NF-kappa B Activity Is Induced by Neural Cell Adhesion Molecule Binding to Neurons and Astrocytes*

Leslie A. KrushelDagger , Bruce A. Cunningham§, Gerald M. EdelmanDagger §, and Kathryn L. Crossin§parallel

From the Dagger  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
Top
Abstract
Introduction
References

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-kappa 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-kappa 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-kappa B-binding complex. NF-kappa 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-kappa B-binding sites. N-CAM binding also resulted in degradation of Ikappa B-alpha protein followed by an increase in the levels of Ikappa B-alpha mRNA and protein. These results indicate that N-CAM homophilic binding at the cell membrane leads to increased NF-kappa B binding to DNA and transcriptional activation in both neurons and astrocytes. Activation of NF-kappa 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
Top
Abstract
Introduction
References

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-kappa B, and thereby alter their transcriptional properties (25, 26). NF-kappa 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 Ikappa B. External stimuli such as cytokines induce the phosphorylation and degradation of Ikappa B allowing for the translocation of NF-kappa B into the nucleus (reviewed in Refs. 28 and 29).

NF-kappa B has recently been found in both neurons and glia (reviewed in Ref. 30). In astrocytes, NF-kappa B is activated by cytokines in response to neural injury (31), and in neurons, NF-kappa B is activated in response to neural activity (32-34). The protein has been located at the synapse (35), and NF-kappa 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-kappa 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-kappa B proteins p50 and p65. This DNA binding was correlated with an increase in transcriptional activation of both a synthetic NF-kappa B reporter construct and an Ikappa B-alpha promoter construct that contains an NF-kappa B DNA-binding site. NF-kappa 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-kappa 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-kappa B-luc reporter construct (36) was a generous gift from Dr. Mercedes Rincon. The Ikappa B-alpha reporter construct was made by producing a double-stranded oligonucleotide corresponding to base pairs -87 to +22 of the porcine Ikappa B-alpha gene (37) with HindIII and Xho restriction sites on the 5' and 3' ends, respectively. This was cloned into pGL3 basic vector (Promega). The Ikappa 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-kappa B oligonucleotides representing the NF-kappa 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-kappa 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-beta -gal, and in some experiments, 5 µg of Ikappa 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-1beta , TNF-alpha , or LPS were added. After 16-20 h the cells were harvested and assayed for luciferase and beta -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-kappa B Oligonucleotide-- To investigate the effects of N-CAM on NF-kappa 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-kappa 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-kappa 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-kappa B DNA-protein complexes (Fig. 1B). These results indicate that N-CAM binding to the astrocyte cell surface increased the formation of NF-kappa B-DNA binding complexes.


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 1.   Addition of N-CAM reagents increases NF-kappa 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-kappa 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-kappa 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.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2.   NF-kappa 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.

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-kappa 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-kappa B but not all transcription factors.

Temporal Analysis of NF-kappa B Activation in Astrocytes and Neurons-- To determine the levels of NF-kappa 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-kappa B/DNA binding in astrocytes was observed as early as 1 h after addition of Ig III (Fig. 2A). The levels of NF-kappa B-DNA complexes were maximal around 6 h and remained at high levels for 24 h. Three separate NF-kappa 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-kappa B/DNA binding. Higher basal levels of NF-kappa B/DNA binding were observed in untreated neurons as compared with astrocytes. Nevertheless, addition of N-CAM produced an increase in NF-kappa B binding 1 h after treatment, and this binding was maximal after 3 h. As in astrocytes, three different NF-kappa 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-kappa B in a time-dependent manner.

Composition of the NF-kappa B Binding Complexes-- The NF-kappa B complex in mammals is comprised of homodimers and heterodimers from a group of five different proteins (28). Antibodies against the NF-kappa 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-kappa 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-kappa B complexes. Antibodies directed to the remaining NF-kappa B proteins did not supershift any of the NF-kappa B binding complexes. Similar supershift results were observed after treatment of astrocytes and neurons with lipopolysaccharide (LPS), interleukin 1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ) (data not shown). These results demonstrate that the NF-kappa B-DNA complexes in both astrocytes and neurons are comprised primarily of p50 and p65 proteins.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   NF-kappa 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-kappa B proteins prior to EMSA.

