The calcium-sensing receptor in bone--mechanistic and therapeutic insights.

REVIEWS The calcium-sensing receptor in bone —mechanistic and therapeutic insights David Goltzman and Geoffrey N. Hendy Abstract | The extracellular calcium-sensing receptor, CaSR, is a member of the G protein-coupled receptor superfamily and has a critical role in modulating Ca2+ homeostasis via its role in the parathyroid glands and kidneys. New evidence suggests that CaSR expression in cartilage and bone also directly regulates skeletal homeostasis. This Review discusses the role of CaSR in chondrocytes, through which CaSR contributes to the development of the cartilaginous growth plate, as well as in osteoblasts and osteoclasts, through which CaSR has effects on skeletal development and bone turnover in young and mature animals. The interaction of skeletal CaSR activation with parathyroid hormone (PTH), which is secreted by the parathyroid gland, can lead to net bone formation in trabecular bone or net bone resorption in cortical bone. Allosteric modulators of CaSR are beneficial in some clinical conditions, with effects that are mediated by the ability of these agents to alter levels of PTH and improve Ca2+ homeostasis. However, further insights into the action of CaSR in bone cells might lead to CaSR-based drugs that maximize not only the effects of the receptor on the parathyroid glands and kidneys but also on bone. Goltzman, D. & Hendy, G. N. Nat. Rev. Endocrinol. advance online publication 10 March 2015; doi:10.1038/nrendo.2015.30

Introduction

Department of Medicine, McGill University, 687 Pine Avenue West, Montreal, QC H3A 1A1, Canada (D.G., G.N.H.). Correspondence to: G.N.H. geoffrey.hendy@ mcgill.ca

Extracellular Ca2+ homeostasis is modulated by para thyroid hormone (PTH) and the active form of vitamin D, 1,25-dihydroxyvitamin D3, which regulate Ca2+ flux in the kidneys, intestine and bone. The extracellular calcium-sensing receptor (CaSR), a cation-s ensing G protein-coupled receptor (GPCR), and possibly other Ca2+‑sensing receptors determine the levels of Ca2+ in the extracellular fluid ([Ca2+]e) and also critically contribute to regulation of these levels.1 Nevertheless, experimen tal findings have identified CaSR in a number of tissues not classically involved in regulating levels of [Ca2+]e and these observations have expanded the role of this receptor to include skeletal homeostasis. The skeletal cells that express CaSR and the roles of CaSR in these cells are now well defined. In addition, extensive experience with positive and negative modu lators of CaSR has been obtained, which has enabled evaluation of the actual versus potential performance of these modulators in the clinical setting. In this Review, we discuss the functions and modulation of the activ ity of CaSR as they relate to extracellular Ca2+ homeo stasis, to chondrocytes of the developing cartilaginous growth plate, and to the bone-forming osteoblasts and bone-resorbing osteoclasts of the skeleton. The differ ential effects of activating CaSR in the developing skel eton versus the adult skeleton are considered, as well as the relationship between CaSR activation and PTH action, which can lead to net bone formation in trabecu lar (cancellous or spongy) bone or net bone resorption Competing interests The authors declare no competing interests.

in cortical (compact) bone. Small-molecule allosteric modulators of CaSR (both positive and negative) have been developed and current clinical and experimental experience with these modulators will form the basis for developing a new generation of compounds that potentially have specific beneficial effects on bone.

Extracellular calcium-sensing receptor Function In humans, CASR (located on chromosome 3) encodes CaSR, 2 a class C GPCR expressed on the cell mem brane as a disulphide-linked constitutive homodimer.3,4 Although CaSR is found in a variety of tissues, the highest expression of the receptor is found in the para thyroid glands and kidneys. CaSR exhibits pleiotropic interactions with Gαq/11, Gαi/o and Gα12/13 in diverse cell types and with Gαs in specific cell types.5,6 Such eclec tic cell-dependent and context-dependent interactions facilitate selective regulation of the wide array of cellular effects associated with CaSR. CaSR-mediated increases in renal Ca2+ excretion and perhaps other actions, such as effects on the gut, serve to limit increases in [Ca2+]e levels (hypercalcaemia) that result from hypercalcaemic challenges; suppression of PTH levels does not seem to be an absolute require ment in mitigating these increases.7–9 By contrast, for defence against decreased [Ca2+]e levels (hypocalcae mia), CaSR-mediated PTH secretion, PTH gene expres sion and cellular proliferation in the parathyroid gland are required. Binding of PTH to the GPCR PTH/PTHrelated peptide receptor (PTH/PTHrP receptor), facili tates increased renal reabsorption of Ca2+ and stimulates

