Introduction: The calcium sensing receptor presence (CaSR) at the surface of the osteocytes has never been clearly investigated. The CaSR are known to be express on osteoblasts. Osteocytes being old osteoblasts embedded in the matrix, this expression of CaSR is likely, and could constitute a key role to calcium signalling.
Strontium ranelate (SrRan) has shown to activate osteoblasts by fixation on CaSR (Chattopadhyay N 2007, Biochem Pharmacol; Hurtel-Lemaire AS 2009, J Biol Chem). Our aim has been to investigate this expression of CaSR on the osteocyte, and its modulation by SrRan.
Materials and methods: We used MLOY4 cell cultures (osteocytes) and the RAW cells, not treated with SrRan, constitute the controls and as known to express CaSR. Several SrRan concentrations (0, 0.1, 1, and 5 mM) have been tested at different times (0, 1, 2, 3, 5, 7, and 14 days). To assess CaSR expression on the membrane and inside cell, we used a CaSR mouse primary antibody (ThermoScientific). After, we revealed the primary antibody by a fluorescent secondary antibody (phycoerythrin) via a flow cytometry.
Results: First, CaSR were present on the MLOY4 membrane and still with a higher level in the total cell (membrane and inside cell) which confirmed its presence both on the membrane and inside the cell. CaSR expression levels of MLOY4 were approximately twice the RAW cell expression with 1 mM SrRan and three times with 5 mM SrRan. Second, a dose-effect of SrRan was identified. CaSR levels increased significantly with 1 and 5 mM concentrations, and at 5 and 7 days of SrRan exposure (5 days: 1 mM+30% and 5 mM+70% compared to 0 mM, and 7 days: 1 mM+175% and 5 mM+380% compared to 0 mM).
In conclusion, our data have shown that: i) CaSR is expressed both on and inside the osteocytes and ii) its expression is modulated by SrRan, with a dose-dependant increase.
18 - 21 May 2013
European Calcified Tissue Society