FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.06-6304fje.
This Article
Right arrow Abstract Freely available
Right arrow Summary
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.06-6304fjev1
20/14/2562    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mentaverri, R.
Right arrow Articles by Brown, E. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mentaverri, R.
Right arrow Articles by Brown, E. M.
(The FASEB Journal. 2006;20:2562-2564.)
© 2006 FASEB

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

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

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

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

{ddagger} 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

ABSTRACT

Intracellular transduction pathways that are dependent on activation of the CaR by Cao2+ have been studied extensively in parathyroid and other cell types, and include cytosolic calcium, phospholipases C, A2, and D, protein kinase C isoforms and the cAMP/protein kinase A system. In this study, using bone marrow cells isolated from CaR–/– mice as well as DN-CaR-transfected RAW 264.7 cells, we provide evidence that expression of the CaR plays an important role in osteoclast differentiation. We also establish that activation of the CaR and resultant stimulation of PLC are involved in high Cao2+-induced apoptosis of mature rabbit osteoclasts. Similar to RANKL, Cao2+ (20 mM) appeared to trigger rapid and significant nuclear translocation of NF-{kappa}B in a CaR- and PLC-dependent manner. In summary, our data suggest that stimulation of the CaR may play a pivotal role in the control of both osteoclast differentiation and apoptosis in the systems studied here through a signaling pathway involving activation of the CaR, phospholipase C, and NF-{kappa}B.—Mentaverri, R., Yano, S., Chattopadhyay, N., Petit, L., Kifor, O., Kamel, S., Terwilliger, E. F., Brazier, M., Brown, E. M. The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis.


Key Words: phospholipase C • NF-{kappa}B • caspases

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 (1) . Over the past decade, a tumor necrosis factor (TNF) family member called receptor activator of NF-{kappa}B ligand (RANKL) and its receptor (RANK) have been shown to control osteoclast differentiation, activation, and survival (2 3 4 5) . The actual existence of osteoclasts appears to be dependent on RANK-RANKL signaling, since mice lacking either the RANK or the RANKL genes appear to be devoid of osteoclasts (4 , 6) . RANKL, a protein expressed by osteoblasts and activated T-cells, acts directly on osteoclast precursors and mature osteoclasts, respectively, to stimulate their formation and activity. Signaling through RANK involves recruitment of cytosolic TNF receptor-associated factors (TRAF) 2 and 6, which in turn activate activating protein (AP) –1 and NF-{kappa}B, respectively. Phosphorylation of I{kappa}B permits NF-{kappa}B to translocate to the nucleus, where it regulates the expression of a variety of genes involved in inflammation and immunity, cell proliferation, response to stress, and apoptosis. Osteoprotegerin (OPG), a soluble decoy receptor produced mainly by osteoblasts, binds RANKL, thereby preventing RANK-mediated signaling (7) .

Precise regulation of the extracellular ionized calcium concentration (Cao2+) is a high priority for multicellular organisms owing to the key roles that calcium plays in numerous cellular processes, such as maintaining membrane potential and controlling hormonal secretion, cellular proliferation, differentiation, and survival (8 , 9) . It is now well established that increasing Cao2+ to levels comparable to those resulting from local bone resorption (10) inhibits osteoclast differentiation and osteoclastic bone resorption. Thus osteoclasts can "sense" increasing levels of Cao2+, which in turn trigger a rapid rise in the cytosolic calcium concentration, disassembly of podosomes, and osteoclast apoptosis (11 12 13 14) . Several mechanisms have been invoked as mediators of Cao2+-induced effects on the osteoclast, including the putative roles of the ryanodine receptor (15) , a transient potential receptor channel (16) , and the calcium sensing receptor (CaR) as sensors of Cao2+ (17 , 18) . However, relatively little is known about the precise cellular mechanisms underlying the effects of Cao2+ on osteoclast function.

In this study we focused our attention on assessing the role played by the CaR in two processes that are intimately involved in regulating osteoclastic activities and, in turn, the extent of bone resorption (i.e., osteoclast differentiation and apoptosis). Using well-characterized osteoclast models, namely murine bone marrow cells (19) or RAW 264.7 cells that are differentiated in vitro to osteoclasts (20) as well as mature rabbit osteoclasts (21) , we demonstrate here that both mature osteoclasts and preosteoclasts express the CaR and that lack of a functional CaR, or antagonizing endogenous CaR function though overexpression of a dominant negative form of the CaR (DN-CaR), reduces both the formation of osteoclasts as well as Cao2+-induced osteoclast apoptosis.

MATERIALS AND METHODS

In vitro osteoclastogenesis
In vitro osteoclastogenesis was studied using a protocol adapted from the one described by T. Koga et al. (20) . Briefly, after sacrifice, bone marrow cells were mechanically flushed from the medullary cavities of the long bones of CaR–/– or wild-type (WT) mice and cultured overnight in {alpha}-MEM (Sigma, St. Louis, MO, USA) with 10% fetal calf serum (FCS). Littermates were obtained from breeding of heterozygous mice (CaR+/–) with targeted disruptions of exon 5 of the CaR gene from the laboratory of Drs. John and Christine Seidman (Harvard Medical School, Boston, MA, USA) (22) . Nonadherent bone marrow cells were then seeded on 96-well plates at a density of 30,000 cells/well and cultured for 5 or 6 days in {alpha}-MEM with 10% FCS or in {alpha}-MEM with 10% FCS containing 50 ng/ml soluble recombinant murine (rm) RANKL (R&D Systems, Minneapolis, MN, USA) and 10 ng/ml rmM-CSF (R&D Systems). Medium was replaced on the third day. In each well, TRAP-positive cells were then counted after staining with a leukocyte acid phosphatase kit (Sigma 387-A).

RAW 264.7 cell cultures were performed at a cell density of 3000 cells per cm2, and cells were incubated for 5 days in 10 µl of {alpha}-MEM containing 10% FCS and 50 ng/ml rmRANKL. Culture medium was replaced on the third day, and TRAP-positive multinucleated cells differentiated from the RAW 264.7 cells, referred to hereafter as osteoclast-like cells (OCLs), were counted in each well at the end of the culture period.

Osteoclast isolation and culture
Mature osteoclasts (OCs) were isolated from the long bones of rabbits or mice according to the procedure described by Foged et al. (23) , with slight modifications. Rabbit or mouse long bones were dissected and minced with scissors in {alpha}MEM supplemented with 10% heat-inactivated FCS. Cells were then dissociated from bone fragments by vigorous vortexing and collected by centrifugation (4 min, 500 rpm) before seeding onto 24-well plates or on 12 mm glass coverslips.

