(Received for publication, August 9, 1995)
From the
Current studies indicate that ceramide is involved in the
regulation of important cell functions, namely cell growth,
differentiation, and apoptosis. In the present study, the possible role
of ceramide in the differentiation of neuroblastoma Neuro2a cells was
investigated. The following results were obtained. (a)
Ceramide content of Neuro2a cells, induced to differentiate by retinoic
acid (RA) treatment rapidly increased after addition of RA, was
maintained at high levels in RA-differentiated cells and returned to
the starting levels with removal of RA and reversal of differentiation;
under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with
[H]sphingomyelin or
[
H]sphingosine or L-[
H]serine, the metabolic formation of
ceramide was markedly higher and more rapid in RA-differentiated than
undifferentiated cells. (c) Inhibitors of ceramide
biosynthesis (Fumonisin B1,
-chloroalanine and L-cycloserine) diminished the extent of the differentiating
effect of RA and concomitantly Cer content decreased. (d) The
activity of neutral sphingomyelinase increased after addition of RA,
maintained high levels in RA-differentiated cells, and returned to the
initial levels with removal of RA. (e) Experimental conditions
that cause an elevation of ceramide content (treatment with sphingosine
or ceramide or C
-ceramide or bacterial sphingomyelinase)
inhibited cell proliferation and stimulated neurite outgrowth;
dihydro-analogues of sphingosine, ceramide, and C
-ceramide
had no effect on differentiation. (f) treatment with Fumonisin
B1 completely inhibited sphingosine-induced differentiation. These data
suggest a specific bioregulatory function of ceramide in the control of
Neuro2a cell growth and differentiation and pose the general hypothesis
of a mediator role of ceramide in the differentiation of cells of
neural origin.
Increasing evidence indicates important roles for molecules of
sphingoid nature in the modulation of cell response to different
extracellular signals. These molecules include sphingosine, ceramide,
and some derivatives of them, N-methylated forms of
sphingosine, sphingosine-1-phosphate, and
ceramide-1-phosphate(2, 3) . Ceramide (N-acyl-erythro-sphingosine) has been shown to possess
bioeffector properties and to act as a key molecule in a new signal
transduction pathway, the sphingomyelin pathway or
cycle(4, 5, 6, 7) . In fact, in
several cell lines, especially of the immune system, the activation of
certain growth factor receptors by vitamin D3 and cytokines (tumor
necrosis factor , interleukin-1
, and
-interferon)
induces sphingomyelin hydrolysis by activation of sphingomyelinase,
resulting in the elevation of the intracellular levels of ceramide.
This, in turn, acts as mediator of the elicited physiological effects,
presumably by controlling the activity of specific protein kinases and
protein phosphatases. In particular, ceramide has emerged as a
candidate for regulatory roles in biological processes that are
intimately connected to each other, including cell proliferation,
oncogenesis, differentiation, and apoptosis (reviewed in (4, 5, 6, 7, 8, 9, 10) ).
A process that is based on the regulation of
proliferation/differentiation and differentiation/apoptosis is neural
development. Several cell systems (neurons, glial cells, neurotumoral
cells) that undergo morphological and functional differentiation in
culture are available to study this process in vitro. Some
studies suggest that sphingolipids and sphingoid molecules may be
involved in the regulation of neural development. In fact, exogenously
added glycosphingolipids are capable to affect differentiation of
neurons in primary culture and to induce differentiation of
neuroblastoma cells in vitro (for a review, see (11) ). Moreover, in cultured hippocampal neurons, sphingolipid
biosynthesis is necessary for axonal outgrowth(12) , and
inhibition of sphingolipid biosynthesis and degradation causes opposite
effects on axonal branching(13) . Furthermore, induced
expression of G and/or b-series gangliosides is followed
by differentiation of Neuro2a cells(14) . Finally, in T9 glioma
cells, addition of a cell-permeable ceramide analog
(C
-ceramide) causes growth inhibition and formation of
processes, in analogy with nerve growth factor, which produced the same
effects with a concomitant increase of the cellular level of
ceramide(15) .
