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J. Biol. Chem., Vol. 281, Issue 34, 24695-24703, August 25, 2006
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-induced PGE2 Production*
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From the
Departments of Biochemistry and Molecular Biology, Microbiology and Immunology, **Pathology and Laboratory Medicine, and ||Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 and the ¶Department of Veterans Affairs Medical Center, Charleston, South Carolina 29401
Received for publication, May 17, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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/prostaglandin E2 (PGE2) pathway was investigated. The amphiphilic compound desipramine, a frequently employed inhibitor of acid sphingomyelinase (ASMase), blocked PGE2 production. However, the action of desipramine was independent of its action on ASMase, since neither genetic loss of ASMase (Niemann-Pick fibroblasts) nor knockdown of ASMase using RNA interference affected TNF-induced PGE2 synthesis. Further investigations revealed that desipramine down-regulated acid ceramidase (AC), but not sphingosine kinase, at the protein level. This resulted in a time-dependent drop in sphingosine and S1P levels. Moreover, exogenous administration of either sphingosine or S1P rescued PGE2 biosynthesis after desipramine treatment. Interestingly, knockdown of endogenous AC by RNA interference attenuated cyclooxygenase 2 induction by TNF and subsequent PGE2 biosynthesis. Taken together, these results define a novel role for AC in the TNF/PGE2 pathway. In addition, the results of this study warrant careful reconsideration of desipramine as a specific inhibitor for ASMase. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and IL-1
were shown to induce COX-2 through activating the SK1/S1P pathway, such that knockdown of SK1 by RNA interference blocked the induction by TNF of COX-2 and subsequent PGE2 synthesis (1). Interestingly, S1P synergizes with a parallel ceramide kinase (CK)/ceramide 1-phosphate pathway, which regulates cytosolic phospholipase A2 and arachidonic acid release (2). Besides COX-2 induction, S1P has also been shown to affect neutrophil priming (3), macrophage activation (4), mast cell degranulation (5), and chemotaxis of inflammatory cells. Therefore, the SK1/S1P pathway is emerging as a key regulator of several aspects of the inflammatory cascade (reviewed in Refs. 6-8).
Although SK1 plays a major role, regulation of S1P levels is a function of the net responses of enzymes of the sphingolipid network that influence the levels of ceramide and sphingosine (precursors for S1P synthesis). Ceramide generated de novo or from sphingomyelin (SM) hydrolysis can be deacylated by ceramidases to sphingosine. Consequently, agonist-driven activation of SK1 results in conversion of sphingosine to S1P. Indeed, evidence supporting the existence of such a signaling pathway began to emerge from studies implicating sphingolipids in regulation of COX-2. For example, treatment with sphingolipid metabolites, such as ceramide, sphingosine, or S1P, induces production of PGE2 (9-12). Furthermore, ceramide exogenously added or generated by treatment with sphingomyelinase (SMase) enhanced IL-1-mediated PGE2 production (9). Although these studies implicate SM metabolites in COX-2 regulation, the contribution of the implicated enzymes of sphingolipid metabolism, namely ceramidases and sphingomyelinases, to this process is poorly understood.
In this study, we focused initially on defining the contribution of acid SMase (ASMase) to cytokine-induced generation of S1P. In humans, ASMase deficiency results in Niemann-Pick disease (NPD), a neurodegenerative disorder resulting in pathologic lysosomal accumulation of SM. Whereas numerous studies have suggested ASMase as an important player during receptor- and nonreceptor-mediated cellular stress responses (reviewed in Refs. 13-15), the role of ASMase in inflammatory signaling cascades, such as prostaglandin synthesis, remains unknown. The availability of genetic models (knock-out mice) and cell lines derived from NPD patients allowed better understanding of the role of ASMase in signal transduction pathways. Attempts to determine contributions of ASMase to cell responses have also relied on the use of pharmacological inhibitors, especially tricyclic amines. The addition of the tricyclic amine desipramine to neuroblastoma cells and skin fibroblasts was shown to decrease ASMase activity in a time- and dose-dependent manner (16, 17). Interestingly, there was no change in ASMase activity when desipramine was added to cellular homogenates in vitro, suggesting an indirect effect of the compound. The mechanism of ASMase inhibition by desipramine was addressed in careful studies by Sandhoff and coworkers. The authors demonstrated that desipramine, probably by virtue of its cationic amphiphilic properties, displaces ASMase from a membrane-bound state to the lysosomal lumen, allowing its proteolytic degradation (18). However, it remains unclear whether this process is specific for ASMase or not.
