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Published ahead of print on January 10, 2008, doi:10.1165/rcmb.2007-0274OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 639-646, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0274OC

Apoptotic Sphingolipid Signaling by Ceramides in Lung Endothelial Cells

Terry R. Medler*,1, Daniela N. Petrusca*,2, Patty J. Lee3, Walter C. Hubbard4, Evgeny V. Berdyshev5, Jarrett Skirball1, Krzysztof Kamocki2, Edward Schuchman6, Rubin M. Tuder1,7 and Irina Petrache1,2,7

1 Division of Pulmonary and Critical Care Medicine, 4 Division of Clinical Pharmacology, and 7 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; 2 Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Indiana University, Indianapolis, Indiana; 3 Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, Connecticut; 5 Division of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois; and 6 Division of Molecular Genetics, Mount Sinai School of Medicine, New York, New York

Correspondence and requests for reprints should be addressed to Irina Petrache, M.D., Indiana University, Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Van Nuys Medical Science Building, 635 Barnhill Drive, MS224, Indianapolis, IN 46202-5120. E-mail: ipetrach{at}iupui.edu


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate–chain length (C8:0) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-{alpha}–generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C8:0 ceramide– and TNF-{alpha} (plus cycloheximide)–induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C16:0 ceramide in response to C8:0 and TNF-{alpha} treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-{alpha}–stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.

Key Words: apoptosis • lung • cytokines • signaling • sphingolipids



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Prevention of lung microvascular endothelial apoptosis modulated by serine palmitoyl transferase might evolve as a therapeutic strategy in emphysema.

 
Excessive lung endothelial cell apoptosis may account for the pathogenesis of nonmalignant lung disease, including emphysema (a chronic obstructive pulmonary disease) and several types of acute lung injury (1–4). The molecular mechanisms leading to pulmonary endothelial cell apoptosis include the up-regulation of ceramides, a class of signaling sphingolipids comprising several species differing by fatty acid chain length and saturation (1, 2, 5). Although several downstream mediators of ceramide-induced apoptosis have been identified, little is known regarding the upstream events leading to ceramide production or the specific involvement of individual ceramide species in mediating apoptosis. Ceramides may be generated via two pathways: the de novo pathway, involving upstream activation of the serine palmitoyl transferase (SPT), and the sphingomyelinase pathway, through acid (ASMase) or neutral sphingomyelinase activation. Alternatively, intracellular ceramide levels may also increase by blocking its metabolic clearance. It has been suggested that individual ceramide species could play distinct biological roles (6). Moreover, the specific ceramide species induced by intermediate-chain ceramides or by TNF-{alpha} remain undefined.

Using pharmacological inhibitors in a model of murine emphysema, we have shown that ceramide up-regulation via the de novo pathway was critical for lung cell apoptosis in vivo and that ceramide activation occurred upstream of caspase-3 activation (1). Interestingly, the ASMase (specifically its soluble isoform), but not the neutral sphingomyelinase, was also activated in this model of emphysema and may have accounted for the increased production of endogenous paracellular ceramides in response to exogenous ceramide (1).

These results have raised several questions. First, does the uptake of bioactive (extracellular) ceramide trigger new synthesis of endogenous ceramides to activate caspases in primary lung endothelial cells, and if so, by which pathway? Second, is there a common pathway of ceramide synthesis required for pro-apoptotic signaling in these cells? To address these questions, we analyzed endogenous ceramide species generated in response to extracellular ceramide or TNF-{alpha}, a relevant trigger of endothelial apoptosis. Previously, investigators used exogenous ceramide to mimic the action of the intracellular signaling ceramide. Furthermore, due to limited solubility, mostly very-short chain (C2-C6) ceramides have been studied in cell culture systems. The premise of our work was that bioactive paracellular pools of ceramides may initiate distinct intracellular signaling events when compared with the intracellular-generated ceramides. We approached these experimental questions using small inhibitory RNA strategies coupled with mass spectrometric measurements of ceramide species, and employing longer-chain C8:0 ceramides, which may be more relevant to cellular responses to naturally occurring ceramides. Our results, some of which were previously presented in abstract form (7), indicate that the de novo serine palmitoyl transferase (SPT)-activated pathway of sphingolipid synthesis is necessary for pro-apoptotic intracellular ceramide generation in primary mouse lung endothelial cells in response to both extracellular ceramide and TNF-{alpha}. We then compared the pattern and kinetics of intracellular ceramide species generated by the two stimuli using mass spectrometry.


