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BACKGROUND The WNT signaling pathway is involved in the regulation of bone homeostasis, and the effect of WNT signaling pathway-related gene (WNT16 and LRP5) polymorphisms on osteoporosis risk among Chinese postmenopausal women is still unknown. Hence, we performed a case-control study to assess the association of WNT signaling pathway-related gene polymorphisms and osteoporosis risk. METHODS A total of 1026 women (515 osteoporosis patients and 511 controls) of postmenopausal age who were randomly sampled from Xi'an 630 Hospital (Shaanxi Province, China) were involved in this study. Seven genetic polymorphisms in WNT16 (rs3779381, rs3801387, rs917727 and rs7776725) and LRP5 (rs2291467, rs11228240 and rs12272917) were selected and genotyped using the Agena MassARRAY iPLEX system. The association of the genetic polymorphisms and osteoporosis risk was assessed by odds ratios and 95% confidence intervals. The multifactor dimensionality reduction (MDR) method was conducted to analyze single nucleotide polymorphism (SNP)-SNP interaction. RESULTS We found that LRP5 polymorphisms (rs2291467, rs11228240 and rs12272917) were significantly associated with a decreased risk of osteoporosis in homozygote, recessive and additive models (p 24 (p

The conversion of mechanical energy into biochemical changes within living cells is process known as mechanotransduction. Bone is a quintessential tissue for studying the molecular mechanisms of mechanotransduction, as the skeleton's mechanical competence is crucial for vertebrate movement. Bone cell mechanotransduction is facilitated by a number of cell biological pathways, one of the most prominent of which is the Wnt signaling cascade. The Wnt co-receptor Lrp5 has been identified as a crucial protein for mechanical signaling in bone, and modifiers of Lrp5 activity play important roles in mediating signaling efficiency through Lrp5, including sclerostin, Dkk1, and the co-receptor Lrp4. Mechanical regulation of sclerostin is mediated by certain members of the Hdac family. Other mechanisms that influence Wnt signaling-some of which are mechanoresponsive-are coming to light, including R-spondins and their role in organizing the Rnf43/Znrf3 and Lgr4/5/6 complex that liberates Lrp5. While the identity of the key Wnt proteins involved in bone cell mechanical signaling are elusive, the likely pool of key players is narrowing. Identification of Wnt-based molecular targets that can be modulated pharmacologically to make mechanical stimulation (e.g., exercise) more beneficial is an emerging approach to improving skeletal integrity and reducing fracture risk.

Objective To explore genetic polymorphisms of the Wnt/β-catenin signalling pathway in primary SS (PSS). Methods We included 98 patients with PSS and 165 healthy volunteers. Genomic DNA was extracted from peripheral blood samples. Through an open-array platform of low density, we genotyped 25 polymorphisms from 14 genes (WISP1, DKK1, SOST, FRZB, LRP1, LRP4, LRP5, LRP6, GSKB, ADAMTS5, GDF5, FMN2, ADIPOQ and COL11A1) involved in the Wnt/β-catenin signalling pathway. We compared the allelic and genotypic frequencies with Fisher’s exact test and logistic regression analysis adjusted by age, gender and individual admixture, as well as bootstrap-resampling analysis. We assessed the gene–gene interaction by the multifactor dimensionality reduction method. Results We found a positive significant association with four polymorphisms: LRP5 rs606989, FRZB rs409238, GSK3B rs2037547 and ADIPOQ rs2241766. All of them conferred risk for PSS, being the highest among subjects carrying three to four risk alleles (P < 0.001). According to a multifactor dimensionality reduction analysis, the best models included the LRP5 (rs606989), FRZB (rs409238) and ADIPOQ (rs2241766) polymorphisms. Conclusion LRP5, FRZB and ADIPOQ genes related in the Wnt/β-catenin signalling pathway increased the risk of PSS. Further research is needed to establish their functional role in this clinical entity.

Bone abnormalities as a consequence of osteoblast deregulation are associated with several diseases such as diabetes and chronic kidney disease. Important role for oxidized low density lipoproteins (oxLDL) in the pathophysiology of bone disorders has been reported. However, little is known about the effects and mechanisms of oxLDL on the process of osteoblastogenesis in human mesenchymal stem cells (MSCs). We show that oxLDL concentrations of ~ 10-25 µg protein (0.43-1.0 µM MDA/mg protein) inhibited the differentiation of MSCs to osteoblasts. We demonstrate that the underlying mechanism entails the suppression of the Wnt signaling through the down-regulation of β-catenin. Further, we show the association of scavenger receptor CD36 with the receptors LRP5/6 and Frizzled in mediating the oxLDL effects on the differentiation of MSCs to pre-osteoblasts. Inhibiting CD36 restored osteoblasts differentiation in the presence of oxLDL. Our findings suggest that oxLDL interferes with the canonical Wnt signaling pathway in a CD36 dependent manner leading to an inhibition of osteoblastogenesis.

Hematopoietic stem cells (HSCs) have the capacity for self-renewal to maintain the HSCs' pool and the ability for multilineage differentiation, which are responsible for sustained production of multiple blood lineages. The regulation of HSC development is controlled precisely by complex signal networks and hematopoietic microenvironment, which has been termed the HSCs' niche. The Wnt signaling pathway is one of a variety of signaling pathways that have been involved in HSC self-renewal and maintenance. Previous studies are indeterminant on the regulation of adult HSCs upon canonical Wnt signaling pathways because of the different experimental systems and models used. In this study, we generated the conditional knockout Wnt coreceptor low-density lipoprotein receptor-related protein 5 (Lrp5) and low-density lipoprotein receptor-related protein 6 (Lrp6) mice in adult hematopoiesis via Vav-Cre Loxp system. Inactivation of Lrp5 and -6 in a hematopoietic system diminished the pool of HSCs, but there were no obvious defects in mature immune cells. Lrp5 and -6 double deficiency HSCs showed intrinsic defects in self-renewal and differentiation due to reduced proliferation and increased quiescence of the cell cycle. Analysis of HSC gene expression suggested that the quiescence regulators were significantly up-regulated, such as Egr1, Cdkn1a, Nr4a1, Gata2, Junb and Btg2, and the positive cell cycle regulators were correspondingly down-regulated, such as Ccna2 and Ranbp1. Taken together, we investigated the roles of Lrp5 and -6 in HSCs by functional and bioinformatic assays, and we demonstrated that Lrp5 and -6 are required for the self-renewal and differentiation of adult HSCs. The canonical Wnt pathway may contribute to maintaining the HSC pool and regulate the differentiation of adult HSCs by controlling cell cycle gene regulatory module.-Liu, J., Cui, Z., Wang, F., Yao, Y., Yu, G., Liu, J., Cao, D., Niu, S., You, M., Sun, Z., Lian, D., Zhao, T., Kang, Y., Zhao, Y., Xue, H.-H., Yu, S. Lrp5 and Lrp6 are required for maintaining self-renewal and differentiation of hematopoietic stem cells.

