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

https://doi.org/10.1016/j.freeradbiomed.2020.04.008Get rights and content
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Highlights

  • Inorganic phosphate (Pi) generates reactive oxygen species (ROS) in committed osteogenic cells.

  • Both exogenous ROS and Pi-induced intracellular ROS utilize Erk1/2 signaling mediator.

  • Only exogenous ROS induces stress activated kinases P38 and JNK.

  • In contrast to exogenous ROS, Pi-induced endogenous ROS is not detrimental to osteogenic cells.

  • ROS generated in response to Pi contributes to physiological function of osteogenic cells.

Abstract

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 Pi stimulates their major function (mineralization), however in osteogenic precursors and endothelial cells Pi cytotoxicity has been reported. Interestingly, Pi cytotoxic effects have been associated with ROS production in the pathological vascular mineralization. In this study, we investigated a molecular mechanistic link between elevated Pi 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 Pi enhances intracellular ROS production. Furthermore, using a combination of mineralization assays and gene expression analyses, we determined that Pi-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 Pi, we identified differences in signaling routes that determine physiologic versus toxic effect of ROS on osteogenic cells. Specifically, while both extracellular and Pi-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 Pi-induced mineralization through the molecular pathway that is distinct from ROS-induced cytotoxic effects.

Graphical abstract

The proposed mechanism underlying differential osteogenic cells responses to Pi-induced intracellular ROS and exogenous ROS: Pi-induced ROS (green) increase mineralization by activating Erk1/2 MAPKs and increasing osteogenic gene expression without inducing osteogenic cell death. Exogenous ROS (red) activates Erk1/2 MAPKs as well stress-associated MAPKs (P38 and JNK1/2), which may drive cytotoxic effect of the extracellular ROS.

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Keywords

Reactive oxygen species (ROS)
Inorganic phosphate (Pi)
Mineralization
Signaling
Osteogenic cells
Mitogen activated protein kinases (MAPKs)

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