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Endocrine Research Unit Department of Medicine San Francisco Department of Veterans Affairs Medical Center University of California, San Francisco, California 94121
Address all correspondence and requests for reprints to: Dolores Shoback, 111N, Endocrine Research Unit, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: dolores{at}itsa.ucsf.edu
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Introduction |
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The careful morphologic and histomorphometric studies of the growth plate and skeleton in CaR knockout (-/-) mice by Garner et al. (1) suggest unanticipated functions for CaRs in cartilage and bone development. CaR -/- mice have profound hyperparathyroidism and hypercalcemia but do not show the expected hyperparathyroid bone changes. Instead, they exhibit classic features of rickets including growth retardation, expanded growth plates with reduced calcification, widened metaphyses, the "rachitic rosary" rib deformity, and impaired bone mineralization. Manifestations of the latter disturbance include hyperosteoidosis, a prolonged mineralization lag time, and delayed endochondral bone formation. These findings suggest that CaRs play a role, directly or indirectly, in the orderly mineralization of bone and growth plate cartilage.
Which bone cells express CaRs? In initial studies, House et al. (2) reported that CaR protein and transcripts were expressed in several cell types from bone marrow including a population that expresses the enzyme alkaline phosphatase, a marker of the osteoblast lineage. Subsequently, other investigators detected CaR RNA by Northern analysis and RT-PCR and CaR protein by immunoblotting and immunocytochemistry in several standard osteoblastic cell lines (3). Studies examining bovine and rat bone sections demonstrated that osteoblasts, osteocytes, and some osteoclasts express CaRs by in situ hybridization and immunocytochemistry (4). High extracellular Ca2+ and other divalent and polyvalent cations stimulated proliferation and chemotaxis in several of the osteoblastic cell lines (3). Interpreting these responses to Ca2+ in the context of bone remodeling, one might envision the following scenario—that extracellular Ca2+-sensing mechanisms in the membranes of osteoblasts enable the cells to home to sites of active bone resorption, where local Ca2+ levels can reach as high as 8–40 mM. There the osteoblasts are stimulated to divide and then participate in the repair of the resorption cavity, thus completing the cycle of bone remodeling.
Not all investigators, however, agree that osteoblasts express CaRs. While there is consensus that osteoblasts sense changes in the extracellular [Ca2+] and proliferate in response to high extracellular [Ca2+], two groups have failed to detect CaR transcripts or protein in osteoblasts or osteoblastic cell lines (5, 6, 7, 8). Furthermore, Pi et al. (8) reported that virally transformed osteoblasts from CaR -/- mice show proliferative and signaling responses to high extracellular [Ca2+], despite the absence of full-length CaRs. This experiment does not rule out an important role for Ca2+-sensing in osteoblastic function. These results may be ambiguous. By virtue of how it was engineered, the CaR -/- mouse can express CaR splice variants in certain tissues. For example, skin (9), kidney (9), and growth plate cartilage (manuscript submitted) from these mice express CaR transcripts with exon 5 spliced out. This exon was the site into which the neomycin cassette was inserted to create the knockout (10). Whether this alternatively spliced form of the CaR can sense changes in the extracellular [Ca2+] and mediate signal transduction has yet to be fully explored. What can be concluded from the osteoblast studies is that, regardless of the molecular mechanism(s) involved, extracellular Ca2+-sensing by osteoblasts is likely to modulate key steps in bone formation and repair.
Whether changes in the extracellular [Ca2+] act via CaRs to play a part in regulating bone formation and remodeling in vivo is not established. Infants with neonatal severe hyperparathyroidism (NSHPT), typically due to two mutant CaR alleles, have severe bone demineralization at birth but not the typical growth plate deformities of rickets. Once the hypercalcemia and hyperparathyroidism are cured by surgery, these children grow normally, and their bones heal. Their skeletal and growth plate cartilage abnormalities have generally been ascribed to severe hyperparathyroidism and not to rickets (11), although there is little in the way of histomorphometric data that address this point. Clearly, the loss of CaR function in the parathyroid glands and kidneys of children with NSHPT causes their systemic mineral disturbance. Some of the CaR mutants that cause NSHPT, however, retain some residual signaling capability. This may be sufficient, in tissues like bone and cartilage, to provide enough Ca2+-sensing once the hyperparathyroidism in these children is cured. Alternatively, other as yet unidentified Ca2+-sensing molecules in bone and cartilage may compensate for the lack of CaRs. The human model of the CaR "knockout," therefore, does not resolve all the issues related to Ca2+-sensing mechanisms and possible actions of CaRs in cartilage and bone.
