SAMMSON is highly expressed in liver cancer and liver TICs. SAMMSON silenced cells show impaired self-renewal capacity, while, its overexpression induces enhanced self-renewal. SAMMSON drives the activation of Wnt/β-catenin signaling, and thus promotes liver TIC self-renewal. SAMMSON interacts with EZH2, a core component of PRC2 complex, and inhibits the expression of CTNNBIP1 through EZH2 dependent manner. SAMMSON binds to CTNNBIP1 promoter and recruits EZH2 to CTNNBIP1 promoter. What’s more, targeting liver TICs through SAMMSON, EZH2 and Wnt/β-catenin signaling impaired liver TIC self-renewal, decreased tumor propagation and severity. Taken together, SAMMSON drives liver TIC self-renewal through EZH2-dependent Wnt/β-catenin activation.Liver cancer is one of the most serious cancers all over the world. Liver tumor initiating cells (TICs) account for tumor initiation and metastasis. However, the regulatory mechanism of liver TICs remains unclear. Here we found long noncoding RNA  Liver cancer, including hepatocellular carcinoma (HCC) and cholangiocarcinoma, is one of the most serious cancers all over the world ; while, Wnt/β-catenin signaling, one of the most important signaling in liver TICs, is regulated by many modulators . The welcis or in trans. Meanwhile, lncRNAs also interact with some key signaling molecules, and participate in the activation of signaling pathways through regulating the stabilities or activities of interaction proteins [Long noncoding RNAs (LncRNAs) are defined as RNA transcripts that contain more than 200 nucleotides in length without protein coding potential . For lonproteins –24. AlthSAMMSON is a long noncoding RNA located on chromosome 3p13-3p14, a frequent amplified region in melanomas [SAMMSON is a target of transcription factor SOX10 and interacts with p32 to enhance its oncogenic function [SAMMSON recruits p32 to mitochondrial localization and silencing of SAMMSON disrupts mitochondrial function of melanoma cells, conferring SAMMSON a new target for melanoma elimination. A recent work also revealed that SAMMSON is required for melanoma cell viability [SAMMSON in liver cancer and liver TICs is unclear. Here we found high expression of SAMMSON in liver cancer and liver TICs. SAMMSON initiates Wnt/β-catenin activation and the self-renewal of liver TICs. SAMMSON interacts with and recruits EZH2 to the promoter of CTNNBIP1, and inhibits its transcription initiation.elanomas . SAMMSONfunction . SAMMSONiability . HoweverSAMMSON plays an essential role in melanoma cell viability and metabolic vulnerability, while, its role in liver tumorigenesis and liver TIC self-renewal is unknown. Accordingly, we detected the expression levels of SAMMSON in liver cancer. SAMMSON is highly expressed in HCC samples, especially in advanced HCC samples , and found SAMMSON was highly expressed in spheres , followed by sphere formation. SAMMSON silenced cells showed impaired sphere formation  treated cells, SAMMSON overexpression had no influence on liver TIC self-renewal, suggesting that SAMMSON induced liver TIC through Wnt/β-catenin signaling  of SAMMSON is required for the interaction between SAMMSON and EZH2 , so we detected these modification of CTNNBIP1 promoter. As the results indicated, both in SAMMSON silenced and EZH2 inhibited cells, impaired enrichment of repressive histone modification were detected, further confirming the role of SAMMSON and EZH2 in transcriptional repression of CTNNBIP1  in liver cancer and liver TICs. Through interacting with EZH2 and inhibiting the transcription activation of CTNNBIP1, SAMMSON participates in Wnt/β-catenin activation, liver TICs self-renewal, liver cancer invasion and propagation. Additionally, we found SAMMSON-EZH2-Wnt/β-catenin axis served as a target for liver TIC elimination.This study explored the role of SAMMSON and EZH2, are involved in liver TIC self-renewal.Wnt/β-catenin signaling is the one of most deeply investigated pathways in TICs, especially in liver cancer and colorectal cancer , 27. As SAMMSON binds to CTNNBIP1 promoter and recruits EZH2 to CTNNBIP1 promoter, represses the transcription initiation of CTNNBIP1 and finally activates Wnt/β-catenin signaling. Our discovery reveals a new layer for Wnt/β-catenin regulation.β-catenin is core component of Wnt/β-catenin signaling, and β-catenin stability regulation and activity regulation are precisely regulated. Upon Wnt activation, APC complex, a degrading machine of β-catenin, is disrupted . β-catenSAMMSON is located on mitochondria and interacted with p38 in melanoma cells [SAMMSON’s nuclear location in liver TICs. Meanwhile, SAMMSON involved in mitochondrial function and viability of melanoma cells [SAMMSON participates in liver TIC self-renewal. There are so many differences between our finding and previous work. In our opinion, at least two reasons account for these difference. One is the different tumor types. Liver tumor and melanoma are very different, and many regulatory networks may differ in different tumors [SAMMSON is truly located in cell nucleus and interacts with EZH2.In previous report, ma cells ; here wema cells , 26; hert tumors . The othEZH2 is the core component of PRC2, one of the most important chromatin remodeling complexes . EZH2 me293T cells (ATCC CRL-3216), used for lentivirus production, were purchased from ATCC. Cells were maintained in DMEM medium, supplemented with 10% FBS (Invitrogen), 100 μg/ml penicillin, and 100 U/ml streptomycin.Human liver cancer samples were obtained from the department of Pathology with informed consent, with the approval of the Institutional Review Board. All experiments were approved by the institutional committee of Institute of the First Affiliated Hospital of Nanchang University.Anti-histone H3 (cat. no. 4499), anti-CCND2 (cat. no. 3741), anti-EZH2 (cat. no. 5246) antibodies were from Cell Signaling Technology. Anti-β-catenin (cat. no. ab32572), anti-CTNNBIP1 (cat. no. ab129011) antibodies were obtained from Abcam. Anti-β-actin (cat. no. A1978) were obtained from Sigma-Aldrich. Phycoerythrin (PE)-conjugated CD133 (cat. no. 130098826) was purchased from MiltenyiBiotec. Anti-EEA1 (sc-53939), anti-Myc (sc-4084) antibodies were purchased from Santa Cruz Biotechnology. Alexa488-conjugated donkey anti-mouse IgG (cat. no. R37114) and Alexa594-conjugated donkey anti-rabbit IgG (cat. no. R37119) were from Molecular Probes. DAPI (cat. no. 28718-90-3) were from Sigma-Aldrich. The LightShift Chemiluminescent RNA EMSA kit (cat. no. 20158) and Chemiluminescent Nucleic Acid Detection Module (cat. no. 89880) were from Thermo Scientific. T7 RNA polymerase (cat. no. 10881767001) and Biotin RNA Labeling Mix (cat. no. 11685597910) were from Roche.SAMMSON silenced cells. 36 h later, cellular luciferase activities were detected with dual-detection luciferase detection kit .TOPFlash reporter plasmid  and mutant FOPFlash reporter plasmid , along with thymidine kinase (TK), were transfected into For sphere formation assay, 5000 primary cells were added in Ultra Low Attachment 6-well plates (Corning Company) for incubation with DMEM/F12 (Life Technologies) supplemented with B27, N2, 20 ng/ml bFGF  and 20 ng/ml EGF . Two weeks later, the spheres were collected and counted for sphere initiating ratios.SAMMSON silenced spheres were incubated in 1% formaldehyde at 37°C for crosslinking, followed by incubation in SDS lysis buffer and subsequent ultrasonic for shearing DNA to segments of 200-1,000 bp in length. EZH2 antibody was added into samples for DNA segment enrichment.We performed ChIP assays according to the manual of Upstate Biotechnology, Inc. Briefly, SAMMSON were generated in vitro with biotin RNA labeling mix. The labeled RNA transcript was incubated with oncospheres lysate, followed by streptavidin beads enrichment and SDS–PAGE. The interaction protein was detected with Western blot or mass spectra.For RNA pulldown assays, biotin-labeled SAMMSON enrichment was examined using real-time PCR.For RIP, liver oncospheres were incubated with 1% formaldehyde for crosslinking, and crushed with RNase-free RIPA buffer. The Supernatants were incubated with EZH2 antibody and subsequent protein A/G beads for RNA enrichment, and 6SAMMSON silenced or overexpressed cells were subcutaneously injected into BALB/c nude mice for 1 month. Then tumors were obtained for weight detection. For tumor initiating assays, 10, 1×102, 1×103, 1×104, and 1×105SAMMSON silenced cells were subcutaneously injected into BALB/c nude mice. Tumor initiation was observed 3 month later. For tumor propagation and initiating assay, 6-week-old BALB/c nude mice were used. For every samples, 6 mice were used.For tumor proliferation assay, 1×10Two-tailed Student’s t tests were used for statistical analysis. P <0.05 was considered to be statistically significant.


Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins  and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis. In situ transition of fibroblasts into α‐smooth muscle actin (α‐SMA)‐positive myofibroblasts, transdifferentiation of epithelial cells, tissue accumulation of bone marrow‐derived progenitor cells trafficking from the circulation are some of the putative mechanisms underlying the expansion of the pool of biosynthetically activated mesenchymal cells Pulmonary fibrosis (PF) is a progressive interstitial lung disease characterized by fibroblast proliferation and excess deposition of extracellular matrix proteins, which lead to lung function distorted in vivoOn the other hand, the possible mechanisms of myofibroblast elimination or dedifferentiation are still almost an uncharted territory Smads family proteins play a key role in the process of the transfer of TGF‐beta signalling from cell surface receptors to the nucleus, and signal transduction mediated by different Smad and different TGF family members Canonical Wnt/β‐catenin signalling is one of the major oncogenic pathways that provide essential developmental signals during embryogenesis. Meanwhile, the importance of dysregulated Wnt/β‐catenin signalling in a variety of human diseases has been long appreciated In this study, we used alveolar epithelial cell (AEC) and fibroblasts to investigate TGF‐β/β‐catenin pathway convergence, as well as its consequences on transcription of α‐SMA and collagen I, target genes associated with myofibroblast proliferation. This article describes the mechanisms through which epithelial cells and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin and highlights its role in lung fibrosis. Understanding the mechanism on the basis of myofibroblast proliferation may provide useful strategies for preventing or reducing fibrosis.2. When the cells reached 70–80% confluence and then cultured in 1640 medium without FBS for 6 hrs before stimulated with recombinant human TGF‐β1  for 24 hrs. Then, treated with ICG‐001 10 μM  for 6 hrs was added to the medium at the indicated concentrations and time‐points. Nuclear and cytoplasmic protein extracts from A549 and HELF cells for Western blot analysis.Human pulmonary epithelial cell line A549 and human embryonic lung fibroblast HELF were obtained from ATCC  and cultured in 1640 medium  in the presence of 10% foetal bovine serum  at 37°C in a humidified atmosphere with 5% COFor β‐catenin knockdown, A549 and HELF cells were transfected with small‐interfering RNA (siRNA) targeting the human β‐catenin gene or control non‐targeting siRNA using lipofectamine 2000 . The β‐catenin siRNA and negative control siRNA were designed and synthesized by GenePharma  according to human‐specific sequences. The primer sequences of β‐catenin siRNA were as follows: β‐catenin forward, 5′‐GCAGUUGUAAACUUGAUUATT‐3′, β‐catenin reverse, 5′‐UAAUCAAGUUUACAACUGCTT‐3′, which were transfected into A549 and HELF with lipofectamine 2000 according to the manufacturer's instructions. To identify whether the gene is silenced completely, reverse‐transcription polymerase chain reaction (RT‐PCR) was used. Total mRNA was extracted from cells using TRIzol reagent (Invitrogen) and cDNA was synthesized from mRNA using Prime Script RT‐PCR synthesis kit  following the manufacturer's instructions. RT‐PCR was performed under standard protocol. The following primers were synthesized by Sangon Biotech Co., Ltd : β‐catenin, the forward primer was 5′‐GGGTCCTCTGTGAACTTGCT‐3′ and the reverse was 5′‐AATCTTGTGGCTTGTCCTCA‐3′, and the amplicon size was 162 bp. β‐actin, the forward primer was 5′‐TCAGGTCATCACTATCGGCAAT‐3′ and the reverse was 5′‐AAAGAAAGGGTGTAAAACGCA‐3′, and the amplicon size was 432 bp. Results were presented as relative mRNA levels normalized against those of β‐actin. After 6 hrs, medium was replaced with fresh medium supplemented with or without TGF‐β1 for an additional 24 hrs. Proteins were harvested for detection by Western blot analysis.Forty Sprague Dawley rats weighing 180–220 g were purchased from the Experimental Animal Center of Anhui Medical University, Hefei, Anhui, China, and assigned to two groups randomly. Lung injury in the model group was induced by the endotracheal injection of 5 mg/kg bleomycin , while the control group received the same volume of saline instead. The day of intratracheal injection with BLM or saline was designated day 0. At 14 and 28 days after BLM injection, the rats were anaesthetized with 10% chloral hydrate (2.5 ml/kg) intraperitoneally, and then the animals were killed and the lungs were removed. All rats experimental procedures were approved by Anhui Medical University Animal Care Committee, and followed the protocol outlined in The Guide for the Care and Use of Laboratory Animals published by the USA National Institute of Health .H&E and immunohistochemical (IHC) staining were performed as described previously Lung homogenates and cell lysates were collected in RIPA Lysis Buffer  containing 1 mM proteinase inhibitor phenylmethylsulfonyl fluoride . Nuclear and Cytoplasmic Protein Extraction Kits were used to collect protein in the nuclear . The extracts were mixed with sample buffer in a ratio of 4:1 and denatured by boiling. Then, the equivalent amount of protein was separated using 12% SDS‐PAGE and transferred to PVDF membranes . After blocked with 5% non‐fat milk  for 2 hrs, the membranes were incubated overnight at 4°C with the primary antibodies anti‐E‐cadherin , anti‐α‐SMA , anti‐vimentin , anti‐Col‐I , anti‐Smad2/3 , anti‐p‐Smad2/3 , anti‐β‐catenin , anti‐H3 , anti‐GAPDH  and anti‐β‐actin  antibodies, which were all from USA. On the next day, the blots were incubated with anti‐rabbit or antimouse IgG horseradish peroxidase‐conjugated secondary antibodies  for 1 hr at room temperature. Finally, the signal was detected with enhanced chemiluminescence reagent . Intensity was quantified using Scion Image software . All experiments were performed independently at least three times. β‐actin or GAPDH was used as an equal protein loading control.t‐test. Groups containing multiple comparisons were analysed using anova, and values of P < 0.05 were considered to be statistically significant.Data are expressed as the mean ± standard deviation (S.D.). Differences in measured variables between experimental and control groups were assessed using Student's in vivo, 40 rats received intratracheal injection of saline ± bleomycin (5 mg/kg). After 14 and 28 days, immunostaining of lung paraffin section with E‐cadherin  for 4 hrs before treated them with TGF‐β1 (10 ng/ml) for 24 hrs. As shown in Figure Several studies have demonstrated that alveolar type‐II epithelial cells in response to TGF‐β can undergo the EMT and differentiate into myofibroblasts in vitro is well documented. To evaluate the effect of β‐catenin on activating TGF‐β1/Smad2/3 signalling, A549 and HELF cells were exposed to a constant dose of ICG‐001 (10 μM) for the indicated times. The results show that the expression of smad2/3 proteins inhibited by ICG‐001 significantly , and the ‘Smad2/3′ indicates the total Smad2/3 protein (including the p‐Smad2/3 and the Smad2/3 not phosphorylated in the cells). The results show that TGF‐β1 directly activates Smad2/3 phosphorylation and β‐catenin siRNA effectively inhibits the activation of Smad2/3 (Fig. Pulmonary fibrosis is characterized by excessive proliferation of fibroblasts and concomitant collagen accumulation within the alveolar and interstitial compartments of the lung In the present study, we investigated the direct interaction of TGF‐β1 with fibroblasts or epithelial cells, and the potential cellular effects caused by TGF‐β1 in fibroblasts or epithelial cells. Although the present data show that TGF‐β1 directly induced the phosphorylation of Smads, the mechanism underlying TGF‐β1‐activated phosphorylation of Smads is still unresolved. However, it has not been elucidated whether β‐catenin involves the recognized TGF‐β1/Smad2/3 signalling that contributes to myofibroblast proliferation in PF via Smad‐dependent TGF‐β signalling while suppressing AECs or fibroblast differentiation and inducing their differentiation into myofibroblasts. The net effect of these combined stimulatory and inhibitory activities is to promote fibrogenesis Our results indicate that TGF‐β1 is a multifunctional cytokine that plays important roles in the fibroblast‐to‐myofibroblast transition and EMT. The canonical TGF‐β signalling pathway involves TGF‐β receptor‐mediated phosphorylation of the Smad proteins including Smad2 and Smad3, which dimerize together, and after phosphorylation translocate to the nucleus to induce expression of target genes. As is stated above, the new target for antifibrotic urgent need to find except for targeting TGF‐β1. Although dysregulation of Wnt signalling is implicated in tumorigenesis, the potential relevance of Wnt signalling in fibrogenesis has only recently begun to be appreciated, and the potential mechanisms remain to be elucidated. Accumulated data have shown that canonical Wnt/β‐catenin pathway is involved in the induction of myofibroblast proliferation In summary, the demonstration that impaired Wnt antagonism is associated with Wnt/β‐catenin pathway hyperactivation in lung tissue obtained from a subset of rats with PF. Canonical Wnt/β‐catenin signalling involved in fibroblasts and AECs stimulated their proliferation, migration and myofibroblast proliferation. Induction of interaction between β‐catenin by TGF‐β and inhibition of this interaction and of myofibroblast proliferation by ICG‐001 and β‐catenin siRNA suggest that β‐catenin is required for regulation of fibrotic reaction by Smad2/3. Therefore, the Wnt/β‐catenin pathway is a promising target for antifibrotic therapeutic approaches. The exact molecular mechanism responsible for the repression of expression of contractility‐related genes after Wnt/β‐catenin signalling perturbation in myofibroblasts is unknown and its elucidation represents an attractive challenge for future researches.Liang Xu, L.C.Li participated in research design; L.C.Li, D.L.Li and Zhihui Zhang conducted experiments; X.T.Mo, W.C.Zhou, P.Zhao contributed new reagents or analytic tools; L.C.Li, W.H.Cui, X.T.Mo performed data analysis; Liang Xu, J.Gao wrote or contributed to writing of the manuscript.All authors disclose that they do not have any commercial association that might pose a conflict of interest in connection with the manuscript.


