Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900
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ABSTRACT |
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Tissue injury in
inflammation involves the release of several cytokines that activate
sphingomyelinases and generate ceramide. In the lung, the impaired
metabolism of surfactant phosphatidylcholine (PC) accompanies this
acute and chronic injury. These effects are long-lived and extend
beyond the time frame over which tumor necrosis factor (TNF)- and
interleukin-1
are elevated. In this paper, we demonstrate that in
H441 lung cells these two processes, cytokine-induced metabolism of
sphingomyelin and the inhibition of PC metabolism, are directly
interrelated. First, metabolites of sphingomyelin hydrolysis themselves
inhibit key enzymes necessary for restoring homeostasis between
sphingomyelin and its metabolites. Ceramide stimulates
sphingomyelinases as effectively as TNF-
, thereby amplifying the
sphingomyelinase activation, and TNF-
, ceramide, and sphingosine all
inhibit PC:ceramide phosphocholine transferase (sphingomyelin
synthase), the enzyme that restores homeostasis between sphingomyelin
and ceramide pools. Second, ceramide inhibits PC synthesis, probably
because of its effects on CTP:phosphocholine cytidylyltransferase, the
rate-limiting enzymatic step in de novo PC synthesis. The data
presented here suggest that TNF-
may be an inhibitor of phospholipid
metabolism in inflammatory tissue injury. These actions may be
amplified because of the ability of metabolites of sphingomyelin to
inhibit the pathways that should restore the normal
ceramide-sphingomyelin homeostasis.
sphingosine; tumor necrosis factor-; pulmonary surfactant; cytidine 5'-triphosphate
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INTRODUCTION |
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THE SPHINGOMYELIN PATHWAY interacts in a large number of cellular activities including differentiation and mitosis, gene transcription, and cell death (26). These multiple actions result in part from the ubiquitous presence of receptors that initiate the signaling pathway, activated by several ligands. These include cytokines that are released at the onset of inflammatory injury (26). The central molecule in this pathway is ceramide (15).
There are numerous reports, many in the older literature, that show
that changes in cellular metabolism accompany inflammation (12,
27); indeed, some of the earliest reports on tumor necrosis factor (TNF)- emphasized its effects in promoting cachexia
(6). In the inflammatory response of the lung resulting
from acute traumatic injury or infection, these events may affect the
metabolism of phospholipids required for the maintenance of the proper
amount and composition of pulmonary surfactant (30). The
resulting condition leads to alveolar collapse, interference with gas
transfer, hypoxia, and, in 40-60% of the patients, death
(25).
Recent work (1, 34) indicates that TNF- inhibits
phosphatidylcholine (PC) synthesis, but there is very little published work on the mechanism by which TNF-
exerts this effect. It has been
shown that TNF-
given to type II cells results in increased levels
of ceramide and sphingosine (22), suggesting that TNF-
could be acting through one of these sphingolipids. Furthermore, in another recent study, Mallampalli and coworkers
(21) showed that TNF-
inhibited PC synthesis,
principally by its actions on CTP:phosphocholine cytidylyltransferase
(CT). These changes appeared to be mediated through a decrease
in the amount of CT, possibly through ubiquitin-proteasome processing.
The work described in this and the companion paper (3)
confirms and extends these observations. In this paper, we concentrate
on describing the role of the sphingomyelin metabolites formed as a
result of the stimulation of TNF-
on this overall process. In the
companion paper, we investigate the signaling pathway used by ceramide
to inhibit CT activity.
We report here that ceramide, generated as a consequence of signaling
initiated by TNF-, may have a central role in these metabolic
perturbations. We present evidence that ceramide or a metabolite of
ceramide inhibits regulatory events that would be expected to restore
the normal balance between sphingomyelin and ceramide concentrations.
In this regard, we report that TNF-
and ceramide and sphingosine,
products of sphingomyelin metabolism initiated by TNF-
, inhibit the
key enzyme responsible for the restoration of sphingomyelin-ceramide
homeostasis, PC:ceramide phosphocholinetransferase (sphingomyelin
synthase). Furthermore, ceramide activates acidic and neutral
sphingomyelinases rather than inhibiting the products. These dual
actions of ceramide (and/or ceramide metabolites) promote elevated
ceramide pools, thereby reinforcing the sustained injury. We then show
that at least two metabolites of sphingomyelin hydrolysis, ceramide and
sphingosine, mimic exactly the actions of TNF-
(23) in
that they diminish the synthesis of PC, reduce the content of cellular
PC, and inhibit CT, the rate-limiting enzymatic step in PC synthesis
(29). These results suggest that the effects of TNF-
are through ceramide pathway intermediates and that the pathways
contain self-reinforcing controls that could prolong the elevated
levels of ceramide and the undesirable metabolic effects.
