Multinucleated osteoclast formation in vivo and in vitro by P2X7 receptor-deficient mice.

The P2X7 receptor is a member of the family of P2X purinergic receptors, which upon sustained activation forms large pores in the plasma membrane. In cells of hematopoietic origin, P2X7 receptor activation has been shown to lead to multiple downstream events, including cytokine release, cell permeabilization, and apoptosis. This receptor has also been implicated in the generation of multinucleated giant cells, polykaryons, and osteoclasts. We have recently demonstrated that a blockade of this receptor inhibits osteoclast formation in vitro; therefore, we examined mice deficient in the P2X7 receptor in the context of bone. These mice were healthy and displayed no overt skeletal problems. Furthermore, we were able to demonstrate their ability to form multinucleated cells, in particular osteoclasts, both in vivo and in vitro. We also demonstrate the ability of P2X7R-/- multinucleated osteoclasts, upon stimulation with maitotoxin (MTX), to form pores in the plasma membrane in vitro. These findings are consistent with the existence of an endogenous pore structure present in osteoclast precursor cells that can be activated either by the P2X7 receptor, or in its absence, by alternative signals to mediate fusion and pore formation. These data provide further insight into the mode of action of the P2X7 receptor.

Blockade of the pore-forming P2X7 receptor inhibits formation of multinucleated human osteoclasts in vitro.

Osteoclasts are large, multinucleated, terminally differentiated cells formed by the fusion of mononuclear hemopoietic precursors. Their function is the resorption of bone, which is an essential part of the growth, modeling and remodeling of the skeleton. Though some osteoclast differentiation factors have recently been identified, the molecular basis for the fusion process that leads to multinucleation is poorly understood. The ATP-gated P2X7 receptor is a plasma membrane receptor belonging to the family of P2X purinergic receptors. It is known to be expressed by cells of hemopoietic origin where its activation leads to multiple downstream events including cytokine release, cell permeabilization and apoptosis. More recently this receptor has been implicated in the generation of multinucleated giant cells and polykaryons. Here we show that human osteoclasts express P2X7 receptors in vitro and in vivo, and that these receptors are functional in vitro, as assessed by pore-formation studies. More importantly, blockade of the P2X7 receptor with the antagonist oxidized ATP or a blocking monoclonal antibody significantly inhibits the fusion of osteoclast precursors to form multinucleated osteoclasts. Taken in combination with previous results from our laboratory demonstrating P2X7 receptor-mediated apoptosis and inhibition of bone resorption in vitro, these data suggest an important role for the P2X7 receptor in the regulation of the osteoclast population. The P2X7 receptor provides a significant new target for modulating osteoclast function in diseases characterized by increased osteoclast number and excessive bone turnover.

Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblast-expressed receptor activator of nuclear factor-kappa B ligand.

Nucleotides such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) exist in the extracellular environment where they are agonists at P2 receptors. Both P2Y G-protein-coupled receptors and P2X ligand-gated ion channels are expressed by osteoblasts and osteoclasts, reflected in the diverse nucleotide-induced effects reported to occur in bone. Previous reports have implicated ATP as a proresorptive agent; however, these studies were unable to determine whether ATP mediated its actions directly on osteoclasts, or indirectly via osteoblasts. The development of techniques to generate human osteoclasts in vitro has allowed us to further investigate the intriguing role of extracellular nucleotides with regard to osteoclast activity. This study reports that nearly all P2-receptor-subtype mRNAs were expressed throughout human osteoclast development, and provides evidence for functional P2 receptor expression by these cells. In cultures of human osteoclasts alone, neither ATP nor UTP affected the quantity of resorption by these cells; however, in cocultures of osteoblast-like UMR-106 cells and human osteoclasts, ATP, but not UTP, greatly enhanced resorption, indicating a role for osteoblasts in mediating the proresorptive effects of ATP. Furthermore, ATP, but not UTP, elevated receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein expression by UMR-106 cells. These data are consistent with observations that UMR-106 cells predominantly express P2Y(1) with low expression of P2Y(2), thereby explaining the response to ATP and not UTP, and further substantiating the involvement of osteoblasts in ATP-induced effects on osteoclasts. These results significantly advance our understanding of the role of P2 receptors in bone, and indicate that local-acting ATP may play a pivotal role in osteoclast activation at bone-resorbing sites by inducing elevated expression of RANKL.

