Title: Ceramide structural features required to stimulate ABCA1-mediated cholesterol efflux to apolipoprotein A-I
Abstract: Ceramide is a component of the sphingomyelin cycle and a well-established lipid signaling molecule. We recently reported that ceramide specifically increased ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I), a critical process that leads to the formation of cardioprotective HDL. In this report, we characterize the structural features of ceramide required for this effect. C2 dihydroceramide, which contains a fully saturated acyl chain and is commonly used as a negative control for ceramide apoptotic signaling, stimulated a 2- to 5-fold increase in ABCA1-mediated cholesterol efflux to apoA-I over a 0–60 μM concentration range without the cell toxicity apparent with native C2 ceramide. Compared with C2 ceramide, C6 and C8 ceramides with medium-length N-acyl chains showed a similar extent of efflux stimulation (a 2- to 5-fold increase) but at a higher onset concentration than the less hydrophobic C2 ceramide. In contrast, the reduced and methylated ceramide analogs, N,N-dimethyl sphingosine and N,N,N-trimethyl sphingosine, failed to stimulate cholesterol efflux. We found that changes in the native spatial orientation at either of two chiral carbon centers (or both) resulted in an ∼50% decrease compared with native ceramide-stimulated cholesterol efflux. These data show that the overall ceramide shape and the amide bond are critical for the cholesterol efflux effect and suggest that ceramide acts through a protein-mediated pathway to affect ABCA1 activity. Ceramide is a component of the sphingomyelin cycle and a well-established lipid signaling molecule. We recently reported that ceramide specifically increased ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I), a critical process that leads to the formation of cardioprotective HDL. In this report, we characterize the structural features of ceramide required for this effect. C2 dihydroceramide, which contains a fully saturated acyl chain and is commonly used as a negative control for ceramide apoptotic signaling, stimulated a 2- to 5-fold increase in ABCA1-mediated cholesterol efflux to apoA-I over a 0–60 μM concentration range without the cell toxicity apparent with native C2 ceramide. Compared with C2 ceramide, C6 and C8 ceramides with medium-length N-acyl chains showed a similar extent of efflux stimulation (a 2- to 5-fold increase) but at a higher onset concentration than the less hydrophobic C2 ceramide. In contrast, the reduced and methylated ceramide analogs, N,N-dimethyl sphingosine and N,N,N-trimethyl sphingosine, failed to stimulate cholesterol efflux. We found that changes in the native spatial orientation at either of two chiral carbon centers (or both) resulted in an ∼50% decrease compared with native ceramide-stimulated cholesterol efflux. These data show that the overall ceramide shape and the amide bond are critical for the cholesterol efflux effect and suggest that ceramide acts through a protein-mediated pathway to affect ABCA1 activity. Ceramide, a critical intermediate in the sphingomyelin cycle, is well established as a lipid signaling molecule (1Futerman A.H. Hannun Y.A. The complex life of simple sphingolipids..EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (530) Google Scholar, 2Kolesnick R.N. Goni F.M. Alonso A. Compartmentalization of ceramide signaling: physical foundations and biological effects..J. Cell. Physiol. 2000; 184: 285-300Crossref PubMed Scopus (377) Google Scholar, 3Hannun Y.A. Obeid L.M. The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind..J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar, 4Pettus B.J. Chalfant C.E. Hannun Y.A. Ceramide in apoptosis: an overview and current perspectives..Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (668) Google Scholar) involved in regulating apoptosis and cellular stress responses (1Futerman A.H. Hannun Y.A. The complex life of simple sphingolipids..EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (530) Google Scholar, 5Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Programmed cell death induced by ceramide..Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1604) Google Scholar, 6Hannun Y.A. Functions of ceramide in coordinating cellular responses to stress..Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1491) Google Scholar, 7Okazaki T. Bell R.M. Hannun Y.A. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation..J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 8Bielawska A. Linardic C.M. Hannun Y.A. Modulation of cell growth and differentiation by ceramide..FEBS Lett. 1992; 307: 211-214Crossref PubMed Scopus (89) Google Scholar). Many extracellular agents that induce apoptosis cause the accumulation of ceramide, including 1α,25-dihydroxyvitamin D3, tumor necrosis factor-α, Fas ligands, and nerve growth factor (9Hannun Y.A. The sphingomyelin cycle and the second messenger function of ceramide..J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar, 10Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. Role of ceramide as a lipid mediator of 1 α,25-dihydroxyvitamin D3-induced HL-60 cell differentiation..J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar, 11Mathias S. Dressler K.A. Kolesnick R.N. Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha..Proc. Natl. Acad. Sci. USA. 1991; 88: 10009-10013Crossref PubMed Scopus (348) Google Scholar, 12Dressler K.A. Mathias S. Kolesnick R.N. Tumor necrosis factor-alpha activates the sphingomyelin signal transduction pathway in a cell-free system..Science. 1992; 255: 1715-1718Crossref PubMed Scopus (367) Google Scholar). Moreover, exogenous ceramide treatment induces internucleosomal DNA fragmentation, a hallmark of apoptosis onset (5Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Programmed cell death induced by ceramide..Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1604) Google Scholar). Because of this critical role for ceramide in inducing apoptosis, it is perhaps unsurprising that some tumors, particularly multidrug-resistant tumors, exhibit reduced ceramide concentrations and convert ceramide to glucosylsphingolipids that do not activate the apoptotic program (13Radin N.S. Designing anticancer drugs via the Achilles heel: ceramide, allylic ketones, and mitochondria..Bioorg. Med. Chem. 2003; 11: 2123-2142Crossref PubMed Scopus (0) Google Scholar, 14Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. Accumulation of glucosylceramides in multidrug-resistant cancer cells..J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 15Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells..J. Biol. Chem. 1997; 272: 1682-1687Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Recently, ceramide dysregulation was also implicated in Alzheimer's disease as a mediator of nerve degeneration (16Cutler R.G. Kelly J. Storie K. Pedersen W.A. Tammara A. Hatanpaa K. Troncoso J.C. Mattson M.P. Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease..Proc. Natl. Acad. Sci. USA. 2004; 101: 2070-2075Crossref PubMed Scopus (815) Google Scholar). In terms of novel ceramide functions, we recently found that treating certain cultured cell lines with ceramide significantly increased their ability to efflux cellular cholesterol (17Witting S.R. Maiorano J.N. Davidson W.S. Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1..J. Biol. Chem. 2003; 278: 40121-40127Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). This increased cholesterol efflux was not an artifact of ceramide-induced apoptosis or cytotoxicity. Rather, ceramide upregulated cholesterol efflux only in cell lines expressing ABCA1, a membrane-associated ATPase that transfers cellular lipids to lipid-poor apolipoproteins such as apolipoprotein A-I (apoA-I) (18Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. et al.The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease..Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1338) Google Scholar, 19Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. et al.Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1..Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1258) Google Scholar, 20Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. et al.Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency..Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1496) Google Scholar). Ceramide was found to specifically increase ABCA1 at the cell surface, with a corresponding increase in cellular apoA-I binding (17Witting S.R. Maiorano J.N. Davidson W.S. Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1..J. Biol. Chem. 2003; 278: 40121-40127Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). As cardiovascular diseases stemming from atherosclerosis are a leading cause of death in industrialized countries (211997 Heart and Stroke Statistical Update. December 1996. American Heart Association, Dallas, TX.Google Scholar), the ABCA1-apoA-I reaction is especially important as it leads to the formation of cardioprotective HDL. Unfortunately, most existing strategies for upregulating ABCA1 expression, such as cAMP treatment (22Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages..J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) and liver and retinoic acid X receptor agonists (23Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers..Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1146) Google Scholar), have highly pleiotropic effects that may counteract the antiatherogenic benefits of increasing ABCA1. Thus, it is important to investigate the mechanism of the ceramide effect as a potential treatment strategy. There are two general possibilities for how ceramide exerts its myriad signaling effects, all of which are believed to originate from membranes due to its extreme hydrophobicity (2Kolesnick R.N. Goni F.M. Alonso A. Compartmentalization of ceramide signaling: physical foundations and biological effects..J. Cell. Physiol. 2000; 184: 285-300Crossref PubMed Scopus (377) Google Scholar). The first is that ceramide alters cellular signals by physically affecting membrane lipid packing to indirectly affect target proteins. Evidence that ceramide can act physically to affect signaling proteins comes from studies showing that ceramide can perturb phospholipid bilayers to alter protein kinase C activity (24Huang H.W. Goldberg E.M. Zidovetzki R. Ceramides modulate protein kinase C activity and perturb the structure of phosphatidylcholine/phosphatidylserine bilayers..Biophys. J. 1999; 77: 1489-1497Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A second possibility is that ceramide can act through a biological mechanism and bind directly to target proteins associated with the membrane. Ceramide has been shown in vitro and in vivo to activate both phosphatase protein phosphatase 2A (PP2A) [ceramide-activated protein phosphatase (CAPP)] and Kinase Suppressor of Ras [ceramide-activated protein kinase (CAPK)] (25Dobrowsky R.T. Kamibayashi C. Mumby M.C. Hannun Y.A. Ceramide activates heterotrimeric protein phosphatase 2A..J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar, 26Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid..J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 27Zhang Y. Yao B. Delikat S. Bayoumy S. Lin X.H. Basu S. McGinley M. Chan-Hui P.Y. Lichenstein H. Kolesnick R. Kinase suppressor of Ras is ceramide-activated protein kinase..Cell. 1997; 89: 63-72Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 28Chalfant C.E. Szulc Z. Roddy P. Bielawska A. Hannun Y.A. The structural requirements for ceramide activation of serine-threonine protein phosphatases..J. Lipid Res. 2004; 45: 496-506Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 29Wolff R.A. Dobrowsky R.T. Bielawska A. Obeid L.M. Hannun Y.A. Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction..J. Biol. Chem. 1994; 269: 19605-19609Abstract Full Text PDF PubMed Google Scholar). Ceramide stimulation of CAPP or CAPK can then propagate signals through the protein kinase Cα, c-Jun, and Bcl-2 or Ras, Raf, and extracellular signal-regulated kinase (ERK) signaling pathways, respectively (3Hannun Y.A. Obeid L.M. The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind..J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar). To further characterize the actions of ceramide that lead to its effects on ABCA1, we set out to determine the structural features of ceramide that are required to enhance cholesterol efflux. This was accomplished by systematically modulating the chemical structure of ceramide and correlating those changes to the promotion of cholesterol efflux from cultured CHO cells. The data indicate that the ceramide-amide bond and overall molecular shape are critical for the cholesterol efflux effect. CHO cells from the American Type Culture Collection (Manassas, VA) were used for all experiments. CHO cells were maintained in growth medium, Ham's F12 with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptavidin (all cell culture reagents were from Invitrogen, Carlsbad, CA) at 37°C in 5% CO2. [1α,2α(n)-3H]cholesterol was used to radiolabel cells for cholesterol efflux experiments (GE Healthcare, Waukesha, WI). C2, C6, and C8 ceramides (N-acetyl, N-hexanoyl, and N-octanoyl sphingosine) and fatty acid-free BSA were from Calbiochem (San Diego, CA). C2 dihydroceramide (N-acetyl sphinganine) and l-erythro (2R,3S), d-threo (2R,3R), and l-threo (2S,3S) C2 ceramides were from Matreya (Pleasant Gap, PA). N,N-dimethyl sphingosine (DMS) and N,N,N-trimethyl sphingosine (TMS) were from Avanti Polar Lipids (Alabaster, AL). Scintiverse