Role Of Canonical Wnt Signaling Research Articles

322P Exploring the role of canonical WNT signaling in gallbladder cancer progression and therapeutic implications of XAV939

322P Exploring the role of canonical WNT signaling in gallbladder cancer progression and therapeutic implications of XAV939

Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

β-catenin mediates growth defects induced by centrosome loss in a subset of APC mutant colorectal cancer independently of p53.

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, β-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway.

Notoginsenoside R1 Promotes Proliferation and Osteogenic Differentiation of hPDLSCs via Wnt/β-Catenin Signaling Pathway.

To investigate the roles of Notoginsenoside R1 (NG-R1) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and explore its possible mechanism. hPDLSCs were isolated and, then characterized by flow cytometry. Cell-counting kit-8 (CCK-8) and colony assays were used to validate the effect of different NG-R1 concentrations on hPDLSCs proliferation and the optimal concentration was determined. Quantitative detection of alkaline phosphatase (ALP) activity at optimal concentration and the mineralization of the cells was investigated by Alizarin Red S staining. qRT-PCR and Western blot were utilized to examine the factors expression levels of ALP, Runx Family Transcription Factor 2 (RUNX2), Collagen I (Col-1) and catenin beta 1 (CTNNB1; β-catenin). In addition, the tankyrase inhibitor XAV-939 was used to explore NG-R1's role in canonical Wnt signaling. hPDLSCs were positive for surface antigens CD90 while negative for CD34 and CD45, which indicated that we have successfully isolated the hPDLSCs. Furthermore, a concentration of 20μmol NG-R1 dramatically enhanced hPDLSCs proliferation, ALP activity, and mineral deposition. ALP, RUNX2, COL-1, and β-catenin expression were all rised in comparison to control group. After XAV-939 was added to disrupt the canonical Wnt signaling, the impact of NG-R1 appeared to be reversed. These findings suggest that NG-R1 can stimulate osteogenic differentiation of hPDLSCs, which is probably attributable to canonical Wnt signaling activation.

Canonical Wnt signaling enhances pro-inflammatory response to titanium by macrophages

Biomaterial characteristics like surface roughness and wettability can determine the phenotype of macrophages following implantation. We have demonstrated that inhibiting Wnt ligand secretion abolishes macrophage polarization in vitro and in vivo; however, the role of canonical Wnt signaling in macrophage activation in response to physical and chemical biomaterial cues is unknown. The aim of this study was to understand whether canonical Wnt signaling affects the response of macrophages to titanium (Ti) surface roughness or wettability in vitro and in vivo. Activating canonical Wnt signaling increased expression of toll-like receptors and interleukin receptors and secreted pro-inflammatory cytokines and reduced anti-inflammatory cytokines on Ti, regardless of surface properties. Inhibiting canonical Wnt signaling reduced pro-inflammatory cytokines on all Ti surfaces and increased anti-inflammatory cytokines on rough or rough-hydrophilic Ti. In vivo, activating canonical Wnt signaling increased total macrophages, pro-inflammatory macrophages, and T cells and decreased anti-inflammatory macrophages on both smooth and rough-hydrophilic implants. Functionally, canonical Wnt activation increases pro-inflammatory macrophage response to cell and cell-extracellular matrix lysates. These results demonstrate that activating canonical Wnt signaling primes macrophages to a pro-inflammatory phenotype that affects their response to Ti implants in vitro and in vivo.

Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation.

Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.

Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts.

ABSTRACTBovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper.

Inhibition versus activation of canonical Wnt-signaling, to promote chondrogenic differentiation of Mesenchymal Stem Cells. A review.

Canonical Wnt signaling regulation is essential for controlling stemness and differentiation of mesenchymal stem cells (MSCs). However, the mechanism through which canonical Wnt-dependent MSC lineage commitment leads to chondrogenesis is controversial. Some studies hypothesize that inhibition of canonical Wnt signaling induces MSC chondrogenic differentiation, while others support that the pathway should be activated to achieve MSC chondrogenesis. The purpose of the present review is to analyze data from recent studies to elucidate parameters regarding the role of canonical Wnt signaling in MSC chondrogenic differentiation.

Canonical Wnt Signaling Pathway on Polarity Formation of Utricle Hair Cells.

