a series of pre-clinical studies to develop iCRT3 as a drug candidate for treating sepsis. To limit the interference from other treatments, antibiotic administration to these animals was avoided for this mechanistic study. Also, to reduce the variability in statistic analyses, only male mice were used in this study. To conduct any future pre-clinical studies to evaluate the efficacy of iCRT3, based on the positive outcomes observed in this study, the antibiotic administration and mixed gender issues also need to be considered to mimic the clinical setting. In this study, we have demonstrated the effectiveness of iCRT3 treatment on the reduction of inflammation, apoptosis, and lung injury in short-term. Whether its effect can extend to long-term benefit needs to be further determined in a survival study.In conclusion, data provided in this study identifies that iCRT3 blocks the Wnt/β-catenin activity in macrophages to inhibit cytokine production and NFκB signaling. Treatment with iCRT3 significantly attenuates the sepsis-induced organ injury and systemic inflammation. In particular iCRT3 improves the histopathology of lung tissue and decreases lung collagen deposition. iCRT3 does so by effectively inhibiting the sepsis-induced excessive cytokine production, apoptosis and neutrophil infiltration in the lungs. Thus, Wnt/β-catenin signaling may be a potential therapeutic agent for sepsis.2.Mouse macrophage cell line RAW264.7 was purchased from ATCC . RAW264.7 cells were cultured in DMEM medium  containing 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO5 cells per ml. Cells were transiently co-transfected with 250 ng of TOP-TK-Luc or FOP-TK-Luc  and 25 ng pRL-TK  reporter plasmids, using the Lipofectamine 3000 Reagent  according to the manufacturer’s instructions. At 24 h after transfection, cells were pre-treated with iCRT3  or vehicle for 50 min and then stimulated with LPS (1 ng/ml) for another 24 h. The cells were lysed 48 h post-transfection and luciferase activity was measured with a Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s instructions. TOP-TK-Luc contains optimal and FOP-TK-Luc contains mutated TCF-binding sites placed upstream of a firefly luciferase reporter gene. The TOP and FOP firefly luciferase activity was normalized to Renilla luciferase activity from the cotransfected pRL-TK plasmid used as an internal control for transfection efficiency. All experiments were performed in triplicate at least twice.RAW264.7 cells were seeded the day before transfection at a density of 1.24 × 10Cell viability in each well was determined using a cell proliferation assay kit according to the manufacturer’s instruction . Briefly, 20 μl of the assay reagent were added to each well of the 96 well plate containing 100 μl of cells in culture medium and incubated at 37 °C for 1–4 h in a humidified 5% CO2 atmosphere. The absorbance was recorded at 490 nm.Male C57BL/6 mice (20 to 25 g) purchased from Taconic Biosciences  were used in all experiments at 8–12 weeks age. These mice were housed in a temperature-controlled room on a 12 h light/dark cycle in the animal facility within the Feinstein Institute for Medical Research  to acclimate to the environment for at least 5 days before being used for experiments and fed a standard laboratory diet. All experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health  and were approved by the Institutional Animal Care and Use Committee (IACUC) at the Feinstein Institute for Medical Research. All efforts were made to minimize suffering. Sepsis was induced in mice using CLP procedure. Briefly, the mice were anesthetized by isoflurane inhalation, and the abdomen was shaved and cleaned with 10% povidone iodine before operation. A 2-cm midline laparotomy was performed to expose the cecum. The cecum was tightly ligated (1 cm from the cecum tip) with a 4–0 silk suture distal to the ileocecal valve, double punctured with a 22-gauge needle, gently squeezed to expel small amount of feces and returned to the peritoneal cavity. The laparotomy site was then closed with a 6–0 silk suture in two layers and the animals were immediately resuscitated with 1 ml of normal saline injected subcutaneously. The sham animals underwent the same procedure but the cecum was neither ligated nor punctured. At 20 h after CLP or sham operation, mice were anesthetized and blood, lungs were collected. Blood samples were centrifuged to collect plasma and a section of lung tissue was preserved in formalin for histopathological analysis. The rest of lung tissues were immediately frozen in liquid nitrogen, and all the samples were stored at −80 °C until analysis.Mice were randomly allocated to three groups: sham (n = 5 mice), vehicle and treatment (n = 8 mice per group). iCRT3  was reconstituted with cell culture grade 100% DMSO as 50 mg/ml stock. 