Cancer stem cells (CSCs) represent a unique subset of cells within a tumor thatpossess self-renewal capacity and pluripotency, and can drive tumor initiation andmaintenance. First identified in hematological malignancies, CSCs are now thought toplay an important role in a wide variety of solid tumors such as NSCLC, breast andcolorectal cancer. The role of CSCs in driving tumor formation illustrates thedysregulation of differentiation in tumorigenesis. The Wnt, Notch and Hedgehog (HH)pathways are developmental pathways that are commonly activated in many types ofcancer. While substantial progress has been made in developing therapeutics targetingNotch and HH, the Wnt pathway has remained an elusive therapeutic target. This reviewwill focus on the clinical relevance of the Wnt pathway in CSCs and tumor cellbiology, as well as points of therapeutic intervention and recent advances intargeting Wnt/β-catenin signaling. Cancer stem cells (CSCs) represent the apex in the hierarchical model of tumor genesis,heterogeneity and metastasis -4. AnaloDue to their similarities to normal stem cells, CSCs are predicted to rely on pathwaysthat govern development, self-renewal and cell fate. In embryonic stem cells, theseprocesses are in large part regulated by three signaling programs: the Wnt, Notch andHedgehog (HH) pathways -10. It iSince the developmental pathways important to normal stem cells are also important toCSCs, a great deal of time has gone into developing therapeutic agents targeting thesepathways. The main focus of this review is the Wnt/β-catenin pathway. However,due to the similarities in signaling components and crosstalk among these pathways, itis important to provide a brief review of the Notch and HH signaling cascades, whereprogress has outpaced efforts in the Wnt pathway. These pathways share overarchingthemes such as myriad permutations of ligand/receptor interactions that ultimatelyimpinge and rely heavily upon a central molecule in the signal transduction cascade:lessons learned from approaches successful in targeting Notch and HH pathways mayprovide valuable insight in how to approach the Wnt signaling pathway.Notch signaling regulates numerous processes in both embryonic development and in adulttissue renewal . During The numerous steps required to translate ligand binding to target gene activationprovide a series of potential points of therapeutic intervention in the Notch signalingpathway. As a result, there are currently numerous preclinical therapeutics underevaluation, as well as several clinical trials involving Notch pathway inhibitors.Preclinical agents including monoclonal antibodies (mAbs) targeted against Notch andNotch ligands are meant to disrupt ligand/receptor interaction events ,31. WhilUnder normal conditions, HH signaling plays important roles in embryonic development andis also involved in tissue regeneration in adults ,37. ActiDysregulation of nearly every step of the HH signaling pathway has been linked to cancerprogression. For example, mutations in Ptch1, Smo, Gli1 and Gli3 are linked to BCC,medullablastoma and pancreatic cancer ,43,46-48Elucidation of the Wnt signaling pathway was built upon the seminal observation that theDrosophila segment polarity gene Wingless had a common origin with the murine oncogeneInt-1 . This di2+ pathway, regulateprocesses such as cell dorsoventral patterning and neuronal migration, function throughβ-catenin independent mechanisms, and remain better characterized in lowerorganisms [Wnts are secreted glycoproteins which bind to cell surface receptors to initiatesignaling cascades important in many physiological settings, as described above . Wnt sigrganisms -66. Canorganisms -72. Thisrganisms ,73-75.It has been appreciated for decades that dysregulation of the mechanisms that regulateβ-catenin signaling is a common feature across a broad spectrum of human cancersFigure . HoweverIn the absence of Wnt stimulus, β-catenin is held in an inactive state by amultimeric “destruction” complex comprised of adenomatous polyposis coli(APC), Axin, glycogen synthase kinase 3β (GSK3β) and casein kinase1α (CK1α) . APC andAn additional layer of regulation that keeps β-catenin levels low in cells is theexpression and secretion of antagonists of the Wnt pathway. These come in two flavors,proteins that bind Wnt ligands, and proteins that bind Wnt receptors . MembersCanonical Wnt/β-catenin signaling is initiated when a Wnt ligand engages itsco-receptors, Fzd and LRP5/6, forming a ternary complex on the extracellular membrane.Fzd, made up of 10 family members, is a seven transmembrane receptor that binds Wntsthrough an extracellular cysteine-rich domain (CRD) . LRP5/6 Wnt binding to Fzd-LRP5/6 initiates a cascade of events that results in disruption ofthe β-catenin destruction complex, leading to β-catenin stabilization andnuclear translocation. The early events initiated by Wnt-Dvl-LRP binding remainincompletely understood. However, current data demonstrate that Wnt binding results inphosphorylation of the cytoplasmic tail of LRP5/6 within a PPSP domain in its C-terminus. This phThe relevance of Wnt signaling in human cancers is highlighted by the frequency withwhich this pathway is aberrantly activated across a vast range of malignancies. Thefirst described, and perhaps best well known role for Wnt/β-catenin signaling isin colon cancer, where nearly 90% of these tumors harbor mutations that result inβ-catenin mutation -100. TheInterestingly, a growing body of evidence illustrates a critical role ofβ-catenin in CSCs -105. ForThe brief overview of canonical Wnt signaling described above provides a glimpse intothe complexity of this system. However, the array of numerous ligands, receptors,kinases, signal transducers and transcription factor complexes also provide anopportunity for multiple modes of therapeutic targeting and intervention. We willtherefore discuss three major areas of targeting the Wnt pathway which have shownpromise in recent years: receptor/ligand interactions, cytosolic signaling components,and nuclear signaling components.Mutations downstream of Wnt receptors, such as those found in APC or β-catenin,were the first examples of aberrant Wnt signaling in human cancers. Some cancers,however, demonstrate hallmarks of constitutive Wnt signaling in the absence ofdownstream mutations. Triple negative breast cancers and non-small cell lung cancers(NSCLCs) have been demonstrated to harbor high levels of uncomplexed cytosolicβ-catenin and exhibit a high basal level of Wnt/β-catenin transcriptionalactivation ,111. Thiin vitro results are encouraging,and warrant further exploration into the development of specific Wnt antibodies.Given the sheer number of Wnt growth factors, and functional redundancy demonstrated byknock-out mouse models, it is tempting to speculate that antibodies directed against anyparticular Wnt may not be a viable approach for inhibiting the Wnt pathway. However, aseries of studies by the Jablons group has demonstrated that certain tumor models relyheavily on specific Wnts. Monoclonal antibodies against Wnt1 and Wnt2 drive apoptosis ina variety of tumor models, including melanoma, NSCLC, mesothelioma, sarcoma, breast andCRC cells ,114-118.invitro, as well as in multiple xenograft models [While tumors which rely exclusively on a specific Wnt may be amenable to monoclonalantibody targeting, tumors that are driven by multiple Wnt ligands would not beeffectively targeted in this manner. In this scenario, a pan-Wnt inhibitor may prove tobe more efficacious. A recent study from Genentech demonstrated that a soluble ligandbinding domain of Fzd8, Fzd8-CRD-Fc, inhibited autocrine Wnt signaling t models . Mice trAn alternative approach to inhibiting ligand/receptor interactions would be to targetthe Wnt co-receptors, LRP5/6 and members of the Fzd family. The LRP family ofco-receptors is comprised of 2 highly homologous members, LRP5 and LRP6. These are longsingle-pass transmembrane receptors, and endogenous Wnt inhibitors of the Dkk familybind LRP5/6 to block Wnt signaling ,120. ThuThe Fzds are family of seven transmembrane receptors comprised of 10 members. Fzd familymembers share a high degree of homology in their extracellular cysteine-rich domain(CRD), which is the region responsible for Wnt-Fzd interactions . While nBiologics approaches interfering with Wnt/co-receptor interactions have shown promise,as illustrated above. The intracellular signaling components of the Wnt pathway alsoprovide myriad targets for therapeutic intervention, and are ripe for targeting witheither small molecules or perhaps protein mimetics designed to impact critical stepsalong the Wnt signaling cascade.Constitutive β-catenin signaling, due to inactivating mutations in APC oractivating mutations within β-catenin itself, plays a critical role in thedevelopment of certain cancers. Colorectal cancer is the best example, as nearly 90% ofall CRCs harbor mutations driving β-catenin signaling. These mutations lead tothe stabilization and accumulation of β-catenin and subsequent translocation intothe nucleus. Preventing β-catenin stabilization and accumulation are of obviousinterest, though this has proven to be an extremely difficult task, perhaps becauseβ-catenin does not possess enzymatic activity. However, a greater understandingof the β-catenin destruction complex and the auxiliary proteins involved in itsregulation provide new possibilities for therapeutic intervention to regulate this stepof the Wnt signaling pathway.The most upstream intracellular components of the Wnt pathway are at the receptor level.LRP5/6 phosphorylation plays a critical role in initiating the Wnt signaling cascade. While tA critical component of the β-catenin destruction complex is the scaffoldingprotein Axin. Axin overexpression can inhibit Wnt signaling, and Axin undergoes geneticinactivation in various cancers -128. Thiin vitro, CWP232291 demonstrated anti-proliferative effectsin various cell lines, and inhibited transcription of β-catenin target genes. Inan in vivo AML model, CWP232291 inhibited tumor progression andexhibited a favorable safety profile, and is currently scheduled for Phase I clinicaltrials in AML and multiple myeloma in 2010. While its mechanism of action remains to beelucidated, this compound was reported to be active in the context of both wild-type andmutant β-catenin, raising the possibility of anti-tumor effects across a broadrange of cancers.Ultimately, these myriad upstream signaling events impinge upon β-catenin,leading to its stabilization, accumulation and nuclear translocation. Despite intenseresearch efforts, progress has been slow in directly and selectively targetingβ-catenin. Theriac Pharmaceutical Corporation recently described a small moleculeinhibitor of the Wnt pathway which is proposed to function via inducing β-catenindestabilization . CWP2322Several agents with diverse, or even unknown, mechanisms of action have shown activityin Wnt/β-catenin driven cancers and cancer cell lines. Aspirin and non-steroidalanti-inflammatory drugs (NSAIDs) have recently shown promise in clinical trials atpreventing polyp formation in colon cancer patients without mutations in APC, andregular use of these agents has been correlated with a decreased occurrence of cancerssuch as breast and lung cancer -134. WhiAn additional component of Wnt signaling, especially important in those tumors in whichparacrine/autocrine Wnt signaling drives activation of the pathway, is the