(Received for publication, August 9, 1995; and in revised form, October 17, 1995)
From the
The roles of sphingosine and sphingosine 1-phosphate in
Ca signaling following platelet-derived growth factor
(PDGF) receptor stimulation were investigated in the oligodendrocyte
cell line CEINGE cl3, using single-cell fura-2 microfluorimetry and
videoimaging. Two different Ca
responses were
observed, which differed in their delays and kinetics. The first
response, which occurred after a shorter delay, exhibited a single
Ca
peak often followed by a plateau, while the second
type of response was characterized by a longer delay and by
Ca
spikes with different frequencies and amplitudes.
The latter phenomenon was never observed after stimulation of G
protein-coupled receptors for ATP, ET-1, and BK. The incubation with
the inhibitor of sphingosine kinase, DL-threo-dihydrosphingosine, significantly increased
the percentage of cells responding to PDGF-BB exposure with
Ca
spikes (87 versus 47%), while it did not
modify the Ca
response elicited by exposure to ATP,
ET-1, or BK. Exposure to exogenous 10 µM sphingosine or 1
µM sphingosine 1-phosphate produced oscillatory and
non-oscillatory Ca
responses, respectively, similar
to those elicited by PDGF-BB. A second application of PDGF-BB, 30 min
after the first, was normally ineffective in producing a Ca
response. However, if the second exposure was preceded by the
inhibition of sphingosine 1-phosphate formation, an oscillatory
Ca
response occurred in all cells. We conclude that
intracellular levels of sphingosine and sphingosine 1-phosphate may
differentially modulate Ca
signaling triggered by
PDGF receptor stimulation in CEINGE cl3-transformed oligodendrocytes.
Polypeptide growth factors regulate cellular functions by
binding to transmembrane receptors, which possess tyrosine kinase
activity (RTKs)(1, 2) . PDGF ()is able to
promote growth in fibroblasts(3) , smooth muscle
cells(4) , and glial cells(5) . One of the first
cellular events that occurs following the stimulation of PDGF receptors
is an increase in
[Ca
]
(6, 7) .
This increase has been shown to be correlated with PDGF-induced cell
proliferation(8, 9, 10) . The
[Ca
]
increase caused
by PDGF receptor stimulation is mainly the result of
Ca
mobilization from intracellular stores, a
mechanism recognized as being a consequence of an IP
formation, at least in smooth muscle
cells(10, 11) . In the case of PDGF receptors, this
involves the
isoform of phospholipase
C(1, 12) , which is directly phosphorylated on both
serine and tyrosine residues by the activated receptor(13) .
Overexpression of this enzyme potentiates IP
production
following PDGF exposure in fibroblasts(14) .
It has recently
become clear, however, that a second and possibly equally important
biochemical pathway may be involved in PDGF-evoked Ca signaling. It was initially shown that the sphingomyelin
metabolite sphingosine was able to mobilize intracellular
Ca
when exogenously added to smooth muscle cells. It
has also been hypothesized that a further modification of the molecule
is necessary for its Ca
mobilizing
action(15) . A subsequent study demonstrated that SPP produced
from sphingosine is a potent mitogen for 3T3 fibroblasts. Like
sphingosine, SPP mobilizes Ca
by an intracellular
mechanism (16) . Several further studies have confirmed that
sphingosine and SPP are able to mobilize Ca
from
intracellular stores in different cell types(17, 18) .
The recent observation that intracellular levels of sphingosine and SPP
increase after PDGF and fetal calf serum stimulation of Swiss 3T3
fibroblasts is consistent with these sphingolipids mediating the
mitogenic action of PDGF(19) . Furthermore, sphingosine kinase,
the enzyme responsible for converting sphingosine to SPP, shows a
transient increase in activity after PDGF stimulation. Blocking this
enzyme with the competitive inhibitor DL-threo-dihydrosphingosine markedly reduces the DNA
synthesis induced by PDGF(19) . This suggests that SPP is the
main sphingolipid in the signal transduction pathway, which follows
PDGF receptor stimulation in these cells. It is possible that both
sphingolipids might mobilize Ca
through a modulatory
action on the IP
pathway(18) , or they could act in
a completely independent fashion, as recently suggested for SPP in
fibroblasts(20) . However, the exact mechanism by which these
sphingolipids act still needs to be elucidated. It is also important to
understand whether each lipid has an active role in intracellular
signaling or whether SPP is the effective mediator and sphingosine is
only an intermediate metabolite.