On these premises, we decided to carry out a systematic investigation on the involvement of ceramide as a bioregulator in neural differentiation and the associated processes of proliferation and apoptosis. In the present study, we investigated the role played by ceramide in the differentiation of neuroblastoma Neuro2a cells. Initially, we determined the ceramide concentration and the metabolic routes leading to ceramide in Neuro2a cells induced to differentiate by treatment with retinoic acid, under strictly standardized conditions. Then, the ceramide levels of Neuro2a cells were increased by different treatments, and the effects on differentiation were observed. The data obtained strongly suggest that ceramide is involved in the regulation of Neuro2a cell differentiation.
Figure 1:
Content of
endogenous Cer and Sph in Neuro2a cells during RA-induced
differentiation. For details on the culture conditions, see
``Experimental Procedures.'' In some experiments (-RA, dotted line) RA was removed after 24 h, and incubation
continued for a further 24 h. Data are the mean values ± S.D. of
three experiments in duplicate. , control; *, +RA;
,
-RA.
Figure 7: Effect of Fumonisin B1 on Neuro2a differentiation. Cells were incubated for 8 h with 1 µM Sph (Sp) or 20 µM RA in the absence or presence of 25 µM Fumonisin B1 (FB). C, control, untreated cells. Data are expressed as % of cells bearing long neurites ± S.D. For further details see ``Experimental Procedures.''
Figure 2:
Incorporation of radioactivity into the
total lipid extract, Sph, and water after feeding undifferentiated
(control) and differentiated (RA-treated) Neuro2a cells with 40 nM [H]Sph for different times. Data are the
mean values ± S.D. of three experiments in duplicate. White
bars, control; stippled bars,
RA-treated.
Figure 3:
Incorporation of radioactivity into
different metabolites after feeding undifferentiated (control) and
differentiated (RA-treated) Neuro2a cells for different times with 40
nM [H]Sph. Data are the mean values
± S.D. of three experiments in duplicate. Asterisk, p < 0.01, RA-treated (stippled bar) versus control (white bar) at the same pulse
time.
In pulse-chase
experiments with L-[H]serine (Table 2), control and RA-differentiated Neuro2a cells
incorporated similar amounts of radioactivity into total sphingolipids
(mainly Cer, SM, gangliosides, and neutral glycosphingolipids). At all
investigated times [
H]Cer represented the major
H-sphingolipid (from 52 to 87% of total sphingolipids) and
was produced in significantly higher amounts by differentiated than
undifferentiated cells. On the basis of these results, the effect of
inhibitors of serine-palmitoyl transferase (a key enzyme in
sphingolipid biosynthesis) on RA-induced differentiation was
investigated. As shown in Table 3, treatment of Neuro2a cells
with 2.5 mM
-chloroalanine or L-cycloserine
resulted in a time-dependent, substantial, although not complete,
inhibition of RA-induced differentiation.
The feeding experiments
with [Sph-H]SM provided similar results (Fig. 4). After 2 h of feeding with 4 µM
[Sph-
H]SM, followed or not by 4 h chase,
[
H]Cer represented the major radiolabeled
metabolite in both undifferentiated and RA-differentiated cells, but
its metabolic formation was significantly higher in the differentiated
ones (2.4-fold and 1.8-fold at 0 and 4 h chase, respectively) (Fig. 4). Conversely, other
H-metabolites produced
during [Sph-
H]SM metabolism (mainly
Glc-Cer, gangliosides, Sph, and water) were markedly lower in
differentiated than control cells (Fig. 4). Also in these
experiments
H
O (which could be properly
measured after 4 h chase) constituted a very minor metabolite (about
6.5 and 2.5% of total incorporated radioactivity in undifferentiated
and RA-differentiated cells, respectively). When
[Sph-
H]SM was administered under
conditions that block endocytosis or lysosomal degradation,
[
H]Cer formation was only partially reduced in
both control and RA-differentiated cells (Table 4). This
indicates that only a portion of
[Sph-
H]SM is internalized into cells and
processed in the lysosomes, the remainder being produced at an
extralysosomal level (possibly the plasma membrane). The amount of
[
H]Cer produced in the presence of chloroquine or
at 4 °C was much higher (2.7- and 1.9-fold, respectively) in RA
differentiated than undifferentiated cells. On these premises, and
since neuroblastoma cells are known to contain an
Mg
-stimulated N-SM-ase(41, 42) , we
investigated the possible role of this enzyme in Cer formation during
RA-induced Neuro2a cell differentiation. As shown in Fig. 5, the
activity of Mg
-stimulated N-SM-ase, increased during
RA-induced differentiation, was maximal in the fully differentiated
cells and decreased upon removal of RA, paralleling reversal of cell
differentiation. Fumonisin B1, at the concentrations used with cells in
culture, did not affect the in vitro assay of N-SM-ase.
