(Received for publication, December 4, 1996, and in revised form, January 30, 1997)
From the § Durham Veterans Administration Geriatric
Research Education and Clinical Center and the
Departments of Medicine and Cell Biology, Duke
University Medical Center, Durham, North Carolina 27710
The activation of sphingomyelinase and generation of ceramide have been implicated as important regulatory pathways in cell growth and apoptosis. Bacterial sphingomyelinase has been used in many cell systems to mimic the activation of endogenous sphingomyelinase. These studies, however, have been complicated by the inability of exogenously applied bacterial sphingomyelinase to perform many of the effects of short chain cell permeable ceramides, indicating that there may be a distinct signal transducing pool of sphingomyelin not accessible to exogenous sphingomyelinase or that endogenous ceramide is not sufficient to induce these changes. We cloned the Bacillus cereus sphingomyelinase gene by polymerase chain reaction and subcloned it into a mammalian expression vector under the control of an inducible promoter. Upon stable transfection and induction of B. cereus sphingomyelinase, there were increases in neutral sphingomyelinase activity, cellular ceramide levels, cleavage of the death substrate poly(ADP-ribosyl)polymerase, and cell death. In contrast, exogenously applied B. cereus sphingomyelinase, despite causing higher elevations in ceramide levels, was unable to induce poly(ADP-ribosyl)polymerase cleavage or cell death. These results support the existence of a signal transducing pool of sphingomyelin that is distinct from the pool accessible to exogenous sphingomyelinase.
It is now well appreciated that ceramide, the product of
sphingomyelin hydrolysis, may play an important role in apoptosis (1)
and other cellular responses including terminal differentiation (2),
cell cycle arrest (3), and cellular senescence (4). Evidence to support
this comes from studies using cytokines (such as
TNF1 (5), interleukin 1
(6, 7), and
nerve growth factor (8)) or chemotherapeutic agents (such as
vincristine (9), cytosine arabinoside (10), and daunorubicin (11)),
which are known to induce apoptosis, demonstrating that these agents
induce elevations of intracellular ceramide. In addition exogenously applied short chain cell-permeable ceramides are able to mimic these
inducers and cause apoptosis. Finally, studies with inhibitors of
ceramide metabolism, such as PDMP (12) and D-MAPP (13), show that these compounds also cause apoptosis, probably as a consequence of elevating ceramide levels.
Sphingomyelinase has been implicated as the cause of elevation of
intracellular ceramide levels whereby neutral sphingomyelinase activity
has been demonstrated to increase in response to TNF as well as
other inducers of apoptosis (3, 6, 10, 14, 15). However, other sources
of ceramide generation have been proposed, including de
novo synthesis of ceramide (11) as well as sphingomyelin
hydrolysis by acid pH optimum sphingomyelinase (16).
Bacterial sphingomyelinase from Bacillus cereus functions at neutral pH (17) and thus has been considered a useful tool in tissue culture experiments to induce elevation of cellular ceramide levels in an attempt to mimic the biological effects of activation of cellular sphingomyelinase (18-21). This approach has the advantage of generating ceramide in the membrane at physiologically relevant concentrations and avoids some of the drawbacks of using exogenous ceramides. However, we have found that in many of these experiments, exogenously applied bacterial sphingomyelinase lacks substantial activity in inducing apoptosis and other responses. This suggests one of two possibilities: 1) that ceramide is not sufficient to induce those responses or 2) that there are distinct intracellular compartments where sphingomyelinase is activated leading to localized ceramide generation, thus implying that certain pools of ceramide are the biologically relevant or active pools.
In this study, we wanted to examine these possibilities and to determine whether exogenously applied bacterial sphingomyelinase is biologically different from activation of "cellular" sphingomyelinase, thus yielding distinct pools of ceramide. To achieve this goal, we stably transfected Molt-4 leukemia cells with B. cereus sphingomyelinase under the control of an inducible promoter. We demonstrate that we could induce apoptosis only upon induction of transfected sphingomyelinase activity and not when bacterial sphingomyelinase was used exogenously. These data support the hypothesis that ceramide is biologically active and that the generation of ceramide intracellularly is distinct from ceramide generated at the outer leaflet of the plasma membrane.
Materials
B. cereus was obtained from American Type Culture Collection (ATCC 14579). Restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase, and RNA molecular weight markers were obtained from Boehringer Mannheim. Nick translation kit was purchased from Life Technologies, Inc. Taq polymerase was purchased from Perkin-Elmer Corp. Nitrocellulose and Zeta-probe blotting membranes were purchased from Bio-Rad. Anti-PARP anti-serum was purchased from Enzyme Systems Products (Dublin, CA). G418, RPMI 1640 medium, and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. Hygromycin B was purchased from Calbiochem.