Our study was aimed to investigate the effects of lgals3a (Gal-3 encoding gene) on the development of zebrafish embryo and its underlying mechanisms. Morpholino (MO) technology was used to inhibit the expression of zebrafish lgals3a, and the effect of lgals3a gene knockdown on zebrafish embryo development and the number of monocyte macrophages was observed. Effect of lgals3a-e3i3-MO on apoptosis of zebrafish was detected by acridine orange staining. In addition, the mRNA expression levels of Wnt/β-catenin signaling pathway-related genes were detected by RT-qPCR. Compared with control-MO group, the zebrafish embryos injected with lgals3a-e3i3-MO had obvious defects in the head, eyes, and tail, and pericardial edema. Lgals3a-e3i3-MO significantly reduced the number of mononuclear macrophages in zebrafish embryos compared with the control-MO group. The results of acridine orange staining showed that compared with the control-MO group, lgals3a-e3i3-MO promoted cardiomyocyte apoptosis in zebrafish. Furthermore, lgals3a-e3i3-MO significantly up-regulated the expression of dkk1b, wnt9a, lrp5, fzd7a, β-catenin, Gsk-3β, mycn, myca in the Wnt/β-catenin pathway, and decreased the expression of lef1. These results indicate that lgals3a-e3i3-MO inhibits zebrafish embryo development, reduces the number of mononuclear macrophages, activates Wnt/β-catenin signaling pathway and promotes cardiomyocyte apoptosis.

Background Frizzled (FZD) proteins function as receptors for WNT ligands. Members in FZD family including FZD2, FZD4, FZD7, FZD8 and FZD10 have been demonstrated to mediate cancer cell epithelial-mesenchymal transition (EMT). Methods CCLE and TCGA databases were interrogated to reveal the association of FZD5 with EMT. EMT was analyzed by investigating the alterations in CDH1 (E-cadherin), VIM (Vimentin) and ZEB1 expression, cell migration and cell morphology. Transcriptional modulation was determined by ChIP in combination with Real-time PCR. Survival was analyzed by Kaplan–Meier method. Results In contrast to other FZDs, FZD5 was identified to prevent EMT in gastric cancer. FZD5 maintains epithelial-like phenotype and is negatively modulated by transcription factors SNAI2 and TEAD1. Epithelial-specific factor ELF3 is a downstream effecter, and protein kinase C (PKC) links FZD5 to ELF3. ELF3 represses ZEB1 expression, further guarding against EMT. Moreover, FZD5 signaling requires its co-receptor LRP5 and WNT7B is a putative ligand for FZD5. FZD5 and ELF3 are associated with longer survival, whereas SNAI2 and TEAD1 are associated with shorter survival. Conclusions Taken together, FZD5-ELF3 signaling blocks EMT, and plays a potential tumor-suppressing role in gastric cancer.

The cytoplasmic β4-subunit of the voltage-gated calcium channels has been shown be involved in the regulation of gene transcription. This subunit interacts with the transcription factor TCF4 and inhibits the Wnt/β-catenin signaling pathway. These results may also explain the inhibitory effect of the β4-subunit on cell proliferation., The β4 isoform of the β-subunits of voltage-gated calcium channel regulates cell proliferation and cell cycle progression. Herein we show that coexpression of the β4-subunit with actors of the canonical Wnt/β-catenin signaling pathway in a hepatoma cell line inhibits Wnt-responsive gene transcription and decreases cell division, in agreement with the role of the Wnt pathway in cell proliferation. β4-subunit–mediated inhibition of Wnt signaling is observed in the presence of LiCl, an inhibitor of glycogen synthase kinase (GSK3) that promotes β-catenin translocation to the nucleus. Expression of β4-subunit mutants that lost the ability to translocate to the nucleus has no effect on Wnt signaling, suggesting that β4-subunit inhibition of Wnt signaling occurs downstream from GSK3 and requires targeting of β4-subunit to the nucleus. β4-subunit coimmunoprecipitates with the TCF4 transcription factor and overexpression of TCF4 reverses the effect of β4-subunit on the Wnt pathway. We thus propose that the interaction of nuclear β4-subunit with TCF4 prevents β-catenin binding to TCF4 and leads to the inhibition of the Wnt-responsive gene transcription. Thereby, our results show that β4-subunit is a TCF4 repressor and therefore appears as an interesting candidate for the regulation of this pathway in neurons where β4-subunit is specifically expressed.

Molecular underpinning of mycobacteria-induced CNS-pathology is not well understood. In the present study, zebrafish were infected with Mycobacterium fortuitum and the prognosis of CNS-pathogenesis studied. We observed M. fortuitum triggers extensive brain-pathology. Evans blue extravasation demonstrated compromised blood-brain barrier (BBB) integrity. Further, decreased expression in tight-junction (TJ) and adherens junction complex (AJC) genes were noted in infected brain. Wnt-signaling has emerged as a major player in host-mycobacterial immunity but its involvement/role in brain-infection is not well studied. Sustained expression of wnt2, wnt3a, fzd5, lrp5/6 and β-catenin, with concordant decline in degradation complex components axin, gsk3β and β-catenin regulator capn2a were observed. The surge in ifng1 and tnfa expression preceding il10 and il4 suggested cytokine-interplay critical in M. fortuitum-induced brain-pathology. Therefore, we suggest adult zebrafish as a viable model for studying CNS-pathology and using the same, conclude that M. fortuitum infection is associated with repressed TJ-AJC gene expression and compromised BBB permeability. Our results implicate Wnt/β-catenin pathway in M. fortuitum-induced CNS-pathology wherein Th1-type signals facilitate bacterial clearance and Th2-type signals prevent the disease sequel.

