From the Laboratory of Neurosciences, NIA Gerontology
Research Center, National Institutes of Health, Baltimore, Maryland
21224, the § Department of Pharmacology and Therapeutics,
University of Manitoba Faculty of Medicine, Winnipeg, Manitoba R3E 0T6,
Canada, and the
Department of Neuroscience, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
Received for publication, February 12, 2001
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ABSTRACT |
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The transcription factor nuclear
factor- The transcription factor
NF- Stimuli that can activate NF- Recent studies have shown that impairment of ER function with
accumulation of proteins in the ER and the consequent release of
Ca2+ from this organelle stimulates NF- Cortical Cell Cultures and Experimental Treatments--
Cerebral
cortices were removed from embryonic day 18 Harlan Sprague-Dawley rats
(Harlan Sprague Dawley, Inc., Indianapolis, IN). Cells were
dissociated by mild trypsinization and trituration as described
previously (32) and were seeded into 60-mm polyethyleneimine-coated culture dishes containing Eagle's minimum essential medium
supplemented with 26 mM NaHCO3, 40 mM glucose, 20 mM KCl, 1 mM
sodium pyruvate, 10% (v/v) heat-inactivated fetal bovine serum
(Sigma), and 0.001% gentamycin sulfate. After a 3-5-h incubation
period to allow for cell attachment, the medium was replaced with 2 ml
of neurobasal medium with B27 supplements (Life
Technologies, Inc.). Experimental treatments were performed on
7-9-day-old neuronal cultures in which ~95% of the cells were
neurons and the remaining cells were astrocytes. IP3,
glutamate, and ryanodine were purchased from Sigma; xestospongin
C (XeC) was purchased from Calbiochem; TNF- Total Cell Extract Preparation and Electrophoretic Mobility Shift
Assay (EMSA)--
Total cell extracts were prepared as described (33).
Briefly, cells were harvested, washed twice with ice-cold
phosphate-buffered saline, and lysed for 30 min at 4 °C in Totex
buffer (350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 5 mM dithiothreitol, 0.1%
phenylmethylsulfonyl fluoride, 1% aprotinin, 4 µg/ml leupeptin, and
20 mM HEPES, pH 7.9). Samples were centrifuged at
14,000 × g for 10 min, and aliquots of the supernatant
were collected and stored at Preparation of Cytoplasmic and Microsomal
Fractions--
Cerebral cortices from adult female Harlan
Sprague-Dawley rats were homogenized (20 strokes at 300 rpm) with a
Teflon homogenizer in ice-cold buffer containing 1 mM EDTA,
0.32 M sucrose, 0.1 mM dithiothreitol, and 1 mM HEPES, pH 7.4, as described previously (34), and
cytoplasmic and microsomal fractions were isolated by differential
centrifugation. Briefly, tissue fragments and cellular debris were
removed by centrifugation at 500 × g for 10 min, and
supernatants were centrifuged at 20,000 × g for 20 min
to pellet intact mitochondria and nuclei. The supernatant was then
centrifuged at 100,000 × g for 1 h to obtain the
cytoplasmic fraction and the microsomal pellet enriched in ER and Golgi
membranes. Microsomal extracts (MSEs) were prepared by suspending
microsomes in ice-cold buffer (1:100, v/v) and centrifuging at
100,000 × g for 1 h. Microsomes were then
resuspended in buffer (1:2, v/v) and treated with various agents that
affect ER calcium release. At the end of the treatment, the suspension
was centrifuged at 100,000 × g for 1 h, and
supernatants were used as MSEs. For experiments involving incubation of
MSE with the cytoplasmic extract, 3 µl of MSE was added to 10 µl of
cytoplasmic extract, and mixtures were incubated at room temperature
for 1 h.
Immunoblot Analysis and Measurement of Intracellular Free Calcium
Levels--
Immunoblot analyses were performed using methods described
previously (35). Briefly, lanes of a 15% acrylamide gel were loaded
with cell homogenate, and proteins (100 µg of protein/lane) were
separated by electrophoresis and transferred to a nitrocellulose sheet.
The nitrocellulose sheet was reacted with primary antibody against
grp78 (1:4000 dilution; Stressgen, Inc.), and the primary antibody was
detected using horseradish peroxidase-conjugated secondary antibody
(1:20,000 dilution; Jackson ImmunoResearch Laboratories, Inc.) and a
chemiluminescence-based detection kit (ECL kit, Amersham Pharmacia
Biotech). [Ca2+]i in cultured neurons was
quantified by fluorescence imaging of the calcium indicator dye fura-2
as described previously (32). Briefly, cells were incubated for 30 min
in the presence of 2 µM fura-2 acetoxymethyl ester
(Molecular Probes, Inc.), washed with Locke's buffer, and incubated
for 40 min prior to imaging. For measurements of the intraluminal
Ca2+ concentration, microsomes were incubated for 30 min in
the presence of fura-FF acetoxymethyl ester. Cells and microsomes were
imaged on a Zeiss Axiovert microscope (40× oil immersion objective)
coupled to an Attofluor imaging system. [Ca2+]i
in 12-20 neuronal cell bodies or four to six microsomal clusters per
microscope field was monitored prior to and after exposure of cells to glutamate.
Inhibition of IP3 Receptor-mediated Calcium Release
Decreases and Thapsigargin Increases NF- Inhibition of IP3 Receptor-mediated Calcium Release
Inhibits Inducible NF- Microsomal Extract Regulates Cytoplasmic NF-
To determine the identity of the major NF-
Microsomal preparations were treated with thapsigargin and calcium in
the absence or presence of XeC for 1 h and centrifuged, and 3 µl
of supernatant (MSE) was added to 10 µl of the cytoplasmic fraction.
