The canonical Wnt signaling pathway (or Wnt/β-catenin pathway) plays a pivotal role in embryonic development and adult homeostasis; deregulation of the Wnt pathway contributes to the initiation and progression of human diseases including cancer. Despite its importance in human biology and disease, how regulation of the Wnt/β-catenin pathway is achieved remains largely undefined. Increasing evidence suggests that post-translational modifications (PTMs) of Wnt pathway components are essential for the activation of the Wnt/β-catenin pathway. PTMs create a highly dynamic relay system that responds to Wnt stimulation without requiring de novo protein synthesis and offer a platform for non-Wnt pathway components to be involved in the regulation of Wnt signaling, hence providing alternative opportunities for targeting the Wnt pathway. This review highlights the current status of PTM-mediated regulation of the Wnt/β-catenin pathway with a focus on factors involved in Wnt-mediated stabilization of β-catenin. Wnt proteins belong to an evolutionarily conserved family of secreted cystein-rich glycoproteins. Wnts can activate β-catenin-dependent canonical Wnt pathway and β-catenin-independent non-canonical Wnt pathways, including planar cell polarity pathway and calcium pathway -3. InterA key feature of the canonical Wnt pathway is the regulated degradation of transcription coactivator β-catenin by the β-catenin destruction complex, consisting of Glycogen Synthase Kinase 3α and 3β (GSK3α and GSK3β), Casein Kinase 1 (CK1), Adenomatous Polyposis Coli (APC), scaffold protein Axin and transcription co-factor β-catenin . In the In addition to core components of the Wnt pathway , nonMost components in the Wnt/β-catenin pathway including Wnt proteins undergo one or more covalent modifications. For PTMs of Wnt proteins including glycosylation and palmitoylation, we refer the reader to two excellent reviews ,24. In tAddition of a phosphate group to amino acid residues on serine, threonine or tyrosine residues, is one of the most important and well-studied post-translational modifications in eukaryotes. As one of the first PTMs to be described, phosphorylation plays critical roles in the regulation of many cellular processes; abnormal phosphorylation results in a variety of human diseases . Many coIn the absence of Wnt, CK1α phosphorylates β-catenin at Ser45, which precedes and is required for subsequent phosphorylation of β-catenin at Ser33, Ser37 and Thr41 by GSK3 . Phosphoin vitro and in vivo studies show that dually phosphorylated PPPSPxS peptides are sufficient to inhibit GSK3 kinase activity towards β-catenin and other physiological GSK3 target sites including tau and glycogen synthase [Several models have been proposed to explain Wnt-mediated inhibition of β-catenin phosphorylation by GSK3: (i) Disruption of the destruction complex. Wnt induces rapid disruption of Axin/GSK3 interactions, which separates GSK3 from its substrate β-catenin, thus inhibiting β-catenin phosphorylation and causing initial stabilization of β-catenin . (ii) Insynthase ,127.  and co-receptor LRP5/6 initiates a signaling cascade resulting in stabilization of β-catenin and the activation of β-catenin-dependent transcription ,135,136.2 formation [2 formation, which promotes LRP6 aggregation, LRP6 phosphorylation and Axin recruitment by unclear mechanism.Different models have been proposed to depict the process: (i) Initiation and amplification of LRP6 phosphorylation . In the ormation . Upon WnAs discussed above, Dvl plays a critical role in the assembly LRP6 coreceptor complex and LRP6 phosphorylation. Dvl itself is a phosphorylation substrate: phosphorylation of Dvl can be catalyzed by RIPK4 (receptor-interacting serine/threonine-protein kinase 4), PAR-1 (Partitioning-defective 1), CK2 and CK1 -77. It i21A/21A/9A/9A knockin embryonic stem (ES) cells [Gsk3α and Gsk3β have redundant function in the Wnt/β-catenin pathway . How canS) cells  appear tin vitro substrate [in vitro kinase assay using myelin basic protein (MBP) or tau as substrates [There are several additional phosphorylation sites on GSK3 that have been reported to be associated with the regulation of β-catenin level in various biological contexts, but their role in Wnt signaling remains undetermined and elusive. Phosphorylation of GSK3β at threonine 43 by Erk , primes GSK3β for phosphorylation at Ser9 by p90RSK, and mediates HBV-X protein (HBX)-induced upregulates β-catenin in human hepatocellular carcinoma cells . Phosphoubstrate ,159. In ubstrate . Howeverbstrates ,161. Conbstrates ,162, othbstrates .In contract to the N-terminus phosphorylation by CK1 and GSK3 that triggers β-catenin ubiquitination and degradation, phosphorylation of several sites on β-catenin C-terminus  appears to stabilize β-catenin and affect its nuclear accumulation -90, leadUbiquitin is an 8.5 kDa regulatory protein found in almost all tissues of eukaryotic organisms. Ubiquitination is a PTM in which an ubiquitin protein is attached to a substrate protein through an enzymatic process requiring three types of enzymes: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases E3s) ,169. As s ,169. Aβ-TrCP-ubiquitin ligase complex subsequently attaches K48-linked polyubiquitin chains onto lysine residues 19 and 49 at the N-terminus of β-catenin [Modulation of the abundance of the Wnt pathway components through ubiquitination-mediated proteasomal and lysosomal degradation plays a critical role in the regulation of Wnt signaling. A characteristic feature of the canonical Wnt pathway is tight regulation of the level of β-catenin controlled by CK1- and GSK3β-mediated phosphorylation and subsequent proteolytic degradation. In the absence of Wnt, phosphorylation of the N-terminus of β-catenin—Ser45 by CK1α, followed by GSK3-mediated phosphorylation of Ser33, Ser37 and Thr41— triggers the recruitment of the β-TrCP ,86,123, -catenin ,93, caus-catenin . How thi-catenin . Like β--catenin ,183, it -catenin . This ma-catenin . The sta-catenin -99. Both-catenin , whereas-catenin ,99, suggAxin (Axin1 and Axin2) is a scaffold protein that directly interacts with other core components of the destruction complex ,184,185.β-TrCP E3 complex to form a supercomplex. The CSN supercomplex regulates the balance between β-catenin and APC: while it stimulates β-catenin degradation, USP15 associated with the CSN stabilizes APC. Upon Wnt stimulation, the CSN complex dissociates from SCFβ-TrCP and the APC-Axin complexes, rendering APC susceptible for proteolysis. This model suggests that Wnt-induced degradation of APC promotes β-catenin stabilization, which is not consistent with earlier studies showing stabilization of APC upon Wnt signaling [Considered as a Wnt pathway negative regulator, the scaffold protein APC facilitates GSK3-mediated phosphorylation of β-catenin . APC is ignaling ,190.Dvl  is the decision point for downstream canonical and non-canonical Wnt signaling branches and plays a critical role in the relay of signals from the LRP6 receptor complex to downstream effectors in the Wnt/β-catenin pathway -194. TheNEDD4L  is a member of the NEDD4 family ubiquitin ligases, directly binds to Dvl2 through the WW3 domain of NEDD4L and the PY motif of Dvl2, and targets Dvl2 for proteasomal degradation through K6-, K27-, and K29-linked atypical ubiquitination . By promThe involvement of multiple ligases highlights the importance of tight control of Dvl level in cells. But how these E3 ubiquitin ligases—KLHL12, ITCH, NEDD4L, VHL and Malin—distinguish themselves from each other as an ubiquitin ligase for Dvls remains unclear. It is possible that they act on Dvl in a different format or different isoform of Dvl. For example, ITCH only ubiquitinates phosphorylated Dvl, whereas pVHL and KLHL12 ubiquitinates both phosphorylated and unphosphorylated Dvl, and KLHL12 seemed to prefer targeting Dvl3 over Dvl2 . It is aThe levels of Wnt receptor LRP6 and Fz, not surprisingly, greatly impact the activation of the pathway ,196. TheUbiquitination commonly drives cell-surface receptor internalization and lysosomal degradation . The ubiDrosophila cells, surprisingly, it acts below the stabilization of β-catenin. How Trabid-mediated deubiquitination of APC links to the activity of the TCF–β-catenin transcription complex remains unsolved. Two later studies shed more light on the molecular mechanisms underlying K63-linked ubiquitination of APC in Wnt signaling [The most studied function of ubiquitination in the Wnt pathway relates to protein turnover. However, emerging evidence indicates that nonproteolytic function of polyubiquitination of core Wnt pathways proteins through lysine 63 plays an important role in the regulation of the pathway ,79,198. ignaling ,198. APCignaling . TogetheBoth positive and negative roles of K63-lined ubiquitination of Dvl in the Wnt regulation have been reported ,79. A prUbiquitination of Axin by ubiquitin ligases Smurf2 and RNF146 mediates Axin degradation ,45,47. SIn addition to ubiquitin, there is a growing family of ubiquitin-like proteins (UBLs) that modify cellular targets in a pathway parallel to, but distinct from, that of ubiquitin. SUMO  proteins are a family of small proteins that are around 100 amino acids in length and 12 kDa in mass. Mammalian SUMO has four isoforms: SUMO1, SUMO2, SUMO3, and SUMO4. SUMO2 and SUMO3 share 95% sequence homology and are distinct from SUMO-1(often collectively referred to as SUMO2/3). Sumoylation refers to the process that small ubiquitin-related modifier (SUMO) is covalently attached to the target proteins through sequential enzymatic reactions involving the activity of SUMO activating enzyme (E1), SUMO conjugating enzyme (E2) and SUMO ligase (E3) ,201. SumThe identification of Axam (Axin