Sex Hormone-binding Globulin mRNA Research Articles

Effect of placental sex hormone-binding globulin single nucleotide polymorphism rs6259 on protein and function in gestational diabetes mellitus.

Sex hormone-binding globulin (SHBG) has a key role in the occurrence and development of the gestational diab-etes mellitus(GDM). Single nucleotide polymorphism(SNP) rs6259 is a functional site in SHBGgene, which is suspected to regulate the SHBG level. The present study explored the placental SHBGSNPrs6259 distribution in Chinese pregnant women and the influence on placental SHBG concentrations, to assess the relationship of SHBGrs6259 in the occurrence and development of GDM. We screened the SHBG rs6259 allele in 210healthy and 180GDM gravidas by PCR-RFLP and restriction enzyme and measured placental SHBG concentrations in each genotypic group with western blot analysis. The mechanisms of SHBGrs6259 function were analyzed by cell culture, recombinant lentivirus transfection, real-time PCR, and western blot analysis. We found the differences of SHBGAsn327 allele frequency and the genotype distribution in GDM and control groups were statistically significant (P<0.05). Western blot analysis results showed that the Asn327 allele group was associated with a higher placental SHBG level than the Asp327 allele homozygote group (P<0.05). In HTR8-SVneo cell transfection, the positive transfection groups (SHBG-rs6259Asn) led to an obviously higher tendency of SHBG mRNA and protein expression than the negative control groups (SHBG-rs6259Asp), the normal cell group, and the blank control group (blank lentivirus LV-5)(P<0.05). Our data, therefore, reflected that SHBGSNPrs6259 causes changes in placental SHBG concentration and may play a functional role in the molecular mechanisms of GDM etiology.

SHBG expression is correlated with PI3K/AKT pathway activity in a cellular model of human insulin resistance

Decreased sex hormone-binding globulin (SHBG) expression is an independent risk factor for gestational diabetes mellitus(GDM).However, the mechanisms that link low SHBG expression and insulin resistance in GDM is unclear. In this study, we investigated the placenta SHBG in the PI3K/AKT pathway to reveal the mechanism that links decreased SHBG to insulin resistance. A insulin resistance cells model was established by the method of insulin stimulation. Two groups were set up, HTR8/Svneo cells and insulin-resistance cells of HTR8/SVneo. The expression of SHBG and PI3K/AKT associated factors were detected using real-time PCR and western blotting and their correlations were analyzed. The results showed that SHBG protein and mRNA levels in insulin resistance cells were both significantly lower. Along with decreased SHBG expression, the mRNA and protein levels of IRS-1, IRS-2, PI3Kp85α and GLUT-3, GLUT-4 decreased significantly. However, the expression of GLUT-1 increased significantly. Pearson correlation analysis showed that SHBG mRNA expression was positively correlated with IRS-1, IRS-2 and PI3Kp85α mRNA levels. According to the results, low SHBG expression not only participates in the development of local insulin resistance, but may also play an important role in PI3K/AKT pathway-mediated systemic insulin resistance and gestational diabetes.

Expression and correlation of sex hormone-binding globulin and insulin signal transduction and glucose transporter proteins in gestational diabetes mellitus placental tissue