After 1 h at room temperature, samples were examined for NF-
A recent report suggested that NF- A Signal Other than Calcium Mediates NF- NF- We found that a concentration of XeC reported to selectively block
IP3 calcium channels in a noncompetitive manner (36-38) can decrease the basal level of NF- Activation of TNF receptors results in recruitment of TNF
receptor-associated death domain, which acts as an adaptor for TNF receptor-associated factor-2, which in turn mediates NF- The cell-free system that was used as a model in our studies consisted
of a membrane-free cytoplasmic fraction and an extract taken from
ER-rich microsomes after treatment with agents that affect ER calcium
pools. We manipulated the ER calcium regulatory proteins with specific
agents: thapsigargin to irreversibly inhibit Ca2+-ATPases
and to decrease intramicrosomal calcium maximally, XeC to specifically
block calcium release from IP3 stores, IP3 to mimic the in vivo system of IP3 receptor
activation, and ryanodine to examine the other major pool of ER
calcium. Treatment of microsomes with thapsigargin resulted in release
into MSE of a factor(s) that activates cytoplasmic NF- Addition of thapsigargin and XeC directly to the cytoplasmic extract
did not alter NF- Increasing data implicate cell calcium-regulating mechanisms as having
a central role in governing NF-B (NF-
B) plays critical roles in neuronal survival and
plasticity and in activation of immune responses. The activation of
NF-
B has been closely associated with changes in intracellular
calcium levels, but the relationship between the two remains unclear.
Here we report that inhibition of endoplasmic reticulum (ER)
D-myo-inositol 1,4,5-trisphosphate (IP3)-gated calcium release caused decreased basal NF-
B
DNA-binding activity in cultured rat cortical neurons. Activation of
NF-
B in response to tumor necrosis factor-
and glutamate was
completely abolished when IP3 receptors were blocked, and
NF-
B activation in response to depletion of ER calcium by
thapsigargin treatment was also decreased by IP3 receptor
blockade. We further investigated the relationship between
IP3 receptor activation and NF-
B activity using a
cell-free system. Microsomes enriched in the ER were isolated from
adult rat cerebral cortex, resuspended, and treated with agents that
induce or inhibit ER calcium release. They were then recentrifuged, and
the supernatant was added to cytoplasmic extract isolated from the same
source tissue. We found that microsomes released an NF-
B-stimulating
signal in response to activation of IP3 receptors or
inhibition of the ER Ca2+-ATPase, but not in response to
ryanodine. Studies of intact cells and cell-free preparations indicated
that the signal released from the ER was not calcium and was heat- and
trypsin-sensitive. Our data suggest that activation of IP3
receptors is required for a major component of both constitutive and
inducible NF-
B binding activity in neurons and that decreasing ER
intraluminal calcium levels triggers release of a diffusible
NF-
B-activating signal from the ER.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B1 is a member of the
NF-
B/Rel family, which includes p50, p52, p65 (RelA), c-Rel, and
RelB proteins (1, 2). Prototypical NF-
B is a p50-p65 heterodimer
that is retained in the cytoplasm of unstimulated cells in an inactive form by the inhibitory protein I
B
. In response to various
stimuli, I
B
is phosphorylated, rapidly ubiquitinated, and
subsequently proteolyzed by a 26 S proteasome complex. The degradation
of I
B
unmasks the nuclear localization signal of the NF-
B
heterodimer, which then translocates into the nucleus, where it binds
to its cognate sequence on DNA and thereby regulates gene
transcription. In the brain, NF-
B is constitutively active in many
neurons (3), wherein its activity may be further increased by
excitation (4, 5) and ischemia (6, 7) and in neurodegenerative
disorders (8-10). Because activation of NF-
B is associated with
cell injury and death, it has been proposed that this transcription
factor contributes to the neuronal death process (6, 11). However, results from cell culture and in vivo studies have
demonstrated that activation of NF-
B in neurons represents a highly
protective response that promotes cell survival and plasticity
(12-16).
B include TNF-
(12, 17, 18),
interleukin-1
(19), glutamate (20), nerve growth factor (21, 22),
and secreted amyloid precursor protein (23). Increased NF-
B activity
may also occur in response to oxidative stress, perturbed calcium
homeostasis, and DNA damage (2, 24-26). However, the mechanisms that
regulate NF-
B activity in neurons under basal conditions and in
response to various physiological and pathological conditions are not
known. Changes in the concentration of intracellular free
calcium ([Ca2+]i) regulate numerous functions in
neurons, including synaptic plasticity and cell survival (27, 28).
Influx through the plasma membrane has long been regarded as the main
source of Ca2+-mediated intracellular signals. However, the
significance of Ca2+ released from internal stores such as
the ER has become increasingly apparent. In neurons, the ER is
distributed throughout the cytoplasm and represents a large and
releasable pool of calcium. Increased [Ca2+]i can
stimulate release of Ca2+ stored in the ER, a phenomenon
known as calcium-dependent calcium release that is
regulated by the ER-resident IP3- and ryanodine-sensitive receptor calcium channels (29). The intracellular signaling molecule
IP3, the ligand for ER IP3 receptors, is
generated by activation of phospholipase C and cleavage of
phosphoinositol bisphosphate into diacylglycerol and IP3.
Calcium regulates the sensitivity of these channels to IP3,
increasing the calcium current (28). Therefore, alterations in
[Ca2+]i will affect the activity of the ER
IP3 receptor and thus affect cellular events regulated by
this receptor.
B
DNA-binding activity and NF-
B-dependent gene
expression (30, 31). Because IP3 receptors are highly
sensitive to many of the same stimuli that activate NF-
B, and
alterations in ER function are associated with increased NF-
B
binding and transcription, we examined the relationship between ER
IP3 receptors and NF-
B activation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was purchased from
Pepro-Tech Inc. (Rocky Hill, NJ); and thapsigargin and BAPTA free acid
were purchased from Molecular Probes, Inc. (Eugene, OR). Each reagent
was prepared as a 500× or 1000× stock in an appropriate solvent
(saline or dimethyl sulfoxide).
70 °C until taken for assay. The
protein content of the extract (supernatant) was measured by the
Bradford method (Bio-Rad). For EMSA, equal amounts of protein were
incubated in a 20-µl reaction mixture containing 20 µg of bovine
serum albumin; 1 µg of poly(dI-dC); 2 µl of buffer containing 20%
glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25%
Nonidet P-40, 2 mM dithiothreitol, 0.1%
phenylmethylsulfonyl fluoride, and 20 mM HEPES, pH 7.9; 4 µl of buffer containing 20% Ficoll 400, 300 mM KCl, 10 mM dithiothreitol, 0.1% phenylmethylsulfonyl fluoride, and
100 mM HEPES, pH 7.9; and 20,000-50,000 cpm of
32P-labeled oligonucleotide (Promega, Madison, WI)
corresponding to either an NF-
B-binding site (5'-AGT TGA GGG
GAC TTT CCC AGG C-3') or a CREB-binding site (5'-AGA GAT TGC CTG ACG
TCA GAG AGC TAG-3'). After 20 min at room temperature, reaction
products were separated on a 7% nondenaturing polyacrylamide gel.
