Ouabain's Influence on TRPV4 Channels of Epithelial Cells: An Exploration of TRPV4 Activity, Expression, and Signaling Pathways.

Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell-cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.

Expression, purification, and refolding of the recombinant extracellular domain ß1-subunit of the dog Na+/K+-ATPase of the epithelial cells.

The ß1-subunit of the Na+/K+-ATPase is a cell membrane protein, beyond its classic functions, it is also a cell adhesion molecule. ß1-subunits on the lateral membrane of dog kidney epithelial cells trans-interact with ß1-subunits from another neighboring cells. The ß-ß interaction is essential for the formation and stabilization of intercellular junctions. Previous studies on site-directed mutagenesis and in silico revealed that the interaction interface involves residues 198-207 and 221-229. However, it is necessary to report the interaction interface at the structural level experimentally. Here, we describe the successful cloning, overexpression in E. coli, and purification of the extracellular domain of the ß1-subunit from inclusion bodies. Experimental characterization by size exclusion chromatography and DLS indicated similar hydrodynamic properties of the protein refolded. Structural analysis by circular dichroism and Raman spectroscopy revealed the secondary structures in the folded protein of type ß-sheet, α-helix, random coil, and turn. We also performed ß1-ß1 interaction assays with the recombinant protein, showing dimers' formation (6xHisß1-ß1). Given our results, the recombinant extracellular domain of the ß1-subunit is highly similar to the native protein, therefore the current work in our laboratory aims to characterize at the atomic level the interaction interface between EDß1.

The ß2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule.

The ß2 subunit of Na+, K+-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While ß1 isoform have been shown to trans-interact in a species-specific mode with the ß1 subunit on the epithelial neighboring cell, the ß2 subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of ß2 or described its adhesion mechanism. Until now, the interactions pronounced for ß2/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether ß2 is a cell-cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell-cell aggregation, and protein-protein interaction assays. We observed that the glycosylated extracellular domain of ß2/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human ß2 subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein-protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant ß2 subunit show that the ß2 subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human ß2 subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).

E7 oncoprotein from human papillomavirus 16 alters claudins expression and the sealing of epithelial tight junctions.

Tight junctions (TJs) are cell­cell adhesion structures frequently altered by oncogenic transformation. In the present study the role of human papillomavirus (HPV) 16 E7 oncoprotein on the sealing of TJs was investigated and also the expression level of claudins in mouse cervix and in epithelial Madin­Darby Canine Kidney (MDCK) cells. It was found that there was reduced expression of claudins ­1 and ­10 in the cervix of 7­month­old transgenic K14E7 mice treated with 17ß­estradiol (E2), with invasive cancer. In addition, there was also a transient increase in claudin­1 expression in the cervix of 2­month­old K14E7 mice, and claudin­10 accumulated at the border of cells in the upper layer of the cervix in FvB mice treated with E2, and in K14E7 mice treated with or without E2. These changes were accompanied by an augmented paracellular permeability of the cervix in 2­ and 7­month­old FvB mice treated with E2, which became more pronounced in K14E7 mice treated with or without E2. In MDCK cells the stable expression of E7 increased the space between adjacent cells and altered the architecture of the monolayers, induced the development of an acute peak of transepithelial electrical resistance accompanied by a reduced expression of claudins ­1, ­2 and ­10, and an increase in claudin­4. Moreover, E7 enhances the ability of MDCK cells to migrate through a 3D matrix and induces cell stiffening and stress fiber formation. These observations revealed that cell transformation induced by HPV16 E7 oncoprotein was accompanied by changes in the pattern of expression of claudins and the degree of sealing of epithelial TJs.

A Model for the Homotypic Interaction between Na+,K+-ATPase ß1 Subunits Reveals the Role of Extracellular Residues 221-229 in Its Ig-Like Domain.

The Na+, K+-ATPase transports Na+ and K+ across the membrane of all animal cells. In addition to its ion transporting function, the Na+, K+-ATPase acts as a homotypic epithelial cell adhesion molecule via its ß1 subunit. The extracellular region of the Na+, K+-ATPase ß1 subunit includes a single globular immunoglobulin-like domain. We performed Molecular Dynamics simulations of the ectodomain of the ß1 subunit and a refined protein-protein docking prediction. Our results show that the ß1 subunit Ig-like domain maintains an independent structure and dimerizes in an antiparallel fashion. Analysis of the putative interface identified segment Lys221-Tyr229. We generated triple mutations on YFP-ß1 subunit fusion proteins to assess the contribution of these residues. CHO fibroblasts transfected with mutant ß1 subunits showed a significantly decreased cell-cell adhesion. Association of ß1 subunits in vitro was also reduced, as determined by pull-down assays. Altogether, we conclude that two Na+, K+-ATPase molecules recognize each other by a large interface spanning residues 221-229 and 198-207 on their ß1 subunits.

