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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.

Original publication

DOI

10.1002/jcb.20009

Type

Journal article

Journal

J Cell Biochem

Publication Date

01/04/2004

Volume

91

Pages

962 - 972

Keywords

Acid Phosphatase, Acids, Actins, Antigens, CD14, Autocrine Communication, Bone Resorption, Bone and Bones, Carrier Proteins, Cell Adhesion, Cell Differentiation, Cell Movement, Collagen, Cyclic GMP, Dentin, Glass, Humans, Immunohistochemistry, Integrin alphaVbeta3, Isoenzymes, Leukocytes, Macrophage Colony-Stimulating Factor, Membrane Glycoproteins, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Microscopy, Interference, Nitric Oxide, Nitroprusside, Osteoclasts, Paracrine Communication, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, S-Nitroso-N-Acetylpenicillamine, Tartrate-Resistant Acid Phosphatase, omega-N-Methylarginine