(Received for publication, January 21, 1997, and in revised form, March 21, 1997)
From the Departments of Medicine and Cell Biology,
Duke University Medical Center, Durham, North Carolina 27710, the
§ Laboratoire de Biochimie, "Maladies Metaboliques,"
Institut Louis Bugnard, 31054 Toulouse, France, and the
¶ Department of Biochemistry, Setsunan University, Hirakata,
Osaka 573-01, Japan
The role of cytosolic phospholipase
A2 (cPLA2) in the regulation of ceramide
formation was examined in a cell line (L929) responsive to the
cytotoxic action of tumor necrosis factor (TNF
). In L929 cells,
the addition of TNF
resulted in the release of arachidonate, which
was followed by a prolonged accumulation of ceramide occurring over
5-12 h and reaching 250% over base line. The formation of ceramide
was accompanied by the hydrolysis of sphingomyelin and the activation
of three distinct sphingomyelinases (neutral
Mg2+-dependent, neutral
Mg2+-independent, and acidic enzymes). The variant cell
line C12, which lacks cPLA2, is resistant to the cytotoxic
action of TNF
. TNF
was able to activate nuclear factor
B in
both the wild-type L929 cells and the C12 cells. However, TNF
was
unable to cause the release of arachidonate or the accumulation of
ceramide in C12 cells. C6-ceramide overcame the resistance
to TNF
and caused cell death in C12 cells to a level similar to that
in L929 cells. The introduction of the cPLA2 gene into C12
cells resulted in partial restoration of TNF
-induced arachidonate
release, ceramide accumulation, and cytotoxicity. This study suggests
that cPLA2 is a necessary component in the pathways leading
to ceramide accumulation and cell death.
The sphingomyelin (SM)1 cycle, first
described by Okazaki et al. (1), has gained recognition over
the past few years as a key mechanism for regulating anti-mitogenic
signals. Activation of this cycle through the regulation of a
signal-induced sphingomyelinase (SMase) results in generation of the
lipid second messenger ceramide. Ceramide then modulates a number of
biological fates, including growth inhibition (1-3), differentiation
(2), apoptosis (4-6), and cell cycle arrest (7). Although recent
studies have begun to catalogue inducers such as TNF,
interleukin-1
, nerve growth factor, and Fas that are capable of
signaling through the SM cycle (see Refs. 5, 6, and 8 for reviews), the
mechanisms by which these inducers stimulate SMase activity remain
poorly understood.
TNF, through interaction with either a 55- or 75-kDa TNF receptor
(9, 10), impacts upon a myriad of intracellular signaling cascades,
including protein phosphorylation cascades, transcription factors, and
lipid messengers (11). Two classes of lipid mediators have been
implicated in TNF
signaling, glycerophospholipid metabolites and
sphingolipid metabolites (11, 12), and recent evidence suggests that
these two classes of lipids may interact (13). In HL-60 cells, a linear
correlation was established among TNF
stimulation, AA generation,
and SM cycle activation: TNF
-stimulated AA liberation preceded
ceramide generation, and AA reproduced the effects of TNF
on the SM
cycle (13). Although these studies suggested that AA and/or its
metabolites may be involved in activation of SMase, the physiologic
role of the PLA2/AA pathway in regulating SMase activity
has not been determined.
In this study, we examined the role of PLA2 in SMase
activation in the L929 murine fibroblast cell line. In L929 cells,
TNF treatment is known to produce potent cytotoxic effects (14). TNF
-resistant L929 cells have also been generated via clonal selection from resistant populations of L929 mouse fibroblasts grown in
the presence of TNF
(14, 15). One of these cell lines, C12, differed
from the original L929 cells by the absence of cPLA2 and by
the lack of inducibility of AA in response to TNF
. However, in all
parameters of TNF
receptor binding and internalization, this
resistant cell line was found to be analogous to the parental L929
line.
Using these two cell lines, L929 and C12, we investigated the necessity
of cPLA2 activation and AA generation for TNF-induced ceramide generation. In the L929 model system, we found that the kinetics of cytokine-induced lipid mobilization occurred much later
than previously documented in HL-60 (13, 16) and U937 (17) cells.
