Statins and the cholesterol biosynthesis pathway.

<p>Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-mediated formation of mevalonate, which is the rate-limiting step of the cholesterol biosynthesis pathway. Farnesylpyrophosphate (-PP) and its derivative, geranylgeranyl-PP, are lipids that posttranslationally modify immunologically important guanosine triphosphate (GTP)-binding proteins, such as Ras and Rho. This isoprenylation permits the subsequent activation and membrane translocation of these proteins, which is necessary for a number of their cellular functions. Dolichol production is dependent on the presence of farnesyl-PP, and dolichol is a necessary substrate for the <i>N</i>-glycosylation of immunologically important transmembrane glycoproteins such as CD147. Inhibitors of the production of specific intermediates downstream of mevalonate include the farnesyl transferase inhibitor (FTI)-277, the geranylgeranyl transferase inhibitor (GGTI)-298, and tunicamycin, a selective inhibitor of <i>N</i>-glycosylation.</p

Downregulation of <i>de novo</i> synthesized CD147 glycoprotein at the cell surface after statin treatment.

<p>Levels of biotinylated cell surface proteins were compared with cellular total protein levels (in whole cell lysates) by immunoblot. A: biotinylated cell surface proteins; B: whole cell lysates. Note that the lowly glycosylated (LG) form of CD147 was not present on the cell surface and that the expression of the highly glycosylated (HG) form of CD147 was reduced predominantly on the cell surface. Experiments were conducted in duplicate; one representative immunoblot is shown. C: Quantitative analysis of bands shown in (A) and (B). The value of the band intensity of PMA-differentiated cells was set as 1.0, respectively. Black bars: HG CD147 on the cell surface. Grey bars: HG CD147 in whole cell lysate. White bars: LG CD147 in whole cell lysate. Basal, untreated cells; PMA, PMA-differentiated cells; PS, pravastatin; AS, atorvastatin; FS, fluvastatin; TUN, tunicamycin. * p < 0.05, compared with PMA-differentiated cells whose value was set as 1.0.</p

Effects of statins and controls on the glycosylation status of CD147.

<p>Total cell lysates were subjected to immunoblotting to assess the glycosylation status of CD147. The effects of fluvastatin, rescue compounds and inhibitors of crucial steps in the cholesterol biosynthesis pathway are shown. Alpha tubulin served as a loading control for the immunoblots. Experiments were conducted in duplicate; one representative immunoblot is shown (A). B: Quantitative analysis of LG CD147 bands shown in (A). The value of the band intensity of PMA-differentiated cells was set as 1.0. Diamonds and thirteenth column: non-glycosylated core protein (27 kDa) after tunicamycin treatment. Basal, untreated cells; PMA, PMA-differentiated cells; FS, cells treated with fluvastatin (1 μM or 10 μM). Rescue compounds: MEV, mevalonate; SQU, squalene; CHOL, cholesterol. Cholesterol pathway inhibitors: FTI-277; GGTI-298; FPT-1; AP-9, antagonistic peptide 9; TUN, tunicamycin. * on top of columns: p < 0.05, compared with PMA-differentiated cells whose value was set as 1.0. * on horizontal lines: p < 0.05, fluvastatin plus rescue compounds compared with fluvastatin only.</p

Alterations in CD14 expression in PMA-differentiated THP-1 cells induced by statin and control treatment.

<p>The cell surface expression of CD14 in THP-1 cells treated with statins, rescue compounds, and cholesterol pathway inhibitors was analyzed by flow cytometry. THP-1 cells were left untreated or treated with PMA in the presence or absence of statins or controls. The changes in CD14 expression are shown in overlaid histograms. A: PMA-differentiated cells versus untreated cells (basal). B: PMA-differentiated cells versus cells treated with PMA in the presence of atorvastatin (AS), pravastatin (PS), and fluvastatin (FS). C-E: PMA-differentiated cells versus cells treated with FS: FS-treated cells rescued with (C) dolichol (DOL), (D) farnesylpyrophosphate (FPP), or (E) geranylgeranylpyrophosphate (GGPP). F: PMA-differentiated cells versus cells treated with the cholesterol pathway inhibitors FTI-277, GGTI-298 or tunicamycin (TUN). Gray histograms, isotype controls. Experiments were conducted in triplicate; one representative result is shown.</p

Effects of statins and controls on surface versus total CD147 expression.

<p>Cell permeabilization studies were conducted to confirm cellular retention of CD147. Results are shown as overlaid histograms. Light lines: surface-expression of CD147 in unpermeabilized THP-1 cells; bold lines: total cellular expression of CD147 in permeabilized cells. A: PMA-differentiated cells. B: PMA-differentiated cells treated with fluvastatin (FS). C: fluvastatin-treated, PMA-differentiated cells rescued with farnesylpyrophosphate (FPP). D: PMA-differentiated cells treated with tunicamycin (TUN). Gray histograms, isotype controls. Experiments were conducted in triplicate; one representative result is shown.</p

Alterations in CD147 expression on PMA-differentiated THP-1 cells upon statin and control treatments.

<p>The cell surface expression of CD147 on THP-1 cells treated with statins, rescue compounds, and cholesterol pathway inhibitors was analyzed by flow cytometry. THP-1 cells were left untreated or treated with 200 nM PMA for 24 h in the presence or absence of statins or controls. Changes in CD147 expression are shown in overlaid histograms. A: PMA-differentiated cells versus untreated cells (basal). B: PMA-differentiated cells versus cells treated with PMA in the presence of atorvastatin (AS), pravastatin (PS), or fluvastatin (FS). C-D: Rescue experiments: C: PMA-differentiated cells versus cells treated with fluvastatin (FS) and fluvastatin-treated cells rescued with dolichol (DOL). D: PMA-differentiated cells versus cells treated with fluvastatin (FS) and fluvastatin-treated cells rescued with farnesylpyrophosphate (FPP). E: PMA-differentiated cells versus cells treated with fluvastatin (FS) and fluvastatin-treated cells rescued with geranylgeranylpyrophosphate (GGPP). F: PMA-differentiated cells versus cells treated with the cholesterol pathway inhibitors FTI-277, GGTI-298 or tunicamycin (TUN). Gray histograms represent isotype controls. Experiments were conducted in triplicate; one representative result is shown.</p

Alteration of the morphology of PMA-differentiated THP-1 cells by treatment with statins and various controls.

<p>THP-1 cells were (A) left untreated or (B-L) treated with 200 nM PMA for 24 h (B) without statins or in the presence of (C) 10 μM pravastatin (PS); (D) 10 μM atorvastatin (AS); (E) 10 μM fluvastatin (FS); (F) 10 μM fluvastatin plus 100 μM mevalonate (MEV); (G) 10 μM fluvastatin plus 10 μM farnesylpyrophosphate (FPP); (H) 10 μM fluvastatin plus 10 μM geranylgeranylpyrophosphate (GGPP); (I) 10 μM fluvastatin plus 10 μM dolichol (DOL); (J) 10 μg/ml tunicamycin (TUN); (K) 10 μM of the farnesyl transferase inhibitor (FTI)-277; or (L) 10 μM of the geranylgeranyl transferase inhibitor (GGTI)-298. Cellular morphology was assessed by light microscopy at 50x magnification. Numbers represent percentage of THP-1 cells with adherent, amoeboid morphology indicating differentiation (examples are marked by arrows). Experiments were conducted in triplicate; one representative result is shown.</p