Overall, TSN is capable of inhibiting the lipogenesis of adipocytes by activating the Wnt/β-catenin pathway, suggesting potential application of TSN as a natural anti-obesity agent.Toosendanin (TSN), a triterpenoid extracted from Melia toosendan, has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, and anti-arthritic activities. However, its anti-adipogenic effect remains unknown. Here, we found that TSN dose-dependently attenuated lipid accumulation in preadipocytes 3T3-L1 as evidenced by Oil Red O staining. TSN also significantly downregulated mRNA and protein levels of adipocytokines (adiponectin and leptin), CCAAT/enhancer binding proteins α (C/EBP-α), peroxisome proliferator-activated receptor γ (PPAR-γ), fatty acid synthase, and acetyl-CoA carboxylase in adipocytes. To understand the mechanism, we observed that TSN effectively activated Wnt/β-catenin pathway, in which TSN increased low density lipoprotein receptor related protein 6, disheveled 2, β-catenin, and cyclin D1 expression levels, while it inactivated glycogen synthase kinase 3β by enhancing its phosphorylation. Moreover, TSN reduced weight of gonadal white fat and serum triacylglycerol (TAG) content in high-fat diet (HFD)-fed mice. Interestingly, the  Obesity is originated from the excessive lipid accumulation of adipose tissue associated with adipogenesis. The adipogenesis is a complex process involving preadipocyte proliferation, differentiation, intracellular lipid accumulation, and changes in genes expression3. Among these processes, adipocyte differentiation is tightly regulated by lots of key adipogenic transcription factors, including nuclear receptor peroxisome proliferator activated receptor gamma (PPAR-γ) and members of the CCAAT/enhancer binding protein (C/EBP) family5. These transcription factors are indispensable for expression of adipocyte-specific genes, such as adipocyte fatty acid binding protein (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), leptin, and adiponectin6.Obesity promotes the prevalence of a variety of diseases including type 2 diabetes, hyperlipidemia, hypertension, cardiac injury, and cancer7. In the canonical Wnt/β-catenin signaling, Wnt ligands bind to frizzled receptors and low density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors. Following this interaction, disheveled (DVL) gets phosphorylated and the glycogen synthase kinase 3 (GSK3)-AXIN-adenomatous polyposis coli (APC) complex is disrupted, which promotes the stabilization and nuclear translocation of β-catenin7. In the nucleus, β-catenin positively regulates its target gene cyclin D1 (CCND1), which downregulates major adipogenic transcription factors, such as PPAR-γ and C/EBP-α9. Therefore, activating β-catenin pathway and its components could be considered as effective strategies to combat obesity.Increasing evidences suggest that the wingless-type MMTV integration site (Wnt)/β-catenin serves as a negative regulator for adipocyte differentiation10; allopurinol, quercetin, and rutin ameliorate lipid accumulation in fructose-fed rats11; thiacremonone attenuates lipid accumulation partially mediated via AMPK activation in 3T3-L1 adipocytes12. The fruit of MeliatoosendanSieb et Zucc, also known as Chuan Lian-Zi in Chinese medicine, has significant analgesic effects13; in addition, it can be used to kill parasites such as roundworms14. Toosendanin (TSN) is the main active component of the fruit of MeliatoosendanSieb et Zucc; it can selectively block acetylcholine (Ach) release and has antibotulismic effects both in vivo and in vitro17. In recent years, TSN has been demonstrated to induce apoptosis on a variety of cancer cells18. We previously described that TSN, when used at low concentrations, was able to reverse lung cancer cells resistance toward TRAIL-mediated apoptosis by upregulating TRAIL receptors19. All of the data suggest potential application of TSN in cancer treatment.Natural products that modulate expression of proteins involved in adipogenesis have attracted much attention. For example, pterostilbene inhibits adipocyte differentiation by targeting C/EBP homologous protein-10 (CHOP-10) in 3T3-L1MeliatoosendanSieb et Zucc is also an important ingredient in some Chinese prescriptions to treat fat liver diseases, suggesting it may influence lipid metabolism. However, the anti-adipogenic effect of TSN has not yet been investigated. To fill this gap and test the hypothesis, we performed this study, and found that TSN can significantly attenuate adipogenesis through activating the Wnt/β-catenin signaling.Interestingly, the fruit of TM 2000 transfection reagent was purchased from Invitrogen .The chemical structure of TSN is shown in Fig. 12. 3T3-L1 preadipocytes were cultured in DMEM (10% FBS). To induce differentiation, two days post-confluence (day 0) cells were incubated with induction medium supplemented with MDI  and 10% FBS for 48 h. The medium was then replaced with DMEM containing 5 μg/mL insulin and 10% FBS for another 48 h (day 2). On 4th day, the cells were maintained in DMEM/10% FBS until most of the cells were differentiated into mature adipocytes with abundant lipid droplets at 6th day. TSN was added to the culture medium at different concentrations during the adipogenic period (6 days). Cells cultured in DMEM with 10% FBS but without induction medium were regarded as the undifferentiated group (UND).The 3T3-L1 preadipocytes were differentiated as previously described12. Preadipocytes were seeded and induced to differentiate in induction medium with increasing concentrations of TSN for 6 days. On 6th day, the treatment medium was removed and replaced with 100 μL 10% FBS fresh medium and 20 μL MTT cell viability reagents. Then, cells were incubated at 37 °C in a 5% CO2 atmosphere for 1 h. The absorbance at 490 nm was measured on a M200 PRO multifunctional microplate reader .The cell viability was determined by MTT assay in 96-well plates12. At 6th day, cells were washed and fixed in 4% paraformaldehyde for 30 min, stained with Oil Red O working solution and incubated for another 20 min at room temperature. After washed three times with PBS, cells were photographed by a light microscope . Next, quantitation was carried out by extracting Oil Red O-stained lipid droplets with 100% isopropanol and the OD at 540 nm was measured with a M200 PRO multifunctional microplate reader . Finally, lipid accumulation for each TSN-administrated group is expressed relative to that of MDI-differentiated cells.The cellular lipid content was evidenced by Oil Red O staining as previously described20. Total RNA was extracted from 3T3-L1 adipocytes and gonadal fat tissue using TRIZOL reagent. The cDNA was obtained by reverse transcription in a 20 μL reaction containing 2 μg of total RNA, oligo (dT), and reverse transcription premix. Next, normal amplification consisted of 28–30 cycles as follows: denaturing at 94 °C for 30 s, annealing at 56 °C for 1 min and extending at 72 °C for 30 s, followed by a final 10 min incubation at 72 °C. The PCR was performed using a Bio-Rad T100TM Thermal Cycler , PCR products were electrophoresed by 2% agarose gel electrophoresis and visualized using a Tanon 2000 imaging system .The reverse transcription-polymerase chain reaction (RT-PCR) and the quantitative real-time PCR (Q-PCR) were performed as previously describedThe Quantitative real-time PCR (Q-PCR) reactions were performed with the SYBR green PCR system in an ABI 7500 thermal cycler. The cycling conditions were as follows: 95 °C for 3 min; followed by 40 cycles involving denaturing at 95 °C for 10 s, annealing at 60 °C for 5 s and extension at 72 °C for 10 s. Expression of mRNAs was normalized by the mRNA levels of β-actin, which was used as an internal control. The primers were shown as follows: C/EBP-α: sense, 5′-GCGCAAGAGCCGAGATAAAG-3′, antisense, 5′-CGGTCATTGTCACTGGTCAACT-3′; C/EBP-β: sense, 5′-CGCAGACAGTGGTGAGCTT-3′, antisense, 5′-CTTCTGCTGCATCTCCTGGT-3′; C/EBP-δ: sense, 5′-CAAGCTGAGCGACGAGTACA-3′, antisense, 5′-AGCTGCTCCACCTTCTTCTG-3′; PPAR-γ: sense, 5′-TGAACGTGAAGCCCATCGAG-3′, antisense, 5′-CTTGGCGAACAGCTGAGAGG-3′; FAS: sense, 5′-ATCCTGGAACGAGAACACGATCT-3′, antisense, 5′-AGAGACGTGTCACTCCTGGACTT-3′; ACC: sense, 5′-AGCTGATCCTGCGAACCT-3′, antisense, 5′-GCCAAGCGGATGTAAACT-3′; Adiponectin: sense, 5′-GGCAGGCATCCCAGGACATC-3′, antisense, 5′-TCTCACCCTTAGGACCAAGAAGAC-3′; Leptin: sense, 5′-GAGACCCCTGTGTCGGTTC-3′, antisense, 5′-CTGCGTGTGTGAAATGTCATTG-3′; LRP6: sense, ACCTCAATGCGATTTGTTCC, antisense, GGTGTCAAAGAAGCCTCTGC; DVL2: sense, GCTTCCACATGGCCATGGGC, antisense, CACTGCTGGTGAGAGTCACAG; β-Catenin: sense, GCCAAGTGGGTGGTATAGAG, antisense, CTGGGTATCCTGATGTGC; CCND1: sense, AAATCGTGGCCACCTGGAT, antisense, CATCCGCCTCTGGCATTTTG; β-Actin: sense, AGAACATCATCCATGCATCCA, antisense, GCCTGCTTCACCACCTTCTTG.20. 3T3-L1 adipocytes were lysed using RIPA lysis buffer with a protease inhibitor cocktail. Gonadal fat tissue was lysed by the total protein extraction kit for adipose tissues from Invent Biotechnologies . The protein concentrations of lysate were determined by using the Bradford protein assay kit. Equal amounts of protein (30 to 60 μg) in each sample were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and then incubated overnight with primary antibodies against C/EBP-α, PPAR-γ, FAS, ACC, adiponectin, leptin, β-catenin, phosphorylated GSK3β, and α-tubulin (1:1000 dilution) at 4 °C. After washing three times in Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody. Target proteins were detected with the enhanced chemiluminescence (ECL) detection system and visualized with the Tanon imaging system and the gene snap program .The western blot was performed as previously describedTM 2000 transfection reagent. Two days after confluence, 3T3-L1 cells were cultured in serum-free medium for 1 h and transfected with 60 nM of β-catenin siRNA or control siRNA using the transfection reagent. After 9 h, the transfected cells were differentiated as described above.β-catenin knockdown by siRNA transfection was performed as described in instruction of lipofectamine® Reporter Assay kit  following the manufacturer’s instructions.The 3T3-L1 adipocytes in 24-well plates were transfected with empty vector (pGL3 basic) or the β-catenin promoter (−220 bp to +10 bp) plasmid, which was cloned into pGL3 basic. After transfection for 10 h, the cells were incubated for another 10 h. Then, luciferase activity was determined using a Dual Luciferase21. The gonadal fat tissues and liver from mice were fixed in 4% formalin buffer and the fixed specimens were processed to paraffin blocks, sectioned, and subject to hematoxylin-eosin (H & E) staining for histological analysis according to the standard protocols.The H & E staining was performed as previously described22. The mice were fed with a 45% high fat diet (HFD) from FBSH  for one month and a half, the mice were grouped randomly into NFD + placebo, HFD + placebo, HFD + TSN (0.1 mg/kg·d) and 8 mice for each group. The effect of TSN on the levels of ALT and AST (U/L) in liver was examined by ELISA kit according to the manufacturer instruction. Body weight, food intake, and weight of gonadal white fat (gWAT) were calculated. The triacylglycerol (TAG) content in serum and gonadal fat tissue was determined in duplicate using the triglyceride (GPO-Trinder) kit. The gonadal fat tissues were subjected to hematoxylin-eosin (H & E) staining for histological analysis as described above. The mRNA expressions of C/EBP-α, PPAR-γ, and β-catenin were examined by real-time PCR. Total protein of gonadal white fat was extracted by total protein extraction kit for adipose tissues from Invent Biotechnologies, and the protein samples were subjected to western blot analysis.Eight weeks old male C57 BL/C6 mice used in this study were approved by the Animal Care and Protection Committee of Nanjing University-Gulou hospital (SYXK 2004-0013). The authors confirmed that all animals received human care and all animal experiments were performed in accordance with the relevant guidelines and regulations. This experiment was conducted as previously described#p < 0.001 and Фp < 0.001). P < 0.05 was considered