(Received for publication, April 27, 1995; and in revised form, June 28, 1995)
From the
We examined neurotrophin-induced sphingomyelin hydrolysis in
cells expressing solely the low affinity neurotrophin receptor,
p75, and in PC12 cells that co-express
p75
and Trk receptors. Nerve growth factor
(NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and
NT-5 stimulated sphingomyelin hydrolysis with similar kinetics in
p75
-NIH-3T3 cells. Although brain-derived
neurotrophic factor (10 ng/ml) was slightly more potent than NGF at
inducing sphingomyelin hydrolysis, NT-3 and NT-5 induced significant
hydrolysis (30-35%) at 0.1 to 1 ng/ml in
p75
-NIH-3T3 cells. NT-3 did not induce
sphingomyelin hydrolysis in Trk C-NIH-3T3 cells nor in cells expressing
a mutated p75
containing a 57-amino acid
cytoplasmic deletion, thus demonstrating the role of p75
in this signal transduction pathway. In
p75
-NIH-3T3 cells, neurotrophin-induced
sphingomyelin hydrolysis 1) localized to an internal pool of
sphingomyelin, 2) was not a consequence of receptor internalization,
and 3) showed no specificity with respect to the molecular species of
sphingomyelin hydrolyzed. In contrast to cells expressing solely
p75
, NGF (100 ng/ml) did not induce
sphingomyelin hydrolysis in PC12 cells. Interestingly, NT-3 (10 ng/ml)
induced the same extent of sphingomyelin hydrolysis in PC12 cells as
was apparent in p75
-NIH-3T3 cells. However, in
the presence of NGF, NT-3 was unable to induce sphingomyelin
hydrolysis, raising the possibility that Trk was modulating
p75
-dependent sphingomyelin hydrolysis.
Inhibition of Trk tyrosine kinase activity with 200 nM K252a
enabled both NGF and NT-3 in the presence of NGF to induce
sphingomyelin hydrolysis. These data support that p75
serves as a common signaling receptor for neurotrophins
through induction of sphingomyelin hydrolysis and that cross-talk
pathways exist between Trk and p75
-dependent
signaling pathways.
The neurotrophins are a family of growth factors critical for
the survival and development of specific populations of neurons within
the central and peripheral nervous systems(1) . Nerve growth
factor (NGF), ()the prototypic neurotrophin, is the best
characterized member of this family of growth factors that also
includes brain-derived neurotrophic factor (BDNF), and neurotrophin-3
(NT-3), -4, and -5(2, 3, 4) .
Neurotrophins interact with two classes of cellular receptors
possessing both high and low affinity binding
characteristics(5, 6, 7) . Recent studies
suggest that formation of high affinity binding sites requires the
expression of a member of the trk gene family (8, 9) . The trk gene family encodes several
receptor-linked tyrosine kinases, Trk A, Trk B, and Trk C, which
preferentially interact with NGF, BDNF/NT-4/NT-5, and NT-3,
respectively(10, 11, 12, 13, 14, 15, 16) .
Similar to other receptor-linked tyrosine
kinases(17, 18) , neurotrophins induce Trk receptor
dimerization and autophosphorylation (19) . Activation of Trk
tyrosine kinase subsequently initiates multiple phosphorylation events
regulating the activity of the MAP kinase cascade(20) ,
phospholipase C(21) , and
phosphatidylinositol-3-kinase(22) . Activation of these
downstream signaling components by Trk is critical for NGF-induced
neurite outgrowth in PC12 cells(23) .
In contrast to the
rather restricted binding of neurotrophins to their respective Trk
receptor, all of these molecules bind with lower affinity to a
transmembrane protein known as the low affinity neurotrophin receptor,
p75(24, 25, 26) .
p75
lacks kinase activity and possesses no
structural motifs recognized to couple to established signal
transduction pathways(27, 28, 29) . Although
p75
possesses a consensus sequence for the
potential binding of G-proteins(30) , there is little evidence
supporting a p75
-dependent G-protein-mediated
signal.