N-CAM Binding Leads to Increased NF-kappa B-dependent Transcription-- To examine whether the increased levels of nuclear NF-kappa B binding activity led to concomitant increases in transcription, two different NF-kappa B reporter constructs were used. A luciferase reporter construct containing two tandem consensus NF-kappa B DNA-binding sites upstream of a minimal Fos promoter (36) (NF-kappa B-luc) was transfected into astrocytes. Addition of Ig III or agents known to stimulate NF-kappa B activity, including IL-1beta , TNF-alpha , and LPS, all produced significant increases in luciferase activity from the NF-kappa 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-kappa B (data not shown).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Addition of Ig III to astrocytes and neurons activates transcription of an NF-kappa B/reporter construct, and activation is prevented by co-transfection with an Ikappa B-alpha construct containing a deletion of the amino terminus of the protein. Astrocytes (A) and neurons (B) were electroporated with NF-kappa B-luc, CMV-beta -gal, and either pcDNA3 or Ikappa B-mut (5 µg each vector). After recovery, cells were treated with Ig III (10 µg/ml), LPS (5 µg/ml), IL-1beta (1 ng/ml), and TNF-alpha (1 ng/ml) for approximately 18 h and then harvested for luciferase and beta -galactosidase activity. The results are shown as the ratio of the luciferase activity to the beta -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.

The activation of NF-kappa B is dependent on the dissociation of the NF-kappa B proteins from the inhibitory Ikappa B proteins in the cytoplasm (reviewed in Ref. 28). Upon phosphorylation of two amino-terminal sites in Ikappa B-alpha , the Ikappa B-alpha protein dissociates from the complex and is then degraded (28). However, a form of Ikappa B-alpha with the amino terminus deleted does not dissociate and therefore prevents NF-kappa B nuclear translocation (38). Co-transfection of NF-kappa B-luc and an Ikappa B-alpha construct having the amino-terminal 36 amino acids deleted (Ikappa 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-1beta , or TNF-alpha stimulated luciferase expression from the NF-kappa B-luc construct (Fig. 4B). The basal level of NF-kappa 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 Ikappa B-mut was co-transfected with NF-kappa B-luc. These results together suggest that N-CAM binding stimulates NF-kappa B-dependent transcription in both astrocytes and neurons.

One of the genes transcribed in response to NF-kappa B activation is Ikappa B-alpha . A luciferase construct (Ikappa B-alpha -luc) containing a portion of a native gene that contains an NF-kappa B-binding site, the porcine Ikappa B-alpha promoter (-87/+22) (37), was transfected into both astrocytes (Fig. 5A) and neurons (Fig. 5B). Addition of N-CAM as well as LPS, IL-1beta , and TNF-alpha increased luciferase activity from this construct, and the increase was abolished when Ikappa B-mut was co-transfected. These results suggest that N-CAM binding can induce transcription of genes containing NF-kappa B sites in their promoters.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Addition of N-CAM to astrocytes and neurons increases the transcriptional activity of a truncated Ikappa B-alpha promoter construct that is blocked by co-transfection with Ikappa B-mut. Astrocytes (A) and neurons (B) were electroporated with 5 µg of a construct containing a portion of the Ikappa B-alpha promoter (-87/+22) that has a functional NF-kappa B-binding site upstream of a luciferase gene. The cells were co-transfected with 5 µg of CMV-beta -gal and either 5 µg of Ikappa 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 beta -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.

