Diverse modes of action of progesterone and its metabolites

ft. Steroid Biochem. Molec. Biol. Vol. 56, Nos. 1-6, pp. 209-219, 1996

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D i v e r s e M o d e s of A c t i o n of P r o g e s t e r o n e and its M e t a b o l i t e s V i r e n d r a B. M a h e s h , * D a r r e l l W. B r a n n and L a w r e n c e B. H e n d r y Department of Phys(ology and Endocrinology, Medical College of Georgia, Augusta, GA 30912-3000, U.S.A. P r o g e s t e r o n e a n d its m e t a b o l i t e s h a v e a v a r i e t y o f d i v e r s e effects in t he b r a i n , u t e r u s , s m o o t h m u s c l e , s p e r m a n d t he oocyte. T h e effects i n c l u d e changes in e l e c t r o p h y s i o l o g i c a l e x c i t a b i l i t y , i n d u c t i o n o f anest]~esia, r e g u l a t i o n o f g o n a d o t r o p i n s e c r e t i o n , r e g u l a t i o n o f e s t r o g e n r e c e p t o r s , modulation of uterine contractility and induction of acrosome reaction and oocyte maturation. The l a t e n c y o f th e effects v a r y f r o m s e v e r a l seconds to s e v e r a l h o u r s . Thus, it is not s u r p r i s i n g t h a t m u l t i p l e m e c h a n i s m s o f a c t i o n a r e involved. T h e classical m e c h a n i s m o f s t e r o i d h o r m o n e a c t i o n o f i n t r a c e l l u l a r r e c e p t o r b i n d i n g has b e e n s u p p l e m e n t e d by the possibility o f t he s t e r o i d a c t i n g as a t r a n s c r i p t i o n f a c t o r a f t e r t he b i n d i n g o f t he r e c e p t o r p r o t e i n to DNA. O t h e r m e c h a n i s m s i n c l u d e i n f l u e n c e o f t h e s t e r o i d s on m e m b r a n e fl ui di t y a n d act i ng t h r o u g h o t h e r cell signalling s y s t e m s , m e m b r a n e r e c e p t o r s a n d GABAA r e c e p t o r s . O f p a r t i c u l a r i n t e r e s t a r e m u l t i p l e m e c h a n i s m s f o r th e s a m e t y p e s o f action. F o r e x a m p l e t h e effect o f p r o g e s t e r o n e on g o n d a d o t r o p i n rel ease is l a r g e l y e x e r t e d via t h e classical i n t r a c e l l u l a r r e c e p t o r as well as m e m b r a n e r e c e p t o r s , w h e r e a s 3~,5~-tet r a h y d r o p r o g e s t e r o n e - i n d u c e d L H r e l e a s e o c c u r s via t he GABAA r e c e p t o r s y s t e m . T h e i n h i b i t i o n o f u t e r i n e c o n t r a c t i l i t y by p r o g e s t e r o n e is r e g u l a t e d by p r o g e s t e r o n e r e c e p t o r s while t he a c t i o n o f 3 a t,5 ~t- tetr ah y d ropr oges t e r one on u t e r i n e c o n t r a c t i l i t y is r e g u l a t e d by GABAA r e c e p t o r s . T h e r e g u l a t i o n o f t h e d i f f e r e n c e s in t he p a t t e r n o f p r o g e s t e r o n e effects on e s t r o g e n r e c e p t o r d y n a m i c s in th e a n t e r i o r p i t u i t a r y a n d t he u t e r u s in t he s a m e a n i m a l a r e also o f c o n s i d e r a b l e i n t e r e s t .

J. Steroid Biochem. Molec. Biol., Vol. 56, Nos. 1-6, pp. 209-219, 1996

slices [12] and membrane bound cAMP in the amphibian oocyte [13]. These are just a few examples of the effects of progesterone in which the latency of effect may vary from seconds to hours and the effects may be of short duration or sustained. The effects of progesterone may also differ in different tissues in the same animal and the same effect may occur using similar or different mechanisms. In addition, progesterone metabolites may share some of the effects of progesterone while they may also exert their own characteristic biological effects. This paper reviews the diverse biological effects of progesterone and its metabolites and the multiple mechanisms by which such effects are manifested.

