Essential Role for Estrogen Receptor in Stromal- Epithelial Regulation ...

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Endocrinology 148(2):566 –574 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-0906

Essential Role for Estrogen Receptor ␤ in StromalEpithelial Regulation of Prostatic Hyperplasia Stephen J. McPherson, Stuart J. Ellem, Evan R. Simpson, Vladimir Patchev, Karl-Heinrich Fritzemeier, and Gail P. Risbridger Centre for Urological Research (S.J.M., S.J.E., G.P.R.), Monash Institute of Medical Research, Monash University, Clayton 3168, Australia; Prince Henry’s Institute (E.R.S.), Melbourne 3168, Australia; and Schering AG, CRBA Gynecology and Andrology (V.P., K.-H.F.), 13342 Berlin, Germany Estrogens, acting via estrogen receptors (ER) ␣ and ␤, exert direct and indirect actions on prostate growth and differentiation. Previous studies using animal models to determine the role of ER␤ in the prostate have been problematic because the centrally mediated response to estrogen results in reduced androgen levels and prostatic epithelial regression, potentially masking any direct effects via ER␤. This study overcomes this problem by using the estrogen-deficient aromatase knockout mouse and tissue recombination to provide new insight into estrogen action on prostate growth and pathology. Homo- and heterotypic aromatase knockout tissue recombinants revealed stromal aromatase deficiency induced hyperplasia in normal prostatic epithelium due to disruption of paracrine interaction between stroma and epithelia. Treatment of tissue recombinants with an ER␤-specific agonist demonstrated that stimulation of ER␤ elicits antiproliferative

responses in epithelium that are not influenced by alterations to systemic androgen levels or the activation of ER␣. Additionally, work performed with intact aromatase knockout mice demonstrated that the administration of an ER␤-specific agonist ablated preexisting prostatic epithelial hyperplasia, whereas an ER␣-specific agonist did not. Therefore, failed activation of ER␤, resulting from local stromal aromatase deficiency, in conjunction with increased androgen levels, results in increased epithelial cell proliferation and prostatic hyperplasia. These data demonstrate essential and beneficial effects of estrogens that are necessary for normal growth of the prostate and distinguishes them from those that adversely alter prostate growth and differentiation. This highlights the potential of selective estrogen-receptor modulators, rather than aromatase inhibitors, for the management of dysregulated prostate growth. (Endocrinology 148: 566 –574, 2007)

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Aromatase deficiency results in hypertrophy and hyperplasia of the prostate gland at maturity (11), providing a novel way of examining whether ER␤ has antiproliferative activity within the prostate gland. Because aromatase is expressed in the stroma and ER␤ receptors are located predominantly in the epithelia of the prostate (8, 12), we have proposed that the action of estrogen via ER␤ involves stromal-epithelial cell signaling (13). Additionally, we believe that the action of estrogen via ER␤, like ER␣, permanently and irreversibly alters prostate epithelial cell differentiation during development (6, 7). Mesenchymal-epithelial cell interactions are vital for the correct development of the male reproductive tract, including the prostate gland (14). Tissue recombination techniques have successfully been used to define mechanisms of stromal signaling during development and differentiation of the epithelia, particularly the role of androgens acting via the stromal androgen receptor (AR) to induce epithelial differentiation and proliferation (15, 16). Taking a similar approach, we used prostate tissue from aromatase knockout (ArKO) mice to test the effect of a deficiency of prostatic stromal aromatase on prostate development and the subsequent activation of epithelial ER␤ using a selective ER␤ agonist. This study shows that aromatase deficiency, and therefore the absence of local estrogen and failure to activate ER␤, has a significant impact on prostate development. It alters the inductive and instructive properties of the neonatal prostatic stroma, leading to perturbation of stromal-epithelial cell signaling and aberrant prostatic growth. In the absence of ER␤

UMEROUS MAMMALIAN physiological systems in both males and females are regulated by androgens and estrogens, including reproductive tract function, development, and disease. In the male, androgens are generally considered to be proliferative, whereas estrogens have dual actions. In the prostate gland, estrogen has direct and indirect effects on epithelial cell differentiation and proliferation (1–3). Indirectly, estrogens suppress the release of pituitary LH, reducing synthesis of testicular androgens, which lowers systemic androgen levels and induces apoptosis and prostatic epithelial atrophy (4, 5). Concomitantly, acting locally via estrogen receptor (ER)␣ within the prostatic stroma, estrogens stimulate aberrant epithelial cell differentiation and proliferation, leading to squamous metaplasia (6, 7). More recently, an antiproliferative action of estrogen was suggested to occur via activation of the epithelial ER␤ (8 –10). However, those studies used intact wildtype (wt) or transgenic male mice, which remain sensitive to the inhibition of pituitary function by estrogen. This outcome, mediated by ER␣, can result in prostate glandular regression, making antiproliferative effects caused by the activation of ER␤ difficult to identify. First Published Online October 26, 2006 Abbreviations: AR, Androgen receptor; ArKO, aromatase knockout; ER, estrogen receptor; PCNA, proliferating cell nuclear antigen; SCID, severe combined immunodeficient; SERM, selective ER modulator; SVM, seminal vesicle mesenchyme; wt, wild type. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

