Steroids 82 (2014) 14–22

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Steroids

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Review

Steroid signaling: Ligand-binding promiscuity, molecular symmetry,and the need for gating

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⇑ Corresponding author at: Pieta Research, PO Box 27069, Edinburgh EH10 5YW, UK. Tel.: +44 131 478 0684.E-mail addresses: rlathe@pieta-research.org (R. Lathe), ykotelev@staffmail.ed.ac.uk (Y. Kotelevtsev).

Richard Lathe a,b,c,⇑, Yuri Kotelevtsev a,b,d,e

a State University of Pushchino, Prospekt Nauki, Pushchino 142290, Moscow Region, Russiab Pushchino Branch of the Institute of Bio-Organic Chemistry, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russiac Pieta Research, PO Box 27069, Edinburgh EH10 5YW, UKd Biomedical Centre for Research Education and Innovation (CREI), Skolkovo Institute of Science and Technology, 143025 Skolkovo, Russiae Queens Medical Research Institute, University of Edinburgh, Little France, Edinburgh EH16 4TJ, UK

a r t i c l e i n f o

Article history:Received 14 June 2013Received in revised form 3 December 2013Accepted 6 January 2014Available online 21 January 2014

Keywords:SymmetryPromiscuityLigand-binding

a b s t r a c t

Steroid/sterol-binding receptors and enzymes are remarkably promiscuous in the range of ligands theycan bind to and, in the case of enzymes, modify – raising the question of how specific receptor activa-tion is achieved in vivo. Estrogen receptors (ER) are modulated by 27-hydroxycholesterol and 5a-androstane-3b,17b-diol (Adiol), in addition to estradiol (E2), and respond to diverse small moleculessuch as bisphenol A. Steroid-modifying enzymes are also highly promiscuous in ligand binding andmetabolism. The specificity problem is compounded by the fact that the steroid core (hydrogenatedcyclopentophenanthrene ring system) has several planes of symmetry. Ligand binding can be in sym-metrical East–West (rotation) and North–South (inversion) orientations. Hydroxysteroid dehydrogen-ases (HSDs) can modify symmetrical 7 and 11, also 3 and 17/20, positions, exemplified here by yeast3a,20b-HSD and mammalian 11b-HSD and 17b-HSD enzymes. Faced with promiscuity and symmetry,other strategies are clearly necessary to promote signaling selectivity in vivo. Gating regulates hormoneaccess via enzymes that preferentially inactivate (or activate) a subclass of ligands, thereby governingwhich ligands gain receptor access – exemplified by 11b-HSD gating cortisol access to the mineralocor-ticoid receptor, and P450 CYP7B1 gating Adiol access to ER. Counter-intuitively, the specificity of ste-roid/sterol action is achieved not by intrinsic binding selectivity but by the combination of localmetabolism and binding affinity.

� 2014 Elsevier Inc. All rights reserved.

Contents

1. The paradigm: specificity of steroid–protein interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152. What does a steroid-binding polypeptide recognize? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.1. Promiscuity of receptor binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.2. What is the primary ER ligand? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.3. A further problem – steroid symmetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3. Promiscuity and symmetry: steroid-metabolizing enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.1. 11b-HSD (HSD11B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.2. HSD17B10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.3. ACAT1 – one enzyme, two binding sites, multiple ligands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.4. Other examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4. Lack of specificity – the need for gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.1. HSD11B and the mineralocorticoid receptor (MR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.2. CYP7B1 and estrogen receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.3. Reverse (‘positive’) gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.4. Different ligands, different effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.5. Targeted transport and delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1. The paradigm: specificity of steroid–protein interactions

The current paradigm is that steroids (and related moleculesincluding sterols) induce their biological effects by binding selec-tively to cognate receptor targets (the ‘lock and key’ model). How-ever, there is increasing realization that the stereochemistry ofligand binding to nuclear receptors and metabolizing enzymes isfar more flexible than previously thought. This conclusion is borneout by evolutionary and functional studies.

