Mechanistically, TEAD4 associates with TCF4 to form a complex and cobind target genes. VGLL4 targets this TEAD4–TCF4 complex to interfere the functional interplay between TEAD4 and TCF4, suppressing the transactivation of TCF4. Collectively, our study indicates that Wnt/β-catenin and Hippo-YAP signalling are directly linked at transcription factor-level, and VGLL4 can target a TEAD4–TCF4 complex to co-regulate both pathways.Concerted co-regulation of multiple signalling pathways is crucial for tissue homoeostasis and tumorigenesis. Here we report that VGLL4, a previously identified YAP antagonist, also functions as a regulator of Wnt/β-catenin signalling. The expression of VGLL4 is significantly downregulated in clinical colorectal carcinoma (CRC) specimens, positively associated with patient survival rate, and inversely correlated with the expression of Wnt target genes in CRCs. Knockdown of VGLL4 enhances proliferation and tumour formation of CRC cells. A designed peptide mimicking the function of VGLL4 effectively inhibits CRC progression in a  The Wnt/β-catenin and YAP signaling pathways have fundamental roles in cancer. Here, the authors show that VGLL4, a known YAP antagonist, also negatively regulates Wnt/β-catenin signaling by targeting TEAD-DNA-TCF4 complex, thereby inhibiting colorectal cancer growth. The evolutionarily conserved Wnt/β-catenin and Hippo-YAP signalling pathways play fundamental roles in individual development and tissue homoeostasis12345689Dysregulation of Wnt/β-catenin or Hippo-YAP signalling pathways has multiple pathological consequences. For example, >90% of colorectal cancer (CRC) patients show aberrant activation of the Wnt/β-catenin signalling pathway, resulting in sustained accumulation of β-catenin in the nucleus, and suggesting that transactivation of β-catenin-TCF4 target genes represents a primary initial event in CRC ref. . Other m7131519SAV results in robust transcriptional upregulation of Wnt/β-catenin target genessox2 and snail2. Recently, Piccolo and colleagues proposed that YAP/TAZ could be a constitutive component of the β-catenin destruction complexThere is a growing amount of evidence for multi-point crosstalk between the Wnt/β-catenin and Hippo-YAP signalling pathways. Most studies to date indicate that YAP/TAZ can act as direct mediators between these pathways. For example, the Hippo-YAP pathway has been reported to be involved in the regulation of Wnt/β-catenin signalling through the interaction of YAP/TAZ with β-catenin and/or DVL refs , 24. In Cooperation between transcription factors is a common theme, and DNA-dependent formation of transcription factor pairs can alter their binding specificityIn this work, we report the clinical relevance of VGLL4 in CRC and its regulatory effect in the Wnt/β-catenin signalling pathway. The expression of VGLL4 is significantly downregulated in CRC patients, and inversely correlated with the expression of Wnt/β-catenin target genes. Loss of VGLL4 is associated with decreased survival rates of CRC patients, highlighting a critical role for VGLL4 in tumorigenesis. Surprisingly, the Hippo pathway transcription factor TEAD4 directly associates with the Wnt pathway transcription factor TCF4 via their DNA-binding domains, forming a complex on target genes. VGLL4 binds to this TEAD4-TCF4 complex to inhibit transactivation of both TCF4 and TEAD4. These findings not only identify VGLL4 as a coregulator of both Wnt/β-catenin and Hippo-YAP signalling, but also reveal a functional interplay between the transcription factors of these two pathways.P<0.01). Moreover, VGLL4 staining was significantly associated with tumour size, lymph node metastasis and TNM stage (P<0.05), but not significantly correlated to age, gender and tumour metastasis  of patients with normal colons exhibited high levels of nuclear VGLL4 (++ and +++) in their colons, whereas only 48% (29 out of 60) of patients with CRC had high nuclear expression of VGLL4 in the cancerous samples from their colons . The pattastasis . Consisttastasis . Taken thttp://www.ncbi.nlm.nih.gov/gds) was employedVGLL4 were negatively correlated with tumour stage  Database . A univariate analysis (paired t test) indicated that the mRNA levels of VGLL4 were decreased in 17 out of 30 (57%) CRC samples relative to normal tissue (P=0.0356)  , confirm samples .CCND1, Axin2 and c-MYC significantly increased as the cancer progressed, whereas the mRNA levels of VGLL4 at the late stage of CRC (stage III and IV) were significantly less than the mRNA levels at the early stage (stage III and IV) of the disease  assay in 293T cells. As shown in  tumours . These rAxin2, CCND1 and c-MYC, again suggesting an inhibitory role of VGLL4 in the Wnt/β-catenin pathway  RNA vectors that efficiently knocked down human VGLL4 expression . In cont pathway .As Wnt3a-induced nuclear accumulation of β-catenin is a hallmark for the activation of the Wnt/β-catenin pathway, we next examined whether VGLL4 could affect the cytosolic/nuclear localization of β-catenin upon Wnt3a stimulation in HEK293T cells. Western blotting analysis of nuclear and cytosolic β-catenin revealed that VGLL4 did not significantly affect Wnt3a-induced translocation of β-catenin to the nucleus . ConsistCTNNB1 or APC, therefore leading to constitutive activation of the Wnt/β-catenin pathway. Similar to the observations in Wnt3a-stimulated 293T cells, the TOP-FLASH luciferase reporter activity and transcription of Wnt target genes  were decreased by overexpression of VGLL4, but increased by knockdown of VGLL4 , 39 genes were negatively regulated by VGLL4, that is, 34.2% of Wnt target genes were negatively regulated by VGLL4  contain an N-terminal TEA domain responsible for DNA binding, and a C-terminal YAP-binding domain (YBD) responsible for binding YAP/TAZ and VGLL proteins . Since V16 cells . AltogetIf the interaction of VGLL4 with TEADs is essential for VGLL4-mediated negative regulation of TCF4 transactivation, one may expect that TEADs could play an important role in Wnt/β-catenin signalling. To verify the functional relevance of TEADs in Wnt/β-catenin signalling, we performed TOP-FLASH luciferase reporter assay in cells depleted of TEAD1-4 . NotablyAxin2 . Consistent with this observation, transcription of the Wnt target genes Axin2, CCND1 and c-MYC was markedly enhanced in tumours in which VGLL4 was depleted  mutant mice (APCmin/+), a well-known mouse model for colorectal and intestinal cancer, to further corroborate the function of VGLL4 in CRC. APCmin/+ mice at 4 weeks of age were treated with the Super-TDU peptide by tail vein injection for 8 weeks, and then the mice were killed (−1 5-fluorouracil (5-FU), a conventional drug used in cancer therapy. Adenomas were counted manually, as described in the Methods section, using a dissecting microscope. As shown in APCmin/+ mice exhibited significantly more and larger spontaneous adenomas than did wildtype control mice; while treatment with Super-TDU and 5-FU alleviated such situation. After treatment with the Super-TDU peptide, the number and average size of adenomas were significantly decreased, by ∼65%, as compared with those treated with a control peptide , which we developed previously for the study of Hippo-YAP signallinge killed . In para peptide . Meanwhi peptide . Since tAPCmin/+ mice, as compared with those treated with a control peptide  in which β-catenin is hyperactivated, exemplifying a potential targeted therapy pointing to the crosstalk node between Wnt and Hippo pathways. It remains to be determined whether VGLL4 functions as a turn-off checkpoint during the late stage of Wnt signalling, and whether there is another co-regulator of Wnt/β-catenin and Hippo-YAP signalling that also targets the TCF4–TEAD4 complex via a similar mechanism. Investigations in these regards should help advance the development of new strategies for treating cancer.Colorectal carcinoma is the third most common type of cancer and the third most prevalent cause of death by cancer in the worldThirty and five paired fresh colorectal carcinoma and normal tissue samples from patients, who underwent surgery between October 2010 and September 2012, were collected for quantitative RT-PCR and western blot analysis, respectively. On the day of tumour resection, the specimen was received in the laboratory immediately following surgery. The specimen was bathed in sterile saline in a sterile container. Research samples were collected during this dissection, including procurement of tissue for confirmation of diagnosis by pathology. The disease stage of each patient was classified or reclassified according to the 2009 AJCC staging systemCRC and normal tissue microarray sections were prepared by Shanghai Outdo Biotech Co. Ltd. . This tissue array contains tissues from 40 human patients containing normal colons and 60 patients with CRC to examine the expression profiles of VGLL4 by IHC. For IHC, TMA sections were incubated with anti-VGLL4 antibody . VGLL4 staining were scored by two independent pathologists, blinded to the clinical characteristics of the patients. The scoring system was based on the staining intensity and extent. Staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Staining extent was dependent on the percentage of positive cells (examined in 200 cells) were divided into 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). According to the staining intensity and the staining extent scores, the IHC result was classified as 0–1, negative (−); 2–4, weakly positive (+); 5–8, moderately positive (++) and 9–12, strongly positive (+++).−1 of penicillin, and 100 μg ml−1 of streptomycin at 37 °C with 5% CO2 in a humidified incubator . During the study, all cell cultures were periodically tested for mycoplasma by using MycoAlert Mycoplasma Detection Kit . For western blot, antibodies specific for VGLL4 , TEAD4 , Histone H3  and β-catenin  were purchased from Abcam ; those for FLAG  and α-tubulin  were from Sigma ; and those for TCF4 , c-Jun  and GST  were bought from Santa Cruz Biotechnology .HEK293T cells, L cells , SW480 cells and HCT116 cells were obtained from Shanghai Life Academy of Sciences cell library . All cells were grown in DMEM medium , and maintained in culture supplemented with 10% heat-inactivated fetal calf serum, 100 ug mlMammalian expression vectors for VGLL4 and its HF4A mutant were described previouslyDuplexes of siRNA targeting TEAD1, TEAD2, TEAD3, TEAD4, TCF4 and negative control were synthesized by Genepharma . The siRNA sequences are as following. For human TEAD1 (targeting its CDS sequence), the forward oligo used were 5′–GGCAUGCCAACCAUUCUUATT–3′, and the reverse oligo were 5′–UAAGAAUGGUUGGCAUGCCTT–3′. For human TEAD2 (targeting its CDS sequence), the forward oligo used were 5′–GCCAGAUGCAGUUGAUUCUTT–3′ and the reverse oligo were 5′–AGAAUCAACUGCAUCUGGCTT–3′. For human TEAD3 (targeting its CDS sequence), the forward oligo used were 5′–CAGCCACAUACAGGUUCUATT–3′, and the reverse oligo were 5′–UAGAACCUGUAUGUGGCUGTT–3′. For human TEAD4 (targeting its UTR sequence), the forward oligo used were 5′–GGAACAAACUGUGCCUGAATT–3′, and the reverse oligo were 5′–UUCAGGCACAGUUUGUUCCTT–3′. For human TCF4 (targeting its UTR sequence), the forward oligo used were 5′–GGGACAUGCAUGGAAUCAUTT–3′, and the reverse oligo were 5′–CCCUGUACGUACCUUAGUATT–3′.For negative control, the forward oligo used were 5′–UUCUCCGAACGUGUCACGUTT–3′, the reverse oligo were 5′–ACGUGACACGUUCGGAGAATT–3′.