ObjectiveThe aim of the present study was to investigate the probable pathogenesis of gestational diabetes mellitus (GDM) by analyzing the correlation between sex hormone-binding globulin (SHBG) secreted by the placenta during pregnancy and insulin signaling components and glucose transporter proteins (GLUTs) in the placental tissue. Design and methodsPlacental tissue was collected from full-term and non-obese [body mass index <25kg/m2] pregnant women; 10 diagnosed with GDM and 10 with normal pregnancy. We used real-time polymerase chain reaction (PCR), immunohistochemistry and western blotting to detect expression of protein and mRNA of SHBG and insulin signaling components and GLUTs in placental tissue. ResultsIn the placental tissue of non-obese women, there was a decrease in expression of SHBG protein and mRNA, with a concurrent decrease in expression of GLUT-4 protein and mRNA in women with GDM compared with normal controls. There was a decrease in GLUT-3 and insulin receptor substrate (IRS)-1 protein expression and lower IRS-2 mRNA expression was also observed in GDM placental tissue. Linear correlation analyses showed a positive correlation between SHBG and IRS-2 mRNA (P=0.038, R2=0.2178, y=0.249x+1.4208); positive correlation between SHBG and phosphatidylinositol 3-kinase (PI3K) p85α mRNA (P=0.035, R2=0.224, y=0.3506x+0.7433); positive correlation between SHBG and GLUT-4 mRNA (P=0.000, R2=0.5174, y=1.3822+1.7811x); positive correlation between IRS-2 and GLUT-4 mRNA (P=0.002, R2=0.4064, y=−0.8272+2.9592x); negative correlation between IRS-1 and PI3K p85α mRNA (P=0.005, R2=0.366, y=2.4492–0.1929x); negative correlation between IRS-1 and GLUT-3 mRNA (P=0.027, R2=0.243, y=0.9254–0.0714x); and positive correlation between IRS-2 and GLUT-1 mRNA (P=0.004, R2=0.3794, y=0.0225+0.6298x). ConclusionThe results confirm that defective receptors for insulin signal transduction and GLUT proteins are present in GDM placental tissue. Decreasing expression of SHBG may participate in regulation of insulin signaling, leading to a concomitant decrease in expression of relevant insulin signaling components in placental tissue, implying insulin resistance and eventual development of GDM.

Sex Hormone-Binding Globulin Gene Expression and Insulin Resistance

The plasma level of sex hormone binding globulin (SHBG), a glycoprotein produced by hepatocytes, is subject to genetic, hormonal, metabolic, and nutritional regulation, and is a marker for the development of the metabolic syndrome and diabetes. Because the mechanism for these associations is unclear, and no studies of SHBG gene expression in humans have been published, SHBG mRNA was measured in human liver samples and related to anthropometric data. Inpatients at a private, nonprofit, university-associated hospital were studied. Subjects were fifty five adult men and women undergoing hepatic resection as treatment for cancer. Main outcome measures were SHBG mRNA and serum SHBG levels. SHBG mRNA was a strong predictor of serum SHBG with higher levels of the mRNA and protein in women than in men. The relationship between SHBG mRNA and circulating SHBG differed in males and females consistent with a sex difference in post-transcriptional regulation. A strong positive correlation was found between the level of the mRNA for the transcription factor HNF4α and SHBG mRNA. Insulin resistance (IR), assessed by homeostatis model assessment, was related inversely to SHBG mRNA and to HNF4α mRNA as well as to circulating SHBG levels. These mRNAs, as well as serum SHBG, were higher when the hepatic triglyceride concentration was low, and decreased with increasing body mass index but were unrelated to age. Fat accumulation in liver and IR are important determinants of SHBG gene expression and thereby circulating SHBG levels that are perhaps mediated through effects on the transcription factor HNF4α. These findings provide a potential mechanism to explain why low SHBG predicts the development of type 2 diabetes.

SHBG in GDM maternal serum, placental tissues and umbilical cord serum expression changes and its significance

AimsTo compare the expression of sex hormone binding globulin (SHBG) in normal placental tissues and serum with placental tissues from patients with gestational diabetes mellitus (GDM) and to deduce the mechanism affecting placental SHBG in GDM. MethodsEnzyme-linked immunosorbent assay (ELISA) method were used to detect SHBG levels changes in normal and GDM maternal serum, umbilical cord serum and placental. We measured SHBG mRNA and protein using reverse-transcription polymerase chain reaction, and Western blotting, respectively, in normal and GDM placental tissues. ResultsIn maternal serum, compared with the control group, GDM group: glucose, insulin increased, SHBG decreased. In the placenta, compared with the control group, GDM group: SHBG, SHBG mRNA and SHBG protein decreased. There is no correlation between placenta SHBG and maternal serum SHBG respectively in control and GDM group, but in GDM group maternal serum insulin and placenta SHBG are linear correlation. ConclusionsGDM serum and placental SHBG levels are reduced, hyperinsulinemia may lead to a reduction of SHBG in circulating blood, but also damage the placenta cells which led to placental synthesis and secretion of SHBG decrease.