INTRODUCTION T h e hormone progesterone exerts a wide spectrum of biological activity in a variety of tissues. T he effects of progesterone could be stimulatory or inhibitory depending upon the tissue involved, the dose of progesterone used and its time of administration. Among the many stimulatory effects of progesterone are the enhancement of gonadotropin releasing hormone (GnRH) [1], dopamine [2] and gonadotropin secretion [3, 4 for review] and the induction of lordosis [5], uteroglobin synthesis [6], acrosomal reaction and Ca 2÷ influx in sperm [7, 8] and amphibian oocyte maturation [9]. On the other hand progesterone inhibits uterine contractility [10], electrophysiological excitability in the h~master dorsal midbrain neurons [11], gonadotropin secretion dependent on the dose and/or time of administration [3, 4 for review], norepinephrine induced cAMP induction in hypothalamic

REGULATION OF THE PREOVULATORY TYPE OF GONADOTROPIN SURGE BY PROGESTERONE AND ITS METABOLITES

Effects of progesterone on gonadotropin secretion Proceedings of the 12th International Symposium of The Journal of Steroid Biochemistry & Molecular Biology, Berlin, Germany, 21-24 May 1995. *Correspondence to V. B. Mahesh. 209

The role of estradiol in triggering the preovulatory type of gonadotropin surge has been established by the removal of the estrogen effects by ovariectomy, and the

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use of estradiol antibodies or estradiol antagonists resulting in the abolition of the surge and its reinstatement by estradiol administration. This subject has been extensively reviewed [3, 4, 14, 15]. Nevertheless, it is clear that estradiol is not the only steroid responsible for regulating the preovulatory gonadotropin surge. In the ovariectomized rat and the rabbit, estradiol failed to bring about a gonadotropin surge that was comparable to the preovulatory surge in magnitude and duration and progesterone was necessary for the full surge and restoring the sensitivity of the pituitary to G nRH comparable to that found on the afternoon of proestrus [16-19]. Finally, if the adrenal, which is also a source of progesterone, was removed in addition to the ovary, estradiol could no longer induce an L H surge [16]. T he facilitative effect [18, 20-24] and the inhibitory effects [20, 22-26] of progesterone have been demonstrated in several studies to be dependent upon the time of its administration. Estradiol priming in the ovariectornized rat is essential for progesterone to exert its action on gonadotropin secretion [21, 23, 24, 27]. This is due to the fact that estrogens are required for the induction of hypothalamic and anterior pituitary progesterone receptors [28]. Using very low doses of estradiol in ovariectomized immature rats (0.1/~g/kg body weight for 4 days), Mahesh and coworkers were able to induce a preovulatory type gonadotropin surge by the administration of a single injection of progesterone [23,24,29]. In subsequent experiments, the use of 2 #g of estradiol in ethanol-saline at 1700 h for 2 days in ovariectomized immature rats followed by a single injection of progesterone on the third day brought about a minimal, if any, L H release by estrogen alone while a preovulatory type L H and F S H surge was induced by progesterone [30-33]. The mechanisms involved in the progesteroneinduced gonadotropin surge include the rapid release of G n R H [29], decreased degradation of hypothalamic G n R H [34], progesterone-induced decrease in anterior pituitary occupied nuclear estrogen receptors [28, 35, 36] as well as the direct effect of progesterone on the pituitary in enhancing sensitivity to G n R H [37]. The effect of progesterone on hypothalamic G n R H release is mediated through excitatory amino acids [38 for review]. Th e physiological importance of progesterone in attenuating the gonadotropin surge is readily demonstrated by the use of the progesterone antagonist RU486 [3, 39] and 3/3-hydroxysteroid dehydrogenase inhibitors trilostane [4] and epostane [40]. Effect of progesterone secretion

metabolites

on gonadotropin

Progesterone is actively metabolized in the body to a variety of metabolites. 5e-Pregnane-3,20-dione (5~-dihydroprogesterone; 5e- D H P) and 3e-hydroxy5e-pregnan-20-one (3e,5e-THP) are secreted by the ovary [41, 42], and progesterone is actively metabolized

