Furthermore, we found that over-expression of Cyr61 in HepG2 cells promoted the progression of HCC xenografts in SCID mice. These findings indicate that Cyr61 is a direct target of β-catenin signaling in HCC and may play an important role in the progression of HCC.Abnormal activation of the canonical Wnt signaling pathway has been implicated in carcinogenesis. Transcription of Wnt target genes is regulated by nuclear β-catenin, whose over-expression is observed in Hepatocellular Carcinoma (HCC) tissue. Cyr61, a member of the CCN complex family of multifunctional proteins, is also found over-expressed in many types of tumor and plays dramatically different roles in tumorigenesis. In this study, we investigated the relationship between Cyr61 and β-catenin in HCC. We found that while Cyr61 protein was not expressed at a detectable level in the liver tissue of healthy individuals, its expression level was elevated in the HCC and HCC adjacent tissues and was markedly increased in cancer-adjacent hepatic cirrhosis tissue. Over-expression of Cyr61 was positively correlated with increased levels of β-catenin in human HCC samples. Activation of β-catenin signaling elevated the mRNA level of Cyr61 in HepG2 cells, while inhibition of β-catenin signaling reduced both mRNA and protein levels of Cyr61. We identified two TCF4-binding elements in the promoter region of human Cyr61 gene and demonstrated that β-catenin/TCF4 complex specifically bound to the Cyr61 promoter  Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third deadliest cancer worldwide. Nearly 78% of the global HCC were reported in Asian countries Multiple factors and signaling pathways have been shown to contribute to the tumorigenesis of HCC Cysteine-rich protein 61 (Cyr61/CCN1), a member of the CCN growth factor family, is a secreted, integrin-binding protein that regulates multiple cellular activities such as cell adhesion, migration, proliferation, survival and apoptosis in vitroin vivo. Our finding strongly suggests that Cyr61 is regulated by β-catenin in HCC and has multiple functions in the genesis and the progression of HCC.There is evidence that expression of some members of CCN family, namely CTGF/CCN2 and WISP-1/CCN4, are regulated by Wnt/β-catenin signaling pathway We previously reported that elevated Cyr61 mRNA levels were found in the HCC tissue compared to those found in the normal tissue by RT-PCR analysis. Moreover, we observed that the expression of Cyr61 was closely associated with some clinical manifestations, including the number of cancer nodules, degree of tumor differentiation, and venous infiltration. However, the expression did not correlate with some other clinical observations, such as the size of tumor, metastasis, cancer embolus and AFP concentration P<0.05), and was mainly located in cytoplasm (P<0.01). This phenomenon was also observed within the same tumor where stronger staining was found in more differentiated tumor cells (P<0.01).There are reports by other laboratories indicating that expression of Cyr61 is down regulated or is not significantly different in HCC tissue compared with that in the normal tissue ytoplasm : A. In tor cells : A-5. FuP<0.01)  .The correlation of expression between β-catenin and Cyr61 in HCC samples leads us to postulate that β-catenin may regulate Cyr61 expression. HCC cell line HepG2 was used to determine whether the expression of Cyr61 could be influenced by β-catenin. We infected HepG2 cells with adenovirus expressing β-catenin, and assessed the Cry61 mRNA levels at various time points post infection. An increased expression of β-catenin was observed at 48, 72 and 96 hrs after adenovirus Adβ-catenin infection, which was not seen in cells infected with a control virus, AdGFP . AssessmTo further demonstrate that Cyr61 expression is directly related to the levels of β-catenin, we studied the effect of reduced expression of β-catenin on Cyr61 expression. HepG2 cells were infected with adenovirus expressing siRNA for β-catenin (Adsiβ-catenin) or non-specific siRNA (AdSES-hus), expression levels of β-catenin as well as Cyr61 were assessed by RT-PCR and western blot. We found that while both the mRNA and protein levels of β-catenin were reduced by introducing β-catenin siRNA via adenovirus, they were not affected by the non-specific siRNA . In cellWe then studied whether β-catenin is directly responsible for transcriptional activation of Cry61. β-catenin activates transcription of its target genes through forming a protein complex with TCF, which binds to the promoter region of the target genes. We utilized a dominant negative form of TCF4(dnTCF4) that fails to interact with β-catenin and results in decreased transactivation activity for β-catenin/TCF4 complex and reduced target gene expression. HepG2 cells were co-infected with Luciferase reporter construct AdTOP-Luc and adenovirus expressing dnTCF4 (AddnTCF4) or control virus expressing GFP (AdGFP). A significant down-regulation in TOP-Luc reporter activity was apparent after cells were infected with AddnTCF4, but not the control AdGFP, indicating that the transactivation activity of β-catenin is inhibited in the presence of dnTCF4 .We assessed the expression levels of Cyr61 in these cells by western blot. In cells infected with AddnTCF4, where the transactivation activity of β-catenin was inhibited, the Cyr61 protein level was also reduced. This was not seen in cells infected with control AdGFP where the β-catenin activity was not affected . These fIt has been shown that a consensus TCF4 binding element (TBE) is presented in the target genes of β-catenin/TCF4 complex ChIP assay was employed to determine whether β-catenin/TCF4 directly bind to the promoter region of Cyr61 in HepG2 cells. Agarose gel analysis of PCR products showed that anti-β-catenin antibody effectively immunoprecipitated the Cyr61 promoter containing the two TBEs , lane B.To determine if over-expression of β-catenin affected Cyr61 promoter activity directly, the fragment extending from −750 bp to −500 bp upstream of the transcriptional start site of human Cyr61 promoter was cloned into pGL3 to generate a luciferase reporter construct (pGL3-TBE). As shown in Furthermore, to determine the role of TBE motifs in regulating Cyr61 gene transcription, site-directed mutation of one or double TBE sites was generated in the context of pGL3 luciferase reporter plasmid. Cyr61 promoter activity, which was increased by β-catenin in 293 cells, was abrogated when one or both of the TBE sites were mutated . Cyr61 pin vivo with an animal model. HepG2 cells were infected with AdRFP or AdCyr61 at the same infection ratio for 36 hrs before subcutaneous implantation. Tumor sizes were measured every 3 days after implantation. As seen in in vivo than that of the HepG2 cells infected with AdRFP. Over-expression of Cyr61 increased the growth rate by 22% compared with the RFP control group (P = 0.02). Specifically, the doubling times for the tumor mass of Cyr61 over-expressed cells and RFP control cells were 2.53±0.16 days and 3.47±0.27 days respectively in the HepG2 xenografts in SCID mice.Previously, we found that over-expressing the exogenous Cyr61 in HepG2 cells by recombinant adenovirus vector leads to increased proliferation and migration ability in HepG2 cells H&E staining of the xenograft tumor tissue revealed that there were hyperplasia in fibrous connective tissue, infiltration of inflammatory cells, and multinuclear tumor cells . To explGiven the importance of Wnt/β-catenin pathway in normal embryonic and adult liver development, it is not surprising to see that its activity is perturbed in HCC. About 50%–70% of HCC are found to have an increased level of β-catenin in the cytoplasm or nucleus, which is believed to provide growth advantage for tumor cells. Many of the target genes of Wnt/β-catenin signaling pathway are involved in promoting cell proliferation.In the present study, we showed that Cyr61 is over-expressed in HCC, and that it is one of the target genes for Wnt/β-catenin pathway. We also demonstrated that increased expression of Cyr61 promoted the growth of HepG2 cell xenografts in SCID mice.The roles of the Cyr61 in cancer development are complex. A higher level of Cyr61 is found in breast cancer and it is shown to induce estrogen-independence and to promote invasiveness of breast cancer In our study, it is worth noticing that Cyr61 is expressed markedly higher in cancer-adjacent hepatic cirrhosis tissue, which was recognized as precancerous lesions, than in the tumor tissue itself. These results suggest that the abnormal expression of Cyr61 may be closely related to the development of HCC and hepatic cirrhosis, and Cyr61 may be involved in the progression of hepatic cirrhosis to HCC. It is also noted that the expression of Cyr61 is reduced in poorly differentiated HCC. Similar findings are seen in colorectal cancer The exact role of Cyr61 in human cancers remains largely undefined. Both inhibitory and promoting effects of Cyr61 on cancers have been reported. We demonstrated in this study that Cyr61 protein expression was increased in HCC, which is consistent with our previous work and that of the others in vitro but also in vivo. Animal experiments are in general considered as a better assessment for tumor development than the in vitro experiments. Overall, we believe that our conclusion is supported by our in vitro and in vivo findings.Feng et al showed that Cyr61 suppressed proliferation of HCC cell line, and negatively regulated cell motility and invasiveness. The author concluded that Cyr61 acts as a tumor suppressor in HCC γβ3, α6β1, αγβ5 and αIIβ3, which leads to a variety of biological activities, including cell adhesion, migration, and invasion Cyr61 is known to interact with various integrins, including αin vitro and the progression of HCC xenografts in SCID mice. Based on our results, it is reasonable to hypothesize that early interference with Cyr61 signaling pathway may prevent the transition from hepatic dysplasia to HCC. Future studies about the complex role and mechanism of Cyr61 in tumorigenesis and progression of HCC are needed.In summary, our study indicates that Cyr61 is a target gene of β-catenin signaling in HCC. Over-expression of Cyr61 is induced by abnormal expression of β-catenin and promotes the growth and migration of HCC cell line The experimental protocols were approved by the Ethical Committee of Third Military Medical University (TMMU). Verbal consent was obtained from all patients and guardians prior to the procedure. Written consent, which contains many personal information, may impact the lives of patients. So, we think that written consent was not necessary. This decision was approved by the Ethical Committee of Third Military Medical University.All animal protocols were approved by the Ethical Committee of Third Military Medical University .All patients samples were collected from the First Affiliated Hospital of TMMU. Complete clinical data and pathological grade were also available for all patients. Among the collected samples, 35 cases  of HCC, with patients aging from 28 to 73 years old (an average of 49.8), were paraffin-embedded tissues. 30 cases  of hepatic cirrhosis of paraffin-embedded cancer-adjacent tissues, aging from 39 to 67 years old (an average of 50) were collected from the Department of Pathology. All these specimens were subjected to histological examination and immunohistochemistry studies. 