Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

Corticosteroid-Binding Globulin (SERPINA6) Consolidates Sexual Dimorphism of Adult Rat Liver.

Produced by the liver, corticosteroid-binding globulin (CBG) regulates the plasma distribution and actions of glucocorticoids. A sex difference in pituitary growth hormone secretion patterns established during puberty in rats results in increased hepatic CBG production and 2-fold higher plasma corticosterone levels in females. Glucocorticoids control hepatic development and metabolic activities, and we have therefore examined how disrupting the SerpinA6 gene encoding CBG influences plasma corticosterone dynamics, as well as liver gene expression in male and female rats before and after puberty. Comparisons of corticosterone plasma clearance and hepatic uptake in adult rats, with or without CBG, indicated that CBG limits corticosterone clearance by reducing its hepatic uptake. Hepatic transcriptomic profiling revealed minor sex differences (207 differentially expressed genes) and minimal effect of CBG deficiency in 30-day-old rats before puberty. While liver transcriptomes in 60-day-old males lacking CBG remained essentially unchanged, 2710 genes were differentially expressed in wild-type female vs male livers at this age. Importantly, ∼10% of these genes lost their sexually dimorphic expression in adult females lacking CBG, including those related to cholesterol biosynthesis, inflammation, and lipid and amino acid catabolism. Another 203 genes were altered by the loss of CBG specifically in adult females, including those related to xenobiotic metabolism, circadian rhythm, and gluconeogenesis. Our findings reveal that CBG consolidates the sexual dimorphism of the rat liver initiated by sex differences in growth hormone secretion patterns and provide insight into how CBG deficiencies are linked to glucocorticoid-dependent diseases.

Plasma cortisol-linked gene networks in hepatic and adipose tissues implicate corticosteroid-binding globulin in modulating tissue glucocorticoid action and cardiovascular risk.

Genome-wide association meta-analysis (GWAMA) by the Cortisol Network (CORNET) consortium identified genetic variants spanning the SERPINA6/SERPINA1 locus on chromosome 14 associated with morning plasma cortisol, cardiovascular disease (CVD), and SERPINA6 mRNA expression encoding corticosteroid-binding globulin (CBG) in the liver. These and other findings indicate that higher plasma cortisol levels are causally associated with CVD; however, the mechanisms by which variations in CBG lead to CVD are undetermined. Using genomic and transcriptomic data from The Stockholm Tartu Atherosclerosis Reverse Networks Engineering Task (STARNET) study, we identified plasma cortisol-linked single-nucleotide polymorphisms (SNPs) that are trans-associated with genes from seven different vascular and metabolic tissues, finding the highest representation of trans-genes in the liver, subcutaneous fat, and visceral abdominal fat, [false discovery rate (FDR) = 15%]. We identified a subset of cortisol-associated trans-genes that are putatively regulated by the glucocorticoid receptor (GR), the primary transcription factor activated by cortisol. Using causal inference, we identified GR-regulated trans-genes that are responsible for the regulation of tissue-specific gene networks. Cis-expression Quantitative Trait Loci (eQTLs) were used as genetic instruments for identification of pairwise causal relationships from which gene networks could be reconstructed. Gene networks were identified in the liver, subcutaneous fat, and visceral abdominal fat, including a high confidence gene network specific to subcutaneous adipose (FDR = 10%) under the regulation of the interferon regulatory transcription factor, IRF2. These data identify a plausible pathway through which variation in the liver CBG production perturbs cortisol-regulated gene networks in peripheral tissues and thereby promote CVD.

Corticosteroid-binding Globulin (SERPINA6) Establishes Postpubertal Sex Differences in Rat Adrenal Development.