Although the kinetics of activation were protracted in the L929 system,
we found that, similar to HL-60 and U937 cells, the mechanism of
ceramide generation was still through the activation of SMase and the
subsequent hydrolysis of SM. Furthermore, we found that the generation
of ceramide in response to TNF
did not occur in the resistant line,
which was incapable of liberating AA following cytokine treatment.
Finally, we found that TNF
-induced AA generation, ceramide
generation, and cytotoxicity could be partly re-established in a C12
variant containing a cPLA2 expression vector. This study
implicates cPLA2 activation and AA generation as necessary
precursors to TNF
-induced activation of the SM cycle. The
implications of these findings are discussed.
Materials
The L929 cell line and its C12 variant have been previously
described (14, 15). Dulbecco's modified Eagle's medium and kanamycin
sulfate were purchased from Life Technologies, Inc. Heat-inactivated
fetal calf serum was purchased from Summit Biotechnologies (Fort
Collins, CO). Arachidonic acid was purchased from BIOMOL Research
Laboratories Inc. (Plymouth Meeting, PA). [3H]Choline
chloride and [3H]arachidonic acid were purchased from
DuPont NEN. TNF was a gift from Hoffmann-La Roche (Basel,
Switzerland). All other reagents were obtained from Sigma.
Methods
Cell CultureAll cells were maintained for up to 20 passages at 37 °C in a 5% CO2 incubator. For general maintenance, L929 and C12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 60 mg/liter kanamycin sulfate. L929/neo and CPL4 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 0.8 mg/ml G418. For studies, cells were plated at 2 × 104/well in 12-well plates, at 5 × 104/well in 6-well plates, or at 1 × 105/well in 10-cm Petri plates. Cells were allowed to grow to 50-70% confluence and then washed, refed, rested for 4 h, and treated as indicated. Time-matched controls were always run concurrently.
Arachidonic Acid ReleaseCells seeded in 6- or 12-well
plates were grown for 2 days and then labeled with 1 µCi/ml
[3H]arachidonic acid for 24 h. Post-labeling, cells
were washed, refed, and rested for 4 h. Cells were then treated as
indicated, and 1.0-1.2 ml of culture medium was harvested from each
treatment well. Non-adherent cells were pelleted out of the harvested
medium, and 400-µl aliquots were counted to determine the levels of
released label. The total counts associated with cells were ~150,000
dpm, and 7% of the total label (10,500 dpm) was released in response to TNF. The medium was also subjected to TLC to assess whether metabolites such as phospholipids or acylglycerols accounted for the
observed counts. We found that minimal counts, if any, were associated
with such metabolites.
Cells grown in 10-cm Petri plates were grown for 2-4 days, refed, rested, and treated. Following the indicated times of treatment, the media were removed from the plates, and adherent cells were washed with PBS. Cells were then scraped, and the lipids were extracted via the method of Bligh and Dyer (18). In instances where cell floaters were analyzed, the treatment media and the PBS wash were pooled and spun down. The resultant pellet was then considered the "floater" population, and lipids were harvested from these cells. Extracted lipids were dried, resuspended in chloroform, and aliquoted for phosphate (19) and diacylglycerol kinase (13) analyses as described previously. Ceramide and diacylglycerol levels were quantitated using external standards, and the resultant values were normalized against total lipid phosphate.
SM QuantitationCells grown in 10-cm Petri plates for 2-4 days were treated as described for the ceramide measurements. Lipids were extracted via the method of Bligh and Dyer (18), and SM was quantitated by the bacterial SMase method described previously (13).
SMase Isolation and AssayCells were seeded at 3 × 105/30-cm Petri plate in 30 ml of regular growth medium.
Cells were allowed to grow for 3-4 days and then washed, refed, and
treated. Following the indicated treatment times, cells were scraped
into a minimal volume of serum-free medium and pelleted. Retrieved
cells were resuspended in cold lysis buffer (20) and lysed via three
cycles of freeze-thawing (one cycle = 3 min in a methanol/dry ice
bath, 3 min at room temperature, and vortexing). By this protocol,
95% of the cells were lysed. Cells were spun at 2100 rpm (1000 × g) for 10 min to remove nuclei and the few unlysed cells.
