New Insights in Bone Biology: Unmasking Skeletal Effects of the Extracellular Calcium–Sensing Receptor

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Science Signaling  02 Sep 2008:
Vol. 1, Issue 35, pp. pe40
DOI: 10.1126/scisignal.135pe40


Experiments performed in mice in which expression of the extracellular calcium–sensing receptor (CaSR) was completely nullified specifically in parathyroid cells, chondrocytes, or cells of the osteoblast lineage have identified phenotypes that indicate a key role for the CaSR in embryonic development of the skeleton, postnatal bone formation, and osteoblast differentiation that are independent of the calcitropic hormone axis. These long-awaited studies further clarify the signaling relationships between the parathyroid gland, kidney, and metabolic bone disease in patients with mutations in the gene encoding the CaSR, and they provide new insights into understanding the signaling pathways involving the CaSR in skeletal cells.

For over half a century, mechanisms contributing to the deposition and removal of bone mineral have been studied (1, 2). Calcification of cartilage is required for normal endochondral bone formation, the process of long bone growth whereby calcified cartilage is resorbed and replaced by bone tissue (3). Chondrocytes (cartilage-producing cells) in growing long bones undergo a maturation sequence and are organized in zones of proliferating prehypertrophic and hypertrophic cells in a mineralized matrix (designated the growth plate). Thus, calcium deposition at the growth plate is essential for normal limb development and bone growth. Likewise, calcification of bone matrix produced by osteoblasts is necessary for the differentiation of surface osteoblasts to osteocytes, the cells that constitute an interconnected cellular network within mineralized bone. The connective tissue function of bone requires the formation of the appropriate macro- and microarchitecture of bone, which is defined by the amount and organization of calcium phosphate (hydroxyapatite) crystals within the bone matrix. The mineral and matrix quality together contribute to the mechanical properties of bone needed to support our weight and locomotion with muscle (4). Bone also has the metabolic function of maintaining the concentrations of serum Ca2+ and phosphorus within a tight physiological range. This is accomplished by removal of bone mineral through acid-mediated solubilization of the mineral and breakdown of the bone proteins by bone-resorbing cells called osteoclasts.

The sensing of low concentrations of serum Ca2+ ([Ca2+]e) by extracellular calcium–sensing receptor (CaSR) in the chief cells of the parathyroid gland triggers the secretion of parathyroid hormone (PTH) and initiates a cascade of events that stimulates Ca2+ reabsorption by the kidney, enhances renal synthesis of 1,25(OH)2D3 (also called calcitriol, the active form of vitamin D3) so as to increase Ca2+ absorption across the gut, and promotes bone resorption by osteoclasts—all of which are required for normalizing Ca2+ homeostasis (Fig. 1). Thus, an exquisite integration of hormonal, cellular, molecular, and chemical events surrounding the deposition and movement from bone tissue of Ca2+ is established. In the article by Chang and colleagues (5), new insights into the regulation of Ca2+ homeostasis reveal yet another level of coordination between the parathyroid endocrine organ and bone, which is mediated by CaSR, a guanine nucleotide–binding protein (G protein)–coupled receptor.

Fig. 1

Classic Ca2+ homeostasis (dashed arrows) and novel developmental functions (arrows) of CaSR have been revealed by cell type–specific null mutations of Casr in the mouse. CaSR is found in bone, kidney, and gut, which are the three main Ca2+-mobilizing organs. The normal homeostatic signaling pathways between these organs and the parathyroid gland have been detailed previously (9). The functions performed by CaSR in each organ are outlined in boxes. To maintain normal Ca2+ homeostasis, CaSR in parathyroid cells (PTCs) senses alterations in [Ca2+]e. The release of parathyroid hormone (PTH) enables bone and kidney to respond in a manner to normalize [Ca2+]e, through the activation of key responses in kidney [production of 1,25(OH)2D3 and reabsorption of Ca2+], intestine [Ca2+ absorption through the increased abundance of 1,25(OH)2D3], and bone matrix resorption through PTH (not shown). The direct role of CaSR in the intestine is questionable because an intestine-specific knockout of Casr has not been performed. Targeted knockout of Casr through the crossing of Casr floxed mice with mice expressing Cre under the control of tissue-specific promoters has identified novel functions for CaSR in skeletal development. Ablation of Casr in the parathyroid gland resulted in the expected phenotypes that occur in patients with inactivating mutations in Casr, such as hyperparathyroidism and hypercalcemia. Deletion of Casr in chondrocytes demonstrated a requirement for CaSR in early skeletal development, whereas a role for CaSR in promoting bone cell differentiation was determined by deletion of Casr in cells of the osteoblast lineage.

With the discovery of CaSR in 1993 (6) came an exciting turning point in the study of Ca2+ homeostasis biology, and investigations were initiated to determine whether CaSR or other Ca2+ sensors might also contribute to the regulation of bone mineral deposition and resorption. Biochemical studies of bone tissue identified correlations between hydroxyapatite content and the accumulation of specialized noncollagenous Ca2+-binding proteins and phosphoproteins forming the bone extracellular matrix (ECM) (7). Such observations often raised questions as to the possible existence of a master regulator that provides the capacity to sense Ca2+ abundance in the bone milieu to drive bone formation, to inhibit bone resorption, or both, or whether this regulator simply monitors Ca2+ availability in bone microenvironments. CaSRs are broadly expressed in many tissues, and recent studies have identified that they can sense the broader nutritional status of cells by interacting with amino acids in a function related to that of sensing Ca2+ (8).

