Pulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.
All experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [3H]-thymidine incorporation assay.
Increased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation.
This study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.
Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/β-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised β-catenin or β-catenin shRNA, suggesting it does not involve Wnt/β-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca2+ and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of β-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer.
sFRP1; prostate cancer; Wnt signal; Frizzled; Wnt5a
In breast cancer, deregulation of the WNT signaling pathway occurs by autocrine mechanisms. WNT ligands and Frizzled receptors are coexpressed in primary breast tumors and cancer cell lines. Moreover, many breast tumors show hypermethylation of the secreted Frizzled-related protein 1 (sFRP1) promoter region, causing low expression of this WNT antagonist. We have previously shown that the WNT pathway influences proliferation of breast cancer cell lines via activation of canonical signaling and epidermal growth factor receptor transactivation, and that interference with WNT signaling reduces proliferation. Here we examine the role of WNT signaling in breast tumor cell migration and on xenograft outgrowth.
The breast cancer cell line MDA-MB-231 was used to study WNT signaling. We examined the effects of activating or blocking the WNT pathway on cell motility by treatment with WNT ligands or by ectopic sFPR1 expression, respectively. The ability of sFRP1-expressing MDA-MB-231 cells to grow as xenografts was also tested. Microarray analyses were carried out to identify targets with roles in MDA-MB-231/sFRP1 tumor growth inhibition.
We show that WNT stimulates the migratory ability of MDA-MB-231 cells. Furthermore, ectopic expression of sFRP1 in MDA-MB-231 cells blocks canonical WNT signaling and decreases their migratory potential. Moreover, the ability of MDA-MB-231/sFRP1-expressing cells to grow as xenografts in mammary glands and to form lung metastases is dramatically impaired. Microarray analyses led to the identification of two genes, CCND1 and CDKN1A, whose expression level is selectively altered in vivo in sFRP1-expressing tumors. The encoded proteins cyclin D1 and p21Cip1 were downregulated and upregulated, respectively, in sFRP1-expressing tumors, suggesting that they are downstream mediators of WNT signaling.
Our results show that the WNT pathway influences multiple biological properties of MDA-MB-231 breast cancer cells. WNT stimulates tumor cell motility; conversely sFRP1-mediated WNT pathway blockade reduces motility. Moreover, ectopic sFRP1 expression in MDA-MB-231 cells has a strong negative impact on tumor outgrowth and blocked lung metastases. These results suggest that interference with WNT signaling using sFRP1 to block the ligand- receptor interaction may be a valid therapeutic approach in breast cancer.
Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good model for branching studies since it recapitulates early mammalian pulmonary development.
The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis.
Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133+CD10- hematopoietic progenitor cells and CD10+ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells.
These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.
The role of persistent activation of pancreatic stellate cells (PSCs) in the fibrosis associated with chronic pancreatitis (CP) is increasingly being recognized. Recent studies have shown that Wnt signaling is involved in the development of fibrosis in multiple organs, however, the role of specific Wnts in pancreatic fibrosis remains unknown. We investigated the role of Wnt signaling during PSC activation in CP and the effect of β-catenin inhibition and Dickkopf-related protein 1 (Dkk1) restoration on the phenotype of PSCs. CP was induced in mice by repetitive caerulein injection and mouse PSCs were isolated and activated in vitro. The expression of Wnts, β-catenin, secreted frizzled-related proteins (sFRPs) and Dkks was analyzed by quantitative RT-PCR and western blotting. The canonical Wnt signaling pathway was examined by immunofluorescence and western blot detection of nuclear β-catenin expression. The effect of recombinant mouse Dkk-1 (rmDkk-1) on cell proliferation and apoptosis was assessed by flow cytometry, immunofluorescence, immunocytochemistry and Cell Counting Kit-8 (CCK-8) analysis. The expression of β-catenin, collagen1α1, TGFβRII, PDGFRβ and α-SMA in PSCs treated with different concentrations of rmDkk-1 or siRNA against β-catenin was determined by quantitative RT-PCR and western blotting. Wnt2 was the only Wnt whose expression was significantly upregulated in response to PSC activation, and Wnt2 and β-catenin protein levels were significantly increased in the pancreas of CP mice, whereas Dkk-1 expression was evidently decreased. Nuclear β-catenin levels were markedly increased in activated PSCs, and rmDkk-1 suppressed the nuclear translocation of β-catenin and the proliferation and extracellular matrix production of PSCs through the downregulation of PDGFRβ and TGFβRII. Upregulation of Dkk-1 expression increased apoptosis in cultured PSCs. These results indicate that Wnt signaling may mediate the profibrotic effect of PSC activation, and Wnt2/Dkk-1 could be potential therapeutic targets for CP.
