Fresh water acclimation elicits a decrease in plasma corticosteroids in the euryhaline Atlantic stingray, Dasyatis sabina.

It is thought that the elasmobranch corticosteroid hormone 1α-hydroxycorticosterone (1α-B) functions as both a glucocorticoid (GC) and mineralocorticoid (MC). Classical antinatriuretic MC activities would run counter to the osmoregulatory strategy of euryhaline elasmobranchs acclimating to fresh water (FW). Therefore we hypothesize that FW acclimation will be accompanied by a decrease in plasma corticosteroids in these animals. However, events that activate the "fight-or-flight" response could mask changes associated with acclimation to lower salinities. To better define the MC role of corticosteroids in elasmobranchs, we designed a transfer system that allows the acclimation of Atlantic stingrays (Dasyatis sabina) from seawater (SW) to FW over 12h while minimizing other extraneous stressors. Blood and interrenal glands were sampled from one group of stingrays 24h after FW transfer, while another group was sampled two weeks after FW transfer. Two other groups served as mock-transfer controls in that they were treated and sampled in the same way, but remained in SW for the entire period. Plasma corticosteroids, osmolality, chloride, and urea were significantly lower in FW-acclimated stingrays (compared to mock-transfer stingrays) 24h after FW transfer. This pattern remained after two weeks in FW, with the exception that plasma corticosteroids returned to pre-acclimation levels. There were no significant differences between experimental groups in interrenal levels of mRNAs encoding key steroidogenic proteins (steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme). Temporally decreased corticosteroid levels during FW acclimation are consistent with the unique strategy of euryhaline elasmobranchs, whereby lower plasma osmolality is maintained in FW vs. SW environments to reduce hydromineral gradients.

Molecular classification of an elasmobranch angiotensin receptor: quantification of angiotensin receptor and natriuretic peptide receptor mRNAs in saltwater and freshwater populations of the Atlantic stingray.

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 AT(1) vs. AT(2) proteins. This classification is further supported by molecular analysis of AT(1) and AT(2) proteins demonstrating conservation of AT(1)-specific amino acid residues and motifs in D. sabina AT. Molecular classification of the elasmobranch angiotensin receptor as an AT(1)-like protein provides fundamental insight into the evolution of the vertebrate RAS.

Regulation of mRNAs encoding the steroidogenic acute regulatory protein and cholesterol side-chain cleavage enzyme in the elasmobranch interrenal gland.

The rate-limiting and regulated step in steroidogenesis, the conversion of cholesterol to pregnenolone, is facilitated by the steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage (P450scc). We have isolated cDNAs encoding StAR and P450scc from the Atlantic stingray, Dasyatis sabina, and characterized the steroidogenic activity of the encoded proteins using a heterologous expression system. Green monkey kidney (COS-1) cells cotransfected with D. sabina StAR and human P450scc/adrenodoxin reductase/adrenodoxin fusion (F2) constructs produced significantly more pregnenolone than cells transfected with the F2 construct alone. COS-1 cells transfected with a modified F2 construct (F2DS) in which human P450scc is replaced by D. sabina P450scc had higher rates than cells transfected with D. sabina P450scc alone. In other vertebrates, the stress peptide adrenocorticotropic hormone (ACTH) elicits its effects on corticosteroidogenesis in part through regulation of StAR and P450scc mRNAs. In vitro incubation of D. sabina interrenal tissue with porcine ACTH significantly increased intracellular cAMP and corticosteroid production. As demonstrated by quantitative PCR, ACTH also induced significant increases in mRNA abundance of both StAR and P450scc. Our results suggest that, as in higher vertebrates, chronic ACTH-induced glucocorticoid synthesis in elasmobranchs is mediated by regulation of primary steroidogenic mRNAs. This study is the first to demonstrate steroidogenic activity of an elasmobranch P450scc protein and express a composite elasmobranch steroidogenic pathway in a heterologous cell line. Also, the regulation of StAR and P450scc mRNAs has not previously been demonstrated in elasmobranch fishes.

Adaptation of a corticosterone ELISA to demonstrate sequence-specific effects of angiotensin II peptides and C-type natriuretic peptide on 1alpha-hydroxycorticosterone synthesis and steroidogenic mRNAs in the elasmobranch interrenal gland.

It is thought that a single corticosteroid, 1alpha-hydroxycorticosterone (1alpha-B), is both a glucocorticoid and mineralocorticoid in the elasmobranch fishes. We investigated the putative mineralocorticoid role of 1alpha-B by examining regulation of interrenal 1alpha-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 1alpha-B. In interrenal cultures, the antinatriuretic peptide angiotensin II (ANG II) was potently steroidogenic, whereas C-type natriuretic peptide had no effect on 1alpha-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.