Although several studies have
examined the intracellular Ca signaling triggered by
PDGF in vascular smooth muscle cells (10) and 3T3
fibroblasts(6) , data regarding this transductional mechanism
in oligodendroglial cells are not available. The development and
proliferation of oligodendrocytes are critically regulated by
PDGF(21, 22, 23) , which, besides its
mitogenic and chemotactic activity, stimulates c-Jun and c-Fos
expression in oligodendrocyte progenitor cells(24) .
In this
study, single-cell microfluorimetry and videoimaging were used to
investigate Ca signaling as a result of PDGF receptor
stimulation in the cell line CEINGE cl3. These cells, immortalized with
polyoma middle T antigen, have been identified as oligodendrocytes by
using antibodies directed against galactocerebroside C(25) , a
specific marker for oligodendroglia.
These cells were
cultured as a monolayer in polystyrene dishes and maintained in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) containing 10% heat-inactivated fetal calf serum (Life
Technologies, Inc.) and 50 µg/ml gentamycin. Cells were grown in a
humidified incubator at 36.5 °C in a 5% CO atmosphere
and were fed twice weekly. All experiments were performed with cells
from passages 60-80.
The single-cell videoimaging was performed as described previously (26) with some modifications. Coverslips were mounted on a coverslip chamber (Medical Systems Co., Greenvale, NY), and the fluorescence measurements were performed at 23-26 °C. Cells were continuously superfused using a peristaltic pump (Gilson) with a flow rate of 650 µl/min, and the perfusion medium was directed on cells under observation by a microtube positioned by a macromanipulator (Narishige, Japan). The removal of experimental solutions from the coverslip chamber (500 µl, volume) was achieved using a microaspirator (Medical System Co., Greenvale, NY) connected to a vacuum pump, and the entire volume of the chamber turned over in less than 60 s. A two-way valve (Thomson, Springfield VA) controlled the flow from a separate injection loop, which was used to perfuse cells with different experimental solutions.
Ca-free experiments were performed using a
nominally Ca
-free standard buffer containing 0.2
mM EGTA. Before starting the experiment, cells were perfused
with this buffer for 2-3 min to completely remove Ca
from the extracellular environment. Fura-2 fluorescence was
imaged with an inverted Nikon Diaphot microscope using a Nikon
20
/1.3 NA Fluor DL objective lens. The cells were illuminated
with a 100-watt Xenon lamp (Oriel Corp., Stratford, CT) with quartz
collector lenses. A shutter and a filter wheel containing the two
different interference filters (340 and 380 nm) were controlled by a
computer. Emitted light was passed through a 400-nm dichroic mirror,
filtered at 480 nm, and collected by a video camera (MTI, Michigan
City, IN) connected with a KS 1381 light intensifier (Videoscope
Intl.). Images of as many as 10-30 cells/field were averaged
using an Avio Image S (Nippon Avionic) real time image processor set at
16 frames for each data point and digitized in an image analyzer
(Applied-Imaging Ltd., Dukesway Gateshead, United Kingdom) connected to
a computer equipped with Tardis software. Each cell in the image was
independently analyzed for each time point in the captured sequence.
All individual [Ca
]
traces
shown are from a single cell and are representative responses for a
given field of cells. It should be mentioned that, as previously
reported, some CEINGE cl3 cells show spontaneous changes in the
[Ca
]
, represented by
Ca
spikes with different amplitudes and
frequencies(27) . Although this phenomenon has not been
frequently observed, a previous data acquisition of 5 min was performed
before each experiment, and those cells showing such spontaneous
Ca
spiking activity were not considered for the
analysis.
For the calibration of fluorescent signals we used cells
loaded with fura-2; R and R
are, respectively, the ratios at saturating and zero
[Ca
]
and were obtained
perfusing cells with a salt solution containing 10 mM CaCl
and 5 µM ionomycin for 3 min and
subsequently with a Ca
free salt solution containing
10 mM EGTA for 20 min. The values of the obtained R
and R
, expressed as
gray level mean, were used to calculate the calibration curve using the
Tardis software. Intracellular calcium concentration was determined as
previously reported(28) .
Figure 1:
Non-oscillatory Ca
responses induced by PDGF-BB. Single-cell analysis is shown of fura-2
fluorescence in cells exposed for 5 min to 10 ng/ml PDGF-BB (added at
the arrow). An acquisition time of one data point every 5 s
was used. A, a single increase in
[Ca
]
was followed by a
small plateau, which outlasted PDGF-BB removal; B, a single
increase in [Ca
]
was
followed by a return to basal levels while PDGF-BB was still present; C, a [Ca
]
trace obtained with an acquisition time of one data point
every 800 ms during a non-oscillatory
response.
