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University of Arkansas for Medical Sciences, Division of Nephrology, and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas.
Correspondence to Dr. Sudhir V. Shah, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Slot 501, Little Rock, AR 72205. Phone: 501-257-5832; Fax: 501-257-5827; E-mail: shahsudhirv{at}uams.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was therefore undertaken to examine the role of ceramide synthase for ceramide generation in renal tubular epithelial cells exposed to H2O2. We also examined the cause-effect relationship among ceramide generation, DNA damage, and cell death in oxidant injury to renal tubular epithelial cells. Our data provide strong evidence that H2O2 increases ceramide synthase activity, resulting in enhanced ceramide generation, which is a key modulator of DNA damage and cell death in oxidant injury to renal epithelial tubule cells.
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Materials and Methods |
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-32P] ATP
and [N-methyl 14C] sphingomyelin were obtained from Amersham Life
Science, Inc. (Arlington Heights, IL). All other agents were purchased from
Sigma Chemical Co. (St. Louis, MO). Silica gel 60 plates for thin-layer
chromatography (TLC) were purchased from Whatman (Hillsboro, OR).
Cell Culture
LLC-PK1 cells obtained from the American Type Culture Collection
(Rockville, MD) were cultured as in our previous studies
(11,12,13,14,15).
Cultures were maintained in a humidified incubator gassed with 5%
CO2-95% air at 37°C and fed at intervals of 48 to 72 h. Cells
were used 2 days after confluence.
Experimental Protocol
To examine the effect of H2O2 on ceramide generation,
cells were rinsed with serum-free Dulbecco's modified Eagle's medium (DMEM) at
pH 7.4 and exposed to 1 mM H2O2 for different time
intervals as indicated, and ceramide generation was measured as described
below. The dose of H2O2 used and the time of exposure in
the study were based on our previous studies
(11,12).
To determine which pathway—namely, hydrolysis of sphingomyelin by
sphingomyelinases or ceramide generation by ceramide synthase—is
involved in oxidant-induced ceramide generation, we measured the activities of
ceramide synthase and sphingomyelinases as well as sphingomyelin content in
the cells exposed to H2O2, as described below. To
examine the role of ceramide synthase in oxidant injury, we used a specific
inhibitor of ceramide synthase, fumonisin B1, which blocks sphinganine
N-acyltransferase, resulting in a decrease in ceramide
(9,10,16,17,18).
To examine the effect of fumonisin B1 on H2O2-induced
ceramide generation as well as DNA damage and cell death, LLC-PK1 cells were
washed with serum-free DMEM, preincubated with 50 µM fumonisin B1 for 30
min, and then exposed to H2O2. The dose of fumonisin B1
was chosen on the basis of our recent studies
(14).
Assay for Ceramide
The lipids were extracted according to the method of Bligh and Dyer
(19). Lipids in the
organic-phase extract were dried under nitrogen and subjected to mild alkaline
hydrolysis (0.1 N methanolic KOH for 1 h at 37°C) to remove
glycerophospholipids (9).
Samples were re-extracted, and the organic phase was dried under nitrogen.
Ceramide was quantified by in vitro diacylglycerol (DG) kinase assay,
as described elsewhere (20)
and used in our recent studies
(14), with minor modification.
In brief, the dried extracted lipids under nitrogen were dissolved in 20 µl
of 7.5% n-octyl-ß-glucopyranoside, 25 mM
dioleoylphosphatidylglycerol, and1 mM diethylenetriaminepentaacetic acid
(DETAPAC), pH 7.0. Then the following solutions were added in order: 50 µl
of reaction buffer containing 100 mM imidazole HCl (pH 6.6), 100 mM NaCl, 25
mM MgCl2, 2 mM ethyleneglycolbis(B-aminoethyl
ether)-NN-tetraacetic acid (pH 6.6), 2 µl of 100 mM dithiothreitol
in 1 mM DETAPAC (pH 7.0), 10 µl of DG kinase (5 µg/10 µl) from E.
coli strain N4830/pJW10 diluted in 100 mM imidazole-HCl and 1 mM DETAPAC
(pH 6.6), and 8 µl of water. The reaction was initiated by the addition of
10 µl each of unlabeled 10 mM ATP and [-32P] ATP (4.5 µCi per
sample) in 20 mM imidazole (pH 6.6), 1 mM DETAPAC, and incubation at 25°C
for 45 min. The reaction was stopped by addition of 0.7 ml 1% (wt/vol)
perchloric acid. The lipids were extracted with 3 ml of methanol/chloroform
(2/1 vol/vol), 1 ml of chloroform, and 1 ml of 1% perchloric acid. The lower
chloroform phase was washed with 2 ml of 1% perchloric acid/methanol (7/1
vol/vol) and then used for measurement of ceramide. The standard ceramide type
III (from bovine brain) were also treated in the same manner. Ceramide
1-phosphate was resolved by TLC on silica gel 60 plates by use of a solvent
system of chloroform/acetone/methanol/acetic acid/water (10.5/5/2/2/1,
vol/vol/vol/vol/vol). The TLC plate was dried and autoradiographed overnight.