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REVIEWS Key points ■■ The extracellular calcium-sensing receptor (CaSR) expressed in the parathyroid glands and kidneys modulates blood levels of Ca2+; CaSR is also expressed on bone cells, where it regulates skeletal homeostasis ■■ In chondrocytes, CaSR contributes to development of the cartilaginous growth plate, whereas in osteoblasts CaSR is required for the proliferation and differentiation of these cells, and for bone matrix production ■■ In young animals, activation of CaSR in osteoclasts inhibits bone resorption, which results in enhanced bone anabolism ■■ In old animals, although activation of osteoblasts augments bone formation, this activation also increases the expression of receptor activator of nuclear factor κB ligand and bone resorption by osteoclasts ■■ The relationship between activation of CaSR in the skeleton and levels of parathyroid hormone can lead to net bone formation in trabecular bone and net bone resorption in cortical bone ■■ Although currently available allosteric modulators of CaSR in the parathyroid gland are beneficial in some clinical conditions, future CaSR-based drugs might be developed that have improved effects on bone

the activity of the enzyme 25-hydroxyvitamin D‑1α hydroxylase (also known as 25-OHD‑1α hydroxylase and encoded by CYP27B1 in humans), which con verts inactive 25-hydroxyvitamin D3 to its active form, 1,25-dihydroxyvitamin D3. This active form has effects predominantly through the 1,25-dihydroxyvitamin D3 receptor (also known as vitamin D3 receptor) to augment intestinal Ca2+ absorption. PTH also increases bone turnover and results in net bone resorption, particu larly of cortical bone, which results in mobilization of skeletal Ca2+.10 However, PTH also increases secretion of FGF23, which can result in decreased production and clearance of 1,25-dihydroxyvitamin D3, thus limiting further increases in levels of [Ca2+]e.11 Increased levels of [Ca2+]e in the presence of high-normal or elevated serum levels of phosphorus can also increase FGF23 secretion, although this action does not seem to be mediated by CaSR.12 Nevertheless, CaSR contributes to mitigating both hypocalcaemia and hypercalcaemia.13 Although Ca2+-bound CaSR might stimulate the pro duction of calcitonin, which could have a physiological role in inhibiting bone resorption and cause hypo calcaemia in some species, calcitonin is highly unlikely to have a similar role in humans. Following total thyroid ectomy (for example, for thyroid cancer), in which C cells (the source of calcitonin) are removed as well as other thyroid components, decreased Ca2+ levels due to hypoparathyroidism might occur rather than increased Ca2+ levels owing to calcitonin deficiency.14 Additionally, in cases of C-cell tumours (known as medullary thyroid carcinomas) calcitonin is abundantly produced but, again, no obvious abnormalities in Ca2+ homeostasis are evident.15 Consequently, release of calcitonin by Ca2+stimulated CaSR activity is unlikely to substantially contribute to Ca2+ homeostasis in humans.

Polymorphisms in humans The central role of CaSR in [Ca 2+]e homeostasis in humans was highlighted by the identification of poly morphisms in CASR, which are associated with serum levels of Ca 2+. 16 The CASR single nucleotide

polymorphism rs1801725 was associated with serum levels of Ca2+, with increased serum levels of PTH and with decreased serum levels of phosphorus in a genomewide study.17 Casr expression in mouse tibia was markedly upregulated in response to a diet low in calcium,18 which is consistent with 1,25-dihydroxyvitamin D3-mediated transactivation of Casr by vitamin D receptor–retinoid X receptor heterodimers.19 This single nucleotide polymor phism was also associated with BMD.18 Overall, these findings suggest an important role for CaSR, not only in mineral homeostasis, but also in skeletal homeostasis.

Modulation Some modulators of CaSR have been developed, includ ing cinacalcet (a type II calcimimetic in clinical use that binds to a site distinct from that bound by the physio logical ligand), that amplify the sensitivity of CaSR to [Ca2+]e levels and thus function as positive allosteric modulators. 20 Calcilytic compounds are agents that have been developed as negative allosteric modulators or antagonists of CaSR to stimulate secretion of PTH and take advantage of the skeletal anabolic capacity of PTH that occurs when its levels are intermittently rather than continuously increased. Calcilytics were, therefore, initially used as orally active anabolic agents for the treatment of osteoporosis.21 In some studies in ovariec tomized rats, calcilytic compounds such as SB‑423557 (a pro-drug of SB‑423562) transiently increased plasma levels of PTH and stimulated bone formation.22 By con trast, administration of NPS 2143 to osteopenic rats did not have net anabolic effects in bone, despite increased plasma levels of PTH and the presence of estradiol, an inhibitor of bone resorption.23 Consequently, the poten tial of calcilytics to indirectly stimulate bone formation is uncertain. In addition to PTH, calcilytic action might also release the other contents of the chief cell secre tory granules of the parathyroid gland, which include C‑terminal fragments of PTH24 and chromogranin A,25 and cathepsin-type enzymes.26 However, any potential positive or negative skeletal effects of these other secreted factors remain to be determined. Skeletal effects Studies in vitro and in animal models in vivo and ex vivo have provided considerable insight into the skeletal roles of CaSR. Nevertheless, no consensus exists on the effects of CaSR in the skeleton. Cartilage Phosphorus, 1,25-dihydroxyvitamin D3, PTHrP and other local and systemic regulators have been implicated in normal development and mineralization of the growth plate. Low serum levels of phosphate can inhibit apopto sis of hypertrophic chondrocytes and alter development of the cartilaginous growth plate, which leads to rickets.27 Studies in Cyp27b1–/– mice indicate that normal growth and morphology of the growth plate might also require the action of 1,25-dihydroxyvitamin D3.28 Furthermore, deletion of Pthlh (which encodes PTHrP in mice) pro duces abnormal epiphyseal cartilage development and

