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Effects of Ca²⁺ sensing receptor activation in the growth plate¹

SHUFANG WU^*, TERESA PALESE, OM PRAKASH MISHRA, MARIA DELIVORIA-PAPADOPOULOS and FRANCESCO DE LUCA^*,2

^* Section of Endocrinology and Diabetes and
Neonatal Medicine, Department of Pediatrics, Drexel University College of Medicine, St. Christopher’s Hospital for Children, Philadelphia, Pennsylvania, USA; and
Division of Pediatric Endocrinology, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland, USA

²Correspondence: St. Christopher’s Hospital for Children, Erie Ave. at Front St., Philadelphia, PA 19134, USA. E-mail: francesco.deluca{at}drexel.edu

SPECIFIC AIMS

Regarding longitudinal bone growth, it has been assumed that calcium ion is important exclusively for cartilage and bone matrix mineralization. Based on previous evidence, we hypothesized that calcium ion, through the activation of Ca²⁺ sensing receptor (CaR) in the growth plate, regulates proliferation and hypertrophy/differentiation of growth plate chondrocytes and, in turn, the rate of longitudinal bone growth.

PRINCIPAL FINDINGS

1. CaR activation in the growth plate stimulates longitudinal bone growth
Fetal rat metatarsal bones (20 days postconception) were cultured for 7 days in serum-free medium containing 0, 0.1, 0.3, or 1 mM Ca²⁺ (n=14–23/group). Ca²⁺ induced a significant, concentration-dependent increase in metatarsal longitudinal growth (P<0.01 by ANOVA, Fig. 1 ). To verify that extracellular Ca²⁺ was acting through the CaR, we cultured metatarsal bones with NPS-R-568, a CaR agonist. In the presence of 10 nM NPS-R-568, bone rudiments grew more than control bones (139% of control, n=70–71/group, P<0.001, Fig. 1 ).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effects of extracellular Ca2+ and NPS-R-568 on longitudinal bone growth (mean±SE). Bone length was measured using an eyepiece micrometer in a dissecting microscope. A) Fetal rat metatarsals (dpc20, n=14-23/group) were cultured for 7 days in serum-free medium containing 0, 0.1, 0.3, or 1 mM calcium chloride. B) Fetal rat metatarsals (dpc20, n=70-71/group) were cultured for 7 days in serum-free medium containing 0 or 10 nM NPS-R-568.

2. CaR activation in the growth plate stimulates chondrocyte hypertrophy and chondrocyte hypertrophy/differentiation
Since the rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis, we evaluated the effects of CaR activation on chondrocyte hypertrophy/differentiation and chondrocyte proliferation, the two main components of growth plate chondrogenesis. To assess chondrocyte hypertrophy/differentiation, we examined the bone rudiments histologically. After 7 days in culture, treatment with NPS-R-568 increased the height of the growth plate hypertrophic zone (148% of control, n=21/group, P<0.001, Fig. 2 ). In the cells of the hypertrophic zone, NPS-R-568 also stimulated expression of collagen X, a marker of chondrocyte differentiation (assessed by immunohistochemistry). We next studied chondrocyte proliferation by measuring ³H-thymidine incorporation into bone rudiments at the end of the culture period (7 days). NPS-R-568 (10 nM) significantly increased ³H-thymidine incorporation into the metatarsal bones (118% of control, n=13/group, P<0.05, Fig. 2 ), suggesting an increased cell replication rate in the metatarsal bone. The proliferative zone height of the bones treated with NPS-R-568 was greater than that of control, which suggests that the increased cell replication rate led to an increased number of chondrocyte in the proliferative zone (Fig. 2) . NPS-R-568 did not significantly affect the height of the epiphyseal zone (Fig. 2) or the ossification center (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of NPS-R-568 on growth plate histology and total 3H-thymidine incorporation (mean±SE). A) Fetal rat metatarsals (dpc20, n=8–9/group) were cultured for 7 days in serum-free medium with or without NPS-R-568 (10 nM). After routine histological processing, bones were embedded in paraffin and 5 µm longitudinal sections were obtained. The height of the growth plate epiphyseal, proliferative, and hypertrophic zones was measured by a single observer blinded to the treatment regimen. *P <0.001 (vs. control). B) During the last 3 h of the 7 day culture period, 3H-thymidine was added to the culture medium. Bones (dpc20, n=13/group) were washed and solubilized, then total 3H-thymidine incorporation was measured by liquid scintillation counting. *P < 0.05 (vs. control).

