| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(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
[
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 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 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.
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 [
Figure 3:
Incorporation of radioactivity into
different metabolites after feeding undifferentiated (control) and
differentiated (RA-treated) Neuro2a cells for different times with 40
nM [
In pulse-chase
experiments with L-[
The feeding experiments
with [Sph-
Figure 4:
Metabolism of exogenous SM in control and
RA-differentiated Neuro2a cells. Cells were fed with 4 µM
[Sph-
Figure 5:
Mg
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
Figure 10:
Effect of different treatments on
[
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 GH The results obtained with the
[Sph- 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 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.
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 26868-26875
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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.
, 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) ).
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) .
Materials
All reagents were of
analytical grade, and solvents were redistilled before use.
Dulbecco's modified Eagle's medium (DMEM) (
)and
FCS (heat-inactivated before use) were from Seromed (Biochrom KG,
Berlin). Crystalline bovine serum albumin, N-acetylneuraminic
acid, bovine brain sphingomyelin (SM), D-erythrosphingosine
(C-18) (Sph), DL-threo-dihydrosphingosine (C-18) (DL-threosphinganine), ceramide (N-acyl-D-erythro-C-18-sphingosine) (Cer), N-palmitoyl-DL-dihydrosphingosine (dihydro-Cer),
retinoic acid (RA), Fumonisin B1, Staphylococcus aureus sphingomyelinase (SM-ase), -chloro-L-alanine, and L-cycloserine were from Sigma; N-acetyl-erythrosphingosine (C-Cer) and standard
neutral glycosphingolipids (Glc-Cer, Lac-Cer, and
Gb
Ose-Cer) were from Matreya Inc. (Pleasant Gap, PA); N-acetyl-dihydrosphingosine (C-dihydro-Cer) was
from Calbiochem; [H]NaBH (6.5
Ci/mmol), [H]acetic anhydride (0.5 Ci/mmol), L-[3-H]serine (30 Ci/mmol),
[-
P]ATP (0.5-3 Ci/mmol), and
[Me-
H]thymidine (25 Ci/mmol) were from
Amersham International (Amersham, Bucks, United Kingdom); HPTLC silica
gel plates were from Merck (Darmstadt, Germany); Escherichia coli
sn-1,2-diacylglycerol kinase was from Calbiochem. Sphingomyelin,
radiolabeled at C-3 of the long chain base
([Sph-H]SM), and D-erythrosphingosine, tritiated at C-3
([H]Sph) were prepared and purified as described
previously(16, 17, 18) . Their specific
radioactivity was 0.35 and 1.1 Ci/mmol, respectively; the radiochemical
purity, assessed by HPTLC and autoradioscanning, was higher than 98% in
both cases. Standard gangliosides, GgOse-Cer and H-sphingolipids (Cer, Glc-cer and gangliosides) were
obtained as previously
reported(18, 19, 20, 21) .Cell Cultures
The murine neuroblastoma
cell line, clone NB2a (Neuro2a, CCL-131, American Cell Type Culture
Collection), was used. Cells were cultured in Falcon dishes in DMEM
supplemented with 10% FCS, 4 mML-glutamine, 1 mM sodium pyruvate, 100 units/ml potassium penicillin G, and 100
µg/ml streptomycin sulfate in 5% CO, 95% air-humidified
atmosphere. To induce neurite outgrowth, cells were plated at a 1.8
10/cm cell density, and 48 h after
plating the medium was replaced with 2% FCS-DMEM containing 20
µM retinoic acid(22) , the incubation being
continued for different times up to 48 h. Control experiment showed
that incubation up to 48 h with 2% FCS-DMEM, in the absence of retinoic
acid, caused only a very modest outgrowth of processes.Metabolism of Exogenously Added
[Sph-
The ability of
Neuro2a cells to metabolically process SM or Sph or serine added to the
culture medium was assessed on control and RA-differentiated cells
(after 24 h of treatment). At the time of experiments, cells were fed
with 4 µM [Sph-H]Sphingomyelin And
[
H]Sphingosine and L-[
H]Serine
H]SM (1.4
µCi/ml in DMEM) or 40 nM [H]Sph (44
nCi/ml in 2% FCS-DMEM) or 200 nML-[H]serine (6 Ci/ml in a serine-free
medium) for different times, as previously reported(18) . In
some experiments, cells were submitted, after pulse, to a 2-4-h
period of chase in 10% FCS-DMEM devoid of H molecules. At
the end of the pulse or pulse-chase period, cells were consecutively
washed with 10% FCS-DMEM (twice) and phosphate-buffered saline (twice),
harvested by scraping, and lyophilized(20) . The influence of
endocytosis and lysosomal activity on the metabolic processing was
assessed by performing experiments at 4 °C (condition that blocks
endocytosis) or in the presence of 50 µM chloroquine (drug