Studies on an ancient metazoon, the sponge Amphimedon queen-slandica, revealed two nuclear hormone receptors that more closelyresemble the modern retinoid-X receptor (RXR) than the group oftrue steroid receptors; it was argued that the ancestral ligand, whosebinding arose through a process of ‘molecular tinkering’, was possi-bly a long-chain fatty acid [1]. True steroid-type receptors arosemore recently. Genome sequence comparisons have revealed thatthe first nuclear steroid-type receptor most closely resembled theestrogen receptor (ER), and studies in ancient fishes argue that gen-ome duplication then subsequently led to the evolution of a secondreceptor that most closely resembled the progesterone receptor (PR)[2,3]. Reconstruction of the putative ancestral ER-like receptor at thefoot of the evolutionary tree allowed demonstration that this poly-peptide responded best to E2 [3], although alternative ligands (suchas 3-hydroxysterols) were not tested.

However, there is no relationship in the position in the evolu-tionary tree and the chemical nature of the current ligand, leadingto the speculation that ligand-binding dependency emerged sev-eral times independently [4,5] and that specific ligand binding ar-ose relatively late. The most likely conclusion is that earlyreceptors responded to a range of molecules. For example, theemergence of E2 as a selective ligand for ER must have arisen latebecause there is no easy synthetic route for the generation of E2from likely precursors. In another example, the reconstructedancestor of the mineralocorticoid and glucocorticoid receptors(MR and GR, respectively) in jawless fishes responds best to aldo-sterone, despite evidence that the molecule was absent at thisevolutionary stage, arguing that the ancestor receptor bound toa range of related molecules that pre-dated aldosterone [6]. Aswe will see below, the capacity to bind to a range of relatedmolecules is conserved through to the present day, with impor-tant implications for the selectivity of hormone signalingmechanisms.

Fig. 1. Structure of estradiol. (A) Ball and stick model; (B) spacefill (Va

2. What does a steroid-binding polypeptide recognize?

The structure of the steroid nucleus does not easily lend itself tohighly specific molecular contacts. As shown in Fig. 1, there are fewmolecular groups in a molecule such as estradiol that permit ste-reospecific interactions, raising the question of what specific con-tacts steroid receptors make with the target ligand. X-ray studieshave shown that, following receptor binding, steroids are largelyburied within the folded polypeptide, but the assumption thatprimary interactions are spread uniformly across the moleculedoes not appear to be correct. Instead, initial binding is likely tobe driven by a small number of contacts within restricted domainsof the molecule, with weak stabilizing contacts across themolecular surface – that only in a second step lead to complexrefolding [7].

This conclusion is perhaps inevitable, regarding steroid-bindingreceptors and modifying enzymes, for two reasons. First, if thebinding polypeptide made extensive high-affinity contacts acrossthe whole ligand then the molecule would no longer dissociateonce a local hormone surge had abated, or following reaction com-pletion. Second, ligand-binding specificity for modern steroids isoften governed by a small number of molecular differences, some-times a single group. If the whole molecule were to be recognizedby the binding polypeptide then the impact of a single groupwould be diminished, and binding specificity would be lost.Clearly, some compromises must be made, but it appears that ste-roid/sterol receptors make initial high-affinity contacts with only afraction of the ligand, and therefore interact with a spectrum ofrelated molecules.

2.1. Promiscuity of receptor binding

We define here ‘promiscuity’ as the capacity of an enzyme orreceptor to bind to (and in the case of enzymes, modify) a rangeof different substrates, often in different configurations and at dif-ferent positions. The term ‘promiscuity’ has been widely used inthis context (e.g., [8,9]) and the concept is akin to that of ‘biologicalmessiness’ [10].

The mammalian ER affords a good example. It was proposed thatbinding is driven by the steroidal A ring alone, and that the activity ofthe molecule in terms of receptor activation is driven by the D ring:Duax’s 1978 ‘A-ring binding/D-ring acting’ model for estrogens and

n der Waals surface). Modeling performed using JSmol/JMol [92].

16 R. Lathe, Y. Kotelevtsev / Steroids 82 (2014) 14–22

progestins (Fig. 2) [11]. Although this early model is a simplification,more extensive studies confirm that the only tight-binding pocketencompasses the phenolic A ring, and the overall scope of the li-gand-binding pocket, that is only opened upon ligand binding, canaccommodate bulky substituents – ERa can therefore bind to a widediversity of molecules ([7] for discussion).

