Centrosomal enrichment and proteasomal degradation of SYS-1/β-catenin requires the microtubule motor dynein

The Caenorhabditis elegans Wnt/β-catenin asymmetry (WβA) pathway utilizes asymmetric regulation of SYS-1/β-catenin and POP-1/TCF coactivators. WβA differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased toward their appropriate cell fate. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Owing to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNA interference (RNAi)-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered “centrosomal.”     

Functional analyses of vertebrate TCF proteins in C. elegans embryos

In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.     

Ethacrynic Acid Exhibits Selective Toxicity to Chronic Lymphocytic Leukemia Cells by Inhibition of the Wnt/β-Catenin Pathway

Background

Aberrant activation of Wnt/β-catenin signaling promotes the development of several cancers. It has been demonstrated that the Wnt signaling pathway is activated in chronic lymphocytic leukemia (CLL) cells, and that uncontrolled Wnt/β-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy. Thus, the Wnt signaling pathway is an attractive candidate for developing targeted therapies for CLL.

Methodology/Principal Findings

The diuretic agent ethacrynic acid (EA) was identified as a Wnt inhibitor using a cell-based Wnt reporter assay. In vitro assays further confirmed the inhibitory effect of EA on Wnt/β-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/β-catenin target genes, including LEF-1, cyclin D1 and fibronectin. Immune co-precipitation experiments demonstrated that EA could directly bind to LEF-1 protein and destabilize the LEF-1/β-catenin complex. N-acetyl-L-cysteine (NAC), which can react with the α, β-unsaturated ketone in EA, but not other anti-oxidants, prevented the drug''s inhibition of Wnt/β-catenin activation and its ability to induce apoptosis in CLL cells.

Conclusions/Significance

Our studies indicate that EA selectively suppresses CLL survival due to inhibition of Wnt/β-catenin signaling. Antagonizing Wnt signaling in CLL with EA or related drugs may represent an effective treatment of this disease.     

IQGAP1 Protein Regulates Nuclear Localization of β-Catenin via Importin-β5 Protein in Wnt Signaling

In the canonical Wnt signaling pathway, the translocation of β-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of β-catenin function via importin-β5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of β-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-β5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and β-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of β-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of β-catenin in the canonical Wnt signaling pathway.     

kin-19/casein kinase Iα has dual functions in regulating asymmetric division and terminal differentiation in C. elegans epidermal stem cells

Casein Kinase I (CKI) is a conserved component of the Wnt signaling pathway that regulates cell fate determination in metazoans. We show that post-embryonic asymmetric division and fate specification of C. elegans epidermal stem cells are controlled by a non-canonical Wnt/β-catenin signaling pathway, involving the β-catenins WRM-1 and SYS-1, and that C. elegans kin-19/CKIα functions in this pathway. Furthermore, we find that kin-19 is the only member of the Wnt asymmetry pathway that functions with, or in parallel to, the heterochronic temporal patterning pathway to control withdrawal from self-renewal and subsequent terminal differentiation of epidermal stem cells. We show that, except in the case of kin-19, the Wnt asymmetry pathway and the heterochronic pathway function separately and in parallel to control different aspects of epidermal stem cell fate specification. However, given the function of kin-19/CKIα in both pathways, and that CKI, Wnt signaling pathway and heterochronic pathway genes are widely conserved in animals, our findings suggest that CKIα may function as a regulatory hub through which asymmetric division and terminal differentiation are coordinated in adult stem cells of vertebrates.Key words: C. elegans, kin-19, casein kinase Ialpha (CKIα), Wnt, stem cell, asymmetric cell division, heterochronic, temporal identity, terminal differentiation, self-renewal     

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2-L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.     

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency.     

Imbalance of Wnt/Dkk Negative Feedback Promotes Persistent Activation of Pancreatic Stellate Cells in Chronic Pancreatitis

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.     

The Chlamydia pneumoniae Inclusion Membrane Protein Cpn1027 Interacts with Host Cell Wnt Signaling Pathway Regulator Cytoplasmic Activation/Proliferation-Associated Protein 2 (Caprin2)

We previously identified hypothetical protein Cpn1027 as a novel inclusion membrane protein that is unique to Chlamydia pneumoniae. In the current study, using a yeast-two hybrid screen assay, we identified host cell cytoplasmic activation/proliferation-associated protein 2 (Caprin2) as an interacting partner of Cpn1027. The interaction was confirmed and mapped to the C-termini of both Cpn1027 and Caprin2 using co-immunoprecipitation and GST pull-down assays. A RFP-Caprin2 fusion protein was recruited to the chlamydial inclusion and so was the endogenous GSK3β, a critical component of the β-catenin destruction complex in the Wnt signaling pathway. Cpn1027 also co-precipitated GSK3β. Caprin2 is a key regulator of the Wnt signaling pathway by promoting the recruitment of the β-catenin destruction complex to the cytoplasmic membrane in the presence of Wnt signaling while GSK3β is required for priming β-catenin for degradation in the absence of Wnt signaling. The Cpn1027 interactions with Caprin2 and GSK3β may allow C. pneumoniae to actively sequester the β-catenin destruction complex so that β-catenin is maintained even in the absence of extracellular Wnt activation signals. The maintained β-catenin can trans-activate Wnt target genes including Bcl-2, which may contribute to the chlamydial antiapoptotic activity. We found that the C. pneumoniae-infected cells were more resistant to apoptosis induction and the anti-apoptotic activity was dependent on β-catenin. Thus, the current study suggests that the chlamydial inclusion protein Cpn1027 may be able to manipulate host Wnt signaling pathway for enhancing the chlamydial anti-apoptotic activity.     

