title: Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro.
abstract: Osteoblasts form an epithelium-like layer with tight junctions separating bone matrix from extracellular fluid. During mineral deposition, calcium and phosphate precipitation in hydroxyapatite liberates 0.8 mole of H(+) per mole Ca(+2). Thus, acid export is needed for mineral formation. We examined ion transport supporting osteoblast vectorial mineral deposition. Previously we established that Na/H exchangers 1 and 6 are highly expressed at secretory osteoblast basolateral surfaces and neutralize massive acid loads. The Na/H exchanger regulatory factor-1 (NHERF1), a pdz-organizing protein, occurs at mineralizing osteoblast basolateral surfaces. We hypothesized that high-capacity proton transport from matrix into osteoblast cytosol must exist to support acid transcytosis for mineral deposition. Gene screening in mineralizing osteoblasts showed dramatic expression of chloride-proton antiporters ClC-3 and ClC-5. Antibody localization showed that ClC-3 and ClC-5 occur at the apical secretory surface facing the bone matrix and in membranes of buried osteocytes. Surprisingly, the Clcn3(-/-) mouse has only mildly disordered mineralization. However, Clcn3(-/-) osteoblasts have large compensatory increases in ClC-5 expression. Clcn3(-/-) osteoblasts mineralize in vitro in a striking and novel trabecular pattern; wild-type osteoblasts form bone nodules. In mesenchymal stem cells from Clcn3(-/-) mice, lentiviral ClC-5 shRNA created Clcn3(-/-), ClC-5 knockdown cells, validated by western blot and PCR. Osteoblasts from these cells produced no mineral under conditions where wild-type or Clcn3(-/-) cells mineralize well. We conclude that regulated acid export, mediated by chloride-proton exchange, is essential to drive normal bone mineralization, and that CLC transporters also regulate fine patterning of bone.
authors.affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Quitterie C
authors.initials: QC
authors.lastname: Larrouture
authors.affiliation: Department of Neurobiology, Pharmacology & Physiology, University of Chicago, Chicago, Illinois.
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Nelson
authors.affiliation: Departments of Pathology and of Microbiology, Immunology & Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Robinson
authors.affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Irina
authors.initials: I
authors.lastname: Tourkova
authors.affiliation: Department of Cell Biology, Washington University, Saint Louis, Missouri.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Schlesinger
authors.affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania Veteran's Affairs Medical Center, Pittsburgh, Pennsylvania hcblair@pitt.edu.
authors.forename: Harry C
authors.initials: HC
authors.lastname: Blair
keywords: Chloride–proton antiporter; ClC‐3; ClC‐5; mineral transport; osteoblast