NF-
B Activity Is Induced by Neural Cell Adhesion Molecule
Binding to Neurons and Astrocytes*
Leslie A.
Krushel
,
Bruce A.
Cunningham§,
Gerald M.
Edelman
§¶, and
Kathryn L.
Crossin§
From the
Neurosciences Institute,
San Diego, California 92121 and the § Department of
Neurobiology, The Scripps Research Institute and the ¶ Skaggs
Institute for Chemical Biology, La Jolla, California 92037
 |
ABSTRACT |
The neural cell adhesion molecule, N-CAM, is
expressed on the surface of astrocytes and neurons, and N-CAM
homophilic binding has been shown to alter gene expression in both of
these cell types. To determine mechanisms by which N-CAM regulates gene
expression, we have analyzed DNA binding of and transcriptional
activation by NF-
B after N-CAM binding to the cell surface. Addition
of purified N-CAM, the recombinant third immunoglobulin domain of N-CAM, or N-CAM antibodies either to neonatal rat forebrain astrocytes or to cerebellar granule neurons increased NF-
B/DNA binding activity in nuclear extracts as measured by electrophoretic mobility shift assays. Analysis using supershifting antibodies indicated that, in both
cell types, p50 and p65 but not p52, c-Rel, or Rel B were contained in
the NF-
B-binding complex. NF-
B-mediated transcription was
increased after N-CAM binding to astrocytes and neurons as demonstrated
by the activation of two different luciferase reporter constructs
containing NF-
B-binding sites. N-CAM binding also resulted in
degradation of I
B-
protein followed by an increase in the levels
of I
B-
mRNA and protein. These results indicate that N-CAM
homophilic binding at the cell membrane leads to increased NF-
B
binding to DNA and transcriptional activation in both neurons and
astrocytes. Activation of NF-
B, however, did not influence the
previously reported ability of N-CAM to inhibit astrocyte proliferation. These observations together support the hypothesis that
N-CAM binding activates multiple pathways leading to changes in gene
expression in both astrocytes and neurons.
 |
INTRODUCTION |
Several families of adhesion molecules contribute to the
mechanical stability of cell-cell and cell-extracellular matrix
interactions (1). Evidence is accumulating that, in addition to their
mechanical properties, adhesion molecules function to initiate
cytoplasmic signaling cascades (reviewed in Refs. 2-6). The role of
these cascades in initiating changes in gene transcription is well
described for certain extracellular stimuli such as growth factors (7, 8), but little is known about the nuclear events that result from cell
adhesion molecule binding.
The neural cell adhesion molecule
(N-CAM)1 is a member of the
immunoglobulin-like (Ig) superfamily of adhesion molecules, and it
contains five Ig repeats and two repeats similar to the type three
domains in fibronectin (FN) (9). N-CAM mediates adhesion predominantly
through homophilic binding that appears to involve all of the Ig
domains (10, 11). During development, N-CAM affects cell migration,
neurite outgrowth, and target recognition (reviewed in Refs. 1, 12, and
13). In the adult, N-CAM is expressed at synaptic junctions and is
thought to modulate synaptic function (reviewed in Refs. 14 and 15);
moreover, its promoter appears to be activated in response to neural
stimulation (16). In response to neural injury, N-CAM can inhibit the
proliferative response by astrocytes (17) and promote axonal
regeneration (18-20).
The cellular processes affected by N-CAM homophilic binding are likely
to require alterations in gene transcription. In support of this
notion, N-CAM interactions at the cell surface of neurons (21) and
astrocytes (22) have been shown to alter gene expression. The
identification of transcription factors activated by N-CAM should
therefore aid in the elucidation of intracellular signaling pathways
and genes that are regulated by cell adhesion.
Our previous studies have indicated that N-CAM homophilic binding
inhibits proliferation of neonatal astrocytes in vitro (23) and adult astrocytes in vivo after injury (17). These
studies also revealed that the glucocorticoid receptor (GR), a
transcription factor activated by steroid binding, plays a role in
N-CAM-mediated signaling (22, 24). For example, N-CAM binding to
astrocytes led to activation of reporter constructs driven by two
copies of a consensus glucocorticoid response element (22). In
addition, the GR antagonist RU-486 partially blocked the activities of
N-CAM. These studies support the hypothesis that N-CAM binding to
astrocytes results in alterations in transcription factors leading to
changes in gene transcription.
To explore this hypothesis further, we have begun to search for changes
in other transcription factors in astrocytes and neurons that are
altered in response to N-CAM binding. In addition to its ability to
bind DNA, the GR can also bind other transcription factors, including
NF-
B, and thereby alter their transcriptional properties (25, 26).