Figure 2:
Oscillatory Ca responses
induced by PDGF-BB. In 43% of responding cells, a 5-min exposure to 10
ng/ml PDGF-BB (added at the arrow) evoked a Ca
spiking activity with different frequencies and amplitudes. The
response shown in A was typical. Less than 5% of oscillating
cells showed only two spikes during the 14 min following the PDGF-BB
addition (B). The delay preceding the Ca
responses was 251 ± 7 s, significantly longer than the
delay of 166 ± 4 s preceding non-oscillatory responses (Fig. 1).
To exclude the
possibility that the non-oscillatory response consisted of a
Ca spiking response at very high frequency, an
acquisition time of 800 ms was adopted. Using this faster analysis,
non-oscillatory Ca
responses with a shorter delay
could still be demonstrated (Fig. 1C). Furthermore,
following the initial non-oscillatory response, no further increases in
[Ca
]
were observed during the
remaining data acquisition period.
A very small percentage of
oscillatory cells (<5%) displayed only two Ca spikes in the 14 min following the PDGF-BB addition (Fig. 2B). However, even in this case, the average
delay of the Ca
response was similar to that observed
in cells responding with multiple Ca
spikes.
Perfusion of the growth factor for a shorter period of time (1 min) did not significantly modify the percentage of cells responding with non-oscillatory and oscillatory patterns and the relative time course of these responses (63%, delay = 133 ± 7 s and 37%, delay = 266 ± 22 s, respectively; n = 55).
Figure 3:
Effect of extracellular Ca removal on PDGF-BB-induced Ca
signaling. Prior
to exposure to 10 ng/ml PDGF-BB (added at the arrow), cells
were perfused for 3 min with a Ca
free solution
containing 200 µM EGTA. Each panel represents a
typical response of cells exposed to PDGF-BB in absence of
extracellular Ca
(empty circles) compared to
an analogous response obtained in different cells when extracellular
Ca
was present (filled circles). A,
a non-oscillatory response represented by a monophasic increase in
[Ca
]
, not followed by
the plateau phase when the extracellular Ca
was
removed; B, an oscillatory Ca
response with
spikes of reduced amplitude when cells were exposed to PDGF-BB after
extracellular Ca
removal. Panel C compares
the response of two typical oscillatory cells and shows that in the
presence of extracellular Ca
, the spiking activity
lasts longer than following extracellular Ca
removal.
In these series of experiments, the images were acquired for longer
time periods (21 min instead of 14 min). Data are from 110 cells
analyzed in six separate experiments.
Figure 4:
Typical Ca responses
evoked by stimulation of G protein-coupled receptors for BK, ET-1, and
ATP. Exposure of cells to 1 µM BK, 100 nM ET-1,
and 100 µM ATP is shown. Perfusion of each agonist started
at 30 s and was maintained for 5 min.
Figure 5:
Effect of sphingosine kinase inhibition on
PDGF-BB-induced Ca responses. Upper panel,
percentage of non-oscillatory (NOX) and oscillatory (OX) cells in response to 10 ng/ml PDGF-BB with (&cjs2109;) or
without (&cjs2108;) a previous 5-min incubation with the sphingosine
kinase inhibitor DL-threo-dihydrosphingosine (10
µM); lower panel, a typical oscillatory
Ca
response in a cell incubated with 10
µMDL-threo-dihydrosphingosine prior to
being exposed for 5 min to 10 ng/ml PDGF-BB. The inhibitor was perfused
during the entire experiment. Data are from 62 cells analyzed in five
separate experiments.
In contrast, a
10-min incubation with 10 µMDL-threo-dihydrosphingosine did not modify the
Ca responses evoked by following exposure to 100
µM ATP, 100 nM ET-1, or 1 µM BK
(data not shown).
Finally, when cells were exposed to 10
µMDL-threo-dihydrosphingosine alone, no
changes in the [Ca]
were
observed (data not shown).
Figure 6:
Effects of perfusion with exogenous
sphingosine and SPP. A, oscillatory Ca responses caused by exposing cells for 5 min to 10 µM sphingosine, following a previous 10-min incubation with 10
µMDL-threo-dihydrosphingosine. Data are
from 53 cells analyzed in four different experiments. B, a
monophasic Ca
increase elicited perfusing cells for 5
min to 1 µM SPP. Each substance was added to the cells as
indicated by the arrow. Data are from 38 cells analyzed in
three separate experiments.
Figure 7:
Effect of two consecutive additions of
PDGF-BB on Ca signaling. After an initial
Ca
response elicited by a 5-min exposure to PDGF-BB
in non-oscillatory (A) and oscillatory cells (C), a
second addition to the same cells, after a 30-min interval, was unable
to cause a further [Ca
]
increase (B and D). Panels A and B show the only cells out of 42 cells analyzed in three
separate experiments that responded with a single Ca
peak to the second PDGF-BB addition.
