(Received for publication, September 18, 1995; and in revised form, December 4, 1995)
From the
Regulation of the cytosolic free Ca concentration by nerve growth factor was investigated in
C6-2B glioma cells newly expressing the high affinity nerve
growth factor receptor trkA, using Fura-2 fluorescence ratio
imaging. In these cells, nerve growth factor (50 ng/ml) evoked a novel
3-fold increase in cytosolic free Ca
concentration, while no measurable Ca
response
was observed in wild type or mock-transfected cells lacking a
functional trkA receptor. K-252a, a tyrosine kinase inhibitor
which prevents nerve growth factor-mediated responses in C6-2B
cells expressing trkA, also blocked the rise in cytosolic free
Ca
concentration by nerve growth factor. Moreover,
basic fibroblast growth factor, which in these cells elicits
biochemical changes similar to nerve growth factor, failed to affect
cytosolic free Ca
concentration, further supporting
the specificity of nerve growth factor/trkA receptor in
mediating a Ca
response. While insensitive to
chelation of extracellular Ca
, the response was
abolished following depletion of Ca
stores or
blockade of intracellular Ca
release, providing
strong evidence that intracellular Ca
is the main
source for nerve growth factor-evoked cytosolic free Ca
concentration increase. Nerve growth factor increased the
cytosolic free Ca
concentration also in NIH3T3 cells
overexpressing trkA but devoid of p75 nerve growth factor
receptor. Our data suggest that trkA but not p75 is required
for nerve growth factor-evoked Ca
signaling.
Neurotrophic factors are required for the development, survival,
and maintenance of distinct populations of neurons. Among various
growth factors which have been proposed to function as neurotrophic
agents in the central nervous system is nerve growth factor
(NGF)()(1) , a prototype member of the neurotrophin
family of growth factors which includes brain-derived growth factor,
neurotrophin-3, and neurotrophin 4/5 (reviewed in (2) ). NGF is
required for the differentiation and survival of sympathetic and some
sensory neurons in the peripheral nervous system and provides trophic
support for the cholinergic neurons of the basal forebrain (reviewed in (3) ).
NGF exerts its neurotrophic activity by binding to a
receptor complex comprised of a low affinity component,
p75(4, 5) , which has been postulated
to interact with G-proteins(6, 7) , and an high
affinity component, trkA(8, 9, 10, 11) , which
contains a cytoplasmic domain with tyrosine kinase
activity(12) . trkA, the product of trkA
protooncogene(13) , undergoes autophosphorylation upon NGF
binding(12) , and it has been shown to be essential for the
biological activity of NGF(14, 15) .
A great deal
of information is now available on the various biological responses
elicited by NGF. However, at present, the role played by either
receptor component in the NGF signal transduction mechanism(s) as well
as the second messenger(s) leading to specific NGF responses are still
under investigation. Controversial results have been reported showing
that a number of second messengers are affected by NGF, such as
cAMP(7, 16, 17) , cGMP(18) ,
phosphoinositides(7, 19) , arachidonic
acid(20, 21) , and glycosylphosphatidylinositol
metabolites(22) . The role of Ca as a
putative mediator of NGF responses has also been investigated; however,
the data gathered so far are highly contradictory. The original report
that NGF caused a small Ca
efflux (23) was
not supported by subsequent studies (24) . Likewise, the more
recent findings (25, 26) that NGF evokes a small and
rapid increase in cytosolic free Ca
concentration
([Ca
]
) have not been
confirmed (27) . In addition, NGF has been proposed to increase
Ca
uptake, possibly through a unique Ca
channel(28, 29) .
All of the above studies
have been performed in PC12 cells (a pheochromocytoma cell line widely
used as a model for studying NGF-induced responses) which express both
low and high affinity NGF receptor components(30) . C6-2B
cells, a rat glioma cell line expressing p75(31) but devoid of the high affinity receptor trkA(32) , are unresponsive to NGF. Upon stable
transfection with trkA rat cDNA, C6-2B cells
(C6trk
) exhibit novel NGF-mediated biochemical
responses (c-fos induction and phosphorylation of trkA) and morphological changes (increased length of process
extension)(32) . NIH3T3 cells, devoid of
p75
(9) , display a novel responsiveness to NGF
when expressing trkA (trkA NIH3T3)(33) .
Therefore, we reasoned that C6trk
and trkA
NIH3T3 cells could represent two useful models to evaluate the role
played by either receptor component in the modulation of the
intracellular Ca
dynamics by NGF. The data presented
indicate that (i) in both C6trk
and trkA
NIH3T3 cell lines NGF increases
[Ca
]
by mobilizing
intracellular Ca
, and (ii) trkA, but not
p75
, is essential for the NGF-evoked Ca
response.