In support, ligand-binding promiscuity for ER is demonstratedby studies on molecules that have only limited similarity to E2. Adiversity of alkylphenolic compounds can bind to ER and activatetranscription [12]. The pesticide DDT (dichlorodiphenyltrichloro-ethane), one of the first known xenoestrogens [13], is known to ex-ert potent biological effects on the reproductive tract [14]. Othermolecules with ER endocrine effects include phytoestrogens anddioxins [15] and these interfere with diverse aspects of estrogenicsignaling [16]. In a comprehensive study, Blair et al. [17] screened188 environmental chemicals. In a competition assay, 100 werefound to bind to ER, and of these 26 were classified as strong bind-ers. The study confirmed that an aromatic A ring is absolutely re-quired; binding was increased by the presence of two rings and,although a 3-OH group markedly increased binding, this was notabsolutely essential [17]. These conclusions are reinforced by a re-cent study on an ancestral ER dubbed AncSR1 [9]. The small molec-ular sizes of some of these molecules (e.g., bisphenol A and phenolred; Fig. 3) [18] support the contention that initial ligand bindingto ER is governed by a very limited molecular domain.

2.2. What is the primary ER ligand?

If at first contact ER only binds with high affinity to part of themolecule, notably the hydroxylated A ring, the logical consequenceis that ER in vivo must bind to a range of molecules similar to E2,raising the question – what is the true ER ligand? There is extensiveevidence that other steroids and sterols, including androstanediol

Fig. 2. Schematic of estrogen binding to its receptor emphasizing the primaryrecognition of the phenolic A ring. From [11], with permission. For more detaileddescription of ER ligand binding, where initial contacts with the A-ring cleftprecipitate refolding of the molecule, see [7].

Fig. 3. Limited similarities between estrogen mimetics and estradiol. (a) Estradiol,(b) bisphenol A, (c) phenol red.

(5a-androstane-3b,17b-diol; Adiol), androstenediol (androst-5-ene-3b,17b-diol), and 27-hydroxycholesterol (27OHC) (reviewedin [19]), are active as ER ligands. As we will see later, Adiol is a phys-iological ligand for ERb in the prostate, and 27OHC is a physiologicalmodulator of both ERa and ERb. Although the affinity of the recep-tors for 27OHC is far lower than for E2, the concentrations of27OHC are markedly higher – the molecule is thus likely to competewith E2 for receptor binding.

From an evolutionary perspective the preference of ER for E2 isperplexing because E2 is only produced via a chain of conversions(via androgens) and is therefore unlikely to have been the ancestralligand for ER. On these grounds, sterols (and 3b-hydroxysteroids)are more likely contenders as ancestral ligands. Regarding ER andE2, it seems likely that (as with aldosterone and MR, above) the li-gand evolved after the receptor.

Interestingly, it has been speculated (as for other receptors, seeSection 4.4) that there may be ligand-specific functional differ-ences: ERb complexed to Adiol may adopt a conformation that re-cruits the co-repressor CtBP (C-terminal binding protein), leadingto anti-inflammatory effects [20] that are absent when ERb bindsE2. This raises the interesting question of whether a single receptor(ER) can fulfil different roles depending on the nature (and orienta-tion, see below) of the ligand.

2.3. A further problem – steroid symmetry

The problem of promiscuous binding to a variety of moleculeswith only limited similarity to the lead ligand is accentuated bythe fact that the steroidal molecule itself has multiple planes ofsymmetry (Fig. 4). This leads to the speculation that the ligandmolecule can bind to its target, be it a steroid-type nuclear receptoror a metabolizing enzyme, in more than one orientation.

Modeling studies have revealed that androstenediol is likely todock into human ERa in two different configurations, one in whichis as for E2 (76/100 poses) and a second in which the entire mole-cule is ‘North–South’ (up–down) inverted (24/100 poses) [21]. Thisraises the intriguing possibility that binding of a single molecule indifferent orientations could have different biological activities.

These promiscuity and symmetry issues reported for ER arelikely to be only the tip of the iceberg; similar considerations nodoubt apply to other receptors (discussed by Baker [22]).

Fig. 4. Symmetrical positions on the steroid nucleus via rotation or inversion,exemplified by estradiol. Substituents above the plane of the steroid are describedas b and are shown as a solid line ( ); those below the plane are described as aand are shown by a broken line ( ). Planes of rotational symmetry are indicated,emphasizing the similarity of the 3a and 17b positions (180� rotation around the zaxis), 11b and 7a (180� rotation around the x axis), and 11b and 7b (180� rotationaround the y axis).