Associating Molecule) as a novel Axin-binding protein and a negative regulator of Wnt/β-catenin pathway provides the first evidence that sumoylation is involved in the regulation of Wnt signaling . Axam beSumoylation of the scaffold protein Axin at its C-terminal six amino acids stretch (C6 motif), a motif that is critical for Axin interaction with three SUMO-1 E3s, PIAS1, PIASxβ and PIASy, prevents its polyubiquitination, thus increasing its stability ,208. GivProtein acetylation is a process that an acetyl group is transferred to the ϵ-amino group of an lysine residue of target protein . Acetyladrosophila CREB-binding protein (dCBP) binds to dTCF, acetylates lysine 25 in the Armadillo (β-catenin in drosophila)-binding domain of dTCF, which in turn lowers the affinity of Armadillo binding to dTCF, thereby repressing TCF [Members of the TCF/LEF family are also substrates of acetylation -116. It sing TCF . In contsing TCF , thus vain vitro. It is of great interest to understand the role of ADP-ribosylation of GSK3β in Wnt signaling in the future.ADP-ribosylation, which refers to the enzymatic transfer of one or more ADP-ribose from NAD + to the acceptor proteins , has beePTMs often interplay to work in concert . The intAlthough crosstalk between PTMs has emerging as an important regulatory mechanism in signal transduction ,231-233,Another example of PTM crosstalk in the Wnt/β-catenin pathway is the poly-ADP-ribosylation-mediated polyubiquitination of Axin . A recenBiological and clinical study over the past few years has greatly expanded our understanding of the complex Wnt signaling network, but there are still many fundamental aspects of the Wnt-related biology to be discovered and understood. Characterization, dynamic detection and quantification of chemical modifications of the pathway components are essential for us to gain deeper insight into biological control of the pathway. Although identification of PTMs involved in the Wnt/β-catenin pathway and validation of their function are far from completion—in fact a large number of PTMs are not all validated as physiological relevant, the concept that relaying Wnt signal requires dynamic changes in PTM states of the pathway components has been emerged. We now know that instead of relying solely on one particular modification, the Wnt pathway is controlled by the coordinated actions of phosphorylation, ubiquitination and other PTMs. However, little is known as to how PTM coordination is effectively achieved at the molecular and cellular level. For example, systematic analysis of PTM changes with respect to space and time remains an important future goal for PTM research in the Wnt field, and how signaling pathways or upstream enzymes that catalyze these PTMs interact combinatorially, hierarchically or reciprocally to ensure the sequence of the occurrence of the PTMs and kinetics of their durations during both “Wnt on and Wnt off” situations remains largely unexplored.As the importance of PTMs has increasingly been appreciated, there is an increasing need for improved technologies that enable researchers to measure the state and function of PTMs in physiological situations. Characterizing the function of PTM is technically challenging due to the dynamic and reversible nature of PTM, and the complicated interplays between PTMs. A research trend in the field of PTM is the mouse knockin technology. Knockin methodology, coupled with gene knockouts and specific pharmacological inhibitors, is a powerful approach to dissect the physiological roles of individual modification at given sties on a given protein. However, it is an imperfect model because it knocks the protein permanently in one form—the protein with mimicked modification or the protein lacking modification, therefore it cannot recapitulate the dynamic and reversible nature of the PTMs.Aberrant activation of Wnt/beta-catenin pathway contributes to development of different human cancers, especially colorectal cancers. As pointed by Nusse and Varmus , “among The authors declare that they have no competing interests.CG, GX, and JH wrote the review. All authors read and approved the final manuscript.


Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if β-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established β-catenin-deficient (β-catΔ/Δ) mouse embryo-derived stem cells and showed that β-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs). However, teratomas formed from embryo-derived β-catΔ/Δ ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional β-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that β-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight β-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.The canonical Wnt/β-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. β-Catenin, encoded by the  In the absence of a Wnt signal, β-catenin is rapidly degraded by a multi-protein complex including Axin, adenomatous polyposis coli (APC) and glycogen synthase kinase 3 (GSK3). Wnt proteins stimulate signaling through various frizzled receptors, low density lipoprotein receptor-related protein 5/6 (LRP 5/6) co-receptors and disheveled to inactivate the β-catenin-degradation complex, and the stabilized β-catenin associates with transcription factors of the Tcf (T-cell factor)/Lef (lymphoid enhancer factor) family resulting in the transcriptional activation of target genes Ctnnb1 deficient mouse ESC lines were established by deleting functional exons in cultured mouse ESC lines, and it still remains to be determined whether β-catenin plays an important role in the transformation of preimplantation embryos into pluripotent stem cells.Previous studies based on both loss- and gain-of-function mutations of β-catenin in mice have established the importance of the role of Wnt/β-catenin signaling in the regulation of the vertebral axis, germ layer specification and tumorigenesis Δ/Δ mESCs) with the deletion of exons 2–6 using mice containing the loss-of-function mutation in β-catenin to produce mutant oocyte and sperm through the germ cell-specific Cre-loxP system. β-catΔ/Δ mESCs are a material which can be used to address the role of β-catenin in the acquisition of pluripotency including self-renewal and differentiation from preimplantation embryos. It is also noteworthy that β-catΔ/Δ mESCs lack the residual β-catenin of either maternal or paternal origin. Our results show that β-catenin is not essential for acquiring and maintaining proliferative self-renewal, but that it is required for normal multilineage differentiation. Interestingly, these embryo-derived β-catΔ/Δ mESCs contributed to oncogenic germ cell tumors in a teratoma assay. These results were corroborated by our rescued β-catΔ/Δ mESCs experiments. Our findings show that β-catenin plays an important role in multilineage differentiation and tumorigenesis in germ cell tumors. Our embryo-derived β-catΔ/Δ mESCs also provide a novel research tool for immature germ cell tumors.Our recent work demonstrated that β-catenin plays a role in sperm-oocyte membrane adhesion and the transition of the membrane upon mouse fertilization, using homogenous defects of β-catenin in both oocyte and sperm cells Ctnnb1) ZP3−creTg), which was kindly provided by Dr. Barbara B. Knowles . The F1 offspring, β-cateninfloxed/floxedTgZP3−cre (β-catfl/Δ), were propagated through brother-sister mating. The presence of the cre-recombinase gene in these offspring was detected by polymerase chain reaction (PCR) analysis using the following set of primers: Cre-S  and Cre-A . This primer set yielded a band of 389 bp. Furthermore, to produce conditionally β-catenin-deficient sperm, β-cateninfloxed/floxed (β-catfl/fl) mice were mated with mice expressing cre-recombinase driven by protamine promoter floxed/floxedTgprotamin−cre male mice, and confirmed that the β-catenin gene was deleted in their epididymal sperm (data not shown). Then, to obtain completely β-catenin-deficient embryos, these two strains of mice were crossed. The β-catenin-deficient (β-catΔ/Δ) fertilized eggs were collected and cultured to the blastocyst stage at 37°C and 5% CO2 in air.To produce oocytes with a single gene deleted, floxed mutant mice on a C57BL/6J background with exon 2–6 flanked by loxP on the β-catenin gene  supplemented with 1,000 U/ml LIF, 3 µM CHIR99021 (Wako) and 1 µM PD0325901 (Wako) on Matrigel-coated  dishes fl/fl and β-catfl/Δ mESC lines were derived directly either from β-catfl/fl or β-catfl/Δ blastocysts, respectively. The NCH4.3 ESC line (C57BL/6J) Embryo-derived stem cells were established using mouse ESCs derivation medium of knockout DMEM  supplemented with 15% knockout serum replacement, 1 x non-essential amino acids (NEAA), 0.05 mM 2-mercaptoethanol, 2 mM GlutaMax, 100 U/mL penicillin G, 10 mg/mL streptomycin , 1000 U/mL of leukemia inhibitory factor (LIF)  and the MEK kinase inhibitor PD98059 . The blastocyst embryos developed from β-catenin-deficient fertilized eggs were individually treated with EmbryoMax Acidic Tyrode’s Solution  to remove the zona pellucidae and plated on a feeder layer. Mouse embryonic fibroblasts (MEF) were inactivated by gamma irradiation in gelatinized culture dishes with mouse ESCs derivation medium. At 48 hours after plating, one half volume of fresh medium was added to each culture well. The medium was changed daily and inner cell mass-derived outgrowths were dislodged with a Pasteur pipette, washed in a drop of fresh mouse ESC derivation medium and incubated for 5 min in StemPro Accutase (Life Technologies) at 37°C. The clumps were gently dissociated with a Pasteur pipette and transferred onto a fresh plate of MEF To detect different combinations of fl/fl, fl/Δ and Δ/Δ β-catenin alleles, the genomic PCR was performed using the