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.     

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.     

Isoquercitrin Suppresses Colon Cancer Cell Growth in Vitro by Targeting the Wnt/β-Catenin Signaling Pathway

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent.     

Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

Background

During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified.

Objectives

The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice.

Methods

Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice.

Results

Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis.

Conclusion

Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.     

Remote Activation of the Wnt/β-Catenin Signalling Pathway Using Functionalised Magnetic Particles

Wnt signalling pathways play crucial roles in developmental biology, stem cell fate and tissue patterning and have become an attractive therapeutic target in the fields of tissue engineering and regenerative medicine. Wnt signalling has also been shown to play a role in human Mesenchymal Stem Cell (hMSC) fate, which have shown potential as a cell therapy in bone and cartilage tissue engineering. Previous work has shown that biocompatible magnetic nanoparticles (MNP) can be used to stimulate specific mechanosensitive membrane receptors and ion channels in vitro and in vivo. Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC. Frizzled receptors were tagged using anti-Frizzled functionalised MNP (Fz-MNP). A commercially available oscillating magnetic bioreactor (MICA Biosystems) was used to mechanically stimulate Frizzled receptors remotely. Our results demonstrate that Fz-MNP can activate Wnt/β-catenin signalling at key checkpoints in the signalling pathway. Immunocytochemistry indicated nuclear localisation of the Wnt intracellular messenger β-catenin after treatment with Fz-MNP. A Wnt signalling TCF/LEF responsive luciferase reporter transfected into hMSC was used to assess terminal signal activation at the nucleus. We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array. Western blot analysis was used to probe the mechanism of signalling activation and indicated that Fz-MNP signal through an LRP independent mechanism. Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP. This study provides proof of principle that Wnt signalling and Frizzled receptors are mechanosensitive and can be remotely activated in vitro. Using magnetic nanoparticle technology it may be possible to modulate Wnt signalling pathways and thus control stem cell fate for therapeutic purposes.     

Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma – Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3

It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.     

Activation of Wnt/β-Catenin Pathway in Monocytes Derived from Chronic Kidney Disease Patients

Patients with chronic kidney disease (CKD) have significantly increased morbidity and mortality resulting from infections and cardiovascular diseases. Since monocytes play an essential role in host immunity, this study was directed to explore the gene expression profile in order to identify differences in activated pathways in monocytes relevant to the pathophysiology of atherosclerosis and increased susceptibility to infections. Monocytes from CKD patients (stages 4 and 5, estimated GFR <20 ml/min/1.73 m²) and healthy donors were collected from peripheral blood. Microarray gene expression profile was performed and data were interpreted by GeneSpring software and by PANTHER tool. Western blot was done to validate the pathway members. The results demonstrated that 600 and 272 genes were differentially up- and down regulated respectively in the patient group. Pathways involved in the inflammatory response were highly expressed and the Wnt/β-catenin signaling pathway was the most significant pathway expressed in the patient group. Since this pathway has been attributed to a variety of inflammatory manifestations, the current findings may contribute to dysfunctional monocytes in CKD patients. Strategies to interfere with this pathway may improve host immunity and prevent cardiovascular complications in CKD patients.     

Nonsteroidal Anti-inflammatory Drugs Diclofenac and Celecoxib Attenuates Wnt/β-Catenin/Tcf Signaling Pathway in Human Glioblastoma Cells

Glioblastoma, the most common and aggressive primary brain tumors, carry a bleak prognosis and often recur even after standard treatment modalities. Emerging evidence suggests that deregulation of the Wnt/β-catenin/Tcf signaling pathway contributes to glioblastoma progression. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit tumor cell proliferation by suppressing Wnt/β-catenin/Tcf signaling in various human malignancies. In this study, we sought to inhibit Wnt/β-catenin/Tcf signaling in glioblastoma cells by the NSAIDs diclofenac and celecoxib. Both diclofenac and celecoxib significantly reduced the proliferation, colony formation and migration of human glioblastoma cells. Diclofenac and celecoxib downregulated β-catenin/Tcf reporter activity. Western and qRT-PCR analysis showed that diclofenac and celecoxib reduced the expression of β-catenin target genes Axin2, cyclin D1 and c-Myc. In addition, the cytoplasmic accumulation and nuclear translocation of β-catenin was significantly reduced following diclofenac and celecoxib treatment. Furthermore, diclofenac and celecoxib significantly increased phosphorylation of β-catenin and reduced the phosphorylation of GSK3β. These results clearly indicated that diclofenac and celecoxib are potential therapeutic agents against glioblastoma cells that act by suppressing the activation of Wnt/β-catenin/Tcf signaling.     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.     