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EXPERIMENTAL PROCEDURES |
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Cell culture. We used H441 cells for these experiments. NCI-H441 [American Type Culture Collection (ATCC)] is a human adenocarcinoma thought to be derived from small-airway epithelial cells. These cells synthesize several of the constituents of surfactant (31) and have been widely used for studies in this area (5, 44). The cells were obtained from ATCC at the 50th passage, expanded in 10% fetal bovine serum (FBS)-McCoy's 5A medium with 50 µg/ml of gentamicin, frozen, and maintained in small aliquots in liquid nitrogen. Cells used for these experiments were maintained in the same medium. Experiments were conducted in six-well plates when cells had reached ~50-60% confluence. In all protocols, the medium was changed to McCoy's 5A without FBS 24 h before the start of the experiment.
These experiments studied the effects of TNF-Sphingomyelinase (EC 3.1.4.12) activity.
We measured acidic and neutral sphingomyelinase activity in H441 cells
using the method described by Quintern and Sandhoff (32).
Assay of neutral sphingomyelinase activity was performed in a buffer
containing 20 mM HEPES (pH 7.4), 10 mM MgCl2, 2 mM EDTA, 5 mM dithiothreitol, 10 mM -glycerophosphate, 750 µM ATP, 1 mM
phenylmethylsulfonyl fluoride, 10 µM pepstatin, 10 µM leupeptin, and 0.2% Triton X-100. After incubation for 5 min on ice, cells were
homogenized by repeated passage through an 18-gauge needle. Nuclei and
cell debris were removed by centrifugation at 800 g. The
protein concentration in the cell lysate was measured with a
bicinchoninic acid assay (Pierce) with BSA as standard. Fifty micrograms of protein were incubated for 2 h in a shaking water bath at 37°C in a buffer (100 µl final volume) containing 20 mM HEPES (pH 7.0), 1 mM MgCl2, and 0.02 µCi
[N-methyl-14C]sphingomyelin. At the
end of the reaction period, the
[14C]phosphosphocholine produced from
[14C]sphingomyelin was extracted with 800 µl of
chloroform-methanol (2:1 vol/vol) and 250 µl of water.
[14C]phosphosphocholine in the upper water phase
was determined by scintillation counting. Acidic sphingomyelinase
activity was measured identically but in a buffer of 250 mM sodium
acetate and 1 mM EDTA (pH 5.0).
PC:ceramide phosphocholinetransferase activity. We assayed phosphocholinetransferase activity according to Marggraf and Kanfer (24). Cells were lysed in hypotonic, cold 1 mM MgCl2 and homogenized with a Dounce homogenizer. After a low-speed centrifugation for 5 min to pellet cell debris, the supernatant was further centrifuged at 100,000 g for 30 min at 4°C. The pellet containing the membrane fraction was used for the enzyme assay. Phosphocholine transferase activity was determined by measuring the quantity of 3H-labeled sphingomyelin produced from phosphatidyl[N-methyl-3H]choline substrate. The reaction mixture contained 0.05 µCi of phosphatidyl[N-methyl-3H]choline, 10 mM HEPES (pH 7.4), 3 mM MnCl2, 1% fatty acid-free BSA, and 50 µg of membrane protein in a total volume of 100 µl. In some experiments, we also added 2 mg/ml of C8 ceramide to ensure that a substrate limitation would not affect the results (24). The incubations were carried out for 6 h in a shaking water bath at 37°C and were terminated by the addition of 1 ml of 0.2 M methanolic NaOH. The contents were heated at 55°C for 30 min to hydrolyze glycerolipids and cooled to 20°C. Fifteen micrograms of cold sphingomyelin carrier were added to each sample, immediately followed by the addition of 0.5 ml of 0.45 M HCl. The lower organic phase containing sphingomyelin was separated by TLC on silica gel G with two solvent systems: solvent 1, chloroform-methanol (95:5 vol/vol), and solvent 2, methanol-2-propanol-0.25% KCl-triethylamine (9:25:6:18 vol/vol). Radiolabeled sphingomyelin was scraped and counted by liquid scintillation counting. Activity in the cells at time 0 was taken as the control value.
CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) activity. We used the method of Cornell (9). We scraped cells and resuspended them in 0.5 ml of a buffer of 20 mM Tris, pH 7.0, 0.1 M NaCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. We homogenized the cell suspension with a Dounce homogenizer and sonicated it for 10 min at 4°C. The lysate was centrifuged for 5 min at 800 g, and the supernatant was further centrifuged at 100,000 g for 60 min. The membrane fraction was resuspended in 75 µl of homogenization buffer and used in the enzyme assay. The reaction contained 50 mM Tris · HCl, pH 8.5, 10 mM MgCl2, 0.5 mM EGTA, 2.4 mM sn-1,2-diolein in 0.15% Tween 20, 0.05 mCi of CDP[methyl-14C]choline, and 50 µl of membrane suspension in a final volume of 100 µl. We incubated the reaction mixture at 37°C in a shaking water bath for 30 min and terminated the reaction with the addition of 1.5 ml of methanol-chloroform (2:1 vol/vol). We extracted 14C-labeled PC into the organic phase of a Bligh-Dyer solvent partition and quantified it with scintillation counting. The results were normalized for the amount of protein in the assay mixture.
CT (EC 2.7.7.15) activity. The assay for CT activity was done on the membrane fraction with the method of Vance et al. (41) as modified by Weinhold and Feldman (43). We incubated 5-20 µg of protein in a shaking water bath for 1 h at 37°C in the reaction buffer [50 mM Tris-Cl (pH 6.5), 12 mM magnesium acetate, 10 µl of [methyl-14C]phosphocholine, and 3 mM CTP] in a total volume of 100 µl. At the end of the reaction time, we added 400 µl of water and 500 µl of charcoal saturated with 20 mM phosphosphocholine. After a vigorous mixing, the contents were centrifuged at 2,800 rpm, and the supernatants were passed over small charcoal columns. The charcoal, which adsorbs [14C]CDPcholine, was then washed with 1 ml of water (3 times) to remove nonspecific radioactivity. The adsorbed radioactive CDPcholine was eluted with 500 µl of formic acid and counted in a scintillation counter. The assay was linear over a concentration of 0-7 µg of protein (r2 = 0.9). In initial experiments, we compared activities in the membrane fraction either with or without the addition of 100 µM sonicated egg PC-oleic acid (1:1 molar ratio) to the assay mixture. The results in both protocols were nearly identical, and the data reported here are from assays without added liposomes. Liposomes added to the cytosolic fractions markedly enhanced measured activities as expected.
Phospholipase C (EC 3.1.4.3) and phospholipase A2 (EC 3.1.1.4) activities. The method used was according to Krug and Kent (19). We scraped and homogenized cells in a buffer of 0.4 M Tris · HCl, pH 7.3, 50 mM CaCl2, 1 mg/ml of BSA, and 0.6 M NH4SO4. We incubated ~50 µg of cell protein with a substrate of 1,2-di[1-C14]hexadecanoyl-L-3 PC and conducted the reaction for 1 h at 37°C. We terminated the reaction by adding 0.2 M Na2EDTA, pH 7.3, and extracted the lipids. We added carrier lipids of diacylglycerol (DAG) and lysophosphatidylcholine (lysoPC) and resolved them with TLC on silica gel thin-layer plates. DAG was separated with the chromatographic system of Freeman and West (13), with a solvent system of ethyl ether-benzene-ethanol-acetic acid [40:50:2:0.2] followed by ethyl ether-hexane [6:94]. 1,2-DAG was clearly separated from 1,3-DAG and cholesterol in this solvent system. The lipids were visualized by spraying with 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS), and the 1,2-DAG was spot scraped and counted. LysoPC was separated with the method of Touchstone et al. (40) as described in Quantification of phospholipids. Phospholipase (PL) C was assayed as the amount of radioactive DAG formed per hour per microgram of protein; PLA2 was assayed as the amount of lysoPC.
Chromatography of sphingolipids. Sphingomyelin, ceramide, and sphingosine, when needed together for the analysis of an experiment, were isolated by the following procedures. Extracted lipids were subjected to mild base hydrolysis as described by Marggraf and Kanfer (24). Sphingolipids were isolated from hydrolyzed glycerolipids by solvent partition and separated by TLC with chlororoform-acetone-methanol-acetic acid-water (150:40:20:20:10 vol/vol). Sphingomyelin, ceramide, and sphingosine pools were localized by TNS spray and, if radioactive, were scraped and quantified by measuring the amount of radioactivity. In this chromatographic system, sphingomyelin, C6 ceramide or endogenous ceramides, C2 ceramide, and sphingosine were resolved.