Encoded by SerpinA6, plasma corticosteroid-binding globulin (CBG) transports glucocorticoids and regulates their access to cells. We determined how CBG influences plasma corticosterone and adrenal development in rats during the pubertal to adult transition using CRISPR/cas9 to disrupt SerpinA6 gene expression. In the absence of CBG, total plasma corticosterone levels were ∼80% lower in adult rats of both sexes, with a greater absolute reduction in females than in males. Notably, free corticosterone and adrenocorticotropic hormone were comparable between all groups. Between 30 and 90 days of age, wild-type female rats showed increases in adrenal weight and the size of the corticosterone-producing region, the zona fasciculata (zf), in tandem with increases in plasma CBG and corticosterone concentrations, whereas no such changes were observed in males. This sex difference was lost in rats without CBG, such that adrenal growth and zf expansion were similar between sexes. The sex-specific effects of CBG on adrenal morphology were accompanied by remarkable changes in gene expression: ∼40% of the adrenal transcriptome was altered in females lacking CBG, whereas almost no effect was seen in males. Over half of the adrenal genes that normally exhibit sexually dimorphic expression after puberty were similarly expressed in males and females without CBG, including those responsible for cholesterol biosynthesis and mobilization, steroidogenesis, and growth. Rat adrenal SerpinA6 transcript levels were very low or undetectable. Thus, sex differences in adrenal growth, morphology and gene expression profiles that emerge during puberty in rats are dependent on concomitant increases in plasma CBG produced by the liver.

Stress-related genetic components in attention-deficit/hyperactivity disorder (ADHD): Effects of the SERPINA6 and SERPINA1 genetic markers in a family-based brazilian sample.

SERPINA6 and SERPINA1 were recently identified as the main genes associated with plasma cortisol concentration in humans. Although dysregulation in the Hypothalamus-Pituitary-Adrenal (HPA) axis has been observed in Attention Deficit/Hyperactivity Disorder (ADHD), the molecular mechanisms underlying this relationship are still unclear. Evaluation of the SERPINA6/SERPINA1 gene cluster in ADHD may provide relevant information to uncover them. We tested the association between the SERPINA6/SERPINA1 locus, including 95 single nucleotide polymorphisms (SNPs), and ADHD, using data from a Brazilian clinical sample of 259 ADHD probands and their parents. The single SNP association was tested using binary logistic regression, and we performed Classification and Regression Tree (CART) analysis to evaluate genotype combinations' effects on ADHD susceptibility. We assessed SNPs' regulatory effects through the Genotype-Tissue Expression (GTEx) v8 tool, and performed a complementary look-up analysis in the largest ADHD GWAS to date. There was a suggestive association between ADHD and eight variants located in the SERPINA6 region and one in the intergenic region between SERPINA6 and SERPINA1 after correction for multiple tests (p < 0.032). CART analysis showed that the combined effects of genotype GG in rs2144833 and CC in rs10129500 were associated with ADHD (OR = 1.78; CI95% = 1.24-2.55). The GTEx assigned the SNPs as eQTLs for genes in different tissues, including SERPINA6, and the look-up analysis revealed two SNPs associated with ADHD. These results suggest a shared genetic component between cortisol levels and ADHD. HPA dysregulation/altered stress response in ADHD might be mediated by upregulation of corticosteroid binding globulin (CBG, encoded by SERPINA6) expression.

GR-mediated anti-inflammation of α-boswellic acid: Insights from in vitro and in silico studies.

Although multiple bioactivities of α-boswellic acid have been reported, the molecular mechanism of its anti-inflammatory action is not yet clear. Hence, glucocorticoid receptor (GR)-mediated anti-inflammation of α-boswellic acid was investigated in this work. Fluorescence polarization assay suggested that α-boswellic acid bound to GR with IC50 value of 658.00 ± 0.21 µM. Upon binding to α-boswellic acid, GR translocated from cytoplasm into nucleus of HeLa cells, facilitating sequential transcriptional regulation of GR-related genes. Luciferase reporter assay suggested that α-boswellic acid lacked GR transcriptional activity, indicating its potential as a dissociative GR ligand. Interestingly, α-boswellic acid selectively modulated the anti-inflammatory gene CBG (marker for GR transrepression), while leaving the "side-effect" gene TAT (marker for GR transactivation) unaffected in HepG2 cells. Furthermore, α-boswellic acid inhibited lipopolysaccharide-stimulated cytokines production in U937 macrophages, confirming its anti-inflammation property in vitro. Molecular docking showed that both hydrogen-bonding and hydrophobic interactions helped to stabilize α-boswellic acid-GR binding. Their binding stability was further confirmed in a 70-ns dynamics simulation. In summary, α-boswellic acid could bind to and translocate GR but did not induce glucocorticoid response element-mediated transcription. Since α-boswellic acid showed the dissociated characteristic that separated transrepression from transactivation, it might be a selective GR modulator against inflammatory disorders.