−1) for further incubation. The medium that contained G418 was changed every two to three days. After two weeks, isolated colonies began to appear. In three weeks, a pool of G418-resistant cells was obtained for further studies. Luciferase activities were determined using the Dual-Luciferase Assay System (Promega).Transient transfection of cells was performed using Lipofectamine 2,000 from Invitrogen  according to the manufacturer's instructions. To select stable transfectants, the cells were transfected and incubated overnight, and then switched to a medium containing G418  using the comparative Ct quantization method. Real-time PCR Master Mix (Toyobo) was used to detect and quantify the expression level of the target gene. GAPDH was used as an internal control. The primers used were as follows:hVGLL4: 5′–AACTGCAACCTCTCGCACTG–3′ (F), 5′–GAGTGGGTGTCGCTGTTGAA–3′ (R);hAxin2: 5′–CTGGCTTTGGTGAACTGTTG–3′ (F), 5′–AGTTGCTCACAGCCAAGACA–3′ (R);hAxin2 (ChIP): 5′–CGCCTTTGAAGTGCACAGT–3′(F), 5′–ACCCAGGTCCTGTTTCCAG–3′(R); hCCND1: 5′–GCTGCGAAGTGGAAACCATC–3′ (F), 5′–CCTCCTTCTGCACACATTTGAA–3′ (R); hc-MYC: 5′–GGCTCCTGGCAAAAGGTCA–3′ (F), 5′–CTGCGTAGTTGTGCTGATGT–3′ (R); hGAPDH: 5′–GACGTGAGCATGTACCCTAGC–3′ (F), 5′–GCGTAGCCATTCCAGTCCT–3′ (R); mAxin2: 5′- ATGAGTAGCGCCGTGTTAGTG -3′ (F), 5′- GGGCATAGGTTTGGTGGACT -3′ (R); mCCND1: 5′- GCGTACCCTGACACCAATCTC -3′(F), 5′- ACTTGAAGTAAGATACGGAGGGC–3′(R); mc-myc: 5′- ATGCCCCTCAACGTGAACTTC–3′ (F), 5′- GTCGCAGATGAAATAGGGCTG–3′ (R); mGAPDH: 5′–AATGGATTTGGACGCATTGGT–3′(F), 5′–TTTGCACTGGTACGTGTTGAT–3′(R). Note: F, forward; R, reverse.Microarray assay was performed by knockdown and overexpression of VGLL4 in HCT116 cells. In either case, the microarray data presented in the manuscript is average value from two replicates. Specifically, HCT116 were transfected with the indicated plasmids or siRNAs . Following 48 h transfection, RNA was extracted using TRIZOL Reagent  following the manufacturer's instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2,100 . Qualified total RNA was further purified by RNeasy mini kit  and RNase-Free DNase Set . Total RNA was amplified and labelled by Low Input Quick Amp Labelling Kit, One-Color , following the manufacturer's instructions. Labelled cRNA were then purified by RNeasy mini kit . Each slide was hybridized with Cy3-labelled cRNA and hybridized overnight to a Human Gene Expression 4 × 44 K microarray. After hybridization, slides were scanned by Agilent Microarray Scanner .−1 × 100.Cells were transfected with the indicated plasmids. An ATP-based cell viability assay was used for detecting cell proliferation. The ATP content was measured in accordance with the protocol of the CellTiter-Glo luminescent cell viability assay kit (Promega). Briefly, 100 μl of assay reagent was added to the wells, and mixed for 2 min at room temperature. After 10 min, intracellular ATP content was measured using a multilabel luminescence counter . Cell viability was calculated using the following equation: % Cell viability=–value (blank)) × −value (blank))4 cells were seeded on soft agar in six-well plates, and colonies with a diameter of >0.05 mm were counted 14 days after seeding.Cells were stable-transfected with indicated plasmids. A total of 106; SW480 2 × 106). Mice were killed after four weeks and tumour volumes were then measured. Double-blind study has been designed and followed during the animal assays. All animals were randomly assigned to treatment groups in all experiments and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Biochemistry and Cell Biology. The approval ID for using the animals was No. 081 by the Animal Core Facility of SIBCB.Six-week-old healthy male nude mice (BALB/cA-nu/nu) were obtained from the Shanghai Experimental Animal Center and maintained in pathogen-free conditions. During the tumour formation assay, cancer cell lines transfected with VGLL4 shRNA were injected into the flank of the mice  were injected once daily with 0.1 ml of Super-TDU in saline via the tail vein. Mice were randomized to receive either 50or 500 μg kg−1 per day of Super-TDU peptide or control peptide. In addition, mice were treated with 50 mg kg−1 5-fluorouracil (5-FU) intravenously as a positive control.L cells were plated on coverslips in six-well plates and transfected with the indicated plasmids. Twenty-four hours later, cells were treated with or without Wnt3a for 2 h. Coverslips with the cells were washed once with PBS and fixed in 4% formaldehyde in PBS for 15 min. After permeabilization with Triton X-100 (0.25%) in PBS for 15 min, cells were blocked with PBS containing BSA (5%) for 1 h and then incubated with primary antibodies for 1 h. After three separate washes, cells were incubated with a secondary antibody for another hour and then stained with DAPI for 2 min. The coverslips were washed extensively and fixed on slides. Images were captured using a Leica laser scanning confocal microscope (Leica TCS SP2 AOBS).The downstream sequence of SOX2 gene containing TEAD and TCF binding sites were amplified by PCR. Target DNA were detected by agarose gel and purified by Gel Extraction Kit (Tiangen). The primers used were as follows: SOX2 sense: TAATATAAATTGAATGAATAAGAG; SOX2 antisense: CTCATGCACACAAAGGCACTG.Escherichia coli (E. coli) BL21 (DE3) CodonPlus cells. The protein expression was induced by 0.4 mM isopropyl β-D-thiogalactopyranoside at A600=0.8 in Terrific Broth medium and the cells were cultured for an additional 18 h at 16 °C. The cells were then harvested by using centrifugation and then resuspended in the lysis buffer . The cell homogenates were lysed by using a high-pressure homogenizer (JNBIO3000plus) at 1,800 bar and cell debris was removed by centrifugation at 20,000g for 40 min. The soluble fraction was loaded onto a Ni or Glutathione Sepharose  or Amylose Resin (NEB) pre-equilibrated with lysis buffer and the proteins were eluted with an elution buffer  plus 400 mM imidazole, 20 mM reduced glutathione or 20 mM maltose. Untagged proteins were generated by TEV protease treatment. Tagged proteins were concentrated and applied to a HiLoad 16/60 Superdex 200 pg column  pre-equilibrated with a buffer consisting of 20 mM Hepes, 100 mM NaCl, and 1 mM DTT, pH 7.5. Untagged TEAD4 was applied to the same column pre-equilibrated with a buffer consisting of 20 mM Hepes, 250 mM NaCl and 1 mM DTT, pH 7.5. The purity of the proteins was monitored by SDS–polyacrylamide gel electrophoresis (SDS–PAGE).Human TEAD4 , TEAD4 , TEAD4 , VGLL4 (203–256), TCF4 , TCF4 (8–54), TCF4 (326–411) and β-catenin were cloned into pET28a with a His-SUMO, MBP, GST and His tag respectively followed by a tobacco etch virus (TEV) protease cleavage site at the N terminus and expressed in −1) after transfection or stimulation, and incubated overnight with indicated antibodies (0.6 μg mg−1 protein) together with Protein A/G beads (Santa Cruz). Beads were then washed three times with lysis buffer, and immunoprecipitates were eluted with SDS loading buffer and resolved in SDS–PAGE gels. The proteins were transferred to PVDF membrane (Bio-Rad) and further incubated with the indicated antibodies.For immunoprecipitation experiments, whole cell extracts were prepared with or without DNase I  containing proteinase inhibitors. Lysates were sonicated to yield 300–1,000 bp DNA fragments. After elimination of cell debris by centrifugation, the sample was diluted with 1.8 ml of ChIP dilution buffer and was pre-cleared with 10 μl protein A-Sepharose (50% slurry) for 30 min at 4 °C with agitation. The sample (0.1 ml) was saved to assess input DNA. 2–5 μg of the antibodies TCF4  and TEAD4  was incubated with the sheared cross-linked chromatin to immunoprecipitate the indicated complexes. Input and immunoprecipitated DNA were subjected to Sybergreen Q PCR cycles with primers overlapping the gene body and upstream and downstream regulatory regions of human AXIN2.HEK293T cells were suspended in 5 × volume of cell lysis buffer  and incubated for 5 min on ice. The suspension was centrifuged at 700Interaction analysis was performed using an Octet Red 96 instrument (ForteBio). Wild-type TCF4 was labelled by biotin in 20 mM Hepes pH 7.5, 100 mM NaCl, 1 mM DTT, and biotinylated proteins were immobilized on streptavidin (SA) biosensors and incubated with TEAD4 (TEA) proteins in 1 × kinetics buffer. Data were analysed using Octet Data Analysis Software 7.0 (ForteBio).t test or unpaired t test. For correlation, the Spearman rank correlation was used for continuous variables. Fisher's exact test was used to identify statistical significance of correlation between VGLL4 staining (IHC) and clinicopathological factors. Survival curves were calculated according to the Kaplan-Meier method; survival analysis was performed using the logrank test. P<0.05 was considered to indicate a significant difference.Both cellular and animal studies tended to be underpowered. Estimated of sample size for a planned comparison of two independent means using a two-tailed test were undertaken using an online calculator and in the SAS statistical software package (9.1.3). Data are expressed as mean±s.d. or median and interquartile range for continuous variables and as frequencies and proportions for categorical variables. Continuous data were compared by using paired The RNA microarray data that support the findings of this study have been deposited in the NCBI Gene Expression Omnibus (GEO) with the accession code GSE81665. All other all relevant data are available from the authors.How to cite this article: Jiao, S. et al. VGLL4 targets a TCF4–TEAD4 complex to coregulate Wnt and Hippo signalling in colorectal cancer. Nat. Commun.8, 14058 doi: 10.1038/ncomms14058 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures


Amorpha fruticosa L. Our data demonstrated that dalbinol inhibited the growth of HepG2, HepG2/ADM and Huh7 cells in a concentration-dependent manner. Pharmacological experiments also showed that dalbinol suppressed growth and induced apoptosis in these HCC cell lines in vitro. Furthermore, we found that dalbinol promoted β-catenin degradation, which was mediated by the ubiquitin-proteasome pathway. To summarize, our results illustrate that dalbinol inhibited HCC cell growth by facilitating β-catenin degradation through the ubiquitin-proteasome pathway. Hence, we propose that dalbinol will be a promising agent for the treatment of HCC subtypes with aberrant Wnt/β-catenin pathway activation.Hepatocellular carcinoma (HCC) is a highly malignant tumor, and the main cause of treatment failure is malignant proliferation. Aberrations in Wnt/β-catenin signaling are associated with HCC development. Despite the improvements in overall survival made over the past decade from the advent of molecularly targeted therapies, these treatments do not have efficacy in all patients with different pathogeneses. Therefore, there is a demand for novel chemotherapeutic agents for HCC. To this end, we built a natural compound library and screened out a rotenoid named dalbinol from the seeds of  Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. It ranks 6th in terms of global incidence, and is steadily increasing in Western countries. The rise in the incidence of HCC is caused by the recent increase in hepatitis C virus (HCV) infection, a subtype of the hepatitis virus that is associated with human cancers . AlthougDrosophila to mammals. In its quiescent state, low β-catenin levels are maintained through an ubiquitin-proteasome degradation pathway following adenomatous polyposis coli (APC)-mediated transportation from the nucleus to the cytoplasm [Aberrations in the canonical Wnt signaling pathway contribute to congenital malformations, osteoporosis and cancers –7, incluytoplasm . In maliytoplasm , β-catenytoplasm  and Axinytoplasm . In factytoplasm , 16. β-cytoplasm .HCC diagnoses are clinically confirmed only at advanced stages, meaning that in most cases, surgical resection is not suitable , 19. As Amorpha fruticosa L. seeds [Pseudomonas aeruginosa [In recent years, many rotenoid derivatives have been described as promising anti-tumor agents. Among these, deguelin has attracted the most interest. Deguelin not only upregulates IGFBP3  and reprL. seeds . In spitruginosa , dalbinoAmorpha fruticosa L. seeds, and discovered its anti-cancer effects on HepG2, HepG2/ADM and Huh7 cells. Our results revealed dalbinol-induced apoptosis in HCC cell lines. What is more, it inhibited HCC cell growth by promoting the ubiquitin–proteasome-mediated degradation of β-catenin. Thus, dalbinol may be a promising agent for the treatment of HCC and other cancers with aberrant Wnt/β-catenin signaling.In this study, we extracted and purified dalbinol from dried Amorpha fruticosa L. seeds and has a similar chemical structure to rotenone  were added to cells as a control. Dalbinol inhibited the growth of HepG2 Figure , HepG2/A2 Figure , HepG2/A2 Figure  and Huh72 Figure  cells.To evaluate whether dalbinol induced apoptosis, we conducted flow cytometry assay after Annexin V/propidium iodide double staining of dalbinol-challenged HCC cells. These data suggested a trend towards apoptosis induction in HepG2, HepG2/ADM and Huh7 cells in a concentration-dependent manner after dalbinol treatment, however the values were not significantly different Figure . We nextThe CCK-8 assays described above demonstrated that dalbinol inhibited cell proliferation in HCC cell lines, but the underlying mechanisms remained to be elucidated. It has been reported that deguelin, another rotenoid, suppresses growth of cancer cells by inhibiting Wnt signaling , 37. TheNuclear β-catenin accumulation has been shown to propel the development of many tumors. We next examined whether dalbinol exerted its anti-cancer effect by decreasing the accumulation of nuclear β-catenin. To this end, we made nuclear and cytoplasmic fractions and analyzed β-catenin levels in each. Both fractions showed reduced β-catenin levels following dalbinol treatment, though cytoplasmic β-catenin was reduced more significantly than nuclear β-catenin Figure . ImmunofAs shown in Figure Furthermore, to test whether dalbinol promotes cytoplasmic β-catenin degradation, we employed a co-immunoprecipitation assay. This showed that the interaction between β-TrCP, an E3 ubiquitin ligase involved directly in β-catenin degradation, and β-catenin increased in HepG2/ADM cells treated with dalbinol Figure , right. Data from several laboratories have shown that rotenoids of different origins, such as deguelin, boeravinone and 6-deoxyclitoriacetal exert cytostatic effects on cancer cells derived from various tissues. However, dalbinol, another rotenoid derivative, has not been reported for its anti-cancer activity. In this study, we tested the anti-cancer activity of dalbinol in the HCC cell lines HepG2, HepG2/ADM and Huh7. Particularly, we uncovered that dalbinol exerted an anti-proliferative activity by promoting β-catenin degradation, thus blocking Wnt signaling. Our data suggested dalbinol possessed a lower cytotoxicity than rotenone in immortalized normal hepatocytes Figure . With reUpon dalbinol challenge, apoptotic cell death was triggered. This was demonstrated by flow cytometry using an Annexin V probe; cleavage of pro-caspase-3 and PARP was also noted. Downregulation of survivin , Mcl-1  and uprede novo protein synthesis. Besides, dalbinol-induced decrease in the GSK-3β (pSer9) level meant GSK-3β was activated, which resulted in phosphorylation of β-catenin . Adriamycin resistant HepG2/ADM cells were kindly provided by Dr. Caiguo Ye . HepG2, HepG2/ADM and Huh7 cells were cultured with DMEM  with 10% fetal bovine serum  in a 5% COA fruticosa L. by Dr. Xin Wu  using previously described procedures [A fruticosa L. seeds were deposited in the Guangdong Key Laboratory for Research and Development of Natural Drugs , and the voucher specimen number was 20130910. Rotenone and CHX were from MP Biomedicals Corporation , propidium iodide was from Sigma-Aldrich , MG132 was from Calbiochem  and DAPI was from Beyotime . Specific anti-Caspase-3 p17 (Cat: 25546-1-AP), Dvl-3 (Cat: 13444-1-AP), Cyclin D1 (Cat: 60186-1-lg) and IgG (Cat: B900610) antibodies were from Proteintech . Antibodies against Cleaved PARP (#5625), Survivin (#2808), Mcl-1 (#5453), Bim (#2933), Bax (#5023), Ubiquitin (#3936), β-catenin (#8480), GSK-3β (pSer9) (#9322), β-TrCP (#11984) and β-actin (#3700) were from Cell Signaling Technology . Protein A/G PLUS-Agarose (sc-2003) and the antibody against Dvl-2 (sc-13974) were from Santa Cruz Biotechnology . Annexin V-FITC (Cat: 51-65874X) and antibodies against GSK-3β (Cat: 610201) and c-Myc (Cat: 551101) were obtained from BD Biosciences Pharmingen . Histone 3 antibody (orb136531) was bought from Biorbyt . Goat Anti-Rabbit IgG (H+L), DyLight (tm) 488 Conjugated (No. 35552) was from Pierce Biotechnology .The natural compound dalbinol was extracted and purified from the seeds of ocedures . The A f3cells were seeded in each well of 96-well microplates and co-incubated with dalbinol at different concentrations for 72 h. Cell viability was evaluated as a percentage of the control cells, which were treated with equal volumes of DMSO (<0.1%) in DMEM. Three hours prior to culture termination, 10 μL of CCK-8 liquid was added to each well, and optical density was read on a 96-well microplate reader at 450 nm. IC50 values were calculated using GraphPad Prism 5.0 software .The Cell Counting Kit-8 (CCK-8) assay was used to evaluate the effect of dalbinol on cell viability. Briefly, 2×10For colony formation assays, 500 cells/well were seeded in a 12-well plate. After over-night incubation, the cells were exposed to concentration gradients of dalbinol and cultured for 9 days. The medium mixed with compound was replaced on the 4th day. On day 9, cells were washed with PBS, fixed in ice-cold methanol for 10 min, and stained with crystal violet for 15 min at room temperature. After rinsing with distilled water twice and drying at room temperature, images of the colonies were scanned using an HP scanner. Representative images were shown. Colonies were counted using an inverted phase-contrast microscope, and aggregates of ≥50 cells were identified as a colony. All numbers from three independent experiments were normalized to the average of controls using GraphPad Prism 5.0 software.2). Next, the cells were resuspended in 100 μL binding buffer containing 3% (v/v) Annexin V-FITC and incubated at room temperature for 20 min in the dark. After incubation, the cells were resuspended in 400 μL binding buffer. PI was added before FACS analysis. Apoptosis rates were analyzed with WinMDI 2.9 software (http://facs.scripps.edu/).Apoptosis was detected by FACS after Annexin V/PI double staining. Cells were cultured in the presence of different concentrations of dalbinol for 24 h. Then they were pelleted by centrifugation, washed twice in cold PBS, then washed in 500 μL binding buffer  according to the manufacturer's instructions. The lysate collected after ultracentrifugation was quantitated with the BCA Protein Assay Kit, and total protein was separated by SDS-PAGE, then electro-blotted to nitrocellulose filters. The filters were blocked with 5% non-fat milk in PBS before probing with specific primary antibodies at different conditions. The filters were then washed with 1% Tween 20 in PBS, and conjugated with the appropriate secondary antibodies, and developed.Cells cultured in 35 mm dishes with 14 mm glass bottom wells were washed with PBS twice, fixed for 15 min in PBS containing 4% paraformaldehyde, and permeabilized with 0.5% NP-40 and 1% BSA in PBS for 15 min. After washing with PBS for 5 min thrice and blocking with 5% BSA for 1 h, the cells were incubated with β-catenin primary antibody in a humidity chamber at 4°C overnight, followed by washing with PBS for 5 min thrice and incubation with DyLight (tm) 488-conjugated (green fluorescence representing β-catenin) secondary antibody together with DAPI at 4°C for 1 h in the dark. The cells were mounted onto slides and visualized using a Leica TCS SP5 II confocal laser-scanning microscope image system .2, 1% NP-40, 10% Glucose) on ice for 1 h. After centrifuging at 15,000 ×g for 10 min at 4°C, supernatant was collected and 600 μg total protein in 800 μL lysis buffer was incubated with 6 μg primary antibody  at 4°C overnight with rocking (IgG control). Meanwhile, 50 μg total protein was saved and mixed with loading buffer as input for western blotting. For the co-IPs, 60 μL of prewashed protein A/G agarose beads were incubated with 600 μg samples at 4°C for 1 h with rocking. After incubation, the immune complexes were washed three times with lysis buffer, and proteins were eluted by boiling in loading buffer, followed by separation by 10% SDS-PAGE and western blot analysis using the corresponding antibodies.For co-IPs, cells cultured on 60 mm dishes were washed with PBS and lysed with lysis buffer  was used to perform all statistical analyses. Student's t test was employed to compare data between two groups, and comparisons among multiple groups employed one-way ANOVA with Tukey's post hoc intergroup comparisons.