Regular use of β2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP–PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways.
Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37°C), a β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP–PKA cascade was assessed in complete bronchi and in cultured epithelial cells.
Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP–PKA cascade.
Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.
Wnt signaling plays critical roles in mammalian lung development. However, Wnt signaling in the development of the zebrafish swimbladder, which is considered as a counterpart of mammalian lungs, have not been explored. To investigate the potential conservation of signaling events in early development of the lung and swimbladder, we wish to address the question whether Wnt signaling plays a role in swimbladder development.
For analysis of zebrafish swimbladder development, we first identified, by whole-mount in situ hybridization (WISH), has2 as a mesenchymal marker, sox2 as the earliest epithelial marker, as well as hprt1l and elovl1a as the earliest mesothelial markers. We also demonstrated that genes encoding Wnt signaling members Wnt5b, Fz2, Fz7b, Lef1, Tcf3 were expressed in different layers of swimbladder. Then we utilized the heat-shock inducible transgenic lines hs:Dkk1-GFP and hs:ΔTcf-GFP to temporarily block canonical Wnt signaling. Inhibition of canonical Wnt signaling at various time points disturbed precursor cells specification, organization, anterioposterior patterning, and smooth muscle differentiation in all three tissue layers of swimbladder. These observations were also confirmed by using a chemical inhibitor (IWR-1) of Wnt signaling. In addition, we found that Hedgehog (Hh) signaling was activated by canonical Wnt signaling and imposed a negative feedback on the latter.
We first provided a new set of gene markers for the three tissue layers of swimbladder in zebrafish and demonstrated the expression of several key genes of Wnt signaling pathway in developing swimbladder. Our functional analysis data indicated that Wnt/β-catenin signaling is required for swimbladder early development and we also provided evidence for the crosstalk between Wnt and Hh signaling in early swimbladder development.
sFRP-3 is a soluble antagonist of Wnts, widely expressed in developing embryos. The Wnt gene family comprises cysteine-rich secreted ligands that regulate cell proliferation, differentiation, organogenesis and oncogenesis of different organisms ranging from worms to mammals. In the canonical signal transduction pathway Wnt proteins bind to the extracellular domain of Frizzled receptors and consequently recruit Dishevelled (Dsh) to the cell membrane. In addition to Wnt membrane receptors belonging to the Frizzled family, several other molecules have been described which share homology in the CRD domain and lack the putative trans-membrane domain, such as sFRP molecules (soluble Frizzled Related Protein). Among them, sFRP-3 was originally isolated from bovine articular cartilage and also as a component of the Spemann organizer. sFRP-3 blocks Wnt-8 induced axis duplication in Xenopus embryos and binds to the surface of cells expressing a membrane-anchored form of Wnt-1. Injection of sFRP-3 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks.