following primer: . The resulting products of 221, 324 and 500 bp corresponded to the wild-type allele, the floxed allele and the deleted allele, respectively in vitro, 1×104 feeder-independent mESCs were plated onto each well of 96-well plate with a low attachment surface  and cultured in differentiation medium containing Knockout DMEM (Life technologies) with 20% FBS , 1×NEAA, 2 mM GlutaMax, 100 U/mL penicillin G and 10 mg/mL streptomycin , to generate embryoid bodies (EBs).For induction of differentiation fl/fl (fl/fl#1 and 2), β-catfl/Δ (fl/Δ#2 and 3), β-catenin-reintroduced β-catΔ/Δ and β-catΔ/Δ  mESCs lines were suspended at 5×106 cells/ml in mESC medium, and 200 µl of the cell suspension (1×106 cells) were injected subcutaneously into the dorsal flank of nude mice  using a 25-G needle. Three to four weeks after injection, tumors were surgically dissected from the mice. Samples were fixed in phosphate-buffered saline (PBS) containing 4% formaldehyde and embedded in paraffin and subjected to histological examination with hematoxylin and eosin (HE) staining. Small samples of the tissue samples were also collected for RNA isolation.For the tumor formation assay, NCH4.3, β-catBstBI and BamHI digestion, and inserted into the BstBI and BamHI site of pIREShyg3 vector (Takara Bio), to create pCMV-mCherry-hyg. To add the 2A peptide sequence to the 3′-end, mouse β-catenin was amplified by PCR with primer pairs 5′-Nhe-Koz-Catnb (gctagcaccatggctactcaagctgacctgatggagttg) and Catnb-T2A-Cla-3′ (atcgattgggccaggattctcctcgacgtcaccgcatgttagcagacttcctctgccctctccggagcccaggtcagtatcaaaccaggccagc) and cloned into pCR2.1-TOPO vector, to create TOPO-Catnb-2A. This β-catenin-2A fragment was excised from TOPO-Catnb-2A by NheI and ClaI digestion, and inserted into the NheI and BstBI sites of pCMV-mCherry-hyg vector, to create pCMV-Catnb-2A-mCherry-hyg. To generate a piggyBac vector carrying a CAG promoter–driven β-catenin-2A-mCherry, β-catenin-2A-mCherry fragment was first amplified by PCR with primer pairs of 5′-Xho-Koz-Catnb (ctcgagaccatggctactcaagctgacctg) and Catnb-Bam-Cla-3′ (atcgatggatccttacttgtacagctcgtcc), then cloned into pCR2.1-TOPO vector, to create TOPO-Catnb-2A-mCherry. This β-catenin-2A-mCherry fragment was excised from TOPO-Catnb-2A-mCherry by XhoI and ClaI digestion, and inserted into the XhoI and ClaI sites of the pPB-CAG-EBNXN vector, kindly gifted by Dr. A Bradley  The β-catenin expression vector was constructed as follows. The mCherry was amplified by PCR with primer pairs 5′-BstBI-mCherry (ttcgaaatggtgagcaagggcgagga) and mCherry-Bam-3′ (ggatccttacttgtacagctcgtcca) to add BstBI and BamHI sites to the 5′- and 3′-sites of mCherry , and cloned into pCR2.1-TOPO vector (Life Technologies), to create TOPO-mCherry. This mCherry fragment was excised from TOPO-mCherry by Δ/Δ mESCs were washed with PBS once, detached by Accutase and suspended in mESC derivation medium. Cells were dissociated into a single-cell suspension by vigorous pipetting and counted. Pellets of 2×106 cells were made and mixed with 1 µg of the β-catenin expression vector and 1 µg of the hyperactive piggyBac transposase expression vector (pCMV-hyPBase), kindly gifted by Dr. A Bradley, in 100 µl of mouse ES cell nucleofector solution . The cell suspension was transferred to a cuvette and electroporated using an Amaxa Nucleofector device (Lonza) with program A30 following the manufacturer’s protocol. The electroporated cells were plated onto 60 mm dishes on MEF in mESC medium. The mCherry-positive colonies were picked up and the clonal lines were maintained as rescued β-catΔ/Δ mESC (res-β-catΔ/Δ mESC).β-catChromosome karyotyping was carried out at the Nihon Gene Research Laboratories, Miyagi, Japan).Alkaline phosphatase (ALP) staining for mouse ESCs was carried out using an alkaline phosphatase kit  according to the manufacturer’s protocol. Immunohistochemistry was carried out on mouse ESCs or EBs seeded on gelatin covered 35 mm glass coverslips  after fixation with 4% paraformaldehyde, followed by permeabilization in PBS with 0.5% Triton-X and blocking with PBS containing 5% normal serum appropriate for each antibody. Incubation with primary antibodies against Oct3/4 , Nanog , Sox2 , E-cadherin , β-catenin , α-catenin , Tuj-1 , Desmin , Afp  and Plakoglobin  was carried out overnight at 4°C. Incubation with corresponding secondary anti-mouse, anti-rabbit and anti-goat antibodies coupled with Alexa 488 or Alexa 546 (BD Biosciences) containing 4′,6-diamidino-2-phenylindole (DAPI) for nuclei staining was carried out for one hour at room temperature in the dark in PBS containing 0.1% bovine serum albumin. Staining was observed under a LSM 720 laser scanning confocal microscope.Tumor samples were fixed in PBS containing 4% formaldehyde and embedded in paraffin. Sections were stained with the following primary antibodies: anti-keratin5/8 , Desmin , NeuN , Oct3/4 , pan- Cytokeratin  , Sall4 , β-catenin .RNA was isolated using the RNeasy Mini Kit . Single strand complementary DNA was synthesized from 1–2 µg of total RNA in 20 µl of reaction mixture containing oligo-dT or random primers using the Superscript III first strand cDNA synthesis system (Life Technologies). We used two types of chemistries for the quantitative reverse transcription polymerase chain reaction (qRT-PCR); TaqMan probe-based [TaqMan Array Mouse Stem Cell Pluripotency Panel (P/N 4385363): Life Technologies] and SYBR Green-based (Platinum SYBR Green qPCR SuperMix-UDG: Life Technologies) systems. Transcript levels were determined using the ABI PRISM Sequence Detection System 7900HT (Life Technologies). All qRT-PCRs using SYBR Green were carried out in triplicate, and relative quantification was carried out using Gapdh as a reference gene. Actb, Gapdh and Eef1a1 were used as endogenous controls when we analyzed gene expression using the TaqMan Array. Hierarchical clustering analyses were performed using Ct values for gene expression data with MEV v4.8 statistical analysis software. The primers used were as follow: Oct3/4, F tgttcccgtcactgctctgg and R ttgccttggctcacagcatc; Nanog, F cctgattcttctaccagtccca and R ggcctgagagaacacagtcc; Lefty1, F gctacaacacagccatgcca and R cctcttttgcctccggagag; Afp, F ccatcacctttacccagtttgt and R cccatcgccagagtttttctt; T, F ctcggattcacatcgtgagag and R aaggctttagcaaatgggttgta; NeuroD1, F acagacgctctgcaaaggttt and R ggactggtaggagtagggatg; Ctnnb1, F ctcaccaccgcgagggcttg and R gcagtccaccagctaggcgc; Tcf3, F gacagaagtggaatttgtgtccg and R agtgcctgctactttctacgat; Sox2, F gcggagtggaaacttttgtcc and R cgggaagcgtgtacttatcctt; Klf4, F aacctttcacactgtcttcccacg and R cccttggactcttcctttctcctg; Axin2, F gcaggagcctcacccttc and R tgccagtttctttggctctt; Sox17, F agccatttcctccgtggtgt and R aacactgcttctggccctcag; Nestin, F tgcatttccttgggataccag and R cttcagaaaggctgtcacaggag; Gapdh, F tgcgacttcaacagcaactc and R cttgctcagtgtccttgctg.Western blots were carried out as described by Ishii et al. with slight modification in vivo contribution of β-catΔ/Δ mESCs, β-catΔ/Δ mESCs, were transfected with the constitutive enhanced green fluorescence protein (EGFP) expression vector (pCAG-EGFP) Δ/Δ mESC (EGFP-β-catΔ/Δ mESC) line, which was continuously cultured on feeder layers in mESC derivation medium with LIF. EGFP-β-catΔ/Δ mESCs were injected into blastocysts of ICR mice, then transferred to the uteri of pseudopregnant ICR mice, as described previously Δ/Δ mESCs with mCherry fluorescence were used for blastocyst injections as donor cells. Embryos were dissected either on embryonic day (E) 10.5 or 12.5 and the contribution of mESCs to embryos was assessed using a fluorescence stereomicroscope .To visualize the ad libitum. All the transgenic mice were monitored for signs of suffering including tumor formation and weakening at least every two days. All surgery was performed under isoflurane anesthesia. Mice were euthanized by carbon dioxide inhalation at humane endpoints or before the collection of embryos and tissue samples as recommended by the American Veterinary Medical Association Panel on Euthanasia. All experiments were subject to the 3 R consideration  and all efforts were made to minimize animal suffering, and to reduce the number of animals used.All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the National Research Institute for Child Health and Development . Mice were housed in plastic cages lined with soft wood chips. The cages were kept in a conventional, air-conditioned mouse room under a 12 h light/dark cycle and the mice received food and water in vitro. The success rate for achieving blastocyst development was around 80%. We established 12 independent stem cell lines from β-catenin-deficient blastocysts and referred to them as β-catΔ/Δ mESCs (Δ/Δ1–12)  . PCR gen-catenin . In addis tested . Furtherfl mESCs . Plakogl/Δ mESCs . β-catΔ/Δ/Δ mESCs were morphologically similar to β-catfl/fl mESCs, expressed Alkaline Phosphatase, and were karyotypically normal  analysis and western blotting  inhibitor, PD0325901, and GSK3β inhibitor, CHIR99021 Δ/Δ, under the serum- and feeder-free conditions using the 2i+LIF system, β-catfl/Δ, and β-catfl/fl mESCs showed a similar rate of cell proliferation  is a direct transcriptional target of β-catenin We sought to determine whether β-catl/fl EBs . The extl/fl EBs . At the tΔ/Δ EBs . Our resΔ/Δ mESCs in vivo, we performed a chimera assay by blastocyst injection using EGFP-β-catΔ/Δ, β-catfl/fl and β-catfl/Δ mESC lines. EGFP-β-catΔ/Δ mESCs were stably maintained with constitutive EGFP expression  composed of seminoma, embryonal carcinoma, and choriocarcinoma characters  by mCherry expression : The examined β-catΔ/Δ mESC lines had normal karyotypes. The β-catΔ/Δ (Δ/Δ2) mESC line showed a normal karyotype of 40XX. (B): Western blots of Nanog and Oct3/4 in wild-type (NCH4.3 and R1), β-catfl/fl (fl/fl2), β-catfl/Δ (fl/Δ2 and fl/Δ3), β-catΔ/Δ  mESCs and MEF. (C): Growth curve of β-catfl/fl (fl/fl2), β-catfl/Δ (fl/Δ3), β-catΔ/Δ (Δ/Δ6 and Δ/Δ8) and res-β-catΔ/Δ (rsc) mESC lines over a period of 5 days in feeder-free and serum-free culture (2i+LIF). Cell population doublings are plotted against days .