NF-
B was originally identified as a transcription factor that was
constitutively bound to the enhancer region in the light chain gene in
B lymphocytes (27) but was subsequently found in other cells in a
latent state bound to I
B. External stimuli such as cytokines induce
the phosphorylation and degradation of I
B allowing for the
translocation of NF-
B into the nucleus (reviewed in Refs. 28 and
29).
NF-
B has recently been found in both neurons and glia (reviewed in
Ref. 30). In astrocytes, NF-
B is activated by cytokines in response
to neural injury (31), and in neurons, NF-
B is activated in response
to neural activity (32-34). The protein has been located at the
synapse (35), and NF-
B may therefore function as an important
signaling molecule in the nervous system.
In the present study, we examined whether N-CAM binding altered NF-
B
DNA binding and transcriptional activation. Addition of N-CAM reagents
to primary cultures of neonatal rat cerebellar granule neurons or
forebrain astrocytes led to increased DNA binding of the NF-
B
proteins p50 and p65. This DNA binding was correlated with an increase
in transcriptional activation of both a synthetic NF-
B reporter
construct and an I
B-
promoter construct that contains an NF-
B
DNA-binding site. NF-
B/DNA binding did not affect the ability of
N-CAM to inhibit astrocyte proliferation. The present findings,
together with previous results, suggest that multiple pathways are
stimulated after N-CAM homophilic binding, resulting in differential
gene transcription and different cellular behaviors.
 |
MATERIALS AND METHODS |
Reagents--
Purified N-CAM, N-CAM recombinant proteins, and
N-CAM antibodies were described previously (11, 24). To remove any
possible endotoxins, recombinant proteins were purified using an
endotoxin removing gel (Pierce). NF-
B antibodies for EMSA supershift
analysis and for Western blots were obtained from Santa Cruz
Biotechnology. Antibodies directed against glial fibrillary acidic
protein and neuron-specific enolase were obtained from Dako. BAY
11-7082 and BAY 11-7085 were obtained from Biomol. The NF-
B-luc
reporter construct (36) was a generous gift from Dr. Mercedes Rincon. The I
B-
reporter construct was made by producing a
double-stranded oligonucleotide corresponding to base pairs
87 to +22
of the porcine I
B-
gene (37) with HindIII and
Xho restriction sites on the 5' and 3' ends, respectively.
This was cloned into pGL3 basic vector (Promega). The I
B mutant
construct which contains a deletion of the base pairs encoding the
first 36 amino acids (38) was a generous gift from Dr. Dean Ballard.
The NF-
B oligonucleotides representing the NF-
B DNA binding
sequence 5' GGG GAC TTT CCC for EMSA were produced on an
oligonucleotide synthesizer (Applied Biosystems Inc.) and annealed. The
EGR and OCT double-stranded oligonucleotides and the NF-
B mutant
double-stranded oligonucleotide in which the G (underlined above) was
replaced by C were obtained from Santa Cruz Biotechnology, Inc.
Cell Culture--
Primary cultures of astrocytes were obtained
from the forebrains of postnatal day 3-4 rats as described (23).
Immunocytochemistry of the cells demonstrated that greater than 98% of
the cells were positive for the glial marker glial fibrillary acidic
protein and less than 2% were reactive with markers for
oligodendrocytes, microglia, and fibroblasts. Neurons were obtained
from the cerebella of postnatal days 3-4 rats as described (39). The
neurons were plated on laminin-coated substrates in 10% fetal bovine
serum/DMEM overnight and then the media was replaced with Neurobasal
media (Life Technologies, Inc.) and B27 supplement (Life Technologies, Inc.).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays--
Nuclear extracts were prepared from astrocytes and
neurons as described (40). EMSA was performed as described (41).
Transcriptional Activation Assays--
Primary astrocytes were
electroporated after 7-10 days in vitro as described
previously (22). Neurons were electroporated immediately after
isolation from neonatal animals. Each suspension of cells was
electroporated with 5 µg of a luciferase reporter construct, 5 µg
of CMV-
-gal, and in some experiments, 5 µg of I
B-mut or 5 µg
of pcDNA3. The cells were plated in 10% fetal bovine serum/DMEM,
and after overnight incubation the medium was changed to DMEM
(astrocytes) or to Neurobasal/B27 (neurons). After 2 days for the
astrocytes and 8 h for the neurons, N-CAM, IL-1
, TNF-
, or
LPS were added. After 16-20 h the cells were harvested and assayed for
luciferase and
-galactosidase activity as previous described
(22).