Here we report that sFRP-3 specifically blocks EGF-induced fibroblast proliferation and foci formation. Over-expression of sFRP-3 reverts EGF-mediated inhibition of hair follicle development in the mouse ectoderm while its ablation in Xenopus maintains EGF-mediated inhibition of ectoderm differentiation. Conversely, over-expression of EGF reverts the inhibition of somitic myogenesis and axis truncation in Xenopus and mouse embryos caused by sFRP-3. In vitro experiments demonstrated a direct binding of EGF to sFRP-3 both on heparin and on the surface of CHO cells where the molecule had been membrane anchored.
sFRP-3 and EGF reciprocally inhibit their effects on cell proliferation, differentiation and morphogenesis and indeed are expressed in contiguous domains of the embryo, suggesting that in addition to their canonical ligands (Wnt and EGF receptor, respectively) these molecules bind to each other and regulate their activities during embryogenesis.
Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.
Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.
These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.
De-regulation of the wingless and integration site growth factor (WNT) signaling pathway via mutations in APC and Axin, proteins that target β-catenin for destruction, have been linked to various types of human cancer. These genetic alterations rarely, if ever, are observed in breast tumors. However, various lines of evidence suggest that WNT signaling may also be de-regulated in breast cancer. Most breast tumors show hypermethylation of the promoter region of secreted Frizzled-related protein 1 (sFRP1), a negative WNT pathway regulator, leading to downregulation of its expression. As a consequence, WNT signaling is enhanced and may contribute to proliferation of human breast tumor cells. We previously demonstrated that, in addition to the canonical WNT/β-catenin pathway, WNT signaling activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mouse mammary epithelial cells via epidermal growth factor receptor (EGFR) transactivation.
Using the WNT modulator sFRP1 and short interfering RNA-mediated Dishevelled (DVL) knockdown, we interfered with autocrine WNT signaling at the ligand-receptor level. The impact on proliferation was measured by cell counting, YOPRO, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay; β-catenin, EGFR, ERK1/2 activation, and PARP (poly [ADP-ribose]polymerase) cleavages were assessed by Western blotting after treatment of human breast cancer cell lines with conditioned media, purified proteins, small-molecule inhibitors, or blocking antibodies.
Phospho-DVL and stabilized β-catenin are present in many breast tumor cell lines, indicating autocrine WNT signaling activity. Interfering with this loop decreases active β-catenin levels, lowers ERK1/2 activity, blocks proliferation, and induces apoptosis in MDA-MB-231, BT474, SkBr3, JIMT-1, and MCF-7 cells. The effects of WNT signaling are mediated partly by EGFR transactivation in human breast cancer cells in a metalloprotease- and Src-dependent manner. Furthermore, Wnt1 rescues estrogen receptor-positive (ER+) breast cancer cells from the anti-proliferative effects of 4-hydroxytamoxifen (4-HT) and this activity can be blocked by an EGFR tyrosine kinase inhibitor.
Our data show that interference with autocrine WNT signaling in human breast cancer reduces proliferation and survival of human breast cancer cells and rescues ER+ tumor cells from 4-HT by activation of the canonical WNT pathway and EGFR transactivation. These findings suggest that interference with WNT signaling at the ligand-receptor level in combination with other targeted therapies may improve the efficiency of breast cancer treatments.
The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes.
We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses.
We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized β-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts.
These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype.
Skeletal muscle; Fetal myoblasts; Canonical Wnt signaling; BMP4 signaling; Differentiation; Slow muscle specification
It has been two decades since investigators discovered the link between the Drosophila wingless (Wg) gene and the vertebrate oncogene int-1, thus establishing the family of signaling proteins known as Wnts. Since the inception of the Wnt signaling field, there have been 19 Wnt isoforms identified in humans. These secreted glycoproteins can activate at least two distinct signaling pathways in vertebrate cells, leading to cellular changes that regulate a vast array of biological processes, including embryonic development, cell fate, cell proliferation, cell migration, stem cell maintenance, tumor suppression, and oncogenesis. In certain contexts, one subset of Wnt isoforms activates the canonical Wnt/β-catenin pathway that is characterized by the activation of certain β-catenin-responsive target genes in response to the binding of Wnt ligand to its cognate receptors. Similarly, a second subset of Wnt isoforms activates β-catenin-independent pathways, including the Wnt/ calcium (Wnt/Ca) pathway and the Wnt/planar cell polarity (Wnt/PCP) pathway, in certain cellular contexts. In addition, research has identified several secreted proteins known to regulate Wnt signaling, including the Dickkopf (DKK) family, secreted Frizzled-related proteins (sFRPs), and Wnt inhibitory factor-1 (WIF-1). The advent of technologies that can provide genome-wide expression data continues to implicate Wnts and proteins that regulate Wnt signaling pathways in a growing number of disease processes. The aim of this review is to provide a context on the Wnt field that will facilitate the interpretation and study of Wnt signaling in the context of human disease.
