Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines

The secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at ∼0.5 μm) activate Ca²⁺ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1α) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca²⁺ rise and chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo.Trefoil factor 2 (TFF2),² previously known as spasmolytic polypeptide, is a unique member of the trefoil family that is expressed primarily in gastric mucous neck cells and is up-regulated in the setting of chronic inflammation. Experimental induction of ulceration in the rat stomach leads to rapid up-regulation of TFF2 expression with high levels observed 30 min after ulceration with persistence for up to 10 days (1). TFF2 is secreted into the mucus layer of the gastrointestinal tract of mammals where it stabilizes the mucin gel layer and stimulates migration of epithelial cells (2–4), suggesting an important role in restitution and in maintenance of the integrity of the gut. Exogenous administration of recombinant TFF2, either orally or intravenously, provides mucosal protection in several rodent models of acute gastric or intestinal injury (5, 6). A TFF2^-/- knock-out mouse model has confirmed the importance of TFF2 in the protection of gastrointestinal mucosa against chronic injury (7).It is widely accepted that trefoil factors exert their biological action through a cell surface receptor. This suggestion comes from studies on binding of ¹²⁵I-labeled TFF2 that demonstrated specific binding sites in the gastric glands, intestine, and colon that could be displaced by non-radioactive TFF2 (6, 8–10). Structural studies have revealed potential binding sites for receptors for all members of the trefoil factor family (11, 12). In concordance with this hypothesis, several membrane proteins were found to interact with TFF2. First it was shown that recombinant human TFF2 (and TFF3) could bind to a 28-kDa peptide from membrane fractions of rat jejunum and two human adenocarcinoma cell lines, MCF-7 and Colony-29 (13). Later it was found that recombinant TFF3 fused with biotin selectively bound with a 50-kDa protein from the membrane of rat small intestinal cells (14). However, these 28- and 50-kDa proteins were characterized only by their molecular size without further identification. Two TFF2-binding proteins that have been characterized include a 140-kDa protein, the β subunit of the fibronectin receptor, and a 224-kDa protein called muclin (15). Another TFF2-binding protein was isolated by probing two-dimensional blots of mouse stomach with a murine TFF2 fusion protein, leading to the identification of the gastric foveolar protein blottin, a murine homolog of the human peptide TFIZ1(16). Although these three proteins have now been well characterized, none of them has been shown to mediate responses to TFF2, and no activated signaling cascades have been shown.Despite the absence of an identified cell surface receptor for TFF2, there is nevertheless clear evidence that TFF2 and TFF3 rapidly activate signal transduction pathways (17, 18). TFF3 prevents cell death via activation of the serine/threonine kinase AKT in colon cancer cell lines (19). The TFF3 protein also activates STAT3 signaling in human colorectal cancer cells, thus providing cells with invasion potential (20). TFF3 treatment leads to EGF receptor activation and β-catenin phosphorylation in HT-29 cells (21) and to transient phosphorylation of ERK1/2 in oral keratinocytes (22). With respect to TFF2, recombinant peptide enhances the migration of human bronchial epithelial cell line BEAS-2B (4). TFF2 has been shown to induce phosphorylation of c-Jun NH₂-terminal kinase (JNK) and ERK1/2. Consistent with this observation, the motogenic effect of TFF2 is significantly inhibited by antagonists of ERK kinases and protein kinase C but not by inhibitors of p38 mitogen-activated protein kinase (MAPK). It is believed that the motogenic effect of trefoil factors and of TFF2 in particular, could contribute to in vivo restitution of gastric epithelium by enhancing cell migration.Although previous studies have suggested that TFF2 functions primarily in cytoprotection, accumulating evidence now suggests that TFF2 may also play a role in the regulation of host immunity. For example, recombinant TFF2 reduces inflammation in rat and mouse models of colitis (23, 24). In addition, TFF2 was detected in rat lymphoid tissues (spleen, lymph nodes, and bone marrow) (25). Recently we and others found TFF2 mRNA expression in primary and secondary lymphopoietic organs (26, 27). These data suggest that TFF2 may play some function in the immune system. In concordance with these findings, we detected an exacerbated inflammatory response to acute injury in TFF2 knock-out animals (27, 28). These observations prompted us to look at the possible function of TFF2 in immune cells. Unexpectedly we found that TFF2 modulates Ca²⁺ and AKT signaling in lymphoblastic Jurkat cells and that these effects appear to be mediated through the CXCR4 receptor.     

