Hepatic Corticosteroid-binding Globulin Research Articles

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.

Abstract 700: Genetic Deletion Of Neutrophil Elastase Does Not Alter Lesions In Mice With Atherosclerosis Induced By Aav8-mediated Over-expression Of PCSK9

Introduction: Elevated circulating glucocorticoids are causally linked to increased cardiovascular disease. Atherosclerosis is a key cardiovascular pathology, yet the contribution of glucocorticoids underlying atherogenesis is unclear. Corticosteroid binding globulin (CBG) regulates glucocorticoid action through binding, thus controlling its systemic bioavailability. Cleavage of CBG by neutrophil elastase (NE) is proposed to enhance bioavailability of glucocorticoids by reducing its binding affinity. Strikingly, Apoe -/- mice lacking NE (ELA -/- ) display reduced atherosclerosis. Hypothesis: This investigation addresses the hypothesis that NE influences atherogenesis through CBG cleavage, increasing glucocorticoid bioavailability at sites of plaque development. Methods: Adult (8-12wk old) male C57BL/6J, ELA -/- and ELA +/+ mice were injected with AAV8-PCSK9 (IP; 1x10 12 vector genomes) and fed a western diet (21% fat; 0.21% cholesterol) for 18wks (N=10-13). Control mice received diet without viral injection (N=10). Results: In C57BL/6J mice, histological analysis of brachiocephalic arteries demonstrated lesion development with AAV8-PCSK9-treatment, in parallel with increased (P=0.015) circulating NE protein levels (58.0 ± 5.5ng/ml) compared to diet-control mice (NE levels 32.8 ± 6.2ng/ml). AAV8-PCSK9 treatment did not alter circulating glucocorticoids (Corticosterone AM levels P=0.94; AAV8-PCSK9 65 ± 16nM; Control 74 ± 36nM) but hepatic CBG (Serpina6) transcript levels were reduced (1-fold; P=0.01; AAV8-PCSK9 0.57 ± 0.04; Control 1.13 ± 0.20). Interestingly, no differences in lesion size (P=0.8539; 23.2 ± 2.5%) or collagen content (P=0.5156; 17.64 ± 5.6%), or in circulating glucocorticoid levels (corticosterone AM levels; P=0.36; 225 ± 59nM) were observed in ELA -/- mice compared to controls (lesion size 24.2 ± 6.3%; collagen 12 ± 5%; glucocorticoid levels 142 ± 29nM). Conclusions: In contrast to published data using Apoe -/- mice, these results demonstrate that NE does not influence lesion size in this model of atherosclerosis. This suggests that NE inhibition would not be a useful therapeutic target in atherosclerosis. Future studies will directly address the role of CBG in the development of atherosclerosis.

Opposite Expression of Hepatic and Pulmonary Corticosteroid-Binding Globulin in Cystic Fibrosis Patients.

Cystic fibrosis (CF) is characterized by a chronic pulmonary inflammation. In CF, glucocorticoids (GC) are widely used, but their efficacy and benefit/risk ratio are still debated. In plasma, corticosteroid-binding globulin (CBG) binds 90% of GC and delivers them to the inflammatory site. The main goal of this work was to study CBG expression in CF patients in order to determine whether CBG could be used to optimize GC treatment. The expression of CBG was measured in liver samples from CF cirrhotic and non-CF cirrhotic patients by qPCR and Western blot and in lung samples from non-CF and CF patients by qPCR. CBG binding assays with 3H-cortisol and the measurement of the elastase/α1-antitrypsin complex were performed using the plasmas. CBG expression increased in the liver at the transcript and protein level but not in the plasma of CF patients. This is possibly due to an increase of plasmatic elastase. We demonstrated that pulmonary CBG was expressed in the bronchi and bronchioles and its expression decreased in the CF lungs, at both levels studied. Despite the opposite expression of hepatic and pulmonary CBG in CF patients, the concentration of CBG in the plasma was normal. Thus, CBG might be useful to deliver an optimized synthetic GC displaying high affinity for CBG to the main inflammatory site in the context of CF, e.g., the lung.