to 5 e - D H P and 3e,5~-THP by the hypothalamus and the pituitary [43-45]. These metabolites accumulate in the hypothalamus and the pituitary as well [46]. T hey have been shown to have a stimulatory and/or suppressive effect on gonadotropin secretion which depends upon the animal model used and the dose employed [47-49]. Using the low dose (0.1 #g/kg bodyweight) estrogenprimed immature rat model, Murphy and Mahesh demonstrated that 5 e - D H P brought about a selective release of F S H [50] whereas 3e,5~-THP brought about a selective release of L H [51]. T he selective effects of 5c~-DHP and 3~,5~-THP were confirmed by using pregnant mare's serum gonadotropin-primed immature rats exposed to constant light [52]. These observations led to the intriguing possibility that the gonadotropin releasing activity of progesterone is mediated through the metabolism of progesterone. In order to test this possibility, the 5~-reductase inhibitor was used in vivo experiments [53] and in dispersed pituitary cell cultures in vitro [37]. T he 5~-reductase inhibitor attenuated the FSH-releasing activity of progesterone in vitro [37] and in vivo [53] without altering L H release. Thus, the 5e-reduction of progesterone appeared to be important for its action of FSH release. The release of L H stimulated by 3 e , 5 e - T H P raised questions regarding its mechanism of action because unlike progesterone and 5~-DHP, 3e,5~-THP does not bind to the progesterone receptor in the anterior pituitary and the uterus [54, 55]. Work by Majewska et al. [56] in 1986 had demonstrated that 3~,5~-THP was a potent barbiturate-like modulator of the 7aminobutyric acidA (GABAA) receptors in the brain. This: led Brann et al. [57] to examine whether the gon~idotropin releasing activity of 3 e , 5 e - T H P was mediated through the GABAA receptor system. T h e action of 3e,5~-THP on L H release was unaltered by the use of the antiprogestin RU486. However, the GABAA antagonist, picrotoxin attenuated the 3~,5eT H P induced L H surge [57]. These observations provide evidence that the gonadotropin releasing effects of progesterone and 3 e , 5 e - T H P are manifested using different mechanisms. INHIBITION OF UTERINE CONTRACTILITY BY PROGESTERONE AND ITS M E T A B O L I T E S T he observation that progesterone and its 3~t,5~reduced metabolite 3ct,5~-THP brought about L H release in vivo utilizing different receptor systems, namely the progesterone receptor and the GABAA receptor was of considerable interest. T o further examine similar biological response with different mechanisms of action, the effect of progesterone and its metabolites on uterine contractility were studied. T he action of progesterone on inhibiting uterine contractility from the diestrous-II rat is well

Modes of Action of Progesterone and its Metabolites recognized [10, 58]. Several progesterone metabolites are also active in this regard [58] and they are also known to have potent anesthetic effects [59, 60]. Progesterone and 3 of its metabolites 5fl-pregnane-3,20dione, 3fl-hydroxy-5fl-pregnan-20-one and 3~,5ctT H P were studied by us for their effects of uterine contractility [61]. T h e three metabolites of progesterone were more active than progesterone in inhibiting uterine contractility. T h e effects on uterine contractility of progesterone and 5fl-pregnane-3,20-dione, both of which possess a 3-ketone group, were inhibited by the progesterone antagonist RU486 but not the GABA a antagonist picrotoxin [61]. On the other hand, the effects of compounds with a 3-hydroxyl group namely 3fl-hydroxy-5fl-pregnan-20-one and 3ct,5ctT H P were inhibited by the GABA A antagonist picrotoxin but not the antiprogestin RU486. These results clearly demonstrate that different mechanisms can be involved in exerting similar biological effects. DIRECT E F F E C T S OF P R O G E S T E R O N E ON T H E OVARIAN FOLLICLE This section is written to emphasize that the action of progesterone in producing a particular biological response may occur at multiple sites. T h e use of the progesterone antagonist in the cycling as well as the P M S G - p r i m e d immature rat attenuated the preovulatory gonadotropin surge and reduced the n u m b e r of ova per ovulating rat [39]. This, however, does not rule out direct effects o1" progesterone on the ovary. In immature rats treated with P M S G and ovulated by the exogenous administration of h C G , the 3fl-hydroxysteroid dehydrogenase inhibitor epostane reduced the n u m b e r of ovulations [62]. Since the gonadotropins responsible for follicle development and ovulation were exogenous, the observations represent direct actions of progesterone on the ovarian follicle. T h e ovulation inhibition effects of epostane could be counteracted by the administration of progesterone [62]. Progesterone appears to stimul~Lte ovarian proteolytic enzyme activity involved with follicle rupture [63]. Thus, multiple sites of action of progesterone may be present for the facilitation of ovulation. EXAMPLES OF DIFFERENCES IN THE E F F E C T S OF P R O G E S T E R O N E AND ITS M E T A B O L I T E S Although several progesterone metabolites have similarities to the effects of progesterone, such as their actions on gonadotropin release and uterine contractility, this p h e n o m e n o n is not universally true. Since a n u m b e r of effects of progesterone on the uterus and endometrium are estrogen dependent, uteroglobin synthesis induction by progesterone has been frequently used as a marker for progesterone action [6]. Unlike progesterone, 5 e - D H P , which has been shown to stimulate selective F S H release, was found to be