Astrocyte Proliferation Assays--
Astrocytes were plated at a
density of 1 × 105 cells/well in 96-well plates in
10% fetal bovine serum/DMEM overnight. The media were changed to
serum-free media for 48 h, after which additions of reagents to
the cells was done. Four h later, [3H]thymidine was added
(10 µCi/ml) for an additional 16 h. Incorporation of
[3H]thymidine was measured as described previously
(23).
Differential Display--
Differential display analysis was
carried out using total RNA from untreated or Ig III-treated astrocytes
and cerebellar neurons isolated as described previously (22). The
RT-PCR reaction conditions were previously described (42) using
H-T11G as the anchored 3' primer and AP5 as the 5'
arbitrary primer (43). The RT-PCR products were resolved on a 6%
acrylamide/urea gel using the Genomyx sequencer that has been optimized
for differential display. Dried gels were exposed overnight to x-ray
film, and bands of interest were cut from the gel, reamplified by PCR
using the same primers used for the differential display, and sequenced
using Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech).
 |
RESULTS |
N-CAM Binding to the Astrocyte Cell Surface Increases Nuclear
Protein Binding to a Consensus NF-
B Oligonucleotide--
To
investigate the effects of N-CAM on NF-
B/DNA binding, astrocytes
derived from neonatal rat forebrains were cultured for 6 h in the
presence of one of three different N-CAM reagents: N-CAM (5 ng/ml)
purified from early postnatal rat brain, the recombinant third Ig
domain (Ig III) of N-CAM (10 µg/ml), or a polyclonal antibody against
N-CAM (500 µg/ml). Nuclear extracts were obtained from these cells
after treatment and subjected to electrophoretic mobility shift assays
(EMSA) using a 32P-labeled double-stranded oligonucleotide
probe containing a consensus NF-
B-binding site. Two DNA-protein
binding complexes were observed in extracts from astrocytes treated
with each of the three N-CAM reagents (Fig.
1A). In contrast, extracts
from untreated astrocytes showed little or no DNA binding. In all
cases, the binding was prevented in the presence of a 100-fold excess
of unlabeled probe (Fig. 1A, right) but not by an
oligonucleotide containing a mutation in the NF-
B DNA-binding site
(Fig. 2). Recombinant proteins
corresponding either to the two fibronectin repeats in N-CAM or to
fibronectin repeats 8-10 of the fibronectin protein itself (which
contains the RGD cell binding tripeptide) did not affect the formation of NF-
B DNA-protein complexes (Fig. 1B). These results
indicate that N-CAM binding to the astrocyte cell surface increased the formation of NF-
B-DNA binding complexes.

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Fig. 1.
Addition of N-CAM reagents increases NF- B
but not OCT or EGR/DNA binding. A, the recombinant
third Ig domain (Ig III) of N-CAM (10 µg/ml), purified rat N-CAM (5 µg/ml), or a N-CAM polyclonal antibody (500 µg/ml) was added
individually to neonatal rat forebrain astrocytes cultured in
serum-free media. Nuclear extracts were then prepared after 6 h
from treated (+) and untreated ( ) astrocytes, and binding to a
32P-labeled oligonucleotide probe containing the
NF- B-binding site was determined by EMSA. A 100-fold excess of
unlabeled oligonucleotide abolished binding. B, nuclear
extracts derived from untreated astrocytes ( ) or astrocytes treated
with Ig III (10 µg/ml), the recombinant FN domains 1-2 of N-CAM (FN
1-2, 10 µg/ml), or the recombinant FN domains 8-10 of fibronectin
(FN 8-10, 10 µg/ml) were used in a NF- B EMSA as stated
(A). C and D, nuclear extracts were
prepared from untreated ( ) astrocytes or treated with Ig III (10 µg/ml) or basic fibroblast growth factor (bFGF, 20 ng/ml). Binding to
a 32P-labeled oligonucleotide probe containing the OCT
(C) or EGR (D)-binding site was determined by
EMSA.
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Fig. 2.
NF- B/DNA binding peaks within 6 h of
Ig III treatment in both astrocytes and neurons. Nuclear extracts
obtained from astrocytes (A) and cerebellar granule neurons
(B) treated with Ig III (10 µg/ml) (+) for 1-24 h were
compared with extracts from untreated control cultures ( ) incubated
for the same times. Inclusion of a 100-fold excess of unlabeled
oligonucleotide, but not of an oligonucleotide in which the consensus
DNA-binding site was mutated, abolished DNA protein binding.
