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Results for “Osteocalcin”

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At least 19 recordsLinked to original sources

PubMed1997

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.

3T3 Cells↗

PubMed1991

The effect of uninephrectomy on osteocalcin metabolism in sheep: a direct evaluation of renal osteocalcin clearance.

Indirect evidence has suggested that the kidney is a major organ of clearance for osteocalcin, a circulating marker of osteoblast function. The objectives of the present study were (1) to confirm the role of the kidney in osteocalcin clearance (2) to quantify the contribution of extrarenal sites and (3) to investigate the renal mechanism(s) of osteocalcin clearance. Plasma osteocalcin levels, osteocalcin plasma clearance rate (PCR) and plasma production rate (PPR) were determined in oophorectomized (OX) and uninephrectomized oophorectomized (UOX) sheep. The osteocalcin renal extraction efficiency (REE) and the effective renal plasma flow (ERPF) were measured, and the osteocalcin renal clearance rate (RCR) was calculated. The osteocalcin PCR was reduced significantly in UOX compared with OX sheep (2.0 +/- 0.1 (n = 9) vs 2.5 +/- 0.1 litres/h (n = 44); P less than 0.0005). In UOX sheep with plasma creatinine levels less than or equal to 130 mumol/l, the osteocalcin REE was 9 +/- 1.3% and the osteocalcin RCR was 50-91% of osteocalcin PCR (n = 4). In UOX sheep with plasma creatinine levels in the range 100-440 mumol/l, there was a linear relationship between osteocalcin PCR and ERPF; the osteocalcin RCR was related to the osteocalcin PCR (RCR = 0.9 x PCR - 0.50). Intravenous infusion of the synthetic glucocorticoid triamcinolone acetonide (TA) in UOX sheep led to marked decrements in plasma osteocalcin levels and the osteocalcin PPR, and a significant increase in the osteocalcin PCR. These changes were accompanied by a 44% increase in ERPF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: implications for the identification of cells of the osteoblastic lineage in vitro.

Rabbit antisera to bovine osteocalcin were produced independently in two laboratories and their specificities established by western blot analysis. By immunohistochemistry each of the five polyclonal antisera produced an intense cytoplasmic staining in human bone-derived cells. Staining intensity was strongly attenuated by preabsorption of the antisera with osteocalcin. No staining was observed using nonimmune rabbit serum. However, the choice of skin cells as negative controls for osteocalcin synthesis yielded an unexpected positive staining pattern similar to that seen with the bone-derived cells over a range of antiserum dilutions. This was not caused by the uptake of exogenous osteocalcin from the culture medium because a similar pattern of staining was observed when medium was supplemented with osteocalcin-depleted fetal calf serum. Treatment with 1,25-dihydroxyvitamin D3 induced osteocalcin mRNA expression and osteocalcin secretion in cultures of bone-derived cells but not in skin fibroblasts. The results demonstrate that these polyclonal antisera also recognize epitopes shared with other proteins synthesized in culture by skin fibroblasts. Furthermore, three mouse monoclonal antibodies to distinct regions of the osteocalcin molecule show differential staining of human bone-derived cells, skin cells, and osteosarcoma cells (MG63). These observations indicate that the shared epitope residues in the central region of osteocalcin and are consistent with the specific synthesis of osteocalcin by bone cells alone. The observed nonspecificity of many osteocalcin antisera may compromise immunocytochemical studies of the osteoblast phenotype in studies in vitro when based solely on reactivity with inadequately characterized osteocalcin antisera.

Animals↗

PubMed1995

Characterization of structural sequences in the chicken osteocalcin gene: expression of osteocalcin by maturing osteoblasts and by hypertrophic chondrocytes in vitro.

Osteocalcin is one of the major noncollagenous proteins specific to mineralized connective tissues of vertebrates. A cDNA clone encoding the chicken osteocalcin gene was isolated, and the complete coding sequence for the 97-amino-acid pre-pro-osteocalcin was deduced. The 48-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Lys-Arg sequence preceding the NH2-terminal His of the mature 49-amino-acid chicken osteocalcin, which is believed to be necessary for pro-peptide cleavage. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, including Phe at -16, which targets vitamin K-dependent gamma-carboxylation of the three specific Glu residues at positions 17, 21, and 24 in the mature protein. Northern blots of total RNA were prepared from embryonic and adult chicken tissues (bone, brain, heart, intestine, kidney, muscle) and probed with chicken osteocalcin cDNA. The appearance of a single 0.5 kb mRNA species confirms that bone is the major site of osteocalcin expression in vivo. In primary osteoblasts isolated from 17-day embryonic chicken calvaria, an osteocalcin mRNA of similar size is expressed concurrently with culture mineralization in vitro. Hypertrophic chondrocytes from 12-day ventral vertebrae and from the cephalic half of 17-day caudal sternae also express osteocalcin mRNA, but nonhypertrophic chondrocytes from the caudal half of 17-day sternae do not express osteocalcin mRNA.

