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Introduction
Over the past decade, sphingolipid sphingomyelin and
its metabolites, namely, ceramide (Cer), sphingosine (SPH),
and sphingosine-1-phosphate (S1P), have emerged as a new
class of signal-transduction molecules mediating a variety
of cellular processes[1,2]. Cer is known to be associated with
growth arrest and apoptosis[3], whereas S1P is thought to
promote growth and survival[3,4]. It has also been proposed
that the ratio of Cer to S1P within a cell determines the
outcome of an apoptotic stimulus[3_5].
Ceramidase (CDase) deacylates Cer to form SPH, which
can be further phosphorylated to S1P by the sphingosine
kinase (SPHK). Thus far, 3 subtypes of CDase, namely, acid,
neutral, and alkaline CDase, have been cloned and classified
in mammalian cells according to the pH optima required for
their activity. The current understanding is that the
formation of SPH occurs via hydrolysis of Cer by CDase and not
via de novo synthesis; therefore, CDase is vital not only for
Cer degradation, but also for SPH/S1P
generation[6]. Several lines of evidence support the role of CDase in
sphingolipid-mediated cell regulation in response to a variety of stimuli,
including cytokines[7_9], oxidized low density lipoprotein
(oxLDL)[10], growth
factors[11], and advanced glycation end
products[12]. For example, the activation of neutral CDase
(N-CDase) results in a decrease in the intracellular levels of
Cer, thereby protecting rat mesangial cells from
cytokine-induced apoptosis[8,9]. The overexpression of acid CDase
provides protection against cytokine-induced apoptosis in
murine fibrosarcoma L929 cells by the reduction of
intracellular Cer, thereby directing the Cer/S1P rheostat towards cell
survival[13]. Taken together, these results indicate that CDase
may function as a key "switch" in determining the
intracellular levels of Cer and S1P, its downstream active metabolite,
thus, playing a pivotal role in the regulation of cell survival
or apoptosis in response to external
stimuli[6].
Cytokines, such as interleukin (IL)-1β, TNF-α, and
interferon (IFN)-γ, are toxic to pancreatic β-cells and have been
implicated in the pathogenesis of
diabetes[14]. Recent data show that
IL-1β and TNF-α induce early and sustained increases in SPHK activity and S1P levels in rat
β-cell line INS-1 and pancreatic islets. Moreover, exogenous S1P provides
protective effects against cytokine-induced apoptosis in
β-cells[15,16]. Although CDase is known to act as a key "switch"
in the sphingolipid signaling pathway and potentially
protects the cells against cytokine-induced damage, the role of
the CDase pathway in β-cells has not been elucidated. In
this study, we investigated for the first time the activity and
expression of N-CDase in β-cells and its role in the cellular
response to cytokines.
Materials and methods
Reagents Recombinant rat IL-1β, TNF-α, and
IFN-γ, protease inhibitor cocktail, o-phthaldehyde, and the mouse
monoclonal anti-β-actin antibody were obtained from Sigma
(St Louis, MO, USA). The Annexin V_fluorescein-isothiocyanate (FITC) kit was from Bender MedSystems
(Vienna, Austria). TriPure isolation reagent was from Roche
(Indianapolis, IN, USA) and the reverse transcription kit was
from Toyobo (Osaka, Japan).
C17-D-erythro-SPH and
C12-Cer were purchased from Avanti Polar Lipids (Alabaster, AL,
USA). C12-D-erythro-SPH was from Larodan (Malmö,
Sweden). Secondary horseradish peroxidase-conjugated
antibodies (swine antirabbit immunoglobulin [IgG] and
rabbit antimouse IgG) were purchased from DakoCytomation
(Glostrup, Denmark). Triton X-100 and the bicinchoninic
acid (BCA) protein assay kit were from Pierce (Rockford, IL,
USA).
Cell culture The rat insulin-secreting INS-1 cells
(passages 16_33, a kind gift from Prof Xiao HAN, Key
Laboratory of Human Functional Genomics of Jiangsu Province,
Nanjing Medical University, Nanjing, China) were grown in
RPMI-1640 medium supplemented with 10 mmol/L HEPES,
10% fetal calf serum (FCS), 2 mmol/L L-glutamine, 1 mmol/L
sodium pyruvate, 50 µmol/L 2-mercaptoethanol, 100 U/mL
penicillin, and 100 mg/L streptomycin.
Polyclonal antibody preparation An affinity-purified
rabbit antirat N-CDase polyclonal antibody was raised against
the peptide (KNRGYLPGQGPFVANFA) corresponding to
amino acids 311_327 of rat N-CDase (GenBank accession
No NM053646), and the peptide sequence was designed
according to Geoffroy et al[12].