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REVIEWS altered endochondral bone formation.29 CaSR stimulates the release of PTHrP in normal cells and in cancer cells,30 and PTHrP expression in the cartilaginous growth plate is reduced in newborn Casr–/– mice.31 Hence, CaSR might regulate the cartilaginous growth plate, at least in part, by promoting release of PTHrP. In vitro studies have shown that CaSR might also mediate differentiation of chondrocytes in the growth plate.32–34 Furthermore, mice in which chondrocyte- specific deletion of Casr was induced between embryonic day (E) 16 and E18 exhibited delayed development of the growth plate.35 Nevertheless, treatment of Cyp27b1–/– mice (which are deficient in 1,25-dihydroxyvitamin D3 and develop rickets when fed a normal diet) with cina calcet produced no positive effect on growth plate architecture or longitudinal bone growth.36 Thus, stimu lation of CaSR is possibly insufficient to overcome the chondroc yte dysfunction that results from a lack of 1,25-dihydroxyvitamin D3. Alternatively, cinacalcet- induced CaSR signalling pathways in chondrocytes might differ from those in the parathyroid gland; further studies are required to confirm this sugges tion and whether cinacalcet selectively activates signal transduction pathways in parathyroid cells. Casr−/−;Cyp27b1−/− double-knockout mice fed normal chow develop more severe secondary hyperpara thyroidism than Cyp27b1−/− single-knockout mice, 37 which is accompanied by more severe hypophospha taemia as well as more marked abnormalities in the growth plate, less mineral deposition and more retard ation of bone growth. When hypercalcaemia was induced in Casr−/−;Cyp27b1−/− mice by administration of a diet high in calcium and lactose, mineralization of the growth plate and the growth of long bones improved. This amelioration might have been due to non-CaSR-mediated modulation of the growth plate by high levels of [Ca2+]e; however, a hypomorphic Casr allele possibly expressed in these animals as a result of alternative splicing 38 could have facilitated the improvement. Nevertheless, even in hypercalcaemic Casr–/–;Cyp27b1–/– mice, mineralization of the growth plate, although improved, was not normal ized. Consequently, the absence of a fully functional CaSR, presence of severe hypophosphataemia, absence of 1,25-dihydroxyvitamin D3 and possibly absence of PTHrP might all have contributed to residual rachitic abnormalities even in the presence of adequate levels of [Ca2+]e.

role of CaSR in modulating the skeletal effects of PTH, as the typical skeletal phenotype reported after deletion of Pth was no longer observed. Most studies on the role of CaSR in bone have focused on the bone-remodelling process and have shown reductions in trabecular bone volume, abnormalities in trabecular microarchitec ture and increases in cortical porosity; 35,42 however, some evidence indicates that cortical thickness is also reduced, which suggests that CaSR also has negative effects on bone modelling.35 By use of in situ hybridiza tion, Casr mRNA has been found mainly in bone marrow cells, osteoblasts and osteocytes, and infrequently in mature osteoclasts.43

In vitro effects CaSR is functionally active in cells of the osteoblast lineage in vitro, in which high levels of [Ca 2+]e can increase inositol phosphate production, increase intra cellular levels of free Ca2+ and reduce PTH-induced cAMP formation.43 Interestingly, these positive effects on osteoblastic cells can be blocked by the action of calcilytics.44 Exposure of primary osteoblasts or a vari ety of osteoblast-like cells to high levels of [Ca2+]e or polycationic CaSR agonists (such as neomycin or gado linium) stimulates osteoblast proliferation, differentia tion and matrix-mineralization capacity via activation of mitogen-activated protein kinases such as extracellular signal-regulated protein kinases 1 and 2, p38 and/or c‑Jun N‑terminal kinase.44–47 Strontium ranelate seems to reduce the risk of frac tures in patients with osteoporosis by altering bone remodelling and the divalent ion Sr 2+ activates multiple signalling pathways in bone cells, at least some of which occur via CaSR.48 Moreover, Sr 2+ induces osteoblast pro liferation by activating CaSR,49 mediates differentiation of osteoblasts,50 enhances osteoblast-mediated matrix mineralization,51 reduces expression of receptor activa tor of nuclear factor κB ligand (RANKL)52 and increases expression of osteoprotegerin in osteoblasts and/or stromal cells, which can antagonize the ost eoclast- stimulating cytokine RANKL and result in reduced differe ntiation of pre-osteoclasts into osteoclasts. 53 However, pathways other than CaSR must be involved in these processes as the proliferative effect of Sr 2+ is maintained in osteoblasts isolated from Casr–/– mice.54

