From the Hormel Institute, University of Minnesota,
Austin, Minnesota 55912 and the § Department of
Pathophysiology, Henan Medical University,
Zhengzhou 450052, Peoples Republic of China
Received for publication, July 7, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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The sphingomyelin-ceramide pathway is an
evolutionarily conserved ubiquitous signal transduction system that
regulates many cell functions including apoptosis. Sphingomyelin (SM)
is hydrolyzed to ceramide by different sphingomyelinases. Ceramide
serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase)
activity was induced by UVA in normal JY lymphoblasts but was not
detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who
have an inherited deficiency of acid SMase. We also provide evidence
that UVA can induce apoptosis by activating acid SMase in normal JY
cells. In contrast, UVA-induced apoptosis was inhibited in MS1418
cells. Exogenous SMase and its product, ceramide (10-40
µM), induced apoptosis in JY and MS1418 cells, but
the substrate of SMase, SM (20-80 µM), induced apoptosis
only in JY cells. These results suggest that UVA-induced apoptosis by
SM is dependent on acid SMase activity. We also provide evidence that
induction of apoptosis by UVA may occur through activation of JNKs via
the acid SMase pathway.
Cell death is an irreversible process that culminates in cessation
of biological activity (1-3) and can occur through apoptosis or
necrosis (4-11). Apoptosis is an active and physiological mode of cell
death and is well characterized by morphological changes including cell
shrinkage, cytoplasmic blebbing, chromatin condensation, and DNA
fragmentation (2). In recent years, substantial progress has been made
in understanding the multistep regulatory mechanisms that are
associated with the propensity of a cell to respond to various stimuli
with apoptosis (1, 2, 7, 8). The regulatory system involves the
presence of at least two distinct checkpoints, one controlled by the
Bcl-2/Bax family of proteins (5-7) and the other by the
cysteine and possibly serine proteases (2, 7, 8, 12, 20-22). In
addition, mitochondria (9, 13) and the sphingomyelin
(SM)1-ceramide pathway
(11, 12, 14-17) play important roles in apoptotic signal transduction.
These systems interact with the machinery regulating cell proliferation
and DNA repair through several oncogenes and tumor suppressor genes
such as p53 (2). Hence, antitumor strategies based on
modulation of the propensity of the cell to undergo apoptosis attract
great interest in oncology.
Sphingolipids such as SM had been previously regarded as metabolically
inactive, functioning only as structural components of the membrane
(17, 18). However, besides its structural role in biomembranes, SM
plays a pivotal role in signal transduction and regulation of cellular
functions including growth, differentiation, proliferation, and
apoptosis (14-18). A number of studies have demonstrated that
extracellular cytokines and stress stimuli, such as TNF Solar ultraviolet (UV) radiation is known to be one of the most common
environmental carcinogens leading to skin cancer (27-29). Also, UV
exposure induces apoptosis in cultured cells and in vivo (29, 30). Most research has focused on the UVC (200-290 nm) and UVB
(290-320 nm) induction of apoptosis (31, 32), and little is known
about the effect of UVA (320-400 nm), which comprises over 90% of the
solar UV. Here, we observe that acid SMase is activated by UVA, and we
provide evidence that UVA-induced apoptosis is dependent on acid SMase
activity. Exogenous sphingomyelinase and its product, ceramide, also
induce apoptosis independent of activation of intracellular SMase, but
induction of apoptosis by SM is dependent on the SMase activity. Our
data further indicate that UVA-induced apoptosis may occur through
activation of JNKs via the SMase pathway.
Reagents--
Dulbecco's modified Eagle's medium (DMEM), RPMI
1640, penicillin, streptomycin, L-glutamine, and fetal
bovine serum (FBS) were purchased from Life Technologies, Inc.
C2-ceramide
(N-acetyl-D-erythro-sphingosine), a biologically
active cell-permeant ceramide analog, C2-dihydroceramide (N-acetyl-D-erythro-sphinganine) that is
inactive and may be used as a negative control for
C2-ceramide, and SM from bovine brain used as a precursor
to ceramide second messengers via the action of sphingomyelinase, were
purchased from BioMol Inc. (Plymouth Meeting, PA). These three
sphingolipids were dissolved in dimethyl sulfoxide
(Me2SO) from Pierce. Acid SMase, pH 4.5, from human placenta, phosphocholine, aprotinin, leupeptin,
12-O-tetradecanoylphorbol-13-acetate (TPA), PD98059,
SB2020190, and Hoechst 33258 (bisbenzimide,
2-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5-bi-1H-benzimidazole trihydrochloride pentahydrate) were from Sigma.
Cell Culture--
Epstein-Barr virus-transformed normal human
lymphoblast cell lines, JY, or Niemann-Pick lymphoblast cell lines,
MS1418 (23), were a generous gift from Dr. Richard Kolesnick,
Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer
Center, New York. The two cell lines were maintained in a mixture of
RPMI 1640 and DMEM (1:1, v/v) containing 15% FBS, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin, and cultured in a humidified atmosphere of 5%
CO2, 95% air at 37 °C.
