Genetic analysis of serum osteocalcin and bone mineral in multigenerational Afro-Caribbean families.

UNLABELLED: Osteocalcin is a major component of bone matrix. Concentrations of total, carboxylated, and uncarboxylated osteocalcin, are highly heritable and genetically correlated with bone mineral content (BMC) within African ancestry families. INTRODUCTION: Osteocalcin (OC) is a protein constituent of bone matrix and a marker of bone formation. We characterized the heritability of serum OC measures and identified genomic regions potentially involved in the regulation of OC via high-density genome-wide linkage analysis in African ancestry individuals. METHODS: African ancestry individuals (n = 459) were recruited, without regard to health status, from seven probands (mean family size = 66; 4,373 relative pairs). Residual heritability of serum OC measures was estimated and multipoint quantitative trait linkage analysis was performed using pedigree-based maximum likelihood methods. RESULTS: Residual heritabilities of total OC, uncarboxylated OC, carboxylated OC and percent uncarboxylated OC were 0.74 ± 0.10, 0.89 ± 0.08, 0.46 ± 0.10 and 0.41 ± 0.09, respectively. All OC measures were genetically correlated with whole body BMC. We obtained strong evidence of bivariate linkage for percent uncarboxylated OC and whole body BMC on chromosome 17 (logarithm of the odds [LOD] = 3.15, 99 cM). CONCLUSIONS: All forms of OC were highly heritable and genetically correlated with total body BMC in these African ancestry families. The identified linkage region contains several candidate genes for bone and energy metabolism including COL1A1 and TNFRSF11A. Further studies of this genomic region may reveal novel insight into the genetic regulation of OC and bone mineralization.

The (1)H NMR structure of bovine Pb(2+)-osteocalcin and implications for lead toxicity.

Structural information on the effect of Pb(2+) on proteins under physiologically relevant conditions is largely unknown. We have previously shown that low levels of lead increased the amount of osteocalcin bound to hydroxyapatite (BBA 1535:153). This suggested that lead induced a more compact structure in the protein. We have determined the 3D structure of Pb(2+)-osteocalcin (49 amino acids), a bone protein from a target tissue, using (1)H 2D NMR techniques. Lead, at a stoichiometry of only 1:1, induced a similar fold in the protein as that induced by Ca(2+) at a stoichiometry of 3:1. The structure consisted of an unstructured N-terminus and an ordered C-terminal consisting of a hydrophobic core (residues 16-49). The genetic algorithm-molecular dynamics simulation predicted the lead ion was coordinated by the Gla 24 and Gla 21 residues. It is proposed that mineral binding occurs via uncoordinated Gla oxygen ions binding to calcium in hydroxyapatite. A comparison of Pb(2+)- and Ca(2+)-osteocalcin suggests Pb(2+), at a lower stoichiometry, may induce similar conformational changes in proteins and subsequent molecular processes normally controlled by calcium alone. This may contribute to a molecular mechanism of lead toxicity for calcium binding proteins. Lead exposure may alter the amount of mineral bound osteocalcin and contribute to abnormal bone remodeling.

Molecular basis and clinical application of biological markers of bone turnover.

An ideal battery of tests would include indices of bone resorption and formation. They should be unique to bone, reflect total skeletal activity, and should correlate with traditional measures of bone remodeling activity, such as radiocalcium kinetics, histomorphometry, or changes in bone mass. Factors that confound their measurement, such as circadian rhythms, diet, age, sex, bone mass, liver function, and kidney clearance rates, should be clearly defined (Fig. 9). To date, no bone marker has been established to meet all these criteria, and each marker may have its own specific advantages and limitations. There are still questions that must be answered before there can be complete confidence in the information gained from measurement of any of the bone markers. Furthermore, it should be emphasized that none of the markers are diagnostic for any particular bone disease and cannot be used for this purpose in individual patients. Nevertheless, recent advances in research and development have provided assays with increased specificity, sensitivity, and availability. Because of this, bone markers can be used for a variety of important purposes: as tools for basic bone biology research, for defining general physiological phenomenon in clinical studies or drug trials, and for following individual patients.