Although the results from this study showed that desipramine treatment inhibited PGE2 synthesis, we provide evidence that this process is independent of ASMase inactivation. Further investigations revealed that desipramine down-regulated acid ceramidase (AC) at the protein level without influencing total cellular SK1. This resulted in a drop in total cellular levels of sphingosine and S1P. Finally, using RNA interference (RNAi) knockdown, we demonstrate that AC is required in the TNF/PGE2 pathway. Potential mechanisms by which AC may contribute to this response are discussed. We also discuss the potential uses of desipramine as a pharmacologic agent in evaluating sphingolipid-mediated responses.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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was purchased from PeproTech, Inc. (Rocky Hill, NJ). Desipramine was purchased from Sigma. C16-ceramide, sphingosine, and S1P were synthesized in the Lipidomics Core Facility at the Medical University of South Carolina. For ceramidase assays, N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine was purchased from Avanti (Alabaster, AL). COX-2 was probed using specific anti-human polyclonal primary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). AC mouse monoclonal antibody was purchased from BD Biosciences, and SK1 rabbit polyclonal antibody was described previously (19). PGE2 Assay—The PGE2 assay was performed using a prostaglandin E2 monoclonal enzyme immunoassay kit from Cayman Chemicals (Ann Arbor, MI) according to the manufacturer's instructions. Media containing PGE2 collected from the indicated treatment groups competed with PGE2-acetylcholinesterase conjugate for a limited amount of PGE2 monoclonal antibody. The antibody-PGE2 conjugate bound to a goat anti-mouse antibody previously attached to the wells. The plate was washed to remove any unbound reagents, and then the substrate to acetylcholinesterase was provided. The concentration of PGE2 in a sample is inversely proportional to the yellow color produced. The results are expressed as pg/ml calculated according to the PGE2 standard curve.
Western Blotting—L929 fibroblasts subjected to the indicated treatments were scraped on ice-cold phosphate-buffered saline and collected by centrifugation. Cells were lysed using radioimmune precipitation lysis buffer supplemented with a mixture of protease and phosphatase inhibitors (Pierce), and lysates were normalized to protein concentration using Bio-Rad Bradford reagent (Bio-Rad). Samples (30 µg) were boiled in Laemmli sample buffer and separated by SDS-PAGE. This was followed by protein transfer to 0.45-µm nitrocellulose membranes and blocking with 5% milk or bovine albumin solution. Blots were probed by overnight incubation with the indicated antibodies. Membranes were washed three times with 0.1% Tween 20 in Tris-buffered saline before adding appropriate horseradish peroxidase-conjugated secondary antibodies. The ECL immunoblotting detection system (Amersham Biosciences) was used to visualize the bands. Equal loading was checked by probing with actin-specific antibody (Santa Cruz Biotechnology).
In Vitro SMase Assay—SMase assay was carried out by the method described previously (20). L929 cells were lysed in 50 mM Tris buffer supplemented with protease inhibitors using a probe sonicator. Cellular debris was removed after centrifugation at 3000 x g for 10 min. Proteins (50 µg) were adjusted to a total volume of 100 µl, and the reaction was started by adding 100 µl of the reaction mixture containing 1 mM EDTA, 250 mM sodium acetate (pH 5.0), 100 µM [choline-methyl-14C]sphingomyelin, and 0.1% Triton X-100. After incubation at 37 °C for 1 h, the reaction was stopped by adding 1.5 ml of chloroform/methanol (2:1) followed by adding 400 µl of water. Phases were separated by centrifugation at 2000 x g for 5 min. Quantitation of the amount of released radioactive phosphocholine was determined by subjecting 400 µl of the upper phase to scintillation counting.
In Vitro AC Assay—Detection of total cellular AC enzymatic activity was performed according to previously described methods (21-24) with slight modifications. Briefly, fibroblasts were lysed in an acidic buffer (pH 4.5) consisting of 50 mM sodium acetate, 5 mM magnesium chloride, 1 mM EDTA, and 0.5% Triton X-100. Lysates normalized to equal total proteins (50 µg) were incubated in the assay buffer (pH 4.5), consisting of 0.2% Igepal-CA 630, 250 mM sodium acetate, and 150 µM NBD-C12 ceramide in a total volume of 100 µl. The reaction mixture was incubated at 37 °C for 1 h. Conversion of NBD-C12 ceramide to NBD-dodecanoic acid was detected by spotting the organic phase on a TLC plate. Results were quantitated by densitometric analysis using ImageQuant software.
