Glucocorticoids have diverse physiological functions: they affect central nervous system function, intermediary metabolism and restore homeostasis after stress. Secretion of glucocorticoids is regulated by the hypothalamic-pituitary-adrenal (HPA) axis; negative feedback at the hypothalamus and pituitary suppresses glucocorticoid secretion while the hippocampus exerts additional control over HPA axis activity. Glucocorticoids exert most of their actions, including negative feedback, via the glucocorticoid receptor (GR). The glucocorticoid sensitivity of a given cell/tissue is dependent on the level of GR expression. The regulation of the GR gene is complex; GR levels in adult animals are subject to glucocorticoid regulation and can be permanently "programmed" by early life events, with hippocampal GR permanently increased by neonatal handling (via alterations in serotonin turnover) and decreased by prenatal dexamethasone exposure. Evidence suggests that these effects may be mediated through differential regulation of variant exon-1 containing GR mRNAs; in rats the GR gene contains 8 protein-coding exons (exons 2-9) and at least 11 alternate untranslated exons 1 (exons li-ln) which may reflect transcription regulated by alternate promoters. The aim of this thesis was to further investigate the distribution of these variant GR transcripts and examine whether glucocorticoids themselves differentially regulate GR mRNA and its alternate exons 1 in a tissue and region-specific manner. Tissue and region-specific differences in the expression of variant GR mRNA transcripts were found in rat and mouse. Most GR mRNA variants were ubiquitously expressed, but those containing exon 11 were restricted to rat thymus, liver and hippocampus and mouse spleen, while those containing exon 14 were absent from rat cerebral cortex and mouse lung, heart and abdominal fat. In situ mRNA hybridisation on rat brain showed that all the exons 1 studied showed differences in their regional expression when compared to distribution of the total population of GR mRNAs. In contrast, in rat liver and thymus GR mRNA variants showed the same regional distribution as total GR mRNA with highest expression in periportal region of the liver and the thymic cortex. To investigate whether glucocorticoids differentially regulate variant GR mRNAs, in situ mRNA hybridisation was used to investigate the expression variant exons 1 in the hippocampus of adult rats subjected to 72h (ST) or 3-week (LT) adrenalectomy with glucocorticoid replacement. Variant GR mRNAs containing exons 1₅, 1₇, 1₁₀ and 1₁₁ were not affected by adrenalectomy or supraphysiological glucocorticoid replacement in ST or LT animals. However, both adrenalectomy and supraphysiological glucocorticoid replacement significantly upregulated total GR mRNA in the hippocampus of ST animals while adrenalectomy significantly upregulated total GR mRNA in the hippocampus of LT animals. In situ mRNA hybridisation and RNase protection analysis were used to investigate expression of variant GR mRNAs in the liver. Glucocorticoid manipulations did not significantly affect expression of variant GR mRNAs containing exons 1₅, 1₆, 1₁₀ and 1₁₁. However, in ST adrenalectomised animals glucocorticoid replacement significantly downregulated GR mRNA levels compared to adrenalectomised animals given vehicle alone. Adrenalectomy had no effect on total GR mRNA expression in the LT animals, although in these animals the periportal :perivenous ratio of GR expression was significantly increased by adrenalectomy compared to sham. Preliminary data from DNase I hypersensitive site mapping in control animals showed an area of DNase I sensitive chromatin around the position of exon 1₁₀, present in the majority of GR mRNA in the liver. In the thymus, although adrenalectomy with either high and low dose glucocorticoid replacement in ST animals caused a significant downregulation of GR mRNA in the cortex and medulla compared to sham, the expression of exons 1₁, 1₆ and 1₁₀ of the GR gene was not significantly affected by glucocorticoid manipulations. There was no effect of glucocorticoid manipulation on GR or its variants in the LT animals. These results demonstrate tissue-specific differences in the distribution of GR mRNA variants, suggesting that variations in promoter usage may have a role in determining the "set-point" of GR expression in different tissues. The observed tissue-specific effects of glucocorticoids on GR mRNA expression could not be accounted for by changes in expression of any of the variant GR mRNA transcripts studied. This suggests that either the expression of another variant mRNA (known or novel) is regulated by glucocorticoids or that expression of all or a subset of the variant GR mRNAs changed with a magnitude too small to be detected in this study.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:726368 |
| Date | January 2003 |
| Creators | Freeman, Alistair Iain |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/24590 |
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