0163-769X/90/1101-0080I02.00/0 Endocrine Reviews Copyright © 1990 by The Endocrine Society
Vol. 11, No. 1 Printed in U.S.A.
The Functions of Corticosteroid-Binding Globulin and Sex Hormone-Binding Globulin: Recent Advances* WILLIAM ROSNER Department of Medicine, St. Luke's/Roosevelt Hospital Center, and Columbia University, College of Physicians and Surgeons, New York, New York 10019
binding proteins are trapped in the vascular compartment, wherein they provide a buffer reservoir of steroids that can be rapidly made available to the free pool of hormone by simple dissociation. We emphasize "possible" because it is not necessary that the plasma binding proteins' primary function be to serve as buffer reservoirs for the free hormone hypothesis to be correct. Nevertheless, it is undoubtedly this corollary that has caused a number of investigators to seek out other functions for CBG and SHBG. It is easy to conceive that CBG might have been designed to provide a buffer reservoir of cortisol. Cortisol is, after all, classically needed in times of stress, and there are a host of well-known biological mechanisms that activate the hypothalamic-pituitary-adrenocortical axis under these circumstances. Although not examined experimentally in great detail, mechanisms that decrease intracellular free hormone could be activated, so that plasma free cortisol could diffuse down a concentration gradient, into cells, with the subsequent rapid release of CBG-bound cortisol. Although there is little direct evidence to support this sequence of events in stress, neither is there a convincing counterexample. Indeed, in certain types of stress, CBG rapidly disappears from plasma (12, 13); although that observation can be interpreted in a number of ways, it is compatible with CBG freeing all of its bound hormone and making it rapidly available. A probable mechanism for the rapid disappearance of CBG has been published (14) and lends support to this view of the biology (see below). A difficulty with this scheme is that there does not seem to be a crying need for such a mechanism. The hypothalamic-pituitary-adrenal axis is a rapidly responding system, and the organism would gain only a little because of a readily accessible pool of CBG-bound cortisol, available quickly as a one-time spurt. However, there is a potential small biological profit in it. On the other hand, CBG binds progesterone as tightly as it does cortisol, and it is difficult to envision the necessity for having a rapidly available pool of this steroid. Further, the immediate availability of the ste-
Introduction
T
he presence of corticosteroid-binding globulin (CBG) in plasma was detected about 30 yr ago, and of sex hormone-binding globulin (SHBG) about 20 yr ago. In the interval since their discovery, sophisticated binding and RIAs have been devised for their measurement; their concentrations in plasma and other biological fluids, from milk to saliva, have been measured in myriad clinical conditions in humans, and numerous experimental ones in animals; their phyletic distribution has been examined; they have been isolated and their complementary DNAs (cDNAs) cloned; their binding specificities for steroids have been examined in heroic detail; hormonal and drug influences on their plasma concentration in vivo, and their secretion by liver cells in vitro, have been widely explored; they have been the subject of two long, splendid monographs (1, 2) and many shorter reviews (3-11) [wherein the original citations for the first part of this sentence may be found]—and yet we still have not come to a consensus on what it is that they do. Undeniably, they bind certain steroids. But to what biological end? Although there always have been dissenters, the consensus generally has been that only the nonprotein bound, i.e. free, steroid hormones are available for movement out of capillaries and into cells, where they may either initiate a biological response or be cleared from the circulation via a variety of metabolic pathways. This mechanism has been termed the free hormone hypothesis; it has been debated systematically over the last 7-8 yr and, of late, has been the theme of a vigorous debate. This topic is the subject of a separate review in this journal (lla) and will be touched on only briefly, as necessary, in this analysis. A possible corollary to the free hormone hypothesis is that the plasma steroid hormoneAddress requests for reprints to: William Rosner, M.D., St. Luke's/ Roosevelt Hospital Center, 428 West 59th Street, New York, New York 10019. * This work was supported by NIH Grants DK-28562 and DK36714.
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February, 1990
FUNCTIONS OF CBG AND SHBG
roids that are bound to SHBG, testosterone, dihydrotestosterone, and estradiol, would seem to offer no advantage to the organism. This last argument, although strong, is not destructive to those who argue for the importance of CBG as an easily available reservoir of cortisol. To state the obvious, SHBG is not CBG, and the hormones that they bind carry out vastly different roles. If CBG and SHBG have functions in addition to regulating the steady state free hormone concentration in plasma, what might they be?