Ouabain Enhances Cell-Cell Adhesion Mediated by ß1 Subunits of the Na+,K+-ATPase in CHO Fibroblasts.

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine ß1 subunit become adhesive, and those homotypic interactions amongst ß1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between ß1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the ß1 subunit of the Na+,K+-ATPase (CHO ß1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO ß1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the ß1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO ß1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO ß1 cells. Collectively, our findings suggest that the ß1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.

The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the ß2 Subunit.

Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the ß1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic ß1-ß1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its ß2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the ß2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the ß2 subunit. qPCR results showed a time-dependent increase in the level of ß2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the ß2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the ß2 subunit.

Apoptosis of hemocytes from lions-paw scallop Nodipecten subnodosus induced with paralyzing shellfish poison from Gymnodinium catenatum.

The toxic dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons (PSPs) that are consumed and accumulated by bivalves. Previously, we recorded a decrease in hemocytes 24h after injection of PSPs (gonyautoxin 2/3 epimers, GTX2/3) in the adductor muscle in the lions-paw scallop Nodipecten subnodosus. In this work, qualitative and quantitative analyses, in in vivo and in vitro experiments, revealed that the lower count of hemocytes results from cells undergoing typical apoptosis when exposed to GTX 2/3 epimers. This includes visible morphological alterations of the cytoplasmic membrane, damage to the nuclear membrane, condensation of chromatin, DNA fragmentation, and release of DNA fragments into the cytoplasm. Induction of apoptosis was accompanied by phosphatidylserine exposure to the outer cell membrane and activation of cysteine-aspartic proteases, caspase 3 and caspase 8. Addition of an inhibitor of caspase to the medium suppressed activation in hemocytes exposed to the toxins, suggesting that cell death was induced by a caspase-dependent apoptotic pathway. The results are important for future investigation of the scallop's immune system and should provide new insights into apoptotic processes in immune cells of scallops exposed to PSPs.

Epithelial junctions depend on intercellular trans-interactions between the Na,K-ATPase ß1 subunits.

N-Glycans of the Na,K-ATPase ß1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the ß1 subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous ß1 subunit with various YFP-linked ß1 subunits expressed in Madin-Darby canine kidney cells was used to assess ß1-ß1 interactions. The amount of co-precipitated endogenous dog ß1 was greater with dog YFP-ß1 than with rat YFP-ß1, showing that amino acid-mediated interactions are important for ß1-ß1 binding. Co-precipitation of ß1 was also less with the unglycosylated YFP-ß1 than with glycosylated YFP-ß1, indicating a role for N-glycans. Mixing cells expressing dog YFP-ß1 with non-transfected cells increased the amount of co-precipitated ß1, confirming the presence of intercellular (YFP-ß1)-ß1 complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated ß1 subunits. The decrease in ß1 co-precipitation both with rat YFP-ß1 and unglycosylated YFP-ß1 was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. Reducing N-glycan branching by specific inhibitors increased (YFP-ß1)-ß1 co-precipitation and strengthened intercellular junctions. Therefore, interactions between the ß1 subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the ß1 subunit N-glycan structure can regulate stability and tightness of intercellular junctions.

Ouabain modulates epithelial cell tight junction.

Epithelial cells treated with high concentrations of ouabain (e.g., 1 microM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K(+) pumping nor disturb the K(+) balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J(DEX)). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J(DEX), and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.

The polarized distribution of Na+,K+-ATPase: role of the interaction between {beta} subunits.

The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na(+),K(+)-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na(+),K(+)-ATPase polarity depends on interactions between the beta subunits of Na(+),K(+)-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these beta subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.

Tight junction and polarity interaction in the transporting epithelial phenotype.

Development of tight junctions and cell polarity in epithelial cells requires a complex cellular machinery to execute an internal program in response to ambient cues. Tight junctions, a product of this machinery, can act as gates of the paracellular pathway, fences that keep the identity of plasma membrane domains, bridges that communicate neighboring cells. The polarization internal program and machinery are conserved in yeast, worms, flies and mammals, and in cell types as different as epithelia, neurons and lymphocytes. Polarization and tight junctions are dynamic features that change during development, in response to physiological and pharmacological challenges and in pathological situations like infection.

New diseases derived or associated with the tight junction.