   MATERIALS AND METHODS
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Chemicals and Reagents
N-Octanoyl-D-erythro-Sphingosine (C8, as well as standard ceramides including the internal standard C17 ceramide utilized for mass spectrometry), 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled C12 ceramide, and dihydroceramides standards were from Avanti Polar Lipids (Alabaster, AL). Fumonisin B1 (FB1; Cayman, Ann Arbor, MI) and myriocin (Biomol Research Laboratories, Plymouth Meeting, PA) were used to inhibit de novo ceramide synthesis. The following primary antibodies were used: active caspase-3/7 (Cell Signaling Technology, Beverly, MA, and Abcam, Cambridge, MA), caspase-8 (IC12, Cell Signaling), ASMase (Santa Cruz Biotechnologies, Santa Cruz, CA and from E.S.), SPT (Abgent, San Diego, CA), actin (Calbiochem, La Jolla, CA), vinculin (Calbiochem), and GAPDH (Abcam). Human recombinant TNF-{alpha} and all other reagents were from Sigma-Aldrich (St. Louis, MO), unless otherwise specified.

Cell Culture Experiments
Primary mouse lung microvascular endothelial cells were obtained as described (8, 9) and experiments were performed up to passage 18. Cells were maintained in complete culture medium consisting of Dulbecco's modified Eagle's medium (Gibco, Invitrogen Corporation, Carlsbad, CA), 20% FBS, and penicillin/streptomycin (100 U/ml, 0.1 mg/ml; Sigma) at 37°C in 5% CO2 and 95% air. Experiments were performed at 80 to 100% confluence. Ceramides were first dissolved in ethanol and then diluted in 1% FBS–containing cell culture medium to a final ethanol concentration less than 0.1%. All controls were included this vehicle. Cells were treated with either C8:0 ceramide at a final concentration of 10 µM, or TNF-{alpha} (20 ng/ml) in the presence of cycloheximide (1 µg/ml; Sigma). As with human primary lung endothelial cells (10), the pro-apoptotic effects of TNF-{alpha} in mouse lung endothelial were evident only with the addition of cycloheximide, which we added in all experiments involving TNF-{alpha}. Culture media was changed to serum-free media for 1 hour before treatment. After the addition of C8:0 ceramide or TNF-{alpha}, or vehicle, the serum content was increased to 1% for 24 hours and then cells were harvested and lysed in cell lysis buffer (containing HEPES [10 mM], EDTA [2 mM], CHAPS/NP40 [0.1%], DTT [5 mM], PMSF [1 mM], pepstatin A [10 µg/ml], leupeptin [20 µg/ml], and aprotinin [10 µg/ml]). Similarly, NBD-labeled C12:0 ceramide was added for the indicated period of time to cultured endothelial monolayers grown on coverslips. Cells were then washed with PBS, labeled with DAPI, and then fixed in 3% formalin before visualization by fluorescence microscopy.

siRNA Constructs
The siRNA sequences targeting mouse SPT subunit 1 and acid sphingomyelinase were designed using mRNA sequences from Gen-Bank (gi:29244576 and gi:6755581, respectively). Sequences were then BLASTed against the mouse genome to eliminate those with homology to other genes. Scramble sequences were designed and BLASTed to eliminate sequences that were homologous to other mouse genes. For each target, two sequences were chosen to determine the effectiveness of silencing. The chosen sequences are as follows. SPTLC1: 5'-AATACTTTGGATAATCAGACT-3'; ASMase: 5'-AAGTTGATCAAGAGCCAAAAG -3'; scrambled siRNA: 5'-AATTTTACGGTTAAACGAACTCCTGTCTC-3'. Sense and antisense oligonucleotides were obtained from the Johns Hopkins University Genetic Resources CORE Facility. For construction of the siRNA, the Silencer siRNA Construction Kit by Ambion (Austin, TX) was used following the manufacturer's instructions. Endothelial cells (at 30–50% confluence) were transfected using siPortamine (Ambion) as a vehicle for the siRNA duplexes. For determination of transfection efficiency, cells were visualized using fluorescence microscopy after SiGlo (Dharmacon, Lafayette, CO) transfection under conditions similar to those of the targeted siRNAs. Cells were serum-deprived for 4 hours before transfection and remained in serum-free media for an additional 4 hours, followed by addition of full serum media of an equal volume (10% FBS final concentration). Cells were then incubated until optimum silencing was obtained (6 d for SPTLC1 and 3 d for ASM) before apoptosis experiments were conducted.

