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PubMed1994

Circadian and circatrigintan changes in osteoblastic activity assessed by serum osteocalcin. Physiological and methodological aspects.

The author has shown the existence of reproducible circadian changes and significant variation during the circatrigintan rhythm in the osteoblastic marker, serum osteocalcin. The author find that appropriate exploratory data analyses are important and often imperative tools in the demonstration of these low frequent rhythms, and the effects of intervention. The metabolism of osteocalcin needs further investigation since it is essential to consider the kinetic of serum osteocalcin in the interpretation of data. However, based on current knowledge of the kinetics of serum osteocalcin, and the variation in other osteoblastic markers, the author conclude that the results are indicative of the existence of biological rhythms in osteoblastic activity rather than changes in metabolic clearance. The physiological aspects on the circadian rhythm are that osteoblastic activity peaks during the early night and decrease in the morning in both men and women. The circadian osteoblastic activity is relatively stable during seasons and apparantly not acutely related to sleep. Modest doses of alcohol decrease serum osteocalcin within few hours but the circadian rhythm is not different in smokers compared with non-smokers. There are evidences for endogeneous cortisol as regulator of the osteocalcin rhythm especially by mediating the morning decrease in osteocalcin. Osteocalcin synthesis is not completely blocked by glucocorticoids since the effect is blunted by administration of 1,25-(OH)2D3. Although serum osteocalcin may respond to acute doses of several other osteotrophic hormones, it is doubtfull whether endogeneous PTH, 1,25-(OH)2D3, sex hormones, or growth hormone play any role as acute regulators of the circadian osteocalcin rhythm. The circatrigintan rhythm is characterized by highest levels of serum osteocalcin during the luteal phase. There is a significant cross correlation with the follicular increase in serum estradiol but the regulation of serum osteocalcin during the ovulatory cycle remains to be settled. The results, however, support the interaction between sex hormones and bone metabolism. The practical implications are that blood sampling for determination of serum osteocalcin should be strictly standardized with regard to the time of the day and the time since last medication or intervention with factors that may affect osteoblastic activity.

Circadian Rhythm↗

PubMed2004

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone.

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).

Amino Acid Sequence↗

PubMed1987

Amniotic fluid osteocalcin concentrations in pregnancies uncomplicated by fetal bone disease.

Osteocalcin is the major noncollagenous bone protein. Serum osteocalcin concentrations increase in conditions when bone turnover is increased, reflecting enhanced osteoblastic activity. The presence of osteocalcin in amniotic fluid (AF) has not been reported. We undertook this study to determine if osteocalcin is present in AF and to establish standards for AF osteocalcin concentrations, to be used in pregnancies at risk for diseases with increased fetal bone turnover. One hundred and thirty-one samples of AF from 15 to 43 weeks gestation were analyzed for osteocalcin by a radioimmunoassay detecting greater than or equal to 0.1 ng/ml, with a coefficient of variation of 6%. Osteocalcin measured in AF had the same gel-filtration characteristics as serum osteocalcin and the standard curves for serum and AF osteocalcin were parallel. Indications for amniocentesis were genetic study (n = 15) and fetal lung maturation (n = 116). None of the pregnancies studied produced an infant with bone mineral disorder. In 67% of the AF samples, osteocalcin was undetected; in 95% of the samples the concentration was less than 2.4 ng/ml. Concentrations of AF osteocalcin did not correlate with gestational age or birthweight, and were lower than the reported concentrations in cord serum or maternal serum. Thus normal amniotic fluid osteocalcin concentrations are less than 2.4 ng/ml; we speculate that its elevation in fetal bone disorders may provide a genetic counselling tool.

Amniotic Fluid↗

PubMed1989

Osteocalcin during the reproductive cycle in normal and diabetic rats.