The space between neighboring epithelial cells is sealed by the tight junction (TJ). When this seal is leaky, such as in the proximal tubule of the kidney or the gallbladder, substances may cross the epithelium between the cells (paracellular pathway). Yet, when TJs are really hermetic, as is the case in the epithelium of the urinary bladder or the colon, substances can mainly cross the epithelium through the transcellular pathway. The structure of the TJ involves (so far) some 50-odd protein species. Failure of any of these components causes a variety of diseases, some of them so serious that fetuses are not viable. A fast-growing number of diseases are recognized to depend or involve alterations in the TJ. These include autoimmune diseases, in which intestinal TJs allow the passage of antigens from the intestinal flora, challenging the immune system to produce antibodies that may cross react with proteins in the brain, thyroid gland or pancreas. TJs are also involved in cancer development, infections, allergies, etc. The present article does not catalogue all TJ diseases known so far, but describes one of each type as illustration. It also depicts the efforts being made to find pharmaceutical agents that would seal faulty TJs or release their grip to allow for the passage of large molecules through the upper respiratory and digestive tracts, such as insulin, thyroid, appetite-regulatory peptide, etc.

Contacts and cooperation between cells depend on the hormone ouabain.

Cell adhesion is a crucial step in proliferation, differentiation, migration, apoptosis, and metastasis. In previous works we have shown that cell adhesion is modulated by ouabain, a highly specific inhibitor of Na+,K+-ATPase, recently found to be a hormone. In the present work we pursue the investigation of the effect of ouabain on a special type of cell-cell interaction: the rescue of ouabain-sensitive MDCK cells (W) by ouabain-resistant cells (R). In cultured monolayers of pure W cells, ouabain triggers the "P-->A mechanism" (from pump/adhesion) consisting of a cascade of phosphorylations that retrieves adhesion-associated molecules occludin and beta-catenin and results in detachment of the cell. When W cells are instead cocultured with R cells, the P-->A reaction is blocked, and W cells are rescued. Furthermore, in these R/W cocultures ouabain promotes cell-cell communication by means of gap junctions by specifically enhancing the expression of connexin 32 and addressing this molecule to the plasma membrane. Ouabain also promotes the internalization of the beta-subunit of the Na+,K+-ATPase. These observations open the possibility that the crucial processes mentioned at the beginning would be under the control of the hormone ouabain.

Sodium/potassium ATPase (Na+, K+-ATPase) and ouabain/related cardiac glycosides: A new paradigm for development of anti- breast cancer drugs?

Prolonged exposure to 17beta-estradiol (E2) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERalpha and ERbeta. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na+, K+-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and activity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na+, K+-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na+, K+-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.

The polarized expression of Na+,K+-ATPase in epithelia depends on the association between beta-subunits located in neighboring cells.

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta1-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta1-subunit forces CHO-beta cells to coexpress endogenous alpha-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog beta1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.

E-Cadherin and tight junctions between epithelial cells of different animal species.

The assembly and permanent sealing of tight junctions (TJs) depend crucially on cell-cell contacts containing E-cadherin. This poses a puzzling problem because, while TJs can be established between epithelial cells from different tissues and even different animal species ("heterotypic TJs"; Gonzalez-Mariscal et al. 1989, J Membr Biol 107:43), the cell-cell binding mediated by E-cadherin is a highly specific one (Takeichi 1995, Curr Opin Cell Biol 7:619). Yet the demonstration that TJs can be established at heterotypic borders is open to two distinct challenges. First, it is based on transepithelial electrical resistance (TER) and restriction to ruthenium red permeation only, which today are known to be just two of the many characteristics of TJs; and second some attributes of the TJs (e.g. the presence of specific molecules) have been found even in cells that do not establish these structures. This raised the question of whether heterotypic TJs were not true or full TJs. In the present work we demonstrate that heterotypic TJs in mixed monolayers of MDCK cells with a different cell type (LLC-PK1) are true TJs through several criteria, such as TER, the ability to stop the membrane diffusion of fluorescent sphingomyelin from the apical to the lateral domain, the presence of ZO-1, ZO-2, occludin, claudin-1 and claudin-2. We then turn to the presence of E-cadherin at heterotypic borders, and observe that it cannot be detected by the highly specific DECMA-1 antibody, in spite of the fact that this antibody does reveal the presence of E-cadherin at homotypic contacts of the same cell. Yet, ECCD-2, an antibody against another domain of E-cadherin, reveals that this molecule may be present at both types of borders. Thus, E-cadherin is present at heterotypic borders, yet it seems to be in a conformation unable to bind DECMA-1. Our results suggest: (1) that heterotypic borders can establish fully developed TJs; (2) that the sealing of these heterotypic TJs depends on E-cadherin; (3) but that this dependence is mediated through a cascade of chemical reactions involving two different G-proteins, PLC, PKC and calmodulin, which we have characterized elsewhere (Balda et al. 1991, J Membr Biol 122:193); and (4) hence molecules of E-cadherin that trigger junction formation can act from a distant homotypic contact.