Nicotinic acetylcholine receptors modulate osteoclastogenesis

Abstract

Background: Our aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis.

Methods: The presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity.

Results: We could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca(2+) oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine.

Conclusions: We have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo.

Keywords: Acetylcholine receptor; Nicotine; Osteoclastogenesis.

Fig. 1

Osteoclast formation is inhibited by nAChR ligands in vitro. a (Left panel) Immunocytochemical staining of choline acetyl transferase (ChAT) (Millipore, dilution 1:50) in BMMs stimulated with M-CSF and RANKL and (right panel) control staining with secondary antibody (AB) (biotinylated rabbit-antigoat IgG) only. Scale bars = 100 μm. b–g Effect of nAChR ligands on osteoclastogenesis. Images in the bottom panel are representative of corresponding experiments; scale bar = 100 μm; b nicotine; c epibatidine; d PNU282987; e nicotine after pretreatment with 100 μM mecamylamine; f mecamylamine; g alpha-bungarotoxin. BTX alphabungarotoxin; TRAP tartrate-resistant acidic phosphatase

Fig. 2

Nicotine reversibly blocks RANKL-induced osteoclast formation in vitro. a Effect on nicotine on the first (M-CSF-driven) phase of osteoclastogenesis. b Effect on nicotine on the second (M-CSF and RANKL-driven) phase of osteoclastogenesis. c–g Effect of nicotine on expression of RANKL-induced genes: c TRAP; d cathepsin K; e MMP9; f NFATc1; g RANK. h Effect of nicotine on osteoclastogenesis in the presence and absence of M-CSF in the second phase. i Osteoclast formation after 7 days of treatment with 50 ng/ml RANKL and 30 ng/ml M-CSF in the presence/absence of nicotine 500 μg/ml; osteoclast formation after 12 days of treatment with 50 ng/ml RANKL and 30 ng/ml M-CSF with presence/absence of nicotine 500 μg/ml in the first 7 days of the assay. M-CSF Macrophage colony-stimulating factor, MMP9 matrix-metalloprotease 9, NFATc1 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1, Nic nicotine, RANK receptor activator of nuclear factor kappa B, RANKL receptor activator of nuclear factor kappa B ligand, TRAP tartrate-resistant acidic phosphatase

Fig. 3

Nicotine inhibits in vivo osteoclastogenesis. Calvarial bone resorption induced by subcutaneous injection of lipopolysaccharide (LPS) above the calvaria; LPS, phosphate-buffered saline (PBS), nicotine, or nicotine and LPS. Images in the bottom panel are representative of the indicated groups; scale bars = 100 μm. a Area of erosion/total tissue area; b number of osteoclasts

Fig. 4

Nicotinic AChR ligands block the differentiation of TRAP-positive mononuclear cells in vitro. a Nicotine; b epibatidine; c PNU282987; d mecamylamine; e alpha-bungarotoxin; f effect of nicotine on cytoxicity as assessed by MTT assay; g percentage of live (Annexin V low/7-AAD low), apoptotic (Annexin V high/7-AAD low) and dead (Annexin V high/7-AAD high) cells with flow cytometry plots showing representative experiments (right panels). h–j Effect of nicotine on percentage of cells at the end of the in vitro osteoclastogenesis assay staining positive for marker: g F4/80; h CD11b; i GR1 low/CD11b+. 7-AAD 7-Aminoactinomycin-D, OD optical density, TRAP tartrate-resistant acidic phosphatase

Fig. 5

Osteoclastogenesis is partly inhibited in nAChR subunit –/– mice. a, b Osteoclastogenesis in WT, α7–/–, β2–/– and β4–/– mice with or without 500 μg/ml nicotine treatment. Images in the right panel are representative of the corresponding experiments; scale bars = 100 μm. c, e Bone volume/tissue volume; d, f trabecular density; g trabecular thickness; h trabecular separation; i number of osteoclasts/bone perimeter; j osteoclast surface/bone surface. TRAP tartrate-resistant acidic phosphatase, WT wild-type

Fig. 6

Nicotinic AChR ligands inhibit RANKL-induced Ca²⁺ oscillation. a, b Intracellular Ca²⁺ level in BMMs, a cultured in the presence of M-CSF for 72 hours and b cultured in the presence of M-CSF and RANKL for 72 hours. c–g Effect of acute administration of nAChR ligands on intracellular Ca²⁺ level in BMMs cultured in the presence of M-CSF and RANKL for 72 hours. c Nicotine (Nic; 100 μg/ml); d PNU282987 (PNU; 5–10 μM); e acetylcholine (Ach; 10 μM); f alpha-bungarotoxin (BTX: 100 nM); g mecamylamine (Mec; 10 μM) and ACh (10 μM). h Intracellular Ca²⁺ levels in BMMs cultured in the presence of M-CSF, RANKL and nicotine (100 μg/mL) for 72 hours