Endocrine Abstracts

Cellular sensitivity to circulating glucocorticoids is critical and is principally determined by the level of glucocorticoid receptor (GR). GR expression is most potently regulated by glucocorticoids themselves, although the mechanism is unknown. The rat GR gene encodes at least eleven alternate and untranslated exons 1, eight of which lie in a 3kb CpG island close to exon 2. We have recently shown that individual exons 1 are subject to tissue specific expression and regulation. Specifically, exon 1-7 is selectively and permanently up-regulated in the hippocampus by early life events.
To further investigate cell-specific regulatory elements, transient transfection studies were carried out in three cell lines using a set of constructs forming a 3' deletion series of the rat GR promoter fused to a luciferase reporter gene. 'P2' contains the entire promoter and shows the highest activity in all cell lines. 'P1-7' contains the exon 1-7 promoter and shows the highest activity of any individual promoter in B103 (rat neural) and C6 (rat glioma) cells, but not in H4IIE (rat hepatoma) cells (p<0.001). Transfections in B103 cells using a 5' deletion series of P1-7 identified a 134bp region required for high promoter activity (p<0.001). High activity is not maintained if this fragment is reversed, i.e. it does not behave as a classical enhancer. Furthermore, DNaseI footprinting studies of the fragment indicated protein binding at a putative SP-1 site. A 5' deletion series of P2 transfected into B103 cells identified a 526bp region upstream of exon 1-4 required for down-regulating activity following glucocorticoid treatment. DNaseI hypersensitive site mapping in H4IIE cells showed that a 3.6kb fragment encompassing the entire CpG region is highly sensitive to nuclease digestion. As such, the demonstrated cell-specific activity and expression of alternate exons 1 may confer differential regulation of GR.