The resulting homogenate was assayed for SMase activity as described
previously (13). Assay conditions for the three different
sphingomyelinases were as follows: 1) neutral
Mg2+-dependent: 10 nmol of SM (2 × 105 cpm), 0.1% Triton X-100, 0.1 M Tris-HCl,
pH 7.4, and 5 mM MgCl2; 2) neutral
Mg2+-independent: 10 nmol of SM (2 × 105
cpm), 0.1% Triton X-100, and 0.1 M Tris-HCl, pH 7.4; and
3) acidic: 10 nmol of SM (2 × 105 cpm), 0.1% Triton
X-100, and 0.1 M sodium acetate, pH 5.0.
Cells were grown in 6-well plates for 2 days and then washed, refed, rested, and treated. Four hours prior to harvest, 1 µCi/ml [3H]thymidine was added to each well. Following the indicated treatment times, cells were harvested via a modification of a previously described method (21). Briefly, the medium was removed, and cells were washed twice with cold PBS. Cells remaining in wells were washed twice with 5% trichloroacetic acid and solubilized in 0.5 ml of 0.25 N NaOH, and 0.3 ml was collected and counted.
Crystal Violet AssayCells were grown in 6- or 12-well plates for 2 days and then washed, refed, rested, and treated. Following treatment, the medium was removed, and cells were washed with PBS. The amount of cells remaining adhered to the plate was assessed via crystal violet staining as described previously (22).
NF-Cells were grown to 70-80% confluence
and then treated in the presence of regular culture medium. Following
the indicated treatment times, cells were harvested via trypsinization,
and the pellets were washed one time with cold PBS. Nuclear extractions and electrophoretic mobility shift assays were run via a modification of methods previously described (23). Briefly, the cell pellets were
quick-frozen using an ethanol/dry ice bath, and the pellets were then
resuspended in 50-100 µl of hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, and 1 mM dithiothreitol). This hypotonic
lysis yields ~100% lysis of cells. The lysed cells were then spun,
and the nuclear pellet was recovered and resuspended in 15 µl of
hypertonic buffer (20 mM HEPES, pH 7.9, 0.4 M
NaCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride). Extraction of the
nuclear protein was achieved by gentle mixing of this mixture for 30 min at 4 °C. The debris was then spun down, and the resultant
supernatant was diluted with 20-70 µl of dilution buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 20% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride). Approximately 2-µl aliquots were used for the Bio-Rad protein assay, and the remaining portion was quick-frozen and stored at 80 °C until gel shift assays were run. Protein-DNA reactions were performed in a 20-µl volume and contained 8-10 µg of nuclear extract, 1 µg of
poly[d(I·C)], 1 µg of poly[d(N)6], 10 µg of
bovine serum albumin, 20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 5 mM
dithiothreitol, and 10,000-50,000 cpm radiolabeled oligonucleotide
probe (see Ref. 23 for sequences used). Reactions were allowed to
proceed for 20 min and then terminated by the addition of 6 µl of
15% Ficoll. Nondenaturing polyacrylamide gels (5%) that had been
prerun for 1-1.5 h at 200 V were loaded with equal volumes of reaction
mixture and run at 200 V for 1.5-2 h. Gels were then dried and exposed
to film. Shown (Fig. 10) is a representation of an autoradiogram
obtained in this manner.
L929 murine fibroblasts are
acutely sensitive to the cytotoxic effects of TNF. Treatment with
0.1-30 nM TNF
induced significant cell death within
24 h as detected via cell counts, crystal violet assays, and
[3H]thymidine uptake measurements (Fig.
1). Interestingly, cytotoxicity in these cells was
accompanied by the loss of adhesion to the culture plate at later time
points. By 12 h of treatment, a floater population of cells was
starting to appear. Following 24 h of treatment, 97% of the cells
were found in this floater population, of which almost 99% were dead
and only 1% remained viable (Fig. 1B). Of the cells
remaining adhered to the culture plate, 50-80% of the cells remained
viable as determined by trypan blue exclusion. With increased periods
of TNF
treatment, fewer cells remained in the viable/adherent
population, and more cells were found in the dead/floater population.