(TIF)Click here for additional data file.Figure S2fl/fl, β-catfl/Δ and β-catΔ/Δ ESCs.Quantitative PCR examination of β-cat Expression levels of self-renewal marker genes  relative to Gapdh in β-catfl/fl (blue bar: fl/fl1 and fl/fl2), β-catfl/Δ (purple bar: fl/Δ2 and fl/Δ3) and β-catΔ/Δ  mESCs.(TIF)Click here for additional data file.Figure S3Δ/Δ ESCs in serum- and feeder-free conditions of culture.β-cat (A): Phase-contrast images of cellular expansion of β-catfl/fl and β-catΔ/Δ mESCs under serum- and feeder-free conditions using the 2i+LIF system with mitogen-activated protein kinase kinase (MEK) inhibitor (PD0325901) and GSK3β inhibitor (CHIR99021) on days 1, 3 and 5. Scale bars are 200 µm. (B): Quantitative PCR analysis of β-catfl/fl (fl/fl1 and fl/fl2) and β-catΔ/Δ (Δ/Δ1 and Δ/Δ2) mESCs in serum- and feeder-free conditions. Axin2 expression was normalized to Gapdh. In the canonical Wnt/β-catenin signaling cascade, Axin2 acts as the scaffold of the β-catenin destruction complex. Axin2 was not up-regulated in our β-catΔ/Δ mESCs, and so β-catΔ/Δ mESCs are transcriptionally defective in the canonical Wnt/β-catenin pathway.(TIF)Click here for additional data file.Figure S4Δ/Δ ESCs showed restored development potential in the chimera assay.β-catenin-rescued β-cat (A): β-catΔ/Δ mESCs with an integrated piggyBac vector carrying a CAG promoter–driven β-catenin-2A-mCherry (res-β-catΔ/Δ mESCs) expressed red fluorescent protein mCherry. Scale bars are 500 µm. (B): Immunofluorescence staining for β-catenin (red), α-catenin (green), and E-cadherin (green) of res-β-catΔ/Δ mESC colonies as observed under confocal microscopy. Nuclei are stained for DAPI (blue). Scale bars are 20 µm. (C): Chimeras were generated by injection of res-β-catΔ/Δ mESCs into ICR host blastocysts. Chimeric embryos on E10.5 displayed the high contribution of res-β-catΔ/Δ mESCs to the whole body. Scale bars are 500 µm.(TIF)Click here for additional data file.Figure S5Hierarchical clustering analysis of expression data from the TaqMan array across the 96 marker genes. Multiple gene expression analysis of mESC lines and F9 (A) and tumors (B) by quantitative PCR using TaqMan Array Mouse Stem Cell Pluripotency Card (Life technologies). (A): The two subtypes of stem cell lines were clustered into distinct clusters with reversed gene expression patterns. The group of wild-type, res-β-catΔ/Δ and β-catΔ/Δ mESC lines was clustered from F9 EC. (B): Tumor clustering was different from stem cells. β-catΔ/Δ tumors were clustered into the same cluster as tumors derived from F9 EC, and separately clustered from teratomas of wild-type and res-β-catΔ/Δ mESCs. The level of expression of each gene in each sample, relative to the median level of expression of that gene across all the samples, is represented using a red-black-green color scale as shown in the key .(TIF)Click here for additional data file.Figure S6Δ/Δ mESCs.Chimeric embryos at E12.5 generated from EGFP-β-cat Contribution of EGFP-β-catΔ/Δ mESCs to mouse embryonic development. Embryos were analyzed using a ﬂuorescence stereomicroscope on E12.5. Embryos with scattered EGFP fluorescence showed limb malformations (white arrow head). Scale bars are 2 mm.(TIF)Click here for additional data file.Figure S7fl/fl, β-catΔ/Δ and res-β-catImmunofluorescence staining of Plakoglobin in β-catΔ/Δ. Immunofluorescence staining for Plakoglobin (green) and DAPI (blue) of β-catfl/fl, β-catΔ/Δ and res-β-catΔ/Δ mESC colony as observed under confocal microscopy. Scale bars are 20 µm.(TIF)Click here for additional data file.


Mouse genetic study has demonstrated that Axin2 is essential for calvarial development and disease. Haploid deficiency of β-catenin alleviates the calvarial phenotype caused by Axin2 deficiency. This loss-of-function study provides evidence for the requirement of β-catenin in exerting the downstream effects of Axin2.Here we utilize a gain-of-function analysis to further assess the role of β-catenin. A transgenic expression system permitting conditional activation of β-catenin in a spatiotemporal specific manner has been developed. Aberrant stimulation of β-catenin leads to increases in expansion of skeletogenic precursors and the enhancement of bone ossification reminiscent to the loss of Axin2. The constitutively active signal promotes specification of osteoprogenitors, but prevents their maturation into terminally differentiated osteoblasts, along the osteoblast lineage. However, the prevention does not interfere with bone synthesis, suggesting that mineralization occurs without the presence of mature osteoblasts. β-catenin signaling apparently plays a key role in suture development through modulation of calvarial morphogenetic signaling pathways. Furthermore, genetic inactivation of the β-catenin transcriptional target, cyclin D1, impairs expansion of the skeletogenic precursors contributing to deficiencies in calvarial ossification. There is a specific requirement for cyclin D1 in populating osteoprogenitor cell types at various developmental stages.These findings advance our knowledge base of Wnt signaling in calvarial morphogenesis, suggesting a key regulatory pathway of Axin2/β-catenin/cyclin D1 in development of the suture mesenchyme. The mammalian skull consists of neurocranium and viscerocranium that are formed from skeletogenic mesenchyme derived from both mesoderm and neural crest . The craA patent suture is necessary for continuous growth of the skull bones. Defects in either cell proliferation, differentiation, or apoptosis affecting the process of intramembranous ossification have been shown to induce premature fusion of cranial sutures, leading to development of craniosynostosis . AbnormaOur previous work has linked an evolutionary conserved Wnt signal transduction pathway for the first time to craniosynostosis . Mice wiIn this study, we employ a gain-of-function analysis to determine if aberrant activation of β-catenin induces calvarial defects reminiscent to the Axin2 mutant. A transgenic system integrating the tetracycline-dependent activation and Cre-mediated recombination methods has been developed. Our findings demonstrate that inducible stimulation of β-catenin causes severe suture abnormalities resembling those with the Axin2 ablation. The expansion of skeletogenic precursors and their subsequent differentiation are altered, leading to increased bone ossification. β-catenin signaling plays a critical role in development of the suture mesenchyme through modulation of calvarial morphogenetic signaling pathways. Furthermore, analyzing mice with disruption of the β-catenin transcriptional target, cyclin D1, we reveal its essential role in the expansion of skeletal precursors. Cyclin D1 is required for propagation of specific types of osteoprogenitors at various developmental stages.S-M2, under control of the Axin2 promoter, we tested the capability of conditional gene expression in the developing calvaria. Mice carrying the Axin2-rtTA transgene were crossed with TRE-lacZ mice to obtain double transgenic mice , which lacks important phosphorylation sites [Ax2 mouse model permits stimulation of β-catenin in a spatiotemporal specific fashion during formation of the craniofacial skeleton. Inducible expression of sβ-cat in the Axin2-expressing cells had significant effects on craniofacial skeletogenesis . Similar to that observed in the Axin2 nulls, the expression of a direct Wnt target, cyclin D1, was highly stimulated . This was accompanied by stabilization of its target Cdk4, which promotes cell cycle G1/S transition . Statistical analysis showed that these alterations in cell proliferation significantly contribute to the mesenchymal defects of the sβcatAx2 sutures , Fgfr2  and Fgf2 , affected by Axin2 ablation [Ax2 mutants. Stimulated expression of phosphorylated Smad1/5/8 proteins found in the Axin2 nulls was also observed by the expression of sβ-cat . The results imply that β-catenin stimulation is able to recapitulate the effect of the Axin2 ablation, leading to activation of the FGF and BMP pathways . Unfortunately, mice with inducible expression of the dominant β-catenin protein are unable to survive beyond 4 weeks after the initial activation of transgene takes place. The lethality issue, most likely due to the Axin2 promoter being active in other tissues and organs, has prevented us from assessing the long term effect of the transgenic expression. Nonetheless, the new finding nicely complements our previous work using a loss-of-function analysis in which haploid deficiency of β-catenin alleviates the skull defects caused by the Axin2 disruption .We have previously shown that as a transcriptional target of Wnt, cyclin D1 is modulated by alterations of the Axin2/β-catenin regulatory pathway ,7. HowevAs a direct transcriptional target of β-catenin, cyclin D1 acts in this regulatory pathway. However, the loss of cyclin D1 interferes with only the Runx2 negative naïve cells within the suture mesenchyme, but not the Runx2 positive progenitors at the osteogenic fronts at newborns. The skeletogenic signaling pathways of FGF and BMP are also unaffected at the osteogenic fronts. Our data imply that the restoration of skeletogenic activities at the osteogenic fronts of cyclin D1-/- might be a compensation effect of other cyclins with similar function. The loss of cyclin D1 may be compensated by cyclin D2, D3 and E. Nevertheless, we cannot rule