Β-catenin Protein Stability Research Articles

Inhibition of the canonical Wnt pathway by high glucose can be reversed by parathyroid hormone‐related protein in osteoblastic cells

Recent in vivo findings suggest that the bone sparing effect of parathyroid hormone-related protein (PTHrP) in diabetic mice might occur at least in part through targeting a suppressed Wnt/β-catenin pathway in osteoblasts. We here aimed to examine the inhibitory action of a high glucose environment on specific components of the canonical Wnt pathway, and the putative compensatory effects of PTHrP, in osteoblastic cell cultures. Mouse osteoblastic MC3T3-E1 cells and primary cultures of fetal mouse calvaria were exposed to normal (5.5 mM) or high (25 mM) D-glucose (HG), with or without PTHrP (1-36) or PTHrP (107-139) for different times. In some experiments, MC3T3-E1 cells were incubated with the Wnt pathway activators Wnt3a and LiCl, or were transfected with plasmids encoding either a mutated β-catenin that cannot be targeted for degradation or a human PTHrP (-36/+139) cDNA, or the corresponding empty plasmid, in the presence or absence of HG. The gene expression of Wnt3a and low density receptor-like proteins (LRP)-5 and 6, as well as β-catenin protein stabilization and β-catenin-dependent transcription activity were evaluated. Oxidative stress status under HG condition was also assessed. The present data demonstrate that HG can target different components of the canonical Wnt pathway, while β-catenin degradation appears to be a key event leading to inhibition of Wnt/β-catenin signaling in mouse osteoblastic cells. Both PTHrP peptides tested were able to counteract this deleterious action of HG. These in vitro findings also provide new clues to understand the underlying mechanisms whereby PTHrP can increase bone formation.

The β‐catenin pathway contributes to the effects of leptin on SREBP‐1c expression in rat hepatic stellate cells and liver fibrosis

Liver fibrosis is commonly associated with obesity and most obese patients develop hyperleptinaemia. The adipocytokine leptin has a unique role in the development of liver fibrosis. Activation of hepatic stellate cells (HSCs) is a key step in hepatic fibrogenesis and sterol regulatory element-binding protein-1c (SREBP-1c) can inhibit HSC activation. We have shown that leptin strongly inhibits SREBP-1c expression in rat HSCs. Hence, we aimed to clarify whether the β-catenin pathway, the crucial negative regulator of adipocyte differentiation, mediates the effects of leptin on SREBP-1c expression in HSCs and in mouse liver fibrosis. HSCs were prepared from rats and mice. Gene expressions were analysed by real-time PCR, Western blot analysis, immunostaining and transient transfection assays. Leptin increased β-catenin protein but not mRNA levels in cultured HSCs. Leptin induced phosphorylation of glycogen synthase kinase-3β at Ser(9) and subsequent stabilization of β-catenin protein was mediated, at least in part, by ERK and p38 MAPK pathways. The leptin-induced β-catenin pathway reduced SREBP-1c expression and activity but did not affect protein levels of key regulators controlling SREBP-1c activity, and was not involved in leptin inhibition of liver X receptor α. In a mouse model of liver injury, the β-catenin pathway was shown to be involved in leptin-induced liver fibrosis. The β-catenin pathway contributes to leptin regulation of SREBP-1c expression in HSCs and leptin-induced liver fibrosis in mice. These results have potential implications for clarifying the mechanisms of liver fibrogenesis associated with elevated leptin levels.

Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

Background and Aims: Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3βmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.

A Novel Function of YWHAZ/β-Catenin Axis in Promoting Epithelial–Mesenchymal Transition and Lung Cancer Metastasis