Bone No bone phenotype39 or a minimal bone phenotype13 was observed in Casr null mice that were intercrossed with Gcm2-deficient mice, which results in the molecular ablation of parathyroid glands, or by intercrossing with mice with global Pth deletion. However, a bone pheno type has been described in both newborn40 and older mice41 with a global deletion of Pth, as well as in humans with hypoparathyroidism. Consequently, rather than CaSR having no effect in the skeleton, the lack of a bone phenotype39 or the minimal bone phenotype13 in mice deficient in both Casr and Pth seem consistent with the

Neonatal effects Studies in genetic mouse models have indicated that CaSR might also be required for normal development of the fetal and neonatal skeleton in vivo.35 Osteoblastspecific Casr conditional knockout mice have abnor mal skeletal histology at birth and arrested osteoblast differentiation, evident by markedly reduced numbers of osteoblasts and decreased expression of osteoblast markers.44 Interestingly, these mice also had mildly eleva ted serum levels of PTH and Ca2+; however, the origin of this mild hyperparathyroidism is unclear, as indeed is the extent to which it modified the phenotype of the

Bone turnover in animals Osteoblasts



REVIEWS mice. The Casr construct used to produce the osteoblast- specific knockout mice, in which exon 7 which encodes the seven‑transmembrane and C‑terminal domains of CaSR, was deleted, produces a secreted dominant- negative extracellular domain that can inhibit [Ca2+]emediated CaSR signalling in vivo and increase serum levels of PTH.55 Nevertheless, the overall findings suggest that CaSR deficiency has a negative effect on osteoblastic anabolic activity. Endogenous PTH, functioning via direct effects on the PTH/PTHrP receptor, enhances osteoblastic trabecular bone formation in newborn mice.40 High levels of [Ca2+]e also seem to independently increase osteoblastic bone formation in suckling murine neonates. Furthermore, [Ca2+]e might increase bone formation in conjunction with PTH, although the precise mechanism remains to be determined.56 The effects of increased levels of [Ca2+]e are probably transduced by CaSR, because changes in levels of [Ca2+]e have not been consistently reported to influence Ca2+ channel or Ca2+ transporter activity in osteoblasts, and the role of GPCR family C group 6 member A (also known as GPRC6A) as a potential alter native skeletal receptor for [Ca2+]e is uncertain.57–59 By contrast, in mice with targeted deletion of Pth or Pth and Casr, both Ca2+-induced increases in response to changes in levels of dietary calcium that result in bone anabolism and Ca2+-induced enhancement of PTH-stimulated bone anabolism in neonates seem to be mediated by CaSR.60 Effects in adults Positive anabolic effects of the CaSR agonist cinacalcet were not obvious in adult Cyp27b1–/– mice with intact CaSR;33 however, PTH-induced increases in trabecular bone volume were lower in Pth–/–;Casr–/– mice than in Pth–/– mice after 2 weeks of continuous PTH infusion.61 These findings seem to be consistent with a role for CaSR in augmenting PTH-induced bone formation in adults as well as in neonates.

Osteoclasts In vitro effects Studies have demonstrated the presence of CaSR in monocytes and macrophages,62 and also in osteoclasts.63,64 Furthermore, in situ hybridization studies have dem onstrated the presence of Casr mRNA in bone marrow cells and osteoclasts.43 Additional findings support a role for CaSR in enhancing osteoclast differentiation; however, exposure of osteoclasts to very high levels of [Ca2+]e, at concentrations probably present only in the bone microenvironment, results in a dramatic with drawal of mature osteoclasts, followed by a profound inhibition of bone resorption.65–68 Mature osteoclasts also undergo apoptosis in the presence of high levels of [Ca 2+]e, which probably occurs via signalling path ways that involve phospholipase C and nuclear factor NF‑κB;68 a dominant-negative CaSR construct abrogates this effect.69 Cumulatively, these findings suggest that, at least in vitro, activation of CaSR is linked to increased osteoclast differentiation, increased osteoclast apop tosis and inhibition of bone resorption, although the