In Experiment 2 (n = 15), M- and F-waves were recorded from the right APB. Stimulation at 110% MNS at the wrist evoking muscle contraction increased MEP amplitudes after PES compared with those at baseline, whereas DNS at the index finger and 90% MNS at the wrist not evoking muscle contraction decreased MEP amplitudes after PES. M- and F-waves, which reflect spinal cord or muscular and neuromuscular junctions, did not change following PES. These results suggest that muscle contraction and concomitant muscle/joint afferent inputs specifically enhance M1 excitability.Modulation of cortical excitability by sensory inputs is a critical component of sensorimotor integration. Sensory afferents, including muscle and joint afferents, to somatosensory cortex (S1) modulate primary motor cortex (M1) excitability, but the effects of muscle and joint afferents specifically activated by muscle contraction are unknown. We compared motor evoked potentials (MEPs) following median nerve stimulation (MNS) above and below the contraction threshold based on the persistence of M-waves. Peripheral nerve electrical stimulation (PES) conditions, including right MNS at the wrist at 110% motor threshold , right MNS at the index finger (sensory digit nerve stimulation [DNS]) with stimulus intensity approximately 110% MNS (DNS condition), and right MNS at the wrist at 90% MT (90% MNS condition) were applied. PES was administered in a 4 s ON and 6 s OFF cycle for 20 min at 30 Hz. In Experiment 1 ( Converging evidence suggests that afferent somatosensory inputs such as peripheral nerve electrical stimulation (PES), muscle tendon vibration and active and passive movements can induce changes in primary motor cortex (M1) excitability  elicited by transcranial magnetic stimulation (TMS) over the M1 after a prolonged period of PES . In animals, S1 areas 3a and 2 predominantly receive proprioceptive inputs from muscle and joint afferents  participated in this study. Twenty-three subjects were right handed, and three were left handed. Based on health-related and TMS questionnaires  was recorded from the right APB muscle via disposable Ag/AgCl electrodes in a belly-tendon montage. The signals from the EMG electrodes were amplified (×100) by an amplifier , filtered , digitized at 4 kHz using an A/D converter , and stored on a lab computer for later offline analysis .Magnetic stimuli were delivered through a figure-of-eight coil  connected to a Magstim 200 stimulator . The coil was held tangentially to the skull over the left M1 area with the handle pointing posterolaterally at 45° to the sagittal plane. The TMS coil was placed over the left M1 at the position producing the largest MEPs from the right APB muscle (the motor hotspot). Position and orientation of the coil for the motor hotspot were marked according to magnetic resonance imaging (MRI) via Visor2 TMS Neuronavigation , and the coil was held in place to maintain position. T1-weighted MRI was obtained using a 1.5-T system before the experiment . The TMS intensity was set at that inducing peak-to-peak amplitude of ~1 mV in APB at baseline. The TMS intensity is expressed as a percentage of the maximum stimulator output.Continuous electrical stimulation was delivered through bar electrodes to the right median nerve at the wrist (mixed nerve) or the right index finger tip (sensory nerve) by means of an electrical generator . The electrical stimulation (0.2-ms square wave constant current pulses) was delivered for 20 min at 30 Hz in a 4 s ON and 6 s OFF cycle in all PES conditions. The MT was determined based on the minimum stimulus intensity that can elicit M-waves  to the right median nerve at the wrist (mixed nerve) using surface EMG. Three different PES conditions were used: (1) electrical stimulation to the right median nerve at the wrist with intensity set to 110% MT ; (2) electrical stimulation to the right index finger tip with intensity equal to that used in condition (1) DNS; and (3) electrical stimulation to the right median nerve at the wrist with intensity at 90% MT . All subjects felt tactile perception without pain in DNS condition.Electrical pulses (0.2-ms square wave constant current pulses) were delivered through bar electrodes to the right median nerve at the wrist using an electrical generator . M- and F-waves were recorded from right APB using surface EMG. Stimulus intensity was 120% of the stimulus strength required to produce maximum M-wave.Fifteen healthy subjects  participated in Experiment 1. The experimental protocols are shown in Figure Fifteen healthy subjects  participated in Experiment 2. We conducted Experiments 1 and 2 on different days, and the subjects who participated in our study were different for different experiments. Hundred M- and F-waves were recorded with an interstimulus interval of 1 s before (baseline) and 5 and 15 min after PES (post 5 and post 15). The same electrical stimulus intensity was used before and after PES.In these experiments, DNS condition was performed after 110% MNS condition to decide on DNS intensity based on the 110% MNS intensity. Thus, these experiments were conducted in the following order to avoid the effects pertaining to the sequence: 110% MNS, DNS and 90% MNS; 110% MNS, 90% MNS and DNS; or 90% MNS, 110% MNS and DNS.post hoc comparisons. Also, unpaired t-test was used to compare PES intensity between PES conditions in Experiments 1 and 2. Statistical significance for all tests was set at P < 0.05.For Experiment 1, the peak-to-peak amplitudes of 10 of the 12 recorded MEPs (excluding the maximum and minimum) were averaged for each time point .In Experiment 1, the mean PES current amplitudes were 10.3 ± 0.6 mA for 110% MNS, 10.3 ± 0.6 mA for DNS, and 8.1 ± 0.6 mA for 90% MNS. In the Experiment 2, the mean currents for these PES conditions were 8.7 ± 0.6 mA, 8.7 ± 0.6 mA and 7.9 ± 0.7 mA, respectively. There were no significant differences in the PES intensity between experiments in each of the PES conditions  = 0.164, P = 0.849) on the TMS intensity.The TMS intensity was 56.8% ± 1.9% of maximum stimulator output in the 110% MNS PES condition, 57.1% ± 1.9% in the DNS condition, and 57.3% ± 1.9% in the 90% MNS condition. One-way repeated-measures ANOVA showed no significant effects of SESSION  = 14.147, P < 0.001, η2 = 0.503) and an INTERVENTION × TIME interaction  = 9.478, P < 0.001, η2 = 0.404) but no significant main effect of TIME  = 3.137, P = 0.059, η2 = 0.183). Baseline MEP amplitudes did not differ between PES conditions . At each post-PES time point (post 5 and post 15), post hoc tests revealed a significant difference in MEP amplitude between 110% MNS and both DNS and 90% MNS conditions  but no difference between DNS and 90% MNS conditions . Alternatively, in both the DNS and 90% MNS conditions, post hoc tests showed a significant decrease in MEP amplitude at post 5 and post 15 time points compared with baseline  = 2.182, P = 0.132, η2 = 0.135) and TIME  = 0.823, P = 0.389, η2 = 0.056) and no INTERVENTION × TIME interaction  = 2.260, P = 0.120, η2 = 0.139) on M-waves  = 3.000, P = 0.087, η2 = 0.176) and TIME  = 0.242, P = 0.787, η2 = 0.017) and no INTERVENTION × TIME interaction  = 1.554, P = 0.199, η2 = 0.100) on F-waves. For the F/M ratio, two-way repeated-measures ANOVA showed no significant main effects of INTERVENTION  = 1.943, P = 0.178, η2 = 0.122) and TIME  = 0.178, P = 0.838, η2 = 0.013) and no INTERVENTION × TIME interaction  = 1.505, P = 0.213, η2 = 0.097). For F-wave persistence, two-way repeated-measures ANOVA showed significant main effects of INTERVENTION  = 5.060, P = 0.013, η2 = 0.265), but no significant main effects of TIME  = 1.164, P = 0.327, η2 = 0.077) and no INTERVENTION × TIME interaction  = 1.323, P = 0.283, η2 = 0.086; Table Post hoc tests conducted for studying the main effect of INTERVENTION revealed a significant difference in F-wave persistence between DNS and 90% MNS conditions (P = 0.048) but no difference between 110% MNS and both DNS and 90% MNS conditions .Plots of individual M-wave amplitudes Figure  and F-waThis study demonstrates that while mixed nerve electrical stimulation at an intensity above MT (muscle contraction elicited by the stimulation) significantly increases MEP amplitude, indicative of enhanced M1 excitability, the mixed nerve electrical stimulation at an intensity below MT (muscle contraction not elicited by the stimulation) or sensory nerve electrical stimulation decreases MEP amplitude, indicative of reduced M1 excitability. Neither M- nor F-wave amplitude differed between these stimulus conditions, suggesting that the changes occur at the level of the cortex. These results suggested that proprioceptive inputs induced by both mixed nerve electrical stimulation and muscle contraction caused by the electrical stimulation might be involved in the increase in M1 excitability after PES.Previous studies also reported increased MEP amplitude following PES intensities sufficient to produce muscle contraction . Animal studies have shown that cutaneous afferent inputs project mainly to areas 3b and 1  increased MEP, whereas that at an intensity below MT (muscle contraction not elicited by PES) decreased MEP. One possible explanation for PES-induced MEP facilitation is the specific timing of direct Ia fiber activation by PES and somatosensory input from contracting muscles. Saito et al.  reportedSensory nerve and mixed nerve stimulation did not evoke muscle contraction and decreased MEP in this study. Ridding et al.  reportedExperiment 2 was designed to test whether the effect of PES on MEP amplitude was due to excitability changes in cortical, spinal cord, or muscle and neuromuscular junction. No significant changes in M- and F-waves were observed following any of the three PES protocols, consistent with previous studies showing that M-, F- and H-waves were not modified by PES  for clarifying whether inter- and intra-individual variability in the MEP response related to SEP response reflected S1 excitability. The specific neuroplastic mechanism for PES with and without muscle contraction effects on M1 excitability is unknown. However, different brain activities may contribute to M1 excitability increase and decrease from our results. Studies employing multiple methodologies  are planned to elucidate the underlying neuroplastic mechanism induced by PES with and without muscle contraction. Although we compared the effects of mixed nerve stimulation with those of DNS at the same PES intensity, the number and diameter of neural fiber activated by PES were different. We would, therefore, like to explore whether similar MEP depression can be observed even if a wider range of sensitive nerve branches, including those of the middle finger, are stimulated near future.In summary, this study demonstrates that mixed MNS eliciting muscle contraction significantly increases MEP amplitude, indicative of enhanced M1 excitability, while sensory MNS alone and sub-MT mixed MNS significantly decrease MEP, indicating reducing M1 excitability. Indeed, the lack of effect on M- and F-waves confirms that this modulation is at the level of the cortex rather than spinal cord, neuromuscular junction, or muscle. This study shows that sensory inputs specific to muscle contraction can enhance M1 excitability.HO and RS conceived the study and designed the experiments. RS and MN conducted the experiments. SM and SKojima performed interpretation of data. RS and SKotan performed the statistical analysis. KS and YI helped writing the manuscript. HO and RS wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health.Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent  The gut epithelial barrier is selectively permeable to nutrients, minerals, water and selected antigens, while protecting against potentially harmful bacteria, viruses and antigenic materials. Neonatal gut has a relatively high permeability, which is required for transferring of maternal immunoglobulins in colostrum and selected antigens, further facilitating the proper development of the gut immune system ,2. Howevin vivo studies. Compared with in vivo studies, in vitro cell culture is convenient, low cost and time-saving, suitable for using chemical stimulators and inhibitors, and suitable for genetic manipulation without physiological complications. However, cell lines are immortalized cells and their cellular behaviour may differ significantly from normal epithelial cells. The recently developed epithelial organoid culture uses Lgr5 (leucine-rich-repeat-containing G protein-coupled receptor) as a surface marker for isolating gut epithelial stem cells, which further develops into organoid structures using three-dimensional matrigel as a scaffold [in vivo epithelial differentiation during gut development due to the lack of underlying mesenchymal tissues that intricately interact with the epithelium to regulate its development [in vivo studies are either costly or impossible to use chemical inhibitors and activators, or RNA interferences, to manipulate epithelial differentiation without inducing physiological complications or other changes. There is a need to establish an ex vivo model, which can mimic in vivo gut epithelial development but has the convenience and flexibility of in vitro cell culture.Up to now, studies on epithelial differentiation and migration were mainly using epithelial cell lines and scaffold . This orscaffold ,16. Furtelopment ,17. Likeex vivo model for studying gut epithelial differentiation and migration using embryonic (E) 13.5-day fetal small intestine. The model can effectively track the differentiation of gut epithelium real-time, which, in combination with the immunohistochemical and histochemical examination of structures, provides a powerful venue for elucidating key mechanisms regulating gut epithelial development.Here, we established an in vitro and in adult mice [ex vivo gut model.AMP-activated protein kinase (AMPK) is recognized as a master regulator of energy metabolism ,19, and ult mice , but wer2.2.1.#440140, Sigma, St Louis, MO, USA) in acetone for 15 min [−1 fibronectin . Coverslips were dried under a biosafety cabinet. Before culturing, coverslips were placed into 35 mm cell culture dishes , and four sterile 4.7 × 8 mm cloning cylinders  were placed onto the fibronectin-coated area on coverslips.Coverslips  were dipped into acidic alcohol , air dried, then submerged into 2% 3-aminoprophytriethoxysilane . Mice harbouring an Lgr5-EGFP-IRES-creERT2 (Lgr5) allele  were crossed with ROSAmT/mG  mice to obtain conditional knockout mice (Lgr5mT/mG). Epithelial stem cells in ex vivo gut from Lgr5mT/mG mice will convert cellular fluorescent protein from red to green signal in Lgr5-positive cells and their derived cells upon induction of 4-hydroxytamoxifen. The double-fluorescent ex vivo gut facilitated the tracing of epithelial movement and reorganization of Lgr5-derived cells. Mice with AMPKα1-floxed gene  were cross-bred with tamoxifen-inducible Cre mice (129-Gt(ROSA)26Sortm1(cre/ERT)Nat/J, #004847, Jackson Lab) as previously described to obtain AMPKα1 conditional knockout (KO) mice [α1 can be deleted in all tissues of AMPKα1 KO mice. Embryonic (E) staging was started from the morning when a copulatory plug was observed and counted as embryonic day 0.5 (E0.5). Pregnant mice at E13.5 were euthanized for ex vivo gut preparation.C57BL/6 J mice were purchased from Jackson Laboratory (KO) mice . In resp2.3.2 humidified incubator. The cloning cylinders were carefully removed after 24 h of culture. The gut segments were cultured for 10 days, and the culture medium was changed every 3 days. To determine Gata4 expression in different segments of small intestine, ex vivo guts from embryonic proximal or distal small intestine were cultured and collected at 7 days separately. To evaluate the effects of signalling regulators on the differentiation of ex vivo guts, culture medium containing 100 nM LDN-193189 , 10 μM DAPT  or 100 ng ml−1 Wnt 3a and 500 ng ml−1 R-spondin1  was prepared to treat the guts for 7 days. To induce AMPK knockout, 4-hydroxytamoxifen  was used during the first three days of culture.E13.5 embryos were removed aseptically and transferred into ice-cold culture medium  supplemented with 10% fetal bovine serum  and 1% antibiotic–antimycotic solution (Invitrogen). The small intestine was dissected from embryos according to a procedure described previously with modifications . Briefly2.4.ex vivo guts were fixed in freshly prepared 4% formaldehyde for 2 h at room temperature and then rinsed with PBS for three times. Ex vivo guts were submerged in PBS with 30% sucrose for 1 h at room temperature, which served as a cryoprotectant for gut during freezing. The cultured guts were carefully peeled from coverslips with dissecting blades under a stereo microscope, then vertically embedded into Tissue TekR OCT compounds  in embedding moulds (Thermo Fisher Scientific), followed by placement into isopentane (Sigma) pre-cooled by liquid nitrogen for 30 s. The frozen ex vivo gut samples were stored at −80°C and sectioned using a cryostat  to 5 µm thickness and mounted directly onto ultra-stick micro slides , then stored at −80°C till staining.Following the removal of culture medium, 2.5.#MS-113-B, 1 : 50; E-cadherin , Life Technologies #33–4000, 1 : 200; Chromogranin A (enteroendocrine cells), Developmental Studies Hybridoma Bank #CPTC-CHGA-1, 1 : 200; Vimentin (fibroblasts), Cell Signaling Technology #5741, 1 : 200; Ki67 (proliferative cells), Cell Signaling Technology #9449, 1 : 200; CDX2 , Cell Signaling Technology #12306, 1 : 300; β-catenin (adherens junctions), Cell Signaling Technology #9562, 1 : 300; Cytokeratin 8 (columnar epithelium), Developmental Studies Hybridoma Bank #TROMA-I, 1 : 400; Lysozyme (Paneth cells), Thermo Fisher Scientific #PA5-16668, 1 : 800. Then the slides were incubated with respective fluorescent secondary antibodies  diluted in 5% goat serum (1 : 1000) at room temperature for 2 h. Immunofluorescence was imaged using a fluorescence microscope . Negative controls were stained with secondary antibody without primary antibody incubation.Sections were permeabilized in 1% Triton X-100 (Sigma) in PBS at room temperature for 30 min, boiled in 10 mM sodium citrate  for 20 min, incubated with 5% goat serum  in PBS (pH 7.4) for 1 h to block non-specific binding sites, then incubated with primary antibodies overnight at 4°C. The following antibodies were used: Smooth muscle actin (smooth muscle cells), Thermo Fisher Scientific 2.6.For Alcian blue staining, tissue sections were stained with 1% Alcian blue solution  for 30 min at room temperature as previously described . Haemato2.7.ex vivo gut samples for each group using TRIzol (Sigma). Five hundred nanograms of cDNA was synthesized using a Reverse Transcription kit . qPCRs for 5 ng cDNA per reaction were performed on a CFX RT-PCR detection system (Bio-Rad) using SYBR Green (Bio-Rad). The primers were designed to cross two exons to prevent amplification of genomic DNA, and primer sequences are shown in the electronic supplementary material, table S1. Five nanograms of cDNAs per reaction from adult mouse intestine and fat tissue were used as positive control (PC) and negative control (NC), respectively. 18S rRNA was used as an internal control.Total RNA was extracted from 10 2.8.In situ apoptosis was determined using TACS TdT-DAB detection kit  per the manufacturer's instructions. Briefly, ex vivo gut sections were treated with Cytopore and blocked with 3% H2O2. DNA fragments were labelled with biotin and HRP-conjugated streptavidin, then incubated with DAB substrates. The counterstaining was performed with 1% Methyl Green. The images were taken using Leica DM2000 LED light microscope .2.9.Ex vivo gut section obtained from each fetus was considered as an experimental unit. Statistical analysis was performed using Prism 6 . Data were presented as means ± standard error of the means (s.e.m.). Differences between means were determined using two-tailed t-test followed by Duncan's multiple test when appropriate. All experiments were replicated in at least three independent experiments. p ≤ 0.05 was considered to be statistically significant.3.3.1.ex vivo gut was examined over 10 days. Gut sections adhered to coverslips within 24 h, and gradually expanded and spread out over time (a–d), and guts gradually flattened out from a tubular structure to a spindle-shaped structure over 10 days (c–f). Addition of Wnt3a and R-spondin1 could extend the culture viability of ex vivo guts to 3 weeks. To further examine the functionality of epithelial cells, the activity of alkaline phosphatase produced by epithelial absorptive cells was tested . Compared with day 0, the activity increased greatly at 7 days, indicating ex vivo gut remained functional even though cell death slightly occurred.The morphology of ver time . Startinver time . The gutver time a–d, and  10 days . To dete3.2.ex vivo guts at 7 days were stained for the respective differentiated cells. The presence of goblet cells and Paneth cells were visible on the surface of gut section . Enteroendocrine cells were identified in the centre of gut lumen, suggested by chromogranin A staining (c). Absorptive cells, indicated by the columnar epithelial marker cytokeratin 8, formed a tight layer on the outside surface of cultured guts, with a small number concentrated in the centre of the original gut lumen (d). CDX2, the key epithelial transcriptional factor, had a similar distribution to absorptive cells, which might be due to its crucial role in intestinal differentiation (e). By contrast, the remaining components were composed of mesenchymal cells, which were concentrated under the epithelium, providing scaffolds for epithelial cells (f). Fibroblasts formed the first scaffold layer under epithelium, as indicated by vimentin staining (f). Smooth muscle cells formed the second scaffold layer under fibroblasts based on smooth muscle actin staining (f). Myofibroblasts were developed in the spreading area (gut ‘tail’), as suggested by co-staining of vimentin and smooth muscle actin (f). To further test whether interstitial cells of Cajal contribute to spontaneous peristalsis in ex vivo guts [C-kit was tested, which was beyond the detection level . Furthermore, the peristalsis was promoted after treatment with a neurotoxin, botulinum . The expression of Gata4 in ex vivo guts at 7 days from embryonic proximal small intestine was higher than the one from distal small intestine, indicating the segment specificity in cultured gut . Additionally, Wnt activators, BMP inhibitor and Notch inhibitor could alter the expression of differentiation markers of ex vivo gut , suggesting these signalling pathways regulate the fates of intestinal stem