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McPherson et al. • ER␤ Inhibits Prostatic Hyperplasia

signaling, increased cell proliferation results in non-neoplastic increases in epithelial tissue identified as epithelial hyperplasia (17). This study demonstrates that although androgens are essential for the coordinated growth of the prostate, local estrogenic activity is equally essential for the modulation of normal prostate development. Estrogen, acting via ER␤, has a definite antiproliferative effect on prostatic epithelium that is able to prevent and ablate prostatic epithelial hyperplasia. Therefore, estrogens, acting in synergy with androgens and ER␤, are required to regulate the proliferative and antiproliferative changes that occur during normal prostate development and differentiation. Materials and Methods Animals ArKO and severe combined immunodeficient (SCID) mice were housed at Monash Medical Centre under specific pathogen-free conditions with free access to soy-free chow and water. All experiments were approved by the Monash University Animal Ethics Committee and conducted in accordance with the National Health and Medical Research Council Guidelines for the Care and Use of Laboratory Animals. Day of birth was designated d 0.

Tissue recombination Tissue recombinants were prepared as previously described (6, 14, 18) using newborn (d 0) wt or ArKO mouse stroma (S) and adult (16-wk-old) wt or ArKO epithelia (E). As indicated in Fig. 1, tissue recombinants composed of epithelial tissue from adult ArKO or wt prostates recombined with seminal vesicle mesenchyme (SVM) from newborn (d 0) ArKO or wt mice to produce the homotypic combinations wt-S/wt-E

FIG. 1. Formation of homotypic and heterotypic prostate tissue recombinants using ArKO and wt mice. In this study, tissue recombination involved different combinations of neonatal stroma (S) from ArKO or wt mice being recombined with epithelial cells (E) from hyperplastic 16-wk-old adult ArKO or normal wt mouse prostates and grafted under the renal capsules of intact male SCID mice up to four per kidney for 6 wk. At the end of the grafting period, depending on the types of stroma and epithelium used, implanted tissues give rise to homotypic wt-S/wt-E (A) and ArKOS/ArKO-E (B) or heterotypic ArKO-S/wt-E (C) and wt-S/ArKO-E (D) prostatic tissue recombinants. Scale bars, 25 ␮m (recombination); 5 mm (grafting); 400 ␮m (harvesting).

Endocrinology, February 2007, 148(2):566 –574

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and ArKO-S/ArKO-E and heterotypic combinations ArKO-S/wt-E and wt-S/ArKO-E. Up to eight recombinants were grafted under the renal capsules of any one intact host SCID mouse. Because the genotype of neonatal mouse SVM donors was not available until after composition of the tissue recombinants and grafting into host mice recombinants was completed, the homo- and heterotypic grafts were randomly assigned to host mice, thus removing any bias in their positioning. After grafting under the renal capsule of intact male SCID mice, recombinants were harvested after 6 wk and fixed in Bouin’s fixative for analysis. A minimum of eight grafts were obtained for each tissue recombinant group.

Specific ER modulators The ER␤-specific agonist (8␤-ER2) and ER␣-specific agonist (16␣-LE2) were obtained from Dr. Fritzemeier (Schering AG, Berlin, Germany). The chemical characteristics and biologic effects of these compounds, demonstrating selective activation of ERs, have been previously described (19 –21). Doses of ER-specific agonists used throughout this study were based on those previously used in male and female rats (19). ER␤ agonist or placebo was administered to host SCID mice in the form of subcutaneous long-term release pellets (Innovative Research of America, Sarasota, FL) at the time of renal grafting, providing a dose comparable to 300 ␮g/kg䡠d. In experiments using intact adult male ArKO mice, the ER␤ agonist was administered by daily injection at doses of 100 and 30 ␮g/kg䡠d. ER␣ agonist was administered to adult ArKO mice at doses of 3 and 0.3 ␮g/kg䡠d by daily subcutaneous injection. In all cases, treatment with ER agonists was continued for 6 wk before tissue was collected. A minimum of four host SCID mice were used for treatment with either ER␤ agonist or placebo to provide at least eight of each homoor heterotypic tissue recombinant type. For treatment of intact animals, a minimum of six animals was used per treatment group.