Amino Acid Sequence↗

PubMed1996

Osteocalcin detection in aging serum and whole blood: stability of different osteocalcin fractions.

Human serum osteocalcin is a well known bone formation marker. On the basis of their different affinities for hydroxyapatite, the total immunoreactive osteocalcin may be separated into two fractions. Six commercial test kits for osteocalcin were compared. All kits reacted with both osteocalcin fractions but the absolute amounts found in the same serum samples differed widely. During serum storage at room temperature, there was no significant loss of osteocalcin during the first 6 h. After longer storage periods, the recorded decrease of osteocalcin depended on the system used: with two kits, over 80% of the original immunoreactive antigen was left after 9 days. It is considered that the different osteocalcin fractions may become useful as markers for different metabolic bone processes. A more precise definition of the various circulating osteocalcin fractions, and the development of separate tests for each fraction, are requirements for the optimal use of osteocalcin as a diagnostic tool for metabolic bone disorders.

Adult↗

PubMed2002

A urine midmolecule osteocalcin assay shows higher discriminatory power than a serum midmolecule osteocalcin assay during short-term alendronate treatment of osteoporotic patients.

We isolated and characterized a peptide fragment corresponding to amino acid sequence 14-28 of human osteocalcin in urine from Paget's disease, and developed a polyclonal antibody reactive to this peptide in urine. We used this antibody to measure urinary fragments of osteocalcin and compared to efficacy of the urinary osteocalcin assay with a serum osteocalcin (sOC) assay (ELISA-Osteo, Cis-Bio International) to monitor the short-term changes in bone turnover in response to alendronate treatment. The synthetic peptide-based urinary osteocalcin (uOC) radioimmunoassay (RIA) showed an analytical sensitivity of 6.25 ng/mL, standard curve range of 3.12-400 ng/mL, and mean intra- (n = 20) and interassay (n = 30) coefficient of variation (CV) of <15%. Urine osteocalcin concentrations in postmenopausal osteoporotic patients were approximately 90% higher than in normal premenopausal controls. Series of 24 h urine and matched serum samples were collected at baseline, 30 days, and 90 days after treatment of postmenopausal osteoporotic patients with daily dose of 10 mg alendronate. We measured urinary osteocalcin (uOc) levels and urinary N-telopeptide (uNTx, Ostex) in urine samples and serum N-telopeptide (sNTx), C-telopeptide (sCTx, Osteometer), serum osteocalcin (sOC) as well as bone-specific alkaline phosphatase (sALP) (Alkphose-B, Metra Biosystems) in serum samples. The percent change data obtained between baseline and 30 days (n = 18) posttreatment suggested a rapid decline in uOC concentration (-27%, p < 0.01) in response to alendronate treatment, as compared with a marginal and nonsignificant decrease in sOC (-7.2%, p = 0.417) or sALP (-3.4%, p = 0.689), two specific markers of bone formation. As expected, due to the coupling of bone formation and bone resorption, the concentration of all markers showed a 30%-45% decline compared with baseline values after 90 days (n = 16) of treatment. Correlation of markers after a 30 day treatment with alendronate revealed a higher correlation (r = 0.61, p < 0.01) between uOC and uNTx, as compared with sOC (r = 0.03, p = 0.447) or sALP (r = -0.14, p = 0.295) with uNTx. Similarly, correlation coefficients with r values between 0.48 and 0.55 (p < 0.05) were observed between uOC, sNTx, and sCTx, whereas no significant correlation was observed between sOC and sNTx or sCTx. These results provide indirect evidence that fragments measured by the urine assay probably originated from bone resorption, and suggest that the uOC assay could be used to assess short-term changes in bone metabolism with regard to osteocalcin.