which blocks the activity of lysosomal enzymes), as previously
reported(19) . Volatile radioactivity, HO, released in the culture medium, was
determined by fractional distillation of the culture medium under
carefully controlled conditions, collection of the distilled fractions,
and measurement of the radioactivity by liquid scintillation
counting(23) .Lipid Extraction and Quantification
Total
lipids were extracted from lyophilized cells(20) , and, after
partitioning, the organic phase was subjected to mild alkaline
hydrolysis(24) . The obtained aqueous and organic phases were
counted for radioactivity and analyzed by HPTLC. The following solvent
systems (by volume) were used: for the organic phase (Cer, neutral
glycosphingolipids, Sph, and SM) chloroform/methanol/water (55:20:3) or
chloroform/methanol/32% NHOH (40:10:1) or
hexane/chloroform/acetone/acetic acid (10:35:10:1); for the aqueous
phase (gangliosides) chloroform/methanol/0,2% CaCl (55:45:10). After HPTLC, the plates were radioscanned with a
digital autoradiograph (Berthold, Germany) and then submitted to
fluorography(19, 20) . The identification and
quantification of [H]Cer and other H
metabolites was performed as described
previously(19, 20) .Treatment of Cultured Cells with Exogenous
Sphingosine, Natural Ceramide, C
5 -Ceramide, or Bacterial
SM-ase
10-10
cells were
plated in 35-mm Falcon dishes and grown in 10% FCS-DMEM for 24 h before
the experiments. At the time of experiments, dishes were washed three
times with 2% FCS-DMEM and incubated for different times (up to 24 h)
in the same medium (1 ml/dish) containing D-erythro-Sph
(0.1-10 µM) or C-Cer (1-10
µM), or natural Cer (0.5-10 µM) or
bacterial SM-ase (100 milliunits/ml). C-Cer (which is a
cell-permeable analog of Cer) and Sph solutions were prepared by adding
small volumes of stock solutions in absolute ethanol to 2% FCS-DMEM
(the final concentration of ethanol never exceeded 0.1%). The solutions
were allowed to stand at 37 °C for 1 h before treatment of the
cells. Natural Cer was dissolved in ethanol/dodecane, 98:2 (v/v)
according to Ji et al.(25) , and added to the medium
to reach the final wanted concentration. Under these conditions, Cer is
able to penetrate into cells. The final concentration of ethanol and
dodecane never exceeded 0.5 and 0.01%, respectively. Neither ethanol
nor dodecane at the used concentrations caused visible changes of cell
morphology. Bacterial SM-ase was added to the culture medium just
before use. In parallel experiments, 1 µMDL-threosphinganine or dihydro-Cer or
C-dihydro-Cer, dissolved as D-erythro-Sph, Cer, or
C-Cer, was used.Effect of Inhibitors of Sphingosine and Ceramide
Synthesis on Neuro2a Differentiation
The involvement of
serine-palmitoyl transferase in RA-induced differentiation in vivo was assessed by adding 2.5 mM -chloroalanine or L-cycloserine, known inhibitors of this
enzyme(26, 27) , 2 h prior and during RA treatment.
The involvement of Cer synthase in the same process was ascertained
running experiments in the presence of Fumonisin
B1(12, 28) , the known inhibitor of this enzyme.Morphological Differentiation and Measurement Of
Neurite-like Processes Outgrowth
The degree of
morphological differentiation was assessed by phase-contrast
microscopy. In particular, 200-300 cells in 4-5 random
fields in each dish were counted, and cells bearing neurite-like
processes longer than the major cell body diameter (after treatments
not exceeding 8 h), or bearing a neurite with a length at least double
that of cell diameter (after treatment longer than 8 h), were scored as
differentiated. Data were expressed as neurite-bearing cells (short or
long neurites) as percentage of total cells counted. Cell aggregates
were not scored, and cells with more than one neurite were only counted
once. Cell viability was assessed by the trypan blue exclusion method.[
5 H]Thymidine
Incorporation
10-10cells were
grown in 35-mm Falcon dishes in 10% FCS-DMEM. 24 h after plating cells
were cultured in 2% FCS-DMEM containing different sphingoids or
bacterial SM-ase (as described above) for 24 h. The medium was removed
and replaced with 1 ml of DMEM containing 0.5 µCi of
[H]thymidine(29) . After 2 h at 37
°C, cells were harvested with phosphate-buffered saline and treated
with 10% trichloroacetic acid. The insoluble residue, filtered on
microfiber glass filters GF/C (Whatman International Ltd., Maidstone,
UK), was submitted to radioactivity counting.Other Methods
Radioactivity was
determined by liquid scintillation counting, fluorography, and
radiochromatoscanning (Digital Autoradiograph, Berthold,
Germany)(19, 20) . The content of Cer and Sph was
determined by the method of Preiss et al.(30) and
Ohta et al.(31) , respectively. Total protein was
assayed (32) using bovine serum albumin as the standard.