processingand secretion of the Wnt ligands. In the same study mentioned above that led to thediscovery of the Axin destabilizer IWR-1, IWP-1 and IWP-2 were shown to inhibitWnt-driven transactivation activity with similar potency, though through a distinctmechanism . IWPs weUpon entering the nucleus, β-catenin interacts with members of the TCF/LEF familyof transcription factors to drive target gene expression. In the absence ofβ-catenin, TCF/LEF is held in a transcriptionally inactive state throughinteractions with co-repressors such as Groucho and HDACs . β-catenA compelling series of studies by the Kahn group has suggested differential roles forthe highly homologous CBP and p300 in Wnt/β-catenin-driven signaling, especiallywith regard to the role of the Wnt pathway in CSCs ,145-147.Leproucelet and colleagues utilized a high throughput screen to identify a series ofsmall molecules that disrupted β-catenin/TCF interaction . These cinvitro, and inhibited tumor growth in an HCT116 xenograft model. It will beof interest to determine if inhibitors of this component of the β-catenin/TCFtranscriptional complex, as well as the others listed above, serve as novel targets ofinhibition in the Wnt signaling pathway.An expanded base of knowledge regarding critical β-catenin/TCF co-regulators willbe critical in the development of novel antagonists of the Wnt signaling pathway. Forexample, recent reports from the Clevers and Yamada groups have identified Traf2- andNck-Interacting Kinase (TNIK) as a critical mediator of Wnt-driven transcription in CRC,150. In As a result of its role in numerous cancers, the Wnt signaling pathway is a prime targetfor therapeutic intervention. Inhibition of Wnt signaling has proven to be an elusivegoal over the years. However, as new developments emerge in the field of Wnt biology,the field continues to inch closer to an effective strategy of Wnt inhibition.Concurrent with these advances comes the realization that Wnt signaling plays criticalroles in biology of CSCs.A role for Wnt signaling has been demonstrated in a variety of CSC settings, includingcolon, breast and cutaneous CSCs, as well as in hematopoietic stem cells ,151-153.+CD24low stem-like cells inmetastases, suggesting a role for breast CSCs in the metastatic process of this disease[+CD24lowcell-surface marker pattern and the ability to form spheroids in suspension culture- keyproperties of normal and cancer stem cells [Another potential advantage of employing a Wnt-targeted therapy is the potential rolefor CSCs in resistance to classical cytotoxic treatments (i.e. chemotherapy and ionizingradiation) and in metastatic disease. CSCs in a broad range of cancers are relativelymore resistant to these conventional therapeutic approaches than their bulk tumor cellcounterparts, as has been described in CSCs in leukemias and breast, colorectal, andbrain cancers ,159,160. disease,161. Fur disease-165. Dur disease-165. Celem cells . It is t


Chronic obstructive pulmonary disease (COPD) is characterized by abnormal extracellular matrix (ECM) turnover. Recently, activation of the WNT/β-catenin pathway has been associated with abnormal ECM turnover in various chronic diseases. We determined WNT-pathway gene expression in pulmonary fibroblasts of individuals with and without COPD and disentangled the role of β-catenin in fibroblast phenotype and function.We assessed the expression of WNT-pathway genes and the functional role of β-catenin, using MRC-5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD.1, a growth factor important in COPD pathogenesis, induced WNT-5B, FZD8, DVL3 and β-catenin mRNA expression. The induction of WNT-5B, FZD6, FZD8 and DVL3 mRNA by TGF-β1 was higher in fibroblasts of individuals with COPD than without COPD, whilst basal expression was similar. Accordingly, TGF-β1 activated β-catenin signaling, as shown by an increase in transcriptionally active and total β-catenin protein expression. Furthermore, TGF-β1 induced the expression of collagen1α1, α-sm-actin and fibronectin, which was attenuated by β-catenin specific siRNA and by pharmacological inhibition of β-catenin, whereas the TGF-β1-induced expression of PAI-1 was not affected. The induction of transcriptionally active β-catenin and subsequent fibronectin deposition induced by TGF-β1 were enhanced in pulmonary fibroblasts from individuals with COPD.Pulmonary fibroblasts expressed mRNA of genes required for WNT signaling. Stimulation of fibroblasts with TGF-β1 is enhanced in pulmonary fibroblasts from individuals with COPD. This suggests an important role of the WNT/β-catenin pathway in regulating fibroblast phenotype and function in COPD.β-catenin signaling contributes to ECM production by pulmonary fibroblasts and contributes to myofibroblasts differentiation. WNT/β-catenin pathway expression and activation by TGF-β Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation, which is associated with an abnormal inflammatory response of the lungs to noxious particles or gases. Long-term exposure to cigarette smoke is the major risk factor for the development of COPD Fibroblasts play an important role in ECM turnover in the parenchyma and small airways by producing ECM constituents Recently, it was demonstrated that activation of the canonical WNT/β-catenin signaling pathway is associated with fibroblast activation, fibrosis and tissue repair 1-induced human lung fibroblast phenotype and function. Furthermore, we compared the expression of WNT pathway genes and activation of β-catenin in primary pulmonary fibroblasts of individuals with and without COPD.In the present study, we investigated the expression of WNT-pathway genes in human lung fibroblasts and determined the functional role of the transcriptional co-activator β-catenin in regulating TGF-β2, FZD6 and FZD8 as well as the intracellular signaling protein dishevelled (DVL3) and the key-effector of canonical WNT signaling, β-catenin, were abundantly expressed (1 in airway smooth muscle (unpublished data), and based on recent literature indicating the involvement of the selected WNT ligands, FZD receptors and intracellular signaling molecules in cellular processes relevant for fibroblasts function We first investigated WNT pathway gene expression in MRC-5 human lung fibroblasts. A clear mRNA signal was observed for the majority of WNT pathway genes, but with considerable differences in the degree of expression . The WNTxpressed . This su1/smad pathway 1 induced cellular responses and therefore we investigated first the phosphorylation of smad3. MRC-5 fibroblasts were stimulated with TGF-β1 (2 ng/ml) for various time-points resulting in a time-dependent increase in ser423/425-smad3 phosphorylation, which was most profound at the early time-points of 1–2 hours  altered the expression profile of specific WNT-pathway genes , whereas FZD2 mRNA expression was significantly down-regulated to 0.63±0.07-fold compared with untreated fibroblasts. The expression of WNT-5A, WNT-16, FZD6 and DVL3 was unaltered after TGF-β1 stimulation . The clinical characteristics of the subject groups are represented in 1 had no effect on mRNA expression of WNT-5A and WNT-16, but induced mRNA expression of WNT-5B. Interestingly, WNT-5B mRNA expression in TGF-β1 treated fibroblasts was higher in individuals with than without COPD , whilst WNT-16 expression was significantly higher in individuals with COPD stage II  In line out COPD . The mRNout COPD .2, FZD6, and FZD8 was similar in fibroblasts from individuals with and without COPD, independent of GOLD stage  in fibroblasts from individuals with COPD than controls after TGF-β1 stimulation  resulted in a significant increase in protein expression of the differentiation markers α-sm-actin, fibronectin and MMP-2 (1 (48 hours) distinctively induced the formation of stress fibers (F-actin) in these cells, another indication of myofibroblast differentiation (1 for 48 hours shows that the increase of fibronectin and α-sm-actin protein expression is concentration-dependent (1 was investigated in more detail.We next studied activation of the WNT effector β-catenin in response to TGF-βnd MMP-2 . Cytochentiation . Stimulaependent . Interesependent . Therefo1 (2 ng/ml) resulted in a significant, time-dependent increase in total β-catenin protein expression after 24 hours of stimulation compared to untreated fibroblasts  β-catenin, with kinetics similar to the induction of total β-catenin   to silence β-catenin protein expression. After siRNA treatment, total β-catenin expression was reduced to 57±7% in fibroblasts at baseline . TGF-β1 control) , whereascontrol) .1-induced gene and protein expression. Non-targeting siRNA treated fibroblasts were stimulated with TGF-β1 for 24 and 48 hours , resulting in increased expression of α-sm-actin  was not affected (data not shown).Next, we investigated the functional effects of β-catenin silencing on TGF-βsm-actin  and fibrsm-actin . The indsm-actin . Silenci1, without affecting basal expression of either α-sm-actin or fibronectin  complex ronectin .1 also resulted in a significant induction of active β-catenin in fibroblasts from individuals with and without COPD. Interestingly, the induction of active β-catenin was significantly higher in fibroblasts from individuals with COPD than those without COPD  between individuals with and without COPD (data not shown). Thus, β-catenin activation and subsequent fibronectin deposition in response to TGF-β1 is enhanced in lung fibroblasts from COPD patients compared to lung fibroblasts from controls.Fibroblasts from individuals with and without COPD had similar expression of active β-catenin at baseline. Stimulation with TGF-βout COPD . In accoith COPD . The exp8, DVL3 and β-catenin were significantly induced by TGF-β1 in a concentration dependent manner in both MRC-5 and primary human lung fibroblasts. Interestingly, WNT-5B, FZD6 and FZD8 expression were significantly more upregulated in response to TGF-β1 in primary fibroblasts from individuals with than without COPD. Furthermore, we also show that β-catenin, the key effector of canonical WNT signaling, regulates the induction of collagen1α1, α-sm-actin and fibronectin deposition by pulmonary fibroblasts in response to TGF-β1, whereas the expression of PAI-1 is not regulated by β-catenin. Finally, we provide evidence that the induction of transcriptionally active β-catenin and subsequent fibronectin deposition induced by TGF-β1 are significantly enhanced in lung fibroblasts from COPD patients.Pulmonary fibroblasts play a pivotal role in COPD by regulating ECM turnover in the lungs 1 stimulation in activating WNT signaling and regulating transcription of tissue repair genes such as fibronectin, despite the fact that the peripheral lung is the primary site of tissue destruction associated with pulmonary emphysema An active and complex remodeling process is present in the peripheral lung when COPD develops, resulting in small airway fibrosis and a variable degree of emphysema. Fibroblasts are the primary cell type responsible for the production and maintenance of the extracellular matrix. Alterations in fibroblast function may therefore play an important role in COPD. In this respect, the canonical WNT/β-catenin signaling pathway is of particular interest, since this pathway has been linked to tissue repair and remodeling 1We propose that during COPD pathogenesis, irrespective of GOLD stage, fibroblasts from the peripheral lung are promoted to repair tissue