27 cases of surgical resection HCC and HCC adjacent specimens , with age ranging from 26–68 years old (an average of 50), were acquired from the Department of Hepatobiliary Surgery. Five cases of normal liver samples were obtained from deceased liver donors. Liver tissue samples were fixed in 4% formalin for histological examination and immunohistochemistry, and were used to justify for final diagnoses. None of the patients had received preoperative chemotherapy or radiation therapy.The cell lines HepG2 and HEK293 were obtained from Shanghai Cell Bank (Shanghai). HepG2 cells were cultured in RPMI1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin and 10 µg/ml streptomycin at 37°C in a humidified 5% carbon dioxide atmosphere. HEK293 cell line was cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin and 10 µg/ml streptomycin at 37°C in a humidified 5% carbon dioxide atmosphere.Analysis of β-catenin and Cyr61(Abcam) expression was performed on the paraffin section of human liver tissues. The staining procedure was conducted as decribed previously HepG2 cells were seeded in six-well plates. At 80% confluence, cells were infected with adenovirus expressing various genes including Adβ-catenin or AdGFP, Adsiβ-catenin or AdSES-hus (negative control siRNA), AddnTCF4 or AdGFP, AdCyr61 or AdRFP, respectively. After infection, cells were harvested at 48 h, 72 h and 96 h for subsequent analysis.5′-CGGGGTACCATGCCACTGTGGGTATTAATTTG-3′, anti-sense: 5′-TCCCCCGGGTGA AAGAGTAGAACGGGGAAGA-3′. The PCR product was cloned into pGL3 using KpnI and SmaI. Point mutations in the putative TCF-binding site(TBE) were generated from the pGL3-TBE luciferase reporter plasmid according to the manufacturer's recommendations . 293 cells were seeded in twenty-four-well plates and cultured up to 80% confluence, at which point they were infected with Adβ-catenin, AdGFP, or AddnTCF4, and at the same time, transfected with a luciferase reporter construct  using Lipofect2000. HepG2 cells were seeded in twenty-four-well plates and cultured up to 80% confluence, at which point they were transfected with a luciferase reporter construct  using Lipofect2000. Renilla luciferase reporter was used as internal control. Thirty hours after infection, luciferase assay was performed using the Dual Luciferase Assay System Kit (Promega). All experiments were performed in triplicate. Results are shown in means ± S.D.To determine if over-expression of β-catenin affected Cyr61 promoter activity directly, we construct a luciferase reporter pGL3-TBE. The fragment extending from −750 bp to −600 bp upstream of the transcriptional start site of human Cyr61 promoter, which contains two TBEs, was generated by PCR amplification with the following primers: sense: Luciferase reporter construct TOP-Luc containing three copies of the Tcf/Lef sites upstream of a thymidine kinase (TK) promoter and the firefly luciferase gene(AdTOP-Luc) was used to examined the effect of the dominant negative TCF4 (dnTCF4) on β-catenin/TCF4 transactivation activity. HepG2 cells were seeded in 24-well plates. At 80% confluence, cells were infected with AddnTCF4 or AdGFP. Twenty-four hours after infection, cells were infected with AdTOP-Luc. Thirty hours after infection, luciferase assays were performed, using Luciferase Assay System kit, in accordance with the manufacturer's protocols (Promega). All experiments were performed in triplicate. Results are shown in means ± S.D.20) with a Superscript RT-PCR kit (TOYOBO Japan). The generated cDNA was amplified using gene-specific primers: β-catenin 5′-AAAGCGGCTGTTAGTCACTGG-3′ and 5′-GACTTGGGAGGTATCCACATCC-3′; Cyr61 5′-ACTTCATGGTCCCAGTGCTC-3′ and 5′-AAATCCGGGTTTCTTTCACA-3′; GAPDH 5′-CATCATCTTCCAGGAGCG-3′ and 5′-TGACCTTGCCCACAGCCTTG-3′.cDNA was synthesized using oligo (dTCell lysates were prepared with cell lysis buffer containing a protease inhibitor cocktail (Roche). 50 µg of total protein for each sample was loaded onto 8% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with antibody against β-catenin or Cyr61 at a dilution of 1∶1000 or 1∶2000 respectively. After the blots were incubated with horseradish peroxidase-conjugated secondary antibody, immune-reactive signals were detected using ECL kit (Millipore America).5′-ATGCCACTGTGGGTATTAATTTG-3′ and 5′-TGAATGAAAGAGTAGAACGGGGAAG A-3′.Chromatin immunoprecipitation (ChIP) assay was carried out as described previously 6 cells per mouse. Tumor size of each mouse was measured every three days regularly, and the tumor volume was estimated with the formula a×b2×0.5, where a and b represent the maximal and minimal diameters respectively. Mice were euthanized on day 21, and tumor masses were retrieved for histological analysis and immunohistochemical staining of Ki67. Growth curves were plotted using tumor volume for each experimental group at the set points.After infected with AdCyr61 or AdRFP for 36 h, HepG2 cells were subcutaneously injected into the flank of 4-week-old SCID mice , at 5.0×10P<0.05 or P<0.01. The average absorption of the immunohistochemistry staining for the matched patient samples was compared using the K Pearson test at a statistical level of P<0.01. SPSS software version 13.0 was used to analyze these data.Data is expressed as mean ± standard deviation (S.D.). Statistical analysis of the data was performed with the Student paired t test, the 2-tailed Student t test at a statistical level of


Wnt/β-catenin signalling is widely implicated in embryogenesis, tissue homeostasis and tumorigenesis. The key event in Wnt signalling activation is β-catenin accumulation, which is controlled by both its production and degradation. However, much more emphasis has been placed on the understanding of its degradation. Here, we show that the synthesis of β-catenin protein, which requires a group of serine/arginine-rich splicing factors (SRSF), also contributes to its tumorigenic activity. Overexpression of SRSF1 and SRSF9 promote β-catenin accumulation via the recruitment of β-catenin mRNA and by enhancing its translation in an mTOR-dependent manner. We further demonstrate that, like SRSF1, SRSF9 is also an oncogene, and is frequently overexpressed in multiple types of human tumours. Finally, our results suggest that promoting degradation and blocking production of β-catenin synergistically reduce β-catenin levels under pathological conditions and that a combinational therapy could be a promising approach for the treatment of cancer patients. CTNNB1) itself also leads to uncontrolled protein accumulation  are essentially involved in almost every step of the process . Since SRSF9 is closely related to SRSF1, which is studied extensively, we focused our characterization on these two proteins in the following study. Using Wnt-responsive reporter assay, we found that human SRSF1 and SRSF9 were also able to enhance Wnt1- as well as β-catenin-induced reporter expression, whereas SRSF2 could not . Co-transfection of SRSF1 or SRSF9 with this ‘real’ human β-catenin cDNA also produced much more β-catenin protein . Finally, to test whether SRSF1 and SRSF9 are able to elevate endogenous β-catenin levels, we measured β-catenin protein in the cytosolic and nuclear fractions of transfected HEK293T cells. As shown in Since β-catenin accumulation is the key event in Wnt signalling activation, we measured β-catenin protein level after SRSF1 or SRSF9 co-expression. As indicated in The canonical human SR protein family contains 12 members , and found a significant increase (p < 0.05) of SRSF9 (SRp30c) expression in tumour tissues over the expression in corresponding normal tissues in 156 of 360 studies. Fisher's meta analysis indicated that the observed increase in those paired studies was extremely significant . These results demonstrate that SRSF9 is broadly upregulated in tumour samples.Because over-accumulated β-catenin is tumorigenic, and SRSF1 has been suggested as a proto-oncogene . We then applied siRNAs against SRSF1 and SRSF9 to HCT116 cells and measured their proliferation. As shown in Reducing β-catenin level is considered as a very promising approach to target cancerous cell proliferation and thus cancer therapy. Luckily, several small molecules were recently identified to promote β-catenin degradation via diverse mechanisms, which provided candidate leading compounds for drug development Polakis, . FurtherOur results demonstrated that SRSF1 and SRSF9 are able to promote β-catenin protein synthesis. We further showed that these SR proteins are generally involved in Wnt- or upstream mutation-induced β-catenin accumulation. β-catenin therefore is proposed as a target of SR proteins in tumorigenesis upon their overexpression. Regarding β-catenin over-accumulation in cancer cells, our study emphasizes that the impairment of degradation and enhancement of synthesis make simultaneous contributions and therefore, approaches targeting both paths should be considered during therapy.in vitro and to form tumours in nude mice; (iii) down-regulation of SRSF9 in colon cancer cell lines reduced their colony formation ability. With the exception of the above-mentioned SR proteins, the other members of this protein family have not been studied regarding their role in tumorigenesis. Our results suggested that β-catenin could be a target of some SR proteins, and their ability to cause β-catenin accumulation might be an indication of the tumorigenic activity. This is consistent with the correlation that all known oncogenic SR proteins  are able to promote β-catenin accumulation, and all non-tumorigenic ones (SRSF2 and SRSF6) cannot. These results promote us to speculate that there are additional canonical SR proteins that are potentially oncogenic. It would be interesting in the future to systemically verify, among all canonical SR proteins, their expression in tumour samples as well as their tumorigenic activities, assuming specific antibodies are available.Among the 12 canonical SR splicing factors, SRSF1 and SRSF3 have been suggested as proto-oncogenes , SW480 cell line was cultured in L15 medium and HCT116 cell line was cultured in Mccoy's 5A medium. All culture medium were supplemented with 10% foetal bovine serum (FBS), penicillin and streptomycin.Human SRSF1 and SRSF9 plasmids were kindly given by Prof. Benoit Chabot  and Prof. Javier F. Caceres . Other human SR plasmids of proteins were bought from Origene . The expression constructs were generated using PCR and subcloned into pCS2+ vectors with a N-terminal FLAG tag. SRSF1 and SRSF9 were also subcloned into pEF6/Myc-His vector, which were used for establishing stable NIH3T3 cell lines. Using the QuikChange strategy (Stratagene), we generated the point mutations of SRSF1 and SRSF9. All plasmid constructs were verified by sequencing. siRNA synthesis was performed by GenePharma  and the siRNA sequence for human SRSF1 and SRSF9 were as follows, human SRSF1-1: GGAAAGAAGATATGACCTA, SRSF1-2: GAAGTTGGCAGGATTTAAATT; human SRSF9-1: GCTGATGTGCAGAAGGATG, SRSF9-2: GGAATATGCCCTGCGTAAA. Transfection of plasmid and siRNA were performed as described previously  following the instruction of the manufactory. FLAG (Sigma F3165) and SRSF1 (Santa Cruz sc-73026) antibodies were used in RIP assays.Wnt/β-catenin signalling plays significant roles in embryogenesis, tissue homeostasis and tumorigenesis. β-Catenin degradation has been the centre of attention in targeting Wnt signalling-mediated tumorigenesis. However, β-catenin synthesis is undeservedly neglected, which also contributes to β-catenin accumulation in cancer cells. The mechanisms mediating the control of β-catenin synthesis remain largely unknown, an issue we addressed by a