Purified osteoclasts, used hereafter to directly assess the effect of calcium on apoptosis of mature osteoclasts, were obtained by removing osteoblasts as well as stromal cells from the wells using a solution of 0.01% collagenase-dispase prepared with PBS. Purified osteoclasts were then incubated in medium for 2 h. Next cells were cultured in {alpha}-MEM supplemented with 1% FCS and containing various amounts of test substances or transfected with the DN-CaR and cultured in {alpha}-MEM supplemented with 1% FCS containing various amounts of test substances. Cell purity was assessed using tartrate-resistant acid phosphatase (TRAP) staining and was close to 99% in all cases.

To assess nuclear translocation of NF-{kappa}B, rabbit bone cells were submitted to an additional step before being seeded onto 12 mm glass coverslips. As first described by Collin-Osdoby et al. (24) , the bone cell pellet was resuspended in 15 ml of serum-free medium and carefully placed on top of a 70–40% FCS gradient. The preparation was then left undisturbed for 30 min to allow the larger multinucleated osteoclasts to settle under unit gravity and penetrate the FCS layers. Before being seeded onto glass coverslips, the bottom 15 ml of the gradient, which contains predominantly mature osteoclasts, was harvested, centrifuged for 5 min at 700 rpm, and resuspended in {alpha}-MEM supplemented with 10% FCS.

Reverse transcription and quantitative real-time polymerase chain reaction (PCR)
Total RNA was prepared using TRIzol reagent (Life Technologies, San Diego, CA, USA). For both types of PCR, 2 µg of total RNA was used to synthesize single-stranded cDNA (Omniscript reverse transcription kit, Qiagen, Chatsworth, CA, USA).

As described (25) , a "hot start" PCR protocol was used to assess CaR mRNA expression, and intron-spanning primers were employed to prevent amplification of products arising from genomic DNA. Primer sequences were 5'-TCTGTTCTCTTTAGGTCCTGAAACA-3', sense; 5'-TCATTGATGAACAGTCTTTCTCCCT-3', antisense. Bidirectional sequencing of the PCR fragments was performed using the same primer pairs by means of an automated sequencer (AB377; Applied Biosystems, Foster City, CA, USA) and dideoxy terminator Taq technology in the DNA Sequence Faculty of the University of Maine (Orono, ME, USA).

SYBR green chemistry was used to perform quantitative determinations of the mRNAs encoding for TRAP, the CaR, and a housekeeping protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The design of sense and antisense oligonucleotide primers was based on published cDNA sequences using Primer Express (version 2.0.0, Applied Biosystems). Primer sequences are listed in Table 1 . The cDNAs were amplified using an ABI PRISM 7000 sequence detection system (PE Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocol. GAPDH was used to normalize differences in RNA isolation, RNA degradation, and the efficiency of the reverse transcription reaction. Sizes of the PCR products were verified on 2.0% agarose gels and by melting curve analysis.


View this table:
[in this window]
[in a new window]

  		Table 1. Primer pairs used for quantitative real-time PCR

Immunocytochemistry
Cells were fixed with a 3.7% formaldehyde-PBS solution (5 min) and successively stained with the leukocyte acid phosphatase kit, Hoechst 33258 (0.2 mM), and an anti-CaR antibody (Ab) (see below). CaR expression in mouse or rabbit mature osteoclasts was investigated using a rabbit polyclonal antiserum called 4637 or a mouse monoclonal antiserum called LRG (generous gifts from Drs. Karen Krapcho and Edward Nemeth of NPS Pharmaceuticals (Salt Lake City, UT, USA) and Drs. Allen Spiegel and Paul Goldsmith, NIDDK, NIH, respectively), which were raised against peptides corresponding to amino acids 345–359 and 374–391 of the N-terminal extracellular domain of the CaR, respectively (26 , 27) . All slides were finally mounted on coverslips using VECTASHIELD® (Vecta Laboratories, Burlington, CA, USA) and examined using a confocal microscope (Zeiss LSM510).

Western blot analysis
Expression of the CaR in OCLs and mature rabbit OCs was also investigated using Western blot analysis. Samples were subjected to SDS-containing 6.5% PAGE, and detection of appropriately sized bands was performed using 4637 Ab following the protocol described by Kifor et al. (28) . Immunoblots were visualized using an enhanced chemiluminescence (ECL) system (PerkinElmer Life Sciences, Norwalk, CT, USA).

Detection of osteoclast apoptosis
As described by Kameda et al. (29) , after treatment with reagents cells were fixed with 3.7% formaldehyde for 10 min and stained with 0.2 mM Hoechst 33258 for 10 min. Cells were examined under a fluorescence microscope (Olympus BH2) to determine morphological changes of the chromatin, as described previously (30) . At least 100 TRAP-positive multinucleated cells were scored in order to assess the incidence of apoptotic changes in the chromatin, and the extent of apoptosis of mature osteoclasts was expressed as the percentage of apoptotic osteoclasts per total osteoclast number. A phospholipase C (PLC) inhibitor (U73122), and inhibitors of IP3-dependent intracellular signaling [i.e., 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365] were purchased from Tocris Cookson Ltd. (Bristol, UK). Peptide inhibitors of the caspase cascade [i.e., benzyloxycarbonyl-Val-Ala-Asp (Ome)-fluoromethylketone (Z-VAD-FMK), benzyloxycarbonyl-Leu-Glu-His-Asp (Ome)-fluoromethylketone (Z-LEHD-FMK)] used to assess the role of caspases in Cao2+-induced osteoclast apoptosis and an NF-{kappa}B inhibitor (Ro106–9920) were obtained from Calbiochem (San Diego, CA, USA).

NF-{kappa}B localization by immunofluorescence
Rabbit osteoclasts seeded onto glass coverslips were incubated with various test substances in osteoclast culture medium at 37°C. Treatments were started at various times prior to fixation for 5 min with 3.7% formaldehyde, as presented in subsequent sections. Cells were washed twice with PBS and incubated for 10 min in 0.5% Triton-X 100-PBS solution. Osteoclasts were then incubated overnight at 4°C with a mouse anti-p65 primary Ab (Santa Cruz Technology, Santa Cruz, CA, USA; sc-8008), then for 1 h at room temperature with an AlexaFluor-488-conjugated, goat anti-mouse IgG (H+L) secondary Ab. Coverslips were finally mounted on slides using VECTASHIELD® before examination using a confocal microscope.