As part of the inner ear, the vestibular system is responsible for sense of balance, which consists of three semicircular canals, the utricle, and the saccule. Increasing evidence has indicated that the noncanonical Wnt/PCP signaling pathway plays a significant role in the development of the polarity of the inner ear. However, the role of canonical Wnt signaling in the polarity of the vestibule is still not completely clear. In this study, we found that canonical Wnt pathway-related genes are expressed in the early stage of development of the utricle and change dynamically. We conditionally knocked out β-catenin, a canonical Wnt signaling core protein, and found that the cilia orientation of hair cells was disordered with reduced number of hair cells in the utricle. Moreover, regulating the canonical Wnt pathway (Licl and IWP2) in vitro also affected hair cell polarity and indicated that Axin2 may be important in this process. In conclusion, our results not only confirm that the regulation of canonical Wnt signaling affects the number of hair cells in the utricle but also provide evidence for its role in polarity development.

M. fortuitum-induced CNS-pathology: Deciphering the role of canonical Wnt signaling, blood brain barrier components and cytokines

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 role of canonical Wnt signaling in dentin bridge formation

ObjectiveWnt signaling has been reported to be involved in dentin bridge formation. However, the detailed mechanism has not yet been clarified. We elucidated the localization of canonical Wnt signaling molecules during dentin bridge formation. MethodsPulp of the maxillary first molar in mice was exposed and directly capped with MTA cement. Maxillae were collected on the 1st, 4th, 7th, 14th, and 28th days after treatment. After μCT analysis, immunohistochemistry for Wnt3a, Wnt10a, β-catenin, F4/80, and osterix was performed in paraffin-embedded sections. ResultsOn the 4th and 7th days after pulp capping, odontoblasts and dental pulp cells expressed Wnt3a, Wnt10a, and β-catenin. On the 14th day, reactionary dentin was formed around the pulp exposure area. Odontoblasts and dental pulp cells express Wnt3a, Wnt10a, and β-catenin. Additionally, F4/80- and Wnt10a-positive macrophages were observed at the center of the dental pulp. When the dentin bridge was formed on the 28th day, reparative odontoblasts expressed Wnt3a, β-catenin and osterix. ConclusionWnt ligands derived from odontoblasts and dental pulp cells are important for the activation of odontoblasts and the differentiation of reparative odontoblasts during dentin bridge formation. Macrophage-derived Wnts are also involved in reparative odontoblast differentiation.

β-Catenin Regulates Cardiac Energy Metabolism in Sedentary and Trained Mice.

The role of canonical Wnt signaling in metabolic regulation and development of physiological cardiac hypertrophy remains largely unknown. To explore the function of β-catenin in the regulation of cardiac metabolism and physiological cardiac hypertrophy development, we used mice heterozygous for cardiac-specific β-catenin knockout that were subjected to a swimming training model. β-Catenin haploinsufficient mice subjected to endurance training displayed a decreased β-catenin transcriptional activity, attenuated cardiomyocytes hypertrophic growth, and enhanced activation of AMP-activated protein kinase (AMPK), phosphoinositide-3-kinase–Akt (Pi3K–Akt), and mitogen-activated protein kinase/extracellular signal-regulated kinases 1/2 (MAPK/Erk1/2) signaling pathways compared to trained wild type mice. We further observed an increased level of proteins involved in glucose aerobic metabolism and β-oxidation along with perturbed activity of mitochondrial oxidative phosphorylation complexes (OXPHOS) in trained β-catenin haploinsufficient mice. Taken together, Wnt/β-catenin signaling appears to govern metabolic regulatory programs, sustaining metabolic plasticity in adult hearts during the adaptation to endurance training.

The Involvement of Canonical Wnt Signaling in Memory Impairment Induced by Chronic Cerebral Hypoperfusion in Mice.

Chronic cerebral hypoperfusion (CCH) has been proposed to contribute to the progression of memory loss, which is the main symptom of vascular cognitive impairment (VCI). Accumulating evidence indicates that underlying pathophysiology, such as neurodegeneration, may lead to memory loss. However, the underlying molecular basis of memory loss in CCH remains unclear. Here, we investigated the roles of canonical Wnt signaling, which modulates hippocampal function, in a CCH model. CCH was induced by unilateral common carotid artery occlusion (UCCAO). Mice were randomly divided into a sham-operated group or one of three UCCAO groups with different endpoints (1.5, 2.5, and 3.5 months) after UCCAO. Memory function and hippocampal levels of Wnt-related proteins were measured. A Wnt activator, lithium, was administered intraperitoneally to assess memory improvements. In the groups examined 2.5 and 3.5 months after UCCAO, impaired object recognition memory was accompanied by inhibition of Wnt signaling and decreased expression of synaptic/neural activity-related proteins. Recognition memory and Wnt signaling were significantly positive correlated. Moreover, activation of Wnt signaling with lithium significantly attenuated memory loss and recovered synaptic/neural marker expression after UCCAO. Our results suggest that CCH may affect synaptic plasticity via dysregulation of signaling pathways, including canonical Wnt signaling, which could be partly involved in memory loss. As Wnt activator administration alleviated the effects of CCH on memory loss, modulation of Wnt signaling may be a promising therapeutic strategy for VCI.

