title: Sclerostin regulates release of bone mineral by osteocytes by induction of carbonic anhydrase 2.
abstract: The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF-κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL-mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin-stimulated mineral dissolution by human primary osteocyte-like cells (hOCy) and mouse MLO-Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate-resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO-Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co-presence of the carbonic anhydrase inhibitor, acetozolamide. Car2-siRNA knockdown in MLO-Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO-Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co-addition of acetozolamide. These findings suggest a new role for sclerostin in the regulation of perilacunar mineral by osteocytes.
authors.affiliation: Bone Cell Biology Group, Centre for Orthopaedic & Trauma Research, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kogawa
authors.forename: Asiri R
authors.initials: AR
authors.lastname: Wijenayaka
authors.forename: Renee T
authors.initials: RT
authors.lastname: Ormsby
authors.forename: Gethin P
authors.initials: GP
authors.lastname: Thomas
authors.forename: Paul H
authors.initials: PH
authors.lastname: Anderson
authors.forename: Lynda F
authors.initials: LF
authors.lastname: Bonewald
authors.forename: David M
authors.initials: DM
authors.lastname: Findlay
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Atkins
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D001842:Bone and Bones / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000096:biosynthesis*; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D005819:Genetic Markers; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008903:Minerals / Q000378:metabolism*; D010011:Osteocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008903:Minerals; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C422903:SOST protein, human; C486030:Sost protein, mouse; D002256:Carbonic Anhydrases; D002118:Calcium
keywords: CARBONIC ANHYDRASE 2; OSTEOCYTE; OSTEOCYTIC OSTEOLYSIS; SCLEROSTIN; SOST