Thus, loss of adhesion appears to correlate with terminal stages of the
L929 death process induced by TNF
.
TNF
TNF
treatment of L929 cells also led to maximal increases in ceramide
levels within 24 h (Fig. 2A). As early
as 5-6 h following treatment with 5 nM TNF
, increases
in ceramide of 150% were observed (Fig. 2A,
inset). This delayed TNF
-induced ceramide generation differed from the previously reported effects in which cytokine stimulated ceramide production within seconds or minutes of treatment. In fact, L929 cells showed no fluctuation in ceramide over a period of
1-60 min (data not shown), a time frame in which ceramide levels have
been shown to peak and subsequently return to basal levels in HL-60
cells treated with TNF
(13). Furthermore, L929 cells demonstrated
prolonged elevations of ceramide levels. Instead of attaining a peak
level and quickly returning to basal levels, L929 cells were found to
maintain almost an 180% increase in ceramide as long as 48 h
following TNF
treatment.
More important, ceramide elevations in response to TNF preceded the
onset of death. In the adherent population, which showed minimal cell
death, maximal ceramide elevations were observed. Also, ceramide
elevations were observed as early as 5 h following treatment; in
contrast, the presence of floaters was not seen until 12 h (data
not shown). Floater populations maintained high levels of ceramide,
suggesting that basal levels could not be reattained prior to the onset
of death (Fig. 2B).
Since there was such a difference in the kinetics of
TNF-induced ceramide elevation in L929 cells compared with
previously studied cytokine-stimulated systems, it became important to
ascertain whether sphingomyelin was the source of ceramide. We
therefore determined whether SM levels decreased in response to TNF
stimulation. Indeed, we found that, as early as 2 h following
treatment, SM levels had decreased by ~20%, and by 14 h, when
ceramide levels had increased to maximal levels, SM levels dropped by
40% as assessed by loss of label (Fig. 3).
We then investigated whether the difference in the temporal relation
between TNF induction and ceramide elevation could be explained by a
difference in the type of SMase activated. In HL-60 cells, it has been
shown that the rapid elevation of ceramide is the consequence of
activation of a neutral cytosolic SMase activity. In contrast,
stimulation of L929 cells with TNF
was found to increase the
activity of not only the two neutral SMase activities (both
magnesium-independent and magnesium-dependent), but also
the acidic SMase activity (Fig. 4). Although the
elevation of all three activities appeared to be analogous (~2-fold)
in the adherent population of cells, there were some differences among
the three activities in the floater population (Fig. 4). Floater/dead
cells appeared to have the greatest increase in neutral magnesium-independent SMase activity (>3-fold). The neutral
magnesium-dependent SMase was the first to show increased
activity in response to TNF
, and its activity was increased
~2-2.5-fold at 4-6 h following treatment (data not shown).
AA Generation Temporally Precedes Ceramide Generation
As with
other cell systems, TNF stimulation of L929 cells led to an increase
in AA release (Fig. 5). AA release in L929 cells demonstrated delayed kinetics, with the earliest increases observed following only 3-4 h of treatment (Fig. 5B) (14). Following 5-60 min of TNF
treatment, times in which HL-60 cells have been found to elevate AA levels, almost no change in AA release was found
(data not shown). Furthermore, over the time period in which AA release
was observed in L929 cells, the maximal level of AA elevation
superseded the levels observed in other cell systems. Following 4-8 h
of TNF
treatment, an almost 2-fold increase in AA release was
observed.
Similar to HL-60 cells, cytokine-stimulated AA release temporally
preceded ceramide elevations (Fig. 5B versus Fig.
2A, inset). Whereas ceramide increases could not
be seen until 5 h following treatment with 5 nM
TNF, AA release was seen 1-2 h earlier. Temporally, therefore, the
link between AA generation and ceramide generation still holds even
with the prolonged kinetics observed in L929 cells.