family of important RNA binding proteins, SR proteins.In our study, we show that SRSF1 and SRSF9 promote β-catenin accumulation via enhancing β-catenin mRNA translation. Similar with SRSF1, SRSF9 also has oncogenic activity, which at least partially due to promoting β-catenin accumulation. Correspondingly, depletion of either SR proteins reduced colon cancer cell proliferation. Moreover, considering the importance of β-catenin's production and degradation, we demonstrated in principle that promoting degradation and blocking production of β-catenin synergize in reducing cancerous β-catenin level.Our study establishes a novel link between the Wnt/β-catenin signalling and the SR proteins and indicates that some of the SR proteins contribute to β-catenin accumulation. Our results suggest that β-catenin is a major translational target of a subset of SR proteins, mediating their tumorigenic activity. Furthermore, our work demonstrates the importance of β-catenin synthesis during tumour progression and emphasizes that promoting degradation and blockage production of β-catenin protein should be equally considered during therapeutic treatment of Wnt signalling-mediated tumour.Sucrose gradient sedimentation was carried out as previously described  was used to determine the number of viable cells in each well. Triplicates were included for each sample. The absorbance at 492 nm was measured using a 96-well plate reader.4 NIH3T3 cells were suspended in 0.3% low melting point agarose and then laid onto a bottom layer containing 1% agarose in six-well plates with triplicate. Colonies were stained and counted 4 weeks later. For SW620 cells, 500 cells were suspended in 0.3% top agarose and laid on top of the 0.6% bottom agarose in six-well plates with triplicate. Colonies were stained and counted 2 weeks later.NIH3T3 cells were transfected with pEF6/Myc-His-SRSF1 or -SRSF9, selected with blasticidin (10 µg/ml) and a mixture of SRSF1 or SRSF9-over-expressing NIH3T3 cell lines were obtained. 1 × 10Immunohistochemistry was performed with human colon carcinoma  tissue array (BC05118a) and multiple organ tissue array (MC5003a) by Cybrdi, Inc. . Anti-SRSF1 (Invitrogen 325600), SRSF9 (Santa Cruz sc-134036) and β-catenin (BD Biosciences 610154) antibodies were used at 1:50, 1:10 and 1:200 dilutions, respectively. A semi-quantitative analysis in stained sections was performed by an independent pathologist.5 NIH3T3 cells stably expressing pEF6-vectors or stably expressing SR proteins, with eight mice in each group. Tumour size was measured with vernier caliper at Day 7, 13, 16, 19 after implantation, and tumour volume was calculated with the following formula: volume = (width)2 × length/2. Tumour weight was recorded when the mice were received euthanasia at Day 19.BALB/c nude mice were injected subcutaneously with 2.5 × 10Xenopus clones from Xenopus cDNA library; YF and WW designed and supervised the study; YF and WW wrote the manuscript with contributions from BH.YF and BH performed most of the experiments with partial contributions from ZS and JHa; ZS, JHa, JHu and YW verified the initial


Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1), a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2) and IκB kinase β (IKKβ), which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI), the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of  Ubiquitination is one of the most important posttranslation modifications in eukaryotes . Though Kelch-like ECH-associated protein-1(Keap1), a Bric-a-Brac (BTB) protein, is a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex which is in charge of recognizing ubiquitination substrates through protein-protein interaction (PPI). There are four characteristic domains in Keap1 : the BroBesides Nrf2, the DC domain of Keap1 has been reported to bind other proteins, including DJ-1, sequestosome 1, p62, IκB kinase β (IKKβ), Hsp90, p65, Bcl-2/Bcl-xL -21. AmonNrf2 and NF-κB is the core of redox control of gene transcription . Redox sXenopus laevis IKKβ [Considering the core role of IKKβ and Nrf2 in NF-κB signaling and Nrf2-ARE pathway, the selective recognition mechanism of Keap1 with IKKβ or Nrf2 is vital to the crosstalk between NF-κB and Nrf2 signaling. However, compared to Keap1-Nrf2 system, the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In this paper, the structure of human IKKβ modeled from the crystal structure of vis IKKβ  was firsAll structures obtained from protein data bank (PDB) were corrected using clean protein tool in Discovery Studio (DS) 3.0 package . All calculations were conducted using Dawning TC2600 cluster. Except for otherwise mentioned, other parameters were set as default.Homo sapiens IKKβ against the PDB sequences was analyzed by the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/blast/). The Xenopus laevis IKKβ (PDB code: 3QA8) which was identified as the most homologous sequence from PDB . The sequence similarity of 36TGE 39N) were chosen as the residues including in the binding site [The crystal structure of Keap1 DC domain was downloaded from PDB (PDB code: 2FLU with high resolution of 1.50 Å). ZDOCK , a rigiding site . Top 200RDOCK, an algorithm for refinement of docked complexes using the CHARMm-based minimization, was used to optimize and re-rank the docking poses to pick out near-native structures . The docThe preparation of all molecular files and MD simulations were conducted using the GROMACS package, version 4.5.3. Unless otherwise noted, the all-atom OPLS force field were assigned to all molecules and the simple point charge (SPC) water model was applied to solvate the molecules. Counterions were added to the solvent to keep the system neutral. All bond lengths were constrained using the LINCS method , allowinUpon completion of the two equilibration phases, the well-equilibrated system at the desired temperature and pressure was used to run the production MD without position restraints. Other parameters were the same as the NPT ensemble in the equilibration phase. The coordinates were saved every 10 ps. The Keap1 DC domain was simulated for 100 ns and the Keap1-IKKβ complex system was simulated for 50 ns. The trjconv tool within the GROMACS was used to strip out coordinates and correct for periodicity. The production MD without position restraints of Keap1 DC domain and Keap1-IKKβ were repeated for three times. The Gromos method  within GThe Calculate Mutation Energy (Binding) protocol in DS 3.0 was used to evaluate the effect of single-point mutations on the binding affinity of molecular partners in the protein-protein complex. The energetic effect of each mutation on the binding affinity is calculated as the difference between the binding free energy in the mutated structures and the wild type protein. All interaction energy terms are calculated by CHARMm using a Generalized Born implicit solvent model and contain empirically scaled contributions of van der Waals and electrostatic interactions and a non-polar solvation energy term. The input structure of Keap1-IKKβ complex was obtained from the cluster analysis of the MD simulation trajectory. In order to compare the difference of key residues, the crystal structure of Keap1-Nrf2 (PDB code: 2FLU) was used as the input structure to conduct the virtual alanine mutation. In order to avoid the unreasonable clash, the preliminary minimization was carried out for both cases. The residues within 3.5Å around ETGE motif in Keap1 were selected for calculation. The Energy Terms Scaling Factors were set as default.Xenopus laevis. Through the IKKβ of Xenopus laevis is highly homologous to Homo sapiens’  on the binding residues was performed to evaluate the energetic effect of each residue on the binding affinity. The mean values of Virtual Alanine Mutation of Keap1-IKKβ complex obtained from triplicate MD simulations with standard deviation were shown in Taken together, the MD simulations and Virtual Alanine Mutation Scanning combine to indicate the different substrate recognition mechanism of Keap1. In the case of Nrf2-Keap1 system, three arginines of Keap1 play an important role in recognizing the Nrf2 through interacting with the key glutamic acids. Although the arginine-glutamic acid interactions also exist in the IKKβ-Keap1 system, the tyrosine, especially Tyr525, participated interactions dominate the recognition of IKKβ. These differences may be utilized by the organism to fine tune the Keap1-Cul3 E3 ligase complex to modulate Keap1-Nrf2-ARE and IKKβ-Nf-κB pathway simultaneously.Degradation by ubiquitination is a common strategy taken by the key modulators responsible for stress responses ,54,55. TIKKβ and Nrf2 are the primary substrates of Keap1. IKKβ can activate the transcription factor NF-κB that is directly involved in inflammation and tumor progression. Furthermore, IKKβ has been shown to promote tumorigenicity through phosphorylation-mediated inhibition of tumor suppressors and thus is considered as an oncogenic kinase ,57,58. WHerein, depending on the recognition mechanism of Keap1 and IKKβ, we postulate a new regulatory mechanism between Keap1-Nrf2-ARE and IKKβ-NF-κB pathway mediated by Keap1. The results described above reveal that Nrf2 and IKKβ are recognized by Keap1 through similar but not identical mechanism, indicating the competitive binding of IKKβ and Nrf2. In basal conditions, overexpression of IKKβ may result in the derepression of Nrf2 via competive binding Keap1. It is consistent with published result that p65 and most of its upstream molecules repress ARE-Luc reporter and NQO1-Luc reporter activity with varying degrees, while IKKβ is an exception . In induActivation of ARE system by negatively controlling the Keap1 protein holds a great promise for the development of novel class of antiinflammatory and anticancer agents . HoweverIn summary, the intermolecular recognition mechanism of Keap1 and IKKβ was investigated through protein-protein docking, MD simulations and virtual alanine mutation. Key tyrosines were firstly found to be important for the PPI of Keap1-IKKβ. The presented PPI model of IKKβ-Keap1 with structural information will aid in the rational design of small molecular PPI inhibitors to manipulate the transactivation activity of Nrf2 for therapeutic treatments of stress-related diseases. The crosstalk between Keap1-Nrf2-ARE and IKKβ-Nf-κB pathways mediated by Keap1 proposed in this paper also provides valuable insights into the regulatory mechanisms of cellular redox homeostasis.File S1Homo sapiens IKKβ and Xenopus laevis IKKβ (PDB code: 3QA8).Sequence alignment of (DOCX)Click here for additional data file.File S2Homo sapiens IKKβ.Profile-3D analysis of The total Verify Score is 209.87 and verify scores of most residues are greater than 0. Even these residues bearing verify score less than 0 are far away from the binding site.(DOCX)Click here for additional data file.File S3Total energy and potential energy during the MD simulations of Keap1-IKKβ.As shown in figure, total energy and potential energy of systems are constant during the MD simulation.(XLSX)Click here for additional data file.File S4Detailed information of clustering analysis.(DOCX)Click here for additional data file.File S5The mdp files used in the MD simulations.(ZIP)Click here for additional data file.File S6The related structures, including homology model of human IKKβ, the protein-protein results and structures used for alanine scanning calculations obtained from the triplicate MD simulations, used in the paper.(ZIP)Click here for additional data file.


