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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3527-3535
Copyright © 2004 by The Endocrine Society

Estrogen to Antiestrogen with a Single Methylene Group Resulting in an Unusual Steroidal Selective Estrogen Receptor Modulator

Jing-xin Zhang, David C. Labaree, Gil Mor and Richard B. Hochberg

Department of Obstetrics and Gynecology, and Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Dr. Richard B. Hochberg, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520. E-mail: richard.hochberg{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Selective estrogen receptor (ER) modulators (SERMs) are important therapeutic agents for breast cancer prevention and treatment. We have synthesized two analogs, E11–2,1 [methyl-(3,17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate] and E11–2,2 [ethyl-(3,17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate], the methyl and ethyl esters of an estradiol analog, substituted in the B ring at C-11ß with a carboxymethyl group. The shorter methyl ester, E11–2,1, has high ER affinity and high estrogenic potency in the Ishikawa estrogen cell bioassay, whereas the longer ethyl ester, E11–2,2, has even higher ER affinity, but little or no estrogenic activity. We found that this minor change of one methylene group transforms a potent estrogenic agonist into an antagonist in vitro with either ER {alpha} or ß. In the rat, E11–2,2 acts as a SERM in the uterus, where it inhibits estradiol-induced proliferation, and as an estrogen agonist in the liver and skeleton, where it decreases plasma cholesterol and increases bone growth. The characteristic feature of antiestrogens, including SERMs, is a long and polar side-chain that prevents agonist-induced conformation of helix 12 of ER. E11–2,2 with its short, nonpolar side-chain, lacks this critical structure, presenting the possibility that it might act through a unique mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ADDITION TO the many estrogen agonists, potent antagonists have been developed for therapeutic intervention, including some that are tissue specific and act as either agonists or antagonists depending upon the site of action (1, 2). They have been named selective estrogen receptor (ER) modulators (SERMs). As shown in Fig. 1Go, SERMS and pure antiestrogens (either steroidal or nonsteroidal) share the common structural feature of a long-chain extension bearing a polar or charged group close to its terminus. Recently, we synthesized an unusual compound that has the properties of a SERM, but is devoid of their characteristic long-chain and polar substituents. We had been synthesizing novel families of locally active "soft" estrogens, for treatment of vaginal dyspareunia associated with menopause or antiestrogen therapy (3, 4). These compounds are esters of carboxylic acid analogs of estradiol (E2), and the esters are ER ligands capable of estrogenic stimulation, whereas the parent, charged carboxylates, are not. This feature makes them susceptible to hydrolysis by esterases, ubiquitous enzymes that rapidly convert the estrogen esters into their carboxylates, thereby generating inactive metabolites. Because they are metabolically labile, these compounds are estrogenic only in tissues in which they are placed directly; for example, the vagina. Substituents were placed at positions in E2 that are known to minimally interfere with binding (7{alpha}-, 11ß-, 15{alpha}-, and 16{alpha}-). Strangely, some of the derivatives at C-11ß exhibited unusual properties: a dramatic disassociation between ER binding and estrogenic action. The methyl ester, E11–2,1 [methyl-(3,17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate; Fig. 1Go] bound strongly to the ER (Fig. 2AGo), and as expected, it was a strong agonist in the Ishikawa cell estrogen bioassay (Fig. 2BGo). In contrast, the ethyl ester, E11–2,2 [ethyl-(3,17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate; Fig. 1Go] also bound strongly to the ER (slightly better than E11–2,1), but it was almost devoid of activity in the Ishikawa assay (Fig. 2BGo). There was some minor stimulatory activity at very high concentrations of E11–2,2, but it did not approach the maximum induced with E2 or E11–2,1.



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  		FIG. 1. Structures of E11–2,1 and E11–2,2; the SERMs, tamoxifen and raloxifene; the pure antiestrogen, ICI 164,384; and the ERß antagonist/ER{alpha} agonist, R,R-THC.

 


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  		FIG. 2. A, Competition of binding of [3H]E2 to the ER in castrate rat uterine cytosol. B, Estrogenic stimulation of AlkP in Ishikawa cells. C, Inhibition of estrogenic stimulation of AlkP in Ishikawa cells. The cells were stimulated with 10–9 M E2 and the indicated concentration of E11–2,2 or E11–2,1. None of the studies is background subtracted. Note (C) that approximately 10–7 M E11–2,2 completely suppresses E2 stimulation. A and B are drawn from data in representative experiments previously reported (4 ). Error bars show the SEM.