Recent evidence suggests that p75 may
also play a role in regulating cellular responses to neurotrophins. For
example, p75
has been demonstrated to
participate in the formation of high affinity neurotrophin binding
sites(21, 31) . Furthermore, specific domains in
p75
may be involved in modulating the effects of
Trk tyrosine kinase activity on cell growth and
differentiation(32) . Moreover, we have recently demonstrated
that p75
may signal through a Trk-independent
pathway. In this respect, NGF was found to activate a novel lipid
second messenger pathway, known as the sphingomyelin cycle,
specifically through p75
(33) .
NGF-stimulated sphingomyelin metabolism resulted in the production of
the bioactive sphingolipid metabolite ceramide. Ceramide has been
implicated as a mediator in antimitogenic pathways leading to cell
growth inhibition, cell differentiation, and apoptosis (34) .
Indeed, in rat T9 glioma cells, exogenous ceramide mimicked the effect
of NGF on cell growth inhibition and differentiation(33) .
Moreover, T9 glioma cells express p75
but not
Trk A, suggesting that activation of the sphingomyelin cycle may
mediate some of the growth suppressing and differentiative effects of
neurotrophins via p75
(33) .
The
ability of all neurotrophins to interact with p75suggested that neurotrophin-induced sphingomyelin hydrolysis may
be a general signaling mechanism inherent to this family of growth
factors. Therefore, in this study we examined the ability of other
neurotrophins to induce sphingomyelin hydrolysis in fibroblast cells
expressing wild-type or mutated forms of p75
. In
addition, we examined the effects of neurotrophins on sphingomyelin
hydrolysis in cells that co-express both p75
and
Trk A. Co-expression of Trk A with p75
abolished
p75
-dependent sphingomyelin hydrolysis.
Interestingly, activation of other receptor-linked tyrosine kinases had
no effect on p75
-dependent sphingomyelin
hydrolysis. Taken together, our results indicate that the neurotrophins
induce sphingomyelin hydrolysis through p75
and
that co-expression of Trk and p75
modulate
neurotrophin-induced sphingomyelin hydrolysis.
Figure 1:
Neurotrophins induce sphingomyelin
hydrolysis in p75-NIH-3T3 cells. A,
P75
-NIH-3T3 cells were labeled for 3 days with
[
H]choline chloride, and the cells were placed in
serum-free medium containing 50 mM HEPES, pH 7.4, for 4 h.
Cells were treated with the indicated concentrations of neurotrophins
for 12 min in a 37 °C water bath. Lipids were extracted, and
sphingomyelin levels were assessed as described under
``Experimental Procedures.'' Sphingomyelin levels were
normalized to nmol of phospholipid phosphate, and the results expressed
as the percent of control levels of sphingomyelin. Data are mean
± S.E. and are derived from (n = 9) NT-3; (n = 6) BDNF and NGF; and (n = 3) NT-5. Data
were analyzed by analysis of variance with comparison of multiple
means. Asterisks indicates p < 0.05 from control. Arrows indicate p < 0.05 from BDNF at same
concentration. B, time course of NT-3 and BDNF-induced
sphingomyelin hydrolysis. Cells were labeled as above and treated with
10 ng/ml NT-3 or 100 ng/ml BDNF for the indicated times in a 37 °C
water bath. Sphingomyelin levels were normalized to nmol of
phospholipid phosphate, and the results were expressed as the percent
of time-matched control levels of sphingomyelin. Results presented are
the mean ± S.E. of data from two experiments (n = 6).
To ensure
that the percent decrease in radiolabeled sphingomyelin corresponded to
an actual mass decrease, sphingomyelin mass levels were determined
following neurotrophin treatment. Total sphingomyelin mass in
p75-NIH-3T3 cells was 40.2 ± 3 pmol/nmol of
phospholipid phosphate. Thus, based upon the percent of sphingomyelin
hydrolyzed in the labeling studies (30-35%), NT-3 decreased
sphingomyelin levels by about 12-14 pmol/nmol of phospholipid
phosphate. Correspondingly, sphingomyelin mass decreased by 16.5
pmol/nmol of phospholipid phosphate following treatment of
p75
-NIH-3T3 cells with 10 ng/ml NT-3 for 15 min.
Collectively, these data demonstrate that p75
mediates
the effects of neurotrophins on sphingomyelin hydrolysis.