Serum hormones Measurement of serum testosterone levels was performed by ANZAC Research Institute (Sydney, Australia) using methods previ-

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ously described by Ly et al. (22). Briefly, after organic extraction with a 3:2 mixture of hexane and ethyl acetate, the organic fraction was dried overnight and then reconstituted with a 1% gelatin PBS buffer before being processed in a radioimmunoprecipitation assay with a specific antibody raised to testosterone (T3-125; Endocrine Sciences Laboratories, Calabasas, CA) and a liquid chromatography-purified tritiated testosterone. After 16-h incubation at 4 C, free and bound hormones were separated with dextran T70-coated charcoal. The testosterone standard was calibrated against a World Health Organization testosterone preparation (coefficient of variation, 3.1–7.5%).

Immunohistochemistry After fixation, tissue recombinants were embedded and serially sectioned at 5 ␮m. Uniform systematic random sampling was used to select every 10th section for hematoxylin-eosin staining or immunohistochemical analysis. Immunohistochemistry was conducted with antibodies for AR (N-20; Santa Cruz Biotechnology, Santa Cruz, CA), ER␣ (ID5) and proliferating cell nuclear antigen (PCNA) (PC10; Dako Corp., Carpinteria, CA), and ER␤ (Novocastra, Newcastle upon Tyne, UK) using previously described protocols (11, 23). Analyses of AR and PCNA immunolocalization were conducted using CAST software (version 2.1.4; Olympus Corp., Albertslund, Denmark) as previously described (11, 23).


To demonstrate the importance of locally synthesized estrogen on local stromal-epithelial cell interactions, we used tissue recombination to study the effects of estrogen deficiency. First, we compared homotypic tissue recombinants derived from wt mice with those derived from ArKO mice (i.e. wt-S/wt-E, ArKO-S/ArKO-E; Fig. 1, A and B). The wt recombinants gave rise to normal prostate tissue morphology as expected (Fig. 2A), but those derived from ArKO tissues generated prostate tissue with extensive epithelial infolding (Fig. 2B), characteristic of in vivo epithelial hyperplasia, previously reported in the prostates of adult ArKO mice (11). Quantitation showed that epithelial infolding in the ArKO-S/ArKO-E homotypic recombinants was significantly increased, being approximately 3-fold greater compared with wt-S/wt-E recombinants (Fig. 2E). These data demonstrate that the difference in epithelial hyperplasia must be due to the effects of local estrogen deficiency and perturbation of stromal-epithelial cell interactions, because ArKO and wt homotypic recombinants were exposed to the same host environment and hormone levels.

Quantitation of epithelial hyperplasia Epithelial hyperplasia in tissue recombinants was identified according to morphologic criteria specifically defined by Shappell et al. (17) as a non-neoplastic increase in epithelial tissue compared with agematched controls. Practically, epithelial hyperplasia was identified as increased epithelial folding within tissue recombinants and the extent of this epithelial folding was accurately estimated using CAST software, counting frames, and systematic uniform random sampling methods adapted from those used to estimate epithelial morphology in gut (24). Briefly, the incidence of epithelial folds (“branches”) projecting into the prostatic lumen, away from the basement membrane, was counted for every 10th section of a graft (minimum of six sections per graft) with eight grafts analyzed per group (n ⱖ 8). Counts were expressed per unit of section area for each graft with the mean for each tissue graft used for comparison.

Statistics Data were analyzed to determine normality and significant differences were determined by either t test or one-way ANOVA. Significance was accepted at P ⬍ 0.05. Analyses were conducted using Prism 4.00 software (GraphPad Software Inc., San Diego, CA). Data are expressed as mean ⫾ sem unless otherwise noted.

Results Estrogen deficiency alters the inductive and instructive properties of the neonatal stroma

Homotypic tissue recombinants. The regeneration of prostatic tissue using tissue recombination is based on the inductive and instructive properties of neonatal urogenital mesenchyme or seminal vesicle stromal tissue such that it directs the differentiation of prostatic epithelium. These properties are not exhibited by adult prostatic stroma, but both neonatal and adult epithelia are responsive to neonatal stroma (25, 26). Stromal and epithelial prostate tissues are derived from neonatal and adult tissues, respectively, and may be from either wt or ArKO mice (Fig. 1). Using this technique of tissue recombination, Cunha and Risbridger (6, 15, 16) made seminal contributions to our understanding of stromal-epithelial interactions in prostate development and disease, including the elucidation of the roles of the androgen and ER␣ receptors.