statistically significant between groups.The values are presented as means ± SD. Statistical differences between two means were determined using Student’s t-test  using Graphpad prism 5 to calculate p-values , 31.24% (P < 0.01), and 65.5% (P < 0.001) respectively, as compared with the control cells  for 6 days. Cell viability was determined by MTT assay after treatment. As shown in Fig. lls Fig. .th day, the mRNA levels of C/EBP-α and PPAR-γ in TSN-treated cells was nearly 53% and 62% of the control cells. Notably, TSN also demonstrated a weak inhibitory effect on the mRNA expressions of C/EBP-β and C/EBP-δ, the upstream regulators of PPAR-γ and C/EBP-α, but the effect was not significant (P > 0.05). Consistent with the results obtained by real-time PCR, the protein levels of C/EBP-α and PPAR-γ were also reduced by 0.73-fold and 0.52-fold  in TSN (50 nM)-treated cells, as compared with control cells  mRNA and protein levels both in vitro and in vivo, which indicated that TSN inhibited adipogenesis by hindering the transcription factor cascade.The process of preadipocytes differentiation is regulated by a complex network of transcription factors. PPAR-γ and C/EBP-α are the master adipogenic transcription factors mainly found in adipose tissue3. As showed in this study, TSN significantly decreased the expression of FAS and ACC, suggesting that TSN may negatively control lipid accumulation by modulating biosynthesis-related genes in differentiated 3T3-L1 adipocytes.Next, the adipogenic transcription factors modulate the expression of adipocyte-specific genes, including FAS and ACC which are the essential lipogenic enzymes monitoring biosynthesis of fatty acids and triacylglycerols in the late stage of adipogenesis7. As a negative regulator of adipocyte differentiation, β-catenin can directly suppress the activity of PPAR-γ by means of the functional interaction between the TCF/LEF-binding domain of β-catenin and the catenin-binding domain of PPAR-γ26. The β-catenin can also decrease the activity of PPAR-γ and C/EBP-α by increasing CCND1 transcription26. In this study, the expressions of Wnt/β-catenin signaling proteins were upregulated by TSN in well-differentiated 3T3-L1 adipocytes as well as in HFD-fed mice. The β-catenin protein and mRNA levels were elevated by TSN through suppressing the degradation of β-catenin protein and enhancing the transcription of β-catenin mRNA, respectively. Moreover, TSN-induced upregulation of adipogenesis was reversed by β-catenin silencing. Hence, Wnt/β-catenin signaling plays critical role in the anti-adipogenic effect by TSN  may activate the AP-1 and G3BP132, the second messenger Ca2+ can also stimulate the transcriptional activity of AP-133. Interestingly, TSN increases Ca2+ influx in NG108-15 cells via L-type Ca2+ channels34, TSN can also trigger the generation of H2O2 in A549 cells19. Both ROS and Ca2+ are involved in the tumor-sensitizing effect of TSN on non-small-cell lung carcinoma19. Therefore, it seems reasonable to presume that TSN-induced upregulation of β-catenin is also regulated by Ca2+ and ROS signaling. Obviously, more experiments need to be done to clarify this presumption. Finally, the anti-adipogenesis of TSN imply that this chemical may also be useful in the treatment of fatty liver disease (FLD), because lipid accumulation in hepatocyte is a major characteristic of FLD. This concern will be addressed in further studies.It has been reported that the Wnt/β-catenin pathway is activated when the secreted glycoprotein Wnt binds to a cell surface receptor, such as the frizzled receptor and LRP5/6 co-receptorsin vitro and in vivo (Fig. In conclusion, TSN inhibited adipocytes differentiation and lipid accumulation through activating Wnt/β-catenin signaling both ivo Fig. . TSN migData 1


The beneficial effects of tea consumption on cancer prevention have been generally reported, while (−)-Epigallocatechin-3-gallate (EGCG) is the major active component from green tea. Cancer stem cells (CSCs) play a crucial role in the process of cancer development. Targeting CSCs may be an effective way for cancer intervention. However, the effects of EGCG on colorectal CSCs and the underlying mechanisms remain unclear. Spheroid formation assay was used to enrich colorectal CSCs from colorectal cancer cell lines. Immunoblotting analysis and quantitative real-time polymerase chain reaction were used to measure the alterations of critical molecules expression. Immunofluorescence staining analysis was also used to determine the expression of CD133. We revealed that EGCG inhibited the spheroid formation capability of colorectal cancer cells as well as the expression of colorectal CSC markers, along with suppression of cell proliferation and induction of apoptosis. Moreover, we illustrated that EGCG downregulated the activation of Wnt/β-catenin pathway, while upregulation of Wnt/β-catenin diminished the inhibitory effects of EGCG on colorectal CSCs. Taken together, this study suggested that EGCG could be an effective natural compound targeting colorectal CSCs through suppression of Wnt/β-catenin pathway, and thus may be a promising agent for colorectal cancer intervention. Colorectal cancer is the third most common cancer and the most common gastrointestinal cancer in the world. As predicted, more than 1 million people will develop colorectal cancer every year . AccumulWnt/β-catenin pathway is considered one of the most important pathways in the maintenance of CSCs properties . In the (−)-Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol from green tea , a widelTherefore, the present study aimed to investigate the influence of EGCG on colorectal CSCs as well as the underlying mechanisms. Here, we documented that EGCG inhibited colorectal CSCs properties through suppression of Wnt/β-catenin pathway. 2. Both cell lines were grown in RPMI 1640 medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 mg/mL streptomycin . EGCG and dimethyl sulfoxide were purchased from Sigma . LiCl was acquired from Biosharp .Human colon cancer cell lines DLD-1 and SW480 were obtained from American Type Culture Collection and maintained at 37 °C in incubator containing 5% CODLD-1 and SW480 cells were washed and transferred to DMEM-F12 medium  in non-adherent dishes (Costar). This medium contained no serum but 20 ng/mL EGF , 5 μg/mL insulin , 10 ng/mL bFGF , 0.4% BSA , 2% B27 . The spheroids were grown for six days and fed every 48 h. Spheroids were counted under a microscope  if the diameter was greater than 50 μm. Cells were harvested and washed with PBS and lysed with lysis buffer. Concentrations of the protein were determined by the bicinchoninic acid protein assay . Forty micrograms of proteins were subject to 7.5–10% SDS-PAGE and transferred to NC membrane . After being blocked with 5% non-fat milk for 1 h, the membranes were incubated with relevant primary antibody overnight at 4 °C. Then, the membranes were washed and probed with horseradish peroxidase-conjugated secondary antibody. GAPDH served as the loading control. The antibodies used in the present study were purchased from Proteintech .−(ΔΔCt). The PCR primers were synthesized by Beijing Genomics Institute , and the primers used were as follows:CD133-F, 5′-TACAACGCCAAACCACGACTGT-3′;CD133-R, 5′-TCTGAACCAATGGAATTCAAGACCCTTT-3′;CD44-F, 5′-GACACATATTGTTTCAATGCTTCAGC-3′;CD44-R, 5′-GATGCCAAGATGATCAGCCATTCTGGAAT-3′;ALDHA1-F, 5′-GCACGCCAGACTTACCTGTC-3′;ALDHA1-R, 5′-CCTCCTCAGTTGCAGGATTAAAG-3′;Oct4-F, 5′-TGGGATATACACAGGCCGATG-3′;Oct4-R, 5′-TCCTCCACCCACTTCTGAG-3′;Nanog-F, 5′-TTTGTGGGCCTGAAGAAAACT-3′;Nanog-R, 5′-AGGGCTGTCCTGAATAAGCAG-3′;β-catenin-F, 5′-AAGACATCACTGAGCCTCCAT-3′;β-catenin-R, 5′-CGATTTGCGGGACAAAGGGCAA-3′;PCNA-F, 5′-CTGAAGCCGAAACAGCTAGACT-3′;PCNA-R, 5′-TCGTTGATGAGGTCTTGAGTGC-3′;Cyclin D1-F, 5′-AGGCCCTGGCTGCTACAAG-3′;Cyclin D1-R, 5′-ACATCTGAGTGGGTCTGGAG-3′;GAPDH-F, 5′-CAAGGTCACCATGACAACTTTG-3′;GAPDH-R, 5′-GTCCACCACCCTGTTGCTGTAG-3′.Total RNA was isolated with Trizol reagent . Total RNA (1 μg) was transcribed into cDNA with the 5× All-In-One RT MasterMix . The qRT-PCR was performed using an EvaGreen 2× qPCR MasterMix  and ABI 7300 real-time PCR detection system . The levels of mRNA expression for each gene were normalized by its respective GAPDH. Fold changes in gene expression were calculated by a comparative threshold cycle (Ct) method using the formula 2After treatment for six days, the cell spheroids were washed and fixed with paraformaldehyde. The cell spheroids were then blocked with 5% BSA and stained with rabbit CD133 antibody from Proteintech  at 4 °C overnight. After being washed with PBS, the cell spheroids were stained with FITC-conjugated goat-anti-rabbit antibody  for 1 h. The cell spheroids were then counterstained with 4,6′-diamidino-2-phenylindole (DAPI) for 10 min. The fluorescent images were obtained using a fluorescence microscope .t-test and one-way analysis of variance (ANOVA) were performed to compare the difference between two or multiple groups. Significant difference was taken as * p < 0.05 or ** p < 0.01. All analyses were performed with SPSS version 11.0 software.Data are expressed as means ± SD. Student’s Accumulating evidence has revealed that spheroids formed in serum-free medium (SFM) exhibited more stem cell properties . CD133, We next examined the inhibitory effects of EGCG on colorectal cancer spheroids. DLD-1 and SW480 cell spheroids were treated with different concentrations of EGCG for six days. Spheroid formation assay showed that EGCG treatment led to a significant decrease in the number and size of DLD-1 and SW480 cell spheroids in a dose dependent manner A. WesterTo further investigate the effect of EGCG on spheroid growth in vitro, we next evaluated the alteration in cell proliferation as well as apoptosis after EGCG treatment. As shown in To determine the role of Wnt/β-catenin pathway in the inhibitory effects of EGCG on colorectal CSCs, LiCl was applied to activate Wnt/β-catenin pathway. As predicted, With the acknowledgement of cancer stem cell theory, the critical role of CSCs in cancer development, progression, and drug resistance has been demonstrated. Wnt/β-catenin pathway is considered as the vital pathway in maintaining the stemness of CSCs. EGCG, a natural compound from green tea, is identified as a chemopreventive and chemotherapeutic agent for cancer. Herein, we revealed that EGCG inhibited colorectal CSCs through targeting Wnt/β-catenin pathway. These results may provide new insights into the interventional strategies against colorectal CSCs. In the present study, we enriched colorectal CSCs from adherent colorectal cancer cell lines DLD-1 and SW480 using a serum-free-medium stem cell culture system, one of the commonly used methods for the isolation and enrichment of CSCs which is based on CSCs capability to form three-dimensional tumorspheres in vitro. We showed the tumorsphere formation capability of both DLD-1 and SW480 cells cultured in SFM. Meanwhile, the enrichment effect was verified by the changes of the protein and mRNA expression levels of colorectal CSC markers, including CD133, CD44, ALDHA1, Oct-4, and Nanog. CD133, CD44, and ALDHA1 were considered as the CSC markers in colorectal cancer ,6. In adSince CSCs are considered the driving force of cancer initiation, development, and drug resistance, great efforts have been made to develop chemical approaches targeting CSCs ,19. StriWnt/β-catenin pathway is a highly conserved signaling, participating in embryonic development, homeostasis maintenance, and cancer development as well. It has been shown that in colorectal cancer, about 80% of tumors have nuclear accumulation of β-catenin and 90% are endowed with an activated level of Wnt/β-catenin pathway . Wnt/β-cIn conclusion, our study suggested that EGCG inhibited colorectal CSCs through targeting Wnt/β-catenin pathway. These results may provide a new vision on exploring natural compound as well as new strategies for cancer intervention. It should be noted that the inhibitory effects of EGCG on various CSCs may be different, and hence more studies are warranted to evaluate its efficacy in different types of cancers.


