Regulation of intracellular cholesterol distribution by Na/K-ATPase

Abstract

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. We show here that graded knockdown of cellular Na/K-ATPase alpha1 subunit produces a parallel decrease in both caveolin-1 and cholesterol in light fractions of LLC-PK1 cell lysates. This observation is further substantiated by imaging analyses, showing redistribution of cholesterol from the plasma membrane to intracellular compartments in the knockdown cells. Moreover, this regulation is confirmed in alpha1(+/-) mouse liver. Functionally, the knockdown-induced redistribution appears to affect the cholesterol sensing in the endoplasmic reticulum, because it activates the sterol regulatory element-binding protein pathway and increases expression of hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in the liver. Consistently, we detect a modest increase in hepatic cholesterol as well as a reduction in the plasma cholesterol. Mechanistically, alpha1(+/-) livers show increases in cellular Src and ERK activity and redistribution of caveolin-1. Although activation of Src is not required in Na/K-ATPase-mediated regulation of cholesterol distribution, the interaction between the Na/K-ATPase and caveolin-1 is important for this regulation. Taken together, our new findings demonstrate a novel function of the Na/K-ATPase in control of the plasma membrane cholesterol distribution. Moreover, the data also suggest that the plasma membrane Na/K-ATPase-caveolin-1 interaction may represent an important sensing mechanism by which the cells regulate the sterol regulatory element-binding protein pathway.

FIGURE 1.

Down-regulation of Na/K-ATPase α1 reduces both caveolin-1 and cholesterol in caveolar fractions. A, cell lysates from P-11 and PY-17 cells were subjected to sucrose gradient fractionation as described under “Experimental Procedures.” Caveolin-enriched fractions (4/5) together with fractions 6∼11 were taken for Western blot analysis of Na/K-ATPase α1 and caveolin-1. A representative Western blot of five independent experiments is shown. B, cell lysates from P-11 cells were fractionated as in panel A, and all 12 fractions were assayed for cholesterol. The percentage of the cholesterol amount in each fraction was calculated. C, a representative Western blot of three experiments showing the amount of Na/K-ATPase α1 and caveolin-1 in caveolin-enriched fractions (4/5). Cholesterol content was measured from caveolin-enriched fractions. **, p < 0.01 compared with P-11 control cells, n = 5. D, cholesterol content was measured from total cell lysates, no difference was detected, n = 3.

FIGURE 2.

Effects of alterations in Na/K-ATPase or caveolin-1 on cellular cholesterol distribution. A, B, C, D, and E show filipin staining of free cholesterol from P-11, PY-17, C2–9, AAC-19, and mCBM cells, respectively. Arrows point to the intracellular vesicular filipin signals. Scale bar:20 μm.

FIGURE 3.

Effects of changes in Na/K-ATPase amount on cytosol and membrane cholesterol content. Cytosol and membrane fractions were prepared from cell lysates as described under “Experimental Procedures.” Cholesterol was measured from the membrane fractions (A) and the cytosol fractions (B). The ratio between cytosol and membrane cholesterol is also shown (C). *, p < 0.05 or **, p < 0.01 compared with P-11 control cells, n = 5.

FIGURE 4.

The interaction between the Na/K-ATPase α1 and caveolin-1 is important for proper cholesterol distribution. A, P-11 and PY-17 cells were treated with 1 μm PP2, an Src inhibitor for 2 h. Afterward, cell lysates were fractionated into cytosol and membrane fractions and then subjected to cholesterol measurement. Data are mean ± S.E., n = 4. B, caveolin-1 protein level was assayed in P-11 and C2–9 cells by Western blot. C, cell lysates were processed as in Panel A and measured for cholesterol. The cholesterol ratio between cytosol and membrane was calculated. *, p < 0.05 or **, p < 0.01 compared with P-11 control cells, n = 3.

FIGURE 5.

Overexpression of N terminus of Na/K-ATPase α1 redistributes cellular cholesterol. A, a typical confocal image showing the cellular distribution of NT-YFP (bottom left) and the endogenous caveolin-1 protein in the LLC-PK1 cells (top right) or in the NT-YFP cells (bottom right). Immunostaining of caveolin-1 was performed as described under “Experimental Procedures.” B and C, filipin staining of cellular cholesterol from control P-11- and NT-YFP-expressing LLC-PK1 cells. Scale bar:20 μm. D, cell lysates were processed as described in Fig. 3, and the cholesterol ratio between cytosol and membrane was calculated. **, p < 0.01 compared with P-11 control cells, n = 3.

FIGURE 6.

Effects of the expression of α1D371N mutant on cellular cholesterol distribution. Relative cholesterol content was assayed and calculated in the cytosol and membrane fractions from PY-17 cells and AAC-19 cells, which is wild-type α1-transfected PY-17 cells (A) and the vector-transfected and the pump-null D371N transfected PY-17 cells (B). *, p < 0.05 or **, p < 0.01 compared with control cells, n = 6. C, the representative confocal images of three separate experiments showing (left panel) YFP-D371N mutant signal (middle panel) filipin signal and (right panel) merged image from the pump-null D371N-transfected TCN cells. Arrows indicate the plasma membrane signals. Scale bar:20 μm.

FIGURE 7.

Down-regulation of Na/K-ATPase α1 in mice liver leads to cholesterol redistribution. A, Western blot analysis showed the down-regulation of α1 level in the α1^+/– mice liver (n = 5). B, liver samples from both α1^+/+ and α1^+/– mice were processed as in Fig. 3. Cholesterol from cytosol and membrane fractions was measured, and the ratio was calculated. **, p < 0.01 compared with α1^+/+, n = 6.

FIGURE 8.

Down-regulation of Na/K-ATPase α1 in mouse liver leads to the activation of Src and ERK1/2 and redistribution of caveolin-1. A, Western blot analysis showed the increase in active Src (Src-pY418) and pERK1/2 in α1^+/– mice liver samples. The quantitative data are presented as mean ± S.E. n = 6. B, liver samples from α1^+/+ mice and α1^+/– mice were subjected to sucrose density fractionation as in Fig. 1. Caveolin-enriched fractions (4/5) together with fractions 6∼11 were taken for Western blot analysis of caveolin-1. A representative Western blot from three independent experiments is shown. The percentage of signals from caveolin-enriched fractions against total signals is shown below. *, p < 0.05 compared with α1^+/+.

FIGURE 9.

SREBP2 pathway is activated in α1^+/– mice liver. A–C, samples from both α1^+/+ and α1^+/– mice livers were analyzed by Western blot for the active form of SREBP2, HMG-CoA reductase, and LDL receptor, respectively. A representative Western is shown, and quantitative data are collected from 5 α1^+/+ and 5 α1^+/– mice livers. D, the liver weight/body weight ratio was calculated from four α1^+/– mice and seven α1^+/+ mice livers. *, p < 0.05, **, p < 0.01 compared with α1^+/+.