Lipid Extraction and Ceramide Species Measurement by Tandem Mass Spectroscopy
To determine which ceramide species were present in cultured endothelial cells, we used a modification of the combined liquid chromatography-tandem mass spectrometric (LC-MS/MS) technique by Sullards and Merrill (11). Cellular or lung tissue lipids were extracted and lipid content was assessed by measurements of total lipid phosphorus (Pi) measurements (1). After lipid extraction, ceramides were eluted as previously described (1). The following individual molecular species of ceramides were monitored: 14:0, 16:0, 18:0, 18:1, 20:0, 24:0, and 24:1-ceramides, using C17:0 ceramide as internal standard.

Enzymatic Activity Determination
The ASMase activity was measured at pH 5.0 in the absence of added cations as described (1) using the Amplex E Red Sphingomyelinase Assay Kit (Molecular Probes, Eugene, OR) following the manufacturer's instructions. The rate of labeled product formation over 60 minutes was expressed as the sample's OD rate from which the blank's OD rate was subtracted. Hydrogen peroxide and sphingomyelinase were used as positive controls. The SPT activity was measured by cell pulse-labeling with D3-serine and quantitation of dihydrosphingosine and D2-analogs of dihydrosphingosine by liquid chromatography tandem mass spectrometry as described previously (12). The approach is based on the incorporation of D3-serine into sphingoid bases during sphingolipid de novo biosynthesis and detection of M+2 mass shift in the portion of newly formed dihydrosphingosine (we have determined that one deuterium is exchanged with protium during molecule ionization). Briefly, after lipid extraction from cells spiked with C17-sphingosine sphingoid bases were separated using Discovery C18 column (2.1 x 50 mm, 5 µm particle size; Supelco, Bellefonte, PA) employing a gradient from methanol/water/formic acid (61/38/1) containing 5 mM ammonium formate to methanol/acetonitrile/formic acid (39/60/1) containing 5 mM ammonium formate at flow rate of 0.5 ml/minute. The sphingolipids were ionized via positive electrospray ionization with detection via multiple reaction monitoring. Standard curves for each sphingoid base were constructed by adding increasing concentrations of the individual analytes to approximately 50 pmol C17-sphingosine employed as the internal standard.

Apoptosis Assays
Executioner caspase (Caspase-3 and/or -7) activity was measured with ApoONE Homogeneous Caspase-3/7 assay kit (Promega, Madison, WI) as described (1). Human recombinant caspase-3 (Calbiochem) was used as a positive control. Apoptotic nucleosomal release was measured with a nucleosomal ELISA kit (Oncogene; Calbiochem) (1). Apopercentage kit (Biocolor Ltd, Accurate Chemical and Scientific Corporation, Westbury, NY) was used following the manufacturer's instructions. After labeling, the cells were visualized by phase-contrast microscopy. Cell count was performed in triplicate and normalized by DAPI-counterstained nuclei.

Western Blotting
Total endothelial cell lysates were loaded in equal protein amounts (10 µg, unless otherwise noted) determined by BCA (Pierce, Rockford, IL). Proteins were resolved by SDS-PAGE (Novex; Invitrogen), followed by immunoblotting as previously described (1). The chemiluminescent signals were quantified by densitometry (ImageQuant; Amersham, Piscataway, NJ) and normalized by housekeeping proteins (actin, GAPDH, or vinculin).