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The levels of other transcription factor-DNA complexes were not altered
in the presence of N-CAM. EMSA was performed using nuclear extracts
from untreated astrocytes or astrocytes treated with Ig III or basic
fibroblast growth factor (bFGF) and labeled oligonucleotide probes
corresponding to consensus DNA elements recognized by either the
octamer (OCT) or early growth response (EGR) transcription factor
families. The OCT sequence is a binding site for the POU homeodomain
octamer family (reviewed in Ref. 44), and the EGR sequence is a binding
site for members of the EGR gene family, which are immediate early
response genes (reviewed in Ref. 45). The high level of protein
complexes with the OCT oligonucleotide probe observed in the untreated
astrocyte extracts did not change upon addition of Ig III or bFGF (Fig.
1C). In contrast the DNA-protein complex formed with the EGR
consensus sequence was only marginally detectable in the untreated and
Ig III-treated cells but increased dramatically in the bFGF-treated
extracts (Fig. 1D). The combined results demonstrate that
the increase in NF-
B-DNA complexes by N-CAM is due to the binding of
the N-CAM Ig domains and not the FN repeats and that N-CAM binding
activates NF-
B but not all transcription factors.
Temporal Analysis of NF-
B Activation in Astrocytes and
Neurons--
To determine the levels of NF-
B/DNA binding over time,
nuclear extracts were obtained from astrocytes or early postnatal cerebellar granule neurons that had been treated with Ig III for 1-24
h. Increased NF-
B/DNA binding in astrocytes was observed as early as
1 h after addition of Ig III (Fig. 2A). The levels of
NF-
B-DNA complexes were maximal around 6 h and remained at high
levels for 24 h. Three separate NF-
B complexes were observed. At the later time points, it was difficult to differentiate between the
two fastest migrating bands, but it appeared that the fastest migrating
band was absent after 16 h of N-CAM treatment.
In cerebellar granule neurons (Fig. 2B), addition of Ig III
also increased NF-
B/DNA binding. Higher basal levels of NF-
B/DNA binding were observed in untreated neurons as compared with astrocytes. Nevertheless, addition of N-CAM produced an increase in NF-
B binding
1 h after treatment, and this binding was maximal after 3 h.
As in astrocytes, three different NF-
B binding complexes were
observed in neurons, and the levels of the fastest migrating complex
decreased after 16 h. Therefore, in both astrocytes and neurons,
the addition of N-CAM increased the binding activity of NF-
B in a
time-dependent manner.
Composition of the NF-
B Binding Complexes--
The NF-
B
complex in mammals is comprised of homodimers and heterodimers from a
group of five different proteins (28). Antibodies against the NF-
B
proteins were used to supershift components of the complex in
astrocytes and neurons that were treated with Ig III for 6 h (Fig.
3, A and B). An
antibody to p50 shifted almost all of the NF-
B binding complexes in
both astrocytes and neurons. In astrocytes, the p65 antibody
supershifted the slower migrating complex but only slightly diminished
the intensity of the fastest moving complex. In neurons, the p65
antibody supershifted all three NF-
B complexes. Antibodies directed
to the remaining NF-
B proteins did not supershift any of the NF-
B
binding complexes. Similar supershift results were observed after
treatment of astrocytes and neurons with lipopolysaccharide (LPS),
interleukin 1
(IL-1
) and tumor necrosis factor-
(TNF-
)
(data not shown). These results demonstrate that the NF-
B-DNA
complexes in both astrocytes and neurons are comprised primarily of p50
and p65 proteins.

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Fig. 3.
NF- B complex in astrocytes and neurons
after Ig III treatment is comprised primarily of p50 and p65
subunits. Nuclear extracts from astrocytes (A) or
cerebellar granule neurons (B) treated with Ig III for
6 h were incubated for 30 min at room temperature with antibodies
directed against different NF- B proteins prior to EMSA.
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N-CAM Binding Leads to Increased NF-
B-dependent
Transcription--
To examine whether the increased levels of nuclear
NF-
B binding activity led to concomitant increases in transcription,
two different NF-
B reporter constructs were used. A luciferase
reporter construct containing two tandem consensus NF-
B DNA-binding
sites upstream of a minimal Fos promoter (36) (NF-
B-luc) was
transfected into astrocytes. Addition of Ig III or agents known to
stimulate NF-
B activity, including IL-1
, TNF-
, and LPS, all
produced significant increases in luciferase activity from the NF-
B
reporter construct (Fig. 4A).
Addition of either bFGF or a recombinant protein encoding the two
fibronectin type three repeats of N-CAM did not activate NF-
B (data
not shown).

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Fig. 4.
Addition of Ig III to astrocytes and neurons
activates transcription of an NF- B/reporter construct, and
activation is prevented by co-transfection with an I B- construct
containing a deletion of the amino terminus of the protein.
