Hydrogen peroxide-induced poly(ADP-ribosyl)ation regulates osteogenic differentiation-associated cell death

We set out to investigate the role of poly(ADP-ribosylation), the attachment of NAD+-derived (ADP-ribose)n polymers to proteins, in the regulation of osteogenic differentiation of SAOS-2 cells and mesenchymal stem cells. In osteogenic differentiation medium, SAOS-2 cells showed mineralization and expressed alkaline phosphatase and osteoblastic marker genes such as Runx2, osterix, BMP2, and osteopontin. The cells also released hydrogen peroxide, displayed poly(ADP-ribose) polymerase (PARP) activation, and showed commitment to cell death (apoptosis and necrosis). Scavenging reactive oxygen species by glutathione or decomposing hydrogen peroxide by the addition of catalase reduced differentiation, PARP activation, and cell death. We silenced the expression of the main PAR-synthesizing enzyme PARP-1 and the PAR-degrading enzyme poly(ADP-ribose) glycohydrolase (PARG) in SAOS-2 osteosarcoma cells (shPARP-1 and shPARG, respectively). Both shPARP-1- and shPARG-silenced cells exhibited altered differentiation, with the most notable change being increased osteopontin expression but decreased alkaline phosphatase activity. PARP-1 silencing suppressed both apoptotic and necrotic cell death, but the PARP inhibitor PJ34 sensitized cells to cell death, indicating that the effects of PARP-1 silencing are not related to the activity of the enzyme. PARG silencing resulted in more apoptosis and, in the last days of differentiation, a shift from apoptosis toward necrosis. In conclusion our data prove that hydrogen peroxide-induced poly(ADP-ribose) signaling regulates cell death and osteodifferentiation.

Graphical AbstractFigure optionsDownload high-quality image (178 K)Download as PowerPoint slideHighlights
► H2O2 is a key regulator of osteogenic differentiation of SAOS-2 and mesenchymal stem cells.
► H2O2 induces PARylation and cell death during osteogenic differentiation.
► The concerted actions of PARP-1 and PARG regulate osteodifferentiation.
► This regulation involves PARP activity but also has a nonenzymatic component.