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PubMed1992

Inhibition of the morning cortisol peak abolishes the expected morning decrease in serum osteocalcin in normal males: evidence of a controlling effect of serum cortisol on the circadian rhythm in serum osteocalcin.

Osteocalcin (OC) in serum varies in a remarkably constant circadian rhythm with zenith at night and nadir in the morning. The factors controlling this rhythm are unknown, but several studies indicate that serum cortisol could be of major importance. We tested this hypothesis in a double-blind, placebo-controlled, cross-over study comprising 10 normal male volunteers (aged 23-31 yr) by measuring the response in serum OC and cortisol rhythms to a single dose of metyrapone (30 mg/kg body weight) administered at midnight. During placebo, serum cortisol consistently peaked early in the morning before 0730 h. Ingestion of metyrapone at 2400 h significantly postponed and flattened this peak (P less than 0.01). On both occasions, serum OC increased towards peak levels around 0300 h (P less than 0.01) with no overall differences in the OC profiles. However, when the serum OC time series were synchronized according to the individual cortisol nadirs, we found a significant (P less than 0.01) decrease in serum OC on the placebo day approximately 4 h after the cortisol nadir, whereas no significant changes (P greater than 0.50) were seen on the metyrapone day. Moreover, the mean serum OC level tended to be higher (P less than 0.10 in the interval 0-12 h, and P = 0.06 in the interval 4-8 h) on the metyrapone day compared with the placebo day. On the placebo day, the mean level of serum cortisol during the interval 0-4 h correlated inversely with the mean level of serum OC in the interval 4-8 h (r = 0.77, P less than 0.05). This relation was not found on the metyrapone day. In conclusion, administration of metyrapone, which reduced and postponed the early morning cortisol peak, abolished the normal morning decrease in serum OC. This strongly supports that changes in endogenous serum cortisol are of major importance for the circadian rhythm in serum OC.

Adult↗

PubMed1997

[Separation and purefied of calf osteocalcin, set up radioimmunoassay for osteocalcin].

OBJECTIVE AND METHODS: We extracted and purified osteocalcin (BGP) from calf femur by gel filtration, FPLC and HPLC subsequently. The rabbits were immunized by this antigen and produced antibody. With this antibody (final 1: 35,000 dilution) we established RIA for BGP. This assay is stable and sensitive, the kd was 2.29 x 10(11). The intra assay variation was 2.1% and the inter assay variaton was 5.6%. It has no crossreaction with insulin, parathyroid hormone or calcitonin. RESULTS: The serum BGP values were detected in normal subjects and six kinds of bone metabolic diseases. In 81 normal subjects, the average serum BGP level was (5.3 +/- 1.5) ng/ml and in postmenopausal osteoporosis the serum BGP level [(6.2 +/- 1.9) ng/ml, n = 42] was higher than the normal (P < 0.05). The serum BGP levels were increased much in primary hyperparathyroidism [(12.8 +/- 7.0) ng/ml, n = 21, P < 0.001]. Increased values of BGP were also found in patients with chronic renal failure [(8.5 +/- 2.4) ng/ml, n = 18, P < 0.01] and Paget's disease [(6.7 +/- 2.2) ng/ml, n = 12, P < 0.05]. Decreased serum BGP values were found in Cushing syndrome [(2.9 +/- 0.9) ng/ml, n = 16, P < 0.001] and hypoparathyroidism [(3.4 +/- 1.1) ng/ml, n = 42, P < 0.001]. This result showed that in diseases with high bone turnover, the serum BGP levels were increased and in diseases with low bone turnover, the serum BGP levels were decreased. CONCLUSION: The data suggest that serum BGP is a sensitive marker for bone turnover. It is very improtant in studying bone metabolic diseases.

Adult↗

PubMed1994

Osteocalcin: a non-invasive index of metabolic bone disease in patients treated by CAPD.