out the possibility that the lethality associated with the cyclin D1 deletion could result in selected phenotypic representation. This is because only 72% of mice with disruption of cyclin D1 able to survive after birth . The mutUsing sophisticated mouse genetic studies, we demonstrate that aberrant stimulation of β-catenin causes calvarial defects resembling those of the Axin2 mutation. Constitutive activation of β-catenin enhances both expansion of skeletogenic precursors and bone ossification. Together with our previous findings, these results indicate that β-catenin plays an essential role in the calvarial morphogenetic signaling pathways. In addition, as a direct transcriptional target of β-catenin, cyclin D1 is required for the expansion of skeletal precursors during calvarial morphogenesis. This study provides genetic evidence to further support the importance of Wnt signaling in calvarial morphogenesis, suggesting a key regulatory pathway of Axin2/β-catenin/cyclin D1 in development of the suture mesenchyme.Ax2 mouse strain, mice carrying Axin2-rtTA and TRE-Cre transgenes were first bred into the β-catΔEx3Fx heterozygous background [The Axin2-rtTA ,33, TRE-ckground . The exp4) were seeded in 24 well plates, and maintained in differentiation media containing 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate for 6 days, followed by alkaline phosphatase staining as described [Isolation and culture of primary skeletal precursors were performed as described . BrieflySkulls were fixed for skeletal preparation or paraffin embedded, sectioned and stained with hematoxylin/eosin for histology or von Kossa staining as described ,7. Detaip values were obtained using student t test.Immunological staining with avidin:biotinlylated enzyme complex was performed as described ,7. The sAJM, HIY and WH established the mouse models and carried out the genetic studies. AJM, TM and WH designed the experiments. AJM, TM, JF and HIY performed molecular characterizations, cell proliferation and differentiation assays and statistical analyses. AJM, TM and WH wrote the paper. All authors read and approved the final manuscript.Figure S1: The skeletogenic activities are restored at the osteogenic fronts of cyclin D1-/- after birth. Immunostaining analysis reveals that the expression of Fgf2 , Fgfr1 , Fgfr2  and phosphorylated Smad1/5/8  is not affected in the mutant metopic sutures  compared to the control  at newborn. Arrows indicate the osteogenic fronts. Scale bars, 50 μm (A-H).Click here for file


The glucagon receptor (GCGR) is a member of the class B G protein–coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin–mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor–related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter–mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations. G protein–coupled receptors (GPCRs) with seven-transmembrane domains form a large family that respond to extracellular signals by activating heterotrimeric G proteins. The glucagon receptor (GCGR) is a class B GPCR. Its ligand, glucagon, is a 29-amino acid peptide secreted by the islet A cells of the endocrine pancreas. The binding of glucagon to its receptor activates the cAMP/protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) pathways Frizzled (Fz) receptors, which are known as atypical GPCRs, are receptors for the Wnt family of secreted glycoproteins A number of classical GPCRs have been shown to cross-talk or activate the β-catenin pathway in a Wnt-independent manner by various mechanisms. The stimulation of α-adrenergic receptors on cardiomyocytes , or prostaglandin E2 receptors on colon cancer cells  results in the stabilization of β-catenin and the activation of β-catenin signaling s-coupled GPCRs including PTH1R and GCGR It was reported that binding of parathyroid hormone (PTH) to its receptor PTH1R induces activation of the β-catenin pathway through Lrp6 As a classical GPCR, activation of the glucagon receptor causes an increase of intracellular cAMP level, which in turn activates the PKA signaling pathway to activate cAMP-response element (CRE)-mediated gene expression. Using the CRE-Luc reporter construct, we found that HEK293 without GCGR transfection did not respond to GCG1-29 stimulation . After tTo check whether glucagon can activate the β-catenin signaling pathway, we first used HEK293 cells transfected with the GCGR receptor. Treatment with the agonist GCG1-29 increased β-catenin protein levels relative to a control, non-treated sample (NT) or that treated with the antagonist GCG9-29 . As a poActivation of the β-catenin pathway leads to stabilization of β-catenin in the cytosol, which can translocate into the nucleus and associate with TCF transcription factors to activate TCF promoter–mediated gene expression. Because we observed the stabilization of β-catenin protein upon activation of GCGR receptor, we next examined whether activation of GCGR stimulated TCF promoter–mediated luciferase activity, an indicator for an active β-catenin signaling pathway. 293STF cells (HEK293 cells stably transfected with TCF-Luc reporter DNA) We observed an increase in β-catenin protein level and TCF-mediated luciferase activity upon activation of GCGR. Because Lrp5/6 is an essential coreceptor for Wnt/β-catenin signaling, we asked whether cotransfection with Lrp5/6 will potentiate glucagon-induced β-catenin signaling. HEK293 cells transfected with GCGR had a modest increase in β-catenin protein level upon GCG1-29 treatment, whereas cotransfection with GCGR and Lrp5 caused a larger increase . Next, wTo understand the mechanism of glucagon-induced β-catenin signaling, we first asked whether glucagon-induced cAMP/PKA activity is required for activating the β-catenin pathway. HEK293 cells were transfected with GCGR and treated with GCG1-29 and H89, a PKA inhibitor. Inhibition of PKA activity completely blocked the activation of the β-catenin pathway induced by GCG1-29 . In anotWe next asked whether inhibition of Lrp5/6 would reduce glucagon-induced β-catenin signaling, and we used two approaches. First, we used a dominant negative inhibitor of Lrp5/6, the Lrp5 extracellular domain (ECD) Because cotransfection with GCGR and Lrp5 increases glucagon-induced β-catenin stabilization and TCF luciferase activity, we examined whether GCGR and Lrp5 physically interact with each other by immunoprecipitation. HEK293 cells were cotransfected with HA-tagged GCGR and v5-tagged Lrp5. Using western blots, we found that both proteins were well expressed . If we iIn this report, we found that activation of the glucagon receptor not only led to activation of the classic cAMP/PKA pathway , but alsPTH1R, GLP-1R and GCGR all belong to class B GPCR family, which have the closest phylogenetic relationship to Frizzled receptors Using immunoprecipitation, we found that Lrp5/6 physically interacted with GCGR , S1. UsiOur data suggested that association of GCGR and Lrp5 alone is not sufficient for activation of the downstream β-catenin pathway. In addition, ligand binding is required, presumably through inducing conformational changes of GCGR and phosphorylation of Lrp5/6 to activate the downstream β-catenin pathway. However, pre-association of GCGR with Lrp5/6 on the cell surface can greatly facilitate the signaling communications between GCGR and Lrp5/6. So activation of GCGR upon ligand binding can directly cross-talk to Lrp5/6 to transmit downstream β-catenin signaling whereas phosphorylation and activation of Lrp5/6 on the other hand can communicate back to GCGR to boost GCGR mediated cAMP/PKA pathway. This mutual communication is supported by our cell-based reporter data showing that cotransfection of Lrp5 not only enhanced glucagon induced β-catenin signaling  but alsoGlucagon-induced β-catenin signaling is relatively weaker than Wnt protein-induced β-catenin signaling. The relative weak signal was not due to lack of interaction between GCGR and Lrp5/6, but may be intrinsic to GCGR itself. In Frizzled receptors, two residues in the intracellular loops 1 and 3 (Arg263 and Leu443 in human Fz5) and a motif in the C-terminal tail (KTXXXW) play an important role in Dishevelled protein recruitment and Wnt/β-catenin signaling What is the physiological consequence of cross-talk to β-catenin signaling from GCGR? Wnt/β-catenin signaling helps to promote stem cell renewal and in many cases favors proliferation over differentiation. Several lines of evidence suggest that Wnt/β-catenin signaling may help pancreatic cells survive and proliferate. First, Wnt/β-catenin signaling is involved in the genesis of pancreatic islets and the proliferation of pancreatic beta cells Overall, our results and others suggested that the function for Lrp5/6 is broader than being a coreceptor for the canonical Wnt signaling pathway and it can serve as a key scaffold molecule to participate in both β-catenin and cAMP/PKA signaling pathways. By engaging with several different GPCRs, it can regulate a range of important physiological functions including embryonic development, bone formation, and metabolism. Our results may help to explain the pleiotropic phenotypes of Lrp5 and 6 mutations. To better understand the physiological role of Lrp5/6 in metabolism, future studies with Lrp5/6 conditional knockout or mutant mice are needed.All animal work has been carried out in accordance with US National Institutes of Health guidelines. All procedures utilized in this study were evaluating for animal welfare and ethics considerations and were approved by the VARI Institutional Animal Care and Use Committee (VARI-IACUC) prior to initiation (VAI-IACUC protocol ID #09-10-025).LiCl was purchased from Sigma Aldrich. The antibodies used were mouse anti-β-catenin monoclonal antibody (BD