The possibility of the extracellular calcium-sensing receptor (CaR) as the sensor of external calcium (Ca²⁺_O) in osteoblasts, the bone-forming cells, has been proposed. Nevertheless, the osteoblast expression of this receptor is still disputed. We investigated the osteoblast expression and intracellular signalling mechanisms of the CaR in freshly isolated fetal rat calvarial cells (FRC; from animals that were killed humanely) and murine clonal osteoblast cell line 2T3 cells.

We amplified and sequenced bona fide CaR transcripts from both FRC and 2T3 cells by reverse-transcriptase PCR. Specific (i.e. peptide-protectable) CaR immunoreactivity was detected by Western analysis and immunofluorescence microscopy using anti-CaR monoclonal and polyclonal antibodies in these cells. Furthermore we used freshly frozen, non-decalcified preparations of rat femur and human mandible (biopsies obtained with ethical approval) and demonstrated, for the first time, the CaR immunofluorescence in the cells of osteoblast origin.

We then investigated the acute (5-60 min) and short term (24 h) effects of varying [Ca²⁺]_o (0.5, 1.2, 1.8, 2.5 and 5 mM) and of the non-permeant CaR agonist gadolinium (Gd³⁺) (10-100 µM). Treatment with the CaR agonists Ca²⁺_O and Gd^3Æ+_o both result in the phosphorylation of the extracellular signal-regulated kinases (ERK1/2). The effect of 50 µM Gd^3Æ+_o was ablated by the co-incubation with PD98059, an inhibitor of the ERK-activating kinase, MEK. Furthermore this response was partially inhibited by the PI₃ kinase inhibitors wortmannin and LY294002. We also observed increased phosphorylation of the two Akt activation sites, namely Thr 308 and Ser 473 and the phosphorylation of its downstream effector glycogen-synthase kinase β (GSK3β). Together, these responses are consistent with signals of proliferation and cell survival. We then evaluated the activity of the collagen I promoter-driving green fluorescent protein (GFP) in stably transfected 2T3 cells. Our results show that the GFP fluorescence intensity was significantly increased after 24 h incubation with 2.5 mM Ca²⁺_O (ANOVA, n = 4, P < 0.05) and 10, 50 and 100 µM Gd^3Æ+_o (ANOVA, P < 0.01, P < 0.001 and P < 0.05 respectively, n = 4). Furthermore, we show by northern blotting that the 24 h incubation of FRC cells in 2.5 mM Ca²⁺_O increases the production of collagen I mRNA.

We conclude that osteoblasts respond to acute elevations of the [Ca²⁺]_o and that this phenomenon is likely to be mediated by the calcium-sensing receptor, which is expressed in these cells.

This work was funded by the ARC and MRC.