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and assayed as described. The efficiency of gene silencing was assessed by measuring enzymatic activity or protein levels of the target gene. Levels of Sphingolipid Metabolites—Mass spectrometric analysis of lipids was performed using electrospray ionization MS/MS analysis on a Thermo Finnigan TSQ 7000 triple quadruple mass spectrometer, operating in multiple reaction-monitoring positive ionization mode as described previously. Briefly, about 2-3 x 106 cells were fortified with the internal standards (ISs; C17 base D-erythrosphingosine, N-palmitoyl-D-erythro-C13-sphingosine, and heptade-canoyl-D-erythrosphingosine). Calibration curves were constructed by plotting peak area ratios of synthetic standards corresponding to each target analyte with respect to the appropriate internal standard. The target analyte peak areas from the samples were similarly normalized to their respective internal standard and then compared with the calibration curves using a linear regression model. Results were normalized to total protein levels.
Statistical Analysis—Student's t test was performed between control and treated states and/or between treatment and treatment plus RNAi-mediated inhibition states on a minimum of three independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A key role for TNF in the inflammatory cascade is induction of inflammatory mediators, such as the prostaglandin PGE2. Treatment of L929 cells with TNF for 8 h resulted in extracellular release of PGE2 into the medium. Interestingly, pretreatment with desipramine resulted in significant reduction in the levels of PGE2 detected after TNF treatment (Fig. 1A). Since COX-2 is a rate-limiting step during prostaglandin biosynthesis, the effect of desipramine on COX-2 induction was evaluated. As expected, a robust elevation in COX-2 protein levels was observed after 8 h of TNF treatment. However, the COX-2 response was markedly attenuated after pretreatment with desipramine (Fig. 1A).
Next, and in order to determine if desipramine affected the sphingolipid pathway upstream of COX-2, the levels of S1P, which has been shown to be necessary for induction of COX-2 in response to TNF, were analyzed under similar treatment conditions. Mass spectrometric measurements revealed significant elevation in S1P within 10 min of TNF treatment. Interestingly, the S1P response was significantly attenuated in desipramine-pretreated cells (Fig. 1B). This result points to a site of action of desipramine upstream of the generation of S1P.
The above results showing significant effects of desipramine on COX-2 and S1P are potentially consistent with a role for ASMase in the TNF-PGE2 pathway, and we investigated this role using fibroblasts derived from NPD patients who exhibit genetic deficiency in ASMase activity. However, the results showed that NPD fibroblasts responded to TNF with a robust elevation of PGE2 that actually exceeded the response seen in "control" fibroblasts derived from Lesch-Nyhan patients, who have no perturbations in ASMase (Fig. 2A). Moreover, and similar to L929 cells, desipramine treatment was still able to attenuate the PGE2 response in NPD fibroblasts (Fig. 2B). Thus, in contradistinction to the results with desipramine, these results in NPD fibroblasts argue against a role for ASMase in the TNF-induced PGE2.
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response were then evaluated. Thus, cells were transfected with either ASMase RNAi or scrambled sequence for 48 h before PGE2 detection. As shown in Fig. 2D, ASMase knockdown did not exert a significant effect on the PGE2 response. Together with the results from NPD cells, the results from knockdown imply that the PGE2 response does not require the activity of ASMase.
A Functional Lysosomal Compartment Is Required for PGE2 Biosynthesis—Activation of the TNF receptor elicits changes in different cellular compartments, including mitochondria, lysosomes, and nucleus. Since desipramine is a lysomotropic hydrophobic amine, its actions (including effects on ASMase) point to the lysosome. To investigate the role of lysosomal sphingolipid metabolism in prostanoid biosynthesis, we used the lysomotropic agent chloroquine. L929 fibroblasts preincubated with 50 µM chloroquine were treated with or without recombinant human TNF. The production of PGE2 was then analyzed after 8 h. Whereas treatment with TNF enhanced PGE2 production, this response was markedly blocked after pretreatment with chloroquine (Fig. 3A). Since S1P generation has been shown as an indispensable step for cytokine-induced PGE2 production, it became important to measure levels of this proinflammatory sphingolipid. As illustrated in Fig. 3B, preincubation with chloroquine blocked synthesis of S1P after TNF treatment. These findings suggest that a functional lysosomal compartment is required for events downstream of the TNF receptor, leading to S1P and prostanoid production.
