title: Caveolin-1 regulates P2X7 receptor signaling in osteoblasts.
abstract: The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware; and.
authors.forename: Vimal
authors.initials: V
authors.lastname: Gangadharan
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware; and.
authors.forename: Anja
authors.initials: A
authors.lastname: Nohe
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware; and Bioimaging Center, Delaware Biotechnology Institute, Newark, Delaware.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Caplan
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware; and Bioimaging Center, Delaware Biotechnology Institute, Newark, Delaware.
authors.forename: Kirk
authors.initials: K
authors.lastname: Czymmek
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware; and Bioimaging Center, Delaware Biotechnology Institute, Newark, Delaware rlduncan@udel.edu.
authors.forename: Randall L
authors.initials: RL
authors.lastname: Duncan
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D034622:RNA Interference; D058486:Receptors, Purinergic P2X7 / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: C495541:Cav1 protein, mouse; D051242:Caveolin 1; D058911:Purinergic P2X Receptor Agonists; D058920:Purinergic P2X Receptor Antagonists; D058486:Receptors, Purinergic P2X7
keywords: P2X7R; caveolin-1; endocytosis; osteoblasts; purinergic signaling