damage, but that this repair response becomes insufficient in more advanced stages of disease. This can either be because of the development of intrinsic defects in the lung fibroblast (e.g. reduced proliferative capacity of fibroblasts in advanced stages of COPD The smoking histories (pack-years and smoking status) of the individuals with and without COPD were very similar, thus excluding that the observed differences between COPD and controls were primarily due to differences in smoking habits. No separate non-smoking control group (never smokers) was included in the study design. Therefore, this study does not provide insight into the effect of smoking on WNT pathway gene expression by fibroblasts. There is a statistically significant difference in age between individuals with COPD stage II (older) and individuals with either COPD stage IV or no COPD. Since the baseline expression of WNT pathway genes is higher in fibroblasts from COPD stage II compared to the other two groups (significant for WNT-16 and DVL3), we investigated the correlation between WNT pathway gene expression and age in the individuals without COPD . We did 1 treatment, which corresponds with the kinetics of activation of smad3. In addition, other signaling pathways such as ERK1/2 may be activated by TGF-β1 that regulate the WNT pathway gene expression as demonstrated for β-catenin in our recent report 1 in more detail, as we report that TGF-β1 induces WNT and FZD mRNA expression in human lung fibroblasts and activates β-catenin signaling, which contributes to the fibroblast phenotype and function.Tissue repair by fibroblasts is a complex process involving the interplay of various growth factors and intracellular signaling pathways. Recently, crosstalk between WNT signaling pathway and growth factors in fibroblasts has been demonstrated 1 induces β-catenin expression via several intracellular mechanisms. β-Catenin levels are tightly regulated by the constitutively active enzyme GSK-3. A fraction of cellular GSK-3 forms a complex with AXIN, casein kinase I (CK-I) and adenomatous polyposis coli (APC); this complex phosphorylates and subsequently targets β-catenin for proteosomal degradation 1 induces a transient time-dependent phosphorylation of both GSK-3 isoforms in MRC-5 fibroblasts, which might account for the initial increase of β-catenin stability. In addition, enhanced secretion of canonical WNT ligands by fibroblasts in response to TGF-β1 may signal in an autocrine fashion, which then stabilizes β-catenin by disrupting the GSK-3/AXIN/CK-I/APC complex 1 stimulation progresses even when GSK-3 phosphorylation has returned to basal levels, supporting such an autocrine signaling loop. In addition, β-catenin protein expression can be induced by growth factors by de novo synthesis of the protein 1 induces β-catenin expression in pulmonary fibroblasts are therefore not fully understood and require further exploration.Our data suggest that TGF-β1 did not or only modestly affect the mRNA expression of the canonical WNT target genes dickkopf-1 (DKK-1), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) or MMP-2, and attenuated AXIN-2 mRNA expression in MRC-5 fibroblasts  activating the transcription of specific genes 1, and the WNT-signaling pathway in lung fibroblasts is of major interest to understand tissue repair mechanisms.Surprisingly, TGF-βroblasts , even th1 induced collagen Iα1 and PAI-1 mRNA as well as α-sm-actin and fibronectin protein expression, indicative of fibroblast activation 1-induced collagen Iα1, α-sm-actin and fibronectin expression. However, the induction of PAI-1 did not change, indicating that β-catenin directs TGF-β1 signaling to specific intracellular pathways. Further, these data indicate that the responsiveness of the fibroblasts to TGF-β1 was not affected by down regulation of β-catenin. Collectively, these data demonstrate that β-catenin signaling plays an important role in the activation process of pulmonary fibroblasts. This may contribute to COPD pathogenesis, because the activation of β-catenin signaling and subsequent fibronectin deposition in response to TGF-β1 is higher in lung fibroblasts from patients with than without COPD.Our results further show that TGF-β1. In primary fibroblasts of COPD patients, this activation is greatly enhanced compared to healthy controls, as is the induction of β-catenin. This suggests that WNT/β-catenin signaling plays an important role in tissue repair in the lung, and that targeting β-catenin-dependent gene transcription holds promise as a therapeutic intervention in COPD.In conclusion, our results indicate that the WNT/β-catenin signaling pathway is activated in pulmonary fibroblasts in response to the cytokine TGF-βhttp://www.rug.nl/umcg/onderzoek/researchcode/index) and national ethical and professional guidelines .The study protocol was consistent with the Research Code of the University Medical Center Groningen  criteria 1) 96.9% predicted) was derived from noninvolved lung tissue of patients undergoing surgical resection for pulmonary carcinoma. Patients had no airway obstruction and no chronic airway symptoms, such as cough and sputum production. Material was always taken as far away as possible from the tumour, or from a noninvolved lobe. No histopathological lesions were present.Tissue from the control group (median forced expiratory volume in one second (FEV1 52.6% of predicted) was derived from noninvolved lung tissue from patients undergoing resection surgery for pulmonary carcinoma. Histopathologically emphysematous lesions were present, however, of limited and varying severity. Moderate forms of emphysema can be histopathologically demonstrated by finding isolated or free-lying segments of viable alveolar septal tissue or isolated cross sections of pulmonary vessels.Tissue of GOLD stage II COPD patients (median FEV1 17.1% predicted) was obtained from patients with COPD undergoing surgery for lung transplantation or lung volume reduction. All individuals had quitted smoking for at least 1 year before surgery. The resected tissue showed both macroscopically and microscopically severe emphysematous lesions, often accompanied by bullae. Subpleural fibrous areas were avoided.Tissue of GOLD stage IV COPD patients . Isolated cells were characterized as fibroblasts by morphological appearance and expression pattern of specific proteins 1 in Ham's F12 medium supplemented with 0.5% FBS, L-glutamine and antibiotics. When applied, pharmacological inhibitors (i.e. quercetin 40 µM or PKF115–584 100 nM) were added 30 minutes before the addition of TGF-β1.MRC-5 lung fibroblasts later stabilization buffer and homogenized by passing the lysate 10 times through a 20 gauge needle. Lysates were then mixed with an equal volume of 70% ethanol, and total mRNA was purified using RNeasy mini spin columns. The eluted mRNA was quantified using spectrophotometry . Equal amounts of total mRNA (1 µg) were then reverse transcribed and stored at -20°C until further use.Total mRNA was extracted using the RNeasy mini kit . Briefly, cells were harvested in RNAcDNA was subjected to real-time PCR, which was performed with a MyiQ™ Single-Color detection system . In short, 12.5 µl iQ™ SYBR Green Supermix, containing fluorescein to account for well to well variation, 0.1 µM of gene-specific forward and reverse primer and 1 µl of 1∶5 diluted cDNA sample were used in a total volume of 25 µl and added to a 96 well plate. The sequences of the primers used for determining WNT pathway components and WNT target genes are listed in the supporting information tables , S3, S4.gene of interest – Cq18S rRNA; designated as ΔCq). Several housekeeping genes, including β2-microglobulin  and phospholipase A2 , were tested for the influence of the experimental procedure on the expression −(ΔΔCq).Real-time PCR data were analyzed using the comparative cycle threshold (Cq: amplification cycle number) method. Cycle parameters were: denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds for 40 cycles followed by 5 minutes at 72°C. The amount of target gene was normalized to the endogenous reference gene 18S ribosomal RNA  followed by a period of 24 hours in Ham's F12 supplemented with 0.5% FBS, L-glutamine and antibiotics. Consecutively, medium was refreshed and cells were stimulated with TGF-β1 (2 ng/ml) for 48 hours.MRC-5 fibroblasts were grown to ∼90% confluence in 6-well cluster plates and transiently transfected with a 21-bp, double-stranded siRNA targeted against the β-catenin transcript . Cells were transfected in serum-free Ham's F12 without any supplements using 1.5 µg/ml of siRNA in combination with lipofectamine 2000 transfection reagent. Control transfections were performed using a non-silencing control siRNA . After 6 hours of transfection, cells were washed once with warm (37°C) Hank's Balanced Salt Solution . Lysates were then sonicated and protein concentration was determined according to Pierce protein determination according to the manufacturer's instructions. Lysates were stored at −20°C till further use.To obtain whole cell lysates, cells were washed once with ice-cold (4°C) HBSS then lysed in ice-cold sodiumdodecylsulphate (SDS) buffer  and nuclear extracts were prepared using the nuclear extract kit  according to the manufacturer's instructions. Protein concentration in the nuclear extracts was determined according to the Bradford protein assay. Nuclear extracts were stored at −80°C till further use.Confluent MRC-5 fibroblasts were serum deprived in Ham's F12 medium supplemented with 0.5% (v/v) FBS, 2 mM L-glutamine, 100 µg/l streptomycin and 100 U/ml penicillin for a period of 24 hours. Subsequently cells were stimulated for 24 hour with TGF-βEqual amounts of protein (10–20 µg/lane) were subjected to electrophoresis on polyacrylamide gels, transferred to nitrocellulose membranes and analyzed for the proteins of interest using specific primary and HRP-conjugated secondary antibodies. By using enhanced chemiluminescence reagents, bands were either subsequently visualized on film or recorded in the G:BOX iChemi gel documentation system equipped with GeneSnap image acquisistion software . Band intensities were quantified by densitometry using Totallab™ software  or GeneTools analysis software , respectively.1 2 ng/ml for 48 hours, fixed for 15 min at 4°C in cytoskeletal (CB) buffer  containing 3% paraformaldehyde (PFA). Cells were then permeabilized by incubation for 5 min at 4°C in CB buffer containing 3% PFA and 0.3% Triton X-100. For immunofluorescence microscopy, fixed cells were first blocked for 2 hours at room temperature in Cyto-TBS buffer  containing 1% bovine serum albumin (BSA) and 2% normal donkey serum. Incubation with primary antibody  occurred overnight at 4°C in Cyto-TBS containing 0.1% Tween 20 (Cyto-TBST). Incubation with Cy3-conjugated secondary antibody was for 2 h at room temperature in Cyto-TBST. Filamentous actin was stained with Alexa Fluor 488 phalloidin (15 minutes at RT) and nuclei with Hoechst 3342. After staining, coverslips were mounted using ProLong Gold antifade reagent (Invitrogen) and analyzed by using an Olympus AX70 microscope equipped with digital image capture system (ColorView Soft System with Olympus U CMAD2 lens).Lung fibroblasts were plated onto Lab-Tek™ borosilicate chamber slides and treated with TGF-β1 was from R&D systems . All other chemicals were of analytical grade.Mouse anti-α-sm-actin, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody, HRP-conjugated goat anti-rabbit antibody and HRP-conjugated rabbit anti-goat antibody were purchased from Sigma . Goat anti-MMP-2 antibody was purchased from R&D systems . Rabbit anti-GSK-3 antibody, goat anti-fibronectin (C20) antibody, Mouse anti-Lamin A/C antibody, mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody and Rabbit-anti-Smad2/3 antibody were obtained from Santa Cruz Biotechnology . Rabbit anti-phospho-Ser9/21-GSK-3 antibody and rabbit anti-phospho-432/425-smad3 antibody were from Cell Signaling Technology . Mouse anti-total β-catenin antibody was from BD Biosciences . Mouse anti-non-phosphorylated-β-catenin antibody (clone 8E7) was from Millipore . Cy3 conjugated secondary antibodies were obtained from Jackson Immunoresearch . Lipofectamine 2000 transfection reagent and alexa Fluor 488 phalloidin were from Invitrogen . Recombinant human TGF-βFigure S118S ribosomal RNA abundance in primary fibroblasts individuals without and with COPD. Primary lung fibroblasts were isolated from individuals without (control) and with COPD (GOLD stage II and IV) as described in the methods. The fibroblasts were grown to confluence and treated for 4 hours with TGF-β1 (2 ng/ml). Analysis of 18S ribosomal RNA is performed by qRT-PCR analysis with 0.025 µg of cDNA as input. (A) Average 18S rRNA expression in primary human lung fibroblasts and (B) raw Cq-values for all the individual subjects. 