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Despite the therapeutic value of calcitonin in treating bone disease, a biological role of endogenous calcitonin is controversial. Having previously demonstrated that the CTR has a biological role in protecting against calcium stress using a global CTRKO mouse model, the purpose of this study was to determine whether the protection conferred by the CTR during induced hypercalcemia is mediated via CTR expression on osteoclasts. Mice were generated, in which the CTR was deleted specifically within osteoclasts (OCL-CTRKOs) and compared with mice in which the CTR was deleted globally (global CTRKOs). Significantly, peak serum calcium levels following induced hypercalcemia were >18% higher in global-CTRKOs and OCL-CTRKOs than controls (P<0.01) due to increased bone resorption (P<0.05). Peak serum calcium levels relative to controls were greater in global-CTRKO males than OCL-CTRKO males (P<0.001), which may, at least in part, be due to increased reabsorption of calcium in the kidney (P<0.01). Controls for all analyses were sex-matched littermates with normal CTR expression. In conclusion, the CTR protects against hypercalcemia predominantly via its inhibitory action on osteoclasts., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The 'calcitonin family' is a group of peptide hormones that share structural similarities with calcitonin, and includes calcitonin gene-related peptide (CGRP), amylin, adrenomedullin and adrenomedullin 2 (intermedin). These hormones are produced by different tissues, with calcitonin being produced in thyroid C cells, alphaCGRP predominantly in neural tissue, amylin in beta-islet cells of the pancreas and adrenomedullin in many tissues and cell types. Bone appears to be a common target for all the peptides of the calcitonin family, although the specific bone effects of the peptides vary. Administration of calcitonin produces rapid lowering of serum calcium levels, mainly through inhibition of bone resorption by osteoclasts. In vitro and in a number of animal experimental models, amylin and CGRP are also effective in inhibiting osteoclast activity and bone resorption. Amylin, adrenomedullin and CGRP can also affect cells of the osteoblast lineage, inducing osteoblast proliferation and promoting bone formation. Receptors for the peptides of the calcitonin family are formed by heterodimerization of the calcitonin receptor (CTR) or calcitonin receptor-like receptor (CLR) with receptor activity modifying proteins (RAMPs). Although the different combinations of these proteins create receptors with distinct ligand specificities, there is a degree of cross-reactivity and the receptors are able to bind other ligands from the family, usually with lower affinity. Analysis of the expression of the receptors for the calcitonin family in 16 samples of human osteoblasts showed high levels of CLR and RAMP1, low levels of RAMP2 and no expression of RAMP3 or CTR. Recent studies of the bone phenotype of knockout animals lacking the calcitonin, alphaCGRP or amylin gene indicated that in this experimental system the main physiological role of amylin in bone is the inhibition of bone resorption, that of CGRP is the activation of bone formation, while calcitonin, unexpectedly appears to be inhibiting bone formation without affecting bone resorption. Further investigations will be required to determine the mechanisms of action of calcitonin peptides in bone and their significance to human bone physiology.

Previous studies have implicated pro-inflammatory cytokines in the bone loss of estrogen deficiency. The aim of this study was to investigate the expression of key regulatory molecules of bone remodeling in the trabecular bone microenvironment in osteoporosis. Bone samples were taken from the intertrochanteric region of the proximal femur of patients undergoing total hip arthroplasty for a subcapital fragility fracture of the femoral neck (#NOF). For comparison, samples were taken from age-matched control individuals at routine autopsy. Expression of RANKL, RANK, osteoprotegerin (OPG), IL-6, IL-11, osteocalcin (OCN), and calcitonin receptor (CTR) messenger RNA (mRNA) species were analyzed and the data were nonparametrically distributed. The median expression of the proresorptive genes, RANK and IL-6, were significantly elevated in the fracture group compared to an age-matched control group (2.2 [1.9-2.9; 25th-75th percentiles] > 1.0 [0.4-2.1], P < 0.03; 3.9 [1.8-6.2] > 0.8 [0.7-1.5], P < 0.002, respectively). In contrast, there were no significant differences in expression of RANKL, OPG, CTR, or OCN mRNA between the #NOF and control groups. The median RANKL/OPG mRNA ratio was significantly greater in hip fracture bone than in bone from controls (4.8 [3.8-7.6] > 3.2 [2.1-4.0], P < 0.05). IL-6 mRNA levels associated strongly with RANKL mRNA levels in the #NOF group (r = 0.77, P < 0.001), but not in the control group. A strong positive association was found between IL-11 mRNA levels and RANKL mRNA levels in the #NOF group (r = 0.81, P < 0.001), consistent with the apparent coordinated regulation of IL-6 and IL-11 in bone samples from the #NOF group (r = 0.93, P < 0.0001). These data suggest a relative increase in the expression of the molecular promoters of osteoclast formation and activity in #NOF bone, which may lead to the imbalance between bone formation and resorption associated with fragility fracture.

