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Four novel glucocorticoid receptor (hGR) alpha transcripts that have various deletions of exon 2 sequences are identified. Among these hGRalpha splice variants, three of them, 1A1/E2dist hGRalpha, 1A2/E2prox hGRalpha and 1A3/E3 hGRalpha, arise from the hGR 1 A promoter, while 1B/E3 hGRalpha comes from the hGR 1 B promoter.

Steroids are lipophilic hormones that impinge upon virtually every physiological process in vertebrates. The synthesis of the correct type and amount of steroid at the proper time requires the precise regulation of the steroidogenic acute regulatory protein (STAR) and various steroidogenic cytochrome P450 and hydroxysteroid dehydrogenase enzymes. The transcription factors steroidogenic factor-1, DAX-1 and chicken ovalbumin upstream promoter transcription factor are critical to the coordinated expression of steroidogenic genes. Factors that alter the activity of steroidogenic proteins or the expression of the genes encoding them can therefore have dramatic effects on the health and reproductive success of vertebrates. We have isolated cDNAs encoding STAR, steroidogenic enzymes and steroidogenic transcription factors from a teleost, the Atlantic croaker (Micropogonias undulatus) in order to investigate the effects of natural and anthropogenic stressors on gonadal steroidogenesis. The Atlantic croaker is an abundant and ecologically important species for which a wealth of ecological and physiological data exists, making it an excellent model for the study of environmental stress and its effects on reproduction. Atlantic croaker steroidogenic cDNAs encode proteins that are 28-86% identical to corresponding proteins in other species. Using RT-PCR, we detected steroidogenic mRNAs in ovary, testis and head kidney (adrenal cortex homolog). Preliminary evidence indicates that both reproductive hormones (e.g. gonadotropins) and stress hormones (e.g. adrenocorticotropic hormone, cortisol) can regulate gonadal steroidogenesis by modulating the expression and activity of steroidogenic proteins.

At least three different promoter regions (1A, 1B, and 1C) are involved in the expression of the human GR gene. Promoters 1B and 1C are found in a 2800 bp region of DNA immediately upstream of the exon 1C transcriptional initiation site. Transcripts containing either exon 1B or 1C are expressed in a wide variety of human tissues and cultured cells. Luciferase reporter constructs were created containing promoter 1B plus 1C (−2738 to +19), promoter 1B (−2738 to −1046) alone, or promoter 1C (−1045 to +19) alone. All three constructs were equally effective in driving luciferase expression in HeLa (human cervical carcinoma) cells. In Jurkat (human T-cell acute lymphoblastic leukemia) cells, constructs containing promoters 1B plus 1C or promoter 1B were equally active, but the promoters 1B plus 1C construct was 35% more active than the promoter 1C construct. However, in HepG2 (human hepatoma) cells, the promoter 1C construct was as effective as promoters 1B plus 1C and more than twice as effective as promoter 1B. Sequences that reside proximal to the exon 1B transcriptional start site included three Sp1 (FP2-FP4) sites. Another site (FP1) contains the sequence TGATAG, which strongly resembles the consensus binding sequence for the GATA family of transcription factors. However, oligonucleotide competition and supershift analysis of FP1 indicates that this site is not a binding site for GATA proteins. These four sites are in addition to three YY1 and one Sp1 sites previously reported in promoter 1B. In HeLa cells, deletion of the three YY1 sites results in only a 30% loss of activity and substantial loss of activity occurs only after deletion of all four Sp1 sites, indicating the critical importance of Sp1 in GR expression in these cells. In contrast, the elimination of the three YY1 sites results in a dramatic decrease in promoter strength in both HepG2 and Jurkat cells (64 and 77%, respectively), while subsequent deletions of promoter elements do not result in substantial changes in promoter activity in these cell lines. This study shows that both promoters 1B and 1C are important for the ubiquitous expression of the human GR gene. Differences in the utilization of these promoters in various cell types are likely a reflection of different promoter availability and/or the levels of specific transcription factors in the cell. This could contribute to tissue-specific expression of GR levels in different cell types. [Copyright &y& Elsevier]

Alternative splicing of exon 9 in human glucocorticoid receptor (hGR) transcripts yields two native hGR transcripts and proteins, hGRalpha and hGRbeta. We have now identified four novel hGRalpha transcripts that have various deletions of exon 2 sequences. Among these hGRalpha splice variants, three of them, 1A1/E2dist hGRalpha, 1A2/E2prox hGRalpha, and 1A3/E3 hGRalpha, arise from the hGR 1A promoter, while 1B/E3 hGRalpha comes from the hGR 1B promoter. When fused to Flag and enhanced green fluorescent protein (EGFP) tags at the carboxy terminus, all transcript variants can be correctly translated in vitro and in vivo. The Flag-tagged hGRalpha protein variants can functionally bind to a glucocorticoid response element (GRE) and can mediate hormonal stimulation of a pGRE-luciferase reporter gene. Compared to the "classical", native hGRalpha, these four variants exhibit a cell type-specific activation of a reporter gene, and this is influenced by the hGRalpha 3' untranslated region in the hGR transcript. When equal amounts of the cDNAs for these GRalpha variant proteins are transfected into cells, they can exhibit lower or higher transcriptional activation compared to the classical GR. Furthermore, the EGFP-tagged proteins are nuclear localized, even in the absence of hormone. Using quantitative reverse transcription PCR, we found that these transcripts exist at a low level in CEM-C7 cells and IM-9 cells, although the concentrations of the 1A3/E3 hGRalpha and 1B/E3 hGRalpha transcripts are higher than for hGRbeta transcripts, while 1A1/E2dist hGRalphaand 1A2/E2prox hGRalpha transcript levels are comparable to the 1A1 hGRalpha and 1A2 hGRalpha (without the exon 2 deletions) transcript levels, respectively. Because these novel hGR, N-terminal deleted, protein variants have altered biological activity, their expression could potentially affect the hormone sensitivity or resistance in leukemia and be useful in diagnosing hormone-sensitive or -resistant disease.