Mg-stimulated neutral sphingomyelinase (N-SM-ase)
activity was assayed on the cell homogenate as previously
reported(33) . Ganglioside content was determined (34) as lipid bound sialic acid (using N-acetylneuraminic acid as the standard) on the aqueous phase
(see above) desalted by Sephadex G-25 column chromatography. Neutral
glycolipids and sphingomyelin were purified from the organic phase
(after alkaline methanolysis) by Unisil silicic acid column
chromatography(35) . Gangliosides and neutral glycolipids,
separated by HPTLC (see above), were quantified by densitometric
scanning (Camag TLC densitometer) (23) after visualization with
a p-dimethylaminobenzaldehyde reagent (36) or a
diphenylamine reagent(37) , respectively. SM was determined
after perchloric acid digestion(38, 39) . Statistical
significance of differences was determined by the Student t test.
Complex Sphingolipid Content in RA-differentiated
Neuro2a Cells
Data regarding the total amount of
sphingomyelin, gangliosides, and neutral glycosphingolipids as well as
the qualitative pattern of major gangliosides and neutral
glycosphingolipids in control and RA-differentiated Neuro2a cells are
reported in Table 1. As shown, the content of both sphingomyelin
and neutral glycosphingolipids was significantly reduced in
differentiated cells as compared to the control preconfluent growing
cells. Moreover, and in agreement with previous studies(40) ,
significant changes in the ganglioside and neutral glycosphingolipid
pattern were recorded; particularly, RA-differentiated cells had a
lower content of G and higher contents of G
,
G
, G
, and GgOse
-Cer than
undifferentiated cells.
Ceramide and Sphingosine Levels during RA-induced
Differentiation of Neuro2a Cells
After RA treatment in low
(2%) FCS medium, Neuro2a cells underwent differentiation, which started
to be appreciable after 60-120 min and resulted in a very
elaborated network of processes after 24-48 h, in agreement with
reported findings(22) . As shown in Fig. 1, the content
of endogenous Cer (1.02 ± 0.11 nmol/mg protein, in
undifferentiated cells) increased rapidly with time in Neuro2a cells
after RA addition until about 8 h (1.55 ± 0.46 nmol/mg protein).
This level remained almost constant in differentiated cells (1.69
± 0.33 and 1.74 ± 0.36 nmol/mg protein at 24 and 48 h,
respectively). The increase of Cer content could be acknowledged at the
first investigated time after RA treatment (30 min), indicating that
the cell response to RA, in terms of Cer production, was very prompt.
The removal of RA from the medium caused reversal of differentiation,
as expected(22) , with a parallel decrease of the Cer content
to the starting level. It is noteworthy that the Sph content (100
± 12 pmol/mg protein) appeared to be unaffected by RA treatment.
Concomitant treatment of Neuro2a cells with RA and 25 µM Fumonisin B1, the inhibitor of Cer
synthase(12, 28) , reduced but not blocked the process
of differentiation (Fig. 7, lanes RA and RA + Fumonisin B1) and caused a concomitant decrease of Cer content
(1.34 ± 0.12 nmol/mg protein). It is noteworthy that upon RA
plus Fumonisin B1 treatment, the Cer content remained substantially
higher than that of undifferentiated cells.
, control; *, +RA; 
,
-RA.