Adult↗

PubMedDate not supplied

Relationship of osteocalcin and matrix Gla protein gene polymorphisms to serum osteocalcin levels and bone mineral density in postmenopausal Korean women.

OBJECTIVE: To investigate the relationship of osteocalcin and matrix Gla protein (MGP) gene polymorphisms to serum osteocalcin levels, and bone mineral density (BMD) in postmenopausal Korean women. DESIGN: The osteocalcin gene Hind III and MGP gene cytosine-adenine polymorphisms were analyzed in 267 postmenopausal Korean women. Serum osteocalcin, bone alkaline phosphatase, C-telopeptide of type I collagen, and BMD at the lumbar spine and femoral neck were measured. RESULTS: No significant differences in BMD of the lumbar spine and femoral neck were observed across MGP genotypes, whereas a significant lower BMD at the lumbar spine (but not at the femoral neck) was observed in women with the (h) allele (lower case 'h' signifies the presence of the Hind III site) in a dose-response manner. Serum osteocalcin levels among bone turnover markers studied were significantly higher in women without the 210-bp MGP (cytosine-adenine) allele, or with the osteocalcin hh genotype. CONCLUSIONS: The osteocalcin gene Hind III polymorphism is a genetic factor that is associated with BMD of the lumbar spine in Korean women, and Gla gene polymorphisms are associated with higher osteocalcin levels.

Aged↗

PubMed1994

Age- and gender-related changes in the distribution of osteocalcin in the extracellular matrix of normal male and female bone. Possible involvement of osteocalcin in bone remodeling.

With increasing age, bone undergoes changes in remodeling that ultimately compromise the structural integrity of the skeleton. The presence of osteocalcin in bone matrix may alter bone remodeling by promoting osteoclast activity. Whether age- and/or gender-related differences exist in the distribution of osteocalcin within individual bone remodeling units is not known. In this study, we determined the immunohistochemical distribution of osteocalcin in the extracellular matrix of iliac crest bone biopsies obtained from normal male and female volunteers, 20-80 yr old. Four different distribution patterns of osteocalcin within individual osteons were arbitrarily defined as types I, II, III, or IV. The frequency of appearance of each osteon type was determined as a percent of the total osteons per histologic section. The proportion of osteons that stained homogeneously throughout the concentric lamellae (type I) decreased in females and males with increasing age. The proportion of osteons that lack osteocalcin in the matrix immediately adjacent to Haversian canals (type III) increased in females and males with age. Osteons staining intensely in the matrix adjacent to Haversian canals (type II) increased in females and was unchanged in aging males. Osteons that contained osteocalcin-positive resting lines (type IV) increased in bone obtained from males with increasing age but were unchanged in females. Sections of bone immunostained for osteopontin (SPP-I), osteonectin, and decorin did not reveal multiple patterns or alterations in staining with gender or increasing age. We suggest that the morphology of individual bone remodeling units is heterogeneous and the particular morphologic pattern of osteocalcin distribution changes with age and gender. These results suggest that differences in the distribution of osteocalcin in bone matrix may be responsible, in part, for the altered remodeling of bone associated with gender and aging.

Adult↗

PubMed1993

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin.

We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

Animals↗

PubMed1995

Osteocalcin metabolism in late fetal life: fetal and maternal osteocalcin levels.

To clarify the osteocalcin metabolism in the fetus, we determined venous osteocalcin levels of 13 women and umbilical arterial and venous osteocalcin levels of their newborns at delivery. Calcium, phosphorus and alkaline phosphatase levels were also established. Umbilical arterial and venous blood calcium and phosphorus levels were significantly higher than maternal venous blood values (P < 0.001 for all). Umbilical arterial calcium was 2.46 +/- 0.02 mmol/l, phosphorus 1.48 +/- 0.04 mmol/l, umbilical venous calcium 2.50 +/- 0.03 mmol/l, phosphorus 1.45 +/- 0.04 mmol/l, maternal calcium 2.16 +/- 0.03 mmol/l, phosphorus 0.98 +/- 0.04 mmol/l. Both umbilical venous (5.85 +/- 0.66 nmol/l) and arterial (3.49 +/- 0.51 nmol/l) osteocalcin levels were significantly higher than maternal values (1.42 +/- 0.15 nmol/l). The high umbilical venous osteocalcin levels may be due to increased osteocalcin degradation in fetus or placental osteocalcin synthesis.