Quantification of phospholipids. We quantified PC by gas chromatography after extraction, separation by TLC, and quantitative transesterification with an internal standard of diheptadecanoyl PC. PCs were isolated with the following protocol. Lipids in cells and media were extracted and dissolved in a small volume of 1:1 (vol/vol) chloroform-methanol. The phospholipids were separated by silica gel TLC with the following chromatographic system: first, 95:5 (vol/vol) chloroform-methanol to remove neutral lipids followed by the solvent system B of Touchstone et al. (40). Phospholipids were detected by a TNS spray, and the PC band was recovered by scraping and was transesterified with 1% H2SO4 in methanol for 2 h at 70°C together with an internal standard of a known amount of diheptadecanoyl PC. Methyl esters were extracted with hexane, and PC content was quantified by gas-liquid chromatography.
Materials. Reagents were from Sigma or Calbiochem. Radioactive materials were from Amersham or New England Nuclear.
Data analysis. Statistical evaluation was done by ANOVA with the use of Statview (Abacus Concepts, Berkeley, CA). Significance was assumed when P < 0.05, with P < 0.1 shown for informational purposes.
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RESULTS |
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Effects of ceramide on endogenous sphingolipid content.
We first showed that H441 cells respond to TNF- with the expected
activation of sphingomyelinases. The results are shown in Fig.
1. A relatively low concentration of
TNF-
(10 ng/ml) activated both acidic and neutral sphingomyelinases
to >150% of control values within 2 h after administration, and
these activities remained elevated for 4 h. We next investigated
the effects of ceramide on sphingomyelinase activity, thinking that
ceramide might limit the extent of sphingomyelin hydrolysis. The
results in Fig. 1 indicate the opposite: 10 µM C2
ceramide activated both acidic and neutral sphingomyelinases as
effectively as 10 ng/ml of TNF-
, with maximum activation from 2 through 4 h. The effect was dose dependent and specific because
dihydro-C2 ceramide had no effect (dihydro-C2
ceramide, 103% of control value at 2 h).
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Effects of ceramide on PC content and metabolism.
Mallampalli et al. (21) have reported that betamethasone
given to adult rats decreased sphingomyelinase activity in lung tissue,
and, as expected, this effect was associated with an increase in
sphingomyelin and a decrease in sphingosine. Furthermore, they found
that sphingosine was a competitive inhibitor of CT, thereby implying
that an activation of sphingomyelinase would result in an inhibition of
CT. We tested this assumption by performing experiments on the effects
of TNF- on CT activity and on the synthesis of PC. A relatively low
dose of TNF-
(10 ng/ml) reduced CT activity to 50% of control
values within 6 h, and the effect was persistent through at least
24 h. C2 ceramide and sphingosine were equally effective, but dihydro-C2 ceramide had no inhibitive effect
(Fig. 6).
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Are the effects of ceramide a nonspecific inhibition of the enzymes of the Kennedy pathway? We considered that the effects of C2 ceramide on PC synthesis might be from the inhibition of multiple sites in the pathway of de novo synthesis, perhaps symptomatic of a generalized reduction of cell function due to developing apoptosis or necrosis. To assess this, we assayed the activity of the last enzyme in the Kennedy pathway, CDPcholine:1,2-DAG cholinephosphotransferase, reasoning that if the inhibition of PC synthesis by ceramide was nonspecific, this activity should be inhibited as was that of CT. This was not found. C2 ceramide (10 µM) had no demonstrable effect on the activity of CDPcholine:1,2-DAG cholinephosphotransferase at any time period between 2 and 24 h (P > 0.10; data not shown). These results indicate that ceramide does not affect all of the enzymes of the Kennedy pathway and suggest that the results are not due to the nonspecific inhibition of PC from cell death.