In neocortical neurons in vitro, proximity ligation assays confirmed the close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD-95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for the modulation of synapse formation, whereby MET activation induces an alignment of presynaptic and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex.MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume, and excitatory synapse development. In a recent coimmunoprecipitation/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin coimmunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine The gene encoding the MET receptor tyrosine kinase is associated with autism spectrum disorder, and influences typical and atypical synapse development and cortical circuit function. The present studies focus on determining potential molecular mechanisms through which the receptor functions in neocortical neurons during synaptogenesis. The findings show that the MET receptor interacts functionally with other proteins also implicated in promoting new synapse assembly, which is reduced upon disruption of the interactions. Thus, in some instances of autism spectrum disorder, disturbances of these molecular interactions may relate to the pathophysiology of cortical circuit development.MET promoter, which reduces MET transcript and protein levels -mediated neocortical synapse formation. Previous studies have shown the following: (1) that MET and β-catenin are expressed at the developing neocortical synapse ; (2) thain vitro ; (3) thain vitro ; and (4)in vitro , with th42 Y142; . Consist42 Y142; . Here, wJ mice were purchased from The Jackson Laboratory and the day of birth was considered postnatal day 0 (P0). Animals had free access to food and water and were housed in a 13/11 h light/dark cycle. All research procedures using mice were approved by the Institutional Animal Care and Use Committee at Children’s Hospital Los Angeles. All efforts were made to minimize animal suffering and to reduce the number of animals used.Timed-pregnant C57BL/6Mouse β-catenin full-length cDNA was cloned by PCR from an adult mouse brain cDNA library using high proof PfuUltra II Fusion HS DNA Polymerase (Agilent) according to the manufacturer protocol, using the following primer pair: 5' CTAGCTAGCTAGATGGATACGTATCGCTACATAATGGCTACTCAAGC 3' and 5' TGCTCTAGAGCATTACAGGTCAGTATCAAACCAGGCCAGCTGATT 3'. Purified β-catenin cDNA fragments were subcloned into a PCI expression vector (Promega) and transformed into DH5α competent cells (Invitrogen). PCI-β-catenin plasmids were purified using the Zyppy Plasmid Maxiprep Kit (Zymo Research) and PCR site-directed mutagenesis of β-catenin (β-catenin Y142F) performed according to a published strategy  using thThe purified fragments were subcloned into a p3XFLAG-CMV-10 vector (Sigma-Aldrich) to generate p3XFLAG-CMV-10-β-catenin and p3XFLAG-CMV-10-β-cateninY142F plasmids. The fidelity of the entire coding sequences of all plasmids was confirmed by DNA sequencing (Genewiz).in situ hybridization using the RNAscope Multiplex Fluorescent Reagent kit (Advanced Cell Diagnostics) according to manufacturer instructions. RNAscope probes and the regions used to generate the probes were as follows: Met  and β-catenin . Alexa Fluor 488 and Atto550 detection reagents were used to visualize Met and β-catenin, respectively. Images were acquired using a Zeiss LSM 710 confocal microscope with a 20× objective. The imaging parameters and z-axis were adjusted to bring the sample into focus. The parameters were maintained to capture focused optical images in each wavelength.P14 mouse brains were fresh frozen in ice-cold isopentane and sectioned in the coronal plane at 25 μm. Sections were subjected to dual fluorescent 2PO4 0.1 (Baker), CaCl2 0.25 (Baker), MgCl2 0.1, dextrose 1 (VWR). Either 25 ng/ml HGF (R&D Systems) or the same volume of vehicle (PBS) was added for 5 min (Co-IP experiments) or for 5, 10, and 20 min (β-catenin phosphorylation experiment) at 37°C to the synaptosomes. For Co-IP experiments, the synaptosomes were centrifuged at 16,000 × g for 15 min, and the pellets were lysed in Co-IP lysis buffer containing the following (in mM): HEPES 50, pH 7.4, EGTA 2, EDTA 2, NaF 30, sodium orthovanadate 10, β-glycerol phosphate 40, 1% Triton X-100, and a protease inhibitor cocktail. The lysate was centrifuged at 16,000 × g for 30 min, and the resulting supernatant used for Co-IP, with a goat anti-MET antibody (R&D Systems), a mouse anti-β-catenin antibody (BD Biosciences), or a rabbit anti-N-cadherin antibody (Santa Cruz Biotechnology). An equivalent amount of goat, mouse or rabbit IgG antibody (Jackson ImmunoResearch) was used in parallel lysates as a negative control. The Co-IP complexes were bound to protein G plus agarose beads (Pierce), after which the beads were washed in Co-IP lysis buffer plus 150 mM NaCl. The complexes were eluted from the beads by boiling in the final sample buffer  and analyzed by Western blot. For the β-catenin phosphorylation experiment, the synaptosomes were centrifuged at 16,000 × g for 15 min followed by lysis in final sample buffer. Blots were probed with antibodies directed against β-catenin , N-cadherin , synapsin1 , synaptophysin1 , phospho-MET , and MET . Digital images of the Western blots were acquired using a CCD camera coupled to a UVP BioImaging System using VisionWorksLS Image Acquisition software .All reagents for Co-IP and Western blots were from Sigma-Aldrich, unless otherwise noted. Crude synaptosomes were isolated from the neocortex of male and female P14 mice  were andWestern blot analyses of Co-IPs in neocortical synaptosomes in the presence or absence of HGF were performed in three independent experiments. For each blot, representing an independent Co-IP experiment, the density of each immunoreactive band was measured in ImageJ (version 1.46r), and a background subtraction was applied. First, to account for different efficiencies of each pull down in each experiment, a ratio of coimmunoprecipitated protein  in 24-well plates, which were precoated with poly-d-lysine (Sigma-Aldrich). Cells were initially plated in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS). After 4 h, the medium was replaced with neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and l-glutamine (Invitrogen), and one-half of the volume of medium was replaced every 3 d. This condition results in slower growth than when using glial conditioned medium or a glial feeder layer. To achieve the sparse labeling of neurons, at 5 d in vitro (DIV), cultures were transfected with a p3XFLAG-CMV10-β-catenin or p3XFLAG-CMV10-β-catenin Y142F plasmid using a calcium phosphate transfection kit according to manufacturer instructions (Clontech). In some experiments, a synaptophysin-GFP (Syn-GFP) plasmid  was cotransfected to label synaptic vesicles. At 14 DIV, 25 ng/ml HGF or the same volume of PBS was added to the medium for 5, 10, or 30 min (PLA assays) or for 10 min (Syn-GFP cluster assays). The experiments were repeated in at least three independent culturing sessions. At the end of the assay period, coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature and processed for immunocytochemistry.Primary cultures of neocortical neurons were prepared from P1 mice  with thein situ PLA kit (Sigma-Aldrich), as described previously  for 20 min and blocked in blocking buffer (5% FBS in PBST) for 1 h at room temperature. Coverslips were incubated overnight at 4°C in the following primary antibody cocktails diluted in blocking buffer: (1) rabbit anti-bassoon  and mouse anti-flag ; or (2) mouse anti-PSD-95  and rabbit anti-flag (1:1000). Following three washes with PBS, coverslips were incubated for 1 h at room temperature in the following cocktails of Alexa Fluor-labeled secondary antibodies : (1) 546 goat-anti-mouse and 633 goat-anti-rabbit for bassoon/flag staining; or (2) 546 goat anti-rabbit and 633 goat-anti-mouse for PSD-95/flag staining. After three washes in PBS, coverslips were mounted onto glass slides with Prolong Mounting Medium (Life Technologies).The PLA was used to determine spatial proximity between proteins that are immunolabeled with the Duolink eviously . Immunofz-axis were adjusted until an appropriate focus was reached. All images in a given culturing session were captured and analyzed with the same exposure time and settings. Note that the visualization of spines requires a longer exposure than that needed to visualize the labeling of synaptic proteins in the linear range. Because the settings were optimized for the analysis of synapses, there is an apparent absence of dendritic protrusions in the captured images. For PLA analyses, in each culturing session, six to eight fields were randomly imaged for each treatment group. Quantitative measures of MET association with select protein partners were obtained by counts of PLA clusters/area of dendrite using published methods  density (average number of puncta per 100 μm of axon length); (2) integrated density ; (3) major length ; and (4) the size of the puncta.Images were captured using an automated laser-scanning confocal microscope  with a 60× oil-immersion objective. The focal point of the beam and the  methods . For theU test was used to compare differences between PBS- and HGF-treated groups. The unpaired t test with Welch’s correction was applied to compare differences between PBS- and HGF-treated groups if the data were normally distributed. For all tests, p values are reported to the fourth decimal place, and values <0.05 are considered to be significant. Statistical analyses and the preparation of graphs were performed using GraphPad Prism 6.0.Data were expressed as the mean ± SEM. For each experimental manipulation, data were collected from at least three independent culturing sessions. Individual neurons were considered as samples , and samPreviously, a discovery-based Co-IP/MS method was used to detect the MET interactome in neocortical synaptosomes at the peak of synaptogenesis (P14) and identified β-catenin as a member of the MET interactome . In thisF = 9.401, p = 0.0025, pa = 0.8383, df = 174; pb = 0.0002, df = 174). Specifically, after stimulation for 10 min, there was an ∼50% decrease in the density of PLA clusters in HGF-treated compared with PBS-treated cultures  following HGF addition, compared with PBS treatment . These data suggest that there is an increase in a functional MET/synapsin 1 complex following HGF stimulation.Following HGF stimulation, β-catenin dissociates from the MET complex. We hypothesized that the activation of MET could result in the recruitment of other protein partners into the receptor complex. MET is localized in presynaptic and postsynaptic compartments, but with predominant enrichment in presynaptic compartments in the developing neocortex  and cultreatment C,D. In reatment E. Thespe = 0.003, t = 3.882, df = 51; Previous reports demonstrated phosphorylation of β-catenin at Y142 in response to HGF in cancer cells and cultured hippocampal neurons . SimilarF = 9.570, p = 0.0027; pf = 0.8269, df = 84; pg = 0.0013, df = 84; ph = 0.0002, df = 84), but no significant difference in PLA signal in flag-β-cateninY142F-transfected neurons  = 80, pj = 0.0087; U(34) = 84, pk = 0.0129, U(34) = 103, pl = 0.0633). We further characterized Syn-GFP puncta colabeled with bassoon or PSD-95, measuring parameters previously shown to be influenced by β-catenin  = 8.056, p = 0.0056) and Syn-GFP/PSD-95  = 4.141, p = 0.0441) clusters. Pairwise analysis revealed that, in the absence of HGF, there was no significant difference in the density of Syn-GFP/bassoon  and Syn-GFP/PSD-95  clusters in neurons transfected with wild-type β-catenin, but not in flag-β-cateninY142F-transfected neurons , compared with PBS. These results demonstrate that HGF activation of MET promotes an increased density of Syn-GFP/bassoon and Syn-GFP/PSD-95 clusters by regulating the MET/β-catenin complex through the phosphorylation of β-catenin at Y142.Previous studies have demonstrated a role for an N-cadherin/β-catenin complex in synaptic vesicle localization at the synapse . Given oMET (β-catenin (Many genes have been identified as components of molecular networks involved in ASD risk . These pMET  and β-ca-catenin ,b. A pos-catenin . Previou-catenin , followe-catenin . We demo-catenin . Indepen-catenin .Met results in premature maturation, assessed morphologically, electrophysiologically, and by the increased membrane insertion of AMPA receptor subunits (in vitro results in both immature spine growth and electrophysiological