(Received for publication, October 7, 1996, and in revised form, December 3, 1996)
From the Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637
Ca2+ signaling induced by
platelet-derived growth factor (PDGF) was investigated in the
oligodendroglial cell lines CG4 and CEINGE clone 3, using fura-2
microfluorimetry and video imaging. CEINGE cl3 cells, immortalized with
polyoma middle T antigen, were found to uniformly express the polyoma
middle T antigen protein as well as 2,3
-cyclic nucleotide
3
-phosphodiesterase, a specific marker for oligodendroglia. PDGF-BB
induced both oscillatory and non-oscillatory Ca2+ responses
in CEINGE cl3 cells as well as in CG4 cells, grown either as O-2A
progenitors or differentiated oligodendrocytes. However, in CG4 cells
the percentage of oscillatory Ca2+ responses was higher
than that observed in CEINGE cl3 cells. In contrast, oscillatory
Ca2+ responses were not observed in PC-12 cells transfected
with
-PDGF receptor (PDGFR) or in NIH 3T3 fibroblasts. CG4 cells
expressed only the
-PDGFR, whereas CEINGE cl3 cells expressed both
and
isoforms. When CEINGE cl3 cells were exposed to PDGF-AA,
which binds only to the
-PDGFR, the percentage of oscillatory
Ca2+ responses was higher than that observed after PDGF-BB
stimulation. We previously reported that block of the enzyme
sphingosine kinase, and a consequent increase in intracellular
sphingosine levels in CEINGE cl3 cells caused an increase in the
percentage of oscillatory Ca2+ responses induced by
PDGF-BB. However, in CG4 cells block of sphingosine kinase did not
increase the oscillatory Ca2+ response elicited by PDGF-BB,
although the addition of exogenous sphingosine induced an oscillatory
Ca2+ response in 77% of cells studied. We hypothesize that
the
-PDGFR is less effective than the
-PDGFR in stimulating the
activity of sphingosine kinase. The results also suggest that
- and
-PDGFRs may differently regulate sphingolipid metabolism.
PDGF1 plays a fundamental role in the
development of oligodendroglia. Oligodendrocyte progenitors, which
originate from the subventricular zones, undergo several PDGF-sensitive
steps while acquiring the ability to produce myelin during migration
and proliferation (1-4). PDGF is mitogenic and chemotactic for several
cell types, including fibroblasts, smooth muscle, and glial cells
(5-7). Three PDGF isoforms have been identified, being homo- or
heterodimers of related A and B polypeptide chains and named AA, -AB,
and -BB (8). Two separate isoforms of the PDGFR, named and
,
have also been identified (for review see Ref. 8). These receptors consist of transmembrane tyrosine kinases that dimerize upon binding to
the growth factor (9). This is a prerequisite for the reciprocal phosphorylation of the receptors on different tyrosine residues localized in the intracellular portion of the molecule (10, 11). This
process leads to the formation of docking sites for molecules with
catalytic activity such as PLC-
, phosphatidylinositol 3-kinase, and
Ras-GAP or molecules that function as adaptors for other substrates
like Grb2, Nck, and Shc (for review see Ref. 12). It has been shown
that the
-PDGFR binds both PDGF-AA and PDGF-BB with comparable
affinity, whereas the
-PDGFR binds PDGF-BB exclusively (13, 14);
PDGFRs also differ in their functional interaction with other
molecules, like PLC-
1 (15) and in the induction of
several phenomena like chemotaxis, membrane edge ruffling, and
mitogenesis, depending on the cell type studied (for review see Ref.
8).
It has been previously shown that oligodendroglial cells only express
the isoform of PDGFR, which is down-regulated when O-2A glial
progenitors differentiate into oligodendrocytes (16-18).
