HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6304fjev1 20/14/2562    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mentaverri, R. Articles by Brown, E. M. Search for Related Content PubMed PubMed Citation Articles by Mentaverri, R. Articles by Brown, E. M.

The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis

R. Mentaverri^*,,1, S. Yano^*, N. Chattopadhyay^*, L. Petit, O. Kifor^*, S. Kamel, E. F. Terwilliger, M. Brazier and E. M. Brown^*

^* Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA;

Unité de Recherche sur les Mécanismes de la Résorption Osseuse (Université de Picardie) et INSERM, ERI-12, Amiens, France; and

Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA

¹Correspondence: Unité de Recherche sur les Mécanismes de la Résorption Osseuse et INSERM, ERI-12, 1 rue des Louvels, 80037 Amiens, France. E-mail: romuald.mentaverri{at}sa.u-picardie.fr

SPECIFIC AIMS

The aim of the present study was to investigate whether the calcium sensing receptor (CaR) in osteoclast precursors and mature osteoclasts participates in the control of osteoclast differentiation and apoptosis.

PRINCIPAL FINDINGS

1. The CaR is expressed by preosteoclasts as well as mature osteoclasts
To assess the role played by the CaR to sense extracellular calcium (Ca_o²⁺), we focused on whether osteoclast precursors and mature osteoclasts express the CaR. Reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR showed that undifferentiated RAW 264.7 cells and RAW differentiated osteoclast-like cells (OCLs) express CaR mRNA. CaR expression was also assessed by immunostaining and immunoblotting, showing that differentiated and isolated mature osteoclasts are both immunoreactive for the CaR. Most if not all mature osteoclasts isolated from the long bones of CaR^+/+ mice are CaR-positive, whereas CaR immunoreactivity was absent in bone cells isolated from CaR^–/– mice, confirming antibody (Ab) specificity.

2. The CaR is involved in osteoclast differentiation
Because CaR^–/– mice demonstrate severe hyperparathyroidism accompanied by hypercalcemia and hypophosphatemia, it is difficult to reach conclusions as to the role played by the CaR in osteoclasts based solely on histological analysis. We assessed the ability of bone marrow cells (BMC) isolated from CaR^+/+ and CaR^–/– mice to differentiate into tartrate-resistant acid phosphatase-positive cells in the presence of a receptor activator of NF-B ligand (RANKL, 50 ng/ml) and macrophage colony-stimulating factor (M-CSF; 25 ng/ml). Osteoclast differentiation that takes place from BMC isolated from CaR^–/– mice was reduced by 70% vs. BMC isolated from CaR^+/+ mice (P<0.001). Consistent with this result, antagonizing the activity of the CaR in RAW 264.7 cells with a dominant negative form of the CaR (DN-CaR) also reduced osteoclastic differentiation by 50% vs. control, ßbeta;-Gal-transfected RAW 264.7 cells (P<0.001).

3. The CaR is involved in calcium-induced osteoclast apoptosis
We first confirmed that Ca_o²⁺ (1.8 to 20 mM) induces apoptosis of mature rabbit osteoclasts in a dose-dependent manner. Transfection of these osteoclasts with the DN-CaR partially but significantly reduced calcium-induced osteoclast apoptosis, indicating that high Ca_o²⁺-evoked apoptosis of osteoclasts depends at least in part on CaR-dependent Ca_o²⁺ sensing (Fig. 1 ). To confirm the importance of a G-protein-coupled, receptor-based mechanism in regulating osteoclast apoptosis, we investigated the involvement of phospholipase C (PLC) and the inositol 1,4,5-triphosphate (IP₃) signaling pathways using specific pharmacological inhibitors (U73122, 2-APB, and SKF-96365). All three compounds significantly inhibited osteoclast apoptosis, which decreased from 56 ± 2% (cells treated with 20 mM Ca_o²⁺ alone) to 13 ± 3% (U-73122; 10 µM), 30 ± 2% (2-APB; 50 µM), and 22 ± 2% (SKF-96365; 10 µM), respectively. We also showed that inhibitory peptides Z-VAD-fmk and Z-LEHD-fmk significantly and dose-dependently inhibited calcium (20 mM) -elicited apoptosis of osteoclasts, indicating that Ca_o²⁺ induces mature osteoclast apoptosis in a caspase-dependent manner.

