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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Biochim Biophys Acta. 2010 Nov 11;1813(5):979–983. doi: 10.1016/j.bbamcr.2010.11.002

Calcium Signaling in Osteoclasts

Sung-Yong Hwang, James W Putney Jr 1
PMCID: PMC3078988  NIHMSID: NIHMS252679  PMID: 21075150

Abstract

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca2+) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca2+ oscillations occur during RANKL-mediated osteoclastogenesis. Ca2+ oscillations provide a digital Ca2+ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca2+/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca2+ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca2+ oscillations, and we discuss possible molecular players in this specialized Ca2+ response that appears pivotal for normal bone function.

Introduction

Bones are dynamic living organs that are constantly renewed throughout one’s life. This constant and balanced bone turnover relies on the process of bone remodeling mediated by osteoblasts that form bone and osteoclasts that resorb bone [57]. Imbalance between osteoblastic bone production and osteoclastic bone resorption favoring bone resorption is known to occur in many bone diseases such as postmenopausal osteoporosis, arthritis, and bone tumors [36,43]. Accordingly, most drugs used in the treatment of osteoporosis are anit-resorptive in nature. Bisphosphonates, estrogen, and calcitonin are currently the main pharmacological approaches for prevention of bone loss [28,35]. However, there are many side effects from the long-term use of these drugs such as constipation, diarrhea, tumorigenic and cardiovascular effects, and osteonecrosis of the jaw [28,35]. As a result, there have been considerable efforts to develop new therapeutic targets for the treatment or prevention of bone loss.

The activation of the receptor activator of nuclear factor-κB (RANK) by its specific ligand (RANKL) is an essential initiating signal for osteoclastogenesis, One of the key downstream signals in the RANK/RANKL pathway is the Ca2+ dependent calcineurin/NFAT pathway, implicating a significant role for Ca2+ signaling. We will discuss the RANKL-dependent pathway and the role of Ca2+ signaling in more detail below.

Ca2+ mobilization in osteoclasts

Ca2+ serves as a ubiquitous second messenger that can specifically mediate and regulate a variety of downstream signaling pathways [1]. Many different stimuli have been shown to regulate Ca2+ concentrations in osteoclasts. Extracellular acidification caused a decrease in intracellular Ca2+ concentration in isolated chicken osteoclasts which in turn enhanced attachment of cells to bone matrix [45]. Yu and Ferrier [56] reported that ATP triggers a transient rise in intracellular Ca2+ concentrations in rabbit osteoclasts. They concluded that P2 purinergic receptors are involved in this rise of Ca2+. The same group demonstrated that the ATP-induced Ca2+ rise was smaller and more transient in Ca2+ free buffer, suggesting that activation of Ca2+ influx contributes to the Ca2+ signal in osteoclasts. αvβ3 integrin receptors are highly expressed in osteoclasts and known to be important for the function and adhesion of osteoclasts on the bone matrix [8,10,15,16]. Activation of the integrin receptor by specific peptides caused a transient Ca2+ response in the absence of extracellular Ca2+ [38]. Xia and Ferrier [50] reported that mechanical perturbation of osteoclasts induced a Ca2+ mobilization response whose amplitude and duration were dependent on the extracellular Ca2+ concentration. Radding et al. [34] observed intracellular Ca2+ puffs in acid-secreting osteoclasts, which they suggest may be involved in signaling acid secretion for bone resorption.