Figure 2:
Trk C is not coupled to activation of
sphingomyelin hydrolysis. A, Trk C-NIH-3T3 or wild-type
NIH-3T3 cells were labeled for 3 days with
[H]choline chloride, and the cells were placed in
serum-free medium containing 50 mM HEPES, pH 7.4, for 4 h.
Cells were treated with 10 ng/ml NT-3 or PBS for the indicated times in
a 37 °C water bath. Lipids were extracted and sphingomyelin levels
assessed as described under ``Experimental Procedures.''
Sphingomyelin levels were normalized to nmol of phospholipid phosphate,
and the results were expressed as the percent of time matched control
levels of sphingomyelin. Results presented are mean ± S.E. of
data from two experiments (n = 6). B, NT-3
induces autophosphorylation of Trk C. Sub-confluent Trk C-NIH-3T3 cells
were placed in serum-free medium containing 50 mM HEPES, pH
7.4 for 4 h prior to treatment with 0-50 ng/ml NT-3 for 10 min in
a 37 °C water bath. Cells were lysed, and 0.5 mg of protein was
immunoprecipitated with anti-pan Trk antibody 203 for 2 h at 4 °C.
Immune complexes were formed by the addition of protein A-Sepharose,
and proteins were resolved by SDS-polyacrylamide gel electrophoresis.
Tyrosine phosphorylation of Trk C (M
=
145,000) was determined by immunoblotting as described under
``Experimental Procedures.'' N.S. equals nonspecific
binding.
To confirm that the lack of NT-3-induced sphingomyelin hydrolysis was not due to a functional defect in activation of Trk C receptors, the effect of NT-3 on Trk C autophosphorylation was examined. As determined by anti-phosphotyrosine immunoblotting, incubation of Trk C-NIH-3T3 cells for 15 min with 0.1-50 ng/ml of NT-3 increased Trk C autophosphorylation (Fig. 2B). Significant autophosphorylation of Trk C was seen at 10 ng/ml NT-3; a concentration that was ineffective at stimulating sphingomyelin hydrolysis in these cells. Taken together, these results support that Trk C does not couple to sphingomyelin hydrolysis and that the lack of NT-3-induced sphingomyelin hydrolysis in these cells was not due to a functional defect in Trk C.
Figure 3:
NT-3 does not induce sphingomyelin in
cells expressing a mutated form of p75. PS cells were
labeled for 3 days with [
H]choline chloride, and
the cells were placed in serum-free medium containing 50 mM HEPES, pH 7.4 for 4 h. Cells were treated with the indicated
concentrations of NT-3 for 12 min in a 37 °C water bath. Lipids
were extracted, and sphingomyelin levels were assessed as described
under ``Experimental Procedures.'' Sphingomyelin levels were
normalized to nmol of phospholipid phosphate, and the results were
expressed as the percent of control levels of sphingomyelin. Results
are mean ± S.E. of data from two experiments (n = 6).
Treatment of p75-NIH-3T3 cells with NT-3 alone induced
a 26% decrease in sphingomyelin, defining the NT-3 sensitive pool (Fig. 4). Pretreatment of cells with bacterial sphingomyelinase
decreased cellular sphingomyelin levels 52%, defining an outer leaflet
bacterial sphingomyelinase-sensitive pool. If NT-3 is hydrolyzing a
distinct pool of sphingomyelin, then treatment with bacterial
sphingomyelinase plus NT-3 should decrease sphingomyelin levels by
about 75-80% (52 + 26%). Indeed, treatment of
p75
-NIH-3T3 cells with both bacterial sphingomyelinase
and NT-3 decreased sphingomyelin levels by 70%; indicating that the
effects of NT-3 on sphingomyelin are additive to the effects of
bacterial sphingomyelinase. To determine the specificity of this
response, p75
-NIH-3T3 cells were also treated with EGF
± bacterial sphingomyelinase pretreatment. Similar to results
obtained in rat T9 glioma cells(33) , EGF did not induce
sphingomyelin hydrolysis. Subsequent to bacterial sphingomyelinase
treatment, EGF also had no effect on the remaining pool of
sphingomyelin (Fig. 4). Taken together, these results
demonstrate that NT-3 specifically decreases a distinct pool of
sphingomyelin that is not accessible to bacterial sphingomyelinase and
that may reside on the internal leaflet of the plasma
membrane(37) .
