Suppressive function of androgen receptor in bone resorption

Abstract

As locally converted estrogen from testicular testosterone contributes to apparent androgen activity, the physiological significance of androgen receptor (AR) function in the beneficial effects of androgens on skeletal tissues has remained unclear. We show here that inactivation of AR in mice using a Cre-loxP system-mediated gene-targeting technique caused bone loss in males but not in females. Histomorphometric analyses of 8-week-old male AR knockout (ARKO) mice showed high bone turnover with increased bone resorption that resulted in reduced trabecular and cortical bone mass without affecting bone shape. Bone loss in orchidectomized male ARKO mice was only partially prevented by treatment with aromatizable testosterone. Analysis of primary osteoblasts and osteoclasts from ARKO mice revealed that AR function was required for the suppressive effects of androgens on osteoclastogenesis supporting activity of osteoblasts but not on osteoclasts. Furthermore, expression of the receptor activator of NF-kappaB ligand (RANKL) gene, which encodes a major osteoclastogenesis inducer, was found to be up-regulated in osteoblasts from AR-deficient mice. Our results indicate that AR function is indispensable for male-type bone formation and remodeling.

Figures

Fig. 1.

(a) Strategy to generate male and female ARKO mice using CMV-Cre transgenic mice. (b) Growth curves of ARKO and WT littermate mice. (c) Serum hormone levels in 8-week-old ARKO and WT mice. (d) Lack of AR expression in bone of ARKO mice (RT-PCR). (e) Soft x-ray images of femora and tibiae from 8-week-old ARKO and WT mice.

Fig. 2.

Osteopenia in male ARKO mice. (a) Bone loss in femur of 8-week-old male ARKO mice by BMD analysis. (b) Bone length in 8-week-old ARKO and WT littermate mice. (c) Three-dimensional computed tomography images of distal femora and axial sections of distal metaphyses from representative 8-week-old male WT and ARKO littermates. (d) Histological features and histomorphometry of the proximal tibiae from 8-week-old male ARKO and WT mice. For Villanueva–Goldner staining of sections from representative ARKO and WT male littermates, mineralized bone is stained green. BV/TV, trabecular bone volume expressed as a percentage of total tissue volume. (e) Histological features and histomorphometry of the midshaft of femora of 8-week-old mice. Cort.Th., cortex thickness.

Fig. 3.

High bone turnover in male ARKO mice and its partial prevention by an aromatizable androgen. (a) Two calcein-labeled mineralized fronts visualized by fluorescent micrography and bone formation parameters in the proximal tibia of 8-week-old male ARKO and WT littermate mice. Ob.S/BS, percentage of bone surface covered by cuboidal osteoblasts; MAR, mineral apposition rate. (b) Two calcein-labeled mineralized fronts and bone histomorphometric parameters in the midshaft of femora from 8-week-old male ARKO and WT littermate mice. ES/BS, percentage of eroded surface. (c) TRAP staining and bone resorption parameters in the proximal tibiae of 8-week-old male WT and ARKO mice. TRAP-positive osteoclasts on secondary spongiosa are stained red. Oc.S/BS, percentage of bone surface covered by mature osteoclasts; N.Oc/B.Pm, number of mature osteoclasts in 10 cm of bone perimeter. (d) Serum osteocalcin levels and urinary deoxypyridinoline levels of 8-week-old male ARKO and WT mice. (e) BMD values of femora from 8-week-old mice after orchidectomy (ORX) and hormone replacement. T, testosterone.

Fig. 4.

(a) Osteoclastogenesis in bone marrow cell and osteoblast cocultures. TRAP-positive multinucleated osteoclast numbers were counted after 8-day coculture of bone marrow cells and osteoblasts from male ARKO and WT mice in the presence of 10 nM 1α,25(OH)2D3. (b) Pit area resorbed by osteoclasts over an additional 48-h period of coculture on a dentine slice. (c) Osteoclast formation in cultured M-CSF-dependent bone marrow macrophages. (d) Survival rate of isolated osteoclasts formed during coculture of osteoblasts and bone marrow cells. (e) Gene expression of RANKL and G3PDH in cultured primary osteoblasts from male ARKO and WT littermates.

Fig. 5.

Schema of skeletal sex hormone action. In male WT mice, skeletal sex hormone activities are mediated by both AR and ER. In female WT mice, skeletal function of ER is likely to dominate over that of AR as serum levels of AR ligands in females are quite low. In male ARKO mice, testicular testosterone production is severely impaired by hypoplasia of the testes, leading to a lack of skeletal sex hormone actions. In contrast, female ARKO mice may not be greatly affected by disruption of AR signaling.