title: Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures From Rat Calvaria.
abstract: The in vitro osteogenic differentiation has been intensively studied. However, it is not yet clear precisely how osteogenesis can be optimized. Changes in extracellular Ca(2+) concentration ([Ca(2+) ]e ), as well as modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the effects of a combined treatment of ATPγ-S and high [Ca(2+) ]e (5.35 mM) on osteogenic differentiation and function of primary cell cultures from rat calvaria. Our results indicate that ATPγ-S stimulates cell transition from the G0 to S phase of cell cycle, involving the PI3K signaling pathway. Treatment with 10 or 100 µM ATPγ-S and [Ca(2+) ]e (ATP-[Ca(2+) ]e ) for 48 h increases cell number significantly above the control. ATPγ-S treatment in osteogenic medium containing [Ca(2+) ]e stimulates the gene expression of BMP-4, BMP-5, and OPN at 16, 48, and 72 h, respectively, above control. In same conditions, treatment for 6 days with 10 µM UTP or 100 µM UDP significantly increased the ALP activity respect to control. Cells grown in osteogenic medium showed a statistically significant increase in calcium deposits at 15 and 18 days, for 10 µM ATPγ-S treatment, and at 18 and 22 days, for [Ca(2+) ]e treatment, respect to control but ATP-[Ca(2+) ]e treatment shown a significant greater mineralization at 15 days respect to ATPγ-S, and at 18 days respect to both agonists. In conclusion, we demonstrated that an osteogenic medium containing 10 µM ATPγ-S and 5.35 mM [Ca(2+) ]e enhance osteogenesis and mineralization by rat primary calvarial cells cultures. J. Cell. Biochem. 117: 2658-2668, 2016. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, CONICET, San Juan 670, (B8000ICN) Bahía Blanca, Argentina.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Laiuppa
authors.affiliation: Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, CONICET, San Juan 670, (B8000ICN) Bahía Blanca, Argentina. gsantill@criba.edu.ar.
authors.forename: Graciela E
authors.initials: GE
authors.lastname: Santillán
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism*; D055417:Bone Morphogenetic Protein 5 / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004338:Drug Combinations; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012886:Skull / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: C000603843:BMP5 protein, rat; C525811:Bmp4 protein, rat; D055415:Bone Morphogenetic Protein 4; D055417:Bone Morphogenetic Protein 5; D003598:Cytoskeletal Proteins; D004338:Drug Combinations; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; C529605:OPHN1 protein, rat; D012333:RNA, Messenger; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D002118:Calcium
keywords: CALCIUM; EXTRACELLULAR ATP; OSTEOGENESIS; PURINERGIC RECEPTORS