View larger version (13K): [in this window] [in a new window]   Figure 1. Increasing Cao2+ (1.8–20 mM) promotes apoptosis of ßbeta;Gal-transfected mature rabbit osteoclasts, an effect attenuated in DN-CaR-transfected osteoclasts. Data are expressed as mean ± SEM of 3 independent experiments. **P < 0.01 and ***P < 0.001 compared with their respective controls (i.e., ßbeta;Gal- or DN-CaR-transfected osteoclasts cultured for 48 h in the presence of 1.8 mM Cao2+).

4. Calcium sensing through the CaR activates NF-B in mature rabbit osteoclasts
NF-B plays a pivotal role in the control of two key events that regulate the osteoclast life span: osteoclast differentiation and survival. Because nuclear translocation of NF-B was recently shown to be under the control of intracellular calcium signaling in mature osteoclasts after RANKL stimulation, we next investigated whether NF-B activation participates in the osteoclast calcium sensing mechanism. NF-B activation was assessed by the nuclear translocation of NF-B in mature rabbit osteoclasts as assessed by immunofluorescence. When osteoclasts were cultured for up to 12 h with 20 mM Ca_o²⁺, NF-B nuclear translocation was transient and appeared to be as rapid as RANKL-induced nuclear translocation of NF-B (Fig. 2 ). As observed for high Ca_o²⁺-induced osteoclast apoptosis, Ca_o²⁺-elicited NF-B activation was dramatically reduced in DN-CaR-transfected osteoclasts compared with ßbeta;Gal-transfected cells, suggesting that NF-B activation is involved in the calcium "sensing" capacity of osteoclasts. Consistent with our findings described above, pharmacological inhibition of PLC using U73122 (10 µM) also inhibited Ca_o²⁺-induced NF-B nuclear translocation in mature rabbit osteoclasts. When cells were preincubated for 30 min with 5 µM of Ro106–9920, the ability of high extracellular calcium concentrations to stimulate mature rabbit osteoclast apoptosis was abrogated.

View larger version (26K): [in this window] [in a new window]   Figure 2. In response to high Cao2+, NF-B moves from the cytoplasm (A) to the nucleus (B) in mature rabbit osteoclasts. Maximum translocation is observed at 1 h, when 61 ± 7% of the osteoclasts exhibit nuclear localization of NF-B. Nuclear localization of NF-B then slowly decreases to reach a plateau at 6 h (25%), when NF-B activation remains significantly different from that observed in osteoclasts cultured for 1 h in the presence of 1.8 mM Cao2+ (P<0.01). When DN-CaR-transfected rabbit osteoclasts were treated with 20 mM Cao2+ for up to 12 h, Cao2+-induced activation of NF-B was dramatically reduced compared with ßbeta;-Gal-transfected osteoclasts (C).

CONCLUSIONS AND SIGNIFICANCE

Osteoclasts are giant multinucleated cells that arise from the proliferation, differentiation, and fusion of mononuclear hematopoietic progenitors (GM-CFU) through a process that is regulated by both local and systemic factors. It is now well established that raising Ca_o²⁺ to levels comparable to those resulting from local bone resorption inhibits osteoclast differentiation and osteoclastic bone resorption. Osteoclasts can "sense" increasing levels of Ca_o²⁺, which in turn appear to trigger a rapid rise in the cytosolic calcium concentration, disassembly of podosomes, and osteoclast apoptosis. However, relatively little is known about the precise cellular mechanisms underlying the effects of Ca_o²⁺ on osteoclast function. In this study we assessed osteoclastogenesis in vitro using bone marrow cultures from CaR^–/– mice. Using this approach, we established that bone marrow cells isolated from CaR^–/– mice showed a reduced capacity to differentiate into TRAP-positive multinucleated cells. This was confirmed by the decrease in the differentiation of RAW 264.7 cells observed after DN-CaR transfection, providing strong evidence that the CaR exerts a direct effect on osteoclastogenesis.