Methods
PCR Amplification of Genomic DNA from B. cereus and DNA SequencingGenomic DNA was isolated from B. cereus
cultured overnight at 30 °C in brain heart infusion cultured medium
(Sigma). For PCR amplification, the sense and antisense
sequences used were 5-CCTCTAGATGGAGGCATAGAAATAGCGC-3
and
5
-AAACAACTTCATAGAAATAGTCG-3
, respectively. PCR amplification was performed at a denaturing temperature of 94 °C for 1 min
followed by annealing at 37 °C for 2 min, and extension was at
72 °C for 2 min for a total of 30 cycles. The amplified fragment
(1000 base pairs) was separated by electrophoresis with a 0.8% agarose
gel and purified by phenol extraction. For sequence confirmation, the
purified insert was subcloned into the vector pBS (Stratagene), and its
sequence was determined using the version 2.0 DNA Sequencing Kit
(U. S. Biochemical Corp.). The 1-kilobase fragment was then removed
from pBS and blunt-ended by the Klenow fragment, NotI linkers were added, and the fragment was then cloned into a
NotI-linearized pOP13CAT vector (Lac Switch Inducible
Mammalian Expression System from Stratagene). The resultant plasmid was
named Bacterial Sphingomyelinase-13 (BSM-13).
To obtain stable transfectants, Molt-4 cells,
grown in RPMI 1640 containing 10% FBS and 25 mM HEPES,
were first transfected by electroporation with p3SS vector (10 µg
for 1 × 106 cells in 50 ml) using the Bio-Rad
electroporation apparatus. Hygromycin (0.3 mg/ml) was added to the
cells 48 h later and maintained as a selective pressure. The
expression of the Lac repressor was monitored by Western blot with a
polyclonal antibody (Stratagene). Upon obtaining a stable transfectant
of the Lac repressor protein, the BSM-13 plasmid was then introduced by
electroporation. In addition to hygromycin, selection pressure was
further achieved with geneticin (0.2 mg/ml). The expression of the
1-kilobase fragment was examined by Northern blot.
Stably transfected cells (5 × 105 cells/ml) were rested for 2 h in RPMI containing 2% FBS and 25 mM HEPES prior to the addition of desired concentrations of IPTG. At the indicated times, cells were removed and assayed for viability (by trypan blue exclusion), ceramide content, sphingomyelinase activity and PARP proteolysis.
Ceramide MeasurementThe level of ceramide in the transfected cells was determined by the Escherichia coli diacylglycerol kinase assay system essentially as described (22).
Sphingomyelinase Activity AssayFor determining the activity of the neutral, magnesium-dependent sphingomyelinase, cells (2 × 107) were washed three times with ice-cold PBS and disrupted by freezing and thawing (three times in methanol-dry ice bath) in 400 µl of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM EGTA, 1 mM sodium vanadate, 10 mM B-glycerol phosphate, 1 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, 20 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin. The lysate was centrifuged for 10 min at 1,000 × g at 4 °C, and the supernatant (post nuclear homogenate) was centrifuged for 60 min at 100,000 × g at 4 °C. The resulting pellet (membrane fraction) was resuspended in 200 µl of lysis buffer. An aliquot of the membrane preparation was incubated for 30 min at 37 °C with [14C]sphingomyelin (100,000 dpm, 10 nmol) in a mixed micelle assay containing 100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 0.1% Triton X-100 (final volume, 100 µl). The radiolabeled product was extracted as described (3), and the radioactivity was determined by liquid scintillation counting. To determine the acid sphingomyelinase activity, membranes were prepared from cells using a lysis buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride. The activity of acid sphingomyelinase was determined using a mixed micelle assay containing 100 mM sodium acetate, pH 5.0, and 0.1% Triton X-100.
Western Blot for PARP ProteolysisTransfected cells (5 × 106) were pelleted, resuspended in 133 µl of PBS and 23 µl of 5 × SDS-electrophoresis sample buffer, and boiled immediately. 15 µl of the cellular lysate was subjected to electrophoresis with a 6% SDS-polyacrylamide gel. Western blots were performed exactly as described in Ref. 23.
We initially set out to evaluate the ability of exogenous
bacterial sphingomyelinase to generate ceramide and to induce
apoptosis. Molt-4 leukemia cells were treated with B. cereus
sphingomyelinase and evaluated for ceramide generation and for cell
death. Fig. 1A shows that exogenously
administered sphingomyelinase induced significant increases in cellular
ceramide levels. Surprisingly, however, B. cereus
sphingomyelinase failed to induce cell death in these cells at
concentrations of up to 300 mU/ml and for as long as 24 h (Fig.