The differences in bone and cartilage pathology between the CaR -/- mouse model and NSHPT may be more apparent than real. CaR -/- mice die within a few days of birth because of their severe metabolic disturbances. Their early death precludes a developmental assessment of the importance of Ca2+-sensing and CaRs in tissues beyond the early postnatal period. Fragmentary evidence, noted in the present report, suggests that the skeletal phenotype in CaR -/- mice may wane in animals that survive to 12 d of age (1). Thus, while CaRs in bone and cartilage appear to play a central role in mineralization before and immediately after birth, they may be less important in these tissues later in postnatal life. Perhaps there are other sensing-molecules that are expressed in the first several days after birth that compensate for the absence of full-length CaRs. At present, there is no information available to address this issue directly.
Our findings in rat and bovine bone demonstrate the presence of CaR transcripts and protein in osteoblasts by in situ hybridization and immunocytochemistry, respectively (12). It is surprising that Garner et al. (1) detected transcripts encoding the CaR only in bone marrow and growth plate cartilage and not in bone itself. Their negative results could be due to limitations in the sensitivity of RT-PCR to detect CaR expression in a subpopulation of bone cells. The impressive defects in mineralization and bone formation reported in CaR -/- mice support that this is an osteoblast phenotype and that osteoblastic function is crippled. Osteoblasts are the largest cell population in bone and are responsible for the complex cascade of events that lead to normal bone mineralization. A defect in matrix mineralization might logically be predicted to be the result of dysfunctional osteoblasts. In an animal model with severe demineralization, it is in fact difficult to envision an underlying pathogenesis that does not involve osteoblasts in a central way. Finally, it is remarkable that these animals maintain such marked systemic hypercalcemia while their bones are starved of calcium.
How could the targeted deletion of full-length CaRs bring about such a profound block in cartilage and bone mineralization? One possibility is that deleting the molecular mechanism by which chondrocytes and bone cells obtain accurate information about the availability of Ca2+ in the extracellular fluid for mineralization prevents even the initial steps of Ca2+ deposition into the matrix. In the growth plate, CaRs are strongly expressed in hypertrophic chondrocytes (4). Deleting CaRs from these cells may interfere with their ability to mineralize the growth plate and cause the expanded hypertrophic zone and epiphysis (1). A second possibility is that the combination of impaired Ca2+-sensing, due to the deletion of CaRs in a critical cell population, plus chronic and intense stimulation of chondrocytic and osteoblastic PTH receptors, due to florid hyperparathyroidism, produces a global disturbance of chondrocyte and osteoblast function that uncouples these cells from their normal program of mineralization. Indeed, several features of the bone histology in these animals indicate the influence of hyperparathyroidism as well as rickets. A third explanation involves possible alterations in matrix protein composition in the bones of CaR -/- mice. Perhaps the matrix proteins in these bones retard the initiation and propagation of hydroxyapatite crystals that form a mature mineralized extracellular matrix. Consistent with this final scenario, studies with cultured chondrogenic RCJ3.1C5.18 cells in our laboratory have shown that high extracellular [Ca2+] alter the expression of several critical cartilage matrix genes. Growth of these cells at high [Ca2+] suppressed the expression of the cartilage proteoglycan aggrecan, while expressing CaR antisense oligonucleotides enhanced aggrecan expression (12). In addition, the effects of high [Ca2+] on aggrecan expression are interrupted by expressing a dominant negative CaR mutant using an adenoviral gene transfer strategy (manuscript submitted). Aggrecan is known to block mineralization (13). We have further found that growth at high extracellular [Ca2+] enhances mineralization of the extracellular matrix by these cultured chondrogenic cells (our manuscript, submitted). These observations support a role for extracellular Ca2+ and CaRs in cartilage mineralization and provide an underlying mechanism to explain the growth plates defects seen in CaR -/- mice reported by Garner et al. (1). Whether extracellular Ca2+ and or CaRs have similar effects on matrix gene expression and mineralization in bone remain critical issues to resolve. Considerably more data will be necessary to address these possibilities in a definitive way to understand better the cartilage and bone phenotype caused by CaR deletion. Fortunately, mouse models can be developed in which CaRs are selectively knocked out or overexpressed in specific cartilage and bone cell populations to avoid the hypercalcemia and hyperparathyroidism that confound efforts in interpreting the phenotype of the currently available animal models.
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Acknowledgments |
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Footnotes |
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Abbreviations: CaRs, Calcium receptors; NSHPT, neonatal severe hyperparathyroidism.
Received July 6, 2001.
Accepted for publication July 9, 2001.
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