Figure 1:
Effect of NGF on
[Ca]
in C6-2B
mock-transfected and C6trk
cells. Fura 2-AM loaded
C6-2B mock transfected (open squares) and
C6trk
cells (solid squares) were challenged
with NGF (50 ng/ml) and [Ca
]
changes were measured as described under ``Materials
and Methods.'' ATP (100 µM) was then added to mock
transfected cells (open squares). Data are the population
means of the [Ca
]
responses from one coverslip per each culture and are
representative of 5 coverslips (mock transfected cells) and 32
coverslips (C6trk
cells) with 10-50
cells/coverslip imaged in a single field.
Figure 2:
Induction of trkA and Erk
tyrosine phosphorylation by NGF in two C6trk clones.
C6trk
cells from two separate clones (nos. 41 and 43)
were incubated in serum-free medium in the absence(-) or presence
(+) of 50 ng/ml NGF for 5 min at 37 °C and then lysed as
described under ``Materials and Methods.'' The lysates were
immunoprecipitated with either pan-trk polyclonal (lanes
1, 2, 5, and 6) or pan-Erk monoclonal (lanes 3, 4, 7, and 8) antibodies.
The immunoprecipitates were transferred to nitrocellulose and probed
with anti-phosphotyrosine antibody. M= molecular mass
standards in kDa. Lanes 1-4, clone no. 41; lanes
5-8, clone no. 43. The positions of trkA, Erk2, and
IgG are indicated by the arrows on the right.
Figure 3:
Dose-dependent increase in
[Ca]
by NGF in
C6trk
cells. C6trk
cells (clone no.
43) were exposed to various concentrations of NGF (1-50 ng/ml).
The peak [Ca
]
from a
total of 2-16 coverslips (10-50 cells/coverslip imaged in a
single field) was calculated. Results are expressed as percent ±
S.E. of the maximal [Ca
]
response achieved upon challenge with 50 ng/ml NGF (basal:
85 ± 18.6 nM (n = 34), NGF: 236
± 78 nM (n = 16)). Numbers in
parentheses are the number of coverslips examined at each one of
the indicated doses. Five coverslips were used to test for the effect
of 1 ng/ml NGF on
[Ca
]
; however, only
two coverslips displayed few (<2%) responsive
cells.
Figure 4:
Effect of EGTA on the NGF-evoked
[Ca]
rise in
C6trk
cells. Cells were incubated for 1 min with
serum-free Ham's F-10 medium in the absence (control, open
squares) or presence of 2 mM EGTA (solid
squares) and then challenged with NGF (50 ng/ml) always in the
absence or presence of 2 mM EGTA. Data are the population
means of [Ca
]
levels
from one coverslip and are representative of a total of 10 coverslips
per experimental condition (10-50 cells/coverslip in a single
field).
Further support for the hypothesis
that intracellular Ca is the main source for the peak
response to NGF was provided by experiments in which either the
internal stores were depleted of Ca
with the
microsomal Ca
-ATPase inhibitor thapsigargin (TG) (40) or the release of Ca
from the stores was
blocked with dantrolene, a muscle relaxant widely used as an inhibitor
of intracellular [Ca
]
mobilization (36, 41, 42) . Exposure of
C6trk
cells to 100 nM TG elicited a
[Ca
]
increase similar in
magnitude and kinetics to that previously described in wild type
C6-2B cells(36, 42) . When TG-pretreated cells
were challenged with NGF (50-100 ng/ml), no increase in
[Ca
]
was observed (Fig. 5). Because the response to the Ca
ionophore ionomycin (5 µM) was unaffected by TG (Fig. 5), these data provide strong evidence that NGF increases
[Ca
]
by mobilizing the cation
from intracellular stores. In line with the above results, when
C6trk
cells were exposed to NGF first and subsequently
to ATP, which in C6-2B cells induces inositol 1,4,5-trisphosphate
(IP
) formation and releases Ca
from
internal stores(36) , the Ca
response to ATP
was reduced by about 50% in those cells which had responded to NGF,
compared to NGF-unresponsive cells (Fig. 6). Likewise, TG evoked
a 2.6 ± 0.1-fold increase in [Ca
]
in cells which previously responded to NGF,
while it elicited a 5.7 ± 0.8-fold increase in NGF-unresponsive
cells (n = 3). The results obtained with ATP and TG
indicate that NGF evokes Ca
release from
intracellular stores which are included in the IP
- and
TG-sensitive Ca
pools. This is consistent with
previous findings showing that, in PC12 cells, NGF induces hydrolysis
of phosphatidylinositol (7, 19, 43) with
consequent production of IP
which is known to release
Ca
from intracellular stores(39) . Last,
dantrolene, which in C6-2B cells prevents the
[Ca
]
increase evoked by either
agonists of receptors coupled to phosphatidylinositol hydrolysis or
TG(36, 42) , abolished NGF-induced Ca
transient in C6trk
cells without appreciably
affecting the ability of ionomycin to elicit a Ca
entry response (Fig. 7).