Metabolic Source of Ceramide in RA-differentiated
Neuro2a Cells
The pathways of Cer generation in
RA-differentiated cells were inspected by pulsing Neuro2a cells with H precursors of Cer, namely [H]Sph or L-[H]serine or
[Sph-H]SM, and following the formation
of H metabolites. As shown in Fig. 2,
undifferentiated and fully differentiated Neuro2a cells rapidly
incorporated and metabolized [H]Sph in a
time-dependent fashion. The radioactivity present in the total lipid
extract, as well as tritiated water released in the culture medium
(volatile radioactivity), produced by complete Sph degradation, was a
little higher in differentiated than undifferentiated cells and
markedly increased with pulse time in both cell types. Conversely,
[H]Sph diminished with pulse time and represented
only a minor portion of total incorporated radioactivity (radioactivity
in the total lipid extract plus volatile radioactivity) at all the
investigated times (about 5% at 30 min and 2% at 120 min). HO was a minor product of
[H]Sph metabolism, since after 30 and 120 min of
pulse it accounted only for about 8% and 12%, respectively, of the
total incorporated radioactivity in both undifferentiated and
RA-differentiated cells. [H]Cer represented by
far the major radiolabeled metabolite of exogenous
[H]Sph, accounting for more than half of total
incorporated radioactivity in both undifferentiated and
RA-differentiated cells (Fig. 3). It was produced more rapidly
and at a higher extent in differentiated cells, whereas its utilization
for the biosynthesis of complex sphingolipids (Glc-Cer, SM, and
gangliosides) was higher in undifferentiated cells.
H]Sph for different times. Data are the
mean values ± S.D. of three experiments in duplicate. White
bars, control; stippled bars,
RA-treated.
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.
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.
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 HO (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.
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.
-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.
Effect of Induced Increase of Ceramide Content on
Neuro2a Cell Differentiation and Growth
Undifferentiated
Neuro2a cells were treated with Sph, natural Cer, C-Cer, or
bacterial SM-ase, conditions that are known to increase the cellular
Cer level, and the differentiation process was morphologically
assessed. The first set of experiments relied on the metabolic studies
described above, indicating that exogenous Sph is actively taken up by
Neuro2a cells and rapidly and predominantly N-acylated to
ceramide. As shown in Fig. 6a, the addition of
exogenous Sph (0.1-10 µM) resulted in a
dose-dependent increase of the cellular level of Cer. Parallelly,
neurite outgrowth was markedly stimulated in a dose- and time-dependent
manner (Fig. 6, b and c, and Fig. 8, B and C). When Neuro2a cells were treated with 25
µM Fumonisin B1, the differentiating effect of exogenous
Sph was not detectable (Fig. 7, lanes Sp and Sp
+ Fumonisin B1). It is noteworthy (and as reported above)
that treatment with Fumonisin B1 inhibited the differentiating effect
of RA as well, but only partially. Treatment of Neuro2a with 1
µM natural Cer, 1 µM C-Cer, or
bacterial SM-ase (100 milliunits/ml) for 2-4 h also resulted in
the stimulation of neurite outgrowth (up to 2.5-fold with bacterial
SM-ase and natural Cer and 3.5-fold with C-Cer) (Fig. 8, D-F, and 9). In contrast to the marked
inhibitory effect exerted on Sph and RA-induced differentiation,
Fumonisin B1 did not affect the differentiation promoted by bacterial
SM-ase after 4 and 24 h (106 and 93% with respect to SM-ase treated
cells, respectively). As shown in Fig. 10, the administration of
exogenous Sph, or natural Cer or C-Cer or bacterial SM-ase,
at the conditions stimulating neuritogenesis, caused a marked
diminution of [H]thymidine incorporation into
DNA, indicating inhibition of cell proliferation.
-Cer; F, 10 µM natural
Cer.
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.
Specificity of Ceramide-induced Differentiation of
Neuro2a Cells
To evaluate the specificity of
ceramide-induced differentiation, we compared the effects of equimolar
concentrations of different sphingoids on this process. As shown in Table 5, under the experimental conditions used, where Sph,
natural Cer, and C-Cer were active, either a racemic
mixture of threo-dihydro-Sph or C-dihydro-Cer or
dihydro-Cer did not exert any effects on Neuro2a cell differentiation.
C 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.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.
-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.