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unable to stimulate uteroglobin synthesis [64]. Thus, progesterone metabolites may not be active in all tissues. T h e pioneering work of Selye in 1942, showing that steroids could produce anesthetic effects [65], led to the development of a variety of steroidal anesthetics. Several of these anesthetics were the 3e,5~t- and the 3e,5fl-reduced progesterone metabolites which reportedly do not bind to the classical intracellular progesterone receptors [54,55,66]. Recent studies have provided evidence that steroid metabolites, particularly the 3ct-hydroxy ring-A compounds are potent regulators of the GABAA receptor system in the brain [56, 67-70]. T h e major mode of action of these steroids appears to be exerted through the GABAA receptor mediated chloride ion flux [67, 69, 70]. 3ct,5~-THP and 3~t,5ct-tetrahydrodeoxycorticosterone are approximately 10 times more potent than benzodiazepines and 200 times more potent than pentobarbital in potentiating GABA-mediated chloride uptake in rat cerebral cortical synaptoneurosomes [67, 69]. T h e 3ct,5ct-reduced metabolites of progesterone gain added significance because progesterone can be reduced to these metabolites in neurons and glial cells. T h e physiological significance of the regulation of 3 e - h y d r o x y ring A reduced steroids is far-reaching as such interactions could have importance in stress, cycle related seizures, premenstrual syndrome anxiety, postpartum blues, memory, cognition and depression to name only a few [70 for review]. Thus, the 3~-hydroxy ring A reduced metabolites of progesterone have unique biological activities of their own that are of considerable importance. TISSUE SPECIFIC DIFFERENCES IN THE MODE OF ACTION OF P R O G E S T E R O N E Studies by McPherson et al. [23] and McPherson and Mahesh [24] showed that progesterone had a dose-related effect on gonadotropin secretion in the estrogen-primed ovariectomized rat. In this model, the low and the high dose of progesterone stimulated gonadotropin secretion whereas the intermediate dose was ineffective. Work by Smanik et al. [28] and Calderon et al. [35] demonstrated that a reduction of occupied nuclear anterior pituitary but not hypothalamic estrogen receptors was associated with the effect of progesterone in stimulating gonadotropin secretion [3, 4 for review]. Detailed studies by Fuentes et al. [36] showed that by using the 0.8 and 4 mg/kg bodyweight dose of progesterone, the anterior pituitary occupied nuclear estrogen receptors were decreased very dramatically, whereas the intermediate 2.0mg/kg bodyweight dose of progesterone was less effective. However, in the same animal, progesterone decreased the occupied nuclear estrogen receptor of the uterus in a dose-dependent manner [36]. T h e tissue specific changes in anterior pituitary and uterine occupied

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nuclear estrogen receptors were confirmed by Fuentes et al. [71] using another steroid 5 ~ - D H P which gave results similar to that with progesterone. T h e changes in anterior pituitary estrogen receptors brought about by the low dose and high dose of progesterone as compared to the intermediate dose of progesterone were of profound biological significance because the low and high doses of progesterone stimulated gonadotropin release [23, 24] and attenuated estrogeninduced prolactin release [72]. T o date, the mechanisms involved in the multiphasic effects of progesterone on anterior pituitary estrogen receptors are not understood and this subject is being studied further.