Plasma Cortisol and Risk of Atrial Fibrillation: A Mendelian Randomization Study.

CONTEXT: Atrial fibrillation (AF), cardiac arrhythmias, and related risk factors are common in patients with Cushing's syndrome, or clinical chronic hypercortisolism. While hypercortisolism may be associated with AF, this association has not yet been ascertained causally. OBJECTIVE: To determine whether plasma cortisol is causally associated with AF using a 2-sample Mendelian randomization (MR) design. METHODS: Three genetic variants in the SERPINA1/SERPINA6 locus and functionally associated with plasma cortisol were identified in the CORtisol NETwork consortium (12 597 participants). Summary-level genome-wide association study (GWAS) data for the associations between the cortisol-associated variants and AF were obtained from a GWAS meta-analysis of 6 studies (60 620 AF cases and 970 216 noncases) and the FinnGen consortium (17 325 AF cases and 97 214 noncases). The fixed-effects inverse-variance weighted approach accounting for genetic correlations between variants was used for analysis. Multivariable MR analyses were conducted to assess potential mediating effects of systolic blood pressure (SBP) and waist circumference (WC). Summary-level GWAS data for SBP and WC were obtained respectively from the International Consortium of Blood Pressure (757 601 participants) and the Genetic Investigation of ANthropometric Traits consortium (232 101 participants). RESULTS: One standard deviation increase in genetically predicted plasma cortisol was associated with greater risk of AF (odds ratio [OR] 1.20, 95% CI 1.06-1.35). The association attenuated when adjusting for genetically predicted SBP and WC (OR 0.99, 95% CI 0.72-1.38). CONCLUSION: Evidence derived from the MR study suggests a positive association between plasma cortisol and risk of AF, likely mediated through SBP and WC.

Variation in the SERPINA6/SERPINA1 locus alters morning plasma cortisol, hepatic corticosteroid binding globulin expression, gene expression in peripheral tissues, and risk of cardiovascular disease.

The stress hormone cortisol modulates fuel metabolism, cardiovascular homoeostasis, mood, inflammation and cognition. The CORtisol NETwork (CORNET) consortium previously identified a single locus associated with morning plasma cortisol. Identifying additional genetic variants that explain more of the variance in cortisol could provide new insights into cortisol biology and provide statistical power to test the causative role of cortisol in common diseases. The CORNET consortium extended its genome-wide association meta-analysis for morning plasma cortisol from 12,597 to 25,314 subjects and from ~2.2 M to ~7 M SNPs, in 17 population-based cohorts of European ancestries. We confirmed the genetic association with SERPINA6/SERPINA1. This locus contains genes encoding corticosteroid binding globulin (CBG) and α1-antitrypsin. Expression quantitative trait loci (eQTL) analyses undertaken in the STARNET cohort of 600 individuals showed that specific genetic variants within the SERPINA6/SERPINA1 locus influence expression of SERPINA6 rather than SERPINA1 in the liver. Moreover, trans-eQTL analysis demonstrated effects on adipose tissue gene expression, suggesting that variations in CBG levels have an effect on delivery of cortisol to peripheral tissues. Two-sample Mendelian randomisation analyses provided evidence that each genetically-determined standard deviation (SD) increase in morning plasma cortisol was associated with increased odds of chronic ischaemic heart disease (0.32, 95% CI 0.06-0.59) and myocardial infarction (0.21, 95% CI 0.00-0.43) in UK Biobank and similarly in CARDIoGRAMplusC4D. These findings reveal a causative pathway for CBG in determining cortisol action in peripheral tissues and thereby contributing to the aetiology of cardiovascular disease.

NewBG: A surrogate corticosteroid-binding globulin with an unprecedentedly high ligand release efficacy.