Figure 5:
Thapsigargin prevents
[Ca]
increase induced
by NGF in C6trk
cells. Cells were exposed to
thapsigargin (TG, 100 nM; open squares) or vehicle
(0.001% dimethyl sulfoxide; solid squares), washed, and
challenged with NGF (50 ng/ml). Ionomycin (5 µM) was also
added to TG-pretreated cells. The
[Ca
]
profiles shown
are the population means from one coverslip and are representative of
two other experiments (10-50 cells/coverslip in a single
field).
Figure 6:
Effect of NGF on intracellular
Ca mobilization by ATP. C6trk
cells
were exposed to NGF (50 ng/ml) and subsequently to ATP (100
µM). Data are the mean
[Ca
]
responses
recorded from five cells which responded (open squares) and
five cells which did not respond (solid squares) to NGF. Cells
are from the same field and were imaged simultaneously. Similar results
were obtained from several (>10) other coverslips (30-50
cells/coverslip in a single field).
Figure 7:
Blockade of the NGF-evoked
[Ca]
rise by
dantrolene in C6trk
cells. Cells were incubated for 10
min with either dantrolene (DAN, 40 µM; solid
squares) or vehicle (0.4% dimethyl sulfoxide; open
squares), briefly washed and imaged. NGF (50 ng/ml) was then added
and [Ca
]
monitored.
Ionomycin (5 µM) was also added to dantrolene-treated
cells. A 6.3
objective was used to follow simultaneously the
[Ca
]
changes in
80-100 cells in a field. Data are the population means from one
coverslip and are representative of six coverslips per experimental
condition (80-100 cells/coverslip in a single
field).
Figure 8:
Inhibition by K-252a of the NGF-evoked
[Ca]
increase in
C6trk
cells. Fura-2AM loaded cells were incubated with
K-252a (200 nM; solid squares) or vehicle (0.01%
dimethyl sulfoxide; open squares) for 20 min, imaged and
Ca
response to NGF (50 ng/ml) was monitored. TG (100
nM) was then added to K-252a-treated cells. The mean
[Ca
]
response from one
coverslip is shown and is representative of a total of three coverslips
with 10-50 cells/coverslip in a single
field.
Figure 9:
bFGF
fails to increase [Ca]
in C6-2B or C6-2Btrk
cells.
Fura-2AM-loaded, mock-transfected C6-2B cells (A) and
C6trk
cells (B) were exposed to the indicated
compounds, and changes in [Ca
]
were monitored. bFGF and NGF were used at 50 ng/ml; ATP was
used at 100 µM. Data are the population means of the
[Ca
]
levels from one
coverslip per each culture. Similar results were obtained in three
other coverslips (10-50 cells/coverslip in a single field) from
both cell types.
Figure 10:
NGF mobilizes intracellular
Ca in trkA NIH3T3 cells. A, Fura-2-loaded
cells were incubated for 1-2 min with serum-free Dulbecco's
modified Eagle's medium in the absence (control, open
squares) or presence of 2 mM EGTA (solid
squares) and then challenged with NGF (50 ng/ml) in the continuous
absence or presence of EGTA. B, cells were exposed to TG (1
µM; solid squares) or vehicle (0.01% dimethyl
sulfoxide, open squares), washed with drug-free medium, and
challenged with NGF (50 ng/ml). Data are the population means of the
[Ca
]
levels from one
coverslip and are representative of at least four coverslips per each
experimental condition with 1-40 cells imaged per
coverslip.
We have previously shown that C6-2B cells express only
the low affinity component (p75) of the NGF receptor
complex(32) . Consistent with the notion that trkA is
required for the biological activity of NGF(14, 15) ,
C6-2B cells are unresponsive to NGF, as demonstrated by the lack
of induction of c-fos, phosphorylation of NGF target proteins,
and morphological changes, following NGF treatment(32) . All of
these responses, which are among NGF-mediated biological effects, can
be observed in PC12 cells, an NGF responsive cell line expressing both trkA and p75
receptors(30) . In this
report we have shown that wild type and mock-transfected C6-2B
cells expressing p75
but lacking a functional trkA receptor (32) failed to display a measurable
Ca
response upon NGF treatment. However, upon
exposure to ATP, which in C6-2B cells mobilize Ca
from internal stores(36) , or ionomycin, which induces
Ca
entry, Ca
responses comparable
to those previously described (36) were observed, implying that
the lack of changes in the [Ca
]
following NGF cannot be ascribed to inefficiency of the
mechanisms involved in either intracellular Ca
release or Ca
influx.
