Endocrine Abstracts (2006) 11 P756

The glucocorticoid receptor 1a promoter in CD4+CD8+ thymocytes is rapidly upregulated following dexamethasone treatment in vitro

J Nixon1, J Sheridan2, C Blackburn2 & K Chapman1

1Queens Medical Research Institute University of Edinburgh, Edinburgh, United Kingdom; 2Institute For Stem Cell Research University of Edinburgh, Edinburgh, United Kingdom.

Glucocorticoid Receptor (GR) levels are increased in response to glucocorticoids (GC) in T-lymphoma cell lines susceptible to GC-induced cell death (GICD). The GR gene is transcribed from 5 promoters generating alternate first exons, 1A-1E, with 1A expression restricted to immune tissue and the cortex of the brain. Expression of the 1A-containing variant correlates with susceptibility to GICD in mouse thymocytes. Here we describe the time course of GC induction of total GR mRNA as well as 1A and 1B in mouse thymocytes.

Total mouse thymocytes were treated with 10−6 M dexamethasone (dex) for up to 6 h. RNA was extracted and Real-Time PCR assays for total GR mRNA and the 1A and 1B GR mRNA variants performed on cDNA. Total GR mRNA and 1A mRNA levels were increased 3-fold in thymocytes treated for 2 h with dex, compared to untreated samples. Following 4 h dex treatment, total GR mRNA levels in thymocytes were 1.5-fold higher than in untreated cells and remained at this level after 6 h, whereas 1A-containing mRNA was elevated 2.8-fold above control levels after 4 h dex treatment, falling to 2-fold above control after 6 h. To ascertain which population of thymocytes show this change in GR mRNA levels, CD4+CD8+ double positive (DP) and CD4CD8 double negative (DN) thymocytes were FACS sorted and treated with dex over 6 h. DP cells showed an identical increase in total GR mRNA and 1A at 2 h and 6 h to total thymocytes, consistent with the majority of the thymocyte population being DP cells. Levels of 1B-containing GR mRNA were elevated 2-fold above levels in control cells by 2 h dex treatment, falling to control levels after 6 h. In contrast to DP cells, preliminary data from DN cells suggested that 2 h dex treatment caused a 1.5-fold increase in total GR mRNA, returning to control levels by 6 h. No change was detected in levels of 1A-containing GR mRNA in DN cells following either 2 h or 6 h dex treatment. These data demonstrate a rapid and transient increase of GR mRNA in DP thymocytes, which correlates with DP cells being the population most sensitive to GICD.

The decline in total GR mRNA levels between 2 h and 6 h of dex treatment contrasts to the sustained increase seen in a human leukemic T-cell line and may be due to rapid induction of apoptosis in primary DP thymocytes with the highest levels of GR mRNA; this is currently under investigation.

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