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/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.

FIGURE 1.

Effects of ouabain on cell growth in different cells. A, LLC-PK1 cells, BT20 cells, and DU145 cells were subcultured in 96-well plates (10,000 cells/well) and serum-starved. After exposure to different concentrations of ouabain for 24 h, the cells were subjected to MTT assay as described under “Experimental Procedures.” The values are mean ± S.E. The quantification data are from three independent experiments. ^*, p < 0.05. B, the above three cell lines were subcultured in 12-well plates (50,000 cells/well) and serum-starved. After ouabain treatment, three wells of control (Con) and ouabain-treated cells were trypsinized and counted at indicated time points. The same experiments were repeated four times. C, LLC-PK1, BT20, and DU145 cells were serum-starved overnight and treated with ouabain for 24 h. Equal amount of cell lysates were assayed for p21^cip using Western blot.

FIGURE 2.

Effects of ouabain on Na/K-ATPase expression in different cells. A, LLC-PK1, BT20, and DU145 cells were treated with different concentrations of ouabain for either 24 or 72 h. Equal protein amount of cell lysates were analyzed for Na/K-ATPase α1 expression. The upper panel shows representative Western blots of 24 h treatment, and the lower panel shows quantitative data from four or five independent experiments presented in the bar graphs. B, LLC-PK1 and BT20 cells were treated with 10 nm ouabain for 24 h, and the cell lysates (50 μg/lane) were assayed for Na/K-ATPase β1 using Western blot. C, LLC-PK1, BT20, and DU145 cells were exposed to different concentrations of ouabain for 72 h and then subjected to [³H]ouabain binding assays as described under “Experimental Procedures.” ^*, p < 0.05; ^**, p < 0.01. Con, control; Oua, ouabain.

FIGURE 3.

Effects of Na/K-ATPase knockdown on ouabain-induced cell growth regulation. The control (P-11) and Na/K-ATPase knockdown cells (A4–11 and PY-17) were treated with different concentrations of ouabain for 24 h and subjected to MTT assays. n = 10–15 measurements. ^*, p < 0.05; ^**, p < 0.01.

FIGURE 4.

Effects of Na/K-ATPase knockdown on cell growth. A, P-11, PY-17, and AAC-19 cells were subcultured in 12-well plates (50,000 cells/well), and three wells of each were trypsinized and counted at the indicated time points. The data were from three independent experiments. B, P-11, PY-17, and AAC-19 cells were lysed in radioimmune precipitation assay buffer and analyzed by Western blot using an anti-p21^cip antibody. A representative Western blot and the quantification data are shown. ^*, p < 0.05 compared with P11 cells. C, cell cycle distribution was measured as described under “Experimental Procedures.” The same experiments were repeated three times.

FIGURE 5.

Effects of ouabain on α1 mRNA levels. LLC-PK1, BT20, and DU145 cells were treated with 10 nm ouabain for 24 h. Total RNA was extracted and RT-PCR was performed to probe the α1 mRNA as described under “Experimental Procedures”. Data were from four independent experiments. oua, ouabain; con, control.

FIGURE 6.

Ouabain increases endocytosis and degradation of the Na/K-ATPase. A, LLC-PK1 cells, BT20 cells, and DU145 cells were treated with 10 nm ouabain (Oua) for 6 h and fixed with cold methanol. Na/K-ATPase α1 was immunostained and visualized using a Leica confocal microscope. The red arrows indicate the Na/K-ATPase α1-positive vesicles. B and C, LLC-PK1 or BT20 cells were serum-starved for 24 h, exposed to 10 μg/ml cycloheximide (CHX) for 1 h, and then treated with 10 nm ouabain for different times as indicated. The cell lysates were subjected to Western blot analyses of Na/K-ATPase α1. Tubulin was used as an internal control. A representative Western blot, and the quantitative data from four experiments are shown. ^*, p < 0.05. Con, control.

FIGURE 7.

Ouabain up-regulates Na/K-ATPase expression and cell growth through activation of the PI3K/Akt/mTOR pathway. A, LLC-PK1 cells were pretreated with 10 nm rapamycin (Rap) or 50 μm LY294002 for 30 min. Ouabain (Oua, 10 nm) was then added into the medium for an additional 24 h. The cell lysates were analyzed for Na/K-ATPase α1 using Western blot. The data were from four separate experiments. ^*, p < 0.05 in comparison with control. B, LLC-PK1 and BT20 cells were incubated with ouabain for 15 min, and the cell lysates were analyzed for phosphorylated Akt (pAkt). The values are means ± S.E. of three experiments. ^*, p < 0.05. C, DU145 cells were serum-starved and treated with ouabain or 10 nm IGF for 15 min. The pAkt was probed by Western blot, and a representative Western blot from three repeats is shown. D, LLC-PK1 cells were pretreated with 10 nm rapamycin for 30 min and then incubated with ouabain for an additional 24 h. The cell growth was then measured using MTT assays. The quantitative data were from three to five independent experiments. ^*, p < 0.05 in comparison with the control (Con).

FIGURE 8.

Involvement of Src and caveolin-1 in ouabain-induced growth regulation. A, SYF cells and SYF-Src cells were treated with different concentrations of ouabain (Oua) for 72 h and measured for α1 expression. A representative Western blot and quantitative data from four separate experiments are shown. B, P-11 and C2-9 cells were treated with different concentrations of ouabain for 72 h and probed for α1 expression. A representative Western blot and quantitative data from four separate experiments are shown. C, C2-9 cells were treated with different concentrations of ouabain for 15 min and probed for pAkt. A representative Western blot of three independent experiments is shown. D, P-11 and C2-9 cells were exposed to different concentrations of ouabain, and cell growth was measured by MTT assays. Fetal bovine serum was used as a positive control. The data are from five separate experiments. ^*, p < 0.05 in comparison with untreated control (Con) cells.