Somatostatin Is Essential for the Sexual Dimorphism of GH Secretion, Corticosteroid-Binding Globulin Production, and Corticosterone Levels in Mice

Distinct male and female patterns of pituitary GH secretion produce sexually differentiated hepatic gene expression profiles, thereby influencing steroid and xenobiotic metabolism. We used a fully automated system to obtain serial nocturnal blood samples every 15 minutes from cannulated wild-type (WT) and somatostatin knockout (Sst-KO) mice to determine the role of SST, the principal inhibitor of GH release, in the generation of sexually dimorphic GH pulsatility. WT males had lower mean and median GH values, less random GH secretory bursts, and longer trough periods between GH pulses than WT females. Each of these parameters was feminized in male Sst-KO mice, whereas female Sst-KO mice had higher GH levels than all other groups, but GH pulsatility was unaffected. We next performed hepatic mRNA profiling with high-density microarrays. Male Sst-KO mice exhibited a globally feminized pattern of GH-dependent mRNA levels, but female Sst-KO mice were largely unaffected. Among the differentially expressed female-predominant genes was Serpina6, which encodes corticosteroid-binding globulin (CBG). Increased CBG was associated with elevated diurnal peak plasma corticosterone in unstressed WT females and both sexes of Sst-KO mice compared with WT males. Sst-KO mice also had exaggerated ACTH and corticosterone responses to acute restraint stress. However, consistent with their lack of phenotypic signs of excess glucocorticoids, cerebrospinal fluid concentrations of free corticosterone in Sst-KO mice were not elevated. In summary, SST is necessary for the prolonged interpulse troughs that define masculinized pituitary GH secretion. SST also contributes to sexual dimorphism of the hypothalamic-pituitary-adrenal axis via GH-dependent regulation of hepatic CBG production.

Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein that is primarily synthesized in the liver and binds cortisol and progesterone with high affinity. In this study, a CBG secreting hepatocellular carcinoma derived cell line (HepG2) was used to investigate the hormonal regulation of hepatic CBG synthesis. HepG2 cells were grown for 72 h in 30, 300 and 3000 nM concentrations of estradiol (E 2), testosterone (T), insulin, thyroxin (T 4) and dexamethasone (DMZ) and the secreted CBG quantified by a novel enzyme-linked immunosorbent assay (ELISA). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out to determine the effects of these hormones on the relative distribution of CBG glycoforms. Insulin, T 4 and high concentrations of E 2 decreased the secretion of CBG by HepG2 cells ( p < 0.05). Ethanol, the solvent used for E 2, T and DMZ, also significantly attenuated CBG secretion. 2D-PAGE resolved 13–14 glycoforms of CBG produced by HepG2 cells. Insulin caused a reduction in the synthesis of more acidic, while T 4 and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E 2 resulted in the synthesis of additional isoforms of increased acidity, which may represent a type of CBG only seen during pregnancy in vivo. Possible physiological implications of these findings are discussed.

Hepatic glucose regulation and metabolism in adult sheep: effects of prenatal betamethasone

Fetal exposure to synthetic glucocorticoids in sheep results in increased fetal hepatic 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and corticosteroid-binding globulin (CBG) protein levels and insulin resistance in postnatal life. The aim was to determine whether these changes persisted to adulthood and whether alterations in mediators of hepatic glucocorticoid and glucose regulation contributed to changes in metabolism. Pregnant ewes or their fetuses received either repeated intramuscular saline (MS, FS) or betamethasone injections (0.5 mg/kg; M4, F4) at 104, 111, 118, and 124 days of gestation (dG), or a single betamethasone injection at 104 dG followed by saline at 111, 118, and 124 dG (M1, F1). Offspring were catheterized at 2 and 3 yr of age and given an intravenous glucose challenge (0.5 mg/kg). Hepatic tissue was collected at 3.5 yr. At 2 yr of age, basal plasma insulin was elevated in M4 offspring and at 3 yr of age was elevated in F4 offspring. Basal insulin-to-glucose ratio was significantly elevated in M4 offspring at 2 yr of age and elevated in M1, M4, and F4 offspring at 3 yr of age. All betamethasone treatments resulted in significant increases in hepatic glucose-6-phosphatase (G-6-Pase) activity. Hepatic glucocorticoid receptor protein levels were not altered in M1 and M4 offspring but were increased in F1 and F4 offspring. Hepatic CBG protein levels were lower in F4 but not F1 offspring and were unchanged from control in M1 and M4 offspring. Prenatal betamethasone exposure results in elevated hepatic G-6-Pase activity in adulthood and may contribute to long-term changes in metabolism.