Figure 4:
Metabolism of exogenous SM in control and
RA-differentiated Neuro2a cells. Cells were fed with 4 µM
[Sph-H]SM for 2 h followed or not by 4 h
chase in the absence of exogenous SM. The radioactivity incorporated
into Cer, Glc-Cer, gangliosides, Sph, and water was measured. Data are
the mean values ± S.D. of three experiments in duplicate. Asterisk, p < 0.01, RA-treated versus control at the same chase time.
Figure 5:
Mg-dependent N-SM-ase
activity in Neuro2a cells during RA-induced differentiation. Cells were
treated with 20 µM RA and after different times the
activity of Mg
-dependent N-SM-ase was assayed on the
cell homogenate. In some experiments (asterisk), RA was
removed after 24 h, and incubation continued for further 24 h. Data are
expressed as percent of time-matched controls and are the mean values
± S.D. of three experiments in
triplicate.
Figure 6: Effect of exogenous Sph on Cer content and morphological differentiation in Neuro2a cells. Cells were incubated with different concentrations of Sph for 2 h (a and b) or with 1 µM Sph for different times (c). Data are the mean values ± S.D. of three experiments in duplicate.
Figure 8:
Effect of different treatments on the
morphological differentiation of Neuro2a cells. Cells were plated in
10% FCS-DMEM, and after 24 h the medium was replaced with 2% FCS-DMEM
containing the different agents. The incubation was then prolonged for
24 additional hours. A, control cells, 2% FCS; B and C, 5 and 10 µM Sph, respectively; D, 100
milliunits/ml bacterial SM-ase; E, 10 µM
C-Cer; F, 10 µM natural
Cer.
Figure 10:
Effect of different treatments on
[H]thymidine incorporation in Neuro2a cells.
Cells were incubated with 1 µM Sph, 1 µM C
-Cer, 1 µM natural Cer, or 100
milliunits/ml bacterial SM-ase for 24 h under the conditions specified
in the legend to Fig. 8. Cells were pulsed for the last 2 h of
incubation with [
H]thymidine. Data are the mean
values ± S.D. of three experiments in duplicate.
,
control; &cjs2090;, C
-Cer; ⊞, SM-ase; &cjs2108;,
sphingosine; &cjs2110;, ceramide.
The first piece of evidence provided by this study is that
enhanced levels of Cer are characteristic of RA-differentiated Neuro2a
cells. In fact, Cer (but not Sph) content increases during RA-induced
differentiation, is maintained at high levels in differentiated cells,
and returns to the basal values upon reversal of differentiation. At
least two metabolic pathways, de novo Cer biosynthesis and SM
degradation, seem to contribute to increasing the Cer content in
differentiated cells. Both pathways appear to be more efficient in
differentiated cells. Particularly, it is surprising the rapidity and
efficiency by which exogenous Sph is acylated to Cer in
RA-differentiated cells, with a concomitant lesser degree of Cer
metabolic progression to more complex sphingolipids (gangliosides, SM),
thus resulting in Cer accumulation. A similar situation has been
reported to occur in GHC
cells, where treatment
with RA, at concentrations able to inhibit cell proliferation, causes a
significant and prolonged increase of cellular Cer content as a result
of increased Sph N-acylation(43) . Since Sph content
is maintained constant during RA differentiation, an increased
replenishment of Sph pool either by neosynthesis or sphingolipid
degradation is requested in differentiated cells. The data here
presented on L-[
H]serine metabolism and
on the effects of two inhibitors of serine palmitoyltransferase
demonstrate that an increased neosynthesis of Cer does occur in
RA-differentiated cells. This evidence suggests that the activity of
serine palmitoyltransferase, a rate-limiting enzyme in de novo Cer biosynthesis(44) , is enhanced in RA-differentiated
cells.