Wnt/β-catenin signaling is a branch of a functional network that dates back to the first metazoans and it is involved in a broadrange of biological systems including stem cells, embryonic development and adult organs. Deregulation of components involved inWnt/β-catenin signaling has been implicated in a wide spectrum of diseases including a number of cancers and degenerative diseases.The key mediator of Wnt signaling, β-catenin, serves several cellular functions. It functions in a dynamic mode at multiple cellular locations,including the plasma membrane, where β-catenin contributes to the stabilization of intercellular adhesive complexes, the cytoplasmwhere β-catenin levels are regulated and the nucleus where β-catenin is involved in transcriptional regulation and chromatin interactions.Central effectors of β-catenin levels are a family of cysteine-rich secreted glycoproteins, known as Wnt morphogens. Through theLRP5/6-Frizzled receptor complex, Wnts regulate the location and activity of the destruction complex and consequently intracellular β-catenin levels. However, β-catenin levels and their effects on transcriptional programs are also influenced by multiple other factors includinghypoxia, inflammation, hepatocyte growth factor-mediated signaling, and the cell adhesion molecule E-cadherin. The broad implicationsof Wnt/β-catenin signaling in development, in the adult body and in disease render the pathway a prime target for pharmacologicalresearch and development. The intricate regulation of β-catenin at its various locations provides alternative points for therapeuticinterventions. Wnt/β-catenin signaling is a branch of an extensive functional network that developed around a class of proteins - called armadillo proteins - that dates back to the first anaerobic metazoans. Wnt/β-catenin signaling is involved in a broad range of biological systems, including stem cells biology, developmental biology, and adult organ systems. Drosophila, Wingless, was shown to be required for proper wing formation ..DrosophiThe key mediator of Wnt signaling, the armadillo protein β-catenin, is found in a dynamic mode at multiple subcellular localizations, including junctions where it contributes to stabilize cell-cell contacts, the cytoplasm where β-catenin levels are tightly controlled by protein stability regulating processes and the nucleus, where β-catenin is involved in transcriptional regulation and chromatin interactions. Central extracellular regulators of β-catenin levels are the Wnt morphogens. However, multiple other processes, including hepatocyte growth factor, prostaglandines, PKA (Protein Kinase A), E-cadherin, and hypoxia, can also influence β-catenin levels.β-catenin itself is a specialized member of the larger armadillo protein family that consists of three subfamilies: the p120 subfamily, the beta subfamily (β-catenin and plakoglobin) and the more distant alpha subfamily. The functional interplay between members of this protein family is not well understood, but an involvement of p120 and plakoglobin in Wnt/β-catenin signaling has been shown.1).The regulation of the presence and stability of β-catenin and functionally convergent armadillo proteins – in particular p120 - at the various cellular localizations as well as their shuffling within the cell provides alternative intervention points for therapeutic reagents. The broad implications of Wnt/β-catenin signaling in development, the adult body and in disease renders it a prime target for pharmacological research and development. A short overview map for canonical Wnt signaling is presented on Fig.  signaling. The levels of β-catenin at different subcellular localizations are regulated by a variety of processes including site-specific phosphorylation of β-catenin. In particular, the control of the turnover of cytoplasmic β-catenin by the destruction complex and the control of the destruction complex by the Wnt signalosome have been studied extensively. Other important mechanisms regulating subcellular β-catenin thresholds are those controlling its mobilization from adherens junctions and its translocation to the nucleus. One of the central end points of the Wnt/β-catenin signaling pathway is the regulation of transcription through the binding of β-catenin to members of the Tcf-1/lymphoid enhancer factors  family of transcription factors in the nucleus -14. The structure of β-catenin can be divided into three domains: the N-terminal domain, the armadillo domain consisting of 12 armadillo repeats, and the C-terminal domain . ThroughIn this review we will first describe the alterations of β-catenin in the destruction complex and the proteins that are involved in this process. We will then focus on the Wnt signalosome that recruits components of the destruction complex thus inactivating it. Subsequently, we will summarize how the signalosome is removed from the cell surface by endocytosis. Next we will describe the cellular pool of β-catenin at cell junctions and the mobilization of β-catenin from this pool. Then we will focus on the implications of β-catenin in transcription control. Finally, we will summarize some of the pathways that influence Wnt/β-catenin signaling. Several proteins in the Wnt/β-catenin pathway that are implicated in other cellular processes will be briefly described.In the absence of an active Wnt signalosome, cytoplasmic β-catenin associates with the destruction complex. The main known structural components of the destruction complex are APC and Axin. To this structural core, the casein kinases CK1α, δ, and ε (to be referred to collectively as CK1) and GSK3 (glycogen synthase kinase 3) are recruited .In the degradation complex the processing of β-catenin is considered as a phosphorylation-dependent flux along the Axin scaffold protein which is regulated by a stepwise series of phosphorylations triggered by the kinases CK1 and GSK3 , 20. A cIn diverse human cancers Axin mutations are associated with increased levels of β-catenin -30. MutaIn the destruction complex, Axin serves as a coordinating scaffold for the kinases GSK3 and CK1, for the structural protein APC and Dishevelled (Dvl/Dsh) as well as for β-catenin . Early mThere are two Axin proteins in humans - Axin1 , and Axin2 . Each of the Axin genes encodes two isoforms, a and b, which differ by splicing variants . Axin anDrosophila it was shown that the α-subunit of Go physically binds to Axin and recruits it to the plasma membrane . . 388]. IPhosphorylation of E-cadherin by CK1δ at Ser846 also reduces its binding to β-catenin . InteresBesides its implications in junctions, the main effector function of β-catenin is in the nucleus. Here it regulates transcription through interactions with a number of transcription factors, including predominantly Tcf/Lef, Hif-1 and possibly also Oct4. β-catenin may also have a more unspecific role on transcription regulation through interactions with chromatin. The shuttling of cytoplasmic β-catenin to the nucleus and back to the cytoplasm is not entirely understood. A picture emerges, where the nuclear uptake of β-catenin can be enhanced by the context-dependent C-terminal phosphorylation of β-catenin at S675 by PKA. Evidence suggests that the export of β-catenin from the nucleus to the cytoplasm can be GSK3-dependent . Both kihttp://www.stanford.edu/~rnusse/wntwindow.html). All members of the Tcf/Lef family  contain an N-terminal binding domain for β-catenin, followed by a context-dependent regulatory domain (CRD) with binding sites for the co-repressor Groucho (Gro), a HMG-box DNA-binding domain, and a C-terminal domain with binding sites for the co-repressor C-terminal binding protein (CtBP) [In the nucleus, the interaction between β-catenin and the zinc finger transcriptional factors of the Tcf/Lef family has been described and is seen as the classical regulatory unit for Wnt/β-catenin target genes (n (CtBP) , 397. Len (CtBP) . In micen (CtBP) , 398. Fun (CtBP) .In the absence of β-catenin, members of the Tcf/Lef family form a complex with co-repressors such as Groucho, CtBP, and HDAC leading to a repression of the transcription complex , 400-402Xenopus [Xenopus does not affect embryonic axis formation [Also, the armadillo protein plakoglobin is able to associate with the Tcf/Lef transcriptional complex, although with less affinity than β-catenin, and early reports claim that both proteins are able to activate Tcf/Lef reporters -407. PlaXenopus . Furtherormation . p120 also affects Wnt/β-catenin-mediated transcription. In the absence of phosphorylated p120, the zinc finger transcription factor Kaiso binds to the HMG domain of Tcf, forming a co-repressor complex together with histone deacetylase (HDAC). Phosphorylation of p120 causes its dissociation from E-cadherin, its entrance to the nucleus and binding to Kaiso. In consequence, Kaiso loses its role as a co-repressor . Kaiso bThe Tcf/Lef transcriptional complex has a multitude of further binding partners . ProteinTcf/Lef-mediated transcription is target of numerous regulative covalent modifications like phosphorylation, SUMOylation, ubiquitination, and acetylation , 396. ItThe Nemo-like kinase (NLK) phosphorylates amino acids Thr155 and Ser166 of Lef-1 and amino acids Thr178 and Thr189 of Tcf-4 which have been shown to lead to a reduced DNA-binding , 429. ThThe E3 ligase PIASy SUMOylates Lef-1, which could lead - context dependent - to either an activation or to an inhibition of Lef-1 , 433. 50  that targets Hif-1α for rapid degradation in the proteasome . StrikinIn a low oxygen environment – as frequently found in stem cell niches – Hif-1α forms a complex with ARNT (the constitutive active form of Hif-1α) and enters the nucleus where it competes with Tcf/Lef proteins for β-catenin binding . Hence, s which competes with APC for binding to the RGS domain of Axin [Both COX activity and Wnt/β-catenin signaling are interconnected and have been associated with tumorigenesis. An inhibition of the cyclooxygenases COX-1 and COX-2 leads to reduction of prostaglandin synthesis including the pro-inflammantory prostaglandin E2 (PGE2) . PGE2 ac of Axin . Hence,  of Axin . Finally of Axin .Recently a connection between prostaglandin H2 and Wnt/β-catenin signaling was shown on the transcriptional level. Hence, the gene encoding 15-prostaglandin dehydrogenase, which dehydrogenates prostaglandin H2, was shown to be repressed by β-catenin . FurtherA number of anti-inflammatory COX-2 inhibitors were reported to affect Wnt signaling . In thisThe TGFβ signaling pathway belonging to the same protein superfamily as bone morphogenic proteins (BMP) and Nodal, is involved in multiple biological processes including proliferation, apoptosis and cancerogenesis -464. TGFSmad7 is not the only representative of the Smad family that affects β-catenin. Smad4 in complex with its receptor R-Smad interacts with β-catenin in the nucleus. In chondrocytes it was shown that the C-terminal domain of Smad3 interacts with the N-terminal and central domains of β-catenin in a TGF-β-dependent manner .Finally, the TGFβ pathway was shown to induce phosphorylation of β-catenin at Tyr654 through an activation of Src kinase(s), influencing both the presence in junctional complexes, and the nuclear localization of β-catenin . Platelet-derived growth factor (PDGF) regulates cellular division and participates in angiogenesis. PDGF treatment leads to a phosphorylation of the p68 helicase, which facilitates the nuclear translocation of β-catenin and its interaction with the Tcf/Lef complex . InteresBoth Notch and Wnt/β-catenin signaling are interconnected. Recently it was shown that membrane-associated uncleaved Notch directly interacts with β-catenin, serving as a protein trap and down-regulating the cellular levels of β-catenin. This process has been demonstrated to require the endocytic adaptor protein Numb and lysosomal activity . In turnAlthough Wnt/β-catenin is a major signaling pathway with very significant implications in a broad range of diseases, addressing the pathway through small drugs or therapeutic antibodies is still at its infancy. Despite the description of a multitude of interesting bio-targets in the pathway, together with the identification of reagents that interfere with these bio-targets, it is by no means clear which bio-targets in the pathway may give a lead in drug discovery.Creating specific agents to any bio-target is a challenge. In addition, several of the known bio-targets in the Wnt/β-catenin pathway are also implied in a multitude of other pathways raising specificity issues. Since the Wnt/β-catenin signaling pathway is highly complex, a number of back-up mechanisms as well as feedback loops exist. In this context it is also necessary to bear in mind that β-catenin is a member of a larger protein family – the armadillo protein family - and other members of that family, including p120 and plakoglobin, may have functions that are supportive to, or overlapping with β-catenin. Although it is not settled where the best druggable bottlenecks in the pathway may be, or if such bottlenecks exist at all, three main interference points in the pathway are at current predominantly explored: (i) the Wnt signalosome, (ii) the destruction complex and (iii) β-catenin targets and interactions in the nucleus. In a broader sense, other manipulations including altering cellular junctions, influencing prostaglandine-mediated signaling, affecting HGF signaling and mechanisms that influence Hif-1α levels are important in the context of addressing Wnt/β-catenin signaling. 1), a PARP/Tankyrase blocker [Hence, it is not clear to what extent the pathway could be silenced by a single therapeutic agent and whether it may ultimately be necessary to regulate the pathway at two or multiple points simultaneously in an approach that may be termed “cloud inhibition”. Indeed, a combination of an EGFR and Tankyrase inhibition recently revealed a close functional correlation of both pathways and confirmed the synergistic effect of a dual antagonistic treatment in lung cancer cells . Ziboten blocker . However, even if Wnt/β-catenin signaling can be reduced more stringently with combinations of inhibitors, it is important to note that a complete inhibition of Wnt/β-catenin signaling may not be desirable as it may be necessary to maintain a basic Wnt/β-catenin activity in an organism to ensure the viability of natural Wnt dependent cells.Despite of all the described issues, significant excitement is sensed in the field that developing reagents altering Wnt/β-catenin signaling can provide valuable new therapeutic tools which will allow to address disease conditions that have hitherto escaped therapeutic success.


