Calcium‑dependent activation of PHEX, MEPE and DMP1 in osteocytes

Calcium (Ca²⁺) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca²⁺ concentration, and the levels of bone mineralization regulator proteins [phosphate‑regulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLO‑Y4 cells was determined using the live/dead assay following treatment with various Ca²⁺ concentrations (1.8, 6, 12, 18, 24 and 50 mM) for different durations (15 and 60 min, and 24 h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase‑3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15 min, there were more live cells than dead cells; however, after 60 min, the number of dead cells was increased following treatment with 24 and 50 mM Ca²⁺. After 24 h, there were more dead cells than live cells following treatment with 50 mM Ca²⁺. After 24 h of Ca²⁺ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24 mM Ca²⁺. In addition, as Ca²⁺ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24 mM Ca²⁺ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future.