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3. Promiscuity and symmetry: steroid-metabolizing enzymes

Promiscuity extends to steroid/sterol metabolizing enzymes.The view ‘one pocket, one ligand-binding conformation’ was firstchallenged by work on 3a,20b-hydroxysteroid dehydrogenase(3a,20b-HSD) of the yeast Streptomyces hydrogenans. The enzymecatalyzes both 3a and 20b oxidoreduction of steroid substrates.How can a single enzyme modify its substrate at two very differentpositions? Two models were debated: (i) a single substrate-bind-ing site, but with two different pockets accommodating NAD/NADPcofactors, or (ii) substrate binding in two different orientations,with a single cofactor site. X-ray studies revealed a single cofactorsite [23], confirming the Sweet and Samant [24] proposal that thesubstrate can bind in two alternative orientations, such that twodistinct positions are exposed to cofactor-mediated oxidoreduction(see [25]). As discussed below, this appears to be commonplace.

Fig. 5. Molecular modeling of HSD11B1 interactions with 7b-hydroxy DHEA andcorticosterone (11b,21-dihydroxyprogesterone) showing conservation of distancesbetween the target hydroxyl and both C4 of NADP and the catalytic tyrosine(Tyr183) residue. From [32] with permission.

3.1. 11b-HSD (HSD11B)

The property of bifunctionality is shared by the enzymes thatcatalyze the interconversion of ‘inert’ (11-keto; cortisone in hu-mans and 11-dehydrocorticosterone in mice and rats) and ‘active’11b-hydroxylated glucocorticoids (cortisol in humans, corticoste-rone in mice and rats), thereby regulating the biological activityof glucocorticoids [26]. However, the 7a and 11b positions aresymmetrically positioned ([27] and Fig. 4), and some HSD11B en-zymes can also modify the 7-position of the steroid backbone[28–31] – although this may not be true of all HDS11B enzymes be-cause there are suggestions of marked species-specificity in thealternative orientations that can be accommodated.

In exploration of the mechanism, Nashev et al. [32] expressedmouse HSD11B1 together with hexose-6-phosphate dehydroge-nase to drive the production of NADPH cofactor. In addition to cat-alyzing 11b oxidoreduction, HSD11B1 was found to catalyzeefficiently the interconversion of 7-keto and 7-hydroxylated DHEA,at both the 7a and 7b positions. Furthermore, HSD11B1 was foundto catalyze hepatic reduction of 7-oxolithocholic acid [33]. Molec-ular modeling of HSD11B1 has revealed that substrate can bind intwo different orientations and, for example, the 11b and 7b posi-tions adopt an almost identical binding configuration in relation-ship to NADP cofactor and the catalytic tyrosine residue in theenzyme (Fig. 5) [34], despite East–West rotation of the molecule.Activity at 7a may reflect North–South inversion of the molecule(Fig. 4).

Interestingly, the enzymes HSD11B1 and 2 are not the only en-zymes capable of catalyzing 11b oxidoreduction: in mice knocked-out for both enzymes significant HSD11B activity remains (Y.K.,unpublished data). Identification of the other enzymes withHSD11B activity will be of interest.

3.2. HSD17B10

A further case is the Alzheimer disease Ab-binding dehydroge-nase. Yeast two-hybrid screening using Ab as bait identified anew binding partner dubbed ‘ERAB’ [35] or ABAD (Ab-binding alco-hol dehydrogenase). The protein has now been shown to be iden-tical to type 10 17b-HSD (HSD17B10) [36]. HSD17B enzymestypically catalyze the interconversion of estradiol (17b-hydroxy)and estrone (17-oxo), and regulate the bioavailability of active hor-mone in tissues such as ovary [37]. However, this specific isoformis present at highest levels in hippocampus [38] and, moreover, un-like other HSDs, the enzyme is membrane-bound [39] and is prin-cipally confined to mitochondria [40,41]. The true in vivo substratefor the enzyme has not been established. HSD17B10 has othersubstrates in addition to steroids/sterols, and also acts as a

2-methyl-3-hydroxybutyryl CoA dehydrogenase implicated in iso-leucine catabolism [42]; such substrates are presumed to fold toadopt a steroid-like configuration in part of the molecule.