Exendin-4 (Ex-4) is a Glucagon-like peptide 1 (GLP-1) receptor agonist approved for the treatment of Type 2 Diabetes (T2DM), which requires daily subcutaneous administration. In T2DM patients, GLP-1 administration is reported to reduce glycaemia and HbA1c in association with a modest, but significant weight loss. The aim of present study was to characterize the site-specific profile and metabolic effects of Ex-4 levels expressed from salivary glands (SG) in vivo, following adeno-associated virus-mediated (AAV) gene therapy in two different animal models of obesity prone to impaired glucose tolerance and T2DM, specifically, Zucker fa/fa rats and high fed diet (HFD) mice. Following percutaneous injection of AAV5 into the salivary glands, biologically active Ex-4 was detected in the blood of both animal models and expression persisted in salivary gland ductal cell until the end of the study. In treated mice, Ex-4 levels averaged 138.9±42.3 pmol/L on week 6 and in treated rats, mean circulating Ex-4 levels were 238.2±72 pmol/L on week 4 and continued to increase through week 8. Expression of Ex-4 resulted in a significant decreased weight gain in both mice and rats, significant improvement in glycemic control and/or insulin sensitivity as well as visceral adipose tissue adipokine profile. In conclusion, these results suggest that sustained site-specific expression of Ex-4 following AAV5-mediated gene therapy is feasible and may be useful in the treatment of obesity as well as trigger improved metabolic profile. Glucagon-like peptide 1 (GLP-1), a gastrointestinal hormone mainly produced in a nutrient-dependent manner Gene therapy offers the possibility of a long-term expression in the treatment of many chronic diseases, including T2DM Recently, Adeno-associated Viruses (AAVs) have advanced to the forefront of gene therapy, due to their ability to achieve long-term transgene expression in vivo and low immunogenicity SG are recognized as a useful depot organ in gene therapy, having several important features of other endocrine glands, such as high protein production and ability to secrete proteins into the bloodstream In the present study we have characterized metabolic effects and site-specific secretion profile of sustained Ex-4 expression from the SG of both a polygenic and monogenic animal models of obesity prone to impaired glucose tolerance and T2DM. Ex-4 expression resulted in a significant decrease in weight gain in both models, improved glycemic control and/or insulin sensitivity and visceral adipose tissue adipokine profile, thus suggesting long-term benefit following sustained expression.et alRecombinant AAV particles were produced using a four-plasmid procedure as previously described in Di Pasquale G 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U of penicillin/ml, and 0.1 mg of streptomycin/ml. 293T cells were transfected with the AAV5 Ex-4 vector at 103 DNase Resistant Particles (DRP)/ml per cell. Ex-4 levels into the culture medium was assayed using a specific enzyme immunoassay kit .The generated recombinant AAV5 virus encoding the Ex-4 minigene was then tested in vitro. 293T human renal epithelial cells were grown at 37°C under a 5% COin vitro bioactivity assay, supernatant medium from 293T cells transduced with AAV5 Ex-4 (after 96 hours of incubation) was tested on Chinese hamster ovary cell line obtained from American Type Culture Collection  stably transfected with rat GLP-1 receptor (CHO-GLP1R) accordingly to previously reported study from Egan JM et alFor Animal studies were carried out in strict accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for experimental animal care. The study protocol was approved by the Italian National Health Institute Committee on Animal experiments. All surgeries were performed under anesthesia, and all efforts were made to minimize suffering.ad libitum . The HFD supplied 60% of energy as fat and 20% as carbohydrate. Fatty acid composition was as follows: 42.0% saturated fatty acids , 43% monounsaturated fatty acids (oleic acid), and 15% polyunsaturated fatty acid (linoleic acid and linolenic acid). The HFD contained 300 mg cholesterol/kg and its energy density was 21.10 kJ/g. The HFD fed mice are recognized as an efficient and robust animal model for obesity, early prone to impaired glucose tolerance and T2DM development Male 4 week old CD1 mice (n = 20) were purchased from Harlan Laboratories , housed at five animals per group and fed High Fat Diet ad libitum (Purina Rodents Laboratory Diet). Zucker fa/fa rats are conversely a spontaneous monogenic obesity model, characterized by a missense mutation on the leptin receptor gene Male Zucker fa/fa rats (n = 10) purchased from Charles River laboratories  were housed in single cage and received standard chow 12 DRP/ml) vector. Control animals  received 50 µl of AAV-5 vector devoid of Ex-4 transgene (empty vector). On a weekly basis weight, food, water intake, urine volume, glycaemia were monitored throughout the study. In order to evaluate effects of treatment on short term food consumption, a 120 minutes food intake evaluation, after overnight fasting, was also performed in rats. A fixed amount of standard chow was given in individual cages and rodents food intake  was measured every 15 minutes.Submandibulary salivary glands of 8 week old HFD mice (n = 10) and of 9 week old Zucker fa/fa rats (n = 5) were transduced by single percutaneous injection of 50 µl of AAV5 Ex-4 (5×100–120 min  =  (G0+ G15 ) x 15/2+(G15+ G30 ) x 15/2+(G30+ G60 ) x 30/2+(G60+ G120 ) x 60/2.An intraperitoneal insulin tolerance test (ITT) was performed in HFD mice, 6 weeks following vector administration. Each animal was fasted for 4 hours. Following intraperitoneal insulin  injection (1 UI/kg), blood samples from the lateral tail vein were collected to measure glycaemia at 0, 15, 30, 60, 90 and 120 minutes. The glucose areas under the curve  were calculated according to formula: AUC2 (80%) inhalation.Blood samples were withdrawn at baseline and week 6 in HFD mice in order to detect Ex-4, glycaemia, insulinemia, HbA1c, leptin and adiponectin circulating levels and at 0, 4, and 8 week in Zucker fa/fa rats to detect Ex-4 and glucose values. HbA1c and insulinemia were determined at baseline and 8 weeks after vector administration. Blood samples were obtained through jugular sampling conducted in isoflurane-anesthetized animals. At study end, rats and mice were respectively euthanized by COSG, liver, spleen, and pancreas tissues were collected for DNA extraction and vector distribution assay.Ex-4 exhibits 53% structural homology to native GLP-1. Circulating Ex-4 levels were determined by a specific enzyme immunoassay kit , unable to detect endogenous GLP-1 values, according to the manufacturers’ instructions. Minimum detectable concentration was 2.6 pmol/L.5′-TGGAAATCCCCGTGAGTCA-3′) for CMV promoter. Amplification and detection were performed with ABI Prism 7700 Sequence Detection System . A PCR cycling reaction involved an initial hold at 95° for 10 minutes, followed by cycling conditions of 95°C for 15 seconds, 60°C for 1 min for 40 cycles. The viral DNA in each sample was quantified by comparing the fluorescence amplification profiles with a set of DNA standards using AAV5 vector and 100 ng of genomic DNA of untreated animals for each specific tissue. Each measurement was carried out in duplicate. Data are expressed in copies of AAV5 for 100 ng of genomic DNA.In order to assess vector biodistribution at the end point of the study, a DNA isolation kit was used to purify total genomic DNA from SG, liver, spleen and pancreas . Quantitative PCR amplification  of genomic DNA (100 ng) was performed with ABI PRISM 7700 Sequence Detection System  by using the SYBR Green PCR Master Mix and a specific 5′ and 3′ primer pair appropriate  and control (n = 5) HFD mice, fixed in 4% paraformaldehyde for 24 hours at room temperature, cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) for approximately 12 hours at 4°C and then embedded in Killik cryostat embedding medium . Cryosections, 10 µm thick, were collected on polylysine-coated slides.The slides were pre-incubated in 0.5% Triton  and 1.5% Bovine Serum Albumin (BSA)  in PBS for 15 minutes at room temperature to saturate non specific sites. Then the sections were incubated 24 hours at 4°C with primary antibody against Ex-4  at final dilution of 1∶50.Subsequently, the sections were incubated with an Alexa Fluor 488 secondary Donkey anti Rabbit antibody  at a final dilution of 1∶333 for 2 hours at room temperature. The immunoreaction products were observed under an epifluorescence Zeiss Axioskop microscope  at x40 magnification.Serum leptin and adiponectin levels were assayed only in the polygenic model of obesity prone to im paired glucose tolerance and T2DM such as HFD mice using commercially available kit according to manufacturer’s instructions. Sandwich enzyme immunoassay (ELISA) was used for the quantitative measurement of mouse proteins . Intra- and inter-assay coefficient of variation were less than 5%.Total RNA was extracted from 50 mg of mice visceral adipose tissue. Briefly, tissue samples were collected, immediately snap frozen in liquid nitrogen and disrupted by homogenization in QIAzol Lysis Reagent using the TissueLyser . RNA was extracted using RNeasy Lipid Tissue Mini Kit  according to the manufacturer’s instructions. One µg of RNA was treated with TURBO DNA-free™ DNase Kit  and reverse-transcribed into cDNA for 1 h at 37°C in a 50 µl reaction containing 1× RT buffer, 150 ng random hexamers, 0.5 mmol/l dNTPs, 20 units of RNAsin Ribonuclease Inhibitor  and 200 units of M-MLV RT .5′-GCT GGA ATT ACC GCG GCT-3′; leptin forward: 5′-TCC AGA AAG TCC AGG ATG ACA C-3′, reverse: 5′-CAC ATT TTG GGA AGG CAG G-3′; adiponectin forward: 5′-ACA ATG GCA CAC CAG GCC GTG A-3′, reverse: AGC GGC TTC TCC AGG CTC TCC TTT-3′. Each cDNA sample was assayed in duplicate and a no template control was included in every reaction. For each sample, gene expression values were normalized by 18s RNA content and reported as AU ratio.Real Time quantitative PCR was carried out on DNA Engine Opticon™ 2 Continuous Fluorescence Detection System , using Platinum® SYBR® Green qPCR SuperMix-UDG  and 300 nM specific primers for each gene : 18 s forward 5′-CGG CTA CCA CAT CCA AGG AA-3′, reverse + test Kit (Bayer). Insulin levels were determined using a commercially available insulin RIA kit ; the intra-assay