Comments for:

Bone protection by estrens occurs through non–tissue-selective activation of the androgen receptor

Sara H. Windahl, … , Michèle Resche-Rigon, Roland Baron

Sara H. Windahl, … , Michèle Resche-Rigon, Roland Baron

Published September 1, 2006
Citation Information: J Clin Invest. 2006;116(9):2500-2509. https://doi.org/10.1172/JCI28809.

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Research Article Bone biology

Let us not toss out the baby with the bath water

Submitter: Stavros C. Manolagas | ManolagasStavros@uams.edu

University of Arkansas for Medical Sciences

Published October 2, 2006

We have carefully read the recent article by Windahl et al. (1) and the accompanying commentary by Dr. Neill (2). We agree with the bottom line conclusion that estren may not be a suitable clinical candidate for the treatment of postmenopausal osteoporosis because of unwanted effects on reproductive tissues that we did not elicit under the experimental conditions we used four years ago. This having been said, we strongly disagree with the interpretation of the evidence, the black and white conclusions of Windahl et al., the “who is right” cast on the subject, and the editorial suggestion to “call the whole replacement off,” instead of a scholarly attempt to reconcile seemingly incongruent data and advance knowledge in the process.
Indeed, in dismissing estren and exalting the advantages of their SERM, Windahl et al. have bypassed the customary scholarly courtesy of offering an explanation for the significant discrepancies between their findings and those of Kousteni et al. (3), as well as those of several other groups. More important, Windahl and colleagues have ignored a substantial body of basic science and considerable advances towards understanding the mechanism of estrogen action on bone and other tissues, made with the use of estren and other related paradigmatic compounds as tools. To paraphrase Albert Einstein, science needs to be made as simple as possible, but not simpler than that. We are afraid that the treatment of the subject by Windahl et al. and the accompanying commentary has crossed this barrier of oversimplification and in the process discounts the importance of newly discovered mechanisms of steroid hormone action (4 -8). Here is why:
Steroid hormones, including estrogens and androgens, can induce cellular responses, not only through direct activation of gene transcription resulting from cis or trans interactions of their receptors with DNA-binding sequences on target gene promoters, but also indirectly. These latter responses result from extranuclear action of the same hormone receptors causing activation of various cytoplasmic protein kinase cascades, which in turn alter the activity of other transcription factors or co-activators of the receptors themselves (9-16). During the last few years, we and others have advanced the idea that the development of ligands that selectively activate the so-called nongenomic pathways should provide useful tools to investigate the significance of these pathways.
4-Estren-3_,17_-diol (estren) is a synthetic ligand of the estrogen (ER) or androgen (AR) receptor which in standard equilibrium assays binds the ER with an affinity that is about 0.15% of that of 17_ –estradiol (17) and AR with an affinity which is about 2% of that of the potent androgen R1881 (J.A. Katzenellenbogen, University of Illinois, personal communication). In an extensive series of in vitro, as well as in vivo, studies we have found that estren potently activates kinase-mediated actions of the ER or AR and downstream transcriptional events at concentrations 3 to 4 orders of magnitude lower than those required to stimulate classical genotropic transcription (11;17). Such kinase-mediated actions are evidently responsible for the anti- and pro-apoptotic effects of estren, as well as estrogens and androgens, in osteoblasts/osteocytes and osteoclasts, respectively. Most strikingly, estren is equipotent to estradiol or dihydrotestosterone in its in vitro effects on bone cell survival and kinase activation, in spite of the substantially lower affinity for the ER or AR.
The goal of the studies of the Science article by Kousteni et al. was to assess in the mouse model the suitability of this compound for the treatment of postmenopausal osteoporosis (3). Postmenopausal osteoporosis is, by definition, a disease of adulthood in which bone accrual has occurred normally (in the vast majority of cases) and bone loss has occurred long after sexual maturity and peak bone mass has have been established. Mice (C57BL/6 or Swiss Webster) do not reach peak bone mass until the age of 5 to 6 months (18;19). We had, therefore, appropriately selected for the purpose of the Kousteni study Swiss Webster mice between the ages of 6-8 months. We found and reported that estren was at least as effective as estrogen and androgen in preventing the bone from loss that ensued upon gonadectomy in female or male mice while it had no measurable effects on the uterus or seminal vesicle (3). The purpose of the study by Windahl et al. was to “directly compare” estren with a SERM in development by this group of workers. To accomplish their goal, they chose as model 3 month old mice with immature skeletons. We believe that this choice is, under the circumstances, inappropriate, as no one has ever envisioned or suggested using estren or SERMs in growing humans.