Gene delivery by recombinant adeno-associated virus
High-efficiency gene transfer into mature rabbit osteoclasts as well as RAW 264.7 cells was accomplished using a recombinant adeno-associated virus (rAAV) -based method. A bovine CaR sequence with a naturally occurring dominant-negative mutation R186Q (DN-CaR) or the same vector encoding ß-galactosidase cDNA (ß-Gal) (as a control for nonspecific effects of viral infection) was placed under the control of a cytomegalovirus (CMV) immediate-early (CMV-IE) promoter element and packaged in the same vector as described previously (31) . Before being exposed to the virus, RAW 264.7 cells, as well as mature rabbit osteoclasts, were cultured overnight in {alpha}-MEM supplemented with 10% FCS. Cells were then washed once with serum-free {alpha}-MEM, and ~1000 viral particles/cell were used to infect each well (as optimized by pilot studies). Cells were incubated for 90 min in serum-free medium at 37°C in a cell culture incubator. Equal volumes of {alpha}-MEM containing 20% serum were added to the cells to achieve a final serum concentration of 10%. The cells were finally cultured for 36 h before being used for the experiments described later. On each coverslip, at least 80% of the osteoclasts appeared to be ß-Gal-positive, confirming the high efficiency of our rAAV-based approach (data not shown).

Statistical analysis
Results are expressed as the mean ± SEM. The statistical differences among groups were evaluated using the Kruskal-Wallis test. The Mann-Whitney U test was then used to identify differences between the groups when the Kruskal-Wallis test indicated a significant difference (P<0.05).

RESULTS

Preosteoclasts as well as mature osteoclasts express CaR
To assess the role played by the CaR in the capacity of osteoclasts to sense extracellular calcium, we first focused our attention on whether osteoclast precursors and mature osteoclasts express this receptor. Reverse transcription PCR (Fig. 1 A) as well as quantitative real-time PCR (Fig. 1B ) showed that undifferentiated RAW 264.7 cells (RAW) and RAW-differentiated osteoclasts-like (OCLs) express CaR mRNA. It should be noted that OCLs express 15-fold more copies of CaR mRNA than osteoclast precursors (Fig. 1B ). As described (32) , differentiation of OCLs from RAW 264.7 cells was documented by both TRAP staining and amplification of TRAP mRNA by real-time PCR (data not shown).


Figure 1
View larger version (62K):
[in this window]
[in a new window]

  		Figure 1. Analysis of CaR expression at mRNA and protein levels. A) RT-polymerase chain reaction (RT-PCR) amplification of CaR mRNA isolated from RAW 264.7 cells (lane 1 and 2; RAW) and from RAW differentiated osteoclast-like cells (lane 3 and 4; OCLs) obtained after RANKL (50 ng/ml) stimulation revealed that both cell types express CaR mRNA. MW standards and negative control (lane 5). B) Amplification of CaR mRNA by quantitative real-time PCR confirmed that both cells express CaR mRNA and suggests that the mRNA is expressed at a higher level in OCLs than in undifferentiated RAW 264.7 cells. Data are representative of the mean ± SEM of 3 independent experiments. C) Immunostaining of OCLs and D) mature rabbit osteoclasts (OCs) using 4637 rabbit primary anti-CaR Ab and LRG mouse primary anti-CaR Ab, respectively. E) Western blot analysis, using 4637 Ab, confirmed that both OCLs (lanes 1 and 2) and OCs (lanes 4 and 5) express CaR at the protein level. Lane 3 illustrates results using protein isolated from CaR-transfected HEK-293 cells (positive control). Immunoreactivity was detected using the DAKO 3-amino-9-ethyl carbazole Substrate System (DAKO Corp., Carpenteria, CA, USA) and an ECL system (PerkinElmer Life Sciences).

Immunostaining (Fig. 1C, D ) and immunoblotting (Fig. 1E ) were then used to assess the presence or absence of the CaR at the protein level. Both OCLs and mature rabbit osteoclasts appear to be positive for CaR immunoreactivity. Confirming the specificity of the staining, preabsorption of the primary antiserum with the specific peptide against which it was raised dramatically reduced CaR immunoreactivity (data not shown). Expression of the CaR was confirmed by immunostaining of bone cells isolated from the long bones of CaR+/+ mice (Fig. 2 A), which revealed that numerous, if not all, of the isolated bone cells are CaR-positive (red signal). Both CaR–/– and CaR+/+ mice possess mature TRAP-positive multinucleated osteoclasts, which appeared similar in size and number of nuclei (Fig. 2) . We failed to find any CaR immunoreactivity in bone cells isolated from CaR–/– mice (Fig. 2B ), confirming the specificity of the immunostaining for the CaR. Thus, the CaR was shown to be expressed in the plasma membrane of both mature and preosteoclasts. Of note is the apparent localization of the CaR immunoreactivity in the cytosol in this cell type, a finding reported in several other cell types as well (33 34 35) despite the presumed plasma membrane localization of the mature functional protein that mediates the actions of extracellular calcium on these cells.


Figure 2
View larger version (64K):
[in this window]
[in a new window]

  		Figure 2. Bone cells isolated from CaR+/+ and CaR–/– mice exhibit TRAP-positive multinucleated osteoclasts. Hoechst staining for nuclei (blue): bone cells isolated from CaR+/+ (A) and CaR–/– (B) mice showed multinucleated cells. Staining for TRAP activity (black) with a leukocyte acid phosphatase kit (Sigma): as shown in these representative pictures, isolated bone cells from both CaR+/+ and CaR–/– mice showed TRAP-positive cells. Staining for the CaR with an AlexaFluor 546-conjugated, secondary Ab (red signal): as expected, bone cells isolated from mice lacking exon 5 of the CaR, encoding for portion of the extracellular domain of the receptor (CaR–/–), appear negative for 4637 staining (B) while numerous, if not all, cells isolated from CaR+/+ mice were detected as positive (A). Cells derived from both CaR+/+ and CaR–/– mice appear to generate TRAP-positive multinucleated osteoclasts. Confocal images (400x).