Serum osteocalcin has been found to correlate with bone formation. However, present literature gives only limited data on osteocalcin and bone histomorphometry in patients undergoing peritoneal dialysis. This study assessed serum osteocalcin, dialysate osteocalcin, peritoneal clearance of osteocalcin (Clp-osteocalcin) and mass transfer of osteocalcin (MTp-osteocalcin), and evaluated relationships between these values and bone histomorphometry. Eighteen patients were treated by continuous ambulatory peritoneal dialysis (CAPD). Bone biopsies, serum and dialysate osteocalcin, serum levels of parathyroid hormone, alkaline phosphatase, aluminum, phosphate, Ca2+ and vitamin D3 metabolites were measured at the start and in 10 of the patients a year later. Serum osteocalcin was found to be elevated. Osteocalcin was detected in the dialysate resulting in significant values of Clp-osteocalcin and MTp-osteocalcin. Serum and dialysate levels of osteocalcin correlated significantly (r = 0.66, P < 0.001) and like MTp-osteocalcin with serum levels of alkaline phosphatase and PTH. Histomorphometry showed that osteitis fibrosa was the predominant bone disease detected. Serum concentration of osteocalcin correlated with osteoid thickness, eroded and osteoclast surfaces, aluminum staining, and some of the bone dynamic parameters. Dialysate osteocalcin, MTp-osteocalcin, PTH and alkaline phosphatase correlated with practically the same histomorphometric parameters as serum osteocalcin. No correlations were seen between Clp-osteocalcin and any histomorphometric parameters. Serum osteocalcin was elevated above the normal range, and significant positive correlations between serum osteocalcin and bone formation parameters were found. Serum osteocalcin correlated with almost the same histomorphometric parameters as PTH. Thus, serum levels of PTH and osteocalcin gave additional information to one another as non-invasive parameters in this group of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1998

Vitamin K status and bone health: an analysis of methods for determination of undercarboxylated osteocalcin.

Recent studies suggest that fracture risk is associated with increased undercarboxylated osteocalcin. Methods use differences in binding of undercarboxylated and fully carboxylated osteocalcin to hydroxyapatite or barium sulfate. We evaluated these methods and found that results varied with the amount and preparation of the salts. Furthermore, patient samples with differing amounts of total osteocalcin could not be directly compared. Errors in the determination of undercarboxylated osteocalcin were minimized by expressing data as the percent of the total osteocalcin in the sample, and correcting for the basal level of osteocalcin using a polynomial equation derived from multiple binding curves. Errors from 5-15% in estimation of undercarboxylated osteocalcin were observed without both of these corrections. When differing types of assays were employed (RIA, intact, N-terminal), results also were affected. In normal adults and children and in patients on long-term warfarin therapy, the percent osteocalcin not bound to hydroxyapatite was lower when measured with an intact assay than by a polyclonal RIA. Differences were related to the amount of N-terminal osteocalcin fragments, which had low affinity for hydroxyapatite and resulted in variable overestimation of undercarboxylated osteocalcin. In a kit specific for uncarboxylated osteocalcin, we found good discrimination between carboxylated and uncarboxylated intact osteocalcin. However, the assay detected large osteocalcin fragments and overestimated their concentration by up to 350%. Values for uncarboxylated osteocalcin were not different in patients on coumadin compared with normal adults with this kit, but when normalized to the total intact osteocalcin, percent uncarboxylated osteocalcin was greater in patients on coumadin than in controls, as would be expected. Kit values for uncarboxylated osteocalcin in normal children were higher than intact values in the same subject, because of the increased reactivity of the kit toward circulating fragments that were elevated in children. Thus, for estimation of undercarboxylated osteocalcin, care must be taken to standardize the hydroxyapatite or barium sulfite used for binding, to correct for the basal level of osteocalcin in the sample, to use immunoassays that do not detect small fragments, and to express the results as the percent of the total osteocalcin in the sample. Without these precautions, the assessment of undercarboxylated osteocalcin is not reliable.

