title: 1,25-Dihydroxyvitamin D3 and extracellular calcium promote mineral deposition via NPP1 activity in a mature osteoblast cell line MLO-A5.
abstract: While vitamin D supplementation is common, the anabolic mechanisms that improve bone status are poorly understood. Under standard mineralising conditions including media ionised calcium of 1.1 mM, 1,25-dihydroxyvitamin D3 (1,25D) enhanced differentiation and mineral deposition by the mature osteoblast/pre-osteocyte cell line, MLO-A5. This effect was markedly increased with a higher ionised calcium level (1.5 mM). Gene expression analyses revealed that 1,25D-induced mineral deposition was associated with induction of Enpp1 mRNA, coding for nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and NPP1 protein levels. Since MLO-A5 cells express abundant alkaline phosphatase that was not further modified by 1,25D treatment or exposure to increased calcium, this finding suggested that the NPP1 production of pyrophosphate (PPi) may provide alkaline phosphatase with substrate for the generation of inorganic phosphate (Pi). Consistent with this, co-treatment with Enpp1 siRNA or a NPP1 inhibitor, PPADS, abrogated 1,25D-induced mineral deposition. These data demonstrate that 1,25D stimulates osteoblast differentiation and mineral deposition, and interacts with the extracellular calcium concentration. 1,25D regulates Enpp1 expression, which presumably, in the context of adequate tissue non-specific alkaline phosphatase activity, provides Pi to stimulate mineralisation. Our findings suggest a mechanism by which vitamin D with adequate dietary calcium can improve bone mineral status.
authors.affiliation: Bone Cell Biology Group, Centre for Orthopaedic and Trauma Research, University of Adelaide, Adelaide, SA 5005, Australia; Discipline of Medicine, University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Dongqing
authors.initials: D
authors.lastname: Yang
authors.affiliation: Discipline of Medicine, University of Adelaide, Adelaide, SA 5005, Australia; Musculoskeletal Biology Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Turner
authors.affiliation: Bone Cell Biology Group, Centre for Orthopaedic and Trauma Research, University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Asiri R
authors.initials: AR
authors.lastname: Wijenayaka
authors.affiliation: Discipline of Medicine, University of Adelaide, Adelaide, SA 5005, Australia; Musculoskeletal Biology Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Anderson
authors.affiliation: Discipline of Medicine, University of Adelaide, Adelaide, SA 5005, Australia; Musculoskeletal Biology Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia; Endocrine Bone Research, Chemical Pathology, SA Pathology, Adelaide, SA 5000, Australia.
authors.forename: Howard A
authors.initials: HA
authors.lastname: Morris
authors.affiliation: Bone Cell Biology Group, Centre for Orthopaedic and Trauma Research, University of Adelaide, Adelaide, SA 5005, Australia. Electronic address: gerald.atkins@adelaide.edu.au.
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Atkins
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic; D002118:Calcium / Q000494:pharmacology*; D002454:Cell Differentiation; D002460:Cell Line; D015870:Gene Expression; D051379:Mice; D010006:Osteoblasts; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D018167:Receptors, Calcitriol / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D018167:Receptors, Calcitriol; C118756:dihydroxy-vitamin D3; D015675:Osteocalcin; D014807:Vitamin D; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases; D002118:Calcium
keywords: 1,25-Dihydroxyvitamin D; Alkaline phosphatase; Enpp1; Inorganic phosphate; Osteoblast differentiation; Pyrophosphate