YWHAZ, also known as 14-3-3zeta, has been reportedly elevated in many human tumors, including non-small cell lung carcinoma (NSCLC) but little is known about its specific contribution to lung cancer malignancy. Through a combined array-based comparative genomic hybridization and expression microarray analysis, we identified YWHAZ as a potential metastasis enhancer in lung cancer. Ectopic expression of YWHAZ on low invasive cancer cells showed enhanced cell invasion, migration in vitro, and both the tumorigenic and metastatic potentials in vivo. Gene array analysis has indicated these changes associated with an elevation of pathways relevant to epithelial-mesenchymal transition (EMT), with an increase of cell protrusions and branchings. Conversely, knockdown of YWHAZ levels with siRNA or short hairpin RNA (shRNA) in invasive cancer cells led to a reversal of EMT. We observed that high levels of YWHAZ protein are capable of activating β-catenin-mediated transcription by facilitating the accumulation of β-catenin in cytosol and nucleus. Coimmunoprecipitation assays showed a decrease of ubiquitinated β-catenin in presence of the interaction between YWHAZ and β-catenin. This interaction resulted in disassociating β-catenin from the binding of β-TrCP leading to increase β-catenin stability. Using enforced expression of dominant-negative and -positive β-catenin mutants, we confirmed that S552 phosphorylation of β-catenin increases the β-catenin/YWHAZ complex formation, which is important in promoting cell invasiveness and the suppression of ubiquitnated β-catenin. This is the first demonstration showing YWHAZ through its complex with β-catenin in mediating lung cancer malignancy and β-catenin protein stability.

Disruption of a Sox9–β-catenin circuit by mutant Fgfr3 in thanatophoric dysplasia type II

Mutations in fibroblast growth factor (FGF) receptors are responsible for a variety of skeletal birth defects, but the underlying mechanisms responsible remain unclear. Using a mouse model of thanatophoric dysplasia type II in which FGFR3(K650E) expression was directed to the appendicular skeleton, we show that the mutant receptor caused a block in chondrocyte differentiation specifically at the prehypertrophic stage. The differentiation block led to a severe reduction in hypertrophic chondrocytes that normally produce vascular endothelial growth factor, which in turn was associated with poor vascularization of primary ossification centers and disrupted endochondral ossification. We show that the differentiation block and defects in joint formation are associated with persistent expression of the chondrogenic factor Sox9 and down-regulation of β-catenin levels and activity in growth plate chondrocytes. Consistent with these in vivo results, FGFR3(K650E) expression was found to increase Sox9 and decrease β-catenin levels and transcriptional activity in cultured mesenchymal cells. Coexpression of Fgfr3(K650E) and Sox9 in cells resulted in very high levels of Sox9 and cooperative suppression of β-catenin-dependent transcription. Fgfr3(K650E) had opposing effects on Sox9 and β-catenin protein stability with it promoting Sox9 stabilization and β-catenin degradation. Since both Sox9 overexpression and β-catenin deletion independently blocks hypertrophic differentiation of chondrocytes and cause chondrodysplasias similar to those caused by mutations in FGFR3, our results suggest that dysregulation of Sox9 and β-catenin levels and activity in growth plate chondrocytes is an important underlying mechanism in skeletal diseases caused by mutations in FGFR3.

Role of CSN5/JAB1 in Wnt/β-catenin activation in colorectal cancer cells

CSN5/JAB1 is a critical subunit of the COP9 signalosome (CSN) and is overexpressed in many human cancers, but little is known about the role of CSN5 in colorectal cancer (CRC). To explore the functional role of CSN5 in colorectal tumorigenesis, we applied siRNA technology to silence CSN5 in HeLa, SW480, HCT116, HT29, and CaCo2 cells. CSN5 knock-down led to reduced β-catenin and phospho-β-catenin levels and this was paralleled by reduced CRC cell proliferation and reduced apoptosis rates, whereas the short-term β-catenin protein stability was enhanced by CSN5 knock-down in SW480 cells. Together, these data implicate the CSN in the pathogenesis of CRC via regulation of the Wnt/β-catenin pathway.