The introduction of ligand-binding sites into proteins and the engineering of molecular allosteric coupling pathways are topical issues in protein design. Here, we show that these issues can be addressed concurrently, using the serpin human α1-antichymotrypsin (ACT) as a model. We have introduced up to 15 amino acid substitutions into ACT, converting it into a surrogate corticosteroid-binding globulin (CBG), thereby creating a new binding globulin (NewBG). Human CBG and ACT share 46% sequence identity, and CBG served as the blue-print for our design, which was guided by side-chain-packing calculations, ITC measurements and crystal structure determinations. Upon transfer of ligand-interacting residues from CBG to ACT and mutation of specific second shell residues, a NewBG variant was obtained, which binds cortisol with 1.5 µM affinity. This novel serpin (NewBG-III) binds cortisol with a 33-fold lower affinity than CBG, but shares a similar ligand-binding profile and binding mode when probed with different steroid ligands and site-directed mutagenesis. An additional substitution, i.e. A349R, created NewBG-III-allo, which introduced an allosteric coupling between ligand binding and the serpin-like S-to-R transition in ACT. In NewBG-III-allo, the proteinase-triggered S-to-R transition leads to a greater than 200-fold reduction in ligand affinity, and crystal structures suggest that this is mediated by the L55V and A349R substitutions. This reduction significantly exceeds the 10-fold reduction in binding affinity observed in human CBG.

Stress-related genetic polymorphisms in association with peripartum depression symptoms and stress hormones: A longitudinal population-based study.

Individual differences in the response of the stress system to hormonal changes during pregnancy and the postpartum period render some women susceptible to developing depression. The present study sought to investigate peripartum depression and stress hormones in relation to stress-related genotypes. The Edinburgh Postnatal Depression Scale was used to assess peripartum depressive symptoms in a sample of 1629 women, followed from pregnancy week seventeen to six months postpartum. Genotypes of ninety-four haplotype-tag single nucleotide polymorphisms (SNPs) in sixteen genes of the hypothalamus-pituitary-adrenal axis pathway were analyzed and data on psychosocial and demographic factors was collected. In sub-studies, salivary cortisol awakening response in gestational week 35-39, salivary evening cortisol levels in gestational week 36 and postpartum week 6, and blood cortisol and cortisone levels in gestational week 35-39 were analyzed. SNP-set kernel association tests were performed at the gene-level, considering psychosocial and demographic factors, followed by post-hoc analyses of SNPs of significant genes. Statistically significant findings at the 0.05 p-level included SNPs in the hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1) gene in relation to self-rated depression scores in postpartum week six among all participants, and serpin family A member 6 (SERPINA6) gene at the same time-point among women with de novo onset of postpartum depression. SNPs in these genes also associated with stress hormone levels during pregnancy. The present study adds knowledge to the neurobiological basis of peripartum depression by systematically assessing SNPs in stress-regulatory genes and stress-hormone levels in a population-based sample of women.

Corticosteroid-binding globulin, induced in testicular Leydig cells by perfluorooctanoic acid, promotes steroid hormone synthesis.

Perfluorooctanoic acid (PFOA) is an abundant perfluoroalkyl substance widely applied in industrial and consumer products. It is a ubiquitous environmental pollutant and suspected endocrine disruptor. Corticosteroid-binding globulin (CBG) is a monomeric glycoprotein that can bind specifically to anti-inflammatory steroids, such as glucocorticoids and progesterone, in circulation. Our previous proteomic profile analysis revealed that CBG levels increased in testes after PFOA treatment. In the present study, we verified its increase in mouse testes following oral exposure to PFOA (0, 1.25 and 5 mg/kg/day for 28 days) by immunohistochemical analysis and Western blotting. In addition, RNA fluorescence in situ hybridization (FISH) confirmed that testicular CBG was specifically expressed in Leydig cells. Serum CBG levels in all three PFOA groups also increased, accompanied by increased corticosterone in the 5 and 20 mg/kg/day groups and decreased adrenocorticotropic hormone in the 20 mg/kg/day group. Thus, the influence of PFOA on blood CBG may change free steroid hormone concentrations, thereby serving as an endocrine disruptor. A stimulation effect of PFOA on CBG was also observed in vitro using the Leydig tumor mLTC-1 cell line. Overexpression of CBG in mLTC-1 cells increased progesterone release in culture media. In addition, CBG-induced proteins involved in steroidogenesis in mLTC-1 cells, including steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase/17,20 lyase (CYP17A1), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which may be the mechanism behind increased progesterone. Furthermore, the production and release of CBG in mLTC-1 cells were also induced by luteinizing hormone, though this mechanism requires further exploration.