I. The Regulation of the Plasma Concentration of SHBG and CBG As indicated in Introduction, there has been a lively argument concerning the free hormone hypothesis over the past several years. That free steroids can enter cells and initiate hormone actions is incontrovertible; the disagreements center about the quantitative roles of free and bound hormone. As detailed in the review by Mendel (lla), the standard model of free steroid diffusing from the plasma compartment to initiate hormone action is a paradigm that probably serves most steroids in most tissues. That the steroid-binding proteins are the pivotal, albeit not the sole, regulators of the concentration of free steroids is also incontrovertible. Thus, variations in the plasma concentration of these two proteins are, in turn, central to the determination of the concentration of free steroids in plasma, and a brief discussion of their regulation is in order. Although, from the time of their initial discovery, it was believed that CBG and SHBG were synthesized and secreted by the liver; it was not until years later that the hepatic secretion and synthesis of CBG were demonstrated. Weiser et al. (15) showed that rat liver slices, incubated in vitro, synthesized a protein that had the same binding specificity as, and migrated on polyacrylamide gel electrophoresis with, CBG. Subsequently CBG messenger mRNA (mRNA) was demonstrated in the liver of guinea pigs (16) and rats (17), and CBG's secretion by Hep G2 cells was shown (18). At about the same time as these observations regarding CBG were occurring, SHBG also was shown to be secreted by the transformed human liver cell line, Hep G2 (19). Most recently cDNA clones coding for SHBG have been isolated from liver cDNA libraries (20-22). Similarly cDNA clones coding for CBG have been isolated from liver and lung cDNA libraries (23). (The physiological significance of the pulmonary cDNA clone has yet to be determined.) Thus, all the tools are in place to examine the regulation of the synthesis and secretion of both SHBG and CBG, which, together with information on their disposal, will
81
yield an understanding of the regulation of their concentration in plasma. The preceding developments took place against the background of a large body of literature detailing the effects of a variety of substances and clinical conditions on the plasma concentration of both CBG and SHBG (Table). After the initial demonstration (19) of the secretion of SHBG by Hep G2 cells, important information on substances that directly inhibit or stimulate SHBG secretion came to light. Thyroid hormones, at physiological concentrations, cause increased secretion of SHBG by Hep G2 cells in vitro (24-26). These observations explain the very powerful effect of thyrotoxic states in increasing plasma SHBG. The long-known effect of the increase in plasma SHBG secondary to estrogens is also seen after Hep G2 cells are treated with estrogens (2527). However, the required concentration is so markedly supraphysiological that the question arises as to whether the mechanism of the striking increase in SHBG in vivo is due to increased synthesis and secretion. An alternative hypothesis, and evidence to support it, has been put forth for the increase in plasma T4-binding globulin (TBG) seen after estrogen administration and in pregnancy (28). Estrogens result in a greater degree of sialylation of the carbohydrate moiety of TBG, and these more heavily sialylated isoforms are cleared from the plasma more slowly than desialylated ones. Thus, the TABLE 1. Factors impacting on the concentration of SHBG in plasma Refs. Increased SHBG Increased thyroid hormone Increased estrogens Pregnancy Luteal phase of menstrual cycle Exogenous estrogens Cirrhosis of the liver Phenytoin (Dilantin) Tamoxifen Prolonged stress Carcinoma of the prostate Anorexia nervosa Aging in men High carbohydrate diet Decreased SHBG Obesity Syndromes of androgenization in women (PCOS, hirsutism, acne) Testosterone treatment in normal and hypogonadal men Hyperprolactinemia Increased GH Menopause Progestational agents Danazol Glucocorticoids
(30, 32, 95, 96) (30, 97-100) (101, 102) (97, 103-105) (98, 106) (107) (108) (109) (110, 111) (111) (104, 112, 113) (40) (30, 114-117) (30-32, 118)
(33) (26, 34, 35) (30, 119) (120-122) (Ref. 6, p 208) (123-125) (30, 126, 127)
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ROSNER
increased plasma TBG is attributed to an ultimate effect of estrogens on clearance, rather than on synthesis and secretion. Since SHBG is a sialic acid-containing glycoprotein (29), the same mechanism could apply. Surprisingly, it has also been observed (27), and confirmed (26), that androgens, which lower plasma SHBG in vivo (3033), increase the synthesis and secretion of SHBG in vitro. Thus, the association of hyperandrogenic clinical conditions (see Table 1) with a depressed SHBG must be due to either increased clearance or to an as yet to be accounted for mechanism. The [reversible] decline in SHBG seen in hyperprolactinemia (26, 34, 35) has also been duplicated by incubating Hep G2 cells with PRL (26). Finally, insulin has been shown to profoundly lower SHBG secretion in vitro and to block the stimulatory effect of both T4 and estradiol (26). This is a most interesting result because, before it was obtained, there were no reasonable hypotheses available to explain the vanishingly small level of SHBG seen in massive obesity. Obese individuals, including many with polycystic ovarian syndrome, are often hyperinsulinemic; the speculation that it is too much insulin that is responsible for their depressed plasma SHBG is reasonable but remains to be proven. Clearly, the topic is complex, but a challenging beginning has been made. In contrast to the multiplicity