coefficient of variation was 1.4%. Insulin resistance was evaluated by the homeostasis model assessment of insulin resistance (HOMA-IR) index calculated as:  Glycaemic values were determined in the morning, after overnight fasting. Blood was obtained via tail vein and tested, using an Accu-Chek Aviva Nano meter (Roche). HbA1c percentage values were measured on 5 µl of whole blood using an A1CNow The statistical significance of differences between experimental and control groups was analyzed by Student’s t-test. p<0.05 was considered statistically significant. Values are presented as mean ± standard error (SE).In order to facilitate processing and secretion, Ex-4 coding sequence was linked to the secretory signal peptide from nerve growth factor (NGF), which was modified to be cleaved by the furin protease. Initial in vitro experiments suggested the chimeric protein could be secreted from cells in culture. Media collected from AAV5 Ex-4 transduced 293T cells produced an average of 38.3±10.4 pmol/L when assayed for Ex-4 biological activity on Chinese hamster ovary cell line stably transfected with rat GLP-1 receptor (CHO/GLP-1R). The furin overexpression by transfection of a plasmid encoding the protease, increased the active Ex-4 in the medium to 75.6±11.0 pmol/L.in vivo in two different animal models of obesity prone to impaired glucose tolerance and T2DM, following single 50 ul (5×1012 DRP/ml) vector administration into their SG. Our initial in vivo experiments suggested that like erythropoietin (27), NGF-Ex-4 can be produced by the salivary glands and will traffic through the cell via the endocrine pathway resulting in circulating serum levels. In AAV5 Ex-4 HFD mice (n = 10), Ex-4 levels averaged 138.9±42.3 pmol/L at week 6, when assayed by a specific Enzyme Immunoassay (EIA) kit. In AAV5 Ex-4 Zucker fa/fa rats (n = 5), mean circulating Ex-4 level was 238.2±72 pmol/L at week 4 and increased to 3.25 nmol/L at week 8. In control animals, average circulating Ex-4 levels resulted less than 2.6 pmol/L, thus above detection limit, at week 6 in mice and both at week 4 and 8 in rats.Ex-4 secretion was evaluated Biological activity of Ex-4 was also confirmed on CHO/GLP-1R cells (data not shown).In order to assess vector biodistribution, DNA samples were collected from the SG, liver, spleen and pancreas of HFD mice (n = 5 AAV5 Ex-4 treated and n = 5 naïve mice) at the end of the study and vector copy number was determined by quantitative Polymerase Chain Reaction (qPCR) amplification using specific primers for the CMV promoter. Naïve animals yielded background levels of 55±29 copies/100 ng of DNA extracted from SG. In AAV5 Ex-4 HFD mice, a 60-fold increase in vector copy number was detected in SG (3551±1618 copies/100 ng of DNA). The vector copy number detected in other tissues such as liver (154±56 copies/100 ng of DNA), spleen (65±23 copies/100 ng of extracted DNA) and pancreas (104±47 copies/100 ng of DNA) were at or near background levels.Expression was also confirmed by immunohistochemical staining for Ex-4 in SG sections from AAV5 Ex-4 (n = 5) and control (n = 5) HFD mice euthanized at week 6. Ex-4 expression is observed only in the AAV5 Ex-4 treated group. Only salivary ductal cells revealed positive staining, which is consistent with the tissue tropism of the AAV5 vector , AAV5 Ex-4 HFD mice had a significantly lower weight gain in comparison to control animals  compared with control animals . In contrast, no significant difference in adiponectin levels were observed . The reduction in leptin circulating levels correlated with a significant reduction in visceral adipose tissue leptin mRNA expression compared with control animals . No difference in visceral adipose tissue adiponectin mRNA expression  was detected.Mice feed a high fat diet will develop T2DM after 12 weeks. Accordingly, the model animals exhibited throughout the study a mild increase of glycemia and HbA1c, high serum insulin levels and HOMA-IR index. In order to better understand early effects of AAV5 Ex-4 treatment on the development of insulin resistance, insulin tolerance was tested (ITT) following an intraperitoneal insulin injection. At week 6, AAV5 Ex-4 mice showed a greater insulin-induced reduction in glycaemia 15, 30 and 60 minutes following injection compared with control mice Figure 4No significant difference was observed in fasting glycaemia, glycosuria or HbA1c values throughout the study. At the end of the study, AAV5 Ex-4 mice showed significant lower fasting insulin circulating levels and HOMA-IR index compared with control animals Table 2.On the other hand, significant reductions in HbA1c levels and glycosuria were observed in Zucker fa/fa rats. AAV5 Ex-4 treatment resulted in significantly lower HbA1c level as compared with control animals . Glycosuria was detected in 4 control rats 8 weeks after vector administration. No glycosuria was reported in treated rats. Accordingly to the low hypoglycaemic risk profile of Ex-4, no significant difference in fasting glycaemia was observed between treated and control rats during the study. Moreover, a statistically significant difference in fasting insulinemia and HOMA-IR index was detected between treated and control animals at the end of the study Table 2.et al reported that an adenoviral-mediated transduction of SG with a vector encoding GLP-1, can induce short-term moderate reduction of blood glucose in a murine model of diabetes GLP-1 RA are a very interesting therapeutic approach for the treatment of T2DM, showing a remarkable efficacy on glycaemic control and beneficial effects on body weight Our study shows for the first time a sustained secretion of Ex-4 at pharmacological levels. These circulating levels are several fold higher than reported for endogenous human GLP-1 after a meal (40 pmol/l) A reduction of fasting insulin levels, together with an improvement of HOMA insulin resistance index (based on fasting insulin and glucose) was observed in treated rats and mice, suggesting that Ex-4 secretion improves insulin sensitivity; this was confirmed by the results of insulin tolerance test in HFD mice. Although weight loss could explain the enhancement of insulin sensitivity, a direct insulin-sensitizing effect of Ex-4 is also possible. Alternatively, the improvement in insulin sensitivity could be due to the inhibition of glucagon secretion.Although the effect of Ex-4 expression on body weight could have contributed to the improvement in glycaemic control, it is very likely that direct action of Ex-4 on insulin and glucagon secretion, and possibly insulin resistance, played a major role.Furthermore, some limitations of present study should be recognized. 14 week HFD mice and 17 week Zucker fa/fa rats are more experimental model of obesity, and insulin resistance than models of overt diabetes, as indicated by fasting glycemic values throughout the study. Therefore, we performed the insulin sensitivity test in the HFD mouse, because of its greater similarity to polygenic pathogenesis of human obesity compared with Zucker fa/fa rat. However, glycemic control was evaluated through HbA1c (as a surrogate marker), for fasting glycemia and glycosuria detection in both animal models.This study suggests an alternative approach to delivering Ex-4 is possible and can reduce weight gain as well as trigger improved metabolic function in two animal models. Although Ex-4 expression has shown metabolic benefits there is concerns about the long-term safety of this drug and its effect on inducing tumors in rodents


In this study we investigated the effects of canonicalWnt/β-catenin signaling during early embryonic differentiation and hematopoieticspecification using an embryonic stem cell system. Our data clearly demonstratesthat following early differentiation induction, canonical Wnt signaling inducesa strong mesodermal program whilst maintaining a degree of stemness potential.This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog.β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyurylevels, which in-turn lead to Nanog up-regulation. During differentiation,active canonical Wnt signaling also up-regulated key transcription factors andcell specific markers essential for hematopoietic specification, in particulargenes involved in establishing primitive erythropoiesis. This led to asignificant increase in primitive erythroid colony formation. β-cateninsignaling also augmented early hematopoietic and multipotent progenitor (MPP)formation. Following culture in a MPP specific cytokine cocktail, activation ofβ-catenin suppressed differentiation of the early hematopoietic progenitorpopulation, with cells displaying a higher replating capacity and a propensityto form megakaryocytic erythroid progenitors. This bias towards erythroidlineage commitment was also observed when hematopoietic progenitors weredirected to undergo myeloid colony formation. Overall this study underscores theimportance of canonical Wnt/β-catenin signaling in mesodermal specification,primitive erythropoiesis and early hematopietic progenitor formation duringhematopoietic induction.The generation of hematopoietic stem cells (HSCs) during development is a complexprocess linked to morphogenic signals. Understanding this process is importantfor regenerative medicine applications that require  XenopusWnt 4 has been identified as essential for the formation and maintenance of theventral blood islands [+, c-Kit-endothelial-like precursors that express VE-cadherin. Subsequent to this stageβ-catenin signaling is dispensable for HSC establishment but can modulate HSCbehavior as demonstrated by increased reconstitution potential following activationof the pathway [Wnt proteins are highly insoluble glycoproteins, which remain associated with thecell surface or extracellular matrix of the cell following secretion . Consequ islands . Interes islands ,8. This  islands ,11. Wnt  islands  with ana islands . Canonic pathway . These sin vivo canonical Wntsignalling, revealed that mild to moderate activation of the pathway is advantageousresulting in increased clonogenic and differentiation potential with greaterreconstitution potential of HSCs, whereas high levels of activation resulted in adifferentiation block and failure to reconstitute irradiated recipient mice [In adult hematopoiesis, loss- and gain- of function of β-catenin combined withcre-mediated recombination to target the HSC population has produced conflictingviews regarding