(Received for publication, July 24, 1995; and in revised form, November 13, 1995)
From the
The glia-derived, neurotrophic protein S100 has been
implicated in development and maintenance of the nervous system.
However, S100
has also been postulated to play a role in
mechanisms of neuropathology, because of its specific localization and
selective overexpression in Alzheimer's disease. To begin to
address the question of whether S100
can induce potentially toxic
signaling pathways, we examined the effects of the protein on nitric
oxide synthase (NOS) activity in cultures of rat cortical astrocytes.
S100
treatment of astrocytes induced a time- and dose-dependent
increase in accumulation of the NO metabolite, nitrite, in the
conditioned medium. The S100
- stimulated nitrite production was
blocked by cycloheximide and by the NOS inhibitor N-nitro-L-arginine methylester, but not by the
inactive D-isomer of the inhibitor. Direct measurement of NOS
enzymatic activity in cell extracts and analysis of NOS mRNA levels
showed that the NOS activated by S100
addition is the
calcium-independent, inducible isoform. Furthermore, the specificity of
the effects of S100
on activation of NOS was demonstrated by the
inability of S100
and calmodulin to induce an increase in nitrite
levels. Our data indicate that S100
can induce a potent activation
of inducible NOS in astrocytes, an observation that might have
relevance to the role of S100
in neuropathology.
The normal development and maintenance of the brain involves the
temporal and spatial coordination and proper functioning of a number of
intracellular and cell-cell signaling events, and the contribution of
glial cells to these signaling processes is becoming more widely
appreciated. The classical concept of the role of glia in brain
function is rapidly changing with newer evidence of the crucial nature
of these cells in controlling neurotransmitter levels, maintaining
calcium homeostasis, and synthesizing and releasing neurotrophic and
growth factors (for review, see (1) ). One such glia-derived
factor is S100, a protein that promotes neuritic outgrowth of
specific neuronal populations (e.g. cortical(2, 3) , dorsal root ganglia(4) ,
serotonergic(5, 6) , and motoneurons(7) ) and
enhances survival of neurons during development (7, 8) and after insult(9) . S100
is also
a glial mitogen, inducing phosphoinositide hydrolysis, increases in
intracellular calcium, and protooncogene
expression(10, 11) . These trophic functions require
nanomolar concentrations of a disulfide-linked S100
dimer (see (12) ). Thus, S100
may be beneficial during development of
the nervous system, and increased S100
expression and secretion
following acute glial activation in response to central nervous system
injury may be one mechanism the brain uses in attempts to repair
injured neurons.
However, S100 may also reach concentrations
that are deleterious, e.g. in neurodegenerative diseases like
Alzheimer's disease and Down syndrome where chronic glial
activation occurs(13) . It has been found that S100
levels
in severely affected brain regions of Alzheimer's disease
patients are severalfold higher than in age-matched control
samples(13, 14) , that S100
is increased
selectively in regions that exhibit the most neuropathological
involvement(15) , and that S100
overexpression is
correlated with the prevalence of neuritic plaques(16) . These
data raise the possibility that high concentrations of S100
may be
detrimental, an idea supported by the recent demonstration that an S100
isoform can induce apoptosis in PC12 cells through sustained increases
in intracellular calcium(17, 18) .
As a first
approach to addressing the question of whether S100 can induce
potentially toxic signaling pathways, we examined the effect of
S100
on nitric oxide (NO) generation. NO is synthesized by the
enzyme nitric oxide synthase (NOS) (
)through the conversion
of L-arginine to L-citrulline. In the nervous system,
NO has been implicated in the regulation of cerebral blood flow,
synaptic plasticity, and cell growth (see (19) ). A large
amount of data also supports the concept that NO production in the
central nervous system may be involved in the neuropathology associated
with ischemia, traumatic insults, and neurodegeneration (see (19, 20, 21) ). We report here that treatment
of rat cortical astrocytes with S100
results in a stimulation of
NOS activity and generation of NO.
Total RNA (10 µg/lane) was run under denaturing conditions on a
1% agarose-formaldehyde gel, transferred to Hybond-N membranes
(Amersham), and cross-linked in a UV Stratalinker (Stratagene).
Membranes were incubated for 3 h at 42 °C in pre-hybridization
solution (5 SSC (20
SSC = 3 M NaCl, 0.3 M sodium citrate, pH 7.0), 5
Denhardt's solution
(50
Denhardt's = 100 g/liter Ficoll, 100 g/liter
polyvinylpyrrolidone, 100 g/liter bovine serum albumin), 50 mM sodium phosphate, pH 7.0, 1% glycine, 0.3% sodium dodecyl sulfate
(SDS), 50% formamide, 20 µg/ml denatured salmon sperm DNA).