Desipramine Decreases Levels of Sphingosine and S1P—Since in previous studies S1P was implicated in mediating the effects of TNF on PGE2 (1), we investigated whether desipramine modulated sphingolipid levels. After incubation of L929 cells with 10 µM desipramine, sphingolipid levels were analyzed over a period of 4 h by mass spectroscopy. As shown in Fig. 4A, desipramine treatment did not change basal levels of total ceramide. However, the results showed a significant time-dependent drop in the levels of sphingosine and S1P (Fig. 4B). Although this decrease was still observed after up to 4 h of desipramine treatment, the maximal drops in S1P (60%) and sphingosine (40%) were observed within the first 30 and 60 min, respectively. Therefore, desipramine modulates acutely the levels of two bioactive sphingolipids.
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Since S1P biosynthesis requires the presence of its precursor substrate sphingosine and since desipramine induced a significant drop in sphingosine levels, we next evaluated the effects of desipramine on ceramidases. We focused on acid ceramidase, since, similar to ASMase, this enzyme is present in the lysosome and indeed is found to interact with ASMase (25). Western blotting analysis revealed loss of AC protein that correlated with the dose and time of desipramine treatment, as illustrated in Fig. 5, B and C, respectively. Next, we addressed the question whether the observed down-regulation of AC is mediated by a proteolytic process. To that end, desipramine treatment was performed in the presence or absence of the broad range protease inhibitors pepstatin and leupeptin. Interestingly, leupeptin treatment restored basal protein levels of AC, suggesting that the desipramine effect is mediated by a cysteine protease (Fig. 5D). These results have two important implications. They first demonstrate that the effects of desipramine on sphingolipid enzymes are not restricted to ASMase. They also suggest that acid ceramidase may play a role in the PGE2 response to TNF.
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—induced PGE2 Synthesis—Based on the above and in order to evaluate the role of AC in the TNF/PGE2 pathway, we employed RNAi to achieve specific knockdown of AC. To that end, L929 cells were treated with 50 nM scrambled or AC-specific RNAi sequences. After incubation for 48 h, a significant drop (80%) in total AC protein levels was observed (Fig. 6A). The results showed that the basal levels of PGE2 were similar in cells treated with AC or scrambled RNAi. However, knockdown of AC resulted in a significant inhibition of PGE2 production after 8 h of TNF treatment (Fig. 6A). Likewise, induction of COX-2 by TNF was attenuated after loss of AC. On the other hand, targeting neutral ceramidase (NC) with a specific RNAi sequence did not influence PGE2 biosynthesis after TNF treatment (Fig. 6B). Thus, AC appears to contribute to the production of PGE2, probably through its influence on the levels of S1P.
To investigate whether TNF treatment modulates AC activity, an in vitro enzymatic assay was performed. After an acute time course of TNF treatment (20 min), total cellular lysates were incubated with fluorescently labeled C-12 ceramide at pH 4.5. As shown in Fig. 6C, there were no detectable changes in ceramide breakdown to dodecanoic acid after TNF treatment. Therefore, the results suggest that the TNF/PGE2 pathway selectively utilizes sphingosine generated via basal AC activity without activating the enzyme itself.
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plus desipramine. The results showed that exogenous treatment with sphingosine and S1P, but not ceramide, rescued the PGE2 response to TNF after desipramine treatment (Fig. 7). Thus, taken together, the results suggest an important role for acid ceramidase in regulating the PGE2 response to TNF. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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/PGE2 pathway. Using a L929 fibroblast cell culture model, it was found that the tricyclic amine desipramine inhibits COX-2 induction and PGE2 production in response to TNF. Although desipramine treatment down-regulated ASMase activity in a dose- and time-dependent manner, the results suggest that ASMase per se does not participate in this pathway. On the other hand, the results point to AC as the relevant target for this compound in the TNF/PGE2 pathway. Mass spectrometric analysis revealed that desipramine induced a time-dependent drop in sphingosine and S1P cellular levels, without noticeable changes in ceramide. Furthermore, it was observed that desipramine down-regulates AC but not SK1 protein levels. Moreover, using AC specific RNAi, it was shown that AC is important for PGE2 synthesis induced by TNF. Finally, both sphingosine and S1P, but not ceramide, overcame the desipramine effect, thus further localizing the action of desipramine to AC.
