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Molecular Endocrinology, doi:10.1210/me.2003-0491
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Molecular Endocrinology 18 (5): 1120-1130
Copyright © 2004 by The Endocrine Society

Estren (4-Estren-3{alpha},17ß-diol) Is a Prohormone that Regulates Both Androgenic and Estrogenic Transcriptional Effects through the Androgen Receptor

Michael Centrella, Thomas L. McCarthy, Wei-Zhong Chang, David C. Labaree and Richard B. Hochberg

Departments of Surgery (M.C., T.L.M., W.-Z.C.) and Obstetrics, Gynecology and Reproductive Sciences (D.C.L., R.B.H.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Michael Centrella, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208041, New Haven, Connecticut 06520-8041. E-mail: michael.centerlla{at}yale.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alternative mechanisms of steroid action, through both traditional nuclear receptors and indirect pathways of gene activation, are emerging. Recent studies suggest that the synthetic steroid, 4-estrene-3{alpha},17ß-diol (estren), has nongenotropic as well as sex-nonspecific osteogenic effects in ovariectomized and orchidectomized mice. We found limited estrogen receptor-dependent effects by estren on gene expression in primary osteoblast cultures and showed that it binds poorly to estrogen and androgen receptors in vitro. However, estren potently regulated direct and indirect androgen receptor-dependent effects on gene expression by osteoblasts. Consistent with this, osteoblasts produced the potent androgen 19-nortestosterone from estren by way of a 3{alpha}-hydroxysteroid dehydrogenase-like activity. Moreover, recombinant 3{alpha}-hydroxysteroid dehydrogenase (AKR1C9) and osteoblast-derived cell lysate each effectively converted estren to 19-nortestosterone in vitro, and mRNA encoding this enzyme occurs in osteoblasts. In addition to its androgenic activity, estren potently stimulated androgen receptor-dependent effects on gene expression through conventional estrogen-sensitive transcriptional elements in osteoblasts. Therefore, through local metabolism, estren indirectly activates the androgen receptor to regulate both androgen- and estrogen-like transcriptional responses by bone-forming cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INITIAL EVIDENCE SHOWED that sex steroids act through direct genomic interactions by hormone-activated receptors, which contain domains necessary for ligand binding, dimerization, DNA binding, and gene transactivation. However, sex steroids also cause indirect effects on gene expression through complexes formed between their own receptors and other transcription regulators, or through steroid-dependent changes in the activity of other transcription factors (1, 2, 3, 4, 5, 6, 7, 8). Therefore, sex steroids can stimulate or inhibit gene expression when activated steroid receptors form competent homodimers, or associate with other proteins to form active or inactive transcriptional or regulatory complexes.

One well-recognized target tissue for sex steroids is the skeleton. Bone forms and remodels throughout life. However, when osteoblastic bone formation diminishes relative to osteoclastic bone resorption, bone mass is reduced, fractures increase, and mobility and function are impaired. These pathologies are especially notable when sex steroid levels fall in the elderly or after sex organ ablation (9, 10, 11). In this situation, bone loss is thought to follow a release from native constraints on osteoclast development through a sex steroid-dependent decrease in transcription factor CCAAT enhancer binding protein ß (C/EBPß; also termed nuclear factor-IL-6) activity and its enhancing effect on IL-6 expression by osteoblasts (12, 13). Estradiol also suppresses the synthesis of the bone growth factor IGF-I by inhibiting C/EBP{delta}-dependent activation of the IGF-I gene promoter in osteoblasts (14). Imbalances in the rate of bone remodeling after sex steroid withdrawal can be restored by steroid hormone replacement therapy. This occurs, at least in part, through its modulating effects on these and other important osteoblast transcription factors, as well as its opposing effects on osteoblast and osteoclast apoptosis, (6, 7, 8, 14, 15, 16).