Although
temporal correlations establish AA as a precedent to ceramide
generation, the necessity for PLA2 activity/AA generation to modulate TNF-induced ceramide elevation required the use of other
tools. The L929 variant line C12 is known to be defective in
cPLA2 as evaluated by Northern blot analysis, activity
measurements (14), and Western blot analysis (data not shown). In L929
cells, 30 nM TNF
caused a 60% decrease in cell numbers
as early as 16 h following treatment; in contrast, cytokine
treatment of C12 cells resulted in very little cytotoxicity even
following extended treatment (Fig. 6). At best, in the
C12 cell line, a maximal cytotoxic effect of ~35% decrease in
proliferation was observed after prolonged (48 h) stimulation with 30 nM TNF
.
Similar to previous findings (15), we found that the resistance of C12
cells to TNF accompanied the inability to generate AA. Whereas the
parental L929 line showed an elevation of AA release that peaked within
3-5 h of treatment (Fig. 5), C12 cells exhibited little or no increase
in AA even following prolonged stimulation (Fig. 5A). A
decreased amount of total cellular AA could account for the inability
of resistant lines to generate AA in response to TNF
. Thus, it
became important to ascertain whether the mass levels of AA were
different in L929 versus C12 cells. As shown in Table
I, there was very little difference in the basal
mass levels of AA between the two lines, with both L929 and C12 cells containing 50-60 pmol of AA/nmol of phosphate. Similarly, there were
no significant differences in the content of other fatty acids between
these two cell lines (Table I).
|
We next assessed the ability of TNF to stimulate SM
hydrolysis and ceramide generation in the resistant cells. We found
that, similar to AA release, ceramide generation was perturbed in C12 cells. In L929 cells, TNF
was able to induce a 2-fold elevation of
ceramide levels within 14 h of treatment with 30 nM
TNF
. In contrast, C12 cells exhibited no change in ceramide levels
during the same time period (Fig. 7). Neither extended
(24 h) nor short-term treatment with cytokine (data not shown) elicited
any change in ceramide in these resistant cells. Furthermore, we found
that TNF
treatment did not lead to a decrease in SM levels (Fig. 3) or to an elevation of any of the three SMase activities that have been
linked with signaling, the neutral magnesium-independent, neutral
magnesium-dependent, and acidic activities (Fig.
8). The basal specific activities of all three enzymes
(neutral magnesium-independent, ~1000 cpm/mg/h; neutral
magnesium-dependent, ~2000 cpm/mg/h; and acidic,
~20,000 cpm/mg/h) were equivalent in L929 and C12 cells. Indeed,
treatment with TNF
in C12 cells caused a decrease in the activity of
the SMases, possibly a result of activation of anti-apoptotic pathways
in C12 cells (such as NF-
B) that remain responsive in this cell line
(see below).
Ceramide Does Not Induce AA Generation, but Does Induce Cytotoxicity
The above results suggest that the generation of AA
is coupled to ceramide generation. To establish that perturbation of
ceramide generation is not the reason for the aberrant response of C12 cells in generating AA, we determined whether exogenous ceramide addition affected AA levels. In L929 cells, we found that treatment with 10 µM C6-ceramide had no stimulatory
effect on AA release (Fig. 9A). In contrast,
treatment of L929 cells with equivalent concentrations of
C6-ceramide caused death (Fig. 9B).
Concentrations as low as 1 µM C6-ceramide
induced a 30% decrease in proliferation within 24 h, and
concentrations of 10-40 µM caused as much as a 50-90%
decrease in growth within 24 h. Thus, ceramide is able to elicit
the same biological end point as TNF without stimulating AA
production, demonstrating that ceramide generation is not upstream of
AA generation.
Ceramide Overrides the Resistance of C12 Cells
C12 cells, similar to L929 cells, did not release AA in response to 10 µM C6-ceramide (Fig. 9A). However, concentrations of 1-40 µM C6-ceramide were able to elicit cytotoxicity in both of these cells (Fig. 9B). Notably, C6-ceramide was more cytotoxic to C12 cells than to L929 cells. Thus, the defect in C12 cells could be rectified via exogenous treatment with ceramide, suggesting that ceramide functions downstream of PLA2 activation and AA generation.
