Generation of bioengineered feather buds on a reconstructed chick skin from dissociated epithelial and mesenchymal cells

Various kinds of in vitro culture systems of tissues and organs have been developed, and applied to understand multicellular systems during embryonic organogenesis. In the research field of feather bud development, tissue recombination assays using an intact epithelial tissue and mesenchymal tissue/cells have contributed to our understanding the mechanisms of feather bud formation and development. However, there are few methods to generate a skin and its appendages from single cells of both epithelium and mesenchyme. In this study, we have developed a bioengineering method to reconstruct an embryonic dorsal skin after completely dissociating single epithelial and mesenchymal cells from chick skin. Multiple feather buds can form on the reconstructed skin in a single row in vitro. The bioengineered feather buds develop into long feather buds by transplantation onto a chorioallantoic membrane. The bioengineered bud sizes were similar to those of native embryo. The number of bioengineered buds was increased linearly with the initial contact length of epithelial and mesenchymal cell layers where the epithelial‐mesenchymal interactions occur. In addition, the bioengineered bud formation was also disturbed by the inhibition of major signaling pathways including FGF (fibroblast growth factor), Wnt/β‐catenin, Notch and BMP (bone morphogenetic protein). We expect that our bioengineering technique will motivate further extensive research on multicellular developmental systems, such as the formation and sizing of cutaneous appendages, and their regulatory mechanisms.     

Moderate activation of Wnt/β-catenin signaling promotes the survival of rat nucleus pulposus cells via regulating apoptosis,autophagy, and senescence

This study aimed to investigate the specific role of Wnt/β-catenin signaling in compression-induced apoptosis, autophagy, and senescence in rat nucleus pulposus (NP) cells. Initially, the cells underwent various periods of exposure to 1.0 MPa compression. Wnt/β-catenin signaling associated molecules were assessed in detail, and then 0, 24 and 48 hours exposure periods were selected. The cells were then divided into control, Wnt/β-catenin inhibitor (IWP-2), Wnt/β-catenin activator (LiCl), and β-catenin overexpression groups. After 0, 24, and 48 hours of compression, apoptosis, autophagy, and senescence were evaluated by Western blot analysis and real-time polymerase chain reaction and were visually observed by Hoechst33258, monodansylcadaverine, and SA-β-gal stainings, respectively. Additionally, the regulatory effect of Wnt/β-catenin signaling on cell morphology, viability, cell cycle, death ratio, and ultrastructure was detected to thoroughly evaluate the survival capacity of NP cells. The results established that compression elicited a time-dependent activation of Wnt/β-catenin signaling. The IWP-2 treatment decreased cell survival rate, which corresponded to downregulation of autophagy as well as increases in apoptosis and senescence. LiCl treatment enabled more efficient of cell survival accompanied by increased autophagy and downregulated apoptosis and senescence; however, in contrast to LiCl, overexpression of β-catenin aggravated compression-induced NP cells death. In conclusion, moderate activation of Wnt/β-catenin signaling enables more efficient of NP cells survival via downregulation of apoptosis, senescence, and upregulation of autophagy, and overactivation of Wnt/β-catenin signaling achieved the opposite effect. Treatment strategies that aim to regulate Wnt/β-catenin signaling might be a novel target for improving compression-induced NP cells death and potential treatment of intervertebral disc degeneration.     

Mesothelial- and epithelial-derived FGF9 have distinct functions in the regulation of lung development

Fibroblast growth factor (FGF) 9 is a secreted signaling molecule that is expressed in lung mesothelium and epithelium and is required for lung development. Embryos lacking FGF9 show mesenchymal hypoplasia, decreased epithelial branching and, by the end of gestation, hypoplastic lungs that cannot support life. Mesenchymal FGF signaling interacts with β-catenin-mediated WNT signaling in a feed-forward loop that functions to sustain mesenchymal FGF responsiveness and mesenchymal WNT/β-catenin signaling. During pseudoglandular stages of lung development, Wnt2a and Wnt7b are the canonical WNT ligands that activate mesenchymal WNT/β-catenin signaling, whereas FGF9 is the only known ligand that signals to mesenchymal FGF receptors (FGFRs). Here, we demonstrate that mesothelial- and epithelial-derived FGF9, mesenchymal Wnt2a and epithelial Wnt7b have unique functions in lung development in mouse. Mesothelial FGF9 and mesenchymal WNT2A are principally responsible for maintaining mesenchymal FGF-WNT/β-catenin signaling, whereas epithelial FGF9 primarily affects epithelial branching. We show that FGF signaling is primarily responsible for regulating mesenchymal proliferation, whereas β-catenin signaling is a required permissive factor for mesenchymal FGF signaling.     

