Inorganic phosphate induces apoptosis of osteoblast-like cells in culture.
The major goal of this investigation was to test the hypothesis that one of the major products of bone resorption, inorganic phosphate (Pi), activates osteoblast apoptosis. Osteoblast-like cells were isolated from explants of human bone. In monolayer culture, these cells showed an osteogenic phenotype. Thus, the cells exhibited raised alkaline phosphatase activity, expressed osteogenic messenger RNA transcripts, and formed biological mineral. When these cells were treated with 1-7 mmol/L Pi there was a dose- and time-dependent decrease in cell viability. Accordingly, after 48 h, 5 mmol/L Pi reduced the number of viable osteoblast-like cells by 25%; 7 mmol/L Pi reduced the number of cells by 60%. By 96 h, following treatment with 5 mmol/L Pi, the percentage of viable cells was 30%, whereas 7 mmol/L Pi caused an almost complete loss of osteoblast viability. Osteoblast death was blocked by treating the cells with phosphonoformic acid, an inhibitor of the plasma-membrane Na-Pi transporter. Using morphological and end-labeling procedures, we confirmed that cell death was through apoptosis. To probe the mechanism of cell death, osteoblast-like cells were probed with rhodamine 123, a dye that is responsive to the membrane potential. We noted that Pi-treated cells displayed a profound loss of mitochondrial membrane potential, suggesting that the anion activated the death program through the induction of a mitochondrial membrane permeability transition. We conclude that high levels of osteoblast apoptosis observed at sites of bone resorption may be linked to release of Pi from bone mineral.