Phytochemicals Attenuating Aberrant Activation of ��-Catenin in Cancer Cells

Phytochemicals are a rich source of chemoprevention agents but their effects on modulating the Wnt/β-catenin signaling pathway have remained largely uninvestigated. Aberrantly activated Wnt signaling can result in the abnormal stabilization of β-catenin, a key causative step in a broad spectrum of cancers. Here we report the modulation of lithium chloride-activated canonical Wnt/β-catenin signaling by phytochemicals that have antioxidant, anti-inflammatory or chemopreventive properties. The compounds were first screened with a cervical cancer-derived stable Wnt signaling reporter HeLa cell line. Positive hits were subsequently evaluated for β-catenin degradation, suppression of β-catenin nuclear localization and down-regulation of downstream oncogenic targets of Wnt/β-catenin pathway. Our study shows a novel degradation path of β-catenin protein in HeLa cells by Avenanthramide 2p (a polyphenol) and Triptolide (a diterpene triepoxide), respectively from oats and a Chinese medicinal plant. The findings present Avenanthramide 2p as a potential chemopreventive dietary compound that merits further study using in vivo models of cancers; they also provide a new perspective on the mechanism of action of Triptolide.     

Activation of Wnt/β-Catenin Signaling Increases Apoptosis in Melanoma Cells Treated with Trail

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation.     

Cadherin Switching and Activation of ��-Catenin Signaling Underlie Proinvasive Actions of Calcitonin-Calcitonin Receptor Axis in Prostate Cancer

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic β-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular β-catenin, its translocation in the nucleus, and transactivation of β-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/β-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-β-catenin signaling.Prostate cancer (PC)² is the most commonly diagnosed cancer and the second leading cause of cancer deaths in men in the United States (1, 2). Although androgen ablation therapy is effective in men with advanced disease for some time, the disease subsequently progresses to the androgen-independent stage. The population of prostate cells expressing neuroendocrine factors such as calcitonin (CT) also increases during this progression (3–5). At this stage, the disease is metastatic and chemoresistant. Present evidence suggests that cancer metastasis is usually preceded by the disruption of normal cell-cell adhesion and the loss of integrity of the primary tumor site (6, 7). This process may include several genetic, molecular, and morphological changes characterized by epithelial-to-mesenchymal transition (EMT) (8–10). The EMT is characterized by the loss of cell polarity, altered cell-cell and cell-matrix adhesion, and acquisition of migratory, mesenchymal phenotype. Other reported changes include down-regulation of E-cadherin, induction of N-cadherin, release of β-catenin from junctional complexes, and its translocation to the nucleus (11–13). However, the precise molecular mechanisms associated with this process are obscure.Several growth factors, including hepatocyte growth factor, transforming growth factor-β, vascular endothelial growth factor, and epidermal growth factor, have been reported to induce EMT in tumor cell lines (14–16). We have shown that the expression of CT and its G protein-coupled receptor (CTR) is remarkably higher in advanced PCs, and the CT-CTR autocrine axis is a potent stimulator of PC cell tumorigenicity, invasion, and metastasis (4, 17–19). Although CT-stimulated increase in the motility and invasion of PC cells may be mediated by CT-stimulated secretion of matrix metalloproteinases and urokinase-type plasminogen activator, the precise molecular mechanisms preceding these CTR actions remain to be elucidated (18, 20). We tested the hypothesis that CT induces biochemical and morphological changes associated with EMT to increase the invasiveness of PC cells.Our results indicate that activation of the CT-CTR autocrine axis in prostate cancer cells induced several changes associated with EMT such as remodeling of tight and adherens junctions, cadherin switching, and activation of WNT/β-catenin signaling. In contrast, the silencing of the CT-CTR axis reversed this process. Moreover, cyclic AMP-dependent protein kinase (PKA) plays a key role in this CT-CTR-mediated process. This is the first study demonstrating the action of prostate CTR on junctional complexes and WNT/β-catenin signaling of PC cell lines.     