Exploring the mechanistic and temporal regulation of LRP6 endocytosis in canonical WNT signaling.

Endocytosis plays a pivotal regulatory role in canonical WNT signaling. Internalization of the low-density lipoprotein receptor-related protein 6 (LRP6) receptor complex can either promote or attenuate canonical WNT signaling, depending on the employed internalization pathway. Detailed analysis of the mechanism of LRP6 internalization and its temporal regulation is crucial for understanding the different cellular responses to WNT stimulation under varying conditions and in various cell types. Here, we elucidate the mechanisms involved in the internalization of LRP6 and re-evaluate existing, partly contradicting, theories on the regulation of LRP6 receptor internalization. We utilize a computational approach that aims at finding a set of mechanisms that accounts for the temporal dynamics of LRP6 receptor internalization upon WNT stimulation. Starting with a simple simulation model, we successively extend and probe the model's behavior based on quantitative measurements. The final model confirms that LRP6 internalization is clathrin independent in vertebrates, is not restricted to microdomains, and that signalosome formation delays LRP6 internalization within the microdomains. These findings partly revise the current understanding of LRP6 internalization in vertebrates.

The role of Wnt/β‐catenin signaling pathway in lens and retina development

AbstractWnt/β‐catenin signaling pathway functions by regulating the amount of the transcriptional co‐activator β‐catenin that controls key developmental gene expression programs. However, β‐catenin is known to have a dual function in cells. Apart from its key role as a nuclear effector of Wnt/β‐catenin signaling pathway (transcriptional function), β‐catenin is an integral structural component of cadherin‐based adherens junctions (cytoskeletal function). The essential role of β‐catenin during development is demonstrated by the finding that β‐catenin knockout mice fail to develop beyond embryonic day E6.5. Tissue‐specific deletion of β‐catenin gene in lens and neural retina resulted in a number of ocular phenotypes such as formation of ectopic lenses, failure to form lens vesicle, altered fiber cell differentiation and disruption of typical laminated structure of neural retina. In order to distinguish the phenotypes resulting from cytoskeletal or transcriptional function of β‐catenin we used mouse strain, in which the endogenous β‐catenin is replaced by mutant form of the protein, retaining cytoskeletal but lacking transcriptional function. We found that mice deficient for transcriptionally competent β‐catenin manifest abnormal development of lens epithelium and ciliary marginal zone of the neural retina. Our data unequivocally identify specific roles of canonical Wnt signaling for lens and neural retina development.

R-spondins: Multi-mode WNT signaling regulators in adult stem cells.

R-spondins (RSPOs) are secreted cysteine-rich glycoproteins that belong to a superfamily of thrombospondin type 1 repeat-containing proteins. RSPOs together with WNT proteins potentiate canonical WNT/β-catenin signaling activity. Over the last several years, the understanding of the regulatory mechanisms and functional roles of RSPOs in many biological contexts has increased. Particularly, because a leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), a stem cell marker originally identified as a marker for intestinal stem cells, and two closely related proteins, LGR4 and LGR6, were identified as cognate receptors for RSPOs, significant research progress has been made in understanding the functional roles of RSPO/LGR signaling in stem cell biology. Given the crucial roles of canonical WNT signaling in self-renewal and differentiation of various types of stem cells, examination of RSPO function and underlying mechanism in these stem cells has provided new insight into the regulatory roles of WNT signaling in stem cell behavior. In this review, we summarize and discuss recent advances in the understanding of the signaling mechanism and roles of RSPOs in different stem cell contexts.

Dynamics of WNT signaling components in the human ovary from development to adulthood.

