Release of Intact and Fragmented Osteocalcin Molecules from Bone Matrix during Bone Resorption in Vitro
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m314324200
ISSN1083-351X
AutoresKaisa K. Ivaska, Teuvo A. Hentunen, Jukka Vääräniemi, Hannele Ylipahkala, Kim Pettersson, H. Kalervo Väänänen,
Tópico(s)Bone and Dental Protein Studies
ResumoOsteocalcin 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. 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. Osteocalcin (OC) 1The abbreviations used are: OC, osteocalcin; Gla, γ-carboxyglutamic acid; FBS, fetal bovine serum; PTH, parathyroid hormone; BafA1, bafilomycin A1; E64, trans-epoxysuccinyl-L-leucylamido-[4-guanidino]butane; MAb, monoclonal antibody(ies); PBS, phosphate-buffered saline; CTX, C-terminal cross-linked telopeptide of type I collagen; TRACP5b, tartrate-resistant acid phosphatase isoenzyme 5b; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption ionization time of flight mass spectrometry; TRITC, tetramethylrhodamine isothiocyanate. is a 6-kDa noncollagenous protein produced by osteoblasts (1Beresford J.N. Gallagher J.A. Poser J.W. Russell R.G. Metab. Bone Dis. Relat. Res. 1984; 5: 229-234Google Scholar), osteocytes (2Mason D.J. Hillam R.A. Skerry T.M. J. Bone Miner. Res. 1996; 11: 350-357Google Scholar), and odontoblasts (3DiMuzio M.T. Bhown M. Butler W.T. Biochem. J. 1983; 216: 249-257Google Scholar). Osteocalcin messenger RNA has also been detected in tissues other than bone, but it appears to be processed properly only in the bone microenvironment (4Thiede M.A. Smock S.L. Petersen D.N. Grasser W.A. Thompson D.D. Nishimoto S.K. Endocrinology. 1994; 135: 929-937Google Scholar, 5Jung C. Ou Y.C. Yeung F. Frierson Jr., H.F. Kao C. Gene. 2001; 271: 143-150Google Scholar). The structure of osteocalcin is characterized by three glutamic acid residues, which undergo a vitamin K-dependent carboxylation. The γ-carboxyglutamic acid residues (Gla) provide osteocalcin with the ability to bind bone hydroxyapatite with a high affinity (6Dowd T.L. Rosen J.F. Li L. Gundberg C.M. Biochemistry. 2003; 42: 7769-7779Google Scholar, 7Hoang Q.Q. Sicheri F. Howard A.J. Yang D.S. Nature. 2003; 425: 977-980Google Scholar). Osteocalcin is the second most abundant protein in the bone matrix, and it is highly conserved among all vertebrate species (8Hauschka P.V. Lian J.B. Cole D.E. Gundberg C.M. Physiol. Rev. 1989; 69: 990-1047Google Scholar). The biological function of osteocalcin is probably related to the regulation of bone turnover and/or mineralization (9Boskey A.L. Gadaleta S. Gundberg C. Doty S.B. Ducy P. Karsenty G. Bone. 1998; 23: 187-196Google Scholar, 10Ducy P. Desbois C. Boyce B. Pinero G. Story B. Dunstan C. Smith E. Bonadio J. Goldstein S. Gundberg C. Bradley A. Karsenty G. Nature. 1996; 382: 448-452Google Scholar). The expression of osteocalcin is a marker of late osteoblast differentiation and is induced only after the expression of other osteoblastic markers such as alkaline phosphatase and type I collagen (11Owen T.A. Aronow M. Shalhoub V. Barone L.M. Wilming L. Tassinari M.S. Kennedy M.B. Pockwinse S. Lian J.B. Stein G.S. J. Cell. Physiol. 1990; 143: 420-430Google Scholar, 12Bellows C.G. Reimers S.M. Heersche J.N. Cell Tissue Res. 1999; 297: 249-259Google Scholar). Newly synthesized osteocalcin is mostly (60–90%) adsorbed to the bone hydroxyapatite via the Gla residues, but a part of it leaks into the circulation where it can be detected (13Price P.A. Poser J.W. Raman N. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3374-3375Google Scholar, 14Price P.A. Williamson M.K. Lothringer J.W. J. Biol. Chem. 1981; 256: 12760-12766Google Scholar). Although osteoblasts synthesize only intact osteocalcin (15Celeste A.J. Rosen V. Buecker J.L. Kriz R. Wang E.A. Wozney J.M. EMBO J. 1986; 5: 1885-1890Google Scholar), osteocalcin may further undergo intracellular processing or be degraded after secretion, leading to the generation of smaller fragments. Only intact molecules are able to bind to the bone hydroxyapatite, and osteocalcin fragments lose their binding ability probably because of an altered conformation and subsequent loss of affinity for bone mineral (6Dowd T.L. Rosen J.F. Li L. Gundberg C.M. Biochemistry. 2003; 42: 7769-7779Google Scholar, 16Novak J.F. Hayes J.D. Nishimoto S.K. J. Bone Miner. Res. 1997; 12: 1035-1042Google Scholar). Circulating osteocalcin has been widely used in clinical investigations as a marker of bone formation (17Power M.J. Fottrell P.F. Crit. Rev. Clin. Lab. Sci. 1991; 28: 287-335Google Scholar), whereas protein expression has served as an index of osteoblastic phenotype and bone formation in vitro (11Owen T.A. Aronow M. Shalhoub V. Barone L.M. Wilming L. Tassinari M.S. Kennedy M.B. Pockwinse S. Lian J.B. Stein G.S. J. Cell. Physiol. 1990; 143: 420-430Google Scholar). Earlier studies have suggested that circulating osteocalcin originates exclusively from biosynthesis in osteoblasts and not from the breakdown of bone matrix (14Price P.A. Williamson M.K. Lothringer J.W. J. Biol. Chem. 1981; 256: 12760-12766Google Scholar, 18Brown J.P. Delmas P.D. Malaval L. Edouard C. Chapuy M.C. Meunier P.J. Lancet. 1984; 1: 1091-1093Google Scholar, 19Riggs B.L. Tsai K.S. Mann K.G. J. Bone Miner. Res. 1986; 1: 539-542Google Scholar, 20Charles P. Poser J.W. Mosekilde L. Jensen F.T. J. Clin. Invest. 1985; 76: 2254-2258Google Scholar). However, later studies on patients with different metabolic bone diseases have suggested that not all of osteocalcin fragments are derived from the metabolism of osteocalcin in the circulation or peripheral organs but also from osteocalcin embedded in bone (21Gundberg C.M. Weinstein R.S. J. Clin. Invest. 1986; 77: 1762-1767Google Scholar, 22Taylor A.K. Linkhart S. Mohan S. Christenson R.A. Singer F.R. Baylink D.J. J. Clin. Endocrinol. Metab. 1990; 70: 467-472Google Scholar, 23Gundberg C.M. Grant F.D. Conlin P.R. Chen C.J. Brown E.M. Johnson P.J. LeBoff M.S. J. Clin. Endocrinol. Metab. 1991; 72: 438-443Google Scholar). Thus, part of osteocalcin found in the blood may also originate from the resorption process, when osteocalcin embedded in the bone matrix is released during bone degradation (Fig. 1). During bone resorption, osteoclasts secrete protons into the space between the bone surface and cells using vacuolar type H+ATPase, and the acidification results in the dissolution of inorganic mineral. Organic bone matrix degradation is mediated by proteolytic enzymes, primarily cathepsin K, and the released material is endocytosed for further degradation in transcytotic vesicles in the resorbing osteoclast. Eventually, the degraded material is excreted into the extracellular space via a functional secretory domain (24Nesbitt S.A. Horton M.A. Science. 1997; 276: 266-269Google Scholar, 25Salo J. Lehenkari P. Mulari M. Metsikkö K. Väänänen H.K. Science. 1997; 276: 270-273Google Scholar). Because osteocalcin is rather susceptible to proteolysis in vitro (16Novak J.F. Hayes J.D. Nishimoto S.K. J. Bone Miner. Res. 1997; 12: 1035-1042Google Scholar, 26Baumgrass R. Williamson M.K. Price P.A. J. Bone Miner. Res. 1997; 12: 447-455Google Scholar), acids and proteases may also attack osteocalcin during bone degradation. Although the secretion of osteocalcin from osteoblasts has been widely studied, the release of osteocalcin molecules from the bone matrix and their potential contribution to the circulating osteocalcin pool has been discussed (27Christenson R.H. Clin. Biochem. 1997; 30: 573-593Google Scholar, 28Fohr B. Dunstan C.R. Seibel M.J. J. Clin. Endocrinol. Metab. 