A member of the tumor necrosis factor (TNF) family, receptor activator of nuclear factor kappaB ligand (RANKL; also known as ODF, OPGL, and TRANCE), plays critical roles in osteoclast differentiation and activation in the presence of macrophage colony-stimulating factor (M-CSF). Recently, TNF-alpha has also been shown to induce the formation of multinucleated osteoclast-like cells (MNCs) in the presence of M-CSF from mouse macrophages. We demonstrated that mononuclear preosteoclast-like cells (POCs) were formed in the presence of conditioned medium of osteoblastic cells in a rat bone marrow culture depleted of stromal cells. Using this culture system, in this study we examined whether TNF-alpha affects differentiation into POCs from hematopoietic progenitor cells. Human TNF-alpha (hTNF-alpha) markedly stimulated the formation of POCs. Moreover, a concentration as low as 0.005 ng/mL of hTNF-alpha increased the level of mRNA for calcitonin receptor (CTR) and cathepsin-K of POCs. The POCs induced by hTNF-alpha formed MNCs, which showed dentine-resorbing activity after coculture with primary osteoblasts. Stimulation was observed after 24 h of treatment with hTNF-alpha only on day 1 or day 2 of the culture. After 24 h of hTNF-alpha treatment, expression of the receptor activator of nuclear factor kappaB (RANK) mRNA was markedly increased. The addition of soluble RANKL (sRANKL) to the preformed POCs efficiently induced MNCs. Interestingly, treatment of bone marrow cells with hTNF-alpha and sRANKL synergistically augmented the formation of MNCs. This formation was abolished by the addition of human osteoprotegerin (hOPG). These results suggest that cooperation of TNF-alpha and RANKL is important for osteoclastogenesis.

Calcitonin inhibits bone resorption via its receptor (CTR) on osteoclasts. Two hCTR isoforms, hCTR1 and hCTR2, give proteins that differ in their structure and signaling pathways. We investigated whether specific isoforms or quantitative changes in total hCTR mRNA were associated with high bone resorption and turnover in menopause or osteoporosis. The hCTR mRNA in mononuclear blood cells of premenopausal (PreM), healthy (PostM), and osteoporotic (OsteoP) postmenopausal women was assessed using reverse-transcriptase polymerase chain reaction. hCTR1 and hCTR2 were investigated for 59 total RNA samples, and semiquantitative analysis of total hCTR mRNA was performed for 71. Serum calcitonin, free urinary deoxypyridinoline (D-Pyr), serum bone alkaline phosphatase (SBAP), and osteocalcin (SOC) were also evaluated. Serum calcitonin levels did not differ in PostM and OsteoP. The prevalence of each isoform was similar in the three groups. Healthy postmenopausal women and OsteoP with hCTR2 had lower bone turnover (D-Pyr: 6.79 +/- 0.54, n = 25; SBAP: 11.63 +/- 1.47, n = 26; SOC: 8.31 +/- 0.58, n = 26) than those without hCTR2 (D-Pyr: 9.90 +/- 1.95, n = 5; SBAP: 21 +/- 5.19, n = 5; SOC: 11.9 +/- 2.10, n = 5; p < 0.05). Total hCTR mRNA levels were not different in PreM and PostM. By contrast, values were strikingly lower in OsteoP (0.57 +/- 0.17, n = 28) than in PostM (2. 25 +/- 0.61, n = 19, p < 0.05) and negatively correlated with bone markers values in both. We suggest that a specific isoform and amounts of total hCTR mRNA are linked to increased bone resorption in postmenopausal osteoporosis.

The presence of the calcitonin (CT) receptor is a distinguishing characteristic of osteoclasts; however, species variability exists with respect to functional responsiveness to CT. In the present study, CT responsiveness and temporal expression of the CT receptor in differentiating cultures of porcine osteoclasts was examined and compared to murine osteoclasts. In vitro porcine osteoclast differentiation was evaluated using bone marrow cultures from neonatal pigs. Murine osteoclast differentiation was studied using cocultures of murine bone marrow and BALC cells, a calvarial-derived cell line. In the presence of 1,25 (OH)2D3, a time-dependent increase in osteoclast differentiation was observed in porcine and murine cultures. Salmon CT (sCT) and porcine CT (pCT) inhibited 1,25 (OH)2D3-stimulated porcine osteoclast differentiation at 10(-8) and 10(-7) mol/L (60% with 10(-7) mol/L sCT and 85% inhibition with 10(-7) mol/L pCT). Treatment of murine cocultures with sCT (10(-17)-10(-7) mol/L) resulted in a concentration-dependent decrease in osteoclast differentiation with a maximal inhibition of 70%. Osteoclast differentiation was inhibited in a concentration-dependent manner by recombinant human transforming growth factor-beta1 (rhTGF-beta1) in both species. The effects of CT on resorption lacunae formation were determined by culturing in vitro generated porcine or murine osteoclasts on bovine cortical bone slices for 18 h in the presence or absence of CT. With both porcine and murine osteoclasts, a concentration-dependent decrease in resorption lacunae formation was observed between 10(-13) and 10(-7) mol/L sCT with the highest concentrations completely abolishing resorption. However, pCT only inhibited porcine osteoclastic resorption at 10(-7) mol/L. CT receptor messenger ribonucleic acid (mRNA) expression was determined at different time points during in vitro osteoclast differentiation. In porcine cultures, expression of CT receptor mRNA correlated with the presence of osteoclasts. In murine cocultures, mRNA for the CT receptor was observed at each time point examined and was independent of the presence of multinucleated osteoclasts. Thus, porcine and murine differentiating osteoclast cultures express CT receptor mRNA; however, receptor expression correlates with osteoclast formation only in the porcine cultures. In summary, porcine and murine osteoclasts express CT receptor mRNA and functional responsiveness to CT. These findings suggest that the effects of sCT on osteoclast resorption are similar in murine and porcine cells, but that sCT is a less potent inhibitor of porcine than murine osteoclast differentiation.