Expression of Ikappa B-alpha mRNA and Protein after N-CAM Binding-- The activation of the Ikappa B-alpha reporter construct suggested that Ikappa B-alpha 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 Ikappa B-alpha . This result is consistent with the observed ability of N-CAM binding to activate the Ikappa B-alpha promoter/reporter construct and further demonstrated that the transcription of the endogenous Ikappa B-alpha gene was up-regulated in response to N-CAM binding. In non-neural cells, increased Ikappa B-alpha transcription occurs following the degradation of Ikappa 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 Ikappa B-alpha protein (Fig. 7, arrows). A transient decrease in the level of Ikappa B-alpha 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 Ikappa B proteins, similar to that reported in non-neural cells (28, 29).


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 6.   Ikappa B-alpha 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 Ikappa B-alpha .


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 7.   The level of Ikappa B-alpha 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 Ikappa B-alpha antibodies and antibodies to the glial fibrillary acidic protein; the neuronal proteins were immunoblotted with polyclonal Ikappa B-alpha and antibodies to neuron-specific enolase. 125I-protein A was used to detect the antibodies. The Ikappa B-alpha 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.

Inhibition of Astrocyte Proliferation by N-CAM Is Not Influenced by NF-kappa 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-kappa 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-kappa 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 Ikappa B-alpha and subsequently inhibit the nuclear translocation of NF-kappa 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).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 8.   Inhibiting NF-kappa 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.

To determine that BAY 11-7082 and BAY 11-7085 were indeed inhibiting NF-kappa 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-kappa 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-kappa 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-kappa B/DNA binding and NF-kappa B-dependent transcription. The transcription of an NF-kappa B-responsive gene, Ikappa B-alpha , was increased after treatment of cells with N-CAM. Alterations in NF-kappa 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-kappa 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-kappa B activity as measured by EMSA analysis. Of the recombinant proteins tested, the one corresponding to Ig III increased NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa B complex, we found that p50 and p65 were the major protein components in the NF-kappa 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-kappa 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-kappa B-containing promoter/reporter constructs. Differential display analysis and activation of an Ikappa B promoter/reporter construct demonstrated that Ikappa B-alpha 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 Ikappa B-alpha gene (37, 53, 54), many genes expressed in the nervous system contain NF-kappa 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-kappa 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-kappa B activity by receptor binding at the cell surface is the phosphorylation of Ikappa B proteins (reviewed in Refs. 28 and 29). Phosphorylation of Ikappa B leads to its dissociation from the NF-kappa B complex and its subsequent degradation. Free NF-kappa B subunits can then translocate into the nucleus where they increase Ikappa B transcription, thereby forming an autoregulatory loop (57-60). NF-kappa B-dependent transcription activated by N-CAM binding was inhibited by co-expression of a truncated form of Ikappa B taht could not be phosphorylated. N-CAM therefore appears to regulate NF-kappa B activity in neurons and astrocytes in a manner similar to that previously described for other stimuli. Recently, multiple kinases that phosphorylate Ikappa 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-kappa 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-kappa B/DNA binding or NF-kappa B-mediated transcription (data not shown). This indicates that N-CAM does not act through the FGF receptor to activate NF-kappa B.

Cell adhesion mediated by integrins has recently been shown to activate NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa B can occur in neurons in response to neural stimulation (32), to treatment with glutamate (33), or to treatment with TNF-alpha (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-kappa 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-kappa 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-kappa 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.

parallel 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.
    REFERENCES
Top
Abstract
Introduction
References