Multiple immunoreactive forms of osteocalcin in uremic serum.

Circulating osteocalcin, which normally reflects the rate of bone formation, is elevated in uremia. In 18 patients receiving maintenance hemodialysis, serum osteocalcin levels were directly related to the bone formation rate (r = 0.88, P less than 0.001), osteoblastic osteoid surface density (r = 0.65, P less than 0.01), and osteoclastic resorptive surface density (r = 0.75, P less than 0.001). Multiple regression analysis showed that osteocalcin levels remained positively correlated with osteoclastic resorption when the bone formation rate was held constant (P less than 0.01). The intimation that the coupling of bone formation and resorption could not explain the relationship between osteocalcin and resorption led us to determine whether fragments of this abundant matrix protein are released by bone resorption and retained in uremia. Sera from dialysis patients with renal osteodystrophy were fractionated by sequential gel filtration and HPLC, and assayed for immunoreactive osteocalcin. When normal serum was analyzed, a single sharp peak was found. In pooled sera from patients with high osteoclastic resorptive surfaces identified by histomorphometry, we found five additional immunoreactive peaks, while three additional peaks were detected in sera from patients with lower osteoclastic surfaces. Bio-Gel P-10 chromatography showed that these multiple peaks were of lower molecular weight than intact osteocalcin. We suggest that the liberation of bone matrix by osteoclasts contributes to the circulating osteocalcin immunoreactivity in uremia.

The displacement of calcium from osteocalcin at submicromolar concentrations of free lead.

Lead, an environmental toxin, is known to impair some of the functional properties of osteocalcin, a small protein (MW, 5700) active in bone mineralization and resorption. To investigate a possible mechanism of lead toxicity at the molecular level, we have studied the interaction of lead with osteocalcin using 43Ca and 1H NMR. The measured 43Ca NMR linewidth as well as longitudinal relaxation rate (1/T1) of 43CaCl2 progressively increased with increasing amounts of added osteocalcin. A titration measuring 43Ca linewidth as a function of [Ca2+]/[Osteocalcin] ratio could be fitted to a single metal binding site with a dissociation constant of 7 microM. The 43Ca 1/T1 of Ca-osteocalcin decreased in the presence of Pb2+ due to competitive displacement of Ca2+ by Pb2+. The magnitude of decrease in the effect of osteocalcin on 43Ca 1/T1 in the presence of Pb2+ was consistent with the existence of only one tight divalent cation binding site. An analysis of the NMR T1 data in osteocalcin solutions containing both Pb2+ and Ca2+ yielded a Pb-osteocalcin dissociation constant of about 2 nM. The 1H NMR spectra showed Pb-induced changes in the same aliphatic and aromatic resonances of osteocalcin that are also affected by Ca(2+)-binding, supporting interaction of Pb2+ at the Ca2+ site. However, the existence of significant differences between the Pb-osteocalcin and Ca-osteocalcin NMR spectra indicates some differences in the structures of the two complexes. Since Pb2+ inhibits the binding of osteocalcin to hydroxyapatite, the high affinity of Pb2+ for osteocalcin would indicate significant inactivation of osteocalcin even at submicromolar free lead levels. Pb(2+)-induced inactivation of osteocalcin could affect bone mineral dynamics and may be related to the observed inverse correlation between blood Pb(2+)-levels and stature and chest circumference observed in growing children.

The effect of Pb(2+) on the structure and hydroxyapatite binding properties of osteocalcin.

Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb(2+) at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb(2+) on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb(2+) induces a similar structure in osteocalcin as Ca(2+) but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb(2+) to osteocalcin (K(d)=0.085 microM) than Ca(2+) (K(d)=1.25 mM). The hydroxyapatite binding assays show that Pb(2+) causes an increased adsorption to hydroxyapatite, similar to Ca(2+), but at 2-3 orders of magnitude lower concentration. Low Pb(2+) levels (1 microM) in addition to physiological Ca(2+) levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca(2+) alone. These results suggest a molecular mechanism of Pb(2+) toxicity where low Pb(2+) levels can inappropriately perturb Ca(2+) regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb(2+)-intoxicated animals.