An increase in [Ca2+]i is one of the first events that occurs following the stimulation of PDGFRs (19). The role of Ca2+ as second messenger in chemotaxis, cell proliferation, and immediate early gene expression has been well documented (20-23). For example, a clear correlation between changes in mRNA levels of a set of immediate early genes and the modulation of [Ca2+]i induced by glutamate receptor stimulation in oligodendrocyte precursors has been observed (24). Activation of different sets of genes following the stimulation of distinct intracellular Ca2+ signaling pathways has also been described in neurons (25). It is possible that not only the magnitude of the [Ca2+]i increase but also its kinetics could play a crucial role in this process. In fact, different Ca2+ signaling kinetics could activate separate biochemical pathways leading to diverse messages at the nuclear level. Considering the multiple events regulated by PDGF during oligodendroglial development, this possibility appears to be very interesting. However, few studies have investigated PDGF-induced Ca2+ signaling during oligodendroglial differentiation (26). We have previously shown that mT-transformed oligodendroglial CEINGE cl3 cells responded to PDGF-BB exposure with two kinetically distinct types of Ca2+ signals. The relative intracellular levels of the two sphingolipids, sphingosine and sphingosine 1-phosphate (SPP), which are modulated by the activity of the enzyme sphingosine kinase, appeared to be responsible for the oscillatory and non-oscillatory Ca2+ responses, respectively (27).
In this paper, we show that CEINGE cl3 cells express both - and
-PDGFR. The Ca2+ response induced by PDGF and by
exogenous sphingosine and SPP in these cells was compared with that
observed in the oligodendroglial cell line CG4 (28), in PC-12 cells
transfected with the
-PDGFR (29), and in NIH 3T3 fibroblasts. This
was done in order to ascertain whether the patterns of Ca2+
signaling observed in CEINGE cl3 cells are a peculiarity of this clone
or a particular feature of the oligodendroglial lineage. Furthermore,
the consequences of blocking sphingosine kinase on Ca2+
signaling caused by
- or
-PDGFR activation were investigated.
PDGF-BB, PDGF-AA, and Tween 20 were from
Calbiochem-Novabiochem (San Diego, CA). ATP, sphingosine,
DL-threo-dihydrosphingosine, and neomycin
sulfate were from Sigma. Neomycin chloride was used to
avoid possible nonspecific effect of high sulfate concentrations and
was prepared, as described (30), by dissolving neomycin sulfate at 1 M and precipitating the sulfate with 3 volumes of 1 M BaCl2. The supernatant was neutralized to pH
7.4 with 1 M HCl. SPP was from Biomol (Plymouth Meeting,
PA). Fura-2/acetoxymethyl ester (fura-2/AM) was from Molecular Probes
(Eugene, OR). The sources of primary antibodies were as follows:
anti-mT mouse monoclonal antibody (PAb 750) was from Dr. Steve
Dilworth; anti-CNP and anti-GalC mouse monoclonal antibodies were from
Boehringer Mannheim; anti-PDGFR rabbit polyclonal antibody was from
Upstate Biotechnology (Lake Placid, NY); anti-
PDGFR mouse monoclonal
antibody was from Transduction Laboratories (Lexington, KY).
CEINGE clone 3 and NIH 3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% fetal calf serum (Life Technologies, Inc.) and 50 µg/ml gentamycin on uncoated plastic Petri dishes (Falcon).
PC-12 pheochromocytoma cells transfected with the -PDGFR (wild type)
and parental PC-12 cells were grown in DMEM containing 5% newborn calf
serum and 5% horse serum (Life Technologies, Inc.) on
poly-L-lysine-coated (100 µg/ml) plastic Petri dishes
(29).
CG4 cells as O-2A progenitors were grown in DMEM containing 30% of a medium conditioned by the neuroblastoma cell line B-104, biotin, insulin, and a mixture of hormones and growth factors (N1), as described previously (31). To differentiate CG4 to oligodendrocytes, cells were grown in DMEM containing biotin, N1, and a concentration of insulin three times higher than that used for O-2A progenitors (32). Cells were grown on poly-D-ornithine-coated (100 µg/ml) plastic Petri dishes. Fetal calf serum (1%) was eventually added to the growth medium after the 3rd day of differentiation. CG4/oligodendrocytes were maintained in culture from 8 to 12 days, and cell differentiation was evaluated by microscopic observation and by immunofluorescence staining against galactocerebroside C (GalC).