Concentrations of osteocalcin were measured in plasma and bone of normal and diabetic rats during the reproductive cycle and compared with plasma 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) concentrations. The age-dependence of osteocalcin was also examined. Plasma concentrations of osteocalcin levels were low but detectable in 21-day-old fetuses (3.7 +/- 0.3 nmol/l); osteocalcin concentrations were highest in weaning rats (104 +/- 9 nmol/l) and decreased thereafter. In adult rats, plasma concentrations of both osteocalcin and 1,25-(OH)2D3 increased during the last days of normal pregnancy, and even more so in rats fed a diet low in calcium and phosphate. After an early post-partum decline, osteocalcin concentrations in plasma remained at non-pregnant levels in lactating rats fed a high calcium/phosphate diet while their 1,25-(OH)2D3 concentrations were higher than in non-pregnant rats; however, lactating rats fed a low calcium/phosphate diet showed increasing osteocalcin concentrations. In spontaneously diabetic BB rats, plasma osteocalcin concentrations were severely decreased compared with those in non-diabetic rats, more than would have been expected from their decreased 1,25-(OH)2D3 concentrations. Moreover, plasma osteocalcin did not increase during pregnancy or lactation in diabetic rats, even when fed a low calcium/phosphate diet. Fetuses of diabetic rats also had lower plasma osteocalcin levels than fetuses from non-diabetic rats or than weight-matched fetuses from semistarved rats. In contrast to plasma osteocalcin concentrations, bone osteocalcin concentrations and content were not altered by pregnancy, lactation, low calcium/phosphate diet or diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMedDate not supplied

The relationship between osteocalcin levels and sexual stages of puberty in male children.

Osteocalcin is a specific and reliable marker which increases with rapid bone turnover and gives data about bone metabolism. The pubertal growth spurt is also known as a good example of rapid bone turnover. The aim of this 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. The osteocalcin levels and alkaline phosphatase levels were compared during the pubertal growth spurt. Serum osteocalcin and alkaline phosphatase levels were evaluated in 100 eligible healthy male children and adolescents (aged 10 to 17 years). Five groups (n: 20 each) of children and adolescents were formed according to their sexual maturation stages. Finally, the subjects were divided into three main groups in relation to the pubertal growth spurt and sexual maturation stages. Data were evaluated and compared among these three groups: First group = Stage 1 (prepuberty) + Stage 2 (early puberty) consisted of 40 (20 + 20) children and adolescents. Their mean osteocalcin value was 17.2 +/- 6.3 ng/ml and alkaline phosphatase 573.8 +/- 143.9 IU/L. Second group: Stage 3 + Stage 4 consisted of 40 (20 + 20) children and adolescents. These groups were known as the pubertal growth spurt groups. Their mean osteocalcin value was 29.4 +/- 10.6 ng/ml and alkaline phosphatase 728.4 +/- 233.9 IU/L. Third group: In this group, there were 20 children and adolescents who reached Stage 5 of sexual maturation and whose pubertal growth spurt was slowing. Their mean osteocalcin value was 15.3 +/- 5.8 ng/ml and alkaline phosphatase 435.8 +/- 184.8 IU/L. During the pubertal growth spurt, there is a relationship between bone remodelling and increasing osteocalcin and alkaline phosphatase levels. When sexual maturation reaches Stage 4 at 14 years old, osteocalcin and alkaline phosphatase levels make a peak, associated with the rapid growth in height. As sexual maturation reaches Stage 5, osteocalcin and alkaline phosphatase levels gradually decrease with growth maturation and their levels decline to the level of adults at the completion of this period. Our study showed that osteocalcin and alkaline phosphatase levels can be used as markers for evaluation of the growth spurt period.

Adolescent↗

PubMed1985

Studies of hormonal regulation of osteocalcin synthesis in cultured fetal rat calvariae.