Biosciences), mouse anti-β-actin antibody (Abcam), mouse anti-v5 antibody and v5 antibody-conjugated agarose beads (Sigma Aldrich), rabbit anti-GCGR antibody (H-57) (Santa Cruz), and mouse anti-HA antibody (Covance). Forskolin and H89 were obtained from Calbiochem. A Dual Luciferase assay kit was purchased from Promega. GLP-1 peptides, glucagon peptides, and PTH peptides were purchased from peptide 2.0. Dickkopf-1 (DKK1) protein was purchased from R&D systems.Renilla luciferase (Rlu) gene under the control of the thymidine kinase promoter was used as a transfection control (Promega). The TCF-Luc plasmid (M50 Super 8× TOPFlash), with the firefly luciferase gene under the control of the TCF promoter, was obtained from Addgene. Mouse DKK1, Lrp5, Lrp6, and Wnt5-Fz8 plasmids have been described before The TKRlu plasmid with the HEK293, COS-1 and Hep3B cells were from ATCC. 293STF cells (which are HEK293 cells with an integrated “Super-Top-Flash” TCF-luciferase reporter) have been described HEK293 or other cells were cultured in 6-well plates . For experiments requiring transfection, cells were transfected with the indicated DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After transfection, cells were treated with different reagents for the indicated time. Cell pellets were lysed in 90 µl of 1× cell lysis buffer  on ice for 30 min and western blots were performed as described earlier HEK293 cells in 6-well plate were transfected with HA-tagged GCGR with or without v5-tagged Lrp5 plasmids on day 1 with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. On day 2, cells were treated with or without GCG1-29 for 1 h. Cells were subsequently harvested and lysed in 100 µl of 1× cell lysis buffer  on ice for 30 min. Cell debris was removed by centrifugation. The ExactaCruz™ E kit from Santa Cruz was used to immunoprecipitate HA-tagged GCGR from the cell lysate with 2–5 µg mouse anti-HA antibody according to the manufacturer's protocol. For immunoprecipitation with v5 antibody, 50 µl of anti-v5 agarose beads were directly incubated with the cell lysate for 2 h at 4°C with mixing induced by rotation of the tube. The beads were spun down and washed three times with 1× TBST  buffer. Then 50–60 µl of 2× reducing electrophoresis buffer was added to the beads and they were incubated at 37°C for 5 min before loading onto an SDS-gel for western blot analysis.Renilla luciferase) by use of Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were induced with the indicated reagents at 24 h after transfection. Seventeen hours after induction, cells were harvested and lysed in the plate with 100 µl of 1× passive lysis buffer (Promega) at room temperature for 15 min, and the firefly and Renilla luciferase activities were measured on an Envision luminometer (Perkin-Elmer) with the Dual Luciferase assay kit (Promega). Firefly luciferase data were normalized to Renilla luciferase data.To measure the CRE reporter activity, HEK293 cells were transfected with CRE-Luc DNA. To measure the TCF reporter activity, either 293STF cells or HEK293 cells transfected with the TCF-Luc plasmid were used. HEK293 or 293STF cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal bovine serum (FBS). Cells were plated on a 24-well plate for 24 h prior to transfection. Cells were transfected in Opti-MEM I (Invitrogen) with various plasmids+10 ng of control TKRlu plasmid . After 24 h, the cells were transiently transfected with 3 µg of DNA/dish using the diethylaminoethyl (DEAE)-dextran method h (Renilla luciferase-specific substrate)  with the cell suspension. The luminescence signals were collected immediately using a 2103 Envision fluorescence plate reader configured with the <700 nm dichroic mirror and with dual emission filter sets for luminescence  and fluorescence . Fluorescence of the YFP was acquired by exciting the samples at 485 nm and collecting the emission at 525 nm. The BRET ratios were calculated based on the ratio of emission from YFP and Rlu, as described previously BRET experiments were performed using COS-1 cells as described previously 2 values using Prism 4.0. .Saturation BRET studies were also performed as described previously Figure S1Lrp6 physically interacts with GCGR. A). HEK293 cells were transfected with a control vector (GFP), v5-tagged Lrp6, HA-tagged GCGR, or both for 24 h and then were treated with or without 50 nM GCG1-29 for 1 h. Cells were harvested and lysed and used for western blot analysis. The blot was probed with v5 antibody and then stripped and reprobed with HA antibody. The β-actin blot was used as a loading control. B). HEK293 cells were transfected and treated similarly as in A. The cells were harvested and lysed, and equal amounts of lysate were immunoprecipitated with the indicated antibody. For HA antibody, the antibody complex was pulled down by protein G beads. For v5 antibody, it was a single-step pull-down because the antibody was directly conjugated to the agarose beads. After pull-down, the beads were washed three times with 1× TBST and then incubated in 1× SDS sample buffer to release the bound proteins. The lysates were used for western blot analysis and probed with the indicated antibody.(TIF)Click here for additional data file.Figure S2Coexpression of Lrp5 enhanced the CRE Luciferase activity. HEK293 cells were transfected with GCGR or GCGR+Lrp5 plasmids along with CRE-Luc and TKRlu  on day 1. Cells were left untreated or treated with 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the luciferase activity as described.(TIF)Click here for additional data file.Figure S3Sequence alignment of the intracellular loop 1 and 3, C-terminal region of Frizzled receptors and three class B GPCRs. The IC loops and C-terminal region were predicted by the HMMTOP server (TIF)Click here for additional data file.Figure S4Effects of glucagon and Wnt3a on the TCF Luc reporter activity in the 293STF cells expressing GCGR and Lrp5. 293STF cells were transfected with 100 ng each of Lrp5 and GCGR plasmids and 10 ng of TKRlu  on day 1. Cells were either left untreated, or treated with 50 nM GCG1-29, 10% Wnt3a Conditioned Media (CM) or 50 nM GCG1-29+10% Wnt3a CM on day 2. Cells were harvested on day 3 to measure the luciferase activity as described in the “(TIF)Click here for additional data file.Figure S5The immunoprecipitation experiment using HA antibody. HEK293 cells were transfected with 2000 ng of the indicated DNAs and treated with or without 50 nM GCG1-29 for the indicated time, similarly as in (TIF)Click here for additional data file.


Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling. The receptor tyrosine kinase (RTK) and WNT/β-catenin signaling systems represent two major routes for cellular communication that synergistically regulate many essential developmental and regenerative processes, but the mechanisms of their cross-talk remain poorly defined. Inactivation of glycogen synthase kinase 3 (GSK3) is a critical event in WNT/β-catenin signal transduction as GSK3-mediated phosphorylation of β-catenin targets it for degradation In contrast to this simple hypothesis, experimental evidence argues that the PI3K/AKT pathway does not activate WNT/β-catenin. Ectopic AKT activation or insulin treatment , both fail to activate WNT/β-catenin signaling In light of these data, the exact mechanism of RTK and WNT/β-catenin signaling cross-talk remains an open question. We recently demonstrated that ERK MAP kinase activates WNT/β-catenin signaling via phosphorylation of WNT co-receptor low density lipoprotein receptor-related protein 6 (LRP6) rat chondrosarcoma chondrocytes (RCS) for effects of FGF signaling, we found FGF2-mediated upregulation of the Topflash luciferase reporter, which records the transcriptional activation of canonical WNT/β-catenin pathway. FGF2 activated Topflash in all 10 experiments conducted  (When probing  n = 10) . When co n = 10) . This wa n = 10) . The Top n = 10) . We next n = 10) . Importa n = 10) . CollectWe next asked whether the PI3K/AKT pathway accounts for the effect of FGF2 on WNT3a/β-catenin signaling. FGF2 induced some PI3K/AKT activation in RCS cells, as determined by WB for AKT phosphorylation at Ser473 or a cell-free AKT kinase assay. AKT activation was accompanied by increased inhibitory phosphorylation of GSK3α at Ser21 . HoweverExamining the WNT/β-catenin pathway for components that were subject to FGF2-induced phosphorylation revealed WNT co-receptor LRP6 phosphorylation at Ser1490 and Thr1572 . Ser1490FGF2 effect on the WNT3a/β-catenin pathway requires LRP6 phosphorylation at its PPPS/TP motifs . RecentlDetailed analysis of the endogenous LRP6 SDS-PAGE migration pattern shows two discrete protein bands in RCS cells. Interestingly, while treatment with FGF2 caused phosphorylation of both the upper and lower LRP6 bands, WNT3a only induced phosphorylation of the upper form . WNT3a-mTransfected LRP6 also migrated as two bands when expressed in RCS or HEK293 cells. Treatment with the glycosylation-inhibitor tunicamycin, or the Golgi-transport inhibitor brefeldin resulted in disappearance of the upper LRP6 band . De-glycThe ERK MAP kinase represents a common signaling intermediate utilized by many systems. We therefore asked whether the signaling systems unrelated to FGF are also capable to signal via the ERK/LRP6 pathway. First, we focused on small GTPase RAS and RAF-1 kinase that both lie upstream of ERK in the ERK signaling module. Expression of constitutively active forms of RAS (RasV12) or RAF-1 (RafCAAX) lead to ERK activation, and corresponding strong induction of basal or WNT3a-mediated Topflash activity which was sensitive to U0126 . MoreoveWe next asked whether other signaling systems signal utilize ERK/LRP6 pathway. Expression of constitutively active