Astrocytes (A) and neurons (B) were
electroporated with NF- B-luc, CMV- -gal, and either pcDNA3 or
I B-mut (5 µg each vector). After recovery, cells were treated with
Ig III (10 µg/ml), LPS (5 µg/ml), IL-1 (1 ng/ml), and TNF- (1 ng/ml) for approximately 18 h and then harvested for luciferase
and -galactosidase activity. The results are shown as the ratio of
the luciferase activity to the -galactosidase activity for each
treatment condition. The error bars represent the standard
deviation from quadruplicate samples in a representative experiment.
Each experiment was replicated a minimum of five times.
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The activation of NF-
B is dependent on the dissociation of the
NF-
B proteins from the inhibitory I
B proteins in the cytoplasm (reviewed in Ref. 28). Upon phosphorylation of two amino-terminal sites
in I
B-
, the I
B-
protein dissociates from the complex and is
then degraded (28). However, a form of I
B-
with the amino
terminus deleted does not dissociate and therefore prevents NF-
B
nuclear translocation (38). Co-transfection of NF-
B-luc and an
I
B-
construct having the amino-terminal 36 amino acids deleted
(I
B-mut) (38) prevented the response to Ig III and to the other
reagents in astrocytes (Fig. 4A). In addition, the basal
level of activity was also substantially decreased.
In cerebellar neurons, addition of N-CAM, LPS, IL-1
, or TNF-
stimulated luciferase expression from the NF-
B-luc construct (Fig.
4B). The basal level of NF-
B-driven luciferase activity was higher in neurons as compared with astrocytes, and both the basal
level as well as the further induction of luciferase activity were
abolished when the I
B-mut was co-transfected with NF-
B-luc. These
results together suggest that N-CAM binding stimulates
NF-
B-dependent transcription in both astrocytes and neurons.
One of the genes transcribed in response to NF-
B activation is
I
B-
. A luciferase construct (I
B-
-luc) containing a portion of a native gene that contains an NF-
B-binding site, the porcine I
B-
promoter (
87/+22) (37), was transfected into both
astrocytes (Fig. 5A) and
neurons (Fig. 5B). Addition of N-CAM as well as LPS,
IL-1
, and TNF-
increased luciferase activity from this construct,
and the increase was abolished when I
B-mut was co-transfected. These
results suggest that N-CAM binding can induce transcription of genes
containing NF-
B sites in their promoters.

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Fig. 5.
Addition of N-CAM to astrocytes and neurons
increases the transcriptional activity of a truncated I B-
promoter construct that is blocked by co-transfection with
I B-mut. Astrocytes (A) and neurons (B)
were electroporated with 5 µg of a construct containing a portion of
the I B- promoter ( 87/+22) that has a functional NF- B-binding
site upstream of a luciferase gene. The cells were co-transfected with
5 µg of CMV- -gal and either 5 µg of I B-mut or 5 µg of
pcDNA3. The cells were recovered from the transfection, treated,
and assayed as stated under "Materials and Methods" and Fig. 4. The
results are shown as the ratio of the luciferase activity normalized to
the -galactosidase activity for each treatment condition. The
error bars represent the standard deviation from
quadruplicate samples of a representative experiment. Each experiment
was replicated a minimum of five times.
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Expression of I
B-
mRNA and Protein after N-CAM
Binding--
The activation of the I
B-
reporter construct
suggested that I
B-
gene transcription may be up-regulated after
the addition of N-CAM. To examine differential gene expression
following N-CAM treatment, primary cultures of astrocytes and
cerebellar granule neurons were treated with Ig III for 6 h or
remained untreated. The RNA was obtained from these cells and subjected
to differential display analysis. The level of numerous mRNAs
showed changes in their expression levels after treatment with Ig III
(data not shown). Two bands that were expressed at a higher level in
both N-CAM-treated astrocytes and neurons (Fig.
6, arrows) were sequenced and
shown to encode I
B-
. This result is consistent with the observed
ability of N-CAM binding to activate the I
B-
promoter/reporter construct and further demonstrated that the transcription of the endogenous I
B-
gene was up-regulated in response to N-CAM
binding. In non-neural cells, increased I
B-
transcription occurs
following the degradation of I
B proteins (28, 29). A Western blot of extracts obtained from astrocytes or neurons treated with Ig III for
1-24 h was performed to determine the expression levels of I
B-
protein (Fig. 7, arrows). A
transient decrease in the level of I
B-
protein was seen in
astrocytes at 1 h following addition of Ig III and between 3 and
6 h in neurons (see Fig. 2). The blots were also incubated with
antibodies to glial fibrillary acidic protein (Fig. 7A,
arrowhead) or neuron-specific enolase (Fig. 7B,
arrowhead) to demonstrate equivalent protein loading for each condition. These results suggest that in neurons and astrocytes an
autoregulatory loop exists for the turnover of I
B proteins, similar
to that reported in non-neural cells (28, 29).