UVA, UVB, and UVC Irradiation of Cells--
The UVA source used
was a Philips TL100 watt/10R lamp obtained from Ultraviolet Resources
International (Lakewood, OH). UVA irradiation, filtered through about 6 mm of plate glass to eliminate UVB and UVC generated by the UVA lamps,
was administered to cultured cells in the UVA box with two ventilation
fans installed to eliminate UVA thermal stimulation. The doses of UVA
irradiation were 20, 40, 60, or 80 kJ/m2 as designated. The
UVB irradiation was carried out in a UVB chamber that was fitted with a
Kodak Kodacel K6808 filter that eliminates all wavelengths below 290 nm. The UVC radiation was from germicidal lamps. The cultured cells
were or were not starved by replacing medium with 0.5% FBS/DMEM/RPMI
mixed medium (for JY or MS1418 cells), and then cells were exposed to
UVA, UVB, or UVC at various doses as described.
C2-ceramide, SM, and SMase Treatments--
Normal
human lymphoblast JY cells or acid SMase-deficient lymphoblast MS1418
cells (3 × 106 to 5 × 106) (23, 33)
were seeded into 100-mm dishes or wells of 6-well plates and cultured
for 24 h in the mixture of RPMI 1640 and DMEM (1:1, v/v)
containing 15% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg of streptomycin/ml in a humidified
atmosphere of 5% CO2, 95% air at 37 °C. After having
been replaced with freshly mixed medium containing 0.5% FBS, these
cells were cultured for an additional 30 min and were treated with
C2-ceramide (10-40 µM),
C2-dihydroceramide (10-40 µM), SM (20-80
µM), SMase (0.5-2.5 units/ml), or phosphocholine (20 or
200 µM). Me2SO was used as an internal
negative control. These differentially treated cells were incubated and
then harvested after various times as indicated.
Internucleosomal DNA Fragmentation Ladder Assay--
DNA
fragmentation visualized by agarose gel electrophoresis (21, 30, 33) is
considered a biochemical hallmark for apoptosis. Briefly, JY or MS1418
cells (3 × 106 to 5 × 106) were
cultured in 100-mm dishes and treated with UVA, UVB, or UVC irradiation
or with C2-ceramide, C2-dihydroceramide, SM,
SMase, or phosphocholine and then harvested by pipetting and
centrifuging at 1,500 × g. The cells were lysed with
buffer containing 5 mM Tris-HCl, pH 8.0, 20 mM
EDTA, and 0.5% Triton X-100, and left on ice overnight at 4 °C.
After centrifugation at 17,000 × g for 45 min at
4 °C, fragmented DNA in the supernatant fraction was extracted twice
with phenol/chloroform/isopropyl alcohol (25:24:1, v/v) and once with
chloroform and then precipitated overnight at Detection of Nuclear Chromatin Condensation and Nuclear
Fragmentation--
Morphological changes characteristic of apoptosis
were detected with Hoechst staining (34, 35). Briefly, exponentially growing JY or MS1418 cells (1 × 106) were irradiated
with UVA, UVB, or UVC at the doses indicated. At 2-12 h after
irradiation, cells were harvested by centrifuging at 1,500 × g for 5 min at 4 °C and washed twice with ice-cold PBS
and then fixed in 1% formaldehyde in PBS, pH 7.4, for 15 min on ice
and postfixed in 70% ethanol and stored at Assay for Acid SMase Activity in Preparations from Irradiated
Cells--
The enzymatic hydrolysis of SM to ceramide and
phosphocholine by SMase was measured at pH 5.0 with the Amplex Red
reaction kit (Molecular Probes Inc., Eugene, OR) (38). Briefly, after irradiation of JY or MS1418 cells (2.3 × 106) with
UVA (80 kJ/m2), UVB (8 kJ/m2), or UVC (60 J/m2), cells were pelleted by centrifugation at 1,500 × g for 10 min at 4 °C and washed twice with ice-cold
PBS. The cell pellet was resuspended and lysed in 0.6 ml of buffer
containing 50 mM sodium acetate, pH 5.0, 1% Triton X-100,
1 µg/ml aprotinin, 1 mM EDTA, and 100 µg/ml
phenylmethylsulfonyl fluoride for 60 min on ice. Then the supernatant
fraction was saved by centrifugation at 17,000 × g for
10 min at 4 °C to remove nuclei. The protein concentration in the
supernatant fraction was measured with the Bio-Rad Protein Assay. JY or
MS1418 cell membrane-free supernatant fractions (adjusted to pH 5.0)
were assayed for SMase activity in a two-step reaction system. First,
to generate phosphocholine and ceramide, 0.5 mM SM was
added to the supernatant fraction and incubated for 60 min at 37 °C.