levels of [Ca2+]e required for these processes might not be equivalent. Although high concentrations of calcium (>6.0 mM) inhibit osteoclast formation in vitro, the calcimimetic cinacalcet HCl (30–1,000 nM) had no effect on osteo clast formation or bone resorption in vivo.70 Furthermore the calcilytic NPS 2143, which effectively stimulates PTH secretion in vivo, had no effect on bone resorption by iso lated human osteoclasts in vitro.23 Consequently, whether allosteric modulators that act as agonists or antagonists of CaSR in parathyroid cells function similarly in osteo clasts is unclear. Future studies comparing Ca2+-induced CaSR signalling with allosteric-modulator-induced CaSR signalling in parathyroid cells compared with osteoclasts could provide new insights. Neonatal effects Osteoclast number, and osteoclast surface relative to bone surface, were markedly higher in the metaphyseal regions of bone in 2‑week-old Casr–/– mice than in wild-type litter mates.31 Osteoblast-specific deletion of Casr increased expression of RANKL, the number of osteoclasts and cortical porosity in 21-day-old mice, in comparison to wild-type littermates.42 Given that these animals had mild hyperparathyroidism, whether this increased bone resorption is independent of PTH or reflects an inter action between CaSR and PTH is unclear. Studies in Pth–/– and Pth–/–;Casr–/– pups demonstrated that CaSR is needed to support [Ca2+]e-induced inhibition of osteoclastic bone resorption, which occurs at least in part by modulation of the RANKL:osteoprotegerin ratio.60 Consequently, at least in early life, the predominant effect of [Ca2+]e that is mediated by CaSR seems to be the uncoupling of bone formation from bone resorption by inhibition of osteo clastic bone resorption, whilst osteoblastic bone formation is stimulated (Figure 1). Effects in adults Transgenic mice 6 weeks of age or older expressing a constitutively active mutant form of CaSR in mature osteoblasts display increased bone resorption, which is associated with increased levels of RANKL, decreased bone volume and diminished BMD71 (Figure 1). Genetic disruption of Casr in Cyp27b1–/– mice reduced bone resorption owing to the absence of [Ca2+]e-stimulated CaSR activity.37 Thus, part of the calcaemic effect of high levels of circulating 1,25-dihydroxyvitamin D3 possibly occurs by an increase in the levels of [Ca2+]e via increased intestinal calcium absorption, which facilitates increased [Ca2+]e-induced bone resorption via CaSR.72 In addition, CaSR has been reported to modulate PTH-stimulated bone turnover in 8‑week-old mouse models, in which PTH was infused in Pth–/–;Casr–/– mice.61 In Pth–/–;Casr–/– mice, the increases in cortical porosity and decreases in cortical bone volume induced by PTH infusion were reduced in comparison with cortical bone changes induced by PTH in Pth–/– mice.61 Additionally, PTHstimulated RANKL expression and osteoclastogenesis were lower in cultured bone marrow from Pth–/–;Casr–/– mice than PTH responses in cultured bone marrow from



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Figure 1 | Model of CaSR action in bone formation and resorption. Osteoblastogenesis proceeds via commitment of MSCs Natureinto Reviews | Endocrinology to osteoblast precursors that proliferate and differentiate to preosteoblasts, which in turn mature osteoblasts. Mature osteoblasts secrete unmineralized bone matrix (osteoid) that mineralizes to produce bone in which mature osteoblasts become imbedded as osteocytes. During osteoblast stimulation, the expression of R is increased in osteoblastic cells and that of its decoy receptor O is decreased, resulting in an increased R:O ratio that promotes all steps of osteoclastogenesis; bone resorption and bone formation are therefore coupled. Osteoclastogenesis proceeds from HPCs through mononuclear osteoclast precursors, which fuse to form inactive osteoclasts that can become active bone-resorbing osteoclasts. In cells of the osteoblast lineage, Ca2+-stimulated CaSR promotes osteoblastogenesis and bone formation.31,36,44–48 a | In young animals, Ca2+-stimulated CaSR decreases the R:O ratio,48 which induces apoptosis in mature osteoclasts,65–68 prevents bone resorption and uncouples bone resorption from bone formation, leading to net bone accrual. b | In older animals, Ca2+‑stimulated CaSR also promotes osteoblastogenesis and bone formation.36,61 However, Ca2+-stimulated CaSR increases the R:O ratio, which enhances osteoclastogenesis35,71 and increases bone resorption. Bone formation and bone resorption are thus coupled; however, net bone formation can predominate in trabecular bone and net bone resorption can predominate in cortical bone, thus enhancing the effects of PTH in both compartments. 61 Abbreviations: CaSR, calcium‑sensing receptor; HPC, haematopoietic progenitor cell; MSC, mesenchymal stromal cell; O, osteoprotegerin; PTH, parathyroid hormone; R, RANKL (also known as receptor activator of nuclear factor κB ligand).