2003; 88: 5059-5075Google Scholar, 29Chen J.T. Hosoda K. Hasumi K. Ogata E. Shiraki M. J. Bone Miner. Res. 1996; 11: 1784-1792Google Scholar) but not clearly documented. If such a contribution exists, the circulating osteocalcin should rather be considered an indicator of bone turnover and not merely a marker of bone formation. The purpose of the present study was to investigate whether osteocalcin detectable by immunoassays is released from bone during osteoclastic bone resorption, in addition to osteocalcin synthesized during bone formation. Furthermore, our aim was to evaluate different immunoassays for their capability of detecting bone matrix-derived osteocalcin in osteoclast cultures and to gain insight into the molecular forms of osteocalcin released during bone resorption in vitro. Reagents—α-Modified minimum essential medium, fetal bovine serum (FBS), 1 m HEPES solution, and antibiotics (penicillin and streptomycin) were purchased from Invitrogen. Macrophage colony-stimulating factor was purchased from R & D Systems, and the receptor activator of nuclear factor κB ligand (RANKL) and tumor necrosis factor α were from Peprotech. Dexamethasone, parathyroid hormone (PTH), bafilomycin A1 (BafA1, an inhibitor for vacuolar type H+ATPase), trans-epoxysuccinyl-l-leucylamido-[4-guanidino]butane (E64, an inhibitor for cysteine proteases), Hoechst 33258, and leukocyte acid phosphatase kit 387-A were purchased from Sigma. Calcium was determined with Calcium Roche/Hitachi reagents from Roche Applied Science. Streptavidin-coated microtitration plates, Assay® buffer, Delfia® wash solution, Delfia® enhancement solution, and Victor2 Multilabel Counter were from PerkinElmer Life Sciences, and synthetic human osteocalcin (residues 1–49 with Gla at positions 17, 21, and 24) was purchased from Advanced Chemtech (Louisville, KY). The monoclonal antibodies (MAb) for osteocalcin have been described in detail previously (30Hellman J. Käkönen S.M. Matikainen M.T. Karp M. Lövgren T. Väänänen H.K. Pettersson K. J. Bone Miner. Res. 1996; 11: 1165-1175Google Scholar). Briefly, MAb 3G8 requires the full-length molecule for recognition, MAb 8H12 binds to residues in region 7–19 and MAbs 2H9 and 3H8 have an epitope on the residues spanning positions 20–43. In addition, MAb 3H8 favors the Gla-containing forms of osteocalcin. The MAbs were raised either against bovine osteocalcin (3G8 and 3H8) or the fusion protein of glutathione S-transferase and human osteocalcin (8H12 and 2H9). The Alexa 647-labeled goat anti-mouse antibody, TRITC-labeled phalloidin, and succimidyl ester of carboxyfluorescein were purchased from Molecular Probes (Eugene, OR). Osteocalcin Immunoassays—MAb 3G8 or 8H12 were used as biotinylated capture antibodies and MAb 2H9 or 3H8 as europium-labeled tracer antibodies resulting in three different two-site combinations: 3G8/2H9 (I-OC, for intact OC), 8H12/2H9 (M-OC, for the majority of OC), and 8H12/3H8 (T-OC, for total OC). The antibodies were biotinylated with 50-fold molar excesses of biotin-isothiocyanate and labeled with 200-fold molar excesses of europium(III) chelate as described previously (30Hellman J. Käkönen S.M. Matikainen M.T. Karp M. Lövgren T. Väänänen H.K. Pettersson K. J. Bone Miner. Res. 1996; 11: 1165-1175Google Scholar). Synthetic human osteocalcin 1–49 was used as a calibrator. Samples or calibrators (10 μl of each) were added to the wells of streptavidin-coated plates. A mixture containing 100 ng of bio-MAb and 100 ng of Eu-MAb in 50 μl of Assay® buffer containing 5 mmol/liter EDTA was added to each well. After2hof shaking at room temperature (22 °C), the plates were washed six times with Delfia® wash solution, and 200 μl of Delfia® enhancement solution was added to each well. After 30 min of shaking, time-resolved fluorescence was measured using the Victor Multilabel