Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling

Abstract

Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcβ1-3Galβ1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism.

Keywords: Golgi; UGCG; Verotoxin-cholera toxin retrograde transport; glucosylceramide; lactotriaosylceramide.

Fig. 1.

Statins increase GlcCer and Lc3Cer in A431 cells. (A) Total GSL extracts from 10⁶ A431G cells cultured with Crestor™ pill extract (theoretical rosuvastatin concentration 20 µM) or 5 µM lovastatin for 72 h were separated by TLC in solvent C and detected by orcinol spray for carbohydrate. Migration of standards is indicated; GlcCer, lactosylceramide, Gb₃ and Gb₄ (globo-series GSLs, globotriaosyl or tetraosylceramide, respectively), GM3 (monosialoganglioside). Statins substantially increase GlcCer and a ceramide trihexoside. (B) Neutral GSLs from A431S cells grown ± 10 µM lovastatin or U18666A for 48 h were separated in solvent B and detected with orcinol. GSL changes induced by U18666A are distinct from statin effects. (C) The TLC trihexoside region is shown; A431S cells were treated with lovastatin or vehicle control as in (B), then TLC plates were probed with anti-Lc₃Cer, anti-Gg₃, or VT1 B-subunit for Gb₃. Antibody–ligand binding to standard GSLs is shown left. (D) MALDI MS of neutral GSLs of A431S cells treated as in (B). The ceramide trihexoside (Gb₃) C24:1/24:0 fatty acid region is shown, and sodiated peaks for C24:0 (m/z 1158.8) and C24:1 (1156.8) are labeled. On lovastatin treatment, the sodiated C24:0/C24:1 peaks of Lc3Cer are apparent (m/z 1199.8/1197.9, respectively). (E) TLC analysis of cholesterol and phospholipid fractions of A431S cells treated as in (B). Lipids were identified by co-migration with standards. Cholesterol from 2 × 10⁵ cells was run in solvent D and detected with FeCl₃ spray; PE, PC, and SM from 5 × 10⁵ cells run in solvent B were detected by iodine vapor. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 2.

Lovastatin elevates GlcCer in all cell lines tested. (A, upper panel) Total saponified lipids, from cells treated 48 h ± 5–10 µM lovastatin, were separated by TLC in solvent B. GSLs are detected by orcinol spray, migration of standards is shown on left. GlcCer is increased in all cell lines tested. (A, lower panel) TLC immunoblot; replicate plate probed with anti-Lc3Cer shows increased/induced Lc3Cer in ACHN, DU145, HeLa, U87MG, HEK, and PC-3 cells. (B) Long-term, low-dose lovastatin titration in PC-3 cells shows both GlcCer and Lc3Cer (detected with orcinol and anti-Lc3Cer, respectively) are elevated at 500 nM lovastatin dose. (C) Time course treatment with 10 µM lovastatin. GSL changes are apparent at 24 h in PC-3 cells and 48 h in A431S cells. GSLs from 10⁶ cells separated by TLC in solvent (C). (D) Identification of slow-migrating lactosamine-containing species. Total neutral GSL fraction from A431G cells treated 48 h ± 20 µM rosuvastatin was separated by TLC in solvent C and probed with anti-Lc3Cer (MAC-1; terminal GlcNAcβ1-3Gal), anti-nLc4 (FE-A5; terminal Galβ1-4GlcNAcβ1-3) or anti-SSEA1 (Lewis × determinant; Galβ1-4(Fucα1-3)GlcNAcβ1-3). (E) Identification of GM3 in A431G cells. The acidic fraction of cells from panel (D) was detected with orcinol or probed with mAb anti-GM3 (clone DH2). Three major acidic GSLs were detected including GM3, which was increased in statin-treated cells. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 3.

Statin-induced increase in GlcCer results from prenylation-dependent GCS elevation. (A) ACHN cells were cultured with 10 µM lovastatin, 20 µM rosuvastatin or 10 µM atorovastatin for 48 h. GlcCer was detected by TLC/orcinol (upper panel), GCS by western blot (middle panel; 20 µg protein lysate) and GCS enzyme activity was monitored by conversion of NBD-Cer to NBD-GlcCer (bottom panel; 3 h reaction, GlcCer:Cer band intensity ratios are shown). (B) Lc3Cer synthase activity, monitored by conversion of NBD-LacCer to NBD-Lc3Cer, increased in lysates of PC-3 cells treated with low dose (0.5 or 1.0 µM) lovastatin for 4 days, or acute treatment (10 µM, 48 h). (C) ACHN cells were treated with 20 µM rosuvastatin ± 10 µM GGPP for 48 h and analyzed for GCS enzyme activity. Bar graph shows average GlcCer:Cer ratio±range of duplicate samples. (D) Western blot of ACHN cell lysates, treatment as in (C). (E) TLC analysis of GSLs from A431S cells treated with vehicle control or 10 µM lovastatin ± 10 µM GGPP for 48 h (orcinol detection). This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 4.