DIVERSITIES IN THE MECHANISM OF ACTION OF STEROIDS Considerable evidence has accumulated to indicate that steroid hormones may use a n u m b e r of diverse mechanisms of action to account for their multiple actions which include rapid effects, acute effects and sustained effects [73 for review]. T h e various mechanisms are summarized in Fig. 1. Mechanism I: Classical intracellular receptor mediated mechanism

T h e classical mechanism of steroid hormone action involving intracellular binding with the hormone receptor, dissociation of the heat shock protein, dimerization, phosphorylation, binding to D N A , recruitment and stabilization of transcription factors at the promoter region and activation of R N A polymerase

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Mechanism 2 proposes that the steroid could insert in the phospholipid bilayers of membranes which would alter the fluidity of the membrane and alter ion fluidity. Although several examples have been cited in a variety of tissues [74-76], it is unclear to what degree, if any, this effect would contribute to the overall biological action of the steroid. Furthermore, it is hard to reconcile how steroid hormones could exert tissue specific effects if regulation of cell membrane fluidity was a major mechanism of membrane effects of steroids since all membranes would be expected to be susceptible to such non-specific fluidity changes. T h e high specificity of many of the steroid effects is also difficult to reconcile with the major mechanism being a nonspecific perturbation of membrane fluidity. Because of these questions, recent attention has turned to the search for membrane receptors for steroid hormones. Mechanism

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Mechanism 3 deals with the activation of steroid hormone like effects with second messenger systems. Phosphorylation is an important step in gene activation by steroid-receptor complexes and dibutyrl cAMP can elicit mating behavior in female rats similar to the effects of progesterone [77]. Progesterone-induced lordosis behavior is also facilitated by phosphodiesterase inhibitors [78]. 8 - B r o m o - c A M P has been shown to mediate progesterone receptor-dependent transcription in the absence of progesterone [79]. T h e expression of progesterone receptor-dependent transcription is also stimulated by okadiac acid, an inhibitor of protein phosphatases [79]. T h e activation of steroid receptormediated transcription by a variety of growth factors such as E G F [80, 81] and IGF-1 [82] has been demonstrated by the use of antiestrogens which were able to antagonize EGF-stimulated uterine growth and E G F and IGF-1 stimulated estrogen mediated transcription using estrogen receptor gene constructs in transfected cells. Furthermore, when progesterone receptornegative monkey kidney cells were cotransfected with a chicken progesterone receptor expression vector and a reporter plasmid, dopamine increased the progesterone receptor-mediated transcription to that comparable with progesterone induced transcription [83]. These are a few examples of multiple pathways of steroid hormone receptor activation. T h e physiological significance of these pathways has not been established. Mechanism 4: Steroid action exerted at the cell membrane

Fig. 1. V a r i o u s m e c h a n i s m s

of a c t i o n t h a t e x p l a i n r a p i d , a c u t e a n d s u s t a i n e d effects o f s t e r o i d h o r m o n e s ( F r o m B r a n n et

aL [73]).

Mechanism 4 deals with the binding of steroids with membrane receptors for which evidence has accumulated during the past 15 years [84-89]. Both neural and

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Modes of Action of Progesterone and its Metabolites 5'

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Fig. 2. Diagram of site in DNA, 5"-dTdG-3".5'-dCdA-3', which accommodates ligands in the steroid/thyroid superfamily acid 3~,,5~t-THP. Each ligand forms a unique pattern of stereospecific donor/acceptor linkages to DNA at the positions n u m b e r e d and listed in the table. The dotted line on the diagram and the double line in the table divide linkages on opposite DNA strands. The numbering of atoms on the DNA designate: the phosphate oxygens on adjacent strands which can act as either proton acceptors (PO-; Nos 1, 6) or proton donors (POH; Nos 2, 7); proton acceptor at the 04 of thymine (No. 4); proton acceptor at the 5' phosphate of thymine (PO-; No. 5); proton acceptors at the 04 of the deoxyribose sugars attached to guanine (No. 3) and adenine (No. 8); proton donor of the Nil 2of adenine (No. 9). The fits of ligands evaluated by computer modeling are designated with a *. Water molecules appear to be required for certain linkages to the base pairs, e.g. the 1l~-hydroxyl group of cortisol to the 04 of thymine; although not listed, initial computer modeling indicates the hydrogen bond of the 17~-hydroxyl of cortisol which forms a hydrogen bond to No. 3 may form a preferred linkage to the N-7 of guanine.