Counter. The calibration curve covered a range from 0.4 to 59 ng/ml. Analytical detection limits were defined as the concentration corresponding to the mean value + 3 S.D. of 12 determinations of the zero calibrator and were set for the I-OC, M-OC, and T-OC assays as 0.02, 0.06, and 0.50 ng/ml, respectively. The withinassay and between-assay coefficient of variations (CVs) were determined using a control sample prepared from FBS and were found to be less than 10% (n = 12). Osteoclast Cultures—A mixed rodent bone cell population was cultured on bovine bone slices as described in detail previously (31Lakkakorpi P. Tuukkanen J. Hentunen T. Järvelin K. Väänänen K. J. Bone Miner. Res. 1989; 4: 817-825Google Scholar) and originally introduced by Boyde et al. (32Boyde A. Ali N.N. Jones S.J. Br. Dent. J. 1984; 156: 216-220Google Scholar) and Chambers et al. (33Chambers T.J. Revell P.A. Fuller K. Athanasou N.A. J. Cell Sci. 1984; 66: 383-399Google Scholar). Briefly, osteoclasts were mechanically isolated from the long bones of 1-day-old Sprague-Dawley rats and allowed to attach to devitalized slices of bovine cortical bone (thickness, ∼0.15 mm) for 30 min, after which the nonadherent cells were washed away. The osteoclasts were cultured on 24-well plates in α-modified minimum essential medium (1 ml/well) supplemented with 10% osteocalcin-depleted FBS, 20 mm HEPES, and 100 units/ml penicillin, 100 μg/ml streptomycin for 3–5 days at +37 °C and 5% CO2. Controls consisting either of bone slices alone or mixed bone cell population plated on glass coverslips were included in each experiment. PTH (10 nm), BafA1 (3 nm), and E64 (50 μm) were added at the beginning of the culture when indicated, and medium samples (30–50 μl/well) were collected daily and stored at –20 °C until analyzed. Human osteoclasts were induced to differentiate from peripheral blood mononuclear cells as published elsewhere (34Hentunen T. Väänänen H.K. J. Bone Miner. Res. 2001; 16: S377Google Scholar). Briefly, mononuclear cells were isolated from human peripheral blood using the Ficoll-Paque™ technique (Amersham Biotech). The cells were washed four times with phosphate-buffered saline (PBS), and 1,000,000 cells/bone slice were allowed to adhere for 2 h. The nonadherent cells were washed away, and the monocytes adhered on the bone were cultured in α-modified minimum essential medium supplemented with 10% regular FBS, 20 mm HEPES, antibiotics, 10 ng/ml of macrophage colony-stimulating factor, 20 ng/ml of RANKL, 10 ng/ml of tumor necrosis factor α, and 10–8m dexamethasone for 12 days. Half of the medium was replaced with fresh medium containing 2-fold concentrations of cytokines every 4 days. Additionally, to study the release of the inorganic matrix in the absence of osteoclasts, some bovine bone slices were exposed to 0.6 m HCl at +4 °C for 24 h. Osteocalcin-depleted Fetal Bovine Serum—The FBS used in the rat osteoclast cultures was depleted of bovine osteocalcin prior to use. Equal amounts of MAbs 8H12, 2H9, and 3H8 (1 mg of MAb mixture/1 ml of matrix) were coupled to a gel matrix (Affi-Gel 10; Bio-Rad) according to the manufacturer's instructions using sterile reagents. FBS (14 ml) was mixed with the coupled matrix (1 ml) in an end-over-end rotator for 1 h at +4 °C and centrifuged for 10 min at 1000 rpm. The supernatant, i.e. osteocalcin-depleted FBS, was collected and stored at –20 °C. The matrix was washed two times with PBS and osteocalcineluted with 0.5 m glycine-HCl, pH 2.5, in an end-over-end rotator for 15 min at +4 °C. The matrix was then washed twice with PBS prior to the preparation of the next batch. Evaluation of Osteoclast Cultures—The osteocalcin immunoassays I-OC, M-OC, and T-OC described above and a competitive osteocalcin enzyme-linked immunosorbent assay (Rat-MID osteocalcin