Polygenic risk score of SERPINA6/SERPINA1 associates with diurnal and stress-induced HPA axis activity in children.

PURPOSE: Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood. Variation in genes SERPINA6 encoding for CBG, SERPINA2 and SERPINA1 (serpin family A member 6, 2, and 1) have been shown to influence morning plasma cortisol and CBG in adults. However, association of this genetic variation with diurnal and stress-induced salivary cortisol remain unknown. This study aims to investigate the effect of genetic variation in SERPINA6/2/1 loci on diurnal and stress-induced salivary cortisol in children. METHODS: We studied 186, 8-year-old children with genome-wide genotyping. We generated weighted polygenic risk score (PRS) based on 6 genome-wide significant SNPs (rs11621961, rs11629171, rs7161521, rs2749527, rs3762132, rs4900229) derived from the CORNET meta-analyses. Salivary cortisol was measured across one day and in response to the Trier Social Stress Test for Children (TSST-C). RESULTS: Mixed models, adjusted for covariates, showed that the PRS x sampling time interactions associated with diurnal (P < 0.001) and stress-induced (P = 0.009) salivary cortisol. In the high PRS group (dichotomized at median) the diurnal salivary cortisol pattern decreased less from awakening to bedtime than in the low PRS group (standardized estimates of sampling time -0.64 vs. -0.73, P < 0.0001 for both estimates). In response to stress, salivary cortisol increased in the high PRS group while it remained unchanged in the low PRS group (standardized estimates of sampling time 0.12, P = 0.015 vs. -0.06, P = 0.16). These results were mainly driven by minor alleles of rs7161521 (SERPINA6) and rs4900229 (SERPINA1). CONCLUSIONS: Genetic variation in SERPINA6/2/1loci may underpin higher hypothalamic-pituitary-adrenocortical axis activity in children.

Leydig cell genes change their expression and association with polysomes in a stage-specific manner in the adult mouse testis.

Spermatogenesis in mammals occurs in a very highly organized manner within the seminiferous epithelium regulated by different cell types in the testis. Testosterone produced by Leydig cells regulates blood-testis barrier formation, meiosis, spermiogenesis, and spermiation. However, it is unknown whether Leydig cell function changes with the different stages of the seminiferous epithelium. This study utilized the WIN 18,446 and retinoic acid (RA) treatment regime combined with the RiboTag mouse methodology to synchronize male germ cell development and allow for the in vivo mapping of the Leydig cell translatome across the different stages of one cycle of the seminiferous epithelium. Using microarrays analysis, we identified 11 Leydig cell-enriched genes that were expressed in stage-specific manner such as the glucocorticoid synthesis and transport genes, Cyp21a1 and Serpina6. In addition, there were nine Leydig cell transcripts that change their association with polysomes in correlation with the different stages of the spermatogenic cycle including Egr1. Interestingly, the signal intensity of EGR1 and CYP21 varied among Leydig cells in the adult asynchronous testis. However, testosterone levels across the different stages of germ cell development did not cycle. These data show, for the first time, that Leydig cell gene expression changes in a stage-specific manner during the cycle of the seminiferous epithelium and indicate that a heterogeneous Leydig cell population exists in the adult mouse testis.

The role of corticosteroid-binding globulin in the evaluation of adrenal insufficiency.

Cortisol is a hydrophobic molecule that is largely bound to corticosteroid-binding globulin (CBG) in the circulation. In the assessment of adrenal insufficiency, many clinicians measure a total serum cortisol level, which assumes that CBG is present in normal concentrations and with a normal binding affinity for cortisol. CBG concentration and affinity are affected by a number of common factors including oral contraceptive pills (OCPs), fever and infection, as well as rare mutations in the serine protease inhibitor A6 (SERPINA6) gene, and as such, total cortisol levels might not be the ideal way to assess adrenal function in all clinical circumstances. This paper reviews the limitations of immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the measurement of total cortisol, the challenges of measuring free serum cortisol directly as well as the difficulties in calculating an estimated free cortisol from total cortisol, CBG and albumin concentrations. Newer approaches to the evaluation of adrenal insufficiency, including the measurement of cortisol and cortisone in the saliva, are discussed and a possible future role for these tests is proposed.

