FTY720 stimulates 27-hydroxycholesterol production and confers atheroprotective effects in human primary macrophages

Abstract

Rationale: The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known.

Objective: We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages.

Methods and results: FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann-Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor-induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not.

Conclusions: We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.

Figure 1. FTY720 alters cholesterol homeostasis and improves survival of human macrophage foam cells

A. Macrophages were pre-treated for 24 h ± 1 μmol/L FTY720 and foam cell formation induced by addition of 50 μg/mL acLDL for 24 h or 72 h in the continued presence or absence of FTY720. Cellular free cholesterol and cholesteryl esters were determined from at least 12 independent donors per condition (C = control, FTY = FTY720). B. Cholesterol esterification was measured as [³H]oleic acid incorporation into cholesteryl esters (CE). Cells were treated ± 1 μmol/L FTY720 for 24 h, followed by a 6-h incorporation with [³H]oleic acid in the presence of 50 μg/mL acLDL. Measurements are from three independent donors. C. Determination of ACAT activity from cell lysates. Macrophages were treated for 24 h ± 1 μmol/L FTY720 prior to cell lysis and in vitro ACAT activity measured in the continued presence or absence of 1 μmol/L FTY720. Results are from 6 independent donors. D. Macrophages were treated for 24 h ± 1 μmol/L FTY720 followed by loading with 50 μg/mL acLDL in the presence of the ACAT inhibitor PKF 58-035 (10 μg/mL) for 16 h. Cell viability was then measured from three donors in triplicate. E. Macrophages were treated ± 1μmol/L FTY720 for 24 h and loaded with 50 μg/mL acLDL in the presence of PKF 58-035 for 8 h. Externalized phosphatidylserine was stained with FITC-conjugated Annexin V, and the fraction of positive cells was counted. A minimum of 180 cells from three separate fields were quantified. F. Macrophages were treated for 24 h ± 1 μmol/L FTY720, and then incubated for 48 h with 100 μg/mL acLDL and PKF 58-035. Cellular CHOP levels were quantified by Western blotting from three independent donors.

Figure 2. FTY720 induces re-routing of acLDL derived cholesterol

Macrophages were pre-treated for 24 h ± 1 μmol/L FTY720. Cells were then pulse labeled with 50 μg/mL [³H]cholesterol-labeled acLDL for 4 h, followed by a 3-h chase in the presence of 10 μg/mL apoA-I. Lipids were extracted from cells immediately after the pulse, or from cells and medium after the chase. The amounts of cellular free cholesterol (Chol) (A), cellular cholesteryl esters (CE) (B) and cholesterol effluxed to apoA-I (C) were measured by scintillation counting. A and B show pooled data from 9 donors, C shows the results from individual donors separately. D. Macrophages were treated for 24 h ± 1 μmol/L FTY720 and loaded with 50 μg/mL acLDL for 72 h, followed by cholesterol efflux to 10 μg/mL apoA-I for 72 h ± 1 μmol/L FTY720. Cellular free cholesterol and cholesteryl esters were determined before and after cholesterol efflux from at least 6 donors per experimental condition.

Figure 3. Upregulation of proteins involved in endosomal cholesterol trafficking in FTY720 treated cells

Representative Western blots and quantification of NPC1, NPC2, LAMP1 and ABCA1 levels from macrophages. Cells were treated for 24 h ± 1 μmol/L FTY720. Then, foam cell formation was induced by addition of 50 μg/mL acLDL for 24 h. At this stage, not loaded (-) and acLDL-loaded (acLDL) cells were harvested. A parallel set of acLDL loaded macrophages were allowed to undergo cholesterol efflux to 10 μg/mL apoA-I for 24 h (acLDL→apoA-I) before harvesting for analysis. Bars show the relative quantities of the proteins from at least 3 independent donors.

Figure 4. Late endosomal organelles are affected by FTY720

A. Macrophages were treated ± 1 μmol/L FTY720 for 48 h and 50 μg/mL acLDL for the last 24 h of incubation. Cells were fixed and stained with filipin and anti-LAMP-1 antibodies. B. Electron microscopic images of cells treated ± 1 μmol/L FTY720 for 24 h, followed by a 2-h pulse with 50 μg/mL DiI-acLDL and a 4-h chase. The number of multilamellar bodies was quantified from 44 control cells and 48 FTY720 treated cells. Scale bar, 500 nm. C. Macrophages were treated for 24 h ± 1 μmol/L FTY720 prior to fixing and processing for electron microscopy. The percentage of mitochondria with endosomal contacts (example indicated by arrowheads) was quantified from 21 and 19 randomly chosen visual fields from control and FTY720 treated cells, respectively. Scale bar, 250 nm.

Figure 5. Cellular targeting and effects of BODIPY-FTY720

A. Cholesterol esterification in macrophages was measured by [³H]oleic acid incorporation into cholesteryl esters. The cells were pre-treated with ± 1 μmol/L FTY720 or 1 μM BODIPY-FTY720 for 24 h, followed by a 6-h incubation with [³H]oleic acid in the presence of 50 μg/mL acLDL ± FTY720 or BODIPY-FTY720. Measurements are from three individual donors. B. Macrophages were incubated for 24 h with 1 μmol/L BODIPY-FTY720 ± 5 μmol/L DMS. Lipids from the cell pellet and from 1/8 of the medium were analyzed by TLC. The upper arrow indicates the position of BODIPY-FTY720 and the lower arrow that of BODIPY-FTY720-P. C. Macrophages were labeled with μmol/L BODIPY-FTY720 for 16 h and imaged live by confocal microscopy. A confocal section from the basal part of the cells is shown. Scale bar, 10 μm. D. Macrophages were labeled with 0.5 μmol/L BODIPY-FTY720 for 24 h followed by a 2-h pulse and a 2-h chase with DiI-acLDL. Wide field microscopy images show BODIPY-FTY720 and DiI-acLDL distribution in macrophages. The insets are composite time lapse sequences showing the movement of BODIPY-FTY720 and DiI-acLDL during 10 s (higher magnification of the area indicated). Images were acquired at a rate of 5 Hz, arrowheads indicate structures with high colocalization of BODIPY-FTY720 and DiI-acLDL. Scale bar, 10 μm.

Figure 6. BODIPY-FTY720 trafficking in NPC1-deficient cells

Wild-type and NPC1-deficient (M12) CHO cells were labeled with 1 mg/mL rhodamine-dextran for 24 h followed by incubation with 1 μmol/L BODIPY-cholesterol or 1 μmol/L BODIPY-FTY720 for 24 h and chasing for 0 h or 24 h. Living cells were imaged and BODIPY intensity in dextran-positive organelles was quantified directly after loading (0 h) and following the 24 h chase. Quantification from eight independent measurements and representative fluorescence micrographs are shown. Scale bar, 10 μm.

Figure 7. FTY720 stimulates 27-hydroxycholesterol production and upregulates ABCA1 independently of S1P receptor activation

A. Macrophages were pre-treated ± 1 μmol/L S1P for 24 h, followed by a 6-h incubation with [³H]oleic acid in the presence of 50 μg/mL acLDL ± S1P. Cholesterol esterification was measured by [³H]oleic acid incorporation into cholesteryl esters (CE) from six individual donors (left panel). Cells were treated for 24 h ± 1 μmol/L S1P or FTY720 and ABCA1 expression analyzed by Western blotting. Results are from three individual donors (middle and right panels). B. Macrophages electroporated with control siRNA or S1P₁ siRNA were treated ± 1μmol/L FTY720 for 24 h. Cell lysates were subjected to Western blotting using S1P₁ and ABCA1 antibodies. S1P₁ siRNA reduced S1P₁ protein levels by ∼70% (left panel). ABCA1 levels were quantified from three individuals (middle panel). Cholesterol esterification was measured from a parallel set of siRNA and FTY720 treated macrophages (right panel). C. Quantification of 25- and 27-hydroxycholesterol (HC) content in macrophages treated for 24 h ± 1 μmol/L FTY720. Results are from six individual donors.

Figure 8. Mechanism for FTY720 induced upregulation of ABCA1

A. Macrophages were treated for 24 h ± 1 μmol/L FTY720 together with 10 μmol/L T0901317 or 5 μmol/L UVI3003 as indicated. Representative Western blot and quantification of ABCA1 levels from three donors in duplicate. B. Control siRNA or NPC1 siRNA treated macrophages were treated ± 1 μmol/L FTY720 for 24 h. The cellular levels of NPC1 and ABCA1 were analyzed by Western blotting and ABCA1 was quantified from three individual donors in duplicate. C. Macrophages were treated for 24 h ± 1 μmol/L FTY720. Surface proteins were biotinylated, pulled down with streptavidin agarose, and surface ABCA1 analyzed by Western blotting. Ont-tenth of cell lysate was removed before pull-down of the biotinylated proteins to reflect the total cellular amount of ABCA1. The level of ABCA1 on the surface was 15-20% of total ABCA1 in both control and FTY720 treated cells. Macrophages from a total of five donors were analyzed. D. Proposed model of FTY720 action in macrophage atheroprotection. 27-HC, 27-hydroxycholesterol. The thick arrows indicate the intracellular routing of cholesterol.