of specific agents that affect plasma SHBG, plasma CBG appears impervious to most specific influences; however, it is altered in a number of pathological states. There is almost no underlying biological logic that connects these conditions. Hence, we are left with a potpourri of conditions wherein CBG is altered, but, for the most part, no fundamental common mechanism for these changes is discernible. Like SHBG, estrogens whether endogenous (pregnancy) or exogenous cause a substantial increase in CBG (36, 37). Unlike SHBG there is no difference in plasma CBG concentration between men and women, and it does not vary during the menstrual cycle. Pharmacological amounts of glucocorticoids, whether endogenous (Cushing's syndrome) or exogenous, result in a depressed CBG (38, 39). As is the case for SHBG, there are modest dietary influences on CBG (40). However, whereas a high carbohydrate diet causes a 35-40% increase in plasma SHBG, plasma CBG falls by about 20% under the same conditions (40). As already mentioned plasma CBG decreases drastically in septic shock (12,13). Doe et al. (41) made the interesting observation that CBG was depressed in pernicious anemia, but not folate deficiency, and that treatment of the disease with vitamin B12 reversed the change within 2 weeks. They speculated about the possible importance of vitamin B i2 in hepatic CBG synthesis. De Moor and his colleagues (42, 43) have examined plasma CBG in a variety of hematological disorders and found abnormally high concentrations of
Vol. 11, No. 1
CBG in about 15% of these. The major abnormalities occurred in malignant lymphoproliferative disorders, particularly acute lymphoblastic leukemia. There are other scattered reports of relatively unimpressive changes in CBG, and these have been reviewed recently (44). Although CBG has been shown to be secreted by the Hep G2 cell line (18), unlike the situation for SHBG, data are not yet available to assist in finding the common thread among the foregoing. It is worth noting, however, that unlike TBG (28), the difference in glycosylation between CBG isolated from nonpregnant and pregnant rabbits does not effect its rate of clearance from plasma (44a).
II. Models That Do Not Require the Interaction of the Binding Proteins with Cells A. SHBG functions to regulate androgen-estrogen balance
Because testosterone binds more tightly to SHBG than does estradiol, while estradiol binds more tightly to albumin than does testosterone, it would be expected that changes in the concentration of SHBG would differentially affect the free concentration of these steroids. Experimental evidence confirmed this expectation (45) and led to the formulation of the model illustrated in Fig. 1 (from Refs. 11 and 45). It is apparent from the figure that this model predicts that, all other things being equal, the ratio of free testosterone to free estradiol is altered as the concentration of SHBG is changed. The biological importance of this change depends upon whether androgen or estrogen target tissues respond primarily to the absolute concentration of these steroids or to their ratio. A response to their ratio forms the central assumption in the concept of "androgen-estrogen balance," a widely quoted notion used to explain many poorly understood biological observations. For instance, the fact that individuals with testicular feminization have female-sized breasts has been attributed to the fact that normal male circulating levels of estrogens can cause this to occur in the face of complete androgen resistance. The difficulty with this attractive concept is that there is little direct evidence to support it, e.g. full doseresponse curves measuring an androgen response in the presence of varying amounts of estrogen or vice versa. Whether or not the concept of androgen-estrogen balance is correct, the instantaneous effect of changing the concentration of SHBG differentially affects the concentration of free estradiol and testosterone. If the free hormone hypothesis is correct, however, what follows is probably different in men and women. For instance, an increase in SHBG leads to a proportionately greater fall in free testosterone than in free estradiol as illustrated in Fig. 1A. In men this would be followed by increased testosterone secretion; in women, whose circulating tes-
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FUNCTIONS OF CBG AND SHBG
February, 1990 Unbound testosterone
Unbound oestrodiol
A. Oestrogen servo
— Oesfradiol Increases SHBG
hormone available to them. The model has the appeal of allowing differential amounts of steroid to be made available at different sites, in the face of a concentration of free hormone that is the same everywhere in the arterial tree. As indicated above, the topic is reviewed in detail elsewhere in the symposium on plasma binding-proteins (11a). C. CBG delivers bound cortisol to sites of inflammation
B. Balance
n
C. Androgen servo
Tes-tosterone
83
•-
Decreases SHBG
FIG. 1. The proposed role of SHBG in regulating relative unbound testosterone (T) and estradiol (E2) concentrations. SHBG is denoted by the fulcrum, and unbound T and E2 by the boxes on each end of the see-saw. A, Increased E2 production; unbound E2 rises, and by stimulating SHBG production causes a differential fall in unbound T. Here SHBG change acts as an estrogen servo. B, Balance. C, Increased androgen production. Unbound T rises, and effect is then amplified by a fall in SHBG, which tips the balance further in favor of T (androgen servo). Normal adult female situation = A; normal adult male situation = C. Any third factor (such as thyroid hormones) which alters the SHBG concentration will also be expected to alter the ratio of unbound T to E2. [Reproduced with permission from Refs. 45 and 11.]