Hepatic corticosteroid-binding globulin (CBG) messenger RNA expression and plasma CBG concentrations in young pigs in response to heat and social stress1,2

Plasma cortisol, porcine corticosteroid-binding globulin (pCBG), hepatic CBG expression, and other physiological and behavioral measures of stress were studied in pigs in response to elevated temperature in conjunction with establishing a social hierarchy. Twenty-four crossbred pigs were weaned at 25 d of age (three or six pigs from six sows) and housed in littermate groups at 23 +/- 2 degrees C. At 57 d of age (d 0), animals were weighed and placed under general anesthesia for collection of blood (10 mL) and liver (approximately 100 mg) samples. On d 1, three unacquainted pigs of similar BW (23 +/- 1 kg) from different litters were allotted to each of eight nursery pens within two environmentally controlled rooms (12 pigs per room). From d 1 to 7, one room was maintained at 23 +/- 2 degrees C (CON) and the other at 33 +/- 2 degrees C (HEAT). Both rooms were kept at 23 +/- 2 degrees C from d 8 to 14. Animals were videotaped for 72 h beginning on d 1 and 8 to document behavioral changes in response to room temperature. The social hierarchy of pigs within each pen was based on fight activity recorded on d 1 to 3. Blood and liver tissue were collected again on d 7 and 14. The ADG for HEAT pigs increased (P < 0.05) over d 8 to 14 compared with d 1 to 7. In contrast to CON pigs, HEAT pigs displayed increased (P < 0.01) drinking but decreased feeding and lying in contact with other pigs from d 1 to 3, and similar drinking and feeding but increased (P < 0.01) lying with contact behaviors from d 8 to 10. With the exception of subordinate pigs exhibiting less (P < 0.05) frequent standing/walking behavior than the dominant or intermediate pigs on d 1 to 3, frequency of behaviors for both recorded time periods did not differ among pigs due to social status, regardless of treatment. The concentration of plasma haptoglobin in HEAT pigs on d 7 compared with d 0 increased (467 vs. 763 mg/L; P < 0.05), whereas cortisol and pCBG decreased (274 vs. 235 nmol/L and 11.4 vs. 9.9 mg/L, respectively; P < 0.05) as a result of treatment. The free cortisol index (total cortisol/pCBG) was greater (P < 0.05) in HEAT pigs on d 14 than on d 0 or 7. Hepatic CBG mRNA level was not affected by treatment. On d 14, HEAT pigs had plasma cortisol, pCBG, and haptoglobin concentrations similar to those of CON pigs. These results indicate that measured behavioral and physiological responses were not related to social status, and decreased circulating levels of cortisol and pCBG in pigs following a 7-d exposure to elevated temperature may not be determined by hepatic CBG mRNA expression.

Corticosterone binding globulin regulation and thymus changes after thermal injury in mice

Thermal injury is extremely stressful, and data characterizing the systemic endocrine stress response to this injury are sparse. The objective of this study was to measure the effects of thermal injury on mice on corticosterone (Cort) levels in relation with corticosteroid-binding globulin (CBG) and thymus cell populations. The endocrine stress response was determined by measuring total Cort, free Cort, CBG binding capacity, liver CBG mRNA, and circulating CBG levels at 1, 2, 5, and 10 days postburn. Thymus cell populations were also analyzed. After thermal injury, a rapid increase of total Cort was observed in the first 48 h. This was associated with a decrease of hepatic CBG mRNA, protein levels, and binding capacity. Percentage of free Cort in the burn group peaked at day 2 postburn with a dramatic (+500%) increase. This correlated with a significant decrease of thymus cellularity (50% less). Phenotypic analyses showed that corticosensitive cells were significantly altered. After treatment (5 days), both endocrine and immune parameters returned to control levels. Our results demonstrate that, after a thermal injury, CBG is mainly responsible for Cort's action on corticosensitive immune cells.

