Article Figures & Data
Figures
- FIG. 1.
Effect of therapeutic levels of lovastatin on cellular cholesterol and Ras prenylation. BHK cells (a) and HUVECs (c) were cultured in growth medium containing 10% DCS, 10% FCS, or 10% LPDS in the presence of lovastatin for 48 h. BHK cells (b) and HUVECs (d) were cultured in growth medium containing 10% LPDS supplemented with 100 or 240 mg/dl purified LDL-C. Total cellular cholesterol levels were measured. The graphs show means ± standard errors of the means (SEM) for 3 independent experiments. Differences between lovastatin-untreated and lovastatin-treated cells under each culture condition in panels a to d were assessed using one-way analysis-of-variance (ANOVA) tests, and significant differences are indicated (*, P < 0.05). (e) HUVECs were treated with various concentrations of lovastatin in the presence of 10% LPDS for 48 h. Control cells were cultured in standard growth medium containing 10% FCS. Cells were lysed in 1% Triton X-114 and lysates separated into detergent-enriched (Det.) and aqueous (Aq) phases by warming. After separation of the two phases by centrifugation, a 1× volume of the detergent-enriched phase (20 μl) containing hydrophobic proteins and a 2× volume of the aqueous phase (40 μl) containing hydrophilic proteins were blotted with an anti-pan Ras, anti-Rho, or anti-Rap1A/B antibody. A representative blot and mean percentages of the detergent fraction for 3 independent experiments are shown.
- FIG. 2.
Therapeutic levels of lovastatin modulate ERK and Akt activation. BHK cells (a and e) and HUVECs (b and f) were cultured in growth medium containing 10% DCS, 10% FCS, or 10% LPDS in the presence of lovastatin for 48 h. Cell lysates were assayed for ppERK or pAkt (S473) using phospho-specific antibodies and quantitative immunoblotting. The graphs show mean levels of ppERK or pAkt, relative to levels in cells grown in 10% DCS or FCS without lovastatin, ± SEM for 3 independent experiments. Differences between lovastatin-untreated and lovastatin-treated cells under each culture condition were assessed using one-way ANOVA tests, and significant differences are indicated (*, P < 0.05). (c and d) Total cellular cholesterol levels in Fig. 1 were plotted against the corresponding mean ppERK levels shown in panels a and b. The line was fitted by linear regression.
- FIG. 3.
Activation of Ras signaling by lovastatin is a consequence of cholesterol depletion. (a) BHK cells treated with lovastatin in the presence of 10% LPDS for 48 h were incubated with 25 μM water-soluble free cholesterol (+CHOL) or vehicle (CON) at 37°C for 20 min and harvested. Total Ras-GTP levels were measured in an RBD pulldown assay and compared to the level for cells grown in DCS. The graph shows mean fold increases in Ras-GTP levels ± SEM for 3 independent experiments. Differences between lovastatin-treated and control cells in the presence and absence of free cholesterol were assessed using one-way ANOVA tests. Significant differences are indicated (*, P < 0.05). A representative blot is shown. (b) BHK cells were treated with various concentrations of lovastatin in the presence of 10% LPDS for 48 h. Control cells were cultured in standard growth medium containing 10% DCS. H-, N-, and K-Ras-GTP loadings were measured using an RBD pulldown assay. A blot representative of 4 (H- and N-Ras) or 3 (K-Ras) independent experiments is shown.
- FIG. 4.
EGF receptor expression is downregulated by lovastatin treatment. BHK cells were treated with lovastatin in the presence of 10% LPDS for 48 h. Control cells were cultured in standard growth medium containing 10% DCS. Total EGFR and phospho-EGFR (Y1068) levels were measured by quantitative immunoblotting. The graph in panel a shows mean EGFR and pEGFR levels ± SEM relative to levels in cells grown in control medium. An immunoblot representative of 3 independent experiments is also shown. ERK2 levels are used as a loading control. The specific activity of the EGFR (b) was estimated as the normalized ratio of phospho-EGFR to total EGFR using values obtained from panel a. For panels a and b, differences between lovastatin-treated and control cells were assessed using one-way ANOVA tests. Significant differences are indicated (*, P < 0.05; **, P < 0.01). (c) BHK cells were cultured in growth medium containing 10% DCS or 10% LPDS in the presence of lovastatin. Note that these growth conditions replicate those in Fig. 1a, where the presence of DCS maintains normal cell cholesterol levels in the presence of lovastatin. Total EGFR levels were measured by quantitative immunoblotting. The graph shows mean EGFR levels ± SEM relative to levels in cells grown in DCS. Differences between cells cultured in DCS and LPDS at each concentration of lovastatin were assessed using two-tailed t tests. Significant differences are indicated (*, P < 0.05; **, P < 0.01). An immunoblot representative of 3 independent experiments is also shown. ERK2 levels are used as a loading control.
- FIG. 5.