SPARC Suppresses Apoptosis of Idiopathic Pulmonary Fibrosis Fibroblasts through Constitutive Activation of ��-Catenin

Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the accumulation of scar tissue in the lung interstitium. A hallmark of the disease is areas of injury to type II alveolar epithelial cells with attendant accumulation of fibroblasts in areas called fibroblastic foci. In an effort to better characterize the lung fibroblast phenotype in IPF patients, we isolated fibroblasts from patients with IPF and looked for activation of signaling proteins, which could help explain the exaggerated fibrogenic response in IPF. We found that IPF fibroblasts constitutively expressed increased basal levels of SPARC, plasminogen activator inhibitor-1 (PAI-1), and active β-catenin compared with control cells. Control of basal PAI-1 expression in IPF fibroblasts was regulated by SPARC-mediated activation of Akt, leading to inhibition of glycogen synthase kinase-3β and activation of β-catenin. Additionally, IPF fibroblasts (but not control fibroblasts) were resistant to plasminogen-induced apoptosis and were sensitized to plasminogen-mediated apoptosis by inhibition of SPARC or β-catenin. These findings uncover a newly discovered regulatory pathway in IPF fibroblasts that is characterized by elevated SPARC, giving rise to activated β-catenin, which regulates expression of downstream genes, such as PAI-1, and confers an apoptosis-resistant phenotype. Disruption of this pathway may represent a novel therapeutic target in IPF.     

T Cell Receptor-dependent Tyrosine Phosphorylation of ��2-Chimaerin Modulates Its Rac-GAP Function in T Cells

The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported β2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of β2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of β2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of β2-chimaerin and identifies a novel mechanism for its inactivation.T cell activation requires presentation of an antigen by antigen-presenting cells (APC)² to the T cell receptor (TCR); this event involves the reorganization of several scaffolds and signaling proteins, leading to formation of the immunological synapse (IS) (1). Correct protein redistribution during synapse formation is critical for an efficient T cell response, and it is largely regulated by actin polymerization at the T cell/APC contact site as a result of TCR-regulated Rac-dependent signals (2, 3). Like other Rho GTPases, Rac cycles between a GTP-bound active state and a GDP-bound inactive state. This continuous recycling is regulated by the concerted action of two proteins as follows: GEF, which activates Rac by mediating GDP/GTP exchange (4), and GAP, which induces Rac inactivation by accelerating intrinsic Rac GTPase activity, converting GTP to GDP (5).Vav-1 is the best studied GEF for Rac, and it has critical roles in T cell-dependent functions (6). In naive, unstimulated T cells, Vav-1 is in an inactive state through autoinhibition, as the GEF domain is blocked by the N-terminal region (7). This autoinhibition is relieved by TCR-mediated tyrosine phosphorylation (8, 9). Thymocytes from Vav-1-deficient mice have a developmental block, and their mature T cells show severe defects in IS formation, as well as reduced Ca²⁺ influx, IL-2 production, T cell proliferation, and cytotoxic activity (10–13). Although several studies have shown a key role for Vav-1, the mechanisms that govern Rac inactivation downstream of the TCR remain elusive.The chimaerins are a family of Rho-GAP, with specific activity for Rac. In addition to their catalytic domain, they have an N-terminal SH2 domain and a C1 domain required for interaction with the lipid messenger diacylglycerol (DAG) and with phorbol esters (14). There are two mammalian chimaerin genes (CHN1 and CHN2), which encode the full-lengthα2-(ARHGAP2) and β2-chimaerins (ARHGAP3), and at least one splice variant each (α1 and β1) that lack the SH2 domain. The α-chimaerins are expressed abundantly in brain and are linked to neuritogenesis and axon guidance (15–20). β2-Chimaerin expression is ubiquitous (21) and is involved in EGF-dependent Rac regulation (22, 23). Experiments in T cells showed that β2-chimaerin participates in chemokine-dependent regulation of T cell migration and adhesion (24). A very recent study implicates chimaerins in the modulation of Rac activity during T cell synapse formation, suggesting that this protein family contributes to DAG-mediated regulation of cytoskeletal remodeling during T cell activation (25).Determination of the β2-chimaerin crystal structure provided important clues regarding its mechanism of action. In the absence of stimulation, the protein is in an inactive state in which the N-terminal domain maintains a “closed” conformation, blocking Rac binding and concealing the C1 domain (26). These structural data were fully supported by experiments in live T lymphocytes showing that phorbol myristate acetate (PMA)-dependent translocation of β2-chimaerin was less effective than that of its isolated C1 domain (24). These data not only confirmed the lack of accessibility of the β2-chimaerin C1 domain but also suggested that there are negative regulatory mechanisms that promote β2-chimaerin release from the membrane.DAG-dependent signaling is critical for the modulation of T cell functions, by virtue of its ability to bind and regulate C1 domain-containing proteins such as protein kinase Cθ, protein kinase D, and RasGRP1 (27). An important issue is to determine how the different DAG-binding proteins discriminate between distinct DAG pools, and how DAG activates certain C1-containing proteins and not others. Some mechanisms that allow discrimination between DAG receptors include the distinct affinity of C1 domains for different DAG pools, association of C1 domain-containing proteins to specific scaffolds, and/or structural determinants in these proteins that limit C1 domain accessibility to membrane DAG (28–30).To explore the events that contribute to the specific regulation of β2-chimaerin, we studied β2-chimaerin phosphorylation in the context of TCR stimulation. We show that β2-chimaerin is phosphorylated in tyrosine residues after TCR stimulation, and we identify Lck as the Tyr kinase responsible for this phosphorylation. Generation of point mutants identified Tyr-153, at the hinge of the SH2 and C1 domains, as the main tyrosine residue phosphorylated in response to TCR stimulation. Cells expressing a β2-chimaerin mutant defective for Tyr-153 phosphorylation show anomalies in TCR clustering, conjugate formation, NF-AT activation, and IL-2 production that correlate with elevated Rac-GAP activity in this mutant. Subcellular localization analysis of the β2-chimaerin mutants reveals that impairment of β2-chimaerin phosphorylation at Tyr-153 promotes C1-mediated β2-chimaerin stabilization at the plasma membrane, providing a mechanistic explanation for its higher Rac-GAP activity. In summary, our results demonstrate for the first time that tyrosine kinase-mediated negative regulation of β2-chimaerin is elicited by physiological stimulation in T lymphocytes, and suggest that TCR stimulation provides both positive and negative signals for β2-chimaerin activation.     

