Osteoblasts are bone-forming cells dependent upon the fluctuations in [Ca²⁺]_o regulated by the calciotropic hormones (e.g. parathyroid hormone). We investigated the sensing of Ca²⁺_O by freshly isolated fetal rat calvarial cells (FRC; from animals that were humanely killed) and the involvement of the Ca²⁺_O-sensing receptor (CaR) in this mechanism by using CaR agonists. Specifically, we investigated short- (24, 48 and 72 h) and long-term (5-23 days) effects of [Ca²⁺]_o (0.5, 1.2, 1.8 and 2.5 mM) and of the non-permeant CaR agonist gadolinium (Gd^3Æ+_o ; 10-100 µM) on FRC proliferation, differentiation and the production of mineralised nodules. [Ca²⁺]_o of 1.2 mM served as a control concentration.

Increasing [Ca²⁺]_o from 1.2 to 1.8 and 2.5 mM and treatment with 25 and 50 µM Gd^3Æ+_o increased FRC cell proliferation at days 1-10 of the treatment with Ca²⁺_O (P < 0.05 for 1.8 mM Ca²⁺_O at day 1 and 3; P < 0.01 for 1.8 and 2.5 mM Ca²⁺_O at day 5; P < 0.001 for 1.8 and 2.5 mM Ca²⁺_O at days 7 and 10. All n = 3; ANOVA), at day 5, 7, 10 and 14 of the treatment with 50 µM Gd^3Æ+_o and at day 14 for 25 µM Gd^3Æ+_o (both P < 0.05, n = 3, ANOVA). Subsequently we measured changes in the mRNA expression levels of osteoblast differentiation markers in response to increasing [Ca²⁺]_o and 50 µM Gd^3Æ+_o . Core-binding factor1 (cbfa1), osteocalcin (OC) and osteopontin (OP) mRNA levels increased in the presence of 1.8 and 2.5 mM [Ca²⁺]_o and 50 µM [Gd³⁺]_o at days 12 and 18. No changes in the mRNA levels were observed for tissue non-specific alkaline phosphatase (TNAP) and for the housekeeping gene β-actin. We showed that TNAP enzyme activity (nmoles of paranitrophenol per µg protein) was dependent on a narrow range of [Ca²⁺]_o, with [Ca²⁺]_o lower than 1.2 mM and greater than 1.5 mM producing significant inhibitory effects at 7 days of treatment (P < 0.01; n = 6, ANOVA). Finally, both Ca²⁺_O (1.8 and 2.5 mM) (n = 6) and Gd^3Æ+_o (50µM) (n = 3) increased the production and the area of mineralised nodules (P < 0.05, ANOVA). The FRC responses to low [Ca²⁺]_o (0.5 mM) included an increase in proliferation at short-term time points, a decrease in proliferation and mineralised nodule formation and an increase in the expression of osteocalcin mRNA in the long-term observations.

Our results indicate that osteoblasts sense both high and low [Ca²⁺]_o, and that small deviations of [Ca²⁺]_o from the physiological range have a significant impact on osteoblast proliferation and differentiation, independently of other paracrine and endocrine factors. The effects of increasing [Ca²⁺]_o are likely to be mediated by the calcium-sensing receptor, which is expressed in these cells (see Dvorak et al. 2003).

This work was funded by the MRC and ARC.