 
The Ishikawa cell estrogen bioassay, which was developed in this laboratory, has been widely used because it closely mirrors the in vivo behavior of estrogens (5). In most cases there is parallelism between ER affinity and Ishikawa cell potency. Consequently, disassociation between ER binding and biological stimulation is unusual for estrogen agonists, but it is a fundamental property of estrogen antagonists and indicates that E11–2,2 might be one. If so, this seemingly minor modification of a one-carbon methyl ester (E11–2,1) into a two-carbon ethyl ester (E11–2,2) has transformed a relatively strong agonist into an antagonist. However, E11–2,2 is devoid of the long polar side-chain that is a hallmark of an antiestrogen and would be a highly unusual estrogen antagonist. This led us to undertake the study reported in this paper to investigate the possibility that E11–2,2 is an antiestrogen or a SERM.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
E11–2,1 and E11–2,2 [systematic names are, respectively, methyl (3, 17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate; ethyl (3,17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate] were synthesized as we previously described (4). Statistical analyses (ANOVA) were performed using PRISM (GraphPad, Inc., San Diego, CA).

Ishikawa cell assay

The Ishikawa cell assay was performed as we previously described (5). In short, the cells were grown in 96-well plates in estrogen-free medium (phenol red free, with charcoal-stripped calf serum) in the presence of (stimulatory assay) test compounds as well as E2 and estrone (E1; Steraloids, Inc., Newport, RI) at concentrations that were varied over several log orders. For the antiestrogen assay, a range of concentrations of E11–2,1 or E11–2,2 was added concurrently with 1 nM E2. The treated cells were grown for 3 d. To determine alkaline phosphatase (AlkP) activity, the cells were frozen, defrosted, and incubated with the chromogenic substrate, p-nitrophenylphosphate, at room temperature. The hydrolysis product, p-nitrophenol, was measured kinetically at 405 nm. The Ki was determined by comparison with the Ka of E2 (determined in parallel) using the curve-fitting program PRISM.

ER{alpha}, ERß, and ER element (ERE)-transfected JAR cells

The specificity of the antiestrogenic activity relative to the ER subtypes, ER{alpha} and ERß, was determined in the human choriocarcinoma JAR cell line transfected with plasmids containing a consensus ERE fused to a firefly luciferase reporter gene and separately with the expression vectors for either human ER{alpha} or human ERß. JAR cells were routinely cultured in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum, 0.5% nonessential amino acids (Life Technologies, Inc.), and 1% PEST (100 U penicillin/ml and 100 µg streptomycin/ml). Cells were seeded in six-well plates 24 h before transfection. Transfections using the Mirus Trans IT (Mirus Corp., Madison, WI) reagent were performed as described by the manufacturer in a serum- and antibiotic-free mixture of phenol-red free OptiMEM with 0.75 µg 3xERE-TATA-Luc reporter (6, 7) and 0.1–0.4 µg pCXN2 human ER{alpha} or pCXN2 h-ERß as indicated (8). The pCXN2 h-ER{alpha} (9) and pCXN2 h-ERß (10) were gifts from Prof. Satoshi Inoue (University of Tokyo, Tokyo, Japan) (11). The ERE-reporting vector was constructed by introducing an HpaI/BglII fragment containing 3x ERE-TATA into SmaI/BglII of the pGL3-Luc basic vector (Invitrogen, Carlsbad, CA). Medium was changed to a phenol red-free RPMI containing 10% dextran-coated charcoal-treated calf serum, 0.5% nonessential amino acids, and no PEST. After 24 h, E2 and the indicated concentrations of E11–2,2 or vehicle (0.1% ethanol) were added simultaneously. Cells were incubated for 12 h at 37 C in 5% CO2. The cells were harvested in 10 mM Tris-HCl/10 mM EDTA/150 mM NaCl and centrifuged for 4 min at 4000 rpm, supernatant was removed, and cell pellets were lysed in Lysis Buffer 2 (Bio-Orbit, Turku, Finland). Luciferase activity was measured using the GenGlow system (Promega, Madison, WI).

