11-Deoxycortisol controls hydromineral balance in the most basal osmoregulating vertebrate, sea lamprey (Petromyzon marinus).

It is unknown whether and how osmoregulation is controlled by corticosteroid signaling in the phylogenetically basal vertebrate group Agnatha, including lampreys and hagfishes. It is known that a truncated steroid biosynthetic pathway in lampreys produces two predominant circulating corticosteroids, 11-deoxycortisol (S) and 11-deoxycorticosterone (DOC). Furthermore, lampreys express only a single, ancestral corticosteroid receptor (CR). Whether S and/or DOC interact with the CR to control osmoregulation in lampreys is still unknown. We examined the role of the endogenous corticosteroids in vivo and ex vivo in sea lamprey (Petromyzon marinus) during the critical metamorphic period during which sea lamprey increase osmoregulatory capacity and acquire seawater (SW) tolerance. We demonstrate in vivo that increases in circulating [S] and gill CR abundance are associated with increases in osmoregulatory capacity during metamorphosis. We further show that in vivo and ex vivo treatment with S increases activity and expression of gill active ion transporters and improves SW tolerance, and that only S (and not DOC) has regulatory control over active ion transport in the gills. Lastly, we show that the lamprey CR expresses an ancestral, spironolactone-as-agonist structural motif and that spironolactone treatment in vivo increases osmoregulatory capacity. Together, these results demonstrate that S is an osmoregulatory hormone in lamprey and that receptor-mediated discriminative corticosteroid regulation of hydromineral balance is an evolutionarily basal trait among vertebrates.

Modulation in the mRNA expression of ecdysone receptor gene in aquatic midge, Chironomus riparius upon exposure to nonylphenol and silver nanoparticles.

Chironomus riparius, a non-biting midge (Chironomidae, Diptera), is extensively used as a model organism in aquatic ecotoxicological studies, although little is known about its genome sequences. Ecdysteroids are steroid hormones that play an important role in development, growth, moulting of larva, and reproduction in Chironomus spp. The effect of ecdysteroids is mediated by their binding to the ecdysteroid receptor (EcR). To study the effect of environmental stressors, nonylphenol and silver nanoparticles (AgNPs), on the modulation of EcR mRNA, in this study, full length cDNA of C. riparius ecdysone receptor (CrEcR) was identified from the Expressed Sequence Tags (ESTs) database and expression of the corresponding mRNA was analyzed following exposure to nonylphenol and AgNPs. The CrEcR cDNA was 2548 base pairs (bp) in length, with a 5' untranslated region (UTR) of 242 bp and a 3' UTR of 684 bp. The open reading frame contains 1617 nucleotides, encoding 539 amino acids with a predicted molecular weight of 61 kDa and pI of 5.89, and revealed the presence of several domains associated with DNA binding, dimerization, ligand binding and transcriptional activation characteristic of steroid receptor family members. It was found that the expression level of CrEcR was significantly up-regulated on exposure to nonylphenol and significant up or down regulation was observed on exposure to AgNPs. These finding shows that nonylphenol as well as AgNPs could modulate the ecdysone nuclear receptor and may have significant implications in different developmental stages in C. riparius.

Hypericum perforatum differentially affects corticosteroid receptor-mRNA expression in human monocytic U-937 cells.

A dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis represents a prominent finding in major depression, possibly related to a dysfunction of the corticosteroid receptor system. Antidepressants are involved in the restoration of the altered feed-back mechanism of the HPA-axis, probably via normalization of corticosteroid receptor functions. Since Hypericum perforatum has antidepressive properties, we here examined its putative actions on glucocorticosteroid receptor mRNA levels in human blood cells as a peripheral model for neuroendocrine effects in human brain cells. Our data show that Hypericum (LI 160) affects the cellular mRNA levels of both, the glucocorticoid receptor (GR)-α and its inhibitory counterpart, the GR-ß, at clinically-relevant concentrations. Under these conditions, a bimodal effect was observed. Dose-response studies suggest a rather small effective concentration range and time-effect data show a primary and transient up-regulation of GR-α mRNA levels and a down-regulation of GR-ß mRNA levels after 16 h of treatment. The sodium channel blocker benzamil neutralized the effects of Hypericum, pointing to an at least partial mechanism of action via this pathway. In conclusion, Hypericum treatment differentially affects GR-mRNA levels in the human system. Our data suggest a bimodal effect on GR, resulting in a time-and dose-related modification of GR-mediated cellular effects. Such a mechanism has been alleged as an important way of action for a number of antidepressants. It is the first time that a specific effect on both receptors, especially on the subtype of GR-ß, is shown under antidepressive treatment in a human system under in vitro conditions.

Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors.

Recent sequencing of amphioxus and sea urchin genomes has provided important data for understanding the origins of enzymes that synthesize adrenal and sex steroids and the receptors that mediate physiological response to these vertebrate steroids. Phylogenetic analyses reveal that CYP11A and CYP19, which are involved in the synthesis of adrenal and sex steroids, first appear in the common ancestor of amphioxus and vertebrates. This correlates with recent evidence for the first appearance in amphioxus of receptors with close similarity to vertebrate steroid receptors. Other CYP450 enzymes involved in steroid synthesis can be traced back to invertebrates, in which they have at least two functions: detoxifying xenobiotics and catalyzing the synthesis of sterols that activate nuclear receptors. CYP450 metabolism of hydrophobic xenobiotics may have been a key event in the origin of ligand-activated steroid receptors from constitutively active nuclear receptors.

Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway.

BACKGROUND: 20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis. PRINCIPAL FINDINGS: Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes. CONCLUSIONS: We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.

The brown shrimp (Crangon crangon L.) ecdysteroid receptor complex: cloning, structural modeling of the ligand-binding domain and functional expression in an EcR-deficient Drosophila cell line.

cDNAs encoding ecdysteroid receptor (EcR) and retinoid X receptor (RXR) were cloned and sequenced from brown shrimp Crangon crangon (Crustacea: Decapoda), a common faunal species and commercially important in the North-West European coastal waters. A 3D model of the ligand-binding domain (LBD) of EcR was created and docking of ponasterone A (PonA) was simulated in silico. Finally, we report the transfection of expression plasmids for these receptors in the mutant Drosophila L57-3-11 cell line. Through an ecdysteroid responsive reporter assay we clearly prove the functionality of shrimp ecdysteroid receptor in the transfected L57-3-11 cell line. Our results indicate that the Drosophila L57-3-11 cell line and in silico LBD modeling can be used to study the function of crustacean ecdysteroid receptors and be applied to assess endocrine disrupting effects on non-target crustacean species.

Genetic polymorphisms of CYP1A2, CYP3A4, CYP3A5, pregnane/steroid X receptor and constitutive androstane receptor in 207 healthy Spanish volunteers.

BACKGROUND: Variability of cytochrome P450 (CYP) in humans is largely related to the pharmacological and toxicological effects of drugs and chemicals. Identification of single nucleotide polymorphisms (SNPs) could be important for knowing their involvement in many drugs metabolism. The goal of this study was to analyze the genotype frequency of 10 SNPs related to mirtazapine metabolism [CYP3A4*17, CYP3A4*18, CYP3A5*3A, CYP1A2*1F, pregnane/steroid X receptor (PXR) (rs3814055, rs38114057, rs3814058) and constitutive androstane receptor (CAR) (rs4073054, rs2307424, rs2502815)]. METHODS: The study was carried out in 207 healthy Spanish volunteers that had participated in phase I clinical trials. Other studies were performed: Hardy-Weinberg equilibrium, haplotype estimation and linkage disequilibrium. RESULTS: No mutation related to CYP3A4*17 and CYP3A4*18 was found. Therefore, we analyzed data for the other eight SNPs. Allele frequencies were in equilibrium with the Hardy-Weinberg equation. Six haplotypes were determined for three PXR SNPs, and four for CAR SNPs. Tests for linkage disequilibrium showed a high association between PXR (rs38114057) and PXR (rs3814058) (p= 0.001), and between the three CAR SNPs (p=0.001), which could be useful for identification of tag SNPs. CONCLUSIONS: In the present study, the genotype frequencies of some SNPs related to mirtazapine metabolism in Spaniards were analyzed and showed that our study population is representative of HapMap European population. The results obtained could be analyzed with pharmacokinetic parameters of mirtazapine to elucidate the genotype-phenotype relationship, the involvement of these SNPs in metabolic reactions, drug interactions, and prediction of treatment response.

