Cation Sensing Receptors in Bone: A Novel Paradigm for Regulating Bone Remodeling? (1997)

BONE UNDERGOES a remodeling process consisting of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. This regenerative process involves the coordinated activities of osteoclasts, which are of hematopoietic origin, and osteoblasts, which are derived from mesenchymal stem cells. These cells, respectively, remove and replace mineralized bone in a discrete microenvironment on bone surfaces. Hormones, local and matrix-derived growth factors and cytokines, as well as cell-to-cell interactions modulate osteoblast and osteoclast function and mediate cross-talk between osteoblasts and osteoclasts. A recent advance in our understanding of bone metabolism is the recognition that calcium released from bone may directly regulate bone cell function and participate in coordinating the activities of osteoblasts and osteoclasts in concert with calcitropic peptides. Several lines of evidence indicate that extracellular calcium in the osseous microenvironment regulates bone cell function. In vivo and in vitro studies demonstrate that calcium and other divalent and trivalent cations stimulate bone formation and inhibit bone resorption. For example, calcium added to bone organ cultures decreases bone dissolution.1 In addition, direct application of calcium and the divalent cation strontium to avelolar bone stimulates bone formation in rats.2 Both the dietary supplementation of strontium and its systemic administration increases vertebral trabecular bone volume and prevents bone loss in rat models.3,4 Similarly, the intravenous administration of the trivalent cation aluminum stimulates bone formation in excess of bone resorption and increases spinal bone density in dogs.5-7 Successful induction of bone formation also has been observed by direct injection of other trivalent cations such as gadolinium and yttrium onto calvaria (Juerg Gasser, personal communication). Thus, divalent and trivalent cations uncouple bone formation and bone resorption, leading to a net increase in bone mass under certain experimental conditions. Additional in vitro studies demonstrate that these divalent and trivalent cations have direct actions on osteoblast and osteoclasts. Isolated osteoclasts respond to increments in extracellular calcium by a reduction in bone resorptive activity through a voltage-insensitive calcium signaling pathway.8 A cation sensing mechanism is also present in osteoblasts. In this regard, osteoblasts demonstrate a proliferative and chemotactic response to high concentrations of extracellular calcium.9-12 Strontium also enhances osteoblast precursor replication in vitro.13 Moreover, the trivalent cations aluminum and gadolinium as well as organic polycations, such as neomycin, stimulate preosteoblast DNA synthesis and early response gene expression in vitro.9 The response of osteoblasts to extracellular calcium and other polycationic ligands appears to be mediated through the activation of G-protein coupled signaling cascades.9,14 Moreover, cations act synergistically with insulin-like growth factor (IGF)-I to stimulate DNA synthesis9 and parathyroid hormone (PTH) to stimulate cyclic adenosine monophosphate (cAMP) accumulation in cultured osteoblasts,15 indicating possible interactions with osseous growth factors. Calcium as well as aluminum also stimulate the autocrine production of IGF-I in osteoblasts in vitro.16,17 Together, these observations provide compelling but indirect evidence for the presence of cation sensing mechanisms in osteoblasts and osteoclasts. Finally, calcium, the physiologic ligand for these putative cation sensing receptors, is present in the local bone environment at concentrations sufficient to elicit a response in osteoclastic and osteoblastic precursors. Microelectrode studies indicate that calcium concentrations of between 8 and 40 mM exist within resorption lacunae.18 The identification of several calcium sensing receptors in many tissues and the recognition that sensing of extracellular calcium is a general paradigm whereby cells respond to changes in extracellular calcium concentrations further support the possibility that calcium modulates bone cell function through cell surface extracellular calcium sensing receptors. The molecular characterization of the putative bone calcium sensing receptors remains uncertain. To date, three distinct calcium sensing receptors have been identified that are possible candidates for bone calcium receptors: the calcium sensing receptor (CaR),19 a cell surface Type II ryanodine-like receptor,20 and a 500 kD protein belonging to the low density lipoprotein (LDL) receptor protein superfamily.21 This lipoprotein-like calcium receptor has been found in parathyroid cells, proximal renal tubule cells, and cytotrophoblasts.20,22 Its physiologic function remains unknown and currently