By using 3 month old mice C57BL/6 mice (which weigh between 20 and 25 grams) as compared to 6-8 month Swiss Webster mice (which weigh 45-50 grams) Windahl and colleagues have more than doubled the dose of estren we had used, on a weight basis, and more than doubled on the basis of the metabolic rate (20). Moreover, as it has been established carefully by Modder and colleagues (21), the uterus of the growing mouse is more sensitive to estrogen than the uterus of the mature mouse. Specifically, while 5 ng/g/day of estradiol is needed to prevent loss of uterine weight in 3 month old ovariectomized C56BL/6 mice, an 8-fold higher dose, 40 ng/g/day, is needed to prevent loss of uterine weight in 6 month old C57BL/6 mice. Yet, the same dose of estrogen (5 ng/g/day) is sufficient to prevent loss of bone in the growing and mature C57BL/6 mice, strongly supporting the contention that the differential sensitivity of uterus and bone to estrogen replacement in adulthood may provide a window of a therapeutic opportunity for dissociating effects of estrogen and estrogen- like compounds on the two tissues. Notably, administration of a very low dose of estradiol to postmenopausal women for 2 years decreased bone turnover without causing endometrial changes (22). In the data depicted in supplemental figure 3 of the Windahl paper, it is shown that decreasing the dose of estren to 25 _g/mouse/day significantly decreased the effect of the compound on seminal vesicle weight, but had no significant effect on bone volume, supporting the contention for a differential sensitivity of reproductive organs versus bone. Yet, the authors chose to interpret statistically insignificant data (on the effects of estren on bone) as significant using the rationale that “were somewhat decreased,” to buttress their conclusion that the effects of estren on bone and seminal vesicles cannot be separated
In a report to the American Society of Bone and Mineral Research which is published as an abstract (23) this same group of workers found that 4-Estren 3_-17_ diol, an isomer of estren, when given by injection to ovariectomized mice maintained bone at control levels but, similar to the SERM PSK3471, the estren isomer “…induced very moderate increases in seminal vesicle and uterus weights compared to GDX [gonadectomized] controls.” It seems to us that this observation has confirmed the essential finding of the Kousteni paper that estren works on bone without affecting reproductive organs, at least under certain experimental conditions. While one can argue whether this reflects different sensitivity of the two tissues to estren, or whether the size of the window of this therapeutic opportunity with estren is small or large, as will be discussed below, the cardinal fact remains that there is indeed such a window. Omitting this information from the Windahl paper is disturbing.
In contrast to the findings of Windahl et al. that estren and estradiol are equally effective on the murine uterus and that the effects of the former are the result of its androgenic properties, Moverare et al. have reported that estren had only 16% of the activity of 17_-estradiol in 11 month old mice of a mixed C57BL/6J/129 background (24). Moreover, in the Moverare studies the effects of estren on the uterus and bone were completely abrogated in mice lacking the estrogen receptor, but having intact androgen receptors, leading these authors to conclude that estren is a weak estrogen. This incongruency between the Windahl results and those of Moverare were also ignored in the discussion of the Windahl paper, even though the Moverare article was cited in their paper. In another study of the effects of estren on the uterus of 10 to 12 week old C57BL/6 mice, Hewitt and colleagues found that estren behaves as a weak estrogen, and that its transcriptional profile is indeed distinct from that of DHT or 19-nortestosterone (25). In that same study, a 3-fold higher dose of estren than the dose we have previously reported to exert a beneficial effect on bone without increasing uterine weight (3), produced only 50% of the effect of E2 on uterine weight of adult ovx mice in a three day uterine bioassay; but at the dose we had used in the Kousteni report was ineffective. Yet, estren stimulated the expression of rapid – presumably kinase-regulated – response genes as robustly as E2, but was a weak activator of the expression of late – ER/DNA-regulated – response genes.