Heterotypic tissue recombinants

The pivotal role of the stroma in the induction of epithelial hyperplasia was further demonstrated by comparing the heterotypic tissue recombinants (ArKO-S/wt-E, wt-S/ ArKO-E) (Fig. 1, C and D) with the homotypic recombinants. Epithelial hyperplasia was morphologically identifiable in ArKO-S/wt-E recombinants (Fig. 2C), being significantly greater than that seen in wt-S/wt-E recombinants and comparable to that seen in ArKO-S/ArKO-E grafts (Fig. 2E). These data indicate that the absence of aromatase from the prostatic stroma, and thus estrogen synthesis, is necessary for the initiation of epithelial hyperplasia in mature (wt) epithelia. A further comparison was made of heterotypic wt-S/ ArKO-E recombinants (Fig. 2D) with homotypic recombinant tissues. This recombinant is composed of neonatal inductive stroma from wt mice and adult ArKO epithelia that exhibit epithelial hyperplasia as we have previously described (11). Thus, this heterotypic recombinant tested the ability of the wt neonatal stroma to redirect hyperplasia in adult epithelial tissue (Fig. 1). The data obtained from heterotypic wt-S/ArKO-E tissue recombinants show that the inductive and instructive capacity of normal wt stroma was unable to reverse the hyperplastic phenotype at least within the 6-wk time frame of this experiment (Fig. 2E). It is possible that wt stroma may be able to ablate the hyperplastic pathology of the ArKO epithelium if the duration of epithelial exposure to signals from the wt stroma is extended; however, this remains to be determined. Overall, these data demonstrate that homotypic ArKO-S/ ArKO-E recombinations accurately reconstitute the pathology observed in the prostates of intact ArKO mice. Furthermore, these data demonstrate that the absence of aromatase activity from the stroma results in the induction of prostatic epithelial hyperplasia within normal prostatic epithelium. The recombinant tissues were exposed to identical systemic hormones from the host mice in which the grafted tissues were maintained; thus, these results show that there is a



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perturbation in local stromal epithelial-cell signaling related to estrogen deficiency. Immunohistochemical localization of steroid hormone receptors and proliferation in tissue recombinants

To further examine the local cell-cell signaling in these tissue recombinants, we investigated whether differences in hormone receptor expression could account for the different tissue responses to the same systemic hormone levels. The presence of AR is essential for normal prostatic differentiation (25) and initial examination identified AR immunoreactivity in all tissue recombinants; however, there were no obvious differences in the pattern of AR localization (Fig. 3, A–D). Although AR was localized to both stroma and epithelium, subsequent quantitation confirmed no significant differences in AR localization between tissue recombinants (Table 1). Localization of ER␤ showed immunoreactivity was almost completely confined to the epithelial tissue of each graft (Fig. 3, E–H). Unlike ER␤, ER␣ could not be detected in any tissue recombinant nor was there any evidence of PR (data not shown). Despite the absence of any obvious differences in the localization of steroid hormone receptors in the tissue recombinants, PCNA immunoreactivity was detectable in all tissue recombinants and was localized predominantly in the epithelium (Fig. 3, I–L). Quantitation of PCNA localization demonstrated significantly increased expression in all recombinants containing ArKO stroma and/or epithelium compared with the homotypic wt-S/wt-E controls (Table 2). Proliferation was significantly increased in wt epithelium after recombination with ArKO-S (ArKO-S/wt-E). In contrast, the proliferation in hyperplastic ArKO-E was unaffected by wt-S (wt-S/ArKO-E), exhibiting similar levels of proliferative activity to that seem in ArKO-S/wt-E and ArKO-S/ArKO-E recombinants (Table 2). Failure of stromal signals to stimulate ER␤ activity results in induction of prostatic epithelial hyperplasia

FIG. 2. Local stromal-epithelial cell interactions direct prostatic hyperplasia independent of systemic hormones. Tissue recombinants derived from wt-S/wt-E (A), ArKO-S/ArKO-E (B), ArKO-S/wt-E (C), or wt-S/ ArKO-E (D) from prostate with differing degrees of epithelial hyperplasia as demonstrated by epithelial infolding. E, The extent of epithelial infolding (estimated as the number of branches per millimeter squared) was significantly increased in all homo-heterotypic ArKO tissue recombinants (open bars) compared with wt-S/wt-E controls (solid bar) despite exposure to the same hormonal milieu. Values represent mean ⫾ SEM, n ⱖ 8 (P ⬍ 0.05); significant difference indicated by different superscripts. Scale bars, 100 ␮m (A–D); 400 ␮m (insets).

ER␣ and ER␤ are believed to promote different proliferative responses in prostatic tissues. ER␣ is required for epithelial differentiation and proliferation (6), whereas ER␤ is believed to be antiproliferative (8 –10). However, the levels of androgens were not reported in these latter studies and if reduced would lower epithelial cell proliferation and induce apoptosis. The results of the recombination experiments presented here did not show squamous metaplasia and implicate ER␤ rather than ER␣ in mediating aberrant stromal-epithelial cell signaling leading to epithelial hyperplasia. In the absence of stromal aromatase activity and with reduced estrogen synthesis within the tissue, we postulated that a failure to locally activate ER␤ caused epithelial hyperplasia in the tissue recombinants. To test this postulate, we again used the aromatase-deficient prostatic tissue recombination model to reveal the cellcell interactions, independent of systemic hormone levels. The tissue recombinants composed of neonatal stroma from ArKO mice (ArKO-S) with either wt or ArKO epithelium show epithelial hyperplasia so we administered an ER␤-