the importance of β-catenin for normal HSC function. Conditionaldeletion of β-catenin using Vav-1-Cre impaired HSC self-renewal ,16,suppent mice . in vitro modelfor studying both the initiation of primitive hematopoiesis and later stages whenmore definitive hematopoiesis becomes established [Although these studies provide important information on the role of canonicalWnt/β-catenin signaling in steady state homeostasis the ability to study earlyembryonic hematopoiesis has been hindered by the inability to specificallyover-express/delete key components of this pathway during embryogenesis. Mouseembryonic stem (ES) cells therefore provide an ideal ablished ,27-29. Fablished . Mesoderablished –34with ablished ,33,34. Uablished -37.in vitro model.Activation of the pathway at different stages of differentiation was achieved usingcomplementary pharmacological and genetic approaches. We demonstrate that β-catenindependent signaling induces a strong mesodermal program whilst maintaining a degreeof stemness potential during early differentiation induction. This is accompanied bya strong induction of genes involved in primitive hematopoietic development. Whendirected to undergo hematopoietic differentiation, β-catenin signaling enhanced thisprocess by promoting early hematopoietic and MPP, megakaryocytic erythroidprogenitors (MEP) and erythroid colony formation. Overall, we demonstrate that thecanonical Wnt pathway enhances developmental hematopoiesis processes, especiallyprimitive and more definitive erythropoiesis.To characterise the role of the canonical Wnt/β-catenin signal transduction pathwayin early cell specification and more specifically early hematopoieticdifferentiation, we have utilised ES cells as an Dominant positive ΔGSKβ-catenin (DP-βC), with the CK1 and GSK-3 binding sitesSerine 33, 37, 45 and threonine 41 mutated to alanine by site-directedmutagenesis,  was cloned intopUHD10-3 neomycin and transfected into E14tga murine ES cells expressing thetetracycline-sensitive transactivator, tTA. The β-catenin mutation resulted in adominant positive form, (DP-βC), resistant to proteosomal degradation. Culture,selection and screening of clones were performed as previously described . For the50 of the pharmacological inhibitors6-bromoindirubin-3’oxime/BIO, and XAV939 . Self-renewal of parentalES cells plus the pharmacological inhibitor 5 μM BIO, 5 μM & 10 μM XAV, ordimethyl sulfoxide alone and DP-βC ES cells plus and minus tetracycline wereanalyzed using alkaline phosphatase staining. Cells were washed, fixed inmethanol and then stained for 15 minutes with 1 mg/mL Fast Red TR salt TM(Sigma) dissolved in 0.1 M Tris pH 9.2 containing 200 µg/mL Napthol AS-MXphosphate. XTT bioreduction assays and trypan blue exclusion were performed as previouslydescribed  to asses-ΔΔCT method was used to calculate relative expression levelsfor each gene. RNA was reverse-transcribed using a High Capacity cDNA ReverseTranscription kit (Applied Biosystems) and PCR performed using the AppliedBiosystems® TaqMan® Mouse Stem Cell Pluripotency Array (4385363) as permanufacturer instructions. Data was quantified using RQ Manager Analysissoftware. Relative gene expression was calculated using the 2-ΔΔCTmethod. Genes included for analysis had a CT value ranging between 18-35, withthe ΔCT calculated using the average CT from five endogenous controls asreference genes. Following calibration using the control samples (-DP-βC or-BIO) the RQ ratio (arbitory units) of the test sample (+DP-βC or +BIO) wereplotted as fold increase/decrease as a measure of mRNA gene expression.Transcriptional changes of >2.0 fold change were included in theanalysis.Total RNA was prepared using RNAeasy Plus extraction kit (Qiagen). RNA (1 μg) wasreverse-transcribed using Superscript reverse transcriptase and oligo dT primers(Invitrogen Life Technologies). Semi-quantitative PCR was performed using 2 μLcDNA and standard conditions using gene-specific primers with non-saturatingcycle-numbers (24-32 cycles). Quantitative PCR was performed using 2 μL cDNAwith gene specific primers  and 2x TImmunoblotting was performed for; Phospho-STAT 3 (9131), Phospho-Akt substrate(9614), GAPDH (2118), TCF1, TCF4 & LEF1 9383) , STAT 3 (sc-7179), Nanog , Total GSK 3(05-412), Active β-catenin , Total β-catenin(610154), anti-GSK3β Tyr 216  and Brachyury  as previously described [3 (Cell S7 cells per mL of FACS Buffer (PBS with 2% FCS and 0.02% sodiumazide) and incubation with 1 µg of Rat Anti-Mouse CD16/CD32 Fc Block™  for 30 min, 4°C. For intracellular staining, the cellswere harvested, blocked, washed x 2 with FACS Buffer prior to fixing andpermeabilization using FIX & PERM® Kit (BD) as per manufacturer’sinstructions. Labelling was performed using 0.5 μg of each flurochromeconjugated antibody or relevant isotype control for 1 h, 4°C in the dark.Secondary staining was performed for a further 30 min for any indirect stains.Brachyury (Abcam 20680), Nanog (560277), Oct3/4 (9006287), Sox2 (9006407), CD11b(552850), Flk1 (555308), CD24 (55313137), CD41 (556437), CD44 (560451), CD45(557235), c-Kit (553356), Sca1 (558162), Gr1 (553129) and Flt3 (553842) all fromBD, CD71 (113802), Ter119 , FITC Secondary (BD 554020),Step-Avidin Secondary (BD 554064) were used. Following washing, cells wereanalyzed on a FACSCanto™ II flow cytometer (BD) and data analysed using FlowJosoftware (Treestar). For isolation of hematopoietic populations cells wereimmunostained and isolated on a FACSAria™ cell sorter flow cytometer (BD).For immunostaining analysis, cells were harvested, resuspended at 0.5 x106 cells were formaldehydetreated to cross-link DNA-protein interactions and reaction stopped usingGlycine. Cells were lysed in cell lysis buffer and nuclei were pelleted,resuspended in nuclei lysis buffer (NLB) and incubated for 10 min, 4°C. IPdilution buffer (IPDB) was added and nuclei sonication on ice to obtain 300-1000bp fragments of sheared DNA. Fragments were collected by centrifugation,resuspended in NLB:IPDB and chromatin precleared using 100 µl Rabbit serum, 1 hat 4°C. Protein G-agarose (100 μl of the bed volume) was added and incubatedovernight at 4°C. Antibody-agarose complexes were pelleted and the chromatinsupernatant divided into five portions, one was stored as input (positivecontrol) and the other four immunoprecipitated with 2 µg; Stat 3, Brachyury,TCF/LEF-1 or IgG (as a negative control) antibody at 4°C overnight. ProteinG-agarose (50 μl of the bed volume) was added and incubated for 3 h at 4°C. TheProtien G-agarose-complexes were collected by centrifugation, washed x 2 in lowsalt IP wash Buffer x 2 in high salt IP wash buffer and x 2 with TE buffer, pH8.0. To reverse the cross links, IP Elution buffer was added. The samples wereincubated with RNase A at 65°C for 6 h and proteinase K treated overnight at45°C. DNA was extracted using phenol chloroform and analysis performed using PCRwith ChIP specific primers . Colonies were scored following 7 days in culture. For MPP colonyformation [4/mL in methylcult supplemented with 1% BSA fraction V andcytokines to promote myeloid progenitor colony formation; 25 ng/mL GM-CSF, 100ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL IL-11, 10 ng/mL TPO, 10 ng/mL Flt3(PeproTech™), and 1 U/mL EPO (R&D Systems Ltd.) for 7 days. The colonieswere replated in the same cocktail for a further 7 days to assess replatingability. To promote granulocyte/macrophage differentiation, early hematopoieticprogenitors (Day 8) were replated at 2.5 × 104/mL in methylcult with1% BSA and cytokines to promote both myeloid and erythroid colony formation; 25ng/mL GM-CSF, 25 ng/mL G-CSF, 10 ng/mL SCF, 10 ng/mL IL-3 and 2 U/mL EPO for 7days. Hematopoietic colonies were scored using a Nikon Eclipse TS100 microscopeand digital camera system. Cells were harvested at day 0 (ES), day 4 (earlydifferentiation induction) day 8 (early hematopoietic progenitor) and day 15 for RNA extraction and FACS analysis.Primary EB (Day 4) and early hematopoietic progenitor formation Day 8) werecarried out as previously described  werecar,41 and aormation , early h45+3.1%, DP-βC75+2.1%, BIO 86+3.2%, XAV 26+1.5%,n=3). However canonical Wnt/β-catenin signaling was unable tosustain self-renewal with cells undergoing a degree of differentiation by 96 hof LIF withdrawal. BIO exhibited a greater propensity to suppressdifferentiation than DP-βC, indicative of GSK-3 ability to modulate otherpathways such as the PI3 kinase pathway [Nanog, Nr5a2, Oct3/4, Rex1,Tcf and Cdx2 were up-regulated by DP-βC expressionand GSK-3 inhibition (BIO). Furthermore β-catenin activation strongly induced amesodermal/mesoendodermal program with characteristic markersBrachyury (T), Pecam 1, Nodal, MyoD1 and Tcfcp2ll up-regulated. This was accompaniedby a corresponding suppression of endodermal genes . Semi-quantitative PCRconfirmed changes in expression of key genes significantly altered followingcanonical Wnt/β-catenin signaling. In addition Sall 1 andID 1 genes important for suppressing differentiation weredown-regulated following DP-βC expression and BIO treatment. As predicted XAVtreatment had the opposite effect, down-regulating Nanog, Oct3/4,Brachyury, Nodal and BMP4 expression  or by generating stable ES cell linesusing the tetracycline regulated system to inducibly express DP-βC with CK1 andGSK-3 phosphorylation sites mutated to prevent proteosomal degradation . Negativ pathway . To detepression . Flow cypression & S1B. TTo explore the complex interplay involved in stem cell maintenance duringdifferentiation processes we examined the relationship between β-cateninsignaling and its ability to regulate TCF/LEF, Brachyury, STAT3 and Nanoglevels. Immunoblotting analysis revealed that DP-βC and BIOup-regulated/sustained higher protein levels of TCF/LEF, Brachyury, STAT3 andNanog during differentiation induction  . Flow cSca1, c-Kit andHoxB4, genes involved in HSC formation and self-renewalwere up-regulated by activation of the pathway. In addition the specificerythroid lineage marker, GATA1 and the common lymphoid myeloidprogenitor marker, PU-1 were also up-regulated. Althoughdetected GATA2, Fog1 and EpoR genes involvedin erythroid specification were unchanged whereas SCL anothermaster regulator of hematopoiesis was undetected ). The total cell population generated was analysed forthe key endothelial markers Flk1, CD44 and the mesodermal marker brachyury byflow cytometry and revealed that a greater percentage of cells expressed