The reaction was then placed on ice and the fluorogenic probe Amplex
Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), which is sensitive to
H2O2, was added and further incubated at 37 °C for 60 min to generate H2O2 (through
alkaline phosphatase hydrolysis of phosphocholine and choline oxidation
by choline oxidase to generate betaine and
H2O2). H2O2 in the
presence of horseradish peroxidase reacts with Amplex Red to generate
the fluorescent resorufin. Each reaction mixture contained 5 µM Amplex Red reagent, 1 unit/ml horseradish peroxidase,
0.1 unit/ml choline oxidase, 4 units/ml alkaline phosphatase, all
supplied with the kit. For a detailed description of the assay see Zhou
et al. (38). The fluorescence intensity was measured
immediately at 610 nm (excitation at 560 nm) with a SPEX Fluoromax
instrument (Instruments S.A., Inc. Edina, NJ), temperature-controlled
to 37 ± 0.1 °C (Neslab, RTE-111, Neslab Instruments,
Portsmouth, NH).
Measurement of Cellular Ceramide Levels--
JY or MS1418 cells
were starved for 12 h in medium containing 0.5% FBS. The cells
were then harvested at 5, 15, or 30 min following irradiation with UVA
(80 kJ/m2), UVB (8 kJ/m2), or UVC (60 J/m2). Lipids were extracted from the cell pellets with
chloroform:methanol (2:1, v/v) (39). The lipids dissolved in chloroform
were applied to solid phase extraction cartridges (50 mg Extract-Clean
Silica, Alltech, Deerfield, IL), and ceramides were eluted with 2.5 ml of chloroform:methanol (98:2, v/v) after the neutral lipids had been
eluted with 2 ml of chloroform. After acid hydrolysis of the ceramide
fraction (5% v/v HCl:methanol, 70 °C, 3 h), free sphingosine
was extracted into chloroform in the presence of the internal standard,
0.1 µg of C16 dihydrosphingosine (Matreya, Pleasant Gap, PA). The
extracts were dried and derivatized with tertiary
butyldimethylchlorosilane/imidazole reagent (Alltech). Tertiary
butyldimethylchlorosilane/imidazole derivatives were analyzed by gas
chromatography/mass spectrometry (Hewlett-Packard 5892 GC, 5972 Mass
Selective Detector, and 7673 Autosampler, HP5 MS capillary column) in
the selected ion-monitoring mode. The base peaks were monitored as
follows: m/z 472 for C16 dihydrosphingosine (M-57; loss of
tert-butyl moiety) and m/z 353 for sphingosine (cleavage between C2 and C3). The amount of ceramides was calculated from the ratio of peak areas and normalized to cell
number.2
Phosphorylation of ERKs, JNKs, and p38--
Mitogen-activated
protein kinases (MAPKs), including ERKs, JNKs and p38 kinases, are
known to be activated via phosphorylation (40). Therefore, the
phosphorylated levels of MAPKs reflect MAPKs activity. Here, immunoblot
analysis of phosphorylated proteins for ERKs, JNKs, and p38 kinase was
carried out using the specific antibodies against phosphorylated sites
of ERKs (Tyr-204 of p44 and p42), JNKs (Thr-183/Tyr-185), and p38
kinase (Thr-180/Tyr-182) (New England Biolabs, Inc., Beverly, MA).
Briefly, JY or MS1418 cells (3 × 106) growing
exponentially were seeded in a 100-mm dish and starved for 24 h in
a mixed medium of DMEM and RPMI 1640 (1:1, v/v) containing 0.5% FBS.
After irradiation with UVA (80 kJ/m2), UVB (8 kJ/m2), or UVC (60 J/m2), the cell pellets were
harvested by centrifugation at 1,500 × g and then
washed once with ice-cold PBS and lysed in 300 µl of SDS sample lysis
buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS,
10% (v/v) glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue. The lysed samples were sonicated for 5-10 s. Samples
containing equal amounts of protein (Bio-Rad protein assay) were loaded
in each lane of an 8% SDS-polyacrylamide gel for electrophoresis and
subsequently transferred onto Immobilon-P transfer membrane. The
phosphorylated MAPK proteins were detected by Western immunoblotting using a chemiluminescent detection system and visualized using the
Storm840 PhosphorImaging system. Intensity of bands in Western blots
was calculated and analyzed using the Image-QuaNTTM
software (Molecular Dynamics, Sunnyvale, CA).
