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.     

Transmembrane protein 97 exhibits oncogenic properties via enhancing LRP6-mediated Wnt signaling in breast cancer

Upregulation of transmembrane protein 97 (TMEM97) has been associated with progression and poor outcome in multiple human cancers, including breast cancer. Recent studies suggest that TMEM97 may be involved in the activation of the Wnt/β-catenin pathway. However, the molecular mechanism of TMEM97 action on Wnt/β-catenin signaling is completely unclear. In the current study, TMEM97 was identified as an LRP6-interacting protein. TMEM97 could interact with LRP6 intracellular domain and enhance LRP6-mediated Wnt signaling in a CK1δ/ε-dependent manner. The binding of TMEM97 to LRP6 facilitated the recruitment of CK1δ/ε to LRP6 complex, resulting in LRP6 phosphorylation at Ser 1490 and the stabilization of β-catenin. In breast cancer cells, knockout of TMEM97 attenuated the Wnt/β-catenin signaling cascade via regulating LRP6 phosphorylation, leading to a decrease in the expression of Wnt target genes AXIN2, LEF1, and survivin. TMEM97 deficiency also suppressed cell viability, proliferation, colony formation, migration, invasion, and stemness properties in breast cancer cells. Importantly, TMEM97 knockout suppressed tumor growth through downregulating the Wnt/β-catenin signaling pathway in a breast cancer xenograft model. Taken together, our results revealed that TMEM97 is a positive modulator of canonical Wnt signaling. TMEM97-mediated Wnt signaling is implicated in the tumorigenesis of breast cancer, and its targeted inhibition may be a promising therapeutic strategy for breast cancer.Subject terms: Protein-protein interaction networks, Breast cancer     

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.     

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.