title: Autocrine and paracrine nitric oxide regulate attachment of human osteoclasts.
abstract: Nitric oxide (NO) can reduce bone loss in chronic bone diseases. NO inhibits or kills osteoclasts, but the mechanism of action of NO in human bone turnover is not clear. To address this, we studied effects of NO on attachment and motility of human osteoclasts on mineralized and tissue culture substrates under defined conditions. Osteoclasts were differentiated in vitro from CD14 selected monocytes in RANKL and CSF-1, and characterized by cathepsin K expression, tartrate-resistant acid phosphatase (TRAP) activity, acid secretion, and lacunar resorption. Cell attachment was labeled with monoclonal antibody 23C6, specific for a binding domain of a key osteoclast attachment protein, the CD51/CD61 integrin dimer (alpha(v)beta(3)), with or without cell permeabilization. A ring of integrin attachment during bone degradation delimits an extracellular acid compartment, while alpha(v)beta(3) forms focal attachments on non-resorbable substrates. On resorbable substrate but not non-resorbable substrate, alpha(v)beta(3) labeling required cell permeabilization, in keeping with the membrane-matrix apposition that excludes large molecules and allows extracellular acidification. Acid secretion was labeled with the fluorescent weak base indicator lysotracker. NO donors, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), downmodulated acid secretion simultaneously with cytoskeletal rearrangement, with alpha(v)beta(3) redistributed to a discontinuous pattern that labeled, on bone substrate, without membrane permeabilization. These effects were reversible, and an inhibitor of NO synthesis, N(G)-monomethyl-L-arginine (l-NMMA), increased acid secretion and decreased heterogeneity of attachment structures, showing that NO is an autocrine regulator of attachment. A hydrolysis-resistant activating cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate replicated effects of NO donors, while an inhibiting analog, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer, opposed them. On tissue culture or mineralized substrates, NO or cGMP analogs directly regulated motility; after washout cells reattached and survived for days. We conclude that NO is produced by human osteoclasts and regulates acid secretion and cellular motility, in keeping with autocrine and paracrine NO regulation of the resorption cycle.
authors.affiliation: Department of Pathology, University of Pittsburgh, and Veteran's Affairs Medical Center, Pittsburgh, Pennsylvania 15243, USA.
authors.forename: Beatrice B
authors.initials: BB
authors.lastname: Yaroslavskiy
authors.forename: Yanan
authors.initials: Y
authors.lastname: Li
authors.forename: David J P
authors.initials: DJ
authors.lastname: Ferguson
authors.forename: Sara E
authors.initials: SE
authors.lastname: Kalla
authors.forename: Jennifer I
authors.initials: JI
authors.lastname: Oakley
authors.forename: Harry C
authors.initials: HC
authors.lastname: Blair
mesh_terms: D000135:Acid Phosphatase / Q000032:analysis; D000143:Acids / Q000378:metabolism; D000199:Actins / Q000032:analysis; D019898:Autocrine Communication / Q000502:physiology; D001862:Bone Resorption / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D001842:Bone and Bones / Q000378:metabolism; D002352:Carrier Proteins / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D003094:Collagen / Q000032:analysis; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D003804:Dentin / Q000378:metabolism; D005898:Glass; D006801:Humans; D007150:Immunohistochemistry; D039302:Integrin alphaVbeta3 / Q000032:analysis; D007527:Isoenzymes / Q000032:analysis; D007962:Leukocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D009599:Nitroprusside / Q000494:pharmacology; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019899:Paracrine Communication / Q000502:physiology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D000071681:Tartrate-Resistant Acid Phosphatase; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D000143:Acids; D000199:Actins; D002352:Carrier Proteins; D039302:Integrin alphaVbeta3; D007527:Isoenzymes; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; D009599:Nitroprusside; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D016173:Macrophage Colony-Stimulating Factor; D003094:Collagen; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D006152:Cyclic GMP