The results obtained with the
[Sph-H]SM feeding experiments showed
that also SM degradation contributes to enhance the Cer level in
differentiated Neuro2a cells. Cer formation in differentiated cells
remains markedly elevated also when endocytosis or lysosomal
degradation are inhibited, especially in RA-differentiated cells. This
indicates that an extralysosomal, possibly plasma membrane-bound SM-ase
is mainly responsible for the increased SM degradation in
RA-differentiated cells. Consistent with this interpretation is the
evidence, here provided, that the activity of the
Mg
-dependent N-SM-ase, an enzyme especially
concentrated in neural tissues (45) and in cells of neuronal
origin(41, 42) , increases during RA-induced
differentiation of Neuro2a cells. It is also worth noting that previous
studies have shown that N-SM-ase increases in rat brain parallelly with
neuronal maturation (46) . A further support to the notion that
Cer increase concomitant to RA-induced differentiation is due to
stimulation of both de novo biosynthesis and SM degradation
comes from the observation that treatment with Fumonisin B1, which
inhibits ceramide synthase (one of the enzymes of the biosynthetic
route) (12, 28) but does not affect the activity of
N-SM-ase, markedly reduced, but not suppressed, both the formation of
neurite-like processes and the increase of Cer level induced by RA
treatment. The increase of Cer content owing to RA-induced
differentiation was about 70% and was accompanied by an overall
decrease of the sphingolipid content (particularly SM but also neutral
glycosphingolipids) of differentiated as compared to undifferentiated
cells, as well as a decrease of the metabolic involvement of Cer in the
biosynthesis of complex sphingolipids. It will be interesting to
ascertain whether a particular pool of Cer, separated from the one used
for biosynthetic purposes, is involved in differentiation and thus
submitted to enhancement. In this case, the increase of
``active'' Cer might be still higher.
The second,
important, piece of evidence provided by this study is that conditions
leading to enhance the Cer content of undifferentiated Neuro2a cells,
in the absence of inducers like RA, succeeded in stimulating
neuritogenesis, concomitantly with inhibition of cell proliferation. In
fact, supplying of exogenous Sph, natural Cer, or C-Cer or
treatment with bacterial SM-ase is followed by induction of neurite
formation and inhibition of thymidine incorporation into DNA. We
observed that Fumonisin B1, which blocks Cer biosynthesis from Sph or
dihydro-Sph(12, 28) , completely inhibited Neuro2a
cell differentiation upon treatment with exogenous Sph but did not
affect the differentiating effect promoted by bacterial SM-ase
treatment. Notably, the stimulation of Neuro2a cell neuritogenesis
following treatments that enhance the Cer content appeared to be rather
specific since dihydroderivatives of Sph, Cer, and C
-Cer
did not exert such effect. This body of observations strangely suggests
that the increase of Cer originated from neosynthesis and/or SM
degradation is instrumental to Neuro2a cell differentiation. A support
to this view comes from the finding that nerve growth factor causes
growth inhibition and formation of processes in T9 glioma cells, with
concomitant increase of cellular Cer(15) . Furthermore, in
hippocampal neurons, Fumonisin B1 treatment inhibits axonal growth,
together with Cer biosynthesis, and Cer derivatives are able to reverse
this effect and to cause a significant increase in axonal
length(12) . Moreover, exogenous sphingosylphosphocholine,
which promotes neuritogenesis in different neuroblastoma cells
including Neuro2a, is rapidly processed by cells, Cer being the main
metabolic product(47) . In agreement with this hypothesis is
also the finding that in leukemia cells SM hydrolysis, following
activation of N-SM-ase, triggers cell differentiation(48) . The
reported evidence that exogenous Cer was unable to induce neurite
outgrowth(49) , and Sph-inhibited neuritogenesis (50) in neuroblastoma cells, seemingly contrasting our results,
can be explained on the basis of the different culture and general
experimental conditions used by those investigators. Moreover, no
evidence was provided by these authors for any increase of Cer levels
under the adopted experimental conditions.
It is worth stressing that in Neuro2a cells the increased level of Cer in RA-induced differentiation occurs very early, persists along cell differentiation, but returns to the basal values upon reversal of differentiation. Therefore, Cer more than a trigger of differentiation seems to be a necessary instrument for differentiation. Hence, its role would be that of a bioregulator, its constant presence being needed for the expression of a particular functional state of the cell. If so, the enzymes directly involved in Cer formation and utilization should be considered as suitable targets of influences governing the transition of neural cells from the stage of proliferation to that of differentiation.
In conclusion, this work provides solid evidence for a bioregulatory implication of ceramide in the differentiation of Neuro2a cells and poses the general question of a mediator role of ceramide in the control and maintenance of differentiation in cells of neural origin.