cells during differentiation.To characterize the differentiation and distribution of intestinal epithelial cells in cultured guts,  section a,b. Entestaining c. Absorput lumen d. CDX2, ntiation e. By conal cells f. Fibrobstaining f. Smoothstaining f. Myofible actin f. To furivo guts , mRNA ex3.3.ex vivo gut, which were unexpected, because epithelial cells were anticipated to concentrate inside the gut lumen. To address the originality of the surface epithelial layers, ex vivo guts isolated from intestinal stem cell-derived fluorescent mice (Lgr5mT/mG) were further used for tracing epithelial movement and reorganization.We observed the presence of epithelial layers on the surface of x vivo guts of Lgr5mT/mG showed exclusively red fluorescence (a). During 9 days of culture, residual cells with red fluorescence remained clearly visible (a–d). Of note, newly Lgr5-derived epithelial cells with green fluorescence appeared and concentrated at the two ends of gut sections at day 3 , and further progressively spread along the surface of the gut at day 6 , leading to almost complete coverage of gut at day 9 . To identify Lgr5+-clusters, we tested the distribution of columnar epithelial cells using cytokeratin 8 staining. We found that Lgr5+-clusters were localized below the differentiated epithelial layer , and reconfirmed that differentiated epithelial cells spread on the surface of the gut (figures 4e and 3d). We further examined the movement of Lgr5-derived cells at the bottom of ex vivo gut . From day 1 to day 10, Lgr5-derived cells started to concentrate at two open ends of ex vivo guts, gradually spread into a large area and formed an epithelial layer on the surface of ex vivo guts . mRNA expression of Foxl1 was further detected in ex vivo gut, suggesting Foxl1-expressing mesenchymal cells might facilitate the formation of stem cell niche [At day 0, erescence a. During visible a–d. Of nll niche   and AMPKα1 conditional knockout (AMPKα1 KO) mice. Alcian blue staining indicated that the prevalence of goblet cells was remarkably decreased in AMPKα1 KO ex vivo gut compared with that of WT (a). Lyz1 was higher in AMPK KO group, while Cdh1, Glut1 and Glut2 were higher in WT group at mRNA level (b).The above established at of WT a. Lyz1 wNA level b.Figureex vivo gut contracted and underwent peristalsis much stronger than that of AMPK KO at 7 days of culture . Because smooth muscle cells contribute to gut peristalsis, we further tested smooth muscle actin. In WT ex vivo guts, smooth muscle actin initiated to localize under the epithelial layer, and gradually spread along the contour of ex vivo gut; while in AMPK KO ex vivo guts, smooth muscle actin became increasingly disorganized over time (figure 6d).Under the normal physiological condition, guts undergo peristalsis. We found that WT ex vivo guts. Both epithelial cells (cytokeratin 8 positive) and intestinal transcription factor (CDX2 positive) progressively moved from gut lumen to surface in WT ex vivo gut (a). However, this movement was absent in AMPK KO gut where both types of cells remained inside gut lumen (a), indicating that AMPKα1 deficiency unequivocally impeded the formation and overall arrangement of epithelial layers, which might be due to the downregulated mRNA expression of epithelial transcription factors (b).We further tested the effects of AMPK on epithelial cells of vivo gut a. Howeveut lumen a, indica factors b.Figurea, E-cadherin and β-catenin in WT ex vivo guts gradually moved towards the exterior layer, and completely covered the surface layer over time; this phenomenon was absent in AMPK KO ex vivo guts . Since barrier function is also related to intestinal proliferation, we further tested Ki-67 (b). Ki67-positive cells were reduced due to AMPKα1 deficiency, which was consistent with the downregulated expression of Ctnnb1 and Lgr5 (figure 7b). Additionally, the apoptosis significantly induced after AMPK deficiency (c). Pro-apoptotic markers BAX and Casp3 were upregulated in AMPK KO gut, while anti-apoptotic marker Bcl-2 was downregulated (d). These data collectively demonstrated the necessity of AMPKα1 in proper gut health, and established ex vivo guts as a proper model for examining gut epithelial differentiation and formation.To further determine the role of AMPK in barrier function, E-cadherin and β-catenin, the markers for intestinal paracellular junction, were examined. Consistent with what we observed in figure 7ivo guts a,b. Sinced Ki-67 b. Ki67-pficiency c. Pro-apegulated d. These 4.in vivo model is costly, time-consuming and often lacks specific niche for toxicological, pharmacological and nutraceutical studies. The recently developed organoid model from mouse intestinal stem cells [Epithelial cell lines and mice are commonly used models for studying epithelial health. An em cells  or from em cells  allows tem cells .ex vivo gut culture system established in this study allows epithelium developing in a native gut niche without costly growth factors or other promoters. All steps of gut epithelial differentiation and migration can be closely monitored, which is difficult to do with live animals. Furthermore, inhibitors, activators, toxicants or carcinogens can be applied to ex vivo gut to study their biological effects on gut differentiation. This model was successfully applied to one nutraceutical study to determine the effects of purple potato extract on intestinal differentiation [ex vivo gut culture is that gut sections are vulnerable to waggle and fall off from the coverslip during the first three days of culturing. Thus, careful handling is needed when moving culture dishes. Also, the defined crypt-villus structure could not be found in ex vivo guts. Thus, intestinal morphogenesis may not be applicable to this model.The ntiation . The shoex vivo gut culture model. During murine intestinal development, the major differentiated cells, such as absorptive enterocytes, goblet cells and enteroendocrine cells, can be detected [ex vivo guts has been detected. The difference between ex vivo model and in vivo is that ex vivo guts rearrange during culture, with epithelial cells distributing on the surface layer and mesenchymal cells concentrated under the epithelium as scaffolds.During early gut development, the intestinal epithelial structure including crypts and villi starts to form around E14, a critical time window for intestinal development . Thus, tdetected , while Pdetected . CDX2, Edetected . Alcian detected . In thismT/mGex vivo gut was used to trace intestinal stem cells and their derived cells. Lgr5-derived intestinal cells ‘climb’ onto the surface of ex vivo gut, showing the migration of intestinal cells. On the other hand, intestinal stem cells give rise to enterocytes, goblet cells, enteroendocrine cells and Paneth cells, which were also discovered in ex vivo guts at 7 days post-incubation. Of note, Paneth cells, contributing to the niche of intestinal stem cells, normally start to differentiate from P14 in mice [Lgr5 in mice . Paneth  in mice .ex vivo guts, the mesenchymal layers are clearly visible, which located below the epithelial layer; smooth muscle cells, fibroblasts and myofibroblasts were further detected. Importantly, the ex vivo gut demonstrates spontaneous peristalsis, very similar to the gut movement in vivo. Interstitial cells of Cajal (ICC), one type of interstitial cell in the gut, contribute to gut peristalsis, and the absence of ICC is related to motility disorders [ex vivo guts, indicating that peristalsis in ex vivo gut is not due to ICC functionality. Likewise, botulinum toxin could even increase muscular activity in a spinal cord–muscle coculture system [ex vivo gut has mesenchymal layers which can be useful for studying epithelial motility.The mesenchymal layers under the epithelial layer are the sources of growth factors and cytokines regulating epithelial differentiation ,42. Therisorders . Howevere system . In shorα1 in embryonic gut epithelial development. We observed an astonishing difference between WT and AMPKα1 KO ex vivo gut. Our results are supported by recent reports showing the positive roles of AMPK in gut epithelial barrier function [α1 in proper peristalsis and overall health of fetal gut. AMPK specific deficiency in intestinal epithelial cells promoted proliferation of crypt cells in adult mice [ex vivo gut suppressed the proliferation and induced apoptosis, possibly due to the subsequent apoptosis induced by inadequate or lagging differentiation. Consistently, AMPK mutants in Drosophila cause abnormal epithelial integrity, defective mitotic cell division and even embryonic lethality [α1 KO ex vivo gut is partially associated with the downregulation of intestinal transcription factors, such as CDX2 [To demonstrate the effectiveness of the model, we evaluated the role of AMPKfunction ,45, as wfunction ,46–48. Afunction , while tult mice , while Aethality . The def as CDX2 . However as CDX2 .ex vivo gut culture system not only maintains the physiological structure of the gut but can effectively track intestinal epithelial differentiation and migration. Using the established ex vivo gut model, we further demonstrated a critical regulatory role of AMPKα1 in epithelial differentiation. This model has potential applications in examining impacts of nutrients, microbial metabolites, toxicants and antigenic materials on gut epithelial development, as well as exploring mechanisms regulating differentiation and migration.In summary,


Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as  APC and CTNNB1 (the gene encoding β-catenin) mutations in the Wnt/β-catenin pathway and RAS, RAF, and upstream epidermal growth factor receptor (EGFR) mutations in the RAS-ERK pathway have been known to play important roles in the tumorigenesis. In human colorectal cancer (CRC), series of gene mutations are involved in the tumorigenesis, and the accumulation of genetic and epigenetic alterations are involved in the tumorigenesis.2 The tumor suppressor APC functions as a “gatekeeper” and its gene mutations occur at the initiation stage of the colorectal tumorigenesis, and KRAS mutations occur at the early adenoma stage and play roles in the progression of the tumorigenesis.2 The APC and KRAS mutations frequently occurs simultaneously; however, interaction of these two gene mutations in the tumorigenesis is poorly understood. Over the past decade, significant progress has been made in understanding the mechanism of cross-talk between the Wnt/β-catenin and RAS-ERK pathways in cancer. APC and KRas or CTNNB1 and HRas mutations resulted in the cooperatively progressed tumorigenesis in mouse genetic studies.5 In addition, the CRC patients with both APC and KRAS mutations reveled much severe cancer phenotypes compared with their single mutation.2 Furthermore, both APC and KRAS mutations resulted in the activation of cancer stem cells (CSCs) even progression to metastasis in the mouse xenograft model.6 Overall, the Wnt/β-catenin and RAS-ERK pathways interact in the various stages of the colorectal tumorigenesis and combined mutations of these two pathway genes are critical in the malignant transformation of CRC. In this review, we are going to discuss the current understanding for the roles of the cross-talk between the Wnt/β-catenin and RAS-ERK pathways in tumorigenesis especially for CRC, and also will discuss the mechanical