Preparation and Analysis of Normal, jnk1 Activation of Acid SMase by UVA, UVB, and UVC
Irradiation--
Acid SMase plays an important role in cellular
response to extracellular stimuli by transmitting the signal into cells
through the acid SMase pathway (11, 12, 14-18, 23, 24). The
AmplexTM SMase assay kit (38) was used for measuring acid
SMase activity in vitro in UVA-, UVB-, and UVC-irradiated
cell preparations. As shown in Fig. 1, in
normal JY cells, acid SMase was activated almost immediately after UVA,
UVB, or UVC irradiation, but in acid SMase-deficient MS1418 cells the
induction was comparatively weaker. In normal JY cells, the peak of
acid SMase activity appeared at 15 min following UVA irradiation, and
the activity was maintained at least 30 min. In JY cells treated with
UVB or UVC, the activation peak occurred almost immediately at 5 min
following irradiation and was maintained at that level or slightly
higher for at least 30 min. In MS1418 cells, the peaks induced by UVA
or UVB occur only at 15 min and by UVC only at 5 min and then the
activity goes back to unstimulated levels. The peak level of acid SMase activity in normal JY cells was at least 3-4-fold greater than that
observed in SMase-deficient MS1418 cells (Fig. 1).
Increased Cellular Ceramide Level following UVA, UVB, or UVC
Irradiation--
Ceramide is generated by the hydrolysis of SM by
SMase (14-22). Ceramide levels were measured by
gaschromatography/mass spectrometry2 In JY cells,
ceramide levels increased almost immediately after UVA, UVB, or UVC
irradiation, but no increase was observed in the SMase-deficient MS1418
cells (Fig. 2). These data and those in
Fig. 1 indicate that the increase in ceramide in JY cells following exposure to UV irradiation is caused by acid SMase activation.
UVA-induced Apoptosis Occurs in Normal SMase Cells but Is Inhibited
in SMase-deficient Cells--
Apoptosis was analyzed according to
morphological changes observed in apoptotic nuclei and DNA
fragmentation laddering. As shown in Fig.
3A, UVA induced typical
apoptosis in normal SMase JY cells, but significantly less DNA
laddering induced by UVA was observed in SMase-deficient MS1418 cells.
UVA-induced apoptosis was dose- (Fig. 3, A and C)
and time-dependent (Fig. 3B, left panel) in SMase-normal JY cells. At 10 h following
irradiation of cells with 40-80 kJ/m2 of UVA, DNA
fragmentation laddering was evident in normal JY cells but almost not
present in MS1418 cells (Fig. 3A). The number of apoptotic
nuclei determined by Hoechst staining was directly related to doses of
UVA irradiation (Fig. 3C). Typical apoptosis occurred in JY
cells at 6 h after UVA irradiation (80 kJ/m2) and then
increased until 12 h (Fig. 3B, left panel).
In contrast, the time-dependent apoptotic response was
significantly diminished in MS1418 cells (Fig. 3B,
right panel). On the other hand, UVB- or UVC-induced
apoptosis was not significantly different in either normal SMase or
SMase-deficient cells (Fig. 3, A-C). These data suggest
that UVA- but not UVB- or UVC-induced apoptosis may depend on
activation of the acid SMase pathway.
Exogenous Acid SMase and Ceramide Can Induce Apoptosis in Both
Normal and SMase-deficient Cells--
Intracellular levels of ceramide
have been shown to increase in response to various extracellular
stimuli including UV exposure (12, 20, 42), and ceramide has been
reported to induce apoptosis in target cells (15, 42). Here, our data
show that apoptosis was induced after treatment of both normal JY and
SMase-deficient MS1418 cells with exogenous SMase (Fig.
4B) or cell-permeant
C2-ceramide (Fig. 4A). However, apoptosis did
not occur in either cell line after treatment with
C2-dihydroceramide, an internal negative control (Fig.
4A). Additionally, phosphocholine, another hydrolysis product of SMase, did not induce apoptosis in either cell line (data
not shown). These data suggest that SMase and ceramide, but not
phosphocholine, are implicated in mediating apoptosis.
Exogenous SM Can Induce Apoptosis in Normal SMase Cells but Not in
SMase-deficient Cells--
SM is hydrolyzed by SMase to generate
ceramide and phosphocholine. As shown in Fig. 4, A and
B, after treatment of cells with SM, apoptosis was induced
in normal SMase cells (upper and lower left
panels) but not in SMase-deficient cells (upper and
lower right panels). These data indicate that acid SMase is
required for SM-induced apoptosis.
UVA-induced Phosphorylation of JNKs Is
SMase-dependent--
Activation of JNKs by UVB and UVC has
been shown to be SMase-dependent (43). Here, we found that
UVA-induced phosphorylation of JNKs was also
SMase-dependent (Fig. 5,
A and B) and the phosphorylation coincided with
activation of acid SMase by UVA (Fig. 1). In addition, UVB- or
UVC-induced phosphorylation of JNKs occurred in JY cells (Fig. 5,
A and B), but in SMase-deficient MS1418 cells,
JNKs phosphorylation was not significantly induced by UVA, UVB, or UVC
(Fig. 5, A and B). These data suggest that SMase
is required for activation of JNKs by UVA, UVB, or UVC, further
indicating that JNKs are downstream kinases of the SMase signaling
pathway. Additionally, TPA-induced phosphorylation of JNKs was observed
in both cell lines (Fig. 5A), suggesting that TPA-induced
activation of JNKs may be independent of SMase.