Loss of ��-Catenin Decreases the Strength of Single E-cadherin Bonds between Human Cancer Cells

The progression of several human cancers correlates with the loss of cytoplasmic protein α-catenin from E-cadherin-rich intercellular junctions and loss of adhesion. However, the potential role of α-catenin in directly modulating the adhesive function of individual E-cadherin molecules in human cancer is unknown. Here we use single-molecule force spectroscopy to probe the tensile strength, unstressed bond lifetime, and interaction energy between E-cadherins expressed on the surface of live human parental breast cancer cells lacking α-catenin and these cells where α-catenin is re-expressed. We find that the tensile strength and the lifetime of single E-cadherin/E-cadherin bonds between parental cells are significantly lower over a wide range of loading rates. Statistical analysis of the force displacement spectra reveals that single cadherin bonds between cancer cells feature an exceedingly low energy barrier against tensile forces and low molecular stiffness. Disassembly of filamentous actin using latrunculin B has no significant effect on the strength of single intercellular E-cadherin bonds. The absence of α-catenin causes a dominant negative effect on both global cell-cell adhesion and single E-cadherin bond strength. These results suggest that the loss of α-catenin alone drastically reduces the adhesive force between individual cadherin pairs on adjoining cells, explain the global loss of cell adhesion in human breast cancer cells, and show that the forced expression of α-catenin in cancer cells can restore both higher intercellular avidity and intercellular E-cadherin bond strength.The reduction of intercellular adhesion in a solid tumor is a critical step in the progression of tumor cells to metastasis (1). How normal cells lose their ability to form strong adhesions within a tissue is not well understood (2, 3). The loss of adhesion between adjoining epithelial cells and the ensuing onset of metastasis occur through an epithelial-to-mesenchymal transition that often correlates with the loss of cytoplasmic protein α-catenin and a poor prognosis in a wide range of cancers, including breast (4), esophageal (5), gastric (6, 7), cervical (8), and colorectal cancer (9). In normal epithelial tissues, α-catenin localizes to junctions that organize at the interface between adjacent epithelial cells through clustering of cell surface adhesion transmembrane molecule cadherin and its association to the cytoskeleton (10, 11). On the extracellular side, structural studies suggest that cadherin molecules form molecular pairs that interact with cadherin pairs on an adjacent cell through their distal Ca²⁺-binding domains (12). On the intracellular side, cadherin pairs are connected to the cytoskeleton network through specific linker proteins. Until recently it was believed that one critical linker protein between the cytoplasmic domain of cadherin and the actin cytoskeleton was α-catenin, because it can both bind filamentous actin (F-actin) and E-cadherin through β-catenin (13, 14). However, a recent study indicates that α-catenin can either bind the E-cadherin-β-catenin complex as monomer or cross-link actin filaments as homodimer but cannot bind both E-cadherin-β-catenin and F-actin simultaneously (15). Therefore, whether the loss of α-catenin plays a direct role in the loss of adhesion in human cancer cells is unclear.Our recent data using engineered Chinese hamster ovarian cells suggest that α-catenin mediates the rapid strengthening of individual intercellular E-cadherin/E-cadherin bonds following initial molecular recognition between cells bearing E-cadherin molecules (16). Furthermore, α-catenin mediates the formation of additional E-cadherin/E-cadherin bonds once a first bond is formed between adjoining cells to form a nascent intercellular junction (16). Here we hypothesize that the loss of cytoplasmic protein α-catenin in human cancer cells greatly affects the ability of E-cadherin molecules on the surface of these cells to form firm adhesion by reducing the strength of individual intercellular E-cadherin/E-cadherin bonds.Our strategy is to compare parental breast cancer cells that lack α-catenin (MDA-MB-468 cells; denoted here MDA468) with these cells when α-catenin is introduced and exploit high resolution live cell single-molecule force spectroscopy (17) to probe the strength of individual E-cadherin/E-cadherin bonds between adjacent cells (18). The cells are juxtaposed for a controlled time of contact, the probability of successful interactions is subsequently measured, and the mechanical properties (tensile strength, molecular stiffness, and reactive compliance) and biochemical properties (interaction energy, dissociation rate, and bond lifetime) of single intercellular E-cadherin/E-cadherin bonds are analyzed.Our main hypothesis cannot be readily tested using purified proteins. Our ability to measure molecular interactions between live cells (17) rather than recombinant proteins ensures that the proper orientation of cadherin on the cell surfaces and its post-translational modifications are physiological. Moreover, using living cells ensures that the cytoplasmic domain of transmembrane receptors (here human E-cadherin) can interact with cytoplasmic proteins (in particular β-catenin and α-catenin), thereby allowing cell signaling pathways that can influence cell adhesion to function normally.     