The band corresponding to ceramide 1-phosphate was scraped, and incorporation
of 32P into ceramide 1-phosphate was quantified by Cerenkov
scintillation counting. The level of ceramide was determined by comparison
with a standard curve generated concomitantly of known amounts of ceramide.
Data are expressed as pmol ceramide/nmol of total lipid phosphate, determined
according to the method of Ames and Dubin
(21).
Measurement of Sphingomyelin Content
The lipids were extracted, dried under nitrogen as described above, and
resolved on TLC plate by use of chloroform/methanol/acetic acid/water
(50/30/8/5, vol/vol/vol/vol), as described elsewhere
(9). The standards of known
amounts of sphingomyelin were also run on TLC. The spot corresponding to
sphingomyelin was visualized by use of iodine vapor, scraped, and transferred
into glass tube. The lipids from silica were extracted three times with 500
µl of chloroform/methanol (2/1 vol/vol), and the phosphate content in the
combined extracts was measured. The sphingomyelin content was quantified by
use of a standard curve of phosphate amount for sphingomyelin and normalized
to total phosphate.
Preparation of the Subcellular Fractions to Measure Ceramide
Synthase
Two subcellular fractions were tested: mitochondrial and microsomal. The
fractions were prepared as described elsewhere
(22), with minor modification.
Cells were scraped and pelleted by centrifugation at 1000 x g
at 4°C. Cells were washed twice with cold phosphate-buffered saline and
resuspended in 300 µl of homogenizing buffer (20 mM Hepes [pH 7.4], 0.25 M
sucrose, and 10 µg/ml leupeptin and 10 µg/mg antipain). Cells were
homogenized and spun at 800 x g for 10 min to remove nuclei and
debris. The supernatant was centrifuged at 10,000 x g for 30
min to precipitate mitochondria, and the pellet was resuspended in the
homogenizing buffer. The supernatant was further centrifuged at 100,000
x g for 60 min. The transparent microsomal membrane pellet was
resuspended in the homogenizing buffer, which contained 0.2% Triton X-100, and
used for the ceramide synthase assay as described below. Protein contents were
determined by use of the BCA protein assay kit, according to the
manufacturer's instructions (Pierce, Rockford, IL).
Assay for Ceramide Synthase
Ceramide synthase activity in LLC-PK1 cells was measured as described
elsewhere (9), with minor
modifications. If not specifically changed, the conditions of the reaction
were as follows. The same amount of protein (50 to 100 µg) from each sample
was incubated in a final volume of 1-ml reaction mixture that contained 2 mM
MgCl2, 20 mM Hepes (pH 7.4), 20 µM defatted bovine serum
albumin, 20 µM dihydrosphingosine (sphinganine), 70 µM unlabeled
palmitoyl—CoA, and 0.2 µCi of [1-14C] palmitoyl—CoA
(specific activity, 53.6 mCi/mmol) for 60 min at 37°C. The reaction was
stopped by extraction of lipids by use of 2 ml of chloroform/methanol (1/2
vol/vol). The lower phase was dried under nitrogen and applied to a 20-cm
silica gel TLC plate. Radiolabeled dihydroceramide was resolved by use of a
solvent system of chloroform/methanol/1 N ammonium hydroxide (40/10/1
vol/vol/vol), identified by iodine vapor staining on the basis of comigration
with ceramide type III standards, and quantified by a liquid scintillation
counting. Data are expressed as the percentage of control at time point 0.
Assay for Acid or Neutral Sphingomyelinase
At the end of experiment, serum-free medium was removed, and the cells were
washed two times with phosphate-buffered saline at 4°C. Protein extract
from cells was prepared as follows. Cells were lysed in 200 µl of either
neutral buffer containing 20 mM Hepes (pH 7.4), 10 mM MgCl2, 2 mM
ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 100 mM Na3
VO4, 100 mM NaMO4, 10 mM ß-glycerophosphate, 750
µM ATP, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM of
pepstatin, and 0.5% CHAPS or acid buffer that contained 0.2% Triton X100, 0.5%
CHAPS, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM
pepstatin. After incubation for 20 min at 4°C, cells were homogenized. To
obtain the protein extracts for neutral sphingomyelinase assay, centrifugation
at 800 x g was used
(23). Protein extracts for
acid sphingomyelinase assay were obtained by centrifugation at 16,000 x
g for 5 min, as suggested by Wiegmann et al.