Lack of AA Generation Does Not Perturb Other TNFSince disruption of PLA2
activity/AA generation should exclusively affect downstream events
regulated by AA, other signaling events not associated with AA should
remain unperturbed. We therefore determined whether TNF signaling to
NF-
B remained intact. TNF
-induced NF-
B activation in L929
cells exhibited very different kinetics from the mobilization of AA or
ceramide. Nuclear translocation of NF-
B was observed via gel shift
analysis as early as 5 min following stimulation with 0.2 nM TNF
(Fig. 10A). Treatment
of L929 cells over a period of hours showed that nuclear translocation of NF-
B was maintained over 4 h, but unlike AA and ceramide
liberation, began to decline markedly by 12 h. More important, C12
cells, similar to L929 cells, showed nuclear translocated NF-
B
within 15 min of treatment with 0.5 nM TNF (Fig.
10B). The level of NF-
B activation seen in the
TNF
-sensitive versus -resistant fibroblasts was nearly
identical. Thus, NF-
B stimulation, a TNF
-responsive signaling
event, appears to be intact even in cells incapable of signaling
through PLA2 and AA. These results establish a specificity to the perturbation of ceramide generation in resistant cells.
C12 cells, which had been found to lack
TNF-responsive cPLA2 activity, were transfected with an
expression plasmid containing cPLA2 to generate the CPL4
cell line (15). These cells express PLA2 activity (14) and
cPLA2 protein as evaluated by Western blot analysis (data
not shown). These CPL4 cells were utilized to assess the necessity of
cPLA2 activity for ceramide generation. When treated with
TNF
, CPL4 cells responded similarly to both the parental L929 and
control vector-transfected L929/neo cell lines; however, CPL4 cells
displayed delayed kinetics and decreased responsiveness to TNF
.
Concentrations of 5 nM TNF
stimulated only 60% death
within 24 h (versus 90% death in L929/neo cells) in
CPL4 cells (Fig. 11A). CPL4 cells, like L929
cells, also responded to TNF
by stimulating AA release. At 1 h
of TNF
treatment, when the vector (positive) controls were beginning
to show a response (138%), CPL4 cells showed lower than basal levels
of AA release (90%). However, by 4 h of treatment with 5 nM TNF
, CPL4 cells showed levels of AA release (150%)
comparable to those seen in L929/neo cells (190%) (Fig.
11B). CPL4 cells also responded to exogenous ceramide
treatment much like the other lines, with doses of 1-40
µM having potent effects within 12-24 h (Fig.
11C). Finally, CPL4 cells were able to respond to TNF
treatment with generation of ceramide. Ceramide generation corresponded
to the other changes in CPL4; thus, elevations occurred later than in
L929/neo cells and never reached the full extent of response attained
in the vector controls. Following 5 h of treatment with 5 nM TNF
, L929/neo cells showed a response of an almost
125% ceramide increase; in contrast, CPL4 cells exhibited basal levels
of ceramide at this time. By 24 h, CPL4 cells still showed
decreased responsiveness compared with L929/neo cells; however, they
did produce significantly more (180%) ceramide than control untreated
cells (Fig. 11D). Since C12 cells did not show any level of
ceramide responsiveness to cytokine, the increase in ceramide
generation can be attributed to the expression of cPLA2.
The attenuation in the responses of CPL4 cells may be related to the
incomplete restoration of PLA2 levels in this cell line as
seen by activity measurements (14) and by Western blot analysis (data
not shown). Since CPL4 cells showed significant enhancement of
cytokine-induced lipid generation and growth inhibition compared with
the precursor C12 line, which differs only in the expression of
cPLA2, these results establish the necessity of
cPLA2 activity/AA generation for ceramide generation and
growth inhibition.
This study demonstrates the importance of PLA2 to
ceramide generation and cytotoxicity. Previous studies in the HL-60
cell system suggested a link between AA and the SM cycle in TNF
signaling (13); however, the L929 system, used here, has allowed the
further development of these initial studies. L929 cells vary markedly from the HL-60 model in two critical respects. First, the temporal correlation between receptor activation and lipid mobilization (both AA
release and ceramide release) is greatly attenuated in L929 cells,
taking hours as opposed to minutes. Second, the magnitude of change in
ceramide elicited by TNF
stimulation of L929 cells (2-4-fold
changes) exceeds the levels attainable upon cytokine stimulation of
HL-60 cells (at best, 1.5-1.8-fold changes) (13, 16). Despite these
differences, both systems show TNF
-induced PLA2
activation/AA generation to occur prior to SM hydrolysis and ceramide
generation. Thus, the L929 model demonstrates that AA-mediated
signaling to ceramide is not restricted to one cell system, but is
indeed a cascade that may have greater implications.