Importantly, HSD1710 is capable of modifying both the 3a and17b positions of steroid substrates. Several studies have addressedthe substrate specificity of the enzyme [40,43,44]. Modificationscatalyzed include 3a and 17b oxidoreduction, but there is alsosignificant activity at the 20, 21, and 7 (both a and b) positions.Modification at 3a is of considerable interest because 3a-hydrox-ysteroids (and potentially sterols) have potent anesthetic activityand directly target GABA and intracellular (mitochondrial)receptors that control neuronal activation (reviewed in [45]).Generally, different HSD17 enzymes are reported, in addition tocatalyzing interconversions at the 17 position, to catalyzeconversions at the 3 and 7 positions, and also at the 20 (in20-hydroxyprogesterone), 21 (in 11-dehydrocorticosterone) and24 (in 3-ketostearoyl-CoA) positions [46].

3.3. ACAT1 – one enzyme, two binding sites, multiple ligands

ACAT, also known as sterol O-acyltransferase, affords anotherexample. ACAT1 is responsible for converting cholesterol to cho-lesteryl esters, thereby promoting cholesterol sequestration infatty droplets, and has been implicated in foam-cell formationand atherogenesis. In addition to modifying cholesterol (predomi-nantly at the 3b position), substrates include lathosterol, cholesta-nol, 7-dehydrocholesterol, 7a-hydroxycholesterol, and 25-hydroxycholesterol, as well as a range of dietary plant sterols[47–50]. ACAT can also metabolize the steroid pregnenolone [51].

ACAT1 contains not just one, but two, binding sites for sterols.One represents the enzyme active site; the second site governsallosteric activation of the enzyme. Allosteric activation is medi-ated largely by side-chain oxidized cholesterols, and not by closeanalogs such as 7-ketocholesterol [48]. However, once cholesterolis bound to the allosteric site, the enzyme becomes highly promis-cuous towards different substrate sterols [48]. In fact, ACAT1 activ-ity on pregnenolone (above) is crucially dependent on cholesterol.

18 R. Lathe, Y. Kotelevtsev / Steroids 82 (2014) 14–22

In the absence of cholesterol, pregnenolone is a poor substrate but,in the presence of cholesterol, becomes an excellent substrate [51].

ACAT is also modulated by, and may metabolize, estrogens. Inaddition to binding the anti-estrogen tamoxifen, ACAT activity isinhibited by E2 itself, but only at concentrations in the 10 lMrange [52] (E2 generally circulates in the nM range). Conversely,the prototypical ACAT inhibitor Sah 58-035 is a potent ER agonist[53].

Steroid symmetry considerations would predict that ACAT islikely to esterify E2 at the 17b position (and potentially at the 3 po-sition). 17b esters of E2 are known to accumulate in associationwith low-density lipoprotein [54] and have been specifically impli-cated in mammary tumorigenesis [55]. The related enzyme leci-thin/cholesterol acyltransferase (LCAT) has been reported tocatalyze preferential esterification of E2 at the 17b position, inaddition to modifying Adiol at both 3b and 17b [56]. It is thereforelikely that ACAT also modifies E2 at 17b but this remains to bedemonstrated.

3.4. Other examples

These cases are not unusual: the capacity to bind to and/ormodify steroid substrates at different positions appears to be wide-spread. As a further illustration, some 3b-HSD enzymes can modifythe 17b position (reviewed in [57]) and a small number of modifi-cations can convert an enzyme catalyzing 3a oxidoreduction toone catalyzing 17b or 20a conversions [58]. Indeed, HSD11B ap-pears to have evolved from an ancestral HSD17B2-type enzyme[59]. Furthermore, some enzymes are able to modify the choles-terol side-chain (positions 20–27), suggesting that these carbonsmight possibly fold back to adopt a compact configuration resem-bling the 3D structure of the steroid nucleus.

Together, these studies confirm that steroid-binding enzymesmake initial high-affinity contacts with only a small fraction ofthe molecule and, depending on the orientation in which the sub-strate inserts into the binding site, catalyze modifications at differ-ent positions on the steroid nucleus. Notably, these molecules canadopt alternative configurations, both via rotation (East–West) andinversion (North–South or up–down; Fig. 4). Of these differentpositions, the 3a and 17b (or perhaps 20b) appear to have the clos-est resemblances, as do the 7a/b and 11b positions (Fig. 4).