thesemarkers following β-catenin expression or BIO treatment than in the controlcultures, indicative of enhanced hemangioblast formation [ii), Brachyury andFlk1 expression in line with the increase shown by flowcytometry. In addition GATA 3 & 4 were up-regulated andNanog and Oct3/4 down-regulated,confirming enhanced differentiation (iii)). Flowcytometry analysis was also performed to determine the percentage of cellsexpressing the early hematopoietic progenitor markers CD41, CD45, c-Kit, Sca1and Flt3. Gating on the CD41+, CD45+ population revealedthat these early hematopoietic progenitors also expressed the HSC markers Sca1and c-Kit  hematopoieticprogenitor populations had alteration in their lineage priming potentialfollowing activation of the canonical Wnt pathway we isolated this population byFACs sorting for further analysis (i)). First we confirmed that theactivated cells still had higher levels of total and active β-catenin expression and master regulators ofhematopoietic lineage commitment  compared to the control populations (iii)); as well as genes involved in establishing primitiveand more definitive erythropoiesis (iii & iv)). A similar gene profilewas observed when the total population containing both the hemangioblast/earlyhematopoietic population was analysed in our cultures for CD41 and CD71 expression. Results indicate a significantincrease in the percentage of cells expressing these MEP markers in the DP-βCand BIO treated cultures (loc-Kithi), confirmc-Kithi).GATA1,Klf-1, Fog1 and EPO-R along withthe adult globin genes Hbb-b1, Hbb-b2, whereas the monocyticspecific genes Egr1 & 2 (To determine whether active β-catenin blocked granulocyte-macrophage (GM) colonyformation we employed a cytokine cocktail which strongly promotes CFU-GM whilstpermitting BFU-E and GEMM formation . HoweverEgr1 & 2  weredowOct3/4,Nanog, Sox2 and Rex1allup-regulated. Genes involved in mesodermal patterning were also up-regulatedfollowing activation of the β-catenin pathway. In particular the T-box targetsEomesogermin (Eomes) andBraychury (T) were significantly up-regulated.During mouse development Eomes is the earliest regulator ofmesoderm formation being required for both embryonic and extraembryonic mesodermformation [T overlapswith Eomes, with T also implicated in laterprocesses such as left/right axis determination and somite segmentation [Fgf4 expressed inintermediate mesoderm [GATA 4 and 6 expressed in lateral mesoderm during organogenesis[Lefty 1 & 2 andNodal involved in left/right axis determination, and lateralplate mesoderm formation during gastrulation [Nanog, Rex1, Lefty1 andLefty2 all down-regulated [Nanog. Using flow cytometry andChIP technology we demonstrate that a complex relationship exists between β-cateninsignaling and LEF/TCF/Brachyury/Nanog levels. Following β-catenin activation,TCF/LEF could potentially directly bind to its consensus sites in the Brachyurypromoter [The canonical Wnt/GSK3β/β-catenin pathway is known to act synergistically with theLIF/STAT-3 pathway to improve ES cell self-renewal -50. Howeormation . T overlentation . In addimesoderm ,GATA 4 ogenesis,55, Leftrulation ,57. A prpromoter  leading promoter ,58.Our promoter  whereas + mesoderm followed by a reduction in CD41+ primitiveerythroid colony formation [SCL and T3 globin transcription [Kit,CD34 and Pecam 1, genes which mark the onset ofprimitive hematopoietic cell establishment from extraembryonic mesoderm and YSdevelopment [GATA1, Pecam 1,Tie2 and LMO2 with no change in GATA2,Fog1 and EpoR levels. It is known that primitiveerythroid cells arise from GATA1 and 2 expressingcells in the YS which also have endothelial potential and express the endothelialmarkers Flk1, Tie2 and Pecam 1 [GATA2 null mice areembryonic lethal, due to severe anemia during the early phase of YS hematopoiesis(E10-11) [GATA2 precedes GATA1 and must decrease asGATA1 expression increases to enable erythroid differentiation.Another transcription factor essential for primitive hematopoiesis and induced byGATA2 is LMO2. LMO2 null mice die around E9 of severe anemia, witha lack of any YS hematopoiesis [GATA1and SCL to facilitate DNA binding and erythroid gene transcription[EKLF, Fog, EpoRand heme biosynthesis enzymes [Hbz and Hbb-βH1 which are then superseded bythe Hba-α1, Hba-α2 and ɛy- globingenes, as proerythroblasts at E7.5 transition to reticulocytes at E15.5 [Recent studies report Wnt, along with Activin and BMP signals are important forhematopoietic development, with inhibition of Wnt signaling preventing the formationof Flk1ormation . Converscription . Wnt famcription ,13,26,60cription , howeverelopment . Along w Pecam 1 . GATA2 n(E10-11) . Expressopoiesis ,64. TCF opoiesis . LMO2 thcription. Key ery enzymes ,68. Consat E15.5 . Our datat E15.5 . Taken t+c-Kit+ population followed bygene profiling revealed that transcription factors involved in HSC specification andself-renewal were strongly up-regulated in these early hematopoietic progenitors byβ-catenin signaling, including SCL/Tal1, essential for initiatingthe hematopoietic program at this stage [The transition from primitive to more definitive hematopoiesis involves the formationof hematopoietic progenitors with self-renewing potential. In order to explore howβ-catenin influenced this process we directed ES cells to form hemangioblast/earlyhematopoietic progenitors. Active β-catenin significantly augmented this processwith 60-75% of the cells expressing the early HSC/progenitor markers CD41, c-Kit,Sca1 and Flt3 compared to ~50% in the control cultures, with 40% of the cells alsoexpressing the pan-hematopoietic marker CD45 compared to 25% in the controlcultures. Isolation of the Sca1is stage .Genes eloCD41hiCD71hi) within both theSca1hic-Kitlo and Sca1loc-Kithipopulations. Following MPP replating, β-catenin signaling sustained the number ofSca1hic-Kitlo cells. This is in accordance with the mousemodels where conditional expression of active β-catenin in the hematopoietic systemresults in a large expansion of Sca1+c-Kit+ LT-HSC. Analysisrevealed that these HSCs were unable to sustain long-term reconstitution abilityfollowing transplantation. This was due to β-catenin pushing cells into cycleincreasing proliferation and leading to the accumulation of undifferentiatedprogenitors which eventually resulted in HSC exhaustion. In addition the progenitorsgenerated exhibited defective GM colony forming ability and enhanced potential toundergo erythroid differentiation in vitro and invivo reflected by higher erythroid cell numbers in the bone marrow andspleen [invitro model cytokine signaling through G-CSF and GM-CSF was able topartially overcome this block in GM colony formation, supporting the idea thatalthough β-catenin promotes an erythroid transcriptional program, the HSC/progenitorpopulations still retain the capacity to undergo granulocyte/macrophagedifferentiation. In order to determine how influential canonical Wnt signaling was in predisposingcells to undergo erythropoiesis, the early hematopoietic progenitor population werecultured in a cytokine cocktail to promote MPP formation . Active dspleen ,22. Recedspleen . In our Overall we demonstrate the transcriptional changes canonical Wnt signaling alters toorchestrate early developmental hematopoietic stages. Our data clearly defines arole for β-catenin in directing ES cells to form mesodermal progenitors andenhancing the formation of hematopoietic progenitors with erythrocytic potential. Inaddition β-catenin induced expression of genes involved in maintaining stem cellpotential during these early differentiation decisions, an essential processnecessary for sustaining the rapid proliferation required during the establishmentof the hematopoietic system. Figure S1Wnt signaling up-regulates brachyury levels in the absence ofLIF (A). Dot plots and graphs showing DP-βCexpression or BIO treatment increases the percentage of cells expressingBrachyury over 72 and 96 hours following LIF withdrawal. . Activatingthe β-cateninpathwaysustainsNanog, andOct3/4expressionfollowingLIF removal (B). Representative histogramsshowing DP-βC expression or BIO treatment sustains the percentage of cellsexpressing Nanog and Oct3/4 over 72 and 96 hours to similar levels asobserved at 20h following LIF withdrawal. (TIF)Click here for additional data file.Figure S2β-catenin activation enhances hemangioblast and hematopioetic markersduring differentiation. FACS analysis to measure percentage ofcells expressing hemangioblast and hematopoietic markers following DP-βCexpression or BIO treatment. Activation of the pathway up-regulates themesodermal marker Brachyury along with mesoendodermal Flk1 and CD44.Analysis also shows that HSC markers (Sca1 & c-Kit), myeloid progenitormarker (Flt3) and hematopoietic cell marker (CD45) are all up-regulated byactivation of the canonical Wnt pathway . (TIF)Click here for additional data file.Figure S3Early hematopoietic progenitors up-regulate early and late erythroidgenes following canonical signalling. RT-PCR analysis for thetotal hemangioblast/hematopoietic progenitor populations(i) key hematopoietic genes (ii) erythroid/globin genes.The control cells (DP-βC + tet or E14 ES cells -BIO) were used ascalibrators and the fold change was calculated using the 2 -ΔΔCTmethod . (TIF)Click here for additional data file.Figure S4Activation of the canonical Wnt pathway increases MPP and MEPgeneration. Multi-parameter flow cytometry analysis of the MPPpopulations following DP-βC expression and BIO treatment. The dot plotsrepresent the expression profiles for CD24, Ter119, Gr1 and Flt3 after thefirst round of MPP formation and following replating for an additional 7days in the MPP cytokine cocktail. Representative dot plots shown, n=3. (TIF)Click here for additional data file.Table S1Table showing the CT, ΔΔCT and Fold change values for ChIP data.Gels depicting levels of Brachyury or Nanog following IP for the TCF or theBrachyury promoter respectively between treated (DP-βC or BIO) and controlsamples with Rabbit IgG used as a negative control.(TIF)Click here for additional data file.Table S2Table showing the forward and the reverse primers used in the ChIPassays in this study and the sizes for each product with respect to thestart site.(TIF)Click here for additional data file.Table S3Tables showing the primer sequence, accession numbers and the cyclingconditions for the forward and reverse primers used in thisstudy.(TIF)Click here for additional data file.