BD Cocktail, toluene, and all other chemicals were of the highest grade from Fisher (Hampton, NH). Liquid scintillation counts were measured with the Perkin-Elmer 1900CA TriCarb Liquid Scintillation Analyzer. CHO cells were grown to confluence on 12-well plates in growth medium and labeled with [3H]cholesterol by incubating with basal medium, Ham's F12, and 0.2% BSA supplemented with [3H]cholesterol for 24 h (1 μCi/ml). Monolayers were then treated with sphingolipid (added as ethanol solution; final concentration, 0.5–60 μM) in basal medium for 12–16 h. After rinsing twice with basal medium, cells were incubated with apoA-I (10–15 μg/ml) in basal medium with sphingolipid (0.5–60 μM) for 4 h. Medium was collected and 0.45 μM was filtered (Millipore, Billerica, MA). Cellular lipids were extracted with isopropanol, which was then evaporated with positive air pressure and dissolved in toluene. Medium and cell-associated [3H]cholesterol were measured by adding aliquots of each sample to scintillation fluid followed by liquid scintillation counting. Fold change efflux (vs. unstimulated cholesterol efflux to apoA-I) was calculated as follows: fold change efflux = (((total media counts)/(total media counts + total cellular counts)) × 100)sphingolipid-treated cells ÷ (((total media counts)/(total media counts + total cellular counts)) × 100)untreated cells efflux to apoA-I. Human apoA-I was purified to homogeneity from plasma HDL (1.21 < density > 1.062 g/ml) as reported previously (30Davidson W.S. Hilliard G.M. The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass spectrometry study..J. Biol. Chem. 2003; 278: 27199-27207Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 31Silva R.A. Hilliard G.M. Li L. Segrest J.P. Davidson W.S. A mass spectrometric determination of the conformation of dimeric apolipoprotein A-I in discoidal high density lipoproteins..Biochemistry. 2005; 44: 8600-8607Crossref PubMed Scopus (98) Google Scholar). ABCA1 present at the cell surface was measured using the biotin labeling-streptavidin capture method with slight modifications (17Witting S.R. Maiorano J.N. Davidson W.S. Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1..J. Biol. Chem. 2003; 278: 40121-40127Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 32Feng B. Tabas I. ABCA1-mediated cholesterol efflux is defective in free cholesterol-loaded macrophages. Mechanism involves enhanced ABCA1 degradation in a process requiring full NPC1 activity..J. Biol. Chem. 2002; 277: 43271-43280Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Briefly, CHO cells were grown to confluence in T75 flasks and treated for 12–16 h with sphingolipid in basal medium (final sphingolipid concentrations, 5 μM for DMS and TMS and 20 μM for ceramide and dihydroceramide; added as ethanol solution). Cells were chilled on ice in a cold room to 4°C and then washed five times with ice-cold PBS (1.06 mM potassium phosphate monobasic, 2.97 mM sodium phosphate dibasic, and 155.17 mM sodium chloride, pH 7.4; Invitrogen). Surface proteins were biotinylated through two 20 min incubations with 0.5 mg/ml sulfo-NHS-SS-biotin (Pierce) in PBS on a platform orbital rotator at 4°C. Any remaining biotin reagent was quenched by two 5 min incubations with 50 mM Tris in PBS, pH 7.4, again on a platform orbital rotator at 4°C. Cells were then washed once with ice-cold PBS, scraped into 1 ml of PBS, and pelleted by centrifugation. Pellets were homogenized in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μM leupeptin) and lysed during 30 min of incubation on ice. Cellular debris was cleared by centrifugation at 4°C, and supernatant was transferred to a fresh Eppendorf tube. Protein concentration in the supernatants was determined using the Markwell-Lowry assay (33Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples..Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5308) Google Scholar). Equal protein amounts (75–300 μg of protein) were added to 85 μl of Ultralink Plus immobilized streptavidin gel (Pierce) in Handee Spin Cups (Pierce) and incubated overnight with gentle shaking at 4°C. Eluant was removed from streptavidin gel by centrifugation at room temperature [eluant contains nonbiotinylated (i.e., intracellular) proteins]. The gel was washed five times with 300 μl of lysis buffer that was subsequently removed by centrifugation. Surface proteins were collected by adding 2-mercaptoethanol-enriched loading dye (30–35 μl; 60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 0.1% bromophenol blue, and 350 mM 