Figure 4:
NT-3 hydrolyzes a bacterial
sphingomyelinase-resistant pool of sphingomyelin.
p75-NIH-3T3 cells were labeled with
[
H]choline chloride for 3 days and placed in
serum-free medium containing 50 mM HEPES, pH 7.4, for 4 h.
Cells were then treated with 100 milliunits/ml bacterial
sphingomyelinase or vehicle for 30 min at 37 °C. The cells were
then transferred to a 37 °C water bath and treated with PBS, 10
ng/ml NT-3, or 10 ng/ml EGF for 12 min. Lipids were extracted, and
sphingomyelin was quantitated as described under ``Experimental
Procedures.'' Sphingomyelin levels were normalized to total
phosphatidylcholine counts/min, and the results were expressed as the
percent of control. Results are the mean ± S.E. (n = 3) of data from one representative experiment performed
twice.
Figure 5:
NT-3
does not hydrolyze a specific molecular species of sphingomyelin.
p75-NIH-3T3 cells (A) or PC12 cells (B) were labeled with [
H]choline
chloride for 3 days and placed in serum-free medium containing 50
mM HEPES, pH 7.4, or DMEM containing 3% serum for 4 h,
respectively. Cells were then treated with PBS or 10 ng/ml NT-3 in a 37
°C water bath, and the lipids were extracted. Aliquots were used
for determination of total radioactivity, and the samples were matched
for total counts/min. Glycerophospholipids were removed by mild base
hydrolysis, and the base-resistant lipids were extracted. Aliquots of
the organic phase were quantitatively transferred and evaporated under
nitrogen, and the lipid residue was dissolved in 50 µl of
chloroform. The molecular species of sphingomyelin were analyzed by
RP-HPLC and eluted isocratically with methanol, 5 mM potassium
phosphate, pH 7.0 (98:2) at a flow rate of 1 ml/min. One-ml fractions
were collected, and the radioactivity was
quantitated.
Figure 6:
NGF and NT-3 have differential effects on
activation of sphingomyelin hydrolysis in PC12 cells. PC12 cells were
labeled with [H]choline chloride for 3 days and
placed in DMEM containing 3% serum for 4 h. The cells were transferred
to a 37 °C water bath and treated with PBS, 100 ng/ml NGF, or 10
ng/ml NT-3 for the indicated times. The lipids were extracted, and
sphingomyelin was quantitated as described under ``Experimental
Procedures.'' Sphingomyelin levels were normalized to total
phospholipid phosphate, and the results were expressed as the percent
of time matched control values. Results shown are mean ± S.E.
and derive from six experiments (n = 18) for NGF and
two experiments (n = 6) for
NT-3.
The absence of NGF-induced
sphingomyelin hydrolysis in PC12 cells was unexpected since this
concentration of NGF effectively induced sphingomyelin hydrolysis in
both T9 glioma cells and p75-NIH-3T3 cells. However, both
of these cell lines express solely p75
(33) ,
while PC12 cells express p75
at approximately 36-fold the
level of Trk A. To determine if the presence of Trk A was affecting
NGF-induced p75
-dependent sphingomyelin hydrolysis, PC12
cells were incubated with NT-3. PC12 cells lack Trk C receptors and are
unresponsive to NT-3 in terms of tyrosine phosphorylation and the
ability to develop neurites(49) . However, treatment of PC12
cells with 10 ng/ml NT-3 induced significant sphingomyelin hydrolysis
over a time course similar to that observed in
p75
-NIH-3T3 cells ( Fig. 6and Fig. 5B). Although NT-3 can bind to Trk A(6) ,
NT-3-induced sphingomyelin hydrolysis was not due to activation of Trk.
NT-3 (10 ng/ml) had no effect on cellular tyrosine phosphorylation in
PC12 cells (data not shown).