Adult↗

PubMed1994

Development of a heterologous radioimmunoassay for canine osteocalcin.

The aim of this study was to develop a routine and reliable radioimmunoassay (RIA) for dog osteocalcin. Two peaks of dog osteocalcin were purified to apparent homogeneity according to N-terminal sequence analysis. Amino acid composition analysis suggested that the second peak was intact dog osteocalcin whereas the first peak could be a truncated molecule. High titer (> 1:5,000) anti-dog osteocalcin antisera were produced in rabbits. The antiserum recognized dog and rat osteocalcins but not that in serum of human, bovine, rabbit, mouse, guinea pig, or goat. A homologous RIA using anti-dog osteocalcin as the antibody and dog osteocalcin as the tracer and standard was developed. Taking advantages of the facts that (1) anti-dog osteocalcin cross-reacted in parallel with rat osteocalcin and (2) purified rat osteocalcin is commercially available, we devised an approach that used rat osteocalcin as the tracer and standard, and anti-dog osteocalcin as the antibody to develop a heterologous RIA. This assay recognized dog serum osteocalcin and diluted in parallel with rat and dog osteocalcins. Quantitation was done using rat osteocalcin to construct standard curves, and results were expressed in ng/ml of rat osteocalcin-equivalent. The detection limit of the assay was 5 ng/ml rat osteocalcin-equivalent, and half-maximal displacement was seen at 30-40 ng/ml rat osteocalcin-equivalent. The inter- and intraassay variations were 16.1% and 8.5%, respectively. The assay accurately determined the amount of exogenously added dog osteocalcin in serum. The results quantitated with this RIA correlated well (r = 0.975, n = 86) with those obtained with the homologous RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed2000

Comparison of the analytical and clinical performance characteristics of an N-MID versus an intact osteocalcin immunoradiometric assay.

Osteocalcin is the most abundant non-collagenous protein in bone, reflecting its formation. It was reported that the instability of intact osteocalcin results from the cleavage of the C-terminal sequence of osteocalcin to produce a large N-terminal osteocalcin fragment. A two-site immunoassay for detecting both the N-terminal osteocalcin fragment and the intact osteocalcin was developed that were both independent of an unstable C-terminal sequence. The aim of this study is to investigate the performance of an N-MID osteocalcin immunoradiometric assay and to compare it with an intact osteocalcin assay. Ten serum samples were repeatedly frozen and thawed up to seven times. The variability of the values of N-MID osteocalcin was less than that of the intact osteocalcin. For stability of osteocalcin in serum after storage, the mean value of N-MID was 94. 3% of the initial value after 7 days at 4 degrees C, whereas the intact was 73.4%. The reduction of intact values were significantly larger than that of N-MID after 2, 5 and 7 days. At -30 degrees C, the values of N-MID did not change for up to 10 weeks. The concentrations of osteocalcin measured by an N-MID osteocalcin and an intact osteocalcin were investigated in 27 premenopausal subjects, 27 postmenopausal subjects, and 68 osteoporotic patients (23 with vertebral fractures and 45 with hip fractures). The percent mean increase of osteocalcin in postmenopausal subjects over premenopausal subjects was 98% in N-MID versus 42% in the intact assay. The z-scores of N-MID and intact showed similar results in all groups. N-MID osteocalcin significantly correlated with intact osteocalcin (r=0.755), and other biochemical markers for bone formation, such as bone specific alkaline phosphatase (r=0.606) and C-terminal propeptide of type I procollagen (PICP) (r=0.568). An N-MID IRMA had better stability during storage than intact and had the discriminative ability which is similar to the intact assay in postmenopause and osteoporosis. Therefore, an N-MID osteocalcin IRMA could improve the clinical utility and evaluation of osteocalcin.

Adult↗

PubMed1998

Diurnal variation in total and undercarboxylated osteocalcin: influence of increased dietary phylloquinone.