This raises several interesting questions. For example, doesEBI2, a receptor for 7a-hydroxylated sterols [60,61], respond to11b-hydroxylated molecules? Do LXRs, that bind to 22, 24, 25and/or 27-oxidized cholesterols [62–64], also bind to 17b/3a-hydroxylated molecules?

Fig. 6. Gating of cortisol access to the mineralocorticoid receptor (MR) by 11b-HSD. Insediffer between human and rodent, but gating takes place in both cases.

4. Lack of specificity – the need for gating

The lack of selectivity, at first sight, would appear to be incom-patible with accurate hormone signaling. Therefore, other mecha-nisms must be exploited to boost selectivity. We discuss ‘gating’,where combinations of two (or more) ligand-binding polypeptidesare used to achieve the required in vivo selectivity in target tissues.We define gating as a process in which local enzyme action in atarget tissue dictates whether a systemically delivered moleculecan, or cannot, gain access to a target binding site. We distinguishbetween gating and detoxification (oxidative and other metabo-lisms principally mediated by the liver); although, given the poten-tial to regulate hormone availability, it would be surprising ifhepatic metabolism does not in some instances play a specific reg-ulatory role (not reviewed here). In the following we focus on twoexamples where gating has been demonstrated.

4.1. HSD11B and the mineralocorticoid receptor (MR)

As noted earlier, HSD11B enzymes catalyze the interconversionof ‘inert’ (11-keto; cortisone/11-dehydrocorticosterone) and ‘ac-tive’ 11b-hydroxylated glucocorticoids (cortisol/corticosterone).The ability of the enzyme to gate the action of glucocorticoidsand mineralocorticoids emerged from studies of patients withapparent mineralocorticoid excess as a result of deficiency ofHSD11B2 [65]. Briefly, kidney MRs are activated by both aldoste-rone and cortisol, molecules that differ centrally in oxidation ofaldosterone at the 18 position, three carbons away from the 11 po-sition. For this reason aldosterone is not a substrate for HSD11B2,whereas cortisol is an excellent substrate. HSD11B2 thus inacti-vates cortisol, but not aldosterone.

Normally, the action of local HSD11B2 prevents cortisol actionat MR, and the receptor therefore responds only to aldosterone –but, in patients lacking this enzyme, cortisol is able to activaterenal MR, leading to apparent mineralocorticoid excess ([65],reviewed in [26,66,67]). Selective activation of MR by aldosterone– and not by cortisol – is achieved not by the intrinsic ligand-bind-ing selectivity of the receptor but by the substrate specificity oflocally expressed HSD11B enzymes (Fig. 6).

4.2. CYP7B1 and estrogen receptors

One of the two major ERs in mammals, ERb, binds to both E2and the widely circulating steroid Adiol. However, ERb respondspoorly to 7a-hydroxylated Adiol, whose formation by hydroxyl-ation of Adiol is catalyzed by the oxysterol 7a-hydroxylase CYP7B1

rt panel adapted from Kotelevtsev et al. [93]. Note that the exact hormonal ligands

R. Lathe, Y. Kotelevtsev / Steroids 82 (2014) 14–22 19

(noting, importantly, that CYP7B1 is only poorly active against E2itself). In the developing rodent prostate, ERb activation is neces-sary to control cellular proliferation [68]. Inactivation of this CYPled to hyperactivation of ERb by Adiol and markedly reduced cellu-lar proliferation [68]. Similar gating takes place in females, and fe-male Cyp7b1�/� mice display early uterine and mammary glandproliferation attributed to hyperactivation of ERb by Adiol [69]. Aparallel CNS hyperproliferation phenotype was seen in Cyp7b1�/�

mice owing to overactivation of brain ERb by Adiol [70]. Thus,the specificity of ERb activation by E2, and not by Adiol (or indeedby androstenediol or 27OHC), is ensured by the substrate specific-ity of locally expressed CYP7B1, in conjunction with the intrinsicactivity of the receptor (Fig. 7).