Competition experiments were performed by incubating extracts with
labeled oligonucleotide probe in the presence of 100-fold excess
unlabeled NF-
B, CREB, or AP-1 oligonucleotide. To characterize the
NF-
B complexes, supershift assays were performed. Extracts were
preincubated for 45 min at room temperature with antibodies recognizing
either p50 or p65 subunits of NF-
B (Santa Cruz Biotechnology, Santa Cruz, CA). The mixtures were further incubated with
32P-labeled probe and resolved as described above.
Radioactivity of dried gels was detected by exposure to Kodak X-Omat
film, and images on the developed film were scanned into a computer
using a UMAX 1200s scanner. Densitometry was performed using Image
software (Scion Corp., Frederick, MD). Paint Shop Pro software (Jasc
Inc., Minneapolis, MN) was used for preparation of the final figures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B Activity in Cortical
Neurons--
The possible involvement of ER pools of calcium regulated
by IP3 receptors in controlling basal levels of NF-
B
activation was investigated using XeC, a membrane-permeable blocker of
IP3-induced Ca2+ release (36-38). Conversely,
to release the total pool of ER luminal calcium, we used thapsigargin,
a specific inhibitor of the ER Ca2+-ATPase (39, 40).
Cultured primary cortical neurons were treated with 1 µM
XeC, 100 nM thapsigargin, or vehicle for 6 h, and
protein extracts were analyzed for NF-
B DNA-binding activity. XeC
caused a significant decrease in the basal levels of activated NF-
B relative to control levels (Fig. 1,
a and b). The decrease in NF-
B activation was
not due to interference of XeC with the assay used to detect NF-
B
activity as determined by addition of XeC to the reaction (data not
shown). As previously reported (41), treatment with thapsigargin
produced a large and significant increase in NF-
B activity (Fig. 1,
a and b). To determine if the effects of XeC and
thapsigargin were specific for NF-
B, we measured the levels of CREB
DNA-binding activity. CREB is a transcription factor thought to play a
role in synaptic plasticity and neuronal survival (42, 43), and
activation of CREB is thought to be primarily regulated by calcium
(44). In contrast to their effects on NF-
B activity, XeC and
thapsigargin each produced a small increase in CREB binding activity
(Fig. 1a). We next determined whether IP3
receptor blockade would alter the thapsigargin-induced activation of
NF-
B. XeC was added to the cortical cultures 30 min before thapsigargin, and binding activity was analyzed after 6 h;
IP3 receptor blockade inhibited the ability of thapsigargin
treatment to activate NF-
B (Fig.
2a).
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Fig. 1.
Effects of xestospongin C and thapsigargin on
basal levels of NF- B and CREB DNA-binding
activities in cerebral cortical cell cultures. a, neurons
were treated with vehicle (control (C)), 100 nM
thapsigargin (Tg), or 1 µM xestospongin C
(X). After 6 h, total cell extracts were prepared, and
EMSAs were performed using oligonucleotides specific for NF-
B- or
CREB-binding sites. b, films from four separate gel-shift
assays were scanned, and densitometric analyses were performed. Data
are the means ± S.E. (n = 4). *,
p < 0.05 versus control (Con)
(analysis of variance with Scheffe's post-hoc test).
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Fig. 2.
Xestospongin C inhibits induction of
NF- B activation. a, shown are the
effects of 100 nM thapsigargin in the absence
(Tg) or presence (Tg/X) of 1 µM XeC
on NF-
B DNA-binding activity. b-d, following treatment
with either vehicle or 1 µM XeC for 30 min, cells were
incubated with TNF-
, thapsigargin, or glutamate for 6 h; total
cell extracts were prepared; and EMSAs were performed using
oligonucleotides specific for NF-
B- or CREB-binding sites.
b, shown are the effects of 100 ng/ml TNF-
in the absence
or presence of XeC (X) on NF-
B and CREB binding
activities. c, films from three separate gel-shift assays of
cells treated with vehicle (control (C)), 100 ng/ml TNF-
(T), or 1 µM XeC for 30 min prior to TNF-
treatment (T+X) were scanned, and densitometric analyses
were performed. Data are the means ± S.E. (n = 3). *, p < 0.01 compared with control
(Con); #, p < 0.01 compared with TNF-
alone (analysis of variance with Scheffe's post-hoc test).
d, shown are the effects of 20 µM glutamate in
the absence (G) or presence (G/X) of 1 µM XeC on NF-
B DNA-binding activity.
B Activity in Cortical Neurons--
NF-
B
is not only a constitutively active transcription factor, but is also
highly inducible in neurons by several stimuli, including the
excitatory neurotransmitter glutamate (20, 45, 46) and the
pro-inflammatory cytokine TNF-
(12). We therefore determined whether
activation of IP3 receptor-mediated calcium channels is
necessary for NF-
B DNA-binding activity induced by glutamate and
TNF-
. Cells were pretreated for 30 min with vehicle or XeC and were
then exposed to 100 ng/ml TNF-
or 20 µM glutamate for
6 h. TNF-
produced a significant increase in NF-
B activity that was completely abolished by pretreatment with XeC (Fig. 2, b and c). This effect was specific for NF-
B
because neither TNF-
nor XeC affected the binding activity of CREB
(Fig. 2b). Induction of NF-
B DNA-binding activity was
observed with glutamate treatment, and pretreatment with XeC completely
abolished the activation of NF-
B by this excitatory neurotransmitter
(Fig. 2d). Cells pretreated with XeC prior to exposure to
glutamate demonstrated a level of NF-
B activity lower than that of
control neurons exposed to glutamate. Measurement of
[Ca2+]i in neurons by imaging of the calcium
indicator dye fura-2 showed that XeC treatment resulted in only a small
attenuation of the glutamate-induced increase in
[Ca2+]i. Values were as follows: control, 86 ± 14 nM; 1 µM XeC (5-min exposure), 96 ± 24 nM; 20 µM glutamate (peak response), 618 ± 87 nM; and 1 µM XeC plus 20 µM glutamate (peak response), 410 ± 49 nM (values are the means ± S.D. of determinations
made in four cultures with measurements made in at least 12 neurons/culture).