Separating Growth from Elastic Deformation during Cell Enlargement

Plants change size by deforming reversibly (elastically) whenever turgor pressure changes, and by growing. The elastic deformation is independent of growth because it occurs in nongrowing cells. Its occurrence with growth has prevented growth from being observed alone. We investigated whether the two processes could be separated in internode cells of Chara corallina Klien ex Willd., em R.D.W. by injecting or removing cell solution with a pressure probe to change turgor while the cell length was continuously measured. Cell size changed immediately when turgor changed, and growth rates appeared to be altered. Low temperature eliminated growth but did not alter the elastic effects. This allowed elastic deformation measured at low temperature to be subtracted from elongation at warm temperature in the same cell. After the subtraction, growth alone could be observed for the first time. Alterations in turgor caused growth to change rapidly to a new, steady rate with no evidence of rapid adjustments in wall properties. This turgor response, together with the marked sensitivity of growth to temperature, suggested that the growth rate was not controlled by inert polymer extension but rather by biochemical reactions that include a turgor-sensitive step.     

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca²⁺ channels. Inhibition of the oxidative response impaired Cl⁻ efflux, K⁺ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.     

Heparan Sulfate Biosynthesis Enzymes in Embryonic Stem Cell Biology

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst and can give rise to all cell types in the body. The fate of ES cells depends on the signals they receive from their surrounding environment, which either promote self-renewal or initiate differentiation. Heparan sulfate proteoglycans are macromolecules found on the cell surface and in the extracellular matrix. Acting as low-affinity receptors on the cell surface, heparan sulfate (HS) side chains modulate the functions of numerous growth factors and morphogens, having wide impact on the extracellular information received by cells. ES cells lacking HS fail to differentiate but can be induced to do so by adding heparin. ES cells defective in various components of the HS biosynthesis machinery, thus expressing differently flawed HS, exhibit lineage-specific effects. Here we discuss recent studies on the biological functions of HS in ES cell developmental processes. Since ES cells have significant potential applications in tissue/cell engineering for cell replacement therapies, understanding the functional mechanisms of HS in manipulating ES cell growth in vitro is of utmost importance, if the stem cell regenerative medicine from scientific fiction ever will be made real.     

Bile Acid-induced Epidermal Growth Factor Receptor Activation in Quiescent Rat Hepatic Stellate Cells Can Trigger Both Proliferation and Apoptosis

