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Endocrine Research Unit, Department of Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco, San Francisco, California 94121
Address all correspondence and requests for reprints to: Dr. Dolores Shoback, Endocrine Research Unit, 111N, San Francisco Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: dolores.shoback{at}ucsf.edu.
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Abstract |
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B ligand), the major stimulator of osteoclast differentiation and activation, which is the likely underlying mechanism for the bone loss. The phenotype of Act-CaR mice is not attributable to systemic changes in serum [Ca2+] or PTH levels. We provide the first in vivo evidence that increased signaling by the CaR in mature osteoblasts can enhance bone resorption and further propose that fluctuations in the [Ca2+] within the bone microenvironment may modulate remodeling via the CaR. |
Introduction |
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Extracellular [Ca2+] ([Ca2+]e) has been proposed to fluctuate dramatically within the BMU, from approximately 0.5 mM during bone formation, when the extracellular matrix is being mineralized (2), to greater than or equal to 2 mM during bone resorption and matrix degradation (3). Numerous in vitro studies have shown that osteoblasts sense and respond to fluctuations in [Ca2+]e independently of systemic factors (for review, see Ref. 4). We and others have shown that high [Ca2+]e concentrations affect osteoblasts at many stages of differentiation to promote cell chemotaxis, proliferation, maturation, gene expression, and matrix mineralization (5, 6, 7, 8, 9, 10). [Ca2+]e may, therefore, regulate remodeling through distinct actions on bone cells, at different stages of their development. There have, however, been no in vivo studies designed to test the hypothesis that osteoblasts sense and respond to changes in the [Ca2+]e within the BMU, independently of systemic [Ca2+]e.
The extracellular Ca2+-sensing receptor (CaR) is a G protein-coupled receptor that detects and responds to changes in [Ca2+]e (11). Important physiological functions of the CaR have been shown in the parathyroid and kidney cells, in which the CaR controls PTH secretion and urinary Ca2+ excretion (12). Studies of mice in which the full-length CaR gene is ablated confirm the nonredundant role of the parathyroid and kidney CaRs in the maintenance of Ca2+ homeostasis (13, 14, 15). We and others have shown CaR expression and activity in cells of the osteoblastic lineage (5, 6, 7, 8, 9, 10, 16, 17). Although the CaR is a clear candidate for mediating Ca2+ sensing by osteoblasts, its role in vivo remains controversial (15, 18, 19). Assessment of the functions attributable to the osteoblast CaR, using the existing global CaR knockout mouse models, is complicated by the severe metabolic disturbances that these animals demonstrate [hypercalcemia and hyperparathyroidism (13, 14, 15) and the potential compensation by the CaR splice variant expression that has been described in several cell types from these animals (20, 21)].
We developed a transgenic mouse model expressing constitutively active CaRs (Act-CaRs) targeted to mature osteoblasts. Our goal was to use this model to directly test the hypothesis that increased responsiveness of osteoblasts to [Ca2+]e affects their function via activation of the CaR.
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Materials and Methods |
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Microcomputed tomography (µCT)
All µCT measurements were made using a SCANCO VivaCT 40 scanner (SCANCO Medical, Bassersdorf, Switzerland). For in vivo µCT, animals were anesthetized with 2% isoflurane (Baxter Healthcare Corporation, Deerfield, IL) in oxygen and scanned every 6 wk, from 6 to 30 wk of age. For cancellous bone, 160 serial cross-sectional scans (1.05 mm) of the secondary spongiosa of the left distal femoral metaphysis were obtained, from the end of the growth plate extending proximally to the shaft. The isotropic voxel (volumic pixel) size was 10.5 µm, and x-ray energy was 55 kV. For cortical bone, 60 serial cross-sections (0.21 mm) of the tibial diaphysis were scanned, starting from the tibio-fibular junction and extending proximally. The voxel had an isotropic size of 21 µm and x-ray energy of 55 kV.
For analysis of µCT images, a global threshold was applied to segment mineralized from soft tissue (marrow). The threshold of 27.5 and 40.0% of the grayscale was set for analysis of cancellous and cortical bone, respectively. Linear attenuation was calibrated using hydroxyapatite. Image analysis was performed using software provided by SCANCO Medical.
