Involvement of Na/K-ATPase in hydrogen peroxide-induced activation of the Src/ERK pathway in LLC-PK1 cells

We have shown that Na/K-ATPase interacts with Src. Here, we test the role of this interaction in H₂O₂-induced activation of Src and ERK. We found that exposure of LLC-PK1 cells to H₂O₂ generated by the addition of glucose oxidase into the culture medium activated Src and ERK1/2. It also caused a modest reduction in the number of surface Na/K-ATPases and in ouabain-sensitive Rb⁺ uptake. These effects of H₂O₂ seem similar to those induced by ouabain, a specific ligand of Na/K-ATPase, in LLC-PK1 cells. In accordance, we found that the effects of H₂O₂ on Src and ERK1/2 were inhibited in α1 Na/K-ATPase-knockdown PY-17 cells. Whereas expression of wild-type α1 or the A420P mutant α1 defective in Src regulation rescued the pumping activity in PY-17 cells, only α1, and not the A420P mutant, was able to restore the H₂O₂-induced activation of protein kinases. Consistent with this, disrupting the formation of the Na/K-ATPase/Src complex with pNaKtide attenuated the effects of H₂O₂ on the kinases. Moreover, a direct effect of H₂O₂ on Na/K-ATPase-mediated regulation of Src was demonstrated. Finally, H₂O₂ reduced the expression of E-cadherin through the Na/K-ATPase/Src-mediated signaling pathway. Taken together, the data suggest that the Na/K-ATPase/Src complex may serve as one of the receptor mechanisms for H₂O₂ to regulate Src/ERK protein kinases and consequently the phenotype of renal epithelial cells.     

Identification of a potential receptor that couples ion transport to protein kinase activity

In our previous studies, we have demonstrated that the Src-coupled α1 Na/K-ATPase works as a receptor for cardiotonic steroids, such as ouabain, to regulate cellular protein kinase cascades. Here, we explore further the structural determinants of the interaction between the α1 Na/K-ATPase and Src and demonstrate that the Src-coupled α1 Na/K-ATPase allows the cell to decode the transmembrane transport activity of the Na/K-ATPase to turn on/off protein kinases. The α1 Na/K-ATPase undergoes E1/E2 conformational transition during an ion pumping cycle. The amount of E1 and E2 Na/K-ATPase is regulated by extracellular K(+) and intracellular Na(+). Using purified enzyme preparations we find that the E1 Na/K-ATPase can bind both the Src SH2 and kinase domains simultaneously and keep Src in an inactive state. Conversely, the E1 to E2 transition releases the kinase domain and activates the associated Src. Moreover, we demonstrate that changes in E1/E2 Na/K-ATPase by either Na(+) or K(+) are capable of regulating Src and Src effectors in live cells. Together, the data suggest that the Src-coupled α1 Na/K-ATPase may act as a Na(+)/K(+) receptor, allowing salt to regulate cellular function through Src and Src effectors.     

Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.     

NaKtide, a Na/K-ATPase-derived Peptide Src Inhibitor, Antagonizes Ouabain-activated Signal Transduction in Cultured Cells

