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Signals Resulting From the Expression and Interaction of Morphoregulatory Molecules

K.L. Crossin, L.A. Krushel, B.A. Cunningham, B.H. Holst, P.W. Vanderklish, G.R. Phillips, G.M. Edelman

Understanding embryonic development requires knowledge both of how genes control the patterned organization of tissues in the embryo and of how gene networks operate in individual cells as a consequence of the feedback the networks receive from cellular interactions. Interactions among cells and interactions of cells with the extracellular matrix are mediated by cell adhesion molecules (CAMs), substrate adhesion molecules (SAMs), and the receptors for these 2 types of molecules. CAMs and SAMs regulate cellular proliferation, movement, and differentiation and, in the nervous system, neurite guidance and extension. One of our major goals is to understand the influence of CAM and SAM binding on subsequent morphogenesis and gene expression. Our recent work has focused on understanding the changes that result when cells interact via neural CAM (N-CAM) or with tenascin, a molecule in the extracellular matrix. We are also investigating changes in the expression of CAM genes and other genes in response to neural activity.

N-CAM is a transmembrane glycoprotein of the immunoglobulin superfamily that is expressed by both neurons and astrocytes. N-CAM levels increase after injury, suggesting that regulation of N-CAM expression is important in nerve regeneration. To examine the influence of neural activity on the expression of N-CAM, we used homologous recombination to insert the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of ß-galactosidase under the control of the N-CAM promoter.

Animals homozygous for the disrupted allele expressed no N-CAM mRNA or protein, but the pattern of expression of ß-galactosidase in heterozygous and homozygous embryos was similar to that of N-CAM RNA in wild-type animals. The homozygotes had the morphologic abnormalities observed in N-CAM knockout mice by other investigators, but the hippocampal long-term potentiation in the Schaffer collaterals was identical in homozygous, heterozygous, and wild-type animals.

Heterozygous mice were used to examine changes in the regulation of the N-CAM promoter in response to enhanced synaptic transmission. Synaptic transmission was altered by using an experimental drug, ampakine, which is an allosteric modulator of glutamate receptors of the (R,S)-/alpha/-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype that enhances normal glutamate-mediated synaptic transmission. Treatment of the mice with ampakine increased the expression of ß-galactosidase in vivo and in tissue slices in vitro. Similar treatments also increased the expression of N-CAM RNA. The effects of ampakine in tissue slices were strongly reduced in the presence of CNQX, an AMPA antagonist. Taken together, these results indicate that facilitation of AMPA receptor--mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that synthesis of N-CAM is regulated in part by synaptic activity.

Signaling to the nucleus occurs not only after synaptic activity but also after CAM binding at the cell surface. In previous studies, we showed that N-CAM inhibits astrocyte proliferation in vitro and in vivo and that N-CAM can inhibit growth factor--stimulated astrocyte proliferation and alter gene expression through changes in the activity of glucocorticoid receptors. To extend these studies, we explored signaling pathways stimulated by growth factors that might be influenced by N-CAM binding.

One such pathway involves activation of mitogen-activated protein (MAP) kinase. In astrocytes, addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor. Of more importance, the presence of RU-486, a glucocorticoid antagonist, prevented inhibition by N-CAM. Similar to our previous findings on the effect of N-CAM on proliferation and gene expression, these results indicate that the influence of N-CAM on MAP kinase activity requires the functioning of the glucocorticoid receptor. These results have prompted us to consider effects of N-CAM on other transcription factors, such as activator protein-1 and nuclear factor /kappa/B, that are also influenced by the glucocorticoid receptor.

Tenascin is an extracellular matrix protein synthesized by glia that influences neurite outgrowth and cell migration during development. It is also highly expressed by glial tumors (gliomas) and may influence metastasis. We studied the regions within the tenascin protein that are involved in the migration of glioma cells. Three independent regions within the fibronectin-like repeats of the protein affected migration and attachment of the cells. In other studies, one of these domains bound to the integrin ₈ß₁. Together our studies indicate that cells integrate their responses to various domains of tenascin through multiple receptors to effect cell type--specific responses.

The results of these and ongoing investigations should provide a deeper understanding of the modulation of gene expression by CAMs and by neural activity. These studies should complement research on the regulation of the synthesis of CAMs and SAMs by the products of homeotic genes.