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47^phox and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Cζ, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H₂O₂), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.Hydrophobic bile acids play a major role in the pathogenesis of cholestatic liver disease and are potent inducers of hepatocyte apoptosis by triggering a ligand-independent activation of the CD95² death receptor (1–5). The underlying molecular mechanisms are complex and involve a Yes-dependent, but ligand-independent activation of the epidermal growth factor receptor (EGFR), which catalyzes CD95-tyrosine phosphorylation as a prerequisite for CD95 oligomerization, formation of the death-inducing signaling complex (DISC), and apoptosis induction (6, 7). Bile acids also activate EGFR in cholangiocytes (8) and activated hepatic stellate cells (HSC) (9), however, the mechanisms underlying bile acid-induced EGFR activation in HSC remained unclear (9). Surprisingly, bile acid-induced EGFR activation in HSC does not trigger apoptosis but results in a stimulation of cell proliferation (9). The behavior of quiescent HSC toward CD95 ligand (CD95L) is also unusual. CD95L, which is a potent inducer of hepatocyte apoptosis (10–12), triggers activation of the EGFR in quiescent HSC, stimulates HSC proliferation, and simultaneously inhibits CD95-dependent death signaling through CD95-tyrosine nitration (13). Similar observations were made with other death receptor ligands, i.e. tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) (13). The mitogenic action of CD95L in quiescent, 1–2-day cultured HSC is because of a c-Src-dependent shedding of EGF and subsequent auto/paracrine activation of the EGFR (13). This unusual behavior of quiescent HSC toward death receptor ligands may relate to the recent findings that quiescent HSC might represent a stem/progenitor cell compartment in the liver with a capacity to differentiate not only into myofibroblasts but also toward hepatocyte- and endothelial-like cells (14). Thus, stimulation of HSC proliferation and resistance toward apoptosis in the hostile cytokine milieu accompanying liver injury may help HSC to play their role in liver regeneration. During cholestatic liver injury quiescent HSC are exposed to increased concentrations of circulating bile acids, but it is not known whether this may lead to HSC proliferation (as shown for activated HSC) (9), HSC apoptosis (as shown for hepatocytes) (1–7), or both of them. Therefore, the aim of the current study was (a) to identify the molecular mechanisms underlying bile acid-induced EGFR activation and (b) to elucidate whether bile acid-induced signaling can couple to both cell proliferation and cell death in quiescent HSC.The present study shows that cholestatic bile acids trigger a rapid NADPH oxidase activation in quiescent HSC, which leads to a Yes-mediated EGFR phosphorylation and HSC proliferation. In contrast to hepatocytes, hydrophobic bile acids do not induce a JNK signal in HSC. However, when JNK activation is induced by coadministration of either cycloheximide (CHX) or hydrogen peroxide (H₂O₂), the bile acid-induced mitogenic signal is shifted to an apoptotic one.     

Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was αII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium dependence of the founder members of the family, the ubiquitously expressed enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain family includes more than a dozen members with sequence relatedness to the catalytic subunits of calpain 1 and 2. Calpains have a modular domain architecture. By convention, the family is subdivided into classical and nonclassical calpains, according to the presence or absence, respectively, of a calcium-binding penta-EF-hand module in domain IV of the protein (1). Classical calpains include calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7, 10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an essential role for calpains during embryonic development. Knock-out of the small regulatory subunit (Capn4) results in embryonic lethality (2, 3). Similarly, inactivation of the Capn2 gene blocks development between the morula and blastocyst stage (4). In humans, mutations in CAPN3 underlie limb-girdle muscular dystrophy-2A, and polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus (5, 6).Even under conditions of calcium overload, where calpains are presumably activated maximally, only a subset (<5%) of cellular proteins are hydrolyzed (7). Calpains typically cleave their substrates at a limited number of sites to generate large polypeptide fragments that, in many cases, retain bioactivity. Thus, under physiological conditions, calpains probably participate in the regulation of protein function rather than in non-specific protein degradation.More than 100 proteins have been shown to serve as calpain substrates in vitro, including cytoskeletal proteins (8), signal transduction molecules (9), ion channels (10), and receptors (11). In vivo, calpains are believed to function in myoblast fusion (12), long term potentiation (13), and cellular mobility (14). Unregulated calpain activity, secondary to intracellular calcium overload, is associated with several pathological conditions, including Alzheimer disease (15), animal models of cataract (16), myocardial (17), and cerebral ischemia (18).In addition to their domain structure, calpains are often classified according to their tissue expression patterns. Calpain 1, 2, and 10 are widely expressed in mammalian tissues, but other members of the calpain family show tissue-specific expression patterns. Calpain 8, for example, is a stomach-specific calpain (19), whereas expression of calpain 9 is restricted to tissues of the digestive tract (20). The expression of calpain 3 was originally thought to be limited to skeletal muscle (21), but splice variants of calpain 3 have since been detected in a range of tissues. At least 12 isoforms of calpain 3 have been described in rodents (22), of which several are expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88 (retina) (23).Calpains have been studied intensively in the ocular lens because of their suspected involvement in lens opacification (cataract). Calpain-mediated proteolysis of lens crystallin proteins causes increased light scatter (24). Unregulated activation of calpains is observed in rodent cataract models (25), where calpain-mediated degradation of crystallin proteins (26) and cytoskeletal elements (27) is commonly observed. Calpain inhibitors are effective in delaying or preventing cataract in vitro (28, 29) and in vivo (30, 31).It is likely, however, that calpains have important physiological roles in the lens beyond their involvement in tissue pathology. Terminal differentiation of lens fiber cells involves a series of profound morphological and biochemical transformations. For example, differentiating lens fiber cells undergo an enormous (>100-fold) increase in cell length, accompanied by extensive remodeling of the plasma membrane system (32). Early in the differentiation process, fusion pores are established between cells, as neighboring fibers are incorporated into the lens syncytium (33). A later stage of fiber cell differentiation involves the dissolution of all intracellular organelles, a process that is thought to eliminate light-scattering particles from the light path and contribute to the transparency of the tissue (34). Any or all of these phenomena might require the developmentally regulated activation of calpains. This is consistent with our previous observation that in calpain 3 knock-out mice the transition zone is altered, suggesting a change in the differentiation program (35).In the current study, therefore, we examined the depth-dependent expression pattern and activity of calpains in the mouse lens. Fluorogenic substrates were microinjected into the intact lens to visualize calpain activity directly, and proteomic approaches were used to identify endogenous calpain substrates. The cleavage pattern of one of these, αII-spectrin, was examined in detail. Immunocytochemical and immunoblot analysis with wild type and calpain 3-null lenses indicated that αII-spectrin is a specific calpain 3 substrate in maturing lens fiber cells. Together, the data suggest that calpains are activated relatively late in fiber cell differentiation and may contribute to the remodeling of the membrane cytoskeleton that accompanies fiber cell maturation.     

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca²⁺. The complex formed in the presence of Ca²⁺ was more stable than that without Ca²⁺. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca²⁺ and B. Removal of the Ca²⁺ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca²⁺ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca²⁺ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca²⁺ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca²⁺_, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca²⁺ but also borate and Ca²⁺ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.Boron (B) is an essential element for higher plant growth, although its primary function is not known (Loomis and Durst, 1992). Determining the sites of B in cells is required to identify its function. In cultured tobacco cells more than 80% of cellular B is in the cell wall (Matoh et al., 1993), whereas the membrane fraction (Kobayashi et al., 1997) and protoplasts (Matoh et al., 1992) do not contain a significant amount of B. In radish (Raphanus sativus L. cv Aokubi-daikon) root cell walls, B cross-links two RG-II regions of pectic polysaccharides through a borate-diol ester (Kobayashi et al., 1995, 1996). The association of B with RG-II has been confirmed in sugar beet (Ishii and Matsunaga, 1996), bamboo (Kaneko et al., 1997), sycamore and pea (O''Neill et al., 1996), and red wine (Pellerin et al., 1996). In cultured tobacco cells the B associated with RG-II accounts for about 80% of the cell wall B (Kobayashi et al., 1997) and RG-II may be the exclusive carrier of B in higher plant cell walls (Matoh et al., 1996). Germanic acid, which partly substitutes for B in the growth of the B-deprived plants (Skok, 1957), also cross-links two RG-II chains (Kobayashi et al., 1997). These results suggest that the physiological role of B is to cross-link cell wall pectic polysaccharides in the RG-II region and thereby form a pectic network.It is believed that in the cell wall pectic polysaccharides are cross-linked with Ca²⁺, which binds to carboxyl groups of the polygalacturonic acid regions (Jarvis, 1984). Thus, the ability of B and Ca²⁺ to cross-link cell wall pectic polysaccharides needs to be evaluated. In this report we describe the B-RG-II complex of radish root and the role of B-RG-II and Ca²⁺ in the formation of a pectic network.     

