Plasma steroid-binding proteins: primary gatekeepers of steroid hormone actionBiologically active steroids are transported in the blood by albumin, sex hormone-binding globulin SHBGand corticosteroid-binding globulin CBG. Albumin binds steroids with limited specificity and low affinity, but its high concentration in blood buffers major fluctuations in steroid concentrations and their free fractions. Both are glycoproteins that are structurally unrelated, and they function in different ways that extend beyond their cortisol binding globulin and testosterone or buffering functions in the blood. Plasma SHBG and CBG production by the liver varies during development and different physiological or pathophysiological bindibg, and abnormalities in the plasma levels of SHBG and CBG or their abilities to muscle pharm anavar steroids are associated with a variety of pathologies. Cortisol binding globulin and testosterone how the unique structures of SHBG and CBG determine their specialized functions, how oxandrolone cycle in their plasma levels are controlled, and how they function outside the blood circulation provides insight into how they control restosterone freedom of steroids to act in health and disease.
Sex-Hormone-Binding Globulin is an Oestrogen Amplifier | Nature
Biologically active steroids are transported in the blood by albumin, sex hormone-binding globulin SHBG , and corticosteroid-binding globulin CBG. Albumin binds steroids with limited specificity and low affinity, but its high concentration in blood buffers major fluctuations in steroid concentrations and their free fractions.
Both are glycoproteins that are structurally unrelated, and they function in different ways that extend beyond their transportation or buffering functions in the blood. Plasma SHBG and CBG production by the liver varies during development and different physiological or pathophysiological conditions, and abnormalities in the plasma levels of SHBG and CBG or their abilities to bind steroids are associated with a variety of pathologies.
Understanding how the unique structures of SHBG and CBG determine their specialized functions, how changes in their plasma levels are controlled, and how they function outside the blood circulation provides insight into how they control the freedom of steroids to act in health and disease.
Upon their release from steroidogenic cells, biologically active steroids are transported in the blood largely by albumin, sex hormone-binding globulin SHBG , and corticosteroid-binding globulin CBG. Although CBG and SHBG are present in much lower concentrations in plasma than albumin, their high affinity and specificity for steroids enables them to play much more dynamic roles in determining the plasma concentrations of their main ligands.
In addition, they control the amounts of free steroids that passively diffuse into cells, and they accomplish this in distinct and diverse ways Hammond , Perogamvros et al. The liver is responsible for plasma SHBG and CBG production, but their genes are also expressed in several other tissues where their protein products function differently than in the blood Hammond , The free hormone hypothesis provides a foundation for understanding how steroids act at the target cell level by postulating that only free steroids that are not bound by proteins passively diffuse through the plasma membranes of cells Mendel Steroids that are loosely and non-specifically bound to albumin have also been proposed to be accessible to tissues Pardridge , but steroids still have to dissociate from albumin before they diffuse into cells and exert their activities.
At present, the proposition that only free steroids diffuse into cells therefore still best explains the clinical manifestations of either steroid hormone excess or deficiency, and knowledge of free steroid concentrations in plasma is critical to understanding their biological activities. While measurements of free steroid concentrations remain the most robust indicator of the biological activities of plasma steroids Vermeulen et al.
This is because steroid-target cells in multicellular organ systems are often compartmentalized and separated from the blood vasculature. Moreover, tissues and organ systems vary enormously in terms of their vascular permeability and the nature of their blood supply, including blood flow and transit time. Extreme examples include the highly fenestrated aspect of the blood vasculature in the liver, where hepatocytes are essentially bathed in blood, vs cells within the brain and testis that are separated by blood barriers.
In addition, sex steroid-sensitive epithelial cells in organs such as the prostate, breast, and endometrium are separated from blood capillaries by complex basement membranes, and are compartmentalized together with other cell types e. Thus, the locations of target cells in relation to the blood supply, the endothelial vascular permeability, the composition of the extravascular fluids and extracellular matrix, as well as the juxtaposition of different cell types within a tissue all dictate the ultimate ability of steroids to access their target cells.
This review provides examples of how albumin, CBG, and SHBG function in concert with each other, as well as separately, to control the actions of steroid hormones in both the blood and extravascular tissue compartments. Albumin is the most abundant protein in the blood and it binds steroids and other small lipophilic molecules non-specifically.
