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

Abstract

Here we show that ouabain-induced cell growth regulation is intrinsically coupled to changes in the cellular amount of Na/KATPase 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 ouabaininduced 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 ϩ and K ϩ 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-...

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

“…Binding of ouabain to the ␣1 Na/K-ATPase stimulated Src within minutes and increased the expression of ␣1 Na/KATPase in hours (5,20). Indeed, these changes in response to ouabain (100 M) were observed in the control AAC-19 cells (Fig.…”

“…Cell Counting Assay-Cell growth was measured as previously described (20). Cells were seeded at a density of 20,000/ well in 12-well plates in DMEM containing 10% FBS.…”

“…[ 3 H] Ouabain Binding-To measure the surface expression of the endogenous pig Na/K-ATPase, [ 3 H]ouabain binding assay was performed as described (20). Cells were cultured in 12-well plates until confluent and serum-starved overnight.…”

Modulation of Na+-K+-ATPase cell surface abundance through structural determinants on the α1-subunit

“…Additional Na ϩ -K ϩ -ATPase ion-pumping capacity at the cell surface may help preserve intracellular ion homeostasis during I-R, but studies like our initial characterization of the ␣1-L499V mutant itself (44) or the graded knockdown of ␣1 subunit (31) have shown that surface expression does not necessarily correlate with increased ion-pumping function. In fact, other non-ion-pumping functions of Na ϩ -K ϩ -ATPase may be involved, such as survival signaling or preservation of the integrity of intracellular structures (7,8,45). Clearly, further investigation in cells exposed to I-R is needed to clarify the relative contribution of Na ϩ -K ϩ -ATPase ion-pumping and non-ion-pumping functions in the protection afforded by increased cell surface expression.…”

“…-ATPase at the cell surface is important to both ion-pumping and receptor functions, and modulation of cellular Na ϩ -K ϩ -ATPase activity through changes in cell surface expression has been reported in response to major physiological or pathophysiological stimuli. Such stimuli include CTS themselves (32,45), the parathyroid hormone (24), dopamine (4), insulin (3,12,18), hypoxia (14), and hypercapnia (46). Over the past 15 years, investigations using heterologous expression systems have focused on the identification of key structural determinants along the Na ϩ -K ϩ -ATPase ␣1 polypeptide that influence its expression at the cell surface under basal conditions or in response to specific stimuli.…”