Aluminum Trichloride Inhibited Osteoblastic Proliferation and Downregulated the Wnt/β-Catenin Pathway

Aluminum (Al) exposure inhibits bone formation. Osteoblastic proliferation promotes bone formation. Therefore, we inferred that Al may inhibit bone formation by the inhibition of osteoblastic proliferation. However, the effects and molecular mechanisms of Al on osteoblastic proliferation are still under investigation. Osteoblastic proliferation can be regulated by Wnt/β-catenin signaling pathway. To investigate the effects of Al on osteoblastic proliferation and whether Wnt/β-catenin signaling pathway is involved in it, osteoblasts from neonatal rats were cultured and exposed to 0, 0.4 mM (1/20 IC₅₀), 0.8 mM (1/10 IC₅₀), and 1.6 mM (1/5 IC₅₀) of aluminum trichloride (AlCl₃) for 24 h, respectively. The osteoblastic proliferation rates; Wnt3a, lipoprotein receptor-related protein 5 (LRP-5), T cell factor 1 (TCF-1), cyclin D1, and c-Myc messenger RNA (mRNA) expressions; and p-glycogen synthase kinase 3β (GSK3β), GSK3β, and β-catenin protein expressions indicated that AlCl₃ inhibited osteoblastic proliferation and downregulated Wnt/β-catenin signaling pathway. In addition, the AlCl₃ concentration was negatively correlated with osteoblastic proliferation rates and the mRNA expressions of Wnt3a, c-Myc, and cyclin D1, while the osteoblastic proliferation rates were positively correlated with mRNA expressions of Wnt3a, c-Myc, and cyclin D1. Taken together, these findings indicated that AlCl₃ inhibits osteoblastic proliferation may be associated with the inactivation of Wnt/β-catenin signaling pathway.     

Aberrant Activation of the WNT/β-Catenin Signaling Pathway in Lupus Nephritis

Objective

The canonical WNT pathway has been implicated as playing important roles in the pathogenesis of a variety of kidney diseases. Recently, WNT pathway activity was reported to be elevated in the renal tissue of a lupus mouse model. This study aimed to evaluate the potential role of the WNT pathway in the pathogenesis of human lupus nephritis.

Methods

The expression of β-catenin was evaluated in renal biopsy specimens from lupus nephritis patients and control kidney tissues by immunohistochemistry and western blotting. Real-time polymerase chain reaction (RT-PCR) was used to detect RNA expression of β-catenin, Dkk-1 and Axin2. Plasma concentrations of Dkk-1 were measured by ELISA.

Results

Immunohistochemistry and western blotting revealed increased expression of β-catenin in the kidneys of patients with lupus nephritis compared with control kidney tissues (p<0.05), accompanied by an increase in mRNA expression of β-catenin (p<0.01) and axin2 (p<0.05).β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis (p<0.01) at the mRNA expression level; the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury. Greater plasma Dkk-1 concentrations were found in LN patients compared with controls (p<0.05). Plasma Dkk-1 concentrations also correlated negatively with anti-dsDNA antibody levels and positively with serum C3 levels.

Conclusions

The canonical WNT/β-catenin signaling pathway was activated in lupus nephritis patients, accompanied by an increase in plasma levels of Dkk-1. Altered WNT/β-catenin signaling was related to the pathogenesis of lupus nephritis and might play a role in renal fibrosis.