Plasma levels of cortisol and corticosteroid-binding globulin (CBG) and hepatic CBG mRNA expression in pre- and postnatal pigs

The relationships among hepatic corticosteroid-binding globulin (CBG) mRNA expression and plasma concentrations of cortisol and CBG was evaluated in fetal pigs ( n=7–14 per age) on days 50, 70, 80, 90, and 104 of gestation and postnatal pigs ( n=8 per age) on days 1, 3, 10, 20, 30, and 40 following birth. In fetal pigs, hepatic CBG mRNA expression was highest ( P<0.01) on day 50 as compared to days 90 and 104, exhibiting an overall negative relationship ( r=−0.63; P<0.01) with estimated gestation age. Plasma porcine CBG (pCBG) concentration was correlated ( r=0.34; P<0.05) with hepatic CBG mRNA level. Plasma cortisol concentrations were not different over this same period. In postnatal pigs, hepatic CBG mRNA expression increased ( P<0.01) from days 3 to 40. The pCBG concentration increased ( P<0.01) from days 1 (6.1±3.4 μg/ml) to 10 (15.1±3.7 μg/ml), while plasma cortisol concentration remained constant. An understanding of the relation between hepatic CBG mRNA and circulating pCBG concentrations may provide insight into the mechanisms determining the bioavailability of cortisol necessary in prenatal development and the conservation of cortisol during postnatal development in the pig.

Glucocorticoids induce corticosteroid-binding globulin biosynthesis by immature mouse liver and kidney.

Marked changes in mouse corticosteroid-binding globulin (CBG) gene expression in the liver and kidney occur postnatally. To study the influence of glucocorticoids on the initiation of mouse CBG biosynthesis in these tissues during the first two weeks after birth, we administered dexamethasone (0.5 microgram/g body wt/day) to 4- and 11-day-old pups for three days. This resulted in higher serum CBG and hepatic CBG mRNA levels in animals, irrespective of their ages. Higher relative amounts of CBG mRNA in the kidneys of 14-day-old pups after three days of dexamethasone treatment co-incided with higher amounts of intact and proteolytically cleaved CGB in their urine, and both are indicative of increased CGB production by the developing renal tubules. When an additional group of 11-day-old pups (n = 4) was treated with 0.25 microgram dexamethasone/g body weight per day for five days, this also resulted in significantly higher levels of serum CBG (P < 0.01), hepatic CBG mRNA (P < 0.01) and renal CBG mRNA (P < 0.05), compared to littermates treated with the oil vehicle alone. In contrast, serum CBG levels progressively decreased in adult female mice during five days of treatment with 0.5 microgram dexamethasone/g body weight per day. Taken together, these data indicate that glucocorticoids induce murine CBG gene expression in the immature liver and kidney, and support the concept that the effects of glucocorticoids on CBG gene expression are developmentally stage-specific.

Developmental regulation of corticosteroid-binding globulin biosynthesis in the baboon fetus.