18S rRNA expression at baseline is indicated by open circles (○) and after TGF-β1 stimulation  by closed circles (•). Median of each group is indicated by -----.(TIF)Click here for additional data file.Figure S2No age-dependent effects on WNT pathway gene expression in pulmonary fibroblasts of individuals without COPD. Expression of WNT-5B, DVL3 and FZD8 as a function of age of the individual primary lung fibroblasts isolated from individuals without COPD (control) as described in the methods. The fibroblasts were grown to confluence and subsequently mRNA was isolated. Analysis of WNT pathway gene expression is performed by qRT-PCR and corrected for 18S rRNA expression. The uninterrupted line indicates the linear regression.(TIF)Click here for additional data file.Figure S3Effect of TGF-β stimulation on canonical WNT target genes in human lung fibroblasts. qRT-PCR analysis of α-sm-actin (positive control), AXIN-2, vascular endothelial growth factor (VEGF), dickkopf-1 (DKK-1), interleukin-8 (IL-8) and matrix metalloproteinase-2 (MMP-2) in MRC-5 fibroblasts after 24 h of TGF-β1 (2 ng/ml) stimulation. Expression of canonical WNT target genes by TGF-β1 is corrected for 18S rRNA and expressed relative to untreated MRC-5 fibroblasts (control). Data represents mean ± s.e.m. of 5–10 independent experiments. *p<0.05, ***p<0.001 compared to untreated MRC-5 fibroblasts (two-tailed student's t-test for paired observations).(TIF)Click here for additional data file.Table S1Primers used for determination of WNT ligands by qRT-PCR analysis.(DOCX)Click here for additional data file.Table S2Primers used for determination of the dishevelled protein family by qRT-PCR analysis.(DOCX)Click here for additional data file.Table S3Primers used for determination of FZD receptors by qRT-PCR analysis.(DOCX)Click here for additional data file.Table S4Primers used for determination of LRP-coreceptors, WNT target genes and housekeeping genes by qRT-PCR analysis.(DOCX)Click here for additional data file.


Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells.Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF , a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27 V600E inhibitor) and ipilimumab  have improved outcomes for patients with metastatic melanoma. Despite these advances, few alternative therapies exist for advanced melanoma, and these available treatments work for only a limited time. Thus there is a need for a greater understanding of melanoma biology and new treatments.The incidence of melanoma is rising faster than that of any other cancer 3) groups to the guanidino nitrogen atoms of arginine amino acid residues. Currently, eleven PRMT proteins have been identified in humans. These proteins are homologous and share a central catalytic domain. PRMT enzymes methylate their targets in either an asymmetric (Type I PRMTs) or symmetric configuration (Type II) Post-translational protein modification is involved at all levels of cellular regulation. Protein arginine methyltransferases (PRMT) catalyze the attachment of methyl  but accelerated growth of others . PRMT5 depletion also reduced MITF expression, and increased p27in situ and Clark's stage I-IV), and metastatic melanoma tumors  have also been detected in melanocytic nevi, suggesting early events trigger aberrant growth or transformation PRMT5 protein expression in human melanoma specimens was evaluated by IHC in 248 de-identified tissues from individual patient samples and two separate tissue microarrays. Clinical outcomes data was not available for these samples. Included in these samples were normal skin, melanocytic nevi, primary malignant melanoma tumors  and cytoplasmic staining (Spearman correlation 0.59 (array 1) and 0.81 (array 2); p<0.0001) (not shown).The staining intensity of PRMT5 was also assessed. Due to heterogeneity of tissue quality and processing, data from each of two tissue arrays were analyzed independently. Overall, there was no difference in staining intensity between tissue types . However, a positive correlation between staining intensity and the percentage PRMT5+ cells was observed in the two arrays. This was true for both nuclear (Spearman correlation 0.79 (array 1) and 0.80 (array 2), Marked heterogeneity in PRMT5 localization was observed across patient samples . RegardlBRAF or NRAS mutational status  . All linl status . Subcelll status . Of the in vivo, A375 or Hs294T cells were implanted subcutaneously into athymic mice. IHC analysis confirmed PRMT5 expression in tumors from both cell lines. In both A375 and Hs294T tumors, PRMT5 expression was highest in the cytoplasm .Human melanoma cell lines were obtained from the ATCC , Dr. Soldano Ferrone  or Dr. Meenhard Herlyn . Immortalized human epidermal melanocytes (HEM) were purchased from ScienCell Inc. . The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 , PRMT5 , β-actin ; GAPDH (Sigma # G8795); lamin B , MITF , MITF , cyclin D1 (Abcam # ab6152), cyclin D1 , Mep50 , Mep50 , STAT3 , and p27http://www.tissue-array.net), arrays from the AIDS and Cancer Specimen Resource  (http://acsr.ucsf.edu)), or de-identified specimens from OSUWMC. Regarding specimens from the Ohio State University Wexner Medical Center, the need for patient consent was waived by the Institutional Review Board, as no patient identifiers were associated with the tissues. All animal studies were approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC).All studies were approved by the Institutional Review Board of The Ohio State University Wexner Medical Center . PRMT5 IHC data were obtained from 21 normal epidermis (including melanocytes and keratinocytes), 26 melanocytic nevi, 135 malignant, and 66 metastatic melanoma human specimens. Formalin-fixed paraffin embedded samples were obtained as commercially available tissue microarrays  was used at a dilution of 1∶70. The secondary detection system used was Mach 4 Alkaline Phosphatase  with Vulcan Fast Red chromogen . Serial sections were stained in duplicate on a Dako Autostainer , counterstained (hematoxylin), and mounted. Each specimen was analyzed in a blinded fashion under direction of a board-certified dermatopathologist . The percentage of PRMT5+ cells in each sample was recorded, then sorted into four categories: negative (0–5% of cells in sample), low (6–25%), medium (26–75%), or high (>75%) percentage of PRMT5+ cells. Samples were sub-categorized via percentage of PRMT5+ in nucleus or cytoplasm. PRMT5 staining intensity in the nucleus and cytoplasm was qualitatively assessed. PRMT5 negative samples were recorded as zero; positive samples were scored from 1 to 5, where 1 =  low intensity and 5 =  highest intensity. Serial sections from arrays and specimens were stained in duplicate. Twenty-two specimens were discarded due to inadequate, lost, or damaged tissue, poor quality, or discordant results between duplicates as determined by the dermatopathologist.Cell lysates were subjected to immunoblot analysis as described 6–1×107 cells per lysate. Protein (8–10 µg) was analyzed by SDS-PAGE, and validated using nuclear and cytoplasmic markers lamin B or GAPDH, respectively.Subcellular fractionation was performed using the NE-PER buffer kit (Thermo-Scientific #78833) per manufacturer's instructions using 5×106 cells in 100 µL PBS) were injected subcutaneously into female athymic Balb/cnu/nu mice as described All animal studies were approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC). A375 or Hs294T human melanoma cells (2×106–1×107 cells were lysed in MPER lysis buffer (Thermo-Scientific #78501). Lysates were pre-cleared for 30 min at 4°C on a rotating platform using True-Blot IgG beads . Samples were centrifuged at 10,000× g for 3 min, and supernatants incubated with IgG, or primary antibody against Mep50, cyclin D1, or STAT3 for 1h at 4°C rotating. Samples were incubated with IgG beads for 1h at 4°C then centrifuged at 10,000× g for 3 min at 4°C. Beads were washed 3× in MPER buffer, and proteins were reduced (β-mercaptoethanol) prior to immunoblot.5×10PRMT5 was depleted from cells using siRNA duplexes  targeting exon 15 of PRMT5 transcript: 5′-CCGCUAUUGCACCUUGGAA-3′ Forty-eight hours following transfection of melanoma cells with either control siRNA or PRMT5 siRNA (see methods), total cellular RNA was processed using TRIzol (Invitrogen) per manufacturer's instructions. Single-tube TaqMan miRNA assays for each miR of interest (Applied Biosystems) were used to detect and quantify mature miRNAs as described Cell proliferation was measured as optical density (O.D.) at 570 nm via MTT -2, 5-diphenyltetrazolium bromide) assay; ATCC). Assays were performed in replicates of five wells/condition.Fisher's exact test was used to compare the dichotomous outcome of PRMT5 protein >5% vs. ≤5% in patient specimens  by specimen type. Wilcoxon rank sum test was used to compare percentage of PRMT5+ cells in subcellular compartments by specimen type and Spearman's correlation to evaluate relationships between staining intensity and percentage of PRMT5+ cells.Figure S1PRMT5 protein does not associate with MITF protein. IP against the MITF protein or IgG control was performed in 1106Mel and MeWo melanoma cell lines, and subjected to immunoblot using antibodies against MITF and PRMT5. The MITF antibody used here for IP recognizes both isoforms of MITF, which differ in their N-terminal regions.(TIF)Click here for additional data file.Figure S2PRMT5 does not associate with BRAF or CRAF in unstimulated melanoma cells. IP against BRAF, CRAF or IgG control was performed in representative human melanoma cell lines and subjected to immunoblot using antibodies against BRAF, CRAF or PRMT5.(TIF)Click here for additional data file.Table S1Distribution of human melanoma and normal skin samples obtained for assessment of PRMT5 protein.(DOCX)Click here for additional data file.Table S2Genotype of BRAF and NRAS within human melanoma cell lines as determined by Sanger Sequencing with the ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1.(DOCX)Click here for additional data file.