We determined the effect of basic fibroblast growth factor (bFGF) on osteoclast-like cell (OCL) formation in bone marrow cultures using C57BL/6 mice. Cells were cultured for 7 days with or without bFGF at various concentrations or 10(-8) mol/L 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. bFGF dose-dependently increased OCL formation per well (10(-10) mol/ L = 40 +/- 2; 10(-9) mol/L = 146 +/- 13; 10(-8) mol/L = 156 +/- 12) compared with control (< 7 per well). The effects of bFGF at 10(-9) and 10(-8) mol/L were similar to that of 10(-8) mol/L 1,25(OH)2D3 (154 +/- 11 per well). OCLs formed by bFGF were multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive, expressed calcitonin receptors, and formed characteristic resorption pits. We also determined whether bFGF enhanced OCL formation during the early proliferative or late differentiating phases of the cultures. When bFGF (10(-8) mol/L) was added only on days 1-2 or days 3-4 of 6 day cultures, there was a significant increase in OCL formation. In contrast, when bFGF was added only on days 5-6 few OCLs formed. Addition of bFGF at days 1-6 or days 1-2 and days 5-6 caused similar increases in OCL formation, which were greater than OCL formation induced by treatment for days 1-2 or days 1-4. We examined the production of prostaglandin E2 (PGE2) in the cultures because bFGF is a potent stimulator of PGE2 synthesis in bone, and PGE2 stimulates OCL formation. bFGF treatment significantly increased PGE2 levels in 7 day cultures (controls = 1.4 +/- 0.1 nmol/L, 10(-8) mol/L bFGF = 132.5 +/- 0.7 nmol/L). In addition, treatment of marrow cultures with the prostaglandin synthesis inhibitors, indomethacin or NS-398 (both at 10(-6) mol/L), completely blocked bFGF-induced OCL formation. We conclude that bFGF stimulates OCL formation in C57BL/6 bone marrow cultures by mechanisms that require prostaglandin synthesis. This pathway is likely to be one mechanism by which bFGF stimulates resorption.

Cells exhibiting osteoclast characteristics of calcitonin receptors (CTRs) and tartrate-resistant acid phosphatase (TRAP) histochemistry are formed in murine bone marrow cultures treated with 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We have previously demonstrated that CTR mRNA is highly expressed in these cultures. The aim of this study was to investigate homologous regulation of the CTR, and regulation of TRAP expression in osteoclast-like cells after brief treatment with salmon CT (sCT). Murine bone marrow cells were cultured in 9 cm dishes in the presence of 10 nmol/L 1,25-(OH)2D3. On day 6 of culture, when multinucleated cells were abundant, the cells were treated with 1 nmol/L sCT for 1 h. Both control and treated cells were then harvested at intervals up to 72 h posttreatment, and both CTR and TRAP mRNA levels assessed by reverse-transcription PCR (RT-PCR). In parallel cultures, cells with CTR expression detectable by autoradiography, and TRAP positivity by histochemistry, were counted. The effects of brief sCT treatment could be seen 6 h after treatment when the CTR RT-PCR product was markedly reduced. Total recovery of CTR mRNA levels had not occurred even after 72 h. Calcitonin treatment had little effect on TRAP mRNA levels. There was no difference in the numbers of multinucleated TRAP(+) osteoclast-like cells between treated and control cells. These results indicate that brief sCT treatment, while not influencing multinucleated osteoclast-like cell number, causes specific, acute reduction of CTR mRNA in bone marrow culture-derived osteoclasts. The prolonged decrease in CTR mRNA levels suggests that recovery may require new osteoclast formation, and indicates that regulation of the CTR in cells of the osteoclast lineage is different from that in nonosteoclastic cells and tissues.