Its plasma concentrations are normally relatively constant and approximately times greater than those of the other major steroid-binding proteins Dunn et al. However, reductions in plasma albumin concentrations, which are often seen in patients with severe malnutrition, cirrhosis, the nephrotic syndrome, and other critical illnesses, have been predicted to alter the plasma distribution of testosterone Dunn et al.
Although the mathematical models used in latter study indicated that changes in plasma albumin levels predict only a small effect on the plasma distribution of cortisol, a recent study in critically ill patients has indicated that this introduces a bias in calculations of plasma free cortisol levels Molenaar et al. Under most conditions, however, where plasma albumin levels are with normal ranges, its main function is to buffer changes in the plasma distribution of steroids when their concentrations increase transiently, or when the production or function of CBG or SHBG change under different physiological conditions or during disease.
Apart from fish, all other vertebrate classes have a plasma protein that binds glucocorticoids and progesterone with high affinity Westphal However, as described in detail below, the specific cleavage of CBG by proteases within a distinct structural domain serves to promote the targeted delivery of CBG ligands to their sites of action.
The most obvious function of CBG in the blood is to transport glucocorticoids Brien , Perogamvros et al. A Influence of CBG on the plasma distribution of cortisol. B Estimated proportional occupancy of plasma CBG by its major ligands, cortisol and progesterone, in blood samples taken from women before and during pregnancy, and the invervillous compartment of the placenta at term. Note that the cortisol distribution in plasma with inactive CBG is expected to resemble that in patients homozygous for naturally occurring CBG variants with undetectable steroid-binding activity, for example, CBG GV Perogamvros et al.
B Proportional occupancy of CBG in serum from women during the luteal phase of the menstrual cycle vs the third trimester of pregnancy as estimated computationally from data of serum CBG, cortisol and progesterone levels Dunn et al.
In addition to the liver, Serpina6 is expressed at relatively high levels in several other tissues, such as the endocrine pancreas and proximal convoluted tubules of the kidney during early development in mice Scrocchi et al. For instance, the CBG produced by the developing rodent kidney is secreted luminally into the proximal convoluted tubules Scrocchi et al.
However, during the second and third trimesters of human pregnancy, the large amounts of progesterone produced by the placental trophoblast are capable of displacing glucocorticoids from CBG, and under these circumstances CBG will assume the role of a major plasma progesterone transport protein at least during late gestation Fig.
In support of this, it has been reported that plasma CBG may influence circulating progesterone levels during human pregnancy and serves as a local regulator of progesterone levels and activity at the maternal—fetal interface Benassayag et al. This was further illustrated in a study of pregnant Chinese women with a relatively common frequency of 1: In the latter study, CBG levels correlated with amounts of circulating progesterone during the first two trimesters of pregnancy, as well as the amounts of progesterone in the intervillous blood Lei et al.
As also reported previously Benassayag et al. Moreover, plasma levels of CBG in some intervillous blood samples at term were similar to the corresponding maternal circulating levels, while in others they were as much as four times lower Lin et al.
Importantly, in those intervillous samples with very low CBG levels, the levels of progesterone were about two to three times higher than in the peripheral blood Lin et al. When this is considered in relation to the estimates of the proportional occupancy of CBG by progesterone in maternal blood in late gestation Fig. Although such large differences in intervillous blood levels of CBG must also translate into large differences in the free fractions of both cortisol and progesterone at the maternal interface at term, the physiological significance of this remains obscure, especially because there were no obvious differences in pregnancy outcomes or the health of neonates in CBG-deficient pregnant women as compared with women with normal plasma CBG levels Lei et al.
This latter observation is of interest because stress during pregnancy causes elevations in plasma cortisol, and has been associated with female skewing of the sex ratio at birth Navara , which may be exacerbated in the offspring of CBG-deficient women. It should also be noted that CBG may act differently in regulating progesterone and or cortisol bioavailability at human fetal—maternal interface in different species, including rodents, where the placenta does not make large amounts of progesterone.
Crystal structure analyses of human and rat CBG showed that the steroid-binding site is located close to the surface of the protein Klieber et al. In addition, numerous SerpinA6 mutations have been reported in animals that were used to study the actions of glucocorticoid in health and disease Moisan Knowledge of these CBG variants is important because the algorithms used to calculate free glucocorticoid concentrations in blood samples rely on the assumption that the steroid-binding affinities of CBG in a particular species are constant, and this highlights the need to develop accurate methods to directly determine free plasma glucocorticoid concentrations.