Both mature osteoclasts and preosteoclasts appeared to express the CaR, suggesting that stimulation of the CaR acts not only on osteoclast differentiation but may also regulate osteoclast life span. Confirming this hypothesis, we showed that up to 20 mM of Ca_o²⁺ rapidly promotes the apoptosis of mature osteoclasts through a classical caspase-3- and –9-dependent mechanism. We also found that calcium-induced osteoclast apoptosis at least partially involves the CaR, which on activation may trigger a PLC-dependent release of intracellular calcium stores that leads to osteoclast apoptosis. In support of these data, we demonstrated that high Ca_o²⁺ triggers nuclear translocation of NF-B in mature rabbit osteoclasts and that Ca_o²⁺-induced NF-B activation is downstream of CaR and PLC stimulation. We conclude that Ca_o²⁺-induced NF-B activation may be linked to the induction of apoptosis of mature osteoclasts. Thus, Ca_o²⁺ sensing through the CaR may play a key role in NF-B nuclear translocation and so affect not only osteoclast apoptosis, but likely also osteoclast differentiation. From the data we gathered regarding the role played by the CaR in RANKL-induced osteoclastogenesis and osteoclast apoptosis, it can be speculated that activation of the CaR may participate physiologically both in promoting and inhibiting differentiation over ranges of calcium concentration that differ from one another. Therefore, knockout the CaR results in loss of both effects. This might provide a mechanism for initially permitting osteoclastogenesis to proceed, then inhibiting it as bone resorption increases and the local calcium concentration rises.

In summary, our data clearly demonstrate that the CaR is intimately involved in processes that control osteoclast differentiation and osteoclast apoptosis in the systems investigated here through a signaling pathway involving activation of the CaR, phospholipase C, and NF-B. As observed in other tissues, such as the parathyroid glands, calcium sensing through the CaR may play a pivotal role in the release of calcium from bone, and therefore control two of the most important steps that regulate the overall activity of osteoclasts (Fig. 3 ).

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing the role played by CaR in osteoclast precursors and mature osteoclasts. Expressed by osteoclast precursors and mature osteoclasts, CaR appears to play a key role in osteoclast differentiation and apoptosis. On activation by increases in the extracellular calcium concentration known to occur in association with osteoclastic bone resorption, CaR stimulates phospholipase C, which is responsible for promoting translocation of NF-B from the cytoplasm to the nucleus in mature osteoclasts. Most likely in association with other transcription factors, Cao2+-induced activation of NF-B then leads mature osteoclasts into apoptosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6304fje

This article has been cited by other articles:

				J. E. Fonseca Rebalancing bone turnover in favour of formation with strontium ranelate: implications for bone strength Rheumatology, July 1, 2008; 47(suppl_4): iv17 - iv19. [Abstract] [Full Text] [PDF]

				S. Yang, W. Chen, P. Stashenko, and Y.-P. Li Specificity of RGS10A as a key component in the RANKL signaling mechanism for osteoclast differentiation J. Cell Sci., October 1, 2007; 120(19): 3362 - 3371. [Abstract] [Full Text] [PDF]

				M. M. Dvorak, T.-H. Chen, B. Orwoll, C. Garvey, W. Chang, D. D. Bikle, and D. M. Shoback Constitutive Activity of the Osteoblast Ca2+-Sensing Receptor Promotes Loss of Cancellous Bone Endocrinology, July 1, 2007; 148(7): 3156 - 3163. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6304fjev1 20/14/2562    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mentaverri, R. Articles by Brown, E. M. Search for Related Content PubMed PubMed Citation Articles by Mentaverri, R. Articles by Brown, E. M.