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.

TRPV4 activity regulates nuclear Ca2+ and transcriptional functions of ß-catenin in a renal epithelial cell model.

TRPV4 is a nonselective cationic channel responsive to several physical and chemical stimuli. Defects in TRPV4 channel function result in human diseases, such as skeletal dysplasias, arthropathies, and peripheral neuropathies. Nonetheless, little is known about the role of TRPV4 in other cellular functions, such as nuclear Ca2+ homeostasis or Ca2+ -regulated transcription. Here, we confirmed the presence of the full-length TRPV4 channel in the nuclei of nonpolarized Madin-Darby canine kidney cells. Confocal Ca2+ imaging showed that activation of the channel increases cytoplasmic and nuclear Ca2+ leading to translocation of TRPV4 out of the nucleus together with ß-catenin, a transcriptional regulator in the Wnt signaling pathway fundamental in embryogenesis, organogenesis, and cellular homeostasis. TRPV4 inhibits ß-catenin transcriptional activity through a direct interaction dependent upon channel activity. This interaction also occurs in undifferentiated osteoblastoma and neuroblastoma cell models. Our results suggest a mechanism in which TRPV4 may regulate differentiation in several cellular contexts.

Regulation of Na/K-ATPase expression by cholesterol: isoform specificity and the molecular mechanism.

We have reported that the reduction in plasma membrane cholesterol could decrease cellular Na/K-ATPase α1-expression through a Src-dependent pathway. However, it is unclear whether cholesterol could regulate other Na/K-ATPase α-isoforms and the molecular mechanisms of this regulation are not fully understood. Here we used cells expressing different Na/K-ATPase α isoforms and found that membrane cholesterol reduction by U18666A decreased expression of the α1-isoform but not the α2- or α3-isoform. Imaging analyses showed the cellular redistribution of α1 and α3 but not α2. Moreover, U18666A led to redistribution of α1 to late endosomes/lysosomes, while the proteasome inhibitor blocked α1-reduction by U18666A. These results suggest that the regulation of the Na/K-ATPase α-subunit by cholesterol is isoform specific and α1 is unique in this regulation through the endocytosis-proteasome pathway. Mechanistically, loss-of-Src binding mutation of A425P in α1 lost its capacity for regulation by cholesterol. Meanwhile, gain-of-Src binding mutations in α2 partially restored the regulation. Furthermore, through studies in caveolin-1 knockdown cells, as well as subcellular distribution studies in cell lines with different α-isoforms, we found that Na/K-ATPase, Src, and caveolin-1 worked together for the cholesterol regulation. Taken together, these new findings reveal that the putative Src-binding domain and the intact Na/K-ATPase/Src/caveolin-1 complex are indispensable for the isoform-specific regulation of Na/K-ATPase by cholesterol.

Ouabain Modulates the Adherens Junction in Renal Epithelial Cells.

BACKGROUND/AIMS: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. METHODS: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, ß-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²âº removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. RESULTS: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, ß-catenin and γ-catenin and displaced ß-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and ß-catenin in the plasma membrane after Ca²âº replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of ß-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase. CONCLUSION: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.

Isoform-specific role of Na/K-ATPase α1 in skeletal muscle.

The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15%) isoform and α2 comprising the bulk of the Na/K-ATPase pool. The acute and isoform-specific role of α2 in muscle performance and resistance to fatigue is well known, but the isoform-specific role of α1 has not been as thoroughly investigated. In vitro, we reported that α1 has a role in promoting cell growth that is not supported by α2. To assess whether α1 serves this isoform-specific trophic role in the skeletal muscle, we used Na/K-ATPase α1-haploinsufficient (α1+/-) mice. A 30% decrease of Na/K-ATPase α1 protein expression without change in α2 induced a modest yet significant decrease of 10% weight in the oxidative soleus muscle. In contrast, the mixed plantaris and glycolytic extensor digitorum longus weights were not significantly affected, likely because of their very low expression level of α1 compared with the soleus. The soleus mass reduction occurred without change in total Na/K-ATPase activity or glycogen metabolism. Serum analytes including K+, fat tissue mass, and exercise capacity were not altered in α1+/- mice. The impact of α1 content on soleus muscle mass is consistent with a Na/K-ATPase α1-specific role in skeletal muscle growth that cannot be fulfilled by α2. The preserved running capacity in α1+/- is in sharp contrast with previously reported consequences of genetic manipulation of α2. Taken together, these results lend further support to the concept of distinct isoform-specific functions of Na/K-ATPase α1 and α2 in skeletal muscle.

Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase.

The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase ß1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na+ affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na+ was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1.

Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells.