Figure 7:
Inhibition of Trk tyrosine kinase enables
NGF to stimulate sphingomyelin hydrolysis in PC12 cells. PC12 cells
were labeled with [H]choline chloride for 3 days
and placed in DMEM containing 3% serum for 4 h. The cells were then
preincubated with 0, 2 nM, 200 nM, or 2 µM K252a for 1 h. The cells were transferred to a 37 °C water
bath and treated with PBS, 100 ng/ml NGF, 10 ng/ml NT-3, or NGF +
NT-3 for 15 min. The lipids were extracted, and sphingomyelin was
quantitated. Sphingomyelin levels were normalized to total
phosphatidylcholine counts/min, and the results were expressed as the
percent of time matched control values. Results shown are mean ±
S.E. and derive from five experiments (n =
15).
Next, to determine if the
inhibition of p75-dependent sphingomyelin hydrolysis was
due to a general inhibitory signal generated by receptor-linked
tyrosine kinases, PC12 cells were incubated with either EGF or
platelet-derived growth factor in the presence or absence of NT-3. If
activation of cellular tyrosine kinase activity is sufficient to
inhibit p75
-dependent sphingomyelin hydrolysis, then EGF
and platelet-derived growth factor should inhibit NT-3-induced
sphingomyelin hydrolysis. However, both ligands had no effect on
NT-3-induced sphingomyelin hydrolysis (Fig. 8). These results
suggest that Trk tyrosine kinase activity was specifically modulating
p75
-dependent sphingomyelin hydrolysis.
Figure 8:
The
EGFR and platelet-derived growth factor-receptor do not modulate
NT-3-induced sphingomyelin hydrolysis. PC12 cells were labeled with
[H]choline chloride for 3 days and placed in DMEM
containing 3% serum for 4 h. The cells were transferred to a 37 °C
water bath and treated for 15 min with PBS, 10 ng/ml EGF, or 10 ng/ml
platelet-derived growth factor in the presence or absence of 10 ng/ml
NT-3. The lipids were extracted, and sphingomyelin was quantitated.
Sphingomyelin levels were normalized to total phosphatidylcholine
counts/min, and the results were expressed as the percent of time
matched control values. Results shown are mean ± S.E. from two
experiments (n = 6).
Taken
together, these data support that specific cross-talk pathways exist
between Trk and p75-dependent signaling pathways.
Our data establish that neurotrophin-induced sphingomyelin
hydrolysis is mediated solely through p75. Although NGF,
BDNF, NT-3, and NT-5 bind to p75
with rather equivalent
affinities(25, 26) , the neurotrophins displayed a
differential ability to induce sphingomyelin hydrolysis in
p75
-NIH-3T3 cells. In this respect, both NT-3 and NT-5
were about 100-fold more potent at inducing sphingomyelin hydrolysis
than BDNF and NGF. However, BDNF, NT-3, and NGF (33) showed
similar kinetics for inducing sphingomyelin hydrolysis in
p75
-NIH-3T3 cells.
That p75 can
recognize the various neurotrophins as similar, but not identical,
molecules has been previously
noted(25, 26, 51, 52) . For example,
NGF, BDNF, and NT-3 show distinct rates of dissociation from
p75
(25, 26) . Therefore, it is possible
that differences in the dissociation rates of the neurotrophins for
p75
may affect their ability to induce sphingomyelin
hydrolysis.
An additional distinction between the neurotrophins is
that NT-3 and BDNF exhibit positive cooperativity in their binding to
p75 at low ligand
concentrations(25, 26) . As such, at low NT-3
concentrations (0.1-1 ng/ml), receptor occupancy of p75
would be predicted to be low. However, significant sphingomyelin
hydrolysis occurred at these concentrations of NT-3, suggesting that
only a small percentage of p75
receptors may need to be
occupied to activate sphingomyelin hydrolysis. Such a mechanism may be
analogous to neurotrophin-dependent neuronal survival where viability
is enhanced well below full occupancy of high affinity
receptors(6, 49) . Alternatively, a highly localized
pool of p75
may be involved in signal transduction (see
below).
Although NT-3 induced significant sphingomyelin hydrolysis
at ligand concentrations near the K for Trk C (1.8
10
M;
0.5
ng/ml)(15, 26) , NT-3 did not induce sphingomyelin
hydrolysis in fibroblasts expressing Trk C. These data strongly suggest
that Trk C does not directly activate neurotrophin-induced
sphingomyelin hydrolysis.