UVA-, UVB-, UVC-induced Phosphorylation of ERKs Is Inhibited in
Normal SMase Cells Compared with SMase-deficient MS1418
Cells--
Although ERKs were reported not to be activated by UVA
irradiation (44), our data showed that UVA, like UVB and UVC, induced phosphorylation of ERKs in SMase-deficient MS1418 cells, and the phosphorylation was blocked in SMase-normal JY cells compared with
control values (Fig. 6, A and
B). The data suggest that ERKs phosphorylation may not be
related to UV-induced activation of SMase signaling in JY cells.
UVA-, but Not UVB- or UVC-induced Phosphorylation of p38 Kinase, Is
Also Inhibited in Normal SMase Cells--
Activation of p38 kinase was
shown to be induced by UVA (44). Here, our data showed that UVA-induced
phosphorylation of p38 kinase occurred in both JY and MS1418 cells, but
the phosphorylation appeared to be less in JY cells (Fig.
7, A and B). On the
other hand, UVB- or UVC-induced phosphorylation of p38 kinase was not significantly different overall in these two cell lines (Fig. 7,
A and B). These data also suggest that
UVA-induced p38 kinase activation is not related to UV-induced
activation of the SMase signaling pathway.
PD98059 and SB202190 Do Not Inhibit UVA-induced Apoptosis in JY
Cells--
Whether activation of ERKs or p38 kinase is reported to be
involved in apoptosis or anti-apoptosis depends on cell type and the
kind of stimulation (45-47). Here, to study further the role of
ERKs and p38 kinase in UVA-induced apoptosis, we used PD98059, a
selective inhibitor of MEK1 resulting in inhibition of ERK activation (48), and SB202190, a selective inhibitor of p38 kinase activation (49). Our data showed that PD98059 and SB202190 did not inhibit UVA-induced apoptosis in JY cells (Fig.
8, left panel). On the other
hand, UVA-induced apoptosis was observed in SMase-deficient MS1418
cells after pretreatment with PD98059 or SB202190 (Fig. 8, right
panel). These data suggest that ERKs and p38 kinase are not
required for UVA-induced apoptosis in normal JY cells.
UVA-induced Apoptosis Is Blocked in jnk1 Exposure to UV radiation can cause cell cycle arrest (50, 51),
alterations in mitochondrial membrane permeability (30), and cell death
by necrosis or apoptosis (42, 51). In the present study, we observed
that UVA, like UVB and UVC, induced apoptosis in normal lymphoblast
cells (JY) but not in MS1418 lymphoblasts from Niemann-Pick type D
patients who have an inherited deficiency of acid SMase. UVA radiation
is known to induce an array of stress proteins quite distinct from
those induced by UVB or UVC (51). UVB and UVC were clearly shown to
mimic growth factor responses and stimulate signal transduction
including the SMase pathway (12, 20, 32, 43). Noncytotoxic exposure to
UVA can also up-regulate several signal molecules (32, 42), but the
role of the SMase pathway in UVA-induced apoptosis is not well understood.
In this study, we provide evidence that UVA, UVB, and UVC can all
activate acid SMase (Fig. 1) and lead to an increase in ceramide levels
(Fig. 2) in normal JY cells. Analysis of DNA fragmentation laddering
and morphological changes in apoptotic cells showed that sublethal
doses of UVA (20-80 kJ/m2), similar to lethal doses of UVB
and UVC, can also induce apoptosis in normal JY cells, but UVA-induced
apoptosis is prevented in acid SMase-deficient MS1418 cells. On the
other hand, the difference in UVB- or UVC-induced apoptosis was less
apparent in normal and SMase-deficient cells. These results suggest
that UVA-induced apoptosis occurs through activation of acid SMase,
whereas UVB- or UVC-induced apoptosis occurs through SMase-independent
pathways. However, additional pathways involved in mediating
UVA-induced apoptosis cannot be disregarded, including neutral SMase
(25, 26), stress-activated protein kinase/JNKs (12), caspases (30), and
mitochondria (30), because UVA-induced apoptosis is not completely
blocked in acid SMase-deficient MS1418 cells.