Extracellular nucleotide signaling: a mechanism for integrating local and systemic responses in the activation of bone remodeling.

Bone turnover occurs at discreet sites in the remodeling skeleton. The focal nature of this process indicates that local cues may facilitate the activation of bone cells by systemic factors. Nucleotides such as adenosine triphosphate (ATP) are locally released, short-lived, yet potent extracellular signaling molecules. These ligands act at a large family of receptors-the P2 receptors, which are subdivided into P2Y and P2X subtypes based on mechanism of signal transduction. Nucleotides enter the extracellular milieu via non-lytic and lytic mechanisms where they activate multiple P2 receptor types expressed by both osteoblasts and osteoclasts. In this review the release of ATP by bone cells is discussed in the context of activation of bone remodeling. We provide compelling evidence that nucleotides, acting via P2Y receptors, are potent potentiators of parathyroid hormone-induced signaling and transcriptional activation in osteoblasts. The provision of a mechanism to induce activation of osteoblasts above a threshold attained by systemic factors alone may facilitate focal remodeling and address the paradox of why systemic regulators like PTH exert effects at discreet sites.

Expression of a P2X7 receptor by a subpopulation of human osteoblasts.

There is now conclusive evidence that extracellular nucleotides acting via cell surface P2 receptors are important local modulators of bone cell function. Multiple subtypes of P2 receptors have been localized to bone, where their activation modulates multiple processes including osteoblast proliferation, osteoblast-mediated bone formation, and osteoclast formation and resorptive capacity. Locally released nucleotides also have been shown to sensitize surrounding cells to the action of systemic factors such as parathyroid hormone (PTH). In nonskeletal tissue recent attention has focused on one particular P2 receptor, the P2X7 receptor (previously termed P2Z), and its ability to form nonselective aqueous pores in the plasma membrane on prolonged stimulation. Expression of this receptor originally was thought to be restricted to cells of hemopoietic origin, in which it has been implicated in cell fusion, apoptosis, and release of proinflammatory cytokines. However, recent reports have indicated expression of this receptor in cells of stromal origin. In this study, we investigated the expression of the P2X7 receptor in two human osteosarcoma cell lines, as well as several populations of primary human bone-derived cells (HBDCs) at the levels of messenger RNA (mRNA) and protein. We found that there is a subpopulation of osteoblasts that expresses the P2X7 receptor and that these receptors are functional as assessed by monitoring ethidium bromide uptake following pore formation. Inhibition of delayed lactate dehydrogenase (LDH) release in response to the specific agonist 2',3'-(4-benzoyl)-benzoyl-adenosine triphosphate (BzATP) by the nonspecific P2X receptor antagonist PPADS confirmed a receptor-mediated event. After treatment with BzATP SaOS-2 cells exhibited dramatic morphological changes consistent with those observed after P2X7-mediated apoptosis in hemopoietic cells. Dual staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and a P2X7-specific monoclonal antibody confirmed the induction of apoptosis in osteoblasts expressing the P2X7 receptor. These data show for the first time the expression of functional P2X7 receptors in a subpopulation of osteoblasts, activation of which can result in ATP-mediated apoptosis.

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone.

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.

A CD36-binding peptide from thrombospondin-1 can stimulate resorption by osteoclasts in vitro.

Thrombospondin-1 (TSP-1), purified from platelets, stimulates resorption by avian osteoclasts in an in vitro resorption assay. TSP-1 binds to a number of different cellular receptors via different domains of the molecule and several short receptor-binding sequences have been identified within the TSP-1 molecule. In this study, we have used synthetic peptides representing these various sequences in order to identify the cellular receptor and TSP domain responsible for stimulation of resorption. We show that one peptide CSVTCG, which represents the CD36-binding region of TSP-1, stimulates resorption in a fashion similar to the intact molecule, while the peptides RGDS, RFYVVMWK, and RFYVVM, representing other cell-binding domains of TSP, have no effect on resorption. Using RT-PCR and immunoblotting, we further demonstrate expression of CD36 in human osteoclastoma (giant cell tumour), primary human bone derived cells, and clonal osteoblastic cells. These studies suggest that CD36 is involved in regulation of resorption by osteoclasts and is the receptor responsible for the resorption-promoting effects of TSP-1.