The synthesis of osteocalcin, the major non-collagenous protein of adult bone, was examined in cultures of 21-day fetal rat calvariae. Osteocalcin was measured by a sensitive and specific radioimmunoassay. Osteocalcin concentration in unincubated calvariae was 14.5 +/- 0.5 ng/calvaria. After incubation, there was a continuous increase in bone and medium osteocalcin, and by 96 h the values were about 100% higher than in unincubated calvariae. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) at 10(-11) to 10(-8)M increased osteocalcin synthesis. The effect appeared as early as 6 h after treatment and was primarily observed in the culture medium, and 1,25-(OH)2D3 stimulated osteocalcin up to 9-fold by 96 h. Concomitant with the effect on osteocalcin synthesis, 1,25-(OH)2D3 inhibited collagen synthesis. Cycloheximide markedly decreased osteocalcin concentrations in control and 1,25-(OH)2D3-treated calvariae. The stimulatory effect on osteocalcin synthesis was specific to 1,25-(OH)2D3 since 24,25-dihydroxyvitamin D3, parathyroid hormone, epidermal growth factor, and prostaglandin E2 did not stimulate osteocalcin synthesis, and parathyroid hormone and epidermal growth factor opposed the 1,25-(OH)2D3 stimulatory effect. Insulin did not alter osteocalcin concentration by itself but enhanced the effect of 1,25-(OH)2D3. In conclusion, 1,25-(OH)2D3 stimulates osteocalcin synthesis in cultures of normal calvariae, but this effect is not shared by other hormones known to affect bone metabolism.

Animals↗

PubMed1996

Abnormal modulation of serum osteocalcin by dietary phosphate and 1,25-dihydroxyvitamin D3 in the hypophosphatemic mouse.

We evaluated in normal and hypophosphatemic (Hyp) mice whether changes in serum levels of osteocalcin in response to dietary phosphate supplementation, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration were related to perturbations in calcium phosphate homeostasis. In normal mice, serum osteocalcin levels were not altered by phosphate supplementation. In contrast, phosphate supplementation in Hyp mice led to a 2-fold decrease in serum osteocalcin to normal levels after 3 days and to an increase in osteocalcin levels after 14 days. The decrease in osteocalcin was associated with normophosphatemia, severe hypocalcemia, and marked increases in circulating 1,25(OH)2D3 levels, whereas the increase in osteocalcin levels was associated with normophosphatemia and no change in serum calcium and 1,25(OH)2D3. Administration of PTH decreased serum osteocalcin in both genotypes. Infusion of 1,25(OH)2D3 for 3 days elicited increases in serum osteocalcin and calcium levels in normal mice, whereas in Hyp mice it produced significant decreases in osteocalcin levels and no change in serum calcium. However, with a more prolonged infusion of 1,25(OH)2D3, hypercalcemia and increases in serum osteocalcin were induced in mutant mice. Our results suggest that the abnormal osteocalcin response of Hyp mice is not directly attributable to an osteoblast dysfunction but is secondary, at least in part, to perturbations in factors that modulate the osteoblast activity, especially serum calcium and/or PTH.

Analysis of Variance↗

PubMed1989

Osteocalcin serum levels in patients following renal transplantation.

Osteocalcin serum levels reflect bone turnover. In renal insufficiency secondary hyperparathyroidism and reduced renal clearance might be responsible for elevated serum levels of osteocalcin. Renal transplantation might improve renal osteodystrophy and therefore could influence osteocalcin serum levels. We determined the influence of renal transplantation on osteocalcin levels in 37 consecutive patients (25m/12f) by RIA. Blood samples were collected prior to, 3 days, 28 days, 6 months and 12 months after renal transplantation. Prior to renal transplantation osteocalcin levels were significantly elevated (mean +/- s: 23.4 +/- 12.8 ng/ml) compared to healthy volunteers (4.1 +/- 1.4 ng/ml). Following renal transplantation osteocalcin decreased significantly (9.4 +/- 8.9 ng ml) 3 days and (7.1 +/- 7.8 ng/ml) 28 days. However, 6 and 12 months following renal transplantation the mean osteocalcin level increased again (8.3 +/- 5.7 ng/ml, 12.1 +/- 15.4 ng/ml). At 6 months 11 and at 12 months only 6 of 37 patients had osteocalcin levels in the normal range. 12 months following renal transplantation 21 out of 37 patients with elevated osteocalcin levels had parathyroid hormone levels above the normal range. Additionally to increased osteocalcin levels patients prior to renal transplantation had elevated alkaline phosphatase. Alkaline phosphatase had following renal transplantation a similar pattern as osteocalcin with initial decrease and secondary increase 6 and 12 months after renal transplantation. Parathyroid hormone was elevated in all patients before renal transplantation. Following renal transplantation mean parathyroid hormone levels tell significantly, however remained above normal range in 57% of these 37 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

Development of a new radioimmunoassay for human osteocalcin: evidence for a midmolecule epitope.