Silybin Synergizes with Wnt3a in Activation of the Wnt/β-catenin Signaling Pathway through Stabilization of Intracellular β-Catenin Protein

The Wnt/β-catenin signaling pathway regulates diverse developmental processes and adult tissue homeostasis. Inappropriate regulation of this pathway has been associated with human diseases, such as cancers, osteoporosis, and Alzheimer`s disease. Using a cell-based chemical screening with natural compounds, we discovered silybin, a plant flavonoid isolated from the Silybum marianum, which activated the Wnt/β-catenin signaling pathway in a synergy with Wnt3a-conditioned medium (Wnt3a-CM). In the presence of Wnt3a-CM, silybin up-regulated β-catenin response transcription (CRT) in HEK293-FL reporter cells and 3T3-L1 preadipocytes through stabilization of intracellular β-catenin protein. Silybin and Wnt3a-CM synergistically reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptorγ (PPARγ) and CAATT enhancer-binding protein α (C/EBPα) in 3T3-L1 preadipocytes, accompanied by the activation of Wnt/β-catenin signaling pathway. Taken together, our findings indicate that silybin is a small-molecule synergist of the Wnt/β-catenin signaling pathway and can be used as a controllable reagent for investigating biological processes that involve the Wnt/β-catenin signaling pathway.

Abstract 1003: The inhibitory role of quercetin-induced Dickkopf-1 on the growth of 4T1 breast cancer cell line

Abstract Introduction: The highly conserved Wnt/β-catenin canonical signaling pathway plays a crucial role in the normal development and tumor development. Although β-catenin is stabilized in about 50% of breast cancer, mutations in the Wnt/β-catenin signaling pathway are relatively rare. Dickkopf-1 (Dkk-1) was first identified as a secreted inhibitor of the canonical Wnt/β-catenin signaling pathway and increased Dkk-1 results in the inhibition of β-catenin dependent target gene expression. In this study, we investigated the effect of quercetin, a flavonoid, to regulate 4T1 breast cancer cells by inducing the expression of Dkk-1. Methods: Using a MTT assay, the inhibition of cell proliferation by quercetin (0-40μM) on 4T1 cancer cells was measured. Effects of quercetin (20μM) on Dkk-1 mRNA expression were detected by quantitative real-time RT-PCR. To test the regulation of Wnt/β-catenin signaling activity, 4T1 cells were transiently transfected with β-catenin repoter gene (Topflash). After treatment of quercetin(20μM) for 48 hours, transcriptional activity of β-catenin was estimated by luciferase assay. The protein stability of β-catenin was analyzed by Western blotting. To confirm the inhibitory effect of Dkk-1, MTT assay was performed after treatment of human-recombinant Dkk-1 (h-r-Dkk-1) on 4T1 cells. Results: The growth of 4T1 cells, treated with the indicated amount of quercetin (0-40μM) for 48 hours, were reduced in a dose-dependent manner (p=0.0002). Real-time RT-PCR revealed that the expression of Dkk-1 mRNA was significantly increased by about five-fold at 20μM of quercetin. Within 48 hours of treatment, a 20μM concentration of quercetin reduced β-catenin transcriptional activity by approximately 2-fold (p=0.0037). Protein stability of β-catenin was down-regulated by quercetin (20μM) in a time-dependent manner. Also the addition of h-r-Dkk-1 to 4T1 cell line showed a similar role in the inhibition of cell proliferation (p=0.0320). Conclusion: The present study investigated the possible existence of autocrine Wnt/β-catenin signaling for regulating breast cancer cell growth. quercetin showed a potent inhibitory role in the Wnt/β-catenin signaling activity as well as the growth of 4T1 breast cancer cells. Moreover, Dkk-1, as a negative regulator for Wnt/β-catenin signaling, was confirmed to be induced by quercetin. Therefore the present study suggest that Wnt/β-catenin signaling may be a promising target pathway for innovative treatment strategies of breast cancer. Aknowledgement : This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-000-1266). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1003. doi:10.1158/1538-7445.AM2011-1003

Wnt/β‐catenin signaling is stimulated by α‐melanocyte‐stimulating hormone in melanoma and melanocyte cells: implication in cell differentiation