Statistical Analysis
Mean values between two groups were compared using Student's t test. For three or more groups, comparisons were made by ANOVA, followed by post hoc t test. Statistical significance was set at P < 0.05.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Kinetics of Intracellular Ceramide Production and Caspase-3 Activation in Lung Endothelial Cells Treated with Exogenous Ceramides and TNF-{alpha}
Because the longer-chain ceramides' poor solubility limits the exogenous delivery of ceramides, most investigators use short-chain ceramides. However, the effects of very short–chain ceramides, such as 2-carbon length fatty acid chain (C2:0), may be nonphysiological and/or nonspecific. We therefore chose ceramides of 8 to 12 carbon-length fatty acid chains, which more closely resemble endogenous ceramides while still being deliverable and permeating endothelial cells, as shown here and in our previous studies (1). We and others have shown that administration of short-chain ceramides triggers apoptosis not only by intracellular exchange of a short-chain fatty acid with long-chain fatty acids, but also by synthesis of endogenous ceramides via cell-specific pathways (1, 13). To investigate which pathway of ceramide synthesis mediates the pro-apoptotic effects induced by extracellular intermediate-chain ceramides in lung endothelium, we first examined the time-course of cellular uptake and apoptosis triggered by C8:0 ceramide. These responses were compared with that induced by the TNF-{alpha}, a cytokine well known to induce endogenous pro-apoptotic ceramide signaling. Treatment of primary mouse lung endothelial cells with exogenous C8:0 ceramide induced a rapid elevation in total endogenous ceramides (C14:0 and higher), as early as 2 hours and remaining sustained for the first 24 hours of treatment (Figure 1a). Treatment with TNF-{alpha} (20 ng/ml) plus cycloheximide (known to cause apoptosis in these cells) elevated endogenous ceramide species between 6 h and 24 hours of treatment (Figure 1a). This up-regulation of endogenous ceramides preceded or was concurrent with the activation of caspase-3 in C8:0 ceramide- (Figure 1b) and TNF-{alpha}–treated cells (Figure 1c), respectively. Microscopically, both C8:0 ceramide and TNF-{alpha} treatments induced endothelial cells apoptosis at 16 hours (see Figure E1a in the online supplement). Similar results were obtained with fluorescently labeled C12:0 ceramide, which was incorporated by endothelial cells as early as 2 hours. C12:0 ceramide triggered typical apoptotic chromatin condensation at 18 hours (DAPI staining, Figure E1b). Next, to identify which enzymatic pathway of sphingolipid synthesis was responsible for the pro-apoptotic effects of exogenous ceramide, we employed siRNA specifically designed to SPTLC1 and ASMase.


Figure 1
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  		Figure 1. Kinetics of endogenous ceramide and caspase-3 up-regulation induced by exogenous ceramide (C8:0 Cer) and TNF-{alpha} in primary lung endothelial cells. (a) Endogenous ceramides were measured by mass spectrometry, then normalized by lipid phosphorus (Pi). The data are presented as fold change compared with controls. In shaded circles are shown vehicle controls compared with untreated controls. When compared with controls, both exogenous C8:0 ceramide (10 µM) and TNF-{alpha} (20 ng/ml with cycloheximide) increased endogenous ceramide levels (mean ± SEM; *P < 0.05 versus controls, Student's t test). (b) Caspase-3 activity (normalized by protein concentration and expressed as fold change compared with control) in endothelial cells treated with exogenous C8:0 ceramide (10 µM) (mean ± SEM; *P < 0.05 versus controls, Student's t test). (c) Caspase-3 activity (normalized by protein concentration and expressed as fold change compared with control) in endothelial cells treated with TNF-{alpha} (20 ng/ml with cycloheximide) (mean ± SEM; *P < 0.05 versus controls, Student's t test).

 
Inhibitory Effect of Silencing RNA for SPT and ASMase
SPT, the rate-limiting enzyme in the de novo pathway of ceramide synthesis, contains two subunits, of which the SPT subunit 1 (SPTLC1) is essential for enzymatic activity (14). Primary mouse lung endothelial cells were treated with vehicle (SiPortamine), nonspecific fluorescently labeled siRNA (siGlo) (Figure 2a), scrambled siRNA, or SPTLC1 siRNA. As expected, cells treated with the SPTLC1 siRNA exhibited reduced basal SPT protein levels by immunoblot (Figure 2b) and markedly reduced the SPT enzymatic activity (Figure 2b) 6 days after siRNA transfections. In contrast, similar treatment with nonspecific, scrambled siRNA failed to inhibit SPTLC1 protein expression (Figure 2c). The delayed reductions in SPT activity and SPTLC1 protein levels induced by SPTLC1 siRNA are likely explained by a long half-life of SPT protein in these cells. ASMase siRNA, but not control transfection reagent, significantly reduced basal ASMase activity and protein levels (Figure 2d) at 72 hours after transfection. Similar treatment with scrambled siRNA failed to inhibit ASMase protein expression (Figure 2e).