A diurnal variation exists in blood levels of the vitamin K-dependent bone protein osteocalcin. However, it is not known whether the carboxylated and undercarboxylated constituents of osteocalcin also vary. Therefore, osteocalcin and undercarboxylated osteocalcin were measured in specimens collected every 4 hours over a 24-hour period in nine healthy subjects (five males, four females) ages 20-33 years who were consuming a mixed diet containing 100 microg of phylloquinone. Osteocalcin and undercarboxylated osteocalcin were measured by radioimmunoassay (RIA) before and after treatment with barium sulfate. Although the percent undercarboxylated osteocalcin did not change, a diurnal variation was observed in total osteocalcin, carboxylated osteocalcin, and undercarboxylated osteocalcin, with peak concentrations at 4 a.m. and the lowest concentrations between 12 p.m. and 4 p.m. The difference between the total osteocalcin peak and trough concentrations averaged 28 +/- 7 (SEM)%. There were no gender differences in these rhythms. The effect of dietary phylloquinone as a modulator of these rhythms was evaluated in a randomized study by increasing phylloquinone intake to 420 microg/day with fortified corn oil, split between the lunch and dinner meals. Total and carboxylated osteocalcin fluctuations and concentrations were not affected by the dietary treatment. The diurnal variation in undercarboxylated osteocalcin was abolished with supplementation and concentrations at 8 a.m. (14 hours following supplementation) (2.3 +/- 0.2 ng/ml) were significantly lower than the unsupplemented levels (2.7 +/- 0.2 ng/mL, P = 0.006). The percentage of undercarboxylated osteocalcin was similarly decreased after supplementation (19.7 +/- 1.3%) in relation to the mixed diet cycle (24.2 +/- 1.6%, P = 0.006) at 8 a.m. on the second day. Dietary supplementation induced a fluctuation in percentage undercarboxylated osteocalcin with a decline in levels starting at approximately 12 a.m. Therefore, additional dietary phylloquinone does not appear to modulate the total osteocalcin diurnal rhythm, but can influence its undercarboxylated component.

Adult↗

PubMed1991

Influence of dexamethasone on the vitamin D-mediated regulation of osteocalcin gene expression.

The influence of dexamethasone on expression of the osteocalcin gene which encodes the most abundant non-collagenous and only reported bone-specific protein was examined in ROS 17/2.8 osteosarcoma cells which express a broad spectrum of genes related to bone formation. Consistent with previous reports, quantitation of cellular osteocalcin mRNA levels by Northern blot analysis, osteocalcin gene transcription by activity of the osteocalcin gene promoter fused to a chloramphenicol acetyl-transferase (CAT) mRNA coding sequence following transfection into ROS 17/2.8 cells, and osteocalcin biosynthesis by radioimmunoassay indicate that dexamethasone in a concentration range of 10(-6) to 10(-9) M only modestly modifies basal levels of osteocalcin gene expression. However, dexamethasone significantly inhibits these parameters of the vitamin D-induced upregulation of osteocalcin gene expression in both proliferating and in confluent ROS 17/2.8 cells. In this study, we observed that the extent to which abrogation of the vitamin D response occurs is dependent on basal levels of osteocalcin gene expression as reflected by a complete inhibition of the vitamin D-induced upregulation in a ROS 17/2.8K subline with low basal expression and only a partial reduction of the vitamin D stimulation in a ROS 17/2.8C subline with eightfold higher levels of basal expression. This effect of glucocorticoid appears to be at the transcriptional and post-transcriptional levels as demonstrated by a parallel decline in the cellular representation of osteocalcin mRNA, osteocalcin gene promoter activity, and osteocalcin biosynthesis. The complexity of the glucocorticoid effect on vitamin D-mediated transcriptional properties of the osteocalcin gene is indicated by persistence of sequence-specific protein-DNA interactions at two principal osteocalcin gene promoter regulatory elements, the osteocalcin (CCAAT) box which modulates basal level of transcription, and the vitamin D responsive element, where vitamin D-mediated enhancement of osteocalcin gene transcription is controlled.