tosterone concentration does not contribute to regulation of the hypothalamic-pituitary-gonadal axis, such compensation would not occur. An analogous argument could be made for situations involving alterations of free estrogens in men. Thus, although not explicit in this model, it is clear that changes in free hormone ratios are differentially compensated for in men and women. On the whole, it seems doubtful that SHBG evolved to regulate androgen-estrogen balance. B. SHBG and CBG selectively deliver their bound steroids to tissues This model postulates that both SHBG and CBG are subject to express forces in the microcirculation that promote the dissociation of bound steroids (46). These forces are not distributed uniformly in the microcirculation of all organs, thus furnishing a mechanism whereby specific tissues can help regulate the amount of steroid
Human CBG cDNA has been cloned from liver, and its primary structure has been deduced (23). Although the protein was not expressed and shown to bind cortisol, the evidence that the correct cDNA was obtained was quite strong, and included an exact match with the known N-terminal sequence of CBG. Several important facts emerged from comparing the sequence to large protein data bases. There was no sequence homology with any steroid hormone receptor, or with SHBG. However, there was a greater than 30% homology with the serpin (serine protease inhibitor) superfamily of proteins. The proteins in this family share a typical tertiary structure that accounts for their marked inhibition of serine proteases (47). They also share the property that they are cleaved by their target enzymes. When CBG is incubated with neutrophil elastase (14), a serine protease that is released by neutrophils in large amounts at sites of inflammation, there is a fairly quick release of a 4K peptide from its sequence, homologous to that released by other serpins upon incubation with an appropriate serine protease. The enzymatic cleavage of CBG leads to a 10-fold decrease (4 C) in CBG's affinity for cortisol (14) (Fig. 2, from Ref. 14). These data are consistent with a model that would allow the rapid delivery of cortisol to sites of inflammation by a mechanism involving specific cleavage of CBG by neutrophil elastase, followed by a decrease in CBG's association constant for cortisol, and hence relatively more free cortisol. These observations are brand-new, and their physiological role needs further exploration. Is this a one-time rapid "dumping" of cortisol at an inflammatory site? If so, not much would be accomplished. On the other hand, enlisting CBG-bound cortisol to serve as a reservoir to permit the free cortisol concentration to remain high at sites of inflammation, while maintaining basal free cortisol concentrations elsewhere, is an attractive way to integrate these experiments into a general view of CBG's function. This ability of CBG to fractionate the availability of cortisol on a site by site basis is a long-standing hypothesis (48,49) with which these observations are consistent. III. Models That Require That CBG and SHBG Interact with Cells The possibility, that the view that CBG and SHBG served only a reservoir function was mistaken, was hinted
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ROSNER
84
Vol. 11, No. 1 Native CBG K a =1.4xi0 9 M" 1
35
FIG. 2. Scatchard plot for CBG showing the decrease in cortisol binding affinity subsequent to cleavage. Equilibrium dialysis was performed at 4 C in phosphate buffer, pH 7.4. Dialysis bags contained 3 ml CBG (70 nM), and the outer phase (10 ml) contained [l,2-3H]cortisol (20 nCi ml"1) and unlabeled cortisol (1-22 nM). After equilibrium with shaking for 72 h, aliquots of inside and outside phases were counted for radioactivity. Results were corrected for nonspecific binding in the presence of a 100-fold molar excess of cortisol over CBG and for the presence of endogenous cortisol in the CBG preparation. [Reproduced with permission from P. A. Pembertow et at: Nature 336:275, 1988 (14).]
30
25
35 2 0
15
10
Cleaved CBG K a =1.6x10 8 M" 1
0.5
1.0
15
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Bound cortisol (10"8M)
at by immunocytochemical and biochemical data purporting to demonstrate the intracellular presence of SHBG and CBG in various androgen, estrogen, glucocorticoid, and progesterone target organs. In the past 12 yr there have been reports on the immunocytochemical demonstration: of SHBG in monkey prostate, epididymis, and testis (50), in human testis and epididymis (51), and in mammary carcinoma cells (52); and of CBG in liver (53), uterus (54), kidney (55), lymphocytes (55), a variety of rat tissues (56), and in those pituitary cells (corticotropes) that secrete ACTH (57). A number of biochemically based studies also supported the presence of CBG or a CBG-like binder in various glucocorticoid and progesterone target organs: pituitary (58, 59); kidney (60); uterus (61-63); muscle (64); lung (65); breast cancer (66); and lymphocytes (55). An SHBG-like binder was also reported to be present in human prostatic cytosol (67, 68). Some of these reports were flawed by the omission of critical controls and/or a lack of evidence that the observations were not due to contamination with plasma, but the "where there is smoke, there is fire" approach to discovery encouraged a number of investigators to pursue alternatives to the reservoir model of the serum steroid-binding proteins. A. CBG interacts with membranes The bulk of reports on the presence of intracellular CBG finally set off a search for a mechanism for its entry into cells. Strel'chyonok et al. (69) attempted to examine the binding of CBG to hepatic membranes, but inexpl-
icably used [125I]asialoCBG as a probe rather than [125I] CBG. The data in that report showed specific binding of [125I]asialoCBG to human hepatic membranes. However, native CBG was almost 4 orders of magnitude less effective than asialoCBG itself in the displacement of [125I] asialoCBG. Thus, the observed binding activity could have been attributed most reasonably to the interaction of [125I]asialoCBG with the well-known liver membrane asialoglycoprotein receptor (70). Displacement of the [125I]asialoCBG could easily have been due to a trace (
Vol. 11, No. 1 Printed in U.S.A.