Cholecystokinin: molecular cloning and immunohistochemical localization in the gastrointestinal tract of larval red drum, Sciaenops ocellatus (L.).

The current study sought to clarify the role of cholecystokinin (CCK) in the digestion of larval red drum (Sciaenops ocellatus) in order to better characterize the processes limiting the utilization of microparticulate diets at first feeding. The red drum CCK cDNA, isolated from adult anterior intestine and pyloric caeca, contains a 414 base pair (bp) open reading frame encoding a deduced amino acid sequence of 138 residues which is highly similar to preprocholecystokinin from other vertebrates. The mature CCK octapeptide has the same amino acid sequence as that found in mammals and in Atlantic herring (Clupea harengus). Tissue distribution analysis of adult and juvenile red drum using primers specific for red drum CCK mRNA revealed bright bands in samples from the brain, pyloric caeca, anterior intestine, and gonad with fainter bands seen in all other tissues. Immunohistochemical analysis of larval red drum showed that CCK-immunoreactive (CCK-IR) cells were present as early as 3 days post hatch (DPH) in some fish and were present in all fish by 6 DPH. CCK-IR cells were found in the anterior midgut in early larvae and had spread to the first bend of the gut by day 6. In older larvae (18+ DPH), CCK-IR cells were found in large numbers in the anterior intestine and in the developing pyloric caeca. The sequence and distribution of CCK mRNA along with the presence of CCK-IR cells in early red drum larvae suggest that CCK is present and may be capable of regulating pancreatic secretion in early red drum larvae.

The onset of cortisol synthesis and the stress response is independent of changes in CYP11B or CYP21 mRNA levels in larval red drum (Sciaenops ocellatus).

Although cortisol plays an important role in teleost development, the onset of cortisol production and the cortisol stress response in teleosts remain poorly understood. Here we have reported basal cortisol levels and the development of the cortisol stress response in larval red drum (Sciaenops ocellatus). We isolated partial nucleic acid sequences encoding two key corticosteroidogenic enzymes, CYP11B and CYP21 and assessed ontogenetic patterns of their mRNA levels relative to basal and stress-induced cortisol production. Basal cortisol was first detected 3 days post-hatch (DPH) and reached a maximum at 9 DPH. Cortisol did not increase in response to an acute stressor prior to 6 DPH. From 6 DPH forward, stress caused significant increases in larval cortisol content. Stress-induced cortisol levels in 6-9 DPH larvae were highest 1h post-stress. In larvae 11 DPH and older, the highest cortisol measurements occurred 0.5h post-stress. Elevated cortisol was still evident after 3h in 6 DPH larvae. From 11 DPH onward, basal cortisol levels were reestablished in larvae by 1h post-stress. CYP11B and CYP21 transcripts were detected in red drum 12h prior to hatching and in all post-hatch larvae examined. Changes in CYP11B and CYP21 mRNA levels did not occur in association with the ontogenetic appearance of cortisol, or the onset of the stress response. As larvae developed, the dynamics of the cortisol stress response matured from a low magnitude, slow recovery response, to a response similar to that observed in juvenile and adult fish.

Hormonal regulation of the steroidogenic acute regulatory protein (StAR) in gonadal tissues of the Atlantic croaker (Micropogonias undulatus).

The steroidogenic acute regulatory protein (StAR), a member of the StAR-related lipid transfer domain (START) family, is critical to regulated steroidogenesis in vertebrates. We have isolated a cDNA encoding StAR from a well-studied model of teleost physiology, the Atlantic croaker Micropogonias undulatus. This cDNA (1204 nucleotides total length) contains an open reading frame of 858 nucleotides encoding a protein of 286 amino acids. Molecular phylogenetic analysis indicates the putative Atlantic croaker StAR protein is more closely related to StAR proteins (62-85% identity) than to the related START protein MLN-64 (28-31% identity). Green monkey kidney cells (COS-1) cotransfected with Atlantic croaker StAR and human cholesterol side chain cleavage (SCC) expression constructs are able to produce significantly more pregnenolone than cells transfected with SCC alone. StAR mRNA is detected in the Atlantic croaker head kidney by reverse transcriptase-polymerase chain reaction (RT-PCR) and in the kidney and hypothalamus in some individuals. Gonadal StAR gene expression is below the level of detection by RT-PCR in most individuals, but can be detected using fluorescent probes in quantitative RT-PCR. StAR mRNA is not detected in the Atlantic croaker brain. Six hour in vitro treatment of Atlantic croaker ovarian follicles with human chorionic gonadotropin (hCG) is insufficient to significantly alter StAR mRNA levels; however, 24 h hCG treatment induces StAR mRNA levels 17-fold over untreated controls. Neither 6 nor 24 h treatment with hCG significantly alters StAR mRNA levels in Atlantic croaker testicular minces. Likewise, 6h in vitro treatment with estradiol, testosterone or the maturation-inducing steroid 17,20beta,21-trihydroxy-4-pregnen-3-one is without effect on gonadal StAR mRNA levels.

