title: Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation.
abstract: Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown that after exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (β-glycerophosphate/ascorbic acid/dexamethasone). In parallel, with the increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomes upregulated. This effect, measured on transcriptional level is also substantiated by immunohistological studies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonic anhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-deposit producing SaOS-2 cells by scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed an accumulation of the signals for the element carbon and, as expected, also for phosphorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-II activity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and a subsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest that Ca-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We propose that activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiency diseases.
authors: ERC Advanced Investigator Grant Research Group at Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Duesbergweg 6, D-55128 Mainz, Germany. wmueller@mail.uni-mainz.de
authors: Werner E G
authors: WE
authors: Müller
authors: Heinz C
authors: HC
authors: Schröder
authors: Ute
authors: U
authors: Schlossmacher
authors: Vlad A
authors: VA
authors: Grebenjuk
authors: Hiroshi
authors: H
authors: Ushijima
authors: Xiaohong
authors: X
authors: Wang
mesh_terms: D000086:Acetazolamide / Q000009:adverse effects; D001205:Ascorbic Acid / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism*; D001862:Bone Resorption / Q000188:drug therapy; D002130:Calcium Phosphates / Q000378:metabolism*; D002257:Carbonic Anhydrase Inhibitors / Q000009:adverse effects; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003907:Dexamethasone / Q000378:metabolism; D004926:Escherichia coli; D018507:Gene Expression Regulation, Developmental; D005994:Glycerophosphates / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D015854:Up-Regulation; D014965:X-Rays
chemical_list: D001639:Bicarbonates; D002130:Calcium Phosphates; D002257:Carbonic Anhydrase Inhibitors; D005994:Glycerophosphates; D003907:Dexamethasone; D002256:Carbonic Anhydrases; D000086:Acetazolamide; D001205:Ascorbic Acid; C031463:beta-glycerophosphoric acid
keywords: Bone metabolism; Ca-carbonate; Carbonic anhydrase; Hydroxyapatite; Osteoblasts; SaOS-2 cells