Nuclear factor of activated T cells (NFAT) proteins repress canonical Wnt signaling via its interaction with Dishevelled (Dvl) protein and participate in regulating neural progenitor cell proliferation and differentiation

The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.     

Activated β-catenin forces N2A cell-derived neurons back to tumor-like neuroblasts and positively correlates with a risk for human neuroblastoma

Neuroblastoma is an embryonic malignancy arising from neuroblasts. The mechanisms that regulate the origination of neuroblastoma are still not very clear. In this study, we revealed that 6-bromoindirubin 3'-oxime (BIO), a specific GSK-3β inhibitor, promoted N2A cells-derived neurons to become tumor-like neuroblasts. Moreover, constitutively activated β-catenin (S33Y) also promoted this process, whereas, silencing endogenous expression of β-catenin abolished BIO-induced effects. These results implicated the potential relationship between the Wnt/β-catenin signaling and neuroblastoma formation. Indeed, we found that the amount of β-catenin in nucleus, which indicated the activation of Wnt/β-catnin signaling, was accumulated in human neuroblastoma specimens and positively correlated with clinical risk of neuroblastoma. These results give us a new sight into the neuroblastoma initiation and progression, and provide a potential drug target for neuroblastoma treatment.     

Structural and functional characterization of the Wnt inhibitor APC membrane recruitment 1 (Amer1)

Amer1/WTX binds to the tumor suppressor adenomatous polyposis coli and acts as an inhibitor of Wnt signaling by inducing β-catenin degradation. We show here that Amer1 directly interacts with the armadillo repeats of β-catenin via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and that Amer1 assembles the β-catenin destruction complex at the plasma membrane by recruiting β-catenin, adenomatous polyposis coli, and Axin/Conductin. Deletion or specific mutations of the membrane binding domain of Amer1 abolish its membrane localization and abrogate negative control of Wnt signaling, which can be restored by artificial targeting of Amer1 to the plasma membrane. In line, a natural splice variant of Amer1 lacking the plasma membrane localization domain is deficient for Wnt inhibition. Knockdown of Amer1 leads to the activation of Wnt target genes, preferentially in dense compared with sparse cell cultures, suggesting that Amer1 function is regulated by cell contacts. Amer1 stabilizes Axin and counteracts Wnt-induced degradation of Axin, which requires membrane localization of Amer1. The data suggest that Amer1 exerts its negative regulatory role in Wnt signaling by acting as a scaffold protein for the β-catenin destruction complex and promoting stabilization of Axin at the plasma membrane.     

Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/β-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and β-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.     

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.     

Regulation of β-catenin nuclear dynamics by GSK-3β involves a LEF-1 positive feedback loop

Nuclear localization of β-catenin is integral to its role in Wnt signaling and cancer. Cellular stimulation by Wnt or lithium chloride (LiCl) inactivates glycogen synthase kinase-3β (GSK-3β), causing nuclear accumulation of β-catenin and transactivation of genes that transform cells. β-catenin is a shuttling protein; however, the mechanism by which GSK-3β regulates β-catenin nuclear dynamics is poorly understood. Here, fluorescence recovery after photobleaching assays were used to measure the β-catenin-green fluorescent protein dynamics in NIH 3T3 cells before and after GSK-3β inhibition. We show for the first time that LiCl and Wnt3a cause a specific increase in β-catenin nuclear retention in live cells and in fixed cells after detergent extraction. Moreover, LiCl reduced the rate of nuclear export but did not affect import, hence biasing β-catenin transport toward the nucleus. Interestingly, the S45A mutation, which blocks β-catenin phosphorylation by GSK-3β, did not alter nuclear retention or transport, implying that GSK-3β acts through an independent regulator. We compared five nuclear binding partners and identified LEF-1 as the key mediator of Wnt3a and LiCl-induced nuclear retention of β-catenin. Thus, Wnt stimulation triggered a LEF-1 positive feedback loop to enhance the nuclear chromatin-retained pool of β-catenin by 100-300%. These findings shed new light on regulation of β-catenin nuclear dynamics.     

