Nicotinic acetylcholine receptors modulate osteoclastogenesis

Acetylcholine (ACh) was the first neurotransmitter to be discovered [1]. It is an essential mediator of both the central and the peripheral nervous system [2, 3]. ACh acts on two main types of receptors: nicotinic acetylcholine receptors (nAChRs), which are ligand-gated ionotropic receptors, and muscarinic acetylcholine receptors (mAChRs), which are metabotropic G-protein coupled receptors [4]. Neuronal nAChRs are pentamers composed of either homomeric or heteromeric combinations of twelve different nicotinic receptor subunits: α2 through α10 and β2 through β4 [5]. In addition to its role in the nervous system, ACh is increasingly recognized as an important modulator in various non-neuronal tissues [6‐8].

nAChRs have also been identified on cells known to be essential for the maintenance of bone homeostasis, such as osteoblasts and osteoclasts [9‐12]. In addition, other components involved in cholinergic transmission, including the vesicular ACh transporter, but also the enzyme choline acetyltransferase needed for the synthesis of Ach, have been reported to be present in murine osteoblasts [13]. Moreover, recent studies have revealed the presence of cholinergic innervation in bone marrow, suggesting a potential regulatory role of ACh on all bone marrow cells including osteoclasts, but possibly also osteoblasts [12, 14, 15].

Osteoclasts are formed by the fusion of hematopoetic mononuclear cells [16]. This process requires the presence of macrophage colony-stimulating factor (M-SCSF) and receptor activator of nuclear factor kappa B ligand (RANKL) [16]. Recently, several studies have implicated nAChRs and mAChRs in regulating osteoclastogenesis; however, their effects on osteoclastogenesis have not yet been fully elucidated [17‐20]. nAChR agonists, such as nicotine or carbamylcholine, have been shown to reduce the formation of osteoclasts in vitro [12, 17], while other studies have reported a stimulating effect of nAChR agonists on osteoclastogenesis [18, 21, 22]. Moreover, nicotine was also shown to affect osteoblast differentiation and function [23‐25]. Mice lacking the α2 nAChR subunit or the M2 mAChR have increased bone resorption and low bone mass due to increased osteoclastogenesis [12, 19], while female α7 knockout mice exhibited significantly increased bending stiffness and cortical thickness, as well as reduced gene expression of the osteoclast marker cathepsin K [20].

The potential relevance of cholinergic regulation in bone homeostasis under pathological conditions, such as chronic inflammation, was further highlighted by investigations into α7-deficient mice which exhibit increased bone loss as compared to wild-type (WT) mice in an animal model of arthritis [26].

Our aim was to investigate the role of nAChRs in osteoclasts in detail, and to examine the effect of these receptors on osteoclastogenesis and bone turnover in WT and knockout mice lacking individual nAChR subunits.

The RANK receptor and its ligand, RANKL, play key roles in osteoclastogenesis both within the framework of physiological bone turnover and in various diseases characterized by inflammatory bone loss [16]. The central role of RANKL in osteoclast formation and bone degradation is further underlined by successful therapeutic approaches targeting this pathway. Denosumab, a RANKL-specific monoclonal antibody, is not only an effective treatment for postmenopausal osteoporosis, but also reduces local bone loss in patients with rheumatoid arthritis [37, 38]. The finding that nAChR activation can interfere with RANK signaling is supported by the following findings: 1) when nicotine was added only to the first (solely M-CSF-driven) phase of osteoclast differentiation, it had no effect on OC generation; 2) when nicotine was present during the second phase in parallel with MCSF and RANKL, it caused an inhibitory effect; 3) even when M-CSF was absent from the second phase, nicotine was able to reduce osteoclastogenesis; and 4) the relative expression of RANKL-dependent gene, including TRAP, cathepsin K, matrix metalloproteinase 9 and NFATc1, was decreased in OCs treated with nicotine.