The CaR is involved in osteoclast differentiation
Because CaR–/– mice demonstrate severe hyperparathyroidism accompanied by hypercalcemia and hypophosphatemia (36) , it is difficult to draw definitive conclusions concerning the role played by the CaR in osteoclasts based solely on histological analysis. In this study, we assessed the ability of bone marrow cells (BMC) isolated from both CaR+/+ and CaR–/– mice to differentiate into TRAP-positive osteoclasts. Equal numbers of BMC isolated from CaR+/+ and CaR–/– mice were cultured for 5 days in the presence or absence of RANKL (50 ng/ml) and M-CSF (25 ng/ml). As shown in Fig. 3 A, the osteoclastic differentiation that occurs from BMC isolated from CaR–/– mice was reduced by 70% compared with that taking place in BMC isolated from CaR+/+ mice (P<0.001). Consistent with this result, antagonizing the action of the CaR in RAW 264.7 cells with the DN-CaR reduced osteoclastic differentiation by 50% compared with ß-Gal transfected RAW264.7 cells (Fig. 3B , P<0.001). However, in both cases osteoclastic differentiation was not completely abrogated, suggesting that CaR deficiency impairs but does not block osteoclastogenesis.


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Figure 3. Role played by CaR in osteoclast differentiation in vitro. A) Bone marrow cells (BMC) isolated from CaR–/– and CaR+/+ mice were cultured for 5 days in the presence of RANKL (50 ng/ml) and M-CSF (25 ng/ml). Data are expressed as the percentage of TRAP-positive cells derived in each experiment from CaR+/+ BMC cultured with RANKL and M-CSF (109 to 365 TRAP-positive cells were observed in each well). Data are representative of 4 independent experiments (n=12). ***P < 0.001 compared with the number of TRAP-positive cells derived from CaR+/+ mice. B) ß-Gal- or DN-CaR-transfected RAW 264.7 cells (ß-Gal RAW or DN-CaR RAW) were cultured for 5 days in the presence of 50 ng/ml RANKL. Data are expressed as the percentage of TRAP-positive cells counted in each experiment from ß-Gal-transfected RAW cells when cultured for 5 days with RANKL (138 to 302 TRAP-positive multinucleated cells were observed in each well). Data are representative of 3 independent experiments (n=12). ***P < 0.001 compared with ß-Gal-transfected RAW cells cultured for 5 days in the presence of RANKL (50 ng/ml).

The CaR is involved in calcium-induced osteoclast apoptosis
In these experiments we first confirmed that Cao2+ (from 1.8 to 20 mM) induces apoptosis of mature rabbit osteoclasts in a dose-dependent manner (Fig. 4 A). Indeed, DN-CaR transfection of the mature rabbit osteoclasts partially but significantly abrogated the calcium-induced osteoclast apoptosis, indicating that Cao2+-elicited apoptosis of osteoclasts depends at least in part on CaR-dependent Cao2+ sensing. As shown in Fig. 4A , osteoclast apoptosis in the DN-CaR-transfected cells was reduced by 40% compared with that in ß-Gal-transfected osteoclasts, when cells were cultured in the presence of 20 mM Cao2+ (P=0.015). To confirm the importance of a G-protein-coupled, receptor-based mechanism in the regulation of osteoclast apoptosis, we investigated the involvement of the PLC and 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 was reduced from 56 ± 2% (cells treated with 20 mM Cao2+ alone) to 13 ± 3% (U-73122; 10 µM), 30 ± 2% (2-APB; 50 µM), and 22 ± 2% (SKF-96365; 10 µM), respectively (Fig. 4B ). We also showed that the specific caspase inhibitory peptides, Z-VAD-fmk (Fig. 4C ) and Z-LEHD-fmk (Fig. 4D ), significantly and dose-dependently inhibited calcium (20 mM) -evoked apoptosis of osteoclasts, indicating that Cao2+ induces apoptosis of mature osteoclasts in a caspase-dependent manner.


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		Figure 4. Role played by CaR in Cao2+-induced apoptosis of rabbit osteoclasts. A) Increasing levels of Cao2+ (from 1.8 mM to 20 mM) stimulate, to varying extents, apoptosis of ß-Gal- and DN-CaR-transfected mature rabbit osteoclasts. Data are expressed as means ± SEM of 3 independent experiments. **P < 0.01 and ***P < 0.001 compared with their respective controls (i.e., ß-Gal or DN-CaR-transfected osteoclasts cultured for 48 h in the presence of 1.8 mM Cao2+). B) We also assessed the role of the PLC and intracellular calcium signaling in Cao2+-induced osteoclast apoptosis using three well-described pharmacological blockers [i.e., U73122 (10 µM), 2-APB (50 µM) and SKF-96365 (10 µM)]. Data are expressed as means ± SEM of 3 independent experiments. ***P < 0.001 compared with osteoclasts cultured for 48 h in the presence of 20 mM Cao2+. C, D) Effects of Z-VAD-fmk (caspase 3 inhibitory peptide) or Z-LEHD-fmk (caspase 9 inhibitory peptide) on Cao2+-induced apoptosis of rabbit osteoclasts. Data are expressed as means ± SEM of 2 independent experiments. *P < 0.05 and ***P < 0.001 compared with cultures treated with Cao2+ (20 mM) alone.

