Bone phenotype in melanocortin 2 receptor-deficient mice

Abstract

Considering that stress condition associated with osteoporosis, the hypothalamic-pituitary-adrenal (HPA) axis, which is essential for central stress response system, is implicated in regulating bone mass accrual. Melanocortin 2 receptor (MC2R), the receptor of adrenocorticotropic hormone is expressed in both adrenal gland cells and bone cells. To elucidate the role of HPA axis in bone metabolism, we assessed the skeletal phenotype of MC2R deficient mice (MC2R ^-/- mice). We first examined bone mineral density and cortical thickness of femur using dual x-ray absorptiometry and micro-computed tomography. We then conducted histomorphometric analysis to calculate the static and dynamic parameters of vertebrae in MC2R ^-/- mice. The levels of osteoblastic marker genes were examined by quantitative PCR in primary osteoblasts derived from MC2R ^-/- mice. Based on these observations, bone mineral density of femur in MC2R ^-/- mice was increasing relative to litter controls. Meanwhile, the thickness of cortical bone of femur in MC2R ^-/- mice was remarkably elevated. Moreover, serum osteocalcin level was drastically raised in MC2R ^-/- mice. However, bone histomorphometry revealed that static and dynamic parameters reflecting bone formation and resorption were unchanged in vertebrae of MC2R ^-/- mice compared to the control, indicating that MC2R function may be specific to appendicular bone than axis bone. Taken together, the HPA axis due to deletion of MC2R is involved in bone metabolism.

Keywords: ACTH, adrenocorticotropic hormone; Bone metabolism; FGD, familial glucocorticoid deficiency; GCs, Glucocorticoids; HPA axis, hypothalamic-pituitary-adrenal axis; HSD, hydroxysteroid dehydrogenase; Hypothalamic-pituitary-adrenal axis; MC2R, melanocortin 2 receptor; Melanocortin 2 receptor; Osteocalcin.

Fig. 1

High bone mass in association with thickening of cortical bone in MC2R^−/− mice femur DXA analysis and micro-computed tomography scanning of femur in MC2R^+/− and MC2R^−/− mice (each N = 6). (A) Bone mineral density in male MC2R^+/− and MC2R^−/− mice at 3 and 6 months, (B) Bone mineral density in female MC2R^+/− and MC2R^−/− mice at 3 and 6 months, (C) Cortical bone thickness in male MC2R^−/− and MC2R^+/− mice at 6 months, (D) Bone volume per tissue volume (BV/TV) in male MC2R^−/− and MC2R^+/− mice at 6 months. MO. month-old. #, P < 0.05; ###, P < 0.001. Data are expressed as means ± SEM.

Fig. 2

Normal skeletal phenotype in prenatal period of MC2R^−/− mice Skeletons from E18.5 embryos of MC2R^−/− and MC2R^+/− mice (each N = 4) were double-stained with alcian blue and alizarin red. Enlarged head region of the skeleton and embryos.

Fig. 3

Normal cancellous bone in MC2R^−/− mice Histomorphometric analysis of the vertebrae of male MC2R^+/− and MC2R^−/− mice (each N = 6) at 6 months. Bone volume per tissue volume (BV/TV), Trabecular thickness (Tb. Th), Mineral apposition rate (MAR), Osteoblast surface area per bone surface area (Ob.S/BS), Osteoclast number per bone perimeter (No. Oc./BPm).

Fig. 4

Serum osteocalcin level was increased in MC2R^−/− mice (A) Serum osteocalcin in male MC2R^−/− and MC2R^+/− mice (each N = 5) at 6 months, (B) Urinary elimination of deoxypyridinoline (DPD) in male MC2R^+/− and MC2R^−/− mice (each N = 5) at 6 months, #, P < 0.05. Data are expressed as means ± SEM.

Fig. 5

No distinguishable effect of osteoblastic gene expression on primary osteoblasts derived from MC2R^−/− mice (A) Gene expression of Akp2 in MC2R^+/− and MC2R^−/− mice, (B) Gene expression of Bglap in male MC2R^+/− and MC2R^−/− mice. We performed in triplicate wells and repeated more than 2 times.