RNA isolation and quantitative RT_PCR The INS-1 cells
were seeded in 60 mm dishes and cultured for 2 d in complete
medium. Then the medium was replaced with RPMI
supplemented with 0.5% FCS for 4 h, followed by incubation with
or without a cytokine mixture (5 ng/mL IL-1β, 10 ng/mL
TNF-α, and 50 ng/mL IFN-γ). During the indicated time periods,
total cellular RNA was extracted with TriPure isolation reagent,
and 1 µg of RNA was reverse transcribed. Quantitative PCR
was then performed using a 7900HT real-time PCR system
(Applied Biosystems, Foster, CA, USA) and carried out in a
25 µL reaction mixture consisting of 2 µL cDNA, 12.5 µL
2×Master mix, 0.625 µL of the probe (20 µmol/L), and 1.125
µL of 20 µmol/L forward and reverse primers. The reactions were
performed with 40 cycles (30 s at 95 °C and 1 min at 60
°C). The primers used for the real-time PCR were as follows: N-CDase,
5'-CCCAGGGCGGCTTTACA-3' (forward) and 5'-TGGGTGAACATGACGGATGT-3' (reverse), and GAPDH,
5'-CAAGTTCAACGGCACAGTCAA-3' (forward) and 5'-TGGTGAAGACGCCAGTAGACTC-3' (reverse). The GAPDH
gene was amplified for each sample and used as an internal
control gene to calculate the relative level of
expression for the N-CDase gene using the
2-ΔΔCt method[17].
Assessment of apoptosis and necrosis For the apoptosis
assessment, the INS-1 cells were cultured in medium without
FCS for 4 h before exposure to cytokines for 24 h. After
treatment, the cells were collected and washed with cold
phosphate-buffered saline and incubated for 10 min with
Annexin V, then propidium iodide (PI) in the appropriate
buffer was added according to the manufacturer's instructions. The analysis was performed with a FACScan
flow cytometer (BD Biosciences, San Jose, CA, USA) using
the CellQuest software (BD Biosciences). This method has
been successfully used to assess apoptosis/necrosis in
INS-1 cells and rat and human
β-cells[18,19].
Western blotting analysis Cellular proteins were
prepared from the INS-1 cells, and immunoblots were performed
as previously described[12] with some modifications. Briefly,
the INS-1 cells were collected and resuspended in buffer A
(25 mmol/L HEPES, pH 7.5, 5 mmol/L EDTA, 0.5% Triton
X-100, 1.5 mmol/L sodium fluoride, 1 mmol/L sodium vanadate,
and 10 µL/mL protease inhibitor mixture) and homogenized
by brief sonication. The homogenate was centrifuged for 10
min at 10 000×g at 4 °C, and the supernatant was taken for
protein determination. The cellular proteins were then
electrophoresed in a 7.5% SDS_PAGE gel and transferred onto
Hybond ECL membranes (Amersham Biosciences, Piscataway, NJ, USA). The enhanced chemiluminescence
(ECL) membranes were incubated with primary antibodies
against N-CDase (1:500) overnight at 4 °C, followed by
incubation with horseradish peroxidase-conjugated secondary
antibodies (1:2000) for 2 h at room temperature. The signals
were detected with the ECL system. To control lane loading,
the same membranes were probed with anti-β-actin
antibodies (1:5000) after being washed with washing buffer. The
signals were quantified by scanning densitometry using
National Institutes of Health Image J software (NIH,
Bethesda, MD, USA). The results from each experimental group were
expressed as relative integrated intensity compared with that
of the control INS-1 cells measured with the same batch.
N-CDase activity assay N-CDase activity was determined
by the released sphingoid base from Cer using a HPLC
assay adopted from Xu et
al[20]. The INS-1 cells were collected
and resuspended in buffer A and centrifuged for 15 min at 10
000×g at 4 °C. The supernatant was collected, and the
protein concentration was determined by the BCA method.
Each sample containing 100 µg of protein was then assayed
for N-CDase activity by incubation with 100 µmol/L
C12-Cer in 0.2 mol/L HEPES buffer (pH 7.5) with 0.2% Triton X-100
and 5 mmol/L CaCl2 for 2 h at 37 °C. SPH formation was linear
over this time of incubation and proportional to the amount
of sample protein for up to 400 µg/assay. The reactions were
stopped by extraction with chloroform and methanol. The
o-phthaldehyde derivative of released SPH was determined by
HPLC with C12-D-erythro-SPH as an external standard and
C17-D-erythro-SPH as an internal standard. HPLC was
conducted using the Agilent 1050-HPLC model fitted with a
eclipse XDB-C18 column (Agilent Technologies, Palo Alto,
CA, USA). The solvent was methanol_potassium phosphate
buffer (90:10 v/v) and the flow rate was 0.8 mL/min. A HP1046
fluorescence detector was used with an excitation at 345 nm
and emission at 455 nm.