IL1β Down-regulation of Sex Hormone-Binding Globulin Production by Decreasing HNF-4α Via MEK-1/2 and JNK MAPK Pathways

Patients suffering from low-grade chronic inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, diabetes, and obesity, have low plasma sex hormone-binding globulin (SHBG) levels. These diseases are characterized among other features by high plasma IL1β levels. The aim of the present study is to explore whether IL1β could regulate hepatic SHBG production to account for low SHBG levels in these diseases. We provide evidence that daily IL1β treatment reduces SHBG production in HepG2 cells by the down-regulation of HNF-4A via the MAPK kinase (MEK)-1/2 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways through the activation c-Jun transcription factors. The human SHBG promoter sequence contains two putative activator protein 1 (AP1) binding sites recognized by c-Jun transcription factors, but they are not necessary for the IL1β-induced down-regulation of SHBG promoter activity in luciferase reporter gene assays. Daily treatment with IL1β reduces hepatic nuclear factor (HNF)-4α mRNA and protein levels via the MEK-1/2 and JNK MAPK signaling pathways. Moreover, IL1β rapidly decreased HNF-4α mRNA and protein levels while increased phospho-c-Jun protein levels after the treatment. Finally, daily IL1β treatment of human SHBG transgenic mice reduced plasma SHBG and SHBG mRNA levels. Moreover, IL1β treatment also reduced HNF-4α mRNA and protein levels while increased hepatic phospho-c-Jun protein levels. Our results show that IL1β reduces hepatic SHBG production by decreasing HNF-4α via MEK-1/2 and JNK MAPK pathways. In addition, our findings suggest that IL1β could be involved the low plasma SHBG levels reported in chronic low-grade inflammatory diseases.

Expression changes of sex hormone binding globulin in GDM placental tissues

To compare the expression of sex hormone binding globulin (SHBG) in normal placental tissues with placental tissues from patients with gestational diabetes mellitus (GDM) and to deduce the mechanism affecting placental SHBG in GDM. We detected SHBG localization and measured SHBG mRNA and protein using immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blotting, respectively, in normal and GDM placental tissues. The distribution of SHBG in placental cells was examined using immune electron microscopy. Compared to controls, placental tissues from patients in the GDM group displayed disordered cell surface microvilli that were decreased in quantity, swollen, and had narrowed and broken gap junctions. Intracellular abnormalities included expanded rough endoplasmic reticula, swollen mitochondria, and irregular nuclear morphologies with non-uniform chromatin. SHBG localized primarily to trophoblast cell membranes and cytoplasm. SHBG was strongly expressed on the microvilli side and weakly expressed on the basement membrane with uneven staining. SHBG also was expressed in villous stromal cells and vascular endothelial cells. Compared to the controls, placental tissues from the GDM group displayed significantly decreased immunostaining rates for SHBG, as well as significantly lower levels of SHBG mRNA and protein expression (P<0.05). SHBG was detected in placental trophoblast cells from patients with GDM, and the synthesis and secretion of SHBG were reduced when trophoblast cells were irregular. A decrease in SHBG could affect placental function or aggravate GDM. Our results suggest that placental SHBG plays an important role in the pathogenesis of GDM.

The ability of hydroxylated estrogens (2-OH-E2 and 4-OH-E2) to increase of SHBG gene, protein expression and intracellular levels in MCF-7 cells line