Adolescent↗

PubMed1992

Vitamin D and osteocalcin levels in liver transplant recipients. Is osteocalcin a reliable marker of bone turnover in such cases?

Patients with advanced liver disease are at increased risk for the development of hepatic osteodystrophy in the form of either osteomalacia or osteoporosis. The pathogenesis of these two bone diseases is multifactorial and includes, among other factors, alterations in vitamin D metabolism, malnutrition and hypogonadism. Little is known regarding vitamin D metabolism and the osteoblastic activity in liver transplant recipients. In order to clarify these issues, vitamin D metabolites and osteocalcin levels were measured prior to and 30 days following liver transplantation in 30 cirrhotic patients of various etiologies. While the mean plasma concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D of the entire group of 30 patients were significantly greater prior to orthotopic liver transplantation (OLTx) as compared to those after OLTx (11.5 +/- 8.6 vs. 7.4 +/- 5.8 ng/ml, p = 0.0066 and 41.0 +/- 34.6 vs. 20.4 +/- 11.0 pg/ml, p = 0.0003, respectively), no significant changes in osteocalcin concentrations pre- or post-transplantation could be demonstrated (5.2 +/- 3.0 vs. 6.4 +/- 4.1 ng/ml, p = 0.51). Furthermore, no correlation between the plasma concentration of osteocalcin and either vitamin D metabolite, the prothrombin time or cyclosporine levels was found. The reasons for the normal levels of osteocalcin prior to OLTx can be explained by the fact that in vitamin-K-deficient states osteocalcin is predominantly decarboxylated and, therefore, a smaller proportion is bound to bone and/or the synthesis of osteocalcin is partially modulated by 1,25-dihydroxyvitamin D, the level of which has been found to be normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1998

Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin.

Osteocalcin, the gamma-carboxyglutamic acid-containing protein, which in most species is the predominant noncollagenous protein of bone and dentin, has been postulated to play roles in bone formation and remodeling. Recently, genetic studies showed that osteocalcin acts as an inhibitor of osteoblast function. Based on von Kossa staining and measurement of mineral apposition rates in tetracycline-labeled bones, osteocalcin knockout animals were reported to have no detectable alterations in bone mineralization. To test the hypothesis that, in addition to regulating osteoblastic activity, osteocalcin is involved in regulating mineral properties, a more sensitive assay of mineralization, Fourier transform infrared microspectroscopy (FT-IRM) was used to study thin sections of femora of 4-week-, 6-month- (intact and ovariectomized), and 9-month-old wild-type and osteocalcin-knockout mice. FT-IRM spectra provided spatially resolved measures of relative mineral and carbonate contents, and parameters indicative of apatite crystal size and perfection. No differences were detected in the mineral properties of the 4-week-old knockout and wild-type mice indicating that the mineralization process was not altered at this time point. Six-month-old wild-type animals had higher mineral contents (mineral:matrix ratios) in cortical as compared with trabecular bones; mineral contents in knockout and wild-type bones were not different. At each age studied, carbonate:phosphate ratios tended to be greater in the wild-type as compared with knockout animals. Detailed analysis of the phosphate nu1,nu3 vibrations in the spectra from 6-month-old wild-type animals indicated that the crystals were larger/more perfect in the cortical as opposed to the trabecular bones. In contrast, in the knockout animals' bones at 6 months, there were no differences between trabecular and cortical bone in terms of carbonate content or crystallite size and perfection. Spectral parameters of the cortical and trabecular bone of the knockout animals resembled those in the wild-type trabecular bone and differed from wild-type cortical bone. In ovariectomized 6-month-old animals, the mineral content (mineral:matrix ratio) in the wild-type cortices increased from periosteum to endosteum, whereas, in the knockout animals' bones, the mineral:matrix ratio was constant. Ovariectomized knockout cortices had lower carbonate:phosphate ratios than wild-type, and crystallite size and perfection resembled that in wild-type trabeculae, and did not increase from periosteum to endosteum. These spatially resolved data provide evidence that osteocalcin is required to stimulate bone mineral maturation.

Age Factors↗

PubMed1993

Production of monoclonal and polyclonal antibodies against human osteocalcin sequences and development of a two-site ELISA for intact human osteocalcin.