All cells were maintained in a humidified incubator at 36.5 °C in a 10% CO2 atmosphere and were fed twice weekly.
ImmunofluorescenceCells were grown on 15-mm round glass coverslips coated with polylysine and fixed with 4% paraformaldehyde added to the growth medium for 10 min (2% final concentration). Cells were then treated with fresh paraformaldehyde (4%) for 20 min and after being washed were incubated for 1 h in phosphate-buffered saline (PBS) containing 10% normal goat serum (Jackson Immunoresearch, West Grove PA) and 0.05% Tween 20. Cells were then incubated for 1 h with the primary antibodies at the following dilutions: anti-mT 1:100; anti-CNP 10 µg/ml. A biotinylated secondary antibody was incubated for 1 h, followed by a 30-min incubation with streptavidin conjugated with indocarbocyanine (CY3) (Jackson Immunoresearch, West Grove PA). Nuclear counterstaining was obtained using Hoechst 33342 (Molecular Probes, Eugene, CA). Coverslips were mounted using an aqueous mounting solution (10% glycerol in PBS) containing 2.5% (w/v) of 1,4-diazabicyclo[2.2.2.]octane (Sigma) as antifading agent. Cell fluorescence was detected using an Axioskop microscope equipped for epifluorescence (Zeiss, Germany).
Western Blot AnalysisCells growing in 100-mm tissue
culture dishes were washed twice with cold PBS and then scraped from
the dish. After a brief centrifugation, the pellet was resuspended in a
lysis buffer of the following composition: 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM dithiothreitol, 1% Nonidet P-40,
2 µg/ml aprotinin, 20 µM leupeptin, 1 mM
[4-(2-aminoethyl)benzenesulfonylfluoride, HCl] (Calbiochem). The
protein concentration in the clarified lysate was determined by Bio-Rad
protein assay. Cell lysates containing equal amounts of protein were
resolved on a SDS-polyacrylamide gel electrophoresis according to the
method of Laemmli (33) using 5, 10, and 12.5% polyacrylamide gel for
PDGFRs, mT, and CNP, respectively. The separated proteins were
electroblotted to Hybond-enhanced chemiluminescence nitrocellulose
membrane (Amersham Corp.) or Hybond-PVDF (Amersham Corp.) for PDGFRs.
Membranes were then incubated for 1 h in PBS containing 5% milk
powder, 2% bovine serum albumin, and 0.1% Tween 20. This buffer was
also used for the incubation with primary antibodies at the following
dilutions: anti-mT 1:1000, anti-CNP 10 µg/ml, anti-PDGFR
(Transduction Lab) 1 µg/ml, anti-
PDGFR (Upstate) 1:2000. Results
with anti-PDGFRs were also compared with anti-
PDGFR (1890) and with
anti-
PDGFR (7649) rabbit polyclonal antibodies from Dr. Daniel F. Bowen-Pope, both used at 1:2000 dilution. After a wash with PBS
containing 0.1% Tween 20, a secondary antibody conjugated with
horseradish peroxidase (Promega) was used at 1:10.000 dilution. The
binding of the antibodies to the membrane was detected using the
enhanced chemiluminescence system (Amersham Corp.).
Cells were grown on 25-mm no. 1 glass coverslips coated with poly-L-lysine (100 µg/ml) and were loaded with 2 µM fura-2 acetoxymethyl ester (fura-2/AM) using a balanced salt solution (standard buffer) of the following composition (in mM): NaCl 157, KCl 5, MgSO4 0.4, MgCl2 0.5, KH2PO4 0.6, NaHCO3 3, HEPES 20, glucose 10, CaCl2 2, and bovine serum albumin 0.2% (osmolality 320-340 mosm/kg), pH adjusted to 7.4 with Tris 1 M. Cells were incubated with fura-2/AM for 25-45 min at room temperature to avoid probe compartmentalization and then incubated for an additional 10-15 min with standard buffer to allow the complete de-esterification of the fluorescent probe. The single cell video imaging of as many as 10-30 cells/field and the calibration of fluorescent signals were performed as described previously (27).