The G protein-coupled receptors Frizzled1-10 (FZD1-10) act as molecular checkpoints mediating intracellular signaling induced by 19 mammalian, secreted Wingless/Int-1 lipoglycoproteins (WNTs). Despite the vital roles of these signaling components in health and disease, our knowledge about WNT/FZD selectivity, and the mechanisms of receptor activation and intracellular signal propagation by individual ligand/receptor pairs is limited due to the current lack of suitable biophysical techniques. Here, we developed fluorescence-based biosensors that detect WNT-induced FZD conformational changes in living cells in order to assess WNT action via FZDs at the most proximal level, i.e. the receptor conformation. By testing a panel of recombinant ligands on conformational biosensors representing all four homology clusters of FZDs, we discover yet unappreciated selectivities of WNTs to their receptors and, surprisingly, identify distinct ligand-induced receptor conformations. Furthermore, we demonstrate that FZDs can undergo conformational changes upon WNT binding without being dependent on the WNT co-receptors LRP5/6. This sensor toolbox provides an advanced platform for a thorough investigation of the 190 possible WNT/FZD pairings and for future screening campaigns targeting synthetic FZD ligands. Furthermore, our findings shed new light on the complexity of the WNT/FZD signaling system and have substantial implications for our understanding of fundamental biological processes including embryonal development and tumorigenesis.

Entropy rate is increased by several metabolic and thermodynamics abnormalities in neurodegenerative diseases (NDs). Changes in Gibbs energy, heat production, ionic conductance or intracellular acidity are irreversibles processes which driven modifications of the entropy rate. The present review focusses on the thermodynamic implications in the reprogramming of cellular energy metabolism enabling in Alzheimer's disease (AD) through the opposite interplay of the molecular signaling pathways WNT/β-catenin and PPARγ. In AD, WNT/β-catenin pathway is downregulated while PPARγ is upregulated. Thermodynamics behaviors of metabolic enzymes are modified by dysregulation of the canonical WNT/β-catenin pathway. Downregulation of WNT/β-catenin pathway leads to oxidative stress and cell death through inactivation of glycolytic enzymes such as Glut, PKM2, PDK1, MCT-1, LDH-A but activation of PDH. In addition, in NDs, PPARγ is dysregulated whereas it contributes to the regulation of several key circadian genes. AD is considered as a dissipative structure that exchanges energy or matter with its environment far from the thermodynamic equilibrium. Far-from-equilibrium thermodynamics are notions driven by circadian rhythms. Circadian rhythms directly participate in regulating the molecular pathways WNT/β-catenin and PPARγ involved in the reprogramming of cellular energy metabolism enabling AD processes.

Metastasis significantly reduces the survival rate of osteosarcoma (OS) patients. Therefore, identification of novel targets remains extremely important to prevent metastasis and treat OS. In this report, we show that SPARCL1 is downregulated in OS by epigenetic methylation of promoter DNA. In vitro and in vivo experiments revealed that SPARCL1 inhibits OS metastasis. We further demonstrated that SPARCL1-activated WNT/β-catenin signaling by physical interaction with various frizzled receptors and lipoprotein receptor-related protein 5/6, leading to WNT-receptor complex stabilization. Activation of WNT/β-catenin signaling contributes to the SPARCL1-mediated inhibitory effects on OS metastasis. Furthermore, we uncovered a paracrine effect of SPARCL1 on macrophage recruitment through activated WNT/β-catenin signaling-mediated secretion of chemokine ligand5 from OS cells. These findings suggest that the targeting of SPARCL1 as a new anti-metastatic strategy for OS patients.

Wnt signalling pathways have key roles in joint development, homeostasis and disease, particularly in osteoarthritis. New data is starting to reveal the importance of tightly regulating canonical Wnt signalling pathway activation to maintain homeostasis and health in articular cartilage. In addition to the presence of different Wnt antagonists that limit pathway activation in articular cartilage, the reciprocal crosstalk between the canonical and non-canonical cascades and competitive antagonism between different Wnt ligands seem to be critical in restraining excessive Wnt pathway activation. Changes in transcriptional complex assembly upon Wnt pathway activation, epigenetic modulation of target gene transcription, in particular through histone modifications, and complex interactions between the Wnt signalling pathway and other signalling pathways, are also instrumental in adjusting Wnt signalling. In this Review, the cellular and molecular mechanisms involved in fine-tuning canonical Wnt signalling in the joint are updated, with a focus on the articular cartilage. The interventions for preventing or treating osteoarthritis are also discussed, which should aim to limit disease-associated excessive canonical Wnt activity to avoid joint damage.

Summary Wnt signaling controls multiple biological process, particularly the embryonic development of metazoans. Sustained expression of Wnt signaling components in the mature mammalian CNS and their apparent deregulation in certain neuropathologies suggest that it also plays a part beyond embryonic development to regulate normal brain function. We describe a noncanonical Wnt/Ca 2+ signaling cascade that regulates the electrophysiological intrinsic properties of rat neurons, resulting in sustained membrane depolarization and the mobilization of Ca 2+ from internal stores. These effects require tyrosine kinase-like orphan receptor 2 (RoR2), activation of PLC, and voltage-gated Ca 2+ channels. Activation of this signaling cascade then promotes surface expression of N-methyl-D-aspartate receptors (NMDARs) through a SNARE-dependent mechanism. This neuronal Wnt/Ca 2+ signaling pathway represents a mechanism for Wnt ligands to regulate normal brain processes in the mature animal and provides a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders where NMDARs are compromised.