The Functions of Corticosteroid-Binding Globulin and Sex Hormone-Binding Globulin: Recent Advances* WILLIAM ROSNER Department of Medicine, St. Luke's/Roosevelt Hospital Center, and Columbia University, College of Physicians and Surgeons, New York, New York 10019
binding proteins are trapped in the vascular compartment, wherein they provide a buffer reservoir of steroids that can be rapidly made available to the free pool of hormone by simple dissociation. We emphasize "possible" because it is not necessary that the plasma binding proteins' primary function be to serve as buffer reservoirs for the free hormone hypothesis to be correct. Nevertheless, it is undoubtedly this corollary that has caused a number of investigators to seek out other functions for CBG and SHBG. It is easy to conceive that CBG might have been designed to provide a buffer reservoir of cortisol. Cortisol is, after all, classically needed in times of stress, and there are a host of well-known biological mechanisms that activate the hypothalamic-pituitary-adrenocortical axis under these circumstances. Although not examined experimentally in great detail, mechanisms that decrease intracellular free hormone could be activated, so that plasma free cortisol could diffuse down a concentration gradient, into cells, with the subsequent rapid release of CBG-bound cortisol. Although there is little direct evidence to support this sequence of events in stress, neither is there a convincing counterexample. Indeed, in certain types of stress, CBG rapidly disappears from plasma (12, 13); although that observation can be interpreted in a number of ways, it is compatible with CBG freeing all of its bound hormone and making it rapidly available. A probable mechanism for the rapid disappearance of CBG has been published (14) and lends support to this view of the biology (see below). A difficulty with this scheme is that there does not seem to be a crying need for such a mechanism. The hypothalamic-pituitary-adrenal axis is a rapidly responding system, and the organism would gain only a little because of a readily accessible pool of CBG-bound cortisol, available quickly as a one-time spurt. However, there is a potential small biological profit in it. On the other hand, CBG binds progesterone as tightly as it does cortisol, and it is difficult to envision the necessity for having a rapidly available pool of this steroid. Further, the immediate availability of the ste-
Introduction
T
he presence of corticosteroid-binding globulin (CBG) in plasma was detected about 30 yr ago, and of sex hormone-binding globulin (SHBG) about 20 yr ago. In the interval since their discovery, sophisticated binding and RIAs have been devised for their measurement; their concentrations in plasma and other biological fluids, from milk to saliva, have been measured in myriad clinical conditions in humans, and numerous experimental ones in animals; their phyletic distribution has been examined; they have been isolated and their complementary DNAs (cDNAs) cloned; their binding specificities for steroids have been examined in heroic detail; hormonal and drug influences on their plasma concentration in vivo, and their secretion by liver cells in vitro, have been widely explored; they have been the subject of two long, splendid monographs (1, 2) and many shorter reviews (3-11) [wherein the original citations for the first part of this sentence may be found]—and yet we still have not come to a consensus on what it is that they do. Undeniably, they bind certain steroids. But to what biological end? Although there always have been dissenters, the consensus generally has been that only the nonprotein bound, i.e. free, steroid hormones are available for movement out of capillaries and into cells, where they may either initiate a biological response or be cleared from the circulation via a variety of metabolic pathways. This mechanism has been termed the free hormone hypothesis; it has been debated systematically over the last 7-8 yr and, of late, has been the theme of a vigorous debate. This topic is the subject of a separate review in this journal (lla) and will be touched on only briefly, as necessary, in this analysis. A possible corollary to the free hormone hypothesis is that the plasma steroid hormoneAddress requests for reprints to: William Rosner, M.D., St. Luke's/ Roosevelt Hospital Center, 428 West 59th Street, New York, New York 10019. * This work was supported by NIH Grants DK-28562 and DK36714.
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February, 1990
FUNCTIONS OF CBG AND SHBG
roids that are bound to SHBG, testosterone, dihydrotestosterone, and estradiol, would seem to offer no advantage to the organism. This last argument, although strong, is not destructive to those who argue for the importance of CBG as an easily available reservoir of cortisol. To state the obvious, SHBG is not CBG, and the hormones that they bind carry out vastly different roles. If CBG and SHBG have functions in addition to regulating the steady state free hormone concentration in plasma, what might they be?