Pth–/– mice.61 Overall, these observations are consistent with a role for CaSR in augmenting PTH-induced corti cal bone loss. Stimulation of CaSR by Ca2+ occurs even at low levels of Ca2+and increases in a dose-dependent manner as levels of Ca2+ increase.73 Consequently, acti vation of CaSR to facilitate osteoclast-mediated bone resorption might occur in the bone milieu as Ca2+ is released from bone by the action of PTH. Furthermore, in the bone microenvironment, the levels of Ca2+ might be much higher during resorption than the levels of this cation in the blood.74 Increases in levels of [Ca2+]e in the bone microenvironment, which result from PTHenhanced renal reabsorption of Ca2+ and from 1,25- dihydroxyvitamin D3-induced gut absorption of Ca2+, might amplify cortical bone resorption by stimulating CaSR (Figure 2). Furthermore, the degree of cortical bone loss (a major determinant of long bone fractures) in primary hyperparathyroidism might be influenced by the degree of existing hypercalcaemia as a result of negative [Ca2+]e effects on cortical bone, which occur via CaSR. Overall, the findings suggest that, at least in young adult animals, activation of CaSR increases osteoclastic bone resorption (Figure 1) and amplifies the effects of circulating PTH (Figure 2).

Bone mineralization Mice with osteoblast-specific deletion of Casr have undermineralized skeletons (osteomalacia),35 which suggests a direct role for CaSR in bone mineralization. Increased expression of genes encoding proteins that inhibit matrix mineralization (such as progressive ankylo sis protein homologue, ectonucleotide pyrophosphatase/ phosphodiesterase family member 1 and osteopontin) was observed in conditional osteoblast-specific Casr–/– mice, which indicates that these genes might con tribute to the reduced bone mineralization observed.42 Furthermore, improved mineralization with the cal cimimetic cinacalcet has been reported in Cyp27b1−/− mice,36 which is consistent with a direct role for CaSR in bone mineralization. Systemic deletion of Casr in mice results in hyper parathyroidism and osteomalacia;35,75–77 however, elimi nation of the hyperparathyroidism by also genetically deleting PTH in these mice seems to normalize the skeletal phenotype,39 which suggests that the increased levels of unmineralized bone matrix (osteoid) might be a direct consequence of elevated PTH levels. Indeed, infusion of PTH into wild-type or Pth–/– mice might increase production of osteoid,61 which probably takes



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Figure 2 | Model of bone resorptive action of skeletal CaSR in Ca2+ homeostasis. Nature Reviews | Endocrinology Decreased levels of [Ca2+]e lead to reduced activation of CaSR in the parathyroid gland (1) and increased release of PTH (2), which converts inactive 25-hydroxyvitamin D3 to active 1,25-dihydroxyvitamin D3, leading to increased intestinal Ca2+ absorption (3) and increased levels of [Ca2+]e. Elevated levels of PTH also enhance Ca2+ reabsorption in the kidney (4) and further increase levels of [Ca2+]e (5), as well as increasing bone turnover (6). These effects result in net bone resorption (primarily in cortical bone) and release of Ca2+, thereby increasing levels of [Ca2+]e (7). Elevated levels of [Ca2+]e can stimulate CaSR in the bone, further increasing bone turnover and net bone resorption61 as part of a feedforward system. Cumulative increases in levels of [Ca2+]e then activate CaSR in the parathyroid gland (8), thereby inhibiting the further release of PTH. Excessive levels of [Ca2+]e activate CaSR in the kidney (9) to increase levels of [Ca2+]u. Dashed lines represent contributing factors. Abbreviations: [Ca2+]e, Ca2+ in extracellular fluid; [Ca2+]u, Ca2+ in urine; CaSR, calcium-sensing receptor; PTH, parathyroid hormone.

place, in part, by induction of hypophosphataemia and, in part, by enhancement of the expression of inhibitors of mineralization such as matrix Gla protein.78 However, the volume of osteoid induced by PTH infusion was greater in Pth−/−;Casr−/− mice than in wild-type or Pth−/− mice,61 which also suggests a role for CaSR in facilitating bone mineralization. Nevertheless, elevated serum levels of PTH, in the absence of substantial osteomalacia, have been observed in murine models of primary hyperparathyroidism79 and in patients with hyperparathyroidism80 despite the pres ence of hypophosphataemia. Improved bone mineral ization occurred in these hypercalcaemic mice81 and also in Casr−/−;Cyp27b1−/− mice made hypercalcaemic by dietary means.37 Considering this amelioration occurred despite the absence of CaSR, the possibility cannot be excluded that other receptors or nonreceptor-mediated mechanisms are involved in the mineralization of bone mediated by increased levels of [Ca2+]e. CaSR might, therefore, directly participate in enhancing bone mineral ization, even though this effect could be mitigated by very high levels of PTH and/or hypophosphataemia. Furthermore, other mechanisms of mineralization might