In this study we have raised polyclonal and monoclonal antibodies against human osteocalcin sequences coupled to bovine serum albumin (BSA). The antibodies were used for the development of a novel two-site ELISA on microtiter plates for intact human osteocalcin. The epitope, recognized by the monoclonal hybridoma product Os 31/2 that was used as the capture antibody, was located on the amino terminal sequence 1-29. The antibodies used for detection recognized the sequence 1-49. This method failed to detect any tryptic peptides derived from human osteocalcin sequence 1-49 nor did it detect peptide 1-29, thus demonstrating high specificity for the intact osteocalcin 1-49 only. This is a novel technique for the determination of intact osteocalcin. We have also validated the assay parameters for routine use. This direct and relatively simple procedure promises to be a powerful diagnostic and investigative tool.

Adult↗

PubMed1987

Collagen and non-collagenous protein content (osteocalcin, sialoprotein, proteoglycan) in the iliac crest bone and serum osteocalcin in women with and without hand osteoarthritis.

Quantitative and qualitative variations in bone composition were studied in postmortem iliac crest bone specimens from 32 women aged 60 to 75, grouped according to the presence of osteoarthritis at the hand joints. The EDTA extraction yield of macromolecules was significantly greater in the osteoarthritis group. Collagen and osteocalcin content were significantly increased and proteoglycan content decreased in the osteoarthritis group. No difference in sialoprotein content was found. In a clinical study of 20 patients with generalised osteoarthritis, we found compared to 15 controls also significantly increased serum levels of osteocalcin. These results indicate in an unselected group of cases that differences in bone matrix constituents exist between elderly women with and without an osteoarthritic constitution. The finding that these alterations are present even far away from the articular lesion, and the fact that the serum osteocalcin levels are elevated, indicate that osteoarthritis is part of a more generalised bone disease and not a purely cartilage disease.

Aged↗

PubMed2004

Impaired gamma carboxylation of osteocalcin in elderly women with type II diabetes mellitus: relationship between increase in undercarboxylated osteocalcin levels and low bone mineral density.

We conducted a cross-sectional examination of the role of serum vitamin K levels as they relate to bone metabolism in elderly women with type II diabetes mellitus (DM). Eighty-five elderly women with type II DM were enrolled. Three fractions of vitamin K, phylloquinone (PK), menaquinone 4 (menatetrenone; MK 4), and menaquinone 7 (MK 7), along with undercarboxylated osteocalcin (UcOC), intact osteocalcin (IOC), urinary deoxypyridinoline (udpd), urinary type I collagen N-telopeptide (NTx), and intact parathyroid hormone (IPTH) were measured. Bone mineral density was measured in the lumbar spine (LSBMD) by dual-energy X-ray absorptiometry (DXA), and T scores or Z scores were calculated. The patients were divided into two groups by T score, under -2.5 (osteoporotic group) and over -2.5 (non-osteoporotic group). UcOC levels in osteoporotics patients were significantly higher than those in the non-osteoporotic group (3.09 +/- 3.94 vs 1.82 +/- 1.76 ng/ml, P = 0.02). The correlation between Z score and logarithmic UcOC/IOC levels in type II DM showed a negative trend ( P = 0.07) and a significantly and negatively association with logarithmic NTx ( r = -0.38; P = 0.001). In osteoporotic DM, the UcOC/IOC ratio was significantly correlated with the Z score ( r = -0.61; P << 0.05). Furthermore, logarithmic UcOC/IOC showed a negative correlation with logarithmic MK 7 ( r = -0.50; P = 0.001). In conclusion, the reduction in LSBMD in elderly women with type II DM may be associated, in part, with a defect in Gamma-glutamylcarboxylation by vitamin K.

Aged↗

PubMed1993

Secretion of osteocalcin and its propeptide from human osteoblastic cells: dissociation of the secretory patterns of osteocalcin and its propeptide.