there are no data showing that the LDL-like receptor is expressed in bone cells. An extracellular membrane ryanodine-like receptor is the leading candidate for the osteoclast calcium receptor.20 In this issue of JBMR, House et al. suggest that CaR may be present in bone cell precursors.23 CaR is the prototypic sensor for extracellular calcium that was originally isolated from bovine parathyroid glands and subsequently also identified in the kidney, brain, and keratenocytes, as well as detected in low abundance in other tissues such as thyroid C-cells, pituitary, ovary, pancreas, small intestine, spinal cord, lung, and retina.19,24-26 CaR encodes a 120 kD seven transmembrane domain G protein–coupled receptor belonging to Group II of the metabotropic glutamate (mGluR) receptor family (http://receptor.mgh.harvard.edu). Trivalent and divalent cations as well as the polycationic antibiotics activate CaR (with apparent EC50s: Gd3+ [20 μM] > neomycin [60 μM] > Ca2+ [3 mM] > Mg2+ [10 mM]).19 In addition, chiral amine derivatives (calcimimetics) specifically interact with distinct sites in the CaR receptor and facilitate receptor activation by cations.25 CaR activation inhibits cAMP accumulation, and stimulates inositol phosphate turnover and intracellular calcium in parathyroid cells. At present, the known physiologic role of CaR is to transduce the effects of extracellular calcium on PTH secretion in the parathyroid gland. Its role in regulating calcium homeostasis has been confirmed by the discovery that inactivating mutations in the CaR gene causes familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism27 and by the demonstration that targeted ablation of CaR in mice results in hypercalcemia and elevated PTH levels.28 The findings of House et al. extend the function of CaR to the possible regulation of cells within the hematopoietic and mesenchymal lineages in bone marrow.23 Using reverse-transcribed polymerase chain reaction (RT-PCR), in situ and Northern hybridization and immunohistochemistry, these investigators unequivocally demonstrate the presence of CaR in a mixture of low density unfractionated mononuclear cells isolated from whole human bone marrow. It is unclear, however, which of the multitude of marrow cell constituents express CaR. In particular, it remains uncertain whether osteoblast precursors express this calcium sensing receptor. The data localizing CaR to specific cell types is based on experiments in which anitsera to regions of CaR that share homology to metabotropic glutamate receptors are employed. The significance of CaR immunoreactivity in cells within the macrophage/monocyte lineage and alkaline phosphatase (ALP) expressing mononuclear cells is open to alternative interpretations. It is possible the CaR antibody recognizes ALP positive cells that are not preosteoblasts or cross-reacts with a related but functionally distinct receptor. These observations will require further experimentation in order to independently confirm them. Allowing for the possible lack of immunohistochemical specificity, CaR immunoreactivity is present in both ALP and esterase positive cells within the bone marrow that include bone cells precursors, erythroid precursors, and megakaryocytes. House et al.23 did not attempt to identify CaR in purified populations of authenticated osteoblast precursors which can be isolated from bone by a variety of selection techniques. Osteoblast precursors make up a small percentage of marrow cells, and ALP is not sufficient to identify osteoblast precursors. Though these findings suggest that CaR may regulate bone cell function, additional data indicate that the bone cell receptor is functionally similar but molecularly distinct from the known CaR (vide infra). These questions not withstanding, the presence of CaR in bone marrow cells adds additional information regarding the possible role of calcium sensing in bone, particularly cells of hematopoetic origin. Ryanodine receptors are a family of microsomal proteins that regulate release of calcium from intracellular stores. Recent functional studies and immunohistochemistry analysis have identified an isoform of the microsomal ryanodine receptor that is expressed on the surface of osteoclasts.8,20,29 The finding of a calcium receptor that is structurally distinct from CaR in osteoclasts is consistent with the functional differences that exist in the osteoclast response to cations. The osteoclast cation sensing receptor has a lower apparent affinity for Ca2+ (15 mM) and other cations (e.g., Ba2+ [20 mM] and Mg2+ [12.5 mM]) and also displays differences in cation specificities compared with CaR (i.e., the CaR agonist neomycin does not activate the osteoclast receptor, whereas nickel activates osteoclasts but not CaR). Finally, CaR calcimimetic compounds are ineffective in altering osteoclast function, and mRNA for CaR has not been