  1. Edelman, G. M., and Crossin, K. L. (1991) Annu. Rev. Biochem. 60, 155-190[CrossRef][Medline] [Order article via Infotrieve]
  2. Doherty, P., Smith, P., and Walsh, F. S. (1996) Persp. Dev. Neurobiol. 4, 157-168
  3. Yamada, K. M. (1997) Matrix Biol. 16, 137-141[CrossRef][Medline] [Order article via Infotrieve]
  4. Yap, A. S., Brieher, W. M., and Gumbiner, B. M. (1997) Annu. Rev. Dev. Biol. 13, 119-146[CrossRef][Medline] [Order article via Infotrieve]
  5. Crockett-Torabi, E. (1998) J. Leukocyte Biol. 63, 1-14[Abstract]
  6. Hayflick, J. S., Kilgannon, P., and Gallatin, W. M. (1998) Immunol. Res. 17, 313-327[Medline] [Order article via Infotrieve]
  7. Jaye, M., Schlessinger, J., and Dionne, C. A. (1992) Biochim. Biophys. Acta 1135, 185-199[Medline] [Order article via Infotrieve]
  8. Denhardt, D. T. (1996) Biochem. J. 318, 729-747[Medline] [Order article via Infotrieve]
  9. Cunningham, B. A., Hemperly, J. J., Murray, B. A., Prediger, E. A., Brackenbury, R., and Edelman, G. M. (1987) Science 236, 799-806[Medline] [Order article via Infotrieve]
  10. Zhou, H., Fuks, A., Alcaraz, G., Bolling, T. J., and Stanners, C. P. (1993) J. Cell Biol. 122, 951-960[Abstract]
  11. Ranheim, T. S., Edelman, G. M., and Cunningham, B. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4071-4075[Abstract/Free Full Text]
  12. Walsh, F. S., and Doherty, P. (1997) Annu. Rev. Cell Dev. Biol. 13, 425-456[CrossRef][Medline] [Order article via Infotrieve]
  13. Edelman, G. M. (1992) Dev. Dyn. 193, 2-10[Medline] [Order article via Infotrieve]
  14. Fields, D., and Itoh, K. (1996) Trends Neurosci. 19, 473-480[CrossRef][Medline] [Order article via Infotrieve]
  15. Rose, S. P. R. (1996) J. Physiol. (Paris) 90, 387-391[CrossRef][Medline] [Order article via Infotrieve]
  16. Holst, B. D., Vanderklish, P. W., Krushel, L. A., Zhou, W., Langdon, R. B., McWhirter, J. R., Edelman, G. M., and Crossin, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2597-2602[Abstract/Free Full Text]
  17. Krushel, L. A., Sporns, O., Cunningham, B. A., Crossin, K. L., and Edelman, G. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4323-4327[Abstract]
  18. Martini, R. (1994) J. Neurocytol. 23, 1-28[Medline] [Order article via Infotrieve]
  19. Daniloff, J. K., Shoemaker, R. S., Lee, A. F., Strain, G. M., and Remsen, L. G. (1995) Rest. Neurol. Neurosci. 7, 137-144
  20. Fu, S. Y., and Gordon, T. (1997) Mol. Neurobiol. 14, 67-116[Medline] [Order article via Infotrieve]
  21. Mauro, V. P., Wood, I. C., Krushel, L., Crossin, K. L., and Edelman, G. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2868-2872[Abstract]
  22. Crossin, K. L., Tai, M.-H., Krushel, L. A., Mauro, V. P., and Edelman, G. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2687-2692[Abstract/Free Full Text]
  23. Sporns, O., Edelman, G. M., and Crossin, K. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 542-546[Abstract]
  24. Krushel, L. A., Tai, M.-H., Cunningham, B. A., Edelman, G. M., and Crossin, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2586-2592[Abstract/Free Full Text]
  25. McKay, L. I., and Cidlowski, J. A. (1998) Mol. Endocrinol. 12, 45-56[Abstract/Free Full Text]
  26. Ray, A., and Prefontaine, K. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 752-756[Abstract]
  27. Sen, R., and Baltimore, D. (1986) Cell 47, 921-928[Medline] [Order article via Infotrieve]
  28. Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve]