Loss of the transcription factor p45 NF-E2 results in a developmental arrest of megakaryocyte differentiation and the onset of a high bone mass phenotype.

NF-E2 is a transcription factor required for megakaryocyte differentiation. The phenotype of mice deficient in p45 NF-E2 has been characterized by increased numbers of immature megakaryocytes and the absence of functional platelets. These mice also exhibited a high bone mass phenotype with up to a 6-fold increase in trabecular bone volume and a 3- to 5-fold increase in the bone formation rate. Our data indicated that both osteoblast and osteoclast numbers were increased in vivo with a 4- to 10-fold increase in osteoblast number/tissue area and approximately a 5-fold increase in osteoclast number/tissue area. Serum osteocalcin levels were also increased in NF-E2-deficient mice, corroborating the histomorphometric data and confirming that the osteoblasts were functional. Urinary cross-links levels were measured to confirm osteoclast activity. Interestingly, the increased bone was observed only in bony sites of hematopoiesis, and was not seen in flat bones such as calvariae. We showed that cells of the osteoblast lineage do not express NF-E2 mRNA. The increased bone phenotype was adoptively transferred into irradiated wild-type mice using spleen cells from NF-E2-deficient mice. These observations suggest that a megakaryocyte-osteoblast interaction occurs which is anabolic for bone.

The osteocalcin propeptide is not secreted in vivo or in vitro.

Osteocalcin is initially synthesized as an 11 kD molecule consisting of a 23-residue translocation signal peptide that is cleaved during translation, a 26-residue propeptide that targets the protein for gamma-carboxylation, and the 49-residue mature protein. Although the majority of newly synthesized osteocalcin is deposited into bone matrix, a small amount can be detected in blood, and it is this characteristic that has led to its current clinical use as a specific index of osteoblastic activity. Nothing is known, however, about the fate of the propeptide. If osteocalcin and the propeptide are cosecreted, then the concentration of the propeptide could also be useful as a marker of osteoblastic function and, further, may be superior to osteocalcin because it would be unaffected by binding to bone. To test this hypothesis, we synthesized a peptide corresponding to 21 residues of the osteocalcin propeptide from humans and produced a polyclonal antibody to this peptide. Human sera were screened for the presence of the propeptide, and the human osteosarcoma cell line MG-63 was tested for secretion of the propeptide. We could not detect any osteocalcin propeptide in sera from normal adults or individuals with renal failure or primary hyperparathyroidism or those on long-term coumadin therapy. Likewise there was no propeptide present in media from cells grown in the presence of vitamin K, 1,25-(OH)2D3, warfarin, or warfarin plus 1,25-(OH)2D3. In contrast, the cell extract, characterized by high-performance liquid chromatography, contained mature osteocalcin, free propeptide, and the proosteocalcin precursor when cells were grown in the presence of 1,25-(OH)2D3 alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone status of senescent male rats: chemical, morphometric, and mechanical analysis.

The bone status of male rats 6, 12, and 24 months of age (n = 10) was examined. Femur calcium (Ca), phosphorus (P), and osteocalcin contents; serum chemistry; and mechanical properties of the bone were measured and correlated. Diaphyseal Ca, P, and osteocalcin contents were not different in animals 6 and 12 months of age but decreased significantly at 24 months: -7.4% for Ca, -4.2% for P, and -24% for osteocalcin compared to 12 months. Femurs from 24-month-old (senescent) rats were characterized by a scalloped appearance of the midfemoral endosteal surface and by cortical porosities. These age-associated changes coincided with nearly two-fold increases in serum immunoreactive parathyroid hormone (PTH) and osteocalcin. Serum Ca did not change with age, whereas serum P decreased (-14.8%) from 6 to 24 months. Maximum breaking force required to fracture femurs at midshaft did not change with age. Hence, the strength of the femur as an intact organ was not compromised with age despite the loss of diaphyseal Ca and P in the senescent animal. However, ultimate stress, a parameter that normalizes for differences in bone geometry and size, decreased 35% in femurs from 12- compared with 24-month-old animals. These mechanical results might be explained by the morphometric finding that, in contrast to the small but progressive age-associated increases in femur weight and length, the cortical and medullary areas increased at least two-fold. Therefore, the strength of the intact femur was maintained by architectural compensations, even though normalized tissue strength decreased with age. These findings suggest that bone status was compromised in the aged male rat.