We have previously shown that the Na/K-ATPase binds and inhibits Src. Here, we report the molecular mechanism of Na/K-ATPase-mediated Src regulation and the generation of a novel peptide Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades. First, the Na/K-ATPase inhibits Src kinase through the N terminus of the nucleotide-binding domain of the α1 subunit. Second, detailed mapping leads to the identification of a 20-amino acid peptide (NaKtide) that inhibits Src (IC₅₀ = 70 nm) in an ATP concentration-independent manner. Moreover, NaKtide does not directly affect the ERK and protein kinase C family of kinases. It inhibits Lyn with a much lower potency (IC₅₀ = 2.5 μm). Third, highly positively charged leader peptide conjugates including HIV-Tat-NaKtide (pNaKtide) readily enter cultured cells. Finally, the following functional studies of pNaKtide demonstrate that this conjugate can specifically target the Na/K-ATPase-interacting pool of Src and act as a potent ouabain antagonist in cultured cells: 1) pNaKtide, unlike PP2, resides in the membranes. Consistently, it affects the basal Src activity much less than that of PP2. 2) pNaKtide is effective in disrupting the formation of the Na/K-ATPase/Src receptor complex in a dose-dependent manner. Consequently, it blocks ouabain-induced activation of Src, ERK, and hypertrophic growth in cardiac myocytes. 3) Unlike PP2, pNaKtide does not affect IGF-induced ERK activation in cardiac myocytes. Taken together, we suggest that pNaKtide may be used as a novel antagonist of ouabain for probing the physiological and pathological significance of the newly appreciated signaling function of Na/K-ATPase and cardiotonic steroids.The Na/K-ATPase is expressed in most eukaryotic cells and is essential for maintaining the transmembrane ion gradient by pumping Na⁺ out of and K⁺ into cells (1). Structurally, the enzyme consists of two non-covalently linked α and β subunits. Similar to other P-ATPases, the Na/K-ATPase α subunit has 10 transmembrane domains with both the N and C termini located in the cytoplasm (2, 3). Based on the published crystal structures of Na/K-ATPase (4), the α subunit consists of several well-characterized domains. The actuator (A)² domain consists of the N terminus and the second cytosolic domain (CD2) connected to transmembrane helices M2 and M3, and the highly conserved discontinuous phosphorylation (P) domain is close to the plasma membrane, while the nucleotide-binding (N) domain is relatively isolated (2). There is a significant amount of movement of both the A and N domains during the ion-pumping cycle as in the SR Ca²⁺-ATPase (4–6). It appears that the A domain rotates, while the N domain closes during the transport cycle. Interestingly, these domains have also been implicated in interacting with many protein partners, including inositol 1,4,5-trisphosphate receptors, phosphoinositide 3-kinase, phospholipase C-γ (PLC-γ), ankyrin, and cofilin (7–12).Src, a member of the Src family non-receptor kinases, plays an important role in the signal transduction pathways of many extracellular stimuli such as cytokines, growth factors, and stress responses (13) and has been considered as a promising target for therapeutic intervention in certain cancers (14) and bone diseases (15). Several endogenous inhibitors of Src have been documented previously, including the C-terminal Src kinase, CSK-homologous kinase, Wiscott-Aldrich syndrome protein, RACK1, and caveolin (16–19).Previously, we and others (20) have demonstrated that binding of cardiotonic steroids (CTS) such as ouabain to the Na/K-ATPase stimulates multiple protein kinase cascades. Moreover, the knock-out of Src prevents these cascades from being activated (10, 21, 22). More recently, we have observed that the Na/K-ATPase interacts directly with Src via at least two binding motifs. One of these interactions is between the CD2 of the α1 subunit and the Src SH2, and the other involves the third cytosolic domain (CD3) of the α1 subunit and the Src kinase domain. We propose that the formation of the Na/K-ATPase/Src complex serves as a receptor for ouabain to stimulate the aforementioned protein kinase cascades. Specifically, the CD3-Src kinase interaction maintains Src in an inactive form whereas the binding of ouabain to the Na/K-ATPase disrupts this interaction, resulting in the assembly and activation of different pathways including ERK cascades, PLC/PKC pathway and mitochondrial production of reactive oxygen species (23). Thus, the Na/K-ATPase functions as an endogenous negative Src regulator. This proposition is consistent with the fact that the basal Src activity is inversely correlated to the amount of Na/K-ATPase α1 subunit in both cultured cells (24) and in α1 heterozygous mouse tissues (25). Therefore, to better understand how the molecular interactions between the Na/K-ATPase and Src regulate Src activity, we have further mapped the Src-binding domains in the CD3 of α1. These studies led to the identification of a peptide Src inhibitor (pNaKtide) and the demonstration that pNaKtide can act as a novel ouabain antagonist capable of inhibiting ouabain-induced activation of protein kinase cascades and hypertrophic growth in cardiac myocytes.     

SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase

Our previous studies have suggested that the α1 Na/K-ATPase interacts with Src to form a receptor complex. In vitro binding assays indicate an interaction between second cytosolic domain (CD2) of Na/K-ATPase α1 subunit and Src SH2 domain. Since SH2 domain targets Src to specific signaling complexes, we expressed CD2 as a cytosolic protein and studied whether it could act as a Src SH2 ligand in LLC-PK1 cells. Co-immunoprecipitation analyses indicated a direct binding of CD2 to Src, consistent with the in vitro binding data. Functionally, CD2 expression increased basal Src activity, suggesting a Src SH2 ligand-like property of CD2. Consistently, we found that CD2 expression attenuated several signaling pathways where Src plays an important role. For instance, although it increased surface expression of Na/K-ATPase, it decreased ouabain-induced activation of Src and ERK by blocking the formation of Na/K-ATPase/Src complex. Moreover, it also attenuated cell attachment-induced activation of Src/FAK. Consequently, CD2 delayed cell spreading, and inhibited cell proliferation. Furthermore, these effects appear to be Src-specific because CD2 expression had no effect on EGF-induced activation of EGF receptor and ERK. Hence, the new findings indicate the importance of Na/K-ATPase/Src interaction in ouabain-induced signal transduction, and support the proposition that the CD2 peptide may be utilized as a Src SH2 ligand capable of blocking Src-dependent signaling pathways via a different mechanism from a general Src kinase inhibitor.     