Figure 8:
Effect of sphingosine kinase inhibition
on Ca responses induced by a second PDGF-BB exposure.
After an initial Ca
response produced by PDGF-BB (A and B), the same cells were incubated for 10 min
with 10 µMDL-threo-dihydrosphingosine
during the 30-min interval preceding a second PDGF-BB exposure, which
is shown in panels D and E. Both non-oscillatory and
oscillatory cells responded to a second PDGF-BB addition in an
oscillatory fashion. Panel C shows one single cell out of six
that was unresponsive to a first PDGF-BB exposure but showed an
oscillatory Ca
response to the second PDGF-BB
addition after incubation with DL-threo-dihydrosphingosine (F). Data are
from 55 cells analyzed in three separate
experiments.
This study shows that in cells belonging to the
oligodendrocyte lineage, PDGF-BB induces two distinct patterns of
Ca signaling, possibly as a consequence of different
modulatory roles played by sphingosine and SPP.
Both Ca response patterns were partially dependent on the presence of
extracellular Ca
. Removal of extracellular
Ca
reduced both their magnitude and duration (Fig. 3), suggesting that some Ca
influx
following PDGF-BB exposure must take place. This influx may occur via
the ``capacitative'' pathway for refilling intracellular
Ca
pools, which can be induced by their
depletion(29, 30) .
The non-oscillatory pattern
observed in CEINGE cl3 cells exposed to PDGF-BB is very similar to
analogous Ca responses induced by the same growth
factor in fibroblasts (6) and vascular smooth muscle
cells(7) . However, the oscillatory Ca
responses induced by PDGF-BB in CEINGE cl3 cells have never been
described in any other cell type. Ca
spikes have
frequently been observed upon stimulation of G protein-coupled
receptors and are generally considered to be a consequence of some
interplay between Ca
influx and Ca
mobilization from intracellular stores(30) . However, exposure
of CEINGE cl3 cells to three different agonists at G protein-coupled
receptors (Fig. 4), or to ryanodine or caffeine, was unable to
elicit oscillatory Ca
responses. Thus, this
phenomenon seems to be peculiar to PDGF-BB stimulation of this cell
type. Furthermore, it also suggests that the mechanisms involved in the
genesis of Ca
oscillations are unrelated to
Ca
mobilization from ryanodine/caffeine-sensitive
intracellular pools.
We might hypothesize that the oscillatory
Ca response elicited by PDGF-BB in CEINGE cl3 cells
involves a signaling pathway different from the activation of
phospholipase C by G proteins. Indeed, the evidence suggests that the
two different Ca
responses observed after PDGF-BB
exposure may be the result of a common biochemical pathway. The
different delays associated with the two responses could result from
the different time required for the production of two different second
messengers such as sphingosine and SPP. Indeed, an increase in the
production of both of these substances has been described in 3T3
fibroblasts in response to PDGF-BB(19) .
Sphingosine kinase
has been proposed to catalyze the generation of SPP from
sphingosine(31) . 10 µMDL-threo-dihydrosphingosine inhibited the activity of
this enzyme by 50% in isolated platelets. In the same system,
generation of SPP in response to exogenous sphingosine was inhibited up
to 25%(32) . Similar results were obtained in 3T3 fibroblasts,
where 10 µMDL-threo-dihydrosphingosine
inhibited the production of SPP induced by exogenous sphingosine by 63%
and also completely eliminated the generation of SPP elicited by
PDGF-BB(19) . Incubation of CEINGE cl3 cells with DL-threo-dihydrosphingosine did not affect the
Ca response to ATP, BK, and ET-1. This suggests that
sphingolipids are not involved in the Ca
signaling
following G protein-coupled receptor stimulation, as previously
observed in fibroblasts(19) . However, sphingosine kinase
inhibition caused a significant increase in the percentage of cells
responding to PDGF-BB with [Ca
]
oscillations (Fig. 5). Interestingly, the delay preceding
this response was identical to that observed in cells spontaneously
responding to PDGF-BB in an oscillatory fashion. These data strongly
suggest that an increase in intracellular sphingosine levels or the
consequent reduction in SPP levels facilitates the occurrence of
oscillatory Ca
responses to PDGF-BB. To further
verify this hypothesis, exogenous sphingosine and SPP were separately
added to the cells. Sphingosine and SPP caused Ca
responses that were oscillatory and non-oscillatory, respectively (Fig. 6). SPP exposure never caused an oscillatory
Ca
response in any of the responsive cells analyzed.