base for the interaction between the two pathways highlighted by the stability regulation of RAS via the Wnt/β-catenin pathway. Finally, we will discuss our promising small molecular approach controlling CRC where both β-catenin and RAS especially mutant KRAS levels were increased by their stabilization by APC loss in human CRC.Cancer is a complex disease involving multiple mutations of genes including those involved in the many different signaling pathways. However, the relationships between mutations of the genes involving different pathways are poorly understood. The Wnt/β-catenin and RAS-ERK pathways are major pathways involving human cancers, and abnormalities in these two pathways genes are well characterized, such as 7 Moreover, Wnt/β-catenin signaling has crucial roles in the daily processes of tissue maintenance and regeneration in the hair, skin and intestine, and so on.8 The Wnt/β-catenin pathway is initiated by Wnt family ligands, secreted lipoglycoproteins that act to the frizzled (FZD) family receptors.9 The key signaling molecule β-catenin determining the activation status of this pathway subjected to the stability regulation at the cytosol. In the resting status without Wnt stimuli, the cytoplasmic β-catenin form the ‘destruction complex’ composed with the casein kinase 1α (CK1α), glycogen synthase kinase 3β (GSK3β), APC, and Axin, and its level is maintained as low by the series of events including priming phosphorylation by CK1α at Ser45 and subsequently at Thr41, Ser37, and Ser33 by GSK3β,10 and recruitment of the β-TrCP E3 linker followed by degradation through the polyubiquitination-mediated proteasomal degradation.11 When the secreted Wnt ligands are accumulated, those interact to the FZD receptor together with the lipoprotein receptor-related protein (LRP)-5/6 co-receptors result in activation of dishevelled (DVL) protein  or lymphoid enhancer factor (LEF), and activate expression of the target genes involving various pathophysiologies including the genes involving proliferation and transformation, such as c-MYC, c-Jun, CCND1 (the gene encoding cyclin D1), EGFR, CD44, CD133, and leucine-rich repeat-containing receptor 5 (LGR5) and so on , such as binding of epidermal growth factor (EGF) to the EGFR result in its dimerization and following autophosphorylation at the tyrosine amino acid residues provide docking sites for several SH2 domain-containing adaptor proteins, such as growth factor receptor bound protein 2 (Grb2), Shc, phospholipase C gamma (PLCγ), p110 catalytic subunit of PI3 kinase -144 and Thr-148 by GSK3β, and subsequently the β-transducin repeat-containing protein (β-TrCP) E3 linker is recruited to the phosphorylated Ras and degraded by polyubiqutination-dependent proteasomal degradation  were selected. As a secondary screening, the compounds reduced both β-catenin and Ras levels were further screened by immunoblotting. Subsequently, potential toxic compounds were excluded from the candidates by using highly fragile neural stem cells for further characterization  of 2.1 and 0.5 µM, respectively, destabilized both β-catenin and RAS, and were further characterized their functions.43 KY1220 and KY1022 inhibited both ERK and Akt pathways, and suppressed proliferation and transformation of CRC cells. Especially, these compounds effectively suppressed growth and transformation of various CRC cell lines which harboring either wild-type or mutant KRAS via degradation of both β-catenin and RAS via the enhancement of their polyubiquitination.43 KY1022 inhibited EMT, motility, invasion with induction of apoptosis of CRC cells harboring both APC and KRAS mutations.43 In addition, the small intestinal tumors of ApcMin/+/KRasG12DLA2 mice were also suppressed by KY1220 and KY1022.43To identify compounds destabilize both β-catenin and RAS via targeting the Wnt/β-catenin pathway, we initially screened a small molecule library by using HEK293 cells harboring the pTOPflash reporter which sensitively response to the β-catenin/TCF transcription factor in their chromosome.ion Fig. . ThroughAPC and KRAS mutations.41 All the compounds improved their functions has NO2 moiety at the phenyl ring , not with the inactive control biotinylated compound (LJE-H-274) following confirmation of the specific interaction of LJE-H-225 with recombinant Axin  for KY1797K-Axin binding as 2.9 × 10−7 M. The regulator of G-protein signaling (RGS) domain of Axin (RGS-Axin) was characterized as a site of KYA1797K interaction by analyzing the RGS domain deleted Axin mutant followed by abolishment of its RAS degrading ability. We further predicted several amino acid residues involving KYA1797K by chemical shift perturbations using NMR spectra and confirmed lysinen-147 (K147) as a most important residue in the function by KYA1797K.41 This K147 in the RGS domain of Axin is essential for KYA1797K binding, and it directly forms hydrogen bond with the NO2 at the phenyl ring  mediated membranous localization and alternative approaches, such as synthetic lethality and small interfering RNA mediated reduction of Ras, no effective pharmacologic inhibitors of the Ras have reached the clinic, prompting the widely held perception that RAS proteins are “undruggable”.53 As one of the continuous efforts to develop small molecules control oncogenic RAS, ARS-853  inhibitors, such as IWR-1 and WAV939 are known to suppress Wnt/β-catenin signaling pathway through Axin stabilization via inhibition of TNKS-mediated poly-ADP-ribosylation (PARsylation)  Fig. .58,59 TNnce Fig. .41 Diffeins Fig. . KYA1797APC and KRAS mutations in CRC. Pathological significance of these two mutations was evidenced by correlative increment of both β-catenin and RAS in tumor tissues of CRC patients harboring an APC mutation in most cases. On the base of these results together with identification of the detailed mechanism of common stability regulations of both β-catenin and RAS lead us to develop small molecules degrading both β-catenin and RAS protein. KYA1797K, a representative such compounds illustrated its mode of action binding the RGS domain of Axin for GSK3β activation, suppressed the growth of various CRCs, including those resistant to the EGFR targeting therapies by KRAS mutations.The genetics of multistep colorectal carcinogenesis has been extensively studied over the past decades, and frequent alterations of Wnt/β-catenin and RAS/ERK pathway genes in different cancers especially human CRC has been recognized. In addition, cooperative interactions between mutations of the Wnt/β-catenin and Ras-ERK pathway genes have been shown by various genetic studies mostly using the mouse system. The most frequent mutations in the two pathway genes involving their interaction in the tumorigenesis is the APC or KRAS mutations but also by level increments of both β-catenin and RAS. However, considering the small molecular approach degrading β-catenin and RAS is not effective in cancer cells with non-degradable β-catenin mutation,41 for the best outcome, the strategy should be carefully applied based on the individual’s genetics following precision oncology concept.61Overall, the idea for the simultaneous destabilization of β-catenin and RAS via targeting Wnt/β-catenin pathway may serve as an effective anti-cancer strategy for CRCs and other cancer types attributed not only by


Phases of proliferation and differentiation were defined. Markers of squamous metaplasia and epithelial ciliation were followed while enhancing β‐catenin signaling by blocking glycogen synthase kinase 3β with SB216763 and shRNA as well as inhibiting canonical WNT signaling with apical application of Dickkopf 1 (Dkk1). Our findings indicate that enhanced β‐catenin signaling decreases the number of ciliated cells and causes squamous changes in the epithelium, whereas treatment with DDk1 leads to an increased number of ciliated cells.Wnt signaling is essential for the differentiation of airway epithelial cells during development. Here, we examined the role of Wnt signaling during redifferentiation of ciliated airway epithelial cells  ALI, air liquid interfaceBrdU, 5‐bromo‐2′‐deoxyuridineDkk1, Dickkopf 1FOXJ1, forkhead box 1Fz, frizzledGSK3β, glycogen synthase kinase 3βKD, knock downLRP5/6, low‐density lipoprotein receptor‐related proteins 5 or 6MCC, mucociliary clearanceNHBE, normal human bronchial epitheliumPCP, planar cell polarityPFA, paraformaldehydeTCF, T cell factorSimilar to skin, the airway epithelial surface is directly exposed to the environment. The airway surface can be injured by inhalation of dust, cigarette smoke, and infectious agents. Chronic exposure to irritants causes squamous metaplasia, goblet and basal cell hyperplasia, and atrophy of the epithelium Mucociliary clearance (MCC) is a key mechanism for protecting the airways from inhaled irritants The genesis of cilia is a complex process. It involves apical organization of an actin network Xenopuset al. Wnt signaling is important for embryonic development Dickkopf‐1 (Dkk1) was identified as an inhibitor of canonical Wnt signaling Here, we examined the effect of Wnt signal modulation by Dkk1 and inhibition of GSK3β on the differentiation of ciliated airway epithelial cells in human air liquid interface (ALI) cultures.Human recombinant Dkk1 protein was purchased from R&D Systems Inc. . All other chemicals were acquired from Sigma‐Aldrich , if not specified otherwise.Human airways were obtained from organ donors whose lungs were rejected for transplant. Consent for research was obtained by the Life Alliance Organ Recovery Agency of the University of Miami with local IRB‐approved written consents conforming to the standards set by the Declaration of Helsinki. From these lungs, airway epithelial cells were isolated, expanded on collagen I‐coated dishes and differentiated at an ALI on collagen IV‐coated T‐clear filters with 0.4 μm pores  as previously described Normal human airway epithelial cells grown on Transwell filters at the ALI were fixed with 4% paraformaldehyde (PFA) m sodium citrate buffer (pH 6.0). After blocking for 1 h with gelatin 1% (wt/vol) in PBS, sections were incubated overnight at 4 °C with rabbit anti‐Dkk1 antibody or nonimmune rabbit IgG as a control  before staining with Alexa 555 anti‐rabbit IgG (1 mg·mL−1). Cilia were labeled with mouse antiacetylated α‐tubulin antibodies (1 : 1000) and stained with Alexa 647 anti‐mouse IgG (1 mg·mL−1). Nuclei were visualized with DAPI and slides were mounted with Gel/Mount .Tracheal rings from three human lungs from nonsmokers were fixed in 4% PFA, embedded in paraffin and sectioned. Sections were processed as previously described m BrdU in DPBS for 12 h before fixation with 4% PFA, treatment with 1M and 2M HCl for 10 min each at 37 °C and staining with anti‐BrdU antibodies  using a fluorescent secondary Alexa antibody (Molecular Probes). Proliferation index was determined as the ratio of nuclei stained with anti‐BrdU and total nuclei stained with DAPI.To quantify proliferation, cells were labeled with 100 μm Tris, pH 8.5, and 0.1 mm EDTA in the presence of protease inhibitors and cleared from debris by centrifugation. Ten microgram of protein was separated using SDS/PAGE gels  and electro‐blotted onto Immobilon P membranes . The membranes were blocked with 5% nonfat dry milk in PBS and incubated with the primary antibody for 2 h at room temperature, washed with PBS and 0.05% Tween and