Reactive oxygen species (ROS) generation as an underlying mechanism of inorganic phosphate (Pi)-induced mineralization of osteogenic cells

Reactive Oxygen Species (ROS) are a natural byproduct of oxygen metabolism. At physiological levels, ROS regulate multiple cellular processes like proliferation, migration, and differentiation. Increased levels of ROS are associated with pathological conditions, such as inflammation and vascular calcification, where they elicit cytotoxic effects. These contrasting outcomes of ROS have also been reported in osteogenic precursor cells. However, the role of ROS in committed osteogenic cells has not been investigated. Cytotoxic and physiologic effects have also been demonstrated for extracellular phosphate (Pi). Specifically, in committed osteogenic cells P-i stimulates their major function (mineralization), however in osteogenic precursors and endothelial cells P-i cytotoxicity has been reported. Interestingly, P-i cytotoxic effects have been associated with ROS production in the pathological vascular mineralization. In this study, we investigated a molecular mechanistic link between elevated P-i and ROS production in the context of the mineralization function of committed osteogenic cells. Using committed osteogenic cells, 17IIA11 odontoblast-like cell and MLO-A5 osteoblast cell lines, we have unveil that P-i enhances intracellular ROS production. Furthermore, using a combination of mineralization assays and gene expression analyses, we determined that P-i-induced intracellular ROS supports the physiological mineralization process. In contrast, the exogenous ROS, provided in a form of H2O2, was detrimental for osteogenic cells. By comparing molecular signaling cascades induced by extracellular ROS and P-i, we identified differences in signaling routes that determine physiologic versus toxic effect of ROS on osteogenic cells. Specifically, while both extracellular and P-i-induced intracellular ROS utilize Erk1/2 signaling mediator, only extracellular ROS induces stress-activated mitogen-activated protein kinases P38 and JNK that are associated with cell death. In summary, our results uncovered a physiological role of ROS in the P-i-induced mineralization through the molecular pathway that is distinct from ROS-induced cytotoxic effects.