Statistical AnalysisData values expressed as mean ± S.E. Student's t test or Mann-Whitney non-parametric test were used, and statistical significance was defined as a p value of 0.01 or less.
The CEINGE cl3 cell line was obtained by infecting E14 rat brain cells with a murine leukemia virus carrying the gene encoding the polyoma mT antigen (34). Although CEINGE cl3 cells have been previously found to express a retroviral mRNA hybridizing to the polyoma mT gene (34), we decided to verify that the protein was effectively expressed.
An equal amount of protein (60 µg) from a total cell lysate of CEINGE
cl3 cells and NIH 3T3 fibroblasts (as a negative control) was loaded
onto a 10% polyacrylamide gel. As shown in Fig. 1, CEINGE cl3 cells expressed a protein with a molecular weight of approximately 55,000, whereas in NIH 3T3 fibroblasts the protein was
not detected. Immunofluorescent staining obtained using CEINGE cl3
cells shows that a significant amount of the protein was detectable in
every cell in the field (Fig. 1). No staining was obtained using NIH
3T3 fibroblasts (not shown).
The expression of the oligodendroglial marker GalC by CEINGE cl3 cells
has been previously reported (34). To further characterize the clone,
we investigated the expression of CNP as a specific marker, since this
enzyme is expressed early and at high levels during the development and
differentiation of oligodendrocytes and appears to be involved in the
biogenesis of myelin (35, 36). For this purpose, an equal amount of
protein (40 µg) from the total cell lysate of CEINGE cl3 cells, NIH
3T3 fibroblasts (as a negative control), and CG4 cells differentiated
to oligodendrocytes (as a positive control) was loaded onto a 12.5%
polyacrylamide gel. Fig. 2 shows that both CEINGE cl3
cells and CG4/oligodendrocytes expressed a protein with a similar
molecular weight of approximately 45,000-50,000. The amount of protein
expressed appeared to be significantly higher in CG4/oligodendrocytes
when compared with CEINGE cl3 cells, whereas NIH 3T3 fibroblasts did
not express this protein at all. These results were confirmed by
immunofluorescence staining, showing that CEINGE cl3 cells stained
positively for CNP (Fig. 2). Similar positive staining was obtained
using CG4/oligodendrocytes, whereas NIH 3T3 fibroblasts appeared
completely negative (not shown).
Single Cell Video Imaging of the Ca2+ Response Induced by PDGF-BB in CEINGE Clone 3 and CG4 Cells
As described
previously (27), exposure of CEINGE cl3 cells to 10 ng/ml PDGF-BB for 5 min was able to induce Ca2+ responses with two different
types of kinetics. The first response was characterized by an early
single increase in [Ca2+]i, which was frequently
followed by a plateau. In contrast, the pattern of the second
Ca2+ response was oscillatory, with several spikes of
different frequencies and magnitudes beginning significantly later than
the non-oscillatory response (Fig. 3, A and
B). Similarly, the growth factor caused [Ca2+]i increases, both in CG4/O-2A progenitors
and CG4/oligodendrocytes, with a similar dual pattern (Fig. 3,
C and D). The number of cells responsive to the agonist
was significantly lower in CG4 cells in comparison to CEINGE cl3 cells
(see Table I).
|
Finally, the percentage of oscillatory cells in CG4/O-2A progenitors and CG4/oligodendrocytes was higher than in CEINGE cl3 cells (Table I), and the delays preceding the onset of the two distinct Ca2+ responses were significantly different, as observed in CEINGE cl3 cells.
Single Cell Video Imaging of the Ca2+ Response Induced by PDGF-BB in NIH 3T3 Fibroblasts andWe investigated whether Ca2+ responses with
different kinetics were also induced by PDGF-BB in other cell types
such as NIH 3T3 fibroblasts and PC-12 cells transfected with the isoform of PDGFR (29). 3T3 fibroblasts responded with a single
Ca2+ rise frequently followed by a plateau
(Fig. 4B), as previously reported by others
(37). Comparable responses were observed in PC-12 cells, although the
magnitude of the [Ca2+]i increase was smaller
than in 3T3 fibroblasts (Fig. 4A). However, the oscillatory
pattern observed in CEINGE cl3 and CG4 cells was never detected in NIH
3T3 or PC-12 cells.