properties. The data together are consistent with a unique dual role for MET during synapse development: initiating synapse formation at early stages and maintaining an immature functional state until MET signaling is eliminated. In vivo, MET receptor activation is robust during the period of rapid synapse formation between P7 and P14, but then rapidly falls to near-negligible levels by P16 (The dissociation of the MET/β-catenin complex is required for the HGF-induced increase in the density of Syn-GFP/bassoon and Syn-GFP/PSD-95 clusters in neocortical neurons. This increase, which is observed over a short assay period of 10 min, is suggestive of an increased number of nascent synapses in the presence of HGF and is reminiscent of increased synapse density, defined by synapsin 1 and PSD-95 colocalization, in the same culture paradigm after stimulation with HGF for 24 h . It shousubunits . Overexps by P16 , a time s by P16 .in vivo, both β-catenin and MET are also localized at postsynaptic sites in the neocortex and hippocampus (in vivo (in vivo. However, we note that β-catenin and MET have been demonstrated independently to modulate several features of postsynaptic development. For example, β-catenin regulates dendritic morphogenesis, dendritic spine density, and postsynaptic structure and function (In the current study, we focused on the role of MET/β-catenin complex in modulating presynaptic development. Specifically, HGF increases synaptic vesicles clustering at the active zone and at the synapse through regulation of MET/β-catenin complex by the phosphorylation of β-catenin at Y142. We also showed that, under basal culture condition, β-catenin as well as β-catenin Y142F itself could promote synapse formation. This may reflect the role of β-catenin independent of HGF/MET signaling and phosphorylation of β-catenin at Y142 as discussed previously , as wellpocampus . Thus, t(in vivo . While ifunction . Similarfunction . Thus, iin vitro raise the issue of defining the cellular and circuit context in which this signaling system may operate in vivo. At P14, we found that there are both colabeled and single-labeled neurons expressing Met and β-catenin transcripts in layers II-III and V-VI. The double-labeled neurons located superficially are almost entirely intrinsic intratelencephalic cortico-cortical or callosal neurons, whereas deep layer MET+ neurons could be both callosal and cortico-fugal in nature. During the active period of synaptogenesis in the mouse neocortex, Hgf mRNA is evident mostly in deep layers of the neocortex at P14 (in vivo. Determining the specific subpopulation identities is currently under investigation.The analyses of HGF-induced regulation of MET/β-catenin functional interactions in neocortical neurons x at P14 . Thus, bin situ techniques will provide opportunities to more carefully characterize the coexpression of multiple members of the MET interactome, 11% of which have been associated with neurodevelopmental disorders (Our current findings are consistent with converging evidence that the developing neocortical synapse is disrupted in ASD, with many ASD risk genes having implicated or demonstrated roles in synapse development and plasticity . Less prisorders , in disc


To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting  or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity.Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of  This effect is achieved by sequestering β-catenin in a submembrane location, thereby preventing its translocation to the nucleus where, in normal conditions, it activates the transcription of growth-promoting target genes.2 Not surprisingly, as a result of these activities, cadherins exert a recognized onco-suppressor effect that can be observed in different cell types, depending on their expression pattern.3 In the last decade, a novel cadherin, named μ-protocadherin (MUCDHL) to underline the hybrid nature of its extracellular portion, which contains four cadherin-like and three mucin-like domains, has been isolated and characterized.4 This protein maintains all the general features of cadherins and carries, in its intra-cellular portion, four prolin-rich domains together with a single PDZ domain, previously shown to bind β-catenin. A survey of MUCDHL distribution revealed that significant levels of its expression were exclusively detected in a couple of anatomical sites, including colorectal mucosa.5 This observation suggested a possible role in colorectal carcinogenesis that was subsequently supported by experimental results. Indeed, interaction of MUCDHL with β-catenin, which is constitutively activated in colorectal cancer (CRC), was confirmed and MUCDHL expression exhibited a remarkable downregulation in >70% of CRC cases, in association with higher proliferation indexes.6 Notably, data obtained in our laboratory showed that treatment of CRC cells with an anti-inflammatory drug named mesalazine (5-ASA), that is able to inhibit β-catenin signaling in vitro and to exert anti-CRC chemoprevention activity in vivo,7 markedly induces MUCDHL expression.8 This finding might, in our opinion, reflect two distinct explanations: (i) 5-ASA could induce MUCDHL expression, sequester β-catenin on the cell membrane and inhibit its signaling pathway; or (ii) 5-ASA could inhibit β-catenin, leading to an upregulation of MUCDHL, dependent on the fact that expression of this protein is negatively regulated by β-catenin signaling  came from the observation that MUCDHL downregulation always accompanies adenomatous polyposis coli (APC) inactivation in CRC. In fact, immuno-histochemical data obtained in our laboratory showed that 11 out of 21 analyzed CRC cases were negative for MUCDHL and APC expression, whereas the remaining exhibited a double positivity for the same proteins  transcription factor. Cell counts, obtained in a preliminary dose-response experiment, indicated that a complete inhibition of proliferation was reached with a 30–60 μM concentration after 96 h of treatment, in CaCo2 cells, and 15–30 μM concentration after 48 h of treatment, in HCT116 cells , a transcriptional activator of MUCDHL.To further support the hypothesis that expression of MUCDHL is negatively regulated by active 16 cells . Thus, uIn the CaCo2 cell line, at the highest FH535 concentration, transcript levels of the four analyzed genes were induced 3.4-, 2.2-, 8.6- and 1.9-fold, respectively, with virtually all variations exhibiting statistical significance . A similAs expected, protein levels of TCF4 were strongly downregulated in both FH535-treated CRC cell lines and, in general, gene expression changes detected by western blot were more pronounced than those observed by QRT-PCR , panel cβ-catenin signaling pathway exerts a negative regulation on the expression of MUCDHL and CDX2.Together, this analysis suggests that the To confirm the data obtained with the FH535 compound using a more specific approach, the same cell lines were transfected with a mix of three TCF4 siRNA oligonucleotides and the effects of this treatment on gene expression were evaluated by QRT-PCR and western blotting.Transfection with the anti-TCF4 siRNA induced an apparently modest downregulation of its target TCF4 mRNA of 32 and 50% in CaCo2 and HCT116 cells, respectively, as assessed by QRT-PCR ; howeverP-values   .β-catenin signaling, unlike the CRC cell lines where this pathway is constitutively activated as consequence of APC mutation. This condition was obtained upon treatment with 5 or 10 mM LiCl, which is known to inhibit the GSK3β enzyme, leading to stabilization and activation of β-catenin. Then, QRT-PCR analysis was carried out as described in the previous experiment. With the only exception of the Met gene, the results observed were, as expected, opposite to those elicited by withdrawal of the β-catenin agonists. In fact, at the highest LiCl concentration, there was a 46% decrease for MUCDHL, 61% for Keratin-20, 48% for E-cadherin, 60% for CDX2 and 42% for p21waf1, whereas, under the same conditions, the CD44 gene showed a 2.6-fold increase of its transcript levels   .β-catenin activity, correlate with those of the CDX2 transcription factor, suggesting its involvement in the regulation of MUCDHL levels. To verify this possibility, the CaCo2 cells were transfected with a scrambled control siRNA or with a mix of three anti-CDX2 siRNAs for 12 h, and then cultured for an additional 4-day period in the absence or presence of 20 mM 5-ASA. The decision to use this compound was based on its anti-CRC chemoprevention capacity, previously related to its ability to inhibit β-catenin activity and induce MUCDHL expression.In the studies presented here, variations of MUCDHL expression in response to modulators of As expected, CaCo2 cells transfected with control siRNA and exposed to 5-ASA, exhibited a 2.8-and 3.4-fold increase of CDX2 and MUCDHL mRNA, respectively, detected by QRT-PCR . Instead10 E-cadherin is considered the main adhesion molecule in epithelial tissues and loss of its expression, when observed, has been ascribed to epigenetic alterations (gene promoter hypermethylation) or to transcriptional repression mediated by Snail family members.11 These proteins can be divided into two subgroups that are able to repress E-cadherin expression directly, binding to its promoter region , or indirectly, with a different mechanism . Interestingly, a number of reports have highlighted the ability of the β-catenin pathway to activate Snail family transcription repressors.13 This effect is reached through a couple of distinct molecular mechanisms: (i) activation of Snail and Slug as consequence of Wnt-induced GSK3β inhibition, leading to their stabilization and nuclear translocation; and (ii) direct transcriptional activation of Twist and ITF2 by β-catenin/TCF4.17 However, experimental evidence supporting the existence of a molecular cascade, linking β-catenin signaling to Snail proteins and to E-cadherin expression, has almost exclusively been obtained in breast cancer and its normal counterpart.15 On the other hand, loss of E-cadherin expression is rarely detected in CRC where downregulation of MUCDHL is, instead, a common event.6 In this report, we clearly demonstrated, for the first time, that MUCDHL expression is negatively regulated by β-catenin signaling. This finding was obtained in epithelial cancer cells as well as in normal colon enterocytes, represented by CRC cell lines and colonic organoids, respectively. Moreover, treatments inhibiting the entire β-catenin pathway (Wnt deprivation), or GSK3β activity (LiCl), or β-catenin-dependent transcription , all affected MUCDHL expression, suggesting that this effect could be ascribed to a transcription repressor induced by β-catenin, such as the ITF2 member of the Snail family. Computational analysis of ‘Chip-Seq' data contained inside the ENCODE project database, allowed us to retrieve, among Snail family proteins able to bind MUCDHL promoter, the Zeb1 repressor. However, QRT-PCR analysis on HCT116 cells transfected with an anti-TCF4 siRNA oligonucleotide or treated with the FH535 compound, failed to detect an upregulated expression of genes coding for either ITF2 or Zeb1, suggesting that they do not have an important role in β-catenin regulation of MUCDHL expression (data not shown). Conversely, variations of MUCDHL expression, in response to stimuli modulating the activity of the β-catenin pathway, consistently showed a direct correlation with those of the transcription activator CDX2. A previous report has demonstrated that the transcription of MUCDHL gene is directly controlled by this transcription factor through a specific DNA-binding site in its promoter region.18 Taken together, these observations allow us to speculate that the negative regulation of MUCDHL expression by β-catenin might be actually exerted via CDX2, through a transcription repressor that remains to be identified. This hypothesis is supported by the evidence that CDX2 silencing interferes with the capacity of 5-ASA, a β-catenin inhibitor with anti-CRC chemoprevention activity, to upregulate MUCDHL expression. In aggregate, our data suggest that the silencing of MUCDHL expression observed in CRC is consequent to the constitutive activation of the β-catenin pathway, which is typical of these tumors. The development of compounds able to restore the expression of MUCDHL, thus stimulating its anti-tumor (anti-proliferative and anti-metastatic) properties, opens important possibilities for chemoprevention and/or therapy of CRC. The rationale