RNA interference The gene silencing of rat N-CDase
was performed essentially according to a standard
protocol[21]. The efficiency of gene
silencing was assessed by enzymatic
activity or by measuring the protein level of the target gene
using Western blotting. Briefly, the INS-1 cells were plated
onto 60 mm dishes. The cells were then transfected with
scrambled control siRNA (SCR; Ambion catalog
No AM4611) or prevalidated rat N-CDase-specific siRNA (Ambion
catalog No AM16704, all from Ambion, Austin, TX, USA).
Transfection was performed using HiPerfect transfection reagent
(Qiagen, Hilden, Germany) according to the manufacturer's
directions. The final concentration of siRNA was 20 nmol/L.
At 36 h post-transfection, the cells were treated with
cytokines for the indicated time period and then collected
for Western blotting, activity assay, and flow cytometry.
Statistical analysis Data are expressed as mean±SD.
Significance was tested by Student's t-test or ANOVA, and
P<0.05 was considered to be statistically significant.
Results
Cytokines induced the chronic activation of N-CDase
To investigate whether N-CDase activity was present in the
INS-1 cells and affected by cytokines, the INS-1 cells were
stimulated for different time periods with a cytokine mixture
(5 ng/mL IL-1β, 10 ng/mL TNF-α, and 50 ng/mL IFN-γ) that
was reported to induce apoptosis in pancreatic β-cells and
INS-1 cells[18,22]. N-CDase activity was measured by
quantitating the released SPH. The results showed that N-CDase
was active in the INS-1 cells (18.2±2.5 pmol
SPH·min_1·mg_1 protein in the control cells). Moreover, the treatment of the
INS-1 cells with cytokines resulted in a time-dependent
delay in the activation of N-CDase. As a result, the activation
of N-CDase was first observed at 8 h after stimulation
(129%±16% of that of the control), reached a maximum at 16
h (183%±33% of that of the control), and remained elevated at
24 h (170%±21% of that of the control; Figure 1).
Cytokines upregulated N-CDase protein
expression To test whether the effect of cytokines on N-CDase activity
could be attributed to an increased N-CDase protein content,
the INS-1 cells were treated with cytokines at different time
points, and the N-CDase protein levels were quantified.
Western blotting showed that the antibody against N-CDase
recognized a protein with an apparent molecular mass of
110_120 kDa, as previously described in rat mesangial
cells[8,12]. The N-CDase expression increased
significantly at 8 h (171%±30% of that of the control) and further increased at
16 h (293%±54% of control), and remained elevated at
24 h (255%±67% of that of the control; Figure 2).
Cytokines upregulated N-CDase mRNA expression
To confirm whether there was also an upregulation of the
N-CDase mRNA induced by cytokines, total RNA was isolated from
the INS-1 cells at the indicated time points and subjected to
quantitative RT_PCR. A marked increase in N-CDase mRNA
was observed as early as 4 h after treatment. The mRNA
levels peaked at 12 h and remained elevated at 24 h (Figure
3).
Inhibition of N-CDase activity by siRNA enhanced
cytokine-induced apoptosis To determine whether the
inhibition of CDase activity potentiated the cytotoxic response
induced by cytokines, N-CDase was inhibited by RNA
interference in the presence and absence of cytokines for 24 h.
The basal and cytokine-induced expression and activity of
N-CDase were markedly inhibited in the INS-1 cells
transfected with N-CDase siRNA compared to the untransfected
cells or the cells transfected with SCR (Figure 4A, 4B).
Furthermore, although the treatment of the INS-1 cells with
N-CDase siRNA alone did not induce significant apoptosis,
it enhanced cytokine-induced apoptosis (20.2%±2.9%) and
necrosis (9.5%±1.9%) compared to the untransfected cells
(apoptosis: 14.6%±1.7% and necrosis: 5.0%±1.6%) and the
negative control transfected cells (apoptosis: 12.7%±2.5%
and necrosis: 6.3%±1.3%; (Figure 5A, 5B).
Discussion
CDase is an essential enzyme that catalyzes the
formation of SPH from Cer and plays a crucial role in
sphingolipid-mediated cell regulation. To our knowledge, this study is the
first to characterize N-CDase expression and activity, as well
as its role in the cellular response to cytokines in the
pancreatic β-cell line INS-1, a known in vitro model currently used
for the study of pancreatic β-cell function and apoptosis.