B Activity in an ER
Calcium Channel-dependent Manner--
Our results to this
point indicated that ER IP3-sensitive calcium pools can
regulate NF-
B activity in neurons. To gain further insight into the
mechanism behind this, we used an in vitro cell-free system
to separate cytoplasmic pools of inactive NF-
B from the ER,
therefore allowing us to study ER-derived signals that might modulate
NF-
B activity. Microsomal preparations were isolated as illustrated
(Fig. 3a), and tests were
conducted to establish their content of ER. Immunoblot analysis (Fig.
3b) of grp78, an ER-resident chaperone protein, performed on
the various fractions demonstrated that the microsomal fraction was
highly enriched in this ER marker. Moreover, measurements of the
concentration of intramicrosomal calcium and the subsequent reduction
in this value with exposure of the microsomes to IP3
demonstrated high levels of functional IP3 receptors in
this preparation (Fig. 3c).
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Fig. 3.
Subcellular fractionation protocol and
characterization of microsomes and cytosolic
NF- B proteins. a, shown are the
microsomal, MSE, and cytoplasmic extract isolation protocols.
XsC, xestospongin C; Thaps, thapsigargin.
b, equal amounts of protein from the subcellular fractions
were separated by SDS-polyacrylamide gel electrophoresis and probed
with an antibody against the ER-resident protein grp78. Similar results
were obtained in a separate experiment. Super, supernatant.
c, the intraluminal calcium concentration was measured prior
to and after exposure to 10 µM IP3. Values
(nanomolar) are the average of measurements made in two separate
experiments. d, supernatant fractions from the cortex
following a 100,000 × g centrifugation for 1 h
(cytoplasmic fraction (Cyt)) were incubated for 45 min with
anti-p50 antibody (Ab), anti-p65 antibody, or a combination
of anti-p50 and anti-p65 antibodies. Binding activity and band position
were then analyzed by EMSA. The four sites of NF-
B binding
identified are referred to as band A (A), band B
(B), supershifted band 1 (ss 1), and supershifted
band 2 (ss 2).
B-binding complexes found
in the cytoplasmic fraction, supernatants from the 100,000 × g centrifugation were incubated with anti-p50 antibody,
anti-p65 antibody, or a combination of anti-p50 and anti-p65 antibodies for 45 min. There were two major bands identified in these
experiments, bands A and B (Fig. 3d). Addition of anti-p50
antibody shifted band A in its entirety to a much higher position,
called supershift 1, and shifted the lower band to a slightly higher
position, called supershift 2. Band A was also greatly diminished in
intensity by anti-p50 antibody treatment. Incubation with anti-p65
antibody caused a disappearance of band A, but had no apparent effect
on band B. Incubation with both anti-p50 and anti-p65 antibodies caused
a complete shift upward of band A to the supershift 1 position and
shifted band B slightly upwards and reduced its intensity. Thus, band A
is the p50-p65 heterodimer, and band B contains p50-p50 homodimers. A
binding pattern identical to that seen in the cytoplasmic extracts of
this study was previously seen in whole cell extracts of cultured rat
cortical neurons (47).
B
DNA-binding activity. Significant increases in NF-
B activity were
observed following addition of MSE, thus indicating the presence of a
microsome-derived NF-
B-stimulating signal (Fig. 4, a and b). MSE
from thapsigargin-treated microsomes caused a significant enhancement
of this binding activity (Fig. 4, a and b). MSE
from XeC-treated microsomes did not elevate NF-
B activity, and
treatment with XeC reduced by ~50% the thapsigargin-stimulated NF-
B activity (Fig. 4, a and b).
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Fig. 4.
Evidence that an
NF- B-activating factor is released from the ER
in response to calcium release. a, shown are the effects of
the microsomal extract on NF-
B binding activity. Microsomes were
pretreated for 30 min with vehicle or 1 µM XeC
(X) and then incubated for 1 h with vehicle (control
(C)), 100 nM thapsigargin (Tg), or 10 µM Ca2+. The microsome suspension was
centrifuged at 100,000 × g, and 3 µl of MSE was
added to 10 µl of cytoplasmic fraction. After incubation for 1 h
at 37 °C, NF-
B DNA-binding activity was determined. As a control,
the cytoplasmic fraction without addition of microsomes
(Cyt) was also analyzed. b, exposures from four
different experiments were scanned, and densitometry was performed.
Data are the means ± S.E. (n = 4). *,
p < 0.05, and **, p < 0.01 compared
with the cytoplasm alone; +, p < 0.05 compared with
thapsigargin alone; #, p < 0.05 compared with
cytoplasmic extract/MSE (Cyt/MSE) (analysis of variance with
Scheffe's post-hoc test). c, the cytoplasmic fraction, MSE,
or pelleted microsomes (Pellet) were treated with vehicle, 1 µM XeC, or 100 nM thapsigargin for 1 h
prior to being analyzed for NF-
B DNA-binding activity.
B may localize to the ER (48),
raising the possibility that the NF-
B-stimulating agent released by
microsomes was the activated transcription factor itself. To examine
this possibility, EMSAs were performed on cytoplasmic fractions, MSEs,
and microsomal pellets following treatment with vehicle, thapsigargin,
or XeC. Although there was binding activity found in MSE and
microsomes, this binding was >10-fold less than that seen in the
cytoplasm. Neither thapsigargin nor XeC had any effect on NF-
B
DNA-binding activity levels in any of the three fractions tested (Fig.
4c). We then assessed the necessity of the ER in the
microsomal preparation for NF-
B activation by treating isolated
cytoplasmic extracts from whole adult rat cortices with 100 nM thapsigargin or 1 µM XeC for 1 h at
room temperature prior to analysis by EMSA. As shown in Fig.
5a, neither thapsigargin nor
XeC directly affected NF-
B activity relative to control levels, thus
confirming that the presence of the ER is necessary for the activation
of NF-
B.
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Fig. 5.
Activation of NF- B
in response to calcium release from the ER is not mediated by calcium
itself. a, aliquots of cytoplasmic fractions were left
untreated (control (C)) or were treated with 100 nM thapsigargin (Tg) or 1 µM
xestospongin C (X) for 1 h, and NF-
B DNA-binding
activity was determined by EMSA. b, aliquots of cytoplasmic
fractions were left untreated (control (Con)) or were
treated with calcium at concentrations from 1 µM to 10 mM for 1 h, and NF-
B binding activity was
determined by EMSA. c, aliquots of cytoplasmic fractions
were left untreated or were treated for 1 h with 10 µM Ca2+, 10 µM BAPTA
(B), or Ca2+ plus BAPTA, and NF-
B DNA-binding
activity was determined.