Klotz et al. (44) reported that UVA did not activate ERKs,
and we found that UVA, like UVB and UVC, did not induce phosphorylation (Fig. 6) and activation (data not shown) of ERKs in SMase-normal JY
cells. However, UVA strongly induced ERKs phosphorylation in SMase-deficient MS1418 cells suggesting that in the absence of SMase,
UVA may stimulate alternate pathways (e.g. ERKs) that may protect against UVA-induced apoptosis in these cells. We also found
that UVA, as well as UVB and UVC, induced phosphorylation and
activation of p38 kinase (Fig. 7). However, UVA-induced phosphorylation of p38 kinase appeared to be less in normal SMase JY cells compared with SMase-deficient MS1418 cells, whereas UVB- or UVC-induced phosphorylation was similar between the two cell lines (Fig. 7). Neither inhibition of UVA-induced ERKs activation by PD98059 (48) nor
inhibition of p38 kinase activation by SB202190 (49) blocked UVA-induced apoptosis in normal JY cells (Fig. 8, left
panel). However, MS1418 cells pretreated with PD98059 or SB202190
now showed typical apoptosis following UVA irradiation (Fig. 8,
right panel). These results further confirm that activation
and phosphorylation of ERKs and p38 kinase do not appear to be involved
in UVA-induced apoptosis in normal JY cells, but in the absence of
SMase, UVA irradiation may induce ERKs and p38 kinase leading to
inhibition of apoptosis.
Acid SMase was previously shown to be involved in UVB- and UVC-induced
activation of JNKs (43). In the present study, we observed that UVA,
like UVB and UVC, induced phosphorylation of JNKs in acid SMase normal
JY cells and that the phosphorylation of JNKs was markedly inhibited in
SMase-deficient cells (Fig. 5). However, TPA-induced JNKs
phosphorylation was not different between the two cell lines. These
results suggest that UVA-, UVB-, or UVC-, but not TPA-induced
phosphorylation and activation of JNKs, is acid
SMase-dependent. In addition, we observed that UVA-induced apoptosis was completely blocked in jnk1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
interleukin-1
, FAS ligand, heat shock,
-radiation (23), and UVC
irradiation (12, 14), cause the activation of sphingomyelinase (SMase)
and the release of ceramide. Ceramide, a product of SMase-catalyzed
hydrolysis of SM, was shown to act as a lipid second messenger or
biomodulator of diverse stress-related responses including cell cycle
arrest, cell senescence, and apoptosis (14-22). This pathway is
referred to as the SM cycle, SM-ceramide pathway (11, 17), or the SMase
pathway. To date, at least seven classes of mammalian SMases have been
described, differing in subcellular location, pH optimum, cation
dependence, and roles in cell regulation (11, 15, 17). Two forms of
SMases, distinguishable by their pH optima, are capable of initiating
signal transduction (12). The acid SMase (pH optimum 4.5-5.0) is
activated in cells exposed to ionizing radiation, FAS, CD28,
interleukin-1, or TNF
(23, 24). The neutral SMase (pH optimum 7.4)
has been implicated in mediating apoptosis in cells exposed to serum
starvation, anti-FAS antibody, vitamin D, TNF
, or cytosine
arabinoside (25, 26).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C with 100%
ethanol and 5 M NaCl. The DNA pellet was saved by
centrifuging at 17,000 × g for 45 min at 4 °C and
then washed once with 70% ethanol and resuspended in Tris-HCl, pH 8.0, with 100 µg/ml RNase at 37 °C for 2 h. The DNA fragments were
separated by 1.8% agarose gel electrophoresis. DNA laddering in the
gel was stained with ethidium bromide and photographed in UV light.
20 °C up to 4 days.
Again, the cells were washed twice in PBS, and pellets were resuspended
in Hoechst staining buffer containing 20 µg/ml Hoechst 33258 and 2%
Me2SO in PBS and incubated for 30 min at 37 °C in the
dark. Nuclei were visualized using a fluorescence microscope. In each
sample, a minimum of 400 cells was counted. The condensed, compacted,
and fragmented nuclei were counted and expressed as a percentage of the
total nuclei.
/
, and
jnk2
/
Primary Embryo Fibroblasts--
Embryonic fibroblasts from
normal, jnk1
/
, and jnk2
/
knockout mice
were isolated and prepared according to the procedure of Loo and Cotman
(41). Cells were established in culture in DMEM supplemented with 10%
FBS, 2 mM L-glutamine, 100 units/ml penicillin,
and 100 µg/ml streptomycin in a humidified atmosphere of 5%
CO2 at 37 °C. For analysis of apoptosis, the cells were irradiated with UVA in serum-free DMEM, and the cells were lysed, and
the DNA laddering assay was performed as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of acid SMase activity in
UV-irradiated cells. JY and MS1418 cells (2.3 × 106) were grown in a mixture of RPMI 1640 and DMEM (1:1,
v/v) containing 15% FBS. The cells were starved for 12 h in
medium containing 0.5% FBS and then irradiated with UVA (80 kJ/m2), UVB (8 kJ/m2), or UVC (60 J/m2). After lysis of cells, protein concentration and acid
SMase activity in supernatant fractions were measured as described
under "Materials and Methods." The figure shows the relative
fluorescence units in JY and MS1418 cells normalized to mg of cellular
protein. The value for control cells was subtracted from each sample.
Each bar indicates the mean and S.D. from four assay
samples.
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Fig. 2.