A recent study revealed that in INS-1 cells and islets,
IL-1β and TNF-α induced early and sustained increases in SPHK
activity and S1P levels, a downstream metabolite in the
sphingolipid metabolic pathway[15]. Recently, Laychock
et al reported that exogenous S1P provides protective effects
against cytokine-induced apoptosis in
β-cells[16]. These findings prompted us to further investigate whether CDase, the
rate-limiting enzyme in the sphingolipid pathway, also played
a role in the pathological response of β-cells to cytokines.
CDase, although not described in β-cells, is believed to
function as a key "switch", determining the intracellular levels of
Cer and S1P and providing protection against cytokine
toxicity[6]. In contrast to early increased SPHK activity and S1P
levels as previously reported[15], our results showed
that cytokines did not induce rapid activation of N-CDase. The
prolonged treatment of the INS-1 cells with cytokines
resulted in the delayed activation of N-CDase, and this
activity increased from 8 to 24 h, reaching a peak at 16 h.
It has been shown that N-CDase is stimulated in response
to cytokines in various cell types[7_9]. Here, we confirmed
the chronic activation of N-CDase induced by cytokines in
INS-1 cells, which might result from the increased N-CDase
mRNA and protein expression. Additionally, a quantitative
discrepancy between the increase in the activity and the
elevation in the protein level of N-CDase was observed in
this study. We speculated that it might be related to the
regulation mechanism of N-CDase. In addition to being
regulated by gene transcription, the N-CDase expression and
activity were probably also modulated by phosphorylation
per se, either by protein kinase C or tyrosine
kinase[6,7,23]. Thus, although not described in
β-cells, the possibility that N-CDase was also regulated by
phosphorylation/dephosphorylation cannot be ruled out in this study.
Previous data show that cytokine-induced increases in
SPHK activity and S1P levels in INS-1 cells appear to occur
in the following 2 phases: an early phase independent of
new protein synthesis, and a late phase (after treatment for 8
h) dependent on new protein synthesis, as well as the
induction of new enzymes[15]. Current understanding indicates
that CDase is vital not only for Cer degradation, but also for
SPH/S1P generation. Further, the concomitant activation of
CDase and SPHK has been observed in response to several
stimuli, including cytokines, oxLDL, growth factors, and
advanced glycation end products in a variety of cell
types[7_12]. Thus, our findings regarding the cytokine-induced chronic
activation of N-CDase in INS-1 cells, which was associated
with an increased amount of N-CDase protein, raised the
possibility that N-CDase activation may be involved in the
increase in SPHK activity and S1P levels induced by
cytokines in the late phase. Further elucidation of the
quantitative relationship between the N-CDase activation and S1P
generation in response to cytokines is clearly warranted.
Furthermore, we observed that when N-CDase activity
was inhibited by N-CDase siRNA transfection,
cytokine-induced apoptosis in INS-1 cells markedly increased. These
results suggest that N-CDase potentially provides
protection against cytokine toxicity. Our findings are also
consistent with previous reports stating that
TNF-α/IL-1β induces the activation of N-CDase in rat mesangial cells, and when
its activity is inhibited, cytokine-induced cell apoptosis can
occur[8,9]. Cer has been implicated in cell death following a
myriad of cellular stresses as it mediates several apoptosis
signaling pathways, such as the activation of caspase-3,
inhibition of phosphatidylinositol 3-kinase/the protein
kinase B (PKB) pathway either by the activation of a
phosphatase and subsequent dephosphorylation of PKB, and an
increase in mitochondrial
permeability[3,5,24]. However, CDase activation results in a decrease in the intracellular Cer levels
and a concomitant increase in S1P, which promotes growth
and survival and opposes the pro-apoptotic effects of Cer,
leading to apoptosis suppression. Thus, the mechanism by
which CDase exerts its protective effects appears to involve
the regulation of the cellular Cer/S1P
ratio[6,24]. Osawa et al further demonstrated that the overexpression of N-CDase
prevents hepatocyte apoptosis induced by TNF-α through
reducing C16-Cer and promoting the activation of AKT
through S1P formation[25]. Therefore, the chronic activation
of N-CDase may reduce intracellular Cer and the
ratio of Cer/S1P, mediating the protective effects against
cytokine-induced apoptosis in INS-1 cells.
In summary, N-CDase is active in INS-1 cells, and its
activity is chronically increased by cytokine treatment.
Furthermore, the finding that the inhibition of N-CDase
activity enhances cytokine-induced apoptosis suggests that
it potentially protects β-cells from cytokine toxicity. However,
the regulatory mechanisms underlying N-CDase activity and
cellular Cer/S1P levels in response to cytokines remain to be
determined.
Acknowledgment
We thank Yi-te HSU (Medical University of South
Carolina, USA) for carefully reading the manuscript.
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