Extra-nuclear effects of estrogen on cortical bone in males require ERαAF-1

Abstract

Estradiol (E2) signaling via estrogen receptor alpha (ERα) is important for the male skeleton as demonstrated by ERα inactivation in both mice and man. ERα mediates estrogenic effects not only by translocating to the nucleus and affecting gene transcription but also by extra-nuclear actions e.g., triggering cytoplasmic signaling cascades. ERα contains various domains, and the role of activation function 1 (ERαAF-1) is known to be tissue specific. The aim of this study was to determine the importance of extra-nuclear estrogen effects for the skeleton in males and to determine the role of ERαAF-1 for mediating these effects. Five-month-old male wild-type (WT) and ERαAF-1-inactivated (ERαAF-1⁰) mice were orchidectomized and treated with equimolar doses of 17β-estradiol (E2) or an estrogen dendrimer conjugate (EDC), which is incapable of entering the nucleus and thereby only initiates extra-nuclear ER actions or their corresponding vehicles for 3.5 weeks. As expected, E2 treatment increased cortical thickness and trabecular bone volume per total volume (BV/TV) in WT males. EDC treatment increased cortical thickness in WT males, whereas no effect was detected in trabecular bone. In ERαAF-1⁰ males, E2 treatment increased cortical thickness, but did not affect trabecular bone. Interestingly, the effect of EDC on cortical bone was abolished in ERαAF-1⁰ mice. In conclusion, extra-nuclear estrogen signaling affects cortical bone mass in males, and this effect is dependent on a functional ERαAF-1. Increased knowledge regarding estrogen signaling mechanisms in the regulation of the male skeleton may aid the development of new treatment options for male osteoporosis.

Keywords: cortical bone; estrogen; estrogen dendrimer conjugate; male skeleton.

Figure 1

ERαAF-1 is important for the extra-nuclear estrogenic effects on cortical bone in males. Cortical thickness (Ct.Th) (A) and density (Ct.Den) (B) were analyzed in tibia of orchidectomized WT and ERαAF-1⁰ male mice treated with Veh, E2, DC or EDC for 3.5 weeks. *P < 0.05 and ***P < 0.001, Student’s t test. Data are presented as mean ± s.e.m. (n = 9–11).

Figure 2

EDC-initiated extra-nuclear signaling does not affect trabecular bone in males. Trabecular bone volume per total volume (BV/TV) (A), thickness (Tb.Th) (B), trabecular number (Tb.N) (C) and trabecular separation (Tb.Sp) (D) in vertebrae (L₅) of orchidectomized WT and ERαAF-1⁰ male mice treated with Veh, E2, DC or EDC for 3.5 weeks. (E) Representative scans of trabecular bone in vertebrae L₅. **P < 0.01 and ***P < 0.001, Student’s t test. Data are presented as mean ± s.e.m. (n = 9–11).

Figure 3

Summary of E2 and EDC effects in WT and ERαAF-1⁰ males. E2 treatment affects both cortical and trabecular bone in WT males and has a moderate effect on cortical, but not trabecular, bone in ERαAF-1⁰ males. EDC treatment only affects cortical bone in WT males, and this effect is abolished in ERαAF-1⁰ mice. (A) ++, normal effect; +, moderate effect; 0, no effect. (B) Schematic presentation of the main proposed effects of EDC and E2 in WT and ERaAF-1⁰ male mice.