variant of non-receptor tyrosine kinase SRC (SRC-Y529F) in RCS cells resulted in ERK activation accompanied by potent induction of basal or WNT3a-mediated Topflash activity, which was inhibited by U0126 . MoreoveAmong the FGFRs, RCS cells express FGFR2 and FGFR3, and we showed the effect of endogenous, wt FGFR2 and FGFR3 activation on WNT/β-catenin signaling . To testAmong the FGFR2 mutants, expression of C342R, C342Y and Y375C FGFR2 resulted in spontaneous ERK activation and corresponding LRP6 phosphorylation at Thr1572 . MoreoveWhen used alone, FGF2 increased basal Topflash levels in RCS cells  as did oA critical event in WNT/β-catenin signaling appears to be the phosphorylation within the intracellular PPPS/TP motifs of LRP6. Removal of any of the five PPPS/TP motifs impairs WNT signaling; removal of all five motifs results in complete loss of WNT/β-catenin signal transduction How does the RTK/ERK-mediated LRP6 phosphorylation activate WNT/β-catenin signaling? Activation of FGFR2 and FGFR3, via cell treatment with FGF2 ligand, induced total LRP6 phosphorylation at levels similar to WNT3a , yet it WNT-induced LRP6 phosphorylation requires signalosome assembly and thus can only target the fully mature, transmembrane form of LRP6. This differs from ERK-mediated LRP6 phosphorylation, since ERK is a cytosolic kinase that can phosphorylate both the mature (transmembrane) LRP6 and immature (intracellular) LRP6 during its Golgi-based cell membrane translocation/maturation pathway . We specWhen used alone, FGF2 increased basal Topflash levels in RCS cells. Although this induction appeared weak compared to Topflash activation caused by WNT3a or FGF2/WNT3a, it represented signal increase by more than 100% , when coOur data predict that activation of WNT/β-catenin signaling via LRP6 phosphorylation may represent a common signaling theme since many diverse signaling systems utilize ERK MAP kinase. Moreover, we show that activating mutants of FGFR2 and FGFR3 signal via ERK/LRP6 pathway. These mutants are associated with more than 10 human conditions, including syndromes affecting skeleton , skin , and cancer development  RCS and HEK293 cells were propagated in DMEM media, supplemented with 10% FBS and antibiotics . The growth factors and chemicals were obtained from the following manufacturers: FGF1, FGF2, WNT3a, WNT5a, DKK1, EGF, NGF ; heparin (Invitrogene); wortmannin, LY294002, U0126 ; tunicamycin ; anisomycin ; SU5402, brefeldin A . For de-glycosylation, cells were lysed in 0.5% NP40 lysis buffer supplemented with protease inhibitors , and 25 µl of lysate was incubated with 3 µl of N-Glycosidase F (Roche) for 16 hours at 37°C.3VO4. Protein samples were resolved by SDS-PAGE, transferred onto a PVDF membrane and visualized by chemiluminiscence . Integrated optical density (I.O.D.) of the WB signal was quantified by Scion Image software . The following antibodies were used: β-catenin ; P-LRP6T1572 ; ERK1/2, P-ERK1/2T202/Y204, JNK, P-JNKT183/Y185, p38, P-p38T180/Y182, P-ELK1S383, AKT, P-AKTS473, GFP, GSK3, P-GSK3αS21, P-GSK3α/βS21/9, LRP6, P-LRP6S1490, P-β-cateninS33/37, P-β-cateninS33/37/T41 ; P-β-cateninY142 ; ACTIN, FGFR2, FGFR3, TRKA, EGFR ; V5 (Invitrogene); α-tubulin . AKT and ERK immunoprecipitations and kinase assays were performed according to the manufacturer’s protocol . For the recombinant ERK kinase assay, the reactions were carried-out with 300 ng of recombinant ERK and 600 ng of recombinant LRP6 in 50 µl of kinase buffer  in the presence of 200 µM ATP for 30 minutes at 30°C. Recombinant LRP6 was described earlier Cells were lysed in buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 25 mM NaF, supplemented with proteinase inhibitors and 10 mM NaCells were fixed in 4% paraformaldehyde, mounted in Vectashield medium containing DAPI for nuclear staining , and stained with β-catenin-AlexaFluor 488  antibody, according to manufacturer’s protocol. Confocal fluorescence and two photon laser scanned images were taken on a Leica TCS-SP MP confocal microscope .Renilla vector was 3∶1. The luciferase activity was determined using a Dual-Luciferase Reporter Assay (Promega). The FGFR3 vectors and vectors carrying different LRP6 variants were described previously Cells were transfected using the FuGENE6, according to manufacturer’s protocol (Roche). For the luciferase reporter assays, the µg ratio between the Topflash luciferase vector (obtained from R. Moon) and pRL-TK  control Figure S1FGF2 upregulates WNT/β-catenin signaling independent of the PI3K/AKT pathway. (A) RCS cells were treated with FGF2 (10 ng/ml) for the indicated times and analyzed for activatory Ser473 phosphorylation of AKT by WB. (B) AKT was immunoprecipitated from FGF2-treated cells and used in a kinase assay with recombinant GSK3 as a substrate. C1, C2 - untreated controls, samples with omitted ATP serve as a negative control for the kinase assay. Note the slight FGF2-mediated activation of AKT determined by both Ser473 WB and a kinase assay (quantified by densitometry), with corresponding increase in AKT-mediated inhibitory phosphorylation of GSK3α/β at Ser21/9. (C) Cells were transfected with Topflash firefly luciferase (F-Luc) and control Renilla luciferase (R-Luc) vectors, pretreated with PI3K/AKT inhibitors (Wortmannin and LY294002) for 1 hour prior to FGF2 (10 ng/ml) and WNT3a (40 ng/ml), and analyzed for luciferase activity 20 hours later. Both inhibitors showed little effect on FGF2 and/or WNT3a-mediated Topflash activation, when compared with ERK pathway inhibition (Renilla luciferase (R-Luc) vectors, treated with FGF2 and/or WNT3a, and analyzed for luciferase activity 20 hours later. Note the lack of ca-AKT-GFP effect on both FGF2 and WNT3a-mediated Topflash activation.hibition . (D) Cel(PDF)Click here for additional data file.Figure S2Analysis FGFR3 and LRP6 transgene expression in RCS cells. (A) Cells were transfected with high amounts of either V5-tagged LRP6 (upper panel) or FGFR3 (lower panel) vectors (6 µg of plasmid per 1×105 cells), and analyzed for the indicated molecules 48 hours later. K508M - kinase inactive FGFR3 mutant. Note the high amounts of Ser1490 phosphorylation of wild-type LRP6, indicating its constitutive activation in an overexpressed state. Also note the ERK activation in cells overexpressing highly active FGFR3 mutant K650E (lower blot). (B) Cells were transfected with the indicated FGFR3 variant together with LRP6 (0.4 µg of FGFR3 vector + 2.4 µg of LRP6 vector per 1×105 cells), Topflash firefly luciferase (F-Luc) and control Renilla luciferase (R-Luc) vectors, grown for 48 hours, analyzed for indicated molecules by WB, and used for one of the three Topflash experiments presented in (C). Spontaneous wt LRP6 phosphorylation, as well as ERK activation by FGFR3-K650E are detectable even at low amount of the transfected vectors (quantified by densitometry). (C) Cells were transfected with the indicated FGFR3 variant together with LRP6, Topflash luciferase (F-Luc) and control Renilla luciferase (R-Luc) vectors, grown for 48 hours, and analyzed for luciferase activity. Data represent an average from three independent experiments, with the indicated standard deviations . K508M - kinase inactive FGFR3 mutant.(PDF)Click here for additional data file.Figure S3Constitutively active forms of RAF, RAS and SRC signal via ERK/LRP6 pathway.  RCS cells were transfected with empty plasmid or plasmid encoding RafCAAX, RAS-V12 or SRC-Y529F, grown for 48 hours, and analyzed for indicated molecules by WB.  Cells were transfected as indicated together with Topflash firefly luciferase (F-Luc) and control Renilla luciferase (R-Luc) vectors, grown for 24 hours, treated with U0126 (20 µM) one hour before WNT3a (40 ng/ml), and analyzed for luciferase activity 20 hours later. Data represent an average from three or four transfections (each measured twice). Potent Topflash activation mediated by RafCAAX, RAS-V12 or SRC-Y529F was significantly rescued by U0126 .  Cells were transfected with RafCAAX, RAS-V12 or SRC-Y529F together with wt LRP6 or LRP6-5A mutant, Topflash luciferase (F-Luc) and control Renilla luciferase (R-Luc) vectors, grown for 48 hours, and analyzed for luciferase activity. Data represent an average from three or four transfections (each measured twice), with the indicated standard deviations. Statistically significant differences are indicated .(PDF)Click here for additional data file.Figure S4Effect of activating FGFR3 mutants on basal levels of Topflash activity. Cells were transfected with the indicated FGFR3 vectors together with Topflash firefly luciferase (F-Luc) and control Renilla luciferase (R-Luc) vectors, and analyzed for luciferase activity 48 hours later. Data represent an average from four transfections (each measured twice), with the indicated standard deviations . Results are representative of four experiments. Note the differences in the basal Topflash transactivation mediated by FGFR3 mutants, which correspond to relative levels of FGFR3 activation by each particular mutation (PDF)Click here for additional data file.Figure S5Effect of FGF signaling on WNT/β-catenin signaling in PC12 cells. Cells were transfected with Topflash vector, the indicated FGFR3 expression plasmid, and Renilla luciferase vector at a ratio of 1∶1∶0.1. Cells were transfected with a total of 10.5 µg DNA using Lipofectamine 2000 (Invitrogen). The following day, cells were serum-starved 3 hours, treated with 40 ng/ml FGF1 and/or 20 ng/ml WNT3a, and analyzed for luciferase activity 24 hours later. F- FGF1, W - WNT3a, wild-type - wt FGFR3, kinase dead - K508M-FGFR3, K650E - K650E-FGFR3 mutant. Data represent three independent experiments. Statistically significant differences are indicated .(PDF)Click here for additional data file.