  29. Baeuerle, P. A. (1998) Curr. Biol. 8, R19-R22[Medline] [Order article via Infotrieve]
  30. O'Neill, L. A. J., and Kaltschmidt, C. (1997) Trends Neurosci. 20, 252-258[CrossRef][Medline] [Order article via Infotrieve]
  31. Perez-Otano, I., McMillian, M. K., Chen, J., Bing, G., Hong, J. S., and Pennypacker, K. R. (1996) Glia 16, 306-315[CrossRef][Medline] [Order article via Infotrieve]
  32. Meberg, P. J., Kinney, W. R., Valcourt, E. G., and Routtenberg, A. (1996) Mol. Brain Res. 38, 179-190[Medline] [Order article via Infotrieve]
  33. Kaltschmidt, C., Kaltschmidt, B., and Baeuerle, P. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9618-9622[Abstract]
  34. Furukawa, K., and Mattson, M. P. (1998) J. Neurochem. 70, 1876-1886[Medline] [Order article via Infotrieve]
  35. Kaltschmidt, C., Kaltschmidt, B., and Baeuerle, P. A. (1993) Mech. Dev. 43, 135-147[CrossRef][Medline] [Order article via Infotrieve]
  36. Kopp, E., and Ghosh, S. (1994) Science 265, 956-959[Medline] [Order article via Infotrieve]
  37. de Martin, R., Vanhove, B., Cheng, Q., Hofer, E., Csizmadia, V., Winkler, H., and Bach, F. H. (1993) EMBO J. 12, 2773-2779[Abstract]
  38. Brockman, J. A., Scherer, D. C., McKinsey, T. A., Hall, S. M., Qi, X., Lee, W. Y., and Ballard, D. W. (1995) Mol. Cell. Biol. 15, 2809-2818[Abstract]
  39. Schousboe, A., Meier, E., Drejer, J., and Hertz, L. (1989) in A Dissection and Tissue Culture Manual of the Nervous System (Shahar, A., de Vellis, J., Vernadakis, A., and Haber, B., eds), pp. 203-206, Alan R. Liss, Inc., New York
  40. Schreiber, E., Matthias, P., Müller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419-6423[Medline] [Order article via Infotrieve]
  41. Holst, B. D., Goomer, R. S., Wood, I. C., Edelman, G. M., and Jones, F. S. (1994) J. Biol. Chem. 269, 22245-22252[Abstract/Free Full Text]
  42. Pompeiano, M., Cirelli, C., and Tononi, G. (1998) in Molecular Regulation of Arousal States (Lydic, R., ed), pp. 157-165, CRC Press, Inc., Boca Raton, FL
  43. Liang, P., Zhu, W., Zhang, X., Guo, Z., O'Connell, P., Averboukh, L., Wang, F., and Pardee, A. B. (1994) Nucleic Acids Res. 22, 5763-5764[Medline] [Order article via Infotrieve]
  44. Scholer, H. R. (1991) Trends Genet. 7, 323-329[Medline] [Order article via Infotrieve]
  45. Beckmann, A. M., and Wilce, P. A. (1997) Neurochem. Int. 31, 477-510[CrossRef][Medline] [Order article via Infotrieve]
  46. Pierce, J. W., Schoenleber, R., Jesmok, G., Best, J., Moore, S. A., Collins, T., and Gerritsen, M. E. (1997) J. Biol. Chem. 272, 21096-21103[Abstract/Free Full Text]
  47. Kamiguchi, H., and Lemmon, V. (1997) J. Neurosci. Res. 49, 1-8[CrossRef][Medline] [Order article via Infotrieve]
  48. Bazzoni, G., and Hemler, M. E. (1998) Trends Biochem. Sci. 23, 30-34[CrossRef][Medline] [Order article via Infotrieve]
  49. Thompson, J. E., Phillips, R. J., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1995) Cell 80, 573-582[Medline] [Order article via Infotrieve]
  50. Moynagh, P. N., Williams, D. C., and O'Neill, L. A. (1993) Biochem. J. 294, 343-347[Medline] [Order article via Infotrieve]
  51. Kaltschmidt, C., Kaltschmidt, B., Neumann, H., Wekerle, H., and Baeuerle, P. A. (1994) Mol. Cell. Biol. 14, 3981-3992[Abstract]
  52. Schmidt-Ullrich, R., Mémet, S., Lilienbaum, A., Feuillard, J., Raphaël, M., and Israël, A. (1996) Development 122, 2117-2128[Abstract/Free Full Text]