Figure 2
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  		Figure 2. Effect of targeted siRNA treatment on SPT (serine palmitoyl transferase) and ASMase (acid sphingomyelinase) in lung endothelial cells. (a) Immunofluorescence micrograph of primary mouse lung endothelial cells treated with vehicle alone, siPortamine (left) and vehicle with fluorescently-labeled siRNA, SiGlo (right), showing a high efficiency (> 90%) of siRNA transfection (figures representative of n = 3). (b) SPT activity was measured as D3-serine incorporation (% of total labeled serine administered to cells) by mass spectrometric analysis of labeled dihydrosphinganine and sphingosine (n = 3). Cells were assessed 6 days after treatment with SPTLC1 siRNA (50 nM). Inset: Upper immunoblot of SPT subunit 1 (57 kD) performed 6 days after treatment with SPTLC1 (SPT long-chain base subunit 1) siRNA at the indicated concentrations. Lanes: 1, untreated cell lysate; 2, siPortamine; 3, SPTLC1 siRNA, 50 nM; and 4, SPTLC1 siRNA, 100 nM. The lower immunoblot is for actin, shown as loading control. (c) Densitometry measurements of SPTLC immunoblots normalized by vinculin density as loading control of untreated cell lysates (CTL) and scramble siRNA (Scr SiRNA). Mean ± SEM (n = 3). (d) ASMase activity was measured fluorometrically at pH 5.0, as described in MATERIALS AND METHODS, then normalized by protein concentration (n = 3). The experiments were performed in endothelial cells treated with the indicated concentrations of ASMase siRNA for 72 hours; (*P < 0.05, t test). Inset: representative (n = 3) immunoblot of ASMase (57 kD) 72 hours after treatment with ASMase siRNA at the indicated concentrations. Lanes: 1 and 2, untreated cell lysates; 3, Vehicle; 4, ASMase siRNA, 25 nM; and 5, ASMase siRNA, 50 nM. The same membrane was immunoblotted for GAPDH, as loading control. (e) Densitometry measurements of ASMase immunoblots normalized by vinculin density as loading control of untreated cell lysates (CTL) and scramble siRNA (Scr SiRNA). Mean ± SEM (n = 3).

 
Exogenous Ceramide–Induced Caspase-3 Activation Requires De Novo Sphingolipid Synthesis in Lung Endothelial Cells
In response to C8:0 ceramide administration, primary lung endothelial cells significantly activated the executioner caspases-3/7 (Figure 3a) and the precursor caspase-8 (Figure E1e). Pretreatment of endothelial cells with inhibitors of de novo sphingolipid synthesis such as SPTLC1 siRNA (Figure 3a) or the pharmacological inhibitor fumonisin B1 (data not shown) blocked the activation of effector caspases in response to ceramide. In contrast, treatment with ASMase siRNA did not inhibit ceramide-induced caspase-3/7 activity (Figure 3b) or nucleosomal ELISA (data not shown), suggesting that the sustained (16–24 h), pro-apoptotic responses to exogenous ceramides in lung endothelial cells are primarily mediated by de novo sphingolipid synthesis, rather than sphingomyelin hydrolysis. Consistent with the notion that exogenous C8:0 ceramide stimulated de novo ceramide synthesis, we measured a 75% increase in dihydroceramides, the ceramide precursors in the de novo, SPT-regulated pathway compared with control cells (8.5 nmol/mmol lipid Pi in C8-treated cells versus 4.8 nmol/mmol lipid Pi in vehicle-treated cells, P = 0.01, data not shown). Moreover, the increase in intracellular ceramides (C14:0 and above) was blocked by treatment with SPTLC1 siRNA (Figure 3c).


Figure 3
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  		Figure 3. Effect of ceramide synthesis inhibition on effector caspase activation induced by extracellular ceramide in pulmonary endothelium. Primary mouse lung endothelial cells were pretreated with (a) SPTLC1 siRNA (50 nM, 6 d) or (b) ASMase siRNA (50 nM, 72 h) before C8-ceramide treatment (10 µM, 18 h; Caspase-3/7 activity was normalized by protein concentration; Veh (vehicle siPortamine; mean + SD). Note the robust inhibition of C8-ceramide–induced caspase activation by the specific inhibition of SPTLC1 compared with the vehicle control. (c) Effect of SPTLC1 siRNA on endogenous endothelial cell ceramide content (C14:0 and higher fatty acid chain length) measured by mass spectrometry at 16 hours (mean ± SD, *P < 0.05 versus control cells; #P < 0.05 versus control C8-ceramide–treated cells).

