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-kappaB 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-kappaB/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-kappaB-binding complex. NF-kappaB-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-kappaB-binding sites. N-CAM binding also resulted in degradation of IkappaB-alpha protein followed by an increase in the levels of IkappaB-alpha mRNA and protein. These results indicate that N-CAM homophilic binding at the cell membrane leads to increased NF-kappaB binding to DNA and transcriptional activation in both neurons and astrocytes. Activation of NF-kappaB, 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.
Highlights
Several families of adhesion molecules contribute to the mechanical stability of cell-cell and cell-extracellular matrix interactions [1]
neural cell adhesion molecule (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 g/ml), or a polyclonal antibody against N-CAM (500 g/ml)
Recombinant proteins corresponding either to the two fibronectin repeats in N-CAM or to fibronectin repeats 8 –10 of the fibronectin protein itself 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
Summary
Several families of adhesion molecules contribute to the mechanical stability of cell-cell and cell-extracellular matrix interactions [1]. 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. 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 revealed that the glucocorticoid receptor (GR), a transcription factor activated by steroid binding, plays a role in N-CAMmediated signaling [22, 24]. 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. External stimuli such as cytokines induce the phosphorylation and degradation of IB allowing for the translocation of NF-B into the nucleus (reviewed in Refs. 28 and 29)
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