(23) and
Jaffrézou et al.
(24). The supernatants were
kept at -80°C until used for the assay.
The activity of acid or neutral sphingomyelinase was measured as described elsewhere (23,24,25,26), with modification (7). For the acid sphingomyelinase assay, 10 nmoles of unlabeled sphingomyelin and 0.02 µCi of [N-methyl 14C] sphingomyelin (specific activity, 56.0 mCi/mmol) were added to a glass tube, and the lipids were dried under N2. Then 50 µl of acidic buffer containing 250 mM sodium acetate, 1 mM ethylenediaminetetraacetic acid (pH 5.0), and protein extract (30 to 50 µg) were added, and the volume was brought up to 150 µl. The samples were then incubated for 90 min and 2 h at 37°C for acid and neutral sphingomyelin assay, respectively. For the neutral sphingomyelinase assay, the reaction was carried out in neutral buffer that contained 20 mM Hepes (pH 7.4) and 1 mM MgCl2 (pH 7.4). At the end of incubation, the reaction was stopped by the addition of 0.8 ml of chloroform/methanol (2/1 vol/vol) and 0.25 ml of H2O. The samples were then vortexed, centrifuged, and the upper phase (0.3 ml), which contained radioactive phosphatidyllcholine, a product of hydrolysis of [14C] sphingomyelin, was counted by scintillation counting. The data are expressed as pmol of [14C] sphingomyelin hydrolyzed/mg of protein per h. In the used cells, the activity of acid sphingomyelinase in 16,000 x g supernatants was not different from the activity in 800 x g supernatants, as had been observed in some studies (27,28,29,30,31,32).
Determination of DNA Strand Breaks
The residual double-strand DNA was measured by the alkaline unwinding assay
and determination of ethidium bromide fluorescence used in our previous
studies
(11,13,14,15).
In Situ Detection of DNA Fragmentation
The terminal deoxynucleotidyl transferase nick end labeling technique was
used to detect in situ DNA strand breaks, as described in our
previous studies (15). Cells
were grown to confluence on two-chamber tissue culture slides. At the end of
the experiment, the cells were fixed in 4% paraformaldehyde and then treated
in 1% H2O2 in phosphate-buffered saline for 10 min to
remove endogenous peroxidase activity. After labeling with digoxigenin-labeled
nucleotides, the cells were treated with peroxidase-conjugated
anti-digoxigenin antibody and stained with peroxide substrate by use of the
in situ ApopTag Plus labeling method (Oncor, Gaithersburg, MD).
Determination of Cell Injury
Cell viability was determined using trypan blue exclusion, as described
elsewhere
(11,12,13,14,15).
Irreversible cell death was measured by use of lactate dehydrogenase (LDH)
release (LDH kit, DG-1340-K, Sigma).
Statistical Analyses
Results are means ± SEM. Statistical significance was determined by
unpaired t test and ANOVA. P < 0.05 was considered to be
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of H2O2 on Ceramide Synthase Activity in
LLC-PK1 Cells
Preliminary experiments were performed to test the optimal conditions
(concentration of substrate and incubation time) for the ceramide synthase
activity assay in LLC-PK1 cells. We observed that the maximum rate of the
enzymatic reaction was reached with 20 µM dihydrosphingosine and 60 min
incubation, as had been suggested elsewhere by Bose et al.
(9)
(Figure 2A). Under these
conditions, the enzyme activity was analyzed in mitochondrial and microsomal
fractions (Figure 2B), where
high ceramide synthase activity is usually observed
(29,30).
Apparent kinetic parameters were calculated by use of the Lineweaver-Burk
equation. The ceramide synthase reaction in mitochondrial fraction was
characterized by Vmax = 0.62 pmol/mg per min and
Km = 0.19 µM. In microsomal fractions,
Vmax was 1.07 pmol/mg per min and Km
was 0.09 µM. Thus, in further experiments we used microsomal fraction
because it contained a higher activity of ceramide synthase.