Expression of Both
We investigated the expression of
and
receptors for PDGF in CG4 and CEINGE cl3 oligodendroglial
cells. Positive control NIH 3T3 fibroblasts that express both PDGFR
isoforms (38) and negative control parental PC-12 cells which have no
detectable expression of PDGFRs (39) were also included in the
immunoblots. Fig. 5 shows that the
isoform of PDGFR
was detected in both CG4/O-2A progenitors and CG4/oligodendrocytes as
well as in CEINGE cl3 cells. The latter cells showed an amount of
protein that was comparable to that found in 3T3 fibroblasts, whereas
CG4 cells expressed an even higher level of the protein regardless of
their state of differentiation. In all cell types two separate proteins with an apparent molecular weight of about 180,000 and 160,000 were
detected. It is likely that a mature glycosylated form and a partially
modified form of the receptor are expressed as described previously in
primary oligodendroglia (16). However, neither protein was detected in
PC-12 cells. Although the
isoform of PDGFR was not detected in CG4
cells, in agreement with previous studies in primary oligodendroglia
(16), CEINGE cl3 cells clearly expressed the
isoform of the PDGFR.
The protein which has an apparent molecular weight of 190,000 was also
present in 3T3 fibroblasts, whereas it was undetectable in PC-12 cells
(Fig. 5).
Effect of Neomycin on PDGF-BB-induced Ca2+ Response in CG4 and CEINGE cl3 Cells
A pharmacological approach was adopted
for studying the consequences of the difference in PDGFR expression in
CG4 and CEINGE cl3 cells. The aminoglycoside neomycin is able to
inhibit the binding of PDGF-BB to the receptor, whereas binding to
the
receptor is not affected by this drug (30). When
CG4/oligodendrocytes were preincubated with 5 mM neomycin
for 15 min before starting perfusion with PDGF-BB (10 ng/ml), the
Ca2+ response was totally abolished in all the 46 cells
analyzed (Fig. 6A). However, subsequent
exposure of the same cells to 100 µM ATP elicited a
[Ca2+]i increase, usually a rapid peak followed
by a plateau, in more than 90% of cells studied. This demonstrates
that neomycin neither inhibited PLC
nor exerted any
toxic effect. However, preincubation of CEINGE cl3 cells with neomycin
did not inhibit the Ca2+ signaling induced by PDGF-BB
stimulation (Fig. 6B), and the usual pattern of both
oscillatory and non-oscillatory Ca2+ response was
observed.
Exposure of CEINGE cl3 Cells to PDGF-AA
When CEINGE cl3 cells
were perfused for 5 min with 20 ng/ml PDGF-AA, which is able to bind
only the isoform of PDGFR, 149 out of 247 cells studied (60%)
showed a [Ca2+]i increase (data not shown). In
69% of the responsive cells, the kinetics were oscillatory and
comparable with those induced by PDGF-BB exposure but preceded by a
significantly shorter delay (146 ± 7 versus 278 ± 9 s). Similarly, a shorter delay was also detected in the
remaining 31% of the cells responding in a non-oscillatory fashion,
when compared with the non-oscillatory response induced by PDGF-BB in
CEINGE cl3 cells (93 ± 7 versus 200 ± 7 s).
CEINGE cl3 cells were perfused with either 20 ng/ml PDGF-AA or
10 ng/ml PDGF-BB for 5 min during a 15-min experiment. After a 20-min
interval, they were restimulated with the same or the other PDGF
isoform. A homologous second stimulation with PDGF-AA was unable to
induce a Ca2+ response in all the analyzed cells which were
responsive to the first pulse (Fig. 7). These results
are similar to those we previously observed after a homologous second
stimulation with PDGF-BB (27). PDGF-AA also failed to induce a
Ca2+ response when perfused after PDGF-BB (Fig. 7).