identified in rabbit osteoclast or human osteoclastoma cells.25 Antisense strategies and/or targeted disruption of the ryanodine receptor have not been reported. Thus, the functional role of this receptor in mediating cation responses in osteoclasts remains to be established. The identification of CaR in monocytes/macrophage lineages by immunohistochemistry by House et al.23 also raises the possibility that the CaR or related receptor might be present in hematopoietic lineages giving rise to osteoclasts. However, other studies have failed to identify the CaR in isolated mononuclear cells that release IL–6 in response to increments of extracellular calcium.30 Further studies will be needed to determine the molecular structure and function of the putative calcium receptor(s) in osteoclast precursors. Although the present studies identified CaR-like receptor by immunohistochemistry in ALP positive cells in the marrow, there is compelling evidence that the functionally important cation receptor in osteoblasts is distinct from CaR. In this regard, clinical disorders caused by an inactivating mutation of CaR do not appear to have a bone remodeling abnormality or an osseous phenotype independent of that caused by alterations in PTH.27 In addition, no bone phenotype was reported in the CaR knockout mouse.28 Moreover, other studies have failed to show CaR expression in osteoblasts or osteoblast precursors. CaR is not present in MC3T3-E1 preosteoblasts that display a cation sensing response in vitro.9 The presence of CaR in ALP positive bone marrow cells, though suggestive, is not sufficient to establish that osteoblast precursors express CaR. Confirmation that CaR is present in true osteoblastic precursors or cells within the osteoblast lineage awaits the study of isolated homogenous populations of early stomal cells.31,32 Indeed, additional data suggest that the osteoblast cation sensing receptor is functionally distinct from the known CaR. For example, the cation specificity for the putative osteoblast cation sensing receptor differs from that of CaR.9,14 Aluminum, which stimulates the putative osteoblastic cation receptor, does not stimulate CaR transfected into HEK cells under the conditions studied and specific phenylalkylamines, which are selective pharmacological enhancers of CaR function, fail to modulate cation stimulatory responses in osteoblasts (unpublished observation). Finally, magnesium, which is an agonist for CaR, does not stimulate DNA synthesis or chemotaxis in osteoblast cultures.9 The signal transduction cascades that are coupled to receptor activation also appear to be different in the osteoblast cation sensing receptor.9,11 Though cations appear to activate Gαi and Gαq in osteoblasts, unlike CaR in other cells, PI-PLC dependent activation of inositol phosphate (IP3) and intracellular calcium release does not occur in osteoblasts in response to cation stimulation.9,14,15 PI3-kinase is not involved in extracellular calcium stimulation of osteoblasts.11 Protein kinase C (PKC)-dependent pathways, however, appear to mediate the replicative response to cations in osteoblasts, which suggest activation of phosphatidylcholine-phospholipase C (PC-PLC)– or phospholipase D (PLD)-dependent pathways.14,15 Thus, the osteoblast calcium receptor has many features that discriminate it from known CaR. Recently, a sequence homologous but distinct from CaR has been identified in MC3T3-E1 osteoblasts.33 Whether this putative G protein–coupled receptor-like sequence is the osteoblast cation receptor remains to be established. In conclusion, studies investigating the molecular mechanism whereby bone cells respond to extracellular cations have led to the hypothesis that calcium released by osteoclast-mediated bone resorption results in feedback inhibition of osteoclast activity and stimulation of proliferation and recruitment of osteoblastic precursors. Although this concept has not been proven, there is compelling evidence for distinct extracellular calcium sensing mechanisms in osteoblasts and osteoclasts that regulate bone metabolism. These putative osteoblast and osteoclast cation sensing receptors are likely involved in the physiologic regulation of coupled bone formation and may also be targets for the pharmacologic actions of possible novel agonists capable of stimulating de novo bone formation and suppressing osteoclast-mediated bone resorption. Further studies will be needed to confirm the molecular identity of the putative cation receptor(s) in bone marrow cells, confirm cell-specific expression, and determine their functional role in regulating bone remodeling. This work was supported by grants RO1-AR37308 and RO1-AR43468 from the National Institutes of Health, National Institutes of Arthritis and Musculoskeletal and Skin Diseases. We thank Jane Hollingsworth for secretarial assistance in preparation of this manuscript.