Sex Hormone-Binding Globulin (SHBG) - specific carrier for sex steroids - regulates hormone bioavailable fraction and estrogen signaling system in breast cancer cells. This study was conducted to elucidate the effects of hydroxylated estrogen (E2) metabolites (2-OH-E2 and 4-OH-E2) on sex hormone binding globulin (SHBG) mRNA and protein expression as well as on intracellular and extracellular SHBG levels. MCF-7 human breast cancer cells were cultured with 2-OH-E2 or 4-OH-E2 in concentration of 1, 10 and 100 nM for 24 h, 17β-estradiol being used as a positive control. SHBG levels were measured in medium and cells by ELISA, SHBG mRNA expression was evaluated by real-time-PCR and protein expression by Western blot analysis. 4-OH-E2 in high doses and 2-OH-E2 in the highest dose, while 17β-estradiol in all doses used increased intracellular but not extracellular SHBG levels. Both metabolites increased SHBG mRNA expression, the rank order of potency being E2 > 4-OH-E2 > 2-OH-E2. Both E2 and its metabolites increased SHBG protein expression. Although the metabolites showed a lower potency than 17β-estradiol, further studies are needed to clarify whether hydroxylated metabolites of E2 are potent ligands for SHBG.

Human SHBG mRNA Translation Is Modulated by Alternative 5′-Non-Coding Exons 1A and 1B

BackgroundThe human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5′ UTR exons 1A and 1B differently regulate SHBG translation.ResultsCis-regulatory elements that could potentially regulate translation were identified within the 5′UTRs of SHBG TU-1A and TU–1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5′UTR sequence did show SHBG translation, whereas inclusion of the 5′UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5′UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B.ConclusionsThe use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.

2021 Imaging tumour hypoxia: the need to combine techniques

The aim of the present study was to investigate the probable pathogenesis of gestational diabetes mellitus (GDM) by analyzing the correlation between sex hormone-binding globulin (SHBG) secreted by the placenta during pregnancy and insulin signaling components and glucose transporter proteins (GLUTs) in the placental tissue.Placental tissue was collected from full-term and non-obese [body mass index <25 kg/m2] pregnant women; 10 diagnosed with GDM and 10 with normal pregnancy. We used real-time polymerase chain reaction (PCR), immunohistochemistry and western blotting to detect expression of protein and mRNA of SHBG and insulin signaling components and GLUTs in placental tissue.In the placental tissue of non-obese women, there was a decrease in expression of SHBG protein and mRNA, with a concurrent decrease in expression of GLUT-4 protein and mRNA in women with GDM compared with normal controls. There was a decrease in GLUT-3 and insulin receptor substrate (IRS)-1 protein expression and lower IRS-2 mRNA expression was also observed in GDM placental tissue. Linear correlation analyses showed a positive correlation between SHBG and IRS-2 mRNA (P = 0.038, R2 = 0.2178, y = 0.249x + 1.4208); positive correlation between SHBG and phosphatidylinositol 3-kinase (PI3K) p85α mRNA (P = 0.035, R2 = 0.224, y = 0.3506x + 0.7433); positive correlation between SHBG and GLUT-4 mRNA (P = 0.000, R2 = 0.5174, y = 1.3822 + 1.7811x); positive correlation between IRS-2 and GLUT-4 mRNA (P = 0.002, R2 = 0.4064, y = −0.8272 + 2.9592x); negative correlation between IRS-1 and PI3K p85α mRNA (P = 0.005, R2 = 0.366, y = 2.4492–0.1929x); negative correlation between IRS-1 and GLUT-3 mRNA (P = 0.027, R2 = 0.243, y = 0.9254–0.0714x); and positive correlation between IRS-2 and GLUT-1 mRNA (P = 0.004, R2 = 0.3794, y = 0.0225 + 0.6298x).The results confirm that defective receptors for insulin signal transduction and GLUT proteins are present in GDM placental tissue. Decreasing expression of SHBG may participate in regulation of insulin signaling, leading to a concomitant decrease in expression of relevant insulin signaling components in placental tissue, implying insulin resistance and eventual development of GDM.