A major finding of this study is that regulation of PGE2 synthesis by TNF is independent of ASMase. This conclusion is supported by the following observations. 1) Treatment of NPD fibroblasts with TNF resulted in pronounced synthesis of PGE2 that exceeded the levels secreted by control fibroblasts. 2) There was no significant change in the levels of PGE2 after knockdown of ASMase using specific RNAi sequence. These results are consistent with an earlier study by Manthey and Schuchman (26) examining the role of ASMase in lipopolysaccharide signaling. The authors reported that lipopolysaccharide-induced gene expression of specific cytokines (TNF, IL-1, and interferon-
) is intact in ASMase-/- macrophages. Furthermore, TNF induced similar degradation of I
B in ASMase-/- and ASMase+/+ macrophages (26). Thus, taken together, the results suggest that ASMase does not appear to play key roles in cytokine-induced inflammatory responses.
However, our results do not exclude a role for other SMases in general during inflammatory signaling cascades. For instance, treatment of human colon cancer cells with exogenous neutral SMase resulted in a 6-fold increase in IL-8 as opposed to the 1.5-fold elevation seen with exogenous ASMase treatment (27). Also, the cytokine IL-1 has been shown to activate exogenous neutral SMase, which appears to be involved in signaling from the IL-1 receptor (28). Therefore, ceramide generated by different SMases may participate differentially in inflammation and apoptosis.
In addition to previous results implicating SK1 in TNF-induced formation of PGE2, the current results implicate an important and novel role for AC in the TNF/PGE2 pathway. This is evidenced by the following observations: 1) desipramine down-regulated AC at the same dose at which it blocked PGE2 synthesis; 2) exogenous sphingosine rescued PGE2 synthesis after desipramine treatment; and 3) knockdown of AC by a specific RNAi sequence attenuated the TNF-induced PGE2 synthesis.
Ceramidases are key enzymes regulating cellular levels of the bioactive sphingolipids ceramide, sphingosine, and S1P. There are three major classes of ceramidases that have been described with different pH optima (acid, neutral, and alkaline). The earliest ceramidase to be identified was the lysosomal acidic enzyme, whose deficiency leads to Farber disease (22). Activation of acid ceramidase has been linked to resistance of ceramide-mediated death pathways. For instance, overexpression of AC was shown to protect cells from TNF-induced apoptosis (29). Similarly, survival of alveolar macrophages and specific prostate cancers is associated with enhanced AC activity (30, 31). Conversely, inhibition of AC induces apoptosis of prostates cancer cells in xenograft as well as cell culture models (32). The present study extends the current understanding of the antiapoptotic role of AC. In this context, our data highlight AC as an essential player in cytokine-induced inflammatory responses. Indeed, recent models of tumorigenesis converge on the importance of sustained inflammation in tumor promotion within tissues, such as liver, colon, and others (reviewed in Ref. 33). Therefore, the prosurvival phenotype promoted by AC might be explained by its role in inflammatory cascades as suggested by the current results.
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activates AC, which then would generate sphingosine. The subsequent action of SK1 would then result in the formation of S1P. Alternatively, AC is required for the provision of basal sphingosine as a substrate for the action of SK1 (i.e. according to this scenario, there is no requirement for activation of AC in response to TNF). We favor the second hypothesis for the following reasons. 1) There is as of yet no evidence for acute activation of AC by TNF within the time frame of induction of S1P (Fig. 6C), and 2) if anything, there is a drop in sphingosine levels at 10 min following TNF, concomitant with S1P formation (1). This would argue that the primary action of TNF is on SK1, which would deplete sphingosine (substrate) and increase S1P (product). Had AC been the primary target, one would expect sphingosine levels to increase (or at least not decrease) and then drive the formation of S1P. 3) The ability of desipramine to acutely induce a drop in sphingosine levels suggests that AC is responsible for "maintenance" of sphingosine levels under basal conditions. Moreover, it is well established that lysosomal ceramide cannot exit to the cytosol unless it is deacylated to sphingosine (34). Having in mind that SK1 is a cytosolic enzyme, it becomes plausible to hypothesize that deacylation of ceramide to sphingosine by AC may be a prerequisite to S1P generation during inflammatory cascades (Fig. 8). Whereas the current data clearly demonstrate that AC is upstream of cytokine-induced S1P production, alternative mechanisms by which AC regulates the TNF/PGE2 pathway could not be ruled out at this point. For instance, plasma membrane availability of TNF receptors is a limiting factor for TNF signaling. Upon binding to its ligand, the TNF receptor internalizes within minutes and fuses with the endolysosomal compartment (35, 36). Although this process seems to be essential for TNF-induced apoptosis, it remains to be investigated whether a similar process is required for inflammatory signaling downstream of the TNF receptor. Moreover, treatment with lysomotropic agents (chloroquine or ammonium chloride) decreases the number of cell surface TNF receptors (37, 38). Interestingly, inhibition of lysosomal proteases did not have the same effect, thus raising the possibility that trafficking of the TNF receptor could be regulated by lysosomal lipases rather than proteases.