The most common mechanism of Ca2+ mobilization by extracellular stimuli involves activation of phospholipase C (PLC)-coupled receptors, leading to the production of inositol-1,4,5- trisphosphate (IP3). IP3 binds to and activates the IP3 receptor (IP3R) resulting in Ca2+ release from the endoplasmic reticulum (ER) [1]. IP3 has been shown to induce Ca2+ release from bone cells including osteoblasts and osteoclasts [11]. Yaroslavskiy et al. [52] demonstrated that the IP3R type 1 is required for activation of Ca2+-dependent μ-calpain and nitric oxide-induced Ca2+ mobilization in osteoclasts. Morikawa et al. [31], using RT-PCR and immunofluorescence, reported the presence in rat osteoclasts of all three isotypes of IP3R. Interestingly, Morikawa et al. found that IP3R type 3 is localized to podosomes where osteoclasts adhere to bone, suggesting a potential role of IP3R in the formation or function of podosomes. Malgaroli et al. [24] reported that osteoclast cells sense high extracellular Ca2+ and respond with increased intracellular Ca2+ transients that may be linked to activation of PLC. Similarly, Zaidi et al. [58] reported that high extracellular Ca2+ induces elevation of intracellular Ca2+ in isolated rat osteoclasts. They also suggest that extracellular Ca2+ regulates bone resorption activity of osteoclasts. Subsequently it was confirmed by Seuwen et al. [37] that high extracellular Ca2+ elicits Ca2+ release associated with production of inositol phosphate in osteoclast-like cells, suggesting the involvement of Ca2+ sensing receptors in Ca2+ signaling in osteoclasts. By use of a Ca2+ receptor knockout mouse as well as a Ca2+ receptor dominant negative construct, Mentaverri et al. [27] provided evidence that Ca2+ sensing receptors play a critical role in osteoclast differentiation and apoptosis.

Involvement of the ryanodine receptor (RyR), an intracellular Ca2+ release channel, in the activation of Ca2+ mobilization in osteoclasts was investigated by several groups. Zaidi et al. [60] reported that Ni2+ induced cytosolic Ca2+ release in rat osteoclasts and this response was blocked by ryanodine, suggesting the presence of Ca2+ releasing ryanodine receptors. Similarly, Shankar [40] showed that low concentrations of caffeine, a RyR agonist, induce cytosolic Ca2+ mobilization in isolated rat osteoclasts. One group has suggested that RyRs are expressed in the plasma membrane of osteoclasts, functioning as extracellular Ca2+ sensing receptor [30,61]. However, this idea has not as yet gained general acceptance [6].

Thus, various stimuli activated Ca2+ signaling in osteoclasts, and the signal appears to be comprised of Ca2+ released from intracellular stores, and also entering the cell across the plasma membrane. One major mechanism for activating Ca2+ entry across the plasma membrane is the store-operated pathway [33]. To determine the role played by store-operated channels in osteoclasts, Zaidi et al. [59] examined the effects of thapsigargin, a membrane permeant inhibitor of the ER Ca2+ transporting ATPase. When applied to osteoclasts, thapsigargin increased intracellular Ca2+ concentration in a Ca2+ free buffer, indicating discharge of Ca2+ from internal stores. . The thapsigargin-induced Ca2+ elevation was augmented upon restoration of extracellular Ca2+, indicating a stimulated Ca2+ influx in osteoclasts. Activation of Ca2+ influx by thapsigargin is generally taken as evidence for the store-operated Ca2+ entry pathway [3]. This type of Ca2+ entry is blocked by several pharmacological agents such as 2-aminoethyldiphenyl borate, SKF-96365, and low concentration (1–5 μM) of Gadolinium (Gd3+), and as discussed below, these agents have been shown to affect osteoclast signaling function [18,26]. Consistent with Zaidi’s observation, Shankar et al. [39] showed that store-depletion by the Ca2+ ionophore, ionomycin, elicits a rapid and transient Ca2+ mobilization in Ca2+ free media, which becomes sustained after restoration of extracellular Ca2+, indicating that two phase of Ca2+ signaling occurs in osteoclasts, Ca2+ release followed by activation of Ca2+ influx upon store depletion.