Regardless of its beneficial effects on the skeleton, pharmacological treatment with native sex steroids risks the prospect of inappropriate or uncontrolled gene activation in hormone-sensitive tissues throughout the body (17, 18). Consequently, synthetic sex steroid-like molecules that exhibit a possibly more tissue-restricted or function-restricted focus attract considerable interest. One such compound, 4-estrene-3{alpha},17ß-diol (estren), was recently shown to restore bone in both ovariectomized and orchidectomized mice. Those investigations showed that estren failed to bind efficiently to the estrogen receptor (ER{alpha}), leading to the speculation that estren functioned indirectly through the activation of other transcription factors in bone-forming cells (6, 7). In this study we asked whether estren could directly activate gene transcription in isolated osteoblasts. We also examined the possibility of indirect effects by estren on transcription factors C/EBP and Runx2, the expression and activity of which are associated with gene expression by differentiated osteoblasts (19, 20, 21), and whether any of these effects required specific steroid receptors. Our findings reveal surprising, complex, and possibly adverse outcomes associated with systemic estren therapy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Weak ER{alpha}-Dependent Effects on Gene Expression in Estren-Treated Osteoblasts
Primary cultures of fetal rat osteoblasts express little or no endogenous sex steroid receptors before late stage differentiation in vitro (22, 23) but respond rapidly to these hormones after transfection with expression plasmids encoding ER{alpha} or androgen receptor (AR) (8, 14). In this way they replicate, at least in part, the sex steroid-sensitive status of osteoblasts in more mature organisms (24). Importantly, they also provide a sensitive system to examine independent aspects of either ER{alpha} or AR on osteoblast activity in a system that is uncomplicated by endogenous sex steroid receptor expression. Osteoblasts that were transfected to express ER{alpha} and a reporter plasmid driven by consensus estrogen response elements (EREs) are induced by as little as 0.1 nM of estradiol (8, 14). Under these conditions, they were insensitive to as much as 10 nM of dihydrotestosterone (DHT), and were modestly activated by 10 nM of the synthetic steroid estren (Fig. 1AGo). By relation to estradiol, estren was approximately 300-fold weaker in activity.



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  		Fig. 1. Estren Has Minimal ER{alpha}-Dependent Effects on Gene Expression in Osteoblasts

A, Osteoblasts were cotransfected to express plasmids encoding ER{alpha} and a reporter driven by consensus ERE. Cells were then treated with estren (N), estradiol (E), or dihydrotestosterone (D) as indicated. B, Osteoblasts were cotransfected to express plasmids encoding ER{alpha} and reporter IGF1711b-Luc, driven by a 1.7-kb fragment of the rat IGF-I gene promoter and a 0.3-kb portion of exon I where a C/EBP-sensitive enhancer resides. Cells were then treated with vehicle (0), E, or N without (–) or with (+) PGE2 to increase endogenous C/EBP activation. C, Osteoblasts were cotransfected to express plasmids encoding ER{alpha}, M1-Runx2, in which Runx2 was fused to the GAL4 DNA binding domain, and reporter plasmid 5XGAL4, driven bv five copies of a GAL4 response element. Cells were then treated with vehicle (0), E, or N. E significantly increased gene expression by ERE and M1-Runx2 and significantly suppressed the stimulatory effect of PGE2 on IGF1711b-Luc in ER{alpha}-transfected cells. The weak effect of 10 nM N on ERE-driven gene expression was insignificant by comparison to E. D, Ligand binding in extract from rat uteri containing ER{alpha} was determined with [3H]estradiol with unlabeled E or N, as indicated.

 
Earlier studies revealed that estradiol, through ER{alpha}, differentially regulates the transcription potential of other osteoblast transcription factors. In this regard, estradiol potently suppresses the activity of transcription factors C/EBPß and C/EBP{delta} (12, 13, 14) whereas it enhances the activity of the osteoblast-restricted transcription factor Runx2 (8). In contrast to estradiol, estren at 10 nM failed to suppress the stimulatory effect of prostaglandin E2 (PGE2), which activates endogenous C/EBPs to drive IGF-I gene promoter activity (14, 25) (Fig. 1BGo). Estren also failed to replicate the stimulatory effect of estradiol on Runx2 transcriptional activity in ER{alpha} transfected osteoblasts (Fig. 1CGo). Therefore, the effect of hormone-activated ER{alpha} is sex steroid specific and mimicked only in small part by estren. Consistent with these and previous studies (7), estren bound weakly in vitro to ER{alpha}, with approximately 300-fold lower affinity relative to estradiol (Fig. 1DGo and Table 1Go).


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  		Table 1. Relative Binding by Steroids to ER{alpha} and AR in Vitro

 
Potent Androgen-Like Activity in Estren-Treated Osteoblast Cultures
In contrast to results with ER{alpha}, when osteoblasts were transfected to express AR, estren at 0.1–10 nM strongly activated gene expression through consensus androgen response elements (Fig. 2AGo). In AR-expressing cells, estren at 10 nM also suppressed PGE2-dependent activation of the IGF-I gene promoter with a potency equivalent to dihydrotestosterone (DHT) (Fig. 2BGo). Estren had no effect in the absence of AR and, similar to our earlier studies with the potent androgen methyltrienolone (8), estren did not activate either exogenous or endogenous Runx2-dependent gene expression in the presence of AR (Fig. 2CGo). Therefore, estren also caused indirect effects on osteoblast transcription factor activity that replicate those induced by native androgen. Regardless of its AR-dependent biochemical potential, however, estren also bound weakly in vitro to AR, with approximately 200-fold lower affinity relative to DHT (Fig. 2DGo and Table 1Go).