Calcium sensing through the CaR activates NF-{kappa}B in mature rabbit osteoclasts
Komarova et al. recently demonstrated that RANKL-induced nuclear translocation of NF-{kappa}B is regulated by a PLC-dependent mechanism (37) . Because of the role played by NF-{kappa}B in both osteoclast differentiation and survival, we speculated that NF-{kappa}B activation may play a key role as a mediator of the osteoclast calcium sensing mechanism. As described before, NF-{kappa}B activation was assessed by the nuclear translocation of NF-{kappa}B in mature rabbit osteoclasts as assessed by immunofluorescence (37) . The entire osteoclast population was examined (usually 100–200 osteoclasts per coverslip), and mature osteoclasts were rated as positive for nuclear translocation of NF-{kappa}B when the NF-{kappa}B fluorescent labeling of one or more of the nuclei exceeded that of the cytoplasm (Fig. 5 A, B). RANKL (50 ng/ml) transiently and significantly increased the percentage of ß-Gal-transfected osteoclasts showing nuclear localization of NF-{kappa}B. As shown in Fig. 5C , under these culture conditions NF-{kappa}B activation is maximal at 1 h (52±9%, P<0.001), then gradually decreases until 12 h, when it reaches a plateau where ~20% of the osteoclasts remain positive for nuclear translocation of NF-{kappa}B. As shown in Fig. 5D , when osteoclasts were cultured for up to 12 h with 20 mM Cao2+ alone, nuclear translocation of NF-{kappa}B was transient and appeared to be as rapid as during RANKL-induced nuclear translocation of NF-{kappa}B. Maximum activation was observed at 1 h, when 61 ± 7% of the osteoclasts exhibit nuclear translocation of NF-{kappa}B (P<0.001). The nuclear translocation of NF-{kappa}B then slowly decreased to reach a plateau at 6 h ({approx}25%), when NF-{kappa}B activation remained significantly different from that observed in osteoclasts cultured for 1 h in the presence of 1.8 mM Cao2+ (P<0.01). CaR-DN transfection of the cells dramatically reduced the high Cao2+-induced NF-{kappa}B activation (Fig. 5D ). Thus, 1 h after addition of high Cao2+ (20 mM), the percentage of osteoclasts showing nuclear localization of NF-{kappa}B significantly decreased from 61 ± 7% to 15 ± 1% (P<0.01). When osteoclasts were pretreated for 30 min with U73122 (10 µM), high Cao2+-induced NF-{kappa}B activation was significantly reduced compared with that present in cells treated with 20 mM Cao2+ (Fig. 5E ). U73343 (10 µM), a less potent pharmacological inhibitor of PLC, was without effect on the high Cao2+-induced nuclear translocation of NF-{kappa}B (data not shown). As shown in Fig. 6 , when cells were preincubated for 30 min with 5 µM of a well-known inhibitor of NF-{kappa}B (Ro106–9920), the ability of high extracellular calcium concentrations to stimulate mature rabbit osteoclast apoptosis was completely abrogated.


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		Figure 5. Role played by CaR in nuclear translocation of NF-{kappa}B. As illustrated in panels A, B (confocal imagery: 200x), osteoclasts were rated positive for nuclear translocation of NF-{kappa}B only when the NF-{kappa}B fluorescent labeling of one or more of the nuclei exceeded that of the cytoplasm (B). C) ß-Gal-transfected rabbit osteoclasts were treated with RANKL (50 ng/ml) or its vehicle for various times for up to 12 h. Samples were then fixed at the indicated times, and p65 localization was determined by immunofluorescence. Data are expressed as means ± SEM of the percentage of osteoclasts exhibiting nuclear localization of NF-{kappa}B on each coverslip and are representative of 3–6 independent experiments. **P < 0.01 and ***P < 0.001 compared with vehicle-treated ß-Gal-transfected osteoclasts. D) ß-Gal- or DN-CaR-transfected rabbit osteoclasts were treated with 20 mM Cao2+ or its vehicle for up to 12 h. Data are representative of 3–8 independent experiments. **P < 0.01 and ***P < 0.001 compared with ß-Gal-transfected osteoclasts cultured with 1.8 mM Cao2+ (bGal+vehicle). $$P < 0.01 compared with ß-Gal-transfected osteoclasts cultured with 20 mM Cao2+ (bGal+Ca 20 mM). E) ß-Gal-transfected rabbit osteoclasts were simultaneously treated with 20 mM Cao2+ and U73122 (10 µM) or its vehicle. Data are representative of 3–8 independent experiments.**P < 0.01 and ***P < 0.001 compared with ß-Gal-transfected osteoclasts cultured with 1.8 mM Cao2+ (bGal+vehicle). $P < 0.05 compared with ß-Gal-transfected osteoclasts cultured with 20 mM Cao2+ (bGal+Ca 20 mM).


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Figure 6. Role played by NF-{kappa}B activation in Cao2+-induced apoptosis of rabbit osteoclasts. We assessed the role of a well-known inhibitor of NF-{kappa}B activation (Ro106–99200) in Cao2+-induced osteoclast apoptosis. Data are expressed as means ± SEM of 3 independent experiments. ***P < 0.001 compared with osteoclasts cultured for 48 h in the presence of Cao2+.

DISCUSSION

It is now well accepted that the balance of proliferation, differentiation, and apoptosis of bone cells determines the size of the osteoclast or osteoblast populations at any given time in the life of the bone. Since these initial observations, several positive or negative regulators of osteoclast differentiation and apoptosis were brought to light, including hormones, cytokines, and growth factors. We provide evidence here suggesting that CaR stimulation, as occurs when cells are submitted to an increased level of Cao2+, should be considered in a similar manner.

Expression of the CaR in osteoclasts was first described by Kameda et al. (17 ), who pointed out in 1998 that CaR expression in mature osteoclasts may play a functional role in bone resorption through a direct effect of extracellular calcium on various activities of osteoclasts. Despite the importance of the skeleton in Cao2+ homeostasis, to date only indirect evidence has confirmed such a hypothesis (14 , 18 , 38 , 39) . Inconsistent with these in vitro data, histological analysis of bones from CaR–/– mice rescued by inactivation of the PTH gene failed to reveal any evidence for CaR’s involvement in the regulation of osteoclast activity and in osteoblast recruitment in vivo (40) , explaining why the CaR has been thought to play a minor, if any, role in osteoclast biology (40 , 41) .

Due to the severe hyperparathyroidism present in CaR–/– mice (40) and the resultant effects of PTH on osteoclastogenesis (42) , we chose to assess osteoclastogenesis in vitro using bone marrow cultures from CaR–/– mice. Utilizing this approach, we clearly established that bone marrow cells isolated from CaR–/– mice show a reduced capacity to differentiate into TRAP-positive multinucleated cells. This result was confirmed by a decrease in the differentiation of RAW 264.7 cells to multinucleated, osteoclast-like cells observed after CaR-DN transfection, providing strong evidence that the CaR exerts a direct effect on osteoclastogenesis.

Mature osteoclasts and preosteoclasts both appeared to express CaR, suggesting that stimulation of the CaR acts not only on osteoclast differentiation but may also regulate bone resorption and the osteoclast life span. Confirming such a hypothesis, we showed that increasing levels of Cao2+ up to 20 mM rapidly led to apoptosis of mature osteoclasts through a classical caspase-3 and –9-dependent mechanism. Calcium (20 mM) -induced osteoclast apoptosis at least partially involves a G-protein-coupled receptor, the CaR, which may trigger a PLC-dependent release of intracellular calcium stores and induce osteoclast apoptosis when stimulated. These results agree completely with those published by Malgaroli et al. (11 ) and Bennett et al. (16 ), who have shown that osteoclasts "sense" elevated extracellular calcium through a process involving PLC activation and the associated rise in intracellular calcium (Cai2+). Nonetheless, probably because of the presence of residual dimeric WT CaR, which remains active in mature rabbit osteoclasts even after DN-CaR transfection, Cao2+-induced osteoclast apoptosis was not prevented entirely. 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 in both promoting and inhibiting differentiation over ranges of calcium concentration that differ from one another. Therefore, knockout of 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. The proapoptotic action of high calcium concentrations could further reduce the pool of osteoclasts in this latter setting. It would be of interest in future studies to look at genes upstream of the cellular events regulated in the osteoclast by the CaR, namely, osteoclastic differentiation, its inhibition by calcium, and apoptosis.