In contrast, 61% of the cells previously exposed to PDGF-AA were able to respond with a [Ca2+]i increase to a subsequent stimulation with PDGF-BB.
Effect of DL-threo-Dihydrosphingosine on PDGF-BB-induced Ca2+ SignalingIncubation for 15 min
with the inhibitor of the sphingosine kinase,
DL-threo-dihydrosphingosine (10 µM) (40), did not increase the percentage of oscillatory
Ca2+ responses induced by 10 ng/ml PDGF-BB in CG4/O-2A
progenitors and CG4/oligodendrocytes, in contrast to results previously
observed in CEINGE cl3 (27). In CEINGE cl3 cells incubated with
DL-threo-dihydrosphingosine, the percentage of
oscillatory responses evoked by PDGF-BB was 87 ± 4 (70% of
responsive cells in 62 cells analyzed), and in CG4/O-2A progenitors and
CG4/oligodendrocytes oscillatory kinetics were observed in 49 ± 9 and 33 ± 8% of cells, respectively (Fig. 8; see
also Table I). The percentage of responsive CG4/O-2A progenitors was
51% in 39 cells analyzed with a delay of 268 ± 35 s, while the percentage of responsive CG4/oligodendrocytes was 23% in 31 cells
analyzed with a delay of 255 ± 48 s.
DL-threo-Dihydrosphingosine was also ineffective
in inducing an oscillatory Ca2+ response to PDGF-BB in NIH
3T3 and -PDGFR-transfected PC-12 cells. Despite a 15-min incubation
with the sphingosine kinase inhibitor, all the cells analyzed responded
uniformly in a non-oscillatory fashion to PDGF-BB exposure (data not
shown).
Effect of Sphingosine and Sphingosine 1-Phosphate on [Ca2+]i in CG4/Oligodendrocytes, NIH 3T3 Fibroblasts, and
Exogenous
sphingosine was perfused onto the cells after a 15-min incubation with
10 µM DL-threo-dihydrosphingosine
to block its conversion to SPP. A 5-min perfusion with 10 µM sphingosine elicited an oscillatory Ca2+
response in the majority of the CG4/oligodendrocytes studied (Table II). NIH 3T3 fibroblasts and
-PDGFR-transfected PC-12 cells responded with a single
[Ca2+]i increase, never showing any oscillatory
Ca2+ signaling (data not shown).
|
Exposure of cells for 5 min to 1 µM SPP produced a
non-oscillatory Ca2+ response in 82% of the
CG4/oligodendrocytes (Table II) and in all the responsive NIH 3T3
fibroblasts and -PDGFR-transfected PC-12 cells analyzed (data not
shown).
In these experiments we studied the Ca2+ responses
elicited by stimulation of PDGFRs in the CG4 cell line and compared
them with analogous effects induced in CEINGE cl3 cells and in cells of
non-oligodendroglial origin such as NIH 3T3 fibroblasts and -PDGFR-transfected PC-12 cells. Our results clearly demonstrate the
oligodendroglial origin of CEINGE cl3 cells and also suggest that the
oscillatory Ca2+ response induced by PDGF is a peculiarity
of oligodendroglia. The results also suggest that
-PDGFR may be less
effective than
-PDGFR in stimulating the enzyme sphingosine
kinase.
First we characterized the CEINGE clone 3 cells. Western blot analysis for the mT antigen clearly showed expression of the protein in the CEINGE cl3 cell line. Uniform detection of the protein in all cells was evident using immunofluorescence (Fig. 1). Thus the differences in PDGF-BB-induced Ca2+ responses previously described (27) are not a consequence of unequal protein levels in the cell population. It has been previously shown that CEINGE cl3 cells stain positively for GalC antigen, a specific immunological marker for oligodendrocytes (34). As we now also show the CEINGE cl3 cell line also expresses the specific oligodendroglial marker CNP. Two isoforms of this enzyme have been identified, with molecular weights of 46,000 and 48,000, both being derived from a single gene (41). In O-2A precursors only the mRNA encoding the larger isoform has been found, whereas in differentiated oligodendrocytes both CNP mRNAs have been described (42). Western blot analysis showed that a single protein band was identified in CEINGE cl3 cells, showing a molecular weight of approximately 45,000-50,000 and corresponding to a similar band identified in CG4/oligodendrocytes (Fig. 2).
