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Regulation, Function, and Signaling Mechanisms of Cell Adhesion Molecules

The fundamental processes of embryogenesis have evolved to translate the linear genetic code reproducibly into a 3-dimensional organism. Early in development, dividing cells derived from the fertilized egg adhere into specific aggregates. The gene program of each aggregate is then altered, moving the cells down differentiation pathways leading to specific tissues and organs. My colleagues and I have characterized adhesion proteins on the surfaces of cells that allow aggregation, and we have shown that different tissues have different combinations of these cell adhesion molecules or CAMs. One of our objectives is to describe the signals that regulate the expression of gene programs when cells aggregate via specific CAMs.

In addition to forming tissues and organs, development must proceed in such a way that after morphogenetic movements each collective or aggregate of cells is in its proper place in the overall body plan. Transcription factors such as the protein products of genes called Hox and Pax, which are expressed in a place-dependent fashion, are among the regulators of this process. These factors act by turning sets of other genes on or off at critical places along the axis of the embryo. The finding that CAM genes are targets for proteins encoded by Hox and Pax indicates that regulation of CAM gene expression may play a role in tissue morphogenesis in specific regions of the embryo. Our second major goal is to define the cellular processes and cellular components in addition to products of Hox and Pax that are responsible for changing the expression of CAM genes during developmental processes.

Genes are controlled through regions of the DNA called promoters, and transcription factors can bind to specific parts of the promoter DNA to activate or repress gene expression. In previous studies, we found that several proteins encoded by Hox and Pax alter the expression of the neural CAM called N-CAM both in cells and in transgenic mice. More recently, we studied regulation of the N-CAM promoter by neural activity.

To examine the influence of neural activity on the expression of N-CAM, we inserted the bacterial gene lacZ, as a reporter, between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the N-CAM 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 ß-galactosidase expression in heterozygous and homozygous embryos was similar to that of N-CAM RNA in wild-type animals. The homozygotes had most of the abnormalities observed in N-CAM knockout mice described by others; the major exception was that hippocampal long-term potentiation in the Schaffer collaterals was identical in our 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 the experimental drug ampakine, an allosteric modulator of glutamate receptors sensitive to the agonist (R,S)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 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 mRNA. 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 N-CAM synthesis is regulated in part by synaptic activity.

In previous studies, we detected a regulatory region common to the promoter of 2 neural CAMs: Ng-CAM and L1. These CAMs are expressed at high levels in the nervous system and are important for the outgrowth and bundling of neuronal processes during development. We used the L1 promoter and found that a segment of the gene spanning the region from the promoter to the fourth exon (approximately 18 kb) was required to give a neurally restricted expression pattern in transgenic mice. Deletion of a single 21-bp regulatory element called the neural restrictive silencer element (NRSE) within the second intron resulted in extraneural expression of the L1 gene, primarily in mesenchymal derivatives of the neural crest. These experiments indicated that tissue-specific expression of L1 is modulated by the NRSE.

Although the gene for L1 is first expressed at the time of neural differentiation, it is expressed most abundantly during postnatal development when extensive neural outgrowth and formation of synaptic connections occur. These features of L1 expression motivated us to examine how the NRSE controls the expression of L1 during postnatal development. We found that during postnatal development and in adults the NRSE can function both as a silencer and as an enhancer. Expression of ß-galactosidase in neurons within the cortex, striatum, thalamus, and hippocampus and in the peripheral glia that ensheathe olfactory and cochlear nerves was greater in newborn mice with the NRSE-mutated L1lacZ reporter gene than in mice with the wild-type L1lacZ transgene. Later, during postnatal development and in adulthood, mice with the NRSE-mutant transgene had a loss of expression of ß-galactosidase in several neural structures, whereas mice with the wild-type transgene did not. Collectively, these data indicate that the NRSE plays a dual role as a silencer and an enhancer in modulating expression of the gene for L1 in the nervous system.

To determine the signals that occur as a result of the aggregation of cells by CAMs, we have exploited the ability of brain glial cells (astrocytes) to divide in tissue culture. Previously, we showed that N-CAM inhibits astrocyte proliferation in vitro and in vivo and that it can also inhibit proliferation of astrocytes induced by growth factor 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, an antagonist of the glucocorticoid receptor, 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 the 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.

Publications

Edelman, G.M., Jones, F.S. Gene regulation of cell adhesion: A key step in neural morphogenesis. Brain Res. Rev., in press.

Holst, B.D., Vanderklish, P.W., Krushel, L.A., Zhou, W., Langdon, R.B., McWhirter, J.R., Edelman, G.M., Crossin, K.L. Allosteric modulation of AMPA-type glutamate receptors increases activity of the promoter for the neural cell adhesion molecule, N-CAM. Proc. Natl. Acad. Sci. U.S.A. 95:2597, 1998.

Kallunki, P., Edelman, G.M., Jones, F.S. The neural restrictive silencer element can act as both a repressor and enhancer of L1 cell adhesion molecule gene expression during postnatal development. Proc. Natl. Acad. Sci. U.S.A. 95:3233, 1998.

Krushel, L.A., Tai, M.-H., Cunningham, B.A., Edelman, G.M., Crossin, K.L. Neural cell adhesion molecule (N-CAM) domains and intracellular signaling pathways involved in the inhibition of astrocyte proliferation. Proc. Natl. Acad. Sci. U.S.A. 95:2592, 1998.