RANKL-evoked Ca2+ mobilization in osteoclasts

Two hematopoietic factors, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor, are essential for osteoclastogenesis, [5,17,21,23,53,55]. Mature and functional osteoclasts are formed from bone marrow-derived monocyte/macrophage precursor cells in the presence of these two required factors. Importantly, Takayanagi [44] reported that cytosolic Ca2+ oscillations occur not in response to IL-1 but rather to RANKL during osteoclastogenesis, suggesting the presence of signaling pathways specifically activated by RANKL. These same authors reported that, on the basis of genome wide screening, NFATc1 is specifically up-regulated by RANKL. Interestingly, NFATc1 is known to be regulated by Ca2+/calmodulin-dependent calcineurin. They proposed that Ca2+ oscillations provide a prolonged digital Ca2+ signal which activates calcineurin, leading to up-regulation (and autoamplification) of NFATc1 and thereby promotes osteoclastogenesis. In support of this idea, transient Ca2+ mobilization by Ca2+ ionophores such as ionomycin failed to up-regulate NFATc1 [44]. Furthermore, they showed that ectopic over-expression of NFATc1 is sufficient to induce osteoclastogenesis even in the absence of RANKL. Subsequently, many other groups confirmed the importance of RANKL-induced Ca2+ oscillations in osteoclastogenesis. Yang and Li [51] showed that genetic ablation of regulator of G-protein signaling 10 (RGS10) abolishes RANKL-induced Ca2+ oscillations, leading to impaired up-regulation of NFATc1 and osteoclastogenesis. These authors demonstrated that over-expression of NFATc1 partially rescues the impaired osteoclastogenesis in RGS10−/− in the absence of RANKL. By using a proteomic technique, Yoon et al. [54] found that Lyn, a Src family tyrosine kinase, was down-regulated in RANKL-induced osteoclastogenesis which might suggest a role of Lyn as a negative regulator during osteoclast differentiation. Consistent with this idea, the same group observed that knockdown of Lyn resulted in an increase in NFATc1 expression accompanying Ca2+ oscillations. Knockdown of Lyn also promoted the formation of both TRAP-positive multinucleated osteoclasts and resorption pits [54]. In addition, several studies have indicated that RANKL induces a more immediate and transient Ca2+ elevation in isolated, mature osteoclasts. Komarova et al. [20] observed that RANKL triggers an intracellular Ca2+ rise in isolated rat osteoclasts. The Ca2+ rise was apparently derived solely from an intracellular Ca2+ source, and signaled translocation of NFκB and enhanced osteoclast survival. Chamoux et al. [7] reported that RANKL elicited a rapid and sustained intracellular Ca2+ rise in osteoclasts cultured from human blood. In this case, extracellular Ca2+ influx appeared to be the major source of the Ca2+ signal.

Role of Ca2+ influx in RANKL-evoked Ca2+ signaling

As described earlier, cytosolic Ca2+ oscillations occur during RANKL-mediated osteoclastogenesis [44]. These oscillations are initiated by activation of co-stimulatory receptors such as the osteoclast-associated receptor and TREM2 after binding of RANKL to RANK [19]. Subsequently, these receptors recruit the spleen tyrosine kinase, which activates PLCγ by phosphorylation in a concert with Tec-family kinases [41,42]. Activation of PLCγ triggers the production of IP3, resulting in release of Ca2+ from the ER. It is noteworthy that in most cell types, activation of receptors coupled to PLC by high concentrations of agonists triggers Ca2+ release from the ER followed by Ca2+ influx through store-operated Ca2+ entry (SOCE) [1,33]. Lower and more physiological concentrations of receptor agonists induce repetitive Ca2+ oscillations [2,46], similar to those seen in RANKL-mediated osteoclast differentiation. SOCE is necessary to refill the store in order to maintain Ca2+ oscillations, which run down in the absence of SOCE [4]. Alternatively, SOCE in some instances may directly provide activator Ca2+ to trigger downstream responses [9]. In either case, store-operated Ca2+ (SOC) channel would be expected to play an important physiological role in RANKL-induced Ca2+ signaling. Accordingly, Mentaverri et al. [26] reported that inhibition of SOCE by two relatively non-specific SOC channel blockers, 2-aminoethyldiphenyl borate and SKF-96365, diminished bone resorption activity of osteoclasts. Furthermore, a low concentration of Gd3+, which is a relatively specific SOC blocker, abolished RANKL-induced Ca2+ oscillations [18]. The same group also demonstrated that knockdown of STIM1 which is a recently identified SOC protein, significantly reduces the frequency of RANKL-induced Ca2+ oscillations, suggesting that SOCE is an important component of the Ca2+ oscillations/calcineurin/NFATc1-dependent signaling complex induced by RANKL.