Expression of Mutant α1 Na/K-ATPase Defective in Conformational Transition Attenuates Src-mediated Signal Transduction

The α1 Na/K-ATPase possesses both pumping and signaling functions. Using purified enzyme we found that the α1 Na/K-ATPase might interact with and regulate Src activity in a conformation-dependent manner. Here we further explored the importance of the conformational transition capability of α1 Na/K-ATPase in regulation of Src-related signal transduction in cell culture. We first rescued the α1-knockdown cells by wild-type rat α1 or α1 mutants (I279A and F286A) that are known to be defective in conformational transition. Stable cell lines with comparable expression of wild type α1, I279A, and F286A were characterized. As expected, the defects in conformation transition resulted in comparable degree of inhibition of pumping activity in the mutant-rescued cell lines. However, I279A was more effective in inhibiting basal Src activity than either the wild-type or the F286A. Although much higher ouabain concentration was required to stimulate Src in I279A-rescued cells, extracellular K⁺ was comparably effective in regulating Src in both control and I279A cells. In contrast, ouabain and extracellular K⁺ failed to produce detectable changes in Src activity in F286A-rescued cells. Furthermore, expression of either mutant inhibited integrin-induced activation of Src/FAK pathways and slowed cell spreading processes. Finally, the expression of these mutants inhibited cell growth, with I279A being more potent than that of F286A. Taken together, the new findings suggest that the α1 Na/K-ATPase may be a key player in dynamic regulation of cellular Src activity and that the capability of normal conformation transition is essential for both pumping and signaling functions of α1 Na/K-ATPase.     

Regulation of alpha1 Na/K-ATPase expression by cholesterol

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.     

Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation,MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling

Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3 mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α₂ isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present cell culture study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK_1/2 and PI3K/AKT; interaction between EGF receptor, α₁, and α₂ isoforms of Na,K-ATPase, Src, ERK_1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that (i) ammonia increases EGF receptor phosphorylation at EGFR⁸⁴⁵ and EGFR¹⁰⁶⁸; (ii) ammonia-induced ERK_1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK_1/2 phosphorylation; (iv) ammonia stimulates association between the α₁ Na,K-ATPase isoform, Src, EGF receptor, ERK_1/2, AKT and caveolin-1; (v) ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and (vii) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK_1/2, but in response to 3 mM ammonia it does not appear until after 12 h. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.     

Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.     

The Na,K-ATPase receptor complex: its organization and membership

A major difference between the Na,K-ATPase ion pump and other P-type ATPases is its ability to bind cardiotonic steroids such as ouabain. Na,K-ATPase also interacts with many membrane and cytosolic proteins. In addition to their role in Na,K-ATPase regulation, it became apparent that some of the newly identified interactions are capable of organizing the Na,K-ATPase into various signaling complexes. This new function confers a ligand-like effect to cardiotonic steroids on cellular signal transduction. This article reviews these new developments and provides a comparison of Na,K-ATPase-mediated signal transduction with other receptors and ion transporters.     

The other functions of the sodium pump

Na/K-ATPase (the sodium pump) was discovered in the 1950s as the plasma membrane enzyme that carries out the coupled active transports of Na⁺ and K⁺ across the membranes of nearly all eukaryotic cells. It was not until the 1990s when it was shown that besides pumping ions, Na/K-ATPase is also capable of stimulus-induced interactions with neighboring proteins that lead to activations of signal transduction pathways causing cell growth. This article is an attempt to review the progress of the research on these signaling functions of sodium pump during the past 2–3 decades. The covered topics include (a) the controversial digitalis-induced growth activations through the epidermal growth factor receptor and Src kinase in cardiac myocytes and several other cell types; (b) the extensive findings on digitalis-induced growth activations in cardiac myocytes and other cell types through phosphatidylinositol 3-kinases; and (c) a number of interesting but insufficiently studied signaling functions of the sodium pump.     

Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.     

Identification of a pool of non-pumping Na/K-ATPase

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. Here, we present experimental evidence demonstrating that over half of the plasma membrane Na/K-ATPase in LLC-PK1 cells is performing cellular functions other than ion pumping. This "non-pumping" pool of Na/K-ATPase, like the pumping pump, binds ouabain. Depletion of either cholesterol or caveolin-1 moves some of the "non-pumping" Na/K-ATPase into the pumping pool. Graded knock-down of the alpha1 subunit of the Na/K-ATPase eventually results in loss of this "non-pumping" pool while preserving the pumping pool. Our prior studies indicate that a loss of the non-pumping pool is associated with a loss of receptor function as evidenced by the failure of ouabain administration to induce the activation of Src and/or ERK. Therefore, our new findings suggest that a substantial amount of surface-expressed Na/K-ATPase, at least in some types of cells, may function as non-canonical ouabain-binding receptors.     

Ouabain-Induced Alterations of the Apical Junctional Complex Involve α1 and β1 Na,K-ATPase Downregulation and ERK1/2 Activation Independent of Caveolae in Colorectal Cancer Cells

Studies have reported that Na,K-ATPase interacts with E-cadherin to stabilize (AJs) and regulate the expression of claudins, the main proteins present in the tight junction (TJ) in epithelial cells containing caveolae. However, the role of this ATPase in the regulation of the AJ and TJ proteins in colorectal cancer cells as well as the molecular events underlying this event in a caveolae-independent system remain undefined. In the present study, we used ouabain, a classic drug known to inhibit Na,K-ATPase, and Caco-2 cells, which are a well-established human colorectal cancer model that does not exhibit caveolae. We demonstrated that ouabain treatment resulted in a reduction of the β1 Na,K-ATPase protein and cell redistribution of the AJ proteins E-cadherin and β-catenin, as well as the α1 Na,K-ATPase subunit. Furthermore, ouabain increased claudin-3 protein levels, impaired the TJ barrier function and increased cell viability and proliferation during the early stages of treatment. Additionally, the observed ouabain-induced events were dependent on the activation of ERK1/2 signaling; but in contrast to previous studies, this signaling cascade was caveolae-independent. In conclusion, our findings strongly suggest that α1 and β1 Na,K-ATPase downregulation and ERK1/2 activation induced by ouabain are interlinked events that play an important role during cell–cell adhesion loss, which is an important step during the tumor progression of colorectal carcinomas.     

Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular (22)Na(+) transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular (22)Na(+) transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.     

Molecular mechanism of cofilin dephosphorylation by ouabain

We previously reported that phosphorylated cofilin-triosephosphate isomerase (TPI) complex interacts with Na,K-ATPase and enhances the pump activity through the phosphorylation of cofilin via Rho-mediated signaling pathway. In this study, we tested the hypothesis that the dephosphorylation of cofilin may be induced through Na,K-ATPase inhibition by ouabain. The phosphorylation level of cofilin by ouabain which decreases in a time- and dose-dependent manner in various human cell lines, remains unchanged by pretreatment with Src inhibitor, PP2; epidermal growth factor receptor (EGFR) inhibitor, AG1478; Raf-1 kinase (Raf) inhibitor, GW5074; and ERK kinase (MEK) inhibitor, PD98059, and by transfection of Ras dominant negative mutant (RasN17). This suggests that ouabain dephosphorylates cofilin through the Src/EGFR/Ras/Raf/MEK pathway. Ouabain activates Ras/Raf/MEK pathway, but down-regulates Rho kinase (ROCK)/LIM kinase (LIMK)/cofilin pathway, implying that there may be a cross-talk by ouabain between the Ras/Raf/MEK and the ROCK/LIMK/cofilin pathways. Immunofluorescence and flow cytometry suggest that ouabain-induced active form of cofilin may be involved in cytoskeletal reorganization and cell volume regulation. Thus, these findings demonstrate a new molecular mechanism for the dephosphorylation of cofilin through the inhibition of Na,K-ATPase by ouabain.     

Human Breast Tumor Cells Are More Resistant to Cardiac Glycoside Toxicity Than Non-Tumorigenic Breast Cells

Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.     

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.