A second class of nuclear receptors for oxysterols: Regulation of RORalpha and RORgamma activity by 24S-hydroxycholesterol (cerebrosterol).

The retinoic acid receptor-related orphan receptors alpha and gamma (RORalpha [NR1F1] and RORgamma [NR1F3]) are members of the nuclear hormone receptor superfamily. These 2 receptors regulate many physiological processes including development, metabolism and immunity. We recently found that certain oxysterols, namely the 7-substituted oxysterols, bound to the ligand binding domains (LBDs) of RORalpha and RORgamma with high affinity, altered the LBD conformation and reduced coactivator binding resulting in suppression of the constitutive transcriptional activity of these two receptors. Here, we show that another oxysterol, 24S-hydroxycholesterol (24S-OHC), is also a high affinity ligand for RORalpha and RORgamma (K(i) approximately 25 nM). 24S-OHC is also known as cerebrosterol due to its high level in the brain where it plays an essential role as an intermediate in cholesterol elimination from the CNS. 24S-OHC functions as a RORalpha/gamma inverse agonist suppressing the constitutive transcriptional activity of these receptors in cotransfection assays. Additionally, 24S-OHC suppressed the expression of several RORalpha target genes including BMAL1 and REV-ERBalpha in a ROR-dependent manner. We also demonstrate that 24S-OHC decreases the ability of RORalpha to recruit the coactivator SRC-2 when bound to the BMAL1 promoter. We also noted that 24(S), 25-epoxycholesterol selectively suppressed the activity of RORgamma. These data indicate that RORalpha and RORgamma may serve as sensors of oxsterols. Thus, RORalpha and RORgamma display an overlapping ligand preference with another class of oxysterol nuclear receptors, the liver X receptors (LXRalpha [NR1H3] and LXRbeta [NR1H2]).

Corticosteroid receptors involved in stress regulation in common carp, Cyprinus carpio.

In higher vertebrates, mineralo- (aldosterone) and glucocorticoids (cortisol/corticosterone) exert their multiple actions via specific transcription factors, glucocorticoid (GR) and mineralocorticoid (MR) receptors. Teleostean fishes lack aldosterone and mineral regulatory processes seem under dominant control by cortisol. Despite the absence of the classical mineralocorticoid aldosterone, teleostean fishes do have an MR with cortisol and possibly 11-deoxycorticosterone (DOC) (as alternative for aldosterone) as predominant ligands. We studied corticoid receptors in common carp (Cyprinus carpio L). Through homology cloning and bioinformatic analysis, we found duplicated GR genes and a single MR gene. The GR genes likely result from a major genomic duplication event in the teleostean lineage; we propose that the gene for a second MR was lost. Transactivation studies show that the carp GRs and MR have comparable affinity for cortisol; the MR has significantly higher sensitivity to DOC, and this favours a role for DOC as MR ligand in fish physiology. mRNA of the GRs and the MR is expressed in forebrain (in pallial areas homologous to mammalian hippocampus), corticotrophin-releasing hormone (CRH) cells in the pre-optic nucleus (NPO) and pituitary pars distalis ACTH cells, three key neural/endocrine components of the stress axis. After exposure to prolonged and strong (not to mild acute) stressors, mRNA levels of both GRs and MR become down-regulated in the brain, but not in the NPO CRH cells or pituitary ACTH cells. Our data predicts a function in stress physiology for all CRs and suggest telencephalon as a first line cortisol target in stress.

[Steroid receptor superfamily and its mechanism of action].

Steroid receptors are evoluted from the common ancestor and have the same structure as steroid receptor superfamily. The nuclear receptors are characterized by a central DNA binding domain with specific DNA sequences known as hormone response element, ligand binding domain in the C terminal and N terminal domain with the tissue specificity. Nuclear receptor superfamily is divided into four classes based on the dimerization and DNA binding properties, homodimers as steroid hormone receptor, heterodimers with RXR, homodimers and monomers. Most of orphan receptors fall into the last 2 classes. The coregulators as coactivators and corepressor are determined. The mechanism of action of nuclear receptor on chromatin in the nucleus are clarified precisely from the stand point of the clasters of coregulators and the complex of nuclear receptor and coregulators. The crosstalk of nuclear receptor and peptide hormone signal in the cell is important in the autocrine and paracrine action. Non-genomic action of steroid hormone is also proposed.

