Low NO concentrations inhibit osteoclast formation in mouse marrow cultures by cGMP-dependent mechanism

Abstract

High concentrations of nitric oxide (NO) inhibit bone resorption by mature osteoclasts. We examined the effects of low NO concentrations on osteoclast formation in mouse bone marrow cultures. The NO releasers sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine inhibited the formation of multinucleated cells expressing tartrate-resistant acid phosphatase (a marker for osteoclasts) when administered during the last 3 days of 6-day cultures (differentiation stage) but not during the first 3 days (proliferation stage). SNP (1 microM) completely inhibited pit formation on dentine wafers when added to cultures during osteoclast formation, but 100 microM SNP was required to inhibit pitting by mature osteoclasts. Conversely, the NO synthase inhibitors aminoguanidine and nitro-L-arginine methyl ester both increased osteoclast formation. Inhibition of osteoclast formation by NO likely was guanosine 3',5'-cyclic monophosphate (cGMP) dependent, as SNP increased cGMP in marrow cultures, and 1 mM 8-bromo-cGMP or dibutyryl-cGMP reduced osteoclast formation when administered during the differentiation stage. The cGMP-specific type V phosphodiesterase inhibitor, zaprinast (M & B 22948) also inhibited osteoclast formation (half-maximal inhibitory constant, 100 microM) only when added during the differentiation stage. We conclude that the differentiation stage of osteoclast formation is inhibited by increases in cGMP levels elicited by NO.

RANKL Stimulates Inducible Nitric-oxide Synthase Expression and Nitric Oxide Production in Developing Osteoclasts

“…Whether this NO causes the reduction in c-Fos protein or whether c-Fos declines in an NO-independent manner is a subject for future inquiry. In previous work, NO (supplied by an exogenous NO donor) was reported to diminish OC formation via a cGMP-dependent mechanism in 1,25-dihydroxyvitamin D 3 -differentiated murine marrow cultures (11). In contrast, no evidence was obtained here for the involvement of a cGMP-dependent mechanism to mediate the inhibitory actions of endogenously stimulated iNOS-derived NO in RANKL-differentiated murine RAW cells.…”

“…This negative feedback pathway involves RANKL induction of interferon (IFN)-␤ in a c-Fos-dependent manner, followed by IFN-␤ inhibition of RANKL-induced c-Fos expression necessary for OC formation (5). OC formation and bone resorption are also inhibited by elevated levels of the multifunctional signal molecule nitric oxide (NO) in vivo and in vitro (7)(8)(9)(10)(11)(12)(13). NO is produced from L-arginine in an oxidative reaction catalyzed by NO synthase isoenzymes that are either constitutively expressed and calcium-activated (endothelial and neuronal NO synthase isoforms) or transcriptionally induced (inducible NO synthase (iNOS) isoform) in response to inflammatory stimuli (14).…”

“…Previously, our group (8,12,15) and others (9,16) have shown that OCs and related OC-like cells (as well as other bone cells) express iNOS and release NO in a regulated manner. NO produced endogenously or supplied by NO donors exerts potent biphasic actions that profoundly affect the recruitment, proliferation, differentiation, activity, and/or survival of OCs and osteoblasts, their precursors, and other cells within bone (8,11,17,18). Whereas low levels of NO may support osteoblast bone formation and OC-mediated bone remodeling (both basal and cytokine-induced) (9, 19 -22), high NO levels and NO-generating compounds inhibit OC formation and bone resorption and prevent bone loss, for example, in severe inflammation or estrogen-deficient animals (8, 10 -13, 17-24).…”

PDE1B2 regulates cGMP and a subset of the phenotypic characteristics acquired upon macrophage differentiation from a monocyte

“…Similarly, monocyte differentiation in the presence of adenylyl cyclase agonists such as PGE 2 (14)(15)(16) or the guanylyl cyclase agonist atrial natriuretic factor (ANF) (17) can polarize dendritic cells toward promoting Th1 or Th2 responses, respectively. Likewise, generation of osteoclasts from bone marrow precursors is potentiated by treatments that increase either cAMP or cGMP such as PGE 2 (18,19) and nitric oxide (20). Finally, differentiation of monocyte cell lines, such as the HL-60 (21-23) and U937 (24,25) lines, can be initiated by increased cAMP or cGMP levels.…”

Different levels of the neuronal nitric oxide synthase isoform modulate the rate of osteoclastic differentiation of TIB‐71 and CRL‐2278 RAW 264.7 murine cell clones

“…Moreover, deficient iNOS (van't Hof et al, 2000) mice showed an inhibition of osteoclastic functional activity, while eNOS knockout mice showed an impaired osteoblast function (Aguirre et al, 2001). Insufficient cellular NO concentration reduced consistently osteoclast differentiation from mouse bone marrow cells (Holliday et al, 1997), while other study reported that NO induced a reduction of osteoclast formation (van't Hof and . In order to get over these contradictions, some authors have recently proposed an interesting explanation of these studies suggesting that low-intermediate levels of NO induce osteoclast activity and survival while either the absence or the excess of NO inhibits osteoclast activation and survival (Brandi et al, 1995).…”