It is intriguing to speculate that
differences in the potencies of the neurotrophins to activate
sphingomyelin hydrolysis may reside in differences in the amino acids
that influence neurotrophin binding to p75. Lysines 32,
34, and 95 are critical for the recognition of NGF by
p75
(24, 51, 52) . Although BDNF
lacks Lys-32 and Lys-34, three positively charged residues at positions
95, 96, and 97 sufficiently compensate for this absence(52) ,
enabling BDNF to compete equally with NGF for binding to
p75
(25) . However, BDNF was slightly more potent
than NGF at inducing sphingomyelin hydrolysis. In contrast to BDNF,
NT-3 and NT-4 have a conserved substitution of one or two Arg residues
for Lys-32 and Lys-34, respectively(52) . It is intriguing that
both of these neurotrophins were 100-fold more potent at inducing
sphingomyelin hydrolysis than NGF and BDNF.
In terms of structural
requirements of p75, which may affect sphingomyelin
breakdown, deletion of a 57-amino acid region within the cytoplasmic
domain of p75
abolished NT-3-induced sphingomyelin
hydrolysis. This deletion removed amino acids 249-305 within the
cytoplasmic domain and partially disrupted the highly conserved
juxtamembrane region of p75
(35) . The lack of
NT-3-induced sphingomyelin hydrolysis in cells expressing this
p75
mutant suggests that this region of the cytoplasmic
domain is necessary for stimulation of sphingomyelinase activity.
Whether this region regulates direct coupling to sphingomyelinase or to
intermediary effector proteins remains to be determined.
An
additional consequence of this cytoplasmic deletion is the removal of
Cys-279, which is a probable site of palmitoylation in
p75(53) . The functional role of p75
palmitoylation is uncertain. However, receptor palmitoylation is
associated with targeting of certain proteins to caveolae(54) ,
small plasmallemal invaginations particularly enriched in sphingolipids (55, 56) . In this respect, we have localized a pool
of p75
to caveolae and have found that NT-3 induced
significant sphingomyelin hydrolysis in this membrane fraction. (
)Therefore palmitoylation may help target p75
to caveolae which contain an NT-3-sensitive pool of
sphingomyelin. However, it remains to be determined if receptor
palmitoylation is necessary for coupling of p75
to
sphingomyelin hydrolysis.
As previously mentioned, p75 contains an 11-amino acid sequence (amino acids 370-381)
homologous to the 14-amino acid wasp venom peptide
mastoparan(30) . However, the cytoplasmic deletion of the
p75
mutant does not encompass the mastoparan-like
sequence, suggesting that the presence of this sequence is not
sufficient to induce sphingomyelinase activation.
The cellular
location of the neurotrophin-sensitive pool of sphingomyelin was
addressed by exploiting the inability of bacterial sphingomyelinase to
hydrolyze pools of sphingomyelin, which do not reside on the external
leaflet of the plasma membrane. Treatment with bacterial
sphingomyelinase hydrolyzed approximately 50% of total cellular
sphingomyelin from p75-NIH-3T3 cells. This result is
similar to those obtained from HL-60 leukemia cells(37) . Thus,
about 50% of the cellular sphingomyelin resides in a bacterial
sphingomyelinase resistant pool. In HL-60 cells, the bacterial
sphingomyelinase-resistant pool is the site of sphingomyelin hydrolysis
induced by both tumor necrosis factor-
and vitamin D
(37) . Similarly, NT-3 hydrolyzed a portion of the
bacterial sphingomyelinase-resistant pool of sphingomyelin.
Based
upon subcellular fractionation, the agonist-sensitive pool of
sphingomyelin resides in the plasma membrane(37) . However,
since endosomes co-fractionate with plasma membrane, it was possible
that agonist-sensitive sphingomyelin hydrolysis may occur in endosomal
vesicles following receptor internalization. Although the
internalization of p75 is slow and unaffected by ligand
binding(57) , it is possible that receptor internalization via
the endosomal pathway was a potential site for sphingomyelin
degradation. Treatment of cells in hyperosmotic medium has been shown
to effectively block the internalization of the transferrin,
interleukin-1
, and tumor necrosis factor-
receptors(46, 47, 48) . However,
hyperosmolarity had no effect on NT-3-induced sphingomyelin hydrolysis,
suggesting that receptor sequestration into endosomes is not a major
site of p75
-dependent sphingomyelin hydrolysis.