Recently, we have also found that plasma CBG deficiencies in different colonies of Sprague—Dawley rats, which are widely used in studies of glucocorticoid-dependent stress responses and inflammation, are associated with a greater sensitivity to an acute inflammatory challenge Bodnar et al.
A role for CBG in controlling the activities of glucocorticoids during infectious and inflammatory diseases was suspected from early studies that revealed dynamic reductions in plasma CBG levels in patients with acute infections, traumatic injuries, or severe inflammation Savu et al.
This also helps to understand why specific proteases target CBG and disrupt its ability to bind steroids, thereby facilitating the release of anti-inflammatory steroids at sites of tissue damage or inflammation Hammond et al. It is now known that several endogenous proteases, including neutrophil elastase Hammond et al. While the significance of RCL cleavage by chymotrypsin is unclear, neutrophil elastase and LasB are present at sites of inflammation and infection, and their ability to specifically target and cleave the RCL of CBG is thought to promote the release of glucocorticoids at these locations.
Plasma CBG levels decrease rapidly in patients and animals undergoing acute inflammation Savu et al. Furthermore, any increases in adrenal glucocorticoid production driven by adrenocorticotrophic hormone ACTH -mediated responses to stress can be expected to overwhelm the reduced CBG steroid-binding capacity and accentuate systemic increases in plasma free cortisol levels.
At some point during the recovery from inflammation, plasma CBG levels are expected to gradually rebound to maintain a normal homeostatic balance of plasma glucocorticoid levels and bioavailability. Importantly, this model provides a framework for studies of how plasma CBG might be used as a biomarker of the severity of inflammation, and the time course of infectious or acute inflammatory diseases, as well as studies of how pre-existing deficiencies in either the production of CBG or its steroid-binding properties might contribute to poor responses to these diseases.
Despite species differences in the way the SHBG gene is expressed in the liver during postnatal life, all mammals produce plasma SHBG during critical phases of gonadal and reproductive tract development Hammond In human blood, high SHBG levels during childhood likely serve to restrict the premature actions of sex steroids until SHBG declines in both sexes as puberty advances Hammond , Hammond et al.
The steroid occupancy of SHBG is further reduced in women taking oral contraceptives that promote large increases in serum SHBG levels, while simultaneously preventing ovarian sex steroid production. It is assumed that the increases in SHBG levels in women taking oral contraceptives reflect increased hepatic SHBG production, because similar five- to ten-fold increases in serum SHBG levels occur in concert with increases in estrogen levels during human pregnancy Anderson Increases in serum SHBG in women under these conditions undoubtedly influence the plasma distribution of both androgens and estrogens, and this property has been exploited therapeutically to reduce androgen exposures in women with symptoms of hyperandrogenism Dewis et al.
Although the significance of the large increases in maternal plasma SHBG is unclear, transient androgenization has been reported in a pregnant woman with a SHBG deficiency. The fact that androgenic symptoms resolved in this patient, postpartum, suggested that this may be due to an exposure to fetal adrenal androgens that escape placental metabolism, and which would normally be bound by elevated SHBG levels during pregnancy Hogeveen et al.
Our understanding of the structure and function of SHBG advanced considerably after the crystal structure of the N-terminal laminin G-like domain of SHBG was resolved in complex with a variety of sex steroid ligands Grishkovskaya et al. These high-resolution structures confirmed that androgens and estrogens interact competitively with the same steroid-binding site, but are positioned in opposite and inverted orientations Grishkovskaya et al.
The crystal structures also revealed the location of a calcium-binding site suspected as being essential for both the dimerization and steroid binding of SHBG, and provided insight into how the chelation of calcium in EDTA-treated plasma disrupts these critical structural and functional properties of the molecule Grishkovskaya et al.
In addition, they showed that a zinc atom, positioned at what appears to be an entrance to the steroid-binding site of human SHBG, reduces its affinity for estrogens specifically Avvakumov et al. This zinc-binding site of plasma SHBG is unlikely to be fully occupied because free zinc concentrations in plasma are very low, but it may be occupied in extravascular tissue compartments, such as the prostate and the male reproductive tract, where zinc levels are exceptionally high. It has been proposed that SHBG leaves the blood circulation in some tissues and interacts directly with proteins on the plasma membranes of specific cell types, and that this may contribute to either the delivery of SHBG-bound sex steroids via endocytotic mechanisms or to cell membrane receptor-mediated signaling Rosner et al.