Sox7 gene. Differentiation of P19[shβ-cat] cells in the presence of RA resulted in the upregulation or lack of repression of all of the precursor genes, on day 5 and/or 9, with the exception of Foxc2. However, expression of Sox7, Gli2, the myogenic regulatory factors and terminal differentiation markers remained inhibited on day 9 and overall skeletal myogenesis was reduced. Thus, β-catenin is essential for in vitro formation of premyogenic mesoderm, leading to skeletal myogenesis. RA can at least partially compensate for the loss of β-catenin in the expression of many myogenic precursor genes, but not for myoblast gene expression or overall myogenesis.Previous studies have shown that P19 cells expressing a dominant negative β-catenin mutant (β-cat/EnR) cannot undergo myogenic differentiation in the presence or absence of muscle-inducing levels of retinoic acid (RA). While RA could upregulate premyogenic mesoderm expression, including Pax3/7 and Meox1, only Pax3/7 and Gli2 could be upregulated by RA in the presence of β-cat/EnR. However, the use of a dominant negative construct that cannot be compensated by other factors is limiting due to the possibility of negative chromatin remodelling overriding compensatory mechanisms. In this study, we set out to determine if β-catenin function is essential for myogenesis with and without RA, by creating P19 cells with reduced β-catenin transcriptional activity using an shRNA approach, termed P19[shβ-cat] cells. The loss of β-catenin resulted in a reduction of skeletal myogenesis in the absence of RA as early as premyogenic mesoderm, with the loss of Pax3/7, Eya2, Six1, Meox1, Gli2, Foxc1/2, and Sox7 transcript levels. Chromatin immunoprecipitation identified an association of β-catenin with the promoter region of the  In P19 cells, Meox1 and Gli2 overexpression activated each other’s expression and dominant negative mutants of Meox1/EnR or Gli2/EnR downregulated Pax3 and inhibited skeletal myogenesis XenopusFoxc1 expression regulates the expression of Pax7 in chick intermediate mesoderm The details and complexity of how these positive regulatory loops interact with each other during the formation of myogenic progenitor cells still remains unclear. However, it is known that these genes regulate the commitment of skeletal muscle progenitors to skeletal muscle by activating the expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, Myogenin and MRF4 The Vitamin A derivative, all-trans retinoic acid (RA), is a morphogen that plays an important role during skeletal myogenesis and is formed through the oxidation of all-trans retinal by the enzyme retinal dehydrogenase (RALDH) . These constructs contain a promoter with 8 copies of the TCF/LEF binding sites driving expression of the firefly luciferase gene. The Super8XFOPFLASH construct contains mutated TCF/LEF binding sites, rendering them inactive.5′-TTTGGTTATCAAACCCTAGCCTTCTCAAGAGAAAGGCTAGGGTTTGATAACGCTTTTTT-3′ and 5′-CTAGAAAAAAGCGTTATCAAACCCTAGCCTTTCTCTTGAGAAG GCTAGGGTTTGATAAC-3′) or from 312 bp to 334 bp (5′-TTTGAATCCATTCTG GTGCCACCTTCAAGAGAGGTGGCACCAGAATGGATTCCTTTTTT-3′ and 5′-CT AGAAAAAAGGAATCCATTCTGGTGCCACCTCTCTTGAAGGTGGCACCAGAATGGATT-3′). The annealed oligonucleotides were cloned into the BbsI and XbaI restriction sites of the mU6pro vector, a generous gift from David Turner  5′-TTTGACAAGATGAAGAGCACCAATTCAAGAGATTGGTG CTCTTCATCTTGTTGTTTTTT -3′ and 5′- CTAGAAAAAACAACAAGATGAAGAGCA CCAATCTCTTGAATTGGTGCTCTTCATCTTGT -3′.DNA constructs encoding short hairpin RNAs (shRNAs) complementary to two regions within the mRNA of mouse β-catenin (NM_007614) were created using annealed oligonucleotides encoding sequences from 3056 bp to 3076 bp  with α-minimum essential medium (MEM)  containing 10% fetal bovine serum  The P19 cell lines were differentiated following a 9 day protocol P19 cells were transfected with short hairpin constructs by using the FuGene transfection reagent . The cells were co-transfected with either 0.8 µg of the shβ-catenin or shControl constructs and with 0.8 µg of a plasmid conferring puromycin resistance. Colonies were selected with puromycin for 10 days and subsequently pooled, termed P19[shβ-cat] cells or P19[shControl] cells. Experiments shown represent results from 2 independent pooled populations generated from 2 different shRNA sequences targeting β-catenin, repeated 2 or 3 times.P19 cell lines were transiently transfected at ∼50–60% confluency in monolayer in 60 mm tissue culture dishes  with 1.0 µg of Renilla luciferase and either Super8XTOPFLASH or the Super8XFOPFLASH reporter constructs using the Fugene transfection reagent . The following day, the cells were aggregated and treated with or without 20 mM LiCl in 60 mm petri dishes coated with 1% agarose. The cells were harvested two days after transfection using the Dual Luciferase Kit  and luciferase activity was measured using a GLOMAX 96 microplate luminometer with dual injectors . Luciferase activity was normalized to Renilla activity and represented as fold changes over Super8XFOPFLASH transfected control cell luciferase activity.On day 9, P19 cultures growing on gelatin-coated coverslips were fixed by treatment with methanol. Fixed cells were incubated overnight with MF20 monoclonal antibodies against the muscle-specific marker, Myosin Heavy chain (MHC) as described RNA was isolated from cells using the RNeasy kit  on days 0, 5 and 9 of the differentiation. 1 µg of RNA from each sample was reverse transcribed using the Quantitect Reverse Transcription kit . QPCR was carried out using either a Mastercycler realplex or ABI 7300 machine  and analyzed with either the Realplex software  or using the SDS software . For real-time detection of mRNA expression, 1/20 or 1/40 of the total first strand synthesis product as a template for PCR amplification using either the FastStart SYBR Green with ROX  or the GoTaq QPCR Master Mix  respectively. Each reaction was carried out in duplicate as described 2, 0.1% NP40, 10% glycerol) with a 25G 1 ½ needle (BD Transduction) and incubated 10 min on ice. The lysed cells were centrifuged at 1430 g for 10 min at 4°C. The supernatant was collected as the cytoplasmic fraction. The remaining nuclear pellet was resuspended in 2.25 ml of S1 buffer  and was layered over a 2.25 ml cushion of S3 buffer . Cellular fractions were solubilized in RIPA buffer . Nuclear lysate was sonicated on ice using a microtip with 21×10 s pulses at 25% amplitude with a 10 s pause in between each pulse . The lysates were then clarified by centrifugation at 2800 g for 10 min at 4°C. Protein concentrations were determined using a standard Bradford assay (Biorad). Nuclear protein (15 µg) was diluted to a final concentration of 1× LDS buffer (Invitrogen) and separated on a 4–12% NuPAGE Bis-Tris Precast Gel. Resolved proteins were transferred to a nitrocellulose membrane, which was blocked with 5% Milk in 0.5% TBST and reacted with antibodies against mouse β-catenin (Sigma) or mouse RNA-polII , at a dilution of 1∶2000 or 1∶1000, respectively. Chemiluminescence was generated using appropriate secondary horseradish peroxidase-conjugated antibodies (Thermoscientific), followed by incubation with the Luminata™ Crescendo substrate  and detected and quantified using the Fuji LAS 4000 mini imaging system and Multigauge quantitation software.P19[shControl] and P19[shβ-cat] cells were differentiated with LiCl or DMSO as described above and harvested on days 1 or 5, respectively, by scraping cells in sterile PBS. Cells were collected and pellets were resuspended in 3 ml of ice-cold Buffer B .P19 cells were differentiated for two days in the presence of DMSO or DMSO+RA and processed according to methods described previously The Student’s t-test or one-way ANOVA’s were used to calculate the statistical significance of differences between means, where a *p-value ≤0.05 was considered significant.Panel I). β-catenin transcript levels were significantly reduced on days 0 and 9, with and without RA treatment, in P19[shβ-cat] cells when compared to P19[shControl] cells (Panel I). The expression of β-catenin transcripts on day 5 was not downregulated in P19[shβ-cat] cells treated with DMSO and was slightly upregulated with DMSO and RA. Thus, β-catenin transcript levels were reduced in P19[shβ-cat] cells at the beginning and end of the differentiation time course under both treatment conditions.P19[shβ-cat] cell lines were created to determine if β-catenin expression was essential for efficient skeletal myogenesis in P19 cells. The efficacy of downregulation of β-catenin mRNA, protein, and transcriptional activity was determined in P19[shβ-cat] cells compared to P19[shControl] cells, using QPCR, western blot analysis of nuclear extracts, and luciferase activity, respectively . P19[shβl] cells , Panel IPanels II and III). The abundance of nuclear β-catenin protein was significantly reduced, by approximately 40%, in P19[shβ-cat] cells compared to P19[shControl] cells (Panel III). To test whether the knockdown had a significant effect on β-catenin transcriptional activity, luciferase assays were performed. P19[shβ-cat] and P19[shControl] cells were transiently transfected with either the Wnt-responsive reporter construct Super8XTOPFLASH or the mutant Wnt-nonresponsive reporter construct Super8XFOPFLASH and were aggregated for one day with or without LiCl, a known inducer of Wnt signalling To determine whether the β-catenin mRNA levels correlated with β-catenin protein, western blot analysis was performed on nuclear extracts from cells differentiated for 5 days with DMSO during the aggregation phase, and quantified , termed MF20, and the percentage of total cells expressing MHC was calculated (Panels I and II). Cultures were co-stained with an antibody to desmin (green). Since the structural striated muscle genes are present in both developing cardiac and skeletal muscle, MHC-positive cells were identified as skeletal muscle by their bipolar morphology, which contrasts the rounded shape of cardiomyocytes Since P19[shβ-cat] cells were shown to have highly reduced β-catenin nuclear activity, we set out to determine how this affected Panel II), whereas MHC3 was downregulated by ∼55%. Therefore, β-catenin was required for efficient in vitro skeletal myogenesis as early as the skeletal muscle progenitor stage, since the loss of β-catenin resulted in a downregulation of all skeletal muscle progenitor genes tested.To decipher the stage at which skeletal myogenesis was inhibited, we examined changes in gene expression by QPCR analysis during myogenic differentiation of P19[shβ-cat] cells. On day 5 of differentiation, expression of genes representing the somitic/premyogenic mesoderm stages, such as Meox1, Gli2, Pax3, Six1, Foxc1 and Foxc2 were examined and, on day 9 of differentiation, genes representing the myoblast stage, such as MyoD, myogenin, and Myf-5 were examined Previously we had shown