Axin pathway activity regulates in vivo pY654-β-catenin accumulation and pulmonary fibrosis

Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3β1-mediated cross-talk between TGFβ1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-β-catenin accumulation, but whether pY654-β-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of β-catenin in fibrosis, we explored pY654-β-catenin generation and function. α3β1 was required for TGFβ1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFβ1 stimulated β-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT β-catenin as well as Y654E or Y654F β-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-β-catenin was sensitive to the axin β-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-β-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-β-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total β-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFβ1-induced pY654-β-catenin, provide evidence that pY654-β-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.     

Oncogenic function of DACT1 in colon cancer through the regulation of β-catenin

The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.     

Smad3 Prevents ��-Catenin Degradation and Facilitates ��-Catenin Nuclear Translocation in Chondrocytes

Our previous study demonstrated that transforming growth factor (TGF)-β activates β-catenin signaling through Smad3 interaction with β-catenin in chondrocytes. In the present studies, we further investigated the detailed molecular mechanism of the cross-talk between TGF-β/Smad3 and Wnt/β-catenin signaling pathways. We found that C-terminal Smad3 interacted with both the N-terminal region and the middle region of β-catenin protein in a TGF-β-dependent manner. Both Smad3 and Smad4 were required for the interaction with β-catenin and protected β-catenin from an ubiquitin-proteasome-dependent degradation. In addition, the formation of the Smad3-Smad4-β-catenin protein complex also mediated β-catenin nuclear translocation. This Smad3-mediated regulatory mechanism of β-catenin protein stability enhanced the activity of β-catenin to activate downstream target genes during chondrogenesis. Our findings demonstrate a novel mechanism between TGF-β and Wnt/β-catenin signaling pathways during chondrocyte development.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.     

The Amotl2 gene inhibits Wnt/β-catenin signaling and regulates embryonic development in zebrafish

The Motin family proteins can regulate cell polarity, cell mobility, and proliferation during embryonic development by controlling distinct signaling pathways. In this study, we demonstrate that amotl2 knockdown in zebrafish wild-type embryos results in embryonic dorsalization, and this effect can be antagonized by co-knockdown of the dorsal inducer β-catenin2. Overexpression of amotl2 in masterblind (mbl) homozygous embryos, in which canonical Wnt signaling is up-regulated due to an axin1 mutation, transforms eyeless phenotype into smaller eyes, whereas co-knockdown of amot, amotl1, and amotl2 leads to development of smaller eyes in mbl heterozygotes. In cultured mammalian cells, Motin family members all possess the ability to attenuate Wnt/β-catenin signaling. Focusing on Amotl2, we show that Amotl2 can associate with and trap β-catenin in the Rab11-positive recycling endosomes, and as a result, the amount of β-catenin in the cytosol and nucleus is reduced. Thus, our findings provide novel insights into functions of Motin family members and regulation of Wnt/β-catenin signaling.     

Dishevelled: The hub of Wnt signaling

Wnt signaling controls a variety of developmental and homeostatic events. As a key component of Wnt signaling, Dishevelled (Dvl/Dsh) protein relays Wnt signals from receptors to downstream effectors. In the canonical Wnt pathway that depends on the nuclear translocation of β-catenin, Dvl is recruited by the receptor Frizzled and prevents the constitutive destruction of cytosolic β-catenin. In the non-canonical Wnt pathways such as Wnt-Frizzled/PCP (planar cell polarity) signaling, Dvl signals via the Daam1-RhoA axis and the Rac1 axis. In addition, Dvl plays important roles in Wnt-GSK3β-microtubule signaling, Wnt-calcium signaling, Wnt-RYK signaling, Wnt-atypical PKC signaling, etc. Dvl also functions to mediate receptor endocytosis. To fulfill its multifaceted functions, it is not surprising that Dvl associates with various kinds of proteins. Its activity is also modulated dynamically by phosphorylation, ubiquitination and degradation. In this review, we summarize the current understanding of Dvl functions in Wnt signal transduction and its biological functions in mouse development, and also discuss the molecular mechanisms of its actions.