Bone status of senescent female rats: chemical, morphometric, and biomechanical analyses.

The bone status of female rats, 6, 12, and 24 months of age was examined. Femur Ca, Pi, and osteocalcin contents, as well as biomechanical properties, were measured and correlated to physical indices and serum chemistry. Diaphyseal Ca, Pi, and osteocalcin did not change significantly with increasing age. Serum Ca and Pi concentrations were not altered in the aged rat. Immunoreactive parathyroid hormone (PTH) levels increased significantly with age, when analyzed by linear regression. Serum osteocalcin decreased progressively from 6 to 12 months (-21%) and from 12 to 24 months (-23%). Maximum breaking force required to fracture femurs at midshaft did not change with senescence. Hence, the strength of the femurs as an intact organ was not compromised in aging. However, ultimate stress, a parameter that normalizes for differences in bone geometry and size, decreased 14% from 12 to 24 months. Changes in other biomechanical parameters, including yield and ultimate deformation, strain, and modulus of elasticity, were relatively small, but statistically significant, or were negligible. Morphometric measurements indicated a progressive age-related increase in second moment of area and cortical area. Medullary area did not change with age. Therefore, strength of the intact femur was maintained by architectural compensations, although normalized tissue strength decreased in senescence. The bone status and Ca/Pi homeostasis of the female rat were compared to similar findings, reported previously, for the male animal. The results suggest that bone status and mineral metabolism were compromised in the aged female rat, but the magnitude of change was less than that found for the senescent male rat.

Diet- or warfarin-induced vitamin K insufficiency elevates circulating undercarboxylated osteocalcin without altering skeletal status in growing female rats.

To further characterize the skeletal role of vitamin K (K), markers of bone turnover, density, and strength were evaluated in rats with diet- or warfarin (W)-induced K insufficiency. One hundred two, 7-week-old, female rats were randomly assigned to low K (phylloquinone [K1], 20 microg/kg diet), control K (K1, 1300 microg/kg diet), low-dose W (W, 1.5 mg/kg control diet), or high-dose W plus K (W/K1, 10/100 mg/kg diet). Femur bone mineral content (BMC) and bone mineral density (BMD), plasma prothrombin time (PT) and prothrombin concentration (PC), and serum total alkaline phosphatase (ALP) and skeletal alkaline phosphatase (sALP) were measured at baseline and days 20, 40, 60, and 80. Serum total osteocalcin (OC) and undercarboxylated osteocalcin (ucOC) and femur length (FL) were measured at baseline and day 80. Left femur OC was measured and biomechanical testing of the right femur and third lumbar vertebral body was performed at day 80. Low dietary K elevated circulating ucOC (17% higher than control; p < 0.0001) at day 80. Furthermore, in both W groups, essentially all circulating OC was undercarboxylated and femur OC was lower than control (p < 0.0001). However, there was no change in femur percent ucOC, suggesting deposition of less newly synthesized OC. No between group differences were observed in PT, ALP, sALP, FL, BMC, BMD, or bone strength. In conclusion, skeletal K insufficiency can be induced by W or diet manipulation. This does not hinder peak bone mass attainment in female rats; however, W causes less newly synthesized OC to be deposited in bone.

A comparison of the effects of parathyroid hormone and parathyroid hormone-related protein on osteocalcin in the rat.