Osteocalcin is a vitamin K-dependent bone-specific protein that can be found circulating in the serum. The circulating levels of osteocalcin have been shown to be an index of bone turnover. Existing radioimmunoassays for osteocalcin have been shown to be specific for C-terminal epitopes, a region that is identical in the human and bovine osteocalcin. There are, however, five amino acids different in the N-terminal region of the molecule. We describe here an immunoassay for a midmolecule epitope of osteocalcin using osteocalcin purified from human femoral head bone powder. Antibody specificity was determined using tryptic digests and a synthetic fragment of human osteocalcin. This assay has only a partial crossreactivity with bovine osteocalcin. This is the first report of an assay against a midmolecular epitope of osteocalcin involving a region in which the human and bovine osteocalcins differ. Osteocalcin levels determined by this assay have a significant correlation with both the total serum alkaline phosphatase and the serum skeletal alkaline phosphatase levels in normal adult human serum and, to a greater degree, in sera of patients with conditions associated with increased bone turnover (Paget's disease, hyperparathyroidism, and newborn sera). These correlations are greater than those previously reported for C-terminal assays, suggesting the possibility that different regions of the molecule may elicit different information concerning bone turnover.

Adult↗

PubMed1991

Modulation of 1,25(OH)2D3-induced osteocalcin synthesis in human osteosarcoma cells by other steroidal hormones.

We have previously shown that osteocalcin synthesis is readily induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in MG-63 human osteosarcoma cells (Mahonen et al. (1990) Biochim. Biophys. Acta 1048, 30-37). In the present study, the regulation of osteocalcin synthesis by other hormones of the steroid-thyroid hormone family (retinoic acid, 17 beta-estradiol, triiodothyronine, and dexamethasone) was examined. We found that the other hormones alone had no effects on medium osteocalcin and osteocalcin mRNA concentrations by 96 h of treatment. Compared with 1,25(OH)2D3, however, the combination of 1,25(OH)2D3 with dexamethasone resulted in a greatly reduced medium osteocalcin concentration. Also estradiol and triiodothyronine diminished the stimulatory effect of 1,25(OH)2D3. In contrast, the combination of 1,25(OH)2D3 with retinoic acid resulted in an increased medium osteocalcin concentration. The inhibition of osteocalcin synthesis by dexamethasone and triiodothyronine was accompanied by decreased osteocalcin mRNA levels. Retinoic acid and estradiol, however, did not influence the 1,25(OH)2D3-induced osteocalcin mRNA levels. To examine the specificity of the hormonal effects, the activity of alkaline phosphatase was determined. Both baseline and 1,25(OH)2D3-stimulated alkaline phosphatase activity was found to be inhibited by all other hormones. These results suggest that the steroidal hormones specifically affect osteocalcin synthesis in osteoblastic bone cells, and that complex interactions occur at the level of transcription and/or translation resulting in each case in a finely adjusted rate of osteocalcin synthesis.

Alkaline Phosphatase↗

PubMed2003

Structure, activity, and distribution of fish osteocalcin.

Osteocalcin (bone Gla protein) is an extracellular matrix protein synthesized by osteoblasts that is a marker of bone. Osteocalcin probably originated in the ancestors of Teleostei or bony fish and of the Tetrapoda or amphibians, reptiles, birds, and mammals. We have characterized the Cyprinus carpio (carp) osteocalcin for mineral binding to hydroxyapatite, amino acid sequence, and extent of secondary structure. Hydroxyapatite binding is enhanced in the presence of calcium. The alpha-helical content of teleost osteocalcin increases and beta-sheet structure decreases upon calcium binding, similar to findings in calf osteocalcin. The gene structure and primary sequence of prepro-osteocalcin from 2 pufferfish compared with carp shows that there are many conserved features in teleost osteocalcin genes. Using an immunoassay for carp osteocalcin, we determined that the relative content of osteocalcin is highest in dorsal fin spines and other bones and lowest in scales. The carp osteocalcin antibodies, cross-reactive to other species of fish, were used to study the role of osteocalcin in teleost model systems.