Changes in Sodium Pump Expression Dictate the Effects of Ouabain on Cell Growth

Here we show that ouabain-induced cell growth regulation is intrinsically coupled to changes in the cellular amount of Na/K-ATPase via the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. Ouabain increases the endocytosis and degradation of Na/K-ATPase in LLC-PK1, human breast (BT20), and prostate (DU145) cancer cells. However, ouabain stimulates the PI3K/Akt/mTOR pathway and consequently up-regulates the expression of Na/K-ATPase in LLC-PK1 but not BT20 and DU145 cells. This up-regulation is sufficient to replete the plasma membrane pool of Na/K-ATPase and to stimulate cell proliferation in LLC-PK1 cells. On the other hand, ouabain causes a gradual depletion of Na/K-ATPase and an increased expression of cell cycle inhibitor p21^cip, which consequently inhibits cell proliferation in BT20 and DU145 cells. Consistently, we observe that small interfering RNA-mediated knockdown of Na/K-ATPase is sufficient to induce the expression of p21^cip and slow the proliferation of LLC-PK1 cells. Moreover, this knockdown converts the growth stimulatory effect of ouabain to growth inhibition in LLC-PK1 cells. Mechanistically, both Src and caveolin-1 are required for ouabain-induced activation of Akt and up-regulation of Na/K-ATPase. Furthermore, inhibition of the PI3K/Akt/mTOR pathway by rapamycin completely blocks ouabain-induced expression of Na/K-ATPase and converts ouabain-induced growth stimulation to growth inhibition in LLC-PK1 cells. Taken together, we conclude that changes in the expression of Na/K-ATPase dictate the growth regulatory effects of ouabain on cells.The Na/K-ATPase, a member of P-type ATPase family, was discovered as an energy transducing ion pump. It transports Na⁺ and K⁺ across the cell membrane and maintains ion homeostasis in animal cells (1, 2). Recent studies indicate that the Na/K-ATPase is also an important receptor that can transduce ligand binding into the activation of protein kinase cascades (3). Specifically, the Na/K-ATPase interacts with Src, which provides at least two important cellular regulations (4, 5). First, association with Na/K-ATPase keeps Src in an inactive state. Thus, the Na/K-ATPase serves as a native negative Src regulator (4). Second, this interaction forms a functional receptor complex for cardiotonic steroids (CTS)³ (3), a group of well characterized ligands of the Na/K-ATPase. Cardiotonic steroids include cardenolides (e.g. ouabain) and bufadienolides (e.g. marinobufagenin) (6). Although CTS are known cardiac drugs, some of them have now been identified as endogenous steroid hormones (6–8). Binding of CTS to the receptor complex activates the Na/K-ATPase-associated Src. Subsequently, the activated Src transactivates other tyrosine kinases, and together they recruit and further phosphorylate multiple membrane and soluble proteins, which results in the activation of protein kinase cascades and the generation of second messengers (3, 4, 6). Ultimately, this chain of signaling events would alter cellular functions and cell growth in a cell-specific manner (5, 9–12). For instance, we and others have demonstrated that ouabain-induced activation of ERK and PI3K/Akt/mTOR pathways are responsible for cell growth stimulation in transformed cell lines, in primary cultures, as well as in vivo (13–18).It has also been recognized for a long time that CTS inhibit cell growth in many cancer cells (19–24). Of particular significance are studies that indicate the beneficial effects of CTS therapy in women with breast cancer (25–29). Consistently, recent in vitro and in vivo studies have identified several new CTS compounds that exhibit anti-cancer activities (30–32). Oleandrin, for example, is in clinical trials in the United States as an anti-cancer remedy for human cancers (31, 33). Although ouabain inhibits the pumping function of the Na/K-ATPase, it is important to note that the growth inhibitory effect of ouabain can occur at doses that neither cause significant changes in intracellular Na⁺ and K⁺ nor affect cell viability. Rather, much like its effect on cell growth stimulation, ouabain induces cell growth inhibition through the activation of protein kinases and the generation of second messengers (19–23, 34). For example, a recent report showed that these nontoxic concentrations of ouabain stimulated Src, resulting in activation of the epidermal growth factor receptor/ERK pathway and induction of the expression of cell cycle inhibitor p21^cip and cell growth arrest (34). Thus, it becomes important to understand the molecular mechanisms that govern different fates of cells in response to CTS stimulation.Prior studies have demonstrated that CTS induce endocytosis of the Na/K-ATPase and regulate its cellular expression via receptor-mediated signal transduction (35, 36). Because the Na/K-ATPase has both pumping and signaling functions, it is conceivable that changes in the amount of cellular Na/K-ATPase could have significant consequences on cell growth. Therefore, we have conducted the following experiments to reveal the role of cellular Na/K-ATPase in ouabain-induced cell growth regulation.     