  53. Le Bail, O., Schmidt-Ullrich, R., and Israël, A. (1993) EMBO J. 12, 5043-5049[Abstract]
  54. Ito, C. Y., Kazantsev, A. G., and Baldwin, J., A. S. (1994) Nucleic Acids Res. 22, 3787-3792[Abstract]
  55. Hall, A. V., Antoniou, H., Wang, Y., Cheung, A. H., Arbus, A. M., Olson, S. L., Lu, W. C., Kau, C.-L., and Marsden, P. A. (1994) J. Biol. Chem. 269, 33082-33090[Abstract/Free Full Text]
  56. Rattner, A., Korner, M., Rosen, H., Bauerle, P. A., and Citri, Y. (1991) Mol. Cell. Biol. 11, 1017-1022[Medline] [Order article via Infotrieve]
  57. Brown, K., Park, S., Kanno, T., Franzoso, G., and Siebenlist, U. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2532-2536[Abstract]
  58. Sun, S.-C., Ganchi, P. A., Ballard, D. W., and Greene, W. C. (1993) Science 259, 1912-1915[Medline] [Order article via Infotrieve]
  59. Cheng, Q., Cant, C. A., Moll, T., Hofer-Warbinek, R., Wagner, E., Birnstiel, M. L., Bach, F. H., and de Martin, R. (1994) J. Biol. Chem. 269, 13551-13557[Abstract/Free Full Text]
  60. Chiao, P. J., Miyamoto, S., and Verma, I. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 28-32[Abstract]
  61. Schouten, G. J., Vertegaal, A. C. O., Whiteside, S. T., Israël, A., Toebes, M., Dorsman, J. C., van der Eb, A. J., and Zantema, A. (1997) EMBO J. 16, 31333-3144
  62. DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-558[CrossRef][Medline] [Order article via Infotrieve]
  63. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve]
  64. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) Science 278, 860-866[Abstract/Free Full Text]
  65. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., and Okumura, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3537-3542[Abstract/Free Full Text]
  66. Bowie, A. G., Moynagh, P. N., and O'Neill, L. A. J. (1998) J. Biol. Chem. 272, 25941-25950[Abstract/Free Full Text]
  67. Montaner, S., Perona, R., Saniger, L., and Lacal, J. C. (1998) J. Biol. Chem. 273, 12779-12785[Abstract/Free Full Text]
  68. Mattson, M. P. (1998) Int. Rev. Neurobiol. 42, 103-167[Medline] [Order article via Infotrieve]
  69. Maness, P. F., Beggs, H. E., Klinz, S. G., and Morse, W. R. (1996) Persp. Dev. Neurobiol. 4, 169-181
  70. Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli, C. M. (1998) J. Cell Biol. 141, 1083-1093[Abstract/Free Full Text]
  71. Kheramand, F., Werner, E., Tremble, P., Symons, M., and Werb, Z. (1998) Science 280, 898-902[Abstract/Free Full Text]
  72. Beggs, H. E., Baragona, S. C., Hemperly, J. J., and Maness, P. F. (1997) J. Biol. Chem. 272, 8310-8319[Abstract/Free Full Text]
  73. Hohashi, N., Hayashi, T., Fusaki, N., Takeuchi, M., Higurashi, M., Okamoto, T., Semba, K., and Yamamoto, T. (1995) Int. Immunol 7, 1851-1859[Abstract]
  74. Imbert, V., Farahifar, D., Auberger, P., Mary, D., Rossi, B., and Peyron, J. F. (1996) J. Inflamm. 46, 65-77[Medline] [Order article via Infotrieve]
  75. Guerrini, L., Blasi, F., and Denis-Donini, S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9077-9081[Abstract]
  76. Freudenthal, R., Locatelli, F., Hermitte, G., Maldonado, H., Lafourcade, C., Delorenzi, A., and Romano, A. (1998) Neurosci. Lett. 242, 143-146[CrossRef][Medline] [Order article via Infotrieve]
  77. Crino, P., Khodakhah, K., Becker, K., Ginsberg, S., Hemby, S., and Eberwine, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2313-2318[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.