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Induces Apoptosis in Immortalized Hypothalamic Neurons: Involvement of Ceramide-Generating Pathways
Institutes of Pharmacology and Respiratory Diseases (C.V.), University of Catania School of Medicine, 95125 Catania; and the Department of Internal Medicine, Section of Pharmacology, University of Pavia (P.L.C.), Pavia, Italy
Address all correspondence and requests for reprints to: Dr. Maria Angela Sortino, Institute of Pharmacology, University of Catania School of Medicine, Viale Andrea Doria 6, 95125 Catania, Italy. E-mail: msortino{at}mbox.unict.it
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Abstract |
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on
the growth and viability of GT1–7 hypothalamic neurons and the
intracellular transduction pathways involved in these effects. TNF
caused a reduction of cell number and an induction of apoptotic death.
These effects were mimicked by cell-permeable analogs of ceramide and
by neutral or acidic sphingomyelinase. Exposure to acidic
sphingomyelinase induced a persistent (up to 48 h) reduction of
cell growth and apoptosis, whereas the effect of neutral
sphingomyelinase was time limited. The involvement of acidic
sphingomyelinase in TNF action was demonstrated by the partial
prevention of ceramide generation, apoptosis, and reduced cell growth
by the inhibitor of the acidic sphingomyelinase-generating pathway,
D609, whereas the involvement of ceramide was proved by complete
prevention of TNF-induced effects by treatment with okadaic acid at
concentrations inhibiting ceramide-dependent protein phosphatase. The
present data indicate that TNF, through activation of
ceramide-generating pathways, is able to affect GT1–7 cell viability,
suggesting an additional effect that may contribute to the global
action of this cytokine on neuroendocrine activities. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) as well
as other cytokines are known to affect neuroendocrine secretory
activities (1, 2); this phenomenon may be relevant in the alterations
of endocrine parameters, including disruption of reproductive
function, that accompany infectious states. The
hypothalamus-pituitary-gonadal axis, in fact, exhibits marked
sensitivity to the effects of various cytokines (3, 4), and TNF can
affect the activity of this axis exerting its action at different
levels. Thus, TNF mediates lipopolysaccharide-induced suppression of
the GnRH pulse generator activity (5) and reduces basal and stimulated
LH release by acting either at central level (6) or at the pituitary
(7). In addition, TNF is able to stimulate apoptotic death in
ovarian follicles, suggesting a causative role for this cytokine in the
genesis of follicular atresia (8).
Besides a direct effect on neuroendocrine secretory activity, TNF
may also act by regulating neuronal viability at the hypothalamus. In
fact, in other cellular systems, TNF is known to act as a trophic,
toxic, or differentiating agent (9). At the central nervous system, the
responses to TNF exhibit large variability (10), and neurotoxic
(11, 12, 13, 14, 15, 16, 17) as well as neuroprotective (18, 19, 20) effects of this cytokine
in neuronal cultures have been reported. This appears particularly
intriguing, as definition of the action of TNF on hypothalamic
neuronal viability could provide a significant contribution to the
understanding of the global action of this cytokine at the hypothalamus
and of the possible role of TNF at the intersection between the
neuroendocrine and the immune systems. The relevance of these phenomena
may be related not only to the pathological events that lead eventually
to the impairment of neuroendocrine activity, but also to physiological
conditions that control development and maturation of selected central
nervous system areas, including the hypothalamus. In this respect, the
dual action exerted by TNF may be critical, as it may combine a
general neurotropic effect with the induction of programmed cell death
that takes place during the course of normal development. The
complexity of the response to TNF may be partly related to the
activation of two distinct receptors that mediate TNF signaling:
TNFR1 (p55), whose activation is known to generate intracellular
signals that are responsible also for cell death, and TNFR2 (p75),
whose role has not been completely characterized, but which probably
mediates proliferation and survival events (21). Activation of TNFR1
leads to the hydrolysis of sphingomyelin and the generation of
ceramide, an intracellular second messenger involved in survival and
death phenomena (22, 23). Focusing our attention on this particular
TNF receptor subtype, we have studied the effect of TNF in a
hypothalamic cell population. The availability of the GT1–7 cell line
(24) allowed us to study the action of TNF on cell viability
directly in GnRH-secreting neurons. The choice of this homogeneous cell
population was based on the double opportunity to use an experimental
model that allowed the evaluation of cell viability (25, 26) and the
investigation of intracellular mechanisms. Hence, in GT1–7 cells we
have studied the action of TNF on neuronal viability and
proliferation, TNF-activated transducing mechanisms, and, in
parallel, the effects produced by intracellular mediators that may be
responsible for the transduction of TNF signaling in GnRH-producing
neurons.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell counting
GT1–7 cells were plated into 24-well multiwell plates in
FCS-containing DMEM for 24 h and then maintained in the presence
of the tested drugs for 3–12 h (short term studies) or 24–96 h (long
term studies). Cells were then harvested with a 0.01% trypsin solution
and counted with the aid of a hemocytometer.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay
The MTT cell proliferation assay is based on the conversion of a
diphenyltetrazolium salt into blue formazan detectable in an
enzyme-linked immunosorbent assay plate reader. After exposure to
various treatments, GT1–7 cells were incubated with MTT (0.9 mg/ml,
final concentration) for 2 h at 37 C and then solubilized with
isopropanolol containing 0.1 N HCl. Formazan production was
evaluated in a plate reader with a 560-nm test wavelength and a 690-nm
reference wavelength.
[3H]Thymidine incorporation
GT1–7 cells were plated into 24-well multiwell plates and
exposed to different agents for various lengths of time.
[3H]Methylthymidine (Amersham Pharmacia Biotech, Milan, Italy; SA, 20 Ci/mmol; 1 µCi/ml) was
added during the last 6 h of incubation. Cells were then extracted
with 1 N HClO4, and the incorporated
radioactivity was determined by scintillation counting.
Immunocytochemistry
GT1–7 cells were stained for TNFR1 and TNFR2 using rabbit
polyclonal antibodies specifically recognizing each subtype. Cells were
fixed with 4% paraformaldehyde and exposed to the primary antibody
(Sanbio, Uden, The Netherlands; 1 µg/ml) for 1 h at room
temperature before exposure to antirabbit IgG for 1 h. After
reaction with avidin-biotin-horseradish peroxidase (Elite ABC
Vectastain, Vector Laboratories, Inc.,
Burlingame, CA), staining was developed by exposure to 0.05%
diaminobenzidine-0.01% H2O2.
Flow cytometry
For specific detection of TNFR1/R2, growing GT1–7 cells were
fixed with 4% paraformaldehyde for 30 min, repeatedly washed, and
subsequently treated with anti-TNFR1/R2 (5 µg/ml·30 min) and
fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG (1:100 for
30 min). All incubations were carried out at 4 C. Controls included
omission of the primary antibody and substitution with nonimmune serum.
Samples were analyzed with an ELITE flow cytometer (Coulter
Electronics, Hialeah, FL) with an excitation wavelength of 488 nm and
monitoring of fluorescence at 525 nm. At least 10,000 forward and side
scatter gated events/sample were evaluated.
Ceramide-1-phosphate measurement
GT1–7 cells were cultured in 35-mm dishes and exposed to TNF
for the time indicated. Lipids were extracted and subjected to mild
alkaline hydrolysis, and ceramide levels were measured using a modified
diacylglycerol kinase assay (27) with a commercially available kit
(Amersham Pharmacia Biotech).
Evaluation of apoptotic death
Quantitative analysis of DNA fragmentation was performed with
the cell death detection enzyme-linked immunosorbent assay based on the
photometric sandwich immunoassay of cytoplasmic histone-associated DNA
fragments (Roche Molecular Biochemicals, Mannheim,
Germany).
For cytofluorometric analysis, after fixation with 70% ethanol overnight at -20 C, cells were incubated with ribonuclease (100 µg/ml) for 2 h at 37 C and stained with the nuclear dye propidium iodide (final concentration, 50 µg/ml). Analysis was carried out on a Coulter ELITE flow cytometer and was restricted to cells with diploid and hypodiploid DNA contents.
Laddered patterns of DNA fragmentation were resolved by conventional gel electrophoresis on 1.5% agarose gel impregnated with ethidium bromide and visualized by UV illumination.
Drugs
Unless otherwise specified, all chemicals used were obtained
from Sigma Chemical Co. (St. Louis, MO).
D-Erythro-sphingosine N-octanoyl (C8-ceramide;
Calbiochem, La Jolla, CA) was dissolved in
dimethylsulfoxide and stored at -80 C. Okadaic acid
(Calbiochem, La Jolla, CA) solubilized in water was stored
at -20 C. Neutral sphingomyelinase (N SMase) from Staphylococcus
aureus (Sigma Chemical Co.) was provided in a
solution containing 50% glycerol and 0.25 M phosphate
buffer, pH 7.5, and stored at 4 C. Acidic sphingomyelinase from human
placenta (Sigma Chemical Co.) was provided in 50%
glycerol, 25 mM potassium phosphate, 0.1% Triton X-100,
and 0.05 mM phenylmethylsulfonylfluoride, pH 4.5, and
stored at -20 C. Human TNF was obtained from PeproTech EC Ltd.
(London, UK).
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Results |
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receptors,
TNFR1 and TNFR2, in GT1–7 cells was analyzed by flow cytometry (Fig. 1
), immunocytochemistry (Fig. 1), and
Western blot analysis (data not shown) using antibodies specifically
recognizing each receptor subtype. In immunohistochemical
studies, staining was marked and diffuse within the cell body, but was
also present in neuritic extensions. Interestingly, the expression was
more pronounced for TNFR1 and was selective for a defined cell
population, whereas a small percentage of cells did not show positive
immunostaining.
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(20 ng/ml)
produced a significant reduction of cell number that was already
present after 24 h of incubation and persisted throughout all time
points examined (Fig. 2A). Indeed, a
marked reduction of cell number was already evident after short term
(6- to 12-h) exposure to TNF, as measured by the MTT proliferation
assay (Fig. 2B). The inhibitory effect of TNF was concentration
dependent, significant at 1 ng/ml, and maximal between 10–100 ng/ml
(Fig. 2, inset).
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is correlated to
induction of apoptotic cell death. Characteristic features of
apoptosis, such as DNA laddering, in fact appeared after only 6 h
of exposure to TNF and were particularly evident after 24–48 h
(Fig. 3A). Induction of apoptosis was
confirmed by cytofluorometric analysis; Fig. 3B reveals a pronounced
predyploid cell population in TNF-treated GT1–7 cells stained with
the nucleic acid probe, propidium iodide.
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produced a
significant stimulation of ceramide formation, as assessed by
measurement of the metabolite ceramide-1-phosphate (Fig. 4); this effect exhibited time
dependency, being present after 5 min, peaking at 15 min, and remaining
elevated up to 60 min.
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). However, for longer
time points (72 and 96 h), the behavior of the two compounds was
divergent, as C8-ceramide was still able to maintain a reduced cell
number, whereas GT1–7 cells were no longer sensitive to the inhibitory
action of N SMase, and their number was significantly increased (Fig. 5A). Accordingly, GT1–7 cells exposed to N SMase very rapidly escaped
the inhibitory action of the enzyme and exhibited an increased
proliferation rate, as shown by enhanced [3H]thymidine
incorporation, evident after 48–96 h of incubation (Fig. 5B). The
behavior of C8-ceramide was again different, as a reduction of
[3H]thymidine incorporation could be observed at all time
points examined (Fig. 5B). The lack of effect of long term treatment
with N SMase on GT1–7 cell proliferation might be ascribed to rapid
metabolism and/or inactivation of the enzyme; in support of this
possibility, reexposure of GT1–7 cells to N SMase after 48 h
(total exposure time, 96 h) produced a reduction of GT1–7 cell
number, comparable to that induced by a single treatment for 48 h
(Table 1). The inhibitory action of N
SMase was very rapid; the MTT proliferation assay revealed a very early
reduction of the number of cells that was already significant after
3 h of incubation and was maximal at 12 h (Fig. 6). This prompt effect is suggestive of
induction of cell death; accordingly, short term (6–12 h) treatment
with N SMase (200 mU/ml) determined the appearance of clear signs of
apoptotic death. These were evaluated by measurement of oligonucleosome
formation (Fig. 7A) and analysis of DNA
laddering (Fig. 7B). The time-course studies indicated that induction
of apoptosis by N SMase was very rapid (6–12 h) and was not present
after 48 h (Fig. 7). Conversely, as shown for cell counting,
C8-ceramide (25 µM) treatment produced a marked and
sustained (up to 48 h) apoptotic effect (Fig. 7B).
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is known to activate an acidic sphingomyelinase (Ac SMase)
located in specific intracellular compartments (28). Thus, we analyzed
the effect of this enzyme on the proliferation and viability of GT1–7
cells. Treatment with 200 mU/ml Ac SMase induced a time-dependent
inhibition of cell number. A significant inhibition, as assessed by the
MTT proliferation assay, was present after only 6 h of treatment
and progressively increased, reaching a maximum after 48 h (Fig. 8A). This effect was accompanied by the
appearance of DNA fragmentation, indicative of apoptotic death. Figure 8B reports a representative DNA laddering observed in GT1–7 cells
exposed to Ac SMase for 48 h.
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on
GT1–7 cell viability, we used the xanthate D609, an inhibitor of
phosphatidylcholine-specific phospholipase C (29), a key enzyme in the
activation of Ac SMase (but not N SMase) by TNF (30). Pretreatment
of GT1–7 cells with D609 (5 µg/ml) reduced by about 50% the
accumulation of ceramide-1-phosphate induced by a 15-min exposure to 20
ng/ml TNF (Table 2). Similarly,
preincubation of GT1–7 cells with D609 for 1 h before the
addition of TNF for 48 h significantly reduced the inhibitory
effect of the cytokine on cell number and partially prevented
TNF-induced apoptosis (Table 2). The latter evaluation was carried
out by flow cytometry, which allowed a quantitative analysis of the
protective effect.
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, GT1–7 cells were preincubated
with okadaic acid. This compound is a potent inhibitor of various
protein phosphatases (33); however, at low concentrations, it appears
to act as a relatively specific inhibitor of ceramide-activated protein
phosphatase (31). Okadaic acid, in a range of concentrations shown to
inhibit both protein phosphatases 1 and 2 (>5 nM) caused a
concentration-dependent reduction of cell number (
32% reduction at
10 nM and >70% reduction at 100 nM). In
contrast, smaller concentrations (0.5–1 nM) did not affect
cell number or viability, but completely prevented the inhibitory
effect of TNF on cell number and the induction of apoptosis (Table 3), as assessed by cell counting and
cytofluorometric analysis, respectively. In addition, to exclude
further the involvement of protein phosphatase 1 in okadaic acid
action, GT1–7 cells were pretreated with 1 nM tautomycin
(a compound that, at this concentration, is known to specifically
affect protein phosphatase 1) (34). Under these conditions, the
inhibitory effect of TNF on GT1–7 cell proliferation was not
affected (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is recognized as one of the key molecules mediating
neuroimmune interactions; at the hypothalamus this results in
modulation of hormonal secretion (1, 2, 3, 5, 6), but a role for this
cytokine in hypothalamic neuronal viability can also be hypothesized.
The action of TNF at the central nervous system has been mainly
related to its ability to modulate neuronal survival, but very little
is known about the intracellular events that mediate cytokine action at
this level. In this respect, the immortalized hypothalamic GT1–7 cells
represent a useful neuronal in vitro model, as they express
receptors for TNF, TNFR1, and TNFR2. TNF recognizes, in fact, two
distinct receptors (21), one of which, the TNFR1 or p55, belongs to the
nerve growth factor/TNF receptor family characterized by the presence
of a specific intracellular sequence responsible for the transduction
of a death signal to the cell (35). Treatment of GT1–7 neurons with
TNF caused rapid and prolonged reduction of cell number, an effect
accompanied by induction of apoptotic death. This confirms previous
reports showing that TNF induces apoptosis in neurons (13, 14, 15) and
is involved in neurodegeneration processes (11, 16, 17, 36), but is in
contrast with a large body of evidence that indicates a neuroprotective
activity for this cytokine (18, 19, 20).
Activation of TNFR1 is linked to stimulation of a neutral and an acidic
sphingomyelinase that differ on the basis of their location in distinct
cellular compartments, but that are both responsible for generation of
the intracellular messenger, ceramide (22, 23, 28, 37). Exposure of
GT1–7 cells to TNF caused the accumulation of ceramide, as assessed
by the formation of ceramide-1-phosphate. Ceramide is known to induce
apoptosis in a series of different cellular systems, but its role at
the central nervous system is still controversial. Ceramide has, in
fact, been shown to induce apoptosis in cultured mesencephalic neurons
(38), but it also exerts neuroprotective activity in cultured neurons
deprived of trophic support (39) or exposed to excitotoxic or oxidative
insult (40). In addition, ceramide induces neuronal differentiation
(41) and regulates the balance between neuritic formation and apoptosis
in hippocampal cultures (42). Treatment of GT1–7 cells with
cell-permeable ceramide analogs induced a marked reduction of cell
number and the appearance of distinct features of apoptotic death at
all time points examined (either short or long term treatments). To
evaluate the relative contribution of the ceramide-generating pathways
in the induction of apoptotic death in GT1–7 cells, cultures were
exposed to N or Ac SMase. Treatment with both enzymes produced a
reduction of cell number and induction of apoptotic cell death.
However, the effects observed were temporally divergent, as the action
of N SMase was rapid in its onset but restricted to a short period of
time, whereas Ac SMase induced a reduction of cell number and apoptotic
death that were sustained with time (up to 48 h). This different
time-related behavior may be ascribed to specific, prompt metabolism of
the neutral enzyme whose action is rapidly achieved and completed or,
alternatively, it may be due to the activation of intracellular
pathways able to counterbalance the effect of N SMase on neuronal
viability. One such example is represented by activation of protein
kinase C, whose action on cell survival in our system (data not shown)
as well as in other cellular systems (43) is that of counteracting the
effect of N SMase. However, in our conditions, the involvement of
protein kinase C is partially ruled out by the fact that GT1–7 cells
are still responsive to the action of N SMase once the initial effect
has ended, as demonstrated by the reduction of viable cells observed
after repeated (twice, every 48 h) treatment with the drug.
Interestingly, GT1–7 cells exhibited a very prompt capacity to recover
after completion of N SMase action, and they responded with an
increased proliferation rate starting at 48 h, when cell number
was still decreased in N SMase-treated cultures, and a significant
enhancement at 72 h. Hence, the time-limited action of N SMase on
GT1–7 cell growth and viability revealed the rapid reversibility of
the effect observed. Indeed, the current knowledge of TNFR1 signaling
suggests that activation of Ac SMase is responsible for the
transduction of the death signal, whereas the neutral enzyme would,
instead, mediate proliferation and survival events (44). Based on these
results, an alternative interpretation of the effect observed could be
made. The nature of the early N SMase response could be due to the
enormous amount of ceramide generated within the cell by exogenous
addition of the enzyme (data not shown); under these conditions, the
true response of increased cell growth would be completely masked and
appear late.
In our hands, the activation of Ac SMase seems to be only partially
involved in the action of TNF on GT1–7 cell number and viability as
demonstrated by the partial reduction of TNF-induced effects by
treatment with D609, an inhibitor of phosphatidylcholine-specific
phospholipase C (29, 30) whose stimulation activates the pathway
specifically involving Ac SMase. The contribution of N SMase on TNF
action cannot be extrapolated from the present findings, as no
pharmacological tools are currently available to specifically modulate
this pathway. However, an increased GT1–7 cell proliferation after
TNF treatment has never been observed. It is important to underline
that activation of the sphingolipid metabolism leads to the production
of sphingosine-1-phosphate, a second messenger involved in the
regulation of cellular proliferation and survival (45), and
sphingosine, whose role as an intracellular regulator of cellular
differentiation and apoptosis is now emerging (46, 47). It is, then,
possible that the involvement of the sphingomyelin pathway in
TNF-induced apoptosis may result from a balance between the
generation of different messengers (ceramide, sphingosine, and
sphingosine-1-phosphate), all able to regulate cellular viability. In
the present study we have focused our attention primarily on the role
of ceramide, as ceramide analogs were able to mimic the TNF effects.
In addition, the involvement of ceramide in the action of TNF in
GT1–7 cells is further supported by the complete prevention of the
TNF effect by treatment with low concentrations of okadaic acid.
However, an involvement of sphingosine in TNF-induced apoptosis in
GT1–7 cells cannot be ruled out and requires further investigation. In
the context of a complete picture of TNF action in GT1–7 cells,
although this would open a new, vast issue, well beyond the confines of
the present study, a role for the caspase cascade could be envisaged.
Caspases may, in fact, take part in the action of TNF merely as
executioners of apoptosis (48), but they may also be more deeply
involved in TNF signaling events by regulating intracellular
ceramide production (49, 50).
In conclusion, in immortalized hypothalamic neurons, TNF exerts a
cytotoxic effect, presumably through an increased production of
ceramide. This activity on hypothalamic neuronal viability together
with the modulation of neurohormone secretion may critically contribute
to the impairment of gonadal axis function occurring during infectious
states.
Received March 8, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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