Extracellular ATP activates multiple signalling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells.

In the human breast cancer cell line MCF-7, the nucleotides ATP gamma S and UTP, acting extracellularly through the purinergic receptor P2Y(2), lead to elevated intracellular calcium levels and increased proliferation. ATP gamma S and UTP treatment of MCF-7 cells activated transcription of the immediate early gene c-fos, an important component in the response to proliferative stimulation. c-fos induction was enhanced by co-treatment with ATP gamma S and a variety of proliferative agents including growth factors, tumour promoters and stress. Stimulation with ATP gamma S or epidermal growth factor (EGF) led to extracellular signal-regulated kinase (ERK) activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated fos expression and notably led to increased levels of ERK, CREB and EGF receptor phosphorylation, as well as hyperphosphorylation of ternary complex factor. Nevertheless, the ERK pathway does not fully account for this synergy, since fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that these two agonists signal differently to this immediate early gene. Thus, extracellular nucleotides co-operate with growth factors to activate genes linked to the proliferative response in MCF-7 cells through activation of specific purinergic receptors, which thereby represent important potential targets for arresting the neoplastic progression of breast cancer cells.

Expression of members of the thrombospondin family by human skeletal tissues and cultured cells.

We have previously shown that the multifunctional platelet glycoprotein thrombospondin-1 (TSP-1) promotes resorption in an in vitro resorption assay. However, TSP-1 is one of a family of multifunctional TSP molecules, and the current study was undertaken to investigate whether it is TSP-1 or another TSP family member which may be involved in regulation of resorption in vivo. RT-PCR was performed on cultured human bone cells, cultured human chondrocytes, and three separate samples of human osteoclastoma tissue using primers specific for each TSP family member. mRNA for TSP-2 was detected in almost all samples, and significantly in all osteoclastomas in the above tissues, while TSP-1 was detected less frequently and was only seen in one of three osteoclastomas. TSP-3, -4, and COMP were detected only in a minority of cases. These results indicate that TSP-2 is the most common TSP family member found in skeletal tissues and that TSP-2, rather than TSP-1, may be the molecule responsible for promoting resorption in vivo.

Regulation of epidermal homeostasis through P2Y2 receptors.

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase.

Extracellular nucleotides acting through specific P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue, the human osteosarcoma cell line SaOS-2 and primary osteoblasts express P2Y1 and P2Y2 receptors, respectively. Their activation by nucleotide agonists (ADP and ATP for P2Y1; ATP and UTP for P2Y2) elevates [Ca2+]i and moderately induces expression of the c-fos proto-oncogene. A synergistic effect on c-fos induction is observed by combining ATP and parathyroid hormone, a key bone cell regulator. Parathyroid hormone elevates intracellular cAMP levels and correspondingly activates a stably integrated reporter gene driven by the Ca2+/cAMP-responsive element of the human c-fos promoter. Nucleotides have little effect on either cAMP levels or this reporter, instead activating luciferase controlled by the full c-fos promoter. This induction is reproduced by a stably integrated serum response element reporter independently of mitogen-activated protein kinase activation and ternary complex factor phosphorylation. This novel example of synergy between the cAMP-dependent protein kinase/CaCRE signaling module and a non-mitogen-activated protein kinase/ternary complex factor pathway that targets the serum response element shows that extracellular ATP, via P2Y receptors, can potentiate strong responses to ubiquitous growth and differentiative factors.

G-protein coupled receptors in bone.

The skeleton is a dynamic structure that undergoes continuous remodeling, a prerequisite to meeting the constant loading demands placed upon it. This process is controlled by a multitude of systemic and local factors which interact with receptors presented on the surface of both osteoblasts and osteoclasts; the osteogenic and osteolytic cells of bone. The seven transmembrane G-protein coupled superfamily of receptors are amongst the most important expressed by bone cells. Many local and systemic factors, including prostaglandins and parathyroid hormone, initiate cellular processes via interaction with members of this receptor family. The diversity of signals and signaling cross talk generated by activated G-protein receptor complexes, facilitates a huge range of downstream responses essential in the remodeling of the skeleton. Indeed, agonist-activated signaling crosstalk provides a mechanism for integrating the activities of local and systemic factors, an essential requirement of focal remodeling. This review has focused on those currently known seven transmembrane receptors expressed by bone cells that couple to G-proteins, and describes the nature of receptor-G protein interaction and the resultant functional consequences of effector activation within bone cells.