Blotting, Northern↗

PubMed1994

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.

Animals↗

PubMed2004

Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro.

Osteocalcin detected from serum samples is considered a specific marker of osteoblast activity and bone formation rate. However, osteocalcin embedded in bone matrix must also be released during bone resorption. To understand the contribution of each type of bone cell in circulating osteocalcin levels, we used immunoassays detecting different molecular forms of osteocalcin to monitor bone resorption in vitro. Osteoclasts were obtained from rat long bones and cultured on bovine bone slices using osteocalcin-depleted fetal bovine serum. In addition, human osteoclasts differentiated from peripheral blood mononuclear cells were used. Both rat and human osteoclasts released osteocalcin from bovine bone into medium. The amount of osteocalcin increased in the presence of parathyroid hormone, a stimulator of resorption, and decreased in the presence of bafilomycin A1, an inhibitor of resorption. The amount of osteocalcin in the medium correlated with a well characterized marker of bone resorption, the C-terminal telopeptide of type I collagen (r > 0.9, p < 0.0001). The heterogeneity of released osteocalcin was determined using reverse phase high performance liquid chromatography, and several molecular forms of osteocalcin, including intact molecule, were identified in the culture medium. In conclusion, osteocalcin is released from the bone matrix during bone resorption as intact molecules and fragments. In addition to the conventional use as a marker of bone formation, osteocalcin can be used as a marker of bone resorption in vitro. Furthermore, bone matrix-derived osteocalcin may contribute to circulating osteocalcin levels, suggesting that serum osteocalcin should be considered as a marker of bone turnover rather than bone formation.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

Vitamin K2 enhances osteocalcin accumulation in the extracellular matrix of human osteoblasts in vitro.

The role of vitamin K in osteocalcin accumulation in the extracellular matrix of normal human osteoblasts in culture was investigated by using a human intact osteocalcin-specific assay system. Human osteoblasts produced osteocalcin by treatment with 10(-9) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 20 days in culture. With the addition of vitamin K2 (1.5-5.0 microM), osteocalcin accumulation in the extracellular matrix of the osteoblasts was increased, but the osteocalcin content in the conditioned medium decreased, in comparison with that treated with 10-9 M 1,25(OH)2D3 alone. The enhancement of osteocalcin accumulation induced by vitamin K2 was dependent on the duration of the treatment. The vitamin K2 plus 1,25(OH)2D3-induced osteocalcin accumulation was blocked by the addition of warfarin 2 days before the vitamin treatment. At that time, warfarin significantly reduced the mineralization by osteoblasts in vitro. Osteocalcin accumulated in the extracellular matrix was almost completely precipitated by a low concentration of hydroxyapatite, 10 mg/ml. Moreover, the gamma-carboxyglutamic acid (Gla)-containing osteocalcin level was increased by the vitamin K2 plus 1,25(OH)2D3 treatment. These results proved that vitamin K2 increased Gla-containing osteocalcin, which accumulated osteocalcin in the extracellular matrix, and facilitated mineralization in vitro. Vitamin K2 also enhanced the 1,25(OH)2D3-induced osteocalcin mRNA level, but vitamin K2 alone did not show osteocalcin mRNA expression. We thus demonstrated that vitamin K2 enhanced not only the accumulation of Gla osteocalcin, but also the osteocalcin production induced by 1,25(OH)2D3 in human osteoblasts in culture.

Bone Density↗

PubMed1990

Localization of endogenous osteocalcin in neonatal rat bone and its absence in articular cartilage: effect of warfarin treatment.