underlying this pharmacological approach also implies a possible role of MUCDHL as biomarker to estimate the extent to which these compounds may be effective. The negative effect on MUCDHL expression exerted by constitutive β-catenin activation observed in CRC may be simply the consequence of a physiological mechanism already operating in normal colon enterocytes. Based on the capacity of MUCDHL to sequester and inhibit β-catenin at the cell membrane, this biological activity might represent the result of a positive feedback amplifying the final effects of stimuli that either activate or inhibit the β-catenin signaling pathway. The inverse correlation existing along the axis of colon crypt between the nuclear localization of β-catenin (observed at the bottom) and the expression of MUCDHL (detected on the top), in our opinion, provides further support for this hypothesis.Metastasis is the result of a complex process by which cancer cells detach from the initial tumor mass and acquire the capacity to migrate to distinct anatomical sites where they give rise to one or more secondary tumors. Reduced expression of cadherins is critically important in this process, owing to their role in promoting cell–cell adhesion in normal conditions.HCT116 and CaCo2 cells were obtained from ATCC  and cultured in DMEM supplemented with 10% fetal bovine serum , 2 mM-Glutamine  and 100 U/ml Penicillin/Streptomycin (EuroClone).FH535 was purchased from Sigma-Aldrich and dissolved in DMSO at a 30 mM concentration. Treatment with this compound was carried out at times and concentrations indicated in Results and Figures. Control cells were exposed to an equivalent amount of vehicle. Cell counts were performed at 24 h intervals using the Trypan blue exclusion assay. All the experiments were repeated at least three times.Experiments of mRNA silencing were conducted transfecting a mix of three different siRNA oligonucleotides directed against TCF4 or CDX2 (Sigma-Aldrich) at a concentration of 100 nM with the RNAiMAX reagent . The transfection efficiency of CaCo2 cells was potentiated using the reverse transfection procedure.Cells undergoing RNA extraction were trypsinized, washed once in PBS (EuroClone), re-suspended in lysis buffer and extracted with Qiagen total RNA purification kits , following the manufacturer instructions. Total RNA was quantified using a NanoDrop 2000 spectrophotometer  and 100 ng were used to synthesize cDNA with the High Capacity cDNA Retro-transcription Kit (Invitrogen). Quantitative Real-Time PCR (QRT-PCR) was then conducted using an ABI PRISM 7900 detection system (Applied Biosystems). All primers and probes used for mRNA amplification were designed by Applied Biosystems. Each cDNA sample was run in triplicate using the Taqman Universal PCR Master Mix (Invitrogen) and glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Quantification of RT-PCR signals was performed using the Ct relative quantification method. This procedure calculates the relative changes in gene expression of the target gene normalized with an endogenous control and compared with a calibrator sample.19 In brief, electrophoresis was performed on sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electro-blotting to nitrocellulose sheets. Blotted membranes were pre-blocked with a solution containing 5% nonfat milk  in 0.05% TBST and incubated with a primary antibody specific for each analyzed protein, and with a common secondary antibody conjugated to horseradish peroxidase.Preparation of nuclear and cytoplasmic protein extracts and western blot analysis were carried out as previously described.The following primary antibodies and the respective dilutions were used for western blot analysis: mouse anti-MUCDHL monoclonal antibody (MoAb)  1:500 in TBST 3% milk; rabbit anti-CDX2 MoAb  1:1000 in TBST 1% milk; mouse anti-E-Cadherin MoAb  1:1000 in TBST 5% milk; rabbit anti-TCF4 MoAb  1:1000 in TBST 5% BSA.Expression of actin was also evaluated with a mouse pan-actin MoAb  to normalize the protein content of the various analyzed samples.As secondary antibodies, a goat anti-mouse IgG (Santa Cruz Biotechnology) or a goat anti-rabbit IgG , both conjugated with horseradish peroxidase, were used respectively at 1:10 000 and 1:15 000 dilutions. Detection of western blot signals was carried out using the Westar EtaC enhanced chemi-luminescent substrate . Densitometric analysis was accomplished using the ImageJ Software.Samples of normal colon mucosa were obtained from the Bio-Bank of Modena (BBM) . After surgical resection, the cancerous mucosa was used for histopathological analysis and for the BBM sampling, whereas a small fragment of healthy tissue was used for experiments and the rest was discharged.9 In brief, colonic crypts were separated from the underlying stroma through chelating agents, washed thoroughly and embedded in matrigel. After matrigel polymerization, the crypts were overlaid with complete growth medium which was composed of HAM's F12 supplemented with: antibiotics, 5% FBS (Sigma-Aldrich), Glutamax 1 × (Life Technologies), 10 mM HEPES, N-2 Supplement (Life Technologies) 1 ×, B-27 supplement without retinoic acid (Life Technologies) 1 × , 10 mM Nicotinamide (Sigma-Aldrich), 1 mM N-Acetyl-l-cysteine (Sigma-Aldrich), 1 μg/ml RSPO1 , 100 ng/ml human EGF (R&D System), 100 ng/ml human Noggin (R&D System), 1 μg/ml Gastrin I (Sigma), 100 ng/ml WNT3a (R&D System), 500 nM LY2157299 , 10 μM SB202190 (Sigma), and 0.01 μM PGE2 (Sigma). For the first 48–72 h of culture, the crypts were left in complete growth medium, with the addition of the Rock inhibitor Y-27623 (10 μM Sigma-Aldrich), in order to allow the formation of organoids.Culture of human colonic organoids was carried out as described by Peter Jung.In the following days, they were subjected to various treatments. Incubation with LiCl (Sigma) was conducted at 5 and 10 mM concentrations of the compound. Reduced levels of WNT3a were obtained lowering its concentration to 20 or 10 ng/ml, corresponding to 1/5 and 1/10 of the standard amount. The differentiation condition was achieved in complete absence of WNT3A, PGE2 and SB202190.After 72 h of treatment, organoids were collected from matrigel with Cell Dissociation Solution (BD Transduction), washed with cold PBS and RNA was extracted as previously described.μm).Morphological analysis of organoids was conducted using the ImageJ software on pictures obtained by light-inverted microscope examination. Circularity was assessed outlining their outer edge with the freehand selection tool and expressed as Units (U) , whereas the thickness of their epithelial lining was measured using the straight line selection tool, and expressed in micrometers .All experiments were repeated at least three times, unless otherwise stated, and the results presented in terms of mean±S.E.M. values. Pairwise comparisons were carried out using the Student's


Endothelial mesenchymal transition (EndMT) plays a critical role in the pathogenesis and progression of interstitial and perivascular fibrosis after acute myocardial infarction (AMI). Pigment epithelium-derived factor (PEDF) is shown to be a new therapeutic target owing to its protective role in cardiovascular disease. In this study, we tested the hypothesis that PEDF is an endogenous inhibitor of EndMT and represented a novel mechanism for its protective effects against overactive cardiac fibrosis after AMI. Masson’s trichrome (MTC) staining and picrosirius red staining revealed decreased interstitial and perivascular fibrosis in rats overexpressing PEDF. The protective effect of PEDF against EndMT was confirmed by co-labeling of cells with the myofibroblast and endothelial cell markers. In the endothelial cells of microvessels in the ischemic myocardium, the inhibitory effect of PEDF against nuclear translocation of β-catenin was observed through confocal microscopic imaging. The correlation between antifibrotic effect of PEDF and inactivation of β-catenin was confirmed by co-transfecting cells with lentivirus carrying PEDF or PEDF RNAi and plasmids harboring β-catenin siRNA(r) or constitutive activation of mutant β-catenin. Taken together, these results establish a novel finding that PEDF could inhibit EndMT related cardiac fibrosis after AMI by a mechanism dependent on disruption of β-catenin activation and translocation. Fibrosis in a healing infarct is part of the reparative process, characterized by the fibroblasts accumulation and redundant deposition of extracellular matrix (ECM), and formation of a fibrous scar maintains the integrity of the chamber. Obviously, an overactive fibrotic response, or increased interstitial and perivascular fibrosis in non-infarcted areas would cause a delayed recovery of the ischemic myocardium1et al. has demonstrated that PEDF inhibits cardiac fibrosis and several relevant factors such as type III collagenPEDF, a non-inhibitory member of the serpin family, is widely expressed in diverse human tissues and more prominently in heart tissue5Traditionally, adult fibroblasts are considered to be derived directly from embryonic mesenchymal cells82112EndMT during embryonic heart development is regulated to a great extent by the TGF-β signaling pathways151618202123In this study, we provided evidence of the expression of PEDF linked to diffuse cardiac fibrosis in rats with AMI. By interfering or overexpressing PEDF in myocardium along the infarct border, we aimed to observe the effect of PEDF on interstitial and perivascular fibrosis. And then we explored whether PEDF could ameliorate EndMT-induced cardiac fibrosis through inhibition of β-catenin-signaling pathway.The fibrotic area and PEDF protein expression were determined in normal and ischemic hearts at 1, 2, 4, or 8 weeks after LAD ligation. MTC staining  revealedTo verify the role of PEDF in ischemic heart, we delivered lentivirus carrying PEDF, PEDF RNAi or LV-CON049 (Vector) by using intramyocardial injections to overexpress or knockdown PEDF in an AMI rat model. The results indicated that PEDF protein expression in the siPEDF group was significantly inhibited compared with the control vector group at 4 weeks after the delivery of the lentivirus (P < 0.05). Whereas in the PEDF group, we found that overexpression of PEDF protein was evident .Based on the results above, we selected the 4-week after AMI as the optimal time point to determine the effect of PEDF on the process of cardiac fibrosis  and cardMTC staining  and SiriEndMT, one of the critical sources of fibroblast in the fibrotic process, was detected to investigate the exact effect of PEDF on inhibiting cardiac fibrosis. Immunofluorescence determination revealed the co-labeling of cells with the myofibroblast and endothelial cell markers, indicating an early stage of EndMT. We performed double labeling for α-SMA or FSP1 and the endothelial marker Lectin to identify endothelial cells (Lectin+), fibroblasts (α-SMA+ or FSP1+) and cells of endothelial origin that gained of a mesenchymal phenotype 2We further examined the effect of PEDF on the aberrant nuclear translocation of β-catenin in microvascular endothelial cells post-AMI, which is essentially involved in the progression of EndMT as previous studies have reported. As we know, β-catenin is a main functional protein of endothelial adherens junctions. The results of confocal microscopic images showed that β-catenin located on the membrane of cells in the shame group. After AMI, a nuclear localization of β-catenin was observed, and PEDF siRNA administration increased the number of such cells, whereas the percentage of nuclear β-catenin-accumulating cells was significantly decreased in the PEDF group . To gathTo further confirm a direct modulation of EndMT by PEDF, we sought to establish stable cell lines overexpressing or silencing PEDF in RCMECs, and cells were exposed to TGF-β1 and evaluated for morphological, phenotypic and functional changes referred to EndMT. As shown in Given a critical role for β-catenin activation in mediating EndMTTo directly investigate whether β-catenin transcriptional activity is involved in the inhibition effect of PEDF on EndMT, we co-transfected cells with lentivirus carrying PEDF or PEDF RNAi and plasmids harboring β-catenin siRNA (r) or constitutive activation of mutant β-catenin. The expression of wild-type β-catenin or constitutive activation of mutant β-catenin was confirmed by western blot analysis . In TGF-Recent investigations have suggested that PEDF treatment can potently inhibit the tissue remodeling and cardiac fibrosis in the rat AMI modelin