Wnt signaling plays a role in diverse processes such as cell proliferation, differentiation, migration or cell polarity. Dysfunction of Wnt signaling is associated with human diseases and aging. Wnts can activate several interacting intracellular signaling pathways, in particular the so called canonical and non-canonical pathways. The canonical Wnt response leads to a stabilization of cytoplasmic β –catenin whereas non-canonical Wnt signaling can result in the activation of calcium–calmodulin dependent kinase II (CamKII). Earlier data revealed that those signaling pathways can inhibit each other in a concentration dependent manner. During Xenopus laevis dorsal axis formation, Wnt11 has been shown to activate both, β − catenin signaling as well as CamKII activity. In line, Wnt11 is required for dorsal as well as ventral cell fates. Here, we show that the concentration dependent mutual inhibition of CamKII and β − catenin signaling is sufficient to obtain a switch-like behavior as opposed to a graded response. We present a model that faithfully recapitulates the activity of Wnt11 during dorso-ventral axis formation in Xenopus laevis on the basis of the Wnt switch behavior.

Renin-angiotensin system (RAS) plays a key role in the development and progression of chronic kidney disease (CKD). Recent studies have demonstrated activation of Wnt/β-catenin pathway by RAS in CKD. However, the underlying mechanisms of RAS and Wnt/β-catenin signaling interaction and their contribution to the pathogenesis of CKD have not been fully elucidated. Present study is designed to investigate the role of RAS/Wnt/β-catenin axis activation in tubulo-interstitial fibrosis and glomerulosclerosis by the cultured HK-2 and podocytes. HK-2 cells and podocytes are treated by angiotensin II (Ang II). Ang II up-regulates expression of various Wnt mRNA and active β-catenin protein in HK-2 cells and podocytes in the time- and dose-dependent manners. In addition, Ang II induces injury, oxidative stress and inflammation and impaired Nrf2 activation in HK-2 cells and podocytes. This was accompanied by up-regulations of RAS components as well as Wnt1, activated β-catenin and its target proteins. RAS/Wnt/β-catenin axis activation results in epithelial-to-mesenchymal transition in HK-2 cells and injuries podocytes. The effect of Ang II is inhibited by losartan and ICG-001, a Wnt/β-catenin inhibitor. We further found that treatment with natural products, ergone, alisol B 23-acetate and pachymic acid B inhibit extracellular matrix accumulation in HK-2 cells and attenuated podocyte injury, in part, by inhibiting Ang II induced RAS/Wnt/β-catenin axis activation. In summary, activation of RAS/Wnt/β-catenin axis results in podocytes and tubular epithelial cell, injury and up-regulations of oxidative, inflammatory and fibrotic pathways. These adverse effects are ameliorated by ergone, alisol B 23-acetate and pachymic acid B. Therefore, these natural products could be considered as novel Wnt/β-catenin signaling inhibitors and anti-fibrotic agents.

Chronic obstructive pulmonary disease (COPD), in particular emphysema, is characterized by loss of parenchymal alveolar tissue and impaired tissue repair. Wingless and INT-1 (WNT)/β-catenin signaling is reduced in COPD; however, the mechanisms thereof, specifically the role of the frizzled (FZD) family of WNT receptors, remain unexplored.To identify and functionally characterize specific FZD receptors that control downstream WNT signaling in impaired lung repair in COPD.FZD expression was analyzed in lung homogenates and alveolar epithelial type II (ATII) cells of never-smokers, smokers, patients with COPD, and two experimental COPD models by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on FZD4, WNT/β-catenin signaling, and elastogenic components were investigated in primary ATII cells in vitro and in three-dimensional lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of FZD4 signaling on alveolar epithelial cell wound healing and repair, as well as on expression of elastogenic components.FZD4 expression was reduced in human and experimental COPD lung tissues as well as in primary human ATII cells from patients with COPD. Cigarette smoke exposure down-regulated FZD4 expression in vitro and in vivo, along with reduced WNT/β-catenin activity. Inhibition of FZD4 decreased WNT/β-catenin-driven epithelial cell proliferation and wound closure, and it interfered with ATII-to-ATI cell transdifferentiation and organoid formation, which were augmented by FZD4 overexpression. Moreover, FZD4 restoration by overexpression or pharmacological induction led to induction of WNT/β-catenin signaling and expression of elastogenic components in three-dimensional lung tissue cultures ex vivo.Reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity.

Hepatitis B virus (HBV) has a global distribution and is one of the leading causes of hepatocellular carcinoma. The precise mechanism of pathogenicity of HBV-associated hepatocellular carcinoma (HCC) is not yet fully understood. Viral-related proteins are known to take control of several cellular pathways like Wnt/β-catenin, TGF-β, Raf/MAPK and ROS for the virus's own replication. This affects cellular persistence, multiplication, migration, alteration and genomic instability. The Wnt/FZD/β-catenin signaling pathway plays a significant role in the pathology and physiology of the liver and has been identified as a main factor in HCC development. The role of β-catenin is linked mainly to the canonical pathway of the signaling system. Progression of liver diseases is known to be accompanied by disturbances in β-catenin expression (mainly overexpression), with its cytoplasmic or nuclear translocation. In recent years, studies have documented that the HBV X protein and hepatitis B surface antigen (HBsAg) can act as pathogenic factors that are involved in the modulation and induction of canonical Wnt signaling pathway. In the present review we explore the interaction of HBV genome products with components of the Wnt/β-catenin signaling pathway that results in the enhancement of the pathway and leads to hepatocarcinogenesis.

Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

Wnt/β-catenin signaling is activated when extracellular Wnt ligands bind Frizzled (FZD) receptors at the cell membrane. Wnts bind FZD cysteine-rich domains (CRDs) with high affinity through a palmitoylated N-terminal “thumb” and a disulfide-stabilized C-terminal “index finger,” yet how these binding events trigger receptor activation and intracellular signaling remains unclear. Here we report the crystal structure of the Frizzled-4 (FZD4) CRD in complex with palmitoleic acid, which reveals a CRD tetramer consisting of two cross-braced CRD dimers. Each dimer is stabilized by interactions of one hydrophobic palmitoleic acid tail with two CRD palmitoleoyl-binding grooves oriented end to end, suggesting that the Wnt palmitoleoyl group stimulates CRD–CRD interaction. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that WNT5A stimulates dimerization of membrane-anchored FZD4 CRDs and oligomerization of full-length FZD4, which requires the integrity of CRD palmitoleoyl-binding residues. These results suggest that FZD receptors may form signalosomes in response to Wnt binding through the CRDs and that the Wnt palmitoleoyl group is important in promoting these interactions. These results complement our understanding of lipoprotein receptor-related proteins 5 and 6 (LRP5/6), Dishevelled, and Axin signalosome assembly and provide a more complete model for Wnt signalosome assembly both intracellularly and at the membrane.

The signaling pathways that sustain the disease process of chronic rhinosinusitis with nasal polyps (CRSwNP) remain poorly understood. We sought to determine the expression levels of Wnt signaling genes in CRSwNP and to study the role of the Wnt pathway in inflammation and epithelial remodeling in the nasal mucosa. Microarrays and real time-quantitative polymerase chain reaction comparing gene expression in matched NPs and inferior turbinates revealed that WNT2B, WNT3A, WNT4, WNT7A, WNT7B, and FZD2 were up-regulated and that FZD1, LRP5, LRP6, and WIF1 were down-regulated in NPs. Immunolabeling showed robust expression of Wnt ligands, nuclear β-catenin, and Axin-2 in NP tissue, suggesting that Wnt/β-catenin signaling is activated in NPs. We used primary human nasal epithelial cell (HNEpC) cultures to test the functional consequences of Wnt pathway activation. Monolayer HNEpCs treated with recombinant human WNT (rhWNT) 3A, but not with rhWNT4, had altered epithelial morphology and decreased adhesion, without loss of viability. We found that neither rhWNT3A nor rhWNT4 treatment induced proliferation. The expression and release of inflammatory cytokines IL-6 and granulocyte-macrophage colony-stimulating factor were increased after rhWNT3A exposure of HNEpCs. When differentiated at an air-liquid interface, rhWNT3A- and WNT agonist-, but not rhWNT4-treated HNEpCs, had abnormal epithelial architecture, failed to undergo motile ciliogenesis, and had defective noncanonical Wnt (planar cell polarity) signaling. On the basis of these results, we propose a model in which Wnt/β-catenin signaling sustains mucosal inflammation and leads to a spectrum of changes consistent with those seen during epithelial remodeling in NPs.

Aberrant activation of Wnt/β-catenin signaling is closely associated with the development of various human cancers, especially colorectal cancers (CRC). The ubiquitin proteasome system (UPS) is essential in the regulation of Wnt signaling and inhibitors targeting the UPS could have great potential in CRC therapy. Ubiquitin-specific protease 7 (USP7), a deubiquitinating enzyme, plays a significant role in neoplastic diseases due to its well-known function of regulating the MDM2-p53 complex. Inspired by our recent study identifying the positive role of USP7 in the Wnt signaling, we report here that USP7 is overexpressed in colorectal carcinoma cell lines and tissues, which is closely related with the poor prognosis. USP7 knockdown inhibits the proliferation of CRC cells with different p53 status, and USP7 inhibition by its inhibitor P5091 attenuates the activity of Wnt signaling via enhanced ubiquitination and the subsequent degradation of β-catenin. In vitro, P5091 inhibited the proliferation and induced apoptosis of CRC cells. P5091 also suppressed in vivo tumor growth in the HCT116 xenograft mouse model, which is consistently associated with reduced expression of β-catenin and Wnt target genes. In conclusion, our preclinical study indicated that USP7 could be a potential drug target and its inhibitor P5091 deserves further development as anticancer agent for Wnt hyper-activated CRC therapy.

In this study, TNF-α was found to activate the WNT/β-catenin pathway in BEAS-2B human bronchial epithelial cells. Levels of phospho-LRP6, Dvl-2, and phospho-GSK-3β were elevated, while that of Axin was reduced by TNF-α treatment. Nuclear translocation of β-catenin and the reporter activity of a β-catenin-responsive promoter were increased by TNF-α treatment. Under the same experimental conditions, TNF-α activated the NF-κB signaling, which includes the phosphorylation and degradation of IκB and nuclear translocation and target DNA binding of NF-κB, and it was found that an inhibitor of NF-κB activation, JSH-23, inhibited TNF-α-induced Wnt signaling as well as NF-κB signaling. It was also found that recombinant Wnt proteins induced NF-κB nuclear translocations and its target DNA binding, suggesting that Wnt signaling and NF-κB signaling were inter-connected. TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression were significantly suppressed by the transfection of β-catenin siRNA compared to that of control siRNA. Transfection of a β-catenin expression plasmid augmented the TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression. These results clearly demonstrated that the WNT/β-catenin pathway modulates the inflammatory response induced by TNF-α, suggesting that this pathway may be a useful target for the effective treatment of bronchial inflammation.