P2Y2 receptors are expressed by human osteoclasts of giant cell tumor but do not mediate ATP-induced bone resorption.

Extracellular nucleotides acting through P2 receptors elicit a range of responses in many cell types. Previously, we have cloned the G-protein coupled P2Y2 receptor from a human osteoclastoma complementary deoxyribonucleic acid (cDNA) library and demonstrated its expression by reverse transcription linked (RT)-PCR and Southern analysis in a number of skeletal tissues, including a purified population of giant cells. In this study we have localized the expression of P2Y2 receptor transcripts to osteoclasts of giant cell tumor of bone by in situ hybridization. In osteoblasts and other cell types, the P2Y2 receptor is coupled to Ins(1,4,5)P3-mediated Ca2+ release from intracellular stores. In this study, the P2Y2 receptor agonists adenosine triphosphate (ATP) and uridine triphosphate (UTP) did not increase cytosolic free calcium concentration ([Ca2+]i) in giant cells isolated from osteoclastoma, while the G-protein coupled calcium sensing receptor agonist, Ni2+, elevated [Ca2+]i in the same cells. These data indicate that P2Y2 receptor transcripts expressed by giant cells are not presented at the surface of cells as functional receptors, or alternatively, functional receptors are coupled to an effector other than [Ca2+]i. ATPgammaS (10 micromol/L), but not UTP (10 micromol/L), significantly stimulated resorption by an enriched giant cell population. These results indicate that ATP-induced effects on resorption, following direct osteoclastic activation, are mediated by a P2 receptor other than the P2Y2 subtype. Nucleotides, released locally in the bone microenvironment in response to acute trauma or transient physical stress, will interact with a complement of P2 receptors expressed by both osteoclasts and osteoblasts to influence the remodeling process.

The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma.

Human cathepsin K is a novel cysteine protease previously reported to be restricted in its expression to osteoclasts. Immunolocalization of cathepsin K in breast tumor bone metastases revealed that the invading breast cancer cells expressed this protease, albeit at a lower intensity than in osteoclasts. In situ hybridization and immunolocalization studies were subsequently conducted to demonstrate cathepsin K mRNA and protein expression in samples of primary breast carcinoma. Expression of cathepsin K mRNA was confirmed by reverse transcription PCR and Southern analysis in a number of human breast cancer cell lines and in primary human breast tumors and their metastases. As this protease is known to degrade extracellular matrix, including bone matrix proteins, it is possible that cathepsin K may contribute to the invasive potential of breast cancer cells, including those that metastasize to bone. Thus, cathepsin K may be a potential target leading to the design of novel drugs for cancer therapy.

Effects of PTH on PTHrP gene expression in human osteoblasts: up-regulation with the kinetics of an immediate early gene.

Parathyroid hormone-related peptide (PTHrP) is a local regulator of human bone turnover, which shares some sequence homology with the systemic hormone parathyroid hormone (PTH). The two proteins exert cellular effects through interaction with a common G protein-coupled PTH/PTHrP receptor on target cells. Whilst the PTH gene has a relatively simple structure the PTHrP gene is complex, alternative splicing of which can generate multiple mRNA species encoding PTHrP of 139, 141 and 173 amino acids. To date little is known regarding the extent to which PTH and PTHrP interact to modulate bone cell function. In this study we have used the quantitative technique of Real-Time polymerase chain reaction (PCR) to investigate the ability of PTH to induce PTHrP expression in SaOS-2 cells and in primary human osteoblasts. In addition, we have used the semi-quantitative techniques of PCR followed by Southern analysis and scanning densitometry to investigate the effects of PTH(1-34) on expression of mRNA species encoding the three PTHrP isoforms. We report a 50 fold increase in PTHrP mRNA expression 30 min after treatment with 100 ng/ml human recombinant PTH (1-34) in SaOS-2 cells, and a 38 fold rise in human osteoblasts 45-90 min post-PTH treatment. mRNA species encoding for PTHrP 1-139, 1-141 and 1-173 were all induced in human osteoblasts 45 min after exposure to PTH. Whilst the 1-139 mRNA species exhibited a sustained expression, both the 1-141 and 1-173 isoforms showed a biphasic induction with a second peak 6 hr post PTH treatment. These data demonstrate that PTH induces expression of the PTHrP gene in both SaOS-2 and primary human osteoblasts with the kinetics of an immediate early gene. Up-regulation of the PTHrP gene in response to PTH may be an important physiological mechanism by which this systemic factor effects a localised response in bone.

Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies.

1. Human osteoblasts responded to the application of extracellular nucleotides, acting at P2-receptors, with increases in cytosolic free calcium concentration ([Ca2+]i). 2. In populations of human osteoblasts, adenosine 5'-diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5'-triphosphate (ATP). 3. ATP and uridine 5'-triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested. 4. However, from the results of single cell studies with ADP (and 2-methylthioATP (2-meSATP)) two groups of cells were delineated; one group responded to ADP (or 2-meSATP) with a rise in [Ca2+]i indistinguishable from that evoked by ATP; whereas the second group failed completely to respond to ADP (or 2-meSATP). 5. Therefore heterogeneity of receptor expression exists within this population of human osteoblasts. The limited distribution of the ADP-responsive receptor underlies the small response to ADP, compared with ATP, recorded in populations of human osteoblasts. This heterogeneity may reflect differences in the differentiation status of individual cells.

Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P2-purinoceptors.

Nucleotides such as ATP can act as extracellular effector molecules by interaction with specific cellular receptors known as P2-purinoceptors. Recently, we cloned the human P2U purinoceptor from osteoclastoma and demonstrated its expression in skeletal tissues. In the current study we have investigated the expression of P2U purinoceptors in human breast tumour cell lines and examined functional effects of extracellular nucleotides on these cells. By reverse transcription-linked polymerase chain reaction (RT-PCR) the expression of mRNA for P2U purinoceptors was demonstrated in four human breast cancer cell lines, Hs578T, MCF-7, SK-Br3 and T47-D. In MCF-7 cells, extracellular ATP (1-100 microM) elevated intracellular free calcium concentration [Ca2+]i, indicating that these cells express functional P2-purinoceptors. UTP elevated [Ca2+]i in an identical manner to ATP, whereas 2-methylthioATP was completely ineffective, and ADP only partially effective. This pharmacological profile suggests that the P2U subtype may be the only P2-purinoceptor expressed by these cells. The functional significance of P2U purinoceptor expression by MCF-7 cells was investigated by analysing the effects of extracellular ATP on cell proliferation. The slowly hydrolysed analogue of ATP, ATPgammaS (which was also shown to elevate [Ca2+]i), induced proliferation of MCF-7 cells when added daily to serum-free cultures over a period of 3 days. ATPgammaS-induced proliferation was demonstrated by three separate methods, detection by scintillation counting of [3H]thymidine incorporation, immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation and direct counting of cell numbers. These data suggest that ATP, possibly released at sites of tissue injury or inflammation, may be capable of growth factor action in promotion of tumour proliferation or progression.

Identification and cloning of human P2U purinoceptor present in osteoclastoma, bone, and osteoblasts.

Extracellular ATP acting through purinoceptors may be an important factor in the modulation of bone turnover. In this study we cloned and sequenced the P2U purinoceptor from osteoclastoma, confirming the recently published human sequence. Furthermore, by the reverse transcriptase-linked polymerase chain reaction (RT-PCR) and Southern blotting we demonstrated expression of P2U receptor mRNA in bone, primary cultures of human bone-derived cells, and two osteosarcoma cell lines, Saos2 and Te85. P2U receptor transcripts were identified in alkaline phosphatase-positive human bone-derived cells isolated by flow cytometry providing strong evidence for the expression of the P2U purinoceptor in mature osteoblasts. P2U receptor transcripts were also detected in a purified giant cell population isolated from osteoclastoma, indicating that this receptor is also expressed by osteoclasts. These data suggest that purinergic agonists may play a role in the regulation of bone metabolism.