Immunocytochemistry after cryoultramicrotomy was used to localize endogenous osteocalcin in bone (calvaria, femoral diaphysis) and epiphyseal femoral cartilage from 8-day-old rats treated (or mot) for 7 days with warfarin. Ultrathin frozen sections were incubated with goat antiserum against rat osteocalcin at high dilutions (2 x 10(-4) to 2 x 10(-6). In calvaria and femur of untreated rats, endogenous osteocalcin was observed in osteoblasts (cytoplasm and nucleus) and in the collagenous matrix. Osteocalcin appeared progressively in osteoblasts and bone matrix in the mineralization front, then increased in the regions of extended calcification. Osteocalcin was also detected in osteocytes but was not as abundant as in osteoblasts. In bone samples of warfarin-treated rats, endogenous osteocalcin was only detected in bone matrix but not in osteoblasts. Furthermore, osteocalcin was only observed if antiserum was not very dilute (2 x 10(-2). In cartilage (hypertrophied and degenerative zones), osteocalcin was not observed in matrix and chondrocytes. However, it was found in the vicinity of matrix vesicles at the initial loci of calcification. Osteocalcin was never detected in the cartilage of warfarin-treated rats. Our results provide ultrastructural immunocytological evidence for the localization of endogenous osteocalcin in osteoblasts, the presence of osteocalcin in bone matrix and a direct gradient between the presence of osteocalcin and the calcification process. Osteocalcin is absent from cartilage, except possibly close to calcifying matrix vesicles. Warfarin inhibits the formation of osteocalcin.

Animals↗

PubMed1991

Regulation of osteocalcin secretion by human primary bone cells and by the human osteosarcoma cell line MG-63.

We present evidence that the regulation of osteocalcin secretion by PTH and PGE2 in normal human bone cells can be produced in the human osteoblast-like cell line MG-63. Both cell cultures showed time- and dose-dependent stimulation of osteocalcin secretion in response to 1,25(OH)2D3. Bovine parathyroid hormone (PTH) amino acid fragment 1-34 (40 nM) and prostaglandin E2 (PGE2, 5 nM) significantly inhibited 1,25(OH)2D3-induced osteocalcin secretion by these cells. The inhibition reached 20 and 36%, respectively. In contrast, PTH 3-34 had no effect on osteocalcin secretion. Both cell cultures produced cAMP in response to PTH. Dexamethasone (Dex) (100 nM) potentiated PTH-induced (40 nM) cAMP synthesis in subconfluent MG-63 cells (1.5-fold increase, P less than 0.05). This treatment with Dex resulted in a greater inhibition of 1,25(OH)2D3-induced osteocalcin secretion (-30%, P less than 0.005) by PTH in MG-63 cells as compared to cells exposed to PTH and 1,25(OH)2D3 alone. Pretreatment of subconfluent MG-63 cells with Dex (100 nM) for 48 h also increased 1,25(OH)2D3-induced osteocalcin secretion by 40% (P less than 0.025). In contrast, treatments of confluent MG-63 cells with Dex inhibited osteocalcin secretion regardless of the 1,25(OH)2D3 doses used. Forskolin (10(-7)-10(-5) M) and dibutyryl cAMP (10(-6)-(10(-3) M) both reproduced the effects observed with PTH and PGE2 in the two cell cultures. Forskolin's action was time-dependent: addition of forskolin (10(-6) M) 12 h after 1,25(OH)2D3 (50 nM) resulted in a progressively weaker inhibition of osteocalcin secretion. Increasing the extracellular calcium concentration of the incubation media resulted in a dose-dependent increase in osteocalcin secretion (P less than 0.01). These results indicate that PTH and PGE2 inhibit osteocalcin secretion by a mechanism involving cAMP production. In contrast, an increase in extracellular calcium stimulated osteocalcin release. Thus the human osteosarcoma cell line MG-63 is a useful osteoblast-like cell model to study the regulation of osteocalcin secretion. Furthermore, a factor (or factors) between hormone-receptor coupling and gene induction can regulate the expression of the osteocalcin gene or affect pre- or posttranslational mechanisms implicated in osteocalcin synthesis and secretion.