Models of chronic kidney disease The percentage of bone surface covered by osteoclasts and osteoblasts is generally increased in bones of uraemic rats with secondary hyperparathyroidism.81–83 Peritrabecular fibrosis and potentially decreased cortical volumetric (v) BMD might also occur in these rats, possibly as a result of the effects of PTH. The phenylalkylamine calcimi metic NPS R‑568 reduced elevated serum levels of PTH, reduced peritrabecular fibrosis and increased cortical vBMD in nephrectomy-induced uraemic rats; however, no decreases in osteoclast or osteoblast surfaces were observed.81 Increased bone resorption (estimated as the percentage of bone surface covered by osteoclasts) and trabecular spacing, and reduced trabecular number and bone volume were observed in adenine-induced uraemic rats with secondary hyperparathyroidism. 82 Treatment of these uraemic rats with the arylalkylamine calcimimetic AMG 641 reduced serum levels of PTH and prevented changes in trabecular bone; however, no signif icant reduction in resorbing bone surface was observed.82 The calcimimetic cinacalcet reduced levels of PTH and increased urinary Ca2+ excretion, but increased bone resorption and reduced bone volume in nephrectomyinduced uraemic rats.83 Furthermore, cinacalcet increased the number and activity of osteoclasts in adult Cyp27b1–/– mice with normal renal function.36 The mechanism of this cinacalcet-induced bone resorption in PTH-sufficient– vitamin D-deficient states requires further investigation. Overall, these studies are not consistent with suppression of bone turnover by calcimimetics in animal models of chronic kidney disease.

Bone turnover in humans CASR mutations Patients with homozygous inactivating mutations in CASR have marked hyperparathyroidism(neonatal severe hyperparathyroidism) and extensive loss of bone at birth; demineralization and bone fractures can also occur.84,85 This presentation is consistent with the effects of increased circulating levels of PTH causing bone resorption and demineralization; however, accel erated osteoclast resorption and decreased bone forma tion owing to loss of skeletal CaSR could contribute to the bone abnormalities, as observed in neonatal rodents deficient in CaSR.40 Increased circulating levels of PTH are present in patients with heterozygous inactivating mutations in CASR who present with familial hypocalciuric hyper calcaemia (FHH); these patients might also have slightly increased bone turnover.86,87 In another study, higher total and trabecular hip vBMD and lower cortical vBMD and hip bone volume were found in patients with FHH than in age-matched and sex-matched control patients; no differences in bone strength were observed between groups.88 These changes are consistent with the skeletal effects of increased levels of PTH; however, bone loss and fractures seem to be less prominent in patients with FHH



REVIEWS than in patients with primary hyperparathyroidism.89–92 Overall, the human data are consistent with those from mice, which suggests that the major effects of sustained circulating levels of PTH on increased bone turnover and bone loss are lessened when CaSR is absent from the bone.61 Several case reports have documented that CaSR activity in some patients with FHH and neonatal hyper parathyroidism (who are heterozygous for CASR inacti vating mutations) can be allosterically modulated with cinacalcet to achieve clinical benefits in terms of control ling hypercalcaemia and reducing levels of PTH. 93–95 Whether or not cinacalcet also improves skeletal status in cinacalcet-treated patients remains to be determined. A patient with autosomal dominant hypocalcaemia (resulting from an activating mutation in CaSR) who had hypoparathyroidism as a manifestation of his disease was treated continuously with intermittent PTH injections from the age of 6 years until 20 years of age.96 Trabecular bone volume was dramatically increased in this patient and a shift toward lower bone mineraliza tion was observed. The excellent anabolic response to intermittent PTH therapy might be due to augmenta tion of the anabolic effects of PTH in trabecular bone by activated CaSR, as has been observed in mouse models.61 The reduced bone mineralization is unlikely to be secondary to the increased CaSR activity and, therefore, might be due to direct effects of exogenous PTH.

Primary hyperparathyroidism Cinacalcet is approved for use in patients with primary hyperparathyroidism. In a year-long placebo-controlled trial of cinacalcet in patients with primary hyper parathyroidism, cinacalcet treatment decreased serum levels of Ca 2+ and reduced serum levels of PTH by ~20%.97 Nevertheless, levels of markers of bone turn over increased slightly, albeit significantly; BMD at the distal third of the radius, total hip and lumbar spine was unaffected. 97 In an open-label extension study of 4.5 years duration, although significant lowering of PTH levels was observed with cinacalcet treatment, no changes in BMD occurred at any of the sites measured.98 In smaller studies of patients with hyperparathyroidism who were treated with cinacalcet, significant decreases in circulating levels of PTH and serum levels of Ca2+ were also observed but, again, significant increases in BMD were not noted.99 In contrast to these studies with cinacalcet, significant improvements in BMD at the spine and hip were reported 1 year after lowering of circulating levels of PTH by parathyroidectomy, irre spective of whether surgery was performed for hyper calcaemic hyperparathyroidism or for the less severe normocalcaemic hyperparathyroidism.100–107 The increase in levels of bone markers and failure to increase BMD following cinacalcet treatment might, therefore, be consistent with a direct effect of cinacalcet on CaSR in osseous cells that increases bone turn over and leads to bone loss and mitigates the effects of decreased circulating levels of PTH. These effects are consistent with results from studies exploring the skeletal

function of CaSR in animal models61 (in which CaSR might enhance PTH-induced bone turnover) and with the association between a CASR polymorphism (which results in an Ala986Ser variant) and vertebral fractures in patients with primary hyperparathyroidism.108