5 and 10 mg/kg body weight (BW) concentrations of iCRT3 were made by diluting stock in sterile normal saline with 5% DMSO. At 5 h after CLP, 5% DMSO in normal saline (vehicle) or iCRT3 at 5 or 10 mg/kg BW doses in 200 μl volume was delivered by intraperitoneal injection using 25 G × 7/8″ hypodermic needle . The investigator performing the animal experiments was blinded to the treatment assignment to eliminate any bias.TNF-α levels in the supernatants from RAW264.7 cultures and IL-6, TNF-α, and IL-1β levels in the plasma samples were quantified by using the specific mouse enzyme-linked immunosorbent assay (ELISA) kits . Plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were measured by using the commercial assay kits  according to the manufacturer’s instructions.The lung tissues were fixed in 10% formalin, paraffin embedded, cut into 5 μm sections, transferred to glass slides and stained with hematoxylin and eosin (H&E) and Masson’s-Trichrome. Morphologic changes in the lung tissues were examined under a light microscope, and evaluated by two independent investigators blinded to the treatment assignment. Lung injury was assessed according to the following pathological features: (i) alveolar mambrane thickening, (ii) vascular congestion, (iii) intra-alveolar hemorrhage, and (iv) interstitial and alveolar neutrophil infiltration. The semi-quantitative scores from 0 to 3 were designated based on absence or presence and severity of each of these features ranging from mild, moderate to severe and a cumulative total histology score was determined.Lung tissue sections were deparaffinized, rehydrated, treated with Proteinase K and stained with TUNEL staining kit  according to the manufacturer’s instructions to detect the presence of apoptotic cells. The negative control was performed by incubating slides in the mixture containing only deoxynucleotidyl transferase. TUNEL-positive cells were counted under a fluorescence microscope across 10 microscopic fields per section at 200 × magnification.DC protein assay . Total cell lysates were fractionated on Bis-Tris gels (4 to 12%) and transferred to nitrocellulose membrane. The membranes were then blocked with 0.1% casein  in phosphate-buffered saline and probed with anti-IκB  or anti-β-actin  antibodies. After the wash, the membranes were incubated with the appropriate fluorescence-labeled secondary antibodies  and scanned by the LI-COR Odyssey Fc Imager (LI-COR Biosciences).RAW264.7 cells were homogenized in lysis buffer  containing a protease inhibitor cocktail  by sonication. Protein concentrations were determined by T) method, normalized to mouse β-actin mRNA was used. Relative expression of mRNA was expressed as the fold change compared to the sham. The primers used for this study are listed in Table TRIzol  was used to extract total RNA from the lung tissue homogenates. RNA was reverse-transcribed into cDNA using murine leukemia virus reverse transcriptase . A PCR reaction was carried out in 20 μl of a final volume containing 0.06 μM of each forward and reverse primer, cDNA, and 10 μl SYBR Green PCR Master Mix . Amplification was conducted in an Applied Biosystems StepOnePlus real-time PCR machine under the thermal profile of 50 °C for 2 min, 95 °C for 10 min followed by 45 cycles of 95 °C for 15 sec and 60 °C for 1 min. For relative quantification, ΔΔCT comparative threshold cycle  and expressed as mean ± standard error of the mean (SEM). Multiple groups were compared with one-way analysis of variance (ANOVA) using Student-Newman-Keuls’ (SNK) test. Differences in values were considered significant if All data generated or analyzed during this study are included in this published article.