title: Ion transporters involved in acidification of the resorption lacuna in osteoclasts.
abstract: Osteoclasts possess a large amount of ion transporters, which participate in bone resorption; of these, the vacuolar-adenosine trisphosphatase (V-ATPase) and the chloride-proton antiporter ClC-7 acidify the resorption lacuna. However, whether other ion transporters participate in this process is currently not well understood. We used a battery of ion channel inhibitors, human osteoclasts, and their subcellular compartments to perform an unbiased analysis of the importance of the different ion transporters for acidification of the resorption lacuna in osteoclasts. CD14(+) monocytes from human peripheral blood were isolated, and mature osteoclasts were generated using RANKL and M-CSF. The human osteoclasts were (1) used for acridine orange assays for evaluation of lysosomal acidification, (2) used for bone resorption assays, (3) used for generation of osteoclasts membranes for acid influx experiments, or (4) lysed in trizol for mRNA isolation for Affymetrix array analysis. Inhibitors targeted toward most of the ion transporters showed low potency in the acidification-based assays, although some inhibitors, such as carbonic anhydrase II and the sodium-hydrogen exchanger (NHE) inhibitors, reduced resorption potently. In contrast, inhibitors targeted at V-ATPase and ClC-7 potently inhibited both acidification and resorption, as expected. We here show evidence that acidification of the resorption lacuna is mainly mediated by V-ATPase and ClC-7. Furthermore, a group of other ion transporters, including carbonic anhydrase II, the NHEs, and potassium-chloride cotransporters, are all involved in resorption but do not seem to directly be involved in acidification of the lysosomes.
authors.affiliation: Nordic Bioscience A/S, Herlev Hovedgade 207, 2730 Herlev, Denmark. kh@nordicbioscience.com
authors.forename: Kim
authors.initials: K
authors.lastname: Henriksen
authors.forename: Mette G
authors.initials: MG
authors.lastname: Sørensen
authors.forename: Vicki K
authors.initials: VK
authors.lastname: Jensen
authors.forename: Morten H
authors.initials: MH
authors.lastname: Dziegiel
authors.forename: Olivier
authors.initials: O
authors.lastname: Nosjean
authors.forename: Morten A
authors.initials: MA
authors.lastname: Karsdal
mesh_terms: D000143:Acids / Q000378:metabolism; D001862:Bone Resorption / Q000378:metabolism*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000143:Acids; C097540:CLCN7 protein, human; D018118:Chloride Channels; D025262:Vacuolar Proton-Translocating ATPases