However, the [Ca
]
increase
elicited by exogenous SPP was characterized by a faster Ca
peak and the absence of a plateau, thereby differing kinetically
from the non-oscillatory response observed in the 57% of cells exposed
to PDGF-BB. This discrepancy might be due to a further signal
transduction mechanism, such as the IP
pathway, which also
participated in mobilizing Ca
during the
non-oscillatory response induced by PDGF. It has also been shown that
SPP can be produced by a sphingosine kinase localized at the level of
the endoplasmic reticulum(33) . This might allow for more
effective Ca
mobilizing actions of SPP produced by
PDGF receptor activation in comparison to that shown by exogenously
added SPP.
On the other hand, the oscillatory responses caused by perfusion with exogenous sphingosine exactly mimicked those induced by PDGF-BB. The shorter delays that preceded both responses, in comparison to the analogous responses elicited by PDGF-BB, can be easily explained by the fact that in these experiments all the steps involving binding of PDGF-BB to its receptor, receptor activation, and sphingomyelinase recruitment do not take place.
Previous studies have suggested that
SPP is the effective Ca releasing metabolite, while
sphingosine only represented an intermediate precursor for sphingosine
kinase activity (15, 20, 33) . However,
another study performed on dermal fibroblasts in the presence of DL-threo-dihydrosphingosine, showed that sphingosine
could indeed mobilize Ca
from intracellular
stores(18) . It should also be noted that no studies on the
role of sphingolipids in PDGF-stimulated Ca
signaling
have been previously performed using oligodendroglial cells. It seems
reasonable to hypothesize that in these cells the mechanism of
Ca
signaling following PDGF receptor stimulation
differs in some respects to that observed in 3T3 fibroblasts and
vascular smooth muscle cells.
Sphingosine kinase activity has been
shown to be stimulated by PDGF-BB in 3T3 fibroblasts, suggesting that
this growth factor may be able to stimulate sphingosine production from
sphingomyelin as well as to modulate the synthesis of SPP(19) .
Thus, the two separate Ca responses produced by
PDGF-BB in CEINGE cl3 cells could be due to different rates of
sphingosine kinase activity. In cells showing oscillatory
Ca
responses, sphingosine kinase might be resistant
to the stimulatory action of PDGF-BB, causing a progressive elevation
in sphingosine levels. This could explain the longer delay preceding
the oscillatory Ca
response due to the slow and
continuous accumulation of sphingosine until it reached an effective
Ca
-releasing threshold. In contrast, in
non-oscillatory cells, a sphingosine kinase sensitive to the
stimulatory action of PDGF-BB could more rapidly transform sphingosine
to SPP, increasing its concentration and lowering that of sphingosine.
It should be noted that in fibroblasts, PDGF-BB induces a maximal
increase in SPP levels after about 3 min (19) and that this
time course is in perfect accordance with the duration of delay
preceding the non-oscillatory response observed in the present study.
A point of particular interest is that most of the cells responding
with a non-oscillatory pattern could be persuaded to oscillate if the
intracellular levels of sphingosine were augmented (or those of SPP
were reduced). Experiments such as those in Fig. 8show that
inhibition of SPP formation produced an oscillatory response in cells
that had responded with a non-oscillatory pattern to a previous PDGF-BB
addition. Furthermore, it seems that a threshold level may exist in the
Ca-releasing concentration of sphingosine. This is
suggested by (i) the unresponsiveness of cells to the second PDGF-BB
addition in the absence of sphingosine kinase inhibition (when
sphingosine levels were increased by inhibition of its transformation
in SPP, a second PDGF-BB pulse elicited a Ca
response) and (ii) some cells unresponsive to an initial PDGF-BB
exposure showed a Ca
response to a second exposure
when sphingosine levels were increased by DL-threo-dihydrosphingosine incubation. This
indicates that sphingosine production induced by PDGF-BB in some cells
may not be sufficient to trigger a Ca
response.
In
conclusion, the results presented here show the involvement of
sphingosine and SPP in the Ca responses induced by
PDGF-BB in cells of the oligodendrocyte lineage. PDGF stimulation of
oligodendroglial cells has a mitogenic effect (21) but is also
of fundamental importance in development of oligodendrocyte progenitors (23, 34) . Thus, the different Ca
mobilization patterns evoked by sphingosine and SPP may reflect
the complex actions of PDGF in these cells. Several reports have
recently shown that increases in intracellular Ca
can
activate separate biochemical pathways, such as those regulating the
expression of immediate early genes(35, 36) . The data
reported here suggest that growth factors may cause distinct
cytoplasmic and/or nuclear events depending on the kinetics of
Ca
signaling produced.