n o n - n e u r a l m e m b r ; m e steroid receptors have b e e n reported, a n d i n m o s t cases the steroids b i n d to the m e m b r a n e receptors w i t h m o d e s t affinity a n d w i t h specificity. T o w l e a n d Sze first d e s c r i b e d m e m b r a n e b i n d i n g sites for steroids in the b r a i n in 1983 [84]. R a m i r e z a n d coworkers [86, 87] have r e c e n t l y r e p o r t e d that radioactive tagged p r o g e s t e r o n e c o n j u g a t e d to b o v i n e s e r u m a l b u m i n (BSA) at the 11-position b i n d s to s y n a p t o s o m a l m e m b r a n e p r e p a r a t i o n s f r o m the

m e d i a l basal h y p o t h a l a m u s / p r e o p t i c area a n d the corpus s t r i a t u m . T h i s b i n d i n g can be displaced b y competition with unlabelled progesterone-BSA c o n j u g a t e s at the 11- or 3 - p o s i t i o n . T h e m e m b r a n e b i n d i n g b y p r o g e s t e r o n e - l l - B S A appears to be r e g u lated b y estradiol as specific b i n d i n g is decreased b y 8 0 % 14 days after o v a r i e c t o m y a n d restored b y estradiol r e p l a c e m e n t [87]. P r o g e s t e r o n e has also b e e n r e p o r t e d to b i n d to h u m a n s p e r m a t o z o a a n d this effect

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Modes of Action of Progesterone and its Metabolites appears to be important for the induction of calcium influx and the acrosomal reaction [98, 100]. Electron microscope autoradiographic studies have also revealed plasma membrane binding sites for estradiol in human spermatozoa although the importance and function of such sites remains to be determined [101,102]. Other studies have reported membrane binding sites for estrogens in breast cancer cells [93, 103, 104], liver [92] and uterus [105, 106]. T h u s , there is ample evidence in the literature for ste:roid bindings sites in the plasma membrane of a variety of tissues which may play a role in mediating many oJ~ the rapid non-genomic effects of steroids.

Mechanism 5: Steroid effects exerted by interaction with G A B A receptors Mechanism 5 deals with steroid hormones interacting with the GABAA receptor system to exert their biological effects. Examples of the effect of 3~,5~-THP on L H release [57] and uterine contractility [61] have been cited earlier in this paper. It has recently been reported that G T I - 1 cells convert progesterone to 3~,5~-THP, possess GABAA receptors and release L H R H in response Eo 3 ~ , 5 ~ - T H P [107]. Mechanisms 2, 4 and 5 can also explain the rapid effects of steroids that may take place within seconds to minutes, without entering into the cell and without binding with classical intracellular receptors and are not blocked by protein synthesis inhibitors.

Mechanism 6: The l~gand insertion mechanism Mechanism 6 is an extension of the classical genomic mechanism in that it proposes that the binding of hormonal ligands tc their cognate receptors and the interaction of the ligand-receptor complexes with D N A is followed by an additional step, namely, insertion of the steroids between base pairs. T h e insertion is facilitated by conformational changes in the receptor induced by ligand and occurs at the hormone responsive elements in D N A (HREs) which are known to bind receptors. In this manner, the specificity of hormonal responses are largely the results of steroid-receptor interactions whereas the magnitude of responses are governed by ligand insertion thereby insuring maximal transcriptional activity/efficiency. T h e rationale and evidence for this mechanism which involves extensive experimental and theoretical findings have been reviewed in previous papers [73, 108-110 and reJ~erences therein] and thus will be discussed only briefly below. Prior to the discovery of receptor proteins, potential interactions between steroids and nucleic acids components were c,f considerable interest as exem-