We compared the effects of parathyroid hormone (PTH1-34) and parathyroid hormone-related protein (PTHrp1-34) on osteocalcin release in the isolated rat hindlimb and in intact and thyroparathyroidectomized (TPTX) rats. PTH1-34 suppressed osteocalcin release from perfused rat hindquarters, while PTHrp1-34 had no effect on osteocalcin release, despite comparable stimulation of cAMP production. Similarly, serum osteocalcin declined in the intact and TPTX animals by 5 h after a single dose of PTH1-34, while there was no response to PTHrp1-34. Chronic administration of PTH1-34 or PTHrp1-34 produced comparable hypercalcemia and hypophosphatemia in sham-operated and TPTX animals. Chronic PTH1-34 administration produced significant increases in serum osteocalcin both in the sham-operated rats and in the TPTX animals. However, in animals chronically treated with PTHrp1-34, there was no change at any time point in osteocalcin in either sham-operated or TPTX rats. These differences could be inherent or merely due to potency differences between the two peptides.

Biochemical parameters associated with low bone density in healthy men and women.

A causal role in age-related bone loss has been attributed to alterations in vitamin D status, the bone mineral regulating hormones, and/or renal function. We assessed biochemical parameters of bone metabolism and renal function in healthy subsets of young and old men (n = 191) and women (n = 120) and evaluated the relationships between these parameters and bone mineral density (BMD) in the radius, spine, and femur. There were no significant associations between BMD at any site and serum 25-OHD, 1,25-(OH)2D, PTH, or creatinine clearance in either young men or in young or old women, after controlling for age. In old men, however, lower radius BMD was significantly related to higher PTH and higher 1,25-(OH)2D and marginally related to lower 25-OHD values. In young men, there were unexpected but significant associations between lower femoral neck BMD and higher serum osteocalcin and urinary calcium/creatinine excretion after age adjustment. In old women, lower spine and radius BMD was also significantly correlated with higher serum osteocalcin. In this healthy, vitamin D-replete population, there were significant cross-sectional declines in BMD in the femur in young and old men and at all sites in old women. Elevated remodeling may be an important feature that contributes to reduced femoral BMD in young men and reduced spine and radius BMD in old women. However, compromised renal function or levels of 1,25-(OH)2D or elevated PTH appear to be neither necessary nor relevant as determinants of osteopenia in the spine or femur in these normal, healthy men and women.

Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes.

A line of transgenic mice expressing a human osteocalcin genomic fragment (hOClocus) and a murine MC3T3-E1 cell line containing a stably integrated human osteocalcin promoter construct have been developed to characterize the osteogenic and hormonal regulation of human osteocalcin in vivo and in vitro. In this study, we used these models to demonstrate a species difference in the regulation of the mouse and human osteocalcin genes by vitamin D. Repeated administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to mice carrying the hOClocus transgene resulted in striking increases in serum human osteocalcin, whereas serum mouse osteocalcin levels were unchanged after 24 h and only modestly increased 48 h after the second dose of hormone. 1,25(OH)2D3 increased human calvarial mRNA expression by 1.8-fold and slightly decreased mouse osteocalcin mRNA levels by approximately 1.2-fold. Furthermore, treatment of primary calvarial osteoblasts from these mice with 1,25(OH)2D3 increased human osteocalcin production but inhibited mouse osteocalcin protein accumulation. To investigate further the mechanism for the apparent species difference in vitamin D3 induction of mouse and human osteocalcin, we examined the effect of 1,25(OH)2D3 in an MC3T3-E1 cell line (MC4) containing a stably integrated 3900 bp osteocalcin promoter-luciferase construct. Treatment of MC4 cells with ascorbic acid resulted in parallel increases of the endogenous mouse osteocalcin protein and luciferase reporter activity over a 12-day period. Continuous exposure of MC4 cells to 1,25(OH)2D3 resulted in time-and dose-dependent increases in the activity of the phOC3900 luciferase construct. By contrast, the hormone had no effect on mouse osteocalcin protein concentrations and inhibited its induction by ascorbic acid. However, when cells were treated acutely with 1,25(OH)2D3 at later times during growth in ascorbic acid, the induction of mouse osteocalcin protein was only partially inhibited. In conclusion, our results indicate that common osteogenic signals regulate both mouse and human osteocalcin gene expression, but the mouse gene is resistant to induction by vitamin D. This species difference in vitamin D regulation of osteocalcin appears to result from the failure of 1,25(OH)2D3 to transcriptionally activate the mouse osteocalcin gene.