EcR expression in the prothoracicotropic hormone-producing neurosecretory cells of the Bombyx mori brain.

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses in the developing cells of the silkworm Bombyx mori. Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B. mori brain. Whole-mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the prothoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secretion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis.

Expression of steroid hormone receptors in the fetal sheep brain during the critical period for sexual differentiation.

The objective of this study was to examine the expression of receptors for androgen, estrogen, and progesterone in the fetal sheep brain during the critical period for sexual differentiation. We isolated mRNA from the hypothalamus-preoptic area (HPOA), amygdala (AMYG), medulla (MD), frontal cortex (FCTX) and olfactory bulbs (OB) of fetal sheep that were delivered on day 64 of gestation. Using a ribonuclease protection assay and species-specific cRNA probes, we measured mRNA expression levels of androgen receptor (AR), estrogen receptor alpha (ERalpha) and progesterone receptor (PR). ERalpha and AR mRNA were expressed in all of the tissues tested and highest in the HPOA. PR mRNA was measured in HPOA and AMYG only and was significantly higher in male than in female fetuses. We conclude that the fetal brain is a target site for circulating steroid hormones. These data have implications for the steroid dependent development of sexually dimorphic brain functions in sheep.

Peripheral nerves: a target for the action of neuroactive steroids.

Peripheral nervous system possesses both classical and non-classical steroid receptors and consequently may represent a target for the action of neuroactive steroids. The present review summarizes the state of art of this intriguing field of research reporting data which indicate that neuroactive steroids, like for instance progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3alpha-diol, stimulate the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein P0 (P0) and the peripheral myelin protein 22 (PMP22). Interestingly, the mechanisms by which neuroactive steroids exert their effects involve classical steroid receptors, like for instance progesterone and androgen receptors, in case of P0 and non-classical steroid receptors, like GABA(A) receptor, in case of PMP22. Moreover, neuroactive steroids not only control the expression of these specific myelin proteins, but also influence the morphology of myelin sheaths and axons suggesting that these molecules may represent an interesting new therapeutic approach to maintain peripheral nerve integrity during neurodegenerative events.

Evolution of the pregnane x receptor: adaptation to cross-species differences in biliary bile salts.

The pregnane X receptor (PXR) regulates the metabolism and elimination of bile salts, steroids, and xenobiotics. The sequence of the PXR ligand-binding domain diverges extensively between different animals, suggesting interspecies differences in ligands. Of the endogenous ligands known to activate PXR, biliary bile salts vary the most across vertebrate species, ranging from 27-carbon (C27) bile alcohol sulfates (early fish, amphibians) to C24 bile acids (birds, mammals). Using a luciferase-based reporter assay, human PXR was activated by a wide variety of bile salts. In contrast, zebrafish PXR was activated efficiently only by cyprinol sulfate, the major zebrafish bile salt, but not by recent bile acids. Chicken, mouse, rat, and rabbit PXRs were all activated by species-specific bile acids and by early fish bile alcohol sulfates. In addition, phylogenetic analysis using maximum likelihood demonstrated evidence for nonneutral evolution of the PXR ligand-binding domain. PXR activation by bile salts has expanded from narrow specificity for C27 bile alcohol sulfates (early fish) to a broader specificity for recent bile acids (birds, mammals). PXR specificity for bile salts has thus paralleled the increasing complexity of the bile salt synthetic pathway during vertebrate evolution, an unusual example of ligand-receptor coevolution in the nuclear hormone receptor superfamily.

Steroid receptor control of reproductive behavior.

Steroid hormone receptors in the brain were thought to be only activated by steroid hormones. Once steroid binds to the receptor, it would act on DNA to regulate gene transcription. Recent data indicate that steroid receptor action is more complex. Steroid receptor activity in the brain is under the control of co-regulatory proteins, such as coactivators. It is the expression of these additional proteins that modulate the activity of steroid receptors. Furthermore, steroid receptors are not only activated by steroid, but can also be activated by neurotransmitters in the absence of steroid. For example, progestin receptors in rodent brain are sensitive to progesterone and to social cues in the environment. This review discusses these emerging mechanisms for steroid receptor control in developing and adult brain.