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Fig. 6.
I B- RNA levels increase in astrocytes
and neurons after treatment with N-CAM. Astrocyte and cerebellar
granule neurons were treated with Ig III (10 µg/ml) for 6 h or
remained untreated ( ). Total RNA was obtained from the cells, and
RT-PCR was performed with HT11G and AP5 primers
(GenHunter). The bands denoted by arrowheads were excised
from the gel and sequenced. They were both shown to encode
I B- .
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Fig. 7.
The level of I B- protein in astrocytes
and neurons is transiently decreased after treatment with Ig III.
Astrocytes (A) or cerebellar granule neurons (B)
were exposed to Ig III (10 µg/ml) in serum-free media. After 1-24 h,
the cells were harvested, and cell extracts were obtained as described
under "Material and Methods." Twenty micrograms of each sample were
loaded on a 10% polyacrylamide gel, subjected to electrophoresis, and
transferred to a polyvinylidene difluoride membrane. The astrocyte
proteins were immunoblotted with polyclonal I B- antibodies and
antibodies to the glial fibrillary acidic protein; the neuronal
proteins were immunoblotted with polyclonal I B- and antibodies to
neuron-specific enolase. 125I-protein A was used to detect
the antibodies. The I B- binding (~38 kDa) is denoted by
arrows, and the glial fibrillary acidic protein (~52 kDa)
and neuron-specific enolase (~46 kDa) binding is shown by
arrowheads.
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Inhibition of Astrocyte Proliferation by N-CAM Is Not Influenced by
NF-
B Activation--
Our previous findings indicated that N-CAM
binding to astrocytes led to decreased proliferation (22-24). It was
therefore possible that activation of NF-
B by N-CAM binding might
have a modulatory role on the anti-proliferative effects of N-CAM. To
determine whether the inhibition of astrocyte proliferation by N-CAM
was altered by NF-
B, an astrocyte proliferation assay was performed. Astrocytes were exposed for 16 h to Ig III (1-10 µg/ml) in the presence or absence of either of the two reagents, BAY 11-7082 and BAY
11-7085, which inhibit the phosphorylation of I
B-
and subsequently inhibit the nuclear translocation of NF-
B (46). The
ability of N-CAM to inhibit proliferation was not changed in the
presence of 50 µM BAY 11-7082 or BAY 11-7085 (Fig.
8A). Moreover, the addition of
these agents alone did not alter the basal level of
[3H]thymidine incorporation (data not shown).

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Fig. 8.
Inhibiting NF- B activation does not alter
the ability of Ig III to inhibit astrocyte proliferation.
A, astrocytes were plated in 96 wells (1 × 105/ml) in serum-containing media. After 24 h the
media were replaced with serum-free media for 48 h. Astrocytes
were then treated with Ig III (1, 3, or 10 µg/ml) or combinations of
Ig III with BAY 11-7082 (50 µM) or BAY 11-7085 (50 µM). Four h after treatment, [3H]thymidine
was added, and the cells were harvested 16 h later. B,
EMSA was performed on nuclear extracts of astrocytes treated with Ig
III (10 µg/ml), BAY 11-7082 (50 µM), BAY 11-7085 (50 µM), or combinations of Ig III with BAY 11-7082 or BAY
11-7085.
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To determine that BAY 11-7082 and BAY 11-7085 were indeed inhibiting
NF-
B translocation and DNA binding, EMSA was performed on astrocytes
treated with Ig III alone or in the presence of BAY 11-7082 and BAY
11-7085 (Fig. 8B). NF-
B/DNA binding was greatly reduced
when astrocytes were treated with both Ig III and BAY 11-7082 or BAY
11-7085. However, the amount of DNA binding that remained in the
BAY-treated cells was greater than in untreated astrocytes. These
results suggest that the activation of NF-
B is not solely involved
in the inhibition of astrocyte proliferation by N-CAM and indicate
therefore that multiple independent intracellular pathways are
activated by N-CAM binding.
 |
DISCUSSION |
Interactions of Ig-like CAMs at the cell surface trigger
intracellular signaling cascades that activate multiple second
messengers (reviewed in Refs. 2, 6, and 47). Little is known, however, about nuclear events that occur downstream of these signals. The present findings demonstrate that N-CAM binding to astrocytes and
neurons in vitro increased NF-
B/DNA binding and
NF-
B-dependent transcription. The transcription of an
NF-
B-responsive gene, I
B-
, was increased after treatment of
cells with N-CAM. Alterations in NF-
B/DNA binding had no effect on
the ability of N-CAM to inhibit proliferation of astrocytes, an
inhibition known to be influenced by activation of the glucocorticoid
receptor by N-CAM (22, 24). These results indicate that multiple
intracellular signaling pathways are activated by N-CAM binding and
lead to changes in cell proliferation and gene expression.