Testicular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.
Human embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-trans retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.
Canonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.
During induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.
Alzheimer's disease (AD) is the most common type of age-related dementia. The disease is characterized by a progressive loss of cognitive abilities, severe neurodegeneration, synaptic loss and mitochondrial dysfunction. The Wnt signaling pathway participates in the development of the central nervous system and growing evidence indicates that Wnts also regulate the function of the adult nervous system. We report here, that indirect activation of canonical Wnt/β-catenin signaling using Bromoindirubin-30-Oxime (6-BIO), an inhibitor of glycogen synthase kinase-3β, protects hippocampal neurons from amyloid-β (Aβ) oligomers with the concomitant blockade of neuronal apoptosis. More importantly, activation with Wnt-5a, a non-canonical Wnt ligand, results in the modulation of mitochondrial dynamics, preventing the changes induced by Aβ oligomers (Aβo) in mitochondrial fission-fusion dynamics and modulates Bcl-2 increases induced by oligomers. The canonical Wnt-3a ligand neither the secreted Frizzled-Related Protein (sFRP), a Wnt scavenger, did not prevent these effects. In contrast, some of the Aβ oligomer effects were blocked by Ryanodine. We conclude that canonical Wnt/β-catenin signaling controls neuronal survival, and that non-canonical Wnt/Ca2+signaling modulates mitochondrial dysfunction. Since mitochondrial dysfunction is present in neurodegenerative diseases, the therapeutic possibilities of the activation of Wnt signaling are evident.
Wnt-5a signaling; Wnt/ Ca2+; mitochondria; Aβ oligomers; hippocampal neurons
Secreted Frizzled-Related Proteins (sFRP) are involved in embryonic development as well as pathological conditions including bone and myocardial disorders and cancer. Because of their sequence homology with the Wnt-binding domain of Frizzled, they have generally been considered antagonists of canonical Wnt signaling. However, additional activities of various sFRPs including both synergism and mimicry of Wnt signaling as well as functions other than modulation of Wnt signaling have been reported. Using human embryonic kidney cells (HEK293A), we found that sFRP2 enhanced Wnt3a-dependent phosphorylation of LRP6 as well as both cytosolic β-catenin levels and its nuclear translocation. While addition of recombinant sFRP2 had no activity by itself, Top/Fop luciferase reporter assays showed a dose-dependent increase of Wnt3a-mediated transcriptional activity. sFRP2 enhancement of Wnt3a signaling was abolished by treatment with the Wnt antagonist, Dickkopf-1 (DKK1). Wnt-signaling pathway qPCR arrays showed that sFRP2 enhanced the Wnt3a-mediated transcriptional up-regulation of several genes regulated by Wnt3a including its antagonists, DKK1 and Naked cuticle-1 homolog (NKD1). These results support sFRP2’s role as an enhancer of Wnt/β-catenin signaling, a result with biological impact for both normal development and diverse pathologies such as tumorigenesis.