B Activation in Response
to ER Calcium Depletion--
We next asked whether NF-
B could be
induced by calcium directly. Aliquots of the cytoplasmic fraction were
treated with calcium at concentrations ranging from 1 µM
to 10 mM for 1 h, followed by analysis of NF-
B
binding by EMSA. As shown in Fig. 5b, calcium did not
affect NF-
B DNA-binding activity at any concentration tested.
These same extracts were analyzed for CREB binding activity; calcium
greatly enhanced CREB binding, which likely results from stimulation of
calcium-activated kinases (49). These data suggest that although the
elevation of calcium in the cytoplasm by itself is able to induce CREB,
it is not sufficient to cause NF-
B activation. Conversely, chelation
of calcium did not produce a decline in NF-
B DNA-binding activity.
Aliquots of the cytoplasmic fraction were treated for 1 h with 10 µM calcium, a 10 µM concentration of the
calcium chelator BAPTA free acid, or a combination of the two. As shown
in Fig. 5c, BAPTA alone or in combination with calcium had
no effect on the levels of NF-
B activity present in the untreated cytoplasm. We next treated microsomes with thapsigargin for 1 h in
presence of BAPTA. Calcium chelation did not affect the ability of MSE
from thapsigargin-treated microsomes to activate NF-
B (Fig.
6a). To test the relative
importance of the IP3- and ryanodine-sensitive calcium
pools, microsomes were treated with 10 nM ryanodine for 1 h, and then MSE was isolated and added to the cytoplasmic
extract for 1 h. The concentration of ryanodine used was based on
data in the literature and our previous study demonstrating the
presence of functional ryanodine receptors in intact neurons and
microsomes from similar neuronal cell cultures (50). MSE from
ryanodine-treated microsomes exhibited no greater NF-
B-stimulating
activity than did MSE from untreated microsomes (Fig. 6a).
Since XeC was very effective in blocking NF-
B activation, we sought
to determine if the NF-
B-activating factor could be generated by
direct addition of IP3 to microsomes. As shown in Fig. 6
(b and c), IP3 addition led to an
increased level of NF-
B activity that was similar to that seen with
thapsigargin; pretreatment with XeC abolished this effect. Treatment of
MSE from IP3-treated microsomes with BAPTA had no effect on
the ability of the extract to stimulate NF-
B binding in cytoplasmic
extracts (data not shown). The NF-
B-activating factor released from
microsomes was inactivated by heating MSE to 100 °C (Fig. 6,
a-c) and was also inactivated by trypsin treatment (Fig.
6a). Collectively, these results indicate that a diffusible factor that is heat- and trypsin-sensitive is released from the ER, in
response to stimuli that induce calcium release from
IP3-sensitive stores, and is capable of activating
NF-
B.
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Fig. 6.
The NF- B-activating
factor released from microsomes is heat-stable and trypsin-sensitive.
a, microsome suspensions were treated for 1 h with
vehicle (control (C)) or 100 nM thapsigargin
after either no pretreatment (Tg) or pretreatment for 30 min
with 10 µM BAPTA (Tg/B) (left
panel). In a separate experiment, microsome suspensions were
treated for 1 h with vehicle or 10 nM ryanodine
following either no pretreatment (R) or pretreatment for 30 min with 10 µM XeC (R+X) (right
panel). MSE was then added to the cytoplasmic fraction for 1 h, and NF-
B binding activity was determined. As a control, an
aliquot of the cytoplasmic fraction not exposed to MSE was also
analyzed (Cyt). Separate aliquots of MSE from
thapsigargin-treated microsomes were either heated at 100 °C for 5 min (Tg/100°) or with trypsin (Tg/T; 0.2%,
~400 units for 5 min at 37 °C; reaction was stopped by addition of
0.2% trypsin inhibitor and transfer in ice). b, microsome
suspensions were treated for 1 h with vehicle or 10 µM IP3 following either no pretreatment
(IP3) or pretreatment for 30 min with 1 µM XeC (X/IP3). A separate aliquot of
MSE from IP3-treated microsomes was heated at 100 °C for
5 min (IP3/100°). MSE was then added to
the cytoplasmic fraction for 1 h, and NF-
B binding activity was
determined by EMSA. c, films from three different EMSAs were
scanned, and densitometric analyses were performed. Data are the
means ± S.E. (n = 3). *, p < 0.01 compared with control (Con); **, p < 0.01 compared with IP3 alone (analysis of variance with
Scheffe's post-hoc test).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B is believed to play important roles in brain development
and in neurodegenerative disorders (2, 51). In unstimulated cells,
NF-
B subunits are present in the cytoplasm, including neurites
and synapses, in an inactive form complexed with I
B
(45, 52).
Increases in cytosolic calcium levels had previously been associated
with activation of NF-
B (41, 53-57); but it was not established if
and how calcium itself activates NF-
B, and increased
[Ca2+]i is an event common to several different
NF-
B-activating pathways (24). Studies of other transcription
factors such as CREB have shown that the source of calcium entry may
affect which genes are transcribed through distinct DNA regulatory
elements (44). To help elucidate the relationship between ER calcium release through IP3 receptors specifically and NF-
B
activation, we conducted a series of experiments in cortical neurons
and found that 1) inhibition of IP3 receptor channels
reduces both basal and thapsigargin-induced NF-
B DNA-binding
activity; 2) the decrease in NF-
B activity is independent of changes
in intracellular calcium levels; 3) extracts from microsomes release a
diffusible NF-
B-stimulating signal that is augmented by
IP3 and thapsigargin and diminished by XeC; and 4) the
ER-derived NF-
B-activating signal is not calcium. This factor
appears to be regulated by IP3 receptor activity and/or the
filling state of the IP3-sensitive pools of intracellular calcium. Thus, our data indicate that it is not the calcium release from ER stores that activates cytoplasmic NF-
B, but rather the decrease in intraluminal calcium that signals release of a diffusible NF-
B-activating factor from the ER.
