Search

Glutamine synthetase is a key enzyme in the recycling of the neurotransmitter glutamate. Expression of this enzyme is regulated by glucocorticoids, which induce a high level of glutamine synthetase in neural but not in various non-neural tissues. This is despite the fact that non-neural cells express functional glucocorticoid receptor molecules capable of inducing other target genes. Sequencing and functional analysis of the upstream region of the glutamine synthetase gene identified, 5' to the glucocorticoid response element (GRE), a 21-base pair glutamine synthetase silencer element (GSSE), which showed considerable homology with the neural restrictive silencer element NRSE. The GSSE was able to markedly repress the induction of gene transcription by glucocorticoids in non-neural cells and in embryonic neural retina. The repressive activity of the GSSE could be conferred on a heterologous GRE promoter and was orientation- and position-independent with respect to the transcriptional start site, but appeared to depend on a location proximal to the GRE. Gel-shift assays revealed that non-neural cells and cells of early embryonic retina contain a high level of GSSE binding activity and that this level declines progressively with age. Our results suggest that the GSSE might be involved in the restriction of glutamine synthetase induction by glucocorticoids to differentiated neural tissues.

In chicken embryo retina, competence for induction of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] gene by glucocorticoid hormones increases progressively with development; this competence is minimal in 6-day retina (E6) and high by day 10 (E10). Because the level of glucocorticoid receptors (GRs) in the retina does not increase during that time, we investigated whether the transcriptional activity of GR increased between days 6 and 10 of development. The glucocorticoid-inducible chloramphenicol acetyltransferase (CAT) constructs 2GRE-37TK and p delta G46TCO, which contain glucocorticoid-responsive elements attached to a TATA box and to the thymidine kinase promoter, respectively, were transfected into E6 and E10 retinas, and their inducibility was examined. CAT expression could be induced in the transfected E10 retina but was not induced in the transfected E6 retina. However, induction was obtained also in E6 retina after cotransfection with a GR expression vector. Noninducible CAT constructs (pRSV-CAT, pSV2CAT, and pBLCAT2) were expressed at both ages at similar levels. The CAT construct pGS2.1CAT, which is controlled by the upstream sequence of the chicken glutamine synthetase gene, could be induced in E10 retina but was not induced in E6 retina; however, cotransfection with the GR expression vector resulted in induction of pGS2.1CAT also in E6 retina. We interpret these results as showing that the transcriptional activity of GR in embryonic retina is developmentally controlled and suggest that its increase is causally implicated in the development of competence for glutamine synthetase induction.

Inducibility by glucocorticoids of the glutamine synthetase gene in chicken embryo retina and the transcriptional activity of the glucocorticoid receptor (GR) greatly increase between embryonic days 6 and 10 (E6, E10), although the level of GR does not markedly change during that time. This apparent discrepancy was investigated by examining the pattern of GR expression in undifferentiated E6 retina and in E10 retina, which consists mostly of differentiated cells. Two GR isoforms, 90 and 95 kDa, were found to be expressed at both of these ages at a similar total level but in different proportions: in E6 retina the level of the 90-kDa isoform was higher, whereas in E10 retina the 95-kDa receptor was higher. However, following treatment of the retinas with cortisol, the 95-kDa isoform became the predominant receptor at both ages. Immunohistochemical analysis revealed that the cellular localization of GR markedly changed in the course of development: in the undifferentiated E6 retina GR was expressed in virtually all cells, whereas in the more differentiated E10 and E12 retina, GR was detected only in Müller glia cells. The latter represent approximately 20% of the cells in this tissue and are the only cells in which glucocorticoid hormone induces the glutamine synthetase gene. We suggest that the compartmentalization of GR in Müller glia is a major aspect of the mechanism that modulates receptor activity during retina development and results in the temporal increase in the inducibility of glutamine synthetase and its specific localization in Müller glia cells.