1B).
Cellular sphingomyelinase activity has been demonstrated to increase in
response to inducers of apoptosis and is believed to be the cause of
intracellular ceramide generation. Exogenously used bacterial
sphingomyelinase, despite causing elevation of ceramide levels,
apparently did not induce cell death. Linardic and Hannun have
demonstrated that exogenous bacterial sphingomyelinase could not reach
the so called signaling pool of sphingomyelin, which they suggested was
on the inner leaflet of the plasma membrane (21). We therefore elected
to stably transfect bacterial sphingomyelinase into cells and study its
effects on ceramide generation and cell death when it was an integral
part of the cell membrane. To do so we harvested genomic DNA from
B. cereus and used it as a template to amplify the B. cereus sphingomyelinase gene by the polymerase chain reaction
using the oligonucleotides described under "Experimental Procedures." We then subcloned the gene and sequenced it to
demonstrate no PCR-induced mutations. The gene was cloned into the
eukaryotic lac-operator-containing vector pOP13CAT from the
Lac Switch inducible mammalian expression system (see "Methods").
This vector was then used to transfect Molt-4 cells that had already
been stably transfected with the eukaryotic Lac repressor-expressing
vector p3SS.
We then showed that sphingomyelinase activity was repressed in the
presence of the Lac repressor. Upon induction by IPTG, this repression
was removed, and enzyme activity was expressed. Sphingomyelinase
activity was significantly increased within 1 h of IPTG treatment
and peaked at 250% of control levels by 4 h (Fig.
2A). The induced sphingomyelinase activity
was a neutral pH optimum membrane activity and was linear with protein
concentration (Fig. 2B). Acidic sphingomyelinase activity
did not change upon IPTG induction (Fig. 2C). Concomitant
with the increase in neutral sphingomyelinase activity, there was an
increase in cellular ceramide levels such that ceramide levels were
induced by IPTG (Fig. 2D) and were significantly higher than
levels in vector transfected cells (Fig. 2E), indicating
that they were due to sphingomyelinase induction and not a consequence
of IPTG treatment.
Because sphingomyelinase activity was increased and cellular ceramide
levels were elevated in response to induction of the bacterial
sphingomyelinase gene, we next evaluated its biological effects. Fig.
3 demonstrates that as early as 2 h after IPTG
induction of sphingomyelinase activity, there was 20% cell death as
measured by trypan blue exclusion. By 8 h, close to 60% of the
cells were unable to exclude trypan blue as compared with less than
10% of vector transfected cells.
To demonstrate if the cell death occurring in response to induction of
B. cereus sphingomyelinase was apoptotic, we assayed for
cleavage of the known death substrate PARP (24). Recently we have
demonstrated that ceramide induces PARP cleavage in Molt-4 cells (23),
an effect also seen with chemotherapeutic agents and considered as an
indicator of apoptosis. Fig. 4 shows that upon ceramide
treatment, PARP was cleaved as expected to the characteristic 85-kDa
fragment. Exogenous bacterial sphingomyelinase (300 mU/ml) treatment
failed to induce PARP cleavage. Induction of transfected sphingomyelinase, on the other hand, induced significant cleavage of
PARP, such that partial PARP cleavage occurred within 3 h (data not shown), and significant cleavage of PARP was seen by 6 and 15 h of IPTG treatment, implying that this enzyme induces cell death by
apoptosis. It is interesting to note that there is a faint base-line
cleavage of PARP in transfected cells even before induction by IPTG,
indicating that this promoter system is slightly leaky.
It has become apparent from several studies that ceramide is sufficient to drive many aspects of cell growth suppression. This is based primarily on studies utilizing short chain ceramide analogues (1, 2, 4) as well as studies that modulate metabolism of ceramide by inhibitors such as PDMP (12) or D-MAPP (13). In our hands, modulating ceramide levels by exogenous bacterial sphingomyelinase failed to exert the biologic effects of growth regulation expected from increases in cellular levels of ceramide. This could be due to one of two reasons: first, that ceramide is not sufficient on its own to produce biological effects, or second, that ceramide functions in unique cellular compartments that the exogenous application of bacterial sphingomyelinase does not appear to modulate.