that Gli2, Meox1, Pax3, Six1 and Foxc1/2 could be upregulated by expression of a constitutively activated β-catenin Foxc1, although Foxc1 overexpression could not initiate the pathway of skeletal myogenesis in P19 cells Sox7 and Pax3 were examined via chromatin immunoprecipitation experiments (ChIP). The 5 kb regions upstream of Pax3 and Sox7 were examined for conserved LEF/TCF binding sites using MuLAN software as described Sox7 upstream region . The enrichment of immunoprecipitated sites at the LBE1 region on day 2 was statistically significant when compared to the enrichment at LBE2 and Pax3, as well as to the enrichment identified on day 0 for all the loci. Thus, β-catenin is associated with a Sox7 gene regulatory region in a population of aggregated P19 cells.To perform the ChIP-QPCR, chromatin was isolated from P19 cells grown in monolayer or after mesoderm induction following aggregation with DMSO for two days. Subsequently, chromatin was extracted and immunoprecipitated with an antibody against β-catenin, followed by QPCR using primer pairs that flank LBE1, LBE2, or the conserved LEF site in the Previously we had shown that RA could not bypass the inhibition of skeletal myogenesis by a dominant negative β-catenin/engrailed fusion protein Since RA is able to recover, at least partially, the expression of several skeletal muscle progenitor genes in P19[shβ-cat] cells, we sought to determine if there was recovery or enhanced expression of myoblast or terminal differentiation markers. As shown by QPCR analysis on day 9 of differentiation, MRF transcript levels and terminal differentiation markers were still reduced in P19[shβ-cat] cells when compared to P19[shControl] cells during RA-induced differentiation . Reduced Foxc2 expression in both the absence and presence of RA in P19[shβ-catenin] cells suggests that canonical Wnt signalling is essential for its expression and that Foxc2 is not regulated by RA signalling, consistent with its predominant role in regulating vascular smooth muscle development Our findings examined transcription factors not studied previously in P19[β-cat/EnR] cells, showing that Six1, Eya2, Foxc1, and Sox7 expression can also be recovered in P19[shβ-cat] cells by RA signalling. The involvement of Six1 and Eya2 in a positive regulatory loop with Pax3 and Pax7 may partially explain their increased transcription Xenopus, Myf5 expression is highly reduced, when canonical Wnt signalling is inhibited, and TCF/LEF sites have been identified in the early epaxial enhancer of Myf5, further suggesting a regulatory role by β-catenin Although the expression of several skeletal muscle precursor genes are upregulated in the presence of RA, MRF and terminal marker expression was significantly downregulated in P19[shβ-catenin] cells, which agrees with findings in P19[β-cat/EnR] cells In summary, these results demonstrate that the transcriptional activity of β-catenin is required for efficient skeletal muscle precursor gene expression leading to myogenesis in P19 cells. Furthermore, β-catenin can be replaced by RA signalling for at least partial expression of many muscle precursor genes but this did not lead to expression of MRFs or terminal muscle differentiation markers. The enhancement of premyogenic mesoderm genes by RA when nuclear β-catenin is reduced suggests that the RA and Wnt signalling pathways are intricately connected and work together in a complex manner to regulate gene expression during embryonic skeletal muscle development. Insight into the intersection of signalling pathways that regulate key transcriptional networks may lead to novel approaches to treat skeletal muscle diseases.Figure S1RA enhanced the expression of MRFs and MHC in P19[shControl] cells. P19[shControl] cells were differentiated in the presence of 1% DMSO with or without 3 nM RA as described in (PDF)Click here for additional data file.


Observers often need to attentively track moving objects. In everyday life, such objects are often visually distinctive. Previous studies have shown that tracking accuracy is increased when the targets contain a visual feature  not possessed by the distractors. Conversely, a gain in tracking accuracy was not observed when the targets differed from the distractors by only a conjunction of features (Makovski and Jiang,  How does an observer make sense of an environment in which objects are constantly changing locations? One important strategy is to keep track of the locations of a handful of the most important objects. In this article we study this tracking ability and investigate to what extent visual distinctiveness can assist object tracking.The ability of humans to keep track of moving objects has typically been studied using the multiple object tracking (MOT) paradigm  not shared by any one distractor, attending to the target features increases the neural activation of the target-responding neurons relative to the neural activation of the distractor-responding neurons. During tracking, an important possible consequence is that in situations where the target might be confused with a distractor if the objects were identical, this greater neural activation of the target-responding neurons prevents the loss of the target.When participants attend to a Although it is already known that this type of target-distractor relationship benefits visual search . They all reported normal or corrected-to-normal visual acuity and reported not being colorblind. All observers provided informed written consent and the study was approved by the Departmental Human Ethics Advisory Group in the School of Psychological Sciences at the University of Melbourne.Subjects viewed a 21″ CRT monitor at a resolution of 1280 × 1024 pixels with a 85 Hz frame rate from a distance of approximately 60 cm. Stimuli were presented in MATLAB  using Psychophysics toolbox . In both these conditions the targets had a feature not shared with the distractors. The purpose of these two conditions was to test the previous finding that featural distinctiveness enhances tracking accuracy relative to the homogenous condition . The point of this condition was to confirm that non-guidable targets do not enhance tracking accuracy relative to the homogenous condition.In the size-color conjunction condition the targets contained a single combination of features not shared by any one distractor. For example, the targets could be small red squares and the distractors could be small green squares and large red squares. Alternatively, the targets might be large blue squares and the distractors could be small blue squares and large yellow squares. All possible combinations of sizes and colors were equally likely to be tested subject to the constraint that the targets always contained a unique set of features not shared by any one distractor. Thus, the targets were always guidable. The point of this condition was to test our hypothesis that conjunction targets enhance tracking accuracy relative to the homogenous condition only when the targets are guidable, i.e., attending to the target features will direct attention preferentially to the targets as opposed to the distractors.  all the distractors were always the same size and color, (b) half the targets had the same color as the distractors but were a different size, and (c) the remaining targets were the same size as the distractors but were a different color. For example, two targets might be small red squares and the other two targets might be large green squares. In this case, the distractors might be all large red squares. Because the targets contained a unique set of features  not shared by any one distractor, the feature-based attention hypothesis predicts that by attending to these two features, attention could be directed preferentially to the targets, thereby increasing tracking accuracy relative to the homogenous condition.An important prediction of our feature-based attention hypothesis is that it should be possible to preferentially direct attention to the targets, even when the targets are not all the same. The conjunction-distinct condition tested a corollary prediction. It used exactly the same target set as the size or color-distinct condition . The crucial difference was that the distractors were no longer homogenous and taken as a set contained the same set of features as the targets, though arranged differently so that no distractor was identical to any target. Half the distractors were always one color and size and the remaining distractors were always a different color and size. For example, two targets might be small red squares and the other two targets might be large green squares. In this case, half the distractors would be small green squares and the remainder would be large red squares. Since the targets and the distractors both contained the same four features , the feature-based attention hypothesis would not predict tracking accuracy to be greater in this condition than in the homogenous condition. Consistent with this prediction, Makovski and Jiang . During the masking, all the squares became white, so that after the masks were removed they were all identical. The observer then used a computer mouse to click on all four targets. The observer did not have to indicate which target had which attributes, so did not have to distinguish between the targets. After the trial ended the observer was informed how many targets he/she got correct. Accuracy was defined as the average proportion of correctly identified targets. As 4 of the 12 squares were targets, chance accuracy was 33%. The experiment began with 10 practice trials followed by 280 test trials, evenly divided between the seven conditions, presented in a random order.F = 111, p < 0.001. We performed planned repeated measures t-tests to test the above predictions.The results are shown in Figure color-distinct condition t(14) = 25.0, p < 0.001 and for the size-distinct condition t(14) = 7.58, p < 0.001. These results confirm previous findings that featural distinctiveness aids tracking  = 8.18, p < 0.001] and that in the color–color conjunction condition . Accuracy in the color–color conjunction condition was slightly greater than that in the homogeneous condition , though this difference was very small (51 versus 47%). This last result is somewhat surprising as the feature-based attention hypothesis would predict the two conditions to be equal.As predicted by the feature-based attention hypothesis, accuracy in the size or color-distinct condition was also significantly greater than that in the homogeneous condition  and that in the conjunction-distinct condition .As predicted by the feature-based attention hypothesis, accuracy in the conjunction-distinct condition was slightly greater than that in the homogeneous condition  – 57 versus 47%. This final result is not predicted by the feature-based attention hypothesis but could explained