Gating extends to other ER ligands. 27OHC is now recognized tobe a selective estrogen receptor modulator (SERM) at both ERa andERb [71,72]. Although it has a lower binding affinity for ERs thandoes E2, circulating concentrations are a little under the lM range,and local tissue levels (such as in atherosclerotic plaques) can evenreach mM concentrations. In vascular endothelial cells 27OHC isnow known to antagonize transcriptional activation by ERs, and27OHC binds to recombinant ERa and ERb with Ki values of 1.2and 0.42 lM respectively [72,73]. However, CYP7B1-mediatedmetabolism of 27OHC [74–76] gates access to ERs. Following vas-cular injury, E2 promotes repair of endothelial denudation in wild-type mice, but repair was significantly reduced in Cyp7b1�/� mice,arguing that lack of CYP7B1 allows 27OHC to antagonize ER activ-ity [73].

Gating seems to be the rule rather than the exception. To pickone further example, progesterone binds more tightly to MR thandoes aldosterone, but has only low activation activity, leading topotent in vitro anti-mineralocorticoid activity [77,78]. The sameis not seen in vivo, suggesting that PROG action at MR is locallygated by metabolizing enzymes [79]. Although the key enzymesinvolved have not been identified, evidence is emerging thatCYP27 also participates in gating PROG access to MR [80]. Someprogestins are also ligands of AR [81], unlike natural PROG itself(which does not bind significantly to AR), but it is not known ifgating processes also operate at this target.

4.3. Reverse (‘positive’) gating

The action of HSD11B or CYP7B prevents steroid/sterol access tonuclear receptors, respectively MR and ER, but in other cases enzy-matic conversion is required to generate ligand. For example, theEpstein–Barr virus-induced gene 2 (EBI2) G protein-coupled

Fig. 7. Gating of 5a-androstane-3b,17b-diol (Adiol) access to ERb by the

receptor is required for immune cell migration. The most potentactivators are 7a-hydroxylated 25-hydroxycholesterols [60,61].In this case the combined activities of two enzymes, CYP7B1 andcholesterol 25-hydroxylase (CH25H), are required to generateEBI2 ligand, and failure of immune cell migration was observedin mice deficient in CH25H [60].

LXR (liver X receptor) positive gating has also been demon-strated. LXRa and LXRb are members of the nuclear family of tran-scription factors that regulate cellular cholesterol efflux. LXRactivation promotes export of excess cholesterol from peripheraltissues to the liver and bile for excretion. The best ligands are cho-lesterols oxidized at the 22, 24, and/or 25 positions [62], but thesesterols are present only at low concentrations, and 27OHC is morelikely to be the endogenous ligand [63,64]. The conclusion that theactivity of cholesterol 27-hydroxylase, CYP27, is required for posi-tive gating of LXR was borne out by inspection of fibroblasts cul-tured from a patient with CYP27 deficiency (cerebrotendinousxanthomatosis, CTX) where upregulation of a LXR target gene in re-sponse to cholesterol loading was markedly impaired [63]. Inter-estingly, Janowski et al. [62] reported that 22(S)-hydroxycholesterol binds to both LXR subtypes in vitro, but does not mod-ulate LXR activity in cells; they speculated that selective ACAT-mediated metabolism of 22(S)-hydroxycholesterol may gatereceptor access [62].

Gating appears to be a generic feature of steroid/sterol biology.To give one invertebrate example, in Caenorhabditis elegans the ste-rol dafachronic acids (DAs) regulate lifespan via the nuclear recep-tor DAF-12 (a homolog of LXR and FXR). DAF-12 activation requiresa short but pivotal pathway of metabolic conversions from choles-terol to DAs, and the activity of this pathway is governed byenvironmental stress – thus attuning development/diapause,reproduction, and longevity to environmental signals (e.g. [82]).It remains an open question whether environmental stresses alsooperate via steroid/sterol pathways to regulate growth and agingin mammals.

4.4. Different ligands, different effects

The possibility was discussed (Section 2.2, above) that bindingof different ligands to ER (E2 vs Adiol) might exert different func-tions. This concept has been amply confirmed for MR. Indeed,although mutation studies have demonstrated that specificity foraldosterone (versus cortisol/corticosterone) is achieved byHSD11B-mediated gating, this is unlikely to be the whole story.(i) Cortisol/corticosterone circulates at up to 100-fold higher levels

action of CYP7B1. Insert panel adapted from Sugiyama et al. [70].