I. The Regulation of the Plasma Concentration of SHBG and CBG As indicated in Introduction, there has been a lively argument concerning the free hormone hypothesis over the past several years. That free steroids can enter cells and initiate hormone actions is incontrovertible; the disagreements center about the quantitative roles of free and bound hormone. As detailed in the review by Mendel (lla), the standard model of free steroid diffusing from the plasma compartment to initiate hormone action is a paradigm that probably serves most steroids in most tissues. That the steroid-binding proteins are the pivotal, albeit not the sole, regulators of the concentration of free steroids is also incontrovertible. Thus, variations in the plasma concentration of these two proteins are, in turn, central to the determination of the concentration of free steroids in plasma, and a brief discussion of their regulation is in order. Although, from the time of their initial discovery, it was believed that CBG and SHBG were synthesized and secreted by the liver; it was not until years later that the hepatic secretion and synthesis of CBG were demonstrated. Weiser et al. (15) showed that rat liver slices, incubated in vitro, synthesized a protein that had the same binding specificity as, and migrated on polyacrylamide gel electrophoresis with, CBG. Subsequently CBG messenger mRNA (mRNA) was demonstrated in the liver of guinea pigs (16) and rats (17), and CBG's secretion by Hep G2 cells was shown (18). At about the same time as these observations regarding CBG were occurring, SHBG also was shown to be secreted by the transformed human liver cell line, Hep G2 (19). Most recently cDNA clones coding for SHBG have been isolated from liver cDNA libraries (20-22). Similarly cDNA clones coding for CBG have been isolated from liver and lung cDNA libraries (23). (The physiological significance of the pulmonary cDNA clone has yet to be determined.) Thus, all the tools are in place to examine the regulation of the synthesis and secretion of both SHBG and CBG, which, together with information on their disposal, will
81
yield an understanding of the regulation of their concentration in plasma. The preceding developments took place against the background of a large body of literature detailing the effects of a variety of substances and clinical conditions on the plasma concentration of both CBG and SHBG (Table). After the initial demonstration (19) of the secretion of SHBG by Hep G2 cells, important information on substances that directly inhibit or stimulate SHBG secretion came to light. Thyroid hormones, at physiological concentrations, cause increased secretion of SHBG by Hep G2 cells in vitro (24-26). These observations explain the very powerful effect of thyrotoxic states in increasing plasma SHBG. The long-known effect of the increase in plasma SHBG secondary to estrogens is also seen after Hep G2 cells are treated with estrogens (2527). However, the required concentration is so markedly supraphysiological that the question arises as to whether the mechanism of the striking increase in SHBG in vivo is due to increased synthesis and secretion. An alternative hypothesis, and evidence to support it, has been put forth for the increase in plasma T4-binding globulin (TBG) seen after estrogen administration and in pregnancy (28). Estrogens result in a greater degree of sialylation of the carbohydrate moiety of TBG, and these more heavily sialylated isoforms are cleared from the plasma more slowly than desialylated ones. Thus, the TABLE 1. Factors impacting on the concentration of SHBG in plasma Refs. Increased SHBG Increased thyroid hormone Increased estrogens Pregnancy Luteal phase of menstrual cycle Exogenous estrogens Cirrhosis of the liver Phenytoin (Dilantin) Tamoxifen Prolonged stress Carcinoma of the prostate Anorexia nervosa Aging in men High carbohydrate diet Decreased SHBG Obesity Syndromes of androgenization in women (PCOS, hirsutism, acne) Testosterone treatment in normal and hypogonadal men Hyperprolactinemia Increased GH Menopause Progestational agents Danazol Glucocorticoids
(30, 32, 95, 96) (30, 97-100) (101, 102) (97, 103-105) (98, 106) (107) (108) (109) (110, 111) (111) (104, 112, 113) (40) (30, 114-117) (30-32, 118)
(33) (26, 34, 35) (30, 119) (120-122) (Ref. 6, p 208) (123-125) (30, 126, 127)
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ROSNER
increased plasma TBG is attributed to an ultimate effect of estrogens on clearance, rather than on synthesis and secretion. Since SHBG is a sialic acid-containing glycoprotein (29), the same mechanism could apply. Surprisingly, it has also been observed (27), and confirmed (26), that androgens, which lower plasma SHBG in vivo (3033), increase the synthesis and secretion of SHBG in vitro. Thus, the association of hyperandrogenic clinical conditions (see Table 1) with a depressed SHBG must be due to either increased clearance or to an as yet to be accounted for mechanism. The [reversible] decline in SHBG seen in hyperprolactinemia (26, 34, 35) has also been duplicated by incubating Hep G2 cells with PRL (26). Finally, insulin has been shown to profoundly lower SHBG secretion in vitro and to block the stimulatory effect of both T4 and estradiol (26). This is a most interesting result because, before it was obtained, there were no reasonable hypotheses available to explain the vanishingly small level of SHBG seen in massive obesity. Obese individuals, including many with polycystic ovarian syndrome, are often hyperinsulinemic; the speculation that it is too much insulin that is responsible for their depressed plasma SHBG is reasonable but remains to be proven. Clearly, the topic is complex, but a challenging beginning has been made. In contrast to the multiplicity of specific agents that affect plasma SHBG, plasma CBG appears impervious to most specific influences; however, it is altered in a number of pathological states. There is almost no underlying biological logic that connects these conditions. Hence, we are left with a potpourri of conditions wherein CBG is altered, but, for the most part, no fundamental common mechanism for these changes is discernible. Like SHBG, estrogens whether endogenous (pregnancy) or exogenous cause a substantial increase in CBG (36, 37). Unlike SHBG there is no difference in plasma CBG concentration between men and women, and it does not vary during the menstrual cycle. Pharmacological amounts of glucocorticoids, whether endogenous (Cushing's syndrome) or exogenous, result in a depressed CBG (38, 39). As is the case for SHBG, there are modest dietary influences on CBG (40). However, whereas a high carbohydrate diet causes a 35-40% increase in plasma SHBG, plasma CBG falls by about 20% under the same conditions (40). As already mentioned plasma CBG decreases drastically in septic shock (12,13). Doe et al. (41) made the interesting observation that CBG was depressed in pernicious anemia, but not folate deficiency, and that treatment of the disease with vitamin B12 reversed the change within 2 weeks. They speculated about the possible importance of vitamin B i2 in hepatic CBG synthesis. De Moor and his colleagues (42, 43) have examined plasma CBG in a variety of hematological disorders and found abnormally high concentrations of
Vol. 11, No. 1
CBG in about 15% of these. The major abnormalities occurred in malignant lymphoproliferative disorders, particularly acute lymphoblastic leukemia. There are other scattered reports of relatively unimpressive changes in CBG, and these have been reviewed recently (44). Although CBG has been shown to be secreted by the Hep G2 cell line (18), unlike the situation for SHBG, data are not yet available to assist in finding the common thread among the foregoing. It is worth noting, however, that unlike TBG (28), the difference in glycosylation between CBG isolated from nonpregnant and pregnant rabbits does not effect its rate of clearance from plasma (44a).