by postulating that target recovery provides a small benefit to tracking accuracy, as detailed in the Section Surprisingly, tracking accuracy in the part and whole components separately . The method of this experiment was similar to that of the previous experiment, with a multi-colored mask covering the objects at the end of the trial, after which the squares reappeared in white, so that they were all identical. The difference from Experiment 1 was that at the end of each trial one item was ringed and the participant was asked only whether or not the ringed item was a target. Conversely, in Experiment 1 participants were asked to click on all the targets. Only asking one question per condition allowed us to compare conditions with different numbers of targets in an unbiased fashion. At the end of each trial the observer was informed whether he/she was correct or not.part–part color conjunction condition and the part-whole color conjunction condition. In both of these conditions the observer was required to track five targets. There were also two homogeneous conditions in which all objects were uniform in color and size. In one, the observer tracked five targets and in the other the observer tracked four targets. The experiment started with 10 practice trials and then there were 200 trials, equally divided between the four conditions.In total, there were four conditions Figure . There wpart-whole conjunction condition than in both the part–part conjunction condition and in the homogenous 5-target condition. As shown by Figure The feature-based attention hypothesis predicts that accuracy should be greater in the homogenous 5-target condition, t(14) = 3.67, p = 0.003. For the part–part conjunction condition, t(14) = 3.08, p = 0.008. For the part-whole conjunction condition, t(14) = 7.16, p < 0.001. For the homogenous 4-target condition, t(14) = 2.31, p = 0.037.Tracking accuracy in each of the four conditions was significantly greater than chance: for the F = 15.7, p < 0.001]. We performed planned repeated measures t-tests to test the predictions described in the Section part–part conjunction condition was again slightly greater than that in the homogeneous 5-target condition, but this difference was not significant . This result is in keeping with our finding from Experiment 1 that accuracy in these two conditions are very similar, showing that part–part color conjunctions do not substantially aid tracking. Conversely, accuracy in the part-whole color conjunction condition was significantly greater than that in the homogeneous 5-target condition  and that of the part–part color conjunction condition . This is in keeping with the prediction of the feature-based attention hypothesis that part-whole conjunctions should aid tracking. To put in perspective how much part-whole conjunctions can aid tracking we compared accuracy in this condition to that in the homogeneous 4-target condition. Accuracy was significantly greater in the part-whole color conjunction condition , even though in this condition observers were required to track one additional target. This finding argues against the track-and-search hypothesis described previously.A repeated measures ANOVA found a significant effect of condition [size-distinct condition and the color-distinct condition. Consistent with Makovski and Jiang  than in the non-guidable situation  or in the homogeneous condition. This contradicts a previous conclusion that conjunction targets cannot increase tracking accuracy  greater than that in the homogenous condition.The above results can all be accounted for by our feature-based attention hypothesis. However, there are some results that are not consistent with this hypothesis. For example, it cannot explain why in Experiment 1 tracking accuracy in the no role in object tracking. Target recovery may play a minor role .According to the model of multiple identity tracking (MOMIT; Oksama and Hyona, size-color conjunction condition than in the homogeneous condition, but it is also not clear how it could explain why performance is greater in the size-color conjunction condition than in the color–color conjunction condition.A model by Vul et al.  is similMakovski and Jiang , p. 193 In conclusion, this is the first study to demonstrate that conjunction targets can increase tracking accuracy. Our findings indicate that this advantage is greatest when all the conjunction targets contain the same combination of features not shared by any one distractor. However, even when this condition is not satisfied, conjunction targets can still aid tracking, albeit to a lesser extent. Our results do not imply that the attentional tracking system has access to bound target representations and instead we have argued for a feature-based attention account.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The transcription factor SOX2, which is involved in the induction of pluripotent stem cells and contributes to colorectal carcinogenesis, is associated with a poor prognosis in colon cancer (CC). Furthermore, SOX2 is a repressor of the transcriptional activity of β-catenin in vitro. Since the majority of CC develop via an activation of the Wnt/β-catenin signalling pathway, indicated by nuclear expression of β-catenin, we wanted to investigate the expression patterns of SOX2 and β-catenin and correlate them with the occurrence of lymph node and distant metastases as indicators of malignant progression.The expression of SOX2 and β-catenin was investigated in a case control study utilizing a matched pair collection (N = 114) of right-sided CCs with either corresponding distant metastases (N = 57) or without distant spread (N = 57) by applying immunohistochemistry.p = 0.006) and distant metastases (p = 0.022). Nuclear β-catenin expression correlated significantly only with distant metastases (p = 0.001). Less than 10% of cases showed a coexpression of high levels of β-catenin and SOX2. The positivity for both markers was also associated with a very high risk for lymph-node metastases (p = 0.007) and distant spread (p = 0.028).Elevated protein expression of SOX2 significantly correlated with the presence of lymph node- (We demonstrated that increased expression of either SOX2 or nuclear β-catenin are associated with distant metastases in right-sided CC. Additionally, SOX2 is also associated with lymph-node metastases. These data underline the importance of stemness-associated markers for the identification of CC with high risk for distant spread. Colon cancer (CC) is one of the most common human malignancies and one of the major causes of cancer related death worldwide . About 6The transcription factor SOX2 is involved together with c-Myc, KLF4 and Oct3/4 in the induction and maintenance of pluripotent stem cells -18. ReceFormalin fixed-paraffin embedded (FFPE) samples of right-sided CCs from 114 patients that underwent curative surgical tumour resection at the Ludwig-Maximilians-Universität (LMU) München between 1994 and 2005 were recovered from the archives of the Institute of Pathology. The corresponding clinico-pathological datasets were obtained from the Munich Cancer Registry   before adding primary SOX2 specific rabbit monoclonal antibody  for 60 min at room temperature. Thereafter, a development step was introduced by adding detection-system  and subsequently substrate-chromogen containing AEC + system  according to the respective manufacturer's protocols. Finally, slides were counterstained using Hematoxylin . To control for unspecific binding of antibodies, isotype controls were included. Normal stomach tissues with a well described SOX2 staining pattern were used as the positive controls .For the detection of β-catenin prediluted anti-β-catenin mouse monoclonal antibody  was used as the primary antibody. Staining was done employing a Ventana Benchmark XT autostainer using the XT UltraView diaminobenzidine kit  following the manufacturer's instructions.Evaluation of SOX2 immunostaining was done by two observers  separately for the nucleus and the cytoplasm. The results for nuclear staining were evaluated for both, percentage and intensity of stained nuclei. Intensity values were categorized as follows: no staining - 0, weak - 1, moderate - 2, and strong - 3. When less than 10% of cells were stained and/or the intensity was missing (0) or weak (1), immunostaining was reported to be negative . The cytResults of the β-catenin specific immunohistochemistry were also evaluated by two observers  separately for the subcellular localization (cytoplasm and nucleus). For nuclear β-catenin expression the quantity of stained tumour cells throughout the whole tumour but not the intensity of staining was considered. The score for nuclear β-catenin expression was as follows: negative - 0, < 30% - 1+, 30-60% - 2+, > 60% positive cells - 3+. For cytoplasmic β-catenin expression the intensity of specific staining was analysed and graded into no - 0, weak - 1, moderate - 2 or strong cytoplasmic staining - 3. For subsequent statistical analysis the cases were classified into low (scores 0 and 1) and high grade (scores 2 and 3) extent for nuclear and cytoplasmic protein expression, respectively.2-test using SPSS v. 18.0 . For all tests a two-sided α-error of less than 5% (p < 0.05) was considered statistically significant.Significance of correlations of the immunhistochemical analyses were calculated using the χp = 0.006), distant metastases (p = 0.022) and histopathological tumour grade (p = 0.043) according to WHO. No correlations with tumour size (according to UICC), gender, or age were found  or without metastases and a disease free survival of more than 5 years (N = 57) was subjected to immunohistochemistry  showed nuclear expression within the stem cell niche of the colonic crypts Figure . No corrp = 0.001), but not with lymph node metastases, tumour stage, histopathological tumour grade, gender, or patient's age (Table Strong nuclear β-catenin expression was found in 33.3% of cases (N = 38) and correlated significantly with the occurrence of distant metastases p = 0.00, but notp = 0.02) and distant spread (seven of eight cases (87.5%) showed distant metastases, p = 0.001; Table Combined high scores of SOX2 and nuclear β-catenin expression were present in 8 of 114 cases (7.0%), thus representing about one third of all SOX2 positive cases. In this subgroup a significant correlation with nodal status .Nevertheless, no correlation between SOX2 expression and nuclear or cytoplasmic β-catenin protein levels with each other was observed  and stem cell formation during embryonic development, the combination of EMT and stem cell competence during malignant progression of CC, both regulated by β-catenin, may result in "migrating tumour stem cells", driving tumour invasion and metastases ,9. β-cat2-test, data not shown). However, similar to β-catenin combined scores of high SOX2 and high