Pre-hybridization solution was removed and membranes were incubated for
18 h at 42 °C in hybridization solution (5
SSC, 1
Denhardt's solution, 20 mM sodium phosphate, pH 7.0,
0.3% SDS, 50% formamide, 20 µg/ml denatured salmon sperm DNA, 10%
dextran sulfate) containing
P-labeled iNOS cDNA probe. The
iNOS cDNA clone was pASTNOS4, corresponding to the rat iNOS cDNA bases
3007-3943 ((31) ; gift of Dr. Elena Galea, Cornell
University), and was labeled with [
-
P]dCTP
by using a Promega Prime-a-Gene random-primed labeling kit. After
hybridization, membranes were washed once for 5 min at room temperature
in 2
SSC; twice for 30 min at 65 °C in 1
SSC, 0.5%
SDS; and once for 10 min at room temperature in 0.1
SSC, 0.1%
SDS. Membranes were exposed to film, and band intensity on the
autoradiograms was quantitated on an imaging densitometer (Bio-Rad,
model GS-670). To correct for potential differences in RNA loading,
membranes were subsequently hybridized with a cyclophilin cDNA probe
(pSP65.1B15; (32) ) and results were expressed as a ratio of
density of iNOS mRNA versus cyclophilin mRNA.
The ability of S100 to stimulate NO production was
determined initially by measuring the accumulation of nitrite (a stable
NO metabolite) in the conditioned medium of astrocytes following
exposure to S100
. Tertiary cultures of neonatal rat cortical
astrocytes were prepared as described under ``Experimental
Procedures.'' These cultures contained approximately 98%
astrocytes, as assessed by positive staining for the astrocyte
intermediate filament protein, GFAP (data not shown). This result was
consistent with the observation that only 2-3% of the cells
showed positive staining for OX-42 (data not shown), a marker for
microglia(33) . Fig. 1shows the time course of nitrite
accumulation following exposure of these cells to S100
(40
µg/ml S100
for 6-72 h). S100
-treated astrocytes
showed increased nitrite accumulation in their conditioned medium
compared to control cultures treated with storage buffer alone. The
increased nitrite levels were evident after a 12 h exposure of cells to
S100
and reached a plateau at about 48 h. The maximal level of
nitrite accumulation in response to S100
was 3-4-fold higher
than that generated by exposure of cells to buffer alone.
Figure 1:
Time
course of S100-stimulated nitrite accumulation in rat astrocyte
cultures. Cells were incubated with 40 µg/ml S100
(squares) or control storage buffer alone (circles)
for the indicated times, and nitrite concentrations in the conditioned
media were determined. Data shown are from one of three independent
experiments, each done in triplicate. Values shown are the mean
± S.E. from the triplicate determinations. Error bars are shown
only when larger than the symbol. S100
-stimulated nitrite
accumulation was significantly different from control values (p < 0.05) at 24-, 48-, and 72-h treatment
times.
The
response to S100 was dose-dependent in the range of 1-40
µg/ml S100
, assayed after 48 h treatment of cells (Fig. 2). The concentration of S100
required to elicit 50%
of the maximal nitrite increase was 20-25 µg/ml, and the
maximal response was achieved by 35-40 µg/ml S100
.
Figure 2:
Dose-response curve of
S100-stimulated nitrite accumulation in rat astrocyte cultures.
Cells were incubated with increasing concentrations of S100
for 48
h, and nitrite concentrations in the conditioned media determined. Data
shown are from one of four independent experiments, each done in
triplicate. Values shown are the mean ± S.E. from the triplicate
determinations. Error bars are shown only when larger than the symbol.
S100
-stimulated nitrite accumulation was significantly different
from control values (p < 0.01) at doses
5
µg/ml.
To
test whether the S100 -induced nitrite accumulation was dependent
on NOS activity, we examined the effect of S100
in the presence of
the NOS inhibitor, L-NAME. Incubation of cells for 48 h with
S100
in the presence of L-NAME almost completely
abolished the nitrite accumulation, whereas the inactive inhibitor
isoform, D-NAME, was ineffective (Fig. 3). The basal
level of nitrite accumulated during 48 h was also affected by L-NAME but not D-NAME.
Figure 3:
Inhibition of S100-stimulated nitrite
accumulation by NOS inhibitor. Cells were incubated for 48 h with
S100
(40 µg/ml) or control storage buffer in the presence or
absence of the NOS inhibitor L-NAME (1 mM) or the
inactive isomer, D-NAME (1 mM), and nitrite
concentrations in the conditioned media determined. Data shown are the
mean ± S.E. from three independent experiments done in
triplicate. *, p < 0.05.
