CD14
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. Copyright 2004
Wiley-Liss, Inc.
PMID: 15034931 [PubMed - in process]