assay; Nordic Biosciences) were used to measure osteocalcin concentration in the medium. The amount of degraded bone matrix was assayed by measuring the C-terminal cross-linked telopeptide of type I collagen (CTX) from the medium (CrossLaps for Culture; Nordic Biosciences), and the activity of tartrate-resistant acid phosphatase isoenzyme 5b (TRACP5b) in the medium was assessed as described previously (35Alatalo S.L. Halleen J.M. Hentunen T.A. Mönkkönen J. Väänänen H.K. Clin. Chem. 2000; 46: 1751-1754Google Scholar). The osteoclasts were fixed with 3% paraformaldehyde and cells were stained for TRACP enzyme activity with a leukocyte acid phosphatase kit. The nuclei were visualized with Hoechst staining, and the TRACP-positive multinucleated cells (at least 3 nuclei) were counted as osteoclasts. In addition to the medium, the osteocalcin and calcium levels were also determined from the supernatants collected from the HCl-treated bone slices. Immunostainings—The organic bone matrix components were visualized with fluorescein-labeled bone. The bone slices were incubated in a bicarbonate solution (pH 8.3) containing a succimidyl ester of carboxyfluorescein for 2 h with gentle stirring and then washed with PBS before usage. Rat osteoclasts were cultured on labeled bone slices for 48 h, fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min on ice, and washed once with 2% bovine serum albumin in PBS (BSA-PBS). The anti-osteocalcin MAb 3H8 (1000 ng/bone slice) diluted in 0.5% BSA-PBS was added and incubated for 45 min at room temperature. After washing with BSA-PBS, the cells were incubated with the Alexa 647-conjugated anti-mouse antibody and TRITC-phalloidin in BSA-PBS for 45 min at room temperature. After washing, the cells were evaluated with a Leica TCS-SP confocal laser scanning microscope equipped with an Argon-Krypton laser (Leica Microsystems). Fluorescein-labeled samples were visualized using a 495–530-nm filter, TRITC-labeled samples were visualized using a 580–630-nm filter, and Alexa 647-labeled samples were visualized using a 660–740-nm filter. Confocal images of triple staining were acquired by a sequential scanning method, i.e. all channels were scanned separately to avoid overlapping. Fractionation of Osteocalcin from Cell Culture Media and Bovine Serum—The culture medium (4 ml) collected from the rat osteoclasts cultured for 5 days with 10 nm PTH and the supernatant obtained from the bone slices incubated with HCl at +4 °C for 24 h were used for the analysis. Further, osteocalcin was isolated from fetal bovine serum using the Affi-Gel 10 matrix coupled to the MAbs 8H12, 2H9, and 3H8 as described above. The samples (the culture medium or the supernatant as such and the osteocalcin isolated from the serum) were extracted in solid phase extraction cartridges (Sep-Pak Plus C18; Millipore) using 40% acetonitrile for elution. The extracted material was fractionated on a Vydac C4 reverse phase high performance liquid chromatography (HPLC) column (2.1 × 150 mm) equipped with a Vydac C4 guard column (both from The Sep/a/ra/tions Group). The solvent gradient used was 2–30% B (0–23 min) (A = 0.1% trifluoroacetic acid/water and B = 0.08% trifluoroacetic acid/acetonitrile), 30–60% B (23–45 min), 60–80% B (45–65 min), 80% B (65–70 min), and 80–2% B (70–75 min) with a flow rate of 150 μl/minute. Fractions (50 μl) were collected during elution, diluted in Assay® buffer and analyzed for osteocalcin with the I-OC, M-OC, and T-OC assays. Fractions collected from the bovine serum fractionation were also analyzed with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-MS) and N-terminal sequencing as described previously (36Ivaska K.K. Hellman J. Likojärvi J. Käkönen S.M. Gerdhem P. Åkesson K. Obrant K.J. Pettersson K. Väänänen H.K. Biochem. Biophys. Res. Commun. 