However, the molecular identity of the channels responsible for the Ca2+ influx is far from a settled issue. Several recent studies have focused on transient receptor potential (TRP) channels as candidates for the channels underlying Ca2+ influx in RANKL-induced Ca2+ oscillations. The vanilloid TRP5 (TRPV5) channels are apparently expressed in human and murine bone samples and in cultured osteoclasts [47]. The TRPV5 was localized to the ruffled border membrane of osteoclasts. Using a mouse model lacking the TRPV5 gene, Van der Eerden et al. [47] concluded that TRPV5 plays a critical role in the function of osteoclasts since in vitro resorption activity was attenuated in TRPV5−/− mice. However, the TRPV5−/− mice actually displayed enhanced osteoclastogenesis [47]. Nonetheless, Hoenderop et al. [14] observed that mice lacking TRPV5 exhibited a decrease in trabecular and cortical thickness of long bones. Furthermore, Masuyama et al. [25] reported an increase in bone mass in TRPV4−/− mice which they attributed to impaired resorption activity of osteoclasts. The mRNA levels of NFATc1 were attenuated in cultured osteoclasts derived from TRPV4−/− mice, while osteoblast phenotypes were not affected, suggesting TRPV4 solely contributes to the differentiation and function of osteoclasts. However, the same group found that TRPV4 was not necessary to generate or to maintain Ca2+ oscillations in osteoclasts since there was no significant difference in the characteristic of Ca2+ oscillations between WT and TRPV4−/− mice (percentage of oscillating cells, frequency and amplitude of Ca2+ oscillations). They concluded that TRPV4 is more likely involved in Ca2+ influx in large and mature osteoclasts after Ca2+ oscillations have disappeared. They used 4α-PDD, a specific TRPV4 agonist to show that TRPV4-mediated Ca2+ response peaks at the later stage of osteoclasts, suggesting there might be another channel underlying Ca2+ influx at the early stages of osteoclast differentiation. Chamoux et al. [7] showed that knockdown of TRPV5 using TRPV5-targeted siRNA leads to inhibition of the RANKL-induced Ca2+ influx in human osteoclasts, which they suggest that TRPV5 is a major player responsible for the RANKL-induced intracellular Ca2+ rise in human osteoclasts. Despite the apparent role of TRPV5 in RANKL-induced Ca2+ signaling in osteoclasts, knockdown of TRPV5 actually promoted bone resorption. On this basis, Chamoux et al. [7] have suggested that TRPV5 may function as a negative regulator of bone homeostasis, similar to the inhibitory role of Lyn on the resorptive activity of osteoclasts [54]. In addition to the Ca2+ channels described above, Moonga et al., clearly demonstrated the expression of a Na+/Ca2+ exchanger that contributes to the functional bone resorbing activity of isolated rat osteoclasts [29].

Concluding remarks

Changes in intracellular Ca2+ concentrations are known to function as universal triggers of diverse signaling pathways, including enzyme activation, cell survival and differentiation. Accordingly, alterations in intracellular Ca2+ concentrations by different stimuli also appear to regulate differentiation and functions of osteoclasts. A summary of the variety of stimuli that can affect Ca2+ signaling in osteoclasts is given in Table 1. There has been much progress in understanding the molecular basis for differentiation and activation of osteoclasts in the last decade following the discovery of RANKL [21,23,53]. Yet, many questions still remain, especially regarding the function of Ca2+ signaling. For example, bone mass was increased in TRPV4−/− mice despite the fact that there was no effect on RANKL-induced Ca2+ oscillations in osteoclasts [25]. Interestingly, Kuroda et al. [22] reported that RANKL-induced Ca2+ oscillations are lost in osteoclasts from IP3R type 2−/− mice, resulting in abolished osteoclastogenesis. However, the osteoclastogenesis returned in the absence of Ca2+ oscillations when the osteoclasts from IP3R type 2−/− mice were co-cultured with osteoblasts. Furthermore, when osteoclastogenesis was induced in IP3R type 2−/− cells lacking Ca2+ oscillations, i.e., when mediated by co-culture with osteoblasts, activation and translocation of NFATc1 were still induced, albeit partially. This oscillation-independent induction of NFATc1 was observed even in the presence of FK506, a calcineurin inhibitor. Collectively, these findings suggest the existence of a possible alternative pathway that is Ca2+ oscillations/calcineurin-independent and activated by interaction of osteoblasts and osteoclasts.

Table 1.

Summary of Documented Calcium Signaling in Osteoclasts.