CEINGE cl3 cells responded to PDGF-BB exposure with Ca2+
signals characterized by oscillatory or non-oscillatory kinetics. We have shown that this may be due to different intracellular levels of
the two sphingolipids, sphingosine and SPP (27). It was interesting to
ascertain whether this phenomenon was a general feature of cells of the
oligodendroglial lineage or was just a peculiarity of CEINGE cl3 cells,
related for example to mT expression. This possibility was suggested by
the fact that mT antigen is able to bind intracellular substrates like
phosphatidylinositol 3-kinase and PLC-1 (43, 44) and that its
expression in NIH 3T3 fibroblasts confers an enhanced responsiveness to
growth factors (45). However, the glial cell line CG4 also responded to
PDGF-BB exposure with dual oscillatory and non-oscillatory kinetics, as
observed in CEINGE cl3 cells (Fig. 3). In contrast an oscillatory
Ca2+ response was never observed when NIH 3T3 fibroblasts,
as well as PC-12 cells transfected with
-PDGFR, were exposed to
PDGF-BB or to exogenous sphingosine. These data indicate that the dual Ca2+ signaling induced by PDGFR stimulation and the
modulatory action of sphingosine and SPP are indeed properties of cells
of the oligodendroglial lineage. The most likely explanation for this
is that in oligodendroglial cells, Ca2+ mobilization
elicited by PDGFR stimulation is much more sensitive to intracellular
levels of sphingosine and SPP than in other cell types. On the other
hand, in the light of our results, the possibility of a modulatory
action operated by sphingolipids on other pathways, such as
PLC
-inositol trisphosphate, cannot be ruled out.
It has been reported that cells of the oligodendroglial lineage only
express the isoform of PDGFR (17, 46) and that oligodendrocytes
show a lower level of mRNA for this receptor and protein expression
compared with O-2A progenitors (16). In agreement with these
observations, we found that CG4 cells grown both as O-2A progenitors
and oligodendrocytes expressed
-PDGFR. However, no decrease in
-PDGFR protein level was observed in CG4/oligodendrocytes, as also
suggested by the similar percentages of CG4/O-2A progenitors and
CG4/oligodendrocytes that responded with a
[Ca2+]i increase to PDGF-BB (Table I). This could
be explained by a peculiarity of this cell line in which
-PDGFR
expression has never been previously investigated or by the fact that
after the 3rd day of differentiation to oligodendrocytes 1% of fetal calf serum was added to the culture medium to increase the cell survival (28, 47). The presence of basic fibroblast growth factor in
the serum may have contributed to an up-regulation of
-PDGFR
expression, as previously reported in primary oligodendroglia (16).
Interestingly, CEINGE cl3 cells also expressed the
isoform of
PDGFR. This finding was confirmed by experiments performed using
neomycin as an inhibitor of PDGF-BB binding to the
-PDGFR (30).
Thus, neomycin completely blocked PDGF-BB-induced Ca2+
signaling in CG4 cells, whereas it was ineffective in CEINGE cl3 cells
(Fig. 6), suggesting that the Ca2+ response observed in
these latter conditions was due solely to
-PDGFR activation. In
light of these results, we exposed CEINGE cl3 cells to PDGF-AA, in
order to selectively stimulate the
-PDGFR and verify whether this
could evoke a Ca2+ response different from that observed
when both
- and
-PDGFRs were stimulated by PDGF-BB. In these
experiments we also observed both oscillatory and non-oscillatory
Ca2+ responses, although the percentage of oscillatory
cells was higher and the delays preceding both Ca2+
responses were shorter than those observed using PDGF-BB.
Interestingly, a second homologous PDGF stimulation of the same CEINGE
cl3 cells was ineffective in increasing the
[Ca2+]i, regardless of the PDGF isoform used.
