title: Oxidation inhibits PTH receptor signaling and trafficking.
abstract: Reactive Oxygen Species (ROS) increase during aging, potentially affecting many tissues including brain, heart, and bone. ROS alter signaling pathways and constitute potential therapeutic targets to limit oxidative damaging effects in aging-associated diseases. Parathyroid hormone receptors (PTHR) are widely expressed and PTH is the only anabolic therapy for osteoporosis. The effects of oxidative stress on PTHR signaling and trafficking have not been elucidated. Here, we used Fluorescence Resonance Energy Transfer (FRET)-based cAMP, ERK, and calcium fluorescent biosensors to analyze the effects of ROS on PTHR signaling and trafficking by live-cell imaging. PTHR internalization and recycling were measured in HEK-293 cells stably transfected with HA-PTHR. PTH increased cAMP production, ERK phosphorylation, and elevated intracellular calcium. Pre-incubation with H2O2reduced all PTH-dependent signaling pathways. These inhibitory effects were not a result of PTH oxidation since PTH incubated with H2O2triggered similar responses. PTH promoted internalization and recycling of the PTHR. Both events were significantly reduced by H2O2pre-incubation. These findings highlight the role of oxidation on PTHR signaling and trafficking, and suggest the relevance of ROS as a putative target in diseases associated with oxidative stress such as age-related osteoporosis.
authors.affiliation: Instituto de Medicina Molecular Aplicada (IMMA)-Universidad San Pablo CEU, Madrid, Spain; Laboratorio de Metabolismo Mineral y Óseo, Instituto de Investigación Sanitaria (IIS)-Fundación Jiménez Díaz, UAM and Red Temática de Investigación Cooperativa de Envejecimiento y Fragilidad (RETICEF)-Instituto de Salud Carlos III, Madrid, Spain; Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Ardura
authors.affiliation: Instituto de Medicina Molecular Aplicada (IMMA)-Universidad San Pablo CEU, Madrid, Spain; Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Verónica
authors.initials: V
authors.lastname: Alonso
authors.affiliation: Laboratorio de Metabolismo Mineral y Óseo, Instituto de Investigación Sanitaria (IIS)-Fundación Jiménez Díaz, UAM and Red Temática de Investigación Cooperativa de Envejecimiento y Fragilidad (RETICEF)-Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Pedro
authors.initials: P
authors.lastname: Esbrit
authors.affiliation: Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: paf10@pitt.edu.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Friedman
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D018384:Oxidative Stress; D021381:Protein Transport; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D018016:Receptors, Parathyroid Hormone; D006861:Hydrogen Peroxide; D000242:Cyclic AMP
keywords: Hydrogen peroxide (PubChem CID:784); Oxidative stress; PTH; PTH receptor; Signaling; Trafficking