215

plified by the early findings of Munck [111,112]. In fact, Huggins and Yang showed that the steroid shape was almost congruent with that of D N A base pairs [113]. Despite numerous experimental and theoretical studies of steroid-nucleic acid complexes, binding of steroids to D N A has been shown to be universally weak [114-117 and references therein]. At the same time, while binding of steroids to their receptors has been shown to be specific, the strength of binding has not correlated well with hormonal activity [118-126]. Additional paradoxical evidence includes the finding that truncated receptors in which the ligand binding domain has been removed still increase transcription in the presence of ligand [127]. Moreover, certain ligands which compete with hormone but covalently bind with receptor have been surprisingly shown to be hormone antagonists rather than agonists [128]. These observations suggest that the role of the hormone extends beyond causing conformational changes in the receptor protein. T h a t hormonal ligands and certain antagonists can come in contact with D N A is supported by observations of covalent adducts formed with D N A both in vitro and in vivo as well as genotoxic damage caused by such ligands [129-132]. A second line of evidence consistent with the insertion hypothesis comes from various modeling observations made over the past 20 years that have been confirmed using computer graphics coupled with energy calculations. Namely, steroid hormones and other ligands in the steroid/thyroid superfamily have been shown to fit between base pairs in partially unwound double stranded D N A [73, 108-110 and references therein]. Each ligand fits particularly well into a common site 5 ' - d T d G - 3 ' . 5 ' - d C d A - 3 ' yet possesses unique stereospecific hydrogen bonds to D N A (Fig. 2), Molecules which possess the same linkages have the same hormonal activity and degree of fit into D N A correlates with the magnitude of hormonal response. Changes in the structures of either the natural ligands or D N A result in an abrupt loss of fit clearly demonstrating these observations are not fortuitous. Intermediates along the biosynthetic pathways of the steroid hormones are increasing fits into D N A whereas those in the catabolic inactivation pathways are increasingly poor fits [133]. Very recently it has become possible to examine the interface of the D N A binding domains of various receptors and their respective HREs [134]. T h e site 5 ' - d T d G - 3 ' . 5 ' - d C d A - 3 ' which fits the hormones was found in the first two bases of half site sequences of all of the HREs in the steroid/thyroid receptor family [110]. Also of interest was that the ligands which are known to fall into two classes based upon the sequence of the remaining bases of the half sites also fall into the

Fig. 3. Computer skeltal m o d e l s (A) and space filling m o d e l s (B) in stereo depicting the fit of 3a,5~-THP into partially unwound DNA at 5"-dTdG-3".5"-dCdA-3" (cf. Fig. 2). The view is f r o m the major groove with letters designating the bases and arrows depicting the location of stereospecific hydrogen bonds.

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same two classes based upon c o m m o n stereochemical linkages in the u n w o u n d site in D N A . Moreover, the interface beween the protein and the H R E s is cleft shaped with the sequence 5 ' - d T d G - 3 ' . 5 ' - d C d A - Y uniquely exposed. D o c k i n g studies demonstrate that hormonal ligands can fit into the cleft and contact these bases. Taken as a whole, experimental and theoretical findings coupled with recent modeling of the h o r m o n e receptor-gene interface provide strong evidence for the ligand insertion hypothesis. T h e h o r m o n e progesterone which is of specific interest to this communication is an excellent fit into D N A and can form two stereospecific linkages to D N A via the 3 and 20 carbonyl groups to protonated phosphate oxygens on adjacent strands CpA and T p G , respectively [135] (Fig. 2). T h e active metabolite 3 ~ , 5 ~ - T H P also fits into the site well and has a c o m m o n linkage with progesterone via the 20 carbonyl group to T p G (Figs 2 and 3) [109, 110, 133]. Unlike progesterone, 3 ~ , 5 7 - T H P has a hydroxyl group in the 3 position requiring a linkage to a negatively charged phosphate at CpA. W h e n measured with energy calculations, 3 a , 5 ~ - T H P had an overall fit of - 5 2 kcal which was 7 kcal better than progesterone. Of particular interest is that 3 ~ , 5 ~ - T H P stands apart from all ligands in the steroid/thyroid superfamily in the manner in which it forms donor/acceptor linkages to phosphate groups on adjacent D N A strands. Unlike the other ligands, it also does not have a k n o w n genomic receptor. T h e s e observations raise several questions. Is the lack of a classical receptor for 3 ~ , 5 ~ - T H P related to its unique fit in D N A compared to other ligands in the steroid/thyroid superfamily? Can 3 ~ , 5 ~ - T H P act at the D N A level without receptor or is its action associated with an yet undiscovered receptor, e.g. an orphan receptor? D o e s 3 ~ , 5 ~ - T H P act at the G A B A receptor because genomic recognition of the h o r m o n e is not possible due to lack of a genomic receptor? At present, it is not feasible to answer these questions. Nevertheless, it is reasonable to assume that interactions of 3 a , 5 a - T H P with G A B A receptors are likely to be the primary mechanism to explain its effects on chloride channels.

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Acknowledgements--This research was supported by research grant HD16688 from the National Institute of Child Health and H u m a n Development, National Institutes of Health, Bethesda M D and with support for computer modelling studies from the Georgia Reseach Alliance.

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