B activation. It has been shown
that the constitutively active form of NF-
B in neurons is
transcriptionally active and may drive basal expression of several
different genes (58). Although the basis of this constitutive activity
has been unclear, our data suggest that it is dependent upon
IP3 receptor activation. It has been previously shown that there is a spontaneous release of calcium from
IP3-sensitive calcium stores (59) resulting from a basal
level of IP3 production and the ER luminal content of
calcium. It should be emphasized that IP3 receptors can be
activated in response to a broad range of stimuli present in neuronal
cultures. IP3 is endogenously produced in cultures as
result of the actions of growth factors, neuropeptides, and other
bio-molecules coupled to phospholipase C-linked
GTP-binding proteins. Activation of purinergic receptors and
glutamate receptors, depolarization of the membranes, and synaptic
activity will all activate the IP3 receptors present in the
ER. According to our data, if the constitutive activity of
IP3 receptors is blocked, the basal activation of NF-
B
in neurons is largely abolished, strongly suggesting that the two
events are functionally related.
B activation (60-62). In our model, XeC significantly inhibited the ability of
TNF-
to induce NF-
B. TNF-
does not evoke calcium transients over short time periods (63); however, it does activate phospholipase C
(64) and can thereby activate ER IP3 receptors even in the absence of elevated intracellular calcium levels (65). Supporting this
is the recent finding of Liu et al. (66) that activation of
NF-
B in astrocytes by TNF-
is dependent upon metabotropic P2Y
receptors. Glutamate, which causes a large influx of calcium in
neurons, also greatly induces NF-
B DNA-binding activity (20, 67).
XeC was able to totally abolish this induction by glutamate, although
XeC treatment only partially attenuated the increase in intracellular
calcium levels caused by glutamate. Glutamate induces ER calcium
release by increasing intracellular calcium (68-70) and by stimulating
IP3 production through phospholipase C activation (71, 72).
Therefore, each of the inducers of NF-
B examined in this study
(glutamate, TNF-
, and thapsigargin) have in common the ability to
initiate calcium release from ER IP3-sensitive stores.
B to a level
3-fold that of the control level. This level of activation is
comparable to that seen in intact cultured cells exposed to agents that
release ER calcium (Refs. 30 and 73 and this study). Conversely,
treatment of microsomes with XeC totally abolished the stimulatory
effect of MSE, resulting in NF-
B binding levels that were no
different from the cytoplasm alone.
B DNA-binding activity, indicating that these
factors must work through microsomes to stimulate NF-
B. Furthermore,
the fact that neither calcium nor BAPTA was able to directly affect
NF-
B DNA-binding activity shows that the calcium released from the
microsomes is not the factor that regulates NF-
B. Indeed, the role
calcium plays in this paradigm seems to be as an intraluminal signal,
such that the depletion of calcium from the IP3 pool is a
signal for release of the NF-
B-activating factor. This is supported
by the observation that direct activation of IP3 receptors
on microsomes using IP3 itself led to MSE that had
NF-
B-stimulating properties similar to those seen with thapsigargin. In addition, pretreatment of microsomes with XeC completely abolished this effect. Release of calcium by ER IP3 receptors results
in a decrease in the pool of calcium in those ER stores, and it is this
calcium depletion that apparently leads to release of the diffusible
factor that stimulates NF-
B activity. Microsomes pretreated with XeC
followed by calcium produced no NF-
B-stimulating activity, indicating that although calcium can directly modulate ER calcium release, it has no effect when IP3 receptors are blocked.
Addition of ryanodine to microsomes did not result in an increase in
NF-
B-stimulating activity in MSE, indicating that it is the ER
IP3 pool specifically that regulates release of this
factor. These findings reveal a heretofore unknown mechanism for
modulation of NF-
B activity by the ER and suggest the presence of a
novel factor released by microsomes that activates NF-
B. The
identity of this factor remains to be determined, but it could
conceivably be a protein because it is heat- and trypsin-sensitive and
because previous studies have shown that luminal proteins are released
from the ER in response to calcium depletion (74).
B activity. We have shown in our
in vitro studies that calcium per se does not
change NF-
B binding. However, our cell-free system examined only the NF-
B-signaling pathway from the ER to NF-
B activation. Indeed, in
the whole cell, the hypothesis that it is the filling state of the ER
IP3 pool that signals NF-
B activation would place
calcium as a major upstream effector. Both IP3 and
ryanodine receptors are sensitive to [Ca2+]i, as
is the ER Ca2+-ATPase. In addition, IP3
production is sensitive to calcium levels (75, 76). In fact, in the
absence of calcium, phospholipase C-mediated production of
IP3 drops precipitously (77). Our data suggest that an
array of signals ranging from neurotransmitters to neurotrophic factors
and cytokines may modulate NF-
B activity, in part, by affecting ER
calcium release. Both ER calcium release (78-80) and NF-
B
activation (13, 16, 52, 81) play pivotal roles in regulating synaptic
plasticity and survival of neurons during development of injury and
disease conditions. In addition, an ER overload (unfolded protein)
response occurs in cells exposed to a variety of stressors (82) and
results in NF-
B activation by a mechanism involving release of
calcium from the ER (41). Our findings therefore identify a novel
signaling pathway whereby activation of IP3 receptors
results in generation of a diffusible signal that activates a
transcription factor known to play important roles in neuronal
plasticity and survival.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants NRSA467305 (to G. W. G.) and NS39184 (to J. D. G.) and by NIA, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These two authors contributed equally to this work.
** To whom correspondence should be addressed: Lab. of Neurosciences, NIA, GRC 4F02, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8463; Fax: 410-558-8465; E-mail: mattsonm@grc.nia.nih.gov.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M101315200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor-
B;
TNF, tumor necrosis factor;
[Ca2+]i, intracellular free calcium concentration;
ER, endoplasmic reticulum;
IP3, D-myo-inositol
1,4,5-trisphosphate;
XeC, xestospongin C;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
EMSA, electrophoretic mobility shift assay;
CREB, cAMP-responsive element-binding protein;
MSE, microsomal
extract.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Baeuerle, P. A., and Baltimore, D. (1996) Cell 87, 13-20[Medline] [Order article via Infotrieve] |
2. | Mattson, M. P., Culmsee, C., Yu, Z., and Camandola, S. (2000) J. Neurochem. 74, 443-456[CrossRef][Medline] [Order article via Infotrieve] |
3. | Kaltschmidt, C., Kaltschmidt, B., Neumann, H., Wekerle, H., and Baeuerle, P. A. (1994) Mol. Cell. Biol. 14, 3981-3992[Abstract] |
4. | Prasad, A. V., Pilcher, W. H., and Joseph, S. A. (1994) Neurosci. Lett. 170, 145-148[Medline] [Order article via Infotrieve] |
5. | Rong, Y., and Baudry, M. (1996) J. Neurochem. 67, 662-668[Medline] [Order article via Infotrieve] |
6. | Clemens, J. A., Stephenson, D. T., Dixon, E. P., Smalstig, E. B., Mincy, R. E., Rash, K. S., and Little, S. P. (1997) Brain Res. Mol. Brain Res. 48, 187-196[CrossRef][Medline] [Order article via Infotrieve] |
7. | Schneider, A., Martin-Villalba, A., Weih, F., Vogel, J., Wirth, T., and Schwaninger, M. (1999) Nat. Med. 5, 554-559[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Hunot, S.,
Brugg, B.,
Ricard, D.,
Michel, P. P.,
Muriel, M. P.,
Ruberg, M.,
Faucheux, B. A.,
Agid, Y.,
and Hirsch, E. C.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7531-7536 |
9. |
Kaltschmidt, B.,
Uherek, M.,
Volk, B.,
Baeuerle, P. A.,
and Kaltschmidt, C.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2642-2647 |
10. |
Akama, K. T.,
Albanese, C.,
Pestell, R. G.,
and Van Eldik, L. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5795-5800 |
11. |
Grilli, M.,
Pizzi, M.,
Memo, M.,
and Spano, P.