2003; 306: 973-980Google Scholar). Statistical Analysis—Comparison between groups was performed with the nonparametric Wilcoxon's test using the Statistical Analysis System Enterprise Guide 2 program (SAS Institute). Bonferroni adjustment was used in multiple comparisons, and a p value of less than 0.05 was considered statistically significant. Because of normal distribution, one-way analysis of variance was used in the comparison of cultures performed with normal versus osteocalcin-depleted FBS, and Pearson correlation coefficients were used in correlation studies. All of the results are presented as the means ± S.E. Osteocalcin (T-OC assay) was detected in the culture medium of rat osteoclasts after 2–3 days of culture on bovine bone, and the amount of osteocalcin increased in a time-dependent manner (Fig. 2A). The concentration of osteocalcin was higher in the osteoclast cultures than in the corresponding controls, and the difference was statistically significant after 3 days of culture (p = 0.0002 for the bone only control and p = 0.0007 for the cells only control, n = 36) and even more pronounced at the end of the culture period (p < 0.0001 for the bone only and p = 0.0014 for the cells only, n = 30). The release of osteocalcin into the medium was significantly increased when PTH, a known stimulator of bone resorption, was added to the medium, and osteocalcin was almost undetectable when BafA1, a potent inhibitor of the vacuolar type H+ATPase and bone resorption, was included in the culture medium (Fig. 2B). Osteocalcin levels in the BafA1-treated cultures were similar to those of the controls at each time point (p values > 0.05). In addition to the T-OC assay, osteocalcin was also detected with the I-OC and M-OC assays, and the levels of all detectable forms of the protein were significantly reduced in the presence of BafA1 (I-OC, p = 0.012; M-OC, p < 0.0001; and T-OC, p < 0.0001; n = 17; Fig. 3). Furthermore, an increase in osteocalcin in response to PTH was significant for all of the three assays (p values less than 0.0001; data not shown). Stimulation with PTH also resulted in the detection of osteocalcin when a competitive osteocalcin enzyme-linked immunosorbent assay was used (212 ± 21 ng/ml, n = 5). However, in the unstimulated cultures, the osteocalcin levels detected with this assay (22.8 ± 9.1 ng/ml, n = 5) did not differ from the reported detection limit (21.1 ng/ml). Osteocalcin was detected inside the resorbing osteoclasts by staining with a monoclonal antibody against a midmolecular epitope (MAb 3H8, epitope in the fragment 20–43) (Fig. 4, B and D). The bone matrix endocytosed from fluorescein-labeled matrix was also clearly detectable inside the cells (Fig. 4, A and C) and vesicles containing labeled bone partially co-localized with the vesicles positive for osteocalcin.Fig. 3Osteocalcin was detected in a medium with immunoassays for various molecular forms of osteocalcin. The release of osteocalcin and the effect of BafA1 was detected by all three assays I-OC, M-OC, and T-OC. Significances of the bone control, the cell control, and the BafA1-treated culture (columns with patterns) compared with untreated osteoclasts cultured on bone (white columns) are shown as follows: a, p < 0.001; b, p < 0.01; c, p < 0.05 (nonparametric Wilcoxon's test with Bonferroni adjustment; n = 9 for bone only, n = 6 for cells only, n = 17 for osteoclasts on bone, and n = 16 for BafA1).View Large Image Figure ViewerDownload (PPT)Fig. 4Osteocalcin is localized inside bone-resorbing osteoclasts. Fluorescein-labeled bone (A and C) and osteocalcin (B and D) can be found partially in the same compartments inside resorbing cells. Rat osteoclasts were stained for osteocalcin after a 2-day culture on fluorescein-labeled bovine bone slices and z sections (A