Histomorphometry
Mice were injected with calcein (15 mg/kg, sc) and demeclocycline (20 mg/kg, sc), 7 and 2 d before they were killed, respectively, to label mineralizing surfaces. Distal femurs were isolated from 6-wk-old wild-type (wt) and Act-CaR littermates, fixed in 10% phosphate-buffered formalin, dehydrated, defatted, and embedded in plastic (methyl methacrylate; Sigma, St. Louis, MO). For osteoblast and osteoid surface quantification, plastic sections (4 µm) were cut, mounted on gelatinized slides, deplasticized, stained according to the Von Kossa method, and counterstained with tetrachrome (Polysciences, Warrington, PA). Quantification of fluorochrome-labeled surfaces was performed under UV illumination, and data were used to calculate mineral apposition rate (MAR) and bone formation rate (BFR). Osteoclasts were identified as tartrate-resistant acid phosphatase (TRAP)-positive red cells, after TRAP activation by naphthol-1-phosphate sodium and counterstaining with fast violet (Sigma). Only cells in direct contact with a resorption pit (indentation in the mineralized matrix) were quantified. Erosion surfaces below osteoclasts were delineated and quantified. Images were captured (Zeiss microscope; MicroImaging, Thornwood, NY) and analyzed (Bioquant, Nashville, TN) in a manner blinded to genotype.
Tissue culture
Calvarial osteoblasts were isolated from 5- to 7-d-old mice and cultured as described previously (24), with the following modifications: 3 d after initial plating, the cells were passaged once and grown to confluency, and RNA was extracted, as described below. Bone marrow cells were isolated from 6- to 10-wk-old mice and cultured (25) for 7 d before crude membrane isolation, as described below.
Quantitative real-time PCR (qPCR)
Femora of 6-wk-old wt and Act-CaR littermates were isolated, and bone marrow was flushed out with PBS, before snap freezing in liquid nitrogen. For some experiments, femora were separated into epiphyseal/metaphyseal and diaphyseal compartments. For RNA isolation, the bones were powdered using a multisample biopulverizer (RPI, Mt. Prospect, IL) and homogenized with a rotor-stator homogenizer (Polytron PT 3000; Brinkmann Instruments, Westbury, NY) in RNA-Stat reagent (Tel-Test, Friendswood, TX). For total RNA isolation from cultured calvarial osteoblasts, cells were scraped in RNA-Stat reagent and freeze thawed, and RNA was isolated according to the instructions of the manufacturer. cDNAs were synthesized with Moloney murine leukemia virus RT (Invitrogen, Carlsbad, CA), and gene expression was quantified using probe-based TaqMan qPCR kits, ABI PRISM 7900HT Sequence Detection System, and SDS software (Applied Biosystems, Foster City, CA). A threshold cycle (number of PCR cycles required to generate a fluorescent signal exceeding a preset threshold) was determined for each gene of interest and normalized to the threshold cycle for a housekeeping gene (L19) in the same sample. Primers and probes for Act-CaR (5'-AGGCCAGCTGCTCGAGAGT-3' and 5'-CTTGAGTCTTCAGAAGTCACATCATG-3') and RANK-L (receptor activator of nuclear factor-B ligand) (5'-GGCCACAGCGCTTCTCAG-3' and 5'-GAGTGACTTTATGGGAACCCGAT-3') were custom made by Integrated DNA Technologies (Skokie, IL) according to published nucleotide sequences.
Immunoblotting
Crude membrane proteins were prepared from cultured bone marrow cells from wt and Act-CaR mice, human embryonic kidney 293 (HEK-293) cells expressing the bovine parathyroid CaR, and mouse brain, as described previously (6). Immunoblotting with a polyclonal anti-CaR antiserum (26) and peroxidase-conjugated antirabbit IgG secondary antibodies (GE Healthcare, Little Chalfont, UK) was performed as described previously (26). Specificity of the antiserum was confirmed by the absence of signal after preincubating anti-CaR antiserum with the peptide against which it was raised.