 
Comparison with TNF-{alpha}–Induced Caspase-3 Activation in Lung Endothelial Cells
The pro-inflammatory cytokine TNF-{alpha} triggers endothelial cell apoptosis either alone (e.g., in bovine pulmonary artery endothelial cells) (15–17), or when supplied concomitantly with the protein synthesis inhibitor cycloheximide (15, 18). TNF-{alpha} induces endothelial cell apoptosis by stimulating endogenous ceramide synthesis (5, 19). Our primary objective was to determine whether exogenous ceramides induce endogenous ceramide synthesis by enzymatic pathways similar to those of TNF-{alpha}. We used SPT and ASMase siRNAs at the concentrations and time points shown above to effectively inhibit ceramide synthesis, before challenging endothelial cells with TNF-{alpha} (20 ng/ml plus cycloheximide; see MATERIALS AND METHODS). The targeted genetic (Figure 4a) and pharmacological (data not shown) inhibition of SPT using SPTLC siRNA or inhibitors (fumonisin B1 or myriocin), respectively, significantly attenuated TNF-{alpha}–induced caspase-3, a marker of apoptosis. In contrast, siRNA-mediated knockdown of ASMase (Figure 4b) did not ameliorate TNF-{alpha}–induced apoptosis. The addition of SPTLC siRNA significantly inhibited TNF-{alpha}–induced ceramides at 16 hours (Figure 4c, P < 0.001). ASMase inhibition decreased baseline ceramide levels, but only minimally attenuated the TNF-{alpha}–induced late ceramide elevation (by 17%, P = 0.11, Figure 4d). Scramble siRNA or vehicle treatment had a slight, but insignificant inhibitory effect on TNF-{alpha}–induced ceramides (Figures 4c and 4d, respectively). These results indicate a negligible contribution of the ASMase pathway to caspase-3 activation and ceramide production induced by TNF-{alpha} at 16 hours. They also suggest that both exogenous C8:0 ceramide and TNF-{alpha} augment de novo sphingolipid synthesis to induce caspase-3 activation in lung endothelial cells. We next addressed whether the ceramide species profiles in response to exogenous ceramide and TNF-{alpha} have a pattern similar to that predicted by the activation of a common enzymatic pathway.


Figure 4
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  		Figure 4. Effect of ceramide synthesis inhibition on TNF-{alpha}–induced effector caspase activation in pulmonary endothelial cells. Primary mouse lung endothelial cells were pretreated with (a) SPTLC1 siRNA (50 nM, 6 d) or (b) ASMase siRNA (50 nM, 72 h) before TNF-{alpha} treatment (20 ng/ml, with cycloheximide). Caspase-3/7 activity was normalized by protein concentration; Veh (vehicle siPortamine). (c, d) Effect of siRNA inhibitory treatments on intracellular ceramides (normalized by lipid phosphorus). Effect of Scramble siRNA (Scr siRNA, 50 nM, 6 d) or SPTLC1 siRNA (50 nM, 6 d) on TNF-{alpha}-induced ceramides (c) compared with the effect of ASMase siRNA (50 nM, 3 d) (d). SPTLC1, but not ASMase inhibition significantly reduced ceramide up-regulation in response to TNF-{alpha} (20 ng/ml, with cycloheximide, 16 h); (mean ± SD, *P < 0.05 versus control untreated cells; #P < 0.05 versus control TNF-{alpha}–treated cells).

 
Patterns of Intracellular Ceramide Species Synthesized in Response to Extracellular Ceramide and TNF-{alpha}
In the context of lung endothelial cell apoptosis, we identified a robust increase in both long-chain (C14-C18) and very long–chain (C20 and higher) ceramides induced by TNF-{alpha} and extracellular ceramide (Figure 5a). The TNF-{alpha}– or C8:0 ceramide–induced synthesis of ceramide species containing very long–chain fatty acids appeared primarily under the control of the de novo pathway (Figure 5b). In contrast, the inhibition of ASMase had a very modest inhibitory effect on ceramide synthesis induced by the two treatments, manifested primarily on the long-chain ceramides. Of note, the vehicle employed for intracellular delivery of siRNA, siPortamine, did not significantly change overall ceramide content compared with other control conditions (such as untreated cells or cells treated with ethanol [0.1%] as a vehicle for ceramide), with the exception of C24:1, which was decreased by siPortamine by 29% (not shown). Therefore, the marked decrease in ceramide species induced by SPTLC1 siRNA (Figure 4c) cannot be attributed solely to its vehicle, siPortamine.


