During ageing, an increase of reactive oxygen species (ROS) occurs, affecting several processes involved in bone homeostasis, namely osteoblast and osteoclast apoptosis, osteoblastogenesis and adipogenesis. Various signalling pathways are known to be enhanced or decreased by ROS and constitute potential therapeutic targets to limit oxidative damage effects in ageing-associated diseases. Transient administration of parathyroid hormone (PTH), a master regulator of bone remodelling, currently represents the only anabolic therapy in osteoporosis. However, the molecular mechanisms underlying the anabolic features of PTH are ill defined. In osteoblasts, PTH binds to the PTH type 1 receptor (PTH1R), a G protein-coupled receptor, and triggers classic G-protein signalling pathways. The aim of the present study was to analyse the effects of ROS on PTH1R signalling and trafficking. We used fluorescence resonance energy transfer (FRET)-based cAMP (Epac) and ERK biosensors, and the calcium fluorescent dye Fluo-4 to analyse by microscope live cell imaging cAMP, ERK and calcium signalling, respectively, triggered by PTH (1-34) in PTH1R-overexpressing human embryonic kidney (HEK)-293 cells in the presence or absence of H2O2. PTHR internalisation and recycling was measured in HEK-293 cells transiently transfected with HA-PTHR using an ELISA protocol based on an anti-HA antibody and an anti-IgG conjugated with alkaline phosphatase. An increase in cAMP production, ERK phosphorylation and accumulation of intracellular calcium was observed upon PTH (1-34) stimulation of HEK-293 cells. Preincubation of these cells with 1-500 μM H2O2 substantially inhibited all of these PTH (1-34)-dependent signalling pathways. These inhibitory effects were not a result of PTH (1-34) oxidation since PTH (1-34) incubated or not with H2O2 triggered similar cAMP responses. In addition, PTH (1-34) ligand induced about 25% internalisation and subsequent recycling of the PTH1R, and both events were significantly reduced by H2O2 preincubation in these cells. These findings highlight the role of H2O2 as an inhibitor of PTH signalling, and suggest the relevance of ROS as a putative target in bone diseases associated to oxidative stress such as age-related osteopenia.
Disclosure: The authors declared no competing interests. This work was supported by RETICEF (RD12/0043/0008).