2-mercaptoethanol), incubating for 30 min in a 37°C water bath, followed by centrifugation. Proteins were separated by SDS-PAGE [20–30 μl loaded, 4–15% gel (Bio-Rad); 200 V, 60 min], transferred to a polyvinylidene difluoride (PVDF) membrane (35–50 V, 120 min), and visualized on an immunoblot as described below. The PVDF membrane was blocked for 1 h in blocking buffer (20 mM Tris, pH 7.6, 137 mM NaCl, 1% Tween-20, and 10% dried milk). Blocked blots were incubated for 2 h with 1:500 Novus rabbit ABCA1 antiserum at room temperature, washed, incubated with Pharmacia HRP-conjugated anti-rabbit secondary antibody for 1 h at room temperature, and visualized using Pierce Super Signal reagents. Sample protein concentration was determined using the Markwell modification of the Lowry protein assay (33Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples..Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5308) Google Scholar). We chose CHO cells as the model system for this work because these cells express significant basal levels of ABCA1 without the need to artificially stimulate expression with cAMP analogs or nuclear receptor agonists. We first set out to compare efflux stimulated by C2 ceramide and C2 dihydroceramide, in which the double bond between carbons 4 and 5 of ceramide is fully saturated (Fig. 1 ). C2 dihydroceramide and C2 ceramide have similar cellular uptake properties, yet dihydroceramide does not exhibit the apoptotic or cytotoxic effects of ceramide and is often used as a negative control for ceramide apoptosis experiments (5Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Programmed cell death induced by ceramide..Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1604) Google Scholar, 27Zhang Y. Yao B. Delikat S. Bayoumy S. Lin X.H. Basu S. McGinley M. Chan-Hui P.Y. Lichenstein H. Kolesnick R. Kinase suppressor of Ras is ceramide-activated protein kinase..Cell. 1997; 89: 63-72Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 34Bielawska A. Crane H.M. Liotta D. Obeid L.M. Hannun Y.A. Selectivity of ceramide-mediated biology. Lack of activity of erythro-dihydroceramide..J. Biol. Chem. 1993; 268: 26226-26232Abstract Full Text PDF PubMed Google Scholar, 35Simon Jr., C.G. Gear A.R. Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation..Biochemistry. 1998; 37: 2059-2069Crossref PubMed Scopus (55) Google Scholar). As shown in Fig. 2 , C2 dihydroceramide stimulated cholesterol efflux to lipid-free apoA-I to the same extent as C2 ceramide at concentrations up to ∼20 μM. At the peak effective concentration for both compounds, cholesterol efflux to apoA-I increased 4-fold compared with that of untreated cells. Interestingly, C2 dihydroceramide continued to increase cholesterol efflux at higher concentrations, whereas for C2 ceramide the effect rapidly decreased. Further experiments revealed that this ceramide-mediated decline was a result of cell toxicity (i.e., many cells had detached from the cell wells) (data not shown).Fig. 2.Dose-response cholesterol efflux experiment for C2 ceramide and C2 dihydroceramide. CHO monolayers were labeled with [3H]cholesterol and then treated with either C2 ceramide (closed circles) or C2 dihydroceramide (open circles) over a 0–60 μM range for 13 h. After treatment, the cells were washed and incubated with medium containing lipid-free human apolipoprotein A-I (apoA-I; 15 μg/ml) for 4 h, and radioactivity was counted in both the efflux medium and the cells. Efflux (minus background efflux to BSA) is presented as fold change compared with efflux to lipid-free apoA-I (dashed line). Each point is the mean of three replicates ± SD. FC, free cholesterol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to varying the saturation of the ceramide double bond, we also manipulated the length of the N-acyl chain (Fig. 1). C2 ceramide is a water-soluble ceramide analog attributable to the near absence of an N-acyl chain, and its biological relevance has been debated versus longer chain ceramides that more closely resemble naturally occurring ceramides (25Dobrowsky R.T. Kamibayashi C. Mumby M.C. Hannun Y.A. Ceramide activates heterotrimeric protein phosphatase 2A..J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar, 34Bielawska A. Crane H.M. Liotta D. Obeid L.M. Hannun Y.A. Selectivity of ceramide-mediated biology. Lack of activity of erythro-dihydroceramide..J. Biol. Chem. 1993; 268: 26226-26232Abstract Full Text PDF PubMed Google Scholar, 36Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. Role for ceramide in cell