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

Abstract

The use of estrogens and androgens to prevent bone loss is limited by their unwanted side effects, especially in reproductive organs and breast. Selective estrogen receptor modulators (SERMs) partially avoid such unwanted effects, but their efficacy on bone is only moderate compared with that of estradiol or androgens. Estrens have been suggested to not only prevent bone loss but also exert anabolic effects on bone while avoiding unwanted effects on reproductive organs. In this study, we compared the effects of a SERM (PSK3471) and 2 estrens (estren-alpha and estren-beta) on bone and reproductive organs to determine whether estrens are safe and act via the estrogen receptors and/or the androgen receptor (AR). Estrens and PSK3471 prevented gonadectomy-induced bone loss in male and female mice, but none showed true anabolic effects. Unlike SERMs, the estrens induced reproductive organ hypertrophy in both male and female mice and enhanced MCF-7 cell proliferation in vitro. Estrens directly activated transcription in several cell lines, albeit at much higher concentrations than estradiol or the SERM, and acted for the most part through the AR. We conclude that the estrens act mostly through the AR and, in mice, do not fulfill the preclinical efficacy or safety criteria required for the treatment or prevention of osteoporosis.

Figure 1. Chemical structure of the SERM compound PSK3471.

Figure 2. Estren-α prevents bone loss in OVX female mice.

(A) Representative μCT sections of tibiae from sham-operated or OVX mice treated with implanted slow-release pellets delivering E₂, PSK3471, or estren-α. (B) Quantification of μCT analysis. Values are mean ± SEM. ^†P < 0.001 versus OVX mice; ^#P < 0.01 versus sham-operated mice; ^##P < 0.01 versus E₂-treated mice. BV/TV, bone volume/tissue volume.

Figure 3. Estren-α reduces bone resorption and bone formation in OVX female mice.

Histomorphometric analysis was performed on tibiae, and urinary Dpyr concentration, a biochemical index of bone resorption, was measured in urine. All compounds were delivered by subcutaneous slow-release pellets. In contrast to E₂ and PSK3471 treatment, estren-α treatment decreased bone formation compared with OVX alone, as shown by the decreased osteoblast surface (OBS) as a percentage of trabecular bone surface (BS) (A) and the bone formation rate (BFR/BS, μm³/μm²/yr) (B). PSK3471 did not modify the bone formation; osteoblast surface and bone formation rate remained high in treated animals and were not significantly altered compared with those in OVX mice. Estren-α treatment also decreased the bone resorption compared with that in OVX mice as shown by the lowered osteoclast number (N.Oc/BS) (C) and urinary Dpyr cross-links (D). PSK3471 also decreased the osteoclast numbers compared with OVX alone, whereas under these experimental conditions, E₂ reduced Dpyr but not the number of osteoclasts. *P < 0.05 and **P < 0.01 versus OVX mice.

Figure 4. Estren-α has deleterious effects on the uterus in WT mice.

(A) Representative uteri from the mice in Figure 2. (B) Histomorphometric quantification of total uterus, myometrium, and endometrium areas. Estren-α was as uterotrophic as E₂, while PSK3471 induced only very moderate uterotrophic effects. **P < 0.01 versus OVX mice.

Figure 5. Estren-α, but not PSK3471, stimulates breast cancer cell proliferation.

(A) Estren-α significantly enhanced cellular proliferation in MCF-7 cells. Indeed, at 10 nM, estren-α and E₂ displayed the same efficacy. PSK3471, in contrast, did not display any agonist activity on MCF-7 proliferation. (B) The experiment was performed as in A, but the ability of each compound to antagonize E₂-induced cell proliferation was tested by cotreating the cells with E₂ (0.1 nM) and increasing doses of compounds, as indicated. PSK3471, but not estren-α, antagonized E₂-induced proliferation of MCF-7 cells. E₂, filled diamonds; estren-α, filled triangles; PSK3471, filled squares.

Figure 6. Estren-α effects on uterus are mediated by ERs and AR.

WT C57BL/6 mice were subjected to sham operation, OVX, or OVX and treatment with estren-α pellets with or without subcutaneous coadministration of the anti-estrogen RU58668 (AE) or the anti-androgen RU58642 (AA). Cotreatment with anti-estrogen decreased the uterotrophic effects of estren by approximately 30%, while cotreatment with the anti-androgen nearly abolished the uterotrophic effects of estren-α. ^†P < 0.001 versus untreated OVX mice.

Figure 7. Estren-α prevents bone loss in ORX WT male mice.

DHT, PSK3471, and estren-α are all fully protective against ORX-induced bone loss. (A) Representative μCT sections of tibiae from sham-operated or ORX mice treated with implanted slow-release pellets delivering DHT, PSK3471, or estren-α. (B) The histomorphometric quantifications of μCT data. Values are mean ± SEM; ^†P < 0.001 versus ORX mice.

Figure 8. Estren-α reduces both bone resorption and bone formation in ORX male mice.

Histomorphometric analysis was performed on tibiae from the mice described in Figure 7. Both estren-α and DHT reduced the bone formation parameters osteoblast surface (OBS/BS) (A) and bone formation rate (B) and the bone resorption parameters osteoclast number (C) and urinary Dpyr cross-links (D) in ORX male mice to levels at or below the levels seen in sham-operated animals. In contrast, PSK3471 had little effect on the ORX-induced increases in bone formation and bone resorption. *P < 0.05 and **P < 0.01 versus ORX mice.

Figure 9. Estren-α protects bone in ORX ERα^–/– male mice by restoring bone turnover.

ERα^–/– mice were subjected to sham operation, ORX, or ORX and treatment with DHT or estren-α, each with or without coadministration of the anti-androgen RU58642. DHT and estren prevented the ORX-induced decreases in cancellous bone volume (A) and increases in osteoblast number (N.Ob/BS) (B), serum osteocalcin (OCN) (C), bone formation rate (D), and osteoclast number (E). The effects of both DHT and estren-α were abolished by the simultaneous delivery of the anti-androgen RU58642. *P < 0.05 and ^†P < 0.001 versus ORX control mice.

Figure 10. Estren-α increases the weight of seminal vesicles in ORX WT and ERα^–/– male mice.

Male WT and ERα^–/– mice were subjected to sham operation or ORX and treatment with DHT, estren-α, PSK3471, and/or the anti-androgen RU58642 as described in Methods. The seminal vesicles were weighed directly after the sacrifice. (A) Representative seminal vesicles from untreated and treated WT mice. (B) Quantification of the seminal vesicle wet weight in WT mice. (C) Quantification of the seminal vesicle wet weight in ERα^–/– mice. ORX significantly reduced the seminal vesicle weight in both wild-type and ERα^–/– mice. Both estren-α and DHT induced seminal vesicle hypertrophy. These hypertrophic effects were abolished by coadministration of the anti-androgen RU58642 in ERα^–/– mice. ^†P < 0.001 versus matched ORX controls.