(1996)
Science
274,
1383-1385 |
12. | Barger, S. W., Horster, D., Furukawa, K., Goodman, Y., Krieglstein, J., and Mattson, M. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9328-9332[Abstract] |
13. | Mattson, M. P., Goodman, Y., Luo, H., Fu, W., and Furukawa, K. (1997) J Neurosci. Res. 49, 681-697[CrossRef][Medline] [Order article via Infotrieve] |
14. | Taglialatela, G., Robinson, R., and Perez-Polo, J. R. (1997) J. Neurosci. Res. 47, 155-156[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Yu, Z.,
Zhou, D.,
Bruce-Keller, A. J.,
Kindy, M. S.,
and Mattson, M. P.
(1999)
J. Neurosci.
19,
8856-8865 |
16. | Albensi, B. C., and Mattson, M. P. (2000) Synapse 35, 151-159[CrossRef][Medline] [Order article via Infotrieve] |
17. | Hazan, U., Thomas, D., Alcami, J., Bachelerie, F., Israel, N., Yssel, H., Virelizier, J. L., and Arenzana-Seisdedos, F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7861-7865[Abstract] |
18. | Swingler, S., Easton, A., and Morris, A. (1992) AIDS Res. Hum. Retroviruses 8, 487-493[Medline] [Order article via Infotrieve] |
19. | Nonaka, M., and Huang, Z. M. (1990) Mol. Cell. Biol. 10, 6283-6289[Medline] [Order article via Infotrieve] |
20. | Kaltschmidt, C., Kaltschmidt, B., and Baeuerle, P. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9618-9622[Abstract] |
21. | Carter, B. D., Kaltschmidt, C., Kaltschmidt, B., Offenhauser, N., Bohm- Matthaei, R., Baeuerle, P. A., and Barde, Y. A. (1996) Science 272, 542-545[Abstract] |
22. |
Maggirwar, S. B.,
Sarmiere, P. D.,
Dewhurst, S.,
and Freeman, R. S.
(1998)
J. Neurosci.
18,
10356-10365 |
23. | Barger, S. W., and Mattson, M. P. (1996) Mol. Brain Res. 40, 116-126[Medline] [Order article via Infotrieve] |
24. | Ginn-Pease, M. E., and Whisler, R. L. (1998) Free Radic. Biol. Med. 25, 346-361[CrossRef][Medline] [Order article via Infotrieve] |
25. | Gius, D., Botero, A., Shah, S., and Curry, H. A. (1999) Toxicol. Lett. 106, 93-106[CrossRef][Medline] [Order article via Infotrieve] |
26. | Mercurio, F., and Manning, A. M. (1999) Oncogene 18, 6163-6171[CrossRef][Medline] [Order article via Infotrieve] |
27. | Kennedy, M. B. (1989) Trends Neurosci. 12, 417-420[CrossRef][Medline] [Order article via Infotrieve] |
28. | Berridge, M. J. (1998) Neuron 21, 13-26[Medline] [Order article via Infotrieve] |
29. | Verkhratsky, A., and Shmigol, A. (1996) Cell Calcium 19, 1-14[Medline] [Order article via Infotrieve] |
30. | Pahl, H. L., and Baeuerle, P. A. (1995) EMBO J. 14, 2580-2588[Abstract] |
31. | Pahl, H. L., and Baeuerle, P. A. (1997) Trends Biochem. Sci. 22, 63-67[CrossRef][Medline] [Order article via Infotrieve] |
32. | Mattson, M. P., Lovell, M. A., Furukawa, K, and Markesbery, W. R. (1995) J. Neurochem. 65, 1740-1751[Medline] [Order article via Infotrieve] |
33. | Baeuerle, P. A., and Baltimore, D. (1988) Cell 53, 211-217[Medline] [Order article via Infotrieve] |
34. | Chen, P. Y., Csutora, P., Veyna-Burke, N. A., and Marchase, R. B. (1998) Diabetes 47, 874-881[Abstract] |
35. |
Glazner, G. W.,
Chan, S. L.,
Lu, C.,
and Mattson, M. P.
(2000)
J. Neurosci.
20,
3641-3649 |
36. | Gafni, J., Munsch, J. A., Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733[Medline] [Order article via Infotrieve] |
37. |
Hu, Q.,
Deshpande, S.,
Irani, K.,
and Ziegelstein, R. C.
(1999)
J. Biol. Chem.
274,
33995-33998 |
38. |
Ma, H.-T.,
Patterson, R. L.,
van Rossum, D. B.,
Birnbaumer, L.,
Mikoshiba, K.,
and Gill, D. L.
(2000)
Science
287,
1647-1651 |
39. | Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., and Dawson, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2466-2470[Abstract] |
40. |
Davidson, G. A.,
and Varhol, R. J.
(1995)
J. Biol. Chem.
270,
11731-11734 |
41. | Pahl, H. L., and Baeuerle, P. A. (1996) FEBS Lett. 392, 129-136[CrossRef][Medline] [Order article via Infotrieve] |
42. | Stevens, C. F. (1994) Neuron 13, 769-770[Medline] [Order article via Infotrieve] |
43. | Huang, E. P., and Stevens, C. F. (1998) Essays Biochem. 33, 165-178[Medline] [Order article via Infotrieve] |
44. | Bading, H., Ginty, D. D., and Greenberg, M. E. (1993) Science 260, 181-186[Medline] [Order article via Infotrieve] |
45. | Kaltschmidt, C., Kaltschmidt, B., and Baeuerle, P. A. (1993) Mech. Dev. 43, 135-147[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Guerrini, L.,
Molteni, A.,
Wirth, T.,
Kistler, B.,
and Blasi, F.