Wnt3a; sFRP2; Wnt signaling; LRP6/5; β-catenin
Pulmonary vascular remodeling and oxidative stress are common to many adult lung diseases. However, little is known about the relevance of lung mesenchymal stem cells (MSCs) in these processes. We tested the hypothesis that dysfunctional lung MSCs directly participate in remodeling of the microcirculation. We employed a genetic model to deplete extracellular superoxide dismutase (EC-SOD) in lung MSCs coupled with lineage tracing analysis. We crossed floxpsod3 and mT/mG reporter mice to a strain expressing Cre recombinase under the control of the ABCG2 promoter. We demonstrated In vivo that depletion of EC-SOD in lung MSCs resulted in their contribution to microvascular remodeling in the smooth muscle actin positive layer. We further characterized lung MSCs to be multipotent vascular precursors, capable of myofibroblast, endothelial and pericyte differentiation in vitro. EC-SOD deficiency in cultured lung MSCs accelerated proliferation and apoptosis, restricted colony-forming ability, multilineage differentiation potential and promoted the transition to a contractile phenotype. Further studies correlated cell dysfunction to alterations in canonical Wnt/β-catenin signaling, which were more evident under conditions of oxidative stress. Our data establish that lung MSCs are a multipotent vascular precursor population, a population which has the capacity to participate in vascular remodeling and their function is likely regulated in part by the Wnt/β-catenin signaling pathway. These studies highlight an important role for microenviromental regulation of multipotent MSC function as well as their potential to contribute to tissue remodeling.
endothelial cell; extracellular superoxide dismutase; myofibroblast; niche; pericytes; pulmonary arterial hypertension; resident lung mesenchymal stem cells
Wnt signaling regulates a variety of cellular processes during embryonic development and in the adult. Many of these activities are mediated by the Frizzled family of seven-pass transmembrane receptors, which bind Wnts via a conserved cysteine-rich domain (CRD). Secreted Frizzled-related proteins (sFRPs) contain an amino-terminal, Frizzled-like CRD and a carboxyl-terminal, heparin-binding netrin-like domain. Previous studies identified sFRPs as soluble Wnt antagonists that bind directly to Wnts and prevent their interaction with Frizzleds. However, subsequent observations suggested that sFRPs and Frizzleds form homodimers and heterodimers via their respective CRDs, and that sFRPs can stimulate signal transduction. Here, we present evidence that sFRP1 either inhibits or enhances signaling in the Wnt3a/β-catenin pathway, depending on its concentration and the cellular context. Nanomolar concentrations of sFRP1 increased Wnt3a signaling, while higher concentrations blocked it in HEK293 cells expressing a SuperTopFlash reporter. sFRP1 primarily augmented Wnt3a/β-catenin signaling in C57MG cells, but it behaved as an antagonist in L929 fibroblasts. sFRP1 enhanced reporter activity in L cells that were engineered to stably express Frizzled 5, though not Frizzled 2. This implied that the Frizzled expression pattern could determine the response to sFRP1. Similar results were obtained with sFRP2 in HEK293, C57MG and L cell reporter assays. CRDsFRP1 mimicked the potentiating effect of sFRP1 inmultiple settings, contradicting initial expectations that this domain would inhibit Wnt signaling. Moreover, CRDsFRP1 showed little avidity for Wnt3a compared to sFRP1, implying that the mechanism for potentiation by CRDsFRP1 probably does not require an interaction with Wnt protein. Together, these findings demonstrate that sFRPs can either promote or suppress Wnt/β-catenin signaling, depending on cellular context, concentration and most likely the expression pattern of Fzd receptors.
β-Catenin; Cysteine-rich domain; Frizzled; sFRP; Wnt
Genetic studies have identified a high bone mass of phenotype in both human and mouse when canonical Wnt signaling is increased. Secreted frizzled related protein1 (sFRP1) is one of several Wnt antagonists and among the loss-of-function mouse models in which 32-week old mice exhibit a high bone mass phenotype. Here we show that impact fracture healing is enhanced in this mouse model of increased Wnt signaling at a physiologic level in young (8 week) sFRP1−/− mice which do not yet exhibit significant increase in BMD. The loss of sFRP1 function in vivo improves fracture repair by promoting early bone union without adverse effects on the quality of bone tissue reflected by increased mechanical strength. We observe a dramatic reduction of the cartilage callous, increased intramembranous bone formation with bone bridging by 14 days, and early bone remodeling during the 28 day fracture repair process in the sFRP1−/− mice. Our molecular analyses of gene markers indicate that the effect of sFRP1 loss-of-function during fracture repair is to accelerate bone healing after formation of the initial hematoma by directing mesenchymal stem cells into the osteoblast lineage via the canonical pathway. Further evidence to support this conclusion is the observation of maximal sFRP1 levels in the cartilaginous callus of a WT mouse, hence in sFRP1−/− mouse progenitor cells are shifted directly into osteoblast lineage. Thus, developing an antagonist to specifically inhibit sFRP1 represents a safe target for stimulating fracture repair and bone formation in metabolic bone disorders, osteoporosis and aging.
Canonical Wnt signaling; sFRP1; Fracture repair; Mechanical strength; Mesenchymal stem cells; Accelerated osteogenesis
Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein indispensable for enamel formation. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) induces osteogenesis in various cell types. Previously, we demonstrated that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists. There is a reciprocal relationship between osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). Wnt10b-mediated activation of canonical Wnt signaling has been shown to regulate mesenchymal stem cell fate. Using the bipotential bone marrow stromal cell line ST2, we have demonstrated that LRAP activates the canonical Wnt/β-catenin signaling pathway. A specific Wnt inhibitor sFRP-1 abolishes the effect of LRAP on the stimulation of osteogenesis and the inhibition of adipogenesis of ST2 cells. LRAP treatment elevates the Wnt10b expression level whereas Wnt10b knockdown by siRNA abrogates the effect of LRAP. We show here that LRAP promotes osteogenesis of mesenchymal stem cells at the expense of adipogenesis through upregulating Wnt10b expression to activate Wnt signaling.
Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.
Craniofacial abnormalities are among the most common birth defects. Understanding the molecular mechanisms underlying craniofacial disorders is crucial for developing treatment strategies. Much of the craniofacial skeleton arises from specialized embryonic structures known as pharyngeal arches. Patterning of these arches requires precise spatial and temporal expression of multiple genes, which is coordinated between tissues by secreted signals. Wnts are secreted ligands expressed throughout the pharyngeal arches yet their role in craniofacial patterning remains unclear. In this study we examine the role of Wnts in craniofacial patterning using transgenic zebrafish to inhibit downstream Wnt signaling. We show that Wnt signaling deficient embryos have lower jaw specific defects, which strongly resembles loss-of-function phenotypes in both the Bmp and Edn1 signaling pathways. Through rescue experiments we find that Wnts are upstream regulators of both Bmp and Edn1 signaling. We thus have uncovered a crucial requirement for Wnt signaling in craniofacial patterning.
In mice, in utero exposure to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) reduces the number of dorsolateral prostatic buds resulting in a smaller dorsolateral prostate and prevents formation of ventral buds culminating in ventral prostate agenesis. The genes and signaling pathways affected by TCDD that are responsible for disrupting prostate development are largely unknown. Here we show that treatment of urogenital sinus (UGS) organ cultures with known inhibitors of canonical Wnt signaling also inhibits prostatic bud formation. In support of the hypothesis that TCDD decreases canonical Wnt signaling, we identify inhibitory effects of TCDD on multiple components of the canonical Wnt signaling pathway in the UGS that temporally coincide with the inhibitory effect of TCDD on prostatic bud formation: (1) expression of R-spondins (Rspo2 and Rspo3) that promote canonical Wnt signaling is reduced; (2) expression of Lef1, Tcf1, and Wif1, established canonical Wnt target genes, is decreased; (3) expression of Lgr5, a RSPO receptor that activates canonical Wnt signaling, is reduced; and (4) expression of Dickkopfs (Dkks), inhibitors of canonical Wnt signaling, is not increased by TCDD. Thus, the TCDD-induced reduction in canonical Wnt signaling is associated with a decrease in activators (Rspo2 and Rspo3) rather than an increase in inhibitors (Dkk1 and Dkk2) of the pathway. This study focuses on determining whether treatment of TCDD-exposed UGS organ cultures with RSPO2 and/or RSPO3 is capable of rescuing the inhibitory effects of TCDD on canonical Wnt signaling and prostatic bud formation. We discovered that each RSPO alone or in combination partially rescues TCDD inhibition of both canonical Wnt signaling and prostatic bud formation.