This suggests desensitization of the transductional mechanisms linking
PDGFRs with the Ca2+ signaling machinery. On the other
hand, PDGF-BB exposure following a previous PDGF-AA pulse induced a
Ca2+ response in the majority of the cells (Fig. 7). These
data can be explained by invoking a selective desensitization of
-PDGFR by PDGF-AA and further supports the presence of
-PDGFRs in
CEINGE cl3 cells.
When PDGF-BB exposure of CG4 cells was preceded by incubation with
DL-threo-dihydrosphingosine, no increase in the
percentage of oscillatory cells was observed in contrast to CEINGE cl3
cells (Fig. 8). Nevertheless, when exogenous sphingosine was perfused alone an oscillatory Ca2+ response was detected in most of
the cells, whereas exposure to SPP mostly produced non-oscillatory
responses (Table II). These data suggest that although sphingosine and
SPP are able to elicit two distinct types of Ca2+ response,
as observed in CEINGE cl3 cells (27), block of sphingosine kinase did
not modulate the Ca2+ signaling following the stimulation
of -PDGFR. This result is difficult to explain in the light of
information available on the mechanism of coupling of PDGFRs to the
sphingolipid metabolic pathway. Although a clear correlation between
PDGFR stimulation and increases in the intracellular levels of
ceramide, sphingosine, and SPP have been reported (48-50), the
biochemical mechanisms underlying these phenomena are still unclear.
Furthermore, although stimulation of sphingosine kinase activity has
been observed during
-PDGFR stimulation (51), no data are available
regarding the
isoform of this receptor. A possible explanation of
our observations could involve lack of stimulation of sphingosine
kinase activity by
-PDGFR. This could result in higher intracellular
levels of sphingosine and account for a higher percentage of
oscillatory cells (see Fig. 9). It is interesting to
note that after exposure to PDGF-BB of both CG4/O-2A progenitors and
CG4/oligodendrocytes, which express only the
-PDGFR, the percentage
of oscillatory cells was higher than that observed when CEINGE cl3
cells were stimulated with the same PDGF isoform. This could be because
the
-PDGFR in CEINGE cl3 stimulates sphingosine kinase to produce more SPP, thus accounting for the higher number of non-oscillatory responses. This hypothesis is supported by the observation that when
CEINGE cl3 cells were stimulated with PDGF-AA instead of PDGF-BB, a
higher percentage of oscillatory cells were detected. However, it seems
reasonable to hypothesize that despite the absence of stimulation of
sphingosine kinase activity, some SPP is still produced, accounting for
the non-oscillatory Ca2+ responses observed during
-PDGFR activation.
The possibility that different PDGF-induced Ca2+ responses in oligodendroglia are coupled to distinct cellular phenomena such as mitogenesis, chemotaxis, and differentiation seems very attractive. Future studies will be essential in order to correlate the kinetics of Ca2+ signaling induced by PDGFRs to particular cytoplasmic and nuclear events.
We thank Dr. Olimpia Meucci Department of
Pharmacological and Physiological Sciences, University of Chicago for
valuable suggestions; Dr. Vittorio Gallo and Dr. James T. Russell,
Laboratory of Cellular and Molecular Neurophysiology, NICHD,
Bethesda, for critically reading the manuscript; Dr. Steve
Dilworth, Department of Chemical Pathology, Royal Postgraduate Medical
School, Hammersmith Hospital, London, for the anti-middle T mouse
monoclonal antibody (PAb 750); Dr. Daniel F. Bowen-Pope, Department of
Medicine, University of Washington, Seattle, and Dr. H. Clive
Palfrey, Department of Pharmacological and Physiological Sciences,
University of Chicago, for the rabbit anti- (1890) and anti-
(7649) PDGFR; Dr. Lynn E. Heasley, University of Colorado Health
Science Center, Denver, CO, and Dr. H. Clive Palfrey, for PC-12 cells
transfected with
-PDGFR; Jeanpyo Lee for culturing PC-12 cells and
Umar Shakur for the technical assistance with the other cell
cultures.