(1997)
J. Neurosci.
17,
6057-6063 |
47. | Glazner, G. W., Camandola, S., and Mattson, M. P. (2000) J. Neurochem. 75, 101-108[CrossRef][Medline] [Order article via Infotrieve] |
48. | May, W., and Bisby, M. A. (1998) Brain Res. 797, 243-254[CrossRef][Medline] [Order article via Infotrieve] |
49. | Deisseroth, K., Bito, H., and Tsien, R. W. (1996) Neuron 16, 89-101[Medline] [Order article via Infotrieve] |
50. |
Chan, S. L.,
Mayne, M.,
Holden, C. P.,
Geiger, J. D.,
and Mattson, M. P.
(2000)
J. Biol. Chem.
275,
18195-18200 |
51. | O'Neill, L. A. J., and Kaltschmidt, C. (1997) Trends Neurosci. 20, 252-258[CrossRef][Medline] [Order article via Infotrieve] |
52. | Meberg, P. J., Kinney, W. R., Valcourt, E. G., and Routtenberg, A. (1996) Mol. Brain Res. 38, 179-190[Medline] [Order article via Infotrieve] |
53. | Kanno, T., and Siebenlist, U. (1996) J. Immunol. 157, 5277-5283[Abstract] |
54. | Sen, C. K., Roy, S., and Packer, L. (1996) FEBS Lett. 385, 58-62[CrossRef][Medline] [Order article via Infotrieve] |
55. | Shatrov, V. A., Lehmann, V., and Chouaib, S. (1997) Biochem. Biophys. Res. Commun. 234, 121-124[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Lazaar, A. L.,
Amrani, Y.,
Hsu, J.,
Panettieri, R. A., Jr.,
Fanslow, W. C.,
Albelda, S. M.,
and Pure, E.
(1998)
J. Immunol.
161,
3120-3127 |
57. |
Quinlan, K. L.,
Naik, S. M.,
Cannon, G.,
Armstrong, C. A.,
Bunnett, N. W.,
Ansel, J. C.,
and Caughman, S. W.
(1999)
J. Immunol.
163,
5656-5665 |
58. | Rattner, A., Korner, M., Walker, M. D., and Citri, Y. (1993) EMBO J. 12, 4261-4267[Abstract] |
59. | Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244[CrossRef][Medline] [Order article via Infotrieve] |
60. | Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78, 681-692[Medline] [Order article via Infotrieve] |
61. | Hsu, H., Shu, S. B., Pan, M. G., Baichwal, V., and Goeddel, D. V. (1996) Cell 84, 299-308[Medline] [Order article via Infotrieve] |
62. | Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495-504[Medline] [Order article via Infotrieve] |
63. | Amrani, Y., Martinet, N., and Bronner, C. (1995) Br. J. Pharmacol. 114, 4-5[Abstract] |
64. | Schutze, S., Wiegmann, K., Machleidt, T., and Kronke, M. (1995) Immunobiology 93, 193-203 |
65. | Bouchelouche, P. N., Bendtzen, K., Bak, S., and Nielsen, O. H. (1990) Cell. Signal. 2, 479-487[Medline] [Order article via Infotrieve] |
66. |
Liu, J. S. H.,
John, G. R.,
Sikora, A.,
Sunheee, C. L.,
and Brosnan, C. F.
(2000)
J. Neurosci.
20,
5292-5299 |
67. | Guerrini, L., Blasi, F., and Denis-Donini, D. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9077-9081[Abstract] |
68. |
Kato, B. M.,
and Rubel, E. W.
(1999)
J. Neurophysiol.
81,
1587-1596 |
69. | Nakamura, T., Barbara, J. G., Nakamura, K., and Ross, W. N. (1999) Neuron 24, 727-737[Medline] [Order article via Infotrieve] |
70. |
Nakamura, T.,
Nakamura, K.,
Lasser-Ross, N.,
Barbara, J. G.,
Sandler, V. M.,
and Ross, W. N.
(2000)
J. Neurosci.
20,
8365-8376 |
71. | Recasens, M., and Vignes, M. (1995) Ann. N. Y. Acad. Sci. 757, 418-429[Medline] [Order article via Infotrieve] |
72. | Liu, H. N., Molina-Holgado, E., and Almazan, G. (1997) Eur. J. Pharmacol. 338, 277-287[CrossRef][Medline] [Order article via Infotrieve] |
73. | Sei, Y., and Reich, H. (1995) Immunol. Lett. 45, 75-80[CrossRef][Medline] [Order article via Infotrieve] |
74. | Booth, C., and Koch, G. L. E. (1989) Cell 59, 729-737[Medline] [Order article via Infotrieve] |
75. | del Rio, E., Nicholls, D. G., and Downes, C. P. (1994) J. Neurochem. 63, 535-543[Medline] [Order article via Infotrieve] |
76. |
Kim, Y. H.,
Park, T. J.,
Lee, Y. H.,
Baek, K. J.,
Suh, P. G.,
Ryu, S. H.,
and Kim, K. T.
(1999)
J. Biol. Chem.
274,
26127-26134 |
77. | Hughes, A. R., and Putney, J. W., Jr. (1990) Environ. Health Perspect. 84, 141-147[Medline] [Order article via Infotrieve] |
78. |
Reyes, M.,
and Stanton, P. K.
(1996)
J. Neurosci.
16,
5951-5960 |
79. |
Harris, K. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12213-12215 |
80. | Yao, C. J., Lin, C. W., and Lin-Shiau, S. Y. (1999) J. Neurochem. 73, 457-465[CrossRef][Medline] [Order article via Infotrieve] |
81. | Hamanoue, M., Middleton, G., Wyatt, S., Jaffray, E., Hay, R. T., and Davies, A. M. (1999) Mol. Cell. Neurosci. 14, 28-40[CrossRef][Medline] [Order article via Infotrieve] |
82. | Chapman, R., Sidrauski, C., and Walter, P. (1998) Annu. Rev. Cell Dev. Biol. 14, 459-485[CrossRef][Medline] [Order article via Infotrieve] |