BACKGROUND/AIMS: The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. METHODS: We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. RESULTS: Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. CONCLUSION: Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

Ouabain increases gap junctional communication in epithelial cells.

BACKGROUND/AIMS: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). METHODS: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. RESULTS: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. CONCLUSION: Ouabain 10 nM increases GJC in MDCK cells.

Ouabain modulates ciliogenesis in epithelial cells.

The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na(+) and H(2)O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na(+),K(+)-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.

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.

Low dose HSP90 inhibition with AUY922 blunts rapid evolution of metastatic and drug resistant phenotypes induced by TGF-ß and paclitaxel in A549 cells.

OBJECTIVES: Despite advances in cancer treatment, drug resistance and metastasis continue to contribute to treatment failure. Since drug resistance and metastasis in cancer are features that often occur toward the late stages in the disease after withstanding numerous selective pressures, they may rely on a shared adaptive mechanism in order to persist. The heat shock response is one of the most well conserved adaptive responses to cellular stress found in nature. A major player in the heat shock response is HSP90, with some studies suggesting that it can facilitate the molecular evolution of drug resistance and metastasis in cancer. Non-small cell lung cancers (NSCLCs) are strongly associated with drug resistance and metastasis either at the time of diagnosis or early in the treatment process. MATERIALS AND METHODS: We explored the role of HSP90 in the evolution of metastatic and drug resistant features in NSCLC by treating A549 cells with AUY922, a clinically relevant HSP90 inhibitor, and inducing metastatic and drug resistant phenotypes via treatment with TGF-ß and paclitaxel, respectively. We measured phenotypic plasticity in E-Cadherin, a marker for epithelial to mesenchymal transition and two ABC transporters associated with drug resistant lung cancers. RESULTS: We found that metastatic and efflux dependent drug resistant features negatively correlated with AUY922 treatment. We followed our results with functional assays relevant to metastasis and ABC transporters to confirm our results. Specifically we found the expression of E-cadherin was significantly increased in A549 cultures pretreated with AUY922 prior to exposure to paclitaxel, while expression of the drug transporters ABCB1 and ABCC1 was significantly reduced under similar conditions. CONCLUSION: Together our data indicates that HSP90 inhibition with AUY922 can limit the acquisition of metastatic and drug resistant phenotypes in A549 cells at low, clinically appropriate doses.

A caveolin binding motif in Na/K-ATPase is required for stem cell differentiation and organogenesis in mammals and C. elegans.

Several signaling events have been recognized as essential for regulating cell lineage specification and organogenesis in animals. We find that the gain of an amino-terminal caveolin binding motif (CBM) in the α subunit of the Na/K-adenosine triphosphatase (ATPase) (NKA) is required for the early stages of organogenesis in both mice and Caenorhabditis elegans. The evolutionary gain of the CBM occurred at the same time as the acquisition of the binding sites for Na+/K+. Loss of this CBM does not affect cell lineage specification or the initiation of organogenesis, but arrests further organ development. Mechanistically, this CBM is essential for the dynamic operation of Wnt and the timely up-regulation of transcriptional factors during organogenesis. These results indicate that the NKA was evolved as a dual functional protein that works in concert with Wnt as a hitherto unrecognized common mechanism to enable stem cell differentiation and organogenesis in multicellular organisms within the animal kingdom.

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.

Ouabain modulates cell contacts as well as functions that depend on cell adhesion.

Ouabain, a toxic of vegetal origin used for centuries to treat heart failure, has recently been demonstrated to have an endogenous counterpart, most probably ouabain itself, which behaves as a hormone. Therefore, the challenge now is to discover the physiological role of hormone ouabain. We have recently shown that it modulates cell contacts such as gap junctions, which communicate neighboring cells, as well as tight junctions (TJs), which are one of the two differentiated features of epithelial cells, the other being apical/basolateral polarity. The importance of cell contacts can be hardly overestimated, since the most complex object in the universe, the brain, assembles itself depending on what cells contacts what other(s) how, when, and how is the molecular composition and special arrangement of the contacts involved. In the present chapter, we detail the protocols used to demonstrate the effect of ouabain on the molecular structure and functional properties of one of those cell-cell contacts: the TJ.

Na,K-ATPase is the putative membrane receptor of hormone ouabain.

At 10 nM, ouabain elicits changes in cell contacts, which are independent and usually in opposite direction to effects occurring at µM levels, suggesting that these depend on entirely different mechanisms.1 However, this does not discard the possibility that in both instances ouabain would act on the same receptor. We demonstrate that such is the case by comparing the response of wild and ouabain-resistant MDCK cells on a very special type of cell contact, the tight junction (TJ).

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.

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.