)-Cer, N-acetyl-erythrosphingosine; Sph, D-erythrosphingosine; SM, sphingomyelin; Glc-Cer,
glucosylceramide; Lac-Cer, lactosylceramide; GgOse-Cer,
GalNAc1-4Gal1-4Glc1-1 ceramide;
GbOseCer,
GalNAc1-3Gal1-4Gal1-4Glc1-1
ceramide; SM-ase, sphingomyelinase; N-SM-ase, neutral sphingomyelinase;
FCS, fetal calf serum; HPTLC, high performance thin layer
chromatography. Gangliosides are named according to
Svennerholm(1) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Krebs, I. Medugorac, S. Rother, K. Strasser, and M. Forster A missense mutation in the 3-ketodihydrosphingosine reductase FVT1 as candidate causal mutation for bovine spinal muscular atrophy PNAS, April 17, 2007; 104(16): 6746 - 6751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Masukawa, H. Tsujimura, and H. Narita Liquid chromatography-mass spectrometry for comprehensive profiling of ceramide molecules in human hair J. Lipid Res., July 1, 2006; 47(7): 1559 - 1571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Shinjyo and K. Kita Up-Regulation of Heme Biosynthesis during Differentiation of Neuro2a Cells. J. Biochem., March 1, 2006; 139(3): 373 - 381. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Baldini, A. M. Martelli, G. Tabellini, C. Horn, K. Machaca, P. Narducci, and G. Baldini Rabphilin Localizes with the Cell Actin Cytoskeleton and Stimulates Association of Granules with F-actin Cross-linked by {alpha}-Actinin J. Biol. Chem., October 14, 2005; 280(41): 34974 - 34984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bulfone, P. Carotenuto, A. Faedo, V. Aglio, L. Garzia, A. M. Bello, A. Basile, A. Andre, M. Cocchia, O. Guardiola, et al. Telencephalic Embryonic Subtractive Sequences: A Unique Collection of Neurodevelopmental Genes J. Neurosci., August 17, 2005; 25(33): 7586 - 7600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Prinetti, L. Basso, V. Appierto, M. G. Villani, M. Valsecchi, N. Loberto, S. Prioni, V. Chigorno, E. Cavadini, F. Formelli, et al. Altered Sphingolipid Metabolism in N-(4-Hydroxyphenyl)- retinamide-resistant A2780 Human Ovarian Carcinoma Cells J. Biol. Chem., February 14, 2003; 278(8): 5574 - 5583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ramm, Y. Alexandrov, A. Cholewinski, Y. Cybuch, R. Nadon, and B. J. Soltys Original Papers: Automated Screening of Neurite Outgrowth J Biomol Screen, February 1, 2003; 8(1): 7 - 18. [Abstract] [PDF]
|
|
|
|
|
|
C. Sato, T. Matsuda, and K. Kitajima Neuronal Differentiation-dependent Expression of the Disialic Acid Epitope on CD166 and Its Involvement in Neurite Formation in Neuro2A Cells J. Biol. Chem., November 15, 2002; 277(47): 45299 - 45305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Murate, M. Suzuki, M. Hattori, A. Takagi, T. Kojima, T. Tanizawa, H. Asano, T. Hotta, H. Saito, S. Yoshida, et al. Up-regulation of Acid Sphingomyelinase during Retinoic Acid-induced Myeloid Differentiation of NB4, a Human Acute Promyelocytic Leukemia Cell Line J. Biol. Chem., March 15, 2002; 277(12): 9936 - 9943. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sajithlal, H. Huttunen, H. Rauvala, and G. Munch Receptor for Advanced Glycation End Products Plays a More Important Role in Cellular Survival than in Neurite Outgrowth during Retinoic Acid-induced Differentiation of Neuroblastoma Cells J. Biol. Chem., February 22, 2002; 277(9): 6888 - 6897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Abnet, C. B. Borkowf, Y.-L. Qiao, P. S. Albert, E. Wang, A. H. Merrill Jr., S. D. Mark, Z.-W. Dong, P. R. Taylor, and S. M. Dawsey A Cross-Sectional Study of Human Serum Sphingolipids, Diet and Physiologic Parameters J. Nutr., October 1, 2001; 131(10): 2748 - 2752. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. MISASI, M. SORICE, L. DI MARZIO, W. M. CAMPANA, S. MOLINARI, M. G. CIFONE, A. PAVAN, G. M. PONTIERI, and J. S. O'BRIEN Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase FASEB J, February 1, 2001; 15(2): 467 - 474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. M. Hernandez, D. J. Discher, N. H. Bishopric, and K. A. Webster Rapid Activation of Neutral Sphingomyelinase by Hypoxia-Reoxygenation of Cardiac Myocytes Circ. Res., February 4, 2000; 86(2): 198 - 204. [Abstract] [Full Text] [PDF]
|
|
ABSTRACT