Figure 5
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  		Figure 5. Patterns of intracellular ceramide species induced by TNF-{alpha} or exogenous ceramides in lung endothelial cells. (a) Effect of TNF-{alpha} (20 ng/ml with cycloheximide, 16 h) or extracellular C8:0 ceramide (10 µM, 16 h) treatment on endogenous long-chain (C14:0 - C18:0) and very long–chain ceramides (C20–24). (b) Inhibitory effect of SPTLC1 and ASMase siRNAs on long-chain and very long–chain ceramides induced by TNF-{alpha} or C8:0 ceramide. Note the robust inhibition of very-long-chain ceramides induction by SPTLC1 siRNA (n = 2). (c) Pie charts of relative expression profiles of individual ceramide species comprising total ceramides measured by mass spectrometry. Ceramide species expression profiles at baseline and in response to C8:0 and TNF-{alpha} treatment. Depicted in the lower pie charts are the effects of SPTLC1 siRNA treatment on ceramide species induced by the two stimuli. (d) Changes in the relative abundance of C16:0 (% of total ceramides) induced by C8:0 and TNF-{alpha} treatment and the effect of SPTLC1 siRNA. The control values include untreated control, vehicle-treated control and scramble siRNA (mean ± SEM; *P < 0.05 versus controls, #P < 0.05 versus C8:0 and TNF-{alpha} treatment, respectively, Student's t test).

 
Of the ceramide species studied, C16:0 and C20:0 ceramides demonstrated absolute concentrations similarly regulated by TNF-{alpha} and C8:0 ceramide. The absolute concentrations of these two ceramide species were elevated by the treatments in an SPT-dependent manner (not shown). However, when measuring the relative expression of ceramide species, C16:0 had the single largest increase in abundance relative to the other ceramide species induced by TNF-{alpha} and C8:0 ceramide (Figure 5c). The relative increase in C16:0 induced by TNF-{alpha} and extracellular C8:0 ceramide was markedly attenuated by SPTLC1 siRNA (Figure 5c), suggesting this species of ceramide might be the target for SPT regulation in response to both TNF-{alpha} and extracellular ceramide.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
These results implicate de novo ceramide synthesis as a critical component of the pro-apoptotic action of extracellular ceramides on endothelial cells. A similar requirement for de novo sphingolipid synthesis was observed for late (16 h) apoptosis induced by TNF-{alpha} in lung endothelial cells. Furthermore, the differential profiles of endogenous ceramides released by these two stimuli associate the relative and absolute increases in C16:0 ceramides with caspase activation in primary lung endothelial cells. Recently, ASMase-dependent increases in ceramide C16:0 have been implicated in TNF-{alpha}–induced apoptosis in hepatocytes (20), underscoring the cell-type specificity of these responses. Ceramide up-regulation is relevant to a wide range of diseases, including radiation-induced tissue injury, Alzheimer's disease, and pulmonary emphysema. In the latter, injury of lung capillary endothelial cells by cigarette smoke itself, oxidants, or circulating pro-inflammatory cytokines may initiate the apoptotic destruction of alveolar septae, a distinctive feature of pulmonary emphysema. TNF-{alpha} and ceramide have both been shown to cause endothelial cell apoptosis and have both been implicated in the pathogenesis of emphysema (21). TNF-{alpha} increases intracellular ROS and ceramide levels, but as previously pointed out by several investigators, the pro-apoptotic signaling events that lie downstream of ceramide may be distinct from TNF-{alpha} (5, 19). This divergence is especially evident in the context of endothelial cell activation by TNF-{alpha}, where pro-apoptotic pathways are inhibited and pro-inflammatory pathways are activated (22). In the context of a pro-apoptotic, ceramide-generating TNF-{alpha} response, however, TNF-{alpha} and ceramides were used almost interchangeably in the study of ceramide-dependent apoptosis. TNF-{alpha}–stimulated ceramides may in fact facilitate pro-apoptotic TNF-{alpha} effects, through fusion of lipid microdomains and clustering of TNF receptors (23). Therefore, work from others and our laboratories suggested that both TNF-{alpha} and extra- (or para)-cellular ceramides may amplify the injury of microvascular endothelial cells. We propose that the injury-amplifying effects of these molecules are mediated by the generation of specific ceramide species.