The discovery of inherited diseases of Ca2+ sensing that were due to mutations in the Casr gene contributed to proving the physiological importance of CaSR in Ca2+ homeostasis. Syndromes arising from either activating or inactivating mutations of Casr have been described, which reset CaSR-regulated [Ca2+]e downward or upward, respectively. Activating mutations cause autosomal dominant hypocalcemia (ADH), a condition of mild to moderate hypocalcemia that can be accompanied by hypercalciuria, particularly during treatment with Ca2+ and vitamin D, which therefore must be monitored carefully for toxicity. In contrast, loss-of-function mutations in Casr cause familial benign hypocalciuric hypercalcemia, a generally benign condition of mild to moderate hypercalcemia that usually does not require specific therapy, in individuals with one mutated allele and neonatal severe hyperparathyroidism (HPT) in homozygotes, which can be lethal in some cases and may require parathyroidectomy early in life to treat the marked hypercalcemia and hyperparathyroidism. These syndromes identified CaSR as the key regulator not only of PTH secretion, by stimulating PTH secretion when [Ca2+]e is low and inhibiting it when [Ca2+]e is high, but also of renal Ca2+ excretion, which decreases when [Ca2+]e is low and increases when [Ca2+]e is high.

The first mouse model in which the Casr gene was knocked out, which was generated in 1995, mimicked the inactivating mutations in Casr of the human genetic disorders. It was generally assumed that the mineralization defect present in the homozygous knockout animals (Casr−/−) was a result of severe HPT (9, 10). Because the bone defects could be rescued by preventing HPT and because CaSR was not identified in skeletal cells until 1999 (11), direct functions of CaSR in the skeleton were not immediately addressed. Not surprisingly, mouse phenotypes are not always what they seem to be, and pursuit of the molecular properties of Casr revealed an alternatively spliced product lacking exon 5 that was generated from the construct designed to disrupt Casr expression (12). This alternatively spliced product was identified in multiple tissues including skeleton, skin, and kidney (12, 13). Hence, it was then realized that this alternative receptor form could potentially provide partial or full functional compensation in some tissues of Casr−/− mice, although it has not been possible to express the alternate in a functional form in heterologous cell systems. One important aspect of the studies conducted by Chang et al. (5) is the generation of a mouse with a floxed Casr in which lox P sites flank exon 7 of Casr, which encodes the seven transmembrane domains and C-terminal tail of CaSR. From this mouse, a series of five conditional Casr knockout mouse strains were generated that lacked the alternative CaSR form (and so were complete knockouts), which were used to investigate tissue-specific functions of CaSR in cartilage and bone during embryonic development and in the postnatal skeleton.

The characterization of the phenotypes of these mice has made a substantial impact from several perspectives. One is the contribution of CaSR to survival and normal development of the growth plate. Targeted ablation of Casr in chondrocytes, through the expression of Cre recombinase under the control of the type II collagen (Col 2) promoter, produced lethality by embryonic day 13, whereas mice with chondrocyte-specific knockout of Casr at late embryonic stages, which was due to tamoxifen-inducible expression of Cre under the control of the Col 2 promoter, were viable and displayed delayed growth plate development. Mice with parathyroid cell (PTC)– or osteoblast-specific deletion of Casr exhibited profound bone defects. Casr floxed mice in which the expression of Cre was under the control of the 2.3-kb collagen type I promoter, which is expressed in early- and late-stage cells of the osteoblast lineage and also in other tissues in early embryos, exhibited severely undermineralized skeletons by postnatal day 3, which worsened until the mice died at 3 weeks. In Casr floxed mice in which Cre was under the control of the Osterix promoter, which is more specific to committed osteoblast lineage cells, a similar phenotype was found, although mouse viability was not compromised. Thus, from these studies, a key role has been established for CaSR in normal bone development.

Complete ablation of Casr in PTCs resulted in severe hypercalcemia and hyperparathyroidism, which was comparable to or more severe than that of the generalized Casr−/− mouse. As expected, mice with PTC-specific knockout of Casr were severely hypercalciuric, because the CaSR in the kidney normally triggers renal Ca2+ excretion in response to hypercalcemia. Curiously, the control mice for this model, which are homozygous for the floxed Casr allele but lack expression of Cre, were hypercalcemic. In addition, despite having a twofold increase in the abundance of full-length Casr mRNA, which might have been expected to result in lower serum Ca2+ concentrations by increasing the abundance of cell-surface CaSR protein, the heterozygous mice, which contained one floxed Casr allele and expressed Cre in the parathyroid gland, were more hypercalcemic than were the control mice. These data raise the question of whether the floxed Casr allele in some way modifies Casr expression, thus rendering the control mice hypercalcemic.

An intriguing finding of Chang and colleagues is that complete deletion of Casr in PTCs affected the bone. This interesting observation raises the question of why abundance of CaSR is decreased in bone when Casr is specifically knocked out only in PTCs. One could speculate that [Ca2+]e (that is, the hypercalcemic phenotype in the mouse) might regulate expression of Casr in bone by a feedback loop. Further, the authors showed by analyses of molecular markers of the bone phenotype that knockout of Casr in cells of the osteoblast lineage inhibited the production of insulin-like growth factor 1 (IGF-1), which is needed for osteoblast survival, and increased the production of the cytokine interleukin-10 (IL-10), which is a proapoptotic factor. Thus, mutations in Casr in patients could have far-reaching effects, and exploring the downstream pathways by which CaSR controls the expression of genes that regulate osteoblastogenesis could lead to novel therapies that expand the options for treating skeletal disorders. With the series of studies undertaken by Chang et al. (5), we can now think of CaSR in a different light in regulating bone development and potentially controlling turnover of the skeleton in the adult.


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