The novel expression
of trkA in C6-2B cells induces NGF
responsiveness(32) . C6trk cells undergo a
transient [Ca
]
increase when
exposed to NGF. Although the kinetics of the
[Ca
]
rise by NGF are similar to
those previously described in PC12 cells(25) , both the potency
and the efficiency of NGF in increasing
[Ca
]
were greater in
C6trk
cells (+200% at
0.2 nM) than
in PC12 cells (+50% at >3 nM). In C6trk
cells, the expression of p75
mRNA is nearly
equivalent to that found in PC12
cells(30, 31, 32) , while trkA mRNA
levels are at least 5-10-fold higher than in PC12
cells(32) . Thus, the overexpression of the newly synthesized
high affinity receptor in C6trk
cells could explain
the greater Ca
response evoked by NGF in these cells
compared to PC12 cells.
Previous findings in PC12 cells showed that
the small and transient increase in
[Ca]
evoked by NGF was mainly
due to Ca
entry(25, 28) , possibly
through a unique Ca
channel(29) . Our study
shows that NGF evokes an increases in
[Ca
]
also in
Ca
-depleted medium, but it fails to do so when
intracellular Ca
cannot be mobilized due to either
depletion of the stores with TG or blockade of the release from the
stores with dantrolene. We therefore propose that, in C6trk
cells, NGF elicits [Ca
]
rise by causing intracellular Ca
mobilization
rather than extracellular Ca
influx. However, we
cannot rule out the involvement of Ca
channels in the
NGF mediated [Ca
]
rise in other
systems because C6-2B cells do not express functional voltage
dependent Ca
channels as KCl depolarization failed to
change [Ca
]
. (
)
The exact mechanism utilized by NGF in evoking
Ca response does not appear to be easily definable.
In C6trk
cells, K-252a, an inhibitor of the tyrosine
kinase activity associated with trkA(12, 51) , blocked NGF-evoked
[Ca
]
rise. Therefore, it is
reasonable to infer that the novel Ca
response to NGF
observed in C6trk
cells is mediated by trkA.
NGF stimulates phosphorylation of phospholipase C
on tyrosine as
well as serine residues (52) by a kinase activity associated
with trkA (53) . Activated phospholipase C-
is
known to lead to phosphatidylinositol hydrolysis with accumulation of
diacylglycerol and IP
. The latter, acting at receptors
located on the endoplasmic reticulum, induces release of
Ca
(39) , suggesting that phospholipase
C-
signaling might mediate the NGF-evoked
[Ca
]
rise. However, bFGF,
which, similarly to NGF, activates phospholipase C-
in
C6trk
and trk NIH3T3 cells (data not shown),
failed to induce an appreciable Ca
response in these
same cells. Thus, it appears that
[Ca
]
rise is a unique response
to trkA activation. Interestingly enough, in C6trk
cells, NGF elicits morphological changes and is a weaker mitogen
than bFGF(32) . Thus, although only speculative at present, the
hypothesis that [Ca
]
rise
elicited by NGF could be a crucial player in triggering the cascade of
molecular events leading to cell differentiation is appealing.
Recent data have shown that, although p75 is not
required for NGF biological activity(54) , the low affinity
receptor appears to be involved in the binding of NGF and signal
transduction of trkA(55, 56, 57, 58, 59) .
From our results, trkA appears to be solely responsible and
sufficient for NGF-evoked [Ca
]
increase since Ca
signaling triggered by NGF
occurs also in trkA NIH3T3 cells lacking p75
.
In a recent report(7) , p75
has been proposed to
mediate, through a pertussis toxin sensitive G-protein, the increase in
cAMP accumulation caused by NGF in PC12 cells. p75
is
also involved in the NGF-mediated activation of sphingomyelin
cycle(60) . Thus, p75
might be involved in
signal transduction pathways other then Ca
, while trkA in signaling other then cAMP. Interestingly, and in
agreement with our interpretation, K-252a blocked NGF-induced IP
accumulation(7, 45) , while it did not affect
the NGF-elicited cAMP formation(7) .
In conclusion, we have
demonstrated that (i) NGF is able to largely modulate
[Ca]
by a mechanism which
involves mobilization of intracellullar Ca
and (ii) trkA and not p75
is required and sufficient to
mediate this response. Important questions which could be addressed
using C6trk
cells as a model include the relationship
(if any) between Ca
signal and morphological changes,
as well as cell differentiation.