Incubation of cells with
S100 in the presence of the protein synthesis inhibitor
cycloheximide prevented the S100
stimulation of nitrite
accumulation (Table 1). Moreover, the specificity of the response
to S100
was examined by comparing the ability of two other related
calcium binding proteins, S100
and calmodulin, to enhance nitrite
accumulation. Table 1shows that neither S100
nor calmodulin
stimulated nitrite accumulation, under conditions where the LPS
positive control induced nitrite accumulation to a similar extent as
S100
. To exclude the possibility that the S100
stimulation of
nitrite accumulation was a result of contaminating LPS in the
recombinant S100
preparation, we isolated S100
from bovine
brain tissue and we took an E. coli lysate lacking the S100
expression vector through the same purification protocol as S100. We
found that bovine brain S100
induced nitrite accumulation to a
similar extent as recombinant S100
, and that the E. coli lysate showed no induction above the buffer control (data not
shown).
The ability of S100 to enhance nitrite accumulation
that was inhibited by NOS inhibitors suggested an activation of NOS
enzyme activity. To test this directly, we examined NOS enzyme activity
in cytosolic extracts of astrocytes treated with S100
for 24 h, by
measuring the conversion of L-arginine to L-citrulline. As shown in Fig. 4, S100
treatment
of astrocytes resulted in a
4-fold stimulation of NOS activity.
The S100
-evoked NOS activity was independent of the presence of
calcium in the enzyme assay, suggesting that the NOS stimulated by
S100
is the inducible enzyme (iNOS). This was further confirmed by
the demonstration that S100
-treated astrocytes showed increased
levels of iNOS mRNA, as measured by Northern blot analysis (Fig. 5).
Figure 4:
S100 stimulation of inducible iNOS
activity in rat astrocyte cultures. Cells were incubated for 24 h with
S100
(40 µg/ml) or control storage buffer, and NOS activity in
cytosolic extracts determined. Enzyme assays were done in the presence
or absence of calcium as described under ``Experimental
Procedures.'' Data shown are from one of four independent
experiments, each done in triplicate. Values shown are the mean
± S.E. from the triplicate determinations. S100
-stimulated
NOS activity was significantly different from control (p <
0.05).
Figure 5:
S100 stimulation of iNOS mRNA. Cells
were incubated for 24 h with S100
(40 µg/ml) or control
storage buffer, and iNOS mRNA levels were determined by Northern blot
analysis of 10 µg/lane total RNA, as described under
``Experimental Procedures.'' The blot was re-probed with
cyclophilin cDNA, and the iNOS mRNA levels were expressed as the ratio
of density of iNOS mRNA versus cyclophilin mRNA. Inset shows the iNOS mRNA bands from control (C) versus S100
-treated (S)
astrocytes.
We have demonstrated that treatment of astrocytes with
S100 enhances NOS activity and release of NO, as demonstrated by
the increase in accumulation of the stable NO metabolite nitrite in the
conditioned medium. Measurement of NOS enzyme activity and mRNA levels
confirms that the NOS stimulated by S100
addition is the
calcium-independent inducible iNOS isoform. The specific NOS inhibitor L-NAME, but not the inactive D-NAME, suppressed the
S100
-induced iNOS activity. Moreover, the specificity of the
effects of S100
on iNOS activation was demonstrated by the
inability of S100
or calmodulin to induce an increase in NO. These
data demonstrate that S100
induces a stimulation of astrocytic
iNOS activity and generation of NO.
The NOS enzyme responsible for
synthesizing NO from arginine exists in three major forms: two
constitutive cNOS isoforms (eNOS and nNOS) that are calcium-calmodulin
dependent and present in a variety of cells, including endothelial
cells, neurons, platelets, and astrocytes; and an inducible form (iNOS)
that is calcium-independent and expressed after gene induction in a
variety of cells, including macrophages, endothelial cells, and
astrocytes (see (34) ). Induction of iNOS in astrocytes has
been well documented previously by exposure of cells in vitro to bacterial endotoxin (LPS) or combinations of cytokines, such as
interleukin-1, interferon-
or tumor necrosis
factor-
(1, 35) . Our data represent the first
report that S100
is another potent inducer of astrocytic iNOS
activity. The S100
-stimulated NO production exhibits similar
characteristics to those reported for induction by LPS or other
cytokines in terms of long (hour) time frame and narrow dose-response
curve(31, 35) . The response occurs over several hours
of continuous application of S100
, and nitrite concentrations
continue to increase up to 48 h after S100
exposure. Similar to
IL-1
(35) , the effect of S100
on iNOS activity was
dose-dependent through a small concentration range. The magnitude of
the nitrite accumulation in response to S100
also varied somewhat
among cultures and among S100
preparations, suggesting that
endogenous coordinators or specific S100
conformational states
might be required for maximal S100
induction of iNOS.