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We next examined whether H2O2-induced enhanced generation of ceramide is due to activation of ceramide synthase or sphingomyelinases. To examine whether oxidant stress results in ceramide synthase activation, we determined the time course of the effect of H2O2 on ceramide synthase activation in LLC-PK1 cells. The exposure of cells to H2O2 resulted in a rapid increase of ceramide synthase activity in microsomes at 5 min (119 ± 3% of control, n = 8, P < 0.005) that continuously increased up to 60 min (145 ± 7% of control, n = 8, P < 0.0001, Figure 3). These data indicate that the H2O2-induced ceramide generation is due to the activation of ceramide synthase in LLC-PK1 cells.
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Effect of H2O2 on Sphingomyelin Content and
Sphingomyelinase Activity in LLC-PK1 Cells
To determine the contribution of sphingomyelinases to
H2O2-induced increase in ceramide, we measured the
sphingomyelin content in LLC-PK1 cells exposed to H2O2.
As shown in Figure 4A, there
was no difference in sphingomyelin content between control cells and cells
exposed to H2O2, which suggests little contribution of
sphingomyelinases to oxidant-induced ceramide generation in renal tubular
epithelial cells. We also measured the activity of acid or neutral
sphingomyelinase in cells exposed to H2O2. In contrast
to previous studies, which suggested that the contribution of
sphingomyelinases to H2O2-induced ceramide generation
(6), exposure of LLC-PK1 cells
to H2O2 did not result in any significant change in acid
or neutral sphingomyelinase activity
(Figure 4, B and C). Taken
together, these data strongly indicate that H2O2 is a
regulator of ceramide synthase but not of sphingomyelinase in renal tubular
epithelial cells. The data also indicate that the pathway that triggers
ceramide generation varies with cell types even in response to the same
stimuli.
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Effect of Inhibitor of Ceramide Synthase, Fumonisin B1, on
H2O2-Induced Ceramide Generation
In a separate study, we examined the ability of a specific inhibitor of
ceramide synthase, fumonisin B1, to suppress
H2O2-induced ceramide generation in LLC-PK1 cells. As
shown in Figure 5, there was no
significant difference in the ceramide level between control cells pretreated
with fumonisin B1 and those without it (19.6 ± 0.4 and 20.4 ±
4.2 pmol/nmol phosphate, respectively, n = 3). However, fumonisin B1
did indeed prevent H2O2-induced ceramide generation (at
60 min, control: 20.4 ± 4.2 pmol/nmol phosphate;
H2O2 alone, 45.2 ± 3.3 pmol/nmol phosphate;
H2O2 + fumonisin B1, 21.2 ± 2.8 pmol/nmol
phosphate; n = 3, P < 0.005).
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Effect of Fumonisin B1 on H2O2-Induced DNA
Damage
To determine the role of ceramide/ceramide synthase in oxidant-induced DNA
damage, we examined the effect of the inhibition of ceramide synthase using
fumonisin B1 on H2O2-induced DNA strand breaks. For that
study, we used a 60-min time point after exposure of cells to
H2O2
(11). As shown in
Figure 6, fumonisin B1 was
highly protective against H2O2-induced DNA strand breaks
(residual double-strand DNA at 60 min: control, 86 ± 1%;
H2O2 alone, 53 ± 2%; H2O2 +
fumonisin B1, 81 ± 7%; n = 3 to 4, P < 0.01). In a
separate study, we examined the effect of fumonisin B1 on
H2O2-induced DNA fragmentation
(Figure 7). Fumonisin B1 also
provided a marked protection against H2O2-induced DNA
fragmentation in LLC-PK1 cells. Taken together, these data suggest that the
inhibition of ceramide synthase is highly protective against
H2O2-induced DNA damage to renal tubular epithelial
cells.