As observed in the assay of osteoclast apoptosis, we showed that Cao2+-induced NF-{kappa}B activation is downstream of the stimulation of both CaR and PLC in mature rabbit osteoclasts. Hence, based our data, Cao2+-evoked activation of NF-{kappa}B could be linked to induction of apoptosis of mature osteoclasts (Fig. 7 ). Whereas NF-{kappa}B is most commonly involved in suppressing apoptosis by transactivating the expression of antiapoptotic genes, it is not surprising it could also promote programmed cell death in mature osteoclasts. Thus, several lines of evidence gathered during the past decade indicate that NF-{kappa}B can enhance the expression of death-promoting genes such as p53, c-Myc, Bcl-xS, or Fas in response to certain death-inducing signals and in certain cell types (43 44 45 46) . To date, we cannot fully explain how the high Cao2+-induced NF-{kappa}B activation mediates osteoclast apoptosis while RANKL-stimulated NF-{kappa}B nuclear translocation does not (data not shown), although high Cao2+ and RANKL could clearly differ in the full range of signaling pathways they activate. That NF-{kappa}B is the only transcription factor responsible for the control of mature osteoclasts apoptosis seems inconceivable. Spatiotemporal activation of other transcription factors, such as AP-1 and NFAT, needs to be further studied in order to provide more compelling data regarding their relative importance in the high calcium-induced apoptosis of mature osteoclasts.


Figure 7
View larger version (42K):
[in this window]
[in a new window]

  		Figure 7. Schematic diagram summarizing the role played by the CaR in osteoclast precursors and mature osteoclasts. Expressed by osteoclast precursors and mature osteoclasts, the CaR appears to play a key role in both the differentiation and apoptosis of osteoclasts. Upon stimulation by extracellular calcium, the CaR activates phospholipase C, which is responsible for translocation of NF-{kappa}B from the cytoplasm to the nucleus in mature osteoclasts. Most likely in association with other transcription factors, Cao2+ induced activation of NF-{kappa}B, then led mature osteoclasts into apoptosis.

Consistent with data recently published by Xu et al. (39 ) in which the reciprocal regulation between high Cao2+ and RANKL signaling was initially highlighted, our data strongly suggest that a tight partnership exists between RANKL and the CaR in osteoclasts. It can be speculated that Cao2+ sensing through the CaR may play a key role in NF-{kappa}B nuclear translocation, and therefore affects both osteoclast differentiation and apoptosis. Molecular interactions, which may exist between the CaR, RANK, and perhaps other proteins in both mature osteoclasts and preosteoclasts, need to be further investigated in order to provide greater insight into the molecular mechanisms that regulate the osteoclast life span under physiological conditions.

In addition to CaR, members of subfamily C of the superfamily of G-protein-coupled receptors such as mGluRs (47) , GABABRs (48) , and, more recently, GPRC6A (49) , have been shown to sense extracellular calcium apparently due to conserved binding sites in their large extracellular domains (50) . GPRC6A was recently shown to respond not only to basic amino acids, but also to extracellular calcium and calcimimetics, indicating it could potentially mediate extracellular calcium sensing in some of the same tissues in which the CaR is expressed (49) . However, differences exist between the CaR and other putative calcium sensors, such as their apparent affinity for calcium. For example, high concentrations of Cao2+ of up to 40 mM were necessary to fully activate GPRC6A whereas a lower concentration of 5 mM Cao2+ can maximally activate CaR, suggesting that GPRC6A may preferentially exert its actions in tissues where high local extracellular concentrations exist, such as in bone. From our data we cannot exclude the possibility that GPRC6A plays a role in calcium-induced NF-{kappa}B nuclear translocation. Further studies are needed to determine whether GPRC6A heterodimerizes with the CaR on bone cells and if it is inhibited by the dominant negative CaR used in our studies. However, we unequivocally demonstrated that bone marrow cells isolated from CaR–/– mice exhibit a reduced ability to differentiate into mature osteoclasts, strongly suggesting that even in the putative presence of GPRC6A, CaR deficiency directly affects osteoclast function.

In summary, our data clearly demonstrate that the CaR is intimately involved in processes that control both osteoclast differentiation and osteoclast apoptosis. Hence, as observed in other tissues involved in calcium homeostasis, such as parathyroid glands, calcium sensing through the CaR may play a pivotal role in the release of calcium from bone and thereby control two of the most important steps that regulate osteoclast activities.

ACKNOWLEDGMENTS

We especially thank Robert Butters for his technical assistance. Confocal microscopy was carried out at the Brigham and Women’s Hospital Confocal Microscopy Core Facility (Boston, MA, USA).

Received for publication April 21, 2006. Accepted for publication August 7, 2006.

