Estrogen receptor-α expression in neuronal cells affects bone mass

Abstract

It has generally been assumed that bone mass is controlled by endocrine mechanisms and the local bone environment. Recent findings demonstrate that central pathways are involved in the regulation of bone mass. Estrogen is involved in the regulation of bone homeostasis and the CNS is also a target for estrogen actions. The aim of this study was to investigate in vivo the role of central estrogen receptor-α (ERα) expression for bone mass. Nestin-Cre mice were crossed with ERα(flox) mice to generate mice lacking ERα expression specifically in nervous tissue (nestin-ERα(-/-)). Bone mineral density was increased in both the trabecular and cortical bone compartments in nestin-ERα(-/-) mice compared with controls. Femoral bone strength was increased in nestin-ERα(-/-) mice, as demonstrated by increased stiffness and maximal load of failure. The high bone mass phenotype in nestin-ERα(-/-) mice was mainly caused by increased bone formation. Serum leptin levels were elevated as a result of increased leptin expression in white adipose tissue (WAT) and slightly increased amount of WAT in nestin-ERα(-/-) mice. Leptin receptor mRNA levels were reduced in the hypothalamus but not in bone. In conclusion, inactivation of central ERα signaling results in increased bone mass, demonstrating that the balance between peripheral stimulatory and central inhibitory ERα actions is important for the regulation of bone mass. We propose that the increased bone mass in nestin-ERα(-/-) mice is mediated via decreased central leptin sensitivity and thereby increased secretion of leptin from WAT, which, in turn, results in increased peripheral leptin-induced bone formation.

Fig. 1.

Specificity of ERα deletion in nestin-ERα^−/− females. RNA was prepared from the hypothalamic region of the brain and the indicated nonneuronal tissues from 3-mo-old nestin-ERα^−/− and control mice. (A) Cre expression, analyzed using real-time PCR, was specifically detected in the brain (n = 4). (B) ERα mRNA levels, shown as percentage of control, were specifically reduced in the brain (n = 6–10). (C) Nestin-ERα^−/− females do not exert disturbed sex steroid levels. Serum levels of estradiol, testosterone and LH were measured in nestin-ERα^−/− females, ERα^−/− females, and their corresponding control females. (n = 4–13). Values are given as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice, Student's t test. ND, not detectable.

Fig. 2.

Deletion of ERα in nervous tissue results in increased bone mass. pQCT analysis of the tibia demonstrated increased trabecular volumetric bone mineral density (Tibia Trab. BMD) (A), increased cortical volumetric BMD (Tibia Cort. BMD) (B), and increased cortical thickness (Tibia Cort. Thk.) (C) in 3-mo-old nestin-ERα^−/− mice compared with controls (n = 6). (D) Representative images of μCT analyses of trabecular bone in the proximal metaphysis of the tibia and in vertebrae L5. Bone volume per total volume (BV/TV) (E), trabecular thickness (Tb.Th) (F), and trabecular number (Tb.N) (G) of trabecular bone in the proximal metaphysis of the tibia and in vertebrae L5 (n = 5–8). Three-point bending of femur demonstrated increased (H) stiffness and (I) maximal load (Max. load) at failure in nestin-ERα^−/− mice (n = 5). (J) Dynamic histomorphometric analysis of tibia giving bone formation rate per tissue volume (BFR/TV; n = 4–5). (K) Effect of ovx on bone mass in nestin-ERα^−/− and control females. Three-month-old female mice were ovariectomized or sham-operated and terminated after 4 wk (n = 5–8). Values are given as mean ± SEM; *P < 0.05, **P < 0.01 vs. control; ^#P < 0.05, ^###P < 0.001 vs. sham, Student's t test.

Fig. 3.

Possible pathways for the central ERα effect on bone. (A) Peripheral (serum) and central serotonin levels, measured using ELISA and HPLC, respectively (n = 8–10). Urinary catecholamine secretion (ng/mL) related to urinary creatinine (Cr) (mg/dL) content (n = 7). UCP1 mRNA expression in BAT (n = 5–9). Leptin receptor (LepR), SOCS3, and PTP1b mRNA expression in hypothalamus (n = 5–7). (B) Serum levels of leptin (n = 9–10). (C) Leptin mRNA expression in WAT (n = 6–7). (D) Retroperitoneal fat mass (mg) (n = 6–7). (E) mRNA expression in bone (tibia) of IL-6 and CIITA (n = 9). (F) T-cell frequency (CD3⁺ cells) in bone marrow (BM) (n = 9–10) and (G) T-cell proliferation in spleen (n = 6–10). (H) Schematic presentation of the main proposed mechanism for the central ERα effect on bone (see Discussion). Values are given as mean ± SEM; *P < 0.05, **P < 0.01 vs. control, Student's t test.