Wnt/β-catenin signaling plays important roles in many developmental processes including neural crest-derived melanocyte development and migration. However, the effective contribution of Wnt/β-catenin pathway in melanogenesis in adult human melanocytes has not been fully elucidated. Here, we report that in melanoma cells and in normal human melanocytes, melanogenesis stimulation by α-melanocyte-stimulating hormone (α-MSH) induces phosphorylation of β-catenin-Ser675 and stabilization of β-catenin protein. Activation of protein kinase A by α-MSH attenuates glycogen synthase kinase-3β, which regulates ubiquitin-dependent degradation of β-catenin, suggesting a coordinated mechanism of β-catenin activity stimulation. Consistent with increased nuclear β-catenin, cyclic adenosine monophosphate (cAMP) elevation facilitates β-catenin-dependent transactivation of many Wnt target genes. Moreover, chromatin immunoprecipitation assays demonstrated an increased association of β-catenin with the proximal promoter of microphthalmia-associated transcription factor, the master regulator of pigmentation. These results demonstrate the existence of cross talk between the cAMP and Wnt pathways in melanocytes, suggesting that β-catenin could play a key role in the physiological regulation of epidermal melanogenesis.

The MIF Homologue D-Dopachrome Tautomerase Promotes COX-2 Expression through β-Catenin–Dependent and –Independent Mechanisms

The cytokine/growth factor, macrophage migration inhibitory factor (MIF), contributes to pathologies associated with immune, inflammatory, and neoplastic disease processes. Several studies have shown an important contributing role for MIF-dependent COX-2 expression in the progression of these disorders. We now report that the MIF homologue, D-dopachrome tautomerase (D-DT), is both sufficient and necessary for maximal COX-2 expression in colorectal adenocarcinoma cell lines. D-DT-dependent COX-2 transcription is mediated in part by β-catenin protein stabilization and subsequent transcription. Also contributing to D-DTs regulation of COX-2 expression are the activities of both c-jun-N-terminal kinase and the MIF-interacting protein, Jab1/CSN5. Interestingly, D-DT-dependent β-catenin stabilization is regulated by COX-2 expression, suggesting the existence of an amplification loop between COX-2- and β-catenin-mediated transcription in these cells. Because both COX-2- and β-catenin-mediated transcription are important contributors to colorectal cancer (CRC) disease maintenance and progression, these findings suggest a unique and novel regulatory role for MIF family members in CRC pathogenesis.

Ca2+/calmodulin-stimulated PDE1 regulates the beta-catenin/TCF signaling through PP2A B56 gamma subunit in proliferating vascular smooth muscle cells.

The phenotypic change of vascular smooth muscle cells (VSMCs), from a 'contractile' phenotype to a 'synthetic' phenotype, is crucial for pathogenic vascular remodeling in vascular diseases such as atherosclerosis and restenosis. Ca(2+)/calmodulin-stimulated phosphodiesterase 1 (PDE1) isozymes, including PDE1A and PDE1C, play integral roles in regulating the proliferation of synthetic VSMCs. However, the underlying molecular mechanism(s) remain unknown. In this study, we explore the role and mechanism of PDE1 isoforms in regulating β-catenin/T-cell factor (TCF) signaling in VSMCs, a pathway important for vascular remodeling through promoting VSMC growth and survival. We found that inhibition of PDE1 activity markedly attenuated β-catenin/TCF signaling by downregulating β-catenin protein. The effect of PDE1 inhibition on β-catenin protein reduction is exerted via promoting glycogen synthase kinase 3 (GSK3)β activation, β-catenin phosphorylation and subsequent β-catenin protein degradation. Moreover, PDE1 inhibition specifically upregulated phosphatase protein phosphatase 2A (PP2A) B56γ subunit gene expression, which is responsible for the effects of PDE1 inhibition on GSK3β and β-catenin/TCF signaling. Furthermore, the effect of PDE1 inhibition on β-catenin was specifically mediated by PDE1A but not PDE1C isozyme. Interestingly, in synthetic VSMCs, PP2A B56γ, phospho-GSK3β and phospho-β-catenin were all found in the nucleus, suggesting that PDE1A regulates nuclear β-catenin protein stability through the nuclear PP2A-GSK3β-β-catenin signaling axis. Taken together, these findings provide direct evidence for the first time that PP2A B56γ is a critical mediator for PDE1A in the regulation of β-catenin signaling in proliferating VSMCs.