Tissue- and sex-specific regulation of CYP19A1 expression in the Atlantic croaker (Micropogonias undulatus).

To better define the tissue- and sex-specific roles of aromatase in fishes, we have isolated a CYP19A1 cDNA sequence from a well-developed model of teleost reproduction, the Atlantic croaker (Micropogonias undulatus). This cDNA encodes a protein which has high identity (57-90%) to known CYP19A1 proteins and segregates with teleost CYP19A1 proteins in molecular phylogenetic analysis. In both sexes, the gene encoding Atlantic croaker CYP19A1 is expressed primarily in gonadal tissue, but also in the brain and other tissues at much lower levels, as determined relative to ribosomal 18S RNA expression by real-time quantitative RT-PCR. In females, the highest levels of CYP19A1 mRNA are found in the developing ovary compared to spawning, regressing and resting ovaries. In contrast, testicular CYP19A1 expression is lowest in developing testes and increases in spawning and regressing testes, although there were no statistically significant differences between stages. Brain CYP19A1 mRNA levels are lower in animals with developing gonads compared to spawning fish. In vitro treatment with human chorionic gonadotropin (10 IU/ml) for 6 or 24h increases CYP19A1 mRNA approximately 16- and 43-fold, respectively, in isolated Atlantic croaker ovarian follicles, but has no effect on CYP19A1 mRNA in testicular or brain minces. Six hour in vitro treatment with sex steroids (estradiol, testosterone or 17,20 beta,21-trihydroxy-4-pregnen-3-one; 290 nM) does not alter CYP19A1 mRNA in ovary, testis or brain. The regulation of CYP19A1 in the Atlantic croaker therefore differs in a tissue- and sex-specific manner.

Human glucocorticoid receptor alpha transcript splice variants with exon 2 deletions: evidence for tissue- and cell type-specific functions.

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.

Interaction between the interferon signaling pathway and the human glucocorticoid receptor gene 1A promoter.

The newly described 1A promoter of the human glucocorticoid receptor (hGR) gene contains an interferon (IFN) regulatory factor element (IRF-E), a binding motif for the family of proteins termed IFN regulatory factors (IRFs) that are regulated by IFNs. To examine the in vivo role of IFNs in hGR gene regulation, human T cell lines (CEM-C7 and Jurkat) were treated with IFN gamma. IFN gamma rapidly induces the expression of IRF-1 proteins in a dose- and time-dependent manner. Luciferase expression is induced by IFN treatment in Jurkat cells transfected with an hGR 1A promoter IRF-E/luciferase reporter gene, but induction is lost with deletion of the IRF-E. Electrophoretic mobility shift and supershift analyses indicate an increase in the binding of IRF-1 to oligonucleotides containing the hGR 1A promoter IRF-E after IFN gamma treatment, whereas IRF-2 binding to this oligonucleotide is unchanged. Human IRF-1 and IRF-2 proteins expressed in Chinese hamster ovary cells bind to the hGR 1A promoter IRF-E; however, only IRF-1 activates transcription. Although IFNs clearly activate a transfected reporter gene containing the hGR 1A promoter in T cells, they do not alter the sensitivity of CEM-C7 cells to glucocorticoid-induced apoptosis. Additional studies revealed that the glucocorticoid steroid hormone, dexamethasone (DEX), completely blocks IFN induction of IRF-1 mRNA levels. This could explain the lack of any greater apoptotic response to a combination of DEX plus IFN compared with the response to DEX alone. In addition, treatment with IFN gamma alone does not alter endogenous GR mRNA levels (including exon 1A-containing transcripts derived from the hGR 1A promoter) in T lymphoblast cells, even though IRF-1 levels are induced. The difference in IRF-1-driven transcription between the hGR 1A reporter construct and the endogenous hGR 1A promoter could potentially be due to epigenetic effects, such as methylation.

Characterization of promoter 1B in the human glucocorticoid receptor gene.

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.