To better characterize the stimulus-specificity of ceramide synthesis, we employed genetic manipulation using small inhibitory RNAs. Pharmacological inhibitors, while relatively specific, commonly interfere with other cellular pathways and at high concentrations may exhibit cellular toxicity. In contrast, siRNAs are highly specific (24), and compared with other molecular manipulations of enzymatic activities in the sphingolipid pathways (e.g., ASMase knockout) they have the advantage of a timed onset of action, thus limiting the potential effect of long-term accumulated lipids in the cells. Furthermore, siRNA treatments are well tolerated in vivo in the lung (4), thus having the prospective for clinical applications. Using this effective tool, we have demonstrated that TNF-{alpha} required de novo sphingolipid synthesis, rather than ASMase activity for caspase-3/7 activation. This result is in contrast with findings in HUVECs and other cell types (5, 25–27), and may reflect organ, species, or inhibitor specificity. For example, similar to lung endothelial cells, de novo ceramide synthesis is required for tumor-induced apoptosis in dendritic cells (28). Furthermore, our results suggest a prolonged half-life of endothelial SPT protein and perhaps the need for prolonged inhibitory strategies before functional SPT assays are performed. It is conceivable that the de novo ceramide synthesis shown here to be essential for triggering apoptosis may be associated with either a very rapid (minutes) and/or late (beyond 18 h) wave of sphingomyelin breakdown, further contributing to lung injury. This hypothesis is supported by work implicating sphingomyelinases in the paracrine amplification of lung apoptosis (1) and in lung inflammation (29). Our results do not rule out the involvement of neutral sphingomyelinase. We focused our work on SPT and ASMase because of their involvement in an apoptosis-dependent murine emphysema model (1). Furthermore, SPT inhibition completely blocked TNF-{alpha} and ceramide-induced caspase-3 activation, suggesting its activation was necessary and sufficient for this response. The relevance of this finding to the in vivo effects of the cytokine awaits further investigation.

As novel investigations into individual ceramide species generation and function emerge, more precise mechanistic conclusions about the role of specific ceramide species in disease will be possible. Our data suggest that very long–chain ceramides and C16:0 are predominantly synthesized by lung endothelial cells via SPT activation and that they might be specifically involved in the activation of executioner caspases. The role of the shorter-chain ceramides prominently induced by C8:0 remains unclear. Distinct ceramide species may differ in subcellular compartmentalization and assume specific signaling roles. Furthermore, individual ceramide species may be differentially regulated through the activation of specific ceramide synthase enzymes. The longevity assurance homolog LASS and various ceramide synthase gene homologs encode distinct ceramide synthase isoforms that become activated downstream of SPT, or are part of the recycling pathway (30, 31). Interestingly, distinct ceramide synthase isoforms are responsible for the generation of specific-length ceramides. For example, the overexpression of ceramide synthase 1 increased C18:0-ceramide levels preferentially, ceramide synthase 2 and 4 increased levels of longer ceramides such as C22:0- and C24:0-ceramides, whereas ceramide synthase 5 and 6 produced predominantly shorter ceramide species (C14:0- and C16:0-ceramides) (32). Future investigations will determine whether specific ceramide synthases are up-regulated differentially by stimuli such as TNF-{alpha} or exogenous ceramides.

In conclusion, our data highlight the importance of SPT in the initiation of lung endothelial cell injury and death. To our knowledge, this is the first study of the pro-apoptotic sphingolipid signaling mechanism and ceramide species signature of paracellular and intracellular generated ceramides in lung endothelial cells. This study has relevance to the development of emphysema, since both ceramides and TNF-{alpha} are pathogenic in experimental models of emphysema, in which they may cause excessive apoptosis of capillary endothelial cells with loss of alveolar structures. Prevention of excessive lung microvascular endothelial cell death modulated by sustained SPT stimulation might therefore represent an attractive therapeutic target.


   Acknowledgments
 
The authors gratefully acknowledge the expert assistance of Patrick Singleton, Ph.D., in the design of siRNA and Kelly Schweitzer, Ph.D., for critical reading of the manuscript.


   Footnotes
 
* These authors contributed equally to this manuscript. Back

This work was funded by ATS/Alpha One Foundation Research Grant and NIH RO1 HL077328 (to I.P.); NIH RO1HL66554 (to R.M.T.); and NIH 1 S10 RR16798 for purchase of LC-MS/MS instrumentation (to W.C.H.).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2007-0274OC on January 10, 2008

Conflict of interest statement: R.M.T. received an unrestricted postdoctoral support grant from Quark Biotech for studies involving RTP801 in cigarette smoke–induced emphysema, $2,500 for speaker fees in an international conference sponsored by AstraZeneca, and $1,500 from the Rush Medical Center's CME speakers training workshop titled, Simply Speaking. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 19, 2007

Accepted in final form January 8, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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