A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica.

A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen-dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S-transferase pull-down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate.

Steroid hormone receptors: evolution, ligands, and molecular basis of biologic function.

The characterization of the superfamily of nuclear receptors, in particular the steroid/retinoid/thyroid hormone receptors, has resulted in a more complete understanding of how a repertoire of hormonally and nutritionally derived lipophilic ligands controls cell functions to effect development and homeostasis. As transducers of hormonal signaling in the nucleus, this superfamily of DNA-binding proteins appears to represent a crucial link in the emergence of multicellular organisms. Because nuclear receptors bind and are conformationally activated by a chemically diverse array of ligands, yet are closely related in general structure, they present an intriguing example of paralogous evolution. It is hypothesized that an ancient prototype receptor evolved into an intricate set of dimerizing isoforms, capable of recognizing an ensemble of hormone-responsive element motifs in DNA, and exerting ligand-directed combinatorial control of gene expression. The effector domains of nuclear receptors mediate transcriptional activation by recruiting coregulatory multisubunit complexes that remodel chromatin, target the initiation site, and stabilize the RNA polymerase II machinery for repeated rounds of transcription of the regulated gene. Because some nuclear receptors also function in gene repression, while others are constitutive activators, this superfamily of proteins provides a number of avenues for investigating hormonal regulation of gene expression. This review surveys briefly the latest findings in the nuclear receptor field and identifies particular areas where future studies should be fruitful. J. Cell. Biochem. Suppls. 32/33:110-122, 1999.

Ten years after: reclassification of steroid-responsive genes.

Although several hundred genes are directly or indirectly regulated by steroid hormones, significant gaps exist in our understanding of the relevant mechanisms, particularly for those genes that do not directly bind intracellular receptors or that exhibit delayed changes in transcription rates upon receptor binding. To assist in defining the mechanism of action of steroid hormones, we are proposing that a standard nomenclature be adopted for classifying steroid-responsive genes, based upon whether the receptors directly bind to the target genes and the kinetics of the response. Three categories are proposed: primary response genes, delayed primary response genes, and secondary response genes.

Chronic social stress produces reductions in available splenic type II corticosteroid receptor binding and plasma corticosteroid binding globulin levels.

Adult male and female rats were housed for 2 weeks in a Visible Burrow System resulting in the development of strong dominant-subordinate relationships among the male rats. Neuroendocrine measures indicated that the subordinate rats, and to a lesser extent dominant rats, experienced chronic HPA axis hyperstimulation during the 2 week experience. This paper focuses on the consequences of this chronic social stress on cytosolic type II corticosteroid receptor binding in the spleen. In the first study, rats were adrenalectomized 18 h prior to sacrifice in order to measure total cellular receptor protein levels in each animal. In spite of the severity of the social stress, there was no decrease in splenic type II corticosteroid receptor binding levels in these short-term adrenalectomized animals. In the second study, rats were left adrenal-intact. Corticosteroid receptor levels in these adrenal-intact animals reflect the level of receptors (available receptors) that were unoccupied by endogenous hormone at the time of sacrifice. Both subordinate and dominant rats had fewer available splenic type II receptors than control rats, suggesting that a greater proportion of receptors in subordinate and dominant rats were occupied and activated by endogenous hormone at the time of sacrifice than in control rats. The differences in available receptor levels were not a function of total plasma corticosterone levels at the time of sacrifice (mean corticosterone levels were the same for control and subordinate rats). Instead, the differences in available receptor levels may have been a function of plasma corticosteroid binding globulin (CBG) levels which regulate free corticosterone levels. There was a large reduction in plasma CBG levels of subordinate (-70%) and dominant (-40%) rats relative to control rats, and there was a significant correlation between plasma CBG level and available type II receptors in the spleen. These results suggest that a decrease in CBG levels as a result of chronic social stress led to greater access of free corticosterone hormone to type II receptors in the spleen than is typically present in rats under basal or acute stress conditions. This result illustrates one mechanism by which chronic stress may have a greater impact than acute stress on splenic immune function.