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DISCUSSION |
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In this paper, we present data that directly link two commonly
observed features of chronic tissue injury, the elaboration of
increased amounts of cytokines and the perturbation of metabolic regulation. We conclude that TNF- activates pathways that reduce PC
concentration in H441 cells and that markedly inhibit the de novo
synthesis of new PC. Metabolic products of sphingomyelin probably
modulate these effects on PC synthesis, and in these cells, the
signaling likely involves the inhibition of CT activity. This
regulation through CT may not be ubiquitous for all cells. In a recent
paper, Bladergroen et al. (7) studied the effects of
C6 ceramide on PC synthesis in Rat-2 fibroblasts. As in our findings, these investigators found that ceramide (C6
ceramide) inhibited PC synthesis, although doses of 25 µM or higher
were required. Directly in opposition to our results, however, they found that C6 ceramide had no effect on CT; rather it
inhibited CDPcholine:1,2-DAG cholinephosphotransferase by >50%. We
have no explanation for these differing results. They are unlikely to
be a result of technical differences, at least as discerned by the
description of the enzyme assay methodology, which was identical to
ours. We note that Bladergroen et al. used a fibroblast, whereas our
studies were done with immortalized epithelial cells. This raises the
interesting possibility that the mode of action of ceramide on PC
synthesis may differ with cell type, a surprising but certainly not
impossible phenomenon.
We used three separate protocols to demonstrate that sphingomyelin pools are reduced and endogenous ceramide is elevated in cells treated with either C2 ceramide or sphingosine. These protocols were based on steady-state labeling of sphingolipid pools with L-[3-3H]- serine, a technique shown to duplicate results obtained by the conventional assay of sphingolipid mass (10, 11, 36, 38). The findings and conclusions drawn from all three protocols were completely consistent. We have not identified which of the sphingomyelin metabolites is responsible for the inhibition of sphingomyelin resynthesis; indeed, several may be acting. Changes in sphingosine pools were not evident after steady-state labeling for 24 h (Figs. 4 and 5) or with quasi-steady-state labeling for 4 h with L-[3H]serine and treatment with C2 ceramide (data not shown). However, this may not be expected if the sphingosine were to act through the initiation of a signaling pathway or be metabolized to another active sphingomyelin metabolite. In such circumstances, the changes in sphingosine mass might be small and transient. For instance, sphingosine phosphate has been shown to affect the activities of cell cycle intermediates and to regulate mitosis and may participate in other signaling pathways, but the actual sphingosine-derived molecules have not been identified (36).
A perplexing aspect of a purported signaling scheme involving TNF-
is the relatively short time span in which increased levels of active
TNF-
are observed (for example see Ref. 33). TNF-
and interleukin (IL)-1
both generally peak within 1 h, whereas the inhibition of surfactant synthesis is notable for days. TNF-
and
IL-1
have frequently been shown to initiate transcription of other
growth factors (37), and we have shown that this may be
responsible for the increased amounts of hepatocyte growth factor seen in chronic lung injury in rats in response to
100% O2 (42). The elaboration of new
regulating factors, therefore, may be an explanation of the longer
range effects of TNF-
. The results presented here, however, provide
yet another mechanism for the duration and intensity of the actions of
TNF-
. Ceramide itself, or a further metabolic product of ceramide,
modulates the principal enzymes needed for the restoration of
sphingomyelin-ceramide homeostasis; i.e., it further activates
sphingomyelinases and inhibits PC:ceramide phosphocholine
transferase. We have not identified which intermediate(s) is
responsible for these actions; it could be sphingosine or one further
downstream. Whatever the ceramide metabolite, these actions would be
expected to prolong the elevation in the ceramide pool and,
consequently, extend the time and duration of the ceramide effect. In
one set of experiments, we measured the resynthesis of sphingomyelin
from ceramide when C2 ceramide was given as a purported
metabolic perturbant and then removed. The inhibited resynthesis was
evident through at least 8 h after removal of the C2
ceramide from the cell medium (we did not follow the experiment beyond
8 h).
An increasing number of reports are emerging that indicate that changes
in PC metabolism are a common feature of the pathophysiology of
experimental chronic lung injury (18) or adult respiratory distress syndrome, especially when it is associated with
inflammation induced by infection (for examples, see Refs.
14, 28, 31). A concomitant
elevation of cytokines is generally observed (35). The
data presented here suggest that the presence of TNF- and IL-1
may not only be coincidental but rather causal and self-reinforcing. Moreover, these effects may not be unique to lung cells. The pathways of sphingomyelin metabolism are common to all cells (15,
26), and many cells use the Kennedy pathway for the de novo
synthesis of PC. We expect, therefore, that other tissues
experiencing inflammatory injury may also share the changes in PC and
sphingolipid content and metabolism that were found in lung cells.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-52664.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. J. King, Dept. of Physiology, Univ. of Texas Health Science Ctr., 7703 Floyd Curl Dr., San Antonio, TX 78284-7756 (E-mail: kingr{at}uthscsa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 November 2000; accepted in final form 7 February 2001.
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