Herein we demonstrate that nAChRs are crucially involved in RANKL-induced osteoclastogenesis by inhibiting Ca²⁺ oscillations, which in turn blocks RANKL-induced activation of c-fos and NFATc1. In contrast to our findings, it has been recently suggested that nAChR agonists have no effect on osteoclastogenesis in vitro [12]; however, the ligands were used at lower concentrations than we utilized in our study. Tanaka et al. administered nicotine at doses comparable to those used here, and observed a reduction in the number of large OCs and the resorptive index, but an increase in the number of small OCs suggesting a reduction in fusion [17]. We were also able to observe an increase in the number of osteoclasts upon administration of low-dose nicotine and PNU282987. Thus nicotinic agonists at low concentration may favor the generation of osteoclasts, whereas high concentrations inhibit osteoclastogenesis. Interestingly it has been suggested that different nAChRs may also convey opposing effects as evidenced by recent studies, which described increased in vitro osteoclastogenesis as well as a significant, though modest, decrease in the systemic bone mass of α2–/– mice [12] and increased bending stiffness and cortical thickness in female α7–/– mice, while male counterparts showed no difference as compared to WT [20]. However, male α7 β2–/– exhibited a significant increase in BMD when compared to WT mice [39]. Interestingly, we found an, albeit modest, increased bone mass in male α7–/– mice, which we could not demonstrate in female α7–/– mice. Gender disparity with regard to bone phenotype is not uncommon and has been described among others in CB1–/– or GPR55–/– mice [40, 41]. In both of these cases males exhibited an osteopetrotic phenotype which females did not, and which was not otherwise explainable. In line with previous studies, which showed no change in gene expression of osteoblast markers, we found no change in the osteoblast phenotype in α7–/– mice [19, 20]. Taken together it appears that high levels of nAChR stimulation may have an overall negative effect on osteoclastogenesis but actions mediated via the α7 receptor may indeed be different and positive in nature.

Our observation that nAChR antagonists also inhibit osteoclastogenesis, coupled with our results showing reduced osteoclastogenesis in α7–/– mice, can be interpreted in the context of desensitization, a key property of nAChRs. Depending on the receptor subtype and the type of agonist, as well as the duration of exposure to nAChRs, agonists have been shown to alter the affinity state of the receptor inducing desensitization, leading to a functional blockade of the receptor [42, 43]. In particular, nicotine and certain experimental α7-selective partial agonists are known to produce a transient activation of α7 receptors followed by a period of prolonged residual inhibition or desensitization [44]. While we derived the dosage of nicotine from published studies [17, 18], due to the fact that we used high doses we cannot exclude the possibility that the inhibition seen with the high-dose nAChR ligands is due to either effects on mAChRs, non-receptor mediated effects (such as the blocking of additional ion channels), or to other factors involved in the regulation of osteoclastogenesis.

Oscillating levels of intracellular calcium appear to be critical for osteoclastogenesis primarily by inducing the dephosphorylation and shuttling of NFATc1 into the nucleus, where it plays a decisive role in the regulation of osteoclastogenesis [32‐34]. After observing the marked acute effect of both nAChR agonists and antagonists on Ca²⁺ oscillation, we could show that, indeed, the nAChR agonist nicotine inhibited RANKL-induced expression and production of NFATc1 and also had a similar effect on c-fos, an early inducer of NFATc1 [45].

The fact that cholinergic signaling has an effect on osteoclastogenesis is also supported by studies which have demonstrated that ACh is produced by cells of the immunoinflammatory sytem, including lymphocytes [46], and the fact that bone marrow also possesses its own sensory and autonomic innervation including cholinergic neurons [12, 14, 15]. This suggests that, under certain circumstances, in addition to ACh released from BMMs, differentiating OCs may also be exposed to ACh released from neurons, which could then have various effects on their nAChRs.

Our results suggest that cholinergic agonists inhibit RANKL-induced osteclastogenesis by interfering with intracellular calcium levels and consequently with the NFATc1 signaling pathway. Of note, this complete blockade of osteoclastogenesis in vitro was neither due to toxic effects nor associated with apoptosis, and was shown to be reversible. However, while high levels of nAChR activity appear to inhibit RANKL-induced osteoclastogenesis, actions mediated by certain nAChRs, in particular the α7 homomeric receptor, may favor it. Further studies are needed to determine the gender-specific effect of α7 and other nAChRs and mAChRs on bone homeostasis.