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FIG. 3. Immunolocalization of steroid hormone receptors and proliferation in tissue recombinants. Immunoreactivity for AR (A–D) was detectable in the stroma and epithelium of all tissue recombinants (brown stained cells). Similarly, ER␤ immunoreactivity (brown stain) was detected in all tissue recombinants (E–H); however, localization was restricted to prostatic epithelial cells only. Proliferating cells expressing PCNA could again be detected in all tissue recombinants and was localized to both stromal and epithelial compartments; however, the number of PCNA expressing cells appeared to be reduced in wt-S/ wt-E tissue recombinants (I) compared with ArKO-S/ArKO-E (J), ArKO-S/wt-E (K), and wtS/ArKO-E (L) tissue recombinants. Scale bar, 50 ␮m.

specific agonist to the host SCID mice bearing these homotypic tissue recombinants (ArKO-S/ArKO-E and wt-S/wtE). Compared with placebo-treated controls (Fig. 4A), exposure to ER␤ agonist had no obvious effect on the morphology of wt-S/wt-E tissues (Fig. 4B). However, compared with placebo treated ArKO-S/ArKO-E controls (Fig. 4C), treatment with an ER␤ agonist abrogated epithelial hyperplasia in ArKO-S/ArKO-E recombinants (Fig. 4D) so that tissues were indistinguishable from wt-S/wt-E tissue recombinants. This inhibition of hyperplasia was also observed in heterotypic ArKO-S/wt-E and wt-S/ArKO-E tissues (data not shown). The total level of PCNA expression was significantly reduced in all ArKO-S/ArKO-E, ArKO-S/wt-E, and wt-S/ArKO-E tissue recombinants (Table 2). This significant response reflected changes observed in proliferation within the epithelial compartments of each of these grafts confirming the reduction in epithelial hyperplasia after exposure to the ER␤ agonist was due to reduced proliferation of prostatic epithelial cells. Proliferation was significantly higher in the stroma of wtS/ArKO-E recombinants compared with that in wt-S/wt-E recombinants (Table 2) and is probably due to the reciprocal epithelial-stromal cell signaling from the hyperplastic epithelium. Treatment with ER␤ agonist did not suppress proliferation in the stromal tissue of any recombinant consistent with ER␤ being predominantly localized in the epithelium. TABLE 1. Immunohistochemical localization of AR in tissue recombinants Recombinant

wt-E/wt-S ArKO-E/ArKO-S ArKO-E/wt-S wt-E /ArKO-S

Percent compartment AR positive Epithelial

Stromal

Total

15.5 ⫾ 0.1 11.4 ⫾ 1.9 10.3 ⫾ 4.0 13.2 ⫾ 3.8

31.6 ⫾ 2.3 24.3 ⫾ 3.3 28.6 ⫾ 6.1 27.0 ⫾ 12.3

18.5 ⫾ 0.2 13.5 ⫾ 2.7 13.5 ⫾ 2.7 14.6 ⫾ 4.4

No significant differences in stromal, epithelial, or total AR localization could be detected between tissue recombinants following stereological quantitation of AR localization. Values represent mean ⫾ SEM, n ⱖ 6 (P ⬍ 0.05).

These data provide further evidence that the stimulation of epithelial ER␤ is a major factor in regulating prostatic epithelial proliferation, but this occurs at a local tissue level and involves stromal-epithelial cell interactions within prostatic tissue. Although the systemic administration of ER␤ agonist was able to ablate epithelial hyperplasia resulting from local deficiency in estrogen synthesis, it had no significant effect on homotypic wt/wt recombinants; neither did it alter the weight of SV, testis, or prostate lobes in the host mice (Table 3). Attenuation of prostatic epithelial hyperplasia by specific activation of ER␤

The preceding results showed systemic administration of an ER␤-specific agonist was able to abrogate aberrant local signaling between epithelium and stroma resulting from aroTABLE 2. Quantitation of PCNA localization in tissue recombinants Tissue

wt-S/wt-E

Treatment

Placebo ⫹ER␤ ArKO-S/ArKO-E Placebo ⫹ER␤ ArKO-S/wt-E Placebo ⫹ER␤ wt-S/ArKO-E Placebo ⫹ER␤

% Tissue PCNA positive Epithelium

Stroma

Total

16.2 ⫾ 2.5a 17.7 ⫾ 0.3a 27.5 ⫾ 2.5b 16.5 ⫾ 0.8a 28.9 ⫾ 2.9b 18.9 ⫾ 0.8a 31.2 ⫾ 3.8b 20.5 ⫾ 0.4a