Genomic determinants of long-term cardiometabolic complications in childhood acute lymphoblastic leukemia survivors.

BACKGROUND: While cure rates for childhood acute lymphoblastic leukemia (cALL) now exceed 80%, over 60% of survivors will face treatment-related long-term sequelae, including cardiometabolic complications such as obesity, insulin resistance, dyslipidemia and hypertension. Although genetic susceptibility contributes to the development of these problems, there are very few studies that have so far addressed this issue in a cALL survivorship context. METHODS: In this study, we aimed at evaluating the associations between common and rare genetic variants and long-term cardiometabolic complications in survivors of cALL. We examined the cardiometabolic profile and performed whole-exome sequencing in 209 cALL survivors from the PETALE cohort. Variants associated with cardiometabolic outcomes were identified using PLINK (common) or SKAT (common and rare) and a logistic regression was used to evaluate their impact in multivariate models. RESULTS: Our results showed that rare and common variants in the BAD and FCRL3 genes were associated (p<0.05) with an extreme cardiometabolic phenotype (3 or more cardiometabolic risk factors). Common variants in OGFOD3 and APOB as well as rare and common BAD variants were significantly (p<0.05) associated with dyslipidemia. Common BAD and SERPINA6 variants were associated (p<0.05) with obesity and insulin resistance, respectively. CONCLUSIONS: In summary, we identified genetic susceptibility loci as contributing factors to the development of late treatment-related cardiometabolic complications in cALL survivors. These biomarkers could be used as early detection strategies to identify susceptible individuals and implement appropriate measures and follow-up to prevent the development of risk factors in this high-risk population.

A naturally hypersensitive glucocorticoid receptor elicits a compensatory reduction of hypothalamus-pituitary-adrenal axis activity early in ontogeny.

We comprehensively characterized the effects of a unique natural gain-of-function mutation in the glucocorticoid receptor (GR), GRAla610Val, in domestic pigs to expand current knowledge of the phenotypic consequences of GR hypersensitivity. Cortisol levels were consistently reduced in one-week-old piglets, at weaning and in peripubertal age, probably due to a reduced adrenal capacity to produce glucocorticoids (GC), which was indicated by an adrenocortical thinning in GRAla610Val carriers. Adrenocorticotrophic hormone (ACTH) levels were significantly reduced in one-week-old piglets only. Expression analyses in peripubertal age revealed significant downregulation of hypothalamic expression of CRH and AVP, the latter only in females, and upregulation of hepatic expression of SERPINA6, by GRAla610Val Transcriptional repression of proinflammatory genes in peripheral blood mononuclear cells (PBMCs) from GRAla610Val carriers was more sensitive to dexamethasone treatment ex vivo However, no significant effects on growth, body composition, blood chemistry or cell counts were observed under baseline conditions. These results suggest that GRAla610Val-induced GR hypersensitivity elicits a compensatory reduction in endogenous, bioactive glucocorticoid levels via readjustment of the hypothalamus-pituitary-adrenal (HPA) axis early in ontogeny to maintain an adequate response, but carriers are more sensitive to exogenous GC. Therefore, GRAla610Val pigs represent a valuable animal model to explore GR-mediated mechanisms of HPA axis regulation and responses to glucocorticoid-based drugs.

Corticosteroid-Binding Globulin: A Review of Basic and Clinical Advances.

Corticosteroid-binding globulin (CBG, transcortin) is the primary cortisol binding protein. It is a non-inhibitory serine protease inhibitor, capable of conformational change from a high cortisol-binding affinity form to a low affinity form upon cleavage of its reactive centre loop by various proteases, such as neutrophil elastase. The burgeoning inflammatory role of CBG applies to acute, severe inflammation where depletion is associated with mortality, and to chronic inflammation where defects in cortisol delivery may perpetuate inflammation. Naturally occurring human mutations influence a wide range of CBG properties and point toward a role in hitherto unexplained chronic musculoskeletal pain and fatigue disorders as well as potentially affecting fertility outcomes including offspring gender. In vitro and knock-out animal models of CBG propose a role for CBG in cortisol transport to the brain, providing a foundation for understanding the human observations in those with CBG mutations and sex differences in stress-related mood and behaviour. Finally, CBG measurement has a practical role in the estimation of free cortisol, useful in clinical circumstances where CBG levels or cortisol binding affinity is reduced. Taken together, novel data suggest a role for cortisol in targeted cortisol delivery, with implications in acute and chronic inflammation, as well as roles in metabolism and neurocognitive function, implying that CBG is a multifaceted component in the mechanisms of hypothalamic-pituitary-adrenal axis related homeostasis.