Thyroid hormones act indirectly to increase sex hormone-binding globulin production by liver via hepatocyte nuclear factor-4α

Thyroid hormones increase hepatic sex hormone-binding globulin (SHBG) production, which is also regulated by hepatocyte nuclear factor-4alpha (HNF-4alpha) in response to changes in the metabolic state of the liver. Since the human SHBG promoter lacks a typical thyroid hormone response element, and because thyroid hormones influence metabolic state, we set out to determine whether thyroid hormones mediate SHBG expression indirectly via changes in HNF-4alpha levels in HepG2 human hepatoblastoma cells, and in the livers of transgenic mice that express a 4.3 kb human SHBG transgene under the control of its own 0.8 kb promoter sequence. Thyroid hormones (triiodothyronine (T(3)) and thyroxine (T(4))) increase SHBG accumulation in HepG2 cell culture medium over 5 days, and increase cellular SHBG mRNA levels. In addition, T(4) treatment of HepG2 cells for 5 days increased HNF-4alpha mRNA and HNF-4alpha levels in concert with decreased cellular palmitate levels. Plasma SHBG levels were also increased in mice expressing a human SHBG transgene after 5 days treatment with T(3) along with increased hepatic HNF-4alpha levels. In HepG2 cells, the human SHBG promoter failed to respond acutely (within 24 h) to T(4) treatment, but a 4-day pre-treatment with T(4) resulted in a robust response that was prevented by co-treatment with HNF-4alpha siRNA, or by blocking the beta-oxidation of palmitate through co-treatment with the carnitine palmitoyltransferase I inhibitor, etomoxir. These data lead us to conclude that thyroid hormones increase SHBG production indirectly by increasing HNF-4alpha gene expression, and by reducing cellular palmitate levels that further contribute to increased HNF-4alpha levels in hepatocytes.

Sex Hormone-Binding Globulin in Fish Gills Is a Portal for Sex Steroids Breached by Xenobiotics

As in most vertebrates, plasma sex hormone-binding globulin (SHBG) is produced in fish liver and regulates sex steroid access to target tissues. Low levels of SHBG mRNA are present in zebra fish gills but are unlikely to account for the high amounts of immunoreactive SHBG in filaments and lamellae. Although the uptake of steroids by fish from water has been reported to correlate with their affinity for SHBG, it is not known how this occurs. Our studies of zebra fish SHBG have revealed its preference for biological active androgen (testosterone), as well as for androstenedione, a sex steroid precursor that also acts as a pheromone in some fish. In addition to natural steroids, zebra fish SHBG has a high affinity for synthetic steroids, such as ethinylestradiol and progestins (levonorgestrel and norethindrone), that are present in waste water systems. Because steroids can pass across fish gills, we examined whether SHBG serves as a portal for natural and synthetic steroids controlling their flux between the blood and aquatic environment. The results indicate that SHBG ligands are rapidly and specifically removed from water by the fish through their gills, whereas the accumulated steroids are released slowly. The capacity of fish to sequester SHBG ligands from water is similar between sexes, independent of size, and characterized by a wide dynamic range. We conclude that SHBG controls the flux of sex steroids across fish gills and that this highly specialized function can be hijacked by xenobiotic ligands of fish SHBGs.

Sex hormone-binding globulin expression in sea bass ( Dicentrarchus labrax L.) throughout development and the reproductive season

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in the blood of vertebrate species, including fish, and regulates the bioavailability and metabolic clearance of these steroids. Liver is the major site of plasma SHBG synthesis, while an SHBG homologue, known as the androgen-binding protein, is produced in testes. When shbg gene expression was examined throughout European sea bass ( Dicentrarchus labrax L.) development, SHBG mRNA was clearly detectable at 7 days post-fertilization and persisted throughout embryonic development. In male and female sea bass, the liver is the principal site of shbg gene expression, as determined by SHBG mRNA analyses. Immunoreactive SHBG is present in the liver and villous stroma of the intestine in both sexes. It is also present in the interstitial space between testicular lobules, and the connective tissue surrounding the ovary in the non-reproductive season and around post-vitellogenic oocytes. Plasma SHBG levels were measured over a 10-month period as male sea bass undergo sexual maturation. Immature females of the same age were also studied over the same time interval. The mean ± S.E.M. plasma SHBG levels in 2-year-old males and females are lower (80 ± 15 nM and 82 ± 16 nM, respectively) during the winter reproductive season (December–March) than the spring (April–June) months (144 ± 32 nM and 193 ± 18 nM, respectively). In both sexes, plasma SHBG levels start to decline 1–2 months before the reproductive season, coincident with a period of rapid weight gain, while increases after the reproductive season are not accompanied by significant changes in body weight. In addition, plasma SHBG in triploid (sterile) and diploid (fertile) male sea bass do not differ during the first spawning season. These data suggest that the decrease in plasma SHBG levels during sexual maturation in sea bass is related to nutritional or metabolic effects in relation to water temperatures and food intake, rather than changes in gonadal sex steroid production.