Stimulus Type of Ca2+ response Significance of Ca2+ response References
Isolated chicken osteoclast Acidification Decrease in [Ca2+]i Promote podosome formation [45]
Isolated rabbit osteoclast ATP (50 μM) Rapid and transient increase in [Ca2+]i ND [56]
Isolated rat osteoclast Peptides that bind integrin Rapid and transient increase in [Ca2+]i around nucleus ND [38]
Isolated rabbit osteoclast Mechanical perturbation Rapid and transient increase in [Ca2+]i ND [50]
Bone-attached chicken osteoclast Acid secretion [Ca2+]i puffs Osteoclastic acid secretion [34]
Human osteosarcoma lines, isolated rat osteoblastic and osteoclastic cells IP3 Transient increase in [Ca2+]i ND [11]
Isolated rat and chicken osteoclast High extracellular Ca2+ Rapid, various types of increase in [Ca2+]i Osteoclast retraction [24]
Isolated rat osteoclast High extracellular Ca2+ Rapid and sustained increase in [Ca2+]i Resorption activity [58]
Osteoclast-like cells GCT23 High extracellular Ca2+ Rapid increase in [Ca2+]i ND [37]
Isolated rat osteoclast Caffeine Rapid and transient increase in [Ca2+]i ND [40]
RANKL-differentiated osteoclast RANKL Delayed and spontaneous [Ca2+]i oscillations Osteoclast differentiation [44]
RANKL-differentiated osteoclast RANKL Delayed and spontaneous [Ca2+]i oscillations Osteoclast differentiation [51]
RANKL-differentiated osteoclast RANKL Delayed and spontaneous [Ca2+]i oscillations Osteoclast differentiation [54]
Isolated rat osteoclast RANKL Rapid and transient increase in [Ca2+]i Promotion of cell survival by nuclear translocation of NFκB [20]
Human osteoclast RANKL Rapid and sustained increase in [Ca2+]i Resorption activity [7]
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ND: not determined

RANKL: receptor activator of nuclear factor-κB ligand

An obvious take-home message from much of the above discussion is the degree of uncertainty with regard to the Ca2+ signaling mechanisms involved in osteoclastogenesis, especially with regard to the route of Ca2+ entry across the plasma membrane. Although there has been limited work in this area, there is ample evidence for the SOCE pathway in osteoclasts [26]. In other cells of the hematopoietic lineage SOCE is known to play an essential role in activating NFAT signaling [12,32]. In just the past few years, the molecular components underlying SOCE have been revealed, in particular the components of the SOCE channels, Orai1, 2 and 3 [13,49,62]. Genetic deletion of Orai1 in mice abolishes SOCE in some, but not all, hematopoietic cells [48]. Zhou et al.[63] recently reported that knockdown of Orai1 abrogates the osteoclastogenesis of human monocytes by suppressing multinucleation of precursor cells, suggesting the involvement of Orai1 channels in osteoclastogenesis. However, many questions still remain yet. What is the mechanism by which Orai1 regulates osteoclastogenesis? It is possible that Ca2+ influx through Orai1 channels activates NFAT, but this has not been demonstrated. How does Orai1 influence the cell-cell fusion of the osteoclasts? Is either Orai2 or 3 also involved? Is Orai1 needed for RANKL-induced Ca2+ oscillations? And what role do the store-operated Orai channels play in the process of bone resorption?

Further progress in understanding the significance of SOCE and Orai channels in Ca2+ oscillations/calcinerin/NFATc1-dependent osteoclastogenesis may provide a more complete molecular picture of the mechanisms underlying Ca2+ signaling in bone. It will help our insight in developing new therapeutic approaches for treatment of many bone diseases in which excessive osteoclastic resorption is involved.

Research Highlights.

Calcium signaling plays a significant role in the process of osteoclastogenesis.

The RANKL receptor utilizes calcium signaling and activation of NFAT to drive differentiation of osteoclasts.

Recent studies demonstrate that a key component of osteoclast calcium signaling involves influx through plasma membrane channels, including members of the TRP channel superfamily, as well as store-operated channels.

Acknowledgments

Drs. David Armstrong and Stephen Shears read the manuscript and provided useful critiques. Work from the authors’ laboratory described in this review was supported by the Intramural Program, National Institutes of Health.

Footnotes

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