REFERENCES

  1. Roodman, G. D. (1999) Cell biology of the osteoclast. Exp. Hematol. 27,1229-1241[CrossRef][Medline]
  2. Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A., Elliott, G., Scully, S., et al (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93,165-176[CrossRef][Medline]
  3. Burgess, T. L., Qian, Y., Kaufman, S., Ring, B. D., Van, G., Capparelli, C., Kelley, M., Hsu, H., Boyle, W. J., Dunstan, C. R., et al (1999) The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J. Cell Biol. 145,527-538[Abstract/Free Full Text]
  4. Kong, Y. Y., Yoshida, H., Sarosi, I., Tan, H. L., Timms, E., Capparelli, C., Morony, S., Oliveira-dos-Santos, A. J., Van, G., Itie, A., et al (1999) OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397,315-323[CrossRef][Medline]
  5. Lacey, D. L., Tan, H. L., Lu, J., Kaufman, S., Van, G., Qiu, W., Rattan, A., Scully, S., Fletcher, F., Juan, T., et al (2000) Osteoprotegerin ligand modulates murine osteoclast survival in vitro and in vivo. Am. J. Pathol. 157,435-448[Abstract/Free Full Text]
  6. Li, J., Sarosi, I., Yan, X. Q., Morony, S., Capparelli, C., Tan, H. L., McCabe, S., Elliott, R., Scully, S., Van, G., et al (2000) RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc. Natl. Acad. Sci. U. S. A. 97,1566-1571[Abstract/Free Full Text]
  7. Anderson, H. C. (1997) An antagonist of osteoclast integrins prevents experimental osteoporosis. J. Clin. Invest. 99,2059[Medline]
  8. Spurr, N. K. (2003) Genetics of calcium sensing—regulation of calcium levels in the body. Curr. Opin. Pharmacol. 3,291-294[CrossRef][Medline]
  9. Hofer, A. M. (2005) Another dimension to calcium signaling: a look at extracellular calcium. J. Cell Sci. 118,855-862[Abstract/Free Full Text]
  10. Silver, I. A., Murrills, R. J., Etherington, D. J. (1988) Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp. Cell Res. 175,266-276[CrossRef][Medline]
  11. Malgaroli, A., Meldolesi, J., Zallone, A. Z., Teti, A. (1989) Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin. J. Biol. Chem. 264,14342-14347[Abstract/Free Full Text]
  12. Miyauchi, A., Hruska, K. A., Greenfield, E. M., Duncan, R., Alvarez, J., Barattolo, R., Colucci, S., Zambonin-Zallone, A., Teitelbaum, S. L., Teti, A. (1990) Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption. J. Cell Biol. 111,2543-2552[Abstract/Free Full Text]
  13. Zaidi, M., Moonga, B. S., Adebanjo, O. A. (1999) Novel mechanisms of calcium handling by the osteoclast: a review-hypothesis. Proc. Assoc. Am. Physicians 111,319-327[CrossRef][Medline]
  14. Lorget, F., Kamel, S., Mentaverri, R., Wattel, A., Naassila, M., Maamer, M., Brazier, M. (2000) High extracellular calcium concentrations directly stimulate osteoclast apoptosis. Biochem. Biophys. Res. Commun. 268,899-903[CrossRef][Medline]
  15. Zaidi, M., Shankar, V. S., Tunwell, R., Adebanjo, O. A., Mackrill, J., Pazianas, M., O’Connell, D., Simon, B. J., Rifkin, B. R., Venkitaraman, A. R., et al (1995) A ryanodine receptor-like molecule expressed in the osteoclast plasma membrane functions in extracellular Ca2+ sensing. J. Clin. Invest. 96,1582-1590[Medline]
  16. Bennett, B. D., Alvarez, U., Hruska, K. A. (2001) Receptor-operated osteoclast calcium sensing. Endocrinology 142,1968-1974[Abstract/Free Full Text]
  17. Kameda, T., Mano, H., Yamada, Y., Takai, H., Amizuka, N., Kobori, M., Izumi, N., Kawashima, H., Ozawa, H., Ikeda, K., et al (1998) Calcium sensing receptor in mature osteoclasts, which are bone resorbing cells. Biochem. Biophys. Res. Commun. 245,419-422[CrossRef][Medline]
  18. Kanatani, M., Sugimoto, T., Kanzawa, M., Yano, S., Chihara, K. (1999) High extracellular calcium inhibits osteoclast-like cell formation by directly acting on the calcium sensing receptor existing in osteoclast precursor cells. Biochem. Biophys. Res. Commun. 261,144-148[CrossRef][Medline]
  19. Scheven, B. A., Visser, J. W., Nijweide, P. J. (1986) In vitro osteoclast generation from different bone marrow fractions, including a highly enriched haematopoietic stem cell population. Nature 321,79-81[CrossRef][Medline]
  20. Koga, T., Inui, M., Inoue, K., Kim, S., Suematsu, A., Kobayashi, E., Iwata, T., Ohnishi, H., Matozaki, T., Kodama, T., et al (2004) Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 428,758-763[CrossRef][Medline]
  21. Kanaoka, K., Kobayashi, Y., Hashimoto, F., Nakashima, T., Shibata, M., Kobayashi, K., Kato, Y., Sakai, H. (2000) A common downstream signaling activity of osteoclast survival factors that prevent nitric oxide-promoted osteoclast apoptosis. Endocrinology 141,2995-3005[Abstract/Free Full Text]
  22. Ho, C., Conner, D. A., Pollak, M. R., Ladd, D. J., Kifor, O., Warren, H. B., Brown, E. M., Seidman, J. G., Seidman, C. E. (1995) A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat. Genet. 11,389-394[CrossRef][Medline]
  23. Foged, N. T., Delaisse, J. M., Hou, P., Lou, H., Sato, T., Winding, B., Bonde, M. (1996) Quantification of the collagenolytic activity of isolated osteoclasts by enzyme-linked immunosorbent assay. J. Bone Miner. Res. 11,226-237[Medline]
  24. Collin-Osdoby, P., Anderson, F., Osdoby, P. (2003) Primary isolation and culture of chicken osteoclasts. Methods Mol. Med. 80,65-88[Medline]
  25. Chattopadhyay, N., Yano, S., Tfelt-Hansen, J., Rooney, P., Kanuparthi, D., Bandyopadhyay, S., Ren, X., Terwilliger, E., Brown, E. M. (2004) Mitogenic action of calcium sensing receptor on rat calvarial osteoblasts. Endocrinology 145,3451-3462[Abstract/Free Full Text]
  26. Yamaguchi, T., Kifor, O., Chattopadhyay, N., Brown, E. M. (1998) Expression of extracellular calcium (Cao2+) sensing receptor in the clonal osteoblast-like cell lines, UMR-106 and SAOS-2. Biochem. Biophys. Res. Commun. 243,753-757[CrossRef][Medline]