Identification of Avian Corticosteroid-binding Globulin (SerpinA6) Reveals the Molecular Basis of Evolutionary Adaptations in SerpinA6 Structure and Function as a Steroid-binding Protein.

Corticosteroid-binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG steroid-binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only ∼40% identity, and their steroid-binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG steroid-binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its steroid-binding affinity, but we present evidence that it may participate in protein folding and steroid-binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct steroid-binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts steroid binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex steroids in these species.

Variation in corticosterone response and corticosteroid binding-globulin during different breeding sub-stages in Eurasian tree sparrow (Passer montanus).

In free-living animals, it has been well demonstrated that the intensity of the adrenocortical response to acute restraint stress can vary with reproductive investment during breeding. The parental care hypothesis posits that the stress response is negatively correlated with parental investment in avian species. To further test this hypothesis, we examined changes in both free and total corticosterone (CORT) at baseline and stress-induced levels (maximal CORT) and corticosteroid-binding globulin (CBG) capacities, in both sexes of a multi-brooded Eurasian tree sparrows (Passer montanus), during the nest building, the early nestling, the later egg-laying, and the later nestling stages. Our results showed Eurasian tree sparrows did not exhibit any differences between sexes in CORT and CBG levels during the egg-laying or nestling stages. Both sexes had lowered CBG capacities and females exhibited lower maximal CORT during the early compared to later nestling stages. In addition, both sexes had lower maximal free CORT levels during the nest building stage than those of the early nestling stages, and males expressed higher total maximal CORT levels than females during nest building stage. The variation in CORT response and CBG levels during different breeding sub-stages in Eurasian tree sparrow may correlate with their energetic situations and parental investments. J. Exp. Zool. 325A:75-83, 2016. © 2015 Wiley Periodicals, Inc.

Steroids and endocrine disruptors--History, recent state of art and open questions.

This introductory chapter provides an overview of the levels and sites at which endocrine disruptors (EDs) affect steroid actions. In contrast to the special issue of Journal of Steroid Biochemistry and Molecular Biology published three years ago and devoted to EDs as such, this paper focuses on steroids. We tried to point to more recent findings and opened questions. EDs interfere with steroid biosynthesis and metabolism either as inhibitors of relevant enzymes, or at the level of their expression. Particular attention was paid to enzymes metabolizing steroid hormones to biologically active products in target cells, such as aromatase, 5α-reductase and 3ß-, 11ß- and 17ß-hydroxysteroid dehydrogenases. An important target for EDs is also steroid acute regulatory protein (StAR), responsible for steroid precursor trafficking to mitochondria. EDs influence receptor-mediated steroid actions at both genomic and non-genomic levels. The remarkable differences in response to various steroid-receptor ligands led to a more detailed investigation of events following steroid/disruptor binding to the receptors and to the mapping of the signaling cascades and nuclear factors involved. A virtual screening of a large array of EDs with steroid receptors, known as in silico methods (≡computer simulation), is another promising approach for studying quantitative structure activity relationships and docking. New data may be expected on the effect of EDs on steroid hormone binding to selective plasma transport proteins, namely transcortin and sex hormone-binding globulin. Little information is available so far on the effects of EDs on the major hypothalamo-pituitary-adrenal/gonadal axes, of which the kisspeptin/GPR54 system is of particular importance. Kisspeptins act as stimulators for hormone-induced gonadotropin secretion and their expression is regulated by sex steroids via a feed-back mechanism. Kisspeptin is now believed to be one of the key factors triggering puberty in mammals, and various EDs affect its expression and function. Finally, advances in analytics of EDs, especially those persisting in the environment, in various body fluids (plasma, urine, seminal fluid, and follicular fluid) are mentioned. Surprisingly, relatively scarce information is available on the simultaneous determination of EDs and steroids in the same biological material. This article is part of a Special Issue entitled 'Endocrine disruptors & steroids'.