Amino Acid Sequence↗

PubMed1998

Evaluation of osteocalcin and pyridinium crosslinks of bone collagen as markers of bone turnover in gingival crevicular fluid during different stages of orthodontic treatment.

Osteocalcin (Oc) and the collagen cross-links pyridinoline (Pyr) and deoxypyridinoline (dPyr) are used as markers of bone turnover in metabolic bone diseases. The aims of this study were: 1) to establish if Oc, Pyr and dPyr can be detected in GCF and 2) using the orthodontic tooth movement model of alveolar bone resorption to evaluate GCF levels of osteocalcin and these collagen cross-links as markers of bone breakdown. Plaque, colour and bleeding indices, probing measurements and GCF samples were collected at two sites in each of 20 adolescents, during 4 stages of fixed appliance therapy: (1) prior to appliance fit, (2) post appliance fit, (3) during active retraction of the maxillary canines, (4) during retention. GCF was collected onto filter paper strips and the volume determined by weighing. An ELISA kit was used for the detection of osteocalcin, whereas Pyr and dPyr were assayed using high performance liquid chromotography (HPLC). Wilcoxon signed ranks test and Bonferroni correction revealed statistically significant increases in plaque (p= 0.012), GCF volume (p=0.024) and osteocalcin concentration (p=0.012), between stages 1 and 2. There were no statistically significant differences between the other variables at this stage or between any of the variables at stages 2 and 3, or between stages 3 and 4. All but 3 of the GCF samples yielded detectable osteocalcin, with large site and subject variation. The median values of osteocalcin and osteocalcin concentration of all the samples were 87.5 pg and 66 pg/microl, with a range of 0-1,248 pg, 0-1,572 pg/microl. The detection of osteocalcin in GCF during every stage, the wide variation between subjects, and the lack of a consistent pattern related to stages of orthodontic treatment, suggests that osteocalcin may merely be a constituent of GCF associated with the developing dentition, which would reduce its potential as a marker of bone turnover in this group. None of the 16 GCF samples analysed for Pyr and dPyr gave a positive result. This study confirms that fitting an orthodontic appliance results in plaque accumulation and increased gingival inflammation, and that GCF volume is the most sensitive indicator of that inflammation. Osteocalcin was detected in GCF collected from adolescents, whereas Pyr and dPyr could not be detected. Further work is required to establish whether GCF osteocalcin levels can be used as a marker of bone turnover, and whether improvements in the sensitivity of detecting Pyr & dPyr make further study of these promising bone markers worthwhile.

Adolescent↗

PubMed1992

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.

Animals↗

PubMed1991

Serum osteocalcin in patients taking L-thyroxine who have subclinical hyperthyroidism.

Serum osteocalcin, an index of osteoblastic activity, is increased in hyperthyroidism. Serum osteocalcin levels are negatively correlated with bone density in patients with overt hyperthyroidism. Osteocalcin levels are also elevated in patients with multinodular goiter and subclinical hyperthyroidism. We therefore measured serum osteocalcin levels in patients taking T4 to determine if they correlated with the degree of TSH suppression. Despite an upward trend in serum osteocalcin measurements with decreasing TSH concentrations, there was no significant difference in serum osteocalcin among groups of patients with normal (0.5-5.0 mu/L), mildly reduced (0.1-0.5 mU/L), or undetectable serum TSH (less than 0.01 mU/L). However, a weak negative correlation was seen between serum TSH and osteocalcin concentrations (r = 0.29, slope = -0.28, P less than 0.05). Osteocalcin did not correlate with either serum free T4 or free T3 concentrations. Serum PTH concentrations were not different among the three patient groups. Our data suggest that osteocalcin is not a useful clinical marker for increased bone turnover in patients with subclinical hyperthyroidism due to T4 therapy. However, the trend towards higher osteocalcin levels in patients with suppressed serum TSH values, and the weak negative correlation between serum TSH and osteocalcin are consistent with findings of reduced bone density in these patients.

