Search

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]

Abstract: Among the most conserved osmoregulatory hormone systems in vertebrates are the renin–angiotensin system (RAS) and the natriuretic peptides (NPs). We examined the RAS and NP system in the euryhaline Atlantic stingray, Dasyatis sabina (Lesueur). To determine the relative sensitivity of target organs to these hormonal systems, we isolated cDNA sequences encoding the D. sabina angiotensin receptor (AT) and natriuretic peptide type-B receptor (NPR-B). We then determined the tissue-specific expression of their mRNAs in saltwater D. sabina from local Texas waters and an isolated freshwater population in Lake Monroe, Florida. AT mRNA was most abundant in interrenal tissue from both populations. NPR-B mRNA was most abundant in rectal gland tissue from both populations, and also highly abundant in the kidney of saltwater D. sabina. This study is the first to report the sequence of an elasmobranch angiotensin receptor, and phylogenetic analysis indicates that the D. sabina receptor is more similar to AT1 vs. AT2 proteins. This classification is further supported by molecular analysis of AT1 and AT2 proteins demonstrating conservation of AT1-specific amino acid residues and motifs in D. sabina AT. Molecular classification of the elasmobranch angiotensin receptor as an AT1-like protein provides fundamental insight into the evolution of the vertebrate RAS. [ABSTRACT FROM AUTHOR]

Abstract: It is thought that a single corticosteroid, 1α-hydroxycorticosterone (1α-B), is both a glucocorticoid and mineralocorticoid in the elasmobranch fishes. We investigated the putative mineralocorticoid role of 1α-B by examining regulation of interrenal 1α-B synthesis by osmoregulatory hormones in the euryhaline stingray Dasyatis sabina. Using synthesized steroid, a commercial enzyme-linked immunoassay was validated for the quantification of 1α-B. In interrenal cultures, the antinatriuretic peptide angiotensin II (ANG II) was potently steroidogenic, whereas C-type natriuretic peptide had no effect on 1α-B titers. However, both peptides significantly decreased abundance of rate-limiting steroidogenic mRNAs (steroidogenic acute regulatory protein, StAR; cholesterol side-chain cleavage, P450scc). We also isolated cDNAs encoding ANG II from three species of elasmobranch, verifying heterogeneity among elasmobranch peptides at the first amino acid position. Potential implications of this heterogeneity were investigated by examining the effects of homologous and heterologous ANG II on interrenal steroid production and steroidogenic mRNAs. Changes at amino acid position three, but not position one, of ANG II significantly affected steroidogenic potency. Conversely, changes at position one, but not position three, significantly affected the potency of ANG II to alter levels of steroidogenic mRNAs. This study is the first to demonstrate regulation of elasmobranch steroidogenic mRNAs by osmoregulatory peptides. [Copyright &y& Elsevier]