The observed acute depletion of sphingosine with desipramine treatment raises a very interesting metabolic implication. If the action of desipramine on sphingosine is primarily due to the inhibition of AC, then three conclusions emerge. First, AC contributes to relatively rapid flux through sphingosine in the cell. Thus, desipramine caused rapid depletion of sphingosine within 60 min, suggesting active and ongoing lysosomal hydrolysis of ceramide by AC. Second, lysosomal sphingosine appears to be a major source for basal S1P. Thus, the action of desipramine resulted in significant depletion of basal S1P (without affecting SK1 levels). Third, lysosomally derived sphingosine appears to account for 
40% of total basal sphingosine. This also suggests that other enzymes and pathways may contribute to sphingosine formation. Full verification of these conclusions awaits the development of more specific pharmacologic inhibitors of AC, since they would be best probed with acute inhibition of AC.
The mechanism of AC down-regulation by desipramine has been addressed. In agreement with what has been previously reported for ASMase (18), it was found that down-regulation of AC by desipramine is mediated by lysosomal proteases. In particular, the cysteine protease inhibitor leupeptin rescued AC from desipramine-induced degradation. Similar results were obtained upon studying the effects of desipramine in prostate cancer cells.3 Indeed, AC and ASMase are not only related by their biochemical and topological properties, but also He et al. (25) reported that the two enzymes closely interact with each other. Interestingly, overexpression of AC led to increased extracellular secretion of ASMase. Furthermore, the authors showed that ASMase coimmunoprecipitated with AC from extracellular media (25). Taken together, these data invite the proposal that desipramine down-regulates AC and ASMase by a shared mechanism.
Another major conclusion from this study relates to the use of desipramine as a specific inhibitor of ASMase. The present findings are distinct from and augment earlier reports showing that desipramine inactivates ASMase but not other specific lysosomal enzymes (17). Whereas desipramine inhibited PGE2 synthesis at the same dose at which it down-regulated ASMase, further investigation showed that desipramine also down-regulated AC but not SK1 at the protein level, and this was accompanied by a loss of sphingosine and S1P. Indeed, the conclusions of this study would have been misleading had we relied on desipramine as a specific ASMase inhibitor. Thus, although the use of desipramine as a pharmacologic inhibitor for ASMase seems convenient, results obtained with this compound should be interpreted carefully. For instance, it may be plausible to exclude the requirement for ASMase if a certain response is not affected by desipramine treatment. However, inhibition of a specific process by desipramine does not provide conclusive evidence for the involvement of ASMase. To that end, we recommend confirmation of these results using other approaches, such as RNAi or mutant mice. Overall, it may be proposed that demonstration of inhibition of a process by desipramine is required to implicate ASMase but not sufficient.
In conclusion, using pharmacologic inhibitors, genetic models (NPD fibroblasts) and RNAi technology, we demonstrate that AC but not ASMase is required for TNF induction of PGE2. Finally, the study warrants reconsideration of desipramine as a specific inhibitor for ASMase.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology Medical University of South Carolina, 175 Ashley Ave., P.O. Box 250509, Charleston, SC 29425. Tel.: 843-792-9318; Fax: 843-792-4322; E-mail: hannun{at}musc.edu.
2 The abbreviations used are: SK1, sphingosine kinase 1; TNF, tumor necrosis factor; COX-2, cyclooxygenase 2; S1P, sphingosine 1-phosphate; ASMase, acid sphingomyelinase; AC, acid ceramidase; RNAi, RNA interference; NPD, Niemann-Pick disease; IL, interleukin; PGE2, prostaglandin E2; CK, ceramide kinase; SM, sphingomyelin; SMase, sphingomyelinase.
3 S. Elojeimy, Y. Zeidan, Y. Hannun, and J. Norris, submitted for publication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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