Bone and Bones↗

PubMed1996

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.

Animals↗

PubMed1997

Human and murine osteocalcin gene expression: conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo.

Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6+/-3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2+/-0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2+/-7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5+/-0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly upregulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.

Animals↗

PubMedDate not supplied

Osteocalcin. A biochemical marker of bone turnover during puberty.

During bone remodelling, osteocalcin is produced by osteoblasts and its level increases during the events characterized by rapid bone turnover. Osteocalcin is a bone matrix protein, which is specific for bone metabolism and it is not influenced by metabolic bone disorders. Osteocalcin is an important marker of bone turnover in physiological and pathological conditions. Physiologically, serum osteocalcin was increased in children, particularly during the first year of life and during puberty, when evolution of the concentration was related to rapidity of physical growth. Evidence of a correlation with growth rates comes from the observation that serum osteocalcin levels parallel the height velocity curve, with higher values in childhood and during adolescence, that later fall to adult values. There are previous studies reporting that there is age- and sex-dependent change in serum osteocalcin levels in children and adolescents with a pattern resembling height velocity curves for children and serum osteocalcin elevation coincides with the pubertal growth spurt. These findings demonstrate that pubertal development and sex should be taken into account rather than chronological age when serum levels of osteocalcin are evaluated. In most of the studies relationships among osteocalcin and chronological age and bone age, but not pubertal developmental stage (sexual maturation stage) were investigated. The aim of our study was to determine whether osteocalcin is a useful marker for the pubertal growth spurt period. In this study, osteocalcin levels in male adolescents were examined in relation to their sexual maturation stage and age. According to our findings, the follow up of osteocalcin levels in relation to sexual maturation stages could be a new method to determine the phase of the pubertal growth spurt. An increase or decrease in osteocalcin levels on consecutive measurements may indicate the child's entering accelerated or decelerated stages of the growth spurt, respectively. We emphasize that the follow up of adolescent growth is made by determination of the sexual maturation stage, and not by age. Osteocalcin is a highly specific, reliable and useful marker for evaluation of the growth spurt and is not influenced by nonosseous disorders.

Adolescent↗

PubMed1996

Measurement of intact rat osteocalcin in osteoblast (ROS17/2.8) cells and in ovariectomized rats with a sandwich enzyme immunoassay.

We developed a sandwich enzyme immunoassay system for intact rat osteocalcin to improve the region specificity for the detection of this molecule. We synthesized two peptides of N-terminal 20 residues and C-terminal 10 residues of rat osteocalcin. After conjugating these peptides with carrier protein, we obtained anti-N- and anti-C-terminal rat osteocalcin antibodies in rabbits raised against these two peptides, respectively. By using these antibodies, we measured intact rat osteocalcin levels in a two-site immunoassay manner. These antibodies did not show the cross-reactivity to human osteocalcin. The immunoreactive peak corresponding to the intact molecules was detected by our intact osteocalcin method after high-performance liquid chromatographic fractionation of osteocalcin fragments in plasma from uremic rats. Furthermore, the intact rat osteocalcin was stable over 8 hours at 25 degrees C. Intact rat osteocalcin levels extracellularly secreted from ROS 17/2.8 cells were measured by this method, showing time- and dose-dependent significant increases when administered 1,25(OH)2D3. The inhibition for the secretion of intact osteocalcin by actinomycin D was also detected quantitatively with this method. In ovariectomized rats, intact osteocalcin levels in plasma were acutely elevated after ovariectomy, and its elevation was significantly depressed by 17beta-estradiol administration. These data suggest that this sandwich method is able to measure the intact form of osteocalcin secreted by osteoblasts. As the antibodies identify the specific regions of osteocalcin molecule, this method would be useful for sensitive estimation of bone turnover for various experimental conditions in rats.

Animals↗