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Effect of Fumonisin B1 on H2O2-Induced Cell
Death
Finally, we examined the effect of fumonisin B1 on
H2O2-induced cell death. In our previous study
(12), we showed that 60 min of
H2O2 treatment resulted in a significant cell death of
LLC-PK1 cells. Thus, we used a 60-min time point after exposure to
H2O2. Irreversible cell death was accessed by use of LDH
release. In preliminary experiments, we measured LDH release by LLC-PK1 cells
treated with 1 mM H2O2 within 24 h after treatment. Our
observation was that the release started to increase after 1 h of treatment
(10%) and reached its apparent plateau (30% to 40%) between 3 and 24 h after
treatment. As shown in Figure
8, fumonisin B1 did not increase the death of untreated cells
(1.05 ± 0.35% versus 1.50 ± 0.35% in control). However,
it prevented H2O2-induced cell death
(H2O2 alone, 10.24 ± 0.89%;
H2O2 + fumonisin B1, 2.90 ± 0.64%, n =
10 to 16). A similar pattern was obtained when cell death was measured by use
of trypan blue staining (data not shown). Therefore, fumonisin B1 provided a
significant protection against cell death in the used model. Taken together,
our data provide the first evidence that H2O2 is a
regulator of ceramide synthase rather than sphingomyelinases and that
H2O2-induced enhanced generation of ceramide through the
activation of ceramide synthase is a determinant of DNA damage and cell death
in oxidant injury to renal tubular epithelial cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have shown that exposure of LLC-PK1 cells to H2O2 resulted in a rapid increase in ceramide generation and activation of ceramide synthase but not sphingomyelinases, which preceded any evidence of DNA damage and cell death. Exposure of LLC-PK1 cells to H2O2 did not result in any significant change in activity of sphingomyelinases. This is supported by the finding that there was no difference in sphingomyelin content between control cells and cells exposed to H2O2. Taken together, these data indicate that the target of the enzyme responsible for ceramide generation in oxidant stress is ceramide synthase rather than sphingomyelinases. Our observation that H2O2 resulted in an increase in ceramide level without any significant increase in activity of sphingomyelinases is similar to that reported by Zager et al. (7), in which iron loading resulted in increase in ceramide level in human proximal tubular cells. However, in their study, pretreatment with fumonisin B1 failed to prevent ceramide generation, and ceramidase activity was inhibited under their condition. On the basis of those data, they concluded that the inhibition of ceramidase is responsible for the rise in ceramide in response to iron-mediated oxidant stress. Although the reasons for the differences are not known, the different response of ceramide generation in response to various stimuli by different cell types or conditions studied have been reported. For example, H2O2 has been shown to activate sphingomyelinases (6) in other cells, and the anti-cancer agent, daunorubicin (9), in which reactive oxygen metabolites have been implicated (32), has been shown to result in ceramide synthase activation with a concomitant increase in sphingomyelin content.
To examine a role of ceramide synthase in regulation of ceramide, which results in DNA damage and cell death in oxidant injury, we used a specific inhibitor of ceramide synthase, fumonisin B1. This compound has very similar structure to sphingosine or sphinganine, which is a substrate for ceramide synthase, and thus blocks ceramide synthase, resulting in a decrease in intracellular ceramide level (9,10,16,17,18). Because of the nature of the action of fumonisin B1, it is highly unlikely for this compound to inhibit sphingomyelinases, which is supported by the inability of fumonisin B1 to prevent cell death in a model in which sphingomyelinases have been implicated (4,9). Recent studies have shown the protective effect of fumonisin B1 against apoptotic cell death in response to stimuli (9,10,16) in which ceramide synthase is involved for ceramide generation. In fact, our data confirmed the ability of fumonisin B1 to suppress H2O2-induced ceramide generation in LLC-PK1 cells and showed that fumonisin B1 provided a marked protection against H2O2-induced DNA damage and cell death. In a recent study, we showed that exogenous ceramide results in DNA damage and cell death in LLC-PK1 cells (14) similar to the effect of H2O2. Taken together, these data strongly indicate that ceramide synthase plays a major role of oxidant-induced ceramide generation, which results in DNA damage and cell death in oxidant injury to renal tubular epithelial cells.
Oxidant stress has been shown to result in DNA fragmentation in various types of cells (33), and scavengers of reactive oxygen metabolites prevent oxidant-induced DNA fragmentation (34,35). We have previously shown that oxidant stress induces endonuclease activation as an early event, leading to DNA damage and cell death in renal tubular epithelial cells (11). Although the sequential consequences of ceramide on endonuclease activation and cell death still remain to be elucidated, our data indicate that activation of the ceramide synthase-dependent pathway is one of the mechanisms by which oxidant stress causes endonuclease activation, leading to DNA damage and cell death in renal tubular epithelial cells.
In summary, the present study has demonstrated that H2O2 increased ceramide synthase activity but not sphingomyelinases, which resulted in enhanced ceramide production before any evidence of DNA damage and cell death in LLC-PK1 cells. The inhibition of ceramide synthase was able to suppress oxidant-induced ceramide production and prevented DNA damage and cell death. Our data provided the first evidence that oxidant stress is a mediator of ceramide synthase activity, resulting in enhanced ceramide generation in oxidant injury to renal epithelial tubule cells.
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, control cells; 
,
H2O2-treated cells.
], and 20 [
] µM). (B) Incorporation of
14C-palmitoyl CoA to form ceramide by ceramide synthase was
compared by use of mitochondrial (