Increase in ceramide level in UV-irradiated
JY cells. JY or MS1418 cells (3 × 106) were
starved for 12 h in medium containing 0.5% FBS and then
irradiated with UVA, UVB, or UVC as described in Fig. 1. Extraction of
lipids and analysis of ceramides were performed as described under
"Materials and Methods." The ceramide level was normalized to cell
number and is shown as % of control (value of 100). The figure shows
that ceramide levels increase in UVA-, UVB-, or UVC-irradiated JY cells
but not in MS1418 cells following irradiation. Each point represents
the mean from triplicate incubations.
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[in a new window]
Fig. 3.
UVA induces apoptosis in normal SMase JY
cells. JY and MS1418 cells (3 × 106) were grown
for 24 h in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing
15% FBS and then irradiated with UVA, UVB, or UVC at the doses
indicated. Following irradiation, the cells were harvested at the time
points indicated. Fragmented DNA in supernatant fractions was extracted
and separated after lysis and centrifugation, and quantitative analysis
of apoptotic cells was determined by DNA laddering analysis
(A and B) and Hoechst staining (C) as
described under "Materials and Methods." The upper
(A) and lower (C) panels
show that the dose-dependent apoptosis induced by UVA
occurs in JY cells but not in MS1418 cells. On the other hand, a
similar level of apoptosis induced by UVB and UVC occurs in both cell
types. B, the middle left panel shows that
apoptosis induced by UVA, UVB, or UVC is time-dependent in
JY cells. The lower right panel shows that only apoptosis
induced by UVB or UVC is time-dependent, and UVA-induced
apoptosis is less significant in MS1418 cells.
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[in a new window]
Fig. 4.
Exogenous ceramide and acid SMase can induce
apoptosis in both normal JY and SMase-deficient MS1418 cells, but SM
only induces apoptosis in JY cells. JY and MS1418 cells (3 × 106) were cultured in a mixture of RPMI 1640 and DMEM (1:1,
v/v) containing 15% FBS and then were treated for 14 h with
C2-ceramide, C2-dihydroceramide, SM, or acid
SMase at the doses indicated. Fragmented DNA was extracted and
separated as described under "Materials and Methods." A,
the upper two panels show that C2-ceramide but
not C2-dihydroceramide can induce apoptosis in both JY and
MS1418 cells. B, the lower two panels show that
SM only induces apoptosis in normal JY cells (lower left
panel) and not in SMase-deficient MS1418 cells (lower right
panel). On the other hand, addition of exogenous SMase induces
apoptosis in both cell types (lower two panels).
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[in a new window]
Fig. 5.
Phosphorylation of JNKs is induced in
UV-irradiated JY cells but not in MS1418 cells. JY or
MS1418 cells (3 × 106) were starved for 24 h in
a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 0.5% FBS. Cells
were irradiated with UVA (80 kJ/m2), UVB (8 kJ/m2), or UVC (60 J/m2) and then harvested by
centrifugation at time points as indicated. After lysis and sonication,
samples containing equal amounts of protein were separated by 8%
SDS-PAGE and analyzed by Western immunoblotting using anti-phospho-JNKs
antibody as described under "Materials and Methods." Nonirradiated
cells were used as a negative control. A, The figure shows
that phosphorylation of JNKs was induced in UVA-, UVB-, or
UVC-irradiated JY cells but almost not in MS1418 cells. B,
intensity of bands was quantitated with Image-QuanNTTM
software. Each value indicates the mean and S.D. from three independent
experiments and is expressed as percentage of increase in
phosphorylation compared with control.
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[in a new window]
Fig. 6.
UV-induced phosphorylation of ERKs is
inhibited in JY cells. JY or MS1418 cells (3 × 106) were starved for 24 h in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 0.5% FBS. Cells were irradiated with
UVA (80 kJ/m2), UVB (8 kJ/m2), or UVC (60 J/m2) and then harvested by centrifugation at time points
as indicated. After lysis and sonication, samples containing equal
amounts of protein were separated by 8% SDS-PAGE and analyzed by
Western immunoblotting using anti-phospho-ERKs antibody as described
under "Materials and Methods." Nonirradiated cells were used as a
negative control. A shows that UVA-, UVB-, or UVC-induced
phosphorylation of ERKs was induced in MS1418 cells but was markedly
less in JY cells. B, intensity of bands was quantitated with
Image-QuanNTTM software. Each value indicates the mean and
S.D. from three independent experiments and is expressed as percentage
of increase in phosphorylation compared with control.
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[in a new window]
Fig. 7.
UVA- but not UVB- or UVC-induced
phosphorylation of p38 kinase is inhibited in JY cells. JY or
MS1418 cells (3 × 106) were starved for 24 h in
a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 0.5% FBS. Cells
were irradiated with UVA (80 kJ/m2), UVB (8 kJ/m2), or UVC (60 J/m2) and then harvested by
centrifugation at time points as indicated. After lysis and sonication,
samples containing equal amounts of protein were separated by 8%
SDS-PAGE and analyzed by Western immunoblotting using anti-phospho-p38
kinase antibody as described under "Materials and Methods."