ABCB1, which gives rise to the multidrug resistance (MDR) phenomenon. MDR is characterized by resistance to nonrelated drugs, and P-glycoprotein (encoded by ABCB1) has been implicated as the major cause of its emergence. Wnt signaling has been demonstrated to be important in several aspects of CML. Recently, Wnt signaling was linked to ABCB1 regulation through its canonical pathway, which is mediated by β-catenin, in other types of cancer. In this study, we investigated the involvement of the Wnt/β-catenin pathway in the regulation of ABCB1 transcription in CML, as the basal promoter of ABCB1 has several β-catenin binding sites. β-catenin is the mediator of canonical Wnt signaling, which is important for CML progression.The advanced phases of chronic myeloid leukemia (CML) are known to be more resistant to therapy. This resistance has been associated with the overexpression of In this work we used the K562 cell line and its derived MDR-resistant cell line Lucena (K562/VCR) as CML study models. Real time PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), flow cytometry (FACS), western blot, immunofluorescence, RNA knockdown (siRNA) and Luciferase reporter approaches were used.ABCB1 in both cell lines in vitro, but its binding was more pronounced in the resistant cell line in vivo. Lucena cells also exhibited higher β-catenin levels compared to its parental cell line. Wnt1 and β-catenin depletion and overexpression of nuclear β-catenin, together with TCF binding sites activation demonstrated that ABCB1 is positively regulated by the canonical pathway of Wnt signaling.β-catenin was present in the protein complex on the basal promoter of ABCB1 in CML.These results suggest, for the first time, that the Wnt/β-catenin pathway regulates  Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL constitutive tyrosine kinase (TK) oncoprotein, the result of the balanced reciprocal translocation of chromosomes 9 and 22 q34;q11)) [1) [1]. BBCR-ABL amplification. Nevertheless, some patients do not present either mechanism or respond to therapy, suggesting other mechanisms, the so-called BCR-ABL-independent mechanisms. Among them is the multidrug resistance (MDR) phenotype that is dependent on the expression of proteins that function as extrusive pumps  ATP and T4 polynucleotide kinase. For binding reactions, 7 μg of nuclear protein extracts, prepared as previously described [TCAAAGGG-3′Dephosphorylated oligonucleotides (25 pmol) were end-labeled with γ-P ATP andescribed . Opt 5′-ABCB1 promoter, protein extracts were incubated for 2 h with 1 μg of the anti-β-catenin (Sigma) and anti-Smad8 (Santa Cruz Technologies) antibodies at 4 °C before the addition of the probe. Anti-Smad8 was used as a negative control.For supershift analysis of the www.abcam.com) protocol. The immunoprecipitated DNA was amplified for sequences containing binding sites by using the following ABCB1 promoter sequence primers: ABCB1p (F) 5′-CAACTCGTCAAAGGAATTAT-3′ and ABCB1p (R) 5′-TTGTACCTTTGATCAACACC-3′.Chromatin from K562 and Lucena cells was fractionated by incubation of purified nuclei with micrococcal nuclease and its immunoprecipitation with anti-β-catenin antibody was performed as described previously . DNA extQuantification was evaluated by RT-qPCR analysis. Immunoprecipitation of Protein A (Santa Cruz Technologies) was used for non-specific binding, and Smad8 (Santa Cruz Technologies) was used as a positive control.ABCB1WNT1β-catenin and β-ACTIN mRNA levels was performed by RT-qPCR. Two micrograms of Trizol (Invitrogen) extracted RNA from cell lines was treat with DNAse Amplification Grade I (Invitrogen) and reverse-transcribed with Superscript II Reverse Transcriptase® (Invitrogen). cDNAs dilutions (1:100) were mixed with SYBR Green PCR Master Mix® (Applied Biosystems) and the following primers: ABCB1: (F) 5′-CCC ATC ATT GCA ATA GCA GG-3′ and (R) 5′-GTT CAA ACT TCT GCT CCT GA-3′; WNT1: (F) 5′-TGG TTT GCA AAG ACC ACC TCC A-3′, and (R) 5′-TGA TTC CAG GAG GCA AAC GCA T-3′; β-CATENIN: (F) 5´-AAG ACA TCA CTG AGC CTG CCAT-3´ and (R) 5´-CGA TTT GCG GGA CAA AGG GCA A-3´; β-ACTIN: (F) 5′-ACC TGA GAA CTC CAC TAC CCT-3′ and (R) 5′-GGT CCC ACC CAT GTT CCA G-3′. RT-qPCR was performed in a Rotor Gene 6000 thermocycler (Corbett) with 50 cycles of 20 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. For each sample, the expression of target genes was normalized to β-actin mRNA levels. Changes in the mRNA levels of genes were evaluated [Analysis of valuated .5 cells/mL. The toxicity of the assay was evaluated by FACS analysis, and ABCB1 mRNA levels were analyzed by RT-qPCR.Cell cultures were exposed to 10 mM LiCl . Treatmevia the analysis of propidium iodide (PI) staining (Sigma-Aldrich). Briefly, K562 and Lucena cells (approximately 3.0 x 105 cells) treated with 10 mM LiCl (24 h and 48 h) were harvested and washed in 500 μL of PBS. PI (1.5 μg/mL) was added to the incubated tubes prior to FACS analysis. PI(−) cells were considered viable. All procedures were performed according to the manufacturer’s protocol. For β-catenin expression detection, cells were harvested (approximately 2.5 x 105 cells) and fixed with PBS/1% Formol. As follow, they were permeabilized with 0.5% Tween 20 to allow intracellular staining and labeled with anti-β-catenin polyclonal antibody (Sigma). Results are expressed as mean relative fluorescence intensity (MRFI), which was calculated by subtracting the mean fluorescence intensity (MFI) for specific antibody by the MFI of the respective secondary antibody (which served as negative isotype control). Ten thousand events were analyzed for each sample in a FACSCalibur Flow Cytometer (Becton Dickinson). The data were analyzed using CellQuest v.3.1 software (Becton Dickinson). All experiments were performed in triplicate.Viability was evaluated 4 cells per well) were split in 24-well plates at 60% confluence in RPMI medium 1 day prior to transfection. The TriFECTa kit contains control sequences for RNAi experiments, including a fluorescently labeled transfection control duplex and a scrambled universal negative control RNA duplex that is absent in human, mouse and rat genomes. Fluorescence microscopy was used to monitor the transfection efficiency according to the manufacturer’s recommendations. Only experiments in which transfection efficiencies were ≥ 80% were evaluated. mRNA levels were measured 48 h after transfection. Duplexes were evaluated at 10 nM. All transfections were minimally performed in duplicate, and the data were averaged. Wnt1 and β-Catenin depletion and RT-qPCR analyses were performed as described above.All RNA oligonucleotides described in this study were synthesized and purified using high-performance liquid chromatography at Integrated DNA Technologies , and the duplex sequences are available upon request. siRNA and transfections were performed following the manufacturer’s protocols of the TriFECTa Dicer-Substrate RNAi kit (Integrated DNA Technologies) and the Trifectin reagent (Integrated DNA Technologies). K562 and Lucena cells (5.5 × 10Cell lysates from K562 and Lucena cells (control - untreated - and treated with LiCl 10 mM) were run on 15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bioead) and incubated with β-catenin (Santa Cruz Technologies), P-GSK3α/β  and α-tubulin (Sigma) antibodies. Antibody binding was detected using enhanced chemiluminescence ECL Plus Western Blotting detection Reagents (GE).Cytospin preparations of K562 and Lucena cells were fixed with methanol, permeabilized with 0.2% triton X-100 PBS, and incubated for 1 h at room temperature with 1% bovine serum albumin (BSA) and 2.0% FBS blocking buffer under shaking. Slides were rinsed once with PBS and 0.05% Tween 20 and then incubated with appropriately diluted primary antibodies in PBS. Cells were incubated with anti-β-catenin polyclonal antibody (Sigma) overnight at 4 °C. After incubation, the slides were rinsed three times and incubated with Alexa® 546 conjugated anti-rabbit IgG (Molecular Probes) for 1 h at room temperature. Sections from each sample were incubated with secondary antibody and served as negative isotype control. The slides were rinsed three times, air-dried and mounted in an antifading medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Labs). Expression and localization of the proteins were observed with a Leica TCS-SP5 AOBS confocal laser scanning microscope (Leica), for capturing representative images of each sample.ABCB1 reporter constructs were synthesized by Gene Art (Germany) and cloned into the firefly pGL3-Basic vector (Promega) upstream of the Luciferase reporter gene. The constructs named pGL3α, containing just the basal promoter (−1019/+1); pGL3β, containing the basal promoter and one TCF binding site (−1067/+1) and pGL3γ, containing the basal promoter and three TCF binding sites (−3187/+1) were inserted into KpnI and BgLII restriction sites of pGL3-basic.The 5 cells from both cell lines (with or without LiCl 10 mM treatment) were co-transfected using Lipofectamine LTX 2000 (Invitrogen) with 1 μg of each Luciferase construct and 100 ng of pRL-SV40 vector (Promega), according to the manufacturer’s instructions. Firefly and Renilla Luciferase activities were measured in cell lysates 48 hours after transfection using the DualGlo Luciferase Assay System (Promega) on a Veritas TM Microplate Luminometer (Turner Biosystems), following the manufacturer’s protocol. All experiments were performed in triplicate. Ratios of Renilla luciferase readings to firefly luciferase readings were taken for each experiment and triplicates were averaged. The average values of the tested constructs were normalized to the activity of the empty pGL3-basic vector, which was arbitrarily set at value 1.For the transient assays, 1.0 x 10t-test. P- Values less than 0.05 were considered as statistically significant . Statistical analyses and graphical representations were performed using GraphPad Prism™ software (GraphPad).Comparison between K562 and Lucena results from different assays was performed by an unpaired ABCB1 promoter.Protein binding to the seven oligonucleotides containing TCF consensus binding sites  was determined by EMSA . We also performed competition assays to verify whether protein complex binding was specific for the TCF consensus binding site. Competition analyses using a 200-fold excess of unlabeled S4, S5 and Opt oligonucleotides demonstrated that the specific binding was reduced . As LiCl treatment did not alter cell viability, we evaluated WNT1 and β-catenin depletion in K562 and Lucena cell lines using RT-qPCR. Using a siRNA approach after 48 h of transfection, a reduction in WNT1 expression of more than 85% and β-catenin of more than70% were achieved in both cell lines when compared to scrambled control sequence-treated cells  and non-MDR cell lines  as models of CML, we demonstrated through EMSA and ChIP analyses that β-catenin binds to the TCF/LEF consensus binding site in the ABCB1 promoter. RT-qPCR analyses indicated that this binding occurred at 2-fold higher levels in Lucena cells than in K562 cells. As it has been demonstrated that the BCR-ABL protein can establish β-catenin expression in CML via TK-mediated phosphorylation [ABCB1 transcription, as previously evidenced in other types of cancer. Interestingly we could demonstrate by RT-qPCR, western blot and FACS, β-catenin higher expression in the MDR cell line. Furthermore, it is known that BCR is a negative regulator of the Wnt/β-catenin pathway. The fusion gene BCR-ABL formed in CML decreases BCR transcription and BCR translation, and thus, there is no more BCR available to complex with β-catenin, leading to the translocation of β-catenin to the nucleus and Wnt/β-catenin pathway activation [In this work, using MDR  share several characteristics despite exhibiting remarkable differences. Self-renewal is an essential stem cell property, but self-renewal pathway activation has also been increasingly recognized as a hallmark of cancer. Interestingly, HSC and CML stem cells also exhibit increased levels of drug efflux-related molecules such as the product of to drugs ,54.ABCB1 transcriptional activation in CML.By the data presented in this work, we provided evidence that the canonical pathway of Wnt signalling is involved in The authors declare that they have no competing interests.SC performed the experiments, statistical analysis and drafted the manuscript. RB participated and assisted the electrophoretic mobility shift assays and Luciferase experiments. BR participated in flow cytometry experiments. MTLCB performed immunofluorescence assays. LP and EA made substantial contributions to the study conception and design and critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.