and B) and x-y sections from the nuclear level (C and D) were obtained with a confocal laser scanning microscope. Actin staining was used to visualize cell boundaries (indicated with a dotted line) and to identify osteoclasts characterized by actin rings.View Large Image Figure ViewerDownload (PPT) The osteocalcin detected in the culture medium of rat osteoclasts had a statistically significant positive correlation to the bone resorption rate as measured by CTX. The correlation coefficient for T-OC and CTX was 0.949 (p < 0.0001, n = 11) at the end of the culture period (day 5) in the cultures performed with osteocalcin-depleted FBS and not treated with stimulators or inhibitors (Fig. 5A). Osteocalcin detected in human osteoclast cultures also had a statistically positive correlation to bone resorption as evaluated by the CTX assay (Fig. 5B). The correlation coefficient at the end of the culture (day 12) was highest for T-OC (r = 0.934, p < 0.0001, n = 48) but also highly significant for the other osteocalcin assays (I-OC, r = 0.916, p < 0.0001; M-OC, r = 0.923, p < 0.0001) and osteocalcin enzyme-linked immunosorbent assay (p = 0.902, p < 0.0001). The treatment of osteoclast cultures with two inhibitors of bone resorption, BafA1 and E64, resulted in distinct responses in putative bone degradation markers (Fig. 6). The amount of all detectable forms of osteocalcin was significantly reduced in the presence of BafA1 compared with the untreated cultures. In particular, the amount of M-OC and T-OC were decreased to ∼10% (p < 0.0001, n = 17) and also I-OC levels reduced to about 40% (p = 0.019, n = 17). In the presence of E64, the amount of osteocalcin was also reduced, but the inhibition was less pronounced. The M-OC and T-OC levels decreased to about 30–40% (p = 0.021 and 0.011, respectively), and a minor and nonsignificant decrease to about 75% was observed in I-OC levels. Thus, the levels of M-OC were significantly different after treatment with BafA1 and E64 (p < 0.0001) as were the levels of T-OC after similar treatments (p = 0.010). A similar, although not significant, trend was observed for I-OC. In contrast, the treatment with BafA1 or E64 resulted in a pronounced reduction in the CTX levels to less than 5% (p < 0.0001 and p = 0.0018, respectively), and the inhibitory effects of both compounds on CTX levels were indistinguishable from each other. The fractionation of osteocalcin from the osteoclast culture medium resulted in one predominant peak that was detectable by all three assays I-OC, M-OC, and T-OC. Also two minor peaks eluting earlier in fractionation were identified, and neither of them contained intact osteocalcin (I-OC). (Fig. 7A). In contrast, only one single peak was observed when osteocalcin released by the acid treatment of bone was fractionated. This peak was detectable with all three assays, including I-OC. (Fig. 7B). The elution profile of bovine serum osteocalcin was more heterogeneous and consisted of at least four main peaks eluting approximately at 43, 45, 47, and 49 min. The peaks at 43 and 45 min were predominantly positive for T-OC, and the peaks at 47 and 49 min were detectable by all three assays I-OC, M-OC, and T-OC (Fig. 7C). The MALDI-MS analysis of these four peaks revealed several prominent ions with molecular masses between 2713 and 5720 Da. The predominant ions identified with MALDI, the N-terminal sequencing results, and the corresponding bovine osteocalcin fragments are summarized in Table I.Table IMALDI-MS analysis of bovine serum osteocalcinHPLC elution timeN-terminal sequencingObserved M+H+Bovine OC fragmentTheoretical M+H+aBecause γ-carboxylation is destroyed in MALDI-MS and not included in observed ions (51,52), all calculations of theoretical molecular
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