6.2 ⫾ 3.8a 3.3 ⫾ 0.8a 26.5 ⫾ 3.5b 22.6 ⫾ 4.3b 23.4 ⫾ 0.4b 17.5 ⫾ 1.9b 21.3 ⫾ 3.2b 19.8 ⫾ 3.9b

14.9 ⫾ 2.6a 21.1 ⫾ 1.1a 27.3 ⫾ 2.4b 17.2 ⫾ 1.2a 28.3 ⫾ 1.8b 18.7 ⫾ 1.0a 30.1 ⫾ 3.8b 20.4 ⫾ 0.8a

PCNA expression in placebo-treated ArKO-S/ArKO-E, ArKO-S/ wt-E, and wt-S/ArKO-E tissue recombinants was significantly elevated in epithelium and stroma compared with wt-S/wt-E recombinants. Treatment with ER␤ agonist significantly reduced the percentage of cells proliferating in epithelium of ArKO-S/ArKO-E, ArKO-S/wt-E, and wt-S/ArKO-E tissue recombinants. Stromal proliferation was not significantly altered by ER␤ agonist, but this lack of response did not significantly alter the total levels of proliferation apparent in each group of recombinants. Values indicate mean ⫾ SEM, n ⫽ 8. Groups with the same superscript are not significantly different (P ⬍ 0.05).



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Discussion

FIG. 4. ER␤ agonist reduces epithelial hyperplasia in ArKO prostate tissue recombinants. A, wt-S/wt-E recombinant from vehicle-treated host showing normal epithelium. B, wt-S/wt-E recombinant from ER␤ agonist-treated host showing normal epithelium. C, ArKO-S/ArKO-E recombinant from vehicle-treated host showing epithelial hyperplasia. D, ArKO-S/ArKO-E recombinant from an ER␤ agonist-treated host shows loss of epithelial hyperplasia and is comparable to wt-S/ wt-E control. Scale bars, 100 ␮m (A–D); 400 ␮m (insets).

matase deficiency. Thus, we tested the effects of administration of a specific ER␤ agonist to intact ArKO male mice to determine if this selective ER modulator (SERM) could reverse existing prostate epithelial hyperplasia and/or hypertrophy. Over 6 wk of administration, the effects of the ER␤ SERM were compared with those of an ER␣-specific agonist. Histologic comparison to wt tissue (Fig. 5D) showed that epithelial hyperplasia normally present in ArKO prostate (Fig. 5E) was attenuated and areas of ArKO tissue were morphologically indistinguishable from wt controls (Fig. 5F). In contrast, ArKO mice receiving an ER␣ agonist showed no attenuating influence on prostatic hyperplasia (Fig. 5G) and exposure to this compound resulted in an inflammatory response evident from the infiltration of inflammatory cells into tissues, a response not seen in ArKO prostate tissue itself (data not shown). Neither ER␣ nor ER␤ agonist showed significant effects on serum testosterone levels compared with control levels, although the responses were highly variable, as is commonly observed (Fig. 5A). Administration of both agonists reduced prostate weight (Fig. 5B), but only ER␣ agonist treatment reduced SV weight (Fig. 5C). The weight of the seminal vesicles is a good indicator of androgen levels and suggests the ER␣ agonist, rather than the ER␤ agonist, lowered systemic androgen levels, although this was not detected in the serum assay.

Estrogens exert both direct and indirect actions on prostate growth and differentiation. The indirect, centrally mediated response to estrogen administration (via ER␣) suppresses androgen levels which in turn reduces epithelial cell proliferation. Consequently, this will mask any putative antiproliferative effects that may be mediated directly by ER␤. To overcome this, we have used the estrogen-deficient, ArKO mouse in addition to ER␤ and ER␣ SERMs to unequivocally demonstrate that ER␤ is a key factor in the regulation of prostatic epithelial proliferation and growth. This study reveals that a failure to activate prostatic ER␤ leads to epithelial hyperplasia, is specifically the result of altered stromal epithelial cell signaling, is independent of systemic hormones, and is reversible after administration of an ER␤ SERM. Elevated levels of systemic estrogen in aging men are associated with the onset of benign and malignant prostate disease (27–29). Estrogen action, mediated via ER␣, will cause aberrant cellular differentiation and proliferation with progression to prostatic hyperplasia, neoplasia, and dysplasia (1–3). Aromatase inhibitors have been tested as treatments for prostate disease, particularly cancer, yet have been surprisingly ineffective in reducing disease progression (30 – 32). Aromatase inhibition, however, will eliminate all estrogen action in the prostate, both direct and indirect, beneficial and adverse. Thus, any beneficial effects of estrogen acting via ER␤ will be lost (8 –10), which may account for the ineffectiveness of aromatase inhibitors in treating prostate disease. Therefore, suppression of estrogenic activity in the prostate may be detrimental, rather than beneficial, in combating prostate disease. Unlike ER␣, the stimulation of ER␤ has been suggested to be antiproliferative. However, this effect has not been demonstrated independent of altered systemic androgen levels, which may also cause a reduction in proliferation. This study provides the first evidence that the ER␤-mediated effects on prostate epithelial cell proliferation are independent of systemic hormone levels. The absence of aromatase activity in both the stroma and epithelium of prostate tissue recombinants results in epithelial hyperplasia comparable to that seen in intact ArKO mice and in men with benign prostatic hyperplasia (17). These data demonstrate that the absence of local aromatase expression in the prostate is a key factor in determining epithelial cell hyperplasia. Based on our prior report demonstrating aromatase expression in the stroma, but not epithelia, of nonmalignant human prostate (13), it was assumed that the stroma was the major site of aromatase activity in the rodent prostate. Consistent with this assumption, tissue recombinants comprising wt epithelium and aromatase-deficient stroma (ArKO-S ⫹ wt-E) developed epi-