II. Models That Do Not Require the Interaction of the Binding Proteins with Cells A. SHBG functions to regulate androgen-estrogen balance
Because testosterone binds more tightly to SHBG than does estradiol, while estradiol binds more tightly to albumin than does testosterone, it would be expected that changes in the concentration of SHBG would differentially affect the free concentration of these steroids. Experimental evidence confirmed this expectation (45) and led to the formulation of the model illustrated in Fig. 1 (from Refs. 11 and 45). It is apparent from the figure that this model predicts that, all other things being equal, the ratio of free testosterone to free estradiol is altered as the concentration of SHBG is changed. The biological importance of this change depends upon whether androgen or estrogen target tissues respond primarily to the absolute concentration of these steroids or to their ratio. A response to their ratio forms the central assumption in the concept of "androgen-estrogen balance," a widely quoted notion used to explain many poorly understood biological observations. For instance, the fact that individuals with testicular feminization have female-sized breasts has been attributed to the fact that normal male circulating levels of estrogens can cause this to occur in the face of complete androgen resistance. The difficulty with this attractive concept is that there is little direct evidence to support it, e.g. full doseresponse curves measuring an androgen response in the presence of varying amounts of estrogen or vice versa. Whether or not the concept of androgen-estrogen balance is correct, the instantaneous effect of changing the concentration of SHBG differentially affects the concentration of free estradiol and testosterone. If the free hormone hypothesis is correct, however, what follows is probably different in men and women. For instance, an increase in SHBG leads to a proportionately greater fall in free testosterone than in free estradiol as illustrated in Fig. 1A. In men this would be followed by increased testosterone secretion; in women, whose circulating tes-
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FUNCTIONS OF CBG AND SHBG
February, 1990 Unbound testosterone
Unbound oestrodiol
A. Oestrogen servo
— Oesfradiol Increases SHBG
hormone available to them. The model has the appeal of allowing differential amounts of steroid to be made available at different sites, in the face of a concentration of free hormone that is the same everywhere in the arterial tree. As indicated above, the topic is reviewed in detail elsewhere in the symposium on plasma binding-proteins (11a). C. CBG delivers bound cortisol to sites of inflammation
B. Balance
n
C. Androgen servo
Tes-tosterone
83
•-
Decreases SHBG
FIG. 1. The proposed role of SHBG in regulating relative unbound testosterone (T) and estradiol (E2) concentrations. SHBG is denoted by the fulcrum, and unbound T and E2 by the boxes on each end of the see-saw. A, Increased E2 production; unbound E2 rises, and by stimulating SHBG production causes a differential fall in unbound T. Here SHBG change acts as an estrogen servo. B, Balance. C, Increased androgen production. Unbound T rises, and effect is then amplified by a fall in SHBG, which tips the balance further in favor of T (androgen servo). Normal adult female situation = A; normal adult male situation = C. Any third factor (such as thyroid hormones) which alters the SHBG concentration will also be expected to alter the ratio of unbound T to E2. [Reproduced with permission from Refs. 45 and 11.]