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  		Fig. 2. Estren Activates AR-Dependent Effects on Gene Expression in Osteoblasts

A, Osteoblasts were cotransfected to express plasmids encoding AR and a reporter driven by consensus ARE. Cells were then treated with N, E, or D, as in Fig. 1AGo. B, Osteoblasts were cotransfected to express plasmids encoding AR and reporter IGF1711b-Luc. Cells were then treated with vehicle (0), E, or N without (–) or with (+) PGE2, as in Fig. 1BGo. C, Osteoblasts were cotransfected to express plasmids encoding AR, M1-Runx2, and reporter plasmid 5XGAL4, as in Fig. 1CGo, or AR and reporter plasmid SXN1C, which contains 2 Runx-sensitive cis-acting response elements, to assess effects on exogenous or endogenous Runx2. Cells were then treated with vehicle (0), E, or N. D and N significantly increased gene expression by ARE, and significantly suppressed the stimulatory effect of PGE2 on IGF1711b-Luc in AR-transfected cells. D, Ligand binding in extract from rat prostate containing AR was determined with [3H]DHT with unlabeled D or N, as indicated.

 
Androgen Formation by Estren-Treated Osteoblasts
Because estren induces potent AR-dependent androgen-like activity at 0.1–1 nM (Fig. 2AGo), concentrations at which virtually no binding to AR occurs (Fig. 2DGo), we asked whether estren could be metabolized to an androgen-like steroid (Fig. 3AGo). Culture medium from osteoblasts incubated with 18 nmol estren, to permit sensitive detection of possible metabolites, produced a peak of UV-absorbing material that eluted by HPLC with the same retention time as 19-nortestosterone (19-NT) of 7.2–7.4 min (Fig. 3BGo). The novel metabolite isolated in this way was further identified as 19-NT by mass spectral analysis (Fig. 3CGo). Within 4–8 h of incubation with estren, approximately 40% of the starting material in osteoblast culture medium could be accounted for by the appearance of 19-NT, and this approached 95% by 24 h (Fig. 3Go, B and C). No 19-NT accumulated in culture medium from vehicle-treated or heat-inactivated (90 C for 5 min) and estren-treated osteoblasts after as long as 24 h of incubation (Fig. 3DGo), or when estren was incubated in a culture plate with no cells. When osteoblasts were instead incubated with 18 nmol of authentic 19-NT, essentially the entire amount of 19-NT was recovered in the medium of 24-h treated cells (data not shown). Moreover, although AR was required for estren-dependent gene expression, 19-NT was produced by osteoblasts even in the absence of AR expression. These results show that osteoblasts constitutively express an enzymatic activity that converts estren to 19-NT. They also indicate that the 19-NT produced in this way then directly activates AR-dependent gene expression, rather than other downstream biochemical events induced by either ER or AR activation or by a 19-NT-derived product. Importantly, direct treatment of osteoblasts with 19-NT replicated the effect of estren on AR-dependent gene expression driven by androgen response elements (AREs) and, like DHT, had no effect on ER{alpha}-dependent gene expression driven by ERE (Fig. 3EGo). In agreement with these results, 19-NT bound to AR with an affinity that was approximately 40% of DHT (26) and bound insignificantly to ER{alpha} (Table 1Go).



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  		Fig. 3. Production of 19-NT by Estren-Treated Osteoblasts

A, The relationship between estren and 19-NT is shown under oxidative conditions with 3{alpha}-HSD. B, HPLC of medium from osteoblasts incubated with estren, supplemented with 11-deoxycortisone (DOC) as a recovery standard. DOC elutes at 4.8 ml, and both authentic 19-NT and the product from estren-treated osteoblasts elute at 7.2–7.4 ml. C, Mass spectra analysis of the steroid produced in culture medium from osteoblasts incubated with estren reveals identity with authentic 19-NT. The diagnostic peak m/z 275 MH+ and base peak m/z 109 result from fission of the 6–7 and 9–10 allylic bonds (indicated by arrows), with hydrogen transfer to the ion fragment. D, Amounts of 19-NT produced at various times after estren addition. H-I indicates cells that were heat inactivated by incubating at 90 C for 5 min before addition of estren. E, Osteoblasts cotransfected to express plasmids encoding ER{alpha} or AR and reporters driven by consensus ERE or ARE were treated with 19-NT, as in Fig. 1AGo. 19-NT significantly increased gene expression in AR/ARE-transfected cells.