The present study determined the roles of estrogen and cortisol in maternal and fetal corticosteroid-binding globulin (CBG) levels and fetal hepatic messenger RNA (mRNA) expression in the baboon. Samples of fetal liver, kidney, and brain were obtained from untreated control animals at early (day 60; n = 4), mid (day 100; n = 8), and late (day 165; n = 5) gestation (term = day 184). Maternal and umbilical blood samples were collected on day 100 from baboons in which betamethasone was administered sc to the mother (n = 6) on days 60-99 of gestation and on day 165 from animals (n = 4) in which the fetus was administered betamethasone on days 150-164 of gestation. Maternal serum cortisol concentrations were similar at mid (43 +/- 2 micrograms/dl) and late (42 +/- 3 micrograms/dl) gestation and decreased (P < 0.05) at midgestation (1 +/- 1 micrograms/dl) and term (31 +/- 4 micrograms/dl) after betamethasone treatment. Umbilical serum cortisol levels were also reduced (P < 0.05) at both mid (1 +/- 1 micrograms/dl) and late (14 +/- 5 micrograms/dl) gestation by betamethasone treatment. Fetal serum CBG levels in untreated animals were lower (P < 0.05) on day 165 (444 +/- 29 pmol/ml) than on day 100 (844 +/- 35 pmol/ml) and increased (P < 0.05) at midgestation (1098 +/- 64 pmol/ml), but not at term (551 +/- 24 pmol/ml), after betamethasone treatment. In contrast, maternal serum CBG levels (range, 528-770 pmol/ml) were not altered by gestational age or betamethasone. The human CBG complementary DNA hybridized to a single mRNA species of 1.8 kilobases in baboon fetal liver; however, CBG was not expressed in fetal kidney and was detectable in fetal brain and pancreas only by reverse transcription-PCR. In untreated baboon fetuses, the mRNA levels of hepatic CBG, expressed as a ratio of 18S RNA, progressively decreased (P < 0.05) in early (1.83 +/- 0.17), mid (0.97 +/- 0.12), and late (0.51 +/- 0.04) gestation. These results demonstrate that fetal hepatic CBG mRNA expression and serum CBG concentrations were elevated early in baboon gestation and exhibited a progressive decline during the course of advancing pregnancy. We suggest that the increased levels of fetal CBG in the early stages of gestation reflect stimulation of hepatic CBG synthesis by maternal cortisol, which we previously demonstrated to occur in the fetus as a result of preferential 11 beta-hydroxysteroid dehydrogenase-catalyzed glucocorticoid reduction across the placenta. The decline in fetal CBG may reflect the developmental increase in catabolism of cortisol to bioinactive cortisone in target tissues of the fetus such as the liver.

Corticosteroid-binding globulin (CBG) production by hepatic and extra-hepatic sites in the ovine fetus; effects of CBG on glucocorticoid negative feedback on pituitary cells in vitro

Plasma cortisol levels increase in fetal sheep during late gestation and this is associated with an increase in plasma corticosteroid-binding globulin (CBG) concentrations. However, the relative tissue sources of plasma CBG, the ontogeny of its biosynthesis and glycoform composition have not been established in the ovine fetus. Therefore we examined whether changes in plasma corticosteroid binding capacity (CBC) in fetal sheep during late gestation were associated with different patterns of glycosylation and reflected changes in tissue CBG expression. Since free cortisol is considered the bioactive fraction, we measured changes in the percent and absolute free cortisol in fetal plasma during late gestation. In order to examine whether CBG alters cortisol negative feedback at the level of the fetal pituitary, we also examined the effect of exogenous CBG in mediating the glucocorticoid-induced suppression of basal and corticotrophin-releasing hormone (CRH)-stimulated ACTH release from fetal pituitary cells in culture. The mean free cortisol concentration in plasma was not different between days 15 and 20 prior to parturition, and between 5 and 10 days prepartum, although it did rise between these times. Plasma CBC in chronically catheterized fetuses rose from 23.3 +/- 4.6 ng/ml at day 115 to 86.5 +/- 20.8 ng/ml at term and then decreased rapidly after birth. Between day 125 and day 140 of pregnancy approximately 10% of fetal plasma CBG was retarded by Concanavalin-A chromatography. This proportion increased at birth and attained adult values of > 70% by one month of age. By Northern blotting the relative levels of CBG mRNA in the fetal liver did not change between days 100 and 125, then increased significantly at day 140, but declined at term and in newborn lambs. CBG mRNA was undetectable in total RNA from lung, kidney, hypothalamus and placentomes, but was present in the fetal pituitary at days 125 and 140. Reverse transcription-PCR was used to confirm the presence of CBG mRNA in pituitary tissue from term fetuses. In cultures of term fetal pituitary cells, added CBG attenuated the cortisol- but not the dexamethasone-mediated suppression of basal and CRH-stimulated ACTH release. We conclude that in fetal sheep there is an increase in the corticosteroid binding capacity of plasma during late pregnancy which regulates, in part, free cortisol levels in the circulation. The liver is the major site of CBG biosynthesis in the fetus and at least until day 140 of gestation the rise in plasma CBC is associated with an increase in hepatic CBG mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticosteroid-binding globulin (CBG) in fetal development.

In fetal sheep the prepartum increase in plasma cortisol concentration is associated with an increase in high affinity corticosteroid binding activity in plasma. This appears to reflect an increase in corticosteroid-binding globulin (CBG) biosynthesis from the fetal liver, and evidence is presented that hepatic CBG gene expression is increased by exposure to glucocorticoids in the fetus. Immunoreactive CBG is found in other fetal tissues, and CBG mRNA is present in fetal pituitary. CBG reduces the ability of cortisol to exert negative feedback on basal or CRH-stimulated ACTH output by fetal sheep pituitary cells in culture. We suggest that CBG interacts with cortisol in a manner that maintains a low negative feedback on the pituitary, and perhaps hypothalamus. This constitutes a component of the cascade of events that is associated with hypothalamic-pituitary-adrenal activation in the late gestation fetus, and with the onset of parturition.

Low-dose cortisol infusion increases plasma corticosteroid-binding globulin (CBG) and the amount of hepatic CBG mRNA in fetal sheep on day 100 of gestation.

Synthetic glucocorticoids stimulate the production of corticosteroid-binding globulin (CBG) by the liver of the sheep fetus near term (day 145). We have examined whether physiological changes in plasma cortisol alter plasma CBG concentrations, patterns of glycosylation and the amount of hepatic CBG mRNA at earlier times during pregnancy (day 100), prior to the activation of fetal hypothalamic-pituitary-adrenal function. Cortisol was infused into chronically catheterized sheep fetuses in amounts that raised the plasma cortisol concentration by about 15 nmol/l. This treatment resulted in a significant increase in the plasma corticosteroid-binding capacity and in the amount of CBG mRNA in the fetal liver, but did not alter the proportion of CBG retained using Concanavalin A chromatography. We conclude that the CBG gene in the liver of fetal sheep responds to physiological changes in plasma concentration of cortisol and we speculate that the rise in plasma CBG concentration is important in diminishing the negative feedback effect of circulating cortisol on the fetal pituitary and hypothalamus.

Glucocorticoid-induced increase in plasma corticosteroid-binding globulin levels in fetal sheep is associated with increased biosynthesis and alterations in glycosylation.

In fetal sheep, there is a concomitant prepartum rise in cortisol and corticosteroid-binding globulin (CBG) that maintains a low free plasma cortisol level and allows for a low negative feedback effect of cortisol on the secretion of ACTH from the fetal pituitary. However, the stimulus for the prepartum increase in CBG and the mechanism(s) of this effect are not known. It has been proposed that glucocorticoids increase CBG concentrations, and therefore, we infused fetal sheep with the synthetic glucocorticoid dexamethasone (DEX; 2 micrograms/min over 15 min every 2 h for 96 h, n = 5) or saline (n = 5). The plasma corticosteroid-binding capacity increased from 30.0 +/- 2.4 to 55.6 +/- 7.7 and 92.6 +/- 11.1 ng/ml at 48 and 96 h, respectively, of DEX infusion. To examine possible mechanisms of increasing fetal plasma CBG, we first cloned and sequenced a sheep CBG cDNA and purified the protein. This allowed us to deduce the primary structure of ovine CBG and to demonstrate that hepatic CBG mRNA abundance (single transcript of 1.8 kilobases) rose from 0.9 +/- 0.2 to 3.6 +/- 1.6 arbitrary units after 96 h of DEX treatment. Fetal DEX treatment produced a significant increase (7.1 +/- 1.2% to 13.1 +/- 1.4%) in the Concanavalin-A-binding forms of CBG that predominate in adult sheep plasma. There was negligible transfer of purified [125I]CBG from the ewe to fetal plasma, urine, or amniotic fluid. We also injected adult sheep with DEX (10 mg/day for 4 days) and demonstrated a significant decrease in plasma corticosteroid-binding capacity by 24 h, which remained suppressed for the duration of the study. After 96 h of DEX treatment, there was also a significant decrease in adult hepatic CBG mRNA abundance. We conclude that glucocorticoids increase fetal plasma CBG in part by increased hepatic biosynthesis. It may also be accentuated by a change in the glycosylation of CBG, but cannot be attributed to transplacental transfer. Furthermore, glucocorticoid treatment exerts opposite effects on CBG biosynthesis in fetal and adult sheep.

Hormonal regulation of corticosteroid-binding globulin biosynthesis in the male rat.

Corticosteroid-binding globulin (CBG) is an acidic glycoprotein produced primarily by the liver and is the major glucocorticoid transport protein in rat blood. Various hormone treatments are known to modify plasma CBG levels and may thereby influence the bioavailability of glucocorticoids. We have, therefore, examined serum CBG and hepatic CBG mRNA levels in male rats after sc steroid (25 micrograms/100 g BW twice daily of dexamethasone, prednisolone, corticosterone, estrone, estradiol, ethinyl estradiol, progesterone, or 5 alpha-dihydrotestosterone) or T4 (10 micrograms/100 g BW twice daily) administration. After dexamethasone treatment, serum CBG levels decreased significantly (P less than 0.002) within 48 h and were 26% of those in vehicle-treated animals by 72 h. At this time, CBG mRNA was undetectable in liver RNA extracts by Northern blotting or solution hybridization, and nuclear run-off experiments indicated that dexamethasone reduces CBG gene transcription in the liver by at least 14-fold. Treatment with ethinyl estradiol also reduced serum CBG levels significantly (P less than 0.015) compared to pretreatment values. No other significant changes in serum CBG levels were observed after any other steroid hormone treatment, and alterations in hepatic CBG mRNA levels were only observed after dexamethasone treatment. T4 administration for 72 h increased serum CBG and hepatic CBG mRNA levels by 66% and 39%, respectively, but did not influence the rate of CBG gene transcription. Thus, increased CBG biosynthesis after thyroid hormone treatment appears to be due to an increase in CBG mRNA stability. No change in the Concanavalin-A-binding properties of CBG molecules was observed after treatment with either dexamethasone or T4, and this suggests that these hormones do not influence the type of oligosaccharide chains associated with this protein. We, therefore, conclude that dexamethasone and T4 regulate hepatic CBG biosynthesis in the rat by modulating CBG mRNA synthesis and stability, respectively.

Ontogeny of Corticosteroid-Binding Globulin Biosynthesis in the Rat*

In plasma, glucocorticoids are transported by corticosteroid-binding globulin (CBG), which is synthesized primarily in the liver. Plasma levels of maternal and fetal CBG fluctuate during gestation, and this may be due to changes in the biosynthesis and/or clearance of the protein. We have, therefore, studied the ontogeny of CBG biosynthesis in the rat by using a solution hybridization assay to measure hepatic CBG mRNA levels. The results indicate that the concentration of CBG mRNA is exceptionally high in 15-day-old fetal livers (55.1 pg CBG mRNA/micrograms total RNA), but declines to very low levels at birth (day 21). During the same period, CBG mRNA levels in maternal livers remained relatively constant (18.9-23.1 pg CBG mRNA/micrograms total RNA). Hepatic CBG mRNA levels were barely detectable 1 week after birth, and a sex difference was apparent by 2 weeks of age, with higher levels in female livers. Although adult CBG mRNA levels were attained by 3 weeks of age, serum CBG concentrations did not reach adult values for an additional 3 weeks. To determine whether age-related differences in the clearance of CBG are responsible for this, CBG from infant or adult animals was radiolabeled and administered iv to infant and adult rats. When this was done, the half-life of CBG in infants (approximately 6.9 h) was consistently less than that in adults (approximately 14.5 h) regardless of the source of the labeled CBG, and we conclude that variations in CBG biosynthesis and clearance may influence glucocorticoid action during fetal and postnatal development.