Total inositol phosphate (InsP) activity assay
wt and Act-CaR cDNAs were subcloned into pcDNA3.1-hygro vectors to generate wt-CaR/pcDNA3.1-hygro and the Act-CaR/pcDNA3.1-hygro constructs. The plasmids and empty vector were transiently transfected into HEK-293 cells by the calcium phosphate method as described previously (27). Levels of total InsP in transfected HEK-293 cells were determined after labeling membrane polyphosphoinositides with [3H]myoinositol and incubating the cells at different [Ca2+]e (0.1–2.5 mM) for 60 min as described previously (27). Effects on total [3H]InsP accumulation are presented as the fold increase over the basal levels in cells maintained at 0.1 mM Ca2+.
Serum biochemistries
Serum was collected from 6- and 12-wk-old mice and assayed for total calcium (Trace DMA Arsenazo III; Thermo Electron Corp., Melbourne, Australia), intact PTH (mouse intact PTH ELISA; Immutopics, San Clemente, CA), OC (mouse osteocalcin; Biomedical Technologies, Stoughton, MA), and pyridinoline crosslinks (Metra Serum PYD; Quidel Corporation, San Diego, CA) according to instructions of the manufacturers.
Statistical analysis
Data from two groups were compared using unpaired Student’s t test. Data from groups were compared using ANOVA with Tukey’s post hoc analysis. Significance was assigned for P < 0.05.
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Results |
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) were established. We confirmed the constitutive activity of the Act-CaR transgene by studying responsiveness of HEK-293 cells expressing the transgene to changes in [Ca2+]e. Total InsP accumulation, an indicator of phospholipase C activity, was significantly enhanced in cells expressing the Act-CaR compared with those expressing the wt CaR (P < 0.0001) (Fig. 1B). Targeting of the transgene specifically to bone was confirmed by qPCR analysis of reverse-transcribed RNA extracted from femora and calvaria (Fig. 1C). Both the epiphyseal/metaphyseal and diaphyseal femoral compartments, which primarily contain cancellous and cortical bone, respectively, exhibited significant transgene expression (Fig. 1C). Immunoblotting of crude membrane fractions of cultured bone marrow cells from Act-CaR mice revealed immunoreactive bands corresponding to the bovine CaR (160 and >210 kDa) (Fig. 1D, lanes 2 and 4). These were comparable in size with specific immunoreactive CaR species in lysates from HEK-293 cells expressing the bovine parathyroid CaR (Fig. 1D, lane 6). Immunoreactivity for the endogenous mouse CaR was noted in membrane fractions of bone marrow cells at approximately 120 and 140 kDa, representing glycosylated forms of the native murine CaR, and was present in both Act-CaR (Fig. 1D, lanes 2 and 4) and wt (Fig. 1D, lanes 1 and 3) mice, as well as in the mouse brain (Fig. 1D, lane 5).
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Serum biochemical analysis
There were no significant differences in levels of total serum calcium or intact PTH between wt and transgenic mice (Table 1). Likewise, levels of serum OC, a marker of bone formation, were equivalent in wt and Act-CaR mice of 6 and 12 wk of age (Table 2). Pyridinoline crosslinks, markers of bone resorption, were unchanged at 6 wk of age but rose slightly in Act-CaR mice at 12 wk of age (P < 0.05) (Table 2).
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and 3). wt controls exhibited consistent age-related bone loss from 6 wk of age. Overall, osteopenia developed in cancellous (Fig. 2) but not cortical bone in transgenic males and females. The bone volume fraction and total bone mineral density (BMD) were significantly reduced in the cancellous bone of Act-CaR mice (Fig. 3, A and B), whereas segmented BMD (i.e. mineral content of bone) was unchanged (data not shown). Changes were evident from the beginning of the study and continued throughout the 30 wk of observation. Structural indices revealed progressive degeneration of trabecular architecture, reflected in the decrease in trabecular number (Fig. 3C) and connectivity density (Fig. 3F) and an increase in trabecular spacing (Fig. 3D), which were pronounced from 12 wk of age onward. Trabecular thickness remained unchanged in males but decreased in transgenic females at 30 wk of age (Fig. 3E). We confirmed all of the in vivo µCT findings by high-resolution in vitro µCT of femora from 12-wk-old wt and Act-CaR mice of both genders (data not shown).