Antagonistic crosstalk of Wnt/β-catenin/Bmp signaling within the Apical Ectodermal Ridge (AER) regulates interdigit formation

Digit and interdigit (D/ID) development is one of the important research fields in molecular developmental biology. Interdigital cell death (ICD) is a morphogenetic event which has been considered as an essential process for D/ID formation. Although some growth factors including Bmp and Fgf signaling can modulate ICD, growth factor crosstalk regulating ICD is poorly understood. Wnt canonical pathway and Bmp signal crosstalk has been considered as the essential growth factor crosstalk in organogenesis. To elucidate the crosstalk to regulate the D/ID formation, we analyzed conditional mutant mice with limb bud ectoderm expressing constitutively activated β-catenin signaling. We showed that modulation of Wnt/β-catenin signal in the limb ectoderm including the AER regulates ID apoptosis. We also demonstrated that Wnt/β-catenin signaling in the ectoderm can positively regulate Fgf8 possibly antagonizing the epithelial derived Bmp signaling. Human birth defects for digit abnormalities have been known to be affected by multiple parameters. Elucidation of the potential mechanisms underlying such D/ID development is an urgent medical issue to be solved. This work would be one of the first studies showing essential growth factor cascades in the D/ID formation.     

Requirement of Wnt/β-catenin signaling in pronephric kidney development

The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/β-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/β-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/β-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/β-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/β-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/β-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.     

Down-regulation of Wnt10a by RNA interference inhibits proliferation and promotes apoptosis in mouse embryonic palatal mesenchymal cells through Wnt/β-catenin signaling pathway

Cleft palate is one of the most common birth defects. Both environmental and genetic factors are involved in this disorder. Here, we investigated the function of Wnt10a in proliferation and apoptosis of mouse embryonic palatal mesenchymal (MEPM) cells. Expression of Wnt10a was down-regulated at both the mRNA and protein levels in transfected MEPM cells containing Wnt10a-specific small hairpin RNA (shRNA) plasmid. Down-regulation of Wnt10a inhibited cell proliferation and induced cell cycle arrest in the S phase in MEPM cells. Moreover, apoptosis was significantly increased in MEPM cells of Wnt10a gene silencing. Finally, the expression of β-catenin was markedly reduced in MEPM cells transfected with shRNA plasmid, indicating that the canonical Wnt/β-catenin signaling pathway was involved in the alterations of cell proliferation and apoptosis induced by Wnt10a knockdown. Thus, our findings reveal that Wnt10a regulates proliferation and apoptosis of MEPM cells at least partially through the canonical Wnt/β-catenin signaling pathway.     

Study of Wnt2 secreted by A-549 cells in paracrine activation of β-catenin in co-cultured mesenchymal stem cells

The canonical Wnt signal pathway is a key regulator of self-renewal and cell fate determination in various types of stem cells. The total pool of β-catenin consists of two different forms: the signaling form of the protein transmits the Wnt signals from the cell membrane to the target genes, whereas the membrane β-catenin is involved in formation of cell-to-cell contact at cadherin junctions. Earlier we developed an in vitro model of epithelial differentiation of mesenchymal stem cells (MSCs) co-cultured with epithelial A-549 cells. The purpose of the present work was to study the role of Wnt2 secreted by the A-549 cells in paracrine induction of β-catenin in co-cultured MSCs. Using the somatic gene knockdown technique, we obtained A-549 cell cultures with down-regulated WNT2. The MSCs co-cultured with the control A-549 cells displayed an increase in the levels of total cellular and signaling β-catenin and transactivation of a reporter construction containing the Lef/Tcf protein family binding sites. In contrast, β-catenin was not induced in the MSCs co-cultured with the A-549 cells with down-regulated WNT2 expression, but the total protein level was increased. We suggest that Wnt2 secreted by A-549 cells induces in co-cultured MSCs the Wnt/β-catenin signaling pathway, whereas the associated increase in total β-catenin level should be due to another mechanism.     

Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE.     

Convergence of Wnt and Notch signaling controls ovarian cancer cell survival

In the last 40 years ovarian cancer mortality rates have slightly declined and, consequently, it continues to be the fifth cause of cancer death in women. In the present study, we showed that β-catenin signaling is involved in the functions of ovarian cancer cells and interacts with the Notch system. Wnt and Notch systems showed to be prosurvival for ovarian cancer cells and their inhibition impaired cell proliferation and migration. We also demonstrated that the inhibition of β-catenin by means of two molecules, XAV939 and ICG-001, decreased the proliferation of the IGROV1 and SKOV3 ovarian cancer cell lines and that ICG-001 increased the percentage of IGROV1 cells undergoing apoptosis. The simultaneous inhibition of β-catenin and Notch signaling, by using the DAPT inhibitor, decreased ovarian cancer cell proliferation to the same extent as targeting only the Wnt/β-catenin pathway. A similar effect was observed in IGROV1 cell migration with ICG-001 and DAPT. ICG-001 increased the Notch target genes Hes-1 and Hey-1 and increased Jagged1 expression. However, no changes were observed in Dll4 or Notch 1 and 4 expressions. Our results suggest that Notch and β-catenin signaling co-operate in ovarian cancer to ensure the proliferation and migration of cells and that this could be achieved, at least partly, by the upregulation of Notch Jagged1 ligand in the absence of Wnt signaling. We showed that the Wnt pathway crosstalks with Notch in ovarian cancer cell functions, which may have implications in ovarian cancer therapeutics.     

Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/β-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/β-catenin signaling were determined, suggested down-regulation of Wnt/β-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3α can inhibit the epithelial differentiation of MSCs. A loss of β-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated β-catenin expression and subsequently decreased β-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/β-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.     

Nuclear translocation of β-catenin during mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype

Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.     

Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation

Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/β-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/β-catenin activity, patterned upregulation of epidermal β-catenin activity and Edar expression are absent. Conversely, forced activation of β-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/β-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.     

Nigericin inhibits epithelial ovarian cancer metastasis by suppressing the cell cycle and epithelial−mesenchymal transition

Epithelial ovarian cancer (EOC) has the highest mortality among various types of gynecological malignancies. Most patients die of metastasis and recurrence due to cisplatin resistance. Thus, it is urgent to develop novel therapies to cure this disease. CCK-8 assay showed that nigericin exhibited strong cytotoxicity on A2780 and SKOV3 cell lines. Flow cytometry indicated that nigericin could induce cell cycle arrest at G0/G1 phase and promote cell apoptosis. Boyden chamber assay revealed that nigericin could inhibit migration and invasion in a dose-dependent manner by suppressing epithelial–mesenchymal transition (EMT) in EOC cells. These effects were mediated, at least partly, by the Wnt/β-catenin signaling pathway. Our results demonstrated that nigericin could inhibit EMT during cell invasion and metastasis through the canonical Wnt/β-catenin signaling pathway. Nigericin may prove to be a novel therapeutic strategy that is effective in patients with metastatic EOC.     

Fas-associated Factor 1 Is a Scaffold Protein That Promotes β-Transducin Repeat-containing Protein (β-TrCP)-mediated β-Catenin Ubiquitination and Degradation

FAS-associated factor 1 (FAF1) antagonizes Wnt signaling by stimulating β-catenin degradation. However, the molecular mechanism underlying this effect is unknown. Here, we demonstrate that the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP) is required for FAF1 to suppress Wnt signaling and that FAF1 specifically associates with the SCF (Skp1-Cul1-F-box protein)-β-TrCP complex. Depletion of β-TrCP reduced FAF1-mediated β-catenin polyubiquitination and impaired FAF1 in antagonizing Wnt/β-catenin signaling. FAF1 was shown to act as a scaffold for β-catenin and β-TrCP and thereby to potentiate β-TrCP-mediated β-catenin ubiquitination and degradation. Data mining revealed that FAF1 expression is statistically down-regulated in human breast carcinoma compared with normal breast tissue. Consistent with this, FAF1 expression is higher in epithelial-like MCF7 than mesenchymal-like MDA-MB-231 human breast cancer cells. Depletion of FAF1 in MCF7 cells resulted in increased β-catenin accumulation and signaling. Importantly, FAF1 knockdown promoted a decrease in epithelial E-cadherin and an increase in mesenchymal vimentin expression, indicative for an epithelial to mesenchymal transition. Moreover, ectopic FAF1 expression reduces breast cancer cell migration in vitro and invasion/metastasis in vivo. Thus, our studies strengthen a tumor-suppressive function for FAF1.     

Leptin-induced epithelial-mesenchymal transition in breast cancer cells requires β-catenin activation via Akt/GSK3- and MTA1/Wnt1 protein-dependent pathways

Perturbations in the adipocytokine profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1), and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with the small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion, and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of the GSK3β-LKB1-Axin complex that leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of the Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation, and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors shows increased expression of Wnt1, pGSK3β, and vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized cross-talk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells.     

Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC(50) values less than 1 μM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.