cycle arrest..J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 37Shabbits J.A. Mayer L.D. Intracellular delivery of ceramide lipids via liposomes enhances apoptosis in vitro..Biochim. Biophys. Acta. 2003; 1612: 98-106Crossref PubMed Scopus (89) Google Scholar). Figure 3 shows that the C6 and C8 medium N-acyl chain lengths also are capable of stimulating cholesterol efflux. In contrast to C2 ceramide, which reached its peak effect at ∼20 μM (Fig. 2), C6 ceramide reached a peak at 30 μM and C8 ceramide reached a peak at ∼40 μM. Also, the C2 treatment showed a more gradual increase in cholesterol efflux with increased concentration. The increased N-acyl chain length has been demonstrated to affect cellular uptake in breast cancer cell lines, with 3-fold more C6 ceramide appearing in cells than C16 ceramide (37Shabbits J.A. Mayer L.D. Intracellular delivery of ceramide lipids via liposomes enhances apoptosis in vitro..Biochim. Biophys. Acta. 2003; 1612: 98-106Crossref PubMed Scopus (89) Google Scholar). Such differences in uptake between the C6-C8 ceramides and the C2 ceramide could account for the higher effective concentration for the longer chain ceramides. We also varied the structure of ceramide at the nitrogen functionality using methylated sphingosines, which closely resemble the sphingolipid sphingosine (Fig. 1). CHO cell monolayers were treated with DMS and TMS over the same concentration range as for the C2 ceramide treatment (Fig. 4A ). We found no appreciable increase in cholesterol efflux over that stimulated by lipid-free apoA-I, and at DMS and TMS concentrations of 20–35 μM, efflux had actually decreased to or below zero. This could be attributable to cytotoxic effects of the methylated sphingosines, as both DMS and TMS have been shown to inhibit cultured cell growth at 3–5 μM and the fluorescent TMS analog has been shown to induce significant amounts of apoptosis at 5 μM (38Endo K. Igarashi Y. Nisar M. Zhou Q.H. Hakomori S. Cell membrane signaling as target in cancer therapy: inhibitory effect of N,N-dimethyl and N,N,N-trimethyl sphingosine derivatives on in vitro and in vivo growth of human tumor cells in nude mice..Cancer Res. 1991; 51: 1613-1618PubMed Google Scholar, 39Dagan A. Wang C. Fibach E. Gatt S. Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death..Biochim. Biophys. Acta. 2003; 1633: 161-169Crossref PubMed Scopus (53) Google Scholar, 40Courage C. Budworth J. Gescher A. Comparison of ability of protein kinase C inhibitors to arrest cell growth and to alter cellular protein kinase C localisation..Br. J. Cancer. 1995; 71: 697-704Crossref PubMed Scopus (72) Google Scholar). Therefore, we performed another set of cholesterol efflux experiments at much lower concentrations of DMS and TMS (0–10 μM range). In this concentration regime below cytotoxic levels, both DMS and TMS failed to mediate an increase in cholesterol efflux and, in fact, inhibited efflux to apoA-I (Fig. 4B). Our previous studies demonstrated that ceramide-stimulated cholesterol efflux was accompanied by an increase in cell surface ABCA1 and a corresponding increase in cell surface-bound apoA-I (17Witting S.R. Maiorano J.N. Davidson W.S. Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1..J. Biol. Chem. 2003; 278: 40121-40127Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Consistent with the cholesterol efflux data, DMS or TMS treatment failed to stimulate the presence of ABCA1 at the cell surface like C2 ceramide (Fig. 4C). It is well known from organic chemistry that stereoisomers exhibit similar physical properties, such as solubility and molecular packing. For example, enantiomers of lipids have been shown to have identical TLC behavior (41Johnson J.E. Zimmerman M.L. Daleke D.L. Newton A.C. Lipid structure and not membrane structure is the major determinant in the regulation of protein kinase C by phosphatidylserine..Biochemistry. 1998; 37: 12020-12025Crossref PubMed Scopus (40) Google Scholar), as have enantiomers of the ceramide mimic 2-amino-1-phenyl-1-propanol (42Bielawska A. Linardic C.M. Hannun Y.A. Ceramide-mediated biology. Determination of structural and stereospecific requirements through the use of N-acyl-phenylaminoalcohol analogs..J. Biol. Chem. 1992; 267: 18493-18497Abstract Full Text PDF PubMed Google Scholar). Despite sharing these purely physical properties, enantiomers differ in spatial organization and have dissimilar shape-dependent properties, such as interaction with proteins. To determine whether ceramide stimulates cholesterol efflux by physic