prostate; urogenital sinus; TCDD; R-spondin; canonical Wnt; mouse; development; bud.
Increased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.
Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.
TNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.
Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.
The Wnt signaling pathway inhibitor Dickkopf-2 (Dkk2) regulates osteoblast differentiation on microstructured titanium (Ti) surfaces, suggesting involvement of Wnt signaling in this process. To test this, human osteoblast-like MG63 cells were cultured on tissue culture polystyrene or Ti (smooth PT (Ra = 0.2 μm), sand-blasted and acid-etched SLA (Ra = 3.22 μm), modSLA (hydrophilic SLA)). Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR. Non-canonical pathway ligands, receptors and intracellular signaling molecules, as well as bone morphogenetic proteins BMP2 and BMP4, were upregulated on SLA and modSLA, whereas canonical pathway members were downregulated. To confirm that non-canonical signaling was involved, cells were cultured daily with exogenous Wnt3a (canonical pathway) or Wnt5a (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3a or Wnt5a to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface. Wnt5a, but not Wnt3a, increased MG63 cell differentiation and BMP2 and BMP4 proteins, suggesting Wnt5a promotes osteogenic differentiation through production of BMPs. Effects of exogenous and endogenous Wnt5a were synergistic with surface microstructure, suggesting the response also depends on cell maturation state. These results indicate a major role for the non-canonical, calcium-dependent Wnt pathway in differentiation of osteoblasts on microstructured titanium surfaces during implant osseointegration.
Cell signaling; Titanium surface roughness; Osteoblast differentiation; Gene expression; Regulatory factors
Thymic epithelial cell (TEC) microenvironments are essential for the
recruitment of T cell precursors from the bone marrow, as well as the
subsequent expansion and selection of thymocytes resulting in a mature
self-tolerant T cell repertoire. The molecular mechanisms, which control
both the initial development and subsequent maintenance of these critical
microenvironments, are poorly defined. Wnt signaling has been shown to be
important to the development of several epithelial tissues and organs.
Regulation of Wnt signaling has also been shown to impact both early
thymocyte and thymic epithelial development. However, early blocks in thymic
organogenesis or death of the mice have prevented analysis of a role of
canonical Wnt signaling in the maintenance of TECs in the postnatal
Here we demonstrate that tetracycline-regulated expression of the canonical
Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult
mice, results in rapid thymic degeneration characterized by a loss of
ΔNP63+ Foxn1+ and
Aire+ TECs, loss of K5K8DP TECs thought to represent
or contain an immature TEC progenitor, decreased TEC proliferation and the
development of cystic structures, similar to an aged thymus. Removal of DKK1
from DKK1-involuted mice results in full recovery, suggesting that canonical
Wnt signaling is required for the differentiation or proliferation of TEC
populations needed for maintenance of properly organized adult thymic
Taken together, the results of this study demonstrate that canonical Wnt
signaling within TECs is required for the maintenance of epithelial
microenvironments in the postnatal thymus, possibly through effects on TEC
progenitor/stem cell populations. Downstream targets of Wnt signaling, which
are responsible for maintenance of these TEC progenitors may provide useful
targets for therapies aimed at counteracting age associated thymic
involution or the premature thymic degeneration associated with cancer
therapy and bone marrow transplants.