tosterone concentration does not contribute to regulation of the hypothalamic-pituitary-gonadal axis, such compensation would not occur. An analogous argument could be made for situations involving alterations of free estrogens in men. Thus, although not explicit in this model, it is clear that changes in free hormone ratios are differentially compensated for in men and women. On the whole, it seems doubtful that SHBG evolved to regulate androgen-estrogen balance. B. SHBG and CBG selectively deliver their bound steroids to tissues This model postulates that both SHBG and CBG are subject to express forces in the microcirculation that promote the dissociation of bound steroids (46). These forces are not distributed uniformly in the microcirculation of all organs, thus furnishing a mechanism whereby specific tissues can help regulate the amount of steroid
Human CBG cDNA has been cloned from liver, and its primary structure has been deduced (23). Although the protein was not expressed and shown to bind cortisol, the evidence that the correct cDNA was obtained was quite strong, and included an exact match with the known N-terminal sequence of CBG. Several important facts emerged from comparing the sequence to large protein data bases. There was no sequence homology with any steroid hormone receptor, or with SHBG. However, there was a greater than 30% homology with the serpin (serine protease inhibitor) superfamily of proteins. The proteins in this family share a typical tertiary structure that accounts for their marked inhibition of serine proteases (47). They also share the property that they are cleaved by their target enzymes. When CBG is incubated with neutrophil elastase (14), a serine protease that is released by neutrophils in large amounts at sites of inflammation, there is a fairly quick release of a 4K peptide from its sequence, homologous to that released by other serpins upon incubation with an appropriate serine protease. The enzymatic cleavage of CBG leads to a 10-fold decrease (4 C) in CBG's affinity for cortisol (14) (Fig. 2, from Ref. 14). These data are consistent with a model that would allow the rapid delivery of cortisol to sites of inflammation by a mechanism involving specific cleavage of CBG by neutrophil elastase, followed by a decrease in CBG's association constant for cortisol, and hence relatively more free cortisol. These observations are brand-new, and their physiological role needs further exploration. Is this a one-time rapid "dumping" of cortisol at an inflammatory site? If so, not much would be accomplished. On the other hand, enlisting CBG-bound cortisol to serve as a reservoir to permit the free cortisol concentration to remain high at sites of inflammation, while maintaining basal free cortisol concentrations elsewhere, is an attractive way to integrate these experiments into a general view of CBG's function. This ability of CBG to fractionate the availability of cortisol on a site by site basis is a long-standing hypothesis (48,49) with which these observations are consistent. III. Models That Require That CBG and SHBG Interact with Cells The possibility, that the view that CBG and SHBG served only a reservoir function was mistaken, was hinted
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ROSNER
84
Vol. 11, No. 1 Native CBG K a =1.4xi0 9 M" 1
35
FIG. 2. Scatchard plot for CBG showing the decrease in cortisol binding affinity subsequent to cleavage. Equilibrium dialysis was performed at 4 C in phosphate buffer, pH 7.4. Dialysis bags contained 3 ml CBG (70 nM), and the outer phase (10 ml) contained [l,2-3H]cortisol (20 nCi ml"1) and unlabeled cortisol (1-22 nM). After equilibrium with shaking for 72 h, aliquots of inside and outside phases were counted for radioactivity. Results were corrected for nonspecific binding in the presence of a 100-fold molar excess of cortisol over CBG and for the presence of endogenous cortisol in the CBG preparation. [Reproduced with permission from P. A. Pembertow et at: Nature 336:275, 1988 (14).]
30
25
35 2 0
15
10
Cleaved CBG K a =1.6x10 8 M" 1
0.5
1.0
15
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Bound cortisol (10"8M)
at by immunocytochemical and biochemical data purporting to demonstrate the intracellular presence of SHBG and CBG in various androgen, estrogen, glucocorticoid, and progesterone target organs. In the past 12 yr there have been reports on the immunocytochemical demonstration: of SHBG in monkey prostate, epididymis, and testis (50), in human testis and epididymis (51), and in mammary carcinoma cells (52); and of CBG in liver (53), uterus (54), kidney (55), lymphocytes (55), a variety of rat tissues (56), and in those pituitary cells (corticotropes) that secrete ACTH (57). A number of biochemically based studies also supported the presence of CBG or a CBG-like binder in various glucocorticoid and progesterone target organs: pituitary (58, 59); kidney (60); uterus (61-63); muscle (64); lung (65); breast cancer (66); and lymphocytes (55). An SHBG-like binder was also reported to be present in human prostatic cytosol (67, 68). Some of these reports were flawed by the omission of critical controls and/or a lack of evidence that the observations were not due to contamination with plasma, but the "where there is smoke, there is fire" approach to discovery encouraged a number of investigators to pursue alternatives to the reservoir model of the serum steroid-binding proteins. A. CBG interacts with membranes The bulk of reports on the presence of intracellular CBG finally set off a search for a mechanism for its entry into cells. Strel'chyonok et al. (69) attempted to examine the binding of CBG to hepatic membranes, but inexpl-
icably used [125I]asialoCBG as a probe rather than [125I] CBG. The data in that report showed specific binding of [125I]asialoCBG to human hepatic membranes. However, native CBG was almost 4 orders of magnitude less effective than asialoCBG itself in the displacement of [125I] asialoCBG. Thus, the observed binding activity could have been attributed most reasonably to the interaction of [125I]asialoCBG with the well-known liver membrane asialoglycoprotein receptor (70). Displacement of the [125I]asialoCBG could easily have been due to a trace (