Isorhamnetin-induced anti-adipogenesis is mediated by stabilization of β-catenin protein

Aims Previous studies have shown that isorhamnetin has anti-adipogenic effects in mouse 3T3-L1 cells. This study was conducted to elucidate the inhibitory mechanisms of isorhamnetin during adipogenic differentiation of human adipose tissue-derived stem cells (hAMSCs). Main methods The effect of isorhamnetin on adipogenic differentiation of hAMSCs was quantified by Oil Red O staining and a triglyceride assay. In addition, real-time PCR and Western blot were used to determine the expression of adipogenesis-related genes. Key findings Isorhamnetin inhibited the adipocyte differentiation of hAMSCs. Additionally, when the effects of Wnt antagonists that promote adipogenesis were evaluated, isorhamnetin was found to down-regulate the mRNA levels of sFRP1 and Dkk1, but had no effect on the mRNA levels of sFRP2, sFRP3, sFRP4 and Dkk3. Isorhamnetin also inhibited the expression of Wnt receptor and co-receptor genes. Furthermore, isorhamnetin increased the protein levels of β-catenin, an effector molecule of Wnt signaling, but had no effect on the mRNA levels of β-catenin. The phosphorylation level of GSK 3β was also increased by isorhamnetin. These results were confirmed by the fact that the expression of c-myc, cyclin D1 and PPARδ, which are target genes of β-catenin, was upregulated by isorhamnetin. Moreover, isorhamnetin reduced the mRNA expression levels of C/EBPα and PPARγ, which are known to be inhibited by c-myc or by cyclin D1 and PPARδ, respectively. Significance Our results indicate that isorhamnetin inhibits the adipogenic differentiation of hAMSCs and that its mechanisms are mediated by the stabilization of β-catenin.

Rad6B Is a Positive Regulator of β-Catenin Stabilization

Mutations in beta-catenin or other Wnt pathway components that cause beta-catenin accumulation occur rarely in breast cancer. However, there is some evidence of beta-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of beta-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces beta-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, beta-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus-Wnt-1 mammary tumor-derived cells, implicating Rad6B in physiologic regulation of beta-catenin stability and activity. Ubiquitinated beta-catenin was detectable in chromatin immunoprecipitations performed with beta-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of beta-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular beta-catenin but with minimal effects on cell membrane-associated beta-catenin. Measurement of beta-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular beta-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates beta-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of beta-catenin, reveal a positive regulatory feedback loop between Rad6B and beta-catenin and a novel mechanism of beta-catenin stabilization/activation in breast cancer cells.

Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular β-catenin protein

The Wnt/β-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/β-catenin signaling pathway. BIM increased β-catenin responsive transcription (CRT) and up-regulated intracellular β-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor γ (PPARγ) and CAATT enhancer-binding protein α (C/EBPα) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of β-catenin protein in 3T3-L1 preadipocyte cells.

AU-rich elements and alternative splicing in the β-catenin 3′UTR can influence the human β-catenin mRNA stability

β-Catenin, the central player of the Wnt signaling cascade, is a well-known oncogene. The regulation of β-catenin protein stability has been studied extensively while other mechanisms that control cellular levels of β-catenin have hardly been addressed. In this study, we show that there are three β-catenin mRNA splice variants that differ solely in their 3′-untranslated region (3′UTR) due to alternative splicing or retaining of an intron. The three isoforms were found to be ubiquitously expressed though in different quantities. Upon induction of the β-catenin protein in peripheral blood mononuclear leukocytes (PBMC), the β-catenin mRNA is induced in an isoform-specific manner. All three variants occur in the cytoplasm and contribute to the synthesis of β-catenin acting as a transcriptional coactivator but have different cytoplasmic stabilities in Hela cells. AU-rich elements (AREs), sequence elements implicated in the regulation of mRNA stability, are found in each of the three transcripts. Surprisingly, the AREs contribute to stabilization of the β-catenin mRNA transcripts in a splicing-dependent manner. The isoform most affected is the one found to be most induced when β-catenin protein accumulates. These results suggest that alternative splicing and AREs can act together in regulating β-catenin mRNA stability and thereby provide a step of controlling the cellular β-catenin concentration.