incubated with a secondary horseradish peroxidase‐labeled antibody . Signal was detected and quantified by chemiluminescence  on a ChemiDoc XRS system (Bio‐Rad). Mouse anti‐β‐actin  was used for loading control. The signals were quantified using image lab software (Bio‐Rad).Total cell protein was prepared from ALI cultured cells by lysis with 1% SDS in 10 mCt method or as a fraction of GAPDH expression as 2−ΔCt × 1000.Total RNA was isolated from ALI cultures using RNeasy mini kit . RNA was quantified utilizing a NanoDrop 1000 . cDNA was made from total RNA using the iScript Kit (Bio‐Rad). mRNAs of interest were quantified by real‐time quantitative RT‐PCR using a Bio‐Rad iCycler and TaqMan gene expression assays (Life Technologies). The following assays were used: GAPDH (4352934E), Cyclin D1 (Hs00765553_m1), β‐catenin (Hs00355049_m1), FOXJ1 (Hs00230964_m1), Wnt4 (Hs00229142_m1), Wnt7a (Hs01114990_m1), GSK3β (Hs01047719_m1), involucrin (Hs00846307_m1), and E‐cadherin (Hs01023894_m1). Changes in expression were calculated using the ΔΔLentiviral shRNA targeting GSK3β (RHS4531) and a nontargeting control vector (RHS4349) were bought from Open Biosystems . Virus was used as described prism 5  was used for statistical calculations. P < 0.05 was accepted as significant.Results were compared by one‐way ANOVA and, if a significant difference was found, a parametric or nonparametric analysis for comparison of individual groups was done, for which significance was reported in the figure legends. For some of the measures, t‐tests were used to compare groups as indicated in the figure legends. n = 50 visual fields 40× for each time point from two different lungs), averages of 2.9 ± 0.2, 3.8 ± 0.4, 4.1 ± 0.4, and 2.5 ± 0.4 mitoses per field were found on days 4, 5, 7, and 13, respectively, but none on days 16, 19, or 25 . Proliferation was also characterized by BrdU incorporation  peaked with 66–76% BrdU positive nuclei compared to DAPI stained per visual field on days 2, 3, and 4. Days 13, 16, and 20 had a significantly lower BrdU staining with 1%, 0.5%, and 0.1%, suggesting there is little cell proliferation at this time. To gauge the time course of differentiation, the ciliated cell population was examined by staining for cilia‐enriched acetylated α‐tubulin at the apical surface and counting ciliated cells per visual field. Ciliated cells were detected on day 16 and remained present until day 25 with 27.9 ± 3 vs 105.4 ± 11.5 vs 76.5 ± 9.4 for days 16, 20, and 25, significantly increased compared to the previous days .To study the differentiation of human airway epithelial cells, undifferentiated normal human bronchial epithelium (NHBE) cells were seeded onto Transwell filters (defined as day 0) and proliferated for 4–5 days submerged before media were removed from the upper chamber to initiate the ALI. We evaluated proliferation using two methods. First, mitotic activity was assessed by counting nuclei with mitotic spindles, visualized by immunofluorescent staining for acetylated α‐tubulin. Counting the number of mitotic spindles (These data suggest that NHBE cells go through two different phases during differentiation: a proliferative first phase with an increase in cell number during the first 2 weeks of culture and a second phase with ciliated cell differentiation with little cell proliferation after 16 days in culture.http://www.ncbi.nlm.nih.gov/gds/?term=GDS2615) n = 3 cell cultures from three different lungs; Fig. n = 3 from three different lungs, Fig. As there are 19 different Wnt genes, microarray gene expression data during mucociliary differentiation were examined , using two different concentrations (n = 45 from three different lungs). Addition of 0.5 μm FH535 decreased proliferation by ~ 32% (47.8% vs 32.6% BrdU positive cells) and addition of 1.5 μm FH535 decreased it by ~ 69% (47.8% vs 14.8% BrdU positive cells). To determine the effect of FH535 on ciliated cell differentiation, the level of FOXJ1 mRNA was measured on days 4, 12, and 24. Blocking β‐catenin with 1.5 μm FH535 resulted in this experiment in cell death between day 4 and 12. Cells treated with 0.5 μm FH535 had a lower level of FOXJ1 mRNA expression on day 12 , but a similar level on day 24 . These results suggest that the β‐catenin pathway is linked to and modulates proliferation of NHBE cells.To further investigate the role of β‐catenin signaling during NHBE proliferation and differentiation, cells were treated throughout differentiation (day 1–24) with FH535, a nuclear Wnt/β‐catenin and PPARγ signaling inhibitor m FH535 had an undistinguishable FOXJ1 mRNA level and ciliation compared to untreated controls . Cells treated with 1.5 μm FH535 differentiated into ciliated cells as well, but had a decreased FOXJ1 expression on 18 days  and 24 days  compared to the control  on day 16 compared to day 4 (3.91 ± 0.26 vs 0.92 ± 0.31 fold of day 1). Expression on day 22 was also significantly (P < 0.05) increased compared to day 10 .Based on the observed differential expression patterns of Wnt7a and Wnt4 during differentiation, we looked at expression of Dkk1 known to inhibit Wnt/β‐catenin signaling in the lung. Immunofluorescent staining of differentiated ALI cultures and human tracheal sections , an inhibitor of GSK3β that stimulates β‐catenin‐associated Wnt signaling −1), a canonical Wnt signaling inhibitor 2 Transwell filters to minimize the effect of submersion, which has been associated with impaired ciliogenesis n = 4 from two different lungs; P < 0.05). These results suggest that canonical Wnt signaling inhibits FOXJ1 expression and must be suppressed to permit ciliated cell differentiation.After establishing the patterns of proliferation and differentiation during airway epithelial cell differentiation at the ALI as well as associating β‐catenin signaling with the proliferative phase and Dkk1 with the differentiation phase, we asked whether a switch in Wnt signaling is necessary for ciliary differentiation. To address this question, NHBE cells were treated both apically and basolaterally for the entire 24 days of differentiation with media alone or media supplemented with SB216763  between SB 216763‐treated cells and other groups  compared to Dkk1‐treated cells (0.91 ± 0.2 fold change) and control cells . In addition, FOXJ1 mRNA expression was significantly decreased (0.18 ± 0.06 fold change) in GSK3β shRNA transduced cells vs NS cultures .To confirm that the results with SB216763 were related to inhibition of GSK3β and not another kinase, GSK3β knock down (KD) was performed with lentiviral‐mediated shRNA expression. qRT‐PCR of GSK3β mRNA confirmed KD in cells expressing GSK3β shRNA (0.22 ± 0.06 fold change) compared to cells expressing nontargeting shRNA .Since manipulating canonical Wnt signaling affected ciliated cell differentiation, we examined the effect of β‐catenin modulation by Dkk1 and SB216763 on the squamous metaplasia marker involucrin and the epithelial marker E‐cadherin. Immunofluorescent staining after 24 days showed increased involucrin expression when stimulating the β‐catenin pathway with SB216763. On the other hand, Dkk1 decreased involucrin expression . On the other hand, SB216763 decreased the protein expression of E‐cadherin on day 24 (0.6 ± 0.82 fold) compared to control conditions (1 ± 0.09 fold) with Dkk1 of . Effects of SB216763 and Dkk1 on the structure of the airway epithelium are demonstrated in Figure S1.Opposite effects were observed with the expression of the epithelial marker E‐cadherin Fig. . QuantitIn conclusion, these experiments suggest that the fate of airway epithelial cell differentiation depends on appropriate Wnt signaling. Stimulation of the canonical Wnt pathway with SB216763 drives the airway epithelium into a squamous cell phenotype as seen by increased involucrin and decreased E‐cadherin expression.Our data suggest an important role for Wnt signaling during differentiation of the airway epithelium to a ciliated cell phenotype. We found that continuous enhancement of β‐catenin signaling by inhibition of GSK3β inhibits ciliogenesis and promotes squamous metaplasia, whereas decreasing β‐catenin pathway signaling with Dkk1 enhances differentiation of the ciliated cell population. Blocking signaling with FH535 showed that β‐catenin is essential during the proliferation but not during the differentiation phase. These data suggest the need for a switch from β‐catenin‐dependent to β‐catenin‐independent Wnt signaling during airway epithelial cell differentiation Fig. .Wnt signaling is an important regulator of proliferation and differentiation processes Exploring the influence of Wnt signaling during differentiation, we found an early phase with increased expression of canonical Wnt signaling element expressions, including Cyclin D1 followed by a second phase characterized by decreased expression of canonical Wnt signaling elements but a dramatic increase of the ciliated cell fate marker FOXJ1 Fig. A. These To further evaluate the importance of β‐catenin for early fate determination as previously described One could argue that the FH535 effect is based on cell toxicity. This is unlikely, as the addition of the same concentration of FH535 restricted to the differentiation phase (Fig. FH535 is known to affect PPARγ signaling by preventing the recruitment of β‐catenin to PPAR‐γ and thus inhibiting β‐catenin/PPAR‐γ interaction The expression pattern of Wnt7a and Wnt4 Fig. A showed Wnt4 is classically described as a noncanonical signaling molecule via activation of mitogen‐activated protein kinase 8 Inhibition of GSK3β with SB216763 blocks intracellular β‐catenin degradation and stimulates canonical signaling, independent of Wnt signals. Therefore, Dkk1 cannot inhibit canonical signaling in the presence of SB216763. The fact that development of cilia does not occur under these circumstances underlines the importance of the inhibition of the β‐catenin signal for ciliated cell development. This also implies that activation of the noncanonical pathway by Dkk1, independent of its blocking effect on the canonical Wnt pathway, cannot rescue the ciliated cell population.Wnt signaling has been shown to be important for the regulation of stem cell proliferation Stimulation of canonical Wnt signaling by overexpressing β‐catenin leads to squamous metaplasia in a mouse model In conclusion, our data suggest a biphasic Wnt signaling pattern during airway epithelial repair with a switch of signaling from an initial β‐catenin‐dependent to a β‐catenin‐independent pattern before differentiation of ciliated cells occurs. Permanent stimulation of the β‐catenin signaling via GSK3β inhibition leads to squamous metaplasia without differentiation of ciliated cells whereas suppression of canonical signals with Dkk1 enhances the ciliated cell population, supporting the importance of a switch in Wnt signaling.AS: Wrote the grant and initiated the project. Planning, executing and analysis of experiments. Making figures and writing drafts of manuscript. JS: Planning, executing and analysis of experiments. Writing and review manuscript. LN: Executing and analysis of experiments. NB: Executing experiments. Making figure and review manuscript. NF: Mentoring project and review manuscript. MS: Mentoring project and review manuscript.Fig. S1. Confocal imaging of the squamous pattern of cultures treated with SB216763.Click here for additional data file.