Changes of Sex Hormone-binding Globulin/SHBG Expression in the Hypothalamo-hypophyseal System of Rats During Pregnancy, Parturition and Lactation

Sex hormone-binding globulin (SHBG) is expressed in hypothalamic magnocellular neurons. High co-localization rates of SHBG with oxytocin have been observed in the hypothalamus, indicating that SHBG plays a role in pregnancy, parturition and lactation. Further studies have shown that hypothalamic SHBG expression is malleable to changing steroid conditions. In this study, we have examined SHBG levels in the supraoptic and paraventricular hypothalamic nuclei and in the posterior pituitary lobe of late pregnant, parturient and early lactating rats by IN SITU hybridization, immunocytochemistry, and ELISA. Immunocytochemical and biochemical analysis showed that the SHBG levels increased during late pregnancy in hypothalamic nuclei. During parturition, SHBG levels fell in the magnocellular nuclei but increased in the posterior pituitary lobe. SHBG levels increase again during lactation. At day six of lactation, there was no significant difference in SHBG levels compared to normal cycling female rats, which served as control in this study. IN SITU hybridization showed increased SHBG mRNA signal during late pregnancy. The highest SHBG expression was observed during parturition. Our data indicate that hypothalamic SHBG expression changes during pregnancy, parturition and lactation, parallel to ovarian steroid and co-localized OT levels. This may in part be linked to known steroid actions on synthesis and secretion of magnocellular hypothalamic peptide hormones, important for the control of parturition and lactation.

Molecular and functional characterization of sex hormone binding globulin in zebrafish.

SHBG (sex hormone binding globulin) transports androgens and estrogens in the blood of vertebrates including fish. Orthologs of SHBG in fish are poorly defined, and we have now obtained a zebrafish SHBG cDNA and characterized the zebrafish SHBG gene and protein through molecular biological, biochemical, and informatics approaches. Amino-terminal analysis of zebrafish SHBG indicated that its deduced precursor sequence includes a 25-residue secretion polypeptide and exhibits 22-27% homology with mammalian SHBG sequences and 41% with a deduced fugufish SHBG sequence. The 356-residue mature zebrafish SHBG (39,243 Da) sequence comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites; and exists as a 105,000 +/- 8700 Da homodimer. Zebrafish SHBG exhibits a high affinity and specificity for sex steroids. An RT-PCR indicated that SHBG mRNA first appears in zebrafish larva, and SHBG mRNA was localized within the liver and gut at this stage of development by whole-mount in situ hybridization. In adult fish, SHBG mRNA was found in liver, testis, and gut. In the liver, immunoreactive SHBG was present in hepatocytes and concentrated in intrahepatic bile duct cells, whereas in the testis it was confined to cells surrounding the seminiferous tubule cysts. In the intestine, immunoreactive SHBG was present in the stroma and epithelial cells of the villous projections and the surrounding muscle. The production and presence of SHBG in the gut of developing and adult zebrafish suggests a novel role for this protein in regulating sex steroid action at this site.

Estradiol Control of Expression and Levels of Estradiol-Binding Proteins in the Medial Preoptic Area, Medial Hypothalamus and Pituitary