Every osteotropic agent has vasculotropic actions. I have yet to see this principle violated. The reason most likely lies within the intimate relationships between bone matrix biology and the arterial vasculature in vertebrate physiology.1 Prototypic bone anabolic hormones, such as parathyroid hormone, recruit osteoblast-derived signals to realign marrow vascular proximity to sites of bone formation.2 These responses are shaped by parathyroid hormone–regulated mechanical cues sensed by osteocytes,3,4 the parenchymal cells imbedded with bone matrix that regulate osteogenic Wnt actions via the release of sclerostin5—an inhibitor of the Wnt coreceptors low-density lipoprotein receptor–related proteins (LRP), LRP5 and LRP6.6,7 The vasculature also provides the sustentacular niche for the osteoprogenitor throughout the skeletal lifespan.8 Conversely, the osteoblasts and progenitors of bone marrow create the hematopoietic microenvironment9 and are regulated gatekeepers of hematopoietic cell egress into the circulation.10 In response to global physiological demands, the vasculature also provides the conduit for movement of calcium and phosphate in and out of the mineralized repository that is the calcified skeletal extracellular matrix.1 See accompanying article on page 553 Some the earliest connections between vascular health and osteotropic signaling were discovered by Bostrom et al.11 In 1993, they demonstrated the expression of the powerful bone morphogenetic protein 2 in the calcifying carotid plaques of patients afflicted with atherosclerosis.11 Many laboratories, including ours, discovered that the Wnt signaling cascades, initially identified as important in craniofacial bone and tooth formation, also participated in vascular mineralization and arteriosclerotic stiffening of conduit vessels.12–18 In both bone19 and aortic vasculature,20 Wnt signaling is an important mediator of osteogenic differentiation downstream of bone morphogenetic protein stimulation. The Wnt family of polypeptide ligands signal via the frizzled family of G-protein–coupled plasma membrane receptors, …

Childhood asthma, an airway inflammatory disease, is a serious threat to the child's quality of life. Recently, TIPE2 expression was reported to be decreased in children with asthma. Therefore, additional studies focusing on TIPE2 might provide an approach for treating childhood asthma. In this study, we found that TIPE2 was poorly expressed in hyperstretched human bronchial epithelial cells (BEAS-2B). TIPE2 overexpression also significantly suppressed the stretch-induced secretion of asthma-related inflammatory factors (TNF-α, TSLP, MMP-9, and VEGF). In contrast, TIPE2 inhibition significantly promoted the secretion of TNF-α, TSLP, MMP-9, and VEGF. Furthermore, overexpression of TIPE2 remarkably inhibited the activation of Wnt/β-catenin in hyperstretched BEAS-2B cells, while siTIPE2 activated Wnt/β-catenin in hyperstretched BEAS-2B cells. Further analysis showed that the Wnt/β-catenin signal inhibitor Dkk-1 could further enhance the TIPE2-induced suppression of Wnt/β-catenin signaling, which also suppressed the siTIPE2-induced secretion of TNF-α, TSLP, MMP-9, and VEGF in hyperstretched BEAS-2B cells. Dkk-1 reversed the effects of siRNA-TIPE2 on Wnt/β-catenin signaling and inflammatory cytokines. In summary, we have exhibited that TIPE2 inhibited the expression of asthma-related inflammatory factors in hyperstretched BEAS-2B cells by suppressing the Wnt/β-catenin signaling pathway. TIPE2 may be involved in airway inflammation during asthma attack, and it may be used as a potential therapeutic target for bronchial epithelial inflammation in childhood asthma.

During the last three decades, our understanding about Wnt signaling has progressed greatly, especially with regards to the molecular mechanism of intracellular transmission of this signaling, as well as its physiological roles. In parallel, the molecular nature of Wnt proteins has gradually but surely been clarified. Wnt proteins are post-translationaly modified with fatty acid and glycosaminoglycans, resulting in constraint of the 3D structure and behavior of the proteins. Specific binding proteins or extracellular vesicles, which appear to shield the lipid moiety from the aquatic environment, enable Wnt proteins to be transported in the extracellular space. Equally, Wnt-interacting proteins in the extracellular space, including heparan sulfate proteoglycan, are also involved in its spreading. Recent studies also show that intercellular transmission of Wnt proteins occurs by cell migration and extension of cell protrusions. Here, we will show the molecular and cellular bases of the trafficking of Wnt proteins and discuss questions that remain to be answered.

Background: Myocardial infarction is one of the leading causes of morbidity and mortality worldwide, triggering irreversible myocardial cell damage and heart failure. The role of low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) as coreceptors of the Wnt/β-catenin pathway in the adult heart remain unknown. Insulin-like growth factor binding protein 4 and dickkopf-related protein 1 (Dkk1) are 2 secreted LRP5/6 binding proteins that play a crucial role in heart development through preventing Wnt/β-catenin pathway activation. However, their roles in the adult heart remain unexplored. Methods: To understand the role of LRP5/6 and β-catenin in the adult heart, we constructed conditional cardiomyocyte-specific LRP5/6 and β-catenin knockout mice and induced surgical myocardial infarction. We also directly injected recombinant proteins of insulin-like growth factor binding protein 4 and Dkk1 into the heart immediately following myocardial infarction to further examine the mechanisms through which these proteins regulate LRP5/6 and β-catenin. Results: Deletion of LRP5/6 promoted cardiac ischemic insults. Conversely, deficiency of β-catenin, a downstream target of LRP5/6, was beneficial in ischemic injury. It is interesting to note that although both insulin-like growth factor binding protein 4 and Dkk1 are secreted Wnt/β-catenin pathway inhibitors, insulin-like growth factor binding protein 4 protected the ischemic heart by inhibiting β-catenin, whereas Dkk1 enhanced the injury response mainly through inducing LRP5/6 endocytosis and degradation. Conclusions: Our findings reveal previously unidentified dual roles of LRP5/6 involved in the cardiomyocyte response to ischemic injury. These findings suggest new therapeutic strategies in ischemic heart disease by fine-tuning LRP5/6 and β-catenin signaling within the Wnt/β-catenin pathway.

Wnt signaling plays a central role in tumor initiation and tumor progression. Mutations in Wnt pathway components, such as the tumor suppressor APC, lead to malignant transformation. While previous studies focused on Wnt-related changes in cancer cells, the impact of aberrant Wnt signaling on the tumor microenvironment is only beginning to emerge. In order to investigate the role of increased Wnt secretion on tumor growth and the microenvironment, we generated a novel germ cell tumor model by overexpressing the Wnt secretion factor Evi/Wls in mouse embryonic stem cells. Evi-overexpressing teratoma were characterized by enhanced tumor growth in supporting a tumor-promoting role of Wnt secretion. Interestingly, enhanced Evi expression correlated with impaired immune cell recruitment. Specifically, T- and B-cell infiltration was reduced in Evi-overexpressing teratomas, which was independent of teratoma size and differentiation. Our study suggests that Wnt secretion impairs immunosurveillance. Since immune cell infiltration has been shown to have prognostic value, the levels of secreted Wnt activity might impact the efficiency of cancer immunotherapy.