In vivo estrogenic/antiestrogenic activity

All animal procedures were approved by the Yale University Institutional animal care and use committee.

Western blot analysis of ER{alpha}

ER{alpha} was determined by Western blotting, performed essentially as we previously described (7). Ishikawa cells treated separately with vehicle, 10–7 or 10–8 M E2, and E11–2,2 were grown for 3 d under conditions described above for the AlkP assay. Afterward the cells were washed three times with PBS and lysed using 1% Nonidet P-40 and 0.1% sodium dodecyl sulfate in the presence of protease inhibitors. Proteins (25 µg/well) were separated by SDS-PAGE on ice using 10% polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were stained with Ponceau Red before the antibody incubation to ensure proper transfer. Immunoblotting was performed after blocking the membranes with 5% powdered milk in water. The blots were incubated first with the ER{alpha} monoclonal antibody clone 6F11 (Novocastra, Newcastle, UK) overnight at 4 C. ER{alpha} was detected using peroxidase-labeled horse antimouse secondary antibody (Vector Laboratories, Burlingame, CA) and Chemiluminescence Reagent Plus (PerkinElmer, Wellesley, MA). The intensity of the signal was analyzed using a digital imaging analysis system (1D Image Analysis software; Scientific Imaging Kodak Co., Rochester, NY). ß-Actin was used as an internal control to normalize the amount of protein loaded in the gels.

Uterotrophic stimulation in immature rats

The uterotrophic assay was performed in immature rats as previously described (12). Female Sprague Dawley rats, 22 d old, were injected sc daily for 3 d with E2 (20 ng, total dose), E11–2,2 (1 and 10 µg), or a mixture of E2 and E11–2,2. Control animals received vehicle (0.1 ml sesame oil). On the fourth day, animals were killed, and uteri were removed, dissected, blotted, and weighed. Each compound was assayed in two separate experiments, with five or six animals per group in each.

Tissue-selective effects in ovariectomized rats

To determine whether E11–1,2 had tissue-selective effects, ovariectomized, approximately 250-g female Sprague Dawley rats were injected with 400 ng/kg E2, three different concentrations of E11–2,2 (20, 60, and 600 µg/kg), or vehicle alone (0.1 ml sesame oil) sc for 35 d. The following day the animals were killed by exsanguination while under ether anesthesia.

Cholesterol. Serum was obtained, and the total cholesterol concentration was determined by a commercial chromogenic assay (Roche, Indianapolis IN).

Uteri. The uteri were dissected, weighed, fixed in formalin, and imbedded in paraffin, and 5-µm sections were prepared. Endometrial luminal epithelium and glandular cell height were measured using the Openlab image analysis system (Improvision, Lexington, MA). Cellular height (in micrometers) was based on calibration with an ocular micrometer (x400) on the microscope. Three to six regions were analyzed in each slide.

Bone. The tibia were dissected free of extraneous tissue and then analyzed histomorphometrically as follows. The bones were fixed in 70% ethanol, dehydrated in graded ethanol, and cleared in toluene. The specimens were then infiltrated with increasing concentrations of methymethacrylate (MMA) and embedded in MMA, as previously described (13). After polymerization, MMA blocks were cut to size, sanded, and polished to the appropriate level. Sections of 4–5 µm were cut, mounted on gelatin-coated slides, and stained with toluidine blue. The bone was analyzed in a blinded manner for standard histomorphometrical measures (14) using the computerized Osteomeasure analysis system (Osteometrics, Atlanta, GA).

The histomorphometric measurements of bone and uterus were performed in tissue sections selected randomly by an independent individual blinded to the treatment groups. The results were compared with control animals receiving sesame oil alone and to the group that was injected with E2.

In a separate experiment, 250-g ovariectomized rats were injected sc with 400 ng/kg E2, 600 µg/kg E11–2,2, or vehicle alone (0.1 ml sesame oil) for 8 d. Steroid administration was started immediately after surgery (d 0). For acclimatization, the animals were handled several times a day, every day. On the evening of the seventh day, food was removed; the next morning the animals were restrained, and core body temperature was measured rectally with a rat probe (Physitemp Instruments, Inc., Clifton, NJ). Afterward they were anesthetized with ether, weighed, and killed by exsanguination. Uteri were weighed as described above, dried overnight in an oven at 105 C, and then weighed again to obtain dry weight. Serum cholesterol levels were determined as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ishikawa cells

The antiestrogenic activity of the two E2 analogs was determined in the Ishikawa cell bioassay by adding the carboxylic acid ester concurrently with 1 nM E2. E11–2,1 does not inhibit the estrogenic effect of E2 on AlkP (Fig. 2CGo). In contrast, E11–2,2, causes a marked inhibition of estradiol, which at approximately 10–7 M reduces estrogenic stimulation to baseline (Fig. 2CGo). The Ki of E11–2,2 determined in four separate experiments performed in duplicate is 3.9 ± 1.4 nM.