The brains of mammals have at least three estradiol-binding proteins: estradiol receptor-α (ERα), ERβ, and sex hormone-binding globulin (SHBG). In this study we compare the effects of estradiol treatment on the expression of mRNA for these three estradiol-binding proteins in two reproductively important brain areas, the medial preoptic area-anterior hypothalamus (MPOA-AH) and medial hypothalamus (MH) as well as in the hippocampus in ovariectomized rats, using the reverse transcriptase-polymerase chain reaction (RT-PCR). We also used surface-enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry (MS) to analyze the effects of estradiol in ovariectomized rats on SHBG levels in the MPOA-MH as well as the neurohypophysis. In vivo estradiol treatment in ovariectomized rats eliminated or significantly reduced expression of all three estradiol-binding proteins in both the MPOA-AH and MH. This change in ERα, ERβ, and SHBG expression did not occur in the hippocampus. Both Northern blot and DNA sequence analysis confirmed the results of the RT-PCR for SHBG. SELDI-TOF MS analysis demonstrated that in vivo estradiol treatments resulted in dramatically decreased levels of SHBG in the hypothalamus and that a reduction in SHBG mRNA by estradiol treatment also resulted in a reduction in SHBG protein levels. Estradiol treatment also eliminated detectable SHBG from the neurohypophysis, suggesting that estradiol controls SHBG levels in this release site. That in vivo estradiol treatments had the same inhibitory effects on mRNA levels for SHBG and both ERs suggests similar translational control mechanisms for all three steroid-binding proteins in the brain. That estradiol treatments also reduced pituitary SHBG suggests that such treatment releases SHBG from the neurohypophysis.

Sex Hormone-binding Globulin in the Human Prostate Is Locally Synthesized and May Act as an Autocrine/Paracrine Effector

Sex hormone-binding globulin (SHBG) is a plasma protein synthesized and secreted by the liver. Its initial description stemmed from its ability to bind estrogens and androgens and its capacity to regulate the free concentration of the steroids that bind to it. Additionally, it participates in signal transduction for certain steroid hormones at the cell membrane. It binds with high affinity to a specific membrane receptor (R(SHBG)) in prostate stromal and epithelial cells, wherein the SHBG.R(SHBG) complex forms. An appropriate steroid binds to this complex and results in increases of intracellular cAMP. These two disparate functions of SHBG, regulation of the concentration of free steroids in plasma and signal transduction in selected tissues, raise the question of how its synthesis and secretion might be regulated so as to best perform these two disparate functions. In this paper we demonstrate that SHBG is produced in human prostate cancer cell lines (LNCaP, DU 145, and PC 3) as well as in cultured human prostate epithelial and stromal cells. In addition, in tissue sections of human prostate, we demonstrate the presence of SHBG (immunocytochemistry) and SHBG mRNA (in situ hybridization). These observations are consistent with the hypothesis that SHBG, destined to participate in signaling at the cell membrane, is locally regulated and produced.

Rabbit sex hormone-binding globulin: expression in the liver and testis during postnatal development and structural characterization by truncated proteins.

Although sex hormone binding globulin (SHBG) is found in the blood plasma of adult humans and rabbits and the gene is expressed in their livers, it is not detected in the plasma of adult rodents nor is it expressed in adult rodent livers. Thus the rabbit represents a good model to study the metabolism and function of SHBG in the blood. We have used a cloned rabbit SHBG cDNA to detect mRNA expression in rabbits during the postnatal period, and to construct truncated SHBG proteins for structure/function analysis. The SHBG mRNA appeared in the testis as early as 3 days after birth. The level increased gradually in abundance throughout postnatal development, and attained a maximum at 12 weeks of age when the gonads were fully matured. In contrast, SHBG mRNA in the livers of male and female animals increased to a maximum by 4 weeks of age, and were maintained at this level until 12 weeks before subsiding to the initial levels. The increase and decrease in SHBG mRNA levels in the liver were accompanied by similar changes in serum SHBG. This suggests that SHBG in the blood circulation comes from the liver and this might also provide a source of SHBG for the male reproductive tract before formation of the blood-testis barrier. To elucidate the minimal sequence of rabbit SHBG responsible for steroid-binding, a panel of 13 truncated SHBG proteins was constructed, expressed in Escherichia coli, and biochemically purified for study. It was shown that the complete protein sequence of rabbit SHBG was important for maintaining a stable steroid-protein complex. Unlike human SHBG for which a truncated protein of the first 206 residues of the 373 amino acid protein can still bind steroid, removal of 43 or more residues from the C-terminus of rabbit SHBG completely abolished steroid-binding.

Expression and Regulation of Human Sex Hormone-Binding Globulin Transgenes in Mice during Development1

Human sex hormone-binding globulin (SHBG) is produced by hepatocytes and transports sex steroids in the blood. The rat gene encoding SHBG is expressed transiently in the liver during fetal life, but it is not expressed in the liver postnatally, and the small amounts of SHBG in rat blood are derived from gonadal sources. To study the biosynthesis and function of human SHBG in an in vivo context, we have produced several lines of transgenic mice that contain either 11 kb (shbg11) or 4.3 kb (shbg4) portions of the human shbg locus. The expression and regulation of these transgenes have now been studied during fetal and postnatal development. In situ hybridization of an shbg11 transgenic mouse fetus at 17.5 days postcoitus located human shbg transcripts only in duodenal epithelial cells and hepatocytes. Temporal differences in the hepatic expression of mouse shbg and human shbg transgenes during late fetal development were reflected in corresponding differences in mouse and human SHBG levels in fetal and neonatal mouse blood. Serum concentrations of human SHBG increased during the first weeks of life regardless of gender until about 20 days of age in shbg11 mice, but after this time they continued to increase only in the males. This sexual dimorphism was reflected in corresponding differences in human SHBG messenger RNA (mRNA) abundance in the livers of these animals. However, it was not observed in shbg4 mice, in which hepatic production of plasma SHBG continued to increase after puberty regardless of gender. Serum testosterone and SHBG levels correlated in all sexually mature shbg transgenic mice. Human shbg transcripts were detectable only in testes of shbg11 mice and increased progressively in abundance from 10 days of age until the animal reached sexual maturity at 30 days of age, with appreciable increases occurring well before any changes in serum testosterone concentration. In the kidney, SHBG mRNA levels accumulated earlier in shbg11 than in shbg4 mice, and the expression of both types of transgenes was sexually dimorphic, with much higher SHBG mRNA levels in the kidneys of male mice. As increases in SHBG mRNA in the male kidneys coincided with increases in serum testosterone during sexual maturation, we reasoned that shbg transgene expression is androgen dependent in the kidney. This was confirmed by demonstrating that a decrease in SHBG mRNA abundance in male mouse kidneys after castration could be reversed by 5alpha-dihydrotestosterone treatment. Moreover, exogenous androgen increased human SHBG mRNA levels in the kidneys of female mice. In summary, comparisons of how different human shbg transgenes are expressed in vivo provides information about the positions of potential regulatory sequences that may control the hormonal regulation and tissue-specific expression of this gene during development.

Sex hormone binding globulin expression and colocalization with estrogen receptor in the human Fallopian tube

The detection of sex hormone binding globulin (SHBG) or SHBG mRNA in several sex steroid target tissues, has raised the possibility that SHBG modulates the action of sex steroids outside the vascular compartment. The presence of SHBG mRNA was investigated by RT-PCR in the poly (A +) RNA fraction of the human Fallopian tube. Human and rat liver were used as positive and negative control tissues, respectively. The electrophoretic analysis of the amplified PCR products showed bands at 219 bp, corresponding to the expected size of the SHBG cDNA, in the Fallopian tube and human liver but not in rat liver, indicating that SHBG might be sinthezised by the Fallopian tube. The cellular localization of SHBG and of estrogen receptor (ER) was examined by immunohystochemistry in consecutive sections of Fallopian tube tissues for individual staining or double immunostaining in the same section. Specific immunostaining of SHBG was present in the epithelial, vascular and muscle cells of the ampullary and isthmic region. In epithelial cells, immunoreactive SHBG was present in the apical end with the highest concentration close to the luminal membrane. The ER was localized in the nuclei of epithelial, stromal and muscle cells of the ampulla and isthmus. Double immunostaining showed that SHBG and ER are colocalized principally in epithelial cells of the ampulla and in muscle cells of the isthmus. In conclusion, the detection of SHBG and SHBG mRNA and the localization of SHBG in estrogen target cells was shown. These findings support the hypothesis that SHBG might regulate sex steroid action at the tissue level.