The Carboxyl-terminal Domain of Atypical Protein Kinase C�� Binds to Ceramide and Regulates Junction Formation in Epithelial Cells

Atypical protein kinase Cs (PKCs) (aPKCζ and λ/ι) have emerged as important binding partners for ceramide, a membrane-resident cell signaling lipid that is involved in the regulation of apoptosis as well as cell polarity. Using ceramide overlay assays with proteolytic fragments of PKCζ and vesicle binding assays with ectopically expressed protein, we show that a protein fragment comprising the carboxyl-terminal 20-kDa sequence of PKCζ (C20ζ, amino acids 405–592) bound to C16:0 ceramide. This sequence is not identical to the C1 domain (amino acids 131–180), which has been suggested to serve as a potential ceramide binding domain. Using immunocytochemistry, we found that a C20ζ protein fragment ectopically expressed in two epithelial cell types (neural progenitors and Madin-Darby canine kidney cells) co-distributed with ceramide. Stable expression of C20ζ-EGFP in Madin-Darby canine kidney cells disrupted the formation of adherens and tight junctions and impaired the epithelium integrity by reducing transepithelial electrical resistance. Disruption of cell adhesion and loss of transepithelial electrical resistance was prevented by incubation with C16:0 ceramide. Our results show, for the first time, that there is a novel ceramide binding domain (C20ζ) in the carboxyl terminus of aPKC. Our results also show that the interaction of ceramide with this binding domain is essential for cell-to-cell contacts in epithelia. Therefore, ceramide interaction with the C20ζ binding domain is a potential mechanism by which ceramide and aPKC regulate the formation of junctional complexes in epithelial cells.Epithelial cells play essential roles in multicellular organisms by forming physiological and mechanical barriers and controlling tissue architecture, because they acquire apicobasal and cell-to-cell (planar) polarity (1, 2). Adherens junctions (AJs)² and tight junctions (TJs) are major structures responsible for cell-to-cell adhesion in epithelial cells (3). The regulation of junction formation requires endocytosis, redistribution, and recycling of junctional proteins, such as E-cadherin (4), and ZO-1. Many factors, including EGF, EGFR, Src kinase, Rho family GTPases Cdc42 and Rac1, and atypical PKC (aPKC), have been found to regulate junction formation (5–9). In Madin-Darby canine kidney (MDCK) cells, Cdc42 modulates AJs by regulating E-cadherin ubiquitination and degradation (9), whereas aPKC directly localized at TJs is required for the asymmetric differentiation of the premature junction complex during epithelial cell polarization (1, 10).The protein kinase C (PKC) family comprises serine/threonine kinases, which consist of a carboxyl-terminal catalytic domain and an amino-terminal regulatory domain (Fig. 1A). The regulatory domain includes an inhibitory pseudosubstrate domain and allosteric sites for activation by phosphatidylserine and, depending on the isoform, calcium (C2 domain) and/or diacylglycerol (C1 domain). aPKC is a subfamily of PKC, which consists of the isoforms ζ and λ/ι. The aPKC isoforms contain only half of the C1 domain, and hence, their activity is not affected by calcium or diacylglycerol/phorbol esters (see Fig. 1A and Refs. 11–13).Open in a separate windowFIGURE 1.Binding of ceramide to the COOH terminus of PKCζ. A, primary structure of aPKC, the