Nonirradiated cells were used as a negative control. A shows
that phosphorylation of p38 kinase was induced in UVA, UVB, or
UVC-irradiated JY and MS1418 cells, and the phosphorylation by UVA but
not UVB or UVC was less in JY cells. B, intensity of bands
was quantitated with Image-QuanNTTM software. Each value
indicates the mean and S.D. from three independent experiments and is
expressed as percentage of increase in phosphorylation compared with
control.
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[in a new window]
Fig. 8.
UVA-induced apoptosis is not inhibited by
PD98059 or SB202190. JY or MS1418 cells (3 × 106) were pretreated for 1.5 h with Me2SO
as control, or with PD98059 or SB202190 and then irradiated with UVA
(80 kJ/m2). Twelve hours after irradiation, cells were
harvested by centrifugation. Fragmented DNA was extracted and separated
as described under "Materials and Methods." Nonirradiated cells
were used as a negative control, and cells treated only with UVA
irradiation were the positive control. The figure shows that PD98059
and SB202190 do not inhibit UVA-induced apoptosis in JY cells
(left panel), and UVA-induced apoptosis occurs after
pretreatment of MS1418 with PD98059 or SB202190 (right
panel).
/
and jnk2
/
Cells--
Activation of JNKs is known to result in phosphorylation of
c-Jun and to be involved in the induction of apoptosis (12, 45, 46).
JNKs were activated by UVA via the acid SMase pathway as described
above (Fig. 5). Here our data showed further that UVA-induced apoptosis
was markedly blocked in jnk1
/
and jnk2
/
cells compared with wild-type jnk+/+ cells (Fig.
9B), although induction of
acid SMAse activity occurred immediately following UVA irradiation of
the three cell lines (Fig. 9A). Taken together, our data
indicate that UVA-induced apoptosis occurs through the SMase signaling
activation of JNK1 and JNK2 and not through activation of ERKs or p38
kinases.
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Fig. 9.
UVA-induced apoptosis is abolished in
jnk1 /
and jnk2
/
cells
albeit UVA induces acid smase activity. Primary embryonic
fibroblasts were prepared from wild-type and jnk1
/
and
jnk2
/
knockout mice. The cells were starved for 12 h in
serum-free DMEM and then irradiated with UVA (80 kJ/m2).
A, the cells were harvested at indicated times following
irradiation. Then acid SMase activities in supernatant fractions were
determined as described in Fig. 1, except that the fluorescence
intensity was measured with a Labsystems Fluoroskan (Franklin, MA) with
filter combination 527 nm (excitation) and 620 nm (emission). The
UVA-induced SMase activity was normalized to nonirradiated control
(value of 100) and is shown as percent. The data are representative of
three independent experiments, and each bar represents the
mean and S.D. from triplicate samples. B, 14 h
following irradiation, detached and attached cells were harvested by
scraping and centrifuging. Fragmented DNA in supernatant fractions was
extracted and separated as described under "Materials and Methods."
Nonirradiated cells were used as negative control. The figures show
that induction of acid SMase activity by UVA occurs (A) but
UVA-induced apoptosis is completely blocked (B) in
jnk1
/
and jnk2
/
cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
and
jnk2
/
cells (Fig. 9B) further indicating that
JNKs are a downstream kinase family of the acid SMase pathway (45, 46).
Overall, these results strongly suggest that UVA-induced apoptosis in
normal SMase JY cells occurs primarily through activation of JNKs via the acid SMase pathway.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Xun Li and Zbigniew Darzynkiewicz at the Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, NY, for providing some details on a direct DNA strand break labeling (36, 37). We also thank Drs. Nanyue Chen and Qing-Bai She for their help on the assays for the DNA fragmentation ladder assay; Randy Krebsbach for assistance with the ceramide assays, and Andria Hansen for secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by The Hormel Foundation, National Institute of Health Grants CA77646, CA81064, GM45741, and GM45928, and the Academy of Finland.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.
¶ To whom correspondence should be addressed: The Hormel Institute, University of Minnesota, 801 16th Ave. NE, Austin, MN 55912. Tel.: 507-437-9640; Fax: 507-437-9606; E-mail: zgdong@smig.net.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M006000200
2 P. C. Schmid, E. V. Berdyshev, R. J. Krebsbach, and H. H. O. Schmid, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SM, sphingomyelin;
SMase, sphingomyelinase;
UVA, ultraviolet light A;
DMEM, Dulbecco's
modified Eagle's medium;
FBS, fetal bovine serum;
Me2SO, dimethyl sulfoxide;
PBS, phosphate-buffered saline;
MAPKs, mitogen-activated protein kinases;
ERKs, extracellular signal-regulated
kinases;
JNKs, c-Jun N-terminal kinases;
p38, p38 MAPK or p38 kinases;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
TNF, transforming growth factor-
.
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