WNT signaling has been shown to play a pivotal role in mammalian gonad development and sex differentiation; however, its role in the developing human ovary has not been investigated. We analyzed a quantitative mass spectrometry dataset to determine the expression of WNT signaling components between 47 and 137 days of development and in adult ovarian cortex tissue. WNT signaling was identified within the top ten canonical pathways of proteins detected at every developmental stage examined. We further examined the specific localization of WNT signaling components glycogen synthase kinase 3 (GSK3B), frizzled 2 (FZD2), and β-catenin (CTNNB1) within ovarian tissue. GSK3B was nearly ubiquitously expressed during fetal development, while FZD2 was specific to germ cell nests during early development. β-catenin exhibited translocation from primarily membrane bound during early ovarian development to cytoplasmic and nuclear staining specifically in early primordial follicles in the fetal ovary. This cytoplasmic and nuclear β-catenin persisted in primordial follicles in adult ovarian tissue, but returned to membrane-bound localization in secondary follicles. We conclude that WNT signaling components are expressed in the human ovary from early to mid-gestation and remain in the adult ovary, and observed evidence for canonical WNT signaling only in the oocytes of primordial follicles. Together, these data are indicative of a role for canonical WNT signaling via β-catenin nuclear translocation during human follicle formation and follicle maintenance.

Wnt signaling regulates neurogenesis

Background Wnt signaling is a highly conserved pathway and plays a critical role in neurogenesis. Objective To explain the role of canonical Wnt signaling on neurogenesis. Content This review summarized the latest general studies of canonical Wnt/β-catenin signaling on neurogenesis including proliferation, differentiation, asymmetric cell division of neural stem cells and adult neurogenesis. Trend Understanding how Wnt signaling effects neurogenesis will be helpful for new strategies of clinical usage. Key words: Wnt; β-catenin; Signaling pathway; Neurogenesis

Canonical Wnt signaling regulates patterning, differentiation and nucleogenesis in mouse hypothalamus and prethalamus

The hypothalamus is a small, but anatomically and functionally complex region of the brain whose development is poorly understood. In this study, we have explored its development by studying the canonical Wnt signaling pathway, generating gain and loss of function mutations of beta-catenin (Ctnnb1) in both hypothalamic and prethalamic neuroepithelium. Deletion of Ctnnb1 resulted in an anteriorized and hypoplastic hypothalamus. Posterior structures were lost or reduced, and anterior structures were expanded. In contrast, overexpression of a constitutively active mutant form of Ctnnb1 resulted in severe hyperplasia of prethalamus and hypothalamus, and expanded expression of a subset of posterior and premamillary hypothalamic markers. Moderate defects in differentiation of Arx-positive GABAergic neural precursors were observed in both prethalamus and hypothalamus of Ctnnb1 loss of function mutants, while in gain of function mutants, their differentiation was completely suppressed, although markers of prethalamic progenitors were preserved. Multiple other region-specific markers, including several specific posterior hypothalamic structures, were also suppressed in Ctnnb1 gain of function mutations. Severe, region-specific defects in hypothalamic nucleogenesis were also observed in both gain and loss of function mutations of Ctnnb1. Finally, both gain and loss of function of Ctnnb1 also produced severe, non-cell autonomous disruptions of pituitary development. These findings demonstrate a central and multifaceted role for canonical Wnt signaling in regulating growth, patterning, differentiation and nucleogenesis in multiple diencephalic regions.

Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector.

After injury, mesenchymal progenitors in the kidney interstitium differentiate into myofibroblasts, cells that have a critical role in kidney fibrogenesis. The ability to deliver genetic material to myofibroblast progenitors could allow new therapeutic approaches to treat kidney fibrosis. Preclinical and clinical studies show that adeno-associated viruses (AAVs) efficiently and safely transduce various tissue targets in vivo; however, protocols for transduction of kidney mesenchymal cells have not been established. We evaluated the transduction profiles of various pseudotyped AAV vectors expressing either GFP or Cre recombinase reporters in mouse kidney and human kidney organoids. Of the six AAVs tested, a synthetic AAV called Anc80 showed specific and high-efficiency transduction of kidney stroma and mesangial cells. We characterized the cell specificity, dose dependence, and expression kinetics and showed the efficacy of this approach by knocking out Gli2 from kidney mesenchymal cells by injection of Anc80-Cre virus into either homozygous or heterozygous Gli2-floxed mice. After unilateral ureteral obstruction, the homozygous Gli2-floxed mice had less fibrosis than the Gli2 heterozygotes had. We observed the same antifibrotic effect in β-catenin-floxed mice injected with Anc80-Cre virus before obstructive injury, strongly supporting a central role for canonical Wnt signaling in kidney myofibroblast activation. Finally, we showed that the Anc80 synthetic virus can transduce the mesenchymal lineage in human kidney organoids. These studies establish a novel method for inducible knockout of floxed genes in mouse mesangium, pericytes, and perivascular fibroblasts and are the foundation for future gene therapy approaches to treat kidney fibrosis.