Specific immunoassay systems for intact human osteocalcin (I-OC) and its 26-residue propeptide have been newly developed to assess their usefulness as biochemical markers of bone metabolism. Using human cultured osteoblastic periosteal cells, we monitored 24 h secretion of these molecules from the osteoblastic cells and also examined the deposition of Ca, P, and I-OC on the extracellular matrix. At day 5, both I-OC and its propeptide were secreted by osteoblastic cells in a concentration-dependent manner by treatment with 1,25-(OH)2D3. This propeptide was not detected in the serum of adult subjects but was detected in the serum of normal children, which confirmed this in vitro result of propeptide secretion. The secretion of I-OC into medium transiently decreased at day 11, when the rapid accumulation of I-OC, Ca, and P, namely mineralization, was observed on the extracellular matrix of osteoblastic cells, although secretion of the propeptide constantly increased throughout the culture period. Therefore, the ratio of the amount of propeptide to I-OC in the supernatant markedly increased when mineralization started. These data demonstrate the superior specificity of propeptide as a marker of osteoblastic function in vitro compared with I-OC and that monitoring the changes in propeptide to I-OC ratios in the culture supernatant may be useful for predicting the timing of mineralization on the extracellular matrix of osteoblastic cells.

Adolescent↗

PubMed1996

Synergistic induction of osteocalcin gene expression: identification of a bipartite element conferring fibroblast growth factor 2 and cyclic AMP responsiveness in the rat osteocalcin promoter.

Fibroblast growth factors (FGFs) are important regulators of calvarial osteoblast growth and differentiation. We have studied the regulation of the osteoblast-specific gene osteocalcin (OC) by FGF2 in phenotypically immature MC3T3-E1 calvarial osteoblastic cells. FGF2 markedly induces OC mRNA accumulation in MC3T3-E1 cells in the presence of forskolin (FSK). Similarly, OC promoter activity (luciferase reporter) is up-regulated 6-10-fold by FGF2/FSK or by FGF2/8-bromo cyclic AMP. Half-maximal induction of OC promoter activity occurs at 1 nM FGF2. By 5' deletion analysis and dinucleotide point mutations, we map one component of this FGF2/FSK response to a GCAGTCA motif in the region -144 to -138 relative to the OC transcription initiation site. The OC promoter region -154 to -90 confers FGF2/FSK responsiveness on the Rous sarcoma virus minimal promoter. By 3' and internal deletion analyses, the region between -90 to -99 is also found to be necessary for FGF2/FSK synergy (encodes a PuGGTCA motif previously identified as a component of FSK induction). A DNA binding activity that recognizes the region -148 to -125 of the rat OC promoter is induced in crude nuclear extracts from MC3T3-E1 cells treated with FGF2 or FGF2/FSK. This binding activity is sequence-specific and does not recognize the TCAGTCA DNA cognate of AP1. Members of the ATF, Fos, and Jun family are not immunologically detected in this inducible DNA binding activity. However, transient co-expression of ATF3 but not ATF2 selectively attenuates the FGF2 component of induction. Thus, a novel FGF2-regulated DNA-protein interaction in the OC promoter participates in the transcriptional control of OC expression by FGF and cyclic AMP in MC3T3-E1 calvarial osteoblasts.

3T3 Cells↗

PubMed1990

Multiple osteocalcin fragments in human urine and serum as detected by a midmolecule osteocalcin radioimmunoassay.

Reliable markers of bone formation are essential to the investigation of metabolic bone disorders. In this regard, evidence indicates that circulating levels of human osteocalcin (OC) correlate with the skeletal isoenzyme of alkaline phosphatase and can be used as an index of bone formation. A disadvantage of using serum OC as a marker of formation is its diurnal variation. To address this problem we carried out our studies to determine the usefulness of urine in the assessment of bone turnover. Using a midmolecule specific human OC RIA, we were able to detect OC in urine of normal adults (42 mugeq/g creatinine), normal children (849 mu/geq/g creatinine), and Paget's disease patients (613 mugeq/g creatinine). Immunoreactive fragments of OC in human urine and human serum were separated by high pressure liquid chromatography. Multiple fragments were found in normal adult urine that were not detected in normal adult serum. Uremic and Paget's disease sera contain several immunoreactive forms of OC, other than the intact molecule, not found in normal adult serum. Additionally, both Paget's disease sera and urine contained a specific peak of immunoreactive material, eluting at 25% acetonitrile, that was not found in any other serum or urine tested. Urinary OC (uOC) correlated with both skeletal alkaline phosphatase (r = 0.91) and serum OC (r = 0.83), indices of skeletal formation. While uOC has a diurnal variation similar to that of serum OC, determinations of 24-h uOC give integrated values of daily bone turnover rates. Z-Score analysis indicates that uOC (z = 14.04) is better able to distinguish between normal children with high bone turnover and normal adults than either skeletal alkaline phosphatase (z = 8.87) or serum OC (z = 9.01).

Adult↗