N-CAM homophilic binding appears to be responsible for the increase in
NF-
B activity inasmuch as multiple N-CAM reagents, including
purified rat N-CAM, recombinant Ig III, and a rabbit polyclonal
antibody directed against N-CAM, all were able to increase NF-
B
activity as measured by EMSA analysis. Of the recombinant proteins
tested, the one corresponding to Ig III increased NF-
B activity,
whereas the one corresponding to N-CAM FN 1-2 did not. This finding is
consistent with studies indicating that N-CAM homophilic binding occurs
through the interaction of the Ig and not the FN domains (10, 11). It
is of interest that an individual monovalent Ig III domain, an N-CAM
antibody, and intact N-CAM all had similar effects on NF-
B/DNA.
Binding of all three molecules to N-CAM on the cell surface apparently
causes similar changes in the protein leading to an activated signaling
state. The ability of an antibody to induce a conformational change has
been observed after binding of integrin antibodies (reviewed in Ref.
48). Future studies will be aimed at understanding the changes in the N-CAM molecule, in particular the cytoplasmic domain, by the binding of
these multiple N-CAM reagents.
The increase in NF-
B/DNA binding in both neurons and astrocytes
occurred within 1 h after addition of N-CAM and remained at high
levels up to 24 h after treatment. The duration of the elevated
NF-
B activity is comparable to that observed after addition of
cytokines to pre-B cells and C6 glioma cells (49, 50). Among the five
known proteins that form the hetero- or homodimers of the NF-
B
complex, we found that p50 and p65 were the major protein components in
the NF-
B-DNA binding complex in astrocytes and neurons stimulated by
N-CAM (Fig. 3) or by cytokines (data not shown). These findings are in
agreement with previous reports suggesting that p50 and p65 are the
major NF-
B protein components expressed in the central nervous
system (51, 52).
Consistent with the EMSA analysis, addition of N-CAM to both astrocytes
and neurons resulted in transcriptional activation of
NF-
B-containing promoter/reporter constructs. Differential display
analysis and activation of an I
B promoter/reporter construct demonstrated that I
B-
was transcribed in both astrocytes and neurons in response to N-CAM binding. Differential display analysis also indicated, however, that there are several other mRNAs that are activated in astrocytes and neurons, and some were cell
type-specific. In addition to the I
B-
gene (37, 53, 54), many
genes expressed in the nervous system contain NF-
B-binding sites in
their promoters, including the neuronal isoform of nitric oxide
synthase (55), proenkephalin (56), and prodynorphin (30). Whether these
genes are targets of NF-
B or other transcription factors whose
activity is regulated by N-CAM binding in astrocytes or neurons remains to be determined.
Studies using lymphocytes demonstrated that the critical element in the
regulation of NF-
B activity by receptor binding at the cell surface
is the phosphorylation of I
B proteins (reviewed in Refs. 28 and 29).
Phosphorylation of I
B leads to its dissociation from the NF-
B
complex and its subsequent degradation. Free NF-
B subunits can then
translocate into the nucleus where they increase I
B transcription,
thereby forming an autoregulatory loop (57-60). NF-
B-dependent
transcription activated by N-CAM binding was inhibited by co-expression
of a truncated form of I
B taht could not be phosphorylated. N-CAM
therefore appears to regulate NF-
B activity in neurons and
astrocytes in a manner similar to that previously described for other
stimuli. Recently, multiple kinases that phosphorylate I
B proteins
have been identified (61-64), and the intracellular pathways that lead
to activation of these kinases are numerous (65), including activation
of Rho GTPases, lipid peroxidation, and activation of protein kinases
(66-68). As yet our data do not indicate which among these pathways
are affected by N-CAM binding to influence NF-
B activity. However,
our previous studies have shown that N-CAM binding led to decreased
growth factor-stimulated mitogen-activated protein kinase activity in
astrocytes (24).