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, A and B), cortical thickness (Fig. 4C), porosity (Fig. 4D), and BMD (tissue and segmented; data not shown) of both males (Fig. 4) and females (data not shown).
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and Table 3). Dynamic histomorphometry parameters showed a modest reduction in osteoblast efficiency, reflected in decreased MAR in the Act-CaR mice (P < 0.05). Recruitment of osteoblasts appeared to be unchanged; there were no differences in the percentage of mineralizing surface. wt and Act-CaR mice also exhibited comparable BFRs as well as static osteoblast parameters, including number of osteoblasts per bone surface and percentage of osteoid surface (Table 3).
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). To determine whether increased RANK-L expression might be involved in the stimulation of bone resorption, we examined its mRNA expression levels in bones of the Act-CaR mice. Compared with wt controls, we observed a significant increase in the levels of RANK-L mRNA by qPCR, in both femora isolated from 6-wk-old mice (Fig. 6A) and cultured calvarial osteoblasts isolated from Act-CaR mice (Fig. 6B).
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Discussion |
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Selective targeting of this transgene was achieved by the OC promoter, ensuring that our model was appropriate for study of CaR function in mature osteoblasts. Specific expression of the OC promoter-driven genes in mature osteoblasts, and to a lesser extent in osteocytes, has been well characterized (23, 29). Critically important to the testing of our hypothesis, that locally elevated [Ca2+]e activates signaling and downstream events in osteoblasts, was the confirmation that Act-CaR mice do not display metabolic disturbances. Levels of total serum calcium and intact PTH were comparable with wt controls. Our findings are, therefore, likely attributable to mechanisms operating within the BMU and not to changes in the systemic circulation. This stands in stark contrast to CaR–/– murine models that exhibit marked metabolic disturbances that could themselves have independent effects on bone cell function (13, 15, 30). Indeed, the Act-CaR mouse is the first model available for study of osteoblast-specific CaR physiology in vivo.
Act-CaR mice exhibited decreased bone volume as well as BMD, evident from 6 wk of age, indicating they did not maintain their peak bone mass comparable with the wt controls. Both in vivo and in vitro µCT revealed an impressive deterioration in trabecular architecture, reflected in decreased connectivity density of the trabecular network, leading to the osteopenic phenotype. The changes in skeletal parameters were progressive and became pronounced from 12 wk of age. We observed no significant or consistent gender differences in the skeletal parameters, indicating that sex hormones are not involved in the development of the phenotype.
The skeletal changes were apparent only in cancellous and not in cortical bone. Considering that we detected transgene expression in both the cortical and cancellous compartments, this dichotomy is probably attributable to higher CaR activity in the cells in cancellous bone as opposed to in cortical bone. We did not, however, directly address this possibility. The concept of "functional compartmentalization" within the skeleton is well established (for review, see Ref. 31). To preserve systemic Ca2+ homeostasis, remodeling is primarily modulated at cancellous sites because of the proximity to the circulation and high surface areas compared with cortical sites (32). Our findings indicate that the osteoblast CaR functions more actively in the cancellous compartment.
To elucidate the underlying mechanisms responsible for cancellous bone loss, we quantified cellular numbers and activities in femora of Act-CaR mice and their littermate controls at 6 wk of age, when the phenotype was starting to develop. Increased osteoclastogenesis and osteoclastic activity in Act-CaR mice was evident from the elevated osteoclast numbers and percentage of erosion surface, respectively. In cultured calvarial osteoblasts and femora from Act-CaR mice, there was increased RANK-L mRNA expression, indicating that constitutive CaR signaling in mature osteoblasts affects this important local regulator of remodeling. RANK-L up-regulation would be predicted to generate more osteoclasts and increase their lifespan and resorptive capacity. Over time, this would promote bone loss and osteopenia. This concept is further supported by increased serum levels of pyridinoline crosslinks, a reflection of bone resorption, in Act-CaR animals, although changes in this parameter were quite subtle.