We have been unable to confirm these observations, but our studies of mice expressing human SHBG transgenes have shown that SHBG does exit the blood vessels in some tissues, and accumulates within extracellular tissue compartments, such as the stroma of the endometrium and epididymis Ng et al. Moreover, we obtained evidence that this involves a steroid ligand-dependent interaction between SHBG and two members of the fibulin family of extracellular matrix-associated proteins, fibulin-1D and fibulin-2 Ng et al.
The biological significance of this remains to be determined, but it provides in vivo evidence that SHBG has the capacity to act in extravascular compartments, extending its functions beyond that of a transport protein that regulates free sex steroids levels in the blood.
Ever since it was realized that the proportions of free testosterone and estradiol in blood samples are inversely related to those of SHBG Anderson , serum SHBG and testosterone measurements have been used in algorithms to calculate free testosterone levels in patients with suspected hyperandrogenism or hypoandrogenism Vermeulen et al.
The mathematical models used to calculate free plasma androgen or estrogen levels currently rely on SHBG measurements obtained using immunoassays Vermeulen et al. These findings highlight the pressing need for sensitive mass spectrometric methods to measure both total and free sex steroid levels in the blood. This is emerging as an important issue because several other relatively common variations in the human SHBG coding sequence are linked to abnormal plasma SHBG levels Table 2.
They include a common non-synonymous SNP rs that causes a substitution of Asp with an Asn residue DN and creates an extra N-linked glycosylation site Power et al.
Individuals who carry the rs SNP have slightly elevated SHBG levels that have been negatively associated with the risk of developing breast cancer Forsti et al. There is also a growing awareness that abnormal plasma SHBG levels, and the subsequent changes in the plasma levels and distribution of sex steroids, are not only predictive of numerous clinical conditions, including low bone density Slemenda et al.
Despite the large number of SHBG measurements performed for diagnostic purposes, it is remarkable that there have been only two reports of complete SHBG deficiencies in humans. As expected, plasma testosterone concentrations in the male proband were well below the normal range, yet his free testosterone levels were normal.
Clinical assessments indicated fatigue, overt muscle weakness, and low body weight, and other symptoms of hypoandrogenism, but gonadal development and sperm production and function appeared to be normal.
However, it was noted that this phenotype might be related to possible consanguinity in this pedigree Vos et al. This provided the first explanation for why low plasma SHBG is a hallmark of the metabolic syndrome and its associated diseases, such as type 2 diabetes and cardiovascular disease Selva et al.
In this regard, it is interesting that luteinizing hormone LH levels rise in hyperthyroxinemic men Ruder et al. Thus, the low plasma levels of adiponectin that are typically seen in overweight individuals at risk of having the metabolic syndrome Kadowaki et al. Their effects on hepatic SHBG production may be influenced by increases in plasma adiponectin that in turn may increase SHBG production, as previously mentioned. This is probably relevant, because pioglitazone and other insulin sensitizers most likely alter the hepatic complement of these orphan NHRs, and these changes may certainly contribute to the modest increases in plasma SHBG levels observed after their administration.
Hormonal, pharmaceutical, metabolic, and nutritional modifiers of hepatic SHBG production. Plasma CBG and SHBG are structurally unrelated and function in very different ways that extend well beyond simple transportation or buffering functions in the blood.
Knowledge of the structures together with the identification of naturally occurring variants of CBG and SHBG provide additional insight into their production and functions. They also illustrate the limitations of current methods for measuring their plasma concentrations, which are used in algorithms to calculate free steroid levels, and highlight the need for more direct methods to measure plasma free steroid concentrations.
Recent insight into the molecular mechanisms responsible for regulation of hepatic CBG and SHBG production explain how abnormalities in their plasma levels are linked to the risk as well as the consequences of a variety of diseases related to abnormal steroid hormone exposures, and how they may be utilized as biomarkers of disease onset, severity, or recovery.
Finally, this review provides several arguments for why CBG and SHBG should be regarded as the primary gatekeepers of steroid hormone action in the blood and extravascular tissue compartments. The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review. This study received support from the Canada Research Chairs Program.
Support from the Canada Research Chairs Program is also gratefully acknowledged. National Center for Biotechnology Information , U.
The Journal of Endocrinology.