Adult↗

PubMed1998

Regulation of osteocalcin production and bone resorption by 1,25-dihydroxyvitamin D3 in mouse long bones: interaction with the bone-derived growth factors TGF-beta and IGF-I.

Bone cells produce multiple growth factors that have effects on bone metabolism and can be incorporated into the bone matrix. Interplay between these bone-derived growth factors and calciotropic hormones has been demonstrated in cultured bone cells. The present study was designed to extend these observations by examining the interactions between either transforming growth factor-beta (TGF-beta) or insulin-like growth factor-I (IGF-I) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a mouse long bone culture model with respect to osteocalcin production and bone resorption. In contrast to the stimulation in rat and human, in the fetal mouse long bone cultures, 1,25(OH)2D3 caused a dose-dependent inhibition of osteocalcin production. Both the osteocalcin content in the culture medium and in the extracts of the long bones was reduced by 1,25(OH)2D3. This effect was not specific for fetal bone because 1,25(OH)2D3 also reduced osteocalcin production by the neonatal mouse osteoblast cell line MC3T3. TGF-beta inhibited whereas IGF-I dose-dependently increased osteocalcin production in mouse long bones. The combination of TGF-beta and 1,25(OH)2D3 did not result in a significantly different effect compared with each of these compounds alone. The IGF-I effect was completely blocked by 1,25(OH)2D3. In the same long bones as used for the osteocalcin measurements, we performed bone resorption analyses. Opposite to its effect on osteocalcin, 1,25(OH)2D3 dose-dependently stimulated bone resorption. TGF-beta reduced and IGF-I did not change basal (i.e., in the absence of hormones) bone resorption. Our results show that 1,25(OH)2D3-enhanced bone resorption is dose-dependently inhibited by TGF-beta and IGF-I. Regression analysis demonstrated a significant negative correlation between 1,25(OH)2D3-induced bone resorption and osteocalcin production. The specificity for their effect on 1,25(OH)2D3-stimulated bone resorption was assessed by testing the effects of TGF-beta and IGF-I in combination with parathyroid hormone (PTH). Like 1,25(OH)2D3, PTH dose-dependently stimulates bone resorption. However, PTH-stimulated bone resorption was not affected by TGF-beta. Like 1,25(OH)2D3-stimulated bone resorption, IGF-I inhibited the PTH effect but at a 10-fold higher concentration compared with 1,25(OH)2D3. In conclusion, the present study demonstrates growth factor-specific interactions with 1,25(OH)2D3 in the control of osteocalcin production and bone. With respect to bone resorption, these interactions are also hormone specific. The present data thereby support and extend the previous observations that interactions between 1,25(OH)2D3 and bone-derived growth factors play an important role in the control of bone metabolism. These data together with the fact that TGF-beta and IGF-I are present in the bone matrix and potentially can be released during bone resorption support the concept that growth factors may control the effects of calciotropic hormones in bone in a localized and possibly temporal manner. Finally, in contrast to human and rat, in mice 1,25(OH)2D3 reduces osteocalcin production and this reduction is paralleled by stimulation of bone resorption by 1,25(OH)2D3. These data thereby show a dissociation between osteocalcin production and bone resorption.

Animals↗

PubMed1995

Osteocalcin expression in primary bone tumors--in situ hybridization and immunohistochemical study.