vivo in order to investigate its antifibrotic effects and elucidate possible mechanisms. In this study, we found that PEDF attenuated the development of cardiac fibrosis, presenting a definite anti-fibrotic effect in an AMI rat model. Furthermore, PEDF effectively reduced ECM deposition around the peri-vascular region located in the border of the infarcted area and protected the vascular integrity. Pathophysiologically, a stable number and function of vessels is indispensable for maintaining and restoring cardiac performance in the ischemic heart. Actually, our previous study found that PEDF could improve rat cardiac function by reducing apoptosis, suppressing vascular permeability and limiting infarct size after AMIIn our study, we evidenced a high expression of endogenous PEDF in the normal myocardium and PEDF expression gradually decreased during AMI, which was consistent with previous studyin vivo showed that PEDF remarkably reduced EndMT and cardiac fibrotic size in peri-vascular region, creating a favorable condition for subsequent recovery of impaired cardiac function.As we know, the formation of the excessive ECM is mainly generated by cardiac myofibroblastsin vitro was confirmed by knocking down or overexpressing PEDF. These observations suggested that PEDF served as an endogenous antifibrogenic factor.Amongst the numerous cytokines that regulate epithelial–mesenchymal transition, TGF-β1 has been known to be the most important oneAnother important finding of this study is that the inhibition effect of PEDF on EndMT is mediated predominantly through a β-catenin dependent pathway. As shown in 3031In conclusions, our study firstly proves that PEDF could inhibit EndMT related cardiac fibrosis after AMI by a mechanism dependent on disruption of β-catenin activation and translocation. These findings are beneficial for further understanding the cardioprotective mechanisms of PEDF and also present a potentiality for establishing a promising strategy for therapy of cardiac fibrosis secondary to AMI.Recombinant rat PEDF (GenBankTM Accession Number NM_177927) was synthesized by Cusabio Biotech, Co., Ltd. . In brief, 293T cells were transfected with the recombinant vector pGEX 6P-1 GST-tagged PEDF. GST-tagged PEDF proteins were purified by high pressure liquid chromatography purification (>90% purity) and amino-terminal sequence determination. The resulting proteins were soluble in aqueous solutions.12 Tu/mL. Plasmid carrying β-catenin siRNA(r) were purchased from Santa Cruz Biotechnology . Plasmid carrying active mutant form of β-catenin (pENTR-N90-beta-catenin) was a gift from Xin Chen Recombinant lentivirus was prepared as described previouslySprague-Dawley (SD) male rats  were obtained from the Experimental Animal Center of Xuzhou Medical College and housed in a controlled environment. All of the experiments conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The animal care and experimental protocols were approved by the Xuzhou Medical College Committee on Animal Care. All of the experiments conformed to the international guidelines on the ethical use of animals.7 TU) prepared in 20 μL Enhanced Infection Solution  with a 20-μL syringe and 25-gauge needle along the infarct border immediately after surgery. At 4 weeks after LAD artery ligation, the expression of PEDF in the border zone areas surrounding the infarction was confirmed by western blot analysis  coronary artery as described previouslyanalysis . MortaliEchocardiography was conducted under sedation by sodium pentobarbital , as described previouslyRCMECs  were used between the third and fifth passage and cultured in endothelial cell medium  supplemented with 5% fetal bovine serum , 1% endothelial cell growth supplement  and 1% penicillin/streptomycin solution. EndMT induction was performed by RCMECs with 10 ng/mL TGF-β1 6 RCMECs were seeded into 60-mm plastic dishes. After the cells reached about 30–35% confluence, PEDF-lentivirus or PEDF-RNAi-lentivirus or LV-CON049 (vector) were transfected following the manufacturer’s protocol at the desired multiplicity of infection (MOI = 20). After 8 hours, transfection medium was removed and fresh medium was added. After an additional 64 hours, GFP co-expression on the construct was used to determine efficiency of viral transduction and the expression of PEDF in the stable cell lines was confirmed by western blot analysis  carrying β-catenin siRNA or active mutant form of β-catenin or empty plasmid using the Lipofectamine 2000  reagent according to the instructions specified by the manufacturer. After treatment for 72 h, GFP co-expression on the construct was used to determine efficiency of viral transduction and the expression of wild-type β-catenin or constitutive activation of mutant β-catenin were confirmed by western blot analysis .Cardiac fibrosis was determined by Masson’s trichrome (MTC) staining . Interstitial collagen was visualized using picrosirius red staining For heart tissue staining, frozen myocardial tissue was horizontally sliced into 5 μm section and mounted on glass slide. Both sections and cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with Triton X-100 (0.1%) and blocked with solution containing 5% bovine serum before applying primary antibody. Specimens were incubated with anti-α-smooth muscle actin (α-SMA) , anti-fibroblast- specific factor 1 (FSP1) , anti-VE-cadherin , anti-CD31 and anti-β-catenin  overnight at 4 °C, washed three times in PBS and incubated with the secondary antibody  under light-protected conditions for one hour at room temperature and counterstained using 4,6-diamino-2-phenyl indole (DAPI) . Cardiovascular endothelial cells were identified by staining with the lectin BS1-B4  under light-protected conditions at room temperature for 3 hours at and the cover slips were mounted on slides using 50% glycerin. Then, the sections and cells were observed under a fluorescence microscope  or confocal laser scanning microscope .Rat tissue protein was extracted from the myocardium of the peri-infarct zone using a lysis buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate, 1% protease, (pH 7.5), and complete protease inhibitor cocktail . For whole cell lysate, cells were lysed with Cell Total Protein Extraction Kit  containing cocktail of phosphatase inhibitors and protease inhibitors. Membrane proteins were extracted using Membrane and Cytoplasmic Protein Extraction Kit  and nuclear proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit  according to the manufacturer’s instructions.+/K+-ATPase ; Lamin B . The membranes were washed three times with 0.1% Tween 20/Tris-buffered saline and the appropriate florescence conjugated secondary antibody was added for 1 hour. Washed again, and they were subsequently scanned by Odyssey Infrared Imaging System .Immunoblotting was performed as previously described. In brief, equivalent amount of protein was fractionated on a 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to 8 μm nitrocellulose membranes . Nonspecific binding was blocked by incubation with 5% non-fat milk in 0.1% Tween 20/Tris-buffered saline for 30 minutes, followed by overnight incubation at 4 °C with the primary antibody(s). The PEDF antibody  was used at a dilution of 1:500; VE-cadherin at 1:500; FSP1 at 1:500; α-SMA at 1:1000; β-catenin at 1:500; active-β-catenin  at 1:500; β-actin  at 1:1000; Na4 cells in serum free media were seeded into the wells of the upper compartment and 600 μl ECM containing 20% FBS was placed into the wells of the lower compartment of the transwell chamber, which was then incubated at 37 °C in 5% CO2 atmosphere. After 12 hours, cells on the upper side of the membrane were taken off with cotton-tipped swabs. The migrated cells on the lower side of the membrane were fixed and stained with 0.4% Trypan Blue and counted with a microscope.RCMECs were pretreated with 10 ng/ml TGF-β1 for 3 days before starting the experiment. Migration assays were performed in the 24-well chamber: a polycarbonate membrane  divided the divided the chamber into upper and lower compartments. A total of 5 × 10All of the data were expressed as the means ± s.d and were processed with Prism software . One-way analysis of variance followed by Student–Newman–Keuls test was performed for statistical comparisons. Values of P < 0.05 were considered to be statistically significant.How to cite this article: Zhang, H. et al. Pigment epithelium-derived factor attenuates myocardial fibrosis via inhibiting Endothelial-to-Mesenchymal Transition in rats with acute myocardial infarction. Sci. Rep.7, 41932; doi: 10.1038/srep41932 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Microbiome science is already in the fast lane, and computations share the credit. How can computations further speed up the pace of microbiome research as it dashes farther, scaling higher summits? What can computations accomplish? What questions can computations help address and what types of algorithms and software should computational biologists aim to develop? These are tantalizing questions that many of us are facing, probably in particular new groups aiming to venture into a relatively nascent and fast-developing area that promises rapid accumulation of data, emergence of alluring new concepts, and significant discoveries. It almost seems like every day, inspiring observations come to light.Clostridium difficile infection, which has led to therapeutic interventions with remarkable success [Consider, for example, the possible link that was observed between gut bacteria of mice and Parkinson disease, in which changes in the bacteria populating the gut apparently appear to be associated with a decline in motor skills. How can this be? It turns out that 70% of all neurons in the peripheral nervous system are located in the gut, and these are directly connected to the central nervous system through the vagus nerve . A remar success . The mic success . Tumor g success .So how can computation accelerate research in the examples above? Broadly, computations can make headway in problems ranging from characterizing taxonomic diversity, classification of microbial species, and tracing their evolution. Computational methods are emerging to facilitate the detection and quantification of diverse patterns among these data, as well as the construction of microbial networks and cross interactions between members of microbial communities. Computation can tackle complex data  on the interactions between microbial communities and their hosts, towards the most challenging question of the quantification of the impact of the human microbiome on our health, as in the examples above.Beyond the discovery of fundamental biological understanding that computations may enable, the development and applications of efficient algorithms to analyze and sift through data relating to infections and surgical and other medical procedures is yet another complex aspect that may critically affect human health. Uncovering trends in antibiotic resistance may impact our healthcare ecosystem—which computations can help decipher. Computational biology can also develop and apply statistical methods to bacterial communities evolving in different environments in diverse populations to obtain correlations and trends in disparate ecosystems. These and additional emerging data may offer new insights into disease processes and microbiome-inspired therapeutic strategies—insights that may provide leads that can be explored experimentally. To date, next-generation sequencing and computation have already made big strides to elucidate the impact of human genomic variation; for the microbiome, the impact of this variation is yet to be surveyed. Taken together, these may also bear on questions such as how medical approaches influence selection pressure on the human microbiome, which may influence treatment decisions.PLOS Computational Biology is planning to catalyze such developments, to stimulate an area that is already surging. In this endeavor, PLOS Computational Biology collaborates with other PLOS community journals to lead to an upswing in microbiome research for the good of the community. Primary among these is PLOS Pathogens. We invite submissions of diverse manuscripts, research, methods, and those describing new and powerful software tools. Together, we look forward to making a difference.New computational approaches are already changing medicine and traditional biology. Computational biologists can spur cutting-edge microbiome research. As the leading journal in computational biology,