Studies in neuronal cells have suggested that intracellular signaling
pathways downstream of FGF receptor and Fyn tyrosine kinase activation
influence the ability of N-CAM to promote neurite extension (2, 69). It
was proposed that the activity of N-CAM was mediated by a cis
interaction with the FGF receptor that led to increased FGF receptor
tyrosine kinase activity. In our studies, addition of bFGF to
astrocytes and neurons produced little or no increase of NF-
B/DNA
binding or NF-
B-mediated transcription (data not shown). This
indicates that N-CAM does not act through the FGF receptor to activate
NF-
B.
Cell adhesion mediated by integrins has recently been shown to activate
NF-
B in endothelial cells via pathways involving Ras and Src
tyrosine kinase (70) and in fibroblasts via activation of Rac1 (71).
Fyn, a member of the Src family of tyrosine kinases, has been proposed
to associate with the N-CAM cytoplasmic domain in neurons (72). In
T-cells, activation of Fyn by stimulation of the T-cell antigen
receptor leads to increased NF-
B activity (73, 74). These signaling
intermediates are all possible candidates for mediating N-CAM binding
and require further study.
The regulation of gene expression is dependent upon the combinatorial
binding of multiple transcription factors to promoter elements. In the
present report, we demonstrate that NF-
B, but not EGR or OCT DNA
binding levels, was altered by N-CAM binding. Our previous studies
indicated that N-CAM homophilic binding in astrocytes activates the GR,
a transcription factor (22-24), and protein interactions between GR
and NF-
B have been shown to influence the transcriptional activities
of both factors in several cell types (25, 26). Preliminary studies in
astrocytes and neurons suggest that antagonism of the GR by RU 486 did
not, however, affect the ability of N-CAM to activate NF-
B (data not
shown). In contrast, RU 486 blocked the ability of N-CAM to inhibit
proliferation, to stimulate GRE-regulated gene expression, and to
inhibit FGF-induced mitogen-activated protein kinase activity (22, 24).
Moreover, the present findings show that blockade of NF-
B
translocation by BAY 11-7082 or BAY 11-7085 had no effect on the
anti-proliferative activity of N-CAM. Together these findings reinforce
the conclusion that N-CAM activates multiple transcription factors
through distinct intracellular pathways that differentially influence
gene expression and cell proliferation.
Both N-CAM and NF-
B have been identified at the synapse, and it has
been proposed that they contribute to neural plasticity and thereby
affect processes such as learning and memory (32, 35, 75). The
localization of N-CAM at the synaptic cleft (35) makes it an attractive
candidate for signaling alterations in the structure and function of
the synapse. Activation of NF-
B can occur in neurons in response to
neural stimulation (32), to treatment with glutamate (33), or to
treatment with TNF-
(34). This response has been correlated with
alterations in activity of voltage-dependent calcium
channels and excitatory amino acid receptor channels (34). Moreover,
activation of NF-
B has been correlated with long term memory
consolidation in the crab (76). The possibility therefore arises that
N-CAM interactions at the synapse could activate NF-
B, which then
may act as a long range signaling molecule by migrating into the
nucleus, as has been proposed for the transcription factor cAMP
response element-binding protein (77). Further studies of the
intracellular signaling pathways downstream of N-CAM binding and
identification of NF-
B target genes will help to determine the
mechanisms by which cell adhesion leads to alterations in gene
expression and cellular behavior.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Mercedes Rincon and Dean
Ballard for DNA constructs; Lisa Remedios, Melanie King, Anna Tran, and
Stacey Olson for excellent technical assistance; and Drs. Frederick
Jones, Vincent Mauro, and Joseph Gally for critical reading of the
manuscript. Kathryn L. Crossin, Bruce A. Cunningham, and Gerald M. Edelman are consultants to Becton Dickinson.
 |
FOOTNOTES |
*
This work was supported by U. S. Public Health Service
Grants HD09635 (to G. M. E.), HD16550 (to B. A. C.), and NS/OD
34874 (to K. L. C.) and a grant from the G. Harold and Leila Y. Mathers Foundation (to G. M. E.).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.
To whom correspondence should be addressed: Dept. of
Neurobiology SBR-14, The Scripps Research Institute, 10550 North Torrey Pines Rd., SBR-14, La Jolla, CA 92037. Tel.: 619-784-2623; Fax: 619-784-2646; E-mail: kcrossin{at}scripps.edu.
The abbreviations used are:
N-CAM, neural cell
adhesion molecule; NF, nuclear factor; EMSA, electrophoretic mobility
shift assay; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor
necrosis factor; luc, luciferase; bFGF, basic fibroblast growth factor; Ig, immunoglobulin-like; GR, glucocorticoid receptor; EGR, early growth
response; DMEM, Dulbecco's modified Eagle's medium; RT-PCR, reverse
transcriptase-polymerase chain reaction; OCT, octamer; FN, fibronectin.
 |
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