Furthermore, the L929 model has been used here to extend our studies
from the correlative level to establish the necessity of AA liberation
for ceramide generation. These studies were possible because of the
availability of L929 clones resistant to the cytotoxic effects of
TNF that are defective in cPLA2. We found that, whereas L929 cells responded to TNF
stimulation by elevating AA levels within 2-4 h, C12 cells showed little if any elevation of AA release. Likewise, we found that, unlike the parental L929 line, this resistant line was incapable of responding to TNF
through the production of
ceramide. In contrast, TNF
-stimulated NF-
B activation appeared to
be intact, suggesting that only AA-dependent cascades were affected in the resistant cells. Thus, initial observations with the
resistant lines illustrated the specificity of the interplay between AA
and ceramide. We further established the necessity for AA generation
through utilization of a variant strain of C12, the CPL4 line, which
differed from C12 only with respect to the presence of an expression
plasmid containing the murine cPLA2 gene. We found that,
through recapitulating PLA2 activity in the CPL4 lines, we
were able to re-establish 1) cytokine-induced AA generation, 2)
TNF
-induced ceramide generation, and 3) TNF
-induced growth
inhibition. Although the CPL4 system did not fully restore cytokine
responsiveness, it did serve to demonstrate the interconnection among
AA, ceramide, and growth.
A number of key points emerge from this study. First, it becomes
apparent that the kinetics of ceramide generation are dependent upon
the type of SMase activated. Whereas rapid ceramide mobilization has
been attributed to the activation of one enzyme (depending on the
system, a neutral, cytosolic, or acidic SMase), more prolonged ceramide
generation appears to be the consequence of multiple activities perhaps
acting together: a neutral Mg2+-dependent
SMase, a neutral Mg2+-independent SMase, and an acidic
SMase. Whether all three of these enzyme activities can be regulated by
AA remains to be determined. Also, since the long-term accumulation of
ceramide involves multiple enzymes, multiple pools of SM could be
implicated. At a first level of examination, however, the ceramide
generated in both the L929 and HL-60 systems appears to be similar (as
determined by TLC) (data not shown). Whether all three SMase activities
found to be stimulated by TNF in L929 cells can act on the same pool of SM, however, remains unknown.
Second, these results raise the question of how PLA2/AA couples to SMase. The exact links between PLA2 activation/AA generation and ceramide generation remain elusive. Furthermore, other components that may be involved in regulating the signaling cascade between TNF receptor stimulation and SMase activation remain to be determined. It is important to note in this context that Fas ligand-induced death, in contrast to TNF, may not require cPLA2 (27).
Finally, the prolonged kinetics of ceramide generation raise a possibility that ceramide may function as a long-term regulator of cell growth/viability. Indeed, most studies examining the changes in ceramide associated with cell death or growth suppression disclose similar long-term changes in ceramide. These include serum withdrawal, fas stimulation, and dexamethasone-induced apoptosis (7, 24, 25). These studies have raised the question whether ceramide generation is a precedent to cell death and differentiation or whether ceramide elevations are a consequence of the biological fate and serve more as a marker of phenotype. The kinetics established in this study clearly show that both SMase activation and ceramide elevation precede the appearance of a dead/floater population of cells. The observed increase in ceramide in the adherent population of cells demonstrates cytokine-responsive SM hydrolysis prior to the onset of death, suggesting that ceramide precedes cell death. Ongoing studies also support a role for ceramide prior to the onset of actual cell death. In cells overexpressing the anti-apoptotic protein Bcl2, it appears that ceramide generation in response to chemotherapeutic agents is not perturbed, although cell death is greatly reduced (26). Such studies continue to expand our understanding of where AA and ceramide fit in the overall scheme between cell stimulation and onset of response, whether it be death, differentiation, or cell cycle arrest.