Targeted expression of calcitonin gene-related peptide to osteoblasts increases bone density in mice.

The neuropeptide calcitonin gene-related peptide (CGRP) is concentrated in fine sensory nerve endings innervating all tissues, including bone. CGRP inhibits osteoclasts, stimulates insulin-like growth factor I and inhibits tumor necrosis factor alpha production by osteoblasts in vitro. To investigate the role of CGRP in bone in vivo, mice were engineered to express CGRP in osteoblasts by placing the human CGRP gene under the control of the rat osteocalcin promoter (Ost-CGRP tg+ mice). Calvaria cultures from transgene positive (tg+), but not tg- mice, produced bioactive CGRP. Trabecular bone density and bone volume, determined by peripheral quantitative computed tomography and bone histomorphometry, respectively, were higher in tg+ than tg- littermates. This increase in bone volume was associated with an increased bone formation rate. Trabecular bone density decreased in tg+ mice as a result of ovariectomy, but remained higher than in sham tg- mice. Targeting CGRP to osteoblasts appears to favor the establishment of a higher trabecular bone mass in mice.

Parenteral pamidronate prevents thyroid hormone-induced bone loss in rats.

Pamidronate (APD) is a bisphosphonate that prevents bone loss from a variety of causes. We studied the role of APD in preventing thyroid hormone-induced bone loss. A total of 32 rats were assigned to one of four treatment groups: (1) -APD/triiodothyronine (-T3), (2) -APD/+T3, (3) +APD/-T3, or (4) +APD/+T3. In the first of two studies, the rats received APD for the first week and T3 for the second week, and then their blood was analyzed for alkaline phosphatase and osteocalcin. Alkaline phosphatase and osteocalcin were significantly higher (p < 0.05) in hyperthyroid rats (-APD/+T3, 3.9 +/- 0.25 mukat/liter and 23 +/- 1.6 nM, respectively) than in control animals (2.53 +/- 0.28 mukat/liter and 18.3 +/- 1.4 nM, respectively). Hyperthyroid rats pretreated with APD (+APD/+T3) had levels of alkaline phosphatase and osteocalcin no different from controls. In a second study, rats were divided into the same four groups, except they received APD/placebo and T3/placebo concomitantly for 3 weeks. At the end of the study, bone mineral density (BMD) of the femur, spine, and whole body was measured by dual-energy x-ray absorptiometry, and the calcium content of the femora was measured directly. In hyperthyroid rats (-APD/+T3) BMD was significantly lower than in controls in the spine (0.201 +/- 0.004 versus 0.214 +/- 0.002 g/cm2, p < 0.05) and femur (0.204 +/- 0.003 versus 0.218 +/- 0.002, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Osteocalcin abnormalities in Hyp mice reflect altered genetic expression and are not due to altered clearance, affinity for mineral, or ambient phosphorus levels.