Our data demonstrate clearly that bacterial sphingomyelinase, when applied exogenously to cells, fails to induce many of the biologic effects normally seen in association with activation of endogenous sphingomyelinase or induced by cell-permeable ceramides. On the other hand, upon stable transfection of bacterial sphingomyelinase into cells and induction of gene expression, we demonstrate the activation of a functional sphingomyelinase protein and a significant elevation of ceramide levels. Although the levels of ceramide achieved by inducing bacterial sphingomyelinase intracellularly are lower than those achieved by using exogenous bacterial sphingomyelinase, it appears that the ceramide generated from transfected sphingomyelinase but not that generated from exogenous sphingomyelinase leads to apoptosis.
Interestingly, several studies to date have demonstrated conflicting
reports on the ability of exogenously applied bacterial sphingomyelinase to induce cellular effects. For example, Ji et al. have demonstrated that bacterial sphingomyelinase can mimic TNF and C2-ceramide (albeit very modestly) and induce
tyrosine phosphorylation of a 23-kDa nuclear protein (25). Raines
et al. have demonstrated that bacterial sphingomyelinase can
mimic TNF and induce MAP kinase activation in HL60 cells (20).
Sasaki et al. have also demonstrated that bacterial
sphingomyelinase induces MAP kinase activity, as does exogenous use of
ceramide in NIH 3T3 cells (26); however, in most studies, exogenous
ceramides do not appear to activate MAP kinase and preferentially
activate jun kinase (or stress-activated protein kinase)
(27). Santana et al. have shown that bacterial
sphingomyelinase as well as exogenous ceramide can partially mimic
TNF
action in granulosa cells and cause inhibition of P-450
aromatase activity (28). Tamura et al. have demonstrated
that bacterial sphingomyelinase can mimic NGF and inhibit neurite
outgrowth in PC12 cells, but the effects of ceramides were not
evaluated (19). Riboni et al. have demonstrated that
bacterial sphingomyelinase as well as exogenous ceramide can mimic
retinoic acid and cause inhibition of cell proliferation, differentiation, and stimulation of neurite outgrowth in neuroblastoma cells (29). On the other hand, Walev et al. have
demonstrated only selective cytotoxic effects of bacterial
sphingomyelinase on monocytic cells but not on human granulocytes,
fibroblasts, lymphocytes, or erythrocytes (30); ceramide effects were
also not tested. In our hands bacterial SMase failed to induce
apoptosis in U937 cells, a monocytic leukemia cell line. Borchardt
et al. demonstrated that bacterial sphingomyelinase caused
elevation of ceramide levels but failed to inhibit growth of human
T-cells (31). This resembles the failure of bacterial sphingomyelinase to induce growth arrest and cell death that we see. Again, these studies may be due to differential responses of different cell types or
due to differential responses of different intracellular pathways to
several potential pools of ceramide generation. Clearly there is a
difference in cellular responses between exogenously applied bacterial
SMase and short chain cell-permeable ceramides, particularly on
modulation of growth arrest and apoptosis.
There are several implications to these results. First, ceramide
generated by activation of transfected sphingomyelinase appears to be
sufficient to induce cellular effects of growth suppression. Second,
these results support the existence of different intracellular pools of
ceramide, such that when cells are treated with bacterial sphingomyelinase, it is able to cleave the sphingomyelin from the outer
leaflet of the plasma membrane, but apparently there remains a part of
sphingomyelin that is not accessible to exogenously applied
sphingomyelinase. In fact, this is supported by evidence from HL60
cells (21) and in fibroblasts (32) where the pool of sphingomyelin that
is hydrolyzed in response to inducers (such as vit D3 or
TNF) appears to be on the inner leaflet of the plasma membrane (or
another compartment distinct from the outer leaflet pool of
sphingomyelin) and not accessible to exogenous sphingomyelinase. This
pool appears to be the signal transducing pool because when sphingomyelinase is transfected into cells, it is able to induce its
cellular effects, implying that ceramide is generated
intracellularly.
If exogenous sphingomyelinase cleaves sphingomyelin and generates ceramide as seen by our data, then how come that ceramide appears to be biologically inactive? Several possibilities emerge. First, it is possible that the ceramide generated in the cell membrane is unable to flip and enter the cell, despite evidence to the contrary (33). Second, ceramide may be metabolized quickly prior to its reaching the cellular compartment where it performs its biological activity. Third, the molecular species of this ceramide is distinct from the signaling ceramide. Finally, the "signaling" ceramide is generated and is active in a compartment distinct from the plasma membrane or in a specialized compartment within the plasma membrane. Obviously, extensive further investigation is required to sort out these possibilities.
We acknowledge Dr. David Perry for careful review of the manuscript and Don Garrett and Rita Fortune for expert secretarial assistance.