We
considered the possibility that our results might reflect an activation
of NOS activity in microglia, rather than astrocytes, because microglia
express high levels of iNOS activity(36) . However, this
possibility seems unlikely because our tertiary cultures are 98%
astrocytes, as determined by GFAP and OX-42 immunoreactivity, and
because the intensity of the iNOS mRNA on Northern blots and the iNOS
enzyme activity from cytosolic extracts are too high to reflect a
signal from only 2% of the cells in the culture. Thus, our data support
an S100
-induced activation of iNOS activity in astrocytes.
The
mechanisms involved in activation of iNOS by S100 addition are
unknown at present. Our observation that S100
stimulates iNOS mRNA
levels suggests that regulation may be at the level of transcription of
the iNOS gene, as has been shown previously for iNOS stimulation by LPS
and interferon-
(34, 37, 38) . However,
iNOS has also been found to be regulated at the level of mRNA and
protein stability(39, 40) . Definition of the
mechanisms by which S100
induces iNOS activity, and whether
S100
interacts with other cytokines to modulate iNOS activity
await further investigation.
The consequences of S100-induced
NO release from astrocytes are not known. There is a wealth of
sometimes conflicting evidence in the literature that NO can be
beneficial or detrimental to nervous system function (see (19) and (21) ). For example, it has been found that NO
can act as a diffusible messenger to mediate cell-cell signaling
pathways involved in synaptic plasticity and regulation of cerebral
blood flow. However, there is also a large amount of biochemical and
pharmacological data to suggest that NO is involved in the
neuropathology associated with traumatic or ischemic insults,
autoimmune diseases, and neurodegenerative disorders. Relevant to our
studies, it has been reported (41) that stimulation of
astrocytic iNOS activity and release of NO leads to enhanced NMDA
receptor-mediated neurotoxicity. We are currently pursuing studies with
astrocytic/neuronal co-cultures to evaluate the effects of
S100
-stimulated NO release on the neuronal cell. In this regard,
it is interesting to note that in some experiments, the
S100
-treated astrocytes exhibited morphological changes consistent
with toxicity, such as release of lactate dehydrogenase and cell
rounding and detachment from the substrate (data not shown). The reason
for this observation has not been defined yet, but appeared to
correlate with the levels of nitrite accumulated in response to
S100
. It is probable that the toxic potential of NO in vivo depends, at least in part, on the concentration released from
cells. However, the biology of NO is complex and the susceptibility of
a cell to NO-mediated toxicity likely depends on a number of variables,
such as acute versus chronic exposure to NO, the array of
redox forms in which NO can exist, availability of reactive oxygen
species and anti-oxidant defenses, changes in ion homeostasis, presence
of other cytoprotective or cytotoxic agents, and the immune status of
the organism.
Our data demonstrate that treatment of astrocytes with
S100 results in a potent induction of iNOS activity and NO
production. Stimulation of iNOS required micromolar concentrations of
S100
, unlike the nanomolar concentrations required for
neurotrophic and mitogenic effects. The requirement of higher
concentrations of S100
for activation of iNOS suggests that
S100
might possess dual roles in regulation of cell function,
being beneficial to cells at low doses and detrimental at higher doses.
If S100
stimulates astrocytic iNOS activity in vivo, this
might be relevant to the role of S100
in neurodegenerative
disorders like Alzheimer's disease and Down syndrome, where
S100
levels are increased severalfold in temporal lobe samples
compared to age-matched control samples(13) . It has been found
that S100
levels are elevated in specific brain regions from
Alzheimer patients(15) , that the overexpression of S100
correlates with the pattern of regional neuropathology and neuritic
plaque involvement(15, 16) , and that S100
is
localized primarily in activated astrocytes surrounding neuritic
plaques(15) . It should also be noted that the local
concentration of S100
in astrocytes surrounding neuritic plaques
in Alzheimer's disease has not been determined, although the
tissue levels are in the micromolar range (15) . The
relationship between increased S100
levels and neuropathology is
not known. It may be that the up-regulation of S100
after acute
central nervous system injury or perhaps in neurodegenerative disease
is part of a compensatory response the brain uses in attempts to repair
injured neurons, through an action of S100
in its neurotrophic and
neuroprotective roles. However, the chronic overexpression of S100
or expression of S100
above some threshold level as in
Alzheimer's disease may have deleterious consequences leading to
neuronal dysfunction and eventual death, perhaps through altered
calcium homeostasis or inappropriate neuritogenesis. Our data that
S100
can stimulate astrocytes to produce NO provide another
possible mechanistic scenario by which the high levels of S100
seen in Alzheimer's disease and other neurodegenerative disorders
might contribute to neuropathology.