20 R. Lathe, Y. Kotelevtsev / Steroids 82 (2014) 14–22

than aldosterone, and HSD11B2 would therefore need to be >99%effective in inactivating cortisol to prevent entirely its action atMR. (ii) Some tissues (e.g., hippocampus and macrophages) expresshigh levels of MR but do not express detectable HSD11B2, but – de-spite the absence of the gating enzyme – receptor signaling is prin-cipally in response to aldosterone (and not, except in stresssituations, in response to cortisol/corticosterone). How is thisachieved? As recently reviewed [66], in addition to differentialbinding kinetics, the conformation of the ligand-bound receptor(and the effects it exerts via downstream effectors) may differmaterially according to the ligand. A single receptor (in this caseMR) can therefore produce a range of responses depending onthe nature of the ligand [66] and, potentially, on its binding orien-tation, further challenging the ‘lock and key’ paradigm.

4.5. Targeted transport and delivery

Enzymatic gating is one mechanism by which specificity can beenhanced in vivo. Clearly tissue- and cell-specific expression pat-terns of metabolizing enzymes and receptor targets also play a piv-otal role in ensuring that ligands act in the right place, and at theright time (not reviewed here). Selective delivery is another potentmechanism. In addition to a range of steroid/sterol binding pro-teins [including low density lipoproteins (LDL), high density lipo-proteins (HDL), their integral apolipoproteins, as well as theiruptake receptors] and metabolizing enzymes (e.g., sulfotransferas-es, methyltransferases, hydroxylases, conjugating enzymes), manyof which act systemically via the liver, a further mechanism forfocusing activity involves selective trans-cellular transport (e.g.,[83]) and intracellular targeting. The lipid-transfer sterol carrierproteins (SCPs) and oxysterol binding proteins (OSBPs) are a casein point. OSBPs contain two membrane-binding surfaces, and func-tion to transfer sterols from one intracellular membrane to another[84–86]. In human there is a family of 12 OSBPs [87,88]. Emergingevidence indicates that each has a distinct profile of sterol bindingand, significantly, subcellular localization [89]. This means thateach binding molecule is differentially targeted within the cell. Evi-dence for selective intracellular transport and delivery of steroidsis less well documented, although SCP2 (also known as nonspecificlipid-transfer protein) is highly promiscuous in the lipid moieties ittransports [90].

The evolutionary impact of such transport proteins is high-lighted by the finding that HSD17B4 is a chimeric protein that con-tains both an HSD domain and a C-terminal region with highsimilarity to SCP2 [91]. It is possible, but so far unproven, thatOSBPs and other transport proteins pre-select particular substratesand orientations for presentation to receptors and metabolizingenzymes, conferring a further layer of molecular selectivity.

5. Concluding remarks

Polycyclic molecules such as steroids and sterols would appearto be well adapted for signaling purposes. They are derived fromone of the most abundant cellular components, cholesterol, and li-pid solubility and structural rigidity lend themselves to signaling –perhaps explaining the ubiquity of this type of molecule in a sys-temic signaling role. However, there are few molecular groupsavailable for highly specific interactions. In consequence, bindingpolypeptides are remarkably promiscuous and interact with multi-ple ligands, with docking taking place in alternative orientations. Itis this adaptability of steroids which makes them natural ligands ofmultiple receptors. However, the selectivity of hormone actionseen today is only achieved through tissue-specific ligand metabo-lism, or ‘gating’ (combined with other mechanisms includingselective delivery). In short, the ‘lock and key’ model is incomplete

– there are (i) multiple keys, and the guardians of the keys (gatingenzymes) govern whether the lock is opened and, indeed, (ii) dif-ferent keys may open the lock in different ways. The design ofpharmaceutical steroidal molecules needs to take into accountboth local (and systemic) metabolism and transport, as well as dif-ferential ligand-binding conformations, and not only bindingaffinity.

Acknowledgments

A preliminary version of this paper was presented at the 2012meeting of the European Network on Oxysterol Research (ENOR)in Djion, France. This work was funded, in part, by a Governmentof Russia Grant (11.G34.31.0032/14.12.2010) to Y.K. We thankRebecca Pruss (Marseille) for critical reading of the manuscriptand many suggestions, and anonymous reviewers for furtherhelpful insights.

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