3. CaR activation stimulates Ca² influx into growth plate chondrocytes
Since CaR activation in other cell types is known to result in increased Ca²⁺ influx from the extracellular to the intracellular compartment through the cell membrane, we measured ⁴⁵Ca-labeled influx into chondrocytes isolated from the rat metatarsals’ growth plates. At the end of the culture period, ⁴⁵Ca influx into cells derived from the NPS-R-568-treated metatarsals was threefold higher than ⁴⁵Ca influx measured in the control cells (P<0.05). This finding confirms the activation of CaR by calcimimetic NPS-R-568.

CONCLUSIONS

CaR is expressed in both the proliferative and hypertrophic zones of the growth plate, where longitudinal bone growth takes place. Yet it is not known whether CaR regulates growth plate chondrogenesis, the primary determinant of longitudinal bone growth. In cultured fetal rat metatarsal rudiments, which comprise two cartilaginous regions resembling the mature growth plate, Ca²⁺- and NPS-R-568-mediated activation of CaR caused a significant increase in chondrocyte proliferation as assessed by total ³H-thymidine incorporation and growth plate histology. The NPS-R-568-mediated activation of the CaR also led to the marked expansion of the growth plate hypertrophic zone as assessed by histological studies. In NPS-R-568-treated bone rudiments, the increased expression of collagen X, a marker for terminally differentiated chondrocytes, indicated that the large cells forming the expanded hypertrophic zone were differentiated chondrocytes. Cellular enlargement of the growth plate chondrocytes adjacent to the bone ossification center is critical for longitudinal bone growth. A widened hypertrophy zone can be due to an increased number of proliferating cells undergoing hypertrophy/differentiation or to a decreased number of hypertrophic cells being replaced by bone tissue. The lack of any NPS-568 effect on the size of the metatarsal ossification center renders the former explanation more likely. Earlier evidence suggests that increased intracellular calcium in growth plate chondrocytes is associated with increased cell size and increased expression of markers of chondrocyte differentiation. Our observation of an increased calcium influx in the cells isolated by NPS-R-568-treated metatarsals suggests that CaR activation stimulates growth plate hypertrophy and collagen X expression by increasing calcium influx and intracellular calcium in chondrocytes.

Recent studies suggest a functional role for CaR in skeletal growth. CaR expression has been shown by in situ hybridization and immunocytochemistry in rat growth plate proliferative and hypertrophic zones. CaR null mice exhibit growth retardation and an expanded growth plate. The increased height of the growth plate hypertrophic zone in these mice would suggest an inhibitory role for CaR on growth plate chondrogenesis, which is in conflict with our observations. On the other hand, the phenotype of these mice could be confounded by their severe hyperparathyroidism and the accompanying hypercalcemia and hypophosphatemia. The predominant rachitic changes seen in these animals indicate that the widening of the hypertrophic zone is secondary to the impaired mineralization of cartilage and bone tissue rather than to a primary defect of growth plate chondrogenesis.

It has been shown that extracellular Ca²⁺ regulates multiple chondrocyte functions. In chick embryo chondrocytes, elevated extracellular Ca²⁺ concentrations (5 or 10 mM) increased collagen X expression but did not affect cell proliferation. However, since the concentrations used in this study are significantly higher than that of serum-ionized Ca²⁺ measured in vivo, the findings observed in cultured chondrocytes may not reflect the physiological role of extracellular Ca²⁺ in the intact growth plate. In a chondrogenic cell line, Chang et al. showed that extracellular Ca²⁺ suppressed cartilage formation and the expression of collagens II and X. The discrepancy between these authors’ and our observations may be due to the different Ca²⁺ concentrations (1–4 mM vs. 1.8 mM, respectively) and different models (clonal cells derived from fetal calvaria vs. whole fetal long bone, respectively) used. Finally, the previous studies did not directly address the effects of CaR activation on longitudinal bone growth.

In conclusion, our findings indicate that the activation of CaR in the growth plate stimulates longitudinal bone growth by increasing chondrocyte proliferation and chondrocyte hypertrophy. These stimulatory effects on growth plate chondrogenesis may result from increased calcium influx through the cell membrane and a subsequent increase of intracellular calcium. Thus, our findings and those of others seem to suggest that CaR (and extracellular calcium, its main agonist) affects both components of longitudinal bone growth, chondrogenesis and ossification. Additional studies (i.e., targeted deletion of CaR gene expression in the growth plate) are needed to further define the physiological role of CaR in the skeletal system.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0294fje

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