Liora Shoshani

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

SummaryProlonged exposure to 17β-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), ERα and ERβ. 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.

Ouabain modulates epithelial cell tight junction

Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) 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 (JDEX). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, JDEX, 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 β Subunits

Na+,K+-ATPase polarity depends on the interaction between the β subunits of Na+,K+-ATPases located on neighboring cells. In the present work, we use energy transfer methods (FRET), in vivo to demonstrate that these β subunits interact directly at the intercellular space of epithelial cells.

The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 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 β1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic β1-β1 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 β2 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 β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 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 β2 subunit.

Na + /K + -ATPase Drives Most Asymmetric Transports and Modulates the Phenotype of Epithelial Cells

Usually the history of an enzyme is the narrative of the works to isolate and purify it, measure its molecular weight, determine its crystal configuration, measure its activity, and so on, along, say, 2 or 3 years. On the contrary, the history of Na+/K+-ATPase is a tortuous road full of pitfalls, skirmishes with physical chemistry, thermodynamics, and even philosophy. Fortunately it had a happy end, because it was the first known molecule to produce vectorial movement of ions using chemical energy; to cyclically modify its selectivity; to participate in the action potential; to act as a self-adhesion molecule at cell-cell contacts; to act as receptor of the hormone ouabain, whose main physiological role is to modulate cell contacts; and to generate a Na+ gradient that enables co- and counter-transporters to transport net amount of ions, sugars, and amino acids, i.e., to act as secondary pumps. It is sufficient to say that one of its crucial properties, i.e., to be expressed polarizedly at the intercellular membrane of transporting epithelial cells, involves the β-subunit of the pump that happens to be an adhesion molecule which plays a crucial role in that polarization mechanism.

The Polarized Distribution of the Na+,K+-ATPase

The life of a cell depends on the perennial inflow of metabolites and outflow of catabolites, ultimately driven by membrane pumps or by the electrochemical potential gradients that these pumps generate. Metazoans have cells in which pumps have an additional function: they accumulate in a certain domain of the membrane to induce polarity. Surprisingly, the polarized distribution of Na+,K+-ATPase does not arise only from canonical signals or classical mechanisms but also from the peculiar affinities between its own subunits. For example, subunits α and β have an affinity for each other that binds them together right after synthesis, and they then migrate through the endoplasmic reticulum and the Golgi apparatus and are delivered to the plasma membrane. In keeping with this role of subunit affinities, we have shown that the polarized distribution of the whole enzyme at the plasma membrane facing the intercellular space arises from the very specific affinity of one β subunit for another. In addition to being distributed in a polarized manner, Na+,K+-ATPase participates in cell polarization by acting as a receptor for the ouabain hormone, thereby promoting ciliogenesis; obviously, the enzyme can act as a receptor because this is polarized toward the blood side where hormones come from. In this chapter, we review the polarized distribution of Na+,K+-ATPase and suggest that the very existence of higher metazoans depends on this polarized expression of pumps.

Regulation of Tight Junctions’ Functional Integrity

The growing molecular complexity of the tight junction (TJ), its ability to modulate the degree of sealing according to physiological requirements, and the fact that its transepithelial electrical resistance (TER) ranges over several orders of magnitude, indicate that there must be a number of agents modulating its permeability. The interest to find these agents stems from an urgency to make the TJ tighter (e.g., to prevent antigen absorption in autoimmune diseases), or to make it leakier (e.g., to allow the absorption of orally administered pharmaceutical drugs). In the present chapter we discuss three forms of modifying the degree of sealing of the TJ: (1) A peptidic factor extracted from urine that makes the TJ tighter, decreases the cellular content of claudin-2, and prompts the relocalization of claudin-4; (2) An experimentally induced modification of the lipidic composition of the plasma membrane that changes some basic attributes of the TJ; and (3) ouabain, a substance that specifically inhibits Na+,K+-ATPase both as an enzyme and as a ion pump, and induces an inotropic activity in heart muscle fibers. Yet we discuss here a newly found property of ouabain: the triggering of a cascade of phosphorylations that results in the opening of the TJ, as part of an overall cell detachment. Interestingly, at concentrations of ouabain within physiological ranges that do not fully detach the cell, the release of the grip induced by this substance sends specific nuclear addressed molecules (NACos) from the diverse sites of attachment to the nucleus, where they modulate the expression of genes that influence proliferation and differentiation.

Evolution of the Transporting Epithelium Phenotype

Metazoans and transporting epithelia (TE) kept a strict correlation throughout evolution because a cell lodged in an intimate tissue and surrounded by an extracellular space less than a micron thick would quickly perish were it not for the intense and highly selective exchange of substances across TE. The main cellular features of TE are tight junctions and apical/basolateral polarity involving close to a hundred molecular species exquisitely assembled. Even when at the dawn of metazoan, junctions and polarity must have been much simpler, it is hard to imagine how the molecules that are involved might have coincided in the same organism and within a few minutes. The present chapter attempts to solve this conundrum by discussing several clues. 1. Polarity, as well as certain molecules involved in its generation and maintenance, are even present in unicellulars. 2. Molecular species belonging to septate and occluding junctions can be found in unicellulars, albeit fulfilling different roles.3 Primitive metazoan might have had very simple epithelia of a transient nature, that helped to retain nutrients and signal molecules for short periods, then opened to allow the whole mass of cells to be flushed by the environment (“Thrifty sponge”). 4. Early metazoan might have compensated the inefficiency of their primitive epithelia with a large surface-to-volume ratio. 5. Finally, the possibility exists that cells might have proliferated without completely detaching from each other, and preserving the orientation of their mitotic spindle, thereby generating an ample overall polarized epithelium that would create an internal environment even before an internal body of somatic cells would grow inside (“the mare nostrum metazoan”).