The Hyp mouse manifests rickets and renal wasting of phosphorus. We previously reported elevated circulating osteocalcin in Hyp mice, and a paradoxical decrease in response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. To investigate these abnormalities further, we characterized in detail the response of circulating osteocalcin to 1,25-(OH)2D3 and compared skeletal osteocalcin in normal and Hyp mice. The affinity of osteocalcin for hydroxyapatite and the protein's clearance were compared in Hyp and normal animals. Finally, the response of osteocalcin messenger RNA (mRNA) to 1,25-(OH)2D3 was examined in normal mice, Hyp mice, and normal mice subjected to dietary phosphorus deprivation. Multiple (n = 3-6) daily doses of 1,25-(OH)2D3 are required to increase serum osteocalcin levels in normal C57 BL/6 mice; the effect is apparent by 6 h and persists for at least 24 h after injection. Single doses of up to 50 ng have no significant effect. In contrast, an approximately 50% decrement in circulating osteocalcin occurs after a single dose of 1,25-(OH)2D3 in Hyp mice. Osteocalcin clearance in Hyp mice appears to be normal. Bone osteocalcin per U calcium or phosphorus is normal in Hyp mice, suggesting that its affinity for hydroxyapatite is normal. Osteocalcin mRNA from Hyp mice is expressed in greater abundance than that from normal animals, reflecting the differences in circulating levels of the protein. Similarly, osteocalcin mRNA from Hyp mice decreases in response to 1,25-(OH)2D3, whereas an increase in osteocalcin message is seen in normal animals. These studies indicate that normal mice are relatively resistant to 1,25-(OH)2D3 stimulation of osteocalcin production. Furthermore, the differences between Hyp and normal mice in circulating osteocalcin reflect differences in the regulation of gene expression.

Development and validation of a radioimmunoassay for mouse osteocalcin: paradoxical response in the Hyp mouse.

The hypophosphatemic (Hyp) mouse is a model for human familial hypophosphatemic rickets. To test the hypothesis that there is an osteoblastic defect in these animals, serum osteocalcin levels were measured in Hyp mice and their normal littermates. Furthermore, the effects of phosphorus deprivation, phosphorus loading, and 1,25-dihydroxyvitamin D3 administration on serum osteocalcin levels were examined. Osteocalcin was purified from mouse hindlimbs, and a polyclonal antibody to this material was produced in a goat. The antibody recognized native and decarboxylated mouse osteocalcin, but could not recognize osteocalcin from several other species. A RIA was developed which had a minimal detection limit of 0.4 nmol/liter (2.2 micrograms/liter) and half-maximal displacement at 2.7-3.3 nmol/liter (14.8-18.2 micrograms/liter). The intraassay coefficient of variation was 6.4%, while the interassay coefficient of variation was 12%. Dilutions of mouse serum samples varied by less than 15%. Analytical recovery was typically greater than 90%. Serum osteocalcin concentrations in Hyp and normal mice were shown to decrease with age. However, circulating osteocalcin levels in Hyp mice were higher than those in their normal littermates regardless of the age of the animal (P less than 0.001). One week of a high phosphorus diet resulted in an increase in serum phosphate in normal and Hyp mice, but serum osteocalcin concentrations were unaffected. On the other hand, dietary phosphorus deprivation for 4 weeks resulted in comparable hypophosphatemia in both Hyp and normal mice, and serum osteocalcin increased in both groups of animals. Intraperitoneal injection of 30 ng/day 1,25-dihydroxyvitamin D3 for 7 days resulted in a 215 +/- 33% increase in serum osteocalcin in normal animals, while the same regimen produced a 250 +/- 29% decrease in the Hyp mouse. Our results are consistent with the hypothesis that abnormal osteoblastic activity is present in Hyp mice. Furthermore, hypophosphatemia may be a general regulator of osteocalcin synthesis or secretion in the mouse.

Osteocalcin production in primary osteoblast cultures derived from normal and Hyp mice.

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17-25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

Osteocalcin in human serum: a circadian rhythm.

Osteocalcin, the vitamin K-dependent protein synthesized in bone, is found in blood. The level of circulating osteocalcin has recently been used as an indicator of the rate of bone turnover. We measured serum osteocalcin during 24-h periods in 6 normal 20- to 30-yr-old men and 4 women. Blood was sampled via an indwelling venous catheter every 30 or 60 min for 24 h. Circadian rhythmicity in circulating osteocalcin was found in 9 of the 10 individuals studied. Osteocalcin levels fell during the morning, rose in the afternoon and early evening, and reached a peak nocturnally. There were no consistent correlations between osteocalcin concentrations and circulating levels of ionized calcium, total calcium, or inorganic phosphate in the subjects tested. This study illustrates the importance of regulating the time of blood sampling for osteocalcin determinations in clinical investigations of metabolic bone disease.