 
Analogous to the 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD)-like activity found in intact cell cultures (Fig. 3Go), recombinant rat 3{alpha}-HSD (AKR1C9) rapidly converted estren to 19-NT in vitro. Initial studies showed that the reaction was complete within 5 min of incubation at 37 C with 5 µg of enzyme, and after 15 min with 0.1–1.0 µg of enzyme. No conversion occurred in the absence of enzyme or when cofactor nicotinamide-adenine dinucleotide phosphate (NADP) was omitted, and the reaction was limited by the addition of indomethacin (27) (Fig. 4AGo). Primary cultures of osteoblasts expressed mRNA encoding the full-length 951-bp coding sequence of 3{alpha}-HSD/AKR1C9. By RT-PCR and sequence analysis, the osteoblast-derived product was indistinguishable from mRNA isolated from rat liver (Fig. 4BGo), where its expression was first characterized (28). Consistent with this, 3{alpha}-HSD activity was present in a postmitochondrial supernatant from osteoblasts. Analogous to results with recombinant 3{alpha}-HSD, the osteoblast-derived enzymatic activity was severely reduced by the absence of NADP or the addition of indomethacin (Fig. 4CGo).



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  		Fig. 4. 3{alpha}-HSD (AKR1C9) Converts Estren to 19-NT in Vitro and Is Expressed by Osteoblasts

A, Estren (3.6 nmol) was incubated for 15 min at 37 C with the amounts of recombinant 3{alpha}-HSD shown. Parallel samples omitted cofactor NADP or included indomethacin (indo, 50 µM), to inhibit 3{alpha}-HSD activity, as indicated. The amount of 19-NT production was measured by HPLC as in Fig. 3BGo. B, Total RNA isolated from rat liver or primary cultures of osteoblasts (obs) was analyzed by RT-PCR using primers corresponding to the 5'- and 3'-ends of the entire 951-bp coding sequence of rat 3{alpha}-HSD (AKR C19). A sizing ladder (stds) is shown on the left, and the arrowhead indicates the major PCR product. C, Estren was incubated for 0 or 30 min at 37 C with 20 µg of total protein from osteoblast-derived lysate. Parallel samples omitted NADP or included indomethacin, and the amount of 19-NT was measured by HPLC, as in panel A. The addition of indomethacin or the omission of NADP significantly suppressed 19-NT production by recombinant 3{alpha}-HSD and osteoblast-derived lysate.

 
Activation of ERE through AR in Estren-Treated Osteoblasts
Many sex hormone-sensitive target cells express both ER and AR (29). Therefore, we asked whether the AR-dependent effect of estren that we noted could be interpreted in some way as sex nonspecific. To address this, we tested estren on gene expression through ER{alpha} and ARE, or through AR and ERE, instead of the conventional sex steroid receptor and response element pairs. Neither estren, nor any androgen or estrogen that we examined, could activate ER{alpha} to induce ARE-driven gene expression. In contrast, estren, 19-NT, and DHT each potently activated AR to induce ERE-driven gene expression, whereas as much as 10 nM of estradiol was without effect. Surprisingly, in this context DHT exhibited approximately 30- to 100-fold lower potency relative to estren or 19-NT. This indicated that exposure to estren could highly effectively induce AR to exhibit a so-called sex-nonspecific hormone receptor-like activity (Fig. 5AGo). The specific androgen antagonist, flutamide (30), suppressed stimulation by 1 nM estren on ARE- or ERE-driven gene expression through AR by approximately 80–90%, whereas the specific estrogen antagonist, ICI 182,780 (31), had no effect (Fig. 5BGo). Even with the high concentration of 10 nM of estren, flutamide suppressed hormone activity by 50% and ICI 182,780 remained ineffective (data not shown). Therefore, estren at highly effective low concentrations appears to cause the majority of its effects on both ARE and ERE cis-acting elements through hormone-activated AR rather than through residual or newly induced ER expression.



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  		Fig. 5. Estren Enhances AR-Dependent Gene Expression by ERE in Osteoblasts

A, Osteoblasts were cotransfected to express plasmids encoding ER{alpha} and a reporter driven by consensus ARE, or AR and a reporter driven by consensus ERE. Cells were then treated with N, E, D, or 19-NT (NT) as in Figs. 1AGo and 3EGo. B, AR/ARE or AR/ERE transfected cells were treated with estren without (0) or with flutamide (F) or ICI 182,780 (I). N, D, and 19-NT significantly enhanced ERE- or ARE-driven gene expression in AR transfected cells. Flutamide significantly suppressed the stimulatory effect of estren.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Sex steroid-like compounds have often been investigated for possibly beneficial biological activities, especially as adjuvants for age-dependent decreases in ambient sex steroid status. Importantly, agents like these with focused effects on specific organ systems or specific biochemical processes would be useful to avoid adverse outcomes that might occur in response to native steroid hormone therapy. Initial studies suggested that one candidate compound, designated by Kousteni et al. (7) simply as estren, helps to maintain bone mass in sex organ-ablated mice in an apparent nongenotropic and sex-nonspecific way. Our current studies in isolated osteoblast cell cultures showed very weak estrogen-like genotropic effects by high concentrations of estren, consistent with its low affinity for ER{alpha}. Nonetheless, exposure to estren strongly activated AR-dependent gene expression through ARE even though it also bound poorly to AR. This paradox was resolved by the appearance of 19-NT (also known as nandrolone), a potent androgen derived from estren by 3{alpha}-HSD enzymatic activity, in medium from osteoblasts incubated with estren. Therefore, local metabolism may efficiently convert estren to 19-NT within the skeletal microenvironment where it directly activates gene expression. This may also occur in other tissues even in the absence of detectable levels of circulating 19-NT, but this has not yet been assessed.

It is important to note that many of our studies relied on exogenous gene transfection, which required the use of osteoblasts at a point in culture when they do not express detectable levels of sex steroid receptors (22, 23, 32, 33). To circumvent this, we transiently transfected osteoblasts to express ER{alpha} or AR. Other studies addressed this deficiency with osteoblast-like cell lines that were stably transfected to express sex steroid receptors under selective antibiotic pressure, or by transforming viral gene elements. We chose to avoid problems associated with antibiotic toxicity, unknown effects derived from stable gene integration, and the phenotypic drift that often occurs when osteoblasts are held in continuous culture. It is unknown how the levels of sex steroid receptors in transiently or stably transfected osteoblasts actually compare with cells in adult bone because, for example, the amounts of ER{alpha} in vivo vary considerably with age as well as with anatomical location (34, 35, 36, 37). We have not yet performed important studies to assess gene expression by estren in more differentiated osteoblasts or in vivo. Due to the presence of both ER{alpha} and AR in those situations, however, it would not have been possible to detect the complex effects of estren on gene expression, principally through AR, in osteoblasts. Nonetheless, gene expression by homologous sex steroid and sex steroid receptor pairs in our receptor-transfected cells was highly selective and sensitive to activation by low, physiological hormone levels (38, 39). Different effects on gene expression directly through sex steroid response elements or indirectly through other transcription factors could perhaps occur in osteoblasts transfected to express sex steroid receptors that are principally either soluble or cell membrane associated. However, when ER{alpha} is restrained by the addition of an epitope that targets it to the cell membrane, it is fully functional as a genomic transcription activator in the presence of estradiol (40). This suggests that the transcriptional effects we observe might also occur through either membrane-bound or soluble AR, but at present this remains unresolved.

The nearly stoichiometric appearance of 19-NT in cultures of estren-treated osteoblasts demonstrates that this substrate is readily oxidized. A-ring unsaturated steroids such as estren are not commonly thought of as substrates for 3{alpha}-HSDs. More common are the 5{alpha}- and 5ß-saturated steroids that can be reduced to form urinary 3{alpha}-hydroxy metabolites such as pregnanediol, androsterone, and etiocholanolone (41). In this regard, the {Delta}4-3-ketone progesterone can be converted to the so-called "neurosteroid" 3{alpha}-hydroxy-4-pregnen-20-one by 3{alpha}-HSD type 2, albeit to relatively low levels (42). Allylic alcohols such as estren are more readily oxidized than their saturated counterparts (43). Enzymatically, the absence of the C-19 methyl group from the A,B-ring junction in estren eliminates a critical element that could hinder enzymatic attack in biological steroid substrates, making estren a facile substrate for oxidation in this way. Importantly, the virtually complete conversion of estren to 19-NT by osteoblasts demonstrates that it is indeed metabolically labile. In agreement with this, we found that recombinant 3{alpha}-HSD, formally designated as AKR1C9, rapidly converts estren to 19-NT in vitro, consistent with the reaction that we observed with osteoblasts or their extracts. Individual isoforms of 3{alpha}-HSD are widely expressed in somewhat tissue-restricted ways. They exhibit bidirectional oxidative and reductive activities, although in some instances one or the other potential predominates (41). Although we cannot state for certain that this reaction is catalyzed in osteoblasts by only one of the several known 3{alpha}-HSDs, we found that rat osteoblasts express mRNA and in vitro enzymatic activity consistent with 3{alpha}-HSD/AKR1C9. Earlier studies showed that osteoblasts also express other HSD enzymes associated with steroid metabolism (44, 45, 46), but this appears to be the first evidence for 3{alpha}-HSD activity and expression by bone-forming cells.

In addition to its androgenic potential, estren potently activated AR-dependent gene expression through ERE in isolated osteoblasts. At least by way of this unexpected activity, estren appears to fulfill the criteria for a sex-nonspecific hormone. Estrogen-like biological effects through AR were also noted in rodents treated with 7{alpha}-methyl-19-nortestosterone (47). Heterologous gene activation by AR in this way may be physiologically and pharmacologically significant, linking androgenic stimulation to an estrogenic response. Although the estren metabolite 19-NT has a moderately weaker affinity for AR by comparison to DHT, its gene activation potential on ERE is greater. Differences between some steroids and their mimetics may relate, in part, to variations in ligand-induced receptor conformation. This then may affect their affinity for specific cis-acting genomic elements or for one or more of the many coregulators now known to interact with and to control specific aspects of steroid receptor-dependent gene expression (48, 49, 50, 51, 52, 53). At least 35 individual coregulators are currently associated with AR activity (54), and it will become essential in future studies to determine whether any of these molecules alter gene expression in ligand- or response element-selective ways. However this occurs in the situation with estren, it seems important to consider that some direct estrogen-like responses may occur in a tissue-specific manner through the AR by steroids that have little or no affinity for ER. In contrast to these observations, estren failed to activate ERE-dependent gene expression in uterine cells from ovariectomized mice (7). Many cells types, including those in the uterus, normally express both ERs and AR, and AR expression in the immature uterus is induced by estrogen (29, 55). Therefore, when native ambient estrogen levels are depleted by ovariectomy, uterine cells are likely to be AR deficient and appear resistant to the androgenic effects of estren treatment. With regard to male reproductive tissue, previous studies showed that estren induced a 2- to 3-fold increase in seminal vesicle weight, which was relatively less than the stimulatory effect of DHT (7). However, this is analogous to the androgenic effect of 19-NT in castrated rats (56).

In osteoblasts that express AR, estren suppressed cAMP-dependent activation of the IGF-I gene promoter. This is analogous to the effect of estradiol by way of ER{alpha}, principally through inhibition of C/EBP{delta} activity (14, 57). Unlike estradiol, estren, at least at the concentrations that we tested, did not significantly affect Runx2-dependent gene expression. However, estrogenic activation of Runx2 requires ER{alpha}, whereas the synthetic androgen methyltrienolone (8), DHT, and 19-NT (data not shown) by themselves all failed to increase endogenous or transfected Runx2 activity in osteoblasts that express functional AR or ER{alpha}.

In agreement with the likelihood that 19-NT production accounts for the biological activity of estren on the skeleton in sex organ-ablated mice, direct treatment with 19-NT enhances aspects of bone status in elderly male and female humans, and in castrated primates and rats (58, 59, 60, 61, 62). The strong androgenic potential of estren that we note is also consistent with greater body hair or vocal depth, attributes that would be difficult to detect in rodents, that occurred in half of the women enrolled in a study of 19-NT as an antiosteoporosis drug (58). Still other concerns, such as the adenomyosis-like uterine architecture pathologies that occurred in ovariectomized monkeys treated with 19-NT (63), also bear consideration from possible estren therapy. Importantly, most of the progestins found in combination oral contraceptives are derived from 19-NT (64), suggesting that complex pharmacological effects might also occur by further estren metabolism in nonskeletal tissue. Finally, the unexpected estren-dependent activation of ERE-driven gene expression in cells that express AR, which occurs with far greater potency relative to DHT, predicts the possibility of some troublesome feminizing effects in males, which still await examination.

In summary, we show that low concentrations of estren activate osteoblasts in a genotropic and AR-dependent way, consistent with its rapid conversion to the potent androgen 19-NT. Transcriptional events induced by estren metabolism occur through both ARE and ERE, explaining, in part, its apparent sex-nonspecific biological activity. Our results have critical implications for both beneficial and adverse effects that could occur in response to systemic estren therapy. This would need to be monitored in particular where androgen- and estrogen-sensitive gene expression coexist in cells that express AR. Importantly, our findings emphasize the need to understand the potential of metabolic activation of candidate compounds that have possible merit for steroid hormone replacement therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells
Primary osteoblast-enriched cultures were prepared from parietal bones of 22-d-old Sprague Dawley rat fetuses (Charles River Laboratories, Wilmington, MA) by methods approved by the Yale Institutional Animal Care and Use Committee. Bone sutures were dissected, and cells were released from the bone fragments by five sequential collagenase digestions. Cells pooled from the last three digestions express many biochemical features that typify differentiating osteoblasts, including high levels of nuclear factor Runx2, PTH receptor, type I collagen synthesis, and alkaline phosphatase activity (19, 65, 66). They also exhibit an increase in osteocalcin expression in response to vitamin D3, differential sensitivity to TGF-ß, bone morphogenetic protein 2, and various prostaglandins, and form mineralized nodules in vitro (67, 68, 69, 70, 71). Cells were plated at 4000/cm2 in DMEM supplemented with 10% fetal bovine serum.

Plasmids
We created reporter plasmid 4XARE containing four AREs (5'-GGTTCTTGGAGTACT-3') derived from the rat probasin gene promoter cloned within a minimal Rous sarcoma virus promoter plasmid (8, 72), and reporter plasmid SXN1C containing two Runx response elements (5'-GGCCGCG-3') derived from the rat TGF-ß receptor I gene promoter (73). Our studies benefited from the generous gifts of reporter and expression plasmids from other investigators. Dr. Stuart Adler (Washington University, St. Louis, MO) provided an expression plasmid for human ER{alpha} and a reporter plasmid driven by ERE (5'-AGGTCACAGTGACCT-3') derived from the frog vitellogenin promoter cloned upstream of a minimal prolactin gene promoter (74). Dr. Chawnshang Chang (University of Rochester, Rochester, NY) provided an expression plasmid for rat AR. Dr. Ivan J. Sadowski (University of British Columbia, Vancouver, Canada) provided cloning vector M1 with the GAL4 DNA binding domain that we used to create an expression plasmid encoding a Runx2-GAL4 DNA binding domain fusion protein designated as M1-Runx2. Dr. Richard A. Maurer (Oregon Health Sciences University, Portland, OR) provided reporter plasmid 5XGAL4-E1b-Luciferase, with five GAL4 response elements (5XGAL4). Dr. Peter A. Rotwein (Oregon Health Sciences University) provided reporter plasmid IGF1711b-Luc, driven by the promoter and a fragment of exon I encoding the rat IGF-I gene containing a potent C/EBP-sensitive response element (5'-CGCAATCG-3'). The expression and activity of all of these constructs in primary osteoblast cultures have been reported previously (8, 14, 20).

C/EBP and Runx2 Activity
Gene expression by transcription factors in the C/EBP gene family was determined with reporter plasmid IGF1711b-Luc with its inherent C/EBP-sensitive response element. Its activity is induced by hormones such as PGE2 that increase cAMP, activate protein kinase A, and cause the activation and translocation of endogenous C/EBPß and C/EBP{delta} in osteoblasts (14, 21, 25). Gene activation potential by the osteoblast-restricted transcription factor Runx2 was determined with expression plasmid M1-Runx2, which activates reporter plasmid 5XGAL4 (8). Effects on endogenous Runx2 activity in osteoblasts were determined with reporter plasmid SXN1C (19, 73).

Transfections
Promoter-reporter constructs, gene expression plasmids, or empty parental vectors were pretitrated for optimal expression efficiency and transfected with reagent TransIT LT1 (Mirus Corp., Madison, WI). Cells at 50–70% culture confluence (25,000–30,000/cm2) were exposed to an optimal amount of expression plasmid (10–20 ng/cm2) or reporter plasmid (10–50 ng/cm2) in medium supplemented with 0.8% fetal bovine serum for 16 h, and then supplemented to obtain a final concentration of 5% serum. Cells were cultured for 48 h, treated for 24 h with steroids (0.1–10 nM) or the antagonists flutamide at 20 µM (30) or ICI 182,780 at 0.1 µM (31) in serum-free medium, rinsed, and lysed. Nuclear-free supernatants were analyzed for reporter gene activity and corrected for protein content. To account for competition among plasmids for limiting transcription components, control cells were transfected with a compensating amount of empty vector. Transfection efficiency was assessed in parallel with positive and negative reporter plasmids as previously described (8, 20).

Ligand Binding
Binding affinity for ER was determined using cytosol prepared from uteri of Sprague Dawley rats that were ovariectomized 24 h before death. Aliquots of the cytosol were incubated with 1 nM [3H]estradiol without or with nonradioactive steroids, by methods previously described (75). Similarly, binding affinity for AR was determined by competition for 2 nM [3H]5{alpha}-DHT in cytosol prepared from prostate of rats that were orchidectomized 24 h before death (76). Incubations were performed overnight on ice, and bound and free ligands were separated by charcoal absorption and quantified by liquid scintillation counting. Nonradioactive steroid concentrations were from 1 pM to 10 µM. Displacement was analyzed with a curve-fitting program (Prism, GraphPad Software, Inc., San Diego, CA).

Steroid Analysis
To assess possible estren metabolites, osteoblasts were incubated with 18 nmol of estren in 5 ml of medium for 24 h. Medium was diluted with 4 vol methanol, extracted with hexanes to remove lipids, and evaporated under vacuum, and the aqueous residue was extracted with ether. Ether extracts were evaporated under a N2 stream and analyzed by thin layer chromatography on Merck silica gel plates F254 (EM Science, Gibbstown, NJ) in ethyl acetate-hexanes (2:1). A strong UV absorbing spot at Rf 0.4 suggested an {alpha},ß-unsaturated ketone, predictive of the {Delta}4-3-ketone, 19-NT. To characterize the product further, medium from cells incubated with estren was extracted as above, with the addition of 6 nmol of 11-deoxycorticosterone as a recovery standard. The residue was analyzed by HPLC by reference to authentic 19-NT, using a LiChrospher 100 Diol column (Merck KGaA, Darmstadt, Germany) eluted at 1 ml/min with CH2Cl2-isooctane (4:1) and monitored by UV light absorption at 240 nm. The osteoblast-derived compound that eluted by HPLC at 7.5 min was identified more definitively by analysis with a PE Sciex API-2000 tandem triple-quadrupole mass spectrometer (PerkinElmer Corp., Norwalk, CT), essentially as described (76). The parent ion m/z = 275 MH+ was scanned for identity.

In Vitro 3{alpha}-HSD Analysis
Reactions comprised 3.6 nmol of estren in 0.1 ml of a solution containing 100 mM potassium phosphate (pH 7.0), 2.3 mM NADP, 4% acetonitrile, and either recombinant rat 3{alpha}-HSD (AKR1C9), generously provided by Dr. Trevor M. Penning (University of Pennsylvania School of Medicine, Philadelphia, PA) or osteoblast-derived cell lysate. For the latter, cells were lysed in hypotonic buffer (14) with a Dounce type homogenizer, and the supernatant obtained by 45 min of centrifugation at 16,000 x g and 4 C was used as enzyme source. Reactions were terminated with ethyl acetate containing 11-deoxycorticosterone as a recovery standard. Production of 19-NT was determined by HPLC as described above.

mRNA Analysis
Total RNA was extracted from primary rat osteoblast cultures and from rat liver with acid-guanidine-monothiocyanate, precipitated with isopropyl alcohol, and dissolved in sterile water (20). Samples (3 µg) were assessed for mRNA using the One-Step RT-PCR kit (Roche Diagnostics Corp. Gmbh, Indianapolis, IN), and forward (5'-ATGGATTCCATATCTCTGCGTGTAGC-3') and reverse (5'-CTATTCATCAGTAAATGGATGATTGGG-3') primers that corresponded to the full-length coding sequence of rat 3{alpha}-HSD (AKR1C9), obtained from GenBank (accession no. NM_138547). Samples were fractionated in parallel with a sizing ladder through 1% agarose and visualized by ethidium staining. The sequence of the osteoblast-derived PCR product was determined by the Keck Foundation Biotechnology Resource Laboratory at Yale.

Statistics
Statistical differences were assessed by one-way ANOVA and Student-Newman-Keuls post hoc analysis, using SigmaStat software (Jandel Corp., San Rafael, CA) from a minimum of nine replicate samples and three studies with different cell preparations. A significant difference was assumed by a P value of <0.05.

Note Added in Proof
After our manuscript was submitted, Moverare et al. (77) reported that estren activates ER{alpha}-dependent gene expression with 1750-fold lower potency relative to estradiol. By comparison, we show that estren activates AR dependent gene expression with 1500-fold greater potency relative to its ER{alpha}-dependent effect, suggesting a predominant mode of action through AR.


   ACKNOWLEDGMENTS
 
We are grateful to Stuart Adler (Washington University, St. Louis, MO), Chawnshang Chang (University of Rochester, Rochester, NY), Yoshiaki Ito (Kyoto University, Kyoto, Japan), Ivan J. Sadowski (University of British Columbia, Vancouver, Canada), and Richard A. Maurer and Peter A. Rotwein (Oregon Health Sciences University, Portland, OR) for some of the reporter and expression plasmids used in these studies, and to Dr. Trevor M. Penning (University of Pennsylvania, Philadelphia, PA) for recombinant rat 3{alpha}-HSD (AKR1C9). We also thank Drs. Caren Gundberg and Joseph A. Madri (Yale University, New Haven, CT) for their critical reading of our manuscript.


   FOOTNOTES
 
This work was supported by Public Health Service Awards AR39201 from National Institute of Arthritis and Musculoskeletal and Skin Diseases, DK56310 from National Institue of Diabetes and Digestive and Kidney Diseases, CA37799 from National Cancer Institute, and HL61432 from National Heart, Lung, and Blood Institute.

Abbreviations: AR, Androgen receptor; ARE, androgen response element; C/EBPß, CCAAT enhancer binding protein ß; DHT, dihydrotestosterone; ER, estrogen receptor; ERE, estrogen response element; 3{alpha}-HSD, 3{alpha}-hydroxysteroid dehydrogenase; NADP, nicotinamide-adenine dinucleotide phosphate; 19-NT, 19-nortestosterone; PGE2, prostaglandin E2.

Received for publication December 19, 2003. Accepted for publication January 28, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  AR
Ligands:   17β-Estradiol  |  Dihydrotestosterone



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