Wnt/β-catenin has been described as crucial for dorsal-ventral and antero-posterior patterning, playing multiple roles at different stages of development. Cholesterol-rich membrane microdomains (CRMMs), cholesterol- and sphingolipid-enriched domains of the plasma membrane, are known as platforms for signaling pathways. Although we have demonstrated the importance of the CRMMs for head development, how they participate in prechordal plate formation and embryo axis patterning remains an open question. Moreover, the participation of the CRMMs in the Wnt/β-catenin signaling pathway activity in vivo is unclear, particularly during embryonic development. In this study, we demonstrated that CRMMs disruption by methyl-beta-cyclodextrin (MβCD) potentiates the activation of the Wnt/β-catenin signaling pathway in vitro and in vivo during embryonic development, causing head defects by expanding the Wnt expression domain. Furthermore, we also found that the action of CRMMs depends on the microenvironmental context because it also works in conjunction with dkk1, when dkk1 is overexpressed. Thus, we propose CRMMs as a further mechanism of prechordal plate protection against the Wnt signals secreted by posterolateral cells, complementing the action of secreted antagonists.

Summary Much of our understanding about how intestinal stem and progenitor cells are regulated comes from studying the late fetal stages of development and the adult intestine. In this light, little is known about intestine development prior to the formation of stereotypical villus structures with columnar epithelium, a stage when the epithelium is pseudostratified and appears to be a relatively uniform population of progenitor cells with high proliferative capacity. Here, we investigated a role for WNT/β-CATENIN signaling during the pseudostratified stages of development (E13.5, E14.5) and following villus formation (E15.5) in mice. In contrast to the well-described role for WNT/β-CATENIN signaling as a regulator of stem/progenitor cells in the late fetal and adult gut, conditional epithelial deletion of β-catenin or the Frizzled co-receptors Lrp5 and Lrp6 had no effect on epithelial progenitor cell proliferation in the pseudostratified epithelium. Mutant embryos displayed obvious developmental defects, including loss of proliferation and disruptions in villus formation starting only at E15.5. Mechanistically, our data suggest that WNT signaling-mediated proliferation at the time of villus formation is driven by mesenchymal, but not epithelial, WNT ligand secretion., Highlights • WNT/β-CATENIN signaling is not required for proliferation during pseudostratified growth • Deleting epithelial β-catenin causes loss of proliferation during villus morphogenesis • Loss of WNT/β-CATENIN signaling leads to perturbations in villus formation • Mesenchymal, not epithelial, WNT ligands are required for epithelial proliferation, In this article, Spence and colleagues show that WNT/β-CATENIN signaling is dispensable for epithelial progenitor cell proliferation during the pseudostratified stage of intestine development, whereas active signaling is required for proliferation and proper villus formation once villus morphogenesis begins. Mechanistically, WNT/β-CATENIN-mediated proliferation is driven by mesenchymal, but not epithelial, WNT ligand secretion.

Fluoride (F) is capable of promoting abnormal proliferation and differentiation in primary cultured mouse osteoblasts (OB cells), although the underlying mechanism responsible remains rare. This study aimed to explore the roles of wingless and INT-1 (Wnt) signaling pathways and screen appropriate doses of calcium (Ca2+) to alleviate the sodium fluoride (NaF)-induced OB cell toxicity. For this, we evaluated the effect of dickkopf-related protein 1 (DKK1) and Ca2+ on mRNA levels of wingless/integrated 3a (Wnt3a), low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled 1 (Dv1), glycogen synthase kinase 3β (GSK3β), β-catenin, lymphoid enhancer binding factor 1 (LEF1), and cellular myelocytomatosis oncogene (cMYC), as well as Ccnd1 (Cyclin D1) in OB cells challenged with 10-6 mol/L NaF for 24 h. The demonstrated data showed that F significantly increased the OB cell proliferation rate. Ectogenic 0.5 mg/L DKK1 significantly inhibited the proliferation of OB cells induced by F. The mRNA expression levels of Wnt3a, LRP5, Dv1, LEF1, β-catenin, cMYC, and Ccnd1 were significantly increased in the F group, while significantly decreased in the 10-6 mol/L NaF + 0.5 mg/L DKK1 (FY) group. The mRNA expression levels of Wnt3a, LRP5, β-catenin, and cMYC were significantly decreased in the 10-6 mol/L NaF + 2 mmol/L CaCl2 (F+CaII) group. The protein expression levels of Wnt3a, Cyclin D1, cMYC, and β-catenin were significantly increased in the F group, whereas they were decreased in the F+CaII group. However, the mRNA and protein expression levels of GSK3β were significantly decreased in the F group while significantly increased in the F+CaII group. In summary, F activated the canonical Wnt/β-catenin pathway and changed the related gene expression and β-catenin protein location in OB cells, promoting cell proliferation. Ca2+ supplementation (2 mmol/L) reversed the expression levels of genes and proteins related to the canonical Wnt/β-catenin pathway.