The ovariectomized 3 month old C57BL/6 mice of the study by Windahl et al. were very peculiar in their failure to exhibit any of the expected changes that occur after loss of sex steroids in mice and humans, including increased osteoblast surface, bone formation rate, osteoclast number, and deoxypyridinoline excretion (a marker of bone resorption). Most surprisingly, estradiol replacement increased, rather than decreased, the number of osteoclasts, which is the opposite of what has been established in the literature of the last 15 years in both animals and humans (26-28). Even more bizarre, despite the increase in osteoclast numbers, Windahl et al. found (Figure 1) that estrogen replacement in their model increased trabecular bone volume as compared to the sham controls. Based on this observation, they concluded that estrogens (and their SERMs) are, therefore, anabolic on bone. This is categorically an erroneous and misleading conclusion, as can be testified by a plethora of basic and clinical studies and over 60 years of clinical experience with estrogen replacement, which has conclusively established that estrogens are anticatabolic/anti-remodeling agents (29). In other words, estrogens (at replacement doses) decrease the rate of bone turnover by suppressing both the increased bone formation and bone resorption that ensues following menopause, and are not bone anabolic agents. Regrettably, the commentary by Dr. Neill has endorsed their erroneous conclusion for “an anabolic effect” of estrogen on bone as a matter of fact— but fact, it is not. Equally puzzling, PSK3471, the SERM that Windahl et al. claim is like estradiol anabolic on bone, had no effect on any parameter of bone turnover. That PSK3471 had the undesirable effect for a SERM to stimulate uterine growth in both the myometrium and endometrium (Figure 4 of Windahl et al) was completely ignored when exalting its safety profile in the discussion of the Windahl paper.
Subsequent to our report in Science, others found that the effects of estren may result from its ability to weakly activate classical genotropic actions of the AR (24;25;30-32), perhaps through conversion to the androgenic metabolite 19-nortestosterone (24;30;31;33). Prompted by these contrasting observations, in unpublished studies shown for the first time here, we have compared the effects of estren to those of estradiol, dihydrotestosterone (DHT), and 19-nortestosterone on uterine weight. Specifically, 6.5 month old Swiss Webster female mice were Sham-operated or ovariectomized. Immediately following surgery 60-day release pellets containing vehicle, 7.6 mg estren, 0.025 mg of 17_-estradiol, 7.6 mg of 19 -nortestosterone (19-NT), or 10 mg of dihydrotestosterone (DHT) were implanted and six weeks later the animals were sacrificed and the uteri were excised and immediately photographed and weighed. In contrast to estradiol, DHT, or 19-nortestosterone, estren, at the dose used in our Science report, did not stimulate uterine growth.
Subsequent to our report by Kousteni et al., we have also performed dose response studies (unpublished) using estren at 100-, 300-, 1000-, and 3000-fold the replacement dose of estradiol. We have confirmed the lack of a uterine effect at the dose we had used in our published studies. Nonetheless, in full agreement with the relative binding affinities of estren for the ER (300- fold lower affinity as compared to estradiol (3)) and the AR (approximately 1/40 that of R1881 or 1/25 that of DHT), we did find that at high enough concentrations, for example 3,000 – 7,000-fold higher than estradiol, estren may also exert genotropic activity in vitro. Consistent with its affinity for the ER, at a dose that exceeded by 10- fold the Kd for the ER (3000-fold the replacement dose of E2), estren restored uterine weight. Similarly, in a 1-week experiment in rats, at the 3000-fold higher that E2 replacement dose of estren exhibited 30% of the effect of E2 on uterine weight. Hence, the difference between the findings of Windahl et al. and Kousteni et al. can be fully accounted for by the difference in the size and age of the animals.