Wnt signaling controls temporal identities of seam cells in Caenorhabditis elegans

The Wnt signaling pathway regulates multiple aspects of the development of stem cell-like epithelial seam cells in Caenorhabditis elegans, including cell fate specification and symmetric/asymmetric division. In this study, we demonstrate that lit-1, encoding the Nemo-like kinase in the Wnt/β-catenin asymmetry pathway, plays a role in specifying temporal identities of seam cells. Loss of function of lit-1 suppresses defects in retarded heterochronic mutants and enhances defects in precocious heterochronic mutants. Overexpressing lit-1 causes heterochronic defects opposite to those in lit-1(lf) mutants. LIT-1 exhibits a periodic expression pattern in seam cells within each larval stage. The kinase activity of LIT-1 is essential for its role in the heterochronic pathway. lit-1 specifies the temporal fate of seam cells likely by modulating miRNA-mediated silencing of target heterochronic genes. We further show that loss of function of other components of Wnt signaling, including mom-4, wrm-1, apr-1, and pop-1, also causes heterochronic defects in sensitized genetic backgrounds. Our study reveals a novel function of Wnt signaling in controlling the timing of seam cell development in C. elegans.     

Loss of DSTYK activates Wnt/β-catenin signaling and glycolysis in lung adenocarcinoma

Aberrant activation of Wnt/β-catenin signaling and dysregulation of metabolism have been frequently observed in lung cancer. However, the molecular mechanism by which Wnt/β-catenin signaling is regulated and the link between Wnt/β-catenin signaling and cancer metabolism are not fully understood. In this study, we showed that the loss of dual serine/threonine tyrosine protein kinase (DSTYK) led to the activation of Wnt/β-catenin signaling and upregulation of its target gene, lactate dehydrogenase (LDHA), and thus the elevation of lactate. DSTYK phosphorylated the N-terminal domain of β-catenin and inhibited Wnt/β-catenin signaling, which led to the inhibition of cell growth, colony formation and tumorigenesis in a lung adenocarcinoma mouse model. DSTYK was downregulated in lung cancer tissues, and its expression was positively correlated with the survival of patients with lung adenocarcinoma. Taken together, these results demonstrate that the loss of DSTYK activates Wnt/β-catenin/LDHA signaling to promote the tumorigenesis of lung cancer and that DSTYK may be a therapeutic target.Subject terms: Cancer metabolism, Non-small-cell lung cancer, Non-small-cell lung cancer     

Pin1-mediated Modification Prolongs the Nuclear Retention of β-Catenin in Wnt3a-induced Osteoblast Differentiation

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis.     

The Wnt/β-Catenin Pathway Interacts Differentially with PTHrP Signaling to Control Chondrocyte Hypertrophy and Final Maturation

Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through β-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/β-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/β-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.     

Plasminogen Activator Inhibitor-1 Is a Transcriptional Target of the Canonical Pathway of Wnt/β-Catenin Signaling

Plasminogen activator inhibitor-1 (PAI-1) is a multifunctional glycoprotein that plays a critical role in the pathogenesis of chronic kidney and cardiovascular diseases. Although transforming growth factor (TGF)-β1 is a known inducer of PAI-1, how it controls PAI-1 expression remains enigmatic. Here we investigated the mechanism underlying TGF-β1 regulation of PAI-1 in kidney tubular epithelial cells (HKC-8). Surprisingly, overexpression of Smad2 or Smad3 in HKC-8 cells blocked PAI-1 induction by TGF-β1, whereas knockdown of them sensitized the cells to TGF-β1 stimulation, suggesting that Smad signaling is not responsible for PAI-1 induction. Blockade of several TGF-β1 downstream pathways such as p38 MAPK or JNK, but not phosphatidylinositol 3-kinase/Akt and ERK1/2, only partially inhibited PAI-1 expression. TGF-β1 stimulated β-catenin activation in tubular epithelial cells, and ectopic expression of β-catenin induced PAI-1 expression, whereas inhibition of β-catenin abolished its induction. A functional T cell factor/lymphoid enhancer-binding factor-binding site was identified in the promoter region of the PAI-1 gene, which interacted with T cell factor upon β-catenin activation. Deletion or site-directed mutation of this site abolished PAI-1 response to β-catenin or TGF-β1 stimulation. Similarly, ectopic expression of Wnt1 also activated PAI-1 expression and promoter activity. In vivo, PAI-1 was induced in kidney tubular epithelia in obstructive nephropathy. Delivery of Wnt1 gene activated β-catenin and promoted PAI-1 expression after obstructive injury, whereas blockade of Wnt/β-catenin signaling by Dickkopf-1 gene inhibited PAI-1 induction. Collectively, these studies identify PAI-1 as a direct downstream target of Wnt/β-catenin signaling and demonstrate that PAI-1 induction could play a role in mediating the fibrogenic action of this signaling.