PLD activity is required for Ras and ERK activation in response to cholesterol depletion. BHK cells were treated with lovastatin for 48 h in the presence of 10% LPDS. Control cells were cultured in 10% DCS. For the final 4 h of incubation, n-butanol (BtOH) or t-BtOH was added to the growth medium to give a final concentration of 1% (vol/vol). Ras-GTP levels were measured using an RBD pulldown assay (a) and ppERK levels measured by quantitative immunoblotting (b). A representative blot for each experiment is shown, with total Ras and ERK2 used as loading controls. Growth in n-butanol inhibits phosphatidic acid production by PLD, whereas growth in t-butanol does not (Materials and Methods). The graphs show mean levels ± SEM for 3 independent experiments. Differences between lovastatin-treated and control cells grown in n-BtOH or t-BtOH were assessed using one-way ANOVA tests. Significant differences are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
- FIG. 6.
Cholesterol depletion stimulates phosphatidic acid production at the plasma membrane. (a) BHK cells were treated with lovastatin for 32 h in the presence of 10% LPDS. Control cells were cultured in standard growth medium containing 10% DCS. In each case, cells were then labeled with [3H]palmitate for a further 16 h. Cellular lipids were extracted and resolved by TLC. The graph shows mean phosphatidic acid (PA) level as a percentage of total phospholipids ± SEM for 3 independent experiments. Differences between lovastatin-treated and control cells were assessed using one-way ANOVA tests, and significant differences are indicated (*, P < 0.05). (b) Intact plasma membrane sheets were generated from BHK cells expressing mGFP-NES-Spo20 and cultured for 48 h in 300 nM lovastatin (LOV) in 10% LPDS or in normal medium containing 10% DCS. Plasma membrane sheets were labeled with anti-GFP antibody conjugated to 5-nm gold particles. The graph shows the mean number of gold particles/μm2 (± SEM). Differences between lovastatin-treated and control cells were assessed using two-tailed t tests. Significant differences are indicated (***, P < 0.001). (c) BHK cells coexpressing mGFP-NES-Spo20 and mRFP-NES-Spo20 or mRFP were cultured in normal medium containing 10% DCS or 10% LPDS with 300 nM lovastatin for 48 h. Cells were imaged in a wide-field FLIM microscope. The graph represents the mean fluorescence lifetime of GFP (± SEM). Differences between lovastatin-treated and control cells were assessed using two-tailed t tests. Significant differences are indicated. BHK cells expressing mGFP-NES-Spo20 alone cultured in standard growth medium was used to determine initial mGFP lifetime. In this assay a reduction in mGFP lifetime indicates increased FRET between mGFP and mRFP.
- FIG. 7.
Lovastatin-induced EGFR downregulation is blocked by EGFR inhibition. BHK cells were cultured for 48 h in normal medium containing 10% DCS or 10% LPDS in the presence of lovastatin with or without 0.5 μM AG1478. Cell lysates were assayed for total EGFR (a and b) or ppERK (c and d). The graphs show mean levels of total EGFR or ppERK, relative to levels in AG1478-treated cells grown with lovastatin under each culture condition, ± SEM for 3 independent experiments. Differences between AG1478-untreated and AG1478-treated cells at each lovastatin concentration were assessed using two-tailed t tests. Significant differences are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (e) BHK cells were cultured for 48 h in 10% LPDS plus 300 nM lovastatin or 10% DCS plus 300 nM lovastatin with or without 0.5 μM AG1478. These growth conditions replicate those represented in Fig. 1a, where the presence of DCS maintains normal cell cholesterol levels in the presence of lovastatin. Control cells were cultured in standard growth medium containing 10% DCS. Total Ras-GTP levels were measured. The graphs show means ± SEM of results from 3 independent experiments. Differences between lovastatin-treated and control cells were assessed using one-way ANOVA tests. Significant differences are indicated (**, P < 0.01).
- FIG. 8.
Lovastatin-induced activation of PLD2 and EGFR are independent events. BHK cells stably expressing GFP-mPLD2 WT, GFP-mPLD2 K758R, or empty pEF6 vector were treated with 300 nM lovastatin for 48 h. Control cells were cultured in standard growth medium containing 10% DCS. Ras-GTP (a) ppERK (b) and pAkt (S473) (c) levels were measured by quantitative immunoblotting. The graphs show mean values ± SEM for 3 independent experiments. The effect of ectopic expression of mPLD2 WT or mPLD2 K758R with lovastatin against pEF6 was assessed using one-way ANOVA tests. Significant differences are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (d) BHK cells stably expressing GFP-mPLD2 K758R or empty pEF6 vector were treated for 48 h with lovastatin in 10% LPDS. Cell lysates were assayed for total EGFR by quantitative immunoblotting. The graphs show means ± SEM for 3 independent experiments. No significant difference in EGFR levels between pEF6-transfected and GFP-mPLD2 K758R-expressing cells were detected using two-tailed t tests.