ER{alpha}- and ERß-transfected JAR cells

To determine whether E11–2,2 inhibited the estrogenic action of E2 in either ER subtype specifically, JAR cells stimulated with 10–9 M E2 and transfected with either ER{alpha} or ERß were concomitantly incubated with varying concentrations of E11–2,2. As shown in Fig. 3Go, E11–2,2 completely abolished, with approximately the same potency, the estrogenic stimulation of the luciferase reporter in cells containing either ER{alpha} or ERß. Consequently, E11–2,2 acts as an antiestrogen with both ER subtypes.



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  		FIG. 3. Antiestrogenic action of E11–2,2 in JAR cells transfected with an ERE-Luc and ER{alpha} (A) and ERß (B). The cells were grown for 12 h in the presence of 10–9 M E2 and the indicated concentrations of E11–2,2. In both panels, 100% is the luciferase response normalized to 10–9 M E2 alone. Error bars show the SD.

 
Effect of E11–2,2 on ER{alpha} in Ishikawa cells

Ishikawa cells were grown in the presence of vehicle, 10–7 and 10–8 M E2, and 10–7 and 10–8 M E11–2,2. After 3 d, ER{alpha} protein content was determined by Western blotting (Fig. 4Go); compared with the vehicle control (without added steroid) the levels were: 10–8 M E2, 72%; 10–7 M E2, 71%; 10–8 M E11–2,2, 107%; and 10–7 M E11–2,2, 104%.



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  		FIG. 4. Western blot analysis of ER{alpha}. Ishikawa cells were grown for 3 d with the following treatments: 1) vehicle control, 2) 10–8 M E2, 3) 10–8 M E11–2,2, 4) 10–7 M E2, and 5) 10–7 M E11–2,2.

 
Uterotrophic effect in the immature rat

The estrogenic and antiestrogenic actions of E11–2,2 at two doses given alone or simultaneously with 20 ng E2 were determined in the classical estrogen bioassay, uterotrophic stimulation in the immature rat (12). Twenty nanograms of E2 is a relatively low dose, given that 5 ng E2 is the lowest dose that consistently produces statistically significant uterotrophic stimulation (3). As shown in Fig. 5Go, the 1-µg dose of E11–2,2 produced a very small uterotrophic response that was not statistically different from the control. This dose of E11–2,2 partially inhibited the uterotrophic stimulation of E2 (37%; P < 0.001). A larger dose (10 µg) of E11–2,2 produced a small, but statistically significant, increase in uterine weight (P < 0.01). However, as shown in Fig. 5Go, this dose of E11–2,2 completely inhibited E2 stimulation of the uterus (P < 0.001), e.g. the uterine weight of the group given the combination of 10 µg E11–2,2 plus E2 was the same as that of animals receiving 10 µg E11–2,2 alone. Although E11–2,2 was slightly uterotrophic, this effect, unlike that of E2, was not due to the stimulation of cell growth (see below). E11–2,2 completely abolished the action of E2 on the uterus.



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  		FIG. 5. Uterotrophic action of E11–2,2 in the immature rat. Twenty-one-day-old rats were injected sc (total dose) with 20 ng E2 with or without the indicated dose of E11–2,2 in 0.1 ml sesame oil or with vehicle alone (control) for 3 d. The 1-µg dose of E11–2,2 did not stimulate the uterus, but produced a statistically significant decrease (P < 0.01) in E2 stimulation. E11–2,2 at 10 µg caused a statistically significant increase (P < 0.01) in uterine weight, but completely inhibited the effect of E2 (P < 0.01). Error bars show the SD.

 
Ovariectomized rat (35-d treatment)

To test for possible tissue-selective effects of E11–2,2, body weight, uterine weight, plasma cholesterol, and various bone parameters were measured in ovariectomized adult female rats given three different doses of E11–2,2, E2, or vehicle (sesame oil). This experimental design is similar to that previously reported for raloxifene (15), except in that study the compounds were administered orally and compared with ethinyl estradiol, whereas in this study the steroids were administered sc and compared with E2. Oral administration was avoided because E11–2,2 is an ester and is easily cleaved by hydrolytic enzymes. It is unlikely to survive in the gastric environment. Subcutaneous administration of estrogens does not abrogate hepatic stimulation, which results in the lowering of blood cholesterol (16).

Body weight. Compared with the ovariectomized control, only E2 and the highest dose of E11–2,2 (600 µg/kg) produced a statistically significant decrease in body weight (Table 1Go). The weights of the animals treated with the two lower doses of E11–2,2 were the same as those of the controls.


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  		TABLE 1. Effect of E11-2,2 on body weight, uterus, and bone in the ovariectomized rat

 
Uterus. Of the three doses of E11–2,2 only two of them, the lowest and the highest, produced a statistically significant increase in uterine weight (20 µg/kg, P < 0.01; 600 µg/kg, P < 0.05; Table 1Go). As evident from the data in Table 1Go, the uterotrophic weight response to E11–2,2 was not dose dependent, because the uteri in the animals receiving the lowest dose of E11–2,2 weighed more (324 mg) than those in the animals receiving the highest dose (306 mg). The uteri from the animals receiving the intermediate dose of E11–2,2 (60 µg/kg) were not significantly different from those of the controls. None of these groups approached the uterine stimulation observed in the E2-treated group (475 mg). In addition to uterine weight, histomorphometric analysis of the height of cells in the lumen and gland of the uterus was performed (Table 1Go). As shown, E2 more than doubled cell height in the lumen, whereas the two lower doses of E11–2,2 had no effect, and the increase in cell height of the lumen in the group receiving the highest dose (600 µg/kg) was not statistically significant. In the gland, E2 almost doubled the cell height, and again, there was no statistically significant effect of any of the doses of E11–2,2.

Plasma cholesterol levels. E2 (400 ng/kg) decreased plasma cholesterol levels to a little more than 70% of those in ovariectomized controls (Fig. 6Go). Likewise, E11–2,2 decreased plasma cholesterol at the two higher doses (60 and 600 µg/kg; P < 0.01). The cholesterol level in the group receiving the lowest dose of E11–2,2 (20 µg/kg), although lower, was not significantly different from the control level. Compared with the animals receiving E2, the effect of the 60 µg/kg dose of E11–2,2 was approximately the same, whereas the cholesterol level in the 600 µg/kg group was lower than that in the E2 group (P < 0.05).



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  		FIG. 6. Effect of E11–2,2 on serum cholesterol in ovariectomized rats. Mature ovariectomized rats were injected sc daily with E2 or E11–2,2 at the indicated dose in 0.1 ml sesame oil for 35 d. *, P < 0.01. Error bars show the SD.

 
Bone. A large number of bone indexes were assessed in the ovariectomized rat by histomorphometric analysis. In all, eight of them showed significant (P < 0.01) differences between the ovariectomized controls and the E2-treated animals, including bone volume, osteoid volume, total osteoid surface, osteoid surface, osteoid volume density, osteoid surface density, trabecular separation, and trabecular number. Three of them are presented in Table 1Go. Only the estrogen-like effect on trabecular space was statistically significant at all three doses of E11–2,2. Stimulation of trabecular number was statistically significant only at the highest dose of E11–2,2. At the two lower doses of E11–2,2, trabecular number was higher than the ovariectomized control value, but this trend was not statistically significant. The same trend was evident for bone volume with E11–2,2. Of the remaining bone indexes (above) in which E2 produced a statistical effect at P < 0.01, none of them showed a statistically significant response to any dose of E11–2,2. E2 significantly stimulated five other indexes (P < 0.05): osteoid volume, osteoid /lamellar osteoid surface, osteoclast surface, osteoclast bone surface density, and osteoblast surface density. Three of these five had trends with E11–2,2 similar to E2, but none was statistically significant. In all, none of this latter group was significantly affected by E11–2,2.

Ovariectomized rat (8-d treatment)

Another study was performed in ovariectomized rats that were treated with the highest dose of E11–2,2 used in the previous experiment (600 µg/kg) and compared with E2-treated and control groups (Fig. 7Go). In this experiment, this dose of E11–2,2 again was slightly uterotrophic compared with the controls. Dry uterine weight was also slightly elevated. The increase in both uterine parameters was considerably less than that the E2-treated animals. As previously reported, E2 increased the core body temperature compared with the ovariectomized controls (17). The core body temperature of the E11–2,2 group was higher than that of the controls, but this was not statistically different from either the control or the E2 groups. In contrast, although E2 treatment lowered both body weight and plasma cholesterol levels, the effect of E11–2,2 was greater on both parameters.



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  		FIG. 7. Effect of E11–2,2 (600 µg/kg) and E2 (400 ng/kg) treatment for 8 d in ovariectomized rats. *, P < 0.001; **, P < 0.01; ***, P < 0.05. Error bars show the SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In addition to the classical ER, now termed {alpha} (ER{alpha}), another receptor has been discovered, ß (ERß) that is present in relatively large amounts in the prostate and ovary (10). Although the physiological role of ERß is not totally clear, peripheral monocytes that express ERß predominantly respond to estrogen stimulation by undergoing apoptosis (7), and ERß knockout mice develop a myeloproliferative disease similar to myeloid leukemia (18). Thus, estrogens acting through ERß influence physiological processes that are not usually associated with these ovarian hormones. It is reported (patent application) that 11ß-analogs of E2, which are similar in substituent length to E11–2,2, but substituted instead with pure alkanes and alkenes from C5 to C9 in length, are antagonists with ERß, but agonists with ER{alpha} (19) in cells transfected separately with the two ER subtypes and a reporter gene. If E11–2,2 acts through the same mechanism, its antiestrogenic effect would be transmitted through ERß. However, it seemed likely to us that the inhibitory effect of E11–2,2 on Ishikawa cell AlkP is different and that it is directed through ER{alpha}. Although the Ishikawa cell contains both ER{alpha} and ERß (20), various ligands, such as 16{alpha}-substituted estradiol analogs (3, 5), which bind to ER{alpha} preferentially (21), produce a stimulatory effect on Ishikawa cell AlkP appropriate to their ER{alpha} affinity (3), whereas phytoestrogens, which bind to ERß preferentially (22), produce a poor response (6, 23). We found that the pure ER{alpha} agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (24), is very potent in the stimulation of Ishikawa cell AlkP and that the ERß agonist 2,3-bis(4-hydroxyphenyl)propionitrile (25) is almost 500-fold less active (unpublished observations). This strongly suggests that Ishikawa AlkP stimulation acts preferentially through ER{alpha}. Nevertheless, because E11–2,2 binds to ERß with high affinity (4), and the two ER subtypes can form heterodimers (26), the possibility existed that the antiestrogenic action of E11–2,2 could be caused by the formation of an inhibitory complex, a heterodimer, ERß-E11–2,2/ER{alpha}-E2, and also, if E11–2,2 is an ER{alpha} agonist, the heterodimer ERß-E11–2,2/ER{alpha}-E11–2,2. Consequently, we investigated the action of E11–2,2 in JAR cells that had been separately transfected with ER{alpha} and ERß and an ERE linked to a Luc reporter gene. E11–2,2 inhibits E2-stimulated transcription of the reporter gene with both ER subtypes. Clearly, if the E211ß-substituted alkanes and alkenes (above) are ER{alpha} agonists and, conversely, ERß antagonists, then E11–2,2 which is an ER{alpha} antagonist, is acting through a markedly different mechanism with ER{alpha}.

The antiestrogenic action of E11–2,2 was investigated further in the classical estrogen bioassay by determining its effect on the uterotrophic stimulation of E2 in the immature rat, an ER{alpha} model. The rat uterus has a preponderance of ER{alpha} compared with ERß (23), and the uterotrophic response to estrogens has been shown to be ER{alpha} ligand selective (27). Additionally, the uterus of the ER{alpha} knockout mouse does not respond to E2 (28). As shown in Fig. 5Go, E11–2,2 inhibits the uterotrophic stimulation of E2. Clearly, E11–2,2 possesses antiestrogenic action in the uterus, an ER{alpha}-selective action.

The slight stimulatory activity of E11–2,2 alone in the Ishikawa assay and the uterotrophic assay more closely resembles that produced by SERMs rather than that of pure antiestrogens. which have no stimulatory action (29). This was tested in vivo in the ovariectomized rat. As in the immature rat, E11–2,2 again caused a slight increase in uterine weight. However, this was not dose dependent, and the small stimulation seen in the group receiving the intermediate dose (60 µg/kg) was not statistically different from that in the ovariectomized control. Moreover, histomorphometric analysis showed that the uterus in E11–2,2-treated animals was not different from that in the control at any of the doses. One likely explanation for the increase in uterine weight in the E11–2,2 groups that is not paralleled by an increase in cell size is that the weight gain is due predominantly to water imbibition, which has been shown to occur through a pathway distinct from that of classical estrogen action (30). However, uterine dry weight is elevated over the control group in the animals treated with 600 µg/kg E11–2,2 for 8 d, which eliminates this possibility. A more likely explanation is that the difference in uterine weight is too small to be detected morphologically. In contrast to the uterus, E11–2,2 produces a dose-dependent decrease in plasma cholesterol. The decrease in plasma cholesterol is a well known estrogenic action on the liver, which in the rat causes an increase in hepatic low density lipoprotein receptors with a concomitant clearance of low and high density lipoprotein cholesterol (31). Thus, in contrast to the uterus, E11–2,2 induces an estrogenic effect in the liver. Although E11–2,2 has some effect on the bone (Table 1Go), it is modest. In the 8-d study, the same effect of E11–2,2 on body weight and cholesterol level was observed. In this experiment the estrogenic effect on core body temperature was measured, and although E2 increased the temperature significantly, the rise observed with E11–2,2 was not statistically significant. The cause of this weak effect in bone and body temperature is not known, but some of the possibilities are: tissue-specific transcriptional activity, low ER levels, or local metabolic inactivation of this labile steroid. These studies demonstrate that E11–2,2 has the properties of a SERM: it is antiestrogenic in the Ishikawa cell, in JAR cells transfected with ER{alpha} or ERß, and in vivo in the uterus, but it is estrogenic in liver and bone.

For all antiestrogens (ER{alpha}), the key structural feature in their biological action is their long and polar side-chain. It prevents helix 12 in the ER from attaining the conformation required for coactivator binding, which leads to gene transactivation (32). As shown in Fig. 1Go, the difference between E11–2,2 and other antiestrogens is not subtle. The side-chain is uncharged, nonpolar, and small compared with the other antiestrogens. The ester group in the side-chain of E11–2,2 is present in exactly the same position and at the same distance from the steroid nucleus as it is in E11–2,1, which is a potent estrogen agonist. Thus, the ester group by itself is obviously not the cause of the antiestrogenic action. Structurally, the difference between the estrogen E11–2,1 and the antiestrogen E11–2,2 is minor, a single methylene group. This difference between a methyl ester and an ethyl ester increases the length of the side-chain by one carbon atom, from four to five nonhydrogen atoms. This seemingly small difference produces a dramatic change from an estrogen to an antiestrogen. Small structural modifications such as this that lead to large biological changes are called an activity cliff (33). Apparently, the increase from four to five atoms in the 11ß side-chain produces an activity cliff in the interactions of E11–2,1 and E11–2,2 with the ER. We found that moving the ester group further from the ring by increasing the carboxylic acid group by one methylene unit, as in E2-11ß-yl-propionate, has little effect on ER binding of the methyl (E11–3,1) or ethyl (E11–3,2) esters. Both have high affinity for ER, and again, both are almost devoid of estrogenic activity in the Ishikawa assay (4). Like E11–2,2, these two E2 analogs are potent antiestrogens in the Ishikawa cell assay (not shown). Thus, the changeover to an antiestrogen is not simply due to the difference between a methyl and an ethyl ester, nor is it restricted to a unique length of five atoms. In the latter compound, E11–3,2, the length of the side-chain is six (nonhydrogen) atoms (five carbons and one oxygen). Consequently, it appears that five atoms is the minimum length, but not the exclusive length, that is required for the side-chain to impart antiestrogenic properties to the steroidal structure.

The x-ray crystal structure of the ER ligand binding domain (LBD) in complex with several agonists and antagonists has been analyzed (32, 34). Comparisons of the conformations of these structures have provided strong evidence of the mechanisms involved. The binding of an agonist to ER involves a complicated complex of several different regions and helixes of the LBD. Similar conformational changes occur upon binding of various antiestrogens and SERMs. However, with these ligands, helix 12, the most C-terminal helix in the LBD, does not attain the proper orientation. The long side-chain present in SERMs extends out of the ER binding pocket and prevents helix 12 from assuming the agonist-activated conformation (32, 34). It assumes an alternate conformation, which prevents binding to the ER of various coactivators that are necessary for transcriptional activation (32, 34). Pure antiestrogens act through a different, but related, mechanism. The crystal structure of the pure antiestrogen, ICI 164,384, bound to the LBD of ERß has been determined, and in this complex the side-chain protruding from the steroid nucleus at C-7{alpha} also prevents helix 12 from attaining its proper orientation (35). The side-chain of ICI 164,384 binds directly to the coactivator recruitment site of the LBD and physically stops the agonist positioning of helix 12. This results in the destabilization of helix 12, which leads to proteolysis of the antiestrogen-ER complex and, thus, depletion of cellular ER (36). As would be expected for a SERM, E11–2,2 had no effect on the concentration of ER{alpha} in Ishikawa cells.

If the long and polar side-chain is the critical factor in blocking estrogen gene activation, how does a steroid with a relatively small and nonpolar side-chain, such as E11–2,2, function as an antiestrogen (SERM)? Given the structures of the other antiestrogens, it is likely that E11–2,2 does not have the characteristics that are required to prevent helix 12 from adopting the ligand-activated conformation. As noted above, E2 analogs with 11ß side-chains consisting of alkanes and alkenes five carbon atoms long (E11–5) or longer, compounds similar to E11–2,2, have been reported to be estrogen antagonists, but only with ERß (19). In contrast to E11–2,2, these are ER{alpha} agonists. This differential behavior between the two ER subtypes is not unprecedented. The R,R enantiomer of tetrahydrochrysene (R,R-THC) has two ethyl groups as side-chains, and it, too, is an ERß antagonist and an ER{alpha} agonist (37). The crystal structure of the THC-ERß complex has been determined and found to be very different from that produced by the interaction of the long side-chain antagonists (38). THC, lacking a bulky side-chain, cannot prevent helix 12 in ERß from adopting the agonist-activated conformation; instead, it appears to stabilize helix 12 in an inactive conformation. Thus, the mechanism of THC estrogen antagonism of ERß has been termed passive antagonism, differentiating it from antiestrogenic mechanisms that directly prevent helix 12 agonist positioning, termed active antagonism. Perhaps the 11ß-pentyl analog of E2, E11–5, which also has a short side-chain, acts as ERß-antiestrogens through a passive mechanism.

Obviously, there are strong similarities between E11–2,2 and E11–5. That E11–2,2 is an antagonist of ER{alpha}, whereas E11–5 is an agonist, strongly suggests that the oxygen atoms in the ester group are responsible for this difference, possibly through hydrogen binding to amino acids in either helix 12 or to the groove in the LBD to which helix 12 must bind in the agonist state. Currently we are performing structure-activity relationship studies to determine the specific structural requirements that are necessary for ER{alpha} antagonism. It should be possible to design SERMs that are similar to E11–2,2, but contain metabolically stable substituents. Importantly, such estrogenic ligands with apparently different mechanisms of antagonism could exert novel biological and therapeutic effects (38).


   Acknowledgments
 
We are grateful for the excellent technical assistance of Toni Reynolds and Christopher Forestiere.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants CA-37799 and HL-61432 (to R.B.H.) and CA-92435 (to G.M.) and by the Physiology Core of the Yale Core Center for Musculoskeletal Disorders (P30 Core Center Award AR46032).

Abbreviations: AlkP, Alkaline phosphatase; E1, estrone; E11–2,1, methyl-(3,17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate; E11–2,2, ethyl-(3,17ß-dihydroxyestra-1,3,5(10)-triene-11ß-yl)acetate; E2, estradiol; ER, estrogen receptor; ERE, ER element; LBD, ligand binding domain; MMA, methymethacrylate; SERM, selective ER modulator; THC, tetrahydrochrysene.

Received November 18, 2003.

Accepted March 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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