Neurotrophins did not specifically hydrolyze one molecular species of sphingomyelin. This result was independent of the diversity in the molecular species composition of sphingomyelin within a given cell. Diversity in the molecular species composition of sphingomyelin may have direct consequences for the bioactivity of the product of its degradation, ceramide. In this respect, the molecular species composition of ceramide has been shown to differentially stimulate protein phosphatase 2A activity(58, 59) .
NGF
induced sphingomyelin hydrolysis in two cell lines which express solely
p75, i.e. p75
-NIH-3T3 cells and
rat T9 glioma cells(33) . However, in PC12 cells, which
co-express Trk A and p75
, NGF had little effect on
sphingomyelin hydrolysis. On the other hand, NT-3 induced significant
changes in sphingomyelin levels over a time course very similar to that
seen in NT-3-treated p75
-NIH-3T3 cells. Interestingly,
although NT-3 induced sphingomyelin hydrolysis in PC12 cells, it has no
effect on tyrosine phosphorylation and does not induce differentiation
in these cells(49) ; suggesting that NT-3-induced sphingomyelin
signaling is not sufficient for differentiation of PC12 cells.
Moreover, the lack of sphingomyelin hydrolysis by NGF supports that
sphingomyelin signaling is not involved in NGF-induced differentiation
of PC12 cells.
The biologic role of NT-3-induced sphingomyelin
metabolism in PC12 cells remains to be determined. It is possible that
the product of sphingomyelin hydrolysis, ceramide, may initiate a
pathway that inhibits NGF-induced neurite development in PC12 cells. In
this respect, treatment of PC12 cells with either bacterial
sphingomyelinase (60) or exogenous ceramide inhibits NGF-induced neurite outgrowth, although we were unable
to observe significant inhibition of NGF-induced neurite outgrowth in
the presence of 10 ng/ml NT-3. However, since the presence of NGF
inhibits NT-3-induced sphingomyelin hydrolysis in PC12 cells (Fig. 7), NT-3 may be unable to generate the bioactive mediator
ceramide. Ceramide may attenuate NGF-induced neurite outgrowth through
modulation of cellular protein phosphorylation/dephosphorylation
cascades(58, 59, 61, 62) .
Recent
studies have demonstrated that p75 can modulate and/or
potentiate the activity of Trk receptors. For example, overexpression
of p75
relative to Trk enhances tyrosine
autophosphorylation of Trk and increases neuronal maturation in MAH
cells(32) . Furthermore, p75
was found to
potentiate masked autocrine loops in transfected
fibrolasts(31) . Our study suggests that the reciprocal
situation also exists, i.e. that Trk tyrosine kinase can
specifically modulate p75
signaling. This regulation at
least requires the tyrosine kinase activity of Trk, but it may also
encompass other structural features of the Trk receptor.
At this
point, we can not determine if Trk is directly or indirectly affecting
p75-dependent sphingomyelin hydrolysis. p75
is not significantly phosphorylated on tyrosine following NGF
treatment of PC12 cells, suggesting that direct phosphorylation of
p75
by Trk is an unlikely point of regulation. It is
possible that tyrosine phosphorylation/dephosphorylation of the
signal-activated neutral sphingomyelinase inhibits its activity.
However, other then cation requirements, little is known about factors
regulating this enzyme (63) . Alternatively, Trk may not
inhibit NGF-induced sphingomyelin hydrolysis but stimulate rapid
sphingomyelin resynthesis leading to no net change in overall
sphingomyelin levels. However, that NT-3 induces sphingomyelin
hydrolysis in PC12 cells suggests that Trk does not constitutively
inhibit sphingomyelinase or stimulate sphingomyelin resynthesis.
Finally, Trk may indirectly modulate p75
-dependent
signaling via coupling to immediate downstream effectors such as
phosphatidylinositol 3-kinase, phospholipase C-
, or proteins in
the mitogen-activated or stress-activated protein kinase
cascades(23) . Regardless, our data provide biochemical
evidence that neurotrophin receptors may undergo extensive cross-talk
in coordinating signal transduction and support the notion that
p75
has a functional signaling capacity.