Osteoblast parameters, assessed by histomorphometry, were essentially unchanged in transgenic vs. wt mice. Osteoblast numbers and their recruitment, evident from percentage of mineralized surface and percentage of osteoid surface, were comparable in cancellous bone from wt and Act-CaR mice, consistent with the lack of expression of the transgene in proliferating osteoblasts. Dynamic histomorphometry did suggest a marginally decreased bone-forming efficiency, likely attributable to altered regulation of the function of mature osteoblasts expressing the transgene, evident from a reduction in MAR. An equivalent decrease in MAR was also consistently observed in 12-wk-old mice (data not shown). Inadequate osteoblastic responses to ongoing bone resorption could be contributing to the development of the osteopenic phenotype. Nevertheless, these changes do not seem to significantly affect BFRs at either time point. In support of these findings, we observed similar levels of serum OC, a marker of bone formation, in wt and Act-CaR mice.
Changes in [Ca2+]e have been proposed to affect several cell types in bone. High [Ca2+]e, acting via the CaR, has been shown to inhibit osteoclast function (28), stimulate preosteoblast chemotaxis to the site of resorbed bone (8), and support both osteoblast proliferation (5, 9) and bone formation (5, 10). As mineralization progresses, CaR signaling may also act to prevent excessive accumulation of bone, by stimulating production of inhibitors of mineralization [e.g. osteopontin (5)]. Such countermanding events may reduce the bone-forming efficiency of mature osteoblasts, which we observed in this study.
Our findings provide evidence that CaR activation in mature osteoblasts supports osteoclastogenesis and promotes bone resorption in vivo. This is supported by our previous reports of Ca2+-induced RANK-L expression by osteoblastic cells and the resultant osteoclastogenesis (33). We speculate that such recruitment and differentiation of osteoclasts may serve as a part of a feedback loop, which ensures that short-lived osteoclasts (days) are replaced and available within the BMU throughout its lifespan (months). In our model of constitutive activation of CaR in osteoblasts, the net skeletal phenotype effect was enhanced osteoclastogenesis, likely driven by chronically elevated RANK-L levels. Direct inhibition of osteoclastic resorption, which might occur concomitantly in the presence of high [Ca2+]e, was not present because of the transgene targeting strategy.
Could such a putative feedback loop contribute to mechanisms responsible for pathologic bone resorption? Local release of paracrine factors fuel the excessive resorption seen in solid tumors that metastasize to bone, as well as in multiple myeloma. These factors have been proposed to drive a "vicious cycle" of bone resorption and tumor growth (34, 35). Our findings suggest that the local hypercalcemia may itself be involved in perpetuation of the vicious cycle, via stimulation of RANK-L production and support of osteoclastogenesis. The CaR is clearly a potential mechanism for mediating such responses.
Our observations indicate that the CaR in mature osteoblasts regulates bone resorption via its stimulatory effects on osteoclasts. These studies provide direct in vivo evidence, demonstrating for the first time that CaR signaling in mature osteoblasts is involved in controlling the maintenance of bone mass and bone turnover.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors of this manuscript have nothing to declare.
First Published Online April 5, 2007
Abbreviations: Act-CaR, Constitutively active mutant CaR; BFR, bone formation rate; BMD, bone mineral density; BMU, bone multicellular unit; [Ca2+]e, extracellular [Ca2+]; CaR, extracellular Ca2+-sensing receptor; µCT, microcomputed tomography; HEK-293, human embryonic kidney 293 cells; InsP, inositol phosphate; MAR, mineral apposition rate; OC, osteocalcin; qPCR, quantitative real-time PCR; RANK-L, receptor activator of nuclear factor-B ligand; TRAP, tartrate-resistant acid phosphatase; wt, wild type.
Received February 1, 2007.
Accepted for publication March 23, 2007.
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