(Received for publication, March 8, 1995; and in revised form, August 9, 1995)
From the
In order to better understand the significance of tumor necrosis
factor- (TNF-
) and interleukin-1
(IL-1
)-receptor
internalization in the sphingomyelin pathway signal transduction, we
investigated receptor signaling under conditions in which receptor
internalization is blocked. We demonstrate that human recombinant
TNF-
and IL-1
both induced sphingomyelin and
phosphatidylcholine hydrolysis at either 4, 14, or 37 °C in human
skin fibroblasts and U937 monocytic cells. Cytokine-induced
sphingomyelin degradation also occurred when endocytosis was inhibited
by incubating the cells in hypertonic medium. While internalization was
not required for the production of ceramide, activation of the
transcription factor NF-
B was strongly reduced when cells were
stimulated with TNF at low temperature or in hypertonic medium. Under
these conditions, activation of NF-
B by the cell-permeant
C
-ceramide (N-acetylsphingosine), by exogenous
sphingomyelinase or by phorbol myristate acetate was also inhibited.
These results suggest that low temperature and hypertonicity, two
inhibitors of receptor internalization: (i) do not affect the
TNF-
- or IL-1
-induced sphingomyelin hydrolysis, but (ii) do
inhibit a step distal to ceramide of the intracellular signaling
pathway leading to NF-
B activation.
Accumulating evidence suggests an important role for
sphingomyelin (ceramide phosphocholine, SPM) ()hydrolysis
and ceramide generation in cytokine
signaling(1, 2, 3, 4) . Indeed, the
action of tumor necrosis factor (TNF-
) and interleukin-1
(IL-1
), i.e. two unrelated cytokines which share many
biological activities(5) , seems to be mediated by this
recently identified SPM
pathway(6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
This pathway also appears to transduce the cellular responses to other
extracellular agents, such as dihydroxyvitamin D3(16) ,
-interferon(6) , nerve growth factor(17) ,
ionizing radiation(18) , and CD95 (Fas antigen)
engagement(19) . Thus, through the generation of ceramide, the
SPM hydrolysis has emerged as an initial key signal in the regulation
of diverse cell functions, including
differentiation(6, 16) , inhibition(20) , or
stimulation (21) of cell growth and
apoptosis(11, 18, 19, 22) . Current
arguments for the importance of the SPM pathway in TNF-
or
IL-1
cell signaling are based on the observations that, like the
cytokine, ceramide can stimulate a membrane-bound serine/threonine
kinase(7) , a cytosolic protein phosphatase(23) , a
mitogen-activated protein kinase (24) and promote
down-regulation of c-myc proto-oncogene RNA levels (6) , enhancement of cyclooxygenase gene
expression(14) , and activation of transcription factors such
as nuclear factor
B (NF-
B) (9, 13, 25, 26) and
AP-1(27) .
The specific binding of TNF- and IL-1
to cell surface receptors appears to be the first step for mediating
most of the biological responses to these
cytokines(5, 28, 29) . Binding of TNF-
and IL-1
is then followed by internalization of the
cytokine-receptor complex by receptor-mediated
endocytosis(30, 31) . However, whether internalization
of these two ligands is a necessary step for induction of cellular
responses in general is still under debate(28, 29) .
For instance, internalization of TNF-
seems to be required for
mediating its cytotoxic effects(32) , but not for inducing
phosphorylation of a 26-kDa cytosolic protein involved in TNF
signaling(33) . Internalization of IL-1
is also believed
to be important for its physiological responses, but it has been
reported that IL-1 can stimulate rapid diacylglycerol production
without being internalized (34) . Elsewhere, the role of
internalization in the triggering of SPM pathway has never been
investigated.
The present study was undertaken to determine the
importance of cytokine and cytokine receptor internalization in the
stimulation of the SPM pathway. We show that, under a variety of
conditions that rule out receptor-mediated endocytosis, both TNF-
and IL-1
can trigger SPM hydrolysis but not the
ceramide-stimulated activation of NF-
B.
Figure 1: Low temperature and hypertonicity inhibit the uptake of fluorescent LDL by human skin fibroblasts. After a 24-h preincubation in RPMI 1640 medium devoid of lipoproteins (containing 2% Ultroser), normal skin fibroblasts were incubated for 30 min at 4 °C, at 14 °C (B), or at 37 °C in the absence (A) or presence of 0.4 M sucrose (C) or 0.31 M NaCl (D). Then, DiI-labeled LDL (40 µg/ml final concentration) was added to the cells and incubation continued for 60 min under the same conditions. After washing, cells were viewed as described under ``Experimental Procedures.'' Cells incubated at 4 °C showed no fluorescence (data not shown).
Figure 2:
TNF- and IL-1
induce
phosphatidylcholine and SPM turnover in normal skin fibroblasts at 14
and 4 °C. Normal skin fibroblasts were labeled with
[
H]choline as described under ``Experimental
Procedures.'' After 48-h labeling, the medium was removed and
cells chased in medium containing 1% fetal calf serum. Prior to
addition of 2 nM TNF-
or 6 pM IL-1
, cells
were preincubated for 30 min at the indicated temperature. A control
experiment was simultaneously carried out in the absence of cytokine.
At the indicated time points, incubations were stopped and
phosphatidylcholine (A and C) and SPM (B and D) levels (dpm/mg of cell protein) determined as described
under ``Experimental Procedures.'' Total cell-associated
radioactivity averaged 2,157,500 ± 174,600 dpm/mg protein. The
phospholipid levels are expressed as percentage of the value observed
at time 0. In the presence of TNF, the values correspond to the mean
± S.E. of four independent experiments (one representative
experiment is shown for IL-1).
Since endocytosis is abolished at 4 °C ((43) ; see also
our own data with fluorescently labeled LDL), the effect of cytokines
on the activation of SPM hydrolysis was also assessed at 4 °C.
Quite unexpectedly, at this temperature, both TNF- and IL-1
were able to promote SPM (and phosphatidylcholine) degradation on skin
fibroblasts (Fig. 2, C and D). Of note, the
time course and extent of response were very similar to those obtained
at 37 °C.
Further evidence that the SPM pathway could be
activated at low temperature was provided by measurement of ceramide
production. Skin fibroblasts were metabolically labeled to equilibrium
with [H]palmitic acid and then stimulated with
TNF-
. As shown in Fig. 3, addition of TNF at both 37 and 4
°C resulted in ceramide generation. Peak effect was observed at 30
min, i.e. concomitant to maximal hydrolysis of SPM (see Fig. 2). Comparable results were obtained after stimulation at
14 °C (data not shown).
Figure 3:
TNF- induces ceramide production in
skin fibroblasts at 4 °C. Human normal skin fibroblasts were
labeled with [
H]palmitic acid as described under
``Experimental Procedures.'' After 48-h labeling, the medium
was removed and cells chased in medium containing 1% fetal calf serum.
Prior to addition of 2 nM TNF-
, cells were preincubated
for 30 min at the indicated temperature. At the indicated time points,
incubations were stopped, total lipids were extracted, separated by
thin-layer chromatography, and ceramide content (dpm/mg of cell
protein) determined as described under ``Experimental
Procedures.'' Values are expressed as percentage of radioactivity
at time 0 (basal levels of ceramide averaged 89,690 ± 9870
dpm/mg of protein). The data are from one representative experiment out
of two or three independent experiments.
While the above studies were performed
on human skin fibroblasts, subsequent experiments investigated the
cytokine action on a different cell type, the human U937 monocytic cell
line, in which previous works have studied the SPM
pathway(9, 11, 13, 19) . In U937
cells, TNF- activates two successive cycles of
choline-phospholipid hydrolysis. As already
reported(9, 10, 13, 46) ,
stimulation of phosphatidylcholine degradation in U937 cells occurred
at about 2 min and then at about 20 min after addition of TNF (Fig. 4A), maximal degradation being usually observed
in the second cycle. TNF-
also stimulated two cycles of SPM
hydrolysis which closely paralleled those of phosphatidylcholine (Fig. 4A). (
)
Figure 4:
TNF- induces phosphatidylcholine and
SPM turnover in U937 cells at 14 and 4 °C. Human monocytic U937
cells labeled with [
H]choline for 48 h were
preincubated for 30 min at 37 °C (A), 14 °C (B), or 4 °C (C). After incubation with 3 nM TNF-
at the indicated temperature and for the indicated
periods, phosphatidylcholine (PC, open symbols) and
SPM (solid symbols) levels (dpm/mg of cell protein) were
determined as described under ``Experimental Procedures.''
Total cell-associated radioactivity averaged 2,193,000 ± 396,000
dpm/mg of protein. The phospholipid levels are expressed as percentage
of the value observed at time 0 and correspond to the mean ±
S.E. of at least five separate experiments.
The results of Fig. 4, B and C, clearly demonstrate that
activation of SPM (and phosphatidylcholine) degradation in response to
TNF- at 14 or 4 °C was as effective as at 37 °C. Such
effects were further confirmed by determining the mass of cellular
phospholipids and SPM. Lipid phosphorus assays indicated that at 4
°C both total phospholipids and SPM contents similarly decreased by
about 15 and 30% at the early and late cycles, respectively (data not
shown).
Figure 5:
TNF- and IL-1
induce
phosphatidylcholine and SPM turnover in skin fibroblasts incubated in
hypertonic media. Normal skin fibroblasts were labeled with
[
H]choline as described under ``Experimental
Procedures.'' After 48-h labeling, the medium was removed and
cells chased in medium containing 1% fetal calf serum. Prior to
addition of 2 nM TNF-
or 6 pM IL-1
, cells
were preincubated for 30 min in the presence of 0.31 M NaCl (A and B) or 0.4 M sucrose (C and D). A control experiment was simultaneously carried out in the
absence of cytokine. At the indicated time points, incubations were
stopped and phosphatidylcholine (A and C) and SPM (B and D) levels determined as described under
``Experimental Procedures.'' The phospholipid levels (dpm/mg
of cell protein) are expressed as percentage of the value observed at
time 0 and correspond to the mean ± S.E. of three independent
experiments (one representative experiment is shown for
IL-1).
Figure 6:
Effect of low temperature and hypertonic
media on the activation of NF-B. In A, normal skin
fibroblasts were left untreated (lane 1) or treated for 30 min
with 2 nM TNF-
at 37 °C (lanes 2-7), 4
°C (lane 8), 14 °C (lane 10) or in the
presence of 0.4 M sucrose (lane 9). Nuclear extracts
were prepared as described under ``Experimental Procedures,''
and equal amounts were incubated with
[
-
P]ATP-labeled NF-
B oligonucleotide
probe. Specificity was determined by competitive experiments in the
presence of 25- or 50-fold excess unlabeled NF-
B (lanes 3 and 4) or AP1 (lanes 5 and 6) consensus
oligonucleotides. Similar results were obtained on U937 cells. In B, fibroblasts were left untreated (lane 1) or
treated for the indicated times with 2 nM TNF-
at 37
°C in the absence (lanes 2, 4, 6, 8, and 10) or
presence of 0.31 M NaCl (lanes 3, 5, 7, 9, and 11). In C, U937 cells were left untreated (lane
1) or incubated for 30 min with 3 nM TNF-
at 37
°C (lane 2) or treated for 60 min with 10 µM
C
-ceramide at 37 °C (lane 3) or with 30
µM C
-ceramide at 37 °C (lane 4),
at 4 °C (lane 5), or in the presence of 0.4 M sucrose (lane 6). In D, fibroblasts were left
untreated (lane 2) or incubated for 30 min with 2 nM TNF-
at 37 °C (lane 1) or treated with 100
milliunits/ml sphingomyelinase from B. cereus (SPMase) for 30 min at 37 °C (lane 3), at 4
°C (lane 4), or in the presence of 0.4 M sucrose (lane 5) or 0.31 M NaCl (lane 6). In E, U937 cells were left untreated (lane 1) or
incubated for 30 min with 2 nM TNF-
at 37 °C (lane 2) or treated with 0.2 µM phorbol myristate
acetate (PMA) for 3 h at 37 °C (lane 3), at 4
°C (lane 4), or in the presence of 0.31 M NaCl (lane 5) or 0.4 M sucrose (lane 6). The gels
are representative of two to six separate
experiments.
To further
elucidate the absence of NF-B activation under conditions which
block endocytosis, cells were treated directly with
C
-ceramide, a cell-permeant ceramide analog(6, 11, 14, 18, 20, 21) or
with bacterial sphingomyelinase to generate ceramide in cell
membranes(6, 15, 25) . C
-ceramide
activated NF-
B nuclear translocation in skin fibroblasts or U937
cells (Fig. 6C, lanes 3 and 4). Incubation of
the cells with C
-ceramide at 4 °C or in hypertonic
media resulted in significant inhibition of NF-
B activation (Fig. 6C, lanes 5 and 6); similar data were
obtained when treating the cells by exogenous sphingomyelinase (Fig. 6D). Interestingly, NF-
B nuclear
translocation by phorbol myristate acetate, a well known activator of
NF-
B (see, for instance, (41) ), was also inhibited by low
temperature and hypertonicity (Fig. 6E, lanes
4-6). Thus, it can be concluded from these studies that
these inhibitors also affect a step distal to both TNF and ceramide,
which is required for NF-
B activation.
A large number of biochemical events induced by TNF- or
IL-1
have been reported to be mimicked by ceramide, supporting the
assertion that the so-called SPM pathway could mediate the action of
these two cytokines(1, 2, 3, 4) .
The binding of TNF-
and IL-1
to their respective receptors is
known to be rapidly followed by endocytosis (30, 31) .
However, the role of receptor internalization in TNF-
or IL-1
signal transduction in general, and in stimulation of the SPM pathway
in particular, remains to be clearly established. We report here that
the SPM pathway can be triggered under experimental conditions that
rule out receptor internalization.
Previous reports have clearly
demonstrated that receptor-mediated endocytosis is
temperature-dependent (43) and inhibited by
hypertonicity(44, 45) . Use of these treatments was
selected in our study because they are effective (see our data) and
noninvasive methods to inhibit endocytosis, whereas experiments using
metabolic poisons may be limited in their interpretation due to
nonspecific effects unrelated to endocytosis. In addition, lowering the
temperature below 17 °C or incubation in hypertonic medium have
been reported to severely reduce the cellular internalization of
TNF- (30, 47, 48) and
IL-1
(31, 49) . In contrast, we demonstrate that
under these conditions TNF-
and IL-1
activate SPM hydrolysis
as well in two different cell types. At low temperature or in
hypertonic medium, the kinetics and extent of SPM (and
phosphatidylcholine) degradation, and ceramide generation, were
remarkably similar to those observed under control conditions.
Thus,
the present data strongly suggest that cytokine-receptor
internalization is not required for activation of the SPM pathway. This
contrasts with the general belief that internalization of TNF is
important for induction of
cytolysis(28, 32, 50) . TNF-induced gene
expression in cultured human endothelial cells has also been reported
to be dependent on endocytosis(48) . However, there are reports
describing biologic effects of cytokines under conditions where
receptor internalization is excluded. For instance, microinjected TNF
seems to exhibit cytotoxic activity(51) . At low temperature,
TNF can also stimulate the phosphorylation of a p26 cytosolic
protein(33) . The role of internalization in IL-1
signaling also remains unclear(49) . A subclone of EL4 cells
has been described that binds but does not respond to IL-1 possibly due
to its inability to internalize the cytokine(52) . However,
Rosoff et al.(34) have reported that IL-1
induces a very rapid increase in diacylglycerol in Jurkat cells in the
absence of detectable specific binding of the cytokine. In addition,
internalization of IL-1
does not correlate with its ability to
transduce the IL-1 signal for the induction of IL-2 gene expression, an
important physiological effect of the hormone, since mutant receptors
lacking most of the cytoplasmic domain still can bind and internalize
IL-1, but they do not allow activation of the IL-2
promoter(53) . Other instances where cytokine receptor
internalization is not required for signal transduction include protein
tyrosine phosphorylation induced by macrophage-colony-stimulating
factor (54) or granulocyte-macrophage-colony-stimulating
factor(55) .
Our findings on activation of the SPM pathway
in the absence of endocytosis have important biological implications.
First, they suggest that SPM (and phosphatidylcholine) hydrolysis is a
proximal event in TNF- or IL-1
signal transduction, which
likely occurs in close vicinity of the cytokine receptor, i.e. at the plasma membrane. Such a subcellular topology for
cytokine-induced ceramide generation is perfectly consistent with
previous suggestions for a tight coupling of the SPM pathway to
TNF-
and IL-1
receptors (8, 15) and with
recent observations that the signaling pool of SPM localizes to the
plasma membrane(56) . (
)The second implication of
our results is that the sphingomyelinase which is activated upon
cytokine binding should be located at or near the plasma membrane. Two
enzymes could potentially account for SPM hydrolysis at this
subcellular site: the plasma membrane bound, neutral,
magnesium-dependent sphingomyelinase(57) , which has been
reported to be stimulated by TNF and vitamin
D3(2, 3, 13, 16, 58) , and
the (possibly translocated) cytosolic, neutral, magnesium in dependent
sphingomyelinase(4, 59) . Whereas the question of
which of these (or another, yet uncharacterized) neutral
sphingomyelinases is actually activated still remains to be elucidated,
our data make very unlikely the hypothesis (9, 13) for
the involvement of an endolysosomal sphingomyelinase in cytokine
signaling. This conclusion that acid sphingomyelinase is probably not
implicated in TNF-
or IL-1
signal transduction is
corroborated by two independent observations which examined different
actions of the cytokines(26, 39) . Finally, the
evidence that at 4 °C, or in hypertonic medium, the SPM decrease
induced by addition of TNF-
or IL-1
is followed by a recovery
to the starting levels indicates that such a recovery is probably due
to the reversibility of the SPM cycle rather than to the loss of
cytokine activity (desensitization) by down-regulation of the cytokine
receptors. Indeed, down-modulation of the TNF receptors is a result of
the internalization of p60 receptor (60) . Since the p60
receptor triggers the SPM
pathway(10, 13, 61) , it is therefore likely
that, under conditions where internalization is blocked, the
reversibility of SPM degradation is not due to down-regulation of the
TNF receptor.
Another interesting feature of the present study is
the surprising observation that SPM (and phosphatidylcholine)
hydrolysis occurred at 4 °C (or 14 °C) as well as at 37 °C.
Although many types of enzyme activity are reduced at 4 °C, there
are some well documented examples of enzymes fully active at low
temperature, e.g. tyrosine kinases (see (55) and
references therein). However, to our knowledge, there has been no
report of mammalian phospholipases in intact cells retaining their
activity at 4 °C. Of note, some enzymes of lipid metabolism are
active at low temperature, either in vitro or in
vivo, such as snake venom (62) or Escherichia coli()phospholipases A2, and rat hormone-sensitive
lipase(63) . The fact that the phospholipid-degrading enzymes
stimulated by cytokines are insensitive to low temperatures suggests
that the SPM pathway might represent a crucial signal transduction
pathway, possibly present in poikilotherms or at least able to support
a cold adaptability.
Cytokine-dependent activation of transcription
factors such as NF-B is a central mechanism involved in
transduction of the multiple biological actions of TNF-
and
IL-1
(28, 29, 41) . SPM hydrolysis and
ceramide generation have recently been proposed as mediators of
NF-
B signaling(9, 25) , although ceramide might
not be required nor sufficient for NF-
B
activation(4, 20, 27, 64, 65) .
Under the conditions we used to inhibit receptor internalization,
although cytokines were fully capable of inducing the degradation of
SPM and the production of ceramide, NF-
B translocation to the
nucleus was inhibited. (
)This result could be in agreement
with the observation that TNF-induced expression of various genes was
decreased by inhibiting cytokine endocytosis(48) .
Several
hypotheses may account for the strong inhibition of NF-B
activation while the SPM pathway is activated. The first possibility is
that the steps between ceramide and NF-
B activation are inhibited
by cold and hypertonicity. Indeed, NF-
B activation triggered by
exogeneous sphingomyelinase or by C
-ceramide is also quite
limited under these conditions. One could argue that low temperatures
might inhibit the internalization of ceramide; however, hypertonic
medium does not affect fluid-phase endocytosis(44) . The
observation that phorbol ester stimulation of NF-
B was also
impaired underlines the sensitivity of NF-
B activation to both by
low temperatures and hypertonicity. The second possibility that only
the nuclear translocation step is affected cannot yet be ruled out.
Finally, a third hypothesis is that the signaling component between TNF
and NF-
B nuclear translocation is independent of the signaling
connection between TNF and SPM hydrolysis, which is in agreement with
recent suggestions(20, 64, 65) . However, in
our experimental cell systems both the C
-ceramide analog
and the ceramide produced in cell membranes by bacterial
sphingomyelinase were able to induce some activation of NF-
B.
Whether ceramide is the only mediator of TNF in NF-
B activation
still remains to be established.
In summary, several lines of
evidence support the conclusion that cytokine receptor internalization
is not required for activation of the SPM signal transduction pathway.
Our study also indicates that low temperatures and hypertonicity
inhibit not only receptor-mediated endocytosis but also some distal
step(s) required for the activation of the NF-B transcription
factor.