Endothelin-1 Promotes Epithelial-to-Mesenchymal Transition in Human Ovarian Cancer Cells

Despite considerable efforts to improve early detection and advances in chemotherapy, metastatic relapses remain a major challenge in the management of ovarian cancer. The endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis has been shown to have a significant role in ovarian carcinoma by promoting tumorigenesis. Here we show that the ET-1/ET(A)R autocrine pathway drives epithelial-to-mesenchymal transition (EMT) in ovarian tumor cells by inducing a fibroblastoid and invasive phenotype, down-regulation of E-cadherin, increased levels of beta-catenin, Snail, and other mesenchymal markers, and suppression of E-cadherin promoter activity. Activation of ET(A)R by ET-1 triggers an integrin-linked kinase (ILK)-mediated signaling pathway leading to glycogen synthase kinase-3beta (GSK-3beta) inhibition, Snail and beta-catenin stabilization, and regulation of transcriptional programs that control EMT. Transfection of dominant negative ILK or exposure to an ILK inhibitor suppresses the ET-1-induced phosphorylation of GSK-3beta as well as Snail and beta-catenin protein stability, activity, and invasiveness, indicating that ET-1/ET(A)R-induced EMT-promoting effects depend on ILK. ET(A)R blockade by specific antagonists or reduction by ET(A)R RNA interference reverses EMT and cell invasion by inhibiting autocrine signaling pathways. In ovarian carcinoma xenografts, ABT-627, a specific ET(A)R antagonist, suppresses EMT determinants and tumor growth. In human ovarian cancers, ET(A)R expression is associated with E-cadherin down-regulation, N-cadherin expression, and tumor grade. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression and identify novel targets of therapeutic intervention.

Cloning of the rat β-catenin gene ( Ctnnb1) promoter and its functional analysis compared with the Catnb and CTNNB1 promoters

Considerable recent interest has focused on the stabilization and accumulation of β-catenin protein in human and animal tumors and the corresponding activation of downstream β-catenin/TCF/LEF target genes. However, there is only sparse information on the regulation of β-catenin expression at the transcriptional level and its possible involvement in physiological and pathophysiological processes. We report here the cloning and characterization of a 3.6-kb promoter fragment from the rat β-catenin gene, Ctnnb1, and its comparison with corresponding promoters from the mouse and human genes, Catnb and CTNNB1. Several AP1 binding sites were confirmed in the promoters of all three species using mobility shift and reporter assays, and one putative TCF/LEF site also was observed in the promoter of CTNNB1. Subsequently, protein/DNA array analyses identified numerous other transcription factors through their high-affinity binding to the Ctnnb1 promoter, including E2F1, NFκB, MEF1, Pax5, ISRE2, Smad3/4, GATA, and ZIC. The strong binding of E2F1 and NFκB is especially noteworthy, because the former transcription factor is regulated by cyclin D1, a downstream target of β-catenin/TCF/LEF signaling, whereas the latter transcription factor has been implicated in “cross talk” between the Wnt and the NFκB signaling pathways. These results are discussed in terms of their implications for human cancer development and specifically the various tumors in which β-catenin mRNA is overexpressed, as well as for embryonic development, when reversible changes in β-catenin expression occur in response to secreted Wnt ligands. The findings reported here should provide important avenues for further research focused on the regulation of Ctnnb1 activity, including the ability of β-catenin/Tcf downstream targets to modulate β-catenin expression at the transcriptional level.

Requirement for intracellular calcium modulation in zebrafish dorsal–ventral patterning

The phosphoinositide (PI) cycle is an important signal transduction pathway that, upon activation, generates intracellular second messengers and leads to calcium release. To determine whether PI cycle-mediated intracellular calcium release is required for body plan formation, we systematically dissect PI cycle function in the zebrafish (Danio rerio). We inhibit PI cycle function at three different steps and deplete internal calcium stores, demonstrating an impact on endogenous calcium release and Wnt/β-catenin signaling. Inhibition of endogenous calcium modulation induces hyperdorsalized phenotypes in a dose-dependent manner. Ectopic dorsal-signaling centers are generated in PI cycle-inhibited embryos as demonstrated by altered β-catenin subcellular localization and ectopic expression of β-catenin target genes. These results provide evidence that modulation of calcium release is critical for early embryonic patterning and acts by influencing the stabilization of β-catenin protein.