TABLE 3. Weights of host mouse organs after ER␤ agonist administration to tissue recombinants Treatment

Body (g)

VP (mg)

AP (mg)

LP (mg)

DP (mg)

SV (mg)

Testis (mg)

⫹ Placebo ⫹ ER␤

53.2 ⫾ 0.6 53.5 ⫾ 5.1

5.3 ⫾ 0.4 5.3 ⫾ 1.5

27.8 ⫾ 2.5 25.6 ⫾ 4.8

2.8 ⫾ 2.3 3.1 ⫾ 0.1

8.4 ⫾ 1.9 7.1 ⫾ 1.9

232.9 ⫾ 81.8 251.8 ⫾ 63.8

149.1 ⫾ 23.5 152.3 ⫾ 17.5

Administration of ER␤ agonist (300 ␮g/kg per day) to intact male SCID mice bearing tissue recombinants did not significantly alter body weights or weights of SV, testis, ventral, anterior, lateral, or dorsal prostate lobes (VP, AP, LP, and DP, respectively) compared with placebo treated SCID mice also bearing tissue recombinants. Values represent mean ⫾ SEM, n ⱖ 6 (P ⬍ 0.05).

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FIG. 5. ER␤ agonist reduces prostate prostatic epithelial hyperplasia in intact ArKO mice. A, Serum T levels were not altered in ArKO mice treated with vehicle control (P, solid bar) (ER␣ 0.3 or 3.0 ␮g/kg䡠d; open bars) or ER␤ agonist (30 or 100 ␮g/kg䡠d; shaded bars). B, VP weights were significantly reduced by ER␣ (3.0 ␮g) and ER␤ agonist (30 and 100 ␮g). C, SV weights were significantly reduced by ER␣ but not ER␤ agonist. Compared with normal wt prostates (D), ArKO prostates (E) demonstrate a hyperplastic epithelial morphology throughout the tissue (E; arrows). F, ArKO tissue treated with ER␤ agonist (100 ␮g) show epithelial morphology comparable to wt prostate. G, ArKO recombinant treated with ER␣ agonist (3.0 ␮g) show no reduction in epithelial hyperplasia. Values represent mean ⫾ SEM; *, Significance P ⬍ 0.05, n ⱖ 6 compared with ArKO control. Scale bar, 50 ␮m (D–G).

thelial hyperplasia, whereas wt stroma recombined with wt epithelia (wt-S ⫹ wt-E) did not. This directly implicates the stroma as a key determinant of epithelial pathology with the loss of stromal aromatase causing epithelial hyperplasia. The ability of the stroma to direct epithelial development is not a new concept and has been studied and reported previously (34 –37). Abnormal stromal characteristics lead to abnormalities in epithelial cell differentiation such as the lack of stromal AR expression contributing to aberrant epithelial cell differentiation during development or in cancer (34 –37). The converse situation, in which normal stroma may reverse and/or abrogate an existing abnormal epithelial pathology, has not been reported nor was it observed in the current study. When normal stroma was recombined with hyperplastic ArKO epithelia (wt-S ⫹ ArKO-E), hyperplasia persisted. Although it is possible that a longer experimental time frame was required for the reversal of hyperplasia, this is unlikely. Additionally, Cunha and colleagues have demonstrated that the signaling between the stroma and the epithelium is reciprocal, and once deregulated, a vicious cycle of miscommunication ensues that serves to exacerbate the pathology (35, 36). This was readily apparent in the current study in which increased cell proliferation was observed in the stroma of tissue recombinants composed of wt stroma and ArKO epithelium (wt-S/ArKO-E). The data presented provide new insight into the pivotal role of estrogen in stromal-epithelial cell signaling during prostate development. The disruption of stromal aromatase activity (and consequently estrogen synthesis) perturbs regulatory signals acting on the epithelium, resulting in epithelial hyperplasia that is independent of systemic hormone levels. However, the mechanism behind this effect is unclear. ER␣ induces atrophy via the suppression of androgen synthesis as well as promoting aberrant proliferation of the prostatic epithelium (squamous metaplasia). Loss or suppression of ER␣ activity, therefore, would be more likely to result in a normal epithelium than a hyperplastic one. Alternately, if ER␤ is a negative regulator of prostate growth

