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Research Topics
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 Research Topics    |    Major Focus Areas
Research Topics
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Genetic Studies
Project Summary

Title: Molecular Regulation of Corticosteroid Receptor Expression in Stress-Responsive Cells
Synopsis: This study seeks an explanation of the body's mechanisms for dealing with stress by analyzing its effects in those stress-responsive tissues of the pituitary, hypothalamus, and adrenal glands.
Overall Project Objective: Elucidate the mechanisms of regulating human glucocorticoid receptor (hGR) expression in tissues of the hypothalamic-pituitary-adrenal (HPA) axis. Abnormal HPA axis response is often seen in major depression, and in posttraumatic stress disorder (PTSD), and hGR may be involved in these alterations.
Status/Results to Date: Analysis of human glucocorticoid receptor (hGR) mRNA revealed a high degree of heterogeneity in the 5'-UTR. Nine exon 1 isoforms were identified. All non-protein coding exons 1 were spliced to a common exon 2. Four of these (1A, 1B, 1E, and 1H) are located in a 3 kb GC-rich region, immediately upstream from intron A, containing hGR promoter activity. With the exception of mRNA containing exon 1H, all isoforms originating from this region are ubiquitously expressed. The two most highly expressed isoforms (1A and 1B) were coordinately autoregulated in cultured T and B cells, suggesting promoter elements contained within the 3 kb GC-rich region are responsible for basal level hGR expression. Two exon 1 sequences were mapped to the region previously identified as intron A. One of these, contiguous with exon 2, was highly expressed in the adrenal gland, and in bone marrow. Three additional exon 1 sequences, located at least 22 (1G), 245 (1I), and 647 (1C) kb upstream from the 3 kb GC-rich region, were identified. The most distal sequence was identical to the 5'-end of the mRNA for the p62 subunit of dynactin, suggesting it is derived from a rare trans-splicing event. Expression of the isoform containing upstream exon 1G was tissue-specific; the highest levels of expression were in pituitary, spleen and liver. A different pattern of expression was observed for upstream exon 1I, suggesting that expression from putative promoters both upstream and downstream of the 3 kb GC-rich region may be responsible for tissue-specific hGR expression and regulation. To identify cis-acting regulatory elements responsible for the regulation of hGR promoter activity, a variety of cell lines corresponding to stress-responsive tissues have been transfected with promoter constructs for: the 3 kb GC-rich region containing promoter activity for exons 1A, 1B, 1E and 1H; the upstream exon 1G; and the upstream exon 1I. Constructs containing the 3 kb GC-rich region demonstrated promoter activity in nearly all cell types, confirming that this promoter is most probably responsible for basal hGR expression. To date, we have been unable to demonstrate promoter activity for the construct corresponding to upstream exon 1G, and have therefore been unable to identify any associated regulatory elements. However, initial experiments with promoter constructs for exon 1I have indicated that promoter activity can be observed in transfected B cells. Thus, we are confident that within the next six months we will have made significant progress towards completion of the specific aim of identifying important cis-acting regulatory elements.
Project:DoD-80
Agency:Department Of Defense
Location:USUHS
P.I. Name:Jeffrey Harmon, Ph. D.
Research Type:Mechanistic
Research Focus:Brain & Nervous System
Focus Category:Genetic Studies
Status:Ongoing
Study Start Date:February 01,1999
Estimated Completion Date:January 31,2002
Specific Aims: The specific goals are to: 1) determine the extent of alternative hGR promoter utilization, and identify and map each promoter; 2) characterize the cis-acting regulatory elements associated with each hGR promoter; and 3) identify tissue specific trans-acting factors responsible for differential (positive or negative) hGR autoregulation.
Methodology: Human glucocorticoid receptors (hGR) mRNA 5'-ends will be used to identify corresponding genomic sequences. For sequences not present in available genomic clones, human chromosome libraries will be screened. Functional promoter activity will be evaluated by using chimeric reporter genes transfected into a panel of cell lines representing various stress-responsive tissues (pituitary, hypothalamus, adrenal). Cis-acting regulatory elements will be identified using transfection assays with normal and mutant promoters to assess the effects of adrenocorticotrophin (ACTH), and corticotropin releasing hormone (CRH) on promoter activity. Tissue-specific trans-acting factors will be characterized using nuclear extracts prepared from appropriate cell lines to identify physical interactions with relevant cis-acting promoter elements.
Most Recent Publications:

Hillmann AG, Ramdas J, Multanen K, Norman MR, Harmon JM. Glucocorticoid receptor gene mutations in leukemic cells acquired in vitro and in vivo. Cancer Research, 60(7):2056-62, Apr 2000. Abstract

Ramdas J, Harmon JM. Glucocorticoid-induced apoptosis and regulation of NF-kappaB activity in human leukemic T cells. Endocrinology, 139(9):3813-21, Sep 1998. Abstract

Ramdas J, Liu W, Harmon JM. Glucocorticoid-induced cell death requires autoinduction of glucocorticoid receptor expression in human leukemic T cells. Cancer Research, 59(6):1378-85, Mar 1999. Abstract

Yamashita H, Nevalainen MT, Xu J, LeBaron MJ, Wagner K, Erwin RA, Harmon J, Hennighausen L, Kirken RA, Rui H. Role of serine phosphorylation of Stat5a in prolactin-stimulated beta-casein gene expression. Mol. Cell Endocrinol, 25;183(1-2):151-63, Oct 2001. Abstract