Osteocalcin is one of the most abundant noncollagenous proteins found in adult bone. It is a highly conserved gamma-carboxyglutamic acid-containing protein that is believed to be produced exclusively by osteoblasts. In this study, intracellular and extracellular localization of osteocalcin in osteosarcoma was examined with anti-osteocalcin antibody and in situ hybridization using a synthetic oligonucleotide. Immunohistochemically, osteoblastic osteosarcomas were all positive for osteocalcin. The chondroblastic osteosarcomas were positive on the neoplastic chondrocytes. The five fibroblastic osteosarcomas out of seven were positive for osteocalcin immunostaining over the neoplastic spindle cells. Five cases of osteoblastic osteosarcomas out of seven were positive for osteocalcin in situ hybridization. Two cases of chondroblastic osteosarcomas and three cases of fibroblastic osteosarcomas were positive for in situ demonstration of osteocalcin. The malignant tumor giant cells were positive for osteocalcin immunostaining 83%. They were also positive for in situ hybridization. The benign giant cells in five giant cell tumors and five aneurysmal bone cysts were negative for osteocalcin immunostaining. The benign giant cells in three chondroblastoma and three Paget's disease were positive for osteocalcin. In this study, the osteocalcin in situ hybridization and immunostaining has very important meaning for making differential diagnoses of, especially giant cell rich bone forming tumors.

Base Sequence↗

PubMed1994

Six osteocalcin assays compared.

We evaluated comparatively six commercially available osteocalcin kits: ELSA-OST-NAT IRMA (CIS), ELSA-Osteo IRMA (CIS), Osteocalcin IRMA (Nichols), OSTK-PR RIA (CIS), OSCA Test Osteocalcin RIA (Henning), and Osteocalcin RIA (Nichols). All of them presented acceptable values of variance, sensitivity, and serial dilutions. However, only the ELSA-OST-NAT, ELSA-Osteo, and OSCA Test kits had analytical recoveries near 100% for added purified human osteocalcin; the other assays showed important differences in standardization. After treating samples with antibody against amino acids 43-49 of human osteocalcin, only the ELSA-OST-NAT IRMA detected no residual osteocalcin. Stability of serum values at room temperature and at 4 degrees C is good in the ELSA-Osteo and Osteocalcin IRMAs and in the Henning RIA if serum samples are collected in special tubes available with the kit. Differences in stability and after antibody treatment can be due to the degradation of intact osteocalcin molecules in serum and to osteocalcin fragments detected by assays. Results by different assays were well correlated for samples from controls and postmenopausal osteoporotic women but not for patients with impaired renal function or Paget disease of bone, probably because of osteocalcin fragments accumulated in serum of these patients and differences in specificity of the assays.

Antibody Specificity↗

PubMed1997

State of methylation of the human osteocalcin gene in bone-derived and other types of cells.

DNA methylation is a general mechanism of controlling tissue-specific gene expression. Osteocalcin is a bone matrix protein whose expression is limited almost entirely to osteoblasts. We were interested in determining whether the state of methylation of the osteocalcin gene plays a role in its expression by studying human bone-derived (MG-63, U2-Os, SaOs-2) and other types (normal lymphocytes, A-498, Hep G2) of cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that osteocalcin mRNA production is stimulated by 1,25(OH)2D3 in MG-63 and induced in SaOs-2 but not in U2-Os osteoblast-like osteosarcoma cells. Genomic analysis of the human osteocalcin gene showed that the local surroundings of this single-copy gene are identical in all cell lines studied. Using an isoschizomeric pair of restriction enzymes and Southern analysis, we found that the osteocalcin gene is identically methylated in all three osteosarcoma cell lines. The same sites are also methylated in human normal lymphocytes and A-498 kidney cells, whereas the degree of methylation is higher in Hep G2 human hepatocellular carcinoma cells. Furthermore, the osteocalcin gene was identically protected against enzymatic digestion at the chromatin level in normal lymphocytes and in all cell lines studied. Induction of hypomethylation of DNA by 5-azacytidine treatment did not cause an induction of osteocalcin synthesis in these cell lines. On the contrary, it attenuated the induction by 1,25(OH)2D3 in MG-63 cells. In gel mobility shift assays, human vitamin D receptor and the AP-1 transcription factor bound to an unmethylated response element oligonucleotide of the osteocalcin gene with greater affinity than to an in vitro methylated response element. These results indicate that the in vivo methylation state of the osteocalcin gene at sites determined in this study does not correlate with the inducibility of this gene. Nevertheless, the in vitro results clearly indicated that hypomethylation of critical regions of the osteocalcin gene promoter is a potential mechanism influencing effective binding of specific nuclear factors and, consequently, gene expression.

Antimetabolites, Antineoplastic↗