Human endometrium undergoes cycles of proliferation and differentiation throughout the reproductive years of women. The endometrial stem/progenitor cells contribute to this regenerative process. They lie in the basalis layer of the endometrium next to the myometrium. We hypothesized that human myometrial cells provide niche signals regulating the activities of endometrial mesenchymal stem-like cells (eMSCs). In vitro coculture of myometrial cells enhanced the colony-forming and self-renewal ability of eMSCs. The cocultured eMSCs retained their multipotent characteristic and exhibited a greater total cell output when compared with medium alone culture. The expression of active β-catenin in eMSCs increased significantly after coculture with myometrial cells, suggesting activation of WNT/β-catenin signaling. Secretory factors in spent medium from myometrial cell culture produced the same stimulatory effects on eMSCs. The involvement of WNT/β-catenin signaling in self-renewal of eMSCs was confirmed with the use of WNT activator (Wnt3A conditioned medium) and WNT inhibitors (XAV939 and inhibitor of Wnt Production-2 [IWP-2]). The myometrial cells expressed more WNT5A than other WNT ligands. Recombinant WNT5A stimulated whereas anti-WNT5A antibody suppressed the colony formation, self-renewal, and T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcriptional activities of eMSCs. Moreover, eMSCs expressed FZD4 and LRP5. WNT5A is known to activate the canonical WNT signaling in the presence of these receptor components. WNT antagonist, DKK1, binds to LRP5/6. Consistently, DKK1 treatment nullified the stimulatory effect of myometrial cell coculture. In conclusion, our findings show that the myometrial cells are niche components of eMSCs, modulating the self-renewal activity of eMSCs by WNT5A-dependent activation of WNT/β-catenin signaling. Stem Cells 2019;37:1455–1466

Aims/Introduction: Several lines of evidence suggest that dysregulation of the WNT signaling pathway is involved in the pathogenesis of type 2 diabetes. This study was performed to elucidate the effects of a high-fat/high-sucrose (HF/HS) diet on pancreatic islet functions in relation to modulation of WNT ligand expression in β-cells. Materials and methods Mice were fed either standard mouse chow or a HF/HS diet from 8 weeks of age. At 20 weeks of age, intraperitoneal glucose tolerance tests were performed in both groups of mice, followed by euthanasia and isolation of pancreatic islets. WNT-related gene expression in islets and MIN6 cells was measured by quantitative real-time RT-PCR. To explore the direct effects of WNT signals on pancreatic β-cells, MIN6 cells were exposed to recombinant mouse WNT4 protein (rmWNT4) for 48 h, and glucose-induced insulin secretion was measured. Furthermore, Wnt4 siRNAs were transfected into MIN6 cells, and cell viability and insulin secretion were measured in control and Wnt4 siRNA-transfected MIN6 cells. Results Mice fed the HF/HS diet were heavier and their plasma glucose and insulin levels were higher compared with mice fed standard chow. Wnt4, Wnt5b, Ror1, and Ror2 expression was upregulated, while Fzd4, Fzd5, Fzd6, Lrp5, and Lrp6 expression was downregulated in the islets of mice fed the HF/HS diet. Wnt4 was the most abundantly expressed WNT ligand in β-cells, and its expression was increased by the HF/HS diet. Although exposure to recombinant mouse WNT4 protein for 48 h did not alter glucose-induced insulin secretion, it was significantly reduced by knockdown of Wnt4 in MIN6 cells. Conclusions We demonstrated that the HF/HS diet-induced increase of WNT4 signaling in β-cells is involved in augmentation of glucose-induced insulin secretion and impaired β-cell proliferation. These results strongly indicate an essential role of WNT4 in the regulation of β-cell functions in mouse pancreatic islets.

Significant progress has been made to identify the cells and signaling molecules involved in the mechanobiological regulation of bone remodeling. It is now well accepted that osteocytes act as mechanosensory cells in bone expressing several signaling molecules such as nitric oxide (NO) and sclerostin (Scl) which are able to control bone remodeling responses. In this paper, we present a comprehensive multiscale computational model of bone remodeling which incorporates biochemical osteocyte feedback. The mechanostat theory is quantitatively incorporated into the model using mechanical feedback to control expression levels of NO and Scl. The catabolic signaling pathway RANK–RANKL–OPG is co-regulated via (continuous) PTH and NO, while the anabolic Wnt signaling pathway is described via competitive binding reactions between Wnt, Scl and the Wnt receptors LRP5/6. Using this novel model of bone remodeling, we investigate the effects of changes in the mechanical loading and hormonal environment on bone balance. Our numerical simulations show that we can calibrate the mechanostat anabolic and catabolic regulatory mechanisms so that they are mutually exclusive. This is consistent with previous models that use a Wolff-type law to regulate bone resorption and formation separately. Furthermore, mechanical feedback provides an effective mechanism to obtain physiological bone loss responses due to mechanical disuse and/or osteoporosis.

Cajanolactone A (CLA) is a stilbenoid discovered by us from Cajanus cajan (L.) Millsp. In our study, CLA was found to promote osteoblast differentiation in human bone marrow mesenchymal stem cells (hBMSCs), as judged by increased cellular alkaline phosphatase activity and extracellular calcium deposits, and elevated protein expression of Runx2, collagen-1, bone morphogenetic protein-2, and osteopontin. Mechanistic studies revealed that hBMSCs undergoing osteoblast differentiation expressed upregulated mRNA levels of Wnt3a, Wnt10b, LRP5/6, Frizzled 4, &beta, catenin, Runx2, and Osterix from the early stage of differentiation, indicating the role of activated Wnt/&beta, catenin signaling pathway in osteoblast differentiation. Addition of CLA to the differentiation medium further increased the mRNA level of Wnt3a, Wnt10b, Frizzled 4, LRP5, and &beta, catenin, inferring that CLA worked by stimulating Wnt/LRP5/&beta, catenin signaling. Wnt inhibitor dickkopf-1 antagonized CLA-promoted osteoblastogenesis, indicating that CLA did not target the downstream of canonical Wnt signaling pathway. Treatment with CLA caused no changes in mRNA expression level, as well as protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), indicating that CLA did not affect the OPG/RANKL axis. Our results showed that CLA, which promoted osteoblast differentiation in hBMSCs, through activating Wnt/LRP5/&beta, catenin signaling transduction, is a promising anti-osteoporotic drug candidate.

Wingless-related integration site (WNT)/β-catenin signaling regulates diverse physiological functions including tissue regeneration. Activation of WNT signaling can be inhibited by various agents. Lee et al. investigate the interaction of CXXC-type zinc finger protein 5 (CXXC5) with Dishevelled as one such negative regulator of WNT in hair follicle regeneration.

Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro. GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer. Implications: This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach. Mol Cancer Res; 14(9); 859–68. ©2016 AACR.