Effect of statin treatment on sphingolipid metabolite levels. (A) Summary of sphingolipid metabolism pathways. CerS, ceramide synthases 1–5, which vary in fatty acid chain length preference; DES, dhCer desaturase; SMS or SMase, sphingomyelin synthase or sphingomyelinase, respectively; Cerase, ceramidase; GCC; glucocerobrosidase (cytosolic and lysosomal forms). Extracts of ACHN cells treated with 20 µM rosuvastatin for 0, 24 or 48 h were analyzed by quantitative LC–MS-MS for; (B) precursor Sa and dhCer species and (C) total Cer, GlcCer and SM. In (D) the individual fatty acid species for Cer, GlcCer and SM are shown. Quantitative measurement of some species was not available at the time of analysis; all species analyzed are shown. Bar graphs represent average ng/10⁶ cells ± range of duplicate samples divided by two. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 5.

Rab prenylation inhibitor replicates statin effects on GSLs. (A) Scheme for effect of statins or specific inhibitors of farnesyl transferase, geranylgeranyl transferase −1 or geranylgeranyl transferase II on protein prenylation. (B) Effects of specific prenylation inhibitors on GSL and GCS expression were determined. The Rab geranylgeranyl transferase inhibitor, 3-PEHPC, was effective to increase GlcCer (TLC, upper panel) and GCS (western blot, lower panel). ACHN cell treatment was 48 h with 3 mM 3-PEHPC, 15 µM FT1-277, 10 µM GGTI-2133 or 20 µM rosuvastatin. Inhibition of prenylation by 3-PEHPC and FTI-277 are indicated by appearance of the slower migrating, unprenylated forms of Rab6 (25 kD) and the co-chaperone HDJ2 (40 kD), respectively. Inhibition of GGTase I is confirmed using an antibody specific for unprenylated Rap1A (21 kD). Blots were probed with anti-GAPDH to verify equal sample loading and transfer. (C) Neutral GSLs of A431S cells treated with the inhibitor panel as in (A). A dose–response between 0 and 10 µm lovastatin was seen for GlcCer and Lc3Cer elevation and 1 mM 3-PEHPC caused similar increases within this range. (D) Lc3CerS enzyme activity in lysates of A431S cells treated as in (B). Band intensity (arbitrary units) of NBD-Lc3Cer was quantified using Image J; error bars represent the range of duplicate samples divided by two. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 6.

Golgi GCS is relocalized after statin or 3-PEHPC treatment. (A) After permeablization with 50 µg/mL filipin cells were double-labeled with rabbit anti-GCS 1.2 antiserum and mAb GM130 (cis-Golgi) or mAb GS15 (medial Golgi). GCS was localized in punctate Golgi structures partially overlapping with GS15, and proximal but largely distinct from GM130 and filipin-labeled TGN (pseudo-colored red in the bottom panel for clarity). Scale bar = 6 µm. (B) ACHN cells were treated 48 h with vehicle, 10 µM rosuvastatin, 1 mM 3-PEHPC, 2 µM P4 or 30 min with 10 µM BFA, prior to fixation and labeling. After statin or 3-PEHPC treatment, GCS became more widely distributed, the TGN cholesterol pool (but not PM cholesterol) was absent, anti-GM130-labeled dispersed Golgi “mini-stacks”. Scale bar = 10 µm.

Fig. 7.

Rab prenylation inhibitor mimics statins to alter intracellular traffic. (A) ACHN cells were pre-treated 48 h with vehicle, rosuvastatin, 1 or 3 mM 3-PEHPC, 15 µM FTI-277 or 10 µM GGT1-2133, then Alexa488-VT1 B subunit and Cy3-CT B subunit were internalized together at 37°C for 1 h. TGN cholesterol and the Golgi targeting of VT-B and CT-B are apparent in control, GGTI-2133 and FTI-277 treated cells. In statin or 3-PEHPC-treated cells, intracellular cholesterol is dispersed and internalized VT-B and CT-B became more diffuse within the cell and retention at the plasma membrane was increased. Scale bar = 21 µm. (B) Solid lines/square symbols, VT1; dashed lines/triangles, VT2; solid symbols, 1 pg/mL VT; open symbols, 100 pg/mL VT.