Chronic kidney disease Cinacalcet has been shown to reduce PTH levels in patients with chronic kidney disease109 and to reduce the risk of requiring surgical parathyroidectomy. 110 Several small studies in patients on haemodialysis have reported that cinacalcet treatment decreased serum levels of PTH and also decreased levels of bone turn over markers;111–114 a few of these patients developed adynamic bone.113 In a larger study, cinacalcet reduced levels of PTH by ~50% and decreased several histo morphometric indices of bone turnover, although gen erally by smaller amounts.115 In most of these studies, however, vitamin D and/or phosphate binders were used concurrently and these combinations might have confounded interpretation of the effects of cinacalcet on bone. Consequently, the potential of cinacalcet to improve skeletal abnormalities that result from hyper parathyroidism in patients with chronic kidney disease remains to be determined. Osteoporosis Results have been reported for two calcilytics that have progressed to phase II studies in postmenopau sal women with osteoporosis (BMD T score ≤2.5).116,117 Short-term antagonism of CaSR in the parathyroid gland results in a transient release of endogenous PTH and it has been proposed that this transient release could mimic the effects of intermittent administration of exo genous PTH—a powerful osteoanabolic agent. However, the results of studies in humans have not been positive. Ronacaleret preferentially increased trabecular vBMD, which was offset by small decreases in BMD at cortical sites.116 The failure of this treatment was interpreted as being consistent with the initiation of mild ronacaleretinduced hyperparathyroidism, which results from longterm (6–8 h) increases in plasma levels of secreted PTH, which have a catabolic effect.116 However, the duration of having increased plasma levels of PTH induced by ronacaleret is comparable to that elicited by exogenously administered PTH and the magnitude of the PTH levels achieved with exogenous subcutaneous PTH adminis tration is even higher, yet exogenous PTH is profoundly anabolic.118 Following administration of MK‑5442 (also known as JTT‑305 and encaleret) to postmenopausal women with osteoporosis, a dose-dependent increase in levels of PTH occurred that lasted >3.5 h.117 Small but significant increases in levels of markers of bone for mation were observed after 6 months. Although levels of bone resorption markers initially decreased, they were also significantly increased by 6 months. BMD did not increase at either trabecular or cortical sites in bone. The failure of this treatment was attributed to the development of mild hyperparathyroidism and, consequently, further development of both ronacaleret



REVIEWS and MK‑5442 as potential treatments for osteoporosis was discontinued. Calcilytics are tissue-nonselective and, hence, probably also act as antagonists of CaSR in skeletal cells. On the basis of results from animal studies, calcilytics possibly diminish the ability of transiently increased levels of PTH to stimulate bone formation, either by directly inhibit ing osteoblast activity and/or by reducing the efficacy of endogenously released PTH.

Conclusions

If activation of CaSR in osteoblasts enhances the resorp tive effect of PTH by increasing osteoclast activity via increasing the RANKL:osteoprotegerin ratio, then the residual PTH that is secreted after calcimimetic admin istration would have a disproportionate effect. Therefore, it might be beneficial to antagonize CaSR in osteoblasts. Ideal CaSR modulators for hyperparathyroid states might therefore be those that not only stimulate CaSR in the parathyroid gland (thus reducing PTH secretion) but also antagonize CaSR in osteoblasts, thus impeding the 1.

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accelerated bone turnover (including that induced by PTH) that leads to bone loss. By contrast, ideal CaSR modulators for osteoporosis might be those that tran siently inhibit CaSR in the parathyroid gland (to promote transient release of PTH) but stimulate CaSR in osteo blasts, thus augmenting the PTH-induced anabolic effect on osteoblasts. Whether further studies on the compara tive mechanism of action of Ca2+ on CaSR in parathy roid cells as opposed to in osteoblasts can define discrete pathways amenable to the development of appropriately biased agonists remains to be determined. Review criteria A search for original articles focusing on calcium-sensing receptor and bone was performed in MEDLINE and PubMed using the following search terms: “calciumsensing receptor”, “bone”, “cartilage”, “osteoblast”, “osteoclast”, “calcimimetic” and “calcilytic”, alone or in combination. All articles were English language, full-text papers. The reference lists of identified articles were searched for additional relevant papers.

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