(8 –10), then the loss of local estrogen metabolism would result in reduced ER␤ activation and consequently increased cell proliferation. Consistent with this idea, the administration of an ER␤-specific agonist (ER␤ SERM) inhibited the development of epithelial hyperplasia (ArKO-S/wt-E recombinants) and abrogated the existing hyperplasia in recombinants prepared with ArKO epithelia (ArKO-S/ ArKO-E and wt-S/ArKO-E recombinants). Administration of the ER␤ SERM also resulted in the ablation of existing epithelial hyperplasia in intact ArKO mice. Significantly, this occurred without any suppression of serum testosterone levels or other adverse affects on the hormone-sensitive tissues of the reproductive tract. This in vivo response is in direct contrast to the effects of an ER␣ agonist which, despite reducing the weight of the prostate and other reproductive organs, had no effect on the hyperplastic pathology of the epithelia. These data strongly implicate ER␤ stimulation as a major regulatory factor of prostatic epithelial cell proliferation. However, it is not yet clear exactly which endogenous ligands activate ER␤ in vivo. It has been previously suggested that metabolites of reduced androgens such as 5␣-androstane-3␤,17␤-diol (3␤Adiol) may act as preferred ligands for ER␤, yet the results of the current study are inconsistent with this premise (9). The metabolism of testosterone results in both reduced androgens as well as estrogens via the reductase and aromatase enzymes respectively. As demonstrated, the failed ER␤ activation in the ArKO mouse leads to the development of epithelial hyperplasia. Because reductase activity is not affected in this model (11), it would appear that locally synthesized estrogens are the preferred ligand for ER␤ and are crucial for the prevention of epithelial hyperplasia. Although reduced androgens (like 3␤Adiol) may bind to ER␤, they would appear to be much less effective in this role than estrogens. Studies using exogenous estrogens have demonstrated both direct and indirect effects on prostatic differentiation and proliferation (Fig. 6A). Indirectly, estrogenic suppres-



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information to the existing paradigm (Fig. 6B). First, it demonstrates an essential role for aromatase in stromal-epithelial cell signaling whereby locally synthesized estrogen activates ER␤ to prevent epithelial hyperplasia. Second, it provides direct evidence that ER␤ is antiproliferative without the complicating factor of altered systemic androgen levels. This study clearly demonstrates that local estrogen synthesis is critical for normal growth of the prostate. Conversely, it is well known that too much estrogen has adverse effects on prostate pathology. More effective therapies will need to establish the right balance of estrogen, an outcome that cannot be achieved by aromatase inhibitors that block both the beneficial and adverse effects of estrogen via ER␤ and ER␣, respectively. This study suggests that ER␤-specific agonists may be of considerable benefit in the treatment of prostate disease together with antagonists to ER␣. Acknowledgments Received July 7, 2006. Accepted October 16, 2006. Address all correspondence and requests for reprints to: Gail P. Risbridger, Centre for Urological Research, Monash Institute of Medical Research, Monash University, Clayton 3168, Australia. E-mail: [email protected]. Disclosure Summary: V.P. and K.-H.F. are currently employed by Schering AG.

References

FIG. 6. Estrogenic regulation of prostatic proliferation and differentiation. A, Exogenously administered estrogen can both directly and indirectly effect prostate differentiation and proliferation in intact male mice. Indirectly, suppression of LH release by the pituitary reduces testicular androgen synthesis lowering systemic androgen levels, thus inhibiting normal epithelial cell proliferation and differentiation. Acting locally via ER␣ in the prostatic stroma, estrogens directly stimulate aberrant epithelial cell differentiation and proliferation leading to squamous metaplasia. Therefore, the balance between androgens and estrogens is such that the proliferative action of androgens is reduced at the same time as aberrant proliferation by local estrogen is stimulated. B, Using estrogen deficient aromatase knockout mice the endogenous effects of estrogen deficiency reveal a different balance between androgens and estrogens in regulation of cellular proliferation. Aromatase in the prostatic stroma converts testosterone to estrogen, which is then able to act directly on ER␤ in the prostatic epithelium to inhibit differentiation and proliferation despite maintaining androgen levels. Therefore, aromatase deficiency establishes an abnormal balance between local androgen and estrogen reducing the antiproliferative effects of estrogen via ER␤ while retaining the proliferative effect of androgens.

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