caspase 3 proteolytic fragment ζCasp II, and the NH₂-terminal deletion mutant C20ζ-EGFP. B, 2 μg of recombinant His-tagged PKCζ was proteolytically digested by 20 ng of recombinant caspase 3. Proteolysis by caspase 3 occurred first after amino acid 239 (4-h incubation) and then after amino acid 459 (10-h incubation, ζCasp II). C, binding to ceramide spotted on nitrocellulose (overlay assay). FL PKCζ and the COOH-terminal proteolytic fragment ζCasp II bound to C16 ceramide. D, C16 ceramide vesicle binding assay (LIMAC). Ectopically expressed C20ζ-EGFP prepared from a cell lysate was bound to ceramide vesicles; EGFP was not. Protein was detected using anti-aPKC and anti-GFP antibodies. Lanes 1–3, loading input for ceramide vesicles; lanes 4–6, eluate of vesicle binding columns (output). Lanes 7 (input) and 8 (output) show that PKCζ-EGFP did not bind to vesicles prepared with sphingomyelin (SM) instead of ceramide. E, subcellular fractionation of cells expressing FL PKCζ-EGFP or C20ζ-EGFP.Apart from its function in apoptosis (13–15) and cell growth (16), aPKC has been found to play a pivotal role in cell polarity, both in neuroepithelial cells (17–20) or other epithelial cell types (1, 10). Consistently, the gene knock-out of aPKC shows loss of cell junction formation and detachment of neural progenitor cells from the neuroepithelium (8, 21–23). We and others have found that the sphingolipid ceramide activates aPKC, recruits it to structured microdomains, and regulates cell polarity and motility (24–28). Using lipid vesicle-mediated affinity chromatography (LIMAC) assays, we showed for the first time that ceramide directly binds to aPKC (25). Yet which domain of aPKC binds to ceramide is not known.Using lipid overlay and LIMAC assays, we show here that a COOH-terminal 20-kDa domain of PKCζ (C20ζ) binds to ceramide. Similar to its full-length counterpart, the C20ζ protein fragment resides in cellular membranes, where it co-distributes with ceramide in both C17.2 (neural progenitor) and MDCK cells. To study the function of this ceramide binding domain, we established a stably transfected MDCK cell line expressing C20ζ-EGFP. In these cells, the protein level of E-cadherin is reduced, and the cellular distribution of E-cadherin, ZO-1, and β-catenin is disrupted when compared with EGFP-transfected cell lines. Further, transepithelial electrical resistance (TER) assays show that the C20ζ-EGFP cell line has reduced impedance when compared with the control cell line expressing EGFP. This finding suggests that the C20ζ protein fragment is a dominant negative mutant of PKCζ. The effects of this dominant negative mutant can be, at least partially, rescued by incubation with C16:0 ceramide, suggesting that ceramide regulates aPKC and aPKC-dependent cell junction formation by interaction with the COOH-terminal domain.     

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.     

Roles of Volume-sensitive Cl<Superscript>−</Superscript> Channel in Cisplatin-induced Apoptosis in Human Epidermoid Cancer Cells

The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl⁻/HCO₃⁻ exchangers and Cl⁻ channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO₃⁻, we investigated the relation between cisplatin-induced apoptosis and Cl⁻ channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl⁻ current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl⁻ channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.