Using HeLa cells transduced with wild type ER_ or the ligand binding domain of ER_ localized to the cell membrane, the OB-6 osteoblastic cell line, MCF-7 breast carcinoma cells and uteri from mice treated with 17_- estradiol (E2) or estren we have shown that nongenotropic ER actions regulated a population of genes distinct from those regulated by genotropic ER actions (34). In addition, other work from our group indicates that selective activation of kinase-mediated actions of the estrogen receptor (ER) with estren induces osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner (Kousteni et al. submitted to MCB). This effect can be reproduced using recently synthesized abiotic, nondegradable poly(amido)amine dendrimer macromolecules that are conjugated to multiple estrogen molecules through chemically robust linkages. Because of their charge and size, these estrogen-dendrimer conjugates (EDCs) remain outside the nucleus. Like estren, these compounds stimulate ERK, Shc, and Src phosphorylation in MCF-7 breast cancer cells at low concentrations, yet they are very ineffective in stimulating transcription of endogenous estrogen target genes, being approximately 10,000-fold less potent than estradiol in genomic actions. In contrast to estradiol, EDC was not effective in stimulating breast cancer cell proliferation (35). Conversely, in contrast to estren or EDC, the two synthetic compounds that stimulate kinase-mediated ER actions 1,000 – 10,000 times more potently than direct DNA interactions, Estradiol, 19-nortestosterone, or DHT could not replicate these effects at concentrations as high as 5 orders of magnitude greater than those of estren. In fact, in sharp contrast to estren, E2 suppresses exogenous or endogenous BMP-induced osteoblast commitment and differentiation. Consistent with the in vitro findings, estren, but not E2, stimulated Wnt/_-catenin-mediated transcription in TCF -lacZ transgenic mice. Moreover, E2 stimulated BMP signaling, in mice in which ER_ lacks DNA binding activity and classical ERE-mediated transcription (ER_NERKI/-), but not in wild type controls. This evidence reveals for the first time the existence of a large signalosome, in which inputs from the ER, kinases, BMPs, and Wnt-signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription. In line with our results, White and colleagues have found that estren is a weak regulator of AR-mediated transcription but a moderate promoter of Xenopus oocyte maturation, a process that involves nongenotopic actions of androgens (36;37). In contrast, 19-nortestosterone is a highly potent regulator of AR-mediated transcription but a very weak activator of Xenopus oocyte maturation.
Finally, we have compared the effects of estren to those of intermittent PTH, a proven bone anabolic agent, on the skeleton of mice and rats and have been unable to show that estren has bone anabolic properties comparable of those of PTH, as determined by BMD or _CT. Whether the apparent inability of estren to manifest in vivo a bone anabolic effect, consistent with its pro-differentiation effects on osteoblast progenitors in vitro, is due to its more potent anti- remodeling/anti-resorptive action that overwhelms or conceals its putative bone forming property, or is due to unknown pharmacokinetic shortcomings, suboptimal route or even frequency of administration remains unknown.
In closing, collective evidence from us and others has led us to propose that estren: a) exerts its effects as a result of the selective activation of kinase-mediated actions of the ER or AR; b) has a biologic profile that is distinct from estradiol, SERMs or androgens; and c) under conditions in which it exhibits a desirable effect on bone, estren exerts minimal or blunted effects on reproductive organs. Based on these lines of evidence, we had suggested earlier that estren represents the prototype of a function-specific ER/AR ligand with unique biologic properties, not exhibited by the natural ligands of these receptors. Three significant advances argue in favor of this contention. First, cell-impermeable estrogen conjugates produce identical effects to those of estren, while they are ineffective in stimulating transcription of endogenous ERE- containing genes (38). Second, screening of focused libraries has led to the identification of additional synthetic compounds that like estren can dissociate kinase- from ERE-mediated transcription, even though, unlike estren, they have comparable affinity for ER to that of estradiol, and no AR binding capability (39). Thus, the function-dissociating properties of estren and other such compounds are unrelated to weak or strong binding to the ER or the AR. Instead, as we had proposed originally, these compounds most likely induce distinct conformation of the receptor protein in such a manner that can initiate kinase cascades, but not classical cis- or trans-mediated transcription. Third, function-specific ligands of the ER, other than estren, have been identified and they also lack uterotropic effects but retain other non-reproductive actions such as immunomodulation (40) and ischemic neuroprotection (41). Hence, despite the dismissive conclusion of the Windahl article about a single compound (estren), extensive literature supports our view that selective activators of ER- mediated kinase cascades may indeed represent a novel class of pharmacotherapeutics with the potential of biologic outcomes distinct from those produced with natural ligands that activate genotropic and nongenotropic signals alike, or synthetic ligands that activate only the former.
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