CD133 showed strong predictive value according to the risk of distant spread  and the presence of lymph-node metastases . In summary, our data underline the importance of stemness-associated markers for the prediction of distant spread and the prognostification of CC. These observations also indicate that markers and marker-combinations for a stemness phenotype could be relevant for identification of CCs with high risk for distant spread and subsequently could have major impact on clinical decisions according to the radicalness of loco-regional surgery and adjuvant chemotherapy.This is also supported by our finding that SOX2 expression can be found within the stem cell niche in a small proportion of normal colonic crypts (Figure Through in vitro studies it was shown that SOX2 represses the transcriptional activity of the β-catenin/TCF complex, thus affecting the expression of oncogenic β-catenin driven transcription ,21,22. TTaken together, our results suggest that SOX2 protein expression may be a possible prognostic marker in the management of CC patients. Nevertheless, this observation requires validation in larger, randomized patient cohorts including also the left side of the colon and the rectum before SOX2 immunohistochemistry should be used in the clinical setting.In summary, we report that overexpression of SOX2 significantly correlated with lymph-node and distant metastases in right-sided CCs. Since both types of metastases are independent predictors of bad prognosis our results are in concordance with the reduced survival reported in the literature. In the same collection, we revealed that nuclear β-catenin was associated with distant metastases. Furthermore, coexpression of both markers was significantly associated with distant spread and lymph-node metastases in our collection of CCs.The authors declare that they have no competing interests.JN: Participated in the design and coordination of the study, evaluated the β-catenin and SOX2 immunohistochemistry, performed the statistical analysis and drafted the manuscript. FB: Evaluated the SOX2 immunohistochemistry, participated in the selection of the case control collection. DH: Participated in the design and selection of the case control collection and helped to draft the manuscript. LK: Participated in the selection of the case control collection and evaluated the β-catenin immunohistochemistry. JE: Participated in the design of the case control collection and provided clinical data from the Munich Cancer Registry. RML: Validated the SOX2 antibody for immunohistichemistry. MG: Participated in the design and coordination of the study and helped to draft the manuscript. TK: Conceived of the study, participated in the design, provided funding and helped to draft the manuscript. AJ: Is the principal investigator, conceived of the study and participated in its design and coordination, provided funding and drafted the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/518/prepub


The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional importance in human disease. In contrast, very little is known about the roles of chondroitin sulfate molecules and sulfation patterns during mammalian development and cell lineage specification. Here, we report a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through modulation of Wnt/beta-catenin signaling. Lineage marker analysis demonstrates that enzymatic elimination of endogenous chondroitin sulfates leads to defects specifically in cardiac differentiation. This is accompanied by a reduction in the number of beating cardiac foci. Mechanistically, we show that endogenous chondroitin sulfate controls cardiac differentiation in a temporal biphasic manner through inhibition of the Wnt/beta-catenin pathway, a known regulatory pathway for the cardiac lineage. Treatment with a specific exogenous chondroitin sulfate, CS-E, could mimic these biphasic effects on cardiac differentiation and Wnt/beta-catenin signaling. These results establish chondroitin sulfate and its sulfation balance as important regulators of cardiac cell lineage decisions through control of the Wnt/beta-catenin pathway. Our work suggests that targeting the chondroitin biosynthesis and sulfation machinery is a novel promising avenue in regenerative strategies after heart injury. Chondroitin-4-sulfotransferase-1 (C4st-1) gene Carbohydrate sulfotransferase 3 (CHST3) gene, involved in the production of CS, have been shown to be associated with Larsen syndrome, humero-spinal dysostosis CHST3 knock-out mice and have neither skeletal nor cardiac defects The glycosaminoglycan chondroitin sulfate (CS) consists of linear chains of repeating disaccharide units covalently linked to cell surface and secreted proteins to form chondroitin sulfate proteoglycans The Wnt/beta-catenin signaling pathway plays critical roles in many developmental processes, and aberrant Wnt/beta-catenin pathway activity is causally associated with many human diseases, including cancers Interestingly, CS has recently been shown to control the Wnt/beta-catenin pathway. CS-E, but not other CS forms, can bind Wnt3a ligand with high affinity Here, we initially set out to investigate the roles of CS in ES cell differentiation in embryoid body (EB) cultures. We demonstrate by lineage marker analysis that enzymatic elimination of endogenous chondroitin sulfate by the bacterial enzyme Chondroitinase ABC (ChABC) leads to a defect in cardiac differentiation, and causes a reduction in the number of functional cardiac foci. We further show that elimination of CS in EB cultures activates the Wnt/beta-catenin pathway, a known regulatory pathway with a biphasic function in cardiac lineage determination. Limiting ChABC treatment to only early or late stages of ES cell differentiation mimics the biphasic effects of Wnt/beta-catenin pathway activation on cardiac differentiation. Temporally restricted treatment with a specific exogenous chondroitin sulfate, CS-E, inhibits Wnt/beta-catenin signaling and phenocopies the biphasic effects of endogenous CS on cardiac differentiation and Wnt/beta-catenin signaling. Together, these results establish a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through inhibition of the Wnt/beta-catenin pathway.Chondroitinase ABC (protease-free) and CS-E were obtained from The Associates Of Cape Cod, USA. C4S was obtained from Sigma Inc., USA.R1 ES cells and EBs were cultured as previously described Immunofluorescence microscopy was performed as previously described RNA was prepared using TRI Reagent or TRIZOL according to the manufacturer’s instruction. Subsequently, 1.5 microgram of RNA were reversed transcribed using MuMLV-Reverse Transcriptase , followed by real time amplification using a 2xSYBR Green PCR Master Mix  on an Applied Biosystems 7500 Real Time PCR platform in 15 microliter reactions using an annealing temperature of 60°C.The following primer pairs were used (5′ to 3′):HPRT:ATGCCGACCCGCAGTCCCAGC and CGAGCAAGTCTTTCAGTCCTGTCCOct4:TTGGGCTAGAGAAGGATGTGGTT and GGAAAAGGGACTGAGTAGAGTGTGGNanog:GTGCTGAGCCCTTCTGAATC and GAACTCTCCTCCATTCTGAACBrachyury:CTGGCCACACCAGCATGCTGCC and GTACCATTGCTCACAGACCAGAGACTGMesogenin:CCGGGATCCTGGGTGAGACCTTCCTCAGC and TGCCAAGCTTGGCCTGGGCTCTCTCCCGCFgf5:GATTGTAGGAATACGAGGAGTTTTCAGCAAC and TCTTGGAATCTCTCCCTGAACTTACAGTCAAfp:AGCAAAAGCCTGAACTGACAGAGGAGCAG and GTTTTGGAAATCAACTTTGGACCCTCTTCTGTGGata4:AAGAGATGCGCCCCATCAAGACA and TGGGGACAGCTTCAGAGCAGACAGNkx2.5:GGCGTCGGGGACTTGAACACC and CGCACTCACTTTAATGGGAAGMlc-2v:TGGGTAATGATGTGGACCAA and GGGAGGTTCTCCAAAGAGGA.Wnt3a and control conditioned media were prepared from commercially available L cells stably transfected with a Wnt3a expression plasmid, or non-transfected control L cells , as previously described Protein lysates from EBs were isolated in RIPA lysis buffer as previously described ES cells or EBs were transiently transfected with firefly TOPFLASH Experiments were performed in triplicates; appropriate statistical analysis was conducted for comparisons among groups (Student’s t-test).In order to investigate the roles of endogenous chondroitin sulfates in ES cell differentiation, we employed a previously established EB flat culture system Nanog and Oct4  were affected by ChABC treatment  spiked on day 3 in control cultures, and then decreased drastically in later time points. Treatment with ChABC showed the same pattern, with somewhat reduced expression on day 3 when compared to controls. Expression of the endoderm marker alpha-fetoprotein (Afp) was initiated only after day 6, and rose steadily thereafter in control cultures. The same pattern was present with ChABC treatment; however, Afp expression was reduced by 20% at day 12. Expression of the early and axial mesoderm marker Brachyury spiked on day 6, and was indistinguishable between control and ChABC-treated cultures. Mesogenin as a marker of paraxial mesoderm also spikes on day 6, but expression did not decline as fast as expression of Brachyury. Treatment with ChABC lead to an increased expression of Mesogenin on day 9, but was equivalent to controls on day 12. We next investigated markers of cardiac differentiation in our experimental setup. The transcription factors Gata4 and Nkx2.5 are early determinants of cardiac differentiation. Expression of these two factors started between days 3 and 6 and peaked on day 9, followed by a reduction on day 12 in control conditions. Interestingly, treatment with ChABC led to an approximately 50% reduction in Gata4 and Nkx2.5 expression on day 12 (myosin light chain -2 ventricle (Mlc-2v), a marker of differentiated cardiomyocytes. Expression started only after day 6, which is consistent with the onset of cardiomyocyte differentiation in these EB cultures, and increased dramatically on day 12 in control cultures. EB cultures treated with ChABC displayed a severe reduction of Mlc-2v expression on day 12 when compared to controls  gene have been shown to be associated with Omani-type spondyloepiphyseal dysplasia. A subset of these patients exhibit mild cardiac abnormalities, including mitral, tricuspid and aortic regurgitations CHST3 knock-out mice and have neither skeletal nor cardiac defects Several studies have highlighted cardiac expression domains of CS and enzymes involved in CS biosynthesis. For example, we showed previously that in vitro and/or in vivo after heart injury.While the roles of CS in mammalian heart development are not well understood, increasing evidence supports the notion that CS-containing proteoglycans (CSPGs) plays critical roles during cardiovascular injury. The CS side chains of the CSPG Biglycan can control elastin assembly in vascular walls From a signaling point of view, this work has important pharmacological implications. Aberrantly activated Wnt/beta-catenin signaling has been associated in a number of human diseases and malignancies, including cancer, fibrosis, and cardiovascular disease