  27. Goldsmith, P. K., Fan, G., Miller, J. L., Rogers, K. V., Spiegel, A. M. (1997) Monoclonal antibodies against synthetic peptides corresponding to the extracellular domain of the human Ca2+ receptor: characterization and use in studying concanavalin A inhibition. J. Bone Miner Res. 12,1780-1788[CrossRef][Medline]
  28. Kifor, O., Kifor, I., Moore, F. D., Jr, Butters, R. R., Jr, Brown, E. M. (2003) m-Calpain colocalizes with the calcium sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR. J. Biol. Chem. 278,31167-31176[Abstract/Free Full Text]
  29. Kameda, T., Ishikawa, H., Tsutsui, T. (1995) Detection and characterization of apoptosis in osteoclasts in vitro. Biochem. Biophys. Res. Commun. 207,753-760[CrossRef][Medline]
  30. Mentaverri, R., Kamel, S., Wattel, A., Prouillet, C., Sevenet, N., Petit, J. P., Tordjmann, T., Brazier, M. (2003) Regulation of bone resorption and osteoclast survival by nitric oxide: possible involvement of NMDA-receptor. J. Cell. Biochem. 88,1145-1156[CrossRef][Medline]
  31. Tfelt-Hansen, J., MacLeod, R. J., Chattopadhyay, N., Yano, S., Quinn, S., Ren, X., Terwilliger, E. F., Schwarz, P., Brown, E. M. (2003) Calcium sensing receptor stimulates PTHrP release by pathways dependent on PKC, p38 MAPK, JNK, and ERK1/2 in H-500 cells. Am. J. Physiol. 285,E329-E337
  32. Yano, S., Mentaverri, R., Kanuparthi, D., Bandyopadhyay, S., Rivera, A., Brown, E. M., Chattopadhyay, N. (2005) Functional expression of beta-chemokine receptors in osteoblasts: role of regulated upon activation, normal T cell expressed and secreted (RANTES) in osteoblasts and regulation of its secretion by osteoblasts and osteoclasts. Endocrinology 146,2324-2335[Abstract/Free Full Text]
  33. Ziegelstein, R. C., Xiong, Y., He, C., Hu, Q. (2006) Expression of a functional extracellular calcium sensing receptor in human aortic endothelial cells. Biochem. Biophys. Res. Commun. 342,153-163[CrossRef][Medline]
  34. Bruce, J. I., Yang, X., Ferguson, C. J., Elliott, A. C., Steward, M. C., Case, R. M., Riccardi, D. (1999) Molecular and functional identification of a Ca2+ (polyvalent cation) sensing receptor in rat pancreas. J. Biol. Chem. 274,20561-20568[Abstract/Free Full Text]
  35. Chattopadhyay, N., Cheng, I., Rogers, K., Riccardi, D., Hall, A., Diaz, R., Hebert, S. C., Soybel, D. I., Brown, E. M. (1998) Identification and localization of extracellular Ca2+ sensing receptor in rat intestine. Am. J. Physiol. 274,G122-G130
  36. Anderson, J. J., Garner, S. C. (1998) Phytoestrogens and bone. Baillieres Clin. Endocrinol. Metab. 12,543-557[CrossRef][Medline]
  37. Komarova, S. V., Pilkington, M. F., Weidema, A. F., Dixon, S. J., Sims, S. M. (2003) RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts. J. Biol. Chem. 278,8286-8293[Abstract/Free Full Text]
  38. Takahashi, E., Mukohyama, H., Aoki, K., Duarte, W. R., Lerner, U. H., Ohya, K., Omura, K., Kasugai, S. (2002) High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture. J. Med. Dent. Sci. 49,109-120[Medline]
  39. Xu, J., Wang, C., Han, R., Pavlos, N., Phan, T., Steer, J. H., Bakker, A. J., Joyce, D. A., Zheng, M. H. (2005) Evidence of reciprocal regulation between the high extracellular calcium and RANKL signal transduction pathways in RAW cell derived osteoclasts. J. Cell. Physiol. 202,554-562[CrossRef][Medline]
  40. Garner, S. C., Pi, M., Tu, Q., Quarles, L. D. (2001) Rickets in cation sensing receptor-deficient mice: an unexpected skeletal phenotype. Endocrinology 142,3996-4005[Abstract/Free Full Text]
  41. Zaidi, M., Adebanjo, O. A., Moonga, B. S., Sun, L., Huang, C. L. (1999) Emerging insights into the role of calcium ions in osteoclast regulation. J. Bone Miner. Res. 14,669-674[CrossRef][Medline]
  42. Boyce, B. F., Xing, L., Shakespeare, W., Wang, Y., Dalgarno, D., Iuliucci, J., Sawyer, T. (2003) Regulation of bone remodeling and emerging breakthrough drugs for osteoporosis and osteolytic bone metastases. Kidney Int. Suppl. ,S2-S5
  43. Wu, H., Lozano, G. (1994) NF-kappa B activation of p53. A potential mechanism for suppressing cell growth in response to stress. J. Biol. Chem. 269,20067-20074[Abstract/Free Full Text]
  44. La Rosa, F. A., Pierce, J. W., Sonenshein, G. E. (1994) Differential regulation of the c-myc oncogene promoter by the NF-kappa B rel family of transcription factors. Mol. Cell. Biol. 14,1039-1044[Abstract/Free Full Text]
  45. Chen, X., Kandasamy, K., Srivastava, R. K. (2003) Differential roles of RelA (p65) and c-Rel subunits of nuclear factor kappa B in tumor necrosis factor-related apoptosis-inducing ligand signaling. Cancer Res. 63,1059-1066[Abstract/Free Full Text]
  46. Chan, H., Bartos, D. P., Owen-Schaub, L. B. (1999) Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50–p65 recruitment. Mol. Cell. Biol. 19,2098-2108[Abstract/Free Full Text]
  47. Kubo, Y., Miyashita, T., Murata, Y. (1998) Structural basis for a Ca2+ sensing function of the metabotropic glutamate receptors. Science 279,1722-1725[Abstract/Free Full Text]
  48. Saunders, R., Nahorski, S. R., Challiss, R. A. (1998) A modulatory effect of extracellular Ca2+ on type 1alpha metabotropic glutamate receptor-mediated signalling. Neuropharmacology 37,273-276[CrossRef][Medline]
  49. Pi, M., Faber, P., Ekema, G., Jackson, P. D., Ting, A., Wang, N., Fontilla-Poole, M., Mays, R. W., Brunden, K. R., Harrington, J. J., Quarles, L. D. (2005) Identification of a novel extracellular cation sensing G-protein coupled receptor. J. Biol. Chem. 280,40201-40209[Abstract/Free Full Text]
  50. Hofer, A. M., Brown, E. M. (2003) Extracellular calcium sensing and signalling. Nat. Rev. Mol. Cell. Biol. 4,530-538[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Rheumatology (Oxford)Home page
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]

Home page
 J. Cell Sci.Home page
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]

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

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS