Corticosteroid-Binding Globulin is expressed in the adrenal gland and its absence impairs corticosterone synthesis and secretion in a sex-dependent manner.

Corticosteroid-binding globulin (CBG) is synthesized by the liver and secreted into the bloodstream where binds to glucocorticoids. Thus CBG has the role of glucocorticoid transport and free hormone control. In addition, CBG has been detected in some extrahepatic tissues without a known role. CBG-deficient mice show decreased total corticosterone levels with missing of classical sexual dimorphism, increased free corticosterone, higher adrenal gland size and altered HPA axis response to stress. Our aim was to ascertain whether CBG deficiency could affect the endocrine synthetic activity of adrenal gland and if the adrenal gland produces CBG. We determined the expression in adrenal gland of proteins involved in the cholesterol uptake and its transport to mitochondria and the main enzymes involved in the corticosterone, aldosterone and catecholamine synthesis. The results showed that CBG is synthesized in the adrenal gland. CBG-deficiency reduced the expression of ACTH receptor, SRB1 and the main genes involved in the adrenal hormones synthesis, stronger in females resulting in the loss of sexual dimorphism in corticosteroid adrenal synthesis, despite corticosterone content in adrenal glands from CBG-deficient females was similar to wildtype ones. In conclusion, these results point to an unexplored and relevant role of CBG in the adrenal gland functionality related to corticosterone production and release.

Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPß.

Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPß, able to tether to the GR, as well as HNF3α involved in GR signaling, are present. C/EBPß, but not HNF3α, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg. Furthermore, knockdown of C/EBPß protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPß's involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPß and GR to the Cbg promoter, while C/EBPß knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPß.

Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus).

Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears.

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

Expression of corticosteroid binding globulin in the rat central nervous system.

Immunoreactivity for corticosteroid binding globulin was observed in the hypothalamus of intact male rats in the magnocellular nuclei and in single neurons in the periventricular nucleus and the lateral hypothalamus. The suprachiasmatic and the arcuate nuclei contained parvocellular neurons with specific immunoreactivity. Extensive networks of immunopositive fibers were observed in the lateral hypothalamus, the preoptic region, the bed nucleus of the stria terminalis and along the third ventricle. Immunostained axons often exhibited varicosities. The internal and the external layer of the median eminence showed numerous bundles of immunostained axons. Herring bodies in the posterior pituitary lobe contained specific immunoreactivity while pituicytes remained unstained. A portion of the Purkinje cells in the cerebellum and mossy fibers in the cerebellar granular layer stained for corticosteroid binding globulin. Some of the pyramidal cells in the hippocampus were corticosteroid binding globulin positive. Immunostained fibers occurred in the mesencephalon in the periaqueductal grey and in the medulla oblongata. A small fraction of the ependymal cells was also stained. In the spinal cord we observed specific immunoreactivity in a portion of the neurons in the dorsal horn. With polymerase chain reaction we confirmed the presence of the respective transcripts in the different brain regions. The multiple locations of corticosteroid binding globulin throughout the central nervous system suggest multiple functional properties, including neuroendocrine and neurohumoral functions.

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 (E2), testosterone (T), insulin, thyroxin (T4) 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, T4 and high concentrations of E2 decreased the secretion of CBG by HepG2 cells (p<0.05). Ethanol, the solvent used for E2, 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 T4 and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E2 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 corticosteroid-binding globulin (CBG) messenger RNA expression and plasma CBG concentrations in young pigs in response to heat and social stress.

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.

Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue.

Corticosterone binding (CB) capacity was determined in visceral and subcutaneous white adipose tissue (WAT), as well as in plasma of lean Zucker rats. Perfusion of rats with saline eliminated most liver and kidney corticosterone binding but did not affect CB in WAT. The cytosol extracts of isolated cells, however, did not bind corticosterone in detectable amounts. By means of a RT-PCR procedure it was found that corticosterone-binding globulin (CBG) was expressed in WAT. By immunohistochemical detection in WAT sections, CBG was seen in a thin layer surrounding the cells near the plasma membrane. These data suggest that the CBG layer surrounding the cells may act as a protective barrier limiting the access of glucocorticoids to adipocytes.

Testosterone is required for corticosteroid-binding globulin upregulation by morphine to be fully manifested.

We previously reported that morphine increases the concentration of corticosteroid-binding globulin (CBG) in blood of male, but not female, rats. This pronounced sexual dimorphism suggested that CBG upregulation by morphine might be androgen-dependent. In the current studies, we found that castration, whether performed just before or just after puberty or in adulthood, increased the concentration of CBG in adult male rats. Naltrexone did not prevent this increase and, therefore, it does not appear to be attributable to the release of endogenous opioids. Exposure to morphine for 1 week in adulthood increased ( approximately 100%) the concentration of CBG in intact, i.e., sham-castrated, males. The CBG levels of castrated rats treated with morphine did not differ from those of intact rats treated with morphine. However, because castration increased the concentration of CBG, the difference between the placebo and morphine groups decreased with time after castration. At 4 weeks after castration, the difference between the morphine and placebo groups (19%) was no longer statistically significant. Testosterone replacement prevented the rise in CBG levels following castration and maintained the magnitude of the difference between placebo and morphine-treated rats within the normal range. Thus, testosterone appears necessary for morphine effects on CBG to be fully manifested.

Effects of sex steroid hormones on corticosteroid-binding globulin gene expression in human endometrial cancer cell line Ishikawa.

The effect of progestins on intracellular corticosteroid-binding globulin (CBG) mRNA expression in an endometrial cancer cell line (Ishikawa) was examined in an attempt to understand the biological effects of high-dose progestins in the treatment of well-differentiated uterine endometrial cancers. Oestradiol-17 beta (E2) significantly increased CBG mRNA expression in a dose-dependent manner, while a high dose of progesterone with or without E2 suppressed it significantly. Furthermore, a high dose of progesterone or medroxyprogesterone acetate (MPA) suppressed CBG mRNA expression to a greater degree than did chlormadinone acetate or 17 alpha-hydroxyprogesterone caproate with or without E2. These findings suggest that the effects of high-dose progestins on cancer cells may be mediated via suppression of intracellular CBG.

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.

Expression of sex hormone-binding globulin and corticosteroid-binding globulin mRNAs in corpus luteum of human subjects.

To understand the biology of sex steroids in human ovarian corpus luteum, the expression of intracellular sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) mRNAs as a manifestation of intracellular SHBG and CBG expression was determined. The expression of SHBG and CBG mRNAs was detected in all samples analyzed. Luteal SHBG mRNA level showed no significant change during the endometrial secretory phase of the menstrual cycle. On the other hand, luteal CBG mRNA level was significantly higher (p < 0.05) at the mid-secretory phase than that at the early and late secretory phases of the endometrium. These findings suggest that human ovarian corpus luteum synthesizes SHBG and CBG intracellularly, CBG being plausibly involved in the functional life span of corpus luteum.

Evidence that brief stress may induce the acute phase response in rats.

Exposing rats to a single session of inescapable tail shock (IS) reduces corticosteroid binding globulin (CBG) 24 h later (Fleshner et al., Endocrinology 136: 5336-5342, 1995). The present experiments examined whether reductions in CBG are differentially affected by controllable vs. identical uncontrollable tail shock, are mediated by IS-induced glucocorticoid elevation, or reflect IS-induced activation of the acute phase response and whether IS produces fever. The results demonstrate that 1) equivalent reductions in CBG are observed in response to escapable tail shock or yoked IS, 2) IS-induced CBG reduction is not blocked by adrenalectomy in rats that receive basal corticosteroid replacement or by pretreatment with RU-38486, and 3) IS appears to activate the acute phase response, since IS reduces serum levels of an acute-phase negative reactant (CBG), increases serum levels of acute-phase positive reactants (haptoglobin and alpha 1-acid glycoprotein), and increases core body temperature 20-24 h later.

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.

Pituitary adenylate cyclase-activating polypeptide induces expression of corticosteroid-binding globulin in cultured fetal hepatocytes: synergy with tri-iodothyronine.

The purpose of the present study was to determine whether functional receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are expressed in cultured rat fetal hepatocytes and eventually play a role in regulating gene expression of corticosteroid-binding globulin (CBG). We found PACAP38 and PACAP27 to elevate cAMP levels in hepatocytes in a dose-dependent manner, with a plateau being achieved at 10 nM and EC50 values of about 0.5-1 nM. PACAP failed to alter the turnover of inositol phosphates, whereas PACAP and VIP stimulated cAMP accumulation in an equipotent manner, suggesting the presence in these cells of type II receptor isoforms. As revealed by measurements of both CBG mRNA levels and concentrations of binding sites, long-term treatment of fetal cells with 10 nM PACAP, although resulting in partial desensitization of peptide-induced cAMP accumulation, caused a significant 3-fold elevation in CBG synthesis. This stimulatory influence of PACAP was mimicked by the cell permeant N6,2'-O-dibutyryladenosine 3',5'-phosphate (dbcAMP). Treatment of hepatocytes with tri-iodothyronine (T3) enhanced CBG expression and, most interestingly, appeared to synergize with PACAP to elicit a 2-3-fold amplification of CBG synthesis. This study thus provides first evidence for the up-regulation by PACAP and cAMP of CBG expression in fetal hepatocytes and for T3's playing a synergistic role in enhancing PACAP-induced synthesis of the binder.

Regulation of corticosteroid-binding globulin synthesis by 1alpha,25-dihyroxy-vitamin D3 (calcitriol), 9-cis-retinoic acid and triiodothyronine in cultured rat fetal hepatocytes.

Evidence regarding the nature of the regulatory factors which directly act upon liver cells and extra-hepatic tissues to alter CBG synthesis is scarce. The present study used cultured rat fetal hepatocytes to investigate the involvement and possible interplay in this process of several members of the nuclear receptors superfamily: vitamin D (VDR), retinoic acids (RAR/RXR) and thyroid hormones (TR). Treatment of cells with 1alpha,25-(OH)2D3 (1,25-D) elicited a dose-dependent inhibition of basal CBG concentration in culture medium. Maximum inhibition to about 15% of control level was achieved with 0.1-1.0 nM, with an IC50 of 3.8 x 10(-12) M and with no significant change in binding affinity. Differential activation of RAR and RXR with either 9-cis-retinoic acid (9-cis-RA) or the RAR-selective synthetic retinoid TTNPB revealed that high doses of both drugs diminished CBG expression, though the former proved about 10-times more potent than the latter in this regard. Amplification by triiodothyronine (T3) of CBG synthesis failed to block the inhibitory effects of either 1,25-D or retinoids, as revealed by both binding capacity and mRNA measurements. Relative to CBG, 1,25-D similarly depressed the synthesis of alpha-fetoprotein (AFP), while on the contrary, retinoids and T3 were shown to cause opposite effects, as 9-cis-RA and TTNPB elevated and T3 decreased AFP expression. The present findings identify for the first time ligands of VDR and RAR/RXR as powerful negative regulators of both basal and T3-stimulated CBG biosynthesis in fetal hepatocytes and suggest lack of a functional interplay between TR and VR or RAR/RXR in these processes.

Effects of incremental cortisol and adrenalectomy on plasma corticosteroid binding capacity in fetal sheep.

The effects of incremental cortisol infusion or fetal adrenalectomy on plasma corticosteroid-binding capacity (CBC) were examined in sheep fetuses during late gestation (term approximately 150 days). Cortisol, infused from day 120 at 1.5 mg/day for the first 3 days, 2.5 mg/day for the next 5 days, and 3.5 mg/day for the final 2 days, stimulated a significant rise in plasma CBC and immunoreactive corticosteroid binding globulin (CBG). There was a significant positive correlation between individual values for total plasma cortisol concentrations and CBC values. In contrast, fetal adrenalectomy at day 115 prevented the rise in plasma CBC found in intact fetuses at term. These experiments show that exogenous cortisol, given in a manner that mimics the prepartum rise in fetal plasma cortisol, stimulates CBG biosynthesis, whereas abolition of the cortisol rise prevents the increase in CBG. The study provides strong support for the proposal that the prepartum increase in CBG biosynthesis in fetal sheep occurs in response to the progressive rise in adrenal cortisol output by the fetus towards term.

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)

Antagonistic effects of retinoic acid and triiodothyronine in the expression of corticoid-binding globulin (CBG) by cultured fetal hepatocytes.

Cultures of rat fetal hepatocytes were used to investigate the effects and interplay of triiodothyronine (T3) and retinoic acid (RA) in the regulation of gene expression of CBG, compared to that of alpha-fetoprotein (AFP). The cultured cells showed cytological features typical to hepatocytes and actually synthesized CBG and AFP, as evidenced from in situ hybridization with specific radioactive probes. Time course studies indicated that CBG (but not AFP) binding capacity in culture medium and cell mRNA levels disappeared with a half-life of about 2 days, thereby reflecting the decrease previously seen in hepatic CBG mRNA contents during embryonic life. The Kd values for CBG binding were unchanged under these conditions. Culturing of hepatocytes in the presence of T3 resulted in dose-dependent stimulations of both medium CBG and cell mRNA levels, with an EC50 concentration of about 10(-9) M. In sharp contrast, RA caused a reduction in CBG biosynthesis (IC50 = 1.7 x 10(-7) M) and, in addition, antagonized the stimulatory influence of T3. Under the same experimental conditions, AFP synthesis failed to be affected in a similar fashion. We conclude that thyroid hormones and RA directly act on hepatocytes to specifically regulate the expression of CBG in an antagonistic way.

Stimulation of sex hormone-binding globulin mRNA and attenuation of corticosteroid-binding globulin mRNA by triiodothyronine in human hepatoma cells.

We examined the time course and dose response of the triiodothyronine (T3) effect on mRNAs for sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) in cells of the human hepatoma line HepG2. After 7 h of exposure to a saturating dose of T3, SHBG mRNA was unchanged but increased to 1.5 +/- 0.1 times the unstimulated control at 22 h. Maximal stimulation (2.3 +/- 0.6) was observed at 2-3 days. Corticosteroid-binding globulin mRNA was unchanged for 22 h after exposure to T3 but diminished thereafter to 64% by day 3. At 3-4 days of exposure, the changes in both SHBG mRNA and CBG mRNA were dose-responsive to the T3 concentration. For both mRNAs, half-maximal response occurred between 10 and 20 pmol/l bioavailable T3. Cortisol-binding proteins secreted by HepG2 cells after 3 days in culture also were T3 dose-responsive. No re-uptake of secreted CBG by the cells was observed, suggesting that the T3 effect on CBG secretion occurs during production of the mature protein. These data suggest that T3 stimulates the expression of the SHBG gene and attenuates the expression of the CBG gene. The effects of T3 on these genes are consistent with the increase in circulating SHBG and decrease in circulating CBG observed in hyperthyroidism. The HepG2 cells may be a useful human cell line in which to study the diversity of the molecular mechanisms of T3 action.