A Multistep Model for Paclitaxel-Induced Apoptosis in Human Breast Cancer Cell Lines

Despite extensive previous investigation, the events occurring between paclitaxel-induced mitotic arrest and the subsequent onset of apoptosis remain incompletely understood. In the present study, the sequential morphological and biochemical changes that occur after paclitaxel treatment were examined in MDA-MB-468 (p53 mutant) and MCF-7 (p53 wild-type) breast cancer cells. Flow cytometry indicated that paclitaxel induces tetraploidy that persists until the onset of apoptosis in both cell lines. Light and electron microscopy indicated that the cells transiently arrest in mitosis and then enter a multinucleated interphase state characterized by the absence of punctate staining for CENP-F, a G(2) marker, but the presence of cyclin E, a G(1) cyclin, and p21(waf1/cip1), a cyclin-dependent kinase inhibitor. Despite high p21(waf1/cip1) levels, paclitaxel-treated cells incorporated thymidine into DNA. Aphidicolin inhibited this DNA synthesis but not the subsequent onset of apoptosis. Conversely, the broad-spectrum caspase inhibitor benzyloxycarbonyl-val-ala-asp(OMe)-fluoromethylketone inhibited apoptosis and enhanced the number of multinucleated cells but did not facilitate generation of octaploid cells. These results are consistent with a multistep model in which breast cancer cells exposed to paclitaxel undergo an aberrant mitotic exit; proceed through a tetraploid, multinucleated G(1) state; initiate an aphidicolin-suppressible process of DNA repair; and subsequently undergo apoptosis.     

VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death

Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP (valosin-containing protein) gene. The disease is characterized neuropathologically by frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are distinct from those seen in other sporadic and familial FTLD-U entities. The major component of the ubiquitinated inclusions of FTLD with VCP mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most familial forms of FTLD-U. Understanding the relationship between individual gene defects and pathologic TDP-43 will facilitate the characterization of the mechanisms leading to neurodegeneration. Using cell culture models, we have investigated the role of mutant VCP in intracellular trafficking, proteasomal function, and cell death and demonstrate that mutations in the VCP gene 1) alter localization of TDP-43 between the nucleus and cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum stress, 4) increase markers of apoptosis, and 5) impair cell viability. These results suggest that VCP mutation-induced neurodegeneration is mediated by several mechanisms.Frontotemporal lobar degeneration (FTLD)² accounts for 10% of all late onset dementias and is the third most frequent neurodegenerative disease after Alzheimer disease and dementia with Lewy bodies (1). FTLD with ubiquitin-immunoreactive inclusions is genetically, clinically, and neuropathologically heterogeneous (2, 3). FTLD-U comprises several distinct entities, including sporadic forms and familial cases caused by mutations in the genes encoding VCP (valosin-containing protein), GRN (progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9 (2, 3). Frontotemporal dementia with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP gene located on chromosome 9p13-p12 (4-10) (Fig. 1). This multisystem disease is characterized by progressive muscle weakness and atrophy, increased osteoclastic bone resorption, and early onset frontotemporal dementia, also called FTLD (9, 11). Mutations in VCP are also associated with dilatative cardiomyopathy with ubiquitin-positive inclusions (12). Neuropathologic features of FTLD with VCP mutation include frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U). The majority of aggregates are ubiquitin- and TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal cytoplasmic inclusions (NCIs). A small number of inclusions are VCP-immunoreactive (5, 13). Pathologic TDP-43 in inclusions links a spectrum of diseases in which TDP-43 pathology is a primary feature, including FTLD-U, motor neuron disease, including amyotrophic lateral sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and Paget disease of bone, as well as an expanding spectrum of other disorders in which TDP-43 pathology is secondary (14, 15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing protein. CDC48 (magenta), located within the N terminus (residues 22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1 (23-25, 28). There are two AAA-ATPase domains (AAA; blue) at residues 240-283 and 516-569, which are joined by two linker regions (L1 and L2; red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical signature similar to that seen in other sporadic and familial cases of FTLD-U, including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease, FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome 9p (3, 16). TDP-43 proteinopathy in these disorders is characterized by hyperphosphorylation of TDP-43, ubiquitination, and cleavage to form C-terminal fragments detected only in insoluble brain extracts from affected brain regions (16). Identification of TDP-43 as the major component of the ubiquitin-immunoreactive inclusions of FTLD with VCP mutation supports the hypothesis that VCP gene mutations cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase associated with diverse cellular activities) superfamily. The N-terminal domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell division cycle protein 48)) and two AAA-ATPase domains that form a hexameric complex (Fig. 1) (17). Recently, it has been shown that the N-terminal domain of VCP binds phosphoinositides (18, 19). AKT (activated serine-threonine protein kinase) phosphorylates VCP and is required for constitutive VCP function (20, 21). AKT is activated through phospholipid binding and phosphorylation via the phosphoinositide 3-kinase signaling pathway, which is involved in cell survival (22). The lipid binding domain may recruit VCP to the cell membrane where it is phosphorylated by AKT (19).The diversity of VCP functions is modulated, in part, by a variety of intracellular cofactors, including p47, gp78, and Npl4-Ufd1 (23). Cofactor p47 has been shown to play a role in the maintenance and biogenesis of both the endoplasmic reticulum (ER) and Golgi apparatus (24). The structure of p47 contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and together they act as a chaperone to deliver membrane fusion machinery to the site of adjacent membranes (25). The function of the p47-VCP complex is dependent upon cell division cycle 2 (CDC2) serine-threonine kinase phosphorylation of p47 (26, 27). Also, VCP has been found to interact with the cytosolic tail of gp78, an ER membrane-spanning E3 ubiquitin ligase that exclusively binds VCP and enhances ER-associated degradation (ERAD) (28). The Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting of proteins through the ubiquitin-proteasome system (29, 30). The cell survival response of this complex has been found to be important in DNA damage repair though activation by phosphorylation and its recruitment to double-stranded breaks (20, 31). The Npl4-Ufd1-VCP cytosolic complex is also recruited to the ER membrane, interacting with Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At the ER membrane, these misfolded proteins are targeted to the proteasome via ERAD (32-34). VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome system, implicating VCP in the cell survival pathway and neuroprotection (21, 35-37).To investigate the mechanism of neurodegeneration caused by VCP mutations, we first tested the hypothesis that VCP mutations decrease cell viability in vitro using a neuroblastoma SHSY-5Y cell line and then investigated cellular pathways that are known to lead to neurodegeneration, including decrease in proteasome activity, caspase-mediated degeneration, and a change in cellular localization of TDP-43.