Serum Corticosteroid-binding Globulin (CBG) and Hepatic CBG mRNA Relationships during Hamster Pregnancy: Contribution of Decidualization1

We measured serum corticosteroid-binding globulin (CBG) and hepatic CBG mRNA from individual hamsters throughout pregnancy and during decidualization. Serum CBG levels were determined by 3H-cortisol binding assay, and hepatic CBG mRNA levels were measured by Northern blots and solution hybridization assays, using a 32P-labeled cRNA probe derived from a rat CBG cDNA. There was a positive relationship between hepatic CBG mRNA levels and serum CBG levels during pregnancy. Both parameters increased significantly from the time of mating (cycle Day 4) to pregnancy Day 4, showing that CBG synthesis and secretion increased prior to implantation (Day 4). After implantation, serum CBG and hepatic CBG mRNA rose further from pregnancy Day 4 to a peak on Day 14, then decreased before parturition on Day 16. The prepartum decline in hepatic CBG mRNA preceded the fall in serum CBG. Decidualization on pseudopregnancy Day 4 resulted in an increase in serum CBG and hepatic CBG mRNA. Hepatic CBG mRNA increased from Day 5 to Day 7, and serum CBG increased progressively from Day 5 through Day 9 after uterine decidualization in the hamster. The present results demonstrate that the pattern of serum CBG observed in the pregnant hamster follows closely that of hepatic CBG mRNA. A signal emanating from uterine decidual tissue appears to be important in the regulation of hepatic CBG synthesis and secretion during midpregnancy, but other unknown factors appear to be involved in controlling CBG during the early and late stages of pregnancy.

Molecular studies of corticosteroid binding globulin structure, biosynthesis and function

Phylogenetic comparisons of the primary structure of corticosteroid binding globulin (CBG) have revealed several conserved domains that include sites for N-glycosylation and a region which probably represents a portion of the steroid binding site. The major site of CBG biosynthesis in adults is clearly the liver, and the human CBG gene promoter contains sequence elements that interact with liver-specific transcription factors. Low levels of CBG gene expression have been detected in other tissues, and these may be important for fetal development during late gestation when hepatic CBG mRNA levels are low. Studies of the ontogeny of CBG biosynthesis in the rat have also indicated that plasma CBG levels may be influenced by a more rapid clearance of the protein during pubertal development. Analyses of the structural organization and chromosomal location of the human CBG gene have further confirmed its close relationship with the serine proteinase inhibitors, and suggests that CBG, α 1-proteinase inhibitor and α 1-antichymotrypsin evolved relatively recently by gene duplication. The functional significance of this relationship has been examined and our studies suggest that a specific interaction between CBG and elastase on the surface of neutrophils may represent a physiologically important event that promotes the delivery of glucocorticoids to these cells at sites of inflammation.

Characterization of the Corticosteroid-Binding Globulin Messenger Ribonucleic Acid Response in the Pregnant Hamster*

In this study we measured corticosteroid-binding globulin (CBG) mRNA levels in liver and various nonhepatic tissues of pregnant and nonpregnant hamsters. The N-terminal amino acid sequence (37 residues) of hamster CBG was determined and compared with published cDNA-deduced sequence information for rat and human CBG. Hamster CBG showed considerable sequence homology with both rat (70%) and human (59%) CBG. Because of the high level of homology, we were able to use a cRNA prepared from a rat CBG cDNA as a probe in Northern blot and solution hybridization analyses. Northern blots of hamster and rat liver RNA extracts revealed that the rat CBG cDNA probe hybridized to RNAs that were the same size in rats and hamsters. Further, the Northern blot showed that pregnant hamster liver contained substantially more CBG mRNA than nonpregnant hamster liver. The relative amounts of CBG mRNA in pregnant and nonpregnant hamster livers were compared using a solution hybridization assay. Slope-ratio analysis of the hybridization data revealed that pregnant hamster liver (day 14) contained 40-fold more CBG mRNA than nonpregnant hamster liver. When other tissues (kidney, spleen, small intestine, and decidual tissue) were assayed for CBG mRNA, a small amount of hybridization was detected by solution hybridization. However, Northern blot analysis of RNA extracts from nonhepatic tissues showed that the hybridizable sequences did not migrate at the same position as mature CBG mRNA. These results indicate that the observed increase in serum CBG during hamster pregnancy is largely attributable to an increase in hepatic CBG mRNA.